Title of Invention

NOVEL HUMAN COAGULATION FACTOR VII DERIVATIVES

Abstract The present invention relates to a Factor VII polypeptide comprising the amino add sequence of SEQ 10 NO: 1 or a vari- ant thereof, wherein an amino acid has been substituted with a different amino add, wherein said different amino acid Is capable of being conjugated with a chemical group and wheretn said Factor V.II polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIl.
Full Text

COAGULATION FACTOR VII DERIVATIVES
FIELD OF THE INVENTION
The present invention relates to novel human coagulation Factor VII derivatives. Factor VII polypeptides, as well as polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions comprising Factor VII derivatives, uses and methods of treatment
BACKGROUND OF THE INVENTION
Blood coagulation Is a process consisting of a complex Interaction of various blood components (or factors) that eventually gives raise to a fibrin clot Generally, the blood components; which participate in what has been referred to as the coagulation 'cascade', are enzymatlcally inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself Is an activated dotting factor). Coagulation factors that have undergone such a conversion are generally refierred to as 'active factors', and are designated by the addition of theletter "a" to the name of the coagulation factor (e.g. Factor Vila).
Initiation of the haemostatib process is method by the formation of a demplex between tissue factor, exposed as a result of injury to the vessel wall, and Factor Vila. This complex then converts Factors IX and X to their active fomis. Factor Xa converts limited amounts of prothrombin to thrombin on the tissue factor-bearing cell. Thrombin activates platelets and Factors V and VIII into Factors Va and Villa, both cofactors in the further process leading to the full thrombin burst This process includes generation of Factor Xa by Factor IXa. On complex with factor Villa) and occurs on the surface of activated platelets. Thrombin finally converts fibrinogen to fibrin resulting In fonnation of a fibrin dot.
Factor VII is a trace plasma glycoprotein thai circulates in blood as a single-chain zymogen The zymogen Is catalytically Inactive. Single-chain Factor VII may be converted to two-chain Factor Vila by Factor Xa, Factor Xlla, Factor IXa. Factor Vila thrombin in vitro. Factor Xa is believed to be the major physiological activator of Factor VII. The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal Argi6rltei53 peptide bond.
It is often desirable to stimulate or to selectively block the coagulation cascade in a subject Factor Vila has been used to control bleeding disorders that have several causes such as dotting factor deficiencies (e.g. haemophilia A and B or deficiency of coagulation Factors XI or VII) or dotting factor inhibitors. Factor Vila has also been used to control ex-

cessive bleeding occunring in subjects with a normally functioning blood dotting cascade (no clotting factor deficiendes or inhibitors against any of the coagulation factors). Such bleeding may, for example, be caused by a defective platelet fundion, thrombocytopenia or von Wille-brand's disease. Bleeding is also a major problem in connection with surgery and other forms of tissue damage.
European Patent No. 200,421 (ZymoGenetics) relates to the nucleotide sequerice^ encoding human Fador VII and the recombinant expression of Fador VII in mammalian cells.
Dickinson et al. {Proc. Natl, Acad. ScL USA 93,14379-14384,1996) relates to Factor VII polypeptides wherein Lys157, Val158, Glu296. Met298, Asp334. Ser336 or Lys337 have been individually replaced by Ala. Iwanaga et al. (TTrromix.Hgemost (supplement au-' gust 1999), 486, abstract 1474) relates to FactorVlla variants wherein residues 316-320 are deleted or residues 311-322 are replaced with the ccresponding residues from trypsin.
Anticoagulants such as heparin, coumarin, derivatives of coumarin. indandione derivatives, or other agents may be used to selectively block the coagulation cascade in a patient, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated in- .
travascular coagulation (DIC).and a host of other medical disorder. For example,.heparin
treatment or extracorporeal treatment with citrate ion (U.S. Pat No. 4,500,309) may be used In dialysis to prevent coagulation during thecourse of treatment. Heparin is also used in preventing deep vein thrombosis in patients undergoing surgery.
Treatment with heparin and other anticoagulants may, however, have undesirable side effects. Available anticoagulants generally act throughout thebody, rather than acting specificaily at a dot site. Heparin, for example, may cause heavy bleeding. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly deared from the blood. necessitating frequent administration. Because heparin acts as a cofactor for antithrombln 111 (AT 111), and AT 111 is rapidly depleted in DIG treatment, it Is often difficult to maintain theproper heparin dosage, necessitating continuous monitoring of AT III and heparin levels. Heparin Is also ineffective if AT III depletion is extreme. Further, prolonged use of heparin may also increase platelet aggregation and reduce platelet count, and has been Implicated In the development of osteoponosis. Indandione derivatives may also have toxic side effects.
In addition to the articoagufants briefly described above, several naturally occurring proteins have been found to have anticoagulant activity. For example. Reutelingsperger (U.S. Pat. No. 4.736,018) isolated anticoagulant proteins from bovine aorta and human umbilical vein arteries. Maki etal. (U.S. Pat No. 4,732,691) disclose human plabenta-derived anticoagulant proteins. In addition, AT III has been proposed as a therapeutic anticoagulant

(Schipper et a!.. Lancet 1 (8069): 854-856 (1978); Jordan, U.S. Pat No. 4.386,025; Bock et a!.. U.S. Pat No. 4.517.294).
In about 30% or more of patients treated by angioplasty, endarterectomy or bypass grafts, thrombosis and/or smooth muscle cell proliferation in the intima causes re-occlusion of the vessel and consequent failure of the reconstructive surgery. This closure of the vessel subsequent to surgery is known as restenosis. Restenosis is thought to result from a complex interaction of bidogical processes including platelet deposition and thrombus formation, release of chemotactic and mitogenic factors, and the migration and proliferation of vascular smooth muscle cells into the intima of the dilated arterial segment.
The inhibition of platelet accumulation at sites of mechanical injury can limit the rate of restenosis in human subjects. While platelet accumulation occurs at sites of acute vascu- lar injuries, thegeneration of thrombin at these sites may be responsible for the activation of the platelets and their subsequent accumulation.
International Application No. WO-92/15686 relates to inactivated Factor Vila, poly-nucleic acid and mammalian cell lines for the production of inactivated Factor Vila, and compositions comprising inactivated Factor Vila for inhibiting blood coagulation.
International Application No. WO 94/27631 relates to methods for inhibiting vascular restenosis, tissue factor activity, and platelet deposition.
International Application No. WO 96/12800 relates to a method for treatment of acute closure of a coronary artery comprising to the individual a composition which comprises inactivated Factor Vila In conjunction with tissue plasminogen activator or streptokinase.
Most proteins intiroduced into the circulation, are cleared quickly from tine mammalian subject by the kidneys. This problem may be partially overcome by administering a larger amount of the protein or through repeated administration. However, higher doses of the protein can elicit antibodies which can bind and inactivate the protein and/or facilitate the clearance of the protein from thesubject's body. Repeated administration of the therapeutic protein is essentially ineffective and can be dangerous as it can elicit an allergic response.
Various attempts to solve the problems associated with protein therapies include microencapsulation, liposome delivery systems, administration effusion proteins, and chemical modification. The most promising of these to date is modification of the therapeutic protein by covalent attachment of potyalkylene oxide polymers, particulariy polyethylene glycols (PEG). For example. U.S. Pat No. 4,179.337 discloses the use of PEG or polypropylene glycol coupled to proteins to provide a physiologically active non-immunogenic water soluble polypeptide composition. Nucd et al. describe several proteins which have been modified by addition of PEG including adenosine deamidase. L-asparaginase, interferon alpha 2b (IFN-

a2b), superoxide dismutase, streptokinase, tissue plasminogen activator (tPA), urokinase, uricase, hemoglobin, interieukins, interferons, TGF-beta, EGF, and other growth factors (Nucd et a!., 1991. adv.Drug. Delivery Rev. 4:133-151). Attempts such as these have resulted in somewhat longer half-life of the proteins and reduction of protein Immunogenicity.
Typically, PEGylation of proteins involves activating PEG with a functional group which will react with lysine residues on the surface of the protein. If the modification of the protein goes to completion, the activity of the protein is usually lost Modification procedures. which allow partial PEGylation of the protein usually result in only about 50% loss of activity and greatly increased serum half-life, so that the overall effective dose of the protein is lower.
Recent developments in protein PEGylation methods employ activated PEG reagents which react with thiol groups of the protein, resulting in covalent attachment of PEG to a cysteine, which residue was inserted in place of a naturally occurring lysine of the protein. Shaw et al. (U.S. Pat No. 5,166,322) describe specific variants of IL-3 which have a cysteine introduced at specific sites within the naturally occurring amino acid sequence, Sulfhydryf reactive compounds (e.g. activated polyethylene glycol) are then attached to these cysteines by reaction with the IL-3 variant Katre et al. (U.S. Pat. No. 5.206.344) describe specific IL-2
variants which contain a cysteine introduced at a specific site within the naturally occurring

amino acid sequence. The IL-2 variant is subsequently reacted with an activated polythyl
ene glycol reagent to attach this moiety to a cysteine.
There is still a need in the art for improved Factor VII polypeptides having prolonged procoagulant or anticoagulant activity. In particular, there is a need for Factor VII polypeptides which has increased serum half-life without the undesirable side effects such as systemic activation of the coagulation system and bleeding, respectively, associated with conventional therapies, and which can be administered at relatively low doses so that repeated administrations of a larger amount of the protein are avoided.
DESCRIPTION OF THE INVENTION
The present invention relates to novel coagulation Factor VII polypeptides with the same or increased activity compared to wild type Factor Vila and to Factor VII derivatives having Increased serum half-lifes.
Areas in the Factor Vila molecule has been Identified where changes to the primary structure as well as other modifications are allowed without Influencing or reducing the biological activity of Factor Vila. The areas within the structure of Factor Vila, which have been identified not to be involved in the binding to tissue factor or to Factor X includes the amino acid positions from 247 - 260 and from 393 - 406 of SEQ ID NO: 1. Specifically the amino

acids in positions Q250, R396, and P406 of the sequence of SEQ ID NO: 1, have been ana-ysed for the introduction of cysteine (Cys) residues. The introduction of a Cys residues is "oilowed by the conjugation with a chemical group, e.g. polyethylene glycol (PEG) in order to ncrease the half-life in circulation of the Factor VII derivative. A cysteine has also been intro juced in the C-terminal sequence of SEQ ID NO: 1 (refen-ed to as 407C), which is followed by the conjugation of PEG. Also this addition of a cysteine in the C-terminal sequence of 3EQ ID NO: 1 is without reduction in proteolytic activity of Factor Vila.polypeptides. These -actor VII derivatives, e.g. a Factor VII polypeptide conjugated with a PEG molecule, are :herapeuticaily useful in situations where a prolonged effect of Factor VII polypeptides is desirable, e.g. in situations where repeated administration or administration of a larger amount of the Factor VII polypeptide is inconvenient or problematic. Furthermore, tine Factor Vila p0lypeptid.es of the present invention with introduced amino adds (e.g, a Cys residue) capa-ple of being conjugated with a chemical group at positions in the Factor Vila molecule, which do no influence the proteolytic activity, may be used to introoduce any functional group of a xnjugate of Factor VII.
In a first aspect, the present invention relates to a Factor VII polypeptide comprising Jie amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has yeen substituted with a different amino acid, wherein the different anrtino acid is'capable of Deing conjugated with a chemical group and wherein the Factor VII polypeptide has substan-jally the same activity or Increased activity compared to recombinant wild type human Factor yila.
In a second aspect, the invention relates to a Factor Vli polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid X)rresponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ D NO: 1 has been substituted with a different amino acid. It Is to be understood that any amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 can be substituted with a different amino acid without substantially reduction in activity of tine Factor /I I polypeptide.
In a third aspect, the invention relates to a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid. It is to be understood, that the first letter in R396, 3250 and P406 represent the amino add naturally present at the indicated position of SEQ
DNP:J.
In a further aspect the invention relates to a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable

of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa, It is to be understood, that the amino add may be inserted within the sequence of SEQ ID NO: 1, without replacing any amino acid. The insertion of an amino acid may be at the same position within the sequence of SEQ ID NO: 1, where an amino acid is further substituted. Thus, in one embodiment the amino acid insertion is followed ty an amino add substitution or vice versa.
In a further aspect, the invention relates to a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof.
The term "an amino add" as used herein means one or more amino acrds. It is to be understood that the amino add replacing the amino add in or being inserted into or being added to the Factor VII polypeptide is capable of being conjugated with any chemical group that will increase the actual molecular weight of the Factor VII polypeptide. This conjugation with the chemical group indudes but are not limited to covalent attachment of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, colominic acids or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives.
Preferably the chemical group is a biocompatible, non-toxic, non-immunogenic and water-soluble polymer. Preferably the chemical group is water-soluble in all prcpcrtions.
This amino add substitution, insertion, or addition and conjugation with a chemical group is without substantial reduction of procoagulant activity of the activated form of the Factor VII derivative compared with recombinant wild type human Factor VIIa.
The term 'Factor VII polypeptide" as used herein means any protein comprising the amino acid sequence 1-406 of native human Factor VII (SEQ ID NO: 1) or variants thereof. This indudes but are not limited to hunnan Fador VII, human Factor VIIa and variants thereof.
The tenns "factor Vll". or "FVir as used herein means a product consisting of the unactivated form (fador VII). The term "factor VIIa", or"FVIIa" as used herein means a product consisting of the activated form (factor VIIa). This indudes proteins that have the amino acid sequence 1-406 of native human Factor VII or Fador VIIa, it also indudes proteins with a slightly modified amino acid sequence, for instance, a modified N-terminal end including N-

