*Purpose: Owing to their potent immune-modulatory properties, regulatory T cells (Tregs) have been explored as novel cell therapy to induce allograft tolerance, with first clinical trials conducted in kidney and liver transplant patients. Preclinical data revealed, however, a preferential trafficking of antigen-specific Tregs to the site of inflammation with enhanced suppressive function. Genetically engineering Tregs to express a synthetic chimeric antigen receptor (CAR) is a highly effective tool to confer antigen-specificity. HLA-A2 is the target molecule currently employed in the clinical development of CAR-Tregs, which restricts its applicability to HLA-A2 mismatched donor-recipient combinations. To fully exploit the potential of CAR-Tregs as anti-inflammatory treatment for a wider range of patients, we propose to develop Tregs with specificity for the liver. We hypothesise that these Tregs will preferentially home to the transplanted liver and create a compartmentalized immune-tolerant environment.

*Methods: Nine second-generation CAR constructs were designed, each differing in the single-chain variable fragment composition, hinge domain or protein tag, but all specific for antigens preferentially expressed by parenchymal liver cells. Human CD4⁺CD25⁺CD127^–Tregs were FACS isolated and stably transduced using lentiviruses. The CAR-Treg’s antigen-specificity and function were then investigated with activation, proliferation and suppression assays in vitro using antigen-positive cell lines. In vivo, the hepatic tissue homing capacity, persistence and survival of CAR-Tregs were evaluated in immunocompromised NSG mice.

*Results: Two out of the nine liver-specific CARs were selected according to their specificity and activation profile following antigen recognition. In comparison to polyclonal Tregs, CAR-Tregs showed an antigen-dependant increase in activation marker, proliferative capacity as well as suppressive function in vitro. Following infusion into NSG mice, CAR-Tregs were preferentially migrating to the liver and intra-hepatic persistence was detected for up to 20 days post-infusion.

*Conclusions: Organ-specific CAR-Tregs are an alternative approach to favour Treg recruitment to the target tissue with wide applicability in transplantation as well as other inflammatory liver diseases.

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