Dendritic cell immunotherapy in ovarian cancer

Transcription

ABSTRACTS 2016
14TH ANNUAL MEETING
MAY 10–12, 2016
Rheingoldhalle Congress Center
Mainz, Germany
CIMT Abstract List
Therapeutic Vaccination
Abstract List (001 - 018)
No.:
Presenter: Short talk:
Title:
001
Variation
Allagui-
in susceptibility for human malignant melanomas to oncolytic measles
virus
002
Angerer-
Blood
DC preparations generated using automated CliniMACS Prodigy CD1c/
CD304 enrichment and activation System efficiently activate CD8+ antigenspecific T-cells
003
Aurisicchio-
Epitope-minigenes
004
Baert-
Dendritic
for optimal induction of the immune response against tumor
associated antigens
cell immunotherapy in ovarian cancer: an immunosuppressive chal-
lenge
005
Banki-
Combination
of oncolytic virotherapy and DC-based immunotherapy for the
treatment of melanoma
006
Bialkowski-
Intralymphatic
007
Bigalke-
WT-1
008
009
Bulgarelli-
The
010
Bunseyes
Immune
011
Buonaguro-
Discovery
012
Capasso-
An Epitope
013
Cebulayes
TLR9
014
Conner-
Immune
015
016
Dal Col-
Coding-
017
de Haar-
Development
018
Deiser-
Next-generation
mRNA vaccine induces CD8 T-cell responses that inhibit the
growth of mucosally located tumors
and PRAME mRNA transfected TLR 7/8 polarized fast DCs can raise specific immune responses in AML patients that correlate with clinical outcome
-
This abstract has been withdrawn
double face of dendritic cell vaccination in metastatic melanoma: inducing
intratumor immune response can switch tumor cells toward dedifferentiated
state
responses to a mutation-specific peptide vaccine targeting IDH1R132H
in patients with IDH1R132H-mutated gliomas
to first-in-man studies of a multi-peptide-based hepatocellular carcinoma vaccine adjuvanted with CV8102 (RNAdjuvant®) - HEPAVAC
Discovery and Improvement System (EDIS) to study MHC-I epitopes
and improve their sequences
stimulation is required for recall of functional immune memory response
against neo-antigen relapse in liver
responses following intrapleural administration of the oncolytic HSV
Seprehvir in patients with malignant pleural mesothelioma
-
and non coding-RNA profiling of active dendritic cells following stimulation with highly immunogenic tumor cell lysates
of a GMP production protocol for a cord blood-derived dendritic
cell-based vaccine to prevent relapses after hematopoietic cell transplantation in
children with AML
dendritic cell vaccination in postremission therapy of AML:
results of a clinical phase I trial
CIMT Abstract List
No.:
Title:
019
Theranostic
Dewitte-
020
Dorer-
MERIT:
mRNA-loaded microbubbles for ultrasound-assisted dendritic cell
based cancer vaccination
Individualized cancer vaccines for the treatment of TNBC – a phase I
trial
021
Dörrie-
A phase
I/II clinical trial on malignant melanoma with in vitro optimized
mRNA-electroporated dendritic cells as therapeutic vaccine
022
Eyrich-
Dendritic
023
Eyrich-
Characterization
024
Feger-
ORFV Vector
025
Fotaki-
Allogeneic
026
Frøsig-
The Ellegaard
cell vaccination with partial Treg depletion in relapsed glioblastoma –
results from the pilot phase of the HIT-HGG Rez Immunvac study
of TLR3/8-PGE2 versus TNFα/IL-1ß matured dendritic cells
produced for clinical vaccination trials: impact of maturation on migration and
T-cell priming/crosspresentation capacities
Vaccines – therapeutic potency in robust CRPV rabbit tumor model
dendritic cells (AlloDCs) transduced with an infection-enhanced
adenovirus as adjuvant for cancer immunotherapy
Göttingen minipig as a large animal model for anti-cancer vaccina-
tion
027
Gaudernack-
UV1
– a second-generation, peptide-based, therapeutic cancer vaccine targeting
the reverse transcriptase subunit of human telomerase (hTERT)
028
Gerer-
Immunotherapy
029
Grees-
Development
of the Merkel Cell Carcinoma by vaccination with optimized
DCs transfected with the viral oncogenic driver – the large T antigen
of dendritic cell vaccination for combined melanoma immuno-
therapy
030
Grenov-
Developing
a cancer vaccine for two-dimensional T cell activation using the
Invariant chain
031
Hammerich-
Flt3L-based
032
Haradayes
A novel
033
Heidenreich-
RNAdjuvant®,
034
Høgset-
Photochemical
035
Holmberg-
Peptide
036
Hooijberg-
Extent
in situ vaccination for the treatment of lymphoma
combination immunotherapy consisting of tumor-associated macrophage-targeted vaccine, TLR agonist, and neoantigen-specific T cell transfer
cures tumor highly resistant to immune checkpoint blockade
a novel, highly-potent RNA-based adjuvant, combines strong immunostimulatory capacities with a favorable safety profile
internalization – light-induced enhancement of MHC Class I
antigen presentation, giving strong enhancement of cytotoxic T-cell responses to
vaccination
vaccination against cancer testis antigens in combination with hypomethylating treatment for patients with Myelodysplastic Syndrome and Acute
Myeloid Leukemia: An ongoing phase I study
and location of tumor infiltrating lymphocytes in microsatellite stable
colon cancer predict outcome to adjuvant Active Specific Immunotherapy
CIMT Abstract List
No.:
Title:
037
BRAF and
Hoyer-
MEK inhibitors influence human immune cell phenotype and func-
tion
038
Jabulowsky-
A first-in-human
phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients
039
Kramer-
Design
040
Kuttruff-Coqui-
041
Kyzirakos-
A pipeline
042
Lichty-
Oncolytic
043
Löffleryes
Personalized
044
Lybaert-
Innovative
045
Mazorrayes
Immunological
046
Mikyšková-
Cancer
047
Milleryes
IVAC®
048
Moiseyes
Improved
049
Montico-
Exploiting
050
Mottas-
Mixed
051
Müller-
The mechanism
052
Nair-
Oncolytic
053
Nelde-
Identification
054
Orlinger-
Development
055
Peres-
Polymeric
of reversible antigen-adjuvant conjugates for triggered release inside
antigen-presenting cells
GAPVAC-101 phase I trial: First data of an innovative actively personalized peptide vaccination trial in patients with newly diagnosed glioblastoma
for fast track identification of candidate neoantigens from cancer
exome sequencing data
viral immunotherapy of HPV+ cancer
multi-peptide vaccination induces immune responses associated
with long term survival in a patient with metastatic intrahepatic cholangiocarcinoma
generic strategies for the encapsulation of patient-specific cancer
antigens into immune-modulating particles
results obtained in castration-resistant prostate cancer patients
treated with an EGFR-based vaccine.
immunotherapy using dendritic cells pulsed with tumor cells killed by
high hydrostatic pressure in murine models for prostate cancer
MUTANOME – a first-in-human phase I clinical trial targeting individual
mutant neoantigens for the treatment of melanoma
mutanome-directed cancer immunotherapy by immunoinformatic
analysis of cancer neo-epitopes for regulatory T cell activation potential
immunogenic cell death features for improved dendritic cell-based
therapeutic vaccine against mantle cell lymphoma
ligand coated gold nanoparticles as carrier of R848 for cancer immunotherapy
of immune stimulation by Orf virus – a novel viral vector for
therapeutic cancer vaccines
poliovirus activates antigen-presenting cells and promotes anti-cancer
responses in vitro and in vivo
and characterization of HLA class I-restricted MYD88 L265Pderived peptides as tumor-specific targets for immunotherapy
of novel replication-defective lymphocytic choriomeningitis virus
vectors expressing HPV-16 antigens for immunotherapy
nanoparticle-based vaccine to target dendritic cells and the tumor
microenvironment
CIMT Abstract List
No.:
Title:
056
A phase
Podola-
057
Podrazil-
Immunological
058
Rabsteyn-
iVacALL:
059
Rabu-
Optimizing
060
Ramachandran-
061
Rammensee-A
062
063
Rothe-
Enhancing
064
Sainz-
Promising
065
Sanders-
Xenogeneic
2a trial and related preclinical studies to investigate the immunologic
impact, anti-tumor efficacy and safety of VXM01, an oral T-cell inducing vaccine, in late stage colorectal cancer patients
parameters in phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) in patients with rising PSA after primary prostatectomy or salvage radiotherapy for prostate cancer
A personalized peptide-vaccination design platform for pediatric acute
lymphoblastic leukemia patients based on patient-individual tumor-specific
variants
synthetic long peptide-based anti-tumor vaccination using protease
sensitive linkers
-
Preclinical evaluation of triple microRNA-attenuated oncolytic Semliki forest
virus in glioma and neuroblastoma
new synthetic lipopeptide is a superior adjuvant for peptide vaccination
dendritic cell-induced T-cell responses by immunomodulating agents
melanoma therapeutic cancer vaccine based on hybrid lipid-polymeric nanoparticles
vascular endothelial growth factor-2 vaccination in tumor bearing
mice
066
Schütz-
Immunomodulatory
capacity of CD47 functionalized artificial antigen-presenting cells (aAPCCD47+)
067
Seth-
Synergistic
068
Urbiola-
LCMV-GP
069
Van Acker-
Superior
combination of vasculature disrupting agent with TLR7/8 agonist:
Promising strategy for melanoma therapy
pseudotyped oncolytic vesicular stomatitis virus for the treatment of
prostate cancer
innate immune effector cell recruitment by interleukin-15 dendritic
cells
Type I IFN induced upon particle mediated intravenous delivery of antigen
mRNA enhances specific immune responses
070
Van der Jeught-
071
Verbeke-
Messenger
072
Vormehr-
Neo-epitopes
073
Wollmann-
LCMV-GP
074
Yangyes
Immunotherapy
RNA DOTAP-Cholesterol lipoplexes containing TLR agonists allow
single step antigen-loading and maturation of dendritic cells
generated by insertions, deletions and gene fusions as targets for
personalized tumor vaccination
pseudotyped oncolytic vesicular stomatitis virus for the treatment of
ovarian cancer
with INO-3112 (HPV16 and HPV18 plasmids + IL-12 DNA) in
Human Papillomavirus (HPV) associated Head and Neck Squamous Cell Carcinoma (HNSCCa): Interim results
CIMT Abstract List
Cellular Therapy
No.:
Title:
075
Identifying
Allard-
076
Amann-
Targeting
077
Audehm-
Comparison
078
Berger-
Generation
079
Bianchi-
Development
080
Bonte-
Functional
081
Brey-
Targeting
082
Campillo-Davo-
083
Cappuzzello-
Enhancing
084
Chaperot-
Potential
085
Cripe-
Seprehvir,
086
Dutoit-
MET-specific
CARs for cell therapy of patients with GBM
087
Fåne-
Development
of novel chimeric antigen receptors (CAR) to treat B-cell malig-
rare, high avidity self/tumor-specific CD8 T cells in cancer patients
simultaneously non-mutated HLA.A2-restricted MDM2 and p53
tumor-associated antigens as a novel double-edged swords approach for TCR
gene therapy for multiple myeloma
of two allorestricted T-cell receptors targeting two different
Myeloperoxidase-derived HLA-B*07:02-restricted peptide epitopes with different
MHC affinities with respect for their therapeutic potential
of chimeric antigen receptor - modified memory stem cell CD8+ T
lymphocytes from naive precursors by modulation of Wnt/ß-catenin pathway or
inhibition of Akt-signaling
of imaging strategies for investigation of TCR with defined antitumor reactivity in vivo
evaluation of tumor antigen specific T-cells generated from TCR
transduced human hematopoietic stem cells
HCMV-infected fibroblasts with bi-specific CAR-T cells
RNAi-mediated silencing of endogenous TCR enhances tumor killing activity of
TCR-engineered WT1 peptide-specific CD8+ T cells
Cytokine-Induced Killer cell activity with Her2-specific Fc-engineered antibodies and antibody derivatives
immunogenicity of PUVA-induced cell death
an oncolytic herpes immunotherapeutic, enhances GD2-directed
Chimeric Antigen Receptor (CAR) T-cell therapy in GD2-expressing solid tumor
xenografts
nancies
088
Friese-
Constructing
artificial antigen-presenting cells for improved T-cell function in
adoptive T-cell therapy of melanoma
089
Gary-
Insights
090
Gomez-Eerlandyes
091
Hillerdal-
Characterization
092
Hodgins-
Liposomes
093
Inderberg-
Adoptive
into the preventive/preemptive adoptive transfer of CMV- and EBV-specific peptide-stimulated T cells after allogeneic stem cell transplantation as part
of the phase I/IIa clinical trial MULTIVIR-01
Adoptive transfer of autologous T cells modified with a MART-1 specific TCR in
advanced stage melanoma patients
of the avidity of TCR-engineered T cells with novel and established approaches
encapsulating zoledronic acid for cancer immunotherapy and their
effect on the in vivo biodistribution of Vg9/Vd2 T cells in different tumour models
immunotherapy with a little help from CD4 T cells
CIMT Abstract List
Cellular Therapy
No.:
Title:
094
In vitro
Jamitzky-
095
Janssen-
Rapid
096
Jin-
Safe engineering
097
Kayser-
CD4+
098
Kirkin-
Development
099
Klaver-
Plasma
100
Kraus-
Functional
101
Kremer-
CXCR2
102
Kunert-
TCRs
103
Kunert-
Intra-tumoral
104
Lameris-
Activation
105
Legscha-
Targeting
106
MacLeod-
Integration
107
Mall-
Mapping
stimulation conditions affect the immune phenotype of both CD4+ and
CD8+ T cells expressing a GD2-specific chimeric antigen receptor
recovery of innate immune cells after αβ T-cell depleted allogeneic stem
cell transplantation from matched related and unrelated donors
of CAR T cells for adoptive cell therapy of cancer using longterm episomal gene transfer
T-helper-1 cells against the tumor antigens WT1, NY-ESO-1, ROR1,
MAGE-A3 and Survivin for adoptive transfer to treat cancer
of multi-groove tissue culture flasks for growing of lymphocytes
used in adoptive immunotherapy
IFNγ and IL-6 levels correlate with peripheral T-cell numbers in RCC
patients treated with CAR T-cells
characterization of different variants of a PD-1-CD28-fusion receptor
chemokine receptor transduction of human NK cells to improve migration to solid tumors
for MAGE-C2, in combination with epigenetic drug treatment of target
cells, yield tumor-selective therapeutic T cells
production of IL18, but not IL12, by ‘smart’ T cells is non-toxic
and counteracts immune evasion of solid tumors, prolonging survival
of invariant natural killer T-cells by a unique anti-CD1d single domain antibody induces potent tumor destruction in vitro
tumor suppressor p53 isoforms as a novel approach to improve T-cell
based immunotherapy
of a CD19 CAR gene into the TCR alpha chain locus streamlines
production of allogeneic gene-edited CAR T cells
of T-cell receptor-engineered human T cells at the tumor site by Immuno-
PET
108
McCreedyyes
Allogeneic
CAR-T cells gene edited to insert an anti-CD19 CAR into the TCR
alpha locus target and kill CD19+ Raji lymphoma tumors in vitro and in vivo
without causing GvHD
109
Mensali-
Csk overexpression
110
Mroz-
Individualized
111
Oberoi-
Generation
112
makes T cell dummy
immunotherapy of ovarian cancer by targeting Claudin-6 with
CAR-engineered T cells
of tumor-specific NK cells by differentiation of CAR-gene transduced
hematopoietic progenitors
-
CIMT Abstract List
Cellular Therapy
No.:
Title:
113
Exploiting
Owens-
114
Pfeifferyes
Towards
115
Raemdonck-
Exploring
116
117
Rataj-
Arming
118
Sandri-
Feasibility
119
Schaft-
Receptor-transfected
120
Schooryes
On- and
121
Schörgyes
Combining
122
Schütt-
Two are
123
Schütz-
Nanoparticle
124
Simonyes
Retrieval
125
Singhyes
Novel immunotherapies
126
Solum-
Serum
127
128
Such-
Characterization
129
Taborska-
Human
130
Tosi-
Identification
131
Tubb-
Targeting
132
Uslu-
Generating
133
Voss-
A novel
tumour infiltrating lymphocytes (TILs) as a therapeutic strategy in
ovarian cancer – a proof of concept study
-
in vivo delivery of chimeric antigen receptors
novel siRNA delivery approaches with cytotoxic T cells
T cells with activating FcγRIIIa receptors for antibody redirected lysis of
cancer cells
of telomerase-specific adoptive T-cell therapy for hematologic and
solid malignancies
γ/δ T cells; the new magic bullets against melanoma?
off target toxicity profiling for adoptive cell therapy by mass spectrometry-based immunopeptidome analysis of primary human normal tissues
tumor antigen (TA) specific Th1 cells with immune checkpoint
blocking antibodies induces tumor regression in advanced carcinomas
better than one?! Improving safety for CAR T cell therapy
based antigen-specific redirection of T cells to tumors
of functional TCRs from single neo-antigen-specific T cells: Toward
individualized TCR-engineered therapies
for recurrent glioblastoma: The efficacy of CD133 BiTEs
and CAR T cells in preclinical models
replacement might substitute human serum in the GMP production of
Dendritic Cells
-
of recognition profiles of TCRs by a novel DNA-barcode based
multiplex technology
dendritic cells pulsed with high hydrostatic pressure-inactivated prostate cancer cells and matured with poly(I:C) induce autologous lymphocytes to
ex vivo recognize and kill prostate cancer cells
of a HLA-A*0201-restricted immunogenic epitope from the universal tumor antigen DEPDC1
of recurrent somatic cancer mutations for T cell receptor gene therapy
T cells expressing two additional receptors (TETARs) by combining a chimeric antigen receptor and a conventional T-cell receptor for multi-hit
cancer immunotherapy
stabilized single chain TCR format allows for the generation of virus/
tumor antigen-bispecific human T-cells and prevents mispairing with endogenous TCRs
CIMT Abstract List
Cellular Therapy
No.:
Title:
134
A universal
Wälchli-
135
Weinstein-Marom-
136
Wennhold-
Tumorantigen-Specific
137
Westergaard-Preclinical
138
Zhang-
Targeted
killer T-cell for adoptive cell therapy of cancer
Enhancing the effector functions of T cells with a combination of new mRNA
adjuvants for improving adoptive cell therapy
CD40-activated B cells for cancer immunotherapy
development of Tumor-Infiltrating Lymphocytes (TILs) based Adoptive Cell Transfer Immunotherapy (ACT) for patients with advanced ovarian
cancer
NK cells display potent activity against glioblastoma and induce protective antitumor immunity
CIMT Abstract List
Immunomonitoring
No.:
Title:
139
Massive
Andersen-
140
Bentzenyes
Next-generation
141
Challis-
CIP NK
142
Chandran-
Automated
143
Chiang-
Radiation-expanded
144
Coosemans-
Preliminary
multiplexing: DNA barcode Dextramers for T cell epitope discovery and
epitope profiling
detection of cancer-responsive T cells using DNA barcodelabeled peptide-Major Histocompatibility Complex I multimers
proficiency panel 2016: Reducing inter-lab variation in NK activation
and functional markers, CIP
flow cytometry analysis by ReFlow
myeloid-derived suppressor cells are responsible for local
failure of radiation therapy
results of a prospective immunomonitoring trial in ovarian cancer
patients
145
de Goeje-
Immune
monitoring of lung cancer patients to predict clinical outcome using an
automated pipeline for flow cytometry data analysis
146
de Koningyes
CD4+
147
Galaine-
Immunoprevalence
148
Gouttefangeas-
149
Krebs-
Evaluation
150
Lyngaa-
Type,
151
Mandruzzato-Results
152
Niedermannyes
Noninvasive
153
Omokoko-
NGS-based
154
Peper-
Peptide-specific
T-cell immunomonitoring after hematopoietic cell transplantation: identifying patients at risk for virus-predicted adverse outcomes
and magnitude of HLA-DP4 versus HLA-DR-restricted spontaneous CD4 Th1 responses against telomerase in cancer patients
Immunomonitoring and immunoguiding: update on the CIP activities, CIP
of novel predictive marker molecules in malignant melanoma immunotherapy
frequency and breadth of tumor associated antigen reactivity in tumor
infiltrating lymphocyte from metastatic melanoma patients
from the first phase of a harmonization effort for the phenotyping of human myeloid-derived suppressor cells, CIP
ImmunoPET imaging of the PD-1/PD-L1 checkpoint in naïve and
irradiated tumor-bearing mice
αβTCR repertoire analysis in tumor and blood from three melanoma
patients pre and post IVAC® MUTANOME vaccination
T-cell responses against tumor-specific HLA ligands in ovarian
cancer
antigen specific Treg from the bone marrow migrate towards increased
S1P and CCL2 gradients established in the blood of breast cancer patients
155
Rathinasamy-Tumor
156
Rodrigues-Santosyes
157
Schmidtyes
Isolation
Immune monitoring of natural killer cells in chronic myeloid leukemia: split
anergy status depend on tyrosine kinase inhibitor therapy
and analysis of tumor-specific CD8 and CD4 T cells with high affinity,
reversible pMHC multimers
CIMT Abstract List
Immunomonitoring
No.:
Title:
158
PD-1 blockade
Simon-
159
Stam-
Systemic
160
Tudor-
An optimized
161
Turksma-
Antigen-specific
162
Vigano-
Clinical
163
Welters-
Detection
induces quantitative and qualitative changes within a vast and
common antigen-specific T cell repertoire in melanoma treated patients
WT-1 specific T cell reactivity in relation to immune status and
survival following ablative treatment of locally advanced pancreatic cancer by
irreversible electroporation
IFN-γ ELISpot assay to determine CMV protein-reactive effector
cells of cell- mediated immunity
T cell immunomonitoring by HLA tetramer combinatorial
coding for CD8 T cells and CD40L expression on antigen-specific CD4 T cells
immunomonitoring strategies assessing on-target and off-target effects
of anti-CD73 mAbs - The TumAdoR collaborative project
and functional assessment of regulatory T cells in clinical samples,
CIP
164
Welters-
A kit
for the preparation of T-cell Receptor Engineered Reference Sample (TERS)
to control T-cell assay performance, CIP
165
Wistuba-Hamprecht-
166
Zelle-Rieser-
Bone
Associations of peripheral blood Vδ1+ γδ T-cells with overall survival of
melanoma patients
marrow T cells from myeloma patients exhibit features of both T-cell exhaustion and senescence
CIMT Abstract List
New Targets & New Leads
No.:
Title:
167
Identifying
Ashfield-
168
Ayersyes
Relationship
169
Bassani-Sternbergyes
170
Beck-
Validation
171
Bekeschus-
ROS-based
172
Braitbard-
Signal
173
Bräunlein-
Immunogenicity
174
Buettner-
The
175
Charpentier-
Within
176
Deumelandt-
Ex
177
Di Marco-
A “multi-omics
178
Doorduijn-
A novel
179
Doorduijn-
TAP-independent
tumour-specific Class I MHC peptide epitopes by Mass Spectrometry
between immune gene signatures and clinical response to PD-1
blockade with pembrolizumab in patients with advanced solid tumors
Direct identification of neo-epitopes using in-depth immunopeptidomics of
melanoma tissues for the development of anti-tumor immunotherapies
of a clinical 1400-gene assay for genomic profiling of cancer from
DNA and RNA
cancer therapies – a role for cold physical plasma against pancreatic
malignancies in vitro and in vivo
peptide derived monoclonal antibodies impair mmtv-associated tumor
growth
assessment of mutated HLA-ligands identified on melanoma
tissue probes by mass spectrometry
HLA-associated phosphoproteome as a new target for immunotherapy
against hepatocellular carcinoma
the family of MELOE antigens, IRES-dependent translation conditions
exclusive expression in melanoma cells and immunogenicity
vivo expansion of human glioma-infiltrating lymphocytes alters the exhaustion phenotype of T cells
approach” for the identification of T cell epitopes in clear cell
renal cell carcinoma
role for CD4+ T cells in clearance of highly aggressive MHC-I low tumors mediated via NK cells
self-peptides enhance T cell recognition of immune-escaped
tumors
engineering using CRISPR/Cas9: Targeting MMP23 in melanoma
180
Halldórsdóttir-Genetic
181
Ileckayes
Antigen-armed
182
Kedde-
A novel
183
Krächan-
A novel
antibodies in the treatment of B-cell lymphoma
highly tumor-specific antibody for acute myeloid leukemia and
myelodysplastic syndrome targeting a sialylated epitope of CD43
TLR7 agonist reverses NK cell anergy and cures lymphoma-bearing
mice
184
Kretschmer-
Effector
mechanisms of IgA antibodies against CD20 include recruitment of
myeloid cells for ADCC and the alternative complement pathway
185
Kreuzbergyes
IMAB027-DM1
186
Kwekkeboom-Tumor
and IMAB027-vcMMAE, CLDN6-specific antibody-drug conjugates, are effective against human CLDN6-positive cancer cells in vitro and in
vivo
expression of immune inhibitory molecules and TIL counts predict
pancreatic cancer survival
CIMT Abstract List
No.:
Title:
187
Targeting
Lee-
188
Leon-
Human
189
Leon-
Blocking
190
Marillier-
PF-06840003:
191
192
Marschall-
Protecting
193
Menevse-
Discovering
194
Michels-
TiMi1
195
Mitnacht-Kraus-
196
Okadayes
Novel
197
Olwill-
Costimulatory
198
Paret-
Immunogenic
199
Pfeiferyes
Sialyl
200
Platzer-
Cytoreductive
201
Posselt-
Targeting
of reactive oxygen species can be a potential therapeutic strategy for
cancer treatment
IL-2 agonist exhibits a higher antitumor efficacy and lower toxicity than
wild type IL-2 in different preclinical contexts
IL-2 signal in vivo with IL-2 antagonist reduces tumour growth
through the control of regulatory T cell accumulation
a highly selective IDO1 inhibitor that shows good in vivo efficacy
in combination with immune checkpoint inhibitors, and favorable predicted human pharmacokinetic properties
-
immune cells from activation-induced apoptosis via the CD95Lblocking compound APG101
novel targets in a high-throughput fashion: RNAi screen for pancreatic ductal adenocarcinoma (PDAC) associated immune modulators
is a novel immune checkpoint in solid tumors differentially regulating
cAMP-depending signaling in tumor-infiltrating lymphocytes
IMAB362-vcMMAE , a CLDN18.2-specific antibody-drug conjugate, is effective
against human gastric cancer cells in vitro and in vivo
and shared neoantigen for glioma T cell therapy derived from histone 3
variant H3.3 K27M mutation
T-cell engagement by the CD137/HER2 bispecific, PRS-343, leads
to anti-tumor effect and increased tumor infiltrating lymphocytes in a humanized mouse model
lipids of pediatric brain cancer
Glycolipid Stage-Specific Embryonic Antigen 4 (SSEA4) – a novel target for
CAR T cell therapy of solid cancers
and Immunmodulatory drugs influence bispecific CD33/CD3
BiTE® antibody construct (AMG 330) mediated cytotoxicity against Acute Myeloid Leukemia (AML)
DNA damage response genes to improve radiotherapy of pancreatic
cancer
202
Ramskov-
Evaluating
prediction strategies for identification of immunogenic mutationderived neo-epitopes in melanoma
203
Riemer-
Evaluation
204
Ruzicka-
Immunotherapy
205
Schnieders-
4-1BBL
of T cell epitope prediction servers
targeting RIG-I in a mouse model of acute myeloid leukemia
synergizes with IL-12 and IL-2 in the induction of a defensive immune
signature in the human urinary bladder carcinoma microenvironment
CIMT Abstract List
No.:
Title:
206
The
Schuster-
207
Schwenck-
Clinical
208
Shuttleworth-KA2237
209
210
Suarez-Carmona-
211
Thierauf-
Checkpoint-Inhibition
212
Trezise-
Quantitative
213
van Helden-
Rapid
214
Walter-
Anti-tumor
215
Zelba-
In renal
immunopeptidomic landscape of ovarian carcinoma
non invasive in vivo imaging of the differentially expressed tumor associated antigen PSMA by a specific Positron Emission Tomography (PET) ligand
and KA2507: Novel, oral cancer immunotherapeutics targeting PI3Kp110β/p110δ and HDAC6 with single-agent and combination activity
-
Ovarian carcinoma explant culture: model development and application in drug
testing
for advanced mucosal melanoma
live-cell imaging assays for immunotherapy: chemotaxis, T-cell
killing & phagocytosis
generation of T cell receptor like antibodies using genetically reprogrammed memory B cells of immunized rabbits
activity of IMAB027 antibody as a single agent and in combination
with chemotherapy in testicular cancer
cell and prostate cancer a large fraction of the tumor-infiltrated T-cells
cannot be targeted by current checkpoint inhibition approaches
CIMT Abstract List
Improving Immunity
No.:
Title:
216
Second
Beha-
217
Bou Nasser Eddine-
218
Buonaguro-
Effects
219
Clemenz-
Dermaject
generation of IL-15-based tri-functional antibody fusion proteins with
costimulatory TNF-superfamily ligands for cancer therapy
Optimal triggering of anti-tumor CD4+ TH cells by tumor cells expressing
CIITA-driven MHC class II I-A-only molecules
of RNA-based RNAdjuvant® on PBMCs from liver cancer patients in an
ex vivo model
– a novel, convenient intradermal injection device for intracutaneous
injections
220
Colombettiyes
PD-L1
checkpoint blockade enhances anti-tumor activity of CEA TCB, a novel
T-cell bispecific antibody for the treatment of solid tumors
221
Cripeyes
Seprehvir,
222
de Gruijlyes
Local
223
Donnellan-
IMCgp100
224
Eissleryes
Release
225
Holland-
Comparison
226
Hotz-
Reprogramming
an oncolytic herpes viroimmunotherapeutic, enhances therapeutic
efficacy of T cell checkpoint inhibition in solid tumors by increasing T cell
recruitment and remodeling the immunosuppressive microenvironment
adjuvant treatment of clinical stage I-II melanoma with CpG-B/GM-CSF
improves distant recurrence-free survival: long-term follow-up of three randomized controlled phase II trials
ImmTAC: A TCR-based bi-specific immunotherapy for the treatment
of advanced melanoma
of IFN-γ induced chemokines provides the key to efficient combination
immunotherapy of anti-PD-1 antibody with CSF-1R inhibitor
of phase I/II trials regarding antigen-specific versus non-specific
anticancer immunotherapies
of TLR7 signaling enhances antitumor NK and cytotoxic T cell
responses
227
Kapp-
In vitro
and in vivo evaluation of the TLR9 agonist EnanDIM for cancer immunotherapy
228
Kayali-
Platelet-derived
229
Kikodze-
Influence
230
Koksal-
Memory-like
231
KwekkeboomyesFunctionality
232
Kwekkeboom-Blocking
233
O'Donnell-
Probing
microparticles differentially regulates macrophage polarization
of radiofrequency thermal ablation on CD4+ T cell subsets in the
patients with liver cancer
T cells transduced with tumor-specific epitope elicited pronounced
cytotoxic potential
of tumor-infiltrating T cells in hepatocellular carcinoma can be
enhanced by blocking several co-inhibitory pathways
PD-L1 and LAG-3 can revitalize the functionality of tumor-infiltrating
T cells in liver metastasis from colorectal cancer
the increase in neoantigen burden at recurrence in ovarian cancer
CIMT Abstract List
Improving Immunity
No.:
Title:
234
TNFa
Parviainen-
235
Rekdal-
The oncolytic
236
Richardsyes
Hexavalent
237
Sanders-
Immunological,
238
Sapski-
Combinatorial
239
Spagnuolo-
Modulation
240
Tagliamonte-
Efficacy
241
Tähtinen-
T-cell
242
Theurich-
Local
243
Tognarelli-
NK
244
Zhu-
Combination
and IL-2 armed oncolytic adenovirus induces antitumor immune response
and protects from tumor rechallenge in Syrian hamsters
peptide LTX-315 enhances T cell clonality and induces synergy
with CTLA-4 blockade
agonists targeting receptors of the tumor necrosis factor superfamily: TRAIL, CD40L, CD27L and beyond
anti-angiogenic and clinical effects of intratumoral interleukin-12 electrogene therapy plus metronomic cyclophosphamide in dogs with
spontaneous cancer
approaches with costimulatory antibody fusion proteins addressing immunosuppression by IL-10, TGF-beta and immune checkpoints
of T cell recruitment into tumors through synergy between HMGB1
and CXCL12
of a novel multi-drug metronomic chemotherapy combined with a peptide vaccine on tumor challenge in mice
therapy enabling adenoviruses coding for IL-2 and TNF-a systematically
activate tumor-reactive TILs in metastatic, solid cancer
tumor treatment in combination with systemic ipilimumab immunotherapy prolongs overall survival in patients with advanced malignant melanoma
cell characteristics and anti-tumor efficacy in multiple myeloma and lymphoma patients before and after autologous stem cell transplantation
immunotherapy of an inducible, autochthonous, low mutational
load murine lung cancer model expressing human CEA as a tumor-associated
self-antigen
CIMT Abstract List
Tumor Biology and Interaction with the Immune System
No.:
Title:
245
TRPV1
Akman-
246
Al Absi-
Actin
247
Alonsoyes
"Infectious"
248
Arakelian-
Hypoxia
249
Baert-
Tumour-associated
250
Berthel-
Spatial
251
Beyranvand Nejad-
252
Bjerregaard-
MuPeXI:
253
Blattner-
The role
254
Boegel-
Determination
255
Buoncervello-IFN-α
256
Calvo-
Prognostic
257
Das-
Generation
258
de Bruyn-
Treatment
259
Dekenyes
Synergy
260
Dosset-
PD-L1
261
Eggink-
POLE
262
Elkord-
GARP/LAP
263
Erin-
CD200 mimetic
and TrkA agonists alter cytokine secretions of mix leukocyte cultures
obtained from tumor-bearing mice
cytoskeleton remodeling: a novel mechanism for tumor cells to escape
from natural killer cell-mediated cell death
tolerance transforms tumor antigen specific naive CD4 T cells into
induced Tregs in a spontaneous lung tumor model
in the tumor microenvironment: A major regulator of the anti-tumor
immune response
macrophage phenotype makes low grade ovarian cancer a
possible target for immunotherapy
heterogeneity of T cell distribution patterns at the invasive margin of
colorectal cancer liver metastases
T cell costimulatory pathways are required for cisplatin-based chemotherapy
A tool for prediction of neo-epitopes from tumor sequencing data
of CCR5 on MDSC in their recruitment and activation in melanoma
microenvironment
of HLA type and expression from whole transcriptome sequencing data (RNA-Seq)
potentiates the direct and immune-mediated antitumor effects of epigenetic drugs on both metastatic and stem cells of colorectal cancer
role of local immune infiltrate in patients with colon cancer
of MHC class I and class II deficient tumor cell lines using the
CRISPR/Cas9 system
regimen, surgical outcome and T cell differentiation influence prognostic benefit of tumor-infiltrating lymphocytes in high grade serous ovarian
cancer
of anti-PD-1 in combination with targeted therapy is mediated by
CD8+ T cells
tumor expression as an adaptive immune resistance mechanism to counter the antitumor effect of immunogenic chemotherapies
proofreading domain mutations elicit an antitumor immune response in
endometrial cancer
expression on FoxP3+/-Helios+/- Treg subsets in patients with pancreatic cancer and liver metastases from colorectal cancer
PEG-M49 increases therapeutic effects of pegylated liposomal
doxorubicin on poorly differentiated breast carcinoma: Possible role of in-vivo
increased anti-tumoral immune response
CIMT Abstract List
No.:
Title:
264
The effects
Erin-
265
Erin-
Inhibition
266
Erin-
Effects of
267
Fernandes-
Characterization
of PU-H71, a novel HSP90 inhibitor, and radiotherapy co-treatment
in metastatic breast carcinoma: Changes in IL-6 and macrophage inflammatory
protein 2
of PKC activity with Byrostatin alters secretion of inflammatory
chemokines
phosphoramidon on TNF-a and IFN-g release from mix leukocyte
culture obtained from tumor-bearing mice
of the immunogenicity of pancreatic cells in response to elec-
trochemotherapy
268
Fischer-
Tumor-infiltrating
B lymphocytes independently predict outcome in patients
with non-small cell lung cancer and consist mostly of effector subsets
269
Fischer-
Bifunctional
270
Foerster-
The
271
Foerster-
Allogeneic
272
Forlani-
Block
273
Furnessyes
Characterisation
274
Gabriele-
On-chip
275
Georganaki-
Sunitinib
276
Gorris-
Development
277
Hassel-
Investigation
278
Hu-
Role of tumor-derived
279
Kienzle-
Targeting
280
Kim-
A novel
281
Knott-
Tumor-derived
peptide-MHC class I antibody fusions redirect peptide-specific
CD8+ T cells to eliminate tumor cells in vivo
immunome of hepatocellular carcinoma – an in silico analysis
Balb/c mice are more susceptible to B16F10 liver metastasis than
syngeneic C57/Bl6 mice despite a M1-polarized anti-tumor immune response
of the HTLV-1 Tax-1 oncogene-dependent NF-kB activation by the MHC
class II transactivator CIITA. Implications for the control of oncogenic potential
of HTLV-1 infection
of immune and tumour-specific neoantigen landscapes informs
optimal therapeutic targeting in non-small cell lung cancer
dialogue between immune cells and cancer: tracking human dendritic
cell migration and tumor antigen capture upon drug treatment
enhances the anti-tumor responses of agonistic CD40-antibody therapy by reducing MDSCs and synergistically improving endothelial activation and
T-cell recruitment
of multiplex fluorescent IHC immune cell panels to predict immunotherapy outcome
of tumor-reactive T-cell repertoire in the immune infiltrate of
metastatic melanoma under immune checkpoint inhibition
exosomes in immunosuppression in malignant melanoma
Cancer: Encapsulation of TNF-α in pH-sensitive PEI-PEG copolymer
gated dendritic mesoporous silica nanoparticle
model of murine hepatocarcinogenesis in biliary fibrotic mice resembling the multistage process of human primary liver cancer
IL-1 mediates intratumoral immunosuppression via the Tregattracting chemokine CCL22
CIMT Abstract List
No.:
Title:
282
Escape
Koch-
283
Komdeur-
CD103
284
Konkol-
Presence
285
Kwekkeboom-PD-L1,
286
Lennerz-
Ex vivo
287
Low-Marchelli- Patient-derived
288
Marcq-
Into the
289
Metzger-
Surface
290
Momose-
Identification
291
Mullins-
A Crohn´s
292
Nesmiyanov-
mIRNA-155
293
Ozdemir-
Investigation
294
Özgül Özdemir-
295
Park-
Evaluation
296
Parri-
Identifying
297
Parrot-
CD40L+CD4+CD8+
of head and neck squamous cell carcinoma from NKG2D-dependent NK
cell immunosurveillance can be restored by NKG2D ligand depletion
defines intraepithelial CD8+ PD1+ tumor-infiltrating lymphocytes of
prognostic significance in endometrial adenocarcinoma
of immune infiltrates in early phases of prostate cancer: Development
of a preclinical efficacy model to promote immunotherapy development
Galectin-9 and CD8+ TIL are associated with patient survival in Hepatocellular Carcinoma
high-throughput T cell receptor profiling of a melanoma patient’s peripheral tumor antigen-specific T cell repertoire
tumor xenografts in humanized NSG and NSG-SGM3 mice: A
model to study immune responses in cancer therapy
deep: closer look at immune cells and immune checkpoint expression
in human malignant pleural mesothelioma
staining of PD-1 discriminates viable cell populations
of cytotoxic miRNAs in human T cell-released exosomes against
mesenchymal stem cells
related colonic carcinoma cell line showing features of immunoselection is recognized by re-activated autologous tumor-infiltrating lymphocytes as
well as CIK cells
shuttling through gap junctions facilitates CLL progression
of paracrine immunomodulatory effects of mesenchymal stem
cells on the CD4+ T cell subsets
The immunohistochemical investigation of CD44, CD133, NANOG, OCT 3/4,
HLA-G and HLA expressions in the advanced stage breast cancer
of potential factors contributing to immunosuppression in the PDL1
positive tumor microenvironment
the kinases and phosphatases regulating STAT3 with potential dual
anti-cancer and immunotherapeutic effects
intra-tumor double positive T cells : a helper player in
melanoma
298
-
This abstract is withdrawn
299
Prokopi-
Immune
300
Qureshi-
Characterization
evasion by melanoma: Modification of the skin and lymph node immune cell network in a spontaneous melanoma mouse model
of the cancer immune microenvironment of Mdr2(Abcb4)-/mice treated with Diethylnitrosamine and Phenobarbital – A novel model close
to human Hepatocracinogenesis
CIMT Abstract List
No.:
Title:
301
Increased
Qureshi-
302
Ramjiawanyes
cxcr4
303
Röhle-
Characterization
304
Sainiyes
Identification
305
Sapega-
IL-12
306
Sauer-
HLA class
307
Schmidt-
Tumor
308
Schotte-
A patient
309
Schrörs-
Complex
310
Schupp-
Activating
311
Seo-
The role
312
Shatnyeva-
BAG6
313
Siozopoulou-
Desmoid
314
Skadborg-
Characterization
315
Solinas-
Characterization
316
Sonner-
No role
317
Steinhoff-
PD-L1
318
Stoitzner-
Cooperation
319
Tanriover-
The
CD4+ and CD8+ lymphocytic infiltration in patients with triple negative breast cancer suggests susceptibility to immune therapy
inhibition in tumor microenvironment facilitates anti-program death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice
and specificity analysis of tumor infiltrating lymphocytes in
ovarian carcinoma
and characterization of neoepitopes associated with individual
mutational landscape in non-small cell lung cancer
therapy suppresses TC-1 tumor growth accelerated by admixture of the
docetaxel-treated senescent tumor cells
II antigen expression in cervical intraepithelial neoplasia and invasive cancer
and host cell PD-L1 expression is required to mediate suppression of
anti-tumor immunity
derived antibody targeting CD9 inhibits melanoma metastasis
deletion event at B2M locus in a human melanoma patient treated with
IVAC MUTANOME
and repolarizing immune suppressive tumor associated macrophages
using siRNA encapsulated in nano-sized carriers to initiate an anti-tumor immune response against melanoma
of CD8+ T cell-released exosomes on the down-regulation of tumor
invasion and metastasis by elimination of stromal mesenchymal cells
and CBP/p300 regulate ESCRT-mediated exosomes release and protein
sorting
tumors: the importance of the immune cell determination to the development of new treatment options
of inhibitory molecules on tumor-infiltrating lymphocytes in
malignant melanoma
of PD-L1 and PD-1 expression in tumor infiltrating lymphocytes and tertiary lymphoid structures in paired primary tumors and metastases from breast cancer patients
of the stress kinase GCN2 in T cell-mediated tumor rejection as intratumoral tryptophan levels are maintained
upregulation following RLR and TLR-based immunotherapy in a mouse
model of gastric cancer
of Langerhans cells and NK cells guarding the epidermis during
chemical carcinogenesis
expression of Gr1+ and S100A8/A9+ cells in primary tumors and visceral
organs invaded by breast carcinoma cells
CIMT Abstract List
No.:
Title:
320
Alteration
Taranikanti-
321
ten Buren-
Genetic
322
Thomé-
Glioma
323
Treder-
Anti-tumor
324
Vascotto-
Induction
325
Verdegaalyes
A changing
326
Vetter-
Tolerogenic
327
Voigt-
Impact
328
Wölfl-
A dual
329
Wulf-Goldenberg-
330
Zayoud-
The role
331
Zhao-
Genetic
of host immunity with stress in breast cancer patients
engineering in a non-traditional astrocytoma prone mouse background
N-Myc downstream regulated gene 1 (NDRG1) shapes the tumor microenvironment
efficacy by the bispecific tetravalent CD30/CD16A TandAb AFM13
is characterized by strong cross-talk from innate to adaptive immunity and is
enhanced by immune checkpoint inhibitor anti-PD-1
of a well-defined immune response using a novel systemically applied
Toll-Like Receptor 7 agonist in mice and men
neo-antigen landscape in human melanoma under T cell pressure
effects of GM-CSF through expansion of regulatory T-cells and
induction of the Treg-associated chemokine CCL22
of Interleukin-22 on two murine models of lung and breast cancer
role for IL12 in T-cell receptor-dependent and –independent tumor cell
killing: regulation of a DNAM1 mediated, PTPRC/CD45-dependent mechanism
in human effector T-cells
Patient-derived tumor xenografts in humanized mice: a preclinical model for
the development of innovative immunotherapeutics
of the IL-22/IL-22R1 axis in Pancreatic ductal adenocarcinoma
heterogeneity of intra-patient metastases restricts T-cell recognition of
malignant melanoma
001 – 074
Therapeutic
Vaccination
001 | THERAPEUTIC VACCINATION
Variation in susceptibility for human malignant
melanomas to oncolytic measles virus
Allagui F.1,2, Panterne C.1,2, Pouliquen D.1,2, Tangy F.3, Labarrière N.1,2, Achard C.1,2, Dréno B.4,
Khammari A.4, Fonteneau J.-F.1,2, Grégoire M.1,2, Boisgerault N.1,2
INSERM, UMR892, CNRS, UMR6299, Nantes, France,
1
University of Nantes, Nantes, France,
2
CNRS, UMR3569, Institut Pasteur,Viral Genomics and Vaccination Unit, Paris, France,
3
Nantes University Hospital, Department of Dermatology, Nantes, France
4
Oncolytic viruses are developed as novel strategies
production depending on the cell line. Cells resistant
to treat aggressive cancers. Live-attenuated strains of
to MV were also treated with the IFN pathway in-
measles virus (MV) are ideal candidates for oncolytic
hibitor ruxolitinib and became more sensitive to MV,
virotherapy with an excellent safety record. MV is
thus confirming that the innate antiviral signaling is
known to target CD46, which is generally overex-
the critical parameter for oncolytic MV sensitivity.
pressed by cancer cells and is the major entry re-
In conclusion, our data confirm oncolytic MV as a
ceptor for the virus. MV is also highlighted to have
viable therapeutic option for malignant melanoma.
immunogenic properties.Thus it can be harnessed in
The key role of the innate type I IFN response in the
immunotherapeutic approaches.
efficacy of this approach suggests that immunomod-
In the work presented herein, we analyzed the
ulatory therapies should be evaluated in combination
oncolytic effect of MV against a panel of human
with oncolytic viruses. As an example, histone dea-
melanoma cell lines established in our laboratory.
cetylase inhibitors (HDACi) that have been described
These cell lines exhibit varying levels of sensitivity
to dampen innate immunity, could be helpful for im-
to MV infection that cannot be fully explained by
proving oncolysis and therapeutic outcome.
their level of expression of CD46 receptor at their
surface. In melanoma xenograft mouse models,
MV treatment induced important tumor regressions for sensitive cell lines but other were completely insensitive resulting in rapid tumor growth.
We recently demonstrated that the antiviral type I
interferon (IFN) response was critical to determine
the sensitivity of human malignant pleural mesothelioma cells to MV. Thus we analyzed the type I
IFN response in our panel of melanoma cells and
we found that resistant cells had a fully functional
pathway that was activated upon MV infection. On
the contrary, seven out of ten sensitive cell lines
showed defects in this pathway. When pre-treated
with IFN-α or IFN-β, some of these sensitive cell lines
became resistant to MV, suggesting that these defects
could be either upstream or downstream of type IFN
Blood DC preparations generated using automated CliniMACS
Prodigy CD1c/CD304 enrichment and activation System
efficiently activate CD8+ antigen-specific T-cells
Angerer C.1, Schöggl C.1, Schreibelt G.2, Pots J.M.2, De Vries I.J.M.2, Dzionek A.1, Brüning M.1
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany,
1
Radboud University Medical Center, Department of Tumor Immunology, Nijmegen, Netherlands
2
The innate and adaptive immune functions of plas-
Moreover, a detailed functional characterization of
macytoid and myeloid DCs (pDCs and mDCs) make
BDCs of two donors was performed.
them an attractive tool for anti-cancer therapy.
Purity, recovery and phenotype:
Clinical efficacy of vaccination with activated pDCs
Using the CliniMACS Prodigy CD1c/CD304 System,
or mDCs loaded with tumor-peptides was already
BDCs were routinely enriched to a purity of 85% and
demonstrated in phaseI/II studies in melanoma pa-
a recovery of 90%. Isolated BDC were cultured over-
tients resulting in successful induction of anti-tumor
night in the presence of GM-CSF, IL-3, loaded with
immune response and improved overall survival
peptide pools (PepTivators) and activated by the use
(Radboud university medical center, Jolanda M. de
of TLR ligands. Upon cultivation the expression of
Vries). In addition, data from the mouse system
receptors involved in lymph node homing (CCR7)
suggest that IFN-alpha producing pDCs are capable
and the formation of immunological synapsis (CD86,
of trans-activating mDCs thereby enhancing antigen
CD80 and CD83) was induced. Viability of the cells
cross-presentation of mDCs followed by improved
was at 88% in average. The activated BDCs could be
tumor response. A combination of both natural blood
frozen and thawed without induction of alterations
DC subsets therefore provides a promising vaccina-
in their phenotype.
tion approach in cancer immune therapy.
Functional characterization:
The preparation of BDC-vaccines consisting of
Isolated BDCs from two HLA-A2.1+, CMV+ donors
CD304+ (BDCA-4+) pDCs and CD1c+ (BDCA-1+)
were loaded with pp65 PepTivator and co-cultured
mDCs requires a separation system which enables
with autologous pan T-cells. After 6 hours of cultivation
the pre-depletion of monocytes and B-cells and the
re-stimulated CD8+ T-cells produced IFNg and TNFa
subsequent enrichment of BDCs. BDCs additionally
as determined by intracellular staining and up-regu-
need to be cultured overnight for activation and
lated the expression of activation markers. Moreover,
antigen loading. To meet the regulatory require-
BDCs loaded with PepTivator induced strong prolifera-
ments for cell-based therapeutics we have integrated
tion of pp65-tetramer+ CD8+ T-cells indicating their
all manufacturing steps in a closed system operated
capability to induce antigen-specific T-cell responses.
by an automated cell-processing instrument, the
Our data shows the feasibility of the fully automated
CliniMACS ProdigyTM.
production of a BDC-based vaccine using the Clini-
Here we show a summary of the BDC enrichment and
MACS Prodigy instrument, which is currently tested
culture performance and a phenotypic characteriza-
in clinical trials.
tion of BDCs of 6 independent production batches.
Epitope-minigenes for optimal induction of the immune
response against tumor associated antigens
Luberto L.1, Bandini S.1,2, Petrazzuolo A.1, Palombo F.1, Buonaguro L.3, Ciliberto G.3, Aurisicchio L.1
Takis, Rome, Italy,
1
Biogem, Ariano Irpino, Italy,
2
IRCSS Istituto Nazionale Tumori Fondazione Pascale, Naples, Italy
3
We have recently established a workflow that allows
In conclusion, we show that minigenes delivered via
the identification of T cell epitopes within Tumor
DNA-EGT and based on predicted and/or experimen-
Associated Antigens (TAAs) and the construction
tally identified epitopes are powerful tools to induce
of genetic cancer vaccine based on the use of mini-
immune responses and combat cancer. Combina-
genes.
tion studies of minigenes with peptide vaccination,
The T-cell epitope in silico prediction approach is
chemotherapy and immune checkpoint blockade
based on three criteria: 1) binding to MHC Class I
may define new therapeutic opportunities for cancer
alleles; 2) uniqueness to the antigen of interest; 3)
patients.
increased likelihood of natural processing. The combination of in silico prediction and a biochemical
binding/stability assay resulted in an accurate identification of novel TAA-derived epitopes. Predicted
T cell epitopes were connected by furin sensitive
linkers and linked to human tissue plasminogen activator (TPA) signal and E. Coli enterotoxin B subunit,
to construct an optimal minigene scaffold used as
vaccine candidate.
The present study was aimed at evaluating HER2/neu
and hTERT (telomerase) minigenes with the same
technology platform. First of all, minigenes delivered
via Electro Gene Transfer (DNA-EGT) were more immunogenic than genetic vectors encoding the fulllength protein or peptides injected subcutaneously
and they were able to break immune tolerance in wild
type and HLA-A0201 transgenic mice. Moreover, a B
cell epitope selected within HER2 was able to induce
antibodies and provide significant tumor protection
in a HER2-driven transgenic mouse model. Finally,
we demonstrated that the heterologous prime/
boost modality with Long Synthetic Peptides (LSPs)
and minigenes provided a strong synergic effect.
Dendritic cell immunotherapy in ovarian cancer: an
immunosuppressive challenge
Baert T.1,2, Garg A.3, Van Hoylandt A.1,2, Vergote I.1,2, Coosemans A.1,2
KULeuven, Department of Oncology, Laboratory of Gynaecologic Oncology, ImmunOvar Research Group,
1
Leuven, Belgium,
UZ Leuven, Department of Gynaecology and Obstetrics, Leuven Cancer Institute, Leuven, Belgium,
2
KULeuven, Department of Cellular and Molecular Medicine, Laboratory for Cell Death Research and Therapy,
3
Leuven, Belgium
Introduction: Dendritic cell (DC) immunotherapy is
mice. Subjectively, in the therapeutic set up, the
an efficient way to create tumor reactive T cells in
early deaths seemed to be avoided due to DC vacci-
vivo and has proven its efficacy in different tumor
nation and a few long term survivors were observed.
types including endometrial cancer. We investigat-
This was absent in the prophylactic experiments.
ed the effect of DC immunotherapy in a luciferase-
However, the onset of ascites in vaccinated animals
tagged murine model for ovarian cancer.
was much earlier compared to the control group.
Materials and methods: Six to eight weeks old female
In the sc model however, there was a statistical
C57BL/6J-Tyrc-2J/J mice were inoculated either with
significant difference (p=0,0001) in tumor growth
5 x 106 ID8-fLuc cells intraperitoneal (ip) or with 2 x
between the vaccinated group and the tumor bearing
6
10 ID8-fLuc cells subcutaneously (sc). DC were grown
controls. The DC vaccine was able to suppress tumor
from bone marrow derived stem cells in the presence
growth.
of GM-CSF (Granulocyte macrophage colony stimu-
Conclusion: DC vaccination is efficient to reduce
lating factor). After seven days, immature DC were
tumor growth of in a sc mouse model for ovarian
loaded with ID8-fLuc cells treated with Hypericin
cancer. Results in the orthotopic setting are dis-
based photodynamic therapy (Hyp-PDT), followed
appointing. This can probably be explained by a
by three freeze-thaw cycles and subsequently matu-
strong immunosuppressive microenvironment that
rated with lipopolysaccharide (LPS). In the therapeu-
is present at peritoneal metastatic spread. To achieve
tic model, DC were administered once every week sc
efficient DC immunotherapy we will need to combine
at day 21, 28, 35 after ip or sc tumor inoculation. In
DC immunotherapy with other strategies to over-
the prophylactic model, DC were administered once
come immunosuppression.
every week sc at day -14 and -7 before ip tumor inoculation. Weight was followed three to six times a
week. Tumor growth for the ip models was evaluated
weekly, using bioluminescence imaging (BLI). In the
sc model, tumor growth was evaluated by measuring
the largest diameter of the tumor 3x/week using a
vernier caliper. Mice were sacrificed according to our
own published protocol
Results: There was no change in mean survival in the
therapeutic and prophylactic experiments between
the tumor bearing controls and the DC vaccinated
Combination of oncolytic virotherapy and DC-based
immunotherapy for the treatment of melanoma
Banki Z.1, Koske I.1, Barnstorf I.1, Tripp C.2, Stoizner P.2, Romani N.2, Wollmann G.1, Kimpel J.1,
Holm-von Laer D.1
Division of Virology, Medical University of Innsbruck, Innsbruck, Austria,
1
Department of Dermatology and Venereology, Medical University of Innsbruck, Innsbruck, Austria
2
VSV-GP, a novel chimeric Vesicular Stomatitis Virus
combination treatment correlated with increased
(VSV) pseudotyped with the glycoprotein of the lym-
numbers of tumor infiltrating lymphocytes (TIL) and
phocytic choriomeningitis virus represents a prom-
elevated Tconv/Treg and CD8+/Treg ratios. Further-
ising oncolytic virus (OV) that preferentially targets
more, depletion of CD8+ T cells but not NK cells
and kills cancer cells. Release of tumor antigens and
abrogated the therapeutic effect of DCVacc/VSV-GP.
activation of immune response by OV therapy might
Taken together, the combination of VSV-GP and DC-
support dendritic cell (DC)-mediated anti-tumor im-
based immunotherapy might represent a promising
munity. Thus in our study we analyzed the efficacy
therapeutic option for the treatment of melanoma.
and immune mechanisms of the combination of
VSV-GP oncolytic virotherapy with DC-based immunotherapy. Combination of VSV-GP therapy and DCbased vaccination was investigated in the syngeneic
subcutaneous B16-OVA melanoma model. SIINFEKLloaded CpG-activated DCs (DCVacc) and VSV-GP
were applied intra- and peritumorally and immune
responses were analyzed in the spleen and tumor
tissues. The DCVacc/VSV-GP combination therapy
resulted in a significantly improved survival compared to single treatments. Surviving mice from the
DCVacc/VSV-GP treated group showed a long lasting
anti-tumor immunity against B16-OVA and partial
anti-tumor immunity against non-OVA B16 melanoma in rechallenge experiments. Analyzing specific
cytotoxic T lymphocyte (CTL) responses induced by
DCVacc and VSV-GP single and combination treatments we found that both DCVacc and DCVacc/
VSV-GP induced comparable levels of OVA-specific
CD8+ T cell responses. In addition a strong VSV N
peptide-specific CD8+ T cell response was found
upon VSV-GP and DCVacc/VSV-GP treatments. The
improved therapeutic effect by the DCVacc/VSV-GP
Intralymphatic mRNA vaccine induces CD8 T-cell responses that
inhibit the growth of mucosally located tumors
Bialkowski L.1, van Weijnen A.1, Van der Jeught K.1, Renmans D.1, Daszkiewicz L.1, Heirman C.1, Stangé G.2,
Breckpot K.1, Aerts J.1, Thielemans K.1
Vrije Universiteit Brussel, Laboratory of Molecular and Cellular Therapy, Brussels, Belgium,
1
Vrije Universiteit Brussel, Diabetes Research Center, Brussels, Belgium
2
Despite promising preclinical data, the translational
success rate of therapeutic vaccines against HPV-related malignancies is still limited. This can in part be
attributed to the lack of appropriate mouse models,
as rapidly growing ectopic tumors are the most commonly used models for preclinical studies. In this
work, we demonstrate that the tumor microenvironment of TC-1 tumors differs significantly depending
on the anatomical location of tumor lesions (i.e. subcutaneously, in the lungs and in the genital tract).
Our data demonstrate that E7-TriMix mRNA vaccineinduced CD8+ T lymphocytes migrate into the tumor
nest and control tumor growth, although they do not
express the so-called mucosa-associated markers
such as CD103 or CD49a. We additionally show that
despite the presence of the antigen-specific T cells in
the tumor lesions, the therapeutic outcomes in the
genital tract model remain limited. Here, we report
that such a hostile tumor microenvironment can be
reversed by cisplatin treatment, leading to a complete regression of clinically relevant tumors when
combined with mRNA immunization. We thereby
demonstrate the necessity of utilizing clinically relevant models for preclinical evaluation of anticancer
therapies and the importance of a simultaneous combination of anticancer immune response induction
with targeting of tumor environment.
WT-1 and PRAME mRNA transfected TLR 7/8 polarized fast
DCs can raise specific immune responses in AML patients that
correlate with clinical outcome
Bigalke I.1, Fløisand Y.2, Solum G.1, Hønnåshagen K.1, Skoge L.1, Sæbøe-Larssen S.1, Schendel D.3, Kvalheim G.1
Oslo University Hospital, The Norwegian Radium Hospital, Department of Cellular Therapy, Oslo, Norway,
1
Oslo University Hospital, Oslo, Norway,
2
Medigene Immunotherapies GmbH, Martinsried, Germany
3
Elderly patients with acute myeloid leukemia (AML)
suggesting that an epitope spreading had taken
often do not tolerate high dose chemotherapy and are
place. WT-1 signal in BM shows fluctuation in levels
currently lacking curative treatment options.
between each samples but WT-1 is negative in pe-
Immunotherapy with DC vaccines following induc-
ripheral blood. The Pt is still in morphological remis-
tion chemotherapy has been shown by others to have
sion 21 months after start of vaccination.
clinical effects in some AML patients. Five AML pa-
Pt 2 showed initially a WT-1 response. Due to a Bell’s
tients not eligible for bone marrow (BM) transplan-
Palsy the patient was given high doses of cortisone.
tation and with reduced conditioning/consolidation
Immediately thereafter immune response was lost
therapy were treated under hospital exemption with
and WT-1 increased in BM accompanied by a clinical
DC vaccines targeting WT-1 and PRAME.
relapse. In spite of that this patient initially was not
Following informed consent and hematopoietic re-
eligible for transplantation he was now offered BM
covery after induction chemotherapy monocytes
transplantation and is currently in remission.
were collected by apheresis and elutriation and
Pt 3 has a fluctuating elevated WT-1 signal in BM
matured with a previous described cocktail contain-
but is still in morphological remission under vaccine
ing the TLR7/8 ligand R848 resulting in DCs with
treatment for 15 months. Immune responses are also
a polarized release of IL-12p70 and low IL-10 (Sub-
fluctuating below the detection limit of our assay.
klewe et al. 2014).
Pt. 4 showed no specific immune responses and re-
2.5 or 5E+6 DCs per antigen were injected intrader-
lapsed after 6 months of DC vaccination. DC treat-
mal once weekly for 4 weeks (wks), in wk 6 and there-
ment was continued in combination with 5-Azacy-
after in monthly intervals. Blood and bone marrow
tidine. The patient was brought into remission and
(BM) samples were collected at regular intervals and
has been treated with this combination therapy for
minimal residual disease (MRD) was measured in
10 months.
BM by quantitative PCR of WT-1 expression and mor-
Pt. 5 is in remission now for 5 months since start
phology.
of vaccination and assessment of immune responses
Specific T cell responses were assessed by analysis
follows.
of intracellular interferon gamma expression after
Altogether, these results show that fast TLR- polar-
stimulation with peptides of WT-1 and PRAME and
ized DCs can induce or enhance specific T cell re-
hTERT and survivin as vaccine unrelated antigens.
sponses with a patient individual pattern. Clinical
Patient (Pt) 1 mounted a strong response against
responses are related to immune responses and can
PRAME 5 weeks after start of vaccination combined
result in prolonged survival in AML patients not eli-
with an unexpected increase in hTERT response,
gible for curative treatment.
The double face of dendritic cell vaccination in metastatic
melanoma: inducing intratumor immune response can switch
tumor cells toward dedifferentiated state
Bulgarelli J.1, Ancarani V.1, Pancisi E.1, Petrini M.1, Riccobon A.1, Fiammenghi L.1, Cassan S.1, Soldati V.1, Ridolfi L.1,
De Rosa F.1, Gentili G.2, Amadori D.3, Ridolfi R.1, Guidoboni M.1, Granato A.M.1
IRST-IRCCS, Immunotherapy and Somatic Cell Therapy Unit, Meldola, Italy,
1
IRST-IRCCS, Unit of Biostatistics and Clinical Trial, Meldola, Italy,
2
IRST-IRCCS, Department of Medical Oncology, Meldola, Italy
3
DC-based vaccination is one of the most tolerable
by melanoma stem cells, was observed in one third
immunotherapeutic approach and is capable of in-
of patients after vaccination. In addition, patients
ducing strong tumor-focused immune responses.
with lower expression of both MAA and MAGEA1
However, the majority of immunological responders
experienced a long survival. In almost all patients
will eventually undergo late relapse due to mecha-
HLA class I expression remained unchanged after
nisms still largely unknown. To elucidate the com-
vaccination.
plexity of mechanisms involved in late progression
Firstly, our data show that DC vaccination induces a
after immunologically effective DC vaccination, we
decrease of intratumoral TREGs together with an in-
characterized the tumor microenvironment in mela-
crease of activated cytotoxic T lymphocytes (CTLs),
noma tissues taken before and after vaccination.
indicating changes conducive to Th1-type immune
We evaluated changes in tumor-infiltrating T lym-
response in tumor microenvironment. Secondly, our
phocytes as well as in the expression of Melanoma
findings suggest that late relapse after DC vaccina-
Associated Antigens (MAA) and of class I HLA mol-
tion might be sustained by immune-mediated dedif-
ecules (HLA-I) in tumor biopsies from 12 metastatic
ferentiation of melanoma cells. Accordingly, patients
melanoma patients, by immunohistochemistry.
who experienced a particularly favorable clinical
Results showed that DC vaccination induces a sig-
outcome showed the concomitant decrease of gp100
nificant high increase of the intratumoral content
and MAGEA1, suggesting that concurrent targeting
of CD8+ T cells in almost all patients analyzed
of both differentiated and stem-like melanoma cells
+
2
(184±208 vs 295±187 CD8 T cells/mm , p=0.0434).
Moreover, higher numbers of GrB+ T cells were
observed in postvaccine tumor tissue (79±82 vs
179±85 GrB+ cells/mm 2, p=0.0483) indicating that
the majority of the vaccine-induced intratumoral T
cells have an activated/cytotoxic phenotype. Interestingly, the number of intratumoral FoxP3+ TREGs
significantly decrease after vaccination (119±78 vs
47±22 FOXP3+ cells/mm 2, p=0.0015), although no
relevant difference was found between progressing
and nonprogressing lesions. Instead, concurrent decrease of MAA antigens and increase of MAGEA1,
which has been found to be preferentially expressed
may have occurred.
Immune responses to a mutation-specific peptide vaccine
targeting IDH1R132H in patients with IDH1R132H-mutated gliomas
Bunse T.1, Bunse L.1,2, Sanghvi K.1, Sahm F.3,4, Omokoko T.5, Simon P.5, Schmitt A.6, Hückelhoven A.6, Stevanovic S.7,
Laumann M.2, von Deimling A.3,4, Sahin U.5, Schmitt M.6, Wick W.2,8, Platten M.1,2
German Cancer Research Center (DKFZ), DKTK Clinical Cooperation Unit Neuroimmunology and Brain Tumor
1
Immunology, Heidelberg, Germany,
Heidelberg University Medical Center; National Center for Tumor Diseases, Department of Neurology, Heidelberg,
2
Germany,
University Hospital Heidelberg, Department of Neuropathology, Heidelberg, Germany,
3
German Cancer Research Center (DKFZ), Clinical Cooperation Unit Neuropathology, Heidelberg, Germany,
4
BioNTech Cell & Gene Therapies GmbH, Mainz, Germany,
5
University Clinic Heidelberg, Department of Internal Medicine V, Heidelberg, Germany,
6
Interfaculty Institute for Cell Biology, University of Tübingen, Department of Immunology, Tübingen, Germany,
7
German Cancer Research Center (DKFZ), Clinical Cooperation Unit Neurooncology, Heidelberg, Germany
8
ORAL
TALK
SHORT
2016
Immunotherapeutic concepts for brain tumors have
nant IDH1R132H+ astrocytomas at eight German
been hampered by lack of selective measures to
sites: NOA-16 (NCT02454634). The vaccine is made
target the CNS and lack of truly tumor-specific anti-
of an IDH1R132H peptide emulsified in mineral oil
gens. But the concept of an immune privileged CNS
and administered with topical imiquimod. Vaccina-
has to be questioned due to survey of the brain by
tion is implemented into primary standard therapy.
specific T-cells, CNS autoimmunity and presence of
Primary end points are safety and immunogenicity
lymphatics. As tumor-specific antigens, mutated an-
as measured by IDH1R132H-specific antibodies and
tigens have come into focus for all tumor entities.
T cell responses. As of February 2016, 11 patients
Mutations in the gene for isocitrate dehydrogenase
have been enrolled in the trial; no severe adverse
1 frequently occur in diffuse gliomas, mostly as-
events have been reported.
trocytomas, resulting in a point mutation (mostly
We report here on 3 patients treated with the vaccine
IDH1R132H). IDH1R132H is an ideal tumor-specific
on a compassionate use basis. No patient experi-
antigen, because it is tumor-specific, homogenous-
enced a regime-limiting toxicity as defined in the
ly expressed and considered a driving mutation in
trial protocol. Two patients received all 8 doses of
glioma. Preclinical studies have shown that muta-
the vaccine; both developed IDH1-specific antibody
tion-specific Th cell responses spontaneously occur
responses, which were mutation-specific early after
in patients with IDH1-mutated gliomas and that a
vaccination. Of these, one patient developed a vacci-
peptide vaccine encoding IDH1R132H is therapeu-
nation-induced mutation-specific cellular response.
tic in a humanized mouse tumor model. Based on
The second patient had a high baseline mutation-spe-
these data we have initiated a multicenter, first-in-
cific T cell response, which was temporarily boosted
man, phase I clinical vaccine trial, which is planned
by the vaccine. IFN-γ catch assays from PBMCs of
to enroll 39 patients with newly diagnosed malig-
this patient revealed a Th cell-mediated response.
To identify and clone IDH1R132H-specific TCR(s)
for adoptive T cell therapy, single T cells from this
patient specifically responding to IDH1R132H were
sorted and subjected to TCR sequencing. 11 TCRs
have been identified so far and will be cloned and
validated. In addition, ImmunoSEQ® analyses were
performed from patients with T cell responses in
order to detect clonality and identify specifically
expanded TCRs after vaccination. In summary, we
demonstrate for the first time the induction of a
mutation-specific humoral and cellular immune response to the IDH1R132H neoepitope after peptide
vaccination of patients with IDH1R132H-mutated
gliomas. The ongoing trial will analyze safety and
immunogenicity of the vaccine in a defined patient
cohort, which also allows for collecting data on therapeutic efficacy.
Discovery to first-in-man studies of a multi-peptide-based
hepatocellular carcinoma vaccine adjuvanted with CV8102
(RNAdjuvant®) - HEPAVAC
Mayer-Mokler A.1, Accolla R.2, Ma Y.T.3, Heidenreich R.4, Izzo F.5, Koenigsrainer A.6, Loeffler M.6, Flohr C.1,
Mueller P.1, Rammensee H.-G.6, Sangro B.7, Francque S.8, Valmori D.9, Weinschenk T.1, Reinhardt C.1,
Gnad-Vogt U.4, Singh-Jasuja H.1, Buonaguro L.5
Immatics Biotechnologies GmbH, Tuebingen,
1
Germany,
Istituto Nazionale per lo Studio e la Cura dei Tumori,
5
‘Fondazione Pascale’, Naples, Italy,
Università dell’Insubria, Varese, Italy,
6
University of Birmingham, Birmingham, United
7
Kingdom,
8
CureVAC AG, Tuebingen, Germany,
9
2
3
University of Tuebingen, Tuebingen, Germany,
Universidad de Navarra, Pamplona, Spain,
University of Antwerp, Antwerp, Belgium,
4
Hepatocellular
(HCC)/normal
University of Nantes, Nantes, France
tissue
CV8102 adjuvant (RNAdjuvant®) following a single
matched samples have been collected for HLA im-
adjacent
pre-vaccination infusion of low-dose cyclophospha-
munopeptidome analysis. 17 HCC samples from
mide acting as an immunomodulator. The study
HLA-A*02+ patients and 15 samples from HLA-A*24+
drugs are applied without concomitant anti-tumor
patients have been analysed by mass spectrometry
therapy with the intention to reduce risk of tumor re-
(LC-MS/MS). RNA-expression profiles have been
currence/progression in patients who have received
established for 12 HCC samples. HLA-presentation/
all indicated standard treatments. The primary end-
expression of peptides on primary HCC samples (as
points are safety, tolerability, and immunogenicity.
well as mRNA expression) were compared to normal
Secondary/exploratory endpoints are additional im-
tissue samples from relevant organs (including heart,
munological parameters in blood (e.g. regulatory
brain, lung, kidney, liver, nerve, skin etc.) present in
T-cells, myeloid-derived suppressor cells, impact
the Immatics’ database.
of the standard therapy on the natural immune re-
A total of 16 peptides have been selected and con-
sponse), infiltrating T-lymphocytes in tumor tissue,
firmed for immunogenicity for the HepaVac vaccine
biomarkers in blood and tissue, disease-free sur-
and are currently synthesized according to GMP
vival/progression-free survival and overall survival.
standard. Of these, 7 are restricted to HLA-A*02;
Once safety of this vaccination approach has been
5 to HLA-A*24 and 4 to HLA class II. Formulation
determined in the first 10-20 patients the addition of a
development studies have been undertaken leading
checkpoint inhibitor will be considered. Suitable pa-
to a suitable and stable pharmaceutical form. An
tients enrolled in Tuebingen are invited to participate
analytical method was developed which allows the
in a trial extension investigating an actively person-
characterization of each individual peptide within
alized vaccine (APVAC) plus CV8102.
the HepaVac vaccine (IMA970A). At present, pre-
The HepaVac project started in September 2013 and is
clinical studies assessing the combination of the
supported by the European Commission’s 7th Frame-
immunological RNA-based adjuvant (RNAdjuvant®)
work Program under the Grant Agreement Nr. 602893
with the peptide-based HepaVac vaccine IMA970 are
(www.hepavac.eu). The clinical trial HepaVac-101
conducted.
will be conduct in 7 centers located in 6 European
A single-arm, first-in-man trial entitled HepaVac-101
countries, i.e. Italy (Naples and Varese), Germany
is designed to investigate in patients with very early,
(Tübingen), UK (Birmingham), Spain (Pamplona),
early and intermediate stage of HCC the off-the-shelf
Belgium (Antwerpen) and France (Nantes).
multi-peptide-based HCC vaccine (IMA970) plus the
An Epitope Discovery and Improvement System (EDIS) to study
MHC-I epitopes and improve their sequences
Capasso C.1, Magarkar A.1, Cervera Carrascon V.2, Müller M.3, Garofalo M.1, Kuryk L.4, Hirvinen M.1, Bunker A.1,
Cerullo V.1
University of Helsinki, Division of Pharmaceutical Biosciences, Helsinki, Finland,
1
TILT Biotherapeutics Ltd, Helsinki, Finland,
2
Ludwig-Maximilians-Universität München, Munich, Germany,
3
Oncos Therapeutics, Helsinki, Finland
4
Cancer vaccines represent an attractive approach to
target specific antigens and re-direct the immune
system towards malignant cells. However, they
often lack of proper adjuvants and most tumors
establish tolerance against their antigens. In our
study, we address these challenges by using immunogenic adenoviruses as adjuvants for heteroclitic
peptides that might break the established tolerance.
The EDIS framework uses in silico binding and immunogenicity predictions and refines them by molecular dynamic simulations. We started by studying the
model epitope SIINFEKL and two analogues, which
were predicted to have improved immunogenicity.
We confirmed experimentally their increased affinity for the murine H2Kb and ELISPOT assays were
carried out to confirm the cross-reactivity between
the peptides. In addition, the two analogues showed
an higher efficacy against established B16OVA
tumors compared to the native epitope.
Next we sought to test our approach by studying
the TRP2 epitope SVYDFFVWL. By using the EDIS
framework we selected two improved analogues and
confirmed their increased affinity for H2Kb. Then we
investigated their efficacy against established B16F10
tumors. ELISPOT assays were performed to study the
cross-reactivity of T-cells against the peptides.
In our study we highlight how the integration of different in silico tools and parameters might increase
the accuracy of the prediction of heteroclitic peptides.
TLR9 stimulation is required for recall of functional immune
memory response against neo-antigen relapse in liver
Cebula M.1, Riehn M.1, Hillebrand U.1, Schirmbeck R.2, Kreppel F.2, Hauser H.1, Wirth D.1
Helmholtz Centre for Infection Research, Model Systems for Infection and Immunity, Braunschweig, Germany,
1
University of Ulm, Department of Internal Medicine, Ulm, Germany
2
ORAL
TALK
SHORT
2016
Therapeutic vaccination and immunomodulatory
clearance could be measured; indicating that in these
intervention are promising treatment strategies for
conditions T cells became exhausted. In contrast,
cancer patients. However, current protocols fre-
T cell effector functions and complete clearance of
quently do not induce efficacious responses capable
ovalbumin expressing hepatocytes are observed in
of eradicating transformed cells and controlling the
mice displaying low antigen density prior to vaccina-
tumor growth or its relapses. One of the main reasons
tion. These data indicate that the density of antigen
for this limitation is the fact that the immune re-
expressing hepatocytes governs the final outcome of
sponses against cancer cells (either endogenous or
the therapeutic vaccination as such.
induced by therapeutic vaccinations) are often ham-
In order to investigate protective capacity of immune
pered by a severe exhaustion of specific T cells due to
memory induced in mice that efficiently responded
tumor environment but as well due to tissue specific
against low antigen frequency the mice were treated
tolerance mechanisms.
with tamoxifen inducing neo-antigen relapse in 50%
In this study we investigated the protective capacity
of hepatocytes. Interestingly the preexisting func-
of therapeutic vaccine against neo-antigen expres-
tional immunity was not protective against this high
sion in liver - an organ promoting strong tolerance.
antigen challenge as no antigen clearance could be
A conditional mouse model RosaOVA X AlbCreERT2
measured. This emphasizes the dominating tolero-
was employed to provide hepatocyte specific ovalbu-
genic potential of antigen in the liver depending on
min neo-antigen expression upon Tamoxifen induced
its dose. Nevertheless, by coinciding high antigen
T2
activation of CreER recombination. Based on specif-
relapse with TLR9 ligand stimulation in presence
ic expression cassette design neo-antigen expression
of preexisting immunity, clearance of high antigen
is restricted to a fraction of hepatocytes resulting in
load could be achieved. Based on these findings we
a mosaic pattern of antigen distribution. The degree
suggest that TLR9 ligand can be used as potent mod-
of mosaicism is adjustable and depends on tamoxifen
ulator of intrahepatic immunity that overrides liver
dosage. For therapeutic vaccination, a DNA plasmid
specific regulatory cues and might ensure elimina-
or adenoviral vector encoding the ovalbumin neo-
tion of immunologically defined targets such as in-
antigen was injected intramuscularly in mice with
fected or cancerous cells.
10% and 50% of hepatocytes expressing the antigen,
respectively. Independently of the antigen density,
we observed intrahepatic accumulation of antigen
specific T cells. In mice with 50% of OVA expressing
hepatocytes, neither antigen reduction nor antigen
Immune responses following intrapleural administration of the
oncolytic HSV Seprehvir in patients with malignant pleural
mesothelioma
Learmonth K.1, Braidwood L.1, Woll P.2, Bolyard C.3, Kaur B.3, Blyth K.4, Conner J.1
Virttu Biologics, Glasgow, United Kingdom,
1
University of Sheffield/Sheffield Teaching Hospitals, Sheffield, United Kingdom,
2
Ohio State University, Columbus, United States,
3
Queen Elizabeth University Hospital, Glasgow, United Kingdom
4
Seprehvir is an oncolytic immunotherapeutic herpes
up to 28 days post-administration. Increased levels of
simplex virus type 1 mutant deleted in the gene en-
HMGB1 and HSP70 were detected in pleural fluids
coding the neurovirulence factor ICP34.5. Mutants
during this time indicating the potential for immuno-
lacking ICP34.5 are selectively replication competent
logical cell death associated with Seprehvir oncolysis.
in cancer cells and induce anti-tumour immune re-
Robust Th1 responses with increased IFNγ, IP-10, MIG,
sponses. Data supporting immune efficacy stimulat-
I-TAC and TNFα were observed in most patients after
ed by treatment with Seprehvir includes pre-clinical
Seprehvir administration with additional IL-2, IL-10 and
evidence of Th1 cytokine/chemokine responses that
IL-12 responses most prominent in patients receiving 4
facilitate systemic anti-tumour immune responses
doses. There was evidence of immune cell infiltration
via cytotoxic T cells that also reduce the establish-
into pleural fluids after Seprehvir treatment and in-
ment of metastases and protect from re-challenge.
creased levels of Granzyme B in pleural fluids indicate
Recent evidence from mesothelioma patients post-
immune cell-mediated cytotoxicty.
Seprehvir treatment supports this immunotherapy
Analysis of plasma samples indicated strong anti-HSV
activity with robust Th1 cytokine responses detected
IgG responses post-Seprehvir administration, particular-
in pleural fluids, evidence of immune cell infiltration
ly after 2 and 4 doses. Analysis of pleural fluid samples
and activity and the development of a novel anti-
also indicated anti-HSV IgG responses post-Seprehvir
tumour IgG immune response.
administration. Crucially, in most patients, there was a
A phase I/IIa trial to determine the safety and po-
novel anti-tumour IgG response as detected by immuno-
tential for efficacy of Seprehvir given intrapleurally
blotting against extracts from MPM cell lines indicating
to patients with MPM is currently ongoing. Patients
tumour-directed immune responses. Further studies on
7
receive 1x10 iu Seprehvir through their pleural cath-
the identities of the infiltrating immune cells and their
eter on one, two or four occasions a week apart, in
targets are ongoing.
three separate patient cohorts. To date 10 patients
Our trial demonstrates that oncolytic Seprehvir has im-
have been treated, 3 in the one and two dose and
munotherapeutic potential capable of inducing novel
4 in the four dose cohorts and Seprehvir has been
anti-tumour immune responses in mesothelioma pa-
well-tolerated with few adverse events in any pa-
tients. This also confirms pre-clinical studies that clearly
tients. Pleural fluid and plasma samples have been
demonstrated Seprehvir´s immunotherapeutic mode of
collected pre- and post treatment and analysed to
action.
assess patient responses to Seprehvir administration.
Seprehvir replicated/persisted in most patients with HSV
DNA detected in the pleural fluids for, in some cases,
Coding- and non coding-RNA profiling of active dendritic cells
following stimulation with highly immunogenic tumor cell lysates
Ravo M.1, Montico B.2, Tarallo R.1, Giurato G.1, Memoli D.1, Martorelli D.2, Weisz A.1, Dolcetti R.2,3, Dal Col J.2
University of Salerno, Laboratory of Molecular Medicine and Genomics, Baronissi, Italy,
1
Centro di Riferimento Oncologico, National Cancer Institute - IRCCS, Aviano, Cancer Bio-Immunotherapy
2
Unit, Translational Research Dept., Aviano, Italy,
The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia
3
A range of different cancer immunotherapeutic
RA/IFN-treated tumor lysate-loaded IFN-DCs was as-
strategies involving dendritic cells (DCs) have been
sociated to a higher activation of NF-kB pathway and
used to elicit tumor-specific T-cell mediated immune
an increased release of pro-inflammatory cytokines.
responses but, despite the potentiality of DC-based
RNA-Seq analysis identified 1,711 mRNAs differ-
vaccines, results in clinical trials remain unsatisfac-
entially expressed in RA/IFN-treated tumor lysate-
tory. A particular issue resides in the definition of an
loaded IFN-DCs compared to IFN-DCs pulsed with
‘appropriately activated’ DC that exhibits optimum
untreated lysates (FDR adjusted P value of ≤0.05 and
stimulatory capacities. Therefore, the ability to pre-
FC greater than 1.5-fold in at least 1 comparison).
screen DC vaccines before transplantation back into
Functional annotation analysis of the differentially
patients is crucial. Using as a model a DC-based
expressed transcriptome indicates an enrichment of
vaccine recently optimized in our laboratory, tran-
several canonical pathways most significantly af-
scriptional and small non coding RNA profiling of
fected by the treatment such as Dendritic Cell Matu-
functional DCs was performed by RNA sequenc-
ration, Toll-like Receptor Signaling, IL-6 Signaling,
ing. To this end, highly active, partially mature DCs
LXR/RXR Activation. Interestingly, by small non
(IFN-DCs) were generated by culturing for three days
coding RNA sequencing we found also that the de-
human monocytes in the presence of GM-CSF and
regulated transcripts involved in above mentioned
interferon(IFN)-a. IFN-DCs represent a novel class of
pathways were targeted by several differentially ex-
cell-based immunotherapeutic agents, endowed with
pressed miRNAs.
a high migratory behavior and immuno-stimulatory
These results suggest that defining a transcriptional
ability. IFN-DCs were loaded with tumor cell lysates
signature capable of predicting DC stimulatory abil-
obtained from untreated/viable or treated/apoptotic
itiy and functions might represent a useful way to
lymphoma cells. Immunogenic tumor cell apoptosis
assess DC-based vaccine potency.
was induced by combined treatment with 9-cis-retinoic acid and interferon-a (RA/IFN), a highly effective
modality to induce immunogenic cell death ex vivo
that we recently developed. Importantly, IFN-DCs
loaded with lysates from RA/IFN-treated lymphoma
cell lines induce tumor-specific cytotoxic T lymphocytes (CTLs) with enhanced killing efficiency, compared to DC pulsed with lysates from untreated or
γ-irradiated cells. The improved immunogenicity of
Supported by Italian Ministry of Health (GR-2011-02350476)
and Italian Association for Cancer Research (IG-17426)
Development of a GMP production protocol for a cord blood-derived
dendritic cell-based vaccine to prevent relapses after hematopoietic
cell transplantation in children with AML
de Haar C.1, Dunnebach E.1, Plantinga M.1, van Til N.1, Nierkens S.1, Boelens J.J.1
UMC Utrecht, Laboratory for Translational Immunology, Utrecht, Netherlands
1
Introduction: Pediatric patients with refractory/re-
CBDC vaccine containing ampules are thawed and
lapsed acute myeloid leukemia (AML) have only one
washed before being analyzed phenotypically and
treatment option: hematopoietic cell transplantation
functionally.
(HCT). Using cord blood (CB)-derived stem cells,
Results: Starting with approximately 0.3x10^6 CD34+
instead of cells from bone marrow cells or periph-
cells we were able to generate sufficient vaccine per
eral blood, results in less relapses (increased anti-
patient for the three round of vaccination planned
tumor reactivity) and less graft-versus-host disease
in our clinical trial. The CBDCs in our vaccine show
(increased safety). Although this treatment is poten-
upregulated co-stimulatory molecules after matura-
tially curative, still more than half of the children
tion and showed enhanced CCR7-dependent migra-
die from relapses. As such, we want prevent relapses
tion towards CCL19 in a trans-well migrations assay.
by inducing anti-AML immunity using a CB-derived
CD83 expression was used to assess the amount of
dendritic cell (CBDC) vaccine. We have therefore
mature CBDC in our vaccine. In addition, CBDCs ex-
optimized and validated the GMP production of the
pressed WT1 protein after electroporation with WT1-
CBDC vaccine needed for our upcoming clinical trial.
mRNA. The WT1-loaded CBDCs were not only able to
Methods: We have successfully translated and
stimulate T cells in a mixed lymphocyte reaction but
further optimized our pre-clinical protocol for gen-
in an antigen-specific setting as well.
erations of the CBDC vaccine from CD34+ CB stem
Conclusions: We are able to set-up the GMP produc-
cells into a GMP production process. This GMP pro-
tion process of a WT1-loaded CBDC vaccine with
tocol enables us to generate sufficient CBDC vaccine
the goal to stimulate the anti-tumor reactivity of the
cells when using only the 20% of the CB unit as a
newly developing immune system in AML patients
source of C34+ cells. After CliniMACS CD34-isola-
after CB-HCT in a phase I/II clinical trial starting late
tion the cells undergo expansion for two weeks in
2016/early 2017.
culture bags using medium containing FLT3L, SCF,
IL-3 and IL-6. Next, the cells are differentiated into
DCs using medium containing FLT3L, SCF, GM-CSF
and IL-4. The CBDCs are then matured using proinflammatory cytokines and loaded with Wilms’ Tumor
1 (WT1) antigen by electroporation with WT1 encoding mRNA and pulsing with a WT1 15-mer-peptide
pool. After 4 hours recovery, cells are cryopreserved
until time of validation or intradermal vaccination.
Next-generation dendritic cell vaccination in postremission
therapy of AML: results of a clinical phase I trial
Deiser K.1,2, Lichtenegger F.S.1,2, Schnorfeil F.M.1,2, Köhnke T.1, Altmann T.1, Bücklein V.1, Moosmann A.3,
Brüggemann M.4, Heemskerk M.H.M.5, Wagner B.6, Hiddemann W.1, Bigalke I.7, Kvalheim G.7, Subklewe M.1,2
Klinikum der Universität München, Department of Internal Medicine III, Munich, Germany,
1
Helmholtz Zentrum München, Clinical Cooperation Group Immunotherapy, Munich, Germany,
2
Helmholtz Zentrum München, DZIF Research Group Host control of viral latency and reactivation, Munich,
3
Germany,
University Hospital Schleswig-Holstein, Department of Hematology, Kiel, Germany,
4
Leiden University Medical Center, Department of Hematology, Leiden, Netherlands,
5
Klinikum der Universität München, Department of Transfusion Medicine, Cellular Therapeutics and
6
Hemostaseology, Munich, Germany,
Oslo University Hospital - The Norwegian Radium Hospital, Department of Cellular Therapy, Oslo, Norway
7
Postremission therapy for acute myeloid leukemia
In total, 12 patients have been enrolled into the study.
(AML) is critical for elimination of minimal re-
The first 6 patients were analysed in phase I for safety
sidual disease (MRD). In patients not eligible for al-
and toxicity of the DC vaccine. Based on the results,
logeneic stem cell transplantation, alternative treat-
phase II has been initiated. DCs of sufficient number
ment options are needed. Therapeutic vaccination
and quality were generated from leukapheresis in
with autologous dendritic cells (DCs) loaded with
10/11 cases. DCs exhibited an immune-stimulatory
leukemia-associated antigens (LAAs) is a promising
profile based on high surface expression of positive
treatment strategy to induce anti-leukemic immune
costimulatory molecules, the capacity to secrete IL-
responses and to eradicate chemorefractory cells.
12p70, the migration towards a chemokine gradient
We have developed a GMP-compliant 3-day protocol
and processing and presentation of antigen. In 9/9
7 8
including a TLR / agonist to differentiate mono-
vaccinated patients, we observed delayed-type hyper-
cytes of intensively pretreated AML patients into
sensitivity (DTH) responses at the vaccination site,
next-generation DCs.
accompanied by slight erythema and indurations at
A phase I/II proof-of-concept study has been initiated
the injection site, but no grade III/IV toxicities. TCR
using next-generation DCs as postremission therapy of
repertoire analysis by NGS revealed an enrichment
AML patients with a non-favorable risk profile in CR or
of particular clonotypes at DTH sites. In addition, we
CRi after intensive induction therapy (NCT01734304).
detected DC vaccination-specific T cell responses in
DCs are loaded with in vitro transcribed RNA encod-
4/5 patients by multimer staining: Increased frequen-
ing the LAAs WT1 and PRAME as well as CMVpp65
cies of WT1-specific T cells in one patient and strong
as adjuvant and surrogate antigen. Patients are vac-
expansion/induction of CMVpp65-specific T cells in
cinated intradermally with 5x106 DCs of each antigen
one CMV-seropositive and two CMV-seronegative pa-
species up to 10 times within 26 weeks. The primary
tients. Furthermore, we detected increased numbers
endpoint of the phase I/II trial is feasibility and safety
of vaccination-specific IFN-gamma secreting T cells
of the vaccination. Secondary endpoints are immuno-
by ELISPOT analysis: 7/7 patients showed responses
logical responses and disease control.
to CMVpp65 and 2/7 exhibited responses to PRAME
and WT1, respectively. In an individual treatment
attempt, an enrolled patient with impending relapse
was treated with a combination of DC vaccination
and 5-azacytidine, resulting in MRD conversion.
Long-term disease control and immunological responses are studied in the ongoing phase II trial.
We conclude that vaccination with next-generation
LAA-expressing DCs in AML is feasible, safe and
induces anti-leukemia-specific immune responses
in vivo.
Theranostic mRNA-loaded microbubbles for ultrasound-assisted
dendritic cell based cancer vaccination
Dewitte H.1,2, Van Lint S.2, Vanderperren K.3, Thielemans K.2, De Smedt S.C.1, Breckpot K.2, Lentacker I.1
Ghent University, Lab for General Biochemistry & Physical Pharmacy, Gent, Belgium,
1
Vrije Universiteit Brussel, Laboratory for Molecular & Cellular Therapy, Jette, Belgium,
2
Ghent University, Department of Veterinary Medical Imaging and Small Animal Orthopaedics,
3
Merelbeke, Belgium
Introduction: This study aims to investigate a method
was studied after s.c. injection of the contrast agents
to simultaneously load dendritic cells (DCs) with tumor
in dogs, after which contrast-enhanced ultrasound
antigen and immunostimulatory mRNA, by the use
imaging (CEUS) was performed using a Philips iU-22.
of mRNA-sonoporation. Sonoporation makes use of
Results: We previously reported on the in vitro mR-
microscopic gas bubbles that respond to pressure dif-
NA-sonoporation of murine DCs, with transfection
ferences created by ultrasound waves. By adding mR-
efficiencies up to 27% without compromising cell vi-
NA-loaded microbubbles (MBs) to DCs and exposing
ability. The potential of this technique was further as-
them to ultrasound, the microbubbles locally implode,
sessed in vivo by vaccinating E.G7-OVA-bearing mice
causing local cell membrane poration (sonoporation)
with mRNA-sonoporated DCs. When OVA mRNA-so-
while releasing the mRNA for uptake through the
noporated DCs, but especially OVA+TriMix-sonopo-
created pores. As such, this project aims to investigate
rated DCs were used, tumor growth was significantly
theranostic mRNA-loaded MBs for ultrasound-guided,
reduced. For OVA+TriMix, tumors even completely
ultrasound-triggered antigen-loading of DCs, with the
regressed in 30% of the animals. Moreover, rechal-
ultimate goal to perform this transfection within the
lenge with tumor cells did not lead to tumor growth,
lymph nodes in vivo.
indicating long-lasting immunological protection.
Methods: mRNA-loaded MBs were prepared by at-
In order to assess the theranostic potential of these
taching mRNA-lipid complexes onto lipid microbub-
mRNA-loaded MBs, we performed a CEUS study in
bles via avidin-biotin linkages. MBs loaded with
dogs. After s.c. injection, the MBs rapidly drained
mRNA encoding a tumor antigen (ovalbumin, OVA)
to the lymph nodes. Moreover the contrast enhance-
and TriMix (a mixture of 3 mRNAs that modulate the
ment provided by the microbubbles revealed detailed
DC’s functionality) were used to transfect (sonopo-
information on the lymphatic anatomy.
rate) murine DCs in vitro. These mRNA-sonoporated
Conclusions: mRNA-loaded MBs can be used to
DCs were then used as therapeutic vaccines in E.G7-
transfer antigen and immunostimulating TriMix
OVA (OVA-expressing lymphoma)-bearing mice. Sur-
mRNA into DCs in vitro. The resulting mRNA-so-
viving animals were rechallenged with the same, or
noporated DCs can evoke potent antitumor immune
with different (MO4, OVA-expressing melanoma)
responses resulting in tumor regression and immu-
tumor cells to look at antigen-specific immunologi-
nological memory. In addition, rapid MB migration to
cal memory.
the lymph nodes can be imaged via CEUS.
In order to evaluate the potential of the mRNA-load-
Acknowledgements: Heleen Dewitte & Ine Lentacker
ed microbubbles to reach their anatomical targets
are postdoctoral fellows of FWO-Vlaanderen. This
in vivo, lymphatic drainage of mRNA-loaded MBs
project was funded via FWO grant G016513N.
MERIT: Individualized cancer vaccines for the treatment of
TNBC - a phase I trial
Dorer K.1, Heesch S.1, Bukur V.1, Buck J.1, Diekmann J.1, Diken M.2, Ewen K.1, Haas H.1, Kemmer-Brück A.1,
Kloke B.-P.1, Kreiter S.1, Kuhn A.N.1, Kuehlcke K.3, Loewer M.2, Paruzynski A.1, Schwarck D.1, Schmidt M.4, Andre F.5,
De Greve J.6, Kuendig T.7, Lindman H.8, Pascolo S.7, Sjöblom T.9, Thielemans K.6, Zitvogel L.5, Türeci Ö.10, Sahin U.1
BioNTech Group Mainz, Mainz, Germany,
7
TRON - Translational Oncology at the University
8
1
2
Medical Center Mainz, Mainz, Germany,
University Hospital of Zurich, Zurich, Switzerland,
Uppsala University Hospital, SWEDEN, Uppsala,
Sweden,
Eufets GmbH, Idar-Oberstein, Germany,
9
University Hospital Mainz, Mainz, Germany,
10
3
4
Gustave Roussy Comprehensive Cancer Center,
5
Uppsala University, Uppsala, Sweden,
CI3 (Cluster of Individualized Immunointervention),
Mainz, Germany
Villejuif Cedex, France,
Vrije Universiteit Brussel, Brussel, Belgium,
6
The majority of metastatic cancers remain incurable
after surgery and adjuvant chemotherapy will be allo-
since the current methods of treatment often do not
cated to one of two study arms. Patients in ARM1 will
address the inter-individual heterogeneity of cancer.
receive eight vaccination cycles with a personalized
Individualized approaches targeting each individual
set of shared tumor antigens from the WAREHOUSE
patient’s tumor may bring significant improvement.
that correspond to the patient tumor’s antigen-expres-
The Mutanome Engineered RNA Immuno-Therapy
sion profile. Patients in ARM2 will be first treated with
(MERIT) consortium is clinically validating a pioneer-
the personalized WAREHOUSE vaccine approach fol-
ing, individualized messenger RNA-based immuno-
lowed by six vaccination cycles of on-demand manu-
therapy concept for the treatment of triple-negative
factured MUTANOME vaccine encoding the unique
breast cancer (TNBC). MERIT combines two per-
mutation signature of the individual patient. Patients
sonalized treatment concepts: (i) vaccines contain-
will receive the vaccine in parallel to radiotherapy,
ing pre-synthesized mRNAs from an mRNA vaccine
which is standard of care for TNBC patients. The clini-
warehouse (MERIT WAREHOUSE) that encode shared
cal trial is approved in all participating countries; the
breast cancer tumor antigens expressed in the respec-
study start is planned for Q2 2016. The consortium has
tive patient’s tumor; (ii) mRNAs engineered on-de-
set up a multi-disciplinary clinical workflow and trial
mand that encode neo-antigens defined by patient-
design tailored to this unique therapeutic concept,
specific mutations, which will be identified by next
which covers the whole individualized drug develop-
generation sequencing (NGS) and ranked according
ment cycle from target discovery, validation to GMP
to the predicted immunogenicity of the correspond-
manufacturing and drug release for each individual
ing epitope (MERIT MUTANOME). The mRNAs are
patient. We will present the therapeutic concept and
administered intravenously as a nanoparticulate lipo-
study protocol as well as the methodologies required
plex formulation, which protects RNA from degrada-
for this highly innovative phase I trial. The individu-
tion, activates innate immunity, transfects APCs and
alized immunotherapy overcomes the current limi-
consequently induces highly potent antigen-specific T
tations of fixed, off-the-shelf therapeutics and thus
cell responses. A multi-center phase I trial conducted
might increase the clinical benefit for TNBC patients.
in four European countries assesses the feasibility,
This project is a collaborative effort of five partners
safety and biological efficacy of this personalized im-
from academia and industry funded by the European
munotherapy. TNBC patients (pT1cN0M0 - TxNxM0)
Commission’s FP7 and led by BioNTech AG.
A phase I/II clinical trial on malignant melanoma with in vitro
optimized mRNA-electroporated dendritic cells as therapeutic
vaccine
Dörrie J.1, Schaft N.1, Hoyer S.1, Gross S.1, Gerer K.F.1, Lehmann C.H.K.1, Dudziak D.1, Kummer M.1, Erdmann M.1,
Schliep S.1, Schuler G.1, Schuler-Thurner B.1
Universitätsklinikum Erlangen, Dermatology, Erlangen, Germany
1
Dendritic cells (DCs) are the most sophisticated adju-
The vaccine was given i.v. and the mean survival was
vant in therapeutic cancer vaccination. Usually, DCs
18 months. Surprisingly, no difference between those
are generated from the patient’s blood monocytes
with and without ELS-expression was perceived.
(moDCs), matured with cytokines, and loaded with
Meanwhile we and others observed that DCs - even
tumor-associated antigens, but despite promising
after cytokine-maturation - benefit from additional
results, there is room for improvement. While others
activating signals to foster the priming and expan-
sought alternative sources and new procedures
sion of memory effector CD8+ T cells. In contrast to
for DC generation, we concentrated on improving
maturation, this activation was transient. Therefore
cytokine-matured moDCs by functional manipula-
we expressed in the 3rd cohort (31 patients, 19 evalu-
tion via mRNA-electroporation. During a phase I/II
able), activating proteins inside the DCs to ensure
clinical trial with three cohorts, DCs were constantly
their presence at the time of injection. Due to the pre-
improved on the basis of the preclinical research,
vious results, we also stuck to i.v injection. Half of the
while the clinical results from the trial guided the
patients were vaccinated with IITP-matured moDCs,
bench-work. In all cohorts we used moDCs electropo-
electroporated MMS-RNA and CD40L-RNA, while
rated with mRNA encoding the melanoma antigens
the others received moDCs, which were immature
MelanA, Mage-A3, and Survivin (MMS-RNA). In the
electroporated with MMS-RNA in combination with
st
1 cohort (17 patients, 9 evaluable) the DCs were
“Trimix”-RNA (developed by Thielemans et al.) en-
matured with a cytokine cocktail of IL-1ß, IL-6, TNF,
coding constitutively active TLR4, CD40L and CD70).
and PGE2 (IITP), and electroporated with MMS-RNA.
The mean survival of CD40L-DC-treated patients was
moDCs of every 2nd patient were additionally loaded
36.5 months, while that of Trimix-DC-treated ones
with KLH and injected i.d. The mean survival was
was 19 months. Specific immune responses to the
12 months and no influence of the KLH emerged. For
vaccination antigens were frequently observed, but
nd
the 2 cohort, the electroporation was improved, re-
yet no correlation with survival became apparent. In-
sulting in 2x higher antigen expression and allowing
terestingly, we observed a beneficial effect of eosino-
the additional introduction of a functional recombi-
philia throughout the trial. We are now performing
nant E/L-selectin (ELS) to permit DC migration from
an array-based systematic approach to understand
blood into the lymph nodes, thus making IV injec-
the molecular basis of the functional differences of
tion reasonable. Within the 2nd cohort (34 patients,
the DCs used in this trial.
19 evaluable), moDCs were matured with IITP and
electroporated with MMS-RNA, but ELS-RNA was
co-electroporated into the cells of every 2nd patient.
NS and JD contributed equally
Dendritic cell vaccination with partial Treg depletion in relapsed
glioblastoma - results from the pilot phase of the HIT-HGG Rez
Immunvac study
Eyrich M.1, Krauss J.2, Löhr M.3, Technau A.1, Rachor J.1, Monoranu C.4, Warmuth-Metz M.5, Wölfl M.1, Kramm C.6,
Schlegel P.G.1
University Children’s Hospital Würzburg, Laboratory of Stem Cell Processing and Cellular Therapy, Würzburg,
1
Germany,
University Medical Center Würzburg, Pediatric Neurosurgery, Würzburg, Germany,
2
University Medical Center Würzburg, Neurosurgery, Würzburg, Germany,
3
University of Würzburg, Neuropathology, Würzburg, Germany,
4
University Medical Center Würzburg, Neuroradiology, Würzburg, Germany,
5
University Medical Center Göttingen, Pediatric Oncology, Göttingen, Germany
6
Efficacy of therapeutic dendritic cell vaccines (DCV)
sponse of varying magnitude and duration towards
can be limited by immunosuppressive mechanisms
the autologous tumor lysate in a IFNg-PCR assay. Fur-
such as regulatory T cells (Treg) overrepesented in
thermore, we observed an increase in VLA4+ T-cells
the micromilieu of the tumor. Here, we investigated
induced by the vaccine, both in the CD4 as well as
whether a reduction of Treg with metronomic cyclo-
the CD8 compartment. So far, all patients relapsed
phosphamide (metrCyc) might be a feasible option
after a mean of 5.9 months, however, overall survival
to improve vaccine efficacy. 8 patients with replased
was 18.1 months. One patient is disease-free after
glioblastoma were treated in the pilot phase of the
reoperation and continuation of vaccination for 37
HIT-HGG Rez Immunovac study (6 pediatric, 2 adult
months now. 6-month overall survival was 100%. We
patients). After reoperation and monocyte-aphere-
conclude that DC vaccination in combination with
sis, patients received 4 weekly doses of autologous,
partial Treg depletion with metrCyc is feasible, safe,
TNFa/IL-1ß matured DCs pulsed with tumor lysate.
and possibly related with a higher than expected
Thereafter 4 monthy, and subsequently threemonth-
frequency of positive IFNg-responses towards tumor
ly boosts with tumor lysate were given. The intra-
tissue. These pilot data warrant verification in the
dermal injection site was prepared with topical im-
full HIT-HGG Rez Immunovac trial (Eudra-CT 2013-
iquimod. Additionally, patients were pretreated with
000419-26), which will start to recruit patients soon.
oral metrCyc 2-4 weeks before the first vaccination.
MetrCyc was well tolerated with one mild and transient leukopenia in a patient, who received a parallel re-irradiation boost. All patients received at least
7 vaccines (4xDCs, 3xlysate boosts). Treg frequency
decreased by 36%, but returned to normal levels after
cessation of metrCyc. Importantly, 6/6 analyzed patients showed a positive (>1.5fold increase) IFNg-re-
Characterization of TLR3/8-PGE2 versus TNFα/IL-1ß matured
dendritic cells produced for clinical vaccination trials: impact of
maturation on migration and T-cell priming/crosspresentation
capacities
Technau A.1, Gierlich P.1, Lex V.1, Glunz A.1, Sauer S.2, Trautwein N.3, Grigoleit G.-U.4, Wölfl M.1, Schlegel P.G.1,
Eyrich M.1
University Children’s Hospital Würzburg, Laboratory of Stem Cell Processing and Cellular Therapy, Würzburg,
1
Germany,
University Medical Center Würzburg, IZKF Microarray Core Unit, Würzburg, Germany,
2
University of Tübingen, Department of Immunology, Tübingen, Germany,
3
University Medical Clinic II Würzburg, Laboratory of Stem Cell Processing and Cellular Therapy, Würzburg,
4
Germany
Background: Vaccination with tumor-antigen loaded
spectively). Substitution of PGE 2 by IFNγ further in-
dendritic cells (DCs) represent a promising strategy in
creased IL-12 production (8000 pg/ml), but also en-
cancer immunotherapy. However, efficacy of DCs in
hanced IL-10, so that the IL-12/IL-10 ratio was more
clinical trials has been limited so far. This might be due
favorable in the TLR-PGE 2 group. Priming efficacy of
to the fact that the optimal way to mature DCs for thera-
specific CTLs from naïve CD8+ T-cells was compa-
peutic vaccinations has not been established so far.
rable in both groups, when Melan-A was used as a
Methods: We validated two methods of DC matura-
model antigen (39±27 vs. 42±24% after 11 days of
tion which are currently used in clinical vaccination
culture with TLR-PGE 2 vs. TNFα/IL-1ß matured DCs,
trials: the classical way of maturation via cytokines
respectively). Also TCR avidity of primed CTLs was
(TNFα 1000U/ml, IL-1ß 2000 U/ml), and the recently
not different between the groups. When using two
proposed maturation cocktail using Toll-like receptor
glioma associated epitopes with a lower precursor
3/8 stimulation together with PGE2 which results in
frequency in the naïve CD8+ pool (NLGN4X131-139 and
DC1 cells with preserved migratory capacity (poly
PTP1347-1355), there was a trend towards higher frequen-
I:C 20 µg/ml, R848 3 µg/ml, PGE2 10 µg/ml). DCs
cies of specific CTLs in the TLR-PGE2 group. Finally,
were generated from monocytes over 7 days with
in a first round of experiments, TLR-PGE 2 matured
IL-4/GM-CSF followed by 48h maturation with the
DCs showed lower crosspresentation capabilites,
respective maturation cocktails. Then, DCs were
when DCs were loaded with CMV pp65 protein and
analyzed with respect to their phenotype, migration
assayed for IFNγ-stimulation in a CMVpp65 specific
behaviour, cytokine production, T-cell priming and
responder CD8+ T-cell line. However, these latter
crosspresentation capacity.
data await confirmation in further experiments.
Results: TLR-PGE 2 matured DCs showed elevated
Conclusion: Taken together, our results show that
expression of costimulatory molecules such as CD86
maturation of DCs with a TLR-PGE2 cocktail results
and CD80, and appeared more mature (higher CD83
in DCs with superior migration as well as T-cell
and HLA-DR). As expected, DCs matured with the
stimulatory capacities. However, caution might be
PGE 2-containing cocktail showed a significantly
indicated when antigen-processing and crosspresen-
higher migration capacity. Interestingly, only DCs
tation capabilities are required, e.g. in vaccination
expressing highest levels of costimulatory molecules
strategies using tumor-lysate loaded DCs. Therefore,
were able to migrate towards a CCL19/21 gradient.
in future clinical trials the choice of the DC matura-
TLR-PGE 2 matured DCs secreted more IL-12 than
tion cocktail should be tailored to the requested in
TFNα/IL-1ß-stimulated DCs (2000 vs. 50 pg/ml, re-
vivo functions.
ORFV Vector Vaccines - therapeutic potency in robust CRPV
rabbit tumor model
Feger T.1, Amann R.1, Iftner T.2, Schneider M.2, Rammensee H.-G.1
Universität Tübingen, Immunologie, Tübingen, Germany,
1
Universität Tübingen, Experimentelle Virologie, Tübingen, Germany
2
Virus vector vaccines are well known for the induc-
CRPV-DNA via gene gun. After papilloma had estab-
tion of strong immune responses against the insert-
lished the rabbits were vaccinated with 5*107 pfu of
ed antigen. The intrinsic adjuvant function of viral
recombinant ORFV expressing the CRPV proteins E1,
vectors induces strong humoral and cellular respons-
E2, lE6 and E7, respectively. A significant (p< 0,001)
es without additional administration of adjuvants.
reduction of tumor growth was observed already
Despite these massive advantages, several draw-
two weeks post vaccination. The overall response
backs impair the use of viruses as vector vaccines. (i)
rate was 83%. Responses were accompanied by a
Induced immune responses are directed against the
massive infiltration of lymphocytes into the develop-
viral backbone (ii) repeated boost immunizations are
ing tumor. Blood was taken in two weeks intervals
impaired by a growing response against viral back-
and monitored for antigen specific T cells by RT-PCR.
bone proteins (iii) existing prevalence (iii) limited
size of inserted genes and others.
We established an ORFV vector vaccine platform
that allows the fast development of new recombinants within 4 weeks. These recombinants allow
the simultaneous expression of several large target
antigens. Immunization with ORFV vector vaccines
cause a massive induction of the cellular and humoral
immune system and produce a long lasting, balanced immune response. Additionally, the induced
immune response is directed mainly against the inserted foreign antigens but not the viral backbone.
This allows repeated boost immunizations or immunizations against different target antigens. Our
replication deficient ORFV vector vaccines show an
excellent safety profile and are well tolerated.
Here, we used the challenging CRPV (cotton tail
rabbit papilloma) rabbit tumor model to demonstrate
a therapeutic efficacy and tumor growth inhibition
by ORFV vector recombinant vaccination. Outbred
New Zealand white rabbits were challenged with
Allogeneic dendritic cells (AlloDCs) transduced with an infectionenhanced adenovirus as adjuvant for cancer immunotherapy
Fotaki G.1, Jin C.1, Karlsson-Parra A.1, Yu D.1, Essand M.1
Uppsala University, Immunology, Genetics and Pathology, Uppsala, Sweden
1
AlloDC cancer immunotherapy utilizes the allo-
compartment and delay tumor growth in a B16 mela-
geneic reaction as an immunostimulatory adju-
noma model, in comparison with the mature murine
vant. Intratumoral alloDC vaccination reverses the
DCs alone. Concluding, the Ad5f35PTD transduced
immune suppressed tumor micro-environment and
alloDCs expressing gp100 showed potency in elicit-
activates anti-tumor immune responses. Herein, we
ing tumor-specific immune responses in comparison
aim to examine the use of Ad5f35PTD, an infection-
to the alloDCs alone.
enhanced serotype 5 adenovirus containing fibers
from serotype 35 and cell-penetrating peptides on
the hexons, as a vector to deliver tumor-associated
antigens to monocyte-derived immature DCs in combination with an established DC maturation cocktail.
Ad5f35PTD-transduced, cocktail-matured DCs expressed co-stimulatory and activation molecules and
secreted the Th-1 type cytokine IL-12, in vitro. Natural
killer (NK) cells migrated better towards medium
from Ad5f35PTD-transduced, cocktail-matured DCs
than medium from only cocktail-matured DCs, which
was in accordance with a higher CXCL-10 chemokine
level. The alloDC effect was examined in vivo using
mature murine DCs of BALB/c origin, transduced
with Ad5f35PTD encoding the melanoma-associated
antigen gp100, injected subcutaneously into C57BL/6
mice. They induced a higher migration of host DCs
in the draining lymph node, in comparison to the
mature murine DCs alone. The migrated host DCs
efficiently presented the captured gp100 and activated gp100-specific CD8+ T-cells. Moreover, alloDCs
transduced with the gp100-encoding Ad5f35PTD
reduced the immunosuppressive environment when
injected intratumoraly by reducing the ratio of monocytic to granulocytic myeloid-derived suppressor cell
The Ellegaard Göttingen minipig as a large animal model for
anti-cancer vaccination
Frøsig T.M.1, Overgaard N.H.1, Sørensen M.R.1, Buus S.2, Andersen M.H.3, Christensen D.4, Jungersen G.1
Technical University of Denmark, National Veterinary Institute, Section of Immunology and Vaccinology,
1
Frederiksberg, Denmark,
University of Copenhagen, Faculty of Health and Medical Sciences, Laboratory of Experimental Immunology,
2
Department of International Health, Immunology and Microbiology, Copenhagen, Denmark,
Copenhagen University Hospital, Center for Cancer Immune Therapy (CCIT), Department of Hematology,
3
Herlev, Denmark,
State Serum Institute, Department of Infectious Disease Immunology, Copenhagen, Denmark
4
The relevance of rodents as model animals for studies
pigs were divided in three groups with 1 µg/peptide,
of human vaccinology and immunology has been
10 µg/peptide and 100 µg/peptide, respectively, and
questioned from many sides and numerous clinical
immunized seven times each. We previously showed
studies based on data from preclinical rodent studies
in a mouse study that the intraperitoneal adminis-
have failed in the past. Major differences between
tration route is advantageous for inducing cytotoxic
man and rodents in immunology, physiology and
CD8 T cells; here we investigate this route of admin-
size make it difficult to extrapolate from preclinical
istration in the minipigs. A challenge for our proto-
treatment protocols optimized on rodents to the clin-
col is the aim of inducing immunity against endog-
ical setting. The pig size, immune and physiological
enous peptides and as a control of the immunization
systems are much more similar to humans and we
we included tetanus toxoid in all groups in similar
aim to establish the pig as a supplementally large
amounts as the IDO peptides.
animal model for immune therapy against cancer.
