The Diversity of Anti-Factor VIII B-Cell Epitopes in Hemophilia a Patients with Inhibitors

Abstract The most significant complication in the management of patients with hemophilia A continues to be the development of anti-factor VIII antibodies in response to infusions of the fVIII protein. Patients with hemophilia A typically develop a polyclonal B-cell response to fVIII with antibodies to the A2 and C2 domains being the most prevalent. Within the A2 and C2 domains, we have identified non-overlapping epitopes with unique characteristics. There are 5 non-overlapping epitopes in the A2 domain and 3 non-overlapping epitopes in the C2 domain. Within the C2 domain, antibody epitope was more important than inhibitory titer in predicting response to fVIII treatment in a murine in vivo bleeding model. Similar findings were seen within the A2 domain. In previous studies we developed a competition ELISA that assessed the ability of patient plasma to compete with biotinylated monoclonal antibodies (MAbs) with known epitopes for binding to fVIII. Competition with at least 1 of 3 non-overlapping anti-fVIII C2 domain MAbs was seen in 19 of 26 (73%) patient plasmas. The high volume of plasma needed for each monoclonal antibody assessed restricts the use of this assay to map a limited number of epitopes from a single plasma sample. Given the number of non-overlapping epitopes identified on the fVIII protein, the goal of this study was to modify the previous competition ELISA to better define the diversity of the immune response to fVIII using a clinically feasible volume of plasma. Plasma samples and limited clinical data from hemophilia A patients with inhibitors who were HIV negative were obtained from the Biologic Specimen and Data Repository Information Center (BioLINCC) of the NHLBI. A single sample was available from 27 patients while 2 samples from different time periods were available from an additional 23 patients. Epitope mapping was performed using a modified competition ELISA utilizing a single biotinylated MAb concentration with patient plasma (~50 µl total for all experiments) diluted 1:40. The rate of MAb binding in the presence of patient plasma was compared to the rate of MAb binding in the presence of control severe hemophilia A plasma without inhibitors. Competition was considered present when the binding was reduced more than 2 standard deviations below control. The anti-fVIII MAbs in this study were 4 anti-A2 antibodies with non-overlapping epitopes (A2-A, A2-B, A2-D, A2-E) and 3 anti-C2 antibodies with non-overlapping epitopes(C2-A, C2-B, C2-C) as well as one inhibitory antibody from both the A3 and C1 domains. A modified Bethesda assay was performed on each sample which allowed us to group the plasmas into 3 inhibitory titer ranges: < 6 BU/ml, 6-16 BU/ml, and >16 BU/ml. Of the 73 plasma samples 20 showed competition with at least 1 MAb as shown in Table 1. Six of 36 plasmas (17%) with titers of <6 BU/ml showed competition with a single MAb. Three of 20 plasmas (15%) with titers of 6-16 BU/ml showed competition with either 1 or 2 MAbs. Eleven of 17 plasmas with titers of >16 BU/ml showed competition with between 1 and 9 MAbs. No competition was detected in the 6 patients with moderate or mild hemophilia A. In 12 of the 23 patients with 2 samples available, epitopes were detected in at least 1 sample but none of these patients had identical epitope spectra in both samples. Despite the lower overall detection rate compared to the previous assay, our results highlight the diversity of epitope spectra present in patient plasmas with high inhibitory titers. This diversity underscores the need to better understand the makeup of the immune response to fVIII in patients with hemophilia A at the individual epitope level. Given the success of detecting each of the 9 MAb epitopes in at least one patient plasma, work is ongoing to improve the sensitivity of this assay to further define differences in response between patients. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

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Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model.

Blood ◽

10.1182/blood.v114.22.27.27 ◽

2009 ◽

Vol 114 (22) ◽

pp. 27-27

Author(s):

Shannon Meeks ◽

Ernest T Parker ◽

Amy L. Dunn ◽

John F Healey ◽

Pete Lollar

Keyword(s):

Immune Response ◽

Immune System ◽

Factor Viii ◽

Binding Site ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Foreign Protein ◽

C2 Domain ◽

Wild Type ◽

Antibody Titers

Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.

