Mapping real-time metabolic kinetics of expanded CAR T cells using hyperpolarized 13C-glucose and metabolomics

Chimeric Antigen Receptor (CAR) T cell therapy has advanced cancer treatment, providing options for patients with refractory hematological malignancies. Understanding the metabolic changes in CAR T cells during their activation, proliferation, and therapeutic application is essential for improving efficacy. In this study, human CD19 CAR T cells were expanded from three healthy donors under a standard manufacturing protocol. Hyperpolarized ¹³C dDNP-NMR spectroscopy was used to measure glycolytic flux at key time points (days 1, 7, 14, and 21), and ¹H NMR metabolomics was employed to monitor nutrient depletion in the culture medium. The results showed that CAR T cells underwent a metabolic transition from oxidative phosphorylation to aerobic glycolysis by day 7, followed by a return to oxidative phosphorylation by day 21. Glucose depletion was most pronounced during the first week, whereas amino acids were substantially consumed, with a decline in consumption observed after day 12. Real-time NMR spectroscopy revealed large metabolic flux changes that metabolomics did not capture. This study demonstrates the dynamic metabolic plasticity of CAR T cells and highlights the utility of hyperpolarized ¹³C-NMR for tracking glycolytic activity. Addressing metabolic bottlenecks, such as nutrient depletion during early expansion, may improve CAR T cell manufacturing and therapeutic outcomes.