CD3ζ ITAM diversity determines chimeric antigen receptor force and function

Abstract Chimeric antigen receptor (CAR) T cell immunotherapy is a powerful treatment for hematologic malignancies, however clinical responses vary depending on CAR T cell function and persistence. The optimization of CAR design has gained considerable interest with a new focus on calibrating CAR intracellular signals via the immunoreceptor tyrosine-based activation motifs (ITAMs) to increase fitness of CAR T cells. Accumulating evidence suggests that unique ITAM motifs at positions 1, 2 or 3 (ITAMs-A, B, and C respectively) of the CD3ζ signaling domain may have non-redundant roles in signal strength, T cell functionality, persistence, and memory formation. However, the individual contributions of each CD3ζ ITAM in the transmission of force and the activation of mechanosensitive signals is yet unexplored. We have found that human CARs expressing specific ITAM sequences (zeta-A only, zeta-B only or zeta-C only) generate differing amounts of Force and Bond Lifetime after interacting with CD19. Therefore, a CAR may act as a mechanosensor similar to a TCR discriminating the quality of the interaction with antigen as cellular derived forces are applied to the bond. The higher transmission of force directly correlates with the better anti-tumor efficacy in vitro and in vivo. Hence, understanding the mechanotransduction in CARs has important implications in “tuning” the activation of a CAR T cell without changing specificity. Supported by the Seed Grant, Department of Pathology, University of Utah