Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors

Abstract The antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa. Group BC antibodies are the most common and display high specific inhibitory activity and type II kinetics. The C2 epitope groups recognized by 26 polyclonal human anti-fVIII inhibitor plasmas were identified by a novel competition enzyme-linked immunosorbent assay using group-specific murine monoclonal antibodies. Most of the anti-C2 inhibitor plasmas inhibited the binding of both classical and nonclassical antibodies. These results suggest that nonclassical anti-C2 antibodies contribute significantly to the pathogenicity of fVIII inhibitors.

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Identification of Three Non-Overlapping Epitopes in the C2 Domain of Factor VIII.

Blood ◽

10.1182/blood.v108.11.1007.1007 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1007-1007

Author(s):

Shannon Meeks ◽

John F. Healey ◽

John (Pete) S. Lollar

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Structural Complexity ◽

Basis Set ◽

Type I ◽

C2 Domain ◽

Type Ii ◽

Secondary Antibodies ◽

Von Willebrand ◽

Willebrand Factor

Abstract Factor VIII (fVIII) inhibitory antibodies (fVIII inhibitors) are a significant source of morbidity in patients with hemophilia A. Approximately 30% of patients with severe hemophilia A will develop inhibitors. Most inhibitors are directed against either the A2 or C2 domains of fVIII. The repertoire of antibodies to the C2 domain is functionally complex, including antibodies that inhibit fVIII binding to phospholipid and von Willebrand factor and display either type I or type II kinetics. The goal of this study was to investigate the diversity of the immune response to the C2 domain of human fVIII in a murine hemophilia A model and to identify a set of non-overlapping epitopes recognized by these fVIII inhibitors. A panel of 55 murine anti-C2 antibodies was obtained, which included 53 antibodies from anti-fVIII hybridomas produced in our laboratory and previously described antibodies ESH-4 and ESH-8. Nine of the hybridomas were cloned by limiting dilution and the corresponding monoclonal IgG antibodies were purified. IgG from the remaining 44 hybridomas was isolated directly from the hybridoma supernatants and biotinylated. The 9 monoclonal antibodies along with ESH-4 and ESH-8 were used as primary antibodies in a competition ELISA using human fVIII as the antigen. The panel of 55 biotinylated antibodies was used as secondary antibodies. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively. A basis set of 3 antibodies, 1B5-1B, 3E6-1B, and (2)117-1B, was defined, which consisted of a set of non-overlapping antibodies that as a group competed for binding of human fVIII with all other antibodies. 1B5-1B and 3E6-1B exhibited type I kinetics with Bethesda titers of 950 and 30, respectively. The Bethesda titer of (2)117-1B could not be determined due to its type II behavior. ESH-4 and ESH-8 had the same epitope profiles as 3E6-1B and (2)117-1B, respectively. Of the 52 non-basis set antibodies, 85% overlapped with 1B5-1B, 29% with 3E6-1B, and 56% with (2)117-1B. The majority of the antibodies overlapped with more than one member of the basis set, leading to the identification of five classes of antibodies (see figure). Because of the large number of antibodies characterized, it is unlikely that the frequency of antibodies in any additional classes is high. The elucidation of the structural complexity of the anti-fVIII C2 repertoire should be useful in the characterization of the pathogenicity of C2 inhibitors. Figure Figure

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Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation

Blood ◽

10.1182/blood-2007-06-096842 ◽

2007 ◽

Vol 110 (13) ◽

pp. 4234-4242 ◽

Cited By ~ 65

Author(s):

Shannon L. Meeks ◽

John F. Healey ◽

Ernest T. Parker ◽

Rachel T. Barrow ◽

Pete Lollar

Keyword(s):

Factor Viii ◽

Factor Xa ◽

Enzyme Linked Immunosorbent Assay ◽

Phospholipid Membranes ◽

C2 Domain ◽

Fviii Inhibitor ◽

Domain Antibody ◽

Epitope Structure ◽

Common Group ◽

Von Willebrand

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding β-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 β-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape.

