ES2361495T3 - VARIATIONS OF FVII OR FVIIA THAT HAVE IMPROVED COAGULATION ACTIVITY. - Google Patents

Abstract

A variant of Factor VII or Factor VIIa polypeptide, said variant having an amino acid sequence that differs from human Factor VII or human Factor VIIa having the amino acid sequence shown in SEQ ID NO: 1 in no more than 15 residues amino acids, wherein said variant sequence comprises the L65Q substitution and has improved coagulation activity or reduced coagulation time compared to hFVIIa or rhFVIIa.

Description

Field of the Invention

The present invention relates to novel FVII or FVIIa variants comprising replacement L65Q. Such variants have improved coagulation activity. The present invention also relates to the use of said polypeptide variants in therapy, in particular for the treatment of a variety of disorders related to coagulation.

Background of the invention

Blood coagulation is a process that involves a complex interaction of several blood components (or factors) that eventually result in a fibrin clot. In general, the components of blood that participate in what has been referred to as a "coagulation cascade" are proenzymes or cytogens, that is, enzymatically inactive proteins that are converted into an active form by the action of an activator. One of these coagulation factors is FVII.

FVII is a vitamin K-dependent plasma protein synthesized in the liver and secreted in the blood as a single-chain protein with a molecular weight of 53 kDa (Broze and Majerus, J Biol Chem 1980; 255: 1242-1247). The FVII cytogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site, R152-I153, which results in two chains linked by a single disulfide bridge. FVIIa in complex with tissue factor (FT), the FVIIa complex, is capable of converting factor IX and factor X into its activated forms, followed by reactions that lead to rapid thrombin production and fibrin formation (Østerud and Rapaport, Proc Natl Acad Sci USA 1977; 74: 5260-5264).

FVII undergoes modifications after translation, including vitamin K-dependent carboxylation, resulting in ten residues of -carboxyglutamic acid in the N-terminal region of the molecule. Thus, the numbers of residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO: 1 are residues of box carboxyglutamic acids in the Gla domain important for FVII activity. Other modifications after translation include the attachment of a sugar moiety to two N-glycosylation sites that occur naturally at position 145 and 322, respectively, and to two O-glycosylation sites that occur naturally at position 52 and 60 respectively.

The gene encoding human FVII (hFVII) has been mapped to chromosome 13 in q34-qter 9 (from Grouchy et al., Hum Genet 1984; 66: 230-233). It contains nine exons and has an extension of 12.8 kb (O’Hara et al., Proc Natl Acad Sci USA 1987; 84: 5158-5162). This gene organization and protein structure of FVII are similar to those of other vitamin K-dependent procoagulant proteins, with exons Ia and 1b encoding the signal sequence, exon 2, the propeptide and the Gla domain; exon 3 a short hydrophobic region; exons 4 and 5 domains of the epidermal growth factor type, and exon 6 to 8 in the catalytic domain of serine protease (Yoshitake et al., Biochemistry 1985; 24: 3736-3750).

There are reports on experimental three-dimensional structures of hFVIIa (Pike et al., PNAS USA 1999; 96: 8925-30 and Kemball-Cook et al., J Struct Biol 1999; 127: 213-223), of hFVIIa in complex with tissue factor soluble using X-ray crystallographic procedures (Banner et al., Nature 1996; 380: 41 and Zhang et al., J Mol Biol 1999; 285: 2089), and smaller fragments of hFVII (Muranyi et al., Biochemistry 1998 ; 37: 10605 and Kao et al., Biochemistry 1999; 38: 7097).

Some variants of designed FVII proteins have been reported (Dickinson and Ruf, J Biol Chem 1997; 272: 1987519879; Kemball-Cook et al., J Biol Chem 1998; 273: 8516-8521; Bharadwaj et al., J Biol Chem 1996; 271: 30685-30691; Ruf et al., Biochemistry 1999; 38: 1957-1966, US 5,560,580; US 5,288,629; WO 01/83725; WO 02/22776; WO 02/077218; WO 03 / 027147; WO 02/38162; WO 03/037932; WO 99/20767; WO 00/66753 and WO 01/58935).

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

Commercial preparations of human recombinant FVIIa are marketed as NovoSeven®. NovoSeven® is indicated for the treatment of bleeding episodes in patients with hemophilia A or B. NovoSeven® is the only rFVIIa for the effective and reliable treatment available in the market for bleeding episodes.

In WO 91/11514 an inactive form of FVII has been reported in which arginine 152 and / or isoleucine 153 is / are modified. These amino acids are located at the inactivation site. WO 96/12800 describes the inactivation of FVIIa by a serine protease inhibitor. Petersen et al., Eur J Biochem 1999; 261: 124-129 have described the inactivation by carbamylation of FVIIa in the α-amino acid group I153. The inactivated form is able to compete with the natural FVII or FVIIa for binding to TF and inhibit coagulation activity. The use of the inactivated form of FVIIa for the treatment of patients who are in hypercoagulable states, such as patients with sepsis, at risk of myocardial infarction or thrombotic stroke has been suggested.

WO 98/32466 suggests that FVII, among many other proteins, may be PEGylated, but does not contain any information in this regard.

WO 01/58935 discloses a new strategy to develop FVII or FVIIa molecules that have, among others, an increased half-life.

A half-life of circulating rFVIIa of 2.3 hours has been reported in "Summary Basis for Approval for NovoSeven®", FDA reference number 96-0597. Relatively high doses and frequent administration are necessary to achieve and sustain the desired therapeutic or prophylactic effect. Consequently, it is difficult to obtain adequate dose regulation, and the need for frequent intravenous administrations imposes restrictions on the patient's way of life

Treatment with rFVIIa could become more effective if a form of FVIIa designed in such a way as to improve its binding to FT could be used. Without wishing to be bound by a specific theory, said FVIIa variant could improve the treatment by the following mechanism: a modified FVIIa molecule with increased affinity for FT could allow a more effective treatment of bleeding due to its ability to replace inactive FVII or FVIIA inactivated in the FT mediating through this means a stronger amplification of the coagulation pathway and would therefore accelerate the clot formation process and possibly even mediate the formation of a stronger clot. Thus, such improved FVIIa could be more effective in stopping uncontrolled bleeding, for example, in trauma patients.

This increased efficiency will be located in sites of damaged tissue because this is the only place where cells (endothelial cells) that support active TF are present. Thus, in addition to the increased efficacy, a modified FVIIa with increased affinity for the FT will constitute a safer procoagulant treatment due to the location of the activity at the sites of tissue injury, that is, in the cells that are exposed from the endothelium, that is, at sites where an increase in procoagulant activity is desirable.

Accordingly, the main object of the present invention is to provide FVII / FVIIa variants with improved coagulation efficiency, such as improved coagulation activity (reduced coagulation time) and / or an ability to generate stronger clots. Variants can be further designed to obtain an increased phospholipid membrane binding affinity. Such variants will increase the efficiency of FVIIa even further because the molecule can direct the FT present in the platelets through its fusion with the so-called germinated microparticles from, for example, monocytes that produce FT. This addressing will simultaneously locate the increase in the generation of Factor X with the rest of the coagulation cascade, that is, at the site of thrombin and fibrin formation.

Another problem with the current rFVIIa treatment is the relative instability of the molecule against proteolytic degradation. Proteolytic degradation is a major obstacle to obtaining a solution in solution as opposed to a lyophilized product. The advantage of obtaining a stable soluble preparation is based on easier handling for the patient, and, in the case of emergencies, faster action, which can potentially save life. Attempts have been made known to prevent proteolytic degradation by site-directed mutagenesis at the main proteolytic sites in WO 88/10295. Another attempt to prepare stabilized liquid formulations of FVII / FVIIa is described in WO 03/055512.

Thus, a further object of the present invention is to provide FVII / FVIIA variants that, in addition to the aforementioned improved properties, are more stable against proteolytic degradation, that is, possess reduced sensitivity against proteolytic degradation.

WO 02/38162 refers to the modification of FVIIa to increase the amidolytic activity in the absence of tissue factor and affinity for tissue factor when compared with the natural factor ViIa, although without altering the proteolytic activity when it binds to the tissue factor.

WO 01/589 refers to FVII or FVIIa polypeptide conjugates comprising at least one non-polypeptide moiety covalently linked to a polypeptide, in which the amino acid sequence of the polypeptide differs from that of natural FVII or FVIIa in that at least an amino acid residue comprising a binding group of said non-polypeptide moiety has been introduced or removed.

Cheung Wing-Fai et al. (1995), Thrombosis Research, 80, 419-427, refer to the location of a metal dependent epitope in FVII.

Dickinson et al. (1996), Proc Natl Acad Sci USA. 93, 14379-14384, report a study that refers to the identification of surface debris involved in the union of the tissue factor and the catalytic function of FVII. Dickinson et al., Report that Gln-64, Ile-69, Phe-71, Arg-79, Arg-277, Met-306 and Asp-30 have an energetic influence.

Dickinson et al. (1997) Journal of Biological Chemistry, 272, 19875-19879, report a study of affinities for zymogens and species of natural FVII enzymes and mutants and residues of the protease domain that come into contact with FT. The authors conclude that the binding of FT is not influenced by the activation of the zymogen, and that there is a two-way conformational interdependence between the Met306 moiety and the active site of FVII.

Petrovan et al. (2001) J Biol Chem. 276, 6616-6620, report that the Met 156 moiety contributes to the labile conformation of the coagulation factor VIIa enzyme.

Sridhara et al. (1996), Am J Hematol., 53, 66-71, is a study of the effect of the R152Q and R79Q mutations in an ELISA trial that concludes that both states of factor VII activation and mutation of the Amino acid residues within the epidermal growth factor type domain can alter the affinity of factor VII for tissue factor.

WO 00/66753 refers to the membrane binding affinity of vitamin K-dependent polypeptides, including FVII. Amino acid substitutions are mentioned in amino acid 5, 9, 11, 12, 29, 33, 34, 35, or

36.

US 5,580,560 refers to the modification of factor VII / VIIa for stabilization against proteolytic cleavage.

A molecule with a longer half-life in circulation will decrease the number of administrations needed. Given the association of the current FVIIa product with frequent injections, and of the potential to obtain more optimal therapeutic FVIIa levels with simultaneous enhanced therapeutic effect, there is a clear need for FVII or FVIIa molecules with an increased half-life in circulation. One means of increasing the circulating half-life of a protein is to ensure that the renal clearance of the protein is reduced. This can be achieved by conjugating the protein with a chemical moiety that is capable of conferring reduced renal clearance to the protein. In addition, the binding of a chemical moiety to the protein or the substitution of amino acids exposed to proteolysis can effectively block a proteolytic contact envelope that leads to proteolytic degradation of the protein. Polyethylene glycol (PEG) is one such chemical residue that has been used in the preparation of protein therapeutic products.

Thus, a further objective of the present invention is to provide FVII / FVIIA variants that, in addition to the aforementioned improved properties, have an increased in vivo functional half-life and an increased serum half-life.

The aforementioned objectives are met by means of the FVII / FVIIa variants disclosed in this document.

Brief disclosure of the invention

The present invention provides improved recombinant FVII or FVIIa variants comprising the L65Q substitution. This amino acid substitution at the FT binding site of the FVII molecule results in improved coagulation activity. Variants of the invention differ from human Factor VII or human Factor VIIa (SEQ ID NO: 1) in no more than 15 amino acid residues.

In interesting embodiments, the FVII or FVIIa variant has been further modified so that the resulting variant has an enhanced phospholipid membrane binding affinity, increased in vivo functional half-life and / or increased plasma half-life. In other additional embodiments, the variant has been further modified such that it has increased bioavailability and / or reduced sensitivity to proteolytic degradation. Accordingly, medical treatment with said variant offers numerous advantages over the currently available rFVIIa compound, such as a lower dosage, faster action in uncontrolled bleeding and, optionally, longer duration between injections.

In one aspect, the invention relates to a polypeptide variant of Factor VII (FVII) or Factor VIIa (FVIIa) having an amino acid sequence comprising 1-15 amino acid modifications with respect to human Factor VII (HFVII) or Factor Human VIIa (hFVIIa) having the amino acid sequence shown in SEQ ID NO: 1, wherein said variant sequence comprises the L65Q substitution

with the proviso that said variant is not

[K32E + D33N + A34T + K38T + L39E] hFVII or

[A 1Y + K32E + D33N + A34T + K38T + L39E] hFVII or

[A1Y + A3S + F4GK + K32E + D33N + A34T + K38T + L39E] hFVII or [A1Y + L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII or

[A1Y + A3S + F4GK + L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII or

[A3S + F4GK + K32E + D33N + A34T + K38T + L39E] hFVII or

[A3S + F4GK + L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII or

[L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII or

[I42N] hFVII / hFVIIa or [I42S] hFVII / hFVIIa or [I42A] hFVII / hFVIIa or

[I42Q] hFVII / hFVIIa.

In a second aspect, the invention relates to a variant of Factor VIIa polypeptide (FVIIa) having an amino acid sequence comprising 1-15 amino acid modifications with respect to human Factor VIIa (hFVIIa) having the amino acid sequence that is shown in SEQ ID NO: 1, wherein said variant sequence comprises the substitution. The FVII variants comprising the L39E mutation disclosed by Cheung and Stafford in Throm Res 1995; 79; 199-206 are not, within the scope of the present application. Similarly, the FVII / FVIIa variants comprising the I42N / S / A / Q mutations disclosed in US 5,580,560 are not included in the scope of the present application.

Another aspect of the invention relates to a nucleotide sequence encoding the variant of the invention.

In a further aspect, the invention relates to an expression vector comprising the nucleotide sequence of the invention.

In a more additional aspect, the invention relates to a host cell comprising the nucleotide sequence of the invention or the expression vector of the invention.

In another more additional aspect, the invention relates to a pharmaceutical composition comprising the variant of the invention, and an acceptable pharmaceutical carrier or excipient.

Another aspect of the invention relates to the variant of the invention, or the pharmaceutical composition of the invention, for use as a medicament.

Additional aspects of the present invention will be apparent from the following specification as well as from the appended claims.

Brief description of the drawings

Fig. 1 shows the coagulation time versus the concentration of [L65Q] rhFVIIa, [S43Q] rhFVIIa, [L39E] rhFVIIa and [E82Q] rhFVIIa when tested in the "Full Blood Test". The results of rhFVIIa (activated with OSII) and NovoSeven® are included for comparison. 0 rhFVIIa (activated with OSII); ♦ NovoSeven®; ● [L65Q] rhFVIIa; Δ [S43Q] rhFVIIa; x [L39E] rhFVIIa; OR [E82Q] rhFVIIa.

Fig. 2 shows the coagulation time versus the concentration of [I42R] rhFVIIa and [L39Q] rhFVIIa when tested in the "Full Blood Test". The result of NovoSeven® is included for comparison. ♦ NovoSeven®; 0 [I42R] rhFVIIa; ● [L39Q] rhFVIIa.

Fig. 3 shows the coagulation time versus the concentration of [F71D] rhFVIIa, [K62E] rhFVIIa, [F71Y] rhFVIIa, [L65S] rhFVIIa and [F71E] rhFVIIa when tested in the "Full Blood Test". The result of NovoSeven® is included for comparison. ♦ NovoSeven®; ◊ [F71D] rhFVIIa; ● [K62E] rhFVIIa; or [F71Y] rhFVIIa; x [L65S] rhFVIIa;

▲ [F71E] rhFVIIa.

Detailed disclosure of the invention

Definitions

In the context of the present application and of the invention, the following definitions apply:

The term "conjugate" (or interchangeably, "variant of conjugated polypeptide") is intended to indicate a heterogeneous molecule (in the sense of composite or chimeric material) formed by the covalent bond of one or more polypeptide (s) with one or more non-polypeptide moieties such as polymer molecules, lipophilic compounds, sugar moieties

or organic derivatizing agents. Preferably, the conjugate is soluble at relevant concentrations and conditions, that is, soluble in physiological fluids such as blood. Examples of conjugated polypeptide variants of the invention include glycosylated and / or PEGylated polypeptides.

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The term "covalent bond" or "covalently linked" means that the polypeptide variant and the non-polypeptide moiety both covalently bind directly to each other, or covalently bind indirectly to each other through a moiety or moieties. intervention, such as bridge, separator, or rest or link remains.

When used herein, the term "non-polypeptide moiety" means a molecule that is capable of conjugating with a linker group of the polypeptide variant of the invention. Preferred examples of such molecules include polymer molecules, sugar moieties, lipophilic compounds, or organic derivatizing agents, in particular sugar moieties. When used in the context of a polypeptide variant of the invention, it will be understood that the non-polypeptide moiety is linked to the polypeptide portion of the polypeptide variant through a linking group of the polypeptide variant. As explained above, the non-polypeptide moiety can be covalently linked directly to the linkage group or covalently linked to the linkage group indirectly through an intervening moiety or moieties, such as a bridge, separator or rest or link remains

A "polymer molecule" is a molecule formed by the covalent bond of two or more monomers, in which none of the monomers is an amino acid residue. The term "polymer" can be used interchangeably with the term "polymer molecule." It is also intended that the term cover the carbohydrate molecules bound by glycosylation in vitro, that is, a synthetic glycosylation carried out in vitro normally which involves covalently binding a carbohydrate molecule to a linker group of the polypeptide variant, optionally using a crosslinking agent. In vitro glycosylation is further described in detail below.

The term "sugar moiety" is intended to indicate a carbohydrate-containing molecule comprising one or more monosaccharide moieties, capable of binding to the polypeptide variant (to produce a conjugate of the polypeptide variant in the form of a glycosylated polypeptide variant ) by glycosylation in vivo. The term "glycosylation in vivo" is intended to mean any linkage of a sugar moiety that occurs in vivo, that is, during post-translational processing in a glycosylation cell used for expression of the polypeptide variant, for example, by means N-linked or O-linked glycosylation. The exact structure of the oligosaccharide depends, to a large extent, on the glycosylation organism in question.

An "N-glycosylation site" has the sequence NXS / T / C, in which X is any amino acid residue except proline, N is asparagine and D / T / C is both serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine. Preferably, the amino acid residue in position +3 with respect to the asparagine residue is not a proline residue.

An "O-glycosylation site" is the OH group of a serine or threonine moiety.

The term "linking group" is intended to indicate a functional group of the polypeptide variant, in particular of one of its amino acid residues or a carbohydrate moiety, capable of binding to a non-polypeptide moiety such as a polymer molecule, a molecule lipophilic, a sugar moiety or an organic derivatizing agent. Useful binding groups and their corresponding non-polypeptide moieties are evident from the following table.

Link group

Amino acid Examples of non-polypeptide moieties Conjugation procedure (PEG activated Reference

-NH2

End N, Lys Polymer, for example, PEG with amide or imine group mPEG Tresilated with mPEG-SPA Shearwater Inc. Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9 (3.4): 249-304 (1992)

-COOH

End C, Asp, Glu Polymer, for example, PEG, carbohydrate moiety with ester or amide group In vitro coupling with mPEG-Hz Shearwater Inc.

