HK1095357B

HK1095357B - Factor vii or viia gla domain variants

Info

Publication number: HK1095357B
Application number: HK07100447.2A
Authority: HK
Inventors: Jesper Mortensen Haaning; Kim Vilbour Andersen; Claus Bornaes
Original assignee: 拜耳医药保健有限公司
Priority date: 2003-06-19
Filing date: 2004-06-18
Publication date: 2012-09-14

Description

GLA domain variants of factor VII or VIIa

Technical Field

The present invention relates to novel Gla domain variants of factor vii (fvii) or factor viia (fviia) polypeptides, and the use of said polypeptide variants in therapy, in particular in the treatment of various coagulation-related diseases.

Background

Coagulation is a process consisting of a complex interaction between various blood components (or factors) that ultimately leads to the formation of a fibrin clot. The blood component that is normally involved in what is known as the coagulation "cascade" is a proenzyme or zymogen, i.e., a protein without enzymatic activity, which is converted to an active form by the action of an activator. FVII is one of these coagulation factors.

FVII is a vitamin K-dependent plasma protein which is synthesized in the liver and secreted into the blood as a single-chain glycoprotein with a molecular weight of 53kDa (size & Majerus, J.biol.chem 1980; 255: 1242-. FVII zymogen is hydrolyzed by proteases at a single site R152-I153, resulting in a two-chain linked by a disulfide bond, which is converted to the active form (FVIIa). FVIIa complexes with tissue factor (FVIIa complexes) convert both factor IX and factor X into their active forms, and subsequent reactions lead to rapid thrombin generation and fibrin formation (Osterid & Rapaport, Proc Natl Acad Sci USA 1977; 74: 5260-.

FVII undergoes post-translational modifications, including vitamin K-dependent carboxylation, resulting in the production of 10 gamma carboxyglutamic acid residues in the N-terminal region of the molecule. Thus, SEQ ID NO: residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 in 1 are the gamma carboxyglutamic acid residues of the Gla domain that are important for FVII activity. Other post-translational modifications include two naturally occurring N-glycosylation sites at positions 145 and 322, and two naturally occurring O-glycosylation sites at positions 52 and 60, respectively, to which sugar components are attached.

The gene encoding human FVII (hFVII) is mapped to chromosome 13 q34-qter9(deGrouchy et al, Hum Genet 1984; 66: 230-. It comprises 9 exons and is 12.8Kb in length (O' Hara et al, Proc Natl Acad Sci USA 1987; 84: 5158-. The gene organization and protein structure of FVII are similar to those of other vitamin K-dependent procoagulant proteins, exons 1a and 1b encoding signal sequences; exon 2 encodes a polypeptide and a Gla domain; exon 3 encodes a short hydrophobic region; exons 4 and 5 encode epidermal growth factor-like domains; and exons 6 to 8 encode the catalytic domain of a serine protease (Yoshitake et al, Biochemistry 1985; 24: 3736-3750).

Methods of X-ray crystallography (Banner et al, Nature, 1996; 380: 41 and Zhang et al, J.Mol.biol, 1999; 285: 2089) have been reported for experimental three-dimensional structures of hFVIIa (Pike et al, PNAS.U.S.A., 1999; 96: 8925-30 and Kemball-Cook et al, J.struct.biol, 1999; 127-.

Relatively few reports have been made on engineered FVII mutants (Dickinson & Ruf, J Biochem, 1997; 272: 19875-19879, Kemball-Cook et al, J Biol Chem, 1998; 273: 8516-8521, Bharadwaj et al, J Biol Chem, 1996; 271: 30685-8591, Ruf et al, biochemsky, 1999; 38: 1957-1966).

FVII expression in BHK or other mammalian cells has been reported (WO92/15686, WO91/11514, WO88/10295), as well as FVII and kex2 endoprotease co-expression in eukaryotic cells (WO 00/28065).

Commercial formulations of human recombinant FVIIaThe name of (a) is sold.Indicated for the treatment of bleeding episodes in hemophilia a or B patients.Is the only effective and reliable rFVIIa available on the market to treat bleeding episodes.

An inactive form of FVII is reported in WO91/1154, wherein arginine at 152 and/or isoleucine at 153 are modified. These amino acids are located at the activation site. WO96/12800 describes the inactivation of FVIIa by a serine protease inhibitor; petersen et al describe the inactivation of FVIIa by carbamoylation of the alpha amino acid group at I153 (Eur J Biochem, 1999; 261: 124-129). This inactive form can compete with wild-type FVII or FVIIa for binding to tissue factor and inhibiting clotting activity. This suggests that this inactivated form of FVIIa may be useful in treating patients who are highly prone to develop blood coagulation, such as patients with sepsis, who are prone to the onset of myocardial infarction or thrombotic stroke.

For the treatment of uncontrolled bleeding, such as trauma, FVIIa is believed to be capable of activating FX to FXa without binding to tissue factor, and the activation response is believed to be found primarily in activated platelets (Hedher et al blood catalysis & Fibrinolysis, 2000; 11; 107-. However, hFVIIa or rhFVIIa have low activity on FX in the absence of tissue factor, and thus treatment of uncontrolled bleeding, for example in trauma patients, requires high and multiple doses of hFVIIa or rhFVIIa, respectively. Thus, to more effectively treat uncontrolled bleeding (minimize blood loss), there is a need for improved FVIIa molecules that have high activity for FX in the absence of tissue factor. The improved FVIIa molecules show lower clotting events (faster acting/increased clotting activity) when used for uncontrolled blood loss compared to rhFVIIa.

Gla domain variants of FVII/FVIIa are disclosed in WO 99/20767, US 6,017,882 and WO 00/66753, wherein some residues located in the Gla domain are identified as important for phospholipid membrane binding and thus for FX activation. In particular, residues 10 and 32 were found to be critical, and increased phospholipid membrane binding affinity, and thus enhanced FX activation, could be achieved by making mutations P10Q and K32E. Specifically, FX activation was found to be enhanced compared to rhFVIIa at marginal coagulation conditions such as in the presence of low levels of tissue factor.

WO01/58935 discloses a new strategy for the development of FVII or FVIIa molecules with increased half-life etc. by site-directed glycosylation or pegylation.

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

WO 2004/029091 discloses FVII or FVIIa variants with specific modifications located at the tissue factor binding site.

The inventors have now identified other residues in the Gla domain that may further increase phospholipid membrane binding affinity and thereby further enhance FX activation. FVII or FVIIa variants of the invention also reduce tissue factor binding affinity.

It is an object of the present invention to provide improved FVII or FVIIa molecules (FVII or FVIIa variants) which are capable of activating FX to FXa more efficiently than hFVIIa, rhFVIIa or [ P10Q + K32E ] rhFVIIa. In particular, it is an object of the present invention to provide improved FVII or FVIIa molecules (FVII or FVIIa variants) which are capable of activating FX to FXa more efficiently than hFVIIa, rhFVIIa or [ P10Q + K32E ] rhFVIIa in the absence of tissue factor. These objects are illustrated by the FVII or FVIIa variants provided by the present invention.

Disclosure of Invention

The present invention relates in a first aspect to factor vii (fvii) or factor viia (fviia) polypeptide variants having an amino acid sequence comprising a polypeptide having an amino acid sequence which is SEQ ID NO: 1 of human factor vii (hfviii) or human factor viia (hfviia), wherein a hydrophobic amino acid residue is introduced by an amino acid substitution in position 34.

A second aspect of the invention relates to factor vii (fvii) or factor viia (fviia) polypeptide variants having an amino acid sequence comprising a polypeptide having an amino acid sequence which is modified with respect to SEQ ID NO: 1, or 1-15 amino acid modifications of human factor vii (hfviii) or human factor viia (hfviia), wherein the amino acid sequence comprises an amino acid substitution in position 36.

