Redirecting Cholesterol Metabolism During Ex Vivo Biomanufacturing Enhances the Metabolic Fitness and Antitumor Efficacy of CAR-T Cells.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized the treatment of hematologic malignancies; however, its efficacy in solid tumors remains limited, partly due to T cell exhaustion during ex vivo manufacturing. Emerging evidence suggests that cholesterol metabolism plays a critical role in T cell differentiation and function, yet its impact during CAR-T cell production is poorly understood. We investigated the effects of cholesterol modulation during ex vivo CAR-T cell expansion by using low-dose fluvastatin (FL), a clinically approved HMG-CoA reductase inhibitor. We found that cholesterol accumulation during ex vivo expansion promotes CAR-T cell exhaustion. Low-dose FL reduces cholesterol to physiological levels, preserving a less-differentiated, memory-enriched phenotype and attenuating exhaustion, thereby enhancing CAR-T cell cytotoxicity and persistence without affecting viability. In multiple xenograft models, FL-primed CAR-T cells demonstrate superior in vivo expansion, persistence, and tumor control. Mechanistically, FL enhances ERK1/2 phosphorylation to remodel CAR-T cell metabolism from glycolysis to oxidative phosphorylation. Inhibiting ERK1/2 or ATP synthesis abrogates these benefits, indicating that ERK1/2-dependent mitochondrial metabolism is required for CAR-T cell functional improvements conferred by FL. These findings establish cholesterol metabolism as a tunable axis during CAR-T cell manufacturing and propose a clinically feasible, GMP-compatible strategy to enhance CAR-T cell fitness and therapeutic efficacy.