Biofarmaci verdi

Biofarmaci verdi
Eugenio Benvenuto
Laboratorio Biotecnologie
Unità Tecnica Biologia delle Radiazioni e
Salute dell’Uomo
ENEA, Roma, Italy
Firenze, 4 ottobre, 2013
Plant as Natural
Bioreactectors
De materia medica
è un trattato di medicina e
botanica del I secolo d.C.,
scritto dal medico greco
Pedanius Dioscorides o
Dioscoride.
Descrizione di 500 piante
Insieme alla preparazione di
circa 1000 semplici semplici
rimedi farmaceutici
Rimane come testo base per
1500 anni
Plant as Natural
Bioreactors
Taxus
What is Taxol?
Taxol is an anti-cancer ("antineoplastic"
or "cytotoxic")
chemotherapy drug. Taxol is classified
as a "plant alkaloid," a "taxane”
"antimicrotubule agent.
Paclitaxel
Plant as Natural
Bioreactors
Catharanthus roseus
Vinca alkaloids:
Vincristine
Vinblastine
Vinorelbine
Plant as Natural
Bioreactors
Digitalis purpurea
Digitalis lanata
Cardiac glycosides:
digitoxin
digoxin
Plant as Natural
Bioreactors
Artemisia annua
antimalarial drug
Artemisinin
Plant as Natural
Bioreactors
< 1000 dalton molecules
> 100 PLANT-DERIVED PHARMACEUTICALS
Paclitaxel
Vincristine
Artemisinin
WORLDWIDE
74% DISCOVERED FROM MEDICINAL PLANTS
Digitoxin
‘Recombinant Herbal Medicines’
‘Molecular farming’
Large scale production of
biomolecules through genetic
modification of plant or
organelle genome
The terms refer to agricultural
applications due to the use of
crops as biofactories for the
production of high-added value
molecules
“La pianta come biofabbrica “ Metodi di Fermentazione Classica Produzione su larga scale di molecole ad alto valore aggiunto Vs Studio comparativo tra i diversi sistemi per la
produzione di biofarmaci
Recombinant
subunit vaccines
Recombinant
antibodies
Human
biopharmaceuticals
Proteine di interesse farmaceutico
prodotte in pianta
Protein
Growth hormone
Human serum albumin
-interferon
Erytropoietin
Human-secretd lkaline phosphatase
Aprotinin
Collagen
1-antitrypsin
IgG1 (Phosphonate ester)
IgM (neuropeptide hapten)
SigA/G
Host plant system
tabacco, sunflower
tabacco, potato
rice, turnip
tobacco
tobacco
maize
tobacco
rice
tobacco
tobacco
tobacco
scFv-bryodin 1 immunotoxin (CD 40)
IgG (herpes virus simplex)
LSC (herpes virus simplex)
Hepatitis B virus envelope protein
Rabies virus glycoprotein
Escherichia coli heat-labile endotoxin
Norwark virus capsid protein
Diabetes autoantigen
Cholera toxin B subunit
Cholera toxin B and A2 subunits + rotavirus
endotoxin + E. coli fimbrial antigen
Porcine transmissible gastroenteritis virus
glycoprotein S
tobacco
soybean
Chlamidomonas reinhardtii
tobacco
tomato
tabacco, potato
potato
tabacco, potato
tabacco, potato
potato
Comments
In chloroplast ~ 7% TSP
Full size, in chloroplast ~ 11% TSP
First pharmaceutical protein produced in rice
In suspension cells
In roots and leaves
In seeds
Correct modification of structural-protein polymer
In rice suspension cells
Correct assembling by crossing plants
Accumulation in chloroplast
Complex assembling of a secretory antibody by plant
crossing
Recombinant antibody in cell-suspension culture
In seeds
In algae
In clinical trial
Potential edile vaccine
In clinical trial
In clinical trial
In leaves and roots
In chloroplast
Multivalent recombinant antigen for enteric disease
tabacco, mais
For animal vaccination
Adattato da Ma et al. 2003, Nature Reviews Genetics 4, 794-805
Proteine ricombinanti prodotte in pianta
in sperimentazione clinica
Everett et al. 2012, BioProcess International, 10(1): 16-26
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Trasformazione del cloroplasto  circa 20% TSP
Trasformazione nucleare
 0.5% - 2% TSP (semi)
Sistemi transienti *
 27% TSP
corrispondenti a 15-20 mg proteina purificata/kg foglie
* Lombardi R, Villani ME, Di Carli M, Brunetti P, Benvenuto E, Donini M.
Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration.
Transgenic Res. 2010;19(6):1083-97.
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Fattori che influenzano i livelli di espressione:
•  caratteristiche della proteina
•  codon usage
•  sequenze regolatorie utilizzate
•  sistema di espressione utilizzato (stabile o transiente)
•  tipo di DNA trasformato (nucleare o plastidico)
•  numero di copie inserite (per la trasformazione nucleare)
I vantaggi della produzione in pianta
Sistema di espressione eucariotico:
•  assemblaggio di proteine complesse come gli anticorpi
•  modificazioni post-traduzionali
Batteri:
Assenza di glicosilazione
Lieviti:
Aggiungono N-glicani altamente immunogenici costituiti da
catene di mannosio lunghe fino a 100 residui
Cellule di mammifero:
Possono contenere zuccheri ‘non umani’, come l’acido
N-glicosilneuramidico, forma di acido sialico, (in cellule
CHO) o l’α-(1,3)-galattosio terminale (in cellule murine)
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Tempi di produzione ridotti
•  trasformazione stabile: 2-3 mesi
•  trasformazione transiente: 2-3 settimane
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Tempi di produzione ridotti
Costi minori
Stima dei costi di produzione in piante di mais:
10-100 $ per grammo di proteina
equivalenti a:
2-10% fermentatori microbici
0.1% culture di cellule di mammifero
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Tempi di produzione ridotti
Costi minori
Assenza di contaminanti potenzialmente patogenici
Le piante sono esenti dai rischi legati alla utilizzazione di
sistemi di produzione di origine animale
endotossine
virus, prioni, DNA oncogenico
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Tempi di produzione ridotti
Costi minori
Assenza di contaminanti potenzialmente patogenici
Impiego di piante edibili
Ottimizzazione del sistema di produzione
in condizioni controllate
Serra a contenimento di classe 2
Sistema di coltivazione in
condizioni idro-aeroponiche
Plant as Biofactories: Vaccines & Therapeutic
proteins in clinical trial/FDA approval
Phase I trial Phase II trial Phase III trial FDA approval veterinary FDA approval therapeu:c FDA approval vaccine Human Vaccines: Therapeu:c Proteins: Veterinary Vaccines: Therapeu:c enzymes: -­‐ Pandemic and Seasonal Influenza (Medicago, Canada; agro-­‐infiltrated tobacco) -­‐ Norovirus (C. Arntzen, USA; transgenic potato) -­‐ Insulin -­‐ Diabetes (SemBiosys; transgenic safflower) -­‐ Interferon-­‐alpha – HepaDDs C (Biolex, USA; Lemna) -­‐ Newcastle Disease (DowAgroScience, USA; tobacco cells) -­‐ Glucocerebrosidase – Gaucher’s disease (Protalix, Israel; Carrot cell culture; Phase III-­‐FDA’s expanded access program, full licensure being sought) Tecniche di espressione di proteine
eterologhe in pianta
TRASFORMAZIONE
STABILE
gene
TRASFORMAZIONE
TRANSIENTE
(EPICROMOSOMALE)
cloroplasto
citoplasma
nucleo
gene
Sistemi di espressione transiente
Agroinfiltrazione
1 giorno
2 giorni
Agrobacterium
tumefaciens
Virus vegetali
purificazione
Potato Virus X
7-10
giorni
2 giorni
7 giorni
2 giorni
infezione
comparsa sintomi
estrazione
e analisi
mAbH10 yield aLer Agroinfiltra:on of N. benthamiana plants with silencing suppressor p19 from AMCV Environmentally contained greenhouse and vacuum AgroinfiltraDon chamber at ENEA Agroinfltra:on of An:body genes & Silencing Suppressor Circelli P et al 2010
I vantaggi della produzione in pianta
Sistema di espressione eucariotico
Buone rese
Trasformazione del cloroplasto  circa 20% TSP
Trasformazione nucleare
 0.5% - 2% TSP (semi)
Sistemi transienti *
 27% TSP
corrispondenti a 15-20 mg proteina purificata/kg foglie
* Lombardi R, Villani ME, Di Carli M, Brunetti P, Benvenuto E, Donini M.
Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration.
Transgenic Res. 2010;19(6):1083-97.
Plant-based production of xenogenic
proteins
!1. Antibodies
!2. Antigens
Many recombinant antibody formats
have been expressed in plants
Plant-based production of biopharmaceuticals:
two different antibodies Tenascin-C :
!1. Anti-cancer
Antibody
!2. Anti-fungal
Antibody
Tenascin-C is a tumor marker
mAb H10
Alternatively spliced domains
EGF-like domains
Tenascin-C :
Fibronectin type-3 homology repeats
Large hexameric glycoprotein.
Alternative splicing leads to a small and a large isoform
with distinct biological functions.
Large isoform:
  Undetectable in healthy adult tissues;
  Localized around vascular structures in the
tumor stroma of a variety of different
tumors (lung, gliomas, breast cancer)
Selection of an anti-tenascin C antibody
and expression in plant
scFv(H10)
Phage-displayed human scFv(H10)
Human germline constant regions
genes
VH
Fully human IgG1 H10
Plant produced mAb H10
VL
Quantitative and functional ELISA of
IgG(H10) expressing lines on tenascin-C
coated plate.
OD 405
1,2
Best expressor:
0,7% TSP.
1
0,8
0,6
0,4
0,2
0 3*3 5*1 6*3 8*1 7*4 7*6 6*4 8*3 8*4 12*3positive
Transgenic lines
Plant-produced IgG1 is fully functional"
U87 glioblastoma xenograft"
H10 2µg/ml
20x"
Negative control
20x"
RU
400
Chip: IgG(H10) 3000 RU
H10 2µg/ml
20x"
Mouse tenascin-C
Response
300
3 µM
1,5 µM
750 nM
200
Negative control
20x" 100
375 nM
80 nM
0
-100
-100
0
100
200
Time
300
400
s
• KD of 14 nM for recombinant tenascin-C.
mAbH10 yield aLer Agroinfiltra:on of N. benthamiana plants with silencing suppressor p19 from AMCV Western Blot of extracts from leaves
Agroinfiltrated with or without the viral p19
gene silencing suppressor protein
Expression Yield:
640 mg/Kg FW
+p19 –p19 +p19 –p19 +p19 –p19 +p19 –p19 Anti-γ
Anti-λ
Days post Agroinfiltration
•  Sampling time influences antibody
accumulation and integrity
Circelli P. et al 2010
Pilot-scale purification and characterisation of IgG H10 from
vacuum-Agroinfiltrated leaves (250g)
Protein A Purification
Final Yield:
Cation-Exchange Chromatography (CEX)
40mg/Kg
mAb Purity: 99.4%
Endotoxin: < 1 EU/ml
Detection
Size-exclusion Chromatography
Silver staining of the eluted fractions
Lombardi et al 2010 Transgenic Res. 19:1083
Two chimeric mouse–human Ab derived from
an antifungal murine mAb (2G8), in the format
of complete IgG or scFv-Fc, were generated
and produced in plants.
Both recombinant Abs showed to bind the
beta 1,3 glucan (a fungal cell wall component)
which is the target recognized by the original
mAb.
Immunofluorescence staining of major pathogenic fungi,
Candida albicans (a), Aspergillus fumigatus (b) and
Cryptococcus neoformans (c), by the recombinant anti-bglucan Abs.
2G8 Recombinant IgA Formats
Given the effectiveness of such Abs, recombinant
immunoglobulin of type A derived from 2G8 intended for
topical application were also generated.
