The (6-maleimidocaproyl)hydrazone of doxorubicin was synthesized and conjugated to several mAbs, including chimeric BR96, via a Michael addition reaction to thiol-containing mAbs. DTT reduction of disulfides present in the mAb was a reliable and general method for generating a consistent number of reactive SH groups. The conjugates, after purification by Bio-Beads, were free of unreacted linker and/or doxorubicin. All conjugates released doxorubicin under acidic conditions that mimic the lysosomal environment, while they were relatively stable at neutral pH. BR96 conjugates showed antigen-specific cytotoxicity.

Immunoconjugates of monoclonal antibody BR96 and Doxorubicin have been prepared using a novel series of branched hydrazone linkers. Since each linker bound to the mAb carries two DOX molecules, the DOX/mAb molar ratios of these conjugates were approximately 16, twice that of those previously prepared with single-chain hydrazone linkers. The conjugates were stable at a physiological pH of 7, but released DOX rapidly at lysosomal pH 5. The branched series of BR96 conjugates demonstrated antigen-specific cytotoxicity, and were more potent in vitro than the single-chain conjugate on both a DOX (4-14-fold) and mAb (7-23-fold) basis. The results suggest that, by using the branched linker methodology, it is possible to significantly reduce the amount of mAb required to achieve antigen-specific cytotoxic activity. In this paper, the synthesis and in vitro biology of branched chain immunoconjugates are described.