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  1. 1. <ul><li>Monoclonal Antibodies </li></ul><ul><li>Antibodies (Abs). Also known as immunoglobulins (Ig). </li></ul><ul><li>Comprised of 2 heavy chains and 2 light chains </li></ul><ul><li>Monoclonal Abs bind specifically to a single site (epitope) on a particular antigen </li></ul><ul><li>- Abs are produced by B lymphocytes </li></ul><ul><li>Because of their specificity and ease of generation, they are extensively used as therapeutics (“passive immunotherapy”) and as diagnostic and research tools </li></ul><ul><li>-They can be generated in large (unlimited) amounts in vitro </li></ul>
  2. 2. B cells develop in the bone marrow  hematopoietic stem cells and lymphoid stem cells  T-cells and B-cells progenitor = pro-B cell (B220+) precursor = pre-B cells: heavy-chain rearranged immature B cell: IgM + light-chain rearranged mature B cell: IgM + IgD + an antigen encounter in spleen or lymph nodes; goes to periphery Terminally differentiated cell = plasma cell, periphery, Ig secretor Antibodies are made by B-cells
  3. 3. Domain structure of an immunoglobulin molecule disulfide bonds } Fc } Fab F ragment , a ntigen b inding F ragment , c rystallizable
  4. 5. Laboratory fragmentation of antibodies
  5. 6. Ig molecule showing polarity, disulfides, carbohydrate Complementarity determining Region = “CDR” Hypervariable region
  6. 8. Opsonization Complement activation Antibody-dependent cell-mediated cytotoxicity ( ADCC ) Transcytosis Fc functions
  7. 9. Opsonization: Direct uptake of bacteria coated with antibody molecules Complement activation: Activated complement proteins lyse cells by making holes in their mebranes(e.g. bacteria) Antibody-dependent cell-mediated cytotoxicity ( ADCC ): Killer T-cells use antibodies on their surface to target cells with an antigen and kill them. Transcytosis: Antibody-antigen complexes are taken up (endocytosed) on on side of an epithelial cell and directed to theother side, where they are exocytosed Fc functions
  8. 10. Multigene organization of Ig genes – light chains: V, J (variable) and C (constant) – heavy chain: V, D, J, (variable) C (constant) Mechanism of Ab gene rearrangement Recombination signal sequences ( RSS ) –flank V, D, J gene segments – V- RSS ------ RSS -D- RSS --------- RSS -J
  10. 12. Antibodies can participate in host defense in three main ways
  11. 13. Got this far
  12. 14. ADCC = antibody-dependent cell-mediated cytotoxicity
  13. 15. MAb therapy targets Inflammation Autoimmune disease Graft rejection Heart disease (thrombosis) Cancer Viral infection
  14. 16. Therapeutic strategies Mabs straight Mabs fused to other protein binders (e.g., soluble receptors) Mabs fused to cytotoxic agents (toxins, radionuclides) Toxins: ricin (stops protein synthesis) c alicheamicin (DNA breaks) Radionuclides: 90 Y = yttrium 111 I = indium
  15. 17. <ul><li>Problems of mouse MAbs </li></ul><ul><li>Fc portion limited in its ability to interact with Fc receptors of human cells. </li></ul><ul><li>Lower serum half-life </li></ul><ul><li>Development of human anti-mouse antibodies (HAMA) </li></ul><ul><ul><li>Retreatment results in allergy or anaphylactic shock </li></ul></ul><ul><ul><li>Retreatment is less effective </li></ul></ul><ul><li>Solutions via recombinant DNA genetic engineering : </li></ul><ul><li>Chimeric mouse-human antibodies: Hu V-region fused to mouse C regions </li></ul><ul><li>Humanized mouse antibodies, Parts of V-region from human interspersed with mouse CDR V-regions </li></ul><ul><li>Human antibodies (fully), via transgenic mice carrying human immunoglobulin genes (Medarex, Abgenix, Kirin) </li></ul>Breedveld, Lancet 2000 355:9205
  16. 18. MAbs approved for human therapy EGF-R colon cancer HER-2/neu (EGF2) breast cancer CD33 leukemia (AML) VEGF colon cancer IgE asthma Respiratory infection Synciitial Virus IL-2 immunosupressant 26.02.2004 Antineoplastic Humanized Avastin Bevacizumab Genentech 12.02.2004 Antineoplastic Chimeric Erbitux Cetuximab Imclone Systems 27.10.2003 Immunological Humanized Raptiva Efalizumab Genentech 27.06.2003 Antineoplastic Murine BEXXAR Tositumomab-I131 Corixa 20.06.2003 Immunological Humanized Xolair Omalizumab Genentech 31.12.2002 Immunological Human Humira Adalimumab Abbott 19.02.2002 Antineoplastic Murine Zevalin Ibritumomab tiuxetan Biogen IDEC 07.05.2001 Antineoplastic Humanized Campath Alemtuzumab Millennium/ILEX 17.05.2000 Antineoplastic Humanized Mylotarg Gemtuzumab ozogamicin Wyeth 25.09.1998 Antineoplastic Humanized Herceptin Trastuzumab Genentech 24.08.1998 Immunological Chimeric Remicade Infliximab Centocor 19.06.1998 Anti-infective Humanized Synagis Palivizumab MedImmune 12.05.1998 Immunological Chimeric Simulect Basiliximab Novartis 10.12.1997 Immunological Humanized Zenapax Protein Design Daclizumab Labs | 26.11.1997 Antineoplastic Chimeric Rituxan Rituximab Biogen IDEC 22.12.1994 Hemostasis Chimeric ReoPro Abciximab Centocor 19.06.1986 Immunological‡ Murine Orthoclone OKT3 Johnson & Johnson Muromonab-CD3 US approval Therapeutic category mAb type US trade name Sponsor company Generic name
  17. 19. <ul><li>Monoclonal antibody generation </li></ul><ul><li>- Cells needed myeloma cells, mouse spleen cells </li></ul><ul><li>- antigen administration Kohler and Milstein </li></ul><ul><li>- hybridoma formation via cell fusion </li></ul><ul><li>selection mutants required: defer </li></ul><ul><li>- antibody generation cDNA cloning </li></ul><ul><li>- engineered MAbs expression vectors </li></ul><ul><li>- refinement chimeric, humanized, human (defer) </li></ul>
  18. 20. Monoclonal antibodies via cell hybridization Selects for rare hybrid cells
  19. 21. Reduced myeloma hybrid Isoelectric focusing immunoglobulins made in hybridoma cells Unreduced myeloma hybrid Georges Kohler Cesar Milstein
  20. 22. Mab Fusion Proteins Other protein-binding proteins: natural receptors in soluble form Analogous to MAbs and make use of the Fc portion of the antibody molecule: Example: Enbrel (etanercept): Anti-rheumatoid arthritis drug Soluble TNF receptor fused to the Fc IgG 1 domain (TNF= tumor necrosis factor) Ties up TNF, blocking its inflammatory function Fc domain dimerizes the receptor, which increases its affinity for TNF. Fc domain increases the half-life of the protein in the bloodstream Amgen + Wyeth Still experimental: anti HIV drug PRO 542 Soluble CD4 (HIV receptor) fused to IgG 2 . Tetrameric (4 V-regions replaced) Reduced Fc function (since IgG 2 < IgG 1 ), Better half-life Progenics
  21. 23. Single chain antibodies (scFv) Ag binding site 15 AA linker Phage display selection of scFv Source of sequence: PCR from genome or mRNA, add randomization