Prevention of Anti-FVIII Inhibitor Formation Post Protein Infusion in Hemophilic Mice by Prior Feeding of the FVIII-C2 Domain.

The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is a major obstacle to FVIII replacement therapy. Since the oral administration of an antigen may induce antigen-specific immune tolerance (oral tolerance, OT), we have investigated the effects of feeding human FVIII (hFVIII) or the purified C2 domain of the hFVIII protein (hFVIII-C2) to FVIII-deficient BALB/c (BAL-FVIIIKO) mice prior to hFVIII infusion. This project has two related components: (A) the induction of OT via feeding with hFVIII-C2 and (B) the identification of an immunogenic epitope within the C2 domain specific to BAL-FVIIIKO mice that could be used for peptide-specific OT.

We have previously shown that multiple low dose (ug) feedings of hFVIII can induce transient tolerance to hFVIII in mice via active suppression. Other studies indicate that high dose (mg) feedings result in long lasting OT via clonal deletion or anergy. However, due to the high cost and limited availability of recombinant hFVIII, high dose feedings are not feasible. Immunogenic protein domains can also be used for OT, thus we used hFVIII-C2 allowing for the feeding of higher doses (0.1–1mg) than hFVIII (1–10ug). BAL-FVIIIKO mice were fed with a range of hFVIII-C2 and hFVIII doses, and injected subcutaneously (SC) with hFVIII (1ug) or hFVIII-C2 (10ug) with an incomplete adjuvant. Control mice were fed with PBS prior to protein infusion.

Feeding with high doses of hFVIII-C2 prevented inhibitor development after hFVIII injection in 3/8 mice and decreased the inhibitor titer in 4/8 mice. This was accompanied by the production of cytokines IL-4 and IL-10 by splenocytes in vitro. 3/3 mice that were fed high doses of hFVIII-C2 and injected with hFVIII-C2 did not produce anti-FVIII inhibitors after hFVIII-C2 challenge, while control mice produced average inhibitors of 6.9 BU. Mice that were inhibitor negative after feeding with hFVIII or hFVIII-C2 and an injection of hFVIII, were injected with an additional dose of hFVIII four weeks post-feeding. Mice fed high hFVIII-C2 doses remained inhibitor negative, whereas mice fed low hFVIII-C2 or hFVIII doses developed inhibitors. Thus, feeding higher doses of hFVIII-C2 results in longer-term tolerance, compared to lower doses of hFVIII-C2 or hFVIII which results in short-term tolerance. These studies show that OT may be beneficial in preventing anti-FVIII inhibitor formation, and that high doses of fed protein will be required for long-term tolerance.

Studies have shown that peptides spanning antibody epitopes can induce OT. To define strain-specific FVIII-C2 epitopes as targets for OT therapy, site-directed mutagenesis was used to create the mutants Q2311T/V2314A (MUT-hFVIII-C2) within the putative epitope 2311–2316. hFVIII-C2 and MUT-hFVIII-C2 were made using a Pichia pastoris yeast expression system, and were injected SC (10ug) with adjuvant into C57BL/6-FVIIIKO (C57-FVIIIKO) and BAL-FVIIIKO mice. hFVIIIC2 injection resulted in average anti-FVIII inhibitor titers of 5.13 BU and 6.90 BU in C57-FVIIIKO and BAL-FVIIIKO mice respectively. Injection of MUT-hFVIIIC2 resulted in 6.07 BU and 0.35 (0,0,0,1.4) BU in C57-FVIIIKO and BAL-FVIIIKO. These results illustrate that BAL-FVIIIKO mice recognize the region 2311–2316 as an immunogenic epitope, but the C57 strain does not. Thus, Q2311 and V2314 are important in the immunogenicity of this region and a peptide spanning 2311–316 could be used in OT induction in BAL-FVIIIKO mice.