Abstract 1182: New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity

Abstract

Abstract The number of immunotherapeutic strategies for treating cancer have expanded in recent years, increasing the need for assays that can be used to assess their effectiveness. To facilitate studies of these immunotherapies, we developed bioluminescent methods that could rapidly and sensitively measure target cell cytotoxicity. Two approaches were developed. The first method utilizes a bioluminescent enzymatic activity assay for a well-accepted cytotoxicity marker, lactate dehydrogenase (LDH), which is rapidly released from dead cells. With the improved sensitivity of the bioluminescent assay, lower levels of LDH can be measured, resulting in the detection of fewer dead cells (LOD< 10 cells). Cytotoxicity in both 2D and 3D cell cultures can be monitored over time by removing samples of medium at different time points. Here we present examples in which this assay was used to test the effectiveness of immunotherapeutic treatments including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In ADCC models, cell killing was detectable at early time points (by 2 hours). Increases in the cytotoxicity of individual samples could be followed over time, from early time points to 24 hours. In the second approach we developed a novel bioluminescent assay based on the specific labeling of target cells. With this method, intracellular proteins in live target cells are covalently labeled. Upon target cell death, the labeled proteins are released from the cell and the label is quantitated using bioluminescence detection reagents. Using this assay, we were able to quantitate the killing of target cells in both ADCC and CAR-T applications. By adding the detection reagents directly to the medium at the start of the ADCC reaction, cell death was monitored in real-time for up to 6 hours. Neither assay requires genetic engineering and both involve no or minimal target cell preparation. They are applicable to multiple cell types and the non-lytic nature of these approaches provides kinetic capabilities and opportunities for multiplexing. The ability to rapidly quantitate cytotoxicity will facilitate the functional analysis of immunotherapies. Citation Format: Maggie L. Bach, Natasha Karassina, Peter Hofsteen, Donna Leippe, James Cali, Jolanta Vidugiriene. New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity [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 1182.