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Evaluation in Vitro of Adriamycin Immunoconjugates Synthesized Using an Acid-sensitive Hydrazone Linker

Bristol-Myers Squibb Company, Pharmaceutical Research Institute, Wallingford, Connecticut 06492-7660

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 antigennegative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.

¹ To whom requests for reprints should be addressed, at Bristol-Myers Company, Pharmaceutical Research and Development Division, 5 Research Parkway, P. O. Box 5100, Wallingford, CT 06492-7660.

² Present address: Bio-Rad, 1414 Harborway South, Richmond, CA 94804.

Received 6/16/89. Accepted 6/26/90.

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