Derivatives of factor of vii blood coagulation

FIELD: medicine, biotechnologies.

SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.

EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.

20 cl, 2 dwg, 8 ex

 

The SCOPE of the INVENTION

The present invention relates to new derivatives of human factor VII clotting, factor VII polypeptides and polynucleotide constructs encoding such polypeptides, vectors and host cells containing and expressing these polynucleotide, pharmaceutical compositions containing the derivatives of factor VII, applications and treatments.

The BASIS for the creation of INVENTIONS

Blood clotting is a process consisting of a complex interaction of different components (or factors) of blood, which ultimately contribute to the formation of a fibrin clot. Usually components of blood that are involved in what is called "cascade coagulation, are enzymatically inactive proteins (proenzymes or Imogene), which in turn proteolytic enzymes under the action of the activator (which, in turn, is a clotting factor). Coagulation factors that have undergone such a transformation, usually referred to as "active factors", and to designate them usually add the letter "a" to the name of coagulation factor (e.g., factor VIIa).

Initiation of the hemostatic process is mediated by formation of a complex between tissue factor exposed in the da is of the vascular wall, and factor VIIa. Then under the action of this complex is the transformation of factors IX and X in their active forms. Factor Xa convert limited amounts of prothrombin to thrombin on cells bearing tissue factor. Thrombin activates platelets and factors V and VIII, turning them into factors Va and VIIIa, both of which are cofactors in the further process, leading to the full thrombin burst. This process includes the production of factor Xa under the action of factor IXa (in complex with factor VIIIa) and it occurs on the surface of activated platelets. In conclusion, thrombin contributes to the transformation of fibrinogen in fibrin, which leads to the formation of a fibrin clot.

Factor VII is a trace plasma glycoprotein that circulates in the blood in the form of single-stranded Imogene. This zymogen is catalytically inactive. Single-chain factor VII can be converted into double-stranded factor VIIa under the action of factor Xa, factor XIIa, factor IXa, factor VIIa or thrombin in vitro. Factor Xa, apparently, is the major physiological activator of factor VII. Turning imagelogo factor VII activated double-stranded molecule occurs by cleavage of an internal peptide bond Arg152-Ile153.

It is often necessary to selectively stimulate or block the sub is known cascade of blood coagulation. Factor VIIa is used for the control of disorders associated with bleeding that is caused by a number of reasons, such as deficiencies of coagulation factors (e.g. haemophilia A and B or deficiency of coagulation factors XI or VII) or inhibitors of coagulation factors. Factor VIIa is also used to control excessive bleeding occurring in subjects with a normally functioning of the coagulation cascade (no deficiencies of coagulation factors, inhibitors or any of the coagulation factors). Such bleeding may be caused by a defect in the functioning of platelets, thrombocytopenia, or disease von Willebrand's disease. Bleeding is a serious problem in connection with surgical interventions and other forms of tissue damage.

European patent No. 200,421 (ZymoGenetics) is associated with a nucleotide sequence encoding the human factor VII, and recombinant expression of factor VII in mammalian cells.

Publication of Dickinson et al. (Proc. Natl. Acad. Sci. USA 93,14379-14384,1996) associated with factor VII polypeptides in which Lys157, Val158, Glu296, Met298, Asp334, Ser336 or Lys337 individually replaced by Ala. Publication Iwanaga et al. (Thromb. Haemost (supplement august 1999), 466, abstract 1474) is associated with variants of factor VIIa, in which there is a deletion of residues 316-320 or in which the remains of 311-322 replacing the s corresponding residues from trypsin.

Anticoagulants, such as heparin, coumarin, coumarin derivatives, derivatives indandion or other agents can be used for the selective blocking of the coagulation cascade in a patient, for example, during renal dialysis, or in the treatment of deep vein thrombosis, disseminated intravascular coagulation (DIC), as well as other medical disorders. For example, treatment with heparin or in vitro processing of citrate ion (U.S. Patent No. 4,500,309) can be used during dialysis to prevent coagulation during treatment. Heparin is also used to prevent deep vein thrombosis in patients undergoing surgery.

However, treatment with heparin and other anticoagulants may cause unwanted side effects. Available anticoagulants usually manifest their effect throughout the body, and not specifically at the site of formation of a clot. Heparin, for example, can cause heavy bleeding. In addition, having a half-life of about 80 minutes, heparin is rapidly eliminated from the blood, which causes the need for frequent injections. Because heparin acts as a cofactor for anti-thrombin III (AT III), and AT III is quickly exhausted in the treatment of DIC is often difficult to ascertain the correct dose of heparin required for continuous is onitoring AT levels III and heparin. Heparin is ineffective in extreme exhaustion AT III. In addition, prolonged use of heparin may also increase platelet aggregation and reduce the number of platelets, and is also involved in the development of osteoporosis. Derivatives indandion can also cause toxic side effects.

In addition to anticoagulants, briefly described above, it was found that a number of natural proteins formed possesses anticoagulant activity. For Example, Reutelingsperger (U. S. Pat. No. 4,736,018) identified anticoagulant proteins from the aorta of a bull and the umbilical arteries of man. In the publication Maki et al. (U. S. Pat. No. 4,732,891) described anticoagulant proteins derived from human placenta. In addition, AT III has been proposed as a therapeutic anticoagulant (Schipper et al., Lancet 1 (8069): 854-856 (1978); Jordan, U. S. Pat. No. 4,386,025; Bock et al., U. S. Pat. No. 4,517,294).

Approximately 30% or more of patients undergoing angioplasty, endarterectomy or bypass vascular shunt thrombosis and/or proliferation of smooth muscle cells in an intim cause re-occlusion of the vessel and, as a consequence, the impossibility of reconstructive surgery. Such closure of the vessel due to operation known as restenosis. The origin of the phenomenon of restenosis associated with a complex interaction of biological processes, including the deposition of platelets and trombone the Finance, the release of chemotactic and mitogenic factors, as well as migration and proliferation of smooth muscle cells of blood vessels in an intim advanced arterial segment.

Inhibition of the accumulation of platelets at sites of mechanical damage may limit the rate of restenosis in humans. While in sites of acute vascular damage is the accumulation of platelets, thrombin generation in these sites may be responsible for the activation of platelets and their subsequent accumulation.

International application No. WO 92/15686 associated with inaktivirovannye factor VIIa, poliolefinovoy acid and lines of mammalian cells, producing an inactivated factor VIIa, as well as compositions containing an inactivated factor VIIa, for inhibition of blood coagulation.

International application No. WO 94/27631 relates to methods of inhibiting restenosis of blood vessels, activity of tissue factor and deposition of platelets.

International application No. WO 96/12800 associated with treatment of acute closure of a coronary artery, comprising the administration to a patient of a composition, which contains inactivated factor VIIa in combination with tissue plasminogen activator or streptokinase.

The majority of proteins entering the circulation, from mammals rapidly excreted by the kidneys. is that the problem can be partially circumvented by the introduction of large quantities of the protein or by re-introductions. However, higher doses of protein can cause the formation of antibodies, which can bind and inactivate the protein and/or to facilitate the clearance of protein from the body of the patient. Re-introduction of the protein for therapeutic purposes largely ineffective and can be dangerous, as it can cause an allergic reaction.

Various attempts to solve the problems associated with protein therapy include microencapsulation, liposomal delivery systems, the introduction of fused proteins and chemical modification. The most promising of them today is a modification of therapeutic protein by the covalent attachment of polymers of polyalkylene, in particular, polyethylene glycol (PEG). For example, in U.S. patent No. 4,179,337 described the use of PEG or propylene glycol-associated protein, to provide a physiologically active non-immunogenic water soluble polypeptide composition. At Nucci et al. describes several proteins that were modified by addition of PEG, including adenosylmethionine, L-asparaginase, interferon-alpha-2b (IFN-α2b), superoxide dismutase, streptokinase, tissue plasminogen activator (tPA), urokinase, uricase, hemoglobin, interleukins, interferons, TGF-beta, EGF and other growth factors (Nucci et al., 1991, Adv. Drug Delivery Rev. 4: 133-151). The result of such attempts was necrorealist time half-life of these proteins and decrease the immunogenicity of these proteins.

Usually Pegylation of proteins involves activating the PEG functional groups that will react with lysine residues on the surface of the protein. If the protein is completely modified, the protein activity is usually lost. Modification procedures, which allow partial Paglierani protein, usually lead to loss of activity only to approximately 50% and a significant increase in the time of its half-life in serum, so that the total effective dose of the protein decreases.

Modern development methods Paglierani proteins associated with the use of activated PEG reagents that interact with thiol groups of proteins that results in covalent binding of PEG to cysteine, which is built instead of natural formed with lysine. In Shaw et al. (U.S. patent No. 5,166,322) described specific variants of IL-3, which contain the cysteine embedded in specific sites within the nature of the resulting amino acid sequence. Then sulfhydryl reactive compounds (e.g., activated polyethylene glycol) are attached to the specified zisteinom in the reaction with a specific variant of IL-3. At Katre et al. (U.S. patent No. 5,206,344) described specific variants of IL-2, which contain the cysteine embedded in specific sites within the natural is Brazauskas amino acid sequence. In further specific variant of IL-2 reacts with activated polietilenglikoli reagent, attaching this piece to the cysteine.

In the art there is still a need in the superior factor VII polypeptides having prolonged procoagulant or anticoagulant activity. In particular, there is a need to factor VII polypeptides, which would have increased the half-life in serum without any unwanted side effects, such as systemic activation of the coagulation system and bleeding, respectively, associated with traditional therapy, and which could be introduced at relatively low doses, in order to avoid the introduction of more massive doses of protein.

Description of the INVENTION

The present invention relates to new polypeptides of factor VII clotting, having the same or increased activity compared with factor VIIa wild-type and derivative of factor VII, which has increased the half-life in serum.

Were identified region in the molecule of factor VIIa, which can be produced by changes in the primary structure, as well as other modifications which do not cause changes or reduce the biological activity of factor VIIa. The area inside the structure factor VIIa, which were identified as NEWCAST is either in binding to tissue factor or factor X, including amino acids at positions 247-260, and from 393 to 406 of the provisions in the sequence SEQ ID NO: 1. In particular, the amino acids in positions Q250, R396 and P406 sequence SEQ ID NO: 1 were studied from the point of view of the inclusion of cysteine (Cys). The inclusion of cysteine residues was accompanied by conjugation with a chemical group such as polyethylene glycol (PEG), in order to increase the half-life derived factor VII in circulation. Cysteine was introduced at the C-terminal sequence of SEQ ID NO: 1 (referred to as 407C), followed by conjugation with polyethylene glycol. Furthermore, the addition of cysteine in the C-terminal sequence of SEQ ID NO: 1 is not accompanied by a decrease in the proteolytic activity of the polypeptides of factor VIIa. These derivatives of factor VII, such as factor VII polypeptide conjugated with a PEG molecule, are therapeutically applicable in situations where it is desirable prolonged action of factor VII polypeptides, for example, in situations where re-introduction or the introduction of a larger amount of factor VII polypeptide is undesirable or problematic. In addition, the polypeptides of factor VIIa according to the invention with built-amino acids (e.g. cysteine residue)that can konjugierte with a chemical group in the provisions of the molecules of factor VIIa, which do not affect proteo eticheskuyu activity can be used to embed any functional group conjugate of factor VII.

In the first aspect, the present invention relates to a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or its variants, where the amino acid substituted by another amino acid, where another amino acid capable of conjugality with a chemical group and where the factor VII polypeptide essentially has the same or higher activity compared with the activity of recombinant human factor VIIa wild-type.

In the second aspect, the present invention relates to a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or its variants, where the amino acid corresponding to amino acid position selected from 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid. It should be understood that any amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1 may be replaced by another amino acid without a significant reduction of the activity of the factor VII polypeptide.

In the third aspect, the present invention relates to a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or its variants, where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, the replacement of the Jena another amino acid. It should be understood that the first letter in the notation R396, Q250 and P406 is an amino acid found at the specified position in the natural variant sequence of SEQ ID NO: 1.

In another aspect, the invention relates to a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is capable of conjugation with a chemical group that is built inside a sequence of SEQ ID NO: 1 or its variants in the position where the factor VII polypeptide has essentially the same or higher activity compared with the activity of recombinant human factor VIIa wild type. It is understood that the amino acid may be incorporated into the sequence of SEQ ID NO: 1 and without replacement of any amino acids. Insert amino acids can be carried out in the same position within the sequence of SEQ ID NO: 1, where in the future will be made of amino acid replacement. Thus, in one embodiments, the insert will follow amino acid replacement, or Vice versa.

In another aspect, the invention relates to a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is capable of conjugation with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants.

The term "amino acid", ispolzuemyi in the present description, means one or more amino acids. It is understood that the amino acid that replaces the amino acid in the polypeptide factor VII or integrated in this polypeptide, or added to the factor VII polypeptide capable of conjugation with any chemical group that will enhance the true molecular weight of the factor VII polypeptide. Such conjugation with a chemical group includes, but is not limited to covalent attachment of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly(N-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, a copolymer of polypropyleneoxide/ethylene oxide, polypropylenglycol, polyoxyethylene polyols (e.g. glycerol) and polyvinyl alcohol, kolominova acids or other polymers based on carbohydrates, amino acid polymers and bilinovich derivatives.

Preferred chemical group is biocompatible, non-toxic, non-immunogenic, water soluble polymer. Preferably the chemical group is any water-soluble polymer in any proportions.

This amino acid substitution, insertion or additive and conjugation with a chemical group occurs without substantial reduction of the procoagulant activity of the activated form of the derived factor VII compared with the activity, it is recommended bimantoro human factor VIIa wild-type.

Used herein, the term "factor VII polypeptide" refers to any protein containing the amino acid sequence 1-406 of native human factor VII (SEQ ID NO: 1) or its variants. This includes, without limitation, human factor VII, human factor VIIa and its options.

Used herein, the term "factor VII"or "FVII"means a product consisting of a non-activated form (factor VII). Used herein, the term "factor VIIa"or "FVIIa"means a product consisting of activated form (factor VIIa). This includes proteins that have the amino acid sequence 1-406 of native human factor VII or factor Factor VIIa. Also included are and proteins with a slightly modified amino acid sequence, for example, a modified N-end including N-terminal amino acid deletions or additives, such that the resulting proteins substantially retain the activity of factor VIIa. Under the above definitions, the term "factor VII or factor VIIa" also includes natural allelic variations that may exist and occur from one or another of the individual. Can vary the degree and localization of glycosylation or other posttranslational modifications, depending on what the selected host cell, and the cell environment and the possible host cells.