terminal amino acid deletions or additions so long as those proteins substantially retain the activity of Factor VIIa. "Factor Vll" or "Factor VIIa" within the above definition also includes natural allelic variations that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.
The terms "variant or "variants", as used herein, is intended to designate human Fac
tor Vll having the sequence of SEQ ID N.0:1. wherein are or more amino adds of the parent
protein have been substituted by another amino acid and/or wherein one or more amino acids
of the parent protein have been deleted and/or wherein one or more amino adds have been
inserted in protein and/or wherein one or more amino acids have been added to the parent pro
tein. Such addition can take place either at the N-terminal end or at the C-terminal end of the
parent protein or both. .
The term "substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa", as used herein, means an activity more than 70 % of the activity of recombinant wild type human Factor VIIa, In one embodiment the activity is more than 80 % of the activity of recombinant wild type human Factor VIIa. In another embodiment the activity is more than 90 % of the activity of recombinant wild type human Factor VIIa, In a further embodiment the activity Is more than 100 % of the activity of recombinant wild type human Factor VIIa. In a further embodiment the activity is more than 120 % of the activity of recombinant wild type human Factor VIIa, In a further embodiment the activity is more than 200 % of the activity of recombinant wild type human Factor VIIa. In a further embodiment the activity is more than 400 % of the activity of recombinant wild type human Factor VIIa.
The term "Factor Vll derivative" as used herein, is intended to designate a Factor Vll polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, in which one or more of the amino adds of the parent peptide have been chemically modified, e.g. by alkylation, PEGy-lation, acylation, ester formatfon or amide formation or the like. This includes but are not limited to PEGyiated human Factor VIIa, cysteine-PEGylated human Factor VIIa and variants thereof.
The tenri "PEGyiated human Factor VIIa" means Fador VIIa, having a PEG molecule conjugated to an amino add of the human Factor VIIa polypeptide.
The term "cysteine-PEGylated human Factor VIIa " means Factor VIIa having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor VIIa.
The term "a different amino add" as used herein means one or more amino acids that are different from that amino acid naturally present at that position. This includes but are not limited to amino adds that can be encoded by a polynucleotide. Preferably the different

amino add is in natural L-form and can be encoded by a polynudeotide. A specific example being L-cysteine (Cys).
The temi "activity" as used herein means the ability of a Factor VII polypeptide to convert its substrate Fador X to the active Fador Xa. The activity of a Factor VII polypeptide may be measured with the "/n Vitro Proteolysis Assay" (see Example 6).
The term "polyethylene glycol" or TEG" means a polyethylene glycol compound or a derivative thereof, with or without coupling agents, coupling or activating, moeities (e.g., with thiol, triflate, tresyiate. azirdine. oxirane, or preferably with a maleimide moiety). Compounds such as maleimido monomethoxy PEG are exemplary of activated PEG compounds of the invention.
In a further asped, the invention relates to a Factor VII derivative comprising a Factor VII polypeptide having the amino add sequence of SEQ ID NO: 1 ora variant thereof,, wherein an amino acid has been substituted with a different amino add, wherein the different amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Fador VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
The term "a chemical group" as used herein means'one or more chemical groups.
In a further aspect, the invention relates to a Factor VII derivative comprising a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add at a position selected from 247-260, 393-405 or 406 of SEQ ID NO; 1 has been substituted with a different amino add, wherein the different amino acid is conjugated with a chemical group that increases the actual mo-, lecular weight of the Fador VII polypeptide with aboyt 300 daltons to about 100,000 daltons and wherein the Fador VII derivative has substantially the same activity or increased adivrty compared to recombinant wild type human Fador VIIa.
In a further aspect, the invention relates to a Fador VII derivative comprising a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add con'espondlng to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid, wherein the different amino acid is conjugated with a chemical, group that increases the adual molecular weight of the Fador VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa,
In a further aspect, the invention relates to a Fador VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

wherein an amino acid has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased'activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a composition comprising a Factor VII
derivative comprising a Factor VII poiypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein the different amino acid is conjugated with a chemical group that increases the actual molecuiar weight of the Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein the Factor VIl derivative has substantially the samS'activfty or Increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a composition comprising a Factor VIl derivative comprising a Factor VIl polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid, wherein the different amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein the Factor VIl derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention nslates to a composition comprising a Factor VIl derivative comprising a Factor VIl polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant tinereof, wherein an amino acid corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add, wherein Uie different amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VIl derivative has substantially the same activ-ity or increased activity compared to recdmbinant wild type human Factor VIIa.

In a further aspect, the invention relates to a composition comprising a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein the amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VIJ derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VI la.
In a further aspect, the invention relates to a composition comprising a Factor VI! derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been added to theN- or C-terminal of SEQ ID NO: 1 or a variant thereof and wherein theamino add is conjugated with a chemical group that inaeases the actual molecular weight Of the Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a phamiaceuticai composition comprising a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a dif-fereotamino acid, wherein the different amino add Is congated with a chemical group that increases theactual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or iricreased adlvity compared to recombinant wild type human Factor VIIa; and optionally, a pharmaceuticaily acceptable carrier.
In a further aspect, the Invention relates to a phannaceutical ccmposition comprising a Factor VII derivative comprising a Factor VII polypeptide comprising the amino add se^ quence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid coaesponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino add, wherein the different amino acid is conjugated with a chemical group that increases theactual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially thesame activity or increased activity compared to recombinant wild type human Factor VIIa; and optionally, a pharmaceuticaily acceptable carrier.
In a further aspect, Uie inventi'on relates to a phannaceutical composition comprising a Factor VII derivative comprising a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add selected from R396. Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid, wherein the different amino acid is conjugated with a chemical group

that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa; and optionally, a phamiaceutically acceptable carrier.
In a further aspect, the invention relates to a phamTaceutlcal composition comprising a Factor VII derivative comprising a. Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein arramino ad?l has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa; and optionally, a pharmceutically acceptable carrier.
In a further aspect, the invention relates to a pharmaceutical composition comprising a Factor VII derivative comprising a Factor VII polypeptide having the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been added to the N- or C-terminal of SEQ !D NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor Vll derivative his substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa; and optionally, a pharmaceutically acceptable cam'er. In a further aspect, (he invention relates to an inactivated Factor Vll polypeptide, wherein a Factor VII polypeptide is further modified in its catalytic center, which modification inhibits the ability of the Fador Vll polypeptide to activate plasma Fador X or IX In one embodiment the inactivated Factor Vll polypeptide is modified in its catalytic center with a serine protease inhibitor. In a further embodiment the inactivated Factor Vll polypeptide is modified in its catalytic center with a peptide halomethyl ketone seleded from the group consisting of: Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone, D-Phe-Phe-Arg chloromethyl ketone, 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 chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Phe-Arg chloromethyl ketone, Dansyl-D-Phe-Phe-Arg chloromethylketone, Dansyi-Phe-Pro-Arg chloromethylketone. Dansyl-D-Phe-Pro-Arg chloromethylketone, Dansyl-Phe-Pro-Arg chlQromethylketone, Dansyl-D-Phe-Pro-Arg chloromethylketone, Dansyl-L-Glu-Gly-Arg chloromethylketone and Dansyl-D-Glu-Gly-Arg chloromethylketone.

^ .

The temi "inactivated Factor Vll polypeptide" as used herein means a Factor Vll polypeptide with no ability to activate plasma Factor X or IX.
In a further aspect, the invention relates to an inactivated Factor Vll derivative, wherein an inactivated Factor Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof and having a modification in its catalytic center, which modification inhibits the ability of the Factor Vll polypeptide to activate plasma Factor X or IX, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor Vll polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to an inactivated Factor VII derivative,
wherein an inactivated Factor Vll polypeptide comprising the amino acid sequence of SEQ
ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different
amino acid, wherein the different amino acid is capable of being conjugated with a chemical
group and wherein the Factor Vll polypeptide has substantially the same activity or increased
activity compared to recombinant wild type human Factor VIIa and having a modification in
its catalytic center, which modification inhibits the ability of the Factor Vll polypeptide to
activate plasma Factor X or IX, is further conjugated with a chemical group that increases the
actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to
about 100,000 daltons.
In a further aspect, the Invention relates to an inactivated Factor Vll derivative, wherein an inactivated Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260,393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino add and having a modification in its catalytic center, which modification inhibits the ability of the Factor Vll polypeptide to activate plasma Factor X or IX. is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor Vll polypeptide with about 300 daltons to about 100,000 daltors.
In a further aspect, the invention relates to an inactivated Factor Vll derivative. wherein an inactivated Factor Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino acid selected from R396. Q250 or P406 of SEQ ID NO: 1 has been substituted with a different 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor Vll polypeptide with about 300 daltons to about 100,000 daltons. . In a further aspect, the invention relates to an inadivated Factor Vll derivative, wherein an inactivated Factor Vll polypeptide comprising the amino acid sequence of SEQ

ID NO: 1 or a variant thereof, wherein an amino add capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa and having a modification in Its catalytic center, which modification inhibits the ability of theFactor VII polypeptide to activate plasma Factor X or IX, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daitons.
In a further aspect, the invention relates to an Inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant 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, is further conjugated with a chemical group that increases theactual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daitons.
In a further aspect, the invention relates to a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the a'miho acid sequence of SEQ ID NO: 1 or a variant 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 is further conjugated with a chemical group that Increases tfie actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daitons.
In a further aspect, the invention relates to a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been substituted wiUi a different amino add, wherein ti^e different amino acid is capable of being conjugated with a chemical group and wherein the Fador VII polypeptide has substantially the same activity or increased adivity compared to recombinant wild type human Factor VIIa and having a modification in its catalytic center, which modification inhibits the ability of the Fador VII polypeptide to activate plasma Fador X or IX, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further asped. the invention relates to a composition comprising an inadivated Fa'dor VII derivative, wherein an inadivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to

an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof. wherein an amino acid capable of being conjugated with a chemical group has been Inserted within the sequence of SEQ ID NO: 1 or " a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased.activity compared to recombinant wild type human Factor VIIa 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide wfth about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a pharmaceutical composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof and having a modification in its catalytic center, which modification inhibits tine ability of the Factor VII polypeptide to acti-

vate plasma Factor X or IX is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons; and optionally, a phamiaceutically acceptable cam'er.
In a further aspect, the invention relates to a pharmaceutical composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NQ: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein the different amino acid is capable of being conjugated with a chemical group and wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa and having a modification in its catalytic center, which nrodrfication inhibits the ability of theFactor VII polypeptide to activate plasma Factor X or IX. is further conjugated with a chemical group that increases theactual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons; and optionally, a pharmaceutically acceptable carrier.
In a further aspect, theinvention relates to a phannaceutical composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at position setected from 247-260, 393-405 or406*of SEQ1d NO: 1 has been substituted with a different amino acid and having a modification in Its catalytic center, which modification inhibits the ability of the Factor VI! polypeptide to activate plasma FactorX or IX, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100.000 daltons; and optionally, a pharmaceutically acceptable carrier.
" In a further aspect, the invention relates to a pharmaceutical composition comprising an inactivated Factor VII derivative, wherein an Inactivated Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add and having a modification in its catalytic center, which modification inhibits the ability of tine Factor VII polypeptide to activate plasma Factor X or IX, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons; and optionally, a pharmaceutically acceptable earrer.
In a further aspect, the invention relates to a pharmaceutical composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ

ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa 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, is further conjugated with a chemical group that increases the actual molecular weight of theinactivated Factor VII polypeptide with about 300 daitons to about 100,000 daltons; and optionally, a phannaceuticalty acceptable earner.
In a further aspect, the invention relates to a phamnaceutical composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons; and optionally, a pharmaceutically acceptable carrier.
In a further aspect, the invention relates to a polynucleotide construct encoding a Facitor- VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant ther3of, wherein an amino acid has been substituted with a different amino acid, wherein the different amino acid is capable of being conjugated with a chemical group and wherein the Factor VH polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, theInvention relates to a polynucleotide construct encoding a Factor VII poiypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260. 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino*add.
In a further aspect, the invention relates to a polynudeotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has t»een substituted with a different amino add.
In a further aspect, the invention relates to a polynucleotide construct encoding a Factor VII polypeptide comprising Uie amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or q variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.

