scholarly journals Highly efficient and unbiased isolation of anti-factor VIII antibodies from hemophilia A inhibitor patient plasma

2020 ◽  
Author(s):  
Zhiyuan Chen ◽  
Vijay Bhoj ◽  
Benjamin Samelson-Jones ◽  
Hsin-Yao Tang
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Highly Efficient ◽  
Inhibitor Patient

Reduced Antigenicity of Recombinant Ovine Factor VIII in Hemophilia A Inhibitor Patient Plasmas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1124-1124
Author(s):  
Philip M Zakas ◽  
Shannon L. Meeks ◽  
Christopher B Doering
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Amino Acid Level ◽  
Coagulation Factor ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Inhibitor Patient

Abstract Abstract 1124 Hemophilia A is an X-linked recessive disorder caused by deficiencies or functional defects in coagulation factor VIII (fVIII). Approximately 20–30% of patients with severe hemophilia A develop antibodies against fVIII (inhibitors) following fVIII replacement therapy, which presents significant complication to the control of subsequent bleeding episodes. State of the art treatment options for patients with inhibitors include fVIII-bypassing agents such as recombinant factor VIIa or activated prothrombin-complex concentrate. Previously, plasma-derived porcine fVIII was a treatment option for inhibitor patients and was effective due to the reduced antigenicity of porcine fVIII toward anti-human fVIII inhibitors. However due to concerns regarding viral contamination, the plasma-derived porcine fVIII products were discontinued and no alternative fVIII products have been made available to patients with inhibitors. Presently, a recombinant porcine fVIII product (OBI-1, Inspiration Biopharmaceuticals) is being investigated in two phase 3 clinical trials for congenital and acquired hemophilia A. Rationale for the development of such a product consists of the prior success of plasma-derived porcine fVIII and the concept that the most effective and lowest risk treatment for fVIII deficiency, even in the presence of inhibitors, remains a fVIII product. Recently, a line of hemophilia A sheep was reestablished from banked frozen sperm and the ovine fVIII (ofVIII) gene, causal mutation, and protein were genetically and biochemically characterized. B-domain deleted (BDD) ovine fVIII shares 86% identity to human fVIII at the amino acid level and confers phenotypic correction, in vivo, to hemophilia A mice using a tail transaction bleeding model. Recombinant ofVIII was expressed in baby-hamster kidney cells and purified to > 95% homogeneity using a two-step ion exchange chromatography procedure. Highly purified ofVIII displays a specific activity of 18,300 units/mg, which is approximately twice that of recombinant BDD human fVIII. Furthermore, the decay of ofVIII activity following thrombin activation is slower than BDD human fVIII suggesting prolonged activity in vivo. Lastly, ofVIII demonstrates equivalent binding to human von Willebrand factor at physiological concentrations in vitro. A translational aim of the present study was to test the hypothesis that unique sequences within ofVIII confer differential antigenicity compared to human and/or porcine fVIII in congenital and acquired inhibitor patient plasmas. To address this hypothesis, the reactivity of 28 samples (22 congenital patient samples designated 1–22, and 6 acquired hemophilia A patient samples designated A1-A6) from the Emory IRB approved inhibitor bank towards recombinant BDD human, porcine, and ovine fVIII were assessed by enzyme-linked immunosorbant assay (Figure 1). When normalized to the reactivity towards human fVIII, the data revealed reduced reactivity towards ofVIII in 27 of 28 total samples. In only one patient was the reactivity towards ofVIII greater than that towards human fVIII and, in this sample, the reactivity towards porcine fVIII also was greater than 100%. Furthermore, plasma reactivity to ovine fVIII was significantly reduced compared to porcine fVIII (P = 0.025; Mann-Whitney U Test). Median values of the relative cross reactivity towards porcine and ovine fVIII were 54 and 38%, respectively. Preliminary inhibitor analysis (Bethesda assay) of three samples shown to contain titers against human fVIII of 25, 19, and 68 BU/ml, revealed undetectable inhibitor titers towards ofVIII in 2 samples, and a titer of 5 BU/ml in the third, respectively. These results suggest that additional orthologous recombinant fVIII molecules may be enabling to the treatment of patients harboring pathogenic inhibitors to human fVIII. Disclosures: No relevant conflicts of interest to declare.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