We did observe induction of specific T cell respons-
We previously immunized a cohort of healthy lan-
es in all groups using IFN-gamma ELISPOT, with a
drace pigs with overlapping peptides spanning the
tendency of obtaining more responses in the 1 ug/
entire sequences from the cancer-associated pro-
peptide group, but also a possible induction of a more
teins IDO and RhoC, and showed induction of spe-
tolerogenic environment. We will investigate this
cific immune responses. These pigs originated from
further, i.e. through staining for flow cytometry with
a Danish production farm and included some with
our unique swine MHC multimers. Our results show
high background immune signal. This was possibly
the feasibility of using the healthy minipig as a valu-
due to infection with multiple latent pathogens from
able animal model for vaccination against cancer.
the stable and we had to exclude these for subsequent
analyses.
In this current study we switched to using healthy
Ellegaard Göttingen Minipigs as their background
infection level is better controlled and defined, and
in addition these animals are more suited for longterm studies due to their lower growth rate. We used
CAF09 as an adjuvant for a formulation of four 30or 31-mer peptides from the IDO sequence including
11 predicted ligands for SLA-2*03:01, an MHC class
I molecule known to be present in all pigs. 15 mini-
7UV1 - a second-generation, peptide-based, therapeutic cancer
vaccine targeting the reverse transcriptase subunit of human
telomerase (hTERT)
Inderberg E.M.1, Lilleby W.1, Guren T.1, Brunsvik P.F.1, Lislerud K.1, Tornes A.2, Aamdal S.1, Gaudernack G.2
Oslo University Hospital, Oslo, Norway, 2Ultimovacs AS, Oslo, Norway
1
Telomerase represent a near universal target for a
gen sensitive, metastatic prostate cancer [EudraCT
cancer vaccine since it is highly expressed in 85-90%
No. 2012-002411-26] receive UV1 concomitant with
of all cancers, and only weakly in normal tissue rep-
androgen therapy. Analysis of immune response data
resenting a near universal target for a cancer vaccine.
demonstrate effective population immunisation with
Patients with objective clinical responses after
UV1 immune response rates ranging between 76%
vaccination provide a unique opportunity to iden-
and 86% across studies in unselected patients.
tify the immunological characteristics underlying
tumour regression. We screened a hTERT peptide
library using blood samples from cancer patients
experiencing long-term survival following vaccination with different first-generation hTERT vaccines.
The library consisted of long, overlapping hTERT
peptides. Strong immune responses were observed
against multiple novel hTERT epitopes not present in
the vaccines given. Based on these data, three hTERT
peptides shown to elicit strong proliferation responses across several long-term survivors were selected
as components in the UV1 vaccine. UV1 is thus the
first cancer vaccine based on epitope spreading data,
and patient data associating UV1 peptide responses
with survival benefit. UV1 contains multiple epitopes
capable of a broad population coverage. Both Th and
CD8 cells recognize UV1 epitopes and cloned T cells
are multifunctional.
UV1 is currently being investigated in three phase I/
IIa trials where UV1 is given as intradermal injections
with GM-CSF as adjuvant. In melanoma [EudraCT No.
2013-005582-39] UV1 is given in combination with
iIpilimumab. In NSCLC [EudraCT No. 2012-00185220] patients with stage IIIb-IV disease receive UV1 as
monotherapy. Patients with newly diagnosed, andro-
Immunotherapy of the Merkel Cell Carcinoma by vaccination
with optimized DCs transfected with the viral oncogenic driver the large T antigen
Gerer K.F.1,2, Erdmann M.1, Schuler G.1, Schaft N.1, Hoyer S.1, Dörrie J.1
Universitätsklinikum Erlangen, Department of Dermatology, Erlangen, Germany,
1
Friedrich-Alexander-Universität Erlangen-Nürnberg, Division of Genetics, Department of Biology, Erlangen,
2
Germany
In several types of cancer, a viral involvement has
We co-electroporated these optimized caIKK-DCs
been discovered. The contribution of the Merkel cell
with mRNA coding for LT antigen, or LT-DCLamp
polyomavirus (MCV) is associated with the emer-
- the latter one to permit MHC class II presentation.
gence of Merkel cell carcinoma (MCC). Until now, no
These transfected DCs, expressing the LT or the LT-
sufficient therapy is available for MCC. Therapeutic
DCLamp antigens, were used to stimulate autologous
vaccines, like dendritic cell (DC) vaccines, represent
CD8+ T cells or a mixture of CD4+ and CD8+ T cells
an option to generate tumor-specific cytotoxic T cells
for several weeks to measure their ability to induce
(CTLs) to destroy tumors. There the choice of a suit-
an antigen-specific immune response against the LT
able target antigen is crucial. Hence, in this study,
antigens. After at least two rounds of stimulation, the
we examined the immunogenic potency of the viral
T cells stimulated with the LT-transfected optimized
component which causes the malignant transfor-
DCs, recognized the antigen. The stimulation with
mation and which is still present in an established
LT-DCLamp-transfected optimized DCs even led to a
tumor, but not in healthy tissue.
more effective T-cell induction. In most donors, the
MCV can integrate into the host cell genome and
combination of CD4+ and CD8+ T cells was more ef-
express a truncated form of one of its proteins, the
ficient than the use of CD8+ T cells only, but in some
large T antigen (LT), which is the oncogenic driver.
donors the presence of CD4+ T cells decreased the
The LT antigen is a very promising antigen for thera-
specific T-cell response.
peutic DC vaccination, because it is: i) a “foreign”
These results show that optimized DCs transfected
antigen and thus not exposed to self-tolerance mech-
with LT-mRNA were able to present epitopes derived
anisms, ii) it is similar in various patients, and iii) it
thereof. Moreover, these epitopes were immunogen-
is relevant for the oncogenic phenotype avoiding the
ic and induced T-cell responses. In conclusion, we
rise of antigen-loss variants of the tumor.
now possess the technical prerequisites to take this
Although it is possible to generate, mature, and sub-
method into the clinic. This approach offers a new
sequently load autologous DCs with antigen, we ob-
and promising therapy option for a disease without a
served that these cells need an additional activation
current approved or efficient standard therapy.
signal to induce a potent response and even a longlasting immunological memory. This signal was introduced in the cells by constitutively active mutants
of components of the NF-κB signaling pathway
(caIKKs), which highly increased the immunogenicity of the DCs.
NS, SH, and JD share senior authorship
Development of dendritic cell vaccination for combined melanoma immunotherapy
Grees M.1,2, Sharbi-Yunger A.3, Eisenbach L.3, Utikal J.1,2, Umansky V.1,2
German Cancer Research Center (DKFZ), Skin Cancer Unit, Heidelberg, Germany,
1
University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Department of Dermatology,
2
Venereology and Allergology, Mannheim, Germany,
Weizmann Institute of Science, Department of Immunology, Rehovot, Israel
3
Malignant melanoma is known for its fast progres-
related protein (TRP) - 1. Upon the construct elec-
sion and poor response to current treatments. Despite
troporation into DC, cells were injected into naïve
melanoma immunogenicity, the overall results of
C57BL/6 mice. We detected a significant activation of
immunotherapeutic trials are largely disappointing,
CD8 T cell responses, reflected by increased specific
indicating that new approaches for melanoma treat-
killing of target cells in in vitro and in vivo assays.
ment are urgently needed. Tumor escape could be
To complement our DC vaccine repertoire, we have
due to a profound immunosuppression induced by
developed and characterized MHC class II constructs
chronic inflammation in the melanoma microenvi-
encoding for tyrosinase and TRP-1 peptides. A strong
ronment, which is characterized by the long-term se-
stimulation of CD4 T cell response in C57BL/6 mice
cretion of inflammatory mediators. Moreover, the de-
was detected in the proliferation assay. In addition,
velopment of melanoma-specific effector T cells may
DC electroplated with MHC class I restricted con-
be hampered by insufficient tumor antigen delivery,
structs were applied in melanoma-bearing ret trans-
processing and presentation. In the ret transgenic
genic mice to evaluate tumor specific T cell activation
mouse model of spontaneous melanoma that mimics
and anti-tumor efficiency.
human melanoma development, we observed an ac-
Furthermore, developed DC vaccination will be com-
cumulation of inflammatory factors and activated
bined with the neutralization of immunosuppres-
myeloid-derived suppressor cells (MDSCs) in mela-
sion by ultra-low dose paclitaxel. We suggest that
noma lesions, which was associated with tumor pro-
combined melanoma immunotherapy based on the
gression. Upon administration of paclitaxel at ultra-
simultaneous targeting of melanoma escape mecha-
low, non-cytotoxic doses in tumor-bearing mice, we
nisms such as insufficient anti-tumor T cell reactivity
demonstrated a reduction of inflammatory mediators
and immunosuppressive tumor environment could
in melanoma lesions together with decreased MDSC
lead to a significant improvement of existing mela-
frequencies
noma therapies.
and
immunosuppressive
functions,
leading to the restoration of T cell reactivity and prolonged mouse survival.
We have recently established the production of constructs encoding major histocompatibility complex
(MHC) class I molecules that couples the peptide
presentation and activation of dendritic cells (DC).
We have generated such constructs encoding melanoma-associated antigens tyrosinase and tyrosinase
Developing a cancer vaccine for two-dimensional T cell
activation using the Invariant chain
Mensali N.1, Grenov C.A.2,3, Inderberg E.M.1, Kucera A.2,3, Myhre M.R.1, Fredsvik Gregers T.2, Gaudernack G.4,
Kvalheim G.1, Bakke O.2,3, Wälchli S.1,4,5
OUS-KKT, Section for Cellular Therapy, Oslo, Norway,
1
University of Oslo, Department of Molecular Biosciences, Oslo, Norway,
2
University of Oslo, Centre for Immune Regulation, Faculty of Medicine, Oslo, Norway,
3
OUS-IKF, Section for Cancer Immunology, Oslo, Norway,
4
University of Oslo, Centre for Cancer Biomedicine, Oslo, Norway
5
For many years, the focus of cancer immunotherapy
tivation of CD8+ and CD4+ T cells in one shot. Fur-
was on stimulating the activity of CD81 lymphocytes
thermore, the Ii-TGFBRII construct was able to load
to harvest their cytotoxic capacities. However, it has
MHC-I in proteasome deficient T2 cells, suggesting
become more evident that a two-dimensional T cell
a direct loading in the endoplasmic reticulum (ER).
+
+
response involving CD4 T cells as well as CD8 T
Our results identify the CLIP region of Ii as an ideal
cells is necessary for more long-lasting and efficient
vehicle for cancer vaccination, likely leading to an
tumour eradication.
anti-tumour immune response that is co-orchestrat-
We have previously showed that CLIP-modified In-
ed by both CD4+ and CD8+ T cells.
variant chain (Ii) can load MHC class I molecules
(MHC-I) with antigenic peptides. We now show that
expanding the CLIP modified region of Ii to cover
a larger region of the antigen allows simultaneous
MHC-I and MHC class II (MHC-II) loading and consequently CD8+ and CD4+ T cell activation.
The antigenic focus of our study is a public neoantigen created by a frame-shift mutation in the TGFBRII
gene. This mutation is observed in over 70% of colorectal cancer patients with microsatellite instability,
making it a therapeutically relevant target.
Through a series of comparative studies, we have
identified an optimal TGFBRII insert: comprising 23
amino acids from the frameshift mutation. Expression of the selected Ii-TGFBRII construct in target
cells by mRNA electroporation provided strong ac-
Flt3L-based in situ vaccination for the treatment of lymphoma
Hammerich L.1, Brody J.1
Icahn School of Medicine at Mount Sinai, Hematology and Medical Oncology, Tisch Cancer Institute, New
1
York, United States
Background: Low-grade B-cell lymphomas are gen-
Flt3L and XRT with poly-ICLC induced long-lasting
erally incurable, with standard therapies inducing
tumor regression in 40% of mice. Tumor growth at
only temporary remissions. Tumor-targeted vaccines
the untreated site was also delayed by this treatment.
represent promising, novel treatment strategies. In a
I.t. injection of GM-CSF together with Flt3L increased
pre-clinical mouse model, we attempt to develop and
the number of migratory CD103+/TLR3+ and
optimize an in situ vaccine combining recruitment of
IRF8+/TLR3+ (CD103 precursors) DC in spleen and
dendritic cells (DC) and low-dose local radiotherapy
tumor. Consistently, GM-CSF injection enhanced the
(XRT) with intratumoral (i.t) administration of a toll-
efficacy of the Flt3L-primed in situ vaccine leading
like receptor (TLR) agonist.
to complete tumor regression at the treated site and
Methods: Balb/c mice were inoculated with A20
a significant survival benefit compared to the in situ
lymphoma cells subcutaneously on the flank. After 9
vaccine without GM-CSF.
days mice were injected i.t. with Flt3L (30ug/mouse)
Conclusions: In situ vaccination combining intratu-
daily for 9 days, followed by local irradiation (9Gy,
moral Flt3L/GM-CSF injection with local XRT and
single dose) and i.t. injections of poly-ICLC (50ug/
poly-ICLC induces a potent anti-tumor immune re-
mouse) for 5 days. Leukocyte accumulation was
sponse able to induce long-term regression of estab-
analyzed by flow cytometry and animals were moni-
lished lymphoma tumors.
tored for tumor growth and survival. For assessment
of systemic anti-tumor response tumors were inoculated on both flanks, but only one site was treated
as described before. In some groups, GM-CSF was
injected intratumorally at the same time as Flt3L.
To assess uptake of tumor antigens by DC, mCherryexpressing A20 cells were used.
Results: Injection of Flt3L induced potent accumulation of DC at the tumor site, tumor-draining lymph
node and the spleen, with intratumoral injection
being superior to systemic injection. Local XRT increased the amount of mCherry+ DC in the tumor,
indicating increased uptake of dying tumor cells.
While combination of FLt3L and local XRT was not
able to cure established tumors, the combination of
A novel combination immunotherapy consisting of tumorassociated macrophage-targeted vaccine, TLR agonist, and
neoantigen-specific T cell transfer cures tumor highly resistant
to immune checkpoint blockade
Harada N.1, Muraoka D.2, Seo N.1, Akiyoshi K.3, Shiku H.1
Mie University Graduate School of Medicine, Department of Immuno-Gene Therapy, Tsu, Japan,
1
University of Shizuoka, Center for Drug Discovery, Sizuoka, Japan,
2
Kyoto University Graduate School of Engineering, Department of Polymer Chemistry, Kyoto, Japan
3
ORAL
TALK
SHORT
2016
There is a great medical need of novel therapeu-
TLR agonist such as CpG oligoDNA or poly-ICLC
tic strategies effective for the treatment of immune
RNA resulted in the acquisition of antigen presenta-
checkpoint blockade-resistant tumors. We recently
tion activity by TAMs, as assessed by ex vivo co-
found that murine fibrosarcoma CMS5a established
culture with LPA-specific CD8+ T cells. Treatment
in the subcutis of BALB/c mice was completely re-
of established CMS5a tumor (7 days after implanta-
fractory to potent immune checkpoint inhibition
tion) with the CHP:LPA vaccine plus TLR agonist fol-
using anti-PD-1, anti-CTLA-4 and anti-GITR anti-
lowed by adoptive transfer of neoantigen-specific T
bodies. PD-1 and PD-L1 expression and CD8+ T cell
cells resulted in strong suppression of tumor growth
infiltration and activation in this tumor were much
and finally cured the diseased mice. These data evi-
lower as compared to those in immune checkpoint
dently support the clinical application of this novel,
blockade-sensitive murine tumors. Although the
potent combination immunotherapy, designated as
CMS5a tumor had mutation load comparable to the
‘TriCombo ACT’, for the treatment of immune check-
sensitive tumors, CD8+ T cell response to potential
point blockade-resistant tumors.
neoantigens could not be detected. These features
of CMS5a tumor closely resembles those of immune
checkpoint blockade-resistant human tumors. Whole
gene expression analysis at the tumor local site indicated a significant difference in a gene set related
to macrophage activation between the CMS5a tumor
and other sensitive tumors. In accordance with this,
tumor-associated macrophages (TAMs) in the CMS5a
tumor showed inactive status in terms of the expression of CD40, CD80, and MHC-II, and they did not
exert antigen presentation activity. We have developed a macrophage-targeted, nanogel-based vaccine
delivery system comprising of a cholesterol-modified
polysaccharide (so called cholesteryl pullulan or
CHP). Intravenously injected long peptide vaccine
formulated with the CHP nanogel (CHP:LPA vaccine)
was efficiently incorporated into TAMs. Intravenous
administration of the CHP:LPA vaccine with soluble
RNAdjuvant®, a novel, highly-potent RNA-based adjuvant,
combines strong immunostimulatory capacities with a
favorable safety profile
Heidenreich R.1, Tadjalli Mehr K.1, Noth J.1, Koch S.D.1, Döner F.1, Hong H.S.1, Melber K.1, Dähling A.1, Roos T.1,
Lutz J.1, Kowalczyk A.1, Baumhof P.1, Scheel B.1, Voss S.1, Kallen K.-J.1, Fotin-Mleczek M.1, Gnad-Vogt U.1
CureVac AG, Tübingen, Germany
1
Purified recombinant proteins and peptides, which
21, either alone or in combination with 1/20 or 1/10 of
are currently under development in various anti-can-
the licensed Rabipur® dose. In both groups, vaccina-
cer vaccination approaches, lack sufficient immuno-
tions were well tolerated with mild to moderate in-
genicity. Therefore, potent adjuvants are needed to
jection site reactions and flu- like symptoms as main
induce strong and persistent anti-tumor immunity.
side effects. Serum analyte profiling 6 hours and 24
However, currently only few adjuvants are licensed,
hours post RNAdjuvant® treatment revealed a tran-
most of which primarily enhance antibody, but not
sient increase of factors involved in T cell chemotaxis
T cell responses.
but no increase of IL-6. Virus neutralizing antibody
Here, we demonstrate that a novel, well defined, and
titers (VNTs) measured on days 14 and 28 revealed a
thoroughly characterized RNA-based adjuvant me-
significant increase in median VNTs in subjects with
diates balanced and long-lasting humoral and cel-
RNAdjuvant® compared to their respective control
lular immune responses. Our adjuvant significantly
group with 1/10 dose Rabipur® alone.
enhances anti-tumor immunity, and even complete
In summary, our data suggest that RNAdjuvant® rep-
tumor rejection can be achieved as shown for the
resents a novel, highly efficacious adjuvant candi-
syngeneic TC-1 tumor model, a murine model of
date that can enhance cellular and humoral immune
human HPV-induced cervical cancer.
responses.
Our adjuvant acts locally, promoting strong but
transient up-regulation of anti-viral and pro-inflammatory cytokines, CXCR3-ligands and cytoplasmic
RNA sensors at the injection site, avoiding any systemic cytokine release. These changes are followed
by activation of different subsets of immune cells in
the draining lymph nodes. In repeated dose toxicity
studies carried out in mice and pigs no toxicity events
were observed demonstrating an excellent preclinical safety profile. A phase I first in man clinical trial
testing different doses of RNAdjuvant® alone and
in combination with reduced doses of the licensed
rabies vaccine Rabipur® was successfully conducted
in 43 subjects. Healthy volunteers received 2 intramuscular injections of RNAdjuvant® on days 0 and
Photochemical internalization - light-induced enhancement of MHC
Class I antigen presentation, giving strong enhancement of
cytotoxic T-cell responses to vaccination
Høgset A.1, Haug M.2, Brede G.2, Håkerud M.1,3, Nedberg A.G.1,3, Edwards V.1,3, Selbo P.K.1,3, Kundig T.M.4,
Johansen P.4, Otterhaug T.1, Halaas Ø.2
PCI Biotech AS, Oslo, Norway,
1
The Norwegian University of Science and Technology, Department of Cancer Research and Molecular
2
Medicine, Trondheim, Norway,
Oslo University Hospital - The Norwegian Radium Hospital, Department of Radiation Biology, Oslo, Norway,
3
University Hospital Zurich, Department of Dermatology, Zurich, Switzerland
4
For cancer vaccination and immunotherapy it is es-
antigens enhancement of CD8+ T-cell responses
sential to stimulate cytotoxic T-cells (CTLs) to rec-
of up to 100 times have been observed when PCI
ognize and kill the tumour cells. Priming of CTLs
is used in combination with the poly(IC) adjuvant;
is generally mediated through MHC Class I antigen
with a strong synergy between PCI and the adjuvant.
presentation by antigen presenting cells (APCs).
With an HPV long peptide and with several protein
Since the MHC class I presentation machinery is
antigens in addition to the CD8+ T-cell response
localised in the cytosol, MHC class I presentation
a significant stimulation of CD4+ T-cell responses
typically requires cytosolic delivery of the antigen.
can also be observed, and in some cases also an in-
Unfortunately, this is often difficult to achieve with
crease in antibody production.In vivo studies with
exogenously added peptide or protein antigens, since
therapeutic peptide antigen vaccination in a mouse
such antigens are primarily taken up into endocytic
model for HPV-induced cancer show that the use of
vesicles, and then “by default” are routed for MHC
PCI strongly enhances anti-tumour responses to the
Class II presentation. Photochemical internalisa-
vaccine, both when the vaccination is performed
tion (PCI) is a technology that can help solving this
intradermally and when intratumoural administra-
problem by inducing an illumination-mediated per-
tion is employed.The TPCS2a photosensitiser used
meabilisation of the membranes of endocytic vesi-
in PCI is cheap to produce, withstands autoclavation
cles, thereby releasing endocytosed antigens into
and is stable for several years at ambient tempera-
the cytosol. This is achieved by employing a pho-
tures. TPCS2a is currently in clinical development
tosensitising molecule that is designed to localise
for enhancement of the effect of cytotoxic anti-cancer
specifically in endocytic membranes. Upon illumi-
drugs, and it has been shown that TPCS2a can be
nation, the photosensitiser induces photochemical
administered safely to patients in much higher doses
reactions that make the membranes leaky, thereby
than what is needed for the use in immunotherapy.
releasing endocytosed molecules into the cytosol. In
In conclusion, PCI has a completely new mechanism
vitro it has been shown that the use of PCI leads to
of action as a vaccination enhancement technology,
strongly increases MHC Class I presentation APCs.
representing a new and potent tool for stimulation of
In vivo PCI-mediated vaccination is performed by
CTL and in some cases also other types of immune
injecting a mixture of vaccine and photosensitiser
responses. Preparations for a clinical study with PCI-
intradermally, followed by illumination of the injec-
mediated vaccination is on-going.
tion site; and with this regimen, PCI substantially
enhances immune responses to various types of polypeptide- based antigens. Thus, with short peptide
Peptide vaccination against cancer testis antigens in
combination with hypomethylating treatment for patients with
Myelodysplastic Syndrome and Acute Myeloid Leukemia:
An ongoing phase I study
Holmberg S.1, Ortved Gang A.1, Svane I.M.2, Reker Hadrup S.3, Høgh Dufva I.1
Herlev Hospital, Department of Hematology, Herlev, Denmark,
1
Herlev Hospital, Center for Cancer Immune Therapy, Herlev, Denmark,
2
Technical University of Denmark, The National Veterinary Institute, Section for Immunology og Vaccinology,
3
Copenhagen, Denmark
In this phase I trial we will combine the treatment
three CTA’s for which abundant re-expression has
of hypomethylating agents with a peptide vaccine,
been shown following AZA treatment, including NY-
to boost an immune response against four selected
ESO-1, MAGE-A3 and PRAME. WT-1 is additionally
tumor associated antigens which are known to be
included as this protein has proven to be an impor-
regulated by methylation, in patients with high-risk
tant antigen in hematological malignancies and is
myelodysplastic syndrome (MDS) and acute myeloid
likewise upregulated in response to AZA treatment.
leukemia (AML).
The peptides are between 25-29 mer and include a
MDS is a clonal disorder and characterized by in-
broad selection of HLA class I and II epitopes. Each
creasing bone marrow failure due to accumulation
vaccine contains ~50 µg of each peptide and is mixed
of genetic and epigenetic changes in hematopoietic
as a suspension with Montanide ISA-51. The use of
stem cells. In high-risk disease you find chromosom-
synthetic long peptides has shown superior effect in
al breakage, point mutations and promoter hyper-
contrast to minimal peptide sequences. They contain
methylation of tumor suppressor genes, and a high
several CD4 and CD8 T- cell epitopes for a broad
risk of progression to AML. Patients with high-risk
range of HLA types, and thus allowing inclusion of
MDS have a poor prognosis with a median survival
participants without prior selection based on HLA
of around 11 months. For most patients, who are
expression.
not eligible for bone marrow transplantation, hypo-
Inclusion commence after six courses of AZA and
methylating agents, such as azacitidine (AZA), are
following a treatment evaluation. If there is contin-
currently the only treatment option. The demand
ued indication of AZA treatment, a set of three vac-
for more effective therapies in this patient group is
cinations is given together with the following three
huge. Though the mechanism of AZA is not fully elu-
courses of AZA. An additional vaccination is then
cidated re-expression of tumor suppressor genes can
given every six months for two years or until there is
serve as a mechanism for growth arrest. In addition,
unfavorable disease progression.
there is accumulating evidence for an up-regulation
15 patients from the department of Hematology at
of cancer testis antigens (CTA), which could lead to
Herlev hospital, Copenhagen, Denmark, will be in-
increased immune recognition of tumor cells and
cluded starting February 2016. The primary endpoint
immune-mediated tumor cell killing.
is to elucidate whether the combination of AZA and
CTA’s are known to be immunogenic and are only
peptide vaccination is a safe and tolerable treatment,
expressed at immunoprivileged sites and on malig-
but specific immune responses and clinical efficacy
nant cells, making them ideal targets for therapeu-
will also be evaluated.
tic cancer vaccination. We have chosen specifically
Extent and location of tumor infiltrating lymphocytes in
microsatellite stable colon cancer predict outcome to adjuvant
Active Specific Immunotherapy
Turksma A.1, Coupe V.1, Shamier M.1, Lam K.1, de Weger V.1, Belien J.1, van den Eertwegh A.1, Meijer G.1,2, Meijer C.1,
Hooijberg E.1,2
Vrije Universiteit University Medical Center / Cancer Center Amsterdam, Pathology, Amsterdam, Netherlands,
1
Antoni van Leeuwenhoek / Netherlands Cancer Institute, Pathology, Amsterdam, Netherlands
2
Purpose: To determine the prognostic and predic-
intraepithelial T cell infiltrates for clinical outcome
tive value of tumor infiltrating lymphocytes (TIL) in
after ASI treatment. For the analysis we used contin-
colon cancer in a cohort of patients who previously
uous data on T cell counts comparing the treatment
took part in a trial on adjuvant Active Specific Im-
effect of adjuvant Active Specific Immunotherapy in
munotherapy (ASI).
patients with high TIL to the treatment effect in low
Background information: Vermorken et al (Lancet,
TIL.
1999, Vol. 353(9150), p345-350) conducted a multi-
Results: Based on the data presented we concluded
center clinical trial on adjuvant ASI for colon cancer
that 1) high numbers of stromal CD3 T cells have pos-
patients. A vaccine consisting of irradiated autolo-
itive prognostic value measured as DSS for patients
gous tumor cells admixed with the adjuvant Bacil-
with stage II MSS tumors, and 2) high numbers of ep-
lus Calmette-Guérin bacteria has been evaluated.
ithelial CD8 positive T cells have positive prognostic
In that study, a comparison was made between
value measured as RFI for the group of patients with
surgery alone and surgery followed by adjuvant ASI
stage II MSS tumors as well as for the whole group
treatment. The recurrence free interval for patients
(stage II plus stage III together). Furthermore we con-
with stage II tumors was significantly extended for
cluded that high numbers of preexisting stromal CD3
patients treated with surgery plus ASI compared to
positive T cells are of positive predictive value in ad-
surgery alone, but not for stage III patients. A follow
juvant ASI treatment measured as DSS as well as RFI.
up study by de Weger et al (CCR, 2011, Vol. 18(3),
(Note; the results of our study have recently been
p882-889) showed that patients with stage II micros-
published in Clin Cancer Res; 22(2) January 15, 2016).
atellite stable tumors (MSS) benefited most from ASI
Conclusion: ASI therapy contributes to an improved
treatment. The data on patients with microsatellite
DSS and RFI in patients with MSS colon tumors har-
instable tumors (MSI) were inconclusive.
boring high numbers of preexisting stromal CD3+
Current experimental Design: Here we determined
TIL. Validation in future clinical trials is awaited.
the number and location of CD3+ and CD8+ cells
in archival tumor samples of 106 MSS colon cancers.
Disease specific survival (DSS) and recurrence free
interval (RFI) were evaluated in detail at the 5 year
post-treatment time point. First, we investigated the
prognostic value of stromal and intraepithelial T cell
infiltrates independent of treatment arm. Second,
we investigated the predictive value of stromal and
BRAF and MEK inhibitors influence human immune cell
phenotype and function
Hoyer S.1, Eberlein V.1, Schuler G.1, Heinzerling L.1, Schaft N.1, Dörrie J.1
1
BRAF and MEK inhibitors are commonly used to
Therefore we co-cultured gp100/HLA-A2-TCR-trans-
treat melanoma, since BRAF mutations are highly
fected CD8+ T cells and peptide-loaded T2.A1 cells
overrepresented in melanoma and are known to
and determined the cytokine secretion profile, as
serve as driver mutation. Additionally, MEK is tar-
well as CD69 and CD25 expression. Vemurafenib only
geted to circumvent the paradox effect induced by
slightly decreased CD69 expression, but Trametinib
BRAF inhibition. This treatment results only in an
drastically reduced its expression. Thus, Trametinib
increase in the overall survival, but only a small
blocked T-cell activation concerning cytokine secre-
fraction of the patients has durable benefits. Thus,
tion and surface marker expression, though func-
combination therapies might be advantageous. Even
tional avidity was not affected. In contrast, Dab-
though checkpoint blockade together with BRAF/
rafenib only partially influenced the antigen-specific
MEK inhibition was shown to be beneficial in mice,
cytokine secretion by T cells. Concerning surface
administration to human patients induced severe
marker expression Dabrafenib was not able to restore
gastrointestinal toxicity. Hence, we tested in vitro,
the inhibitory effect of Trametinib. When we used
whether the application of BRAF/MEK inhibitors
MCSP-CAR-transfected T cells in stimulation assays,
influences dendritic cell (DC) maturation and T-cell
Vemurafenib and Dabrafenib decreased CD25 up-
activation, thus exploring whether a combination of
regulation, whereas especially Trametinib decreased
BRAF/MEK inhibitors with cellular immunotherapy
CD69 elevation.
might be possible and reasonable.
In conclusion, regarding these in vitro data, com-
We treated DCs during their maturation with BRAF
bination therapies of cellular immunotherapy and
and/or MEK inhibitors and determined the expres-
BRAF/MEK inhibitor treatment seems possible. For
sion of distinct maturation markers and assessed the
DC vaccination a combination with Dabrafenib and
cytokine secretion profile. Vemurafenib inhibited DC
Trametinib and for adoptive T-cell therapy a combi-
maturation as shown by the decreased expression of
nation with Vemurafenib would be recommendable.
CD25 and CD83. Also co-stimulatory molecules like
CD80 and CD40 were slightly affected. Moreover, less
CCR7 was expressed, whereas Trametinib treatment
enhanced CCR7 expression. However, Vemurafenib
treatment also decreased PD-L1 expression and elevated IL-8 secretion levels.
Furthermore we assessed the effects of these inhibitors on T-cell activation and functional avidity.
SH and VE contributed equally
NS and JD share senior authorship
A first-in-human phase I/II clinical trial assessing novel
mRNA-lipoplex nanoparticles for potent cancer immunotherapy
in patients
Jabulowsky R.A.1, Loquai C.2, Diken M.3,4, Kranz L.M.5, Haas H.4, Attig S.3, Buck J.4, Derhovanessian E.1,
Diekmann J.1, Fritz D.4, Jahndel V.1, Kemmer-Brück A.1, Kuehlcke K.6, Kuhn A.N.4, Langguth P.4, Luxemburger U.1,
Meng M.4, Müller F.1, Rae R.3, Sari F.1, Schwarck-Kokarakis D.1, Seck C.1, Spieß K.7, Hassel J.C.8, Utikal J.9,
Kaufmann R.10, Kreiter S.1,3, Huber C.1,3,11, Türeci Ö.11, Sahin U.1,3,5
BioNTech AG, Mainz, Germany,
7
University of Mainz Medical Center, Department of
8
1
2
Dermatology, Mainz, Germany,
TRON - Translational Oncology at the University
3
BioNTech Diagnostics GmbH, Mainz, Germany,
University of Heidelberg, NCT Heidelberg, Department
of Dermatology, Heidelberg, Germany,
German Cancer Research Center (DKFZ), University
9
Medical Center of Johannes Gutenberg University
Medical Center Mannheim, University of Heidelberg,
gGmbH, Mainz, Germany,
Department of Dermatology, Venereology and
BioNTech RNA Pharmaceuticals GmbH, Mainz,
4
Allergology, Mannheim, Heidelberg, Germany,
University of Frankfurt, Department of Dermatology,
Germany,
10
Research Center for Immunotherapy (FZI),
Venereology and Allergology, Frankfurt, Germany,
5
University Medical Center, Mainz, Germany,
EUFETS GmbH, Idar-Oberstein, Germany,
6
CI3 - Cluster of Individualized Immunointervention,
11
Mainz, Germany
Immunotherapeutic approaches have evolved as
ucts advancing from local to systemic targeting of
promising and valid alternatives to available con-
APCs. Here, RNA(LIP) products trigger a Toll-like re-
ventional cancer treatments. Amongst others, vac-
ceptor (TLR)-mediated Interferon-α (IFN-α) release
cination with tumor antigen-encoding RNAs by local
from plasmacytoid dendritic cells (DCs) and mac-
administration is currently successfully employed in
rophages stimulating DC maturation and hence in-
various clinical trials. To allow for a more efficient
ducing innate immune mechanisms as well as potent
targeting of antigen-presenting cells (APCs) and to
vaccine antigen-specific immune responses.
overcome potential technical challenges associated
Notably, BioNTech RNA Pharmaceuticals´ RNA(LIP)
with local administration, we have developed a
formulation is a universally applicable potent novel
novel RNA immunotherapeutic for systemic applica-
vaccine class for intravenous APC targeting and the
tion based on a fixed set of four liposome complexed
induction of potent synchronized adaptive and type-I
RNA drug products (RNA(LIP)), each encoding one
interferon-mediated innate immune responses for
shared melanoma-associated antigen. The ready-to-
cancer immunotherapy.
use RNA(LIP) products are prepared individually in a
To study safety, tolerability, immunogenicity and
straight-forward manner directly prior to use from
evaluate potential clinical activity of this pioneer-
three components being presented in a kit, namely
ing RNA(LIP) immunotherapy concept a multi-center
solutions containing RNA drug product, NaCl diluent,
phase I/II dose escalation trial is currently conducted
and liposome excipient.
in patients with malignant melanoma (NCT02410733)
The novel RNA(LIP) formulation was engineered (i)
demonstrating the swift clinical translation and fea-
to protect RNA from degradation by plasma RNases
sibility of this platform.
and (ii) to enable directed in vivo targeting of APCs
The therapeutic concept, trial design, treatment
in lymphoid compartments, thus (iii) allowing for
status and vaccine-induced immune response data
intravenous administration of multiple RNA prod-
from the first patients treated will be presented.
Design of reversible antigen-adjuvant conjugates for triggered
release inside antigen-presenting cells
Kramer K.1,2, Young S.L.2, Walker G.F.1
University of Otago, School of Pharmacy, Dunedin, New Zealand,
1
University of Otago, Department of Pathology, Dunedin, New Zealand
2
Stable antigen-adjuvant conjugates have shown to
with either PBS, CpG/OVA mixture or a CpG-OVA
elevate immune-responses over mixtures of antigen
conjugate. OTI and OTII cells were co-cultured with
and adjuvant. We hypothesised that antigen-adjuvant
the pulsed BMDCs and immunogenicity was meas-
conjugates designed to be specifically cleaved inside
ured by T-cell proliferation and interferon gamma
the cell may further enhance immunogenicity. Cy-
(IFN-γ) production in the co-culture. The CpG/OVA
tosine-phosphate-guanosine
oligodeoxynucleotide
mixture and the extracellular cleaved conjugate
(CpG) was conjugated onto the model antigen oval-
induced a low percentage of proliferated T-cells as
bumin (OVA) using either stable or reversible linking
well as low IFN-γ production while the stable and
chemistry. The two reversible conjugates were gener-
intracellular cleaved conjugates resulted in a higher
ated with different linkers containing a disulphide
immunogenicity as confirmed by a greater percent-
bond in order to test the sensitivity to cleavage via
age of T-cell proliferation and IFN-γ production. In
disulphide exchange reactions. The stability of the
summary higher immunogenicity correlates with
conjugates was determined by using analytical size-
the stability of conjugates to extracellular GSH con-
exclusion chromatography. Conjugates were incubat-
ditions and to cell culture supernatant.
ed with PBS or PBS containing glutathione (GSH) at
extracellular (10 µM) or intracellular concentrations
(5 mM) for 2 h at 37 °C. Conjugates were additionally incubated overnight at 37 °C in either fresh cell
culture medium (complete Iscove’s Modified Dulbeccos’s Medium, cIMDM) or cell culture supernatant
harvested from murine bone marrow derived dendritic cells (BMDC) which were cultured for 6 days in
cIMDM. Stability studies showed that one disulphide
linker was cleaved under extracellular reducing conditions (10µM GSH) and in the cell culture supernatant. The second disulphide linker was stable to the
extracellular reducing environment and cell culture
supernatant however was cleaved by intracellular reducing conditions (5mM GSH). Immunostimulatory
activity of the conjugates was tested in cell culture
studies using BMDC and T-cells. BMDC were pulsed
GAPVAC-101 phase I trial: First data of an innovative actively
personalized peptide vaccination trial in patients with newly
diagnosed glioblastoma
Kuttruff-Coqui S.1, Frenzel K.2, Hilf N.1, Heesch S.2, Kreiter S.2,3,4, Admon A.5, Bukur V.2, van der Burg S.H.3,6,
Gouttefangeas C.3,7, Kroep J.R.6, Welters M.J.3,6, Piro J.8, Ponsati B.8, Poulsen H.S.9, Lassen U.9, Martinez-Ricarte F.10,
Rodon J.10, Sahuquillo J.10, Stieglbauer M.7, Stevanovic S.7, thor Straten P.9, Skardelly M.7, Tabatabai G.7,
Platten M.11, Capper D.11, Deimling A.11, Dutoit V.12, Okada H.13, Ottensmeier C.14, Feist R.K.1, Fritsche J.1, Laske K.1,
Lewandowski P.1, Löwer M.4, Mendryzk R.1, Meyer M.1, Reinhardt C.1, Paruzynski A.2, Pawlowski N.1, Schoor O.1,
Song C.1, Stevermann L.1, Wagner C.1, Weinschenk T.1, Huber C.3, Rammensee H.-G.7, Dietrich P.-Y.12, Wick W.11,
Sahin U.2, Singh-Jasuja H.1
8
Germany,
9
1
BCN Peptides S.A., Barcelona, Spain,
Center for Cancer Immune Therapy, Copenhagen
2
CIMT - Association for Cancer Immunotherapy,
3
University Hospital, Herlev, Denmark,
Vall d’Hebron University Hospital, Institut Catala de
10
Mainz, Germany,
la Salut, Barcelona, Spain,
TRON -Translational Oncology at the University
11
Medical Center Mainz, Mainz, Germany,
12
TECHNION - Israel Institute of Technology, Haifa,
13
4
5
University Hospital Heidelberg, Heidelberg, Germany,
Université de Genève, Genève, Switzerland,
University of California San Francisco, San Francisco,
Israel,
United States,
Leiden University Medical Center, Leiden, Netherlands,
6
Eberhard Karls Universität Tuebingen, Tuebingen,
7
University of Southampton, Southampton, United
14
Kingdom
Germany,
The Glioma Actively Personalized Vaccine Consorti-
from a pre-manufactured “warehouse”. The ware-
um (GAPVAC; funded by the European Union Frame-
house contains 59 HLA class I-binding and three
work 7 Program) aims at treating newly diagnosed
class II-binding tumor-associated peptides frequent-
glioblastoma (GB) patients with two distinct actively
ly over-presented in GB. APVAC2 is composed of one
personalized vaccines (APVACs).
or two peptides that are de novo synthesized for a
Resected tumor material is analyzed for multiple
given patient and preferentially represent mutation-
biomarkers to characterize the tumor in depth and
bearing neo-epitopes.
to enable the design of APVACs tailored to each in-
After a preparation phase in which the warehouse
dividual patient: Tumor-specific mutations, the HLA
was generated and setup of APVAC selection and
peptidome and gene expression profile are assessed
manufacturing processes took place, the GAPVAC-
by next-generation sequencing, mass spectrometry
101 phase I clinical trial was initiated. Primary end-
and RNA microarray analysis, respectively. Further,
points of the study are assessment of safety, feasibil-
the patient-individual immune status is investigated
ity of APVAC manufacturing and biological activity.
by assessment of leukapheresis samples utilizing an
The trial is conducted at six European centers and
in vitro immunogenicity platform. Data are integrat-
recruits HLA-A*02:01 or A*24:02-positive patients
ed to define two distinct APVACs for each patient:
with newly diagnosed GB after gross total resection.
APVAC1 is composed of up to ten peptides selected
Patients receive APVAC1 and APVAC2 vaccinations
plus immunomodulators (poly-ICLC and GM-CSF)
three and six months post study enrolment, respectively, and concurrent to maintenance temozolomide
(TMZ).
As of November 2015, 11 patients have been enrolled,
of whom six already received APVAC vaccines. Composition and manufacturing are ongoing for four patients. All APVACs were generated in time without
ultimate failures. APVAC1 vaccines differ substantially with 31 out of 59 warehouse peptides have
been selected at least once, indicating the need for
personalization due to tumor heterogeneity even for
non-mutated epitopes. In patients’ tumor samples an
average of 40 non-synonymous mutations (including
known driver mutations) were identified. Injection
site reactions were the most frequent toxicities so far.
One brain edema (Grade 3) and one allergic reaction
(Grade 4) were observed, both potentially related to
the vaccinations. First data on biological activity of
APVACs and updated clinical data will be presented
at the Annual Meeting.
In conclusion, the GAPVAC concept has been successfully translated into the clinics and so far demonstrated to be safe and feasible with its level of
personalization matching the observed tumor heterogeneity.
A pipeline for fast track identification of candidate neoantigens
from cancer exome sequencing data
Kyzirakos C.1, Mohr C.2,3, Armeanu-Ebinger S.1, Feldhahn M.1, Hadaschik D.1, Walzer M.2, Döcker D.1, Menzel M.1,
Nahnsen S.4, Kohlbacher O.2,3,4, Biskup S.1
CeGaT GmbH, Tübingen, Germany,
1
University of Tübingen, Center for Bioinformatics and Dept. of Computer Science, Tübingen, Germany,
2
Max Planck Institute for Developmental Biology, Tübingen, Germany,
3
Quantitative Biology Center (QBiC), Tübingen, Germany
4
Introduction: Virtually every tumor harbors somatic
of the respective variant for tumor development and
mutations. These mutations can lead to mutation-de-
biochemical features.
rived neoantigenic peptides presented on the MHC
Results: Datasets from FFPE and fresh frozen tumor
molecules of the patient’s tumor. T cells are able to
samples were generated. For both sample types, a set
recognize those alterations and may in turn kill the
of highly promising peptides could be compiled. The
transformed cells. The importance of such neoanti-
whole workflow can be accomplished within three
gens in an effective T cell-derived tumor defense has
weeks.
recently been demonstrated in various immunother-
Conclusion: The established workflow provides an
apeutic contexts like immune checkpoint inhibition,
efficient and time saving method for the identifica-
TIL therapy and vaccination. The quality and quan-
tion of tumor-specific mutations and putative patient-
tity of those neoantigens was shown to be of high
individual neoantigens for multiple purposes includ-
prognostic and therapeutic value. To provide a prac-
ing the development of cancer-specific vaccines,
ticable method for the identification of these highly
adoptive T-cell transfer, prediction of clinical utility
individual tumor-specific neoantigens, we have es-
of immune checkpoint inhibitors and immune moni-
tablished an optimized workflow combining exome
toring.
and transcriptome sequencing, database derived expression data, HLA genotyping and peptide epitope
prediction. The workflow is applicable to FFPE as
well as fresh frozen tumor samples.
Workflow: FFPE samples are revised and macrodissected by pathologists to maximize the tumor
content of the sample. Exome sequencing of a tumor
and normal tissue sample is performed and tumorspecific somatic single nucleotide variants and the
patient’s HLA type are determined. Transcriptome
analysis can be performed in parallel using fresh
frozen or RNA-stabilized tumor samples. A complex
bioinformatics pipeline integrates these results and
predicts affected neo-epitopes for the patient’s HLA
type. Resulting peptide sequences are ranked using
expression data, peptide motifs, potential relevance
Oncolytic viral immunotherapy of HPV+ cancer
Lichty B.1, Atherton M.1, Stephenson K.1, Wan Y.1
McMaster University, Hamilton, Canada
1
Human papilloma virus (HPV) is responsible for 5%
etry. Primed mice, boosted with MG1-HPV develop
of the world’s cancer burden. HPV has been etio-
long lasting, marked and specific immunity against
logically implicated in virtually all cases of cervical
E7 and to a lesser extent E6. Boosted mice display
carcinoma and is increasingly responsible for head
very durable, multi-function T cell responses lasting
and neck cancers in the developed world with strains
for months. The prime:boost regimen has therapeu-
16 and 18 considered high risk. It is estimated that
tic efficacy against established subcutaneous TC1
there are annually over 500,000 new cases of cervical
HPV+ tumours (mean volumes of 250 mm 3) result-
cancer worldwide leading to over 250,00 fatalities per
ing in durable cures in this model. A single intrave-
year. Current standard of care for advanced cervi-
nous infusion of MG1-HPV leads to dramatic regres-
cal carcinoma and head and neck cancers involve
sions of very large tumours. Depletion of CD8+ T
chemo-radiation with or without prior surgical resec-
cells impairs the activity of the vaccination protocol
tion resulting in significant morbidity. We are cur-
supporting the key role of cytotoxic lymphocytes in
rently developing an attenuated MG1 Maraba rhab-
our treatment regimen. These data demonstrate the
dovirus as an oncolytic vaccine where we encode
pre-clinical activity of this oncolytic vaccination and
tumour antigens from this oncolytic virus to provide
pave the way for future clinical trials for the treat-
both viral oncolysis as well as tumour vaccination.
ment of advanced metastatic HPV+ cancers.
MG1-MAGE A3 is currently in phase I clinical testing.
We now seek to develop a novel immunotherapeutic for HPV+ cancers. An oncolytic viral immunotherapeutic utilizing MG1 Maraba virus encoding
non-oncogenic HPV antigens based on both E6 and
E7 of strains 16 and 18 has been created (tetravalent
MG1-HPV). In a heterologous prime:boost setting the
use of MG1-HPV led to very large and potent anti-E7
CD8+ T cell responses in mice representing 60% of
peripheral blood CD8+ T cells. Oncolytic vaccination of tumour free mice has generated between 24
and 50 million, combined blood and splenic CD8+
T cells per mouse, determined by interferon gamma
production in response to a single E7 peptide quantified using intracellular staining and flow cytom-
Personalized multi-peptide vaccination induces immune
responses associated with long term survival in a patient
with metastatic intrahepatic cholangiocarcinoma
Löffler M.1,2,3, Chandran P.A.1,3, Laske K.1,3, Schroeder C.4, Bonzheim I.5, Walzer M.1,3,6, Hilke F.J.4, Trautwein N.1,3,
Kowalewski D.J.1,3, Schuster H.1,3, Günder M.1, Mohr C.6, Sturm M.4, Nguyen H.-P.4, Riess O.4, Bauer P.4,
Nahnsen S.7, Nadalin S.2, Zieker D.2, Glatzle J.2, Thiel K.2, Clasen S.8, Bösmüller H.5, Fend F.5, Kohlbacher O.6,
Gouttefangeas C.1,3, Stevanović S.1,3, Königsrainer A.2, Rammensee H.-G.1,3
University of Tübingen, Department of Immunology,
1
Tübingen, Germany,
University of Tübingen, Department of General,
2
University of Tübingen, Institute of Pathology,
5
Tübingen, Germany,
University of Tübingen, Applied Bioinformatics,
6
Visceral and Transplant Surgery, Tübingen, Germany,
Center for Bioinformatics, Quantitative Biology
Deutsches Konsortium für Translationale Krebs-
Center, and Dept. of Computer Science, Tübingen,
3
forschung (DKTK), Deutsches Krebsforschungs
zentrum (DKFZ) Partnerstandort Tübingen, Tübingen,
Germany,
University of Tübingen, Quantitative Biology Center
7
(QBiC), Tübingen, Germany,
Germany,
University of Tübingen, Institute of Medical Genetics
4
and Applied Genomics, Tübingen, Germany,
University of Tübingen, Department of Radiology,
8
Tübingen, Germany
ORAL
TALK
SHORT
2016
Background: Cholangiocarcinomas are rare epithelial
Immunomonitoring and T cell responses: Histologi-
tumors arising from the liver bile ducts and associ-
cal examination of the pulmonary metastasis, excised
ated with a very poor prognosis. Apart from surgery,
seven months after initiation of peptide therapy, in-
no other curative therapies are available and adju-
dicated increased lymphocyte infiltration, CD25 and
vant therapies are lacking. Especially for tumors in
perforin expression and lower FOXP3 expression
the advanced stage or when metastasized, cure is not
compared to the earlier resected liver tumor tissues.
attainable. Immunotherapy looks very promising con-
Three out of seven vaccinated peptides induced spe-
sidering recent clinical findings (Tran E, et al. Science
cific T cell responses detectable in the blood of the
2014). We report the case of a patient with a large uni-
patient during the vaccination course.
locular cholangiocarcinoma diagnosed in 2010 who
Clinical course: Prior to peptide vaccination, the
was treated with a personalized peptide vaccine. Over
tumor recurred thrice after surgical resection and
the course of 3 years, the primary tumor, locally recur-
was resected on every instance. Currently, over five
rent tumors on two instances as well as a pulmonary
years after diagnosis, the patient is alive and well
metastasis were resected and analyzed.
without any detectable tumor manifestation, which
Vaccination: Based on HLA-ligandomics and tran-
is unheard of in metastatic cholangiocarcinoma. No
scriptome sequencing, a vaccine cocktail contain-
adverse events were reported after vaccination except
ing seven peptides (4 HLA-A*03 and 3 MHC-class II
for local granulomas at the vaccination sites. Having
binders) was administered 27 months after initial
seen peptide specific T cell responses and subsequent
diagnosis (in accordance with §35 of the Declaration
long-term tumor-free survival of the patient, we con-
of Helsinki, Seoul 2008) in September 2012 with a
sider our multi-peptide vaccination a safe and promis-
tapering vaccination schedule and using montanide
ing adjuvant approach for cholangiocarcinoma, which
and imiquimod as adjuvants.
warrants formal clinical studies in the future.
Innovative generic strategies for the encapsulation of patientspecific cancer antigens into immune-modulating particles
Lybaert L.1, Ryu N.2, Tom J.2, Aaes T.3, De Vlieghere E.4, Vanparys N.1, De Rycke R.5,6, De Koker S.5, De Wever O.4,
Krysko D.3, Esser-Kahn A.P.2, De Geest B.G.1
University of Ghent, Pharmaceutics, Ghent, Belgium,
1
University of California Irvine, Chemistry, Irvine, United States,
2
University of Ghent, VIB Inflammation Research Center, Ghent, Belgium,
3
University of Ghent, Radiation Oncology and Experimental Cancer Research, Ghent, Belgium,
4
University of Ghent, VIB, Molecular Biomedical Research, Ghent, Belgium,
5
University of Ghent, IRC, Biomedical Molecular Biology, Ghent, Belgium
6
Anti-cancer immune-therapy is currently recognized
onstrate that heat shock prior to cell lysis can be em-
as one of the most promising strategies for treatment
ployed to potentially modulate the immune response.
of metastatic cancer.1 Here we report three innovative
For all strategies, optimization allowed us to obtain
generic strategies for the encapsulation of patient-
stable particles containing a high amount of encap-
specific cancer antigens into particles. This approach
sulated cancer antigens. In addition, cytotoxicity was
covers a broad range of tumor-associated antigens
quantified via a MTT assay and the in vitro uptake ef-
and enables the induction of specific routes of im-
ficiency together with the MHC-I presentation ability
munogenic cell-death potentially leading to more
of dendritic cells was analyzed via flow cytometry.
specific, more efficient and more potent immune ac-
To further increase the immunogenicity of these for-
tivation of the patient.
mulations we functionalized TLR4 and TLR7-ligands
First, we demonstrate in situ encapsulation of live
onto a N-hydroxypropyl methacrylamide based
whole cancer cells into polymeric microcapsules
polymer in close collaboration with the group of
based on layer-by-layer assembly of alternating
Prof. Aaron Esser-Kahn (UC Irvine, LA). This allows
layers of poly(vinylpyrrolidone) and tannic acid fol-
easy co-formulation of the particles with TLR-ligands
lowed by killing of the cancer cells and release of the
while avoiding lack of efficiency and side effects due
cancer cell lysate into the hollow void of the capsules.
to systemic leakage. The maturation potency was as-
Additionally we demonstrated, that heat shock prior
sessed on bmDCs via flow cytometry and on mac-
to cellular encapsulation can be employed to poten-
rophages via RAW blue assays.
tially modulate the immunogenic properties of the
Current experiments involve the co-formulation of
capsules.t
the particles with one or multiple TLR-ligands and
Secondly, we show encapsulation of cancer cells by
investigation of the synergistic immunogenicity po-
means of spray drying in the presence of mannitol
tential with or without the presence of immunogenic
and oppositely charged polyelectrolytes, poly-L-argi-
cell stress/death cytokines in vitro and in vivo.
nine and dextran sulfate. In collaboration with the
group of Dr. Dmitri Krysko (VIB, UGent) we used
References:
an apoptotic colon cancer cell line to investigate the
1
role of immunogenic cell death potentiators in cancer
3
immune-therapy.3
Thirdly, contrary to the in situ encapsulation of
cancer cells, we encapsulated cancer cell lysate into
calcium carbonate microcapsules. Here we also dem-
2
Mellman , G. Coukos , G. Dranoff, Nature 2011, 480, 480
Lybaert L. et. al, Adv Funct Mater 2014, 24, 7139
Garg A.D. et. al, Front Immunol 2015, 6, 588
Immunological results obtained in castration-resistant prostate
cancer patients treated with an EGFR-based vaccine.
Mazorra Z.1, Aira L.E.1, Lavastida A.1, Popa X.1, Mesa M.2, Narjara G.2, Lorenzo-Luaces P.3, Caballero I.4,
Wilkinson B.3, Crombet T.3, Sánchez B.2, Casacó A.3
Center of Molecular Immunology, Clinical Immunology, Havana City, Cuba,
1
Center of Molecular Immunology, Tumor Immunology, Havana City, Cuba,
2
Center of Molecular Immunology, Clinical Direction, Havana City, Cuba,
3
ORAL
TALK
SHORT
Hermanos Ameijeiras’ Hospital, Havana, Cuba
4
2016
Introduction: Metastatic castration-resistant pros-
receiving higher doses of vaccine (600 µg and 800
tate cancer (CRPC) remains incurable due to the lack
µg) showed significant tumor cell recognition and
of effective therapies. Activation of the epidermal
EGFR phosphorylation inhibition by hyper immune
growth factor receptor (EGFR) in prostate cancer
sera. Forty percent of the patients showed a specific
contributes to metastatic progression as well as to
T cell response against EGFR peptides pool in post-
disease relapse. Here, we determined the toxicity and
treatment samples. Although it was not significant,
immunogenicity of an EGFR vaccine in castration-
good immune response seems to be associated with
resistant prostate cancer patients included in a phase
clinical benefit.
I clinical trial.
Conclusions: The EGFR-based vaccine was well tol-
Materials and methods: Castration resistant-pros-
erated and induced high titers of anti-EGFR antibod-
tate cancer patients (n = 24) were intramuscularly
ies and specific T cell response. Despite the small
vaccinated with an EGFR vaccine composed by the
amount of patients, encouraging clinical results were
extracellular domain of the human EGFR molecule
obtained. A new phase II clinical trial is planning.
and very small size proteoliposome from Neisseria
meningitis (VSSP) and Montanide ISA-51 as adjuvants. Patients were included in five groups according to EGFR vaccine doses (100 µg, 200 µg, 400 µg,
600 µg and 800 µg). The primary endpoints were
safety and immunogenicity. The anti-EGFR antibodies were measured by ELISA and the recognition of
EGFR positive tumor cell line by sera was determined
by flow cytometry. The EGFR specific T cell response
was assessed by IFNγ-ELISPOT.
Results: The vaccine was well tolerated. No adverse
events grade III or IV were reported. High titers of
anti-EGFR antibody response were observed in
most of the evaluated patients. There wasn´t significant difference regarding to the titers geometric
mean among the groups of 200, 400, 600 and 800
µg of EGFR vaccine. In the group of 100 µg the antibody titers were significantly lower. Only patients
Cancer immunotherapy using dendritic cells pulsed with tumor
cells killed by high hydrostatic pressure in murine models for
prostate cancer
Mikyšková R.1,2, Štěpánek I.1,2, Indrová M.1,2, Bieblová J.1,2, Šímová J.1,2, Truxová I.3, Moserová I.3, Fučíková J.3,4,
Kanchev I.2,5, Bartůňková J.3,4, Špíšek R.3,4, Reiniš M.1,2
Czech Centre for Phenogenomics, Institute of Molecular Genetics of the ASCR, Immunology Unit, Vestec,
1
Czech Republic,
Institute of Molecular Genetics of the ASCR, v. v. i., Department of Transgenic Models of Diseases, Prague,
2
Czech Republic,
SOTIO, Prague, Czech Republic,
3
Charles University, 2nd Faculty of Medicine and University Hospital Motol, Department of Immunology,
4
Prague, Czech Republic,
Czech Centre for Phenogenomics, Institute of Molecular Genetics of the ASCR, Histopatology Unit, Vestec,
5
Czech Republic
High hydrostatic pressure (HHP) has been shown
etaxel. TRAMP model mimics well human carcino-
to induce immunogenic cell death of cancer cells,
mas, as it develops and progresses through all stages
facilitating their uptake by dendritic cells (DC) and
of carcinogenesis similarly to humans. In another
subsequent presentation of tumor antigens. In our
clinically relevant setting, minimal residual tumor
study, we employed HHP-treated cells for DC-based
disease after surgery, administration of the DC-based
vaccine antigen pulsing. DC co-cultured with HHP-
vaccines pulsed with HHP-inactivated tumor cells
treated tumor cells and matured exhibited higher cell
after surgical removal of TRAMP-C2 tumor slowed
surface expression of maturation markers and pro-
down the growth of recurrent tumor, as compared
duction of IL-12 and other cytokines, as compared to
to operated-only controls. Taken collectively, our
the DC pulsed with irradiated tumor cells. Immuni-
results indicate that DC-based vaccines pulsed with
zation with DC cell-based vaccines pulsed with HHP-
HHP-inactivated tumor cells represent a suitable
treated tumor cells induced high immune responses,
tool for immunotherapy, particularly with regard to
detected by increased spleen cell cytotoxicity and el-
the findings that poorly immunogenic TRAMP-C2
evated IFNg production. In a therapeutic setting, the
tumors were susceptible to this treatment modality.
DC-based vaccine pulsed with HHP-treated tumor
cells combined with docetaxel chemotherapy significantly inhibited growth of TRAMP-C2 and TC-1
tumors, representing a murine model for prostate
and for human papilloma virus-associated tumors,
respectively. Further, DC-based vaccines pulsed with
HHP-inactivated tumor cells were also effective in reducing prostate cancer growth in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model
when used alone or in the combination with doc-
This work was supported by research grant provided by
SOTIO a.s., and in part by MEYS (LM2011032), Academy
of Sciences of the Czech Republic (RVO 68378050), the
project „BIOCEV - Biotechnology and Biomedicine Centre
of the Academy of Sciences and Charles University“
(CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund.
IVAC® MUTANOME - a first-in-human phase I clinical trial
targeting individual mutant neoantigens for the treatment of
melanoma
Miller M.1, Loquai C.2, Kloke B.-P.1, Attig S.3, Bidmon N.1,3, Bolte S.1, Bukur V.1,3, Derhovanessian E.1, Diekmann J.1,
Heesch S.1, Höller C.4, Kühlcke K.5, Langer D.1, Löwer M.3, Müller F.1,3, Ortseifer I.6, Otte B.1, Paruzynski A.1, Rae R.3,
Schrörs B.3, Seck C.1, Spieß K.7, Tadmor A.3, Vogler I.1, Vormehr M.3,6, Kemmer-Brück A.1, Kuhn A.6, Luxemburger U.1,
Kreiter S.1,3, Utikal J.8,9, Huber C.10, Türeci Ö.10, Sahin U.1,3,6
7
Department of Dermatology, University of Mainz,
8
1
2
Mainz, Germany,
TRON-Translational Oncology at the University Medical
3
BioNTech Diagnostics GmbH, Mainz, Germany,
Skin Cancer Unit, German Cancer Research Center
(DKFZ), Heidelberg, Germany,
University Medical Center Mannheim, Ruprecht-
9
Center of the Johannes Gutenberg University Mainz,
Karl University of Heidelberg, Department of
Mainz, Germany,
Dermatology, Venereology and Allergology,
Division of General Dermatology, Department of
4
Dermatology, Medical University of Vienna, Vienna,
Mannheim, Germany,
CI3 - Cluster of Individualized Immunointervention,
10
Mainz, Germany
Austria,
5
BioNTech RNA Pharmaceuticals GmbH, Mainz,
6
Germany,
ORAL
TALK
SHORT
2016
One of the hallmarks of cancer is the inherent insta-
ingly, only patients with a high burden of mutations
bility of the genome leading to multiple genomic al-
and pre-established T-cell responses towards the re-
terations and epigenetic changes that create a unique
spective neoantigens profit from currently approved
molecular profile of a given tumor. Besides initiating
therapies.
and driving carcinogenesis, these processes create
To overcome this restriction, the IVAC® MUTA-
novel, tumor-specific antigens for presentation to
NOME-project harnesses the individual patient’s
the respective patient’s immune system. However,
mutation profile by manufacturing highly potent
spontaneous immune recognition of these neoanti-
neoantigen-encoding RNA vaccines to establish
gens seems to be a rare event with only less than
potent mutation-specific immune responses. To this
1% of mutations inducing a T-cell response in the
end, the individual mutation repertoire is identified
tumor-bearing patient. However, a series of recent
by next-generation-sequencing, potentially immuno-
independent reports revealed that pre-formed specif-
genic mutations are selected and incorporated into a
ic T-cell responses towards these mutation-derived
poly-epitopic RNA vaccine that is tailored to activate
neoantigens are of crucial relevance for the clinical
and expand the individual patient’s neoantigen-spe-
efficacy of immune checkpoint inhibitors. Accord-
cific CD4+ and CD8+ T cells.
A phase I study to test this novel concept of an
active individualized cancer vaccine for the treatment of malignant melanoma was initiated in 2013
(NCT02035956). Notably, BioNTech RNA Pharmaceutical’s IVAC® MUTANOME trial is the first-in-human trial that introduces a fully personalized RNA
vaccine for the treatment of malignant melanoma.
The objective of this clinical trial is to study the feasibility, safety, tolerability, immunogenicity and the
potential clinical activity of the IVAC® MUTANOME
approach.
The recruitment of a patient into the trial triggers
the multi-step IVAC® MUTANOME process covering
(i) the receipt and processing of tumor and blood sample
specimens,
(ii) the identification, prioritization and confirmation of
mutations,
(iii) testing of pre-existing immunity against identified tumor
mutations,
(iv) the selection of mutant neoantigen sequences as vaccine
targets,
(v) design, production of a DNA lead structure,
(vi) GMP manufacturing and release of the patient-specific
mRNA,
(vii) shipment to the clinical trial site and
(viii) the administration of the IMP to patients.
Detailed information on the trial, the recruitment and treatment status as well as data on the assessment of vaccineinduced immune responses will be presented.
Improved mutanome-directed cancer immunotherapy by
immunoinformatic analysis of cancer neo-epitopes for regulatory
T cell activation potential
Moise L.1,2, Richard G.1, Terry F.1, Martin W.1, De Groot A.S.1,2
EpiVax, Inc., Providence, United States,
1
University of Rhode Island, Institute for Immunology and Informatics, Providence, United States
2
ORAL
TALK
SHORT
2016
Advanced computational tools for vaccine design can
These results highlight the benefits of using in silico
be applied to designing individual cancer vaccines.
prediction tools for the selection of neo-epitopes
Tumor-specific mutations discovered using whole-
and how they can improve the design and effica-
exomic sequencing of tumor-normal pairs have now
cy of future cancer vaccines. While retrospective
been shown to be capable of stimulating T cell-me-
in nature, the suite of tools used for these analy-
diated processes that lead to tumor regression. Neo-
ses have been extensively validated in prospective
epitope prediction using computational tools enables
vaccine studies for infectious disease. Removal of
the rapid identification of epitope candidates in the
Treg epitopes, identified by JanusMatrix, has led to
mutanome, but a large proportion of neo-epitopes
the development of more immunogenic vaccine anti-
prove to be not immunogenic. One explanation is
gens. EpiVax’s H7N9 influenza Treg epitope-depleted
that class II major histocompatibility complex (MHC)
vaccine is scheduled for Phase I clinical trial. Direct
epitopes activate regulatory T cells (Tregs) trained
application of JanusMatrix to the neo-epitope-driven
in the thymus on self-antigens, which reduces anti-
cancer vaccine discovery and design pipeline will
tumor activity.
focus candidate selection on higher-value sequences
To address this pitfall, we developed the JanusMatrix
than what conventional T cell epitope mapping algo-
algorithm that parses query sequences into MHC-fac-
rithms generate. Neo-epitopes with low Treg activa-
ing and T cell receptor (TCR)-facing sequences and
tion potential may then be used to support develop-
screens the human epitome to identify MHC ligands
ment of personalized therapies including vaccination
that share TCR faces with human proteins. Tumor-
and in vitro expansion of tumor infiltrating lympho-
specific sequences that share TCR-face patterns with
cytes for adoptive cell transfer.
their wild type counterpart or with multiple human
sequences may cross-react with thymic-derived
Tregs and are thus counter-indicated for immunotherapy. We conducted retrospective analyses of
cancer vaccine efficacy studies performed in mice
[Kreiter et al. 2015 Nature 520, 692-696] showing that
mutanome-directed vaccines effective at preventing
tumor growth contained higher numbers of class II
MHC neo-epitopes, as identified by EpiVax’s iVAX
platform, and had lower potential to cross-react with
other murine proteins.
Exploiting immunogenic cell death features for improved
dendritic cell-based therapeutic vaccine against mantle cell
lymphoma
Montico B.1, Lapenta C.2, Martorelli D.1, Muraro E.1, Comaro E.1, Spada M.2, Donati S.2, Santini S.2, Belardelli F.2,
Dolcetti R.1,3, Dal Col J.1
Centro di Riferimento Oncologico, Department of Translational Reseach, Aviano, Italy,
1
Istituto Superiore di Sanità, Department of Hematology, Oncology and Molecular Medicine, Rome, Italy,
2
The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia
3
Cancer immunotherapy is becoming one of the most
TNFα, IL-1β. Cytotoxic T cells (CTLs) obtained co-
promising strategies for counteracting several malig-
culturing DCs pulsed with RA/IFNα-TCLs were more
nancies. Among approaches available in this field,
efficient in recognize and specifically lyse MCL cells
dendritic cell (DC)-based vaccination is being highly
with respect to those obtained by stimulation with
investigated to exploit the antigen-presenting capa-
untreated TCL-pulsed DCs. Intriguingly, CTLs gener-
bility of these cells, which can boost antigen-specific
ated by RA/IFNα TCL-pulsed DCs, showed enhanced
T-cell responses and generate a long-lasting immu-
efficacy to detect and kill autologous cells exposing
nological memory. Thanks to these properties, DC-
different HLA-A*0201-restricted cyclin D1 epitopes.
based vaccination protocols could be of great help
This is of highly relevance given that cyclin D1 is a
for poorly curable tumours such as mantle cell lym-
hallmark of MCL and a specific tumour-associated
phoma (MCL), characterized by frequent relapses or
antigen. Moreover, exploring the different T sub-
refractory disease. Indeed, at present there is no rec-
populations obtained during CTL generation, we ob-
ognized standard of care in relapsed MCL. Therefore,
served an increase in Th1 and Th17 lymphocytes fol-
the aim of our study is to take advantage from the
lowing the cross-priming with RA/IFNα TCL-pulsed
immunogenic cell death (ICD) induced by 9-cis-reti-
DCs, while a reduction of T regulatory cells was
noic acid (RA) and Interferon(IFN)-α combination to
highlighted in all stimulating conditions. DC-based
generate ex vivo tumour cell lysates (TCL) to be used
vaccine efficacy was also confirmed in vivo taking
as antigen formulation for improved DC-based vac-
advantage from hu-PBL-SCID mouse model. Vaccina-
cines against MCL. Our results demonstrated that the
tion with RA/IFNα-treated TCL-pulsed DCs reduced
combination of RA/IFNα induced a marked apoptosis
tumour growth compared to the control group of
in MCL cell lines and primary cultures. Interestingly
mice but this effect was associated with general signs
RA/IFNα-induced apoptosis shared typical features
of toxicity.
of ICD such as early membrane exposure of Calreti-
Experiments are currently ongoing to better tune our
culin, Heat Shock protein 70 and 90 together with the
in vivo vaccination protocol. In conclusion, results of
decrease of CD47 and late secretion of High Mobility
the present study highlight the therapeutic potential
Group Box 1. Moreover, RA/IFNα-treated cells and the
of RA/IFNα-treated TCL-loaded DC-based vaccine
corresponding TCL were more efficiently recognized
and provide the rationale to assess its tolerability and
and engulfed by mature DCs than untreated controls.
efficacy in clinical studies involving MCL patients.
RA/IFNα-TCL did not enhance the maturation state
of DCs but induced a higher activation of these cells
as shown by the secretion of higher amounts of IL-6,
Mixed ligand coated gold nanoparticles as carrier of R848 for
cancer immunotherapy
Mottas I.1, Bekdemir A.2, Spagnuolo L.1, Stellacci F.2, Bourquin C.1
University of Fribourg, Dep. of Medicine, Chair of Pharmacology, Fribourg, Switzerland,
1
EPFL, Supramolecular Nanomaterials and Interfaces Laboratory (SUNMIL), Lausanne, Switzerland
2
The small molecule TLR7/8 agonist R848 (resiquimod)
tion measured by up-regulation of surface markers
is a highly effective adjuvant that shows promise for
and pro-inflammatory cytokine secretion. Interest-
the immunotherapy of cancer. However, some TLR
ingly, R848 in particulate formulation allowed a re-
agonists have failed in clinical trials because of the
duction in the drug concentration needed to induce
difficult balance between the induction of efficient
a maximum immune response. The particles with
anti-tumor immune responses and safety concerns.
a small gold core (2.5 nm) showed a better uptake
Indeed, the systemic administration of R848 can lead
that correlated with a stronger immune cell activa-
to side effects because it indiscriminately activates
tion than particles with a larger gold core (4.5 nm).
the immune system. Here we propose the use of a na-
Moreover, we confirmed the potential of the particu-
noparticle carrier to modulate R848 biodistribution.
late formulation to induce an adaptive immune re-
Effectively, even without a specific targeting strat-
sponse using a dendritic cell-induced T-cell prolifera-
egy, particulate formulations show passive accumu-
tion assay. We are now planning in vivo experiments
lation in lymph nodes and often leads to a better drug
to analyze the biodistribution of nanoparticles and
uptake by antigen-presenting cells. Based on these
adjuvant.
two principles, we aim to increase the drug concentration in antigen-presenting cells in the lymph node,
thus reducing unspecific systemic effects of the drug.
We synthetized water-soluble gold nanoparticles
coated with mixed carbon-based ligand as carrier for
R848: the nanoparticles were coated with both 1-octanethiol (OT) and 11-mercaptoundecane sulfonate
(MUS) ligands with a controlled stoichiometric ratio
(MUS:OT = 2:1). While sulfonate moieties provide
nanoparticles with high stability in biological media,
the hydrophobic parts of the ligands constitute efficient pockets for R848 loading.
Our results show that none of the gold nanoparticle
formulations were cytotoxic after 24 h exposure to
macrophages or dendritic cells. Compared to cargofree nanoparticles, the R848-loaded particles were
efficient in inducing antigen-presenting cell activa-
This project is supported by the National Center of Competence in Research for Bio-inspired Stimuli-Responsive Materials (www.bioinspired-materials.ch).
The mechanism of immune stimulation by Orf virus - a novel
viral vector for therapeutic cancer vaccines
Müller M.1, Feger T.1, Amann R.1, Rammensee H.-G.1
University of Tuebingen, Department of Immunology, Tuebingen, Germany
1
Viral vector vaccines represent most excellent induc-
With the aid of a recombinant ORFV expressing the
ers of cell-mediated and humoral immune responses.
fluorescent marker protein mCherry, the mode of
Therefore, intensive investigations are performed to
action and the immune cells involved in this immune
improve the use of several virus families as safe and
activation is investigated. In a first step, freshly iso-
efficient viral vectors, not only against diverse infec-
lated human PBMC subpopulations were infected at
tious diseases but also against tumors. During the
different time points and with different multiplicity
last decade we developed a novel vector virus plat-
of infections and the infection rate and cell viability
form using Orf virus (ORFV), a member of the genus
were measured. Thereby we observed that profes-
Parapoxvirus of Poxviridae. The attractiveness of an
sional antigen presenting cells (APC) were most sus-
ORFV vector rely on the following advantages: (i) a
ceptible for infection/uptake of virus. Through the
very restricted host range, (ii) no evidence for viral
inhibition of phagocytosis we could show that ORFV
systemic spread, (iii) the fast induction of humoral
is most likely taken up by APCs.
and cellular immune responses, especially also in
Next we wanted to investigate the activation status of
non-permissive hosts that do not support vector rep-
the APCs. Therefore we analyzed the effect of ORFV
lication, (iv) a short-term vector-specific immunity
on the expression of surface markers which are im-
allowing multiple re-immunizations, and (v) the pos-
portant for antigen-presentation and co-stimulation
sibility to generate recombinants by targeted deletion
of T-cells (e.g. HLA DR, CD40, CD80 and CD86).
of ORFV virulence genes on the basis of the highly
In further experiments we examined the cytokine
attenuated, apathogenic ORFV strain D1701-V.
release to elucidate the effects of ORFV infection on
Recently, we were able to demonstrate the excellent
the immune stimulatory properties of APCs. Addi-
immune stimulating (humoral and cellular) and pro-
tionally, we were able to boost the immune stimula-
tective capacity of ORFV-based recombinants not only
tory capacity of ORFV with the heterologous expres-
against numerous different viral diseases but also in
sion of co-stimulatory molecules and cytokines.
animal tumor models. Obviously, it would be of great
interest, if ORFV might serve as a platform for the development of therapeutic tumor vaccines for humans.
Hence, we would like to investigate the so far poorly
understood mechanism of immune stimulation and
-induction as well as the possibilities to further manipulate the induced immune response by co-expression
of immune regulating factors more precise.
Oncolytic poliovirus activates antigen-presenting cells and
promotes anti-cancer responses in vitro and in vivo
Brown M.C.1,2, Holl E.K.1, Boczkowski D.1, Bigner D.D.1,2, Gromeier M.1,2, Nair S.K.1,2
Duke University School of Medicine, Durham, United States,
1
Preston Robert Tisch Brain Tumor Center at Duke University, Durham, United States
2
Background: We have developed an oncolytic po-
MB231 (CEA+) triple-negative breast cancer cell
liovirus, PVSRIPO, that lyses malignant cells while
line, SUM149 (EGFR+) inflammatory breast cancer
generating inflammation at the site of the tumor.
cell line and LNCaP (PSA+) were used in this study.
PVSRIPO is a recombinant polio:rhinovirus chimera
To analyze oncolytic poliovirus mediated immune
that has been modified to eliminate neuropatho-
reactions in vivo, we treated subcutaneous tumors
genicity. PVSRIPO has cancer tropism due to ectopic
intratumorally with PVSRIPO and evaluated immune
expression of the poliovirus receptor, CD155, in solid
cell infiltration using flow cytometry and immuno-
cancers. Importantly, PVSRIPO therapy is effective
histochemistry (IHC).
in the presence of neutralizing antibodies and an
Results and conclusions: PVSRIPO oncolysate ac-
innate antiviral response. A first-in-human Phase-1
tivates human DCs primarily through direct viral
study with PVSRIPO has shown remarkable promise
infection. PVSRIPO infection of DCs is sublethal;
in patients with recurrent glioblastoma, a uniformly
induces MHC class II and costimulatory molecule
lethal disease. PVSRIPO tumor cell killing is asso-
expression; and leads to IFNβ, IL12, and TNFα pro-
ciated with the induction of danger- and pathogen-
duction. Incubation of DCs with PVSRIPO-induced
associated molecular patterns (DAMPs and PAMPs)
tumor lysate stimulates DC activation and IL12 pro-
and simultaneous non-lethal infection of antigen-
duction. Using an in vitro human immunotherapy
presenting cells (APCs) such as monocytes and den-
assay, we demonstrate that human DCs loaded with
dritic cells (DCs).
PVSRIPO-induced tumor cell lysate stimulate tumor
Methods: To understand key immune events associ-
antigen-specific T cells. Autologous DCs transfected
ated with poliovirus infection of APCs, we examined
with RNA that encodes for CEA, EGFR, MART or
the effects of PVSRIPO on primary human monocyte-
PSA were used to assess tumor antigen-specificity
derived DCs. PVSRIPO-treated DCs were evaluated
of T cells. PVSRIPO treated in vivo tumors showed
for expression of maturation/activation markers. To
increased innate and adaptive immune cell recruit-
determine whether DCs exposed to PVSRIPO-lysed
ment and were accompanied with increased mouse
tumor cells present tumor antigens to T cells, we per-
survival. Our data suggests that along with destruc-
formed an in vitro human assay. Human DCs gener-
tion of tumor cells, oncolytic poliovirus mediates an-
ated from HLA-A2+ donor cells were incubated with
ti-tumor immune events. In ongoing studies we con-
PVSRIPO-induced tumor cell lysate and then used to
tinue to analyze PVSRIPO mediated immune events
stimulate autologous T cells in vitro followed by a
in syngeneic, immunocompetent murine models of
cytotoxic T lymphocyte (CTL) assay. HLA-A2 human
brain and breast cancer.
cell lines DM6 (MART+) melanoma cell line, MDA-
Identification and characterization of HLA class I-restricted
MYD88 L265P-derived peptides as tumor-specific targets for
immunotherapy
Nelde A.1,2, Stickel J.S.1, Kowalewski D.J.2, Wolz O.-O.2, Kanz L.1, Langerak A.W.3, Muggen A.F.3, Bonzheim I.4,
Fend F.4, Rammensee H.-G.2, Stevanović S.2, Weber A.N.R.2
University Hospital Tübingen, Department of Hematology and Oncology, Tübingen, Germany,
1
University of Tübingen, Interfaculty Institute of Cell Biology, Department of Immunology, Tübingen, Germany,
2
Erasmus MC Rotterdam, Department of Immunology, Rotterdam, Netherlands,
3
University Hospital Tübingen, Department of Pathology, Tübingen, Germany
4
Non-Hodgkin lymphomas (NHL) are frequent ma-
be detected after in vitro priming with a maximum
lignancies with considerable mortality. A recur-
frequency of 14.1% peptide-specific CD8+ T cells.
rent somatic and oncogenic driver mutation in the
The functionality and specificity of peptide-specific
Toll-like receptor adaptor gene MYD88, Leu265Pro
CD8+ T cells after aAPC-based in vitro priming was
(L265P) has been identified in up to 90% of certain
validated by intracellular cytokine staining for IFN-γ
NHL subtypes. Genetic alterations affecting a pro-
and TNF-α. We detected specific and functional
tein-coding region have the potential to generate
CD8+ T cell populations after stimulation with the
mutation-derived peptides that are presented by HLA
mutated peptides, but not after stimulation with the
class I proteins and might be recognized by cytotoxic
corresponding wild type peptides. Furthermore, the
is a widely occurring
peptide-specific cytotoxic activity of specific CD8+ T
and tumor-specific mutation, we investigated the po-
cells was demonstrated in a VITAL assay. The poly-
T cells. Because MYD88
-containing peptides for CD8 T
clonal MYD88L265P -specific CD8+ T cells lysed autolo-
cell mediated immunotherapy as a new therapeutic
gous peripheral blood mononuclear cells loaded with
tential of MYD88
L265P
L265P
approach for MYD88
+
L265P+
NHL.
the mutated peptides, but not cells presenting the
Based on in silico prediction we identified potential
wild type peptides.
HLA ligands encompassing the MYD88L265P mutation
In this study, we identified and characterized
for several HLA class I allotypes. Functional charac-
MYD88L265P mutation-derived HLA class I ligands for
L265P
-
T cell mediated immunotherapy. The strong immu-
derived HLA class I ligands with regard to induction
nogenicity of the HLA-B*07 and HLA-B*15-restricted
of T cell responses identified a set of immunogenic
mutation-derived peptides as well as the functional-
terization of the candidate HLA class I MYD88
-
ity and specificity of peptide-specific CD8+ T cells,
mutated NHL patient, memory T cell responses tar-
demonstrated by cytotoxicity assays, underline the
peptides for HLA-B*07 and B*15. In one MYD88
-derived HLA class
potential of the MYD88L265P mutation as tumor-spe-
I ligands were detected by IFN-γ ELISPOT. Efficient
cific target. These data highlight the potential of
T cell priming was demonstrated in vitro using naïve
MYD88L265P mutation-specific immunotherapy as a
T cells of healthy volunteers as well as of MYD88WT
novel broadly applicable and tumor-specific treat-
CLL patients. In detail, three HLAB*07 peptides
ment approach for patients with MYD88L265P+ NHL.
geting three different MYD88
L265P
L265P
and one HLAB*15-restricted peptide were analyzed
using artificial antigen-presenting cell-based (aAPC)
in vitro priming experiments. For all tested peptides
proliferation of peptide-specific CD8+ T cells could
Development of novel replication-defective lymphocytic
choriomeningitis virus vectors expressing HPV-16 antigens for
immunotherapy
Schmidt S.1, Berka U.1, Bonilla W.V.2, Qiu J.3, Ying M.3, Pen S.3, Schwendinger M.1, Pinschewer D.2, Lilja A.1,
Monath T.1, Hung C.-F.3, Orlinger K.1
HOOKIPA Biotech AG, Vienna, Austria,
1
University of Basel, Department of Biomedicine, Basel, Switzerland,
2
Johns Hopkins University, Department of Pathology, Baltimore, United States
3
High-risk human papillomaviruses (HPV) such as
In conclusion, our data demonstrate that replication-
HPV-16, cause over 500.000 cases of cervical, ano-
defective LCMV-based immunotherapy vectors are
genital and oropharyngeal cancer annually. The
highly immunogenic and show excellent therapeu-
viral oncoproteins E6 and E7 represent excellent
tic efficacy in a preclinical model of HPV-induced
targets for immunotherapy, and the abundance of
cancer. Moreover, the ability to efficiently augment
+
tumor-specific CD8 T lymphocytes predicts overall
CD8+ T cell responses in homologous prime-boost
survival in HPV-related cancer patients. Hence, the
treatment regimens represents a key discriminating
induction of E6/E7-specific CD8+ T cells represents
feature of rLCMV vectors for cancer immunotherapy.
a central aim of HPV-related cancer immunotherapy.
Pre-existing vector-neutralizing antibodies (nAb)
can present a major obstacle in development of viral
vector-based immunotherapies. Even in previously
seronegative individuals, vector-nAb can rapidly
develop after administration, thus limiting the effectiveness of subsequent booster administrations. Here
we have evaluated the immunogenicity and therapeutic efficacy of a novel replication-defective E6/
E7-expressing immunotherapy vector (rLCMV-E6/
E7), which is based on Lymphocytic choriomeningitis virus (LCMV). It has been demonstrated before
that rLCMV vectors do not elicit vector neutralizing
antibodies, and hence HPV-16 specific T cell responses in mice could be greatly enhanced in mice by homologous booster vaccinations. Second-generation
vectors co-expressing immunomodulatory molecules
have also been generated. The immunogenicity and
efficacy of these candidate immunotherapy vectors
were analyzed in the syngeneic murine TC-1 tumor
model. Our studies showed potent HPV-specific
CD8+ T cell induction in tumor-bearing mice, resulting in abolition of palpable tumors.
Polymeric nanoparticle-based vaccine to target dendritic cells
and the tumor microenvironment
Peres C.1,2,3, Viana A.S.4, Graça L.2, Préat V.3, Florindo H.F.1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon,
1
Portugal,
Instituto de Medicina Molecular (IMM), Faculty of Medicines, Universidade de Lisboa, Lisbon, Portugal,
2
Louvain Drug Research Institute (LDRI), Faculté de Pharmacie, Université Catholique de Louvain, Brussels,
3
Belgium,
Centro de Química e Bioquímica, Faculty of Sciences, Universidade de Lisboa, Lisbon, Portugal
4
Cancer vaccines have been used as an alternative
efficiency (EE) and loading capacity (LC) were quan-
therapeutic strategy and have already shown promis-
tified by HPLC, while siRNA EE and LC were deter-
ing results. However, only a small number was able
mined by PicoGreen® reagent. Finally, cell viability
to lead to an effective tumor regression, which can
was determined by Alamar Blue® assay. siRNA-NP
be explained by the immunosuppressive proper-
knockdown capacity is currently under evaluation
ties of tumor microenvironment induced namely by
by Western blotting and flow cytometry.
the release of potent immunosuppressor molecules.
Overall, NPs presented a mean diameter close to
Therefore, the elimination of both the tumor itself
200 nm with low polydispersity index (PdI) values
and the tumor microenvironment, without adversely
(≤0.200), ZP close to neutrality, and high EE (>85%)
affecting the desired antitumor effector cells, seems
values for both antigen and siRNA. PLA NPs showed
to be an ideal therapeutic strategy to eradicate this
no cytotoxicity on targeted cells and DCs after 72h of
disease. Thus, the aim of the present study is to
incubation, even at high NP concentration 0.5 mg/
develop a polymeric nanoparticle (NP)-based cancer
mL). Three different chitosan (Cs) derivatives were
vaccine to deliver incorporated tumor-associated an-
used for antigen or siRNA complexation. However,
tigens and/or small interfering RNA (siRNA) to target
no significant differences were observed between
dendritic cells (DCs) and for immunomodulation
the physicochemical properties of those three dif-
by silencing immune-suppressive cytokines within
ferent nanoparticulate systems. Similarly, no signifi-
breast tumor site.
cant differences were detected in NP’s size, surface
Antigen or siRNA-chitosan complexes encapsulated
charge and cytotoxicity when formulated with PVA
in poly(lactide acid) (PLA) NPs have been formulated
and Pluronic as external phase surfactant. Moreover,
by a double emulsion solvent evaporation method.
non-targeted and targeted NPs also presented similar
These NPs were coated with polyvinyl alcohol (PVA)
properties. Therefore, it is possible to state that the
or with block co-polymer Pluronic to improve stabili-
formulation method followed for PLA-based NP prep-
ty under physiological conditions. In order to potenti-
aration is highly reproducible and this nanoparticu-
ate tumor targeting, NP surface was modified by hya-
late system constitutes a promising platform for the
luronic acid (HA), a targeting moiety that specifically
delivery of TAA and immunomodulators to different
recognizes CD44 receptor, overexpressed on several
cells within tumor microenvironment.
tumor cells. NP size, surface charge (ZP) and morphology were analyzed by Dynamic Light Scattering, Laser Doppler Electrophoresis and Atomic Force
Microscopy (AFM), respectively. Antigen entrapment
A phase 2a trial and related preclinical studies to investigate
the immunologic impact, anti-tumor efficacy and safety of
VXM01, an oral T-cell inducing vaccine, in late stage colorectal
cancer patients
Beckhove P.1,2, Grüllich C.1, Bichat F.3, Jenkins R.4, Springer M.5, Wieckowski S.6, Lubenau H.5, Breiner K.6,
Franklin S.4, Glaize A.3, Maubant S.3, Warren M.4, Jäger D.1, Podola L.1,2
National Center for Tumor Diseases (NCT), University Medical Center Heidelberg, Heidelberg, Germany,
1
Regensburg Center for Interventional Immunology (RCI), University Medical Center Regensburg, Regensburg,
2
Germany,
Oncodesign S.A., Dijon, France,
3
Pathology Diagnostics Ltd, Waterbeach, United Kingdom,
4
VAXIMM GmbH, Mannheim, Germany,
5
VAXIMM AG, Basel, Switzerland
6
VXM01 is an orally applied T-cell inducing immu-
immune cells by immunohistochemistry. The mean
notherapy based on live attenuated Salmonella typhi
level of VEGFR-2-specfic CD8+ T cells increased sig-
vector carrying a eukaryotic expression plasmid
nificantly from 0.76% ±0.33 in the control group to
coding for vascular endothelial growth factor recep-
1.42% ±0.67 and 2.80% ±1.05 in mice treated with
tor 2 (VEGFR-2). A recent phase I trial demonstrated
VXM01m and VXM01m+CYP respectively. Mean
safety, immunogenicity and transient anti-angiogen-
tumor volume at day 30 was reduced from 2111 ±
ic activity in advanced pancreatic cancer patients.
507 mm3 in the control group to 1332 ± 627 mm 3
Notably, sustained T-cell responses have been meas-
and 1411 ± 551 mm3 in mice treated with VXM01m
ured in patients treated monthly with VXM01 after
and VXM01m+CYP respectively. The mean propor-
a one-week initiation treatment course. The purpose
tion of intratumoral CD8+, FoxP3+ and PD-1+ cells
of the current studies is to gain more insight into the
increased by a factor of respectively 2.3, 3.1 and 2.3
mode of action of VXM01 in preclinical mouse models
in the VXM01m group, and 3.3, 2.2, and 2.1 in the
of colorectal cancer as well as in patients with meta-
VXM01m+CYP group, as compared to the control
static colorectal cancer.
group.
VXM01m, a murine analog of VXM01, has shown
In parallel, a phase I clinical trial (EudraCT No 2015-
substantial T cell responses and consistent antitumor
003068-34) recently started aiming to evaluate the
activities in different animal tumor models. In a pre-
safety and tolerability of VXM01 treatment in colorec-
liminary experiment, VXM01m was applied to CT26
tal cancer patients with liver metastases and to inves-
colorectal carcinoma tumor-bearing mice. Animals
tigate immunologic and tumor responses. In this trial,
(n=10) received per os administrations with VXM01m
24 patients will receive 4 oral prime doses of VXM01
or with the empty vector (control group) on days 1,
on day 1, 3, 5, and 7 followed by 4-weekly administra-
6
3, 5 and 7, followed by challenge with 1 × 10 CT26
tions up to W64, as add-on to their standard of care
cells s.c. on day 8 and two further administrations
therapy. Besides safety and tolerability, and clinical
with VXM01m on days 14 and 21. In another group,
response, immunological endpoints include peripher-
mice received a pretreatment with cyclophosphamide
al immune response (ELISpot) and tumor infiltrating
(CYP; 100 mg/kg). At day 30, VEGFR-2-specific CD8+
lymphocytes and tumor vasculature by immunohisto-
T cells were measured using VEGFR-2 MHC Pentam-
chemistry on serial liver metastasis biopsies.
ers. Tumor tissues were analyzed for infiltration with
Immunological parameters in phase I/II clinical trial of
dendritic-cell based immunotherapy (DCVAC/PCa) in patients
with rising PSA after primary prostatectomy or salvage
radiotherapy for prostate cancer
Podrazil M.1, Fucikova J.1,2, Jarolim L.3, Bilkova P.2, Hensler M.2, Becht E.4, Gasova Z.5, Kayserova J.1, Horvath R.6,
Fialova A.2, Vavrova K.1, Spisek R.1,2, Bartunkova J.1,2
Charles University, 2nd Faculty of Medicine and University Hospital Motol, Department of Immunology, Prague,
1
Czech Republic,
Sotio, Prague, Czech Republic,
2
Charles University, 2nd Faculty of Medicine and University Hospital Motol, Department of Urology, Prague,
3
Czech Republic,
Laboratory ‘Cancer, Immune Control and Escape’, INSERM U1138, Cordeliers Research Centre; University
4
Pierre and Marie Curie, UMRS 1138; University Paris Descartes, UMRS 1138, Paris, France,
Institute of Hematology and Blood Transfusion, Prague, Czech Republic,
5
Charles University, 2nd Faculty of Medicine and University Hospital Motol, Department of Pediatric and Adult
6
Rheumatology, Prague, Czech Republic
Background: Effect of cancer immunotherapy at the
second cycle. No significant side effects were re-
minimal residual disease stage can be evaluated in
corded. The median PSADT in all treated patients
patients with the biochemical relapse as detected
increased from 5,67 months prior to immunotherapy
by ultrasensitive PSA measurements. We have per-
to 18,85 months after 12 doses. Twelve patients who
formed an open label, single arm phase I/II clinical
continued the immunotherapy with the 2nd cycle
trial in 27 patients with the biochemical relapse of
had median PSADT of 58 month which remained
the prostate cancer using autologous mature den-
stable after the second cycle. We observed signifi-
dritic cells pulsed with killed LNCap prostate cancer
cant changes in the peripheral CD3+ T cells during
cell line, DCVAC/PCa.
the course of the trial. Conversely, the percentage
Methods: A single arm phase I/II trial registered as
of activated CD3+/HLA-DR+ cells as well as CD4+
EudraCT 2009-017259-91 involved 27 patients with
and CD8+ were not significantly changed. Addition-
rising PSA after RP or SRT. Study medication contain-
ally, we haven´t observed any significant decrease
7
ing 1 x 10 autologous dendritic cells pulsed with
in frequencies of regulatory T cells in the peripheral
killed prostate-cancer cell line LNCap (DCVAC/PCa
blood. Twelve out of 27 patients had significantly
manufactured from a leukapheretic product) was ad-
higher numbers of antigen-specific T cells against
ministered s.c. at monthly intervals. The first cycle
PSA before treatment compared with healthy con-
contained at least 12 doses. Twelve of the patients
trols. Similar results were obtained for MAGE-A1
with the best PSA-reponse continued with a second
and MAGE-A3 antigen specific T cells, for which
cycle of immunotherapy. The primary objective of
6 out of 27 had significantly increased numbers of
the study was to assess safety. Secondary objectives
antigen-specific T cells compared with the healthy
were PSA kinetics measured as PSA doubling time
controls. Long-term administration of DCVAC/PCa
(PSADT) and immune responses.
induced a statistically significant increase in the
Results: Twenty five patients were evaluable after
antigen-specific T cells against PSA in both tested
the first DCVAC/PCa cycle and 12 patients after the
time points (DCVAC-4 and DCVAC-12) and against
MAGE-A1 only in first tested time point (DCVAC-4).
Gene expression levels related to CD8/NK cytotoxicity were significantly overexpressed among patients
classified as responders compared to non-responders
before the DCVAC/PCa vaccination.
Conclusions: Long-term immunotherapy of prostate
cancer patients experiencing early sign of PSA recurrence using dendritic cells pulsed by killed LNCAP
cell line was safe, induced immune response and led
to significant extension of PSADT. Further long-term
follow-up may show whether the changes in PSADT
could affect the clinical course in patients with biochemically recurrent prostate cancer.
iVacALL: A personalized peptide-vaccination design platform for
pediatric acute lymphoblastic leukemia patients based on patientindividual tumor-specific variants
Rabsteyn A.1,2, Kyzirakos C.1, Schröder C.3, Sturm M.3, Mohr C.4, Walzer M.4, Pflückhahn U.1, Walter M.3,
Feldhahn M.4, Laske K.2,5, Schlegel P.1, Seitz C.1, Bonin M.3, Stevanovic S.2,5, Bauer P.3, Kohlbacher O.4,
Gouttefangeas C.2,5, Rammensee H.-G.2,5, Handgretinger R.1,2, Lang P.1,2
University Children’s Hospital Tübingen, Department of General Paediatrics, Oncology/Haematology, Tübingen,
1
Germany,
German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Tübingen,
2
Tübingen, Germany,
Institute for Human Genetics, University of Tübingen, Tübingen, Germany,
3
Institute for Applied Bioinformatics, University of Tübingen, Tübingen, Germany,
4
Institute for Cell Biology, University of Tübingen, Department of Immunology, Tübingen, Germany
5
We established a platform for the design of patient-
tides harboring the altered amino acids are subse-
individual peptide vaccination cocktails by combina-
quently predicted in silico by algorithms SYFPEITHI,
tion of whole exome sequencing of tumor and normal
NetMHC and NetMHCpan for the patients’ individual
tissue with in silico epitope prediction algorithms for
HLA type.
individual patient HLA types.
Whole exome sequencing was performed for 25 pa-
Acute lymphoblastic leukemia (ALL) is the most
tients. ALL-specific SNVs were identified using a com-
common pediatric malignancy. Standard chemo-
parative bioinformatics pipeline. We found an average
therapy is a successful treatment in 80% of patients,
of 29 mutations per patient. For all patients, MHC class
only about 20% develop a relapse, however these pa-
I and MHC class II epitopes could be predicted suc-
tients have a dismal prognosis. Prevention of relapse
cessfully.
after first-line chemotherapy or stem cell transplan-
We applied the platform for 5 patients based on com-
tation (SCT) is therefore mandatory. Accumulation
passionate need and designed individual peptide vac-
of somatic mutations is one characteristic feature
cines. In all cases validated mutations could be identi-
of malignant cells. These single nucleotide variants
fied and epitope prediction was performed for MHC I
(SNVs) can lead to altered amino acid sequences of
& II binders. The predicted peptides were synthesized
the translated proteins, which in turn can be pre-
and vaccination cocktails were formulated. The vac-
sented as antigenic peptides on HLA molecules of the
cination schedule provides 16 vaccinations over 33
malignant cells. A peptide vaccination composed of
weeks using GM-CSF and Imiquimod as adjuvant. The
mutated T cell epitopes specific for individual patient
vaccination was generally well tolerated. Response to
tumors is therefore a promising approach to prevent
the vaccination was monitored by detection of T cells
relapse in high-risk patients. For this purpose we
recognizing the vaccinated peptides occurring over
detect nonsynonymous mutations by whole exome
time in peripheral blood of the patients. Monitoring
and transcriptome sequencing of patient leukemic
was performed for each vaccination time point by
blasts and normal reference tissue. HLA binding pep-
prestimulation with the peptides and subsequent in-
tracellular cytokine staining (ICS) of T cells and FACS
analysis. In all 5 patients we could detect a developing
CD4+ T cell response against the vaccinated mutated
MHC II binding peptides.
Whole exome sequencing of pediatric ALL patients
is feasible and yields a small amount of mutations
per patients. However, these few mutations are sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T cell
responses in patients. Moreover, the platform is not
limited to ALL / Leukemia but can also be applied for
solid tumor patients. A phase I/II multicenter clinical
study will start in 2016.
Optimizing synthetic long peptide-based anti-tumor vaccination
using protease sensitive linkers
Rabu C.1,2, Florenceau L.1, Beauvillain C.3, Jeannin P.3, Labarrière N.1, Lang F.1,2
INSERM, UMR892, CNRS, UMR6299, Nantes, France,
1
University of Nantes, School of Pharmacy, Nantes, France,
2
INSERM, UMR892, CNRS, UMR6299, Angers, France
3
It is now established that anti-tumor vaccination
dramatic effect on cross-presentation efficiency. For a
strategies relying solely on short peptides coding for
given class I epitope, some linkers enable more than
class I epitopes activating CD8 T lymphocytes fail
a hundred-fold increase in epitope presentation levels
to elicit strong clinical responses and that recruit-
whilst some others strongly alter the capacity of the
ing CD4 helper T lymphocytes is crucial to enhance
APC to cross-present the class I epitope.
vaccine efficacy. Numerous class II and class I
The choice of an optimized linker that will ensure an
epitopes have been characterized from tumor anti-
optimal presentation of both class I and II epitopes
gens, eliciting specific T cell responses, and several
will thus allow significantly optimizing the thera-
vaccination trials using synthetic long peptides (SLP)
peutic efficiency of therapeutic vaccination based on
reported a enhanced efficiency compared to minimal
SLP for cancer patients.
class I peptides. These epitopes can be either separated on the natural sequence by hundreds of amino
acid or on the contrary, overlapping, that can impair
their processing efficiency. In both cases, it raises
the question of how to best couple a class I and class
II epitope when designing SLPs for therapeutic vaccination.
Our strategy is to combine a defined CD4 class II
epitope to a defined CD8 class I epitope, joining them
with a cathepsin-sensitive linker in order to increase
its intra cellular processing by antigen presenting
cells (APC).
We are using MELOE-1 as a model antigen, from which
we previously characterized an immunodominant
HLA-A*0201 epitope involved in melanoma immunosurveillance and a number of additional epitopes
presented in various HLA class II haplotypes (HLA
DRb1*01, *11, HLADQb1*02 *06 …). We have tested
a serie of linkers and we show that if the coupling
sequence has no major influence on the processing
efficiency of the class II epitopes, it has however a
Preclinical evaluation of triple microRNA-attenuated oncolytic
Semliki forest virus in glioma and neuroblastoma
Ramachandran M.1, Yu D.1, Dyczynski M.1, Baskaran S.1, Nelander S.1, Zhang L.1, Dimberg A.1, Saul S.2, Merits A.2,
Jarblad J.-L.1, Essand M.1
Uppsala University, Immunology Genetics and Pathology, Uppsala, Sweden,
1
University of Tartu, Institute of Technology, Tartu, Estonia
2
Background: Glioblastoma and high-risk neuroblas-
Conclusion: SFV4miRT has completely attenuated
toma are cancers with poor outcome. Immunother-
neurotoxicity, while retaining its oncolytic potential.
apy in the form of neurotropic oncolytic viruses is a
SFV4miRT is an excellent candidate for treatment of
promising therapeutic strategy for these malignan-
gliomas and neuroblastomas with low IFN-β secre-
cies. Here we evaluate the oncolytic potential of the
tion.
type-I interferon (IFN)-insensitive, neuro-pathogenic
Semliki forest virus (SFV)-4 in gliomas and neuroblastomas. To reduce neurotoxicity we constructed
SFV4miRT, which is attenuated in normal CNS cells
by three microRNAs: miR124, miR125, miR134.
Methods: In vitro antitumor activity of SFV4miRT
was examined in mouse and human neuroblastoma
and, glioma cells as well as in patient-derived human
glioma cell cultures (HGCC). In vivo neurotoxicity
and therapeutic efficiency was evaluated in two syngeneic orthotopic glioma models (CT-2A, GL261) and
one syngeneic subcutaneous neuroblastoma model
(NXS2). The role of IFN-β in inhibiting therapeutic
efficiency was investigated.
Results: The introduction of microRNA target sequences significantly reduced neurotoxicity of SFV4.
A single intravenous injection of SFV4miRT prolonged
survival and cured 4 of 8 mice (50%) with NXS2 and
3 of 11 mice (27%) with CT-2A but only 1 of 15 mice
(7%) with GL261 tumor. In vivo efficacy correlated
with in vitro killing of the corresponding cell lines
and to their secretion of IFN-β upon SFV infection,
with very low IFN-β induction for NXS2 and CT-2A
compared to GL261. Killing efficiency of HGCC lines
also depended on IFN-β response and interferon-α/β
receptor (IFNAR)-1 receptor expression.
A new synthetic lipopeptide is a superior adjuvant for peptide
vaccination
Rammensee H.-G.1,2, Chandran A.1,2, Zelba H.1,2, Gouttefangeas C.1,2, Kowalewski D.1,2, Di Marco M.1,2, Haen S.1,2,3,
Löffler M.1,2,4, Klein R.3, Karoline L.1, Artzner K.1, Backert L.1,5, Schwenck J.6,7, la Fougère C.6, Pichler B.7, Kneilling M.7,8,
Metzler G.8, Bauer J.8, Weide B.2,8, Schippert W.8, Stevanovic S.1,2, Wiesmüller K.-H.9
University Tübingen, Immunology, Tübingen, Germany,
1
University Tübingen, DKTK, DKFZ partner site Tübingen, Tübingen, Germany,
2
University Tübingen, Medicine II, Tübingen, Germany,
3
University Tübingen, Surgery, Tübingen, Germany,
4
University Tübingen, Applied Bioinformatics, Center for Bioinformatics and Department of Computer
5
Science, Tübingen, Germany,
University Tübingen, Nuclear Medicine, Tübingen, Germany,
6
University Tübingen, Werner Siemens Imaging Center, Tübingen, Germany,
7
University Tübingen, Dermatology, Tübingen, Germany,
8
EMC microcollections, Tübingen, Germany
9
We previously showed that the bacterial lipopep-
(120-200 spots) and CD4 (400-700 spots/ 300.000
tide Pam3Cys-Ser-Ser, meanwhile known as a TLR2
cells) responses. Pre-vaccination ELISPOT tests were
ligand, acts as a strong adjuvant for the induction of
negative for the class II peptide and weak for the two
virus specific mouse CD8 T cells when covalently
HLA class I peptides (10-20 spots). The granuloma,
coupled to a synthetic peptide (Deres et al., Nature
resected at day 44, contained highly activated CD4
1989). Such conjugates are difficult to purify by
and CD8 TEM cells. Ex vivo IFN- ƴ ELISPOT assay
HPLC, not water-soluble and extremely complex for
resulted in 120 spots for the class I and 400 spots
GMP production. We now designed a synthetic lipo-
(/50.000 cells) for the class II peptide(s) with a back-
peptide, named XS15, which is easy to purify and is
ground of around 40 spots, likely due to remnant
water-soluble. Specific human CD4 and CD8 T cells
vaccine peptides on antigen presenting cells in the
are induced and activated in vivo upon a single s.c.
granuloma. This was verified for all three peptides by
injection with Montanide containing free synthetic
mass spectrometry of granuloma HLA ligands. The
viral peptides when admixed to XS15.
total number of vaccine-antigen specific functional T
An HLA-A*01 restricted adenovirus 10AA peptide,
cells was calculated to be 3,5 mio in the granuloma
an HLA-B*08 influenza 9AA peptide, a promiscu-
and 12 mio in the peripheral blood. Thus, in contrast
ous HLA-DR restricted 15AA EBV peptide (240 µg
to previously reported data in mice and humans, a
each) and 80 µg of XS15 were emulsified in Monta-
human granuloma induced by Montanide/peptide/
nide ISA51 and injected s.c. into an HLA-matched
strong adjuvant is not a destructive sink for the ma-
volunteer in a volume of 400 µl. A granuloma at
jority of antigen specific T cells.
the injection site developed to a volume of about 8
Existing adjuvants potentially useful for peptide vac-
ml, as measured by sonography at days 17 and 41.
cination are of limited availability and/or efficiacy. It
18
FDG-PET/MRI on day 43 indicated it to be highly
remains now to be seen whether XS15 is a useful ad-
metabolically active. Ex vivo IFN-ƴ ELISPOT assays
juvant also for tumor peptide vaccines, in particular
from PBMCs at days 28 and 44 showed strong CD8
in a personalized setting.
Enhancing dendritic cell-induced T-cell responses by
immunomodulating agents
Rothe M.1,2,3, Lichtenegger F.S.1,2, Deiser K.1,2, Schnorfeil F.1,2, Schlüter M.1,2, Neitz J.1,2, Hiddemann W.1,
Subklewe M.1,2
Klinikum der Universität München, Department of Internal Medicine III, Munich, Germany,
1
Helmholtz Zentrum München, Clinical Cooperation Group Immunotherapy, Munich, Germany,
2
Immunotargeting of cancer (i-Target) Doktorandenkolleg, Elitenetzwerk Bayern, Munich, Germany
3
Immune checkpoint modulation represents a strate-
fold, n=6). Combination of anti-LAG-3 and anti-PD-
gy to enhance anti-tumor immune responses. Here
1 induced a 6.9-fold increase (n=6). Lenalidomide
we analyzed the impact of immune checkpoint mod-
induced a 5.4-fold increase in IFN-γ release (n=8).
ulation on T-cell activation by TLR-matured dendritic
All of these agents also enhanced T-cell proliferation.
cells (TLR-3-DCs).
To analyze the impact of checkpoint blockade on
Monocyte-derived DCs were generated in 3 days
primary versus recall immune responses, CD3-pos-
using a TLR7/8 agonist-containing maturation cock-
itive T cells were enriched by MACS beads and
tail. A mature DC phenotype was confirmed by
sorted according to CCR7 and CD45RO expression
analysis of characteristic DC markers (CD14, CD83,
levels into naive (Tnaive), central memory (TCM), effec-
CCR-7, HLA-DR = LAG3 receptor) using flow cy-
tor memory (TEM), and effector memory RA (TEMRA).
tometry. Positive costimulatory molecules were ex-
These T-cell subpopulations were again cocultivated
pressed at a high level [Median specific fluorescence
with autologous DCs and blocking antibodies. Block-
intensity (Median SFI): CD80 32.8; CD86 32.1; n=7
ade of PD-1 increased IFN-γ secretion by Tnaive- and
for both], but inhibitory molecules were also ex-
TEM- subsets significantly, while blockade of LAG-3
pressed to a significant extent (Median SFI: PD-L1
resulted in significantly increased IFN-γ secretion by
6.2, n=10; HVEM 2.0, n=10; ILT-3 2.5, n=7). A DC-T-
Tnaive- and TCM-subsets. These results indicated that
cell coculture system was used to test the relevance
PD-1 and LAG-3 checkpoint inhibitors target differ-
of the interaction with the respective ligands on T
ent T-cell subsets with different effectiveness.
cells. TLR-3-DCs were pulsed with a peptide pool of
Our data suggests that the efficacy of DC vaccination
viral and bacterial antigens (CEFT) and cocultivated
can be enhanced by combination with immunomod-
with autologous T cells for 4 days. T-cell activation
ulating agents. Furthermore, our data supports syn-
was induced by CEFT peptide-pulsed DCs as seen by
ergistic effects of blocking PD-1 and LAG-3 on T cells
IFN-γ secretion (CBA) and T-cell proliferation (CFSE).
possibly due to different effects on Tnaive-, TCM- and
This was accompanied by upregulation of the check-
TEM-subsets.
point molecules PD-1 and LAG-3 on T cells (Δ% positive CD4+/CD8+: PD-1 22.0/7.9, LAG-3 3.6/7.5). To
assess the impact of immunomodulators on T-cell responses, immune checkpoint-blocking antibodies or
lenalidomide were added to the coculture. Elevated
IFN-γ levels were obtained by blocking PD-L1 (1.4fold, n=11), PD-1 (2.0-fold, n=14) and LAG-3 (5.9-
Promising melanoma therapeutic cancer vaccine based on hybrid lipid-polymeric nanoparticles
Sainz V.1,2, Matos A.I.1, Viana A.3, Lopes J.A.1, Brocchini S.2, Zloh M.4, Florindo H.F.1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Department
1
of Galenic Pharmacy and Pharmaceutical Technology, Lisbon, Portugal,
UCL School of Pharmacy, Department of Pharmaceutics, London, United Kingdom,
2
Faculdade de Ciências, Department of Chemistry and Biochemistry, Lisbon, Portugal,
3
University of Hertfordshire, Department of Pharmacy, Hatfield, United Kingdom
4
Hybrid lipid-polymeric nanoparticles (HL-NPs) stand
LC of 4.21 ± 0.07 µg/mg). AFM analysis evidenced
out as potential drug delivery candidates. This study
that the addition of lipids to the PLGA matrix result-
aimed to design a therapeutic HL-NPs cancer vaccine
ed in smoother nanoparticle surfaces. Nanoparticle
able to deliver entrapped antigens and immune
treated cell viability was close to 100 %, and nano-
modulators to dendritic cells (DCs) for eradication of
particle internalization levels by DCs increased with
primary/metastatic cells by strengthening the host
the incubation time and absence of lipids. However,
immune response.
it is expected that the higher EE and LC observed
HL-NPs were prepared by the double emulsion-solvent
for HL-NPs will overcome those lower levels of in-
evaporation method. Two different lipids, 1-Palmi-
ternalization.
toyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC)
In conclusion, a promising hybrid nanoplatform for
and 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol
antigen delivery and DC activation and maturation
(DMPG), were used to modify the Poly(lactic-co-gly-
was developed. In vivo studies, in a metastatic malig-
colic acid) (PLGA) matrix. HL-NPs hydrodynamic
nant melanoma mouse model, are ongoing to evalu-
diameter and polydispersity index were determined
ate if the HL-NPs are able to induce a selective and
by Dynamic Light Scattering; surface morphology
extensive immune response capable to elicit reduc-
was evaluated by Atomic Force Microscopy (AFM)
tion of tumor growth or even its eradication.
and zeta potential was determined by Laser Doppler
Acknowledgements: The authors are grateful to:
Electrophoresis. The entrapment efficiency (EE) and
i) Fundação para a Ciência e a Tecnologia, Minis-
loading capacity (LC) were quantified by fluores-
tério da Ciência e da Tecnologia Portugal for iMed.
cence using OVA Alexa Fluor® 647 conjugate as a
ULisboa grant UID/DTP/04138/2013, UTAP/ICDT/
model antigen. The viability of DCs in the presence of
DTPFTO/0016/2014 research project and PhD grant
the HL-NPs was inferred using AlamarBlue® Assay,
SFRH/BD/87869/2012; and ii) EPSRC (Engineering &
while the internalization of the HL-NPs by these
Physical Sciences Research Council) Centre for Inno-
phagocytic cells was evaluated by flow cytometry.
vative Manufacturing in Emergent Macromolecular
The mean diameter of HL-NPs (137 ± 0.59 nm) was
Therapies.
lower than the one presented by the polymeric ones
(199 ± 11 nm). All formulations presented a monodispersed population, zeta potential close to neutrality and high EE and LC values (polymeric nanoparticles: EE of 69.91 ± 4.57 % (w/v), LC of 3.52 ± 0.24
µg/mg; and HL-NPs: EE of 84.06 ± 1.37 % (w/v),
Xenogeneic vascular endothelial growth factor-2 vaccination in
tumor bearing mice
Denies S.1, Cicchelero L.1, Sanders N.N.1
Ghent University, Laboratory of Gene Therapy, Faculty of Veterinary Medicine, Merelbeke, Belgium
1
In this study a xenogeneic DNA vaccine encoding
vaccinated mice could be demonstrated. Unexpect-
for human vascular endothelial growth factor recep-
edly, the vaccine caused an increased quantity of
tor-2 (hVEGFR-2) was evaluated in two murine tumor
early micrometastases in the liver. Lung metasta-
models, the B16-F10 melanoma and the EO771 breast
ses were not increased by the vaccine. These early
carcinoma model. The hVEGFR-2 DNA vaccine was
liver micrometastes did however not grow into mac-
administered by intradermal injection followed by
roscopic metastases in either control or vaccinated
electroporation. The immunogenicity and the bio-
mice when allowed to develop further after surgical
logical efficacy of the vaccine was tested in (1) a pro-
removal of the primary tumor.
phylactic setting, (2) a therapeutic setting and (3) a
therapeutic setting combined with surgical removal
of the primary tumor. In the prophylactic and therapeutic setting, 14 vaccinated and 14 control mice
were included per tumor model. Ten mice were followed for tumor growth and survival and 4 mice per
group were sacrificed for biological read-outs. The
systemic cellular immune response was measured
by a bioluminescence based cytotoxicity assay with
VEGFR-2 expressing target cells. Humoral immune
responses were quantified by ELISA. Ex vivo bioluminescence imaging of organs was used to detect
(micro)metastases. For the experiment where vaccination was combined with surgery, ten vaccinated
and ten control mice were included per tumor model,
and all ten were sacrificed three weeks after removal
of the primary tumor for ex vivo bioluminescent
quantification of (micro)metastases. A cellular and
humoral immune response was present in prophylactically and therapeutically vaccinated mice, in both
tumor models. Nevertheless, survival in prophylactically vaccinated mice was only moderately increased,
and no beneficial effect on survival in therapeutically
Immunomodulatory capacity of CD47 functionalized artificial
antigen-presenting cells (aAPCCD47+)
Gallenstein N.1, Schappert A.2, Bruns H.3, van Zandbergen G.1, Schütz C.1
Paul-Ehrlich-Institute, Immunology, Langen, Germany,
1
Johann Wolfgang Goethe-University Hospital, Internal Medicine I, Frankfurt am Main, Germany,
2
University of Erlangen, Internal Medicine 5 - Hematology/Oncology, Erlangen, Germany
3
Artificial Antigen-Presenting Cells, aAPC, have suc-
directly translate into different T cell activation, an-
cessfully been used to stimulate antigen-specific T
tigen-specific T cell priming experiments, comparing
cell responses in vitro as well as in vivo. While aAPC
aAPC and aAPC CD47+ in co-cultures with either pre-
compare favorable to autologous dendritic cells in
treated macrophages or preconditioned media, were
vitro, their effect in vivo might be diminished through
performed.
rapid clearance by macrophages. Currently, we could
Our data for the first time show that aAPC function-
demonstrate that classical two-signal aAPC, addition-
alized with CD47 maintain their stimulatory capac-
CD47+
), efficient-
ity in vitro, demonstrate enhanced in vivo efficiency
ly inhibited phagocytosis by macrophages in vitro.
and hold the potential to indirectly modulate T cell
While this effect was CD47 concentration dependent
responses by inhibiting phagocytosis through mac-
their ability to generate and expand antigen-specific
rophages. Thus this next generation aAPC CD47+ might
T cells was not affected. Furthermore, aAPC CD47+ gen-
facilitate the application of the aAPC technology for
erated T cells displayed equivalent killing abilities
future therapeutic vaccination approaches possibly
and polyfunctionality when compared to classical
synergizing with already existing approaches.
ally functionalized with CD47 (aAPC
aAPC generated T cells. In addition, in vivo studies
demonstrated an enhanced stimulatory capacity and
tumor inhibition of aAPC CD47+ over classical aAPC in
conjunction with diverging bio-distribution in different organs. Interestingly, we detected in macrophage
co-cultures with aAPC CD47+ significantly reduced
amounts of immunosuppressive cytokines such as
IL-10 and TGF-β and comparable amounts of other
cytokines such as TNF and IL-1β when compared to
classical aAPC.
