Deimmunization of HLA-DR Epitopes in the C2 Domain of Human FVIII (144.29)

Abstract

Abstract To reduce immunogenicity of therapeutic proteins, recombinant proteins can be modified to lower HLA binding of epitopes, a method (deimmunization) useful with monoclonal antibodies. Since the immune response to human clotting factor VIIII limits therapeutic efficacy, we reported that immunodominant peptides in the C2 domain of fVIII could be modified to reduce MHCII binding to display less antigenicity in vitro in a hemophilic fVIII KO mouse model (H-2b). We have extended these studies in humanized HLA-DR transgenic mice and identified responses to C2 peptides p2191-2210, p2231-51, p2254-69, p2271-89; and p2310-30 in DR2, DR3, DR4 mice, as well as in DR3 mice crossed to fVIII KO hemophilic mice. DR transgenic mice were immunized with the designated original peptides (ORG) or with rFVIII in CFA, and LN cells tested in T-cell proliferation assays against rFVIII and ORG peptides. While p2191 dominantly stimulates H-2b, other peptides are immunodominant in DR2, DR3 and DR4 mice. A different pattern was revealed (DR3 x fVIII KO) mice depending on whether they also express H-2b. Efforts to test the reactivity/antigencity and immunogenicity of modified (MOD) peptides predicted to have lower class II binding will be reported. These results will be utilized to design and produce a functional full-length modified rFVIII that can be used as a therapeutic protein in hemophilia A (Supported by NIH R43 HL088834-01).