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J.D. Lai et al.
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Figure 7. FVIII-specific IgM and IgG in naïve HA mice and healthy humans bind preferentially to BHK-rFVIII. FVIII-specific IgM was detected by indirect ELISA using plasma from naïve (A) HA-R593C mice and (B) HA mice. (C) Competition assay in which the specificity of these IgM antibodies for BHK-rFVIII was assessed by a 30 min preincubation with either other rFVIII concentrates, or human pdVWF (n=3). (D) Glycan occupancy at each N-linked Asn consensus sequence derived from LC- MS/MS analysis. (E) Murine IgM binding to deglycosylated rFVIII products (n=7). (F) FVIII-binding of IgM isolated from healthy humans. (G) Binding of IgG from healthy human plasma to different rFVIII products. (H) Binding of IgG from healthy human plasma to de-N-glycosylated BHK-rFVIII. Line and error bars represent mean and SEM respectively. The Wilcoxon test was used for paired statistical analysis. *P<0.05; **P<0.01; ***P<0.001. BHK: baby hamster kidney cells; CHO: Chinese ham- ster ovary cells; rFVIII: recombinant factor VIII; BDD: B-domain deleted; Ig: immunoglobulin; PNGaseF: Peptide: N-Glycosidase F.
culation in the presence of the protective effect of VWF.48 N-linked glycans may result in steric hindrance to prevent Ig binding. Indeed, removal of N-linked glycans from BHK- and CHO-rFVIII increased IgM binding to a greater extent in the latter, suggesting that glycans on CHO-rFVIII better mask underlying epitopes.
These rFVIII immune complexes may provide an expla- nation for the enhanced immunogenicity documented with BHK-rFVIII through fragment crystallizable (Fc)- mediated uptake by DCs.49 The binding of IgM to antigens further facilitates the binding of mannose-binding lectin, and both can trigger the deposition of complement that greatly increases the binding potential of FVIII to endocyt- ic receptors.50 These data therefore support the observa- tion of increased clearance and immunogenicity of BHK- rFVIII. The presence of FVIII-specific IgM in rFVIII naïve HA mice, and both IgM and IgG in healthy individuals, suggests an innately immunogenic property of FVIII.
Previous studies have reported anti-FVIII antibodies in up to 19% of healthy subjects.26,27,51 These autoantibodies have been mapped to several regions in the FVIII heavy chain, and a single region in the A3 domain of the FVIII light chain.51 Complete glycosylation of the partially occu- pied sites described herein may inhibit these initial immune complexes from forming. Although these anti- rFVIII Igs likely possess low binding affinities, the avidity of IgM binding, in addition to IgG binding, may compen- sate and contribute to cellular uptake of FVIII immune complexes leading to either clearance or antigen presenta- tion.52,53
In this study, our data suggest that N-linked glycans shield underlying FVIII epitopes. We propose that the increased immunogenicity of BHK-rFVIII shown in two murine models of hemophilia A and four separate epi- demiological studies is, in part, related to incomplete N- linked glycosylation that exposes immunogenic epitopes
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haematologica | 2018; 103(11)