Versions of gla domain of factor vii or viia

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to production of versions of the Gla domain of human factor VII or human factor VIIa, and can be used in medicine. Amino acid sequence of the FVII or FVIIa version is obtained, which differs on 1 to 15 amino acid residues with amino acid sequence of the human factor VII (hFVII) or human factor VIIa (hFVIIa), in which a negatively charged amino acid residue is introduced by substitution in position 36. Obtained variants of FVII or FVIIa are used in a composition for treating mammals with diseases or disorders, where blood clotting is desirable.

EFFECT: invention allows for producing versions of FVII or FVIIa with high clotting activity and/or high activation of factor X, compared to natural form of hFVIIa.

42 cl, 3 dwg, 5 tbl, 11 ex

 

The technical field to which the invention relates.

The present invention relates to new polypeptide variants of the Gla domain of factor VII (FVII) or factor VIIa (FVIIa), and the application of such polypeptide variants in therapy, in particular for the treatment of various diseases associated with bleeding disorders.

The level of technology

Blood clotting is a process involving a complex interaction of numerous components (or factors) of blood, which usually leads to the formation of a fibrin clot. Usually blood factors that are involved in the so-called “clotting cascade”, are the precursors of enzymes or Imogene, i.e. enzymatically inactive proteins that are converted to their active form under the action of a specific activator. One of these coagulation factors is factor FVII.

Factor FVII is a vitamin K-dependent blood plasma protein that is synthesized in the liver and secreted into the blood in the form of a single-chain glycoprotein with a molecular mass of 53 kDa (Broze & Majerus, J. Biol hem. 1980; 255:1242-1247). Zymogen FVII is converted into the active form (FVIIa) as a result of proteolytic cleavage at a single site, R152-1153, which leads to the formation of two polypeptide chains linked by a single disulfide bridge. FVIIa in complex with tissue factor (FVIIa complex) capable of converting the two other factors of blood coagulation factor IX (FIX) and factor X (FX)in their active forms, which triggers reactions leading to the rapid formation of thrombin and the formation of fibrin clot (Osterud & Rapaport, Proc Natl Acad Sci USA 1977; 74:5260-5264).

Factor FVII undergoes post-translational modifications, including vitamin K-dependent carboxylation, leading to the formation of ten residues of γ-carboxyglutamic acids at the N-terminal site of the molecule. Thus, residues numbered 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO:1, are remnants of the γ-carboxyglutamic acid in the Gla domain, which is important for the activity of FVII. Other post-translational modifications include attaching a sugar component to the two existing natural sites of N-glycosylation at positions 145 and 322, respectively, to the two natural areas 0-glycosylation sites at positions 52 and 60, respectively.

It is established that the gene encoding the factor FVII person (hFVn), located in chromosome 13 in the locus q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). It contains nine exons and covers an area the size of 12.8 TPN (O Nagy et al., Proc Natl Acad Sci USA 1987; 84:5158-5162). The gene organization and structure of protein molecules FVII is similar to other vitamin K-dependent procoagulant proteins, in which the exons 1A and 1b encode a signal sequence; exon 2 encodes propeptide and Gla domain; exon 3 encodes a short hydrophobic Uch the drain; exons 4 and 5 encode the domain, similar to epidermal growth factor; and exons 6 to 8 encode the catalytic domain of serine proteases (Yoshitake et al., Biochemistry 1985; 24:3736-3750).

In the literature there are reports of a three-dimensional structure of hFVIIa (Pike et al., Natl Acad Sci USA 1999; 96:8925-8930 and Kemball-Cook et al., J. Syruct. Biol., 1999; 127:213-223), and the structure of the complex hFVIIa with soluble tissue factor, obtained by x-ray crystallography (Banner et al., Nature 1996; 380:41 and Zhang et al., J. Mol. Biol. 1999; 285:2089), as well as on the structure of small fragments hFVII (Mihau et al., Biochemistry, 1998; 37:10605 and Kao et al., Biochemistry, 1999; 38:7097).

There are also several reports of FVII variants, obtained by the methods of protein engineering (Dickmson & Ruf, J Biol Chem, 1997; 272:19875-19879; Kemball-Cook et al., J Biol Chem 1998; 273:8516-8521; Bharadwa; et al., J Biol Chem, 1996; 271:30685-30691; Rufet al., Biochemistruy, 1999; 38:1957-1966).

There are reports of FVII expression in cells KSS or in other mammalian cells (WO 92/15686, WO 91/11514 and WO 88/10295) and co-expression of FVII and endoprotease kex2 in eukaryotic cells (WO 00/28065).

Commercial preparations of recombinant human FVIIa (rhFVIIa) sold under the trademark NovoSeven®. NovoSeven®for the treatment of bleeding in patients with hemophilia type a and B. NovoSeven®is the only drug rhFVIIa for efficient and reliable treatment of bleeding, which is available on the market currently.

About inactive form FVI, in which the modified arginine 152 and/or isoleucine 153 (1153), was reported in WO 91/11514. These amino acids are located in the area of activation. In WO 96/12800 described the inactivation of FVIIa inhibitor of serine proteases. About inactivation of FVIIa by carbamylcholine OS-amino acid group 1153 reported in the work of Petersen et al., Eur J Biochem, 1999; 261:124-129. Inactivated form is able to compete with the factors of wild-type FVII or FVIIa for binding to tissue factor and is able to inhibit clotting activity. It is supposed to use inactivated form of FVIIa to treat patients suffering from diseases associated with increased blood clotting, for example, patients with sepsis, the risk of myocardial infarction or suffering from clots.

In connection with the treatment of uncontrolled bleeding, for example, in case of injuries, it is assumed that FVIIa is able to turn in the active FX FXa without binding of tissue factor, and, as expected, this reaction activation occurs primarily on the surface of activated platelets (Hedner et al. Blod Coagulfion & Fibrinolysis, 2000; 11:107-111). However hFVIIa or rhFVIIa in the absence of tissue factor have low activity against FX and, consequently, the prevention of uncontrolled bleeding, for example, in patients with trauma, requires repeated use of relatively high until the hFVIIa or rhFVIIa. Therefore, for more effective treatment of uncontrolled bleeding (to reduce blood loss) need to be improved (improved) FVIIa molecules, which possess high activity against FX in the absence of tissue factor. Such improved FVIIa molecule should exhibit reduced clotting time (faster action/increased clotting activity) compared to rhFVIIa when used in cases of uncontrolled bleeding.

Variants of the Gla domain of factor FVII/FVIIa were disclosed in WO 99/20767, US 6017882 and WO 00/66753, where some residues located in the Gla domain has been found to be essential for binding to phospholipid membranes and, consequently, to activate FX. In particular, it was found that the residues 10 and 32 are critically important and that the increased affinity for binding to phospholipid membranes and, consequently, increased activation of FX can be achieved by mutations P10Q [replacement of the Proline residue (P) at position 10 on the glutamine residue (Q)] and CE [replacement of the lysine residue (K) at position 32 the residue glutamic acid (E)]. In particular, it was found that the FX activation was enhanced compared to rhFVIIa for minor conditions for coagulation, such as conditions in the presence of low levels of tissue factor.

WO 01/58935 raskryvaetsya strategy for obtaining molecules FVII or FVIIa, having inter alia (everything else) increased the half-life due to the directional glucoseamine or attachment of polyethylene glycol.

WO 03/093465 discloses variants of FVII or FVIIa with certain modification in the Gla domain and having one or more sites of N-glycosylation, introduced outside the Gla domain.

WO 2004/029091 discloses variants of FVII or FVIIa with certain modifications in the binding site of tissue factor.

In the present invention identified new residues in the Gla domain, which provides a further increase of the binding affinity of phospholipid membranes and, therefore, a further increase in the activation of FX. Variants of FVII or FVIIa of the present invention may also exhibit reduced binding affinity of tissue factor.

The subject of the present invention to provide improved molecules FVII or FVIIa (variants of FVII or FVIIa), which is able to more effectively activate the conversion of FX to FXa than hFVIIa, rhFVIIa or [P10Q+K32E]rhFVIIa. In particular, the subject of the present invention to provide improved molecules FVII or FVIIa (variants of FVII or FVIIa), which is able to more effectively activate the conversion of FX to FXa than hFVIIa, rhFVIIa or [P10Q+K32E]rhFVIIa in the absence of tissue factor. These goals were set when creating variants of FVII or FVIIa, described emyh in this invention.

Disclosure of inventions

In its first aspect, the present invention relates to a polypeptide variant factor VII (FVII) or factor Vila (FVIIa), having the amino acid sequence comprising 1-15 modifications of amino acids in relation to the factor VII human (bFVII) or factor Vila person (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, where to position 34 introduced by replacing the hydrophobic amino acid residue.

In its second aspect, the present invention relates to a polypeptide variant factor VII (FVII) or factor Vila (FVIIa), having the amino acid sequence comprising 1-15 modifications of amino acids in relation to the factor VII human (hFVII) or factor VIIa person (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, where the amino acid sequence comprises a substitution of the amino acid in position 36.

In its third aspect, the present invention relates to a polypeptide variant factor VII (FVII) or factor VIIa (FVIIa), having the amino acid sequence comprising 3-15 modifications of amino acids in relation to the factor VII human (hFVII) or factor VIIa person (bFVIIa) with the amino acid sequence shown in SEQ ID NO:1, where the amino acid sequence includes the replacement of amino acids at positions 10 and 32 and at least one replacement Amin the acid in position, which is chosen from the group consisting of positions 74, 77, and 116.

The following aspects of the present invention relate to a nucleotide sequence that encodes the polypeptide variants of the present invention, the expression vector comprising the sequence, and host cells comprising the nucleotide sequence or expression vector.

The following aspects of the present invention relate to pharmaceutical compositions comprising the polypeptide variants of the present invention, the use of the polypeptide variants of the present invention or pharmaceutical compositions of the present invention as medicaments and methods of treatment using the polypeptide variants or pharmaceutical compositions of the present invention.

The following aspects of the present invention will become clear from the following description and from the claims.

Brief description of figures

The Figure 1 shows the dependence of the clotting time of the blood concentration for the variants of the present invention under the conditions described in “Analysis of whole blood”.

The Figure 2 shows the maximum speed factor-dependent formation of thrombin for variants of the present invention under the conditions described in “Defining thrombogram”.

The piano is the Gur 3 shows the maximum speed of the phospholipid-dependent formation of thrombin for variants of the present invention under conditions described in “Defining thrombogram”.

The implementation of the invention

Definition

In the context of the present description and claims, the following definitions apply.

The term “FVII or a FVII polypeptide” refers to a molecule FVII (facfor VII)in the form of a single polypeptide chain. An example of a FVII polypeptide is wild-type FVII person. (hFVII), having the amino acid sequence shown in SEQ ID NO:1. It must, however, be understood that the term “FVII polypeptide” also includes hFVII-like molecules, such as fragments or variants in SEQ ID NO:1, in particular, ways in which sequence includes at least one or up to 15, preferably up to 10 modifications of amino acids compared to SEQ ID NO:1.

The term “FVIIa or FVIIa polypeptide” refers to a molecule in its activated FVII (FVII activate) double-stranded state. In those cases, when for a description of the amino acid sequence of FVIIa is used amino acid sequence represented in SEQ ID NO:1, you need to understand that the peptide bond between R152 and I153 single-stranded form was split, and one formed chain includes amino acid residues 1-152, and the other circuit includes balances 153-406.

The terms “rFVII and rFVIIa” refers to polypeptides FVII and FVIIa, obtained with the technique of obtaining recombina is the shaft of proteins (recombinant FVII/FVIIa).

The terms “hFVII” and “hFVIIa” refers to FVII and FVIIa wild-type human (Amdm FVII/FVIIa), respectively, with the amino acid sequence shown in SEQ ID NO:1.

The terms “rhFVII” and “rhFVIIa” refers to FVII and FVIIa wild-type person, having the amino acid sequence shown in SEQ ID NO:1, obtained using recombinant techniques. An example of rhFVIIa is NovoSeven®.

The term “Gla domain” is used to denote amino acid residues occupying the area from 1 to 45 of SEQ ID NO:1.

Accordingly, the term “position outside the Gla domain”encompasses amino acid residues in positions 46-406 in SEQ ID NO:1.

The abbreviation “FX”, “TF” and “TFP%” denote the X factor, tissue factor (tissue factor) and the inhibitor involving tissue factor (tissue factor parhway inhibitor), respectively.

The term “proteiny domain” refers to amino acid residues in the field 153-406, counting from the N-Terminus.

The term “catalytic center (catalytic site, catalytic site) is used to denote providing catalytic activity of the triad of amino acids consisting of S344, D242 and HI 93 polypeptide variant.

The term source(th)” (arent) is used to refer to molecules that will be modified/improved according to the present invention. Although the original polypeptide, which will be subjected to the modification according to the present invention, can be any polypeptide FVII or FVIIa and, therefore, may be of any origin, for example, can be a polypeptide not humans, and other mammals, it is preferable that the original polypeptide was hFVII or hFVIIa.

“Variant” is a polypeptide that differs in one or more amino acid residue from the original polypeptide, usually in 1-15 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues), in the case of differences of 1-10 amino acid residues, for example, 1-8, 1-6, 1-5 or 1-3 amino acid residues. Typically, the original polypeptide is hFVII or hFVII.

The term “conjugate” (or interchangeably “conjugated polypeptide”) is used to indicate a heterogeneous (in the sense of composite or chimeric) molecule formed when a covalent joining of one or more polypeptides to one or more non-polypeptide components, such as molecules, polymers, lipophilic compounds, sugar or agents of organic origin. Preferably, the conjugate was soluble at the concentrations used and the conditions, that is, was soluble in physiological fluids, such as blood. Examples of conjugated polypeptides of the present invention include glycosylated and/or polyethylene glycol (PEG) on peptide.

The term “covalent joining” or “covalently attached” means that the polypeptide variant and a non-polypeptide component or covalently bound directly to each other, either covalently linked to each other directly, but rather, at least one intermediate component, such as a bridge, insert, or a bifunctional linking agent.