Under used herein, the terms "variant" or " variants" refers to human factor VII having the sequence of SEQ ID NO: 1, where one or more amino acids of the parent protein has been replaced with another amino acid and/or where one or more amino acids of the parent protein subjected to deletions and/or where one or more amino acids inserted in the parent protein and/or one or more amino acids added to the parent protein. This Supplement can take place either at the N-end or at the C-end of the parent protein, or on both of them.

The term "essentially the same or higher activity compared with the activity of recombinant human factor VIIa wild-type" refers to the activity that constitutes more than 70% of the activity of recombinant human factor VIIa wild type. In one embodiment, the activity is more than 80% of the activity of recombinant human factor VIIa wild type. In another embodiment, the activity is more than 90% of the activity of recombinant human factor VIIa wild type. In another embodiment, the activity is more than 100% of the activity of recombinant human factor VIIa wild type. In another embodiment, this activity is more than 120% of the activity of recombinant human the economic factor VIIa wild type. In another embodiment, this activity is more than 200% of the activity of recombinant human factor VIIa wild type. In another embodiment, this activity is more than 400% of the activity of recombinant human factor VIIa wild-type.

The term "derived factor VII" here refers to the factor VII polypeptide having the sequence of SEQ ID NO: 1 or its variant, where one or more amino acids of the parent protein is chemically modified, e.g. by alkylation, Paglierani, acylation, the formation of ester or education amide, etc. This includes, but is not limited to, Targeted human factor VIIa, cysteine-Targeted human factor VIIa and its options.

The term "Targeted human factor VIIa" means the factor VIIa with a molecule of polyethylene glycol, conjugate with amino acid polypeptide is human factor VIIa.

The term "cysteine-Targeted human factor VIIa" means the factor VIIa with a molecule of polyethylene glycol, conjugate with the sulfhydryl group of the cysteine embedded in the human factor VIIa.

The term "another amino acid" refers to one or more amino acids that differs from the amino acid, which is found in nature on this is ogenyi. This includes, without limitation, amino acids that can be encoded by polynucleotides. Preferably another amino acid is a natural L-form and can be encoded by polynucleotides. A specific example is L-cysteine (Cys).

The term "activity" refers to the ability of the factor VII polypeptide to convert its substrate, factor X, factor Xa. The activity of the factor VII polypeptide can be measured by analysis of proteolysis in Vitro (see Example 6).

The term "polyethylene glycol"or "PEG"means the connection of polyethylene glycol or its derivative, in the presence or in the absence of binding agents that bind or activate fragments (for example, in the presence of thiol, triflate, cresylate, asinine, oxirane or preferably in the presence of maleimide fragment). Compounds such as maleimide-monomethoxy-PEG, are examples of active compounds of polyethylene glycol according to the invention.

In another aspect, the present invention associated with the derived factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or its variants, where the amino acid substituted by another amino acid, where the specified another amino acid conjugated with a chemical group that increases the IP is different molecular weight of the factor VII polypeptide with 300 daltons to approximately 100000 daltons, and where derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

The term "chemical group" refers to one or more chemical groups.

In another aspect, the invention relates to a derivative of factor VII containing the factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or its variants, where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide with 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a derivative of factor VII containing the factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where another amino acid conjugated to khimicheskoi group, which increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a derivative of factor VII containing the factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is embedded in the sequence of SEQ ID NO: 1 or a variant where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a derivative of factor VII containing the factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is added to the N - or C-Termini of the sequence SEQ ID NO: 1 or a variant where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 Gave the tone, and where derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons, about 100,000 daltons, and where the derived factor VI has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons, about 100,000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is inserted into the sequence of SEQ ID NO: 1 or its variant, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention is Asano with composition containing the derived factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is added to the N - or C-Termini of the sequence SEQ ID NO: 1 or a variant where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a pharmaceutical composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition comprising about what svodnoe factor VII, containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where a different amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinantly the human factor VIIa wild type; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is inserted into the sequence of SEQ ID NO: 1 or its variant, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type; and, optionally, pharmaceutically acceptable media.

In another aspect, the invention relates to a pharmaceutical composition comprising a derivative of factor VII containing the factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared the Oia with recombinant human factor VIIa wild type; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to polypeptide inactivated factor VII, where the factor VII polypeptide is additionally modified in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX. In one embodiment, the polypeptide inactivated factor VII modified in its catalytic center of the inhibitor of serine proteases. In another embodiment, the polypeptide inactivated factor VII modified in its catalytic center valomaciclovir peptide selected from the group consisting of Phe-Phe-Arg-chloromethylketone, Phe-Phe-Arg-chloromethylketone, D-Phe-Phe-Arg-chloromethylketone, D-Phe-Phe-Arg-chloromethylketone, Phe-Pro-Arg-chloromethylketone, D-Phe-Pro-Arg-chloromethylketone, Phe-Pro-Arg-chloromethylketone, D-Phe-Pro-Arg - chloromethylketone, L-Glu-Gly-Arg-chloromethylketone and D-Glu-Gly-Arg - chloromethylketone, Dansyl-Phe-Phe-Arg-chloromethylketone, Dansyl-Phe-Phe-Arg-chloromethylketone, Dansyl-D-Phe-Phe-Arg-chloromethylketone, Dansyl-D-Phe-Phe-Arg-chloromethylketone, Dansyl-Phe-Pro-Arg-chloromethylketone, Dansyl-D-Phe-Pro-Arg-chloromethylketone, Dansyl-Phe-Pro-Arg-chloromethylketone, Dansyl-D-Phe-Pro-Arg-chloromethylketone, Dansyl-L-Glu-Gly-Arg-chloromethylketone and Dansyl-D-Glu-Gly-Arg-chloromethylketone.

The term "polypeptide inactivated factor VII", as used herein, means a polypeptide factor VII, no way is first to activate plasma factor X or IX.

In another aspect, the invention associated with the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a version thereof and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the invention associated with the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where this other amino acid can be conjugated with a chemical group and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and has a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true the molecular weight of iactiveaware what about the factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the invention associated with the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 Daltons.

In another aspect, the invention associated with the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactive the bathing of the factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the invention associated with the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid that is capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and has a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the invention associated with the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid that is capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide AK is to wirawati plasma factor X or IX, additionally anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a version thereof and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and has modificat the Yu in its catalytic center, the modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 Daltons to approximately 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino the slot in position, selected from the provisions R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid that is capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or a variant of this sequence at the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 Dalfopristin to 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid that is capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or variants of this sequence, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons.

In another aspect, the present invention relates to a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a version thereof and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately do daltons; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group, which increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to the amino acid in position, select nom of the provisions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately up to 100000 daltons; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the present invention is related is about with the pharmaceutical composition, contains the derived inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid that is capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or a variant of this sequence at the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type, and having a modification in its catalytic center, which modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid that is capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or variants of this sequence and having a modification in its catalytic center, with this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of inactivated factor VII polypeptide 300 daltons to approximately 100000 daltons; and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a polynucleotide construct that encodes the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the present invention relates to a polynucleotide construct that encodes the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the present invention relates to a polynucleotide construct that encodes a polypeptide factor VII containing s is nokiatool sequence of SEQ ID NO: 1 or its variant, where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the invention relates to a polynucleotide construct that encodes the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a polynucleotide construct that encodes the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its version.

In one aspect the polynucleotide construct is a vector.

The term "polynucleotide" means a single - or double-stranded polymer of deoxyribonucleotide or ribonucleotidic bases read from the 5'-end to 3'-end. Polynucleotide include RNA and DNA can be isolated from natural sources, synthesized in vitro or obtained from the combined the AI natural and artificial molecules. The length of the polynucleotide molecule is given here in nucleotides (indicated by "nt") or in pairs nucleotides (marked "BP"). The term "nucleotide" is used here in cases where the context so permits, to indicate single or double-stranded molecules. When this term is applied to a double-stranded molecules, it is used to denote the full length, and it will be assumed that it is equivalent to the term "nucleotides". Specialists in this field will be obvious that the two strands of double-stranded polynucleotide may differ slightly in length and that its ends can be removed as a result of enzymatic cleavage; thus, not all nucleotides within a double-stranded polynucleotide molecule to be coupled. Typically, these unpaired ends in the length of no more than 20 nucleotides.

The term "vector"as used in the present description, means essentially any nucleic acid capable of amplification in the host cell. Thus, the vector may be a stand-alone can replicate vector, i.e. a vector which exists as an extrachromosomal education, replication which does not depend on the replication of the chromosome, such as a plasmid. Alternatively, the vector may be such that he, being introduced in the host cell, is integrated into the host genome and replicated together with chromium the wash (chromosomes), in which it is integrated. The choice of vector will often depend on the host cell into which it is going to be introduced. Vectors include, but are not limited to this plasmid vectors, phage vectors, viral or cosignee vectors. Typically, the vectors contain an origin of replication and at least one breeding gene, i.e. a gene that encodes a product that can easily be determined or whose presence is essential for cell growth.

In a further aspect, the present invention relates to eukaryotic cell host containing a polynucleotide construct containing the sequence encoding the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where the specified another amino acid capable of conjugality with a chemical group and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In a further aspect, the present invention relates to eukaryotic cell host containing a polynucleotide construct containing the sequence encoding the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or its variant, where AMI is ocelot, corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In a further aspect, the present invention relates to eukaryotic cell host containing a polynucleotide construct containing the sequence encoding the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In a further aspect, the present invention relates to eukaryotic cell host containing a polynucleotide construct containing the sequence encoding the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted within the sequence of SEQ ID NO: 1 or its variants in the position where polynucleotide factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In a further aspect, the present invention relates to eukaryotic cell host containing a polynucleotide construct containing a sequence encoding polypep the d factor VII, containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants.

Here, the term "eukaryotic cell-host" refers to any cell, including hybrid cells, which can be expressed heterologous DNA. Usually the host cell include, but are not limited to, insect cells, yeast cells, mammalian cells, including human cells, such as BHK cells, CHO, HEK and COS. Actually cultured host cells according to the present invention are preferably mammalian cells, more preferably - established lines of mammalian cells, including, but not limited to, cell line CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), kidney (newborn) hamster (BHK) and HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72,1977).

The preferred cell line BHK is a cell line tk-ts13 BHK (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110,1982), hereinafter in this description referred to as cells, BHK 570. The cell line BHK 570 may be obtained from American cell culture collection, 12301 Parklawn Dr., Rockville, MD 20852, under the registration number ATCC CRL 10314. Cell line tk-ts13 BHK also available in the ATCC under registration number CRL 1632.

Others the many suitable cell lines include, not limited to, Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), the human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc. Natal. Acad. Sci. USA 77: 4216-4220,1980). Suitable are also 3T3 cells, Namalwa cells, myeloma cells, and the fusion of myeloma cells with other cells. In one of the embodiments, the eukaryotic host cell derived from a mammal. In another embodiment, the eukaryotic host cell is selected from the group consisting of CHO cells, BHK cells or HEK cells.

In another aspect, the invention relates to transgenic animals expressing a polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to transgenic animals expressing a polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid is position, selected from the provisions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the invention relates to transgenic animals expressing a polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the invention relates to transgenic animals expressing a polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to transgenic animals expressing a polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-is NCO sequence SEQ ID NO: 1 or its version.

In another aspect, the invention relates to a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the invention relates to a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, this from retina associated with a transgenic plant, expressing the polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its version.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing in an appropriate nutrient medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where another amino acid capable of konjugierte chemical group is Oh, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type under conditions where protein synthesis on the basis of the polynucleotide constructs, and obtaining the factor VII polypeptide from the culture medium.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing in an appropriate nutrient medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, under conditions where protein synthesis on the basis of the polynucleotide constructs, and obtaining the factor VII polypeptide from the culture medium.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing in an appropriate nutrient medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing linakis is now the sequence of SEQ ID NO: 1 or its variant, where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, under conditions where protein synthesis on the basis of the polynucleotide constructs, and obtaining the factor VII polypeptide from the culture medium.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing in an appropriate nutrient medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, under conditions in where possible the synthesis of protein-based polynucleotide constructs, and obtaining the factor VII polypeptide from the culture medium.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing in an appropriate nutrient medium eukaryotic hoteisconrad, containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variant, under conditions where protein synthesis on the basis of the polynucleotide constructs, and obtaining the factor VII polypeptide from the culture medium.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes recovering the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes recovering the factor VII polypeptide from milk produced by transgenic animals, e is stresseraser polynucleotide construct, encoding the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes recovering the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes recovering the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide of lady essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type, in conditions where protein synthesis on the basis of the polynucleotide constructs, and obtaining the factor VII polypeptide from the culture medium.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes recovering the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its version.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and factor VII polypeptide and the transgenic plants.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and recovering the factor VII polypeptide from the transgenic plants.

In another aspect, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and recovering the factor VII polypeptide from the transgenic plants.

In another embodiment, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing cells of a transgenic plant expressing the polynucleotide design, the function, encoding the factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, and the restoration of the factor VII polypeptide from the transgenic plants.

In another embodiment, the invention relates to a method of producing the factor VII polypeptide, where the method includes culturing cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variant, and the restoration of the factor VII polypeptide from the transgenic plants.

In another embodiment, the invention relates to a method of producing a derivative of factor VII, which includes the following stages:

a) producing the factor VII polypeptide;

b) conjugation of the factor VII polypeptide with a chemical group;

C) applying the derived factor VII on casinoonlinecasinogaming or gel-filtration column; and

g) elution derived factor VII.

In another embodiment, the invention relates to a method of producing derived inactivated factor VII, which includes the following stages:

a) producing the factor VII polypeptide;

b) modification of the factor VII polypeptide in its catalytic center of the inhibitor of serine proteases;

C) conjugation of inactivated factor VII polypeptide with a chemical group;

d) applying the derived inactivated factor VII on cation-exchange chromatography or gel filtration column; and

d) elution derived inactivated factor VII.

