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Glycosylation modulates FVIII immunogenicity
in CHO-rFVIII but not in BHK-rFVIII (Figure 5B). Similarly, the sialylation of sub-terminal galactose residues is more complete in BHK-rFVIII as evidenced by a high proportion of fully-sialylated glycans (Figure 5C-E). We did not observe differences in the frequency of fucosylated N- linked glycans between products (data not shown).
Glycosylation of rFVIII does not influence binding to or induction of IFNγ production by splenocytes or splenic dendritic cells in vitro
Glycosylation differences between CHO- and BHK- rFVIII may alter the association of FVIII with different cell types or receptors. To address this, we employed a FVIII binding assay to unsorted splenocytes from rFVIII naïve HA or HA-R593C mice. However, we did not observe sig- nificant differences in the binding of the different rFVIII preparations to these cells (Online Supplementary Figure S6A,B). We further assessed downstream immune responses to rFVIII in naïve mixed lymphocyte popula- tions using an interferon (IFN)γ enzyme-linked immunospot (ELISPOT). Splenocytes were isolated from naïve HA mice or HA-R593C mice and stimulated with the two rFVIII proteins for 48 hr. The number of cells secreting IFNγ did not differ between the two rFVIII con- centrates (Online Supplementary Figure S6C). Since these data suggest that the early rFVIII innate immune responses are similar between HA mice and HA-R593C mice, we used HA-R593C mice for subsequent experiments.
Differential FVIII immunogenicity in hemophilia A mouse models is not explained by differences in sialic acid content
Given that the degree of N-linked sialylation was the greatest source of glycan variation between BHK- and
CHO-rFVIII, we removed the terminal sialic acids from BHK-rFVIII using a2-3,6,8 neuraminidase for 18 hr at 37°C. Desialylation was confirmed by lectin binding assay (Online Supplementary Figure S7A). Desialylated FVIII:C procoagulant function was 62.3% of the control, however, FVIII antigenicity was conserved (Online Supplementary Figure S7B,C). The removal of sialic acid from BHK-rFVIII did not influence the binding to spleno-
+
cytes or CD11c dendritic cells (Figure 6A,B). Considering
the potential immunomodulatory role of sialic acid, we next investigated whether different glycoforms of rFVIII could influence the expression of the co-stimulatory mol- ecules, CD80 and CD86, on naïve or LPS-stimulated splenocytes and DCs. While we observed significant upregulation of both CD80 and CD86 in LPS-stimulated conditions, the removal of sialic acid did not influence surface co-stimulatory molecule expression in either splenocytes or DCs (Figure 6C, D). Similarly, HA-R593C mice treated subcutaneously with BHK-rFVIII or its desia- lylated glycoform, as described above, did not exhibit sig- nificant differences in the incidence or the titre of FVIII- specific IgG (Figure 6E, F).
N-linked glycans prevent binding of non-neutralizing IgM and IgG to rFVIII
We next evaluated the interaction of non-neutralizing IgM and IgG on rFVIII, and the ability of N-linked glycans to regulate this interaction. We found that incubation of naïve HA, or HA-R593C mouse plasma on a FVIII-coated microtitre plate resulted in the increased binding of BHK- rFVIII-specific IgM (Figure 7A,B) relative to CHO-rFVIII. FVIII-specific IgG was not detected (data not shown). Of note, competition through preincubation with other forms of FVIII showed that these IgM molecules are spe-
A
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Figure 2. Immunogenic differences between BHK- and CHO-rFVIII in HA mice. (A) F8 exon 16 KO HA mice were immunized intravenously (IV) with 6 IU (0.6 mg; 240 IU/kg) of either BHK- or CHO-rFVIII biweekly for 2 weeks. Blood was collected by cardiac puncture 28 days after the first infusion, and plasma was isolated by cen- trifugation. Samples were assessed for (B) incidence and (C) titres of FVIII-specific IgG. FVIII-responders were analyzed for (D) the incidence (P=0.015) and (E) con- centration of FVIII inhibitors. The horizontal lines and error bars represent the mean and SEM. Mann-Whitney U test and Fisher’s exact test were used where appro- priate. *P<0.05. BHK: baby hamster kidney cells; CHO: Chinese hamster ovary cells: rFVIII: recombinant factor VIII; Ig: immunoglobulin; n.s. not significant; BU: bethesda unit.
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