Abstract 5486: Bioluminescent assays for antibody-drug conjugate development: internalization and Fc-mediated functions

Abstract Antibody-drug conjugates (ADCs) combine targeted delivery with potent cytotoxic payloads, utilizing multiple mechanisms of action (MoA). These include direct cytotoxicity following internalization of the ADC by antigen-expressing cells, killing of bystander cells through cleaved payloads, antigen function blockade, and immune-mediated cytotoxicity (e.g. Antibody-Dependent Cellular Cytotoxicity and Antibody-Dependent Cellular Phagocytosis) driven by the antibody Fc region. Comprehensive characterization and potency testing of ADCs require assays that capture each MoA. To address this need, we developed bioluminescent tools to streamline ADC evaluation, focusing on antibody internalization, Fc-mediated effector functions, and bystander killing. Studying bystander killing is particularly important in immunotherapy strategies for cancer, as it allows ADCs to target heterogenous tumors where not all cells express the antigen. The internalization assay employs luciferase-labeled Fab fragments with a quenching mechanism to measure intracellular antibody localization. The assay was validated using HER2-expressing SKOV3 cells with anti-HER2 monoclonal antibodies as model systems. It shows strong time- and antigen-dependent luminescence, achieving over 100-fold signal increases compared to controls, enabling quantitative ADC screening. Additional assays include Fc receptor (FcR) binding assays and cell-based reporter bioassays for ADCC and ADCP activities. The HiBiT technology is a luminescent tagging system that enables precise and sensitive detection of protein interactions and cellular events, making it a powerful tool for evaluating Target Cell Killing (TCK) by effectors and cytotoxic payloads carried by antibodies. To assess payload efficacy, a HiBiT-enabled bystander killing assay was developed, distinguishing ADCs with varying payload properties. Taken together, the internalization assay, bystander killing assay, and Fc effector bioassays, all based on bioluminescent platforms, offer a comprehensive and high-throughput solution for evaluating ADC functionality. With exceptional sensitivity, broad dynamic range, and efficient workflows, these tools enable precise optimization of therapeutic efficacy, delivering significant value for preclinical ADC development. Citation Format: Julia K. Gilden, Christopher Eggers, Yitong Li, Rod Flemming, Nicholas Hess, Gopal B. Krishnan, Frank Fan, Brock Binkowski, Marjeta Uhr, Mei Cong. Bioluminescent assays for antibody-drug conjugate development: internalization and Fc-mediated functions [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 5486.