Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker

A novel method for linking Adriamycin (ADM) to monoclonal antibodies is described in which the 13-keto position of the anthracycline is used as the attachment site to the linker arm. A new ADM acylhydrazone derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone hydrochloride, which contains a pyridyl-protected disulfide, was synthesized and used for conjugation to monoclonal antibodies (MAbs) that were thiolated with N-succinimidyl 3-(pyridyldithiol)propionate or 2-iminothiolane. This resulted in formation of a linker between MAb and drug that contained a disulfide bond. Conjugation conditions were optimized to yield conjugates with high ADM:MAb molar ratios. The final immunoconjugate yields were found to decrease as the ADM:MAb molar ratio of the conjugates increased. Stability studies indicated that ADM was released from the immunoconjugates at mildly acidic pHs ranging from 4.5-6.5. Treatment of immunoconjugates with mild reducing agent dithiothreitol resulted in release of an acylhydrazone derivative of ADM. Flow-cytometric studies showed that the binding activity of various MAbs following conjugation to ADM was preserved at ADM:MAb molar ratios up to 10. Antibody-directed cytotoxicity was demonstrated under several assay conditions using combinations of antigen-positive and antigen-negative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.