(continuation)

Link group

Amino acid Examples of non-polypeptide moieties Conjugation procedure (PEG activated Reference

-SH

Cys Polymer, for example, PEG, rest In vitro coupling of PEG maleimide and Shearwater Inc. Delgado et al, critical

carbohydrate with disulfide, maleimide or vinyl sulfone group

PEG-vinyl sulfone reviews in Therapeutic Drug Carrier Systems 9 (3,4): 249-304 (1992)

-OH

Be, Thr, Lys, -OH PEG sugar residue with ester, ether, carbamate, carbonate O-linked glycosylation in vivo

-CONH2

Asn as part of a Nglicosylation site Polymer with sugar moiety, for example, PEG N-glycosylation in vivo

Aromatic rest

Phe, Tyr, Trp Carbohydrate rest In vitro coupling

-CONH2

 Gln Carbohydrate rest In vitro coupling Yan and Wold, Biochemistry, 1984, Jul 31; 23 (16): 375965

Aldehyde Ketone

Oxidized oligosaccharide Polymer, for example PEG, PEG-hydrazide PEGylation Andresz et al., 1978, Makromol. Chem. 179: 301, WO 92/16555, WO 00/23114

Guanidino

Arg Carbohydrate rest In vitro coupling Lundblad and Noyes, Chimical Reagents for Protein Modification, CRC Press Inc., Florida, USA.

Imidazole ring

His Carbohydrate rest In vitro coupling As for guanidine

For N-glycosylation in vivo, the term "linking group" is used in an unconventional manner to indicate the amino acid residues that constitute a N-glycosylation site (with the sequence NXS / T / C, in which X is any amino acid residue except proline, N is asparagine and S / T / C is either serine, threonine or cysteine, preferably serine or

5 threonine, and most preferably threonine). Although it is an asparagine residue of the N-glycosylation site it is one to which the sugar moiety is attached during glycosylation, said binding cannot be achieved unless the other amino acid residues of the N-glycosylation site are present.

Accordingly, when the non-polypeptide moiety is a sugar moiety and the conjugation is to be achieved by in vivo glycosylation, it should be understood that the term "amino acid moiety comprising a linker group for the moiety

10 non-polypeptide ”as used in conjunction with alterations in the amino acid sequence of the polypeptide variant means that one or more amino acid residues that constitute an in vivo N-glycosylation site will be altered in such a way that any one of a Functional N-glycosylation site in vivo is introduced into the amino acid sequence or removed from said sequence.

In the present application, amino acid names and atom names are used (e.g., CA, CB, CD, CG, SG, NZ,

15 N, O, C, etc.) as defined by the Protein DataBank (PDB) (www.pdb.org) based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (remains names, names of atoms, etc.), Eur.

J. Biochem., 138, 9-37 (1984) along with its corrections in Eur. J. Biochem., 152, 1 (1985)).

The term "amino acid residue" is intended to indicate an amino acid residue contained in the group consisting of alanine (Ala or A) residues, cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E) , phenylalanine (Phe or F), glycine

20 (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y).

The terminology used to identify amino acid positions is illustrated as follows: G124 indicates that position 124 is occupied by a glycine residue in the amino acid sequence shown in SEQ NO: 1. G124R 25 indicates that the position glycine residue 124 has been replaced with an arginine residue. Alternative substitutions are indicated with

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an "/", for example, N145S / T means an amino acid sequence in which the asparagine at position 145 is substituted with either serine or threonine. Multiple substitutions with a "+" are indicated, for example K143N + N145S / T means an amino acid sequence comprising a substitution of the lysine moiety in position 143 with an asparagine moiety and a substitution of the asparagine moiety in position 145 with a serine moiety or A threonine rest. The insertion of an additional amino acid residue, such as the insertion of an alanine residue after G124 is indicated by G124GA. The insertion of two additional alanine residues after G124 is indicated by G124GAA, etc. When used herein, the term "inserted in X position" or "inserted in X position" means that the amino acid residue (s) is inserted between the amino acid residue. X and X + 1. A deletion of an amino acid residue is indicated by an asterisk. For example, the deletion of the glycine residue at position 124 is indicated by G124 *.

Unless otherwise indicated, the numbering of amino acid residues performed herein is done with respect to the amino acid sequence of natural FVII / FVIIa (SEQ ID NO: 1).

The term "differs from", as used in conjunction with specific mutations is intended to allow additional differences to be present apart from the specified amino acid difference. For example, in addition to the substitutions specified in positions 39, 42, 43, 62, 65, 71, 82 and / or 275, the FVII or FVIIa polypeptide variant may comprise other substitutions. Examples of such modifications or additional differences may include truncation of the N and C-terminus by one or more amino acid residues (for example, 1-10 amino acid residues), or the addition of one or more extra moieties at the N and / or N-terminus. C, for example, the addition of a methionine moiety at the N-terminus, as well as the "conservative amino acid substitutions", that is, substitutions carried out within groups of amino acids with similar characteristics, for example, small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids.

Examples of these conservative substitutions are shown in the following table

one

Alanina (A) Glycine (G) Serina (S) Threonine (T)

2

Aspartic acid (D) Glutamic acid (E)

3

Asparagine (N) Glutamine (Q)

4

Arginine (R) Histidine (H) Lysine (K)

5

Isoleucine (I) Leucine (L) Methionine (M) Valine (V)

6

Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Further examples of further modifications include modifications that provide an increase in an increased in vivo functional half-life or an increased serum half-life. Specific examples of such modifications are discussed below. In addition, the polypeptide variant of the invention may contain additional modifications that provide an increase in binding affinity in an enhanced phospholipid membrane. Specific examples are provided below.

The term "nucleotide sequence" is intended to indicate a consecutive stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic, or any combination thereof.

The term "polymerase chain reaction" or "PCR" generally refers to a method of amplifying a desired nucleotide sequence in vitro, as described, for example, in US 4,683,195. In general, the PCR procedure involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of hybridizing preferably with a nucleic acid template.

The term "vector" refers to a plasmid or other nucleotide sequences that are capable of replicating inside a host cell or integrating into the genome of the host cell, and as such, are useful for performing different functions. together with compatible host cells (a vector-host system): to facilitate cloning of the nucleotide sequence, that is, to produce usable amounts of the sequence, to direct the expression of the gene product encoded by the sequence and to integrate the sequence of nucleotides in the genome of the host cell. The vector will contain different components depending on the function that it performs.

"Cell", "host cell", "cell line" and "cell culture" are used interchangeably herein and it should be understood that all these terms include the progeny resulting from the growth or culture of a cell.

"Transformation" and "transfection" are used interchangeably to refer to the process of introducing DNA into a cell.

"Operably linked" refers to the covalent attachment of two or more nucleotide sequences, by enzymatic ligation or otherwise, in a relative configuration to each other such that the normal function of the cells can be carried out. sequences For example, the nucleotide sequence encoding a presequence or secretory leader is operably linked to a nucleotide sequence encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide: a promoter or enhancer binds operable way with a coding sequence if it affects the transcription of the sequence; A ribosome binding site is operably linked to an encoding sequence if it is positioned in such a way that it facilitates translation. Generally, "operably linked" means that the nucleotide sequences that bind are contiguous and, in the case of the secretory leader, contiguous and in reading phase. Binding is carried out by ligation at convenient restriction sites. If such sites do not exist, then synthetic adapter oligonucleotides are used, together with standardized recombinant DNA procedures.

In the context of the present invention, it is intended that the terms "modification" or "amino acid modification" cover the substitution of an amino acid side chain, the substitution of an amino acid residue, the deletion of an amino acid residue and / or the insertion of an amino acid residue of interest.

The terms "mutation" and "substitution" are used interchangeably herein.

The term "introduces" refers to the introduction of an amino acid residue by replacing an existing amino acid residue, or alternatively by inserting an additional amino acid residue.

The term "delete" refers to the elimination of an amino acid residue by replacing the amino acid residue to be removed by another amino acid residue, or alternatively by deletion (without substitution) of the amino acid residue to be removed.

The term "FVII" or "FVII polypeptide" refers to an FVII molecule provided in single stranded form. An example of an FVII polypeptide is the natural human FVII (hFVII) which has the amino acid sequence shown in SEQ ID NO: 1. It should be understood, however, that the term "FVII polypeptide" also covers hFVII type molecules, such as fragments or variants of SEQ ID NO: 1, in particular variants in which the sequence comprises at least one, such as 1-15, preferably 1-10, amino acid modifications compared to SEQ ID NO: 1.

The term "FVIIa" or "FVIIa polypeptide" refers to a FVIIa molecule provided in its activated double stranded form. When the amino acid sequence of SEQ ID NO is used: to describe the amino acid sequence of FVIIa it will be understood that the peptide bond between R152 and I153 has been cleaved from the single stranded form, and that one of the chains comprises amino acid residues 1- 152, the other amino acid residues 153-406.

The terms "rFVII" and "rFVIIa" refer to FVII and FVIIa molecules produced by recombinant techniques, respectively.

The terms "hFVII" and "hFVIIa" refer to natural human FVII and FVIIa, respectively, which have the amino acid sequence shown in SEQ ID NO: 1.

The terms "rhFVII" and "rhFVIIa" refer to human natural FVII and FVIIa, which have the amino acid sequence shown in SEQ ID NO: 1, produced by recombinant means. An example of rhFVIIa is NovoSeven®.

When used herein, it is intended that the term "Gla domain" encompasses amino acid residues No. 1 to 45 of SEQ ID NO: 1. Accordingly, the expression "position located outside the Gla domain" encompasses amino acid residue 46-406 of SEQ ID NO: 1.

The abbreviations "FX", "TF" and "TFPI" mean Factor X, Tissue Factor and Tissue Pathway Inhibitor, respectively.

The term "protease domain" is used approximately for residues 153-406 counted from the N-terminus.

The term "catalytic site" is used to mean the catalytic triad consisting of S344, D242 and H193 of the FVII / FVIIa molecule.

The term "parental" is intended to indicate the molecule to be modified / improved in accordance with the present invention. Although the parental polypeptide to be modified by the present invention can be any FVII or FVIIa polypeptide, and thus be derived from any origin, for example, a non-human mammalian origin, it is preferred that the parental polypeptide be hFVII or hFVIIa .

A "variant" is a polypeptide that differs in one or more amino acid residues from its parental polypeptide, usually in 1-15 amino acid residues (for example, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues), such as in 1-10 amino acid residues, for example, in 1-8, 1-6, 1-5 or 1-3 amino acid residues. Normally, the parental polypeptide is HFVII or hFVIIa. Thus, a "variant" normally contains 1-15 amino acid modifications (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 modifications of amino acids), such as 1-10 amino acid modifications, for example, 1-8, 1-6, 1-5 or 1-3 amino acid modifications with respect to the parental polypeptide. As explained above, the parental polypeptide is normally hFVII or hFVIIa. It will be understood that a polypeptide variant according to the present invention will differ from SEQ ID NO: 1 in at least one of the following positions: L39, I42, S43, K62, L65, F71, E82 and / or F275.

In the present context, the term "modification" encompasses its insertions, deletions, substitutions and combinations. It will be understood that a polypeptide variant according to the present invention will be modified in at least one position with respect to the parental polypeptide.

The term "amidolytic activity" is intended to mean the activity measured in the "Amidolytic Assay" described herein. In order to present "amidolytic activity" a variant of the invention, in its activated form, must have at least 10% of the amidolytic activity of rhFVIIa when tested in the "Amidolytic Assay" described herein. In a preferred embodiment of the invention the variant, in its activated form, has at least 20% of the amidolytic activity of rhFVIIa, such as at least 30%, for example, at least 40%, more preferably at at least 50%, such as at least 60%, for example, at least 70%, even more preferably at least 80%, such as at least 90% of the amidolytic activity of rhFVIIa when tested in the " Amidolytic Assay ”described herein. In an interesting embodiment, the variant, in its activated form, has substantially the same amidolytic activity as rhFVIIa, such as an amidolytic activity of 75125% of the amidolytic activity of rhFVIIa.

The term "coagulation activity" is used to mean the activity measured in the "Full Blood Test" described herein. It will be understood that the activity measured in the "Complete Blood Test" is the time required to obtain clot formation. In this way, a lower coagulation time corresponds to a greater coagulation activity.

The term "enhanced coagulation activity" is used to indicate that the coagulation time of the polypeptide variant decreases statistically significantly with respect to that generated by rhFVIIa as determined under comparable conditions and when measured in the "Blood Test. Complete ”described in this document.

The term "immunogenicity" as used in conjunction with a given substance is intended to indicate the ability of the substance to induce a response from the immune system. The immune response can be a cell-mediated or antibody-mediated response (see, for example, Roitt: Essential Immunology (8th Edition, Blackwell) for a further definition of immunogenicity). Normally, a reduced reactivity to the antibody will be an indication of reduced immunogenicity. The reduced immunogenicity can be determined by the use of any suitable method known in the art, for example in vivo or in vitro.

The term "functional half-life in vivo" is used in its normal meaning, that is, the time in which 50% of the biological activity of the polypeptide variant will continue to be present in the target body / organ, or the time in the which amidolytic or coagulation activity of the polypeptide variant is 50% of the initial value.

As an alternative to the functional determination of the half-life in vivo, the “serum half-life” can be determined, that is, the time in which 50% of the polypeptide variant circulates in the plasma or bloodstream before clearing . The determination of the serum half-life is often simpler than the determination of the functional half-life in vivo and the magnitude of the serum half-life is usually a good indication of the magnitude of the functional half-life in vivo. Alternatively, the terms for serum half-life include "plasma half-life", "circulation half-life", "serum clearance", "plasma clearance" and "half-life on clearance". The polypeptide variant is clarified by the action of one or more of the reticuloendothelial (SER), kidney, spleen or liver systems, by tissue factor, elimination mediated by the SEC receptor or other receptor, or by specific or non-specific proteolysis. Normally, the clearance depends on the size (with respect to the cut of the glomerular filtration), the load, the bound carbohydrate chains, and the presence of cellular protein receptors. The functionality to be retained is normally selected from the activity of binding to the procoagulant, proteolytic or receptor. The in vivo functional half-life and serum half-life can be determined by any suitable procedure known in the art.

The term "increased" as used with respect to the functional half-life in vivo or serum half-life is used to indicate that the relevant half-life of the polypeptide variant is statistically significantly increased with respect to that of a reference molecule. , such as an hFVIIa or rhFVIIa (for example, NovoSeven®) as determined under comparable conditions (normally determined in an experimental animal, such as rats, rabbits or pigs).

The expression "AUCIV" or "Area under the curve when administered intravenously" is used in its normal meaning, that is, as the area under activity in the serum-time curve, in which the variant of the intravenously administered polypeptide, in particular, when administered intravenously in rats. Normally, the measured activity is the "coagulation activity" as defined hereinbefore. Once the experimental activity-time points have been determined, the AUCIV can be conveniently calculated using a computer program, such as GraphPad Prism 3.01.

It will be understood that in order to have a direct comparison between the AUCIV values of different molecules (for example, between the variants of the invention and rhFVIIa) the same amount of activity must be administered. Consequently, the AUCIV values are usually normalized (that is, the differences in the injected dose are corrected) and expressed as AUCIV / administered dose.

The term "reduced sensitivity to proteolytic degradation" is primarily intended to mean that the polypeptide variant has reduced sensitivity to proteolytic degradation compared to hFVIIa or rhFVIIa (eg, NovoSeven®) as determined under comparable conditions. Preferably, the proteolytic degradation is reduced by at least 10% (for example 10-25% or 10-50%), such as at least 25% (for example, 25-50%, in 25-75% or 25-100%), more preferably at least 35%, such as at least 50% (for example, 50-75% or 50-100%), even more preferably at least 60%, such as at least 75% (for example 75-100%) or even at least 90%. Most preferably, proteolytic degradation is reduced by at least 99%.

The term "renal clearance" is used in its normal meaning to indicate any clearance that takes place by the kidneys, for example, by glomerular filtration, excretion or tubular degradation in the tubular cells. Renal clearance depends on the physical characteristics of the polypeptide, including size (diameter), hydrodynamic volume, symmetry, shape / stiffness, and load. Normally a molecular weight of approximately 67 kDa is considered to be a cut-off value for renal clearance. Renal clearance can be established by any suitable assay, for example, an assay established in vivo. Normally, renal clearance is determined by administering a labeled polypeptide (e.g., radiolabeled or fluorescently labeled) to a patient and measuring the activity of the label in the urine collected from the patient. Renal clearance is determined with respect to the corresponding reference polypeptide, by For example, human natural FVIIa, under comparable conditions. Preferably, the rate of renal clearance of the polypeptide variant is reduced by at least 50%, preferably at least 75%, and most preferably by at least 90% compared to hFVIIa or rhFVIIa (for example, NovoSeven ®).

The terms "at least 25%" of its side chain exposed to the surface of the molecule "and" at least 50% of its side chain exposed to the surface of the molecule "are defined with reference to Example 1, in the that the calculations, etc., are described in detail.

It should be noted that when the expressions "at least 25%" of its side chain exposed to the surface of the molecule "and" at least 50% of its side chain exposed to the surface of the molecule "are used together with the introduction from the N-glycosylation site in vivo, these terms refer to the surface accessibility of the amino acid side chain at the position where the sugar moiety actually binds. In many cases, it will be necessary to introduce a serine or a threonine residue in the +2 position with respect to the asparagine residue to which the sugar residue actually binds (unless, of course, this position is already occupied by a serine residue or one threonine) and these positions, where serine or threonine residues are introduced, are allowed to remain buried, that is, they have less than 25% or 50% of their side chains exposed to the surface of the molecule.

In the present specification and claims, any reference to "a" component, for example, in the context of a non-polypeptide moiety, an amino acid moiety, a substitution, a buffer, etc., is intended to refer to one or more of said components. components, unless defined otherwise or unless it is clear from the particular context if this is not the case. For example, the expression "a component selected from the group consisting of A, B and C" is intended to include all combinations of A, B and C, that is, A, B, C, A + B, A + C, B + C or A + B + C.

A polypeptide, nucleotide sequence or other component is "isolated" when it is partially or completely separated from the components with which it is normally associated (other peptides, polypeptides, proteins (including complexes, for example, polymerases and ribosomes that may accompany a sequence natural), nucleic acids, cells, synthetic reagents, cellular contaminants, cellular components, etc.), for example, as of other components with which it is normally associated in the cell from which it is originally derived. A polypeptide, nucleotide sequence or other component is isolated when partially or completely recovered or separated from other components of its natural environment such that it is the predominant species present in a composition, mixture,

or set of components (that is, on a molar basis it is more abundant than any other individual species in the composition). In some examples, the preparation consists of more than about 60%, more than

about 70%, or more than about 75%, usually more than about 80%, or

preferably more than about 90% of the isolated species. The terms "tissue factor binding site", "active site region" and "edge of the active site binding cleft" are defined with reference to Example 1, in which the aforementioned sites / regions are determined.

The term "hydrophobic amino acid residue" includes the following amino acid residues: Ile, Leu, Met, Val, Phe, Tyr and Trp. The term "charged amino acid residue" encompasses the following amino acid residues: Lys, Arg, His, Asp and Glu. The expression "negatively charged amino acid residue" includes the following amino acid residues: Asp and Glu. The expression "positively charged amino acid residue" includes the following amino acid residues: Lys, Arg and His. The term "polar amino acid residue" encompasses the following amino acid residues: Gly, Ser, Thr, Cys, Tyr, Asn and Gln. The term "mammal" as used herein includes humans, nonhuman primates (by

example, baboons, orangutans, monkeys), mice, pigs, cows, goats, cats, rabbits, rats, guinea pigs, hamsters,

horses, monkeys, sheep, or other non-human mammals. The term "effective amount" means a dosage or quantity sufficient to produce a desired result. The result desired may comprise an objective or subjective improvement in the recipient of the dosage or amount

Variants of the invention

In one aspect, the present invention relates to a variant of Factor VII polypeptide (FVII) or Factor VIIa (FVIIa) which has an amino acid sequence comprising 1-15 amino acid modifications with respect to Factor VII human (HFVII) or human Factor VIIa (hFVIIa) that has the amino acid sequence shown in SEQ ID NO: 1, wherein said variant sequence comprises the L65Q substitution,

with the proviso that said variant is not [K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [A1Y + K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [A1Y + A3S + F4GK + K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [A1Y + L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [A1Y + A3S + F4GK + L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [A3S + F4GK + K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [A3S + F4GK + L8F + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII / hFVIIa or [LBF + R9V + P10Q + K32E + D33N + A34T + K38T + L39E] hFVII / FVIIa or [142N] hFVU // hFVHa or [I42S] hFVII / hFVIIa or [I42A] hFVII / hFVIIa or [I42Q] hFVII / hFVIIa. In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the

L39E replacement.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the L39Q replacement. In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the

L39H replacement.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the I42R replacement. In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the

S43Q replacement. In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the K62E replacement.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the K62R substitution.

