Constitutive RLI Armoring Enhances CAR-NK Cell Effector Functions but Causes Lethal Toxicity In Vivo

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is a promising immunotherapy for hematological malignancies. While engineered interleukin15 (IL15) variants like membrane-bound IL15 (mbIL15) and the IL15/IL15Rα heterodimer (RLI) can enhance NK cell activity, their relative efficacy and safety as armor for CAR-NK cells remain unclear. This study systematically evaluated primary human CAR-NK cells co-expressing an anti-CD19 CAR (19ζ) with soluble IL15, mbIL15, or RLI. We found that 19ζ-RLI CAR-NK cells exhibited superior IL15 secretion, proliferation, cytotoxicity, and migration in vitro, and effectively controlled tumors in vivo. However, all IL15-armored constructs, particularly 19ζ-RLI, induced lethal toxicity in mice, characterized by CAR-NK hyperproliferation and elevated systemic IL15. Transcriptomic analysis revealed that this toxicity correlated with a hyperactive molecular state driven by persistent IL15 signaling. In conclusion, this study suggests that constitutive IL15 armoring can be a potent but risky strategy for enhancing CAR-NK cells, with RLI being the most potent yet toxic exemplar of this general principle. Our findings highlight the necessity of incorporating safety-optimized strategies, such as inducible cytokine expression, into the design of cytokine-armored CAR-NK therapies for clinical translation.