Monoclonal antibody

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Monoclonal antibody

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Monoclonal antibody

  1. 1. Biotechnology IIMonoclonal antibody Southeast University Department of Pharmacy Assignment on: Monoclonal Antibody Submitted to: Abdullah Al Hasan Lecturer Department of Pharmacy Southeast University Submitted by: Md. Waliullah Wali ID# 2010000300031 Batch- 15th (B) Department of Pharmacy Southeast University Date of Submission: 25 December, 2013 1|Page
  2. 2. Biotechnology IIMonoclonal antibody Table of Contents Monoclonal antibody ........................................................................................................................ 3 History of MAb development ............................................................................................................ 4 The types of mAb designed ............................................................................................................... 4 Production of Monoclonal Antibodies .............................................................................................. 4 Advantages ........................................................................................................................................ 7 Disadvantages ................................................................................................................................... 7 Applications of Monoclonal Antibodies ............................................................................................ 7 Monoclonal antibodies for cancer treatment ............................................................................... 8 Monoclonal antibody therapy in rheumatoid arthritis ................................................................. 9 Pharmacology of some Monoclonal antibodies drugs ...................................................................14 Bevacizumab ................................................................................................................................14 Brand Name : Avastin ......................................................................................................................14 Rituximab .....................................................................................................................................19 Indication of Rituximab rheumatoid arthritis treatment ................................................................20 Abciximab .....................................................................................................................................21 Palivizumab ..................................................................................................................................22 Brand names: Synagis® .................................................................................................................22 Side effects of monoclonal antibodies ............................................................................................22 Side effects of licensed monoclonal antibodies..............................................................................23 References .......................................................................................................................................27 2|Page
  3. 3. Biotechnology IIMonoclonal antibody Monoclonal antibody Antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-shaped protein produced by B cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. “An antibody produced by a single clone of cells. Monoclonal antibody is therefore a single pure type of antibody” Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in amino acid sequence. It is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each monoclonal antibody recognizes a specific antigen. Figure 1: Structure of Monoclonal Antibody (Left) and their attachment with Antigen (right) Antibodies are secreted by a type of white blood cell called a plasma cell. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in amino acid sequence 3|Page
  4. 4. Biotechnology IIMonoclonal antibody History of MAb development 1890 Von Behring and kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the “ side-chain theory” 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended. Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment crystalline (Fc) from rabbit y-globulin. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells 1990 Milstein produced the first monoclonal antibodies. The types of mAb designed A. Murine source mAbs: rodent mAbs with excellent affinities and specificities generated using conventional hydrioma technology. Clinical efficacy compromised by HAMA(human anti murine antibody) response, which lead to allergic or immune complex hypersensitivities. B. Chimeric mAbs: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource) C. Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework Production of Monoclonal Antibodies Process by which bulk quantities of targeted antibodies against a specific antigen are produced.Monoclonal antibodies are produced via multiple/identical copies of a certain cell 4|Page