Treatment of Hemophilia A with a Highly Purified Factor VIII Concentrate Prepared by Anti-FVIIIc Immunoaffinity Chromatography

1992 ◽  
Vol 67 (01) ◽  
pp. 019-027 ◽  
Author(s):  
Joseph E Addiego ◽  
Edward Gomperts ◽  
Liu Shu-Len ◽  
Patricia Bailey ◽  
Suzanne G Courter ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Virus Transmission ◽  
Human Albumin ◽  
Immunoaffinity Chromatography ◽  
Exchange Chromatography ◽  
Pharmacokinetic Studies ◽  
Enveloped Viruses ◽  
Clinically Significant ◽  
Detergent Mixture

SummaryTo reduce the risk of pathogenic virus transmission associated with the therapeutic administration of plasma-derived antihemophilic factor (FVIIIc), a process utilizing anti-FVIIIc immunoaffinity chromatography to isolate FVIIIc has been developed. In addition, the starting cryoprecipitate solution has been treated with an organic solvent/detergent mixture to inactivate lipid-enveloped viruses. A final ion exchange chromatography step is used to further remove contaminants, e.g., anti-FVIIIc antibody, potentially leached with FVIIIc during the immunoaffinity step. The purified FVTII is stabilized for lyophili-zation and storage by the addition of human albumin. The monoclonal anti-FVIIIc antibody used in the immunoaffinity step of the process is not detectable in the final preparation. Viral reduction studies performed at specific steps of the process demonstrate that 11 logs of human immunodeficiency virus (HIV) and greater than 4-5 logs of other lipid-enveloped viruses are inactivated within the first 30 s of exposure to the solvent/ detergent mixture and 4-5 logs of various model viruses, e. g. Endomyocarditis virus (EMC), are physically removed during washing of the immunoaffinity column. The lyophilized product is reconstituted using sterile water in a matter of seconds.The pharmacokinetics of Hemofil® M were compared to those obtained using a standard heat-treated concentrate (Hemofil® CT) in five severe factor VIII deficient hemophiliacs in a randomized, cross-over study. No statistically significant differences were observed in mean half life (p >0.6) or median recovery (p = 0.4) between the two preparations. No clinically significant adverse effects were observed in patients receiving either FVIII preparation.In addition, 43 patients at 18 different centers underwent pharmacokinetic studies, with a nominal dose of 50 u/kg FVIIIc Hemofil® M. The mean recovery was 103.6%, and the t 1/2 was 14.6 h. The recovery of FVIII in this group was as expected, providing an increase of assayed FVIII of approximately 2% per unit of FVTII/kg infused.Clinical trials using Hemofil® M have been initiated in 124 hemophilia A patients. The safety and efficacy of Hemofil® M has been established. To date, 0 of 60 patients tested have seroconverted to HIV. None of the previously untreated patients show clinical or laboratory evidence of Non-A, Non-B hepatitis (NANB), with 21 patients remaining negative as far as presence of antibodies to the Hepatitis C virus (a-HCV negative) at least 6 months after the initial infusion. There is no evidence of neoantigenicity, evidenced by seroconversion to murine antibody. An 8.7% (2 of 23) prevalence of anti-FVIIIc inhibitor development has been observed in previously untreated patients with FVIIIc⩽3%, receiving only the monoclonally purified solvent/ detergent treated FVIII concentrate while on study and on poststudy surveillance. All patients demonstrated clinical hemostasis following product use for either on demand bleeding or surgical prophylaxis.

A Survey of the Effectiveness of Prothrombin Complex Concentrates in Controlling Hemorrhage in Patients with Hemophilia and Anti-Factor VIII Antibodies

1980 ◽  
Vol 44 (01) ◽  
pp. 039-042 ◽  
Author(s):  
Philip M Blatt ◽  
Doris Ménaché ◽  
Harold R Roberts
Keyword(s):  
Factor Viii ◽  
Ad Hoc ◽  
Hemophilia A ◽  
Working Party ◽  
International Committee ◽  
Factor Ix ◽  
Prothrombin Complex Concentrates ◽  
Factor Viii Concentrates

SummaryThe treatment of patients with hemophilia A and anti-Factor VIII antibodies is difficult. Between July 1977 and June 1978, a survey was carried out by an ad hoc working party of the subcommittee on Factor IX concentrates of the International Committee on Thrombosis and Hemostasis to assess the effectiveness of Prothrombin Complex Concentrates in controlling hemorrhage in these patients. The results are presented in this paper and, although subjective, support the view that these concentrates are not as effective in patients with inhibitors as Factor VIII concentrates are in patients without inhibitors.