A third aspect of the invention relates to factor vii (fvii) or factor viia (fviia) polypeptide variants having an amino acid sequence comprising a polypeptide having an amino acid sequence which is modified with respect to SEQ ID NO: 1 or human factor viia (hfviia), wherein the amino acid sequence comprises amino acid substitutions in positions 10 and 32 and at least one other amino acid substitution in a position selected from positions 74, 77 or 116.

Other aspects of the invention relate to nucleotide sequences encoding the polypeptide variants of the invention, expression vectors comprising said nucleotide sequences, and host cells comprising said nucleotide sequences or expression vectors.

Other aspects of the invention relate to pharmaceutical compositions comprising a polypeptide variant of the invention, the use of a polypeptide variant of the invention or a pharmaceutical composition of the invention as a medicament, and methods of treatment using a polypeptide variant or a pharmaceutical composition of the invention.

Other aspects of the invention will be apparent from the following description and appended claims.

Drawings

FIG. 1 shows clotting time versus variant concentration of the present invention when measured in a "Whole Blood Assay".

FIG. 2 shows the maximum tissue factor-dependent thrombin generation rate of the variants of the invention as determined in the "Thrombogram Assay".

FIG. 3 shows the maximum phospholipid-dependent thrombin generation rate of the variants of the invention as determined in a "thrombogram assay".

Detailed Description

Definition of

Throughout this application and this invention, the following definitions are used:

the term "FVII" or "FVII polypeptide" refers to a FVII molecule provided in a single chain. An example of a FVII polypeptide is wild-type human FVII (hfvii) having the amino acid sequence of SEQ ID NO: 1. however, it is to be understood that the term "FVII polypeptide" also includes hFVII-like molecules, such as SEQ ID NO: 1, in particular a fragment or variant thereof having a sequence identical to SEQ ID NO: 1 compared to a variant comprising at least one, such as up to 15, preferably up to 10 amino acid modifications.

The term "FVIIa" or "FVIIa polypeptide" refers to a FVIIa molecule that is provided in the form of two activated chains. When SEQ ID NO: 1 for the description of the amino acid sequence of FVIIa, it is understood that the peptide bond between R152 and I153 in the single-chain form is cleaved and that one of the chains comprises amino acid residues 1-152 and the other chain comprises amino acid residue 153-406.

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

The terms "hFVII" and "hFVIIa" refer to wild-type human FVII and FVIIa, respectively, having the amino acid sequences of SEQ ID NO: 1.

the terms "rhFVII" and "rhFVIIa" refer to human wild-type FVII and FVIIa which have the amino acid sequences of SEQ ID NO: 1, prepared by recombinant methods. Examples of RhFVIIa are

The term "Gla domain" is intended herein to include SEQ ID NO: 1, 1-45 amino acid residues.

Thus, the term "position outside the Gla domain" includes SEQ ID NO: 1, amino acid residues 46-406.

The abbreviations "FX", "TF" and "TFPI" refer to factor X, tissue factor and tissue factor pathway inhibitors, respectively.

The term "protease domain" is used at about residues 153-406 from the N-terminus.

The term "catalytic site" is used to refer to the catalytic triad of S344, D242 and H193 of the polypeptide variant.

The term "parent" is intended to refer to a molecule that is to be modified/improved according to the invention. Although the parent polypeptide to be modified by the present invention may be any FVII or FVIIa polypeptide, and thus be derived from any source, e.g. non-human mammalian source, it is preferred that the parent polypeptide is hFVII or hFVIIa.

A "variant" is a polypeptide that differs from its parent polypeptide by one or more amino acid residues, typically in 1-15 amino acid residues (e.g., 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, e.g., in 1-8, 1-6, 1-5, or 1-3 amino acid residues. Typically, the parent polypeptide is hFVII or hFVIIa.

The term "conjugate" (or interchangeably "conjugated polypeptide") means a heterologous (in the sense of a complex or chimeric) molecule formed by the covalent attachment of one or more polypeptides to one or more non-polypeptide components, such as polymer molecules, lipophilic compounds, sugar components and organic derivatizing agents. Preferably, the conjugate is soluble at the relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood. Embodiments of the conjugated polypeptides of the invention include glycosylated and/or pegylated polypeptides.

The term "covalently linked" means that the polypeptide and non-polypeptide components are either directly covalently linked to each other or indirectly covalently linked to each other through at least one intervening component, such as a bridge, spacer or linking moiety.

The term "non-polypeptide moiety" as used herein denotes a molecule, which is distinct from and linked together by peptide bonds to a peptide polymer composed of amino acid monomers, said molecule being capable of coupling to a linking group of a polypeptide variant of the invention. Preferred examples of such molecules include polymeric molecules, sugar components, lipophilic compounds or organic derivatizing agents. When used herein in the conjugates of the invention, it is understood that the non-polypeptide moiety is linked to the polypeptide portion of the conjugated variant via a linking group of the polypeptide. As indicated above, the non-polypeptide moiety may be covalently linked, directly or indirectly, to a linking group.

The term "polymer molecule" is defined as a molecule formed by the covalent attachment of two or more monomers, wherein none of the monomers is an amino acid residue, unless the polymer is human albumin or another plasma-rich protein. The term "polymer" is interchangeable with the term "polymer molecule". The term encompasses carbohydrate molecules attached via in vitro glycosylation, i.e., synthetic glycosylation performed in vitro, which generally involves covalent attachment of a carbohydrate molecule to an attachment group of a polypeptide variant, optionally with the use of a cross-linking agent.

The term "carbohydrate moiety" is intended to refer to a carbohydrate-containing molecule that includes one or more monosaccharide residues capable of associating with a polypeptide variant through in vivo glycosylation (to produce a polypeptide variant conjugate in the form of a glycosylated polypeptide variant). The term "in vivo glycosylation" is intended to mean any linkage of sugar moieties that occurs in vivo, i.e., in post-translational processing in the glycosylated cell used to express the polypeptide variant, e.g., by N-linked or O-linked glycosylation. The exact structure of the oligosaccharide structure depends to a large extent on the glycosylation-type organism of interest.

An "N-glycosylation site" has the sequence N-X-S/T/C, wherein X is any amino acid residue except proline, N is asparagine, and S/T/C is serine, threonine or cysteine, preferably serine or threonine, most preferably threonine. Preferably, the amino acid residue at position +3 relative to asparagine is not a proline residue.

An "O-glycosylation site" is an OH-group of a serine or threonine residue.

The term "linking group" denotes a functional group of a polypeptide variant, in particular of an amino acid or carbohydrate moiety thereof, which is capable of coupling to a non-polypeptide component such as a polymer molecule, a sugar component, a lipophilic compound or an organic derivatizing agent. Useful linkers and their compatible non-polypeptide components are shown in the following figures.

Linking group	Amino acids	Examples of non-polypeptide Components	Coupling method/activated PEG	Reference to
					-NH₂	N-terminal, lysine	Polymers, e.g. PEG, having amide or imine groups	mPEG-SPATresylatedmPEG	Shearwater Inc. Delgado et al, clinical reviews in therapeutic Drug carriers 9(3, 4): 249-304(1992)
-COOH	C-terminal aspartic acid, glutamic acid	Polymers, e.g. PEG, carbohydrate components having ester or amide groups	mPEG-Hz in vitro coupling	Shearwater Inc.
					-SH	Cysteine	Polymers, e.g. PEG, carbohydrate components having disulfide, maleimide or vinylsulfone groups	PEG-vinyl sulfone PEG-maleimide in vitro coupling	Shearwater Inc. Delgado et al, clinical reviews in therapeutic Drug carriers 9(3, 4): 249-304(1992)
-OH	Serine, threonine, lysine, OH-	PEG with sugar component having ester, ether, carbamate, carbonate groups	In vivo O-linked glycosylation
					-CONH₂	Asparagine as part of the N-glycosylation site	Sugar component polymers, e.g. PEG	In vivo N-glycosylation
Aromatic residue	Phenylalanine, tyrosine, tryptophan	Carbohydrate component	In vitro coupling
					-CONH₂	Glutamine	Carbohydrate component	In vitro coupling	Yan & Wold，Biochemistry，1984，Jul 31：23(16)：3759-65
Aldehyde ketones	Oxidized oligosaccharides	Polymers, e.g. PEG, PEG-hydrazide	PEGylation	Andresz et al, 1978, makromol. chem.179: 301, WO92/16555, WO00/23114
					Guanidine (guanidine)	Arginine	Carbohydrate component	In vitro coupling	Lundblad &Noyes, Chemical Reagents for protein modification, CRC PressInc., Florida USA
Imidazole ring	Histidine	Carbohydrate component	In vitro coupling	Homoguanidine

For in vivo N-glycosylation, the term "linker" is used in a non-conventional manner to denote the amino acid residues that constitute the N-glycosylation site (sequence N-X-S/T/C, as described above). Although the asparagine residue of an N-glycosylation site is the residue that is attached to the sugar component during glycosylation, such attachment cannot be accomplished unless other amino acid residues are present at the N-glycosylation site.

