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

monoclonal antibody Polyclonal antibody only one B-lymphocyte clone more B-lymphocyte clone

Polyclonal antibodyMonoclonal antibody (low affinity) Monoclonal antibody (high affinity) Number of recognized antigen determinants several (frequent cross-reactions) one (but frequent cross-reactions) mostly one Specificitypolyspecificoften polyspecificmonospecific AffinityVarying (diverse antibodies) lowhigh Concentration of non- specific immunoglobulines highlow Yieldhighlow Cost of preparationlowhigh StandardisabilityImpossible (or uneasy) easy Amountlimitedlimitless Applicabilitymethod-dependentlowexcellent Features of polyclonal and monoclonal antibodies

Procedure of monoclonal antibody production Hybridoma technology - 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.

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

(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

1.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) Factors influencing the efficacy of monoclonal antibody production

Possible use of monoclonal antibodies - 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

Monoclonal antibodies as drugs? 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?

Evolution of monoclonal antibodies Mouse Chimeric Human Humanized

Humanizing monoclonal antibodies 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.

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

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

- 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 Monoclonal antibodies as drugs

Monoclonal antibody nomenclature 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 systemAbciximab

Monoclonals in tumor therapy 1.„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)

Immunsuppressive monoclonals 1. 1.Anti-TNF-α antibodies infliximab (Remicade): since 1998, chimeric adalimumab (Humira): since 2002, recombinant human 2.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!)

-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 Immunsuppressive monoclonals 2.

Molecular targeted drugs NameTypeTargetIndications Alemtuzumab (Mabcampath) Daclizumab (Zenapax) Basiliximab (Simulect) Rituximab (Rituxan/Mabthera) Trastuzumab (Herceptin) Gemtuzumab Ibritumomab (Y 90 ) Edrecolomab Gefitinib Imatinib Monoclonal antibody, humanized Monoclonal IgG1, chimeric Monoclonal IgG1, humanized Monoclonal IgG4, humanized Calicheamicinnel konjugált Monoclonal IgG1, murine Monoclonal IgG2, murine EGFR-TKI KIT-TKI CD52 IL-2 R CD20 HER2/neu CD33 CD20 EpCAM EGFR TK TK CLL, CML transplantation Lymphoma Breast cancer, NSC lung cancer leukemia lymphoma CRC NSCLC GIST, CML

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 Further possibilities with monoclonals