In one embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing in the appropriate growth medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, under conditions in which the possible synthesis of b is the LCA-based polynucleotide constructs, and recovering the factor VII polypeptide from the culture medium.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing in the appropriate growth medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, under conditions where protein synthesis on the basis of the polynucleotide constructs, and the restoration of the factor VII polypeptide from the culture medium.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing in the appropriate growth medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, under conditions where protein synthesis on the basis of the polynucleotide constructs, and recovering the polypeptide fact the RA VII of the culture medium.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing in the appropriate growth medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, under conditions where protein synthesis on the basis of the polynucleotide constructs, and the restoration of the factor VII polypeptide from the culture medium.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing in the appropriate growth medium eukaryotic host cell containing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-end of sequence ID NO: 1 or its variant, under conditions where protein synthesis OS is ove polynucleotide constructs, and recovering the factor VII polypeptide from the culture medium.

In another embodiment according to the invention, the method for producing the factor VII polypeptide includes the restoration of the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another embodiment according to the invention, the method for producing the factor VII polypeptide includes the restoration of the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another embodiment according to the invention, the method for producing the factor VII polypeptide is engages in the recovery of the factor VII polypeptide from milk, produced transgenic animals expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another embodiment according to the invention, the method for producing the factor VII polypeptide includes the restoration of the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another embodiment according to the invention, the method for producing the factor VII polypeptide includes the restoration of the factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or e is an option where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its version.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing the cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, and the restoration of the factor VII polypeptide from the transgenic plants.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing the cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and recovering the polypeptide VII of transgenic plants.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing the cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and recovering the factor VII polypeptide from the transgenic plants.

In another embodiment according to the invention, the method for producing the factor VII polypeptide comprises culturing the cells of a transgenic plant expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, and the restoration of the factor VII polypeptide from the transgenic plants.

In another embodiment according to the invention, the method for producing the factor VII polypeptide includes cultivation is their cells, transgenic plants, expressing the polynucleotide construct encoding a factor VII polypeptide containing the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variant, and the restoration of the factor VII polypeptide from the transgenic plants.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where this other amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

Under used here, the term "treatment" refers to introduction of an effective amount of therapeutically active compounds according to the invention to prevent any symptoms of the disease or prevent the Oia development of the disease, or order of supervision or relief of such symptoms or already developed painful conditions. Thus, the term "treatment" also includes prophylactic treatment.

The term "amplification of normal hemostasis system" refers to increasing the ability to generate thrombin.

Used here, the term "disorders associated with bleeding reflects any defect, congenital, acquired, or induced, cellular or molecular nature, which manifests itself in the form of bleeding. Examples include deficiencies of coagulation factors (e.g., hemophilia A and B or deficiency of coagulation factors XI or VII), inhibitors of coagulation factors, defective platelet function, thrombocytopenia, or disease von Willebrand's disease.

It is implied that the term "bleeding episodes includes uncontrolled and excessive bleeding, which represents the main problem in connection with surgical stress, and other forms of tissue damage. Uncontrolled and excessive bleeding may occur in subjects with normal coagulation system, as well as in subjects with disorders associated with blood clotting and bleeding. Deficiencies of coagulation factors (e.g., hemophilia A and B or deficiency of coagulation factors XI or VII),inhibitors of coagulation factors can be the cause of the violations, associated with bleeding. Excessive bleeding can also occur in subjects with normal coagulation system, as well as in subjects with a normally functioning cascade of blood clotting (no deficiencies of coagulation factors or inhibitors, active against any of the clotting factors), and can be caused by a defective platelet function, thrombocytopenia, or disease von Willebrand's disease. In such cases, the bleeding may be similar to those bleeding caused by hemophilia, because the hemostatic system, as in the case of hemophilia, is deprived of the necessary clotting "connections" or has abnormal coagulation "connections" (such as platelets or protein factor von Willebrand's disease), which is a cause of major bleeding. The subjects who survived extensive tissue damage associated with surgical exposure or extensive trauma, the normal haemostatic mechanism can be suppressed by the body's need for immediate hamostaseologie, and in response to it, despite the normal mechanism geostationary may develop bleeding. Achieving satisfactory geostationary is also a problem in cases where bleeding in organs such as the brain, the eyes and the area of the inner ear where geostationary surgically limited. The same problem may occur in the selection process of the biopsy material from the different organs (liver, lung, tumor tissue, gastrointestinal tract), as well as in laparoscopic surgery. The common point in all these situations is the difficulty associated with providing hemostasis using surgical techniques (sutures, clamps etc)that also applies to cases where there are diffuse bleeding (hemorrhagic gastritis and profuse uterine bleeding). Acute and profuse bleeding may also occur in subjects with anticoagulation therapy, in which a defective hemostasis induced by this therapy. Such entities may require surgical intervention in cases when there is need to quickly suppress anticoagulant effect. Radical Sagalova prostatectomy is usually undertaken by the procedure in subjects with localized prostate cancer. This surgery is often complicated by the significant, and sometimes massive blood loss. Significant blood loss during prostatectomy are mainly associated with complicated anatomical situation, with different gustavsborgsvagen areas that do not easily lend themselves hemostatic manipulation during surgery and which can Ave the lead to diffuse bleeding from a vast area. Another situation that can cause problems in case of unsatisfactory hemostasis, is when subjects with normal haemostatic mechanism undergo anticoagulation therapy to prevent thromboembolic disease. Such therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, as well as aspirin and other platelet aggregation inhibitors.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where this other amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where this other amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity comparison with recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is inserted into the sequence of SEQ ID NO: 1 or its variant, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII region which gives essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variant, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In one of the embodiments according to the invention, the bleeding associated with hemophilia A or B. In another embodiment, the bleeding is associated with acquired hemophilia with inhibitors. In another embodiment, the bleeding associated with thrombocytopenia. In another embodiment, the bleeding is associated with disease von Willebrand's disease. In another embodiment, the bleeding associated with severe tissue damage. In the other embodiment, bleeding associated with severe trauma. In another embodiment, the bleeding associated with surgery. In another embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is associated with profuse uterine bleeding. In another embodiment, the bleeding is associated with the organs with disabilities for mechanical hemostasis. In another embodiment, the bleeding occurs in the brain, in the area of the inner ear and the eyes. In another embodiment, the bleeding associated with the process of taking a biopsy. In another embodiment, the bleeding associated with anticoagulation therapy.

Under used herein the term "subject" refers to any animal, particularly mammals, and this term, if necessary, can be used along with the term "patient".

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where this other amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide sample is about 300 daltons to approximately 100000 daltons, and where derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where this other amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared with recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid series is here SEQ ID NO: 1 or its variant, where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where this other amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is inserted into the sequence of SEQ ID NO: 1 or its variant, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild type, to obtain medicines for the treatment of episodic blood is echini or to enhance normal hemostasis system.

In another aspect, this invention is related to the application of the derived factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is added to the N - or C-Termini of the sequence SEQ ID NO: 1 or a variant where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type to obtain drugs for the treatment of episodic bleeding or to enhance normal hemostasis system.

In another aspect, the present invention relates to a method of treatment of a subject episodic bleeding or associated with bleeding disorders or with reinforcing normal hemostasis system, where the method includes introduction to the subject, if necessary, a therapeutically or prophylactically effective amount of a derivative of factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where this other s who nakikita conjugated with a chemical group, which increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the present invention relates to a method of treatment of a subject episodic bleeding or associated with bleeding disorders or with reinforcing normal hemostasis system, where the method includes introduction to the subject, if necessary, a therapeutically or prophylactically effective amount of a derivative of factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where is this another amino acid conjugated to a with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another and the too, the present invention relates to a method of treatment of a subject episodic bleeding or associated with bleeding disorders or with reinforcing normal hemostasis system, where the method includes introduction to the subject, if necessary, a therapeutically or prophylactically effective amount of a derivative of factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, where this other amino acid is conjugated with a chemical group that increases the true the molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the present invention relates to a method of treatment of a subject episodic bleeding or associated with bleeding disorders or with reinforcing normal hemostasis system, where the method includes introduction to the subject, if necessary, a therapeutically or prophylactically effective amount p is vizvolnogo factor VII, comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is inserted into the sequence of SEQ ID NO: 1 or its variant, and where the amino acid is conjugated with a chemical group that increases the true molecular weight of the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another aspect, the present invention relates to a method of treatment of a subject episodic bleeding or associated with bleeding disorders or with reinforcing normal hemostasis system, where the method includes introduction to the subject, if necessary, a therapeutically or prophylactically effective amount of a derivative of factor VII comprising a factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid is added to the N - or C-Termini of the sequence SEQ ID NO: 1 or a variant where the amino acid is conjugated with a chemical group that increases the true molecular weight the factor VII polypeptide is about 300 daltons to approximately 100000 daltons, and where the derived factor VII about lady essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In a further aspect, the present invention involves the use of derived inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a version thereof and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons to obtain a medicine for inhibiting the patient's blood clots.

In a further aspect, the present invention involves the use of derived inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group, and where the factor VII polypeptide having essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and having a modification in its catalytic center, and this modification inhib the imagers and the ability of the factor VII polypeptide to activate plasma factor X or IX, additionally anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons to obtain a medicine for inhibiting the patient's blood clots.

In a further aspect, the present invention involves the use of derived inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons to obtain a medicine for inhibiting the patient's blood clots.

In a further aspect, the present invention involves the use of derived inactivated factor VII, where the polypeptide inactivated factor VII, including the impact of amino acid sequence SEQ ID NO: 1 or its variant, where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons to obtain a medicine for inhibiting the patient's blood clots.

In a further aspect, the present invention involves the use of derived inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has the same activity or increased activity compared to recombinant human factor VIIa wild-type, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, additionally conyuge the IAOD with a chemical group, which increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons to obtain a medicine for inhibiting the patient's blood clots.

In a further aspect, the present invention involves the use of derived inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons to obtain a medicine for inhibiting the patient's blood clots.

In a further aspect, the present invention relates to a method of inhibiting thrombosis in a patient, comprising local injection at the site of vessel sensitive patient to thrombus formation, therapeutically effective dose is oppozitsii, includes derived inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a version thereof and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons.

In a further aspect, the present invention relates to a method of inhibiting thrombosis in a patient, comprising local injection at the site of vessel susceptible to thrombus formation in a patient, a therapeutically effective dose of a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid substituted by another amino acid, where is this another amino acid capable of conjugality with a chemical group and where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type and having a modification in St. who eat catalytic center, with this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons.

In a further aspect, the present invention relates to a method of inhibiting thrombosis in a patient, comprising local injection at the site of vessel sensitive patients to thrombosis, a therapeutically effective dose of a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons.

In further aspects the e, the present invention relates to a method of inhibiting thrombosis in a patient, comprising local injection at the site of vessel sensitive patients to thrombosis, a therapeutically effective dose of a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid corresponding to amino acid selected from R396, Q250 or P406 of the sequence SEQ ID NO: 1, has been replaced with another amino acid, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons.

In a further aspect, the present invention relates to a method of inhibiting thrombosis in a patient, comprising local injection at the site of vessel sensitive patients to thrombosis, a therapeutically effective dose of a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII, which includes AMI is kislotno sequence of SEQ ID NO: 1 or its variant, where the amino acid capable of conjugality with a chemical group that is inserted in the sequence of a SEQ ID NO: 1 or its variant in the position where the factor VII polypeptide has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type, and having a modification in its catalytic center, and this modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, in addition anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons.

In a further aspect, the present invention relates to a method of inhibiting thrombosis in a patient, comprising local injection at the site of vessel sensitive patients to thrombosis, a therapeutically effective dose of a composition comprising a derivative inactivated factor VII, where the polypeptide inactivated factor VII comprising the amino acid sequence of SEQ ID NO: 1 or a variant where the amino acid capable of conjugality with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants, and having a modification in its catalytic center, and is shown modification inhibits the ability of the factor VII polypeptide to activate plasma factor X or IX, additionally anywhereman with a chemical group that increases the true molecular weight of the polypeptide inactivated factor VII about 300 daltons to approximately 100000 daltons.

In one of the embodiments according to the invention, the chemical group is essentially neutral.

Here, the term "neutral" refers to biocompatible chemical group, and assume that it is non-toxic, non-immunogenic, water soluble. The chemical group being within this definition are essentially neutral, include, but are not limited to, polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly(N-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, copolymers of polypropyleneoxide/ethylene oxide, polypropylenglycol, polyoxyethylene polyols (e.g. glycerol) and polyvinyl alcohol, kolominova acid or other polymers based on carbohydrates, polymers of amino acids and bitenova derivatives.

In another aspect according to the invention, the chemical group is water-soluble.

In another aspect according to the invention, the chemical group has a molecular weight of from about 1000 daltons to about 80,000 daltons.

In another aspect according to the invention, the chemical group has a molecular weight of from about 5000 daltons to 60,000 is Alton.

In another aspect according to the invention, the chemical group has a molecular weight from about 10,000 daltons to 40,000 daltons.

In another aspect according to the invention, the chemical group has a molecular weight from about 500 daltons to 20,000 daltons.

In another aspect according to the invention, the chemical group has a molecular weight from about 500 daltons to 5,000 daltons.

In another aspect according to the invention, the chemical group has a molecular weight of from about 750 daltons to 5,000 daltons.

In a further aspect according to the invention, the chemical group is a polyethylene glycol.

In a further aspect according to the invention, a chemical group selected from one to seven molecules of polyethylene glycol.

In the preferred embodiment according to the invention, the chemical group represents one molecule of polyethylene glycol.

In another embodiment according to the invention, the chemical group is a monometoksipolietilenglikolya.

In another embodiment according to the invention, the chemical group is a dextran.

In another embodiment according to the invention, the chemical group is a poly(N-vinyl pyrrolidone)polyethylene glycol.

In another embodiment according to the invention, the chemical group is homapal the measures of propylene glycol.

In another embodiment according to the invention, the chemical group is polypropyleneoxide.

In another embodiment according to the invention, the chemical group is polypropylenglycol.

In another embodiment according to the invention, the chemical group is polyoxyethylene polyol.

In another embodiment according to the invention, the chemical group is a polyvinyl alcohol.

In another embodiment according to the invention, the chemical group is clomidbuy acid.

In another embodiment according to the invention, the chemical group is a polymer-based carbohydrate.

In another embodiment according to the invention, the chemical group is a polymer of amino acids.

In another embodiment according to the invention, the chemical group is bitenova derived.

In another embodiment according to the invention, the chemical group conjugated with a free sulfhydryl group present on the amino acid that replaces the amino acid in the polypeptide, are included in the polypeptide or added to the polypeptide.

