Firenze, 4 ottobre, 2013 - TRIWU · 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

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 Dioscor ides o Dioscoride. Descrizione di 500 piante Insieme alla preparazione di circa 1000 semplici semplici rimedi farmaceutici Rimane come testo base per 1500 anni

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


Digitalis purpurea Digitalis lanata Cardiac glycosides: digitoxin digoxin


Artemisia annua antimalarial drug Artemisinin


Artemisinin

Digitoxin

Vincristine Paclitaxel

< 1000 dalton molecules > 100 PLANT-DERIVED PHARMACEUTICALS

WORLDWIDE

74% DISCOVERED FROM MEDICINAL PLANTS

‘Recombinant Herbal Medicines’

Large scale production of biomolecules through genetic mod i f i c a t i o n o f p l a n t o r organelle genome The terms refer to agricultural applications due to the use of crops as biofactories for the production of high-added value molecules

‘Molecular farming’

“La pianta come biofabbrica “

Produzione su larga scale di molecole ad alto valore aggiunto

Vs

Metodi di Fermentazione Classica

Studio comparativo tra i diversi sistemi per la produzione di biofarmaci

Proteine di interesse farmaceutico prodotte in pianta

Adattato da Ma et al. 2003, Nature Reviews Genetics 4, 794-805

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Protein Host plant system Comments Growth hormone tabacco, sunflower In chloroplast ~ 7% TSP Human serum albumin tabacco, potato Full size, in chloroplast ~ 11% TSP

-interferon rice, turnip First pharmaceutical protein produced in rice Erytropoietin tobacco In suspension cells Human-secretd lkaline phosphatase tobacco In roots and leaves Aprotinin maize In seeds Collagen tobacco Correct modification of structural-protein polymer

1-antitrypsin rice In rice suspension cells IgG1 (Phosphonate ester) tobacco Correct assembling by crossing plants IgM (neuropeptide hapten) tobacco Accumulation in chloroplast SigA/G tobacco Complex assembling of a secretory antibody by plant

crossing scFv-bryodin 1 immunotoxin (CD 40) tobacco Recombinant antibody in cell-suspension culture IgG (herpes virus simplex) soybean In seeds LSC (herpes virus simplex) Chlamidomonas reinhardtii In algae Hepatitis B virus envelope protein tobacco In clinical trial Rabies virus glycoprotein tomato Potential edile vaccine Escherichia coli heat-labile endotoxin tabacco, potato In clinical trial Norwark virus capsid protein potato In clinical trial Diabetes autoantigen tabacco, potato In leaves and roots Cholera toxin B subunit tabacco, potato In chloroplast Cholera toxin B and A2 subunits + rotavirus endotoxin + E. coli fimbrial antigen

potato Multivalent recombinant antigen for enteric disease

Porcine transmissible gastroenteritis virus glycoprotein S

tabacco, mais For animal vaccination

Proteine ricombinanti prodotte in pianta in sperimentazione clinica

Everett et al. 2012, BioProcess International, 10(1): 16-26

* 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

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



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)


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)



Buone rese

•  trasformazione stabile: 2-3 mesi

•  trasformazione transiente: 2-3 settimane

Tempi di produzione ridotti



Buone rese

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

Costi minori




Buone rese

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



Buone rese

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

-‐ Insulin -‐ Diabetes (SemBiosys; transgenic safflower) -‐ Interferon-‐alpha – HepaDDs C (Biolex, USA; Lemna)

Human Vaccines: Therapeu:c Proteins: Veterinary Vaccines: Therapeu:c enzymes:

Phase I trial

Phase II trial

Phase III trial

FDA approval veterinary

FDA approval therapeu:c

FDA approval vaccine

Plant as Biofactories: Vaccines & Therapeutic proteins in clinical trial/FDA approval

-‐ Pandemic and Seasonal Influenza (Medicago, Canada; agro-‐infiltrated tobacco) -‐ Norovirus (C. Arntzen, USA; transgenic potato)