The term “non-polypeptide component” is used to refer to molecules, non-peptide polymer, constructed from amino acid monomers linked together by peptide bonds, the molecule which may be associated with a group of polypeptide variants of the present invention, capable of such connection. Preferred examples of such molecules include polymer molecules, sugar, lipophilic components or agents of organic origin. When using the term “conjugated options” in the context of the present invention should be understood that the non-polypeptide component associated with the polypeptide part of a conjugated options through able to join the group of the polypeptide. As noted above, the non-polypeptide component can be covalently associated with a group to join, either directly or through an intermediate component.

“Polymer molecule” is what reculas, formed through covalent connection of two or more monomers, of which no single monomer is not an amino acid residue, except when the polymer is a serum albumin of a person or other protein of blood plasma, present in significant quantity. The term “polymer” may be used interchangeably with the term “polymer molecule”. This term is also used to denote carbohydrate molecules attached in vitro in the process of glycosylation, that is, when the artificial glycosylation, usually conducted in vitro and consists in the covalent joining of molecules of carbohydrate to able to join the group of polypeptide variants, sometimes using bifunctional cross-linking agent.

The term “sugar component” is used to refer to a carbohydrate-containing molecule comprising one or more monosaccharide residues that can be attached to the polypeptide variant (to obtain a conjugate polypeptide variant in the form of a glycosylated polypeptide variant) by glycosylation in vivo. The term “glycosylation in vivo” is used to refer to accession by any sugar component occurring in vivo, i.e. in the process of post-translational processing in providing Glick is solirovanie cells, used for expression of the polypeptide variant, for example, by N-glycosylation or O-glycosylation. The exact structure of the attached oligosaccharide largely depends on the organism used for expression and subsequent glycosylation.

“The site of N-glycosylation has the sequence N-X-S/T/C, where X represents any amino acid residue except Proline, N is asparagine, a S/T/C are serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine. Preferably, the amino acid residue in position +3 in relation to the asparagine was not a Proline.

“The site of O-glycosylation” is HE-a group of serine or threonine residue.

The term “group to join” is used to denote the functional groups of the polypeptide variant, in particular the group of amino acid residue or a carbohydrate component of this option, capable of joining a non-polypeptide component, such as a polymer molecule, a lipophilic molecule, a sugar component, or an organic agent to obtain derivatives. Suitable group to join, and the relevant non-polypeptide components are presented in the table below.

Group long accessionAmino acidExamples polipeptides componentThe method of conjugation/ Activated PEGLink
-NH2N - terminal, LysPolymer, such as PEG, amide or imine groupMPEG-SPA Trailerfunny MPEGNektar Therapeutics Delgado et al, Critical reviews in Therapeutic Drug Carrier Systems 9(3, 4):249-304 (1992)
-COOHC - terminal Asp, GluPolymer, such as PEG, ether or amide group. Carbohydrate componentMPEG-Hz Linking in vivoNektar Therapeutics
-SHCysPolymer, such as PEG, disulfide, maleimides or vinylsulfonate group
Carbohydrate component
PEG-vinylsulfonic PEG-maleimide Binding in vivoNektar Therapeutics Delgado et al, Critical reviews in Therapeutic Drug Carrier Systems 9(3, 4):249-304 (1992)
-HESer, Thr, Lys, OH-The sugar component. PEG with a complex ether, EF is rum, the carbamate, carbonateO-glikozidirovanie in vivo
-CONH2Asn as part of the site of N-glycosylationThe sugar component
The polymer, for example, PEG
N-glycosylation in vivo*
Aromatic residuePhe, Tight, TipCarbohydrate componentBinding in vitro
-CONH2GinCarbohydrate componentBinding in vitroYan and Wold, Biochemistry, 1984, Jul 31; 23(16):3759-3765
Aldehyde KetoneOxidized oligosaccharideThe polymer, for example, PEG-Hydrazide PEGAttaching PEGAndresz et al., 1978, Macromol. hem. 179:301, WO 92/16555, WO 00/23114
GuanidineArgCarbohydrate componentBinding in vitroLundblad and Noyes, Chemical Reagents for
Protein Modification, CRC Press Inc., Florida, USA
Imidazole ringHisCarbohydrate componentBinding in vitroAs for guanidine

For N-glycosylation in vivo, the term “group to join” is used in non-traditional value to indicate the amino acid residues forming part of the site of N-glycosylation (with the sequence N-X-S/T/C, as mentioned above). Although the asparagine residue at the site of N-glycosylation is a residue, to which in the process of glycosylation is attached sugar component, this connection may not occur if there are no other amino acid residues that are part of the site of N-glycosylation.

Thus, when the non-polypeptide component is a sugar component and its conjugation occurs when glycosylation in vivo, the term “amino acid residues that make up a group for attaching non-polypeptide component”used in connection with changes in amino acid sequence, you must understand that one or more amino acid residues constituting the site of N-glycosylation in vivo, have been modified so to enter in the amino acid sequence of the functional site of N-glycosylation in vivo.

In this bid, medium, small the names of the amino acids and denote atoms (for example, CA, CB, CD, CG, SG, NZ, N, O, etc. are used in accordance with the Protein DataBank (PDB) (www.pdb.org based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Ammo Acids and Peptides (residue names, atom names, etc.), Aug. J. Biochem., 138:9-37 (1984), taking into account changes in Aug. J. Biochem., 152, 1 (1985).

The term “amino acid residue” is used to refer to any natural or synthetic amino acid residue, primarily to refer to amino acid residues belonging to the group of the 20 amino acids commonly found in natural proteins, i.e. residue selected from the group consisting of alanine residues (A1A or a), pestaina (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Il or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), Proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (hr or T), valine (Val or V), tryptophan (Thr or W) and tyrosine (Tight or Y).

The terminology used to indicate the position of amino acids, is as follows: G124 means that at position 124 the amino acid sequence shown in SEQ ID NO:1, is a residue of glycine. G124R means that the glycine residue at position 124 has been replaced by an arginine residue. Alternative replacement are identified by testing the ka “/”, for example, N145S/T means the amino acid sequence in which the asparagine residue in position 145 shifted to either serine or threonine. Multiple substitutions are indicated with “+”, for example, K143N+N145S/T means amino acid sequence which includes substitution of the lysine residue at position 143 in the asparagine residue and substitution of the asparagine residue in position 145 on the balance of serine or threonine. Inserting additional amino acid residue, for example, inserting a residue of alanine after G124 referred to as G124GA. Insert two additional alanine residues denoted as G124GAA and so on. Using expressions “insert in position X” or “inserted at the position of X” means that the amino acid residue (residues) inserted (inserted) between amino acid residues X and X+1. Dividing the amino acid residue is indicated by an asterisk. For example, dividing the glycine residue at position 124 is denoted as G124*.

Unless otherwise indicated, the numbering of amino acid residues in the present invention corresponds to the numbering of the amino acid sequence of the polypeptide hFVII/hFVIIa (SEQ ID NO:1).

The term “different from...”, used in connection with specific mutations, is intended to refer to additional differences in addition to the specific differences in amino acid sequence. the example in addition to the modifications made in the Gla domain to increase the activation of FX, the polypeptide may have other modifications that do not necessarily relate to that effect.

Thus, in addition to modifications of amino acids disclosed in the present invention, it is necessary to understand that the amino acid sequence of the polypeptide variants of the present invention, if necessary, may contain other changes, that is, other substitutions, insertions or Delphi. For example, such changes may include shortening the N - and/or C-end of one or more amino acid residue (e.g., 1-10 amino acid residues) or join one or more incremental amino acid residues to the N - and/or-the end, for example, adding a methionine residue to the N-end or introduction of the rest of pestaina near-end and “conservative amino acid substitutions, i.e. substitutions conducted within groups of amino acids with similar characteristics, for example, within groups of amino acids with a little radical, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids.

Examples of such conservative substitutions are shown in the table below.

1Alanine(A) Glycine (G)Series (S)Threonine (T)
2Aspartic acid (D)Glutamic acid (E)
3Asparagine (N)Glutamine (Q)
4Arginine (R)Histidine (H)Lysine (K)
5Isoleucine (I)Leucine (L)Methionine (M)Valine (V)
6Phenylalanine (F)Tyrosine (Y)Tryptophan (W)

Other examples of additional modifications disclosed in the sections “Modifications outside the Gla domain and Other modifications outside the Gla domain”.

The term “nucleotide sequence” is used to denote a continuous plot, consisting of two or more molecules of nucleotides. Nucleotide posledovatel is the social origin may be of genomic, cDNA, RNA, semisynthetic, synthetic, or any combination thereof.

The term “vector” means a plasmid or other nucleotide sequence capable of replication within the host cell or can be integrated into the genome of the host cell and are thus suitable for various functions in the appropriate cell host system (vector-host) to facilitate cloning, nucleotide sequence, i.e. to obtain the necessary quantities of sequences that allow expression of the gene product encoded by this sequence, and for integration of the nucleotide sequence into the genome of the host cell. The vector may contain different components depending on the functions that must be provided.

“Cell”, “a host cell”, “cell line” and “cell culture” are used in the present invention can be used interchangeably, and, as should be understood that these terms include the offspring, resulting from the growing or cultivation of cells.

The terms “transformation” and “transfection” are used interchangeably to refer to the process of introducing DNA into a cell.

The term “functionally linked” refers to covalent connection between two or more nucleotide sequences using an enzymatic Legerova the Oia or other way to obtain this configuration sequences relative to each other, which ensures the normal functioning of all sequences. Usually “functionally linked” means that the nucleotide sequence of connected continuously and, in the case of providing the secretion leader are adjacent within the same reading frame. Linking is provided by ligating the appropriate restriction sites. If such sites are not available, use synthetic oligonucleotide adaptors or linkers using standard methods of work with recombinant DNA.

In the context of the present invention the term “modification” or “modified amino acid” is used to denote the replacement of the side chain of amino acids, substitution of amino acid residue, division amino acid residue or insertion of amino acid residue.

The term “type” refers to the introduction of amino acid residue, in particular, by replacing an existing amino acid residue, or alternatively, by inserting additional amino acid residue.

The term “remove” refers to a deletion of amino acid residue, in particular by replacing the amino acid residue to be removed, another amino acid residue, or alternatively, by dividing (without replacement) amino acid residue that is necessary is IMO to delete.

In the context of the present invention, the term “activity” should be understood as corresponding to the activity measured in the conditions in which this activity is actually measured.

Accordingly, the term “amidopirina activity” (aminodolytic activity) is used to indicate activity, measured here in this Dimension amylolyticus activity”. For the manifestation of “amylolyticus activity variant of the present invention in its activated form must have at least 10% of amylolyticus activity of rhFVIIa when measured under the conditions described in this section “Measuring amylolyticus activity”. In preferred embodiments of the present invention the variant, in its activated form, has at least 20% of amylolyticus activity of rhFVIIa, for example, at least 30% or at least 40%, more preferably at least 50%, for example 60%, or at least 70%, even more preferably at least 80%, such as at least 90% of amylolyticus activity of rhFVIIa when measured under the conditions described in this section “Measuring amylolyticus activity”. In the most interesting incarnations variant, in its activated form, has almost the same rhFVIIa amylolyticus activity, for example, amidolytic is a mini activity at the level of 75-125% of amylolyticus activity of rhFVIIa.

The term “coagulation activity” means the activity described here measured in the Analysis of whole blood”, that is, means the time required for the formation of a clot. Thus, less clotting time corresponds to a higher clotting activity.

The term “increased clotting activity” is used in the case when the clotting time for the polypeptide variant is statistically significantly lower compared with that obtained using rhFVIIa or [P10Q+K32E]rhFVIIa when its dimension in comparable conditions as described here, “the Analysis of whole blood”

In the context of the present invention, the term “activity” is also used in relation to the ability of the options to activate the conversion of FX to FXa. This activity is also referred to as “the capability to activate FX or FXa-forming activity and can be measured, as described below in the Analysis of the TF-independent activation of factor X”.

The term “increased capability to activate FX” or “increased FXa-forming activity” is used to indicate that the variant of the present invention, in its activated form, has a statistically significantly increased ability to convert FX to FXa in comparison with the molecules of the comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa. To the extent to which vari is NT present invention (in its activated form) can increase the rate of transition FX in the active state, it's easy to measure, as described below in the Analysis of the TF-independent activation of factor X”.

The term “immunogenicity” is used in relation to the substance used to denote the ability of a substance to induce an immune system response. The immune response may be cell or response mediated by antibodies (see, for example, Roitt: Essential Immunology (10thEdition, Blackwell) for a more detailed definition immunogenicity). Immunogenicity can be determined using any suitable method known to the experts in this field, for example, in vivo or in vitro.

The term “functional half-life in vivo” is used in its standard sense, i.e. denotes the time during which 50% of the biological activity of the polypeptide is still present in the body/target organ, or the time during which the activity of the polypeptide is 50% of its original value.

Alternatively, the time dimension of the functional half-life in vivo can be measured “time half-life in serum, i.e. the time during which 50% of the polypeptide circulates in the plasma or bloodstream. The definition of time half-life in serum is often more simple than the timing of the functional half-life in vivo, and the time value of half-life in serum is usually good the m indicator time functional half-life in vivo. Alternative term time half-life in serum are the terms “half-time in the plasma, the half-life in circulation”, “clearance serum, plasma clearance” or “half-time”. Remove the polypeptide from the circulation is maintained as a result of the work of one or more of the reticuloendothelial systems (RES), kidney, spleen or liver, due to the removal of tissue factor-mediated SEC receptor or other receptors, or due to specific or non-specific proteolysis. Usually the clearance depends on the size (relative to threshold the permeability of glomerular filtration glomerular filtration), the charge, the presence of attached carbohydrate chains, and the presence of cellular receptors for this protein. Functional quality that you want to keep, usually chosen from procoagulant activity, proteolytic or receptor-binding activity. The functional half-life in vivo and time half-life in serum can be determined using any suitable method known to specialists in this field.

The term “increased” in relation to time functional half-life in vivo or time half-life in serum is used to indicate that the half-time variant polypeptide is statistically significantly higher than that for which olekuly comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa, when determining in comparable conditions (usually determined on experimental animals such as rats, rabbits, pigs or monkeys).

