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Demannosylated recombinant factor viii for the treatment of patients with hemophiila a

一种去糖基化、氨基酸的技术，应用在凝血/纤溶因子、血液疾病、基因工程等方向，能够解决增加发病率和死亡率、影响患者生活质量、治疗成本增加等问题

Inactive Publication Date: 2011-02-09
LFB BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The emergence of FVIII inhibitors not only leads to a 3-fold increase in treatment costs (5), but also seriously affects the quality of life of patients, increasing morbidity and mortality

Method used

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  • Demannosylated recombinant factor viii for the treatment of patients with hemophiila a
  • Demannosylated recombinant factor viii for the treatment of patients with hemophiila a
  • Demannosylated recombinant factor viii for the treatment of patients with hemophiila a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0408] Example 1: Human DCs derived from monocytes

[0409] Peripheral blood was isolated from heparinized buffy coats of healthy adult donors by adhering to plastic cell culture dishes in RPMI1640 medium supplemented with 10% human AB serum, glutamine, and antibiotics for 60 minutes monocytes. Wash gently 3 times with culture medium to remove non-adherent cells. Adherent monocytes were treated in the presence of 500 IU / ml recombinant human interleukin 4 (rhIL-4) (R&D Systems (Lille, France)) supplemented with 1% human AB serum, antibiotics and 1000 IU / ml recombinant human granulocyte- Macrophage colony-stimulating factor (rhGM-CSF) (Immuno Tools (Friesoythe, Germany)) culture medium. Half of the medium, including all supplements, was changed every 2 days. After 5 days of culture, non-adherent cells and loosely attached cells (corresponding to the DC-enriched fraction) were harvested, washed and used for subsequent experiments.

Embodiment 2

[0410] Example 2: Binding of human recombinant full-length FVIII, human recombinant FVIII with B domain deletion to fluorescein

[0411] Human recombinant full-length FVIII (1000IU, Kogenate, Bayer), recombinant human FVIII (BDD-FVIII, 1000IU, Refacto Wyeth) dissolved in water, and containing 5mM CaCl 2 Sodium bicarbonate buffer (pH 9.2) was dialyzed at 4°C, and then coupled with fluorescein-5-isothiocyanate (isomer I, Sigma-Aldrich, Saint Quentin Fallavier, France) at 4°C 7-8 hours. The labeled FVIII was further dialyzed against RPMI1640 medium to remove unconjugated FITC. FVIII-FITC was quantified by Bradford assay using bovine serum albumin as standard.

Embodiment 3

[0412] Example 3: Properties of receptors involved in FVIII endocytosis

[0413] DCs were incubated with 5 mM EDTA, mannan (1 mg / ml) or galactose (1 mg / ml) at 37°C for 30 minutes, and then added with FVIII-FITC (40 μg / ml) for 2 hours. Endocytosis at 4°C served as a control (not shown).

[0414] result

[0415] figure 1 A : In the presence of EDTA, the endocytosis of FVIII-FITC was inhibited by 92±16.5% (P<0.01). This data suggests a role for divalent ion-dependent receptors in the process of FVIII endocytosis by DCs. The polysaccharide mannan (competitive ligand for mannose-sensitive uptake) reduced FVIII-FITC intake by 60±19% (P<0.01), while galactose (competitive ligand for galactose-sensitive uptake) body) had no significant effect.

[0416] figure 1 B : In our experiments, the specificity of mannan for mannose-sensitive CLRs was determined by using FITC-labeled dextran (a typical ligand for mannose-sensitive CLRs (especially CD206)) and fluorescent Yellow (LY),...

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Abstract

There is provided in accordance with the practice of this invention a demannosylated Factor VIII, the immunogenicity of which is substantially decreased or abolished in Human. The modified factor VIII is disclosed together with the modified amino acid sequence, changed by at least one substitution. The modified factor VIII is useful for hemophiliacs, either to avoid or prevent the action of inhibitory anti-FVIII antibodies.

Description

technical field [0001] The present invention relates to modified Factor VIII (FVIII) that is substantially non-immunogenic or has low immunogenicity. The present invention also relates to a nucleic acid construct comprising DNA encoding said modified FVIII, and to a method for expressing and producing said modified FVIII in a host cell or organism. The invention also relates to methods of administering the modified FVIII to a subject for the treatment of bleeding disorders. Background technique [0002] Human Factor VIII:C (FVIII) is a coagulation factor that is absent in the X-chromosome-linked bleeding disorder hemophilia A and is a major cause of hemorrhagic morbidity and mortality in affected males. Typically, people with hemophilia are treated with transfusions of whole blood. More recently, it has been treated with a preparation of FVIII concentrates derived from human plasma. However, the use of plasma-derived products exposes hemophiliacs to the risk of possible v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/37C07K14/755C12N1/21C12N5/10C12P21/00A01K67/027C12N1/15C12N15/12
CPCC07K14/755A61K38/00A61P43/00A61P7/00A61P7/02A61P7/04C12N15/11C12N15/63A61K38/37
Inventor A·S·什图鲁S·拉克罗伊斯-德马兹S·卡韦里S·达斯古普塔J·贝里
Owner LFB BIOTECH
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