Monoclonal antibodies

Monoclonal antibodies,History,Magic Bullets,Types, Production, Hybridoma Technology,Applications, Limitations

1. Monoclonal Antibodies M.Meenakshi , Assistant professor, Department of Microbiology, Sri Ramakrishna College of Arts & Science for Women, Coimbatore

2. HISTORY OF MONOCLONAL ANTIBODIES A technique to produce monoclonal antibodies was devised by Georges Kohler and Cesar Milstein in 1975. The method relies on fusing B cells from an immunized animal (typically a mouse) with an immortal myeloma cell line and growing the cells under conditions in which the unfused normal and tumor cells cannot survive. The resultant fused cells that grow out are called  hybridomas ; each hybridoma makes only one Immunoglobulin, derived from one B cell from the immunized animal. The antibodies secreted by many hybridoma clones are screened for binding to the antigen of interest, and this single clone with the desired specificity is selected and expanded. The products of these individual clones are monoclonal antibodies, each specific for a single epitope on the antigen used to immunize the animal and to identify the immortalized antibody-secreting clones.

3. MAGIC BULLET The magic bullet is a scientific concept developed by a German Nobel laureate Paul Ehrlich in 1900. While working at the Institute of Experimental Therapy, Ehrlich formed an idea that it could be possible to kill specific microbes, which cause diseases in the body, without harming the body itself.

4. INTRODUCTION Monoclonal antibodies ( Mabs ) Antibodies are glycoprotein synthesized in blood against specific antigens to combat and give immunity. Such antibodies are heterogenous and are polyclonal antibodies. Therefore they do not have characteristics of specificity. If a specific lymphocyte after isolation and culture invitro becomes capable of producing a single type of antibody which bears specificity against specific antigen, it is known as monoclonal antibody. These monoclonal antibodies are derived from a single clone of cell which recognize only one kind of antigen. Monoclonal antibodies are produced against a variety of proteins, glycoproteins, glycolipids, nucleic acids and chemically defined groups linked to protein carriers.

5. TYPES OF MONOCLONAL ANTIBODIES Naked Monoclonal Antibodies:  There is no drug or radioactive material attached to them.  Conjugated Monoclonal Antibodies:  Monoclonal antibodies joined to a chemotherapy drug or to a radioactive particle are called conjugated monoclonal antibodies. Bispecific   Monoclonal Antibodies:  These drugs are made up of parts of 2 different monoclonal antibodies, and they can attach to 2 different proteins at the same time. 

6. HYBRIDOMA TECHNOLOGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES: Monoclonal antibodies are produced by hybridoma technology. The term hybridoma is used to fused cells resulting due to fusion of following two types of cells-a lymphocytes and tumor cell. An antibody producing B- lymphocytes ( eg . Spleen cell of mouse immunized with RBCs from sheep) A single myeloma cell ( eg . Bone marrow tumor cell) that can adopted to grow for infinite time in culture The fused product derived the ability of two different types of cells. ie . Ability to produce large amount of pure antibodies as lymphocytes and ability to grow or multiply indefinitely like tumor cell.

7. STEPS INVOLVED IN PRODUCTION OF MONOCLONAL ANTIBODIES Step I: Immunization of rabbit or rat and extraction of B-lymphocytes Step II: fusion of myeloma cell with B-lymphocytes: Step III: selection of hybridoma cell. Step IV: culture of Hybridoma cell: Step V: Inoculation of hybridoma cell into suitable host.

8. STEPS INVOLVED IN PRODUCTION OF MONOCLONAL ANTIBODIES

9. STEPS INVOLVED IN PRODUCTION OF MONOCLONAL ANTIBODIES STEP I: IMMUNIZATION OF RABBIT OR RAT AND EXTRACTION OF B-LYMPHOCYTES In order to isolate B-lymphocyte producing certain antibodies, rabbit or lab rat is immunized through repeated injection of specific antigen (sheep RBCs) A sample of B-cells is extracted from spleen of rabbit or rat STEP II: FUSION OF MYELOMA CELL WITH B-LYMPHOCYTES: The extracted B-lymphocytes is added to a culture of myeloma cell from bone marrow. The intended result is the formation of hybridoma cells formed by fusion of B-cell and myeloma cell. The fusion is done by using Polyethylene glycol (PEG) or by electrophoration or by using phages .