In another embodiment according to the invention, the chemical group conjugated with cysteine.

In another embodiment according to the invention that replaces included or added amino acids is capable of conjugality with a chemical group.

In another embodiment according to the invention, the amino acid capable of conjugality with the chemical group is an amino acid with a free sulfhydryl group.

In another embodiment according to the invention, the amino acid capable of conjugality with the chemical group is cysteine.

In another embodiment according to the invention that replaces included or added amino acid is an amino acid with a free sulfhydryl group, such as cysteine.

In another embodiment, the amino acid of the factor VII polypeptide is inserted at a position selected from positions 247-260, 393-405 or 406 of the sequence SEQ ID NO: 1.

In another embodiment, the amino acid of the factor VII polypeptide corresponding to position R396 sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another embodiment, the amino acid of the factor VII polypeptide corresponding to position Q250 sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another embodiment, the amino acid of the factor VII polypeptide corresponding to position P406 sequence SEQ ID NO: 1, has been replaced with another amino acid.

In another embodiment, associated with the factor VII polypeptide, the additional amino acid capable of conjugality with a chemical group that is inserted in the sequence of SEQ ID NO: 1 or its variant in the position where the polypeptide factor VI has essentially the same activity or increased activity compared to recombinant human factor VIIa wild-type.

In another embodiment, associated with the factor VII polypeptide, another amino acid that can konjugierte with a chemical group has been added to the N - or C-Termini of the sequence SEQ ID NO: 1 or its variants.

In another embodiment, associated with the factor VII polypeptide, the amino acid added to the C-end of the sequence SEQ ID NO: 1.

In another embodiment, associated with the factor VII polypeptide, the amino acid added to the N-end of the sequence SEQ ID NO: 1.

In another embodiment, associated with the factor VII polypeptide, added cysteine.

In another embodiment, associated with the factor VII polypeptide, which comprises cysteine.

In another embodiment, associated with the factor VII polypeptide, the amino acid is selected from the group consisting of K157, V158, E296, and M298, L305, D334, S336, K337 and F374 sequence SEQ ID NO: 1, has been replaced with another amino acid that increases the activity compared to recombinant human factor VIIa wild-type.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to K157 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from G, V, S, T, N, Q, D, and E.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to V158 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from S, T, N, Q, D, and E.

In another embodiment, include the nom with factor VII polypeptide, the amino acid corresponding to V158 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from T and D.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to E296 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from R, K, and V.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to E296 sequence SEQ ID NO: 1, is replaced by the amino acid V.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to M298 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from R, K, Q and N.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to M298 sequence SEQ ID NO: 1, is replaced by the amino acid Q.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to L305 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from A, V, L, I, M, F, W, P, G, S, T, C, Y, N, E, K, R, H, D, and Q.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to L305 sequence SEQ ID NO: 1, is replaced by the amino acid V.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to D334 sequence SEQ ID NO: 1, is replaced by the amino acid E.

In another embodiment, connected the with factor VII polypeptide, the amino acid corresponding S336 sequence SEQ ID NO: 1, is replaced by the amino acid G.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to K337 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from A, G, V, S, T, N, Q, D, and E.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to K337 sequence SEQ ID NO: 1, is replaced by the amino acid A.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to F374 sequence SEQ ID NO: 1, replaced with an amino acid independently selected from A, V, L, I, M, F, W, P, G, S, T, C, Y, N, E, K, R, H, D, and Q.

In another embodiment, associated with the factor VII polypeptide, the amino acid corresponding to F374 sequence SEQ ID NO: 1, is replaced by the amino acid P.

In another embodiment according to the invention, the factor VII polypeptide is human factor VII.

In another embodiment according to the invention, the factor VII polypeptide is human factor VIIa.

In the present description amino acids are represented by abbreviations, as shown in Table 1, adopted by the Commission on Biochemical Items (CBN) IUPAC-IUB. It is understood that the amino acid and the like [connection]with the isomers represented by the name or by using the following abbreviations, which if not stated otherwise, are in the natural L-form. In addition, the left and right ends of the amino acid sequence of the peptide, if not stated otherwise, represent, respectively, of its N - and C-ends.

Asn
Table 1

Abbreviations, which are labeled amino acids
Amino acidThree-letter codeSingle-letter code
GlycineGlyG
ProlineProP
AlanineAlaA
ValineValV
LeucineLeuL
IsoleucineIleI
MethionineMetM
CysteineCysC
PhenylalaninePheF
TyrosineTyrY
TryptophanTrpW
HistidineHisH
LysineLysK
ArginineArgR
GlutamineGlnQ
AsparagineN
Glutamic acidGluE
Aspartic acidAspD
SerineSerS
ThreonineThrT

The present invention relates, as indicated above, also with the method of obtaining a polypeptide is human factor VII. The polypeptides of the human factor VII is preferably produced using recombinant DNA technology. With this purpose, the DNA sequence encoding the human factor VII can be isolated by obtaining genomic libraries or cDNA and screening for DNA sequences that are fully or partially encoding the protein using synthetic oligonucleotide probes, in accordance with standard techniques (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). For purposes associated with the present invention, preferably, the DNA sequence encoding this protein was human nature, i.e. that it was obtained from a cDNA library or DNA in the human genome.

DNA sequences encoding polypeptides of the human factor VII can be obtained and also synthetically using exhaust standard methods, for example, phospho who Medicago method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. In accordance with fostamatinib method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, subjected to annealing, with the subsequent ligation and cloned in appropriate vectors.

The DNA sequence can be obtained by polymerase chain reaction using specific primers, for instance as described in U.S. patent 4,683,202, Saiki et al., Science 239 (1988), 487-491, or Sambrook et al., above.

DNA sequences encoding polypeptides of the human factor VII, typically include in a recombinant vector, which may be any vector that is convenient from the point of view of recombinant DNA technology, and the choice of vector is usually dependent on the host cell into which it is introduced. Thus, the vector may be a stand-alone can replicate vector, i.e. a vector which exists as an extrachromosomal object, the replication of which is not dependent on chromosomal replication, e.g. a plasmid. Alternatively, the vector may be such that when introduced into the host cell integrates into the genome of the host cell and is replicated together with the chromosome (chromosomes), in which it was integrated.

This vector is preferably expressing the vector in which the DNA sequence, encoding the polypeptides of the human factor VII, operatively associated with additional segments required for transcription of the DNA. Usually expressing vector derived from plasmid DNA or viral DNA, or it may contain elements of both. The term "operatively linked" indicates that the segments aranzhirovany so that they operate consistently in accordance with the objectives, for example, in the region of the promoter initiates transcription of the DNA, which occurs along the whole chain DNA sequence that encodes a polypeptide.

The promoter may be any DNA sequence which possesses transcriptional activity in the selected host cell and which can be obtained from the genes encoding either homologous or heterologous proteins for a given host cell.

Examples of suitable promoters for directing the transcription of the DNA encoding the polypeptide is human factor VII, are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), the promoter of MT-1 (gene metallothionein) (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell 41: 521-530,1985) or major late promoter of adenovirus 2 (Kaufman and Sharp, Mol. Cell Biol, 2: 1304-1319, 1982).

An example of a suitable promoter for use in insect cells is the polyhedrin promoter (U.S. patent 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), prom is Thor P10 (J. M. Vlak et al., J. Gen. Virology 69,1988, pp. 765-776), the main promoter of the protein polyhedrosis virus of Autographa californica (European patent 397 485), the promoter of the immediate early gene 1 baculovirus (U.S. patent 5,155,037; U.S. patent 5,162,222) or the promoter delayed-early gene of the baculovirus 39K (U.S. patent 5,155,037; U.S. patent 5,162,222).

Examples of suitable promoters for use in yeast cells as host cells include promoters of glycolytic of yeast genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 promoter (U.S. patent 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654).

Examples of suitable promoters for use in host cells of filamentous fungi are, for example, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of other suitable promoters are the promoters obtained from the gene encoding TAKA-amylase A. oryzae, aspartyl the proteinase Rhizomucor miehei, neutral α-amylase, A. niger, kislotostabilen α-amylase, A. niger, glucoamylase (gluA) A. niger or A. awamori, lipase, Rhizomucor miehei, alkaline protease, A. oryzae, triosephosphate A. oryzae or acetamidate A. nidulans. Preferred are promoters of TAKA-amylase and gluA. Suitable promoters are promoters that are specified, for example the EP, in the European patents EP 238 023 and EP 383 779.

DNA sequences encoding polypeptides of the human factor VII, may also, if necessary, operatively linked to a suitable terminator, such as the terminator of human growth hormone (Palmiter et al., Science 222,1983, pp. 809-814) or the TPI1 terminator (Alber and Kawasaki, J. Mol. Appl. Gen. 1,1982, pp. 419-434)or terminator ADH3 (McKnight et al., The EMBO J. 4,1985, pp. 2093-2099). The vector may also contain a set of sites of RNA splicing, localized downstream from the promoter and upstream from the site of injection such as the sequence of factor VII. Preferred sites of RNA splicing can be derived from adenovirus and/or immunoglobulin genes. In expressing the vector also contains a polyadenylation signal, localized downstream from the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the area 5 adenovirus Elb, the terminator of human growth hormone (DeNoto et al. Nuc. Acids Res. 9: 3719-3730,1981) or the polyadenylation signal of the gene factor VII human or bovine factor VII gene. The expression vectors can also include non-coding viral leader sequence, such as tripartite (tripartite) leader sequence adenovir the CA 2, localized between the promoter and the sites of RNA splicing; and enhancer sequence, such as the SV40 enhancer.

The recombinant vector may also comprise a DNA sequence enabling the vector to replicate specifically in the desired host cell. An example of such a sequence (when the host cell is a cell of a mammal) is the origin of replication of SV40.

When the host-cell is a yeast cell, suitable sequences enabling the vector to replicate, are replication genes REP 1-3 and origin of replication of the yeast plasmid 2μ.

The vector may include breeding marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene encoding dihydrotetrazolo (DHFR), or gene TPISchizosaccharomyces pombe(described by P. R. Russell, Gene 40, 1985, pp. 125-130), or a gene that provides drug resistance, for example resistance to ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. In the case of filamentous fungi selective markers include amdS, pyrG, argB, niaD and sC.

In order to master the cells to direct the polypeptide is human factor VII according to the invention in the secretory pathway, it is necessary to provide a recombinant vector is kratornas signal sequence (also known as a leader sequence, preprofessionals or proposedvalue). The secretory signal sequence is linked to a DNA sequence that encodes the polypeptides of human factor VII in the correct reading frame. Secretory signal sequences are typically located 5'-end of the DNA sequence that encodes the peptide. The secretory signal sequence may thus be normally associated with the protein or may be from a gene encoding another secretory protein.

In the case of secretion from yeast cells secretory signal sequence may encode any signal peptide, which ensures efficient direction of expressed polypeptides of human factor VII in the cell of the secretory pathway. The signal peptide may be the natural signal peptide or a functional part, or it may be a synthetic peptide. It was found that a suitable signal peptides are α-factor signal peptide (see U.S. patent 4,870,008), the signal peptide of salivary amylase mouse (cf. O. Hagenbuchle et al., Nature 289,1981, pp. 643-646), modified carboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670) or signal peptide espartignac protease 3 (YAP3) (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137.

For efficient secretion in yeast, a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence that encodes the polypeptide is human factor VII. The function of the leader peptide is to direct downregulation of the peptide from the endoplasmic reticulum to the Golgi apparatus and then into secretory vesicles, so that he was secreted into the culture medium (i.e. exportation of the polypeptides of the human factor VII through the cell wall or at least through the cellular membrane into periplasmatic space of the yeast cell). The leader peptide may be a leader peptide of the yeast alpha-factor (the use of which is described, for example, in U.S. patent 4,546,082, U.S. patent 4,870,008, and in European patents EP 16 201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic leader peptide, i.e. such a leader peptide, which is not found in nature. Synthetic leader peptides can be, for example, constructed as described in international applications WO 89/02463 or WO 92/11378.

For use in filamentous fungi signal peptide can be obtained, respectively, of the gene encoding the amylase or glucoamylase of Aspergillus sp., of the gene encoding lepotuoli the proteinase Rhizomucor miehei or lipase Humicola lanuginosa. Preferably, if the signal peptide derived from a gene encoding TAKA-amylaseA. oryzaeneutral alpha-amylaseA. niger, kislotostabilen amylaseA. nigeror glucoamylaseA. niger. Acceptable signal peptides are described, for example, in EP 238 023 and EP 215 594.

For use in insect cells, signal peptide can be, respectively, obtained from the gene of the insect (see WO 90/05783), such as signal peptide predshestvennki form adipokinetic hormone LepidopteraManduca sexta(see U.S. patent 5,023,328).

Techniques used for ligation of DNA sequences encoding polypeptides of the human factor VII promoter and - optional - termination and/or secretory signal sequence, respectively, and to embed them into suitable vectors containing the information necessary for replication, are well known to specialists in this field (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

Methods transliterowany of mammalian cells and ekspressirovali DNA sequences embedded in the cells are described, for example, Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-45.

Selective m is rquery can be introduced into the cell in a single plasmid simultaneously with the gene of interest, or they may be introduced into the cell as part of the same plasmid. If part of the same plasmid selective marker and gene of interest can be under the control of the same promoter, with this arrangement of genes is formed dahlstrand messenger. Designs of this type known to specialists in this field (e.g., Levinson and Simonsen, U.S. patent No. 4,713,339). Can also be successfully added to the mixture, which is introduced into the cells, additional DNA, known as DNA carrier.

After the DNA is captured by the cells, the latter growing in a suitable environment for cell growth, usually 1-2 days before the expression of the gene of interest. Under used herein the term "suitable environment for cell growth" means a medium containing nutrients and other components required for cell growth and expression of interest polypeptide is human factor VII. Such an environment typically includes a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, proteins and growth factors. For producing gamma-carboxylating protein environment must contain vitamin K, preferably in a concentration from about 0.1 μg/Melpomene to 5 µg/ml Then produce a selection of medicines for the selection of the growth of cells, which stably Express a selective marker. For cells that were transliterowany amplificare selective marker, the concentration of the drug may be increased for the purpose of selection of a larger number of copies of the cloned sequences, thus increasing the levels of expression. Then the clones of stably transfected cells are screened for expression of interest polypeptide is human factor VII.

