Monoclonal Antibodies

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1 Monoclonal Antibodies Homogeneous antibody preparations produced in the laboratory by hybridomas. Produced by identical type of B cell clones. Recognize a signal epitope on an antigen.

2 Hybridoma Technology Discovered by Kohler and Milstein in 1975 Awarded noble prize in 1984 Hybridomas are prepared by fusing myeloma cells with antibody producing B cells obtained from immunized animals. The hybrid cells: 1. Produce antibodies of predetermined specificity 2. Are immortal

3 Steps for Production of Monoclonal Antibodies 1. Immunization 2. Splenectomy 3. Cell Fusion 4. Selection of Clones 5. Cloning

4 1. Immunization A mouse is immunized with the antigen against which monoclonal antibody is to be synthesized. Antigen is mixed with an adjuvant and repeatedly injected intra dermally at multiple sites and at different times to stimulate the immune response of mouse. 2. Splenectomy The animal is scarified, spleen is removed and B cells are isolated.

5 3. Cell Fusion B cells are fused with myeloma cells. Polyethylene glycol (PEG) is used as a fusing agent. Heterokaryons of myeloma cells and B cells are produced by fusion of plasma members within minutes after exposure to PEG. Some cells remain un fused and they must be destroyed.

6 4. Selection of Clones Myeloma cells used in hybridoma technology are HGPRT (hypoxanthine guanine phosphoribosyl transferase) mutant. HGPRT enzyme is required for synthesis of nucleotides through salvage pathway. Denovo pathway of nucleotide synthesis is also made defective by exposing myeloma cells to aminopterin. Now these cells cannot make nucleotides and depends on supplement for survival.

7 4. Selection of Clones (Cont ) Selection is done in HAT (Hypoxanthine Aminopterin Thymidine) medium. Myeloma cells begin HGPRT mutants don t survive in HAT medium. Mortal B cells are also unable to proliferate indefinitely. The only cells which survive are fused hybridoma cells. HGPRT enzyme is produced by B cells needed for growth in HAT medium.

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10 5. Cloning The selected hybridomas are cloned in vitro to produce multiple copies of monoclonal antibodies from a single parent cell. The antibodies produced are assayed to determine their antigen binding ability by tests like ELISA or immuno-dot blot or Antigen Microarray Assay.

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12 Chimeric and Humanized Monoclonal Antibodies Mouse antibodies can elicit immune response when injected in humans. As a result they can be removed from blood and result in production of human anti mouse antibodies. Human constant domains are added in the mouse monoclonal antibody construct to make chimeric antibodies. Humanized antibodies can be prepared by replacing parts of variable region.

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14 Human Monoclonal Antibodies Produced by transgenic mouse. Human immunoglobulin gene is transferred into mouse genome, the transgenic mouse is then injected with the desired antigen against which antibody is to be produced. In this way murine antibody is fully transformed into human antibody.

15 Applications Monoclonal antibodies have wide applications in: Diagnostics Immunopurification Therapeutics

16 Diagnostic Use Diseases, presence of pathogens, pregnancy, blood groups etc. could be detected using monoclonal antibodies. In order to detect pregnancy monoclonal antibodies specific for human chorionic gonadotropin (HCG) are used. Monoclonal antibodies can be used to detect pathogens or viruses by using them in ELISA (Enzyme linked immunosorbant assay).

17 Immunopurification Purification of compounds very similar in nature. The test sample and monoclonal antibody are loaded in separate wells on agarose gel and electric current is applied. They both move and due to formation of Ag-Ab complex form a precipitin line. By using monoclonal antibodies, interferons could be purified which in turn could be used to inactivate T lymphocytes involved in organ rejection during transplantation.

18 Therapeutic Use In the treatment of cancer monoclonal antibodies have extensive role. They are used in different ways to treat cancer. 1. Naked Monoclonal Antibodies 2. Conjugated Monoclonal Antibodies i. Radiolabelled ii. Chemolabelled

19 1. Naked Monoclonal Antibodies Work on their own. Bind to antigens on cancer cell surface. They destroy the cancer cells in any one of the following ways: 1. Boost the immune response of a person to destroy the cancer cells 2. Target immune system check points to boost the immune response. 3. Other binds and blocks antigens on cancer cells that assist in growth and metastasis of cancer cells.

20 2. Conjugated Monoclonal Antibodies Attached to a radioactive particle or chemotherapeutic drug. Any of these substances can be directly transferred by monoclonal antibodies to cancer cells. They travel throughout the body until they find their target antigen and then deliver the toxic substance where needed. In this way damage to the normal cells of body is prevented.

21 i. Radiolabelled Monoclonal antibody Ibritumomab tiuxetan (Zevalin ) used for the treatment of some types of non Hodgkins lymphoma. It targets the CD20 antigen found on the surface of cancerous B cells and directly delivers the radioactivity to these cells.

22 ii. Chemolabelled monoclonal antibodies Also known as antibody-drug conjugates (ADC s) Have a strong chemotherapeutic drug attached. Which if used alone will cause serious side effects.