Generation and Characterization of FVIII-Specific CAR-Transduced Regulatory T Cells

The development of inhibitory antibodies (inhibitors) against FVIII is the most critical complication in the treatment of hemophilia A patients as hemostasis can no longer be reestablished by FVIII replacement therapy. Immune tolerance induction by frequent FVIII infusions is demanding, costly and not successful in all treated patients leading to an urgent need for the development of new therapeutic approaches for the prevention or treatment of FVIII inhibitors. Regulatory T cells (Tregs) are important for the maintenance of tolerance and have a high therapeutic potential in the context of autoimmune or inflammatory immune disorders. As Tregs are polyclonal, treatment with Treg pools comprises the risk of a general immunosuppression. Thus, the establishment of antigen-specific Tregs could be of great benefit for a broad range of patients, including inhibitor positive hemophilia A patients. To create such specific Tregs, a FVIII-specific scFv isolated out of a synthetic phage display library was used to generate a second generation chimeric antigen receptor (CAR).

To verify the specificity of the CAR for FVIII and the functionality of the recombined cytoplasmic domain (CD28 and CD3zeta), naïve CD4 T cells were retrovirally transduced with the generated CAR construct and a proliferation assay was conducted in the presence of plate-bound or soluble FVIII, as well as soluble FVIII presented by autologous irradiated PBMCs. Proliferation of transduced cells was more effective when FVIII was presented plate-bound or by PBMCs. In a therapeutically-relevant setting, this would be very promising, as transduced T cells should not be activated by soluble FVIII in the bloodstream but rather by FVIII presented on antigen-presenting cells in lymphatic organs.

Next, functionality of the CAR construct in Tregs was addressed. Transduced Tregs showed extracellular expression of the scFv and could be stimulated MHC-independently with FVIII. Such stimulated cells showed increased expression of Treg activation markers LAP and GARP. Thus, by using the generated CAR for transduction of Tregs, it was possible to create FVIII-specific Tregs that can be stimulated MHC-independently, opening new possibilities for therapeutic approaches in hemophilia A patients with FVIII inhibitors.