Abstract 3179: A comprehensive serial killing workflow for assessment of CAR T-cells

Abstract Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T cells) has been highly successful in individuals with hematological malignancies. However, its efficacy against most solid tumors remains elusive due to short-term persistence and inadequate expansion of CAR T-cells within the tumor microenvironment. Traditional in vitro cytolysis assays often fail to reflect the true antitumor potential of the CAR T cells, as they typically involve brief co-culture periods and assess CAR T-cell performance only from a single perspective. To address these limitations, we developed a long-term multiplexed workflow that can robustly and comprehensively assess CAR T cell products. In our study, the EpCAM CAR T-cells were repeatedly challenged with an EpCAM expressing breast cancer cell line, T47D cells, over seven consecutive rounds. During each challenge, the EpCAM T-cells were directly added to the T47 D cells cultured on an E-Plate VIEW 96 (E-Plate) for 24 hours, at the effector to target ratio (E:T) of 0.5 :1. The CAR T-cell cytotoxicity against T47D cells was continuously measured over three days using an xCELLigence RTCA eSight system (eSight). After each co-culture round, the CAR T-cell metabolism, immunophenotype and percentage were evaluated using Agilent XF Pro analyzer and NovoCyte flow cytometer respectively. Following assessment, the challenged CAR T-cells were transferred to a new tumor cell-containing E-Plate to repeat the assay sequence. Our results indicate that: 1) although the percentage of CAR T-cells progressively increased from 25% to 80% by the fifth challenge round, a significant decline was observed in the sixth round; 2) CAR T-cells exhibited a gradual reduction in proliferation and cytotoxicity against tumor cells, along with an increase in the PD1+Tim3+ population across repeated challenges; and 3) CAR T-cells demonstrated a distinct metabolic profile, characterized by progressively decreased maximal respiration, spare respiratory capacity (SRC), and ATP production rate throughout the successive challenges. In conclusion, this multiplexed workflow enables comprehensive evaluation of CAR T-cell proliferation, killing potency, exhaustion phenotype, and metabolic profile over repeated tumor cell challenges, providing a more accurate prediction of their in vivo therapeutic efficacy. Citation Format: Xiaoyu Zhang, Jyothi Thyagabhavan Mony, Natalia Romero. A comprehensive serial killing workflow for assessment of CAR T-cells [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 3179.