A novel and easy-to-use approach for assessment of chimeric antigen receptor (CAR) modified immune cell cytotoxicity

Abstract

Abstract Cancer immunotherapies have shown promising results for treating various cancers. One such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), thus combining tumor antigen specificity with immune cell activation in a single receptor. Currently, assessment of this CAR-modified immune cell cytotoxicity is limited by conventional tools (e.g., standard Chromium-51 release assay), which are time-consuming, costly, and labor-intensive. In this study, we developed a novel, easy-to-use, and non-radioactive approach for assessment of CAR-modified immune cell cytotoxicity on the basis of luciferase bioluminescent signal. After a 4- hour incubation of CAR-modified immune cells in a 96-Well optical-bottom microplate, which was pre-seeded with targeted cell stablely expressed eGFP -firefly luciferase fusion gene (GFP-FFluc), the chemical bioluminescent signal of GFP-FFluc was quantified by a fluorescent microplate reader. The positive correlation between FFLuc signal and number of target cells was observed. The dose-dependent specific lysis is comparable with Chromium-51 release assays. The cytotoxicity activity measured by this approach can be further quantified under a common inverted fluorescence microscope to evaluate the morphology and dynamics of CAR-modified immune cells. In conclusion, this newly developed approach provides the scientific community with a powerful tool to study the CAR-modified immune cell cytotoxicity.