Quadroma bispecific antibodies

Quadromas are bispecific antibodies synthesized by hybrid hybridomas obtained by fusion of two lymphocytes producing antibodies with different specificity. Because in hybrid hybridomas the expressed VH and VK genes of the parental cells are all produced there are ten molecular species of hybrid antibodies which theoretically can be formed as expected from the random association of the product of each gene. However, immunochemical studies of Ig mol ecules produced by quadromas showed that only six-species were secreted and indicated a marginal preference for heavy-light chain homologous combination. The theoretical yield of a bispecific antibody with desired specificity produced by quadromas is 8-17% of the total Ig synthesized.

The production of quadromas is based on the principle of fusing two parental cells, each carrying a different drug resistant marker and selecting the hybrids of both type of cells in the presence of both drugs.

There have been only a few successful attempts to produce quadromas by fusing cells sensitive or resistant to various drugs such as hypoxanthine-aminop-terin-thymidine (HAT)-sensitive, ouabain-resistant, neomycin (G418)-resistant, or resistant to iodoaceta mide damage. Utilization of these drugs requires tedious experiments to select drug-sensitive or -resistant mutants in the population of hybridomas which will be used to generate quadromas. A new and easier method takes advantage of defective retroviruses carrying genes conferring resistance to neomycin or methotrexate.

Each partner of the fusion is infected with a retrovirus which confers a stable drug resistance at a very high frequency. The yield of quadromas using retro-virus-infected cells is generally higher than those using cells sensitive or resistant to various drugs. Utilization of hybridomas producing antibodies with different isotypes or idiotypes facilitates the purification of bispecific antibodies using anti-isotype or anti-idiotype immunoadsorbants.

Bispecific antibodies specific for the TCR of cytotoxic T lymphocytes (CTLs) and for viral antigens budding from the surface of target cells or against tumor antigens have been shown to be very efficient in small concentrations to focus CTL activity on target cells and to cause their lysis. The Fab fragment binding to the TCR is sufficient to activate the CTL, and the other Fab fragment brings the target cell in intimate contact with the CTL thereby causing lysis of the target.

It has been clearly shown that the redirected lysis mediated by bispecific antibodies circumvents the genetic restriction requirement of T cells. The efficacy of bispecific antibodies can be increased by pre-activation of CTLs with specific antigens or super-antigens. Bispecific monoclonal antibodies have numerous advantages when compared with chemically constructed heteroconjugates. These advantages include greater uniformity, stability and purity, and the absence of aggregates which may be cleared from the circulation as immune complexes. They also have a longer half-life in vivo because of their normal structure. In addition, because of the monomeric structure of the combining site it is unlikely that it will cause antigen modulation, a phenomenon which requires cross-linking of receptors or antigens associated with the membrane of effector or target cells.

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