Critical Aspect: Glycosilation
Endoplasmic reticulum
Possible solu:ons: Golgi apparatus
• ER Reten:on Plants
• Plants “silenced” defec:ve in the ability to synthesize fucosyl-­‐transferase e ß(1,2)xylose
xylosyl-­‐transferase α(1,3)fucose
• Expressing human beta(1,4)-­‐galactosyltransferase in plant cells to modify sugars and decrease contents of beta(1,2)-­‐xylose and alpha(1,3)-­‐fucose. Animals
Sialic acid
Galactose
Plant-based production of xenogenic
proteins
!1. Antibodies
!2. Antigens
Vaccini prodotti in piante edibili
purificazione
Patogeno
Vaccino
conservazione
somministrazione
smaltimento
Pianta
Quantità tessuto
vegetale/
somministrazione
Quantità di
Numero di
vaccino/
somministrazioni
somministrazione
patata
100-110 g
~890 g
2-3
lattuga
200-150 g
~60-45 g
2
Virus Epatite B
VLP HBsAg
Virus di Norwalk
(gastroenterite)
VLP NVCP
patata
150 g
215-751 g
2-3
Enterotossina di
E. coli (diarrea)
LT-B
(subunità B)
patata
100 g
970-485 g
3
mais
2,1 mg
~ 1 mg
3
Referenza
Thanavala et al. 2005,
PNAS 102,3378-82
Kapusta et al. 1999,
FASEB J. 13,1796-99
Tacket et al. 2000,
J Infect Disease 182,302-5
Tacket et al. 1998,
Nature Med 4
Tacket et al. 2004,
Vaccine 22,4385-89
Aumento del titolo anticorpale
Potato Virus X (PVX) surface display of
HIV-derived epitope(s)
gp 41
HIV-1
PVX
• Approx. 1300 coat proteins per
PVX particle
• N -terminus of each coat
protein exposed on the outer
surface
Cartoon model of the HIV-1 putative trimeric
envelope spike
‘Broadly Neutralizing Antibodies Targeted to the Membrane-Proximal External Region of Human
Immunodeficiency Virus Type 1 Glycoprotein gp41’ ZWICK et al. J. VIROLOGY(2001) 10892–
10905
Potato Virus X (PVX) surface display of CTL epitope(s)
CVPs activate ASNENMTEM-specific CD8+ T cells
NP epitope
Influenza Virus
PVX
Multiepitope-Targeted Vaccines Based on HSP70
from Plants Biofactories of Recombinant Antigens
Plant Heat Shock proteins 70
do activate immune system!
An immunization strategy based
on these complexes, poorly
explored so far, could help to
overcome the problems related to
epitope identification, resulting in
naturally formulated multiepitope
vaccines.
HPV 16 Piante Boreaaori di vaccini contro il virus del papilloma umano + Nico.ana benthamiana Chlamydomonas
reinhardtii
HPV Saponaria officinalis Valutazione efficacia su modelli pre-­‐clinici Virus vegetali in bio-nanotecnologie
Journal of Biomolecular
Structure and Dynamics
Structure-based design and
experimental engineering
of a plant virus nanoparticle for
the presentation of
immunogenic epitopes and as
a drug carrier
I successi ‘produttivi’ del ‘molecular
farming’
•  Collagene in tabacco transgenico
PMI israeliana
Ruggiero et al. 2000, Triple helix assembly and
processing of human collagen produced in transgenic
tobacco plants, FEBS Letters 469, 132–136
•  Biofarmaceutici per malattie rare in cellule di carota
Israele
Taliglucerasi alfa. Sopperisce alla
carenza dell’enzima glucocerebrosidasi
(malattia di Gaucher, con disfunzioni
nel processo di degradazione cellulare).
•  Vaccini per le pandemie in piante agroinfiltrate
USA
In collaborazione con il Pentagono,
prodotte 10 milioni di dosi di vaccino
contro l’influenza di tipo H1N1 in un
mese.
Thirty years of transgenic plants
Twenty years of Plant Antibody
Engineering @ENEA
Ten years of advanced molecular
farming…..
Imagine a world in which any protein
either naturally occurring or designed
by man could be produced safely,
inexpensively and in almost unlimited
quantities using only simple nutrients,
water and sunlight…..’
Julian Ma et al.
Nature Review Genetics,
October 2003
Recombinant Pharmaceuticals from
Plants for Human Health
2004 - 2009
Pharma-Planta
Aims and Objectives
Pharma-Planta aims to build
a plant based production
platform for pharmaceuticals
in Europe and to enter the
first candidates of this
pipeline into Phase I clinical
trial.
Pharma-Planta-Who are
we?
