MONOCLONAL ANTIBODIES

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MONOCLONAL ANTIBODIES:

MONOCLONAL ANTIBODIES Presented by K. Sreekanth M.Pharm I year II sem 1 Under the guidance of Dr. S. K . Umadevi M.Pharm ., Ph.D., Professor and HOD, ( Deparment of pharmaceutics) Rao’s College of pharmacy .

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2 INTRODUCTION

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Antibodies are produced by a specialized group of cells called B-Lymphocytes. When an foreign antigen enters the body due immune response B-Lymphocytes develops into plasma cells and liberates antibodies or immunoglobulins of various types( Ig A, Ig D, Ig E, Ig G, Ig M). 3

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4 What’s the role of antibody in immune system? Each Antigen has specific antigen determinants ( epitopes ) located on it. The antibodies have complementary determining regions (CDRs). These are mainly responsible for the antibody specificity. Each antigen has several different epitopes on it. They are recognised by many different antibodies. All these antibodies thus produced act on the same antigen. Hence these are designated as polyclonal antibodies.

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5 What’s the need to develop monoclonal antibodies? In general naturally produced antibodies are non-specific and heterogenous in nature. Hence there are several limitations in the use of polyclonal antibodies for therapeutic and diagnostic purposes. Thus there is a need for producing monoclonal antibodies for different antigens. George Kohler and Cesar Milstein got noble prize in 1984 for the production of MAbs in large scale.

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6 What are monoclonal antibodies? MAb is a single type of antibody that is directed against a specific antigenic determinant( epitope ). Monoclonal antibodies are specific to antigen and are homogenous.

History of Mab development:

History of Mab development 1890 Von Behring and kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the “ side-chain theory” 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended. Almost the same time, Porter isolated fragment of antigen binding ( Fab ) and fragment crystalline ( Fc ) from rabbit y-globulin. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine- aminopterin - thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells 1990 Milstein produced the first monoclonal antibodies.

Structure of MAb :

Structure of MAb 8

Antigen- antibody binding:

Antigen- antibody binding 9

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10 Hybridoma technology: In this B-Lymphocytes and myeloma cells are mixed together and exposed to PEG for a short period. The mixture contains hybridoma cells, myeloma cells and lymphocytes. This mixture is washed and cultured in HAT(hypoxanthine aminopterin and thymidine ) medium for 7-10 days. only hybridoma cells remain in the mixture. Principle involved in monoclonal antibodies production

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Immunise Spleen Cell Myeloma Cell Line FUSE HAT sensitive Hybridoma HAT resistant Stable hybrid myeloma producing desired antibody SELECT 11

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12 Immunization Cell fusion Selection of hybridomas Screening the products Cloning and propagation Characterization and storage production of monoclonal antibodies

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13 Immunize an animal usually mouse by injecting with an appropriate antigen along with Freund’s complete or incomplete adjuvant. Adjuvants are non specific potentiators of specific immune responses. Injection of antigens at multiple sites are repeated several times for increased stimulation of antibodies. 3 days prior to killing of animal a final dose is given intravenously. Spleen is aseptically removed and disrupted by mechanical or enzymatic methods to release the cells. By density gradient centrifugation lymphocytes are separated from rest of the cells. Immunization

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14 Lymphocytes are mixed with HGPRT deficient myeloma cells and is exposed to PEG for a short period. The mixture is then washed and kept in a fresh medium. The mixture contains hybridomas , free myeloma cells, and free lymphocytes. Cell fusion

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15 Dihydrofolate Synthesis of nucleotides Tetrahydrofolate Precursors Nucleotides---->DNA Hypoxanthine Thymidine De novo synthesis Salvage pathway Aminopterin HGPRT TK

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16 The above mixture is cultured in HAT medium for 7-10 days. Due to lack of HGPRT enzyme in myeloma cells, salvage pathway is not operative and aminopterin in HAT medium blocks the de novo synthesis of nucleotides. Hence free myeloma cells are dead. As the lymphocytes are short lived they also slowly dissappear . Only the hybridomas that receives HGPRT from lymphocytes are survived. Thus hybridomas are selected by using HAT medium Suspension is diluted so that each aliquot contains one cell each. These are cultured in regular culture medium, produced desired antibody. Selection of hybridomas

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17 Screening is done for antibody specificity. For this we need to test the culture medium from each hybridoma culture for desired antibody specificity. Common tests like ELISA and RIA are used for this. In these tests the antigens are coated to plastic plates. The antibodies specific to the antigens bind to the plates. The remaining are washed off. Thus the hybridomas producing desired antibodies are identified. The antibodies secreted by them are homogenous and specific and are referred as monoclonal antibodies. Screening the products

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18 The single hybrid cell producing the desired antibody are isolated and cloned. Usually two techniques are commonly employed for this a) Limiting dilution method: Suspension of hybridoma cells is serially diluted so the aliquot of each dilution is having one hybrid cell. This ensures that the antibody produced is monoclonal. b) Soft agar method: In this method the hybridoma cells are grown in soft agar. These form colonies and the colonies are monoclonal in nature. Cloning and propagation

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19 Biochemical and biophysical characterization are made for desired specificity. It is important to note the monoclonal antibody is specific for which antigen MAbs must be characterized for their ability to withstand freezing and thawing. Characterization and storage

