Future Prospects for the Use of Monoclonal Antibodies - Research Paper Example

Introduction Antibodies (also known as immunoglobulin) are glycoproteins secreted by specialized B lymphocytes (plasma cells). Antibodies areone of the main effectors of the immune system. These host proteins, which are found in the plasma and extracellular fluids, are the first response to foreign molecules and organisms (Lipman et al, 2005) History of antibodies It was Behring and Kitasato, in 1890, who demonstrated that it is possible to neutralize even a fatal dose of the diphtheria toxin in an animal from the serum obtained from an animal actively immunized against the same. They were able to demonstrate the same results with tetanus toxin. Later, Behring suggested that active immunity could be produced in humans by a combination of diphtheria toxin and antitoxin serum (Llewelyn, Hawkins, Russell, 1992). However, it was Paul Ehrlich whose work made it possible to produce antiserum to diphtheria toxin in a mass scale. The side chain theory of toxicity was proposed by Ehrlich, according to which, “toxins mediated their effects on cells through preformed protein side chains and immunity arose because of overproduction of these side chains” (Llewelyn, Hawkins, Russell, 1992.) César Milstein and Georges Köhler, in 1975, managed to produce in vitro "custom-built" antibodies. They produced a hybridoma by fusing rodent antibody producing cells with tumor cells from mice bone marrow. A hybridoma, provided with the correct nutrients, can grow indefinitely and divide to produce monoclonal antibodies (antibodies of a single type), on a large scale (Saldanha, 2000.) Types of antibody, their structure and effects Five classes of immunoglobulin (Ig) are found in mammals: IgA, IgD, IgE, IgG, and IgM. In some select mammals, IgG and IgA are further subdivided into subclasses (isotypes) due to polymorphisms in the heavy chain (Lipman et al, 2005). The prototype antibody is IgG, which is a glycoprotein having a molecular weight of 150000 Dalton. The molecule has a Y shaped structure, consisting of two identical heavy chain-light chain heterodimers, that is linked together by a disulphide bridge. While the heavy chain comprises three constant immunoglobulin (Ig) domains and one variable (V) domain, the light chains consist of a single constant Ig domain and a single variable domain. The host defense mechanism is initiated by the antigen binding site (Fab) and the Fc region (fragment crystallisable) site (Llewelyn, Hawkins, Russell, 1992.) In order to effectively bind a wide range of antigens, there are numerous mechanisms which come into play. Some of these mechanisms include: production of the antibody’s binding site by the combination of different heavy and light chains, a high somatic mutation rate, genetic recombination within hypervariable regions, and imprecise joining during recombination. Due to these various mechanisms, it is possible to produce a wide range of coding regions and transcription of unique complementarity determining regions (CDRs) (Lipman et al, 2005.) A region known as the hinge connects the two arms (Fab) of the antibody molecule containing the antigen-binding domains and the tail (Fc1) or crystallisable fraction. This area is rich in proline, threonine, and serine, and gives a lateral and rotational movement to the antigen-binding domains. By the means of this, the antibody is able to interact with a variety of antigen presentations (Lipman et al, 2005.) The phenomenon of antigen interaction is crucial to the antibody’s primary defense function and also to its use as a research or therapeutic reagent. The T and/or B cells mediate the specificity of the antibody response. This is achieved by means of membrane-associated receptors that bind antigen of a single specificity (Lipman et al, 2005.) After the specific antigens binds to the antibody and after obtaining the appropriate activating signals, the B lymphocytes starts to divide. This produces memory B cells. In addition, the B lymphocytes terminally differentiate into antibody secreting plasma cell clones. Each of these plasma cell clones produce antibodies that recognize the identical antigenic epitope. These memory B lymphocytes lie in an inactive state till they get later activated by their specific antigen. Thus, the memory B