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

Blood ◽  
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

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

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1151-1153 ◽  
Author(s):  
Shannon L. Meeks ◽  
John F. Healey ◽  
Ernest T. Parker ◽  
Rachel T. Barrow ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Xa ◽  
Enzyme Linked Immunosorbent Assay ◽  
C2 Domain ◽  
Type Ii ◽  
Fviii Inhibitor ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Murine Monoclonal Antibodies

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.

A Subset of High Titer Acquired Hemophilia Patients May Benefit from Treatment with High Dose Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3476-3476
Author(s):  
Shannon Meeks ◽  
John F Healey ◽  
Ernest T Parker ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
High Titer ◽  
High Dose ◽  
Type I ◽  
C2 Domain ◽  
Type Ii ◽  
Acquired Hemophilia ◽  
Proteolytic Activation

Abstract Abstract 3476 Poster Board III-413 Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies (Abs) to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune Abs to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with autoimmune hemophilia often have Abs with type II kinetics in which there is incomplete inactivation of fVIII at saturating concentrations of inhibitor. We have characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of Abs. Groups A, AB, and B are classical anti-C2 Abs that block fVIII and fVIIIa binding to phospholipid. Groups BC and C consist of non-classical anti-C2 Abs that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Subsequently, we identified classical and non-classical anti-C2 Abs in human fVIII inhibitor plasmas. Most murine non-classical Abs have inhibitor titers greater than 10,000 Bethesda units/mg IgG. In a murine in vivo bleeding model, both type I classical C2 Abs, type II non-classical C2 Abs, and a type I anti-A2 Ab produced similar amounts of blood loss that were significantly greater than control mice injected with 180 U/kg of fVIII alone. Increasing the dose of fVIII to 360 U/kg overcame the bleeding diathesis produced by the type II MAbs, but not the type I Abs. These results were consistent with the in vitro Bethesda assay in which a type I anti-A2 Ab, 4A4, completely inhibited both 1 U/mL and 3 U/mL fVIII, while there was 40% residual activity at saturating concentrations of a type II anti-C2 Ab, 2-77, at either concentration of fVIII. To determine if similar in vitro characteristics exist in patients with acquired hemophilia, plasmas from 3 patients with high titer type II inhibitors were studied. All 3 plasmas primarily had C2 domain epitope specificity that included non-classical Abs. Plasma A7 additionally had detectable anti-A2 activity. Recovery of fVIII activity after a 2 h incubation at 37 °C at nominal added concentrations of 1 mL and 3 U/mL fVIII was compared (Table 1). At 3 U/mL added fVIII, recovery of activity in plasmas A4 and A5 was 1.1 U/mL and 0.51 U/mL, respectively, despite the presence of inhibitor titers of 18 and 11 Bethesda units (BU) per mL. The presence of anti-A2 Abs, which typically have type I kinetics, may have contributed to the overall lower recovery of activity in plasma A7. These results suggest that treatment with high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 Abs. Table 1 Patient Plasma Inhibitor Titer (BU/mL) Recovered Activity at 1U/mL FVIII (U/mL) Recovered Activity at 3 U/mL FVIII (U/mL) A4 18 0.31 1.1 A5 11 0.18 0.51 A7 62 0.07 0.12 Disclosures: No relevant conflicts of interest to declare.

Anti-Factor VIII A2 Epitopes In a Murine Bleeding Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1100-1100
Author(s):  
Joshua Eubanks ◽  
Wallace Hunter Baldwin ◽  
Rebecca Markovitz ◽  
Ernest T Parker ◽  
Shannon L. Meeks
Keyword(s):  
Plasma Concentration ◽  
Factor Viii ◽  
B Cell ◽  
Hemophilia A ◽  
High Dose ◽  
Type I ◽  
C2 Domain ◽  
Type Ii ◽  
Acquired Hemophilia

Abstract Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain but not both. We have shown that within the C2 domain of fVIII antibody epitope is more important than inhibitory titer in predicting pathogenicity in a murine in vivo bleeding model. In this project we investigated the pathogenicity of a diverse panel of anti-A2 monoclonal antibodies (Mabs) in the murine in vivo bleeding model. We have previously characterized anti-A2 antibodies into groups A, AB, B, BCD, C, D, DE, and E based on the pattern of overlap on the B-cell epitopes in a competition ELISA. Table 1 shows the characteristics of the anti-A2 Mabs. Mabs were injected retro-orbitally into Exon 16 hemophilic (E16) mice at a dose of 0.5 umg per g body weight (∼ 65nM plasma concentration). Fifteen minutes later, mice were injected with B-domain deleted human fVIII at a dose of 180U/kg (∼ 2.5nM plasma concentration). In addition, a subset of Mabs has also been tested at a high dose of 360U/kg (∼5 nM). Two hours after fVIII injection, the mice were anesthetized and a 4mm tail snip was performed. Blood was collected in a tube of normal saline over 40 minutes and measured. 4A4, 2-76, and 1D4 are all high inhibitory titer, type I Mabs that produced significant bleeding with 180 U/kg fVIII when compared to control. In addition, 2-76 and 4A4 were tested at the higher dose of fVIII and significant bleeding was again seen. In comparison, the high titer type II Mab 2-54 had a similar inhibitory titer but no significant bleeding at either dose of fVIII. B94 is a type II inhibitor with a similar inhibitory profile to 2-54, but maximum inhibition is 45% as compared to 82% for 2-54. B94 also was not pathogenic at either fVIII dose tested. Both 4C7—a non-inhibitory Mab—and B25—a very low titer Mab that would be predicted to have residual fVIII activity at the Mab concentration tested—did not produce significant bleeding. The inhibitory titer alone did not predict bleeding phenotype within a diverse panel of anti-A2 Mabs. This discrepancy combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy. Disclosures: No relevant conflicts of interest to declare.