  5. 5. Biotechnology IIMonoclonal antibody called a hybridoma. To create Hybridoma cells the fusion of 2 cells are needed in order to combine the characteristics of the 2 cells into 1 cell. 1 of the cells is a producing cell antibody which is a B-Lymphocyte used from a laboratory mouse and the other is a tumor cell named myeloma. Tumor cells have the ability to grow indefinitely and at an exceeding rate from normal cell growth. Laboratory produced Hybridoma cells replicate much faster than normal antibody producing cells, and the individual hybridomas produce the specific antibodies for an indefinite period of time. Figure 2: Production of Monoclonal Antibodies Hybridoma cells manufacture the specific monoclonal antibody that was originally produced by the B-Lymphocyte cell. The original B-Lymphocyte cell will produce the Monoclonal antibody depending on the kind of antigen that was injected into the mouse just prior to the harvesting of the B-Lymphocyte cells. A small example is, if the mice were 5|Page
  6. 6. Biotechnology IIMonoclonal antibody injected with a certain virus, the mouse will have B-Lymphocytes that produce those specific virual antibodies. Fusion with a tumor cell to make the hybridoma, result in the production of monoclonal antibodies against the specific virus. The hybridoma cells are placed into media that can help them grow and produce the bulk quantities of monoclonal antibodies. There are 2 ways for growing monoclonal antibodies, 1 is to grow them in laboratory flasks meaning In Vitro, and the other is to grow them in the stomach lining of mice. Injecting the hybridomas into the mice is the familiar method of harvesting monoclonal antibodies. This method is done by mixing spleen cells from the mouse that has been immunized with the desired antigen with myeloma cells.The myeloma cells need to have lost their ability to synthesize HGPRT enzyme (hypoxanthine-guaninephosphoribosyltransferase) which enables the cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Cells have another pathway that they synthesize purines so lack of HGPRT is not a problem for the cell but when cells are exposed to aminopterin they are unable to use this other pathway and are fully dependent on HGPRT for their survival. So to summarize, unfused myeloma cells can’t grow since they lack HGPRT and unfused normal spleen cells can’t grow since they have a limited life-span. Hybridoma cells are grow indefinitely since the spleen-cell copartner supplies HGPRT and the myeloma partner is immortal. The first step is transferring of the cell fusion mixture to HAT culture medium which contains hypoxanthine, aminopterin& pyrimidine thymidine. The 2nd step is testing the supernatants from each culture in order to locate the producing the desired antibody. One must isolate the single cells from each antibody-positive culture and subculture them, this represents the clone which its antibodies are monoclonal. Every single cell culture secretes a specific kind of antibody that is directed against a certain determinant/selected antigen. The 3rd step is scaling up the size of the cultures of the successful clones. Hybridoma cultures can be grown indefinitely in vitro in culture vessels which yield 15-65 µg/ml and in vivo using mouse, where the antibody concentration in the serum 0.5-15 mg/ml. In the past years, animal welfare activists in worldwide are trying to limit the use of mice for the production of monoclonal antibodies. When the monoclonal antibody is produced it can be used as a probe to track down, bind to and purify the specific protein that induced its formation. 6|Page
  7. 7. Biotechnology IIMonoclonal antibody Advantages 1) Homogeneity: Monoclonal antibody represents a single antibody molecule that binds to antigens with the same affinity and promote the same effectors functions. 2) Specificity: The product of a single hybridoma reacts with the same epitope on antigens. 3) Immunizing Antigen: Need not be pure or characterized and is ultimately not needed to produce large quantities of antibody. 4) Selection: It is possible to select for specific epitope specificities and generate antibodies against a wider range of antigenic determinants. 5) Antibody Production: Unlimited quantities of a single well-defined monospecific reagent. Disadvantages 1) Affinity: Average affinity of monoclonal antibodies are generally lower than polyclonal antibodies. 2) Effector Functions: Because antibody is monoclonal, it may not produce the desired biologic response. 3) Specificity: Monoclonals against conformational epitopes on native proteins may lose reactivity with antigens. 4) Cross reactions: Antibodies sometimes display unexpected crossreactions with unrelated antigens. 5) Time and effort commitment: Very Large. Applications of Monoclonal Antibodies Applications of Monoclonal Antibodies Diagnostic ApplicationsBiosensors & Microarrays Therapeutic Applications Transplant rejection Cardiovascular disease Cancer Infectious Diseases Inflammatory disease Clinical Applications. Purification of drugs, imaging the target Future Applications Fight against Bioterrorism 7|Page
  8. 8. Biotechnology IIMonoclonal antibody Autoimmune diseases Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind to and inhibit TNF-α.[31] Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants. Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderateto-severe allergic asthma. Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. A. mAbs act directly when binding to a cancer specific antigen and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. B. mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active conjugates. C. it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell ADEPT- antibody directed enzyme prodrug therapy; ADCC- antibody dependent cell-mediated cytotoxicity; CDC- complement dependent cytotoxicity; MAb- monoclonal antibody; ScFv- single-chain Fv fragment Figure 3 : action of Monoclonal antibodies for cancer treatment 8|Page
