P-140-T - Bioluminescent Assays for Antibody Drug Conjugate Development: Internalization and Bystander Killing Functions

Antibody-drug conjugates (ADCs) act through multiple mechanisms, including direct cytotoxicity via internalization and bystander killing of antigen-negative tumor cells. Existing tools often lack the sensitivity and specificity needed to distinguish these functional properties during preclinical development. We developed two bioluminescent platforms to address these gaps. The Antibody Internalization Assay uses luciferase-labeled Fab fragments with an extracellular quenching mechanism, achieving >100-fold signal increases in HER2-expressing cells. The HiBiT-based Bystander Killing Assay selectively measures antigen-negative bystander cell death in mixed co-culture, successfully distinguishing payload-dependent bystander activity across multiple ADCs — a differentiation unachievable with CellTiter-Glo® 2.0. Together, these no-wash, high-throughput tools enable precise characterization of ADC internalization and bystander killing, offering significant value for preclinical optimization of ADC candidates targeting heterogeneous tumors.