-‐ 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

gene

nucleo

TRASFORMAZIONE STABILE

TRASFORMAZIONE TRANSIENTE

(EPICROMOSOMALE)

gene

cloroplasto

citoplasma

Agrobacterium tumefaciens

Agroinfiltrazione

Sistemi di espressione transiente

Virus vegetali Potato Virus X

comparsa sintomi infezione

estrazione e analisi

purificazione

2 giorni

2 giorni

7-10 giorni

7 giorni

1 giorno

2 giorni

mAbH10 yield aLer Agroinfiltra:on of N. benthamiana plants with silencing suppressor p19 from AMCV

Circelli P et al 2010

Environmentally contained

greenhouse and vacuum

AgroinfiltraDon chamber at ENEA

Agroinfltra:on of An:body genes & Silencing Suppressor

* 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.



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

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

!1. Anti-cancer Antibody

!2. Anti-fungal Antibody

Tenascin-C :

EGF-like domains Fibronectin type-3 homology repeats

Alternatively spliced domains

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)

Tenascin-C is a tumor marker

mAb H10

Large hexameric glycoprotein. Alternative splicing leads to a small and a large isoform with distinct biological functions.

Tenascin-C :

Human germline constant regions genes

Fully human IgG1 H10

Phage-displayed human scFv(H10)

Selection of an anti-tenascin C antibody and expression in plant

Plant produced mAb H10

VH VL

scFv(H10)

Transgenic lines

OD

405

0

0,2

0,4

0,6

0,8

1

1,2

3*3 5*1 6*3 8*1 7*4 7*6 6*4 8*3 8*4 12*3 positive

Quantitative and functional ELISA of IgG(H10) expressing lines on tenascin-C coated plate.

Best expressor: 0,7% TSP.

-100

0

100

200

300

400

-100 0 100 200 300 400

RU

Res

pons

e

s Time

Chip: IgG(H10) 3000 RU

Mouse tenascin-C

80 nM

375 nM

750 nM

3 µM 1,5 µM

• KD of 14 nM for recombinant tenascin-C.

Plant-produced IgG1 is fully functional"

U87 glioblastoma xenograft"

Negative control 20x"

H10 2µg/ml 20x"

Negative control 20x"

H10 2µg/ml 20x"

mAbH10 yield aLer Agroinfiltra:on of N. benthamiana plants with silencing suppressor p19 from AMCV

Expression Yield: 640 mg/Kg FW

Days post Agroinfiltration

+p19 –p19 +p19 –p19 +p19 –p19 +p19 –p19

Western Blot of extracts from leaves Agroinfiltrated with or without the viral p19 gene silencing suppressor protein

Anti-γ Anti-λ

•  Sampling time influences antibody accumulation and integrity

Circelli P. et al 2010

Final Yield: 40mg/Kg

mAb Purity: 99.4%

Endotoxin: < 1 EU/ml Detection

Pilot-scale purification and characterisation of IgG H10 from vacuum-Agroinfiltrated leaves (250g)

Size-exclusion Chromatography

Protein A Purification

Silver staining of the eluted fractions

Cation-Exchange Chromatography (CEX)

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-b-glucan Abs.

Given the effectiveness of such Abs, recombinant immunoglobulin of type A derived from 2G8 intended for topical application were also generated.

2G8 Recombinant IgA Formats

Critical Aspect: Glycosilation Plan

ts

Animals

Endoplasmic reticulum Golgi apparatus

Sialic acid

Galactose

α(1,3)fucose

ß(1,2)xylose

Possible solu:ons:

• ER Reten:on

• Plants “silenced” defec:ve in the ability to synthesize fucosyl-‐transferase e xylosyl-‐transferase

• 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.

