Real-time cytotoxicity assay using whole-well imaging for CAR-T cells targeting non-adherent tumor cells 3083

Description Introduction Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing T (CAR-T) cells have demonstrated remarkable effectiveness in hematologic malignancies. The complexity and variability associated with genetically engineered immune cells necessitates ex-vivo assays that are accurate and reproducible, as highlighted by the FDA, in its guidance for CAR-T development. Here, we present a live-cell imaging assay that quantifies target cell lysis as a measure of CAR-T cytotoxicity. Methodology: Cytotoxicity assays were performed on an Agilent xCELLigence RTCA eSight. Raji cells expressing green fluorescent protein were used as targets for unlabeled CD19-CAR-T and control T cells. CD19-CAR-T cells (E) were added to 12,000 Raji-GFP (T) cells at E:T of 2.0, 1.5, 1.0, 0.5 and 0.25, after 21 hours. Images were acquired at 220ms exposure, every 45 minutes, for 140 hours. Results Cytotoxicity increased significantly with increasing E:T. The maximum cytolysis increased from 68% (0.25) to 98% (2). The time to achieve 40% killing (KT40) increased from 2.5h (2) to 55h (0.25). Specific killing mediated by CD19-CART determined using control T cells as reference, increased significantly from 67% (0.25) to 82% (2.0). Conclusion Whole-well, live-cell imaging data from non-adherent cells can be acquired on RTCA eSight to determine the cytotoxicity potential of CAR-T cells. Topic Categories Technological Innovations in Immunology (TECH)