Therefore, we closely investigated the immunomodulatory capacity of aAPC CD47+ in human primary T
cell and macrophage co-cultures. Both, expression
of co-stimulatory and activation markers and the
secreted cytokine profile was monitored utilizing
ELISA and flow cytometry based techniques. Finally,
to address the question if the detected differences
Synergistic combination of vasculature disrupting agent with
TLR7/8 agonist: Promising strategy for melanoma therapy
Seth A.1,2, Lee H.1, Park C.1, Lee J.-Y.2, Hong K.S.1,2
Korea Basic Science Institute, Bioimaging Research Team, Cheongju, Korea, Republic of,
1
Chungnam National University, Graduate School of Analytical Science and Technology, Daejeon, Korea,
2
Republic of
Gardiquimod is an imidazoquinoline compound and
rate as compared to control groups, which was also
is a potent toll-like receptor 7 and 8 (TLR7/8) agonist.
in good correlation with immuno-chemical analyses
It causes activation of innate immune response and is
from tumor tissue samples. This research highlights
known to have potent anti-viral and anti-tumor effect
combination of vasculature disrupting with immu-
[1, 2]. It activates NFκB and MAP kinase pathways
no-stimulation as a promising approach for manage-
in innate immune cells and is speculated to stimu-
ment of solid tumor.
late antigen presenting cells (APCs) which were rendered tolerant in immuno-suppressed tumor microenvironment. 5,6-Dimethylxanthenone-4-acetic Acid
(DMXAA) exerts its anti-tumor effect by disrupting
the tumor vasculature leading to generation of a necrotic center in a solid tumor. However, the limitation with DMXAA treatment is that the tumor cells
present in the periphery are unaffected by the drug,
leading to incomplete therapy. In this research, combination of gardiquimod with DMXAA was assessed
to target B16 melanoma in a mouse model.
Uniform and spherical gardiquimod encapsulated
PLGA nanoparticles were prepared using single
emulsion method. Their size was ∼193 nm and the
encapsulation efficiency was found to be 11.4 µg/
mg. The role of nanoparticulate formulation was assessed by observing improved activation of BMDCs
in the presence of PLGA-gardiquimod as compared
to free gardiquimod. The nanoparticle uptake by
BMDCs was also confirmed by fluorescence imaging.
Further, PLGA-gardiquimod and DMXAA in the
ratio of 1:10, 1:100, 1:200 and 1:500 synergistically
enhanced cytokine (TNFα and IL-12) secretion from
BMDCs. Mice treated with the combination had significantly lower tumor volume and a higher survival
[1] F. Ma, J. Zhang, J. Zhang, C. Zhang, Cell Mol Immunol, 7
(2010) 381-388.
[2] M
. Buitendijk, S.K. Eszterhas, A.L. Howell, AIDS Research
and Human Retroviruses, 29 (2013) 907-918.
LCMV-GP pseudotyped oncolytic vesicular stomatitis virus for
the treatment of prostate cancer
Urbiola C.R.1, Kimpel J.1, Santer F.2, Culig Z.2, Holm-von Laer D.1, Wollmann G.1
Medical University Innsbruck, Department of Virology, Innsbruck, Austria,
1
Medical University Innsbruck, University Clinic of Urology, Innsbruck, Austria
2
Prostate cancer (PCa) is the second leading cause
revealed that VCaP and TRAMP-C1 were still able
of cancer death in the U.S. and Europe. Diagnosed
to mount an IFN-I induced antiviral response, while
at early stages, prostate cancer can be surgically
defects in the IFN-I signalling pathway were found
removed. However, despite many research efforts,
in VSV-GP susceptible cell lines. Results in cell lines
long-term effective therapies are not available. Onco-
were confirmed in primary cultures derived from pa-
lytic viruses (OV) that preferentially replicate in and
tients who had undergone radical prostatectomy. In
kill tumour cells are a potent novel treatment option
our in vivo studies, VSV-GP was able to cure Du145
for cancer patients after failure of common thera-
subcutaneous tumours in a xenograft model and
peutic strategies such as chemotherapy or surgery.
was able to slow down tumour growth in a TRAMP-
Through cell lysis, OV set free tumour antigens,
C1 subcutaneous syngeneic model, significantly
which in combination with the OV adjuvant effect,
increasing life expectancy of VSV-GP treated mice.
unleashes a strong anti-tumour immune response.
Since TRAMP-C1 are highly responsive to IFN-I sig-
Our group previously reported that oncolytic Ve-
nalling, we used two different approaches to improve
sicular Stomatitis Virus (VSV) pseudotyped with the
therapy outcome, either a combination therapy with
LCMV glycoprotein (VSV-GP) is a promising, highly
the Jak1/2 inhibitor Ruxolitinib, or a knock down
efficient and safe oncolytic virus. Here, we propose
of the IFNAR1 gene in TRAMP-C1 cells. However,
the use of the oncolytic VSV-GP for the treatment of
neither of these approaches resulted in an improved
prostate cancer.
outcome.
We used prostate cancer cell lines and primary cul-
VSV-GP is a promising novel therapeutic for the treat-
tures from patient samples to test the efficacy of
ment of prostate cancer. To optimize the efficiency of
VSV-GP in prostate cancer. We analysed oncolytic
VSV-GP, further studies will be necessary to better
efficiency as well as the role of the innate immune
understand how the oncolytic effect, the IFN-I re-
response in therapy outcome. VSV-GP was further
sponse and anti-tumour immune response interact
tested in vivo both in a xengoraft and a syngeneic
and what strategies will result in enhanced thera-
mouse model. In addition, IFN-I response was modu-
peutic outcome.
lated either using the Jak1/2 inhibitor, Ruxolitinib
(Novartis), or by knocking down the IFNAR1 gene
in the tumour.
VSV-GP exhibited high oncolytic efficiency in vitro,
efficiently killing the majority prostate cancer cell
lines tested. Further analysis of resistant cell lines
Superior innate immune effector cell recruitment by interleukin15 dendritic cells
Van Acker H.H.1, Beretta O.2, Anguille S.1,3, De Caluwé L.1,4, Papagna A.2, Van den Bergh J.M.1, Willemen Y.1,
Goossens H.1, Berneman Z.N.1,3, Van Tendeloo V.F.1, Smits E.L.1,3,5, Foti M.2, Lion E.1,3
Laboratory of Experimental Hematology, Tumor Immunology Group (TIGR), Vaccine & Infectious Disease
1
Institute (VAXINFECTIO), University of Antwerp, Faculty of Medicine and Health Sciences, Edegem, Belgium,
Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy,
2
Center for Cell Therapy & Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium,
3
Institute of Tropical Medicine, Antwerp, Belgium,
4
Center for Oncological Research (CORE), University of Antwerp, Faculty of Medicine and Health Sciences,
5
Antwerp, Belgium
Introduction: A key requisite for the success of a
chemokines involved in anti-tumor immune effector
dendritic cell (DC)-based vaccine in treating malig-
cell attraction, while IL-4 DCs display a more immu-
nancies is the capacity of the DCs to attract immune
noregulatory profile characterized by high expression
effector cells, considering crosstalk with DCs is
of Th2 and regulatory T cell-attracting chemokines.
partially regulated by cell-contact-dependent mech-
A possible explanation for the superior recruitment
anisms. The clinical effectiveness of DC vaccines
of effector lymphocytes by IL-15 DCs could be as-
might therefore, at least partly, rely on their ability to
cribed to the CCL4-CCR5 signalling pathway because
secrete the appropriate chemokines, allowing them
of higher CCL4 chemokine gene expression in IL-15
to effectively recruit, engage, and activate (γδ) T cells
DCs and lowered CCR5 expression on both migrat-
and natural killer (NK) cells. To this extent, we have
ed γδ T cells and NK cells. Following validation of
made a head-to-head comparison of interleukin (IL)-
significant higher CCL4 secretion by IL-15 DCs then
15-cultured DCs and conventional IL-4-cultured DCs
by IL-4 DCs, we demonstrated that neutralization of
with regard to their proficiency in the recruitment of
CCR5 on PBMC prior to migration resulted in a signif-
(innate) immune effector cells.
icant inhibition of γδ T cell and NK cell recruitment
Methods: Short-term monocyte-derived IL-15 DCs
by IL-15 DCs, whereas this was not observed for IL-4
were prepared as previously reported (Anguille et al.
DC-mediated migration.
J Transl Med. 2009), replacing IL-4 with IL-15 for DC
Conclusion: Our results show that IL-15 DCs are su-
differentiation and using a Toll-like receptor 7/8 ago-
perior to IL-4 DCs in terms of attraction of all im-
nist-containing maturation cocktail. The potential of
portant immune effector lymphocytes, namely CD8+
DCs to attract immune cells was studied at RNA and
T cells, γδ T cells and NK cells. Furthermore, our
protein levels with micro-array analysis and ELISA,
data suggest involvement of the CCL4-CCR5 signal-
and functionally, by means of transwell chemotaxis
ing pathway in the improved capacity of IL-15 DCs
assays and flow cytometry.
to recruit antitumor immune effector lymphocytes,
Results: IL-15 DCs and IL-4 DCs attracted distinct
by means of increased expression and secretion of
PBMC populations. Whereas IL-15 DCs significant-
CCL4 by IL-15 DCs. In addition to the previously
ly recruited immune effector lymphocytes, includ-
demonstrated superior T cell- and NK cell stimula-
ing CD8+ T cells, γδ T cells and NK cells, IL-4 DCs
tory properties and direct tumor cell killing capacity
predominantly attracted monocytes and B cells. This
of IL-15 DCs, these findings further underscore their
was in accordance with the gene expression analysis,
immunotherapeutic potential.
revealing that IL-15 DCs exhibit a high expression of
Type I IFN induced upon particle mediated intravenous
delivery of antigen mRNA enhances specific immune responses
Van der Jeught K.1, Broos K.1, Puttemans J.1, Verbeke R.2, De Witte H.2, Heirman C.1, Lentacker I.2, Thielemans K.1,
Breckpot K.1
Vrije Universiteit Brussel, Biomedical Sciences, Jette, Belgium,
1
Ghent University, Ghent, Belgium
2
Protection of mRNA via packaging opens its sys-
es. Surprisingly, in contrast to previous reports, we
temic application for tumor immunotherapy and in
showed that type I IFN is increasing the capacity of
other fields. mRNA is degraded by RNAses, which
packaged antigen mRNA to improve antigen-specif-
are found abundantly throughout the entire body
ic immune responses using IFN-alpha/beta receptor
and more specifically at high amounts in the blood.
knockout mice. These results show that the precise
Therefore, in order to broaden the applications of
role of type I IFN is not yet fully established, and that
mRNA as a clinical compound; packaging of the
further investigation is warranted.
latter is of major interest. This study shows that Lipofectamine® RNAiMAX, a lipoplex that is developed
to package small interfering RNA, is able to complex
messenger RNA (mRNA) into particles and protect
the mRNA from RNAses. Furthermore, we show that
intravenous (IV) delivery of packaged mRNA encoding firefly luciferase results in a very strong splenic
signal as fast as 15 minutes after injection. Hereby,
showing that RNAiMAX packaged mRNA can be
rapidly translated into a functional protein upon
IV delivery. Using CD11c-Diphteria Toxin Receptor
mice we could show that CD11c+ cells are found to
be the dominant cell fraction leading to high bioluminescent signal, as confirmed by flow cytometry.
Next, we showed that IV delivery of small amounts
of antigen encoding mRNA could lead to strong
immune responses. The delivery of multiple antigens
at the same time resulted in a similar specific lysis
as when delivered separately. The latter is of major
interest when translating this approach to the clinic.
In line with other packaging agents, RNAiMAX packaged mRNA elicits type I interferon (IFN) responses. This type I IFN was recently shown to abrogate
the induction of antigen-specific immune respons-
The first both authors contributed equally.
Messenger RNA DOTAP-Cholesterol lipoplexes containing TLR
agonists allow single step antigen-loading and maturation of
dendritic cells
Verbeke R.1, Dewitte H.1, Wayteck L.1, Breckpot K.2, De Smedt S.1, Lentacker I.1
Ghent University, Ghent Research Group on Nanomedicines, Faculty of Pharmacy, Ghent, Belgium,
1
Vrije Universiteit Brussel, Laboratory of Molecular and Cellular Therapy, Department of Biomedical Sciences,
2
Jette, Belgium
In dendritic cell (DC)-based immunotherapy, DCs
derived DCs (BM-DCs) are not properly activated by
are modified with tumor-associated antigens (TAAs)
mRNA lipoplexes (as such) and fail to induce strong
and immune adjuvants so that they can present TAA
CD8+ T cell responses in vitro. However, we give clear
epitopes and spark T cell-mediated immunity against
evidence that co-encapsulation of the TLR agonists
cancer. Recently, there is a growing of interest in
CpG oligodeoxynucleotides or monophosphoryl-lipid
finding ways to modify dendritic cells (DCs) in vivo,
A in DOTAP-cholesterol/mRNA lipoplexes strongly
which holds the promise of targeting the immune
improves their potency to mature BM-DCs, without
players in their natural habitat. In this study, we aim
compromising the transfection efficiency. Most im-
to design immunogenic lipid based carriers which
portantly, this resulted in DCs with a much stronger
package and protect TAA-encoding mRNA in serum-
capacity to activate antigen-specific CD8+ T cells
containing media, in order to induce high antigen ex-
when compared to immature (non-adjuvant treated)
pression levels in DCs in situ, while simultaneously
mRNA-transfected DCs.
initiating a potent immune response.
We investigated two types of lipid formulations for
the delivery of TAA-encoding mRNA; both of them
contain the commonly used DOTAP as cationic lipid
combined with either DOPE or cholesterol as ‘helper
lipid’. While the use of DOTAP-DOPE liposomes as
liposomal carriers in current research on mRNA delivery is widespread, we reveal clear indications that
DOTAP-cholesterol based carriers are more suitable
for the delivery of mRNA in vivo. DOTAP-cholesterol liposomes protect mRNA from degradation by
RNases, they are not prone to aggregate upon exposure to serum, and can achieve efficient transfection
in bone marrow derived DCs (BM-DCs) in the presence of high amounts of serum.
In addition, there is an ongoing debate whether mRNA
nanoparticles can cause the activation of DCs via a
“self-adjuvant effect”, or if extra immune adjuvants
are required. We demonstrate that bone marrow-
Neo-epitopes generated by insertions, deletions and gene fusions
as targets for personalized tumor vaccination
Vormehr M.1, Schrörs B.2, Boegel S.2, Löwer M.2, Diken M.2, Kreiter S.2, Türeci Ö.2, Sahin U.1,2,3
Research Center for Immunotherapy (FZI), Mainz, Germany,
1
TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH,
2
Mainz, Germany,
Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany
3
Accumulating evidence reveals that cancer-associ-
tions featuring a multitude of epitopes predicted to
ated mutations are key targets of tumor specific T
bind to MHC class I and MHC class II. In conclusion,
cells in spontaneous and immunotherapy induced
our data calls for extending the neo-antigen reper-
immune responses against cancer. As single nucleo-
toire for tailored vaccine approaches to indel and
tide variants are the most abundant cancer mutations,
fusion based mutations.
research so far has focused mainly on this subtype.
Mutations that introduce several new amino acids
are even more interesting from an immunological
point of view, as they may simultaneously harbor
multiple T-cell neo-epitopes. For this reason, we investigated the prevalence and immunogenicity of
two other types of mutations, namely small cancerassociated insertions and deletions (indels) and gene
fusions as targets for personalized cancer vaccination. We identified indels and fusions in the next
generation sequencing data of three murine tumor
models. Using pharmacologically optimized RNA
encoding selected mutations as a vaccine format, we
show that a considerable fraction of indel and fusion
based mutations are immunogenic. Moreover, we
reveal that such mutations may encode several T-cell
epitopes. Employing the same predictive algorithms
on sequencing data of corresponding human cancer
types, we identify indel and fusion gene based muta-
LCMV-GP pseudotyped oncolytic vesicular stomatitis virus for
the treatment of ovarian cancer
Kimpel J.1, Urbiola C.1, Dold C.1, Marth C.2, Muik A.3, Holm-von Laer D.1, Wollmann G.1
Medical University Innsbruck, Virology, Innsbruck, Austria,
1
Medical University Innsbruck, Gynecology and Obstetrics, Innsbruck, Austria,
2
Paul Ehrlich Institute, Molecular Biotechnology and Gene Therapy, Langen, Germany
3
Treatment options for advanced ovarian cancer
of VSV-GP with the JAK1/2-inhibitor ruxolitinib was
remain limited. Metastasis commonly occurs in the
successfully tested in both models and found to
peritoneal cavity. First line therapy consisting of de-
enhance the oncolytic effect. The drug inhibited the
bulking surgery and chemotherapy usually fails to
signalling pathway induced by type I IFN and could
cure patients and eventually tumours relapse. One
thus be used to inhibit the antiviral innate immune
very promising new treatment approach is the use
response and enhance intratumoral viral replica-
of oncolytic viruses (OV) that preferentially replicate
tion. Importantly, despite inhibiting the antiviral
in and kill tumour cells. Through cell lysis, OV set
response, no toxicity was observed in mice receiv-
tumour antigens free, which in combination with the
ing up to 109 pfus (plaque forming units) VSV-GP via
OV adjuvant effect, unleashes a strong anti-tumour
intraperitoneal application.
immune response. Our group previously reported
In conclusion, VSV-GP was tested as a potent on-
that oncolytic Vesicular Stomatitis Virus (VSV) pseu-
colytic virus to treat ovarian cancer. Restriction of
dotyped with the LCMV glycoprotein (VSV-GP) is a
viral replication due to the innate immune response
promising, highly efficient and safe oncolytic virus.
could be overcome by the combination treatment of
Here, we propose the use of the oncolytic VSV-GP for
VSV-GP with the Jak-1/2 inhibitor ruxolitinib.
the treatment of ovarian cancer.
Oncolytic activity was assessed in vitro on a variety
of ovarian cancer cell lines and VSV-GP was found
to efficiently infect and lyse most of the cell lines.
However, analysis of the innate immune response of
ovarian cancer cells to VSV-GP revealed IFN type I
production and induction of an antiviral state of the
cells as a potential mechanism leading to shortcomings in virotherapeutic treatment. In vivo, VSV-GP
was tested in a subcutaneous ovarian cancer xenograft mouse model using the A2780 cell line. Treatment led to tumour remission, but in most cases
relapse was observed. In an orthotopic xenograft
mouse model, intraperitoneal injection of the virus
led to significantly prolonged survival compared to
untreated animals. In addition, combination therapy
Immunotherapy with INO-3112 (HPV16 and HPV18 plasmids +
IL-12 DNA) in Human Papillomavirus (HPV) associated Head
and Neck Squamous Cell Carcinoma (HNSCCa): Interim results
Aggarwal C.1, Cohen R.1, Morrow M.2, Kraynyak K.2, Bauml J.1, Weinstein G.1, Boyer J.2, Yan J.2, Mangrolia D.2,
Oyola S.2, Duff S.2, Yang Z.2, Bagarazzi M.2
University of Pennsylvania, Philadelphia, United States,
1
Inovio Pharmaceuticals, Inc., Plymouth Meeting, United States
2
ORAL
TALK
SHORT
2016
Objective: Oropharyngeal HNSCCa is frequently as-
(n=3), dizziness (n=3), dysphagia (n=2), injection
sociated with HPV infection. DNA-based immuno-
site hematoma (n=2) and candidiasis (n=2). There
therapy with plasmids encoding HPV16 and HPV18
were two unrelated SAE cases due to hospitalization:
E6/E7 antigens has been shown to generate robust
Grade 2 post-surgical procedure hemorrhage and
immune responses in women with HPV-driven
Grade 3 acute non-traumatic kidney injury. Enroll-
high-grade cervical dysplasia. We hypothesize that
ment and correlative analysis are ongoing; among
HPV-specific immunotherapy with INO-3112 (6mg
10 pts’ samples tested to date, as compared to base-
VGX-3100 + 1mg INO-9012) in patients with HPV-
line, 10 of 10 evaluable pts showed elevated antigen
associated HNSCCa will generate robust immunity
specific antibody titers at any time point. Nine of 10
which may contribute to disease control.
evaluable pts exhibited increased HPV-specific cel-
Method: This open-label Phase I/IIa trial included
lular responses by IFN-gamma ELISpot. Eight out
adults with HPV-positive (assessed by p16) HNSCCa,
of 9 evaluable pts had HPV-specific CD8+ T cell re-
ECOG PS 0-1. Two cohorts: Cohort 1, patients (pts)
sponses to INO-3112 by flow cytometric analysis and
receive INO-3112 pre and post-surgery; Cohort 2, pts
all 10 pts had positive cellular immune responses in
receive INO-3112 after completion of cisplatin based
at least one assay.
chemoradiation. INO-3112 is delivered IM followed
Conclusion: These interim results demonstrate that
by electroporation with the CELLECTRA® device,
INO-3112 DNA-based immunotherapy can safely gen-
once every 3 weeks for a total of 4 doses. Pts are
erate HPV-specific CD8+ T cell immunity in patients
followed for 2 years. Primary and secondary end-
with HPV-related HNSCCa. All tested pts had positive
points are safety and immune responses. Exploratory
immune responses.(NCT02163057)
endpoints include: anti-tumor effect and progressionfree-survival.
Results: As of January 2016, 20 of 25 pts have been
treated. Cohort 1: n=6, Cohort 2: n=14; 18 males and
2 females; median age 57 years (range 32-76); cancers
at base of tongue=7, tonsil=13; never smoker=8;
median follow-up is 195 days (range 19-430). INO-3112
was well tolerated with no treatment related Grade 3
AE, No Grade 4 or higher AEs. The most common
(10% and above) AEs were injection site pain (n=14),
injection site erythema (n=4), injection site swelling
075 – 138
Cellular
Therapy
075 | CELLULAR THERAPY
Identifying rare, high avidity self/tumor-specific CD8 T cells in
cancer patients
Allard M.1, Couturaud B.1, Schmidt J.2,3, Duong M.N.1, Baumgaertner P.2, Gannon P.O.1, Speiser D.E.1,2,
Hebeisen M.1, Rufer N.1,2
Lausanne University Hospital Center and University of Lausanne, Department of Oncology,
1
Epalinges, Switzerland,
Ludwig Cancer Research Center, Epalinges, Switzerland,
2
TCMetrix Sàrl., Epalinges, Switzerland
3
Rationale: Cytotoxic T cells recognize, via their T-cell
sponse experiments. NTAmer-sorted high avidity T
receptors (TCRs), small antigenic peptides (p) pre-
cells were also superior in controlling tumor growth
sented by major histocompatibility complex (MHC)
in vivo than lower avidity T cells. Remarkably, we
molecules on the surface of infected or malignant
found that the TCR-pMHC avidity repertoire de-
cells. The TCR avidity for pMHC is a key parameter
pended on the type of tumor antigen, as CD8 T cells
for T cell-mediated immunity, with stronger TCR-pM-
specific for the cancer testis antigen NY-ESO-1157-165
HC interactions conferring superior T cell activation
displayed higher avidities than T cells specific for the
and protection from disease than weaker ones. Yet,
differentiation antigen Melan-A 26-35. Yet, both tumor
low avidity is a fundamental feature of most tumor-
antigen-specific TCRs revealed significantly lower
specific CD8 T cells. Consequently, there is a need
avidities than those that bind to persistent herpes
for a robust technology that allows rapid and efficient
virus antigens (CMV/pp65495-503, EBV/BMFL1259-267).
detection and isolation of individual CD8 T cells of
Conclusions: Collectively, our work indicates that
high TCR avidity and enhanced functionality against
NTAmers are effective tools to isolate rare cytotoxic T
malignant cells.
cells with best suitable anti-tumor TCRs and highest
Methodology: To identify these rare T cells, we used
poly-functional qualities against tumor cells, repre-
the recently developed NTAmer technology, which
senting a strong asset for the development of cancer
allows for the direct quantification of TCR-pMHC
immunotherapy. Moreover, NTAmers allowed for the
dissociation kinetics on living tumor-reactive CD8 T
first time to directly compare the binding parameters
cells from peripheral blood (1). TCR avidity analysis
of a large library of antigen-specific T cell clones (n
was combined with various in vitro functional assays
= 250) and to demonstrate strong binding differ-
and in vivo adoptive cell transfer experiments in im-
ences between self/tumor- versus virus-specific rep-
munodeficient NSG mice.
ertoires. These findings provide a fundamental ex-
Results: NTAmer off-rates accurately predicted
planation to the inefficiency of tumor-reactive T cell
multiple functions (i.e. tumor cell killing, cytokine
responses to control and eliminate advanced disease,
secretion and proliferation index) of large panels
namely the lack of anti-cancer cytotoxic T cells of
of tumor-specific T cells isolated from melanoma
high affinities/avidities.
patients following therapeutic vaccination or with
long-lasting natural anti-tumor responses. Our data
substantiate that the TCR-pMHC avidity correlates
systematically with ligand potency (EC50), but not
with maximal biological activity (Emax) in dose-re-
Reference: (1) Hebeisen et al., Cancer Res, 75(10):1983, 2015
Targeting simultaneously non-mutated HLA.A2-restricted MDM2
and p53 tumor-associated antigens as a novel double-edged
swords approach for TCR gene therapy for multiple myeloma
Amann E.1, Antunes E.1, Jacobi B.1, Theobald M.1,2,3, Echchannaoui H.1,4
University Medical Center Mainz, Third Medical Department (Hematology, Oncology and Pneumology), Mainz,
1
Germany,
Johannes Gutenberg University Mainz, Research Center Immunology, Mainz, Germany,
2
German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Frankfurt/Mainz, Germany,
3
German Cancer Research Center (DKFZ) partner site Frankfurt/Mainz, German Cancer Consortium (DKTK)
4
partner site Frankfurt/Mainz, Mainz, Germany
Background: Adoptive T cell receptor (TCR) gene
group. Interestingly, extracted tumor cells exhibited
therapy has shown efficacy in cancer patients. The
a down-regulation of MDM2 expression and concom-
human homologous of the murine double-minute
itantly an up-regulation of p53 antigen expression
2 protein (MDM2) tumor-associated antigen (TAA)
which was associated with a lower recognition of
is overexpressed in a variety of human tumors, in-
these tumor cells by the MDM2-specific TCR. Ac-
cluding soft tissue sarcoma, melanoma and multi-
cordingly, combining MDM2- and p53-specific TCR
ple myeloma (MM). MDM2 protein overexpression
transduced T cells improved tumor control in vivo
is particularly observed in invasive and metastatic
compared to mock-treated mice or treatment with
melanoma and is associated with enhanced prolifer-
only one group of specific TCR transduced T cells.
ation and survival of MM cells. We have generated
In addition we observed an enhanced PD-L1 expres-
and optimized a high-affinity HLA-A*02:01-restricted
sion in ex vivo tumor cells compared to the parental
CD8-dependent MDM2 (81-88)-specific murine TCR
cells and up-regulation of PD-1 in tumor-infiltrating
as a potential therapeutic TCR to target MM.
lymphocytes (TIL).
Methods: The MDM2-specific TCR was modified by
Conclusion: Using MDM2- and p53-specific TCR
codon optimization, addition of a second disulfide
transduced T cells represent a novel approach to
bond between TCR α and β constant domains and
circumvent tumor escape mechanisms like antigen
cloned into a 2A-based bicistronic retroviral vector.
down-regulation in MM. The combination of adop-
Human T cells from healthy donors were retrovirally
tive immunotherapy and checkpoint inhibitors like
transduced with the optimized MDM2-specific TCR
anti-PD-1 antibody could be a potential treatment in
and a single-chain p53 (264-272)-specific TCR (Voss
MM.
et al., Blood 2010) and expression levels were analyzed by flow cytometry. MDM2 and p53 expression in MM cell lines was determined by Western
blot. The therapeutic efficacy of MDM2/p53 dual
TCR modified T cells was evaluated in NOD-scid
IL2R gamma chain null (NSG) mice engrafted with the
HLA-A*02:01-expressing NCI-H929 MM cell line.
Results: In this xenograft MM mouse model we could
observe a prolonged overall survival and tumor-infiltrating T cells in mice which received MDM2-specific
TCR transduced T cells compared to mock-treated
Comparison of two allorestricted T-cell receptors targeting
two different Myeloperoxidase-derived HLA-B*07:02-restricted
peptide epitopes with different MHC affinities with respect for
their therapeutic potential
Audehm S.1, Klar R.1, Bräunlein E.1, Mall S.1, Bianchi H.1, Peschel C.1, Utsch C.1, Busch D.2, Peper J.3, Stevanović S.3
Technical University Munich, Klinikum rechts der Isar, München, Germany,
1
Technical University Munich, Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, München,
2
Germany,
Eberhard Karls Universität Tuebingen, Interfaculty Institute for Cell Biology, Tübingen, Germany
3
The interaction of T-cell receptors (TCR) with major
both TCR was seen in cross-reactivity tests against
histocompatibility complex class I (MHC) molecules
a set of 58 HLA-B*07:02 restricted peptides. Various
and antigenic peptides (TCR-p-MHC) is fundamental
stimulated T cell responses in vitro as well as in vivo
for the recognition of tumor-derived antigenic pep-
using different tumor cell lines indicate that in depth
tides by the adaptive immune system. We previously
investigations in respect to the characterization of
identified two potential peptide epitopes derived
the tri-molecular TCR-p-MHC will be of importance
from the hematopoietic differentiation antigen my-
to understand the efficacy of the TCR restricted to
eloperoxidase (MPO) from primary human tumor
the binders and the value of binding prediction tools
samples by an immunopeptidomic approach. Despite
for selection of target structures in immunotherapy.
the peptide epitopes differ considerably in their affinities to their common restricted human leukocyte
antigen (HLA)-B*07:02, two TCR that specifically
recognize the selected peptide ligands, isolated in a
single HLA-mismatch approach were investigated to
address the question whether only high affinity peptides are suitable targets for clinical translation such
as T-cell therapy or even weak binders could also be
promising peptide candidates.
MHC-peptide (MHC-p) affinity prediction tools classified one of the peptides as a strong binder and the
other with an 8-9 fold reduced binding affinity as a
weak binder. UV-mediated peptide exchange assays
were conducted to verify the substantial difference of
the MHC-p affinity prediction results. Peptide specificity of both TCR was assessed by an alanine scan
resulting in recognition patterns specific only for the
human MPO protein. In case of the TCR recognizing the high avidity peptide no allo-HLA reactivity
among 53 different HLA alleles tested could be observed while the second TCR peptide independently
recognizes a single HLA-B allele. No recognition by
Generation of chimeric antigen receptor - modified memory stem
cell CD8+ T lymphocytes from naive precursors by modulation of
Wnt/ß-catenin pathway or inhibition of Akt-signaling
Berger A.1, Khan S.1, Chmielewski M.2, Abken H.2, Theobald M.1, Hartwig U.F.1
University Medical Center of Johannes Gutenberg-University Mainz, III. Dept. of Medicine - Hematology,
1
Internal Oncology & Pneumology, Mainz, Germany,
University of Cologne, Dept. of Internal Medicine I & Center for Molecular Medicine, Cologne, Germany
2
Adoptive cellular therapy (ACT) of T cell receptor
B-ALL (NALM16) together with TWS119 or Akt in-
(TCR)- or chimeric antigen receptor (CAR)-repro-
hibitor VIII. CD19 CAR expression was determined
grammed T cells has advanced as a personalized
by flow cytometry, and both ELISPOT and cytotoxic-
and effective immunotherapy for leukemia and solid
ity assays were used in functional analyses.
tumors. However, ACT, especially to solid tumors is
Upon repetitive restimulation and TWS119/Akt in-
often hampered by limited T cell engraftment and
hibitor VIII treatment we obtained strong expansion
limited capability of terminally differentiated, high
of T cells with a TSCM/CM phenotype. In contrast, this
avidity effector T cells (TEFF) to establish sustained
effect was less pronounced by naive T cells cultured
antitumor immunity. Recently, long-living stem cell
in the sole presence of interleukin (IL)-2, IL-7, IL-12,
memory T cells (TSCM) with an enhanced capacity
IL-15, and IL-21, confirming that both Wnt/ß-caten-
for self-renewal and plasticity to differentiate into ef-
in and PI3K-Akt-mTOR pathways play a key role in
fectors could be shown to elicit potent antitumor re-
T cell differentiation. In addition, CD8+CD45RA+C-
sponses, prolonged survival and memory. Moreover,
D45RO-CD95+CD62L+CCR7+ TSCM showed high ex-
modulating the Wnt/ß-catenin or PI3K-Akt-mTOR
pression levels of CD19 CAR, elicited strong IFN-γ
signaling pathways in T cells using inhibitors of gly-
release and cytolytic activity to CD19+ NALM-16
cogen-sythase-kinase-3β (TWS119) or Akt (inhibitor
cells. This effect was also seen in CD19 CAR positive
VIII) have emerged as promising approaches to block
total CD8+ T cells although less pronounced.
+
CD8 effector T cell differentiation and to facilitate
Studies to evaluate the therapeutic efficacy of CD19-
the in vitro generation of TSCM and TCM. In the present
CAR redirected TSCM/CM following adoptive trans-
proof of concept study, we therefore investigated the
fer into NALM-16 B-ALL xenografted NSG mice are
+
generation of CD19 CAR expressing CD8 TSCM from
in progress and will be reported.
naive CD8+ T lymphocytes by modulating T-cell dif-
In conclusion, these studies demonstrate that redi-
ferentiation using TWS119 or Akt inhibitor VIII to be
rection of TSCM by retroviral transfer of optimized
used for ACT.
leukemia- or tumor-reactive TCRs or CARs may be a
+
+
Naive CD8 CD45RA T cells isolated from PBMC by
MACS® were polyclonally stimulated with CD3/CD28
Dynabeads in the presence of various cytokines and
retrovirally transduced with a second generation
CD19 CAR three days after activation. To promote
a TSCM/CM phenotype transduced cells were either
polyclonally restimulated or co-cultured with CD19+
promising approach to improve ACT.
Development of imaging strategies for investigation of TCR
with defined antitumor reactivity in vivo
Bianchi H.1, Mall S.1, Beziere N.2, Klemm U.2, Peschel C.1, Ntziachristos V.2, Krackhardt A.M.1
Klinikum rechts der Isar, Technische Universität München, München, Germany,
1
Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Neuherberg, Germany
2
T-cell based immunotherapies are novel and prom-
with respect to the limit of detection by MSOT in
ising therapeutic approaches for a variety of ma-
agarose phantoms and in vivo. For the in vivo analy-
lignant diseases. However, diverse approaches
sis, T cells mixed with matrigel were subcutaneously
including those using T-cell receptor (TCR)- and chi-
injected in the back of a mouse. T cells labeled with
meric antigen receptor (CAR)-transgenic T cells show
DiR presented the most sensitive detection by MSOT
highly different characteristics in vitro and in vivo.
both in phantoms and in vivo, providing a detection
Preclinical in vivo models providing high predictive
sensitivity of up to 2,5x104 cells. However, in case
value with respect to tumor reactivity and toxicity or
of DiR-labeled T cells simultaneously expressing
treatment failure due to tumor evasion are currently
iRFP720, the detection of the DiR signal by MSOT was
missing. For surveillance of therapeutic efficacy of
highly impaired and the sensitivity of the method de-
adoptive T cell transfer, nuclear imaging has been
creased around 10 times. For T cells expressing iRFP
used as a non-invasive and sensitive cell tracking
alone, up to 2,5x106 cells could be detected in vivo by
technology, although limits in spatial resolution are
MSOT. A xenogenic mouse model of myeloid sarcoma
given. We aimed to develop optoacoustic imaging
was used and human central memory T cells (TCM)
as an alternative non-invasive and novel method to
transgenic for the leukemia-specific TCR2.5D6 and
track TCR-transgenic T-cell responses in vivo. Multi-
subsequently labeled with DiR were adoptively
spectral optoacoustical imaging (MSOT) operates in
transferred. MSOT imaging was performed at differ-
the near-infrared (NIR) spectral region and allows
ent time points post TCM transfer in order to inves-
deep penetration in tissue with high resolution.
tigate TCM-distribution in vivo over time. Although
Cell dyes and reporter genes were tested as suitable
tumor rejection was observed, DiR-TCM signal could
tracers for detecting T cells with MSOT. T cells labeled
not be detected in the tumor by MSOT. However, TCM-
with DiR, a stable cell membrane dye, presented
infiltration of the tumor was confirmed by fluores-
bright fluorescence and strong absorption in the NIR
cence microscopy. The weak DiR fluorescence signal
spectrum. As an alternative labeling method, T cell
may be caused by proliferation of the TCR-transgenic
were stably transduced with variations of the re-
TCM within the tumor leading to dilution of the fluo-
porter gene near-infrared fluorescent protein (iRFP),
rescent dye. Thus, novel more sensitive tracers need
in which the variation iRFP720 showed a higher
to be developed in order to use MSOT for a more
brightness and a detectable signal by MSOT due to
sensitive T-cell tracking.
its higher emission in the near-infrared spectrum.
T cells labeled with DiR, T cells expressing iRFP720
and T cells harboring both tracers were compared
Functional evaluation of tumor antigen specific T-cells generated
from TCR transduced human hematopoietic stem cells
Bonte S.1, Snauwaert S.2, Stauss H.3, Heemskerk M.H.M.4, Vandekerckhove B.1, Kerre T.2
Ghent University, Ghent, Belgium,
1
Ghent University Hospital, Ghent, Belgium,
2
University College London, London, United Kingdom,
3
Leiden University Medical Center, Leiden, Netherlands
4
Chemotherapy leads to cure of acute myeloid leuke-
By using a polymorphic target tumor antigen, such
mia (AML) in less than half of the patients. Stem
as minor histocompatibility antigens (MiHA), we
cell transplantation (alloSCT) can be used as an im-
hope to increase the affinity of the in vitro gener-
munotherapeutic treatment to cure the patient, but it
ated tumor antigen-specific T-cells. The polymorphic
carries a high risk of toxicities and mortality, espe-
nature of these MiHA results in a TCR with high af-
cially in older patients with comorbidities. Moreover,
finity, in case of a MiHA mismatch. Donor lympho-
not all patients have a suitable donor.
cyte infusion (DLI), sometimes given after relapse,
Therefore, we have developed a novel immunothera-
show that T-cells recognizing MiHA are responsible
peutic strategy in which we generate T-cells in vitro,
for graft-versus-leukemia (GVL), but also for graft-
starting from TCR transduced hematopoietic precursor
verus-host disease (GVHD). By using T-cells that ex-
cells (HPC) from cord blood or mobilized peripheral
clusively recognize hematopoietic-restricted MiHA,
blood, by culturing the HPC on OP9-DL1, in the pres-
one could separate GVL from GVHD.
ence of SCF, FLT3L and IL-7. This novel immunothera-
We are now using the in vitro OP9-DL1 co-culture
peutic strategy has several advantages over the classi-
system to generate, starting from HPC, T-cells with
cal immunotherapy protocol in which TCR-transduced
a single TCR recognizing HA-2, a hematopoietic-re-
peripheral blood lymphocytes (PBL) are used: a higher
stricted MiHA.
specificity because of the presence of only 1 TCR (com-
In this study we compared the in vitro functional-
pared to an endogenous and a transduced TCR in TCR-
ity of HA-2-specific T-cells and WT1-specific T-cells.
transduced PBL, which could give rise to mispairing of
In an in vitro chromium release assay, HA-2-specific
both TCR α and β chains, and could therefore lead to
T cells showed a higher affinity compared to WT1-
lower affinity and unwanted, possibly hazardous spe-
specific T-cells. We are also setting up a preclinical in
cificities) and longer in vivo persistence because of the
vivo mouse model to evaluate the in vivo functional-
naïve phenotype (compared to the end-stage mature
ity and specificity of in vitro generated tumor anti-
T-cell phenotype in TCR-transduced PBL).
gen-specific T-cells. For this, NSG mice are injected
Using this strategy, with the WT1-TCR, we have gen-
with AML patient samples (which we have typed for
erated WT1-specific T-cells, targeting Wilms’ Tumor
WT1 and HA-2) and, a few weeks later, our in vitro
1 (WT1), a non-polymorphic tumor antigen that is
generated T-cells are injected. The preclinical mouse
overexpressed on 70% of the AMLs. These T-cells are
model is a necessary step before we can bring this
highly specific but have a low affinity due to the fact
novel targeted T-cell immunotherapy to the clinic.
that WT1 is also expressed on normal cells, albeit at
low levels.
Targeting HCMV-infected fibroblasts with bi-specific CAR-T cells
Brey C.1, Proff J.1, Full F.2, Ensser A.2, Holter W.1, Lehner M.1
Children´s Cancer Research Institute, Vienna, Austria,
1
Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
2
We investigate the possibility of an HLA-independent
inhibit HCMV-infection by secretion of IFN-γ and
T cell therapy of infections with human cytomegalo-
TNF, and that designing bi-specific CARs containing
virus (HCMV) and developed a chimeric antigen re-
mutated Fc spacers might be attractive for enhancing
ceptor (CAR) for targeting HCMV glycoprotein B (gB).
CAR function.
This CAR contains an IgG1-Fc spacer domain, which
is known to interact with Fc receptors (FcRs) and to
abrogate persistency and efficacy of CAR-T cells in
preclinical tumour models. We speculate, however,
that in our context this Fc-domain could be beneficial
by enabling additional targeting of HCMV-encoded
FcRs.
When we investigated fibroblasts three days after
infection with HCMV we found strong expression of
gB on the cell surface and high capacity for binding
of IgG1, indicating expression of HCMV-FcRs. T cells
modified with the gB-specific CAR were strongly
activated by these HCMV-infected target cells and
efficiently inhibited further HCMV-infection by secretion of IFN-γ and TNF. By performing blocking
experiments we could demonstrate that the HCMV
encoded FcRs enhanced the activation of the CAR-T
cells and, hence, the secretion of the inhibitory cytokines. In order to exploit this fact therapeutically, we now ask whether we can specifically target
HCMV-FcRs separately from endogenous human
FcRs. Such specific targeting might be accomplished
by using mutated Fc variants, since HCMV-FcR and
human extracellular FcRs have different binding
sites within the Fc domain. This possibility is investigated in current experiments.
In summary our data show that CAR-T cells can
RNAi-mediated silencing of endogenous TCR enhances tumor
killing activity of TCR-engineered WT1 peptide-specific CD8+
T cells
Campillo-Davo D.1, Fujiki F.2, Van den Bergh J.M.J.1, Smits E.L.1,3,4, Berneman Z.N.1,4,5, Sugiyama H.2,
Van Tendeloo V.F.I.1
University of Antwerp, Laboratory of Experimental Hematology, Edegem, Belgium,
1
Osaka University Graduate School of Medicine, Department of Functional Diagnostic Science, Osaka, Japan,
2
University of Antwerp, Center for Oncological Research (CORE), Edegem, Belgium,
3
Antwerp University Hospital, Center for Cell Therapy & Regenerative Medicine, Edegem, Belgium,
4
Antwerp University Hospital, Department of Hematology, Edegem, Belgium
5
The major bottleneck with standard cancer therapies
phocytes. WT1 peptide-specific TCR expression was
is treatment failure leading to progressive disease or
evaluated by HLA-A2/WT1 tetramer analysis. TCR
clinical relapse. The specificity of T cells for their
functionality was analyzed by expression of surface
cognate antigen turns them into an attractive and
activation markers on CD8+ T cells, cytokine release
targeted cancer therapeutic. However, the scarcity
and flow cytometry-based cytotoxicity assay. Here,
of tumor-reactive T cells and the difficulty of their
we show that electroporation of CD8+ T cells with
isolation in sufficient numbers for adoptive immu-
WT1 TCR mRNA leads to transient expression of the
notherapy have impeded to broaden their clinical
TCR. Furthermore, WT1 TCR mRNA-electroporated
application. Gene transfer of a T cell receptor (TCR)
CD8+ T cells can effectively recognize and kill WT1
specific for a tumor-associated antigen into T cells
epitope-bearing tumor cells in an HLA-A*0201-re-
would confer redirected anti-tumor specificity to ef-
stricted fashion. In addition, we show a marked en-
fector T cells for adoptive immune therapy. Here, we
hancement of WT1 peptide-specific TCR expression
sought to isolate and in vitro validate novel WT1 pep-
when combining electroporation of WT1 TCR mRNA
tide-specific TCRs derived from leukemia patients
and siRNAs against endogenous TCR expression. Im-
who demonstrated clinical benefit after receiving a
portantly, this enhanced WT1 peptide-specific TCR
WT1-targeted DC vaccine. We cloned a patient-de-
expression was correlated with a significant increase
rived HLA-A*0201-restricted WT1 peptide-specific
in CD8+ T cell WT1 peptide-specific killing activity,
TCR and validated its expression and function using
expression of CD69 and CD137 activation markers
a TCR-deficient Jurkat J76.7 cell line stably trans-
and cytokine production upon co-culture with WT1
duced with CD8 and an NFAT-driven GFP reporter
epitope-bearing target cells. In conclusion, tumor an-
gene. High-level transgenic TCR expression on Jurkat
tigen-specific killing capacity and T cell activation
J76.7 cells was detected by GFP expression upon TCR
was notably improved when using a novel double
signaling. In order to suppress translation of endog-
RNA electroporation approach based on the combi-
enous TCR mRNA and mispairing of endogenous
nation of codon-optimized WT1-specific TCR mRNA
and transgenic TCR α- and TCR β-chains, siRNAs
and siRNAs that suppress wild type TCR sequences.
targeting TCR constant regions were produced.
These results could pave the way for developing a
Next, we designed a codon-optimized siRNA-resis-
clinically safer strategy for T cell-based adoptive im-
tant TCR construct from the wild type sequence of
munotherapy of patients with WT1-expressing ma-
WT1 peptide-specific TCR. Further, we optimized a
lignancies.
protocol combining TCR siRNA and TCR mRNA electroporations in resting peripheral blood CD8+ lym-
Enhancing Cytokine-Induced Killer cell activity with Her2-specific
Fc-engineered antibodies and antibody derivatives
Cappuzzello E.1, Kellner C.2, Rosato A.1, Peipp M.2
University of Padova, Department of Surgery, Oncology and Gastroenterology, Padova, Italy,
1
Christian-Albrechts-University Kiel, Division of Stem Cell Transplantation and Immunotherapy, Kiel, Germany
2
Purpose: Cytokine-Induced Killer (CIK) cells are an
on target cells specifically redirect CIK cell function
attractive approach for cellular immunotherapy, as
against a specific target, avoiding unwanted non-
they are capable of recognizing tumor cells without
specific activation.
the need of antigen-specific priming and can be ef-
Conclusions: These data lead us to envisage new
ficiently and rapidly expanded in vitro. In this study,
perspectives for adoptive immunotherapy where
we aimed at increasing CIK cell antitumor activity
antigen-specific retargeting of immune cells can be
using Fc-engineered trastuzumab, bispecific anti-
achieved by the combination of non antigen-specific
bodies or recombinant immunoligands, which are
effector cells and tumor-specific antibodies, thus
able to target both a tumor-associated antigen (Her2)
confirming CIK cell as a promising tool for immuno-
and activating receptors expressed by CIK cells (CD3,
therapy approaches.
NKG2D and NKp30).
Methods: Antibody derivatives were produced in a
mammalian expression system and purified by affinity chromatography. CIK cell cytotoxic activity was
assessed against ovarian tumor cells either alone or
in combination with trastuzumab, Fc-engineered
formats of trastuzumab (glyco- and protein-engineered variants), Her2xCD3 bispecific antibody or
recombinant immunoligands.
Results: The presence of the engineered antibodies
significantly enhanced CIK cell activity, inducing a
higher target cell lysis as compared to the wild type
antibody. The engagement of CD3 with a Her-2-targeting bispecific antibody produced an outstanding
enhancement of killing, which resulted in a higher
extent of lysis than that achieved with trastuzumab.
Discussion: The results reported in this work open
additional opportunities to further improve CIK cell
antitumor activity. Importantly, when using bispecific antibodies the concomitant engagement of both a
triggering molecule on CIK cells and a tumor antigen
Potential immunogenicity of PUVA-induced cell death
Coppard C.1, Hannani D.2, Gabert F.1, Plumas J.1, Chaperot L.1
EFS;INSERM-U1209;Université Grenoble-Alpes, Immunobiology and Immunotherapy in Chronic Diseases, La Tronche,
1
France,
PDC line Pharma, La Tronche, France
2
Extracorporeal photopheresis (ECP) is a cellular im-
PUVA induces the up-regulation of Calreticulin at
munotherapy based on the infusion of autologous
the surface of treated cells. The ecto-Calreticulin ex-
peripheral blood mononuclear cells treated ex-vivo
pression is associated with the release of HMGB1 but
by a photosensitizing agent (8-Methoxy-psoralen,
not ATP. Of note, PUVA-treated activated alloreactive
8-MOP) and UVA (Hannani, Transplantation 2010
T cells emit higher levels of DAMPS than resting T
a,b); a process leading to cell apoptosis. This treat-
cells. Interestingly, monocyte-derived dendritic cells
ment can cure T cell lymphomas, graft versus host
(Mo-DCs) were found to preferentially engulf these
disease, and auto-immune diseases. Although ECP is
apoptotic activated alloreactive T cells. Moreover,
routinely used in many clinical centers worldwide, its
Mo-DCs maturate when in contact with activated
mechanism of action (MoA) is still largely unknown.
alloreactive T cells, regardless the PUVA-treatment,
Two different hypotheses have been proposed so far.
suggesting that PUVA induced apoptosis could be an
Indeed, ECP is thought to promote the immunity
immunogenic process.
of specific anti-T cell responses in lymphoma, or to
Experiments analyzing the polarization of naive T
promote regulatory T cells development in auto- or
cells activated by these Mo-DC will be performed to
allo-immune disorders. In order to get further in-
go on investigating PUVA-induced cell death immu-
sights in the understanding of ECP MoA, we charac-
nogenicity. Moreover, we have set up a mouse model
terized in vitro the features of PUVA induced apopto-
in which ECP show clinical efficacy, which will allow
sis (i.e immunogenic or tolerogenic). Apoptotic cells
us to go further deciphering the ICMP mechanisms
are usually tolerogenic, rapidly and silently cleared
of action.
by scavenger cells, however, in particular circumstances, apoptotic cells can emit/release DAMPS
(Dammage-Associated Molecular Patterns) such as
Calreticulin, HMGB-1 and ATP rendering them immunogenic (Hannani, Cancer J, 2011).
PUVA induced apoptosis has been studied by using
activated alloreactive T cells generated by a mixedlymphocyte reaction, mimicking those involved in
GVHD. We have previously shown that activated
alloreactive T cells massively undergo apoptosis,
faster than resting T cells post PUVA (Hannani,
Transplantation 2010b). Our results show now that
Seprehvir, an oncolytic herpes immunotherapeutic, enhances
GD2-directed Chimeric Antigen Receptor (CAR) T-cell therapy in
GD2-expressing solid tumor xenografts
Haworth K.1, Haile S.2, Mackall C.2, Conner J.3, Cripe T.1
Nationwide Children’s Hospital, Ohio State University, Columbus, United States,
1
Stanford University, Palo Alto, United States,
2
Virttu Biologics, Glasgow, United Kingdom
3
While chimeric antigen receptor (CAR) T-cell thera-
models with Seprehvir induces an immune response,
pies have shown remarkable anticancer efficacy in
which includes the T-cell attractant chemokines
patients with relapsed and refractory lymphoid leu-
CXCL-10 (IP-10) and CCL-5 (RANTES) and T-cell ac-
kemias, their effectiveness in patients with solid
tivating cytokines such as IFN-g and TNF-a, while
tumors has been more challenging. Among the bar-
down-regulating such inhibitory cytokines as TGF-b.
riers thought to interfere with CAR T cell efficacy are
Flow cytometry analysis revealed variable tumoral
impaired homing to tumors and poor CAR T cell per-
GD2 surface expression on each of these models,
sistence, likely attributable to the immunosuppres-
while the CAR T-cells displayed high CXCR-3 and
sive microenvironment. Due to their proinflamma-
CCR-5 surface expression, allowing for chemotactic
tory effects, oncolytic viruses are strong candidates
signaling through CXCL-10 and CCL-5, respective-
to potentiate the competence of CAR T cells within
ly. The CAR T-cells displayed increased migration
solid tumors. Seprehvir (HSV1716) is an HSV-1 at-
toward oHSV-infected tumor cells over non-infected
tenuated by deletion of the RL1 gene encoding the
cells. Mice treated with combination therapy had
neurovirulence protein ICP34.5. The virus has a long
significantly delayed tumor growth and prolonged
track record of safety in clinical trials and is current-
survival when compared to CAR treatment alone.
ly being tested in adolescents and young adults with
Despite being athymic nude mice, the majority of
refractory solid tumors (see www.clinicaltrials.gov:
mice cured by combination therapy were resistant to
NCT00931931, NCT02031965). We hypothesized that
tumor rechallenge, suggesting the long-term persis-
intratumoral administration of Seprehvir enhances
tence of CAR T cells. These results indicate that the
GD2-directed CAR T cell efficacy. We characterized
addition of Seprehvir may be a valuable adjunct to
the chemokine and cytokine profiles of human GD2-
CAR T-cell therapy and should be further explored
positive Ewing sarcoma and neuroblastoma cell lines
in clinical trials.
before and after oHSV inoculation. We performed
transwell migration assays of third-generation (containing CD28, OX40, and CD3z signaling domains)
GD2-directed human CAR T-cells before and after
the addition of Seprehvir in these models in vitro.
We then performed in vivo survival studies using
athymic nude mice and cyclophosphamide (CPM)
lymphodepletion prior to CAR therapy. Our results
suggest that infection of these pediatric solid tumor
MET-specific CARs for cell therapy of patients with GBM
Chaitanya K.1, Walker P.R.1, Dietrich P.-Y.2, Dutoit V.1
University of Geneva, Geneva, Switzerland,
1
Geneva University Hospital, Geneva, Switzerland
2
Glioblastoma (GBM) is the deadliest form of primary
IFN-γ and IL-2 after incubation with recombinant
brain tumor with a median survival time of only
c-MET but not with irrelevant prostate specific mem-
15 months. Although conventional therapies have
brane antigen (PSMA) protein, with variable effica-
evolved, they only modestly improve survival, making
cies depending on the hinge lengths. Furthermore,
novel therapeutic strategies an urgent need. Here,
c-MET CARs recognized the c-MET-expressing U251
we aim at generating GBM-specific chimeric antigen
GBM cell line, as determined by secretion of TNF-α,
receptor (CAR) T cells targeting the c-MET protein,
IFN-γ and IL-2. Further functional characterization is
which is expressed at the surface of many malignant
undergoing, including tumor cell killing, which will
cells, including GBM. We isolated c-MET-specific an-
enable us to choose the optimal CAR construct for
tibody variable heavy (V H) and light (V L) chains from
pre-clinical testing in an in vivo xenografted glioma
a commercially available hybridoma using degener-
mouse model.
ate primers and inserted a (Gly4-Ser)3 liker between
the two to obtain the c-MET-specific single chain
variable fragment (scFv). We additionally introduced
mutations in the framework regions of the scFv in
order to augment affinity to the antigen. Then, we
generated different CAR constructs with the aim to
test several hinge lengths (one CD8α hinge and 3 different IgG4 hinges), several costimulatory molecules
(41BB or CD28 cytoplasmic domain) and presence or
absence of suicide genes (iCaspase9 or RQR8) and
IL15/IL-12 cytokines. The c-MET scFv was inserted
in the above mentioned CAR backbones which were
cloned into lentivector harboring the murine stem
cell virus (MSCV) promoter for expression. Lentiviral packaging and production was performed in
HEK293T cells and the resulting viruses were used to
transduce CD3/28 bead-activated T cells, with more
than 85% transduction efficiencies, confirmed by
GFP and surface scFv expression. These c-MET-specific CARs were specifically able to secrete TNF-α,
Development of novel chimeric antigen receptors (CAR) to treat
B-cell malignancies
Fåne A.1, Inderberg E.M.1, Huse K.2,3, Oksvold M.2,3, Myhre M.1, Skorstad G.1, Løset G.Å.4, Smeland E.2,3,
Funderud S.2, Holte H.5, Kvalheim G.1, Myklebust J.H.2,3, Wälchli S.1,2,3
Oslo University Hospital, Department of Cellular Therapy, Oslo, Norway,
1
Oslo University Hospital, Department of Cancer Immunology, Oslo, Norway,
2
Oslo University Hospital, Centre for Cancer Biomedicine, Oslo, Norway,
3
University of Oslo, Center for Immune Regulation, Oslo, Norway,
4
Oslo University Hospital, Department of Oncology, Oslo, Norway
5
Adoptive T-cell therapy using chimeric antigen re-
third generation format) was performed, including
ceptor (CAR) has given impressive clinical results
comparison to a clinical anti-CD19 CAR (FMC63),
in hard to cure haematological cancers. CAR-mod-
with promising results. CD19 and CD37 are expressed
ified T cells targeting the CD19 antigen have shown
on a similar spectrum of malignancies, and our find-
cure rates of 90% in acute lymphatic leukemia. The
ings suggest that CD37-redirected CAR T cells could
identification of new targets on B cells represents a
be used as an alternative to or in combination with
novel strategy for therapy of B-cell malignancies. We
CD19-directed therapies.
have a large collection of hybridomas, each producing monoclonal antibodies, and an antibody phage
library which could be cloned into a CAR scaffold.
Some of these antibodies have already been used in
clinical trials, but never tested as CARs. We therefore
aim at designing CARs targeted against unexploited
B-cell antigens. Identification and validation of new
targets will lead to the development of novel CARs
for the treatment of additional haematological malignancies. CD37 is highly expressed on malignant B
cells in non-Hodgkin lymphoma (NHL) and chronic
lymphocytic leukemia (CLL). It is also expressed in
hairy cell leukemia and lymphoplasmacytic lymphoma. We have developed a novel CAR targeting the
CD37 antigen, which represents a promising therapeutic target for B-cell malignancies. In vitro validation of our anti-CD19 and anti-CD37 CARs (in the
Constructing artificial antigen-presenting cells for improved
T-cell function in adoptive T-cell therapy of melanoma
Friese C.1, Donia M.1,2, thor Straten P.1, Svane I.M.1,2, Met Ö.1,2
Center for Cancer Immune Therapy, Herlev, Denmark,
1
Herlev Hospital, Department of Oncology, Herlev, Denmark
2
Adoptive T-cell therapy (ACT) is a cancer immuno-
The aAPCs currently being established at CCIT are
therapy for metastatic melanoma patients based on
genetically modified with various T-cell co-stimula-
autologous tumor-infiltrating lymphocytes (TILs).
tory molecules and Fc receptors for antibody loading.
TIL therapy takes advantage of naturally existing
Preliminary data testing aAPCs in REPs has shown
tumor-reactive T cells already present within the
clinical grade expansion of tumor-reactive TILs from
tumor which are isolated from surgically resected
patients with metastatic melanoma. The results also
tumor lesions, expanded ex vivo and re-infused into
indicate a higher frequency of CD8+ T cells versus
the patient after lymphodepleting chemotherapy and
CD4+ T cells in the aAPC-expanded TILs in compari-
in combination with recombinant IL-2. With this per-
son to PBMC-supported TIL expansion.
sonalized therapy objective response rates of up to
At present the aAPCs are developed further and opti-
50% including complete tumor regression in 10-20%
mized and their feasibility in expanding TILs for ACT
of the patients have been reported from several inde-
of melanoma as well as renal cancer, ovarian cancer
pendent centers.
and sarcoma is tested in ongoing experiments.
Despite the great potential TIL therapy has shown
in the treatment of metastatic melanoma, some confounding issues are still to be addressed prior to entry
into the standard of care for melanoma patients. An
important area requiring improvement is the technical protocols for expansion of TILs for therapy. At
present, a large number of peripheral blood mononuclear cells (PBMCs) derived from different blood
donors is required to be used as feeders/stimulators
for the rapid-expansion protocol (REP).
Genetically engineered artificial antigen-presenting
cells (aAPCs) that express any desired T-cell activating or co-stimulatory molecule on the cell surface
have the potential to eliminate the need to use PBMCs
from multiple donors and could lead to improved effector-memory qualities with a longer persistence of
TILs in the patients.
Insights into the preventive/preemptive adoptive transfer of
CMV- and EBV-specific peptide-stimulated T cells after allogeneic
stem cell transplantation as part of the phase I/IIa clinical trial
MULTIVIR-01
Gary R.1, Aigner M.1, Moosmann A.2, Ritter J.3, Seitz V.3, Moi S.1, Schaffer S.1, Balzer H.1, Maas S.4, Strobel J.5,
Zimmermann R.5, Zingsem J.5, Gottmann A.1, Kremer A.1, Hennig S.6, Hummel M.3, Mackensen A.1, Gerbitz A.1,7
University Hospital Erlangen, Department of Internal Medicine 5, Erlangen, Germany,
1
Helmholtz Zentrum München, DZIF Research Group Host Control of Viral Latency and Reactivation, Munich,
2
Germany,
Charité Berlin, Institute of Pathology, Berlin, Germany,
3
University Hospital Erlangen, Center for Clinical Studies (CCS), Erlangen, Germany,
4
University Hospital Erlangen, Department of Transfusion Medicine and Hemostaseology, Erlangen, Germany,
5
HS Diagnomics GmbH, Berlin, Germany,
6
Charité University Hospital Berlin, Department of Hematology, Oncology and Tumorimmunology, Berlin,
7
Germany
Reactivation of CMV and EBV negatively impacts on
leukapheresis of the donor, CMV- and EBV-specific
outcome after allogeneic stem cell transplantation
T cells are preferentially expanded from a small
(aSCT). Specific antiviral therapy is only available
fraction of the stem cell graft. A strong expansion
for CMV. With the exception of ganciclovir all drugs
of virus-specific T cells could be observed for the
are being used off-label. 40-50% of patients reacti-
first products analyzed by flow cytometry with HLA
vate CMV following aSCT. For the 20-30% of patients
class I multimers. Reconstitution and cell counts of
reactivating EBV, only the use of rituximab is avail-
leukocytes after aSCT are monitored for both treat-
able to control EBV. Rituximab leads to long term
ment and control group. To obtain further insights
B-cell depletion requiring frequent administration
into the expansion of transferred T cells, the TCR
of immunoglobulins. To cover the unmet medical
beta (TCRb) repertoire of the T-cell product before
need of CMV- and EBV-control after aSCT, we in-
and after adoptive transfer in the patient is monitored
vestigate a cell therapy approach by means of CMV-
by high throughput sequencing. Specificities of TCRb
and EBV-specific peptide-stimulated T cells. We set
sequences can be assigned by determining the reper-
up a prospective randomized controlled phase I/IIa
toire of HLA/peptide-multimer-sorted CD8+ T cells.
multi-center clinical trial to evaluate the preventive
New virus-specific TCRb sequences can be identi-
and preemptive adoptive transfer of this ATMP in pa-
fied thereby. Furthermore, TCR sequences within the
tients after aSCT (EudraCT number 2012-004240-30).
T-cell product can be tracked in the patient. Taken
The multi-center trial is currently recruiting.
together, our first observations demonstrate feasibil-
For manufacturing of the cell product two peptide
ity of our approach under clinical trial conditions.
pools (CMV and EBV) each covering 17 well-defined
HLA class I and class II epitopes for stimulation of
donor derived PBMC are used. PBMC collected by
leukapheresis of mobilized or non-mobilized donors
can be used as starting material. To avoid a second
Adoptive transfer of autologous T cells modified with a MART-1
specific TCR in advanced stage melanoma patients
Gomez-Eerland R.1, van den Berg J.1, van Zon M.1, Bakker N.1, de Boer R.1, Nuijen B.2, Schumacher T.1, Haanen J.3
NKI-AVL, Immunology, Amsterdam, Netherlands,
1
NKI-AVL, Pharmacy, Amsterdam, Netherlands,
2
AVL-NKI, Immunology/Medical Oncology, Amsterdam, Netherlands
3
ORAL
TALK
SHORT
2016
At the NKI-AVL, a TCR gene therapy trial to treat
granulocytopenia and thrombocytopenia approxi-
stage IV melanoma patients is currently recruiting
mately 1.5 month after infusion, of which one patient
patients. The TCR used in this protocol is specific for
is now fully recovered.
the HLA-A*0201 restricted MART-126-35 epitope, which
In addition to the observed toxicity, the CT scan one
is expressed on the majority of melanoma cells.
month post-infusion demonstrated a partial clini-
Unique to this trial is the use of the combination of
cal response in one patient , which is still ongoing 6
anti-CD3/CD28 beads for T cell activation plus IL-7/
months post infusion (50 % tumor reduction of target
IL-15 for subsequent culture and expansion, instead
lesions). This was accompanied by high frequencies
of the more commonly used strategy that utilizes the
of gene-modified T cells (59% of CD3+ cells) in the
combination of anti-CD3 antibody and IL-2. The aim
circulation at one month post-infusion, which were
of this altered production strategy is to generate a
still measurable 6 month post-infusion (8 % of the
“less differentiated“ T cell product that may have a
CD3+ cells).
better engraftment potential and anti-tumor activity.
The second patient in this cohort has a stable disease
Thus far, 6 patients have been treated with in total 3
2 month after infusion with ~3 % CD3+ gene-modi-
different cell doses. Gene modified T cells could be
fied T cells measurable in the blood.
found back in the circulation for up to two months
On the basis of the available data, we conclude that
post infusion even when as little as 5x107 trans-
TCR-modified T cells created by this method can
duced T cells were infused. On-target toxicity against
have a very high engraftment potential and show in
MART-1 expressing melanocytes in the skin, leading
vivo activity at low doses, thereby suggesting the po-
to vitiligo, was observed in all three patients treated
tential value of this strategy in TCR gene therapy that
at this low dose.
target other tumor-associated antigens.
In the two patients treated with the next dose level
(25x107 transduced T cells), severe toxicity was seen,
including hypotension, fever, edema, oliguria, severe
skin rash,uveitis and hearing loss, accompanied with
high levels of IL-6 in the circulation. The patients
were successfully treated with anti-IL-6R antibody,
but required additional steroid therapy for the on-target toxicity (skin, eyes and inner ears). All toxicities
were reversible, except for hearing loss in one ear of
one patient. Moreover both patients also developed
Characterization of the avidity of TCR-engineered T cells with
novel and established approaches
Hillerdal V.1, Boura V.2, Björkelund H.2,3, Andersson K.2,3, Essand M.2
Uppsala University, IGP, Uppsala, Sweden,
1
Uppsala University, Uppsala, Sweden,
2
Ridgeview Instruments, Vänge, Sweden
3
The avidity of genetically-modified T cells used for
higher nanomolar peptide concentration range. That
cancer treatment is crucial for the successful outcome
intermediate activation of the TARP TCR - T cells
of the therapy. We have recently identified a T-cell
may be beneficial in the treatment setting as the cells
receptor that recognizes a peptide from the prostate-
may be less exhausted and resistant to over-activa-
specific antigen TARP. As TARP is specifically ex-
tion.
pressed in prostate and strongly over-expressed on
prostate cancer T cells targeting TARP could have
potential as a therapy for metastatic prostate cancer.
After evaluating the cytotoxic activity of the TARP
TCR it is important to further characterize its avidity
and functional avidity. We have compared TARPTCR transduced T cells to T cells transduced with
TCR recognizing an epitope from the pp65 cytomegalovirus protein, which was also developed by our
group.
We compared the binding of multimers to the
TCR-transduced cells and found that pp65 TCR- T
cells bound to a higher extent to multimers, but both
pp65 TCR- and TARP TCR- transuced T cells bound
multimers independently of the CD8 co-receptor. We
used a novel technology in collaboration with Ridgeview Instruments® to measure the binding of the
T cells to their target cells in real time. We were able
to detect binding of both pp65 TCR- and TARP TCR- T
cells to peptide-pulsed target cells. The results were
compared with classical functional avidity assays
such as IFN-γ ELISA and target killing assays. The
functional avidity of pp65 TCR- transduced T cells
was significantly higher that that of TARP TCR-transduced T cells. Nevertheless, TARP TCR- T cells were
able to produce adequate amounts of IFN-γ in the
Liposomes encapsulating zoledronic acid for cancer
immunotherapy and their effect on the in vivo biodistribution
of Vg9/Vd2 T cells in different tumour models
Hodgins N.1, Wang J.T.-W.1, Parente-Pereira A.2, Al-Jamal W.T.3, Maher J.2, Al-Jamal K.T.1
King’s College London, Institute of Pharmaceutical Sciences, London, United Kingdom,
1
King’s College London, Research Oncology, London, United Kingdom,
2
University of East Anglia, School of Pharmacy, Norwich, United Kingdom
3
Introduction: Zoledronic Acid (ZOL) is a nitro-
after pre-injection with free or liposomal ZOL was
gen-containing bisphosphonate that can inhibit
imaged by SPECT-CT followed by gamma counting.
farnesyl diphosphate synthase (1) and has been
Results and discussion: The Vγ9/Vδ2 T cells were
shown to sensitise tumour cells to destruction by
used in a co-culture model with cancer cell lines to
Vγ9/Vδ2 T cells due to phosphoantigen accumula-
determine the effect that ZOL has on the sensitisation
tion (2). ZOL is rapidly cleared from the circulation
of cancer cells for destruction by Vg9/Vd2 T cells.
and is accumulated by the bone thus limiting its in
It was shown that ZOL or L-ZOL at concentrations
vivo activity (3). Liposomal ZOL (L-ZOL) has been
of 10 µM. had no cytotoxic effects alone. However,
shown to increase the levels of ZOL at tumour sites
when Vγ9/Vδ2 cells were added to cells pre-treated
via the enhanced permeation and retention effect (4)
with ZOL or L-ZOL, a significant reduction in cell vi-
and has been proposed to be used in conjunction
ability to 6-32% and 38 - 91 %, respectively, was ob-
with Vγ9/Vδ2 T cells against a wide range of cancers.
served. In vivo,
Success of the therapy, however, may depend on
accumulation of 6% and 2-3% per gram tumour
several factors such as tumour type and location in
in murine and human solid tumours, respectively.
the body. The latter may influence accessibility of
The biodistribution of
tumour cell to destruction by the T-cells. The aim of
mice pre-treated with ZOL or L-ZOL was also exam-
111
In-labelled liposomes have shown
111
In-labelled Vγ9/Vδ2 cells in
this study is to quantify L-ZOL and Vγ9/Vδ2 T cell
ined. Independent of human tumour type, Vg9/Vd2
accumulation in a range of murine and human sub-
uptake in mice pre-injected with PBS or ZOL was
cutaneously implanted tumours.
1.5% per gram tumour. This increased to 2-2.5% in
Materials and methods: L-ZOL was prepared by thin
mice pre-injected with L-ZOL (p > 0.05 in A375Pβ6,
film hydration using DSPC as the principle lipid. γδ
PANC-1 and PANC0403 and < 0.05 in A375Ppuro).
T cells were isolated from whole blood and used in
Conclusion: The reported data suggests that Vγ9/Vδ2
a co-culture model with cancer cell lines (PANC-1,
T cell migration to solid tumours can be modulated
PANC0403, A375Ppuro and A375Pβ6) pre-treated
with L-ZOL pre-treatment but is tumour type depen-
with ZOL or L-ZOL. Liposomes were formulated
dent. SPECT/CT imaging can be a useful tool to assess
containing 1% DSPE-DTPA and were radiolabelled
suitability of this therapeutic modality to cancer pa-
with
111
In. Their biodistribution in tumour-bearing
SCID/Beige mice (PANC-1, PANC0403, A375Ppuro
and A375Pβ6) and BALB/c mice (CT26 and 4T1) was
imaged by SPECT-CT. 111In tropolone was then used to
radiolabel Vγ9/Vδ2 T cells and their biodistribution
tients and can be put in place as a pre-screening tool.
Adoptive immunotherapy with a little help from CD4 T cells
Wälchli S.1,2, Myhre M.R.1, Lislerud K.1, Mensali N.1, Fåne A.1, Groven A.3,4, Bakke O.3,4, Kvalheim G.1,
Gaudernack G.2, Inderberg E.M.1
Oslo University Hospital - The Norwegian Radium Hospital, Dept. of Cellular Therapy, Oslo, Norway,
1
Oslo University Hospital - The Norwegian Radium Hospital, Institute for Cancer Research, Section for
2
Immunology, Oslo, Norway,
University of Oslo, Dept. of Molecular Biosciences, Oslo, Norway,
3
University of Oslo, Centre for Immune Regulation, Faculty of Medicine, Oslo, Norway
4
T-cell based immunotherapy is an attractive treat-
identifying highly functional HLA class II restricted
ment for advanced cancer. Although the critical
TCRs for adoptive T-cell transfer.
role of CD4 T-cell help in tumour elimination is
The use of these TCRs may have strong therapeutic
well established, the focus of T-cell receptor (TCR)
potential not only in haematopoietic malignancies
transfer has largely exploited HLA class I-restricted
and in melanoma where tumour cells often express
molecules. The importance of CD4 T-cell responses
HLA class II but also in general through recognition
against tumour neoantigens, in addition to their role
of antigens cross-presented by tumour infiltrating
in priming and maintenance of CD8 T cell responses,
APCs.
has also recently been emphasized.
In addition, combining the redirection of T cells with
We have identified and cloned several HLA class
both HLA class I- and class II-restricted TCRs may
II-restricted CD4+ T cells isolated from patients who
further enhance the therapeutic effect in adoptive T
have clinically benefitted from vaccination with long
cell therapy.
peptides or dendritic cells targeting antigens such as
We are presently developing in vivo models to vali-
hTERT, survivin and frameshift mutated TGFβRII. In
date these TCRs and evaluate the impact of CD4 TCR
these patients strong T-cell responses against pep-
redirection in tumour eradication.
tides other than those used for vaccination were detected, suggesting epitope spreading. We validated
these HLA-DR and -DQ restricted T-cell clones and
showed their ability to recognise target cells loaded
with long antigenic peptides at low concentrations
and, if available, direct tumour cell recognition.
We further isolated the TCR coding sequences and
used mRNA electroporation for TCR redirection of
expanded T cells. Both CD8+ and CD4+ T cells expressing the TCRs produced TNF-α, IFN-γ and degranulated (CD107a+) following co-incubation with
peptide-loaded targets.
We have demonstrated that selecting highly functional CD4+ T-cell clones specific for tumour antigens from patients with clinical responses after immunotherapy treatment is a successful method for
In vitro stimulation conditions affect the immune phenotype of
both CD4+ and CD8+ T cells expressing a GD2-specific chimeric
antigen receptor
Ochs L.1, Altvater B.1, Kailayangiri S.1, Spurny C.1, Rossig C.1,2, Jamitzky S.1
University Children’s Hospital, Pediatric Hematology and Oncology, Münster, Germany,
1
Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), Münster, Germany
2
The success of chimeric antigen receptor (CAR) mod-
cultures, we observed a higher proportion of CD8+
ified T cells therapy depends on the use of optimal
TCM after DB stimulation with 19.6% (17.8-26.3%) of
T cell products which ensure a long lasting and ef-
TCM compared to 6.1% (3.7-6.9%) in 3/28 stimulated
ficient tumor control. T cell persistence was found
cultures (p = 0.005). Compared to non-transduced T
to be associated with a central memory immune
cells, CAR-expressing T cells in both types of cultures
phenotype at the time of infusion. In addition, the
had higher proportions of TCM cells which were again
expression of immune-inhibitory receptors on the
noticeably increased in DB compared to 3/28: 60.7%
surface of the effector T cells is an important obsta-
(53.0-69.7%) CD4+ TCM stimulated with DB vs. 33.6%
cle, since interactions with the respective inhibitory
CD4+ (29.9-49.3%) TCM (p=0.02), and 48.0% (34.4-
ligands expressed on tumor cells induce tolerance
65.2%) CD8+ TCM with DB vs. 27.7% (17.7-38.8%)
and exhaustion. T cell products in the clinical trials
CD8+ TCM with 3/28 stimulation (p< 0.01). Expres-
were stimulated under diverse conditions, leading to
sion of classical immune checkpoint receptors, ana-
substantial variations of the composition of the T cell
lyzed by flow cytometry with PD-1, CTLA-4, TIM-3,
product. Here, we compared two different stimulation
and LAG-3 specific antibodies, did not differ between
conditions with respect to the immune phenotypes
CAR transduced CD8+ T cells stimulated under the
of the T cell products: coated anti-CD3/CD28 anti-
two different conditions. In CAR transduced CD4+
bodies [3/28], and Dynabead stimulation of enriched
T cells, expression of TIM-3 was increased after DB
CD3+ T cells [DB]. Peripheral blood T cells from
stimulation with 21.2% (16.5-26.7%) of CD4+ T cells
three healthy donors were stimulated with either of
expressing the exhaustion marker, compared to 9.9%
the two methods, retrovirally transduced with the
(6.6-11.4%) in CD3/28 stimulated T cell cultures (p
GD2-specific CAR GD2-BBζ on day 2, and expanded
= 0.02).
in RPMI/AIMV medium with 50 IU/ml recombinant
In conclusion, we found that the stimulation condi-
human interleukin-2. T cell expansion rates and
tions have a strong impact on the T cell phenotypes
transduction efficiencies were comparable between
of the products, and that CD4+ and CD8+ subsets
the two stimulation conditions. We observed no dif-
are affected differentially. The optimal T cell culture
ferences in the CD4+/CD8+ ratio of all T cell prod-
conditions for a product with sustained persistence
ucts on day 14 after initial stimulation. In contrast,
in vivo will ultimately emerge from clinical trials.
we found noticeable differences in the proportions of
central memory T cells (TCM) between the two types
of cultures, defined by the expression of CD3, CD4,
CD8, CD45RO and CD197. In non-transduced T cell
Rapid recovery of innate immune cells after αβ T-cell depleted
allogeneic stem cell transplantation from matched related and
unrelated donors
Janssen A.1, de Witte M.2, Fleurke G.2, Timmerman L.1, Slaper I.3, Spierings E.1, Kuball J.1,2
University Medical Centre Utrecht, Laboratory of Translational Immunology, Utrecht, Netherlands,
1
University Medical Centre Utrecht, Hematology, Utrecht, Netherlands,
2
University Medical Centre Utrecht, Cell Therapy Facility, Utrecht, Netherlands
3
Introduction: Orchestrating the immune reconstitu-
Results: Primary engraftment (chimerism > 95%)
tion after hematological allogeneic stem cell trans-
was observed in all patients. Immune reconstitution
plantation (allo-SCT) is an attractive option to de-
started with recovery of the NK cells and γδ T-cells to
crease complications, like infections and graft versus
normal levels within the first month after allo-SCT.
host disease (GVHD) without losing the graft versus
As anticipated the adaptive immune system showed
leukemia effect. Novel transplantation strategies
a delayed reconstitution within the first 6 months.
such as depletion of αβ T-cells in the graft is pro-
With NGS of the TCRβ repertoire, after 100 days
posed to decrease the incidence of GVHD, whereas
already a surprising diversity of TCRβ repertoire was
the remaining innate cells such as NK cells and γδ
observed in different T cell subsets. The incidence of
T-cells may provide control of infected and trans-
GVHD > grade II within 100 days in this cohort was
formed cells. This approach has been pioneered in
0%. There was no increase of infections of CMV and
haplo-SCT. In our centre we have extended this strat-
EBV observed compared to the historical cohort. In
egy to matched related donors (MRD) and matched
the period of follow-up (1-12 months) 5 patients de-
unrelated donors (MUD).
ceased and 4 patients had a relapse. Overall survival,
Methods: Patients with hematological malignancies
relapse, and event free survival rates were non inferi-
who received an αβT-cell depleted allo-SCT of a MUD
or when compared to the historical cohort.
or MRD were analyzed. αβT-cell reduction was per-
Conclusion: αβT-cell depleted allo-SCT leads to
formed by negative selection with anti-αβTCR anti-
a rapid reconstitution of the innate immune cells,
bodies in combination with magnetic microbeads
followed by a subsequent recovery of the adap-
(Miltenyi Biotec, Germany). The maximal contami-
tive immune system. Reconstitution of diversity in
nation with αβT-cells was 5x10 /kg. The conditioning
the αβT-cell receptor repertoire varies in different
regimen consisted of: ATG, fludarabine and busilvex.
subsets of T cells as measured with NGS. The low
Immune suppression consisted of 28 days of myco-
incidence of severe GVHD and sufficient control of
phenolic acid. A cohort of 38 patients was analyzed
infections suggest that this transplantation strategy
for immune reconstitution and clinical outcome and
can serve as a platform for subsequent immunolog-
compared to an historical control cohort of recipients
ical interventions such as pre-emptive donor lym-
of a T cell replete allo-SCT. In addition in a subset of
phoid infusions. These preliminary results needs to
patients next generation sequencing (NGS) of the T
be confirmed in extended follow-up and in a planned
cell receptor β chain (TCRβ) was performed using the
multicenter study.
5
Illumina/MiSeq sequencing to determine the diversity
of the TCRβ in T cells after αβT-cell depleted allo-SCT.
Safe engineering of CAR T cells for adoptive cell therapy of
cancer using long-term episomal gene transfer
Jin C.1, Ramachandran M.1, Fotaki G.1, Nilsson B.1, Essand M.1, Yu D.1
Uppsala University, Uppsala, Sweden
1
Success in CAR T cell therapy of cancer is currently relying on long-term gene expression owing to
gamma retrovirus (RV) or lentivirus (LV) integration.
However, uncontrolled RV/LV integration in host cell
genomes has the potential risk of causing insertional
mutagenesis. Herein, we describe a novel episomal
and long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element for
either over-expression or down-regulation of genes.
The insertional events of this vector are below detection level. CD19 CAR T cells engineered with a
NILV-S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV
vector, even after numerous rounds of cell division.
NILV-S/MAR-engineered CD19 CAR T cells exhibited
similar cytotoxic capacity upon CD19+ target cells
recognition as LV-engineered T cells and are as effective in vivo. We propose that NILV-S/MAR vectors
are superior to current options for enabling long-term
gene expression without the risk of insertional mutagenesis and genotoxicity.
CD4+ T-helper-1 cells against the tumor antigens WT1, NYESO-1, ROR1, MAGE-A3 and Survivin for adoptive transfer to
treat cancer
Kayser S.1, Schleicher S.1, Boß C.2, Kyzirakos C.1,3, Stevanović S.4, Lang P.1, Röcken M.2, Handgretinger R.1,
Feuchtinger T.1,5
University Children´s Hospital Tübingen, Oncology and Hematology, Tübingen, Germany,
1
University Hospital Tübingen, Department of Dermatology, Tübingen, Germany,
2
Cegat GmbH, Tübingen, Germany,
3
University Tübingen, Cell Biology, Department of Immunology, Tübingen, Germany,
4
Ludwigs-Maximilians University, Munich, Dr. von Haunersches Kinderspital, Hematology / Oncology,
5
Munich, Germany
Adoptive T cell therapy is a promising option to treat
derived WT1-specific T cells in combination with a
cancer. Anti-tumor effects can be mediated by T cells
WT1 vaccine was accompanied with WT1-specific T
or NK cells. CD8+ T cells have a cytolytic capacity
cell responses and a sustained clinical remission for
that is enhanced by the presence of CD4+ T cells.
now 19 months. As we found that pre-treatment of
Besides their memory and helper function CD4+
sarcoma cells with demethylating agents upregulates
Th1 cells are mediators of senescence in tumor cells
the expression of cancer testis antigens (including de
which can induce a permanent growth arrest. This
novo expression), this would be another interesting
growth arrest is a result of the simultaneous secre-
option for a combination with adoptive immunother-
tion of TNF and IFN-γ by CD4+ T-helper-1 (Th1)
apy against cancer testis antigens. Further improve-
cells. According to these considerations, we devel-
ments can be achieved by combination of the therapy
oped a protocol according to good manufacturing
with antibodies to checkpoint inhibitors like PD1 or
practice (GMP) to generate CD4+ Th1 cells by IFN-γ
LAG3 which resulted in an enhanced proliferation of
enrichment technique against the tumor antigens
the generated antigen specific T cells. Evaluation of
NY-ESO-1, WT1, MAGE-A3, Survivin, ROR-1 and
the tumor antigen and immune checkpoint regulator
PRAME. The generated T-cells showed high numbers
B7H3 demonstrated a strong expression on sarcoma
of tumor antigen-specific IFN-γ+ and TNF+ CD4+
and melanoma cell lines which can be exploited for
cells, analyzed by intracellular flow cytometry and
future adoptive T cell therapies.
multiplex cytokine assays. Co-incubation of supernatants of the T cells with melanoma cells could
induce a growth arrest in the tumor cells, analyzed
in BrdU-Assays. T cells had an effector memory phenotype and proliferated in response to the tumor antigens. Treatment of a WT1+ patient with a relapsed
acute myeloid leukemia patient with stem cell donor
Development of multi-groove tissue culture flasks for growing of
lymphocytes used in adoptive immunotherapy
Kirkin A.1, Dzhandzhugazyan K.1, Jensen M.R.1
CytoVac A/S, Hørsholm, Denmark
1
Adoptive immunotherapy represents a method for
and collecting the cultivated cells.
treatment of cancer and infectious diseases that is
We have used multi-groove flasks for in vitro induc-
rapidly developing. A principal procedure of this
tion of tumor-specific CTL response by cultivating
method is ex vivo cultivation of lymphoid cells for
peripheral blood lymphocytes from healthy donors
the purpose of inducing the immune response and/
with autologous activated CD4+ cells expressing a
or expanding the effector cells. In general, these pro-
variety of cancer/testis antigens. Using cells from
cesses depend on close interaction of lymphocytes
200 ml of blood, the CTLs were equally and success-
with other cells, especially at the initial steps of cul-
fully generated in both standard and new grooved
tivation. Therefore, culture conditions favoring close
flasks. However, generation of CTLs from a smaller
cell interactions should provide better opportunities
(40 ml) blood volume from glioblastoma patients was
for both antigen-specific activation and initial expan-
a challenge using standard flasks and was successful
sion of lymphocytes. Several approaches are used for
in only half of the cases. Use of multi-groove flasks
initial cultivation of lymphocytes for adoptive im-
enabled us to generate CTLs in more than 80% of
munotherapy, including use of 96- or 24-well tissue
cases, which points to a higher efficiency when ini-
culture plates. Efficiency of generation and expan-
tially only a small number of cells are available.
sion of tumor-specific lymphocytes in these plates
In addition to initiating immune responses, these
have been demonstrated. However, these methods
multi-groove flasks will be useful for initial expand-
are highly laborious and subject to risk of contamina-
ing of TILs, especially from the tumor samples with
tion compared to methods employing standard tissue
few infiltrating lymphocytes, as well as for growing
culture flasks.
tumor cells in a more natural, spheroid form. Experi-
We have developed a new design of tissue culture
ments, exploring some of these applications are now
flasks where the surface has a plurality of parallel
in progress.
U-shaped grooves, used for growing the cells favor-
In conclusion, these new flasks have the advantages
ing close cell interactions. Furthermore, exchange of
of multi-well plates by creating conditions for close
part of the medium can be done by tilting the flask
interaction of cells. In addition, the design adds ease
about a first tilting axis arranged parallel with the
of exchanging media, the advantage of a more safe
longitudinal axis of the respective grooves into a
closed system known from tissue culture flasks, and
first tilting position, and then removing the medium
permits use of batch operations on the cultivated
and adding fresh medium. Harvesting of cells is per-
cells.
formed by tilting the flask about a second tilting axis
perpendicular to the longitudinal axis of the grooves,
Plasma IFNγ and IL-6 levels correlate with peripheral T-cell
numbers in RCC patients treated with CAR T-cells
Klaver Y.1, van Steenbergen S.1, Sleijfer S.2, Debets R.1, Lamers C.1
Erasmus MC Cancer Institute, Tumor Immunology, Rotterdam, Netherlands,
1
Erasmus MC, Medical Oncology, Rotterdam, Netherlands
2
Autologous T-cells genetically modified to express a
tested, IFN-γ and IL-6 levels in plasma are potential
Chimeric Antigen Receptor (CAR) against carboxy-
surrogate markers for T-cell persistence. We advo-
anhydrase-IX (CAIX) were administered to twelve
cate plasma cytokine measurements during T-cell
patients with CAIX-positive metastatic renal cell car-
treatment, as this might give valuable information
cinoma (RCC). Here, we questioned whether plasma
about in vivo presence and reactivity of adoptively
cytokine levels following treatment, or in vitro cy-
transferred T-cells.
tokine production from the T-cell infusion products
could serve as surrogate markers for peripheral T-cell
persistence or in vivo T-cell activity. We analyzed
the levels of 27 cytokines in patient plasma as well
as culture supernatant by a multiplex cytokine bead
array, and T-cell persistence by flow cytometry or
Q-PCR. We demonstrated that surface as well as gene
expression of CAR is down-regulated following T-cell
infusion, and peripheral numbers of CAR T-cells are
best captured by flow cytometry (and not by quantifying DNA by qPCR). Therapy was challenged by
liver enzyme abnormalities that were most likely
caused by T-cells recognizing CAIX-expressing bile
duct epithelia, and which were used as a measure for
in vivo T-cell activity.
Notably, plasma IFN-γ and IL-6 levels correlated significantly with the in vivo presence of CAIX CAR
T-cell numbers. IFN-γ and IL-6 are not constitutively
produced by T-cells, as they were not present in the
culture supernatant of T-cells prior to their infusion.
This would argue that IFN-γ and IL-6 in blood originate from infused CAIX CAR T-cells that were triggered by CAIX in vivo. Interestingly, plasma IFN-γ or
IL-6 levels did not correlate with liver enzyme values.
In conclusion, our data show that out of 27 cytokines
Functional characterization of different variants of a
PD-1-CD28-fusion receptor
Kraus F.B.T.1, Rataj F.1, Grassmann S.1, Chaloupka M.1, Endres S.1, Kobold S.1
Ludwig-Maximilians-Universität München, Abteilung für Klinische Pharmakologie, München, Germany
1
Background: Interaction of the Programmed Death
activity was paralleled by enhanced binding of PD-L1
Receptor 1 (PD-1) and its ligand, PD-L1, has been
to PTM in comparison to CTM and CEX.
shown to suppress T cell activity and allows tumors
Mutation of the functional variants within PTM
to evade T cell mediated immune-response. Our
lead to a loss of potency in the mutant-constructs.
group recently showed that antigen-specific T cells
PTM-transduced T cells produced statistically sig-
transduced with a PD-1-CD28 fusion receptor are
nificantly more IFN-γ than PTM-FMNM, PTM-AYAA,
protected from PD-1-mediated inhibition and receive
or PTM-FMNM-AYAA. PTM engagement induced
additional co-stimulation. We now aimed at charac-
proliferation in a PYAP-dependent manner, while
terizing the structural requirements and functional
YMNM was dispensable for the proliferative effect. In
domains needed for the observed anti-tumoural
contrast, production of various cytokines and chemo-
effects.
kines by PTM engagement seems to be dependent on
Methods: Three variants were generated by altering
both motifs, since mutant constructs induced overall
the composition of the extracellular and transmem-
lower cytokine levels compared to PTM. In accor-
brane domain: the PD-1 transmembrane construct
dance to our in-vitro observations - in-vivo tracking
(PTM), the CD28-transmembrane construct (CTM)
experiments showed enrichment of PTM-transduc-
and the CD28 extracellular and transmembrane con-
ed T cells as compared to mutant-constructs within
struct (CEX). Additional three functional variants
tumor tissue.
were generated by inserting point-mutations into the
Conclusion: Activity of PD-1-CD28 fusion receptor
signaling motives of CD28 as follows: mutation of
depends on its PD-1 transmembrane domain as well
YMNM to FMNM (PTM-FMNM), mutation of PYAP to
as on the intracellular functional CD28-domains.
AYAA (PTM-AYAA) and the double mutant PTM-FMNM-AYAA. Functional characterization was carried
out in vitro using ELISA, murine cytokine array and
flow cytometry. In vivo fusion receptor transduced
ovalbumine (OVA)-specific OT1-T cells were transferred into mice bearing Panc02-OVA tumors.
Results: Upon stimulation with anti-CD3 antibodies
and recombinant PD-L1, all receptors were functional as assessed by IFN-γ release and by induction of
proliferation. PTM proved to be superior to CTM and
CEX for all read-outs. Mechanistically, the increased
CXCR2 chemokine receptor transduction of human NK cells to
improve migration to solid tumors
Kremer V.1, Wennerberg E.1,2, Seitz C.1, Lundqvist A.1
Karolinska Institutet, Department of Oncology-Pathology, Stockholm, Sweden,
1
Weill Cornell Medical College, Cornell University, Department of Radiation Oncology, New York, United
2
States
Adoptive transfer of natural killer (NK) cells is being
capacity and functionality of the NK cells compared
increasingly used for cancer treatment; however,
to non-transduced controls, including cytotoxicity
clinical responses have so far been limited to patients
against K562 (20.5±2.1% vs 25.3±0.3% at an effector
with hematological malignancies. An important rate-
to target ratio of 1:1), interferon-gamma production
limiting factor in patients with solid tumors is the ef-
(8.2±2.8% vs 8.8±3.8%) as well as their degranula-
fective homing of infused NK cells to the tumor site.
tion (44.2±18.4% vs 44.3±10.6%).
Various solid tumors, including renal cell carcinoma
Taken together, these results indicate that the intro-
(RCC), readily secrete ligands for the chemokine re-
duction of the CXCR2 gene into NK cells enables their
ceptor CXCR2. We found, however, that upon ex vivo
migration along a tumor-derived chemokine gradient
activation or expansion with IL-2, the expression of
without altering their effector functions, which could
CXCR2 was gradually lost on human primary NK
potentially increase the success of NK cell-based
cells. Moreover, CXCR2 expression was significantly
therapies against solid tumors.
lower in tumor infiltrating NK cells compared with
peripheral blood NK cells in patients with primary
RCC (p=0.0016). We hypothesize that if the expression of CXCR2 can be restored on activated NK cells,
they will have improved ability to migrate toward
RCC tumors.
Using a retroviral system, we successfully transduced human primary NK cells with CXCR2; transduction efficiency was 56±14%. Calcium mobilization, the first step in chemokine receptor signaling,
was higher in CXCR2-transduced compared with
control NGFR-transduced NK cells in response to
recombinant CXCL8 or RCC conditioned medium.
CXCR2-transduced, but not NGFR-transduced NK
cells, showed on average a 2- to 3-fold enhanced migration toward both recombinant CXCR2 ligands and
conditioned medium from RCC cell lines expressing CXCL1 and CXCL8 (p< 0.05). Furthermore, the
transduction did not compromise the proliferation
TCRs for MAGE-C2, in combination with epigenetic drug treatment of target cells, yield tumor-selective therapeutic T cells
Kunert A.1, van Steenbergen-Langeveld S.1, van Brakel M.1, da Silva M.1, Coulie P.G.2, Lamers C.1, Sleijfer S.1,
Debets R.1
Erasmus MC, Medical Oncology, Rotterdam, Netherlands,
1
de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
2
Adoptive T cell therapy has shown significant clini-
from melanoma, head-and-neck, bladder and triple-
cal success for patients with advanced melanoma
negative breast cancers, but not recognition of MHC-
and other tumors. Further development of T cell
eluted peptides nor peptides highly similar to MC2.
therapy requires improved rationales to select effec-
We conclude that T cell therapy benefits from the
tive anti-tumor yet non-self-reactive T cell receptors
combination of choosing an effective and safe target
(TCRs). Here we tested TCRs directed against various
antigen, such as MAGE-C2, and pre-treatment with
epitopes of the protein MAGE-C2 (MC2) as well as
epigenetic drugs.
conditions that allow maximal and tumor-selective
T cell responses.
Ten TCR sequences against four MC2 epitopes were
isolated from melanoma patients who showed clinical responses following vaccinations. Clinical responses were accompanied by significant frequencies of anti-MC2 CD8 T cells in blood and tumor
without apparent side effects. We introduced these
TCRs into T cells, pre-treated tumor cells of different
histological origins with the epigenetic drugs azacytidine and valproate, and tested on- as well as off
target-reactivities of these TCRs.
MAGE-C2 was not expressed in healthy tissue, except
testis tissue, according to q-PCR and immune histochemistry of a large panel of tissues. Pre-treatment of
tumor cells with epigenetic drugs up-regulated MC2
gene expression and enhanced their recognition by
T cells. In contrast, similar pre-treatment of normal
cell lines did not result in recognition by MC2-directed T cells. Interestingly, the expression levels
of MC2, but not those of CD80, CD86, programmed
death-ligand 1 (PD-L1), or PD-L2, correlate with T
cell responsiveness. One TCR resulted in consistent
recognition of all pretreated, MC2-positive cell lines
Intra-tumoral production of IL18, but not IL12, by ‘smart’ T
cells is non-toxic and counteracts immune evasion of solid
tumors, prolonging survival
Kunert A.1, Chmielewski M.2, Berrevoets C.1, Wijers R.1, Abken H.2, Debets R.1
Erasmus MC, Medical Oncology, Rotterdam, Netherlands,
1
University of Cologne, Center for Molecular Medicine Cologne, Cologne, Germany
2
Adoptive therapy with engineered T cells constitutes
iIL18 T cells showed no signs of toxicity and signifi-
a promising treatment approach for patients with
cantly reduced tumor burden, prolonged overall sur-
malignant disease, but is currently challenged by
vival, an effect that appeared related to a dampened
tumor recurrence and incomplete responses. There
expression of co-inhibitory receptors on T cells.
is accumulating evidence suggesting that the tumor
In conclusion, we show that treatment with iIL12 T
micro-environment directs immune evasion and pre-
cells may be harmful, whereas treatment with iIL18
vents intra-tumoral accumulation and activation of
T cells is able to skew the tumor micro-environment
sufficient numbers of T cells. We aim to change the
in favor of an improved anti-immune response.
tumor micro-environment in favor of a successful
immune response, by locally inducing production of
Interleukin (IL)12 and IL18 in an antigen-dependent
fashion via administered T cells.
To this end, we have engineered T cells with a T cell
receptor (TCR) and murine IL12 or IL18 under the
regulation of a nuclear-factor of the activated T-cell
(NFAT)-driven promoter. These T cells produce IL12
and IL18, and consequently enhanced levels of IFNγ,
following co-culture with antigen-positive but not
negative target cells. Adoptive transfer of inducible
(i)IL12 T cells to melanoma-bearing mice resulted in
severe, edema-like toxicity and a reduced overall survival that was accompanied by decreased numbers of
administered T cells and enhanced levels of inflammatory cytokines in blood. In contrast, transfer of
Activation of invariant natural killer T-cells by a unique antiCD1d single domain antibody induces potent tumor destruction
in vitro
Lameris R.1, de Bruin R.1, van Bergen en Henegouwen P.2, Zweegman S.3, Verheul H.1, de Gruijl T.1,
van der Vliet H.1
VU University Medical Centre, Medical Oncology, Amsterdam, Netherlands,
1
Utrecht University, Biology, Utrecht, Netherlands,
2
VU University Medical Centre, Hematology, Amsterdam, Netherlands
3
Invariant natural killer T (iNKT) cells constitute a
showed rapid and selective destruction of primary
unique T-cell population that plays an important role
multiple myeloma cell when the anti-CD1d VHH was
in anti-tumour immunity and immunosurveillance.
added to a co-culture of iNKT cells and ex vivo bone
Interaction between iNKT TCR and (glyco-)lipids pre-
marrow cells. Importantly, α-GalCer was found to be
sented in CD1d may result in robust activation, direct
completely ineffective.
cytotoxic effect and production of a wide range of cy-
The unique ability of this anti-CD1d VHH to boost
tokines thereby inducing effector (e.g. NK and CTL)
the interaction between iNKT and CD1d may cir-
cell activation in an IFN-γ dependent manner. Activa-
cumvent encountered limitations with applied gly-
tion of iNKT by the strong agonistic glycolipid-ligand
colipids. Moreover, it has the potential to overcome
α- galactosylceramide (α-GalCer) induced potent re-
the immunosuppressive tumor (micro)environment
jection of established tumors in mouse. Human ob-
and exploit the full arsenal of iNKT based immuno-
servational studies underscore these findings, since
therapy.
circulating numbers correlate with patients survival
in various malignancies. However, clinical studies
using α-GalCer have so far been disappointing, possibly due to limitations of the used glycolipid.
We developed a unique anti-CD1d variable domain of
heavy chain-only Ab (VHH or nanobody) that in vitro
profoundly activates iNKT cells in a CD1d-dependent
manner. Activation occurs rapidly (within hours), is
contact dependent and is signified by a fast amount
of IFN-γ production. Importantly, irrespective of the
presented exogenous or endogenous substrate in the
groove of CD1d, the anti-CD1d VHH strongly induce
a Th1-biased cytokine response. Moreover, when
added to a co-culture of iNKT cells and CD1d-positive
tumor cells rapid tumor cell lysis was observed at low
effector to target ratios which was more potent than
with α-GalCer. Target cell lysis was at least partly
granzyme B dependent and occurred at nM-range
concentrations. Likewise, preliminary experiments
Targeting tumor suppressor p53 isoforms as a novel approach
to improve T-cell based immunotherapy
Legscha K.-J.1, Antunes E.1, Amann E.1, Theobald M.1,2,3, Echchannaoui H.1
University Medical Center of the Johannes Gutenberg University Mainz, Third Medical Department
1
(Hematology, Oncology and Pneumology), Mainz, Germany,
Research Center Immunology (FZI), Johannes Gutenberg University Mainz, Mainz, Germany,
2
German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Frankfurt/Mainz, Mainz, Germany
3
Background: Adoptive transfer of tumor antigen-spe+
tivation markers like 4-1BB (CD137) and CD27 were
cific (CD8 ) T cells represents a promising approach
downregulated in p53b-modified T cells and upregu-
in the field of cancer immunotherapy. However in
lated in delta133p53-overexpressing T cells. Expres-
many patients the overall benefit is still limited due
sion of the chemokine receptor CXCR3 and homing
to various tumor escape mechanisms. One important
markers like CD62L and CCR7were also reduced in
tumor-mediated immunosuppression mechanism is
p53beta- and increased in delta133p53-T cells. In
the exhaustion of the tumor infiltrating lymphocytes
contrast, the sensitivity to apoptosis induction after
(TILs) after direct interaction with tumor cells and
antigen-specific activation was higher in p53beta and
the tumor microenvironment (TEM). Genetically
lower in delta133p53-T cells which correlated with
modified T cells which become resistant to the tu-
enhanced proliferation capacity in the delta133p53-
mor-derived suppression could therefore improve
modified T cells. Importantly, p53beta-T cells ex-
anti-tumor response after adoptive transfer. Here we
hibited an impaired tumor-specific killing capacity
show for the first time how the modulation of tumor
while T cells overexpressing delta133p53 isoform re-
suppressor p53 isoforms can affect tumor-specific
tained their killing ability.
T-cell exhaustion.
Conclusion: Genetic modulation of p53 isoforms in
Methods: Human T cells form healthy donors were
human CD8+ T cells represents a novel approach to
retrovirally co-transduced with either p53beta or
circumvent tumor-mediated T cell inhibition. Simul-
delta133p53 isoforms, together with a tumor-specific
taneous overexpression of the delta133p53 isoform
T-cell receptor (TCR). Modified T cells were char-
together with a knockdown of p53beta is a promising
acterized for the expression of key activating/in-
approach to prevent TILs exhaustion, enhance anti-
hibitory molecules, homing markers as well as their
tumor response and therefore could improve adop-
sensitivity to apoptosis induction by flow cytometry.
tive T cell therapy immunotherapy.
Additionally the antigen-specific killing capacity was
examined by standard chromium-release assay and
long-term tumor colony-forming assay.
Results: p53beta-overexpressing T cells exhibit an
increased cell-surface expression of the inhibitory receptor programmed cell death protein 1 (PD-1) while
the delta133p53-transfected cells showed reduced
levels of PD-1 after antigen-specific stimulation or
non-specific CD3/CD28 activation. Furthermore ac-
Integration of a CD19 CAR gene into the TCR alpha chain locus
streamlines production of allogeneic gene-edited CAR T cells
MacLeod D.T.1, Antony J.1, Wetzel K.J.1, Moser R.2, Brown A.E.1, Hux J.A.1, Turner C.A.1, Hekele A.1, Lape J.1,
Beard C.W.1, McCreedy B.1, Bartsevich V.V.1, Nicholson M.G.1, Smith J.1, Hirsch M.2, Jantz D.1
Precision BioSciences, Durham, United States,
1
University of North Carolina at Chapel Hill, UNC Gene Therapy Center, Chapel Hill, United States
2
Chimeric antigen receptor (CAR) T cell therapies
cellular machinery can result in gene knockout (KO),
have achieved dramatic results treating hematolog-
and can also result in knock-in of an exogenous gene
ical malignancies by redirecting the specificity of T
through homology directed repair if appropriate ho-
cells to target CD19 expressing cancer cells. Clinical
mologous sequences are appended to the 5’ and 3’
trials reporting these impressive results have used
ends of the donor DNA template. In the absence of a
autologous therapies, which pose significant man-
donor DNA template, treatment with this meganucle-
ufacturing, logistical, and cost issues, complicating
ase resulted in >60% TCR KO cells, with equivalent
standardization and implementation of such ther-
rates of gene-editing in CD4+ and CD8+ cells. To shift
apies on a larger scale. Importantly, production of
repair towards gene knock-in, we treated cells with
therapeutic doses of autologous CAR T cells cannot
our meganuclease, then transduced these cells using
be achieved for a significant number of patients due
an AAV6 vector containing a donor DNA template
to low T cell counts in patients with advanced stage
consisting of a second generation CD19 CAR driven
disease. Furthermore, CAR T cells are typically gen-
by an exogenous promoter and flanked by sequences
erated by using Lenti- or Retro- viral vectors, result-
of homology to the target site in the TCRα sequence.
ing in random integration of the CAR gene, heteroge-
Incorporating this process into a 14 day procedure for
neous expression of the CAR on the cell surface, and
generation and expansion of TCR knockout CAR T
the potential for insertional mutagenesis. We have
cells, we successfully generated a large population of
developed a platform to address these issues by using
TCR knockout cells with >70% stably expressing the
healthy donor PBMCs and a precise genome editing
CD19 CAR. Precise CAR insertion within TCRα was
process to insert a CD19 CAR gene into a defined
confirmed by PCR. Importantly, these CAR T cells
location in the genome, within the T cell receptor
proliferated, released cytokines including IFN-γ and
(TCR) alpha chain locus. The insertion of the CAR
IL-2, and exhibited potent cytotoxic activity when
gene at this site results in gene-edited allogeneic
cultured with CD19+ target cells. Studies to confirm
CAR T cells that do not express an endogenous TCR
in vivo antitumor activity and absence of GvHD are
and therefore should not be capable of eliciting graft
currently being conducted. In summary, we describe
versus host disease upon adoptive transfer. We first
a novel, scalable method to produce allogeneic CD19
produced and validated a highly engineered mega-
CAR T cells with the CAR gene integrated at a defined
nuclease targeting TCRα, and developed a scalable
location within the genome.
process for electroporating T cells with meganuclease mRNA to generate a double-strand break (DSB)
in the TCRα constant region. Repair of this DSB by
Mapping of T-cell receptor-engineered human T cells at the
tumor site by Immuno-PET
Mall S.1, Yusufi N.2, Wagner R.1, Klar R.1, Bianchi H.1, Steiger K.3, Straub M.3, Audehm S.1, Laitinen I.2, Aichler M.4,
Peschel C.1,5, Ziegler S.2, Mustafa M.2, Schwaiger M.2,5, d´Alessandria C.2, Krackhardt A.1,5
Klinikum rechts der Isar, Technical University Munich, III.Medical Department, Munich, Germany,
1
Klinikum rechts der Isar, Technical University Munich, Department of Nuclear Medicine, Munich, Germany,
2
Klinikum rechts der Isar, Technical University Munich, Department of Pathology and Pathological Anatomy,
3
Munich, Germany,
Helmholtz Zentrum München, Research Unit Analytical Pathology, Munich, Germany,
4
German Cancer Consortium (DKTK), Munich, Germany
5
T-cell based immunotherapies have recently demon-
PET/CT images with semi-quantitative evaluation
strated to be highly effective in an increasing number
of T-cell infiltration based on immunohistochemical
of tumor entities. However, sensitive and clinically rel-
analysis allowed mapping of differential T-cell distri-
evant in vivo imaging technologies are still missing.
butions within the tumor. This F(ab`)2 based T-cell-
Such technologies are, however, highly important to
tracking technology enables non-invasive imaging
understand mechanisms of effective tumor rejection
of TCR-transgenic T cells independently from the
or evasion. Adoptive transfer of T cells genetically
TCR-specificity. Furthermore, the in vivo labeling of
modified by T-cell receptors (TCR) is an attractive
cells using this technology opens the possibility to
novel therapeutic strategy for cancer patients and
track T cells on differential time points most criti-
non-invasive imaging technologies for transgenic
cal for decision making processes of immunotherapy.
T-cells are critical for monitoring and optimization
Moreover, the high sensitivity and simple application
of these therapies. We developed a highly sensitive,
make this approach especially attractive for direct
non invasive imaging technology to track human
clinical translation.
