Resin-Supported Site-Specific Antibody Conjugation Method Leads to Antibody-Drug Conjugates with Retained Efficacy and Improved Stability

Herein, we describe an optimized method for the generation of “thiolated Q295” site-specific antibody-drug conjugates (ADCs) with drug-to-antibody ratio (DAR) 2 from nonengineered IgG1 antibodies. Traditional ADCs take advantage of the 4 intrachain disulfide residues as the sites of attachment. While operationally simple to prepare, ADCs that rely on attachment to these endogenous cysteine residues suffer from heterogeneity arising from stochastic mixtures of differently loaded species. Our team recently reported a site-specific thiolation method targeting the conserved Q295 residue in deglycosylated antibodies. This approach involves deglycosylation of Q297 (using PNGase F) to eliminate steric hindrance from the N-glycan, followed by introducing a thiol-containing small molecule, cysteamine, at Q295, using microbial transglutaminase (mTGase). Our original method employed a global reduction/reoxidation to liberate the Q295 thiol for conjugation. However, this process was challenging due to competing reoxidation of the newly introduced Q295 thiol. In order to overcome this issue, we systematically explored various reducing agents and conditions, ultimately resulting in a new process that avoids the need for reduction/reoxidation. This resin-supported method, which is suitable for high-throughput synthesis, relies on the selective reduction of the engineered disulfide by sterically hindered phosphine, monosulfonated triphenylphosphine (TPPMS). Relying on this optimized methodology, we studied a small set of tubulysin ADCs showing that the resulting Q295-conjugated ADCs have favorable biophysical and biological properties as compared to traditional stochastic conjugation.