In all embodiments of the invention, the FVII or FVIIa variant comprises the L65 / Q substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the L65S substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the F71D substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the F71Y substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the F71E substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the F71Q substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the F71N substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the E82Q substitution.

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the E82N substitution

In a further interesting embodiment of the invention, the FVII or FVIIa variant comprises the F275H substitution.

In a very interesting embodiment of the invention, the FVII or FVIIa variant of the invention comprises a substitution selected from the group consisting of F71Y, K62E and S43Q, in particular, selected from the group consisting of K62E and S43Q.

It will be understood that it may be advantageous to combine one or more of the above-mentioned substitutions. Accordingly, in a further interesting embodiment of the invention, the variant comprises at least two (such as two) substitutions at positions selected from the group consisting of L39, I142, S43, K62, L65, F71, E82 and F275, such as substitutions at selected positions among the group consisting of L39 + I42, L39 + S43, L39 + K62, L39 + L65, L39 + F71, L39 + E82, L39 + F275, I42 + S43, I42 + K62 , I42 + L65, I42 + F71, I42 + F71, I42 + E82, 142 + F275, S43 + K62, S43 + L65, S43 + F71, S43 + E82, S43 + F275, K62 + L65, K62 + F71, K62 + E82, K62 + F275, L65 + F71, L65 + E82, L65 + F275, F71 + E82, F71 + F275 and E82 + F275. In accordance with this embodiment of the invention, it is preferred that the variant comprises at least two (such as two) substitutions at selected positions from the group consisting of L65 + F71, L65 + K62, L65 + S43, F71 + K62 , F71 + S43 and K62 + S43, in particular at least two (such as two) substitutions at positions selected from the group consisting of L65 + K62, L65 + S43 and, K62 + S43. More particularly, the variant of the invention may comprise at least two (such as two) substitutions selected from the group consisting of L39E, L39Q, L39H, I42R, S43Q, K62E, K62R, L65Q, L65S, F71D, F71Y, F71E, F71Q, F71N, E82Q, E82N and F275H, preferably at least two (such as two) substitutions selected from the group consisting of L65Q, F71Y, K62E and S43Q, more preferably at least two (such as two) substitutions selected from the group which consists of L65Q, K62E and S43Q. Specific examples include L65Q + F71 Y, L65Q + K62E, L65Q + S43Q, F71Y + K62E, F71Y + S43Q and K62E + S43Q, in particular L65Q + K62E, L65Q + S43Q and K62E + S43Q.

In more additionally interesting embodiments of the invention, the variant comprises at least three (such as three) substitutions at positions selected from the group consisting of L39, I42, S43, K62, F71, E82 and F275, such as substitutions in Selected positions from the group consisting of L39 + I42 + S43, L39 + I42 + K62, L39 + I42 + L65, L39 + I42 + F71, L39 + I42 + E82, L39 + I42 + F275, L39 + 543 + K62, L39 + S43 + L65, L39 + S43 + F71, L39 + K62 + E82, L39 + S43 + F275, L39 + K62 + L65, L39 + K62 + F71, L39 + K62 + E82, L39 + K62 + F275, L39 + L65 + F71, L39 + L65 + E82, L39 + L65 + F275, L39 + F71 + E82, L39 + F71 + F275, L39 + E82 + F275, I42 + S43 + K62, I42 + S43 + L65, I42 + S43 + F71, I42 + S43 + E82, I42 + S43 + F275, I42 + K62 + L65, I42 + K62 + F71, I42 + K62 + E82, I42 + K62 + F275, I42 + L65 + F71, I42 + L65 + E82, I42 + L65 + F275,142 + F71 + E82, I42 + F71 + F275,142 + E82 + F275, S43 + K62 + L, 65, S43 + K62 + F71, S43 + K62 + E82, S43 + K62 + F275, S43 + L65 + F71, S43 + L65 + E82, S43 + L65 + F275, S43 + F71 + E82, S43 + F71 + F275, S43 + E82 + F275, K62 + L65 + F71, K62 + L65 + E82, K62 + L65 + F275, K62 + F71 + E82, K62 + F71 F275, K62 + E82 + F275, L65 + F71 + E82, L65 + F71 + F275, L65 + E82 + F275 and F71 + E82 + F275, preferably, substitutions at selected positions among the group consisting of K62 + L65 + F71, S43 + L65 + F71, S43 + K62 + L65 and S43 + K62 + F71, in particular S43 + K62 + L65.

More particularly, the variant of the invention may comprise at least three (such as three) substitutions selected from the group consisting of F71Y, K62E and S43Q. Specific examples include L65Q + F71Y + K62E, L65Q + F71Y + S43Q, L65Q + K62E + S43Q and F71Y + K62E + S43Q, in particular L65Q + K62E + S43Q.

Variants of the invention possess an improved coagulation activity (or a reduced coagulation time) compared to hFVIIa or rhFVIIa. In a preferred embodiment of the invention, the ratio between the time to reach the clot formation of the variant (tvariant) and the time to reach the clot formation of hFVIIa or rhFVIIa (twt) is mostly 0.9 when It is tested in the “Complete Blood Test” described in this document. More preferably, the ratio (tvariant / twt) is mostly 0.75, such as 0.7, even more preferably, the ratio (tvariant / twt) is mostly 0.6, more preferably the ratio (tvariant / twt ) is mostly 0.5 when tested in the "Full Blood Test" described herein.

In a further interesting embodiment of the invention, the variant comprises 1-10 amino acid modifications (eg substitutions), such as 1-5 amino acid modifications (eg substitutions), for example, 1-3 amino acid modifications (for example, substitutions) with respect to SEQ ID NO: 1.

For example, the variant may contain at least one amino acid modification made in the Gla domain as explained in the section entitled "Modifications in the Gla domain" below, and / or at least one amino acid modification that leads to the introduction of an N-glycosylation site, as explained in the section entitled "introduction of additional sugar moieties" below, and / or at least one amino acid modification that decreases the affinity for binding to TFPI. Examples of the latest modifications are described in the section entitled "Other modifications" below.

Other modifications

As indicated above, the FVII or FVIIa variant of the invention may comprise additional modifications, in particular, additional modifications that confer additional advantageous properties to the FVII or FVIIa molecule. Thus, in addition to one or more of the above-mentioned substitutions, that is, a substitution in one or more of the positions L39,142, S43, K62, L65, F71, E82, F275H, the variant may comprise at least one additional amino acid modification, in particular, at least one additional amino acid substitution

In order to avoid too much disturbance of the structure and function of the FVII or FVIIa polypeptide, the variant of the FVII or FVIIa polypeptide of the invention normally comprises an amino acid sequence that has at least 95% identity with SEQ ID NO: 1 , such as at least 96% identity with SEQ ID NO: 1, for example, at least 97% identity with SEQ ID NO: 1, at least 98% identity with SEQ ID NO: 1, or at minus 99% identity with SEQ ID NO: 1. The identity of the amino acid sequence is conveniently determined from the aligned sequences, using, for example, the ClustalW program, version 1.8, June 1999, using default parameters (Thompson et al., 1994, ClustalW: Improving the sensitivity of progressive alignment of multiple sequences through sequence weighting, position-specific penalty of position and choice of weighting matrix, Nucleic Acids Research, 22 : 4673-4680) or from the PFAM family database version 4.0 (http: Hl2fam.wustl.edu/) (Nucleic Acids Res. January 1, 1999; 27 (1): 260-2) by using GENEDOC version 2.5 (Nicholas, KB, Nicholas HB Jr., and Deerfield, DW II. 1997 GeneDoc: Analysis and Visualization of Genetic Variation, EMBNEW.NEWS 4:14; Nicholas , KB and Nicholas HB Jr. 1997 GeneDoc: Analysis and Visualization of Genetic Variation).

Modifications in the Gla domain

In an interesting embodiment of the invention, the variant further comprises at least one amino acid modification (such as at least one amino acid substitution and / or insertion) in the Gla domain. Preferably, no modifications are made in position 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35.

Without wishing to be bound by any concrete theory, it is currently believed that an increase in coagulation activity can be achieved by enhanced binding affinity of the FVIIa molecule to the phospholipid membranes present on the surface of activated platelets. It is believed that this enhanced affinity results in a higher local concentration of activated FVIIa polypeptide in close proximity to the different coagulation factors, particularly FX. Thus, the activation rate of FX to Fxa will be greater, simply due to a higher molar ratio of the FVII polypeptide activated to FX. The increase in the rate of activation of FX results in a greater amount of active thrombin, and thus a greater rate of crosslinking of the fibrin.

Thus, in a preferred embodiment according to this aspect of the invention, the polypeptide variant has, in its activated form, a binding affinity to a phospholipid membrane enhanced with respect to that of the rhFVIIa polypeptide. Phospholipid membrane binding affinity can be measured by methods known in the art, such as by the tests described in Nelsestuen et al., Biochemistry 1977; 30; 1081910824 or as described in Example 1 in US 6,017,882.

Modifications in the FVII Gla domain that lead to an increased phospholipid membrane binding affinity have been described in the art (see, for example, WO 99/20767 and WO 00/66753). The particularly interesting positions in the Gla domain to be modified are the positions P10, K32, D33, A34 as well as the insertion of an amino acid residue between A3 and F4.

Thus, in a preferred embodiment of the invention, the variant comprises, in addition to one or more of the aforementioned modifications, a replacement in a position selected from the group consisting of P10, K32, D33 and A34 and their combinations

In another interesting embodiment, at least one of said substitutions is combined with an insertion of an amino acid residue between position A3 and F4.

Particularly preferred positions are P10 and K32, that is, in a particularly interesting embodiment of the invention, substitutions are made at positions P10 and K32, preferably P10Q + K32E.

Preferably, the substitution to be made at position 32 is K32E, the substitution to be made at position 10 is P10Q, the substitution to be made at position 33 is D33F, and the substitution to be made. to do in position 34 is A34E. The amino acid residue to be inserted between the A3 and F4 position is preferably a hydrophobic amino acid residue, in particular, the insert is A3AY. In an interesting embodiment of the invention, the variant comprises at least one of the following additional modifications: A3AY, P10Q, K32E, D33F, A34E or combinations thereof. Most preferably, the variant comprises one of the following additional modifications: K32E, P10Q + K32E, A3AY + P10Q + K32E, A3AY + P10Q + K32E + D33F, A3AY + P10Q + K32E + A34E or A3AY + P10Q + K32E + D33F + A34E

Modifications outside the Gla domain

A half-life of circulating rhFVIIa of 2.3 hours has been reported in "Summary Basis for Approval for NovoSeven®", FDA reference number 96-0597. Relatively high doses and frequent administration are necessary to achieve and sustain the desired therapeutic or prophylactic effect. As a consequence, it is difficult to obtain adequate dose regulation and the need for frequent intravenous administrations imposes restrictions on the patient's way of life.

A molecule with a longer circulating half-life and / or increased bioavailability (such as an Area under the Increased Curve compared to rhFVIIa when administered intravenously) would decrease the necessary number of administrations. Given the association of the current rhFVIIa product with frequent injections, and the potential to obtain more optimal therapeutic FVIIa levels with simultaneous enhanced therapeutic effect, there is a clear need for improved FVII or FVIIa type molecules.

Accordingly, a further object of the present invention is to provide FVII or FVIIa molecules (variants of FVII or FVIIa) with an increased half-life and / or an increased bioavailability (such as an Area Under the Increased Curve compared to rhFVIIa, when administered intravenously) and have improved coagulation activity.

Accordingly, in an interesting embodiment of the invention, the variant of the invention further comprises at least one linkage group introduced for a non-polypeptide moiety, wherein said linkage group has been introduced at a position located in the outside of the Gla domain.

Thus, an interesting variant of the invention is a variant that, in its activated form and when compared with rhFVIIa, generates an Area Under the Increased Curve when administered intravenously (AUCIV), in particular, when administered intravenously to rats . More particularly, the interesting variants of the present invention are the mentioned variants in which the ratio between the AUCIV of said variant, in its activated form, and the AUCIV of rhFVIIa is at least 1.25, such as at least 1 , 5, for example, of at least 1.75, more preferably of at least 2, such as at least 3, even more preferably of at least 4, such as at least 5, in particular when administered (intravenously) in rats

This effect may correspond to an increased in vivo functional half-life and / or an increased serum half-life compared to rhFVIIa. Accordingly, in another interesting embodiment of the invention, the relationship between the functional half-life in vivo or the serum half-life of the variant, in its activated form, and the functional half-life in vivo or the serum half-life of rhFVIIa It is at least 1.25. More preferably, the ratio between the relevant half-life of the variant, in its activated form, and the relevant half-life of rhFVIIa is at least 1.5, such as at least 1.75, for example at least 2, even more preferably at least 3, such as at least 4, for example, at least 5.

One way to increase the circulating half-life of a protein is to ensure that the renal clearance of the protein is reduced. This can be achieved by conjugating the protein with a chemical moiety, which is capable of conferring reduced renal clearance to the protein.

In addition, the binding of a chemical moiety to the protein or the substitution of the amino acids exposed to proteolysis can effectively block a contact proteolytic enzyme that leads to proteolytic degradation of the protein. Polyethylene glycol (PEG) is one such chemical residue that has been used in the preparation of protein therapeutic products. WO 98/32466 suggests that FVII, among many other proteins, may be PEGylated, but does not contain any additional information in this regard. WO 01/58935 discloses a new strategy to develop FVII or FVIIa molecules that have among others an increased half-life.

Numerous suitable modifications lead to an increase in AUCIV, in WO 01/58935 the functional half-life in vivo and / or the half-life in serum are disclosed. The variants disclosed in WO 01/58935 are the result of a generally new strategy for developing improved FVII or FVIIa molecules. The specific modifications described in WO 01/58935 can be advantageously combined with the modifications described above herein.

The polypeptide variant can also be linked to a serine proteinase inhibitor to inhibit the catalytic site of the polypeptide variant. Alternatively, one or more of the amino acid residues present in the catalytic site (S344, D242 and H193) can be mutated in order to render the resulting variant inactive. An example of such a mutation includes S344A.

The introduced amino acid residue comprising a linking group for a non-polypeptide moiety is selected on the basis of the nature of the non-polypeptide moiety of choice, in most of the examples, on the basis of the procedure in which the conjugation between the polypeptide variant and the non-polypeptide moiety. For example, when the non-polypeptide moiety is a polymer molecule such as polyethylene glycol or a molecule derived from polyalkylene oxide, the amino acid residues comprising a linking group can be selected from the group consisting of lysine, cysteine, aspartic acid, glutamic acid, histidine, and tyrosine, preferably lysine, cysteine, aspartic acid and glutamic acid, more preferably lysine and cysteine, in particular cysteine.

When a linkage group of a non-polypeptide moiety is to be introduced into the parental polypeptide, the position of the amino acid moiety to be modified is preferably located on the surface of the parental FVII or FVIIa polypeptide, and more preferably occupied by an amino acid moiety. which has at least 25% of its side chain exposed to the surface (as defined in Example 1, herein), preferably at least 50% of its side chain exposed to the surface (as defined in Example 1, in this document). These positions have been identified on the basis of an analysis of a 3D structure of the hFVII or hFVIIa molecule as described in the Materials and Procedures section of this document.

In addition, the position to be modified in accordance with this aspect of the invention is preferably selected from a part of the FVII or FVIIa molecule that is located outside the tissue factor binding site, and / or outside. from the region of the active site, and on the outside of the edge of the groove joining the active site. These sites / regions are identified in Example 1 herein. It should be emphasized, however, that in some situations, for example, in the case where an inactivated polypeptide variant is desired, it may be advantageous to carry out modifications in or near such regions. For example, it is contemplated that one or more linkage groups of non-polypeptide moieties, such as linkage groups for N-glycosylation sites, can be introduced into the region of the active site or at the edge of the site cleft active of the FVII or FVIIa molecule. The region of the active site, the tissue factor binding site and the crevice edge of the active site are defined in Example 1 and consist of the following moieties:

I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201, V221 I229, I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285, Q286, T286, T286 T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358, T359, G360, I361, V362, S363, W364, G365, C368 V376, Y377, T378, R379, V380, Q382, Y383, W386, L387, L400 and F405 (active site region);

L13, K18, F31, E35, R36, L39, F40, I42, S43, S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74, F76, E77, G78, R79, E82, K85, Q88, I90, V92, N93, E94, R271, A274, F275, V276, R277, F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325 and R379 (tissue factor binding region ); Y

N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370 (the edge of the groove joining the active site).

In order to determine an optimal distribution of the linkage groups, the distance between the amino acid residues located on the surface of the FVII or FVIIa polypeptide is calculated based on a 3D structure of the hFVII or hFVIIa polypeptide. More specifically, the distance between the CBs of the amino acid residues comprising said linking groups, or the distance between the functional group (NZ for lysine, CG for aspartic acid, CD for glutamic acid, SG for cysteine) of one and the CB of another amino acid residue comprising a linking group. In the case of glycine, CA is used instead of CB. In the FVII or FVIIa part of the polypeptide variant of the invention, any of said distances preferably has more than 8Å, in particular more than 10Å, in order to avoid or reduce a heterogeneous conjugation.

In the case of the introduction of a linking group, an amino acid residue comprising said group is introduced in the position, preferably by substituting the amino acid residue occupying said position.

The exact number of binding groups present and available for conjugation in the FVII or FVIIa polypeptide is dependent on the desired effect to be achieved by conjugation. The effect to be obtained is, for example, dependent on the nature and degree of conjugation (for example, the identity of the non-polypeptide moiety, the number of desirable or possible non-polypeptide moieties to conjugate the polypeptide variant, in the which should be conjugated, or where conjugation should be avoided, etc).

The functional half-life in vivo is, among others, dependent on the molecular weight of the protein, and the number of binding groups necessary to provide an increased half-life therefore depends on the molecular weight of the non-polypeptide moiety in question. In one embodiment, the polypeptide variant of the invention has a molecular weight of at least 67 kDa, in particular at least 70 kDa, for example, as measured by SDS-PAGE according to Laemmli, UK, Nature Vol 227 (1970), p680-85. The FVII itself has a molecular weight of approximately 53 kDa, and therefore an additional 10-20 kDa is required to obtain the desired effect. This may, for example, be provided by conjugating 2-4 molecules of 10 kDa PEG or as described herein otherwise.

