Native-like aggregates of factor VIII are immunogenic in von Willebrand factor deficient and hemophilia a mice

Abstract

The administration of recombinant factor VIII (FVIII) is the first-line therapy for hemophilia A (HA), but 25%-35% of patients develop an inhibitory antibody response. In general, the presence of aggregates contributes to unwanted immunogenic responses against therapeutic proteins. FVIII has been shown to form both native-like and nonnative aggregates. Previously, we showed that nonnative aggregates of FVIII are less immunogenic than the native protein. Here, we investigated the effect of native-like aggregates of FVIII on immunogenicity in HA and von Willebrand factor knockout (vWF(-/-)) mice. Mice immunized with native-like aggregates showed significantly higher inhibitory antibody titers than animals that received native FVIII. Following restimulation in vitro with native FVIII, the activation of CD4+ T-cells isolated from mice immunized with native-like aggregates is approximately fourfold higher than mice immunized with the native protein. Furthermore, this is associated with increases in the secretion of proinflammatory cytokines IL-6 and IL-17 in the native-like aggregate treatment group. The results indicate that the native-like aggregates of FVIII are more immunogenic than native FVIII for both the B-cell and the T-cell responses.

Figure 1

A: The representative far UV-CD spectrum of rFVIII and aggregated rFVIII (80µg/100µL) in Tris buffer (300 mM NaCl, 25 mM Tris and 5 mM CaCl₂, pH 7.0). The CD spectra were acquired multiple times with multiple samples and representative spectra are shown. B: The representative fluorescence emission spectra of fractionated native and aggregate (80µg/mL) species in Tris buffer (25 mM Tris, 300 mM NaCl, 5 mM CaCl₂, pH 7.0). The samples were excited at 280 nm. The fluorescence spectra were acquired multiple times with multiple samples and representative spectra are shown. C: Size exclusion studies of native, native-like aggregates, and non-native aggregates of FVIII. Fluorescent intensity was monitored at 285ex/335em following separation on a Biosep-SEC-4000-S column run with a 0.5 mL/min Tris buffer elution.

Figure 1

A: The representative far UV-CD spectrum of rFVIII and aggregated rFVIII (80µg/100µL) in Tris buffer (300 mM NaCl, 25 mM Tris and 5 mM CaCl₂, pH 7.0). The CD spectra were acquired multiple times with multiple samples and representative spectra are shown. B: The representative fluorescence emission spectra of fractionated native and aggregate (80µg/mL) species in Tris buffer (25 mM Tris, 300 mM NaCl, 5 mM CaCl₂, pH 7.0). The samples were excited at 280 nm. The fluorescence spectra were acquired multiple times with multiple samples and representative spectra are shown. C: Size exclusion studies of native, native-like aggregates, and non-native aggregates of FVIII. Fluorescent intensity was monitored at 285ex/335em following separation on a Biosep-SEC-4000-S column run with a 0.5 mL/min Tris buffer elution.

Figure 1

A: The representative far UV-CD spectrum of rFVIII and aggregated rFVIII (80µg/100µL) in Tris buffer (300 mM NaCl, 25 mM Tris and 5 mM CaCl₂, pH 7.0). The CD spectra were acquired multiple times with multiple samples and representative spectra are shown. B: The representative fluorescence emission spectra of fractionated native and aggregate (80µg/mL) species in Tris buffer (25 mM Tris, 300 mM NaCl, 5 mM CaCl₂, pH 7.0). The samples were excited at 280 nm. The fluorescence spectra were acquired multiple times with multiple samples and representative spectra are shown. C: Size exclusion studies of native, native-like aggregates, and non-native aggregates of FVIII. Fluorescent intensity was monitored at 285ex/335em following separation on a Biosep-SEC-4000-S column run with a 0.5 mL/min Tris buffer elution.

Figure 2

The mean of inhibitory titers (cross) and individual (closed circles) antibody titers against FVIII in HA mice immunized via s.c. administration of native and native-like aggregates of FVIII. (A) Total anti-FVIII titers as determined by ELISA. (B) Inhibitory anti-FVIII titers as determined by the Nijmegen modification of the Bethesda assay. Significant differences are shown as determined by Kruskal-Wallis ANOVA with Dunn’s post hoc.

Figure 2

The mean of inhibitory titers (cross) and individual (closed circles) antibody titers against FVIII in HA mice immunized via s.c. administration of native and native-like aggregates of FVIII. (A) Total anti-FVIII titers as determined by ELISA. (B) Inhibitory anti-FVIII titers as determined by the Nijmegen modification of the Bethesda assay. Significant differences are shown as determined by Kruskal-Wallis ANOVA with Dunn’s post hoc.

Figure 3

The mean of inhibitory titers (cross) and individual (closed circles) antibody titers against FVIII in vWF^−/− mice immunized via s.c. administration of native, native-like aggregates, and non-native aggregates of FVIII. (A) Total anti-FVIII titers as determined by ELISA. (B) Inhibitory anti-FVIII titers as determined by the Nijmegen modification of the Bethesda assay. Significant differences are shown as determined by Kruskal-Wallis ANOVA with Dunn’s post hoc.

Figure 3

The mean of inhibitory titers (cross) and individual (closed circles) antibody titers against FVIII in vWF^−/− mice immunized via s.c. administration of native, native-like aggregates, and non-native aggregates of FVIII. (A) Total anti-FVIII titers as determined by ELISA. (B) Inhibitory anti-FVIII titers as determined by the Nijmegen modification of the Bethesda assay. Significant differences are shown as determined by Kruskal-Wallis ANOVA with Dunn’s post hoc.

Figure 4

The stimulation indices of different splenocytes from mice immunized with different preparations of FVIII. The mean stimulation index (cross) and individual (filled circles) stimulation indices from individual spleens (n=6) of different treatment groups as determined by ³H-thymidine incorporation T cell proliferation assay in vWF^−/− mice.