ADVANCED MOLECULAR THERAPIES OF THE 21-ST CENTURY II. MEDICINAL PRODUCTS MONOCLONAL ANTIBODIES

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1 ADVANCED MOLECULAR THERAPIES OF THE 21-ST CENTURY II. MEDICINAL PRODUCTS MONOCLONAL ANTIBODIES Iv. Atanasova 1 D. Terziivanov 2 1 Bulgarian Drug Agency 2 Department of Pharmacology and Clinical Pharmacology, MF, Sofia University 03 Keywords: recombinant technologies, monoclonal antibodies, targets of therapy Contact Address: Iv. Atanasova, Bulgarian Drug Agency, 8 Damian Gruev St, Sofia 1303, ivanka.atanasova@mail.bg, tel: Abstract: Scores of monoclonal antibodies are already used in the clinical practice, mostly in the field of oncology and some immuno-allergic diseases with various pathogenesis and localization. Other monoclonal antibodies are under development or in different phases of clinical trials. Their use in clinical medicine is due to their ability to modulate the natural course of the disease by affecting critical pathogenic molecules and their ability to stimulate immune-mediated effector functions. Review Article The knowledge of the target specificity of the antibodies drives Paul Ehrlich to his concept of the magic bullet thoroughly discussed in the previous issue 1. The group of the biotechnological medicinal proteins has contributed to more than 55 billion dollars sales in 2005, which is about 20% of the entire pharmaceutical market that year billion dollars. It is estimated that the sum from their sale will reach 94 billion dollars in 2010, which means that the annual increase 9

2 Monoclonal Antibodies of sales is about 12%. Currently, there are about 20 monoclonal antibodies and Fc-related proteins in the market, as sales of four monoclonal antibodies Enbrel, Mabthera, Remicade and Herceptin, approved for use in the period , have brought 23 billion dollars in The use of monoclonal antibodies as therapeutic agents has become possible with the development of hybridoma technology by Kohler and Milstein in This technology allows the creation of immortal antibody-producing cells, derived from immunized mice and subsequent selection of individual branches of cells that produce antibodies with high affinity and specificity to the target antigen. Earlier studies of murine monoclonal antibodies against tumor antigens have been effective in animal models, but transferred to clinical conditions they have presented a number of problems. The reason for this is the short half-life of the antibodies, the immunogenicity of murine proteins in humans and the suppressed immune-mediated effector functions. The main question that required an answer was whether the monoclonal antibodies targeted at the tumor antigens could trigger an immune response that would lead to clinically significant tumor regression. Many of the original restrictions have been overcome by creating chimeric or humanized monoclonal antibodies. The Chimeric antibodies contain variable portion of a murine antibody (this is part of the antibody that specifically recognizes the target antigen) bound to a constant domain of the skeleton of a human antibody. The Humanized antibody contains sequences from murine antibody only on this particular stretch of the variable domain that interacts with the target antigen. These so-called complimentary-determining regions (CDRs) are transplanted on the human antibody. The Human monoclonal antibodies do not contain murine sequences. Because most or all murine sequences are substituted, chimeric, humanized and fully humanized antibodies are less immunogenic and have a longer half-life due to slower clearance, compared to murine monoclonal antibodies. Endogenous antibodies are immunoglobulins synthesized by the B-lymphocytes. Each B-lymphocyte clone produces unique and specific immunoglobulins. Antibodies have two distinct functions: to bind specific Fig.1 Composition of the different types of monoclonal antibodies and the suffixes corresponding to their names. Fab Fc Human -umab Murine -momab Chimeric -ximab Humanized -zumab antigens and activate the immune system mediators, including complement and effector cells. Antibodies are proteins containing 4 polypeptides with molecular weights between kda. Polypeptide chains contain two identical heavy chains (α, δ, ε, γ, μ) and two identical light chains (λ, κ) that bind and form heterodimers through disulphide bonds, thus composing a three-dimensional y-shaped protein (Fig. 2). The amino groups of each light and heavy chain contain variable area that distinguishes the antibodies in terms of amino acid sequence. The unique specificity of the antibodies depends on the amino acid sequence of CDR area. Together, the CDR areas of light and heavy chains form the antigen-binding site of the antibody. The rest of the antibody molecule, known as constant, shows relatively small differences in amino acid sequence. After binding to the target, the fragment crystallizable regions (Fc) of the antibody can summon the so-called effector cells, such as killer cells, macrophages or neutrophils and/or activate the complement system to destroy the target-related cells. These characteristics refer to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and thus are fundamental aspects of the natural biology of the antibodies used for creating drugs with a very powerful biological activity 4. In addition to the ADCC and CDC, the Fc region is responsible for the long half-life of the molecule, because of the interaction with the FcRn receptors. Finally, the Fc domain interacts with certain bacterial proteins such as the A/G protein, which is a strong stimulus for the organism to produce protective molecules. Like many other mammalian proteins, the antibodies are glycoproteins. Glycosylation plays an important role in the biological activity of antibodies and manipulation of this process is used to improve the pharmaceutical properties of the molecule. Genetic manipulation of the antibodies Fc region is also used for improving the serum half-life, ADCC and CDC activity of the molecule 5. 10

