Abstract 2195: Development of a faster potency assay for evaluation of immune cell-mediated cytotoxicity in vitro

Abstract Assessment of the efficacy of CAR T cell therapy relies on robust in vitro potency assays that evaluate the interaction between CAR T cells and cancer cells. Traditional methods require extended incubation periods of target cells of over 24 hours, limiting throughput and efficiency in assay development. In this study, we aimed to significantly reduce assay time by utilizing an impedance-based system to monitor the cytotoxic activity of CAR T cells against SKOV3 cancer cells. By comparing the standard protocol with an expedited approach that allows for a shorter resting period of target cells, we aim to establish a more efficient framework for potency testing that maintains accuracy, while facilitating faster advancements in CAR T cell therapies. In addition to shortening the overall assay time, adding CAR T cells at an earlier timepoint also ensures that the true Effector to Target (E:T) ratios are maintained by reducing the time for target cell doubling. Here, we describe an in vitro potency assay that shortens the assay time course from 72+ hours to 24 hours for immune cell-mediated cytotoxicity. SKOV3 target cells were seeded at 10k cells per well into a 96-well microplate with embedded electrodes in the substrate that detect the attachment and proliferation of target cells. HER-2 CAR T cells were added at either 4 or 24 hours post SKOV3 cell seeding at multiple E:T ratios (1:10, 1:5, 1:2, 1:1, and 5:1). The killing kinetics among each group were directly compared by calculating the Kill Time 50 (KT50), or the time required for 50% of the target cells to be killed. The KT50 for target cells treated at 4 hours was 91.6 ± 3.7 (1:10), 47.8 ± 22.7 (1:5), 19.8 ± 1.8 (1:2), 16.6 ± 0.9 (1:1), and 9.1 ± 0.7 (5:1) hours, while the KT50 for target cells treated at 24 hours was 150.0 ± 3.6 (1:10), 115.6 ± 7.1 (1:5), 83.8 ± 4.5 (1:2), 48.7 ± 2.5 (1:1), and 29.2 ± 0.8 (5:1) hours. As expected, the well groups treated with CAR T cells at 4 hours demonstrated faster KT50 times due to less SKOV3 cell proliferation and, therefore, more accurate E:T ratios. At 24 hours post CAR T addition, a dose response curve was generated and the EC50 was determined to be an E:T ratio of 1:2.4 for target cells treated 4 hours post seeding and E:T ratio of 1:1 for target cells treated 24 hours post seeding. Overall, the data described illustrates a label-free, in vitro workflow to assess potency of CAR T cells in a quicker, more efficient manner using the Maestro Z impedance assay. Citation Format: Danielle Califano, Ben Streeter, Sierra Barnes, Denise Sullivan, Daniel Millard, Stacie Chvatal. Development of a faster potency assay for evaluation of immune cell-mediated cytotoxicity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2195.