The host cell into which the introduced DNA sequences encoding polypeptides of the human factor VII, may be any cell which is capable of producing excision of the modified polypeptides of the human factor VII, including yeast cells, fungi cells and cells of higher eukaryotes.

Examples of lines of mammalian cells for use in the present invention are cell lines COS-1 (ATCC CRL 1650), cells of hamster kidney (BHK) and the cell line 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol 36: 59-72, 1977). The preferred line BHK is a type tk-ts13 cell line BHK (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982, is hereby incorporated into this description by reference), denoted hereafter as cells BHK 570. The cell line BHK 570 deposited in AME the ikan collection of type culture, 12301 Parklawn Dr., Rockville, Md. 20852, under the identification number ATCC CRL 10314. Line tk-ts13 BHK cells available under identification number ATCC CRL 1632. In addition, in the framework of the present invention can be used also a number of other cell lines, including cells of the Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lung cells (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220,1980).

Examples of suitable yeast cells include cellsSaccharomycesspp. orSchizosaccharomycesspp., in particular, strains ofSaccharomyces cerevisiaeorSaccharomyces kluyveri. The ways of transforming yeast cells with heterologous DNA and obtaining from them heterologous polypeptides are described, for example, in U.S. patents 4,599,311, US 4,931,373, US 4,870,008, 5,037,743 and US 4,845,075, all of which are included in the present description by reference. Transformed cells were selected by phenotype determined using selective marker, usually a resistance to the drug, or by the ability to grow in the absence of any specific nutrient, such as leucine. Preferred vectors for use in yeast cells is the POT1 vector, described in U.S. patent 4,931,373. The DNA sequences coding for the polypeptides of the human factor VII, can the be preceded by a signal sequence and is optional - leader sequence, e.g. as described above. In addition, examples of suitable yeast cells are strains ofKluyveromycessuch asK. lactis, Hansenulafor exampleH. polymorphaorPichiafor example,P. pastoris(see Gleeson et al., J. Gen. Environ. 132,1986, pp. 3459-3465; US 4,882,279).

Examples of other fungal cells are cells of filamentous fungi, for example,Aspergillusspp.,Neurosporaspp.,Fusariumspp. orTrichodermaspp., in particular strains ofA. oryzae, A. nidulansorA. niger. UseAspergillusspp. for protein expression are described, for example, in EP 272 277, EP 238 023, EP 184 438. TransformationF. oxysporumcan be produced, for example, in accordance with the methodology described by Malardier et al., 1989, Gene 78: 147-156. TransformationTrichodermaspp. can be implemented, for example as described in EP 244 234.

When the host cells are filamentous fungi, they can be transformed by using the DNA according to the invention, usually by integrating design DNA into the host chromosome, to obtain the recombinant host cells. This integration is usually considered successful, because the stability of such a DNA sequence in the cell is more likely. Integration into the host chromosome can be produced according to conventional methods, for example, by homologous or heterologous, recombi the promotion.

Transformation of insect cells and production of them in heterologous polypeptides may be taken, as described in patents US 4,745,051; US 4,879,236; US 5,155,037; 5,162,222; EP 397,485, all of which are included in the present description by reference. Lines of insect cells used as hosts, can be cell lineLepidopterasuch as cellsSpodoptera frugiperdaor cellsTrichoplusia ni(see U.S. patent 5,077,214). Suitable culturing conditions can be conditions described, for example, in international applications WO 89/01029 or WO 89/01028, or in any of the above links.

Then the above transformed or transfetsirovannyh cells are cultivated in a suitable nutrient medium under conditions permitting expression of the polypeptide is human factor VII, after which the resulting polypeptide, in whole or in part may be extracted from the culture medium. The medium used for culturing cells, can be any convenient to use the environment suitable for the growth of host cells, such as minimal or complex media containing appropriate supplements. Suitable protection can be purchased as is or to prepare them in accordance with the published recipe (e.g., in catalogues of the American type culture Collection). Produced by cells in peptide human factor VII can then be extracted from the culture medium using standard techniques, including separating the host cells from the medium by centrifugation or filtration, precipitation of water-soluble proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by using various chromatographic methods such as ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like, depending on the type of interest polypeptide.

To obtain the polypeptide of recombinant human factor VII used the cloned DNA sequence of factor VII of the wild type. This sequence may be modified to encode the desired variant of factor VII. Full nucleotide and amino acid sequence of human factor VII is known. Cm. U.S. patent No. 4,784,950, which is included in the present description by reference, and which describes the cloning and expression of recombinant human factor VII. The sequence of bovine factor VII is described in the publication Takeya et al., J. Biol. Chem, 263: 14868-14872 (1988), which is incorporated into this description by reference.

Amino acid changes may be made in various ways. Modification of the DNA sequence can be carried out by site-specific mutagenesis. Technology site-specific Mut the Genesis is well-known in this field and described, for example, Zoller and Smith, DNA 3: 479-488,1984). Thus, using the nucleotide and amino acid sequence of factor VII, you can choose to make changes.

DNA sequences for use within the present invention should generally encode preprepared on the amino end of the protein factor VII to obtain the correct post-translational processing (e.g., gamma-carboxylation of glutamic acid residues) and secretion from the host cell. Preprepared can be reprobation factor VII or other vitamin K-dependent plasma protein, such as factor IX, factor X, prothrombin, protein C or protein S. As should be obvious to specialists in this area, additional modifications in the amino acid sequence of factor VII can be produced if these modifications will not cause significant deterioration in the ability of the protein to function as a coagulation factor. For example, factor VII in the catalytic triad can also be modified in the site activation cleavage, in order to cause the inhibition of the conversion of zymogen factor VII in its activated double-stranded form, as in the General form described in U.S. patent No. 5,288,629 included in the present description by reference.

In the framework of the present invention for production of the polypeptide forehead is ekeskog factor you can use transgenic animal. Preferred is the production of proteins in the mammary glands, using as a host female mammal. Expression in the mammary gland and subsequent secretion of the desired protein in the milk helps to circumvent many of the difficulties associated with the separation of proteins from other sources. Milk is easy to collect, is available in large quantities, and it is easy to characterize biochemically. Moreover, the major milk proteins presents in milk at high concentrations (usually about from 1 to 15 g/l). From a commercial point of view is clearly preferable to use as the master of the representatives of this type, which has a high level of milk production. Despite the fact that can be used and smaller animals such as mice and rats (and they are preferred even at the stage of principle testing), in the framework of the present invention preferably using mammals from the category of large animals, including, but not limited to, pigs, goats, sheep and cattle. The sheep is especially preferred because of such factors as the background of transgenetic of this species, the milk yield, cost, and readily available equipment to collect sheep's milk. Cm. the WIPO publication number WO 88/00239 to compare factors influencing the choice of the IDA owner. Usually requires the breeding of these breeds of domestic animals reared for milk production, such as Eastern sheep East Friesland sheep, or for breeding dairy breed by breeding transgenic lines for the future.

In any case, you should use animals with confirmed health status.

To obtain the expression in the mammary gland is used transcriptional promoter of the gene of milk protein. Genes milk proteins include genes encoding casein (see U.S. patent No. 5,304,489 included in the present description by reference), beta-lactoglobulin, alpha-lactalbumin and sour whey protein. Preferred is the promoter of beta-lactoglobulin (BLG). In the case of sheep gene beta-lactoglobulin usually use the area of at least 406 P.N., proximal with respect to the 5'-flanking sequence of the gene, although the preferred larger 5'-flanking sequence, up to 5 TPN, such as a segment of DNA approximately 4.25 TPN covering the 5'-flanking promoter and non-coding part of the gene of beta-lactoglobulin. Cm. Whitelaw et al., Biochem J. 286: 31-39 (1992). Similar fragments of DNA promoter from other species are also acceptable.

Other areas of the gene of beta-lactoglobulin can also be built into the design then, th is would be given the opportunity to be expressed other genomic regions of the gene. In this area it is generally acknowledged that design, devoid of introns, for example, have low expression compared to the structure that contains the DNA sequence (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840 (1988); Palmiter et al., Proc. Nafl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91/02318; each of these publications is incorporated into this description by reference). In this regard, it is generally preferable, when possible, to use genomic sequence containing all or some of the native introns of the gene encoding protein of interest or polypeptide, so that the additional inclusion of at least some introns, for example, beta-lactoglobulin gene is preferred. One such area is the segment of DNA that provides the splicing of the intron and the polyadenylation of RNA from the 3'-noncoding region of sheep beta-lactoglobulin gene. When replacing the natural 3'-non-coding sequences of a gene, this segment of the Ovine beta-lactoglobulin gene can both enhance, and stabilize the levels of expression of interest protein or polypeptide. Within other embodiments, the area surrounding the initiating ATG sequence that encodes a polypeptide of the human factor VII, replaced according to the corresponding sequences of gene-specific milk protein. This replacement allows the intended tissue-specific initiating environment to enhance expression. It is convenient to replace the full preprofessionals polypeptide is human factor VII and 5'-non-coding sequence such as a gene BLG although in this case will be replaced by a smaller area.

For expression of the polypeptide is human factor VII in transgenic animals, the DNA segment encoding the polypeptide is human factor VII, operatively linked to additional DNA segments required for its expression with obtaining expressing units. Such additional segments include the above promoter and sequences that provide for the termination of transcription and polyadenylation of mRNA. Expressing the unit will also include a segment of DNA that encodes a secretory signal sequence, operatively associated with the segment coding for the polypeptide is human factor VII. The secretory signal sequence may be native secretory signal sequence of the polypeptide is human factor VII or may be of another protein such as milk protein. See, for example, the publication of von Heinje, Nuc. Acids Res. 14: 4683-4690 (1986); and Meade et al., U. S. Pat. No. 4,873,316, which is included in the present description their link.

Design expressing units for use in transgenic animals conveniently be done by embedding sequence that encodes a polypeptide of the human factor VII, a plasmid or phage vector containing the additional DNA segments, although expressing unit can be constructed essentially with any ligiously sequence. In particular, it is convenient to obtain a vector containing a DNA segment encoding a milk protein, and replace the coding sequence of milk protein such polypeptide is human factor VII, thereby creating a fused gene, which includes controlling the expression of the milk protein gene. In any case, the clone expressing the units in plasmids or other vectors facilitates the amplification of the polypeptide is human factor VII. Amplification conveniently be produced in bacterial cells-hosts (for example, E. coli cells), then the vectors will generally include the origin of replication and breeding marker functional in bacterial cells-hosts.

Then expressing the unit fits into fertilized eggs (including early embryonic stage) selected host species. The introduction of heterologous DNA can be produced in one of several ways, including ICRI the injection (see, for example, U.S. patent No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474 being more accurate (1988)) or site-directed integration using embryonic stem cells (ES) (see review by Bradley et al., Bio/Technology 10: 534-539 (1992)). Then eggs were implanted into the oviduct or uterus of pseudoharmonic females, providing them with the opportunity to develop. Child bearing in its germline of the integrated DNA can, for Mendel, to hold this DNA through their offspring and to initiate the development of herds of transgenic animals. Commonly used procedures for obtaining transgenic animals are well known to specialists in this field. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol. Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143 (1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort et al., Bio/Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem. 49: 113-120 (1992); U.S. patent No. 4,873,191 and 4,873,316; publication of international patent applications WO 88/00239, WO 90/05188, WO 92/11757 and application great Britain GB 87/00458, which is incorporated into this description by reference. Technologies include alien DNA sequences in animal and in stem cells of the animal were initially tested on mice. See, for example, Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985); and Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985). These technologies vpos is estii were adapted for use on larger animals, including different types of livestock (see, for example, the publication of international applications WO 88/00239, WO 90/05188 and WO 92/11757; and Simons et al., Bio/Technology 6: 179-183 (1988). Summarizing, it should be noted that, according to the technology in the most effective used to date ways of generating transgenic mice or cattle, of the order of several hundred linear molecules of interest DNA has to be injected into one of the pronuclei of fertilized eggs. You can also use the injection of DNA into the cytoplasm of the zygote. You can also use the production in transgenic plants. The expression can be generalized or focused on a specific organ, such as the tuber. See, for example, Brian Harvey, Nature 344: 469-479 (1990); Edelbaum et al., J. Interferon Res. 12: 449-453 (1992); Sijmons et al., Bio/Technology 8: 217-221 (1990); and the publication of the European patent EP 255,378.

Factor VII produced in accordance with the present invention, can be purified using the method of affinity chromatography or column with the antibody against factor VII. Preferably, immunoadsorption column contained a highly specific monoclonal antibody. The use of calcium-dependent monoclonal antibodies, as described in publications Wakabayashi et al., J. Biol. Chem, 261: 11097-11108, (1986) and Thim et al., Biochem. 27: 7785-7793, (1988), included in the present description by reference, is the OS is especially preferred. Additional cleaning can be undertaken using commonly used chemical procedures for cleaning, such as high performance liquid chromatography. Other cleaning methods, including precipitation with barium citrate, known in the field and can be applied for the purification of factor VII, described above (see Scopes, R., Protein Purification, Springer-Verlag, N. Y., 1982). For pharmaceutical purposes substantially pure factor VII, homogeneous for at least 90 - 95%is preferred, and most preferred is the degree of homogeneity of the order of 98 to 99% and above. Partially or until complete homogeneity is cleared, depending on needs, the factor VII can then be used for therapeutic purposes.

The conversion of single-chain factor VII in the active double-stranded factor VIIa can be achieved with the use of factor XIIa, as described Hedner and Kisiel (1983, J. Clin. Invest. 71: 1836-1841), or other proteases having trypsin-like specificity (Kisiel and Fujikawa, Behring Inst. Mitt. 73: 29-42, 1983). An alternative factor VII can be autoactivated by passing it through an ion-exchange chromatographic column, such as mono-Q.RTM (Pharmacia Fire Chemicals) and other (Bjoern et al., 1986, Research Disclosures 269: 564-565). Molecules of factor VII according to the present invention and pharmaceutical compositions according to the invention t is Auda particularly useful for administration to man for the treatment of various conditions, associated with intravascular coagulation.

The invention also provides suitable approaches for selection of the preferred polypeptides of the factor VIIa and the derived factor VIIa according to the invention. These approaches can be effective in the preliminary testing in vitro.

Thus, in Example 5 in the present description disclosed a simple test (called "hydrolysis analysisIn Vitro"on the activity of the polypeptides of factor VIIa according to the invention. Accordingly, the polypeptides of factor VIIa, which are of particular interest are polypeptides in which the relationship between the activity of the variant and the activity of native human factor VII, as shown in Fig. 1, is of the order of 1.0 or greater when tested using the above hydrolysis analysisIn Vitro".

