Identification of Amino Acid Residues Responsible for the Release of Free Drug from an Antibody-Drug Conjugate Utilizing Lysine-Succinimidyl Ester Chemistry

Unexpected release of free drug was observed during the stability testing of an antibody-drug conjugate (ADC). The ADC was designed to use lysine-succinimidyl ester chemistry to conjugate small molecule cytotoxic drugs to the antibody. To elucidate the mechanism of the release of free drug, a succinimidyl ester analog, 7-hydroxy-4-methyl-3-coumarinylacetic acid N-succinimidyl ester, and a series of peptides were used to probe the potential side reaction of succinimidyl ester with other amino acid residues. Cysteine and tyrosine residues were found to be reactive to succinimidyl ester, and the bonds formed through these reactions were found to be labile. Combining the fluorescent property of the succinimidyl ester analog and mass spectroscopy analysis, specific cysteine and tyrosine residues of the antibody were found to be reactive to succinimidyl ester and the bonds formed through this reaction were susceptible to hydrolysis. (C) 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100: 2518-2525, 2011