lymphocytes are the basis for the cellular basis of memory, which results in a rapid elevation of antibody response when re-exposed to a specific antigen (Lipman et al, 2005) Even though an antibody may be able to directly neutralize a virus or toxin, the Fc mediated defense system is required in order to eliminate the antigen source. By coating (opsonising) the cell with IgG, antibodies sensitize the killer (K) lymphocytes and phagocytes to attack a target cell. Since the surface of the cells has receptors for the Fc region of IgG, they are able to recognize the coated target cells. This is known as antibody dependent cellular cytotoxicity (ADCC). The classical complement pathway is activated by the antigen-antibody combination. When the complement is deposited on the target cell, lysis or further opsonisation is produced. About 70% of the immunoglobulin pool is made up of IgG, which in humans has four IgG subclasses (IgG 1-4) with variable initiation of host defense. Antibody dependent cellular toxicity and complement binding is mediated by IgGl and IgG3, while these functions are relatively deficient in IgG2 and IgG4. About 10% of the immunoglobulin pool is made up of IgM, which is a complement fixing antibody. About 20% of the immunoglobulin pool is made up of IgA (secretory immunoglobulin). IgA is found in secretions like saliva and milk. Less than 1% of the immunoglobulin pool is made up of IgD, which is found on the surface of B cells. The function of IgD is not known. IgE is involved in causing immediate hypersensitivity reactions. This occurs when IgE binds to Fc receptors on the surface of basophils, eosinophils, and mast cells, causing the release of pharmacological mediators. (Llewelyn, Hawkins, Russell, 1992.) Difference between polyclonal and monoclonal antibodies Monoclonal antibodies (MAbs) are antibodies produced by a single B lymphocyte clone, while polyclonal antibodies (PAbs) are produced when each lymphocyte is activated to proliferate and differentiate into plasma cells. The advantages of MAbs are homogeneity and consistency. The evaluation of changes in molecular conformation, protein-protein interactions, phosphorylation states, and identification of single members of protein families is possible because of the monospecificity of MAbs. MAbs allows structural analysis (x-ray crystallography or gene sequencing) to be determined for the antibody on a molecular level. It is also possible to generate MAbs constantly, once the required hybridoma has been generated. In contrast to MAbs, PAbs can be generated much more rapidly, at a lesser expense, and with lesser technical skill; they are more stable over a broad pH and salt concentration. When compared to PAbs, MAbs have better specificity (Lipman et al, 2005.) Production of monoclonals Hybridoma Köhler and Milstein in 1975 produced a hybridoma by a technique known as somatic cell hybridization. They did this by fusing myeloma cells with antibody-secreting cells from an immunized mouse (Users.rcn.com, 2009.) The procedure first involves the mixing of spleen cells of a mouse (immunized with the required antigen) with myeloma cells. In order to facilitate the success of the procedure, either myeloma cells, which have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or myeloma cells that have lost the ability to synthesize any antibody molecules of their own, are used. Successfully produced hybridoma cells are able to grow in an indefinite fashion, since the spleen cell partner supplies HGPRT and the myeloma partner is immortal (Zola, 1987; Users.rcn.com, 2009.) After this, the supernatants from each culture are tested to find those that produce the required antibody. Single cells from each antibody-positive culture are isolated and then subcultured. Each supernatant is again tested for the required antibodies. Each positive subculture (having been started from a single cell) represents a clone and its antibodies are monoclonal. The size of the cultures of the successful clones is then scaled up. Hybridoma cultures can be maintained indefinitely either in vitro (in culture vessels) or in vivo (in mice) and later harvested (Zola, 1987; Users.rcn.com, 2009.) Human monoclonals (HmAbs) can be produced by different methods like immortalization of B cells with Epstein–Barr virus, humanization of antibodies from