The activity of the polypeptides can also be assessed using physiological substrate, such as factor X ("analysis of proteolysisIn Vitro") (see Example 6), approximately at a concentration of 100-1000 nm, where after adding the appropriate chromogenic substrate (e.g., S-2765) determine the level of the resulting factor Xa. Moreover, analysis of the activity can be carried out at physiological temperature.

The ability procoagulant polypeptides of factor VIIa to generate thrombin can also be measured by means of analysis is, comprising all relevant coagulation factors and inhibitors at physiological concentrations (minus factor VIII in terms of the modeling of hemophilia A) and activated platelets (as described on page 543 in the publication Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, which is incorporated into this description by reference).

Derived procoagulant factor VII according to the present invention can be used to control diseases that are associated with bleeding and that can be due to several reasons, such as deficiencies of coagulation factor (e.g., hemophilia A and B or deficiency of coagulation factors XI or VII)or inhibitors of coagulation factors, or they can be used to control excessive bleeding occurring in subjects with a normally functioning cascade of blood clotting (in the absence of the deficiencies of coagulation factors or inhibitors of any of the clotting factors). Bleeding can be caused by a defect of platelet function, thrombocytopenia, or disease von Willebrand's disease. They can be observed also in such entities, which under the action of various stimuli induced increased fibrinolytic activity.

In subjects who had undergone extensive tissue damage in connection with the operation or extensive injury, the mechanisms of the hemostasis under conditions when the body is experiencing a great demand in the immediate hamostaseologie, can be suppressed, and as a result, instead of the normal mechanism geostationary can develop bleeding. Achieving satisfactory geostationary is also a problem in cases where bleeding in organs such as the brain, the eyes and the area of the inner ear, and in addition, problems can arise in cases of diffuse bleeding (hemorrhagic gastritis and profuse uterine bleeding), it is difficult to identify the source of bleeding. The same problem may occur in the process of taking biopsy samples from different organs (liver, lung, tumor tissue, gastrointestinal tract), as well as in laparoscopic surgery. These situations pose challenges associated with providing hemostasis using surgical techniques (sutures, clamps etc). Acute and profuse bleeding may occur also when anticoagulant therapy in subjects who have defective hemostasis induced as a result of ongoing therapy. Such entities may require rapid intervention in case we need to immediately counteract the anticoagulant effect. Another situation that can cause problems in case of unsatisfactory hemostasis is the situation, it is always in subjects with normal mechanism of hemostasis spend anticoagulant therapy for the prevention of thromboembolic disease. Such therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, and aspirin and other platelet aggregation inhibitors.

Systemic activation of the coagulation cascade can lead to disseminated intravascular coagulation (DIC). Such complications, however, did not occur in subjects who were treated with high doses of recombinant factor VIIa, because localized hemostatic process of this type, induced by complex formation between factor VIIa and tissue factor (TF)exposed at the site of damage to the vascular wall. Thus, the derived procoagulant factor VII according to the present invention can be used also in its active form for control of such excess bleeding associated with the normal mechanism of hemostasis.

For treatment in connection with deliberate interventions derived procoagulant factor VII according to the present invention, as a rule, should be administered within approximately 24 hours before the intervention, as well as for 7 or more days after it. The introduction of coagulation factor can be produced in various ways, as described in this application.

Dose derived factor VII varies from about 0.05 mg to 500 mg in de the e l e C preferably from about 1 mg to 200 mg per day, and even more preferably from about 10 mg to about 175 mg / day for patients weighing 70 kg as loading and maintenance doses, depending on severity of condition.

Pharmaceutical compositions originally intended for parenteral administration for prophylactic and/or therapeutic treatment. Preferably the pharmaceutical composition is injected parenterally, for example intravenously, subcutaneously or intramuscularly, or they can be entered by continuous or pulsatile infusion. Compositions for parenteral administration include derived factor VII according to the invention preferably in combination with a pharmaceutically acceptable carrier, in which it is dissolved, preferably with a water carrier. Can be used in a variety of aqueous media such as water, buffered water, 0.4% saline, 0.3% glycine and the like, Derivatives of factor VII according to the invention can also be prepared in liposomal preparations for delivery or targeting to sites of damage. Liposomal drugs in General are described, for example, in U.S. patents 4,837,028, 4,501,728 and 4,975,282. The composition may be sterilized by conventional, well known sterilization methods. The resulting water-Rast the ora can be put up for the use of [liquid] or filtered under aseptic conditions and lyophilized, and before use, will be combined with a sterile aqueous solution. These compositions can contain pharmaceutically acceptable additives, if you want to bring them closer to physiological conditions, such as substance to bring pH and tabularasa agents, as agents for the establishment of toychest solution, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

Concentration derived factor VII in these compositions may vary widely, for example, approximately less than 0.5% by weight, usually about at least 1% by weight, up to 15 or 20% by weight and will be selected primarily on the volume of fluid, viscosity, etc. in accordance with specifically selected by way of introduction.

Thus, a typical pharmaceutical composition for intravenous infusion could be made so that it will store up to 250 ml of sterile ringer's solution and 10 mg of the factor VII polypeptide. Current methods of obtaining parenteral entered compositions are methods that are well known or obvious to a person skilled in this field and are described in more detail, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA (1990).

Compositions containing derivatives of factor VII according infusion is he to the invention, can be entered in the preventive and/or therapeutic medical purposes. In therapeutic applications, compositions are administered, as mentioned above, to a subject already suffering from a disease, in a quantity sufficient to coordinate, facilitate or stop the disease and its complications. Quantity adequate to ensure that these goals were achieved, is defined as "therapeutically effective amount". Professionals in this field should be obvious that the amount effective to achieve this goal will depend on the severity of the disease or injury, as well as of body weight and General condition of the particular subject. Typically, however, the effective amount ranges from about 0.05 mg to about up to 500 mg derived factor VII in the day for a subject weighing 70 kg, with the most common dose is from about 1.0 mg to about 200 mg derived factor VII in the day.

It should be borne in mind that the materials according to this invention can be used in cases of serious diseases or conditions in case of serious damage, such as a situation when there is a real or potential threat to life. In such cases, from the point of view of the minimization of extraneous substances and usually the lack of immunogenicity derived from human factor VII in which Oseni human body, possibly, maybe, and requires that the attending physician has appointed the notoriously excessive number of options compositions of factor VII.

Prophylactic use of a composition containing a derivative of factor VII according to the present invention, are administered to a subject prone to the disease or when there is a risk of damage, to enhance the clotting ability of his own body. This number is defined as a "prophylactically effective dose." In prophylactic applications, it is also the exact number will depend on the state of health and body weight of a specific subject, with the usual doses ranging from about 0.05 mg to about 500 mg per day, with the most common dose is from about 1.0 mg to about 200 mg per day for a subject weighing 70 kg

Single or repeated administration of the compositions can be carried out in doses, the level of which, as well as way of introduction, determined by the treating physician. For ambulatory patients requiring daily maintenance doses derived factor VII can be administered by continuous infusion using, for example, system portable pump.

Local delivery-derived factor VII according to the present invention, such as, for example, local application can be executed, for example, using a spray, perfusion, dwuhvalentnykh catheters, stents, built into vascular grafts or stents, hydrogels used for coating of balloon catheters, or other well known methods. In any case, the pharmaceutical compositions should provide a quantity derived factor VI, sufficient for effective treatment of a specific subject.

Polypeptides inactivated factor VII according to the present invention are able to bind with tissue factor on the cell surface. For example, DEGR-factor VIIa binds to the cell surface with the same or greater affinity than factor VIIa wild type. DEGR-factor VIIa, however, has no enzymatic activity, although it binds to tissue factor and acts as a competitive antagonist of factor VIIa wild type, thus inhibiting subsequent stages of the outer path coagulation, leading to the production of thrombin.

Derived inactivated factor VII applies, in particular, to introduce the person in the treatment of various conditions, including intravascular coagulation. For example, although deep vein thrombosis and pulmonary embolism can be treated with traditionally used anticoagulants, described here, derived inactivated factor VI can be used to prevent thromboembolic complications in patients within the group of high risk, such as patients undergoing surgery, or patients with congestive heart failure. In addition, derived inactivated factor VII can act as antagonists induction of coagulation-mediated tissue factor, thus blocking the production of thrombin and the subsequent deposition of fibrin. As such, derived inactivated factor VII can be used for inhibiting the activity of tissue factor, the resulting, for example, for inhibition of blood coagulation, thrombosis and deposition of platelets.

Derived inactivated factor VII can be useful, in particular, in the treatment of intimal hyperplasia, restenosis as a result of acute vascular injury, deep vein thrombosis, arterial thrombosis, surgical thrombosis, bypass grafting of the coronary arteries (coronary artery bypass graft(CABG), percutaneous (transdermal) coronary angioplasty (percutaneous catheter coronaroplasty) (PTCA), stroke, cancer, tumor metastasis, angiogenesis, ischemia/reperfusion, rheumatoid arthritis, thrombolyse, arteriosclerosis and restenosis after angioplasty, acute and chronic indications such as inflammation, septic shock, septicemia, hypotension that RES is retornam distress syndrome in adults (ARDS), disseminated intravascular coagulation (DIC), pulmonary embolism, deposition of platelets, myocardial infarction, or the prophylactic treatment of mammals with atherosclerotic vessels at risk associated with thrombosis. Acute vascular lesions are lesions that appear quickly (i.e. in the period ranging from days to months), in contrast to chronic vascular lesions (e.g., atherosclerosis), which develop throughout life. Acute vascular lesions often result from surgical procedures such as vascular reconstruction, which uses this technology angioplasty, as endarterectomy, atherectomy, the establishment of a vascular implant, or the like. Hyperplasia may also occur as a delayed reaction, for example, in response to the establishment of a graft or transplant organs. As derived inactivated factor VII are more selective than heparin, usually linking only tissue factor exposed at sites of damage, as well as derived inactivated factor VII do not destroy other coagulation proteins, it appears that the latter will be more effective and less risky from the point of view of possible complications, is knitted with bleeding, than heparin in prophylactic use to prevent deep vein thrombosis.

Derived inactivated factor VII, which support the binding of tissue factor, inhibit the accumulation of platelets at the site of vascular lesions by blocking the production of thrombin and the subsequent deposition of fibrin.

Due to the ability of DEGR-factor VII to block the production of thrombin and limit the deposition of platelets at sites of acute vascular lesion derived inactivated factor VII, which support the activity of the binding of tissue factor, but lack the enzymatic activity of factor VIIa can be used to inhibit restenosis of blood vessels.

Compositions containing the derivatives inactivated factor VII, when they are prepared in pharmaceutical compositions, are used, in particular, in the treatment of patients, where they can be given to patients suffering from various illnesses, for the treatment of conditions associated with coagulation. Such derived inactivated factor VII, are able to bind tissue factor, but having significantly suppressed the ability to catalytic activation of other factors in the coagulation cascade, can have a long half-life in plasma and, thus, accordingly, more continue the sustained fashion period anticoagulant activity compared with other anticoagulants. Among the medical indications for the use of these compositions, you can specify those that are usually treated with anticoagulants, such as deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), deposition of fibrin in the lung and the kidney, associated with gram-negative groove toxins and myocardial infarction. These compositions can be used for inhibiting vascular restenosis resulting from mechanical damage to blood vessels, such as damage caused by balloon angioplasty, endarterectomy, rehabilitation atherectomy, the establishment of the stent, laser therapy or rotablation, or secondarily produced in response to vascular grafts, stents, bypass grafting or transplantation of organs. Thus, these compositions can be used to inhibit the deposition of platelets and associated disorders. Thus, the method of inhibiting coagulation, vascular restenosis or deposition of platelets include, for example, the introduction to the patient a composition containing derivatives inactivated factor VII, such as those that contain at least one amino acid substitution in the catalytic triad of Ser344, Asp242 and His193, in a quantity sufficient for effective inhibition svartisen what I restenosis of blood vessels or the deposition of platelets. These methods also find use in the treatment of acute closure of the subject has coronary artery (eg, in acute myocardial infarction), which includes the introduction of derivatives inactivated factor VII, which include DEGR-factor VII and FFR factor VII, in combination with tissue plasminogen activator or streptokinase, and can accelerate induced tissue plasminogen activator thrombosis. Derived inactivated factor VII appointed before the introduction of thrombolytic agent such as tissue plasminogen activator, in conjunction with him or shortly thereafter.

Compositions derived inactivated factor VII will also find significant use in the prevention of cardiogenic embolism and treatment of thrombotic strokes. Due to their low potential to cause complications associated with bleeding, and due to their selectivity derived factor VII can be administered to stroke victims in order to prevent the spread of the occlusal arterial thrombosis. The number of input derived factor VII will vary from patient to patient, depending on the nature and severity of the stroke, but normally the dose will vary in the range of values suggested below.

Derivatives in aktivirovannogo factor VII and compositions containing them can be used also for the inhibition of harmful phenomena, associated with ischemic reperfusion. Severe ischemia, tissue, organ or limb may occur as a result of reduced blood flow and may be associated with trauma, surgical manipulation or low blood pressure. One of the complications associated with severe ischemia is increased levels of tissue factor in the arterial system. This increased expression of tissue factor, most likely stimulate procoagulant response, primarily in the vascular bed of capillaries. After reperfusion of ischemic tissue can form a clot, which can be either occlusal or reocclusion. Blood clots in the bloodstream and the deposition of platelets along the thrombus leads to the formation of secondary ischemic events in this tissue. Blood clots and the presence of platelets then causes the formation and release of a number bioreactive factors, including those that are formed in the process of blood clotting, such as thrombin and factor X, as well as factors released from activated platelets. These factors, in turn, can induce the formation of additional factors from underlying endothelial cells and smooth muscle cells, or from adjacent mononuclear cells to factors such as TNF-alpha and IL-1. This is factors in turn, can then cause the activation of endothelial cells, resulting in increased levels of various adhesion molecules associated with the binding of monocytes and neutrophils. Linking and transmigrate monocytes and neutrophils, release of these cells is biologically active compounds, including the formation of free oxygen radicals, which can exacerbate the level of activation and damage of endothelial cells. In the end, if the cascade of events occurs unchecked, it can lead to systemic complications and potentially to stimulation of the failure of many organs. By blocking according to the invention of tissue factor in the introduction of a specific inhibitor of the binding of tissue factor/factor VII (e.g., FFR-FVIIa) and, thus, blocking the initiation of the external path of blood clotting can be prevented initiation of cascade phenomena, thus avoiding or minimizing the harmful effects associated with ischemia/reperfusion.

