N-linked glycosylation modulates the immunogenicity of recombinant human factor VIII in hemophilia A mice

Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evidence has highlighted that recombinant factor VIII produced in baby hamster kidney cells is more immunogenic than factor VIII produced in Chinese hamster ovary cells. Glycosylation differences have been hypothesized to influence the immunogenicity of these synthetic concentrates. In two hemophilia A mouse models, baby hamster kidney cell-derived factor VIII elicited a stronger immune response compared Chinese hamster ovary cell-derived factor VIII. Furthermore, factor VIII produced in baby hamster kidney cells exhibited accelerated clearance from circulation independent of von Willebrand factor. Lectin and mass spectrometry analysis of total N-linked glycans revealed differences in high-mannose glycans, sialylation, and the occupancy of glycan sites. Factor VIII desialylation did not influence binding to murine splenocytes or dendritic cells, nor surface co-stimulatory molecule expression. We did however observe increased levels of IgM specific to baby hamster kidney-derived factor VIII in naive hemophilia A mice. De-N-glycosylation enhanced IgM binding, suggesting that N-glycan occupancy masks epitopes. Elevated levels of IgM and IgG specific to baby hamster kidney-derived factor VIII were also observed in healthy individuals, and de-N-glycosylation increased IgG binding. Collectively, our data suggest that factor VIII produced in baby hamster kidney cells is more immunogenic than that produced in Chinese hamster ovary cells, and that incomplete occupancy of N-linked glycosylation sites leads to the formation of IgM- and IgG-factor VIII immune complexes that contribute to the enhanced clearance and immunogenicity in these mouse models of hemophilia A.