Abstract 868: Platform for purifying serially cytotoxic T cells: advancing cell engineering and in vivo applications

Abstract Current techniques for the enrichment of engineered T cells rely on surface markers that do not accurately capture a T cell’s exhibited cytotoxicity, specificity for antigens, or its temporal state in the activation-exhaustion cycle. While microscopy-based methods have been developed to assay live T cells based on their interaction and cytotoxicity against antigen-presenting cells (APCs), no single technique can isolate serially cytotoxic T cells based on the frequency and the cytotoxic outcome of their interaction with specific APCs. The addition of an isolation step based on cell behavior is imperative for pooled screening for optimized engineering of T-cells as well as for the utilization of the enriched cells in vivo. Narwhal Bio Inc.'s A1 platform, an instrument, software, and reagent combination, seeks to overcome this challenge by in vitro cell identification, tagging, and downstream isolation of live cells based on behaviors assessed through microscopy. This live-cell-in, live-cell-out process could enable downstream applications like animal studies, further cell engineering, and multiomics drug and target discovery. We have applied this system to enrich CAR-T cells involved in repeated cytotoxic events. We assayed and tagged T cells based on their serial cytotoxicity in an APC co-culture in real time. We then isolated the tagged T cells, assessed their activation state using CD69 and CD25, and confirmed enrichment for the CAR representation in the more cytotoxic cell population. We assessed the potency of the enriched T cells after sorting, culture, and subsequent co-culture with APCs, showing >3x improvement in potency. Lastly, we illustrated how the process can be performed on the order of millions of cells in a day for various in vivo and in vitro downstream applications, including pooled screening of T-cell engineering and use in animal models. Citation Format: Eric Ferguson, John M. Beierle, Behrad Azimi. Platform for purifying serially cytotoxic T cells: advancing cell engineering and in vivo applications [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 868.