The CAR T-cell paradigm - Adoptive Immunotherapies

March 02, 2016
ADOPT-Summit, London
From a basic research to patients
health: The CAR T-cell paradigm
Prof Zelig Eshhar, PhD
Chair Center of Immunology Research,
Tel-Aviv Sourasky Medical Center
and-
Department of Immunology, The
Weizmann Institute of Science
Chimeric Antibody - T Cell receptor combines Antibody specificity
with T Cell function (the 2-chain generation)
Gross et al. Proc. Natl. Acad. Sci. 86: 10024, 1989
THE MODULAR CHIMERIC RECEPTOR DESIGN
Recognition unit
Antibody or TCR scFv
Ligand (heregulin or MHC: peptide)
Receptor (CD4)
Extracellular
Spacer / Hinge
CD8, IgG1, Ck, Ca, CD28
Transmembrane
Any TM canonic structure
Signaling and Co-Signaling domains
Intracellular
CD28, 4-1BB (CD137), IL-2R
CD3z,/FcR 
Syk PTK,
Eshhar, Z., T. Waks, G. Gross, and D. G. Schindler. 1993. Proc Natl Acad Sci USA 90:720-724.
CAR is a Modular Structure
Signal 2
Signal 1
The Pioneer team
Tova Waks
Gidi Gross
Daniel Schindler
Model 1985…
Dr. Steve Rosenberg co-partner of immune-cell therapy
Historic milestones (date of first publication):
1989- First ‘double-chain’ chimeric antibody receptor: TCRaVH + TCRbVL
Gross et al.
1993- First single chain (scFv) chimeric antibody receptor
Eshhar et al.
1993- First antitumor specific CARs (in-vitro cytotoxicity)
Stancovski (Her2/neu); Hwu et al (a folate receptor); Gross et al
(B cell lymphoma idiotype).
1995-First antitumor reactivity by redirected NK cells
Bach et al.
1996-1998 - Introduction of co-stimulatory signaling into the CAR
Hawkins et al; Finney et al; Sadelain et al.
2000-2006- First tumor-specific CAR in patients
Kershaw et al (a folate receptor, ovarian cancer); Lamers et al (CA-IX,
renal cell carcinoma).
2008-2013- Pilot clinical trials showing complete remission of cancer
patients
Brenner et al; Rosenberg et al; June et al; Jensen/Riddell et al; Brentjens
et al.
Prostate Cancer bone lesions 5 weeks post systemic
T-body treatment
Non-irradiated
Irradiated
HBSS
Anti-TNP
Anti-erbB2
Normal contra
lateral bone
(Irradiated)
Pinthus J.H. et al. et al.
Cancer Res 60:6563, 2000
Pinthus et al. JCI 2004
Systemic Therapy of Prostate Cancer Bone Lesions by
Lymphoablation Followed by ErbB-2 Specific T-Bodies
Pinthus et al. J.Clin.Inv., 2004
SCID mice bearing established bone Hu-PC xenograft were pre-conditioned by
cyclophosphamide and a day later the T-bodies were systemically treated with Tbodies
0
0
50
100
150
Improved survival of mice with systemic Renca-HER2 lung micrometastases
200
250
Days
1.2
control group
activated 10^7 balb Tg-N29*3
iv
naïve 10^7 balb Tg-N29*3 iv
1
0.8
control group
0.6
activated Balb-N29 10^7 TgN29*3
0.4
0.2
0
50
100
150
200
250
0.8
0.6
0.4
0.2
1
0.8
Pre-immunization of
mice
control
group bearing
Renca-erbB2 lung metastases with 0.6
activated Balb-N29 10^7 TgN29*3
either Renca or Renca-erbB2
0.4
enhances the antitumor effect of
0.2
naïve HER2-specific T-bodies.