The term “AUCiv” or “area under the curve when administered intravenously” (Area Under the Curve when administered intravenously) is used in its standard value, that is, to denote the area under the curve of the time dependence of activity in the serum, when the polypeptide variant is administered intravenously, in particular, when administered intravenously to rats. Usually measured activity is a “clotting activity” in accordance with the above definition. After measuring activity in the experiment at certain time points parameter AUCivcan easily be calculated using a computer program such as GraphPad Prism 3.01.

You must understand that for direct comparison of the values of AUCivdifferent molecules (for example, variants of the present invention and molecules comparisons, such as rhFVIIa or [P10Q+K32E]rhFVIIa), you must enter them in the same activity quantities. Therefore, the values of AUCivusually are normalized (that is, adjusted for differences in the input doses are expressed as the ratio of AUCiv/the dose applied.

The term “reduced sensitivity to proteolytic, degradati the first means, what the polypeptide variant has a decreased sensitivity to proteolytic degradation compared to hFVIIa, rhFVIIa or [P10Q+K32E]rhFVIIa when measured in the same conditions. Preferably, the proteolytic degradation was reduced by at least 10% (e.g., 10-25% or 10-50%), for example, at least 25% (e.g., 25-50%or 25-75%, or 25-100%), more preferably not less than 35%, for example, 50% (e.g., 50-75% or 50-100%) and even more preferably not less than 75% (e.g., 75-100%), or even at least 90%.

The term “renal clearance” (renal clerans) is used in its standard value to indicate any clearance occurring in the kidney, for example, when glomerular filtration, tubular secretion (tubular excretion or degradation in the renal tubular cells. Renal clearance depends on the physical characteristics of the polypeptide, including the size (diameter), hydrodynamic volume, symmetry, shape/hardness and charge. Usually molecular weight of about KD considered a critical value for the threshold for renal clearance. Renal clearance can be measured using any suitable method, for example, by the method used to measure in vivo. Usually renal clearance is determined by introducing a labeled polypeptides (e.g., radioactively or fluorescently labeled) to a patient and measuring range the of activity used for the label in the urine, collect from the patient. Reduced renal clearance is determined relative to the polypeptide of comparison, for example, rhFVIIa or [P10Q+K32E]rhFVIIa, when measured in the same conditions. Preferably, renal clearance polypeptide variant was reduced at least 50%, preferably at least 75% and more preferably at least 90% compared to rhFVIIa or [P10Q+K32E]rhFVIIa.

The terms “binding site of tissue factor, active area center” and “surface bonding slit active site” is defined by reference to Example 1.

The term “hydrophobic amino acid residues includes the following amino acid residues: isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tyrosine (Y) and tryptophan (W).

The term “negatively charged amino acid residues includes the following amino acid residues: aspartic acid (D) and glutamic acid (E).

The term “positively charged amino acid residues includes the following amino acid residues: lysine (K), arginine (R) and histidine (H).

Variants of the present invention

The modifications in the Gla domain of the original polypeptide, preferably provide the molecule with an increased affinity for binding to phospholipid membranes with improved ability to activate FX to FXa and/and and increased clotting activity. Variants of the present invention may also have a low affinity for binding to tissue factor and reduced activity after binding of tissue factor.

Not limited to any particular theory, it is currently envisioned that the increased affinity for binding to phospholipid membranes leads to a higher local concentration of activated variants of the polypeptide in the immediate vicinity of other coagulation factors, in particular, FX. Thus, the rate of conversion of FX to FXa will be higher simply due to the higher molar ratio of activated variant FVII and FX. Increased activation speed FX, in turn, will lead to the formation of a larger number of active thrombin, i.e. to increase the rate of polymerization of fibrin.

Consequently, it is assumed that the medical use of polypeptide variants according to the present invention can provide advantages over currently available connection rhFVIIa (NovoSeven®), as it can be used in a lower dose, to have increased efficiency and/or more rapid action.

In addition, it is assumed that the options are not dependent on tissue factor, then there are options that have low activity associated with tissue factor than the factor VIIa wild-type human may have certain advantages in security applications, such as reduced risk of unwanted blood clotting (for example, thrombosis or embolism), in particular, when applied for the treatment of severe uncontrolled bleeding, such as trauma.

Thus, in the preferred embodiment of the present invention the polypeptide variant in its active state when compared to a molecule of comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa, has an increased ability to activate FX, in particular, when measured in the absence of tissue factor, as described below in “Measuring the activation of factor X in the absence of tissue factor. More specifically, it is preferable that the ratio between the ability of the polypeptide variant in its active form to activate FX and the ability of the molecule compare to activate FX was at least 1.25 times in the measurement described here in “Measuring the activation of factor X in the absence of tissue factor. More preferably, this ratio was not less than 1.5, such as at least 1.75 or at least 2 and more preferably at least 3, for example at least 4, and most preferably at least 5.

When a molecule comparison is rbFVIIa, the ratio of the ability of the polypeptide variant in its active is able to activate FX and the ability rhFVIIa activate FX is preferably not less than 5, typically, at least 10, when measured under the conditions described below in “Measuring the activation of factor X in the absence of tissue factor”, for example, at least about 15 or 20.

In another most preferred embodiment of the present invention, variants of the invention have increased clotting activity (i.e. reduced clotting time) compared to rhFVIIa or [P10Q+K32E]rhFVIIa.

In the preferred embodiment of the present invention the ratio between the time to the formation of a clot in the presence of the variant (toptionand the time before the formation of a clot in the presence of rhFVIIa (twtor [P10Q+K32E]rhFVIIa (t10Q+K32E) is not more than 0.9 when measured under the conditions described in the following “Analysis of whole blood”. More preferably, this ratio was not more than 0.75, for example, of 0.7, or even more preferably, this ratio was not more than 0.6, and most preferred that this ratio was not more than 0.5.

One or more of the above-mentioned properties can be achieved using the disclosed modifications here.

Variants of the present invention containing a hydrophobic amino acid residue in position 34

As noted above, the present invention in its first aspect relates to polypeptide variants of FVII or FVIIa having the amino acid p is coherence, comprising 1-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO:1)where to position 34 by replacing entered hydrophobic amino acid residue.

Hydrophobic amino acid residue introduced at position 34 may be selected from the group including I, L, M, V, F, Y and W, preferably I, L and V, in particular L.

In a preferred embodiment variant comprises the amino acid substitution at position 10, in particular P10Q, and/or the amino acid substitution at position 32, in particular CE. In a preferred embodiment of the present invention includes both substitutions at positions 10 and 32, such as P10Q+K32E.

In accordance with this, in one of the most interesting embodiments of the present invention the variant comprises the substitutions P10Q+K32E+A34L.

In a particular interesting embodiment of the present invention also includes inserting at least one (usually one) of amino acid residue in position between 3 and 4 residues. Preferably, the inserted amino acid residue was hydrophobic amino acid residue. The preferred insert is A3AY. In accordance with this, in a particular interesting embodiment of the present invention includes modifications A3AY+P10Q+K32E+A34L.

In addition to any of the above modifications variant may further include replacing the provisions in the AI 33. Preferably, in position 33 by replacing was introduced hydrophobic amino acid residue, in particular D33F.

Domain Gla may also contain modifications at other positions, in particular at positions 8, 11, and 28, such as R28F or R28E. On the other hand, you must understand that the Gla domain should not be modified to such an extent that its ability to bind to membranes was broken. In accordance with this preferred not to have made modifications residues that are exposed to γ-carboxylation, that is, preferably, no modifications on the remaining 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35. Similarly, not usually is preferable that the non-polypeptide components, such as the remains of the sugar and/or groups of PEG were introduced in the Gla domain. Accordingly, it is preferred not to have made modifications, creating in the Gla domain of the site of N-glycosylation in vivo.

Finally, you need to understand what is discussed in this section modification in the Gla domain can advantageously be combined with one or more modifications at positions outside the Gla domain (see below sections entitled “Modification outside the Gla domain and Other modifications outside the Gla domain”).

Variants of the present invention comprising the amino acid substitution at position 36

It is to noted above, in its second aspect, the present invention relates to a polypeptide variant FVII or FVIIa having the amino acid sequence comprising 1-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO:1), where the named amino acid sequence comprises the amino acid substitution at position 36.

Preferably introduced by substitution in position 36 the amino acid residue was negatively charged amino acid residue, for example R36E or R36D, in particular R36E.

In a preferred embodiment variant comprises the amino acid substitution at position 10, in particular P10Q, and/or the amino acid substitution at position 32, in particular CE. In a preferred embodiment of the present invention includes both substitutions at positions 10 and 32, such as P10Q+K32E.

A variant of the present invention may also contain a substitution at position 38. Preferably introduced by substitution in position 38 the amino acid residue was negatively charged amino acid residue, for example CA or K38D, in particular CE.

In accordance with this interesting option is the option that includes the following substitutions: P10Q+K32E+R36E or P10Q+K32E+R36E+K38E.

In a particular interesting embodiment of the present invention also includes amino acid substitution in position 34 (that is, we obtain the variant comprises the substitution of the following residues: 10+32+34+36 or 10+32+34+36+38). Preferably introduced by substitution in position 34 the amino acid residue was negatively charged amino acid residue, for example AA or A34D.

Specific examples of preferred variants are such variants, which include the following replacement: P10Q+K32E+A34E+R36E or P10Q+K32E+A34D+R36E+K38E.

In an interesting embodiment of the present invention also includes inserting at least one (usually one) of amino acid residue in position between 3 and 4 residues. Preferably, the inserted amino acid residue was hydrophobic amino acid residue. The preferred insert is A3AY.

In addition to any of the above modifications variant may further include a substitution at position 33. Preferably, in position 33 by replacing was introduced hydrophobic amino acid residue, in particular D33F.

Domain Gla may also contain modifications at other positions, in particular at positions 8, 11, and 28, such as R28F or R28E. On the other hand, you must understand that the Gla domain should not be modified to such an extent that its ability to bind to membranes was broken. In accordance with this preferred not to have made modifications residues that are exposed to γ-carboxylation, that is, preferably, cobine were modifications at residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35. Similarly, not usually is preferable that the non-polypeptide components, such as the remains of the sugar and/or groups of PEG were introduced in the Gla domain. Accordingly, it is preferred not to have made modifications, creating in the Gla domain of the site of N-glycosylation in vivo.

Finally, you need to understand what is discussed in this section modification in the Gla domain can advantageously be combined with one or more modifications at positions outside the Gla domain (see below sections entitled “Modification outside the Gla domain and Other modifications outside the Gla domain”).

Variants of the present invention, comprising amino acid substitutions at positions 74, 77 or 116

As noted above, in its third aspect, the present invention relates to a polypeptide variant FVII or FVIIa having the amino acid sequence comprising 1-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO:1), where the named amino acid sequence comprises amino acid substitutions at position 10, 32 and at least one amino acid replacement in position, which is chosen from the group comprising position 74, 77, and 116.

In the preferred embodiment the amino acid substitution at position 10 is P10Q and amino acid substitution at position 32 the two who is KE.

It is also preferable to replacement provisions, 74, 77 or 116 were selected from a group comprising of P74S, E77A and E116D.

In an interesting embodiment of the present invention also includes amino acid substitution in position 34. Preferably introduced by substitution in position 34 the amino acid residue was negatively charged amino acid residue, for example AA or A34D, in particular AE.

In another interesting embodiment of the present invention also includes inserting at least one (usually one) of amino acid residue in position between 3 and 4 residues. Preferably, the inserted amino acid residue was hydrophobic amino acid residue. The preferred insert is A3AY.

Thus, specific examples of interesting variants include variants containing the following modifications: A3AY+P10Q+K32E+E116D, A3AY+P10Q+K.32E+E77A and P10Q+K32E+A34E-hP74S.

In addition to any of the above modifications variant may further include a substitution at position 33. Preferably, in position 33 by replacing was introduced hydrophobic amino acid residue, in particular D33F.

Domain Gla may also contain modifications at other positions, in particular at positions 8, 11, and 28, such as R28F or R28E. As mentioned above, the Gla domain should not be modified until the second degree, to its ability to bind to membranes was violated, that is, preferably, no modifications on the remaining 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35, and preferably not produced modifications, creating in the Gla domain of the site of N-glycosylation in vivo.

Finally, you need to understand what is discussed in this section modification in the Gla domain can advantageously be combined with one or more modifications at positions outside the Gla domain (see below sections entitled “Modification outside the Gla domain and Other modifications outside the Gla domain”).

Modifications outside the Gla domain

The time of half-life in circulation rhFVIIa is 2.3 hours, as reported in the Summary Basis for Approval for NovoSeven®" (FDA reference number 96-0597). To achieve and maintain the desired therapeutic or prophylactic effect it is often necessary to take relatively high doses of this compound. As a consequence, there are certain difficulties in the selection of adequate dose and the difficulties associated with frequent intravenous administration, which imposes certain restrictions on the way of life of the patients.

A molecule with a longer half-life and/or increased bioavailability (e.g., increased area under the curve” compared to rhFVIIa when intravenously) will require more recog the application. Taking into account the needs of the frequent introduction and the need to achieve optimal therapeutic levels of FVIIa order to obtain the desired effect completely obvious need for FVII or FVIIa-like molecules on improved properties.

In accordance with this further purpose of this invention to provide improved FVII molecules or FVII (variants of FVII or FVIIa) with improved bioavailability (such as “area under the curve” compared to a molecule of comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa, and intravenous), which are able to activate factor X or factor XA (without binding to tissue factor) is more effective than a molecule of comparison, for example, rhFVIIa or [P10Q+K32E]rhFVIIa (i.e. can be used for uncontrolled bleeding, such as trauma, or for chronic conditions, such as hemophilia, more efficiently).

Thus, an interesting variant of the present invention are such options, which, in its activated state, when compared with a molecule of comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa, provide increased area under the curve” when intravenously (AUCiv). It can be easily identified when administered intravenously in rats. More specifically, interesting variants of the present invention are the camping options such for which the ratio of AUCivnamed option in its activated form and AUCivmolecules of comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa, is at least 1.25, such as at least 1.5, which is about 1.75, more preferably not less than 2, for example, at least 3, even more preferably at least 4, e.g. at least 5, in particular when (intravenous) injection in rats.

