Production of Human Immunoglobulin G Antibodies in Plants

1. Production of Human Immunoglobulin G Antibodies in Plants A comparision of stable and transient expressions Amanda Forni

2. Plantibodies • Agrobacterium-mediated transformation • Transient transgenics • Particle bombardment • Simultaneous introduction of multiple constructs • Stable transgenics Stoger et al. (2002)

3. Advantages Produced in containment Controlled conditions Correctly folded, soluble proteins High levels of expression Cost effective for large scale production Safety issues Disadvantages Sporadic transgene silencing Glycolsylation patterns Inefficient expression Environmental contamination Plantibodies Sharma et al. (2009)

4. Applications Stoger et al. (2002)

5. Applications • Vaccine antigens • Medical diagnostics proteins • Industrial and pharmaceutical proteins • Nutritional supplements: minerals, vitamins, carbohydrates and biopolymers Sharma et al. (2009)

6. Villani et al. (2008) • Stable Trangenics • Stable transformation of the nuclear/chloroplast genome • Transient Transgenics • Viral vectors • Agroinfiltration

7. Tenascin-C (TNC) • Extracellular matrix glycoprotein • Over-expressed in tissue neoplasia • Tumour-associated antigen target for therapy and diagnosis

8. Stable Transgenics • Anti-TNC scFv • VH and VL single chain variable fragments were fused to the human IgG molecule and cloned into a plant binary vector. Villani et al. (2008)

9. Stable Transgenics • Constructs transformed into two different Nicotiana tabacum plants, Petit Havana SR1 and Maryland Mammoth • 100+ plants screened for individual Ig chains by PCR, ELISA, and Western Blotting and four lines selected for cross-pollination • Offspring screened by sandwich ELISA binding to mouse TNC Villani et al. (2008)

10. Stable Transgenics • (a) Non-reducing and Reducing SDS-PAGE: Lanes 1 and 2 purified human IgG reference, Lane 3 purified H10 from transgenic line 7.6 • (b) Size-exclusion chromatography: Red – human IgG reference, Black – H10 Villani et al. (2008)

11. Transient Expression • N. banthamiana plants infected by A. tumefaciens containing the HC and LC constructs • Other plants infected by A. tumefaciens containing HC, LC, and p19 silencing suppressor (artichoke mottled crinkle virus) • Leaves tested on days 1, 2, 3, 5, and 6 Villani et al. (2008)

12. Transient Trangenics • (a) Western Blot : Lanes 1, 3, 5, 7 – Presence of p19, Lanes 2, 4, 6, 8 – No p19 • (b) Lane 1 – Co-agroinfiltrated, Lane 2 – Wild type, Lane 3 – Purified human IgG Villani et al. (2008)

13. Transient Transgenics • (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 chromatography: Red – human IgG reference; Blue – purified H10; Black – Blue Dextrin Villani et al. (2008)

14. Results • Yield after purification • Stable Transgenic Line – 0.6-1.1 mg/kg of fresh plant tissue • Transient Expression with p19 – 50-100 mg/kg of fresh plant tissue • Transient Expression without p19 – 10mg/kg of fresh plant tissue Villani et al. (2008)

15. References • Sharma, A. K., Sharma, M. K. 2009. Plants as bioreactors: Recent developments and emerging opportunities. Biotechnology Advances. 27: 811-832. • Stoger, E., Sack, M., Fischer, R., Christou, P. 2002. Plantibodies: application, advantages and bottlenecks. Current Opinion in Biotechnology. 13: 161-166. • Villani, M. E., Morgun, B., Brunetti, P., Marusic, C., Lombardi, R., Pisoni, I., Bacci, C., Desiderio, A., Benvenuto, E., Donini, M. 2008. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies. Plant Biotechnology Journal. 7: 59-71.