Hybrid-hybridoma

Another variation to conjugate antibodies is to use bispecific antibodies. These are produced using chemical means and recombinant techniques to fuse separate hybridomas into a hybrid hybridoma (Fig. 4.6). Bispecific antibodies use one arm of the Fv to target the antigen or tumor cell and the other arm carries the effector molecule of toxins, radioisotopes, or other drugs. 

Staerz, U.D., and M.J. Bevan, Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc Natl Acad Sci USA, 1986. 83(5) 1453-7. 

Milstein, C. and Cuello, A C (1983) Hybrid hybridomas and their use in lmmuno-chemistry. Nature 305, 537-540. 

C Milstein, AC Cuello. Hybrid hybridoma production of bi-specific monoclonal antibodies. Immunol Today 5 299, 1984. 

Catumaxomab Rat IgG2b/Mouse IgG2a bispecfic Hybrid hybridoma EpCAM/CD3 Binds both EpCAM and CD3 Malignant ascites Removab ... 

Hyamine ld22 Hybrid bearings Hybrid circuit coating Hybrid circuits Hybrid glass Hybridoma cells Hybridomas Hycar... 

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 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. 

In 1975, Kohler and Milstein observed that if an antibody-producing cell was fused with a myeloma tumor cell, a rapidly dividing hybrid was produced that synthesized a monospecific antibody. Each hybridoma formed then became a factory, producing antibodies monospecific to a particular sensitizing antigenic epitope. Cell cloning allows selection of hybrids producing antibodies with the desired characteristics. 

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. 

Polyclonal antibodies can react with many epitopes, whereas MAbs are restricted to one epitope on proteins that do not have repeating sequences.24 By definition, polyclonal immunoassays are generally much more sensitive but less specific than monoclonal assays. Bispecific or hybrid antibodies can be used to increase the affinity. Bispecific antibodies are formed by the fusion of two previously established hybridomas to produce antibodies displaying the binding characteristics of both of the antibodies in one molecule.25... 

To obtain MABs, lymphocytes isolated from the spleen of immunized mice (1) are fused with mouse tumor cells (myeloma cells, 2). This is necessary because antibody-secreting lymphocytes in culture have a lifespan of only a few weeks. Fusion of lymphocytes with tumor cells gives rise to cell hybrids, known as hybridomas, which are potentially immortal. 

Figure 10.3. Schematic representation of monoclonal antibody production using immortalized hybrid cells that secrete antibodies selective for the target antigen. The mortal, immune B cells Isolated from mice immunized with a target antigen are fused with myeloma, immortal B cells that express defective antibodies. The selecting of antigen-specific, immortal hybrid cells (hybridomas) results in identification of unique clones of cells that express antibodies with high specificity and affinity (monoclonal antibodies). These cells are cloned and expanded for large-scale monoclonal antibody preparations.

As noted, one of the remaining challenges in obtaining human hybridoma cells for generahng human monoclonal antibodies is the lack of suitable human myeloma cell lines to generate stable hybrid cells that can be cloned and expanded indefinitely in culture. Many human hybridoma... 

The development of hybridoma technology by Milstein and Kohler in 1975 revolutionized the antibody field and radically increased the purity and specificity of antibodies used in the clinic and for diagnostic tests in the laboratory. Hybridomas consist of antibody-forming cells fused to immortal plasmacytoma cells. Hybrid cells that are stable and produce the required antibody can be subcloned for mass culture for antibody production. Large-scale fermentation facilities are now used for this purpose in the pharmaceutical industry. 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Kohler and Milstein are responsible for the third significant development during this time. In 1975 they demonstrated that cells derived from mouse B lymphocytes, which secrete antibodies, were fused with mouse myeloma tumor cells from a mouse, which can grow indefinitely in culture (see Fig. 3). The resulting fused cell, is a hybrid-myeloma or hybridoma cell that can grow in cell culture and produce large quantities of chemically... 

Fig. 3. To produce monoclonal antibodies, antibody producing spleen cells troll] a mouse that has been immunized against an antigen am mixed with mouse myeloma cells. Under the proper conditions, pairs of the cells fuse to form antibody-producing hybrid-myeloma ( hybridoma ) cells, which can live indefinitely in culture. Individual hybridomas are grown in separate wells, and the antibodies they produce are tested against the antigen. When an effective cell line is identified, it is grown either in cuhure or in the body cavities of mice to produce large quantities of chemically identical, monoclonal antibodies (reprinted with permission from Olsen, 1986, p. 26, Copyright, National Academy Press)...

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