monoclonal antibodies

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TOPIC: MONOCLONAL ANTIBODIES PRESENTER: BHARAT LAL M.PHARM ISF COLLEGE OF PHARMACY

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The Perfect World The Real World COLD FLU CHICKEN POX STOMACH UPSET HELP ME ! HELP ! HELP ME!

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THE IMMUNE SYSTEM DEFINITION: - The integrated body system of organs, tissues, cells & cell products that differentiates self from non – self & neutralizes potentially pathogenic organisms. (The American Heritage Stedman's Medical Dictionary) The Latin term “IMMUNIS” means EXEMPT, referring to protection against foreign agents. 2 major components of the immune system* INNATEPhysical – skin, mucus membraneBiochemical –  lysozymeCellular – macrophages, neutrophils* ADAPTIVEHUMORAL immunity -AntibodiesCELL MEDIATED immunity- T-lymphocyte

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BECOMES FUNCTIONING OF THE IMMUNE SYSTEM HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE CELL MEDIATED IMMUNE RESPONSE ANTIGEN (1ST EXPOSURE) ANTIGENS DISPLAYED BY INFECTED CELLS ACTIVATE CYTOTOXIC T CELL GIVES RISE TO ACTIVE CYTOTOXIC T CELL ENGULFED BY STIMULATES MACROPHAGE APC HELPER T CELLS STIMULATES MEMORY HELPER T CELLS MEMORY T CELLS MEMORY B CELLS PLASMA CELLS STIMULATES STIMULATES B CELLS FREE ANTIGENS DIRECTLY ACTIVATE STIMULATES GIVES RISE TO SECRETE ANTIBODIES STIMULATES STIMUlATES Defend against extracellular pathogens by binding to antigens and making them easier targets for phagocytes and complement Defend against intracellular pathogens and cancer by binding and lysing the infected cells or cancer cells

Function of Antibody : 

Function of Antibody First, antibodies have the unique ability to recognize and attach themselves to substances that cause disease. Second, in recognizing and attaching themselves to these pathogenic molecules, they act as markers, sending signals to other parts of the immune system to attack and eliminate the disease-associated substances. Same response can be induced on purpose for specific selection or detection of Your Favorite Antigen (YFA). Can produce millions of antibodies that recognize YFA. Purpose?

Antigens interact with lymphocytes to initiate the immune response. : 

Antigens interact with lymphocytes to initiate the immune response. Bone marrow produces a large and diverse pool of B and T lymphocytes each with antigen receptor of unique specificity. The specificity of antigen receptors is so enormous that there will be, at least, one B cell and T cell line capable of dealing with almost any antigen. Binding of an antigen to the receptor triggers proliferation (clonal selection) of antibody producing effector B (plasma) and T cells, and long-lived memory cells ready to rapidly respond upon future antigen exposure.

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The graph below shows the time required for antibody production after initial exposure to an antigen (e.g. flu shot). Notice that upon second exposure (second peak of the blue line) to the same antigen the production of antibodies is both faster and dramatically larger (log scale). The reason for the rapid and dramatic response upon second exposure to the same antigen is because of the presence of memory B and T cells produced during the first exposure. Second exposure to A and first exposure to B First exposure to A

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Each B cell produces a unique antibody expressed on its surface. Antibodies are proteins, immunoglobulins, of two “light” and two “heavy” polypeptides held together by disulfide bonds. Each polypeptide has a “constant region” and a unique “variable region” composing the antigen binding site. The range of antibody specificity is accomplished by rearrangements and mutation in genes coding for variable regions. The “constant region” determines the type, location, and action of the antibody in the immune response. Variable Constant

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Antibodies are Proteins that Recognize Specific Antigens The site on the surface of an antigen molecule to which an antibody attaches itself

MONOCLONAL ANTIBODY : 

MONOCLONAL ANTIBODY An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Thus they all bind to the same epitope with the same affinity

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Polyclonal antibodies are antibodies that are derived from different cell lines Ex. Muromonab CD3 , Inactivates T lymphocytes that reject tissue grafts,

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Polyclonal antibodies Monoclonal Antibodies Produced by: Many B cell clones A single B cell clone Bind to: Multiple epitopes of all A single epitope of a single antigens used in the antigen immunization Antibody class: A mixture of different All of a single Ab class Ab classes (isotypes) Ag-binding sites: A mixture of Abs with All Abs have the same antigen different antigen-binding binding site sites Animal source Produced in larger animals Typically produced in mice such as rabbits, sheep, goats, donkeys

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Georges Köhler César Milstein, and Niels Kaj Jerne in 1975 who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery hybridoma technology

Hybridoma technology : 

Hybridoma technology

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PRODUCTION OF MONOCLONAL ANTIBODY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum

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Adjuvants non-specific stimulators of immune response Need to satisfy following two properties: prevent rapid catabolism of antigen -mineral oil or aluminium hydroxide precipitates (ii) substance to stimulate immune response by inducing lymphokines production and a local inflammatory response Heat killed bacteria or lipo poly saccharide (LPS) Freund’s adjuvants(FA)( is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster)), FCA (composed of inactivated and dried mycobacteria (usually M. tuberculosis)) FIA ( lacks the mycobacterial components (hence just the water in oil emulsion)) PRODUCTION OF MONOCLONAL ANTIBODY