Thus, when the non-polypeptide moiety is a saccharide moiety and conjugation is achieved by in vivo N-glycosylation, the term "amino acid residue comprising an attachment group for the non-polypeptide moiety" in relation to a change in the amino acid sequence of the polypeptide of interest is to be understood as meaning that one or more amino acid residues constituting the N-glycosylation site are changed in such a way that a functional N-glycosylation site is introduced into the amino acid sequence.

In this application, amino acid nomenclature and atom nomenclature (e.g., CA, CB, CD, CG, SG, NZ, N, O, C, etc.) are used as defined by Protein DataBank (PDB) (www.pdb.org), which is based on IUPAC nomenclature (IUPAC nomenclature and notation of amino acids and peptides (residue nomenclature, atom nomenclature, etc.), eur.j.biochem., 138, 9-37(1984) and its misunderstanding eur.j.biochem., 152, 1 (1985)).

The term "amino acid residue" denotes an amino acid residue comprising any natural or synthetic amino acid residue and is primarily intended to be comprised in the group of 20 naturally occurring amino acids, i.e. selected from the group consisting of: alanine (Ala or a), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

The terms used to identify amino acid positions are exemplified below: g124 indicates that the 124 th amino acid residue in the amino acid sequence shown in SEQ ID NO.1 is occupied by a glycine residue, and G124R indicates that the 124 th glycine residue is substituted by an arginine residue. Alternative substitutions may be represented by "/", e.g. N145S/T represents an amino acid sequence in which asparagine at position 145 is replaced by serine or threonine. Multiple substitutions are indicated by "+", e.g. K143N + X145S/T indicates an amino acid sequence in which the lysine residue at position 143 is substituted with an asparagine residue, and the asparagine residue at position 145 is substituted with a serine residue or a threonine residue. Insertion of an additional amino acid, such as an alanine residue after G124, is denoted as G124 GA. The insertion of two other alanine residues after G124 is denoted as G124AA, etc. In the present invention, the term "inserted position X" or "inserted at position X" denotes an amino acid residue which is inserted between position X and X + 1. Deletions of amino acid residues are indicated by asterisks. For example, the deletion of glycine 124 is denoted as G124^*。

Unless otherwise indicated, the sequence numbers of amino acid residues herein correspond to SEQ ID NO: 1, wild-type FVII/FVIIa amino acid sequence as shown.

The term "different" as used herein in relation to a particular mutation means that additional differences may be permitted in addition to the particular amino acid difference. For example, in addition to the modification made in the Gla domain intended to increase FX activation, the polypeptide may comprise other modifications not necessarily related to this effect.

Thus, in addition to the amino acid changes disclosed herein, it is understood that the amino acid sequence of the amino acid sequence polypeptides of the variants of the invention may contain other changes, i.e., other substitutions, insertions or deletions, as desired. For example, such changes may include truncation of one or more amino acid residues at the N-and/or C-terminus (e.g., 1-10 amino acid residues), or introduction of one or more additional amino acid residues at the N-and/or C-terminus, such as the addition of a methionine residue at the N-terminus or the introduction of a cysteine residue at or near the C-terminus, as well as "conservative amino acid substitutions," i.e., substitutions made between groups of amino acids having similar properties, e.g., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids.

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

1	Alanine (A) Glycine (G) serine (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)

Other examples of other modifications are also disclosed in the section entitled "modifications outside the Gla Domain" and "other modifications outside the Gla Domain".

The term "nucleotide sequence" denotes a contiguous fragment of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combinations thereof.

The term "vector" refers to a plasmid or other nucleotide sequence that is capable of replication or integration into the host cell genome in a host cell and, thus, serves to perform a variety of functions in conjunction with a compatible host cell (vector-host system) to facilitate cloning of the nucleotide sequence, i.e., to prepare a useful amount of the sequence, to direct expression of a gene product encoded by the sequence, and to integrate the nucleotide sequence into the host cell genome. The carrier will contain different components depending on the function it performs.

"cell," "host cell," "cell line," and "cultured cell" are used interchangeably herein and these terms are to be understood to include the progeny of a cell grown or cultured.

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

"operably linked" means that two or more nucleotide sequences are covalently linked, e.g., by enzymatic ligation, in a conformation that is related to one another such that the sequences function normally. Generally, "operably linked" means that the nucleotide sequences being linked are contiguous and, in the case of a secretion leader, contiguous and in reading order. Ligation is accomplished at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers and standard recombinant DNA methods are used.

The term "modification" or "amino acid modification" of the present invention is intended to include substitution of an amino acid side chain, substitution of an amino acid residue, deletion or insertion of an amino acid residue.

The term "introduction" mainly refers to the introduction of an amino acid residue, in particular by substitution of an existing amino acid residue or by insertion of another amino acid residue.

The term "removal" refers to the removal of an amino acid residue, in particular by substituting the amino acid residue with another amino acid residue, or by deleting (not substituting) the amino acid residue to be removed.

The term "activity" according to the present invention is to be understood as an activity associated with an assay in which the activity is actually determined.

Thus, the term "deamidation activity" is used to refer to the activity determined in the "deamidation assay". To exhibit "deamidation activity", the active form of the variant of the invention should have at least 10% of the deamidation activity of rhFVIIa as determined in the "deamidation assay" described herein. In a preferred embodiment of the variant of the invention, the activated form thereof should have at least 20%, such as at least 30%, for example at least 40%, more preferably at least 50%, such as at least 60%, for example at least 70%, more preferably at least 80%, of the deamidation activity of rhFVIIa as determined in the "deamidation assay" described herein. In an interesting embodiment of this variant, in its activated form, said variant has substantially the same deamidation activity as rhFVIIa, such as 75-125% of the deamidation activity of rhFVIIa.

The term "clotting activity" refers to the activity measured in the "whole blood assay" described herein, i.e., the time required to obtain clot formation. Thus, less clotting time corresponds to higher clotting activity.

The term "increased clotting activity" is used to indicate that the clotting time of the polypeptide variant is statistically significantly reduced compared to that of rhFVIIa or [ P10Q + K32E ] rhFVIIa, measured in the "whole blood assay" of the invention, under comparable conditions.

The term "activity" of the invention also refers to the ability of the variant to activate FX to FXa. This activity is also referred to as "FX activation activity" or "FXa generation activity" and can be measured in the "TF-independent factor X activation assay" described herein.

The term "increased FX activation activity" or "increased FXa generating activity" is used to refer to variants of the invention which, in their activated form, have a statistically significant increase in the ability to activate FX to FXa compared to a reference molecule such as rhFVIIa or [ P10Q + K32E ] rhFVIIa. The extent to which a variant of the invention (in its activated form) has an increased FX activation activity may conveniently be determined in a "TF-independent factor X activation assay" as described herein.

The term "immunogenic" in conjunction with a given agent is intended to mean the ability of the agent to induce an immune system response. The immune response may be a cell-or antibody-mediated response (see, e.g., Roitt: antisense immunology (8th Edition, Blackwell), for further definition of immunogenicity). In general, a decrease in antibody reactivity indicates a decrease in immunogenicity. Immunogenicity can be determined by any method known in the art, such as in vivo or in vitro methods.

