Bispecific Monoclonal Antibodies

Bispecific antibodies that have two different antigenic specificities can be created by hybridoma fusion or fusion of antibodies production by classical hybridomas. In the hybridoma fusion method, two hybridoma cell lines are fused to produce a quadroma. Typically, the technology takes advantage of drug marker cell lines having distinct drug sensitivities or chemical fusion [e.g., HAT(s)/Neo(r) and HAT(r)/Neo(s) are fused, positively selected in the proper media (De Lau et al., 1989). and expanded either in vivo or in vitro.] Although the production of antibodies is simple, purification ofbispecific antibodies poses a major technical challenge. Only 5-15% of the total antibody population is bispecific, reflecting the fact that coupling ofhomologous H chains (De Lau et al., 1991) occurs at a much higher frequency than two different H chains. This small amount of bispecific antibody must be separated from most of the anti-genically inactive but structurally similar antibodies.

Chemical Synthesis of Bispecific Antibodies. Monoclonal antibodies, Fab' or F(ab')2 fragments can be coupled to form bispecific antibodies. The most common linkage method involves covalent binding via SH groups in the hinge region ore amino groups. Cross-linkage of antibodies or Fab' fragments can be facilitated by succinimidyl-3-(2-pyridyl-dithiol)-propionate (SPDP). Although the reaction readily activates SH groups, a population ofrandomly cross-linked antibodies with different specificities is created. This limits the utility of the SPDP-linked antibodies (Fanger et al., 1992). Increased specificity can be achieved by linkage of different purified Fab' fragments with 5,5'-dithiobis(2-nitrobenzoic acid) or o-phenylenedimaleimide (Glennis et al., 1987; Brennan et al., 1985). Using o-phenylenedimaleimide, it is possible to add a third fragment to form trispecific antibodies (Tutt et al., 1991). Bi- and trispecific antibodies have limited clinical use because they have increased immunogenicity and reduced in vivo half-life.

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