De-Immunization of Immunodominant HLA-DR Epitopes In the C2 Domain of Factor VIII

Abstract 2207

Development of anti-factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle of replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce binding of FVIII sequences to HLA, based on predicted anchoring residues. This process is known as ‘de-immunization’. Previously, we found that immunogenic peptide epitopes in the C2 domain of FVIII could be modified to reduce MHC class II binding resulting in lower antigenicity in vitro in hemophilic E16 mice (H-2^b). Here, we extended these studies to HLA-DR transgenic mice and identified immunodominant C2 peptides in E16, DR3 and DR4 mice as well as in DR3 mice crossed to E16 mice (DR3×E16). E16 and DR transgenic mice were immunized with the designated original high class II-binding peptides (ORG) or modified peptides (MOD) emulsified in CFA, and lymph node (LN) cells were tested in T-cell proliferation and ELISpot assays against the ORG and MOD peptides. We found that while p2191-2210 dominantly stimulates H-2^b, p2271-89 is immunodominant in DR3 transgenic animals; DR3×E16 T cells recognized both epitopes as would be predicted. On the other hand, p2231-51 and p2310-30 were immunodominant in DR4 mice. We also found that modifications of dominant epitope peptides 2191, 2271, 2231, 2310 showed lower HLA class II binding and T-cell proliferation in corresponding mouse strains; stimulation of IL17-a and IFNγ cytokine production were reduced with modified 2191 in DR3xE16 mice. Direct immunization with rFVIII containing modified peptides is required to validate potential immunogenicity. These results will be utilized to design and produce a functional full-length modified rFVIII that can be used as a less immunogenic therapeutic protein for hemophilia A patients. (Supported by NIH grant R43 HL088834-01 to ADG.)