“MONOCLONAL ANTIBODIES” Jony Mallik M.Pharm Email: jonymallik@ymail

1. “MONOCLONAL ANTIBODIES”JonyMallikM.PharmEmail: jonymallik@ymail.com

2. Topic to be cover • Introduction • Antibodies • Nomenclature of Antibodies • Production of Monoclonal Antibodies • Application of Monoclonal Antibodies • Conclusion

3. ANTIBODIES Antibodiesare a group of glycoproteins secreted by the plasma cells in the serum and tissue fluids of all the mammals. They may occur free in the serum or else occur on the surface of B cell.

5. Structure of Immunoglobulin • 1.Four (4) polypeptide chains: • 2 identical LIGHT chains and 2 • identical HEAVY chains. • 2.Both light and heavy chains are • held together by COVALENT • DISULFIDE BONDS. • 3.Heavy chains are interconnected • by DISULFIDE LINKAGES in • the HINGE region. • 4.Ig has 2 terminal regions: • Carboxyterminal - with constant • amino acid sequence • (constant region). • Aminoterminal - with varying • antibody specificity (variable • region)

7. Epitopes

8. Types of Antibodies • Monoclonal antibodies (mAb) • Polyclonal antibodies

9. MONOCLONAL. POLYCLONAL. Derived from a single B cell clone Derived from different B Lymphocytes cell lines mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity Batch to Batch variation affecting Ab reactivity & titre NOT Powerful tools for clinical diagnostic tests Enable the development of secure immunoassay systems.

11. DISCOVERY OF MONOCLONAL ANTIBODIES • Immortal Monoclonal antibodies were found in the patients suffering from a disease called Multiple myeloma(a cancer of • B-lymphocytes) by GeorgesKohler and Ceaser Milstein in 1975

12. US Food and Drug Administration nomenclatureof therapeutic antibodies

14. HYBRIDOMA TECHNOLOGY Myeloma cell culture applied to fused cells resulting due to fusion of following two types of cells: • an antibody producing B-lymphocyte cell • a single myeloma cell

15. HYBRIDOMA CREATES MONOCLONAL ANTIBODIES • Monoclonal antibodies are typically made by fusing myeloma cells with the B cells from a mouse that has been immunized with the desired antigen • Recent advances have allowed the use of rabbit B-cells.

16. HYBRIDOMA TECHNOLOGY • 1) Immunize animal (mouse or rabbit) • 2) Isolate spleen cells (containing antibody-producing B cells) • 3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol) • 4) Add HAT(hypoxanthine-aminopterin-thymidine) culture to kill unfused myeloma cells • 5) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells)

17. 6) Screen supernatant of each clone for presence of the desired antibody (ELISA) • 7) Grow the chosen clone of cells in tissue culture indefinitely. • 8) Harvest antibody from the culture supernatant.

19. PROCEDURE STEP -1 Immunization of a mouse.

20. STEP -2Culture separately spleen cells (B lymphocytes) that produce specific antibodies. STEP -3(A)Preparation of Myeloma Cells - treated with 8-azaguanine to ensure sensitivity to HAT

21. STEP -3(B)myeloma cells have two features : (i) Unable to synthesize antibodies and (ii) Deficient in hypoxanthine guanine phosphoribosyltransferase enzyme or HGPRT.

22. STEP -4Fusion of cells using polyethylene glycol (PEG).

23. STEP -5 Selection for the hybrid cells: • Selectively grow hypoxanthine aminopterin thymidine (HAT) medium • Aminopterin block nucleotide synthesis pathway • Another pathway needs HGPRT enzyme.

24. STEP -6 Selection of desired hybridoma cell: • Only one in several hundred cell hybrids will produce antibodies of the desired specificity • Facilitated by preparing single cell colonies

25. STEP -7Culture of selected hybridoma • Monoclonal antibodies in large quantity. • May be frozen for future use. • May also be injected in the body of an animal.

26. STEP -8 Harvesting: • Can be done later from the body fluid.

27. PURIFICATION OF MONOCLONAL ANTIBODIES • In-vitro contaminants – growth factors and hormones etc. • In-vivo contaminants – host antibodies, protease, nuclease, nucleic acids etc.

28. Techniques are: (i) Centrifugation and filteration– 0.45µm. (cell, cell debris, lipids and clotted material). (ii) Ion exchange chromatography – charged impurities. Anionic: nucleic acids and endotoxins (iii) Size exclusion chromatography – Transferrin.

29. iv)AFFINITY CHROMATOGRAPHY • Specific binding interaction • Ligand coupled to solid support • Unbound components washed away • Bound molecules stripped by low pH buffer

30. Problems with using mouse monoclonal antibodies • For ethical reason humans cannot be immunized against Pathogen. • Limited therapeutic use in humans • Severe allergic response . • The fused human lymphocytes-mouse myeloma cells are very unstable. • There are no suitable myeloma cells in human that can replace mouse myeloma cells.

31. References: • http://www.searchpdfengine.com/HYBRIDOMA-TECHNOLOGY-FOR-PRODUCTION-OF-MONOCLONAL-ANTIBODIES.html • grants.nih.gov/grants/policy/antibodies.pdf  • http://www.searchpdfengine.com/Therapeutic-monoclonal-antibodies.html • Biotechnology by U Satyanaryana (Pages:214-219)