The total number of amino acid residues to be modified outside the Gla domain in the parental FVII or FVIIa polypeptide (as compared to the amino acid sequence shown in SEQ ID NO: 1) will not normally exceed 10. Preferably, The FVII or FVIIa variant comprises an amino acid sequence that differs in 1-10 amino acid residues from amino acid residues 46-406 shown in SEQ ID NO: 1, usually in 1-8 or in 2-8 amino acid residues, by for example, in 1-5 or in 2-5 amino acid residues, such as in 1-4 or in 1-3 amino acid residues, for example, in 1, 2 or 3 amino acid residues of amino acid residues 46-406 shown in SEQ ID NO: 1.

Similarly, the polypeptide variant of the invention may contain 1-10 non-polypeptide moieties (additional), usually 1-8 or 2-8 non-polypeptide moieties (additional), preferably 1-5 or 2-5 non-polypeptide moieties (additional) such as 1-4 or 1-3 non-polypeptide moieties (additional), for example, 1, 2 or 3 non-polypeptide moieties (additional) it will be understood that said additional non-polypeptide moieties are covalently linked to a link group located outside of the Gla domain.

Introduction of additional sugar residues

In a preferred embodiment of the invention, a linkage group of a sugar moiety, such as a glycosylation site, in particular an in vivo glycosylation site, such as an N-glycosylation site, has been introduced at a position located outside the domain Gla.

When used in the present context, the term "naturally occurring glycosylation site" covers the glycosylation sites at positions N145, N322, S52 and S60. Similarly, the term "naturally occurring O-glycosylation site" includes positions S52 and S60, while the term "naturally occurring N-glycosylation site" includes positions N145 and N322.

Thus, in a very interesting embodiment of the invention, the non-polypeptide moiety is a sugar moiety and the linker introduced is a glycosylation site, preferably an in vivo glycosylation site, such as an O- site. glycosylation or an N-glycosylation site, in particular an N-glycosylation site. Normally, 110 glycosylation sites have been introduced, in particular N-glycosylation sites, preferably 1-8, 1-6, 1-4 or 1-3 glycosylation sites, in particular, N-glycosylation sites have been introduced into a position (s) located outside the Gla domain. For example, 1, 2 or 3 glycosylation sites, in particular, N-glycosylation sites may have been introduced outside the Gla domain, preferably by substitution. Similarly, the variant may comprise 1-10 sugar residues introduced, preferably 1-8, 1-6, 1-4 or 1-3 sugar residues introduced. For example, the variant may contain 1, 2 or 3 sugar residues introduced.

It will be understood that in order to prepare a polypeptide variant, in which the polypeptide variant comprises one or more glycosylation sites, the polypeptide variant must be expressed in a host cell capable of binding to sugar moieties (oligosaccharides) in the ( the) glycosylation site (s) or alternatively undergo glycosylation in vitro. Examples of glycosylating host cells are provided in the following additional section entitled "Coupling to a sugar moiety."

Examples of positions into which glycosylation sites, in particular N-glycosylation sites, can be introduced include, but are not limited to, positions comprising an amino acid residue having an amino acid residue having at least 25% of its side chain exposed to the surface (as defined in Example 1 herein), as in a position comprising an amino acid residue having at least 50% of its side chain exposed to the surface (as is defined in Example 1 in this document). The position is preferably selected from a part of the molecule that is located outside the site of attachment to the tissue factor and / or the region of the active site and / or the outside of the edge of the slit of the active site. These sites / regions are identified in Example 1 herein. It should be understood that when the term "at least 25% (or at least 50%) of its side chain exposed to the surface" is used together with the introduction of an N-glycosylation site, this term refers to accessibility Superficial side chain amino acid in the position where the rest sugar actually binds. In many cases it will be necessary to introduce a serine residue or a threonine residue in the +2 position with respect to the asparagine residue to which the sugar residue actually binds (unless, of course, this position is already occupied by a serine residue or a threonine residue) and these positions are allowed to be buried in which the serine or threonine remains are introduced, that is, they have less than 25% of their side chains exposed to the surface.

Specific and preferred examples of such substitutions that create an N-glycosylation site include a substitution selected from the group consisting of A51N, G58N, G58N + S60T, T106N, K109N, G124N, K143N + N145T, A175T, I205S, I205T, V253N, T267N, T267N + S269T, S314N + K316S, S314N + K316T, R315N + V317S, R315N + V317T, K316N + G318S, K316N + G318T, G318N, D334N and their combinations. More preferably, the Nglycosylation site is introduced by a substitution selected from the group consisting of A51N, G58N + S60T, T106N, K109N, G124N, K143N + N145T, A175T, I205T, V253N, T267N + S269T, S314N + K316T, R315T, R315T + V317T, K316N + G318T, G318N, D334N and their combinations. Even more preferably, the N-glycosylation site is introduced by a substitution selected from the group consisting of T106N, A175T, I205T, V253N, T267N + S269T and combinations thereof. Most preferably, the N-glycosylation site is introduced by a substitution selected from the groups consisting of T106N, I205T, V253N, T267N + S269T and combinations thereof.

In one embodiment, only one N-glycosylation site has been introduced by substitution. In another embodiment, two or more (such as two) N-glycosylation sites have been introduced by substitution. Examples of preferred substitutions that create two N-glycosylation sites include substitutions selected from the group consisting of A51N + G58N, AS1N + G58N + S60T, A51N + T106N, AS1N + K109N, A51N + G124N, A51N + K143N + N145T, A51N + A175T, AS1N + I205T, A51N + V253N, A51N + T267N + S269T, AS1N + S314N + K316T, A51N + R315N + V317T, A51N + K316N + G318T, A51N + G318N, AS1N + D6 +58 K109N, G58N + G124N, G58N + K143N + N145T, G58N + A175T, G58N + I205T, G58N + V253N, G58N + T267N + S269T, G58N + S314N + K316T, G58N + R315N + G317T, G3NT + G317T, G3N G318N, G58N + D334N, G58N + S60T + T106N, G58N + S60T + K109N, G58N + S60T + G124N, G58N + S60T + K143N + N145T, G58N + S60T + A175T, G58N + S60T + I203T, V253, G25N + S60T + T267N + S269T, G58N + S60T + S314N + K316T, G58N + S60T + R315N + V317T, G58N + S60T + K316N + G318T, G58N + S60T + G318N, G58N + S60T + D334N, T10 + 633, N10N T106N + K143N + N145T, T106N + A175T, T106N + I205T, T106N + V253N, T106N + T267N + S269T, T106N + S314N + K316T, T106N + R315N + V317T, T106N + K316 + T36N + K316N K109N + G124N, K109N + K143N + N145T, K109N + A175T, K109N + I205T, K109N + V253N, K109N + T267N + S269T, K109N + S314N + K316T, K109N + R315N + V317T, K109N + K39N + K39N + K39N + K39N + K39N + K39N + K39N3 G124N + K143N + N145T, G124N + A175T, G124N + I205T, G124N + V253N, G124N + T267N + S269T, G124N + S314N + K316T, G124N + R315N + V317T, G124N + K316 + G34N + K316 + G34N + K316 + G34N + K316 K143N + N145T + A175T, K143N + N145T + I205T, K143N + N145T + V253N, K143N + N145T + T267N + S269T, K143N + N145T + S314N + K316T, K143N + N145T + K3TN + K316T + K3TN + K3163 K143N + N145T + G318N, K143N + N145T + D334N, A175T + I205T, A175T + V253N, A175T + T267N + S269T, A175T + S314N + K316T, A17ST + R315N + V317T18, A175T + G173 D334N, I205T + V253N, I205T + T267N + S269T, I205T + S314N + K316T, I205T + R315N + V317T, I205T + K316N + G318T, I205T + G318N, I205T + D334N, V253N + T267N + S269T, V253N + S314N + K316T, V253N + R315N + V317T, V253N + K316N + G318T, V253N + G318N, V253N + D334N, T267N + S269T + S314N + K316T, T267N + S269T + R315N + V317T, T267N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269N + S269T T267N + S269T + D334N, S314N + K316T + R315N + V317T, S 314N + K316T + G318N, S314N + K316T + D334N, R315N + V317T + K316N + G318T, R315N + V317T + G318N, R315N + V317T + D334N and G318N + D334N. More preferably, the substitutions are selected from the group consisting of T106N + A175T, T106N + I205T, T106N + V253N, T106N + T267N + S269T, A175T + I205T, A175T + V253N, A175T + T267N + S269T25, I205T, I205T + T267N + S269T and V253N + T267N + S269T, even more preferably among the group consisting of T106N + I205T, T106N + V253N, T106N + T267N + S269T, I205T + V253N, I205T + T267N + S269T and V253T and V253T and V253T

In an even more additional embodiment, three or more (such as three) N-glycosylation sites have been introduced by substitution. Examples of preferred substitutions creating three N-glycosylation sites include substitutions selected from the group consisting of T106N + A175T + I205T, T106N + A175T + V253N, T106N + A175T + T267N + S269T, T106N + I205T + V253N, T106N + I205T + T267N + S269T, T106N + V253N + T267N + S269T, A175T + I205T + V253N, A175T + I205T + T267N + S269T, A175T + V253N + T267N + S269T and I205T + V263T + V263T + V263T + V203T + V263T + V263T More preferably, the substitutions are selected from the group consisting of T106N + 1205T + V253N, T106N + 1205T + T267N + S269T, T106N + V253N + T267N + S269T and I205T + V253N + T267N + S269T.

As discussed above, it is preferred that the N-glycosylation site be introduced into a position that is neither part of the tissue factor binding site nor part of the region of the active site and of the edge of the site binding cleft. active as defined in this document. It is envisioned that said glycosylation variants belong primarily to the class of active polypeptide variants as defined hereinbefore.

It will be understood that any of the modifications mentioned in the previous sections can be combined.

Other modifications

In a further embodiment of the present invention, the FVII or FVIIa variant may, in addition to the modifications described in the previous sections, also contain mutations, which are known to increase the intrinsic activity of the polypeptide, for example, such as described in WO 02/22776.

Examples of preferred substitutions include the substitutions selected from the group consisting of V158D, E296D, M298Q, L305V and K337A. More preferably, said substitutions are selected from the group consisting of V158D + E296D + M298Q + L305V + K337A, V158D + E296D + M298Q + K337A, V158D + E296D + M298Q + L305V, V 158D + E296D + M298Q, M298Q, M298Q, M298Q, M298Q, M298Q K337A, L305V and K337A.

In a further embodiment of the present invention, the FVII or FVIIa variant may, in addition to the modifications described in the previous sections, also contain mutations, which produce a decrease in TFPI inhibition. An example includes the K341Q substitution disclosed by Neuenschwander et al., Biochemistry 1995; 34: 8701-8707. Other examples include D196K, D196N, G237L, G237GAA and their combinations.

As already indicated above, the variant may also contain conservative amino acid substitutions.

Specific examples of most preferred variants of the invention

Specific examples of most preferred FVII or FVIIa variants are given below: S43Q, K62E, L65Q, S43Q + K62E, S43Q + L65Q, K62E + L65Q, S43Q + K62E + L65Q, P10Q + K32E + S43Q, P10Q + K32E + K62E, P10Q + K32E + L65Q, P10Q + K32E + S43Q + K62E, P10Q + K32E + S43Q + L65Q, P10Q + K32E + K62E + L65Q, P10Q + K32E + S43Q + K62E + L65Q

It will be understood that any of the aforementioned preferred variants can be combined with at least one additional modification carried out outside the Gla domain. In particular, any of the preferred variants mentioned above may be combined with a substitution (s) selected from the group consisting of T106N, I205T, V253N, T267N + S269T, T106N + I205T, T106N + V253N, T106N + T267N + S269T, I205T + V253N, I205T + T267N + S269T, V253N + T267N + S269T, T106N + I205T + V253N, T106N + I205T + T267N + S269T, T106N + V253T + T256N + V253T + V253T + V253T + V253 S269T.

The rest not polypeptide

Based on the present disclosure, the skilled person will understand that amino acid residues comprising other linkage groups can be introduced by substitution in the parental polypeptide, using the same approach as illustrated above with the N-glycosylation sites. For example, one or more amino acid residues comprising an acid group (glutamic acid or aspartic acid), tyrosine or lysine, can be introduced at the positions discussed above. In particular, one or more cysteine residues can be introduced at the positions discussed above.

As indicated further above, the non-polypeptide moiety of the conjugate variant is preferably selected from the group consisting of a polymer molecule, a lipophilic compound, a sugar moiety (by means of in vivo glycosylation) and an organic derivatizing agent . All of these agents can confer desirable properties to the polypeptide variant, in particular enhanced AUCIV, increased in vivo functional half-life and / or increased plasma half-life. The polypeptide variant is usually conjugated with only one type of non-polypeptide moiety, but it can also be conjugated with two or more different types of non-polypeptide moieties, for example, with a polymer molecule and a sugar moiety, with a lipophilic group and a sugar moiety, with an organic derivatizing agent and a sugar moiety, with a lipophilic group and a polymer molecule, etc. The conjugation of two or more different non-polypeptide moieties can be carried out simultaneously or sequentially.

Methods of preparing a conjugate variant of the invention

In the following sections "Conjugation with a polymer molecule", "Conjugation with a sugar moiety", "Conjugation with an organic derivatizing agent" and "Conjugation with a lipophilic compound", the conjugation with specific types of non-polypeptide moieties is described . In general, a conjugate variant according to the invention can be produced by culturing an appropriate host cell under conductive conditions for expression of the polypeptide variant, and recovering the polypeptide variant, wherein a) the polypeptide variant comprises at least an N or O-glycosylation site and in the host cell is a eukaryotic host cell capable of glycosylation in vivo, and / or b) the polypeptide variant is subjected to conjugation with a non-polypeptide moiety in vitro.

It will be understood that conjugation should be designed in such a way as to produce the optimal molecule with respect to the number of attached non-polypeptide moieties, the size and shape of said molecules (for example, if they are linear or branched), and the site (s) binding in the polypeptide. The molecular weight of the non-polypeptide moiety to be used can, for example, be chosen based on the desired effect to be achieved. For example, if the main objective of conjugation is to achieve a conjugate variant having a high molecular weight (for example, to reduce renal clearance) it is normally desirable to conjugate a few non-polypeptide moieties of high molecular weight as possible to obtain the desired molecular weight. When a high degree of protection is desirable, this can be obtained by using a sufficiently high number of low molecular weight non-polypeptide moieties (for example with a molecular weight of between about 300 Da to about 5 kDa, such as a molecular weight from 300 Da to 2 kDa.

Conjugation with a polymer molecule

The polymer molecule to be coupled with the polypeptide variant can be any suitable polymer molecule, such as a natural or synthetic homopolymer or heteropolymer, usually with a molecular weight in the range of about 300-100,000 Da, such as about 500-20,000 Da, more preferably in the range of about 500-15,000 Da, even more preferably in the range of about 2-12 kDa, such as in the range of about 3-10 kDa. When the term "approximately" is used herein in conjunction with some molecular weight, the word "approximately" indicates an approximate average molecular weight and reflects the fact that there will normally be some molecular weight distribution in a given polymer preparation.

Examples of homopolymers include a polyol (i.e., poly-OH), a polyamine (i.e., poly-NH2) and a polycarboxylic acid (i.e., poly-COOH). A heteropolymer is a polymer comprising different coupling groups. , such as a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), which includes polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEG, polyvinyl alcohol (PVA), polycarboxylate, poly (vinyl pyrrolidone), maleic acid polyethylene coanhydride, polystyrene-maleic acid co-anhydride, dextran, which includes carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and / or increase the functional half-life in vivo, and / or the serum half-life. Another example of a polymer molecule is human albumin or another protein abundant in plasma. In general, polymers derived from polyalkylene glycol, non-toxic, non-antigenic, non-immunogenic, have various water solubility properties, and are easily excreted from living organisms.

PEG is the preferred polymer molecule because it has only few reactive groups capable of crosslinking compared to, for example, polysaccharides such as dextran. In particular, monofunctional PEG, for example, methoxypolyethylene glycol (mPEG), is of interest because its coupling chemistry is relatively simple (only one reactive group is available to conjugate with the linkage groups in the polypeptide, accordingly, as removed the risk of crosslinking, the resulting conjugate variants are more homogeneous and the reaction of the polymer molecules with the polypeptide variant is easier to control.

To carry out the covalent bonding of the polymer molecule (s) with the variant of the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, that is, with functional reactive groups ( Examples of which include primary amino groups, hydrazide (HZ), thio, succinate (SUC), succinimidyl succinate (SS) succinimidyl succinamide (SSA) succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidyl carboxymethylate (SCM), carbonate of benzotriazole (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (THREE). Suitable activated polymer molecules are commercially available, for example from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals plc, UK.

Alternatively, polymer molecules can be activated by conventional methods known in the art, for example, as disclosed in WO 90/13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives).

Specific examples of PEG polymers include the following linear PEGs: NHS-PEG for example, SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG ), and NOR-PEG, BTC-PEG, EPOXPEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, THREE-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS and those disclosed in US 5,932,462 and US 5,643,575

In addition, the following publications disclose useful and / or chemical polymer molecules of PEGylation; US 5,824,778, US 5,476,653, WO 97/32607, EP 0 229 108, EP 0 402 378, US 4,902,502, US 5,281,698, US 5,122,614, US 5,219,564, WO 92/16555 , WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95 / 13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 0 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 0 921 131, US 5,736,625, WO 98/05363, EP 0 809 996, US 5,629,384, WO 96/41813, WO 96/07670, US 5,473,034, US 5,516,673, EP 0 605 963, US 5,382,657, EP 0 510 356, EP 0 400 472, EP 0 183 503 and EP 0 154 316.

Preferred examples of particularly preferred activated PEG polymers for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl disulfide-PEG (OPSS-PEG), preferably orthopyridyl disulfide-mPEG (OPSS-mPEG). Normally, said PEG or mPEG polymers will have a size of about 5 kDa, about 10 kDa, about 12 kDa or about 20 kDa.

The conjugation of the polypeptide variant and the activated polymer molecules is carried out by the use of any conventional procedure, for example, as described in the following references (which also describe the suitable procedures for the activation of the molecules polymer): Harris and Zalipsky, eds., Poly (ethylene glycol) Chemistry and Biological Applications, AZC, Washington; R.F. Taylor, (1991), "Protein immobilization. Fundamental and applications", Marcel Dekker, N.Y .; H.H. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al. (1993), "Immobilized Affinity Ligand Techniques", Academic Press, N.Y.).

The skilled person will be aware that the activation procedure and / or conjugation chemistry to be used depends on the link group (s) of the polypeptide variant (example of which are additionally provided above), as well as the functional groups of the polymer (for example, which are amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide, vinylsulfone or haloacetate). PEGylation can be directed to the conjugation of all available linkage groups in the polypeptide variant (i.e., said linkage groups that are exposed on the surface of the polypeptide) or can be directed to one or more specific linkage groups, by For example, the amino group of the N-terminus described in US 5,985,265 or the cysteine residues. In addition, conjugation can be achieved in one step or in a stepwise manner (for example, as described in WO 99/55377).

For PEGylation with cysteine residues (see above) the FVII or FVIIa variant is usually treated with a reducing agent, such as dithiothreitol (DDT) before PEGylation. The reducing agent is subsequently removed by any conventional procedure, such as by desalination. The conjugation of PEG to a cysteine residue normally takes place in a suitable buffer at pH 6-9 at temperatures ranging from 4 ° C to 25 ° C for periods of up to 16 hours.

It will be understood that PEGylation is designed in such a way that it produces the optimal molecule with respect to the number of bound PEG molecules, the size and shape of said molecules (for example, if they are linear or branched) and the site (the) s) binding in the polypeptide variant. The molecular weight of the polymer to be used can, for example, be chosen on the basis of the desired effect to be achieved.

