A Factor VIII with Site-Specific Pegylation Attenuates the Binding with Inhibitors Results in the Retention of Hemostatic Effect
Authors: Yada Koji , Nogami Keiji , Patel Chandra , Sim Derek , Kauser Katalin , Shima Midori 
Published: October 2019

Factor (F) VIII inhibitors develop as alloantibodies in mainly severe hemophilia A (HA) patients after frequent infusion of FVIII concentrates and also as autoantibodies in previously normal individuals resulting in acquired HA. The appearance of autoantibodies usually results in severe bleeding and complicates the hemostatic treatment. The epitopes recognized by FVIII inhibitors are commonly located in the A2 and/or C2 domain of the FVIII molecule. BAY 94-9027(BAY 94) is a B-domain deleted (BDD) recombinant (r)FVIII with site-specific attachment of polyethylene glycol (PEG) that has shown an extended half-life in HA patients as well as animal models of HA (Coyle et al. JTH 2014). The hemostatic effect of BAY 94 in the presence of the inhibitors remains unknown. In this study, we investigated the effects of BAY 94 on the inhibitory activity of antibodies with various epitopes developed in acquired HA as well as congenital HA. Firstly, we assessed the binding of BAY 94 or BDD to the three monoclonal antibodies (mAbs) recognizing the different epitopes with the different inhibitory kinetics pattern (type1 or 2) (monoA; A1 type1, ESH4;C2 type1 and ESH8;C2 type2), and the seven polyclonal inhibitor IgGs obtained from three congenital HA (Case1(recognizing epitope(s) in A2 and C2 type1), 2(C2 type1), 3(C2 type2)) and four acquired HA (Case 4(A2 and C2 type1), 5(A2), 6(C2 type2), 7(C2 type1)) patients plasmas purified with the protein G column. Antibodies were covalently coupled (~5 ng/mm2) to a sensor chip surface, utilizing surface plasmon resonance (SPR) assay to assess binding. BAY 94 bound to the immobilized anti-A1 mAb, recognizing the acidic region, with modest lower binding affinity (Kd ~4-fold higher) than BDD. An anti-C2 mAb ESH8 also bound to BAY 94 with an ~8-fold higher Kd compared to BDD. Another anti-C2 ESH4 bound to BDD with similar binding affinity to anti-C2 ESH8, however, of interest, this mAb failed to bind to BAY 94. Case 1-IgG, recognizing both A2 and C2 domains, bound to BAY 94 with an ~30-fold lower binding affinity (Kd ~ 1.5nM) compared to BDD (Kd ~0.05nM). Similarly, the binding potentials of BAY 94 to Case 2-IgG (Kd ~ 42nM) was markedly decreased compared to BDD (Kd ~ 4.7nM). In addition, Case 3-IgG bound to BDD (Kd ~ 3.1nM) but little to BAY 94. Four anti-FVIII autoAbs (Case 4,5,6 and 7) bound to BDD with weaker affinities compared to anti-FVIII alloAbs and mAbs. However, all autoAbs almost failed to bind to BAY 94. Further, to confirm the hemostatic function of BAY 94 with inhibitors, we evaluated the FVIII:C with BAY 94 or BDD in the presence of anti-FVIII Abs by a one-stage clotting assay. Anti-A1 mAb, monoA as well as anti-C2 ESH8 decreased the FVIII:C levels in BAY 94 and BDD similarly,in a dose-dependent manner. The inhibition of FVIII:C by anti-C2 mAb ESH4, however, had little effect on BAY 94 activity up to the max IgG concentration of 10 nM employed. Similarly, in anti-C2 alloAb Case 2, possessing the similar characteristic of ESH4, the inhibitory effects of this IgG was weaker for BAY 94 compared to BDD. The four anti-FVIII autoAbs decreased the FVIII:C levels of BAY 94 more modestly than BDD. Particularly the inhibitory effect of anti-C2 autoAb Case 6, as well as mAb ESH4, were quite different between BDD and BAY 94. Taken together, BAY 94 bound to anti-FVIII autoAbs as well as alloAbs with weaker binding affinity compared to BDD, independent of its epitope and kinetic mode, resulting in higher residual FVIII:C levels of BAY 94 relative to BDD. BAY 94-9027 is a site-specific pegylated rFVIII with extended half-life in vivo and the data presented here suggests the attached PEG could facilitate its hemostatic function even in the presence of inhibitors, by attenuating the binding with inhibitors.

Disclosures

Yada: Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:F.Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai Paharmaceutical Co., Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Patel:Bayer HealtheCare LLC: Employment. Sim:Bayer HealtheCare LLC: Employment. Kauser:Bayer HealtheCare LLC: Employment. Shima:Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Kaketsuken: Honoraria; Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Research Funding; Biogen: Honoraria, Research Funding.