Chimeric antigen receptor (CAR) cell therapy has revolutionized the treatment of specific hematologic malignancies. However, a significant portion of patients experience relapse because of antigen loss, antigen downregulation, or T cell exhaustion. These challenges highlight the need for functional assays that can evaluate the killing capacity and persistence of CAR T cells under chronic antigen stimulation. Serial killing assays, which measure the ability of CAR T cells to repeatedly eliminate tumor targets, offer valuable insights into the durability and potency of CAR T cell responses. Here, we present an impedance-based assay using the Real-Time Cell Analysis (RTCA) system to quantify CAR T cell-mediated serial killing in vitro. Tumor cells are repeatedly seeded and allowed to adhere to assay-specific E-plates before the addition of CAR T cells at defined effector-to-target (E:T) ratios. The platform continuously monitors tumor cell viability without labels, capturing dynamic cytotoxicity with high temporal resolution. Core readouts include Cell Index (CI) kinetics, tumor-cell killing rate, and time-to-target clearance. The progressive decline in killing capacity observed upon repeated tumor-target engagements serves as a marker of acquired CAR T cell dysfunction, often termed T cell exhaustion. Together, these metrics allow precise evaluation of CAR T cell function at various E:T ratios and enable direct comparison among different CAR T cell constructs or co-treatments over time. To enhance cost efficiency, we developed a plate-washing procedure that enables the reuse of assay E-plates without compromising assay performance or data integrity. The optimized workflow reduces assay cost while preserving analytical robustness. This approach enables affordable and scalable preclinical assessment of CAR T cell function, facilitating improvements in cell-therapy design.
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