other species, using phage display libraries or generating antibodies recombinantly from isolated single B cells (Smith et al., 2009.) Hybridomas, has many disadvantages like possible instability of the anuploid cell lines, as well as its inability to produce human antibodies and to provide antibodies against toxic or highly conserved antigens (Zhiqiang An, 2009.) Recombinant gene technologies can be used to generate antibodies or antibody fragments and overcome the limitations of hybridomas. The most commonly used recombinant antibody fragments are the Fragment antigen binding (Fab) and the single chain Fragment variable (scFv). The Fab fragment consists of the fd fragment of the heavy chain and the light chain linked by a disulfide bond. In the scFv, the variable region of the heavy chain (VH) and the variable region of the light chain (VL) are connected by a short peptide linker (Zhiqiang An, 2009.) To avoid the instability of hybridoma cell lines, the genes encoding VH and VL of a monoclonal antibody can be cloned into an E.coli expression vector in order to produce antibody fragments in the periplasmatic space of E.coli that preserves the binding specificity of the parental hybridoma antibody. Repeated administration of mouse-derived antibodies causes a human anti-mouse antibody (HAMA) response. This can be overcome by 2 approaches: humanization of mouse antibodies or employing repertoires of human antibody genes. Phage display has become the most widely used and most robust selection method. Phage display is based on peptide display on the surface of filamentous phage. By combining the largest possible human gene libraries with a selection based on single molecule interactions, phage display has allowed antibodies to be generated that could not be obtained from animals. It is currently being adapted for the high throughput generation of binders to the human proteome and in particular, delivers high affinity human antibodies for medical use (Zhiqiang An, 2009.) Uses of monoclonal antibodies I. Immunosuppressive agents Hemolytic disease of the newborn due to rhesus incompatability between the mother (rhesus D negative) and a rhesus D-positive fetus is prevented by the administration of anti-D antibodies to ‘at-risk’ mothers immediately after delivery. These antibodies destroy any rhesus-positive fetal red cells, thus preventing sensitization of the mother’s immune system. Numerous monoclonal antibodies have been developed with the aim of interrupting interaction between antigen-presenting cells, T cells and B cells (Chapel et al., 2006). A major concern of using rodent monoclonal antibodies is the potential for triggering reactions after repeated use, with loss of efficacy due to antibodies to the species part of the therapeutic antibody. Production of human monoclonal antibodies, by transforming B cells with EBV or fusing antibody-producing cells with human cell lines, may overcome this problem. An alternative approach has been to ‘humanize’ mouse monoclonal antibodies genetically by transposing their antigen-binding sites (hypervariable regions) onto a human antibody framework. This will retain the complete range of effector properties of human Fc regions while minimizing the immunogenicity of the mouse component. A good example of targeted immunotherapy is the use of anti-tumor necrosis factor (anti TNF) as a therapeutic agent. TNF blockade can be achieved by either the use of a chimeric anti-TNF antibody (infliximab, adalimumab) or a soluble TNF receptor (etanercept) (Chapel et al., 2006). Some antibodies, which are being used as immunosuppressive agents include: anti-lymphocyte globulin, anti-CD52, anti-CD3, anti-CD4, anti-TNF, CTLA4-Ig, anti-CD40L, anti-CD 20 and anti-CD49d-CD29 (Chapel et al., 2006). Immunomodulation-immunoglobulin replacement is essential for patients with primary antibody deficiency and of proven value in several forms of secondary hypogammaglobulinaemia, especially infants with HIV infection and patients with lymphoproliferative malignancy. Intravenous immunoglobulin (IVIG) has benefits in autoimmune disease. One mechanism of particular interest in autoimmune disease is the role of FcRn, the MHC class-I related Fc receptor for IgG, which protects IgG from lysosomal degradation. Blockade of FcRn by high-dose exogenous IgG is likely to result in