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

Yao, B.; Ye, X.; Kong, Q.; Chen, W.; Li, W.; Feng, C.; He, A.; Li, G.; Chen, L.; Chen, X.; Hu, L.; Xie, L.; Qiu, X.; Wang, X.; Lin, Y.; Cao, Y.; Zhou, J.; Zhang, X.; Wang, H.; Tian, R.

doi:10.1101/2024.12.22.629940

bioRxiv · 10.1101/2024.12.22.629940

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

Yao, B.·Ye, X.·Kong, Q.·Chen, W.·Li, W.·Feng, C.·He, A.·Li, G.·Chen, L.·Chen, X.·Hu, L.·Xie, L.·Qiu, X.·Wang, X.·Lin, Y.·Cao, Y.·Zhou, J.·Zhang, X.·Wang, H.·Tian, R.

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Abstract

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 CD28{zeta} and 4-1BB{zeta} CAR-T cells exhibited comparable pTyr changes, CD28{zeta} 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.

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Yao, B., Ye, X., Kong, Q., Chen, W., Li, W., Feng, C., He, A., Li, G., Chen, L., Chen, X., Hu, L., Xie, L., Qiu, X., Wang, X., Lin, Y., Cao, Y., Zhou, J., Zhang, X., Wang, H., Tian, R.. 2024-12-22. Phosphotyrosine Signal Profiling of Clinical CAR-T Reveals Tonic Signaling Associated with Therapeutic Efficacy. https://doi.org/10.1101/2024.12.22.629940

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