3 Fig.2 Schematic presentation of the IgG antibody structure lots is also a problem. Furthermore, the initial material of human origin can be a source of various infections. The effectiveness of monoclonal antibodies depends on: functions and characteristics of target antigens; density of the cell surface or tissue distribution of the antigen; factors, associated with monoclonal antibodies - specificity and isotype. Monoclonal antibodies are administered mainly as biological treatment in oncology and non-cancer diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing arthritis, Crohn s disease, psoriasis, asthma, multiple sclerosis, graft rejection in transplantation, macular edema and degeneration. Based on their mechanism of action, monoclonal antibodies can be divided into three main classes (Fig. 3). The first class of monoclonal antibodies are acting directly on the target antigen. These monoclonal antibodies block or stimulate specific cell membrane molecules (e.g. signaling growth factor receptors) or ligands (vascular endothelial growth factor-vegf), thereby inhibiting tumor growth or activate effector cells. The second class of monoclonal antibodies are cytotoxic by supporting cytotoxic molecules - immunoconjugates. These monoclonal antibodies are conjugates of various cytotoxic molecules incl. chemotherapeutic agents or radioisotopes, some cellular toxins such as diphtheria toxin or biological agents like interferons. The third class of monoclonal antibodies modulate the immunological mechanisms. In these cases, the antibodies exhibit their cytotoxic effects by ADCC-autoimmune dependent complement cytotoxicity or CDC-complement dependent cytotoxicity. These mono clonal antibodies may also have nonimmunological mechanisms of action, including induction of target cell apoptosis. 7,8 In addition to the reactions towards the antibodies, the variability between the different production Target-specific mechanisms Antigen-antibody reaction neutralizes circulating targets or receptors on the cell surface, depending on the epitope against which the antibody is targeted. Antibody binding to the receptors may prevent activation by natural ligands and it can actually trigger the receptors. Epitopes for antibody binding are very critical, since some tumors may alter the surface proteins by modification and thus prevent the binding and recognition of normal and unmodified antigens. 9,10 Trastusumab is a humanized IgG1 monoclonal antibody with targeted mechanism of action against the HER-2 receptors, which are overexpressed in some breast cancers. The binding of antibody-antigen reaction leads to destruction of the HER-2, which is a sign of block cell cycle progression in G1 phase, leading to inhibition of tumor growth. This antibody is effective in breast cancers with overexpression of HER-2 in addition to the standard chemotherapy. Cetuximab is a chimeric IgG1κ monoclonal antibody against the ligand binding site of EGFR and inhibits competitively the binding of EGF, blocks the binding 11

