monoclonal antibodies and engineered antibodies


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monoclonal antibodies and engineered antibodies

  2. 2. Contents Introduction Advantages and disadvantages Production methods Problems associated Applications Engineering antibodies Conclusion References 2
  3. 3. Introduction Antibody(Ab) is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. An antibody is called monoclonal (mAb) when each immunoglobulin is produced by a single clone of cells and hence is identical to every other molecule in the preparation, in terms of heavy as well as light chain structure. Polyclonal antibodies (pAb) are produced by B-lymphocyte which respond to many epitopes of antigen. 3
  4. 4. Polyclonal antibodies (Polyclonal antiserum) Harvest Ab Monoclonal antibodiesB B B B B B B B 4
  5. 5. Advantages Specificity. Affinity. Potential to generate large quantities of Abs under precisely controlled conditions. In vivo and in vitro production is possible with high production rate. Immortal cell lines.Disadvantages mAb cant differentiate between two antigens if the body is directed to an epitope common to antigens. mAb to viral strains are so specific that they don’t react to other minor strains of same virus. pAb have potential for co-operatively binding to respective antigens, so stabilising the overall affinity and binding forming precipitating complexes while mAbs form network with antigens. 5
  6. 6. Affinity Chromatography 6
  7. 7. Production methods- Hybridoma technology 7
  8. 8. In vivo production Hybridoma cells are injected into pristane primed rodents, the cell line proliferates and are stored in ascitic fluid. 10-50 mL of fluid is collected containing several mg/mL of antibody. mAbs produced this way are considered unsuitable because of viral contamination. Widely used in research applications. The inherent variability in animals can result in lack of consistency, while some lines produce solid rather than diffuse tumors, and some produce none or kill the host. 8
  9. 9. In vitro production Fermentation is most widely employed for production of monoclonal antibodies because of the problems associated with above listed methods. No contamination with normal mouse immunoglobulin is seen with fermentation. Apart from this, bacterial cell cultures, transgenic animals and transgenic plants are also used for production of monoclonal antibodies but to a very limited extent. 9
  10. 10. Problems associated with mAb therapy The main problem is the mouse antibodies are recognised by human immune system as foreign material and the patients develop a immune response against them, producing HAMA (Human Anti-Mouse Antibodies). The doses of monoclonal antibodies to treat chronic diseases are typically large. Stability issues concerned with oxidation, deamidation, aggregation, fragmentation and other forms of chemical modification with alter or probably nullify the antibody function. 10
  11. 11. Stabilisation Freeze dried and again recovered Spray dried Ab added to for moisture uptake. polylactide-co-glycolide (PLGA) Carbohydrates are added to produce microspheres. Ex- Trehalose, sucrose etc., Characterization. Monitoring 11
  12. 12. Applications- Diagnosis and therapy Identification of tumors as they express specific membrane proteins. Measurement of circulation steroid harmones and in differentiating viral strains. Bacterial infections and STD. Drug immunoconjugates. Radioisotopes anchored mAbs.Analytical applications RIA and ELISA. Purification of proteins. 12
  13. 13. Role of mAbs in tumor therapy 13
  14. 14. Commercial mAbs in market 14
  15. 15. Engineering antibodies Antibodies exhibit four main effector functions: antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), and half-life/clearance rate. Each of these effector functions is mediated through interaction with a specific set of receptors and cell types: ADCC and phagocytosis through interaction of cell-bound mAbs with Fc gamma receptors (Fc γ R), CDC through interaction of cell-bound mAbs with the series of soluble blood proteins that constitute the complement system (e.g. C1q, C3, C4, etc.), and half-life/clearance rate through binding of antibodies to the neonatal Fc receptor (FcRn). C1q binding and complement activation. FcγR binding and ADCC. FcRn and half-life/clearance rate. 15
  16. 16. Genetically engineered antibodies Chimeric antibody- As HAMA response of patients is due to Fc portion of murine Abs, murine Fv region was fused with human Fc to produce Chimeric genes. Ex- Infliximab, rituximab, and abciximab Humanised antibody- This consists of fusion of hyper variable region, aminao acids responsible for antigen binding with human antibody thus replacing its own hyper variable region. Ex- Mylotarg®, Herceptin, Xolair® Chimeric antiody Humanised antiody 16
  17. 17. Myelotarg® mAb + Calcichemicin Antibody portion of molecule targets CD33 (a cell surface molecule), abundant on the surface of acute myeloid leukemia cells (AML) & absent from normal blood stem cells. AML cells accumulate in the bone marrow and prevent normal bone marrow from growing an functioning properly. Calcichemicin is a potent anti-cancer drug which intercalates into DNA and breaks it, because of which cells undergo apoptosis. Antibody targets calcichemicin to AML cell specifically through CD33 cell surface molecule Calcichemicin kills AML cells. 17
  18. 18. Calcichemicin intercalates in DNA 18
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  21. 21. Conclusion – Challenges and Opportunities Successful clinical application of these novel agents requires the development of stable formulations that can be used for specific delivery methods. Antibodies, because of their endogenous nature, have built-in features that may pose problems for stability as bio-therapeutics. Lyophilization and pH dependent modification. Several modes of targeting is achieved successfully. Linking up the technology to gene therapy will ensure highly specific treatment Examples of such an approach include studies performed using an antibody directed against the Her2/neu antigen to delivery liposomes containing a chemotherapeutic have been described, antibodies used to deliver cationic liposomes for the administration of nucleic acid material for gene therapy. 21
  22. 22. References L.G. Presta, Engineering antibodies for therapy, Curr. Pharm. Biotechnol. 3 (2002) 237– 256. J.W. Park, K. Hong, P. Carter, H. Asgari, L.Y. Guo, G.A. Keller, C. Wirth, R. Shalaby, C. Kotts, W.I. Wood, et al., Development of anti-p185HER2 immunoliposomes for cancer therapy, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 1327– 1331. A.L. Daugherty, R.J. Mrsny, Formulation and delivery issues for monoclonal antibody therapeutics, Advanced Drug Delivery Reviews. 58 (2006) 686–706. Leonard G. Presta, Engineering therapeutic antibodies to minimize immunogenicity and optimize function, Advanced Drug Delivery Reviews 58 (2006) 640–656. Lee K. Tan and Sherie L. Morrison, Antibody structure and antibody engineering, Advanced Drug Delivery Reviews, 2 (1998) 129-142. 22
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