Abstract 5476: In vitro real-time evaluation of bystander effects of antibody-drug conjugates

Abstract Antibody-drug conjugates (ADCs) represent a pivotal advancement in biotherapy, leveraging targeted internalization to treat tumors effectively. A critical phenomenon associated with ADCs is the bystander effect, where cytotoxic drugs released from lysed cancer cells diffuse into neighboring tumor cells, enhancing therapeutic efficacy—particularly in tumors with heterogeneous antigen expression. Despite its significance, methods for detecting and evaluating the bystander effect of ADCs remain under development. In this study, we present a robust approach to monitor and analyze the bystander effect of ADCs in real-time using live-cell imaging and impedance measurements with the Agilent xCELLigence RTCA eSight system. Two innovative assays—the in vitro co-culture assay and the conditioned medium transfer assay—were established for this purpose. The cytotoxic effects of DS8201, an ADC with bystander activity, and T-DM1, an ADC without bystander activity, were assessed in these assays. In the co-culture assay, HER2-positive SKBR3 cells and HER2-negative MCF7 cells were co-seeded in a biosensor plate (E-Plate VIEW 96) the day before testing. The results revealed that DS8201 treatment led to the death of HER2-negative MCF7 cells in the presence of SKBR3 cells, indicative of a bystander effect. In contrast, T-DM1 did not affect MCF7 viability under similar conditions. Both DS8201 and T-DM1, however, induced cytotoxicity in HER2-positive SKBR3 cells. In the conditioned medium transfer assay, medium from SKBR3 cells treated with DS8201 significantly reduced MCF7 cell viability, demonstrating a bystander effect. Conversely, conditioned medium from T-DM1-treated SKBR3 cells did not impact MCF7 viability. Notably, neither DS8201 nor T-DM1 exhibited direct cytotoxic effects on MCF7 cells in isolation. In conclusion, we describe two robust methodologies for detecting and evaluating the bystander effect of ADCs using the RTCA eSight system. These approaches provide valuable tools for optimizing the efficacy, safety, and pharmacokinetics of therapeutic ADCs during biopharmaceutical development. Citation Format: Xiaoyu Zhang, Yun-fei Pu, Tian Wang, Grace Yang, Peifang Ye, Nancy Li. In vitro real-time evaluation of bystander effects of antibody-drug conjugates [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 5476.