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Glycosylation modulates FVIII immunogenicity
sure to greater amounts of FVIII protein accounts for the increase in immunogenicity in a non-inflammatory steady state is unclear. Consistent with our data, Delignat et al. standardized FVIII:Ag between products using human plasma, and observed enhanced immunogenicity of BHK- rFVIII compared to CHO-rFVIII in HA mice.18
The immunogenic disparity between rFVIII concen- trates was greatest when administered subcutaneously in HA-R593C mice. In F8 exon 16 KO HA mice, central tol- erance for human FVIII is limited, which may explain why differences between very similar proteins are not as appar- ent in this mouse model.34 In humanized F8 exon 16 KO HA-R593C mice, immunological tolerance to human FVIII necessitates an adjuvant to elicit an immune response via intravenous administration, which may result in a general heightened immune reactivity against all antigens, thus preventing the resolution of subtle immunogenic differ- ences.20 Of note, there are major differences in the biodis- tribution of FVIII when administered intravenously, where it complexes with murine VWF and predominantly localizes in the liver and spleen, versus subcutaneous deliv- ery, where VWF association is less likely, and FVIII local- izes to the draining lymph node.35 This likely directs FVIII to different populations of phagocytic cells, particularly DCs and macrophages.36
The removal of FVIII from circulation can lead to clear- ance and/or antigen presentation. Even in the dominating
presence of endogenous murine VWF, BHK-rFVIII was cleared at an accelerated rate, independent of differences in VWF-binding under static conditions. This difference in half-life has not been described in human patients, and may only be apparent in this mouse model due to inter- species differences in the mechanisms of FVIII and VWF clearance. In this scenario, assuming that FVIII does not influence VWF clearance to a significant extent, it is possi- ble that the ~5% of BHK-rFVIII that circulates without VWF is cleared faster, thus shifting the equilibrium to pro- mote further FVIII dissociation from VWF.37 We hypothe- sized that post-translational structures could contribute to the different clearance kinetics of these proteins in mice.
Our analysis of rFVIII glycans confirms the findings of previous studies: that rFVIII possesses predominantly core-fucosylated biantennary complex glycans, a heavily sialylated B domain, high mannose glycans at Asn239 and Asn2118, and unoccupied sites at Asn582, Asn943, Asn1384, and Asn1685.28,38,39 Contrary to previous studies, we did not detect tetraantennary glycans or Neu5Gc gly- cans (aGal cannot be detected directly using our meth- ods). Lectin binding analysis of exposed N- and O-linked glycans and mass spectrometry analysis of total N-linked glycans collectively showed differences in sialic acid and high-mannose glycan content. A similar analysis showed an absence of high mannose glycans as well as greater sia- lylation in CHO-derived rFVII compared to that produced
AB
Figure 4. Lectin array analysis of exposed glycans on rFVIII products. rFVIII products were adsorbed on microtitre plates at 1 mg/mL and assessed using a panel of biotinylated lectins. BSA was adsorbed as a control. Binding was detected using a streptavidin poly-HRP and read at 492 nm. (A) Heat plot demonstrating the medi- an-centred binding values of lectins with varying carbohydrate specificites to different rFVIII products. Values shown are representative of the mean of at least 3 inde- pendent experiments on a single lot of each rFVIII product. Statistical summary available in Online Supplementary Table S1. (B) Lectin binding analysis of different production lots of full-length rFVIII from CHO or BHK cells. Data are representative of at least 3 independent experiments. BHK: baby hamster kidney cells; CHO: Chinese hamster ovary cells: rFVIII: recombinant factor VIII; BSA: bovine serum albumin; BDD: B-domain deleted.
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