Dose derived inactivated factor VII, necessary to prevent deep vein thrombosis, approximately ranges from 50 μg to 500 mg per day, more often from 1 mg to 200 mg per day, and more preferably from about 10 to 175 mg / day for patients weighing 70 kg, and the introduction should begin at least priblizitelen the 6 hours prior to surgery and continue for at least as long while the patient will not be transferred to outpatient mode. Dose derived inactivated factor VII for treatment of restenosis, different patients will vary, but usually it ranges proposed above.

Compositions containing the derivatives inactivated factor VII, usually have to be entered within about 24 hours before surgery, and at least for 7 days or more after him. The introduction can be done in different ways, as will be further described hereinafter. Compositions containing the derivatives inactivated factor VII can be also systemically or locally to establish vascular grafts (e.g., by coating synthetic or modified natural grafts arterial vessels) in places anastomosis, surgical endarterectomy (usually carotid endarterectomy), extracorporeal bypass, etc.

In the treatment of established deep vein thrombosis and/or pulmonary embolism dose derived factor VII ranges from about 50 μg to 500 mg per day, more often from 1 mg to 200 mg per day, and more preferably from about 10 to 175 mg / day for patients weighing 70 kg as loading and maintenance doses, depending on the weight of the patient and severity SOSTOYaNIYa, what is the likelihood of bleeding complications when the infusion is small, the derivatives of the inactivated factor VII can replace or reduce the dose of heparin during surgery or after surgery on thrombectomy or embolectomy.

In cases of acute bacteremia, endotoxemia, or DIC, the patient is given a loading dose derived factor VII comprising at least about 50 μg to 500 mg per day, more often from 1 mg to 200 mg per day, and more preferably from about 10 to 175 mg / day for patients weighing 70 kg and maintenance doses range from 50 μg to 500 mg per day, more often from 1 mg to 200 mg per day for patients weighing 70 kg

Preferably, the derived factor VII preferably have half-life (t1/2)exceeds the half-life of unconjugated factor VII, from which it was obtained. Preferably, the half-life derived factor VII has been increased at least 1.5 - 2 times, more preferably about 2 to 3 times, more preferably approximately 5 to 10 times, optimally about 100 times, usually about 6 times compared with the half-life of unmodified parent factor VII.

Conventional methods of attaching polyethylene glycol to proteins are described in U.S. patent No. 4,179,337, published on 18 December 1979 (the switch is in the present description by reference to the description of the methods of attachment of polyethylene glycol to proteins). In addition, other methods of attachment of polyethylene glycol described in U.S. patent No. 5,122,614, published on 16 June 1992, also included in the present description by reference to the description of the methods of attachment of polyethylene glycol to proteins. Probably PEG-maleimide is the most applicable reagent for Paglierani cysteine, however, for the specific modification of cysteine available and other chemicals.

BRIEF DESCRIPTION of DRAWINGS

The present invention is described hereinafter in more detail in the examples, with reference to the accompanying drawings.

In Fig. 1 shows the structure correctly processioning human factor VII clotting, amino acids 1 to 406, gamma carboxyrhodamine residues Glu (γ) and glycosylation (*). Arrow at amino acid residue 152 indicates the site where single-chain factor VII splits, becoming activated double-stranded factor VII (FVIIa).

Fig. 2. Construction of plasmids for expression of polypeptides of recombinant human factor VII. Plasmid pLN174 expresses human factor VII with an associated propeptide, in the nature associated with factor VII.

Further, the present invention is illustrated in the subsequent examples, which, however, is not to limit the scope of protection of the present invention. The characteristics presented above description and in the subsequent examples, may, separately or in any combination with each other, to serve as the basis for implementing the invention in its various possible options.

EXAMPLES

In the following examples, the following terminology is used for amino acid substitutions. The first letter indicates the amino acid present in the natural position of the sequence SEQ ID NO: 1. The next number indicates the position in the sequence SEQ ID NO: 1. The second letter indicates a different amino acid; replacement of a natural amino acid. An example would be R396C, where arginine at position 396 sequence SEQ ID NO: 1 is replaced by cysteine. In another example, V158T/M298Q, valine at position 158 of the sequence SEQ ID NO: 1 is replaced by threonine, and methionine at position 298 of the sequence SEQ ID NO: 1 is replaced by glutamine in the same polypeptide factor VII.

Example 1

Construction of DNA encoding FVII-(R396C), FVII-(Q250C), FVII-(P406C), FVII-(407C), FVII-(V158T/M298Q), FVII-(L305V/M306D/D309S), FVII-(K337A), FVII-(L305V) and FVII- (F374P)

Construction of DNA encoding the FVII-(R396C), FVII-(Q250C), FVII-(P406C), FVII-(407C) (one additional C-terminal cysteine), FVII-(M298Q), FVII-(L305V/M306D/D309S), FVII- (K337A), FVII-(L305V) and FVII-(F374P), were obtained by the method of site-directed mutagenesis using superscreen, double-stranded DNA vector with an insert of the human factor FVII (pLN174) and two synthetic primers containing the desired the mutation. Were used in the us following primers:

Oligopeptide primers, each complementary to opposite chain insertion vector was extended during the cyclic changes of temperature using DNA polymerase Pfu. When enabled, primers were developed mutated plasmid containing the offset Nicky. After thermal cyclic reaction product was treated with Dpnl, which is specific against methylated and paleometeorology DNA for cleavage of the parental DNA template and selection synthesized DNA containing the mutation.

Procedures for obtaining design DNA with polymerase chain reaction using specific primers are well known to specialists in this field (see PCR Protocols, 1990, Academic ress, San Diego, California, USA).

Example 2

Getting FVII-(R396C)

The BHK cells were transfusional basically as described previously (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241 to 243) to obtain expression of the variant FVII-(R396C). The factor VII polypeptide was subjected to purification as follows.

Conditioned medium was loaded into a 25-ml column of Q Sepharose Fast Flow (Pharmacia Biotech) after adding 5 mm EDTA, 0.1% Triton X-100 and 10 mm Tris, bringing the pH to 8.0 and bring the conductivity to 10-11 MS/cm by adding water. Elution of balkanipolis in gradient from 10 mm Tris, 50 mm NaCl, 0.1% Triton X-100, pH 8.0 10 mm Tris, 50 mm NaCl, 25 mm CaCl2, 0.1% Triton X-100, pH 7.5. The fractions containing FVII-(R396C), collected and applied to a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk, Bagsvaerd, Denmark)associated with CNBr-activated Separate 4B (Pharmacia Biotech). The column was balanced 50 mm Hepes, pH 7.5, containing 10 mm CaCl2, 100 mm NaCl and 0.02% Triton X-100. After washing equilibrating buffer and balanced buffer containing 2 M NaCl, the bound material was suirable balanced buffer containing 10 mm EDTA instead of CaCl2. Before use or storage was added an excess of CaCl2compared with EDTA, or FVII-(R396C) transferred in Ca2+-containing buffer. The yield of the product at each stage controlled by measuring the level of factor VII ELISA method, and the purified protein was subjected to analysis by the method of SDS-PAGE.

Example 3

Getting FVII-(M298Q)

The BHK cells were transfusional basically as described previously (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241 to 243) to obtain expression of the variant FVII-(V158T/M298Q). The factor VII polypeptide was subjected to purification as follows:

Conditioned medium was loaded into a 25-ml column of Q Sepharose Fast Flow (Pharmacia Biotech) after adding 5 mm EDTA, 0,1% Triton X-100 and 10 mm Tris, bringing the pH to 8.0 and bring the conductivity to 10-11 MS/cm by adding water. Elution of the protein vypolnyali gradient from 10 mm Tris, 50 mm NaCl, 0.1% Triton X-100, pH 8.0 10 mm Tris, 1 M NaCl, 5 mm CaCl2, 0,1% Triton X-100, pH 7.5. The fractions containing FVII-(V158T/M298Q), collected, added 10 mm CaCl2and was applied to a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk, Bagsvaerd, Denmark)associated with CNBr-activated Separate 4B (Pharmacia Biotech). The column was balanced 50 mm Hepes, pH 7.5, containing 10 mm CaCl2, 100 mm NaCl and 0.02% Triton X-100. After washing equilibrating buffer and balanced buffer containing 2 M NaCl, the bound material was suirable balanced buffer containing 10 mm EDTA instead of CaCl2. Before use or storage was added an excess of CaCl2compared with EDTA, or FVII-(V158T/M298Q) transferred in Ca2+-containing buffer. The yield of the product at each stage controlled by measuring the level of factor VII ELISA method, and the purified protein was subjected to analysis by the method of SDS-PAGE.

Example 4

Getting FVII-(L305V/M306D/D309S)

The BHK cells were transfusional basically as described previously (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241 to 243) to obtain expression of the variant FVII-(L305V/M306D/D309S). The factor VII polypeptide was subjected to purification as follows:

Conditioned medium was loaded into a 25-ml column of Q Sepharose Fast Flow (Pharmacia Biotech) after adding 5 mm EDTA, 0,1% Triton X-100 and 10 mm Tris, bringing the pH to 8.0 and bring the conductivity to 10-11 MS/cm by relax the of water. Elution of the protein was performed in a gradient from 10 mm Tris, 50 mm NaCl, 0.1% Triton X-100, pH 8.0 10 mm Tris, 1 M NaCl, 5 mm CaCl2, 0,1% Triton X-100, pH 7.5. The fractions containing FVII-(L305V/M306D/D309S), collected, added 10 mm CaCl2and was applied to a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk, Bagsvaerd, Denmark)associated with CNBr-activated Separate 4B (Pharmacia Biotech). The column was balanced 50 mm Hepes, pH 7.5, containing 10 mm CaCl2, 100 mm NaCl and 0.02% Triton X-100. After washing equilibrating buffer and balanced buffer containing 2 M NaCl, the bound material was suirable balanced buffer containing 10 mm EDTA instead of CaCl2. Before use or storage was added an excess of CaCl2compared with EDTA, or FVII-(L305V/M306D/D309S) transferred in Ca2+-containing buffer. The yield of the product at each stage controlled by measuring the level of factor VII ELISA method, and the purified protein was subjected to analysis by the method of SDS-PAGE.

Example 5

Analysis of the hydrolysis ofIn Vitro

Native (wild-type) factor VIIa variant and factor VIIa (both hereinafter will be referred to as "factor VIIa") was also studied by direct comparison of their specific activities. The analysis was performed in microtiter tablet (MaxiSorp, Nunc, Denmark). Chromogenic substrate D-Ile-Pro-Arg-para-nitroanilide (S-2288, Chromogenix, Sweden), final concentration was 1 mm, was added to VIIa (final concentration 100 nm) in 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 5 mm CaC12and 1 mg/ml bovine serum albumin. The absorption at 405 nm continuously studied in the reader tablets SpectraMaxTM340 (Molecular Devices, USA). Absorption, developing during the 20-minute incubation, after subtraction of the absorption values in the control well containing no enzyme was used to calculate the relationship between the activities of the variants of factor VIIa and factor VIIa wild type:

Ratio=(A405 nmvariant of factor VIIa)/(A405 nmfactor VIIa wild type).

Example 6

Analysis of proteolysisIn Vitro

Native (wild-type) factor VIIa variant and factor VIIa (both hereinafter will be referred to as "factor VIIa") was also studied by direct comparison of their specific activities. The analysis was performed in microtiter tablet (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nm) and factor X (0.8 μm) in 100 μl of 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 5 mm CaC12and 1 mg/ml bovine serum albumin, incubated for 15 minutes. Then cleavage of factor X was stopped by adding 50 μl of 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 20 mm EDTA and 1 mg/ml bovine serum albumin. The number of generated factor Xa was measured by adding a chromogenic substrate Z-D-Arg-Gly-Arg-para-nitroanilide (S-2765, Chromogenix, Sweden), final concentration was 0.5 mm. Will the stop at 405 nm continuously studied in the reader tablets SpectraMax TM340 (Molecular Devices, USA). Absorption developed in the course of the 10-minute incubation, after subtraction of the absorption values in the control well, not containing FVIIa, used for calculating the ratio between the proteolytic activities of the variants of factor VIIa and factor VIIa wild type:

Ratio=(A405 nmvariant of factor VIIa)/(A405 nmfactor VIIa wild type).

Example 7

The relative activity of the FVIIa polypeptides measured in the samples described in examples 5 and 6

Example 8

PEG-conjugation variants factors FVII-(R396C), FVII-(Q250C), FVII-(P406C), FVII-(407C)

Variants of factor VIIa, as described in example 1, with a free thiol group that is built into any of these provisions (250, 396, 406 or 407 (last provision was added to the C-terminal position)), was introduced in the reaction with 5-fold molar excess of PEG-vinylsulfonic or PEG-maleimide (alternative can be used any other jet in relation to the sulfhydryl group of the derivative of PEG) in aqueous buffer for three hours for the reaction to its actual completion. The molecular weight of the PEG-derivative is at least 10000. The resulting PEG-FVIIa tested on amylolyticus and proteolytic activity as described in examples 5 and 6, and if the yavlyalas residual activity of human factor FVIIa wild type or if Cys was built in option factor FVIIa with increased activity, activity after reaction with a derivative of the PEG must be higher than the activity of human factor FVIIa wild type. PEG-conjugated FVIIa was separated from unreacted FVIIa variant and derived free PEG using chromatography, such as gel filtration on a column of Superdex-200, etc.

PEG-conjugation of proteins at the base of the cysteine is well known to specialists in this field and are described in several publications, including the publication Goodson, R. J. & Katre, N. V. (1990) Bio/Technology 8,343 and Kogan, T. P. (1992) Synthetic Comm. 22, 2417.

1. The polypeptide factor VII clotting, essentially corresponding to the amino acid sequence of SEQ ID NO: I, where the amino acid corresponding to amino acid position selected from positions Q250, R396 and R sequence SEQ ID NO: I, replaced by cysteine, or where a cysteine added to the C-end of the sequence SEQ ID NO: 1.

2. The polypeptide factor VII clotting according to claim 1, where the amino acid corresponding to R396 sequence SEQ ID NO: I, replaced by cysteine.