  9. 9. Biotechnology IIMonoclonal antibody Strategy of a direct or in direct induction of apoptosis in targeted cancer cells 1. mAbs target growth factor receptors to exert a direct effect on the growth and survival of the cancer cells by antagonizing ligand-receptor signaling. 2. mAbs can target to cell surface antigens and directly elicit apoptotic signaling. Until Feb 28, 2005, 18 mAbs were approved by FDA, which were applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic malignancies and psoriasis. The first approved mAbs was OKT-3, which is a murine IgGa2 protein to deplete T cells in patients with acute rejection of renal allotransplant. Monoclonal antibody therapy in rheumatoid arthritis Rheumatoid arthritis (RA) is a systemic disease and the most prevalent of all autoimmune disorders. Here we review recent advances in the development and availability of biologic agents with a focus on monoclonal antibody or smaller formats of targeted engineered therapeutics including novel, non-antibody-based therapeutics. Monoclonal antibodies bind to their targets with high specificity and therefore have excellent potential as therapeutic agents. Biotechnological advances have allowed the production of large quantities of engineered monoclonal antibodies for therapeutic use. Recent research in rheumatoid arthritis has identified important mediators of synovitis. Monoclonal antibodies targeting these have been tested in clinical trials over the last decade. Anti-cytokine therapies, in particular anti-tumour necrosis factor alpha monoclonal antibodies, suppressed inflammation and produced rapid symptomatic improvement. Antilymphocyte monoclonal antibodies produced long-lasting disease suppression in animal models of rheumatoid arthritis. The use of depleting anti-lymphocyte monoclonal antibodies in rheumatoid arthritis had been disappointing as they did not penetrate the synovial joint in sufficient quantity to suppress disease without producing severe and protracted peripheral blood lymphopenia. Consequently, their use in rheumatoid arthritis had been abandoned. In contrast, clinical trials of non-depleting anti-CD4 monoclonal 9|Page
  10. 10. Biotechnology IIMonoclonal antibody antibodies in rheumatoid arthritis showed that they could suppress synovitis. However, it remains unclear whether they could lead to prolonged disease improvement. Although current therapies can reduce the signs and symptoms of RA for many patients, the quest for a cure (or a more complete blockade of the structural damage) in RA is still ongoing and will need treatment approaches, which are not exclusively confined to blocking a particular cytokine, receptor, or autoreactive B or T cell involved in disease progression. To this end exciting treatment alternatives and drug targets are on the horizon that may become available to patients in the future. The human monoclonal antibodies adalimumab and golimumab; infliximab (a chimeric IgG1 monoclonal antibody); etanercept (a TNF receptor–Fc fusion protein); and certolizumabpegol (a pegylated humanized antibody Fab' fragment). Agents with other targets are the IL-1R antagonist anakinra; abatacept (a CTLA-4–Fc fusion protein), which modulates the T-cell co-stimulatory pathway; the humanized antibody tocilizumab, which binds IL-6R; and rituximab, a chimeric anti-CD20 monoclonal antibody that depletes B-cell populations. Abbreviation: CTLA-4, cytotoxic T lymphocyte antigen 4. 10 | P a g e
  11. 11. Biotechnology IIMonoclonal antibody Figure 4: Summary of biologic therapies used in the treatment of rheumatoid arthritis Evolution of monoclonal anybody 11 | P a g e Figure 5 : Generation of TNA antibodies
  12. 12. Biotechnology IIMonoclonal antibody Chimeric mAbs: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource) Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework Recombinant monoclonal antibodies: Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. Phage display library: construction of VH and VL gene libraries and expression of them on a filamentous bacteriophage. The phage expressing an antigen-bonding domain specific for a particular antigen to screen the mAbs List of FDA’s Approved Monoclonal antibody The first approved mAbs was OKT-3 [1986], which is a murine IgGa2 protein to deplete T cells in patients with acute rejection of renal allotransplant. Until Feb 24, 2013, 312 mAbs were approved by FDA, which were applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic malignancies and psoriasis. Figure 6 : FDA’s Approved Monoclonal antibody 12 | P a g e
  13. 13. Biotechnology IIMonoclonal antibody Antibody Brand name Type Indication Abciximab ReoPro chimeric Cardiovascular disease Adalimumab Humira human Several auto-immune disorders Alemtuzumab Campath humanized Chronic lymphocytic leukemia Basiliximab Simulect chimeric Transplant rejection Belimumab Benlysta human Systemic lupus erythematosus Bevacizumab Avastin humanized Colorectal cancer, Age related macular degeneration BrentuximabVedot in Adcetris Chimeric Hodgkin lymphoma Canakinumab Ilaris Human Cetuximab Erbitux chimeric Colorectal cancer, Head and neck cancer Certolizumabpeg ol Cimzia humanized Crohn's disease Daclizumab Zenapax humanized Transplant rejection Denosumab Prolia , Xgeva Human Postmenopausal osteoporosis Eculizumab Soliris humanized Paroxysmal nocturnal hemoglobinuria Efalizumab Raptiva humanized Psoriasis Palivizumab Synagis humanized Respiratory Syncytial Virus Panitumumab Vectibix human Colorectal cancer Ranibizumab Lucentis humanized Macular degeneration Rituximab Rituxan, Mabthera chimeric Non-Hodgkin lymphoma Tocilizumab Actemra Humanised Rheumatoid arthritis Tositumomab Bexxar murine Non-Hodgkin lymphoma Trastuzumab Herceptin humanized Breast cancer 13 | P a g e
