Antibody and Vaccine production in Plants

1. Antibody and Vaccine production in Plants • Plant biopharming of monoclonal antibodies • Virus Research 111 (2005) 93–100 • Kisung Ko, Hilary Koprowski • Plant-produced vaccines: promise and reality • Edward P Rybicki

2. HUMAN DISEASES DISEASES VACCINES VACCINES PLANT ANTIBODY ANTIBODY PLANT Colorectal Cancer   Respiratory Syncytial  Tobacco Epithelial Tumors (EGF receptor)  Hepatitis B  Lettuce HIV  Spinach Rabies  Spinach Tobacco  Tobacco Anthrax  Tobacco Tomato Diphtheria  Tobacco SARS  Tobacco Tomato Smallpox  Tobacco Tomato PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF INFECTIOUS DISEASES PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF CANCER

3. Antibodies • Magic bullets: • Therapeutic – inhibit target involved in disease progression • Diagnostic • Prevention of disease • Cause cytotoxic death of target cells • Act as carriers for radioisotopes, toxins and drugs to site of disease

4. Anti-rabies virus mAb • After exposure treated with Ab • Used to be made in horses • First mAb made in transgenic plants • 4 genes – 2 H, 2 L • Transgenic plant for each one and crossing plants • Later used single binary vector with two promoters

5. Full-size monoclonal antibodies recently produced in transgenic plants

6. Abs expressed in transgenic plants Constant domains Variable Light chain Variable Heavy chain Recombinant hinge region

7. Functional Abs • Need to be properly folded and assembled • Need disulfide bond formation and glycosylation • Down stream processing: • Purification of Ab mostly with Protein A or G • Glycosylation is different in plants • β 1,2 Xylose and α 1,3 fucose • Retain in ER, only mannose is attached • Shorter half-life of Ab

8. Glycosylation of Ab Full-size Ab Large single-chain Ab Camelid heavy-chain Ab Minibodies and Fab fragments don’t get glycosylated

9. Glycosylation in Golgi

10. Antibodies: a compelling success story • high specificity: in vitro and in vivo diagnostics • low toxicity: therapeutic applications • high drug approval rates (24 approved mAbs) • major products in biotechnology (~240 in clinical trials) • inherently stable human proteins • injectable, topical and oral applications • applicable for chronic conditions • potential long-lasting benefits

11. Production Costs for Antibodies Production costs cost in $ / gram hybridomas 1000 transgenic animals 100 transgenic plants 10 Source: Daniell et al. (2001) TIPS 6, 219-226 E. coli & yeast Transgenicplants Tr. animals andanimal cells

12. Comparison of Mammalian and Plant-produced Antibodies • peptide sequence: identical • correct cleavage of Ig-derived signal peptides • kinetics & affinity: identical • stability in seeds > 30 months • antibody types: plant system more versatile (sIgA) • post-translational processing: different • core glycan identical, terminal sugar different plus xylose & fucose • antigenicity & clearance: apparently identical (shorter half-life)

13. Vaccines • Hepatitis B virus surface antigen HBsAg • Norwalk virus capsid • Vibrio cholerae enterotoxin subunit • Animal vaccines • Mink enteritis virus - MEV • Rabbit haemorrhagic disease virus - RHDV • Foot and mouth disease virus - FMDV

14. Oral vaccines • First thought edible vaccines good idea • Banana a day • Oral vaccination • Might elicit antigenic tolerance • ProdiGene - Got contamination of maize and soybean harvest in 2002 with transmissible gastroenteritis virus TGEV

15. Past and Future of PMPs

16. Plant produced Vaccines • HPV L1 – forms VLPs – can protect • Measles virus haemagglutinin • HBsAg • Porcine TGEV • Tetanus toxin • Vacinia virus B5 • Allergy vaccines • HPV • HIV gp41 • Newcastle disease virus • Rotavirus VP7

17. Cancer Vaccines • LSBC - Large Scale Biology Crop • Individually tailored single chain variable region Ab fragments from patients with Non-Hodgkin lymphomas • Colorectal cancer antigen • Cervical Cancer – HPV E7

18. Vaccines against biothreat agents • Biodefence • Anthrax • Plague • Ricin • Haemorrhagic fever

19. PMP Development - Highlights • Series of plant-derived vaccines from Arizona State University have completed clinical trials • Prodigene has trialled two plant-derived vaccines • LSBC pipeline of cancer vaccines prior to insolvency • Guardian Bioscience coccidiosis vaccine, CFIA phase II ongoing • Fraunhofer CMB, rabies vaccine trialled in humans • DowAgro Newcastle disease vaccine, approved Feb 2006 • Heberbiovac (Cuba) approved antibody for HepB vaccine purification

20. Current Challenges in Molecular Farming • yield of recombinant proteins • real quantitative comparison (TSP vs pg/cell/24h) • protein stability (proteases) • post-translational modification(s) • backcrossing in elite lines • extraction and downstream processing • QA, QC and substantial equivalence • clinical trials & regulatory approval

21. Regulatory Challenges for PMP • loci of transgene insertion • expression properties and levels, including PTM • effects of the transgene on the expression of flanking endogenous genes • master line banking to ensure product consistency • contamination with animal excreta, pesticides, organic fertiliser • procedures for detection and removal of weeds and pests • cultivation variables

22. Roadmap Plants for the Future Plant production platforms • Vitamines • Fatty acids • Enzymes • Bio-polymers • Pigments • Pharmaceutical products • Fibers Health food and quality • Amino acids • Oil • Starch Plant protection Efficient agriculture Stress resistance • Viruses • Nematodes • Fungi • Insects • Cold • Drought • Salinization • Bt technology • Herbicide resistance 1997 2005 2015 2025