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Monoclonal antibodies

Monoclonal antibodies. products of a single B-lymphocyte clone homogeneous (antigene-specificity, affinity, isotype). - in human body: only under pathological circumstances e.g. in gammopathy (malign growth of a certain plasma-cell clone). their advantage versus polyclonal antibodies:

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Monoclonal antibodies

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  1. Monoclonal antibodies • products of a single B-lymphocyte clone • homogeneous (antigene-specificity, affinity, isotype) - in human body: only under pathological circumstances e.g. in gammopathy (malign growth of a certain plasma-cell clone) • their advantage versus polyclonal antibodies: • antibodies of the given specificity and isotype can be • produced in large amount and of the same quality

  2. only oneB-lymphocyte clone monoclonal antibody moreB-lymphocyte clone Polyclonal antibody

  3. Features of polyclonal and monoclonalantibodies

  4. Procedure of monoclonalantibodyproduction Hybridomatechnology • immunisation of a mouse/rat with a specific antigen • removal of the spleen or lymph nodes of the mouse, homogenisation • fusion of mouse plasma cells (with spleen origin) + mouse tumor cells (plasmocytoma/myeloma cells with B-cell origin) • identification of antibody producing clones. The newly formed hybridomas are proliferating continuously and producing antibodies which concentrate in the medium.

  5. Immunisation Spleen B cells, HGPRT+ Myeloma cell HGPRT- PEG fusion HAT selection Testing supernatants for specific antibody production Procedure of monoclonalantibodyproduction II. Selection of hybridoma cells aminopterine * HAT= hypoxanthine, aminopterine, thymidine *Hypoxantine-guanine phosphoribosyltransferase

  6. (1)Immunisation of a mouse(2) Isolation of B cells from the spleen(3) Cultivation of myeloma cells(4) Fusion of myeloma and B cells(5) Separation of cell lines(6) Screening of suitable cell lines(7) in vitro (a) or in vivo (b) multiplication(8) Harvesting

  7. Factorsinfluencingtheefficacy of monoclonalantibodyproduction • Activation of antigen-specific B-lymphocytes • - mouse: inbred lines with characterized genetic information, • small amounts of antigen is required for immunisation • The followings influence the efficacy of hybridoma cell separation: • a) the way of immunisation (using adjuvants, place of injection: intraperitoneal, foot, tail-vein (caudal vein), spleen) • b) number of repeated shots to reinforce (boost) the immune response • c) number of days elapsed between the last vaccination and the fusion (2-4 days) • 2. Fusion partners • Sp2/0-Ag14 tetraploid, non-antibody producing plasmocytoma cells from BALB/c mice • (these plasmocytoma cells have HGPRT /hypoxantine-guanine- • phosphoribosyl-transferase/ and thymidine kinase deficiency)

  8. Possibleuse of monoclonalantibodies • Identifying cell types • Immunohistochemistry • Characterization of lymphomas with CD (cluster of differentiation) markers • Isolation of cells • Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral blood!) • Blood group determination (with anti-A, anti-B and anti-D monoclonals) • Analysis of a mixture of antigens • Identification of cell surface and intracellular antigens • Investigation of T-cell activation • Targeted chemotherapy • CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma • Prevention of organ rejection after transplantation targeting T cells (anti-T cell monoclonals) • Drug elimination with antibodies • Anti-digoxin antibodies for the treatment of digoxin-intoxication

  9. Monoclonalantibodiesasdrugs? In immunized (with human antigens) mice the produced antibodies will contain mouse-specific proteins, and therefore, they will elicit an immune response upon administering in human subjects. (see immunogenicity-determining factors!) How we can solve this problem?