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B-Cell Epitopes Within The A2 and C2 Domains Of Coagulant Factor VIII Refined By Orthologous Protein Antigenicity

Blood ◽

10.1182/blood.v122.21.1101.1101 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1101-1101

Author(s):

Philip M Zakas ◽

Kristine Vanijcharoenkarn ◽

Rebecca Markovitz ◽

Shannon L. Meeks ◽

Christopher B Doering

Keyword(s):

Amino Acids ◽

Amino Acid ◽

B Cell ◽

Hemophilia A ◽

Cross Reactivity ◽

C2 Domain ◽

B Cell Epitopes ◽

C2 Domains ◽

Group A ◽

A2 Domain

Abstract Approximately 20-30% of severe hemophilia A patients develop inhibitors (anti-fVIII antibodies) to human fVIII (hfVIII) with the primary targets being the A2 and C2 domains. Studies of A2 domain-specific murine monoclonal antibodies (MAbs) targeting hfVIII identified 8 distinct groups of inhibitory MAbs displaying varying inhibitor titers and kinetics, with groups A, D, and E possessing the greatest inhibitory activity. Furthermore within the C2 domain, we previously characterized 5 distinct groups, whereby group BC antibodies were the most common and displayed the highest specific inhibitory activity. At present, a recombinant porcine fVIII (pfVIII) product (OBI-1, Baxter International) is being investigated within a pivotal clinical trial for persons with acquired hemophilia A. The rational for a recombinant pfVIII product stems from the limited cross reactivity of pfVIII, and potentially other fVIII orthologs, with antibodies developed against hfVIII and the prior clinical success of plasma-derived pfVIII. We have described the biochemical characterization of both pfVIII and, more recently, ovine fVIII (ofVIII) and now investigate their antigenic properties to define/refine key inhibitory epitopes and determine the amino acid differences that confer reduced antigenicity. Using an ELISA-based assay, 15 A2 and 12 C2 MAbs spanning all inhibitory groups were screened against B-domain deleted (BDD) pfVIII and ofVIII. Only 4 A2 domain targeting MAbs representing groups B, C, DE, and E were found to bind pfVIII and all displayed reduced cross-reactivity (Figure 1). Additionally, 3 MAbs within groups B, C, or E also bound ofVIII. No group A or D MAbs screened bound either ortholog. Of the C2 domain targeting MAbs tested, 3 MAbs (I109, D102, and ESH-8) from groups AB, B and C, respectively, bound pfVIII to lesser degrees. Nine of the 12 C2 targeting MAbs bound ofVIII, albeit at reduced signal strength, suggesting divergence in C2 epitopes between pfVIII and hfVIII as well as between pfVIII and ofVIII. Using these data and previous literature defining the specific epitopes of these MAbs, we suggest that species-specific amino acid sequence differences may be responsible for the differential binding. MAb413, a well characterized A2 group A inhibitor, was found not to bind either ortholog. Alanine scanning mutagenesis within the 484 – 508 epitope demonstrated that single point mutations at R484, Y487, R489, or P492 could each inhibit binding of MAb413 to below 10%. In addition, P485 or S488 mutations each reduced binding to approximately 40% (Lubin et al J Biol Chem 272:48). Both pfVIII and ofVIII share substitutions R484S, Y487H, R489G, and pfVIII contains unique substitutions P485A, S488P, and P492L. Since all but two A2 group A MAbs have been mapped to residues 484-508, and because all group A MAbs compete with each other for their epitope, we hypothesize that these substitutions are responsible for diminished binding of group A MAbs. Group D inhibitory antibodies have been mapped to residues 604-740. Within this region, there are 12 shared amino acid substitutions and 7 amino acids altered in either ortholog but not both. As it was demonstrated that the orthologs display differential cross reactivity, this suggests disparate amino acids alter inhibitor-epitope recognition. Walter et al. (J Biol Chem 2013 288:14) used small angle x-ray scattering (SAXS) to isolate the epitope of MAb 3E6, a C2 MAb group A inhibitor, to exposed loops R2209-S2216 and L2178-D2187. Our data show that this MAb is not cross reactive and both orthologs contain S2216T and S2182N substitutions. Finally, ESH-8, a well-studied C2 group C MAb, binds ofVIII and pfVIII equivalently at 29% of the hfVIII level. The epitope for ESH-8 contains amino acids 2248-2285 and within this region there is only one conserved substitution, F2275L. Therefore, we predict that substitution of this residue could restore or further diminish ESH-8 binding. These data provide evidence that the lack of A2 group A and D epitopes, as well as the C2 domain group BC epitopes within these orthologs may be responsible for the residual procoagulant activity in the context of inhibitor plasma. Furthermore, these data also suggest that 1) B-cell epitopes can be defined or refined through this approach, 2) less antigenic residues can be identified and 3) the information obtained can be utilized to rationally design recombinant fVIII products with reduced antigenicity. Disclosures: No relevant conflicts of interest to declare.

