Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

Walter, Justin D.; Werther, Rachel; Brison, Caileen M.; Cragerud, Rebecca K.; Healey, John F.; Meeks, Shannon L.; Lollar, Pete; Spiegel, P. Clint

doi:10.1182/blood-2013-08-519124

Cited by 30 publications

(51 citation statements)

References 61 publications

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“…32 Similarly, we expect that the B-cell epitope residues identified herein comprise a subset of the actual contact areas between each inhibitory antibody and FVIII. This expectation has been confirmed, in part, by recent crystallographic studies illustrating the epitope recognized by another type BC inhibitor (mAb G99) 33,34 and by a nuclear magnetic resonance/mass spectrometry study that identified several FVIII-C2 surfaces occluded by the binding of 4 neutralizing mAbs. 35 SPR-based assignments of epitope residues presented herein are consistent with the regions identified using the same or similar mAbs and these other techniques.…”

Section: Blood 24 April 2014 X Volume 123 Number 17 B-cell Epitopessupporting

confidence: 52%

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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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Section: Blood 24 April 2014 X Volume 123 Number 17 B-cell Epitopessupporting

confidence: 52%

“…This expectation has been confirmed, in part, by recent crystallographic studies illustrating the epitope recognized by another type BC inhibitor (mAb G99) 33,34 and by a nuclear magnetic resonance/mass spectrometry study that identified several FVIII-C2 surfaces occluded by the binding of 4 neutralizing mAbs.…”

supporting

confidence: 52%

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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“…Epitopes, mechanisms of action and kinetics have been defined for a large collection of anti‐A2 and C2 domain murine monoclonal antibodies (MAbs) demonstrating that the murine hemophilia A model recapitulates many features of the anti‐FVIII immune response observed in humans . Recently, high‐resolution structural data of anti‐hFVIII MAbs in complex with the FVIII C2 domain using small angle X‐ray scattering, X‐ray crystallography, and hydrogen‐deuterium exchange mass spectrometry were obtained and have brought the understanding of inhibitor mechanism of action to the atomic level .…”

Section: Introductionmentioning

confidence: 99%

Expanding the ortholog approach for hemophilia treatment complicated by factor VIII inhibitors

Zakas

Vanijcharoenkarn

Markovitz

et al. 2015

Journal of Thrombosis and Haemostasis

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Summary Background The formation of neutralizing antibodies (inhibitors) directed against human coagulation factor VIII (hFVIII) is a life-threatening pathogenic response that occurs in 20–30% of severe congenital hemophilia A patients and 0.00015% of remaining population (i.e. acquired hemophilia A). Interspecies amino acid sequence disparity among FVIII orthologs represents a promising strategy to mask FVIII from existing inhibitors while retaining procoagulant function. Evidence for the effectiveness of this approach exists in clinical data obtained for porcine FVIII products, which have demonstrated efficacy in the setting of congenital and acquired hemophilia. Objectives In the current study, recombinant (r) ovine FVIII (oFVIII), was evaluated for antigenicity and procoagulant activity in the context of human patient-derived and murine model-generated FVIII inhibitors. Methods The antigenicity of roFVIII was assessed using i) inhibitor patient plasma samples, ii) murine anti-FVIII MAbs, iii) immunized murine hemophilia A plasmas, and iv) an in vivo model of acquired hemophilia A Results Overall, roFVIII demonstrated reduced reactivity to, and inhibition by, anti-hFVIII immunoglobulin in patient plasmas. Additionally, several hFVIII epitopes were predicted and empirically shown not to exist within roFVIII. In a murine hemophilia A model designed to mimic clinical inhibitor formation, it was demonstrated that inhibitor titers to roFVIII were significantly reduced compared to the orthologous immunogens, rhFVIII or rpFVIII. Furthermore in a murine model of acquired hemophilia A, roFVIII administration conferred protection from bleeding following tail transection. Conclusion These data support the investigation of FVIII orthologs as treatment modalities in both the congenital and acquired FVIII inhibitor settings.

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“…[8][9][10] Healey et al 11 demonstrated a predominance of anti-fVIII antibodies to the A2 and C2 domains of fVIII in a murine hemophilia A model, but antibodies to the other domains were also detected. Characterization of the structural and functional properties of anti-A2 and anti-C2 domain monoclonal antibodies (mAbs) has advanced our understanding of the epitope spectrum of anti-fVIII antibodies [12][13][14][15] and their mechanisms of inhibition. [16][17][18][19] However, there is increasing evidence of the C1 domain's contribution to fVIII function and immunogenicity.…”

Section: Introductionmentioning

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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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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Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

Abstract: Key Points Antibodies against the factor VIII C2 domain inhibit procoagulant function. Crystal structure analysis of a C2 domain/antibody ternary complex describes epitopes for classical and nonclassical inhibitory antibodies.

Cited by 30 publications

References 61 publications

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

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

Expanding the ortholog approach for hemophilia treatment complicated by factor VIII inhibitors

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

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