Hybrid human-porcine factor VIII proteins partially escape the inhibitory effects of anti-factor VIII inhibitor alloantibodies having A2 or C2 domain specificity

Abstract

Introduction: Porcine factor (pF)VIII has low cross-reactivity with anti-human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital hemophilia A patients with inhibitor (PwHA-I) are in progress. Most FVIII inhibitors recognize the A2 and/or C2 domain(s), and recombinant human-porcine hybrid (hp)FVIII containing mutations within these domains may escape the neutralizing effects of these inhibitors. Aim: To evaluate the ability of hpFVIII to limit the anti-FVIII activity of inhibitor alloantibodies. Methods: Three hybrid mutants were created by substituting the A2, C2 domain or both in human hFVIII, with the corresponding domain of pFVIII (termed hp(A2), hp(C2), and hp(A2/C2), respectively). The reactivity of these mutants was assessed by one-stage clotting assays (OSA), thrombin generation assays (TGA), and rotational thromboelastometry (ROTEM) using FVIII-deficient samples. Results: OSA demonstrated that mutation restricted the inhibitory effects of both anti-FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor-antibodies (polyAb) from PwHA-I. TGA indicated that peak thrombin with hp(A2) and hp(A2/C2) was not attenuated in the presence of anti-A2 polyAb, but that with hFVIII and hp(C2) was suppressed to levels equivalent to those of FVIII-deficient plasma. With anti-A2/C2 polyAb, the activity of hp(A2/C2) was unaffected. ROTEM demonstrated that the addition of hp(A2) or hp(A2/C2) to anti-A2 polyAb shortened clot times/clot formation times, whilst hFVIII or hp(C2) were ineffective. Similarly with anti-A2/C2 polyAb, hp(A2/C2) restored coagulation potential to a greater extent than hp(A2) and hp(C2). Conclusion: Hybrid FVIII mutations within the porcine A2 and/or C2 domain corresponding to respective inhibitor-epitopes could support effective therapy in PwHAI.