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Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B cells as Ig fusion proteins

Blood ◽

10.1182/blood-2004-11-4274 ◽

2005 ◽

Vol 105 (12) ◽

pp. 4865-4870 ◽

Cited By ~ 125

Author(s):

Tie Chi Lei ◽

David W. Scott

Keyword(s):

B Cells ◽

Factor Viii ◽

B Cell ◽

Hemophilia A ◽

Humoral Response ◽

Retroviral Vectors ◽

Inhibitory Antibody ◽

C2 Domains ◽

Antibody Titers ◽

A2 Domain

Abstract Up to 30% of patients with hemophilia A given therapeutic factor VIII (fVIII) can make inhibitory antibodies, the majority of which are reactive with its C2 and A2 domains. We have previously demonstrated that antigen-specific tolerance to several antigens can be induced by lipopolysaccharide (LPS)-activated B-cell blasts transduced with immunoglobulin (IgG)-antigen fusion constructs. To apply this system to hemophilia A inhibitor formation, we created retroviral vectors expressing fVIII amino acids S2173-Y2332 (C2 domain) and S373-R740 (A2 domain) in frame with an IgG heavy chain backbone. These vectors were transduced into B-cell blasts to induce tolerance in both naive and fVIII-primed hemophilic (E16 fVIII-/-) mice. Thus, treatment of E16 fVIII-/- mice with B cells expressing fVIII C2 and A2 domains led to tolerance in terms of specific humoral response (including inhibitory antibody titers) and cellular responses to fVIII and its C2 or A2 domains. Moreover, a significant reduction in immune responses to fVIII could be achieved in immunized hemophilic mice with existing anti-fVIII titers. This hyporesponsive state persisted for at least 2 months and withstood additional challenge with fVIII. Further experiments, in which mice were treated with a depleting monoclonal anti-CD25, suggested that a regulatory T cell may be required for the tolerogenic effect of transduced B cells. These findings demonstrate that B-cell presentation of fVIII domains on an Ig backbone specifically prevents or decreases existing antibodies in hemophilia A mice. (Blood. 2005;105:4865-4870)

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Anti-Factor VIII C1 Domain Antibodies Are Present in the Plasmas of Patients with Hemophilia and Inhibitors

Blood ◽

10.1182/blood.v124.21.1482.1482 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1482-1482

Author(s):