  10. Human Mouse Chimeric Humanized Evolution of monoclonal antibodies

  11. Humanizingmonoclonalantibodies 1. CAMPATH-1H anti-CD52 monoclonal rat antibody Repeated treatments with non-human antibodies induced strong immune response in patients. (Problem: HAMA = human anti-mouse antibodies) The antigen binding region of the rat antibody is exchanged with the antigen binding region of a human antibody – in the resulting antibody, only the CDR will be rat protein (chimeric antibodies). (Problem: HACA = human anti-chimera antibodies, although they are less immunogenic) 2. In vitro phage display: recombinated VDJ regions are expressed on the surface of the filamentous phage capsid. Then, these phages with high-affinity for a given antigen are separated by affinity chromatography. DNA (encoding the human immunoglobulin genes) from these phages are expressed in transgenic mice. 3. Transgenic mice producing human antibodies: Human germ line (not recombinated) Ig locus can be expressed as a transgene in knock-out mice where the Ig genes of the mice were inactivated. Therefore, human Ig gene recombination occurs in the mouse. Hybridomas can be made from mouse B cells producing human antibodies, thus large amount of monoclonal antibodies can be prepared.

  12. In vitro phagedisplay Expressed human antibody Human genes encoding antibodies Recombination of VDJ gene Characterization (Separation of DNA segments encoding human Igs and multiplicate them in transgene mice) Expressed antibodies on the surface of the phage Phage display vector Gene3 Selective binding to antigen Amplification Washing Recovery Discard

  13. mouse Ig „knock-out” transgene mouse(transferring genes encoding human Ig) VDJ átrendeződés a transzgén egérben

  14. Monoclonalantibodiesasdrugs - Tumor therapy Monoclonals made possible the targeted chemotherapy of tumors. It is cell-type specific, but not specific to malignant cells!) • Immunsuppressive monoclonals Cell-type specific immunsuppression

  15. Monoclonalantibodynomenclature The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary names to a group of medicines called monoclonal antibodies. This scheme is used for the World Health Organization’s International Nonproprietary Names. Components of nomenclature: Example:Abciximabab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody used on the cardiovascular system

  16. Monoclonalsin tumor therapy • „Naked MAb”, unconjugated antibody • Anti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphoma • Anti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemia • Anti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancer • Anti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!) • Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. humán!) • 2. Conjugated antibody • Anti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin) • Anti-CD20 + iodine-131 (tositumomab – Bexxar)

  17. Immunsuppressivemonoclonals 1. • Anti-TNF-α antibodies • infliximab (Remicade): since 1998, chimeric • adalimumab (Humira): since 2002, recombinant human • Etanercept (Enbrel) – dimer fusion protein,TNF-α receptor + Ig Fc-part • (Not monoclonal antibody, containing only the Fc part of Ig) • Indications of anti-TNF-α therapy: • Rheumatoid arthritis • Spondylitis ankylopoetica (SPA - M. Bechterew) • Psoriasis vulgaris, arthritis psoriatica • Crohn-disease, colitis ulcerosa • (usually - still – not in the first line!)

  18. Immunsuppressivemonoclonals 2. • Muromonab-CD3 (OKT-3) mouse IgG2a • Against CD3 pan-T-cell antigen, after transplantation; It is rarely (or not) used nowadays (mouse protein!); ongoing trials in diabetes mellitus, with the humanized version • Omalizumab (Xolair): • Anti-IgE humanized IgG1k monoclonal • Ind.: allergic asthma, Churg-Strauss sy. • Daclizumab (Zenapax): • anti-IL-2 receptor humanized antibody • Ind.: transplantation • basiliximab (Simulect): as daclizumab, but chimeric! • efalizumab (Raptiva): anti-CD11a, humanized, used in psoriasis

  19. Moleculartargeteddrugs

  20. Further possibilities with monoclonals • Radioimmunotherapy • As Zevalin, Bexxar – monoclonal + isotope • Antibody-directed enzyme prodrug therapy (ADEPT) • An enzyme is linked to the antibody, and the enzyme will make citotoxic drug from the later administered prodrug • Immunoliposomes • Targeting nucleotides or drugs in liposomes, linked to an antibody (eg. tumor suppressor gene or tissue-specific gene transfer) • Non-immunological targets • as abciximab (ReoPro): inhibition of thrombocyte-aggregation

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