Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice

Abstract

Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance.

Keywords: Factor VIII; Fc fusion protein; FcRn; Hemophilia A; Immune tolerance; Immunogenicity; Regulatory T cells.

Fig. 1

rFVIIIFc induces immune tolerance to FVIII. A. Schematic for dosing regimen and analysis. HemA mice (8–10 weeks old) were injected with rFVIIIFc, BDD-rFVIII (Xyntha), FL-rFVIII (Advate) or vehicle, at 50, 100, or 250 IU/kg once weekly for 4 weeks (days 0, 7, 14, and 21) followed by two injections 2 weeks apart (days 35 and 53). Blood was collected by retro-orbital bleeding on days 0, 14, 21, 28, and 42, prior to the dosing, for isolating plasma and determining anti-BDD-FVIII total and neutralizing antibody levels. On day 56, animals were sacrificed and spleens isolated to prepare single splenocyte suspensions. (B) Splenocytes were subjected to both FACS analysis and PCR-based gene expression profiling as specified. (C) Total anti-BDD-FVIII IgG levels (μg/ml) in individual animals determined on day 42 (n = 8–13/group) (D) Neutralizing antibody titers in individual animals on day 42 as determined using the Bethesda assay (n = 8–13/group). (E) HemA mice pretreated with 50 IU/kg of rFVIIIFc or vehicle, were rechallenged with 250 IU/kg of rFVIIIFc on day 49, as described in Methods. Results presented are the total anti-FVIII IgG levels (μg/ml) determined on indicated days (n = 8/group) (F) Neutralizing antibody titers (BU) on day 28 in rechallenged mice (n = 8/group). (G and H) HemA mice pretreated with 50 IU/kg of rFVIIIFc were challenged with DNP-OVA in adjuvant (see Section 2) subcutaneously on days 42 and 49. Results presented are anti-OVA (G) and anti-DNP (H) immunoglobulin levels (units/mL) compared to naïve mice receiving the two injections (control) and pre-bleeds of rFVIIIFc-tolerized mice (n = 5/group). The bar represents the median for each treatment group. *p < 0.05; **p < 0.01; n.s. not significant by Mann–Whitney’s T-test.

Fig. 2

rFVIIIFc induces Tregs and associated markers of tolerance. (A) Splenocytes from the 100 IU/kg group were stained for surface CD4 and CD25 followed by intracellular Foxp3 and subjected to FACS analysis. Results represent percent splenocytes positive for CD4, CD25, and Foxp3 ± SEM (n = 7–9; *p < 0.05 vs. vehicle; ^†p < 0.05 vs rFVIIIFc; T-test). (B) CD279 (PD-1) surface staining was determined by co-staining splenocytes from the 100 IU/kg groups with anti-CD279 and anti-CD4 and FACS analysis. Results represent percent of CD4+C279+ splenocytes ± SEM (n = 7–9; *p < 0.05 vs. vehicle; ^†p < 0.05 vs rFVIIIFc; T-test). (C) Splenocytes from mice were co-stained for CD4 and intracellular cytokine TNF-α. Results are percent splenocytes double positive for CD4 and TNF-α ± SEM (n = 6–10; *p < 0.05 vs. vehicle; ^†p < 0.05 vs rFVIIIFc; T-test). (D) Dendritic cell surface expression of CD274 (PD-L1) was determined by staining splenocytes from the 100 IU/kg group for CD274 along with CD11c and MHC Class II (n = 7–9; *p < 0.05 vs. vehicle; ^†p < 0.05 vs rFVIIIFc; T-test). (E) T-cell proliferation was measured using CFSE dye based dilution and FACS. CD4+ T-cells from splenocytes of mice injected with 50 or 250 IU/kg of rFVIIIFc twice, one week apart, were loaded with CFSE and incubated with peritoneal macrophages collected from naïve HemA mice at indicated concentrations of BDD-rFVIII as shown for 96 h at 37 °C. Proliferation was measured as a function of decrease in CFSE MFI. Bars represent decrease in MFI of CFSE relative to vehicle in T-cells ± SEM (*p < 0.05, T-test, n = 3–5). The anti-CD3/CD28 incubations were carried out on CD4+ T-cells derived from the 50 IU/kg group. (F) IFNγ secretion profile from the proliferation studies was measured by ELISA using a MSD (meso scale device) ELISA kit. Bars represent fold above vehicle of IFNγ secretion ± SEM (*p < 0.05, T-test; n = 3–5). (G) CD4+ effector T cells (T_eff) and Treg cells were isolated and reconstituted in vitro at indicated ratios in the presence of antigen presenting CD90.2⁻ cells (see Section 2). IFNγ secretion was measured by ELISA using a MSD (meso scale device) ELISA kit. Bars represent fold above vehicle of IFNγ secretion ± SEM (*p < 0.05 vs vehicle; ^#p < 0.05 vs T_eff, T-test; n = 4).

Fig. 3

Tolerogenic mechanisms activated by rFVIIIFc: (A) heat map depicting the expression profiles of all the genes in the real time PCR array among the three tested groups: vehicle, 50 IU/kg and 250 IU/kg of rFVIIIFc. cDNA from each of the total splenocyte samples was used to monitor the expression of individual genes using a real time PCR array consisting of genes focused on tolerance and anergy associated molecules (n = 8–11/group). (B) Expression profile of candidate genes that were identified as being up- or down-regulated by the 50 IU/kg group in comparison with the 250 IU/kg group. Results shown here illustrate the fold change in expression of genes above vehicle group. The cut-off for fold change in regulation was taken as 2, i.e., fold change above 2 was considered up-regulation and below 0.5 as down-regulation. All the candidate genes belonging to the 50 IU/kg group shown here were significantly regulated (p < 0.05 vs. vehicle as well as the 250 IU/kg group; n = 8–11). Expression levels of some of the candidates (C–H) are confirmed by real time PCR. (I) Real time PCR was carried out to determine levels of TGF-β mRNA transcript. Bars represent 2^−ΔCt values for the three treatments (p < 0.05 vs. vehicle as well as the 250 IU/kg group; n = 8–11).

Fig. 4

rFVIIIFc signals via FcRn and/or Fcγ receptors to induce immune tolerance to rFVIII. (A) Hypothesis for the possible receptor dependent mechanisms for rFVIIIFc to induce tolerance. (B) Total anti-FVIII IgG levels on day 42 in HemA mice injected with 50 or 250 IU/kg of rFVIIIFc, rFVIIIFc-N297A and rFVIIIFc-IHH, in comparison with BDD-rFVIII (Xyntha^®) or FL-rFVIII (Advate^®). Results illustrated here are anti-BDD-FVIII IgG levels (μg/ml) and the median bar is depicted for each group in the study (n = 8–13; *p < 0.05; **p < 0.01; Mann–Whitney test). (C–F) FACS analysis for markers indicated from splenocytes of mice injected with 50 or 250 IU/kg of rFVIIIFc or mutants. Bars depict % splenocytes for the marker tested + S.E.M. (*p < 0.05; T-test; n = 6–11).

Fig. 5

Working model for mechanism of action of rFVIIIFc in induction of immune tolerance to rFVIII.