Selective Reduction of Cysteine Mutant Antibodies for Site-Specific Antibody–Drug Conjugates

Developing site-specific conjugation technologies for antibody–drug conjugates (ADCs) aims to produce more homogeneous and controlled drug-loaded ADCs to reduce variability and thereby improve the therapeutic index. This article presents a technology that uses cysteine mutant antibodies and mild phosphine-based reductants to prepare site-specific ADCs. The two types of cysteine mutant antibodies, designated C6v1 and C6v2, have one of the interchain disulfide-forming cysteines in the Fab region in the light chain (LC214) or in the heavy chain (HC220) substituted by alanine (or other amino acids), respectively. Certain phosphine-based reductants were found to selectively reduce the "unpaired" cysteines, at the heavy chain (HC220) for C6v1 or at the light chain (LC214) for C6v2 while keeping the interchain disulfide bonds in the hinge region intact, resulting in 90% of DAR2 species and more than 95% of the desired specific conjugation at HC or LC following conjugation to maleimide moieties. The reduction method shows consistent selectivity toward various C6v1 or C6v2 antibody backbones. Sensitivity toward buffer pH for some reductants can be used to optimize reductant reactivity and selectivity. The technology can be further expanded to generate site-specific DAR4 or dual-payload ADCs based on C6v1 or C6v2 antibodies. This technology offers a method to control drug-loading and conjugation sites using a mild one-pot process, as compared to the reduction–oxidation methods used in technologies such as THIOMAB, and shows superior DAR profiles and process simplification as compared to other selective reduction methods.