accelerated catabolism of endogenous pathogenic IgG with consequent clinical improvement. In addition to monomeric IgG, IVIG contains several other immunologically active constituents including antibodies to cytokines, anti-idiotypic antibodies, soluble CD4, soluble CD8 and HLA molecules, which may contribute to its immunomodulatory effects at high doses (Chapel et al., 2006). Some autoimmune diseases, which have been treated with IVIG include: Kawasaki’s disease, dermatomyositis, Guillain-Barre syndrome, Lambert-Eaton myasthenic syndrome etc (Chapel et al., 2006). Omalizumab is effective in allergic asthma, since it binds to IgE and thus prevents IgE from binding to mast cells. Daclizumab prevents acute rejection of transplanted kidneys by binding to part of the IL-2 receptor produced at the surface of activated T cells (Users.rcn.com, 2009) II. Angiogenesis Inhibitors Abciximab is useful to prevent reclogging of the coronary arteries in patients who have undergone angioplasty. It does this by inhibiting the clumping of platelets by binding the receptors on their surface, normally linked by fibrinogen (Users.rcn.com, 2009) III. Anti-tumor agents Monoclonal antibodies have an anti-tumor effect. This is achieved by combining the monoclonal antibody to a cytotoxic drug (e.g. methotrexate), a toxin (ricin) or a radioisotope (iodine-131). Thus, the monoclonal antibody can specifically target and kill tumor cells effectively. In addition, imunolocalization of tumor deposits, staging of malignant disease and determining the whole body distribution of amyloid deposits is possible with radiolabelled antibodies. Autologous bone marrow grafting requires removal of bone marrow from the patient prior to supralethal therapy (Chapel et al, 2006). Graft-versus-host disease is avoided but if tumor cells have already metastasized to the bone marrow, they are returned to the patient in a viable form. In order to purge the bone marrow of tumor cells monantibodies can kill targeted cells by subsequent addition of complement. Alternatively, antibody can be linked to toxins like ricin. The cells can also be physically trapped using monoclonal antibodies attached to magnetic beads and removed with cobalt magnets (Chapel et al, 2006.) Various mechanisms are attributed to the therapeutic effect achieved by monoclonal antibodies. The direct effects include the cause of programmed cell death or apoptosis. In addition, the other effects include the blocking of growth factor receptors, which stops tumor cell proliferation. An anti-idiotype antibody formation can be brought about in those cells that express monoclonal antibodies. Some of the indirect effects include antibody-dependent cell mediated cytotoxicity (ADCC). This is a type of antibody-mediated cell kill in which cells having cytotoxicity, such as monocytes and macrophages are recruited by monoclonal antibodies. Complement dependent cytotoxicity (CDC) is a term applied to the condition where monoclonal antibodies bind to complement. This leads to direct cell toxicity (Reff, Hariharan, Braslawsky, 2002.) In the treatment of hematologic malignancies three main classes of therapeutic MAbs have shown their usefulness: a. unconjugated MAb, in which the MAb either directly induces negative growth signal or apoptosis or indirectly activates host defense mechanisms to mediate antitumor activity. b. drug conjugates in which the antibody preferentially delivers a potent cytotoxic drug to the tumor, thus decreasing the systemic toxicity normally associated with conventional drug therapy, and c. radioactive immunotherapy in which the antibody delivers a sterilizing dose of radiation to the tumor (Reff, Hariharan, Braslawsky, 2002). In the treatment of B cell malignancies, there are a number of antigens and corresponding monoclonal antibodies. Some of the most active are: 1. Antigen-CD20; antibody- Rituximab, Tositumomab, Ibritumomab 2. Antigen-CD52; antibody-Alemtuzumab 3. Antigen-CD22; antibody-Epratuzumab Rituximab (IDEC-C2B8) is a chimeric antibody, which targets the CD20 antigen. CD20 has an important functional role in B cell activation, proliferation, and differentiation. “A chimeric antibody is one in which the variable domains that contain the antigen-binding sites are from the species used for immunization, and the constant domains of the protein