Abstract 5632: Enhancing dual-payload ADC discovery projects by overcoming heterogeneity and resistance through dual-payload innovation

Abstract Dual-payload antibody-drug conjugates (ADCs) have emerged as an innovative promising next-generation ADC format, delivering two cytotoxic agents to enhance efficacy through synergistic effects, mitigate resistance, without an accompanying increase in toxicity risk, aiming to address the challenge of poor-responsive cancers and rapid patient relapses. The success of dual-payload ADCs hinges on the appropriate dual-payload combination.DNA-damage response (DDR) inhibitors are increasingly being evaluated as payloads for ADCs, with Topoisomerase 1 (Top 1) inhibitors being the most commonly used class. Top 1 inhibition traps DNA breaks at replication forks, simultaneously blocking the DDR pathway severs the repair escape route, leading to replication catastrophe and selective synthetic-lethal cell death.To identify a rationally designed dual-payload combination that overcomes tumor heterogeneity and conventional resistance of colorectal cancer (CRC), synergistic pairs of Top 1 inhibitors and a group of DDR inhibitors with different mechanisms were screened and evaluated. Single payload activities were first investigated in CRC cell panel, and dual-payload combination synergies were then assessed in some selected CRC cell lines and ADC-related drug-resistant cell lines. A pair of Top 1 inhibitor + CHK inhibitor combination showed significant potent synergy in CRC cell lines and ADC-related drug-resistant cell lines with 2D and 3D cytotoxicity evaluation models. Mechanistic studies of this combination were further explored with cell cycle and apoptosis studies, WB of biomarkers, as well as RNA-Sequencing, revealing that the synergy stemmed from coordinated cell cycle arrest and enhanced apoptosis induction, supported by transcriptomic profiling of DDR pathways. Critically, the dual-payload combination showed no increased toxicity in a group of healthy human cell lines and limited or additive hematotoxicity in the in vitro CD34+ HSPCs myeloid progenitor cell proliferation and differentiation assays, indicating a potentially improved therapeutic window. These study strategies and findings accelerate dual-payload ADC innovative research, especially in the stage of proof of concept for dual-payload combination as a promising strategy to overcome cancer heterogeneity and resistance Citation Format: Ying Meng, Lili Chai, Wei Liu, Xue Yang, Min Kang, Rui Song, Zhengtai Li, Yan Zhang, Zhiwei Bao, Ying Bi, Yixiao Zhao, Wange Fan, Xiao Li, Li Li, Tiejun Bing. Enhancing dual-payload ADC discovery projects by overcoming heterogeneity and resistance through dual-payload innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5632.