Neutralization of antifactor VIII inhibitors by recombinant porcine factor VIII

Key Points• C1 domain antibodies with low inhibitor titers by the Bethesda assay are pathogenic in mice due to increased fVIII clearance.• Monoclonal and patientderived polyclonal anti-fVIII C1 domain antibodies recognize similar B-cell epitopes.Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogendeuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. (Blood. 2016;128(16):2055-2067

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“…30 Specific inhibitory activities were converted to Bethesda Units (BUs)/mg IgG using the known concentration of the anti-C1 mAb.…”

Section: Fviii Inhibitor Assaymentioning

confidence: 99%

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

Batsuli

Deng

Healey

et al. 2016

Blood

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“…Unfortunately, porcine FVIII is no longer commercially available, but a recombinant, porcine, B domain-deleted FVIII molecule (OBI-1, Octagen) has been shown to neutralize antibodies to human FVIII in vitro and is currently in phase II clinical trials. 31 …”

Section: -Deamino-8-d-arginine Vasopressinmentioning

confidence: 99%

Acquired Factor VIII Inhibitors: Pathophysiology and Treatment

Alice¹,

Carrizosa²

2006

Hematology

156

198

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Hemophilia A is classically caused by a congenital deficiency of factor VIII, but an acquired form due to inhibitors to factor VIII (FVIII) typically presents later in life. Patients who develop such acquired factor VIII inhibitors may present with catastrophic bleeding episodes, despite having no prior history of a bleeding disorder. Though the disorder is rare, it is known to cause significant morbidity and mortality. This review will focus on what is currently known about acquired hemophilia A, its pathogenesis, its associated etiologies, and its treatment. Factor VIII and Acquired InhibitorsFactor VIII functions as a cofactor to factor IXa in the tenase complex, and a deficiency of factor VIII thus reduces the generation of thrombin on the surface of activated platelets. Factor VIII is synthesized as a 330-kDa precursor protein with an A 1 -a 1 -A 2 -a 2 -B-a 3 -A 3 -C 1 -C 2 domain structure. 1After proteolytic processing, FVIII associates with von Willebrand Factor (vWF) in heterodimers of a heavy (A 1 -a 1 -A 2 -a 2 ) and a light (a 3 -A 3 -C 1 -C 2 ) chain associated by a metal ion interaction. Most acquired FVIII inhibitors bind to the A2, A3 or C2 domains.2,3 Anti-C2 antibodies disrupt the binding of FVIII to phospholipid and vWF, while antibodies to A2 and A3 interfere with FVIII binding to factor X and factor IXa.In congenital hemophilia A patients treated with factor VIII, alloantibodies to FVIII develop in 20-40% of patients. By contrast, acquired inhibitors/autoantibodies against FVIII in nonhemophiliacs occur in only about one case per million per year. 4 In acquired hemophilia, autoantibodies are characteristically non-complement fixing, nonprecipitating immunoglobulins from the IgG family that bind FVIII in a time-and temperature-dependent manner. 5While most alloantibodies inactivate FVIII in direct proportion to their concentration (first-order kinetics), acquired inhibitors/autoantibodies show a non-linear inactivation pattern (type II or second-order kinetics). The kinetics plots in Figure 1 show a linear inactivation of FVIII by a type 1 alloantibody, with eventual complete inhibition of the FVIII activity. By contrast, the type 2 antibody shows a rapid initial inactivation phase followed by a slower phase of equilibrium where some factor VIII can usually be measured.

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“…Interestingly, porcine FVIII has been used effectively in the clinic as a "bypass" therapy; that is, a therapeutic protein that can evade neutralization by anti-FVIII antibodies in many allo-and autoimmune inhibitor patients. [5][6][7] However, some patients have or could develop antibodies that neutralize porcine FVIII as well, 8 because of antigenic cross-reactivity 9 or because regions in which the porcine sequence differs from the human FVIII sequence stimulate effector T cells, leading to antibody production. Identification of the binding sites (B-cell epitopes) on FVIII that are recognized by inhibitors would allow rational design of novel therapeutic FVIII proteins that are more similar to human FVIII and, hence, likely to be less immunogenic.…”

Section: Introductionmentioning

confidence: 99%

High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

Nguyen

Lewis

Ettinger

et al. 2014

Blood

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• Amino acid residues comprising B-cell epitopes recognized by neutralizing antifactor VIII antibodies (inhibitors) have been identified. • Amino acids contributing significant antigen-antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins.Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigenantibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients. (Blood. 2014;123(17):2732-2739 IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is a serious complication that may be encountered when FVIII replacement therapy is administered to patients with hemophilia A (HA). It affects 25% to 30% of the treated HA population, with a peak occurrence after ;14 FVIII infusions.1-3 Autoimmune responses to FVIII can also occur, 4 and although this happens only rarely, the resulting bleeding phenotype can be severe. Inhibitors can be difficult and extremely expensive to manage clinically. Interestingly, porcine FVIII has been used effectively in the clinic as a "bypass" therapy; that is, a therapeutic protein that can evade neutralization by anti-FVIII antibodies in many allo-and autoimmune inhibitor patients. [5][6][7] However, some patients have or could develop antibodies that neutralize porcine FVIII as well, 8 because of antigenic cross-reactivity 9 or because regions in which the porcine sequence differs from the human FVIII sequence stimulate effector T cells, leading to antibody production. Identification of the binding sites (B-cell epitopes) on FVIII that are recognized by inhibitors would allow rational design of novel therapeutic FVIII...

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Neutralization of antifactor VIII inhibitors by recombinant porcine factor VIII

Cited by 45 publications

References 15 publications

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

Acquired Factor VIII Inhibitors: Pathophysiology and Treatment

High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

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