HLA-A2 CAR/IL-2-CISC engineered Treg display robust in vitro and in vivo antigen-specific regulatory function

Chimeric antigen (Ag) receptor-expressing T regulatory cells (CAR-Treg) offer therapeutic potential for treating autoimmunity, allograft rejection, and graft-versus-host disease (GvHD). HLA-A∗02 (A2) CAR (A2CAR)-expressing natural Treg have shown efficacy in preclinical models and are being evaluated in phase 1/2 trials. In the current study, we utilized homology-directed-repair (HDR)-based gene editing to generate A2CAR-expressing engineered Treg (EngTreg). HDR at the <i>FOXP3</i> locus in bulk CD4⁺ T cells was used to enforce stable co-expression of endogenous FOXP3 and a chemically inducible interleukin (IL)-2 signaling complex (CISC or IL-2 CISC). A2CAR expression was achieved by lentiviral transduction or via dual-HDR editing targeting A2CAR to the <i>TRAC</i> locus. A2CAR⁺ CISC⁺ EngTreg (A2CAR EngTreg) products displayed a Treg immunophenotype, low secretion of pro-inflammatory cytokines in response to stimulation, and low cytotoxicity toward A2⁺ target cells <i>in vitro</i>. In a xenogeneic GvHD model driven by human A2⁺ peripheral blood mononuclear cells, A2CAR EngTreg showed superior therapeutic efficacy compared with polyclonal EngTreg. Further, <i>in vivo</i> activation of the IL-2 CISC improved efficacy at limiting doses of A2CAR EngTreg. Together, these findings demonstrate efficient generation of Ag-specific EngTreg utilizing CAR as the targeting moiety and efficacy of A2CAR EngTreg in preclinical models, suggesting potential therapeutic benefit for CAR-expressing EngTreg in transplantation and autoimmune diseases.