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Factor VIII muteins with reduced immunogenicity

A factor and recombinant factor technology, applied in the direction of factor VII, peptide/protein components, coagulation/fibrinolytic factor, etc., can solve the problems of reduced activity of recombinant protein and decreased productivity

Inactive Publication Date: 2011-07-27
BAYER HEALTHCARE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Production of recombinant proteins with altered glycosylation patterns presents several challenges, including a potential decrease in productive yield from recombinant culture and / or reduced activity of the recombinant protein

Method used

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  • Factor VIII muteins with reduced immunogenicity
  • Factor VIII muteins with reduced immunogenicity
  • Factor VIII muteins with reduced immunogenicity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Endocytosis of FVIII by dendritic cells

[0101] The effect of FVIII glycosylation on DC uptake in vitro was determined. Full-length rFVIII was first labeled for FACS analysis and then deglycosylated. To label rFVIII for FACS analysis, 6 μg of fluorescein isothiocyanate (FITC) in PBS (pH 9) was added to 100 μg of deglycosylated FVIII and allowed to mix for 2 hours at 4°C. By in 20mM HEPES, 150mM NaCl, 2% sucrose, and 100ppm Unconjugated FITC was removed by dialysis against -80 (polyethylene glycol sorbitan monooleate) solution (pH 7.5) at 4°C for 2 hours using a 50K membrane. FVIII concentration was quantified by Bradford assay and FVIII activity was determined by chromogenic assay. Tagged rFVIII is then enzymatically deglycosylated using endoglycosidase F1 (Endo-F1 ), which specifically cleaves N-linked oligosaccharides without denaturing the protein. rFVIII was incubated with Endo-F1 at 37°C for 1 hour. rFVIII was injected into 50K membranes and treat...

Embodiment 2

[0103] Example 2: Expression of FVIII mutein in HKB11 cells

[0104] BDD FVIII and three muteins of this BDD FVIII were expressed in HKB11 cells. BDD FVIII contains a deletion of almost 14 amino acids of the B domain such that the first 4 amino acids of the B domain are linked to the last 10 residues of the B domain. One BDD FVIII mutein contained a single glutamine-to-asparagine substitution at position 239 (N239Q), another contained a glutamine-to-asparagine substitution at position 2118 (N2118Q), and a third contained These two mutations (N239Q / N2118Q).

[0105] Using Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA) HKB11 cells were transiently transfected with BDD FVIII and BDD FVIII mutein expression plasmids according to the manufacturer's instructions. HKB11 cells were transiently transfected with BDD and BDD mutein plasmids, and supernatants from these cells were tested for FVIII activity by chromogenic assay and FVIII concentration by ELISA. The specific activiti...

Embodiment 3

[0106] Example 3: Decreased uptake of FVIII muteins by dendritic cells

[0107] Because dendritic cell (DC) uptake of FVIII is thought to be mediated by the interaction of CD206 with the mannose-terminated glycans on FVIII, DC uptake of the N239Q / N2118Q BDD mutein was tested as described in Example 1 Ability. DCs were prepared as described above. DCs from two donors were pooled and then co-cultured with full-length rFVIII, BDD FVIII (described in Example 2), or the N239Q / N2118Q BDD mutein. Cells were co-cultured for 30 minutes in each well of a 96-well plate. The final volume per well was 100 μL, and the final concentration of rFVIII, BDD, or mutein was 10 nM. Plates were then incubated at 37°C for 30 minutes. A parallel uptake assay was also performed at 4°C as a control. Cells were pelleted by centrifuging the plate at 300 g for 5 minutes at 4°C. Aspirate the medium and replace with ice-cold PBS / 10mM EDTA / 0.01% -80 Wash the cells three times. Then pass through 25 μL...

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Abstract

The present invention relates to modified Factor VIII molecules with reduced N-linked glycosylation and reduced immunogenicity. The invention also relates to methods of using modified Factor VIII molecules, for example, to treat patients afflicted with hemophilia.

Description

[0001] This application claims the benefit of US Provisional Application Serial No. 61 / 075,494, filed June 25, 2008, the contents of which are hereby incorporated by reference in their entirety. field of invention [0002] In general, the present invention relates to mutant Factor VIII molecules (Factor VIII muteins) having mutations in certain uncapped N-linked glycosylation sites. These muteins exhibit reduced uptake by antigen-presenting dendritic cells and reduced immunogenicity when used therapeutically. Background of the invention [0003] Human therapeutic proteins (biologics), isolated from natural sources or synthesized via recombinant methods, can induce an immune response when administered to a human patient. These immune responses can lead to effects ranging from minor skin irritation to reduced efficacy of therapeutic drugs, and in some cases can cause massive organ failure or death. [0004] Approximately 30% of patients treated with recombinant factor VIII (r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06
CPCA61K38/00C07K14/755A61P7/04A61K38/37C12N15/11
Inventor 弗雷德.J.阿斯沃德理查德.哈金斯蒋刘小莱约翰.E.墨菲吴非朱荧
Owner BAYER HEALTHCARE LLC
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