Together with conjugation with only a single binding group in the protein (for example, the N-terminus amino group), it may be advantageous that the polymer molecule, which can be linear or branched, has a high molecular weight, preferably of about 10-25 kDa, such as about 15-25 kDa, for example, about 20 kDa.

Typically, polymer conjugation is carried out under the conditions intended to react most of the available polymer binding groups with the polymer molecules. This is achieved by means of a suitable molar excess of the polymer with respect to the polypeptide. Normally, the molar ratios of the activated polymer molecules with the polypeptide are up to about 1000-1, such as up to about 200-1, or up to about 100-1. In some cases the ratio may be slightly lower, however, such as up to about 50-1, 10-1, 5-1, 2-1 or 1-1, in order to obtain an optimal reaction.

It is contemplated according to the invention to couple the polymer molecules with the polypeptide by means of a linker. The skilled person knows the proper linkers well. A preferred example is cyanuric chloride (Abuchowski et al., J Biol Chem 1977; 252; 3578-3581; US 4,179,337; Shafer et al., J Polym Sci Polym Chem Ed 1986; 24; 375378).

Immediately after conjugation, the residual activated polymer molecules are blocked according to the procedures known in the art, for example, by adding a primary amine to the reaction mixture, and the resulting inactivated polymer molecules are removed by An appropriate procedure.

It will be understood that depending on the circumstances, for example, of the amino acid sequence of the polypeptide variant, the nature of the activated PEG compound being used and the specific conditions of PEGylation, which include the molar ratio of PEG to polypeptide , varying degrees of PEGylation can be obtained, obtaining a higher degree of PEGylation usually with a higher ratio of PEG to polypeptide variant. The polypeptides of the PEGylated variant that are the result of any given PEGylation procedure, will, however, normally comprise a stochastic distribution of the conjugated polypeptide variants having slightly different degrees of PEGylation.

Conjugation with a sugar residue

In order to achieve glycosylation in vivo of an FVII molecule comprising one or more glycosylation sites, the nucleotide sequence encoding the polypeptide variant must be inserted into a glycosylating host of eukaryotic expression. The fungal expression host cell (filamentous fungi or yeast), insect or animal cells or cells of transgenic plants can be selected. In one embodiment, the host cell is a mammalian cell, such as a CHO, BHK or HEK cell, for example, a HEK 293 cell, or an insect cell, such as an SF9 cell, or a yeast cell. , for example, S. cerevisiae or Pichia pastoris, or any of the host cells mentioned hereinafter.

In vitro covalent coupling of sugar moieties (such as dextran) with amino acid moieties of the polypeptide variant can also be used, for example, as described, for example, in WO 87/05330 and in Aplin et al. , CRC Crit Rev. Biochem 1981; 259-306. In vitro coupling of the sugar residues of PEG with Gln residues bound to proteins and peptides can be carried out by transglutamines (TGases). Transglutamines catalyze the transfer of donor amine groups with Gln moieties bound to proteins and peptides in a so-called cross-linking reaction. The donor amine groups may be linked to proteins or peptides such as the ε-amino group in lysine moieties or they may be part of a small or large organic molecule. An example of a small organic molecule that functions as an amino acid donor in TGase catalyzed crosslinking is Putrescina (1,4-diaminobutane). An example of a small organic molecule that functions as a cross-linking donor amino acid catalyzed by TGase is a PEG containing amine (Sato et al., Biochemistry 1996; 35; 13072-13080).

TGases, in general, are very specific enzymes, and not all Gln residues exposed on the surface of a protein are accessible to TGase catalyzed cross-linking with amino-containing substances. On the contrary, only a few Gln residues naturally function as TGase substrates but the exact parameters that determine which of said Gln residues are good TGase substrates remain unknown. Thus, in order to make a protein susceptible to TGase catalyzed crosslinking reactions it is often a prerequisite to add stretches at convenient positions of the known amino acid sequence that work very well as TGase substrates. It is known that some amino acid sequences are or contain excellent natural TGase substrates, for example, substance P, elafin, fibrinogen, fibronectin, α2-plasmin inhibitor, αcaseins, and β-caseins.

Conjugation with an organic derivatization agent of the technique

A covalent modification of the polypeptide variant can be carried out by reacting one or more linking groups of the polypeptide variant with an organic derivatizing agent. Suitable derivatization agents and procedures are well known in the art. For example, cysteinyl moieties are more commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl moieties are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (4.imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa1,3-diazole. Histidyl moieties are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful. The reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino-terminal moieties are reacted with succinic acid or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the loading of the lysinyl moieties. Other suitable reagents for derivatizing α-amino containing moieties include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, Ometilisourea, 2,4-pentanedione and the transaminase catalyzed reaction with glyoxylate. Arginyl moieties are modified by reaction with one or some conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa of the guanidine functional group.

In addition, these reagents can react with the lysine groups as well as with the guanidino group of arginine. The carboxyl (aspartyl or glutamyl) side groups are selectively modified by reaction with carbodiimides (RN = C = N-R '), in which R and R' are different alkyl groups, such as 1-cyclohexyl-3- (2- morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, the aspartyl and glutamyl moieties are converted to asparaginyl and glutaminyl by reaction with ammonium ions.

Conjugation with a lipophilic compound

The polypeptide variant and the lipophilic compound can be conjugated to each other, both directly and through the use of a linker. The lipophilic compound may be a natural compound such as a saturated or unsaturated fatty acid, a fatty acid diketone, a terpene, a prostaglandin, a vitamin, a carotenoid or steroid, or a synthetic compound such as a carbonic acid, an alcohol, an amine and a sulfonic acid with one or more alkyl, aryl, alkenyl or other multiple unsaturated compounds. The conjugation between the polypeptide variant and the lipophilic compound, optionally through a linker can be carried out in accordance with the procedures known in the art, for example, as described by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976 and in WO 96/12505.

Binding of a serine protease inhibitor

Binding of a serine protease inhibitor can be carried out according to the procedure described in WO 96/12800

Conjugation of a tagged polypeptide

In an alternative embodiment, the polypeptide variant is expressed as a fusion protein with a tag, that is, an amino acid sequence or a stretch of peptide normally prepared with 1-30, such as with 120 amino acid residues. While allowing quick and simple purification, the tag is a convenient tool to achieve conjugation between the variant of the tagged polypeptide and the non-polypeptide moiety. In particular, the tag can be used to achieve conjugation in microtiter plates or other vehicles, such as paramagnetic beads, to which the variant of the tagged polypeptide can be immobilized by the tag. The conjugation with the tagged polypeptide variant in, for example, microtiter plates, has the advantage that the tagged polypeptide variant can be immobilized in the microtiter plates directly from the culture broth (in principle without any purification) and subjected to conjugation. Therefore, the total number of procedural steps (from expression to conjugation) can be reduced. In addition, the tag can function as a separator molecule, ensuring improved accessibility to the immobilized polypeptide variant to be conjugated. The conjugation using a labeled polypeptide variant can be with any of the non-polypeptide moieties disclosed herein, for example with a polymer molecule such as PEG.

The identity of the specific tag to be used is not critical as long as the tag is able to express itself with the polypeptide variant and is able to be immobilized on a suitable surface or carrier material. Numerous suitable labels are available, for example, from Unizyme Laboratories, Denmark. Subsequent cleavage of the tag of the polypeptide variant can be achieved through the use of commercially available enzymes.

Inactivation of the FVII / FVIIa variants of the invention

In another interesting embodiment of the invention, the variants disclosed herein can be inactivated. The inactivated form is capable of competing with natural FVII or FVIIa for binding with FT and inhibition of coagulation activity, and it is anticipated that said inactivated variants will be very potent antagonists of the tissue factor. Thus, in another aspect, the present invention relates to the FVII / FVIIa variants described herein in their inactivated forms as well as to said inactivated FVII / FVIIa variants for use as medicaments. More particularly, the inactivated variant of the invention can be used for the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder related to FVIIa / FT in a mammal. For example, the inactivated variant of the invention can be used for the manufacture of a medicament for the treatment or prophylaxis of diseases in which an anticoagulant activity is desirable, such as the prophylaxis or treatment of patients in hypercoagulable states, such as patients with sepsis, deep vein thrombosis, patients at risk of myocardial infections or thrombotic stroke, pulmonary embolism, patients with acute coronary syndromes (myocardial infarction and unstable angina pectoris), patients experiencing coronary heart disease, prevention of cardiac episodes and restenosis in patients receiving angioplasty, patients with peripheral vascular diseases. The inactivated variant of the invention can also be used for the manufacture of a medicament for the treatment of respiratory diseases, tumor growth and metastasis. Similarly, the inactivated variant of the invention can be used in a method to treat a mammal having a disease

or disorder related to FVIIa / FT (such as one or more of the diseases or disorders mentioned above), which comprises administering to an mammal in need thereof an effective amount of said conjugate or inactivated composition.

As used herein, the term "inactivated", when used in conjunction with variants of the invention, is intended to mean a variant that has less than 5% of the coagulation activity of hFVIIa or rhFVIIa when measured in the "Full Blood Test" described in this document.

The variants described herein may be inactivated by methods well known in the art. For example, an active FVII or FVIIa polypeptide can become inactive by carbamylating the α-amino group I153 or complexing the polypeptide with a serine protease inhibitor, according to the procedure described in WO 96/12800. A suitable serine inhibitor protein is selected, for example, from the group consisting of an organophosphorus compound, a sulfanylfluoride, a peptide halomethyl ketone, preferably a Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Glu-chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone or a Phe-Phe-Arg chloromethyl ketone, or an azapeptide.

Alternatively, the variants of the invention can become inactive by removing at least one amino acid residue that occupies a position selected from the group consisting of R152, I153, S344, D242 and H193. Elimination can be carried out by substitution or deletion of one or more of the amino acid residues identified above. Preferably, the removal is carried out by substitution, in particular by conservative substitution. Accordingly, the inactivated FVII or FVIIa polypeptide used herein may comprise one or more of the following substitutions: R152X, I153X, S344X, D242X or H193X, wherein X is any amino acid residue, preferably one that leads to a conservative substitution. For example, the inactivated FVII or FVIIa polypeptide comprises the R152X mutations, wherein X is any amino acid residue other than lysine (because lysine is part of a protease cleavage site). Other examples of specific substitutions include I153A / V / L; S344T / A / G / Y, preferably S344A; D242E / A and / or H193R / A.

Another solution includes carrying out modifications in or near the region of the active site. For example, it is contemplated that one or more linkage groups of the non-polypeptide moieties, such as the linkage groups of the N-glycosylation sites, can be advantageously introduced into the region of the active site or at the edge of the cleft of binding of the active site of the FVII or FVIIa variant. The region of the active site, the tissue factor binding site and the edge of the active site binding cleft are defined in Example I herein.

Thus, specific examples of substitutions that create said N-glycosylation site include the substitutions selected from the group consisting of I153N + G155S / T, Q167N + L169S / T, V168N + L170S / T, L169N + L171S / T , L170N + V172S / T, L171N + N173S / T, A175S / T, A175N + L177S / T, L177N + G179S / T, G179N, G180N + L182S / T, T181N + I183S / T, V188N, V189N + A191 T + A19 , S190N + A192S / T, A191N + H193S / T, H193N + F195S / T, F195N + K197S / T, D196N + I198S / T, K197N + K199S / T, I198N + N200S / T, K199N + W201N / T, W201S / T, W + N203S / T, R202S / T, I205S / T, V228N + 1230S / T, I229N + P231S / T, I230N, P231N, S232N + Y234S / T, T233N + V235S / T, Y234N + P236S / T, V235N + G237 / T, P236N, G237N, T238N + N240S / T, T239N + H241S / T, H241N + I243S / T, D242S / T, I243N + L245S / T, A244N + L246S / T, L245N + R247S / T, L246N + L246N / T, V281N + G283S / T, S282N + W284S / T, G283N + G285S / T, W284N + Q286S / T, G285N + L287S / T, Q286N + L288S / T, D289N + G291S / T, R290N + A292S / T , G291N, A292N + A294S / F, T293N + L295S / T, P321N + I323S / T, T324N + Y326S / T, Y326N + F327S / T, F328N + A330S / T, S339N + K341S / T, K341N + D343S + D343S + D343S , G342N + S 344S / T, D343N + G345S / T, S344N + G346S / T, G345N + P347S / T, P347N + A349S / T, H348N, L358N + G360S / T, T359N + I361S / T, G360N + V362N / T, I361 V362N + W364S / T, S363N + G365S / T, W364N + Q366S / T, G365N + G367S / T, T370N + G372S / T, V376N, Y377N + R379S / T, T378N + V380S / T, V380N + Q382S / Q382S / T Q382N + I384S / T, Y383N + E385S / T, W386N + Q388S / T, L387N + K389S / T, L400N + R402S / T and their combinations. Preferably, the substitution is selected from the group consisting of D289N + G291S / T, R290N + A292S / T, G291N, A292N + A294S / T, T293N + L295S / T, S339N + K341S / T, K341N + D343S / T, G342N + S344S / T, D343N + G345S / T, and their combinations. More preferably, the substitution is selected from the group consisting of D289N + G291T, R290N + A292T, G291N, A292N + A294T, T293N + L295T, S339N + K341T, K341N + D343T, G342N + S344T, D343N and combinations G345T, , in particular G291N.

Normally, an inactivated variant has a significantly reduced coagulation activity compared to natural hFVIIa or rhFVIIa. Preferably, the inactivated variant has less than 4% of the coagulation activity of hFVIIa or rhFVIIa when tested in the Complete Blood Test described herein. More preferably, the inactivated variant has less than 3% of the coagulation activity, such as less than 2% of the coagulation activity, for example, less than 1% of the coagulation activity of hFVIIa or rhFVIIa when tested in the Complete Blood Test described herein.

As will be understood, the details and particularities that refer to the inactivated variants of the invention (for example, preferred substitutions, formulation of variants, etc.) will be the same or analogous to the variant (active) aspect of the invention, when be appropriate Thus, the definitions and details that refer to the inactivated variants of the invention will be applied by changing what should be changed to the active variants disclosed herein, when appropriate.

Methods of preparing a polypeptide variant of the invention

The polypeptide variants of the present invention, optionally in glycosylated form can be produced by any method known in the art. Such procedures include constructing a nucleotide sequence that encodes the polypeptide variant and expresses the sequence in a suitable transformed or transfected host. Preferably, the host cell is a gammacarboxylating host cell such as a mammalian cell. However, the polypeptide variants of the invention can be produced, although less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.

A nucleotide sequence encoding a polypeptide variant of the invention can be constructed by isolating or synthesizing a nucleotide sequence encoding a human natural FVII and then changing the nucleotide sequence in such a way as to introduce (i.e., insertion or the substitution) or elimination) of the relevant amino acid residue (s).

The nucleotide sequence is conveniently modified by site-directed mutagenesis according to conventional procedures. Alternatively, the nucleotide sequence is prepared by chemical synthesis, for example, using an oligonucleotide synthesizer, in which the oligonucleotides are designed based on the amino acid sequence of the desired polypeptide variant, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide variant will be produced For example, some small oligonucleotides encoding portions of the desired polypeptide variant can be synthesized and assembled by PCR, ligation or ligation chain reaction (CSF) (Barany, PNAS 88 : 189-193, 1991). Individual oligonucleotides normally contain 5 'or 3' protrusions for complementary assembly.

Alternative methods of nucleotide sequence modification are available to produce polypeptide variants for high throughput screening, procedures, for example, which involve homologous backcrossing such as that disclosed in US 5,093,257, and procedures involving rearrangement. gene, that is recombination between two or more homologous nucleotide sequences that result in new nucleotide sequences that have numerous nucleotide alterations when compared to the starting nucleotide sequences. Gene rearrangement (also known as DNA rearrangement) involves one or more cycles of random fragmentation and reassembly of nucleotide sequences, followed by screening to select nucleotide sequences encoding polypeptides with desired properties. In order for the rearrangement of the nucleic acid to take place based on homology, the relevant parts of the nucleotide sequences are preferably at least 50% identical, such as at least 60% identical, more preferably at least 70% identical, such as at least 80% identical. Recombination can be carried out in vitro or in vivo.

Examples of suitable in vitro gene rearrangement procedures are disclosed in Stemmer et al. (1994), Proc. Natl Acad. Sci. USA; vol. 91, pp. 10747-10751; Stemmer (1994), Nature, vol. 370, pp. 389-391; Smith (1994), Nature vol. 370, pp. 324-325; Zhao et al., Nat. Biotechnol., March 1998; 16 (3): 258-61; Zhao H. and Arnold, FB, Nucleic Acids Research, 1997, Vol. 25. No. 6 pp. 1307-1308; Shao et al., Nucleic Acids Research, January 15, 1998; 26 (2): pp. 68183; and WO 95/17413.

An example of a suitable in vivo rearrangement procedure is disclosed in WO 97/07205. Other techniques for mutagenicity of nucleic acid sequences are disclosed by in vitro or in vivo recombination, for example, in WO 97/20078, and in US 5,837,458. Examples of specific rearrangement techniques include "family rearrangement," synthetic rearrangement "and" in silico rearrangement. "

Family rearrangement involves subjecting a family of homologous genes of different species to one or more cycles of rearrangement and subsequent screening or selection. Family rearrangement techniques are disclosed, for example, in Crameri et al. (1998), Nature, vol. 391, pp. 288-291; Christians et al. (1999), Nature Biotechnology, vol. 17, pp. 259-264; Chang et al. (1999), Nature Biotechnology, vol. 17, pp. 793-797; and Ness et al. (1999), Nature Biotechnology, vol. 17, 893

896.

Synthetic rearrangement involves providing libraries of overlapping synthetic oligonucleotides based, for example, on sequence alignment of the homologous genes of interest. Synthetically generated oligonucleotides are recombined, and the resulting recombinant nucleic acid sequences are screened and if desired, used for additional rearrangement cycles. Synthetic rearrangement techniques are disclosed in WO 00/42561.

In silico rearrangement refers to a DNA rearrangement procedure, which is carried out or modeled using a computer system, thereby partially or completely avoiding the need to physically manipulate nucleic acids. Techniques for in silico rearrangement are disclosed in WO 00/42560.

Once assembled (by synthesis, site-directed mutagenesis or other procedure), the nucleotide sequence encoding the polypeptide is inserted into a recombinant vector and operably linked to control the sequences necessary for the expression of FVII in the desired transformed host cell. .

[0227] It should be understood of course that not all vectors and expression control sequences work equally well to express the nucleotide sequence encoding the polypeptide variants described herein. None of the guests will work equally well with the same expression system. However, one skilled in the art can make a selection between these vectors, expression control sequences and hosts without unnecessary experimentation. For example, in the selection of a vector, the host must be considered because the vector must replicate in it or be able to integrate into the chromosome. The number of copies of the vector, the ability to control this number of copies, and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. In the selection of an expression control sequence, a variety of factors must also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleotide sequence encoding the polypeptide, particularly with respect to potential secondary structures. Guests should be selected in consideration of their compatibility with the chosen vector, the toxicity of the product encoded by the nucleotide sequence, its secretion characteristics, its ability to fold the polypeptide variant correctly, its fermentation or culture requirements, and the ease of purification of the products encoded by the nucleotide sequence.

The recombinant vector may be a vector that autonomously replicates, that is, a vector, which exists as an extrachromosomal entity, the replication of which is dependent on chromosomal replication, for example, a plasmid. Alternatively, the vector is one that, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome (s) in which it has been integrated.