TCR transgenic T cells by targeting the murine constant TCR beta domain of a murine-human hybrid
TCR by a Zirconium-89 (89Zr)-labeled aTCRmu-F(ab’)2
fragment. Although the aTCRmu-F(ab`)2 fragment
showed a high affinity to its target, binding of the
aTCRmu-F(ab`)2 with or without labeling by 89Zr did
not impair functionality of TCR-transgenic T cells in
vitro and in vivo. We established a xenogenic mouse
model of myeloid sarcoma and adoptively transferred
human central memory T cells (TCM) transgenic for
the leukemia-specific TCR2.5D6. Intravenous application of
89
Zr-labeled aTCRmu-F(ab’)2 resulted in
highly sensitive and specific visualization of TCRtransgenic TCM at the tumor site by PET/CT imaging
which was confirmed by ex vivo analyses. Moreover, we detected diverse T-cell distribution patterns
on the tumor site by PET/CT imaging depending on
the tumor size and rejection phase. Correlation of
Allogeneic CAR-T cells gene edited to insert an anti-CD19 CAR
into the TCR alpha locus target and kill CD19+ Raji lymphoma
tumors in vitro and in vivo without causing GvHD
McCreedy B.1, Antony J.1, Martin A.1, MacLeod D.1, Pham C.1, Wetzel K.1, Moser R.2, Brown A.1, Hux J.A.1, Turner C.1,
Hekele A.1, Lape J.1, Beard C.1, Bartsevich V.1, Nicholson M.1, Smith J.1, Hirsch M.2, Jantz D.1
Precision BioSciences, Inc., Cell Therapy, Durham, United States,
1
University of North Carolina at Chapel Hill, Chapel HIll, United States
2
ORAL
TALK
SHORT
2016
Numerous clinical trials of adoptive cellular therapy
cells in media containing IL-7 and IL-15 resulting in
using autologous T cells transduced to express an
a large population of gene edited CAR-T cells that
anti-CD19 chimeric antigen receptor (CAR-T) have
was >99% CD3-, >85% CAR+ with the majority of
demonstrated dramatic objective responses against
these cells expressing a central memory phenotype
CD19+ B cell tumors including durable complete
(CD45RO+,CD62L+). CAR insertion at the site of DSB
responses in some patients. However, production
in TCRα was confirmed by PCR. These CAR T cells
of autologous CAR-T cell therapies pose significant
proliferated, released cytokines including IFN-γ and
challenges including an inability to generate a suffi-
IL-2, and exhibited potent cytotoxic activity when cul-
cient number of cells for therapeutic administration
tured with CD19+ target cells. When injected i.v. into
in patients with advanced stages of disease as well as
NSG mice bearing disseminated Raji-FFluc tumors
manufacturing, logistical, and cost issues that com-
these gene edited CAR-T cells traveled to tumor sites
plicate standardization and implementation of such
and killed tumor cells as evidenced by significant
therapies on a larger scale. We have developed a plat-
reduction in bioluminescence by IVIS as well signifi-
form to address these issues by using healthy donor
cantly increased survival of CAR-T vs. control-treat-
PBMCs and a highly specific engineered homing en-
ed animals. In addition, the gene edited CAR-T cells
donuclease to knock out expression of the endoge-
did not cause GvHD when injected into sub-lethally
nous TCR followed by insertion of a CAR gene at the
irradiated NSG mice as measured by clinical signs,
site of the double strand break (DSB). The resulting
weight loss, and histology compared with non-ed-
allogeneic CAR T cells express the CAR in a consistent
ited T cells from the same donor. In summary, we
and controllable manner but do not express a TCR
have used a novel gene editing platform to gener-
and therefore should not be capable of eliciting graft
ate allogenic CAR-T cells in which the endogenous
versus host disease (GvHD) upon adoptive transfer.
TCR has been knocked out and an anti-CD19 CAR
+
T cells from healthy donors were
inserted at a defined site in the TCRα locus. These
nucleofected with mRNA encoding a TCRα targeted
gene edited CAR-T cells specifically recognize and
homing endonuclease and then transduced using an
kill CD19+ target cells in vitro and in vivo and do
AAV6 vector containing a donor DNA template con-
not cause GvHD in a xenogeneic mouse model. Addi-
sisting of a second generation anti-CD19 CAR driven
tional studies to evaluate anti-tumor efficacy against
by an exogenous promoter and flanked by sequences
subcutaneous CD19+ tumors are ongoing.
Activated CD3
of homology to the target site in the TCRα sequence.
TCR negative CAR+ T cells were expanded in vitro
by co-culture with mitomycin C-treated IM9 (CD19+)
Csk overexpression makes T cell dummy
Inderberg E.M.1, Mensali N.1, Stenvik B.2, Oksvold M.2, Progida C.3, Bakke O.3, Fallang L.-E.2, Kvalheim G.1,
Myklebust J.H.2, Wälchli S.1,2
Oslo University Hospital-Radiumhospitalet, Dep of Cellular Therapy, Oslo, Norway,
1
OUS-Radiumhospitalet, Institute for Cancer Research / Section of Immunology, Oslo, Norway,
2
University of Oslo, Dep of Biosciences, Oslo, Norway
3
TCR signaling is tightly modulated by positive and
homing, selectivity and antigen specificity of ther-
negative regulators in order to guarantee proper
apeutic TCR redirected T cells for adoptive T cell
signaling upon cognate peptide MHC complex rec-
therapy.
ognition. The same type of regulation occurs when
a therapeutic TCR is expressed in a patient T cell
during adoptive transfer. The idea that enhanced
positive signaling could be exploited to improve
TCR efficacy is in early-stage testing. Interestingly,
negative modulators of signaling could also be exploited. Indeed, a main concern using therapeutic
TCRs is their safety. By over-expressing the c-src
kinase (CSK), a negative regulator of TCR signaling,
we were able to completely inhibit TCR signaling
by interfering with both early and late events in the
TCR signaling cascade. As a consequence the effector functions of the TCR-engineered T cells were
shut down. In contrast, the CSK over-expression
did not affect the capability of the TCR to bind the
cognate peptide/MHC complex. We named these
gene-modified T cells “Dummy T cells” because
although they were able to bind their target cells
they had lost the capability to kill. We therefore
propose that the “dummy T cells” could be used
as a prospective tool for in vivo monitoring of the
Individualized immunotherapy of ovarian cancer by targeting
Claudin-6 with CAR-engineered T cells
Mroz K.1, Hobohm K.1, Reinhard K.1, Simon P.1, Birtel M.2, Tolliver C.1, Klein O.1, Büchling T.2, Jahndel V.3, Voss R.-H.4,
Löw R.5, Külcke K.5, Hoff H.6, Walter K.7, Türeci Ö.8, Sahin U.2,3,4
1
TRON gGmbH, Translational Oncology at the University Medical Center of Johannes Gutenberg-University,
2
Mainz, Germany,
3
Universal Medical Center of Johannes Gutenberg-University, Department of Hematology and Oncology,
4
Mainz, Germany,
5
Erdmann Technologies GmbH, Berlin, Germany,
6
Ganymed Pharmaceuticals AG, Mainz, Germany,
7
CI3 - Cluster of Individualized Immunointervention, Mainz, Germany
8
The potential of engineered antigen-specific T cells to
ed effector functions by luciferase-based cytotoxicity
eradicate tumors provoked the development of novel
and CFSE-based proliferation assays. CAR-expressing
T cell based immunotherapy approaches. These
T cells exhibited specific proliferation in response to
include the adoptive transfer of autologous T cells
CLDN6 expressing target cells. Furthermore, CAR-en-
genetically engineered with tumor antigen-specific
gineered T cells efficiently killed CLDN6-expressing
receptors, such as conventional α/β TCRs or chime-
tumor cells in vitro.
ric antigen receptors (CAR). CARs are recombinant
Preclinical safety testing for adoptive immunothera-
receptors that combine HLA independent scFv-me-
pies using CAR engineered T cells relies on stringent
diated antigen-binding with T cell signaling. We
target validation and a robust pre-clinical specificity
designed a second generation CAR with specificity
testing strategy. These include on-target off-tumor tox-
for Claudin-6 (CLDN6), coupled to CD28 and CD3ζ
icity and off-target toxicity. In order to safety testing
signaling domains. CLDN6 is an onco-fetal gene that
and to determine the lower limit of CLDN6 molecules
belongs to the family of tight junction proteins and
per cells that induces CAR-T cell-mediated cytotoxic-
is expressed in human stem cells and during early
ity and cytokine secretion, we tested a large panel of
stage of epidermal morphogenesis. As it is absent in
different tumor cell lines from different origins with
adult healthy tissues, but overexpressed in different
variable CLDN6 expression.
cancers including ovarian cancer, CLDN6 represents
For detection and quantification of CAR-transduced
an ideal target antigen for immunotherapy based on
T cells a qPCR based assay can be validated and has
CAR-engineered T cells.
been widely used in clinical settings. The approach
For functional CAR validation in vitro we used mRNA
followed in our group is a duplex qPCR comprising the
transfer for rapid expression of the CLND6-CAR in T
amplification of WPRE element and the amplification
lymphocytes. We evaluated the CLDN6-CAR-mediat-
of parts of hChr6 for quality control of genomic DNA.
With this approach we are able to detect down to 100
pg of human genomic DNA in murine samples and
down to 100 copies of integrated vector.
To investigate the therapeutic potency of CLDN6
CAR-T cells in a syngeneic solid tumor mouse model,
the efficiency of the humanized lead structure, was
tested in murine T cells in vitro. So far, CLDN6-CAR
mediated proliferative and cytotoxic activities in genetically modified murine T cells upon antigen recognition which is comparable to human redirected
CLDN6-CAR-T cells. We are currently establishing a
tumor model to prove efficacy and safety aspects of
CLDN6-CAR in vivo.
Generation of tumor-specific NK cells by differentiation of CARgene transduced hematopoietic progenitors
Oberoi P.1, Villena F.1, Stein S.1, Bönig H.2, Wels W.S.1
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany,
1
Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt am Main, Germany
2
Natural killer (NK) cells hold promise for adoptive
were found to be expressed by the ex vivo generated
cancer immunotherapy. Like T cells, the antitumor
CD56+ cells, which were functionally active as con-
activity of NK cells can be enhanced by expression
firmed in cytotoxicity assays using K562 tumor cells
of chimeric antigen receptors (CARs) that facili-
as targets.
tate selective recognition and killing of malignant
To restrict CAR expression to NK cells developing
cells. CARs consist of an extracellular single-chain
during the differentiation process, in parallel we
antibody fragment (scFv) for recognition of a cell
constructed a lentiviral vector encoding an ErbB2/
surface antigen, linked to an intracellular signaling
HER2-specific CAR under the control of an NK-spe-
moiety such as CD3ζ or CD3ζ fused to a costimula-
cific NCR1 promoter. We found lineage-specific activ-
tory protein domain. The engagement of CARs on
ity of the CAR construct in established human NK
NK cells triggers antigen-specific lysis of target cells,
cell lines, while no CAR expression was detected in
hence bypassing the need for the activation of endog-
cells of B-cell or myeloid origin. This CAR vector will
enous cytotoxicity receptors.
be transferred into CD34+ HSCs, CAR expression in
For adoptive immunotherapy, NK cells are usually
NK cells and other lineages obtained after ex vivo
isolated from peripheral blood and expanded ex vivo
differentiation will be analyzed, and resulting CAR-
with cytokines before infusion into patients. Experi-
positive cells will be functionally characterized.
mentally, NK cells have also been derived from hematopoietic stem cells (HSCs) by ex vivo differentiation following different protocols. CAR NK cells may
be generated from CAR gene transduced HSCs following a similar approach. To explore this strategy,
we aim to establish a suitable protocol for ex vivo
expansion and subsequent differentiation of CD34+
HSCs into NK cells. Here, mobilized human CD34+
HSCs isolated from peripheral blood of healthy
donors were cultured ex vivo in a specific cytokine
mix to allow preferential generation of NK cells. We
observed the appearance of CD56+ NK cells starting
at day 27 of the culture period, and the percentage of
these cells in the cell pool increased over time. Additionally, various NK cell-associated surface receptors
Exploiting tumour infiltrating lymphocytes (TILs) as a therapeutic strategy in ovarian cancer - a proof of concept study
Owens G.1,2, Sheard V.1, Price M.2, Edmondson R.2, Gilham D.1
University of Manchester, Clinical and Experimental Immunotherapy, Manchester, United Kingdom,
1
University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom
2
Background: Epithelial ovarian cancer (EOC) is the
showed strong functional activity against autologous
fifth most common cause of cancer-related mortal-
tumours cells at an effector/target ratio of 1:1. 92%
ity in women. Despite advances in surgical tech-
of co-cultures demonstrated IFNᵧ secretion above
niques and improvements in the efficacy of cytotoxic
that of TILs alone. Importantly, we have shown that
treatments, there has been no appreciable increase
expanded TILs retain their anti-tumour function in
in overall survival in the last 30 years. Tradition-
vitro following storage at -80°C for a minimum of 8
ally EOC was not considered to be immunogenic;
weeks. TIL cultures resulted in approximately 40%
however, several studies have identified tumour-
CD4+: 60% CD8+ T cell populations. Both CD4+ and
reactive T cells in tumours and ascites, the presence
CD8+ subsets demonstrated features associated with
of which has been shown to correlate with improved
effector memory phenotypes (CCR7- CD45RO+).
clinical outcomes. Tumour infiltrating lymphocyte
Conclusion: We have demonstrated that TILs can be
(TIL) therapy has shown encouraging results in other
reliably and successfully expanded from EOC biop-
immunogenic tumours and may represent a promis-
sies. Importantly, the expanded TILs maintain au-
ing therapeutic strategy for EOC.
tologous tumour recognition in vitro. Based on our
Aims: We aimed to (1) test the reproducibility of an
preliminary data, we are currently developing phase
established protocol to expand TILs from EOC bi-
I/II trial protocols to evaluate the clinical potency of
opsies, and (2) define the anti-tumour function and
TILs in EOC.
specificity of TILs.
Methods: Tumours, collected at the time of primary
surgery, were disaggregated and mitogenically stimulated with anti-CD3/CD28 Dynabeads and IL-2. Dynabeads were removed after 7 days and TILs were
expanded in IL-2 for a further 12 days. Expansion
was recorded on alternate days. To determine functional activity of expanded TILs against autologous
tumour, IFNγ production was measured by ELISA.
Multi-colour flow cytometry was used to characterise
the phenotype of expanded TILs.
Results: To date, TILs were successfully expanded from 12/12 clinical specimens. Total number
of TILs at day 19 ranged from 1.5 - 39.8 x107. TILs
Towards in vivo delivery of chimeric antigen receptors
Pfeiffer A.1, Bender R.R.1, Zhou Q.1, Wels W.2, Buchholz C.J.1
Paul-Ehrlich-Institute, Langen, Germany,
1
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany
2
ORAL
TALK
SHORT
2016
Chimeric antigen receptors (CARs) represent a prom-
generated CAR T cells were stimulated upon antigen
ising tool for cancer treatment. Retargeted CAR T
recognition and show proliferative capabilities in
cells against CD19 have been shown to bring a signif-
vitro. We currently focus on the capability of CD4-
icant clinical benefit to patients, suffering from acute
LVCD19-CAR
lymphocytic leukemia (ALL) or chronic lymphocytic
in vivo after systemic administration of the vector
leukemia (CLL). However, production of CAR T cells
into huPBL-NSG mice. It will be further investigated
requires extensive and time-consuming procedures
weather in vivo generated CAR T cells have anti-tu-
of cell isolation, sorting, transduction and in vitro ex-
mor potential.
panding of immune cells. Receptor-targeted lentiviral vectors (LV), which can transfer genes selectively
into particular types of lymphocytes would enable
direct in vivo gene delivery thus substantially simplifying production. Of relevance for this project here,
are CD4-LV and CD8-LV that selectively transduce
CD4+ or CD8+ cells, respectively.
Based on pseudotyping lentiviral vectors, we generated high-titer CD4- and CD8-targeted vectors, feasible for efficient in vivo gene delivery. So far, we
were able to demonstrate in vivo gene transfer of a
gfp-luciferase reporter gene for each vector type, respectively. Systemic vector administration into the
tail vein of huPBL-NSG mice resulted in luciferase expression in spleen, lung and skin. Gfp expression was
exclusively found in the targeted subpopulations,
namely CD4+ or CD8+ T cells, as determined by flow
cytometry from spleen, lung and blood derived cells.
Next, we generated CAR T cells by ex vivo transduction of huPBMCs using either CD4- or CD8-LV delivering the CD19 CAR gene. CD8 CAR T cells specifically
and efficiently killed target cells whereas non-target
cells remained unaffected. Furthermore, CD8-LV
and CD8-LVCD19-CAR to generate CAR T cells
Exploring novel siRNA delivery approaches with cytotoxic T
cells
Raemdonck K.1, Wayteck L.1, Dewitte H.1, Xiong R.1, Breckpot K.2, Braeckmans K.1,3, De Smedt S.C.1
Ghent University, Laboratory of General Biochemistry and Physical Pharmacy, Department of
1
Pharmaceutics, Faculty of Pharmaceutical Sciences, Ghent, Belgium,
Vrije Universiteit Brussel, Laboratory of Molecular and Cellular Therapy, Department of Biomedical Sciences,
2
Brussels, Belgium,
Center for Nano-and Biophotonics, Ghent, Belgium
3
The therapeutic potential of small interfering RNA
CTL activity can be markedly reduced by the im-
(siRNA) has long been recognized. To facilitate siRNA
munosuppressive tumor microenvironment. Recent
delivery across the many extra-and intracellular bar-
research elucidated various intracellular inhibitory
riers, they are typically formulated into polymer- or
pathways in CTLs that contribute to the impaired T
lipid-based nanoparticles (i.e. nanomedicines, NMs).
cell-mediated anti-tumor response. Downregulation
[1] This abstract describes two distinct approaches
of such pathways in effector T cells with siRNA could
for siRNA delivery applied on cytotoxic T lympho-
significantly boost the efficacy of T cell therapy. Un-
cytes (CTLs). A first concept involves the exploitation
fortunately, primary T cells are hard-to-transfect
of CTLs as carriers for improved in vivo delivery of
and conventional non-viral transfection agents are
siRNA NMs. Second, we report on gold-induced pho-
largely ineffective. Viral vectors and electroporation
toporation as a novel ex vivo approach to enhance
are efficient but their use is restricted by high costs,
siRNA delivery into isolated CTLs.
safety issues and cytotoxicity. Photoporation is a
In cancer therapy, siRNA NMs generally rely on
recent physical approach that can induce cell mem-
passive accumulation in the tumor tissue based on
brane permeability by focusing nanosecond laser
the highly heterogeneous enhanced permeation and
pulses onto cells with gold nanoparticles (AuNPs)
retention (EPR) effect. An alternative delivery strate-
attached to their cell membrane.[3] We showed that
gy exploits the intrinsic capability of activated T cells
this technique can be used to functionally deliver
to infiltrate the tumor mass. We recently succeed-
siRNA into primary cytotoxic T cells with acceptable
ed in the coupling of siRNA NMs to the surface of
toxicity and without the need for polymer or lipid
tumor-migrating CTLs.[2] Importantly, the attached
excipients.
NMs do not compromise T cell functions like proliferation and cytolytic activity. NMs encapsulating
References:
anti-tumor siRNAs require tumor cell internalization
1. Wittrup, A. and Lieberman, J.: Nat. Rev. Genet. 2015, 16(9):
534-552.
2. Wayteck, L. et al.: Biomaterials 2016, 77: 243-254.
3. Xiong, R. et al.: ACS Nano 2014, 8(6): 6288-6296.
to enable cytosolic drug delivery and enhance the
anti-cancer effect. Therefore, we demonstrated the
reversible anchoring of NMs to the CTL surface via
a reduction sensitive coupling for which triggered
NM release could be shown upon the addition of glutathione. We are currently evaluating this combination therapy in a melanoma mouse model.
Arming T cells with activating FcγRIIIa receptors for antibody
redirected lysis of cancer cells
Rataj F.1, Asang F.1, Endres S.1, Kobold S.1
Klinikum der Universität München, Center of Integrated Protein Science Munich (CIPS-M) and Division of
1
Clinical Pharmacology, Department of Internal Medicine IV, München, Germany
Background: Adoptive T cell therapy (ACT) is under
of transduced T cells with an EGFR+ human pancre-
investigation for the treatment of hematologic ma-
atic cancer cells (PaTu 8988t) led to increased IFNγ
lignancies and for solid tumors. ACT targets single
release in the presence of cetuximab (10 fold induc-
antigens expressed by the cancer cells. It is inher-
tion compared to control, p = 0.0002). This effect
ently limited by antigen-loss variants of tumor cells
was antibody dose-dependent. Expression of high af-
and side effects resulting from off-target expression
finity CD16-fusion receptors led to a significantly in-
of the chosen antigen. Designer T cells which would
creased cytokine release compared to the low affinity
allow for variable antigen targeting limited in time
variant. Antigen-driven activation of transduced T
could have considerable advantages both in terms of
cells using was confirmed using the EGFR+ human
safety and efficacy. We hypothesized that arming T
breast cancer cell line MDA-MB-231. Further anal-
cells with fusion receptors consisting of the extracel-
yses regarding the cytokine secretion, cytotoxicity
lular portion of an antibody-binding-receptor (Fcγ re-
and proliferation of the CD16-fusion receptor-trans-
ceptor IIIA, synonymous CD16) and the intracellular
duced T cells are ongoing. Other therapeutic mAbs
domains of CD28 and CD3z could be combined with
are under current investigation.
clinically approved monospecific antibodies. Such a
Conclusions: Our results indicate that CD16-fusion
combination would allow for variation the tumor tar-
receptor may enhance efficacy and potentially safety
geting antibody, depending on antigen expression by
of ACT.
the tumor cell and the affinity for its Fc part, potentially enhancing efficacy and safety of either therapy
alone.
Methods: Different Fcγ fusion receptors consisting
of the CD16 extracellular domain and intracellular
T cell-activating molecules (CD16-CD28-CD3z) were
generated and were retrovirally transduced into
primary human T lymphocytes. Fusion receptor-expression was validated via flow cytometry. IFNγ
release by transduced T cells in the presence or abscence of cetuximab was analyzed in vitro.
Results: Transduction of CD16-CD28-CD3z fusion receptors in primary, human T lymphocytes mediated
antigen-specific T cell activation. In vitro coculture
Feasibility of telomerase-specific adoptive T-cell therapy for
hematologic and solid malignancies
Sandri S.1, Bobisse S.2, Moxley K.3, Lamolinara A.4, De Sanctis F.1, Boschi F.5, Sbarbati A.6, Fracasso G.1, Ferrarini G.1,
Hendriks R.W.7, Cavallini C.8, Scupoli M.T.1,8, Sartoris S.1, Iezzi M.4, Nishimura M.I.3, Bronte V.1, Ugel S.1
University of Verona, Department of Medicine, Verona, Italy,
1
Veneto Institute of Oncology, Familial Cancer Clinic and Oncoendocrinology, Padova, Italy,
2
Loyola University Medical Center, Department of Surgery, Maywood, United States,
3
CESI Aging Research Center, G. D’Annunzio University, Chieti, Italy,
4
University of Verona, Department of Computer Science, Verona, Italy,
5
University of Verona, Department of Neurological and Movement Sciences, Verona, Italy,
6
Erasmus MC, Department of Pulmonary Medicine, Rotterdam, Netherlands,
7
University of Verona, Interdepartmental Laboratory for Medical Research (LURM), Verona, Italy
8
Telomerase (TERT) is over-expressed in about
logic malignancies, such as B-cell acute lymphocytic
80-90% of primary tumors and contributes to sustain
leukemia (B-ALL) and acute myeloid leukemia (AML).
the transformed phenotype. The identification of
Moreover, TERT-based adoptive immunotherapy also
several TERT epitopes in tumor cells have elevated
efficiently controlled cancer progression of differ-
the status of TERT as a potential universal target for
ent solid transplantable tumors, such as melanoma,
selective and broad adoptive immunotherapy. We ex-
breast carcinoma and colon carcinoma, suggesting
plored the feasibility of telomerase as immunothera-
how the transduced anti-hTERT T-cells could be a
peutic target in a transgenic mouse model (IgH.TEµ)
potential “off-the-shelf” reagent applicable to treat
in which ageing generates B-cell chronic lympho-
many oncologic diseases. Even though engineered T
cytic leukemia (B-CLL) displaying common charac-
cell-based therapies have shown long-term efficacy
+
+
teristics with human B-CLL: expansion of CD19 /5
and promising curative potential for the treatment of
clonal cell population expressing high levels of active
cancer, several “on-target, off-tumor” toxicities have
telomerase, which is recognized both in vitro and
been reported. Since experimental models that can
in vivo by adoptively transferred mTERT-specific
predict potential toxic effects against human cells
cytotoxic T lymphocytes (CTLs). Moreover, even if
are currently not available, we could only investi-
TERT is a self antigen able to promote tolerance, we
gate the toxicity of hTERT-based immunotherapeutic
demonstrated that TERT-specific CTLs are detectable
approach towards the hematopoietic compartment.
in the peripheral blood of a B-CLL patient cohort.
Anti-hTERT T-cells did not result in a self-MHC-re-
However, these anti-TERT CTLs display a low func-
stricted fratricide of T cells and was associated with a
tional avidity, which limits their clinical utility in an
toxicity against mature granulocytes but not towards
adoptive cell transfer (ACT) approach. To overcame
human hematopoietic progenitors, as assessed in hu-
this key obstacle to immunotherapy, we isolated an
manized-immune reconstituted mice. Together these
HLA-A2-restricted TCR with high avidity for human
data support the feasibility of TERT-based adoptive
TERT from vaccinated, HLA-A*0201 transgenic mice.
immunotherapy in clinical oncology, highlighting,
TCR-transduced T cells were able to control in vivo
for the first time, the possibility to utilize a high
human B-CLL progression as well as other hemato-
avidity TCR specific for hTERT.
Receptor-transfected γ/δ T cells; the new magic bullets against
melanoma?
Simon B.1, Harrer D.1, Schuler G.1, Uslu U.1, Hoyer S.1, Dörrie J.1, Schaft N.1
1
Tumor-specific T cells play a critical role in spontane-
tokines, such as IL-2, IL-6, TNF and INFγ. Addition-
ous and therapy-induced tumor rejection. T cells that
ally, receptor-transfected γ/δ T cells efficiently lysed
were retrovirally transfected with tumor-specific
melanoma cells antigen-specifically, while the en-
T-cell receptors (TCRs) or chimeric antigen receptors
dogenous γ/δ TCR was still functional. Furthermore,
(CARs) yielded impressive clinical results after being
we are developing a protocol for the GMP-compliant
transferred into cancer patients, but significant side-
expansion and mRNA-electroporation of γ/δ T cells.
effects occurred.
Hence, we believe that these cells are a promising
To counteract these risks the receptors can be tran-
tool for T-cell-based immunotherapy of melanoma.
siently expressed by mRNA transfection. This,
however, also temporally limits the on-target activity
of conventional utilized CD8+ T cells.
γ/δ T cells constitute a subpopulation of peripheral T
cells, which are potent effectors recognizing metabolically abnormal cells, including tumor cells. Hence,
we equipped these cells with a desired tumor specificity by receptor-mRNA electroporation. Mispairing
of introduced and endogenous γ/δ TCR cannot occur
and as an additional benefit after a first burst of cytotoxic activity is exerted via the exogenous receptor,
these cells will maintain their anti-tumor activity via
the endogenous γ/δ TCR.
We introduced a gp100/HLA-A2-specific TCR and a
melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific CAR, which is CD8 independent,
into γ/δ T cells and observed that both receptors were
expressed efficiently on γ/δ T cells. Moreover, we
directly compared receptor-transfected CD8+ α/β T
cells and γ/δ T cells for their cytokine production and
lytic capacity. Both cell populations recognized endogenously processed gp100-antigen and exogenous
loaded gp100 peptide and consequently produced cy-
JD and NS share senior authorship
On- and off target toxicity profiling for adoptive cell therapy by
mass spectrometry-based immunopeptidome analysis of primary
human normal tissues
Fritsche J.1, Schoor O.1, Kutscher S.1, Mahr A.1, Stevermann L.1, Sonntag A.1, Hoffgaard F.1, Vahrenhorst D.1,
Leibold J.1, Goldfinger V.1, Alten L.1, Bunk S.1, Maurer D.1, Walter S.2, Rammensee H.-G.3, Singh-Jasuja H.1,
Weinschenk T.1
Immatics Biotechnologies GmbH, Tübingen, Germany,
1
Immatics US Inc., Houston, United States,
2
Institute for Cell Biology, University of Tübingen, Immunology, Tübingen, Germany
3
ORAL
TALK
SHORT
2016
A major constraint for the broad and safe application
ferences between normal tissues and tumors as well
of Adoptive Cellular Therapy (ACT) is the limited
as absolute peptide copy numbers per cell. In order
number of validated tumor targets, especially for
to assess the off-target risk for a TCR, we predict all
solid tumors. For T-cell receptor (TCR)-based ap-
theoretical HLA- and TCR-binding peptides in the
proaches, presentation of targeted HLA-peptides on
proteome, ideally based on the binding motif of the
normal tissues can lead to on-target toxicity, such as
TCR, and specifically search for actual peptide pres-
severe inflammatory colitis reported upon re-direct-
entation by normal tissues.
ing T cells to an HLA-A*02 restricted carcinoembry-
When analyzing the above described CEA case, we
onic antigen (CEA) epitope. Independently, off-target
were able to detect the CEA-derived peptide IMIG-
cross-reactivity of TCRs occurred in previous ACT
VLVGV not only on HLA-A*02+ colorectal cancer
trials, e.g. when a MAGEA3-directed TCR cross-rec-
samples, but importantly also on normal colorectal
ognized an HLA-A*01 restricted epitope from titin
samples. In the original study describing the titin
expressed on heart, which led to fatal cardiac tox-
case high experimental efforts and complex cell
icities. Here we present a novel approach allowing
culture models were required to retrospectively
the prediction of severe on- and off-target side effects
identify cross-recognition of the peptide on cardio-
before entering into clinical trials.
myocytes as the cause of toxicity. In contrast, with
We used a target discovery engine (XPRESIDENT)
our approach we could directly identify the critical
combining highly sensitive, quantitative mass spec-
peptide ESDPIVAQY as one of the most abundant-
trometry (LC-MS/MS), RNA-Seq-based differential
ly presented peptides on an HLA-A*01+ primary
transcriptomics, immunology and bioinformatics to
human heart sample. We show that this approach
characterize the human immunopeptidome directly
can lead to relevant results also for other pre-clinical
on shock frozen primary human tissues. Over the
and clinical stage TCR candidates.
last years we have built an according database for >
In conclusion our data demonstrate that ultrasensi-
600 tumor samples from > 20 different tumor types
tive LC-MS/MS of primary tissue may represent a
and, importantly, > 300 samples from > 40 differ-
fast, straightforward and meaningful complemen-
ent normal tissue types, resulting in hundreds of
tary method to common in vitro or animal models
thousands of unique HLA-peptide sequences. These
for the prediction of on- and off-target toxicities in
data allow conclusions on which HLA peptides are
TCR-based immunotherapy approaches.
actually presented on primary normal tissues in a
quantitative manner, taking into account relative dif-
Combining tumor antigen (TA) specific Th1 cells with immune
checkpoint blocking antibodies induces tumor regression in advanced carcinomas
Schörg B.F.1, Brenner E.2, Krüger D.1, Griessinger C.M.1, Braumüller H.2, Fehrenbacher B.2, Schaller M.2, Eichner M.3,
Kohlhofer U.4, Quintanilla-Martinez L.4, Pichler B.J.1, Röcken M.2, Kneilling M.1,2
Eberhard Karls University of Tuebingen, Werner Siemens Imaging Center, Department of Preclinical Imaging
1
and Radiopharmacy, Tübingen, Germany,
Eberhard Karls University of Tuebingen, Department of Dermatology, Tübingen, Germany,
2
Eberhard Karls University of Tuebingen, Department of Clinical Epidemiology and Applied Biometry,
3
Tübingen, Germany,
Eberhard Karls University of Tuebingen, Department of Pathology, Tübingen, Germany
4
ORAL
TALK
SHORT
2016
TA-specific IFN-y secreting CD4+ T cells (Th1) mediate
During CIT treatment, the median BGL of RT2 mice
strong anti-tumoral effects in mice and humans and
(n=30) increased from 80 mg/dl (before WBR) to nearly
can induce senescence in cancer cells in a TNF and
normal values of 91 mg/dl (wk 14) while the BGL of
IFN-y dependent manner (Braumüller et al. Nature
RT2 mice treated with TA-Th1 cells + isotype mAbs
2013). Immune and tumor cells express inhibitory
dropped to 66 mg/dl (n=30; p=0.0036). Thus, treat-
immune checkpoint (ICP) ligands (e.g. programmed
ment with TA-Th1 cells surprisingly delayed tumor pro-
death ligand 1 (PD-L1) and lymphocyte activation gene
gression even at this late state. In RT2 mice treated with
3 (LAG-3)) that paralyze tumor infiltrating T cells. ICP-
LAG-3 + PD-L1 mAbs alone (without TA-Th1 cells) the
blockade by specific antibodies such as PD-L1 and
BGL dropped similar to SHAM-treated (isotype Abs, n
LAG-3 can restore T cell functions. We aimed to es-
= 23) mice to values of ~ 40 mg/dl. As expected we
tablish a novel highly efficient TA-Th1 cell and check-
could extend in a longitudinal study the lifespan of the
point inhibitor-based combined immunotherapy (CIT)
CIT-treated WT mice from 14 to 21 wks. H&E histology
in RIP1-Tag2 (RT2) mice with advanced endogenous
and immunohistochemistry of the CIT-treated WT mice
pancreatic insular cell carcinomas where ICP-blockade
revealed very small tumors and a strong lymphocytic
alone is not sufficient.
infiltrate. In contrast, LAG-3 + PD-L1 mAb treated RT2
RT2 mice usually die at 14 weeks (wks) of age due to
mice exhibited large, vascularized endocrine tumors
low blood glucose levels (BGL). CIT was started in 10-11
with a slight lymphcytic infiltrate. Similar results were
wks old RT2 mice. At this stage BGL are reduced to 2/3
obtained in SHAM-treated RT2 mice without any in-
due to enhanced insulin secretion by the progressed
filtrating lymphocytes. Fluorescence microscopy of
insular cell carcinomas. Mice underwent a single 2 Gy
pancreatic tissue from CIT-treated RT2 exhibited high
whole body radiation (WBR) followed by i.p.-injections
p16 and low Ki67 expression in insular cell carcinomas.
of Tag2- Th1 cells (TA-Th1, 1x weekly) and of LAG-3
In sharp contrast, we observed opposite results (high
and PD-L1 mAbs (1-2 x weekly) or isotype Abs. Tumor
Ki67, low p16) in the RT2 control groups.
progression was monitored twice weekly by measuring
To our knowledge this is the first report combining a
BGL. We sacrificed mice at wk 14 and performed H&E
TA-Th1 cell based immunotherapy with ICP-blockade.
+
histology, immunohistochemistry (CD3 , B220) fluo-
CIT is applicable to reinforce Th1-cell based immuno-
rescent microscopy (p16, Ki67) of the pancreas tumor
therapies and to induce insular cell carcinoma regres-
tissue and flow cytometry to determine the distribution
sion even in mice with progressed carcinomas.
TA-Th1 cells in lymphatic organs and of PD-1, PD-L1,
LAG-3 by immune cells.
Two are better than one?! Improving safety for CAR T cell therapy
Schütt A.1, Makin G.1, Gilham D.E.1
University of Manchester, Institute of Cancer Science, Manchester, United Kingdom
1
Adoptive T cell therapy (ACT) harness patient derived
co-CARs will be co-expressed alongside fully com-
tumour tissue or blood to isolate endogenous T cells
petent anti-tumour receptors (TCR, 1st generation
and either activate directly or endow them with anti-
CAR) and compared to 2nd generation CAR T cells
cancer receptors. Chimeric antigen receptors (CARs)
based on their T cell activation profile. Our objec-
are artificial receptors comprising an antibody
tive is to prove the hypothesis that two anti-cancer
derived antigen binding domain of any specificity
receptors work as well as a 2nd generation CAR but
fused to the T cell receptor (TCR) signalling domain
show reduced toxicity to increase the safety of CAR
st
CD3 zeta, designated as first (1 ) generation CARs.
T cell therapy.
Incorporation of co-stimulatory signalling domains
For in vitro studies T cells from healthy donors have
nd
rd
and 3 generation of CARs. Co-stim-
been genetically engineered using retroviruses to co-
ulation supports and enhances long-time survival
express the various construct combinations (TCR/
and persistence of T cells, which is crucial for the
co-CAR, 1st CAR/co-CAR). To determine T cell ac-
nd
tivation CAR T cells were tested in co-culture with
generation anti-CD19 CARs have shown remarkable
tumour cell lines expressing the corresponding an-
success for the treatment of leukaemia. However, for
tigens or against native antigen. Interferon gamma
broader application some safety issues need to be
(IFN-y) and interleukin (IL)-2 concentrations were
addressed. For example, cytokine release syndrome
assessed as well as killing assays (WST-1) performed
arising from synchronized T cell activation was ob-
to gain insight into the activation profile of CAR T
served in patients. Additionally the lack of exclusive
cells. Initially we are focusing on CD28 as co-stim-
antigens present on tumour cells only is problematic
ulator, as 2nd generation CARs containing CD28 are
because targeting cancer antigens that are present
superior to others with further promising co-CARs
on non-tumour tissue causes side effects termed “on
to be investigated.
define the 2
treatment of cancer patients. Clinical trials using 2
target-off tumour”.
To control and reduce T cell cytotoxicity towards
healthy tissue we are using two, instead of one,
tumour specific receptors. The rationale is to split the
activation signal with full T cell activation depending on both receptors binding their corresponding
targets. We designed co-stimulatory CARs (co-CARs)
lacking CD3 zeta, which are insufficient to initiate
signalling but provide co-stimulatory support. These
Nanoparticle based antigen-specific redirection of T cells to
tumors
Schütz C.1,2, Perica K.1, Varela J.C.3, Haupt C.1, Oelke M.1,4, Schneck J.P.1
Johns Hopkins School of Medicine, Institute of Cell Engineering, Department of Pathology, Baltimore, United
1
States,
Paul-Ehrlich-Institute, Langen, Germany,
2
Hollings Cancer Center, Medical University of South Carolina, Department of Medicine, Division of
3
Hematology and Oncology, Charleston, United States,
NexImmune, Inc., Gaithersburg, United States
4
Immunotherapy is the modulation of a patient’s
tive immunotherapeutic approaches for all cancers
immune system to treat illness. Unfortunately many
that can be targeted with antibodies or antibody-like
T cell based attempts have failed due to the fact that
molecules. Furthermore, ATR could also be used
existing tumor-specific T cells are mostly anergic
in conjunction with virus-specific immunization to
or tolerized and ex vivo generated T cells are often
specifically increase the targeted CTL population.
already of exhausted phenotype. Here we present
Ultimately, we expect ATR and their potential for
a novel nanoparticle based approach to selectively
clinical applications to increase our understanding
target cytotoxic T cells (CTL) and re-direct them to
of tumor immunotherapy through T cell redirection.
kill tumors, termed ATR (Antigen-specific T cell Redirectors).
ATR were generated by coupling either MHC-Ig
dimer or clonotypic anti-TCR antibody 1B2 to target
the effector T cell population and an anti-CD19 to
re-direct those to CD19+ tumor target cells onto 50100nm nanoparticles. Flow cytometry and microscope based data confirm that the described ATR
phenotype efficiently and stably stain tumor and T
cells in a dose dependent manner and ATR mediate
antigen-specific conjugate formation of effector T
cells and tumor target cells. Antigen-specific ATR
mediated re-direction of T cells to tumor target cells
was demonstrated in 51Cr-release killing assays at low
E:T ratios. Variation of ATR target-cell : effector-cell
targeting molecule ratio could further increase efficacy. Finally, intra tumoral ATR injection induced T
cell re-direction and reduced tumor growth in a s.c.
Raji/SCIDbeige treatment model.
In summary this data demonstrates that ATR target
and redirect antigen-specific CTL to tumor cells that
would otherwise not be recognized and mediates
their lysis. ATR can be used to develop new innova-
Retrieval of functional TCRs from single neo-antigen-specific T
cells: Toward individualized TCR-engineered therapies
Simon P.1, Tolliver C.1, Breitkreuz A.1, Felz-Gleitz U.1, Omokoko T.1, Hebich L.1, Steege B.1, Hammerton C.1,
Ortseifer I.2, Godehardt E.2, Derhovanessian E.3, Kasemann B.4, Walter G.4, Schrörs B.4, Löwer M.4, Attig S.4,
Miller M.3, Sahin U.3
1
BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany,
2
BioNTech RNA AG, Mainz, Germany,
3
TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg
4
University gGmbH, Mainz, Germany
ORAL
TALK
SHORT
2016
Increasing clinical evidence supports the hypoth-
from endogenously expressed protein in the context
esis that patient-specific immunogenic products of
of HLA-A*02:01 molecules by autologous melanoma
somatic mutations, so-called neo-antigens, are the
cells and that T cells directed against these mutations
relevant targets for successful immunotherapies. The
could be efficiently amplified by IVAC® MUTANOME
IVAC® MUTANOME clinical trial is a first-in-human
vaccination. Furthermore, these data underline that
study evaluating the safety, tolerability and immu-
in single cases the loss of HLA surface expression, a
nogenicity of intra-nodal administration of a person-
known tumor escape mechanism, could potentially
alized vaccination with IVAC® MUTANOME mRNA
limit therapeutic efficacy of anti-tumor responses by
vaccine in patients with advanced melanoma. The
vaccine-induced tumor-reactive T cells. In total we
IVAC trial is a proof-of-concept study incorporating
identified 12 mutation-specific TCRs from three vac-
15 melanoma patients with a comprehensive bio-
cinated patients. The majority of TCRs exclusively
marker program to analyze in depth the induced T
recognized the mutated sequences, while only one
cell responses and the mode of action of the vaccine.
TCR recognized the respective wild-type version. We
In order to characterize the vaccination- induced mu-
established a process for robust discovery and char-
tation-specific T cell responses we applied BioNTech
acterization of individual neo-antigen specific TCRs.
Cell & Gene Therapies’ TCR discovery & characteriza-
This approach enables rapid extraction of multiple
tion platform to isolate functional TCRs from single T
TCRs from repertoires of individuals and sets the
cells of individual mutation-specific repertoires. We
stage for actively personalized immunotherapeutic
isolated single mutation-reactive T cells from three
strategies.
vaccinated melanoma patients and cloned the corresponding TCR genes. For one patient with T cell
responses against eight neo-antigens after IVAC vaccination, we discovered TCR clonotypes from peripheral blood and tumor-infiltrating lymphocytes that
were finally shown to confer specific recognition and
killing of an autologous melanoma cell line. The latter
required restoration of HLA class I surface expression by beta-2-microglobulin (B2M) RNA transfer as
the cell line was shown to have a complete deletion of
the B2M locus. These studies demonstrate that the respective neo-antigens were processed and presented
Novel immunotherapies for recurrent glioblastoma: The efficacy
of CD133 BiTEs and CAR T cells in preclinical models
Singh S.1, Moffat J.2, Vora P.1, Venugopal C.1, Bramson J.3, Adams J.2, Sidhu S.2
McMaster University, Stem Cell and Cancer Research Institute, Hamilton, Canada,
1
University of Toronto/ Donnelly Centre, Toronto, Canada,
2
McMaster University, Hamitlon, Canada
3
ORAL
TALK
SHORT
2016
Glioblastoma (GBM) is feared for its near uniformly
tions and dual binding specificity to both the tumor
fatal prognosis and is characterized by cellular and
antigen CD133 and the CD3 T cell receptor was con-
genetic heterogeneity. Poor survival of glioblasto-
firmed using flow cytometry. Using both CD133high
ma (GBM) patients correlates with the presence of
and CD133low primary patient-derived GBM lines,
CD133+ brain tumor-initiating cells (BTICs), which
we observed binding of BiTEs to CD133+ cells and
are also implicated in the development of treatment
to CD3+ T cells derived from PBMCs. We co-cultured
resistance in GBM. We have recently demonstrated
CD133high and CD133low lines with CD3+ T cells
that a CD133-driven gene signature is predictive of
in the presence and absence of BiTEs. Strikingly, we
poor overall survival and targeting CD133+ treat-
observed CD133-specific BiTEs redirecting T cells to
ment-refractory disease reservoirs may be an effec-
kill CD133-expressing GBM cells, with greater killing
tive strategy to block GBM recurrence.
efficiency observed in CD133high lines, validat-
The adoptive transfer of T cells genetically modi-
ing BiTE target specificity. In the absence of BiTEs,
fied to express chimeric antigen receptors (CARs)
T-cells did not target CD133+ GBMs, and incubating
and bispecific T-Cell engaging antibodies (BiTEs)
T cells with BiTEs and the CD133high line resulted in
present promising immunotherapeutic approaches
increased surface expression of both T-cell activation
that have not yet been validated for recurrent or re-
markers CD69 and CD25 in both CD4+ and CD8+ T
lapsed GBM. Using CellectSeq, a novel methodology
cell populations.
that combines use of phage-displayed synthetic an-
We have uniquely adapted the existing chemoradio-
tibody libraries and high-throughput DNA sequenc-
therapy protocol for GBM patients for treatment of
ing, human CD133-specific synthetic monoclonal
NOD-SCID mice engrafted with human GBM BTICs.
antibody ‘RW03’ was constructed. The sequence
Our in vivo mouse-adapted therapy patient-derived
for RW03 was cloned into a cDNA encoding a CAR
xenograft (PDX) model has the distinct advantage of
scaffold with CD28 and CD3zeta signaling domains
generating recurrent, human, treatment-refractory
and a myc tag. T-cells were engineered to express
GBM. Within this model, we have initiated treatment
CAR using a lentivirus that also expressed truncated
of recurrent GBM with our novel immunotherapeutic
NGFR as a reporter gene. CD133-specific CAR T cells
modalities directed against CD133+ BTICs, to allow
were cytotoxic to CD133+ GBM cells. Co-culturing
for a direct prospective comparison of toxicity and
CD133 CAR-T cells with GBM cells triggered T cell ac-
efficacy of BiTEs and CAR T cell strategies in a highly
tivation as measured by surface expression of CD69
relevant and innovative model of recurrent GBM.
and CD25. In parallel, we used RW03 IgG to construct
CD133-specific BiTEs in four different conforma-
Serum replacement might substitute human serum in the GMP
production of Dendritic Cells
Solum G.1, Honnashagen K.1, Kvalheim G.1, Bigalke I.1
Oslo University Hospital, Department of Cellular Therapy, Oslo, Norway
1
The use of human serum for GMP production of DCs
tocol. CD80 was more upregulated in immature DCs
is still essential to obtain cells of optimal functional-
(iDCs) in serum free conditions compared to control
ity. A problem we are now facing is the decline in
cells, whilst CD86 was higher upregulated in con-
availability of GMP grade human serum. We have
trols. CD14 was down-regulated in iDCs and mDCs,
therefore tested different alternatives of serum free
but the MFI was higher in SR and serum free media
media/serum replacement for production of DCs.
than in the control. The shift in CD14 from iDCs to
In our current clinical dendritic cell protocol, mono-
mDCs was higher in SR than in the control, indicat-
cytes are differentiated into immature DCs (iDCs)
ing that the downregulation started at an earlier time
with IL-4 and GM-CSF, and matured with a cytokine
point in the control cells. The other DC markers did
cocktail containing IL-1b, TNF-a, IFN-g, R848 and
not show significant variations between the different
PGE2 (Zobywalski et al. 2007, Subklewe et al. 2014) in
media. iDCs cultivated in SR media showed a more
VLE RPMI 1640 media with 1,5 % human AB serum.
mature phenotype than control cells and iDCs culti-
The main characteristics of the mature DCs (mDCs)
vated in T cell media. Signal 3 assays revealed that
are high upregulation of maturation markers, and IL-
the mDCs cultivated in serum free media produced
12p70 release combined with low IL-10 release in an
more IL-10 than IL-12p70. SR showed a concentration
in vitro signal 3 assay.
dependent release of IL-12p70 and IL-10; 2,5-10 % SR
In our experiments we used the standard DC pro-
yielded cells that secreted high amount of IL-12p70
tocol as controls. Two different serum free media
and lower amount of IL-10. The lowest concentration
originally developed for T cell expansion and CST
of SR (1 %) gave more IL-10 release than IL-12p70.
immune cell serum replacement (SR) in concentra-
Control cells produced IL-12p70, but lower amounts
tions ranging from 1-10 % in VLE RPMI medium were
than 2,5-10 % SR.
tested. Phenotyping of immature and mature den-
In summary, the culture conditions tested gave no
dritic cells was done by flow cytometry investigating
significant differences of the phenotype of the DCs.
the markers CD80, CD86, CD40, CD14, CD83, CCR7
Our results in the SR experiments look promising
and HLA-DR. IL-10 and IL-12p70 release was meas-
since those DCs produced high IL-12p70 and low
ured after stimulation of mDCs with CD40 ligand
levels of IL-10. More extensive testing of functional-
using standard ELISA kits.
ity of the different types of DCs is ongoing and results
Frozen monocytes from a healthy donor and from
will be presented.
cancer patients were used as starting material. Yield
and viability of mDCs in serum free alternatives were
comparable to mDCs generated with the standard pro-
Characterization of recognition profiles of TCRs by a novel
DNA-barcode based multiplex technology
Such L.1, Bentzen A.K.1, Lyngaa R.1, Becker J.C.2, Linnemann C.3,4, Hadrup S.R.1
Technical University of Denmark, National Veterinary Institute, Section for Immunology and
1
Vaccinology, Frederiksberg C, Denmark,
University Hospital Essen and University of Duisburg-Essen, Translational Skin Cancer Research, Essen,
2
Germany,
Netherlands Cancer Institute, Department of Immunology, Amsterdam, Netherlands,
3
Kite Pharma Europe, Amsterdam, Netherlands
4
Adoptive cell transfer of TCR-transduced T cells has
of the HLA-B*07-LTA APN -specific T cells towards HLA
shown strong tumor rejection capacity of the TCR-
matched target cells. To characterize the recognition
transduced T cells, but also an inherent risk of cross
profile of this TCR we generated a library of 32 pep-
reactivity that can induce lethal side effects based
tides with alanine substitutions, N- and C-term addi-
on the potency of these T cells upon target recogni-
tions and length variations to identify the preferred
tion. Hence, it is crucial to understand the precise
recognition fingerprint of this TCR. Extending to this
recognition element of the TCR prior to clinical in-
we will generate a larger library of peptides carrying
vestigation. Here, we present a novel tool that allows
this fingerprint, to assess the fine-specificity.
description of an affinity-based hierarchy of pMHC
In summary, this provides proof-of-concept for the
interaction using large libraries of peptide variants
use of a novel technology to identify TCRs recogni-
of the originally identified recognition element. We
tion profiles, and may prove highly valuable for the
make use of DNA-barcode labeled MHC multimers
assessment of TCRs prior to clinical investigations.
that allow simultaneous screening for T cell recognition of multiple (> 1000) different peptide specificities in a single sample. Importantly, the relative
contribution of different pMHC molecules can be assessed using this technology - a feature not possible
by conventional flow-based MHC multimer analyses.
This technology will enable us to analyze the hierarchy for recognition of large libraries of different
peptides and therefore describe the fine-specificity
and redundancy of a given TCR.
We have previously characterized CD8+ T cell responses of Merkel cell Carcinoma (MCC) patients,
directed towards the Merkel Cell Polyomavirus-encoded Large T Antigen (LTA). From these MCC responsive T cells we identified a HLA-B*07 restricted
LTA epitope: APNCYGNIPL, and sequenced the corresponding TCR. By generation of TCR transduced T
cells we demonstrated processing and presentation
of the LTA-derived epitope, as well as lytic activity
Human dendritic cells pulsed with high hydrostatic pressureinactivated prostate cancer cells and matured with poly(I:C)
induce autologous lymphocytes to ex vivo recognize and kill
prostate cancer cells
Taborska P.1, Stakheev D.1, Vavrova K.1, Vrabcova P.1, Bartunkova J.1, Smrz D.1
2nd Faculty of Medicine and University Hospital Motol, Charles University in Prague, Institute of
1
Immunology, Prague, Czech Republic
High hydrostatic pressure (HHP) has previously been
shown to trigger immunogenic cell death in multiple
cancer cell lines. Human dendritic cells (DCs) pulsed
with HHP-inactivated prostate cancer cell line LNCaP
was found to induce proliferation of autologous lymphocytes. These lymphocytes then readily responded
to a re-challenge with the DCs. However, what impact
these lymphocytes might have on living LNCaP cells
is not known. Here we show that monocyte-derived
immature DCs from healthy donors that were pulsed
with HHP-inactivated LNCaP cells and stimulated
with TLR3 agonist polyinosinic:polycytidylic acid
(poly(I:C)) have an enhanced surface upregulation
of DC maturation markers CD80, CD83, CD86 and
HLA-DR, and a capacity to induce a strong expansion of autologous lymphocytes. Such the expanded
lymphocytes were found to elicit a cytotoxic response once exposed to living LNCaP cells but not
to a control ovarian cancer cell line SKOV-3. Importantly, autologous lymphocytes that were not previously induced by the DCs did not show a cytotoxic
response to either of the tested lines. Our data indicate that DCs loaded with HHP-inactivated prostate
cancer cells and matured with poly(I:C) may induce
immune response that leads to expansion of lymphocytes that are able to recognize and kill prostate
cancer cells.
Identification of a HLA-A*0201-restricted immunogenic epitope
from the universal tumor antigen DEPDC1
Tosi A.1, Sommaggio R.1, Cappuzzello E.1, Zanovello P.1, Rosato A.1
University of Padova, Padova, Italy
1
With the discovery of tumor-specific and tumor-as-
nodominant epitope, out of ten candidates tested,
sociated antigens, cancer vaccine strategies as well
was capable of inducing CTL populations producing
as adoptive cell therapy with antigen-specific T cells
IFN-γ and exerting a specific and relevant cytotoxic
have become a promising therapeutic approach. Re-
activity in response not only to peptide-loaded cells
cently, DEPDC1 has been described to play an im-
but also to HLA-A*0201-positive tumor cells that en-
portant role in cancer cell growth/survival, as its
dogenously express the DEPDC1 protein. In in vivo
siRNA-mediated knock down suppresses tumor cell
experiments, the peptide-specific CTL population
growth and increases the number of apoptotic cells,
delayed tumor growth and metastatic process in a
while its overexpression is linked to a poor prog-
human breast cancer xenograft mouse model. These
nosis in patients with different tumor histotypes.
findings indicate that the HLA-A*0201-restricted DE-
These data suggest an important involvement of this
PDC1-derived peptide is a putative tumor antigen
protein in tumor progression, and therefore its im-
that could be exploited in immunotherapy against
munological targeting could represent an important
different tumors overexpressing the DEPDC1 protein.
strategy to counteract tumor growth and metastasis.
Our study aimed at identifying HLA-A*0201-restricted DEPDC1-derived immunogenic peptides, to be
used for the generation of cytotoxic T cells for adoptive therapy. The Oncomine database confirmed the
wide overexpression of DEPDC1 in tumors and its
complete absence in normal tissues, indicating that
it could be well regarded as a universal oncoantigen, and might represent a potential and safe target
for immunotherapy. Thereafter, protein expression
was assessed in a large set of tumor cell lines, and
several HLA-A*0201-restricted candidate peptides
were identified by integrating the results of three
epitope prediction programs (BIMAS, NetMHC and
NetCTL), with the final aim to assess their capacity
to induce peptide-specific cytotoxic T lymphocytes
(CTLs) from peripheral blood mononuclear cells of
HLA-A*0201-positive healthy donors. One immu-
Targeting of recurrent somatic cancer mutations for T cell
receptor gene therapy
Tubb V.1, Long H.1, Lee S.1, Bendle G.1
University of Birmingham, Institute of Immunology and Immunotherapy, Birmingham, United Kingdom
1
The introduction of tumour-specific T cell receptor
cell co-cultures and intracellular cytokine staining,
(TCR) α and β genes can rapidly endow patient T
these TCRs displayed fine peptide specificity for
cells with tumour specificity. The clinical testing of
mutant peptides over their wildtype counterparts.
engineered T cell therapies against cancer is garner-
Some TCRs were also capable of recognising cell
ing increasingly promising results. However, such
lines engineered to express the mutation derived
studies have highlighted the critical importance of
neo-antigens, suggesting that these antigens may be
identifying target antigens both for therapeutic ef-
naturally processed and presented by cancer cells.
ficacy and in preventing life threatening toxicity.
Further assessment of reactivity against a wider
In this respect, neo-antigens arising from somatic
range of mutated cancer cell lines and mass spectral
mutations in cancer cells represent attractive targets
analysis of eluted MHC I peptides, will aid investiga-
as they are a class of truly specific cancer antigens
tion of the applicability of these recurrent mutation-
that are likely to be safe targets. Since some somatic
specific TCRs for the treatment of cancer.
cancer mutations occur recurrently between patients,
neo-antigens derived from such mutations represent
particularly attractive targets for widely-applicable
TCR gene therapy. Therefore, we are investigating
whether recurrent cancer mutations encode immunogenic peptide epitopes presented by common HLA
class I alleles and isolating TCR genes specific for
such neo-antigens.
We therefore generated a panel of putative neo-antigen peptides derived from recurrent somatic mutations and explored if an experimental protocol
that combines MHC class I tetramer technology, cell
sorting methodologies and TCR gene sequencing,
can be used to isolate neo-antigen specific TCR genes
from the blood of healthy donors.
A number of TCRs specific for HLA-A*03:01, HLAA*11:01, and HLA-B*07:02 restricted putative peptides generated by recurrent point mutations and
frameshifts were successfully isolated. Using target
Generating T cells expressing two additional receptors
(TETARs) by combining a chimeric antigen receptor and a
conventional T-cell receptor for multi-hit cancer immunotherapy
Uslu U.1, Schuler G.1, Dörrie J.1, Schaft N.1
Universitätsklinikum Erlangen, Department of Dermatology, Erlangen, Germany
1
Introduction: The adoptive transfer of engineered T
cells antigen-specifically at least as good as T cells
cells represents an important approach in the immu-
transfected with a single TCR or a single CAR. The
notherapy of cancer. However, relapse of the tumor
generation of TETARs using different ratios of RNA
can occur due to immune escape mechanisms de-
encoding the gp100 TCR and the MCSP CAR resulted
veloped by the tumor cells, e.g. antigen loss, down-
in cytokine secretion and cytolytic capacity at all
regulation of the MHC presentation machinery, and
used RNA ratios. Regarding the cytolytic kinetics,
defects in antigen processing. To counteract these
the cytolytic capacity of TETARs was similar at 18
immune escape mechanisms, it would be advanta-
hours, 40 hours, and 66 hours after electroporation of
geous to equip T cells with multiple specificities and
the T cells. A higher cytokine secretion and a better
MHC-independent receptors.
cytolytic capacity by TETARs was observed when the
Methods: To study the possible interference of a
TETARs were compared to a 1:1 mixture of two T-cell
T-cell receptor (TCR) with a chimeric antigen recep-
pools, each T-cell pool transfected with one receptor
tor (CAR) after transfer into one T cell and to examine
only, either the TCR or the CAR. This confirms that a
how to counteract possible competing effects, we
TETAR is indeed able to recognize either one of the
+
generated TETARs, CD8
T cells expressing two
antigens, or both at the same time.
additional receptors, by simultaneous transfection
Conclusions: The generation of dual-specific CD8+ T
with a TCR and a CAR using RNA electroporation.
cells transfected with a CAR and a TCR is feasible. No
The TETARs were equipped with a TCR specific
reciprocal inhibition and even some additive effects
for an epitope from the common melanoma-asso-
regarding functionality were observed. The genera-
ciated antigen glycoprotein 100 (gp100), presented
tion of TETARs helps by-passing major mechanisms
in HLA-A02 and a CAR recognizing the melanoma
by which tumor cells escape immune recognition
surface antigen melanoma-associated chondroitin
and the confirmation that chimeric antigen receptors
sulfate proteoglycan (MCSP).
and T-cell receptors can be functionally combined
Results: Cell surface staining of transfected CD8+
opens up new avenues in cancer immunotherapy.
T cells showed that TETARs can be generated by
simultaneous
transfection
of
receptor-encoding
mRNAs using electroporation. Regarding functionality, antigen-specific cytokine secretion efficiency
of the TETARs was similar to - or even better than
- that of T cells transfected with a single TCR or a
single CAR. Also, TETARs were able to lyse target
A novel stabilized single chain TCR format allows for the
generation of virus/tumor antigen-bispecific human T-cells and
prevents mispairing with endogenous TCRs
Knies D.1, Klobuch S.2, Xue S.-A.3, Birtel M.4, Echchannaoui H.5, Yildiz O.6, Omokoko T.6, Guillaume P.7, Romero P.8,
Stauss H.3, Sahin U.4, Herr W.2, Theobald M.5, Thomas S.2, Voss R.-H.9
Municipal Clinic Karlsruhe, Karlsruhe, Germany,
1
University Hospital & Center of Interventional
2
Immunology, University of Regensburg, Department
of Internal Medicine III, Regensburg, Germany,
University College London, Royal Free Hospital,
3
Institute of Immunity & Transplantation, London,
United Kingdom,
Biopharmaceutical New Technologies (BioNTech)
6
Corporation, Mainz, Germany,
Ludwig Cancer Research Center, TCMetrix, Epalinges,
7
Switzerland,
Ludwig Cancer Research Center, Translational Tumor
8
Immunology Group, Epalinges, Switzerland,
University Medical Center of the Johannes Gutenberg-
9
University Medical Center, Johannes Gutenberg
University, Research Center for Immunotherapy
University Mainz, Translational Oncology (TRON),
(FZI), Mainz, Germany
4
Mainz, Germany,
University Medical Center & University Cancer
5
Center, Johannes Gutenberg University Mainz, Third
Department of Medicine, Mainz, Germany,
One branch of Immunotherapy of cancer aims at
This mutual exclusion allowed for the generation
adoptively transferring T-cells genetically modified
of
with tumor-specific heterodimeric α/β T-cell recep-
tumor associated antigen (TAA)-bispecific T-cells
tors (TCRα/β). However, potential mispairing of in-
to augment T-cell activation in CMV-infected tumor
troduced TCRα/β-chains with endogenous β/α-ones
patients. This kind of a donor lymphocyte infusion
may evoke unpredictable autoimmune reactivities. A
(DLI) is envisioned for the treatment of immunosup-
novel single chain (sc)TCR format relies on the fusion
pressed CMV+ leukemia patients after bone marrow
of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain
transplantation who often suffer from a severe reac-
and coexpression of the TCR Cα-domain capable of re-
tivation of CMV.
cruiting the natural CD3-complex for full and hence,
Moreover, residual mispairing was prevented by
native T-cell signaling. Here, we tested whether such
strenghtening the Vα-Li-Vβ-fragment through the
a gp100(280-288)- or p53(264-272) tumor antigen-spe-
design of a novel disulfide bond between a Vα- and
cific scTCR is still prone to mispairing with TCRα via
a linker-resident residue close to Vβ. Multimer-stain-
RNA-transfer or retroviral transduction. In a human
ings, and cytotoxicity-, Perforin/IFNγ-secretion-, and
Jurkat-76 T-cell line lacking endogenous TCRs,
CFSE-proliferation-assays, the latter towards dendrit-
surface expression and function of a scTCR could
ic cells endogenously processing RNA-electroporated
dc/scTCR-modified
cytomegalovirus
(CMV)/
be reconstituted by any cointroduced TCRα-chain
gp100 antigen, proved the absence of hybrid scTCR/
indicating mispairing to take place on a molecular
TCRα-formation without impairing avidity of scTCR/
basis. In contrast, expression in human endogenous-
Cα in T-cells. Additionally, an unstable cytomega-
ly TCRα/β-positive T-cells revealed that mispairing is
lovirus pp65(495-503)-specific scTCR modified this
largely reduced. Competition experiments in Jurkat-
way acquired enhanced cytotoxicity and may accom-
76 confirmed the preference of dcTCR to selfpair and
plish the generation of even more mutually exclusive
to spare scTCR.
sc/scTCR-engineered CMV/TAA-bispecific T-cells.
In human T-cells, we also observed mispairing of a
human scTCR to take place with just the Cα-domain
of an endogenous TCRα. This side reaction was referred to as TCR Cα-mispairing to distinguish it from
conventional TCRα-mispairing scrutinized here.
TCR Cα-mispairing could not be reproduced for a
high-affinity mouse scTCR. This different behavior
will be explained with a partial unfolding model
by which the human Vα-domain of an endogenous
TCRα transiently senses the presence of a human or
a mouse Vβ-domain in a given scTCR for efficient
intra-species chain pairing.
Conclusively, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive
T-cells.
A universal killer T-cell for adoptive cell therapy of cancer
Inderberg E.M.1, Myklebust J.H.2, Mensali N.1, Skorstad G.1, Myhre M.R.1, Fåne A.1, Gaudernack G.2, Kvalheim G.1,
Wälchli S.1,2
OUS-Radiumhospitalet, Cellular Therapy, Oslo, Norway,
1
OUS-Radiumhospitalet, IKF-Cancer Immunology, Oslo, Norway
2
T cell-based therapy has generated remarkable remis-
from CD8 and CD4 T cells demonstrated that UK-92-
sions in hard-to-beat cancers and represents a large
TCR could be stimulated in a pMHC-specific manner.
part of innovations in immunotherapy. Adoptive
We have now shown in vitro that UK-92 cells are as
T-cell transfer (ACT) is a labour intensive method in
specific and potent as redirected T cells to kill target
terms of logistics and mainly depends on the quality
cells. The use of UK-92 as a universal killer cell line
of the patient’s T cells. We have developed a uni-
can anticipate that this technology, if confirmed ef-
versal cell line for TCR expression by modifying the
ficient in vivo, will speed up ACT production time.
FDA-approved NK cell line, NK-92. One of the ad-
In addition the use of UK-92 will bypass other chal-
vantages of this cell line is that it has retained its
lenges related to the use of autologous patient T cells.
killing capacity and can easily be genetically engineered. However, tumour cell recognition by NK-92
is not specific. This can be overcome by introducing
an antigen receptor, such as a chimeric antigen receptor (CAR) or, as in the current work, a TCR. We
herein present evidence that NK-92 can be modified
to become a T cell-like lymphocyte. Complementing
the inherent killing activity of the NK cells with the
specific targeting of cancer antigens through TCR
could represent a perfect combination to prevent
tumor evasion. We named this novel cell line UK-92,
for Universal Killer derived from NK-92, and showed
that UK-92 expressing a therapeutic TCR conserved
the binding capacity to cognate pMHC. Phosphoflow
cytometry was used to verify that the introduced
TCR in UK-92 would mediate intracellular signaling upon crosslinking or by cognate pMHC binding.
Our data show that both early and late TCR signalling players were activated in a TCR-specific manner
(anti-CD3/anti-CD28 stimulation) and further in a
pMHC specific manner upon specific TCR binding.
Finally, functional assays using both TCR isolated
Enhancing the effector functions of T cells with a combination
of new mRNA adjuvants for improving adoptive cell therapy
Weinstein-Marom H.1,2, Pato A.1,2, Levin N.1,2, Susid K.2, Itzhaki O.3, Besser M.3, Peretz T.1, Lotem M.1, Margalit A.2,4,
Gross G.2,4
Hadassah Hebrew University Hospital, Jerusalem, Israel,
1
MIGAL - Galilee Research Institute, Kiryat Shmona, Israel,
2
Ella Lemelbaum Institute for Melanoma, Sheba Medical Center, Ramat Gan, Israel,
3
Tel-Hai College, Upper Galilee, Israel
4
Different approaches for adoptive cell therapy (ACT)
in peripheral blood-derived human T cells and anti-
of cancer with antitumor T cells following their
melanoma sTILs and yTILs. We have found that: i)
propagation and manipulation ex-vivo are currently
Membrane cytokines could replace soluble IL-2 in
explored worldwide. These include the use of autolo-
supporting proliferation of human CD8 and CD4 T
gous tumor-infiltrating lymphocytes (TILs) selected
cells ex-vivo for at least 6 days post-mRNA trans-
for tumor recognition (sTILs) or short-term cultured,
fection. ii) Binding of membrane cytokines to their
non-selected ´young´ (y)TILs and polyclonal T cells
cell-surface receptors mainly occurred in-cis. iii) The
genetically reprogrammed to express tumor-reactive
mere expression of membrane cytokines, caTLR4 and
TCR or chimeric antigen receptor (CAR). Yet, severe
caCD40 in polyclonal human T cells and anti-mela-
hurdles still limit the clinical outcome and broader
noma TILs induced massive production of IFN-g and
application of ACT. Among these are full T cell differ-
exceptional upregulation of 4-1BB, OX40, CD25, CD28
entiation and exhaustion following lengthy ex-vivo
and CD69. iv) No elevation in the inhibitory molecules
propagation, the presence of inhibitory cells, im-
CTLA4 and PD1 could be detected following mRNA
munosuppressive tumor microenvironment and the
transfection. v) These genes could exert remarkable
dependence of T cell survivability on the systemic
synergistic effects. vi) Three days post-transfection,
administration to patients of high-dose IL-2, which
after the initial effect had already waned, a consider-
is often intolerably toxic.
ably larger fraction of transfected, compared to non-
We have developed three classes of genetic adju-
transfected TILs, responded robustly to autologous
vants for improving T cell-based ACT: 1) Membrane-
melanoma, but not to mismatched melanoma. vii)
anchored cytokines which continuously provide T
Enhanced killing of autologous melanoma cells by
cells with high level of the respective cytokine in-cis,
transfected yTILs was clearly observed 4 days post-
focusing on IL-2, IL-12 (single chain) and IL-15. 2)
transfection and some enhancement could still be
Constitutively-active (ca) toll-like receptors, in par-
detected a week later.
ticular caTLR4. 3) Self-oligomerizing, constitutively-
Taken together, these genes offer a new powerful tool
active tumor necrosis factor receptors, concentrating
for augmenting the anti-tumor reactivity of T cells
on caCD40. We employ electroporation of in-vitro-
and can potentially be implemented in different ap-
transcribed mRNA as a safe and efficient delivery
proaches for cancer ACT.
method which allows the co-expression of multiple
genes.
In a series of experiments we co-expressed each of
the 3 membrane cytokines with caTLR4 and caCD40
Tumorantigen-Specific CD40-activated B cells for cancer
immunotherapy
Wennhold K.1, Thelen M.1, Haustein N.1, Shimabukuro-Vornhagen A.1, von Bergwelt-Baildon M.1
University Hospital Cologne, Cologne Interventional Immunology (CII), Department I of Internal Medicine,
1
Cologne, Germany
The therapeutic activation of T cells has demonstrated significant clinical potential, but for technical and
regulatory reasons clinical application of the most
promising approaches is currently limited. Here, we
report of an alternative strategy for cell therapy that
combines the specific induction of a tumor-directed
T-cell response together with antibody production
without the need of genetic engineering. Murine or
human B cells specific for tumor antigens were isolated by use of antigen-biotin tetramers. Stimulation
via CD40 ligand resulted in the development of an
antigen-presenting phenotype and the induction of
a strong antigen-specific T-cell response in vitro and
in vivo. These cells showed a tumor-specific homing
pattern in mice. Differentiation of OVA-specific B cells
into antibody-secreting plasma cells was achieved
by stimulation with interleukin-21 and CD40 ligand.
Prophylactic and therapeutic treatment of tumorbearing mice with OVA-specific CD40B cells and
antibody-secreting plasma cells led to an anti-tumor
immune response resulting in regression of tumors
and a prolonged survival. Our results provide novel
biological insights into the role of antigen-specific B
cells as antigen-presenting cells and provide the rational for their use for cancer immunotherapy.
Preclinical development of Tumor-Infiltrating Lymphocytes (TILs)
based Adoptive Cell Transfer Immunotherapy (ACT) for patients
with advanced ovarian cancer
Westergaard M.C.W.1, Andersen R.1,2, Kjeldsen J.1, Hasselager T.3, Lajer H.4, Donia M.1,2, Svane I.M.1,2
Center for Cancer Immune Therapy, Copenhagen University Hospital, Department of Hematology, Herlev,
1
Denmark,
Copenhagen University Hospital, Department of Oncology, Herlev, Denmark,
2
Copenhagen University Hospital, Department of Pathology, Herlev, Denmark,
3
Copenhagen University Hospital, Rigshospitalet, Department of Gynaecology, Copenhagen, Denmark
4
Non-melanoma solid malignancies are frequently
obtained from >80 % of the patients across different
infiltrated with T-cells, and the recent successes of
ovarian cancer histologies, including rare variants
adoptive cell transfer immunotherapy (ACT) with
such as ovarian carcinosarcomas.
tumor infiltrating lymphocytes (TILs) in melanoma
These findings support the hypothesis that patients
warrant its testing in other tumor histologies.
with ovarian cancer can benefit from ACT with TILs,
In this preclinical study, we have been investigating
and led to the initiation of a pilot clinical trial at our in-
whether clinical-grade TILs could be manufactured
stitution (clinicaltrials.gov identifier: NCT02482090).
from tumor specimens of patients with advanced
ovarian cancer. Tumor-specificity was assessed with
high-sensitive testing of three individual antitumor
functions after co-culture with autologous tumor
cells.
Tumor specimens were obtained from 35 patients
with advanced ovarian cancer. Minimally expanded TILs (Young TILs) were successfully established
from all patients. Young TILs contained a very high
frequency of CD3+ cells (88%±11.1, range 43-98)
with a highly variable CD4/CD8 ratio (5±6.8, range
0.003-38.64). TILs could be further expanded to
clinical numbers with the rapid expansion protocol
(REP), reaching an average fold expansion over 2000
(2079±1333, range 440-5544).
Importantly, recognition of naturally presented autologous tumor antigens was demonstrated in TILs
Targeted NK cells display potent activity against
glioblastoma and induce protective antitumor immunity
Zhang C.1,2, Burger M.C.2,3,4, Jennewein L.5, Genßler S.1, Schönfeld K.1, Mittelbronn M.5, Tonn T.6, Steinbach J.P.2,3,
Wels W.S.1,2
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany,
1
German Cancer Consortium (DKTK), Heidelberg, Germany,
2
Institute for Neurooncology, Goethe University, Frankfurt am Main, Germany,
3
German Cancer Research Center (DKFZ), Heidelberg, Germany,
4
Edinger Institute, Goethe University, Frankfurt am Main, Germany,
5
German Red Cross Blood Donation Service North-East, Dresden, Germany
6
Significant progress has been made over the last
toma cultures and demonstrated selective in vitro cell
decade towards realizing the potential of natural
killing that was dependent on the ErbB2 expression
killer (NK) cells for cancer immunotherapy. NK cells
by the target cells. Potent in vivo antitumor activ-
can respond rapidly to transformed and stressed
ity of NK-92/5.28.z was observed in orthotopic GBM
cells, and have the intrinsic potential to extravasate
xenograft models in NSG mice, leading to a marked
and reach their targets in almost all body tissues.
extension of symptom-free survival upon repeated
In addition to donor-derived primary NK cells, also
stereotactic injection of CAR NK cells into the tumor
continuously expanding cytotoxic cell lines such as
area. In immunocompetent mice, local therapy with
NK-92 are being considered for adoptive cancer im-
NK-92/5.28.z cells resulted in cures of transplanted
munotherapy. High cytotoxicity of NK-92 has pre-
syngeneic GBM in the majority of animals, induction
viously been shown against malignant cells of he-
of endogenous antitumor immunity and long-term
matologic origin in preclinical studies, and general
protection against tumor rechallenge at distant sites.
safety of infusion of NK-92 cells has been established
Our results suggest adoptive transfer of ErbB2-spe-
in phase I clinical trials. To enhance their thera-
cific NK-92/5.28.z cells as a promising new immu-
peutic utility, here we genetically modified NK-92
notherapy approach for ErbB2-positive glioblastoma.
cells to express a chimeric antigen receptor (CAR),
consisting of an ErbB2-specific scFv antibody fragment fused via a linker to a composite CD28-CD3
zeta signaling domain. GMP-compliant protocols for
vector production, lentiviral transduction and expansion of a genetically modified NK-92 single cell
clone (NK-92/5.28.z) were established. Functional
analysis of NK-92/5.28.z cells revealed high and
stable CAR expression, selective cytotoxicity against
ErbB2-expressing but otherwise NK-resistant tumor
cells of different origins in vitro, as well as homing to
ErbB2-expressing tumors in vivo. Ongoing work now
focuses on the development of these cells for adoptive
immunotherapy of ErbB2-positive glioblastoma. We
evaluated the activity of NK-92/5.28.z cells against a
panel of glioblastoma cell lines and primary glioblas-
139 – 166
Immunomonitoring
139 | IMMUNOMONITORING
Massive multiplexing: DNA barcode Dextramers for T cell
epitope discovery and epitope profiling
Andersen S.1, Hansen B.E.1, Bentzen A.K.2, Jakobsen S.1, Hadrup S.R.2, Pedersen H.1
Immudex, Copenhagen, Denmark,
1
`Technical University of Denmark, Section for Immunology and Vaccinology, National Veterinary Institute,
2
Copenhagen, Denmark
Identification of cancer-specific T cell epitopes is key
to the development of many novel cancer vaccines and
immunotherapies. The epitope diversity of the human
population is large, and the technologies for identifying disease-specific epitopes have been inadequate.
Established approaches involve screening one or a
few T cell epitopes at a time, and require a lot of precious patient blood or TIL sample. We have developed
a process in which DNA barcode Dextramer reagents
are used to simultaneously screen for hundreds or
thousands of T cell epitopes in a small patient sample.
Just a few milliliters of blood, tumor infiltrating lymphocytes (TILS), or other cell sample is enough.
Profiling cancer-specific T cells, emerging during the
course of a cellular immune response to tumor development or destruction, is an important aspect of
personalized immunotherapy. Likewise, determining
the mechanism of action (MoA) and the role of T cells
in checkpoint inhibitor therapy is important.
DNA barcode Dextramers allow comprehensive profiling of the antigen-specific T cell response, by allowing simultaneous quantification of thousands of T cell
specificities in a small cell sample.
Next-generation detection of cancer-responsive T cells using
DNA barcode-labeled peptide-Major Histocompatibility Complex
I multimers
Bentzen A.K.1, Marquard A.M.2, Lyngaa R.1, Saini S.K.1, Ramskov S.1, Donia M.3, thor Straten P.3, Szallasi Z.2,
Svane I.M.3, Jakobsen S.N.1,4, Eklund A.C.2, Hadrup S.R.1
Technical University of Denmark, Section for Immunology and Vaccinology, Frederiksberg C, Denmark,
1
Technical University of Denmark, Center for Biological Sequence Analysis, Lyngby, Denmark,
2
University of Copenhagen, Center for Cancer Immune Therapy, Herlev, Denmark,
3
Immudex, Copenhagen, Denmark
4
ORAL
TALK
SHORT
2016
The identification of antigenic peptides recognized
rable to combinatorial encoding of fluorescently-la-
by T cells is important for understanding disease
beled MHC multimers in terms of sensitivity (0.005%
development and for fostering new therapeutic in-
of CD8 T cells), antigen-specificity and frequency of
terventions able to specifically target cancerous
the detected T cell populations (r2=0.99). Further-
cells. Current cytometry-based approaches are
more, we have demonstrated that the technology can
limited to simultaneous screening of T cell reactiv-
be applied for multiplex T cell detection in small-size
ity towards 10-100 distinct peptide specificities in a
biological samples, such as enzymatically digested
single sample, which poorly match the large diver-
tumor material. We utilized this capacity to study the
sity of T cell recognition in humans. Consequently
dynamics of T cell responses in two melanoma pa-
it has been impossible to comprehensively analyze
tients. Specifically we screened for T cell recognition
T cell responsiveness in cancer and other multifac-
towards a large library of shared melanoma-derived
eted immune-related diseases. We have developed
epitopes in various tissue-samples taken before and
and validated a novel technology that enables par-
after T cell expansion and adoptive T cell transfer.
allel detection of numerous different peptide-MHC
Finally, we have applied this multiplex strategy for
responsive T cells in a single sample, using >1000
simultaneous assessment of target recognition and
different peptide-MHC multimers labeled with indi-
functional capability of cancer-responsive T cells,
vidual DNA barcodes. After isolation of MHC mul-
thus identifying T cells among the antigen specific
timer binding T cells their recognition are revealed
cohort that show genuine reactivity towards their
by amplification and sequencing of the MHC multim-
cognate cancer-derived epitope when stimulated
er-associated DNA barcodes. Among MHC multimer-
with autologous tumor.
binding T cells the relative frequency of sequenced
This technology enables true high-throughput detec-
DNA barcodes originating from a given peptide-MHC
tion of cancer-responsive T cells and will advance our
motif is related to the size of the antigen-responsive
understanding of immune recognition from model
T cell population. We have demonstrated the feasi-
antigens to genome-wide immune assessments on a
bility of using large panels of >1000 DNA-barcoded
personalized basis.
MHC multimers for single-tube detection of rare T
cell populations of virus and cancer-restricted origin
in various tissues. When T cell reactivity towards
such large panels of pMHC multimers was tested in
a cohort of 10 healthy donors and 11 melanoma patients this multi-parallel approach performed compa-
CIP NK proficiency panel 2016: Reducing inter-lab
variation in NK activation and functional markers
Challis R.1, Chudley L.2, Bailey E.1, Gao Y.1, Chandran A.3, Williams T.1, Khakoo S.4, Ottensmeier C.5
Wessex Investigational Services Hub (WISH) laboratory, Southampton, United Kingdom,
1
University of Southampton; NIHR ECMC Centre, Southampton, United Kingdom,
2
Department of Immunology, Interfaculty Institute for Cell Biology, Tuebingen, Germany,
3
Clinical and Experimental Sciences (CES), University of Southampton, Southampton, United Kingdom,
4
NIHR/CRUK Experimental Cancer Medicine Centre, Southampton, United Kingdom
5
An NK harmonisation proficiency panel, organised in
software, which has been developed in association
association with the CIMT Immunoguiding Program
with the CIP program, to compare against the local
(CIP), was run in 2014 to assess inter-lab variations
manual gate analysis.
when phenotyping human NK cells by flow cytome-
The data will be returned to the central lab to be
try. The 21 participants quantified predefined NK cell
collated and the inter-lab variation will be assessed
phenotypes but chose their own staining panel con-
to see if it has been significantly reduced in relation
figuration and stimulation protocols. The findings of
to phase 1. A written report will then be returned to
phase one were that overall there was low inter-lab
the participants, to inform them how their results
variation with NK phenotypic markers (CD56, CD16,
related to the rest of the group. The results may also
NKp46), but high variation with activation (CD69
indicate which stimulation works best for specific
and NKG2D) and functional (IFNγ and CD107a)
markers, allowing the stimulation method chosen in
markers. Based on these findings, a second phase
future studies to be decided based on the markers
of the NK harmonisation panel is now taking place.
of interest.
Phase 2 will aim to reduce inter-lab variation when
phenotyping these activation and functional markers
through the use of predefined protocols.
A set stimulation protocol and staining panel configuration will be decided upon for all participants
to follow. The stimulation methods will be chosen
based on which methods saw the greatest up regulation of activation and functional markers.
11 of the participants (across Europe) from phase 1
have registered an interest in taking part in phase 2.
Matched PBMC samples obtained from buffy coats of
3 UK national blood service donors will be sent out,
as well as the reagents for stimulation and staining
(i.e. a set panel of fluorochrome-antibody conjugates).
Local analysis will be performed by the participants,
following an harmonized predefined gating strategy. Participants will also be asked to upload their
fcs data to ‘ReFlow’, an automated cluster analysis
Automated flow cytometry analysis by ReFlow
Chandran A.1, White S.2, Pedersen N.W.3, Jacobsen K.4, Halgreen C.4, Britten C.M.5,6, van der Burg S.H.7,
Hadrup S.R.8, Gouttefangeas C.1, Chan C.2
Interfaculty Institute for Cell Biology, University of Tübingen, Department of Immunology, Tuebingen, Germany,
1
Duke University Medical Center USA, Department of Biostatistics and Bioinformatics, Durham, United States,
2
National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark,
3
Immudex, Copenhagen, Denmark,
4
University Medical Center of the Johannes Gutenberg, University Mainz, Translational Oncology, Mainz,
5
Germany,
(currently) GlaxoSmithKline, Oncology R&D, Stevenage, United Kingdom,
6
Leiden University Medical Center, Department of Clinical Oncology, Leiden, Netherlands,
7
National Veterinary Institute, Technical university of Denmark, Copenhagen, Denmark
8
ReFlow, an open source software, was created for
CD4- live clusters were then picked for stage 2 clus-
2 reasons. First, to manage multi-parameter flow
tering with multimer-fluorochrome and CD8 staining.
cytometry files (FCS) from multi-centric studies.
We generated cell samples, through spiking, contain-
Second, for automated analysis of flow cytometry
ing predictable numbers of antigen specific T cells and
data eliminating the variability associated with sub-
stained them using HLA-peptide multimers. Manual
jective gating strategies during manual analysis. The
and ReFlow analysis of the FCS files of these samples
first purpose has been previously fulfilled. ReFlow is
correlated very well (R2 > 0.98). Limit of detection by
available for use as a server-based web interface to
ReFlow was as low as 0.01% of CD8+ cells.
upload FCS files onto pre-defined multi-parametric
In a second step, 258 FCS files from an HLA-multimer
panel templates. Once uploaded, ReFlow harmonizes
staining proficiency panel conducted by Immudex
inconsistent metadata annotations, extracts fluoro-
in co-operation with the CIP and obtained from 22
chrome compensation information and prepares a
laboratories were analysed using ReFlow. Each labo-
downloadable clean FCS file.
ratory had stained PBMCs from 2 donors using PE
The second purpose of ReFlow, currently being refined,
conjugated HLA-peptide multimers for 2 viral specifi-
involves its use as an automated FCS file analysis pipe-
cities and a negative control multimer in 2 replicates.
line. ReFlow employs statistical models (hierarchical
All other stained fluorochrome-markers were heter-
DPGMM) to identify unique cell subsets in an automated
ogenous. FCS files were uploaded onto ReFlow and
fashion by naturally generating an aligned data model
were analyzed using the 2-stage clustering strategy.
that captures both commonalities and variations across
Total percentage of multimer-PE+ clusters within
multiple samples. After testing the accuracy, precision
each FCS file was compared to the respective per-
and reproducibility using TCR-engineered samples,
centages obtained after manual analysis by the in-
we proceeded to analyze spiked PBMC samples and
dividual labs. We observed a high linear correlation
proficiency panel data. In order to enrich desired cell
(R 2= 0.89). Additionally, comparison of ReFlow with
populations and eliminate dead cells, we incorporated
central manual analysis of all the FCS files is cur-
a 2-stage clustering strategy. In stage 1, the events were
rently ongoing.
clustered based on their physical scatter (FSC and SSC)
and viability dye, CD4, CD8 staining, etc. CD8+ CD3+
Radiation-expanded myeloid-derived suppressor cells are
responsible for local failure of radiation therapy
Chiang C.-S.1, Fu S.-Y.1, Hong J.-H.2
National Tsing Hua University, Biomedical Engineering and Environmental Sciences, Hsinchu, Taiwan,
1
Republic of China,
Chang-Gung Memorial Hospital, Radiation Oncology, Taoyuan, Taiwan, Republic of China
2
This study aimed to examine the role of CD11b+Gr-1+
tion. The monitor of MDSCs in the blood could be an
myeloid-derived suppressor cells (MDSCS) in tumor
index for local tumor control after radiation therapy
regrowth after radiation therapy. In this study, 4
and assessment for the variation of irradiated tumor
mm diameter murine prostate tumor, TRAMP-C1, in
microenvironment.
the shank was irradiated with single dose of 25Gy
of radiation. Immunohistological staining for tumor
tissues showed that Gr-1+ cells were rapidly recruited
into irradiated tumor in 4 hours and chronologically
aggregated at central necrotic region within chronic
hypoxic region. The flow cytometry could further
divide CD11b+ myeloid cells into 4 subpopulations,
including CD11b+Ly6G-LY6C - monocyte/tumor-associated macrophages (TAMs), CD11b+Ly6G+αly6C+
neutrophilic-MDSCs (N-MDSCs), CD11b+Ly6G-Ly6Chi
monocytic-MDSCs (M-MDSCs) and CD11b+Ly6G-Ly6Clow heterogeneous myeloid-derived cells (H-MDCs),
and found that main MDSCs recruited by local
tumor irradiation into the tumor are N-MDSCs and
M-MDSCs. In addition, the percentage of N-MDSCs,
M-MDSCs and H-MDCs were systemically expanded
in peripheral blood after radiation therapy. The increased percentage of MDSCs were associated with
the induction of proteins of GM-CSF, G-CSF, CCL3,
CCL5, CXCL5, IL6, IL17α and VEGF-α in irradiated
tumors and G-CSF, IL6 and TNF-α in the blood. The
administration of anti-Gr-1 antibody further enhanced the efficacy of radiation therapy, indicating
the pro-tumor property of MDSCs. In conclusion, this
study demonstrates that pro-tumoal MDSCs could
be expended both in irradiated tumors and blood,
which were associated with multiple cytokine induc-
Preliminary results of a prospective immunomonitoring trial in
ovarian cancer patients
Coosemans A.1,2, Baert T.1,2, Van Hoylandt A.1,2, Verschuere T.3, Vergote I.1,2
1
Leuven, Belgium,
2
KULeuven, Department of Microbiology and Immunology, Laboratory of Pediatric Immunology, Leuven,
3
Belgium
Background: Ovarian cancer is the second most lethal
cells was seen in all group at the end of primary
type of gynaecological tumour in women with an in-
therapy, compared to diagnosis.. We observed no
cidence rate of 12.5 per 100 000. Surgery in combina-
differences in T cell population or MDSC when com-
tion with platin-based (neo)-adjuvant chemotherapy
paring samples at diagnosis versus after three cycles
remains the cornerstone of therapy. The behaviour
of neo-adjuvant chemotherapy. Currently, we did not
of the immune system outside the tumour and the
observe immunological changes after primary cy-
tumour microenvironment is largely unknown. We
toreductive surgery.
will investigate the presence of the different immune
Conclusions: These observations already provide us
players in the blood of ovarian cancer patients during
with some insight into the immunobiology of ovarian
the disease course.
cancer patients. We plan to evaluate up to 100 pa-
Materials and methods: Peripheral blood mono-
tients prospectively to allow for a correct mapping of
nuclear cells (PBMC) have been collected prospec-
the immune system in ovarian cancer. This informa-
tively in 70 patients with invasive ovarian cancer
tion will be necessary for patients triage and for the
at diagnosis, after cytoreductive surgery ((interval)
development of new therapeutic strategies.
debulking), after three courses of (neo-)adjuvant
chemotherapy, and at the end of their primary treatment (i.e. four samples per patient). The study is still
ongoing. PBMC will also be collected at the moment
of relapse.
Results: Currently, we analysed PBMC samples of
eight patients. Of these, four had a high-grade serous
histology and were diagnosed at FIGO stage IIIc-IV
(a prognostic inferior group), the other four patients
had a well-differentiated endometrioid histology and
were diagnosed at FIGO stage I-II (a prognostic superior group). We applied three panels: a general T
cell panel, a panel for regulatory T cells and a MDSC
(myeloid derived suppressor cells) panel. Preliminary analysis showed significantly more activated
CD8+ T cells in the prognostic inferior group. Interestingly, an significant increase in regulatory T
Immune monitoring of lung cancer patients to predict clinical
outcome using an automated pipeline for flow cytometry data
analysis
de Goeje P.L.1, van Gassen S.2, Poncin M.1, Kaijen-Lambers M.E.H.1, Bezemer K.1, Saeys Y.2, Hegmans J.P.J.J.1,
Aerts J.G.J.V.1,3
Erasmus MC, Pulmonary Medicine, Rotterdam, Netherlands,
1
VIB, Ghent University, Inflammation Research Center, Ghent, Belgium,
2
Amphia Hospital, Lung diseases, Breda, Netherlands
3
Non-small cell lung cancer (NSCLC) has a very
the clinical value of all possible immune subsets
dismal prognosis, as the majority of patients are
when using manual gating methods. Therefore, we
diagnosed with metastatic disease and response to
used a recently developed algorithm pipeline, called
therapy at that stage is generally poor. Stratifica-
FloReMi, to analyze the data. This pipeline uses auto-
tion of patients at diagnosis or early during treat-
matic gating algorithms defining the optimal split in
ment is urgently needed to guide clinicians to select
each marker, and combines each combination of pos-
the most appropriate treatment for each patient.
itive and negative populations, with each of the mean
The immune system has been shown to play an im-
fluorescence intensities to create a large number of
portant role in the response to conventional cancer
features representing all possible populations. Sub-
treatments. To get a more thorough understanding
sequently, the most predictive features are selected
of the various immune effectors involved, and to es-
to train a model for survival analysis. We expect to
tablish an immune profile that can predict prognosis
show preliminary results of these analyses at the con-
or response to therapy, we determined the cellular
ference. Given the complexity of the immune system
immune dynamics in peripheral blood of lung cancer
and the multiple interactions between the different
patients upon chemotherapy treatment.
cell types, we anticipate that an immune profile will
From a cohort of 209 stage IV NSCLC patients, treated
better reflect the immune status of the patient than
with carboplatin, paclitaxel and bevacizumab, blood
only a single variable. This may give us clues about
samples were collected on baseline and after the first
the underlying working mechanisms of (immune)
and second cycle of treatment (week 3 and 6, respec-
therapies and may guide treatment decisions based
tively). Using flow cytometry, CD4+ and CD8+ T cells,
on patients individual immune cell signatures.
their naïve and memory subsets, and regulatory T
cells were assessed. Moreover, functional markers
like CD25, PD1, CTLA4, CD28 and Ki67 were included, as well as IFNγ production after stimulation. We
found that a large variation existed in the proportions
of T cell subsets between patients, while populations
remained relatively stable over time.
We previously showed that myeloid-derived suppressor cells in this cohort are correlated with a poorer
survival. With an increasing number of markers per
staining, however, it becomes unfeasible to assess
CD4+ T-cell immunomonitoring after hematopoietic cell
transplantation: identifying patients at risk for virus-predicted
adverse outcomes
de Koning C.1, Admiraal R.1, Bierings M.B.2, Lindemans C.A.2, Wensing A.3, Versluys B.2, Wolfs T.4, Nierkens S.1,
Boelens J.J.1,2
UMC Utrecht, Translational Immunology, Utrecht, Netherlands,
1
UMC Utrecht, Pediatric Blood and Marrow Transplantation Program, Utrecht, Netherlands,
2
UMC Utrecht, Department of Virology, Utrecht, Netherlands,
3
UMC Utrecht, Department of Pediatrics, Utrecht, Netherlands
4
ORAL
TALK
SHORT
2016
Background: Immunomonitoring is an important
Results: 273 patients (0.1-22.7 years; median fol-
tool to track immune reconstitution (IR) after hemat-
low-up 58 months) were included. CD4+ T-cell re-
opoietic cell transplantation (HCT). A fast and ade-
constitution (CD4+ IR), defined as having ≥50*106
quate IR is pivotal in preventing adverse clinical out-
CD3+CD4+ cells/L within 100-days, was the only
comes, to which viral reactivations (VR) contribute
predictor for VR. CD4+ IR predicted reactivation of
significantly. To this extend, we applied a unique ap-
AdV (HR 0.995, p=0.022); the chance on reactivation
proach in which VR was evaluated as a time-varying
was reduced 5% with every increase of 10/µl CD4+
variable to predict clinical outcomes. Additionally,
T-cell counts. CD4+ IR could also predict EBV (HR
we related VR to IR data, in which we are to first to
0.994, p=0.029) and HHV6 (HR 0.991, p=0.012), but
evaluate IR data as continuous over-time-predictor.
not CMV (p=0.31) and BK (p=0.27). Additionally,
This enabled us to find robust immunomonitoring
duration of AdV-reactivation was shorter with CD4+
predictors for VR, and to investigate whether IR
IR (p=0.011). AdV predicted lower OS (HR 2.17,
could influence VR-predicted adverse outcomes.
p=0.0039) and higher NRM (HR 2.96, p=0.0008).
Methods: In this retrospective analysis, all patients
EBV and HHV6 were predictors for occurrence of
receiving a first HCT between January-2004 and
GvHD, while CMV and BK did not predict clinical
September-2014 were included. IR (CD3/CD4/CD8
outcomes. Interestingly, concomitant CD4+ IR abol-
T-cells, NK- and B-cells) was measured bi-weekly
ished the negative effect of AdV on survival: OS
until 12 weeks, and monthly thereafter. Additionally,
(p=0.67) and NRM (p=0.64).
we retrospectively performed intracellular cytokine
Conclusion: These results stress the importance of
stainings (ICCS) on these samples to evaluate IFNg,
monitoring CD4+ IR after HCT, which enables the
IL17, IL21, IL9, IL22, IL10, and IL13 production by
identification of patients with early CD4+ IR who are
CD4+ T-cells. Main outcomes of interest were VR of
at lower risk for VR-related adverse outcomes. This
adenovirus (AdV), Epstein-Barr-virus (EBV), human-
may limit the need for toxic anti-viral drugs. On the
herpesvirus 6 (HHV6), cytomegalovirus (CMV), and
other hand, the identification of at-risk patients with
BK-virus, screened weekly. VR of AdV, EBV, HHV6,
a delayed CD4+ IR provides the opportunity to pre-
CMV, and BK, defined as having a viral load >1000
emptively intervene with anti-viral (cell) therapies.
copies/mL. Clinical outcomes included overall-sur-
In addition, the identification of at-risk patients could
vival (OS), event-free-survival, non-relapse-mortality
be evaluated in more detail with functional assess-
(NRM), and graft-versus-host-disease (GvHD). Cox-
ment of CD4+ T-cells using ICCS, providing more
proportional-hazard- and Fine-Gray-competing-risk-
in-depth immunomonitoring.
models were used.
Immunoprevalence and magnitude of HLA-DP4 versus HLA-DRrestricted spontaneous CD4 Th1 responses against telomerase in
cancer patients
Laheurte C.1, Galaine J.2,3, Béziaud L.2, Dosset M.2, Kerzerho J.4, Jacquemard C.1, Gaugler B.2, Ferrand C.2,3,
Dormoy A.3, Aubin F.5, Jacoulet P.6, Westeel V.6, Borg C.2,3,7, Tartour E.8, Godet Y.2, Maillère B.4, Adotévi O.2,7
EFS Bourgogne Franche-Comté, Biomonitoring Platform, Besancon, France,
1
University of Franche-Comté, Inserm UMR1098, Besancon, France,
2
EFS Bourgogne Franche-Comté, Besancon, France,
3
CEA, iBiTecS, Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), Gif Sur Yvette, France,
4
University Hospital of Besançon, Dermatology, Besancon, France,
5
University Hospital of Besançon, Pneumology, Besancon, France,
6
University Hospital of Besançon, Medical Oncology, Besancon, France,
7
Georges Pompidou Hospital, Biological Immunology, Besancon, France
8
Cumulative evidence supports that CD4 Th1 cells
of HLA-DR-restricted spontaneous anti-TERT Th1
play a key role in antitumor immunity. We previ-
immunity compared to HLA-DP restriction. These
ously reported the presence of spontaneous HLA-
results provide a new tool for comprehensive moni-
DR-restricted CD4 Th1 responses against telomer-
toring of antitumor CD4 Th1 response in various
ase reverse transcriptase (TERT) in various cancers
cancers.
by using promiscuous HLA-DR epitopes. Here, we
described novel highly immunogenic HLA-DP4binding epitopes from TERT named TERT541-555,
TERT573-587, TERT613-627 and TERT911-925 and addressed
the question about the immunoprevalence and magnitude of the naturally occurring antitumor CD4 T
cell responses restricted by HLA-DP4 or HLA-DR,
the two most common HLA class II. Direct comparative study of spontaneous anti-TERT CD4 T cell responses in a cohort of 87 lung cancer patients showed
that HLA-DP4 and HLA-DR sustained specific Th1
responses in 10.1% and 25.2% of cancer patients respectively (P=0.01). The magnitude of the HLA-DRrestricted responses was 2 to 3 times significantly
higher than HLA-DP one (P=0.005). Similar results
were found in other cancers such as melanoma,
breast cancer, renal cell carcinoma and colon cancer.
Thus, our results describe for the first time in a large
cohort of cancer patients a high immunoprevalence
Immunomonitoring and immunoguiding: update on the CIP
activities
Gouttefangeas C.1, Britten C.M.2, Chan C.3, Kvistborg P.4, Mandruzzato S.5, Ottensmeier C.H.6,
van der Burg S.H.7, Walter S.8, Hadrup S.9, Welters M.J.P.7
Universität Tübingen, Department of Immunology, Tübingen, Germany,
1
GlaxoSmithKline, Stevenage, United Kingdom,
2
Duke University Medical Center, Department of Biostatistics and Bioinformatics, Durham, United States,
3
Netherlands Cancer Institute, Amsterdam, Netherlands,
4
University of Padova, Department of Surgery, Oncology and Gastroenterology, Padova, Italy,
5
Southampton University Hospitals, Cancer Sciences Division, Southampton, United Kingdom,
6
Leiden University Medical Center, Department of Clinical Oncology, Leiden, Netherlands,
7
Immatics US Inc., Houston, United States,
8
Technical University of Denmark, National Veterinary Institute, Copenhagen, Denmark
9
Immunomonitoring is a key element in the development and evaluation of anti-tumor immunotherapies.
The CIP (CIMT Immunoguiding Program) workgroup
has pioneered the concept of harmonization for in
vitro assays assessing cellular biomarkers. Proficiency panels have been the main tools to reach harmonization, allowing both the identification of factors
involved in inter-laboratory variability and the assessment of individual users´ performance relative
to a group of laboratories. After the harmonization of
the commonly used T-cell assays IFNgamma-ELISpot
and HLA-multimer staining has been achieved, CIP
is now focusing on other immune cell subsets relevant for immunotherapy such as regulatory T cells,
myeloid-derived suppressor cells and natural killer
cells. In addition, the workgroup aims at supporting
technical progresses in the immunomonitoring field.
Research activities such as the development of cellular reference samples and the automated processing
of flow cytometry data are ongoing. Recent results
will be presented.
Evaluation of novel predictive marker molecules in malignant
melanoma immunotherapy
Krebs F.1,2, Mitzel-Kaoukhov H.1, Jetter A.1, Behling J.1, Wickert D.1, Lang B.1, Hahn S.A.1, Meissner M.3, Tenzer S.4,
Grabbe S.1, Loquai C.1, Tuettenberg A.1
University Medical Center Mainz, Dermatology, Mainz, Germany,
1
German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Cancer immunology and
2
immunotherapy, Mainz, Germany,
University of Frankfurt, Dermatology, Venereology and Allergology, Frankfurt am Main, Germany,
3
University Medical Center of the Johannes-Gutenberg University Mainz, Institute for Immunology, Mainz,
4
Germany
Therapeutic approaches targeting the advanced
peutic response in melanoma therapy. Two candi-
stages of malignant melanoma show a poor progno-
dates of this training set could already be validated
sis for long-term survival. Approaches to overcome
in additional assays such as ELISA.
immune tolerance include immune checkpoint in-
To correlate our new found biomarkers to previous
hibitors like the monoclonal-antibody anti-CTLA-4
data on MDSC and Treg, immunomonitoring of pa-
(Ipilimumab) and the anti-Programmed death-ligand
tients included analysis of frequencies and function-
1 (PD-L1) inhibitors Pembrolizumab and Nivolumab.
ality of Treg and MDSC subpopulations in peripheral
These costly treatments aim toward the reactiva-
blood mononuclear cells. Preliminary data showed
tion of T effector cells. Unfortunately, only a part of
a decrease in immunosuppressive Treg as well as
treated patients shows a significant, often delayed,
MDSC levels in responders to Ipilimumab therapy. In
clinical benefit.
contrast, both increased in non-responders.
Importantly, there are currently no reliable soluble
To conclude, we identified new potential biomark-
or cellular biomarkers available to early identify
ers differentiating responders and non-responders
and predict responders. Whereas immunomonitor-
to checkpoint inhibitor therapy. Moreover, we found
ing revealed high frequencies of regulatory T cells
that levels of cells bearing immunosuppressive ca-
(Treg) and myeloid-derived suppressor cells (MDSC)
pacity decrease in responders to Ipilimumab therapy
in melanoma patients, it is not known whether Treg
thereby supporting immune response against the
and MDSC numbers display a predictive value for the
tumor.
prediction of clinical benefit.
In the present study, we aim to identify novel biomarkers to differentiate between responders and
non-responders to immune checkpoint inhibitors
already at the early stages of therapy.
Therefore we analyzed plasma and blood from
40 melanoma patients treated with Ipilimumab,
Nivolumab or Pembrolizumab. Potential biomarkers
were identified using a quantitative proteomic analysis of plasma samples. By comparing protein levels in
responders and non-responders we identified a panel
of new candidates for potential predictive biomarkers
which yet are not discussed in the context of thera-
Type, frequency and breadth of tumor associated antigen
reactivity in tumor infiltrating lymphocyte from metastatic
melanoma patients
Lyngaa R.1, Donia M.2,3, Andersen R.2,3, Bjoern J.2, Sick Andersen R.2, Ellebæk E.2,3, thor Straten P.2, Svane I.M.2,3,
Reker Hadrup S.1
Veterinary Institute, DTU, Fredriksberg C, Denmark,
1
Herlev Hospital, University of Copenhagen, Department of Hematology, Center for Cancer Immune Therapy,
2
Copenhagen, Denmark,
Herlev Hospital, University of Copenhagen, Department of Oncology, Copenhagen, Denmark
3
In this study we have investigated the type, frequen-
to non-ipi treated patients (3.8(ipi pre-treated, n=28)
cy and breadth of antigen responses in metastatic
versus 1.9(non-treated, n=37) unpaired two tailed t
melanoma patients undergoing adoptive cell therapy
test, p< 0.05). In patients who received ipilimumab
(ACT). We have screened for responses in the actual
treatment T cell responses towards cancer testis (CT)
infusion product and in some cases also in simul-
and overexpressed antigens (OE) increase dramati-
taneously expanded TIL cultures. When possible,
cally from 7 to 21 (p=0.0062), and 18 to 36 (p=0.03),
tumor reactivity was also tested either on autologous
respectively for CT - and OE antigens. Thus indicat-
or allogeneic tumor cell lines.
ing that treatment with ipilimumab increases the
Methods: We investigated T cell antigen recognition
breadth of the tumor associated immune responses.
against a panel of 167 TAA A2-restricted melanoma-
When looking in the infusion product from patients
associated peptides in 30 A2 positive melanoma pa-
with clinical response (RECIST) to ACT compared
tients, including the infusion product for 13 patients
to non-responders, we observed a trend towards in-
treated with ACT. Antigen recognition was assessed
creased T cell reactivity to melanoma associated an-
using combinatorial encoding of MHC-multimer
tigens (6.5 vs. 1.4, p=0.078).
complexes and multi-color flow cytrometry. Tumor
Conclusion: In tumor infiltrating lymphocytes from
recognition was assesses following stimulation with
metastatic melanoma patients we found that most
autologous or allogenic HLA matched tumor cell
detectable T cell responses were directed against dif-
lines, and intracellular staining of INFg, TNF and
ferentiation antigens and of low frequency. However,
CD107a.
looking in TILs from patients treated with ipilimum-
Results: In average we found 3.4 melanoma-associat-
ab prior to ACT the antigen responses was signifi-
ed T cell responses per culture (n=51) (range 0-16),
cantly broadened to also include several CT and OE
with most responses having a frequency ≤ 0.1% of
antigen responses and responses were of increased
CD8+ cells (97 responses), 46 responses between
frequency.
0.1-1.0% of CD8+ cells and 31 responses had a frequency of ≥1.0% of CD8+ cells. The majority of the
detected T cell populations recognized differentiation antigens, with MART and gp100 being the dominantly recognized proteins. Several of the patients
also received Ipilimumab at some point before tumor
resection for TIL expansion resulting in a significantly higher number of T cell responses compared
Results from the first phase of a harmonization effort for the
phenotyping of human myeloid-derived suppressor cells
Mandruzzato S.1, Brandau S.2, Britten C.M.3, Bronte V.4, Damuzzo V.1, Gouttefangeas C.5, Maurer D.6,
Ottensmeier C.7, van der Burg S.8, Welters M.J.P.8, Walter S.6
University of Padova, Padova, Italy,
1
University Hospital, Essen, Germany,
2
GlaxoSmithKline, Stevenage, United Kingdom,
3
Verona University Hospital, Verona, Italy,
4
University of Tubingen, Tubingen, Germany,
5
Immatics Biotechnologies GmbH, Tubingen, Germany,
6
University of Southampton, Southampton, United Kingdom,
7
Leiden University Medical Center, Leiden, Netherlands
8
Myeloid-derived suppressor cells (MDSCs) are a
vealed a small intra-laboratory, but very high inter-
heterogeneous group of myeloid cells at different
laboratory variance for all MDSC subsets, especially
stages of differentiation, which are often expanded
for the granulocytic subsets. In particular, the use of
in cancer patients and capable of suppressing anti-
a dead-cell marker altered significantly the reported
tumor immune responses. In recent years, recogni-
percentage of granulocytic MDSCs, confirming that
tion of the clinical relevance of MDSCs has steadily
these cells are especially sensitive to cryopreserva-
increased and there is a growing interest for monitor-
tion and/or thawing. Importantly, the gating strategy
ing circulating MDSCs in cancer patients. However,
was heterogeneous and associated with great inter-
there are still divergences in their phenotypic defini-
center variance.
tion. To overcome this obstacle, the Cancer Immuno-
Overall, our results document a high variability
guiding Program (CIP) is coordinating a proficiency
in MDSC phenotyping in a multi-center setting if
panel program that aims at harmonizing MDSC phe-
no harmonization/standardization measures are
notyping.
applied. Although the observed variability depended
In the first step, an international consortium of 23
on a number of identified parameters, the main iden-
laboratories immune-phenotyped 10 putative MDSC
tified factor associated with variation was the gating
subsets on pre-tested, peripheral blood mononuclear
strategy. Based on these findings we propose further
cells of healthy donors, to assess the level of concord-
efforts to harmonize marker combinations and
ance and to define robust marker combinations for
gating parameters to define strategies for a robust
the identification of circulating MDSCs. At this stage,
and consistent enumeration of MDSC subsets.
no mandatory requirements to standardize either
reagents or protocols were introduced.
For each MDSC subset and each donor, we calculated the intra-assay and inter-assay variance of each
reported MDSC subset frequency. Data analysis re-
Noninvasive ImmunoPET imaging of the PD-1/PD-L1 checkpoint
in naïve and irradiated tumor-bearing mice
Hettich M.1, Braun F.2, Niedermann G.1
University Clinics Freiburg, Dept. of Radiation Oncology, Freiburg, Germany,
1
University Clinics Freiburg, Dept. of Nuclear Medicine, Freiburg, Germany
2
ORAL
TALK
SHORT
2016
There is increasing evidence that antibodies block-
tion analyses. The targets of the PET tracer antibod-
ing the PD-1/PD-L1 checkpoint (either anti-PD-1 or
ies were verified by ex vivo flow cytometric analy-
anti-PD-L1) increase in-field anti-tumor responses to
ses. Visualization of immune-related adverse events
ionizing radiation and enhance abscopal effects on
was also possible. In conclusion, we have developed
non-irradiated metastases. Here, we developed PET
two innovative PET tracers that allow imaging the
tracers based on therapeutic antibodies to visualize
expression of the receptor/ligand pair of the impor-
whole-body expression of PD-1 and PD-L1 in mice
tant PD-1/PD-L1 checkpoint and the biodistribu-
and the biodistribution of the surrogate checkpoint-
tion of surrogate checkpoint-blocking antibodies in
blocking antibodies. Two novel PET tracers were
fully immunocompetent mice. This technology also
developed based on anti-PD-1 and anti-PD-L1 check-
enables whole-body pictures of combination radio/
point-blocking antibodies. Non-invasive PET imaging
immunotherapies.
was performed on naïve and tumor-bearing mice.
Mice bearing s.c. B16 melanomas were treated with
hypofractionated radiation therapy (hRT) in combination with CTLA-4 checkpoint blockade before PET
imaging. PD-1 or PD-L1 knockout mice and PD-L1deficient B16 cells generated using the CRISPR/Cas
technology served as specificity controls. The newly
developed PET tracers allowed the highly specific
and high-resolution imaging of PD-1 and PD-L1 expression. In addition, they permitted the noninvasive
imaging of the biodistribution of the two therapeutic antibodies in both naïve and tumor-bearing mice
treated with hRT and CTLA-4 checkpoint blockade.
Imaging of the respective knockout mice, blocking
experiments with an excess amount of unlabeled
antibodies, and the analysis of animals bearing both
wild-type B16 melanomas and PD-L1-CRISPR knockout melanomas demonstrated the high specificity of
the two newly developed PET tracers. The in vivo
imaging data were confirmed by ex vivo biodistribu-
NGS-based αβTCR repertoire analysis in tumor and blood from
three melanoma patients pre and post IVAC® MUTANOME
vaccination
Omokoko T.1, Schwarz J.1, Hebich L.1, Oelbermann A.1, Simon P.1, Tolliver C.1, Steege B.1, Müller F.2, Seck C.2,
Rohde C.3, Wöll S.3, Miller M.2, Sahin U.1,2
1
2
Ganymed Pharmaceuticals AG, Mainz, Germany
3
BioNTech’s IVAC® MUTANOME clinical trial is a
the combination of NGS-based αβTCR profiling and
first-in-human study evaluating the safety, tolerabili-
our single cell TCR cloning technology is a powerful
ty and immunogenicity of intra-nodal administration
diagnostic tool, which permits both detailed analysis
of a truly personalized vaccine in patients with ma-
of vaccine induced T cell responses and identification
lignant melanoma. The IVAC® MUTANOME vaccine
of interesting TCR candidates for fully individualized
approach is based on targeting multiple immunogen-
adoptive T cell therapy approaches.
ic tumor mutations unique to a given patient´s tumor
using a poly-epitopic RNA-based vaccine manufactured on demand and individually for each patient.
As part of the comprehensive biomarker program we
applied NGS-based αβTCR profiling technology for
in depth analysis of the induced T cell responses and
the clinical mode of action. TCR repertoire analysis
was performed with peripheral blood samples from
three melanoma patients obtained pre and post vaccination. For one patient TCR sequencing was also
done on multiple lymph node metastases excised
before and one metastasis excised after vaccination.
We observed only minor changes in the peripheral
TCR repertoires in response to the vaccination. TCR
repertoires in the different metastases however differed vastly. To gain further insight into the vaccine
induced T cell responses, we performed clonotype
tracking of individual (neo-antigen-specific) TCRs isolated in parallel from single CD4+ and CD8+ tumor
infiltrating lymphocytes (TILs) using BioNTech Cell
& Gene Therapies’ single cell TCR cloning platform.