  14. 14. Biotechnology IIMonoclonal antibody Pharmacology of some Monoclonal antibodies drugs Bevacizumab Brand Name :Avastin Bevacizumab is a humanized monoclonal antibody that recognizes and blocks vascular endothelial growth factor A (VEGF-A). VEGF-A is a chemical signal that stimulates the growth of new blood vessels (angiogenesis). Bevacizumab was the first commercially available angiogenesis inhibitor. It stops tumor growth by preventing the formation of new blood vessels by targeting and inhibiting the function of a natural protein called vascular endothelial growth factor (VEGF) that stimulates new blood vessel formation. The drug was first developed as a genetically engineered version of a mouse antibody that contains both human and mouse components. Genentech is able to produce the antibody in production-scale quantities. Blood vessels grow uncontrollably in cancer, retinal proliferation of diabetes in the eye, and other diseases. Bevacizumab can block VEGF-A from creating new blood vessels. Bevacizumab was the first clinically available angiogenesis inhibitor in the United States. Bevacizumab is currently approved by the U.S. Food and Drug Administration (FDA) for cancers that are metastatic (have spread to other parts of the body). It received its first approval in 2004 for combination use with standard chemotherapy for metastatic colon cancer and non-small cell lung cancer. In 2008, it was approved by the FDA for use in metastatic breast cancer, a decision that generated some controversy as it went against the recommendation of its advisory panel, who objected because it only slowed tumor growth but failed to extend survival. Clinical studies are underway in non-metastatic breast cancer, renal cell carcinoma, glioblastomamultiforme, ovarian cancer, castrate-resistant (formally called hormone refractory) prostate cancer, non-metastatic unresectable liver cancer and metastatic or unresectable locally advanced pancreatic cancer. A study released in April 2009 found that bevacizumab is not effective at preventing recurrences of non-metastatic colon cancer following surgery. In May 2009, it received FDA approval for treatment of recurring glioblastomamultiforme, while treatment for initial growth is still in phase III clinical trial. 14 | P a g e
  15. 15. Biotechnology IIMonoclonal antibody Indications of Avastin Colorectal cancer Bevacizumab was approved by the FDA in February 2004 for use in metastatic colorectal cancer when used with standard chemotherapy treatment (as first-line treatment) and with 5-fluorouracil-based therapy for second-line metastatic colorectal cancer. This recommendation was based on the E3200 trial — addition of bevacizumab to oxaliplatin/5FU/leucovorin (FOLFOX4) therapy. In adjuvant colon cancer, data from two large randomized studies have shown no significant benefit and a potential to cause harm in this setting. It was approved by the EMEA in January 2005 for use in colorectal cancer. Lung cancer In 2006, the FDA approved bevacizumab for use in first-line advanced nonsquamous nonsmall cell lung cancer in combination with carboplatin/paclitaxel chemotherapy. The approval was based on the pivotal study E4599 (conducted by the Eastern Cooperative Oncology Group), which demonstrated a 2-month improvement in overall survival in patients treated with bevacizumab (Sandler, et al. NEJM 2004). A preplanned analysis of histology in E4599 demonstrated a 4-month median survival benefit with bevacizumab for patients with adenocarcinoma (Sandler, et al. JTO 2010); adenocarcinoma represents approximately 85% of all non-squamous cell carcinomas of the lung. A subsequent European clinical trial, AVAiL, was first reported in 2009 and confirmed the significant improvement in progression-free survival shown in E4599 (Reck, et al. Ann. Oncol. 2010). An overall survival benefit was not demonstrated in patients treated with bevacizumab; however, this may be due to the more limited use of bevacizumab as maintenance treatment in AVAiL versus E4599 (this differential effect is also apparent in the European vs US trials of bevacizumab in colorectal cancer: Tyagi and Grothey, Clin Colorectal Cancer, 2006). As an anti-angiogenic agent, there is no mechanistic rationale for stopping bevacizumab before disease progression. Stated another way, the survival benefits achieved with bevacizumab can only be expected when used in accordance with the clinical evidence: continued until disease progression or treatment-limiting side effects. Another large European-based clinical trial with bevacizumab in lung cancer, AVAPERL, was reported in October 2011 (Barlesi, et al. ECCM 2011). First-line patients were treated with bevacizumab plus cisplatin/pemetrexed for four cycles, and then randomized to receive maintenance treatment with either bevacizumab/pemetrexed or bevacizumab alone until 15 | P a g e
