Abstract 2955: Systematic mechanistic profiling of ADC-resistant cancer cell lines.

Abstract In recent years, antibody–drug conjugate (ADC) discovery has become one of the hottest areas for targeted therapy development of diverse cancer types. Regardless of its superiority to other therapeutic modalities, resistance remains one of the major and most common challenges, thereby severely hindering its clinical efficacy. ADC-resistant cell lines have been well demonstrated to be valuable and reliable models to help interrogate ADC-relevant resistance, hence facilitating development of therapies overcoming resistance. To this end, we have successfully developed over a dozen of ADC-resistant cancer cell lines by chronically challenging parental cancer cells with escalating dosages of ADCs for several months. We validated such a gain of resistance by a full-dosage drug sensitivity profiling at the endpoint, demonstrating 10 fold of drug IC50 increase in most of the established resistant cell models compared to their respective parental counterparts. To further unravel the underlying drug resistance mechanisms, we applied RNA-Seq and global proteomics profiling on several resistant models paired with their parental controls, including HCC1954 and NCI-N87 Enhertu-resistant, HCC1806 and Colo-205 Trodelvy-resistant cells. Promisingly, we observed that HER2 expression was dramatically and concordantly (∼30-fold) reduced at both mRNA and protein levels according to RNA-Seq and global proteomics profiling, which was further validated by flowcytometry and Western blot analysis. Subsequent cross-drug sensitivity profiling revealed that HCC1954 Enhertu-resistant cells were resistant to the other two HER2-targeting ADCs, T-DM1 and RC48 rather than their respective payloads, which further consolidated that reduction of HER2 expression other than any adaptive resistance to payload plays a pivotal role in the HCC1954 Enhertu-resistant model. Interestingly, we didn’t observe any robust reduction of antibody target expression in the other three ADC resistant models, implying diverse resistance mechanisms across different ADCs and cancer cell lines. We will continue to do datamining and validation on these models in the near future, aiming to shed light on more ADC resistance mechanisms. Citation Format: Tingting Yuan, Yanqiu Bai, Lehua Cheng, Yanan Xu, Wenzhang Chen, Yan Gao, Zhuo Wen, Qiangqiang Fan, Zhengang Peng. Systematic mechanistic profiling of ADC-resistant cancer cell lines 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 2955.