The vector is preferably an expression vector, in which the nucleotide sequence encoding the polypeptide variant of the invention is operably linked with the additional segments required for transcription of the nucleotide sequence. The vector is normally derived from plasmid or viral DNA. Numerous expression vectors suitable for expression in host cells mentioned herein are commercially available or described in the literature. Expression vectors useful for eukaryotic hosts, include, for example, vectors comprising control sequences of the expression of SV40, bovine papillomavirus, adenovirus and cytomegalovirus Specific vectors are, for example, pCDNA3.1 (+) \ Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, La Jolla, CA, USA.). Useful expression vectors for yeast cells include plasmid 2µ and its derivatives, vector POT1 (US 4,931,373), vector pJSO37 described in Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C (Invitrogen). Useful vectors for insect cells include pVL941, pBG311 (Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance And Expression of the Human Gene In Animal Cells", Cell, 45, pp. 685-98 ( 1986), pBluebac 4.5 and pMelbac (both available from Invitrogen) Expression vectors useful for bacterial hosts include known bacterial plasmids, such as E. coli plasmids, which include pBR322, pET3a and pET12a (both from Novagen Inc., WI , USA), plasmids with a wider range of hosts, such as RP4, phage DNA, for example, the numerous lambda phage derivatives, for example NM989, and other DNA phages, such as M13 and phage of Single stranded DNA strands.

Other vectors for use in this invention include those that allow the nucleotide sequence encoding the polypeptide variant to be amplified in the number of copies. Such amplifiable vectors are well known in the art. They include, for example, vectors capable of amplification by DHFR amplification (see for example, Kaufman, U.S. Patent No. 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression ", Mol. Cell. Biol., 2, pp. 1304-19 (1982)) and amplification by glutamine synthetase (" GS ") (see, for example, US 5,122,464 and EP 338,841).

The recombinant vector may further comprise a DNA sequence that allows the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the origin of SV40 replication. When the host cell is a yeast cell, suitable sequences that allow the vector to replicate are those of the replication REP 1-3 genes of the 2µ yeast plasmid and the origin of replication.

The vector may also comprise a selectable marker, for example the product of a gene of which complements a defect in the host cell, such as a gene encoding dihydrofolate reductase (DHFR) or the TPI gene of Schizosaccharomyces pombe (described by PR Russell , Gene 40, 1985, pp. 125-130), or one that confers resistance to a drug, for example, ampicillin, tetracycline kanamycin, chloramphenicol, neomycin, hygromycin or methotrexate. For Saccharomyces cerevisiae, selectable markers include ura3 and leu2. For filamentous fungi, selectable markers include amdS, pyrG, arcS, niaD and sC.

The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of the polypeptide variant of the invention. Each control sequence may be natural or foreign to the nucleic acid sequence encoding the polypeptide variant. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, an enhancer or activation sequence in the 5 'direction, a signal peptide sequence, and a terminator of the transcription. As minimum. Control sequences include a promoter.

A wide variety of expression control sequences can be used in the present invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the above expression vectors as well as any known sequence that controls the expression of prokaryotic or eukaryotic cell genes or their viruses, and their various combinations.

Examples of suitable control sequences to direct transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, for example, the major late promoter of adenovirus 2, the MT-1 promoter (metallothionein gene), the promoter of the immediate-early human cytomegalovirus (CMV) gene, the human elongation factor Iα (EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter, the Rous Sarcoma Virus (VSR) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, the polyadenylation signals of the SV40 or adenovirus Elb region and the Kozak consensus sequence (Kozak, M. J Mol Biol August 20, 1987; 196 (4): 947-50).

In order to improve expression in mammalian cells, a synthetic intron can be inserted into the 5 ’untranslated region of the nucleotide sequence encoding the polypeptide. An example of a synthetic intron is the synthetic intron from the pCI-Neo plasmid (available from Promega Corporation, WI, USA).

Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the P10 promoter, the autographa californica polyhedrosis virus basic protein promoter, and the SV40 polyadenylation sequence. Examples of control sequences suitable for use in yeast host cells include promoters of the yeast α-pairing system, the promoter of the isomerose phosphate triose (TPI) of yeast, promoters of the yeast glycolytic genes or genes of alcohol dehydrogenase, the ADH2-4c promoter, and the inducible GAL promoter. Examples of control sequences suitable for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding the TAKA triose phosphate isomerase amylase or the Aspergillus oryzae alkaline protease, an A-amylase from A Niger, a glucoamylase from A. niger or A. nidulans, an acetamidase from A. nidulans, an aspartic proteinase or lipase from Rhizomucor miehei, the TPI1 terminator and the ADH3 terminator. Examples of control sequences suitable for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system, and the major promoter regions of the lambda phage.

The presence or absence of a signal peptide will depend, for example on the expression of the host cell used for the production of the polypeptide variant to be expressed (if it is an intracellular or extracellular polypeptide) and if it is desirable to obtain secretion. For use in filamentous fungi, the signal peptide may conveniently be derived from a gene that encodes an amylase or glucoamylase from Aspergillus sp., A gene that encodes a lipase or protease from Rhizomucor miehei or a Humicola lanuginosa lipase. The signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid stable amylase, or A. niger glucoamylase. For use in insect cells, the signal peptide of an insect gene can be conveniently derived (compare WO 90/05783), such as the precursor of the adipokinetic hormone of Lepidopteran manduca sixth, (compare US 5,023 .328), honey honey melitin (Invitrogen), UDP ecdysteroid glucosyltransferase (egt) (Murphy et al., Protein Expression and Purification 4, 349-357 (1993) or human pancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997) A preferred signal peptide for use in mammalian cells is that of hFVII or the signal peptide of the murine Ig kappa light chain (Coloma, M (1992) J. Imm. Methods 152: 89-104) It has been found that signal peptides suitable for use in yeast cells are the signal peptide of the S. cerevisiae factor α (compare US 4,870,008), a signal peptide of the modified carboxypeptidase (compare LA Valls et al., Cell 48, 1987, pp. 887-897), the yeast signal peptide BAR1 (compare WO 87/02670), the signal peptide of aspartic yeast protease 3 (YAP3) (compare M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137), and the TA57 synthetic leader sequence (WO 98/32867). It has been found that the signal peptide suitable for use in E. coli cells is the ompA signal peptide (EP 581821).

The nucleotide sequence of the invention encoding a polypeptide variant, if prepared by site-directed mutagenesis, synthesis, PCR or other methods, may optionally include a nucleotide sequence encoding a signal peptide. The signal peptide is present when the polypeptide variant of the cells in which it is expressed is to be secreted. Said signal peptide, if present, must be one recognized by the cell selected for expression of the polypeptide variant. The signal peptide will normally be one associated with human natural FVII.

Any suitable host can be used to produce the polypeptide variant, including bacteria (although not particularly preferred), fungi (including yeasts), plants, insects, mammals, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. . Examples of bacterial host cells include gram positive bacteria such as Bacillus strains, for example, B. brevis or B. subtilis, or Streptomyces, or gram negative bacteria, such as E. coli strains. The introduction of a vector into a bacterial host cell can, for example, be effected by transformation of protoplasts (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), which uses competent cells (see, for example, Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278). Examples of suitable filamentous fungal host cells include strains of Aspergillus, for example, A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma. Fungal cells can be transformed by a procedure that involves protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. EP 238 023 and US 5,679,543 describe suitable methods of transforming Aspergillus host cells. In Malardier et al., 1989, Gene 78: 147-156 and in WO 96/00787, suitable procedures for transforming Fusarium species are described. Examples of suitable yeast host cells include strains of Saccharomyces, for example. S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. Polymorpha or Yarrowia. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920: and are disclosed in Clontech Laboratories, Inc, Palo Alto, CA, USA (in the product protocol of the Yeast Transformation System Kit Yeastmaker ™). Examples of suitable insect host cells include a Lepidoptera cell line, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusia ni (High Five) cells (US 5,077,214). The transformation of insect cells and the production of heterologous polypeptides of the foregoing can be carried out as described by Invitrogen. Examples of suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (eg, CHO-K1; ATCC CCL-61), green monkey cell lines (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL1651)); mouse cells (for example NS / O), hamster kidney (BHK) kidney cells (for example, ATCC CRL-1632 or ATCC CCL-10), and human cells (for example, HEK 293 (ATCC CRL) -1573)), as well as plant cells in tissue cultures. Additional suitable cell lines are known in the art and are available from public repositories such as the American Type Culture Collection, Rockville, Maryland. Mammalian cells, such as a CHO cell can also be modified to express sialyltransferase, for example, 1,6-sialyltransferase, for example, as described in US 5,047,335, in order to provide glycosylation Enhanced polypeptide variant.

It may be of particular interest in order to increase secretion, to produce the polypeptide variant of the invention together with an endoprotease, in particular a PACE (enzyme that converts the basic amino acid into paired) (for example, as described herein. US 5,986,079), such as Kex2 endoprotease (for example, as described in WO 00/28065).

Procedures for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome mediated transfection, viral vectors and the transfection procedure described by Life Technologies Ltd, Paisley, United Kingdom , which uses Lipofectamine 2000. These procedures are well known in the art and are described, for example, in Ausebel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. Mammalian cell culture is carried out in accordance with established procedures, for example, as disclosed in Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, New Jersey , USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997.

In the production processes of the present invention, the cells are cultured in a nutrient medium suitable for the production of the polypeptide variant using methods known in the art. The cell can be cultured, for example, by flask culture with agitation, small-scale or large-scale fermentation (including continuous, discontinuous, discontinuous, or solid-state fermentation) in laboratory or industrial fermenters carried out in a suitable medium and under conditions that allow the polypeptide to be expressed and / or isolated: The culture takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using methods known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). If the variant of the polypeptide is secreted in the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide variant is not secreted, it can be recovered from cell lysates.

The resulting polypeptide variant can be recovered by methods known in the art. For example, the polypeptide variant can be recovered from the nutrient medium by conventional procedures that include, but are not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

Polypeptides can be purified by a variety of methods known in the art that include, but are not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromato-focalization, and size exclusion), electrophoretic procedures (e.g. , preparative isoelectric focusing), differential solubility (eg, precipitation with ammonium sulfate), HPLC, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989 ).

Single stranded polypeptide variants of the invention can be purified and activated for double stranded polypeptide variants by numerous methods as described in the literature (Broze and Majerus, 1980, J. Biol. Chem. 255: 1242-47 and Hedner and Kisiel, 1983, J. Clin. Invest. 71: 1836-41). Another method in which single stranded polypeptide variants can be purified is by incorporation of Zn ions during purification as described in US 5,700,914.

In a preferred embodiment the polypeptide variant is purified as a single stranded polypeptide variant, which is also optionally PEGylated. The optionally PEGylated single chain polypeptide variant is activated both by the use of an immobilized enzyme (eg, factors IIa, IXa, Xa and XIIa) and by self-activation using a positively charged ion exchange matrix or the like.

It is advantageous to first purify the polypeptide variant in its single-stranded form, PEGylate it below and finally activate it by one of the procedures described above or by self-activation as described by Pedersen et al., 1989, Biochemistry 28: 9331-36 . The advantage of carrying out the PEGylation before activation is that the PEGylation of the new amino end formed by the cleavage of R 152-1153 is avoided. PEGylation of this new amino end would make the molecule inactive due to the formation of a hydrogen bond between D242 and the amino group of I153 is necessary for activity.

Pharmaceutical composition of the invention and its use

In a further aspect, the present invention relates to a composition, in particular a pharmaceutical composition, comprising a polypeptide variant of the invention and a pharmaceutically acceptable carrier or excipient.

The polypeptide variant or the pharmaceutical composition according to the invention can be used as a medicament.

Due to the high coagulation efficiency, the polypeptide variant of the invention, or the pharmaceutical composition of the invention, is particularly useful for the treatment of bleeding, including uncontrollable bleeding episodes, such as uncontrollable bleeding episodes along with trauma, thrombocytopenia. , patients in anticoagulant treatment, and patients with cirrhosis, such as patients with cirrhosis with hemorrhagic varicose veins, or other gastrointestinal hemorrhages and in patients undergoing orthotopic liver transplantation, or liver resection (which allows transfusion-free surgery).

Trauma is defined as a lesion in a living tissue produced by an extrinsic agent. It is the 4th cause that causes death in the United States and represents a great financial burden on the economy.

Trauma is classified as blunt or penetrating trauma Blunt trauma results in internal compression, organ damage and internal bleeding while penetrating trauma (as a consequence of an agent that penetrates the body and destroys tissue, vessels and the organs) results in external bleeding.

Hemorrhage, as a result of trauma, can lead to a cascade of problems. For example, physiological compensation mechanisms are initiated with initial peripheral and mesenteric vasoconstriction to derive blood to the central circulation. If the circulation is not restored, then a hypovolemic shock (multiple organ failure due to improper perfusion) follows. Because the tissues throughout the body become lacking in oxygen, anaerobic metabolism begins. In addition, simultaneous lactic acid results in lowering the pH of the blood and metabolic acidosis develops. If the acidosis is severe and is not corrected, the patient may develop a multi-organ failure and die.

Although most trauma patients suffer hypothermia upon arrival in the emergency room due to environmental conditions in the operating room, inadequate protection, intravenous fluid administration, and continuous blood loss that worsens the hypothermic state. Deficiencies in clotting factors may occur due to blood loss or transfusions. Meanwhile, acidosis and hypothermia interfere with blood clotting mechanisms. In this way, coagulopathy develops, which, at the same time, can mask the sites of bleeding and prevent mechanical control of the bleeding. Hypothermia, coagulopathy and acidosis are often characterized as the "traumatic triad of death."

Trauma can be caused by various events, for example, road traffic accidents that result in many different types of trauma. While some road traffic accidents probably result in penetrating trauma, many road traffic accidents are likely to cause blunt head and body trauma. However, these various types of trauma can all result in coagulopathy in the patient. Road traffic accidents are the leading cause of death by accident in the US. There are about 42,000 deaths caused by them in the United States each year. Many trauma patients die at the scene of the accident, while being treated by auxiliary health personnel, before arriving or being in transit to the emergency room.

Another example includes gunshot wounds. Firearm injuries are traumas that result in massive hemorrhage. They are penetrating and destroy tissue as the bullet passes through the body, whether they are in the torso or in an extremity. In the United States, approximately 40,000 people per year die from gunshot wounds.

An additional example includes falls. The falls result in a profile similar to the type of road traffic accident trauma. Falling onto a solid object or the ground from a height can cause penetrating and blunt decelerative trauma. In the United States, falls are a common cause of death by accident, in an approximate number of 13,000.

Another additional example includes accidents with machinery. A small number of people die in the United States from deaths related to machine accidents, either from an injury or from being trapped in machinery. The figures are small but significant - approximately 2,000.

A more additional example includes wounds from sharp objects. Puncture wounds are penetrating lesions that can also cause massive bleeding. The organs that are most likely damaged in a puncture wound are the liver, small intestine and colon.

Hepatic cirrhosis is the terminal sequel of a prolonged repeated lesion in the hepatic parenchyma. The end result is the formation of broad bands of fibrous tissue that separate regenerative nodules that do not maintain normal organization of the hepatic lobes and thus cause impaired liver function. Patients have prolonged protombin times as a result of the depletion of coagulation factors dependent on vitamin K. Pathologically, liver cirrhosis should be regarded as the final common route of chronic liver injury, which may be the result of any lesson prolonged repeated liver cell. Liver cirrhosis can be caused by direct liver injury, which includes chronic alcoholism, chronic viral hepatitis (types B, C, and D), and autoimmune hepatitis as well as indirect injury due to bile duct damage, which includes primary biliary cirrhosis , primary sclerosing cholangitis and biliary atresia. Less common causes of cirrhosis include direct liver injury caused by inherited disease such as cystic fibrosis, alpha-1-antitrypsin deficiency, hemochromatosis, Wilson disease, galactosemia, and glycogen storage disease.

The transplant is mainly reserved for late-stage cirrhotic patients, in which it is the key intervention to treat the disease. To be eligible for transplant, a patient must be classified as a pediatric B or C, as well as meet additional selection criteria. Last year, in the United States alone, 4,954 transplants were performed.

It has been estimated that there are 6,000 bleeding episodes associated with patients undergoing resection every year. This correlates with the cautious position of this procedure although it seems slightly elevated compared to the number of transplants.

Reliable data on the incidence of varicose hemorrhages are difficult to obtain. The key facts known are that at the time of diagnosis, varicose veins are present in approximately 60% of decompensated patients and 30% of compensated patients and that approximately 30% of these patients with varicose veins will experience bleeding and that each episode of Varicose hemorrhage is associated with a 30% risk of mortality.

Thrombocytopenia is produced by one of three mechanisms, decreased bone marrow production, increased splenic sequestration, or accelerated platelet destruction. Thrombocytopenia is a risk factor for bleeding, and platelet transfusion reduces the incidence of bleeding. The threshold of prophylactic platelet transfusion is 10,000 / µl. In patients without fever or infections, a threshold of 5000 / µl may be sufficient to prevent spontaneous bleeding. For invasive procedures. 50,000 platelets / µl is the usual target level. In patients who develop platelet antibodies after repeated transfusions, it can be extremely difficult to control bleeding.

Thus, in a further aspect, the present invention relates to the use of a polypeptide variant of the invention for the manufacture of a medicament for the treatment of diseases or disorders in which clot formation is desirable. A more additional aspect of the present invention relates to a method for treating a mammal that has a disease or disorder in which clot formation is desirable, which comprises administering to a mammal in need thereof an effective amount of a variant of polypeptide or pharmaceutical composition of the invention.

Examples of diseases / disorders in which clot formation is desirable include, but is not limited to, hemorrhages, which include cerebral hemorrhages, as well as uncontrolled hemorrhages, such as trauma. Additional examples include patients undergoing live transplants, patients undergoing resection, thrombocytopenic patients, cirrhotic patients, patients with varicose hemorrhages, hemophilia A, hemophilia B and von Willebrands disease.

The polypeptide variant of the invention is administered to patients in a therapeutically effective dose, usually one approximately parallel to that used in rFVII therapy such as NovoSeven®, or at a lower dosage. By "therapeutically effective dose" herein is understood to be sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose will depend on the circumstances, and will be verifiable by a person skilled in the art using known techniques. Normally, the dose should be able to avoid or decrease the severity or spread of the condition or indication being treated. It will be apparent to the person skilled in the art that an effective amount of a variant or polypeptide composition of the invention depends, among others, on the disease, the dose, the administration schedule, on whether the variant or the polypeptide composition is administered. alone or together with other therapeutic agents, the plasma half-life of the compositions, and the general health of the patient. Preferably, the variant or polypeptide composition is administered in an effective dose, in particular a dose that is sufficient to normalize the coagulation disorder.

The polypeptide variant of the invention is preferably administered in a composition that includes a pharmaceutically acceptable carrier or excipient. "Pharmaceutically acceptable" means a vehicle or excipient that has no adverse effect on patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

The polypeptide variant of the invention can be formulated in pharmaceutical compositions by well known methods. Suitable formulations are described in Remington’s Pharmaceutical Sciences by E.W. Martin (Mark Publ. Co., 16th Ed., 1980).

Polypeptide variants of the invention can be used "as they are" and / or in the form of their salts. Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as, for example, zinc salts. These salts or complexes may be present as a crystalline and / or amorphous structure.

