Modification of An Interactive Surface in the C1 Domain Reduces Immune Responses to FVIII in a Mouse Model of Hemophilia A

Abstract 199^FN2

The X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. The formation of FVIII-neutralizing antibodies in hemophilia A patients is initiated by the endocytosis of FVIII by professional antigen-presenting cells. Endocytosis of FVIII by human monocyte-derived dendritic cells can be significantly blocked by monoclonal antibody KM33, directed towards the C1 domain of FVIII. We have previously shown that C1 domain residues 2092 and 2093 are required for KM33 binding to FVIII (Meems et al., 2009). However, replacement of both these residues by Ala did not completely abolish KM33 binding. We created an additional C1 domain variant which included an arginine at position 2090. A FVIII variant in which three residues, 2090, 2092 and 2093 were substituted by alanine, showed only minimal binding to KM33. Functional analysis revealed that this variant designated FVIII-2090/2092/2093 retains pro-coagulant activity as measured by a chromogenic assay. FVIII-2090/2092/2093 displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine bone marrow-derived dendritic cells. These results emphasize a role for C1 domain residues 2090, 2092 and 2093 in FVIII endocytosis by antigen-presenting cells. We subsequently investigated the in vivo ability of this variant to induce inhibitors in FVIII^−/− mice (E17KO). We show that E17KO mice treated with the FVIII variant have significantly lower anti-FVIII antibody titers when compared to mice treated with wild type FVIII. In accordance with these findings reduced numbers of FVIII-specific antibody-secreting cells were detected in the spleen of mice treated with FVIII-2090/2092/2093. Also, FVIII-specific CD4⁺ T cell responses of splenocytes derived from FVIII-2090/2092/2093 infused mice were greatly reduced when compared to that of splenocytes derived from wild type FVIII infused mice. These findings show that alanine substitutions at positions 2090, 2092 and 2093 result in a FVIII molecule that is significantly less immunogenic when compared to wild type FVIII. Collectively, our data suggest that FVIII variants displaying a reduced uptake by antigen-presenting cells show a reduced immunogenicity in vivo. Based on our findings we hypothesize that FVIII variants displaying a reduced uptake by antigen-presenting cells provide a novel therapeutic approach to reduce inhibitor development in patients with hemophilia A.