Non-Classical Anti-Factor VIII C2 Domain Antibodies Are Pathogenic in a Murine in Vivo Bleeding Model.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2027-2027
Author(s):  
Shannon Meeks ◽  
John F. Healey ◽  
Ernest T. Parker ◽  
Rachel T. Barrow ◽  
John (Pete) Lollar
Keyword(s):  
Blood Loss ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hemophilia A ◽  
Classical Type ◽  
Type I ◽  
Acquired Hemophilia ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain not both. Classical anti-C2 antibodies inhibit the binding of fVIII to phospholipid membranes and to von Willebrand factor. We recently have identified anti-C2 antibodies that inhibit the activation of fVIII, but do not inhibit the binding of fVIII to phospholipid membranes or to von Willebrand factor. These non-classical inhibitors are found in the plasmas of most inhibitor patients (Meeks, S.L. et al. Blood112, 1151-1153, 2008). The pathogenicity of classical and non-classical murine anti-human fVIII monoclonal antibodies (MAbs) was tested in a murine in vivo bleeding model. MAbs were injected into the tail veins of –hemophilia A mice to a peak plasma concentration of 60 nM followed by an injection human B domain-deleted fVIII to a concentration of 2 nM. At 2 hours the mice were anesthetized and a 4 mm tail snip was made. The amount of blood lost into a collection tube of normal saline over 40 minutes was measured. 4A4 is a type I anti-A2 inhibitor with an inhibitory titer of 40,000 Bethesda units (BU)/mg IgG. I54 and 1B5 are classical type I anti-C2 inhibitors with inhibitory titers of 1300 and 930 BU/mg IgG, respectively. 2-77 is a non-classical type II anti-C2 inhibitor that produces a residual fVIII level of 40% at saturating concentrations and whose titer is 21,000 BU/mg IgG. 2-117 is a non-classical anti-C2 MAb with inhibitory activity less than 0.4 BU/mg IgG. All of these MAbs except 2-117 significantly increased blood loss over control mice injected with fVIII alone (p= 0.01-0.02, Mann-Whitney Test) (Fig .1). The amount of blood loss was similar at these saturating concentrations of antibody despite inhibitory titers ranging from 930-40,000 BU/mg IgG. Increasing the dose of fVIII to 4 nM could overcome the bleeding diathesis produced by the non-classical MAb 2-77, but not the type I antibodies, 4A4 and I54. Similar results were seen in the in vitro Bethesda assay where 4A4 completely inhibited both 1 U/ml and 3 U/ml fVIII at saturating concentrations, while 2-77 had 40% residual activity with either 1 or 3 U/ml fVIII (0.4 U and 1.2 U respectively) (Fig. 2). These results suggest that high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 antibodies. FIGURE 1. FIGURE 1. FIGURE 2. FIGURE 2.

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

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
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.

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 ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 958-965 ◽  
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.

scholarly journals Native-Like Aggregates of Factor VIII Are Immunogenic in von Willebrand Factor Deficient and Hemophilia a Mice

2012 ◽  
Vol 101 (6) ◽  
pp. 2055-2065 ◽  
Author(s):  
Dipak S. Pisal ◽  
Matthew P. Kosloski ◽  
C. Russell Middaugh ◽  
Richard B. Bankert ◽  
Sathy V. Balu-iyer
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hemophilia A ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Multimeric composition of factor VIII/von Willebrand factor following administration of DDAVP: implications for pathophysiology and therapy of von Willebrand's disease subtypes

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1272-1278 ◽  
Author(s):  
ZM Ruggeri ◽  
PM Mannucci ◽  
R Lombardi ◽  
AB Federici ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Resting State ◽  
Bleeding Time ◽  
Type I ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have studied the modifications in the multimeric composition of plasma factor VIII/von Willebrand factor and the bleeding time response following administration of 1-Deamino-[8-D-arginine]-Vasopressin (DDAVP) to patients with different subtypes of von Willebrand's disease. In type I, all multimers were present in plasma in the resting state, though they were decreased in concentration. Administration of DDAVP resulted in an increased concentration of these forms as well as the appearance of larger forms than were previously present. There was concomitant correction of the bleeding time. In type IIA, large multimers were absent in the resting state, and although DDAVP induced an average threefold increase in the plasma concentration of factor VIII/von Willebrand factor, the larger multimers did not appear and the bleeding time, although shortened, was not corrected. In contrast, the larger multimers that were also absent from type IIB plasma in the resting state rapidly appeared following DDAVP administration. However, their appearance was transitory and the bleeding time, as in IIA patients, was shortened but not corrected. The characteristic multimeric composition of platelet factor VIII/von Willebrand factor in given subtypes predicted the alteration in plasma factor VIII/von Willebrand factor induced by DDAVP. These studies provide evidence that the different subtypes of von Willebrand's disease represent distinct abnormalities of factor VIII/von Willebrand factor. They also suggest that complete hemostatic correction following DDAVP can be routinely expected only in type I von Willebrand's disease, and only if factor VIII/von Willebrand factor can be raised to normal levels.

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

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2267-2273 ◽  
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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