We observed that CD4 TILs were predominantly enriched. Furthermore we were able to discriminate
pre-existing and de novo induced neo-antigen specific T cell responses. Taken together, we show that
Peptide-specific T-cell responses against tumor-specific HLA
ligands in ovarian cancer
Peper J.1, Röhle K.1, Schuster H.1, Wagner P.2, Rammensee H.-G.1, Stevanović S.1
1
University Hospital Tübingen, Department of Obstetrics and Gynecology, Tübingen, Germany
2
Introduction: The great majority of patients suffer-
HLA-peptide multimer staining.
ing from ovarian cancer (OvCa) relapse after initial
Results: Up to now, 30 of 37 tested peptides (12 dif-
therapy, making OvCa the most lethal gynecologi-
ferent HLA restrictions) derived from 7 OvCa-exclu-
cal malignancy. The immunogenic nature of OvCa
sively presented antigens were able to induce T-cell
is highlighted by frequent infiltration with immune
responses after in vitro priming in healthy donors.
cells, which represent an independent prognostic
Currently T cells from TILs and corresponding TILs
factor in OvCa patients and argue in favor of the de-
of OvCa patients are tested for their reactivity against
velopment of immunotherapies, including peptide-
OvCa-exclusively presented HLA ligands isolated
based cancer vaccines. Employing HLA ligandome
from their respective tumor tissue.
analysis, we identified HLA ligands exclusively pre-
Conclusion: The majority of non-mutated tumor-
sented on OvCa and not on healthy tissue samples
exclusively presented HLA ligands can induce T-cell
of a variety of organs. Their immunogenicity as well
responses in healthy donors showing their suitabil-
as the presence of preexisting T-cell responses tar-
ity as vaccination antigens. Furthermore, we provide
geting those HLA ligands in OvCa patients is so far
first insight into preexisting T-cell responses against
unknown.
those HLA ligands in OvCa patients.
Materials and methods: HLA ligands previously
shown to be exclusively presented on OvCa tissues
by our group were tested for immunogenicity by performing in vitro priming of CD8+ T cells from healthy
blood donors. PBMCs from several OvCa patients
were collected and corresponding tumor samples
obtained during tumor debulking surgery. Tumor-infiltrating lymphocytes (TILs) were isolated from the
fresh tumor samples while parts of the samples were
cryopreserved for analyzing the presentation of OvCa-exclusively presented HLA ligands by mass spectrometry. PBMCs and TILs were screened for peptide-specific reactivity against HLA ligands for which
presentation was previously confirmed by mass spectrometry on the same patient’s tumor tissue employing IFNγ ELISPOT and / or intracellular cytokine and
Tumor antigen specific Treg from the bone marrow migrate
towards increased S1P and CCL2 gradients established in the
blood of breast cancer patients
Rathinasamy A.1, Boehm H.-H.2, Ge Y.2, Dettling S.3, Umansky L.2, Domschke C.4, Herold-Mende C.3, Schuetz F.4,
Beckhove P.1,2
Regensburg Center for Interventional Immunology (RCI), University Clinic Regensburg, Regensburg, Germany,
1
2
University Hospital Heidelberg, Department of Neurosurgery, Heidelberg, Germany,
3
University Hospital Heidelberg, Department of Gynecology and Obstetrics, Heidelberg, Germany
4
High regulatory T cell (Treg) infiltration in breast
tumors is associated with reduced survival. However,
the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the
tumor tissue remain elusive. We here demonstrate
that pronounced Treg infiltration in human breast
tumors correlates with a selective reduction of tumor
antigen specifc Treg from the bone marrow. Furthermore using MHC-II tumor peptide tetramers we
show that tumor antigen specific bone marrow Treg
selectively express the egress receptor Sphingosine1-phosphate receptor 1 (S1P1) and CCR2. S1P1 and
CCR2 were upregulated in Treg upon TCR stimulation and triggered selective Treg but not Tcon cell
migration in response to S1P and CCL2 gradients
between bone marrow and blood which we found to
be significantly increased in breast cancer patients.
Taken together, our data suggest that tumor antigen
specific Treg population in the bone marrow are mobilized into the blood after TCR stimulation through
elevated S1P and CCL2 gradients in breast cancer
patients.
Immune monitoring of natural killer cells in chronic myeloid
leukemia: split anergy status depend on tyrosine kinase
inhibitor therapy
Rodrigues-Santos P.1,2, Almeida J.2, Couceiro P.2, Alves V.1, Růžičková L.3, Freitas-Tavares P.3, Santos-Rosa M.1
Faculdade de Medicina da Universidade de Coimbra, Instituto de Imunologia, Coimbra, Portugal,
1
Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Laboratório de Imunologia e Oncologia,
2
Coimbra, Portugal,
Centro Hospitalar e Universitário de Coimbra, Serviço de Hematologia Clínica, Coimbra, Portugal
3
ORAL
TALK
SHORT
2016
Introduction: Previous studies indicate that Natural
hibitory (KIR2DL1, KIR2DL2) receptors were found
Killer (NK) cells are deficient in Chronic Myeloid
altered in CML. The expression of KIR2DS1 by CD-
Leukemia (CML) patients, although the mechanisms
56dimCD16+ NK cells was highest in CML patients
behind the dysfunction are not completely under-
undergoing Dasatinib therapy. Treatment also in-
stood. Current therapeutic strategies influence these
creased the NKG2C/NKG2A (activatory/inhibitory)
innate lymphoid cells and successful results may be
ratio. Lower expression of NKp30 and NKp44 was
partially explained by the advantageous effects on
compensated by the increase of NKp46+ NK cells.
their cytotoxicity against cancer cells. Due to recent
Production of IFN-γ and suppression of TGF-β+ and
advances in the knowledge of NK cell´s biology, there
IL-10+ NK cells was also a beneficial effect of treat-
is an increasing interest in mapping NK-cell respons-
ment protocol. IFN-γ production decreased with an
es in cancer.
increased TKI dose.
The aim of the present study was to analyze NK cells
Conclusion: Expansion and activation of NK cells was
in CML patients and the effect of therapy and dose
observed in TKI treated CML. Cytotoxic and regula-
dependent mechanisms on essential features of NK
tory functions of NK cells are TKI dependent defining
cells.
a split anergy status. NK cell receptor repertoire is
Material and methods: In this study, we ana-
modulated by TKIs in CML. Information based on
lyzed blood samples from 67 CML patients treated
immune status of CML could help to define patients
with IFN-α and/or different generations of tyrosine
needing to change TKI and those that are “ready” to
kinase inhibitors (TKI). Extended analysis of NK-cell
stop TKI therapy. NK cells are affected during CML
receptor repertoire and functional properties was
and current therapeutic protocols ameliorate NK-cell
performed by multiparametric flow cytometry, cell
performance. In the future, combination of NK cell-
sorting, Luminex xMAP technology and real-time
based immunotherapy with pharmacological inter-
quantitative PCR.
ventions should be investigated in order to eradicate
Results: Relative frequency of NK cells was found
cancer cells and discontinuation of therapy.
reduced at CML diagnosis and recovered after treat-
Financial Support: FEDER (Programa Operacional
+
ment. CML therapy induces an increase of CD62L
Factores de Competitividade - COMPETE) and FCT
CD56bright NK cells, associated to the capacity of mi-
(Fundação para a Ciência e a Tecnologia) through
gration to secondary lymphoid organs. Activation
project PEst-C/SAU/LA0001/2013-2014.
of NK cells and the increased expression of CD137
and CD137L were interpreted as a significant effect
of therapy response. Activatory (KIR2DS1) and in-
Isolation and analysis of tumor-specific CD8 and CD4 T cells
with high affinity, reversible pMHC multimers
Schmidt J.1, Guillaume P.2, Hebeisen M.3, Rufer N.3, Luescher I.1
Ludwig Center of the University of Lausanne, Epalinges, Switzerland,
1
TCMetrix Ltd, Epalinges, Switzerland,
2
Department of Oncology, Lausanne University Hospital, Lausanne, Switzerland
3
ORAL
TALK
SHORT
2016
Adoptive cell transfer of tumor-specific CD8 and CD4
on a NTAmer scaffold. We show that these “im-
T cells is a promising treatment for late stage cancer.
munopure” pMHC class II multimers can efficiently
Peptide-MHC multimers containing biotinylated
stain tumor-specific CD4 T cells, while conventional
pMHC complexes on fluorescent streptavidin conju-
multimers cannot.
gates are widely used to enumerate, analyze and sort
In summary, we have developed new pMHC class
tumor-specific CD8 and CD4 T cells.
I and II multimers, which enable the isolation of
A shortcoming of pMHC class I multimers is that
highly competent tumor-specific CD8 and CD4 T
they strongly bind to cell-surface TCR and CD8 co-
cells, opening new perspectives for adoptive cell
receptor, thereby inducing frequent CD8 T cell death
transfer therapy.
and thus making it a major challenge to isolate viable
high-affinity T cell clones for expansion. We developed reversible pMHC class I multimers (NTAmers)
built on engineered chelate complexes of nitrilotriacetic acid (NTA) and His tag monomers that instantaneously dissociate upon addition of imidazole.
These NTAmers allow sorting of “untouched” CD8
T cells, preventing cell death of high affinity CD8 T
cells. On live antigen-specific CD8 T cell clones, twocolor NTAmers also allow precise measurement of
pMHC-TCR monomeric dissociation kinetics, which
correlate with T cell responsiveness, making it now
possible to screen and isolate those highly functional
tumor-specific CD8 T cells most suitable for therapeutic use.
Qualitative limitations of pMHC class II reagents
typically reside in the poor loading efficiency of peptides in “empty” class II molecules, leading to only
partially-loaded pMHC monomers and thus inefficient staining of antigen-specific CD4 T cells. We
developed “immunopure” multimers made of purified pMHC monomers loaded with tagged peptides
PD-1 blockade induces quantitative and qualitative changes
within a vast and common antigen-specific T cell repertoire in
melanoma treated patients
Simon S.1,2, Vignard V.1,2,3, Florenceau L.1,2,3, Knol A.-C.1,2,3, Dréno B.1,2,3, Khammari A.1,2,3, Lang F.1,2, Labarrière N.1,2,3
Nantes University, Inserm UMR892 CNRS 6299, Nantes, France,
1
LabEx IGO, Nantes, France,
2
Nantes Hospital, Nantes, France
3
Therapeutic strategies using anti-PD-1 antibody re-
We are currently correlating experimental results
ported unparalleled effectiveness for cancer immu-
with clinical outcomes of treated-patients to identify
notherapy. Understanding mechanisms involved in
new biomarkers associated with anti-PD-1 therapy
clinical benefit remains crucial to improve patients’
efficiency.
management.
In addition to the emergence of neo-antigen spe-
Despite its negative role in anti-tumor immunity,
cific T-cells previously documented upon anti-PD-1
PD-1 first identifies reactive tumor-specific T-cells.
therapy, our work describes qualitative and quantita-
We previously demonstrated that PD-1pos melanoma
tive changes within an antigen-specific T-cell reper-
specific T-cell clones exhibited a better functional
toire and offers new prospects for the monitoring of
avidity than their PD-1neg counterpart. We further
patients upon anti-PD-1 therapy.
documented in vitro that PD-1 blockade during the
Fundings: This work has been carried out thanks
selection and amplification process of melanoma
to the support of the Labex IGO project (n° ANR-
specific T-cells from patients’ PBMC, resulted in
11-LABX-0016-01), the DHU OncoGreffe and the Can-
the proliferation of specific T-cells with a biased
céropôle GO.
TCR Vbeta repertoire exhibiting a better functional
avidity (Simon et al., Oncoimmunol, 2016).
We assumed that this bias in antigen specific T-cell
repertoire also occurs in vivo for patients treated with
anti-PD-1 antibody. We compared Melan-A specific
T-cell repertoire diversity from melanoma patients
before and after anti-PD-1 therapy. We documented,
for all patients tested, a bias in Melan-A-specific
T-cell repertoire after treatment with the preferential amplification of clonotypes highly expressing
PD-1. We characterized the most represented Vb
subtypes (reactivity against melanoma cell lines and
functional avidity) and analyzed results according
to the expression of additional inhibitory receptors
to allow discriminating between highly reactive and
exhausted T-cells.
Systemic WT-1 specific T cell reactivity in relation to immune
status and survival following ablative treatment of locally
advanced pancreatic cancer by irreversible electroporation
Stam A.G.M.1, Scheffer H.J.2, Vroomen L.G.P.H.2, de Bruijn B.1, van den Tol M.P.3, Kazemier G.3, Meijerink M.R.2,
de Gruijl T.D.1
VU University Medical Center, Department of Medical Oncology, Amsterdam, Netherlands,
1
VU University Medical Center, Department of Radiology and Nuclear Medicine, Amsterdam, Netherlands,
2
VU University Medical Center, Department of Surgery, Amsterdam, Netherlands
3
Complete surgical resection at an early stage remains
immune suppression after the IRE procedure. Impor-
the only curative treatment option for pancreatic
tantly, in line with the observed post-IRE increase in
cancer. For patients with locally advanced pancreatic
CD8+ T cell proliferation, we found post-IRE boosting
cancer (LAPC), a novel local ablation technique, i.e.
of a pre-existing WT-1 specific T cell response in two
irreversible electroporation (IRE), may offer a much
out of three patients as well as the de novo induction
needed alternative therapy. IRE eliminates tumor
of these responses in another two patients. These
cells by apoptosis through high-voltage electric
WT-1 T cell responses were related to longer overall
pulses, while leaving much of the vasculature intact,
survival (p=0.055).
allowing for effective immune infiltration.
These findings are consistent with a systemic
To obtain evidence of a possible systemic immune
immune stimulatory effect of IRE and support the
stimulatory effect of the local IRE-mediated ablation
combination of percutaneous IRE with therapeutic
of LAPC, we performed an immune monitoring pilot
immune stimulation.
study in the first ten patients enrolled in a clinical trial
exploring the safety, feasibility and efficacy of the
treatment of LAPC with percutaneous image-guided
IRE (the PANFIRE-I study, NCT01939665, clinicaltrials.gov). Flowcytometric analysis was performed of
the frequency and activation state of various lymphocytic and myeloid subsets in the peripheral blood
of the patients, both pre- and post-treatment. Systemic T cell responses were determined to the pancreatic cancer associated antigens mesothelin and
Wilms Tumor (WT)-1 after in vitro stimulation in an
IFNγ Elispot assay, at baseline and at 2 weeks and 3
months after IRE. This pilot study was not powered
to draw definitive conclusions, but should be regarded as hypothesis-generating.
Our data show a transient decrease in systemic regulatory T cell (Treg) frequencies and a simultaneous
transient increase in activated Ki67+CD8+ T cells,
consistent with the temporary lifting of Treg imposed
An optimized IFN-γ ELISpot assay to determine CMV proteinreactive effector cells of cell- mediated immunity
Barabas S.1, Spindler T.1, Tonar C.1, Widmann T.1, Tudor S.1, Bendfeldt H.1, Deml L.1
Lophius Biosciences, Regensburg, Germany
1
In healthy individuals, Cytomegalovirus (CMV) in-
reactive cells and total PBMC counts was observed
fections are efficiently controlled by CMV-specific
(R 2 for stimulation with app65 and aIE-1 was 0.99
cell-mediated immunity (CMI). However, functional
and 0.97, respectively). Thus, the optimized ELISpot
impairment of the CMI in immunocompromized in-
assay may represent a highly standardized, valuable
dividuals can lead to uncontrolled CMV-replication
tool to monitor the functionality of CMV-specific CMI
and severe clinical complications. Thus a reliable,
in immunocompromized patients.
standardized monitoring of the CMV-specific CMI is
highly relevant for the prognosis of CMV-associated
diseases and personalized therapeutic decisions.
Objective of this study was the optimization and
technical validation of an IFN-γ ELISpot assay for the
sensitive and reliable determination of CMV-reactive
effector cells. Therefore T- activated® immunodominant CMV-proteins aIE-1 and app65 have been used
as stimulants enabling the simultaneous detection
of CMV-responsive T helper (Th) cells, cytotoxic T
cells (CTL) as well as natural killer (NK) and natural
killer T (NKT)- like cells. All basic assay parameters
and reagents were tested and optimized to establish
a user-friendly protocol and maximize the signal-tonoise ratio of the ELISpot-assay.
Applying the optimized CMV ELISpot protocol, all
CMV- seropositive healthy individuals tested showed
a positive test result irrespective of the composition
of their HLA alleles. The assay performance is highly
reproducible with coefficients of variation for intraand inter- assay and inter-operator variations of less
than 22 %. Number of spot forming cells are direct
proportional to deployed PBMC counts in the range of
6 x 104 and 2 x 105 PBMC per well. In addition, a linear
correlation between the amount of CMV protein-
Antigen-specific T cell immunomonitoring by HLA tetramer
combinatorial coding for CD8 T cells and CD40L expression on
antigen-specific CD4 T cells
Turksma A.1, Mok J.Y.2, Maas M.2, Hombrink P.3, van Esch W.2, van Ham M.1, ten Brinke A.1
Sanquin, Blood Supply Foundation, Immunopathology, Amsterdam, Netherlands,
1
Sanquin, Blood Supply Foundation, R&D Div. Reagents, Amsterdam, Netherlands,
2
Sanquin, Blood Supply Foundation, Hematopoiesis, Amsterdam, Netherlands
3
Antigen specific T cell analysis is becoming increas-
staining, thereby making it a more sensitive method.
ingly important for the development of a wide range
We were able to detect influenza A-specific T cells
of therapeutic applications covering cancer, infec-
even at a frequency of 0.004%. Intra and inter assay
tious and autoimmune diseases and vaccine design.
variations were low and demonstrate that HTCC is
Fluorescently-labeled HLA tetramer complexes have
a robust technology. Furthermore, HTCC was suc-
become a cornerstone for monitoring T cell specifi-
cessfully combined with phenotypic markers allow-
cities and frequencies. The availability of HLA te-
ing sensitive identification of naïve, memory, and ef-
tramer combinatorial coding (HTCC) technology,
fector antigen-specific CD8+ T cells. The detection
using dual-color encoded HLA class I tetramer com-
of antigen specific CD4+ T cells by CD40L detec-
plexes and flow cytometry, enables the simultane-
tion was highly sensitive with background levels of
ous detection of up to 28 different antigen-specific T
around 0.1%. The specificity of the CD40L detection
cell responses in a single biological sample. In this
method was validated by sorting and cloning CD40L
way, comprehensive epitope (discovery) screens can
CD4+ T cells after CMV long peptide stimulation.
be performed using limited patient material. Using
CMV specific T cell clones where found even after
class II tetramers to determine CD4+ T cell specifi-
only detecting a two-fold increase of CD40L expres-
cities is less robust than class I tetramer detection.
sion above background.
Therefore, a different approach is used to determine
Both CD4 and CD8 T cells responses are necessary
antigen-specific CD4+ T cells. After antigen-specific
for a good anti-tumor effect. These methods allow a
TCR stimulation CD4+ T cells up regulate CD40L.
broad antigen-specific monitoring of the T cell popu-
Detection of CD40L by flow cytometry can be com-
lations in patients.
bined with functional analysis such as cytokine production.
Peripheral blood mononuclear cells derived from
healthy donors have been used to standardize and
validate the HTCC technology and CD40L assay,
using antigens such as CMV, EBV, influenza and
tetanus toxoid.
The detection of antigen-specific CD8+ T cells by
dual coding (n=26) was found to be as efficient as
detection with conventionally fluorescently-labeled
HLA multimers (n=8) with an even lower background
Clinical immunomonitoring strategies assessing on-target and
off-target effects of anti-CD73 mAbs - The TumAdoR collaborative
project
Vigano S.1,2, Jandus C.2, Harari A.1, Gourdin N.3, Menetrier-Caux C.3, Caux C.3, Romero P.2
Center for Experimental Therapeutics, University of Lausanne, Department of Oncology, Lausanne, Switzerland,
1
Ludwig Cancer Research Center, Lausanne, Switzerland,
2
Centre Leon Bérard, Lyon, France
3
The natural progression of tumors is usually asso-
and validated fluorescently labeled antibody panels
ciated with, and even sustained by, tumor-driven
to evaluate the differentiation and polarization of T
immune-modulating mechanisms. Among these
cells, B cells, NK cells, DCs, monocytes and their
the over-expression of the membrane-bound nu-
activation, cytotoxic and cytokine profiles in com-
cleotidase CD73 by various cell types in the tumor
bination with CD73 and CD39 expression by flow
microenvironment (ecto-5’-nucleotidase) increases
cytometry. The analyses allowed a comprehensive
the concentration of extracellular adenosine (Ado),
evaluation of CD73 and CD39 expression by human
which suppresses both innate and adaptive immune
PBMCs of healthy donors. Immune-monitoring tools
responses and through multiple activities contributes
are crucial in the evaluation of immunotherapy in-
to tumor progression. The conversion of ATP into Ado
terventions to identify relevant efficacy and safety
is tightly regulated by cell membrane ecto-enzymes.
predictive biomarkers.
However, while the conversion of ATP into AMP,
predominantly catalyzed by CD39, is reversible, the
CD73 mediated catalysis of AMP into Ado is virtually
irreversible, thus representing a crucial checkpoint
in conversion of pro-inflammatory ATP into immunosuppressive Ado. Recent successes of anti-CTLA-4,
anti-PD-1 and PD-L1 mAbs open the door to a highly
promising novel class of therapeutics targeting tumor-driven immunosuppressive pathways. In this
context the TumAdoR is a collaborative EU-supported consortium that aims at bringing anti-CD73 mAb
candidates to clinical trial. Such antibodies would
be applicable to a wide range of cancers. Among the
tasks shared by the partners, specific diagnostic tools
are in development to assess target expression and
to monitor relevant immune parameters in patients
before and during the treatment. Antibody panels for
immune-monitoring have been developed to assess
by multicolor flow cytometry on-target and off-target
effects of anti-CD73 Abs. In particular, we developed
The research leading to these results has received funding
from the European Community´s Seventh Framework
Program ([FP7/2007-2013] under grant agreement n°602200.
Detection and functional assessment of regulatory T cells in
clinical samples
Santegoets S.J.A.M.1, Dijkgraaf E.1, Kroep J.1, Welters M.J.P.1, van der Burg S.H.1
Leiden University Medical Center, Clinical Oncology, Leiden, Netherlands
1
Regulatory T cell (Treg)-mediated immunosuppres-
sponder cells through dye dilution in a 5-days assay
sion is considered a major obstacle for successful
and determining responder cell activation (CD25 and
cancer immunotherapy. Given their profound effect
CD69 up-regulation) in a 6-20 hours assay. The 3H-
on the outcome of immunotherapy trials, Tregs are
thymidine-based approach is based on a recent publi-
being studied extensively in clinical trials. However,
cation by Tree et al. (Diabetes 64(11):3891-902, 2015),
the multitude of Treg definitions in the reported
describing a highly efficient protocol for measuring
studies makes correct interpretation of data and com-
the suppressive function of Tregs. With this approach
parisons between studies difficult. Also, unambigu-
we should be able to test the suppressive potential
ous enumeration of Tregs by flow cytometry is ham-
of Tregs using only 10,000 cells in an antigen-pre-
pered by the absence of an exclusive, highly specific
senting cell (APC)-dependent and -independent way.
marker and the inability to directly measure their
We expect that we will be able to show data on the
function in immunomonitoring of clinical trials.
functional assays at the meeting.
The Cancer Immunoguiding Program (CIP) recently
reached consensus with leading experts in the field
concerning the use of an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3,
Ki67 and CD45RA to define human Treg cells. The
use of markers was validated in a series of PBMC
from healthy donors and cancer patients, as well as
in tumor-draining lymph nodes (TDLN) and freshly
isolated tumors. Also a robust gating strategy for the
flow cytometric analysis of Tregs in human samples
was determined.
To prove that the cells identified through this marker
set are indeed Tregs with suppressor function, we are
currently developing assays to assess Treg functionality in the setting of (limited) clinical trial samples. To
do this, we are comparing two flow cytometry-based
approaches and are also setting-up a 3H-thymidinebased approach. The flow cytometic assays are based
on measuring inhibition of the proliferation of re-
A kit for the preparation of T-cell Receptor Engineered Reference
Sample (TERS) to control T-cell assay performance
Bidmon N.1,2, Kind S.2, Welters M.J.P.3, Joseph-Pietras D.4, Laske K.5, Maurer D.6, Hadrup S.R.7, Schreibelt G.8,
Rae R.1, Kern F.9, Sahin U.1,2, Gouttefangeas C.5, van der Burg S.H.3, Britten C.M.1,10
University Medical Center of the Johannes Gutenberg
1
University Mainz, Translational Oncology, Mainz,
6
Germany,
Technical University of Denmark, Immunology and
Germany,
7
BioNTech RNA AG, Mainz, Germany,
2
Leiden University Medical Center, Clinical Oncology,
3
Leiden, Netherlands,
Vaccinology, Frederiksberg C, Denmark,
Nijmegen Centre for Molecular Life Sciences, Tumor
8
Immunology, Nijmegen, Netherlands,
University of Southampton, ECMC, Cancer Sciences
9
Unit, Southampton, United Kingdom,
10
4
JPT Peptide Technologies GmbH, Berlin, Germany,
GlaxoSmithKline, Stevenage, United Kingdom
University of Tuebingen, Immunology, Tuebingen,
5
Germany,
Monitoring of the number and function of T cells has
RNA coding for GFP to optimize the electroporation
evolved as an essential tool in the field of immunother-
settings and a RNA encoding a TCR specific for the
apy. However, the results from different institutions,
tumor antigen NYESO1 to prepare TERS for immu-
and even data obtained over time within the same
nomonitoring. In addition, a centrally manufactured
laboratory, are not comparable. There is an urgent
TERS was provided, which was tested in parallel
need for cell samples that have a defined number of
in two independent experiments by HLA-multimer
antigen-specific functional T cells, which could be
staining. The intra-laboratory variance was very low
used as a standard for controlling performance over
and the inter-laboratory variance was below 10% for
time. We have established T-cell receptor (TCR) engi-
the centrally prepared TERS. The locally prepared
neered reference samples (TERS) by electroporating
TERS showed higher variance; the antigen-specific T
selected TCR-RNA into peripheral blood mononuclear
cell frequency ranged between 3-fold lower and 1.5
cells (PBMC) followed by spiking of these transfected
fold higher from the goal frequency of 1%. Never-
cells into autologous PBMC to generate a cell sample
theless, all participants were able to generate TERS
with a defined frequency of antigen-specific CD8+ T
with a detection frequency that was far above the
cells. The technique is simple and can be applied on a
background staining and comparable within their
larger scale. Moreover, the TERS are stable over time;
hands (low intra-laboratory variance). Following
at least for 2 years. A coordinated on-site production
these promising results, a commercialization of the
of TERS with 4 participants was successful, then a
TERS kit is under development in collaboration with
proficiency panel with 6 participants from 4 coun-
JPT peptide technologies. In conclusion, there is a
tries was conducted. Upfront, the commonly avail-
high concordance of the TERS generated within and
able electroporation devices from 4 vendors were
across institutions irrespective of multi-user diver-
extensively tested and a guideline for each device
sity, which makes this TERS kit approach a highly
prepared. The participants of the proficiency panel
attractive tool to control for assay performance.
generated their own TERS at their institutes using the
provided guideline and TERS kit. The kit included a
Associations of peripheral blood Vδ1+ γδ T-cells with overall
survival of melanoma patients
Wistuba-Hamprecht K.1,2, Martens A.1,2, Hähnel K.2, Garbe C.1, Pawelec G.2, Weide B.1
University Medical Center Tübingen, Department of Dermatology, Tübingen, Germany,
1
University Medical Center Tübingen, Department of Internal Medicine II, Tübingen, Germany
2
Recently, interest has resurged in the possibility of
observations are required to validate our results and
using γδ T-cells in cancer immunotherapy, because
to confirm their prognostic value.
these cells can kill melanoma cells in vitro, and
possess regulatory capabilities. γδ T-cells could potentially influence the efficacy of immunotherapies.
Thus, we have investigated γδ T-cells and their respective differentiation stages in late-stage melanoma patients using cryopreserved PBMC from six
clinical centers. Flow cytometry and a carefully validated set of monoclonal antibodies to assess γδ T-cellphenotypes described in the OMIP-20 panel publication was used for data acquisition and processing.
We identified peripheral frequencies of Vδ1+ T-cells
as being negatively associated with overall survival
(OS) of 27 stage IV melanoma patients with different
treatment background. This finding could be validated in a second independent cohort of 109 stage IV
patients treated with ipilimumab. Moreover, comparing total frequencies of Vδ1+ γδ T-cells in patients
with healthy subjects revealed higher proportions
of these cells in patients. A detailed analysis of the
Vδ1+ subset composition in patients by determining
its differentiation signature identified significantly
higher proportions of late-stage differentiated (CD27CD28-CD45+) Vδ1+ cells in patients with poorer
outcome compared to those that were in the prognostic favorable group.
We therefore suggest Vδ1+ T-cell frequencies as novel
candidate biomarkers in melanoma, as we identified
a significant association of these cells with patients’
OS in two independent studies. Nonetheless, further
Bone marrow T cells from myeloma patients exhibit features of
both T-cell exhaustion and senescence
Zelle-Rieser C.1, Thangavadivel S.1, Willenbacher W.2, Biedermann R.3, Brunner A.4, Greil R.5, Jöhrer K.1
Tyrolean Cancer Research Institute, Innsbruck, Austria,
1
Innsbruck Medical University, Department of Internal Medicine, Innsbruck, Austria,
2
Innsbruck Medical University, Department of Orthopedic Surgery, Innsbruck, Austria,
3
Innsbruck Medical University, Department of Pathology, Innsbruck, Austria,
4
Paracelsus Medical University Salzburg, Laboratory for Immunological and Molecular Cancer Research, IIIrd
5
Medical Department, Salzburg, Austria
Multiple myeloma is a still incurable plasma cell
tumor which relies on the bone marrow microenvironment. Besides mutations fueling growth of the
tumor cells, immune deficiencies play an essential
role in disease establishment and progression. Rearming of T cells could bring great merit to improve
therapeutic outcomes, however, the underlying
defects in T-cell responses are only partially understood so far.
Therefore we analysed T cells in the bone marrow of
myeloma patients and compared the phenotype and
function to healthy, age-matched donors. We found
higher numbers of T cells expressing the molecules
CTLA-4, CD160, CD244, PD-1 and CD57, which are
associated with T-cell exhaustion and T-cell senescence. Furthermore, myeloma bone marrow T-cells
showed a decreased ability to proliferative and also
did not retain the capacity to degranulate in response
to T-cell stimuli, as shown by a reduced transfer of
CD107a to the cell surface upon stimulation. Despite
an increased expression of Tbet, CD8+ T cells failed
to produce the cytokines IFNgamma, TNFalpha and
IL-2, which could even not be restored after in vitro
T-cell activation.
These data confirm the presence of exhausted, terminally differentiated CD8+ T cells in myeloma
patients. Our results suggest that interference with
inhibitory molecules could restore the functional activity of bone marrow T-cells and therefore enhance
the efficacy of current immunotherapeutic strategies
in myeloma patients.
167 – 215
New Targets &
New Leads
167 | NEW TARGETS & NEW LEADS
Identifying tumour-specific Class I MHC peptide epitopes by
Mass Spectrometry
Ternette N.1, Muller J.1, Parizotto E.1, Ramasamy A.1, Talbot D.2, King E.3, Ottensmeier C.4, Ashfield R.1
University of Oxford, Jenner Institute, Oxford, United Kingdom,
1
University of Oxford, Oncology, Oxford, United Kingdom,
2
University of Southampton, Medicine, Southampton, United Kingdom,
3
University of Southampton, Experimental Medicine, Southampton, United Kingdom
4
The development of successful cancer immunothera-
scribed in the literature) and b) novel targets. RNA
py has been hampered by the lack of cancer-specific
analysis is on-going to confirm tumour-specificity
target antigens. Cancer antigens are over-expressed
of these proteins, and our eventual aims are i) to
in tumour cells and processed by the proteasome to
produce new cancer vaccines by expressing the an-
create short peptide epitopes which are presented on
tigens in Adenovirus and MVA-based viral vectors,
the cell surface by Class I MHC. Computer algorithms
which are strong inducers of CD8 T cell responses,
can be used to predict epitopes likely to bind to indi-
and ii) to combine these vaccines in the clinic with
vidual MHC types, but are poor at predicting which
immune-check point inhibitors.
are naturally processed and presented; this issue can
be overcome by direct detection of epitopes using
Mass Spectrometry (MS). We are identifying new
targets for tumour immunotherapy, in particular
cancer vaccines, for NSCLC (non-small cell lung carcinoma) and HNSCC (head and neck squamous cell
carcinoma), using LC-MS/MS to identify Class I MHC
epitopes eluted from flash frozen surgical resections.
We are analysing matched tumour and normal tissue
in order to identify epitopes presented on the tumour
cell surface by MHC molecules, but not at detectable
levels on normal cells; these peptides are the targets
for anti-tumour CD8 T cell responses and are crucial
for the efficacy of antigen-specific cancer immunotherapy. We will present several examples of tumourspecific epitopes from a) known cancer antigens (de-
Relationship between immune gene signatures and clinical
response to PD-1 blockade with pembrolizumab in patients
with advanced solid tumors
Ayers M.1, Lunceford J.1, Nebozhyn M.1, Murphy E.1, Loboda A.1, Albright A.1, Cheng J.1, Kang S.P.1, Ebbinghaus S.1,
Yearley J.1, Shankaran V.2, Seiwert T.3, Ribas A.4, McClanahan T.1
Merck & Co., Inc., Kenilworth, United States,
1
University of Washington, Seattle, United States,
2
University of Chicago, Chicago, United States,
3
ORAL
TALK
SHORT
University of California - Los Angeles, Los Angeles, United States
4
2016
Background: Immune checkpoint inhibition with
10 genes to 6, and the expanded immune signature
anti-PD-1 monoclonal antibodies such as pembroli-
was refined from 28 genes to 18. Two new signatures
zumab has demonstrated robust, durable antitumor
enriched in T-cell markers and major histocompat-
activity against many advanced malignancies. We
ibility complex class I and II genes were enumer-
analyzed immune-related gene expression profiles
ated based on analysis of top-ranked genes on the
in pembrolizumab-treated patients with advanced
platform in melanoma samples: “TCR signaling” (13
solid tumors to identify immune gene signatures cor-
genes) and “de novo” (33 genes). All signatures were
related with clinical benefit.
independently tested in HNSCC and gastric cancer
Methods: RNA was extracted from formalin-fixed, par-
and found to be significantly correlated with clini-
affin-embedded sections of baseline tumor samples
cal benefit. Tumors lacking an immune phenotype,
and analyzed using the NanoString nCounter. Two
as suggested by low values of signature scores, did
signatures were prespecified: the “interferon-gamma
not respond to pembrolizumab. Some nonresponders
(IFN-γ)” and “expanded immune.” A 10-gene prelim-
had scores similar to those of responders, suggesting
inary IFN-γ signature was developed in a discovery
an immune phenotype is necessary but not sufficient
set of 19 patients with melanoma treated with pem-
for response.
brolizumab in the phase 1b KEYNOTE-001 study
Conclusions: Immune-related gene expression signa-
(NCT01295827) and was later expanded to a 28-gene
tures composed of genes associated with T-cell cyto-
preliminary “expanded immune” signature. These
toxic function, antigen presentation machinery, and
signatures were subsequently tested and refined
IFN-γ signaling represent reproducible and sensitive
in an independent cohort of 62 additional patients
tools that define common features of the immune
with melanoma treated in KEYNOTE-001. Further
microenvironment associated with response to pem-
evaluation of the refined signatures was performed
brolizumab across multiple tumor types.
in 43 patients with head and neck squamous cell
carcinoma (HNSCC) and in 33 patients with gastric
cancer enrolled in the phase 1b KEYNOTE-012 study
(NCT01848834).
Results: In the melanoma validation set, the IFN-γ
and expanded immune signatures were significantly
correlated with objective response rate (P = 0.047
and 0.027) and progression-free survival (P = 0.016
and 0.015). The IFN-γ signature was refined from
Direct identification of neo-epitopes using in-depth immunopeptidomics of melanoma tissues for the development of antitumor immunotherapies
Bassani-Sternberg M.1,2, Braunlein E.3, Klar R.3, Engleitner T.4,5, Sinitzyn P.2, Audehm S.3, Straub M.6, Weber J.4,5,
Slotta-Huspenina J.6, Specht K.6, Martignoni M.E.7, Werner A.7, Hein R.8, Busch D.9, Peschel C.3,5, Rad R.4,5,
Cox J.2, Krackhardt A.M.3,5, Mann M.2
UNIL/CHUV, Department of Oncology, Epalinges,
1
Switzerland,
Technical University of Munich, Klinik und
7
Poliklinik fuer Allgemeine Chirurgie, Klinikum rechts
Max Planck Institute of Biochemistry, Proteomics and
2
Signal Transduction, Martinsried, Germany,
Technical University of Munich, Medizinische Klinik
3
III, Klinikum rechts der Isar, Munich, Germany,
Technical University of Munich, Medizinische Klinik II,
4
der Isar, Munich, Germany,
Technical University of Munich, Klinik fuer
8
Dermatologie, Klinikum rechts der Isar, Munich,
Germany,
Technical University of Munich, Institute of
9
Klinikum rechts der Isar, Munich, Germany,
Microbiology, Immunology and Hygiene,
German Cancer Consortium (DKTK), Munich, Germany,
Munich, Germany
5
Technical University of Munich, Institut fuer Allge-
6
meine Pathologie und Pathologische Anatomie,
Munich, Germany,
ORAL
TALK
SHORT
2016
The antigenic landscape of tumors distinguishes
the peptides by a nanoflow HPLC and sprayed them
them from healthy cells and has been the ration-
directly into a Q Exactive HF mass spectrometer. We
ale behind a variety of vaccination trials. Recent
developed a new module in the MaxQuant computa-
data show that activation of the immune system
tional environment that integrates next generation
by immune checkpoint blocking therapies leads to
sequencing data and generates customized patients’
tumor rejection and that recognition of mutated an-
specific reference databases that contain nonsynony-
tigens, known as ‘neo-epitopes’ plays a key role. So
mous somatic mutations for the direct identification
far, discovery of neo-epitopes relies mainly on pre-
of neo-epitopes.
diction-based interrogation of the ‘mutanome’ using
We accurately identified the most comprehensive
genomic information as input, followed by highly la-
immunopeptidome from human melanoma tissues,
borious and time-consuming T cell screening assays.
comprising 95,662 unique peptide ligands with a
With the aim to identify the actual in-vivo presented
false discovery rate of 1%, among them more than
neo-epitopes from human melanoma tumors, we
300 known and novel peptide ligands derived from
applied our recently developed in-depth and stream-
known melanocyte-associated differentiation and
lined MS-based immunopeptidomics approach.
cancer testis antigens. Although we did not spe-
We isolated HLA class I and HLA class II peptidomes
cifically enrich for them, we detected 365 phospho
from 25 melanoma primary tissues. We separated
HLA-I and 26 phospho HLA-II peptides, with 261
of these corresponding to known phosphorylation
sites. Peptide ligands harboring such modifications
may represent attractive target candidates for immunotargeting.
We generated customized reference databases using
exome sequencing data for five patients, and for
the first time, using our discovery approach, we
identifed 11 neo-antigens from three patients. We
confirmed their identification with corresponding
synthetic peptides. Four of eleven mutated ligands
proved to be immunogenic by antigen-specific T-cell
responses, hence are confirmed neo-epitopes. Moreover, tumor-reactive T cells with specificity for two
of the neo-epitopes identified by MS were detected
in the patient`s peripheral blood (see also submitted
abstract by E. Bräunlein).
Taken together, we established a comprehensive resource of the melanoma in-vivo immunopeptidome
with a high number of attractive novel targets. Most
importantly, we showed the feasibility of identifying
the in-vivo and immunogenic neo-epitopes, directly
from tumor tissues. This is a promising approach for
the identification of new targets for the development
of immunotherapies.
Validation of a clinical 1400-gene assay for genomic profiling of
cancer from DNA and RNA
Beck J.1, Clark M.J.1, Lefterova M.1, Helman E.1, Alla R.K.1, Church D.M.1, Boyle S.M.1, Luo S.1, Morra M.1, Harris J.1,
Leng N.1, Haudenschild C.1, Chen R.1, West J.1
Personalis, Inc., Field Application Scientist, Menlo Park, United States
1
We present analytical validation of the ACE Cancer
Panel, the largest augmented panel available for
clinical oncology, targeting 1438 cancer and pharmacogenomic genes for enrichment and DNA/RNA
sequencing. Analytical validation was accomplished
using 28 cancer cell lines and reference standards
with known SNVs and/or indels, 19 with known
CNAs, and 17 with known gene fusions. We simulated tumor heterogeneity with allele frequencies
down to 1%, and tumor purity to 10%. Bioinformatics
analyses were performed in both tumour-only and
tumour-normal modes. Additionally, we validated
for use with clinical formalin fixed paraffin embedded, fresh frozen and blood samples. At read depth
mean of ≥500 reads, assay sensitivity for SNVs was
99.7%, AF ≥ 5% (n = 16132), small indels 99.4% AF ≥
10% (n = 671), CNAs 91.2% (n = 34, copy number 0
or ≥8 in tumor-only cell-lines), and fusion transcripts
95.0% (n = 20). Assay specificity was ≥99% for SNVs
and indels. This comprehensive cancer NGS panel
demonstrates highly uniform coverage, ensuring
high sensitivity and specificity for all variant types. It
represents a versatile diagnostic tool to test a core set
of clinically actionable genes, implicate new cancer
genes as clinically relevant and facilitate discovery of
novel therapeutic targets.
ROS-based cancer therapies - a role for cold physical plasma
against pancreatic malignancies in vitro and in vivo
Bekeschus S.1, Liedtke K.R.2, Partecke L.I.2
Leibniz-Institute for Plasma Science and Technology (INP Greifswald), ZIK plasmatis, Greifswald, Germany,
1
University of Greifswald, Department of General, Visceral, Thoracic and Vascular Surgery, Greifswald,
2
Germany
Recently, cold physical plasma has been increasingly
an anticancer role of plasmatreated medium in the
recognized as a promising option in tumor therapy.
absence of systemic side effects, making plasma a
Aggressive malignancies such as pancreatic cancers
promising new complementary therapeutic option in
cause 250.000 deaths annually, exemplifying the
oncology.
need for new therapeutic approaches. We examined
the therapeutic potential of a cold argon plasma jet
(kINPen MED) which has received accreditation as
medical product in Germany. In vitro, exposure of a
murine pancreatic cancer cell line to plasmatreated
medium significantly decreased the cells’ viability
and proliferation capacity whereas ex vivo expanded, autologous mouse fibroblasts were less affected.
Using a syngeneic mouse tumor model with a peritoneal carcinomatosis, repeated intraperitoneal injection of plasmatreated medium significantly reduced
tumor growth (p< 0.008) and tumor mass (p< 0.029)
as determined via magnetic resonance imaging and
weighing of excised tumor nodes, respectively. Importantly, the treatment group showed improved
median animal survival (61 vs 52 days; p< 0.024).
Immunohistochemistry of tumor nodes of treated
animals demonstrated severe apoptosis (p< 0.001)
and a strongly inhibited cell proliferation. Notably
and using several approaches, we could not detect
systemic side effects in the treatment group. First, apoptosis was not present in control organs such as the
liver or gut. Second, the concentration of inflammatory cytokines in splenocytes or mouse blood plasma
was not altered. Third, the distribution of blood leukocyte populations and various hematological parameters remained unchanged. Our results suggest
Signal peptide derived monoclonal antibodies impair mmtvassociated tumor growth
Braitbard O.1, Abramovitch H.1, Hochman J.1
The Hebrew University of Jerusalem, Cell and Developmental Biology, Jerusalem, Israel
1
Mouse Mammary Tumor Virus (MMTV) causes
mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports
its involvement also in human sporadic breast cancer
(up to 30%)and in primary biliary cirrhosis. Still, it is
not known whether the virus plays a role in disease
pathogenesis or whether it represents an epiphenomenon.
The signal peptide of the envelope precursor protein
of this virus: MMTV-p14 (p14 for short) is a significant target for immune intervention due to its cell
surface expression as well as being a multifunctional
protein, in addition to its traditional role in endoplasmic reticulum targeting.
We have generated a panel of monoclonal anti-p14
antibodies directed against different cell surface
epitopes of p14 (both sequence and conformation
related). Some of these monoclonals demonstrate additive binding to p14 while others seem to be competitive. A combination of 2 such additive monoclonals
(M-202 and M-66) impairs the growth of murine lymphoma and mammary carcinoma cell lines in vivo.
However, when applied separately, these monoclonals are ineffective in vivo. The mechanism of this
anti-tumor effect is presently under study. Based on
the above findings we propose the use of anti-p14 antibodies as a tool for passive immunization towards
MMTV-associated tumors as well as for diagnostic
and targeting purposes of such tumors.
Immunogenicity assessment of mutated HLA-ligands identified
on melanoma tissue probes by mass spectrometry
Bräunlein E.1, Bassani-Sternberg M.2, Klar R.1, Audehm S.1, Engleitner T.3,4, Rämisch S.1, Sinitcyn P.2, Straub M.5,
Weber J.3,4, Slotta-Huspenina J.5, Specht K.5, Martignoni M.E.6, Werner A.6, Hein R.7, Busch D.H.8, Peschel C.1,4,
Rad R.3,4, Cox J.2, Mann M.2, Krackhardt A.M.1,4
Klinikum Rechts der Isar, III. Medizinische Klinik, Technische Universität München, Germany,
1
Max Planck Institute of Biochemistry, Martinsried, Germany,
2
Klinikum Rechts der Isar, II. Medizinische Klinik, Technische Universität München, Germany,
3
German Cancer Consortium (DKTK), München, Germany,
4
Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, Germany,
5
Klinikum Rechts der Isar, Klinik und Poliklinik für Allgemeine Chirurgie, Technische Universität München,
6
Germany,
Klinikum Rechts der Isar, Klinik für Dermatologie, Technische Universität München, Germany,
7
Institute of Microbiology, Immunology and Hygiene, Technische Universität München, Germany
8
Malignant Melanoma (MM) is one of the most ag-
stimulation of leukocytes from healthy donors (HD)
gressive tumor entities with rapid disease progres-
and patients as available. We observed recognition of
sion, although recent advances in therapy options,
four epitopes comprising two reactivities in patient
especially immune checkpoint blockade, markedly
Mel15 as well as two specific responses amongst HD.
improved clinical outcome. Nevertheless, there is
Responses of Mel15 against mutated peptides derived
only limited knowledge about specific target struc-
from SYTL4 and NCAPG2 could be reproducibly de-
tures involved in efficient tumor rejection and deter-
tected in stimulation assays with blood-derived T
minants for variable response rates observed after
cells within a time frame of six months. Notably, a
novel immunotherapies are not yet completely un-
residual lung metastasis of Mel15 removed due to en-
derstood.
hanced signal activity by PET-CT, was recognized by
As response to checkpoint modulation has been as-
in-vitro stimulated and expanded T-cell lines. Analy-
sociated with the mutational load, major approaches
sis of simultaneously isolated tumor-infiltrating lym-
attempt to identify immunogenic mutations by in-sil-
phocytes (TIL) revealed reactivity against one of the
ico epitope prediction followed by large-scale screen-
newly identified neo-epitopes. Other epitopes lacking
ing assays. In order to directly identify mutated
specific immune responses in the autologous setting
peptide ligands, we combined mass spectrometric
were further evaluated with naïve T cells from al-
analysis of the immunopeptidome with whole exome
logeneic HLA-matched HD. T cells with specificity
sequencing in five selected patients (see also submit-
for an AKAP6-derived neo-epitope displayed dis-
ted abstract by M. Bassani-Sternberg). We thereby
tinct qualitative differences between recognition of
identified eleven naturally presented mutated peptide
mutated and wildtype ligand. For this neo-epitope,
ligands, including eight mutated ligands derived
target cells transduced with a minigene coding for an
from a single patient (Mel15) who responded well
epitope-spanning protein fragment were efficiently
to anti-CTLA4-blockade. The immunogenic potential
recognized accomplished by a clearly inferior re-
of all peptide candidates was analyzed by in-vitro
activity against the wildtype construct indicating
correct processing and presentation of the identified
neo-epitope.
Taken together, our data demonstrate the high potential of mutated peptide ligands identified by
mass spectrometry. The high yield in validated
neo-epitopes emphasizes their therapeutic value
for either active vaccination or generation of neoepitope-specific T cells for adoptive transfer. In addition, detailed investigation of the frequencies of
mutation-specific T cells may be highly valuable as
potential surrogate markers for the patient`s specific
clinical course.
EB and MBS: shared first authors
MM and AMK: shared last authors
The HLA-associated phosphoproteome as a new target for
immunotherapy against hepatocellular carcinoma
Buettner N.1, Trantham P.D.2, Penny S.A.3, Curbishley S.4, Steadman L.G.3, Millar D.G.5, Goodyear O.C.3,
Russel M.3, Blahova M.4, Speers E.2, Ruth N.D.3, Wong G.3, Thimme R.1, Adams D.H.4, Shabanowitz J.2,
Hunt D.F.2, Cobbold M.5
Universitätsklinikum Freiburg, Innere Medizin II - Gastroenterologie/Hepatologie, Freiburg, Germany,
1
University of Virginia, Department of Chemistry, Charlottesville, United States,
2
University of Birmingham, Immunology and Infection, Birmingham, United Kingdom,
3
University of Birmingham, Centre for Liver Research, Birmingham, United Kingdom,
4
Massachusetts General Hospital, Center for Cancer Immunology, Boston, United States
5
Background: Hepatocellular carcinoma (HCC) is
with chronic liver disease and HCC and analyzed
the sixth most common cancer in the world with a
using multiplexed intracellular cytokine staining.
growing incidence and mortality in many countries
Additionally, MHC-I-pP-specific CD8+ T cells from
of the world. Since HCC is believed to be immuno-
IHLs and TILs were expanded in a large scale using
genic, immunotherapy is considered a promising
a rapid expansion protocol (REP) with anti-CD3, IL-2
new treatment modality. The identification of spe-
and irradiated feeder cells.
cific tumor-associated antigens provides the basis
Results and conclusion: So far we have identified
for the development of an efficient immunotherapy.
over 300 HCC-associated MHC-I-pP, presented by
However, only very few HCC-specific tumor antigens
various HLA molecules. Many of the novel identi-
have been characterized so far and post-translational
fied MHC-I-pP were derived from proteins, which
modified peptides have been overlooked in the past.
can be directly linked to important cancer-associ-
Dysregulation of signaling pathways in cancers leads
ated signaling-pathways. More MHC-I-pP were dis-
to aberrant and augmented protein phosphorylation
played in a greater diversity on HCC in comparison
and in such way modified proteins can be degraded
to non-cirrhotic and cirrhotic liver tissue. CD8+
to generate cancer-specific phosphopeptides. These
T cell responses against this novel class of tumor-
are presented by MHC class I molecules and recog-
antigens were comparable in quantity and quality
nized by T cells. The aim of this study was to iden-
to those seen against viral control epitopes. CD8+
tify HCC-associated, MHC class I-bound phospho-
T cell responses were found in a large fraction of
peptides (MHC-I-pP) and to assess immunity against
patients with early chronic liver disease, were much
this novel class of antigens in patients with chronic
rarer in patients with end-stage liver cirrhosis and
liver disease and HCC.
were lost after HCC formation. Our results therefore
Methods: For identification, tissues were lysed and
suggest that MHC-I-pP may be the target of cancer
MHC class-I complexes affinity purified. The bound
immune surveillance in liver disease. Furthermore,
phosphopeptides were eluted, enriched and charac-
expansion of MHC-I-pP-specific CD8+ T cells in a
terized using mass spectrometry. We compared MHC
large scale was achieved from a combination of REP
class I-bound phosphopeptides (MHC-I-pP) found on
and repeated rounds of MHC-I-pP-specific stimula-
healthy liver tissue, cirrhotic liver tissue and HCC
tion. Therefore, MHC class-I bound phosphopeptides
from patients undergoing liver transplantation. Im-
represent an attractive target for future cancer immu-
munity against MHC-I-pP was assessed in PBMCs,
notherapies with a possible application in adoptive T
intrahepatic lymphocytes (IHLs) and tumor-infiltrat-
cell therapy.
ing lymphocytes (TILs) from healthy donors, patients
Within the family of MELOE antigens, IRES-dependent
translation conditions exclusive expression in melanoma
cells and immunogenicity
Charpentier M.1,2, Croyal M.3, Carbonnelle D.1,2, Fortun A.1,2, Krempf M.3,4, Labarrière N.1,2, Lang F.1,2
UMR INSERM U892 - CNRS U6299 - University of Nantes, Nantes, France,
1
LabEx IGO, Nantes, France,
2
UMR 1280 INRA - Nantes University, CNRH, Nantes, France,
3
Nantes Hospital, Nantes, France
4
We have previously reported that two melanoma an-
upon restimulation by INFγ intracellular staining. In
tigens, MELOE-1 and MELOE-2 involved in T cell im-
marked contrast with the high frequencies of CD4
munosurveillance of melanoma were translated from
and CD8 T cell responses against MELOE-1 in all 4
the polycistronic mRNA meloe by IRES-dependent
healthy donors, we found no CD8 and very rare CD4
mechanisms. We now explored whether upstream
T cell responses against MELOE-4. This suggests an
ORFs from this mRNA could be translated by a clas-
immune tolerance towards this antigen that would
sical cap-dependent mode and indeed found that
be consistent with its expression in normal melano-
the most upstream ORF, named MELOE-4 (54 aa),
cytes. In conclusion, despite its high expression in
was very efficiently translated in melanoma cells.
melanoma cells, the classically translated MELOE-4
Using melanoma cell lines transfected with various
antigen represents a poor target for T cell immuno-
eGFP-tagged MELOE constructs, we could evidence
therapy because of its expression profile and very low
that the expression of MELOE-4 was more than 100
immunogenicity. On the other hand, IRES-dependent
times higher than that of MELOE-1 in melanoma
MELOE antigens represent the best T cell targets for
cells. We have previously shown that melanocytes
immunotherapy due to their very specific expression
also express meloe mRNA but are not recognized by
by melanoma cells and high immunogenicity. This
MELOE-1 or MELOE-2 specific T cell clones suggest-
prompts us to explore other IRES-dependent antigens
ing that IRES-dependent translation of MELOE-1 and
as target for immunotherapy in cancer.
MELOE-2 is not activated in these cells. In contrast,
we documented the presence of MELOE-4 in 4 melanocyte cell lines by mass spectrometry. This presence argues in favor of MELOE-4 as the physiological
product of meloe mRNA in melanocytes.
We then questioned the immunogenicity of MELOE-4
by exploring the CD8 and CD4 T cell repertoire
against this antigen in healthy subjects in comparison to the frequent T cell repertoire against MELOE-1
that we described previously. Using an in vitro protocol of accelerated DC differentiation and maturation, we stimulated PBMCs from 4 healthy donors
with overlapping peptides from either MELOE-1 or
MELOE-4 and tested CD4 and CD8 T cell reactivities
Ex vivo expansion of human glioma-infiltrating lymphocytes
alters the exhaustion phenotype of T cells
Deumelandt K.1, Bunse T.1, Bunse L.1, Sonner J.K.1, Thomé C.2, Nadji-Ohl M.3, Wick W.2,4, Platten M.1,4
German Cancer Research Center, DKTK CCU Neuroimmunology and Brain Tumor Immunology, Heidelberg,
1
Germany,
German Cancer Research Center, DKTK CCU Neurooncology, Heidelberg, Germany,
2
Katharinenhospital Stuttgart, Neurosurgery Clinic, Neurooncology Group, Stuttgart, Germany,
3
University Hospital Heidelberg, Department of Neurology and National Center for Tumor Diseases, Heidelberg,
4
Germany
There is increasing interest in identifying and char-
pression of the proliferation marker Ki67 as well as
acterizing tumor antigen-specific tumor-infiltrating
the cytotoxic serine protease granzyme B, indicating
T lymphocytes (TILs) reactive to tumor antigens (TA)
reinvigoration of exhausted T cells. In conclusion, our
for further therapy such as adoptive T cell therapy.
results show that a commonly used ex vivo expansion
Antigen-experienced T cells typically upregulate
of patient TILs results in a profound skewing of the
exhaustion markers such as PD-1, which is why
TIL phenotype, particularly markers of exhaustion
protocols for selecting TA-specific T cells from the
and reinvigoration. Hence, direct analysis of freshly
periphery make use of these exhaustion markers to
isolated unperturbed TILs may be necessary to allow
select for relevant antigen-experienced T cells. Com-
a faithful assessment of the relevant TIL profile for
monly used protocols for the TIL isolation make use
further identification of TA-specific TILs.
of an ex vivo expansion in order to achieve reasonable
T cell numbers for further analyses. Whether this
expansion affects the phenotype of TILs, however,
remains unclear. In this study, we investigated the
influence of a commonly used ex vivo expansion protocol for the isolation of human glioblastoma (GBM)infiltrating T lymphocytes on their phenotype. This
protocol involved ex vivo expansion of TILs by culturing tumor fragments with complete medium supplemented with IL-2 and CD3 ligand, whereby TILs
were allowed to migrate out of the tumor tissue and
further expanded using IL-2 for two weeks. We demonstrate that the CD8/CD4 T cell ratio shifts dramatically after ex vivo expansion of GBM TILs towards a
CD4 dominance, when compared to freshly isolated
unperturbed TILs. Furthermore, ex vivo expansion
of GBM TILs resulted in an increase of LAG-3+ and
TIM-3+ CD8 and CD4 T cells. Additionally, the frequency of PD-1+ CD8 T cells decreased considerably
after ex vivo expansion, whereas a greater subset of
the PD-1+eomes+ CD8 T cell population indicated ex-
A “multi-omics approach” for the identification of T cell
epitopes in clear cell renal cell carcinoma
Di Marco M.1, Reustle A.2, Kowalewski D.J.1, Backert L.1, Büttner F.2, Winter S.2, Hennenlotter J.3, Bedke J.3,
Schäffeler E.2, Schwab M.2, Rammensee H.-G.1, Stevanovic S.1
1
Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany,
2
University Hospital of Tübingen, Department of Urology, Tübingen, Germany
3
Renal cell carcinoma (RCC) is considered to be one
methylome, transcriptome and metabolome analysis
of the most immunogenic tumors. This is based on
of all samples are being performed allowing for an
the occurrence of spontaneous regressions even at
integrative analysis of cellular aberrations in ccRCC.
metastatic stages, the high level of tumorinfiltrating
Immunohistochemical and fluorescence staining of
T lymphocytes and sporadic responses to nonspe-
target candidates are being conducted for further val-
cific immunotherapies with IL2 and INFα. Recently,
idation. Integrating these data should allow for the
the α-PD-1 antibody Nivolumab was shown to have
comprehensive characterization of RCC associated
beneficial effects for RCC patients regarding overall
HLA ligands even on a mechanistic level. Finally,
survival and overall response rate. This clearly dem-
TUMAPs from promising targets will be tested for
onstrates the potential of T cell based immunother-
their immunogenicity.
apy in RCC. However, in order to further enhance
So far, HLA ligandome analysis was performed for
the specificity and frequency of these anti-cancer
45 ccRCC and corresponding adjacent benign tissues.
T cell responses, novel, synergistic approaches are
In total, over 33.000 HLA ligands from over 10.000
needed. Personalized approaches, such as peptide
source proteins were detected on HLA class I. For
vaccination or adoptive T cell transfer and combi-
HLA class II, over 12.000 peptides from over 3.200
natorial strategies combining checkpoint inhibition
source proteins were discovered. Many promising
with peptide vaccination hold the potential for the
targets, such as ANGPTL4, HSF4, SULF1, VEGFA,
frequent induction of anti-cancer immune responses.
SLC2A5, LPCAT1, EGLN3, NDULF2A4, CAGE1,
In this context, the identification of suitable T cell
P4HA1&2, PLOD2 or the well-known antigens CA9
epitopes is of paramount importance.
and CA12 were identified. Following integrative anal-
For that issue, we are conducting a comprehensive
ysis the corresponding HLA ligands will be tested
analysis of the HLA ligandome of 60 ccRCC and cor-
in in vitro priming assays to validate their ability to
responding adjacent benign tissues by LC-MS/MS.
induce T cell responses.
The extensive cohort will ensure a comprehensive
overview of tumor-associated peptides (TUMAPs)
and HLA source proteins presented exclusively on
tumor tissue (LiTAAs = Ligandome-defined tumorassociated antigens). To further ensure a tumorexclusive presentation, benign tissues of a range
of normal tissues are utilized for comparative HLA
ligandome profiling. Additionally, exome, DNA
A novel role for CD4+ T cells in clearance of highly aggressive
MHC-I low tumors mediated via NK cells
Doorduijn E.1, Sluijter M.1, van der Burg S.1, van Hall T.1
LUMC, Clinical Oncology, Leiden, Netherlands
1
Aldara® cream consists of a TLR7 agonist (imiquimod) and is currently used for the treatment of genital
warts and basal cell carcinoma of the skin. In the
present study, we examined the efficacy of Aldara® in
the context of the highly aggressive RMA-S tumor, a
lymphoma with very low expression of MHC class I.
Mice were s.c. inoculated with RMA-S cells and palpable tumors were treated by Aldara® cream application on the skin at the contralateral flank. Strikingly,
this distant application resulted in complete tumor
regressions in the majority of the mice and a strong
delay in tumor outgrowth in the other animals.
Analysis of the immune response revealed an overall
but temporal activation of NK cells in blood, lymph
nodes and tumors. Additionally, mice harbored more
granulocytes after treatment. Depletion of CSF-1R+
macrophages, pDCs or CD8+ T cells did not abolish
anti-tumor effects, whereas depletion of NK cells
partly destroyed tumor control. Surprisingly, CD4+
T cell depletion completely abolished tumor control,
even though the RMA-S tumor is MHC-II negative. In
blood of treated mice, CD4+ T cells specific for the
endogenous env-H19 tumor antigen were detected.
CD4-depleted tumors had decreased levels of CXCL9
and CXCL10, chemokines involved in lymphocyte
trafficking. We hypothesize that antigen-specific
CD4+ T cells are crucial for the recruitment of NK
cells to the tumors to exert its anti-tumor properties against the MHC-I low lymphoma. These results
highlight the importance of both innate and adaptive
immunity for optimal tumor control.
TAP-independent self-peptides enhance T cell recognition of
immune-escaped tumors
Doorduijn E.1, Sluijter M.1, Querido B.1, Cunha Oliveira C.1, Ossendorp F.2, van der Burg S.1, van Hall T.1
LUMC, Clinical Oncology, Leiden, Netherlands,
1
LUMC, Immunohematology and Blood Transfusion, Leiden, Netherlands
2
Tumor cells frequently escape from CD8+ T cell
recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the
peptide transporter TAP, results in strongly decreased
surface display of peptide/MHC-I complexes. We
previously revealed that deficiency of TAP induces
a unique alternative peptide repertoire and some of
these peptides are immunogenic neo-antigens. We
named these peptides TEIPP: T-cell epitopes associated with impaired peptide processing. The prototypic TEIPP antigen is derived from the housekeeping
protein Trh4, ubiquitously expressed, but selectively
emerging in MHC-I on TAP-deficient cells. In the
present study, we analyzed thymus selection and peripheral behavior of TEIPP T cells using a TCR transgenic mouse. TEIPP T cells were efficiently selected
in the thymus, egressed with a naive phenotype,
and could be exploited for immunotherapy against
immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-I low). In contrast, overt
thymus deletion and functionally impaired TEIPP T
cells were observed in mice deficient for TAP1 due
to TEIPP antigen presentation on all body cells in
these mice. Our results strongly support the concept
that TEIPPs derive from ubiquitous, nonmutated selfantigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune
evasion. These data suggest that TEIPP-specific CD8+
T cells are promising candidates in the treatment of
tumors that have escaped from conventional immunotherapies.
Genetic engineering using CRISPR/Cas9: Targeting MMP23 in
melanoma
Halldórsdóttir H.R.1, Pedersen S.R.1, thor Straten P.1
Center for Cancer Immune Therapy, Copenhagen University Hospital, Herlev, Denmark
1
Disseminated melanoma is a serious disease, causing
metalloproteinase, and has four protein domains,
the death of thousands of people each year. Advanced
one of which is a ShK toxin-like domain that has been
stages of the disease are not sensitive to conventional
shown to block the Kv1.3 potassium channel. T cells
therapies, but now there seem to be effective and
express this channel, especially effector memory T
potentially curative treatments available. The novel
cells, which upregulate it when activated. Potassium
treatment of adaptive cell transfer (ACT) has shown
channels are important for the activation and pro-
potential cures in 20% of patients with metastatic
liferation of T cells since the channels maintain the
melanoma and partial responses in about 50%. Im-
calcium homeostasis in the cell. We hypothesize that
pressive as these results are, 80% of treated patients
melanoma cells can suppress effector memory T cells
still succumb to the disease. A key reason for lack of
by upregulating MMP23, which can block Kv1.3 po-
efficacy in some patients is immunosuppression by
tassium channels on the T cells, and thereby inhibit
the tumor microenvironment. To this end, it is well
the anti-tumor response.
known that cancer cells may negatively impact on
Using the CRISPR/Cas9 technique, we have success-
the functionality of T cells, and thereby avoid killing
fully created and validated two guide RNAs (gRNA)
by tumor specific T cells. Cancer cells express recep-
for MMP-23. These gRNAs target exon 5 and exon 7,
tors and ligands that bind to surface molecules on T
and by co-transfecting with both, we have effectively
cells, thereby blocking their activation and activating
knocked out exon 6, which encodes the ShK toxin-
inhibitory signaling, hence blunting the anti-tumor
like domain. We have thereby created a MMP23
response. Targeting of these molecules could there-
knock out tumor cell line, incapable of blocking the
fore be very beneficial in the treatment of advanced
Kv1.3 potassium channel. Functional studies will be
melanoma and, when combined with ACT, could lead
conducted to see if T cells have a better anti-tumor
to a larger fraction of cured patients. A number of
response towards the knock out cell line compared
tumor suppressor mechanisms have already been
to wild type. We will then move into a humanized
identified, such as PD-1/PD-L1 and CTL4, but there
mouse model to study the T cell response in vivo.
is no doubt that more are to be found.
This will hopefully result in novel insight into the
MMP23 is a metalloproteinase expressed by a subset
immunosuppressive mechanisms of MMP23.
of melanomas. To this end, it has been shown that
tumors expressing high levels of MMP23 have fewer
tumor-infiltrating lymphocytes (TIL) and that there
is correlation between high levels of MMP23 and an
overall worse clinical outcome. MMP23 is a unique
Antigen-armed antibodies in the treatment of B-cell lymphoma
Ilecka M.1, Delecluse H.-J.1
DKFZ Heidelberg, Heidelberg, Germany
1
ORAL
TALK
SHORT
2016
Approximately 90% of lymphoid neoplasms world-
Herpesviruses have been recently proven to provoke
wide originate from B cells and their incidence
a cytotoxic response exhibited by CD4+ T cells. Our
constantly keeps on rising. Diffuse large B-cell lym-
in vitro data strongly supports the idea that differ-
phoma is the most prominent among B-cell malig-
ent B- lymphoma cell lines are able to internalize,
nancies. Since the discovery of anti-CD20 antibody
process and present the viral epitopes incorporated
(Rituximab) as an effective treatment, the overall
into AgAbs. These epitopes are then recognized by
survival of lymphoma patients has dramatically
cytotoxic CD4+ T cells that have the capacity to
improved. However, lymphoma treatment remains
kill the tumor cells presenting them as shown in
challenging due to heterogeneity among the lym-
our cytotoxicity assays. It is relatively easy to clone
phoma subtypes and the development of resistance
the antigenic sequence into the antibody gene and
to existing therapies.
express the modified immunoglobulin. Moreover,
A promising strategy to combat B-cell neoplasms is
the antibodies can accommodate longer antigenic
to use ‘armed antibodies’- i.e. antibodies conjugated
fragments, which makes it possible to use the therapy
to other agents such as radionuclides, drugs or cy-
in patients with different MHCII haplotypes. We have
tokines. Our group has designed a novel antibody
also shown that stimulation with AgAbs can lead to
arming strategy, which utilizes microbial antigens
activation and expansion of the memory EBV-specific
in order to increase the immune response against
CD4 T cells ex vivo. All the data shown prove that
B- cell tumors. This approach takes the advantage
the therapy we propose has a great potential in lym-
of an existing persistent viral infection in a cancer
phoma immunotherapy.
patient and T cells that have been primed during the
first encounter with the virus. Such antigen- armed
antibodies (AgAbs) are specific against B cell surface
markers and serve as epitope delivery platform. In
detail, this strategy involves introducing T- cell
epitopes from Epstein-Barr virus (EBV) into the antibody molecule in order to elicit a specific T- cell
response directed against the B- cell lymphoma cell
that will present this epitope after AgAbs treatment.
The strategy can be extrapolated to other common
herpesviruses as for instance human cytomegalovirus or herpes simplex viruses.
A novel highly tumor-specific antibody for acute myeloid
leukemia and myelodysplastic syndrome targeting a sialylated
epitope of CD43
Gillissen M.A.1,2, Kedde M.1, Yasuda E.1, de Jong G.1,2, Levie S.E.1, Bakker A.Q.1, Hensbergen P.J.3,
van Helden P.M.1, Spits H.1, Hazenberg M.D.2
AIMM Therapeutics, Amsterdam, Netherlands,
1
Academic Medical Center, University of Amsterdam, Dept. of Hematology, Amsterdam, Netherlands,
2
Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, Netherlands
3
Acute myeloid leukemia (AML) and myelodysplas-
spectively. Target identification using mass spectro-
tic syndrome (MDS) are high-risk diseases with a
metric analysis and epitope mapping strategies using
poor prognosis. Even intensive treatment regimens
FLAG-tagged truncated variants revealed the CD43
cure less than 50% of patients, and for the major-
protein as the target. CD43 is a plasmamembrane
ity of patients - over 65 years of age and/or patients
protein, expressed by all hematopoietic cells, but
with comorbidities - such intensive regimens are not
AT14-013 targets a tumor-specific sialylated epitope
feasible. Novel approaches such as monoclonal anti-
on CD43 that is uniquely and widely expressed on all
body therapy directed against highly specific tumor
types of AML as illustrated by its reactivity with each
targets are urgently needed.
of 48 randomly selected AML and MDS patient blast
We aimed to identify novel therapeutic antibodies
samples from our clinic. AT14-013 induces antibody-
highly specific for AML and discover novel tumor-
dependent cell-mediated cytotoxicity and comple-
specific antigens, widely expressed on AML and MDS
ment dependent cytotoxicity on AML cell lines and
but not on healthy hematopoietic and non-hematopoi-
primary blasts.
etic cells. Therefore, we employed a peripheral blood
In conclusion, we have found a novel tumor-specific
sample from a patient that developed a potent graft
sialylated CD43 epitope that is widely expressed on
versus AML allo-immune response following an al-
AML and MDS blasts. This antibody has high poten-
logeneic hematopoietic stem cell transplantation and
tial as a therapeutic antibody, either as a naked an-
a isolated CD27+ IgG+ memory B lymphocytes. We
tibody or manufactured into an antibody-drug con-
transduced these cells with Bcl-6 and Bcl-xL, gen-
jugate, bispecific T cell engager or chimeric antigen
erating stable proliferating pre-plasmablast B cell
receptor T cell.
clones that abundantly produce antibodies. These
were screened for binding to surface antigens on
AML cell lines.
Here, we identified an IgG1 antibody, AT14-013, that
specifically interacts with a broad panel of AML cell
lines and leukemic blasts isolated from newly diagnosed AML and MDS patients at our clinic. AT14-013
does not interact with healthy hematopoietic nor nonhematopoietic cells. The antibody is of donor origin
and antigen experienced as it contains 26/11 somatic
hypermutations in the heavy and light chains, re-
A novel TLR7 agonist reverses NK cell anergy and cures
lymphoma-bearing mice
Krächan A.1, Wiedemann G.1, Jacobi S.1, Chaloupka M.1, Endres S.1, Kobold S.1
Department of Internal Medicine IV, Klinikum der Universität München, Munich, Center of Integrated Protein
1
Science Munich (CIPS-M) and Division of Clinical Pharmacology, Munich, Germany
Toll-like receptor 7 (TLR7) agonists are potent
immune stimulants able to overcome cancer-associated immune suppression. Due to dose-limiting
systemic toxicities, only the topically applied TLR7
agonist (imiquimod) has been approved for therapy
of skin tumors. There is a need for TLR7 activating
compounds with equivalent efficacy but less toxicity.
SC1, a novel small molecule agonist for TLR7, is a
potent type 1 interferon inducer, comparable to the
reference TLR7 agonist resiquimod yet with lower
induction of proinflammatory cytokines. In mice,
in vivo, SC1 activates NK cells in a TLR7-dependent
manner. Mice bearing the NK cell-sensitive lymphoma RMA-S are cured by repeated s.c. administrations
of SC1 as efficiently as by administration of resiquimod. Mechanistically, SC1 reverses NK-cell anergy
and restores NK cell-mediated tumor cell killing in
an IFN-α dependent manner. TLR7 targeting by SC1based compounds may form an attractive strategy to
activate NK cell responses for cancer therapy.
Effector mechanisms of IgA antibodies against CD20 include
recruitment of myeloid cells for ADCC and the alternative
complement pathway
Kretschmer A.1, Lohse S.2, Jansen J.H.M.3, Shibuya A.4, Cragg M.S.5, Leusen J.3, Valerius T.1
Division of Stem Cell Transplantation and Immunotherapy, 2nd Medical Department, Christian-Albrechts-
1
University, Kiel, Germany,
Institute of Virology, Saarland University Medical Center and Saarland University Faculty of Medicine, Homburg,
2
Germany,
Laboratory for Immunotherapy, Department of Immunology, UMC Utrecht, Utrecht, Netherlands,
3
Department of Immunology, Faculty of Medicine, Center for Tsukuba Advanced Research Alliance (TARA)
4
University of Tsukuba, Tsukuba Science-City, Japan,
Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, Southampton University Hospitals,
5
Southampton, United Kingdom
Introduction: Monoclonal antibodies against CD20
acteristics as well as Fab and Fc- mediated effector
have significantly improved the survival of many
functions. Additionally, in vivo depletion of human
lymphoma patients. Based on different mode of
CD20 transgenic B cells was evaluated in a syngeneic
action CD20 antibodies have been classified into type
B cell depletion model.
I or type II antibodies. Type I mAbs trigger effective
Results and discussion: Both variants mediated
complement-dependent cytotoxicity (CDC), whereas
similarly inefficient Fab-mediated effects on direct
type II antibodies are potent inducers of direct cell
cell death induction and homotypic aggregation com-
death. Both types induce similar levels of antibody-
pared to the type II antibody tositumomab. However,
dependent cellular cytotoxicity (ADCC). Currently
human IgA2 but not IgG1 antibodies against CD20
approved CD20 antibodies are of the human IgG1
effectively triggered ADCC by human PMNs, the
isotype that are known to effectively recruit natural
most numerous myeloid effector cell population. Un-
killer cells for ADCC. Antibodies of IgA isotype are
expectedly, CD20 IgA antibodies also triggered CDC
the most abundantly produced antibody isotype in
of tumor cells. CDC was predominantly mediated by
humans and constitute an integral part of the mucosal
the alternative pathway, as evidenced by the kinetics
immune system. They differ from IgG antibodies in
of lysis, the requirement for higher serum concentra-
their pharmacokinetic properties and immune effec-
tions and inhibition by compstatin. Results from in
tor mechanisms. By recruiting polymorphonuclear
vivo experiments demonstrated effective depletion of
cells (PMNs) as the largest effector cell population for
human CD20 transgenic B cells in a syngeneic B cell
ADCC IgA antibodies expose their immunotherapeu-
depletion model. However, in vivo B cell depletion
tic potential. Here, we compared the IgG1 and IgA2
was not mediated by complement or FcαRI - suggest-
isotype variants of the type I CD20 antibody 1F5.
ing that other, currently undefined mechanisms con-
Methods: Recombinant IgA antibodies against the
tribute to their in vivo efficacy. Together, the present-
CD20 antigen were produced by co-transfecting BHK
ed results suggest that CD20 antibodies of human
cells with vectors encoding the 1F5 variable and Igα
IgA isotype constitute promising immunotherapeutic
heavy and κ light chain constant regions, respec-
reagents with unique effector functions.
tively. The resulting antibody variant was compared
to its IgG1 counterpart regarding biochemical char-
IMAB027-DM1 and IMAB027-vcMMAE, CLDN6-specific
antibody-drug conjugates, are effective against human
CLDN6-positive cancer cells in vitro and in vivo
Kreuzberg M.1, Walter K.1, Mitnacht-Kraus R.1, Le Gall F.1, Rohde C.1, Jacobs S.1, Schmitt R.1, Damke C.1,
Talamini M.1, Sahin U.2,3, Türeci Ö.1
1
2
TRON - Translational Oncology at the University Medical Center of the Johannes Gutenberg University
gGmbH, Mainz, Germany, 3BioNTech AG, Mainz, Germany
ORAL
TALK
SHORT
2016
Antibody-drug conjugates (ADCs) are a promising
optosis of CLDN6-positive tumor cells (40-90% apop-
new treatment modality for cancer patients. Recent-
totic cells after 48-72 h) at concentrations of 0.1-12.5
ly two ADCs have been approved for the treatment
µg/mL. Additionally, IMAB027-vcMMAE exerted by-
of cancer, i.e. brentuximab vedotin (monomethyl
stander killing of CLDN6-negative cells in the pres-
auristatin E [MMAE] via cleavable valine-citrulline
ence of CLDN6-positive cells.
[vc] linker) and trastuzumab emtansine (T-DM1).
In vivo, intravenous (i.v.) administration of IM-
IMAB027, a monoclonal antibody (mAB) against
AB027-DM1 or IMAB027-vcMMAE in nude mice
CLDN6, which is only expressed in tumor cells and
with CLDN6-positive xenograft tumors resulted in
not in any adult human healthy tissue, has promising
dose-dependent tumor growth inhibition, a survival
preclinical activity in human CLDN6-positive cancer
benefit, and complete regression of advanced tumors.
models and is currently under clinical evaluation in
A single i.v. application of 16 mg/kg IMAB027-DM1
ovarian cancer. Conjugation of IMAB027 with cyto-
or 16 mg/kg IMAB027-vcMMAE had significant ther-
toxic compounds such as MMAE or DM1 combines
apeutic effects even in highly aggressive advanced
precision targeting with highly effective cytotoxic
xenograft tumors. In addition, human cancer xeno-
drugs limiting their side effects. Here we describe
grafts with low and/or heterogeneous expression of
the preclinical proof-of-principle of IMAB027-DM1
CLDN6 were efficiently treated with IMAB027-vcM-
and IMAB027-vcMMAE for the treatment of CLDN6-
MAE, most likely due to its bystander activity. 16
positive human cancers.
mg/kg (equivalent to 48 mg/m2 in human) was the
CLDN6-positive human ovarian cancer cells inter-
highest tested dose and was well tolerated.
nalize IMAB027, which is a prerequisite for efficient
Both IMAB027-ADCs demonstrated excellent anti-tu-
ADC-mediated cell killing. IMAB027-DM1 and IM-
mor activity against CLDN6-positive human cancer
AB027-vcMMAE both robustly and specifically bind
cells in vitro and in vivo. IMAB027-vcMMAE had ad-
to target-expressing cells. IMAB027-ADCs retained
ditional bystander activity, killing CLDN6-negative
the ability to mediate immune-related mechanisms:
cells in tumors with heterogeneous antigen expres-
antibody-dependent cellular cytotoxicity and com-
sion. In conclusion, our preclinical data support the
plement-dependent cytotoxicity after conjugation.
clinical development of IMAB027-ADCs as the first
Both IMAB027-ADCs potently and efficiently killed
CLDN6-targeting ADCs for the treatment of CLDN6-
CLDN6-expressing human ovarian and testicular
positive cancers.
cancer cells with EC50 of 10-70 ng/mL and maximum
lyses of 60-100% depending on the target cell line.
IMAB027-DM1 and IMAB027-vcMMAE induced ap-
Tumor expression of immune inhibitory molecules and TIL
counts predict pancreatic cancer survival
Sideras K.1, Biermann K.2, Yap K.2, Mancham S.1, Stoop H.2, Peppelenbosch M.1, van Eijck C.3, Sleijfer S.4,
Kwekkeboom J.5, Bruno M.1
Erasmus MC, Gastroenterology and Hepatology, Rotterdam, Netherlands,
1
Erasmus MC, Pathology, Rotterdam, Netherlands,
2
Erasmus MC, Surgery, Rotterdam, Netherlands,
3
Erasmus MC, Cancer Institute, Rotterdam, Netherlands,
4
Erasmus MC-University Medical Centre, Gastroenterology and Hepatology, Rotterdam, Netherlands
5
Background: Novel systemic treatments for exocrine
creatic cancer survival. All immune inhibitory mol-
pancreatic cancer are strongly needed. Immuno-
ecules, with the exception of IDO, were individually
therapies targeting the immune checkpoints CTLA-4
predictive of pancreatic cancer survival, independ-
and PD-1 have been successful in several types of
ent of known clinicopathologic characteristics. In
cancer, but not in pancreatic cancer. Understanding
addition, margin status, lymph node status, tumor
the mechanisms of immune resistance by pancreas
grade and baseline CA-19.9, were independent pre-
cancer is crucial for development of suitable immu-
dictors of pancreatic cancer survival, while T-score,
notherapeutics. Here we examined the expression of
use of chemotherapy or tumor location (ampulla vs
the immune inhibiting molecules PD-L1, Galectin-9,
pancreas) was not. Expression of 4 or 5 of the above
HVEM, IDO and HLA-G, as well as numbers CD8 and
immune biomarkers predicted a group of patients
Fox-P3 tumor infiltrating lymphocytes (TIL) in exo-
with 70% 3-year survival, while their expression in
crine pancreatic cancer and their association with
combination with clinicopathologic characteristics
clinicopathologic characteristics and outcome.
predicted a small group of patients with over 90%
Methods: Tumor tissues from 226 patients who un-
3-year survival.
derwent exocrine pancreatic cancer resection, were
Conclusion: High tumor expression of immune in-
used to construct tissue microarrays. Immunohisto-
hibitory molecules as well as high CD8/FoxP3 TIL
chemistry using validated antibodies was performed.
ratio, are associated with improved survival in
Expression of immune inhibiting molecules on tumor
exocrine pancreatic cancer patients. Expression of
cells was scored by two independent investigators.
multiple immune inhibitory molecules and TIL-in-
Optimal high vs low values were established by ex-
filtration can have powerful prognostic implications,
amining a grid of cutoffs and choosing the cutoff
independent of known clinicopathologic factors. In
with the lowest -2 log likelihood. Clinicopathologic
addition, immune biomarkers can add significantly
characteristics and use of adjuvant or port-recur-
to the prognostication of pancreatic cancer. Finally,
rence chemotherapy were collected.
high prevalence of PDL-1, Gal-9 and HVEM expres-
Results: PD-L1, Gal-9 and HVEM were expressed in
sion by cancer cells suggest that these molecules may
the tumors of the majority of patients, while tumor
have potential as immunotherapeutic targets in exo-
expression of IDO and HLA-G was confined to mi-
crine pancreas cancer.
norities of patients. High tumor expression of PD-L1
(p=.001), Gal-9 (p=.002), HVEM (p< .001), IDO
(p=.040), HLA-G (p=.003) and high CD8/FoxP3-TIL
ratio (p=.011) were associated with improved pan-
The second and third author equally contributed to the
project
Targeting of reactive oxygen species can be a potential
therapeutic strategy for cancer treatment
Lee Y.-R.1, Chen S.-Y.2, Chen H.-R.3
Ditmanson Medical Foundation Chiayi Christian Hospital, Department of Medical Research, Chiayi City,
1
Taiwan, Republic of China,
Ditmanson Medical Foundation Chiayi Christian Hospital, Department of Chinese Medicine, Chiayi City,
2
Taiwan, Republic of China,
National Chung Cheng University, Department of Life Science, Chiayi, Taiwan, Republic of China
3
Chronic inflammation is induced by chemical, bio-
further DNA alterations to the initiated cell popula-
logical, and physical factors and is in turn associ-
tion. It is well known that elevated ROS in the cells
ated with an increased risk of several human cancers
can cause DNA damage, and thus contribute to tumor
formation. Chronic inflammation has been linked to
development through the regulation of cellular pro-
various steps involved in carcinogenesis, including
liferation, angiogenesis, and metastasis. Moreover,
cellular transformation, promotion, survival, prolif-
ROS is observed in various types of cancer cells, and
eration, invasion, angiogenesis, and metastasis. The
this tends to make these cells and tumors more resist-
link of inflammation and cancer has been demon-
ant to chemotherapy. However, enhancing of ROS in
strated by anti-inflammatory therapies that show ef-
the cancer cells can be used as a novel therapeutic
ficacy in cancer prevention and treatment. During in-
strategy in multiple tumors recently.
flammation, mast cells and leukocytes are recruited
In the present study, we demonstrate that a novel
to the site of inflammation, which leads to increase
ROS inducer can suppress the growth of human oral
uptake of oxygen, and thus, an increased release and
squamous cell carcinoma (OSCC). Moreover, cell
accumulation of active oxygen species (ROS) at the
cycle arrest, apoptosis, and senescence were also ob-
site of damage. Therefore, inflammatory cells may
served in OSCC cells after treatment. However, sup-
increase DNA damage by activating pro-carcinogens
pressing of ROS by N-acetyl-L-cysteine can restore
to DNA-damaging species by ROS-dependent mecha-
cell number and reduce the physiological phenomena
nisms.
including cell cycle arrest, apoptosis and senescence.
ROS is defined as an imbalance between production
Therefore, we have shown a novel agent which tar-
of free radicals and reactive metabolites. Oxidative
geting on ROS induction may use as a therapeutic
stress interacts with three stages of this carcinogen-
agent for human OSCCs. Whether these agents can
esis. During the initiation stage, ROS may produce
inhibit tumor growth in patients remains to be elu-
DNA damage by introducing gene mutations and
cidated.
structural alterations of the DNA. In the promotion
stage, ROS can contribute to abnormal gene expression, blockage of cell- to cell communication, and
modification of second messenger systems, thus resulting in an increase of cell proliferation or a decrease in apoptosis of the initiated cell population.
Finally, oxidative stress may also participate in the
progression stage of the cancer process by adding
Human IL-2 agonist exhibits a higher antitumor efficacy and
lower toxicity than wild type IL-2 in different preclinical
contexts
Carmenate T.1, Montalbo M.1, Casadeus A.V.1, Fuentes D.2, Rojas G.1, Leon K.1
Center of Molecular Immunology, Habana, Cuba,
1
CENPALAB, Habana, Cuba
2
IL-2 has been used for the treatment of melanoma
in mice. Initial data also support the potency of this
and renal cell carcinoma, but this therapy has limited
IL2 mutant, when used in combination with other
efficacy and severe toxicity. It has been shown that
immunotherapies of interest.
part of this limited efficacy is due to the IL-2-driven
preferential expansion of regulatory T cells, which
dampen the antitumor immunity. In this study, we
develop and characterize a human IL-2 mutant which
differs from wtIL-2 by four mutations at the interface
with the a-subunit of IL-2R. In vitro, this IL-2 mutant
induces proliferation of CD8+CD44hi and NK1.1 cells
as efficiently as does wtIL-2, but it shows a reduced
capacity to induce proliferation of CD4+Foxp3+ regulatory T cells. In vivo, the IL-2 mutant induces preferential proliferation of CD8+CD44hi, with a quite
reduced proliferation of CD4+Foxp3+ regulatory T
cells. The IL-2 mutant shows higher antimetastatic/
antitumoral effect than wtIL-2 in several transplantable tumor models: the experimental metastasis
model of MB16F0; the experimental and spontaneous
metastasis models for the mouse pulmonary carcinoma 3LL-D1222; the metastasis mammary carcinoma 4T1 and the experimental lymphoma EG7. Relevantly, the IL-2 mutant also exhibits lower lung and
liver toxicity than wtIL-2 when used at high doses
Blocking IL-2 signal in vivo with IL-2 antagonist reduces tumour
growth through the control of regulatory T cell accumulation
Carmenate T.1, Ortiz Y.1, Garcia K.1, Moreno E.1, Graca L.2, Leon K.1
Center of Molecular Immunology, Habana, Cuba,
1
Institutito de Medicina Molecular, Lisboa, Portugal
2
Interleukin 2 has a primary role in the maintenance
derstand the effect of these mutants and to suggest
of the natural and/or induced tolerance, which is me-
suitable strategies to improve their design as poten-
diated by regulatory T cells. Therefore, it has been
tial drugs. Overall our results shows that it is enough
long hypothesized that therapies blocking IL2 signal
to inhibit transiently IL2 signaling to bias helper and
will weaken Tregs activity and promote an immune
regulatory T cells balance in vivo towards immunity.
response, which might be useful in the therapy of
The obtained IL-2 antagonist or an improved version
cancer. The latter idea has been tested in mice using
with longer lifespans and increased affinity for CD25
either anti-IL-2 or anti-IL-2R mAbs, and showing an
could be useful for cancer therapy based on Regula-
antitumoral effect, which cannot be exclusively at-
tory T cell inhibition.
tributed to the capacity of these agents to block IL2
signal in vivo. In this work we pursue an alternative strategy to evaluate this hypothesis. We design
several IL2 mutants which conserve the capacity to
bind to α and β chains of the IL-2 receptor but not to
the γc chain having therefore drastically affected its
signaling capacity. In vitro they behave as very weak
IL2 agonist, when used to stimulate human or mice
IL2 dependent cell lines (KIT225 and CTLL2); but
they consistently inhibit/antagonize IL2-dependent
T cells proliferation and Treg differentiation in vitro.
Moreover in vivo treatment of tumor bearing mice
with these mutants led to a slower tumor growth and
to less accumulation of Treg in the draining lymphnodes. A mathematical model is used to better un-
PF-06840003: a highly selective IDO1 inhibitor that shows good
in vivo efficacy in combination with immune checkpoint inhibitors, and favorable predicted human pharmacokinetic properties
Tumang J.1, Gomes B.2, Wythes M.1, Crosignani S.2, Bingham P.1, Bottemanne P.2, Cannelle H.2, Cauwenberghs S.2,
Chaplin J.1, Dalvie D.1, Denies S.2, De Maeseneire C.2, Folger P.1, Frederix K.2, Guo J.1, Hardwick J.1, Hook K.1,
Jessen K.1, Kindt E.1, Letellier M.-C.2, Liao K.-H.1, Li W.1, Maegley K.1, Marillier R.2, Miller N.1, Murray B.1, Pirson R.2,
Preillon J.2, Rabolli V.2, Ray C.1, Scales S.1, Srirangam J.1, Solowiej J.1, Streiner N.1, Torti V.1, Tsaparikos K.1, Vicini P.1,
Driessens G.2, Kraus M.1
Pfizer, Oncology, San Diego, United States,
1
iTeos Therapeutics, Gosselies, Belgium
2
Tumors use tryptophan-catabolizing enzymes such
as Indoleamine 2,3-dioxygenase-1 (IDO1) to induce
an immunosuppressive microenvironment. IDO1
expression is upregulated in many cancers and described to be a resistance mechanism to immune
checkpoint therapies. IDO1 is induced in response
to inflammatory stimuli such as IFN-g and promotes
immune tolerance through the catabolism of tryptophan and accumulation of tryptophan catabolites
including kynurenine. IDO1 activity leads to effector
T-cell anergy and enhanced Treg function through
upregulation of FoxP3. As such, IDO1 is a nexus for
the induction of key immunosuppressive mechanisms and represents an important immunotherapeutic target in oncology. We have identified and
characterized a new IDO1 inhibitor. PF-06840003 is
a highly selective orally bioavailable IDO1 inhibitor.
PF-06840003 reversed IDO1-induced T-cell anergy
in vitro. In vivo, PF-06840003 reduced intratumoral kynurenine levels in mice by >80% and inhibited tumor growth in multiple preclinical syngeneic
models in mice, in combination with immune checkpoint inhibitors. PF-06840003 has favorable predicted human pharmacokinetic properties, including a
predicted t1/2 of 16-19 hours. These studies highlight
the strong potential of PF-06840003 as a clinical candidate in Immuno-Oncology.
Protecting immune cells from activation-induced apoptosis
via the CD95L-blocking compound APG101
Marschall V.1, Sykora J.1, Merz C.1, Richards D.1, Schnyder T.1, Fricke H.1, Hill O.1, Thiemann M.1, Gieffers C.1
Apogenix AG, Heidelberg, Germany
1
Background: Targeting so-called immune check-
monocyte-depleted PBMCs together with tumor cell
points has become a promising treatment strategy
lines to monitor tumor cell killing by immune cells.
in oncology. In addition to established checkpoint
Results: Differentiated M1- and M2-type macrophages
ligands, which are frequently upregulated by tumor
express a specific pattern of antigens including CD95.
cells to escape immune surveillance, further signal-
Exposure of macrophages to soluble recombinant
ing pathways, e.g. CD95 signaling, regulate immune
CD95L induces apoptosis in both populations. Higher
cell activity. CD95 (APO-1/Fas) is a member of the
sensitivity of M1- compared to M2-macrophages is
Tumor Necrosis Factor Receptor Super Family and
observed, coinciding with higher expression of CD95
its corresponding CD95-ligand (CD95L) is frequently
on M1-macrophages. Addition of APG101 protects
overexpressed in cancers. Binding of CD95L to CD95
macrophages from CD95L-induced cell death in a
triggers activation-induced apoptosis in immune
dose-dependent manner. Similar results are obtained
cells, making this a putative immune checkpoint.
after treatment of T cells with CD95L and APG101 in
Apogenix has developed APG101, a fully human
apoptosis-assays. In direct co-culture, these treated
fusion protein consisting of the extracellular domain
T cells as well as the monocyte-depleted PBMCs are
of CD95 and an IgG1 Fc-domain, which has been con-
able to induce cell death in several tumor cell lines.
firmed as a potent inhibitor of CD95/CD95L signal-
Conclusion: APG101 is a potent inhibitor of CD95/
ing. Here we examined the mode-of-action of APG101
CD95L signaling in both myeloid and lymphoid cells
in the protection of immune cells from activation-
as it protects these immune cells from undergoing
induced cell death and the subsequent effects on
CD95L-induced apoptosis. Protection of immune
tumor cell killing.
cells from CD95L activation-induced apoptosis po-
Methods: Monocytes isolated from healthy-donor
tentially prolongs the immune response in the tumor
blood samples were differentiated in vitro into either
tissue and favors immune surveillance. The inhibi-
M1- or M2-type macrophages, which was confirmed
tion of tumor-induced CD95L-mediated apoptosis of
by multicolor-flow cytometry (FC). Subsequently,
immune cells using APG101 represents an attractive
we analyzed the respective M1- and M2-type mac-
novel concept for immunotherapeutic treatment of
rophages for apoptosis induction following exposure
tumors.
to recombinant CD95L. Analogous experiments were
performed using purified T cells. Analytical FC was
used to monitor apoptosis by measuring appearance
of cleaved PARP. Real-time cell analysis was performed on direct co-cultures of activated T cells or
Discovering novel targets in a high-throughput fashion: RNAi
screen for pancreatic ductal adenocarcinoma (PDAC) associated
immune modulators
Sorrentino A.1,2, Menevse A.N.2, Michels T.2, Khandelwal N.1, Breinig M.3, Poschke I.4, Volpin V.1, Wagner S.5,
Offringa R.4, Boutros M.3, Beckhove P.1,2
German Cancer Research Center (DKFZ), Translational Immunology, Heidelberg, Germany,
1
Regensburg Center for Interventional Immunology (RCI), Lehrstuhl für Interventionelle Immunologie,
2
Regensburg, Germany,
German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics, Heidelberg, Germany,
3
German Cancer Research Center (DKFZ), Division of Molecular Oncology of Gastrointestinal Tumors, Heidelberg,
4
Germany,
German Cancer Research Center (DKFZ), Joint Immunotherapeutics Laboratory, Heidelberg, Germany
5
Background: As one of the most challenging cancer
surface proteins including GPCRs. Transfected
types to treat, pancreatic ductal adenocarcinoma
tumor cells were co-cultured with patient-derived
(PDAC) is the fourth leading cause of cancer-associ-
HLA-A201+ matched tumor infiltrating lympho-
ated deaths worldwide. Infiltration of tumor-specific
cytes (TILs). The impact of gene knock-down on T
cytotoxic T lymphocytes into PDAC tissue correlates
cell mediated tumor lysis was measured from the
with better prognosis, however PDAC cells are able
remaining luciferase intensity. To rule out whether
to escape immunosurveillance by expressing inhibi-
the gene knock-down has an impact on cell viability
tory immune modulators. Induction of anti-tumor
per se, transfected PANC-1 cells were cultured in the
immunity by immune checkpoint blockade hold
absence of TILs.
promise for many tumor entities, yet so far target-
Results: Based on our primary screen, we identified
ing well characterized molecules such as PD-1, PD-L1
155 candidate genes (hits) whose down-regulation in-
and CTLA-4 did not show clinical benefit in PDAC
creased T cell-mediated tumor lysis more efficiently
patients. Therefore, identification of novel immune
than PD-L1 knockdown. The 155 hits generated by the
modulators is a fundamental need in the field of im-
primary screen were subjected to a secondary screen,
munotherapy.
where 70% of them showed the same phenotype.
Aim: Our main aim is to identify PDAC-associated
Among these candidate genes we selected 4 hits for
immune modulators by conducting a high-through-
further validation. So far, we confirmed the expres-
put RNAi screen and successively validate the roles
sion of the hits at mRNA level and validated siRNAs
of candidate molecules, whose blockade could poten-
on-target effect by qPCR and luciferase-based cyto-
tially induce anti-tumor immunity in PDAC patients.
toxicity assay. We verified increased T cell mediated
Methods: We performed a high-throughput RNAi-
killing of PANC-1 cells upon down-regulation of 4 hits
based screen where stably luciferase expressing
by 51chromium release assay. Additionally we assessed
PANC-1 cells were transfected with a siRNA library
that the enhanced T-cell mediated killing was caused
targeting 2514 genes encoding for kinases and cell
by increased susceptibility to apoptosis of tumor cells
upon hits downregulation. We also confirmed that the
predisposition of tumor cells to apoptosis is mainly
mediated by TNF-α.
Conclusion and outlook: We established a new approach to discover novel immune modulators that
are potential immunotherapeutic targets for treatment of PDAC. We validated the inhibitory role of 4
hits in T-cell mediated killing in vitro and our hits
were found to be involved in TNF-α mediated tumor
cell apoptosis. Further in vivo validation of these
candidate immune checkpoint molecules is still required for clinical studies.
TiMi1 is a novel immune checkpoint in solid tumors
differentially regulating cAMP-depending signaling in
tumor-infiltrating lymphocytes
Michels T.1,2, Hartl C.1, Khandelwal N.1, Breinig M.3, Sorrentino A.1, Volpin V.1, Mäder C.1, Umansky L.1, Poschke I.4,
Offringa R.4, Boutros M.3, Eisenberg G.5, Lotem M.5, Beckhove P.1,2
German Cancer Research Center (DKFZ), Translational Immunology, Heidelberg, Germany,
1
Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany,
2
German Cancer Research Center (DKFZ), Signaling and Functional Genomics, Heidelberg, Germany,
3
German Cancer Research Center (DKFZ), Molecular Oncology of Gastrointestinal Tumors, Heidelberg, Germany,
4
Hadassah Hebrew University Hospital, Sharett Institute of Oncology, Jerusalem, Israel
5
Despite major advances in cancer therapy, tumors
well. Furthermore, we validated the in vivo role of
employ a plethora of suppressive mechanisms to
TiMi1 using a xenograft mouse model in combina-
evade immune destruction not matched by today´s
tion with adoptive cell transfer. TiMi1-mediated in-
immunotherapy treatments. In this study, we estab-
hibition functions by altering the cAMP-dependent
lished and utilized a high throughput RNAi screen-
signaling inside T cells. This effect is mediated by
ing to identify potential immune checkpoints using
PKA, Csk and subsequent inhibition of Lck. Prelimi-
patient-derived
lymphocytes
nary experiments suggest that TiMi1 regulates the
(TILs) in conjunction with primary HLA-matched
gap junction transport of cAMP from the tumor to
melanoma cells. We screened a siRNA library target-
the T cell.
ing more than 2800 surface receptors and kinases to
In summary, we established a novel antigen-specific
explore novel targets for immunotherapy.
screening approach for immune checkpoints and
Briefly, M579-A2-luc melanoma cells were reverse-
identified TiMi1 as a promising candidate. TiMi1 in-
ly transfected with the siRNA library and then co-
hibits T cell responses in melanoma by differential
cultured with MART1- and gp100-specific TILs to
regulation of cAMP-dependent signaling inside TILs.
tumor
infiltrating
determine TIL-mediated tumor lysis. The screening
resulted in a hit list of 73 candidates that negatively
regulated CTL cytotoxicity of which 14 were found
in a related pancreatic adenocarcinoma screening as
well. To streamline the discovery process for large
scale molecule libraries, we established a secondary
screen assaying multiple T cell activation markers,
including effector cytokines.
One of the strongest candidates from our primary
screening (and the PDAC screening) is TiMi1 (name
altered), a surface receptor belonging to the class
of olfactory GPCR signaling via cAMP. We found
that knock-down of TiMi1 increased TIL-mediated
killing, increased TIL activity measured by production of type 1-associated cytokines and reduced TC
apoptosis. TiMi1 abrogation increased killing of pancreatic (PDAC) and colorectal cancer (CRC) cells as
IMAB362-vcMMAE , a CLDN18.2-specific antibody-drug
conjugate, is effective against human gastric cancer cells in
vitro and in vivo
Mitnacht-Kraus R.1, Kreuzberg M.1, Le Gall F.1, Walter K.1, Jacobs S.1, Damke C.1, Rohde C.1, Sahin U.2,3, Türeci Ö.1
1
TRON - Translational Oncology at the University Medical Center of the Johannes Gutenberg University gGmbH,
2
Mainz, Germany,
BioNTech AG, Mainz, Germany
3
IMAB362 is a monoclonal antibody against CLDN18.2
Therapeutic targeting of CLDN18.2-positive cancer
with clinical activity in gastric cancer patients.
cells with IMAB362-vcMMAE prevented tumor
CLDN18.2 is virtually absent from adult healthy
growth in early and significantly inhibited tumor
tissues except for the stomach. Recently antibody-
growth or even resulted in complete tumor regres-
drug conjugates (ADC), i.e. trastuzumab-emtansine
sion in advanced tumor mouse models. This effect
or brentuximab vedotin have gained market approval
translated into a significant survival benefit for the
and are used in the clinic. Combining IMAB362 with
IMAB362-vcMMAE-treated mice. Treatment with
a cytotoxic drug such as monomethyl auristatin E
IMAB362-vcMMAE appeared safe and had no toxic
(MMAE) might enhance its clinical activity and add
effects.
new mechanisms of action. Therefore, we investigated
In summary, IMAB362-vcMMAE is an ADC efficiently
the in vitro and in vivo activity of IMAB362 conjugated
killing target-positive human tumor cells in vitro via
to MMAE via a cathepsin-cleavable valine-citruline
direct cytotoxicity and has bystander activity against
(vc) linker (IMAB362-vcMMAE) in gastric cancer
target-negative cells. In vivo, the ADC was well toler-
models.
ated and demonstrated impressive anti-tumor activ-
IMAB362-vcMMAE specifically killed CLDN18.2-ex-
ity in human gastric cancer mouse models. In conclu-
pressing human gastric cancer cells in vitro (EC50 of
sion, IMAB362-vcMMAE a CLDN18.2-targeting ADC
viability reduction < 200 ng/mL of cells with high
may provide a promising new therapeutic option for
CLDN18.2 expression). Tumor-cell killing positively
patients with gastric cancer and clinical evaluation
correlated with the epitope density on the tumor cell
may be warranted.
(spearman r = -0.7167; P = 0.0369). A mechanism
of action of the cytotoxic drug was the induction of
apoptosis as shown by increased caspase 3/7 activity
and annexin V staining in IMAB362-vcMMAE-treated
tumor cells. The cleavable linker allows the release
of the cytotoxic drug from the dying cells to kill surrounding tumor cells independent of CLDN18.2 expression, a process called bystander activity. The ADC
dose-dependently killed CLDN18.2-negative cells only
in the presence of CLDN18.2-positive cells (reduced
viability < 90% depending on concentration and ratio
of CLDN18.2-positive vs -negative cells).
Novel and shared neoantigen for glioma T cell therapy derived
from histone 3 variant H3.3 K27M mutation
Okada H.1, Kohanbash G.1, Okada K.1, Shrivastav S.1, Lin Y.1, Liu S.1, Pollack I.2, Carcaboso A.3, Hou Y.1
University of California San Francisco, San Francisco, United States,
1
University of Pittsburgh, Pittsburgh, United States,
2
Fundació Sant Joan de Déu, Barcelona, Spain
3
ORAL
TALK
SHORT
2016
Malignant gliomas, such as glioblastoma (GBM) and
cells. Furthermore, CTL clones with high and specific
diffuse intrinsic pontine gliomas (DIPG), are lethal
affinities to HLA-A2-H3.3.K27M-tetramer were suc-
brain tumors in both adults and children. Indeed,
cessfully obtained, and α- and β-chain cDNAs from a
brain tumors are the leading cause of cancer-related
high-affinity T cell receptor (TCR) were cloned into a
mortality and morbidity in children. Children with
lentiviral vector. Human T-cells transduced with the
DIPG have median overall survival of 9 to 10 months
TCR demonstrated an antigen-specific but CD8-inde-
with current treatment. Recent genetic studies have
pendent activation, which has been often observed
revealed that malignant gliomas in children and
with high-avidity TCRs. Furthermore, alanine scan-
young adults often show shared missense mutations,
ning revealed the key amino-acid sequence motif in
which encodes the replication-independent histone
the epitope which was responsible for the TCR reac-
3 variant H3.3. Approximately 30 % of overall GBM
tivity. Available human protein data bases indicate
and over 70% of DIPG cases harbor the amino-acid
that the motif is not shared by any known human
substitution from lysine (K) to methionine (M) at
protein.
the position 27 of H3.3. We evaluated whether H3.3-
These data provide us with a strong basis for develop-
derived peptides that encompass the H3.3 K27M mu-
ing peptide-based vaccines as well as adoptive trans-
tation can induce specific cytotoxic T lymphocyte
fer therapy using autologous T-cells transduced with
(CTL) responses in human leukocyte antigen (HLA)-
the TCR.
A2+ CD8+ T-cells.
Four candidate peptides encompassing different ami-
Funding support: V Foundation (Okada), NIH/
no-acid positions around the H3.3 K27M mutation
NINDS R21 NS083171 (Okada)
were synthesized, and peptide-specific CTL lines and
clones were generated from peripheral blood mononuclear cells of HLA-A2+ donors by in vitro stimulation with each of the synthetic peptides. One of the 4
peptides (the H3.3.K27M epitope, hereafter) induced
CTL lines which recognized not only the synthetic
peptide loaded on T2 cells but also lysed HLA-A2+
DIPG cell lines which endogenously harbor the
H3.3.K27M mutation. On the other hand, CTL lines
did not react to HLA-A2+, H3.3 K27M mutation-negative cells or HLA-A2-negative, H3.3 K27M mutation+
Costimulatory T-cell engagement by the CD137/HER2 bispecific,
PRS-343, leads to anti-tumor effect and increased tumor infiltrating
lymphocytes in a humanized mouse model
Hinner M.J.1, Bel Aiba R.-S.1, Schlosser C.1, Wiedenmann A.1, Allersdorfer A.1, Jaquin T.J.1, Matschiner G.1, Berger S.1,
Moebius U.1, Rothe C.1, Olwill S.A.1
Pieris Pharmaceuticals Inc., Freising, Germany
1
Background: CD137 (4-1BB) is a key costimulatory
HER2-positive xenograft. When tumors had reached
immunoreceptor and a member of the TNF-receptor
a predefined size, mice received human PBMCs via
(TNFR) superfamily. While multiple lines of evi-
an intravenous route and weekly intraperitoneal in-
dence show that CD137 is a highly promising target,
jections of PRS-343 or controls for three weeks. We
current mAb approaches are not designed to achieve
found that PRS-343 led to strong tumor growth inhi-
a tumor-mediated activation and, therefore, may
bition and a significantly better response compared
display toxicity and a limited therapeutic window
to either isotype control or an anti-CD137 benchmark
due to peripheral T cell and NK cell activation. To
mAb. The tumor response was accompanied by a
overcome this limitation, we generated PRS-343, a
higher tumor infiltration with human lymphocytes
CD137/HER2 bispecific that is designed to promote
(hCD45+) compared to the isotype or anti-CD137
CD137 clustering by bridging CD137-positive T cells
benchmark controls. While the anti-CD137 bench-
with HER2-positive tumor cells, thereby providing a
mark accelerated the onset of graft-versus-host-
potent costimulatory signal to tumor antigen-specific
disease and led to expansion of CD8+ T cells in the
T cells.
peripheral blood, this effect was, as desired, absent
Methods: Anticalins® are 18 kD protein therapeutics
for PRS-343. The data therefore support both the en-
derived from human lipocalins. We utilized phage
visaged mode of action of selective, tumor-localized
display to generate an Anticalin protein binding to
costimulatory T cell activation and the possibility
CD137 with high affinity and specificity. PRS-343
that this approach leads to reduced systemic toxicity
was obtained by genetic fusion of the CD137-specific
compared to conventional anti-CD137 mAbs.
Anticalin protein to a variant of a HER2-targeting
Conclusion: We report potent costimulatory T-cell
monoclonal antibody, trastuzumab, with an engi-
engagement of the immunoreceptor CD137 in a
neered IgG4 backbone.
HER2-dependent manner, utilizing the CD137/HER2
Results: The bispecific fusion PRS-343 targets CD137
bispecific, PRS-343. Compared to known CD137-tar-
and HER2 with nearly identical affinities compared
geting antibodies in clinical development, this ap-
to the parental building blocks and is capable of
proach has the potential to provide a more localized
binding both targets simultaneously. We show ex
activation of the immune system with higher efficacy
vivo that T cells are efficiently activated when in-
and reduced peripheral toxicity. The positive func-
cubated with PRS-343 and HER2-positive cells and
tional ex vivo and in vivo data of PRS-343 as well as
that the activation is HER2-dependent. The in vivo
the excellent developability profile support investiga-
activity of PRS-343 was investigated utilizing a hu-
tion of its anti-cancer activity in clinical trials.
manized mouse model and the SK-OV-3 cell line as a
Immunogenic lipids of pediatric brain cancer
Paret C.1, El Malki K.1, Stössel S.1, Kron B.1, Lehmann N.1, Roth L.1, Russo A.1, Neu M.1, Wingerter A.1,
Theruvath J.1, Henninger N.1, Sommer C.2, Kabelitz D.3, Sandhoff R.4, Faber J.1
Medical University of Mainz, Pediatric Oncology, Mainz, Germany,
1
Institute of Neuropathology, Mainz, Germany,
2
Institute of immunology, Kiel, Germany,
3
German Cancer Research Centre, Heidelberg, Germany
4
Currently, there are no effective treatments for re-
CD1d in pediatric brain tumor samples. Moreover, we
current malignant intrinsic childhood brain tumors
have started to analyze the frequency and phenotype
and the need in new therapeutic strategies remains
of iNKT cells and γδ T cell in tumor samples and in
a matter of utmost urgency. Targeted immunother-
the peripheral blood of pediatric patients. Transcrip-
apies and particularly T cells based therapy have
tome analysis of tumor samples has been performed
the potential to improve such outcomes while mini-
and these data will be used to identify deregulated
mizing the treatment-related toxicities affecting the
pathways related to the lipid metabolisms. Because
normal developing brain in children. The Central
several enzymes are involved often in a combinato-
nerve system (CNS) has traditionally been perceived
rial fashion in the synthesis and turnover of single
as being immunologically privileged. In reality, the
lipids we will combine gene expression and quan-
blood-brain barrier is disrupted by tissue injury and
titative lipid analysis. For this, we have developed
inflammation, thereby facilitating entry of immune
protocols for the parallel extraction of RNA and lipids
cells into the CNS. Most T cell therapy trials have
from tumor tissues. Preliminary data show that the
been focused on conventional T cells restricted to a
used protocols allow the identification of the lipid
particular HLA allele thus restricting the number of
composition of tumors by modern liquid chromatog-
patients that can benefit from the therapy. A growing
raphy coupled tandem mass spectrometry (LC-MS/
body of experimental evidence suggests that tumor
MS). The lipid extract will be used to identify lipids
cells can be recognized and eliminated by non-con-
able of triggering the activation of non-conventional
ventional T cells like iNK T cells and γδ T cells. Unlike
T cells leading to a better understanding of how these
αβ T cells, iNKT and γδ T cells recognize their targets
cells operate. Our project is expected to provide new
irrespective of HLA haplotype and therefore offer
insights on the biology of iNKT and γδ T cells and
new possibilities for off-the-shelf, pan-population
on their therapeutic utility for pediatric brain tumors
cancer immunotherapies. iNK T cells and γδ T cells
patients.
recognize lipids when presented on the MHC class-I
related CD1d but so far the nature of the recognized
lipids is largely unknown. Importantly, expression
of CD1d in solid tumors has been described, including brain tumors, however few data are available on
the expression in pediatric brain tumor subtypes. To
address the relevance of non-conventional T cells in
cancer therapy, we are analyzing the expression of
Sialyl Glycolipid Stage-Specific Embryonic Antigen 4 (SSEA4) a novel target for CAR T cell therapy of solid cancers
Pfeifer R.1, Lock D.1, Aloia A.2, Tomiuk S.1, Hellmer J.1, Thie H.1, Bosio A.1, Kaiser A.1, Hardt O.1, Johnston I.C.D.1
Miltenyi Biotec, Bergisch Gladbach, Germany,
1
Institute of Molecular Health Sciences, ETH, Zurich, Switzerland
2
ORAL
TALK
SHORT
2016
The recent breakthroughs in the treatment of lym-
(scFvs) were derived from a murine antibody that
phoid malignancies using chimeric antigen receptor
specifically recognizes the sialyl-glycolipid. Healthy
(CAR)-redirected T cells have generated intense in-
donor T cells were enriched by magnetic cell sorting
terest to transfer this technology to the solid tumor
and activated with TransAct™ Reagent, a colloidal
setting. So far, however, the therapeutic success has
nanomatrix-based activation reagent, before lentivi-
been hampered largely due to the lack of truly tu-
ral transduction of the anti-SSEA4 CAR expression
mor-specific antigens, inefficient T cell homing to the
construct. Engagement of SSEA4 by CAR expressing
tumor site and a hostile microenvironment composed
T cells induced CAR-specific activation as character-
of immunosuppressive modulators. In an attempt to
ized by T cell degranulation, secretion of inflamma-
develop a CAR-based cancer treatment modality for
tory cytokines and an effective killing of SSEA4 ex-
solid tumors that is both effective and curative, we
pressing target cells. have identified the sialyl glycolipid stage-specific
Having assessed the performance of different anti-
embryonic antigen (SSEA4) as an epitope whose
SSEA4 CAR constructs, we next generated a panel
expression strongly correlates with metastasis and
of CARs containing humanized scFvs and assessed
chemoresistence in triple negative breast cancers
their performance in primary T cells. CARs with dif-
(TNBCs). Screening analysis by ourselves and others
fering affinities to SSEA4 may be able to discriminate
has further shown this antigen to have restricted
between normal and tumor cells expressing different
distribution in normal tissues, but to be expressed
levels of the antigen, thus minimizing potential tox-
on a broad array of solid tumors including those
icities. Current studies are now focusing on evaluat-
having poor prognosis with conventional treatment.
ing the in vivo functionality using mouse xenograft
As cancer patients are exposed to multiple rounds of
models.
chemotherapy and SSEA-4 positive cells are found
enriched in residual tumors surviving chemotherapy, a sequential therapeutic approach using chemotherapy followed by CAR T cell therapy holds great
promise to improve treatment outcome and overall
survival of cancer patients.