4 Monoclonal Antibodies Fig. 3 Antibody and target-cell interaction I Naked MAb ADCC CDC Radionuclide Redioimmunoconjugate Cytokine Immunocytokine Biotinylated radioactive ligand Tumor cell Streptavidin 3 Multistep targeting Bispecific MAb Killer cell Cellular immunoconjugates Immunotoxin Liposome scfv-enzyme scfv 2 ADEPT Immunoliposome Prodrug Drug Immunoconjugates of endogenous EGFR ligands, thereby inhibiting the function of the receptor. Subsequently, cetuximab induces absorption of EGFR, which can lead to reduced receptor activity of EGFR. Cetuximab targets the cytotoxic immune effector cells against EGFR-expressing tumor cells (antibodydependent cell-mediated cytotoxicity, ADCC). Cetuximab does not bind to other receptors, belonging to the HER group. The product of the protooncogene KRAS (Kirsten rat sarcoma 2 viral oncogene homologue) is a protein, serving as central descending signal transducer of EGFR. In tumors, the KRAS activation by the EGFR, contributes to EGFR-mediated enhanced proliferation, survival and production of proangiogenic factors. KRAS is one of the most frequently activated oncogenes in human cancers. Both in vitro and in in vivo studies, show that cetuximab inhibits proliferation and induces apoptosis of human tumor cells expressing the EGFR. In vitro, cetuximab inhibits the formation of angiogenic factors by the tumor cells and blocks endothelial cell migration. Panitumumab is a recombinant, fully human IgG2 monoclonal antibody that binds with high affinity and specificity to the human EGFR. Panitumumab binds to the ligand binding domain of EGFR and inhibits the receptor autophosphorylation, induced by all known EGFR ligands. Binding of panitumumab to the EGFR, leads to internalization of the receptor, inhibition of cell growth, induction of apoptosis, reduction of interleukin 8 and the production of vascular endothelial growth factor. The KRAS (Kirsten rat sarcoma 2 viral oncogene homologue) gene encodes a small GTPbinding protein, involved in signal transduction. A number of stimuli, including those of EGFR, activate KRAS, which alternately stimulates other intracellular proteins to increase cell proliferation, cell survival and angiogenesis. The pharmacodynamic effects in in vitro and in vivo studies in animals have shown that panitumumab inhibits the growth and survival of tumor cells that express EGFR. There is no established anti-tumor effect of panitumumab on human tumor xenografts, which lack the expression of EGFR. In experimental animal studies, adding of panitumumab to radiotherapy, chemotherapy or other targeted therapeutic agents, leads to increased anti-tumor effect, compared with 12

5 solitary radiotherapy, chemotherapy or therapeutic agents for targeted therapy. Immune-mediated specific mechanisms Clinical efficacy of certain monoclonal antibodies is achieved by immune-mediated effector mechanisms, ADCC and CDC. 11 Rituximab is a murine/human anti-cd20 IgG1k mono clonal antibody, which target site is CD20, expressed on the surface of > 90% of malignant and normal B lymphocytes. CD20 is expressed with low-density on the cells of the chronic lymphocytic leukemia. This antigen is not expressed on stem cells, does not circulate in the plasma and does not change on the cell surface after binding. 12 The mechanism of action is through ADCC, complement-mediated cytotoxicity, growth inhibition, impairment of cell cycle, and apoptosis by direct binding to CD20. In vitro data show that it can sensitize lymphomas towards the action of cyclophosphamide, doxorubicin, vincristine and prednisone. Rituximab is well tolerated and can be applied to most patients regardless of age and stage of disease. Adverse effects are mainly related to the route of administration in the form of infusion. 13 Alemtuzumab was obtained by the methods of genetic engineering and is a humanized IgG1 kappa monoclonal antibody, specific for the kd lymphocyte surface glycoprotein (CD52), expressed primarily on the surface of normal and malignant peripheral blood B and T lymphoid cells. Alemtuzumab is obtained by adding six complement-determining regions of murine IgG2a monoclonal antibody in human IgG1 immunoglobulin molecule. Alemtuzumab causes lysis of lymphocytes by binding to CD52, a highly expressed, unmodulated antigen, which is located on the surface of virtually all B and T lymphoid cells, as well as on monocytes, macrophages and thymocytes. The antibody mediates the lysis of lymphocytes via 13