This effect usually corresponds to increased time functional half-life in vivo and/or increased time half-life in serum compared with a molecule of comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa. In accordance with this, in another interesting embodiment of the present invention the ratio between the functional half-life in vivo, or by the time half-life in serum variant in its active form and time, the functional half-life in vivo, or by the time half-life in serum molecules comparisons, such as rhFVIIa or [P10Q+K32E]rhFVIIa, is at least 1.25 times. More preferably, the ratio between the corresponding half-life of the option in its activated form, and the corresponding half-life of the molecule comparison, such as rhFVIIa or [P10Q+K32E]rhFVIIa, was at least 1.5, such as at least 1.75 or at least 2, even more preferably at least 3, for example, on ENISA least 4 or at least 5.

One way to increase the half-life of the protein in circulation is reduced for him to renal clearance. This can be achieved by attaching to protein chemical component, for example, polyethylene glycol (PEG), which can provide reduced renal clearance of protein.

Moreover, the accession of the chemical component to a protein or replacement of amino acids available for proteolysis, can effectively block contact with a proteolytic enzyme, which otherwise leads to proteolytic degradation of this protein.

As noted above, is associated with proteolytic degradation of the instability is currently a known issue in the treatment with the use of rhFVIIa. Thus, proteolytic degradation is a major problem to obtain the drug in dissolved form in contrast to the lyophilized product. The advantage of obtaining a stable drug in dissolved form is an easy treatment for the patient and, in the case of urgent need, faster action that could potentially save lives. Attempts to prevent proteolytic degradation by using site-directed mutagenesis on the main sites of cleavage are disclosed in WO 88/10295.

WO 01/58935 discloses a number of suitable modifications, leading to HC is the increase in the AUC ivtime functional half-life in vivo and/or time of half-life in serum. Variants disclosed in WO 01/58935 are the result of a new strategy aimed at obtaining improved molecules FVII or FVIIa, which can also be used as a source of polypeptides FVII or FVIIa in the present invention.

More specifically, by the removal or introduction of amino acid residues, including groups which you can join, or a non-polypeptide components in the original polypeptide FVII or FVIIa is possible to specifically adapt the polypeptide in such a way as to make the molecule more accessible for conjugation with the selected non-polypeptide component to optimize the joining part (for example, to find the optimal location and number of non-polypeptide components on the surface of the polypeptide variant FVII or FVIIa to ensure that only necessary to join groups present in the molecule) and, thus, to obtain a new conjugate molecule, with amylolyticus activity and one or more improved property compared to rhFVIIa.

In an interesting embodiment of the present invention is modified by more than one amino acid residue located outside the Gla domain, for example, such changes include removal is the development or introduction of amino acid residue, including designed to attach to the selected non-polypeptide component group. In addition to the removal and/or introduction of amino acid residues of the polypeptide variant may include other substitutions that are not related to introduction and/or removal of such amino acid residues, including groups for attaching non-polypeptide component.

The polypeptide variant may also be connected with the inhibitor of serine proteases for the inhibition of the catalytic centre of the polypeptide variant. An alternative to this, one or more amino acid residues belonging to the catalytic center (S344, D242 and N), can be motivovany order to obtain in the inactive option. An example of such a mutation is S344A.

Amino acid residue including a group designed for attaching non-polypeptide component is selected based on the nature of the selected non-polypeptide component and, in most cases, based on the method by which you will be connecting polypeptide variant with a non-polypeptide component. For example, when the non-polypeptide component is a polymer molecule, such as polyethylene glycol or a derivative polyalkylated, amino acid residue, including intended for joining a group selected from the group consisting of lysine, cysteine, aspartic acid, glutamic acid, histidine and tyrosine, preferably from lysine, cysteine, aspartic acid and glutamic acid, more preferably from lysine and cysteine, in particular cysteine.

When a group designed for attaching non-polypeptide component must be entered or deleted from the original polypeptide, the position intended for the modification of amino acid residue should preferably be located on the surface of the original polypeptide FVII or FVIIa and more preferably should belong to amino acid residue side chain of which at least 25% exposed on the surface (as described herein in Example 1), preferably a side chain at least 50% must be exposed on the surface (as described herein in Example 1). Such provisions can be determined on the basis of the analysis of 3D structures of molecules hFVII or hFVIIa, as described in WO 01/58935.

Moreover, the position intended for modification, preferably selected from the part of the molecule FVII or FVIIa, which is located outside the binding site of tissue factor and/or outside of the site of the active center, and/or outside surface of the connecting slots of the active centre. These sites/areas identified here, in Example 1 in WO 01/58935.

If is the pressure used for joining the group corresponding to amino acid residue, containing such a group and occupying defined above suitable position, preferably replaced with another amino acid residue that does not have a suitable group to join the discussed non-polypeptide component. Usually intended for removal is an amino acid residue, adherence to which would be undesirable, for example, amino acid residue located at or near a functional site of the polypeptide (hence joining this site may result in inactivation or reduced activity of the conjugate as a result, for example, impaired recognition receptor). In this context, the term “functional site” is used to denote one or more amino acid residue that is essential or somehow otherwise involved in the operation or conduct of FVII or FVIIa. Such amino acid residues are part of the functional sections. Functional area can be determined using methods known to experts in this field, and is preferably determined on the basis of analysis of the structure of FVIIa complex with tissue factor (see Banner et al., Nature 1996; 380:41-46).

In the case of the introduction is designed to attach groups of amino acid residue containing such a group is entered in approaching the position, preferably by replacing the amino acid residue occupying this position.

The exact number of intended for joining groups and suitable position in the polypeptide FVII or FVIIa depend on the desired effect, which is planned to be achieved in the result of the merger. The effect that will be achieved, depends on the nature and extent of conjugation (e.g., the identity of non-polypeptide components, the desired number of non-polypeptide components or features of their accession to the polypeptide variant, where the pairing should occur or where it is necessary to avoid, etc).

The total number of amino acid residues that are modified outside the Gla domain of the original polypeptide FVII or FVIIa (compared to the amino acid sequence represented in SEQ ID NO:1), usually does not exceed 10. Preferably, the variant FVII or FVIIa included amino acid sequence, which differs in 1-10 amino acid residues from amino acid residues 46-406 shown in SEQ ID NO:1, typically 1-8 or 2-8 amino acid residues, such as 1-5 or 2-5 amino acid residues, in particular 1-4 or 1-3 amino acid residue, for example by 1, 2 or 3 amino acid residue from the amino acid residues 46-406 presented in SEQ ID NO:1.

Similarly, the polypeptide is the first variant of the present invention may contain 1-10 (additional) non-polypeptide components, usually 1-8 or 2-8 (additional) non-polypeptide components, preferably 1-5 or 2-5 (optional) non-polypeptide components, for example, 1-4, or 1-3 (additional) non-polypeptide component, in particular 1, 2 or 3 (additional) non-polypeptide component. You must understand that such additional non-polypeptide components covalently attached to designed for attachment groups located outside the Gla domain.

Polypeptide variants of the invention, where the non-polypeptide component is the residue of sugar

In the preferred embodiment of the present invention, the group intended to attach a sugar residue, such as glycosylation site, in particular the site for glycosylation in vivo, such as a site for N-glycosylation in vivo, may be introduced and/or removed, preferably introduced, in a position located outside the Gla domain.

When used in this context, the term “existing natural area pisarovina” includes the sites of glycosylation in the provisions of N145, N322, S52 and S60. The term “existing natural site of O-glycosylation in vivo” includes provisions S52 and S60, whereas the term “existing natural site of N-glycosylation in vivo” includes provisions N145 and N322.

Thus, in a very inter is a dream, the embodiment of the present invention the non-polypeptide component is the residue of sugar and entered the group, designed to attach is a glycosylation site, preferably a glycosylation site in vivo, such as the site of O-glycosylation in vivo or site of N-glycosylation in vivo, in particular the site of N-glycosylation in vivo. Usually can be entered 1-10 glycosylation sites, particularly sites of N-glycosylation in vivo, preferably 1-8, 1-6, 1-4, or 1-3 glycosylation site, in particular the site of N-glycosylation in vivo can be entered in one or more positions located outside the Gla domain.

For example, 1, 2 or 3 glycosylation site, in particular the site of N-glycosylation in vivo can be put outside the Gla domain preferably by replacement.

You must understand that to obtain the polypeptide variant, where the polypeptide variant comprises one or more glycosylation sites, the polypeptide variant must be expressed in the cell host, capable to attach sugar (oligosaccharide) residue in the plot (plots) glycosylation or instead polypeptide variant must be glycosylated in vitro. Examples of providing glycosylation host cells below in the section called “Attach to the sugar residue”.

Examples of provisions that can be introduced glycosylation sites, in particular areas of the N in vivo, include amino acid residues that have at least 25% of their side chain exposed to the surface (which is defined here in Example 1), as, for example, such that at least 50% of the side chain exposed to the surface. The position is preferably chosen in the part of the molecule, which is located outside the binding site of tissue factor, and/or portion of the active centre, and/or outside surface of the connecting slots of the active center, as defined here, in Example 1. It should be clear that when the term “at least 25% or at least 50%) side-chain amino acids exposed on the surface” is used in connection with the introduction site of N-glycosylation in vivo, this term refers to the availability of the surface of the side chain of the amino acid at the position where the actual attached sugar component. In many cases you must enter a serine residue or threonine at position +2 relative to the remainder of asparagine, which really attached sugar component, and these provisions, where the introduced residues serine or threonine can be “recessed” (hidden), that is to be exposed on the surface of less than 25% of their side chains.

Specific and preferred examples of such substitutions, creating a site of N-glycosylation in vivo, replacements, which SEL is select from the group consisting of A51N, G58N, T106N, K109N, 0124N, K143N+N145T, AT, I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T, R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N, D334N and combinations thereof. More preferably, the site of N-glycosylation in vivo injected through the replacement of which is selected from the group consisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, AT, I205T, V253N, T267N+S269T, S314N+K316T, R315N+V317T, K316N+G318T, G318N, D334N and combinations thereof. Even more preferably, the site of N-glycosylation in vivo injected through the replacement of which is selected from the group consisting of T106N, AT, I205T, V253N, T267N+S269T and their combinations, in particular with the introduction of one, two or three of T106N, I205T and V253N.

In one embodiment only a single site of N-glycosylation in vivo was introduced by replacement. In another embodiment, two or more (e.g. two) of the site of N-glycosylation in vivo were introduced by replacement. Examples of preferred substitutions, creating two sites of N-glycosylation in vivo, include substitutions selected from the group consisting of A51N+G58N, A51N+T106N, A51N+K109N, A51N+G124N, A51N+K143N+N145T, A51N+A175T, A51N+I205T, A51N+V253N, A51N+T267N+S269T, A51N+S314N+K316T, A51N+R315N+V317T, A51N+K316N+G318T, A51N+G318N, A51N+D334N, G58N+T106N, G58N+K109N, 058N+0124N, G58N+K143N+N145T, G58N+A175T, 058N+I205T, G58N+V253N, 058N+T267N+S269T, G58N+S314N+K316T, G58N+R315N+V317T, G58N+K316N+G318T, G58N-K318N, G58N+D334N, T106N+K109N, T106N+G124N, T106N+K143N+N145T, T106N+A175T, T106N+I205T, T106N+V253N, T106N+T267N+S269T, T106N+S314N+K316T, T106N+R315N+V317T, T106N+K316N+G318T, T106N+G318N, T106N+D334N, K109N+G124N, K109N+K143N+N145T, K109N+A175T, K109N+I205T, K109N+V253N, K109N+T267N+S269T, K109N+S314N+K316T, K109N+R315N+V317T, K109N+K316N+G318T K109N+G318N, K109N+D334N, G124N+K143N+N145T, G124N+A175T, G124N+I205T, G124N+V253N, G124N+T267N+S269T, G124N+S314N+K316T, G124N+R315N+V317T, G124N+K316N+G318T, G124N+G318N, G124N+D334N, K143N+N145T+A175T, K143N+N145T+I205T, K143N+N145T+V253N, K143N+N145T+T267N+S269T, K143N+N145T+S314N+K316T, K143N+N145T+R315N+V317T, K143N+N145T+K316N+G318T, K143N+N145T+G318N, K143N+N145T+D334N, A175T+I205T, A175T+V253N, A175T+T267N+S269T, A175T+S314N+K316T, A175T+R315N+V317T, A175T+K316N+G318T, A175T-H3318N, A175T+D334N, I205T+V253N, I205T+T267N+S269T, I205T+S314N+K316T, I205T+R315N+V317T, I205T+K316N+G318T, I205T+G318N, I205T+D334N, V253N+T267N+S269T, V253N+S314N+K316T, V253N+R315N+V317T, V253N+K316N+G318T, V253N+G318N, V253N+D334N, T267N+S269T+S314N+K316T, T267N+S269T+R315N+V317T, T267N+S269T+K316N+G318T, T267N+S269T+G318N, T267N+S269T+D334N, S314N+K316T+R315N+V317T, S314N+K316T+G318N, S314N+K316T+D334N, R315N+V317T+K316N+G318T, R315N+V317T+G318N, R315N+V317T+D334N and G318N+D334N. More preferably the substitutions are selected from the group consisting of T106N+A175T, T106N+I205T, T106N+V253N, T106N+T267N+S269T, A175T+I205T, A175T+V253N, A175T+T267N+S269T, I205T+V253N, I205T+T267N+S269T and V253N+T267N+S269T, even more preferably from the group consisting of T106N+I205T, T106N+V253N and I205T+V253N.

In the following embodiment, three or more (e.g. three) of the site of N-glycosylation in vivo were introduced by replacement. Examples of preferred substitutions, creating three sites of N-glycosylation in vivo, include substitutions selected from the group consisting of I205T+V253N+T267N+S269T and T106N+I205T+V253N.

As discussed above, is preferred, where the site of N-glycosylation in vivo injected in a position that does not form part of the binding site of tissue factor, site of the active center or surface bonding gap of the active center, as defined in question is the description.

