Plantibodies

1. Plantibodies By, Himani Dey , Pondicherry Uiversity

2. What are they??? A plantibody is an antibody/proteins produced by genetically modified crops. Uses: as edible vaccines, diagnostic/ therapeutic monoclonal antibodies, for disease resistance in plants. Expression of full-length antibody Expression of antibody fragments ( Fab ) Expression of single chain scFv or single domain (V L ) genes . TYPES

3. WHY TO CHOOSE PLANTS? NO ETHICAL ISSUES LOW CONTAMINATION RISK BY BACTERIAL ENZYMES,TOXINS AND VI RUSES ABILITY TO EXPRESS COMBINED TRANGENES BY SEXUAL CROSSING FLEXIBLE PRODUCTION SIZE, LOW COST CORRECT FOLDING AND MODIFICATION OF PROTEINS IN ER LARGE –SCALE PRODUCTION IN BIOTECORS/AGRI-CULTURE EASY TRANSPORT AS SEEDS, MORE VIABILITY

4. The Plantibody Approach - form Plant Molecular Biology (2000), 43, 419–428

5. COMPONENTS OF A PLANTIBODY GENE Plantibodies gene construct can contain sequence for fragment ( Fab , scFv , whole length antibody). Effective promoters for increased expression of proteins attached upstream of the genes. Eg . Hsp70 promoter is more effective the 35S promoter. Signal peptide sequence – needed for targeting of antibody into ER for proper folding of protein. C-terminal peptide- usually KDEL added to C-terminal of the peptide that can increase expression and retention of the protein in cell.

6. PLANTIBODY PRODUCTION Plant Cell Transformation Agrobacterium Mediated Transfer Of Antibody Genes Direct Gene Transfer By Gene Gun , Liposomes etc. Insertion Of Transformed Cell Into Plant Embryo Plant development and propagation in fields

7. Ti- PLASMID GENE CONSTRUCT

8. Plant transformed with gene for kappa chain of light/heavy chain Plant transformed with gene for gamma chain of light/heavy chain Plant with genes for kappa &gamma chain PLANT BREEDING BY SEXUAL CROSS Advantages: New antibody combinations can be produced. Dual purpose vaccines can be made. Properties like enhanced expression, stability, binding affinity can be achieved using different promoters, signal sequences. Mutations in CDRs of V-regions can enhance binding affinity of antibodies.

9. PLANT TISSUE CULTURE Differentiated plant cells states are grown in bioreactors and foreign proteins harvested from either the biomass or culture liquid . ADVANTAGES : Cell cultures contain fewer biological proteins or molecules which may contaminate the product. Large amounts of proteins obtained in short time & less purification steps. Sexual reproduction is not needed to ensure the lifespan of the species. Transgene stability is also increased. DISADVANTAGE: Cannot be used for edible vaccines.

10. APPLICATIONS THERAPEUTIC APPLICATIONS Treatment of infectious disease, inflammation, autoimmune disease or cancer. Tobacco produced mAb is more viable alternative to mAb produced in mouse ascites fluid for the large amounts needed for purification of hepatitis B vaccine . World's first clinically tested plantibody , CaroRx binds specifically to Streptococcus mutans , the bacteria that cause tooth decay, and prevents the bacteria from adhering to teeth. CaroRx is intended for regular topical preventative administration by both dental hygienists and patients allowing a thorough cleaning and intervention for any existing decay.

11. IMMUNIZATION Potential proteins produced are cytokines, hormones, enzymes, epidermal growth factors, interferons ,, and pharmaceutical foodstuff which are considered for oral immunization. Transgenic plants expressing antigens are used as an inexpensive oral-vaccine production and delivery system so immunization through consumption of an " edible vaccine " to provide passive immunity. Eg . Transgenic banana that eaten,are like subunit preparations in that are engineered to contain antigens subunit but bear no genes that would enable whole pathogens to form.

12. One of these is a full-length IgG specific for EpCAM (a marker of colorectal cancer) developed as the drug Avicidin by NeoRx and Monsanto.It is under pre clinical trial phase II. Other antibodies are CaroRx,scFvT84.66, Anti-HSV, 38C13 and PIPP ( antihCG ). Oral cholera vaccine developed in potato .

13. 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 Tr. animals and animal cells Transgenic plants

14. Functional antibodies need: Need to be properly folded and assembled. Need disulfide bond formation and glycosylation . Glycosylation is different in plants. Only mannose is attached-shorter half-life of Ab.

15. Glycosylation in Golgi

16. 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)

17. FULL-SIZE MONOCLONAL ANTIBODIES RECENTLY PRODUCED IN TRANSGENIC PLANTS

18. REFERENCES Monoclonal antibody engineering in plants Andrew Hiatt      ,  Julian K-C. Ma Department of Cell Biology, The Scripps Rcsearch Institute, La Jolla, CA 92037, USA Plantibodies : applications, advantages and bottlenecks   Molecular Biotechnology Unit, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK b  Department for Molecular Biotechnology, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany PLANTIBODY: AN OVERVIEW( Asian journal of Pharmacy and Life Science, Vol. 1 (1), Jan-Mar, 2011) Priya Jain*, Prasoon Pandey , Dheeraj Jain, Pankaj Dwivedi College of pharmacy, IPS academy, rajendra nagar , Indore, India. 452012