Production of Production of Human Human

Immunoglobulin G Immunoglobulin G Antibodies in Antibodies in

PlantsPlantsA comparision of stable and A comparision of stable and transient expressionstransient expressions

Amanda ForniAmanda Forni

PlantibodiesPlantibodies

Agrobacterium-Agrobacterium-mediated mediated transformationtransformation Transient transgenicsTransient transgenics

Particle bombardment Particle bombardment Simultaneous introduction of multiple Simultaneous introduction of multiple

constructsconstructs Stable transgenicsStable transgenics

Stoger et al. (2002)

PlantibodiesPlantibodies

AdvantagesAdvantages Produced in Produced in

containmentcontainment Controlled conditionsControlled conditions Correctly folded, Correctly folded,

soluble proteinssoluble proteins High levels of High levels of

expressionexpression Cost effective for Cost effective for

large scale large scale productionproduction

Safety issuesSafety issues

DisadvantagesDisadvantages Sporadic transgene Sporadic transgene

silencingsilencing Glycolsylation Glycolsylation

patternspatterns Inefficient Inefficient

expressionexpression Environmental Environmental

contamination contamination

Sharma et al. (2009)

ApplicationsApplications

Stoger et al. (2002)

ApplicationsApplications

Vaccine antigens Medical diagnostics proteins Industrial and pharmaceutical

proteins Nutritional supplements: minerals,

vitamins, carbohydrates and biopolymers

Sharma et al. (2009)

Villani et al. (2008)Villani et al. (2008)

Stable TrangenicsStable Trangenics Stable transformation of the Stable transformation of the

nuclear/chloroplast genomenuclear/chloroplast genome

Transient TransgenicsTransient Transgenics Viral vectorsViral vectors AgroinfiltrationAgroinfiltration

Tenascin-C (TNC)Tenascin-C (TNC)

Extracellular Extracellular matrix matrix glycoprotein glycoprotein

Over-expressed in Over-expressed in tissue neoplasia tissue neoplasia

Tumour-associated Tumour-associated antigen target for antigen target for therapy and therapy and diagnosisdiagnosis

Stable TransgenicsStable Transgenics

Anti-TNC scFv Anti-TNC scFv VVHH and V and VLL single chain variable single chain variable

fragments were fused to the human fragments were fused to the human IgG molecule and cloned into a plant IgG molecule and cloned into a plant binary vector.binary vector.

Villani et al. (2008)

Stable TransgenicsStable Transgenics Constructs transformed into two differentConstructs transformed into two different Nicotiana Nicotiana

tabacumtabacum plants, Petit Havana SR1 and Maryland Mammoth plants, Petit Havana SR1 and Maryland Mammoth 100+ plants screened for individual Ig chains by PCR, 100+ plants screened for individual Ig chains by PCR,

ELISA, and Western Blotting and four lines selected for ELISA, and Western Blotting and four lines selected for cross-pollinationcross-pollination

Offspring screened by sandwich ELISA binding to mouse Offspring screened by sandwich ELISA binding to mouse TNCTNC

Villani et al. (2008)

Stable TransgenicsStable Transgenics

(a) Non-reducing and Reducing SDS-PAGE: (a) Non-reducing and Reducing SDS-PAGE: Lanes 1 and 2 purified human IgG reference, Lanes 1 and 2 purified human IgG reference, Lane 3 purified H10 from transgenic line 7.6Lane 3 purified H10 from transgenic line 7.6

(b) Size-exclusion chromatography: Red – (b) Size-exclusion chromatography: Red – human IgG reference, Black – H10human IgG reference, Black – H10

Villani et al. (2008)

Transient ExpressionTransient Expression

N. banthamiana N. banthamiana plants infected by plants infected by A. A. tumefacienstumefaciens containing the HC and containing the HC and LC constructsLC constructs

Other plants infected by Other plants infected by A. A. tumefacienstumefaciens containing HC, LC, and containing HC, LC, and p19 silencing suppressor (artichoke p19 silencing suppressor (artichoke mottled crinkle virus)mottled crinkle virus)

Leaves tested on days 1, 2, 3, 5, and Leaves tested on days 1, 2, 3, 5, and 66

Villani et al. (2008)

Transient TrangenicsTransient Trangenics

(a) Western Blot : (a) Western Blot : Lanes 1, 3, 5, 7 – Lanes 1, 3, 5, 7 – Presence of p19, Presence of p19, Lanes 2, 4, 6, 8 – Lanes 2, 4, 6, 8 – No p19No p19

(b) Lane 1 – Co-(b) Lane 1 – Co-agroinfiltrated, agroinfiltrated, Lane 2 – Wild type, Lane 2 – Wild type, Lane 3 – Purified Lane 3 – Purified human IgGhuman IgG

Villani et al. (2008)

Transient TransgenicsTransient Transgenics (b) SDS PAGE: (b) SDS PAGE: Lane 1 -

reference human IgG; lanes 2 and 3 - 1 purified H10; lane 4 - reference human IgG; lanes 5 and 6 - purified H10

(c) Size-exclusion Size-exclusion chromatography: Red – chromatography: Red – human IgG reference; human IgG reference; Blue – purified H10; Blue – purified H10; Black – Blue DextrinBlack – Blue Dextrin

Villani et al. (2008)

ResultsResults

Yield after purificationYield after purification Stable Transgenic Line – 0.6-1.1 mg/kg Stable Transgenic Line – 0.6-1.1 mg/kg

of fresh plant tissueof fresh plant tissue Transient Expression with p19 – 50-100 Transient Expression with p19 – 50-100

mg/kg of fresh plant tissuemg/kg of fresh plant tissue Transient Expression without p19 – Transient Expression without p19 –

10mg/kg of fresh plant tissue10mg/kg of fresh plant tissue

Villani et al. (2008)

ReferencesReferences Sharma, A. K., Sharma, M. K. 2009. Sharma, A. K., Sharma, M. K. 2009. Plants as Plants as

bioreactors: Recent developments and emerging bioreactors: Recent developments and emerging opportunities.opportunities. Biotechnology Advances. 27: 811-832. Biotechnology Advances. 27: 811-832.

Stoger, E., Sack, M., Fischer, R., Christou, P. 2002. Stoger, E., Sack, M., Fischer, R., Christou, P. 2002. Plantibodies: application, advantages and bottlenecks.Plantibodies: application, advantages and bottlenecks. Current Opinion in Biotechnology. 13: 161-166.Current Opinion in Biotechnology. 13: 161-166.

Villani, M. E., Morgun, B., Brunetti, P., Marusic, C., Villani, M. E., Morgun, B., Brunetti, P., Marusic, C., Lombardi, R., Pisoni, I., Bacci, C., Desiderio, A., Lombardi, R., Pisoni, I., Bacci, C., Desiderio, A., Benvenuto, E., Donini, M. 2008. Benvenuto, E., Donini, M. 2008. Plant pharming of a Plant pharming of a full-sized, tumour-targeting antibody using different full-sized, tumour-targeting antibody using different expression strategies.expression strategies. Plant Biotechnology Journal. 7: Plant Biotechnology Journal. 7: 59-71.59-71.