Abstract 5665: Targeted cell killing: a versatile platform for cell therapy and cytotoxicity assays in diverse cell lines

Abstract The HiBiT Target Cell Killing Assay (HiBiT TCK Assay) is a streamlined, sensitive method for selectively quantifying target cell lysis in coculture experiments with effector cells. HiBiT is an 11AA peptide that binds with high affinity to LgBiT, forming a bright, luminescent enzyme. For HiBiT TCK, target cells are engineered to express a HiBiT-tagged protein, and release of this protein by dead/dying cells leads to complementation with the cell impermeable LgBiT. This gain-of-signal approach enables precise measurements of target cell killing induced by various effectors, including CAR-T cells and cell killing mediated by bispecific antibodies. Here we demonstrate the use of HiBiT TCK with ADCC, ADCP, TDCC and CAR-T applications. Furthermore, we present data of mRNA transfection to transiently express HiBiT in a variety of target cells, allowing for the rapid and uniform labeling of both dividing and non-dividing cells. This approach extends the HiBiT TCK products beyond use of stable cell lines alone and allows users to screen a variety of cell lines. Citation Format: Nicholas J. Hess, Brock Binkowski, Julia Gilden, Gopal Krishnan, Mei Cong, Marjeta Urh. Targeted cell killing: a versatile platform for cell therapy and cytotoxicity assays in diverse cell lines [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 5665.