Cell based payload release highlights design, site, and cell dependent ratio shifts in dual payload ADCs

Dual-payload antibody-drug conjugates (ADCs) are designed to deliver two cytotoxic payloads in a defined ratio, yet whether this designed composition is maintained upon cellular processing is poorly understood. Conventional enzymatic payload release assays confirm linker cleavability but fail to reflect the biological complexity governing intracellular release. To address this, we established a quantitative cell-based liquid chromatography payload-release assay to monitor intracellular drug liberation after ADC treatment across multiple tumor cell lines. Using this platform, we examined how linker design, conjugation site, drug-to-antibody ratio (DAR), and steric environment affect payload release of mono- and dual-payload ADCs. While all constructs exhibited analytically confirmed enzymatic cleavability, the intracellular release profiles frequently diverged from the designed A:B payload ratios. Notably, the extent and direction of these ratio shifts were cell-type dependent, even for the same ADC construct, indicating that differences in protease abundance, endolysosomal trafficking, and microenvironmental pH strongly modulate payload liberation. Furthermore, conjugation site and DAR configuration introduced steric shielding effects that altered linker accessibility and release kinetics, resulting in distinct and less than optimal intracellular payload ratios despite identical chemical designs. Importantly, cell-based release results correlated closely with cytotoxicity outcomes in animal models, underscoring their functional relevance. Together, these findings reveal that the custom-designed dual-payload ratio does not necessarily reflect the effective intracellular ratio, and that cellular context, linker architecture, and conjugation geometry collectively determine the eventual payload balance. This framework provides a mechanistically informative tool for rational optimization of dual-payload ADC design, enabling more predictive control of intracellular pharmacology.