Abstract
Introduction: T cell hematological malignancies are aggressive blood cancers that remain challenging despite various treatments. Current chimeric antigen receptor (CAR)-T and natural killer (NK) therapies show potential but struggle with nonselective elimination during tumor targeting. Since CAR signal strength is determined by the single-chain variable fragment (scFv) and CAR expression levels, fine-tuning these parameters enables selective recognition of malignant cells while preserving normal cells. Here, we aimed to develop optimized CD5 CAR-NK cells (OptiCAR-NK) to achieve potent anti-tumor activity with minimized off-tumor toxicity. Methods: We engineered CD5 CAR-NK cells with different scFv and CAR expression levels. CAR expression was modulated by single-cell isolation and mRNA transfection to assess activity against both malignant and normal T cells in vitro. Therapeutic efficacy and safety were further validated in xenograft and humanized mouse models. Results: Optimization of scFv and CAR expression levels (OptiCAR-NK) enabled selective recognition of CD5+ malignant T cells while maintaining strong anti-tumor activity with minimal toxicity. Mechanistic analysis revealed that NK cells' innate ability to discriminate malignant from normal T cells depends on fine-tuned CAR signal strength and endogenous ligands on target cells. Discussion: Optimized modulation of scFv and CAR expression is crucial for designing a CAR that achieves high anti-cancer efficacy and is safe in normal cells. Our results suggest a promising avenue for optimized CD5 CAR-NK cell therapy to manage T cell malignancies while minimizing off-tumor effects.