Dominant C2 Domain Epitope Specificity of Inhibitor Antibodies Elicited by a Heat Pasteurized Product, Factor VIII CPS-P, in Previously Treated Hemophilia A Patients without Inhibitors

1998 ◽  
Vol 79 (01) ◽  
pp. 62-68 ◽  
Author(s):  
Yoshikatsu Sawamoto ◽  
Richard Prescott ◽  
Degang Zhong ◽  
Evgueni Saenko ◽  
Eveline Mauser-Bunschoten ◽  
...  
Keyword(s):  
Factor Viii ◽  
Untreated Patient ◽  
Hemophilia A ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Heat Pasteurization ◽  
Recombinant Fviii ◽  
Product Factor ◽  
A2 Domain

SummaryFrom June, 1990, to November, 1991, in The Netherlands and Belgium, 16 previously treated severe hemophilia A patients (PTP) developed inhibitors after exposure to factor VIII CPS-P, a new heat pasteurized product. A previously untreated patient (PUP) also developed an inhibitor to CPS-P. In inhibitor neutralization assays with recombinant fVIII C2 and A2 domain polypeptides, plasmas from 14 PTPs were ≥79% neutralized by C2 and <10% by A2, but the PUP plasma was partially neutralized by C2 (48%) and A2 (28%). Immunoprecipitation assays of the PTP and PUP plasmas with the fVIII heavy chain and with recombinant C2 and A3-C1 polypeptides confirmed that the C2 dominant immune response to CPS-P was found only in the PTPs. Competition of the binding of 2 inhibitors to 125I-CPS-P by unlabeled CPS-P and another plasma fVIII was similar, demonstrating that the antibody response was not directed to epitopes only present in CPS-P. We propose that the immunogenicity of the CPS-P C2 domain was altered by heat pasteurization.

scholarly journals Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain

1999 ◽  
Vol 81 (01) ◽  
pp. 39-44 ◽  
Author(s):  
R. Laub ◽  
Di Giambattista ◽  
P. Fondu ◽  
H.-H. Brackmann ◽  
H. Lenk ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hepatitis A ◽  
Hemophilia A ◽  
Hepatitis A Virus ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Inhibitor Titer

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

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 Multicenter Pharmacosurveillance Study for the Evaluation of the Efficacy and Safety of Recombinant Factor VIII in the Treatment of Patients with Hemophilia A

1997 ◽  
Vol 78 (05) ◽  
pp. 1352-1356 ◽  
Author(s):  
Emel Aygören-Pürsün ◽  
Inge Scharrer ◽  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Parvovirus B19 ◽  
Subjective Evaluation ◽  
Recombinant Factor Viii ◽  
Fviii Inhibitor ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Bleeding Episodes ◽  
Recombinant Fviii

SummaryIn this open multicenter study the safety and efficacy of recombinant factor VIII (rFVIII) was assessed in 39 previously treated patients with hemophilia A (factor VIII basal activity ≤15%).Recombinant FVIII was administered for prophylaxis and treatment of bleeding episodes and for surgical procedures. A total of 3679 infusions of rFVIII were given. Efficacy of rFVIII as assessed by subjective evaluation of response to infusion and mean annual consumption of rFVIII was comparable to that of plasma derived FVIII concentrates. The incremental recovery of FVIII (2.4 ± 0,83%/IU/kg, 2.12 ± 0.61%/IU/kg, resp.) was within the expected range. No clinical significant FVIII inhibitor was detected in this trial. Five of 16 susceptible patients showed a seroconversion for parvovirus B19. However, the results are ambiguous in two cases and might be explained otherwise in one further case. Thus, in two patients a reliable seroconversion for parvovirus B19 was observed.

scholarly journals The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3663-3671 ◽  
Author(s):  
Richard Prescott ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Inge Scharrer ◽  
Inga Marie Nilsson ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Epitope Specificity ◽  
Extensive Analysis ◽  
Number Of Patients ◽  
Recombinant Fviii ◽  
Inhibitor Titer

