Diapositiva 1

Proteine ricombinanti (antigeni e anticorpi)
da pianta e da altri sistemi di espressione
per applicazioni diagnostiche e terapeutiche
Rosella Franconi
BAS BIOTEC GEN
[email protected]
ENEA, 3 Aprile 2009
BREVETTI
BENVENUTO E., FRANCONI R., DESIDERIO A.,
TAVLADORAKI P. (1999) ‘ Stabilizing peptides,
polypeptides and antibodies which include them‘.
Brevetto Europeo 1120464
FRANCONI R, et al. (2001) 'Vaccini
a subunità e procedimenti per la loro
produzione‘. RM2001A000332.
Brevetto Europeo
FRANCONI R. & ILLIANO E. (2007).
‘Proteina E6 di HPV ricombinante, solubile e
in forma biologicamente attiva,
procedimento per la sua preparazione, usi e
vaccini terapeutici che la comprendono’.
Brevetto RM2007A000220
FRANCONI R. et al. (2009). ‘Vaccini basati
su chimere genetiche tra antigeni virali,
tumorali e proteine vegetali’.
In fase di deposito
-S-S- - - -- S-S-S S
-S-S-
Anticorpi ricombinanti
Anticorpo a singola
catena: scFvs <30 K
CDR
NH2
S
VH
S
VL
CL
- S
- S
-
-
- S
- S
-
- S
- S
-
S
- S
-
CH
COOH
Molecola di
anticorpo
‘PHAGE DISPLAY’
1. Manipolazioni genetiche:
repertorio di mutanti
2. Espressione alla superficie del
fago
‘Biopanning’
4. Lavaggi
6. Amplificazione
7. Analisi
dei ligandi
specifici
5. Eluizione
3. I fagi legano
l’antigene
Da questo repertorio (5 x 107 molecole diverse) finora isolati
anticorpi stabili e ad alta affinità contro:
Virus vegetali (es. CMV, PVX, AMCV, TSWV)
Antigeni modello (es. BSA, lisozima, GST)
Oncoproteine (es.E7 di HPV 16)
Esempio applicativo 1.
Immunomodulazione/Immunoterapia
Piante di pomodoro resistenti al virus del mosaico del cetriolo
(CMV)
scFv
WT
Esempio applicativo 2.
Realizzazione di nuovi sistemi diagnostici
Immobilizzazione reversibile su supporti
elettrochimici/ottici
Anticorpi ricombinanti (‘Library F8’ e/o da animali
immunizzati)
Possibili applicazioni industriali
•
Sistemi diagnostici per uso biomedico, industria alimentare (es.
monitoraggio micotossine), ambiente, agrobiotecnologie
•
Sviluppo di una piattaforma tecnologica per produzione a basso
costo su larga scala (da microbi e da pianta)
•
Biofarmaci innovativi
BENVENUTO E., FRANCONI R., DESIDERIO A.,TAVLADORAKI P. (1999) 'Stabilizing peptides,
polypeptides and antibodies which include them‘. Brevetto Europeo 1120464
Expression systems of heterologous proteins
MoreBacteria
than 100 Plant Made
Pharmaceuticals
(PMPs) have
(E. coli)
been produced in plant:
PLANTS
-Antibodies
Yeasts
(S. cerevisiae,
-Therapeutic
molecules
P. pastoris)
(i. e. avidin, trypsin, βglucuronidase, human albumin,
serotonin, etc)
Mammalian Cells
(Cos)
-VACCINES
MICROALGAE
TRANSGENIC PLANT TECHNOLOGY (STABLE expression)
POTENTIAL OF VIRAL VECTORS TECHNOLOGY
(Transient expression)
First generation
‘Full virus’ strategy
•High level of
expression:
up to 5 g protein/Kg
fresh leaf biomass
Second-generation
‘Deconstructed virus’ Strategy
•Speed:
50% TSP in 4-10 days
GENERAL SCHEME FOR RECOMBINANT PROTEIN PRODUCTION IN PLANTS
(INDUSTRIAL PROCESS)
Thus far in Europe there has been no commercial application of PMP
technology (although several products have reached the clinical trial
stage like gastric lipase, lactoferrin ecc.)
