Homogeneous Dual-Payload Antibody-Drug Conjugates Produced by Combined Distinct Conjugation Strategies.

Antibody-drug conjugates (ADCs) represent a promising class of targeted cancer therapies, combining monoclonal antibody specificity with cytotoxic drug potency. Despite significant ADC technology advancements, challenges such as limited efficacy, often attributed to tumor heterogeneity and resistance development, remain present. In this study, we developed homogeneous dual-payload ADCs by combining site-selective conjugation methods: second-generation AJICAP technology for lysine modification and conventional interchain-break conjugation. Using trastuzumab as a model antibody, we successfully synthesized a drug-to-antibody ratio (DAR) of 10 (2 + 8) dual-payload ADC with monomethyl auristatin E and deruxtecan, displaying low aggregation and stable physicochemical properties. The dual-payload ADC exhibited superior in vitro cytotoxicity against HER2-positive SKBR-3 cells, compared to T-DXd, and demonstrated enhanced tumor suppression in vivo in a NCI-N87 xenograft model. These results highlight the potential of multipayload ADCs in enhancing therapeutic efficacy while maintaining stability, thereby providing a new strategy to overcome traditional ADC-related limitations.