Glaivy Batsuli ◽

Courtney Cox ◽

John F. Healey ◽

Pete Lollar ◽

Shannon L Meeks

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Conflicts Of Interest ◽

High Titer ◽

Plasma Samples ◽

Severe Hemophilia ◽

Competition Elisa ◽

Antigenic Properties ◽

C1 Domain ◽

Inhibitor Titer

Abstract Hemophilia A is an X-linked disorder characterized by a deficiency or absence of blood coagulation protein factor VIII (fVIII). Treatment involves replacement of fVIII through infusions for acute bleeding episodes or prevention of bleeding events. Approximately 30% of individuals with severe hemophilia A will develop antibodies to fVIII. Many studies have characterized the antigenic properties of the C2 and A2 domains as these domains are considered the predominant immunogenic domains of the fVIII protein. However, there is increasing evidence that the C1 domain contributes to fVIII function and immune response to fVIII. Our laboratory has produced and purified a murine IgG2ak anti-human C1 domain monoclonal antibody (MAb), designated 2A9. In this study, we characterized the functional properties of MAb 2A9 using standard coagulation testing including its anti-fVIII inhibitor titer by Bethesda assay and its ability to inhibit fVIII binding to von Willebrand factor (VWF) by competition ELISA. In the Bethesda assay, 2A9 has an inhibitor titer of 97 BU/mg and is a type II inhibitor. In addition, MAb 2A9 inhibits fVIII binding to VWF in an ELISA assay with a 50% inhibitory concentration (IC50) of 1 µg/ml. This is in comparison to the potent high-titer inhibitory anti-C2 MAb I89 (IC50 0.02 µg/ml) and a control non-inhibitory anti-A2 MAb ID4 (IC50 > 10 µg/ml). We tested 11 plasma samples from patients with congenital hemophilia with inhibitors in the Emory IRB-approved inhibitor bank. The plasma samples have inhibitory titers ranging from 1 - 188 BU/ml with a median inhibitory titer of 54 BU/ml and mean inhibitory titer 59 BU/ml. The plasmas were tested for the presence of antibodies that compete with anti-C1 domain MAb 2A9 using competition ELISA with fVIII as the antigen. Biotinylated MAb 2A9 was serially diluted and the concentration of antibody required to produce an absorbance at 405 nm of 0.3 was compared between control severe hemophilia A plasma and inhibitor plasma samples. Inhibitor plasma samples that reduced the ELISA titer of MAb 2A9 were considered a competitive inhibitor. Of the 11 inhibitor plasma samples, 4 were found to compete with MAb 2A9. Our study demonstrates that anti-C1 domain antibodies are present in the plasma of patients with hemophilia A and inhibitors. Given the increasing evidence that the C1 domain is important in fVIII function it is likely that these anti-C1 antibodies are clinically relevant. Therefore, domains other than A2 and C2 need to be included in future studies of fVIII B cell epitopes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes

Blood ◽

10.1182/blood.v122.21.2358.2358 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2358-2358

Author(s):

Justin D Walter ◽

Rachel A Werther ◽

Caileen M Brison ◽

John F. Healey ◽

Shannon L. Meeks ◽

...

Keyword(s):

Factor Viii ◽

Ternary Complex ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Structural Basis ◽

C2 Domain ◽

Proteolytic Activation ◽

X Ray ◽

Binding Interface ◽

Inhibitory Antibodies

Abstract The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies. Disclosures: No relevant conflicts of interest to declare.

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Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Blood ◽

10.1182/blood.v128.22.3787.3787 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3787-3787

Author(s):

Pete Lollar ◽

Ernest T. Parker ◽

John F. Healey ◽

Christopher B. Doering

Keyword(s):

Immune Response ◽

Factor Viii ◽

Immune Complexes ◽

Hemophilia A ◽

Analytical Ultracentrifugation ◽

Conflicts Of Interest ◽

Sedimentation Coefficient ◽

Coagulation Factor ◽

Velocity Sedimentation ◽

Molar Excess

Abstract Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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The cDNA and derived amino acid sequence of porcine factor VIII

Blood ◽

10.1182/blood.v88.11.4209.bloodjournal88114209 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4209-4214 ◽

Cited By ~ 26

Author(s):

JF Healey ◽

IM Lubin ◽

P Lollar

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Untranslated Region ◽

Hemophilia A ◽

Sequence Determination ◽

C2 Domains ◽

Protein Alignments ◽

A2 Domain

The cDNA corresponding to 137 bp of the 5′ untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337–372 and 1649–1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

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The Structural and Functional Diversity of the Humoral Immune Response to the A2 Domain of Human Factor VIII

Blood ◽

10.1182/blood.v118.21.2247.2247 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2247-2247

Author(s):

Rebecca Markovitz ◽

John (Pete) S. Lollar ◽

John F. Healey ◽

Ernest T Parker ◽

Shannon Meeks

Keyword(s):