You must understand that any modifications noted in previous sections, can be combined with each other, in addition to the existing can be combined with the above substitutions at position 34 and/or 36, in particular A34E/L and/or R36E, and preferably in combination with the above substitutions at position 10 and/or 32, in particular P10Q and/or CE. Among the installed above modifications necessary for the introduction of site N-glycosylation in vivo, the preferred modifications include one, two or three of T106N, I205T and V253N, in particular two of these modifications, that is, T106N+I205T, T106N+V253N or I205T+V253N.

Therefore, in one preferred embodiment of the invention, the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34E+R36E+T106N+I205T.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34E+R36E+T106N+V253N.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34E+R36E+I205T+V253N.

In the following preferred embodiment FVTI or FVIIa variant comprises the modifications P10Q+K32E+A34L+T106N+I205T.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+T106N+V253N.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+I205T+V253N.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10QK32E+A34L+R36E+T106N+I2Q5T.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+R36E+T106N+V253N.

In the following preferred embodiment of the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+R36E+I205T+V253N.

As also explained above, any one or more of these modifications may in addition be combined with insertion of at least one amino acid residue, typically one amino acid residue between positions 3 and 4, where the built-in residue is preferably a hydrophobic amino acid residue. Most preferably the insert is A3AY. Therefore, in additional preferred embodiments of the invention, the FVII or FVIIa variant comprises the modifications are chosen from:

A3AY+P10Q+K32E+A34E+R36E+T106N+I205T;

A3AY+P10Q+K32E+A34E+R36E+T106N+V253N;

A3AY+P10Q+K32E+A34E+R36E+I205T+V253N;

A3AY+P10Q+K32E+A34L+T106N+I205T;

A3AY+P10Q+K32E+A34L+T106N+V253N;

A3AY+P10Q+K32E+A34L+I205T+V253N;

A3AY+P10Q+K32E+A34L+R36E+T106N+I205T;

A3AY+P10Q+K32E+A34L+R36E+T106N+V253N;

A3AY+P10Q+K32E+A34L+R36E+I205T+V253N.

Other modifications outside the Gla domain

In the following embodiment of the present invention, the FVII or FVIIa variant can, in addition to the modifications described above in the previous sections also contain mutations that are already known to lead to increased activity characteristic of the polypeptide, for example, those described in WO 02/22776.

For example, a variant can contain, m is Nisha least one modification in position, which is selected from the group consisting of 157, 158, 296, 298, 305, 334, 336, 337 and 374. Examples of preferred substitutions include substitutions selected from the group consisting of V158D, E296D, M298Q, L305V and CA. More preferably named substitutions selected from the group consisting of V158D+E296D+M298Q+L305V+K337A, V158D+E296D+M298Q+K337A, V158D+E296D+M298Q+L305V, V158D+E296D+M298Q, M298Q, L305V+K337A, L305V and CA.

In the following embodiment of the present invention, the FVII or FVIIa variant can, in addition to the modifications described above in the previous sections also contain other mutations, such as replacement K341Q disclosed in Neuenschwander et al, Biochemistry, 1995; 34, 8701-8707. Other possible additional replacement include D196K, D196N, G237L, G237GAA and combinations thereof.

For more detailed information on joining variants of FVII and FVIIa non-polypeptide components described in patent applications WO 01/58935 and WO 03/093465, to which reference is made and which are included in this description by reference.

Methods of obtaining United conjugation variant of the invention

Generally conjugated variant according to the invention can be obtained by culturing an appropriate host cell under conditions conducive to expression of the variant polypeptide and recovering the variant polypeptide, where (a) a variant polypeptide contains at least one part of N-or O glycosylases the deposits and a host cell is a eukaryotic cell host, capable of glycosylation in vivo, and/or b) join a variant polypeptide of a non-polypeptide component is carried out in vitro.

Attach to the polymer molecule

A polymer molecule, which is designed for connection with the polypeptide variant may be any suitable polymer molecule, such as a natural or synthetic Homo-polymer or heteropolymer, the molecular weight of which, as a rule, is within approximately 300-100000 Yes, for example 500-20000 Yes, more preferably within about 500-15000 Yes or most preferably within about 2 to 12 kDa, such as in the range of about 3-10 kDa. In this case, when the term “about” is used in respect of a particular molecular weight, the word “about” indicates the approximate average molecular weight and reflects the fact that with this method of preparation of the polymer will be a certain distribution of molecular weight.

Examples of homopolymers include polisport (i.e. poly-OH), polyamine (so-called poly-NH2and polycarboxylic acid (i.e. poly-COOH). Heteropolymer is a polymer comprising different attached groups such as hydroxyl group and amino group.

Examples of suitable polymer molecules include polymer molecules selected from the group consisting of polyalkyleneglycol (PJSC, RAO), who Lucaya polyalkyleneglycol (PAG, PAG), such as polyethylene glycol (PEG, PEG) and polypropylenglycol (BCP PPG), branched polyethylene glycols, polyvinyl alcohol (PVA, PVA), polycarboxylate, poly(vinyl pyrrolidone), anhydride polyethylene-komlinovic acid anhydride polystyrene-komlinovic acid, dextran, including carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and/or increase the functional half-life in vivo and/or time of half-life in serum. Another example of a polymer molecule is human albumin or other plasma protein present in significant quantity. As a rule, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, panthenyl, non-immunogenic, water soluble and easily excreted by living organisms.

PEG is the preferred polymer molecule, as it has only few reactive groups capable of forming crosslinks compared to, for example, polysaccharides such as dextran. In particular, monofunctional PEG, such as methoxypolyethyleneglycol (MPEG, mPEG), is of particular interest because the chemistry of its binding is relatively simple (only one reactive group is available for connection with the side groups of the polypeptide). Therefore, since ri is to the formation of additional crosslinks is excluded, the resulting variants compounds are more homogeneous, and the reaction of the linking of the polymer molecules with the polypeptide variant is easier to control.

To increase the efficiency of the covalent joining of polymer molecules (molecules) to the polypeptide variant of the terminal hydroxyl groups of the polymer molecule must be provided in activated form, i.e. the form with reactive functional groups, examples of which include, primarily, amino groups, hydrazide (HZ), thiol, succinate (SUC), Succinimidyl (SS), succinimidylester (SSA), succinimidylester (SPA), succinimidylester (SBA), succinimidylester (SCM), benzothiazolinone (PTS), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylarsonic (NPC) and TResult (TRES). Suitable activated polymer molecules are commercially available, for example from Nektar Therapeutics, Huntsville, AL, USA, or from PolyMASC Phannaceuticals pie, UK.

Specific examples of activated linear or branched polymer molecules for use in the present invention are disclosed in Nektar Molecule Engineering Catalog 2003 (Nektar Therapeutics), are included in this description by reference.

Specific examples of activated PEG polymers include linear Page (PEGs): NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEO, BTC-EG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and extensive Page, such as PEG2-NHS, and also disclosed in US 5932462 and US 5643575, which are both included in this description by dumping. Additional publications representing suitable polymeric molecules, the chemistry of attaching PEG and methods of joining are listed in WO 01/58935 and WO 03/093465.

Specific examples of activated PEG polymers, particularly preferred compounds with the remnants of pestaina, include the following line Page: vinylsurfer-PEG (VS-PEG), preferably vinylsulfonic-MPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-MPEG (MAL-mPEG) and orthopaedically-PEG (OPSS-PEG), preferably orthopaedically-MPEG (OPSS-mPEG). Typically, such polymers PEG or MPEG will have a size of about 5 kDa, about 10 kDa, about 12 kDa, or about 20 kDa,

The person skilled in the art should know that in each case the activation method and/or chemistry accession depends on intended for joining the group (s) polypeptide variant (examples of which are noted above), as well as from the functional groups of the polymer (for example, which is an amine, a hydroxyl, carboxyla, aldehyde, sulfhydryl, Succinimidyl, maleimide, vinylsulfonate or haloacetic). Attach the PEG can occur on all the available side groups polypeptide variant (that is, on such side groups, which are exposed on the surface of the polypeptide) or may be directed to one or more specific side group, such as N-terminal amino group as described in US 5985265 or the remains of pestaina. Moreover, the joining can be achieved in one step or in a step-by-step manner (for example as described in WO 99/55377).

To attach the PEG to the remains of pestaina (see above) variant FVII or FVIIa, usually worked regenerating agent such as dithiothreitol (DDT), before attaching the PEG. Reducing agent is subsequently removed by any conventional method, such as, for example, desalination. Attach the PEG to the rest of pestaina, usually performed in a suitable buffer at pH 6-9 and temperatures ranging from 4°C to 25°C during incubation for up to 16 hours.

You must understand that the accession of the PEG should be done a certain way in order to obtain the optimal molecules in the ratio of the number of attached PEG molecules, the size and shape of these molecules (e.g., that they may be linear or branched) and plot (plots) joining the polypeptide variant. The molecular weight estimated by the use of the polymer may, for example, be selected depending on the desired effect to be achieved.

In connection with prisoedinenie the m to the only group in the protein molecule (e.g., to N-terminal amino group of the benefit can be the fact that the polymer molecule, which may be linear or branched, has a large molecular weight, preferably about 10-25 kDa, such as about 15-25 kDa, for example, about 20 kDa.

Typically, the accession of the polymer is carried out under such conditions, when the largest possible number of groups available for attachment of the polymer tends to react with a large number of polymer molecules. This is achieved by an appropriate molar excess of polymer relative to the polypeptide. Typically, the molar ratio of activated polymer molecules to the polypeptide reaches about 1000-1, such as before or 200-1 to 100-1. In some cases, the ratio may be somewhat lower, however, to achieve such values, as of about 50-1,10-1, 5-1,2-1 or 1-1, in order to achieve optimal reaction conditions.

In accordance with the present invention it is also possible to attach the polymer molecule to the polypeptide via an intermediate bridge. Suitable bridges are well known to specialists in this area, see also WO 01/58935.

After the reaction join the remaining activated polymer molecule are blocked using methods known to experts in this field of research, for example, by adding Pervin the th amine in the reaction mixture, then inactivated molecules of the polymer are removed using a suitable method.

You need to understand that depending on circumstances, for example, from the amino acid sequence of the polypeptide variant, nature of the used activated connection PEG and specific conditions of accession of PEG, including the molar ratio of PEG and polypeptide can be achieved different levels of attaching PEG; higher levels of attaching PEG, mainly, are achieved with a higher ratio of PEG to the polypeptide variant. Polypeptide variants with attached PEG, the result of any of the options attach the PEG, will, however, in normal circumstances, to include the stochastic distribution of the conjugated polypeptide variants having several different levels of attaching a PEG.

Join carbohydrate component

In order to carry out glycosylation in vivo FVII molecules containing one or more glycosylation sites, a nucleotide sequence encoding a polypeptide variant must be inserted in capable of glycosylation of eukaryotic expressing cell host. Expressing a host cell may be selected from cells of fungi (filamentous fungi or yeast)cells is of becomig or animals or cells of transgenic plants. In one of the embodiments a host cell is a cell of a mammal, such as a cell SNO, cell KSS. or SOME, for example, SOME 293, or an insect cell such as SF9 cell, or a yeast cell, for example, Saccharomyces cerevisiae or Pichia pastoris, or any of the undermentioned host cells.

Covalent addition of carbohydrates (such as dextran) in vitro to amino acid residues of the polypeptide variant can also be used, for example as described in WO 87/05330 and in Aplin et al., CRC Crit Rev. Biochem, pp.259-306, 1981. See also WO 03/093465 for more information regarding in vitro glycosylation variants of FVII or FVIIa.

Join inhibitor of serine proteases

Join inhibitor of serine proteases can be carried out in accordance with the method described in WO 96/12800.

Methods of obtaining polypeptide variant, which is the subject of the present invention

Polypeptide variant, which is the subject of the present invention, not necessarily in glycosylated form, can be obtained using any suitable method known to specialists in this field of research. Such methods include providing a nucleotide sequence that encodes a polypeptide variant, and the expression of this sequence in a suitable transformed or transferease the owner. Preferably, when a host cell is capable of γ-carboxylation a host cell, such as a cell of the mammal. In addition, variants of polypeptides that are the subject of the present invention can also be obtained, albeit with lower efficiency, by chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.

A nucleotide sequence encoding a polypeptide which is the subject of the present invention, can be constructed by selection or synthesis of nucleotide sequence that encodes a source FVII, such as hFVn, the amino acid sequence of which is presented as SEQ ID NO:1, and then changing the nucleotide sequences with the aim of appearing in it (i.e. insertion or substitution) or disappearance (i.e. division or replacement) important amino acid residue (residue).

The nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional methods. Alternatively, the nucleotide sequence can be obtained by chemical synthesis, for example, using a synthesizer oligonucleotides (oligonucleotide synthesizer), where the oligonucleotides are created on the basis of the amino acid sequence of the desired polypeptide and choice which are stated preferably such codons, which are the predominant is not uncommon for a host cell, which will be produced recombinant polypeptide. For example, several small oligonucleotides coding sections of the desired polypeptide may be synthesized and assembled by PCR (PCR, polymerase chain reaction (PCR), ligation or ligase chain reaction (LCR, ligation chainreaction) (Barany, Proc Natl Acad Sci USA, 88:189-193, 1991). Individual oligonucleotides typically have overlapping 5'- or 3'-region for complementary Assembly.

Once created (by synthesis, site-directed mutagenesis or another method), the nucleotide sequence encoding the polypeptide is inserted into a recombinant vector, where it should be paired with functional regulatory sequences necessary for expression of FVII in the desired transformed cells of the host.

Specialists in this field of research will be able to find suitable vectors, a regulatory sequence for expression and cell hosts for expression of the polypeptide. The recombinant vector may be a stand-alone can replicate the vector, i.e. a vector which exists in the cell as an extrachromosomal object, the replication of which is independent of the replication of chromosomes, for example, a plasmid. Alternatively,the vector can be one of these, which when introduced into the cell-master integrates into the genome of the host cell and replicated together with the chromosome into which they are integrated.