and:
Bayer AG (major pharmaceutical company, that in 2006 acquired
ICON Genetics) announced in July ‘08 the opening of a production
facility that will use tobacco to manufacture biopharmaceuticals, the
first of which will be a patient-specific antibody vaccine for NONHODGKIN’S LYMPHOMA THERAPY
Greenhouse for containment of GMO
at ENEA Casaccia, Rome
PLANT-DERIVED HPV ANTIGENS/VACCINES
The Nobel Prize in Medicine 2008
“For his discovery of human papilloma
viruses causing cervical cancer"
“His studies allowed to establish that HPV is the
causative agent of cervical cancer: he is the first who
identified a virus as tumor cause.
“His discovery brought to the characterization of the
HPV infection and carcinogenesis mechanisms”
Harald zur Hausen
Doctor and virologist
German Cancer Research Centre
Heidelberg, Germany
Human Papilloma Virus
(HPV)
Cervical Cancer
Second cause of death in women
FIRST in Developing Countries
80% of cervical cancers worldwide
High-risk HPV
300 millions women infected
Global Incidence of Cervical Cancer (Vaccine, vol.3 - 2005)
HPV16 causes more than 50%
of cervical cancer
HPV VACCINES
- PROPHYLACTIC
Commercial vaccines based on the L1 protein (VLPs):
GARDASIL (Merck): VLPs of L1
CERVARIX (GlaxoSmithKline): VLPs
protein from HPV 6/11/16/18 made in
yeast, aluminum adjuvant
of L1 protein from HPV 16/18 made in
baculovirus, AS04 adjuvant
Expensive!!!!
¾Low-cost prophylactic vaccines.
¾Low-cost antigens for vaccinated people follow-up
(post-marketing monitoring).
HPV VACCINES
- THERAPEUTIC
• Therapy through prophylaxis on the long run:
- Effects on population of HPV prophylactic vaccine will be visible after decades
(20 years = 5 million deaths);
- Only prevent up to 70% of all cervical cancers;
- Cannot control existing HPV infections or lesions.
• Best candidates: E6 and E7 oncoproteins (constitutively
expressed in cervical cancer cells and necessary for
progression and maintenance of the cell malignant phenotype.
• No commercially available vaccines.
• No ‘ideal’ therapeutic vaccine => open field
• Many experimental, E7-based therapeutic vaccines in clinical trial.
Experimental therapeutic vaccine based on
the HPV16 E7 protein expressed in PLANT
(”First generation” viral vector: Potato Virus X-derived vector)
40% of tumor-free mice after immunization with E7-cointaining crude plant extracts
80% of tumor-free mice after immunization with PGIPss-E7-cointaining crude plant extracts
FRANCONI R, et al. (2001) 'Vaccini a subunità e procedimenti per la
loro produzione‘. RM2001A000332. Brevetto Europeo (ENEA/IRE/ISS)
-”Second generation” viral vector: pBI-TMV
-Preclinical experiments with purified protein
-E7 protein fused with a bacterial carrier (lichenase from C. termocellum => PATENTED)
1
2
3 4
5
6 7 8
9 10 11 12 13 14
~ 400 μg purified LicKM-E7 fusion
protein / g leaf
62 kD
49 kD
38 kD
Purified Lic-E7GGG stable up to 7 days R.T.
28 kD
17 kD
BSA 0.25– 5 µg
Lic-E7 Lic-E7GGG
GENERATION OF CLONAL ROOT LINES (‘CLONAL ROOT TECHNOLOGY’)
ESTABLISHMENT
OF MASTER ROOT
CULTURE FOR
SELECTED ROOT
LINE
~ 3 g fusion protein/Kg root
In the best expressing clones
PREPARATION
OF
LEAF DISKS
CO-CULTURE WITH
A. rhizogenes HARBOURING
THE RECOMBINANT VECTOR
LicKM-E7, LicKM-E7GGG
BIOMASS
ACCUMULATION
FOR SELECTED
ROOT LINE
SEPARATION OF
INDIVIDUAL ROOTS
GENERATION OF HAIRY ROOTS EXPRESSING LicKM-E7/E7GGG
• 100%
of
tumor-free
mice after immunization
with
purified
E7-Lic
protein (40 μg tot).