Immune Response ◽

Factor Viii ◽

Functional Diversity ◽

Humoral Immune Response ◽

Hemophilia A ◽

Venn Diagram ◽

Type I ◽

Humoral Immune ◽

Thrombin Cleavage ◽

A2 Domain

Abstract Abstract 2247 Hemophilia A is an X-linked recessive disorder that is caused by a deficiency or defect of factor VIII (fVIII) coagulant protein. The major complication of treatment is the development of anti-fVIII antibodies (inhibitors) in approximately 20–30% of patients with severe hemophilia A. The majority of these inhibitors are directed against the A2 or C2 domains (Prescott R et al. Blood 1997). This study examines the structural and functional diversity of the humoral immune response to the A2 domain of human fVIII. A panel of 24 murine anti-A2 monoclonal antibodies (MAbs) produced in our laboratory plus MAb413 (American Red Cross) and GMA012 (Green Mountain, Burlington, VA) were used in this study. Previous studies have shown that anti-C2 MAbs produced from murine anti-fVIII hybridomas had a similar spectrum of epitopes to those found in inhibitor patient plasmas (Meeks SL et al. Blood 2008). A competition sandwich ELISA with immobilized anti-A2 primary MAb, human fVIII, biotinylated anti-A2 secondary MAb and streptavidin–alkaline phosphatase conjugate for detection was used to determine overlapping epitopes. Each antibody was used as both a capture and detection antibody. 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. Porcine/human hybrid fVIII proteins were employed in a direct ELISA to fine map the epitopes of the anti-A2 MAbs. The results of both the competition and human/porcine mapping ELISAs were compiled into a Venn diagram describing overlapping epitopes for all MAbs. Functional mapping of the MAbs included fVIII inhibitor titers by modified Bethesda assay, inhibition in a purified intrinsic Xase assay, and inhibition of thrombin cleavage of fVIII. Thrombin activation assays were run with varying concentrations of MAbs, and fVIII cleavage by thrombin was analyzed by SDS-PAGE. The competition ELISA results demonstrated 7 non-overlapping epitopes on the A2 domain of human fVIII (Figure 1). In addition, the human/porcine mapping ELISA revealed that the epitopes of the anti-A2 MAbs covered the majority of the A2 domain. The inhibitor titers of the anti-A2 MAbs ranged from non-inhibitory to 40,000 Bethesda units (BU)/mg IgG (Table). The inhibitory MAbs displayed both type I (greater than 95% inhibition at saturating MAb concentrations) and type II-(incomplete inhibition at saturating MAb concentrations) behavior. MAb413, a group D MAb, noncompetitively inhibits factor VIIIa cofactor activity without affecting thrombin cleavage. 2–54, a group G MAb, inhibits thrombin cleavage of both heavy and light chains. In contrast, 1D4, which overlaps groups B, E, and F, only inhibited light chain cleavage. Overall these results indicate that the humoral immune response to the A2 domain of fVIII is complex in terms of both structural and functional epitopes. These anti-A2 MAbs were found to target 7 non-overlapping epitopes spanning the majority of the A2 domain. Elucidation of the structural and functional complexity of the anti-A2 repertoire will lead to a better understanding of the pathogenicity of A2 inhibitors.Table:A2 MAb CharacteristicsMAbInhibitor Titer (BU/mg)GroupStructural EpitopeB25100A444–5082G10500B468–484G323000C468–508MAb41321,000D484–5082–934E541–604B664000F604–7402–5433,000G508–541, 604–740 Disclosures: No relevant conflicts of interest to declare.

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Anti-Factor VIII A2 Epitopes In a Murine Bleeding Model

Blood ◽

10.1182/blood.v122.21.1100.1100 ◽

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.

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Pseudoallergy Induced by Factor VIII Injection in Three Subjects with Hemophilia A

Blood ◽

10.1182/blood.v118.21.4353.4353 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4353-4353

Author(s):

Caroline Pfeiffer ◽

Géraldine Lavigne-Lissalde ◽

Georges-Etienne Rivard

Keyword(s):

Immune Response ◽

Factor Viii ◽

Replacement Therapy ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Optimal Choice ◽

