Fc Cavity Conjugation Improves Developability and In Vivo Efficacy of Antibody Drug Conjugates

Antibody drug conjugates (ADCs) are a promising class of therapeutics in cancer treatment. However, the general design principles of ADCs are yet to be established. We hypothesized that shielding the linker payload within the Fc cavity could mitigate premature exposure of linker payloads during systemic circulation and improve in vivo efficacy. To achieve this goal, we employed engineered cysteines and screened conjugation sites to find an optimal site which can mask a payload in the Fc cavity. Through a cathepsin cleavage assay and hydrophobic interaction chromatography, several conjugation sites were identified to produce drug-to-antibody ratio (DAR) 2 Fc cavity ADCs. Among the identified sites, V264C exhibited the strongest payload-shielding effect. Using a molecular-ruler approach, we further quantified the payload-accommodating capacity of each Fc cavity conjugation site and found substantial site-dependent differences in cavity size. The single mutations were further combined to generate DAR4 ADCs, leading to the identification of a practical site selection guideline for engineered cysteine-based DAR4 Fc cavity ADCs. An intermutation distance of ∼ 20 Å emerged as a practical threshold for selecting compatible mutation pairs in DAR4 Fc cavity ADCs. Both the DAR2 and DAR4 Fc cavity ADCs showed improved in vivo efficacy compared with the randomly conjugated ADCs.