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Glycosylation modulates FVIII immunogenicity
Results
BHK-rFVIII is more immunogenic than CHO-rFVIII in mouse models of hemophilia A
While an International Standard exists for determination of the FVIII:C potency of FVIII concentrates (and products are labeled in International Units), there is no standardized method to accurately quantify FVIII:Ag between products.25 Although there are differences in specific coag- ulant activity between commercial preparations (and between different lots of concentrate) (Online Supplementary Figure S2), we dosed rFVIII by the coagulant activity reported by the manufacturer so as to recapitulate clinical practice and the data reported by previous FVIII inhibitor epidemiological studies. To assess the immuno- genic differences between CHO- and BHK-rFVIII, we administered rFVIII subcutaneously or intravenously to HA-R593C mice biweekly (6 IUs; 240 IU/kg per adminis- tration) for two weeks and analyzed plasma on day 28 (Figure 1A). After subcutaneous immunization, we observed a significant increase in the incidence of anti- FVIII IgG antibodies in mice treated with BHK-rFVIII com- pared to CHO-rFVIII 28 days after the initial injection (93.75% vs. 47.3% respectively, P=0.0042; Figure 1B). Further serological analysis showed that the aFVIII IgG titre was also greater when mice were immunized with BHK-rFVIII (Figure 1C). A similar difference was observed in the incidence of all FVIII inhibitory antibodies (93.75% vs. 36.8%, P=0.0003; Figure 1D). However no significant differences were observed in the inhibitory antibody titres (Figure 1E).
To account for the typical biodistribution of FVIII in the bloodstream, we administered rFVIII intravenously with the first injection containing 1 mg of lipopolysaccharide (LPS) as an adjuvant (Figure 1F). We did not observe any significant difference in the incidence of aFVIII IgG between products in these experiments (Figure 1G). However, BHK-rFVIII elicited higher titres of aFVIII IgG (Figure 1H) among FVIII-responders. Analysis of FVIII inhibitory antibodies showed no differences in incidence (Figure 1I) or inhibitor titre (Figure 1J) using this testing protocol.
All mice developed FVIII-specific antibodies by day 28 (Figure 2B), and there were no differences in the titre of FVIII-specific antibodies (Figure 2C). However, mice immunized with BHK-rFVIII exhibited a higher incidence of all FVIII antibodies with an inhibitory titer >1 bethesda unit (BU; 100% vs. 54.5%; Figure 2D), but no difference in the magnitude among FVIII-responders (Figure 2E).
BHK-rFVIII exhibits accelerated clearance that is independent of binding to murine VWF
We next assessed the clearance of rFVIII from the mouse circulation. Following an intravenous infusion of either BHK-rFVIII or CHO-rFVIII at 200 IU/kg in HA mice, plas- ma FVIII:C was measured by chromogenic assay at the indicated time points and normalized to a 5 min post-infu- sion sample. Our results show a significant increase in the clearance rate of BHK-rFVIII compared to CHO-rFVIII (6.22 hrs vs. 9.53 hrs; P=0.02) (Figure 3A).
Given the dominant influence of VWF on FVIII half-life, we assessed whether the rFVIII products exhibit differen- tial binding to endogenous mouse VWF (Figure 3B). FVIII- binding was reported as a function of the amount of FVIII:Ag at each dilution in the assay. These data suggest
that there is no significant difference in the binding to murine VWF between CHO- and BHK-derived rFVIII, and that the differences observed in clearance and immuno- genicity are independent of VWF and may suggest struc- tural moieties, such as post-translational modifications, that facilitate or inhibit cellular uptake.
rFVIII produced in BHK or CHO cell lines contains significantly different glycosylation profiles
Given the differences in clearance and immunogenicity observed in vivo, we next assessed rFVIII glycosylation by using a panel of lectins to detect specific, exposed, carbo- hydrate linkages on CHO- and BHK-rFVIII. CHO-BDD- rFVIII was used as a control, as it lacks all but six of the potential N-linked sites. Multiple lectins specific for simi- lar structures were used to confirm findings, and the medi- an-centered optical absorbance readings were compared between products using the Student t test (Online Supplementary Table S1). These data suggest that BHK- rFVIII has a higher degree of sialylation and fucosylation, and a lower presence of high-mannose glycans compared to CHO-rFVIII (Figure 4A; Principal component analysis Online Supplementary Figure S3). We further confirmed that these glycan differences were conserved across three dif- ferent lots of rFVIII products (Figure 4B).
Table 1. N-linked glycan structures detected across BHK-, CHO-, and CHO-BDD-rFVIII products.
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