Investigations on the Nature of Hemophilia A

1963 ◽  
Vol 09 (01) ◽  
pp. 030-052 ◽  
Author(s):  
Eberhard Mammen
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Barium Sulfate ◽  
Freeze Drying ◽  
Room Temperature ◽  
Hemophilia A ◽  
Normal Human Plasma ◽  
Normal Human ◽  
And Storage ◽  
Prothrombin Activation

SummaryIn this paper an inhibitor is described that is found in hemophilic plasma and serum different from any till now described inhibitor. The inhibitor only inhibits prothrombin activation in the “intrinsic clotting systems”. This inhibitor is probably not present in normal human plasma or serum. It is destroyed by ether and freeze drying, is labile to acid and storage at room temperature. It is stable upon dialysis and has not been adsorbed on barium sulfate, aluminum hydroxide or kaolin. It precipitates at 50% v/v saturation with alcohol. The nature of this inhibitor seems to be a protein or lipoprotein.Factor VIII was isolated from hemophilic plasma. The amount isolated was the same as from normal plasma and the activity properties were not different. Hemophiliacs have normal amounts of factor VIII.

Deficiencies and Inhibitors of Thromboplastic Activity

1966 ◽  
Vol 16 (03/04) ◽  
pp. 574-585
Author(s):  
G. F Grannis ◽  
L. A Kazal
Keyword(s):  
Factor Viii ◽  
Time Course ◽  
Gamma Globulin ◽  
Hemophilia A ◽  
Naturally Occurring ◽  
Thrombin Activity ◽  
Activity Curve ◽  
Lipoprotein Complex ◽  
Thromboplastic Activity

SummaryThe effects of hereditary deficiencies of thromboplastic proteins (hemophilia A and B) on the time course of thrombin appearance and disappearance in plasma (the thrombin activity curve, TAC) were compared with the effects of a naturally occurring gamma-globulin inhibitor of thromboplastic activity and with an anti-thromboplastic activity derived from cephalin (phosphatidylserine-lipoprotein complex).Both inhibitors inhibit reactions involving the protein in which hemophilia A plasma is deficient (factor VIII).

The Immunological Properties of Factor VIII

1966 ◽  
Vol 16 (03/04) ◽  
pp. 559-573 ◽  
Author(s):  
L Uszyński
Keyword(s):  
Factor Viii ◽  
Inhibitory Activity ◽  
Hemophilia A ◽  
Severe Form ◽  
Molecular Defect ◽  
Bovine Plasma ◽  
Human Factor Viii ◽  
Antigenic Properties ◽  
Coagulation Tests ◽  
Normal Human

SummaryRabbits immunized against human AHG fibrinogen-free preparations, were shown to produce anti-AHG antibodies. The inhibitory activity of these antibodies was tested by thromboplastin generation test, thrombelastography, and the specific anti-AHG antibodies neutralization test. The latter test permitted quantitative determination of antigenic form of factor VIII. The inhibitory activity of anti-FI-O-Ta serum resulted exclusively from the anti-AHG antibodies which in coagulation tests behaved like circulating anticoagulants directed against factor VIII.The anti-AHG antibodies were neutralizable by normal human serum or plasma even contained only trace of AHG activity after storage. There was no antigenic form of factor VIII in the severely affected patients with hemophilia A, von Willebrand’s disease nor in the normal plasma adsorbed on bentonite. The presented results suggest a molecular defect of factor VIII in patients with hemophilia A. The severe form of this disease depends, probably, on a major impairment of AHG biosynthesis, leading to changes in the antigenic properties of the molecule. The AHG from rabbit, porcine and bovine plasma respectively did not neutralize the anti-AHG antibodies formed in rabbits immunized against human factor VIII preparations.

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