Preferably the vector is an expression vector in which the nucleotide sequence encoding a polypeptide variant which is the subject of the present invention, functionally associated with additional segments required for transcription of the nucleotide sequence. Typically, the vector is derived from plasmid or viral DNA. Many suitable expression vectors for expression in cells of the hosts mentioned herein are commercially available or described in the literature. Detailed information of suitable vectors for the expression of FVII can be found in WO 01/58935 included in this description by reference.

The term “regulatory sequence” is used in this case to refer to the totality of components that are necessary or offer advantages in the expression of the polypeptide variant, which is the subject of the present invention. Each regulatory sequence may be a natural or alien with respect to nucleotide sequence that encodes a polypeptide variant. This regulatory sequence includes, but is not ograniczenie is by this leading sequence, the sequence of the polyadenylation signal sequence propeptide, promoter, enhancer or above activator sequence, the sequence of the signal peptide and the terminator of transcription. The minimum version of the regulatory sequence comprises a promoter.

A large variety of regulatory sequences for the expression can be used in the present invention, for example, any of the regulatory sequences disclosed in WO 01/58935 included in this description by reference.

Nucleotide sequence which is the subject of the present invention encoding a polypeptide variant, obtained either through site-directed mutagenesis, synthesis, PCR or other methods, may not necessarily include a nucleotide sequence which encodes a signal peptide. The signal peptide is present in the case, when the variant polypeptide must secretariats from cells in which it is expressed. Such signal peptide, when he has a need to be recognized by cell selected for expression of the variant polypeptide. The signal peptide may be homologous (that is naturally associated with hFVII) or heterologous (i.e. originating from another source on the RH is increased hFVII) to the polypeptide or may be homologous or heterologous to the cell-master, that is, the signal peptide is naturally expressed in the cell host either one of those, which are normally not expressed in the cell host. For more information on suitable signal peptides see WO 01/58935.

Each suitable host may be used for producing the polypeptide variants, including bacteria (although in this case it is not preferred), fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, for example, transgenic animals and plants. Preferred are mammalian cells. Examples of bacterial host cells include gram-positive bacteria such as strains of Bacillus, for example, B. brevis or B.subtilis, Psedomonas or Streptomysces or gram-negative bacteria, such as strains of E. Li. Examples of suitable host cells of filamentous fungi include strains Aspergilus, for example, A. oruzae, A. niger or A. nidulans, Fusarium or richoderma. Examples of suitable host cells of the yeast include strains Szccharomyces, for example, S. cerevisiae, Schizoccharomyces, Klyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. polymorpha or Yarrowia. Examples of suitable host cells insects include cell lines Lepidoptora, such as Spodoptera frugiperda (Sf9 or Sf21) or cells Trichoplusioa (High Five) (US 5077214). Examples of suitable host cells mlekovita what they include a cell line of Chinese hamster ovary (Cho, Chinese hamster ovary) (e.g., Cho-K1; ATS CCL-61), cell line green monkey (COS) (e.g., COS 1 (ATSS CRL-1650), COS 7 (ATSS CRL-1651); cells of the mouse (e.g., NS/0), cell line kidney of the newborn hamster (KSS, baby hsmster kidney) (for example, ADS CRL-1632 or ATSS CCL-10) and human cells (for example, SOME 293 (ATSS CRL-1573)). Additional suitable cell lines are known in this field of research and is available in public depositories such as the American Type Culture Collection, Rockville, Maryland. In addition, mammalian cells, such as cells SNO, can be modified in such a way that will expressively sialyltransferase, for example, 1,6-sialyltransferase, for example as described in US 5047335, in order to ensure a higher level of glycosylation of the polypeptide variant.

To increase the secretion in some cases it is useful to produce a variant polypeptide which is the subject of the present invention, together with endoproteases, in particular together with RACE (paired basic amino asid converting enzyme) (for example as described in US 5986079), such as endoprotease KEH (for example as described in WO 00/28065).

Methods of introduction of exogenous DNA into the above-mentioned cell types, as well as other information concerning the expression, production and purification of FVII variants found in WO 01/58935 included in this description by reference.

Pharmaceutical composition, which pre is the subject of the present invention, and use

In a further aspect the present invention relates to compositions, more specifically, the pharmaceutical composition comprising the polypeptide variant, which is the subject of the present invention, and a pharmaceutically acceptable carrier or excipient.

Polypeptide variant, or a pharmaceutical composition, in accordance with the present invention can be used in medicine.

Thanks to the improved properties mentioned above, the polypeptide variants that are the subject of the present invention or pharmaceutical composition which is the subject of the present invention is suitable, in particular, for the treatment of uncontrolled bleeding in injured patients with thrombocytopenia of patients undergoing treatment with anticoagulants and patients with cirrhosis with VariableName bleeding or other upper gastrointestinal bleeding in patients undergoing orthotopic liver transplantation or liver resection, allowing not to resort to transplant (allowing for transfusion free surgery), or patients with hemophilia.

Under the injury is damage to living tissue caused by an external agent. It is the fourth leading cause of death in the United States, leading to a significant financial burden on the economy.

Injuries meant the process identifies to blunt or penetrating. Blunt trauma causes compression of the viscera, organ damage and internal bleeding, while penetrating injury (as a result of the actions of the subject, penetrating into the body and destroying tissue, vessels and organs) leads to external bleeding.

The injury can be caused by many reasons, for example, road traffic accidents, gunshot wounds, falls, accidents at work (mechanical damage) and stab wounds.

Cirrhosis can be caused by direct damage to the liver, including chronic alcoholism, chronic viral hepatitis (types b, C, and D), and autoimmune hepatitis, as well as indirect damage as damage to the bile duct, including primary biliary cirrhosis, primary sclerosing cholangitis and biliary atresia. Less common causes of cirrhosis include direct damage to the liver due to hereditary diseases such as cystic fibrosis, lack of alpha-1-antitrypsin, genomenos, Wilson disease (Wilson's disease, galactosemia, and excessive accumulation of glycogen in the cells - glycogens (for example, illness Andersen). The main method for the treatment of patients with advanced cirrhosis is transplantation.

Thus, in a further aspect of the present invention cases the polypeptide variant, which is the subject of the present invention, from the viewpoint of the production of medicaments for treatment of diseases or disorders in which it is desirable formation of a blood clot. Another aspect of the present invention relates to a method of treatment of a mammal having a disease or disorder, wherein preferably the formation of a blood clot, which includes the introduction in a mammal in need an effective amount of the polypeptide variant or pharmaceutical composition which is the subject of the present invention.

Examples of diseases/disorders, where it is desirable to increase the efficiency of blood clot formation, include, but are not limited to, bleeding, including bleeding in the brain, as well as severe uncontrolled bleeding in patients, for example, in case of injuries. Further examples include patients undergoing vital transplantation, patients who underwent resection, and patients with varicella bleeding. Other widespread illness/disorder, which is considered Tripeptide which is the subject of the present invention are useful for increasing the efficiency of blood clot formation, is hemophilia, for example disease von Willebrand (vor Willebrand's disease), geofi the Oia And, hemophilia or hemophilia C.

Polypeptide variants that are the subject of the present invention, are administered to the patient in therapeutically effective dose, normally, approximately corresponding to the applied dose therapy with rFVII, such as NovoSeven®or in smaller quantities. By “therapeutically effective dose” is a dose sufficient to obtain the desired effect, compared to the state, when this dose was introduced. The exact dose will depend on the circumstances and will be determined by the person skilled in the art using known techniques. Generally, the dose should provide the ability to prevent or reduce the severity or spread of the condition or symptoms being treated. For specialists in this field will be seen that the effective amount of the polypeptide variant or composition which is the subject of the present invention depends, inter alia from the disease, dose, schedule of administration of medication in cases where the polypeptide variant or composition are introduced individually or together with other therapeutic agents, the time of half-life of the composition in the plasma of the blood and the General health of the patient.

Polypeptide variant, which is the subject of the present invention, prepact the tion is introduced in the form of a composition, including pharmaceutically acceptable carrier or excipient. “Pharmaceutically acceptable” means a carrier or excipient that does not cause any adverse effects in the patient, which he entered. Such pharmaceutically acceptable carriers and excipients, as well as suitable methods of preparation of medicines, is well known to specialists in this field (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A.R.Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L.Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press [2000]).

Polypeptide variant, which is the subject of the present invention may be used “as is” and/or in the form of its salts. Suitable salts include, but are not limited to, salts of alkaline or alkaline earth metals such as sodium, potassium, calcium and magnesium, as well as, for example, zinc salts. These salts or complexes can exist as crystalline and/or amorphous structure.

The pharmaceutical composition which is the subject of the present invention, may be administered individually or together with other therapeutic agents. These agents may be introduced as part of the same pharmaceutical composition or may be administered separately from the polypeptide variant, which is the pre is the subject of the present invention, simultaneously and in accordance with another treatment schedule. In addition, the polypeptide variant or pharmaceutical composition which is the subject of the present invention may be used as adjuvant to other therapies.

The concept of “patient” in the context of the present invention includes both human and other mammals. Thus, the methods applicable to both human therapy and for the needs of veterinary medicine, in particular for the treatment of humans.

A pharmaceutical composition comprising the polypeptide variant, which is the subject of the present invention may be manufactured in a variety of dosage forms such as liquid, gel, as freeze-dried or pressed solid. The preferred form will depend on what exactly is the symptom is treated, and it will be obvious to a person skilled in this field.

In particular, the pharmaceutical composition comprising the polypeptide variant, which is the subject of the present invention may be manufactured in lyophilized or stable soluble form. The polypeptide variant may be dried by a variety of procedures known in the field. The polypeptide variant may be prepared in a stable soluble form through-the Oia or shielding sections proteolytic degradation, as described in this paper. The advantage of obtaining a stable soluble drug is the ease of handling for the patient and, in case of critical situations more quickly, which, potentially, can save lives. The preferred form will depend on what exactly is the symptom is treated, and it will be obvious to a person skilled in this field.

Medications, which is the subject of the present invention may be implemented in a variety of ways, including, but not limited to, such as oral, subcutaneous, intravenous, intracerebrally, intranasal, transdermal, intraperitoneal, intramuscular, intra-lungs, vaginal, rectal, intraocular, or any other suitable method. Drugs may be administered by continuous infusion (drip), although bolus injection is also applicable, using the well-known in this field techniques, such as pumps or implantation. In some cases, the drugs can be applied directly in the form of a solution or spray.

Sources

A preferred example of the pharmaceutical composition is a solution, particularly an aqueous solution, created for parenteral administration. Although in many cases prepared pharmaceutical solutions feature vidcom, suitable for immediate use, such parenteral preparations may also be provided in frozen or dried form. In the first of the above cases, the composition must be thawed before use. The latter form is often used to increase the stability of the active substance contained in the composition, in the case of a variety of storage conditions, as it is recognized for professionals in this field that lyophilized preparations, as a rule, are more stable than their liquid counterparts. Such lyophilized preparations restore before use by adding one or more suitable pharmaceutically acceptable solvent, such as sterile water for injection or sterile physiological saline solution.

If the source of the materials they are made for storage as lyophilized preparations or aqueous solutions by mixing, if it is acceptable, the polypeptide variant having the desired degree of purification, with one or more pharmaceutically acceptable carrier, excipient or stabilizer commonly used in this area (all of which are called “fillers”, ipients), which include, for example, buffering agents, stabilizing agents, to the sideboards, isotonicity, non-ionic surfactants (surfactants or detergents, antioxidants and/or other mixed additives such as substances that adds volume or fillers, chelating agents, antioxidants and the cosolvent.

Detailed information about the preparation of starting materials suitable for injection of FVII variants as well as drugs extended release, can be found in WO 01/58935 and WO 03/093465 included in this description by reference.

The present invention is described below using the following examples, but is not restricted by them.

Materials and methods

The active center

The active center is defined as any remnants having at least one atom that is located at a distance within 10 A from any atom in the catalytic triad (residues HI 93, D242, S344).

The dimension of the reduced sensitivity to proteolytic degradation

Proteolytic degradation can be measured using the analysis described in US 5580560, Example 5, where proteolysis is autoproteolysis.

In addition, reduced proteolysis may be registered in the model in vivo using radioactively-labeled samples and comparison of proteolysis rhFVIIa and polypeptide variants of the present invention by sampling blood and analysis method SDS-electrophoresis polyacrylamide gel (SDS-PAGE) and autoradiography.

Regardless of the analysis used to determine proteolytic degradation, the term “reduced proteolytic degradation” is intended to denote a measurable decrease in the cleavage compared to that obtained for rhFVIIa when measured by scanning the gels obtained after carrying out SDS-PAGE and Coomassie staining, HPLC or measure preserved catalytic activity compared with the wild type when using activity analysis, independent of tissue factor, described below.

Determination of molecular weight polypeptide variants

Molecular weight polypeptide variants define any of the following methods: SDS-PAGE, gel filtration, Western blotting performed using matrix assisted laser desorption mass spectrometry mstrix assisted laser desorption mass spectrometry) or equilibrium centrifugation, for example, SDS-PAGE according Laenmui, U.K., Nature, vol.227 (1970), pp.680-685.

Determination of binding affinity of phospholipid membranes

The binding affinity of phospholipid membranes can be determined as described in Nelsestuen et al., Biochemistry, 1977; 30, 10819-10824 or as described in Example 1 in US 6017882.

Analysis of TF-independent activation of factor X

This analysis was described in detail on page 39826 in the work Nelsestuen et al., J Biol. Chem., 2001; 276:39825-39831.

Briefly, the analyzed molecule (l is Bo hFVIIa, rhFVIIa or polypeptide variant of the invention in the activated form) is mixed with a source of phospholipid (preferably phosphatidylcholine and phosphatidylserine in the ratio 8:2) and reliegion factor X in Tris-buffer containing bovine serum albumin (BSA). After a certain time of incubation the reaction is stopped by the addition of excess EDTA. Then measure the concentration of factor XA from changes in absorption at 405 nm after addition of the chromogenic substrate (S-2222, Chromogenix). After correction for background determine the activity of rhFVIIa (awt), independent of tissue factor as a change in absorption for 10 minutes and the activity of the polypeptide variant of the invention (aoption), independent of tissue factor, also measured as a change in absorption in 10 minutes. The ratio of the activity of the polypeptide variant in the activated form and activity of rhFVIIa is defined as aoption/awt.