The term "functional in vivo half-life" is used in its usual sense, i.e. the time during which the polypeptide still has 50% biological activity in the body/target organ, or the time during which the activity of the polypeptide is 50% of the initial activity.

As an alternative to determining functional in vivo half-life, the "serum half-life" may be determined, i.e.the time during which 50% of the polypeptide circulates in the plasma or bloodstream before being cleared. Determination of serum half-life is often simpler than determination of functional in vivo half-life and the magnitude of serum half-life generally gives a good indication of the magnitude of functional in vivo half-life. Other alternative terms for serum half-life include "plasma half-life", "circulating half-life", "serum clearance", "plasma clearance" and "clearance half-life". The polypeptide is cleared by the action of one or more of the reticuloendothelial system (RES), kidney, spleen or liver, by clearance mediated by tissue factor, SEC receptors or other receptors, or by specific or non-specific proteolysis. Clearance is generally dependent on size (relative to the cut-off for glomerular filtration), charge, attached carbohydrate chains, and the presence or absence of cellular receptors for the protein. The retained function is typically selected from procoagulant, proteolytic or receptor binding activity. Functional in vivo half-life and serum half-life may be determined by any suitable method known in the art.

The term "increased" functional in vivo half-life or serum half-life means that the relevant half-life of the polypeptide variant is statistically significantly increased relative to the relevant half-life of a reference molecule, such as rhFVIIa or [ P10Q + K32E ] rhFVIIa, as determined under comparable conditions (typically determined in test animals, such as rats, rabbits, pigs or monkeys).

The term "AUC_iv"or" area under the curve when administered intravenously "is used in its usual meaning, i.e. as the area under activity in a serum-time curve, wherein the polypeptide variant is administered intravenously, in particular in rats. The activity usually measured is the above-mentioned "clotting activity". Once the activity-time point is determined, AUC can be conveniently calculated by a computer program such as GraphPad Prism 3.01_iv。

It will be understood that direct comparison of different molecules (e.g.variants of the invention with reference molecules such as rhFVIIa or [ P10Q + K32E)]AUC of rhFVIIa)_ivValues, the same amount of activity should be given. Thus, AUC_ivValues are generally normalized (i.e. differences in injected dose are corrected) and expressed as AUC of administration_ivDose/dose.

The term "sensitivity to proteolytic degradation" primarily refers to a polypeptide variant that has a reduced susceptibility to proteolysis as compared to hFVIIa, rhFVIIa, or [ P10Q + K32E ] rhFVIIa, as measured under comparable conditions. Preferably, proteolysis is reduced by at least 10%, such as at least 25% (e.g. by 10-25% or 10-50%), such as at least 25% (e.g. by 25-50%, 25-75%, or 25-100%), more preferably by at least 35%, such as at least 50% (e.g. by 50-75% or 50-100%), more preferably by at least 60%, such as at least 75% (e.g. by 75-100%), or even by at least 90%.

The term "renal clearance" is used in its usual sense, i.e. clearance occurring in the kidney, for example, by glomerular filtration, tubular excretion or degradation in tubular cells. Renal clearance depends on the physical properties of the polypeptide, including size (diameter) fluid volume, symmetry, shape/rigidity, and charge. Generally, a molecular weight of approximately 67kDa is considered as the cut-off for renal clearance. Renal clearance can be established by any suitable assay, such as established in vivo assays. Typically, renal clearance is determined by administering a labeled (e.g., radiolabeled or fluorescently labeled) polypeptide to a patient and measuring the activity of the marker in urine collected from the patient. The decrease in renal clearance is determined by comparison under comparable conditions with the corresponding reference polypeptide rhFVIIa or [ P10Q + K32E ] rhFVIIa. Preferably, the polypeptide variant has a renal clearance reduction of at least 50%, preferably at least 75%, most preferably at least 90% relative to rhFVIIa or [ P10Q + K32E ] rhFVIIa.

The terms "tissue factor binding site", "active site region" and "ridge of active site binding cleft" are defined with reference to example 1.

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

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

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

Variants of the invention

Modifications in the Gla domain of a parent polypeptide preferably result in a molecule with increased phospholipid membrane binding affinity, increased ability to activate FX to FXa, and/or increased clotting activity. The variants of the invention may also have reduced tissue factor binding affinity and reduced activity upon binding to tissue factor.

Without being bound by any particular theory, it is presently believed that enhanced phospholipid membrane binding affinity results in higher local concentrations of other coagulation factors, particularly activated polypeptide variants in the vicinity of FX. Thus, the rate of activation of FX to FXa is higher, simply due to the higher molar ratio of activated FVII variant to FX. An increased rate of activation of FX results in a higher amount of active thrombin and thus a higher rate of cross-linking of fibrin.

Therefore, it is believed that medical treatment with the polypeptide variants of the invention may provide more rhFVIIa compounds than are currently available: () Such as lower dosage, increased effectiveness and/or faster action.

Furthermore, it is believed that tissue factor-independent variants, i.e. variants with reduced activity when combined with tissue factor compared to wild-type human factor VIIa, may offer some safety advantages in terms of reducing the risk of unwanted blood clot formation (e.g. thrombosis or thromboembolism), in particular for the treatment of acute uncontrolled bleeding events such as trauma.

Thus, in a highly preferred embodiment of the invention, the polypeptide variant, in its activated form and when compared to the reference molecule rhFVIIa or [ P10Q + K32E ] rhFVIIa, has increased FX activation activity, particularly when measured in a tissue factor-independent assay such as the "TF-independent factor X activation assay" described herein. More specifically, the ratio of the active form of the polypeptide variant's FX activation activity to that of the reference molecule, as determined in the TF-independent factor X activation assay described herein, is preferably at least 1.25. More preferably, the ratio is at least 1.5, such as at least 1.75, for example at least 2, more preferably at least 3, such as at least 4, most preferably at least 5.

When the reference molecule is rhFVIIa, the ratio of the FX activation activity of the active form of the polypeptide variant to the FX activation activity of the reference molecule, as determined in the "TF-independent factor X activation assay" described herein, is preferably at least about 5, usually at least about 10, such as at least about 15 or 20.

In other highly preferred embodiments of the invention, the variants of the invention are conjugated to rhFVIIa or [ P10Q + K32E [ ]]rhFVIIa has increased clotting activity (i.e. reduced clotting time) compared to rhFVIIa. In a preferred embodiment of the invention, the variant is reached when assayed in the "whole blood assay" described hereinTime to clot formation (t)_Variants) And for rhFVIIa (t)_wt) Or [ P10Q + K32E ]]Time to clot formation in rhFVIIa (t)_P10Q+K32E) The ratio of (A) is at most 0.9. More preferably the ratio is at most 0.75, such as 0.7, more preferably the ratio is at most 0.6, most preferably the ratio is at most 0.5.

One or more of the above properties may be achieved by the modifications described herein.

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

As mentioned above, the first aspect of the invention relates to FVII or FVIIa polypeptide variants having an amino acid sequence comprising 1-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO: 1), wherein a hydrophobic amino acid residue is introduced by amino acid substitution in position 34.

The hydrophobic amino acid residue to be introduced into position 34 may be selected from the group consisting of: i, L, M, V, F, Y and W, preferably I, L and V, in particular L.

In a preferred embodiment, the variant further comprises an amino acid substitution in position 10, in particular P10Q, and/or an amino acid substitution in position 32, in particular K32E. In a particularly preferred embodiment of the invention, the variant comprises substitutions in positions 10 and 32, such as P10Q + K32E.

Thus, in an embodiment of interest of the present invention, the variant comprises the substitution P10Q + K32E + a 34L.

In a particular embodiment of interest of the invention, the variant further comprises an insertion of at least one (typically one) amino acid residue between positions 3 and 4. Preferably the inserted amino acid residue is a hydrophobic amino acid residue. Most preferably the insertion is A3 AY. Thus, in a particularly interesting embodiment of the invention, the variant comprises the modifications A3AY + P10Q + K32E + a 34L.

