Membrane positioning across antigen-induced synaptic contacts tunes CAR-T cell signaling and effector responses

Tumor antigen recognition by chimeric antigen receptors (CAR) triggers phosphorylation of their cytoplasmic portions resulting in CAR-T cell activation. We and others have shown that immunoreceptor triggering depends on the formation of close synaptic contacts, determined by the span of immunoreceptor-ligand complexes, from which large inhibitory phosphatases such as CD45 are sterically excluded. Here, we show, varying CAR-antigen complex span, that CAR-T cell activation depends on a formation of close contacts with target cells. CAR-antigen complexes with a span of 4 immunoglobulin superfamily (IgSF) domains maximize CAR-T cell activation, closely matching the span of endogenous TCR-pMHC complexes. Longer CAR-antigen complexes precipitously reduced triggering and cytokine production, but notably, anti-tumor cytotoxicity was largely preserved due to a ∼10-fold lower signaling threshold for mobilization of cytolytic effector function. Increased intermembrane spacing disrupted short-spanned PD-1-PD- L1 interactions, reducing CAR-T cell exhaustion. Together, our results show that membrane positioning across the immunological synapse can be engineered to generate CAR-T cells with clinically desirable functional profiles in vitro and in vivo .