In a further aspect, the invention relates to a polynucleotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof.
In one embodiment the polynucleotide construct is a vector.
The term "a polynucleotide* denotes a single- or double-stranded polymer of deoxy-riboriucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The temi "nucleotides" is used for both single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the temn "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
The term "vector", as used herein, means any nucleic acid entity capable of the amplification In a host cell. Thus, the vector may be an autonomously replicating vector, I.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated Into the host cell genome and replicated together with the chromosome(s) Into which it has been Integrated. The choice of vector will often depend on the host ceil into which it is to be introduced. Vectors include, but are not limited to plasmid vectors, phage vectors, viruses or cosmid vectors. Vectors usually contains a replication origin and at least one selectable gene, i.e., a gene which encodes a product which is readily detectable or the presence of which is essential for cell growth.
In a further aspect, the invention relates to a eucaryotic host cell comprising a polynucleotide construct comprising a sequence encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been substituted with a different amino acid, wherein the different amino add is capable of being conjugated with a chemical group and wherein the Factor VII polypeptide has substantially the same activity or Increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a eucaryotic host cell comprising a polynucleotide construct comprising a sequence encoding a FadorVII polypeptide compris-

ing the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further aspect, the invention relates to a eucaryotic host cell comprising a polynucleotide construct comprising a sequence encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO; 1 has been substituted with a different amino acid.
In a further aspect, the invention relates to a eucaryotic host cell comprising a polynucleotide construct comprising a sequence encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted.withinjhe sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a eucaryotic host cell comprising a polynucleotide construct comprising a sequence encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an'amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminai of SEQ ID NO: 1 or a variant thereof.
The term "a eucaryotic host cell", as used herein, represent any cell, including hybrid cells, in which heterologous DNA can be expressed. Typical host cells Includes, but are not limited to insect cells, yeast ceils, mammalian cells, including human cells, such as BHK, CHO, HEK, and COS cells. In practidng the present invention, the host cells being cultivated are preferably mammalian cells, more preferably an established mammalian cell line, induding, without limitation, CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), baby hamster kidney (BHK) and HEK293 (e.g., ATCC CRL 1573; Graham et al., 1 Gen, Virol, 36:59-72,1977) cell lines.
A prefenred BHK cell line is the tk" ts13 BHK cell line (Waechter and Baserga, Proc,NatlAcad,ScLUSA 79:1106-1110,1982). hereinafter referred to as BHK 570 cells. The BHK 570 cell line is available from the American Type Culture Colledion, 12301 Parklawn
Dr., Rockville, MD 20852, under ATCC accession number CRL 10314. A tk" ts13 BHK cell line Is also available from the ATCC under accession number CRL 1632.
Other suitable cell lines indude, without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548). TCMK (ATCC CCL 139). Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Uriaub and Chasln,

Proc. Natl. Acad. ScL USA 77:4216-4220,1980). Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of myelomas with other cells. In one embodiment the eucaryotic host cell is of mammalian origin. In a further embodiment the eucaryotic host cell is selected from the group consisting of CHO cells, BHK cells or HEK cells.
In a further aspect, the invention relates to a transgenic animal expressing the polynucleotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been substituted with a different amino acid, wherein the different amino acid is capable of being conjugated with a chemical group and wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a transgenic animal expressing the polynudeotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further aspect, the invention relates to a transgenic animal expressing the polynudeotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further aspect, the invention relates to a transgenic animal expressing the polynudeotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa,
In a further aspect, the invention relates to a transgenic animal expressing the polynucleotide construct encoding a Fador VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add capable of being conjugated with a chemical group has been added to the N- or C-terminai of SEQ ID NO: 1 or a variant thereof.
In a further aspect, the invention relates to a transgenic plant expressing the polynudeotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of
. SEQ ID NO: 1 or a variant thereof wherein an amino acid has been substituted With a differ-
ent amino add, wherein the different amino acid is capable of being conjugated with a

chemical group and wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a transgenic plant expressing the polynucleotide construct encoding a Factor Vil polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid conresponding to an amino add at a position selected from 247-260. 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further aspect, the invention relates to a transgenic plant expressing the polynucleotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add.
In a further aspect, the invention relates to a transgenic plant expressing the polynucleotide constmct encoding a Factor VI! polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been Inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspept, 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 thereof, wherein an amino add capable of being conjugated with a chemical gnxip has been added to the N- or C-terminai of SEQ ID NO: 1 or a variant thereof.
In a further aspect, the invention relates to a method for produdng a Factor VII polypeptide, the method comprising cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynudeotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO; 1 or a variant thereof, wherein an amino add has been substituted with a different amino acid, wherein the different amino acid is capable of being conjugated with a chemical group and wherein the Factor VII polypeptide has substantially thesame activity or increased activity compared to recombinant wild type human Factor VIIa under conditions allowing protein synthesis from the polynucleotide construct and recovering the Factor VII polypeptide from the culture medium!
In a further aspect, the invention relates to a method for produdng a Factor VII polypeptide, tile method comprising cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynucleotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ

ID NO: 1 has been substituted with a different amino add under conditions allowing protein synthesis from the polynucleotide construct and recovering the Factor VII polypeptide from the culture medium.
In a further aspect, the invention relates to a method for producing a Factor VII polypeptide, the method comprising cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynucleotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid under conditions allowing protein synthesis from the polynudeotide construct and recovering the Factor VII polypeptide from the culture medium.
• In a further aspect, the invention relates to a method for produping a Factor VII polypeptide, the method comprising cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynucleotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or Increased activity compared to recombinant wild type human Factor VIIa under conditions allowing protein synthesis from the polynucleotide construct apc recovering the Factor VII polypeptide from the culture medium.
In a further aspect, the invention relates to a method for producing a Factor VII polypeptide, the method comprising cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynudeotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-termlnal o SEQ ID NO: 1 or a variant thereof under conditions allowing protein synthesis from the polynudeotide construct and recovering the Factor VII polypeptide from the culture medium.
In a further aspect, the invention relates to a method for produdng a Factor Vll polypeptide, the method comprising recovering the Factor VII polypeptide from milk produced by a transgenic animal expressing a polynudeotide construct encoding a Factor Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amine acid has been substituted with a different amino add, wherein the different amino acid is capable of being conjugated with a chemical group and wherein the Factor Vll polypeptide has substantially the same activity or increased activity compared to recombinant wild type hu-• man Factor VIIa.

In a further aspect, the invention relates to a method for producing a Factor VII polypeptide, the method connprising recovering the Factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260.393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino add.
• In a further-aspect, the invention relates to a method for producing a Factor Vll polypeptide, the method comprising recovering the Fador Vll polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add seleded from R396. Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add.
In a further aspect, the invention relates to a method for produdng a Factor Vll polypeptide, the method comprising recovering the Factor Vll polypeptide from milk produced by a transgenic animal expressing a po/ynudeotide construct encoding a Factor V/l polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEO ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor Vlia.
In a further aspect, the invention relates to a method for produdng a Factor Vll polypeptide, the method comprising recovering the Factor Vll polypeptide from milk produced by a transgenic animal expressing a potynudeotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof.
In a further asped, the invention relates to a method for producing a Factor Vll polypeptide, the method comprising cultivating a cell of a transgenic plant expressing a polynu-deotide construd encoding a Fador VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant Uiereof, wherein an amino acid has been substituted with a different amino add, wherein tfie different amino add is capable of being conjugated with a chemical group and wherein the Factor Vll polypeptide has substantially Uie same activity or increased adivity compared to recombinant wild type human Factor VIIa, and recovering the Factor Vll polypeptide from the transgenic plant
In a further aspect the invention relates to a method for produdng a Fador Vll polypeptide, the method comprising cultivating a cell of a transgenic plant expressing a polynu-

cleotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid con-esponding to an amino add at a position selected from 247-260,393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid, and recovering the Factor Vll polypeptide from the transgenic plant
In a further aspect, the invention relates to a method for producing a Factor Vll polypeptide, the method comprising cultivating a cell of a transgenic plant expressing a polynu-deotide construct encoding a Fador Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid, and recovering the Factor Vll polypeptide from the transgenic plant.
In a further aspect, the invention relates to a method for producing a Factor VII poly-peptide, the method comprising cultivating a cell of a transgenic plant expressing a polynucleotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor Vll polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa, and recovering the Factor Vll polypeptide from the transgenic plant.
In a further aspect, the invention relates to a method for produdng a Factor Vll polypeptide, the method comprising cultivating a cell of a transgenic plant expressing a polynu-deotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof, and recovering the Factor Vll polypeptide from the transgenic plant.
In a further aspect, the invention relates to a method for producing a Factor VII derivative comprising the steps of:
a) produdng a Factor Vll polypeptide;
b) conjugating the Factor Vll polypeptide with a chemical group;
c) applying the Factor Vll derivative to a cation exchange chromatography or gelfiltration column; and
d) eluting the Factor Vll derivative.
In a further aspect, the invention relates to a method of producing an inactivated Factor Vll derivative comprising the steps of: a) produdng a Factor Vll polypeptide;

b) modifying the Factor VII polypeptide in its catalytic center with a serine protease inhibitor.
c) conjugating the inactivated Factor VII polypeptide with a chemical group;
d) applying the Inactivated Factor VII derivative to a cation exchange chromatography or gelfiltration column; and
e) eluting the inactivated Factor VII derivative.
In one embodiment of the invention the method for producing theFactor VII polypeptide comprises cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynucleotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino add. wherein the different amino add is capable of being conju-gated with a chemical group and wherein the Factor VII polypeptide has substantially thesame activity or increased activity compared to recombinant wild type human Factor VIIa under conditions allowing protein synthesis from the polynucleotide constnjct and recovering the Factor VII polypeptide from the culture medium.
In a further embodiment of the invention themethod for produdng the Factor VII polypeptide comprises cultivating in an appropriate growth medium a eucaryotic host cell comprising the polynucleotide construct encoding a Factor* Vll'polypeptide comprising the amino acid sequen9e of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino add at a position selected from 247-260, 393-406 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid under conditions allowing protein syn-thesis from the polynucleotide construct and recovering the Factor VII polypeptide from the culture medium.
In a further embodiment of the invention the method for produdng the Factor VII polypeptide comprises cultivating in an appropriate growfli medium a eucaryotic host cell comprising the polynucleotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add under conditions allowing protein synthesis from the polynucleotide construct and recovering tine Factor Vll polypeptide from the culture medium.
In a further embodiment of the invention the method for produdng the Factor VII polypeptide comprises cultivating in an appropriate growth medium a eucaryotic host ceil comprising the polynucleotide construd encoding a Fador Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor Vll polypeptide has substantially

the same activity or increased activity compared to recombinant wild type human Factor V under conditions allowing protein synthesis from the polynucleotide construct and recoveri the Factor Vll polypeptide from the culture medium.
In a further embodiment of the of the invention the method for produdng the Fad* Vll polypeptide comprises cultivating in an appropriate growth medium a eucaryotic host c comprising the polynucleotide constiuct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NQ: 1 or a variant thereof, wherein an amino add capabh of being conjugated with a chemical group has been added to theN- or C-terminal of SEQ NO: 1 or a variant thereof under conditions allowing protein synthesis from the polynucleol construct and recovering the Factor Vll polypeptide from theculture medium.
In a further embodiment of the invention the method for producing the Factor VII polypeptide cpmprises/ecovering theFactor Vll polypeptide from milk.prodoeed by a trans geric aninial expressing a polynucleotide construct encoding a Factor Vll polypepti'de com prising theamino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein thedifferent amino acid is cj pable of being conjugated with a chemical group and wherein theFactor VI! polypeptide h£ substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further embodiment of the invention the method for producing the Factor Vll polypeptide comprises recovering the Factor Vll polypeptide from milk produced by a trans genie anirqal expressing a polynucleotide constmct encoding a Factor VII polypeptide com prising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260, 393-405 or 406 c SEQ ID NO: 1 has been substituted with a different amino add.
In a further embodiment of the invention the method for produdng the Factor Vll polypeptide comprises recovering the Factor Vll polypeptide from milk produced by a trans genie animal expressing a polynudeotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add.
In a further embodiment of theinvention the method for producing the Factor Vll polypepti'de comprises recovering the Factor Vll polypeptide from milk produced by a transgenic animal expressing a polynudeotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor Vll polypeptic

has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further embodiment of the invention the method for producing the Fader VII polypeptide comprises recovering the Factor VII polypeptide from milk produced by a transgenic animal expressing a polynucleotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add capable of being conjugated with a chemical group has been added to the N- or C-temiinal of SEQ ID NO: 1 or a variant thereof.
in a further embodiment of the invention the method for produdng the Factor VII polypeptide comprises cultivating a cell of a transgenic plant expressing the polynucleotide construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amin add, wherein the different amino acid is capable of being conjugated with a chemical group and wherein the Factor VII polypeptide has substantially the same activity or increased activ ity compared to recombinant wild type human Factor VIIa, and recovering the Factor VII polypeptide from the transgenic plant
In a further embodiment of the invention the method for producing the Factor VII polypeptide comprises cultivating a cell of a transgenic V'snt expressing a polynucleotide " construct encoding a Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a dif
■ ■ . ■ ■ • ■ .,
ferent amino acid, and recovering the Factor Vll polypeptide from the transgenic plant.
In a further embodiment of the invention the method for produdng the Factor VII polypeptide con^rises cultivating a cell of a transgenic plant expressing a polynudeotide constrjci encoding a Factor Vll polypeptide comprising the amino add sequence of SEQ ID MO: 1 or a variant thereof, wherein an amino acid con'esponding to an amino acid selected from R3G6, Q250 or P406 of SEQ ID NO; 1 has been substituted with a different amino add and recovering the Factor Vll polypeptide from the transgenic plant.
In a further embodiment of the invention the method for producing the Factor Vll polypeptide comprises cultivating a cell of a transgenic plant expressing a polynudeotide construct encoding a Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemi cal group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, v/herein the Factor Vll polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa, and recovering the Factor VI polypeptide from the transgenic plant.

