A Surprising Benefit of Cysteine Capping for Antibody Drug Conjugates

Abstract

With a burst of nine FDA approvals granted since 2017, ADCs are riding a significant market boom and gathering optimism as a promising therapeutic approach [2][3][4][5][6][7][8]. With >80 investigational candidates and around 150 active clinical trials [9], ADCs are among the fastest-growing drug classes in cancer therapeutics targeting both solid tumor indications and hematological malignancies. ADCs are complex molecules composed of three key components: a tumor-associated-antigen (TAA)-targeting monoclonal antibody, a cytotoxic drug payload, and a cleavable or noncleavable linker. Conjugation technology is another important aspect of ADCs, by which payloads and linkers are covalently attached to the antibodies. Conventional drug conjugation methods are either through less precise interchain disulfide Cys or random lysine residue in the antibody components, resulting in a huge product heterogeneity with limited control over the conjugation locations. Next-generation site-specific conjugation technologies (Summarized in Table 1; reviewed by [10][11][12]) define precise and proper drug attachments, which determine drug potency and exposure, allow for an optimized and tunable structure-activity relationship, as well as better characterized and monitored homogeneous drug products. Manufacturing Challenges of Site-specific ADCsHowever, manufacturing high-quality site-specific ADCs for clinical confirmation often encounters significant technical hurdles. For the most established Cys-based site-specific conjugation, it requires a complicated full reduction and reoxidation conjugation process [13], due to a disulfide capping on the engineered unpaired Cys residues (Figure 1, [13][14][15]). This so-called Cys-capping modification is a type of posttranslational modifications (PTMs) that are the disulfides typically with either Cys or glutathione (Figure 1). It is also detectable in endogenous membrane-bound or secreted proteins with solvent-exposed unpaired Cys residues [16][17][18].