is a protein ( Antigen binding ) used by the m to identify and neutralize foreign objects like | d viruses. Each antibody recognizes a specific tunigue to its target. These are present on the B-cellSTRUCTURE OF ANTIBODY rhe 2 Identical Light chains (- 220 amino acid long) Variable domain : Vi Constant domain: C. 2 Identical Heavy chains (440 amino. acid long) Variable domain : V4, 3 Constant domain : Cyt, C.2, G43 Covalent Disulphde bonds between Cysteine residues. Flexible “ Hinge region ”| |G OF MONOCLONAL ERA O In 1890, Von Behring & Kitasato discovered antibodies. 1 1n1900, Ehrlich proposed the “ side-chain theory.” O In 1955, Jerne postulated “Natural selection theory.” which EM. Burnet expended. O In the same same time (1955) , Porter isolated Fragment of antigen binding (Fab) & Fragment crystalline (Fe) fim rabbit y- globulin,On 1964, Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin= thymidine (HAT) selection media Ol 1975,Kohler and Milstein provided the most ‘outstanding proof of the clonal selection theory by fusion of normal and malignant cells i.e., Hybridoma Technology.In 1986-1990, the first monoclonal antibodies reached the tarket- Muromonab- CD3 ( produced by Milstein). O In 1988, Greg Winter et al pioneered the techniques to humanise monoclonal antibodies. O Paul Ehriich at the beginning of the 20th century theorized that a cell under threat grew additional side-chains to bind the toxin, and that these additional side chains broke off to become the antibodies that are circulated through the body. It was these antibodies that Ehrlich first described as the "magic bullets" in search of toxins In 2003, First Fully Human monoclonal antibody ~ Adalimumab = be elles PinarDee a A ae Re system and the discovery of the principle for production of monoclonal GeisANTIBODIES | ed by immunizing an animal with the appropriate antigen- wide array of ‘pe stimulated to produce anti- protein antibodies. ‘obtained from immunized animal referred to as “Polyetonal Serum”MONOCLONAL ANTIBODIES 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. & These are a class of highly specific antibodies produced by the clones ofa single hybrid cell. } They all have identical antigen- binding sites. 4 Bind to the same epitope with same affinity. 4 same antibody class ( isotope)ANTIBODIES feared orators MONOCLONAL ‘Multiple epitopes of all antigens used in the————AAA levator ornate MONOCLONAL Less expensive ‘More expensive Limited supply Infinite supply Easily Rapidly produceda REATES MONC ANTIBODIES a Monoclonal antibodies (mAb) are directed against a specific epitope (antigen, antigenic determinant) . O Typically made by fusing myeloma cells with the spleen B cells from a mouse that has been immunized with the desired antigen or a single Hybridoma cell line.ION OF MON’ ANTIBODIES = eee oes.Antibody titre: reached in Seruning of Mice for Antibody Pro a Serum Antibody Titre Determined (Technique :- ELISA/ Flow cytometry ) Titre too low (Pure Antigen) Titre high BOOST (Pure Antigen)STEP 3:- Preparation of Myeloma Cells ga 8 - Azaguanine Myeloma cells Immortal Tumour of Lymphocytes Myeloma cells HGPRT — High Viability & Rapid Growth_ STE! 4:- Fusion of Myeloma cells with Immune Spleen Cells & Selection of Hybridoma Cells Spleen Cells Myeloma Cells Feeder ells Growth Medium [siete] HYBRIDOMA CELLS ELISA PLATE eT OAL HAT Medium eeeEP 4 :- Cloning of Hybridoma Cell Lines by 4 “Limiting Dilution” or Expansion A. Clone each positive Culture . B. Test each Supernatent for Antibodies J C. Expand positive clones ame TP e Method Harvest monoclonal antibadiesHYBRIDOMA SELECTION The “ HAT Trick ” Myeloma cells have been genetically engineered such that they can_not use hypoxanthine, Aminopterin & thymidine (HAT Medium ) a8 a source of nucleic acid biosynthesis and will de in culture (lack of HGPRT enzyme). Spleen cells ( B cells) have limited life span Only 8 cells that have fused with the engineered myeloma cells will survive in culture when grown, in Hat medium.NOMENCLATURE OF MONOCLONAL | ANTIBODYes Cee CE ee Deere) Ce eee Zr Examples ab- + -ci- + -xi-+ -mab: chimeric monocional antibody used on the cardiovascular system ee ee Le humanized monoclonal antibody Wre-teietee ae ma ele or humanized mab used against a