For this study, we constructed a second generation anti-SSEA4-CAR that contains an IgG1 spacer
domain in conjunction with the CD137 and CD3z
signaling domains. Single chain variable fragments
Cytoreductive and Immunmodulatory drugs influence bispecific
CD33/CD3 BiTE® antibody construct (AMG 330) mediated
cytotoxicity against Acute Myeloid Leukemia (AML)
Platzer J.1,2,3, Krupka C.1,2, Pachzelt L.1,2, Brauneck F.1,2, Lichtenegger F.S.1,2, Kufer P.4, Kischel R.4, Köhnke T.1,2,
Altmann T.1,2, Spiekermann K.1,5, Hiddemann W.1,5, Subklewe M.1,2,5
Klinikum of the LMU Munich, Department of Internal Medicine III, Munich, Germany,
1
Helmholtz Institute Munich, Clinical Co-operation Group Immunotherapy, Munich, Germany,
2
Immunotargeting of cancer (i-Target) Doktorandenkolleg, Elitenetzwerk Bayern, Munich, Germany,
3
AMGEN Research (Munich) GmbH, Munich, Germany,
4
German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
5
Previously, we demonstrated that the CD33/CD3
Pretreatment of T cells with AZA and DEC impaired
BiTE® antibody construct AMG 330 is able to induce
AMG 330 mediated cytotoxicity (%lysis: AZA: untreat-
activation and proliferation of residual autologous
ed (UT): 99.8 vs 1µM: 99.2 vs 5µM: 52.1 vs 10µM: 28.6;
T cells. This results in AMG 330 mediated cytotox-
DEC: UT: 78.9 vs 0.3µM: 57.9 vs 2µM: 48.6 vs 5µM: 5.8)
icity against primary AML (pAML) ce
at
and T-cell proliferation (%proliferation: AZA: UT: 74.0
low E:T ratios. Based on these data, an international
vs 1µM: 62.7 vs 5µM: 35.0 vs 10µM: 6.5; DEC: UT: 46.7
phase I study in relapsed/refractory AML patients has
vs 0.3µM: 32.7 vs 2µM: 42.7 vs. 5µM: 10.2) (n=1-7).
been initiated (2014-004462-20). Clinical experience
The addition of DEX to pAML long term cultures con-
with blinatumomab in acute lymphoblastic leukemia
siderably impaired T-cell proliferation (fold change
has shown a clear correlation of leukemic burden and
CD2+ cells: UT: 12.9 vs DEX: 1.3) and IFNγ cytokine
the occurrence of a cytokine release syndrome (CRS).
secretion (pg/ml: UT: 813 vs DEX: 64) (n=3). Ongoing
A cytoreductive phase prior to BiTE® therapy might be
experiments will show the influence of DEX-induced
beneficial to reduce the severity of the CRS. The aim
T-cell impairment on AMG 330 mediated lysis of AML
of this study was to evaluate the influence of cytore-
cells.
ductive drugs (azacytidine, AZA and decitabine, DEC)
TOC had no negative effect on cytotoxicity (%lysis: UT:
on AMG 330 mediated cytotoxicity. In clinical practice,
99.5 vs TOC: 99.7) or T-cell proliferation (fold change:
BiTE® mediated unwanted toxicity is commonly treated
UT: 5.0 vs TOC: 4.9) neither in cocultures nor in pAML
with steroids (dexamethasone, DEX) or with the IL-6R
cultures (n=3-4). Similarly, secretion of IFNγ (pg/ml:
antibody tocilizumab (TOC). In our study we also ana-
UT: 712 vs TOC:839) was not affected.
lyzed the influence of these drugs on T-cell function.
In summary, pretreatment with AZA and DEC impaired
T cells and AML cells were cocultured in the presence
AMG 330 mediated cytotoxicity and T-cell proliferation
of a control BiTE® antibody construct or AMG 330 for
in a concentration depended manner. Ongoing experi-
3 to 7 days. T cells were preincubated with the specific
ments are performed to validate these data in pAML
drug (72 hours; AZA: 1, 5, 10µM, DEC: 0.3, 2, 5µM)
patient samples.
prior to coculture or simultaneously added (DEX 75ng/
Furthermore, in contrast to TOC the use of DEX consid-
ml, TOC 110µg/ml). Experiments were conducted with
erably decreased AMG 330 mediated T-cell proliferation
AML cell lines and healthy donor (HD) T cells or with
and cytokine secretion. If this translates into reduced
pAML patient samples in AML long-term cultures.
cytotoxicity is currently evaluated.
lls
Lysis of AML cells and T-cell proliferation was assessed
by flow cytometry. Supernatants were collected to
assess cytokine secretion profiles.
Targeting DNA damage response genes to improve radiotherapy of pancreatic cancer
Posselt L.1, Orth M.2, Belka C.2, Kirchleitner S.1, Schuster J.2, Endres S.1, Duewell P.1, Lauber K.2, Schnurr M.1
LMU Munich, Division of Clinical Pharmacology, Munich, Germany,
1
LMU Munich, Department of Radiation Oncology, Munich, Germany
2
Background: Pancreatic cancer with a 5 year sur-
oresistance. Combination treatment with radiother-
vival rate of 7 % is the fourth leading cause of can-
apy and small molecule inhibitors lead to reduced
cer-related death in men and women. Chemo- and
clonogenic survival of human PDAC cell lines. The
radiotherapy as well as surgical resection comprise
data could be confirmed using RNAi-based target
the standard treatment options. However, at the time
gene inhibition. According to The Cancer Genome
point of diagnosis only less than 20 % of the patients
Atlas (TCGA), 11 % of the samples with upregulated
approve for surgical resection. Pancreatic cancer is
DNA-PKcs could be correlated to decreased overall
characterized by high radioresistance leading to nu-
survival. In vivo efficacy of DNA-PKcs inhibition
merous treatment failures. Despite progress in the
combined with fractionated radiotherapy is currently
medical management the death rate for pancreatic
being investigated.
cancer increased by 0.3 % during the last years.
Conclusion: Pancreatic cancer is characterized by
Therefore molecular and translational approaches
aggressive growth and poor prognosis necessitating
are urgently needed in order to develop new thera-
the development of new and effective therapeutic
peutic strategies.
drugs. The combination of fractionated radiotherapy
Methods: Radioresistance of 9 different human PDAC
and inhibition of DNA damage response genes for the
cell lines was assessed by colony formation assays
treatment of pancreatic cancer demonstrates a novel
and radioresistance hits were identified by principal
and promising approach deserving further evalua-
component analysis. Potential candidates of the DNA
tion.
damage response genes (DDR) were analyzed via
qRT-PCR and correlated for potential drivers of radioresistance. The effect of identified candidate target
genes for radioresistance of PDAC cell lines was
tested with specific small molecule inhibitors and
RNA interference (RNAi). Clinical relevance will be
established and evaluated by fractionated CT-based
irradiation in an orthotopic pancreatic tumor model.
Results: Relative expression analysis for DNA damage
response genes (DDR) revealed ataxia-telangiectasia
mutated (ATM) serine/threonine protein kinase and
the catalytic subunit of the DNA-dependent protein
kinase (DNA-PKcs) as one of the key players of radi-
Evaluating prediction strategies for identification of
immunogenic mutation-derived neo-epitopes in melanoma
Ramskov S.1, Bjerregaard A.-M.2, Fugmann T.3, Ritz D.3, Donia M.4, Bentzen A.K.1, Andersen R.4, Szallasi Z.2,
Neri D.3, Svane I.M.4, Eklund A.C.2, Reker Hadrup S.1
Technical University of Denmark / National Veterinary Institute, Section for Immunology and Vaccinology,
1
Frederiksberg C, Denmark,
Technical University of Denmark / Department of Systems Biology, Center for Biological Sequence Analysis,
2
Lyngby, Denmark,
Philochem AG, Otelfingen, Switzerland, 4Herlev University Hospital / Haematological Department, Center for
3
Cancer Immune Therapy, Herlev, Denmark
A number of recent reports point to an important role
an overlap in prediction of 29-81%, 13-68% predict-
of mutation-derived neo-antigens in immune recog-
ed solely from TCL and 0-5% predicted solely from
nition of cancer, as predictors of clinical outcome and
TF. Furthermore, we included both RNA expression
as potential targets in personalized immunothera-
values and immunopeptidome analysis by mass
peutic strategies.
spectrometry, as additional tools to identify potential
Mutagen-induced cancer types as melanoma and
immunogenic neo-epitopes.
lung cancer carry a large number of mutations, from
T cell recognition in autologous patient material of
which putative neo-epitopes can be predicted. Since
each personalized peptide library were investigated
the vast majority of mutations are patient specific,
(ongoing studies) by use of a novel technology based
identification of neo-epitopes requires prediction
on DNA-barcode labeled MHC multimers, enabling
and generation of large personalized peptide librar-
high-throughput screening for >1000 neo-epitope
ies. Previous reports of neo-epitope identification
specific T cell populations in a single sample. This
have however demonstrated that only a minority of
broad assessment of neo-epitope reactivity, with
peptides (< 1%) elicit T cell recognition at a detect-
minimal preselection, allows identification of the
able level. Consequently, there is an unmet need to
strongest predictive strategies for identification of
understand the rules identifying immunogenic neo-
immunogenic neo-epitopes.
epitopes.
Identification of precise and effective prediction ap-
In this study we evaluate different neo-epitope predic-
proaches will provide an important step towards the
tion approaches in three melanoma patients. Peptide
use of neo-epitopes as therapeutic targets and predic-
libraries of 200-800 peptides for each patient were
tors of response to immunotherapy.
generated from whole exome sequencing in combination with HLA binding predictions. A comparison
of prediction from autologous tumor cell lines (TCL)
and snap-frozen tumor fragments (TF) resulted in
Evaluation of T cell epitope prediction servers
Hoppe S.1,2, Winter J.1, Zeller C.1, Blatnik R.1,2, Riemer A.B.1,2
German Cancer Research Center (DKFZ), Immunotherapy and -prevention, Heidelberg, Germany,
1
German Center for Infection Research (DZIF), Molecular Vaccine Design, Heidelberg, Germany
2
To assess if a tumor antigen can be recognized by the
servers, we developed a web-based tool, which sys-
cellular arm of the immune system, it is important to
tematically combines the prediction results of several
determine if peptides derived from this protein can
servers. The web interface allows users to paste the
be presented on major histocompatibility complex
desired protein sequence and select a MHC molecule
(MHC) molecules of a given patient. As it is not pos-
of interest. The output is provided in a table format,
sible to test every potential epitope experimentally,
enabling users to choose the best predicted T cell
several in silico prediction tools for MHC-binding
epitopes and also to compare between different pre-
peptides have been developed, which are based on
diction server results.
motif analysis of known epitopes. However, results
vary between prediction servers, and there are vast
differences in the accuracy of predictions for different MHC alleles.
In this study, we evaluated the performance of eight
MHC class I prediction servers for important human
leukocyte antigen (HLA) alleles by testing a large
number of predicted peptides in competition-based
cellular binding assays. With this data, receiver operating characteristic (ROC) curve analysis was performed and the area under the curve (AROC) was
calculated as a measure of prediction quality. We
not only included 9-mer, but also 8-, 10- and 11-mer
peptides.
While predictions for HLA-A2 and -A3 were very accurate, predictions for other alleles like HLA-A11 and
-A24 were less precise. No single prediction server
outperformed the others, but different prediction
servers were found to be best for different HLA types
and peptide lengths. We thus recommend always
using a minimum of two prediction servers to generate reliable results.
In order to make maximal use of available prediction
Immunotherapy targeting RIG-I in a mouse model of acute
myeloid leukemia
Ruzicka M.1, Heuer E.M.1, Meinl H.1, König L.1, Endres S.1, Subklewe M.2, Lichtenegger F.2, Rothenfusser S.1
Klinikum der Ludwig-Maximilians-Universität München, Department of Medicine IV, Division of Clinical
1
Pharmacology, Munich, Germany,
Klinikum der Ludwig-Maximilians-Universität München, Department of Medicine III, Munich, Germany
2
Acute myeloid leukemia (AML) is a clonal disease of
against re-challenge with C1498 cells. Preliminary
primitive haematopoetic precursor cells with a very
data show a significant beneficial effect on survival
poor prognosis. Although full hematological remis-
when pppRNA treatment is combined with aPD-1 an-
sion can be achieved with intensive chemotherapy,
tibody therapy.
the majority of patients experience relapses, making
Based on these results the use of pppRNA as a treat-
post-remission therapy indispensable. Allogenous
ment component in AML is further evaluated.
stem cell transplantation is the state-of-the-art approach to achieve consolidation. However, the abundance of side effects and the limited availability of
the procedure due to donor shortage stress the importance of alternative treatment strategies.
In the presented study we test short 5’-triphosphatemodified hairpin RNAs (ppp-RNA) triggering the
cytosolic pattern recognition receptor retinoic acidinducible gene I (RIG-I) as a treatment strategy in a
mouse model of AML. RIG-I activation mimics viral
infection and leads to the production of inflammatory cytokines on the one hand and induction of an immunogenic cell death in the target cell on the other.
Beyond the direct reduction of viable AML cells, we
expect this therapy to induce an anti-AML-directed
adaptive immune response similar to an active immunization.
Application of ppp-RNA complexed with a cationic
polymer-based transfection reagent significantly
reduced tumor burden, delayed disease progression
and led to long-term survival in 20 per cent of the
animals in an AML model based on C1498 cells in immunocompetent C57BL/6 mice. Furthermore, pppRNA-treated mice surviving AML-induction establish an immunological memory leading to protection
4-1BBL synergizes with IL-12 and IL-2 in the induction of a
defensive immune signature in the human urinary bladder
carcinoma microenvironment
Biermann-Fleischhauer C.1, Miegel A.1, Leusmann A.1, Kornmann M.1, Artanago K.1, Wendt-Cousin D.1, Schumacher U.2, Hofmann A.3, Hoffmann P.3, Becker B.4, Netsch C.4, Gross A.4, Pauels H.-G.5,
Helftenbein G.1, Schnieders F.1
Provecs Medical GmbH, Hamburg, Germany,
1
University Medical Center Hamburg-Eppendorf, Institute of Anatomy, Hamburg, Germany,
2
University of Bonn, Institute of Human Genetics, Platform Genomics, Bonn, Germany,
3
Asklepios Klinikum Hamburg-Barmbek, Clinic of Urology, Hamburg, Germany,
4
Pauels-Consulting, Steinfurt, Germany
5
Recent success of therapeutics directed against
single target activation already at low dosage.
immune check-points supports the concept of ad-
Using this new multivalent concept the effects on
dressing solely immune cell targets to redirect an
molecular microenvironment reprogramming were
immune response against tumor cells. Therapeutic
evaluated in an ex vivo tumor tissue model of human
targeting of the tumor microenvironment requires
bladder carcinoma. Cystectomy specimens collected
regulation of different immune cell types by com-
from more than 40 patients were stimulated for up
binations of biological signals, hence therapeutic in-
to 2 weeks. Evaluation of more than 400 expression
tervention remains a substantial challenge. A wealth
profiles showed that Immunalon® induced a clear
of clinical studies focuses on monoclonal antibody
transgene-dependent enhancement of CD4+ T helper
technology to address single immune cell stimula-
cell activity and subsequent CD8+ T effector cell
tion, co-stimulatory activation or checkpoint inhibi-
activation, together with enhanced NK cell activity.
tion.
Most prominent molecular components indicative
4-1BBL (CD137L, TNFSF9) is a promising co-stim-
for immune processes are the induced chemokines
ulator targeted by monoclonal antibodies in clini-
CXCL9, CXCL10 together with IFN-y along with the
cal studies. To further extend its scope of immune
repression of IL-10. This profile is indicative for a Th1
microenvironment activation we here analyzed the
immune response. Detailed immune cell analysis
effects of adenoviral co-expression of 4-1BBL with
using expression profiling and bioinformatics shows
Interleukin-2 and single-chain Interleukin-12. These
a Th1 specific microenvironment immune signature
genes are known to be involved in cellular immune
and suggests activation of T helper cells, CD8+ cyto-
responses based on different immune cell types.
toxic T cells, NK cells and differentiation of myeloid
In a co-culture system of human tumor cells and
and dendritic cells.
human peripheral blood mononuclear cells (PBMCs),
As cellular targets for Immunalon®, tumor cells,
4-1BBL was found to induce a clear Interferon-y sig-
stroma and immune cells of lymphocytic and mono-
nature. In this setting, in a dose-dependent manner,
cytic ultrastructure were identified by high-resolu-
4-1BBL stimulates synergistically with Interleukin-2
tion transmission electron microscopy.
and Interleukin-12 the secretion of IFN-y. Whole
Together with the opportunity of local application,
genome mRNA expression profiling and pathway
Immunalon® has the potential for inducing favora-
analyses revealed the induction of activation and
ble and long-lasting immunological changes in solid
differentiation of different target immune cell types.
tumor microenvironments with minimal side-effects
The effects of this novel 3-gene adenoviral therapeu-
as compared to systemic treatment.
tic, termed Immunalon®, exceed those induced by
The immunopeptidomic landscape of ovarian carcinoma
Schuster H.1, Peper J.K.1, Bösmüller H.-C.2, Linus B.1,3, Stevanovic S.1, Rammensee H.-G.1, Staebler A.2, Wagner P.4
1
University Hospital Tübingen, Institute of Pathology, Tübingen, Germany,
2
University of Tübingen, Center of Bioinformatics Tübingen, Tübingen, Germany,
3
4
Epithelial ovarian cancer (EOC) remains the most
tube samples facilitated the identification of antigens
lethal gynecological disease owing to late diagnosis
specifically expressed and presented in the context
and frequent resistance to platinum based chemo-
of ovarian carcinoma.
therapy. The overall prognosis of patients suffering
Additional comparative profiling to the immunopep-
from ovarian cancer remains poor and curative treat-
tidome of a variety of benign tissues including ovary,
ment is only possible by surgical resection at an early
fallopian tube, liver, kidney and colon unveiled a
non-metastatic stage. The lack of treatment options
multitude of ovarian cancer antigens (MUC16, MSLN,
and the high immunogenicity of ovarian cancer have
IDO1, KLK10, FOLR1….) to be frequently presented
long demanded the use of an immunotherapeutic ap-
by HLA class I and class II molecules exclusively
proach.
on ovarian tumor tissue. Most strikingly, ligands
Immunotherapies are starting a revolution in cancer
derived from mucin 16 (CA-125) and mesothelin, a
therapy with the introduction of checkpoint inhibi-
molecular axis of prognostic importance in EOC are
tors finally unleashing the power of the immune
presented in more than 80% of patients independent
system. Therapeutic success however seems to be
of HLA type. Furthermore many established cancer/
correlated with a high mutational load (e.g. mela-
testis antigens (MAGE-A/B, ODF2, DPPA2) were pre-
noma, lung cancer) and the presence of T cells tar-
sented by tumors of individual patients.
geting an appropriate tumor rejection (neo)antigen.
The great majority of HLA presented peptides
Knowledge about which antigens are presented in
derived from these antigens were able to prime naive
tumors with lower mutational load, in particular by
T cells from healthy donors and their recognition in
ovarian cancer cells, capable of exposing them to
patients is currently evaluated. For the first time We
immune cells is in contrast still sporadic. This infor-
were able to identify biomarkers, which can predict
mation is however critically needed for the design
ligand presentation of selected antigens. Thereby, we
of targeted immunotherapies, in order to redirect a
envisage to further facilitate individualized antigen
liberated immune response to its envisaged target.
selection for immunotherapy, directly addressing the
We performed large scale immunopeptidome analy-
antigenic profile of a patient’s tumor.
sis by immunoprecipitation of HLA molecules of
more than 40 ovarian carcinoma samples and subsequent mass spectrometric analysis of HLA ligands,
in order to exhaustively characterize the antigenic
landscape of these tumors. Parallel transcriptome
analysis of ovarian carcinoma and ovary or fallopian
Clinical non invasive in vivo imaging of the differentially expressed tumor associated antigen PSMA by a specific Positron
Emission Tomography (PET) ligand
Schwenck J.1,2, Rempp H.3, Reischl G.2, Schittenhelm J.4, Beschorner R.4, Skardelly M.5, Nikolaou K.3,
Pfannenberg C.3, la Fougere C.1
Eberhard Karls University Tübingen, Department of Nuclear Medicine, Tuebingen, Germany,
1
Eberhard Karls University Tübingen, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Germany,
2
Eberhard Karls University Tübingen, Department of Diagnostic and Interventional Radiology, Tübingen, Germany,
3
Eberhard Karls Universität Tübingen, Department of Pathology and Neuropathology, Tübingen, Germany,
4
Eberhard Karls Universität Tübingen, Department of Neurosurgery, Tübingen, Germany
5
Prostate specific membrane antigen (PSMA) is an
tected lymph nodes exhibited PSMA expression but
upcoming, promising target for immunotherapies. It
no uptake of the proliferation marker
is believed that PSMA is ubiquitously expressed at
While 98% of the suspicious bone lesions expressed
the membrane of prostate carcinomas and its metas-
PSMA, PET imaging revealed 8 PSMA-negative bone
tasis. Nevertheless PSMA expression is described in
lesions in 5 patients, from whom one patient only had
other tumors. As clinical indications for biopsies are
PSMA-negative bone lesions. One prostate cancer
rare, only very few studies proved PSMA expression
patient presented with a highly PSMA expressing
in vivo in different carcinomas and its metastases
liver lesion, which was proven to be a cholangio-
except for prostate cancer.
cellular carcinoma by means of biopsy. Furthermore
Non invasive in vivo imaging using PET with radi-
a patient with glioblastoma was investigated with
olabeled PSMA ligands represents a way to assess
68
PSMA expression in vivo for example before onset
by immunohistology revealing increased PSMA ex-
of expensive immunotherapies with potential severe
pression predominantly in the luminal endothelium
side effects.
of the tumor vasculature, but not in healthy brain
Prostate cancer patients with biochemical relapse (n
tissue and vasculature. Analysis of a primary pros-
= 103) after prostatectomy and/or radiotherapy as
tate tumor and a prostate tumor metastasis revealed
well as one glioblastoma patient underwent a whole
PSMA expression, which was not restricted to the
body PET examination using the PSMA ligand Glu-
tumor vessels.
68
68
11
C-choline.
Ga-PSMA-PET. PSMA expression was confirmed
NH-CO-NH-Lys-(Ahx)-[ Ga-HBED-CC] ( Ga-PSMA;
Thus, PSMA is a tumor associated antigen, which is
60 min p.i.; 165±14 MBq). Suspicious lesions were
expressed in different carcinomas, gliomas, metasta-
evaluated visually and semiquantitatively (SUVavg).
ses and tumor vasculature in a very heterogeneous
In prostate carcinoma patients we additionally ana-
manner. It is not known yet, under which conditions,
lyzed the cell proliferation of carcinomas using the
for example therapeutic approaches, tumors express
PET tracer 11C-choline as diagnostic standard (5 min
or loose PSMA expression. Further studies are needed
p.i.; 624±25 MBq).
to specify under which conditions PSMA is expressed.
In the prostate cancer patients remarkable PSMA ex-
This is an essential prerequisite to develop and use
pression was determined in 94% of the lymph nodes
efficient PSMA-targeting therapies in terms of side
suspicious for metastasis. Nevertheless, 29 lymph
effects, costs and clinical outcome. Non invasive in
nodes (6%) in 10 different patients showed no PSMA
vivo imaging such as PET can serve as a potent di-
11
expression, but C-choline PET indicated a high pro-
agnostic and monitoring tool for PSMA expression in
liferative activity. On the other hand, 39% of the de-
preclinical and clinical research.
KA2237 and KA2507: Novel, oral cancer immunotherapeutics
targeting PI3K-p110β/p110δ and HDAC6 with single-agent and
combination activity
Shuttleworth S.1
Karus Therapeutics Ltd., Abingdon, United Kingdom
1
Two de novo-designed classes of orally-active im-
combination is based upon immunotherapeutic and
munotherapeutics - selectively targeting the PI3K-
cell signalling mechanisms, which will be described.
p110β/p110δ and HDAC6 enzymes - for solid and
KA2237 is entering Phase I studies in patients with
hematological cancer treatment will be described.
lymphoma as a single agent in early Q2 2016, with
The former, exemplified by the clinical candidate
KA2507 clinical trials scheduled to commence later
KA2237, are uniquely-selective inhibitors of the PI3K-
in the year. Further clinical investigations into solid
p110β/p110δ isoforms, displaying immunotherapeu-
tumor immunotherapy - both single agent- and com-
tic activity, and inhibiting primary tumor growth
bination-studies - are planned.
and metastasis. The latter, for which KA2507 is the
candidate compound, are HDAC6-specific inhibitors,
which inhibit tumor growth through regulation of
aggresome formation, and which decrease PD-L1 expression via reduction of STAT3 phosphorylation. In
both cases, selectivity has been achieved over other
enzyme superfamily members, with the aim being
to minimize mechanism-related toxicity, thereby potentially providing opportunities to enable elevated
single dose levels - and broader combination modalities not possible with pan-inhibitors - to be investigated. The PI3K-p110β/p110δ and HDAC6 inhibitors
display potent oral activity as single agents in in vivo
tumor syngeneic models, impacting on growth inhibition, PD and tumor marker responses; the PI3Kp110β/p110δ inhibitors also inhibit metastatic spread
and, following surgical removal of primary tumor,
confer promising inhibition of tumor regrowth.
Additionally, the inhibitors work in combination
with one another, making this co-therapy approach
unique in oncology, and potentially important clinically in overcoming resistance mechanisms in both
solid and hematological cancer. The rationale for this
Ovarian carcinoma explant culture: model development and
application in drug testing
Suarez-Carmona M.1, Heinzelmann A.1, Hampel M.1, Schott S.2, Zörnig I.1, Jäger D.1, Halama N.1
National Center for Tumor Diseases and University Hospital Heidelberg, Medical Oncology, Heidelberg,
1
Germany,
University Clinics Heidelberg, Department of Obstetrics and Gynaecology, Heidelberg, Germany
2
Though considerable efforts have been made in the
revealed several modifications in the tumor micro-
development of new tools to study ovarian cancer in
environment. NIM15 inhibition namely resulted in
the past few years, currently available models are
a decreased expression of several macrophage-re-
still either cell line- or mouse-based. We are devel-
lated factors and multiple angiogenic compounds.
oping an innovative human-based model of whole
Furthermore, various factors involved in T-cell
explant tissue culture using primary ovarian tumors.
chemotaxis were also affected. Though preliminary,
Recently in our laboratory, such a model has suc-
these results suggest a broad effect of NIM15 and
cessfully been established for the study of colorec-
encourage us to further elucidate the role of NIM15
tal cancer-associated liver metastasis (Halama et al.
in cancer.
Cancer Cell 2016). Briefly, human cancer tissue is
taken into culture shortly after surgery and is viable
for several days. Explants are treated with (pre-)
clinical-grade drugs and subsequently processed
for histological analysis as well as for multiplex cytokine analysis. This combined approach enables us
to study the impact of various drugs on the tissue
(in terms of tumor cell death, changes in the infiltration and activation of lymphocytes, macrophage
polarization etc.).
Taking advantage of our tissue culture model, our
ongoing project aims at characterizing the role of
NIM15 in cancer. This soluble factor is highly expressed in liver metastases and ovarian primary
tumors, in contrast to healthy tissues. We hypothesized that it might play a tumor-supportive role and
are currently characterizing NIM15 (i.e. identifying
producing cells and target cells, the receptors and
signaling pathways involved). For this, we have produced three mouse monoclonal anti-NIM15 antibodies. After ovarian carcinoma explants were treated
with each antibody, multiplex cytokine analysis
Checkpoint-Inhibition for advanced mucosal melanoma
Thierauf J.1, Veit J.A.1, Hess J.2,3, Treiber N.4, Hoffmann T.K.1
Department of Oto-Rhino-Laryngology Head and Neck Surgery, Ulm University Medical Center, Germany,
1
Ulm, Germany,
Section Experimental and Translational Head and Neck Oncology, Department of Otorhinolaryngology, Head
2
and Neck Surgery, University Medical Center Heidelberg, Heidelberg, Germany,
Research Group Molecular Mechanisms of Head and Neck Tumors, German Cancer Research Center (DKFZ),
3
Heidelberg, Heidelberg, Germany,
Department of Dermatology, University Medical Center Ulm, Ulm, Germany
4
Background: In advanced cutaneous melanoma,
Results: All patients received at least 2 cycles
anti-PD-1/PD-L1 therapy has shown significant and
of nivolumab or pembrolizumab. There were no
durable response compared to classical systemic
adverse events greater than CTCAE grade 2 and no
therapies with a response rate of more than 30%. In
dose reductions. 5/5 patients showed progressive
advanced or recurrent mucosal melanoma conven-
disease after restaging at 3 months. There were no
tional chemotherapies have shown little benefit so
partial or complete responses or even stable disease
far. The efficacy and safety of an anti-PD-1 therapy
and therefore an ORR of 0%. Immunohistochemical
has not been addressed and because of the rare in-
staining demonstrated PD-L1 expression in less than
cidence of this entity, prospective clinical trials are
5% in all cases.
hard to achieve.
Conclusions: As demonstrated before, a relevant
Material and methods: We analyzed 5 patients with
PD-L1 over-expression in mucosal melanoma could
mucosal melanoma of the head and neck who re-
not be confirmed. Also concurrent therapy with
ceived nivolumab (n=2) or pembrolizumab (n=3)
either nivolumab or pembrolizumab showed no clin-
in an advanced stage. Anti-PD-1/PD-L1 therapy was
ical response, but tumor progression in our cohort.
given as 1st line therapy in 2 patients, 2 received 2nd
These results indicate, that PD-1-inhibitors do not
th
line therapy and one had 4 line treatment after re-
seem to serve as new therapeutic options for this
ceiving classical chemotherapies and an anti-CTLA-4
aggressive tumor entity correlating with the low im-
antibody. Expression of PD-L1 and PD-1 in all tumor
munohistochemical expression.
samples was measured by means of immunohistochemical staining. Nivolumab and pembrolizumab
were administered intravenous every 2 weeks and
every 3 weeks, respectively, using a concentration
of 3mg/kg for nivolumab and 2mg/kg for pembrolizumab.
Quantitative live-cell imaging assays for immunotherapy:
chemotaxis, T-cell killing & phagocytosis
Lovell G.1, Bevan N.1, Szybut C.1, Rodionova N.1, Appledorn D.2, Tikhonenko M.2, O’Clair L.2, O’Callaghan T.1,
Dale T.1, Trezise D.1
Essen BioScience R&D, Welwyn Garden City, United Kingdom,
1
Essen BioScience R&D, Ann Arbor, United States
2
For the body to defend and fight against cancer,
with a pH-sensitive red fluorescent probe. Engulf-
immune cells must recognise, engage, destroy and
ment of these labelled cells by J774.1 macrophages
ultimately remove unwanted tumour cells. Under-
is quantified as the appearance of fluorescence over
standing these processes and interactions at the cel-
time.
lular level is central to identifying and validating
The common features of these assays are (1) they
new drug targets and cellular therapy approaches.
provide a full time-course of the biology (2) the
In this study, we describe a cluster of new assays
images and time-lapse movies add credibility and
for quantifying immune cell biology and interactions
valuable biological insight, and (3) the throughput
with tumour cells. These assays are based on non-
and automated image analysis enhance productiv-
invasive live-cell analysis of cells in 96-well micro-
ity. All experiments are performed with low cell
plates using an IncuCyte ZOOM® automated imaging
numbers and without lifting or washing so are not
system. Phase-contrast or fluorescence images are
perturbing to the cell model. Cells can still be used
gathered from cells over time and processed to
for adjunct and follow on studies. These features
measure cell phenotypes, such as immune cell pro-
position quantitative live cell imaging as a valuable
liferation, migration, cell death etc. Three relevant
tool for immune-cell assays, and provide useful com-
example assays are presented:
plementarity to traditional immune cell assay ap-
(1) Chemotaxis of human T-cells, neutrophils & mac-
proaches such as flow cytometry, cytokine ELISAs
rophages
and other end-point plate reader measurements.
Directional migration of leukocytes toward chemoattractants, quantified using ClearView 96-well assay
plates and label-free phase contrast image analysis.
The kinetics of migration and sensitivity to chemoattractants (e.g. C5a, serum, fMLP) are compared.
(2) Apoptotic tumour cell death via immune-cell killing
SKOV-3 ovarian cancer cells are co-cultured with
CD-3/IL-2 peripheral blood mononuclear cells and
monitored for apoptotic cell death using caspase3/7
fluorescent probes.
(3) Efferocytosis/Phagocytosis of apoptotic immune
cells by macrophages
Apoptotic T-cells (camptothecin, Jurkat) are labelled
Rapid generation of T cell receptor like antibodies using
genetically reprogrammed memory B cells of immunized rabbits
van Helden P.M.1, Böhne M.1, Bakker A.Q.1, Wagner K.1, Kwakkenbos M.J.1, Spits H.1
AIMM Therapeutics, Amsterdam, Netherlands
1
Complexes of HLA molecules and peptides derived
parable antibody panel was obtained including 5
from intracellular proteins are emerging as promis-
different groups of antibodies with different fine spe-
ing targets for tumor specific antibodies. For applica-
cificities. The pMHC antibodies were able to induce
tion such antibodies should be highly specific and
ADCC of peptide-loaded T2 cells and not of cells
have a high affinity. Here we used genetic modifi-
loaded with control peptides. Antibodies were pro-
cation to immortalize B cells of immunized rabbits
duced recombinant in HEK cells to allow analysis at
to generate a large collection of antibodies with dif-
higher antibody concentrations. A subset of the anti-
ferent fine specificities and affinities for the same
bodies showed limited reactivity to irrelevant pMHC
peptide-MHC (pMHC) complex. The availability of
complexes at high antibody concentration and was
a panel of such antibodies will facilitate selection
excluded as leads. The HLA-A2-WT1 lead antibody
of the best antibody characteristics for therapeutic
R1560 was able to recognize the naturally processed
purposes.
WT1 epitope presented by HLA-A2 positive cell lines
We immunized rabbits with pMHC monomers: 2x
and induce ADCC. Humanized versions of the rabbit
HLA-A2-NY-ESO-1 and 2x HLA-A2-WT1. Peripheral
antibodies retained their affinity and specificity for
blood B cells were immortalized by retroviral in-
the pMHC complexes. We anticipate that the avail-
troduction of B cell lymphoma (BCL)-6 and Bcl-xL
ability of many antibodies against the same pMHC
resulting in stable, rapidly proliferating B cells that
offers ample opportunity to select the best ones in
express surface Ig and secrete antibody. We used the
terms of affinity, specificity and functional activity.
B cell receptor expression to sort clones that bind
pMHC pentamers. After 2-3 weeks of culture secreted
antibodies were tested for binding to T2 cells loaded
with peptides. Subsequently, specific antibodies
were tested for loss-of-binding to mutant peptides in
which single amino acid residues were exchanged
for alanine. For NY-ESO-1 we identified 40 antibodies binding specifically to the NY-ESO-1 peptide on
HLA-A2. The alanine scan revealed 4 categories of
pMHC specific antibodies for which binding depends
on different peptide residues. Within each category
various antibodies contained point mutations resulting in subtle affinity differences. For WT1 a com-
Anti-tumor activity of IMAB027 antibody as a single agent and
in combination with chemotherapy in testicular cancer
Walter K.1, Kreuzberg M.1, Jacobs S.1, Schmitt R.1, Talamini M.1, Wöll S.1, Rohde C.1, Sahin U.2,3, Türeci Ö.1
1
TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz,
2
Mainz, Germany,
3
IMAB027, a novel ideal monoclonal antibody
prevented tumor growth and prolonged survival.
(mAB), targets the tight-junction protein, claudin-
One cycle (three injections of 35 mg/kg) of IMAB027
6 (CLDN6). CLDN6 is an embryonic antigen that is
in mice with advanced testicular xenograft tumors
absent in human normal adult tissue but aberrantly
resulted in significant tumor growth inhibition and
expressed in solid tumors including ovarian (50%)
prolonged survival. As the standard of care for tes-
and testicular cancer (90%). IMAB027 has preclini-
ticular cancer patients are platinum-based chemo-
cal activity in ovarian cancer and is currently under
therapies, e.g. cisplatin, etoposide plus bleomycin
clinical evaluation in patients with advanced disease
(PEB), mice with advanced testicular tumors were
(ClinicalTrials.gov ID: NCT02054351). In this study,
treated with chemotherapy in combination with
we investigated the in vitro and in vivo anti-tumor
IMAB027. Survival of tumor-bearing mice was sig-
activity of IMAB027 administered as a single agent
nificantly prolonged with IMAB027 treatment com-
or in combination with chemotherapy using preclini-
bined with cisplatin or PEB compared to animals
cal testicular cancer models.
that received chemotherapy alone. IMAB027 plus
In vitro, IMAB027 bound with high affinity and spec-
PEB resulted in complete inhibition of tumor growth
ificity to endogenously expressed CLDN6 in human
in 73% (11/15) of animals.
testicular cancer cell lines. We investigated two
In conclusion, IMAB027 specifically binds to CLDN6
major mechanisms of action for IMAB027: antibody-
and has in vitro and in vivo anti-tumor activity in
dependent cellular cytotoxicity (ADCC) and com-
preclinical testicular cancer models. IMAB027 ad-
plement-dependent cytotoxicity (CDC). IMAB027
ministered as a single agent shows strong anti-tumor
caused efficient lysis through ADCC (median EC50
activity, which is further augmented in combination
~ 20 ng/mL; maximum lysis ~ 80%) and CDC
with chemotherapeutic agents. Testicular cancer pa-
(median EC50 ~ 700 ng/mL; maximum lysis ~ 60%)
tients may therefore benefit from IMAB027 treatment
of CLDN6-positive testicular cancer cells. IMAB027-
either as a single agent or in combination therapy.
mediated ADCC and CDC activity strictly depended
IMAB027 represents a promising novel therapeutic
on the presence of CLDN6 on the cell surface of
agent requiring further clinical investigation assess-
tumor cells. In vivo, animals with advanced human
ing its use for the treatment of patients with testicu-
testicular carcinoma xenografts, positive for CLDN6
lar cancer.
expression, were treated with IMAB027. Dose-range
finding studies in testicular tumor models, with
low-dose maintenance therapy of IMAB027 (three
injections of 4.375 mg/kg per week), significantly
In renal cell and prostate cancer a large fraction of the tumorinfiltrated T-cells cannot be targeted by current checkpoint
inhibition approaches
Zelba H.1, Hennenlotter J.2, Zettl M.3, Bedke J.2, Stenzl A.2, Rammensee H.-G.1, Gouttefangeas C.1
University of Tübingen, Interfaculty Institute of Cell Biology, Department of Immunology, Tübingen, Germany,
1
University Hospital Tübingen, Department of Urology, Tübingen, Germany,
2
Boehringer Ingelheim RCV GmbH & CoKG, NBE Discovery, Vienna, Austria
3
The blockade of the inhibitory receptors (iR) CTLA4
showed increased frequencies of PD1+, LAG3+ and
and PD1 leads to prolonged survival and significant
TIM3+ cells within TILs compared to autologous
tumor shrinkage in about 40% of treated patients,
PBMCs. The percentage of BTLA+ T cells was de-
which led to the approval of three immune checkpoint
creased in TILs compared to PBMCs, whereas the
inhibitor antibodies for the treatment of metastatic
percentage of CTLA4+ T cells did not differ.
melanoma, non-small cell lung cancer and renal cell
We found that CD4+ and CD8+ TILs most frequent-
carcinoma (RCC). PD1 and CTLA-4 are often upregu-
ly expressed solely PD1 (about 15%), and as iR com-
lated on tumor-infiltrating lymphocytes (TILs) and the
bination PD1 + LAG3 for prostate carcinoma (PCa)
interaction with their respective ligands in the tumor
(6%) and PD1 + BTLA for RCC (4%). However, we
microenvironment contributes to inhibition of anti-
observed that for both cancer entities, the largest
cancer immune responses. Further iRs are intensively
fraction of TILs (about one third) did not express
examined at the moment. Here, we determined the
any of the five investigated iRs.
expression of PD1, CTLA4, TIM3, BTLA and LAG3 and
In vitro stimulation of TILs in the presence or
their corresponding ligands on TILs and autologous
absence of checkpoint inhibitor antibodies revealed
PBMCs obtained from patients with RCC or prostate
that the blockade of solely PD1 led to increased func-
cancer (PCa). We furthermore investigated in vitro
tionality (determined by cytokine release, prolifera-
T cell immune functions in the presence of anti-PD1
tion or degranulation) in about 50% of RCC patients,
mAb alone vs a combination of anti-PD1 plus an ad-
whereas the blockade of PD1 plus TIM3 or LAG3 led
ditional checkpoint inhibitor antibody.
to increased functionality in two third of all patients,
TILs were isolated by a combined mechanical and
although these effects were in general rather moder-
enzymatic dissociation from fresh primary tumor
ate (1.5 to 2.3 fold increase).
tissues. PBMCs were isolated from whole blood. Cells
The combined blockade of different iRs appears a
were stained for multicolor flow cytometry in order
promising therapeutic strategy for the treatment of
to determine the (co)expression of iR and iR-ligands
RCC and PCa. We demonstrated that a considerably
on various immune cell populations. Functional at-
high fraction of the infiltrated T-cells do not express
tributes of TILs were assessed by intracellular cy-
any of the common inhibitory receptors. If these cells
tokine staining after in vitro stimulation with coated
are expressing other (unknown) iRs, they might be
CD3 mAb in the presence or absence of one or two
a suitable target population for future checkpoint in-
checkpoint inhibitor antibodies.
hibition.
While the percentage of iR+ T-cells was low in
PBMCs (except BTLA), both CD4+ and CD8+ T-cells
216 – 244
Improving
Immunity
216 | IMPROVING IMMUNITY
Second generation of IL-15-based tri-functional antibody fusion
proteins with costimulatory TNF-superfamily ligands for cancer
therapy
Beha N.1, Ring S.1, Harder M.1, Kontermann R.1, Müller D.1
University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany
1
Cytokines of the common cytokine receptor γ-chain
achieved by both fusion protein formats in targeted
family as well as costimulatory members of the
as well as non-targeted form in vitro. In addition,
TNF receptor-family have shown great potential to
tri-functional antibody fusion proteins combining
support the generation and development of an an-
RD_IL-15 with scOX40L and scGITRL, respectively,
titumor immune response. Nevertheless, their ap-
were generated and analyzed in vitro. In comparison
plication is generally hampered by systemic toxicity
with the bi-functional RD_IL-15_scFv, tri-function-
and limited efficacy as monotherapeutic strategy. In
al fusion proteins were less active in non-targeted
order to address these problems, we aim to combine
form, but significantly more active in targeted form.
and focus the effect of such molecules to the tumor
Proliferation of CD4+T cells, CD8+T cells and NK
site by developing tri-functional antibody-cytokine
cells were stimulated by all fusion proteins. In tar-
fusion proteins, composed of a tumor directed anti-
geted form strongest effects on immune cell prolifer-
body moiety (scFv), IL-15 fused to part of its receptor
ation were obtained by the RD_IL-15_scFv_scGITRL,
alpha chain (RD_IL-15) and the extracellular domain
clearly shifting the PBMC composition in favor of the
of a costimulatory ligand of the TNF-superfamily.
CD8+T cell population. Thus, the second generation
Thus, for the tri-functional fusion protein RD_IL-15_
of IL-15-based tri-functional antibody fusion pro-
scFv_4-1BBL targeting-mediated improvement of the
teins with costimulatory TNF-superfamily members
immune stimulatory activity in comparison with
emerges as a promising option for further therapeutic
the corresponding bi-functional antibody-cytokine
development.
fusion proteins was demonstrated in vitro and antitumor effects in vivo. Here, we report the development of the second generation of this tri-functional
fusion protein, in which the TNF-superfamily ligand
is introduced in a single-chain (sc) format, i.e. consecutive genetic fusion of three TNF ligand domains
in a row, thereby confining natural occurring trimerization of the ligand into a monomeric molecule
that differs in size and valency from the homotrimeric tri-functional molecule of the first generation.
Although for the second generation molecule lower
antibody valency translated into reduced binding
properties, similar immune stimulatory capacity was
Optimal triggering of anti-tumor CD4+ TH cells by tumor cells
expressing CIITA-driven MHC class II I-A-only molecules
Bou Nasser Eddine F.1, Forlani G.1, Tosi G.1, Accolla R.S.1
University of Insubria, Surgical and Morphological Sciences, Varese, Italy
1
The CD4+ TH cell activation is necessary to trigger
T cells and B cells were transferred from the vacci-
an adaptive immune response against most anti-
nated recipients to naïve recipients that were co-in-
gens and particularly tumor antigens. Activation
jected with the parental tumor cells and the results
can be achieved only after CD4+ TH cells recognize
showed that CD4+ TH cells were the main effectors
the tumor associated antigens on the surface of the
of the immune response against the tumor cells.
antigen presenting cells (APC) within the context
These results demonstrate the validity of triggering
of MHC class II (MHC-II) molecules. We previous-
a specific, long-lasting anti-tumor immune response
ly reported the success in triggering a TH-specific
using CIITA-driven MHC class II positive tumor cells
long lasting anti-tumor immune response in the
of different MHC haplotype as stimulators. Impor-
H-2d BALB/c mouse model by using tumor cells that
tantly, the results establish that expression of a single
acted as APC of their own tumor antigens in vivo
MHC-II restriction element, I-A, in tumor cells is suf-
after being genetically modified to express MHC-II.
ficient to trigger CD4+ TH cell protective immune
Stable MHC-II I-A and I-E expression was achieved
response, strongly suggesting that the relevant tu-
after transfection of tumor cells with the MHC-II gene
mor-associated antigenic repertoire can be displayed
transactivator (CIITA). We have now investigated
without the need of the I-E restriction element.
the pertinence of this approach in the H-2b C57BL/6
mouse model despite the defect in their I-Eα gene
and thus the lack of expression of I-E subset. To this
purpose we injected in vivo the CIITA-driven I-A-only MHC-II-positive LLC (lewis Lung Carcinoma) and
MC38 colon carcinoma in the C57BL/6 mice and
their growth rate along with the recipient’s immune
response were analyzed. The CIITA-transfected,
MHC-II-positive tumor cells were either completely
rejected or showed a significant growth retardation
compared to the MHC-II-negative parental tumor.
The protected mice were re-injected with the parental tumors and a complete rejection was obtained
proving that a specific long lasting immune response
was triggered by the CIITA-transfected tumors. Furthermore, total splenocytes or purified CD4+, CD8+
Effects of RNA-based RNAdjuvant® on PBMCs from liver cancer
patients in an ex vivo model
Circelli L.1, Petrizzo A.1, Tagliamonte M.1, Heidenreich R.2, Tornesello M.L.1, Buonaguro F.M.1, Buonaguro L.1
Istituto Nazionale per lo Studio e la Cura dei Tumori, ‘Fondazione Pascale’, Naples, Italy,
1
CureVAC AG, Tuebingen, Germany
2
Introduction: Evaluation of detailed biological
induced, although a more predominant production
effects of adjuvants on immune cells has been as-
of TNFa and IFNγ was observed in HCC patients vs.
sessed in a limited number of studies performed
healthy controls. The cytokine profile was further
only on samples derived from healthy individuals.
confirmed by gene transcriptional analysis, which
No data are available on samples derived from cancer
showed up-regulation of several genes involved in
patients who may have a severe immune impair-
innate and adaptive immune-related pathways.
ment. A novel RNA-based adjuvant (RNAdjuvant®
Conclusions: The multiparametric analysis showed
developed by CureVac) is made of a synthetic single
that the effects of RNAdjuvant® is comparable
stranded RNA molecule combined with a polymeric
between HCC and healthy subjects, although specific
carrier. It is known to interact with toll-like recep-
differences were observed. The present study is the
tors (TLRs) 7 and 8. The effects of RNAdjuvant® on
first demonstration that cancer patients and healthy
PBMCs of liver cancer patients was assessed in an
subjects are equally responsive to adjuvants, sug-
ex vivo setting, using a multiparametric approach to
gesting that the same vaccine formulation can have
analyze network dynamics of early human immune
similar potency in both groups of subjects.
responses.
Methods: 5 healthy volunteers and 17 HCC patients
were enrolled in this study and PBMCs were obtained. After treatment in culture, the effects of RNAdjuvant® was assessed by evaluation of CD80, CD86
and HLA-DR expression; pattern of cytokine production as well as gene expression profile. Moreover, the
downstream effect on CD4+ T cell phenotyping was
evaluated. The TLR4 ligand LPS was used as comparison.
Results: Treatment with RNAdjuvant® showed comparable effects on PBMCs of both HCC and healthy
subjects, as shown by all the evaluated parameters.
CD80, CD86 and HLA-DR expression was found upregulated in circulating dendritic cells with induction
of a CD4+ T cell differentiation towards an effector
phenotype. A mixed Th1/Th2 cytokine pattern was
Dermaject - a novel, convenient intradermal injection device
for intracutaneous injections
Clemenz M.1,2, Kegel M.1,2, Herrlich S.1, da Luz S.1, Kalla J.3, Baumeister S.3, Trächtler M.1, Zengerle R.1,4
Hahn-Schickard, Villingen-Schwenningen, Germany,
1
Verapido Medical GmbH, Villingen-Schwenningen, Germany,
2
Schwarzwald-Baar Klinikum, Akademisches Lehrkrankenhaus der Universität Freiburg, Villingen-
3
Schwenningen, Germany,
Universität Freiburg, Institut für Mikrosystemtechnik - IMTEK, Freiburg, Germany
4
Purpose: We present Dermaject, a novel, con-
such as significantly more effective vaccine and
venient intradermal injection device suitable for
immunotherapy action, faster bloodstream absorp-
injection of liquid drugs into the top layer of the
tion and higher bioavailability.
skin (intradermal / intracutaneous route) and
Methods: Multiple experimental injections with
for drug targeting of the skin, immune and lym-
stained water and different gelatin solution
phatic system. The newly developed and patent-
volumes of 100 to 300 microliters in ex vivo human
ed cannula insertion mechanism in combination
skin and in ballistic gelatin skin models were per-
with microneedle technology enables easy, safe,
formed with Dermaject. Wheal (blister) size in the
precise, fast, standardized, reproducible and leak-
skin was measured. Histological sections of wheal
proof intradermal injections of larger than previ-
centers and edges were obtained and hematoxy-
ously injectable volumes.
lin and eosin (HE) staining was used to determine
Background: Dermaject was tested in numer-
dimensions, locations and depths of injected in-
ous ex vivo and in vivo experiments, demonstrat-
filtrates.
ing safety, easy handling and leak tightness. The
Results: Data showing high precision, accuracy,
design is user-friendly, small and considered for
consistency, standardization and reproducibility
single use in humans. Even at high back pressures
of intradermal injections on the basis of experi-
caused by highly viscous solutions, no leakage was
ments in ex vivo human skin and ballistic gelatin
observed. Furthermore, specific immune response
with the use of the Dermaject intradermal injec-
of vaccines was shown using the device.
tion device, will be presented.
Dermaject precisely and easily inserts a thin
Conclusion: With the newly introduced, safe,
cannula (e. g. 30 Gauge) into the dermis at a well-
easy to use and economical intradermal injection
defined angle, mimicking the Mantoux method.
device Dermaject, intradermal injections can be
Wrinkle formation usually occurring during the
performed reliably. Therefore, it is supposed to be
insertion process is effectively compensated for by
an ideal and highly clinically relevant solution for
partial retraction of the cannula. Handling of the
all parenteral substances and fluids suitable for ad-
device is simple and insertion can be performed
ministration via the intradermal route.
basically by anyone. A specially developed mechanism effectively protects against needlestick injuries.
Intradermal drug delivery, compared to subcutaneous drug delivery, provides numerous benefits,
PD-L1 checkpoint blockade enhances anti-tumor activity of CEA
TCB, a novel T-cell bispecific antibody for the treatment of solid
tumors
Colombetti S.1, Fauti T.1, Papastogiannidis P.1, Le Clech M.1, Nicolini V.1, Fahrni L.1, Sam J.1, Saro J.1, Karanikas V.1,
Klein C.1, Umana P.1, Gerdes C.1, Bacac M.1
Roche Pharmaceutical Research & Early Development, Roche Innovation Center Zurich, Schlieren, Switzerland
1
ORAL
TALK
SHORT
2016
Cancer immunotherapy has recently shown to rep-
cells expressing PD-1 and to a strong induction of
resent a very promising therapeutic approach able to
PD-L1 expression in tumors, similar to the adaptive
extend overall survival of cancer patients. However,
immune resistance mechanisms. The combination of
current immune-based therapies are only effective
CEA TCB with anti-PD-L1 blocking antibody showed
in a subset of patients and combination strategies
a significant increase of anti-tumor activity as com-
are needed to deepen and to broaden the therapeutic
pared to the respective single agents.
efficacy. One mechanism by which cells in various
These preclinical data indicate that, in line with the
tumor tissues limit the host immune response is via
adaptive immune resistance model, CEA TCB treat-
up-regulation of PD-1 ligand (PD-L1) and its ligation
ment leads to up-regulation of the PD-1/PD-L1 sup-
to PD-1 on activated CD8+ T cells, a mechanism
pressive pathway and that the combination of CEA
called adaptive immune resistance. Recent clinical
TCB with PD-L1 blockade results in enhanced anti-
data have shown that blockade of the PD-1/PD-L1
tumor activity. A clinical trial investigating the com-
axis can mediate potent anti-tumor activity.
bination of CEA TCB and atezolizumab is currently
CEA TCB (RG7802, RO6958688) is a novel T cell
ongoing.
bispecific antibody targeting the carcinoembryonic
antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent in a Phase
I study in patients with advanced and/or metastatic
CEA-expressing tumors.
Here we show that, in vitro, CEA TCB-mediated
killing of tumor cells by T-cells led to up-regulation of
PD-1 receptor on both CD4+ or CD8+ T cell subsets
as well as of PD-L1 on tumor cells, as early as 24 h
following treatment with RO6958688. These results
prompted us to investigate in vivo the potential synergistic anti-tumor activity of CEA TCB treatment
in combination with the blockade of the PD1-PDL-1
axis. In vivo studies performed using high-CEA expressing tumor xenografts in fully stem cell humanized NOG mice demonstrated that CEA TCB treatment led to increased frequency of intra-tumor T
Seprehvir, an oncolytic herpes viroimmunotherapeutic, enhances
therapeutic efficacy of T cell checkpoint inhibition in solid tumors
by increasing T cell recruitment and remodeling the immunosuppressive microenvironment
Chen C.-Y.1, Wang P.-Y.1, Hutzen B.1, Sprague L.1, Swain H.1, Haworth K.1, Love J.1, Conner J.2, Cripe T.3
Nationwide Children’s Hospital, Ohio State University, Columbus, United States,
1
Virttu Biologics, Glasgow, United Kingdom,
2
Nationwide Children’s Hospital, Pediatric Hematology/Oncology/BMT, Columbus, United States
3
ORAL
TALK
SHORT
2016
Most solid tumors are characterized by an immu-
on intratumoral virus spread, recruitment of myeloid
nosuppressive microenvironment, limiting the ef-
and lymphoid cellular subpopulations, and immuno-
fectiveness of antitumor immunotherapeutics. Pro-
cytokines. There were no effects of single or combi-
grammed cell death protein (PD)-1-mediated T cell
nation therapy on virus recovered from tumors, on
suppression via engagement of its ligand, PD-L1, is
myeloid cells populations, or on NK cells. In contrast,
of particular interest due to recent successes in se-
the combination of Seprehvir and anti-PD1 antibody
lected cancers. Oncolytic virotherapy induces tumor
resulted in a more proinflammatory microenviron-
shrinkage via multiple mechanisms including direct
ment characterized by increased CD4+ and CD8+
tumor cell lysis, induction of cytotoxic or apoptosis-
T cells without increased CD4+CD25+Foxp3+ regu-
sensitizing cytokines, and induction of anti-tumor
latory T cells, increased gene expression for T-bet,
T cell responses. We recently demonstrated that
interferon-gamma and iNOS and decreased gene ex-
intratumoral injection of an oncolytic herpes virus
pression for IL-10 and MRC-1, with all changes more
induced T cell mediated growth delays and in some
marked in the more highly immunogenic tumor
cases durable remissions in several immunocompe-
model. Overall, our data suggest the combination of
tent mouse models of rhabdomyosarcoma (Leddon
PD-1 and oncolytic herpes virotherapy may be an ef-
et al., Mol Ther-Oncolytics 1, article number: 14010,
fective treatment strategy for some cancers.
2015). We classified models into high and low immunogenicity based on their differential growth rates
in syngeneic hosts relative to athymic nude mice. In
syngeneic animals, response to single agent Seprehvir (HSV1716), a virus currently in pediatric clinical trials (see www.clinicaltrials.gov: NCT00931931,
NCT02031965), was directly proportional to tumor
immunogenicity, without detectable virus replication. Single agent PD-1 blockade also showed moderate but significant tumor growth delay. Strikingly,
combining these two therapies together substantially prolonged overall survival with several complete
responses lasting beyond 60 days in the most immunogenic model. To investigate the mechanism of
combination, we analyzed the effects of treatment
Local adjuvant treatment of clinical stage I-II melanoma with
CpG-B/GM-CSF improves distant recurrence-free survival:
long-term follow-up of three randomized controlled phase II trials
Koster B.D.1, van den Hout M.F.C.M.1, Sluijter B.J.R.2, Molenkamp B.G.2, Vuylsteke R.J.C.L.M.2, Baars A.1,
van Leeuwen P.A.M.2, Scheper R.J.3, van den Tol M.P.2, van den Eertwegh A.J.M.1, de Gruijl T.D.1
VU University Medical Center, Medical Oncology, Amsterdam, Netherlands,
1
VU University Medical Center, Surgical Oncology, Amsterdam, Netherlands,
2
VU University Medical Center, Pathology, Amsterdam, Netherlands
3
ORAL
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2016
Currently, there is no adjuvant treatment available
p=0.011). The 10-year distant recurrence-free sur-
that has been shown to reduce the chances of re-
vival (DRFS) rate in CpG-B and/or GM-CSF-treated
currence after surgical excision of localized mela-
patients was also significantly higher (94% versus
noma. Between 2001 and 2007 we conducted three
59% p=0.039); of note, all patients who developed
randomized and placebo (saline) controlled phase
distant recurrences eventually succumbed to the
II clinical trials in which we treated clinically stage
disease. We conclude that intradermally adminis-
I-II melanoma patients with 1) GM-CSF (3 µg/kg), 2)
tered CpG-B/GM-CSF is safe and may prolong distant
CpG-B (PF-3512676/CpG7909, 8 mg), and 3) CpG-B,
RFS. Consistent with findings from immune moni-
alone or combined with GM-CSF (2 mg and 100 µg,
toring, prolonged (distant) RFS may result from the
respectively), through 1-4 intradermal injections,
boosting of local and systemic antitumor immunity
directly adjacent to the primary melanoma exci-
through local conditioning of the SLN. These excit-
sion scar, within a week of re-excision and sentinel
ing findings warrant further clinical exploration of
lymph node (SLN) biopsy. Injections were well toler-
local non-toxic immune potentiation in early-stage
ated with only transient and mild flu-like symptoms
melanoma to prevent disease recurrence and meta-
and mild-to-moderate fevers after administration.
static spread.
Primary endpoint of the trials was biological efficacy; immune monitoring revealed recruitment and
activation of dendritic cell subsets in the SLN as well
as increased intra-SLN and systemic rates of melanoma antigen reactive CD8+ T cells. Here, we present
clinical follow-up of patients who were in the treatment arms (treated group n=36) and patients who
received saline (saline group n=28) in these three
trials. Remarkably, in the treated group only two
recurrences occurred whereas ten recurrences were
observed in the saline group after a median follow
up of 89.5 months. This translated into a significantly
higher 10-year recurrence-free survival (RFS) rate in
the treated group (94% versus 48% p=0.0046). Even
for patients with pathologically confirmed stage I-II,
this difference in RFS rate held true (97% versus 52%
IMCgp100 ImmTAC: A TCR-based bi-specific immunotherapy
for the treatment of advanced melanoma
Donnellan Z.1, Bossi G.1, Canestraro M.1, Harper J.1, Dukes J.1, Liddy N.1, Paston S.1, Mahon T.1, Molloy P.1, Sami M.1,
Baston E.1, Cameron B.1, Johnson A.1, Vuidepot A.1, Hassan N.1, Jakobsen B.1
Immunocore Ltd, Abingdon, United Kingdom
1
Tumour specific T cells have the potential to eradicate
program for IMCgp100 in uveal melanoma, which
cancer. However anti-tumour immunity is limited by
will begin in the first quarter of 2016. Immunocore
low TCR affinities, MHC down-regulation by cancer
was accepted on the European Medicines Agency’s
cells and an immunosuppressive tumour microen-
(EMA) Adaptive Pathways pilot programme, and
vironment. As most tumour-associated antigens are
IMCgp100 was recently granted Orphan Drug Desig-
auto-antigens, tumour specific T cells with high af-
nation for the treatment of uveal melanoma.
finity TCRs are likely to be deleted in the thymus.
Here we present in vitro data which shows potent
To overcome these limitations we have developed
IMCgp100 mediated killing of both cutaneous and
novel immune-mobilising monoclonal TCRs against
uveal melanoma. IMCgp100 re-directs T cells from
cancer (ImmTACs). These are soluble bi-functional
late stage cancer patients to target melanoma cells
molecules comprising a high affinity TCR fused to an
even when antigen presentation is low. Target cell
anti-CD3 scFv domain. ImmTACs recognise specific
killing is observed within hours and is specific for
tumour antigens presented by MHC class I and re-
gp100. In addition, killing is associated with the
direct T cells to mediate tumour killing.
release of various pro-inflammatory cytokines and
Our first clinical candidate, IMCgp100, is specific for
chemokines, some of which are reported to play a key
the melanoma-associated antigen gp100. The engi-
role in anti-melanoma responses. In vitro, IMCgp100
neered TCR portion of the drug targets and binds the
mediates faster and more extensive killing of uveal
gp100280-288 epitope, which is overexpressed and pre-
melanoma cells compared to cutaneous melanoma
sented by HLA-A2 on the surface of melanoma cells.
cells. Thus, IMCgp100 demonstrates the potential to
The anti-CD3 scFv portion captures and re-directs T
initiate effective immune responses against cutane-
cells to kill the melanoma cells, while normal antigen
ous and uveal melanoma. Overall these data present
negative cells are not affected. IMCgp100 is currently
ImmTACs as a potent targeted immunotherapy.
in Phase IIa clinical trials in patients with advanced
malignant melanoma. The drug is well tolerated
with evidence of tumour reduction in patients with
metastatic melanoma, including traditionally hard to
treat patients in the uveal melanoma subset. The responses observed in uveal melanoma patients treated
with IMCgp100 prompted further investigations into
the activity of IMCgp100 against uveal melanoma.
Clinical and in vitro data support a full development
Release of IFN-γ induced chemokines provides the key to
efficient combination immunotherapy of anti-PD-1 antibody
with CSF-1R inhibitor
Eissler N.1, Mao Y.2, Johnsen J.I.1, Kiessling R.2, Kogner P.1
Karolinska Institutet, Department of Women´s and Children´s Health, Childhood Cancer Research Unit,
1
Stockholm, Sweden,
Karolinska Institutet, Department of Oncology-Pathology, Stockholm, Sweden
2
ORAL
TALK
SHORT
2016
It is well established that removal of immune sup-
notherapies in the clinic. Combination of checkpoint
pression increases anti-tumor immunity of anti-PD-
blockade antibodies with inhibitors of M-CSF/CSF-1R
1 treatment. We have previously demonstrated that
signaling might increase patients´ response rates,
blockade of PD-1/L1 signaling in combination with
and should therefore be evaluated in clinical trials.
CSF-1R inhibition by BLZ945 (Novartis) leads to superior tumor control in a spontaneous mouse model
of neuroblastoma (TH-MYCN). Microarray analysis of tumor tissues revealed significant increase
of lymphocyte-recruiting chemokines CXCL9, 10
and 11 expressed by myeloid cells, and in line with
these results tumors of combination treated animals
showed increased T-cell infiltration. Blockade of
chemokine receptor CXCR3 hampered T-cell infiltration and abrogated combination treatment efficacy in
vivo. Multivariate analysis of 59 immune cell subsets
in spleens and tumors of treated mice highlighted the
correlation between PD-L1 expressing myeloid cells
and treatment outcome. In vitro, combination of antiPD-1 antibody Nivolumab with BLZ945 increased
proliferation of human T and NK cells significantly
when compared to the single compounds. This effect
was dependent on the presence of myeloid cells in
the culture, and confirmed the observations made
in vivo. We conclude that combination of anti-PD1 antibody with CSF-1R inhibitor leads to efficient
repolarization of suppressive myeloid cells, and in
consequence to chemokine guided effector T-cell
recruitment and anti-tumor activity. These findings
are of direct relevance for the application of immu-
Comparison of phase I/II trials regarding antigen-specific versus
non-specific anticancer immunotherapies
Holland J.1, Zwerver R.1, Grosios N.1, Hoffmans R.1
SMS-oncology, Amsterdam, Netherlands
1
Background:
Antigen-specific
immunotherapy
The route of administration for antigen-non-specific
targets particular tumor associated antigens in
agents was mainly IV compared to antigen-specific
order to address and eradicate solely tumor-marker
(46% vs 13%), whereas for antigen-specific agents it
defined cancer cells. In contrast, antigen-non-spe-
was mainly ID (37% vs 0%). Comparing the choice of
cific agents generally stimulate the immune system
study objectives, it became clear that the numbers of
by for example reversal of immune suppression,
two aspects diverged immensely. In trials with anti-
or activation of innate immunity for a better anti-
gen-specific agents the objectives pharmacokinetics
cancer immune response. We investigated whether
(6% vs 54%) as well as the maximum tolerated dose
these different modes of action are reflected in study
(MTD, 10% vs 46%) were chosen less often compared
designs, objectives and results of Phase I/II studies.
to antigen-non-specific agents. Proportionally, more
Material and methods: A PubMed search for full
antigen-non-specific trials (46% vs 23%) identified
length English articles published from 2010-2014
an optimal dose. From the 34 trials that identified an
was conducted on completed cancer immunotherapy
optimal dose, 85% measured the MTD but only 18%
phase I/II studies. Parameters were extracted and
reached it: 19% of all antigen-non-specific and 1%
entered into a database to compile summary tables.
of all antigen-specific agents. Regarding the clinical
Results: For the stated period we identified 123 full
endpoints antigen-specific agents looked more often
length articles discussing immune-oncology phase
at overall survival in comparison to antigen-non-
I trials. Antigen-specific (79%) agents were investi-
specific agents (48% vs 16%).
gated almost 4 times more than non-specific (21%)
Conclusions: There were differences in patient selec-
agents. On average studies investigating antigen-
tion, objectives and results for studies with antigen-
non-specific agents had a slightly increased sample
specific compared to antigen-non-specific agents.
size (44 vs 29 patients). Moreover, we considered
The main difference between the two groups are
the patient’s selection factors. A significant dif-
the use of single tumor indication (54% vs 6%), bio-
ference in the decision to select single tumor type
marker usage (51% vs 15%), and determining phar-
patients was found between antigen-specific and
macokinetics (6% vs 54%), respectively.
non-specific agents (54% vs 6%). Selection based on
biomarkers (HLA or antigen expression) was used
in 43% of all trials. In trials with antigen-specific
agents, more often biomarkers were used to select
the patient population (51% vs 15%). Mainly latestage patients were used within both agents group.
Reprogramming of TLR7 signaling enhances antitumor NK and
cytotoxic T cell responses
Hotz C.1,2, Treinies M.1, Mottas I.1, Roetzer L.C.3, Oberson A.1, Perdicchio M.1, Spagnuolo L.1, Spinetti T.1, Herbst T.1,
Bourquin C.1
University of Fribourg, Department of Medicine/Chair of Pharmacology, Fribourg, Switzerland,
1
Current Address: BioNTech RNA Pharmaceuticals, Mainz, Germany,
2
LMU Munich, Division of Clinical Pharmacology, Munich, Germany
3
Toll-like receptor (TLR) 7 agonists are effective in
topical application for the immunotherapy of skin
cancers, but their performance for the systemic treatment of solid tumors is limited by the development of
TLR tolerance. In this study we describe a novel strategy to overcome TLR tolerance and enhance TLR7-dependent antitumor immune responses through reprogramming of TLR signaling pathways. The sensitivity
of TLR7 signaling in dendritic cells was increased
by prior stimulation with the dsRNA poly(I:C), that
mimics virally induced immune activation. Timing of
the stimulations was important, as sequential stimulation with poly(I:C) and the TLR7 agonist R848 interspaced by 24 h induced higher MAPK and NFkB
signaling in dendritic cells than the simultaneous application of the same ligands. Dendritic cells activated
by sequential poly(I:C)/R848 stimulation efficiently
induced Th1 differentiation and primed NK-cell and
cytotoxic T-cell responses. We have developed a treatment regimen taking advantage of TLR7 reprogramming that cured over 80% of large tumors in mice
by the action of NK cells and cytotoxic T cells. These
results have direct implications for the use of these
clinically established ligands in the immunotherapy
of cancer.
In vitro and in vivo evaluation of the TLR9 agonist EnanDIM
for cancer immunotherapy
Kapp K.1, Schroff M.1, Wittig B.2, Schmidt M.1
Mologen AG, Berlin, Germany,
1
Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet Berlin, Berlin, Germany
2
Introduction: Preclinical and clinical studies support
Results: EnanDIM581 and EnanDIM532 were chosen
the use of TLR9 agonists as immunomodulators
since they resulted in high secretion of IFN-alpha
showing their anti-tumor effect by enhancing both cel-
and IP-10 from human PBMC and a strong activation
lular and humoral immune responses. Two different
of monocytes, NK cells and plasmacytoid dendritic
families of DNA molecules containing non-methylated
cells (pDC) in vitro. Notably, both showed a distinct
CG-motifs for TLR9 activation have been established:
immune activation pattern, with the highest secre-
Dumbbell-shaped dSLIM® molecules are protected
tion of IFN-alpha by EnanDIM581 and the strongest
against exonucleolytic degradation by their cova-
maturation of TLR9-bearing pDC by EnanDIM532.
lently-closed, natural phosphodiester (PO) backbone.
EnanDIM744, comprising EnanDIM581 with addi-
In contrast, single-stranded, oligodeoxynucleotides
tional 5’-end L-nucleotide protection and exhibiting
(CpG-ODN) are most commonly chemically-stabilized
a pattern similar to EnanDIM581, was selected as
by phosphorothioates (PTO) in their phosphate moi-
third EnanDIM® for in vivo studies. In the maximum
eties. However, PTO modifications produce off-target
feasible dose approach, safety assessments were
effects in immune cell populations and have resulted
performed throughout the study period and no mor-
in an unfavorable risk-to-benefit ratio.
tality, clinical signs and body weight changes were
Methods: Linear single-stranded ODN were synthe-
observed, despite the use of extremely high doses of
sized using L-deoxyribonucleotides at their 3’-ends,
app. 300 to 1700 mg/kg. A gross necropsy consisting
which are the natural enantiomers of D-deoxyribonu-
of macroscopic organ evaluation at day 15 revealed
cleotides, to avoid the off-target effects of PTO-modi-
also no toxicity. Regarding immune activation, in-
fied CpG-ODN. D-deoxyribose form the vast majority
creased levels of IP-10 in serum were observed 24
of deoxyribose in present organisms, thus co-evolved
h after injection but not at day 15 confirming that
nucleases are blind for L-deoxyribose, leaving L-pro-
L-nucleotides in EnanDIM® do not change the kinetic
tected ODN intact. We selected nucleotide sequenc-
profile known from other DNA-based TLR9 agonists.
es of such L-protected, CG-motif containing, sin-
Conclusions: EnanDIM®, a new family of TLR9 ago-
gle-stranded ODN, EnanDIM®, for high secretion of
nists, broadly activates the immune system in vitro.
IFN-alpha and IP-10 from human peripheral blood
Maximal feasible doses of EnanDIM® resulted in no
mononuclear cells (PBMC). In a maximum feasible
signs of toxicity and confirmed immunomodulatory
dose approach in CD-1 mice EnanDIM® doses of 10
effects in vivo. Thus EnanDIM® has the potential for
to 50 mg per mice were injected s.c. to evaluate the
clinical development in the treatment of cancer.
acute toxicity and immunomodulatory properties of
EnanDIM® molecules in vivo.
Platelet-derived microparticles differentially regulates
macrophage polarization
Kayali E.S.1, Gursel I.1
Ihsan Dogramaci Bilkent University, Molecular Biology and Genetics Department, Ankara, Turkey
1
Introduction: Platelet-derived microparticles (PMPs)
analyses by flow cytometer and qPCR analysis of
shed from platelets upon activation and constitute
differentially expressed genes.
almost 90% of the circulating MPs. Due to their
Results: Activated THP1 macrophages efficiently
growth factor and nucleic acid containing cargo,
and in a time-dependent manner binds and internal-
PMPs were associated with the generation of immu-
izes PMPs. Co-incubation of increasing amounts of
nosuppressive micro-environment and may promote
PMP induced secretion of higher anti-inflammatory
tumor growth. However, they are also potential can-
cytokine IL10 from both M1 and M2-polarized mac-
didates for prevention and treatment of autoimmune
rophages. Also, Pam3CSK4 loaded PMPs generated a
diseases. We hypothesized that PMPs can favor al-
more pronounced immune-regulatory environment
ternative M2 macrophage polarization. Additionally,
in comparison with the empty PMPs.
coupling PMPs with TLR1/2 agonist Pam3CSK4 that
Conclusion: Our findings point in the direction that
was shown to induce M2-like macrophage polariza-
PMP treatment of macrophages loaded with suitable
tion from human monocytic myeloid-derived sup-
ligand combinations might regulate M1/M2 type
pressor cells would enhance the immuno-regulatory
macrophage differentiation and efficiently harnessed
environment.
either to control tumor development (M1) or to alle-
Methods: Whole blood was collected from healthy
viate symptoms of auto-immune/auto-inflammatory
donors by venipuncture into Vacutainer tubes con-
diseases (M2).
taining EDTA and platelet isolation was completed
within 1h of blood collection. Platelet-rich plasma and
subsequently, platelets were obtained by differential
centrifugation. PMPs from TRAP6-activated platelets were collected with ultracentrifugation. CD41,
CD63 and Annexin-V were used to assess purity of
the platelets and PMPs. PMPs alone or loaded with
TLR1/2 agonist Pam3CSK4 were co-incubated with
PMA-activated THP1 macrophages together with
M1 and M2 macrophage polarization supplements.
IFNγ and LPS were used for M1 and IL4 was added
to promote M2 differentiation. M1-M2 macrophage
polarization was assessed with cytokine ELISA, intracellular cytokine staining, cell surface marker
Influence of radiofrequency thermal ablation on CD4+ T cell
subsets in the patients with liver cancer
Kikodze N.1, Iobadze M.2, Pantsulaia I.1, Nanava N.1, Lemonjava M.2, Chigvinidze N.2, Mizandari M.3,
Janikashvili N.1, Chikovani T.1
Tbilisi State Medical University, Department of Immunology, Tbilisi, Georgia,
1
Tbilisi State Medical University, Institute of Medical Biotechnology, Tbilisi, Georgia,
2
Tbilisi State Medical University, Department of Interventional Radiology, Tbilisi, Georgia
3
The purpose of immunotherapy is to expose tumor
10-fold increase of CD4low+HLA-DR+ T cells (effector/
under the influence of immune surveillance. The side
activated) compared to control - result of ongoing
effect of relatively new interstitial therapy - radiofre-
anti-tumor immune response. RFA procedure and
quency thermal ablation (RFA) is regarded as wide
surgery did not significantly change the frequencies
spectrum tumor antigen release followed by tumor-
of CD4+CD45RO+ and CD4+HLA-DR+ T cells. The
specific immune response.
percentage of CD39+cells was doubled within the
We aimed to investigate a short-term impact of RFA
patients’ CD4+ T cells. This increase was strongly
on the frequency of different phenotypes of CD4+ T
associated with both - CD4low and CD4high popula-
cells in the peripheral blood of primary and meta-
tions of patients´ T cells. Of note, in both groups,
static liver cancer patients. Our results demonstrate
patients and controls, CD39 expression was largely
that the frequency of total circulating CD4+ T lym-
overlapped with CD4low subset, comprising the acti-
phocytes was comparable between the patients and
vated compartment of this population. RFA treatment
controls. CD4high+ /CD4low+ balance was slightly
favored to the significant reduction the percentage of
. The percentages of CD45RA ,
CD39+CD4+ cells in the total CD4+ T cells, although
CD45RO+, HLA-DR+, CD39+ cells were separately
no changes were observed in the group of patients
quantified within CD4low and CD4high populations.
with surgical resection.
shifted toward CD4
low+
+
+
CD45RA (naïve) cells were significantly reduced in
We suppose that one of the important effect of RFA
the cancer patients. This decrease was strongly as-
on liver tumor relies on the destructive impact of
low
populations
RFA on CD4+CD39+ T cells responsible for genera-
of patients´ T cells. Neither RFA treatment nor liver
tion of adenosine, which results in instant inhibition
resection caused any changes of this parameter.
of immune response, recruitment of Treg cells to the
CD45RO expression was largely overlapped with
tumor site and establishment of immunosuppressive
sociated with both - CD4
low
subsets, comprising activat-
environment. Herein, we describe that RFA exerted
ed and non-activated compartments of T cells. The
the immune modulatory effects on distinct subsets
both CD4
and CD4
high
and CD4
high
CD45RO cells was decreased
of circulating CD4+ T cells in liver cancer patients.
1.5 times in patients compared to control. Whereas
Further investigations are intended to clarify the
the percentage of CD4high+CD45RO+ cells was sig-
long-term advantages of immunomodulatory RFA
nificantly elevated, that may indicate the presence
procedure, in comparison with the surgical interven-
of specific memory anti-tumor T cells in patients.
tion, in the patients with liver cancer.
frequency of CD4
low+
+
HLA-DR marker expression was largely overlapped
with activated CD4low+ compartment.There was
Memory-like T cells transduced with tumor-specific epitope
elicited pronounced cytotoxic potential
Koksal H.1, Gursel I.1
Ihsan Dogramaci Bilkent University, Molecular Biology and Genetics Department, Ankara, Turkey
1
Introduction: Antigen specific adoptive T cell therapy
ligand addition promoted the generation of effector
is one of the effective strategies to induce anti-tumor
T-cells rather than supporting memory like CD8+
immunity. CD8+ T cells undergo homeostatic prolif-
T-cell phenotype differentiation.
eration and gain central memory phenotype under
Conclusion: Currently the in vivo therapeutic capac-
lymphophenic conditions. Recently, it was shown
ity of these T-cells is being tested on B16-OVA bearing
that lymphophenic conditions can be mimicked in
mice. Preliminary findings implicated that in vitro
vitro by the utilization of IL-15. Our main goal was
generated and OT-I transduced memory-like CD8+T
to generate antigen specific memory-like T cells in
cells were indeed displayed an antigen specific cyto-
culture and enhance the breadth of traditional adop-
toxic potential much stronger than conventional ef-
tive T cell therapy by inducing more pronounced
fector T-cells activated by CD3/28 antibodies and DC
anti-tumor effect.
conditioned media.
Methods: Dendritic cells (DC) were generated from
mouse bone marrow (BM) in GM-CSF and IL-4 supplemented culture media for 7 days. DCs were activated with CpG ODN, LPS and cGAMP to induce IL-15
secretion. Mouse spleen CD8+T-cells were purified
via MACS and subsequently were activated with CD3/
CD28 antibodies in the presence of DC conditioned
media. Phenotypes of CD8+T cells were investigated
by flow cytometry. Generated central-memory like
CD8+T cells were then transduced with ecotropic
OT-I TCR retrovirus and their in vitro antigen specific
lytic capacities were tested on B16-OVA expressing
cells.
Results: To our surprise, results revealed that any
type of inflammation signal induced by TLR or STING
ligands drastically reduced the memory phenotype
in vitro. Moreover, previously proposed approach
using anti-CD3/CD28 plus IL-15 system is no superior than only IL-15 supplementation in the absence
of activation inducing antibody couple. Strikingly,
Functionality of tumor-infiltrating T cells in hepatocellular
carcinoma can be enhanced by blocking several co-inhibitory
pathways
Zhou G.1, Sprengers D.1, Boor P.1, Doukas M.2, Polak W.3, de Jonge J.3, Thielemans K.4, IJzermans J.3, Bruno M.1,
Kwekkeboom J.1
1
2
3
Vrije Universiteit, Laboratory of Molecular and Cellular Therapy, Department of
4
Immunology-Physiology, Brussels, Belgium
ORAL
TALK
SHORT
2016
Targeting immune checkpoint co-inhibitory path-
granzyme B, and neither displayed increased effec-
ways has been shown to be a promising novel
tor cytokine production upon polyclonal stimulation,
therapeutic approach for several types of cancer.
suggesting restricted functionality. Blocking PD-L1,
Hepatocellular carcinoma (HCC) is highly resistant
TIM-3, or LAG-3 with neutralizing antibodies in-
to chemotherapy, and is the second most common
creased ex vivo proliferation of CD8+ and CD4+ TIL
cause of cancer-related death in the world. To de-
to polyclonal stimuli and to Glypican-3 or MAGE-C2
termine whether co-inhibitory pathways contribute
presented by mRNA-transfected autologous anti-
to intra-tumoral suppression of T cell responses in
gen-presenting cells, and also increased IFN-γ and
HCC, we used paired samples of leukocytes freshly
TNF-α production of CD8+ TIL in polyclonal and
isolated from resected liver tumors (TIL), tumor-free
HCC TAA-specific peptide stimulation assays. Com-
liver tissues (TFL), and peripheral blood of patients
bining PD-L1 blockade with TIM-3 or LAG-3 block-
with HCC. Expression of co-inhibitory molecules on
ade further enhanced these effects.
T cells was then measured by flow cytometry.
Conclusions: PD-1, TIM-3 and LAG-3 are up-regulat-
We found that expression of PD-1, TIM-3 and LAG-3
ed on tumor-infiltrating TAA-specific T cells in HCC
on CD8+ cytotoxic T cells, and expression of PD-1,
patients. Blocking these co-inhibitory molecules en-
TIM-3 and CTLA-4 on CD4+Foxp3- T helper cells
hances the functionality of tumor-infiltrating CD4+
were significantly higher in TIL than in TFL or in
and CD8+ T cells, while combined blocking shows
the blood. In contrast, no up-regulation of BTLA on
additive effects. Therefore, these three co-inhibitory
T cells in TIL was observed. Using MHC class I dex-
pathways may be promising immunotherapeutic
tramers loaded with immunogenic peptides derived
targets for the most prevalent type of primary liver
from Glypican-3 or MAGE-C2, which are both tu-
cancer.
mor-associated antigens (TAA) that are expressed in
HCC tumors, we found that the majority of TAA-specific CD8+ TIL expressed PD-1, TIM-3 and LAG-3.
In addition, their ligands PD-L1, Galectin-9, MHC-II,
CD80 and CD86 were expressed on dendritic cells,
monocytes and B cells in the tumors. Compared to
the cells without expression, CD8+ and CD4+Foxp3TIL expressing those co-inhibitory receptors displayed a more activated status (HLA-DR+, CD69+).
However, they did not show enhanced expression of
Blocking PD-L1 and LAG-3 can revitalize the functionality
of tumor-infiltrating T cells in liver metastasis from colorectal
cancer
Zhou G.1, Sprengers D.1, Boor P.1, Doukas M.2, Grünhagen D.3, Verhoef C.3, Bruno M.1, Kwekkeboom J.1
1
2
Erasmus MC, Surgery, Rotterdam, Netherlands
3
Targeting co-inhibitory pathways has been shown
status. However, they did not show enhanced expres-
to be a promising novel therapeutic approach for
sion of granzyme B or enhanced cytokine production
several types of cancer, but so far not in colorectal
upon polyclonal stimulation, suggesting restricted
cancer (CRC). This lack of success is probably related
functionality. Blocking PD-L1 and LAG-3 with neu-
to the low levels of co-inhibitory molecule expres-
tralizing antibodies increased ex vivo proliferation
sion on tumor-infiltrating lymphocytes (TIL) in the
and effector cytokine production of CD8+ and CD4+
majority of primary CRC tissues. Liver metastasis
TIL to polyclonal stimuli.
(LM) is a leading cause of CRC-related mortality. LM
Conclusions: PD-1, TIM-3, LAG-3 and CTLA-4 are up-
are present in 15-20% of patients at diagnosis and
regulated on tumor-infiltrating T cells in liver me-
develop in another 60% of patients during the course
tastasis from colorectal cancer, and blocking PD-L1
of the disease. Therefore, our aim was to determine
and LAG-3 can re-vitalize the functionality of tumor-
whether co-inhibitory pathways participate in intra-
infiltrating T cells. Therefore, these two co-inhibitory
tumoral suppression of T cell responses in LM from
molecules may be promising immunotherapeutic
CRC. For this purpose, we used paired samples of
targets for the most prevalent type of secondary liver
leukocytes freshly isolated from resected LM-CRC
cancer.
tissues, tumor-free liver tissues (TFL) and peripheral
blood of patients with LM-CRC. Expression of coinhibitory molecules on T cells was then measured
by flow cytometry.
We found that expression of PD-1, TIM-3 and LAG-3
on CD8+ cytotoxic T cells, and expression of PD-1,
TIM-3 and CTLA-4 on CD4+Foxp3- T helper cells
were significantly higher in tumor than in TFL or
in the blood. In contrast, no up-regulation of BTLA
on TIL was observed. In addition, their ligands
PD-L1, Galectin-9, MHC-II, CD80 and CD86 were expressed on dendritic cells, monocytes and B cells in
the tumors. Compared to the cells without expression, CD8+ and CD4+Foxp3- TIL expressing those
co-inhibitory molecules expressed higher levels
of HLA-DR and CD69, indicating a more activated
Probing the increase in neoantigen burden at recurrence in
ovarian cancer
O’Donnell T.1, Snyder A.2, Ahuja A.1, Hammerbacher J.1
Mount Sinai School of Medicine, Genetics and Genomic Sciences, New York, United States,
1
Memorial Sloan Kettering Cancer Center, New York, United States
2
Somatic mutation burden correlates with response
posure in C. Elegans [Meier 2014]. While some trinu-
to checkpoint blockade immunotherapy [Van Allen
cleotide contexts showed moderate enrichment in the
2015], raising the question of how it is affected by
recurrences (such as C>G in 5’ G and 3’ C context,
conventional chemotherapy. We explored changes in
especially for patients receiving cyclophosphamide),
mutation burden in paired primary and chemother-
overall, the signatures active in the recurrences were
apy-treated recurrence samples in 12 ovarian cancer
similar to those of the primaries. In particular, the
patients from the Australian Ovarian Cancer Study
cisplatin signature was not significantly enriched in
[Patch 2015] who received combination paclitaxel/
the treated samples.
carboplatin and other therapies. While the recurrence samples had significantly more mutations than
References:
the matched primary samples, our results suggest
Van Allen, … Garraway, L. A. (2015). Genomic correlates
of response to CTLA-4 blockade in metastatic melanoma.
Science, 350(6257), 207-211.
the new mutations are largely attributable to the
same mutational processes present in the primary
samples, not direct mutagenic impact of the platinum
therapy.
In a Bayesian multiple regression model, recurrence
was associated with a 35% (95% CI=32-37) increase
in mutations over matched untreated samples. Primaries and recurrences had a mean of 50 and 73
mutations predicted to bind MHC I (possible T cell
neoantigens), respectively, a model-adjusted increase
of 36% (CI=11-67). The new mutations were found
at typical allele frequencies, i.e. were not unusually
sub-clonal.
To assess if therapy is associated with a change in
mutational process, we deconvolved each sample’s
mutations into known signatures based on trinucleotide context [Alexandrov 2013]. We used the 30
signatures curated by COSMIC (http://cancer.sanger.
ac.uk/cosmic/signatures) plus an additional signature from substitutions associated with cisplatin ex-
Patch, A.-M., … Bowtell, D. D. L. (2015). Whole-genome
characterization of chemoresistant ovarian cancer. Nature,
521(7553), 489-494.
Alexandrov, … Stratton, M. R. (2013). Signatures of mutational processes in human cancer. Nature, 500(7463), 415-21.
Meier, B., … Campbell, P. J. (2014). C. elegans whole-genome
sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Research, 24(10),
1624-36.
TNFa and IL-2 armed oncolytic adenovirus induces antitumor
immune response and protects from tumor rechallenge in
Syrian hamsters
Havunen R.1, Siurala M.1,2, Parviainen S.1,2, Behr M.1, Nettelbeck D.3, Ehrhardt A.4, Hemminki A.1,2
University of Helsinki, Cancer Gene Therapy Group, Medicum, Faculty of Medicine, Helsinki, Finland,
1
TILT Biotherapeutics Ltd, Helsinki, Finland,
2
3
University Witten/Herdecke, Institute for Virology and Microbiology, Witten, Germany
4
In the last few years immunotherapy has become
hamsters (Mesocricetus auratus) were treated with
an important part of treating many types of cancer.
Ad5/3-E2F-d24 virus bearing human IL-2, TNFa, or
With regard to oncolytic immunotherapy using repli-
both transgenes. Hamster pancreatic cancer (HapT1)
cation competent viruses, the 2015 approval of T-Vec
was treated with five viral injections. In addition,
(Imlygic) is paving the way toward routine use. T
TILs were extracted from syngenic tumors, expand-
cell therapy is known to benefit a portion of mel-
ed ex vivo and administered as a single injection
anoma and leukemia patients, however the immu-
intratumorally. We saw synergy between unarmed
nosuppressive tumor environment can inhibit the
virus and TILs, while the armed viruses turned out
recruitment and activation of transferred T cells. We
to be even more effective. When the cured animals
aim to use cytokine-bearing oncolytic adenoviruses
were rechallenged with the same cancer cells, pre-
to improve and to extend the usage of adoptive T
vious treatment with cytokine-armed viruses pro-
cell therapy. Administered systemically or intratu-
tected the animals against new tumors. In addition,
morally the virus induces immune responses against
splenocytes derived from animals treated with cy-
the tumor by releasing tumor antigens in the pres-
tokine-bearing viruses proliferated more actively ex
ence of danger signals, while arming the virus with
vivo than the controls. To conclude, these findings
immunostimulatory cytokines augments the effect
support development of clinical trials where T-cell
further. Previously, we have identified interleukin 2
therapy is enhanced with oncolytic adenovirus. We
(IL-2) and Tumor Necrosis Factor alpha (TNFα) as the
believe this approach can enable effective and safe
most promising factors to stimulate the graft used in
T-cell therapy of solid tumors.
adoptive T-cell therapy. IL-2 is a common treatment
for malignant melanoma and renal cell carcinoma
and it has a key role in recruiting and activating T
cells. TNFa has prominent anti-immunosuppressive
actions and it directly promotes tumor cell death by
apoptosis and necrosis. Notably, these cytokines can
cause severe side effects when administered systemically, but armed oncolytic viruses accomplish safe,
local and long-lasting, high-level cytokine expression locally in the tumor.
In our preclinical studies we have shown that adenoviruses enhance adoptive T cell therapy. Syrian
The first trials sponsored by TILT Biotherapeutics is in development.
The oncolytic peptide LTX-315 enhances T cell clonality and
induces synergy with CTLA-4 blockade
Rekdal O.1, Camilio K.1, Yusko E.2, Vignali M.2, Sanders C.2, Benzeno S.2, Nestvold J.3, Saunders A.1, Yamasaki T.4,
Zitvogel L.4, Sveinbjörnsson B.1,5
Lytix Biopharma, Oslo, Norway,
1
Adaptive Biotech, Seattle, United States,
2
University of Oslo, Oslo, Norway,
3
Gustave Roussy Cancer Campus, Villejuif, France,
4
University of Tromso, Tromso, Norway
5
LTX-315, a novel oncolytic peptide is effective against
both drug-resistant and drug sensitive cancer cells
with significant lower toxicity towards normal
cells. Intratumoral treatment with LTX-315 results
in growth inhibition, complete regression and long
lasting tumour specific immune response. The oncolytic effect of LTX-315 involves perturbation of the
plasma membrane and distortion of the mitochondrial membrane with subsequent release of DAMPs
(Damage-Associated Molecular Pattern molecules)
such as ATP, cytochrome C and HMGB1. Multi-domain proteins from the BCL-2 family seem to be partially involved in LTX-315 mediated killing.LTX-315
effectively disintegrates cytoplasmic organelles with
subsequent release of tumour antigens as demonstrated by a greater increase in TIL infiltration, TIL
clonality, and the number of clones with greater
abundance in the tumor microenvironment.
LTX-315`s ability to increase T cell infiltration and
clonality makes it ideal as a combination partner for
other immunotherapies, including immune-checkpoint inhibitors.
In preclinical tumour models, combination of
LTX-315 and immune checkpoint inhibitors (antiCTLA4) demonstrate significant synergy. LTX-315
combined with immune checkpoint inhibitors in a
clinical setting are under planning
Hexavalent agonists targeting receptors of the tumor necrosis
factor superfamily: TRAIL, CD40L, CD27L and beyond
Richards D.1, Gieffers C.1, Marschall V.1, Merz C.1, Schnyder T.1, Sykora J.1, Thiemann M.1, Fricke H.1, Hill O.1
Apogenix AG, Heidelberg, Germany
1
ORAL
TALK
SHORT
2016
Tumor necrosis factor receptor superfamily (TNFRSF)
The described engineering concept has been success-
proteins are of great importance in the anti-tumor
fully translated to TRAIL, CD40L, LIGHT and CD27L
immune process. They are widely expressed by
resulting in hexavalent agonists suitable for further
immune and tumor cells highlighting their impor-
development. Expression of the drug candidates in
tance in many locations and phases of the anti-tumor
CHO suspension cells followed by a lab-scale puri-
immune response. Importantly, signaling through
fication process including affinity chromatography
many TNFRSF members, such as CD40, CD27, OX40,
and SEC-based polishing, resulted in homogenous
HVEM, GITR and 4-1BB, results in co-stimulation.
aggregate-free protein lots. The purified proteins bind
Apogenix is currently developing a novel class of TN-
their respective target-receptors with high affinity. In
FRSF-agonists for the treatment of cancer. These ag-
vivo stability/PK studies have been performed in ad-
onists are molecular mimics of cytokines belonging
dition to in vitro experiments with primary human
to the tumor necrosis factor superfamily (TNFSF).
and mouse lymphoid and myeloid cell populations.
Unlike their natural counterparts, the Apogenix re-
Specifically, scCD27L-RBD-Fc was able to bind CD27
combinant TNFSF proteins consist of one single poly-
expressed on primary human CD4+ and CD8+ T
peptide chain composed of three receptor-binding
cells. Importantly, binding significantly increased
domain-forming subsequences (protomers). These
T cell expansion following activation. Results with
single-chain TNFSF receptor-binding domains (scT-
scCD40L-RBD-Fc showed that treatment induced dif-
NFSF-RBD) preserve the three-dimensional organi-
ferentiation of B cells, enhanced monocyte differen-
zation of the trimeric natural TNF-SF cytokine and
tiation into DCs or M1 macrophages and upregulated
can be used to engineer fully human fusion-proteins
activation markers, thus potentially increasing their
in a modular manner. For example, fusing an IgG1
antigen presentation capabilities.
Fc-domain as a dimerization scaffold to the C-termi-
In light of the promising results obtained with TRAIL,
nus of a scTNFSF-RBD creates a hexavalent agonist
CD40L, LIGHT and CD27L, Apogenix is currently ex-
from two trivalent scTNFSF-RBDs. As a result of
panding the TNFRSF-agonist pipeline to target ad-
this molecular design, each molecule is capable of
ditional cell populations, locations and phases of the
clustering six receptors in a spatially well-defined
immune response in order to develop novel therapies
manner in close proximity to each other. Therefore,
to treat cancer and other conditions.
TNFRSF signaling following treatment with the
Apogenix scTNFSF-RBD-Fc in vivo is independent of
secondary clustering through Fc-γ receptors that is
required for many agonistic anti-TNFRSF antibodies
(e.g., anti-TRAIL-R2 or anti-CD40).
Immunological, anti-angiogenic and clinical effects of
intratumoral interleukin-12 electrogene therapy plus
metronomic cyclophosphamide in dogs with spontaneous cancer
Cicchelero L.1, Denies S.1, Vanderperren K.2, Stock E.2, Van Brantegem L.3, de Rooster H.4, Sanders N.N.1
Ghent University, Laboratory of Gene Therapy, Faculty of Veterinary Medicine, Merelbeke, Belgium,
1
Ghent University, Department of Medical Imaging of Domestic Animals, Faculty of Veterinary Medicine,
2
Merelbeke, Belgium,
Ghent University, Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine,
3
Merelbeke, Belgium,
Ghent University, Department of Medicine and Clinical Biology of Small Animals, Faculty of Veterinary
4
Medicine, Merelbeke, Belgium
In this work the immunological, anti-angiogenetic
giogenic effects of IL-12 gene therapy in all dogs we
and clinical effects of metronomic cyclophospha-
also noticed in almost all dogs clinically relevant im-
mide and 3 consecutive intratumoral interleukin-12
provements in quality-of-life and weight. However,
gene therapy (IL-12 EGT) treatments were evaluated
tumor regression could not be obtained. The labo-
in 6 dogs with spontaneous cancer. A total of 3 tumor
ratory and ultrasound results hold great promise for
biopsies (day 1, 15, 35) and 5 blood samples (day 1,
combinatorial strategies of IL-12 gene therapy and
3, 8, 15, 35) were taken prior, during and after in-
metronomic chemotherapy with conventional anti-
tratumoral IL-12 EGT. In all dogs an initial decrease
tumor (immuno)therapies.
in blood immune cells was followed by an increase
1-3 weeks after treatment initiation. Interestingly,
the decrease in peripheral leukocytes 2 days after
IL-12 EGT coincided with erythema and swelling
of the tumor. In the tumor a transient increase in
IL-12 levels were measured, whereas a permanent
increase in interferon gamma (IFN-γ) and thrombospondin 1 (TSP-1) were determined in contrast to a
permanent decrease in vascular endothelial growth
factor (VEGF). In the serum a transient increase in
IL-12 and interleukin-10 (IL-10) levels were noted in
contrast to a transient decrease in VEGF and TSP-1.
There were no changes in intratumoral IL-10 levels or
serum IFN-γ levels. Microbubble contrast-enhanced
ultrasound (CEUS) was performed prior, during and
3 weeks after the last intratumoral IL-12 EGT treatment to establish the anti-angiogenic effect of IL-12
gene therapy. The treatment resulted in a significant
decrease of tumor relative blood volume and blood
flow speed. All primary tumors continued to progress in time, but the CEUS indicated that this progression was slower than before treatment. Besides
the encouraging immunostimulatory and anti-an-
Combinatorial approaches with costimulatory antibody fusion
proteins addressing immunosuppression by IL-10, TGF-beta and
immune checkpoints
Sapski S.1, Kontermann R.1, Müller D.1
University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany
1
Cancer progression is associated with the develop-
memory cells. On CD8+ T cell subpopulations the
ment of mechanisms that lead to an immunosup-
effect of 4-1BBL costimulation was predominant on
pressive microenvironment, hampering an effec-
memory and effector cells. We could show that the
tive immune response. Thus, the success of cancer
presence of IL-10 and TGF-beta inhibited the bispecif-
immunotherapeutic strategies depends strongly on
ic antibody-mediated stimulation of PBMCs, but this
their capacity to overcome such immune suppress-
effect could be partially prevented by costimulation,
ing conditions. Based on a combinatorial approach
especially with 4-1BBL, where also additional com-
with a bispecific antibody and diverse costimulatory
bination with OX40L or B7.1 could further improve
antibody fusion proteins we have investigated on one
the outcome. These two costimulatory combina-
hand the potential of costimulation to counteract the
tions showed to be very effective in supporting the
immunosuppressive effect mediated by IL-10 and
bispecific antibody-mediated stimulation of T cells,
TGF-beta and on the other hand the combinatorial
despite the high expression of PD-L1 on the tumor
effect with checkpoint inhibitors. In our setting, a
cell line in our setting. Furthermore, the application
bispecific antibody directed against a tumor associ-
of checkpoint inhibitors blocking PD-1 and CTLA-4
ated antigen and CD3 was used to retarget T cells
clearly enhanced the stimulatory effect achieved by
to tumor cells in a MHC-independent manner, trig-
the fusion protein combinations, pointing to further
gering effector cell activation. Considering the im-
immunomodulatory options. Thus, combinatorial
portant role of costimulation in the activation and
approaches involving costimulatory antibody fusion
modulation of T cell response, we combined the
proteins show promise to cope with tumor immu-
bispecific antibody with tumor-directed antibody
nossuppressive factors that warrants further explo-
fusion proteins with costimulatory ligands of the
ration.
B7- ( B7.1) and TNF-superfamily (4-1BBL, OX40L,
LIGHT). In our in vitro setting CD4+ and CD8+ T
cells of naive, memory (CM/EM) and effector phenotype were activated by the bispecific antibody and
proliferation could be further enhanced to diverse
degree by the addition of the costimulatory antibody fusion proteins. Costimulation via OX40L and
B7.1 resulted particularly effective on CD4+ T cell
subpopulations, where additional combination with
4-1BBL could further enhance the effect on naive and
Modulation of T cell recruitment into tumors through synergy
between HMGB1 and CXCL12
Spagnuolo L.1, Boss N.1, Hotz C.1, Oberson A.1, Uguccioni M.2, Bourquin C.1
University of Fribourg, Fribourg, Switzerland,
1
Institute for Research in Biomedicine, Bellinzona, Switzerland
2
An essential step to improve the efficacy of T-cell
Collectively, the results obtained from these experi-
based immunotherapy of cancer is to understand the
ments deliver novel information on the mechanisms
mechanisms that govern the migration of T cells into
that control lymphocyte migration and the recruit-
tumors. In this regard, the combined action of dif-
ment of T cells into tumors, and may provide valua-
ferent chemokines that play a role in the attraction
ble tools to improve T-cell-based treatment of cancer.
and recruitment of T cells is an interesting feature
still not well characterized. It has been reported
that the pro-inflammatory molecule HMGB1 and the
chemokine CXCL12, two molecules that are highly
expressed in different types of tumors, can interact
to promote migration of monocytes and fibroblasts.
In line with this, we are examining whether this interaction can also influence the migration pattern of
lymphocytes and whether it can be pharmacologically targeted to enhance the infiltration of T cells
into tumors. We have observed that HMGB1 increases the in vitro migration towards CXCL12 not only of
myeloid cells, but also of T and B lymphocytes. The
increase in lymphocyte migration can be blocked
by glycyrrhizin, a specific HMGB1 inhibitor, which
has been shown to inhibit also the activity of the
HMGB1/CXCL12 complex. Preliminary observations
suggest that glycyrrhizin can also modulate in vivo
the CXCR4-dependent migration of lymphocytes.
Efficacy of a novel multi-drug metronomic chemotherapy
combined with a peptide vaccine on tumor challenge in mice
Tagliamonte M.1, Petrizzo A.1, Luciano A.1, Rea D.1, Barbieri A.1, Arra C.1, Maiolino P.1, Tornesello M.L.1,
Ciliberto G.1, Buonaguro F.M.1, Buonaguro L.1
Istituto Nazionale per lo Studio e la Cura dei Tumori, ‘Fondazione Pascale’, Naples, Italy
1
Background: the tumor immunosuppressive micro-
chemotherapy evaluated in the present study was
environment represents a major obstacle to an effec-
very effective in counterbalancing the immunosup-
tive tumor-specific cellular immune response.
pressive tumor microenvironment. Consequently,
Method: in the present study, the counterbalance
the intrinsic anti-tumor T cell immunity could
effect of a novel metronomic chemotherapy protocol
exert its function, targeting specific TAA and sig-
on such an immunosuppressive microenvironment
nificantly containing tumor growth. The combina-
was evaluated in a mouse model upon sub-cutane-
tion with a peptide vaccine further amplified such
ous ectopic implantation of B16 melanoma cells. The
an effect. Overall, the results show that the newly
chemotherapy consisted of a novel multi-drug cock-
designed metronomic chemotherapy scheme repre-
tail including taxanes and alkylating agents, admin-
sents a promising adjuvant approach to significantly
istered in a daily metronomic fashion. A multi-pep-
enhance efficacy of intrinsic or vaccine-elicited tu-
tide vaccine was combined to assess improvement in
mor-specific cellular immunity.
immune response and containment of tumor growth.
Results: The newly designed strategy was shown to
be safe, well tolerated and significantly efficacious.
Animals treated with metronomic chemotherapy
showed a remarkable delay in tumor growth and prolonged survival as compared to control group. This
was associated with significant reduction in Tregs
and an intrinsic CD8+ T cell response specific to
B16 naturally expressed Trp2 TAA. The combination
with a multi-peptide vaccine significantly enhanced
the effect. Combinatorial treatment induced a CD4+
T cell reduction and CD8+ T cell increase in the peripheral blood. Furthermore, a significant increase in
TILs associated with reduction in the percentage of
suppressive cells was observed in the tumor lesions.
Finally, the metronomic chemotherapy induced a
significant enhancement of immune response to
vaccine peptides.
Conclusion: The novel multi-drug daily metronomic
T-cell therapy enabling adenoviruses coding for IL-2 and TNF-a
systematically activate tumor-reactive TILs in metastatic, solid
cancer
Tähtinen S.1, Blattner C.2, Vähä-Koskela M.1, Saha D.1, Siurala M.1,3, Rusanen J.1, Parviainen S.1,3, Utikal J.2,
Umansky V.2, Hemminki A.1,3
University of Helsinki, Department of Pathology, Helsinki, Finland,
1
German Cancer Research Center (DKFZ), Clinical Cooperation Unit Dermato-Oncology (G300), Heidelberg,
2
Germany,
TILT Biotherapeutics Ltd, Helsinki, Finland
3
Adoptive T cell therapy (ACT) using genetically mod-
pressor cells (MDSCs) or T regulatory cells (Tregs),
ified T cells has shown exceptional efficacy in the
latter of which has previously been associated with
treatment of CD19+ hematological cancers. However,
systemic IL-2 therapy (Ahmadzadeh and Rosenberg,
far less impressive results have been achieved in the
Blood 2006). Instead, Ad5-IL2 treatment induced
treatment of solid tumors due to local immunosup-
upregulation of IL-2 receptor alpha-chain (CD25) in
pression, rendering tumor-infiltrating T cells (TILs)
conventional CD4+CD25+Foxp3- cells, suggesting
hypofunctional. We have previously shown that ad-
that these helper T cells contributed to CD8+ TIL
enovirus (Ad) infection can enhance the efficacy of
activation. Finally, beneficial ratios between tumor-
ACT (Tähtinen et al, CIR 2015) and that intratumoral
reactive PD-1+ CD8+ TILs and Tregs was observed
administration of immunostimulatory cytokines can
in primary and secondary tumor sites, indicating
result in favorable alteration of tumor microenviron-
that IL-2 and TNF-a coding adenoviruses can modify
ment (Tähtinen et al, PLOS One 2015). To combine
the cellular composition of the tumor microenviron-
the benefits of both approaches, we studied if repli-
ment in favor of adoptively transferred T cells.
cation-deficient Ad5-vectors coding for interleukin-2
In conclusion, IL-2 and TNF-a coding adenoviruses
(IL-2) and tumor necrosis factor alpha (TNF-a) would
can break tumor-associated immunotolerance and
affect the activity of adoptively transferred, TCR-
significantly increase the levels of active, tumor-re-
transgenic TRP-2(180-188) specific T cells in vivo.
active T-cells both in injected cutaneous lesions and
To gain clinically relevant mechanism-of-action data,
in non-injected metastatic lymph nodes. Importantly,
we chose to use ret transgenic mouse model that de-
this triple modality may represent an efficient ap-
velops spontaneous malignant skin melanoma which
proach to achieve “CD19-like” clinical responses in
metastasizes into distant organs. Following ACT and
the treatment of solid, metastatic cancers currently
intratumoral virus injection, a significant increase
incurable by standard therapies.
in activated PD-1+ CD8+ T cells was seen in both
cutaneous lesions and in metastatic lymph nodes.
Interestingly, a reverse correlation between tumor
weight and the number of tumor-reactive PD-1+
TILs (p=0.0015) was observed, indicating that T-cell
hypofunction was overcome and successful tumor
lysis was achieved. Local expression of cytokines
did not affect the levels of immunosuppressive
immune cell subsets such as myeloid-derived sup-
Local tumor treatment in combination with systemic
ipilimumab immunotherapy prolongs overall survival in
patients with advanced malignant melanoma
Theurich S.1,2,3, Rothschild S.I.4, Hoffmann M.5, Fabri M.5, Sommer A.5, Garcia-Marquez M.3, Thelen M.3, Schill C.6,
Merki R.6, Schmid T.7, Koeberle D.7, Zippelius A.4, Baues C.8, Mauch C.5, Tigges C.9, Kreuter A.9, Borggrefe J.10,
Schlaak M.2,5, von Bergwelt-Baildon M.1,2,3
University Hospital Cologne, Department I for
1
Internal Medicine, Cologne, Germany,
University Hospital Cologne, Center for Integrated
2
Oncology (CIO), Cologne, Germany,
University Hospital Cologne, Cologne Interventional
3
Immunology (CII), Cologne, Germany,
University Hospital Basel, Department of Internal
4
Medicine, Medical Oncology, Basel, Switzerland,
University Hospital Cologne, Department of
5
Dermatology, Cologne, Germany,
Cantonal Hospital of Aarau, Division of Hematology
6
and Oncology, Aarau, Switzerland,
St. Claraspital Basel, Medical Oncology, Basel,
7
Switzerland,
University Hospital Cologne, Department of
8
Radiation Therapy, Cologne, Germany,
Helios-Klinik St. Elisabeth, Department of
9
Dermatology, Oberhausen, Germany,
University Hospital Cologne, Institute for Diagnostic
10
and Interventional Radiology, Cologne, Germany
Purpose: Immune checkpoint inhibition with ipili-
we identified significantly increased melanoma-
mumab has revolutionized cancer immunotherapy
specific T-cell responses following ipilimumab+LPT
and significantly improved outcomes of patients
in one exemplary patient. Interestingly, adverse im-
with advanced malignant melanoma. Local pe-
mune-related events were not significantly increased
ripheral treatments (LPT) such as radiotherapy or
by the combination treatment. In a multivariable Cox
electrochemotherapy have been shown to modulate
regression analysis we demonstrate that the effect of
systemic immune responses. Preliminary data have
added LPT on OS remained significant, after adjust-
raised the hypothesis that the combination of sys-
ing for BRAF status, tumor stage, tumor burden and
temic immune checkpoint blockade with LPT could
CNS metastases (adjusted HR=0.56, 95% CI=0.31 to
lead to improved clinical outcomes.
1.01, p=0.05).
Patients and methods: Clinical data of consecutively
Conclusion: Our data suggest that the addition of LPT
treated melanoma patients at four cancer centers in
to ipilimumab is safe and effective in patients with
Germany and Switzerland were analyzed. Patients
metastatic melanoma irrespective of adverse disease
either received ipilimumab or ipilimumab and LPT if
characteristics. We hypothesize that enhancement of
indicated for local tumor control. Additional immune
tumor-specific immune responses is most likely the
assessments were performed in order to identify tu-
underlying mechanism and that this combinatory
mor-specific immune responses during the combina-
approach warrants prospective validation.
tion treatment.
