Production of Monoclonal Antibodies - Coursework Example

Production of Monoclonal Antibodies Over view Antibodies that are produced from a single type of immune cell are called monoclonal antibodies (Kimball, 2007). They are all identical because they are clones of a single parent cell. Monoclonal antibodies are of medical interest now, as they are widely used as diagnostic and research reagents. They can be either used singly or coupled to another molecule to achieve results1. Currently, they are used in the treatment of cancer, transplant rejection, macular degeneration, chronic inflammatory diseases, cardiovascular disease, multiple sclerosis and hepatitis B infection. Though it was Paul Ehrlich who proposed about monoclonal antibodies as early as the beginning of 20th century, it was in1975, that Kohler and Milstein combined the unlimited growth potential of myeloma cells with the predetermined antibody specificity of normal immune spleen cells to produce monoclonal antibodies (Kimball, 2007). In this essay, production of monoclonal antibodies for diagnostic and therapeutic purpose will be discussed with reference to Molecule A secreted by leukemic cells. Basic principles in monoclonal antibody production in vivo: Mouse ascites method To produce monoclonal antibodies for medical use, the vertebrate, usually a mouse, is challenged many times with molecule-A against which monoclonal antibodies are to be produced. After this, from the principle organs for B cell production, the lymph nodes and the spleen, B cells are removed. These are then fused with myeloma tumor cells by making the cell membranes more permeable by using polyethylene glycol or electroporation. These fused cells are called hybridomas. Myeloma tumor cells multiply rapidly and indefinitely, but they cannot produce antibodies because of the nature of the tumor. And the good B cells can produce Molecule-A- specific antibodies. Thus culture of these hybridomas produces large amounts of antibodies against molecule-A. Finally, the antigen specific antibodies are then picked up by using ELISA or immuno-dot blot (McArdle, 1997). Though the myeloma cells have lost their ability to produce any antibodies, some of them can still produce antibodies and these need to be removed from the culture. Hence, prior to fusion, the cells are placed in 8 azaguanine media. Cells which have the enzyme, HGPRT3, necessary for antibody production, are killed in this media and thus eliminated. After fusion, the hybridomas are allowed to grow in HAT4 medium and only those that successfully grow in this culture are used. A layer of briclone or feeder cells is used to enrich the medium. These hybridomas can then be used for monoclonal antibody production against molecule-A either in-vitro or in-vivo (McArdle, 1997). Figure-1: Production of Monoclonal Antibodies (National Academy of Sciences, 1997) Initially, monoclonal antibodies were produced in the above explained method. Though, these antibodies are close to human ones, the human body recognizes them as foreign and produces further antibodies against them, causing systemic inflammation and removing them from circulation. A solution to this would be to use humans, but it is neither practical nor ethical. Also, human body may not produce antibodies against its own tissues. From the late 1980s, recombinant DNA technology is used to produce these antibodies in a large scale. There are 2 methods in this. One is by taking the DNA that encodes the binding portion of monoclonal mouse antibodies and merges it with human antibody producing DNA. Then, mammalian cell cultures will be used to express this DNA and produce these half-mouse and half-human antibodies. In the second method, mice are genetically engineered to produce more human like antibodies. In recent days, genetic engineering is the main method of antibody production for medical use. Recent advances also allow the use of rabbit-B cells (Kimball, 2007). Antibodies which are engineered are commonly expressed in Chinese hamster ovary fibroblast or mouse myeloma cells (Hale, Berrie and Bird, 2004). For generating recombinant antibodies, advanced DNA and protein technologies are being used. For desirable selection of antibodies from large diverse populations, ribosome display which employs cell-free protein display technology is used (Wang, 2007). Mechanism of production of monoclonal antibodies against Molecule A secreted by leukemic cells: in vitro procedure. Mice are first immunized with particles containing molecule-A like like whole membranes, intact cells and microorganisms after preparing with adjuvants like freud adjuvant or homogenizing with gel slice. In most centers, the mice are immunized once in every 2-3 weeks, The antibody titres in the mice, produced against the molecule A are measured and once a desired level is reached, spleen from the mice is removed. Cells from this spleen are used for fusion with the myeloma cells. These cells have a limited life span and they are subjected to fusion with myeloma cells. The result is a hybridoma. This is capable of growing in an unlimited manner. The myeloma cells for this purpose are immortalized cells and are cultured with 8-azaguanine to ensure sensitivity to HAT or hypoxanthine-aminopterin-thymidine medium after cell fusion. Culture in 8-azaguanine is done one week before cell fusion. One advantage with HAT medium is that it allows only hybridoma cells to grow and survive in the culture. Cells grown thus will be distributed in 96 well plates which contain feeder cells derived from the peritoneum of the mice. The cells are obtained through peritoneal washes. These feeder cells supply growth factors for the promotion of growth in hybridoma cells. Feeder cells can also be obtained from the bone marrow of mice. The hybridoma cells in the plates grow in small clusters. This is followed by selection for antigen binding. If antigen binding is not done, cloning may be done later. It is important to optimize growth by mouse ascites