In addition to any of the modifications described above, the variant may comprise further substitutions in position 33. Preferably, the hydrophobic amino acid residue is introduced by substitution in position 33, in particular D33F.

The Gla domain may also comprise modifications in other positions, in particular in positions 8, 11 and 28, such as R28F or R28E. On the other hand, it is understood that the Gla domain should not be modified to such an extent that the membrane binding properties are impaired. Thus, preferably no modification is made in the gamma-carboxylated residues, i.e. preferably in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35. In a similar manner, it is generally preferred not to introduce non-peptide moieties such as sugar moieties and/or PEG groups into the Gla domain. Therefore, it is preferred not to make any modifications in the Gla domain that would create N-glycosylation sites in vivo.

Finally, it will be understood that modifications in the Gla domain described in this section may preferably be combined with one or more modifications in a position outside the Gla domain (see below for sections entitled "modifying outside the Gla domain" and "other modifications outside the Gla domain").

Variants of the invention comprising an amino acid substitution in position 36

As mentioned above, the second aspect of the invention relates to FVII or FVIIa polypeptide variants having an amino acid sequence comprising 1-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO: 1), wherein said amino acid sequence comprises an amino acid substitution in position 36.

Preferably, the amino acid residue to be introduced into position 36 by substitution is a negatively charged amino acid residue, such as R36E or R36D, in particular R36E.

Variants of the invention also comprise a substitution in position 38. Preferably, the negatively charged amino acid residue is introduced into position 38 by substitution, for example K38E or K38D, in particular K38E.

Thus, the variant of interest is a variant comprising the following substitutions: substitution P10Q + K32E + R36E or P10Q + K32E + R36E + K38E.

In a particularly interesting embodiment, the variant further comprises an amino acid substitution in position 34 (i.e.the resulting variant comprises a substitution in the residue 10+32+34+36 or 10+32+34+36+ 38). Preferably, the negatively charged amino acid residue is introduced by a substitution in position 34, such as a34E or a 34D.

Specific examples of preferred variants are those comprising the substitution P10Q + K32E + a34E + R36E or P10Q + K32E + a34D + R36E + K38E.

In an embodiment of interest of the present invention, the variant further comprises at least one (usually one) insertion of an amino acid residue located between positions 3 and 4. Preferably, the inserted amino acid residue is a hydrophobic amino acid residue. Most preferably the insertion is A3 AY.

In addition to any of the modifications described above, the variant also comprises a substitution in position 33. Preferably, the hydrophobic amino acid residue is introduced by a substitution in position 33, in particular D33F.

The Gla domain may also contain modifications in other positions, in particular in positions 8, 11 and 28, such as R28F or R28E. On the other hand, it is understood that the Gla domain should not be modified to such an extent that the membrane binding properties are impaired. Thus, preferably no modification is made in the gamma-carboxylated residues, i.e. preferably in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35. In a similar manner, it is generally preferred not to introduce non-peptide moieties such as sugar moieties and/or PEG groups into the Gla domain. Therefore, it is preferred not to make any modifications in the Gla domain that would create N-glycosylation sites in vivo.

Finally, it will be understood that modifications in the Gla domain described in this section may preferably be combined with one or more modifications in a position outside the Gla domain (see below for sections entitled "modifications outside the Gla domain" and "other modifications outside the Gla domain").

Variants of the invention comprising an amino acid substitution in position 74, 77 or 116

As mentioned above, the third aspect of the present invention relates to FVII or FVIIa polypeptide variants having an amino acid sequence comprising 3-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO: 1), wherein said amino acid sequence comprises amino acid substitutions in positions 10, 32 and at least one amino acid substitution in a position selected from the group consisting of positions 74, 77 and 116.

In a preferred embodiment, the amino acid substitution in position 10 is P10Q and the amino acid substitution in position 32 is K32E.

More preferably, the substitution in position 74, 77 or 116 is selected from: P74S, E77A and E116D.

In an embodiment of interest, the variant further comprises an amino acid substitution in position 34. Preferably, the negatively charged amino acid residue is introduced into position 34, e.g. a34E or a34D, in particular a34E, by substitution.

Thus, specific examples of variants of interest include variants containing modifications A3AY + P10Q + K32E + E116D, A3AY + P10Q + K32E + E77A and P10Q + K32E + a34E + P74S.

The Gla domain may also contain modifications in other positions, in particular in positions 8, 11 and 28, such as R28F or R28E. On the other hand, it is understood that the Gla domain should not be modified to such an extent that the membrane binding properties are impaired, i.e. preferably not modified in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35. Preferably no in vivo N-glycosylation sites are generated in the Gla domain.

Modification outside the Gla Domain

2.3 hours of circulating rhFVIIa half-life in "" summer Basis for approximate for", FDA reference number 96-0597. Relatively high dosages and frequent administration are necessary to achieve and maintain the desired therapeutic or prophylactic effect. As a result, proper dose modulation is difficult to achieve and the need for frequent intravenous administration places restrictions on the lifestyle of the patient.

Having a longer circulating half-life and/or increased bioavailability (such as increased area under the curve when administered intravenously compared to rhFVIIa) will reduce the number of administrations necessary. Due to the current need for frequent injections and the possibility to obtain better therapeutic FVIIa levels while increasing the therapeutic effect, there is a clear need for improved FVII-or FVIIa-like molecules.

It is therefore another object of the present invention to provide improved FVII or FVII molecules (FVII or FVIIa variants) which have increased bioavailability (such as an increased area under the curve when administered intravenously compared to a reference molecule such as rhFVIIa or [ P10Q + K32E ] rhFVIIa) and which are more effective in activating factor X to factor Xa (not binding tissue factor) than a reference molecule such as rhFVIIa or [ P10Q + K32E ] rhFVIIa (thereby being more effective in treating uncontrolled bleeding, such as trauma, or chronic diseases such as hemophilia).

Thus, a variant of the invention of interest is one that, in its activated form, is compared to a reference molecule such asrhFVIIa or [ P10Q + K32E]rhFVIIa gives an increased area under the curve (AUC) when administered intravenously, compared to rhFVIIa_iv). This can be conveniently determined by intravenous administration in rats. More specifically, the variant of interest of the invention is the AUC of said variant in the activated form_ivWith reference molecules, such as rhFVIIa or [ P10Q + K32E]AUC of rhFVIIa_ivIs at least 1.25, such as at least 1.5, e.g. at least 1.75, more preferably at least 2, such as at least 3, more preferably at least 4, such as at least 5, particularly when administered (intravenously) in rats.

This effect generally corresponds to an increased functional in vivo half-life and/or an increased serum half-life compared to a reference molecule, such as rhFVIIa or [ P10Q + K32E ] rhFVIIa. Thus, in other interesting embodiments of the invention, the ratio of the functional in vivo half-life or serum half-life of the variant in the activated form to the functional in vivo half-life or serum half-life of a reference molecule, such as rhFVIIa or [ P10Q + K32E ] rhFVIIa is at least 1.25 more preferably the ratio of the relevant half-life of the variant in the activated form to the relevant half-life of a reference molecule, such as rhFVIIa or [ P10Q + K32E ] rhFVIIa is at least 1.5, such as at least 1.75, e.g. at least 2, more preferably at least 3, such as at least 4, e.g. 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 coupling the protein to a chemical moiety, such as polyethylene glycol (PEG), capable of reducing renal clearance of the protein.

In addition, attachment of a chemical moiety to a protein or substitution of an amino acid exposed to proteolysis can effectively block contact with proteolytic enzymes that would otherwise cause proteolysis of the protein.

As mentioned above, instability due to proteolysis is a known problem for current rhFVIIa treatments. Proteolysis is therefore a major obstacle to obtaining preparations in solution as opposed to lyophilized products. The advantage of obtaining a stable soluble preparation is that it is easier for the patient to use and, in emergency situations, works quickly, which is likely to save lives. Efforts to prevent proteolysis by site-directed mutagenesis at major proteolytic sites have been disclosed in WO 88/10295.

