Th146 - Chimeric Antigen Receptor Regulatory T Cells (CAR-Tregs) Provide Significantly Greater Modulation of Alloimmune Responses than Alternative Alloantigen-Specific Treg Strategies

Conferring alloantigen-specificity to ex vivo expanded CD4+CD25+Foxp3+ Tregs increases their capacity to counteract effector alloimmune responses following adoptive transfer into transplant recipients. This has been achieved in humans by: 1) expanding Tregs in the presence of donor B cells (donor-reactive DR-Tregs); 2) culturing Tregs in the presence of donor material and co-stimulation blockade (CSB-Tregs); and 3) transducing Tregs with a HLA-A2-specific chimeric antigen receptor specific (CAR-Tregs). All 3 modalities of manufactured Treg products are currently undergoing clinical development. We lack, however, a clear understanding of the relative potency of each of these strategies. Our goal here was to directly compare the 3 manufacture approaches both in vitro and in vivo. Antigen-specificity and suppressive function of the cell products were evaluated in vitro by performing activation, proliferation, and T cell suppression assays. Therapeutic efficacy was assessed in vivo in a xenogeneic GvHD model in humanized NSG mice. As compared to polyclonal Tregs, all 3 strategies increased the proportion of alloantigen-specific Tregs within the manufactured cell product, with the highest level being observed in the CAR-Treg products. The differences in the precursor frequency of alloreactive Tregs were consistent with the results of the suppression assays, in which on a cell-per-cell basis CAR-Tregs exhibited the strongest effects, followed by DR-Tregs and CSB-Tregs. In vivo, infusion of CAR-Tregs together with HLA-A2+ PBMCs into irradiated NSG mice resulted in most prolongation of recipient survival. Results conclude that the CAR technology is a more powerful strategy in conferring alloantigen specificity to ex vivo expanded Treg products.