The pharmaceutical composition of the invention can be administered alone or together with other therapeutic agents. These agents can be incorporated as part of the same pharmaceutical composition or they can be administered separately from the polypeptide variant of the invention, both simultaneously and in accordance with other treatment schedules. In addition, the polypeptide variant or pharmaceutical composition of the invention can be used as an adjuvant in other therapies.

A "patient", for the purposes of the present invention, includes humans and other mammals. Thus, the procedures are applicable to therapy in humans and veterinary applications. The pharmaceutical composition comprising the polypeptide variant of the invention can be formulated in a variety of ways, for example, as a liquid, gel, lyophilized, or as a compressed solid. The preferred form will depend on the specific indication being treated and will be apparent to a person skilled in the art.

In particular, the pharmaceutical composition comprising the polypeptide variant of the invention can be formulated in stable lyophilized or soluble form. The polypeptide variant can be lyophilized by a variety of procedures known in the art. The polypeptide variant can be in stable soluble form by removing or protecting the proteolytic degradation sites, as described herein. The advantage of obtaining a stable soluble preparation lies in an easier manipulation for the patient and, in the case of emergencies, a faster action, which can potentially save a life. The preferred form will depend on the specific indication being treated and will be apparent to a person skilled in the art.

Administration of the formulations of the present invention can be carried out in a variety of ways, including, but not limited to, oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, intraocular. , or in any other acceptable way. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implant, in some examples, the formulations can be applied directly as a solution or spray.

Parenteral solutions

A preferred example of a pharmaceutical composition is a solution, especially an aqueous solution, designed for parenteral administration. Although in many cases pharmaceutical solutions in solution are provided in liquid form, suitable for immediate use, such parenteral formulations can also be provided in frozen or lyophilized form. In the first case, the composition must be thawed before use. The latter form is often used to increase the stability of the active compound contained in the composition to a wider variety of storage conditions, as recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid equivalents. Such lyophilized preparations are reconstituted before use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline.

In the case of parenteral solutions, they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide variant having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers normally employed in the technique (all of which are called "excipients"), for example, buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic surfactants or detergents, antioxidants and / or other various additives.

Buffering agents help maintain pH in the range that approximates physiological conditions. They are normally present at a concentration that varies between about 2 mM to about 50 mM. Buffering agents suitable for use in the present invention include organic and inorganic acids and their salts such as citrate buffers (for example, a mixture of monosodium citrate-disodium citrate, a mixture of trisodium citric acid citrate, a mixture of citric acid-monosodium citrate , etc.), succinate buffers (for example, a mixture of succinic acid-monosodium succinate, a mixture of succinic acid-sodium hydroxide, a mixture of succinic acid disodium succinate, etc.), tartrate buffers (for example, a mixture of tartaric acid-sodium tartrate, a mixture of tartaric acid-potassium tartrate, a mixture of tartaric acid-sodium hydroxide, etc.), fumarate buffers (for example, a mixture of fumaric acid-monosodium fumarate, a mixture of fumaric acid disodium fumarate, a mixture of monosodium fumarate-disodium fumarate, etc.), gluconate buffers (for example, a mixture of gluconic acid, sodium gluconate gave, a mixture of gluconic acid-sodium hydroxide, a mixture of gluconic acid-potassium gluconate, etc.), oxalate buffer (for example, a mixture of oxalic acid-sodium oxalate, a mixture of oxalic acid sodium hydroxide, a mixture of oxalic acid-potassium oxalate, etc.), lactate buffers (for example, a mixture of lactic acid-sodium lactate, a mixture of lactic acid-sodium hydroxide, a mixture of lactic acid-potassium lactate, etc.) and acetate buffers (for example, a mixture of acetic acid-sodium acetate, a mixture of acetic acid-sodium hydroxide, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris.

Stabilizers refer to a broad category of excipients, which can vary in function from a loading agent to an additive that solubilizes the therapeutic agent or helps prevent denaturation or adhesion to the vessel wall. Typical stabilizers may be polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol , sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol and the like, which include cyclitols such as inositol; polyethylene glycol; amino acid polymers; reducing agents that contain sulfur, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues), proteins such as human serum albumin, bovine serum albumin, gelatin and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are normally present in the range of 0.1 to 10,000 parts by weight based on the weight in weight of the active protein.

Preservatives are added to retard microbial growth, and they are usually added in amounts of approximately 0.2% -1% (w / v). Preservatives suitable for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecylmethylbenzyl ammonium chloride, benzalkonium halides (e.g., benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonifiers are added to ensure the isotonicity of the liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols may be present in an amount between 0.1% and 25% by weight, usually 1% to 5%, taking into account the relative amounts of the remaining ingredients.

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35

40

Four. Five

Non-ionic surfactants or detergents (also known as "wetting agents") may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide from aggregation induced by agitation, which also allows the formulation to be exposed to stress. of superficial shear without causing denaturation of the polypeptide. Non-ionic surfactants include polysorbates (20, 80, etc.), polyoxammers (184, 188, etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.).

Additional varied excipients include fillers or fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient can also be trapped in prepared microcapsules, for example, by coacervation techniques or by interfacial polymerization, for example, microcapsules of hydroxymethylcellulose, gelatin or polymethylmethacrylate, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington’s Pharmaceutical Sciences, above.

Parenteral formulations to be used for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through sterile filtration membranes.

Continuous Release Preparations

Examples of continuous release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide variant, the matrices having a suitable form such as a film or microcapsules. Examples of continuous release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-ethylene vinyl acetate degradable, degradable copolymers of lactic acid-glycolic acid such as ProLease® or Lupron Depot® technology (injectable microspheres composed of copolymers of lactic acid-glycolic acid and leuprólido acetate), and poly-D - (-) - 3- hydroxybutyric While polymers such as ethylene vinyl acetate and lactic acid glycolic acid allow the release of molecules for long periods such as up to or for 100 days, some hydrogels release proteins for shorter periods of time. When encapsulated polypeptides remain in the body for a long time, they can be denatured or added as a result of exposure to moisture at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational stabilization strategies can be devised depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is the formation of the intermolecular SS bond through the thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl moieties, lyophilizing from acid solutions, controlling the moisture content, using additives appropriate, and developing specific polymer matrix compositions.

The invention is further described in the following non-limiting examples.

Materials and procedures

Accessible surface area (ASA)

The Access computer program (B. Lee and FM Richards, J. Mol. Biol. 55: 379-400 (1971)) version 2 (© 1983 Yale University) is used to calculate the accessible surface area (ASA) of atoms Individuals in the structure. This procedure normally uses a probe size of 1.4 Å and defines the Accessible Surface Area (ASA) as the area formed by the center of the probe. Before this calculation, all water molecules and all hydrogen atoms must be removed from the coordinate set, as well as other atoms not directly related to the protein.

Side Chain Fractional ASA

The fractional ASA of the side chain atoms was calculated by dividing the sum of the ASA of the atoms in the side chain by a value that represents the ASA of the side chain atoms of this type of residue in an X-wing tripeptide. Extended wing (See Hubbard, Campbell and Thomton (1991) J. Mol. Biol. 220, 507-530). For this example the CA atom was considered as part of the side chain of glycine residues but not of the remaining residues. The following table was used as a 100% ASA pattern for the side chain:

To

69.23 Å2 Leu 140.76 Å2

Arg

200.35 Å2 Lys 162.50 Å2

Asn

106.25 Å2 Met 156.08 Å2

Asp

102.06 Å2 Phe 163.90 Å2

Cys

96.69 Å2 Pro 119.65 Å2

Gln

140.58 Å2 Be 78.16 Å2

Glu

134.61 Å2 Thr 101.67 Å2

Gly

 32.28 Å2 Trp 210.89 Å2

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His

147.00 Å2 Tyr 176.61 Å2

Ile

137.91 Å2 Val 114.14 Å2

The remains not detected in the structure are defined as having 100% exposure since they are thought to reside in flexible regions. Gamma-carboxyglutamic acids in positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 are all defined as having 100% exposure.

Determination of distances between atoms

The distance between atoms is more easily determined using a molecular graphics software, for example, InsightII® v. 98.0, MSI INC.

Region of the active site

The region of the active site is defined as any residue that has at least one atom in 10Å around any atom in the catalytic triad (residues H193, D242, S344).

Tissue factor binding site determination

The FT binding site is defined as the point that comprises all the remains that have undergone a change in their accessible surface area after FT binding. This was determined by two ASA calculations; one in the isolated ligand (s) in the ligand (s) / receptor (s) complex and one in complete ligand (s) / receptor (s) complex.

Measurement of reduced sensitivity to proteolytic degradation

Proteolytic degradation can be measured using the assay described in US 5,580,560, Example 5, in which the proteolysis is self-protection.

In addition, reduced proteolysis can be tested in an in vivo model using radiolabeled samples and comparing rhFVIIa proteolysis and the polypeptide variant of the invention by extracting blood samples and subjecting them to SDSPAGE and autoradiography.

Regardless of the test used to determine proteolytic activity, it is intended that "reduced proteolytic degradation" means a measurable reduction in cleavage compared to that obtained by rhFVIIa as measured by gel scanning of stained SDS-PAGE gels with Coomasie, HPLC or as measured by the preserved catalytic activity compared to the natural one using the tissue factor independent activity test described below.

Determination of the molecular weight of the polypeptide variants

The molecular weight of the polypeptide variants was determined by any of SDS-PAGE, gel filtration, Western blots, laser-assisted matrix desorption mass spectrometry or equilibrium centrifugation, for example SDS-PAGE according to Laemmli, United Kingdom, Nature Vol 227 (1970), pp. 680-85.

Determination of affinity to tissue factor binding

The ability of the variants to bind to the tissue factor can be evaluated using one or more of the three BIAcore® tests described in Dickinson et al. Proc. Natl Acad. Sci. USA 1996, 93: 14379-14384; Roberge et al. Biochemistry 2001, 40: 9522-9531; and Ruf et al. Biochemistry 1999, 38 (7): 1957-1966.

Determination of TFPI inhibition

The inhibition of FVII by TFPI can be monitored in the amidolytic assay described in Chang et al. Biochemistry 1999, 38: 10940-10948.

Determination of affinity for TFPI

The ability of the variants to bind TFPI was evaluated using one or more of the three BIAcore® tests described in Dickinson et al. Proc. Natl Acad. Sci. USA 1996, 93: 14379-14384; Roberge et al. Biochemistry 2001, 40: 9522-9531; and Ruf et al. Biochemistry 1999, 38 (7): 1957-1966.

Determination of phospholipid membrane binding affinity

The phospholipid membrane binding affinity can be determined as described in Nelsestuen et al., Biochemistry, 1977; 30; 10819-10824 or as described in Example 1 in US 6,017,882.

FT independent factor X activation test

This essay has been described in detail on page 39826 of Nelsestuen et al., J Biol Chem, 2001; 276: 39825-39831.

Briefly, the molecule to be evaluated (any of hFVIIa, rhFVIIa or the variant of the polypeptide of the invention in its activated form) was mixed with a phospholipid source (preferably phosphatidylcholine and phosphatidylserine in an 8: 2 ratio) and relipidized Factor X in TRIS buffer containing BSA. After a specified incubation time, the reaction was stopped by the addition of excess EDTA. The concentration of factor Xa was then measured from the change in absorbance at 405 nm after the addition of a chromogenic substrate (S-222, Chromogenix). After background correction, the independent activity of the rhFVIIa tissue factor (awt) was determined as the change in absorbance after 10 minutes and the independent activity of the tissue factor of the polypeptide variant of the invention (avariant) was also determined as the change in absorbance after 10 minutes. The relationship between the activity of the polypeptide variant, in its activated form, and the activity of rhFVIIa is defined as avariant / awt.

Coagulation test

The coagulation activity of FVIIa and its variants were measured in one-stage trials and coagulation times were recorded on a Thrombotrack IV coagulometer (Medinor): Human plasma was reconstituted with Factor VII depleted (American Diagnostica) and equilibrated at room temperature for 15-20 minutes. Then 50 microliters of plasma were transferred to the coagulometer cups.

FVIIa and its variants were diluted in glyoxaline buffer (5.7 mM barbiturate, 4.3 mM sodium citrate, 117 mM NaCl, 1 mg / ml BSA, pH 7.35). Samples were added to the cup in 50 ul and incubated at 37 ° C for 2 minutes.

Thromboplastin (Medinor) was reconstituted with water and CaCl2 was added to a final concentration of 4.5 mM. The reaction was started by adding 100 µl of thromboplastin.

The same test was used to measure coagulation activity in the absence of FT without the addition of thromboplastin. Data were analyzed using PRISM software.

Whole blood test

The coagulation activity of FVIIa and its variants was measured in one-stage trials and coagulation times were recorded on a Thrombotrack IV coagulometer (Medinor). 100 µl of FVIIa or its variants were diluted in a buffer containing 10 mM glycylglycine, 50 mM NaCl, 37.5 mM CaCl2, pH 7.35 and transferred to the reaction cup. The coagulation reaction was initiated by the addition of 50 µl of blood containing 0.13 M trisodium citrate 10% as anticoagulant. Data were analyzed using Excel or PRISM software.

Amidolytic assay

The ability of the variants to cleave small peptide substrates can be measured using the chromogenic substrate S-2288 (D-Ile-Pro-Arg-p-nitroanilide). FVIIa was diluted to approximately 10-90 nM in assay buffer (50 mM Na-Hepes pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.1% BSA, IU / ml Heparin). In addition, soluble FT (FTs) was diluted to 50-450 nM in assay buffer: 120 µl of assay buffer was mixed with 20 µl of the FVIIa sample and 20 µl of FTs. After 5 min incubation at room temperature with gentle agitation, followed by 10 min incubation at 37 ° C, the reaction was initiated by the addition of the S-2288 substrate at 1 mM and the absorption at 405 nM was determined at various time points.

ELISA test

FVII / FVIIa concentrations (or the variant) were determined by ELISA. The wells of a microtiter plate were coated with an antibody directed against the protease domain using a solution of 2 µg / ml in PBS (100 µl per well). After overnight coating to T. A. the wells were washed 4 times with THT buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.3 Tween-20 0.05%). Subsequently, 200 µl of 1% casein (diluted from a 2.5% stock solution using 100 mM NaCl, 50 mM Tris-HCl pH 7.2) was added per well for blocking. After 1 h of incubation at T. A., the wells were emptied, and 100 µl of sample (optionally diluted in dilution buffer (THT + 0.1% casein)) was added. After another 1 h incubation at room temperature, the wells were washed 4 times with THT buffer, and 100 µl of a biotin labeled antibody directed against the EGF-like domain (1 µg / ml) was added. After another 1 h incubation at T. A. followed by 4 more washes with THT buffer, 100 µl horseradish streptavidin peroxidase (DAKO A / S, Glostrup, Denmark, diluted 1/10000) was added. After another 1 h incubation at T. A., followed by 4 more washes with THT buffer, 100 µl of TMB (3.3 ', 5,5'-tetramethylbenzidine, Kem-en-Tech A / S, Denmark) was added. After 30 min. incubation to T. A. in the dark, 100 µl of 1 M H2SO4 was added and the OD450 nm was determined. A standard curve was prepared using rhFVIIa (NovoSeven®).

Alternatively, FVII / FVIIa or variants can be quantified through the Gla domain rather than through the protease domain. In this ELISA configuration, the wells were coated overnight with an antibody directed against the EGF-like domain and for detection, a calcium-dependent biotin-labeled monoclonal antibody against the Gla domain was used (2 µg / ml, 100 µl per well). In this configuration, 5 mM CaCl2 was added to the THT and dilution buffers.

Examples

Example 1

The x-ray structure of hFVIIa in complex with a soluble tissue factor by Banner et al., J Mol Biol, 1996; 285: 2089 It is noted that the numbering of residues in the reference does not follow the sequence. Here, the inventors have used sequential numbering according to SEQ ID NO: 1. Gamma-carboxyglutamic acids in positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 are all referred to herein as Glu (three letter abbreviation) or E (one letter abbreviation). The remains 143-152 are not present in the structure.

Surface exposure

Carrying out the calculations of the fractional ASA in the FVII fragments alone in combination with the definition of the accessibility of the non-normalized and / or missing remains described in the procedures, resulted in the following remains that had more than 25% of its side chain exposed to the surface: A1, N2, A3, F4, L5, E6, E7, L8, R9, P10, S12, L13, E14, E16, K18, E19, E20, Q21, S23, F24, E25, E26, R28, E29, F31, K32, D33, A34, E35, R36, K38, L39, W41, I42, S43, S45, G47, D48, Q49, A51, S52, S53, Q56, G58, S60, K62, D63, Q64, L65, Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82, T83, H84, K85, D86, D87, Q88, L89, 90, V92, N93, E94, G97, E99, S103, D104, H105, T106, G107, T108, K109, S111, R113, E116, G117, S119, L120, L121, A122, D123, G124, V125, S126, T128, P129, T130, V131, V131, V131, V131 E132,1140, L141, E142, K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152, G155, K157, V158, P160, K161, E163, L171, N173, G174, A175, N185 T185, I186, H193, K1 97, K199, N200, R202, N203, I205, S214, E215, H216, D217, G218, D219, S222, R224, S232, T233, V235, P236, G237, T238, T239, N240, H249, Q250, P251, V253, T255, D256, E265, R266, T267, E270, R271, F275, V276, R277, F278, L280, L287, L288, D289, R290, G291, A292, T293, L295, E296, N301, M306, T307 Q308, D309, L311, Q312, Q313, R315, K316, V317, G318, D319, S320, P321, N322, T324, E325, Y326, Y332, S333, D334, S336, K337, K341, G342, H351, R353, H351, R353 G354, Q366, G367, T370, V371, G372, R379, E385, Q388, K389, R392, S393, E394, P395, R396, P397, G398, V399, L400, L401, R402, P404 and P406.

The following remains had more than 50% of their side chain exposed to the surface: A1, A3, F4, L5, E6, E7, L8, R9, P10, E14, E16, K18, E19, E20, Q21, S23, E25, E26, E29, K32, A34, E35, R36, K38, L39, I42, S43, G47, D48, A51, S52, S53, Q56, G58, S60, K62, L65, Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82, H84, K85, D86, D87, Q88, L89, I90, V92, N93, E94, G97, T106, G107, T108, K109, S111, E116, S119, L121, A122, D123, G124, V131, E132, L141, E142, K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152, G155, K157, P160, N173, G174, A175, K197 K199, N200, R202, S214, E215, H216, G218, R224, V235, P236, G237, T238, H249, Q250, V253, D256, T267, F275, R277, F278, L288, D289, R290, G291, A292, T293, L295, N301, M306, Q308, D309, L311, Q312, Q313, R315, K316, G318, D319, N322, E325, D334, K341, G354, G367, V371, E385, K389, R392, E394, R396, E394, R396 P397, G398, R402, P404 and P406.

Tissue factor binding site

Carrying out the ASA calculations, the following remains in the human FVII change their ASA in the complex. These residues were defined as constituting the tissue factor binding site: L13, K18, F31, E35, R36, L39, F40, I42, S43, S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74, F76, E77, G78, R79, E82, K85, Q88, I90, V92, N93, E94, R271, A274, F275, V276, R277, F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325 and R379.

Region of the active site

The site region was defined as any residue that had at least one atom within a 10 Å distance of any atom in the catalytic triad (residues H193, D242, S344): I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234 V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328, D328 S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358, T359, G360, I361, V362, S363, W364, G365, C368, V376, Y377, T378, R379, V380, Q3880 Y383, W386, L387, L400 and F405.