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Step 2: - Screening Of Mice For Antibody Production HYBRIDOMA TECHNOLOGY After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre too low BOOST(Pure antigen) Titre High BOOST(Pure antigen) 2 weeks PRODUCTION OF MONOCLONAL ANTIBODY

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Myeloma cells have been genetically engineered (HGPRT-) such that they can not use Hypoxanthine, Aminopterin, and Thymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium Hybridoma Selection The “HAT Trick”

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Step 3: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY Immortal Tumor Of Lymphocytes + 8 - Azaguanine Myeloma Cells High Viability & Rapid Growth HGPRT- Myeloma Cells PRODUCTION OF MONOCLONAL ANTIBODY (Hypoxanthin guanine phospho ribosyl transferase)

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Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells HYBRIDOMA TECHNOLOGY FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas PRODUCTION OF MONOCLONAL ANTIBODY

Selection of Hybridoma Cells : 

Selection of Hybridoma Cells By HAT medium Two pathway for nucleotide synthesis: DE NOVO PATHWAY Phosphoribosyl pyrophoshate + uridylate (Blocked by aminopterin) (Catalysed by HGPRT and enzymes) SALVAGE PATHWAY Hypoxanthine + thymidine nucleotide DNA

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MYELOMA CELLS HAVE LOST the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids The selective culture medium is called HAT medium because it contains Hypoxanthine, Aminopterin, and Thymidine Unfused myeloma cells cannot grow because they lack HGPRT. Unfused normal spleen cells cannot grow indefinitely because of their limited life span.

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Step 5: - Isolation of a monoclonal antibody producing hybridoma cell HYBRIDOMA TECHNOLOGY If all the hybridoma cells that have been selected using HAT – medium are grown together , then a polyclonal antibody mixture would be obtained. Done by diluting a suspention of hybridoma cells to such extent that individual aliquots contain, on an average , only one cell. Such cells are transferred to separate fresh media for growth. onces the correct hybridom has been selected and isolated , It can be stored frozed (in liq. Nitrogen) and culture whenever required. PRODUCTION OF MONOCLONAL ANTIBODY

Cont’d : 

Cont’d

Commercial production of Monoclonal Antibodies : 

Commercial production of Monoclonal Antibodies Microencapsulation technology: Hybridoma cells are grown inside hollow microsphere, the cells multiply and grow to higher cell densites than is possible with any other culture system. Since antibody is retained by the membrane, it does not becom contaminated with other immunoglobins if serum is added in the growth medium. This technology results in production of 100mg to 1 gm antibodies per lit. of culture medium.

Commercial production of Monoclonal Antibodies : 

Commercial production of Monoclonal Antibodies Ascites production in mice: Hybridoma cells are injected into the peritoneal cavity of mice. mice are pretreated by i.p. inj. Of pristane to irritate the peritonial cavity and to establish a conditioned enviroment that facilitates the growth of ascitic tumor. The fluid produced can contain a high concentration of secreated monoclonal antibodies 3 to 15mg/ml can be harvested harvested per mouse

Why should we be interested ? : 

Why should we be interested ? mAbs drive the development of multibillion dollar biotechnology industry. Many of the leading pharmaceutical companies have entered the mAb sector, attracted by quicker and less costly development, higher success rates, premium pricing, and a potentially reduced threat from generics The outlook for monoclonal antibody therapeutics is healthy. The ongoing success of existing products, combined with a bulging pipeline of new products awaiting approval and limited generic erosion, point towards robust growth in this segment

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Pure single Ab After immunization, the mouse spleen contains B cells producing specific antibodies. Each B cell produces only one kind of antibody, which binds to its specific antigen. Conventional antiserum is the mixture of all antibodies produced by B cells from spleen. If a single B cell was picked up and cultured, then it will produce only one kind of antibody. But B cell can not survive well in the culture. Myeloma cell can be cultured in the test tube, but can not produce useful antibody. Each hybridoma line can produce pure single antibody, called monoclonal antibody. If B cell is fused with myeloma, the fused cell might be cultured and produce antibody. Adapted from Milstein (1980) Scientific American, Oct. p.58 1 2 3 4 1 2 3 4 Monoclonal antibodies Cell fusion Spleen cells + Myeloma x Antiseum Antigen Immunization A mixture of all Ab 1 2 3 4 B cell Summary

Refrences : 

Refrences Kelley, Brian (2009). "Industrialization of mAb production technology". MAbs 1 (5): 443–452. doi:10.4161/mabs.1.5.9448 PMC 2759494 PMID 20065641. S.P. Vyas & Dixit “pharmaceutical bitechnology” fifth edition , page no. 485-528. R.P. Singh “Microbiology “ kalyani new delhi ,first edition 2005, page no. 373-381. http://en.wikipedia.org/wiki/Monoclonal_antibody_therapy#cite_ref-21 . http://www.1lec.com/Immunology/Monoclonal%20Antibodies/index.html http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html. http://www.pharmatutor.org/articles/brief-overview-on-immunopharmacology.

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