Tu159 - Armoured CAR Tregs with PD1 Promoter-driven IL-10 Have Enhanced Suppressive Function

Abstract Text: Adoptive transfer of regulatory T cells (Tregs) is currently under clinical investigation to treat autoimmunity and transplant rejection. Clinical trials have confirmed the safety and feasibility of this approach, and mouse studies have shown that Treg therapy has increased efficacy when cells are given disease-relevant specificity via expression of chimeric antigen receptors (CARs). However, CAR-Tregs have yet to induce long-lasting tolerance in mice, thus suggesting the need for additional cell engineering. To this end, we aimed to engineer CAR-Tregs to remove PD1, a negative regulator of Treg activation, and introduce IL-10 production under control of the PDCD1 (PD1) promoter. Human CAR-Tregs were generated through lentiviral transduction with an HLA-A2-specific CAR. Homology directed repair at the PDCD1 locus was subsequently used to knock-out PDCD1 and introduce IL10 and a constitutively-expressed, truncated CD19 (tCD19) reporter. Following transduction, CAR-Tregs maintained high expression of key Treg markers, including FOXP3 and Helios. PD1 deletion enhanced CAR-stimulated Treg activation potential. Knock-in of IL10 into the PDCD1 locus resulted in CAR-stimulated expression of IL-10 at levels exceeding those produced by type 1 regulatory (Tr1) cells. RNA sequencing data showed that Treg identity remained intact and distinct from Tr1 cells. IL-10-expressing CAR-Tregs also had improved suppressive function, with superior control of dendritic cells as well as alloantigen- and islet autoantigen-specific T cell activity. When tested in a humanized mouse model of graft versus host disease, IL-10-expressing CAR-Tregs were stable and potently suppressive. Together, these data show that IL-10-armoured CAR-Tregs could enhance the clinical efficacy of Treg therapies.