  16. 16. Biotechnology IIMonoclonal antibody disease progression. Maintenance treatment with bevacizumab/pemetrexed demonstrated a 50% reduction in risk of progression vs bevacizumab alone (median PFS: 10.2 vs 6.6 months, HR 0.50, p<0.001). The National Comprehensive Cancer Network recommends bevacizumab as standard firstline treatment in combination with any platinum-based chemotherapy, followed by maintenance bevacizumab until disease progression. Bevacizumab is given as an intravenous infusion every three weeks at the dose of either 15 mg/kg or 7.5 mg/kg. The higher dose is usually given with carboplatin-based chemotherapy, whereas the lower dose is usually given with cisplatin-based chemotherapy. Breast cancer In December 2010, the FDA removed the breast cancer indication from bevacizumab, saying that it had not been shown to be safe and effective in breast cancer patients. The combined data from four different clinical trials showed that bevacizumab neither prolonged overall survival nor slowed disease progression sufficiently to outweigh the risk it presents to patients. This only prevented Genentech from marketing bevacizumab for breast cancer. Doctors are free to prescribe bevacizumab off label, although insurance companies are less likely to approve off-label treatments. In June 2011, an FDA panel unanimously rejected an appeal by Roche. A panel of cancer experts ruled for a second time that Avastin, the bestselling cancer drug in the world, should no longer be used in breast cancer patients, clearing the way for the U.S. government to remove its endorsement from the drug. The June 2011 meeting of the FDA's oncologic drug advisory committee was the last step in an appeal by the drug's maker. The committee concluded that breast cancer clinical studies of patients taking Avastin have shown no advantage in survival rates, no improvement in quality of life, and significant side effects. Patient support groups were disappointed by the committee's decision. Avastin will still remain on the market as an approved treatment for certain types of colon, lung, kidney and brain cancer (glioblastomamultiforme). Renal cancers In certain renal (kidney) cancers, Bevacizumab improves the progression free survival time but not survival time. In 2009, the FDA approved bevacizumab for use in metastatic renal cell cancer (a form of kidney cancer).following earlier reports of activity EU approval was granted in 2007. Brain cancers 16 | P a g e
  17. 17. Biotechnology IIMonoclonal antibody The FDA granted accelerated approval of Avastin for the treatment of recurrent glioblastomamultiforme in May 2009. Treatment for initial growth is still in phase III clinical trial. In the September 2009 issue of the Journal of Clinical Oncology, UCLA researchers reported that Avastin improves response and survival in patients with recurrent glioblastoma in comparison to historical controls. That study also found that it may be useful in the treatment of radiation necrosis, since it reduces edema and mass effect and diminishes blood-brain-barrier leakage. In a Phase I clinical trial conducted in 2009–2010, John A. Boockvar's team at Weill Cornell Medical College administered bevacizumab intra-arterially directly into the brain tumors of fourteen patients with recurrent glioblastomamultiforme. Results were sufficiently encouraging to propose a Phase II trial of the technique. Preliminary data of a large multicenter trial reported at the ASCO meeting 2013 suggest that patients with newly diagnosed glioblastoma do not benefit from the addition of bevacizumab to chemoradiation therapy. Investigational uses Bevacizumab did not meet its primary endpoint of extending overall survival (OS) in a recent phase III trial in unresectable gastric cancer (in combination with paclitaxel / Taxol), but it did demonstrate a positive result in treatment of ovarian cancer. A study released in April 2009 found that bevacizumab is not effective at preventing recurrences of non-metastatic colon cancer following surgery. Bevacizumab has demonstrated activity in ovarian cancer,and glioblastomamultiform, a type of brain tumour, when used as a single agent. In 2010 two phase III trials showed a 27% and 54% increase in progression-free survival in ovarian cancer. Bevacizumab has been investigated as a possible treatment of pancreatic cancer, as an addition to chemotherapy, but studies have shown no improvement in survival. It may also cause higher rates of high blood pressure, bleeding in the stomach and intestine, and intestinal perforations. The drug is also undergoing initial trials as an addition to established chemotherapy protocols and surgery in the treatment of pediatric osteosarcoma and other sarcomas, such as leiomyosarcoma. Uses in eye disease 17 | P a g e
  18. 18. Biotechnology IIMonoclonal antibody Many diseases of the eye, such as age-related macular degeneration (AMD) and diabetic retinopathy, damage the retina and cause blindness when blood vessels around the retina grow abnormally and leak fluid, causing the layers of the retina to separate. This abnormal growth is caused by VEGF, so bevacizumab has been successfully used to inhibit VEGF and slow this growth. Bevacizumab has recently been used by ophthalmologists in an off-label use as an intravitreal agent in the treatment of proliferative (neovascular) eye diseases, particularly for choroidal neovascular membrane (CNV) in AMD. Although not currently approved by the FDA for such use, the injection of 1.25-2.5 mg of bevacizumab into the vitreous cavity has been performed without significant intraocular toxicity. Many retina specialists have noted impressive results in the setting of CNV, proliferative diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinopathy of prematurity and macular edema secondary to retinal vein occlusions. When bevacizumab is used in the treatment of macular degeneration, only tiny and relatively inexpensive doses (compared to amounts used in colon and other cancers) are required. Some investigators believe that bevacizumab at a cost of around $42 a dose is as effective as ranibizumab at a cost of over $1,593 a dose. The primary pharmacokinetic difference between intraocular Bevacizumab and Ranibizumab is the very large difference in systemic half-lives, i.e. 2 hours for Ranibizumab versus 20 days for Bevacizumab. Since