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20 Encapsulating the hybridoma cells in alginate gels and using a coating solution containing poly-lysine is employed. These gels allow the nutrients to enter in and antibodies to come out. Damon biotech and cell-tech companies are using this technique for commercial production of MAbs . They employ 100-litres fermenters to yield about 100g of MAbs in about 2 weeks period. Large scale production

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21 MAbs derived from mouse are murine derivatives. As they are not human origin, they show HAMA(human antimouse antibody) response. To overcome this we need to cleave the antibody into its respective Fc and Fab fragments. Fab fragments are less immunogenic and their smaller molecular size may facilitate penetration into tumor tissue and result in a longer half-life. Engineering is needed to reduce the immunogenicity. Engineered antibodies

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22 Chimeric antibodies: Hence the murine antibodies are immunogenic to humans, the obvious solution for this is to clone a fully human antibody. But it has many problems like ethical clearance, difficult to culture, impossible to obtain many of the appropriate antibodies. To over come HAMA(human antimouse antibody) response, a chimeric antibody is prepared with Fc region of human IgG and Fab regions of murine origin by the use of DNA recombinant technology. Engineered antibodies

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Mouse Human Chimeric V domains C domains 23

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24 Humanized antibodies: Though chimeric antibodies elicit less HAMA response than murine antibodies, they are still immunogenic due to their murine regions(30%) It is came to know that a small portion(CDR) of an antibody was actually responsible for antigen binding. By this humanized antibodies are prepared by recombinant DNA technology with majority of human antibody framework and CDR’s of murine antibody. Thus humanized antibodies are 95% homology with human antibodies. Engineered antibodies

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Mouse Human Humanised hypervariable framework 25

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26 Bispecific antibodies: These are specific to two types of antigens. They are constructed by r.DNA technology. Each arm is specific to one type of antigen. Engineered antibodies

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27 Immunoconjugate : For MAb targeted drug delivery, a drug is bound covalently to an antibody that is chosen to target it to the desired site of action. Spacer is present between the antibody and the drug. Polymer may be present to increase the no. of drug molecules attached to the antibody. Drug is non-covalently incorporated into a liposome or microsphere to which the targeting antibody is bound to the surface— immunoliposome or immunomicrosphere resp. Engineered antibodies

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28 Principle involved: As several classes of the drugs lack specificity for diseased cells, they show their action on other sites of action. Ex: cytotoxic action of chemotherapeutic agents is directed against any rapidly proliferating cell population. Hence drug targeting is required to overcome this problem. Targeting is classified into three categories: Passive targeting Physical targeting Active targeting Drug targetting

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29 It is the natural in-vivo distribution pattern of the drug delivery system. It is determined by the inherent properties of the carrier like hydrophobic and hydrophilic surface characteristics, particle size, surface charge, particle number. Ex: passive targeting of the lungs is made by modulating the size of the particles to >7µm passive targeting of the Reticuloendothelial system is made by modulating the size of the particles to 0.2-7µm Passive targeting

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30 In this some characteristics of the environment are utilized for the carrying of the drug to the specific site. Ex: thermal sensitive liposomes (local hyperthemia ) magnetically responsive albumin microspheres (localized magnetic field) Physical targeting

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31 Active targeting is usually done by cell-specific ligands . These are specific to specific cell types. But it is limited to small no. of tumor types. Hence MAb targeting is adopted for active targeting. MAb targeting is done by conjugating the drug antibody of the specific targeting type. Hence antibody drug conjugates are used as active targeting drug delivery systems. Active targeting

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32 Toxin conjugates ( immunotoxins ) EX: diphtheria toxin, Ricin have been conjugated to the tumor specific antibodies Ricin has two chains. Amoung these A-chain is cytotoxic and B-chain is non-specific. Hence B-chain is removed and the toxin is conjugated to tumor specific antibody. Thus we increase the specificity of the toxins by using MAbs as active drug targeting systems. Drug conjugates

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33 Drug immunoconjugates : Agents like chlorambucil , methotrexate and doxorubicin are conjugated with tumor specific antibodies. Ex: doxorubicin-BR96 immunoconjugate for Lewis antigen found on the surface of tumor cells. Drug conjugates

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34 They are homogenous in nature. They are specific to a particular antigen with a particular epitope . Ex: Rituximab ( Rituxan ®, anti-CD20) is a good example – this antibody is used for the treatment of lymphoma. Advantages of Monoclonal antibodies

Monoclonals for tumour therapy: :

Cell Depletion Rituxan , Campath (naked) Myelotarg (drug) Zevalin , Bexxar (radioisotope) Blocking receptors Herceptin Attacking vasculature Avastin , Erbitux Vaccination against idiotype Panorex ? Monoclonals for tumour therapy: 35

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36 As they are specific to a particular antigen, they cannot distinguish molecule as a whole. Some times they cannot distinguish groups of different molecules. Ex:- presence of retro viruses as a part of mammalian chromosomes is not distinguished. Mice used in MAb production carry Adenovirus, Hepatic virus, Retrovirus, reovirus , cytomegalovirus, thymic virus. The presence of some of these viruses is detected in hybridomas . This poses a great danger since there is no guarantee for MAb produced is totally virus free. For this reason US food and drug administration insists that MAb for human use should be totally free from all pathogenic organisms including viruses. Limitations

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37 Biotechnology by U. Satyanarayana Harper's Biochemistry 26th edition www.wikipedia.com www.google.com Bibilography

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38 THANK U

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