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Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A

Blood ◽

10.1182/blood.v93.7.2267 ◽

1999 ◽

Vol 93 (7) ◽

pp. 2267-2273 ◽

Cited By ~ 71

Author(s):

Kathelijne Peerlinck ◽

Marc G. Jacquemin ◽

Jef Arnout ◽

Marc F. Hoylaerts ◽

Jean Guy G. Gilles ◽

...

Keyword(s):

Factor Viii ◽

Enzyme Linked Immunosorbent Assay ◽

Type Ii ◽

Wild Type ◽

Mutation Site ◽

Fviii Activity ◽

Polyclonal Igg ◽

Reduced Rate ◽

Von Willebrand ◽

Willebrand Factor

Abstract Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

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Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A

Blood ◽

10.1182/blood.v93.7.2267.407k21_2267_2273 ◽

1999 ◽

Vol 93 (7) ◽

pp. 2267-2273 ◽

Cited By ~ 1

Author(s):

Kathelijne Peerlinck ◽

Marc G. Jacquemin ◽

Jef Arnout ◽

Marc F. Hoylaerts ◽

Jean Guy G. Gilles ◽

...

Keyword(s):

Factor Viii ◽

Enzyme Linked Immunosorbent Assay ◽

Type Ii ◽

Wild Type ◽

Mutation Site ◽

Fviii Activity ◽

Polyclonal Igg ◽

Reduced Rate ◽

Von Willebrand ◽

Willebrand Factor

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

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A Novel Approach to Immune Tolerance Induction in Hemophilia Α with Factor VIII Inhibitor

Blood ◽

10.1182/blood-2018-99-119800 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5042-5042 ◽

Cited By ~ 1

Author(s):

Courtney D. Thornburg ◽

Jonathan M. Ducore

Keyword(s):