VAC-1
DAY 3
DAY 0
Challenge with
5x103 TC-1* cells
50 mice per group
DAY 18
PRIME
BOOST
DAY 6
DAY 21
VAC-2
Comparison in overall survival between early- (Vac-1) and late- (Vac-2) vaccinated
groups of mice.
Log-rank test revealed a significant difference between the VAC groups and controls
(P < 0.0001) whereas a non statistically significant difference between Vac-1 and
Vac-2 was recorded (p = 0,0857).
Venuti et al., (2009) Vaccine
CONCLUSIONS
LicKM-E7GGG fusion protein is a powerful therapeutic vaccine that
is able to cure established experimental tumours and have a
dramatic effect on the overall survival of the treated animals.
Even the late treatment, when the tumour is already fully
established, is able to induce a therapeutic response.
These studies open the possibility of a Phase I clinical trial with
the purified, plant-derived, harmful version of HPV16 E7
oncoprotein (LicE7GGG)
Future work
Construction of similar fusion vaccines against other high risk HPVs,
like HPV18, 31 and 45.
Evaluation of different prime/boost strategies by using an association
with DNA vaccines
COMBINATION VACCINES: HETEROLOGOUS PRIME-BOOST STRATEGY
Control
100
DNA / LicE7GGG
protein
50
0
0 10 20 30 40 50 60 70
•The protocol using DNA followed by
LicE7GGG protein (even without
adjuvant) is more efficient
Conclusion
FROM BENCHTOP TO BEDSIDE
Effective therapeutic vaccines can be accomplished exploiting the potential
of plant production and /or heterologous administration schedule (the best
for HPV-associated cancer) for future bedside applications
=>
PLANTS AS BIOFACTORIES
The results of our work open the way to the exploitation of other plant- or
plant virus- derived sequences, with immunological features, to obtain
vaccines (DNA or protein) of relevance against HPV
=>
PLANTS AS A SOURCE OF IMMUNO-STIMULATORY
MOLECULES
FRANCONI R., VENUTI A., SPANO’ L., MASSA S. (2009).
‘Vaccini basati su chimere genetiche tra antigeni virali,
tumorali e proteine vegetali’. Brevetto in fase di deposito
(ENEA/IRE/UNIVAQ)
MICROALGAE as bioreactors
Combination of PLANT and
MICRORGANISM advantages:
-Economic (light and minerals)
-Axenic growth (easy regulatory path)
-Rapid growth (8h, vegetatively)
-Easy “scale-up”
-”Generally Recognized As Safe” (GRAS)=>
oral vaccine administration
Chlamydomonas reinhardtii
Unicellular eukaryotic green alga
Nuclear transformation
-easy to perform by the glass beads
method
-Eukaryotic post-transductional
modifications
-Possibility of protein secretion with
specific signal sequences
Chloroplast transformation
-homologous recombination in the
chloroplast genome => no positional effects
- easy to perform (unique copy-40% of the
total cell volume)
-heterologous protein compartmentalization
=> protein accumulation
GST-agarose
Dlg
E6-AP
P53
pDZ1
1111-E6
GST PULL-DOWN
ANALISI
SPETTROSCOPICHE
25°C
1600
1400
FRANCONI R. & ILLIANO E. (2007).