Abstract Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

scholarly journals The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3663-3671 ◽  
Author(s):  
Richard Prescott ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Inge Scharrer ◽  
Inga Marie Nilsson ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Epitope Specificity ◽  
Extensive Analysis ◽  
Number Of Patients ◽  
Recombinant Fviii ◽  
Inhibitor Titer

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

Epitope Specificity of Factor VIII Inhibitor Antibodies

1998 ◽  
Vol 18 (03) ◽  
pp. 121-128 ◽  
Author(s):  
Dorothea Scandella
Keyword(s):  
Factor Viii ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Inhibitor Activity ◽  
Epitope Specificity ◽  
Factor Viii Concentrates ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Viii Inhibitor ◽  
Acidic Region

SummaryAntibodies that inactivate factor VIII develop most frequently in patients with severe hemophilia A, and they present a serious complication in the therapy of such individuals. Most patients with inhibitors have two or more different antibodies capable of factor VIII inactivation in their plasma, as shown by assays in which several factor VIII domain fragments are tested for neutralization of the inhibitor activity. Such neutralization assays of 23 patient plasmas suggest that there are three common epitopes. These epitopes have been localized by several methods to the A2, A3, and C2 factor VIII domains. Less common inhibitor epitopes lie within the heavy chain acidic region residues 336-372 and in a second region of the C2 domain. The light chain acidic region, residues 1649-1689, may also contain an inhibitor epitope, but this remains to be confirmed. The inhibitor epitopes of autoantibody patients are more restricted as half of them have anti-C2 domain antibodies that make up 95% of the inhibitor activity. The remainder have several inhibitors similar to those of the hemophiliacs. The predominant C2 domain epitope specificity was also seen in rare cases of inhibitor development due to two heat pasteurized factor VIII concentrates in previously treated patients without inhibitors. Inhibitors in mild hemophiliacs are rare as these individuals are usually tolerant to factor VIII. When tolerance is overcome in such patients, the immune responses characterized were diverse. In some patients there was a complete loss of tolerance.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2837-2837 ◽  
Author(s):  
Margaret A. Robinson ◽  
Courtney Cox ◽  
Wallace Hunter Baldwin ◽  
Philip Zakas ◽  
Shannon L Meeks
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
B Cell ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
C2 Domain ◽  
Plasma Samples ◽  
C2 Domains ◽  
Competition Elisa ◽  
A2 Domain

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.

Development of neutralizing antibodies in application of various concentrates of blood coagulation factor VIII in the treatment of hemophilia A

2021 ◽  
Author(s):  
Н.И. Зозуля
Keyword(s):  
Factor Viii ◽  
Cell Line ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Human Cell Line ◽  
Chinese Hamster Cell ◽  
Coagulation Factor Viii ◽  
Recombinant Fviii

Серьезным осложнением, связанным с лечением гемофилии А, является развитие ингибиторов. В последние годы был проведён ряд исследований, посвящённых данной проблеме: RODIN, INSIGHT, FranceCoag, SIPPET и NuProtect. В данном обзоре суммируются основные результаты этих исследований. Согласно результатам рандомизированного исследования SIPPET, препараты плазматического фактора свертывания крови VIII (FVIII) обладают меньшей иммуногенностью, чем препараты рекомбинантного FVIII, синтезированного из клеточной линии китайских хомячков, что следует учитывать при выборе стратегии лечения. Согласно результатам исследования NuProtect, опубликованным в 2019 г., концентрат рекомбинантного FVIII, полученный из клеточной линии человека, демонстрирует профиль иммуногенности, сходный с таковым у препаратов плазматического FVIII. У ранее нелеченых пациентов с ненулевыми мутациями при применении симоктоког альфа не наблюдалось образования ингибиторов, также как и в случае применения препаратов плазматического FVIII в исследовании SIPPET. Inhibitor development is a serious complication associated with hemophilia A therapy. A number of studies have been carried out of this issue — RODIN, INSIGHT, FranceCoag, SIPPET, and NuProtect. This review summarizes the main results of these studies. According to the results of the SIPPET randomized trial, plasma-derived coagulation factor VIII (FVIII) products are less immunogenic than recombinant FVIII products synthesized from a Chinese hamster cell line; this fact should be taken into account in choosing a treatment strategy. According to the results of NuProtect study published in 2019, the concentrate of human cell line-derived recombinant FVIII demonstrates immunogenicity profi le similar to the one in plasma-derived FVIII products. Previously untreated patients with non-zero mutations receiving simoctocog alfa did not show development of inhibitors as well as in case of administration of plasma-derived FVIII products in SIPPET study.

