Cancer hybridoma

ProstaScint (murine) Cytogen 1996 Liquid IV injection Imaging agent for prostate cancer Hybridoma Phosphate buffer saline... 

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

The hybridoma cells are initially grown in a medium that will not maintain the growth of the cancer cells these therefore die, as do non-fused lymphocytes, leaving only the fused cells. As the hybridoma cells grow, the supernatant fluid is tested for the presence of antibodies. Those cultures producing the desired antibody are further cloned and either grown in bulk or as tumours in animals and the monoclonal antibodies harvested. 

La Riviere G., Schipper C.A., Gebbinck J.W., Koch G., Kuhn L., Lefkovits 1. Roos E. (1992). Putative invasion-specific proteins in mouse T-cell hybridomas that differ in invasive and metastatic potential. Int. J. Cancer, 9 51(5) 745-53... 

La Riviere G., Schipper C., Collard J. and Roos E. (1988). Invasiveness in hepatocyte and fibroblast monolayers and metastatic potential of T-cell hybridomas in mice. Cancer Research, Vol. 48, 3405-3410. 

The restriction enzymes and hybridomas enabled new types of pharmaceutical products The developments associated with restriction enzymes (facilitate genetic engineering) and hybridomas (produce monoclonal antibodies) opened up vistas for a new range of potential products. Isolated from humans directly, these can now be produced in microorganisms or cell culture, and be used in large concentrations to enhance the body s ability to fight heart disease, cancer, and viral infections against which most antibiotics have little or no effect. 

An immunogen induces antibodies from many B cell clones, producing a polyclonal antibody response. In contrast, the propagation of an isolated B cell clone produces an antibody of single specificity. However, the problem is that in tissue culture medium, B cells die within a few days of their isolation from, for example, a mouse spleen. To circumvent this problem, immortality can be conferred on B cells by means of viral transformation Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is carried out to generate hybrids or hybridomas. Generally, the former procedure is used to immortalize peripheral blood B cells and produce human monoclonal antibodies, while myeloma cells are used to produce murine monoclonal antibodies. 

The problem is that if an individual antibody-producing cell is isolated and grown in culture, its descendants have a limited lifespan that severely limits their use for the routine preparation of monoclonal antibodies. In 1975, Milstein and Kohler discovered how monoclonal antibodies of almost any desired antigen specificity can be produced indefinitely and in large quantities. Their method was to fuse a B lymphocyte producing antibody of the desired specificity with a cell derived from a cancerous lymphocyte tumor, called a myeloma cell, which is immortal. The cell fusion is called a hybridoma, which is both immortal and secretes the same specific antibody originally encoded by the B lymphocyte. 

One of the earliest attempts to exploit hybridoma technology for cancer treatment was the work of Levy and co-workers, who produced mAbs reactive with the idiotype expressed on malignant B lymphocytes obtained from individual patients (157-158). Although the need to produce custom-made mAbs for each patient has severely limited the feasibility of this strategy, these pioneering studies did provide early and convincing evidence that mAb therapy could elicit durable antitumor responses. 

Twenty-five years after the revolutionary description of mAh production via hybridoma technology, mAbs have fully entered the clinical arena as new, unique, and important components of the medical armamentarium for the treatment of a variety of diseases. In fact, some of these mAbs can be regarded as major breakthroughs in the treatment of such disorders as transplant rejection, coronary artery disease, rheumatoid arthritis, non-Hodgkin s lymphoma, and breast cancer. It is virtually certain that this initial set of agents is but the first wave of what will become a broad array of mAh therapeutics for disease targets in all areas of medicine. 

The basic principle of hybridoma technology is shown in Fig. 2.23. B cells obtained from the spleens of mice immunized with the antigen of interest are fused with estabhshed mouse myeloma cells. Myeloma cells are transformed cancerous cells of the B lymphocyte lineage with infinite growth capacity. While many myeloma cell hnes continue to secrete their antibody, others lose the ability to produce antibodies. Spleen cells from immunized animals are fused with non-antibody-producing myeloma cells, resulting in a hybrid cell which inherits the properties of both the fusion partners. Thus the hybrids are capable of continued growth in culture like the myeloma cell and also maintain the antibody production of the spleen B cell. 

Primary andbodies Polyclonal rabbit and-EMA, monoclonal mouse and-CALLA (CDIO) (ascitic fluid, hybridoma PHM6) and monoclonal rat anti-c-erbB2 (Insdtute of Cancer Research clone, BTl/83d). 

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