Background

A portion of individuals with hemophilia A develop neutralizing antibodies called inhibitors to glycoprotein factor VIII (FVIII). There are multiple risk factors that contribute to the risk of inhibitor formation. However, knowledge of the role of FVIII asparagine (N)-linked glycosylation in FVIII immunity is limited.

Objective

To evaluate the effect of site-specific N-linked glycan removal on FVIII biochemical properties, endocytosis by murine bone marrow-derived dendritic cells (BMDCs), and antibody responses.

Methods

Four recombinant B domain deleted (BDD) FVIII variants with single-site amino acid substitutions to remove N-linked glycans were produced for experimental assays.

Results

BDD FVIII-N41G, FVIII-N239A, FVIII-N1810A, and FVIII-N2118A with confirmed removal of N-linked glycans and similar glycosylation profiles to BDD FVIII were produced. There were no differences in thrombin activation or von Willebrand factor binding of FVIII variants compared to BDD FVIII, however, reduced FVIII expression, activity, and specific activity was observed with all variants. BDD FVIII-N41G and FVIII-N1810A had reduced uptake by BMDCs, but there were no differences in antibody development in immunized hemophilia A mice compared to BDD FVIII. Half of a repertoire of 12 domain-specific FVIII MAbs had significantly reduced binding to ≥1 FVIII variant with a 50% decrease in A1 domain MAb 2-116 binding to FVIII-N239A.

Conclusions

Modifications of FVIII N-linked glycans reduced FVIII endocytosis by BMDCs and binding of domain-specific FVIII MAbs, but did not alter de novo antibody production in hemophilia A mice suggesting that N-glycans do not significantly contribute to inhibitor formation.