CAR-mediated release of IL-10 increases the function of regulatory T cells: Relevance for future clinical application

Regulatory T cell (Treg) therapy emerges for various indications associated with a breakdown of immune tolerance. Antigen-specific chimeric antigen receptor (CAR) Tregs are frontrunners for transplantation and autoimmune diseases and are currently being clinically evaluated. We aimed to link CAR-antigen engagement with immunosuppressive cargo release into the local microenvironment to boost efficacy and reduce side effects. We used our HLA-A∗02 CAR and immunosuppressive interleukin-10 (IL-10) as model components to generate human CAR Tregs that release IL-10 upon CAR engagement. These were compared to CAR Tregs with constitutive or no IL-10 expression by evaluating phenotypes, antigen-specific IL-10 release, and suppression of effector cell proliferation in vitro and performance in vivo in a humanized xenogeneic graft-versus-host disease (xeno-GvHD) model. We demonstrated successful multi-construct engineering of CAR Tregs, which released upon CAR engagement 2.5-fold more IL-10 than CAR Tregs lacking the corresponding antigen-specific IL-10 secretion module. Neither phenotype nor function was affected by expressing this module. In the xeno-GvHD model, we showed the beneficial effect of IL-10 release, particularly evident when compared to constitutive IL-10 expression that impaired CAR-Treg efficacy. We provide first proof-of-principle for engineering human CAR Tregs to release an immunosuppressive cytokine upon CAR engagement. This approach will both enhance the potency of CAR Tregs at the intended target sites and limit their off-target effects.