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Factor VII or VIIa Polypeptide Variants

a polypeptide, factor viia technology, applied in the direction of peptides, enzymology, drug compositions, etc., can solve the problems of relative instability of the molecule with respect to proteolytic degradation, difficult to obtain adequate dose regulation, and difficult to achieve adequate dose regulation

Inactive Publication Date: 2006-11-30
BAYER HEALTHCARE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is related to a modified FVII or FVIIa protein that has specific amino acid changes and a sugar molecule attached to it. The invention also includes a nucleotide sequence that encodes this protein, an expression vector that can be used to produce the protein, and a host cell that can be used to produce the protein. The invention also includes a pharmaceutical composition containing the modified protein and a carrier or excipient, and the use of the modified protein or pharmaceutical composition as a medicament. The technical effects of the invention include improved stability, increased solubility, and improved pharmacokinetics of the modified protein.

Problems solved by technology

As a consequence adequate dose regulation is difficult to obtain and the need of frequent intravenous administrations imposes restrictions on the patient's way of living.
As indicated above, another problem in current rhFVIIa treatment is the relative instability of the molecule with respect to proteolytic degradation.
Proteolytic degradation is a major obstacle for obtaining a preparation in solution as opposed to a lyophilized product.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0334] The X-ray structure of hFVIIa in complex with soluble tissue factor by Banner et. al., J Mol Biol, 1996; 285:2089 is used for this example. It is noted that the numbering of residues in the reference does not follow the sequence. Here we have used the sequential numbering according to SEQ ID NO:2. The gamma-carboxy glutamic acids at positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 are all here named GLU (three letter abbreviation) or E (one letter abbreviation). Residues 143-152 are not present in the structure.

Surface Exposure

[0335] Performing fractional ASA calculations on FVII fragments alone combined with the definition of accessibilities of non standard and / or missing residues described in the methods resulted in the following residues having more than 25% of their side chain exposed to the surface: A1, N2, A3, F4, L5, E6, E7, L8, R9, P10, S12, L13, E14, E16, K18, E19, E20, Q21, S23, F24, E25, E26, R8, E29, F31, K32, D33, A34, E35, R36, K38, L39, W41, I42, S43, S45, ...

example 2

Design of an Expression Cassette for Expression of rhFVII in Mammalian Cells

[0340] The DNA sequence shown in SEQ ID NO:1, encompassing the short form of the full length cDNA encoding hFVII with its native short signal peptide (Hagen et al., 1986. PNAS 83:2412), was synthesized in order to facilitate high expression in mammalian cells. First the ATG start codon context was modified according to the Kozak consensus sequence (Kozak, M. J Mol Biol 1987 Aug. 20;196(4):947-50), so that there is a perfect match to the consensus sequence upstream of the ATG start codon. Secondly the open reading frame of the native cDNA was modified by making a bias in the codon usage towards the codons frequently used in highly expressed human genes. Further, two translational stop codons were inserted at the end of the open reading frame in order to facilitate efficient translational stop. The fully synthetic and expression optimized hFVII gene was assembled from 70-mer DNA oligonucleotides and finally ...

example 3

Expression of Polypeptide Variants in CHO K1 Cells

[0343] The cell line CHO K1 (ATCC # CCL-61) is seeded at 50% confluence in T-25 flasks using MEMO, 10% FCS (Gibco / BRL Cat # 10091), P / S and 5 μg / ml phylloquinone and allowed to grow until confluent. The confluent mono cell layer is transfected with 5 μg of the relevant plasmid described above using the Lipofectamine 2000 transfection agent (Life technologies) according to the manufacturer's instructions. Twenty four hours post transfection a sample is drawn and quantified using e.g. an ELISA recognizing the EGF1 domain of hFVII. At this time point relevant selection (e.g. Hygromycin B) may be applied to the cells with the purpose of generating a pool of stable transfectants. When using CHO K1 cells and the Hygromycin B resistance gene as selectable marker on the plasmid, this is usually achieved within one week.

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Abstract

The present invention relates to novel polypeptide variants of factor VII (FVII) or factor VIIa (FVIIa) polypeptides, where said variants comprise an amino acid substitution in position 10 and 32 and where said variants further comprise a sugar moiety covalently attached to an introduced in vivo N-glycosylation site located outside of the Gla domain. Such polypeptide variants are useful in therapy, in particular for the treatment of a variety of coagulation-related disorders, such as trauma.

Description

FIELD OF THE INVENTION [0001] The present invention relates to novel polypeptide variants of factor VII (FVII) or factor VIIa (FVIIa) polypeptides, where said variants comprise an amino acid substitution in position 10 and 32 and where said variants further comprise a sugar moiety covalently attached to an introduced in vivo N-glycosylation site. [0002] The present invention also relates to use of such polypeptide variants in therapy, in particular for the treatment of a variety of coagulation-related disorders. BACKGROUND OF THE INVENTION [0003] Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually results in a fibrin clot. Generally, the blood components participating in what has been referred to as the “coagulation cascade” are proenzymes or zymogens, i.e. enzymatically inactive proteins that are converted into an active form by the action of an activator. One of these coagulation factors is FVII. [0004] FVII i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/37C07K14/765C07H21/04C12P21/04A61K38/00C12N15/09A61K38/38A61K38/43A61P7/04A61P17/02C07K14/745C12N1/15C12N1/19C12N1/21C12N5/10C12N9/64C12N15/57
CPCA61K38/00C12Y304/21021C12N9/6437A61P17/02A61P7/00A61P7/04C07K14/745C12N9/64A61K38/36
Inventor HAANING, JESPERANDERSEN
Owner BAYER HEALTHCARE LLC
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