Chimeric Antigen Receptors Transmit Piconewton Forces that are Coupled with T Cell Function

Abstract

Chimeric antigen receptor (CAR) T cells are engineered to display a receptor that binds antigens expressed on the surface of cancerous cells, which leads to cancer cytotoxicity. Recently, the T cell field has come to recognize the role of small, piconewton level forces in establishing specificity and cytotoxicity in naive T cells with the {beta} TCR, raising the possibility that these forces could be present in CAR T cells. Using DNA-based tension probes, we reveal 8-19 pN mechanical forces with [~]1 sec timescales transmitted by CAR T cells to their target antigens. CAR-antigen force magnitude is independent of CAR expression level and shows heterogeneity across different T cell donors, suggesting utility as a biomarker of T cell fitness. Using an established exhaustion model, we show strong correlation between CAR exhaustion, cytotoxic capacity, and CAR-antigen force, suggesting that CAR mechanics provide a biomarker of CAR potency complementary to functional assays. Pharmacological inhibition studies demonstrate that CAR forces are driven by actin, Zap70 and Src family proximal kinases. Titration of dasatinib, a clinically used tyrosine kinase inhibitor also dampens both CAR-antigen tension and CAR function in a dose-dependent manner. Structural engineering of the CAR confirms that force levels are modulated by the scFv receptor and co-stimulatory domains, but force transmission requires CD3{zeta} ITAMs. Taken together, this work shows that CAR T cells transmit pN force to their cognate antigens which holds potential significance in the design and screening of CAR therapeutic candidates and for predicting treatment outcomes in a personalized fashion.