Abstract B137: Advancing ADC Development: Dual-Payload Toxicity Assessment Using a Cancer Cell Panel and ADC Resistant cell lines

Abstract Antibody-drug conjugates (ADCs), which merge antibody specificity with cytotoxic payloads, represent a promising anticancer therapy. Dual-payload ADCs enhance this approach by enabling the delivery of two cytotoxic agents, offering advantages like synergistic effects, resistance mitigation, and dose flexibility. However, their development remains challenging. Cell panel-based studies and drug-resistant cell lines provide powerful tools to explore dual-payload ADCs by evaluating drug combination synergies and elucidating resistance mechanisms. In this study, we utilized a diverse cell panel encompassing over 50 cell lines from multiple cancer types to develop potential dual-payload combinations. Our approach involved combining topoisomerase I inhibitors, such as Exatecan, Dxd, and SN38, with DNA damage repair inhibitors targeting CHK2. To evaluate the effects of these drug combinations, we employed both traditional dose matrices and innovative 1:1/1:3 combination curves. We also established several ADC and payload-resistant cell lines to assess the efficacy of dual payloads in resistant models and explore the resistance mechanisms of these cell lines. Furthermore, we conducted RNA sequencing (RNAseq) and Whole exon sequencing (WES) and gene knockdown (KD) experiments to investigate the mechanisms underlying drug combinations. Our cell proliferation assays revealed promising synergistic effects in multiple cell lines, providing a foundation for dual-payload ADC design. The investigation into drug resistance mechanisms identified the overexpression of ABC transporters and the reduction in antigen expression as the primary causes of resistance in the resistant cell lines. These findings highlight the potential of dual-payload ADCs and underscores the importance of cell panel and resistant cell line studies in their development. The mechanistic insights gained from RNAseq, gene KD, and resistance studies further support the rationale for combining these agents in dual-payload ADCs, offering valuable insights for future clinical applications. Citation Format: Lili Chai, Xue Yang, Zhu Meng, Tiejun Bing. Advancing ADC Development: Dual-Payload Toxicity Assessment Using a Cancer Cell Panel and ADC Resistant cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B137.