Abstract
CAR T cell (CART) therapy holds promise for cancer treatment, but heterogeneity among products limits clinical effectiveness, making systematic profiling essential to identify predictors of success. Recently, a phase 1 clinical trial investigated whether a constitutively active IL-7 receptor (C7R) could safely improve the function and persistence of GD2-directed CARTs (GD2.CARTs) in pediatric patients with high-grade CNS tumors. We analyzed infusion products from trial participants using a custom-designed 33-color full spectrum flow cytometry (FSFC) panel combined with an image-based tumor killing assay to characterize CART products and evaluate the impact of C7R on GD2.CART performance. Patient-specific variations in T cell composition were linked to therapeutic success, with C7R co-expression enhancing the functional phenotype of GD2.CARTs compared to CAR-only products. Unsupervised clustering identified CD8+ T cells associated with clinical responses, marked by activation, infiltration, resilience, and cytotoxicity. Our FSFC-based profiling approach reveals determinants of CART efficacy and supports strategies to optimize adoptive immunotherapy.