Bioluminescent Assays for Antibody-Drug Conjugate Development: Internalization and Bystander Killing Functions

Presented by: Julia Gilden, Promega Corporation Poster Number: P-103-W Poster Session: Antibody Products and Automation Wednesday, 24 Sept. at 15:00 ----- Antibody-drug conjugates (ADCs) are combine targeted delivery with potent cytotoxic payloads. To optimize ADC design, robust tools are required to assess key mechanisms of action, including antibody internalization, Fc-mediated effector functions, and bystander killing. Here, we present two bioluminescent platforms: (1) an internalization assay that quantifies the endocytosis of antibodies in cells, and (2) a suite of bioluminescent assays to evaluate ADC Fc function and bystander killing. The internalization assay uses Fab fragments labeled with luciferase and uses a quenching mechanism to selectively measure intracellular antibody localization. This method demonstrated strong time- and antigen-dependent luminescence, with a >100-fold signal increase relative to controls, facilitating quantitative ADC screening. Complementary assays include FcR binding assays and cell-based reporter bioassays for antibody dependent cellular cytotoxicity (ADCC) and for antibody dependent cellular phagocytosis (ADCP) activities. Additionally, we have developed a HiBiT-enabled bystander killing assay to discern ADCs with differential payload activity. Together, these assays provide a comprehensive, high-throughput approach to evaluate ADC candidates' functional properties, enabling precise optimization of therapeutic efficacy. Their streamlined workflows, sensitivity, and dynamic range offer significant value for preclinical ADC developers.