Abstract 3195: Advancing CAR T clinical development with high-parameter CyTOF technology

Abstract Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells is considered a recent revolutionary treatment in cancer therapy. CAR T therapy has achieved great success in hematological B cell malignancies. However, it has faced significant challenges in solid tumors due to various factors, such as complex tumor microenvironments, restricted trafficking, impersistent antitumor activity and toxicities. A better understanding of CAR T biology will accelerate development of CAR T therapies with improved antitumor efficacy, durability and decreased toxicities. High-parameter flow cytometry has been a powerful tool to functionally characterize CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. However, fluorescence-based cytometry faces significant challenges with signal overlap and autofluorescence, limiting sensitivity and the number of targets that can be detected in CAR T cells. Consequently, rare cell populations are poorly resolved and functional readouts of CAR T cells are unreliable. CyTOF technology overcomes these limitations with low signal overlap and no autofluorescence. Further, CyTOF technology enables a streamlined and flexible workflow in clinical research using freezing antibody cocktails and stained cell samples. Here, we present a 40-plus-marker CyTOF panel to simultaneously analyze phenotypic and functional protein expression in CAR T cells from in vitro co-culture with tumor cells. CD19 CAR T cells were expanded in vitro and co-cultured with Nalm6 cells at different E:T ratios for 2-5 days. A high-parameter CyTOF panel including over 40 surface, cytoplasmic and nuclear markers was used to stain CAR T cells. Samples co-cultured at different time points were stained, barcoded, frozen and simultaneously acquired on a CyTOF XT system at a later date. The cytotoxicity, activation, proliferation, differentiation and exhaustion of CAR T cells were evaluated. Comprehensive profiling revealed that CAR T cells became activated, proliferated and produced cytokines in in vitro co-culture with tumor cells and showed reduced cytotoxicity and exhaustive phenotype after an extended period of expansion. Overall, we demonstrate that the high-parameter CyTOF panel enables deep functional characterization of CAR T cells by simultaneous detection of surface, cytoplasmic and nuclear markers, supporting the continuous clinical development of CAR T products. Citation Format: Ling Wang, Shakir Hasan, Stephen Li, Michael Cohen, Deeqa Mahamed, Christina Loh. Advancing CAR T clinical development with high-parameter CyTOF technology [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 3195.