Abstract 2961: Developing ADC-resistant tumor models for efficacy evaluation of next-generation anticancer therapies.

Abstract Introduction: Antibody-drug conjugates (ADCs) pair monoclonal antibodies with cytotoxic payloads. Trastuzumab deruxtecan (DS8201) targets HER2, and datopotamab deruxtecan (DS1062) targets TROP2. Both use a topoisomerase I inhibitor (Dxd) via a cleavable linker. After target-mediated internalization, proteases release the payload, causing DNA damage, apoptosis, and bystander killing. However, resistance may stem from antigen loss, altered internalization, impaired payload release, efflux pumps, or apoptosis regulation. We developed ADC-resistant cell and organoid models via chronic dosing, lentivirus transduction, and Cas9 mediated knock out. Methods: First, efflux pump proteins (ABCB1, ABCG2) were introduced into HER2+ HCC1954 cells via lentiviral transduction (HCC1954-ABCB1, HCC1954-ABCG2); overexpression was confirmed by Western blot. Cytotoxicity to Dxd/ADC was evaluated by CellTiterGlo (CTG)--assay. Second, chronic in vitro exposure generated HCC1954-DS8201R and HCC1954-DxdR. HER2 expression was assessed by flow cytometry (FACS), and ADC binding/internalization by IncuCyte imaging. RNA-seq investigates the resistance mechanisms. Finally, TROP2 was knocked out using CRISPR in two DS1062 responsive organoids. Loss of TROP2 was confirmed using immunofluorescence, and ADC sensitivity was tested using CTG assay. Xenograft models of ADC-resistant cells are in development for further in vivo studies. Results: HCC1954-ABCB1 showed remarkable resistance to MMAE (IC50: 85.8 nM vs 0.3 nM parental) but showed no change to Dxd. While, HCC1954-ABCG2 is resistant to Dxd (IC50: 10 μM vs 0.509 μM parental), with minimal MMAE impact. The RC48 (Her2-targeting ADC with MMAE) and DS8201 are confirmed payload-based resistances. HCC1954-ABCB1 is resistant to RC48 (IC50: 100 nM vs 0.288 nM parental), while HCC1954-ABCG2 is resistant to DS8201 (IC50: 100 nM vs 5.2434 nM parental). These findings suggest that efflux pump proteins preferentially transport distinct cytotoxic payloads. Chronic exposure to Dxd generated HCC1954-DxdR cells reduced sensitivity to both Dxd (IC50: 10 μM vs 0.2023 μM parental) and DS8201 (IC50: 100 nM vs 2.1695 nM). Similarly, chronic exposure to DS8201 produced the HCC1954-DS8201R, showing significant resistance (IC50: 70 nM vs 2.1695 nM). In HCC1954-DS8201R with reduced HER2, confirmed by FACS and RNA seq, showed a decrease in HER2 binding/internalization, whereas HCC1954-DxdR exhibited ABCG2 amplification. TROP2 KO efficiency was 30% (LU11786B) and 5% (BR9465B). Cytoxicity of TROP2 KO models resistant to DS1062. with loss of DS1062 uptake and absence of the DNA damage marker yH2AX. Xenograft models of ADC-resistant cells are able to grow in vivo. Conclusion: We developed a panel of ADC-resistant tumor models to study mechanisms of acquired resistance. These models provide a valuable preclinical platform for advancing ADC therapy. Citation Format: Jian Feng, Fuyin Zhao, Xinlong Dong, Aaron Li Hua, Chenpan Nie, Dorrith Verstegen, Caitlyn Hulsebosch, Wubin Qian, Sheng Guo, Jessie (Jingjing) Wang, Lenno Krenning, Marrit Putker, Ludovic Bourre, Peng Wang, Jun Zhou. Developing ADC-resistant tumor models for efficacy evaluation of next-generation anticancer therapies 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 2961.