Single-day nonactivated IL-18-armed CAR T cells establish a durable, stemlike state with enhanced persistence.

abstractChimeric antigen receptor (CAR) T-cell therapies have transformed the treatment of B-cell malignancies, yet challenges including manufacturing delays, T-cell exhaustion, and limited persistence impede broader clinical success. Here, we report the single-day production of nonactivated CAR T cells engineered to secrete interleukin-18 (IL-18), a proinflammatory cytokine that enhances T-cell function. These nonactivated CART19-IL-18 cells (IL-18-secreting anti-CD19 CAR T cells) exhibit robust antitumor efficacy across xenograft models of lymphoma, leukemia, and pancreatic cancer. IL-18 expression enhances the functional advantages of naïve-like nonactivated CAR T cells, resulting in improved persistence, metabolic fitness, and resistance to exhaustion. Single-cell transcriptomic analysis revealed upregulation of IL7R, KLF2, and MCL1, alongside suppression of inhibitory checkpoint genes such as PDCD1, TOX, and HAVCR2. Metabolomic profiling demonstrated enhanced mitochondrial bioenergetics, with increased spare respiratory capacity and accumulation of α-ketoglutarate, malate, and spermine. Functional in vitro and in vivo profiling demonstrated enhanced per-cell cytotoxicity and in vivo durability. We complemented these studies with single-cell transcriptomic and metabolomic analyses to define CAR T-cell biological states beyond what is captured by xenograft tumor clearance. This IL-18-enhanced, activation-free CAR T-cell product offers a clinically actionable platform with the potential to reduce vein-to-vein time while improving product potency and persistence, providing a rationale for clinical testing in patients with tumors refractory to standard CAR T-cell therapy.