10. STEPS INVOLVED IN PRODUCTION OF MONOCLONAL ANTIBODIES STEP III: SELECTION OF HYBRIDOMA CELL The next step is selection of hybridoma cells. The B-lymphocytes contains HPRT1 gene which codes for enzyme Hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The enzyme HGPRT involved in synthesis of nucleotides from Hypoxanthine present in culture medium. Therefore B- cells can grow in medium containing Hypoxanthine amonopterin thymine (HAT media). But myeloma cell lack HPRT1 gene so, it does not produce HGPTR enzyme and it does not grow in HAT medium. The myeloma cell fused with another myeloma cell or those do not fused at all die in HAT medium since they do not utilize Hypoxanthine. Similarly, B- cell that fuse with another B- cell or those do not fuse at all die eventually because they do not have capacity to divide indefinitely, So, only hybridoma cell ie . fused cell between myeloma and B-cell can survive and divide in HAT medium. Screening is done to select hybridoma cells which are the desired cell for monoclonal antibodies production.

11. STEPS INVOLVED IN PRODUCTION OF MONOCLONAL ANTIBODIES STEP IV: CULTURE OF HYBRIDOMA CELL: The selected hybridoma cells are cultured in suitable culture medium, often supplemented with insulin, transferon , ethanol, amine and other additional hormones. Some commonly  used culture media for hybridoma cell for production of monoclonal antibodies are: DMEM (Dulbecco’s modified eagle medium) IMDM ( Iscove’s Modified Dulbecco’s Medium) Ham’s F12 RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium ) STEP V: INOCULATION OF HYBRIDOMA CELL INTO SUITABLE HOST These hybridoma cells are then injected into lab animal so that they starts to produce monoclonal antibodies. These hybridoma cells may be frozen and store for future use.

12. STEPS INVOLVED IN PRODUCTION OF MONOCLONAL ANTIBODIES STEP VI: EXTRACTION AND PURIFICATION OF MONOCLONAL ANTIBODIES: Monoclonal antibodies from host animal is extracted and purified by one of the following methods; Ion exchange chromatography Antigen affinity chromatography Radial immunoassay Immune precipitation

13. COMMERCIALLY AVAILABLE MONOCLONAL ANTIBODIES Monoclonal antibodies used for  autoimmune diseases  include  infliximab  and  adalimumab , which are effective in  rheumatoid arthritis ,  Crohn's disease ,  ulcerative colitis  and  ankylosing spondylitis  by their ability to bind to and inhibit  TNF- α .   Basiliximab  and  daclizumab  inhibit  IL-2  on activated  T cells  and thereby help prevent acute  rejection  of kidney transplants. Omalizumab  inhibits human  immunoglobulin E  ( IgE ) and is useful in treating moderate-to-severe allergic  asthma .

14. APPLICATION OF MONOCLONAL ANTIBODIES: About 200 years have elapsed since Edward Jenner vaccinated a young child against smallpox. Since that time, the field of immunology has evolved at a rapid pace and has yielded many critical developments. Although vaccination has thus far proven to be the most cost-effective method of preventing diseases worldwide, the development of MAbs that use the specificity of immunological responses is one of the most successful applications of immunology to date Disease diagnosis: ELISA to test HIV, hepatitis, Herpes etc RIA- to test viral infection Mabs to Human chorionic gonadotropin Identification of phenotypic markers unique to particular cell types The diagnosis of many infectious and systemic diseases  Tumor identification.  Advances in medical research  Functional analysis of cell surface and secreted molecules . .  

15. APPLICATION OF MONOCLONAL ANTIBODIES In the immunosuppression of organ transplantation: In the normal medical practice, immunosuppressive drugs such as cyclosporin and prednisone are administered to overcome the rejection of organ transplantation. In recent years, MAbs specific to T-lymphocyte surface antigens are being used for this purpose MAbs promote efficient opsonization of pathogenic organisms (by coating with antibody) and enhance phagocytosis.

16. LIMITATIONS Fully human monoclonal antibodies are also in clinical use . These are derived using phage display methods or in mice with B cells expressing human Ig transgenes. Humanized antibodies are far less likely than mouse monoclonal to appear foreign in humans and to induce anti-antibody responses . The annual price of mAb therapies is about $100,000 higher in oncology and hematology than in other disease states," comparing them on a per patient basis, to those for cardiovascular or metabolic disorders, immunology, infectious diseases, allergy, and ophthalmology .

17. REFERENCES Lonberg N, Huszar D (1995). "Human antibodies from transgenic mice".  International Reviews of Immunology .  13  (1): 65–93.  doi : 10.3109/08830189509061738 .  PMID   7494109 . https://microbenotes.com/monoclonal-antibodies-types-uses-and-limitations / https:// www.cancer.org/treatment/treatments-and-side-effects/treatment-types/immunotherapy/monoclonal-antibodies.html https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093874/