Mitochondrial remodeling sustains CAR-NK stemness for superior tumor control

Background: NK cell-based therapies are a promising off-the-shelf strategy for cancer treatment. Their inherent safety and lack of graft-versus-host disease make NK cells an attractive platform for CAR engineering, combining innate cytotoxicity with receptor specificity. However, clinical efficacy in solid tumors is limited by poor persistence and rapid metabolic exhaustion in the tumor microenvironment (TME). Strategies that enhance metabolic fitness and delay differentiation are essential. Inspired by stem-cell culture systems, we tested whether Albumin Polyvinylalcohol Essential Lipids (APEL), a defined medium used to support iPSC differentiation into embryoid bodies and NK cells, could preserve CAR-NK stemness and improve metabolic fitness and function. Methods: CAR-NK cells expanded with APEL (CAR-NK stem ) were compared with CAR-NK cells expanded in conventional NK cell media (CAR-NK conv ). Metabolic profiling included mitochondrial mass and potential profiling, Seahorse assays to measure glycolysis and oxidative phosphorylation, nutrient competition assays to assess metabolic flexibility. CAR-NK cells were cultured with various hematologic and solid tumor cell lines, followed by cytotoxicity evaluation using 2D xCelligence and 3D Incucyte including rechallenge assays to assess serial killing capacity. In vivo efficacy was tested using various xenograft mouse models of hematologic cancers (MOLM14, MM1s) and solid tumors (786-0 ongoing). Mechanistic studies included multiomics profiling by bulk RNA-seq and ATAC-seq, CyTOF, and mitochondrial-transfer assays. Results: Our data show that early metabolic reprogramming of CAR-NK stem during expansion drove the development of enlarged and highly active mitochondria, increased spare respiratory capacity (SRC), and superior metabolic flexibility. This metabolic state stabilized stem-like features and limited terminal differentiation via chromatin remodeling toward persistence and memory-associated programs. Functionally, CAR-NK stem cells showed improved proliferation, sustained serial killing, superior nutrient competition, and enhanced tumor control in vitro and in vivo. Mechanistically, mitochondrial transfer from CAR-NK stem cells enhanced cytotoxicity and persistence in recipient NK cells, highlighting mitochondrial remodeling as a key driver of functional fitness. Conclusion: These findings demonstrate that targeted metabolic reprogramming during manufacturing can generate stem-like, highly persistent CAR-NK cells and offer a broadly applicable, immediately translatable strategy for cancer immunotherapy.