WO01/58935 discloses that various causes of AUC_ivA modification of functional in vivo half-life and/or increased serum half-life. The variants disclosed in WO01/58935 are the result of new strategies commonly used for developing improved FVII or FVIIa molecules, which strategies can also be used for the parent FVII or FVIIa polypeptides of the invention.

More specifically, by removing and/or introducing amino acid residues comprising an attachment group for a non-polypeptide moiety in a FVII or FVIIa polypeptide, the polypeptide can be specifically engineered such that the molecule is more readily coupled to the selected non-polypeptide moiety, thereby optimizing the coupling pattern (e.g. ensuring optimal distribution and number of non-polypeptide moieties on the surface of the FVII or FVIIa polypeptide variant and ensuring that only the attachment group intended to be coupled is present in the molecule), and thereby obtaining novel coupled molecules having deamidation activity and one or more improved properties compared to rhFVIIa.

In an embodiment of interest of the invention, more than one amino acid residue located outside the Gla domain is altered, e.g.the alteration comprises removal and introduction of an amino acid residue comprising a linking group for the selected non-polypeptide moiety. In addition to the removal and/or introduction of amino acid residues, the polypeptide variants may comprise other substitutions unrelated to the introduction and/or removal of amino acid residues comprising a linking group for a non-polypeptide moiety.

The polypeptide variants may also be attached to a serine protease inhibitor to inhibit the catalytic site of the polypeptide variant. Optionally, one or more of the amino acid residues present in the catalytic site (S344, D242 and H193) may be mutated to render the resulting variant inactive. One example of such a mutation is S344A.

Amino acid residues comprising a linking group for the non-polypeptide moiety, whether removed or introduced, are selected based on the nature of the non-polypeptide moiety selected, and in most cases, based on the method by which coupling between the polypeptide variant and the non-polypeptide moiety will be achieved. For example, when the non-polypeptide moiety is a polymer molecule such as a polyethylene glycol or a polyolefin oxide derived molecule, the amino acid residue comprising the linking group is 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, particularly cysteine.

As long as the attachment group of the non-polypeptide moiety is introduced into or removed from the parent polypeptide, the amino acid residue to be modified is preferably located at the surface of the parent FVII or FVIIa polypeptide, more preferably occupied by an amino acid residue having a side chain at least 25% 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 herein). The position has been identified based on analysis of the 3D structure of hFVII or hFVIIa molecules, as described in WO 01/58935.

Furthermore, the site to be modified is preferably selected from a part of the FVII or FVIIa molecule which is located outside the tissue factor binding site, and/or outside the region of the active site, and/or outside the ridge of the active site binding cleft. These sites/regions are identified in example 1 herein as well as in WO 01/58935.

In the case of removal of the attachment group, the relevant amino acid residue comprising such a group and occupying a position as defined above is preferably substituted by a different amino acid residue which does not comprise an attachment group for the non-polypeptide moiety of interest. Typically, the amino acid residue to be removed is a residue for which conjugation is unfavorable, e.g. an amino acid residue located at or near the functional site of the polypeptide (since conjugation at that position results in inactivation or reduced activity of the conjugate due to e.g. impaired receptor recognition). In the context of the present invention, the term "functional site" is intended to mean one or more amino acid residues which are essential for or involved in the function or action of FVII or FVIIa. The amino acid residues are part of a functional site. The functional site is determined by methods known in the art and is preferably identified by analyzing the structure of the FVIIa-tissue factor complex (see Banner et al, Nature 1996; 380: 41-46).

In the case of introducing a linking group, the amino acid residue comprising the group is introduced into the relevant position, preferably by substituting the amino acid residue occupying that position.

The exact number of linking groups present in FVII or FVIIa polypeptides and available for coupling depends on the effect to be achieved by coupling. The effect to be obtained depends, for example, on the nature and extent of conjugation (e.g., the nature of the non-polypeptide moiety, the number of non-polypeptide moieties that need or are likely to be conjugated to the polypeptide variant, where conjugation should be conjugated or should be avoided, etc.).

The total number of amino acid residues to be modified (compared to the amino acid sequence SEQ ID NO: 1) located outside the Gla domain in the parent FVII or FVIIa polypeptide is usually not more than 10. Preferably, the FVII or FVIIa variant comprises an amino acid sequence which is identical to the amino acid sequence of SEQ ID NO: 1, amino acid residues s 46-406 differ in 1-10 amino acid residues, typically in 1-8 or 2-8 amino acid residues, e.g. 1-5 or 2-5 amino acid residues, such as 1-4 or 1-3 amino acid residues, e.g. in comparison to SEQ ID NO: 1 differ by 1, 2 or 3 amino acid residues from amino acid residues 46 to 406.

Similarly, a polypeptide variant of the invention may contain 1-10 (further) non-polypeptide moieties, typically 1-8 or 2-8 (further) non-polypeptide moieties, preferably 1-5 or 2-5 (further) non-polypeptide moieties, such as 1-4 or 1-3 (further) non-polypeptide moieties, e.g. 1, 2 or 3 (further) non-polypeptide moieties. It is understood that the further non-polypeptide moiety is covalently linked to a linking group located outside the Gla domain.

Polypeptide variants of the invention in which the non-polypeptide moiety is a sugar moiety

In a preferred embodiment of the invention, the attachment site of the sugar moiety, such as a glycosylation site, in particular an in vivo glycosylation site, such as an in vivo N-glycosylation site, has been introduced into and/or removed from, preferably introduced into, a position outside the Gla domain.

The term "naturally occurring glycosylation site" according to the present invention includes glycosylation sites at positions N145, N322, S52 and S60. The term "naturally occurring in vivo O-glycosylation site" includes positions S52 and S60, and the term "naturally occurring in vivo 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 introduced linking group is a glycosylation site, preferably an in vivo glycosylation site, such as an in vivo O-glycosylation site or an in vivo N-glycosylation site, in particular an in vivo N-glycosylation site. Typically, the introduced 1-10 glycosylation sites, in particular in vivo N-glycosylation sites, preferably 1-8, 1-6, 1-4 or 1-3 glycosylation sites, in particular in vivo N-glycosylation sites, are introduced at one or more positions outside the Gla domain. For example, 1, 2 or 3 glycosylation sites, in particular in vivo N-glycosylation sites, can be introduced outside the Gla domain, preferably by substitution.

It is understood that polypeptide variants comprising one or more glycosylation sites must be produced which are expressed in a host cell capable of attaching a sugar (oligosaccharide) moiety at the glycosylation site or alternatively in vitro glycosylation. An example of a glycosylated host cell is found below in the section entitled "conjugation to a sugar moiety".

Examples of positions at which glycosylation sites, particularly N-glycosylation sites in vivo, have been introduced include amino acid residues having side chains with at least 25% exposed to the surface (as described in example 1 herein), such as at least 50% exposed to the surface. The location is preferably selected from a portion of the molecule that is outside the tissue factor binding site, and/or the active site region, and/or outside the ridge of the active site binding cleft. It is to be understood that the term "at least 25% (or at least 50%) of its side chains are exposed to the surface" when used in the context of in vivo introduction of an N-glycosylation site refers to the surface accessibility of the amino acid side chains in the position of the sugar moiety to which they are actually attached. In many cases, it is desirable to introduce a serine or threonine residue at position +2 relative to the arginine residue to which the sugar moiety is actually attached, and these positions at which the serine or threonine residue is introduced are allowed to be buried, i.e., less than 25% of its side chain is exposed to the surface.

