Single-day non-activated IL-18-armed CAR T cells with enhanced persistence and durable efficacy Free

Background: Chimeric 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 development and preclinical evaluation of a 1-day, non-activated CAR T cell product engineered to secrete interleukin-18 (CART19-IL18), a pro-inflammatory cytokine that enhances T cell function, manufactured without T cell activation or expansion, and designed for clinical translation in B cell malignancies.

Methods: Human T cells were transduced with a lentiviral vector encoding a CD19- or mesothelin-targeting CARs co-expressing IL-18 or GFP, without prior stimulation and expansion, and harvested after 24 hours of transduction. Phenotypic and functional analyses were conducted in vitro using flow cytometry, cytokine secretion assays, cytotoxicity assays, metabolomics, and Seahorse analysis. In vivo efficacy was evaluated in xenograft models of B cell leukemia (Nalm6), lymphoma (JeKo-1), and pancreatic cancer (AsPC-1). Single-cell RNA-sequencing and metabolomics was performed to profile cell state and exhaustion. Comparison groups included traditionally manufactured activated CAR T cells (3-days), and non-activated CAR T cells without IL-18.

Results: IL-18 expression significantly improved tumor control by non-activated CAR T cells across all models tested, despite lower CAR transduction rates (~10%). IL-18 synergized with the naïve-like phenotype of non-activated T cells, promoting expression of memory and survival-associated genes (IL7R, KLF2, and MCL1) while suppressing of inhibitory checkpoint regulators (TOX, PDCD1, and HAVCR2). These cells retained potent cytolytic function despite reduced terminal differentiation. Gene set enrichment revealed enhanced biosynthetic, mitochondrial, and telomere maintenance pathways. Metabolomic profiling of mouse serum revealed increased serum levels of α-ketoglutarate and spermine in non-activated CAR T19-IL18 treated animals, correlating with upregulation of key metabolic enzymes (SAT1, GLS, SLC38A1) in recovered CAR T cells; supporting oxidative metabolism and redox balance. Seahorse analysis confirmed increased spare respiratory capacity and basal respiration in non-activated CART19-IL18 cells, supporting a bioenergetically favorable metabolic profile.

In leukemia and lymphoma xenograft models, CART19-IL18 demonstrated significantly enhanced tumor clearance and persistence, even when administered at lower doses. IL-18 expression was critical for in vivo efficacy, particularly in the non-activated manufacturing context. Functional assays confirmed superior cytotoxicity and metabolic fitness of CART19-IL18 cells. These findings were consistent across multiple donors and tumor models.

Functionally, these cells preserved cytolytic activity, memory-associated transcriptional programs, and exhaustion resistance. This phenotype emerged in the absence of exogenous stimulation, suggesting that avoiding supraphysiologic TCR signaling preserves intrinsic T cell fitness. The 1-day non-activated manufacturing approach bypasses conventional activation steps, accelerates production, reduces reagent use, and allows for fresh product infusion. These features have the potential to expand access and improve outcomes in patients with rapidly progressing or post-CAR T relapsed malignancies.

Conclusion: We have developed a novel, non-activated CART19-IL18 product using a 1-day manufacturing protocol that preserves key T cell features critical for antitumor immunity. This approach builds directly on the clinical success of the activated CART19-IL18 trial. The rapid, activation-free protocol not only simplifies manufacturing logistics but also yields a more potent product. These results support the clinical translation of 1-day CART19-IL18 cells as a next-generation CAR T cell therapy.