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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 PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF INFECTIOUS DISEASES DISEASES VACCINES PLANT Respiratory Syncytial Hepatitis B Tobacco Lettuce HIV Spinach Rabies Anthrax Diphtheria Spinach Tobacco Tobacco Tomato Tobacco SARS Smallpox ANTIBODY PLANT Tobacco Tobacco Tomato Tobacco Tomato PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF CANCER HUMAN DISEASES VACCINES ANTIBODY Colorectal Cancer Epithelial Tumors (EGF receptor) 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 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 Full-size monoclonal antibodies recently produced in transgenic plants Abs expressed in transgenic plants Constant domains Variable Light chain Variable Heavy chain Recombinant hinge region 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 Glycosylation of Ab Full-size Ab Large single-chain Ab Camelid heavy-chain Ab Minibodies and Fab fragments don’t get glycosylated Glycosylation in Golgi 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 Production Costs for Antibodies Production costs cost in $ /gram hybridomas 1000 transgenic animals transgenic plants 100 10 Source: Daniell et al. (2001) TIPS 6, 219-226 E. coli & yeast Tr. animals and animal cells Transgenic plants 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) 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 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 Past and Future of PMPs 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 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 Vaccines against biothreat agents Biodefence – Anthrax – Plague – Ricin – Haemorrhagic fever 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 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 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 Roadmap Plants for the Future Plant production platforms Health food and quality - Amino acids - Oil - Starch Efficient agriculture - Bt technology - Herbicide resistance 1997 2005 Plant protection - Viruses Nematodes Fungi Insects 2015 - Vitamines Fatty acids Enzymes Bio-polymers Pigments Pharmaceutical products Fibers Stress resistance - Cold - Drought - Salinization 2025