Phosphotyrosine Signal Profiling of Clinical CAR-T Reveals Tonic Signaling Associated with Therapeutic Efficacy

Chimeric Antigen Receptor T (CAR-T) therapies have revolutionized the treatment of cancers such as relapsed and refractory B cell malignancies. However, the precise therapeutic mechanism of CAR-T action remain to be elucidated. In this study, we systematically analyzed CAR signaling via phosphotyrosine (pTyr) proteomics in CAR-T cells from both clinical patients and healthy donors. We found that CAR-T products from clinical patients displayed heightened tyrosine phosphorylation, particularly in the JAK-STAT, MAPK and TCR signaling cascades. We also identified the significantly regulated pTyr sites in primary CAR-T cells under tonic signaling or upon stimulation by antigen-presenting CD19-K562 cells. Although both CD28ζ and 4-1BBζ CAR-T cells exhibited comparable pTyr changes, CD28ζ CAR-T cells displayed a more pronounced activation in the TCR signaling pathway. Additionally, comparative analysis between clinical and primary CAR-T cells suggested that CAR-T products were subject to heightened tonic signaling, which may be related to therapeutic relapse. Our findings reveal the state of clinical CAR-T products and refine the CAR-T signal transduction network, providing comprehensive insights and informed guidance for CAR-T therapies.