Research ArticleCAR T CELLS

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

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Sci. Signal.  21 Aug 2018:
Vol. 11, Issue 544, eaat6753
DOI: 10.1126/scisignal.aat6753

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Strength limits potency

T cells engineered to express chimeric antigen receptors (CARs) may represent a curative treatment for B cell malignancies. To understand how to optimize these therapies, Salter et al. used phosphoproteomics to compare the activities of CARs encoding CD28 or 4-1BB costimulatory domains in primary human T cells. Although CARs with CD28 domains provoked more robust signaling than did CARs with 4-1BB domains (due to constitutive Lck association), both CARs activated overlapping T cell signaling pathways. However, CARs that initiated stronger signals also exhibited increased T cell dysfunction, which reduced their potency in a mouse model of lymphoma. These data indicate that the CAR costimulatory domain does not predict the signaling pathways activated in CAR T cells but instead suggest that reducing the strength of signaling may counterintuitively enhance CAR T cell therapeutic efficacy.

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/CD3ζ or 4-1BB/CD3ζ 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/CD3ζ and 4-1BB/CD3ζ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3ζ 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/CD3ζ CAR T cells preferentially expressed T cell memory–associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3ζ 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.

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