Tu158 - Affinity-tuning of Human anti-HLA-A2 Chimeric Antigen Receptors Enhances Their Function

Abstract Text: Regulatory T cells (Tregs), known for their potent immunomodulatory properties, can be genetically engineered using chimeric antigen receptor (CAR) technology to target organ-specific antigens. CARs exhibit supraphysiological affinity to their target antigens compared to T cell receptors. However, the effect of modifying the CAR’s single-chain variable fragment (scFv) affinity on CAR-Treg functionality remains unclear. In this study, we aimed to evaluate the functional properties of affinity-tuned human anti-HLA-A2 CAR-Tregs. Second-generation anti-HLA-A2 CAR constructs expressing low-affinity (LA) and high-affinity (HA) scFvs were designed. Human HLA-A2 negative CD4+CD25+CD127- Tregs were transduced using lentiviral vectors. The antigen-specific activation and suppressive function of CAR-Tregs were assessed in vitro using cell lines and precision-cut liver slices (PCLS). Therapeutic efficacy was evaluated in an in vivo xeno graft-versus-host disease (GvHD) by infusing CAR-Tregs together with HLA-A2+ PBMCs into irradiated NSG mice. Mechanistic studies focused on CAR avidity, internalization and recycling using dextramer tracking. When cultured with HLA-A2 expressing cells, LA CAR-Tregs exhibited the highest proportion of activated alloantigen-specific cells and suppressive effects. Furthermore, when cultured with PCLS, they showed greater tissue infiltration than other cells. In vivo, LA CAR-Tregs exhibited improved persistence, delayed GvHD onset, and prolonged recipient survival. Mechanistically, HA CAR-Tregs exhibited higher avidity for their target antigen but underwent rapid CAR internalization, and reduced dextramer expression upon exposure to physiological antigen levels, indicating CAR downregulation. Affinity-tuning the CAR of anti-HLA-A2 CAR-Tregs significantly alters their functionality, both in vitro and in vivo, which has implications for the design of engineered Treg therapies for clinical use.