Dr.ssa Rosella Franconi - EneaScuola

Proteine Proteine ricombinanti ricombinanti (antigeni (antigeni e e anticorpi) anticorpi)
da da pianta pianta e e da da altri altri sistemi sistemi di di espressione
espressione
per per applicazioni applicazioni diagnostiche diagnostiche e e terapeutiche
terapeutiche
Rosella
Franconi
BAS BIOTEC GEN
rosella.franconi@enea.it
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-

NH 2
CDR
- S - S - - S - S -
- - S - S -
COOH
- S - S
V H
Molecola di
anticorpo
-
Anticorpi ricombinanti
C H
V L
C L
S
S
Anticorpo a singola
catena: scFvs

1. Manipolazioni genetiche:
repertorio di mutanti
7. Analisi
dei ligandi
specifici
‘PHAGE DISPLAY’
2. Espressione alla superficie del
fago
‘Biopanning’
6. Amplificazione
4. Lavaggi
5. Eluizione
3. I fagi legano
l’antigene

Da questo repertorio (5 x 10 7 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 Esempio applicativo applicativo 1. 1.
Immunomodulazione/Immunoterapia
Immunomodulazione/Immunoterapia
Piante Piante di di pomodoro pomodoro resistenti resistenti al al virus virus del del mosaico mosaico del del cetriolo cetriolo
(CMV)
(CMV)
scFv WT

Esempio Esempio applicativo applicativo 2. 2.
Realizzazione Realizzazione di di nuovi nuovi sistemi sistemi diagnostici diagnostici
Immobilizzazione reversibile reversibile su su supporti supporti
elettrochimici/ottici

•
•
•
Anticorpi Anticorpi ricombinanti ricombinanti (‘Library (‘ Library F8’ F8’ e/o e/o da da animali animali
immunizzati)
immunizzati)
Possibili Possibili applicazioni applicazioni industriali 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
Yeasts
(S. cerevisiae,
P. pastoris)
(i. e. avidin, trypsin, βglucuronidase,
human albumin,
serotonin, etc)
Mammalian Cells
(Cos)
systems
More
Bacteria
than 100 Plant Made
Pharmaceuticals
(E. coli) (PMPs) have
been produced in plant:
-Antibodies
-Therapeutic
-VACCINES
molecules
of heterologous
proteins
PLANTS
MICROALGAE

TRANSGENIC PLANT TECHNOLOGY (STABLE expression)

POTENTIAL OF VIRAL VECTORS TECHNOLOGY
(Transient expression)
•High level of
expression:
up to 5 g protein/Kg
fresh leaf biomass
•Speed:
50% TSP in 4-10 days
First generation
‘Full virus’ strategy
Second-generation
‘Deconstructed virus’ Strategy

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 NON-
HODGKIN’S LYMPHOMA THERAPY

Greenhouse for containment
at ENEA Casaccia, Rome
of GMO
PLANT-DERIVED HPV ANTIGENS/VACCINES

Harald
zur
The Nobel Prize
Hausen
Doctor and virologist
German Cancer Research Centre
Heidelberg, Germany
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”
Human Papilloma Virus
(HPV)

Cervical
Cancer
300 millions women infected
Global Incidence of Cervical Cancer (Vaccine, vol.3 - 2005)
Second cause of death in women
FIRST in Developing Countries
80% of cervical cancers worldwide
High-risk HPV
HPV16 causes more than 50%
of cervical cancer

-
PROPHYLACTIC
Commercial vaccines based on the L1 protein (VLPs):
GARDASIL (Merck): VLPs of L1
protein from HPV 6/11/16/18 made in
yeast, aluminum adjuvant
�Low-cost prophylactic vaccines.
HPV VACCINES
Expensive!!!!
�Low-cost antigens for vaccinated people follow-up
(post-marketing monitoring).
CERVARIX (GlaxoSmithKline): VLPs
of L1 protein from HPV 16/18 made in
baculovirus, AS04 adjuvant

-
THERAPEUTIC
HPV VACCINES
• 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
• No ‘ideal’
• Many
therapeutic
available
vaccines.
vaccine => open field
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)
62 kD
49 kD
38 kD
28 kD
17 kD
1 2 3 4 5 6 7 8 9 10 11 12 13 14
BSA 0.25– 5 µg
Lic-E7
Lic-E7GGG
~ 400 μg purified LicKM-E7 fusion
protein / g leaf
Purified
Lic-E7GGG stable
up to
7 days
R.T.