The edge of the junction groove of the active site

The region of the edge of the junction of the active site was defined by visual inspection of FVIIa structure 1FAK.pdb as: N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370.

Example 2

Design of an expression cassette for the expression of rhFVII in mammalian cells

The DNA sequence shown in SEQ ID NO: 2 was synthesized, which encompasses the short form of the full-length cDNA encoding human blood coagulation factor VII with its natural short signal peptide (Hagen et al., 1986 PNAS 83: 2412), in order to facilitate high expression in mammalian cells. First, the context of the ATG start codon was modified according to the Kozak consensus sequence (Kozak, M. J Mol Biol August 20, 1987; 196 (4): 947-50), so that there is a perfect correspondence with the consensus sequence in the 5 'direction of the ATG start codon. Secondly, the open reading frame of the natural human blood coagulation factor cDNA was modified by biasing the codon utilization towards the codons frequently used in highly expressed human genes. In addition, two translation stop codons were inserted at the end of the open reading frame in order to facilitate effective translation stop. The optimized and fully synthetic human FVII gene was assembled between 70-mer DNA oligonucleotides and was finally amplified using final primers by inserting the BamHI and HindIII sites at the 5 ′ and 3 ′ ends respectively using standardized PCR techniques.

A vector was prepared for the cloning of the generated PCR product that encompassed the factor VII expression cassette by cloning the intron from pCINeo (Promega). The synthetic intron from pCI-Neo was amplified using standard PCR conditions and primers:

CBProFpr174: AGCTGGCTAGCCACTGGGCAGGTAAGTATCA (SEQ ID NO: 3) and

CBProFpr175: TGGCGGGATCCTTAAGAGCTGTAATTGAACT (SEQ ID NO: 4)

resulting in a 332 bp PCR fragment. The fragment was cut with NheI and BamHI before cloning into pCDNA3.1 / HygR (obtained from Invitrogen) resulting in PF No. 34

The human factor VII expression cassette was cloned between the BamHI and HindIII sites of PF No. 34, resulting in plasmid PF No. 226.

In order to allow the cloning of the FVII variant genes between the NheI and PmeI sites of the UCOE-based expression CET720 plasmid, a derivative of PF No. 226 was created that lacked the NheI site. Using PF No. 226 as a template, a DNA fragment was prepared by PCR using primers CBProFpr219 (SEQ ID NO: 5) and CBProFpr499 (SEQ ID NO: 6). This fragment was cut with XbaI and XhoI and cloned between the XbaI and XhoI sites of PF No. 226 resulting in plasmid PF No. 444. Plasmids derived from PF No. 444 were constructed which encoded variants of the invention by site-directed mutagenesis in PCR. standardized using PF nº 444 as a template and primers synthesized in a personalized way. Specific expression plasmids based on CET720 were generated by cleavage of the corresponding PF derivative gene variant No. 444 by NheI and PmeI and then cloned into CET720 cut with NheI and PmeI. For example, derivative pB0088 of CET720 which encoded the S43Q variant of FVII was prepared by cleavage of the gene variant by NheI and PmeI from pB0075 and cloned into CET720 cut with NheI and PmeI.

Example 3

Construction of expression vectors encoding the polypeptide variants of the invention

The following default primers were used for primary PCR: [L39H] rhFVII CBProFpr526: GCTGAGCGGACCAAACACTTTTGGATTAGC (SEQ ID NO: 7 - direct primer) CBProFpr527: GCTAATCCAAAAGTGTTTGGTCCGCTCAGC (SEQ ID NO: 8 - reverse primer) [I42R] rhFVII CBProFpr530: CCAAACTGTTTTGGCGCAGCTATAGCGATG (SEQ ID NO: 9 - direct primer) CBProFpr531: CATCGCTATAGCTGCGCCAAAACAGTTTGG (SEQ ID NO: 10 - reverse primer) [S43Q] rhFVII CBProFpr534: AACTGTTTTGGATTCAGTATAGCGATGGCG (SEQ ID NO: 11 - direct primer) CBProFpr535: CGCCATCGCTATACTGAATCCAAAACAGTT (SEQ ID NO: 12 - reverse primer) [K62E] rhFVII CBProFpr536: AACGGGGGCTCCTGCGAGGACCAGCTGCAG (SEQ ID NO: 13 - direct primer) CBProFpr537: CTGCAGCTGGTCCTCGCAGGAGCCCCCGTT (SEQ ID NO: 14 - reverse primer) [K62R] rhFVII CBProFpr538: GGGGGCTCCTGCCGCGACCAGCTGCAGAGC (SEQ NO: 15 - direct primer) CBProFpr539: GCTCTGCAGCTGGTCGCGGCAGGAGCCCCC (SEQ ID NO: 16 - reverse primer) [F71E] rhFVII CBProFpr540: GAGCTATATCTGCGAGTGCCTGCCTGCCTT (SEQ ID NO: 17 - direct primer) CBProFpr541: AAGGCAGGCAGGCACTCGCAGATATAGCTC (SEQ DI NO: 18 - reverse primer) [E82O] rhFVII CBProFpr542: GGGGCGCAATTGCCAGACCCATAAGGATGA (SEQ NO: 19 - direct primer) CBProFpr543: TCATCCTTATGGGTCTGGCAATTGCGCCCC (SEQ ID NO: 20 - reverse primer) [L39E] rhFVII LoB069: GAGCGGACCAAAGAGTTTTGGATTAGC (SEQ NO: 21 - direct primer) LoB070: GCTAATCCAAAACTCTTTGGTCCGCTG (SEQ DOD NO: 22 - reverse primer) [L39Q] rhFVII LoB071: GAGCGGACCAAACAGTTTTGGATTAGC (SEQ ID NO: 23 - direct primer) LoB072: GCTAATCCAAAACTGTTTGGTCCGCTC (SEQ ID NO: 24 - reverse primer) [L65Q] rhFVII LoB075: CTGCAAAGACCAGCAGCAGAGCTATATCTGC (SEQ ID NO: 25 - direct primer) LoB076: GCAGATATAGCTCTGCTGCTGGTCTTTGCAG (SEQ ID NO: 26 - reverse primer) [L65S] rhFVII LoB077: CTGCAAAGACCAGTCCCAGAGCTATATCTGC (SEQ ID NO: 27 - direct primer) LoB078: GCAGATATAGCTCTGGGACTGGTCTTTGCAG (SEQ ID NO: 28 - reverse primer)

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twenty

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Four. Five

[F71D] rhFVII

LoB079: CAGAGCTATATCTGCGACTGCCTGCCTGCC (SEQ ID NO: 29 - direct primer)

LoB080: GGCAGGCAGGCAGTCGCAGATATAGCTCTG (SEQ ID NO: 30 - reverse primer)

[F71Y] rhFVII

LoB081: CAGAGCTATATCTGCTACTGCCTGCCTGC (SEQ ID NO: 31 - direct primer)

LoB082: GCAGGCAGGCAGTAGCAGATATAGCTCTG (SEQ ID NO: 32 - reverse primer)

[K62E + L65Q] rhFVII

CBProFpr703: GAACGGGGGCTCCTGCGAGGACCAGCAGCAGAGCTATATCTGC (SEQ NO: 33 - direct primer)

CBProFpr704: GCAGATATAGCTCTGCTGCTGGTCCTCGCAGGAGCCCCCGTTC (SEQ ID NO: 34 - reverse primer)

Example 4

Expression of FVII or FVII variants in CHO K1 cells

The CHO K1 cell line (ATCC No. CCL-61) was seeded at a 50% confluence in T-25 flasks using MEMα, 10% FCS (GibcoBRL Cat. No. 10091), P / S and 5 Pg / ml phylloquinone , and allowed to grow until confluence. The confluent cell monolayer was transfected with 5 µl / g of the relevant plasmid described above using the Lipofectamine 2000 transfection agent (Life Technologies) according to the manufacturer's instructions. Twenty-four hours after transfection, a sample was removed and quantified using, for example, an ELISA that recognizes the EGF1 domain of human factor VII. At this time point, a relevant selection (eg Hygromycin B) can be applied to the cells, in order to generate a combination of stable transfectants. When CHO K1 cells and the Hygromycin B resistance gene are used as a selectable marker in the plasmid, this is usually achieved within a week.

Example 5

Generation of polypeptide variants that express CHO-K1 cells stably

A vial of the transfectant CHO-K1 combination was thawed and cells were seeded in a 175 cm2 tissue culture flask containing 25 ml of MEMα, 10% FCS, phylloquinone (5 µg / ml), 100 U / l of penicillin, 100 µg / l streptomycin and grown for 24 hours. Cells were harvested, diluted and plated in 96-well microtiter plates at a cell density of ½-1 cells / well. After a week of growth, colonies of 20-100 cells were present in the wells and those wells containing only one colony were labeled. After two more weeks, the media in all wells containing only one colony was replaced with 200 µl of fresh medium. After 24 hours, a sample of medium was removed and analyzed by, for example, ELISA. High production clones were selected and used to produce FVII or its variants.

Example 6

Small-scale purification of polypeptide variants and subsequent activation with the prothrombin activator

FVII and its variants were purified and activated as follows. The procedure was carried out at 4 ° C. 100 mM NaCl and 10 mM CaCl2 were added to 1200 ml of harvested culture media followed by a pH adjustment to 7.5 and sterile filtration. An affinity column was prepared by coupling a calcium-dependent monoclonal antibody against the Gla domain to the CNFr activated Sepharose FF using approximately 5.5 mg of antibody coupled per ml of resin. The culture media prepared overnight were applied to 2 ml of monoclonal antibody on a pre-equilibrated affinity column with 10 mM Tris, 100 mM NaCl, 35 mM CaCl2, pH 7.5. The affinity matrix of the monoclonal antibody with bound FVII was subsequently unpacked by transferring the column material from the column to an empty tube.

FVII or its variants were activated to FVIIa by incubation with prothrombin activator from Oxyuranus scutellatus (OSII), further binding to the affinity matrix. FVIIa was recovered by repackaging the column material followed by elution using 10 mM Tris, 25 mM NaCl, 5 mM EDTA pH 8.6.

The eluate from the first chromatographic stage was introduced directly into a second and final chromatographic column, which consisted of a pre-equilibrated POROS HQ50 column with 10 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 8.6. FVIIa was eluted from the POROS HQ50 column using 10 mM Tris, 25 mM NaCl, CaCl2 35

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fifteen

twenty

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30

35

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Four. Five

mM, pH 7.5 after washing the column with 10 mM Tris, 25 mM NaCl, pH 8.6. The eluted FVIIa from the POROS HQ50 column was stored at -80 ° C without further modification.

Example 7

Large-scale purification of polypeptide variants and subsequent activation

FVII and FVII variants were purified as follows. The procedure was carried out at 4 ° C. Culture media harvested from large-scale production were ultrafiltered using a Millipore TFF system with Pellicon membranes with a cut of 30 Kda. After the concentration of the medium, 5 mM citrate was added and the pH was adjusted to 8, 6. If necessary, the conductivity was decreased below 10 mS / cm. Subsequently, the sample was applied to a Q-sepharose FF column, equilibrated with 50 mM NaCl, 10 mM Tris pH 8.6. After washing the column with 100 mM NaCl, 10 mM Tris pH 8.6, followed by 150 mM NaCl, 10 mM Tris pH 8.6, FVII was eluted using 10 mM Tris, 25 mM NaCl, 35 mM CaCl 2, pH 8, 6.

For the second chromatographic stage, an affinity column was prepared by coupling a calcium-dependent monoclonal antibody against the Gla domain to the Sepharose FF activated with CNBr. Approximately 5.5 mg of antibody per ml of resin was coupled. The column was equilibrated with 10 mM Tris, 100 mM NaCl, 35 mM CaCl2, pH 7.5. NaCl was added to the sample at a concentration of 100 mM NaCl and the pH was adjusted to 7.4-7.6. After O / N application of the sample, the column was washed with 100 mM NaCl, 35 mM CaCl2, 10 mM Tris pH 7.5, and the FVII protein was eluted with 100 mM NaCl, 50 mM citrate, 75 mM Tris pH 7.5.

For the third chromatographic stage, the conductivity of the sample was decreased below 10 mS / cm, if necessary, and the pH was adjusted to 8.6. The sample was then applied to a Q-sepharose column (equilibrated with 50 mM NaCl, 10 mM Tris pH 8.6) at a density of about 3-5 mg of protein per ml of gel to obtain effective activation. After application, the column was washed with 50 mM NaCl, 10 mM Tris pH 8.6 for approximately 4 hours with a flow of 3-4 column volumes (vc) per hour. The FVII protein was eluted using a gradient of 0-100% 500 mM NaCl, 10 mM Tris pH 8.6 in 40 vc. Fractions containing FVII were combined.

For the final chromatographic stage, the conductivity was decreased below 10 mS / cm. Subsequently, the sample was applied to a Q-sepharose column (equilibrated with 140 mM NaCl, 10 mM glycylglycine pH 8.6) at a concentration of 3-5 mg of protein per ml of gel. The column was then washed with 140 mM NaCl, 10 mM glycylglycine pH 8.6, and FVII eluted with 140 mM NaCl, 15 mM CaCl 2, 10 mM glycylglycine pH 8.6. The eluate was diluted in 10 mM CaCl2 and the pH was adjusted to 6.8-7.2. Finally, 0.01% Tween 80 was added and the pH was adjusted to 5.5 for storage at -80 ° C.

Example 8

Experimental results

Submission of the variants of the invention (purified as described in Example 6 above) to the "Complete Blood Test" revealed that the variants had a significant increase in coagulation activity (or a reduced coagulation time) in comparison to rhFVIIa. In Figs. 1-3 experimental results are shown.

SEQUENCE LIST

<110> Maxygen ApS; Maxygen Holdings

<120> Variants of FVII or FVIIa that have improved coagulation activity

<130> 0267wo310

<140>

<141>

<160> 34

<170> PatentIn Ver. 2.1

<210> 1

<211> 406

<212> PRT

<213> Homo sapiens

<400> 1

image 1

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (115) .. (1335)

<400> 2

image2

image2

image2

<210> 3

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 3

agctggctag ccactgggca ggtaagtatc a

31

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 4

tggcgggatc cttaagagct gtaattgaac t

31

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 5

tcagctcgag agcggtagcg ccc

2. 3

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: primer

<400> 6

cccattctag aaaagcggaa cgccagcaaa ccccaggg

28

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 7

gctgagcgga ccaaacactt ttggattagc

30

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 8

gctaatccaa aagtgtttgg tccgctcagc

30

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 9

ccaaactgtt ttggcgcagc tatagcgatg

30

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 10

catcgctata gctgcgccaa aacagtttgg

30

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 11

aactgttttg gattcagtat agcgatggcg

30

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 12

cgccatcgct atactgaatc caaaacagtt

30

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 13

aacgggggct cctgcgagga ccagctgcag

30

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 14

ctgcagctgg tcctcgcagg agcccccgtt

30

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 15

gggggctcct gccgcgacca gctgcagagc

30

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 16

gctctgcagc tggtcgcggc aggagccccc

30

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 17

gagctatatc tgcgagtgcc tgcctgcctt

30

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 18

aaggcaggca ggcactcgca gatatagctc

30

<210> 19

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 19

ggggcgcaat tgccagaccc ataaggatga

30

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 20

tcatccttat gggtctggcaa ttgcgcccc

30

<210> 21

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 21

gagcggacca aagagttttgg attagc

26

<210> 22

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 22

gctaatccaa aactctttggt ccgctg

26

<210> 23

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 23

gagcggacca aacagttttgg attagc

26

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 24

gctaatccaa aactgtttggt ccgctc 26

<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 25 ctgcaaagac cagcagcagag ctatatctgc 30

<210> 26

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 26 gcagatatag ctctgctgctg gtctttgcag 30

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 27 ctgcaaagac cagtcccagag ctatatctgc 30

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 28 gcagatatag ctctgggactg gtctttgcag 30

<210> 29

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 29

cagagctata tctgcgactgc ctgcctgcc

29

<210> 30

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 30

ggcaggcagg cagtcgcagat atagctctg

29

<210> 31

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 31

cagagctata tctgctactgc ctgcctgc

28

<210> 32

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 32

gcaggcaggc agtagcagata tagctctg

28

<210> 33

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the artificial sequence: Primer

<400> 33

gaacgggggc tcctgcgagga ccagcagcag agctatatct gc

42

<210> 34

<211> 42

<212> DNA

<213> Artificial Sequence

<220> 5 <223> Description of the artificial sequence: Primer

<400> 34 gcagatatag ctctgctgctg gtcctcgcag gagcccccgt tc 42

Claims (19)

1.- A variant of Factor VII or Factor VIIa polypeptide, said variant having an amino acid sequence that differs from human Factor VII or human Factor VIIa having the amino acid sequence shown in SEQ ID NO: 1 in no more of 15 amino acid residues, wherein said variant sequence comprises the L65Q substitution and has improved coagulation activity or reduced coagulation time compared to hFVIIa or rhFVIIa. 2. The variant according to claim 1, further comprising at least one substitution selected from the group consisting of L39E, L39Q, L39H, 142R, S43H, S43Q, K62E, K62R, L65S, F71 D, F71 Y, F71E. F71 Q, F71 N, E82Q, E82N, E82K and F275H. 3. The variant according to claim 2, wherein said at least one substitution is F71Y, K62E or S43Q. 4. The variant according to any of claims 1-3, wherein said variant comprises at least one amino acid substitution in the Gla domain. 5. The variant according to claim 4, wherein said at least one substitution in the Gla domain comprises a substitution in at least one position selected from the group consisting of P10, K32, D33 and A34 6. The variant according to claim 5, wherein said variant further comprises an insertion of at least one amino acid residue between position A3 and F4. 7. The variant according to any of the preceding claims, wherein at least one amino acid residue comprising a binding group of a non-polypeptide moiety has been introduced at a position located outside the Gla domain. 8. The variant according to claim 7, wherein said binding group is a glycosylation site in vivo. 9. The variant according to claim 8, wherein said in vivo glycosylation site is an N-glycosylation site introduced by a substitution selected from the group consisting of A51 N, G58N, G48N + S60T, T106N, K109N, G124N, K143N + N145T, A175T, 1205S, 1205T, V253N, T267N, T267N + S269T, S314N + K316S, S314N + K316T, R315N + V317S, R315N + V317T, K316N + G3183, N316 10. The variant according to any of the preceding claims, wherein said variant further comprises at least one substitution in the position selected from the group consisting of 157, 158, 296, 298, 305, 334, 336, 337 and 374 . 11. The variant according to any of the preceding claims, wherein said variant further comprises at least one substitution selected from the group consisting of K341 Q, D196K, D196N, G237L and G237GAA. 12. The variant according to any of the preceding claims, wherein said variant is in its activated form. 13.- A nucleotide sequence encoding a variant as defined in any of claims 1-12. 14. An expression vector comprising a nucleotide sequence as defined in claim 13. 15. A host cell comprising a nucleotide sequence as defined in claim 13 or an expression vector as defined in claim 14. 16. The host cell according to claim 15, wherein said host cell is a carboxylic gamma cell capable of glycosylation in vivo. 17. A pharmaceutical composition comprising a variant as defined in any of claims 1-12, and a pharmaceutically acceptable carrier or excipient. 18. A variant as defined in any of claims 1-12, or a pharmaceutical composition as defined in claim 17 for use as a medicament. 19. Use of a variant as defined in any of claims 1-12 for the manufacture of a medicament for the treatment of a disease or a disorder in which clot formation is desirable.