Abstract
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.