Severe Hemophilia ◽

Intron 22 Inversion ◽

Before And After ◽

Recombinant Fviii

Abstract Abstract 4353 BACKGROUND Hypersensitivity reactions to factor VIII (FVIII) concentrates are rare but well known complications of hemophilia treatment with FVIII concentrates. Occasional reports have suggested IgE mediation. Many cases have presented circumstantial evidence for the implication of FVIII but have failed to clearly identify FVIII-targeting antibodies (Abs). The absence of evidence for IgE contribution to those reactions, but the presence of FVIII specific IgM and/or IgG1 and/or IgG3, could support the hypothesis of complement mediation. These reactions have been called “complement activation-related pseudoallergy” (CARPA). As we did not study complement we call these reactions CARPA-like. We report 3 cases of CARPA-like reactions to FVIII which we believe could contribute to alert clinicians to this potential serious side effect of FVIII replacement therapy and could shed some light on its pathophysiology. PATIENTS / METHODS Subject A, 5 y. old Caucasian previously untreated patient (PUP) with mild hemophilia A (FVIII 0.12 IU/mL, 6955C>T exon 26 P2300S, H1 haplotype, no inhibitor with Nijmegen < 0.4 unit), developed a CARPA-like reaction with his sixth injection of a von Willebrand factor-containing FVIII concentrate (Wilate®, Octapharma) for traumatic knee hemarthrosis. He never needed further FVIII replacement. Subject B, 5 y. old African PUP with severe hemophilia A (FVIII < 0.01 IU/mL, intron 22 inversion, H1 haplotype, no inhibitor with Nijmegen < 0.4 unit) developed a CARPA-like reaction with his sixth injection of Wilate®. He was switched to a B-domain deleted recombinant FVIII concentrate (Xyntha™, Pfizer) and has yet to relapse with CARPA-like reaction after more than 100 exposure days. Subject C, 15 y. old Caucasian PUP with severe hemophilia A (FVIII < 0.01 IU/mL, intron 22 inversion, H1 haplotype, inhibitor with Nijmegen 10 units) developed a CARPA-like reaction with his second dose of a full length recombinant FVIII concentrate (Kogenate®,Bayer) while on ITI induction program. Blood samples were obtained from each patient shortly before and after their CARPA-like reaction. ELISA assays (AM. Vincent, Haemophila 2009) were used to test the presence of IgG, IgM, IgE Abs reacting with different types of FVIII (Baxter’s Advate, Wilate®, Kogenate®, Xyntha™) in patients’ plasma. The x-MAP technology was used to confirm the presence of IgG and IgM Abs, and to better define their isotypic profile. For that purpose, Abs specific for the heavy chain (HC; MAb 8860) or light chain (LC; MAb ESH8) of the FVIII were first immobilized on magnetic fluorescent beads, and then incubated with EDTA-dissociated FVIII. After incubation with patients’ plasma samples, binding of IgG subclasses (IgG1 to IgG4) and IgM were revealed by specific anti-human phycoerythrin-labeled Abs. RESULTS For patients A and B (with negative Bethesda titre), we identified an immune response characterized by the presence of IgM, as well as IgG (patient A IgG1; patient B IgG1, IgG3), simultaneously directed against the HC and the LC. ELISA assays on these 2 patients showed the presence of IgM and IgG, as well as the absence of IgE compatible with a CARPA-like reaction. Similar results with the 4 different FVIII concentrates are in favour of an immune response specific to FVIII rather than an immune response to anything else than FVIII. To further support this contention, ELISA assays were all negative for patient A’s mother and for his PUP hemophilic brother, as well as for subject B’s mother and for six normal control plasma tested on the same ELISA plate. Patient C (with positive Bethesda titre) presented anti-FVIII IgG (IgG1 to IgG4) and IgM Abs also targeting the HC and the LC simultaneously. These 3 patients developed a CARPA-like reaction associated with injection of FVIII concentrates. Subjects A and C were not rechallenged with FVIII after their reaction and progressively lost their anti-FVIII Abs. Subject B was rechallanged over 100 times with the B-domain deleted Xyntha™ without reaction and with progressive lost of his anti-FVIII Abs, suggesting that his reaction might have been mediated by Abs reacting to the B-domain of FVIII. CONCLUSION CARPA-like reactions to FVIII concentrates can be associated with anti-FVIII Abs. ELISA and x-MAP are useful technologies to investigate this complication of hemophilia treatment and could help select an optimal choice of FVIII concentrate for further replacement therapy. Disclosures: No relevant conflicts of interest to declare.

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