Generation of novel anti-tumor chimeric antigen receptors incorporating downstream T-cell signaling proteins

Abstract

Abstract Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.