Tuning the Affinity of Chimeric Antigen Receptors Enhances the Function of Human Engineered Regulatory T Cells

Chimeric antigen receptors (CAR) incorporating single-chain variable fragments (scFv) exhibit much higher affinity for their cognate antigens than native T cell receptors (TCR). Reducing the affinity of the CAR has been shown to improve the function of effector CAR-T cells by limiting their activation-induced exhaustion while preserving their capacity for serial killing of antigen-rich tumor cells. CAR technologies are increasingly being applied to regulatory T cell (Treg)-based immunotherapies as well. However, the impact of CAR affinity on Treg biology and function remains poorly understood. To address this question, we transduced purified human Tregs with second-generation CAR constructs bearing scFvs with varying affinities toward HLA-A2 and compared their properties both in vitro and in vivo. High-affinity (HA) CAR-Tregs displayed higher avidity and more pronounced CAR downregulation upon antigen engagement. In contrast, low-affinity (LA) CAR-Tregs exhibited enhanced antigen-specific activation and superior suppressive capacity. These differences were confirmed using human and mouse precision-cut liver slices and a xenogeneic graft-versus-host disease (GVHD) murine model. LA CAR-Tregs exhibited greater accumulation/persistence, delayed GVHD onset, and improved survival than HA CAR-Tregs. Our findings, indicating that CAR affinity strongly influences CAR-Treg function, provide important considerations for the optimization of engineered Treg therapies and the benchmarking of existing cell products.