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

Abstract

AbstractIntroductionPorcine factor (pF)VIII has low cross‐reactivity with anti‐human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital haemophilia A patients with inhibitor (PwHA‐I) are in progress. Most polyclonal anti‐hFVIII inhibitors recognize its A2 and/or C2 domain(s), and recombinant human‐porcine hybrid (hp)FVIII proteins may escape neutralization by these inhibitors.AimTo evaluate the ability of hpFVIII to limit the anti‐FVIII activity of inhibitor alloantibodies.MethodsThree hybrid proteins were created by substituting the hFVIII A2, C2 domain or both with the corresponding domains of pFVIII [termed hp(A2), hp(C2) and hp(A2/C2), respectively]. The reactivity of these hybrids was assessed by one‐stage clotting assays (OSA), thrombin generation assays (TGA) and rotational thromboelastometry (ROTEM) by adding them to FVIII‐deficient samples.ResultsOSA demonstrated that the hybrid proteins avoided neutralization by anti‐FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor‐antibodies (polyAb) from PwHA‐I. In TGA, thrombin generation with hp(A2) and hp(A2/C2) was not attenuated in the presence of patient IgG recognizing anti‐A2 domain. In contrast, that with hFVIII and hp(C2) was suppressed by this IgG 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).ConclusionHybrid FVIII proteins containing porcine FVIII A2 and/or C2 domain(s) could support effective therapy in PwHA‐I by avoiding neutralization.