virus (RSV) Pe eee tere)These are derived from ‘Mice, Patients treated ‘wich murine mAbs. develop Human Anti- ‘Mouse Antibody (HAMA) response, Es °° Y-ibritumomab CCHIMERIC “They combine the ‘antigen binding parts (ariabie region) of mouse with effector ats constant resion) HUMANISED ‘These are human antibody with agampementary fetermining region (COR) oF hypervariable ‘egion from non human soiree (rodent) grafted ‘onto human variable region. Ee DactizumabADVANTAGES OF MAB'S Umea ekeEFFECTOR FUNCTIONS It may not produce the desired biological response SPECIFICITY Monoclonals against conformational epitopes on native proteins may lose reactivity wth antigens1 Bitinyated 3 _ Waled Mab raicatvelgad MutistepSrgeting | apepr. antibody ; directed enzyme prodrug therapy; ‘ADCC- antibody dependent cell- F mediated cytotoxicity; (¢DC- complement dependent ‘eytotoxicity; MAb- ‘monoclonal (Celarimmunocorgates) ‘antibody; ScFv single-chain Fu FV ‘fragmentery ri}ANTIBODIES e growing knowledge of antibody gene structure and regulation has possible what Cesar Milstein, one of the inventors of monoclonal body technology, has called ‘man-made antibodies.” It is now possible “design and construct genes that encode immunoglobulin molecules in hich the variable regions come from one species and the constant regions from another. ‘New genes have been created that link nucleotide sequences coding antibody proteins with sequences that encode antibody variable regions for particular antigens. 1 Finally, by replacement of the immunoglobulin loci of one species with at of another, animals of one species have been endowed with the capacity ‘fespond to immunization by producing antibodies encoded by the donor's transplanted Ig ¢neric and Hybrid Monoclonal Antibodies for HUMANISED ANTIBODY production jeering an antibody to clone recombinant DNA containing the r, leader, and variable region sequences from a mouse antibody * and the constant-region exons from a human antibody gene. Production of chimeric mouse human monecional antibodies, Chimeric mouse: human heayy- and light-chain expression vectors are produced, These vectors are transfected into Ab inyeloma cells. Culture in ampicillin medium selects for transfected: rnyeloma cells that secrete the. chimeric antibody,‘of two different antibody molecules which ‘chemically cross linking two different antibodies vem in hybridomas consisting of two different mon ing cell lines that have been fused. ‘A CHIMERIC IMMUNOTOXIN is chimeric monoclonal, antibody in which the terminal Fe domain is replaced by toxin chains (white), ‘A HETEROCONJUGATE in which onehalf of the mouse antibody molecule is specific for a tumor antigen and the other half is specific for t CD3/T-cell receptor complex. ye yee“The capacity of mice to rearrange ly heavy- and lightchain ‘gene segments vas disabled by knocking out the Cand C loc. The antibody-producing ‘capacity of these mice was reconstituted by ‘introducing tong stretches ‘of DNA incorporating a large art of the human germ- line and heavy-chain loch {miniloci). Chimeric mice ‘were then bred to ‘establish a line of transgenic mice bearing both heavy: and tight: ‘chain human minilec. Immunization of these mice results in the production of human antibody specific for the target antigen.‘SIDE - EFFECTS OF MONOCLONAL ANTIBODIES ‘Monoclonal antibodies are given intravenously (injected into a vein). These are often more like an allergic reaction & are more common while the drug {s first being given, Possible side effects can include : & (iis ( -@yeep | Gusrea winpesene ‘mmtaogeiay See | eeeonds | eae Stren kof edn Sian |e” | Seem ‘sieaahecronele de a ier (ee core [Steer Seal — ama SOF [ates Saar ‘Beene Sea == COEF on | Atacama Sctard | Gomme) Samara alle Tamaiaed oerANTI BODIES Fst approved mAbs was OKT-3 [1986] which is a murine IgGaz oo patients with acute rejection of renalAntibody Abciximab Adalimumab Alemtuzumab Basiliximab Belimumab Bevacizumab Brentuximab Vedotin Brand name ReoPro Humira Campath Simulect Benlysta Avastin Adcetris Type Indication chimeric Cardiovascular disease everal auto-immune human BSF humanized fhyenic lymphocytic chimeric Transplant rejection human SYREN aRRUS humanized Gglgertalcanget Aee Chimeric Hodgkin lymphomaThank You