A Platform for the Generation of Site-Specific Antibody–Drug Conjugates That Allows for Selective Reduction of Engineered Cysteines

Abstract

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an <i>in silico</i> screening procedure to select for optimal sites in an antibody to which a hydrophobic linker-drug can be conjugated. Sites were identified inside the cavity that is naturally present in the Fab part of the antibody. Conjugating a linker-drug to these sites demonstrated the ability of the antibody to shield the hydrophobic character of the linker-drug while resulting ADCs maintained their cytotoxic potency <i>in vitro</i>. Comparison of site-specific ADCs versus randomly conjugated ADCs in an <i>in vivo</i> xenograft model revealed improved efficacy and exposure. We also report a selective reducing agent that is able to reduce the engineered cysteines while leaving the interchain disulfides in the oxidized state. This enables us to manufacture site-specific ADCs without introducing impurities associated with the conventional reduction/oxidation procedure for site-specific conjugation.