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

Topic to be cover Antibodies Nomenclature of Antibodies Introduction Antibodies Nomenclature of Antibodies Production of Monoclonal Antibodies Application of Monoclonal Antibodies Conclusion By 2008, mAbs should account for 32 percent of all revenue in the biotech market. mAbs drive the development of multibillion dollar biotechnology industry. The outlook for monoclonal antibody therapeutics is healthy

ANTIBODIES Antibodies are 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. Plasma cell is called plasma B cells,plasmocytes r the white blood cells which produce large volm of antibodies. B-cellr r the lymphocytes that play large role in thehumoral immuno response.

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) Hinge=A joint that holds two parts together so that one can swing relative to the other

Epitopes

Types of Antibodies Monoclonal antibodies (mAb) Polyclonal antibodies Mab=are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines

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.

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 Georges Kohler and Ceaser Milstein in 1975 It is interesting that immortal Mab producing cells do exist in nature.

US Food and Drug Administration nomenclature of therapeutic antibodies chimeric=Being or relating to or like a chimera, (Greek mythology) fire-breathing female monster with a lion's head and a goat's body

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 Myeloma cell=It is a tumor of bone marrow Lymphocyte=it is a type of organular white blood cell in vertibrate immune system B cell= r lymphocytes that play large role in humoral immuno response

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. Mab is a single type of antibody that is directed against a specific antigenic determinanats(epitopes)

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)

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.

PROCEDURE STEP -1 Immunization of a mouse. Mouse is immunized using antigen injection against which monoclonal antibodies are to be produced. Repeated injection can be given of specific antigen for the production of specific antibody, facilitated due to proliferation of the desired B cells.

STEP -3(A) Preparation of Myeloma Cells STEP -2 Culture 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 Spleeen=a large dark red oval organ on the left side of the body b/w stomach & diaphragm Myeloma cell=It is a tumor of bone marrow Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumour of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion Serium antibody titre is determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry(technique for counting microscopic cel and chromosome)

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

STEP -4 Fusion of cells using polyethylene glycol (PEG). antibody producing B-cells are mixed with selected myeloma cells and hypoxanthine guanine phosphoribosyl transferase enzyme Mixr is exposed 2 Poly ethelene glycol Aftr few minuites PEG is removed by washing and cells r kept in fresh medium (cells contain Hybridomas ie fused cell,free myeloma cell & free lymphocytes)

Selectively grow hypoxanthine aminopterin thymidine (HAT) medium STEP -5 Selection for the hybrid cells: Selectively grow hypoxanthine aminopterin thymidine (HAT) medium Aminopterin block nucleotide synthesis pathway Another pathway needs HGPRT enzyme. Fused cells are grown in selective HAT (Hypoxanthine-aminopterin-thymidine) medium. HAT medium contains a drug aminopterin, which blocks one pathway for nucleotide synthesis, making the cells dependent on another pathway that needs HGPRT enzyme absent in myeloma cells. Therefore, myeloma cells that do not fuse with B cells will die since they are HGPRT-.B cells that do not fuse will also die because they lack tumorigenic property of immortal growth. Therefore HAT medium allows selection of hybridoma cells, which inherit HGPRT gene from B cells and tumorigenic property from myeloma cells. It grows in 7-10 days of culture

Facilitated by preparing single cell colonies 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 Select the desired hybridoma for cloning and antibody production; this is facilitated by preparing single cell colonies that will grow and can be used for screening of antibody producing hybridomas

Monoclonal antibodies in large quantity. May be frozen for future use. STEP -7 Culture of selected hybridoma Monoclonal antibodies in large quantity. May be frozen for future use. May also be injected in the body of an animal. culture selected hybridoma cells for the production of monoclonal antibodies in large quantity; these hybridoma cells may be frozen for future use and may also be injected in the body of an animal so that antibodies will be produced in the body.

Can be done later from the body fluid. STEP -8 Harvesting: Can be done later from the body fluid. Recovery of monoclonal antibodies can be done later from the body fluid.

PURIFICATION OF MONOCLONAL ANTIBODIES In-vitro contaminants – growth factors and hormones etc. In-vivo contaminants – host antibodies, protease, nuclease, nucleic acids etc. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria.

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. Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography, using columns such as the ProPac WCX-10. Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). Proteins such as Transferrin can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant

iv)AFFINITY CHROMATOGRAPHY Specific binding interaction Ligand coupled to solid support Unbound components washed away Bound molecules stripped by low pH buffer achieve maximum purity in a single step, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

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.

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)