In a further embodiment of the invention the method for producing the Factor VII polypeptide comprises cultivating a cell of a transgenic plant expressing a polynucleotide construct encoding a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-temiinal of SEQ ID NO: 1 or a variant thereof, and recovering the Factor VII polypeptide from the transgenic plant
In a further aspect, the invention relates to the use cf a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein thedifferent amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100.000 daitons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for theenhancement of the nomnal haemostatic system.
The term "treatmenf, as used herein, means theadministration of an effective amount of a therapeutically active compound of the invention with the purpose of preventing any symptoms or disease state to develop or with the purpose of curing or easing such symptoms or diseasestates already developed. The term "treatment" is thus meant to Include prophylactic treatment.
The temn "enhancement of the normal haemostatic system" means an enhancement o the ability to generate thrombin.
As used herein the temn "bleeding disorders" reflects any defect, congenital, acquired or induced, of cellular or molecular origin that is manifested in bleedings. Examples are clotting factor deficiencies (e.g. haemophilia A and B or deficiency of coagulation Factors XI or VII), clotting factor inhibitors, defective platelet function, thrombocytopenia or von Wlllebrand's disease.
The term "bleeding episodes" is meant to include uncontrolled and excessive bleed ing which is a major problem both in connection with surgery and other fomis of tissue damage. Uncontrolled and excessive bleeding may occur in subjects having a normal coagulatior system and subjects having coagulation or bleeding disorders. Clotting factor deficiencies (haemophilia A and B, deficiency of coagulation factors XI or VII) or clotting factor inhibitors may be the cause of bleeding disorders. Excessive bleedings also occur in subjects with a normally functioning blood clotting cascade (no clotting factor deficiencies or -inhibitors against any of the coagulation factors) and may be caused by a defective platelet function, thrombocytopenia or von Willebrand's disease. In such cases, thebleedings may be similar

to those bleedings caused by haemophilia because the haemostatic system, as in haemophilia, lacks or has abnormal essential clotting "compounds" (such as platelets or von Wille-brand factor protein) that causes major bleedings. In subjects who experience extensive tissue damage in association with surgery or vast trauma, the nomial haemostatic mechanism may be oven^elmed by the demand of immediate haemostasis and they may develop bleeding in spite of a normal haemostatic mechanism. Achieving satisfactory haenrx^stasis also is a problem when bleedings occur in organs such as the brain, inner ear region and eyes with limited possibility for surgical haenDOstasis. The same problem may arise in the process of taking biopsies from various organs (liver, lung, tumour tissue, gastrointestinal tract) as well as in (aparoscopic surgery. Common for all these situations is the difficulty to provide haemostasis by surgical techniques (sutures, dips, etc.) which also is the case when bleeding is diffuse (haemorrhagic gastritis and profuse uterine bleeding); Acute and profuse bleedings may also occur in subjects on anticoagulant therapy in whom a defective haemostasis has been induced by the therapy given. Such subjects may need surgical interventions in case the anticoagulant effect has to be counteracted rapidly. Radical retropubic prostatectomy is a commonly performed procedure for subjects with localized prostate cancer. The operation is frequently complicated by significant and sometimes massive blood loss. The considerable blood loss during prostatectomy is mainly related to the complicated anatomical situation, with various densely vascularized sites that are not easily accessible for surgical haemostasis, and which may result in diffuse bleeding from a large area. Another situation that may cause problems in the case of unsatisfactory haemostasis Is when subjects with a nonnal haemostatic mechanism are given anticoagulant therapy to prevent thromboembolic disease. Such therapy may include heparin, other forms of proteoglycans, warfarin or other fomns of vitamin K-antagonists as well as aspirin and other platelet aggregation inhibitors.
In a further aspect, the Invention relates to the use of a Factor VII derivative comprising a Factor VII polypeptide comprising theamino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino add at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino add, wherein the different amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the nomngl haemostatic system.

In a further aspect the invention relates to the use of a Factor VII derivative comprising a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid, wherein th different amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the normal haemostatic system.
In a further aspect, the invention relates to the use of a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein the Factor VII derivative has substantially the same acti^ ity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the nomnal haemostatic system.
In a further aspect, the invention relates to the use of a Factor VII derivative comprising a Factor VII polypeptide having the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof and wherein the amino acid Is conjugated with a chemical group that increases the actual molecular weight of the Factor Vil polypeptide with about 300 daltons to about 100.000 daltons and wherein the Factor VI! derivative has substantially the same acti> ity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the normal haemostatic system.
In one embodiment of the invention, the bleeding is assodated with haemophilia A or B. In another embodiment, thebleeding is assodated with haemophilia with aquired inhibitors. In another embodiment, the bleeding Is assodated with thrombocytopenia. In another embodiment, thebleeding is assodated with von Willebrand's disease. In another embodiment the bleeding is assodated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is assodated with surgen In anoUier embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment the bleeding is associated with haemorrhagic gastritis. In another embodiment

the bleeding is profuse uterine bleeding. In another embodiment, the bleeding is occum'ng in organs with a limited possibility for mechanical haemostasis. In another embodiment, thebleeding is occuning in the brain, inner ear region or eyes. In another embodiment, the bleeding is associated with the process of taking biopsies. In another embodiment the bleeding is assodated with anticoagulant therapy.
The temn "subject" as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the tenm "patienf.
In a further aspect the invention relates to the use of a Factor VII derivative comprising a Factor VIL polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been substituted with a different amino add. wherein . the different amino add is conjugated with a chemical group that increases th^ actual mo--iecular weight of the Factor VII polypeptide with about 300 daftons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the normal haemostatic system.
In a further aspect the invention relates to the use of a Factor VII derivative comprising a Factor VII pplypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid con-espondlng to an amjno add at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted wiUi a different amino acid, wherein the different amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor Vll polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor Vll derivative has substantially the same activity or increased adivity compared to recombinant wild type human Fador VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the nomial haemostatic system.
In a further aspect, the invention relates to the use of a Fador Vll derivative comprising a Fador VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add, wherein the different amino add is conjugated with a chemical group that increases the adual molecular weight of the Factor Vll polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor Vll derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa for the preparation of a medicament

for the treatment of bleeding episodes or for the enhancement of the nonnal haemostatic system.
In a further aspect, the invention relates to the use of a Factor Vll derivative comprising a Factor Vll polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor Vll polypeptide with about 300 daltons to about 1CX),000 daltons and wherein the Factor Vll derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa for the prepch ration of a medicament for the treatment of bleeding episodes or for the enhancement of the normal haemostatic system.
In a further aspect; the invention relates to the Use of a Factor Vll derivative conv prising a Factor Vll polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been added to the N- or C-termlnal of SEQ ID NO: 1 or a variant thereof and wherein the amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor Vll polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor Vll derivative has substantially the same activity or increased activity compared to recombinant wifd type human Factor VIIa for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement'of the nonnal haemostatic system.
In a further aspect, the invention relates to a method for the treatment of bleeding episodes or bleeding disorders In a subject or for the enhancement of the nonnal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophylactlcafly effective amount of a Factor Vll derivative comprising a Factor Vll polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein the different amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor Vll polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor Vll derivative has substantially the same activity or increased activity compared to reconv binant wild type human Factor VIIa.
In a further aspect, the invention relates to a method for thetreatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the nomial haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a Factor Vll derivative comprising a Factor Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino acid at a position selected from 247-260.

393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid, wherein the different amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a method for the treatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the nonmal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophyiactically effective amount of a Factor VII derivative comprising a Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO; 1 or a variant thereof, wherein an amino acid corresponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino acid, wherein the different amino acid is conjugated with a chemical group that increases the actual molecular weight of the Factor Vli polypeptide with about 300 daltons to about 100,000 daitons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the Invention relates to a method for the treatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the normal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophyiactically effective amount of a Factor VII derivative comprising a Factor VII polypeptide having the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein the amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide wtth-about 300 daitons to aixjut 100,000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further aspect, the invention relates to a method for the treatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the normal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophyiactically effective amount of a Factor VII derivative comprising a Factor Vli polypeptide having the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof and wherein the amino add is conjugated with a chemical group that increases the actual molecular weight of the Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein the Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.

In a further aspect, the invention relates to the use of an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant 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, is further conjugated with a chemical group that Increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus formation in a patient
In a further aspect, the invention relates to the use of an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino add, wherein the different amino add is capable of being conjugated with a chemical group and wherein the Factor Vil polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus formation in a patient.
In a further aspect, the invention relates to the use of an inactivated Factor Vfl derivative, wherein an inactivated Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add at a position selected from 247-260,393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino add and having a modification In its catalytic center, which modification inhibits the ability of the Factor VII polypeptide to activate plasma Fador X or IX, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Fador VII polypeptide with about 300 daltons to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus formation in a patient.
In a further asped, the invention relates to the use of an inactivated Factor VII derivative, wherein an inactivated Factor VH polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add coresponding to an amino acid selected from R396. Q250 or P406 of SEQ ID NO: 1 has been substituted witfi a different amino add 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, is further conjugated with a chemical group that increases tine actual molecular weight of theinactivated Factor Vil polypeptide with about 300 daltons to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus formation in a patient.

In a further aspect, the invention relates to the use of an Inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein the Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus fomiation in a patient.
In a further aspect, the invention relates to the use of an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, whereln an amino add capable of being conjugated with a chemical group has been added to theN- or C-terminal of SEQ ID NO: 1 or a variant thereof and having a modification in its catalytic center, which modification inhibits the ability of theFactor VI! polypeptide to activate plasma Factor X or IX, is further conjugated with a chemical group that increases the actual mdecular weight of the inactivated Factor VII polypeptide with aboqt 300 daltcns to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus formation in a patient.
In a further aspect, the invention relates to a method for Inhibiting thrombus forma-tion in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor Vil polypeptide comprising theamino add sequence of SEQ JD NO: 1 or a variant thereof and having a modification In its catalytic center, which modification inhibits the ability of the Factor VII polypeptide to activate plasma Fador X or iX, is further conjugated with a chemical group that Increases the actual molecular weight of theinactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor Vll derivative, wherein an inactivated Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein the different amino add is capable of being conjugated with a chemical group and wherein the Fador Vll polypeptide

has substantially the same activity or increased activity compared to recombinant wild type hunnan Factor VIIa 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor Vil derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino add correseponding to an amino acid at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has. been substituted with a different 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, is further conjugated with a chemical group that increases the actual molecular weight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a method for inhibiting thrombus formation In a patient comprising administering toplcafly to a vascular site susceptible to thrombus fomnation in thepatient a therapeutically effective dose of a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid con-esponding to an amino acid selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different 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, is further conjugated with a chemical group that increases the actual molecular weight of theinactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In a further aspect, the invention relates to a method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein tfie Factor VII polypeptide has substantially thesame activity or increased activity compared to recombinant wild type human Factor VIIa 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. is further In a further aspect, the invention relates to a method for inhibiting thrombus fomiation in a patient comprising administering topically to a vascular site susceptible to thrornbus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant 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, is further conjugated with a chemical group that increases the actual molecular waight of the inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
In one embodiment of the invention the chemical group is substantially neutral.
The term "neutral" as used herein refers to the chemical group being biocompatible, meaning that It is non-toxic, non-immunogenic and water-soluble. Chemical groups being substantially neutral within this definition includes but are not limited to polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polypropylene glycol, polyoxyethylated polyois (e.g.. glycerol) and polyvinyl alcohol, colominic adds or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives.
In a further embodiment of the invention the chemical group is water-soluble.
In a further embodiment of the invention the chemical group has a molecular weight of about 1,000 daltons to about 80.000 daltons.
In a further embodiment of the invention the chemical gnDup has a molecular weight of about 5.000 daJtons to about 60,000 daltons.
In a further embodiment of the invention the chemical group has a molecular weight of about 10.000 daltons to about 40.000 daltons.
In a further embodiment of the invention the chemical group has a molecular weight of about 500 daltons to about 20,000 daltons.
In a further embodiment of the invention the chemical group has a molecular weight of about 500 daltons to about 5000 daltons.

In a further embodiment of the invention the chemical group has a molecular weight of about 750 daltons to about 5000 daltons.
In a further embodiment of the invention the chemical group is polyethylene glycol.
In a further embodiment of the invention the chemical group is selected from one to six molecules of polyethylene glycol.
In a preferred embodiment of the invention the chemical group is one molecule of polyethylene glyco/.
In a further embodiment of the invention the chemical group is monomethoxy-polyethylene glycol.
In a further embodiment of the invention the chemical group is dextran.
In a further embodiment of the invention the chemical group is poly-(N"Vinyl . pyrrolidone) polyethylene glycol.
In a further embodiment of the invention the chemical group is propylene glycol homopolymers.
In a further embodiment of the invention the chemical group is polypropylene oxide.
In a further embodiment of the invention the chemical group is polypropylene glycol.
In a further embodiment of the invention the chemical group Is a po/yoxyethylated polyol. In a further embodiment of the Invention the chemical group Is polyvinyl alcohol. • In a further embodiment of the invention the chemical group is colominic add.
In a further embodiment of the invention the chemical group is a carbohydrate based polymer.
In a further embodiment of the invention the chemical group is a polymer of amino acids.
In a further embodiment of the invention the chemical group is a biotin derivative.
In a further embodiment of the invention the chemical group is conjugated to a free sulfhydryl group present on the amino add substituted for an amino acid in, inserted in or added to the polypeptide.
In a further embodiment of the invention the chemical group is conjugated to a cysteine.
In one embodiment of the invention, the substituted, inserted or added amino acid is capable of being conjugated with a chemical group.
In a further embodiment of the invention the amino add capable of being conjugated with a chemical group is an amino acid with a free sulfhydryl group.
in a further embodiment of the invention the amino add capable of being conjugated with a chemical group is a cysteine.

