Fusion of lymphocytes

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. 

Hybridoma cells - cells obtained through the fusion of lymphocytes and tumor cells. They are particularly effective for the expression of monoclonal antibodies. 

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. 

Majeau, G.R. et al., Mechanism of lymphocyte function-associated molecule 3-Ig fusion proteins inhibition of T cell responses. Structure/function analysis in vitro and in human CD2 transgenic mice, J. Immunol., 152, 2753, 1994. 

A hybridoma is a cell line created by the fusion of two different lymphocyte cell lines, one of which is derived from a tumour. 

Spleen-derived B lymphocytes are then incubated with mouse myeloma cells in the presence of propylene glycol. This promotes fusion of the cells. The resultant immortalized antibody-producing hybridomas are subsequently selected from unfused cells by culture in a specific... 

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. 

The production in vitro of mAbs with a predetermined specificity has only been possible since the advent of the technology of hybridomas, which was introduced by Kohler and Milstein in 1975. These hybridomas are the products of in vitro fusion of myelomas with normal B lymphocytes. The fusion products preserve the capacity for self-propagation in a culture, as well as the secretion of the antibodies of interest, characteristics inherited from the parent myeloma and the normal B lymphocyte, respectively. The myelomas used in such fusions generally involve cell lines from B-lymphocyte tumors developed in mice or rats (Cotton and Milstein, 1973 Kohler and Milstein, 1975b Shulman et al., 1978), while... 

Approximately a week after plating, all the normal (non-fused) B lymphocytes had already been eliminated naturally from the culture, because normal lymphocytes do not propagate in culture. Only those hybrids resulting from the fusion of the myelomas with the B lymphocytes were able to grow in the culture, and some of them also preserved the ability to secrete antibodies against the antigen used for immunization. 

Vaisbourd M, Ignatovich O, Dremucheva A, Karpas A, Winter G (2001), Molecular characterization of human monoclonal antibodies derived from fusions of tonsil lymphocytes with a human myeloma cell line, Hybrid Hybridomics 20 287-92. 

Other selection systems involve the fusion of proline requiring CHO cells and defective mouse cells in HAT medium lacking proline and the fusion of normal lymphocytes (which do not grow in vitro) with defective mouse cells (which do not grow in HAT medium). It is this latter technique which has allowed the isolation of clones of cells which will synthesise in vitro large amounts of a single antibody (i.e. a monoclonal antibody) (Kohler, 1982). 

Antibodies are expressed by hybridoma cells formed by cell fusion of sensitized animal or human B lymphocytes with myeloma cells, or they are generated by EBV (Epstein-Barr virus) transformation of sensitized B lymphocytes. Other heterologous expression systems such as bacteria, yeast, insect cells, and mammalian cells have also been used for expression of antibodies and their fragments. However, because of renaturation problems, glycosylation, and expression levels, mammalian cells are mostly used for the expression of monoclonal antibodies. More recently, technologies have been extensively developed for the expression of antibodies in transgenic animals and transgenic plants. 

Following fusion, hybridomas must be separated from fusion products of the same cell type (e.g., fusion of two B lymphocytes or two myeloma cells) and cells that did not undergo fusion. Unfused B lymphocytes or the fusion product of two... 

The successful fusion of normal and neoplastic lymphocytes has laid the foundation for the production of a variety of antibody specificities of practical relevance in research and diagnosis. The technical problems associated with this approach should not be underestimated, but one cannot fail to recognize the enormous range of applications that lie ahead once these problems have been overcome. 

---