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

• DOI: 10.1021/acs.bioconjchem.0c00337
• Publication Date: 2020-07-22T15:51:35Z
• Authors: Ruud G. E. Coumans; Gerry J. A. Ariaans; Henri J. Spijker; Pascal Renart Ver...; Patrick H. Beusker; Bas P. A. Kokke; Jan Schouten; Marion Blomenröhr; Miranda M. C. van...; Patrick G. Groothuis; Ruud Ubink; Wim H. A. Dokter; C. Marco Timmers
• Keywords: Models, Molecular; 0301 basic medicine; 0303 health sciences; Antibiotics, Antineoplastic; Immunoconjugates; 3. Good health; Duocarmycins; Mice; 03 medical and health sciences; Cell Line, Tumor; Immunoglobulin G; Neoplasms; Animals; Humans; Cysteine; Hydrophobic and Hydrophilic Interactions; Oxidation-Reduction

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 in silico 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 in vitro. Comparison of site-specific ADCs versus randomly conjugated ADCs in an in vivo 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.