Results: A total of 127 melanoma patients were analyzed who either received ipilimumab (n=82) or
ipilimumab+LPT (n=45). We found that the addition of LPT to ipilimumab significantly prolonged
median overall survival (OS) (93 versus 42 weeks,
p=0.0028). As a potential immunological correlate,
The last both mentioned authors contributed equally
NK cell characteristics and anti-tumor efficacy in multiple myeloma and lymphoma patients before and after autologous stem
cell transplantation
Tognarelli S.1,2, Jacobs B.3,4,5, von Metzler I.6, Serve H.6, Bader P.1,2, Mackensen A.3, Ullrich E.1,2
Johann Wolfgang Goethe University Hospital, Childrens Hospital, Department of Pediatric Stem Cell
1
Transplantation and Immunology, Frankfurt am Main, Germany,
Johann Wolfgang Goethe University, LOEWE Center for Cell and Gene Therapy, Frankfurt am Main, Germany,
2
University Hospital Erlangen, Department of Hematology and Oncology, Erlangen, Germany,
3
Oslo University Hospital – The Norwegian Radium Hospital, Department of Cancer Immunology, Oslo,
4
Norway,
University of Oslo, The KG Jebsen Center for Cancer Immunotherapy, Oslo, Norway,
5
Johann Wolfgang Goethe University Hospital, Department of Hematology and Oncology, Frankfurt am Main,
6
Germany
Natural Killer (NK) cells are innate lymphocytes
of NK cells were CD56+CD16++, but after leukocyte
with a strong anti-tumor ability. In tumor patients,
recovery at TP2 CD56++CD16-/+ NK cells represented
such as multiple myeloma (MM) patients, an elevated
the main subset. Surprisingly, CD57 expression was
number of NK cells after stem cell transplantation
significantly increased within the CD56++CD16+/−
(SCT) correlates with a higher overall survival (OS)
population at TP2, but decreased again from TP2
rate. We aimed to study NK cell characteristics and
to TP3. Interestingly, the KIR expression within the
anti-tumor efficacy in tumor patients (16 MM, 16
CD56++CD16+/− NK cell population increased after
lymphomas) before and after autologous SCT (au-
SCT, with a peak at TP2. In more details, both CD56++
to-SCT). Moreover, cytotoxicity of patient-derived,
NK cell subsets upregulated their KIR2DL2/3/S2 and
cytokine-stimulated NK cells against MM cells has
KIR3DL1 expression levels from TP1 to TP2, whereas
been addressed at specific time points (TPs) at di-
the KIR2DL1/S1 levels remained stable. In addition,
agnosis, before and after auto-SCT. NK cells were
we evaluated NK cell functions upon tumor interac-
isolated from PBMCs and further analyzed by FACS
tion at the three defined TPs. CD56++CD16− NK cells
at three different TPs: TP1, before the start of high
were the main subset to produce IFN-γ upon interac-
dose chemotherapy (HDC); TP2, after leukocyte re-
tion with K562 cells at all three TPs. The percentage
covery (leukocytes >1000/µl) following SCT and
of IFN-γ-positive CD56++CD16− NK cells was slightly
TP3, at least 2 weeks after TP2. For testing NK cell
decreased at TP2, but recovered at TP3. Additionally,
cytotoxicity against MM cells, NK cells were purified
MIP-1β- and CD107a-positive CD56++CD16− cells re-
and expanded in vitro for 1-2 weeks with low doses
mained constant between TP1 and TP2, while their
IL-2 and IL-15.
percentages increased from TP2 to TP3. Moreover, in
CD56++CD16− or CD16+ and CD56+CD16++ NK subsets
a small group of MM patients, we isolated NK cells and
were monitored at the three TPs. At TP1 the majority
expanded them for 1-2 weeks prior to the functional
assays. As expected, the expansion rate was reduced
after chemotherapy but NK cells were still able to
exert an efficient killing of MM cells.
Our data demonstrate that NK cells have an altered
phenotype in tumor patients. Although the more “immature” CD56++CD16-/+ subset was the main NK cell
subset after leukocyte regeneration, it expressed high
levels of CD57 and KIRs, usually associated with a
more mature phenotype. Remarkably, these NK cells
were able to secrete cytokines and displayed a high cytotoxic capacity against different types of tumor cells.
Reference: Jacobs B, Tognarelli S, Poller K, Bader P, Mackensen A and Ullrich E (2015) NK Cell Subgroups, Phenotype,
and Functions After Autologous Stem Cell Transplantation.
Front. Immunol. 6:583. doi: 10.3389/fimmu.2015.00583
Combination immunotherapy of an inducible, autochthonous, low
mutational load murine lung cancer model expressing human
CEA as a tumor-associated self-antigen
Zhu E.F.1,2, Rakhra K.2, Abraham W.2, Moynihan K.D.2,3, Mehta N.2,3, Irvine D.J.2,3,4, Wittrup K.D.1,2,3
Massachusetts Institute of Technology, Chemical Engineering, Cambridge, United States,
1
Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, United
2
States,
Massachusetts Institute of Technology, Biological Engineering, Cambridge, United States,
3
Massachusetts Institute of Technology, Materials Science and Engineering, Cambridge, United States
4
Although immunotherapies such as anti-PD-1 and an-
Due to the increased latency of tumor development
ti-CTLA-4 have experienced unprecedented success in
in the autochthonous model, we first tested our com-
the clinic, they benefit only a subset of cancer patients,
bination therapy in a subcutaneous model of a CEA-
often with malignances presenting high mutational
expressing KP lung tumor cell line. We found that
load. In order to expand the utility of cancer immu-
80% of mice bearing established tumors could be
notherapies to a broader range of tumors, we have
cured with a combination of checkpoint blockade
combined two approaches that we have previously
and agents previously described: 1) anti-CTLA-4, 2)
demonstrated to possess anti-tumor efficacy. The first
anti-PD-1, 3) amphiphile vaccine, 4) MSA/IL-2, and
is a combination of anti-tumor antibodies and an IL-2
5) anti-CEA antibody. Curiously, therapy lacking an-
fusion with mouse serum albumin (MSA/IL-2), which
ti-CTLA-4 was unable to cure mice at all, inducing
induces a strong anti-tumor immune response involv-
only modest delays in tumor growth. Importantly, no
ing both innate and adaptive immunity in concert.
obvious long-term systemic toxicity was observed in
The second is an “amphiphile-vaccine” system, which
mice receiving this regimen.
greatly enhances tumor-specific CD8+ T-cell respons-
We are currently testing the efficacy of our combi-
es. When these treatments are combined, the vaccine
nation immunotherapy in the autochthonous lung
generates a large pool of tumor-specific T-cells that are
tumor model. Preliminary results suggest that the
further activated by the synergistic effects of MSA/
anti-CTLA-4 component is important for efficacy in
IL-2 and anti-tumor antibodies.
the lung tumor model as well. Without anti-CTLA-4,
We tested this therapy in an autochthonous model of
infiltrating TILs are restricted to the peripheries of the
murine lung adenocarcinoma that exhibits very low
tumor mass and CD8/Treg ratios are not sufficiently
mutational load, and thus very few tumor-associated
elevated to break tolerance to the CEA antigen.
antigens. In our model, we introduced human carci-
Our results suggest that combining immunotherapy
noembryonic antigen (CEA), an oncofetal antigen that
strategies can be therapeutically relevant against
is often associated with adenocarcinomas including
tumors exhibiting few neoantigens or even a single
LSL-G12D/+
fl/fl
(KP) mice with
known targetable antigen. Such combinations are
mice transgenic for human CEA, to generate KP-CEA
imperative to increase the number of eligible cancer
mice. These mice are infected intratracheally with a
patients for which immunotherapeutic intervention
lentivirus expressing Cre recombinase and human
is effective.
NSCLC. We crossed Kras
p53
CEA, inducing lung tumors that express CEA as a
tumor-associated self-antigen that can be targeted.
245 – 331
Tumor Biology
and Interaction
with the
Immune System
245 | TUMOR BIOLOGY & INTERACTION WITH THE IMMUNE SYSTEM
TRPV1 and TrkA agonists alter cytokine secretions of mix
leukocyte cultures obtained from tumor-bearing mice
Akman M.1, Erin N.1
Akdeniz University, Department of Medical Pharmacology, Antalya, Turkey
1
Majority of the studies about Transient Receptor
cantly more than either treatment alone. Interesting-
Potential Cation Channel Subfamily V member 1
ly Gambogic amide also increased IFNγ release in
(TRPV1) or as known as capsaicin receptors which we
response to ConA stimulation. Both Gambogic amide
encounter on peripheral and central nervous system
and MSK-195 alone or in combination markedly in-
has focused on neuronal functions of the TRPV1. The
creased IFNγ release in response to ConA and irra-
role of TRPV1 on immune cells was recently appreci-
diated tumor cells suggesting that radiotherapy in-
ated. There are few studies conducted on the role of
creases expression or sensitivity or TrkA and TRPV1
immune TRPV1 channels on anti-tumoral response
receptors. IL-10 secretion of leukocytes challenged
to metastatic breast carcinoma. The purpose of this
with tumor cells was only increased by Gamboic
study is to determine the effects of TRPV1 receptors´
amide treatment. In conclusion our results suggest
activation on immune response to metastatic breast
that both TRPV1 and TrkA receptors are involved in
carcinoma.
regulation of cytokine milieu of mice bearing meta-
Brain (4TBM) and liver (4TLM) metastatic breast car-
static breast carcinoma. Further studies are required
cinoma cells were used to induce tumor formation in
to evaluate exact role of each receptor subtype in
Balb-c mice. A group of mice received radiotherapy.
tumor immunology.
TRPV1 receptor agonists (resiniferatoxin, MSK-195)
and TRPV1 antagonist (capsazapine) were used to
evaluate changes in cytokine response of leukocyte
cultures obtained from tumor bearing animals.
TrkA agonist Gambogic amide was used to sensitize TRPV1 receptors. Mix leukocyte cultures (MLC)
were prepared from spleens and lymph nodes of mice
injected with tumor cells 24 days earlier. Drugs were
used separately or combined on challenged (with lipopolyshaccaride (LPS), Concanavalin A (ConA) and
radiation treated tumor cells) or non-challenged mix
leukocytes. Changes in secreted cytokines (IFN-γ,
IL-10, IL-6, IL-17A, TNF-α) were determined.
Both TRPV1 agonists and TrkA agonist increased
TNFα response to LPS challenge. Gambogic amide-MSK-195 combination increased TNFα signifi-
This study was supported by TÜBİTAK-COST action, Grant
no: 115S943.
Actin cytoskeleton remodeling: a novel mechanism for tumor
cells to escape from natural killer cell-mediated cell death
Al Absi A.1, Hoffman C.1, Chouaib S.2, Janji B.1, Thomas C.1
Luxembourg Institute of Health, Oncology, Luxembourg, Luxembourg,
1
Gustave Roussy Cancer Campus, Paris, France
2
Natural killers cells (NKs) are effectors of the innate
In addition, we aim at identifying actin regulatory
immune system that kill cancer and pathogen-infect-
proteins which might be of potential interest for the
ed cells without pre-stimulation through the direct-
development of strategies aim to revert cancer cell
ed secretion of lytic granule contents. This process
resistance to NKs.
requires the formation of a specialized cell-cell interaction region termed the immunological synapse
(IS), Where NKs and target cells physically interact
through their respective receptors and ligands. Previous reports have established that the formation and
activity of the IS largely rely on sequential rearrangement of actin filaments within NK. Our data provide
evidence that tumor cells can remodel their own
actin cytoskeleton to impair IS function and escape
from NK-mediated cell death.
Using live cell imaging, we showed that actin filaments of NK-resistant tumor cells accumulate at the
region surrounding the IS. The disruption of actin
filaments using of actin disrupting drugs increased
the rate of cell conjugation as well as tumor cell susceptibility to NK-mediated cell death. The analysis
of several breast cancer cell lines revealed a striking correlation between the ability of tumor cells to
undergo epithelial-to-mesenchymal transition and
susceptibility to NK-mediated cell death. Interestingly, all mesenchymal cells exhibited reduced susceptibility to NK-mediated cell death as compared to
more susceptible epithelial cells. We thus propose
that tumor cells can escape from NK cells by fast
and dynamic remodeling of their actin cytoskeleton.
The exact function and the mechanism underlying
such remodeling are currently further investigated.
“Infectious” tolerance transforms tumor antigen specific naive
CD4 T cells into induced Tregs in a spontaneous lung tumor
model
Alonso R.1, Flament H.1, Lemoine S.2, Sedlik C.1, Virginie P.1, Bottasso E.1, Denizeau J.1, Piaggio E.1, Lantz O.1
Institute Curie, Inserm U932, Paris, France,
1
Institute Necker Enfants Malades, Inserm U1151, Paris, France
2
ORAL
TALK
T
R
O
H
S
2016
During tumor development, the immune system is
strated de novo induction of tumor antigen specific
facing with persistent exposure to tumor-associated
Treg from uncommitted naïve CD4 T cells. Finally,
antigens, frequently in a non-inflammatory context,
depletion of the host Treg compartment before the
favoring the establishment of tolerance. Passive (ig-
transfer of tumor specific naive CD4 T cells impaired
norance, anergy or deletion of tumor specific T cells)
the conversion of the latters into iTreg and favored
or active mechanism mediated by regulatory T cells
their activation into full-blown effector cells able to
(Tregs) may be involved in tolerance. CD4 T cells
migrate to the tumor sites. Thus, host Tregs confer
are the main source of Tregs but also display indi-
infectious tolerance to naive tumor specific CD4 T
rect or direct anti-tumor activity. Because of the long
cells arriving into the tumor draining lymph node
life span of immune-dominant MHC-II/peptide com-
reinforcing tumor immune suppression.
plexes, transplantable tumor systems are not suitable
to study the relationship between CD4 T cells and
tumors.
In this work, we studied a genetically induced (“spontaneous”) tumor model of lung adenocarcinoma expressing a MHC-II restricted cytoplasmic antigen, luciferase fused to HY:DBY. We showed that the tumor
antigen reaches the lymph node and activates naive
CD4 T cells (from Marilyn TCR transgenic RAG KO
mice, HY:DBY specific T cells) to proliferate and recirculate. Transcriptome analysis of tumor specific
CD4 T cells showed an enrichment of Treg specific
genes starting at the early stages of tumor development and lasting afterward. We formally demon-
Hypoxia in the tumor microenvironment: A major regulator of
the anti-tumor immune response
Arakelian T.1, Mgrditchian T.1, Berchem G.1, Janji B.1
Luxembourg Institute of Health, Oncology, Luxembourg City, Luxembourg
1
Hypoxia is a common characteristic of solid tumor.
provided evidence that inhibition of autophagy by
It is now well established that hypoxic stress in the
knocking down Beclin1 using CRISPR/Cas9 tech-
tumor microenvironment is a major contributor of
nology, induced a massive infiltration of functional
tumor escape from immune surveillance. In addi-
immune cells into the tumor core. In keeping with
tion to its role in the impairment of the cytotoxic
this, we strongly believe that targeting autophagy
function of immune cells, hypoxia contributes to
improves the efficacy of immune checkpoint inhibi-
the emergence of resistant tumor cells able to evade
tors by regulating the temporal dynamic of immune
fully functional host immune system by activating
cells in the tumor microenvironment, leading to the
autophagy-dependent intrinsic resistance mecha-
modulation of the immune landscape of hypoxic
nism. We have recently showed that hypoxic stress
tumors. This study could provide cutting-edge ap-
upregulates the expression of the Programmed
proaches to improve the efficacy of immune check-
Death-Ligand 1 (PD-L1) on the surface of tumor cells
point inhibitors in non-responsive cancer patients.
thereby inhibiting the anti-tumor immune response.
While immune checkpoint inhibitors including PD-1/
PD-L1-based therapy result in remarkably durable
clinical remissions in some patients with melanoma, others reap a short-term benefit or no benefit at
all. It appears that durable clinical remission using
immune checkpoint inhibitors is likely dependent on
the expression of PD-L1 on the surface of tumor cells
and the presence of tumor-infiltrating lymphocytes
in the patient’s tumor. Therefore, understanding the
molecular mechanisms underlying the expression of
immune checkpoint inhibitors and those regulating
the infiltration of immune cells into the tumor bed
represents a major challenge to improve current immunotherapies in non-responsive cancer patients.
Thus, it stands to reason that switching the hypoxic
immune-suppressive microenvironment to an immune-supportive one is a prerequisite to achieve
successful PD-1/PD-L1 based immunotherapy. We
Tumour-associated macrophage phenotype makes low grade ovarian cancer a possible target for immunotherapy
Baert T.1,2, Mathivet T.3, Van Hoylandt A.1,2, Vergote I.1,2, Coosemans A.1,2
1
Leuven, Belgium,
2
INSERM UMR970, Paris Centre de Recherche Cardiovasculaire (PARCC), Paris, France
3
Background: Ovarian cancer is considered a highly
(CD68 as a general marker for TAMs, MHCII as a
aggressive tumour, killing still 80% of patients. It
marker for M1, the mannose receptor (MRC1) as a
comprises a heterogeneous group of malignancies
marker for M2 and Glucose Transporter 1(Glut1) as a
with a different clinical behaviour. Epithelial ovarian
marker for blood vessels and hypoxia).
carcinoma is the most common, which can be further
Results: This new technique makes it possible to
subdivided into low-grade (LGOC), comprising only
visualise at the same time M1, M2 and their relation-
3,4% of all ovarian tumours, and high-grade ovarian
ship to the intratumoral blood vessels. Blood vessels
carcinoma (HGOC) based on the degree of nuclear
appear to be more tortuous with increasing grade of
atypia and the mitotic rate. Although LGOC is a
the tumor. In close proximity, TAM are located. The
more indolent disease compared to HGOC, patients
total amount of TAM in LGOC is significantly less
still die of the disease after multiple relapses. The
compared to HGOC (p< 0,01). This decrease is due
treatment of LGOC is particularly challenging due
to a significant decrease in M2 in LGOC (p< 0,01). In
its resistance to chemotherapy. Currently a large in-
contrast, in HGOC there is an overbalance of M2 and
ternational multicentre trial is going on to investigate
an almost total lack of M1.
MEK-inhibitors as a possible targeted treatment for
Conclusion: These differences in the tumour micro-
low-grade serous ovarian cancer (ARRAY-162-311).
environment highlight the disparity between HGOC
The influence of the immune system in the develop-
and LGOC The local tumour milieu is much more in
ment of ovarian cancer is however poorly studied.
favour of establishing an immune response in LGOC
The role of tumour-associated macrophages (TAM),
compared to HGOC. Current therapy guidelines for
who in a number of cancers will present initially as
advanced stage ovarian cancer do not take tumour
+
+
an M1 phenotype (CD86 , MHCII ), leading to anti-
grade into account. Our findings suggest that LGOC
tumour immunity by initiating the adaptive immune
is possibly a suitable target for T-cell mediated im-
response, but once hypoxia and immunosuppression
munotherapies. For HGOC immunosuppression due
take the upper hand, will switch to the M2 phenotype
to M2-type macrophages seems an important hurdle
+
+
(CD163 , CD206 ), is not clear.
for T cells to achieve an effective antitumor response.
Materials and methods: We prospectively collected
In this case it would be useful to investigate therapies
13 ovarian cancer samples at diagnosis. Biopsies
that reduce the immunosuppressive effect of the M2
were fixed for 24 hours in paraformaledhyde 4%
macrophages in HGSOC.
and sectioned in 200µm sectioned with a vibratom.
Sections were permeabilized, blocked and incubated
with primary antibodies for immunofluorescence
Spatial heterogeneity of T cell distribution patterns at the
invasive margin of colorectal cancer liver metastases
Berthel A.1, Zörnig I.2, Kahlert C.3, Klupp F.4, Ulrich A.4, Weitz J.3, Jäger D.1,2, Halama N.2
National Center for Tumor Diseases and German Cancer Research Center, Clinical Cooperation Unit Applied
1
Tumor-Immunity, Heidelberg, Germany,
National Center for Tumor Diseases and University Hospital Heidelberg, Department of Medical Oncology,
2
Heidelberg, Germany,
University Hospital Dresden, Department of Surgery, Dresden, Germany,
3
University Hospital Heidelberg, Department of Surgery, Heidelberg, Germany
4
The invasive front of colorectal cancer liver metasta-
by immunotherapies. Furthermore, the analysis of
ses represents a clear border between malignant tissue
reversal of these spatial profiles could serve as a bio-
and adjacent normal liver tissue. As T cell density and
marker for successful therapies.
localization on a broader scale have prognostic and
predictive implications, the detailed resolution of T
cell distribution in the invasive margin was investigated. Using high-throughput whole slide imaging
technology, tissue areas of 1 square millimeter size
were analyzed, showing distinct density patterns of
T cell localization in relation to the malignant tissue
across the invasive margins of samples from different
patients. Unsupervised clustering revealed specific
groups of patients with T cell aggregates limited to a
given distance from the tumor. Furthermore, regions
with T cells in close contact to the tumor usually had
elevated T cell numbers further away from the tumor,
whereas the intermediate area showed limited enrichment. There seems to be a distance of around 10
to 30 micrometer from the tumor where a decrease
in T cells is common. The spatial heterogeneity of
T cell distribution in the invasive margin could be
accounted for interactions with immunosuppressive
cells, cell-matrix interactions or chemotactic fields
potentially generated by tumor or endothelial cells
and is currently investigated. Whether the presence
of the observed patterns has clinical importance and
might change under therapy remains to be seen. In
summary, this detailed analysis of spatial profiles
within the microenvironment reveals insights into
the localization of T cells and possible spatial immunosuppressive hurdles that need to be overcome
T cell costimulatory pathways are required for cisplatin-based
chemotherapy
Beyranvand Nejad E.1, van der Sluis T.C.1, van Duikeren S.1, Yagita H.2, Janssen G.M.3, van Veelen P.A.3,
Melief C.J.M.1,4, van der Burg S.H.5, Arens R.1
Leiden University Medical Center, Immunohaematology and Blood Transfusion, Leiden, Netherlands,
1
Juntendo University, Tokyo, Japan,
2
Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, Netherlands,
3
ISA Pharmaceuticals, Leiden, Netherlands,
4
Leiden University Medical Center, Clinical Oncology, Leiden, Netherlands
5
Chemotherapy combined with immunotherapeutic
direct killing of tumor cells but also requires the in-
approaches can exert synergistic effects on inhibi-
duction of tumor-specific immunity via a mechanism
tion of tumor growth. Conventionally, chemotherapy
involving the influx of inflammatory myeloid cells
was believed to be immunosuppressive and merely
with higher expression of costimulatory molecules.
mediate effects via direct killing of tumor cells. Inter-
Together, our study underlines the importance of
estingly, in recent years several studies have shown
cisplatin-mediated T cell costimulation induction to
that certain chemotherapeutic agents can stimulate
accomplish T cell-dependent tumor destruction.
immune cells but the underlying mechanisms are
largely unknown. Here we show in preclinical tumor
models that full tumor eradication by cisplatin chemotherapy is strictly dose-dependent and requires the
presence of CD8+ T cells. Specifically, we found
that cisplatin induces the accumulation of inflammatory myeloid subsets displaying increased levels
of costimulatory molecules (i.e., CD70, CD80 and
CD86) as a result of mediators that are released upon
cisplatin-mediated tumor damage. Interestingly, cisplatin-induced anti-tumor response was abolished in
mice deficient for CD80 and CD86 costimulatory molecules. Moreover, administration of blocking CTLA-4
antibody increased the survival of cisplatin-treated
wild-type mice while there was no effect of CD27
stimulation, replacing CD70 interaction. This implies
the importance of T cell costimulation in chemotherapy-induced anti-tumor response. Importantly,
cisplatin combined with synthetic long peptide vaccination induced synergistic anti-tumor response in
T cell costimulation dependent manner, thereby alleviating side effects although with similar clinical
response. Based on these observations, we conclude
that effective cisplatin therapy is not only based on
MuPeXI: A tool for prediction of neo-epitopes from tumor
sequencing data
Bjerregaard A.-M.1, Nielsen M.1,2, Hadrup S.R.3, Szallasi Z.1,4, Eklund A.C.1
Technical University of Denmark, Department of Systems Biology, Lyngby, Denmark,
1
Universidad Nacional de San Martín, Instituto de Investigaciones Biotecnológicas, Buenos Aires, Argentina,
2
Technical University of Denmark, Section for Immunology and Vaccinology, Lyngby, Denmark,
3
Harvard Medical School/ Boston Children’s Hospital, Computational Health Informatics Program (CHIP),
4
Boston, United States
Recent successes in several types of immunotherapy
testing of neo-epitope specific T-cell activation from
have demonstrated that exploitation of a patient’s
a population of tumor infiltration T-cells, optimiz-
own immune system is a promising strategy to elim-
ing adoptive T-cell therapy, or facilitates the optimal
inate cancer. Personalization of immunotherapies
combination of peptides for personalized cancer vac-
such as cancer vaccines and adoptive T-cell therapy
cines to boost a patient specific immune response.
will depend on identification of patient-specific neoepitopes that can be specifically targeted. For this
reason, there is a need for a tool to analyze sequencing data, extract the tumor specific peptides and
predict which are likely to be immunogenic. Here
we present MuPeXI, the Mutant Peptide Extractor
and Informer, a pipeline that extracts tumor mutation specific peptides and provides relevant information for peptide selection. MuPeXI takes several
mutation types into account, ensuring a larger output
of tumor specific peptides, including single nucleotide variations (SNVs) and indels with or without
frameshift. Standard practice for immunogenicity
estimations are based on theoretical considerations
guided by predicted features such a binding to patients HLA class I molecules, but we are currently
pursuing a qualified calibration of MuPeXI based on
high-throughput T cell response experiments. Tumor
specific peptide extraction with MuPeXI will enable
The role of CCR5 on MDSC in their recruitment and
activation in melanoma microenvironment
Blattner C.1,2, Karin N.3, Utikal J.1,2, Umansky V.1,2
DKFZ Heidelberg, Dermato-Onkology, Heidelberg, Germany,
1
University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Dermatology, Venereology
2
and Allergology, Mannheim, Germany,
Technion, Rappaport Institute for Medical Research, Department of Immunology, Haifa, Israel
3
Melanoma microenvironment is characterized by a
found to correlate with tumor progression. In addi-
strong immunosuppressive network, where myeloid-
tion, an enrichment of CCR5+ MDSC in melanoma
derived suppressor cells (MDSC) play a major role.
lesions was associated with an increase in the con-
MDSC represent a heterogeneous population of im-
centration of CCR5 ligands (CCL3, CCL4 and CCL5)
mature myeloid cells that fail to differentiate into
in tumor microenvironment as compared to serum.
granulocytes, macrophages or dendritic cells. They
Experiments performed on PBMC isolated from
were shown to inhibit an anti-tumor reactivity of T
melanoma patients at different stages revealed that
and NK cells and stimulate regulatory T cells during
the frequency of CCR5+ monocytic and granulo-
tumor progression. However, the exact mechanism
cytic MDSC is significantly increased as compared
of MDSC recruitment at the tumor site is poorly un-
to healthy donors. We found an elevated production
derstood. We used a ret transgenic mouse model
of NO and ROS and higher expression of ARG-1 by
of spontaneous melanoma that mimics the pathol-
circulating CCR5+ MDSC as compared to their CCR5-
ogy, genetic alterations and clinical development of
counterparts.
human malignant melanoma to study the mecha-
Our data suggest that elevated amounts of CCR5+
nism of MDSC migration.
MDSC in melanoma lesions of ret transgenic mice
A significant accumulation of CCR5 expressing
can be caused by increased concentration of CCR5
MDSC was found in skin melanoma lesions and met-
ligands in tumor tissue that allows a preferential
astatic lymph nodes as compared to the bone marrow
accumulation of these cells in the tumor microen-
+
and peripheral blood. Elevated frequency of CCR5
vironment. Furthermore, elevated expression of the
MDSC in melanoma lesions was correlated with
immunosuppressive factors on CCR5+ MDSC reflect
the tumor progression (indicated by an increase in
their increased immunosuppressive capacity and
tumor weight). Interestingly, CCR5+ MDSC displayed
suggest that CCR5 expression can regulate both the
a stronger immunosuppressive phenotype (indicated
MDSC migration to the tumor site and their immuno-
by the expression of arginase-1, ARG-1, and PD-L1
suppressive activity in the tumor microenvironment.
as well as the production of nitric oxide, NO, and
Therefore, novel strategies of melanoma treatment
reactive oxygen species (ROS) than in their CCR5-
could be based on blocking CCR5/CCR5-ligand in-
counterparts. Furthermore, CCR5+ MDSC infiltrating
teractions.
skin tumors and metastatic LN showed a stronger
immunosuppressive pattern than this subset in the
circulation. An elevated expression of PD-L1, ARG-1,
NO and ROS on tumor-infiltrating CCR5+ MDSC was
Determination of HLA type and expression from whole
transcriptome sequencing data (RNA-Seq)
Boegel S.1,2, Scholtalbers J.1,3, Bukur T.1,2, Löwer M.1, Sorn P.1, Castle J.C.1,4, Sahin U.1,2
TRON Translational Oncology at the University Medical Center of Johannes Gutenberg University Mainz
1
gGmbH, Mainz, Germany,
Universal Medical Center of Johannes Gutenberg-University, Mainz, Germany,
2
Present adress: EMBL, Heidelberg, Germany,
3
Present address: Agenus and 4-Antibody AG, Basel, Switzerland
4
The human leukocyte antigen (HLA) molecules
publicly available. We have developed bioinformatics
display peptide antigens that are derived from intra-
workflows capable of using these datasets to further
cellular (class I) and extracellular (class II) proteins
annotate each cell line, including 4-digit HLA types,
on the surface of vertebrate nucleated cells. Deter-
gene expression levels, and expressed viruses. Here,
mining the sequence of these molecules, HLA typing,
we integrated the cell line-specific mutation informa-
is essential for clinical work (e.g, organ transplanta-
tion with the determined cell line-specific HLA types
tion), cancer and immune system research. Current
and HLA binding prediction algorithms to generate a
HLA typing techniques use labor- and time-intensive
catalog of cell line-specific predicted HLA Class I and
methods. By contrast, Next generation sequencing
Class II neo-antigens. Not only are these underlying
(NGS) is a novel platform that enables rapid genera-
characterizations important, but also the ability to
tion of billions of short nucleic acid sequence reads.
easily query them in an effective user interface is
Using ´whole transcriptome´ sequencing (RNA-Seq
similarly essential. For example, easy identification
profiling) not only generates expression profiles
of a cell line appropriate for a specific experiment
but also nucleotide sequence information. Given
would be enabling, such as quickly filtering for a cell
the large number of RNA-Seq profiles in the public
line with a specific HLA type and a specific gene
domain and our efforts to develop individualized T
expression. Here, we re-analyzed RNA-Seq data of
cell-mediated cancer vaccines, we developed an in-
1,082 cancer cell lines and integrated all results and
silico method, seq2HLA, which utilizes the sequence
available annotation in a centralized cell line an-
content of RNA-Seq reads to determine both HLA
notation database, called the TRON Cell Line Portal
class I and class II type as well as HLA expression.
(http://celllines.tron-mainz.de/).
Here, we show two applications of HLA profiling
In addition, we re-analyzed over 3200 public RNA-Seq
using seq2HLA in conjunction with publically avail-
NGS datasets to generate the first large expression
able RNA-Seq data.
atlas of HLA class I and class II across 35 normal
Cancer cell lines are important tools for cancer and
tissues, including locus-specific expression.
immunological research. While immunological characterization of these cell lines is essential, publicly
available information, especially about HLA type,
HLA expression and putative neo-epitopes, have remained largely incomplete. Thankfully, the transcriptomes of many cell lines have been sequenced, mutations have been annotated, and raw datasets made
IFN-α potentiates the direct and immune-mediated antitumor
effects of epigenetic drugs on both metastatic and stem cells of
colorectal cancer
Buoncervello M.1, Romagnoli G.1, Buccarelli M.1, Fragale A.1, Toschi E.1, Parlato S.1, Lucchetti D.2, Macchia D.1,
Spada M.1, Canini I.1, Sanchez M.1, Falchi M.1, Musella M.1, Biffoni M.1, Belardelli F.1, Capone I.1, Sgambato A.2,
Ricci Vitiani L.1, Gabriele L.1
Istituto Superiore di Sanità, Rome, Italy,
1
Istituto di Patologia Generale, Università Cattolica del Sacro Cuore, Rome, Italy
2
Epigenetic alterations, including dysregulated DNA
the suitability of IFN-α in association with epigenet-
methylation and histone modifications, govern the
ics as a novel and promising therapeutic approach for
progression of colorectal cancer (CRC). Cancer cells
CRC management.
exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals.
Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and
histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged
as a new therapeutic frontier in cancer, expected to
overcome drug resistance associated with current
treatments. In this study, we challenged this idea
and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells
and cancer stem cells (CSCs), via tight control of key
survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach
is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed
key role of IFN-α in inducing both antiproliferative
and pro-apoptotic effects on CSCs of CRC was also
found. Overall, these findings open a new frontier on
Prognostic role of local immune infiltrate in patients with colon
cancer
Calvo A.1, Lahera T.2, Torres G.2, Rengifo C.E.3, Quintero S.4, Escobar X.1, Danta D.5, Vázquez J.M.5
National Institute of Oncology and Radiobiology, Cell Biology and Biobank, Havana, Cuba,
1
National Institute of Oncology and Radiobiology, Laboratory of Basic Research and Immunology, Havana, Cuba,
2
Manuel Fajardo Hospital, Department of Pathology, Havana, Cuba,
3
National Institute of Oncology and Radiobiology, Department of Pathology, Havana, Cuba,
4
National Institute of Oncology and Radiobiology, Department of Surgery, Havana, Cuba
5
Introduction: Until now, TNM classification has
(p=0,011) and CD68 (p=0,042) at stromal level and
been the most important factor to estimate the prog-
CD8 intratumoral (p=0,014) were significant prog-
nosis of colon cancer (CC) patients. However, in
nostic factors for overall survival. Among these
recent years, some studies have demonstrated that
variables, level of CD45RO (HR: 0,187; CI95%: 0,060-
the immune contexture in the tumors can be an es-
0,578; p=0,004) was independent prognostic factor
sential prognostic factor.
on multivariate analysis, by Cox regression.
Aim: To assess the prognostic role of local immune
Conclusions: This study is the first approach, in our
infiltrate and to evaluate its relation with clinico-
country, on the prognostic significance of immu-
pathological features in patients with CC.
nological infiltrate in CC. This assessment, mainly
Methods: Paraffin-embedded specimens were retro-
CD45RO TIL, might be used in the prognostic esti-
spectively collected from 50 patients who underwent
mate of CC, although further studies will be required
resection for CC at National Institute of Oncology,
to validate these findings.
Havana, between 2004 and 2008 years. The density
of cells was determined by immunohistochemistry
technique and its relation with survival and clinicopathologic features was evaluated. Lymphocytes
positive for CD3 (T cell), CD45RO (memory marker),
CD8 and granzime B (T cell cytotoxic) were assessed
according to intraepithelial and stromal location in
the tumor. Macrophage infiltration along the tumor
front was also evaluated using CD68.
Results: Patients with poor grade of differentiation,
mucinous type and clinical stage IV showed low infiltration of immunologic cells. The 5-years overall
survival for patients who had high-density of cells
were better compared with patients who had lowdensity, with significant differences statistical for
intraepithelial CD8 (p=0,035) as well as stromal
CD3 (p=0,001), CD8 (p=0,041), CD45RO (p=0,007)
and CD68 (p=0,041). In univariate survival analysis, the high expression of CD3 (p=0,006), CD45RO
Generation of MHC class I and class II deficient tumor cell lines
using the CRISPR/Cas9 system
Das K.1, Eisel D.1, Nicolaus C.1, Goyal A.2, Diederichs S.2, Dickes E.1, Osen W.1, Eichmüller S.B.1
German Cancer Research Center (DKFZ), GMP and T Cell Therapy Unit, Heidelberg, Germany,
1
German Cancer Research Center (DKFZ), Division of RNA Biology and Cancer, Heidelberg, Germany
2
HLA-transgenic (tg) mouse strains of the newer gen-
to be an efficient straight forward strategy for pro-
eration are often devoid of endogenous MHC mol-
duction of MHC knock out cell lines. These could
ecule expression to ensure exclusive generation of
serve as parental cells for co-transfection of compat-
HLA-restricted T cell responses, ruling out poten-
ible HLA alleles together with human tumor anti-
tial interference with H2-restricted T cell reactions.
gens of interest, thereby facilitating the generation of
Thus, in such mouse strains murine MHC molecules
HLA matched murine tumor models. In addition, our
would be recognized as xenogenic, thereby hamper-
tumor cell lines established might offer a useful tool
ing the use of non-autologous murine tumor lines
to investigate tumor reactive T cell responses that
for tumor transplantation experiments. We thus used
function independently from MHC molecule surface
the CRISPR/Cas9 technology to establish murine
expression by the tumor.
tumor cell lines, devoid of MHC I or MHC II surface
expression, respectively. For targeting the MHC I, the
melanoma cell line B16F10 and the murine breast
cancer cell line EO771 stably expressing the tumor
antigen NY-BR-1 (EO-NY) were transfected with expression plasmid encoding β2m-specific guide (g)
RNA and Cas9. The resulting MHC I negative cells
were sorted by flow cytometry to obtain single cell
clones and loss of susceptibility of peptide pulsed
MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The
β2m knockout (KO) clones did not give rise to tumors
in syngeneic mice (C57BL/6N), unless NK cells were
depleted, suggesting that outgrowth of the β2m KO
cell lines was controlled by NK cells. Using gRNAs
targeting the β-chain encoding locus of the IA b molecule we also generated several B16F10 MHCII KO
clones. Peptide loaded B16F10 MHC II KO cells were
not recognized by OT-II cells and tumor growth was
unaltered compared to parental B16F10 cells. Thus
in our hands, the CRISPR/Cas9 system has proven
Treatment regimen, surgical outcome and T cell differentiation
influence prognostic benefit of tumor-infiltrating lymphocytes
in high grade serous ovarian cancer
de Bruyn M.1, Wouters M.C.A.1, Komdeur F.L.1, Klip H.1, Plat A.1, Kooi N.M.1, Wisman G.B.A.1, Mourits M.J.E.1,
Arts H.J.G.1, Oonk M.H.M.1, Yigit R.1, de Jong S.1, Melief C.J.M.2,3, Hollema H.1, Duiker E.W.1, Daemen T.4, Nijman H.W.1
University of Groningen, University Medical Center Groningen, Groningen, Netherlands,
1
Leiden University Medical Center, Leiden, Netherlands,
2
ISA Pharmaceuticals, Leiden, Netherlands,
3
University of Groningen, Groningen, Netherlands
4
Purpose: Tumor-infiltrating lymphocytes (TIL) are
was of prognostic benefit in patients treated with
associated with a better prognosis in high grade
neo-adjuvant chemotherapy.
serous ovarian cancer (HGSC). However, it is largely
Conclusions: Our findings indicate that treatment
unknown how this prognostic benefit of TIL relates
regimen, surgical result and the differentiation of
to current standard treatment of surgical resection
TIL should all be taken into account when studying
and (neo-)adjuvant chemotherapy. To address this
immune factors in HGSC or, by extension, selecting
outstanding issue, we compared TIL infiltration in
patients for immunotherapy trials.
a unique cohort of advanced stage HGSC cancer patients primarily treated with either surgery or neoadjuvant chemotherapy.
Experimental Design: Tissue Microarray (TMA)
slides containing samples of 171 patients were analyzed for CD8+ TIL by immunohistochemistry.
Freshly isolated CD8+ TIL subsets were characterized by flow cytometry based on differentiation,
activation and exhaustion markers. Relevant T cell
subsets (CD27+) were validated using immunohistochemistry and immunofluorescence.
Results: A prognostic benefit for patients with high
intratumoral CD8+ TIL was observed if primary
surgery had resulted in a complete cytoreduction
(no residual tissue). By contrast, optimal (< 1 cm
of residual tumor) or incomplete cytoreduction fully
abrogated the prognostic effect of CD8+ TIL. Subsequent analysis of primary TIL by flow cytometry and
immunofluorescence identified CD27 as a key marker
for a less-differentiated, yet antigen-experienced and
potentially tumor-reactive CD8+ TIL subset. In line
with this, CD27+ TIL was associated with an improved prognosis even in incompletely-cytoreduced
patients. Neither CD8+ nor CD27+ cell infiltration
Synergy of anti-PD-1 in combination with targeted therapy is
mediated by CD8+ T cells
Deken M.A.1, Gadiot J.1, van Gool M.1, Kroon P.1, Lacroix R.1, Verbrugge I.1, Blank C.U.1
Netherlands Cancer Institute, Immunology, Amsterdam, Netherlands
1
ORAL
TALK
SHORT
2016
Treatment of melanoma has changed dramatically
loss. We observed a delayed tumor outgrowth upon
within the last decade. Immunotherapy by T cell
BRAF inhibition (PLX4720 - vemurafenib analog)
checkpoint inhibitors, like antibodies targeting
+ MEK inhibition (trametinib) compared to BRAFi
CTLA-4 and PD-1/PD-L1 and targeted therapy by in-
alone, whereas the addition of a PI3K inhibitor
hibition of an activated MAPK pathway, have shown
(BKM120), mTOR inhibitor (everolimus) or both to
improved overall survival in randomized trials. The
BRAF + MEK inhibition had limited to no addition-
combination of CTLA-4 and PD-1, as well as com-
al effect on tumor outgrowth. The combination of
V600E
and MEK in the MAPK
BRAFi + MEKi showed the most favorable immune
pathway have further improved patients’ outcome.
infiltration profile compared to single treatments. Ad-
However, long-term benefit of immunotherapeutic
dition of PI3Ki to BRAF + MEKi had a less favorable
approaches is so far achieved only for 20-50% of pa-
profile, even though tumor control was comparable.
tients, whereas targeted therapy has a high response
We therefore hypothesized that BRAFi + MEKi is the
rate but the majority relapses. The resistance to BRAF
most promising combination of targeted therapy to
inhibition is often found to be due to activation of the
be combined with immunotherapy. However, tumor
PI3K/AKT/mTOR pathway, which could be achieved
bearing mice treated with either BRAFi + MEKi or
by the loss of PTEN. The high response rate observed
BRAFi + MEKi + PI3Ki combined with anti-PD-1
upon targeted therapy treatment, but low long-term
antibodies experienced both an improved tumor
duration of response, makes it an attractive combi-
control, mostly attributed by complete responses
nation partner for immunotherapy. Targeted therapy
(CRs), as compared to single BRAFi or MEKi inhi-
can directly stimulate immune cells by altered activ-
bition combined with anti-PD-1. Depletion of CD8+
ity of their targets or more indirectly by inducing an
T cells completely abolished the occurrence of CRs.
anti-tumor immune response by release of antigens
FACS analysis of the TILs showed that addition of
and danger signals. So far, preclinical data is limited
anti-PD1 to BRAF + MEKi resulted in increased in-
and clinical attempts of combining targeted and im-
filtration of effector CD8+ T cells producing INFy.
munotherapy have failed due to toxicities.
These data indicate a crucial role for CD8+ T cells
We studied the effect of targeting the MAPK and
for the synergistic effects of targeted- and immuno-
PI3K/AKT/mTOR pathway on tumor outgrowth and
therapy. In summary, our data provide a rationale
the frequencies of tumor infiltrating lymphocytes
for testing targeted therapy with BRAFi + MEKi in
(TILs) in a preclinical mouse model. This syngeneic
combination with anti-PD-1 immunotherapy in mela-
transplantation model is based on a murine tumor
noma patients
bined targeting of BRAF
cell line harboring the BRAFV600E mutation and PTEN
PD-L1 tumor expression as an adaptive immune resistance
mechanism to counter the antitumor effect of immunogenic
chemotherapies
Dosset M.1,2, Lagrange A.1,2, Rivera Vargas T.1,2, Roussey A.1,2, Ghiringhelli F.1,2,3, Apetoh L.1,2
INSERM U866, Dijon, France,
1
Université de Bourgogne Franche Comté, Dijon, France,
2
Centre Georges François Leclerc, Dijon, France
3
Some chemotherapeutic drugs trigger an immunogenic form of tumor cell death, which elicits CD8 T
cell-dependent anticancer immune responses. Yet,
chemotherapy-driven anticancer immunity fails to
induce permanent tumor regression in mice and
shows limited efficacy in the clinic. The reasons why
CD8 T cell anticancer responses triggered by immunogenic chemotherapy are transient remain elusive.
Here we show in colorectal tumor-bearing mice that
the combined chemotherapy 5-Fluorouracil (5-FU)
and Oxaliplatin (OX) elicits in vivo tumor expression of PD-L1, which drives the dysfunction of CD8
tumor-infiltrating lymphocytes and compromises
their ability to eradicate tumors. An extensive study
with other drugs demonstrated that the induction of
tumor PD-L1 expression by chemotherapies was dependent on their ability to drive immunogenic cell
death, thereby defining PD-L1 tumor expression as
an adaptive immune resistance mechanism to immunogenic chemotherapies. Finally, in two mouse colon
cancer models, therapeutic prevention of CD8 T cell
dysfunction by disruption of PD1/PD-L1 signaling
in mice receiving immunogenic chemotherapies restored CD8 T cell function and led to complete longlasting tumor clearance.
Our study thus points out PD1/PD-L1 pathway as
a major immunosuppressive mechanism developed
by the tumor to impede the anticancer efficacy of
immunogenic chemotherapies, and provide impetus
to combine those drugs with PD-1/PD-L1 signaling
inhibitors.
POLE proofreading domain mutations elicit an antitumor
immune response in endometrial cancer
Eggink F.A.1, van Gool I.C.2, Leary A.3, Pollock P.M.4, Crosbie E.J.5, Mileshkin L.6, Adam J.3, Church D.7,8,
Creutzberg C.L.9, de Bruyn M.1, Nijman H.W.1, Bosse T.2
University of Groningen, University Medical Center
1
Groningen, Obstetrics and Gynecology, Groningen,
Peter MacCallum Cancer Centre, Division of Cancer
6
Medicine, East Melbourne, Australia,
The Wellcome Trust Centre for Human Genetics,
Netherlands,
7
Leiden University Medical Center, Pathology, Leiden,
2
Netherlands,
University of Oxford, Molecular and Population
Genetics Laboratory, Oxford, United Kingdom,
Gustave Roussy, Medical Oncology, Villejuif, France,
3
Queensland University of Technology, Institute
4
of Health and Biomedical Innovation, Brisbane,
Churchill Hospital, Oxford Cancer Center, Oxford,
8
United Kingdom,
Leiden University Medical Center, Clinical Oncology,
9
Leiden, Netherlands
Australia,
University of Manchester, St Marys Hospital,
5
Institute of Cancer Sciences, Manchester, United
Kingdom,
Aim: Recent studies have shown that 7% to 12% of
Results: Compared with other endometrial cancers,
endometrial cancers are ultramutated due to somatic
POLE-mutants displayed increased infiltration of
mutation in the proofreading exonuclease domain of
cytolytic CD8+ T cells, enhanced activation of in-
the DNA replicase POLE. Interestingly, these tumors
tratumoral T-cells and an upregulation of markers
have an excellent prognosis. In view of the emerging
for memory T-cells. This was accompanied by up-
data linking mutation burden, immune response, and
regulation of immune checkpoint inhibitors PD1 and
clinical outcome in cancer, we investigated whether
PD-L1. No evidence of increased B-cell infiltration
POLE-mutant endometrial cancers showed evidence
was observed.
of increased immunogenicity.
Conclusions:
Method: We examined immune infiltration and
domain mutant endometrial cancers in a clinically-
activation according to tumor POLE proofreading
relevant patient cohort are characterized by a robust
domain mutations in a high-risk endometrial cancer
intratumoral T-cell response. This study provides a
cohort, including 14 POLE-mutant tumors. This
plausible mechanism that contributes to the excel-
cohort was selected from partner institutions of the
lent prognosis of these cancers.
TransPORTEC (Post Operative Radiation Therapy in
Endometrial Carcinoma) consortium using inclusion criteria of the PORTEC3 study. Tissue Microarray slides were analyzed for CD8+, CD27+, CD103+,
TIA-1+, T-Bet+, CD20+, CD45RO+, PD-1+ and
PD-L1+ cells in the tumorcore and invasive margin
using immunohistochemistry. POLE-mutant tumors
were compared to 3 other endometrial cancer categories: microsatellite instable (MSI) tumors, microsatellite stable (MSS) tumors and tumors expressing p53.
Ultramutated
POLE
proofreading
GARP/LAP expression on FoxP3+/-Helios+/- Treg subsets in
patients with pancreatic cancer and liver metastases from
colorectal cancer
Elkord E.1,2,3, Abd Al Samid M.2, Chaudhary B.2, Khaled Y.S.3, Ammori B.J.3
United Arab Emirates University, Al Ain, United Arab Emirates,
1
University of Salford, Biomedical Research Centre, Manchester, United Kingdom,
2
University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom
3
Background: Regulatory T cells (Tregs) comprise
and levels of IL-10-secreting CD4+ T cells were el-
numerous heterogeneous subsets with distinct phe-
evated in LICRC patients, especially with higher
notypic and functional features. Identifying Treg
tumor staging.
markers is critical to investigate the role and clini-
Conclusions: This work implies that a combination
cal impact of various Treg subsets in pathological
of Treg-specific markers could be used to more ac-
settings, and also for developing more effective im-
curately determine expanded Treg subsets and to
munotherapies.
understand their contribution in cancer settings,
Methods: We investigated different Treg subsets as
and investigations of Treg levels in different cancers
defined by expression of FoxP3 and Helios transcrip-
should consider diverse Treg-related markers such as
tion factors and GARP and LAP immunosuppressive
GARP, LAP, Helios, and others and not only FoxP3 as
markers in peripheral blood samples isolated from
a sole Treg-specific marker.
patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC), and compared
their levels to control groups.
Results: We have recently shown that non-activated FoxP3-Helios+ and activated FoxP3+/-Helios+
CD4+ T cells express GARP/LAP immunosuppressive markers in healthy donors. In this study, we
report similar observations in the peripheral blood
of patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC). Comparing
levels of different Treg subpopulations in cancer patients and controls, we report that in PC patients, and
unlike LICRC patients, there was no increase in Treg
levels as defined by FoxP3 and Helios. However, defining Tregs based on GARP/LAP expression showed
that FoxP3-LAP+ Tregs in non-activated and activated
settings, and FoxP3+Helios+GARP+LAP+ activated
Tregs were significantly increased in both groups
of patients, compared with controls. Additionally,
GARP-/+LAP+ CD4+ T cells made IL-10, and not IFN-g,
CD200 mimetic PEG-M49 increases therapeutic effects of
pegylated liposomal doxorubicin on poorly differentiated breast
carcinoma: Possible role of in-vivo increased anti-tumoral
immune response
Erin N.1, Podnos A.2, Tanrıöver G.1, Prodeus A.3, Garipey J.3, Gorczynski R.2
Akdeniz University, Antalya, Turkey,
1
Toronto General Hospital, Toronto, Canada,
2
Sunnybrook Cancer Centre, Toronto, Canada
3
CD200 is a widely expressed cell surface glycopro-
in CD200ko mice. The IFN-g response to irradi-
tein which has anti-inflammatory effects. We pre-
ated tumor cells was lowest and the IL-10 response
viously reported that CD200 overexpression in the
highest in MLCs prepared from aptamer-treated
host decreased progression and metastasis of highly
CD200ko mice compared to WT and CD200R1ko
aggressive 4THM murine breast carcinoma. We also
mice. PEG-M49 alone did not alter tumor growth
found that CD200 overexpression increases anti-tu-
and metastatic spread in any groups. Peg-Dox signifi-
moral immune responses. Recently a DNA aptamer,
cantly suppressed tumor growth and metastasis but
PEG-M49 was shown to be effective as a CD200
could not induce complete regression in WT mice.
agonist in skin transplantation. Pegylated liposomal
On the other hand, PEG-M49 and Peg-Dox co-treat-
doxorubicin (Peg-Dox), effective clinically in breast
ment induced complete tumor regression and loss of
cancer, was shown to have immunomodulatory
macroscopic lung metastasis in four out of seven WT
effects in animal models. The aim of this study was
mice. Similar changes were observed in CD200R1ko
to explore a possible synergistic interaction between
mice which may imply a primary effect of Peg-M49
the CD200 mimetic PEG-M49 and Peg-Dox. For this
on non-CD200R1 receptors, as previously noted in
purpose 4THM breast carcinoma cells were injected
transplant rejection. The anti-tumoral effects of co-
into the mammary-pads of three groups of Balb/c
treatment were less prominent in CD200ko animals
mice: wild type (WT), CD200 knockout (CD200ko)
with one of seven CD200ko mice showing complete
and CD200R1 knockout (CD200R1ko). Five days after
tumor regression, but with the overall decrease in
injection of tumor cells, mice were injected with
primary tumor burden and metastasis less than in
Peg-Dox (i.p. once a week for three weeks) and PEG-
WT or CD200R1ko mice. This may reflect an overall
M49, or PEG-cApt (negative control) (intravenously,
enhanced tumor growth and metastasis in CD200ko
every three days for 15 days). Changes in tumor
mice, which masks any therapeutic effects of co-
growth, lung metastasis, and phenotypes of immune
treatment. PEG-M49 + Peg-Dox co-treatment mark-
cells in draining lymph nodes, in spleen and within
edly suppressed IL-6 and IL-10 responses to LPS and
tumor tissues were characterized. Immune cells
irradiated tumor cells in WT and CD200R1ko mice
were characterized using GR1, CD11b, F4/80, CD4,
but not in CD200ko mice. These results demonstrated
CD3, CD8 and CD45 antibodies. Mixed leukocyte cul-
for the first time that CD200 mimetics may modulate
tures (MLCs) was used to characterize the cytokine
the anti-tumoral effects of Peg-Dox. Further studies
milieu in the same mice.
are warranted to determine possible clinical applica-
Tumor growth, metastases, and tumor infiltrat-
tions of these synergistic interactions.
ing GR1+CD11b+ cells were markedly increased
The effects of PU-H71, a novel HSP90 inhibitor, and
radiotherapy co-treatment in metastatic breast carcinoma:
Changes in IL-6 and macrophage inflammatory protein 2
Kale Ş.1, Korcum A.F.2, Erin N.1
Akdeniz University, Medical Pharmacology, Antalya, Turkey,
1
Akdeniz University, Radiation Oncology, Antalya, Turkey
2
HSP90 (Heat shock protein 90) inhibitors are consid-
factor, was also increased under in-vitro conditions
ered as new radiosensitizing agents. PU-H71, a novel
but not under ex-vivo conditions.
HSP90 inhibitor, is under evaluation for treatment of
These results demonstrate for the first time that
advanced cancer in Phase I trials. It is however not
therapeutic effects of PU-H71 and radiotherapy
known whether PU-H71 alters release of inflammato-
cotreatment on metastatic breast carcinoma might
ry mediators such as IL-6 and macrophage inflamma-
be limited by increased IL-6 levels. Hence strategies
tory protein 2 (MIP-2) alone or incombination with
to block IL-6 activity may enhance anti-tumoral/
radiotherapy in breast carcinoma cells metastasized
anti-metastatic effects of PU-H71 and radiotherapy
to vital organs. The goal of the present study was to
cotreatment.
evaluate possible PU-H71 and radiotherapy-induced
changes in MIP-2 and IL-6 secretion of breast carcinoma cells metastasized to vital organs such as liver
and brain.
Effect of PU-H71 alone and incombination with radiotherapy in murine aggressive breast carcinoma
cells metastasized to brain (4TBM), liver (4TLM)
and heart (4THM) were determined. Changes in the
levels of IL-6 and MIP-2 from these cancer cell cultures and ex vivo cultures were detected by enzymelinked immunosorbent assay (ELISA).
PU-H71 and radiation co-treatment increased IL-6 secretion under ex-vivo conditions. Under in-vitro conditions IL-6 secretion from these cell lines were very
low. Level of MIP-2, an angiogenic and inflammatory
This study was supported by Akdeniz University Research
Unit (Grant No: 2014.03.0122.005).
Inhibition of PKC activity with Byrostatin alters secretion of
inflammatory chemokines
Erin N.1, Duymuş Ö.2, Nizam E.2
1
Akdeniz University, Antalya, Turkey
2
CXCR2 interacts with a wide range of inflammatory
of SB 225002 but prevented SB 225002-induced en-
chemokines and CXCR2 antagonists are considered
hancement of chemokine secretion. These findings
for treatment-resistant metastatic carcinomas. We
demonstrate that CXCR2 functions as an autorecep-
previously found that attenuating CXCR2 activity
tor for KC (CXCL1) and byrostatin and or related
with SB225002 markedly suppressed proliferation
molecules might be effective to prevent development
of metastatic breast carcinoma cells but increased
of resistance to growth inhibitory effects of CXCR2
MIP-2 secretion which may be due to the inhibition
antagonists.
of protein kinase C (PKC) activity. These results demonstrated that resistance to anti-proliferative effects
of CXCR2 may also arise from feedback increases
in MIP-2 secretion. Based on these findings it is
hypothesized that CXCR2 inhibitors increase other
CXCR2 ligands such as KC (human CXCL1), and this
increase can be prevented by PKC activation. To test
these hypotheses, we determined the effects of Ingenol-3-angelate (I3A) and Bryostatin, PKC activators,
alone and in combination with SB 225002 on MIP-2
and KC secretion and cell proliferation. Ingenol-3-Angelate was chosen for its known anti-tumoral effects
on skin tumors. We used mouse breast carcinoma
cells metastasize to brain (4TBM) and heart (4THM)
which include cancer stem cell features and metastasize extensively. We also determined kinetics of
KC secretion in 4T1 and non-metastatic 67NR mouse
breast carcinoma cells.
We found that there is a time-dependent increase in
KC secretion and inhibition of CXCR2 activity with SB
225002 further increased KC secretion. Surprisingly,
I3A prevented growth inhibitory effects of CXCR2 antagonist under serum-free conditions. Bryostatin, on
the other hand did not alter growth inhibitory effects
This study was supported by TÜBİTAK, Grant no: 115Z286.
Effects of phosphoramidon on TNF-a and IFN-g release from
mix leukocyte culture obtained from tumor-bearing mice
Erin N.1, Dilmaç S.2, Tanrıöver G.2
1
Akdeniz University, Antalya, Turkey
2
The role of proteases in cancer progression and me-
injected mice. In accordance inhibition of endoge-
tastasis is controversial. We previously found that
nous NEP activity with phosphoramidon increased
Neprilysin (NEP, CD10), involved in hydrolysis of
TNF-a and IFN-g secretion from stimulated MLC. Im-
neuropeptides important in immune regulation de-
portantly recombinant NEP did not hydrolyze either
creased in metastatic breast carcinoma. Furthermore
cytokine under in-vitro conditions. Furthermore
our preliminary studies demonstrated that recombi-
we found that levels of NEP decreased markedly in
nant NEP decreases IL-6 release from mix leukocyte
spleens of tumor-bearing mice compared to control
culture (MLC) challenged with tumor cells suggest-
group demonstrating that decreased NEP activity
ing that NEP might be involved in suppression of ex-
might be responsible for increased systemic inflam-
cessive inflammation. There are conflicting findings
matory response observed in animals injected with
regarding the effects of NEP inhibition with phos-
4THM breast carcinoma cells. These results demon-
phoramidon on inflammation. Hence we here exam-
strated that endogenous NEP activity is important to
ined the immunological consequences of inhibition
limit inflammatory response to aggressive breast car-
of NEP activity. MLC cultures were initiated using
cinoma and NEP inhibitors may potentiate inflam-
spleen and lymph nodes of mice injected with 4THM
matory damage. Furthermore strategies to increase
murine breast carcinoma cells orthotopically 14-25
NEP activity may prevent tumor-induced excessive
days earlier. Tumors formed by 4THM cells induce
inflammation.
intense local and systemic inflammation. Control
MLC used cells from non-injected female mice. MLCs
were stimulated with irradiated tumor cells or LPS or
Con-A. A control group contained unstimulated cells.
Phosphoramidon 10 mM were used to inhibit endogenous NEP activity. Recombinant NEP 10 ng/ml
was also used to determine the effects of exogenous
soluble NEP activity on cytokine response to challenge with tumor cells. We also examined changes in
the level of CD10 expression in spleens of the control
as well as tumor-bearing animals using western blot
and immunohistochemistry.
NEP significantly suppressed LPS-induced TNF-a
and IFN-g release from MLC of control and 4THM-
This study was supported by Akdeniz University Research
Unit, Grant no: TSA-2015-400.
Characterization of the immunogenicity of pancreatic cells in
response to electrochemotherapy
Fernandes P.1, O’Donovan T.R.1, McKenna S.L.1, Soden D.M.1, Forde P.1
Cork Cancer Research Centre, University College Cork, Cork, Ireland
1
Pancreatic cancer is one of the deadliest cancers with
The authors wish to acknowledge funding from the
5-year survival only 3% (1). The majority of cases are
Pancreatic Cancer Research Fund UK and Break-
surgically unresectable and show marked resistance
through Cancer Research.
to conventional forms of chemotherapy. Even with
complete surgical resection, recurrence is common,
Reference:
often confounded with distant metastases (2). Elec-
1. Globocan 2012 - Estimated cancer incidence, mortality and
prevalence worldwide 2012
2. Cancer Statistics Registrations, England (Series MBI), No
41, 2010
3. Mir LM EJC Supplements 2006; 4:38-44
4. Gerlini G, et al.Borgognoni. Oncoimmunology 2012; 1:16551657.
5. Roux S, et al. Cancer Immunology Immunotherapy
2008:57:1291-130
troporation is a physical technology that renders
the cell membrane permeable to otherwise poorly
permeant chemotherapies (7). Electrochemotherapy
(ECT) has been shown to be effective at local tumour
resolution with limited side effects (3). Additionally,
preliminary studies have shown that ECT results in
immune recruitment to the treated tumour but also
reduces distal metastasis in murine models, allowing
for extended survival (4-5). Pancreatic cancer cells
were resuspended in PI-containing electroporation
buffer and electroporated at 0.4-1kV/cm in 4mm cuvettes (8 pulses of 99µs at a frequency of 1Hz) using
a BTX electroporator. FACs - Cells were examined
for PI-mediated fluorescence emission. MorphologyCells were cytospun, stained and type of cell death
visually assessed. C.F.A. - Cell recovery was assessed
by standard clonogenic assay. CRT exposure - Cells
were incubated with anti-calreticulin antibody, followed by a conjugated secondary antibody and investigated by flow cytometry. Electroporation has the
potential to increase the uptake of chemotherapeutic
drugs in pancreatic cancer cells thus potentiating
any cytotoxic effect. Furthermore, electroporation
induces transient morphological changes that may
improve the immunogenicity of cell death.
Tumor-infiltrating B lymphocytes independently predict outcome in patients with non-small cell lung cancer and consist
mostly of effector subsets
Fischer R.1, Scheel A.2, Ansén S.1, Rothschild S.3, Hekmat K.4, Heldwein M.4, Dörr F.4, Kissl M.1, Reuter S.1,
Haustein N.1, Thelen M.1, Michels S.1, Nogova L.1, Büttner R.2, Wolf J.1, von Bergwelt-Baildon M.1
University Hospital Cologne, Department 1 for Internal Medicine, Cologne, Germany,
1
University Hospital Cologne, Institute of Pathology, Cologne, Germany,
2
University Hospital Basel, Medical Oncology, Department of Internal Medicine, Basel, Switzerland,
3
University Hospital Cologne, Department of Cardiac and Thoracic Surgery, Cologne, Germany
4
Introduction: Tumor-infiltrating lymphocytes play
cells were detected in 53.5% (174/325) and 52.6%
an important role in cell-mediated immune-destruc-
(171/325) of the analyzed NSCLC tissue samples, re-
tion of cancer cells and tumor growth control. In
spectively. B cell infiltration was not associated with
non-small cell lung cancer (NSCLC) a prognostic role
clinical or histo-pathological characteristics. CD79a
of T cell subtypes, B lymphocytes, natural killer cells
and MUM1 expression were associated with a trend
and dendritic cells within the tumor stroma has been
towards a better outcome (CD79a: median OS 61 vs.
described. Here, we studied the role of tumor-infil-
46 months, p=0.098; MUM1: median OS 58 vs. 46
trating B cells characterized by CD79a (B-cell antigen
months, p=0.096). In the multivariate analysis B cell
receptor complex-associated protein alpha chain) and
infiltration characterized by positivity for CD79a and
MUM1 surface expression (Multiple myeloma onco-
MUM1 was an independent prognostic marker for
gene 1) in patients with NSCLC and analyzed dis-
survival (p=0.047).
tinct subsets of tumor-infiltrating lymphocytes. To
Median age of the FACS population was 69 years, all
our knowledge, this is the first study characterizing
diagnosed with localized disease (stage I: 50%, stage
subsets of tumor-infiltrating B cells, offering a pos-
II: 25%, stage III: 25%). No substantial lymphocyte
sible explanation for correlation of tumor-infiltrating
infiltration was found in normal lung tissue (< 1% of
B lymphocytes and prognosis.
cells), whereas tumor samples contained a high propor-
Methods: B cell infiltration was quantified using
tion of CD45+ lymphocytes (20%). Tumor-associated
immunohistochemistry (IHC) and antibodies to
B cells were enriched for activated B cells with strong
CD79a (Dako, clone JCB117) and MUM1 (Dako, clone
antigen-presentation capacity. No relevant propor-
MUM1p) on tissue microarrays of paraffin embed-
tion of regulatory B cells was found within the tumor
ded tumor sections (n=325). B-cell subsets were ana-
microenvironment.
lyzed by flow cytometry in resected primary tumors,
Conclusions: In this cohort of patients with localized
normal lung tissue and peripheral blood of 21 NSCLC
and locally advanced stage NSCLC, B cell infiltration
patients.
characterized by immunohistochemical positivity for
Results: Median age of the IHC patient population
CD79a and/or MUM1 represents an independent prog-
was 66 years with 64.6% male patients. 54.7% had
nostic marker. The B cell infiltrate is characterized by
adenocarcinoma and 43% squamous cell histol-
an accumulation of activated B cells with an effector
ogy. 65% of patients were diagnosed with local-
phenotype. This finding supports the hypothesis of a
ized disease (stage I: 42.1%; stage II: 21.8%), 30.6%
B cell-mediated anti-tumor immunity.
locally advanced disease (stage III) and 5.2% were
diagnosed with stage IV. CD79a and MUM1positive
Bifunctional peptide-MHC class I antibody fusions redirect
peptide-specific CD8+ T cells to eliminate tumor cells in vivo
Fischer C.1, Herting F.2, Imhof-Jung S.3, Hoffmann E.1, Umana P.4, Klein C.4, Knoetgen H.5
pRED, Large Molecule Research, Penzberg, Germany,
1
pRED, Pharmacology & Veterinary Affairs, Penzberg, Germany,
2
pRED, Large Molecule Research, Roche Innovation Center Penzberg, Germany,
3
pRED, Cancer Immunotherapy Discovery, Zürich, Switzerland,
4
pRED, Therapeutic Modalities, Basel, Switzerland
5
In the adaptive immune response, virus-specific
+
marker. Vaccination of immunocompetent mice gen-
CD8 memory T cells are reactivated by interaction
erated MCMV m38-specific CD8+ T cells. In cyto-
of their T cell receptor with viral peptide-loaded MHC
toxicity assays with splenocytes of vaccinated mice
class I complexes on the plasma membrane of in-
the surrogate molecules mediated INFγ-activation of
fected cells. Antibody-mediated delivery of recombi-
CD8+ T cells and eradication of tumor cells in vitro.
nant viral peptide-MHC class I complexes can mimic
We performed experimental lung metastasis in vivo
a viral infection of target cells and induce cell lysis
studies with B16-F10 melanoma cells and assessed
+
after recruitment of specific cytotoxic CD8 T cells.
tumor burden by visual metastasis counting and
In contrast to classical T cell recruiters based on CD3
RT-PCR. In a preventive setting we observed that the
involvement, the engagement of T cells via MHC
peptide-MHC class I-IgG fusion molecules engaged
class I complexes activates only a peptide-specific
T cells in a peptide-specific mode and induced com-
+
subpopulation of CD8 T cells. This has the advan-
plete elimination of tumor cells in the circulation
tage of a lower risk of side effects due to inappropri-
before settlement in the lung. In a therapeutic setting
ate T cell activation which may lead to a favorable
a delayed metastasis growth could be achieved. Solid
safety profile. Here, we describe syngeneic surrogate
subcutaneous tumor models with MC38 colon cancer
in vivo models to address potency of antibody-target-
cells are ongoing to evaluate tumor growth kinetics
ed peptide-MHC class I cancer treatment.
after treatment with peptide-MHC class I-IgG fusion
Peptide-MHC class I-IgG fusion proteins could suc+
molecules. Furthermore ultra-microscopy and im-
cessfully recruit pre-existing virus-specific CD8 T
munohistochemistry will be employed to investi-
cells from human donor-derived lymphocytes and
gate tumor penetration of peptide-MHC class I-IgG
effectively trigger eradication of the targeted tumor
fusions and recruitment of specific CD8+ T cells into
cells in vitro (Schmittnaegel et al., Cancer Immunol
the tumor.
Res, 2015). Due to the polymorphism of MHC com-
The results demonstrate that peptide-MHC class I-IgG
plexes the in vivo-potency could only be tested in
fusions showed antitumor efficacy in vivo. Full elimi-
syngeneic
mouse
nation of rapidly growing and immune suppressive
models. We designed fully recombinant surrogate
advanced tumors may require additional immune
fusion proteins containing a full length murine IgG
modulation (e.g. PD-1 blockage) which will be ex-
immunocompetent
surrogate
b
antibody and a murine MHC class I complex (H-2K )
carrying an immunodominant epitope of the murine
Cytomegalovirus (m38: SSPPMFRV). The antibody
was directed against a tumor-associated surface
amined in further combination studies.
The immunome of hepatocellular carcinoma - an in silico
analysis
Foerster F.1,2, Hess M.3, Schuppan D.2,4, Binder H.3, Bockamp E.2
University Medical Center Mainz, Department of Medicine I, Mainz, Germany,
1
University Medical Center Mainz, Institute of Translational Immunology, Mainz, Germany,
2
University Medical Center Mainz, Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI),
3
Mainz, Germany,
Beth Israel Deconess Medical Center / Harvard Medical School, Department of Gastroenterology, Boston,
4
United States
Hepatocellular carcinoma (HCC) is one of the most
and tumour stages were not associated with signifi-
common and deadliest types of cancer worldwide. It is
cant changes in the immunome. In contrast, signifi-
typically diagnosed at a stage when no curative treat-
cant differences between patients with extended and
ments are available. With no new drug having been
poor survival were detected. Thus samples of patients
approved for the treatment of HCC in the past 10 years,
with survival >5 years displayed upregulated markers
novel therapeutic approaches are desperately needed.
of CD8+ T-cells and cytolytic activity (such as CD3,
Cancer immunotherapy experiences a revival and
granzyme A, CCL2 and CCL3).
needs to be exploited for the treatment of HCC. The im-
Samples from healthy livers are more appropriate
munome of HCC has been studied relative to adjacent
for contrasting the immune infiltration of HCC than
liver tissue, which introduces a bias, since the relative
samples from tissue adjacent to HCC. This can be
immune infiltration of HCC usually occurs in an envi-
explained by HCC typically developing in long-term
ronment of inflammation. Recently, immune cell type
inflamed and fibrotic liver tissue characterized by
specific marker genes have been identified that allow
an overall increased immune activity. Etiology and
for an in-silico analysis of cell specific immune al-
tumour stage appear not to influence the immunome
terations (immunome). Here we employ these marker
of HCC. However, increased CD8+ T-cell content and
genes and pathways to characterize and quantify the
cytolytic activity are associated with better survival.
immune infiltration of HCC compared to both adjacent
This observation supports the emerging concept of
liver tissue and liver tissue from individuals without
“cold” and “hot” tumors with regard to responsive-
HCC using RNA sequencing data.
ness to immunotherapy, and suggests that immunome
RNA-seq data of HCC (371 samples), of matched
analysis may lead to novel diagnostic and prognos-
noncancerous tissue adjacent to HCC (50 samples)
tic markers, like CD8+ T cells and granzyme A, that
and healthy liver (34 samples) were downloaded
can also be exploited for the monitoring of immuno-
from public sources (TCGA, GTEX). Based on two
therapies. Moreover, immunotherapeutic interven-
immune cell marker gene-sets (Bindea et al. Immunity
tions targeting T-cells and activating their anti-tumor
2013;39:782-95; Rooney et al. Cell 2015;160:48-61), we
mechanisms should be promising strategies for the
analysed the immunome of HCC relative to adjacent
treatment of HCC, which otherwise displays a highly
tissue and healthy liver, and with regard to etiology,
variant genetic profile which is difficult to address
stage and survival.
with advanced molecular therapeutics such as kinase
A principal component analysis revealed that samples
inhibitors.
from HCC were more like their adjacent tissue than
tissue from healthy livers. Notably, different etiologies
Allogeneic Balb/c mice are more susceptible to B16F10 liver
metastasis than syngeneic C57/Bl6 mice despite a M1-polarized
anti-tumor immune response
Foerster F.1,2, Boegel S.3, Strobl S.2, Kaps L.2, Heck R.2, Kim Y.O.2, Bros M.4, Diken M.3, Sahin U.3, Tüttenberg A.4,
Bockamp E.2, Schuppan D.2,5
University Medical Center Mainz, Department of Medicine I, Mainz, Germany,
1
University Medical Center Mainz, Institute of Translational Immunology, Mainz, Germany,
2
3
Mainz, Germany,
University Medical Center Mainz, Department of Dermatology, Mainz, Germany,
4
Beth Israel Deconess Medical Center / Harvard Medical School, Department of Gastroenterology, Boston,
5
United States
Treatment of metastasized malignancies remains a
Within 2 weeks, mice developed severe B16F10 liver
great clinical challenge resulting in poor prognosis.
metastases. Notably, the allogeneic mouse strain
Liver metastases in particular are survival-limiting in
(Balb/c) was more susceptible than the syngene-
a broad range of malignancies including melanoma.
ic strain (C57/Bl6). Metastasized livers showed a
In order to develop more effective molecular thera-
pattern of predominantly innate immunity with
pies a better understanding of the mechanisms that
macrophages representing the major immune cell
underlie (liver) metastasis is warranted. The B16F10
population. Interestingly, Balb/c mice exhibited a
melanoma cell line is a well described cancer cell line
pattern of M1-, while C57/Bl6 mice showed a pattern
with high metastatic potential that has been success-
of M2-macrophage polarization. RNA sequencing
fully used for human anticancer drug development.
of metastasized liver samples demonstrated that
Since the B16F10 cell line has very low expression of
interferon-γ and its downstream pathway (which is
MHC class I molecules, it is suited for studying the
usually associated with an anti-tumor response) was
innate anti-tumor immune response which has so far
significantly overexpressed in Balb/c mice, whereas
not been explored in the context of the liver.
the Tgf-beta pathway (which is considered immuno-
B16F10 cells stably transfected with a luciferase-ex-
suppressive) was significantly upregulated in C57/
pressing lentivirus were administered via intrasplen-
Bl6 mice.
ic injection to induce liver metastasis in two different
B16F10 is such an aggressive cancer cell line that
strains of wild-type mice (Balb/c and C57/Bl6). Biolu-
immunocompetent allogeneic mice fail rejection -
minescence was recorded 7 and 14 days after injec-
despite interferon-γ signalling and increased antigen
tion using an IVIS in vivo imaging system. 2 weeks
presentation. Therefore the B16F10 liver metasta-
after injection, mice were sacrificed and their meta-
sis model allows detailed insights into the innate
static burden measured. Liver probes were analysed
immune response against invading cancer cells and
with regard to immune cell populations and markers
may be utilized for developing immunotherapies
of M1-/M2-polarization by FACS, RT-qPCR and IHC.
targeting innate immunity. These studies are on the
Whole transcriptome sequencing was performed on
way.
selected samples and their differential gene expression analysed.
Block of the HTLV-1 Tax-1 oncogene-dependent NF-kB activation
by the MHC class II transactivator CIITA. Implications for the
control of oncogenic potential of HTLV-1 infection
Forlani G.1, Abdallah R.1, Accolla R.S.1, Tosi G.1
University of Insubria, Department of Surgical and Morphological Sciences, Varese, Italy
1
Human T cell lymphotropic virus type 1 (HTLV-
that CIITA acts as a restriction factor inhibiting Tax-
1) Tax-1, a key protein in HTLV-1-induced T cell
1-promoted HTLV-1 gene expression and replication,
transformation, deregulates diverse cell signaling
indicate that CIITA is a versatile molecule that might
pathways. Tax-1 constitutively activates the NF-kB
also counteract Tax-1 transforming activity. Unveil-
pathway through binding to NF-kB proteins and ac-
ing the molecular basis of CIITA-mediated inhibition
tivation of the IkB kinase (IKK). Upon IKK-mediated
of Tax-1 functions may be important in defining new
phosphorylation of IkB and consequent IkB degra-
strategies to control HTLV-1 spreading and oncogenic
dation, NF-kB migrates into the nucleus, mediating
potential.
Tax-1-stimulated gene expression. We show that the
transcriptional regulator of major histocompatibility
complex class II genes CIITA (class II transactivator),
endogenously or ectopically expressed in different
cells, inhibits the activation of the canonical NF-kB
pathway by Tax-1 and we mapped the CIITA region
that mediates this effect. CIITA affects the subcellular localization of Tax-1, which is mostly retained
in the cytoplasm, and this correlates with the impaired migration of the NF-kB RelA subunit into the
nucleus. Cytoplasmic and nuclear mutant forms of
CIITA reveal that CIITA exploits different strategies
to suppress Tax-1-mediated NF-kB activation in both
sub-cellular compartments. CIITA interacts with
Tax-1 without preventing Tax-1 binding to both IKKg
and RelA. Nevertheless, CIITA affects Tax-1-induced
IKK activity, causing the retention of the inactive p50/
RelA/IkB complex in the cytoplasm. Nuclear CIITA
associates with Tax-1/RelA in nuclear bodies, blocking Tax-1-dependent activation of NF-kB-responsive
genes. Thus, CIITA inhibits both cytoplasmic and
nuclear steps of Tax-1-mediated NF-kB activation.
These results, together with our previous finding
Characterisation of immune and tumour-specific neoantigen
landscapes informs optimal therapeutic targeting in non-small
cell lung cancer
Furness A.1,2,3, Joshi K.1,3, McGranahan N.4, Rosenthal R.2, Ramskov S.5, Lyngaa R.5, Kumar Saini S.5, Wong Y.N.S.1,
Ben Aissa A.1, Pickering L.3, Turajlic S.3,4, Gore M.3, Larkin J.3, Peggs K.1, Hadrup S.5, Swanton C.2,4, Quezada S.1,2
Cancer Immunology Unit, University College London Cancer Institute, London, United Kingdom,
1
CRUK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, United Kingdom,
2
Department of Medical Oncology, The Royal Marsden NHS Foundation Trust, London, United Kingdom,
3
The Francis Crick Institute, London, United Kingdom,
4
Section for Immunology and Vaccinology, Technical University of Denmark, Copenhagen, Denmark
5
ORAL
TALK
T
R
O
H
S
2016
Modulation of co-inhibitory and co-stimulatory mol-
checkpoint blockade, however identification of shared
ecules on tumour-infiltrating lymphocytes (TILs)
predictive biomarkers has proved challenging. We
has emerged as a promising anti-cancer strategy. In
sought to determine the impact of intra-tumoural
contrast to monoclonal antibodies targeting tumour
heterogeneity (ITH) in the neoantigen landscape on
cells directly, immune modulatory antibodies serve
anti-tumour immunity. Through integrated analysis
to augment and direct endogenous T lymphocyte re-
of ITH and neoantigen burden, we demonstrate a
sponses against cancer. Recent pre-clinical studies
relationship between clonal neoantigen burden and
demonstrate that in addition to immune modula-
overall survival in primary lung adenocarcinomas
tion, the activity of this class of antibodies may also
(n=139). With use of an established neoantigen pre-
depend on capacity to deplete intratumoural regula-
diction pipeline, we identified neoantigen-reactive
tory T cells (Treg). This activity results from higher
CD8+ T cells in two patients with early-stage NSCLC.
expression of target molecules on Treg relative to
Further characterisation of these cellular subsets re-
CD4+ helper and CD8+ cytotoxic T cells. We there-
vealed high expression of co-inhibitory molecules in-
fore sought to characterise the expression of B7 and
cluding programmed cell death (PD-1) and the cytol-
TNFR immune checkpoint molecules on individual
ytic enzyme granzyme B (GzmB). Sensitivity to PD-1
tumour-infiltrating T lymphocyte subsets. Evalua-
blockade in patients with advanced NSCLC (n=31)
tion of CD4+FoxP3+, CD4+FoxP3- and CD8+ subsets
appeared enhanced in tumors enriched for clonal but
in a cohort of 10 patients with early-stage non-small
not subclonal neoantigens. T cells recognising clonal
cell lung cancer revealed striking heterogeneity in
neoantigens were detectable in the peripheral blood
immune checkpoint molecule expression between T
of patients deriving durable clinical benefit. These
cell subsets. Remarkably, the observed heterogene-
data suggest neoantigen heterogeneity may influence
ity was consistent between all studied patients and
immune surveillance, support therapeutic develop-
appeared similar in two separate cohorts of patients
ments targeting clonal neoantigens and identify rel-
with advanced melanoma and renal cell carcinoma.
evant immune regulatory pathways serving to limit
Beyond regulation at the tumour site, identification of
the activity of neoantigen-reactive T cells.
relevant epitopes evoking meaningful anti-tumour T
cell activity is paramount. Tumour-specific neoantigens are enriched in patients responding to immune
On-chip dialogue between immune cells and cancer: tracking
human dendritic cell migration and tumor antigen capture
upon drug treatment
Parlato S.1, De Ninno A.2, Molfetta R.3, Toschi E.1, Salerno D.4, Mencattini A.5, Romagnoli G.1, Fragale A.1,
Buoncervello M.1, Canini I.1, Bentivegna E.6, Roccazzello L.6, Gerardino A.2, Martinelli E.5, Paolini R.3,
Businaro L.2, Gabriele L.1
Istituto Superiore di Sanità, Hematology, Oncology and Molecular Medicine, Rome, Italy,
1
Istituto di Fotonica e Nanotecnologie, Consiglio Nazionale delle Ricerche, Rome, Italy,
2
“Sapienza” University, Department of Molecular Medicine, Rome, Italy,
3
IIT-Sapienza, Center for Life NanoSciences (CLNS), Rome, Italy,
4
University of Rome Tor Vergata, Department of Electronic Engineering, Rome, Italy,
5
“Sapienza” University, Rome, Italy
6
The crosstalk between cancer and immune cells is
demonstrate the functional superiority of a specific
a complex phenomenon required to induce a potent
subset of DCs, a mandatory biological outcome for
and effective antitumor response. Upon therapeutic
the onset of an efficacious antitumor response.
treatment, this event represents a need for fighting
cancer. Among immune cells, dendritic cells (DCs)
own the privileged role to recognize cancer cells,
uptake tumor antigens (Ag) and present them to
lymphocytes, bridging innate and adaptive immune
responses. DCs differentiated in the presence of interferon-alpha (IFN-DC) exhibit a marked phagocytic activity and a special attitude in inducing CD8+
T-cell response. By combining advanced microengineered technologies, confocal microscopy and mathematical algorithms, we developed a new microfluidic platform for tracking human DC behavior towards
cancer cells (CRC) upon therapeutic treatment. Our
approach allowed to evaluate in real time the selective propensity of IFN-DC to migrate in the direction of drug-treated cancer cells, move in a 3D tumor
microenvironment, get in touch with cancer cells
in a guided way and perform Ag uptake. Overall,
our microengineering system recapitulates human
immune cell-tumor interactions on-chip, suitable to
Sunitinib enhances the anti-tumor responses of agonistic CD40antibody therapy by reducing MDSCs and synergistically
improving endothelial activation and T-cell recruitment
Georganaki M.1, van Hooren L.1, Huang H.1, Mangsbo S.1, Dimberg A.1
Uppsala University, Uppsala, Sweden
1
CD40-stimulating tumor immunotherapy has potent
and sunitinib compared to all other groups. Our
anti-tumor effects mainly via Th1 immune respons-
results support that the combination of agonistic
es. However, the efficacy of the treatment is limited
CD40-antibody therapy with sunitinib exerts potent
by the immunosuppressive idiosyncrasy of the tumor
anti-tumor complementary effects on the immuno-
microenvironment. In this study, we demonstrate
suppressive myeloid cell compartment and syner-
that combining agonistic CD40 monoclonal anti-
gistically increase endothelial activation associated
body (mAb) with anti-angiogenic therapy using the
with enhanced cytotoxic T cell recruitment.
tyrosine kinase inhibitor sunitinib decreased tumor
growth and improved survival in B16.F10 melanoma
and T241 fibrosarcoma murine tumor models. Agonistic anti-CD40 mAb lead to increased activation of
CD11c+ dendritic cells in the tumor draining lymph
nodes, as indicated by increased CD86 surface expression, while tumor vessel density was decreased
in sunitinib treated mice. Increased accumulation
of CD11b+Gr1+ myeloid derived suppressor cells
(MDSCs) in the tumor draining lymph nodes after
agonistic anti-CD40 mAb therapy was abolished by
co-treament with sunitinib. Endothelial activation,
represented by upregulation of ICAM-1 and VCAM-1
adhesion molecules, was synergistically increased in
the combination treated tumors. In line with this,
CD8+ T cell infiltration was increased in mice treated
with the combination of agonistic anti-CD40 mAb
Development of multiplex fluorescent IHC immune cell panels
to predict immunotherapy outcome
Gorris M.A.J.1, Verweij D.I.1,2, Bol K.F.1,3, Blokx W.A.M.2, Textor J.1,4, de Vries I.J.M.1,3, Figdor C.G.1
Radboud Institute for Molecular Life Sciences, Tumor Immunology, Nijmegen, Netherlands,
1
Radboudumc, Pathology, Nijmegen, Netherlands,
2
Radboudumc, Medical Oncology, Nijmegen, Netherlands,
3
Utrecht University, Theoretical Biology, Utrecht, Netherlands
4
Purpose: With the emergence of multiple, expen-
mated and that results obtained by the multispectral
sive immunotherapies, there is a stringent need to
fluorescent IHC method are reproducible.
identify biomarkers that predict efficacy of immuno-
Keywords: Multispectral fluorescent immunohis-
therapy. In our previous study we determined that
tochemistry, automated analysis, T cell infiltration,
+
the density and distribution of CD3 T cells within
primary cutaneous melanoma correlates with survival of metastatic melanoma patients after dendritic
cell (DC) based immunotherapy. In the present study
we want to further automate and extend this analysis of tumor microenvironment (TME) to distinguish
types of T cells and other immune cells.
Methods: Multispectral fluorescent immunohistochemistry (IHC) using the Opal tyramide signal
amplification was exploited to develop and optimize
a T cell panel to analyze the TME in patients with
melanoma. To validate the results, primary tumors
of metastatic melanoma patients previously treated
with DC-based immunotherapy will be used.
Results: The T cell panel has been optimized, multispectral fluorescent IHC stainings have been performed and analysis is ongoing. Updated results will
be presented.
Conclusion: We here show that scanning and analysis of fluorescently stained TME sections can be auto-
biomarker, immunotherapy, melanoma. (6)
Investigation of tumor-reactive T-cell repertoire in the immune
infiltrate of metastatic melanoma under immune checkpoint
inhibition
Hassel J.1, Flossdorf M.2, Roth J.1, Lauenstein C.3, Appel L.2, Halama N.4, Faryna M.5, Enk A.1, Offringa R.3, Sahin U.5,6,
Poschke I.3
University Hospital Heidelberg, Department of Dermatology and NCT, Heidelberg, Germany,
1
German Cancer Research Center (DKFZ) and Bioquant, University of Heidelberg, Div of Theoretical Systems
2
Biology, Heidelberg, Germany,
German Cancer Research Center (DKFZ), Div Molecular Oncology of Gastrointestinal Tumors, Heidelberg,
3
Germany,
University Hospital Heidelberg, NCT/Department of Medical Oncology, Heidelberg, Germany,
4
5
6
Mainz, Germany
Just a few years ago the median survival of patients
cation of biomarkers for clinical response through
with metastatic melanoma in stage IV (AJCC 2010)
immunohistochemical analysis of TIL before and
was less than 1 year. With ipilimumab as the first
during treatment with immune checkpoint block-
developed immune checkpoint blocker, a significant
ers and (ii) the identification of tumor-reactive TILs
impact on survival of these patients was achieved
and their use as biomarkers through TCR repertoire
with the potential to lead to long-term survival. Ip-
profiling of the TIL and PBMC compartments before
ilimumab is an antibody (Ab) that activates T cells
and during treatment. Immunohistochemical analy-
by blocking the inhibitory receptor CTLA-4. More re-
sis included CD3, CD4, CD8, FoxP3 (T cells), CD20 (B
cently developed Abs that block the PD1/PD-L1 axis
cells), CD163 (macrophages), PD-1 and PD-L1. TCR
display a greater therapeutic index because their
repertoire profiling by deep TCR sequencing focused
effect is more focused on the tumor microenviron-
on changes before and under immunotherapy as well
ment in which PD-L1 is over-expressed.
as the comparison of the tumor tissue infiltrates with
TIL play a crucial role in the therapeutic impact of
the blood compartment in individual patients.
immune checkpoint blockers. It is already known
that ipilimumab induces an increase in the number
of melanoma-reactive CD8+ T cells in the peripheral
blood. In addition, patients responding to a PD-1 Ab
seem to have a more clonal T cell infiltrate before
start of treatment. We investigated tumor samples as
well as peripheral blood from 16 patients with metastatic melanoma before and during treatment with
immune checkpoint blockers (8 responders, 3 with
stable disease and 5 with progressive disease as best
response to treatment). Aims were (i) the identifi-
Role of tumor-derived exosomes in immunosuppression in
malignant melanoma
Hu X.1, Fleming V.1, Altevogt P.1, Utikal J.1, Umansky V.1
German Cancer Research Center (DKFZ), Clinical Cooperation Unit Dermato-Oncology (G300), Heidelberg,
1
Germany
Malignant melanoma is the most dangerous form of
suppression in malignant melanoma patients emerges
skin cancer and accounts for 75 % of all skin cancer
from a long-term interaction of the immune system
deaths. The accumulation of highly immunosup-
with constantly producing of tumor exosomes. We are
pressive regulatory leucocytes, especially myeloid-
studying also the content of tumor exosomes including
derived suppressor cells (MDSCs), play a significant
proteins (such as cytokines, chemokines and growth
role in resistance to immunotherapy of malignant
factors) and miRNA with regulatory functions. We
melanoma. Exosomes are small membrane vesi-
suggest that exosome investigations will help to es-
cles derived from endosome system and have been
tablish their prognostic value and will be used for the
proved to be essential in intercellular communi-
treatment of malignant melanoma patients.
cation. In addition, tumor-derived exosomes can
promote the progression, invasion and metastasis of
cancer. In particular, they can trigger cytokines and
chemokine production by immune cells. However,
the role of tumor-derived exosomes in immune suppressive mechanisms of malignant melanoma and its
interaction with MDSCs remain to be explored.
We have previously shown that the exosomes from
body fluids induced the secretion of inflammatory
cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-23, CCL5 (RANTES) and IL-6
that was due to NF-κB and STAT3 activation. We will
further elucidate the role of tumor-derived exosomes
in switching the differentiation pathway of circulating
monocytes to MDSCs (CD11b+CD14+CD15-HLA-DR−/low
cells) in patients with malignant melanoma. In addition, we will study the molecular mechanisms of this
conversion and further activation of MDSCs by melanoma-derived exosomes. We are performing the isolation, purification and quantification of tumor-derived
exosomes from plasma of malignant melanoma patients. We will verify if the development of immuno-
Targeting Cancer: Encapsulation of TNF-α in pH-sensitive PEI-PEG
copolymer gated dendritic mesoporous silica nanoparticle
Kienzle A.1, Kurch S.2, Schlöder J.1, Tremel W.2, Jonuleit H.1
University Medical Center of the Johannes Gutenberg-University Mainz, Department of
1
Dermatology, Mainz, Germany,
Johannes Gutenberg-Universität, Institut für Anorganische Chemie und Analytische Chemie, Mainz, Germany
2
Aims: TNF-α was originally described in 1975 as a
tial, dynamic light scattering (DLS) and transmission
molecule demonstrating tumour necrotizing activity.
electron microscopy (TEM) measurements. Follow-
Due to both, its cytotoxic effect on tumour cells and
ing functionalization with anti-EGFR-antibody for
its pro-inflammatory potential, TNF-α has the ability
tumour tissue specific targeting DMSN will be used
to slow tumour growth by promoting tumour cell
for tumour therapy in tumour-bearing NOD/ScidtgH-
death and stimulating the immune system. However,
LA-A2+ mice. Humanization of NOD/ScidtgHLA-A2+
even though TNF-α is one of the most well-known
mice is conducted by injection of peripheral blood
anti-tumour factors, its pharmacological use in vivo
mononuclear cells of human volunteers.
is significantly limited by its highly toxic potential
Results: Encapsulated TNF-α showed an 89 %
on the heart, liver and the vascular system. There-
reduced toxicity compared to free TNF-α. Flow cy-
fore, we synthesized a drug delivery system (DDS)
tometry analysis with pH-sensitive fluorescent dye
combining TNF-α loaded, dye functionalized den-
suggested a lysosomal processing and intracellular
dritic mesoporous silica nanoparticle (DMSN) with
release of TNF-α. TEM, DLS and zeta potential anal-
a pH-sensitive PEI-PEG Copolymer for pore cover-
ysis demonstrated stable and functional particles
age. By utilising MTT assays and flow analysis, we
under normal culture conditions. In vivo experiments
examined encapsulation efficiency, cellular uptake
in tumour-bearing humanized mice investigating
and processing of the particles. The major objective
anti-tumour versus systemic activity of encapsulated
of our study is the transport of TNF-α in a nontox-
TNF-α are ongoing.
ic carrier system followed by a tumour tissue spe-
Conclusion: Here, we demonstrate that the combina-
cific release to induce anti-tumour activity without
tion of DMSN for transport and pH-sensitive PEI-PEG
causing systemic toxicity.
copolymer for pore coverage is a potent system al-
Methods: MTT, Flow Analysis, Humanized Tu+
lowing the transport of toxic anti-tumour biologicals.
mour-bearing NOD/ScidtgHLA-A2 Mice, TEM, DLS,
Our promising preliminary results allow further an-
Zeta Potential
tibody functionalization to target tumour in a cell
MTT assays were used to analyse biological activity
specific manner to limit systemic toxic TNF-α effects
of encapsulated TNF-α. DMSN with pH-sensitive and
in vivo.
pH-insensitive dye functionalization were synthesized and flow cytometry was used to ensure uptake,
intracellular processing and release of the particles in
tumour cells. Solubility and functionalization of the
silica nanoparticles were monitored by zeta poten-
A novel model of murine hepatocarcinogenesis in biliary
fibrotic mice resembling the multistage process of human
primary liver cancer
Kim Y.O.1, Park K.-S.1, Herbel L.2, Qureshi A.1, Foerster F.1,2, Zimmerman T.2, Schuppan D.1,3
University Medical Center of the Johannes Gutenberg University Mainz, Institute of Translational
1
Immunology and Research Center for Immune Therapy (FZI), Mainz, Germany,
University Medical Center of the Johannes Gutenberg University Mainz, Department of Gastroenterology
2
and Hepatology, Med. I, Mainz, Germany,
Beth Israel Deaconess Medical Center, Harvard Medical School, Division of Gastroenterology, Boston,
3
United States
Background and aims: To date, the development of
DEN/PB newborn vs. older mice. While the Mdr2−/−
molecular and immune therapies for primary hepa-
mice administered DEN late developed only few mac-
tocellular carcinoma (HCC) has been hampered by
roscopic lesions at 6 months, the newborn DEN treated
the lack of rodent models that reflect the multistep
Mdr2−/− mice reached this stage already at 3 months
process of HCC development in patients. The Mdr2
of age. At 7 months, the early DEN/PB Mdr2−/− mice
(Abcb4) knockout mouse carries the loss of func-
showed a dramatic increase in the number of tumor
tion genetic mutation of patients with progressive
nodules and their size compared to the untreated
intrahepatic cholestasis type 3. Similar to patients,
Mdr2−/− (p< 0.0001), or wt (p< 0.001) or Mdr2−/− late
−/−
mice develop biliary cirrhosis at age 4-6
onset DEN/PB treated mice at 9 months (p< 0.0001).
months and HCC after 12 months, while chemical
After 9 months, all early DEN/PB Mdr2−/− mice pre-
carcinogenesis with diethylnitrosamine (DEN) and
sented tumors ranging from 5 to 35 mm in diameter,
phenobarbital (PB) induces random mutations, with
whereas the late onset treated mice showed lesions
HCC developing in 50-70% of mice after 10 months.
ranging between 1 and 5 mm. In both models, male
Here, we combined both models to generate a rapid,
mice displayed more tumor mass but lower lethality
predictable model that should resemble human HCC
than female mice. Until 7 months of age, hepatic colla-
in most aspects.
gen in the newborn DEN/PB Mdr2−/− mice was 2.1-fold
Methods: 5-day-old FVB Mdr2−/− and FVB wildtype
increased vs. the untreated Mdr2-/- (p< 0.01) or 2.6-
mice were intraperitoneally injected with a single
fold vs the DEN/PB wt mice (p< 0.01), but decreased
dose of DEN (10µg/g body weight) (n=10-12/group),
markedly (2.8-fold, p< 0.0001) at 9 months, when
followed by 0.05% PB in drinking water from age
there was a further 1.5-fold increase in tumor mass.
3 weeks on. Two more groups DEN at age 9 weeks
Conclusion: We established a short-term and repro-
(with PB 3 weeks later) which was repeated every
ducible model of primary liver cancer in immune
8 weeks. Livers were collected after 3, 5, 7, and 9
competent mice that resembles the multistep patho-
months and 3, 6, 9 months, respectively and assessed
genesis of human HCC, a cancer that usually de-
for the number and size of tumors. Sections were
velops by intrinsic and extrinsic mutational events
stained with H&E and hepatic collagen was quanti-
within a cirrhotic microenvironment. Moreover,
fied.
in this model male gender predisposes to more ad-
Results: The numbers and sizes of HCCs were highly
vanced cancers, as in patients with HCC. This model
increased in the DEN/PB Mdr2−/− vs. the DEN/PB wt
should aid in the preclinical development and valida-
mice at all time points. Notably, there was a highly
tion of directly needed small molecule and immune
increased size and number of tumor nodules in the
therapies for HCC.
Mdr2
Tumor-derived IL-1 mediates intratumoral immunosuppression
via the Treg-attracting chemokine CCL22
Knott M.1, Wiedemann G.1, Vetter V.1, Layritz P.1, Kühnemuth B.1, Rapp M.1, Endres S.1, Anz D.1
Klinikum der Ludwig-Maximilians-Universität München, Department of Clinical Pharmacology, Munich,
1
Germany
Tumors are known to escape the body’s immune
In summary, we elucidated a novel IL-1-dependent
system by various mechanisms such as upregulation
mechanism of intratumoral immunosuppression,
of PD1, downregulation of MHC-I and attraction of
which relies on CCL22 induction in DCs. Although
suppressive cell types including myeloid derived sup-
IL-1 signaling triggers additional anti-tumor effects,
pressor cells (MDSC) and regulatory T cells (Treg).
interruption of tumor derived CCL22 secretion still
Here we propose a novel IL-1-mediated immunosup-
might be a promising strategy in tumor immuno-
pressive mechanism leading to secretion of the Treg-
therapy.
attracting chemokine CCL22 by tumor-infiltrating
immune cells.
Our experiments revealed that conditioned media of
various human and murine tumor cell lines strongly induced CCL22 expression in cultured PBMCs or
isolated splenocytes, respectively. Further analysis
showed that most of the investigated tumor cell lines
either expressed IL-1 or strongly induced IL-1 expression in immune cells. Strinkingly, siRNA mediated
knockdown of IL-1α in tumor cells as well as blockade of IL-1 signaling by the IL-1 receptor antagonist
anakinra significantly inhibited the induction CCL22.
In support of these data, reporter assays utilizing
the proximal promoter of the CCL22 gene revealed
the direct activation of transcription of the CCL22
gene by IL-1 signaling. Furthermore, we identified
dendritic cells (DCs) as the main source of CCL22
both in human and mouse. The functional relevance
of reduced CCL22 secretion on Treg migration was
shown in a Transwell migration assay. Using subcutaneous tumor models we confirmed that blockade of
IL-1 signaling by anakinra reduced the intratumoral
and systemic CCL22 levels.
Escape of head and neck squamous cell carcinoma from NKG2D-dependent NK cell immunosurveillance can be restored by
NKG2D ligand depletion
Weil S.1,2, Memmer S.1,2, Huppert V.3, Giannattasio A.2, Becker T.4, Müller-Runte A.3, Lampe K.4, Beutner D.5,
Lechner A.5, Schubert R.6, Herrmann E.7, Koehl U.8, Walter L.4, Steinle A.9, von Bergwelt M.10, Koch J.1,2
University of Mainz Medical Center, Institute of
1
Medical Microbiology and Hygiene, Mainz,
Germany,
Johann Wolfgang Goethe-University, Institute for
7
Biostatistics and Mathematical Modelling, Frankfurt
am Main, Germany,
Georg-Speyer-Haus, Institute for Tumor Biology and
2
Experimental Therapy, Frankfurt am Main, Germany,
Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany,
3
Deutsches Primatenzentrum, Leibniz-Institut
4
für Primatenforschung, Göttingen, Germany,
Hannover Medical School, Institute for Cellular
8
Therapeutics, IFB-Tx, Hannover, Germany,
Johann Wolfgang Goethe-University, Institute for
9
Molecular Medicine, Frankfurt am Main, Germany,
University of Cologne, Department I of Internal
10
University of Cologne, Medical Faculty, Department
Medicine, Stem Cell Transplantation Program,
of Otorhinolaryngology, Head and Neck Surgery,
Cologne, Germany
5
Cologne, Germany,
Johann Wolfgang Goethe-University Hospital,
6
Pediatric Pulmonology, Allergy and Cystic Fibrosis,
Frankfurt am Main, Germany,
The natural killer group 2D receptor (NKG2D) on
sNKG2DLs. Consequently, monitoring of all sNKG-
natural killer (NK) cells recognizes several structur-
2DLs and TGF-β might provide a novel diagnostic
ally related cellular ligands (NKG2DLs) commonly
and/or prognostic marker combination in HNSCC.
overexpressed on tumor cells. However, soluble
Based on tumor spheroids, we show that inhibition
NKG2DLs (sNKG2DLs), released from malignant cells
of NKG2D-dependent cytotoxicity correlates with
by shedding, can inhibit NKG2D-dependent NK cell
drastically reduced NK cell infiltration. Importantly,
cytotoxicity. Consequently, previous studies showed
these data were in accordance with low infiltration
a correlation of elevated individual sNKG2DL plasma
frequencies found in primary tumors of HNSCC pa-
levels with impaired NK cell cytotoxicity in cancer
tients. In a proof-of-concept study in rhesus monkeys
patients.
(Macaca mulatta) infused with soluble human MICA,
Here we report on the sNKG2DL-dependent tumor
we could show quantitative depletion of sNKG2DLs
immune escape mechanism in HNSCC patients.
by adsorbtion apheresis after two plasma cycles on a
Compared to healthy controls, patient plasma levels
LIFE 18 apheresis unit. Therefore, adsorbtion aphere-
of sMICA, sMICB and sULBP1-3 were significantly
sis of sNKG2DLs and potentially other soluble factors
elevated with a peak maximum in the pre-meta-
prior to cellular cancer therapy with wildtype or en-
static stage III and correlated with tumor progres-
gineered immune cells might help to improve the
sion and disease staging characteristics. Moreover,
efficacy of cellular immunotherapy by restoration of
patients’ plasma containing high sNKG2DL levels
tumor infiltration and cytotoxicity.
and TGF-β inhibited NKG2D-dependent NK cell
cytotoxicity, which was restored after depletion of
CD103 defines intraepithelial CD8+ PD1+ tumor-infiltrating
lymphocytes of prognostic significance in endometrial
adenocarcinoma
Workel H.1, Komdeur F.1, Wouters M.1,2, Plat A.1, Klip H.1, Eggink F.1, Wisman G.1, Arts H.1, Oonk M.1, Mourits
M.1, Yigit R.1, Versluis M.1, Duiker E.3, Hollema H.3, de Bruyn M.1, Nijman H.1
University of Groningen, University Medical Center Groningen, Obstetrics and Gynecology, Groningen,
1
Netherlands,
University of Groningen, University Medical Center Groningen, Medical Microbiology, Groningen, Netherlands,
2
University of Groningen, University Medical Center Groningen, Pathology, Groningen, Netherlands
3
Introduction:
tumor-infil-
Conclusions: Due to the restricted expression on in-
trating lymphocytes (TIL) are associated with a
traepithelial CD8+ T-cells, CD103 may be a suitable
prolonged survival in endometrial cancer (EC). By
biomarker for rapid assessment of immune infiltra-
contrast, stromal infiltration of CD8+ TIL does not
tion of epithelial cancers. Furthermore, this intraepi-
confer prognostic benefit. A single marker to dis-
thelial tumor-reactive subset might be an interesting
criminate these populations would therefore be of
T cell subset for adoptive T-cell transfer and/or target
interest for rapid assessment of the tumor immune
for checkpoint inhibition therapy.
Intraepithelial
CD8+
contexture, ex vivo analysis of intraepithelial and
stromal T-cells on a functional level and/or adoptive
T-cell transfer. Here we determined whether CD103,
the αE subunit of the αEβ7integrin, can be used to
specifically discriminate the epithelial and stromal
CD8+ TIL populations in EC.
Methods: CD103+ TIL were quantified in a cohort
of 305 endometrial cancer patients by immunohistochemistry. Localization of CD103+ cells and coexpression of CD103 with CD3, CD8, CD16 and FoxP3
was assessed by immunofluorescence. Further phenotyping of CD103+ cells was performed by flow cytometry on primary endometrial tumor digests.
Results: CD8+CD103+ cells were preferentially
located in endometrial tumor epithelium, whereas
CD8+CD103- cells were located in stroma. CD103+
lymphocytes
were
predominantly
CD3+CD8+
T-cells and expressed PD1. The presence of a high
CD103+ cell infiltration was associated with an improved prognosis in patients with endometrial adenocarcinoma (p=0.035). Moreover, this beneficial
effect was particularly evident in high-risk adenocarcinoma patients (p=0.031).
Presence of immune infiltrates in early phases of prostate cancer:
Development of a preclinical efficacy model to promote
immunotherapy development
Konkol Y.1,2, Tuomela J.1,2, Suominen M.1, Halleen J.1, Bernoulli J.1
Pharmatest Services Ltd, Turku, Finland,
1
Institute of Biomedicine, University of Turku, Department of Cell Biology and Anatomy, Turku, Finland
2
Immunotherapy for prostate cancer has recently
Imbalance in hormone-milieu induced inflammation
emerged as an attractive treatment strategy. Yet,
in the prostate, followed by formation of prostatic in-
preclinical models where relationship between in-
traepithelial neoplasia (PIN)-like lesions and finally
flammation, stroma, tumor cells and prostate cancer
adenocarcinomas in the periurethral region. Very in-
progression can be studied are limited. In the present
terestingly, inflammatory cells, mainly T-cells were
animal models, mainly human cancer cells are used
noticed in the vicinity of PIN-like lesions. During the
in immunocompromised animals and interaction
progression of prostate cancer, inflammatory cells
between the immune system and cancer cannot be
disappeared from the adenocarcinoma sites. In the
monitored in these models due to the lack of active
prostate, inflammation consisting of perivascular,
immune system. As the requirement to test novel
stromal and periglandular T-lymphocytes and intra-
immunotherapies and especially combination treat-
luminal neutrophils remained.
ments is increasing, a preclinical model that takes
Results of this study indicate significance of hormo-
into account tumor microenvironment and immune
nal milieu, especially estrogens and androgens, in
system would be highly useful to promote develop-
the development of inflammation and progression of
ment of novel therapies to combat against prostate
prostate cancer, with a key role for tumor microenvi-
cancer.
ronment. Presence of lymphocytes in the proximity
Aim of the present study was to reveal if there is
of PIN-like lesions during the early phases of pros-
role between the immune system and development
tate cancer, and their disappearance later in the ad-
of prostate cancer, and secondly, to validate a model
enocarcinomas, indicate interaction between innate
to be utilized later in immunotherapy development.
and adaptive immune system and cancer. Therefore,
Intact 10-12 weeks old male Noble rats were s.c. im-
this preclinical prostate cancer model that combines
planted with slow-releasing estradiol and testoster-
immune system and cancer can be utilized when
one pellets for 6, 13 and 18 weeks. Estimated daily
new immunotherapies, combination treatments and
release for testosterone was 0.8 mg and for estradiol
prevention possibilities against prostate cancer pro-
0.08 mg. Control group animals received placebo
gression are developed.
hormone pellets without hormones. Serum samples
were collected during the study to monitor hormone
levels, and prostates were removed and processed for
histopathological evaluation at the end of the study.
Hormonal treatment caused an increase in estradiol
to testosterone ratio, and the prostates were enlarged.
PD-L1, Galectin-9 and CD8+ TIL are associated with patient
survival in Hepatocellular Carcinoma
Sideras K.1, Biermann K.2, Verheij J.3, Takkenberg B.4, Mancham S.1, Hansen B.1, Schutz H.1, de Man R.1,
Sprengers D.1, Buschow S.1, Verseput M.1, Boor P.1, Pan Q.1, van Gulik T.5, Terkivatan T.6, IJzermans J.6,
Beuers U.4, Sleijfer S.7, Bruno M.1, Kwekkeboom J.8
1
2
Academic Medical Center, University of Amsterdam, Department of Pathology, Amsterdam, Netherlands,
3
Academic Medical Center, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research,
4
Amsterdam, Netherlands,
Academic Medical Center, University of Amsterdam, Department of Surgery, Amsterdam, Netherlands,
5
6
Erasmus MC, Cancer Institute, Rotterdam, Netherlands,
7
Erasmus MC-University Medical Centre, Gastroenterology and Hepatology, Rotterdam, Netherlands
8
Novel systemic treatments for Hepatocellular Car-
Imunohistochemistry showed at least some expres-
cinoma (HCC) are strongly needed. Immunothera-
sion of PD-L1, Galectin-9, HVEM and IDO in tumor
py is a promising strategy that can induce specific
cells in 83%, 79%, 97% and 66% of tumors, respec-
anti-tumor immune responses. Understanding the
tively. Their expression was confirmed at the RNA
mechanisms of immune resistance by HCC is crucial
level. In the discovery cohort low tumor expression
for development of suitable immunotherapeutics.
of PD-L1 (p< .001), Galectin-9 (p< .001) and HVEM
Our aim was to examine the co-expression of the
(p< .001), and low CD8+TIL count (p=.016) were
immune inhibiting molecules PD-L1, Galectin-9,
associated with poor HCC-specific survival. PD-L1,
+
infiltrat-
Galectin-9 and CD8+TIL count were predictive of
ing lymphocytes (TIL) in HCC tumors, in associa-
HCC-specific survival independent of baseline clin-
tion with outcome in patients with resected HCC.
icopathologic characteristics, and the combination
Tissues from patients who underwent HCC resec-
of these markers was a powerful predictor of HCC-
tion in two academic centers were used to construct
specific survival (HR 0.29; p< .001). Expressions of
tissue microarrays containing cores taken from the
these same molecules in TFL tissue was not associ-
tumor area and from the tumor-free liver (TFL) area.
ated with HCC specific survival. These results were
Immunohistochemistry using validated antibod-
confirmed in the validation cohort where low tumor
ies was performed. Scoring of expression in tumor
expression of PD-L1 (p=.018), Galectin-9 (p=.047)
cells was performed by two independent investiga-
and low CD8+ TIL count (p=.092) were again as-
tors. Quantitative RT-PCR was performed on resect-
sociated with worse HCC-specific survival, and the
ed tumor and TFL tissue from 20 HCC patients as
combination of PD-L1, Galectin-9 and CD8 TIL also
additional control. Patients from the first academic
predicted HCC-specific survival independent of clin-
center (n=94) acted as the discovery cohort while
icopathologic characteristics (HR 0.43, p=.02). Low
the patients from the second center (n=60) as the
expression levels of PD-L1 and Gal-9 in combination
validation cohort.
with low CD8+TIL count predicts extremely poor
HVEM and IDO, as well as tumor CD8
HCC-specific survival, and it requires a change in
two of these parameters to significantly improve
prognosis.
Conclusion: PD-L1, Galectin-9, HVEM and IDO are
expressed at varying levels by tumor cells in the
majority of HCC patients. Low expression of PD-L1
and Galectin-9 and low CD8+TIL count are associated with poor HCC-specific survival. Upregulation
of PD-L1 and Galectin-9 expression in tumors in response to immunologic pressure may explain why
their presence is associated with better survival.
Individual immune biomarkers can be successfully
combined to form strong prognostic factors in HCC.
Ex vivo high-throughput T cell receptor profiling of a melanoma
patient’s peripheral tumor antigen-specific T cell repertoire
Lennerz V.1,2, Schrörs B.1, Lübcke S.1, Fatho M.1, Kukla K.1, Brettschneider J.1, Wölfel C.1, Pagel S.1, Zhao F.3,
Schadendorf D.3, Paschen A.3, Wölfel T.1,2
University Medical Center Mainz, Medical Department 3, Mainz, Germany,
1
University Cancer Center (UCT), Research Center for Immunotherapy (FZI), University Medical Center (UMC)
2
of the Johannes Gutenberg-University and German Cancer Consortium (DKTK), Partner Site Frankfurt/
Mainz, Mainz, Germany,
University Hospital, University Duisburg-Essen and German Cancer Consortium (DKTK), Partner Site Essen/
3
Düsseldorf, Department of Dermatology, Essen, Germany
In the melanoma model Ma-Mel-86, melanoma lines
ative frequencies of the respective T cells directly
were derived from four distinct lymph node metas-
ex vivo. For this purpose, 3.6 µg gDNA per PBMC
tases occurring over several years. The lines showed
sample, which is the genomic equivalent of about
various immune escape phenotypes including partial
550,000 cells, were subjected to the ImmunoSEQ
or complete HLA class I-deficiency, or loss of melano-
Assay. Using Adaptive Biotechnologies’ ImmunoSEQ
cytic differentiation antigens. By stimulation of au-
Analyzer platform, the TCR sequence repertoires of
tologous CD8+ T lymphocytes against HLA-proficient
both PBMC samples were analyzed for presence and
and -deficient melanoma lines, mixed lymphocyte-tu-
frequencies of the TCR sequences of interest.
mor cell cultures (MLTC) were generated and tumor-
Of 18 target TCR Vb-regions analyzed, 15 were found
reactive T cell clones were isolated thereof. Among
in PBMC from 2004, while PBMC from 2002 contained
these were CD3+/CD8+ ab-T cells recognizing HLA-
only two of the targeted sequences expressed by an
deficient melanoma lines. MLTC and T cell clones
HLA-restricted, mutation-specific T cell clone and by
were applied to forward (cDNA expression cloning)
an HLA -restricted T cell clone of unknown speci-
and reverse (next generation sequencing) approach-
ficity (CTL4C/44). However, the presence of specific
es for antigen identification. Antigens identified com-
TCR sequences in both samples collected more than
prised four mutated and three structurally normal
two years apart, indicates that the respective T cells
shared melanoma-associated antigens restricted by
were memory cells. Of note, six TCR Vb-sequences
HLA class I, and two antigens (transmembrane pro-
assigned to HLA-independent T cells were detected
teins TRP-2 and CSF2RA) recognized by T cells in an
only in PBMC from 2004 collected six months after
HLA-independent manner. Of 18 melanoma-reactive
the first HLA-negative metastasis became clinically
+
ab-T cell clones directed against at least 12
evident. One HLA-independent TCR and the TCR
distinct antigens, the highly diverse VDJ-junctional
of HLA-restricted CTL4C/44 reached high frequen-
regions containing the complementarity determining
cies (> 1%). TCRs against identified HLA-restricted
region 3 (CDR3) of expressed T cell receptors (TCR)
mutated and shared antigens were detected at inter-
were sequenced.
mediate (0,01-0,1%) to low (< 0,01 %) frequencies.