Bevacizumab was designed as a cancer treatment, the long systemic half-life is considered to be a positive feature (allowing greater exposure time of the tumor to the drug), while the same long half-life is a negative feature in intraocular treatment, since it has no benefit outside of the eye and may in fact be detrimental. Although the systemic exposure with both drugs is very low, Ranibizumab has a much lower average systemic exposure (area under the curve), so it may be conjectured to have a commensurately lesser chance of systemic adverse events. The National Eye Institute (NEI) of the National Institutes of Health (NIH) announced in October 2006 that it would fund a comparative study trial of ranibizumab (Lucentis) and bevacizumab (Avastin) to assess the relative safety and effectiveness in treating AMD.This study, called the Comparison of Age-Related Macular Degeneration Treatment Trials (CATT Study), enrolled about 1,200 patients with newly diagnosed wet AMD, randomly assigning the patients to one of four treatment groups. The CATT Study was conducted at 47 clinical sites throughout the United States, following the patients for 2 years. Initial results of the study showing essentially similar outcomes using either drug at one year were formally published after peer review in the New England Journal of Medicine on May 19, 2011. Similar results in this cohort were maintained at two years, with bevacizumab showing non- 18 | P a g e
  19. 19. Biotechnology IIMonoclonal antibody inferiority to ranibizumab, although a statistically non-significant trend to improved visual outcomes from injections given monthly rather than as required was noted with both drugs. Rituximab Brand names:Rituxan and MabThera Rituximab is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of immune system B cells. Rituximab destroys B cells and is therefore used to treat diseases which are characterized by excessive numbers of B cells, overactive B cells, or dysfunctional B cells. This includes many lymphomas, leukemia’s, transplant rejection, and autoimmune disorders. Mechanism of Action Rituximab binds specifically to the antigen CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35), a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes. The antigen is expressed on > 90% of B-cell non-Hodgkin's lymphomas (NHL), but the antigen is not found on hematopoietic stem cells, pro-B-cells, normal plasma cells or other normal tissues. CD20 regulates an early step(s) in the activation process for cell cycle initiation and differentiation, and possibly functions as a calcium ion channel. CD20 is not shed from the cell surface and does not internalize upon antibody binding. Free CD20 antigen is not found in the circulation. B cells are believed to play a role in the pathogenesis of rheumatoid arthritis (RA) and associated chronic synovitis. In this setting, B cells may be acting at multiple sites in the autoimmune/inflammatory process, including through production of rheumatoid factor (RF) and other autoantibodies, antigen presentation, T-cell activation, and/or proinflammatory cytokine production. Mechanism of Action: The Fab domain of rituximab binds to the CD20 antigen on B lymphocytes, and the Fc domain recruits immune effector functions to mediate B-cell lysis in vitro. Possible mechanisms of cell lysis include complement-dependent cytotoxicity 19 | P a g e
  20. 20. Biotechnology IIMonoclonal antibody (CDC) and antibody-dependent cell mediated cytotoxicity (ADCC). The antibody has been shown to induce apoptosis in the DHL-4 human B-cell lymphoma line. Normal Tissue Cross-reactivity: Rituximab binding was observed on lymphoid cells in the thymus, the white pulp of the spleen, and a majority of B lymphocytes in peripheral blood and lymph nodes. Little or no binding was observed in the non-lymphoid tissues examined. Indication of Rituximab rheumatoid arthritis treatment This study characterized the relationship between clinical response, serum rituximab concentrations, and peripheral B-cell levels in patients with rheumatoid arthritis treated with rituximab. Data were analyzed from a double-blind, phase IIa trial in which 161 patients with active rheumatoid arthritis despite continuing methotrexate were randomized to methotrexate alone (10-25 mg/wk), rituximab alone (single course: 1000 mg administered intravenously on days 1 and 15), rituximab plus cyclophosphamide (750 mg administered intravenously on days 3 and 17), or rituximab plus methotrexate. Serum samples for pharmacokinetic analysis were collected through 24 weeks, and peripheral circulating CD19+ B-cell levels were measured through 48 weeks. All treatments were generally well tolerated, with no clinically relevant excess of adverse events leading to withdrawal among patients who received rituximab compared with those who received methotrexate alone. The proportions of patients who achieved an American College of Rheumatology score of 50 at week 24 were 13% (methotrexate alone), 33% (rituximab alone), 41% (rituximab plus cyclophosphamide), and 43% (rituximab plus methotrexate). Peripheral B-cell depletion occurred by day 15 in all patients treated with rituximab. There was no relationship between B-cell depletion and clinical response. Recovery of peripheral B cells was variable and showed no relationship with return of disease activity in patients who responded to rituximab. The mean terminal half-life of rituximab was 19 to 22 days; pharmacokinetic parameters were similar whether rituximab was administered alone or with methotrexate or cyclophosphamide. Because the level of peripherally circulating B cells does not appear to correlate with a maintained clinical response in patients with rheumatoid arthritis, the timing of rituximab retreatment should be based on clinical symptoms rather than peripheral B-cell levels. 20 | P a g e
