Abstract 1270: An isogeneic Trop-2 expression heterogeneity model for evaluating the bystander killing effect of anti-Trop-2 ADC

Abstract Antibody-drug conjugates (ADCs) have recently made significant advancements in cancer treatment, particularly for solid tumors. Among the multiple mechanisms of action of ADC drugs, the bystander effect—where the cytotoxic payloads released by the antibodies diffuse into and kill nearby tumor cells—plays a crucial role, especially in tumors with heterogeneous antigen expression. Therefore, constructing an antigen heterogeneity model for evaluating this bystander killing effect is essential for the preclinical assessment of ADC drugs. To establish a stable Trop-2 antigen expression heterogeneity model, we deleted Trop-2 in the Trop-2-positive NCI-H292 cell line and selected a clone with a similar in vitro proliferation rate to the parental cells. Additionally, to distinguish Trop-2-negative from Trop-2-positive cells in both in vivo and in vitro experiments, we introduced luciferase and mCherry into the Trop-2-negative cells. In vitro, Trop-2-negative and Trop-2-positive cells were mixed in multiple ratios and treated with graded concentrations of anti-Trop-2 ADC. After five days, flow cytometry quantified Trop-2-negative cells to evaluate the bystander killing effect. in vivo, Trop-2-positive and Trop-2-negative cells were mixed in various ratios and implanted subcutaneously in mice, followed by intravenous drug administration. Tumor volume and bioluminescence were measured to assess the efficacy of the anti-Trop-2 ADC and bystander killing activity. The results showed that the anti-Trop-2 ADC exhibited a significant bystander killing effect, significantly inhibiting Trop-2-negative cells both in vitro and in vivo. In summary, we have successfully established an isogeneic Trop-2 expression heterogeneity model and validated the bystander killing activity of anti-Trop-2 ADCs in vitro and in vivo. This model provides a robust platform for the pharmacological and efficacy assessment of anti-Trop-2 ADC therapies. Citation Format: Jingwei Huang, Xiangyi Wang, Zhixiang Zhang, Jingxuan Yan, Jinyan Ma, Man Zhang, Qingyang Gu. An isogeneic Trop-2 expression heterogeneity model for evaluating the bystander killing effect of anti-Trop-2 ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1270.