In a further embodiment of the invention, the substituted, inserted or added amino add is a sulfhydfyl containing amino add such as a cysteine.
In a further embodiment of the Factor VII polypeptide an amino add has been inserted at a position selected from 247-260,393-405 or 406 of SEQ ID NO: 1.
In a further embodiment of the Factor VII polypeptide, the amino add corresponding to R396 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further embodiment of the Factor VII polypeptide, the amino acid corresponding to Q250 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further embodiment of the Factor VII polypeptide, the amino acid corresponding to P406 of SEQ ID NO: 1 has been substituted with a different amino acid.
In a further embodiment of the Factor VII polypeptide an additional amino acid capable of b.eing conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein tine Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa.
In a further embodiment of the Factor VII polypeptide a further amino acid capable
of being conjugated with a chemical group has been added to theN- or C-terminal of SEQ ID
NO: 1 or a variant thereof.' ^
In a furthef embodiment of the Factor VII polypeptide an amino acid has been added to the C-terminal of SEQ ID NO: 1.
In a.further embodiment of the Factor VII polypeptide an amino acid has been added to the N-terminai of SEQ ID NO: 1.
\n a further embodiment of the Factor VII polypeptide a cysteine has been added.
In a further embodiment of the Factor VII polypeptide a cysteine has been Inserted.
In a further embodiment of tine Factor VII polypeptide an amino add selected from the group consisting of K157. V158, E296. M298, L305, D334. S336, K337, and F374 of SEQ ID NO: 1 has been substituted with another amino acid, which amino add increases the activity compared to recombinant wild type human Factor VIIa.
In a further embodiment of the Factor VII polypeptide theamino acid corresponding to K157 of SEQ ID NO: 1 has been substituted with an amino add Independentiy selected from G. V, S, T, N, Q, D and E.
In a further embodiment of tine Factor VII polypeptide the amino acid corresponding to V158 of SEQ ID NO: 1 has been substituted with an amino acid independently selected from S. T. N. Q. D and E.

In a further embodiment of the Factor VII polypeptide the amino acid corresponding to V158 of SEQ ID NO: 1 has been substituted with an amino acid independently selected from T and D.
In a further embodiment of the Factor VII polypeptide the amino acid corresponding to E296 of SEQ ID NO: 1 has been substituted with an amino acid independently selected from R. K and V.
In a further embodiment of the Factor VII polypeptide the amino acid corresponding to E296 of SEQ ID NO: 1 has been substituted with V.
In a further embodiment of the Factor VII polypeptide the amino acid comesponding to M298 of SEQ ID NO: 1 has been substituted with an amino acid independently selected from R, K, Q and N.
In a further embodiment of the Factor VII polypeptide the amino acid corresponding to M298 of SEQ ID NO: 1 has been substituted with Q.
In a further embodiment of the Factor VII polypeptide the amino acid corresponding to L305 of SEQ ID NO: 1 has been substituted with an amino add independently selected from A. V, U I. M, F. W, P, G, S. T. C. Y. N. E. K, R, H. D and Q.
In a further embodiment of the Factor VII polypeptide the amino add corresponding to L305 of SEQ ID NO: 1 has been substituted with V.
In a further embodiment of the Factor VII polypeptide the amino acid corresponding to 0334 of SEQ ID NO: 1 has been substituted with E.
In a further embodiment of the Factor VII polypeptide the amino add corresponding to S336 of SEQ ID NO: 1 has been substituted with G.
In a further embodiment of the Factor Vll polypeptide the amino acid corresponding to K337 of SEQ ID NO: 1 has been substituted with an amino acid independently selected from A. G.V, S.T,N,Q,D and E.
In a further embodiment of the Factor VII polypeptide the amino acid con-esponding to K337 of SEQ ID NO: 1 has been substituted with A.
In a further embodiment of the Factor Vll polypeptide the amino acid corresponding to F374 of SEQ ID NO: 1 has been substituted with an amino add 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 a further embodiment of the Factor Vll polypeptide the amino add con-esponding to F374 of SEQ ID NO: 1 has been substituted with P.
In a further embodiment of the invention the Fador Vll polypeptide is human Factor
Vll. " '• . *
In a further embodiment of the invention the Factor Vll polypeptide is human Factor VIIa,

In the present specification, amino adds are represented using abbreviations, as indicated fn table 1, approved by lUPAOlUB Commission on Biochemical Nomenclature (CBN). Amino acid and the like having isomers represented by name or the following abbre* viat'ons are in natural L-form unless otherwise Indicated. Further, the left and right ends of an amino add sequence of a peptide are, respectively, the N- and C-temiini unless othenvise specified.

The invention also relates to a method of preparing human Fador VII polypeptides as mentioned above. The human Factor VII polypeptides are preferably produced by recombinant DNA techniques. To this end, DMA sequences encoding human Factor VII may be isolated by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the protein by hybridization using synthetic oligonudeotide probes in accordance with standard techniques (cf. Sambrook et al.. Molecular Cloning: A Laboratory Manual, CoW Spring Harbor Laboratory. Cold Spring Harbor, New Yoric, 1989). For the present purpose, the DNA sequence encoding the protein is preferably of human origin, i.e. derived from a human genomic DNA or cDNA library.
The DNA sequences encoding the human Factor Vli polypeptides may also be prepared synthetically by established standard methods, e.g. the phosphoamidite 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. According to the

phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DMA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
The DNA sequences may also be prepared by polymerase chain reaction using specific primers, for instance as descnl)ed in US 4,683,202, SaiW et al., Science 239 (1988). 487 - 491, or Sambrook et al., supra.
The DNA sequences encoding the human Factor VII polypeptides are usually inserted into a recombinant vector which may be any vector, which n^y conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomai entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Altematively, the vector nnay be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chronrK3Some(s) into which it has been Integrated,
The vector is preferably an expression vector in which the DNA sequence encoding the human Factor VII polypeptides is operably linked to additional segments required for transcription of tine DNA In general, theexpression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for tine polypeptide..
The promoter may be any DNA sequence, which shows transcriptional activity in the hpsf cell of choice and may be derived from genes encoding proteins either homologous or heterologous to thehost cell.
Examples of suitable promoters for directing the transcription of theDNA encoding the human-Factor VII polypeptide in mammalian cells are the SV40 promoter (Subramanl et al., Moi. CeilBioi 1 (1981), 854 -864), theMT-1 (metallothionein gene) promoter (Palmtter et al.. Science 222 (1983), 809 - 814), the CMV promoter (Boshart et al.. Ce//41:521-530.1985) or theadenovirus 2 major late promoter (Kaufman and Sharp. Mo/. Ce//. Biol, 2:1304-1319, 1982).
An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (US 4,745,051; Vasuvedan et al., FEBS Lett, 311, (1992) 7-11), theP10 promoter (J.M. Vlak et al., J. Gen, Virology 69,1988, pp. 765-776). the Autographa califomica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155.037; US 5,162,222), or the baculovims 39K delayed-early gene promoter (US 5,155.037; US 5,162,222).
Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al.. J. Biol. Cham, 255 (1980). 12073 - M030] Aiber and

Kawasaki, J. Mo/. 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 {US 4.599,311) or ADH2-4c (Russell et a!.. Nature 304 (1983), 652 - 654) promoters.
le J.
I
r
are the yeast plasmid 2|i replication genes REP 1-3 and origin of replication.
The vector may also comprise a selectable mari
1985, pp. 125-130). or one which confers resistance to a daig, e.g. ampidllin, kanamycin. tetracydin, chloramphenicol, neomydn, hygromydn or methotrexate. For filamentous fungi, selectable markers include andS, pyrG, argB, niaD or sC.
To direct the human Factor VII polypeptides of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vedor. The secretory signal sequence is joined to the D^4A sequences encoding the human Factor VII polypeptides in the con^ct reading frame. Secretory signal sequences are commonly positioned 5' to the DMA sequence encoding the peptide. The secretory signal sequence may be that, normally assodated with the protein or may be from a gene encoding another secreted protein.
For secretion from yeast cells, the secretory signal sequence may encode any signal peptide, whk;h ensures efficient direction of the expressed human Fador VII polypeptides into the secretory pathway of the cell. The signal peptide may be naturally occuning signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the a-factor signal peptide (cf. US 4.870,008), the signal peptide of mouse salivary amylase (cf. 0. Hagenbuchle etai.. Nature 289,1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. LA Vails et al.. Ce//48,1987. pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670). or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al.. Yeast 6.1990. pp. 127-137).
For effident secretion in yeast, a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the human Factor VII polypeptides. The function of tine leader peptide is to allow the expressed peptide to be directed from tine endoplasmic reticulum to theGolgi apparatus and further to a secretory vesicle for secretion Into the culture medium (i.e. exportation of tine human Fador VII polypeptides across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The leader peptide may be the yeast alpha-fador leader (theuse of which is described in e.g. US 4,546,082, US 4,870.008. EP 16 201. EP 123 294. EP 123 544 and EP 163 529). Alternatively, tine leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found In natijre. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.
For use in filamentous fungi, the signal peptide may convenlentiy be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rh'rzomucor mlehel pase or protease or a Humicola lanuginosa lipase. The signal peptide is preferably derived from a gene encoding 4. oryzae TAKA amylase, A, n/ger neutral a-^mylase, A niger acid-stabie amylase, or A niger glucoamylase. Suitable signal peptides are disclosed in, e.g. EP 238 023 and EP 215 594.



tides is introduced may be any cell, which is capable of producing the posttranslational modified human Factor VII polypeptides and includes yeast, fungi and higher eucaryotic cells.
Examples of mammalian cell lines for use in the present invention are the COS-1 (ATCC CRL1650), baby hamster kidney (BHK) and 293 (ATCC CRL1573; Graham et al.. J.
Gen. Virol. 36:59-72.1977) cell lines. A prefen-ed BHK cell line is the tic ts13 BHK cell line (Waechterand Baserga, Proc. Natl. Acad. ScL USA 79:1106-1110,1982, incorporated herein by reference), hereinafter referred to as BHK 570 cells* The BHK 570 cell line has been deposited with the American Type Culture Coilectlon, 12301 Parklawn Dr., Rockville,
Md. 20852, under ATCC accession number CRL 10314. A tic tsIS BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines may be used within the present invention, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), RafHep II (Rat hepatoma; ATCC CRL 1548), f CMK (ATCC CCL 139). Human lung (ATCC HB 8065). NCTC 1469 (ATCC CCL 9.1). CHO (ATCC CCL 61) and DUKX cells (Uriaub and Chasin. Proc. Natl, Acad. ScL USA 77:4216-4220,1980).
Examples of suitable yeasts cells include cells of Saccharomyces spp. or Schizosac-charomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyven\ Methods for transforming yeast ceils with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in US 4,599,311, US 4.931.373. US 4.870.008. 5,037,743, and US 4,845.075, all of which are hercb/incorporated by reference. Transformed cells are selected by a phenotype d^tenmined by a selectable marker, comrTX)nly daig resistance or the ability to grow in the absence of a particular nutrient, e.g. ieudne. A prefenred vector for use in yeast is the P0T1 vector disclosed in US 4,931,373. The DNA sequences encoding the human Factor VII polypeptides may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above. Further examples of suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenufa, e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson etal.. J. Gen. Microbiol. 132,1986. pp. 3459-3465; US 4,882;279).
Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., In particular strains of A oryzae, A. nidulans or A niger. The use of Aspergillus spp, for the expression of proteins is described in, e.g., EP 272 277, EP 238 023. EP184 438 The transfbnnation of F. oxyspomm may. for instance, be canled out as described by Malardier et al., 1989, Gene 78:147-156. The transformation of Trichoderma spp. may be performed for instance as described in EP 244 234.
When a filamentous fungus is used as the host cell, it may be transfomied with the DNA construct of the invention, conveniently by integrating the DNA construct in the host diromosome to obtain a recombinant host cell. This Integration Is generally considered to be an

the DNA constaicts into the host chromosome may be perfomied according to conventional methods, e.g. by homologous or heterologous recombination.
Transfomnation of Insect cells and production of heterologous polypeptides therein may

DNA sequences for use witlnin the present invention will typically encode a pre-pro peptide at the amino-temninus of the Factor VII protein to obtain proper post-translationai processing (e.g. gamma-carboxyiation of glutamic acid residues) and seaetion from the host

cell. The pre-pro peptide may be that of Factor VII or another vitamin K-dependent plasma protein, such as factor IX. factor X, prothrombin, protein C or protein S, As will be appred-ated by those skilled in the art, additional modifications can be made in the amino acid sequence of Factor VII where those modifications do not significantly impair the ability of the

ing the 5' flanking promoter and non-coding portion of the beta-Iactoglobulin gene. See Whitelaw et ai., Biochem J. 286: 31-39 (1992). Similar fragments oi promoter DNA from other species are also suitable.