0
0
50
100
Days
0
50
100
Days
Days
% Survival of tumor bearing mice
1
0
0
1.2
Tova Waks, Anat Globerson, Assaf
Marcus (2009)
% Survival of tumor bearing mice
1.2
% Survival of tumor bearing mice
Pre-activation
(culture with
ConA) of
naïve Tbodies (from
HER2specific Tg
mice)
improves the
antitumor
activity
150
200
150
200
Neo-Adjuvant Treatment by Intratumoral T-Body Treatment of
s.c. Pancreatic Adenocarcinoma Xenograft
(Amit Maliar et al. (Gastroenterology, 2012)
Treatment of Orthotopic Pancreatic Adenocarcinoma Xenograft by T-Bodies
Control
Treated group
CD24 is Pancreatic adenocarcinoma Cancer Stem Cell Antigen
It isxpressed on 80% of passage 2 of Wapac-4 and
35% of passage-3 of Wapac-5
Early passage Wapac 4 and 5 xenografts were treated with CD24 and HER2-specific
CAR T-Cells
ASGCT 14th Annual Meeting
Seattle
May 18, 2011,
Redirected, Tumor-Specific Allogeneic CAR T-Cells
(“allo-T-Bodies”) for Universal Adoptive Cell
Treatment of Cancer
Assaf Marcus, Tova Waks and Zelig Eshhar
Creating a therapeutic time window to allow effective antitumor reactivity with no
GvH by extending the persistence of the allo-T bodies at the tumor site, using1. mild
and transient depletion of the host’s lymphocytes
2. administration of relatively large numbers of the donor T-bodies
3. extending the persistence of the allo-T bodies at the tumor site & LN
Marcus A., Waks T. and Eshhar Z. Redirected tumor-specific allogeneic T cells for
universal treatment of cancer. Blood 2011, 118:975-83.
Dual Recognition by CAR+TCR Enhances the Antitumor
Activity of T-Bodies
• Recognition of tumor antigen (through
the CAR) together with common tissue
antigen (e.g. opportunistic virus antigen or
alloantigen) through the TCR results
better persistence.
• This is most probably due to chronic
stimulation of the dual-specific T-bodies
by normal APCs resulting in longer gene
expression, reduced apoptosis and
cell expansion.
• Immunization can further enhance this
effect
Berger C, Turtle CJ, Jensen MC, Riddell SR. Adoptive transfer of virus-specific and tumorspecific T cell immunity. Curr Opin Immunol. 2009, 21(2):224-32.
Schematic illustration of the experimental setup.
Assaf Marcus et al. Blood 2011;118:975-983
©2011 by American Society of Hematology
Balance between host’ preconditioning and transferred cell dose determines
the antitumor benefit provided by allogeneic T-bodies.
Assaf Marcus et al. Blood 2011;118:975-983
©2011 by American Society of HematologyS
FTY720 Enhances the Antitumor Response of
Allogeneic T bodies
(Cayman chemical)
FTY720 is a derivative of ISP1 (myriocin), a fungal metabolite of the
Chinese herb Iscaria sinclarii as well as a structural analog of sphingosin.
It is a novel immune moderator that prolongs allograft survival in numerous
models by inhibiting lymphocyte emigration from lymphoid organs. Down
regulation of S1P1 receptors on T and B lymphocytes by FTY720 results
in defective egress from spleen, lymph nodes and Peyer patches
(Marloubain M. et al. Nature 427: 355, 2004)
Allo-reactivity modulates the migration and persistence of allo-T-bodies.
Assaf Marcus et al. Blood 2011;118:975-983
©2011 by American Society of Hematology
FTY720 augments allogeneic but not syngeneic adoptive cell therapy.
Assaf Marcus et al. Blood 2011;118:975-983
©2011 by American Society of Hematology
FTY720 reduces the number of circulating allo-T-bodies in the
recipient mice and increases T-body number in the spleen.
Assaf Marcus et al. Blood 2011;118:975-983
©2011 by American Society of Hematology
The Her2NGTransgenic Model
(Courtesy of Genentech, Finkle D., et al Clin. Cancer Res. 2004, 10(7):2499-511)
• FVB heterozygote mice over-expressing the
human erbB-2 transgene under the MMTV
promoter
• Female mice develop mammary tumors with
metastases at the age of 23-45 weeks
• Lower amounts of Her2/neu are also expressed
in normal lungs, pancreas, liver, kidney
Anat Globerson and Tova Waks
Survival of Her2NG Mice Systemically Injected With
erbB-2 specific CAR T-cells
Homing of luminescence T CARs
CAR T-cell persistence (Day 500)
spleen and bone marrow
blood
lymphocytes
Take-home Lessons (from murine models)
For application in adoptive cell therapy
• Use combination of both CD4+and CD8 naive memory redirected T cells
• Precondition the recipient with lymphodepleting treatments
• Remove/neutralize Tregs
• Grow/infuse T bodies with IL-7, IL-15 and IL-21 or combination thereof
• Activation of naïve T-bodies results in better antitumor response
• Vaccination of recipients prior to T-body administration augments antitumor
response
• For longer persistence it is advantageous to employ dual-specific T cells