‘Proteina E6 di HPV ricombinante, solubile e in forma biologicamente attiva,
procedimento per la sua preparazione, usi e vaccini terapeutici che la
comprendono.’ Brevetto RM2007A000220
O.D. 405 nm
1200
1000
800
600
400
200
0
E6 urea +
Freund
E6 urea +
MF59
E6 nativa + E6 nativa +
Freund
MF59
CTRL +
Freund
CTRL +
MF59
M-3 T2 18E6
M-3 T1 18E6
M-3 T2 11E6
M-3 T1 11E6
M-3 T2 16E6
M-3 T1 16E6
30°C
Ctrl (+)
Ctrl (-)
STUDIO
IMMUNOGENICITÀ
M-3 T2 16E6IVT
DEGRADATION ASSAY
M-3 T2
in vitro TRANSLATION &
M-3 T1 16E6IVT
pα-E6
M-3 T2 16E6IVT
ATTIVITÀ
BIOLOGICA
M-3 T1 16E6IVT
1818-E6
M-3 T1
•No buoni anticorpi
per diagnosi in vivo
(‘imaging’) o in vitro
(immunoistoichimica
su ‘smear’ cellulari)
1616-E6
M-3 T0
•E6 di HPV: una
proteina ‘difficile’
da produrre in
forma ricombinante
16E6 ctrl+
Espressione e purificazione della sequenza originale di E6
di HPV in condizioni native e in forma solubile
da cellule di batterio
‘Antigeni da pianta/microalghe/batteri’ Applicazioni industriali
HPV
Vaccino profilattico
•
•
Formulazioni vaccinali sicure ed economiche (L1/VLPs)
Valutazione post-marketing del vaccino profilattico
Vaccino terapeutico (vegetale/genetico)
•
•
Studi clinici di fase I- Strategie di vaccinazione prime/boost eterologa
(DNA-proteina purificata da pianta) anche in assenza di adiuvanti
Fusioni con proteine vegetali (immunostimolazione)
ALTRO………
v
$ 2200
v
v
Medium-Scale protein Production Services
• Algae
•Plants
v
v
Plant-derived
SARS-CoV)
Vaccines/Antigens (i.e. HPV
HPV
antigens
SARS-CoV
antigens
Use of Plant- or Plant Virus-Derived
Sequences for the Development of new
DNA/Protein Vaccines
Recombinant Proteins and Antibodies for the
Development of new Diagnosis Test (i.e.
SARS-CoV, HPV, mycotoxins)
Recombinant Antibodies to
obtain Plants Resistant to
Viral Infections
Bando DTB- Fondi CIPE - prot. 1039
CPH (Chip Proteomico per HPV): Ricerca e sviluppo di una piattaforma biotecnologica per
la realizzazione di prototipi di diagnostica avanzata
A) Valutazione post-marketing del vaccino preventivo.
Prototipi a basso costo per la ricerca di anticorpi specifici di classe IgG o IgM:
=> monitoraggio della sieroconversione in soggetti vaccinati contro HPV
=> identificazione di infezioni persistenti da HPV o lesioni pre-cancerose
- Chip con VLPs prodotte da cellule di insetto;
- L1, L1 deleta, mutata o fusa con altre proteine prodotta da cellule di insetto o da E. coli e
assemblata in vitro;
- L1 o sue forme derivate prodotte in sistemi vegetali.
B) Diagnosi precoce dei tumori HPV-associati
Purificazione delle oncoproteine E6 ed E7 di HPV ad alto rischio (es. HPV16) in forma nativa.
=> diagnosi e monitoraggio dell’infezione e dello sviluppo del tumore
- Saggi immunologici, immunoblot,immunofluorescenza, ‘chip’ di proteine
Selezione di anticorpi ricombinanti (‘phage display’)
=> diagnosi precoce da affiancare all’HPV test per individuare il virus e gli
antigeni associati a tumore da preparati biologici.
- Kit immunoenzimatici, biosensori, immunosensori
S. MASSA
E. ILLIANO
O. DE MURTAS
C. NOBILI
O. BITTI
- A. VENUTI
IRE – Lab. Virologia - RM
- C. GIORGI
P. DI BONITO
ISS - RM
- L. SPANÒ
Univ. L’Aquila – Dip.to
Biologia cellulare
E. BENVENUTO
A. DESIDERIO
M.E. VILLANI
G. GIULIANO
P. FERRANTE
- L. BANKS
International Center
for Genetic Engineering
and Biotechnology
Padriciano (Trieste)
- V. YUSIBOV
Fraunhofer USA
Center for
Molecular Biotecnology
Newark - DE (USA)