In order to investigate the relative contribution of the
HLA-independent TCRs were detected at high to in-
different T cell specificities to anti-tumor immunity
termediate frequencies. In conclusion, the anti-tumor
and immunoediting, the T cell repertoires of PBMC
TCR repertoire was broadened during the course of
from two time points (May 2002, August 2004) were
disease and was then dominated by HLA-independ-
analyzed by TCR Vb-chain deep sequencing (Adap-
ent TCR.
CD8
tive Biotechnologies, Seattle, USA) showing the rel-
Patient-derived tumor xenografts in humanized NSG and NSGSGM3 mice: A model to study immune responses in cancer
therapy
Yao L.-C.1, Cheng M.1, Wang M.1, Low-Marchelli J.1, Banchereau J.2, Shultz L.3, Palucka K.2, Keck J.G.1
The Jackson Laboratory, Sacramento, United States,
1
The Jackson Laboratory, Farmington, United States,
2
The Jackson Laboratory, Bar Harbor, United States
3
Humanized mice engrafted with tumors enable in
in hu-NSG-SGM3 mice. Human lymphocytes in Hu-
vivo investigation of the interactions between the
NSG-SGM3 mice infiltrated the transplanted tumor
human immune system and human cancer. We have
microenvironment. High PD-1 expression by CD8+ T
recently found that humanized NOD-scid IL2Rγnull
cells and Tregs were present within the breast cancer
(NSG) mice bearing patient-derived xenografts (PDX)
microenvironment suggesting anergy and immuno-
allow efficacy studies of check-point inhibitors. Next
suppression in hu-NSG-SGM3 mice. Treatment with
generation NSG strains include triple transgenic NSG
the anti-PD-1 receptor antibody pembrolizumab
mice expressing human cytokines KITLG, CSF2, and
(Keytruda) significantly reduced tumor growth and
IL-3 (NSG-SGM3). Here we provide a direct compar-
the PD-1 expression level on lymphocytes. Thus,
ison of check-point inhibitors evaluation in NSG and
PDX-bearing hu-NSG-SGM3 mice might serve as a
NSG-SGM3 mice engrafted with CD34+ human he-
new and improved platform for preclinical immu-
matopoietic progenitor cells (HPCs) from the same
no-oncology efficacy studies.
donor and implanted with PDX tumors. Corroborating earlier studies, reconstitution of human immune
system in the blood was faster and more robust in
NSG-SGM3 compared to NSG recipients throughout
the course of the study (18 weeks). Human CD45+
cells reached 25% of total blood cells at week 4 in
hu-NSG-SGM3 mice and at week 9 in hu-NSG mice.
A majority of blood hCD45+ cells (55%) in hu-NSGSGM3 at week 4 were CD33+ myeloid cells. By comparison, efficiency of human CD33+ cells (15%) in
the circulation was poor in hu-NSG mice. Moreover,
circulating hCD3+ T cells reached 10% at week 9 and
included regulatory T cells (Tregs). Hu-NSG mice displayed comparable hCD3+ T cells in the blood only
at 12-15 weeks and did not contain Tregs. PDX tumors
were then engrafted into partially HLA-matched huNSG-SGM3 mice at 9 weeks post engraftment. Two
PDX models and one cancer cell line with high PDL1
levels were used to test respond to anti-PD1 therapy
Into the deep: closer look at immune cells and immune checkpoint expression in human malignant pleural mesothelioma
Marcq E.1, Siozopoulou V.1,2, De Waele J.1, Van Audenaerde J.1, Zwaenepoel K.1,2, Hermans C.1, Hens N.3,4,
Pauwels P.1,2, van Meerbeeck J.1,5, Smits E.1,6
Center for Oncological Research, University of Antwerp, Antwerp, Belgium,
1
Department of Pathology, Antwerp University Hospital, Antwerp, Belgium,
2
Interuniversity Institute for Biostatistics and statistical Bioinformatics, Hasselt University, Diepenbeek,
3
Belgium,
Centre for Health Economics Research and Modeling Infectious Diseases, University of Antwerp, Antwerp,
4
Belgium,
Thoracic Oncology/MOCA, Antwerp University Hospital, Antwerp, Belgium,
5
Laboratory of Experimental Hematology, University of Antwerp, Antwerp, Belgium
6
Objectives: Immune checkpoints, such as programmed
formed to examine correlations between the expression
death-1(PD-1), are responsible for controlling and inacti-
data and several clinicopathological parameters of the
vating the immune system in order to avoid autoimmun-
patients.
ity and prevent tissue damage. Blocking the ligand-im-
Results: IHC showed PD-1 expression on lymphocytes
mune checkpoint interaction already showed promising
in 65% of the untreated samples and 71% of the treated
results in several cancer types. Data derived from a small
samples, while the expression on tumor cells was found
number of mesothelioma patients suggest that blocking
in 10% of the untreated samples. PD-L1 expression on its
immune checkpoints could offer new treatment oppor-
turn was seen on lymphocytes and on tumor cells, for
tunities. Gaining more insight in the immunological
the latter only in untreated tissues. All samples showed
aspect of the tumor microenvironment (TME) in human
CD45RO positive lymphocytes. CD4+ and CD8+ lym-
malignant pleural mesothelioma (MPM) would be of
phocytes were found in the stroma and in hotspots of
great interest to develop an efficient immunotherapy.
the lymphoid aggregates. A subset of the CD4+ cells was
Therefore, we investigated the expression of PD-1 and
also FoxP3+ and more CD4+FoxP3+ cells were found
its ligand PD-L1 in human MPM and identified different
in lymphoid aggregates of the treated versus untreated
subsets of immune cells in the TME.
samples(80% vs 50%). Stromal CD68+ histiocytes and
Methods: Immunohistochemistry (IHC) was used to
macrophages were found in all tissue samples. FCM
examine the expression of several immune cell markers,
showed that there was more immune infiltration in
as well as the expression of PD-1 and PD-L1, in formalin
ascites fluid compared to pleural fluid. PD-1 expression
fixed paraffin embedded (FFPE) tissue of 54 MPM pa-
was found on CD4+ and CD8+ T cells and on natural
tients (40 at diagnosis, 14 treated with chemotherapy).
killer cells. PD-L1 was expressed on dendritic cells, mac-
Identification of different subsets of cells present in MPM
rophages, B-cells and podoplanin+ tumor cells. Statisti-
fluids (ascites and pleura) was done using multicolor
cal analysis showed there was no significant difference
flow cytometry (FCM). Statistical analysis is being per-
between the cellular immune composition of pleural and
ascites fluid. The same holds true for the expression of
immune checkpoints on different subsets of immune
cells.
Conclusion: IHC and FCM revealed the diversity of
immune cells present in MPM tissue and fluid samples.
Our data on high expression of PD-1 and PD-L1 support
further investigation of the effect of immune checkpoint
blockade in MPM, as stand-alone treatment and in combination with other therapies. Reactivating immune
responses that are silenced by immune checkpoint receptor-ligand interaction might offer new opportunities
for the improvement of therapeutic strategies for MPM.
Surface staining of PD-1 discriminates viable cell populations
Metzger P.1, Kirchleitner S.V.1, Ritter I.1, Endres S.1, König L.1, Duewell P.1, Schnurr M.1
Klinikum der Universität München, LMU, Division of Clinical Pharmacology, München, Germany
1
Background: Checkpoint inhibitors, such as anti-PD-
treated with staurosporine were analyzed. Again,
1 mAb, show very promising results in the treatment
PD-1 staining was enriched on apoptotic tumor cells.
of various cancer types. PD-1 is mostly expressed
Conclusion: Both anti-PD-1 mAbs used in this study
on T cells and signaling dampens T cell activation
recognize another so far unidentified marker ex-
leading to anergy and apoptosis. There is increas-
pressed by dying or apoptotic cells. Whether this
ing evidence that PD-1 is also expressed on other
is PD-1 or another so far unrecognized molecule is
immune cells such as myeloid-derived suppressor
focus of ongoing research. In studies applying PD-1
cells (MDSC) as well as cancer cells. However the
staining in tumor tissue, dead cell markers should
function of PD-1 on non-T cells is poorly understood.
be included to rule out a possible bias introduced by
Methods: Primary murine immune cells and differ-
dead or dying cells.
ent cancer cell lines (Panc02 and T110299 pancreatic
cancer and B16F10 melanoma) were stained with
anti-PD-1 mAb and analyzed by FACS analysis. Cell
surface expression was evaluated with two different
antibody clones (29F.1A12 and RMP1-14). To discriminate viable from dead cells, co-staining with 7-AAD,
propidium iodide, fixable viability dye and caspase-8
substrate was done.
Results: As expected, activated T cells expressed
high levels of PD-1. MDSC (CD11b+ Gr1+) isolated
from pancreatic tumors showed distinct PD-1 expression as well. However, PD-1 expression directly correlated with reduced viability and apoptosis markers
such as caspase-8, whereas none of the viable cells
stained positive for PD-1. Treatment of tumor-bearing mice with Poly(I:C), a synthetic RNA analogon,
resulted in increased activation of caspases in MDSC
subpopulations. This was accompanied by positive staining for PD1 and reduced cell viability. To
assess whether PD-1 staining correlates to signs of
cell death, Panc02, T110299 and B16F10 tumor cells
Identification of cytotoxic miRNAs in human T cell-released
exosomes against mesenchymal stem cells
Momose F.1,2, Seo N.1,2, Harada N.1,2, Sawada S.2,3, Akiyoshi K.2,3, Shiku H.1,2
Mie University Graduate School of Medicine, Department of Immuno-Gene Therapy, Tsu, Japan,
1
ERATO Akiyoshi Bio-nanotransporter Project, Japan Science and Technology Agency (JST), Tokyo, Japan,
2
Kyoto University, Graduate School of Engineering, Kyoto, Japan
3
Introduction: Our group has found in murine models
ing the active translocation of endogenous cellular
that CD8+ T cell-released exosomes play as an inhib-
miRNAs into exosome cargos in a G-rich sequence-
itor for tumor progression including invasion and me-
dependent manner when multivesicular bodies were
tastasis by destructing mesenchymal tumor stromal
generated in donor T cells. By the real-time cell anal-
cells in part micro (mi) RNA-mediated manner. In
ysis, we identified two exosomal miRNAs (miR-6089,
this study, we tried to identify cytotoxic miRNAs in
miR-6090) capable of exhibiting cytotoxicity and the
human T cell-released exosome-dominant miRNAs
attenuation of cell growth of MSCs. The total mRNA
against mesenchymal stem cells (MSCs) in vitro.
array of cytotoxic miRNA-introduced MSCs revealed
Methods: Exosomes were purified from the culture
the correlation between MSC cytolysis and interferon
supernatants of CD3- and RetroNectin-stimulated
(IFN)-related gene expressions.
PBMCs (3 healthy donors) by the filtration (450 nm
Conclusion: We demonstrated that anti-MSC miRNAs
and 220 nm) and ultracentrifugation (120,000 g, 70
existed into human T cell-released exosomes consist-
min) after removing cells and debris. Exosome-dom-
ent with the study of murine CD8+ T cell-released ex-
inant miRNAs were selected by comparative analysis
osomes. Our study may propose pivotal insights for
of microarray data (Toray 3D-gene) of the exosomal
clinical application of exosomal miRNAs in patients
miRNAs with the donor T cell miRNAs, and then
with invasive and metastatic cancer.
synthesized to examine cytotoxicity against MSCs.
Cytotoxicity of each synthesized miRNA against
adipose tissue-derived MSCs cells was examined
after transfection with Lipofectamine RNAiMAX (Invitrogen) by a real-time cell analyzer (xCELLigence
[ACEA Biosciences]).
Results: CD8+ T cells enriched in the culture process
of PBMCs produced exosomes of approximately 150
nm size. The CD8+ T cell-released exosomes showed
the expression of not only tetraspanins (CD9, CD63,
CD81) but also CD8 and HLA-I molecules. miRNAs
from CD8+ T cell-released exosomes showed different distribution pattern from donor T cell miRNAs.
Interestingly,
exosome-dominant
miRNAs
were
characterized as guanine (G)-rich miRNAs, indicat-
A Crohn´s related colonic carcinoma cell line showing features of
immunoselection is recognized by re-activated autologous tumorinfiltrating lymphocytes as well as CIK cells
Mullins C.1, Harnack C.1, Kuehn F.2, Krohn M.1, Klar E.2, Linnebacher M.1
University Medicine Rostock, Department of General Surgery, Molecular Oncology and Immunotherapy,
1
Rostock, Germany,
University Medicine Rostock, Department of General Surgery, Rostock, Germany
2
The majority of patients with Crohn´s disease (CD)
main reactivity is directed against the cancer testis
are under immunosuppression which generates a dif-
antigens expressed in high levels is currently under
ferent environment for tumor escape and growth, e.g.
investigation.
azathioprine leading to a mismatch repair deficiency.
A worse clinical outcome for patients with Crohn´s
related carcinoma compared to sporadic colorectal
carcinoma (CRC) was shown. Pre-clinical in vitro
models representing molecular features of CAC are
mandatory for functional testing of immunotherapeutical interventions and to predict responses. We
recently established matching patient-derived tumor
cell line, xenograft (PDX) as well as EBV-immortalized B cells and cultures from tumor-infiltrating lymphocytes from an extremely fast growing CD tumor
of a long-term immunosuppressed patient (HROC69).
The tumor cells express a variety of cancer testis antigens and the immune checkpoint molecules CD47,
CD274 and CD276 but express only marginal levels of
HLA class I. Tumor infiltrating T cells could readily
be expanded in standard conditions as well as using
high amounts of IL-2 (thus these cultures represent
cytokine-induced killer cells). Lymphocytes were
highly activated and recognized autologous tumor
cells in co-culture experiments. Whether or not the
mIRNA-155 shuttling through gap junctions facilitates CLL
progression
Nesmiyanov P.1,2, Strygin A.1,3, Tolkachev B.1, Kaplanov K.4, Dotsenko A.1,3, Strygina A.1,3
Volgograd State Medical University, Fundamental Medicine and Biology, Volgograd, Russian Federation,
1
Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Immunology,
2
Moscow, Russian Federation,
Volgograd Medical Science Center, Pharmacology Department, Laboratory for Genomics and Proteomics,
3
Volgograd, Russian Federation,
Volgograd Regional Clinical Oncology Dispensary No.1, Department of Hematology, Volgograd, Russian
4
Federation
Connexin-mediated gap junctions (GJ) allow ex-
with LSCs in presence of GJi. Control antagomir-treated
change of miRNA (miR) between cells. We have
CLL cells had decreases apoptosis rate. High miR-155
previously demonstrated that Cx43, is decreased in
expression was associated with increased survival.
CLL-derived B cells, however undocking from ZO-1
We further tested whether the transported miRNAs
allows for greater GJ size and increased GJ communi-
are functional in vivo. Same cells after co-cultiva-
cation between adjacent cells. miR-155, miR-21, miR-
tion and separation and with/without antagomir
132, miR-212, miR-181, miR-9/7a may confer carcino-
treatment were analyzed for miR-155 and were xeno-
genic effects in leukemia; leukemia cancer stem cells
grafted to 4-8-week-old NSG mice (10 per each cell
(LSCs) may have characteristic miRNA profile. We
type, non-irradiated) by i.v. injection of approx. 104
have hypothesized that GJ-mediated miRNA shut-
cells/mouse along with CD34+- and CD19+-deplet-
tling between leukemic cells may have functional
ed allogeneic PBMCs. Mice injected with miR-155hi
consequences.
LSCs developed leukemia within 21 days post injec-
In order to test our hypothesis we used IGHV-unmu-
tion (9/10, median peripheral CD19+/CD5+ count
tated B cells from CLL Binet stage C patients. Cells
was 25% of all CD45+ cells), with a median sur-
were sorted into CD34+/CD38- (LSCs) (also CXCR4+)
vival time of 59 days. Mice injected with CLL cells
and CD19+/CD5+/CD34- (CLL) cells. Cells were
pre-cultivated with LSCs developed leukemia within
typed for differential miRNA expression, reveal-
21 days post injection (7/10, median CD19+/CD5+
ing 3-fold higher expression of miR-155 in CD34+/
count was 18% of all CD45+ cells) with a median
CD38- cells (LSCs) compared to CLL cells (P < 0.05).
survival time of 51 days (In 4 mice engraftment was
Cells then were co-cultured for 24-48h to allow GJ
transient, probably because of rejection). In contrast,
formation with/without GJ inhibitors (GJi) (50 µM
intact CLL cells or pre-treated with antagomir did
18α-AGA, 1 mM 1-octanol or 100 µM carbenoxolone).
not initiate leukemia in NSG mice (0/10; follow up
Then cells were separated and cultivated for another
12 weeks). Cells proliferating in vivo expressed a
24 h. CLL cells pre-cultivated with LSCs showed sig-
hCD45+hCD3−hCD19+hCD5+ phenotype, indicat-
nificantly decreased apoptosis rate then intact cells
ing they derived from the CLL PBMC inoculum.
or cells co-cultivated in presence of GJi.
Taken together, this is the first evidence that homocel-
To confirm that GJi block miRNA shuttling we repeat-
lular GJ shuttling of LSC-derived miRNA prolong sur-
ed the experiment, treating cells with 100 nM miR-
vival of B cells in vitro and promote transformation
155-specific or control antagomirs (on ice, 30 min). Ap-
of CD19+ cells to leukemia-initiating cells with LSC
optosis rate of antagomir-treated LSC-co-cultivated CLL
features in vivo.
cells was similar to intact cells or cells co-cultivated
Investigation of paracrine immunomodulatory effects of
mesenchymal stem cells on the CD4+ T cell subsets
Ozdemir A.T.1, Özgül Özdemir R.B.2, Kırmaz C.3, Eker Sarıboyaci A.4, Ünal Halbutoğulları Z.S.5, Özel C.5, Karaöz E.6
Ege University Institute of Health Sciences, Stem Cell, Izmir, Turkey,
1
Manisa State Hospital, Allergy and Clinical Immunology, Manisa, Turkey,
2
Celal Bayar Medical School, Allergy and Clinical Immunology, Manisa, Turkey,
3
Eskişehir Osmangazi University, Cellular Therapy and Stem Cell Production, Application and Research
4
Center, Eskişehir, Turkey,
Kocaeli University, Stem Cell and Gene Therapy Research and Application Center, Kocaeli, Turkey,
5
Liv Hospital, Regenerative Medicine and Stem Cell Production Center, Istanbul, Turkey
6
Background: Mesenchymal stem cells (MSCs) are
were performed by using CD4, CD25, Tbet, Gata3,
adult stem cells that have a broad tissue distribution
Stat3 and FoxP3 mAb for evaluation of frequencies
and multipotent differentiation capabilities. The tu-
of T cell subsets.
morigenic ability of MSCs are controversial and the
Results: The proliferation of T cells were signifi-
knowledge about interactions between MSCs and
cantly suppressed by the MSCs. The levels of T cell
tumor immunity are limited. CD4+ T cell subsets
specific cytokines IFG-g, IL-4 and IL-17a significantly
and their plasticity play important role in the tumor
decreased in the co-culture groups compared to the
immunity. MSCs may affect the plasticity of CD4+ T
control groups, but IL-10 and TGF-b1 increased. HGF
cell subsets via secretion of specific cytokines. In this
significantly increased in co-culture groups compared
study, we aimed to show the paracrine immunomod-
to control groups, however IDO, PGE2 and sHLA-G
ulatory effects of MSCs on the CD4+ T cell subsets
levels showed a slight increase, but these increases
to understand the mechanism of cellular interactions
were not statistically significant. The frequency of
at cytokine level.
CD4+/Tbet+ and CD4+/Gata3+ T cell significantly
Method: Dental and peripheral blood samples were
decreased in co-culture group compared to control
taken from volunteers for the isolation of MSCs and
group, but CD4+/Stat3+ and CD4+/CD25+/FoxP3+
CD4+ T cells. After the activation of CD4+ T cells via
T cell increased.
the PHA, MSCs and CD4+ T cells were co-cultured
Conclusion: The plasticity of Th17 cells tightly regu-
for 4 days with the transwell (indirect) co-culture
lated with environmental signals and tumor micro-
system. WST-1 analysis were performed for evalua-
environment derived signals may effect the Th17
tion of T cell proliferation.The levels of IFN-g, IL-4,
cells. Stat3 is an important transcription factor for
IL-17a, IL-10, TGF-b1, IDO, PGE2, sHLA-G and HGF
Th17 differentiation and activation. In this study, we
were measured in the conditioned culture superna-
showed that the frequency of CD4+/Stat3+ T cells
tant by ELISA method for evaluation of T cells and
have increased, but IL-17a levels decreased with ex-
MSCs activities. The flow cytometry (FCM) analysis
istence of MSCs. Furthermore, we detected signifi-
cantly higher levels of HGF which acts as a ligand
for the c-met that allows activation of Stat3, in coculture group compared to control group. As a conclusion, MSCs successfully suppressed the activated
CD4+ T cells. The HGF expression of MSCs may have
important role for plasticity of Th17 and Treg cells
and also emergence of immunosuppressive effects.
The immunosuppressive effects of MSCs should be
further investigate to understand the contribution of
tumor growth.
The immunohistochemical investigation of CD44, CD133,
NANOG, OCT 3/4, HLA-G and HLA expressions in the advanced stage breast cancer
Özgül Özdemir R.B.1, Ozdemir A.T.2, Kırmaz C.1, Oltulu F.3, Yiğittürk G.3, Kurt K.4
Celal Bayar Medical School, Clinical Immunulogy and Allergy, Manisa, Turkey,
1
Ege University Institute of Health Sciences, Stem Cell, Manisa, Turkey,
2
Ege University Medical School, Histology and Embriolgy, Izmir, Turkey,
3
Merkezefendi State Hospital, Medical Pathology, Manisa, Turkey
4
Tumor initiating (TICs) or Cancer Stem Cells (CSCs)
CSCs markers increased in the advanced stage breast
are directly associated with bad prognosis and they
cancer patients, however the expressions of HLA-G
show multiple adaptations for resistance of the chem-
and HLA-E decreased. Further prospective studies
otoxicity, radiotoxicity and the immune evasion.
are needed to confirm our findings and also under-
These cells show different adaptations to escape
stand relationship with disease prognosis and the
from the immune cells mediated cytotoxicity, such
decreased expression of HLA-G and HLA-E.
as cytotoxic T lymphocytes (CTLs) and Natural Killer
(NK) cells. The down regulation of the major histocompatibility (MHC) antigens of tumor cells provide
protection against the CTLs, but with this adaptation
they become a target for the NK cells. The tumor cells
develop an alternative adaptations to protection from
cytotoxic effect of NK cells. One of these adaptations
is upregulation of nonclassical MHC antigens such as
HLA-G and HLA-E.
In this study, we aim to histologically investigate
HLA-G, HLA-E, CD133, CD44, NANOG and Oct3/4
expressions of advanced stage breast cancer. For this
purpose, we immunohistochemically stained the
paraffin embedded tissue sections from obtained advanced stage breast cancer (n=10) and non-cancer
breast biopsies (n=10). We used the H-score calculation for the semi quantitatively evaluation of
staining intensity of cells. We found that the CD133,
CD44, NANOG and Oct3/4 expressions increased in
the advanced stage breast cancer patients compared
with the control patients, but HLA-G and HLA-E decreased.
Several studies demonstrated that the cytotoxic
effects of CTLs and NK cells can be inhibit by the
HLA-G and HLA-E. According to our findings, the
Evaluation of potential factors contributing to immunosuppression
in the PDL1 positive tumor microenvironment
Park E.1, Wilkens K.2, Ma X.-J.1, Li N.1
Advanced Cell Diagnostics, Hayward, United States,
1
Advanced Cell Diagnostics, Segrate, Italy
2
Checkpoint blockade is being established as a new
markers known to contribute to immune regulation
paradigm in cancer treatment with durable tumor
(including TGFb and chemokines/receptors), and (4)
regression prolonged stabilization of disease in pa-
spatial distribution of regulatory T (Treg) cells that
tients with advanced cancers, including non-small-
are instrumental to the maintenance of immunesup-
cell lung cancers (NSCLC). Programmed death 1
pressive tumor microenvironment.
(PD-1) protein, a T-cell co-inhibitory receptor, and
The approach presented here may reveal complex
one of its ligands, PD-L1, play a pivotal role in the
mechanism behind immunosuppression, contribut-
ability of tumor cells to evade the host´s immune
ed by multiple cell types and factors, and may ulti-
system and blockade of interactions between PD-1
mately provide potential insights to restore immune
and PD-L1 provided long lasting clinical benefits in
function guided by proper therapeutic interventions.
many patients. Despite the clinical efficacy observed
Furthermore, by evaluating co-expression profiles
in many patients, many more cancer patients do not
of multiple therapeutic targets, this approach may
respond to PD-1/PD-L1 checkpoint inhibition. Under-
contribute to identifying candidates for combination
standing the roles and markers of tumor infiltrating
therapies either blocking multiple checkpoints or by
immune cells and stromal cells in the tumor micro-
combining with other targeted therapeutics.
environment, along with PD-L1 expression, may be
important in predicting clinical benefit of immune
checkpoint inhibitors and determining combinational therapies.
In this report, we evaluated gene expression of potential factors contributing to immunosuppression
in the PD-L1 positive tumor environment by applying RNAscope®, a highly sensitive and specific
in-situ RNA detection method. Using archived, formalin-fixed paraffin-embedded tumor specimens
from primary NSCLC patients, we first screened for
PD-L1 expression. With 20 selected cases with PD-L1
positive expression, we then evaluated (1) tumor vs.
immune cell types expressing PD-L1, (2) co-expression of PD-L1 with other immune checkpoint markers
(including PD-1, PD-L2, LAG3, PDCD-1), (3) stromal
Identifying the kinases and phosphatases regulating STAT3
with potential dual anti-cancer and immunotherapeutic effects
Parri E.1, van Adrichem A.1, Kaustio M.1, Turunen L.1, Vähä-Koskela M.1, Wennerberg K.1
University of Helsinki, Institute for Molecular Medicine, Finland (FIMM), Helsinki, Finland
1
STAT3 is a critical immuno-oncology regulator since
one that hyperactivated STAT3 mutants. These seven
it acts both as an oncogene driving tumor progres-
hits are genes that are known to be activated (i.e.
sion and drug resistance as well as many immuno-
CSNK2A1, DDR2) or deregulated (CDK8, CSK) in dif-
suppressive and immunomodulatory processes both
ferent cancers. Notably no significant differences
in the tumor cells and immune cells. Pancreatic
were found between regulation of wild type and
ductal adenocarcinoma (PDAC) is a highly aggres-
the hyperactivated STAT3 mutant. No JAK- family
sive cancer that is characterized by a strongly immu-
kinases, but other previously described regulators
nosuppressive microenvironment. A striking genetic
such as casein kinase 2 (encoded by CSNK2A1) were
feature of PDAC is the early emergence of KRAS muta-
found among the validating hits. Furthermore, the
tions and interestingly, STAT3 plays a major role to
activating knockdown hit was CSK, which phos-
promote the mutant KRAS-driven progression into a
phorylates a negative regulatory site of Src family
cancer. Simultaneously, STAT3 signaling in stromal
tyrosine kinases, which in turn are well known to
fibroblasts and myeloid cells promotes development
positively regulate STAT3 activity. Ongoing studies
of a highly immunosuppressive tumor microenviron-
are focused on the mechanism of STAT3 regulation
ment. Therefore, targeting STAT3 signaling in PDAC
by the hit gene products and whether targeting these
is a promising approach to both make the cancer sus-
kinases can modulate the drug responses and im-
ceptible to both chemotherapy and immunotherapy.
munoregulatory capacity of PDAC cells.
STAT3 activity is regulated through protein phos-
In conclusion, we have found several novel targeta-
phorylation. Interestingly, in PDAC cells STAT3
ble putative STAT3-regulatory proteins that are being
phosphorylation appears to be independent of the
further explored for their role in PDAC tumorigenesis
prototypical STAT-activator JAK-family kinases. To
and immunosuppression
better understand how STAT3 is regulated in PDAC
and how it may be optimally targeted, we set out
to identify protein kinases and phosphatases that
regulate STAT3 activity. We established cell lines
with a STAT3 responsive luciferase reporter element
to stably express STAT3(wt) or hyperactivated
STAT3(Y640F), and screened them against a protein
kinase and phosphatase siRNA library. After primary
and validation siRNA screens we found 6 proteins
whose silencing inhibited the activity of STAT3 and
CD40L+CD4+CD8+ intra-tumor double positive T cells:
a helper player in melanoma
Parrot T.1, Oger R.1, Benlalam H.2, Raingeard de la Blétière D.1,3, Pressier L.1, Khammari A.1,4, Dréno B.1,4,
Labarrière N.1, Guardiola P.1,3, Delneste Y.1, Gervois N.1
Centre de Recherche en Cancérologie Nantes-Angers - UMR 892 INSERM / 6299 CNRS / Université Nantes
1
Angers, Nantes, France,
Centre de Recherche en Cancérologie Nantes-Angers - INSERM U892/CNRS 6299, Nantes, France,
2
SNP Transcriptome & Epigenomics Facility, CHU, Angers, France,
3
Unit of Skin Cancer, CHU, Nantes, France
4
While CD4+CD8+ double positive (DP) T cells account
to memory B cells. In addition, DP T cells induced a
for a small fraction of peripheral blood lymphocytes
full maturation of monocyte-derived dendritic cells
in healthy humans, we previously demonstrated an
based on CD83, CD80, CD86 and HLA-DR increased
accumulation of tumor-reactive DP T cells within
expression. Moreover, these matured dendritic cells
melanoma infiltrating lymphocytes, which corre-
were able to efficiently prime cytotoxic CD8 T cell
lates with advanced cancer stage, supporting a role
responses against the melanoma antigen Melan-A.
of these cells in the regulation of anti-tumor immune
Taken together, our results described a non-conven-
responses. Diverse functions from regulatory to cy-
tional class-I restricted DP T cell population present-
totoxic properties were attributed to DP T cells ac-
ing helper properties in the melanoma infiltrate that
cording to the context of the study. In solid tumor,
could activate in vitro B lymphocytes and dendritic
the functions of these cells remain to be elucidated.
cells and thus potentiate an anti-tumor immune re-
Our previous work showed that similarly to their CD8
sponse.
counterparts, intra-melanoma DP T cells are class-I
restricted but are distinguished by a limited lytic
activity against autologous melanoma cells, ruling
out the hypothesis of a cytotoxic role as their main
function.
Based on a comparative transcriptome analysis
between intra-melanoma single positive (SP) CD4,
SP CD8 and DP T cells, we observed that DP T cells
presented a differential expression of the CD40L, a
key molecule involved in CD4 T cell-mediated help,
leading us to investigate their helper function. Upon
TCR activation, a significant fraction of DP T cells
(70%) was able to induce CD40L expression at an intermediary level compared to SP CD4 (90 %) and SP
CD8 T cells (30%). We investigated the CD40L-mediated helper effect of DP T cells on B lymphocytes and
dendritic cells. First, we observed that the co-culture
of activated DP T cells and CD19+ B lymphocytes led
to a significant B cell proliferation that was limited
Immune evasion by melanoma: Modification of the skin and
lymph node immune cell network in a spontaneous melanoma
mouse model
Prokopi N.1, Mairhofer D.G.1, Tripp C.H.1, Ortner D.1, Komenda K.1, Chen S.2, Clausen B.E.3, Stoitzner P.1
Medical University of Innsbruck, Dermatology, Venereology and Allergollogy, Innsbruck, Austria,
1
Rutgers University, Susan Lehman Cullman Laboratory for Cancer Research, New Jersey, United States,
2
University Medical Center (UMC) of the Johannes Gutenberg-University, Institute for Molecular Medicine,
3
Mainz, Germany
Melanoma is the most fatal type of skin cancer.
Skin dendritic cells (DC) can have a major role in
melanoma development since they are the most important sensors in this tissue; at the same time they
can bridge innate and adaptive immunity and determine the outcome of the immune responses that
are triggered. In this study we use a spontaneous
melanoma mouse model, the tg(Grm1)EPv. In this
model, the ectopic overexpression of metabotropic
glutamate receptor 1 (Grm1) in melanocytes leads to
their uncontrolled proliferation and confers to them
an anti-apoptotic phenotype. We have characterized
the immune cell network changes that occur in the
skin and the tumor draining lymph nodes in regards
to skin DC as well as effector cells. Our results show a
clear decrease in the skin DC, with this event already
occurring very early in tumor development. Understanding the processes underlying these changes will
offer useful information in regards to the role of the
different skin DC subsets in tumor antigen presentation and activation of CD8+ T cells. According to
previous studies, Grm1 overexpression has also been
detected in 60% of patient samples, making our findings even more clinically relevant.
Characterization of the cancer immune microenvironment of
Mdr2(Abcb4)-/- mice treated with Diethylnitrosamine and
Phenobarbital - A novel model close to human Hepatocracinogenesis
Qureshi M.A.1,2, Ashfaq-Khan M.1, Kim Y.O.1, Aslam M.1,3, Heck R.1, Zimmermann T.4, Foerster F.1, Schuppan D.1,5
Institute of Translational Immunology & Research Centre for Immunotherapy (FZI), University Medical Centre
1
of the Johannes Gutenberg University of Mainz, Mainz, Germany,
Dow University of Health Scienes, Karachi, Pakistan,
2
Shaheed Benazir Bhutto Women University, Peshawar, Pakistan,
3
Dept. of Medicine 1, University Medical Centre of the Johannes Gutenberg University of Mainz, Mainz,
4
Germany,
Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical
5
School, Boston, United States
Background: Hepatocellular carcinoma (HCC) is the
th
at 3,5,7 and 9 months and liver samples were for-
most common
malin fixed for histopathological analyses. Tissues
cause of malignancy-associated deaths worldwide.
were stained using H&E, an antibody against the
Alarmingly, treatment options for this highly rele-
myeloid M2-marker YM-1, and Sirius-red for colla-
vant malignancy are limited. HCC is a promising but
gen. Immune cell densities were quantified as cells/
largely unexplored candidate for immunotherapy,
mm 2 using the College of American Pathologists
since it usually develops in an inflammatory envi-
guidelines. Degree of fibrosis was scored according
ronment (modulated by advanced fibrosis/cirrho-
to Ishak criteria. Data were entered and analyzed via
sis). However, current mouse models have limited
SPSS using paired t-test, ANOVA and Chi square test.
resemblance to human HCC, little is known about
Results: Compared to all controls, the Mdr2(Abcb4)-/-
the immune cell complexity of HCC, and appropriate
mice treated with DEN and PB showed a distinct
approaches to immune therapy are lacking.
and significantly different pathology in terms of
Objective: To characterize the liver cancer immune
neoplastic progression, immune cell infiltration and
infiltrate relative to fibrosis/cirrhosis in our recently
degree of fibrosis. Livers from these mice were in-
developed model of HCC using Mdr2(Abcb4)-/- mice
creasingly infiltrated with preneoplastic/neoplastic
(abstract, Kim,YO et al).
hepatoma cells (with pleomorphic hyperchromatic
5
commonest malignancy and 3
rd
Methodology:
Mdr2(Abcb4)-/-
strain)
injected
mice
(FVB
nuclei, prominent nucleoli and abundant eosinophil-
intra-peritoneal
ic cytoplasm), surrounded by congested sinusoidal
diehthylnitrosamine(DEN)(10µg/g bw) at the age
areas, from 3 months on. Microscopically, tumour
of 5-days, followed by 0.05% phenobarbital(PB) in
phenotype in these mice ranged from pseudo-tubu-
drinking water starting at 3-weeks. Mdr2(Abcb4)-/-
lar (at 3months) to trabecular morphology (5months
mice that received no DEN and PB, and FVB wildtype
onwards). There was a prominent peri-portal inflam-
mice w/wt DEN and PB treatment were included as
mation, with acute (neutrophils) and chronic (lym-
controls(n=6 per group). All mice were sacrificed
phocytic) infiltrates already at the age of 3 months,
were
with
and marked necro-inflammation from 5 months
onwards. Notably, there was significantly increased
infiltration of alternatively activated macrophages
(M2 phenotype) in the Mdr2 (Abcb4)-/- DEN and PB
treated mice, largely in the intra-tumoural compartment, as early as 3 months. Moreover, these mice
had a more advanced liver fibrosis ranging from portal-portal and portal-central fibrotic bridging (3&5
months) to severe fibrosis (7&9 months).
Conclusion: We show early tumourgienic, necroinflammatory and fibrotic changes in a novel mouse
model close to human HCC. This model will be exploited to identify novel targets for immune therapy.
Increased CD4+ and CD8+ lymphocytic infiltration in patients
with triple negative breast cancer suggests susceptibility to immune therapy
Qureshi M.A.1,2, Bushra S.2, Khan S.2, Ujjan I.D.3, Mirza T.2, Zahid M.2, Schuppan D.1,4
Institute of Translational Immunology & Research Centre for Immunotherapy (FZI), University Medical Centre
1
of the Johannes Gutenberg University of Mainz, Mainz, Germany,
Dow University of Health Scienes, Karachi, Pakistan,
2
Liaquat University of Medical and Health Sciences, Hyderabad, Pakistan,
3
Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical
4
School, Boston, United States
Background: Patients with triple negative breast
Results: Of the 104 breast cancer patients studied a
cancer (TNBC) have limited conventional therapeutic
total of 27 (25%) had TNBC and 77(74%) non-TNBC.
options. These patients are potential, but largely un-
Patients with TNBC showed significantly increased
fathomed, candidates for immunotherapy. However,
infiltration of lymphocytes (T and B cells) compared
immune cell complexity of TNBC is largely under-
to the patients with non-TNBC, while myelocytic in-
studied and demands further exploration.
filtration was not significantly different in the two
Objective: To investigate tumour associated immune
groups. Within the TNBC group, infiltration of T-
cell densities in patients with breast cancer with a
lymphocytes was significantly higher compared to B-
particular focus on TNBC.
lymphocytes. However, CD4 and CD8 infiltration was
Materials and methods: A total of 104 consecutive
not significantly different within the TNBC group.
breast cancer patients undergoing mastectomy were
Conclusion: Patients with TNBC show increased
recruited in the study after ethical approval. Clini-
lymphocytic (both T and B lymphocytes) infiltration
co-pathological parameters were recorded and H&E
compared to the patients with non-TNBC. Moreover,
staining was performed to investigate tumour mor-
TNBC are heavily infiltrated with T lymphocytes
phology. Receptor status was investigated by IHC
compared to the B lymphocytes. This suggests higher
using antibodies against ER, PgR and Her-2, and
immunogenicity of TNBCs and may indicate a higher
patients were classified as having TNBC or non-TN-
responsiveness of these cancers to immunotherapy.
BC tumours (including Luminal A, Luminal B and
Her2 overexpressing tumours). immune cell infiltration was investigated using special stains (Giemsa
for macrophages, toluidine blue for mast cells) and
antibodies: α-CD3 (T lymphocytes), α-CD20 (B-lymphocytes), α-CD4 (helper T lymphocytes) and α-CD8
(cytotoxic T lymphocytes). Immune cell densities
were quantified as cell/mm2 using the CAP guidelines. Data were entered and analyzed using SPSS
version 16. Correlation of immune cell densities
with tumour sub-types was undertaken using paired
t-test, ANOVA and Chi square test. A p-value of <
0.05 was considered as significant.
cxcr4 inhibition in tumor microenvironment facilitates antiprogram death receptor-1 immunotherapy in sorafenib-treated
hepatocellular carcinoma in mice
Ramjiawan R.1,2, Chen Y.1, Reiberger T.3, Ng R.1, Hato T.1, Huang Y.1, Ochiai H.1, Kitahara S.K.1, Unan E.1, Reddy T.1,
Fan C.1, Huang P.1, Bardeesy N.4, Zhu A.4, Jain R.1, Duda D.1
Harvard Medical School/ Massachusetts General Hospital, Radiation Oncology, Boston, United States,
1
VU University Medical Center, Cancer Center Amsterdam, Medical Oncology, Amsterdam, Netherlands,
2
Medical University of Vienna, Division of Gastroenterology & Hepatology, Vienna, Austria,
3
Harvard Medical School/ Massachusetts General Hospital, Medicine, MGH Cancer Center, Boston, United
4
States
ORAL
TALK
T
R
O
H
S
2016
Sorafenib is the only approved therapy for advanced
that blockade of PD-1 alone revealed modest anti-
hepatocellular carcinoma (HCC), but provides limited
tumor effects in treatment-naïve tumors in ortho-
survival benefits. Recently, immunotherapy has
topic and in genetically engineered models of HCC.
emerged as a promising treatment strategy, but its
However, anti-PD-1 treatment had additional anti-tu-
role remains unclear in HCCs. Using orthotopic HCC
mor activity only when combined with sorafenib and
models, we show that sorafenib increases hypoxia
AMD3100, and not when combined with sorafenib
and decreases micro-vascular density which in turn,
alone. The triple combination treatment activated
promotes immunosuppression, characterized by
CD8+ T-cells, which led to an increase of intratumor-
increased intratumoral expression of the immune
al infiltration and resulted into inhibition in tumor
checkpoint inhibitor programmed death-ligand 1
growth and lung metastases. Modulation and activa-
(PD-L1). Sorafenib treatment also led to an increase
tion of immune responses by combining AMD3100
of T-regulatory cells and M2-type tumor associated
and anti-PD1 may be a novel approach to prevent
macrophages. The recruitment of the immunosup-
tumor evasion from sorafenib treatment in HCC.
pressive cells is mediated in part by hypoxia-induced
upregulation of stromal cell-derived 1 alpha (SDF1a).
Inhibition of the SDF1a; receptor (C-X-C receptor type
4 or CXCR4) using AMD3100 prevented the polarization toward an immunosuppressive microenvironment after sorafenib treatment. The combination inhibited tumor growth, lung metastasis, and improved
survival. However, AMD3100 with sorafenib did not
increase CD8+ T-lymphocyte infiltration into HCC
tumors and failed to activate CD8+ T-cells. In separate experiments, using ultrasound-imaging showed
Characterization and specificity analysis of tumor infiltrating
lymphocytes in ovarian carcinoma
Röhle K.1, Peper J.1, Schuster H.1, Wagner P.2, Rammensee H.-G.1, Stevanović S.1
1
2
Introduction: Ovarian cancer (OvCa) is the most
while in CD8+ TILs elevated levels of the terminal
lethal gynecological cancer in women with an overall
differentiated T-cell (EMRA) phenotype were detect-
poor prognosis due to late diagnosis and frequent re-
ed. Compared to corresponding PBMCs, both popula-
sistance to chemotherapy. OvCa is a highly immu-
tions lacked naïve T cells.
nogenic tumor characterized by frequent infiltration
Conclusion: In summary, we provide first insight
with immune cells, which represent an independent
into the expression of various co-inhibitory receptors
prognostic factor in OvCa patients. Knowledge about
among tumor-infiltrating lymphocytes in ovarian
the composition and phenotype of infiltrating T cells
carcinoma.
as well as their specificity and expression of co-inhibitory receptors is so far missing. This information
is however critically important for the design of novel
immunotherapies including the application of checkpoint inhibitor therapy.
Materials and methods: Tumor-infiltrating lymphocytes (TILs) were isolated from fresh tumor tissue
of ovarian cancer patients undergoing surgery. The
expression of inhibitory molecules, namely LAG3,
CTLA4, PD1 and TIM3, was assessed by flow cytometry. T-cell specificity analysis was performed by IFNγ
ELISPOT.
Results: TILs of each analyzed patient expressed
at least one of the tested co-inhibitory receptors.
Notably, expression of LAG3 and / or PD1 was detected in most patients, whereas the expression of CTLA4
and TIM3 was only weak to absent. Within each respective tumor, CD4+ and CD8+ TILs appeared to
show similar expression patterns of co-inhibitory
surface markers. Furthermore, the memory phenotype of TILs was determined via CCR7 and CD45RO
staining. CD4+ and CD8+ TILs were mainly from
the central memory and effector memory phenotype,
Identification and characterization of neoepitopes associated
with individual mutational landscape in non-small cell lung
cancer
Saini S.K.1, Ramskov S.1, Furness A.J.S.2, Bentzen A.K.1, Lyngaa R.1, McGranahan N.2,3,4, Rosenthal R.2,
Swanton C.2,3,5, Quezada S.A.2, Hadrup S.R.1
National Veterinary Institute, Technical University of Denmark, Section of Immunology and Vaccinology,
1
Frederiksberg C, Denmark,
UCL Cancer Institute, CRUK Lung Cancer Center of Excellence, London, United Kingdom,
2
The Francis Crick Institute, Translational Cancer Therapeutics Laboratory, London, United Kingdom,
3
University College London, Center for Mathematics & Physics in the Life Sciences & Experimental Biology,
4
London, United Kingdom,
UCL Cancer Institute, Translational Cancer Therapeutics Laboratory, London, United Kingdom
5
ORAL
TALK
SHORT
2016
Accumulative reports have recently strengthened
different neoepitope-specific T cell populations in a
the important role of mutation-derived antigens in
single sample. Using this technology, we enabled T
immune recognition of cancer, and their potential
cell-detection in both peripheral blood, in-vitro ex-
use in personalized therapeutic approach. We have
panded tumor infiltrating lymphocytes and even en-
identified cytotoxic CD8+ T-cells reactive to patient
zymatic digests of tumor. This, deeper view provides
specific neoepitopes in non-small cell lung cancer
further insight to T cell recognition of mutation-de-
(NSCLC) samples and associated these to the per-
rived neoepitopes and the potential association with
sonal mutational landscape of the NSCLC. Interest-
mutational characteristics. We enabled detection of
ingly, neoepitopes derived from dominant (clonal)
additional neoepitope-restricted T cell populations
mutations provides the strongest association to clini-
in the previously analyzed patient cohort, using this
cal response following immune-checkpoint inhibi-
novel high-throughput. In conclusion, we success-
tion (McGranahan et, Science 2016). We generated
fully report the possibility of identifying neoepitope-
personalized neo-epitope libraries (varying from 150
restricted T cell responses on a personalized basis in
to 500 epitopes per patient) based on tumor specific
a high throughput manner even in limited biological
mutations and screened tumor infiltrating lympho-
samples. This approach can provide novel insight
cytes for peptide-MHC recognition. We have de-
to the immune recognition associated with immu-
tected several neoepitope-reactive T cell responses
notherapeutic measures, and potentially identify
in NSCLC patients using MHC multimer staining.
immune correlates to clinical successes.
Genomic characterization of the underlying mutations revealed that all of neoepitope-restricted T cell
responses were directed against clonal mutations.
Furthermore, we recently developed a multiplex
technology based on DNA-barcode labeled MHC multimers that allow simultaneous screening for > 1000
IL-12 therapy suppresses TC-1 tumor growth accelerated by
admixture of the docetaxel-treated senescent tumor cells
Sapega O.1,2, Šímová J.1,2, Imrichová T.3, Štěpánek I.1,2, Kyjacová L.3, Mikyšková R.1,2, Indrová M.1,2, Bieblová J.1,2,
Bártek J.3, Hodný Z.3, Reiniš M.1,2
Czech Centre for Phenogenomics, Institute of Molecular Genetics of the ASCR, Immunology Unit, Vestec,
1
Czech Republic,
Institute of Molecular Genetics of the ASCR, v. v. i., Laboratory of Transgenic Models of Diseases, Prague,
2
Czech Republic,
Institute of Molecular Genetics of the ASCR, v.v.i., Laboratory of Genome Integrity, Prague, Czech Republic
3
Cellular senescence is considered to be a principal
and < 70% senescence-associated, β-galactosidase
barrier against tumorigenesis that inhibits acquisi-
positive cells, respectively. Secretome analysis of the
tion of an immortal phenotype. Tumour cell senes-
senescent revealed increased expression of a number
cence can be induced by antitumor therapy, such
of inflammatory and protumorigenic cytokines and
as irradiation or chemotherapy and, under certain
chemokines.
circumstances, also by cytokines (IFNγ and TNFα).
We have hypothesized that immunotherapy with
Senescent cells do not proliferate, though, they can
IL-12 could be effective against both senescent and
survive in the organism for a long time and influ-
presenescent tumor cells by induction of both spe-
ence tumor development. Senescent cells express a
cific and non-specific immune responses and thereby
number of secreted proteins and growth factors that
should inhibit the growth of the senescence-acceler-
may stimulate or inhibit cell proliferation.
ated tumor cells. This accelerated tumor growth was
We can hypothesize that not only senescence induc-
effectively inhibited by cell therapy using irradiated
tion but also subsequent senescent cells elimination
IL-12-producing tumor cells. We established that im-
can be critical for effective antitumor therapy. In
munotherapy, such as the IL-12 treatment, can serve
this study, we evaluated the impact of docetaxel in
as an effective tool for elimination of the detrimental
terms of senescence induction, using two C57BL/6
effects caused by senescent tumor cells.
mice-derived tumor cell lines TC-1 and TRAMP-C2.
We have demonstrated acceleration of tumor growth,
when proliferating TC-1 tumor cells were co-administered into syngeneic mice together with tumor cells
that had been subjected to the senescence-inducing
treatments with docetaxel. After this treatment,
both TC-1 and TRAMP-C2 cells were alive but senescent, as characterized by specific cell morphology, increased SA-β-galactosidase activity, increased
expression of p16 and p21 CDK inhibitors, as well
as of the DNA damage marker γH2AX. Proliferating
cell cultures and senescent cell cultures contained
>80% proliferating and < 2% senescence-associated, β-galactosidase positive cells, DTX-treated (senescent) cell cultures contained < 2% proliferating cells
This work was supported by research grants Nos. 15-24769S
from the Czech Science Foundation and NT14461 from the
Grant Agency of the Ministry of Health of the Czech Republic.
HLA class II antigen expression in cervical intraepithelial
neoplasia and invasive cancer
Sauer M.1,2, Reuschenbach M.1,2, Wentzensen N.3, Ferrone S.4, Lahrmann B.5, Grabe N.5,6, Schmidt D.7,
von Knebel Doeberitz M.1,2, Kloor M.1,2
University of Heidelberg, Institute of Pathology, Applied Tumor Biology, Heidelberg, Germany,
1
German Cancer Research Center (DKFZ), Clinical Cooperation Unit, Heidelberg, Germany,
2
National Cancer Institute, National Institutes of Health, Division of Cancer Epidemiology and Genetics,
3
Rockville, United States,
Massachusetts General Hospital, Harvard Medical School, Department of Surgery, Boston, United States,
4
University of Heidelberg, BIOQUANT, Hamamatsu Tissue Imaging and Analysis Center (TIGA), Heidelberg,
5
Germany,
University Hospital, National Center of Tumor Diseases, Medical Oncology, Heidelberg, Germany,
6
Institute of Pathology, Viersen, Germany
7
Objectives: HLA class I antigen expression on tumor
found in the columnar epithelium and cells of the
cells is essential for the recognition of tumor anti-
squamocolumnar junction zone. HLA class II antigen
gens by the immune system. HLA class II antigens
expression was low in CIN1 (40.9%) and peaked in
normally are expressed by professional antigen-pre-
CIN2 (90.0%), then decreasing again towards CIN3
senting cells, but are also reported to be expressed by
lesions (71.4%) and cancer (63.6%). In CIN3 and
several tumors of non-lymphoid origin. Strong HLA
cancers high CD3+ and CD8+ lymphocyte infiltra-
class II antigen expression has been described for a
tion correlated with lack of or heterogeneous HLA
subset of HPV-associated cervical cancers. To char-
class II antigen expression.
acterize HLA class II antigen expression during HPV-
Conclusions: Our results suggest that HLA class II
induced cervical tumor development, we examined
antigens are commonly expressed in precancerous
HLA class II antigen expression in CIN lesions and
stages and cervical cancers. The low percentage of
cervical cancers and correlated HLA class II expres-
HLA class II positivity in CIN1 is compatible with
sion with immune cell infiltration in the lesions and
the hypothesis that only a subset of CIN1, poten-
the adjacent stroma.
tially those originating from the squamocolumnar
Methods: FFPE tissue sections of CIN1, CIN2, CIN3
junction zone, may overexpress HLA class II anti-
and invasive SCC patients (n=103 in total) were ana-
gens and tend to progress into higher grade CIN. In
lyzed by immunohistochemical staining with mono-
later disease stages, HLA class II antigen-positive cell
clonal antibodies specific for HLA class II antigens
clones may be eliminated in an environment of dense
(LGII-612.14) and for different T cell markers (CD3,
T cell infiltration, which would be compatible with
CD8, Foxp3, Granzyme B, CD3 zeta-chain).
the immunoediting concept.
Results: HLA class II antigen expression was absent
in all samples of normal, non-neoplastic squamous
cervical epithelium adjacent to lesions (n=29).
However, a strong and uniform staining pattern was
Tumor and host cell PD-L1 expression is required to mediate
suppression of anti-tumor immunity
Lau J.1, Sanders L.1, Cheung J.1, Navarro A.2, Haley B.2, Totpal K.2, Lianoglou S.2, McBride J.2, Belvin M.1,
Mellman I.1, Kim J.1, Schmidt M.1
Genentech, Inc., Cancer Immunology, South San Francisco, United States,
1
Genentech, Inc., South San Francisco, United States
2
Numerous mechanisms of immunosuppression are
cells is more prevalent and can be even more pre-
employed by cancerous cells to evade surveillance
dictive of response than PD-L1 expression by tumor
and eradication by the immune system. Among
cells alone, at least in indications such as bladder
these, expression of PD-L1 plays a key role in atten-
cancer.
uating anti-tumor responses in both mouse tumor
The underlying mechanism of this association is
models and human cancer patients. PD-L1, a ligand
unclear but these data challenge the prevailing view
for the inhibitory receptor PD-1 on activated T-cells,
that adaptive expression of PD-L1 by tumor cells is
is thought to be adaptively expressed by tumor cells
the sole source of PD-1 checkpoint control in the
in response to inflammatory cytokines (e.g. IFNg),
tumor microenvironment.
thereby directly inhibiting T cell mediated killing.
To interrogate the mechanism behind this associa-
Novel immuno-therapeutic agents targeting PD-L1/
tion, we evaluate the relative roles of PD-L1 expres-
PD-1 have produced unparalleled, durable clinical
sion by the tumor and by the host’s immune cells
responses in a wide range of solid and hematologic
in the suppression of anti-tumor immune respons-
cancers, even in late stage disease, presumably by
es. Using genetic chimera with distinct deletions of
relieving suppression of primed T cells within the
PD-L1 in the tumor cell or host cell compartment,
tumor microenvironment. Yet, prevalence of non-re-
we find that both tumor and host derived PD-L1 play
sponding patient populations emphasizes the neces-
non-redundant roles in modulating the antigen spe-
sity to better understand the underlying biology and
cific immune response, suggesting a thus far under-
identify drivers of efficacy. In the context of agents
appreciated key role of infiltrating immune cells in
targeting the PD-L1/PD-1 pathway, several efforts
both generating and negatively regulating anti-tumor
have been undertaken to link tumor cell PD-L1 ex-
immunity. The work describes the first detailed anal-
pression to clinical benefit, as the adaptive expres-
ysis of how different cellular sources of PD-L1 modu-
sion of PD-L1 on tumor cells is thought to be the dom-
late the antigen specific immune response, important
inant driver of T-cell suppression. Consistent with
information that might enable a more precise identi-
this is the observation that patients whose tumors
fication of patient populations most likely to benefit
express PD-L1 have a greater likelihood of response
from this exciting new class of therapeutics.
than patients whose tumors are PD-L1-negative. This
is particularly true in the case of non-small cell lung
cancer and metastatic bladder cancer.
However, one unexpected finding is that PD-L1 expression by infiltrating myeloid and other immune
A patient derived antibody targeting CD9 inhibits melanoma
metastasis
Schotte R.1, Pos W.1, Verdegaal E.2, Villaudy J.1, Go D.1, Fatmawati C.1, van Helden P.1, van der Burg S.2, Spits H.1
1
Leiden University Medical Center, Dept. Clinical Oncology, Leiden, Netherlands
2
Background: Adoptive T cell therapy and checkpoint
a novel cell surface epitope on the tetraspanin CD9.
inhibitor antibodies are becoming valuable immu-
CD9 is widely expressed throughout the body but
notherapeutical tools for the treatment of metastatic
generally upregulated on malignant tissues. In line
cancer. It is now well established that immune re-
with this, we found minimal reactivity of AT14-12
actions against cancer cells can be induced. It is
to various healthy tissues including melanocytes.
unknown whether immunotherapy also leads to
In contrast, AT14-12 showed significant stronger
generation of tumor-specific antibodies that mediate
binding to melanoma cells. Of note, the strongest in-
an antitumor effect. Here, we investigated the pos-
teraction was found on melanoma tumor cells from
sibility that an antibody response has contributed to
the original patient. In addition, the antibody reacted
the success of the immunotherapy of a patient with
strongly with a number of other tumor types includ-
metastatic melanoma.
ing colon carcinoma, pancreatic and breast cancer.
Methods: A patient diagnosed with stage IV meta-
Importantly, in melanoma xenografted immunodefi-
static melanoma and brain lesions was successfully
cient mice AT14-12 was able to reduce growth of the
treated by reinfusion of ex vivo autologous expanded
primary tumor and strongly blocked the progression
tumor associated T cells (Verdegaal et al., 2011). This
of metastasis.
patient is still in remission 9 years after treatment
Discussion: Altogether these data suggest that the
and has been shown to develop both CD4 and CD8
antibody contributed to the success of the immuno-
T cell responses against neoantigens (Linneman et
therapy in this patient. We anticipate that this an-
al., 2014; Verdegaal, submitted). We now analyzed
tibody could provide help to tumor-reactive T cells
the memory B cell repertoire from this patient for
in the eradication of circulating tumor cells and/
the presence of tumor-reactive B cells. B cells were
or settlement of metastatic tumor cells in vivo. It is
isolated from peripheral blood and subsequently im-
noteworthy that no antibody-related adverse effects
mortalized by forced expression of Bcl-6 and Bcl-xL
were observed during and after treatment of this
preventing terminal differentiation and apoptosis, re-
patient indicating that the antibody is safe for use in
spectively. This has proven to be an effective method
humans. Our results show that the B cell repertoire
for isolation of unique antibodies (Kwakkenbos et al.,
of a patient who is cured after immunotherapy pro-
2010, 2014). Tumor-reactive B cell clones were iden-
vides a highly attractive source of antibodies with
tified by antibody binding to a panel of melanoma
clinical utility.
cell lines.
Results: We isolated one particular B cell clone that
produced an antibody, named AT14-12, recognizing
Complex deletion event at B2M locus in a human melanoma
patient treated with IVAC MUTANOME
Schrörs B.1, Löwer M.1, Suchan M.1, Gangi Maurici S.1, Boegel S.1, Tadmor A.D.1, Bukur V.1, Albrecht C.1,
Barea Roldan D.1, Walter C.1, Weber D.1, Kasemann B.1, Walter G.1, Attig S.1, Rohde C.2, Wöll S.2, Vogler S.3,
Seck C.3, Müller F.3, Miller M.3, Türeci Ö.2, Sahin U.1,3
1
Mainz, Germany,
2
3
Functional beta-2-microglobulin (B2M) is essential
deletion event might have been acquired during cell
for the expression of MHC class I molecules (MHC
line generation, we searched for it in the metasta-
I) on the surface of nucleated cells. The absence of
sis from which MZ-GaBa-018 was derived. Copy
B2M directly implies loss of antigen presentation
number analyses already indicated the loss of at least
via MHC I and represents a known tumor immune
one B2M copy in this metastasis, while the earlier
escape mechanism. The tumor cell line MZ-GaBa-
prae-treatment metastases were estimated with two
018 derived from melanoma patient PA018, who ob-
copies. PCR and Sanger sequencing confirmed the
tained IVAC MUTANOME (NCT02035956) treatment,
complex deletion event in the post-treatment metas-
was lacking MHC I expression on tumor cell surface
tasis as observed in the cell line proving that the loss
as determined via flow cytometry. RNA and exome
of B2M occurred already in the patient. Moreover,
sequencing had been performed on MZ-GaBa-018
B2M deletions were not detected in 1082 tumor cell
and three metastases of the patient that were excised
lines derived from various cancer entities (e.g. mela-
in January 2013, October 2013 (prae-treatment) and
noma, colon, breast and lung cancer) which further
July 2015 (post-treatment). MZ-GaBa-018 was estab-
showed that this was not a common cell line effect.
lished from the latest metastasis.
The remaining B2M copy detected in the tumor DNA
While RNA-seq data confirmed MHC I-mRNA ex-
of the post-treatment metastasis might have been
pression in all metastases and the cell line, B2M was
attributable to wild-type contamination as a tumor
not expressed in MZ-GaBa-018 and the correspond-
content of only 38% was estimated. qPCR analyses
ing exome data revealed that the complete B2M locus
did not indicate an impaired recruitment of immune
was deleted. The deletion started in an intronic se-
cells in the post-treatment metastasis compared to
quence upstream of B2M well covered in the exome
the prae-treatment sample from January 2013.
capture. Split read and discordant paired-end align-
Ongoing studies are now aiming at determining how
ments were used to deduce the deletion end point
early the loss of B2M occurred during the clinical
located more than 200kb downstream. In addition,
course of the disease and whether IVAC MUTANOME
exome reads were applied to de novo assembly and
treatment is selecting for immune escape variants.
the deletion could be detected in a contig sequence.
These data will provide evidence on whether routine
Split reads as well as the de novo assembly indicated
monitoring of B2M loss or equivalent events is advis-
a complex deletion event. This information was used
able.
to design primers with which the complex deletion
event was confirmed and defined in MZ-GaBa-018 via
PCR followed by Sanger sequencing. As this complex
Activating and repolarizing immune suppressive tumor
associated macrophages using siRNA encapsulated in
nano-sized carriers to initiate an anti-tumor immune response
against melanoma
Schupp J.1, Foerster F.2, Bamberger D.3, Leber N.4, Zentel R.4, Wich P.R.3, Schuppan D.2, Tuettenberg A.1
University Medical Center of the Johannes Gutenberg-University, Department of Dermatology, Mainz,
1
Germany,
University Medical Center of the Johannes Gutenberg-University, Institute of Translational Immunology,
2
Mainz, Germany,
Johannes Gutenberg-University, Institute of Pharmacy and Biochemistry, Mainz, Germany,
3
Johannes Gutenberg-University, Institute of Organic Chemistry, Mainz, Germany
4
Tumor cells escape the patient’s immune system by in-
To screen potential siRNA targets for their ability to
ducing immune suppression in the tumor microenvi-
repolarize M2 macrophages and identify nanoparti-
ronment. Suppression is mediated by cell-cell contact
cles with high transfection rates, we have established
as well as secreted factors such as chemokines and
an in vitro culture of human M1 (LPS and IFN-gam-
cytokines. Tumor associated macrophages (TAM),
ma) and M2 (IL-4) macrophages, derived from mono-
also known as M2-polarized or alternatively acti-
cytes isolated from human PBMC. As a control, THP-1
vated macrophages, are major players of the tumor
cells, a human monocytic cell line, are used. Acid de-
microenvironment and have been shown to promote
gradable cationic dextran particles, which are able to
tumor growth by inducing neoangiogenesis, sup-
efficiently encapsulate siRNA and have a size range
porting metastasis and rendering tumor infiltrating
of 100 to 150 nm, already proved to be a promising
lymphocytes (TIL) suppressive or apoptotic. TAM dif-
candidate because of low toxicity and high uptake
ferentiate from tissue macrophages or blood derived
rates in monocytes and macrophages without influ-
monocytes after exposure to IL-4 and/or IL-13. By
encing the phenotype. In wild-type mice, nanopar-
disrupting the signal pathways responsible for TAM
ticles accumulated preferentially in the liver where
phenotype via siRNA mediated gene knockdown tar-
they showed high uptake rates in liver macrophages
geting receptors (IL-4R & CSFR1) and/or downstream
(70-80%). Following repeated treatments, no toxicity
transcription factors (STAT6, IRF4 & NOR1), we try to
could be detected in serum parameters. In a second
reprogram TAM to classical activated immunostimu-
approach we use mannose-functionalized cationic
latory M1 macrophages. To avoid degradation and
nanohydrogel particles in the size range of 10 to 60
unspecific cell uptake siRNA is bound to or encap-
nm to target the M2 phenotype of macrophages.
sulated in nano-sized carriers. Specific targeting
All things considered the use of nanoparticles as
of TAM can be achieved actively using antibodies
drug delivery systems targeting TAMs promises
against characteristic surface markers of this subset
enormous potential to modulate immune tolerance
and mannose as ligand for CD206 or passively by
towards tumors.
exploiting the enhanced permeability and retention
effect in tumor vessels and the high phagocytic activity of macrophages.
The role of CD8+ T cell-released exosomes on the down-regulation
of tumor invasion and metastasis by elimination of stromal
mesenchymal cells
Seo N.1,2, Shirakura Y.1, Tahara Y.2,3, Harada N.1,2, Akiyoshi K.2,3, Shiku H.1,2
Mie University Graduate School of Medicine, Department of Immuno-Gene Therapy, Mie, Japan,
1
Japan Science and Technology Agency (JST), ERATO Bio-nanotransporter Project, Tokyo, Japan,
2
Kyoto University Graduate School of Engineering, Gradurate School of Engineering, Kyoto, Japan
3
Introduction:
Fibroblastic
mesenchymal
tumor
that the reduced growth and CD140a expression in
stromal cells including mesenchymal stem cells
CD8+ T cell-released exosome-treated tumors caused
(MSCs) and cancer-associated fibroblasts (CAFs)
by interrupting tumor progression after apoptotic
promote strongly tumor progression in part an exo-
death of exosome-engulfed fibroblastic mesenchy-
some-mediated manner. In contrast, there is no report
mal tumor stromal cells including MSCs and CAFs
+
about the role of CD8 T cell-released exosomes on
rather than the direct attenuation of tumor cells. Fur-
the regulation of tumor progression. In this study,
thermore, subcutaneous B16F10 tumor lost invasive
we investigated in murine models whether or not ex-
and lung metastatic properties by i.t. treatment of
osomes from CD8+ T cells including CTLs affect on
CD8+ T cell-released exosomes. CD8+ T cells were
the tumor progression focusing on the modification
shown to deplete mesenchymal tumor stromal cells
of tumor stromal cells.
at 3 days after tumor infiltration, while GW4869 (an
Methods: Supernatants obtained by the cultivation
inhibitor of exosome production)-treated them failed
of CD8+ T cells or control cells were collected, and
to eliminate tumor stroma.
subjected to exosome purification by the filtration
Conclusion: Our findings provide new insights that
and ultracentrifugation (100,000 g, 2 hrs). The puri-
CD8+ T cells play as an anti-stromal effector in an
fied exosomes were injected into d-10 subcutaneous
exosome-mediated manner in addition to the conven-
CMS5a, B16, and B16F10 tumors (1.0-1.5 cm diam-
tional cytolysis of tumor cells by cell-cell interaction.
eter) to investigate growth and CD140a expression
In addition, CD8+ T cell-released exosomes have a
of tumors, and subsequent tumor invasion and me-
possibility to develop effective treatment of patients
tastasis. CMS5a-specific CD8+ T cells treated with/
with advanced cancer.
without GW4869 were transferred i.v. of CMS5a
tumor-bearing mice to examine exosome-dependent
modulation of fibroblastic tumor stromal cells.
Results: CD8+ T cell-released exosomes from the
culture supernatant of CD8+ splenocytes of normal
or CMS5a-specific TCR gene-transfected mice, but
not tumor-bearing mice, attenuated CMS5a and B16
growth in correlation with the downregulation of
CD140a expression. The detailed studies using SYTO
RNASelect-staind exosomes, MSC chimeric mice,
and the cultured bone-derived MSCs demonstrated
BAG6 and CBP/p300 regulate ESCRT-mediated exosomes release
and protein sorting
Shatnyeva O.M.1, Reiners K.S.1, Bhagwat P.1, Kubarenko A.2, Hansen H.P.1, Pogge von Strandmann E.1
University Hospital of Cologne, Innate Immunity Group, Cologne, Germany,
1
University of Bonn, Institute for Clinical Chemistry and Pharmacology, Bonn, Germany
2
According to the immune-surveillance hypothesis,
molecules were diminished in exosomes collected
cancer cells evolve strategies to evade or suppress the
from BAG6-deficient cells suggesting that BAG6
immune system as part of the this disease. We have
impacts on exosome cargo. The data presented iden-
recently shown that the impaired activity of NK cells
tify BAG6 as a novel key component in exosome for-
in chronic lymphocytic leukaemia (CLL) is depend-
mation and loading.
ent on soluble ligands for NK cell receptors (NKRs)
that are released by the malignant B-cells. One of
these soluble ligands is BAG6, which engages the cytotoxic NK cell-receptor only when expressed on the
surface of extracellular vesicles (exosomes). BAG6 is
a multifunctional protein also acting as an intracellular chaperone involved in protein targeting and
protein degradation. Here, we analyzed the role of
BAG6 for the release of immune-activating exosomes
upon DNA damage in CLL.
Immunoprecipitation, in vitro translation and a
yeast-two-hybrid approach revealed that BAG6 binds
directly to p53 and forms a ternary complex with
the actetyltransferase CBP/p300 in response to doxorubicin-induced DNA damage. Induction of DNAdamage triggered the release of exosomes and the
nuclear export of BAG6. Subsequently, BAG6 recruits
the ESCRT-(endosomal sorting complex required for
transport) component HRS and is detectable in a cellular complex with the ESCRT components TSG101
and ALIX. The release of exosomes was dependent
on BAG6 and p53 under basal and stress-related conditions.
Exosomes from BAG6 wild type cells were characterized by an enriched expression of BAG6-interacting
partners and immune regulatory molecules. These
Desmoid tumors: the importance of the immune cell
determination to the development of new treatment options
Siozopoulou V.1,2, Marcq E.2, Zwaenepoel K.1,2, Hermans C.2, Somville J.3,4, Smits E.2,5, Pauwels P.1,2
Department of Pathology, University Hospital of Antwerp, Antwerp, Belgium,
1
Center for Oncological Research, University of Antwerp, Antwerp, Belgium,
2
Department of Orthopedics, University Hospital of Antwerp, Antwerp, Belgium,
3
Faculty of Medicine and Health Sciences,University of Antwerp, Antwerp, Belgium,
4
Laboratory of Experimental Hematology, University of Antwerp, Antwerp, Belgium
5
Objectives: Desmoid tumors (DT) are local aggres-
few CD20 positive B lymphocytes were present.
sive (myo)fibroblastic neoplasms, characterized by
Dual staining showed there were slightly more
infiltrative growth and a tendency toward local re-
CD3+CD8+ cytotoxic T lymphocytes present com-
currence, even after complete surgical removal. They
pared to CD3+CD4+ T-helper lymphocytes. Almost
lack metastatic potential, but can be lethal due to
no CD4/FOXP3 double positive cells were seen. Fur-
local effects of growth. Choosing optimal therapy
thermore, there was strong positivity for CD27 and
for DTs is difficult and close observation (“wait-and-
moderate positivity for CD45RO in almost all cases.
see”) is an acceptable strategy for stable asympto-
No expression of CD56 and NKp46 was observed,
matic desmoids. Surgery remains the mainstay; other
which are both markers for natural killer cells. PD-1
treatments of choice are radiotherapy and systemic
and PD-L1 were expressed in the lymphocytes but
therapy, nevertheless with no promising results.
not on tumor cells. CD68 positive histiocytes were
Morphologically those tumors show an inflamma-
invariably seen in the inflammatory response.
tory response, with inflammatory aggregates to be
Currently, statistical analysis is being performed to
located at the periphery of the tumor adjacent to the
investigate whether there is a correlation between
per-existed structures. Identifying the inflammatory
our immunohistochemistry results and clinicopatho-
microenvironment related to the DTs, might contrib-
logical parameters of the patients. The results of this
ute to the development of a better treatment option
analysis will be presented.
for these tumors.
Conclusion: Till today treatment of DTs still remains
Materials and methods: Paraffin-embedded tissue
a big challenge. No effective therapies are available
sections from 32 patients diagnosed with DTs were
yet. Gaining more insight in the inflammatory infil-
included in this study. Immunohistochemistry was
trate surrounding the tumor might contribute to a
performed for several immune cell markers as well
better understanding of the interaction between the
as for the expression of the immune-checkpoint pro-
immune system and the tumor itself. Eventually this
grammed death (PD-)1 and its ligand PD-L1. Dual
can lead to the development of an immunotherapeu-
staining (DAB and alkaline phosphatase) was used
tic strategy, that might be a more efficient therapeutic
for some of the markers. Results were expressed
option for these patients.
as the percentage of positive cells out of the total
number of counted cells. The intensity of staining as
well as the staining pattern were also determined.
Results: The majority of the inflammatory infiltrate
consisted of CD3 positive T lymphocytes, while only
Characterization of inhibitory molecules on tumor-infiltrating
lymphocytes in malignant melanoma
Skadborg S.K.1, Idorn M.1, thor Straten P.1,2
Herlev Hospital, Center for Cancer Immune Therapy, Herlev, Denmark,
1
University of Copenhagen, Faculty of Health and Medical Sciences, Department of Immunology and
2
Microbiology, Copenhagen, Denmark
Adoptive T cell transfer (ACT) using in vitro expand-
highly expressed on natural killer (NK) cell, on a
ed tumor infiltrating lymphocytes (TILs) has shown
small fraction of our CD8+ T cells and on γδ T cells
encouraging results with a complete response rate
(subtypes yet to be verified) in our TILs.
of 20% and objective response rates of 50% in treat-
The screenings will be followed by functional studies
ment of malignant melanoma.
aiming to improve the cytotoxic capacity of the TILs
Despite the tumor specificity that TILs provide, the
in
tumor microenvironment is known to suppress T
bined blocking of inhibitory molecules. Based on our
cell functionality by a range of mechanisms. Cancer
preliminary findings we will start with the blocking
cells can engage a number of inhibitory molecules on
of NKG2A on the tumor infiltrating NK cells in an-
TILs and thus inhibit killing of the cancer cells. By
tibody-dependent cell-mediated cytotoxicity assays.
blocking this interaction, e.g. using the checkpoint
This study aims to identify the obstacles of the tumor
inhibitors Ipilimumab and Nivolumab, the T cells
microenvironment, which will help gain insight into
can regain their ability to kill.
new potential targets on TILs to enhance the success
We want to characterize the expression of inhibitory
rate of ACT.
molecules on TILs derived from melanoma biopsies,
aiming to identify new candidates for blockade.
Thus far, we have isolated TILs and quantified the
immune cell composition (NK-, T-, B-cells) from 10
melanoma biopsies deriving from different patients.
Next, we want to analyze the expression of a selected
panel of inhibitory molecules on these TIL subsets
using multicolor flow cytometry, including NKG2A,
PDL-1, CD200R, TIGIT, CD96, TIM3, which have been
found to inhibit cytotoxicity of the lymphocytes in
various ways. The expression of these inhibitory
molecules will be measured at different time points
applicable to the protocol used for ACT TILs; before
isolation from tumor biopsies (“ex vivo TILs”), after
isolation (“Young TILs”), and after rapid expansion representing the ACT infusion product (“REPd
TILs”). To this end we have found that NKG2A is
51
Cr-release assays through individual and com-
Characterization of PD-L1 and PD-1 expression in tumor
infiltrating lymphocytes and tertiary lymphoid structures in
paired primary tumors and metastases from breast cancer patients
Solinas C.1, Buisseret L.1, Garaud S.1, de Wind R.2, Van den Eynden G.1,3, Boisson A.1, Brown D.4, Naveaux C.1,
De Silva P.1, Migliori E.1, Noel G.1, Spinette A.2, Craciun L.2, Sotiriou C.4, Larsimont D.2, Willard-Gallo K.1
Institut Jules Bordet, Université Libre de Bruxelles, Molecular Immunology Unit, Bruxelles, Belgium,
1
Institut Jules Bordet, Department of Pathology, Bruxelles, Belgium,
2
GZA Hospitals, Department of Pathology and Cytology, Wilrijk, Belgium,
3
Institut Jules Bordet, Université Libre de Bruxelles, J.C. Heuson Breast Cancer Translational
4
Research Laboratory, Bruxelles, Belgium
Programmed death-1 (PD-1) and its ligand PD-L1
Alternatively, when TIL infiltration in the primary
are immune check-point receptors whose interaction
tumor was < 10% (=TIL -) the %TIL was significantly
inhibits an ongoing immune response. In human
higher at the distant site (p=0.004). A similar trend
cancers their expression is positively correlated
was observed with PD-1 and PD-L1 positive primary
with tumor infiltrating lymphocytes (TIL). PD-1 and
tumors (19% and 16% respectively) with a decrease
PD-L1 expression by immunohistochemistry (IHC)
of positive cases in the paired metastases (3% and
is also associated with the presence of tertiary lym-
7% respectively). A significant increase of %PD-1+
phoid structures (TLS) in primary breast cancer
cells has been observed in secondary lesions arising
(BC). PD-L1 emerged as a predictive biomarker, con-
from PD-1- primary tumors (p=0.004). In all metasta-
troversial in predicting responses to anti-PD-1/PD-L1
ses, %TIL varied between 2-15% and TLS were rarely
drugs probably for the heterogeneity of PD-1/PD-L1
found (12% vs 47% in the primary tumors). Globally
expression and TIL infiltration in primary tumors
12% of metastases were PD-L1+ and 24% were PD-1+.
and secondary lesion(s). We analyzed formalin fixed
A significantly higher level of TIL has been observed
paraffin embedded tissue samples from a retrospec-
in lesions from soft tissues compared to skin, brain
tive cohort of BC patients who underwent adjuvant
or breast relapses, while no significant patterns were
surgery for the primary tumor and metastasectomy
observed for PD-1 and PD-L1 expression. These ob-
(n=23) or biopsy (n=21) for their relapse at a distant
servations suggest that the extent of immune infil-
site(s). A double IHC staining using anti-CD3 (T
tration and PD-1/PD-L1 status in metastatic disease
cells) plus anti-CD20 (B cells), and anti-PD-1 plus
could also depend upon the organ site of relapse, the
anti-PD-L1 antibodies were performed on primary
two latter markers probably reflecting an overtime
and metastases tissue sections. Slides were scored
and over space heterogeneous functional status of
blindly and independently by two trained patholo-
TIL. Taken together, these preliminary data suggest
gists. TIL were evaluated as the % of stroma plus
that the metastatic clone may be more immunosup-
tumor area infiltrated by CD3+ and CD20+ cells. PD-1
pressive than the primary tumor with heterogeneity
and PD-L1 were scored as the percent (%) of posi-
in the extent of an immune response further compli-
tive cells. Our results show that overall %TIL was
cated by the organ site(s) of relapse.
higher in primary tumors compared to their matched
metastases (n=44, p=0.03). When TIL in primary
tumors were ≥10% (=17 TIL+ cases, range 10% to
35%) there was a significant decrease in the %TIL
in the corresponding secondary lesion (p< 0.0001).
No role of the stress kinase GCN2 in T cell-mediated tumor
rejection as intratumoral tryptophan levels are maintained
Sonner J.K.1, Deumelandt K.1, Ott M.1, Thomé C.2, Mohapatra S.3, Schulz S.4, Hopf C.4, Opitz C.3, Wick W.2,5,
Platten M.1,5
German Cancer Research Center, DKTK CCU Neuroimmunology and Brain Tumor Immunology, Heidelberg,
1
Germany,
German Cancer Research Center, DKTK CCU Neurooncology, Heidelberg, Germany,
2
German Cancer Research Center, Junior Group Brain Cancer Metabolism, Heidelberg, Germany,
3
Hochschule Mannheim, Institute for Instrumental Analytics and Bioanalytics, Mannheim, Germany,
4
University Hospital Heidelberg, Department of Neurology and National Center of Tumor Diseases, Heidelberg,
5
Germany
General control non-derepressible 2 (GCN2) is a stress
GCN2 knockout mice (LckCre+;GCN2fl/fl) allowed us
kinase that initiates the integrated stress response
to analyze the impact of GCN2 expression on T cell
upon accumulation of uncharged tRNAs. GCN2 acts
infiltration in the B16 melanoma model. Here, GCN2
as a molecular sensor of amino acid homeostasis
did not play a central role in controlling T cell re-
and mediates phosphorylation of the eukaryotic
sponses to peripheral tumors as T cell-specific GCN2
translation initiation factor 2α (eIF2α) in response to
knockout did neither slow down nor accelerate tumor
amino acid depletion, thereby attenuating ribosomal
growth and had no impact on T cell infiltrates. When
translation. In T cells and other cells GCN2 has been
specifically addressing CD8+ T cell responses using
identified as a molecular tryptophan sensor that me-
the gp100 adoptive transfer model, both T cells from
diates downstream effects of the immunoregulatory
pmel wildtype and pmel GCN2-/- mice efficiently de-
enzyme indoleamine-2,3-dioxygenase (IDO). Its ac-
celerated B16 melanoma growth. Even reinforcement
tivation by IDO-mediated tryptophan depletion mit-
of tryptophan depletion by TDO overexpression in
igates T cell proliferation due to anergy induction.
B16 tumor cells did not alter T cell infiltration as
Recently, expression of tryptophan-2,3-dioxygenase
MALDI imaging demonstrated maintenance of intra-
(TDO), a second enzyme involved in tryptophan ca-
tumoral tryptophan levels despite high tryptophan
tabolism, has been correlated to immune escape in
turnover, which prohibits a drop in tryptophan suf-
human tumors and systemic TDO inhibition restored
ficient to activate GCN2 in intratumoral T cells.
tumor rejection in a preclinical model. However,
In conclusion, despite previous studies that propose
tumoral immune resistance as a result of IDO and
anergy induction of T cells mediated by GCN2 acti-
TDO expression has been mainly attributed to accu-
vation in response to IDO expression, our results do
mulation of kynurenine metabolites, activation of the
not suggest that suppression of antitumor immune
aryl hydrocarbon receptor (AHR) and generation of
responses is driven by tryptophan depletion and sub-
regulatory T cells so far.
sequent GCN2-mediated T cell anergy.
In the current study, we tested the hypothesis that
the GCN2 pathway is essential in T cell-mediated
control of tumor growth. Generation of T cell-specific
PD-L1 upregulation following RLR and TLR-based immunotherapy
in a mouse model of gastric cancer
Steinhoff N.1, Spinetti T.1, Spagnuolo L.1, Mottas I.1, Bourquin C.1
University of Fribourg, Medicine/ Pharmacology, Fribourg, Switzerland
1
The pharmacological activation of TLR and RLR
pathways results in stimulation of dendritic cells
and production of IL-12 and type I IFN, and can lead
to the development of effective anticancer T-cell responses. With the use of RLR and TLR-based treatments, we aim to facilitate CD8+ T-cell infiltration
into gastric tumors in CEA424-SV40 T Ag transgenic mice (CEA424-TAg). These mice spontaneously
develop gastric tumors in the pyloric region with
nearly 100% penetrance at an early age. We observed
intratumoral PD-L1 upregulation following treatment
of CEA424-TAg mice with an RLR agonist (poly(I:C))
and with a TLR7 agonist (R848). In vitro, we found
enhanced mRNA expression levels of PD-L1 following exposure to IFNα or IFNβ of the mGC8 cell line,
which is derived from CEA424-TAg gastric tumors.
Additionally, following IFN-I exposure in vitro we
measured an upregulation of PD-L1 and MHC-I at
the protein level on the surface of mGC8 cells. Our
findings suggest that combination with anti-PD-L1
therapy may enhance the efficacy of RLR/TLR immunotherapy for the treatment of gastric cancer.
Cooperation of Langerhans cells and NK cells guarding the
epidermis during chemical carcinogenesis
Stoitzner P.1, Ortner D.1, Tripp C.H.1, Dubrac S.1, Hermann M.2, Doppler W.3, Komenda K.1, Clausen B.E.4
Medical University Innsbruck, Dermatology, Venereology and Allergology, Innsbruck, Austria,
1
Medical University Innsbruck, Department of Anaesthesiology and Intensive Care Medicine, Innsbruck,
2
Austria,
Medical University Innsbruck, Section for Medical Biochemistry, Innsbruck, Austria,
3
University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine,
4
Mainz, Germany
Immunosurveillance of tissue is an important mech-
an accumulation of DNA-damaged keratinocytes.
anism by which the immune system prevents cancer
Our findings demonstrate that during the inititation
development. Skin treatment with the carcinogen
phase of chemical carcinogenesis LC and TNFalpha
7,12-dimethylbenz(a)anthracene (DMBA) and the
mediate recruitment of epidermal NK cells which
promotor
subsequently eliminate transformed cells to inhibit
12-O-tetra-decanoyl-phorbol-13-acetate
(TPA) is a well established murine model for squamous cell carcinoma (SCC). So far the immunological processes during the promotion phase of chemical carcinogenesis in this model were investigated,
however, information on innate immune responses
during the initiation phase of tumorigenesis was
missing. We demonstrate here that dendritic cells
(DC) of the epidermis, namely Langerhans cells (LC)
and NK cells are essential mediators for rapid clearance of DNA-damaged NKG2D-ligand (NKG2D-L) expressing keratinocytes. The depletion of NK cells or
LC shortly before carcinogen application caused accumulation of DNA-damaged cells and higher tumor
burden. Further experiments revealed that NK cell
accumulation in the epidermis depends on LC and
on TNFalpha. The importance of TNFalpha in the
immunosurveillance of chemically treated skin was
verified by the fact that neutralization of TNFalpha
led to a lower number of NK cells in epidermis and
tumor development.
The expression of Gr1+ and S100A8/A9+ cells in primary tumors
and visceral organs invaded by breast carcinoma cells
Tanriover G.1, Dilmac S.1, Erin N.2
Akdeniz University, Histology and Embriyology, Antalya, Turkey,
1
Akdeniz University, Medical Pharmacology, Antalya, Turkey
2
MDSCs (myeloid-derived suppressor cells) are het-
S100A8/A9 immunoreactivity alone or co-expressed
erogeneous group of immune cells from the myeloid
with Gr1 was found in primary tumors formed by
lineage (a family of cells that originate from bone
4TLM and 4THM cells which was markedly higher
marrow stem cells), to which dendritic cells, mac-
than in primary tumor formed by non-metastatic
rophages and neutrophils also belong. Presence
67NR cells. Similarly lung tissues obtained from
of MDSCs in the tumor microenvironment reflects
mice injected with 4TLM or 4THM cells were invaded
poor prognosis and likely to have a role in immune
by S100A8/A9+ and Gr1+ cells. Double positive cells
suppression. S100A8 and A9 (calgranulin A and cal-
were markedly less in lung tissues of animals injected
granulin B) has been implicated in the abnormal dif-
with 67NR cells. S100A8/A9 + cells were mostly lo-
ferentiation of myeloid cells in the stroma of cancer.
calized in red pulp of spleens. We observed increased
Increased S100A8/A9 expression may also have role
number of neutrophils in pheripheral blood of mice
in formation of metastatic milieu. Therefore, in the
injected with metastatic breast carcinoma cells.
present study we determined co-expression of Gr-1 (a
These results demonstrated that tumor derived
myeloid marker) with S100A8/A9 in visceral tissues
factors secreted from aggressive breast carcinomas
invaded by metastatic breast carcinoma.
may increase S100A8/A9+ cells locally and systemi-
We previously isolated liver (4TLM), heart (4THM)
cally and S100A8/A9+ cells may provide appropriate
metastatic cells murine breast carcinoma. We here
milieu for the formation of metastasis.
also used 67NR non-metastatic cells using orthotopically transplantation in mouse models of breast
cancer. 4TLM, 4THM cells (100.000 cells/mouse) and
67NR cells (1.000.000 cells/mouse) were inoculated
into the right upper mammary pad of 8-10 weeks old
female Balb-c mice. Necropsies were performed 25-27
days after injection. Presence of Gr1 and S100A8/A9
positive cells within the primary tumors as well as
lung and spleen tissues were examined using double
and single immunohistochemical staining. The expression pattern and intensity was evaluated by
image J analysis. We also evaluated phenotypes of
immune cells in peripheral blood smears obtained
tumor-bearing mice.
Alteration of host immunity with stress in breast cancer
patients
Taranikanti M.1, Yasmeen N.1, Panda S.1, Tiyyagura S.2
Shadan Institute of Medical Sciences, Physiology, Hyderabad, India,
1
Shadan Institute of Medical Sciences, Microbiology, Hyderabad, India
2
Background: Stress of any kind can alter the immune
in the early post-operative period in breast cancer
system. Surgery is a form of stress that can have sig-
patients. However, the values returned to near pre-
nificant effects on immune system. There occurs a re-
operative levels after 21 days. A positive correlation
duction in the ability to fight the disease progression
existed between psychological stress of having the
and also metastatic spread. Cell mediated immunity
disease and low host immunity. This indicates that
plays an important role in the control of malignancy.
any intervention that further suppresses immunity in
A reduction in celluar immunity can have adverse
breast cancer patients following surgery should not
effects on the body. The objective of the study was to
be used for at least 3 weeks after surgery.
understand the effects of surgical stress on cell mediated immunity in breast cancer patients.
Methods: 31 women with operable early stage breast
cancer, who underwent surgery and were also receiving adjuvant chemotherapy were included in
the study. Blood samples were collected from the
patients during the pre-operative period and twice
during the post-operative period, after 72 hours and
after 21 days. Peripheral Blood mononuclear cells
were subjected to flow cytometry analysis. The mean
values of lymphocyte sub-populations as percentage
were calculated. A questionnaire was also given to
all study participants to assess the levels of stress
of having breast cancer. An informed consent was
taken from all the participants involved in the study.
Results: The mean values of lymphocyte sub-population were found to be significantly lower (p< 0.5)
Genetic engineering in a non-traditional astrocytoma prone
mouse background
ten Buren E.B.J.1, Ormiston M.1, Korkmaz D.1, Buch T.1, vom Berg J.1
University of Zürich, Institute of Laboratory Animal Science, Zürich, Switzerland
1
Local IL-12 treatment leads to T-cell dependent re-
in less popular mouse backgrounds. We aimed at
jection of C57BL/6 syngeneic GL-261 murine brain
generating the aforementioned classical knock-outs
tumors (vom Berg et al., 2013). This finding is
in VM/Dk, starting with RAG1.
based on studies in classical mutant backgrounds
such as RAG1-/-, RAG2-/-Il2rg-/- and Il15ra-/-. Combination therapy with CTLA-4 blockade in a therapeutic setting led to rejection in a large proportion of
animals in the GL-261 model. This tumor is derived
from chemical carcinogenesis and has to be considered immunogenic. In contrast, other glioblastoma
models such as the spontaneous SMA560 tumor
model (VM/Dk mouse background) - which has
lower immunogenicity and is physiologically more
representative - respond less well to the combination treatment. Therefore, in order to increase translational value, we also want to characterize the
anti-SMA560 immune response during treatment
in RAG1-/-, RAG2-/-Il2rg-/- and Il15ra-/- mutant VM/Dk
backgrounds. Yet, as the VM/Dk background is not
widely used, these genetic mutants are not available
and backcrossing is resource intense and time consuming. Utilizing the CRISPR/Cas9 genome editing
toolbox it is now feasible to do genetic perturbations
Glioma N-Myc downstream regulated gene 1 (NDRG1) shapes
the tumor microenvironment
Thomé C.1, Blaes J.1, Rübmann P.1, Sonner J.2, Deumelandt K.2, Osswald M.1, Jugold M.3, Breckwoldt M.4,
Broggini T.5, Czabanka M.5, Vajkoczy P.5, Winkler F.1,6, Platten M.2,6, Wick W.1,6
German Cancer Consortium, German Cancer Research Center, CCU Neurooncology, Heidelberg, Germany,
1
German Cancer Consortium, German Cancer Research Center, CCU Neuroimmunology and Brain Tumor
2
Immunology, Heidelberg, Germany,
Medical Physics in Radiology, German Cancer Research Center, Project Group Small Animal Imaging Center,
3
Heidelberg, Germany,
University Hospital Heidelberg, Department of Neuroradiology, Heidelberg, Germany,
4
University Medicine Berlin, Neurosurgery Clinic Charité, Berlin, Germany,
5
University Hospital Heidelberg and National Center of Tumor Diseases, Department of Neurology, Heidelberg,
6
Germany
Malignant glioma belongs to the most aggressive
density in the NDRG1 KD microenvironment. Ex vivo
neoplasms in humans. They are highly invasive
flow cytometry analyses of the tumor stromal cells
and the cellular and genetic inter- and intratumor
revealed a significant increase in peripheral mac-
heterogeneity contributes to treatment resistance.
rophages in NDRG1 KD microenvironment compared
The interactions and intercellular communications
to the control microenvironment. The same was
between malignant and non-malignant cells in the
shown for dendritic cells as well as for monocytic
tumor microenvironment are deemed tumor-pro-
myeloid derived suppressor cells (MDSCs). Depletion
moting and critically to improve the understanding
of macrophages however did not alter tumor growth
of the disease. N-myc downstream regulated gene 1
of NDRG1 KD tumors. Cytokine array analysis on
(NDRG1) is a stress inducible gene and key determi-
U87MG NDRG1 KD and control supernatants showed
nant of resistance towards alkylating chemotherapy
a marked increase in CCL2 secretion in the NDRG1
in glioblastoma.
KD cells. Macrophages showed an increased migra-
To analyze the NDRG1 effects on the brain tumor
tion rate towards NDRG1 knockdown environment
microenvironment, we used a human NDRG1 knock-
in vitro.
down (KD) glioma model system. In orthotopic xeno-
Glioma NDRG1 shapes the tumor microenvironment
graft experiments control and KD microenvironments
by regulating angiogenesis and influences mac-
were compared for angiogenesis and infiltrating
rophage recruitment in vivo and in vitro. CCL2 was
immune cells dependent on NDRG1. Clodronate li-
identified as a relevant cytokine dependent of the
posomes were used to deplete macrophages. In vitro
NDRG1 status in this human glioma model. These
and in vivo angiogenesis assays were used to assess
data could help to better understand the relationship
neovascularization.
between NDRG1 and the tumor microenvironment.
Orthotopic tumors showed significant volume differences between control and NDRG1 KD implanted
cells and demonstrated a markedly increased vessel
Anti-tumor efficacy by the bispecific tetravalent CD30/CD16A
TandAb AFM13 is characterized by strong cross-talk from
innate to adaptive immunity and is enhanced by immune
checkpoint inhibitor anti-PD-1
Treder M.1, Zhao X.2, Rajasekaran N.2, Reusch U.1, Marschner J.-P.1, Kohrt H.E.2
Affimed GmbH, Heidelberg, Germany,
1
Center for Clinical Sciences Research Stanford, Stanford, United States
2
AFM13 is an NK-cell engaging CD30/CD16A bi-spe-
of tumor-infiltrating NK-cells, T-cells, myeloid cells
cific tetravalent TandAb antibody currently in Phase
and intra-tumoral cytokines such as IFN-gamma.
2 clinical development in Hodgkin Lymphoma (HL)
In contrast to anti-PD-1 monotherapy, which only
and other CD30+ malignancies. Immune checkpoint
induced T-cell infiltration, AFM13 monotherapy was
inhibitors have demonstrated clinical efficacy in a
able to induce infiltration of NK- and T-cells in the
variety of cancers, including HL. NK-cells are also
tumors, however the combination much further en-
regulated by a number of check-points, prompting us
hanced infiltration of both, NK- and T-cells. AFM13
to investigate the combination of AFM13 with several
resulted in stronger infiltration of macrophages than
immuno-modulatory antibodies to enhance anti-tu-
anti-PD-1, which was also increased by the combi-
mor efficacy. In previous experiments we were able
nation of both agents, therefore further supporting
to demonstrate higher efficacy of AFM13 than several
cross-talk between innate and adaptive immunity.
immuno-modulatory antibodies in monotherapy and
Furthermore, tumor analyses at earlier time-points
strong synergy between AFM13 and an anti-PD-1 an-
showed that the initial immune response is charac-
tibody in vitro, as well as in vivo PDX models with
terized by NK-cell infiltration and activation, as well
+
HL tumors. In order to investigate
as infiltration of macrophages, whilst the adaptive
the underlying immunological mechanisms we em-
immune response by T-cells and activated dendritic
ployed the same PDX model by implanting tumor
cells was more pronounced towards the end. Com-
fragments derived from surgical specimens of HL
bining AFM13 and anti-PD-1 augments infiltration
patients in immuno-deficient mice. After establish-
and activation of all immune subpopulations.
ing tumors, mice were reconstituted with autolo-
In conclusion, our data support strong synergistic
gous patient-derived PBMC and treated with AFM13
anti-tumor efficacy when AFM13 is combined with
alone and in combination with anti-PD-1 weekly for
anti-PD-1 checkpoint blockade in HL PDX models,
a total of three weeks. Tumor size, tumor-infiltrating
mediated by tumor-infiltrating lymphocytes, mac-
human lymphocytes, myeloid cells and intra-tumoral
rophages and dendritic cells, and provide strong
cytokines were evaluated at early, intermediate and
evidence for cross-talk between innate and adaptive
late time-points after treatment start. While mono-
immunity induced by AFM13-recruited human NK-
therapy with AFM13 was reproducibly more potent
cells. These results not only validate the anti-tumor
than anti-PD-1, significant synergy was observed
efficacy of AFM13, but also for other NK-cell recruit-
when both agents were combined. Analysis of the
ing TandAb immunotherapy regimens.
human CD30
tumors at the end of the studies revealed a strong correlation between tumor growth inhibition and levels
Induction of a well-defined immune response using a novel
systemically applied Toll-Like Receptor 7 agonist in mice and
men
Vascotto F.1, Petschenka J.1, Reuter K.2, Hüsemann Y.3, Roth R.2, König A.2, Worm C.2, Brkic M.4, Krimmel N.2,
Thiel A.-K.3, Schmitt U.4, Brill W.3, Diken M.1, Kreiter S.1, Hamm S.5, Strobl S.5, Türeci Ö.6, Sahin U.1,2,4
1
Mainz, Germany,
2
BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany,
3
Research Center for Immunotherapy (FZI), Mainz, Germany,
4
4SC Discovery GmbH, Planegg-Martinsried, Germany,
5
CI3 - Cluster of Individualized Immunointervention, Mainz, Germany
6
Toll-like receptor (TLR) ligation activates both the
to be the TLR7 lead candidate for topical treatment
innate and adaptive immune systems and plays an
of cancers. We found that SC1 treatment increased
important role in antiviral and anti-tumoral immu-
frequency of tumor antigen-specific (gp70) CD8 T
nity.
cells detectable in blood, spleen and tumors, which
So far, the only approved TLR agonists in clinical
are the effector cells mediating anti-tumor therapy.
settings are Imiquimod and Resiquimod (R848). Both
Notably, SC1 treated mice mounted a memory CD8
compounds are applied topically since they display
T cell response, which protected mice against tumor
disadvantageous toxic effects after systemic applica-
re-challenges and conferred tumor control after pro-
tion, while newer agents like 852A and VTX2337 just
phylactic adoptive cell transfer (ACT).
recently completed first clinical studies.
In order to increase solubility and selectivity but also
Here we describe preclinical studies using SC1 and
improve the activity in terms of a more beneficial
SC1.2, novel small molecule TLR7 agonists against
cytokine profile SC1 was chemically adapted to a
several murine tumor models. Using a therapeutic
new lead candidate SC1.2. SC1.2 showed an identical
setting, repetitive intravenous injections of SC1 po-
anti-tumoral effect in CT26-WT tumor-bearing mice.
tently prevented lung metastasis formation of 4T1
Moreover, human whole-blood analysis revealed a
orthotopic breast cancer and delayed primary tumor
potent induction of proinflammatory cytokines and
growth of B16 melanoma. In the CT26 colon carci-
activation of the innate immune system after co-in-
noma model, SC1 conferred a strong anti-tumoral
cubation with SC1.2.
effect, prolonging survival and increasing the anti-
In conclusion, these novel TLR7 agonist SC1 and
tumoral immune response. We could show that sys-
SC1.2, in a non-toxic systemic regimen act as strong
temic application of SC1 displayed a far better anti-
anti-tumoral mono-therapeutic agents, potentiat-
tumoral effect compared to 852A, one of the few TLR7
ing anti-tumoral immune response via CD8 T cells,
ligands already described for systemic injections.
indicating promising implications for treatment of
In addition, intratumoral injection of SC1 potently
several cancers entities.
controlled tumor growth similar as R848, known
A changing neo-antigen landscape in human melanoma under
T cell pressure
Verdegaal E.1, Visser M.1, Harryvan T.1, van Buuren M.2, Andersen R.S.3, Hadrup S.R.3, van der Minne C.1,
Schotte R.4, Spits H.4, Haanen J.2, Kapiteijn E.1, de Miranda N.5, Schumacher T.2, van der Burg S.1
Leiden University Medical Center, Medical Oncology, Leiden, Netherlands,
1
Netherlands Cancer Institute, Immunology, Amsterdam, Netherlands,
2
University Hospital Herlev, Hematology, Copenhagen, Denmark,
3
4
Leiden University Medical Center, Pathology, Leiden, Netherlands
5
ORAL
TALK
SHORT
2016
Recognition of neo-antigens encoded by mutated
lost neo-antigen expression arise, demonstrating the
DNA are considered a driving force behind the clini-
occurrence of neo-antigen editing in human cancer.
cal activity of cancer immunotherapies such as T cell
This underscores the importance to induce a broad
checkpoint blockade and adoptive T cell therapy. In
neo-antigen specific T cell response to obtain thera-
mouse models, T cell pressure has been shown to
peutic efficacy and to avoid tumor resistance.
sculpt the antigenicity of tumors, resulting in the
emergence of tumors that lack defined mutant antigens. Whether the T cell-recognized neo-antigen
repertoire in human cancers is constant over time is
unclear. Therefore, we performed an In depth analysis of the neo-epitope specific T cell responses and
the antigens they recognize in two stage IV melanoma patients with long term survival after adoptive T cell transfer. Expression of genes encoding T
cell-recognized neo-antigens were shown to be selectively lost from sequentially obtained tumor cells,
either by preferential transcription of the non-mutant
copy, or by loss of the mutant allele. Strikingly, loss
of expression of T cell-recognized neo-antigens was
accompanied by development of a novel neo-antigen
specific T cell reactivity within tumor-infiltrating
lymphocytes. Our data illustrate that, as a consequence of the detected neo-epitope-specific T-cell
reactivity, immune-edited variants with reduced or
Tolerogenic effects of GM-CSF through expansion of regulatory
T-cells and induction of the Treg-associated chemokine CCL22
Vetter V.1, Knott M.1, Layritz P.1, Kühnemuth B.1, Endres S.1, Anz D.1
Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Division of Clinical Pharmacology,
1
Munich, Germany
Malignant tumors are known to escape effective anti-
of CCL22 may shift the balance between tolerogenic
tumor immune response through the generation of
and antitumoral properties of GM-CSF and thereby
an immune-suppressive tumor microenvironment
represents a promising additional therapeutic strat-
(TME), for instance by recruiting regulatory T-cells
egy in tumor therapy. In order to elucidate the tolero-
(Tregs). Here we aimed to elucidate the contribution
genic effects of GM-CSF mediated CCL22-induction
of granulocyte macrophage colony-stimulating factor
further, we are keen to investigate tumor growth,
(GM-CSF) - a cytokine known to trigger pro- and anti-
CCL22 levels and Treg infiltration in mice bearing
tumoral effects - in the establishment of an tumor
s.c. GM-CSF overexpressing tumors. Additionally, we
promoting TME.
plan to analyze the effects of CCL22 depletion on the
In order to identify potential tolerogenic effects of
potency of vaccination with GVAX (GM-CSF trans-
GM-CSF we stimulated MACS-sorted CD11c+ den-
duced radiated tumor cells).
dritic cells with recombinant GM-CSF in vitro and
analyzed chemokine expression using qRT-PCR.
We could show that GM-CSF induces a range of
chemokines, among them the two Treg-attracting
chemokines CCL22 and CCL17. In line with the
CCL22 induction in vitro, mice treated with recombinant GM-CSF (i.p.) upregulated CCL22 systemically.
Moreover, FACS-analysis of spleen and lymphnodes
revealed an GM-CSF induced expansion of regulatory T-cells as well as an expansion and activiation
of dendritic cells. The proliferation of Tregs through
GM-CSF was also confirmed in vitro using CFSE-labeled unsorted splenocytes. Finally, we investigated
the effect of tumor-derived GM-CSF in supernatants
of stably transduced GM-CSF overexpressing tumor
cell lines on CCL22 induction and Treg migration in
vitro.
In conclusion, we could highlight that GM-CSF shows
tolerogenic effects through the induction of CCL22
and the expansion of regulatory T-cells. Depletion
Impact of Interleukin-22 on two murine models of lung and
breast cancer
Voigt C.1, May P.1, Gottschlich A.1, Wenk D.1, Endres S.1, Kobold S.1
Ludwig-Maximilians-Universität, Division of Clinical Pharmacology, München, Germany
1
Background: Interleukin-22 (IL-22) is a unique cy-
ble role of IL-22 in murine breast and lung cancer.
tokine expressed by several immune cell subtypes
More in vivo data is needed on the regulation and
and acting exclusively on interleukin-22-receptor-1
the impact of IL-22 in these models. The identifica-
(IL-22-R1) positive non-hematopoietic cells. Recent-
tion of IL-22 as a factor in breast and lung cancer
ly, we have demonstrated that expression of IL-22 is
progression may open new therapeutic opportunities
frequently found in primary human small and large
for these diseases.
cell lung cancer and that IL-22 may promote a more
aggressive disease phenotype. The mechanism, the
source and the role of IL-22 in lung cancer and other
tumor entities like breast cancer remain largely unaddressed.
Methods: The Expression of IL-22 and IL-22-R1 were
analyzed by ELISA and qRT-PCR in two different
cancer cell lines (4T1 and LCCL1). Activation of the
IL-22 pathway was detected by Western blot analysis. Proliferation and migration were investigated by
scratch assay and cell titer blue. Tumor tissue IL-22
content was quantified in subcutaneous cell-line
derived tumors in Balb/c mice with multicolor flow
cytometry.
Results: 4T1 and LCCL1 tumor cells expressed the
IL-22-R1 both on protein and mRNA level. Stimulation with recombinant IL-22 lead to a time dependent
increase in STAT3 phosphorylation on protein level.
Stimulation with IL-22 significantly increased cell
proliferation in both cell lines. Remarkably, 4T1 and
LCCL1 cells in vitro did not produce or secrete IL-22.
However, IL-22 was detected within the tumor microenvironment of subcutaneous tumors in different T,
NK and myeloid cell subpopulations.
Conclusions: Our results point towards a possi-
A dual role for IL12 in T-cell receptor-dependent and -independent tumor cell killing: regulation of a DNAM1 mediated,
PTPRC/CD45-dependent mechanism in human effector T-cells
Braun M.1, Ress M.L.1, Yoo Y.E.1, Scholz C.J.2, Eyrich M.1, Schlegel P.G.1, Wölfl M.1
University Children’s Hospital, Würzburg, Germany,
1
University of Würzburg, Core Unit Systems Medicine, Würzburg, Germany
2
Interleukin 12 (IL12) is a key inflammatory cytokine
critically influencing T-cell responses at the time of
priming and may be exploited for cancer immunotherapy. Here we investigated how IL12, and other
inflammatory cytokines, shape effector functions of
human T-cells. Using a defined culture system, we
followed antigen-specific CD8+ T-cells from their
initial activation as naïve T-cells through their expansion as early memory cells to full differentiation
as clonally expanded effector T-cells. Addition of
IL12 8 days after the initial priming event initiated
two mechanistically separate events: first, IL12 sensitized the T-cell receptor (TCR) for antigen-specific
activation leading to an approximately ten-fold increase in peptide sensitivity and better tumor cell
killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this ‘non-specific’
effect was mediated by DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL-12
regulated, DNAM1-mediated killing is dependent on
src-kinases as well as on PTPRC (CD45) activity.
Thus, besides enhancing TCR-mediated activation,
we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptordependent killing pathway via DNAM1.
Patient-derived tumor xenografts in humanized mice: a
preclinical model for the development of innovative immunotherapeutics
Wulf-Goldenberg A.1, Stecklum M.1, Fichtner I.1, Hoffmann J.1
EPO GmbH, Berlin, Germany
1
Patient-derived xenografts (PDX) from different
companied by an increase of human T cells in the
tumor indications transplanted on immunodeficient
peripheral blood. Ex vivo analysis will be performed
mice have demonstrated strong predictive power
demonstrating the cytotoxicity of these T cells and
for many drug development programs in cancer re-
the functionality of the human immune cells.
search. However, one caveat of PDX models is that
Humanized mice bearing various PDX tumors were
they lack a functional immune system, as a precon-
treated with approved therapeutic checkpoint in-
dition for tumor engraftment in the xenogeneic host
hibitors. Ipilimumab that target the CTLA-4 receptor
mice.
leads to a slight tumor growth delay and an increased
Immunotherapy has emerged as one of the most
percentage of T cells in the blood and in the tumor.
promising avenues for cancer therapy. For translating
Nivolumab that targets the PD-1 receptor showed
immunotherapies into the clinics preclinical animal
similar results. The combination of both inhibitors
models are needed which harbor human immune cell
showed a synergistic effect on tumor growth inhibi-
repertoire for rising an immune response.
tion.
To overcome these constraints our aim is the de-
Our humanized mouse models enable appropriate
velopment of PDX models in mice with a functional
preclinical assessment of immune-based therapeutic
human immune system to improve predictability of
anti-tumor strategies especially when combing the
drug efficacy and safety.
humanized mouse with patient-derived xenografts.
We reconstituted a human immune system in mice
by engrafting human hematopoietic stem cells. We
were able to demonstrate that in these mice a full set
of human immune cells, including T cells, B cells, NK
cells, monocytes and dendritic cells can be analyzed
by flow cytometry.
At the time when the human immune system was
developed, established patient-derived tumors were
transplanted on these reconstituted humanized
mice. Different tumor entities are growing in these
humanized mice either similar to non humanized
mice or slightly slower. During the observation
period no tumor rejections by the human immune
cells were evident, although tumor growth was ac-
The role of the IL-22/IL-22R1 axis in Pancreatic ductal
adenocarcinoma
Zayoud M.1, Marcu-Malina V.1, Atias D.2, Stossel C.2, Golan T.3, Goldstein I.1
Tel Aviv University, Sheba Cancer Research Center; Chaim Sheba Medical Center, Sackler Faculty of
1
Medicine, Ramat Gan, Israel,
Institute of Oncology, Sheba Medical Center, Ramat Gan, Israel,
2
Tel Aviv University, Institute of Oncology, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv, Israel
3
Pancreatic ductal adenocarcinoma (PDAC) remains
with a significant increase in their proliferation after
one of the most lethal types of cancer with poor
the addition of IL-22 to the culture medium. Moreo-
prognosis despite extensive efforts. JAK-STAT3 sign-
ver, isolated mononuclear cells from PDAC patients´
aling plays a significant role in the development and
ascites showed higher percentage of IL-22+ lympho-
progression of pancreatic cancer. IL-22 is a cytokine,
cytes, and importantly factors found in PDAC ascites
which belongs to the IL-10 family, acts via activation
induced significant increase on the polarization of
of Jak/Stat3-dependent signaling cascades and is a
naïve T cells into Th22 cells.
well-described growth factor for epithelial cells.
In summary, we show that IL-22 has a positive effect
To study the role of IL-22 in the tumor microenviron-
on the growth of PDAC that uniformly express the
ment of PDAC patients, focusing on the reciprocal
IL-22RA1, while reciprocally the PDAC environment
interactions between IL-22-producing lymphocytes
contains factors that can potentially instruct tissue
and PDAC cells we analyzed the following param-
infiltrating lymphocytes to produce the pro-prolifera-
eters: the expression of IL-22RA1 chain on both
tive and immunosuppressive cytokine IL-22 to create
primary tumor cells isolated from ascites of PDAC
a positive feedback loop.
patients and various PDAC cell lines; the pro-proliferative effect of IL-22 on these various PDCA cells
in-vitro; the capacity of ascites-derived supernatants
from metastatic PDAC patients to polarize naïve T
cells, in vitro, towards the Th22 phenotype; and detection by intracellular staining the percentage of
IL-22 secreting lymphocytes in the ascites of metastatic PDAC patients.
We found high expression of IL-22RA1 on all the
primary PDAC cells and cell lines tested coupled
Genetic heterogeneity of intra-patient metastases restricts
T-cell recognition of malignant melanoma
Zhao F.1, Sucker A.1, Horn S.1, Heeke C.1, Bielefeld N.1, Lernerz V.2, Wölfel T.2, Schadendorf D.1, Griewank K.1,
Paschen A.1
University Hospital Essen, Dermatology, Essen, Germany,
1
University Medical Center of the Johannes Gutenberg University Mainz, III. Medical Clinic, Mainz, Germany
2
Enhancement of anti-tumor T cell cytotoxicity by
Thus our study clearly demonstrates that intra-pa-
blockade of inhibitory checkpoint signaling has re-
tient genetic heterogeneity of melanoma metastases
cently achieved remarkable progress in melanoma
sets multiple barriers to effective T cell recognition,
immunotherapy. But in order to be effective, T cells
a challenge for melanoma immunotherapy.
have to engage specific antigen-HLA class I complexes on the tumor cells. However, this process might be
affected at multiple levels by the high genetic instability of the tumor.
To analyze this, we set up the melanoma patient
model Ma-Mel-86 consisting of four cell lines (MaMel-86a, Ma-Mel-86b, Ma-Mel-86c, Ma-Mel-86f) established from sequential metastases. Analysis of
HLA class I surface expression revealed that only
melanoma cells obtained from metastases MaMel-86a and Ma-Mel-86c were positive while cells
derived from metastases Ma-Mel-86b and Ma-Mel86f showed a stable HLA class I-negative phenotype.
Interestingly, upon short-term co-culture with autologous CD8+ T cells, Ma-Mel-86a and Ma-Mel-86c
cells each amplified their own specific pool of T cells
with only limited cross-reactivity. Ma-Mel-86a cells
preferentially stimulated T cells directed towards antigens presented by HLA-A24 and HLA-B15. Ma-Mel86c cells were protected from these T cells due to an
HLA haplotype loss. On the other hand, Ma-Mel-86c
cell strongly amplified T cells directed towards the
melanoma differentiation antigens (MDA). Ma-Mel86a cells were resistant to MDA-specific T cells as
they completely abrogated expression of the melanocytic differentiation program.
Imprint
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Dendritic cell immunotherapy in ovarian cancer

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