Scientific Co-ordinator:
Professor Julian Ma
St George’s Hospital
Medical School, London,
UK
Partners:
Friedrich Altmann, Austria
Eugenio Benvenuto, Italy
Ralph Bock, Germany
Marc Boutry, Belgium
Paul Christou, Germany
Udo Conrad, Germany
CSIR, S. Africa
Phil Dale, UK
Jurgen Denecke, UK
Ann Depicker, Belgium
Diamyd Medical AB, Sweden
Phil Dix, Ireland
Jurgen Drossard, Germany
Paul Dupree, UK
Rainer Fischer, Germany
Lorenzo Frigerio, UK
Roger Frutos , France
Paul Garside, UK
John Gray, UK
Chris Hawes, UK
Friedemann Hesse, Austria
Tony Kavanagh, Ireland
Nikos Labrou, Greece
David Lewis, UK
George Lomonossoff, UK
Julian Ma, UK
Richard Mahoney, UK
Mosaic Systems BV, Netherlands
Johnathan Napier, UK
Jean-Marc Neuhaus, Switzerland
Jacqueline Nugent, Ireland
Mario Pezzotti, Italy
PolyMun, Austria
David Robinson, Germany
Henri Salmon, France
Stefan Schillberg, Germany
Eva Stoeger, Germany
Alessandro Vitale, Italy
Christian Vivares, France
Pharma-Planta –
Targets
HIV, rabies, Diabetes.
www.pharma-planta.org
Plants as Bio-­‐fermenters of recombinant medicines Monoclonal an:body produc:on in plants offers rapid produc:on advantages in comparison to mammalian cells, with at least equivalent product safety, purity and potency. Key Words: Bio-­‐Beaer Bio-­‐Similar Humanitarian Use First in human trial < $10 /g Vs Are these sufficient to compete with classical fermenta:on technology? BIOTEC per l’Agroalimentare
!
Thank you for your attention!
!
!
http://biotecnologie.casaccia.enea.it
Molecular farming
nel Laboratorio Biotecnologie
Tematica
Ottimizzazione produzione di proteine in piante attraverso il
sistema PV X
Ottimizzazione produzione di proteine in piante attraverso il
sistema agroinfiltrazion e
Accumulo di proteine in corpi oleo s i
Potenziale azione immunostimolante di componenti
vegetali
Produzione in pianta di un peptide “killer” ad azione
antibiotica.
Produzione in pianta di anticorpi anti- -glucano ad azione
antifungina.
Produzione in pianta di un anticorpo anti-tumorale
Produzione in pianta di un vaccino anti-HPV umano.
Produzione in pianta di antigeni HI V
Produzione in pianta di antigeni del virus dell’influenza.
Produzione in pianta di vaccini contro il virus della febbre
suin a
Pubblicazioni BIORAD-FARM
• Betti et al. Mol Plant Pathol. 2012;13:198-203
• Lico et al. J Gener Virol. 2006;87:3103-12.
• Circelli et al. Bioeng Bugs. 2010;1:221-4.
• Capuano et al. Anal Chem. 2011;83:9267-72.
• Buriani et al. Plant Biotechnol J. 2012;10:363-71.
• Buriani et al. Transgenic Res 2011;20:331-44
• Di Bonito et al. Int J Immunopathol Pharmacol 2009;22:96778.
• Donini et al. Appl Environm microbiol 2005;71:6360-7.
• Capodicasa et al. Plant biotechnol J. 2011;9:776-87
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Lombardi et al. Transgenic Res. 2012;21:1005-21.
Lombardi et al. Transgenic Res. 2010;19:1083-97.
Villani et al. Plant Biotechnol J. 2009;7:59-72.
Massa et al. Hum Vaccin. 2011;7 Suppl:147-55.
Giorgi et al. Expert Rev Vaccines. 2010;9:913-24.
Massa et al. Human Gene Therapy. 2008;19:354-64.
Massa et al. Vaccine. 2007;25:3018-21.
Franconi et al. Int J Immunopathol Pharmacol. 2006;19:18797.
Franconi et al. Cancer Res. 2002;62:3654-8.
Marusic et al. Transgenic Res 2009;18:499-512.
Lombardi et al. BMC Biotechnol. 2009;9:96.
De Virgilio et al. J Experiment Botany. 2008;59:2815-29.
Marusic et al. BMC Biotechnol. 2007;7:12.
Lico C et al. Vaccine. 2009;27:5069-76.
• Marconi et alBMC biotechnol 2006;6:29.