3. The polypeptide factor VII coagulation according to any one of claims 1 or 2, where the amino acid corresponding to Q250 sequence SEQ ID N0: 1, is replaced by cysteine.

4. The polypeptide factor VII coagulation according to any one of claims 1 to 3, where the amino acid corresponding to R sequence SEQ ID NO: 1, is replaced by cysteine.

5. Derived VII blood clotting, containing polypeptide factor VII clotting, essentially corresponding to the amino acid sequence of SEQ ID NO: 1, where the amino acid corresponding to amino acid position selected from positions Q250, R396 and R sequence SEQ ID NO: 1, is replaced by a cysteine, or where a cysteine added to the C-end of the sequence SEQ ID NO: 1, where the specified cysteine anywhereman with a chemical group chemical group is polyethylene glycol, which increases the true molecular weight of the specified polypeptide factor VII clotting 300 Yes to approximately 100000 Yes, and where the specified derived factor VII clotting blood has essentially the same activity or increased half-life in serum compared with recombinant human factor VIIa coagulation wild-type.

6. The derived factor VII clotting according to claim 5, where the specified polypeptide factor VII clotting is a polypeptide according to any one of claims 1 to 4.

7. The derived factor VII coagulation according to any one of pp.5 and 6, where this chemical group is essentially neutral.

8. The derived factor VII coagulation according to any one of pp.5-7, where this chemical group increases the true molecular mass derived factor VII clotting approximately 1000 Yes the ome to 80000 Yes.

9. The derived factor VII coagulation according to any one of pp.5-8, where this chemical group increases the true molecular mass derived factor VII clotting approximately 5,000 Da to about 60000 Yes.

10. The derived factor VII coagulation according to any one of pp.5-9, where this chemical group increases the true molecular mass derived factor VII clotting about 10,000 Da to about 40,000 Da.

11. The derived factor VII coagulation according to any one of pp.5-10, where this chemical group is selected from one to six molecules of polyethylene glycol.

12. The derived factor VII clotting in claim 11, where this chemical group represents one molecule of polyethylene glycol.

13. The derived factor VII coagulation according to any one of pp.5-12, where this chemical group conjugated with a free sulfhydryl group present on the replacement included or added to the polypeptide with the amino acid.

14. The derived factor VII coagulation according to any one of pp.5-13, where this chemical group conjugated with cysteine.

15. Nucleic acid encoding a polypeptide factor VII clotting of the blood, characterized by nucleotide sequence corresponding to the amino acid sequence of the polypeptide according to Liu the WMD one of claims 1 to 4.

16. Expressing the vector, designed for eukaryotic host cells of the mammal that contains a nucleic acid according to item 15.

17. Eukaryotic cell-host mammal for expression of the polypeptide factor VII coagulation according to any one of claims 1 to 4, where specified a host cell contains a nucleic acid encoding a specified polypeptide factor VII clotting.

18. Eukaryotic cell-host mammal in 17, where specified a host cell selected from the group consisting of cells Cho cells KSS or SOME cells.

19. The method of producing the polypeptide of factor VII coagulation according to any one of claims 1 to 4, comprising culturing the eukaryotic host cell of a mammal according to any one of p and 18 in a suitable growth medium under conditions allowing protein synthesis from a specified polynucleotide constructs, and obtaining the specified polypeptide factor VII clotting of the culture medium.

20. The method of producing a derivative of factor VII clotting, which includes stage

a) production of the polypeptide factor VII clotting the method according to claim 19;

b) conjugation of the polypeptide factor VII clotting chemical group;

c) applying the derived factor VII coagulation column casinoonline the chromatography or gel filtration column; and

(d) elution derived factor VII clotting.



 

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12 cl, 6 dwg, 4 tbl, 4 ex

FIELD: technological processes; pharmacology.

SUBSTANCE: antagonist of human interleukine-1 receptor is prepared with the help of recombinant strain E. coli that contains plasmid, which provides production of this protein. For this purpose the strain is cultivated. Then from bacterial cells the target product is separated with application of three-stage chromatography and concentration on hydrophobic sorbent. At that the first and the third stage of mentioned chromatographic purification is performed on cation-exchanging resin, and in the second stage of chromatography the anion-exchanging resin is used.

EFFECT: application of invention allows to prepare antagonist of human receptor of high purity.

7 cl, 1 tbl, 4 ex

FIELD: technological processes; medicine.

SUBSTANCE: invention is related to preparation of recombinant analogues of human gamma-interferon and may be used in medicine for prophylactics and treatment of oncological diseases, neoplasms and inflammatory processes of humans. Highly pure genetically engineered analogue of human gamma-interferon - deltaferon with molecular mass of 16.2 kilo Daltons is produced by microbiological synthesis with further chromatographic purification on "KM"-sepharose - cationic-exchange sorbent with high linear speed of flow and suitable for preparative loads, at different pH values. On the basis of deltaferon, which contains by data of polyacrylamide gel-sodium dodecyl sulfate electrophoresis at least 98% of main substance in the form of monomer that possesses antiproliferative and anti-inflammatory activities inherent in gamma-interferon, medical product is prepared, which also includes low molecular polymer filler-stabiliser (reopolyglukine or polyvinylpyrrolidone) and salt buffer system with pH 7.0-7.1.

EFFECT: highly active and stable preparation of deltaferon is produced.

2 cl, 6 dwg, 1 tbl, 3 ex

FIELD: technological processes.

SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.

EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.

7 cl, 20 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: invention concerns glycoforms of VII factor and compositions of VII factor, characterized by modified configurations on basis of asparaginic oligosaccharide chains. In addition invention includes detection method applied for polypeptide glycoforms of VII factor, receiving method and disease treatment method as well.

EFFECT: identification of biologically active forms of recombinant VII factor.

53 cl, 5ex

FIELD: biotechnology, molecular biology, proteins.

SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.

EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.

24 cl, 21 tbl, 32 ex

FIELD: peptides.

SUBSTANCE: invention relates to biologically active peptides possessing antibacterial effect that can be used in biotechnology and medicine. Peptide latarcin shows the following amino acids sequence: H2N-Gly1-Phe2-Phe3-Gly4-Lys5-Met6-Lys7-Glu8-Tyr9-Phe10-Lys11-Lys12-X13-Gly14-Ala15-Ser16-Phe17-Lys18-Arg19-Arg20-Phe21-Ala22-Asn23-Leu24-Lys25-Lys26-Arg27-Leu28-NH2 wherein X represents phenylalanine Phe13 or tryptophan Trp13 residue, and possesses the broad spectrum of antibacterial activity and expressed toxic effect with respect to human promyelocyte leukemia. Using the invention allows expanding assortment of natural peptide antibiotics not containing cysteine residues and possessing high antibacterial activity.

EFFECT: valuable medicinal properties of peptide.

2 tbl, 5 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: method includes definition of target HLA immunotypr, mitotic activation of unfertilised diploid oocytes after meiosis 1 by means of calcium ionophore for development blastocyst masses, each of them contains internal cellular mass (ICM), homozygous on target HLA immunotype, allocation of homozygous stem cells from ICM, plating of these cells for obtaining of lines of cells and selection of cells, homozygous on target HLA immunotype. The way obtaining of bank of these cells obtained from many donors is also described.

EFFECT: advantage of the invention lies in possibility of obtaining of considerable quantity of histocompatible cells for transplantation.

10 cl, 4 ex, 11 dwg

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and concerns obtaining factor VII protein by method of recombinant DNA. Recombinant plasmid DNA was constructed for expression of blood clotting factor VII in mammalian cells, which is product of ligating of fragment of cDNA of human factor VII gene, flanked by sites of restrictases Xhol and BamHI recognising, with large XhoI/BglII fragment of vector pEFZeo, including genes of resistance to ampicillin and zeocin. As result of BHK cell transformation with new recombinant plasmid, cell line BHK/F7 was obtained, which produces recombinant protein of factor VII with output of up to 40 mkg/ml.

EFFECT: obtaining cell line producing recombinant protein of factor VII.

2 cl, 4 dwg, 4 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of genetic engineering and molecular biology. Recombinant adenovirus vector is obtained, which expresses receptor, conjugated with G-protein, EDG2 and GFP-protein, as well as transfected myocardial mammalian cell and transgenic mammal.

EFFECT: obtaining recombinant adenovirus vector which can be applied in veterinary and medicine as cardio-vascular collapse model.

17 cl, 4 dwg

FIELD: agriculture.

SUBSTANCE: development of a reliable means for the identification of the corresponding transformation event in the plant genome. As a result of agrobacterial transformation of the Lugovskoy potato with a genetic maker containing the gene cryIIIa, and the analysis of the resulting transgenic plants demonstrating resistance to the Colorado beetle, the transgenic line (transformation event) 1210 amk has been selected. In the genomic DNA of the plants belonging to this line a fragment has been identified and sequenced, related to the genetic maker insertion area and being a recombinant sequence, one part of which relates to the genetic maker inserted, and the other part relates to the flanking area of the Lugovskoy potato genomic DNA. It is proposed to use the identified recombinant sequence as an identifier of this unique transformation event.

EFFECT: obtaining transgenic potato that is resistant to Colorado beetle and complies with requirements of biological and food safety on basis of high yield Russian variety.

5 cl, 5 dwg, 7 tbl, 7 ex

FIELD: cosmetology.

SUBSTANCE: strain of cell culture Polyscias filicifolia (MOORE EX ROUNIER) BAILEY BFT 01-95 is received from tylosis of hothouse plant leaf of Botanical Institute Russian Academy of Sciences Saint-Petersburg city. It is deposited in All-Russian collection of cell culture (specialized collection of higher plant cells, K.A. Timiryazev VNIIFR) under № BPKK-BP № 58. Invention can be used for receiving of medicinal preparations, nutritional supplements, products of functional meals, cosmetics.

EFFECT: field of invention application is extended.

1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to genetic engineering. New interferon-binding proteins, which modulate activity of different interferon-α subtypes, as well as interferon-β activity, are obtained. Described is cloning of DNA fragment, which codes interferon-α/β, binding protein IFNAB-BPI, and expression of obtained DNA fragment in host-cells both with formation of respective polypeptide and in form of fused proteins. Practical application of obtained proteins as components of pharmaceutical compositions for inhibiting activity of IFN-α or IFN-β is suggested. Invention can be applied in medicine for inhibiting undesirable impact of IFN-α or IFN-β.

EFFECT: obtaining new interferon-binding proteins, which can find application in medicine for inhibiting undesirable impact of IFN-α or IFN-β.

13 cl, 10 dwg, 6 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: method of obtaining the live organization of cells of a similar tissue is revealed, that is the macromass culture including three-dimensional designs similar to tissue, without the aid of a skeleton or a foreign matrix. The cells are plated in a vessel for cell culture with high density per unit area in a range from 3.33×105 to 3×106 cells per cm2. The three-dimensional organization of the cells received in the specified way similar to a tissue is described. When cultivating cell macromasses it is possible to receive the cells, organized in forms similar tissues without the aid of a matrix, and three-dimensional macroscopical structures similar to tissue. In the given work the organization similar to a tissue and macroscopical structures similar to tissue are obtained from fibroblasts of a skin, the osteogene cells received from fat stromal cells, chondrocytes and from osteoblasts. Thus any skeleton or a foreign matrix for obtaining of a tissue or any other agents is not used (except high density of cell plating) which promote formation of a tissue, tissues have completely cellular parentage.

EFFECT: present invention allows simplifying the method of obtaining of substitutes of a human body tissue.

13 cl, 24 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method of obtaining the live organization of cells of a similar tissue is revealed, that is the macromass culture including three-dimensional designs similar to tissue, without the aid of a skeleton or a foreign matrix. The cells are plated in a vessel for cell culture with high density per unit area in a range from 3.33×105 to 3×106 cells per cm2. The three-dimensional organization of the cells received in the specified way similar to a tissue is described. When cultivating cell macromasses it is possible to receive the cells, organized in forms similar tissues without the aid of a matrix, and three-dimensional macroscopical structures similar to tissue. In the given work the organization similar to a tissue and macroscopical structures similar to tissue are obtained from fibroblasts of a skin, the osteogene cells received from fat stromal cells, chondrocytes and from osteoblasts. Thus any skeleton or a foreign matrix for obtaining of a tissue or any other agents is not used (except high density of cell plating) which promote formation of a tissue, tissues have completely cellular parentage.

EFFECT: present invention allows simplifying the method of obtaining of substitutes of a human body tissue.

13 cl, 24 dwg, 1 tbl

FIELD: medicine; biotechnologies.

SUBSTANCE: ovaries cell line of goat Capra hircus L is obtained in Russian Academy of Agrarian Sciences under № 63. The line is intended for a reproduction of animal viruses - a virus of an aphthous fever of types A, O, Asia-1, a virus of Aujeszky's disease, a virus of a sheep smallpox, a virus of a classical plague of pigs, a pneumovirus of birds, with a view of their detecting, allocation, carrying out of virologic researches, and also for manufacturing of diagnostic and preventive veterinary preparations.

EFFECT: increase of efficiency.

7 tbl, 14 ex

FIELD: medicine; pharmacology.

SUBSTANCE: allocated human monoclonal antibodies which specifically bind a receptor of the epidermal growth factor (EGFR), and also corresponding compositions on the basis of antibodies and a biospecific molecule are described. Human antibodies can be received with use of the transgenic mouse capable to formation of set of isotypes of human monoclonal antibodies by recombination V-D-J and switching of isotypes. The pharmaceutical compositions containing human antibodies for treatment or prevention of diseases, mediated by expression EGFR, the transgenic animals distinct from a human, the specified expressing antibodies, hybridomes and transfectomes which produce human antibodies are also presented. Ways of therapy and diagnostics of the diseases mediated by expression EGFR, with use of human antibodies or their antigen-binding of fragments, and also methods of growth suppression of the cells expressing EGFR, and an induction of cytolysis of the specified cells are described.

EFFECT: invention allows obtaining therapeutic and diagnostic preparations of antibodies with improved properties.

53 cl, 22 dwg, 4 tbl, 11 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of genetic engineering and molecular biology. Recombinant adenovirus vector is obtained, which expresses receptor, conjugated with G-protein, EDG2 and GFP-protein, as well as transfected myocardial mammalian cell and transgenic mammal.

EFFECT: obtaining recombinant adenovirus vector which can be applied in veterinary and medicine as cardio-vascular collapse model.

17 cl, 4 dwg

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