  21. 21. Biotechnology IIMonoclonal antibody Abciximab Brand Name :ReoPro Abciximab is a type of "blood thinner" used to prevent blood clots during certain procedures used to open up the blood vessels in the heart (e.g., balloon angioplasty, coronary stent placement, percutaneous coronary intervention-PCI). It is usually used with heparin and aspirin.Abciximab works by preventing platelets in the blood from sticking to each other. When platelets stick to each other, they may form blood clots that may cause a heart attack or cause the opened blood vessel in the heart to close back up. Indications for use Abciximab is indicated for use in individuals undergoing percutaneous coronary intervention (angioplasty with or without stent placement). The use of abciximab in this setting is associated with a decreased incidence of ischemic complications due to the procedure and a decreased need for repeated coronary artery revascularization in the first month following the procedure.[4] Research also shows that this drug can be of use for patients with diabetes and chronic renal insufficiency. It is not the appropriate drug of choice if a patient is scheduled for an emergency surgery (i.e., heart surgery) because bleeding time may take about 12 hours to normalize. Pharmacokinetics Abciximab has a plasma half-life of about ten minutes, with a second phase half-life of about 30 minutes. However, its effects on platelet function can be seen for up to 48 hours after the infusion has been terminated, and low levels of glycoprotein IIb/IIIa receptor blockade are present for up to 15 days after the infusion is terminated. Abciximab does not require renal dose adjustment. 21 | P a g e
  22. 22. Biotechnology IIMonoclonal antibody Palivizumab Brand names: Synagis® Palivizumab is a monoclonal antibody produced by recombinant DNA technology. It is used in the prevention of respiratory syncytial virus (RSV) infections. It is recommended for infants that are high-risk because of prematurity or other medical problems such as congenital heart disease. Palivizumab is a humanized monoclonal antibody (IgG) directed against an epitope in the A antigenic site of the F protein of RSV. In two Phase III clinical trials in the pediatric population, palivizumab reduced the risk of hospitalization due to RSV infection by 55% and 45%. Palivizumab is dosed once a month via intramuscular (IM) injection, to be administered throughout the duration of the RSV season. Palivizumab targets the fusion protein of RSV, inhibiting its entry into the cell and thereby preventing infection. Uses of Palivizumab Palivizumab is used in certain infants and young children to prevent infections of the breathing tubes and lungs that are caused by a certain virus (respiratory syncytial virus-RSV). It is known as a monoclonal antibody, and it works by preventing the growth of RSV. This medication is not used to treat RSV infection Side effects of monoclonal antibodies Monoclonal antibodies are given intravenously (injected into a vein). Compared with the side effects of chemotherapy, the side effects of naked mAbs are usually fairly mild and are often more like an allergic reaction. These are more common while the drug is first being given. Possible side effects can include:Anaphylaxis anaphylactic reactions Malignancy Serious infections Abnormal liver function, Fever Chills Weakness Headache 22 | P a g e Nausea Vomiting Diarrhea Low blood pressure Rashes Hypersensitivity neutropaenia and lipid deregulation
  23. 23. Biotechnology IIMonoclonal antibody Some mAbs can also have other side effects that are related to the antigens they target. For example, bevacizumab (Avastin®), an mAb that targets tumor blood vessel growth, can cause side effects such as high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage. Side effects of licensed monoclonal antibodies The safety and side effects of monoclonal antibodies Target mAb Type Platelet glycopr otein IIb/IIIa Abciximab (ReoPro; Centocor Ortho Biotech,EliLilly) Chimeric antibody fragment: c7E3 Fab Tumour necrosis factor-α Adalimumab (Humira; Abbott) Certolizumab (Cimzia; UCB) Infliximab (Remicade; Centocor Ortho Biotech) CD52 on Alemtuzumab mature (Campath; B, T and Genzyme) natural killer cells 23 | P a g e FDA approv al 1994 Indications* Selected side effects • Hypersensitivity and immunogenicity • Increased risk of bleeding •Thrombocytopaenia Fully human 2002 Humanized pegylated Chimeric 2008 Prevention of ischaemic cardiac complications of percutaneous coronary interventions and unstable angina •Rheumatoid arthritis • Ankylosing spondylitis • Psoriasis • Psoriatic arthritis • Crohn's disease • Ulcerative colitis Humanized 2001 • Infusion reactions and immunogenicity • Hypersensitivity reactions •Immunosuppression and infections (tuberculosis) • Anaemia, leukopaenia and thrombocytopenia • Worsening heart failure • Malignancy, lymphoma and lymph proliferative disorders • Increased nuclear-specific antibodies • B cell chronic • Infusion reactions lymphocytic • Hypersensitivity and leukaemia immunogenicity • Graft-versus• CRS host disease • Tumourlysis syndrome • Multiple • Immunosuppression and myeloma opportunistic infections • Multiple • Cytopaenias: sclerosis pancytopaenia, • Vasculitis lymphopaenia and • Behçet's disease thrombocytopaenia • Autoimmune haemolyticanaemia • Thyroid disorders 1998