Specific preferred examples of such substitutions which result in vivo N-glycosylation sites include substitutions selected from the group consisting of: a51N, G58N, T106N, K109N, G124N, K143N + N145T, a175T, I205S, I205T, V253N, T267N, T267N + S269T, S314N + K316S, S314S + K316S, R315S + V317S, K316S + G318S, D334S, and combinations thereof. More preferably, the in vivo N-glycosylation site is introduced by a substitution selected from the group consisting of: a51N, G58N, T106N, K109N, G124N, K143N + N145T, a175T, I205T, V253N, T267N + S269T, S314N + K316T, R315N + V317T, K316N + G318T, G318N, D334N and combinations thereof. More preferably, the in vivo N-glycosylation site is introduced by a substitution selected from the group consisting of: T106N, a175T, I205T, V253N, T267N + S269T and combinations thereof, specifically one, two or three of T106N, I205T and V253N.

In one embodiment, only one in vivo N-glycosylation site is introduced by substitution. In other embodiments, two or more (such as two) in vivo N-glycosylation sites are introduced by substitution. Examples of preferred substitutions that result in two in vivo N-glycosylation sites include substitutions selected from the group consisting of: a51 + G58, A51 + T106, A51 + K109, A51 + G124, A51 + K143 + N145, A51 + A175, A51 + I205, A51 + V253, A51 + T267 + S269, A51 + S314 + K316, A51 + R315 + V317, A51 + K316 + G318, A51 + G318, A51 + D334, G58 + T106, G58 + K109, G58 + G124, G58 + K143 + N145, G58 + A175, G58 + I205, G58 + V253, G58 + T267 + S269, G58 + S314 + K316, G58 + R315 + V317, G58 + K316 + G318, G58 + G318, G58 + D334, T106 + G124, T106 + K143 + K316, T106 + K175 + K124, T106 + K175 + T124, G109 + K175, G124, G109 + K175, G124, G109 + K175 + K124, G103, K124, K175 + K124, K175 + K124, K175 + K124, G103, K124, K175 + K175, K316, K175 + K318, K124, K175 + K124, K316, K175 +, g124 + T267 + S269, G124 + S314 + K316, G124 + R315 + V317, G124 + K316 + G318, G124 + G318, G124 + D334, K143 + N145 + A175, K143 + N145 + I205, K143 + N145 + V253, K143 + N145 + T267 + S269, K143 + N145 + S314 + K316, K143 + N145 + R315 + V317, K143 + N145 + K316 + G318, K143 + N145 + G318, K143 + N145 + D334, A175 + I205, A175 + V253, A175 + T267 + S269, A175 + S314 + K316, A175 + R315 + V317, A175 + K316 + G318, A175 + G318, A175 + D334, I205 + V253, I205 + T205 + S316, I175 + S205 + K316, I + R315 + I205 + I316 + V269, I205 + I316 + I205 + V269, I205 + I316 + I318, I316 + I318, I316,

v253 + T267 + S269, V253 + S314 + K316, V253 + R315 + V317, V253 + K316 + G318, V253 + G318, V253 + D334, T267 + S269 + S314 + K316, T267 + S269 + R315 + V317, T267 + S269 + K316 + G318, T267 + S269 + G318, T267 + S269 + D334, S314 + K316 + R315 + V317, S314 + K316 + G318, S314 + K316 + D334, R315 + V317 + K316 + G318, R315 + V317 + G318, R334 + V317 + D and G318 + D334. More preferably, the substitution is selected from: T106N + a175T, T106N + I205T, T106N + V253N, T106N + T267N + S269T, a175T + I205T, a175T + V253N, a175T + T267N + S269T, I205T + V253N, I205T + T267N + S269T and V253N + T267N + S269T, more preferably selected from the group consisting of T106N + I205T, T106N + V253N and I205T + V253N.

In another embodiment, three or more (such as three) in vivo N-glycosylation sites are introduced by substitution. Examples of preferred substitutions that result in three in vivo N-glycosylation sites are selected from: I205T + V253N + T267N + S269T and T106N + I205T + V253N.

As noted above, it is preferred that the in vivo N-glycosylation site be introduced at a location that does not form part of the tissue binding site, active site region, or ridge of the active site binding cleft as described herein.

It will be appreciated that any of the modifications described in the above moieties may be combined with each other and also with the above mentioned substitutions in positions 34 and/or 36, in particular A34E/L and/or R36E, preferably with the above mentioned substitutions in positions 10 and/or 32, in particular P10Q and/or K32E. Among the modifications for introducing N-glycosylation sites in vivo, preferred modifications include one, two or three of T106N, I205T and V253N, in particular two of these modifications, i.e. T106N + I205T, and T106N + V253N or I205T + V253N.

Thus, in a preferred embodiment of the invention, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34E + R36E + T106N + I205T.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34E + R36E + T106N + V253N.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34E + R36E + I205T + V253N.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34L + T106N + I205T.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34L + T106N + V253N.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34L + I205T + V253N.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34L + R36E + T106N + I205T.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34L + R36E + T106N + V253N.

In another preferred embodiment, the FVII or FVIIa variant comprises the modification P10Q + K32E + a34L + R36E + I205T + V253N.

As mentioned above, any one or more of these modifications may also be combined with the insertion of at least one, typically a single, amino acid residue between positions 3 and 4, wherein the inserted residue is preferably a hydrophobic amino acid residue. Most preferably the insertion is A3 AY. Thus, in another preferred embodiment of the invention, the FVII or FVIIa variant comprises a modification selected from the group consisting of:

A3AY+P10Q+K32E+A34E+R36E+T106N+I205T；

A3AY+P10Q+K32E+A34E+R36E+T106N+V253N；

A3AY+P10Q+K32E+A34E+R36E+I205T+V253N；

A3AY+P10Q+K32E+A34L+T106N+I205T；

A3AY+P10Q+K32E+A34L+T106N+V253N；

A3AY+P10Q+K32E+A34L+I205T+V253N；

A3AY+P10Q+K32E+A34L+R36E+T106N+I205T；

A3AY+P10Q+K32E+A34L+R36E+T106N+V253N；

A3AY+P10Q+K32E+A34L+R36E+I205T+V253N。

other modifications outside the Gla Domain

In another embodiment of the invention, the FVII or FVIIa variant may contain, in addition to the modifications described in the above section, mutations known to increase the intrinsic activity of the polypeptide, such as those described in WO 02/22776.

For example, the variant may comprise at least one modification in a position selected from: 157, 158, 296, 298, 305, 334, 336, 337 or 374. Examples of preferred substitutions include substitutions selected from the group consisting of: V158D, E296D, M298Q, L305V or K337A. More preferably, the substitution is selected from: V158D + E296D + M298Q + L305V + K337A,

V158D + E296D + M298Q + K337A, V158D + E296D + M298Q + L305V, V158D + E296D + M298Q, M298Q, L305V + K337A, L305V or K337A.

In another embodiment of the invention, the FVII or FVIIa variant may contain, in addition to the modifications described in the sections above herein, other mutations, such as Neuenschwander et al, Biochemistry, 1995; 34: 8701-8707 for the substitution K341Q. Other possible additional substitutions include D196K, D196N, G237L, G237GAA, and combinations thereof.

Further details on the conjugation of FVII and FVIIa variants to non-polypeptide moieties are given in WO01/58935 and WO 03/093465, which are references and are hereby included by reference.

Method for producing coupled variants of the invention

In general, the coupled variants of the invention can be prepared by: cultivating a suitable host cell under conditions that induce expression of the variant polypeptide, and recovering the variant polypeptide, wherein a) the variant polypeptide comprises at least one N-or O-glycosylation site, the host cell is a eukaryotic host cell capable of glycosylation in vivo, and/or b) the variant polypeptide is coupled to a non-polypeptide moiety in vitro.

Coupling to polymer molecules

The polymer molecule to which the polypeptide is coupled may be any suitable polymer molecule, such as a natural or synthetic homopolymer or heteropolymer, typically having a molecular weight in the range of about 300-100000Da, such as about 500-20000Da, more preferably about 500-15000Da, even more preferably in the range of about 2-15kDa, such as about 3-10 kDa. When the term "about" is used herein in reference to a molecular weight, the term "about" means about the average molecular weight and reflects the fact that there is generally a molecular weight distribution in a given polymer article.

