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CAR T cells containing CD28 versus 4-1BB co-stimulatory domains show distinct metabolic profiles in patients

Researchers from King’s College London and collaborators have investigated how different CAR T products imprint distinct metabolic programs following infusion in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Published in Cell Reports, the study compared axicabtagene ciloleucel (axi-cel, CD28 co-stimulation) and tisagenlecleucel (tisa-cel, 4-1BB co-stimulation) to understand how co-stimulatory domains shape CAR T metabolism in vivo. The authors present this research as the first direct comparison of circulating CAR T-cell metabolic states between products, highlighting that successful therapy may depend less on a single pathway and more so on metabolic plasticity - the ability to flexibly use both glycolysis and oxidative phosphorylation.

CAR T-cell therapy has transformed the treatment of B-cell cancers, but upwards of 50% of patients relapse, often due to loss of persistence or functional exhaustion. Preclinical studies suggest CD28 signaling favors glycolysis and rapid effector activity, whereas 4-1BB signaling promotes mitochondrial fitness and central memory phenotypes. However, how these signatures manifest in patients following infusion has remained unclear. In this study, the investigators profiled metabolic markers, nutrient transporters, mitochondrial polarization, and functional pathway use by flow cytometry and SCENITH in peripheral blood CAR-T cells from 25 patients at days 7 and 14 post-infusion, linking results to six-month clinical outcomes.

The results showcased clear product-specific differences early following infusion: CD28-CAR T cells exhibited higher GLUT-1 expression, phospho-mTOR activity, and glycolysis dependence, while 4-1BB-CAR T cells displayed greater polarized mitochondria and oxidative phosphorylation reliance. Effector phenotypes (CD69, PD-1, CD57) were enriched in CD28 products, whereas memory-like features (CD62L) dominated in 4-1BB products. Of note, patients who achieved six-month responses demonstrated convergent, balanced metabolic profiles regardless of CAR design, while non-responders showcased exaggerated skewing toward glycolysis (CD28) or OXPHOS (4-1BB). These findings suggest that metabolic flexibility, rather than strict commitment to one pathway, underpins durable CAR T responses. Overall, this study introduces metabolic monitoring as a potential biomarker of CAR T efficacy and supports future strategies to engineer or modulate CAR T cells for greater metabolic adaptability.

Reference:

Cook MS, King E, Flaherty KR, et al. CAR-T cells containing CD28 versus 4-1BB co-stimulatory domains show distinct metabolic profiles in patients. Cell Rep. 2025;44(7):115973.

https://doi.org/10.1016/j.celrep.2025.115973