Monoclonal antibodies cell fusion

Key Words Monoclonal antibodies cell fusion hybridoma immunization cell culture myeloma splenocytes. 

Monoclonal antibodies are produced as a result of immortalizing and expanding the individual antibody secreting cells artificially in tissue culture (2). Cells grown in this way all have identical epitope specificity and because they are derived from single clones, their product is known as monoclonal antibody. Cells that secrete monoclonal antibodies are known as hybridomas and are typically derived by fusion of two cell types. B-lymphocytes, which have the capacity to make antibody, are obtained from a donor spleen and are physically fused to a tumor cell line, which is immortal. The resulting hybridom as are immortal and produce antibody into the synthetic medium in which they are growing. 

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. 

Monoclonal antibodies are derived from a single, monospecific B cell clone. Monoclonal antibodies can be obtained from hybridoma cells that result from the fusion of antibody-producing B cells with immortal cells of a myeloma cell line. 

Mammalian cell suspension cultures are the preferred choice for large-scale recombinant protein production in stirred-tank bioreactors. The most widely used systems are Chinese hamster ovary (CHO) cells and the murine myeloma fines NSO and SP2/0. In half of the biological license approvals from 1996-2000, CHO cells were used for the production of monoclonal antibodies and other recombinant glycosylated proteins, including tPA (tissue plasminogen activator) and an IgGl fusion with the tumor necrosis factor (TNF) receptor, the latter marketed as Enbrel [7]. 

Monoclonal antibody technology entails isolation of such B-lymphocytes, with subsequent fusion of these cells with transformed (myeloma) cells. Many of the resultant hybrid cells retain immortal characteristics, while producing large quantities of the monospecific antibody. These hybridoma cells can be cultured long term to effectively produce an inexhaustible supply of the monoclonal antibody of choice. 

Since a monoclonal antibody is a fusion product of a malignant mouse cell and an antibody-producing cell, there is some concern about the safety of the production process itself (Petricciani, 1983). Methods for the production of monoclonal antibodies raise two general safety issues (1) the theoretical risk of transferring in the product factors associated with malignancy (e.g., oncogene factors) and (2) the use of animals for antibody production that are known to harbor a number of microbial agents some of which can produce diseases in humans. 

The basic principle for making the rabbit monoclonal antibody is the same as for mouse monoclonals. Rabbit fusion partner cells can fuse to rabbit B-cells to create the rabbit hybridoma cells. Hybridomas are then screened to select for clones with... 

Hybridoma Cell produced by the fusion of antibody-producing plasma cells with myeloma/carcinoma cells. The resultant hybrids have then the capacity to produce antibody (as determined by the properties of the plasma cells), and can be grown in continuous culture indefinitely owing to the immortality of the myeloma fusion partner. This technique enabled the first continuous supply of monoclonal antibodies to be produced. 

The next development was the production of monoclonal antibodies (MAbs) in the mid-1970s. This uses hybridoma technology, which involves the fusion of antibody-producing B cells to immortal myeloma cells. Figure 4.4 shows the preparation of MAbs using hybridoma techniques. A more detailed discussion of biopharmaceuticals production is presented in Section 10.5. 

The membrane fusion activity is probably a function of the El protein, since the hemolytic activity of Sindbis virus can be inhibited by monoclonal antibodies specific for El (Chanas et al., 1982). Moreover, studies using cDNA molecules coding for the spike proteins have shown that if the spike protein is expressed at the cell surface, fusion between cells is induced at low pH. However, when the p62 protein is expressed alone, no fusion occurs (Kondor-Koch et al., 1983). 

Three TNF antagonists are currently approved for the treatment of RA etaner-cept (ETN), infliximab (INF), and adalimumab. ETN a fusion protein of two identical chains of the recombinant human TNF receptor, p75, fused with the Fc portion of human immunoglobulin (Ig) G1 binds to soluble TNF-a in vivo. INF and adalimumab are both monoclonal antibodies to TNF-a INF is chimeric, and adalimumab is fully humanized. Both bind to soluble TNF-a, preventing TNF-a from binding to its receptors on cell surfaces. INF can also bind transmembrane TNF-a, fix complement, and cause cell lysis. 

Even greater flexibility was achieved by genetic fusion of streptavidin with protein A [153,154]. Protein A specifically binds the Fc domain of IgG immunoglobulins of almost all mammals without inhibiting the antigen binding activity of the antibody. The streptavidin-protein A fusion construct was used for the assembly of complexes of biotinylated P-galactosidase and different monoclonal antibodies specific for tumour cell receptors. As a result these complexes were efficiently delivered into several cancer cell lines [154]. 

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