Palbociclib and rapamycin pretreatment boosted the persistence and functionality of CAR-T cells in solid tumors through metabolic reprogramming

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable success in treating hematologic malignancies; however, its efficacy against solid tumors remains limited due to the immunosuppressive tumor microenvironment. Enhancing CAR-T cell persistence and function through the use of culture media additives offers a potential strategy to overcome these challenges. We conducted a systematic evaluation of CAR-T cell culture media additives and selected five FDA-approved compounds—decitabine, palbociclib, metformin, enasidenib, and rapamycin—based on their diverse mechanisms of action and clinical safety profiles. Anti-mesothelin (MSLN) CAR-T cells were generated and assessed under hypoxic in vitro conditions and in two subcutaneous solid tumor mouse models. The effects of individual and combinatorial additive treatments on CAR-T cell function were evaluated. To elucidate the underlying mechanisms, we performed RNA sequencing and metabolic analyses. All five additives partially reversed hypoxia-induced dysfunction in CAR-T cells, with palbociclib, enasidenib, and rapamycin demonstrating the most significant enhancement of cytotoxicity in both 2D and 3D assays. Notably, the combination of palbociclib and rapamycin (Pal + Rap) yielded pronounced additive improvements in CAR-T function, markedly enhancing CAR-T cell persistence, tumor-killing activity, and resistance to tumor recurrence in both HCC1806 and Capan-2 xenograft models. Transcriptomic and metabolic profiling showed concurrent upregulation of oxidative phosphorylation and improved in vivo anti-tumor function in Pal + Rap-preconditioned hypoxic CAR-T cells. Preconditioning CAR-T cells with palbociclib and rapamycin enhances their metabolic fitness and anti-tumor efficacy under hypoxia, offering a simple and clinically feasible strategy to improve CAR-T therapy against solid tumors.