Analysis of coagulation

The coagulation activity of FVIIa and its variants were measured by one-stage assays and the clotting times were recorded on a Thrombotrack IV coagulometer (Medinor). Plasma man, emaciated against factor VII (American Diagnostica), restored and balanced at room temperature for 15-20 minutes. 50 microliters plasma is then transferred to a Cup of coagulometer.

FVIIa and its variants rasba the Lyali buffer pixelenemy buffer (5.7mm barbiturate, 4.3 mm sodium citrate, 117 mm NaCl, 1 mg/ml BSA, pH 7,35). The samples were made in a Cup in the amount of 50 μl and incubated at 37°C for 2 minutes.

Thromboplastin (Medinor) restored with water and added CaCl2to a final concentration of 4.5 mm. The reaction was started by adding 100 μl of thromboplastin.

For measuring clotting activity in absence of TF was used in the same analysis without the addition of thromboplastin. Data were analyzed using the software PRISM.

Analysis of whole blood

The coagulation activity of FVIIa and its variants were measured by one-stage assays and the clotting times were recorded on coagulometer Thrombotrack IV (Medinor). 100 μl of FVIIa or its variants were diluted in buffer containing 10 mm glycylglycine, 50 mm NaCl, 37.5 mm CaCl2pH of 7.35, and carried in a Cup for the reaction. The clotting reaction was started by adding 50 µl of blood containing 10% 0,13 M trinational salt of citric acid as an anticoagulant. Data were analyzed using the software Excel or PRISM.

Amiloliticeski analysis

The ability of the options to split small peptide substrates can be measured using chromogenic substrate S-2288 (D-Ile-Pro-Arg-para-nitroanilide). FVIIa was diluted to about 10-90 nm in buffer for analysis (50 mm Na-Hepes, pH 7.5, 150 mm NaCl, 5 mm CaCl2, 0.1% BSA, 1 u/ml heparin). In addition, dissolve the th TF (soluble TF, sTF) was diluted to 50-450 nm in buffer for analysis. 120 μl of buffer for analysis mixed with 20 µl of sample with FVIIa and 20 µl of the sTF. After 5 min incubation at room temperature with gentle shaking and subsequent incubation for 10 min at 37°C. the reaction starts with the addition of the substrate S-2288 to a concentration of 1 mm and measured the absorbance at 405 nm at several time points.

Analysis using ELISA method

The concentration of FVII/FVIIa (or variant) is determined by using ELISA method. The microtiter wells are sensibiliser antibodies against proteases domain, using a solution containing 2 μg/ml antibody in PBS (100 μl per cell). After sensitization over night at room temperature (room temperature, R.T.) cells are washed 4 times with TNT buffer (100 mm NaCl, 50 mm Tris-HCl, pH 7,2, 0,05% Tween-20). Then 200 μl of 1% casein (obtained by dilution of the original 2.5% solution of casein solution 100 mm NaCl, 50 mm Tris-HCl, pH 7,2) add a cell to block. After 1 hour incubation at R.T. cell empty, and make them 100 ál of sample, optionally diluted with a buffer for dilution (TNT+0.1% casein). After another incubation for 1 hour at room temperature, the cells are washed 4 times with TNT buffer and add 100 ál of antibody against the EGF-like domain, labeled biotype (1 µg/MP). After another incubation for 1 hour at R.T. followed by 4 additional industrial the key buffer TNT, add 100 ál conjugate streptavidin-horseradish peroxidase (DAKO A/S, Glostrup, Denmark, dilution 1/10000). After another incubation for 1 hour at R.T. followed by 4 additional washing with TNT buffer, add 100 ál of TMB (3,3',5,5'-tetramethylbenzidine, Kem-en-Tech A/S, Denmark). After 30 min incubation at R.T. in the dark add 100 ál of 1 M H2SO4determine the optical density at 450 nm (OD450nm). A standard curve is obtained using rhFVIIa (NovoSeven®).

Alternatively, the number of FVII/FVIIa or variants can be determined through binding Gla domain, and not through proteiny domain. In such analyses using ELISA method cell sensibiliser during the night antibodies against EGF-like domain and for detecting use of calcium-dependent monoclonal antibodies against the Gla-domain labeled with Biotin (2 mg/ml, 100 µl of the cell). When using this option analysis for TNT and buffers for breeding add 5 mm CaCl2.

Definition thrombogram

The effect of hFVIIa, rhFVIIa or FVIIa variants on the formation of thrombin in plasma is checked using a modified version of the analysis, described on page 589 in the work Hemker et al. (2000) Thromb Haemost 83:589-591. Briefly, the analyzed molecule (or hFVIIa, rhFVIIa or variant) is mixed with depleted factor FVII-depleted platelet plasma (platelet poor plasma, the PP) containing or relitivaly recombinant tissue factor (such as of Innovin, Dade Behring), or a phospholipid (phosphatidylcholine and phosphatidylserine in the ratio 8:2 or phosphatidylcholine, phosphatidylserine and phosphatidylserine in the ratio 4:2:4).

The reaction starts with the addition of the substrate of thrombin, which gives fluorescent products, and calcium chloride. Fluorescence is measured continuously and amylolyticus the activity of thrombin determined by calculating the slope of the fluorescence curve (increase in fluorescence over time). This method determines the time at which the maximum amylolyticus activity of thrombin (Tmax), and the rate of formation of thrombin (the maximum increase in the activity of thrombin), and also calculate the total activity of thrombin (area under the curve, area under the urve, AUC).

Frozen plasma with added citrate, emaciated of factor FVII, thawed in the presence of trypsin inhibitor from maize (100 µg/ml serum) for inhibition of paired paths coagulation. In each of the wells of a microplate with 96 cells add 80 ál of plasma and 20 μl of a buffer containing rhFVII or option that should be analyzed in the final concentrations in the range 0.1-100 nm. Recombinant tissue factor human (rTF) add 5 μl of buffer for analysis to a final concentration of 1 PM Buffer for analysis contains 20 mm Hepes, 150 mm NaCl and 60 mg/ml BSA and prepared in distilled water. The reaction starts by adding 20 µl of substrate solution containing 0.1 M l. The tablet used for analysis, and the reagents are pre-heated to 37°C and carry out the reaction at this temperature. Used by fluorimetry is BMG Fluonneter filter for excitation light with a maximum transmission at 390 nm and with a filter for the light emitted with maximum transmittance at 460 nm. Fluorescence was measured in each cell of tablets with 96 cells with transparent bottom at intervals of 20-40 seconds for 30-180 minutes. Data analyzed using the software PRISM.

Determination of the binding of tissue factor by means of surface plasma resonance (Biacore Assay)

To measure the relative binding ability of factor VIIa wild type and its variants with soluble tissue factor used method of surface plasma resonance (surface plasmon resonance). Recombinant soluble tissue factor containing the extracellular domain bound with boards to measure the Biacore chip SM, type 270, using the method of linking NHS/EDC. Binding of soluble tissue factor was carried out at pH 4.5, which provides a strong interaction with the surface of the chip,

In this study, the binding of tissue factor protein factor VII is rowdily when the same concentration of FVIIa or variant, that makes possible to perform a relative comparison of alternatives with the wild type. This concentration was determined from the standard curve for wild-type FVIIa, which was passed over the surface of the chip at concentrations in the range 75-0 µg/ml Destruction of FVIIa was performed by washing the chip 10 mm EDTA. In this way it was found that the concentration of 15 μg/ml provides the linkage in the linear range. Then over the surface of the chip missed the solution of FVIIa variants with a concentration of 15 µg/ml to determine the relative binding ability of FVIIa or variants with tissue factor.

Examples

Example 1

For this example, we used the data of x-ray analysis hFVII in complex with soluble tissue factor, see Banner et al., J Mol Biol, 1996; 285:2089. For more information on the calculations used in this example, see WO 01/58935.

Exposure on the surface

When performing fractional ASA calculations for the following residues were identified that more than 25% of their side chain exposed to the surface: A1, N2, A3, F4, L5, E6, E7, L8, R9, P10, S12, L13, E14, e, K18, E19, E20, Q21, S23, F24, E25, E, R28, E, F31, K, D33, A34, E, R36, C, L39 are effective, W41, I42, S43, S45, G47, D48, Q49, A51 Motorway, S52, S53, Q56, 058, S60, C, D63, Q64, L65, Q66, S67,169, F71, L73, R, A75, E77, G78, R79, E, T, N, K, D86, D87, Q88, L89,190, V92, N93, E, G97, E, S103, D104, N, T, 0107, T, K, Sill, R.113, E, G117, S119, L120, L121, A, D123, 0124, V125, S126, T,R, T, V131, E, 1140, L141, E, K, R144, N145, A, S147, K, R, Q150, G151, R152, G155, K, V158, R160, K, E163, L171, 173, G174, A, No. 184, T, 1186, N, K, K, N200, R202, N203, 1205, S214, E, N, D217, G218, D219, S222, R224, S232, T, V235, R, G237, T, T, N240, N, Q250, R, V253, T, D256, E, R266, T, E, R271, F275, V276, R277, F278, L280, L287, L288, D289, R290, G291, A, T, L295, E, N301, M, T, Q308, D309, L311, Q312, Q313, R315, K, V317, G318, D319, S320, R, N322, T, E, Y326, Y332, S333, D334, S336, K, K, G342, N, R353, 0354, Q366, 0367, T370, V371, 0372, R379, E, Q388, K, R392, S393, E, R395, R396, R, 0398, V399, L400, L401, R402, R and R (A1-S45 localized in the Gla domain, residues in other positions localized outside the Gla domain).

It was determined that the following residues of more than 50% of their side chain exposed to the surface: A1, A3, F4, L5, E6, E7, L8, R9, P10, E14, e, K18, E19, E20, Q21, S23, E25, E, E, K, A34, E, R36, C, L39 are effective, 142, S43, 047, D48, A51 motorway, S52, S53, Q56, 058, S60, C, L65, Q66, S67, 169, F71, L73, R, A75, E77, 078, R79, E, N, K, D86, D87, Q88, L89, 190, V92, N93, E, 097, T, 0107, T, K, Sill, E116, S119, L121, A, D123, 0124, V131, E, L141, E, K, R144, N145, A, S147, K, R, Q150, 0151, R152, 0155, K, R160, 173, 0174, A, C, K, N200, R202, S214, E, N, G218, R224, V235, R, G237, T, N, Q250, V253, D256, T, F275, R277, F278, L288, D289, R290, G291, A, T, L295, N301, M, Q308, D309, L311, Q312, Q313, R315, K, G318, D319, N322, E, D334, K, G354, G367, V371, E, K, R392, E, R396, R, G398, R402, R and R (A1-S43 localized in the Gla domain, residues in other positions localized outside the Gla domain).

The binding site of tissue factor

By calculation ASA was determined teledrama residues, localized in hFVII, change their ASA in the complex. It was found that these residues constitute the binding site of the receptor: L13, K18, F31, E, R36, L39 are effective, F40,142, S43, S60, C, D63, Q64, L65,169, C70, F71, s, L73, R, F76, E77, G78, R79, E, K, Q88,190, V92, N93, E, R271, A, F275, V276, R277, F278, R304, L305, M, T, Q308, D309, Q312, Q313, E and R379.

The active center

The active center is defined as any amino acid residue having at least one atom at a distance within 10 a from any atom of the triad of amino acids, providing catalytic activity (residues HI 93, D242, S344): 1153, Q167, V168, L169, L170, L171, Q176, L177, S, G179, 0180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196, K197,1198, W201, V228,1229,1230, P231, S232, T233, Y234, V235, R, G237, T, T239, N240, H241, D242, 1243, A244, L245, L246, V281, S282, G283, W284, G285, Q286, T, T324, E, Y326, M327, F328, D338, S339, C340, C, G342, D343, S344, G345, G346, P347, H348, L358, T359, G360, 1361, V362, S363, W364, G365, C368, V376, Y377, TA, R379, V380, Q382, Y383, W3.86, L387, L400 and F405.

The surface of the connecting slots of the active centre

Plot surface connecting the gap of the active center (active site binsding cleft) was determined by visual examination of the structure of FVIIa 1FAK.pdb: 173, A, C, N200, N203, D289, R290, D291, A, R and T370.

Example 2

Construction of expression cassettes for expression rhFVII in mammalian cells

The expression cassette for the expression of rhFVII was constructed and cloned as described in Example 2 of patent application WO 01/58935.

P the emer 3

The design of the expression cassette that encodes variants of the present invention

PCR using sequences with additional exposed areas (sequense overhang extension, SOE PCR) was used to create structures that can encode options open reading frames with FVII replaced by codons, using standard methods.

Example 4

Expression of polypeptide variants in cells Cho K1

The cell line Cho K1 (ATCC # CCL-61) seeded at 50% confluence in culture flasks T-25, using α, 10% FCS (Gibco/BRL Cat # 10091), P/S and 5 μg/ml of pipelinea, and give the opportunity to grow to confluence. Fused monoclonal layer transferout 5 micrograms of the desired plasmid described above, using the agent for transfection with Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. 24 hours after transfection take a sample and measure the amount of option when using, for example, the ELISA method using antibodies against the EGF1 domain of factor hFVII. To form a population of stable transfectants in this moment of time can be carried out corresponding to the selection of cells (for example, using Hygromycin). When using cells Cho K1 and gene resistance to Hygromycin B, being part of the plasmid as a selective marker for the presence of plasmids, it usually is achieved within about the Noah of the week.

Example 5

Obtaining cells Cho K1, stably expressing the polypeptide variants

The tube is a pool of transfected cells Cho-K1 thawed and seeded with cells in flasks for growing tissue with an area of 175 cm2containing 25 ml α, 10% FCS, phylloquinone (5 μg/ml), 100 u/l penicillin, 100 μg/l streptomycin, and grown for 24 hours. Cells are harvested, diluted and applied to 96-well microplates at a density of cells1/2-1 cell to cell. After 1 week of growth present in the cells of the colony of 20-100 cells, and those cells that contain only one colony, mark; After the next two weeks Wednesday all cells that contain only one colony, replace 200 ál of fresh medium. After 24 hours take the test environment and analyze, for example, using ELISA method. Highly productive clones are selected and used for producing a large number of FVII or variants.