Plant-based production of xenogenic proteins

!1. Antibodies !2. Antigens

Vaccini prodotti in piante edibili

purificazione conservazione somministrazione smaltimento

Patogeno Vaccino Pianta Quantità tessuto

vegetale/ somministrazione

Quantità di vaccino/

somministrazione

Numero di somministrazioni Referenza

patata 100-110 g ~890 g 2-3 Thanavala et al. 2005, PNAS 102,3378-82 Virus Epatite B VLP HBsAg

lattuga 200-150 g ~60-45 g 2 Kapusta et al. 1999, FASEB J. 13,1796-99

Virus di Norwalk (gastroenterite) VLP NVCP patata 150 g 215-751 g 2-3 Tacket et al. 2000,

J Infect Disease 182,302-5

patata 100 g 970-485 g 3 Tacket et al. 1998, Nature Med 4 Enterotossina di

E. coli (diarrea) LT-B (subunità B) mais 2,1 mg ~ 1 mg 3 Tacket et al. 2004,

Vaccine 22,4385-89

Aumento del titolo anticorpale

• Approx. 1300 coat proteins per PVX particle

• N-terminus of each coat protein exposed on the outer surface

HIV-1

PVX

gp 41

Potato Virus X (PVX) surface display of HIV-derived epitope(s)

‘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

Cartoon model of the HIV-1 putative trimeric envelope spike

Potato Virus X (PVX) surface display of CTL epitope(s)

PVX Influenza Virus

NP epitope

CVPs activate ASNENMTEM-specific CD8+ T cells

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 Saponaria officinalis

Chlamydomonas reinhardtii

Nico.ana benthamiana

HPV 16 Piante Boreaaori di vaccini contro il virus del papilloma umano

Valutazione efficacia su modelli pre-‐clinici

+

Virus vegetali in bio-nanotecnologie

Journa l o f B iomolecu lar 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’

PMI israeliana

Ruggiero et al. 2000, Triple helix assembly and processing of human collagen produced in transgenic tobacco plants, FEBS Letters 469, 132–136

•  Collagene in tabacco transgenico

Israele

Taliglucerasi alfa. Sopperisce alla carenza dell’enzima glucocerebrosidasi (malattia di Gaucher, con disfunzioni nel processo di degradazione cellulare).

•  Biofarmaceutici per malattie rare in cellule di carota

•  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

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.

Recombinant Pharmaceuticals from Plants for Human Health

2004 - 2009

Pharma-Planta-Who are we?

Scientific Co-ordinator: Professor Julian Ma St George’s Hospital

Medical School, London, UK

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

Partners:

www.pharma-planta.org

Pharma-Planta –Targets

HIV, rabies, Diabetes.

Plants as Bio-‐fermenters of recombinant medicines

Vs

Are these sufficient to compete with classical fermenta:on technology?

Key Words: Bio-‐Beaer Bio-‐Similar

Humanitarian Use First in human trial

< $10 /g

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.

BIO

TEC

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l’A

groa

limen

tare

!!!

Thank you for your attention!

http://biotecnologie.casaccia.enea.it

Molecular farming nel Laboratorio Biotecnologie

Tematica Pubblicazioni BIORAD-FARM Ottimizzazione produzione di proteine in piante attraverso il sistema PV X

• Betti et al. Mol Plant Pathol. 2012;13:198-203 • Lico et al. J Gener Virol. 2006;87:3103-12.

Ottimizzazione produzione di proteine in piante attraverso il sistema agroinfiltrazion e

• Circelli et al. Bioeng Bugs. 2010;1:221-4.

Accumulo di proteine in corpi oleos i • Capuano et al. Anal Chem. 2011;83:9267-72.

Potenziale azione immunostimolante di componenti vegetali

• 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;22:967-

78.

Produzione in pianta di un peptide “killer” ad azione antibiotica.

• Donini et al. Appl Environm microbiol 2005;71:6360-7.

Produzione in pianta di anticorpi anti- -glucano ad azione antifungina.

• Capodicasa et al. Plant biotechnol J. 2011;9:776-87

Produzione in pianta di un anticorpo anti-tumorale • 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.

Produzione in pianta di un vaccino anti-HPV umano.

• 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;19:187-

97. • Franconi et al. Cancer Res. 2002;62:3654-8.

Produzione in pianta di antigeni HI V • 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.

Produzione in pianta di antigeni del virus dell’influenza. • Lico C et al. Vaccine. 2009;27:5069-76.

Produzione in pianta di vaccini contro il virus della febbre suin a

• Marconi et alBMC biotechnol 2006;6:29.

Documents

Firenze, 4 ottobre, 2013 - TRIWU · 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