Longitudinal Analysis of Factor VIII Inhibitors in a Previously Untreated Mild Haemophilia A Patient with an Arg593→Cys Substitution

1999 ◽  
Vol 81 (05) ◽  
pp. 723-726 ◽  
Author(s):  
Simone Timmermans ◽  
Ellen Turenhout ◽  
Christine Bank ◽  
Karin Fijnvandraat ◽  
Jan Voorberg ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Longitudinal Analysis ◽  
Factor Viii Inhibitor ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Initial Development ◽  
Inhibitor Development ◽  
Epitope Specificity ◽  
A2 Domain

SummaryRecent studies suggest that certain missense mutations associated with mild to moderate haemophilia A predispose to inhibitor development. In this study, we present a longitudinal analysis of the epitope specificity of an inhibitor that developed in a mild haemophiliac with an Arg593→Cys mutation. Immunoprecipitation studies revealed the presence of antibodies directed towards the light chain and A2 domain of factor VIII. Limited reactivity was observed with metabolically labelled C2 domain. Almost complete inhibitor neutralization was achieved upon addition of A2 domain. Binding of the inhibitor was not affected by the presence of the Arg593→Cys substitution in the recombinant A2 fragment. Evaluation of the epitope specificity of anti-factor VIII antibodies in plasma samples obtained at different time-points of inhibitor development revealed initial development of a low titre inhibitor directed towards the A2 domain and factor VIII light chain. A second period of factor VIII replacement therapy resulted in a dramatic rise in factor VIII inhibitor titre, which maintained their original epitope specificity. Based on the results of this and previous studies (Fijnvandraat et al., 1997; Thompson et al., 1997) it is argued that inhibitor development in patients with the Arg593→Cys mutation may proceed via a similar mechanism.

scholarly journals Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2452-2461 ◽  
Author(s):  
JG Gilles ◽  
J Arnout ◽  
J Vermylen ◽  
JM Saint-Remy
Keyword(s):  
Antibody Response ◽  
Factor Viii ◽  
Specific Antibody ◽  
Hemophilia A ◽  
Significant Proportion ◽  
Functional Method ◽  
Specific Antibody Response ◽  
Functional Assays ◽  
Epitope Specificity ◽  
Chromogenic Assay

Abstract A significant proportion of hemophilia A patients receiving transfusions of factor VIII (FVIII) develop a specific antibody response towards FVIII. These antibodies are usually detected by assays in which they inhibit the function of the molecule, such as the Bethesda clotting test. We have prepared anti-FVIII antibodies by specific immunoadsorption from the plasma of four hemophiliacs with stable inhibitor levels. The isotypic distribution of such antibodies was determined and their capacity to bind to insolubilized FVIII was compared with their inhibitory activity in two functional assays, namely, the Bethesda assay and a chromogenic assay. In addition, the FVIII epitope specificity was determined by competition with monoclonal antibodies for the binding to insolubilized FVIII. We show here that (1) anti-FVIII antibodies are not isotypically restricted; thus, a significant proportion of specific IgG2 was found; (2) antibodies are frequently directed towards epitopes of FVIII that are not directly involved in the function of the molecule and therefore escape detection in the Bethesda method or chromogenic assay; and (3) each patient shows a unique pattern of FVIII epitope recognition. We conclude that evaluation of anti-FVIII antibodies by a functional method does not provide an accurate evaluation of the specific antibody response. These findings have important implications for the comparison of the immunogenicity of FVIII molecules produced by different technologies and for the development of methods to control anti-FVIII antibody production.

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