Multiplexed assessment of CAR T-cells over in vitro tumor cell rechallenges 2880

Abstract Description Chimeric Antigen Receptor (CAR) T cell efficacy against solid tumors remains limited due to poor persistence and expansion within the tumor microenvironment. Traditional in vitro cytolysis assays are often insufficient, as they involve short assay time and assess CAR T-cell performance from a single perspective. To overcome these challenges, we developed a long-term multiplexed workflow to assess CAR T-cell products comprehensively. In our study, EpCAM CAR T-cells were repeatedly challenged with EpCAM-expressing T-47D breast cancer cells over seven rounds. In each round, T-cells were added to tumor cells cultured on an E-Plate VIEW 96 at an effector-to-target ratio of 0.5:1. Cytotoxicity was continuously measured over three days using the xCELLigence RTCA eSight system. Post-challenge, CAR T-cell metabolism, immunophenotype, and percentage were analyzed using Agilent XF Pro and NovoCyte flow cytometer before the next round of challenge. Key findings include: 1) CAR T-cell percentages increased from 25% to 80% by round five but declined significantly in round six; 2) proliferation and cytotoxicity reduced over time, accompanied by an increase in the exhausted PD1+Tim3+ population; and 3) metabolic profiles revealed reduced maximal respiration, spare respiratory capacity, and ATP production rates across challenges. This workflow thoroughly evaluates CAR T-cell functionality, exhaustion, and metabolism, providing better insights into their therapeutic potential in vivo. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)