chains are derived from human isotypes” (Reff, Hariharan, Braslawsky, 2002). Rituximab was one of the first monoclonal antibody to obtain FDA approval. This compound is useful in the treatment of low-grade lymphomas, which is refractory to conventional chemotherapy. In addition, in patients with intermediate grade or diffuse large cell non-Hodgkin lymphoma, rituximab has been used, combined with conventional chemotherapy (Reff, Hariharan, Braslawsky, 2002). In the treatment of solid tumors, two main monoclonal antibodies have been used; these are edrecolomab and trastuzumab. Edrecolomab, which is used in colon and rectal cancer, targets the 17-1A antigen. Trastuzumab, which is used in metastatic breast cancer as a first-line monotherapy, has shown good efficacy and safety (Bishop, n.d) Top 10 monoclonal drugs 1. Enbrel (Etanercept). Companies: Amgen, Wyeth, Takeda. Indications: rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis (Martino, M, 2009.) 2. Remicade (Infliximab). Companies: J&J, Schering Plough, Mitsubishi Tanabe. Indications: plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, adult Crohns disease, pediatric Crohns disease, ulcerative colitis, ankylosing spondylitis (Martino, M, 2009.) 3. Rituxan (Rituximab). Company: Roche. Indications: non-Hodgkins lymphoma and rheumatoid arthritis (Martino, M, 2009.) 4. Avastin (Bevacizumab). Company: Roche. Indications: metastatic colorectal cancer, non-squamous non-small cell lung cancer, metastatic breast cancer, glioblastoma, metastatic renal cell carcinoma (Martino, M, 2009.) 5. Herceptin (Trastuzumab). Company: Roche. Indications: breast cancer 6. Humira (Adalimumab). Company: Abbott. Indications: rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, chronic plaque psoriasis, ankylosing spondylitis, Crohn’s disease (Martino, M, 2009.) 7. Lovenox (Enoxaparin). Company: Sanofi Aventis. Indications: DVT blood clots 8. Lantus (Insulin). Company: Sanofi Aventis. Indications: diabetes (Martino, M, 2009.) 9. Aranesp (Darbepoetin). Company: Amgen. Indications: anemia (Martino, M, 2009.) 10. Gardasil. Company: Merck. Indications: HPV and cervical cancer prevention (Martino, M, 2009.) Future uses of Monoclonal Antibodies Human monoclonals have the advantage of being truly non-immunogneic in patients. This means that they can be used repeatedly for imaging and therapy by using the same antibody labeled with gamma or beta emitters. They can be used to evaluate the effects on tumors of other therapeutic agents or biological response modifiers, with single patients serving as their own control. It will allow multiple administrations of immune conjugates. With a human monoclonal, the antibody labeled with a scanning isotope can be administered and in vivo dose deposition determined. The patient can then be treated or injected with a human immunoconjugate. At a future time, the patient can be again scanned to determine optimal timing for multi-agent administration (Goldenberg, 1995.) The future may see the production of the ‘hybrid hybridoma.’ The secreted ‘hybrid’ MAb (with its two distinct paratopes) will have a multitude of uses, either alone, or linked to a drug or leukokine-carrying vehicle such as the liposome (McCullough & Spier, 1990.) Over the past few decades, there has been an ever increasing use of antimicrobial drugs, globally. As a result of this, MRSA and many other bacteria have developed resistance to most commonly used antibiotics, thus reducing their efficiency. Some drug companies (Sanofi-aventis and Alopexx Pharmaceuticals) are in the process of applying for a license on a first-in-class human monoclonal antibody for the prevention and treatment of S. aureus, S.epidermidis, E. coli, Y. pestis. What they are developing is a human monoclonal antibody, which has the potential to become an alternative to antibiotics against MRSA and other infections. Monoclonal antibodies, unlike antibiotics, will not result in the development of bacterial resistance. “The target of the antibody is a carbohydrate on the bacterial capsule known as PNAG. PNAG has been found to be a critical factor in the virulence and immune response to staphylococcal infections. S. aureus strains that cannot produce PNAG have a significantly reduced ability to cause infections. The antibody is directed against PNAG and works by inducing killing