Antibody drug conjugates (ADCs) have emerged as an important class of therapeutic agents comprised of antigen-targeting antibodies and cytotoxic agents. Increasing effort has been focused on developing site-specific conjugation technologies to produce more homogeneous and controlled drug-loaded ADCs for improvement of the therapeutic index. This talk presents a new 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 or C6v2, have one of the inter-chain disulfide forming cysteines in Fab region in the light chain (LC214) or in the heavy chain (HC220) substituted by alanine (or other amino acids), respectively. By using phosphine-based reductants, it is found that the “unpaired” cysteines, at the heavy chain (HC220) for C6v1 or at the light chain (LC214) for C6v2, can be selectively reduced by certain reductants while keeping the inter-chain disulfide bonds in the hinge region intact. Thiol-reactive drug-linker can then be directly added to generate site-specific ADCs with drug-to-antibody ratio (DAR) of 2. With some reductants, DAR2 species is around 90% and the desired specific conjugation at HC or LC is more than 95%. The reduction method shows consistent selectivity towards various C6v1 or C6v2 antibody backbones. Sensitivity towards buffer pH for some reductants can be used to optimize reductant reactivity and selectivity. The mechanism of reductant selectivity on C6v2 antibody is revealed by kinetic study and analysis. This technology offers a method to control drug loadings and conjugation sites using a mild and one-step 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.

Selective reduction of proteins for site-specific antibody-drug conjugation

ACS Spring 2022

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