  24. 24. Biotechnology Interleu kin-2 receptor -α on activate d lympho cytes Basiliximab (Simulect; Novartis) Daclizumab (Zenapax; Roche) Chimeric 1998 Humanized 1997 Disconti nued in Europe Vascula r endothe lial growth factor Bevacizumab (Avastin; Genentech) Humanized 2004 • Metastatic colorectal cancer • Non-small-cell lung carcinoma • Metastatic breast carcinoma • Metastatic renal carcinoma Ranibizumab (Lucentis; Genentech, Novartis) Humanized 2006 (Fab fragment from bevacizuma b) Humanized 2007 • Injected intravitreally for neovascular (wet) age-related macular degeneration • Paroxysmal nocturnal haemoglobinuria • No longer licensed for chronic plaque psoriasis Comple ment C5 CD11a CD3 antigen on T 24 | P a g e Eculizumab (Soliris; Alexion) Efalizumab (Raptiva; Genentech) MuromonabCD3 (OrthocloneOK Humanized 2003 Recentl y disconti nued Mouse 1986 (no Europe • Prophylaxis of renal transplant allograft rejection • Acute resistant allograft rejection in renal, cardiac IIMonoclonal antibody • Cardiotoxicity • Severe acute hypersensitivity reactions • CRS and immunogenicity • Immunosuppression and infections • Local skin reactions • Warnings when combined with other immunosuppressives • Infusion reactions and immunogenicity • Local complications at tumour site • Arterial and venous thromboembolicevents • Haemorrhage • Severe hypertension • Cardiac failure • Reversible posterior leukoencephalopathy syndrome • Slower wound healing and GI perforation • Conjunctivalhaemorrhage • Intraocular inflammation • Increased intraocular pressure • Retinal detachment • Endophthalmitis • Meningococcal andNeisseria infection • Intravascular haemolysis • First-dose reaction complex • Immunosuppression • Serious opportunistic infections • PML • Guillain–Barré syndrome, encephalitis, meningitis • Immune haemolyticanaemia • Immune thrombocytopaenia • Severe acute infusion reactions • Immunosuppression and
  25. 25. Biotechnology cells T3; Ortho Biotech) Natalizumab (Tysabri; Biogen-Idec, Elan Pharmaceutical s) Immuno Omalizumab globulin (Xolair; E (IgE) Genentech, Novartis) Humanized an Medicin es Authori ty authori zation) 2004 Humanized 2003 Fusion protein on RSV Humanized 1998 α4 integrin Palivizumab (Synagis; Medimmune) CD20 on Rituximab Chimeric B cells (Rituxan/Mabth era; Genentech, Biogen Idec) 1997 EGFR 2006 25 | P a g e Panitumumab (Vectibix; Amgen) Fully human IIMonoclonal antibody and hepatic transplant patients infections • Immunogenicity • Cardiovascular side effects • Hepatitis • Highly active relapsingremitting multiple sclerosis • Severe allergic asthma unresponsive to conventional therapy and with acute exacerbations • Prevention of RSV complications in high-risk infants • Follicular nonHodgkin's lymphoma • CD20+ diffuse large B cell nonHodgkin's lymphoma • Autoimmune haematological disorders • Infusion and hypersensitivity reactions • Immunogenicity • PML (0.1%) with immunosuppressives • Hepatotoxicity • Anaphylaxis (0.1%) • Injection site reactions • Immunogenicity • URTI • Churg–Strauss syndrome (rare) • Monotherapy for EGFR-positive metastatic colorectal carcinoma with non-mutated (wildtype) KRAS after failure of • Anaphylaxis and apnoea (rare) • Fever, injection site reactions • Prominent acute infusion reactions • CRS • Tumourlysis syndrome • Transient hypotension • Immunogenicity • Serum sickness • Severe mucocutaneous reactions • Immunosuppression • Hepatitis B reactivation with fulminant hepatitis • PML • Renal toxicity • Cardiac arrhythmias • Infusion reactions • Skin rashes in most patients (90%) • Diarrhoea (60%), nausea and vomiting • Hypomagnesaemia (2%)
  26. 26. Biotechnology conventional chemotherapy • EGFR-positive metastatic colorectal cancer • Squamous cell carcinoma of head and neck Cetuximab Chimeric (Erbitux; Bristol–Myers Squibb, ImClone Systems, Merck Serono) 2004 Trastuzumab (Herceptin; Genentech) Humanized 1998 • ERBB2-positive breast carcinoma Humanized 2009 • Unresponsive active rheumatoid arthritis • Castleman's disease Interleu Tocilizumab kin-6 (Actemra; receptor Roche, Chugai) IIMonoclonal antibody • Severe infusion reactions • IgE against oligosaccharide and HAMA • Urticaria and dermatological toxicity • Bronchospasm and pulmonary toxicity • Hypomagnesaemia • Hypersensitivity and infusion reactions • Cardiotoxicity with anthracyclines • Skin reactions • Pulmonary toxicity • Hypomagnesaemia • Anaphylaxis and anaphylactoid reactions • UTRI • Headache • Serious infections • Abnormal liver function, neutropaenia and lipid deregulation CRS, cytokine release syndrome; EGFR, epidermal growth factor receptor; ERBB2, also known as HER2/neu; FDA, Food and Drug Administration; GI, gastrointestinal; HAMA, human anti-mouse antibodies; KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue; PML, progressive multifocal leukoencephalopathy; RSV, respiratory syncytial virus; URTI, upper respiratory tract infection.*Some of these indications are not currently licensed. 26 | P a g e
  27. 27. Biotechnology IIMonoclonal antibody References Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129 Dale L Ludwig, et. al. Oncogene(2003) 22, 9097-9106 Jancie, M Recheit, etal. Nature biotechnology, 2005, Sep,Vol. 23, No.9Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005 Schwaber, J; Cohen, EP (1973). "Human x mouse somatic cell hybrid clone secreting immunoglobulins of both parental types". Nature244 (5416): 444–7. doi:10.1038/244444a0. PMID 4200460. edit Köhler, G.; Milstein, C. (1975). "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature256 (5517): 495. doi:10.1038/256495a0. PMID 1172191. edit 27 | P a g e

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