Plantibodies

By,

Himani Dey,

Pondicherry Uiversity

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 (VL) genes.

TYPES

WHY TO CHOOSE PLANTS?

NO ETHICAL ISSUES

LOW CONTAMINATION RISK BY BACTERIALENZYMES,TOXINS AND VIRUSES

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

The Plantibody Approach

- form Plant Molecular Biology (2000), 43, 419–428

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.

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

Ti- PLASMID GENE CONSTRUCT

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.

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.

APPLICATIONSTHERAPEUTIC APPLICATIONSTreatment 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.

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.

• 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 .

Production Costs for AntibodiesProduction 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 andanimal cells

Transgenicplants

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.

Glycosylation in Golgi

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)

FULL-SIZE MONOCLONAL ANTIBODIES RECENTLY PRODUCED IN TRANSGENIC PLANTS

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, UKb 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 DwivediCollege of pharmacy, IPS academy, rajendra nagar, Indore, India. 452012