Tolerogenic Fusion Proteins for Hemophilia Inhibitor Formation

The focus of our laboratory has been to employ a gene therapy approach with transduced B cells to induce tolerance to a variety of antigens and achieve clinical efficacy in disease models for multiple sclerosis, type 1 diabetes, and uveitis, as well as in hemophilia. This process requires processing of the expressed epitopes and MHC class II presentation by the transduced B cells. Moreover, there is clear evidence that regulatory T cells are required for both the induction and maintenance of tolerance by this platform technology. The presence of the IgG scaffold in our construct increases both the efficacy and persistence of tolerance since we fuse the antigen (Ag) of interest to an immunoglobulin (IgG1) heavy chain. A series of peptide epitopes with strong MHC class II binding across multiple HLA haplotypes have been identified in IgG. These promiscuous epitopes are conserved between species. Recent data suggest that these Ig-prevalent epitopes activate regulatory T cells. Thus, they have been called 'Tregitopes'. Our hypothesis is that the efficacy of the IgG scaffold in our gene therapy protocol (and in the tolerogenicity of IgG fusion proteins per se) is due to the presence of these regulatory epitopes. To test this hypothesis and provide the basis for future translation to the clinic, we propose to identify the role of regulatory epitopes in B-cell delivered gene therapy by mutating the putative Tregitope residues so they can no longer bind to MHC class II and test the effect of these mutations on tolerance efficacy in a mouse model for human disease. We will then prepare and analyze the efficacy of IgG domain fusion proteins. We have engineered fusions of ovalbumin and immunodominant FVIII domains with murine CH1, CH2, CH3 and Fv, as well as full length IgG, and will produce these in quantity to identify the minimal tolerogenic fusion protein. We will also mutate the regulatory epitopes in the most effective fusion protein to test whether this ablates tolerogenicity. As a future goal beyond our present scope, we will engineer a recombinant FVIII molecule in which the B domain contains IgG domains or a series of Tregitopes. This could yield a functionally active and tolerogenic FVIII for future therapy. (AHA Program: Grant-in-Aid)