Targeting FVIII-Specific B Cells Using BAR-Transduced Regulatory T Cells

Up to a third of hemophilia A (HA) patients receiving therapeutic FVIII develop neutralizing antibodies termed "inhibitors". Once inhibitors develop, clinical management of HA patients becomes extremely difficult. Thus, a rational solution would be to establish FVIII-specific immune tolerance to FVIII in high risk patients. To address this issue in a mouse model of human HA, we elected to use an antigen-specific regulatory T cell (Treg) approach. Analogous to the chimeric antigen receptor (CAR) strategy successfully used in cancer immunotherapy, we have created a chimeric receptor comprising a protein antigen or its domain, linked with the transmembrane and signal transduction domains, CD28-CD3ζ. We termed this receptor "BAR" for B-cell-targeting antibody receptor. Human Tregs (CD4⁺CD25^hiCD127^low) were retrovirally transduced with a BAR containing FVIII C2 domain (C2-BAR) or FVIII A2 domain (A2-BAR) and expanded successfully in vitro. These cells stained positively with anti-C2 and anti-A2 monoclonal antibodies, respectively, and maintained Treg phenotypic markers in terms of co-expression of Foxp3 and Helios. Control human Tregs were transduced with a BAR containing chicken ovalbumin (OVA-BAR). To test the hypothesis that BAR-transduced Tregs could directly and effectively suppress the activity of specific B cells, a xenogeneic model was employed. On day 0, FVIII^-/- HA mice were injected intravenously with 10⁶ transduced human Tregs. The mice were then immunized subcutaneously on day 1 with FVIII in incomplete Freund's adjuvant, and anti-FVIII antibody development was followed. By two weeks after immunization, anti-FVIII antibodies could be detected in the control mice (n = 4). However, in the experimental group (n = 5) that received a mixture of equal number of C2-and A2-BAR Tregs, anti-FVIII antibody development was reproducibly completely blocked for at least 8 weeks. To examine the possible mechanism of BAR Treg suppression, purified B cells and T cells from "tolerized" (A2+C2-BAR) or "control" (OVA-BAR) recipients were mixed and tested for recall responses to FVIII in vitro. The results suggested that the FVIII-specific B cells were directly tolerized while the T-cell response remained intact. Taken together, we report here a successful approach utilizing FVIII-specific BAR-Tregs to directly target FVIII-specific B cells, an approach which could be adapted to address other adverse immune response as well. (Supported in part by a NIH grant HL127495)