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Coagulation factor VII derivatives

A factor and molecular technology, applied in the field of blood coagulation factor VII derivatives, can solve the problems of increasing platelet aggregation, toxic side effects, increasing serum half-life, etc.

Inactive Publication Date: 2009-12-09
NOVO NORDISK HEALTH CARE AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, prolonged use of heparin may also increase platelet aggregation and decrease platelet count, and is associated with the development of osteoporosis
2,3-dihydro-1,3-indanedione derivatives also have toxic side effects
Modification methods that allow partial PEGylation of the protein typically result in only about a 50% loss of the activity and greatly increase the serum half-life, allowing for a lower overall effective dose of the protein

Method used

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  • Coagulation factor VII derivatives
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0318] Construct coding FVII-(R396C), FVII-(Q250C), FVII-(P406C), FVII-(407C), FVII-(V158T / M298Q), FVII-(L305V / M306D / D309S), FVII-(K337A), FVII-(L305V), and FVII-(F374P) DNA:

[0319] Genes encoding FVII-(R396C), FVII-(Q250C), FVII-(P406C), FVII-(407C) (an added C-terminal Cys), FVII-(M298Q), FVII-(L305V / M306D / D309S), FVII-(K337A), FVII-(L305V), and FVII-(F374P) DNA constructs. Use the following primers:

[0320] For FVII-(R396C):

[0321] 5'-GCG CTC AGA GCC ATG CCC AGG AGT CCT CC-3' (SEQ ID NO: 3)

[0322] 5'-GGA GGA CTC CTG GGC ATG GCT CTG AGC GC-3' (SEQ ID NO: 4)

[0323] For FVII-(Q250C):

[0324] 5'-GCT CCG CCT GCA CTG TCC CGT GGT CCT CAC TGA CC-3' (SEQ ID NO: 5)

[0325] 5'-GGT CAG TGA GGA CCA CGG GAC AGT GCA GGC GGA GC-3' (SEQ ID NO: 6)

[0326] For FVII-(P406C):

[0327] 5'-GCG AGC CCC ATT TTG CTA GAC TAG AGG ATC TGG G-3' (SEQ ID NO: 7)

[0328] 5'-CCC AGA TCC TCT AGT CTA GCA AAA TGG GGC TCG C-3' (SEQ ID NO: 8)

[0329] For FVII-(407C):

[0330] 5'-CCT GCG ...

Embodiment 2

[0349] The preparation of embodiment 2FVII-(R396C)

[0350] BHK cells were transfected for expression of the variant FVII-(R396C) essentially as described previously (Thim et al. (1988) Biochemintry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241-243). Factor VII polypeptides were purified as follows:

[0351] The conditioned medium was loaded onto a 25-ml QSepharose Fast Flow column (Pharmacia) to which 5 mM EDTA, 0.1% Triton X-100, and 10 mM Tris were added, the pH was adjusted to 8.0, and the conductivity was adjusted to 10-11 mS / cm by adding water. Biotech). From 10mM Tris, 50mM NaCl, 0.1% Triton X-100, pH 8.0 to 10mM Tris, 50mM NaCl, 25mM CaCl 2 , 0.1% Triton X-100, pH 7.5 gradient to complete the protein elution. Fractions containing FVII-(R396C) were pooled and loaded onto monoclonal antibody F1A2 (Novo Nordisk, Denmark) on a 25ml column. The column was used to contain 10mM CaCl 2 , 100 mM NaCl and 0.02% Triton X-100 in 50 mM Hepes, pH 7.5 equilibra...

Embodiment 3

[0352] The preparation of embodiment 3FVII-(M298Q)

[0353] BHK cells were transfected for expression of variant FVII-(V158T / M298Q) essentially as previously described (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241-243) . Factor VII polypeptides were purified as follows:

[0354] The conditioned medium was loaded onto a 25-ml QSepharose Fast Flow column (Pharmacia) to which 5 mM EDTA, 0.1% Triton X-100, and 10 mM Tris were added, the pH was adjusted to 8.0, and the conductivity was adjusted to 10-11 mS / cm by adding water. Biotech). From 10mM Tris, 50mM NaCl, 0.1% Triton X-100, pH 8.0 to 10mM Tris, 1M NaCl, 5mM CaCl 2 , 0.1% Triton X-100, pH 7.5 gradient to complete the protein elution. Combine the fractions containing FVII-(V158T / M298Q), add 10mM CaCl 2 , and loaded onto a monoclonal antibody F1A2 (NovoNordisk, Denmark) on a 25ml column. The column was used to contain 10mM CaCl 2 , 100 mM NaCl and 0.02% Triton X-100 in 5...

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Abstract

The present invention relates to novel human blood coagulation factor VII polypeptide, factor VII derivative and polynucleotide construct encoding the polypeptide, vector and host cell containing and expressing the polynucleotide, pharmaceutical composition, use and treatment method.

Description

technical field [0001] The present invention relates to novel human blood coagulation factor VII derivatives, factor VII polypeptides, polynucleotide constructs encoding the polypeptides, vectors and host cells containing and expressing the polynucleotides, and pharmaceutical compositions containing factor VII derivatives , uses and treatments. Background technique [0002] Blood coagulation is a process consisting of complex interactions of various blood components (or factors) that ultimately produce a fibrin clot. Generally, the blood components involved in the so-called coagulation "cascade" are enzymatically inactive proteins (proenzymes or zymogens), which are converted into proteolytic enzymes by the action of activators (which are themselves activated coagulation factors). Coagulation factors that undergo this transition are generally referred to as "active factors" and are designated by adding the letter "a" to the coagulation factor name (eg, Factor VIIa). [000...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A01H5/00A01H4/00A01K67/00A01K67/027A61K38/22A61K38/36A61K38/43A61K38/48A61P7/02A61P7/04C07K14/745C07K14/81C07K19/00C12N5/00C12N5/10C12N15/09C12N15/15C12N15/63C12P21/02
CPCA61K38/4846A61P7/00A61P7/02A61P7/04C12N9/64
Inventor 埃贡·佩森
Owner NOVO NORDISK HEALTH CARE AG
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