GENERATION OF CLONAL ROOT LINES (‘CLONAL ROOT TECHNOLOGY’)
CO-CULTURE WITH
A. rhizogenes HARBOURING
THE RECOMBINANT VECTOR
LicKM-E7, LicKM-E7GGG
~ 3 g fusion protein/Kg root
In the best expressing clones
PREPARATION
OF
LEAF DISKS
BIOMASS
ACCUMULATION
FOR SELECTED
ROOT LINE
SEPARATION OF
INDIVIDUAL ROOTS
GENERATION OF HAIRY ROOTS EXPRESSING LicKM-E7/E7GGG
ESTABLISHMENT
OF MASTER ROOT
CULTURE FOR
SELECTED ROOT
LINE

• 100% of tumor-free
mice after immunization
with purified E7-Lic
protein (40 μg tot).

DAY 0
Challenge with
5x103 TC-1* cells
50 mice per group
VAC-1
DAY 3 DAY 18
PRIME BOOST
DAY 6
VAC-2
DAY 21

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
100 Control
50
0
0 10 20 30 40 50 60 70
DNA / LicE7GGG
protein
•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
The results of our work open the way to the exploitation of other plant- or
plant virusderived sequences, with immunological features, to obtain
vaccines (DNA or protein) of relevance against HPV
=>
=>
PLANTS AS BIOFACTORIES
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)

Nuclear transformation
-easy to perform by the glass beads
method
-Eukaryotic post-transductional
modifications
-Possibility of protein secretion with
specific signal sequences
MICROALGAE as
Combination of PLANT and
MICRORGANISM advantages:
bioreactors
-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
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

Espressione e purificazione della sequenza originale di E6
di HPV in condizioni native e in forma solubile
da cellule di batterio
•E6 di HPV: una
proteina ‘difficile’
da produrre in
forma ricombinante
•No buoni anticorpi
per diagnosi in vivo
(‘imaging’) o in vitro
(immunoistoichimica
su ‘smear’ cellulari)
16-E6 16 E6
18-E6 18 E6
11-E6 11 E6
ANALISI
SPETTROSCOPICHE
FRANCONI R. & ILLIANO E. (2007).
ATTIVITÀ
BIOLOGICA
GST PULL-DOWN
in vitro TRANSLATION &
DEGRADATION ASSAY
STUDIO
IMMUNOGENICITÀ
M-3 T0
M-3 T1
Ctrl (-)
M-3 T2
M-3 T1 16E6IVT
M-3 T2 16E6IVT
16E6 ctrl+
pDZ1
P53
E6-AP
Dlg
GST-agarose
M-3 T1 16E6IVT
25°C 30°C
800
‘Proteina E6 di HPV ricombinante, solubile e in forma biologicamente 600
attiva,
400
procedimento per la sua preparazione, usi e vaccini terapeutici 200 che la
comprendono.’ Brevetto RM2007A000220
O.D. 405 nm
1600
1400
1200
1000
0
E6 urea +
Freund
E6 urea +
MF59
Ctrl (+)
E6 nativa +
Freund
M-3 T2 16E6IVT
M-3 T1 16E6
E6 nativa +
MF59
M-3 T2 16E6
M-3 T1 11E6
CTRL +
Freund
pα-E6
M-3 T2 11E6
M-3 T1 18E6
CTRL +
MF59
M-3 T2 18E6

•
•
•
•
‘Antigeni ‘Antigeni da da pianta/microalghe/batteri’
pianta/microalghe/ batteri’ --
Applicazioni Applicazioni industriali 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………

$ 2200
v
v
v
Medium-Scale protein Production Services
• Algae
•Plants
v
v

Plant-derived Vaccines/Antigens
SARS-CoV)
HPV
antigens
Recombinant Antibodies to
obtain Plants Resistant to
Viral Infections
(i.e. HPV
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)

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 -
- C. GIORGI
P. DI BONITO
ISS - RM
-L. SPANÒ
Univ. L’Aquila – Dip.to
Biologia cellulare
RM
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)