Low-affinity CAR-Tregs show enhanced activation and superior suppression in human and murine models

Chimeric antigen receptor (CAR) T cells are a powerful immunotherapy, but their application to regulatory T cells (Tregs) for autoimmune diseases and transplant rejection is still evolving. While high CAR affinity can lead to exhaustion in effector CAR-T cells, the optimal affinity for CAR-Tregs, which require sustained immunosuppressive function, remains poorly understood. Understanding this balance is crucial to prevent activation-induced exhaustion and maximize their therapeutic potential in conditions like graft-versus-host disease (GVHD).

Researchers transduced purified human Tregs with second-generation CAR constructs featuring single-chain variable fragments (scFvs) engineered for varying affinities to HLA-A2. They compared high-affinity (HA-CAR-Tregs) and low-affinity (LA-CAR-Tregs) in in vitro assays for avidity, CAR downregulation, antigen-specific activation, and suppressive capacity. In vivo function was assessed using human and mouse precision-cut liver slices, and a xenogeneic GVHD murine model, evaluating accumulation, persistence, GVHD onset, and survival.

High-affinity CAR-Tregs displayed higher avidity and more pronounced CAR downregulation upon antigen engagement. In contrast, low-affinity CAR-Tregs exhibited enhanced antigen-specific activation and superior suppressive capacity in vitro. These functional differences were confirmed in vivo. In the xenogeneic GVHD murine model, LA-CAR-Tregs demonstrated greater accumulation and persistence compared to HA-CAR-Tregs. This translated into significant clinical benefits: > LA-CAR-Tregs exhibited delayed GVHD onset and improved survival compared to HA-CAR-Tregs, highlighting the critical role of CAR affinity in therapeutic efficacy. The findings indicate that tuning CAR affinity can profoundly influence CAR-Treg function and therapeutic outcomes.