by the patients own white blood cells” (Fiercebiotech, 2009.) Another new technology, which will be used in the future is antibody-drug conjugate (ADC) technology. ADCs are monoclonal antibodies, which carries potent, cell-killing drugs. Developed by Seattle Genetics, this technology uses antibodies attached to synthetic drugs by linker systems. These linker systems are designed in such a way that they remain stable while passing through the bloodstream, and once inside the target cells, release the required drug. As a result, nearby normal cells are spared and it thus, does not cause any unwanted toxic effects of chemotherapy (Seattle Genetics, n.d.) Conclusion Antibodies or immunoglobulin are specialized B lymphocytes (plasma cells), which are found in the plasma and extracellular fluids. Antibodies are one of the main effectors of the immune system and are the first response to foreign molecules and organisms. After Behring and Kitasato’s work in 1890 with diphtheria and tetanus toxin, it was Paul Ehrlich’s work, which enabled to produce antiserum to diphtheria toxin in a mass scale. He also proposed the side chain theory of toxicity. César Milstein and Georges Köhler, in 1975 produced a hybridoma, capable of indefinitely growing and dividing to produce monoclonal antibodies. In mammals, five classes of immunoglobulin (Ig) are found: IgA, IgD, IgE, IgG, and IgM. The prototype antibody is IgG. Monoclonal antibodies (MAbs) are antibodies produced by a single B lymphocyte clone, while polyclonal antibodies (PAbs) are produced when each lymphocyte is activated to proliferate and differentiate into plasma cells. Monoclonal antibodies have several applications including their use as immunosuppressive agents, antitumor agents, and as angiogenesis inhibitors. The future applications and developments include their use to evaluate the effects on tumors of other therapeutic agents or biological response modifiers, development of a ‘hybrid hybridoma’, development of a human monoclonal antibody, which has the potential to become an alternative to antibiotics against MRSA and other infections, and ADC technology. References Bishop, MR, n.d. Monoclonal antibodies. [Online] Available at: http://www.meds.com/immunotherapy/monoclonal_antibodies.html [Accessed 16 November 2009]. Chapel, H, Haeney, M, Misbah, S, & Snowden, N., 2006. Essentials of clinical Imunology. Blackwell Publishing. Fiercebiotech, 2009. Sanofi-Aventis And U.S Biotechnology Company Alopexx Enter Into A Collaboration Agreement For A Novel Human Monoclonal Antibody. [Online] Available at: http://www.fiercebiotech.com/press-releases [Accessed 20 January 2010] Goldenberg, DM, 1995. Cancer therapy with radiolabeled antibodies. CRC press. Llewelyn, MB, Hawkins, RE, Russell, SJ, 1992. MonoclonalAntibodies in Medicine. BMJ. 305. Lipman, NS, Jackson, LR, Trudel, LJ, Weis-Garcia, F, 2005. Monoclonal Versus McCullough, KC, Spier, RE, 1990. Monoclonal antibodies in biology and biotechnology: theoretical and practical approaches. University Press. Martino, M, 2009. Top 10 Blockbuster Biotech Drugs. [Online] Available at: http://www.fiercebiotech.com/slideshows/top-10-blockbuster-biotech-drugs [Accessed 20 January 2010] Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR Journal: 46(3). Reff, ME, Hariharan, K, Braslawsky, G, 2002. Future of Monoclonal Antibodies in the Treatment of Hematologic Malignancies. Cancer Control: Journal of the Moffitt Cancer Center Saldanha, J, 2000. Historical Perspective to Humanized Antibodies. [Online] Available at: http://people.cryst.bbk.ac.uk/~ubcg07s/History.html [Accessed 16 November 2009]. Seattle Genetics, n.d. Seattle Genetics Announces Antibody-Drug Conjugate Collaboration with GlaxoSmithKline. [Online] Available at: http://phx.corporate-ir.net/phoenix.zhtml?c=124860&p=irol-newsArticle&ID=1368209 [Accessed 20 January 2010] Smith, K, Garman, L, Wrammert, J, Zheng, N, Capra, JD, Ahmed, R, Wilson, PC, 2009. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc. 4(3): 372–384. Users.rcn.com, 2009. Monoclonal Antibodies. [Online] Available at: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html [Accessed 20 January 2010] Zola, H, 1987. Monoclonal antibodies: a manual of techniques. CRC press, Inc. Zhiqiang An, 2009. Therapeutic Monoclonal Antibodies: From Bench to Clinic. Wiley. Read More