other regions of the beta-lactoglobulin gene may also be incorporated in constructs,
as may genomic regions of the gene to be expressed. It is generally accepted in the art that
I"
constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al.. Proa Natl. Acad ScL USA 85:836-840 (1988); Palmiter et al.. Proa Natl. Acad ScL USA 88:478-482 (1991); Whitelaw et al., Transgenic Res. 1:3-13,1991); WO 89/01343; and WO 91/02318, each of which is incorporated herein by reference). In this regard, it is generally prefen-ed, where possible, to use genomic sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of Interest, thus the further inclusion of at least some introns from, e.g, the beta-lactoglobulin gene, is preferred. One such region is a DNA segment which provides for intron splicing and RNA polyadenylation from the 3* non-coding region of the ovine beta-lactoglobulin gene.'When substituted for the natural 3* non-coding sequences of agehe, this ovine beta-lactogiobulin segment can both enhance and stabilize expression levels of the protein or polypeptide of interest. Within other embodiments, the region surrounding the initiation ATG of the sequence encoding the human Factor VII polypeptide is replaced with corresponding sequences from a milk specific protein gene. Such replacement provides a putative tissue-specific initiation environment to enhance expression. It is convenient to replace the entire pre-pro sequence of the human Factor V!l polypeptide and 5' non-coding se-' quences with those of, for example, the BLG gene, although smaller regions may be replaced.
For expression of a human Factor VII polypeptide in transgenic animals, a DNA segment encoding the human Factor VII polypeptide is operabiy linked to additional DNA segments required for its expression to produce expression units. Such additional segments include the above-mentioned promoter, as well as sequences which provide for temiination of transcription and polyadenylation of mRNA, The expression units will further include a DNA segment encoding a secretory signal sequence operabiy linked to the segment encoding the human Factor VII polypeptide. The secretory signal sequence may be a native seae-tory signal sequence of the human Factor VII polypeptide or may be that of another protein, such as a milk protein. See. for example, von Heinje, A/uc. Acids Res. 14:4683-4690 (1986); and Meade et ai., U.S. Pat No. 4.873,316. which are incorporated herein by reference.
Construction of expression units for use in transgenic animals is conveniently carried out by inserting a sequence encoding the human Factor Vll polypeptide into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of the human Factor Vll polypeptide, thereby creating a

gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the expression units in plasmids or other vectors facilitates the amplification of the human Factor VII polypeptide. Amplification is conveniently carried out in bacterial (e.g. E. coll) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.
The expression unit is then introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can be accomplished by one of several routes, including microinjection (e.g. U.S. Pat. No. 4.873,191). retroviral infection (Jaenisch, Science 240:1468-1474 (1988)) or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technology "[Q: 534-539 (1992)). The eggs are then implanted into the oviducts or uteri of pseudopregnant females and allowed to develop. Offspring carrying the introduced DNA in their g*enm line can pass the DNA on to their progeny in the normal. Mendellan fashion, allowing the development of transgenic herds.
General procedures for producing transgenic animals QT^ known in the art. 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 etal.. Biol. Reprod. 32:645-651 (1985); Buhler et al., Bio/Technology 8:140-143 (1990); Ebert et al., Bio/Tect}nologyQ: 835-838 (1991); Krimpenfortetal.. Bio/Technology 9: 844-847 (1991); Wail et al.. J. CelL Biochem, 49:113-120 (1992); U.S. Pat. Nos. 4.873,191 and 4,873.316; WIPO publications WO 88/00239. WO 90/05188. WO 92/11757; and GB 87/00458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed In the mouse. See, e.g., Gordon et al.. Proa Natl. Acad. Sci. USA T7: 7380-7384 (1980); Gordon and Ruddle. Science 214:1244-1246 (1981); Palmiter and Brinster. Ce//41: 343-345 (1985); and Brinster et al.. Proc. Natl, Acad, ScL USA 82: 4438-4442 (1985). These techniques were subsequently adapted for use with larger animals, including livestock species (see e.g., WiPO publications WO 88/00239, WO 90/05188. and WO 92/11757; and Simons et al.. BioH'echnology 6:179-183 (1988). To summarize, in the most efficient route used to date in the generation of transgenic mice or livestock, several hundred linear molecules of the DNA of interest are injected irito one of the pro-nuclei of a fertilized egg according to established techniques. Injection of DNA into the cytoplasm of a zygote can also be employed. Production in transgenic plants may also be employed. Expression may be generalized or directed to a particular organ, such as a tuber. See. Hiatt. Nature 344:469-479 (1990); Edelbaum et al.. J. Interferon Res. 12:449-453 (1992); Sijmons et al.. Bio/Techno/ogy 8:217-221 (1990); and European Patent Office Publication EP 255.378.


tration of 10Q-1000 nM, where the factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765). In addition, the activity aissay may be mn at physiological temperature.


A systemic activation of the coagulation cascade may lead to disseminated intravascular coagulation (DIG). However, such complications have not been seen in subjects treated with high doses of recombinant Factor Vila because of a localised haemostatic process of the kind induced by the complex formation between Factor Vila and TF exposed at the site of vessel wall injury. The procoagulant Factor VII derivatives

according to the invention may thus also be used in their activated form to control such excessive bleedings associated with a normal haemostatic mechanism.
For treatment in connection with deliberate interventions, the procoagulant Factor VII derivatives of the invention will typically be administered within about 24 hours prior to performing the inten/ention, and for as much as 7 days or more thereafter. Administration as a coagulant can be by a variety of routes as described herein.
The dose of the Factor VII derivatives ranges from about 0.05 mg to 500 mg/day, preferably from about 1 mg to 200 mg/day, and more preferably from about 10 mg to about 175 mg/day for a 70 kg subject as loading and maintenance doses, depending on the sevenfy of the condition.
The pharmaceutical compositions are primarily intended for parenteral administration for prophylactic and/or ti^erapeutic treatment. Preferably, the phannaceuti'cal compositions are administered parenterally. i.e.. intravenously, subcutaneously. or intramusculariy, or it may be administered by continuous or pulsatile infusion. The compositi'ons for parenteral administration comprise the Factor VII derivative of the invention In combination with, preferably dissolved In, a pharmaceuti'cally acceptable cam'er. preferably an aqueous carrier. A variety of aqueous caniers may be used, such as water, buffered water. 0,4% saline, 0.3% glydne and the like. The Factor Vfl derivatives of the invention can also be formulated into liposome preparations for delivery or targeting to the sites of injury. Liposome preparations are generally described In. e.g., U.S. 4.837.028, U.S. 4,501.728, and U.S. 4.975,282. The compositions may be sterilised by conventional, well-known sterilisation techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilised, tine lyophillsed preparation being combined with a sterile aqueous solution prior to administi^tion. The compositions may contain pharmaceutically acceptable auxiliary substances as required td approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
The concentration of Factor VII derivative in these formulations can vary widely, i.e., from less than about 0.5% by weight, usually at or at least about 1% by weight to as much as 15 or 20% by weight and will be selected primarily by flukl volumes, viscosities, etc, in accordance with the particular mode of administration selected.
Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 mi of sterile Ringer's solution and 10 mg of the Factor VII polypeptide. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example.

Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton. PA (1990).
The compositions containing the Factor VII derivatives of the present invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a subject already suffering from a disease, as described above, in an amount sufficient to cure, alleviate or partially arrest the disease and its complications. An amount adequate to accomplish this is defined as "^erapeutically effective amounr. As will be understood by the person skilled in the art amounts effective for this purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject In general, however, the effective amount will range from about 0.05 mg up to about 500 mg of the Factor VII derivative per day for a 70 kg subject, with dosages of from about 1.0 mg to about 200 mg of the Factor VII derivative p^r day being more commonly used.
It must be kept in mind that the materials of the present invention may generally be employed in serious disease or injury states, that is, life threatening or potentially life threatening situations, in such cases, in view of the minimisation of extraneous substances and general lack of immunogenicity of human Factor Vll derivatives in humans, it is possible and may be felt desirable by the treating physician to administer a substantial excess of these variant Factor Vil compositions.
In prophylactic applications, compositions containing the Factor Vll derivative of the invention are administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulative capability. Such an amount is defined to be a "prophylactically effective dose." In prophylactic applications, theprecise amounts once again depend on tine subject's state of health and weight, but the dose generally ranges from about 0-05 mg to about 500 mg per day for a 70-kilogram subject, more commonly from about 1-0 mg to about 200 mg per day for a 70-kilogram subject.
Single or multiple administrations of the compositions can be carried out with dose levels and patterns being selected by the treating physician. For ambulatory subjects requiring daily maintenance levels, the Factor Vll derivatives may be administered by continuous infusion using e.g. a portable pump system.
Local delivery of the Factor Vll derivative of the present invention, such as, for example, topical application may be earned out, for example, by means of a spray, perfusion, double balloon catheters, stent incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods. In any event, the phannaceutical compositions should provide a quantity of Factor Vll derivative sufficient to effectively ti-eat the subject.

Inactivated Factor Yll polypeptides of the present invention is able to bind to cell-surface tissue factor. For exampjp. DEGR-Fador Vila binds cell-surface tissue factor with an equivalent or higfier affinity than wild-type Factor Vila. DEGR-Factor Vila, however, has no enzymatic activity, yet it binds to tissue factor and acts as a competitive antagonist for wild-type Factor Vila, thereby inhibiting the subsequent steps in the extrinsic pathway of coagulation leading to the generation of thrombin.

The inactivated Factor VII derivatives may be particulariy useful In the treatment of intimal hyperplasia, restenosis due to acute vascular injury, deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG). percutaneous transdermal coronary angioplastry (PICA), strolce, cancer, tumour metastasis, angiogenesis, ischemia/reperfusion, riieumatoid arthritis, thrombolysis, arteriosclerosis and restenosis following angioplastry, acute and chronic indications such as inflammation, septic chock, septicemia, hypotension, adult respiratory distress syndrome (AROS), disseminated intravascular coagulopathy (DIG), pulmonary embolism, platelet deposition, myocardial infarction, or the prophylactic treatment of mammals with atherosclerotic vessels at risk for thrombosis. Acute vascular injuries are those which occur rapidly (i.e. over days to months), in contrast to chronic vascular injuries (e.g. atherosclerosis) which develop over a lifetime. Acute vascular injuries often result from surgical procedures such as vascular reconstruction, wherein the techniques of angioplasty, endarterectomy, atherectomy, vascular graft emplacement or the like are employed. Hyperplasia may also occur as a delayed response in response to, e.g., graft emplacement or organ transplantation. Since inactivated Factor VII derivatives is more selective than heparin, generally binding only tissue factor which has been exposed at sites of injury, and because inactivated Factor VII derivatives does not destroy other coagulation proteins, it will be more effective and less likely to cause bleeding complications than heparin when used prophylactically for the prevention of deep vein thrombosis.

Inactivated Factor VII derivatives which maintain tissue factor binding inhibit platelet accumulation at the site of vascular injury by blocking tfie production of thrombin and the subsequent deposition of fibrin.
Due to the ability of DEGR-Factor VII to block thrombin generation and limit platelet deposition at sites of acute vascular injury, inactivated Factor VII derivatives which maintain tissue factor binding activity but lack Factor VIIa enzymatic activity can be used to inhibit vascular restenosis.
Compositions comprising inactivated Factor VII derivatives are particularly useful in methods for treating patients when formu/ated into phamiaceutical compositions, where they may be given to individuals suffering from a variety of disease states to treat coagulation-related conditions. Such inactivated Factor VII derivatives, capable of binding tissue factor but hflVjng a substgritially reduced ability to catalyze activation of other factors in the clotting cascade, may possess a longer plasma half-life and thus a conrespondingly longer period of anticoagulative activity when compared to other anticoagulants. Among the medical Indications for the subject compositions are those commonly treated with anticoagulants, such as, for example, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), fibrin deposition in lungs and kidneys associated with gram-negative en-dotoxemia, and myocardial infarction. The compositions can be used to inhibit vascular restenosis as occurs following mechanical vascular injury, such as injury caused by balloon angioplasty, endarterectomy, reductive atherectomy, stent placement, laser therapy or rotab-lation. or as occurs secondary to vascular grafts, stents, bypass grafts or organ transplants. The compositions can thus be used to inhibit platelet deposition and associated disorders. Thus, a method of inhibiting coagulation, vascular restenosis or platelet deposition, for example, comprises administering to a patient a composition comprising inactivated Factor VII derivatives, such as that having at least one amino acid substitution In a catalytic thad of Ser344, Asp242 and His193. in an amount sufficient to effectively inhibit coagulation, vascular restenosis or platelet deposition. The methods also find use in the treatment of acute closure of a coronary artery in an individual (e.g. acute myocardial infarction), which comprises administering the inactivated Factor VII derivatives, which includes DEGR-Factor V(l and FFR-Factor VII, in conjunction with tissue plasminogen activator or streptokinase, and can accelerate tPA induced thrombolysis. The inactivated Factor VII derivatives is given prior to, in conjunction with, or shortly following administration of a thrombolytic agent, such as tissue plasminogen activator.
Compositions of inactivated'Factor VII derivatives will also have substantial utility in the prevention of cardiogenic emboli and in the treatment of thrombotic strokes. Because of its low potential for causing bleeding complications and its selectivity, Factor VII derivatives


/

Composit'ons comprising inactivated Factor VII derivatives will typically be administered within about 24 hours prior to performing an intervention, and for as much as 7 days or more thereafter. Administration can be by a variety of routes as further described herein. The compositions comprising inactivated Factor VII derivatives can also be administered sys-temlcally or locally for the placement of vascular grafts (e.g., by coating synthetic or modified natural arterial vascular grafts), at sites of anastomosis, surgical endarterectomy (typically carotid artery endarterectomy), bypass grafts, and the lilce.
In the treatment of established deep vein thrombosis and/or pulmonary embolism, the dose of Factor VII derivatives ranges from about 50 pg to 500 mg/day, more typically 1 mg to 200 mg/day, and more preferably 10 mg to about 175 mg/day for a 70 l In cases of acute bacteremia, endotoxemia or DIG, the patient is given a loading dose of a Factor VII derivative of at least about 50 ^g to 500 mg/day. more typically 1 mg to 200 mg/day. and more preferably 10 mg to about 175 mg/day for a 70 kg patient, with maintenance doses thereafter in (he range of 50 ^g to 500 mg/day, typically 1 mg to 200 mg/day for a 70 kg patient.
Preferably, the Factor VII derivative has a half-life (Ua) which is enhanced relative to the half-life of the unconjugated Factor VII from which It was derived. Preferably, the half-life of the Factor VII derivative is enhanced by at least 1.5-fold to 2-fold, more preferably by about 2'foldi to 3-fold, even more preferably by about 5-fold to 10-fold, optimally about 100-fold, usually about 5-fold relative to the half-life of the unmodified parent Factor VII.
General methods of attaching polyethylene glycol to proteins are disclosed within U.S. Pat. No. 4,179,337 issued Dec. 18,1979 (incorporated herein by reference to disclose methods of attaching polyethylene glycol to proteins). Further, other methods of attaching polyethylene glycol are disclosed within U.S. Pat No. 5,122,614 issued Jun. 16,1992, also incorporated herein by reference to disclose methods of attaching polyethylene glycol to proteins. Maleimido-PEG is pe.'tiaps the most useful reagent for cysteine-PEGyfation, but other chemistries are available for specific cysteine modification.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is described in further detail in the examples with reference to the appended drawings wherein

