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cific to BHK-rFVIII, and that the interaction is not inhibit- ed by human VWF (Figure 7C).
We next evaluated whether N-linked glycans sterically hinder access of IgM to certain epitopes in the FVIII pro- tein backbone. From our MS data, we observed that there were consistently higher proportions of unoccupied N- glycan sites at Asn900, Asn1255, Asn1259, Asn1282, Asn1300, and Asn1810 in BHK-rFVIII compared to CHO- rFVIII (Figure 7D). We subsequently removed all the N- linked glycans from BHK- and CHO-rFVIII using Peptide: N-Glycosidase F (PNGase F), and observed a greater increase in IgM binding to deglycosylated CHO-rFVIII (Figure 7E) when compared to the native rFVIII glycoform.
To extend these findings to humans, we collected plas- ma from healthy human volunteers and quantified levels of FVIII-specific IgM. We observed an increased binding of IgM to BHK-rFVIII compared to CHO-rFVIII and CHO- BDD-rFVIII (Figure 7F). The presence of FVIII-specific IgG antibodies has previously been reported in healthy indi- viduals.26,27 We generated a standard curve using the recombinant antibody, EL14, which bound equally to rFVIII products (Online Supplementary Figure S8). Upon incubation with plasma from healthy human subjects, we observed that a higher proportion of human IgG bound to BHK-rFVIII compared to CHO-rFVIII (Figure 7G). We fur- ther determined that this binding was enhanced when the N-glycans were removed from rFVIII (Figure 7H).
Discussion
Factor VIII immunogenicity remains a significant con- cern among hemophilia treaters, and mounting evidence suggests that rFVIII products differ from pdFVIII concen- trates as well as among themselves. Non-human cell lines have been shown to add immunogenic non-human glycan structures, Gal(a1-3)Galβ1-GlcNAc-R (aGal) and 5-gly- colylneuraminic acid (Neu5Gc) to rFVIII.28,29 In two murine models of HA, in which both non-human glycans are present, we found that BHK-rFVIII was more immuno- genic than CHO-rFVIII. These data suggest that although Neu5Gc and aGal have the potential to induce FVIII immunity in humans, as seen with the immune responses against cetuximab, there are additional mechanisms that contribute to this response.30,31
As per its routine clinical use, mice were dosed by the procoagulant activity of rFVIII. However, the level of inac- tive FVIII protein in the commercial concentrates likely plays a role in immunogenicity. BHK-rFVIII has been reported to have higher FVIII:C and FVIII:Ag than adver- tised.32 We were unable to observe a similar trend across four lots of rFVIII, perhaps due to different methodologies used. High-dose intensive FVIII treatment has been impli- cated as a risk factor for inhibitor development, however this correlation is likely facilitated by the inflammatory milieu of concurrent surgery or bleeding.33 Whether expo-
A
Figure 3. Characterization of FVIII half-life and VWF binding in hemophilia A mice. (A) HA mice were intravenously infused with 200 IU/kg of either BHK-rFVIII or CHO cell line-derived rFVIII. FVIII activity was measured by chromogenic assay, and normalized to a 5 min time point post-injection. Data are repre- sentative of the aggregate data of 3 independent experiments totalling at least 5 biological replicates per time point and 2 lots of each rFVIII product. Error bars represent SEM. (B) Murine VWF from HA mouse pooled plasma was captured on a microtitre plate coated with a polyclonal rabbit anti-human VWF antibody. Increasing concentrations of either BHK-derived, or CHO-derived rFVIII were incubated with the captured VWF and detected using a polyclonal sheep anti-human FVIII antibody. Data shown is one technical replicate representative of 3 inde- pendent experiments. BHK: baby hamster kidney cells; CHO: Chinese hamster ovary cells; rFVIII: recombinant factor VIII.
P=0.02
B
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haematologica | 2018; 103(11)