The Immune Response to Murine Factor VIII in Single Exon Deletion and Total Gene Deletion Murine Models of Hemophilia a
Authors: Patel Seema R , Baldwin Wallace H. , Cox Courtney , Healey John , Parker Ernest T. , Batsuli Glaivy , Meeks Shannon L. 
Published: November 2019

Introduction: Though a vital therapy for patients with hemophilia A, exposure to factor VIII (fVIII) can lead to the development of neutralizing anti-fVIII IgG. Interestingly, not all patients with hemophilia A form inhibitors, with approximately 20-30% of patients with severe hemophilia A and 5% of patients with mild to moderate hemophilia A developing inhibitors. Several factors have been hypothesized to govern the propensity of patients to develop inhibitors, including the type of F8 gene mutation a patient possesses. Approximately, 60-70% of hemophilia A patients with large deletions (greater than 50 base pairs) of the F8 gene form inhibitors, while only 20-30% of patients with smaller deletions develop inhibitors. However, no significant difference in inhibitor formation has been observed between single exon 16 (E16) and complete (TKO) F8 gene deficient hemophilia A mice exposed to human fVIII (hfVIII). As E16 mice are on a mixed S129-B6 background and strain differences can impact the immunological outcome to a given immunogen, it is possible that the mixed background influenced the humoral immune response to hfVIII in E16 mice. However, E16 mice that are backcrossed >10 generations onto the same B6 background as TKO mice (B6-E16) were found to develop a comparable IgG response to hfVIII as B6-TKO and S129/B6-E16 mice. As human and murine B domain deleted (BDD) fVIII are 85% identical, we hypothesize that the diversity between human and murine fVIII may not allow for elucidation of immune differences that may exist between hemophilia A mice with large or small F8 mutations. Accordingly, we herein sought to compare the relative immunogenicity of murine fVIII (mfVIII) in these distinct hemophilia A mice.
Methods:S129/B6 E16, B6-E16 and B6-TKO mice were administered four weekly retro-orbital infusions of 1 μg recombinant BDD mfVIII or hfVIII. Two weeks later, mice were challenged with a 2 μg dose of recombinant mfVIII or hfVIII. One week prior to challenge and one-week post challenge, plasma was collected for examination of anti-fVIII IgG titers by an enzyme linked immunosorbent assay.
Results:Consistent with previous reports, no statistically significant difference in anti-hfVIII IgG titers were observed between S129/B6-E16, B6-E16 and B6-TKO mice. When exposed to mfVIII the majority of B6-TKO mice developed inhibitors (87.5%), while the single exon B6-E16 or S129/B6-E16 mice had 50% and 56% rates of inhibitor development, respectively. The B6-E16 mice had a lower median anti-mfVIII IgG ELISA titer of 12.3 (range 1 to 190.3) compared to S129/B6-E16 and B6-TKO that had median ELISA titers of 473 and 289, respectively. When assessing the anti-mfVIII IgG titers only in the mice that had detectable titer levels, the IgG titers for S129/B6-E16 mice ranged from 34.2 to 1519.4 and for B6-TKO mice ranged from 31.3 to 1377.
Conclusion: Given the difference in rates of immune response to mfVIII in mice carrying a single exon or total F8 gene deletion, mfVIII may be useful to elucidate the correlation between F8 gene mutations and the propensity of an individual to respond to factor replacement therapy, and in particular modulators of the immune response in lower risk F8 gene mutations.

Disclosures
Batsuli: Genentech: Other: Advisory board participant; Octapharma: Other: Advisory board participant; Bayer: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.