Abstract Antibody–drug conjugates (ADCs), comprising monoclonal antibodies and cytotoxic payloads, present inherent complexities in molecular design. The drug-to-antibody ratio (DAR) represents a critical determinant of ADC behavior, influencing pharmacokinetics and tissue distribution. In vivo studies in tumor-bearing murine models demonstrated comparable efficacy between DAR4 and DAR8 ADCs (DAR4: 6 mg/kg, DAR8: 3 mg/kg), suggesting that DAR alone does not dictate therapeutic outcome. Immunohistochemical (IHC) staining of human IgG, used to map intact ADC localization, demonstrated superior tumor penetration by DAR4 ADCs relative to their DAR8 counterparts. However, this enhanced penetration did not translate to improved therapeutic efficacy. To elucidate the mechanistic underpinning of DAR optimization, we leveraged matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to spatially resolve the free payload distribution in tumors. The optimized MALDI imaging method enabled sensitive detection of released payload, revealing similar intensity and distribution pattern for both DAR4 and DAR8 ADCs at 2 and 24 h post-dosing. This spatiotemporal uniformity of payload correlated with consistent pharmacodynamic (PD) responses. Moreover, equivalent payload signal intensities across DAR variants corroborated quantitative analyses of tumor lysates and subsequent efficacy data, reinforcing the pivotal role of intratumoral payload in driving in vivo efficacy. These findings underscore that DAR optimization is both target-dependent and payload-specific, suggesting that ADC design should prioritize factors governing active payload release over passive distribution metrics. The MALDI-IMS methodology developed in this study provides unprecedented spatial resolution of bioactive payload dynamics, establishing a robust platform for dissecting efficacy determinants in preclinical ADC development. Graphical Abstract
Cai et al. (Thu,) studied this question.