Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function

Abstract

Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3 or 4-1BB/CD3 signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3 and 4-1BB/CD3 CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3 CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell–like phenotype and function. In contrast, 4-1BB/CD3 CAR T cells preferentially expressed T cell memory–associated genes and exhibited sustained antitumor activity against established tumors in vivo. Muta-genesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3 CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus, tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.