Rg. 1 The structure of con'ectly processed human coagulation Factor Vil, amino acids 1 to 406, with gamma carboxylated Glu-residues (7) and glycosylation (*). The amjw at amino add residue 152 shows the site where single-chain Factor VII is deaved to be converted to activated two-chain Factor VII (FVIIa).
Fig. 2 Construction of plasmids for expression of recombinant human Fader VII polypeptides, Plasmid pLN174 express human Factor Vll with conneded propeptide naturally asso-dated with Factor VII.
The present invention Is further illustrated by the following examples which, however, are. not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.
EXAMPLES
The terminology for amino add substitutions used the following examples ^re as follows. The first letter represent the amino acid naturally present at a position of SEQ ID NO: 1. The following number represent the position in SEQ ID NO: 1. The second letter represent the different amino acid substituting for the natural amino acid. An example is R396C, where an arginine at position 396 of SEQ ID NO: 1 is replaced by a cysteine. In another example, V158T/M298Q. the valine in position 158 of SEQ ID NO: 1 is replaced by a threonine and the methionine in position 298 of SEQ ID NO: 1 is replaced by a Glutamine in the same Factor Vil polypeptide.
Example 1
Construction of DNA encoding FVIKR396C), FVIKQ250C), FVIKP406C), FVII-(407C), FVII-(V158T/M298Q). FViI-{L305V/M306D/D309S), FVIHK337A), FVIKL305V). and FVIKF374P):
DNA constructs encoding FVIKR3g6C). FVIKQ250C), FVIKP406C). FVIK407C) (One additional C-temr^inal Cys). FVII-(M298Q). FVIKL305V/M306D/D309S), FVri-{K337A). FVII-(L305V), and FV1I-(F374P) was prepared by site-direded mutagenesis using a supercoiled.

aouDie stranaea UNA vector wttn insert of human FVII (pLN174) and two synthetic primers containing the desired mutation. The following primers were used:
For FViKR396G):
5'-GCG CTG AGA GOO ATG CCC AGG AGT CCT CC-S" (SEQ ID NO: 3)
5'-GGA GGA CTC CTG GGC ATG GCT CTG AGC GC-3* (SEQ ID NO: 4)
For FVIKQ250C):
5'-GCTGCG CCTGCACTG TCC CGTGGTCCTCAC TGACC-3' (SEQ ID NO: 5)
5'-GGT CAG TGA GGA CCA CGG GAC AGT GCA GGC GGA GC-3' (SEQ ID NO: 6)
For FVII-(P406C):
5'-GCG AGC CCC ATTTG CTA GAC TAG AGG ATC TGG G-3' (SEQ ID NO: 7)
5'-CCC AGA TCC TCT AGT CTA GCA AAA TGG GGC TCG C-3' (SEQ ID NO: 8)
For FV1I-(407C):
5'-CCT GCG AGC CCC ATT TCC CTG TTA GAC TAG AGG ATC TGG G-3' (SEQ ID NO: 9)
5'-CCC AGA TCC TCT AGT CTA ACA GGG AAA TGG GGC TCG CAG G-3'
(SEQ ID NO: 10)
For FVII-(M298Q):
5'-GCC CTG GAG CTC CAG GTC CTC AAC GTG CCC-3' (SEQ ID NO: 11)
5'-GGG CAC GTT GAG GAC CTG GAG CTC CAG GGC-3' (SEQ ID NO: 12)
For FVIKL305V):
5'-CGT GCC CCG GGT GAT GAC CCA GGA C-3' (SEQ ID NO: 13)
5'-GTC CTG GGT CAT CAC CCG GGG CAC G-3' (SEQ ID NO: 14)
For FVII-(M306D/D309S):
5'-TCT AGA TAG CCA GTC TTG CCT GCA GCA GTC ACG GAA-3' (SEQ ID NO: 15)
5'-TTC CGT GAC TGC TGC AGG CAA GAC TGG GTA TCT AGA-3' (SEQ ID NO: 16)
For FVIKK337A):
5'-CGG ATG GCA GCG CGG ACT CCT GCA AGG G-3' (SEQ ID NO: 17)
5'-CCC GAG CAG GAG TCC GCG CTG CCA TCC G-3' (SEQ ID NO: 18)

For l=VII-(F374P):
5'-CCG TGG GCC ACC CTG GGG TGT ACA CC-3' (SEQ ID NO: 19)
5'-GGT GTA CAC CCC AGG GTG GCC CAC GG-3' (SEQ ID NO: 20)
The oligonucleotide primers, each complementary to opposite strands of the vector insert, were extended durnig temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicies was generated. Following temperature cycling, the product was treated with Dpnl which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.
Procedures for preparing a DNA constnjct using polymerase chain reaction using specific primers'are well known to persons skilled in the art (cf. PCR Protocols', 1990, Academic ress, San Diego, California, USA).
Example 2
Preparation of FVII-{R396C). BHK cells were transfected essentially as previously described (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett, 385,241-243) to obtain expression of the variant FVII-(R396C). The Factor VII polypeptide vras purified as follows:
Conditioned medium was loaded onto a 25-mI column of Q Sepharose Fast Flow (Pharmacia Biotech) after addition of 5 mM EDTA. 0.1% Triton X-100 and 10 mM Tris, adjustment of pH to 8.0 and adjustment of the conductivity to 10-11 mS/cm by adding water. Elution of the protein was accomplished by a gradient from 10 mM Tris, 50 mM NaCI, 0.1% Triton X-1C0, pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaClz. 0.1% Triton X-100, pH 7.5. The fractions containing FVIKR396C) were pooled, and applied to a 25-ml column containing the monoclonal antibody F1A2 (Novo Nordisk, Bagsvaerd, Denmark) coupled to CNBr-ac-tivated Sepharose 4B (Pharmacia Biotech). The column was equilibrated with 50 mM Hepes, pH 7.5, containing 10 mM CaCl2.100 mM NaCI and 0.02% Triton X-100, After washing with equilibration buffer and equilibration buffer containing 2 M NaCI, bound material was eluted with equilibration buffer containing 10 mM EDTA instead of CaCl2. Before use or storage, excess CaCl2 over EDTA was added or FVIKR396C) was transfen^d to a Ca^*-containing buffer. The yield of each step was followed by factor VII ELISA measurements and the purified protein was analysed by SDS-PAGE.



NaCI, bound materia) was eluted with equilibration buffer containing 10 mlVI EDTA instead of CaCl2. Before use or storage, excess CaCl2 over EDTA was added or P/II-(L305V/M306D/D309S) was transferred to a Ca2-containing buffer. The yield of each step was followed by factor VII ELISA measurements and the purified protein was analysed by SDS-PAGE.


minutes, after subtraction of the absorbance in a blank well containing no FVIIa. is used to calculate the ratio between the proteolytic activities of variant and wild-type Factor VIIa:
Ratio = (A405 nm Factor VIIa variant)/(A«5 nm Factor VIIa wild-type).

Example 7
Relative activities of FVlla polypeptides measured in the assays described in examples 5 and 6

PEG Conjugation of FVIKR396C). FVII-{Q250C), FV1I-{P40€C), FVII-{407C)
The Factor VIIa variants as described in example 1, with a free thiol group introduced at any of the mentioned positions (250.396.406 or 407 (the latter C-terminally extended)) are reacted with a 5-fold molar excess of PEG vinylsutfone or PEG-maleimide (alternatively any other sulfhydryl-reactive PEG derivative may be used) in an aqueous buffer for 3 hours to drive the reaction virtually to completion. The molecular weight of the PEG derivative is at least 10.000. The resulting PEG-FVIIa are tested for amidolytic and proteolytic activity as described in examples 5 and 6 and should retain the activity of wild-type human FVlla, or if a Cys has been introduced into a FVlla variant with increased activity, the activity after reaction with the PEG derivative should remain higher than that of wild-type human FVlla. PEG-conjugated FVlla is separated from unreacted FVlla variant and free PEG derivative by means of chromatography such as gel filtration on a column of Superdex-200 or the like.
PEG Conjugation of proteins at Cys residues is l




CLAIMS
1. A Factor Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino add, wherein said different amino acid is capable of being conjugated with a chemical group and wherein said Factor Vll polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.
2. A Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add at a position selected from 247-260, 393-405 or 406 of SEQ ID NO: 1 has been substituted with a different amino acid. ,
3. A Factor Vll polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid corresponding to an amino add selected from R396, Q250 or P406 of SEQ ID NO: 1 has been substituted with a different amino add.
4. The Fador Vll polypeptide according to any one of the claims 1-3, wherein the amino add conesponding to R396 of SEQ ID NO: 1 has been substituted with a different amino acid.
5. The fector Vll polypeptide according to any one of the claims 1-4, wherein the amino add corresponding to Q250 of SEQ ID NO: 1 has been substituted with a different amino add.
6. the Factor Vll polypeptide according to any one of the claims 1-5, wherein the amino add con-esponding to P406 of SEQ ID NO: 1 has been substituted with a different amino add.
7. The Factor Vll polypeptide according to any one of the claims 2-6. wherein said different amino acid is capable of being conjugated with a chemical group.
8. The Factor Vll polypeptide according to any one of the claims 1-7, wherein said different amino add is a cysteine.
9. The Factor VII polypeptide according to any one of the claims 1-8, wherein an additional amino acid capable of being conjugated with a chemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein said Factor Vll poly-

peptide has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.
10. A Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a diemical group has been inserted within the sequence of SEQ ID NO: 1 or a variant thereof at a position, wherein said Factor VII polypeptide has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.
11. The Factor Vll polypeptide according to any one of the claims 9-10, wherein said amino acid has been inserted at a position selected from 247-260. 393-405 or 406 of SEQ ID NO: 1
12. The Factor VII polypeptide according to any one of the claims 1-11, wherein a further amino acid capable of being conjugated with a chemical group has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof.
13. A Factor Vll polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid capable of being conjugated with a chemical group has been added to the N- or C-tenninal of SEQ ID NO: 1 or a variant thereof.
14. The Factor VII polypeptide according to any one of the claims 12-13. wherein said amino acid has been added to the C-terminal of SEQ ID NO: 1.
15. The Factor Vll polypeptide according to any one of the claims 9-14, wherein said amino acid is a cysteine.
16. The Factor Vll polypeptide according to any one of the claims 1-15, wherein an amino acid selected from the group consisting of K157. V158, E296. M298, L305. D334, S336. K337, and F374 of SEQ ID NO: 1 has been substituted with another amino acid, which amino acid increases the activity compared to recombinant wild type human Factor Vila.
17. The Factor Vll polypeptide according to claim 16. wherein K157 has been substituted with an amino acid independently selected from G. V, S. T, N, Q. D and E.
18. The Factor Vll polypeptide according to any one of the claims 16-17, wherein V158 has been substituted with an amino acid independently selected from S, T, N. Q. D and E,

19. The Factor VII polypeptide according to any one of the claims 16-18, wherein E296 has been substituted with an amino acid independently selected from R, K and V.
20. The Factor VII polypeptide according to any one of the claims 16-19. wherein M298 has been substituted with an amino acid independently selected from R. K, Q and N.
21. The Factor VII polypeptide according to any one of the daims 16-20, wherein L305 has been substituted with an amino acid independently selected from A, V, I. M, F. W. P, G, S, T, C, Y. N, E. K, R. H. D and Q.
22. The Factor VII polypeptide according to any one of the claims 16-21, wherein D334 has been substituted with E.
23. The Factor VII polypeptide according to any one of the claims 16-22, wherein S336 has been substituted with G.
24. The Factor VII polypeptide according to any one of the claims 16-23, wherein K337 has been substituted with an amino add independently selected from A, G, V, S, T, N, Q, D and E.
25. The Factor VII polypeptide according to any one of the claims 16-24. wherein F374 has been substituted with an amino add Independently selected from A, V, L, I, M, W, P. G, S. T, C, Y, N, E. K, R. H, D and Q.
26. The Factor VII polypeptide according to any one of the daims 1-25, wherein said Factor Vil polypeptide is human Factor VII.
27. The Factor VII polypeptide according to any one of the daims 1-25, wherein said Factor VII polypeptide is human Factor Vila.
28. An inactivated Factor Vli polypeptide, wherein a Factor VII polypeptide according to any one of the daims 1-27 is further modrfied in its catalytic center, which modification inhibits the ability of the Factor VII polypeptide to activate plasma Factor X or IX.
29. The inactivated Factor VII polypeptide according to claim 28, wherein said Factor VII

polypeptide is modified in its catalytic center with a serine protease inhibitor.
30. The inactivated Factor VII polypeptide according to claim 29, wherein the protease inhibitor is a peptide halomethyl ketone selected from the group consisting of: Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone, D-Phe-Phe-Arg chloromethyl ketone, 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-Gty-Arg chloromethylketone, Dansyl-Phe-Phe-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Phe-Arg chloromethyl ketone, Dansyl-D-Phe-Phe-Arg chloromethylketone, Dansyl-Phe-Pro-Arg chloromethylketone, Dansy!-D-Phe-Pro-Arg chloromethylketone, Dansyl-Phe-Pro-Arg chloromethylketone, Dansyl-D-Phe-Pro-Arg chloromethylketone, Dansyl-L-Glu-Gly-Arg chloromethylketone and Dansyi-D-Glu-Gly-Arg chloromethylketone,
31. A Factor Vil derivative comprising a Factor Vli polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein said different amino acid is conjugated with a chemical group that Increases the actual molecular weight of said Factor Vll polypeptide with about 300 daltons to about 100,000 daltons and wherein said Factor Vil "derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila,
32. The Factor Vll derivative according to claim 31, wherein said Factor Vll polypeptide is according to any one of the claims 1-9,11-12.14-27.
33. The Factor Vli derivative according to claim 31, wherein said amino add corresponds to an amino acid at a position selected from 247-260. 393-405 or 406 of SEQ ID NO: 1.
34. A Factor VII derivative comprising a Factor Vll polypeptide having the amino add sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been Inserted within the sequence of SEQ ID NO: 1 or a variant thereof and wherein said amino acid Is conjugated with a chemical group that increases the actual molecular weight of said Factor Vll polypeptide with about 300 daltons to about 100,000 daltons and wherein said Factor Vll derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.