6 Monoclonal Antibodies complement fixation and antibody-dependent cellmediated cytotoxicity. The antigen is located in a small percentage (<5%) on granulocytes, but not on erythrocytes and platelets. Conjugated monoclonal antibodies The efforts to increase the cytotoxic activity of monoclonal antibodies with subsequent therapeutic efficacy are focused on conjugates using highly toxic substances, including radionuclides and cytotoxic agents. These conjugates deliver toxic substances to the tumor site 14, 15 that normal tissues do not detain. [90Y]-radiolabelled ibritumomab tiuxetan binds specifically to CD20 expressing B-cells, including malignant cells. The isotope yttrium-90 is a pure ß-emitter with an average length of about 5 mm. It kills both the target and neighboring cells. The conjugated antibody has an apparent affinity, which is constant for the SD20 antigen of approximately 17 nm. The binding pattern is very restricted with no cross-reactivity with other leukocytes or other types of human tissue. Initial treatment with rituximab is necessary to clear the circulating B-cells, thus enabling ibritumomab tiuxetan [90Y] to deliver radiation more specifically on the lymphoma B-cells. The use of monoclonal antibodies for the treatment of non-oncology diseases is based on several mechanisms of action: - suppression of tumor necrosis factor TNF - infliximab, adalimumab; - inhibition of lymphocyte trafficking - the mi- 14

7 gration of activated T-cells in the brain and spinal cord as well as the antigens activated on their surface, the so called integrins, are considered to have a leading role in the pathogenesis of multiple sclerosis - natalizumab is a monoclonal antibody directed against these integrins and its application increases the number of remissions. A significant problem with this therapy is its serious adverse effect - multiple leukoencephalopathy, an opportunistic degenerative infection, affecting the CNS and leading to death. - Immunoglobulin E, which plays a crucial role in allergic response and in the creation of anti-ige antibodies like omalizumab, is successfully used in the treatment of immuno-allergic diseases such as asthma and eczema. The use of monoclonal antibodies as a therapy for various oncology and non-oncology conditions is limited by: high price; their parenteral route of administration, requiring compulsory hospitalization and observation; serious adverse reactions; some limitations in current concepts of pathogenesis of the diseases. The monoclonal antibodies represent significant advances in clinical medicine, but they are a reserve that should be used only when there are no satisfactory 16, 17 therapeutic results with conventional therapy. 15

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9 Bibliography: 1. Terziivanov D. 100 Years Later - Has Paul Ehrlich s Magic Bullet Become Reality? J Clin Med. 2010; 3(2): Reichert JM. Current Pharmaceutical Biotechnology. 2008: 9: Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody-based cancer immunotherapy. Nature. 1975; 256: Christiansen J., Rajasekaren AK. Biological impediments to monoclonal antibody-based immunotherapy. Mol Cancer Ther. 2004; 3: Janeway CA: Immunobiology, the immune system in health and disease 6th edn. New York: Garland Science Publishing, 2005: Sharkey RM, Goldenberg DM. Targeted therapy of cancer: New prospects for antibodies and immunoconjugates CA Cancer J Clin. 2006; 56: Newsome B. and M. Ernstoff. The clinical pharmacology of therapeutic monoclonal antibodies in the treatment of malignancy; have the magic bullets arrived? Br J Clin Pharmacol. 2008, 66, 1: Strome S. et al. The Oncologist. 2007, 12: Oldham R. et al. Monoclonal antibodies in cancer therapy: 25 years of progress. Journal of Clinical Oncology. 2008, 26, 11: Li Y. et al. Summary of the primer on tumor immunology and the biological therapy of cancer. Journal of Translational Medicine. 2009, 7: Galon J. et al. Type, density and location of immune cells within human colorectal cancer tumors predict clinical outcomes. Science. 2006, 313: Golay J. et al. The role of complement in the therapeutic activity of rituximab in a murine B lymphoma model homing in lymph nodes. Haematologica. 2006, 91: Weng W and Levy R. Expression of complement inhibition CD46, CD55 and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-hodgkin lymphoma. Blood 2001, 98: Wu AM. Et al. Arming antibodies. Prospects and challenges for immunoconjugates. Nat. Biotechnol. 2005, 23: Nowakowski GS, Witzig TE. Radio immunotherapy for B cell non-hodgkin lymphoma. Clin Adv Hematol Oncol. 2006, 4: Weinberg WC et al. development and regulation of monoclonal antibody products: Challenges and opportunities. Cancer and metastasis reviews. 2005, 24: European Medicine Agency. European Public Assessment Reports (EMEA, London) 17