expansion method so that the cells can be saved and optimal antibody production can be achieved (National Academy of Science, 1997). Growing of hybridomas in batches and subsequent purification of monoclonal antibodies from the culture medium is the simplest method of production of monoclonal antibodies. Serum free media can be used for the growth of hybridoma cell lines to avoid contamination with bovine immunoglobulin. The cells cultures are allowed to adapt to grow in less than 1 percent fetal-calf serum within four passages, following which the cell cultures are incubated in tissue-culture flasks for 10 days under standard growth conditions. From this, monoclonal antibodies is harvested. The concentration of monoclonal antibodies retrieved thus is less than 20 microg/ml. Increasing the concentration of dissolved oxygen through some methods like roller bottles, gas permeable bag and spinner flasks increases the viability of the cells and yields more monoclonal antibodies. The required monoclonal antibody concentration is 0.1-10 mg/ml. To achieve this concentration, filtration devices can be used. Another method of concentration is passage over columns like protein-G and protein-A. This is the batch culture method and it is cheap, takes about 3 weeks to finish and quantities produced are similar to the mouse-ascites method. Use of semipermeable membrane-based systems allows harvesting of larger amounts of monoclonal antibodies. The systems have 2 chambers. In the larger chamber, culture media is present and in the smaller chamber monoclonal antibodies are collected. The most commonly used membrane systems are mini-PERM and CELLIne. These systems have technical difficulties (National Academy of Science, 1997). The main advantages of in vitro methods are reduction in the use of mice, especially during the antibody-production stage, easy to produce and hence useful for large-scale production by pharmaceutical industry, no need to submit animal protocols, production of quantities as high as in ascitic fluid methods but without contaminants of ascitic fluid. The disadvantages are some hybridomas may not grow well or may be lost in culture. Use of fetal calf serum limits the use of some antibodies. In some cultures, there may be loss of glycosylation of antibody resulting unsuitability of antibody product. Monoclonal antibodies derived through batch-culture supernatant retrieve less antibodies than the ascites method. There may be contamination with dead hybridoma cells or cell products. The monoclonal antibodies produced in vitro have poorer binding affinity and are costlier (National Academy of Science, 1997). Large scale production of monoclonal antibodies Large scale production of monoclonal antibodies against molecule-A is essential for diagnostic and therapeutic purposes. For such commercial purposes, large batches of cells are cultures or large number of mice are injected. The output of production must be stable cell line, large quantities of antibodies and uncontaminated. Cost, turnaround time and compliance with regulations are also important. Both in-vivo and in-vitro methods are used for large scale production. In vivo- production is usually much cheaper. However, considering he effort involved in in-vivo methods, many manufacturers prefer in vitro methods. The therapeutic industry mainly uses serum-free invitro technology, to avoid treatment-related allergic responses which may occur as a result of repeated foreign antigen exposure. Currently, mouse antibody genes are replaced with human DNA. Allergic problems can be preventing by humanizing antibodies and allowing production of antibodies in mice with severe immunodeficiency, or in an in vitro system. In the diagnostic industry, cost is a major factor and thus manufacturing occurs mainly in in vivo-derived products. Optimization of production is done by reducing the invasiveness of hybridoma and by increasing the secretion of monoclonal antibodies (National Academy of Science, 1997). Application 1. Diagnostic purpose Monoclonal antibodies produced against cancer cell antigens like molecule-A can be used to detect the presence of molecule-A. Thus the immuno dot tests and Western blot tests are useful in detecting protein on a membrane, immunochemistry tests detect antigen on fixed tissue sections and immunofluorescence tests detect protein in live cells or frozen tissue (Rang, 2003). 2. Therapeutic purpose The main use of monoclonal antibodies in therapeutic range is treatment for cancer. Monoclonal antibodies against certain cancer-specific antigens like molecule-A can be used to induce immunological response against that particular antigen. They can also tagged along with cytokines, isotopes or toxins and allowed to bind with molecule-A and destroy it or identify it. Some of monoclonal antibodies used for such a purpose are bevacizumab, panitumumab and cetuximab (Rang, 2003). Currently, 21 monoclonal antibody products have been approved by the US Food and Drug Administration, of which 6 have been approved for cancer (Oldham and Dillman, 2008). References Hale G, Berrie E, Bird P. (2004). Design and manufacture of monoclonal antibodies for radioimmunotherapy. Q J Nucl Med Mol Imaging, 48(4), 258-66 Kimball, J. W. (2007). Monoclonal Antibodies. Online biology text book. Retrieved on 22nd April, 2010 from http:/users.rcn.com McArdle. J. (1997). Animal Welfare Information Center Newsletter, Vol. 8: no. 3-4 National Academy of Sciences. (1999). Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies Institute for Laboratory Animal Research. Retrieved on 22nd April, 2010 from http://grants.nih.gov/grants/policy/antibodies.pdf Oldham, R.K., and Dillman, R.O. (2008). Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress. Journal of Clinical Oncology, 26, 1774-1777. Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. Wang M, He M. (2007). The rapid discovery of engineered antibodies. The rapid discovery of engineered antibodies. Idrugs., 10(8), 562-5. Read More