• Allogeneic T-bodies under controlled conditions can serve as a universal source
of donor cells in adjuvant therapy for systemic metastases
• Cancer stem cells are optimal targets for T-body cancer therapy
For safer treatment
• Use inducible suicide genes
• Use site-specific integrating vectors
26
The CAR T-cell approach has been tested in pre-clinical human models
Gliomas, Neuroblastomas
Breast
Kidney
Ovarian
Prostate
Sarcomas
Pancreas
Blood
Colon
Protocol for T-body Preparation & Treatment
Summary of objective response rate in ALL clinical trials with anti-CD19 CAR T cells*
Organization
CAR
Indications
Response rate**
NCI – Surgery
and Pediatric
Oncology
Branches
AntiPediatric Acute B –
CD19(FMC63)- Cell Lymphoblastic
CD28 zeta
Leukemia (ALL)
46 r/r patients
60% Complete Response rate (CR)
University of
Pennsylvania
Anti-CD19
Pediatric B-ALL
(FMC63)-41b-b
zeta
59 r/r Patients
1 Month 55 r/r patients 93% CR
6 Months RFS 76%
12 Months RFS 55%
No relapse past 1 year
18 patients in remission beyond 1 year;
13 without therapy
Memorial
AntiAdult B-ALL
Sloan
CD19(SJ25C1)
Kettering
- CD28 zeta
Cancer Center
46 patients
1M 45/46 CR (97%) CR
6M 20/45 CR (44%)
 12M 9/45 (20%) CR
Overall CR Rate 37/45 (82%)
Overall MRD negative 30/36 (83%)
*Recent information from Fred Hutchinson Cancer Center disclosed that 94% of 35 ALL patients treated with
CAR-modified T-cells; went into remission (though symptoms could reappear). Additionally, more than 40
lymphoma patients, similarly treated, came with remission rates of more than 50%. In NHL group of treated
patients, diminished cancer symptoms was evident in more than 80% of cases. (in the Guardian)
** Aggregate objective response rate = 85% (82/97); CRe- Complete Remission (> 18 months)
Future Challenges for CAR T-Cell Therapy
1.
2.
3.
4.
Make it Safer
Prepare a Universal Donor
Make it handy and Cheaper
Expand the spectrum of CAR strategy to treat solid
tumors
5. Overcome the tumor-suppressive microenvironment
THANKS!!!
•
•
•
•
•
•
•
•
•
•
•
•
•
Tova Waks
Anat Globerson
Amit Maliar
Dan Blat
Charlotte Serves
Assaf Marcus
Liat Elboim
Eran Elinav
Nitzan Adam
Itai Kela
Anna
Dinora Morviski
Vica Melina
Supported by: ATTACK an EC Fp6 Consortium, DoD PCRP & BCRP, ISF
Thanks To The CAR T- Team!!!
Both antibody-based chimeric receptor and TCR-based allo-reactivity
contribute to the antitumor response.
Assaf Marcus et al. Blood 2011;118:975-983
©2011 by American Society of Hematology
Conclusions from the CD19 CAR T-Cell
(the NIH experience)
• Conditioning with cyclophosphamide and flludarabine modifies the immune
environment through induction of molecules that can favor the homeostatic
expansion, activation and trafficking of T cells
• A shorter manufacturing process yielded CAR T cell products with a higher
representation of naïve and central memory T-cells. Post induction, CAR Tcells show a diversified subset composition comprising mainly
differentiated T cells and some central memory or or naïve T cells
• Durable responses can occur without long lasting CAR T-cells in the
circulation, allowing normal B cell recovery
• Post-infusion, CAR T cells show a diversified subset composition comprising
mainly differentiated T cells, and some central memory or naïve T cells.
• Product T cells upregulate T cell activation markers in response to CAR
engagement of target.
• CAR T cell treatment results in rapid elevation and subsequent resolution of
circulating cytokines and chemokines within 3 weeks after treatment.
Conclusions from the CD19 CAR T-Cell
(the NIH experience)
• Conditioning with cyclophosphamide and flludarabine modifies the immune
environment through induction of molecules that can favor the homeostatic
expansion, activation and trafficking of T cells
• A shorter manufacturing process yielded CAR T cell products with a higher
representation of naïve and central memory T-cells. Post induction, CAR Tcells show a diversified subset composition comprising mainly
differentiated T cells and some central memory or naïve T cells
• Durable responses can occur without long lasting CAR T-cells in the
circulation, allowing normal B cell recoveryvs
• Post-infusion, CAR T cells show a diversified subset composition comprising
mainly differentiated T cells, and some central memory or naïve T cells.
• Product T cells upregulate T cell activation markers in response to CAR
engagement of target.
• CAR T cell treatment results in rapid elevation and subsequent resolution of
circulating cytokines and chemokines within 3 weeks after treatment.
Clinical trials at ClinicalTrials.gov evaluating CAR T cells against blood cancers
and solid tumors