Factor Viii ◽

Research Funding ◽

Hemophilia A ◽

Tolerance Induction ◽

Immune Tolerance Induction ◽

Fviii Inhibitor ◽

Fviii Activity ◽

Von Willebrand ◽

Willebrand Factor

Abstract Factor VIII (FVIII) inhibitors are currently the most significant complication of hemophilia A therapy. Immune tolerance induction (ITI) is the treatment of choice for inhibitor eradication. However, the optimal ITI regimen has not been identified. We describe an ITI approach for an adolescent male with severe hemophilia A and recurrent and refractory high-titer FVIII inhibitor. This strategy is now being tested in a randomized, controlled trial (NCT03204539). A 1-year-old white, Hispanic male with severe hemophilia A, intron 22 inversion, developed a FVIII inhibitor while receiving on-demand treatment with recombinant FVIII (rFVIII). Due to life-threatening bleeding despite bypassing agents, he was immediately started on ITI with daily rFVIII along with intravenous immunoglobulin (IVIg), solumedrol, and rituximab. He achieved tolerance after 15 months and was switched to every-other-day dosing for prophylaxis. Unfortunately, on prophylaxis he had breakthrough bleeding and inhibitor recurrence, and has required additional ITI regimens over the past 14 years (Table 1). Most recently, the family agreed to switch to Wilate® [von Willebrand Factor/Coagulation Factor VIII Complex (Human); Octapharma USA, Inc.; Hoboken, NJ; U.S. License No. 1646] as an alternative von Willebrand factor (VWF)/FVIII concentrate since he was unable to achieve tolerance on prior plasma-derived (pd) VWF/FVIII concentrate at 100 international units (IU)/kg daily for ~4 years. Wilate® was started when inhibitor titer was 1.5 Bethesda Units (BU). Initial FVIII recovery was 30% and 24-hour trough level was 1%. After several months, blood samples were sent to a diagnostic laboratory at Haemophilie-Zentrum Rhein Main GmbH for lot selection. Lot selection entails measuring residual FVIII activity when patient plasma is mixed with different lots of VWF/FVIII in vitro. This involves using the modified New Oxford method to measure residual FVIII activity after incubation of a FVIII source (lot) with the inhibitor patient plasma. The inhibitor titer is the reciprocal of the dilution of patient plasma that results in 50% of residual FVIII, similar to the Bethesda Unit. Ideally, the lot providing the highest residual FVIII activity will more effectively challenge the immune system, provide better prevention and control of bleeding, and have shorter time to tolerance. Investigators have demonstrated the utility of lot section in in vitro studies. The patient's plasma was tested against 6 lots of Wilate® and the lot with the lowest inhibitor activity was selected for prescription. This lot was allocated to this patient, and the prescribing physician included the lot number on the factor prescription for distribution to the patient via the patient's specialty pharmacy. The patient received Wilate® from the same lot for ~11 months. During that time, his inhibitor decreased from 2.6 BU to 0 after 5 months. A new plasma sample was tested against an additional 5 lots of Wilate®. Inhibitor was negative at that time and all lots of Wilate® revealed a negative inhibitory activity. One lot was selected for ongoing treatment. After 18 months his 48-hour trough FVIII level was detectable. He had blood drawn for pharmacokinetic analysis, which showed FVIII recovery of 55% and an estimated half-life of 6.75 hours. He was switched to every-other-day dosing and has had only one trauma-induced soft-tissue bleed despite increased physical activity, with negative inhibitor and a 48-hour trough of 2%. Disclosures Thornburg: Shire: Research Funding; CSL Behring: Research Funding; ATHN: Research Funding; Bayer Pharmaceuticals: Research Funding; Octapharma: Research Funding; Bioverativ: Consultancy; Genentech: Speakers Bureau; Biomarin: Consultancy; Bluebird Bio: Consultancy; NovoNordisk: Research Funding; Johns Hopkins All Children's Hospital: Research Funding. Ducore:OPKO: Other: investigator; HemaBiologics: Consultancy, Other: investigator, travel support; Shire: Consultancy, Other: travel support, investigator; Biomarin: Other: investigator; Octapharma: Consultancy, Other: travel support, investigator , Research Funding; Bayer Healthcare: Consultancy, Other: travel support, investigator; Pfizer: Other: investigator; Spark Therapeutics: Consultancy, Other: investigator; CSL Behring: Other: investigator.

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Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650655 ◽

1996 ◽

Vol 76 (05) ◽

pp. 749-754 ◽

Cited By ~ 73

Author(s):

Suzuki Suzuki ◽

Morio Arai ◽

Kagehiro Amano ◽

Kazuhiko Kagawa ◽

Katsuyuki Fukutake

Keyword(s):

Complex Formation ◽

Factor Viii ◽

Von Willebrand Factor ◽

Domain Specificity ◽

Factor Viii Inhibitor ◽

C2 Domain ◽

Recombinant Fviii ◽

Von Willebrand ◽

A2 Domain ◽

Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

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Faculty Opinions recommendation of Alloantibodies to therapeutic factor VIII in hemophilia A: the role of von Willebrand factor in regulating factor VIII immunogenicity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725335385.793503724 ◽

2015 ◽

Author(s):

Gili Kenet

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Hemophilia A ◽

Von Willebrand ◽

Willebrand Factor

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A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor

Blood ◽

10.1182/blood.v96.3.958 ◽

2000 ◽

Vol 96 (3) ◽

pp. 958-965 ◽

Cited By ~ 55

Author(s):

Marc Jacquemin ◽

Renaud Lavend'homme ◽

Abdellah Benhida ◽

Beatrijs Vanzieleghem ◽

Roseline d'Oiron ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Cho Cells ◽

Hemophilia A ◽

Chinese Hamster ◽

Expression Vectors ◽

Scatchard Analysis ◽

C1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

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