Abstract 3181: A novel, bioluminescent assay for the selective detection of target cell killing in mixed cultures

Efforts to develop and commercialize T cell immunotherapies would benefit from sensitive and easy-to-use assays to monitor target cell killing. We have developed an approach to selectively measure the death of target cells mediated by CAR/TCR-T cells or other types of effector cells. The method relies on the release of a HiBiT-tagged protein from target cells following cell lysis. Once released, HiBiT, an 11 a.a. peptide tag, binds to cell-impermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconstitute NanoBiT Luciferase. In the presence of furimazine substrate, the luminescent signal is proportional to the amount of target cell killing, and cell lysis can be quantified at a single time point using Maximum Release and Spontaneous Release controls. The assay is compatible with a wide range of target cells per well (e.g. 100-500,000 cells/well), and low levels of Spontaneous Release allow the sensitive detection of low levels of target cell lysis (e.g. 1-5%). Using a modified format, target cell killing can be measured continuously in a bench-top luminometer for 24 hours or more. We demonstrate these approaches using commercially available CAR-T cells and target cells stably expressing a HiBiT-tagged protein via random integration of plasmid DNA or viral transduction. We also demonstrate the use of a thaw-and-use format to facilitate MOA-based lot release assays. The approach provides simple, non-radioactive assay formats for selectively monitoring target cell killing in mixed culture experiments. Citation Format: Brock Binkowski, Aileen Paguio, Christopher Eggers, Braeden Butler, Michael Beck, Frank Fan. A novel, bioluminescent assay for the selective detection of target cell killing in mixed cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3181.