35. The Factor VI! derivative according to claim 34. wherein said Factor VII polypeptide is according to any one of the claims 9-12,14-27.
36. A Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been added to the N- or C-terminal of SEQ ID NO: 1 or a variant thereof and wherein said amino add is conjugated with a chemical group that increases the actual molecular weight of said Factor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein said Factor Vil derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.
37. The Factor VII derivative according to claim 36, wherejn-said Factor VII polypeptide is according to any one of the claims 12-27.
38. The Factor VII derivative according to any one of the claims 1-37. wherein said chemical group is substantially neutral.
39. The Factor VII derivative according to any one of the claims 31-38, wherein said chemical group inaeaset? the actual molecular weight of the Factor Vil derivative with about 1,000 daitons to about 80,000 daltons.
40. The Factor VII derivative according to any one of the daims 31-39, wherein said chemical group increases the actual molecular weight of the Factor VII derivative with about 5,000 daltons to about 60,000 daltons.
41. The Factor VII derivative according to any one of the claims 31-40, wherein said chemical group increases the actual molecular weight of the Factor VII derivative with about 10,000 daltons to about 40,000 daltons.
42. The Factor VII derivative according to any one of the claims 31-41, wherein said chemical group is polyethylene glycol.
43. The Factor VII derivative according to any one of the claims 31-42, wherein said chemical group is selected from one to six molecules of polyethylene glycol.

44. The Factor Vll derivative according to claim 43, wherein said chemical group is one molecule of polyethylene glycol.
45. The Factor Vli derivative according to any one of the claims 31-44, wherein said chemical group is conjugated to a free sulfhydryl group present on the amino add substituted for an amino add in, inserted in, or added to the polypeptide.
46. The Factor Vll derivative according to any one of the ctaims 31-45, wherein said chemical group is conjugated to a cysteine.
47. An inactivated Factor Vll derivative, wherein an inactivated Factor Vll polypeptide comprising the amino add sequence of SEQ 10 NO: 1 or a variant thereof and having a modification in its catalytic center, which modification inhibits the ability of the Fador Vll polypeptide to activate plasma Factor X or IX is further conjugated with a chemical group that increases the actual molecular weight of said inactivated Factor Vll polypeptide with about 300 daltons to about 100,000 daltons.
48. The Inactivated Factor Vlt derivative according to claim 47, wherein said inactivated Factor Vll polypeptide Is according to any one of the daims 28-30.
49. The inactivated Factor Vll derivative according to any one of the dalms 47-48, wherein said chemical group is substantially neutral.
50. The inactivated Fador Vll derivative according to any one of the claims 47-49, wherein said chemical group increases the actual molecular weight of the inactivated Factor Vll derivative witii atx}ut 1,000 daltons to about 80,000 daltons.
51. The inactivated Factor Vll derivative according to any one of the daims 47-50, wherein said chemical group increases the actual molecular weight of the inactivated Factor Vlt derivative with about 5,000 daltons to about 60,000 daltons.
52. The inactivated Fador Vll derivative according to any one of the daims 47-51, wherein said chemical group increases the actual molecular weight of ttie inactivated Factor Vll derivative witii about 10,000 daltons to about 40,000 daltons.

53. The inactivated Factor VII derivative according to any one of the claims 47-52, wherein said chemical group is polyethylene glycol.
54. The inactivated Factor VII derivative according to any one of the claims 47-53, wherein said chemical group is selected from one to six molecules of polyethylene glycol.
55. The inactivated Factor VII derivative according to claim 54, wherein said chemical group is one molecule of polyethylene glycol.
56. The inactivated Factor VlI derivative according to any one of the claims 47-55, wherein said chemical group is conjugated to a free sulfhydryl group present on the amino acid substituted for an amino add in, inserted in or added to the polypeptide.
•■
57. The inactivated Factor VII derivative according to any one of the claims 47-56, wherein said chemical group is conjugated to a cysteine.
58. A composition comprising a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein said different amino acid is conjugated with a chemical group that increases the actual molecular weight of said Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein said Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.
59. A composition comprising a Factor VII derivative according to any one of the claims 31-46.
60. A composition comprising an inactivated Factor Vli derivative, wherein an inactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant 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 is further conjugated with a chemical group that increases the actual molecular weight of said inactivated Factor VII polypeptide with about 300 daltons to about 100.000 daltons.
61. A composition comprising an inactivated Factor VII derivative according to any one of the claims 47-57.

62. A phannaceutical composition comprising a Factor VII derivative comprising a Factor VII olypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein n amino acid has been substituted with a different amino acid, wherein said different amino dd is conjugated with a chemical group that increases the actual molecular weight of said actor VII polypeptide with about 300 daltons to about 100,000 daltons and wherein said actor Vli derivative has substantially the same activity or increased activity compared to re-Dmbinantwild type human Factor Vila; and optionally, a pharmaceutically acceptable carer.
63. A pharmaceutical composition comprising a Factor VII derivative according to any one of
be dairns 31^46; and optionally, a pharmaceutically acceptable carrier. . . ■ .
4. A pharmaceutical composition comprising an inactivated Factor VII derivative, wherein an
lactivated Factor VII polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a
ariant thereof and having a modification in its catalytic center, which modification inhibits the
bility of the Factor VII polypeptide to activate plasma Factor X or IX is further conjugated
^tth a chemical group that increases the actual molecular weight of said inactivated Factor II polypeptide with about 300 daltons to about 100,000 daltons; and optionally, a phamna-eutically acceptable carrier.
5. A pharmaceutical composition comprising an inactivated Factor Vlt derivative according ) any one of the claims 47-57; and optionally, a pharmaceutically acceptable carrier.
6. A polynucleotide construct encoding a Factor VII polypeptide according to any one of the laims 1-27.
7. The polynucleotide construct according to claim 66. which is a vector.
8. A eucaryotic host cell comprising the polynucleotide construct according to any one of ie claims 66-67.
9. The eucaryotic host cell according to claim 68, which is of mammalian origin.
0. The eucaryotic host cell according to claim 69. wherein said cell is selected from the roup consisting of CHO cells, BHK cells or HEK cells.

71. A transgenic animal expressing the polynucleotide construct according to any one of the claims 66-67,
72. A transgenic plant expressing the polynucleotide construct according to any one of the claims 66-67.
73. A method for producing the Factor VII polypeptide according to any one of the claims 1-27, the method comprising cultivating a eucaryotic host ceil according to any one of the claims 68-70 in an appropriate growth medium under conditions allowing protein synthesis from said polynucleotide construct and recovering said Factor VII polypeptide from the culture medium.
74. A method for producing the Factor Vll polypeptide according to any one of the claims 1-27 the method comprising recovering said Factor VU polypeptide from milk produced by the transgenic animal defined in claim 71.
75. A method for producing the Factor Vll polypeptide according to any one of the claims 1-27. the method comprising cultivating a cell of a transgenic plant according to claim 72, and recovering the Factor Vll polypeptide from the resulting plant.
76. A method of producing a Factor Vll derivative comprising the steps of.

a) producing a Factor Vll polypeptide by a method according to any one of the claims 73-75;
b) conjugating the Factor Vll polypeptide with a chemical group;
c) applying the Factor Vll derivative to a cation exchange chromatography orgelfiltration column; and
d) eluting the Factor Vll derivative.
77. A method of producing an inactivated Factor Vll derivative comprising the steps of:
a) producing a Factor Vll polypeptide by a method according to any one of the claims 73-75;
b) modifying the Factor VII polypeptide in its catalytic center with a serine pnDtease inhibitor;
c) conjugating the inactivated Factor Vll polypeptide with a chemical group;

d) applying the inactivated Factor VII derivative to a cation exchange chromatography or gelfiltration column; and
e) efutlng the inactivated Factor VII derivative.

78. The method according to any one of the claims 76-77, wherein said chemical group has a molecular weight of about 300 daitons to about 100,000 daltons.
79. The method according to any one of the claims 76-78, wherein said chemical group is substantially neutral.
80. The method according to any one of the claims 76-79, wherein said chemical group has a molecular weight of about 1,000 daltons to about 80,000.
81. The method according to any one of the claims 76-80, wherein said chemical group has a molecular weight of about 5.000 daltcns to about 60,000.
82. The method according to any one of the claims 76-81, wherein said chemical group has a molecular weight of about 10,000 daltons to about 40,000.
83. The method according to any one of the claims 76-82, wherein said chemical group is polyethylene glycol.
84. The method according to any one of the claims 76-83, wherein said chemical group is selected from one to six molecules of polyethylene glycol.
85. The method according to any one of the claims 76-84, wherein said chemical group is one molecule of polyethylene glycol.
86. The method according to any one of the claims 76-85, wherein said chemical group is conjugated to a free sulfhydryl group present on the amino acid substituted for an amino acid in, inserted in, or added to the polypeptide.
87. The method according to any one of the daims 76-86, wherein said chemical group is conjugated to a cysteine.

88. Use of a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein said different amino acid is conjugated with a chemical group that increases the actual molecular weight of said Factor VIJ polypeptide with about 300 daltons to about 100,000 daltons and wherein said Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila for the preparation of a medicament for the treatment of bleeding episodes or for the enhancement of the nonnal haemostatic system.
89. Use of a Factor VII derivative according to any one of the claims 31-46 for the preparation of a medicament for the treatinent of bleeding episodes or for the enhancement of the normal haemostatic system. ,
90. Use according to any one of the claims 88-89 for the treatment of haemophilia A or B.
91. A method for the treatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the normal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a Factor VII derivative comprising a Factor VII polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein an amino acid has been substituted with a different amino acid, wherein said different amino acid Is conjugated with a chemical group that increases the actual molecular weight of said Factor VII polypeptide with about 300 daltons to about 100.000 daltons and wherein said Factor VII derivative has substantially the same activity or increased activity compared to recombinant wild type human Factor Vila.
92. A method for the treatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the normal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a Factor VII derivative according to any one of the claims 31-46,
93. Use of an inactivated Factor VII derivative, wherein an inactivated Factor VI! polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof is conjugated with a chemical group that increases the actual molecular weight of said inactivated Factor VII polypeptide with about 800 daltons to about 100,000 daltons for the preparation of a medicament for inhibiting thrombus formation in a patient.

the preparation of a medicament for for inhibiting thrombus fonmation in a patient.
95. A method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to tiirombus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor VII derivative, wherein an inactivated Factor VII polypeptide comprising the amino add sequence of SEQ ID NO: 1 or a variant thereof is conjugated with a chemical group that increases the actual molecular weight of said inactivated Factor VII polypeptide with about 300 daltons to about 100,000 daltons.
96. A method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising an inactivated Factor vn derivative according to any one of the daims 47-57.

97. A Factor VII polypeptide substantially as herein described with reference to the accompanying drawings.
98. A pharmaceutical composition substantially as herein described with reference to the accompanying drawings.


Documents:

1479-chenp-2003 claims-duplicate.pdf

1479-chenp-2003 description (complete)-duplicate.pdf

1479-chenp-2003-claims.pdf

1479-chenp-2003-correspondnece-others.pdf

1479-chenp-2003-correspondnece-po.pdf

1479-chenp-2003-description(complete).pdf

1479-chenp-2003-drawings.pdf

1479-chenp-2003-form 1.pdf

1479-chenp-2003-form 18.pdf

1479-chenp-2003-form 26.pdf

1479-chenp-2003-form 3.pdf

1479-chenp-2003-form 5.pdf

1479-chenp-2003-pct.pdf


Patent Number 230089
Indian Patent Application Number 1479/CHENP/2003
PG Journal Number 13/2009
Publication Date 27-Mar-2009
Grant Date 24-Feb-2009
Date of Filing 19-Sep-2003
Name of Patentee NOVO NORDISK HEALTH CARE AG
Applicant Address Andreasstraase 15, CH-8050 Zurich,
Inventors:
# Inventor's Name Inventor's Address
1 PERSSON, Egon 60, Lundavagen, S-232 52 Akarp,
2 OLE HVILSTED OLSEN BAEKKESKOVVEJ 38, DK-2700 BRONSHOJ,
PCT International Classification Number A61K38/48
PCT International Application Number PCT/DK02/00189
PCT International Filing date 2002-03-21
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PA 2001 00477 2001-03-22 Denmark