Example 6

Purification of polypeptide variants and subsequent activation.

Purification of FVII variants and FVII carried out as follows: the Procedure is carried out at 4°C. the Collected culture liquid after the large-scale cultivation of cells subjected to ultrafiltration using Millipore TFF with a Pellicon membrane in order to isolate proteins larger than 30 kDa. After concentration of the environment add zither is up to 5 mm and the pH was adjusted to 8.6. If necessary, the conductivity is reduced to values below 10 MS/see Then, the sample is applied on the column with O-separate FF, balanced, 50 mm NaCl, 10 mm Tris, pH of 8.6. After washing the column with 100 mm NaCl, 10 mm Tris, pH of 8.6, and then a solution of 150 mm NaCl, 10 mm Tris, pH 8,6, elute FVII, using 10 mm Tris, 25 mm NaCl, 35 mm CaCl2pH of 8.6.

For the second phase chromatography prepared affinity column by binding monoclonal calcium-dependent antibodies against Gla-domain with CNBr-activated separate FF. About 5.5 mg of antibody is associated with 1 ml of resin. Column balance with a solution of 10 mm Tris, 100 mm NaCl, 35 mm CaCl2, pH 7.5. Then the sample add NaCl to a concentration of 100 mm and adjusted pH to 7.4 and 7.6. After O/N sample application the column was washed with a solution of 100 mm NaCl, 35 mm CaCl2, 10 mm Tris, pH 7.5, and elute the protein FVII solution of 100 mm NaCl, 50 mm citrate, 75 mm Tris, pH 7.5.

For the third phase chromatography conductivity of samples needed is reduced to values below 10 MS/cm and adjusted pH to 8.6. Then the sample is applied on a column of Q-separate (balanced 50 mm NaCl, 10 mm Tris, pH 8,6) at a density of about 3-5 mg of protein per 1 ml of gel for effective activation. After application the column was washed with a solution of 50 mm NaCl, 10 mm Tris, pH 8.6 out for about 4 hours with the transmission of 3-4 column volumes per hour. Protein FVII elute using a gradient of 0-100% 500 mM NaCl, 10 The M Tris, the pH of 8.6, skipping over 40 column volumes. The fractions containing FVII, unite.

For the final phase chromatography conductivity of the sample is reduced to values below 10 MS/see Then, the sample is applied on a column of Q-separate (balanced solution of 140 mm NaCl, 10 mm glycylglycine, pH 8.6) at a concentration of 3-5 mg protein per 1 ml of gel. Then the column is washed with a solution of 140 mm NaCl, 10 mm glycylglycine, pH 8.6 and elute FVII solution of 140 mm NaCl, 15 mm CaCl2, 10 mm glycylglycine, pH of 8.6. The eluate was diluted to the concentration of CaCl210 mm and the pH adjusted to a value of 6.8 to 7.2. In conclusion, add Tween-80 concentration of 0.01% and adjusted pH to 5.5 to store samples at -80°C.

Example 7

Experimental results - the ability to activate FX

The study "analysis of the TF-independent activation of factor X" using variants of the invention, the following results were obtained (results expressed as a percentage of the activity options P10Q+K32E, used as control):

Table 1
TF-independent activation FX
Option(aoption/aP10QK32E)* 100
rhFVIIa10/td>
[P10Q+K32E]rhFVIIa (control)100
A3AY+P10Q+K32E+A34L216
P10Q+K32E+D33F+A34E194
P10Q+K32E+A34E+P74S190
10Q+K32E+A34E+R36E+K38E144
P10Q+K32E+A34D+R36E140

As can be seen from the above results, the variants of the invention showed an increased ability to activate FX compared to rhFVIIa, as well as in comparison with [P10Q+K32E]rhFVIIa.

Example 8

Experimental results - clotting activity in the Analysis of whole blood"

Exposing variants of the invention the Analysis study of whole blood revealed that they showed increased clotting activity (i.e. less clotting time) compared to rhFVIIa, as well as [P10Q+K.32E]rhFVIIa. Experimental results are shown in Figure 1 and in Table 2 below.

Table 2
Clotting time (Analysis of whole blood)
Optiontoption/twt
rhFVIIa (control)1
A3AY+P10Q+K32E+E116D0,4
A3AY+P10Q+K32E+A34L0,3
P10Q+K32E+A34E+P74S0,3
A3AY+P10Q+K32E+E77A0,4

Example 9

Experimental results - clotting activity in the experiment the Measurement of the coagulation activity"

When measuring TF-dependent coagulation activity (conditions of experiment Measurement of the coagulation activity" described above in the section Materials and methods), it was obvious that variants of the present invention, having a replacement R36E, show significantly reduced clotting activity compared with rhFVII or other variants of the present invention, as shown in Table 3 below. However, as illustrated in Example 7 above, the variants of replacement R36E, have shown an increased ability to activate factor X in the experiment “Analysis of TF-independent activation of factor X”.

Table 3
The average clotting activity
Option(u/mgoptions/units/mgwt ), (n-2-3)
NovoSeven®(control)52,119(100%)
P10Q+K32E52,714 (101%)
A3AY+P10Q+K32E+A34L56,948 (107%)
P10Q+K32E+A34E+R36E1,439(2,7%)
P10Q+K32E+A34D+R36E+K38E1.232 metric (2,4%)

Example 10

Experimental results the formation of thrombin in the experiment Definition thrombogram"

Using both thrombogram - phospholipid (L)-dependent and dependent on tissue factor (TP) - dependent (see above description Definition thrombogram), determined the maximum rate of formation of thrombin for FVIIa variants with different concentrations of protein. The graphical image according to the maximum speed of formation of thrombin (expressed as units of fluorescence (fluorescence units, FU) for s2) as a function of concentration options in PM, there were obtained the results presented on Figure 2 (the maximum value of the velocity-dependent tissue factor in the generation of thrombin) and Figure 3 (the maximum velocity of the phospholipid-dependent formation of thrombin). From these results it is seen that FVIIa variant P10Q CE AE R36E shows differentiated ability education is obyvaci thrombin, depending on whether the reaction PL-dependent or TF-dependent. Maximum speed TF-dependent formation of thrombin of this option is reduced approximately 10-fold (dashed line on Figure 2) compared with FVIIa variant P10Q CE or A3AY P10Q CE A34L. Also the delay time up to the maximum accumulation, peak height and (to a lesser extent) have smaller AUC values for P10Q CE AE R36E compared with other options (results not shown). Unlike TF-dependent activity of PL-dependent activity options P10Q CE AE R36E is equivalent to the activity of other options analyzed in this example (see Figure 3), i.e. this variant has a full PL-dependent activity, although TF-dependent activity is significantly reduced. In the same experiment variant P10Q CE AE R36E directly compared with option P10Q CE AE P74S, which has the highest rate of TF-dependent formation of thrombin, as shown in Figure 2. Differences in TF binding between these two options (i.e. reduction of TF-binding variant P10Q CE AE R36E) is determined by the availability of replacement R36E, perhaps its joint action with the replacement AE.

Example 11

Experimental results - FVIIa binding to tissue factor in the Biacore Assay

In the analysis of variants of the invention by means of surface plasma resonance on Biacoe system using TF-chip, as described in Materials and Methods, the results were as follows:

Table 4
The average value of the response, ed
Option(n=5)
Wild type (Wt) FVIIa888
P10Q; CE714
A3AY; P10Q; CE; A34L967*
P10Q; CE; AE; R36E414
*n=2

In accordance with the data on the TF-dependent rate of formation of thrombin obtained in the experiment Definition thrombogram” (Example 10), the results presented in Table 4, indicate that replacement R36E reduces binding to tissue factor. In the same analysis on Biacore system FVIIa variants with the same modifications as the options listed in Table 4, in combination with two additional modifications, introducing two sites of glycosylation (T106N and V253N or I205T), were also analyzed for binding to tissue factor. The results are shown below in Table 5.

Table 5
The average response, ed
Option(n=5)
T106N; V253N717
T106N; I205T612
P10Q; CE; T106N; I205T502
P10Q; K32E;T106N; V253N498
A3AY; P10Q; CE; A34L; T106N; V253N522
P10Q; CE; AE; R36E; T106N; I205T216

These results are consistent with the results presented in Table 4 and show that, compared with the same options (or wild type), not having additional glycosylation sites are presented in Table 4, the options presented in Table 5, the presence of two new sites of glycosylation provides (additional) decrease in the binding of tissue factor. As in the case of options, presented in Table 4, the presence replacement R36E in glycosylated variant leads to a significant decrease in the level of binding of tissue factor compared to the binding of tissue factor glycosylated variant of the mi, who do not have this change.

1. The polypeptide variant of factor VII (FVII) or factor VIIa (FVIIa), having the amino acid sequence that differs in 1-15 amino acid residues compared to the amino acid sequence of factor VII human (hFVII) or factor VIIa person (hFVIIa), shown in SEQ ID NO:1 in which a negatively charged amino acid residue has been introduced by substitution in position 36.

2. Polypeptide variant according to claim 1, where a named substitute is R36D.

3. Polypeptide variant according to claim 1, where a named substitute is R36E.

4. Polypeptide variant according to any one of claims 1 to 3, further comprising a replacement of the amino acid in position 34.

5. Polypeptide variant according to claim 4, which includes the replacement of A34L.

6. Polypeptide variant according to claim 4, which includes the replacement of AE.

7. The polypeptide variant of claim 6, including replacement A34E+R36E.

8. Polypeptide variant according to any one of the preceding paragraphs, further comprising replacing amino acids selected from the group consisting of P10Q, CE and their combinations.

9. The polypeptide variant of claim 8, which includes the replacement of CE.

10. The polypeptide variant of claim 8, which includes the replacement of P10Q.

11. The polypeptide variant of claim 8, including replacement P10Q+K32E.

12. Polypeptide variant according to claim 11, including replacement P10Q+K32E+A34+R36E.

13. polypeptidyl variant according to item 11, includes replacement P10Q+K32E+A34L+R36E.

14. Polypeptide variant according to any one of the preceding paragraphs, in which at least one amino acid residue comprising the group for attaching non-polypeptide component entered in the position outside the Gla domain.

15. Polypeptide variant according to 14, where the named group to join is a glycosylation site in vivo.

16. Polypeptide variant according to item 15, where the glycosylation site represents a site of N-glycosylation in vivo, introduced by replacement.

17. Polypeptide variant according to item 16, where the named site of N-glycosylation in vivo introduced by replacement of which is selected from the group consisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, AT, I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T, R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N, D334N and their combinations.

18. Polypeptide variant according to 17, comprising at least one substitution, which is chosen from the group comprising T106N, I205T and V253N.

19. Polypeptide variant on p, including two sites of N-glycosylation in vivo, introduced by a substitution selected from the group consisting of T106N+I205T, T106N+V253N, I205T+V253N.

20. Polypeptide variant according to claim 19, including replacement 10Q+K32E+A34E+R36E+T106N+I205T.

21. Polypeptide variant according to claim 19, including replacement P10Q+K32E+A34E+R36E+T106N+V253N.

22. Polypeptide variant according to claim 19, including replacement P10Q+K32E+A34E+R36E+I205TV253N.

23. Polypeptide variant according to claim 19, including replacement 10Q+K32E+A34L+R36E+106N+I205T.

24. Polypeptide variant according to claim 19, including replacement P10Q+K32E+A34L+R36E+T106N+V253N.

25. Polypeptide variant according to claim 19, including replacement P10Q+K32E+A34L+R36E+I205T+V253N.

26. Polypeptide variant according to any one of the preceding paragraphs, further comprising inserting at least one amino acid residue between positions 3 and 4.

27. Polypeptide variant on p, including the insertion of one amino acid residue between positions 3 and 4.

28. Polypeptide variant on p or 27, where between positions 3 and 4 inserted hydrophobic amino acid residue.

29. Polypeptide variant on p where called insert is A3AY.

30. Polypeptide variant according to clause 29, which includes the insertion of A3AY and substitutions P10Q+K32E+A34E+R36E.

31. Polypeptide variant according to clause 29, which includes the insertion of A3AY and substitutions P10Q+K32E+A34L+R36E.

32. Polypeptide variant according to clause 29, which includes the insertion of A3AY and replacement 10Q+K32E+A34E+R36E+T106N+I205T.

33. Polypeptide variant according to clause 29, which includes the insertion of A3AY and replacement 10Q+K32E+A34E+R36E+T106N+V253N.

34. Polypeptide variant according to clause 29, which includes the insertion of A3AY and substitutions P10Q+K32E+A34E+R36E+I205T+V253N.

35. Polypeptide variant according to clause 29, which includes the insertion of A3AY and substitutions P10Q+K32E+A34L+R36E+T106N+I205T.

36. Polypeptide variant according to clause 29, which includes the insertion of A3AY and substitutions P10Q+K32E+A34L+R36E+T106N+V253N.

37. Polypeptide variant on the .29, including the insertion of A3AY and substitutions P10Q+K32E+A34L+R36E+I205T+V253N.

38. Polypeptide variant according to any one of the preceding paragraphs, where the named option is in its activated form.

39. The composition of the blood-clotting comprising the polypeptide variant according to any one of claims 1 to 38 and at least one pharmaceutically acceptable carrier or excipient.

40. A method of treating mammals having a disease or disorder where it is desirable formation of a blood clot, comprising introducing to a mammal in need, an effective amount of the polypeptide variant according to any one of claims 1 to 38, or a composition according to § 39.

41. The method according to p, where the named disease or disorder selected from the group consisting of hemorrhage, including bleeding in the brain, severe uncontrolled bleeding, such as trauma, hemorrhage in patients after transplantation or resection, bleeding from varicose veins and hemophilia.

42. The method according to paragraph 41, where the named disease or disorder is trauma.

43. The method according to paragraph 41, where the named disease or disorder is hemophilia.



 

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