Monoclonal antibodies myeloma cells

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.

Other species of hybridomas, including human, have been produced but are generally created by the use of viruses conferring cellular immortality. Artificial immunization of the donor is often not practical or ethical and so cell lines are often derived from peripheral lymphocytes obtained from individuals naturally immune to the target substance. Some human monoclonal antibody secreting cell lines have been derived from spontaneously occurring myelomas, but this line of approach frequently is unrewarding as the probability that the antibody will be one of interest is remote. 

Figure 4.33. Preparation of Monoclonal Antibodies. Hybridoma cells are formed by fusion of antibody-producing cells and myeloma cells. The hybrid cells are allowed to proliferate by growing them in selective medium. They are then screened to determine which ones produce antibody of the desired specificity. [After C. Milstein. Monoclonal antibodies. Copyright 1980 by Scientific American, Inc. All rights reserved.]...

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. 

Fusion of human lymphocytes with human lymphoblastoid cell lines is a very inefficient process. Fusion of human lymphocytes with murine myeloma cells lead to very unstable hybrids. Upon fusion, preferential loss of human genetic elements is often observed. Unfortunately, particularly common is the loss of chromosomes 2,14 and 22, which encode antibody light and heavy chain loci. The production yields of human monoclonals upon immortalization of the human B-lymphocyte (by whatever means) are also low. 

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. 

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... 

Karpas, A., A. Dremucheva, and B.H. Czepulkowski, A human myeloma cell line suitable for the generation of human monoclonal antibodies. Proc Natl Acad Sci USA, 2001. 98(4) 1799-804. 

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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