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Catalytic anti-factor VIII allo-antibodies

a technology of catalytic anti-factor and alloantibody, which is applied in the field of catalytic anti-factor viii alloantibodies, can solve the problems of life-threatening and crippling haemorrhagic disease, deficiency or deficiency, etc., and achieve the effect of increasing the catalytic rate of thrombin

Inactive Publication Date: 2009-08-20
KAVERI SRINIVAS +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The patent text describes a discovery of a new mechanism for inhibiting the function of Factor VIII, a protein necessary for blood clotting. The discovery involves the discovery of catalytic anti-Factor VIII allo-antibodies, which are antibodies that can degrade Factor VIII. These allo-antibodies are induced by treatment with Factor VIII and can be found in the plasma of patients with severe haemophilia A. The invention provides a method for identifying these allo-antibodies and characterizing the site(s) in the Factor VIII molecule that they target. The invention also provides new amino acid sequences that can inhibit the function of Factor VIII. Overall, the invention provides new approaches for treating diseases caused by anti-Factor VIII allo-antibodies."

Problems solved by technology

Haemophilia A is an X chromosome-linked recessive disorder resulting in defective or deficient Factor VIII molecules, which, in its severe form, is a life-threatening and crippling haemorrhagic disease.

Method used

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Examples

Experimental program
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Effect test

example i

Affinity-Purification of Anti-Factor VIII Antibodies

[0067]Antibodies were isolated from plasma by ammonium sulphate precipitation. Antibodies reactive with Factor VIII were then affinity-purified on a CNBr-activated Sepharose 4B matrix to which immuno-purified commercial human plasma-derived Factor VIII had been coupled (25000 U / 3 g of gel). The flow-throughs of the columns were collected. After extensive washing with PBS pH 7.4, anti-Factor VIII antibodies were eluted using 0.2 M glycine pH 2.8, dialysed against PBS and concentrated with Centriprep. Flow-throughs and eluates were aliquoted and stored at −20° C. until use. F(ab′)2 fragments of anti-Factor VIII antibodies were prepared as previously described.

[0068]The concentration of anti-Factor VIII IgG was 130, 20 and 280 μg per 10 mg of IgG applied to the column in the case of patients Bor, Che and Wal, respectively, (i.e., 143±130 μg / mI of unfractionated plasma), which is in agreement with previous observations.

example ii

Factor VIII-Neutralising Activity

[0069]The Factor VIII-neutralising activity of anti-Factor VIII antibodies was determined by the method of Kasper et al. and expressed as Bethesda units (BU) (ref). BU were defined as the inverse of the concentration of IgG which causes 50% inhibition of Factor VIII procoagulant activity. Residual Factor VIII activity was measured in a one-stage assay by determination of the activated partial thromboplastin time using human plasma depleted of Factor VIII (Behring) as substrate and human placental Pathromtin® (Behring) as activators. Heated plasma or immunopurified anti-Factor VIII IgG to be tested, were incubated with pooled citrated human plasma for 2 h at 37° C. The clotting time of four serial dilutions of a reference plasma pool (Immuno AG, Wien) was compared with the clotting time of three dilutions of each sample to be tested. Dilutions were carried out in Owren-Koller buffer (Diagnostica Stago). Inter-assay variation ranged between 1 and 2.5%....

example iii

Assay for Hydrolysis of Factor VIII

[0071]Commercial human recombinant Factor VIII was labelled with 125I to a specific activity of 11.6 nCi / μg, by using the iodogen method. [125I]-Factor VIII (1.5 to 150 ng) was incubated in 50 μl of 50 mM tris-HCI pH 7.7, 100 mM glycine, 0.025% Tween-20 and 0.02% NaN3 alone or with 17 to 1667 nM of immuno-purified anti-Factor VIII IgG for 5 min to 10 hours at 38° C. Human monoclonal anti-digoxin IgG M061 (mAb) and normal unfractionated human polyclonal IgG (IVIg, Sandoglobulin®), were used as negative controls. Samples were mixed 1:1 with Laemmli's buffer without mercaptoethanol, and were subjected to SDS electrophoresis without boiling, after loading 20 μl of each sample per lane. Samples were run in parallel on 7.5% and 15% SDS-PAGE under non-reducing conditions, after loading 20 μl of each sample per lane. Migration was performed at room temperature using a mini-PROTEAN II system at 25 mA / gel, until the dye front reached the bottom of the gel. T...

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Abstract

The present invention relates to a method of determining the presence of catalytic anti-Factor VIII allo-antibodies capable of degrading Factor VIII in a mammal, and of characterising the cleavage sites in said Factor VIII molecule by said catalytic anti-Factor VIII allo-antibodies. It also relates to an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor; and to a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination; and further to a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor. Finally, the present invention relates to the application in therapeutics of said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, of a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination, and of a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a method of determining the presence of catalytic anti-Factor VIII allo-antibodies capable of degrading Factor VIII in a mammal, and of characterising the cleavage sites in said Factor VIII molecule by said catalytic anti-Factor VIII allo-antibodies.[0002]The present invention also relates to an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.[0003]The present invention further relates to a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination, and to a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.[0004]Finally, the present invention relates to the application in therapeutics of said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, of a pharmaceutical composition comprisi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K7/06C07K16/36A61K31/135A61K38/08C07C211/43A61P7/02G01N33/483A61K31/185A61K38/00A61K38/43A61K45/00A61P7/00A61P7/04B01J20/281C07K14/755C07K16/18C12Q1/26G01N27/447G01N30/88G01N33/15G01N33/50G01N33/53G01N33/68G01N33/86
CPCA61K38/00G01N33/86G01N33/6854C07K14/755A61P43/00A61P7/00A61P7/02A61P7/04G01N33/68
Inventor KAVERI, SRINIVASLACROIX-DESMAZES, SEBASTIENKAZATCHKINE, MICHEL
Owner KAVERI SRINIVAS
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