Examples of homopolymers include polyols (i.e., poly-OH), polyamines (i.e., poly-NH 2), and polycarboxylic acids (i.e., poly-COOH). Heteropolymers are polymers that contain different coupling groups, such as hydroxyl groups and amino groups.

Examples of suitable polymer molecules include polymer molecules selected from the group consisting of: polyalkylene oxides (PAOs), including polyalkylene glycols (PAGs), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEG, polyvinyl alcohol (PVA), polycarbonate, polyvinylpyrrolidone, polyvinyl co-maleic anhydride, polystyrene co-maleic anhydride, dextran, including carboxymethyl dextran, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vivo half-life and/or serum half-life. Another example of a polymer molecule is human albumin or another abundant (abundant) plasma protein. In general, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have various water-soluble properties, and are readily excreted from living organisms.

PEG is a preferred polymer molecule because it has only a very small number of reactive groups capable of cross-linking compared to, for example, polysaccharides such as dextran. In particular, monofunctional PEGs such as monomethoxypolyethylene glycol (mPEG) are of interest because of their relatively simple chemistry of coupling (only one reactive group is available for coupling to a linking group on a polypeptide). Thus, the risk of cross-linking is eliminated, the resulting conjugated variant is more homogeneous, and the reaction of the polymer molecule with the variant polypeptide is more easily controlled.

In order for a polymer molecule to covalently bind to a variant polypeptide, the hydroxyl end group of the polymer molecule must be in an activated form, i.e., have a reactive functional group (examples of which include primary amine groups, Hydrazide (HZ), sulfhydryl groups, Succinate (SUC), Succinimidyl Succinate (SS), Succinimidyl Succinamide (SSA), Succinimidyl Propionate (SPA), Succinimidyl Butyrate (SBA), Succinimidyl Carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehydes, nitrophenyl carbonate (NPC), and tresylate (tres)). Suitably activated Polymer molecules are commercially available, for example from Shearwater Polymer, inc., Huntsville, AL, USA or from polymac Pharmaceuticals plc, UK.

Specific examples of activated linear and branched polymer molecules useful in the present invention are described in Nektar molecular Engineering Catalog 2003(Nektar Therapeutics), which is incorporated herein by reference.

Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG and MAL-PEG, and branched PEGs such as PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat. No. 5,643,575, both references being incorporated herein by reference. Other publications disclosing useful polymer molecules, PEGylation chemistry, and coupling methods are listed in WO01/58935 and WO 03/093465.

Specific examples of activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinyl sulfone-PEG (VS-PEG), preferably vinyl sulfone-mPEG (VS-mPEG); Maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, the PEG or mPEG polymer is about 5kDa in size, about 10kD, about 12kDa, or about 20kDa in size.

One skilled in the art will know to use activation methods and/or coupling chemistry based on the linker group of the variant polypeptide (examples have been given above) and the functional group of the polymer (e.g., is amino, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimide, maleimide, vinyl sulfone (vinysulfone) or haloacetate). Pegylation may be coupled to all available groups on the variant polypeptide (i.e., attachment groups exposed on the surface of the polypeptide), or may be coupled to one or more specific attachment groups, e.g., an N-terminal amino group (as described in US 5985265) or to a cysteine residue. Furthermore, the coupling may be accomplished in one step or in a multi-step manner (e.g., as described in WO 99/55377).

For PEGylation of cysteine residues (see above), FVII or FVIIa variants are usually treated with a reducing agent such as dithiothreitol prior to PEGylation. The reducing agent is then removed by conventional methods, such as by desalting. Coupling of PEG to cysteine residues is typically carried out in a suitable buffer at pH6-9 at 4 ℃ -25 ℃ for a period of up to 16 hours.

It will be appreciated that pegylation is designed such that an optimal molecule is produced with respect to the number of PEG molecules attached, the size and form of the molecules (e.g. whether they are linear or branched), and the site of attachment in the variant polypeptide. The molecular weight of the polymer to be used can be selected according to the desired effect to be achieved.

For coupling to only a single attachment group on the protein (e.g. the N-terminal amino group), it is preferred that the polymer molecule, which may be linear or branched, has a high molecular weight, preferably about 10-25kDa, such as about 15-25kDa, e.g. about 20 kDa.

Typically, polymer coupling is carried out under conditions aimed at reacting as many available polymer linking groups as possible with the polymer molecule. This is achieved by providing a suitable excess of moles of the polymer relative to moles of polypeptide. Typically, the molar ratio of activated polymer molecule to polypeptide is up to about 1000-1, such as up to about 200-1, or up to about 100-1. In some cases, however, the ratio may be lower, such as up to about 50-1, 10-1, 5-1, 2-1, or 1-1, to obtain an optimal reaction.

It also relates to coupling of polymer molecules to polypeptides via linkers according to the invention. Suitable linkers are known to those skilled in the art; see also WO 01/58935.

After coupling, the residual activated polymer molecules are blocked according to methods known in the art, for example by adding a primary amine to the reaction mixture, and the resulting deactivated polymer molecules are removed by suitable methods.

It will be appreciated that depending on the circumstances, e.g., the amino acid sequence of the variant polypeptide, the nature of the activated PEG compound used and the particular pegylation conditions, including the PEG to polypeptide molar ratio, different degrees of pegylation may be obtained, with higher degrees of pegylation generally being obtained with higher PEG to variant polypeptide ratios. However, pegylated variant polypeptides resulting from any given pegylation process will generally comprise a random distribution of conjugated polypeptide variants that are pegylated to a slightly different degree.

Coupling with sugar Components

To achieve in vivo glycosylation of FVII molecules comprising one or more glycosylation sites, the nucleotide sequence encoding the variant polypeptide must be inserted into a glycosylation-type eukaryotic expression host. The expression host cell may be selected from a fungal (filamentous fungal or yeast), insect, or animal cell, or from a transgenic plant cell. In one embodiment, the host cell is a mammalian cell, such as a CHO cell, BHK cell or HEK cell, e.g., HEK293 cell, or an insect cell, e.g., SF9 cell, or a yeast cell, such as Saccharomyces Cerevisiae (Saccharomyces Cerevisiae) or Pichia pastoris (Pichia pastoris) or any host cell described further below.

In vitro covalent coupling of a sugar moiety, such as dextran, to amino acid residues of the variant polypeptide may be performed as described, for example, in WO 87/05330 and Aplin et al, CRC Crit rev. biochem, pp.259-306, 1981. See also WO 03/093465 for further information on the in vitro glycosylation of FVII or variants of FVIIa.

Ligation of serine protease inhibitors

The attachment of the serpin can be carried out according to the method described in WO 96/12800.

Methods of making polypeptide variants of the invention

Polypeptide variants of the invention, optionally in glycosylated form, may be prepared by any suitable method known in the art. The method comprises constructing a nucleotide sequence encoding the variant polypeptide and expressing the sequence in a suitable transformed or transfected host. Preferably, the host cell is a gamma-carboxylated host cell such as a mammalian cell. However, despite the low efficiency, the variants of the invention may be prepared by chemical synthesis or a combination of chemical synthesis and recombinant DNA techniques.

The nucleotide sequence encoding the polypeptide of the present invention can be constructed by the following method: isolating and synthesizing a polypeptide encoding a parent FVII, such as a polypeptide having the amino acid sequence of SEQ ID NO: 1 and then altering said nucleotide sequence such that introduction (i.e. insertion or substitution) or removal (i.e. deletion or substitution) of the relevant amino acid residue is effected.

The nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with conventional procedures. The nucleotide sequence may alternatively be prepared by chemical synthesis, e.g., by using oligonucleotide synthesis, wherein the oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are advantageous in a host cell producing the recombinant polypeptide. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and assembled by PCR (polymerase chain reaction), ligation or Ligation Chain Reaction (LCR) (Barany, Proc Natl Acad Sci USA 88: 189;. 193, 1991). Individual oligonucleotides typically contain 5 'and 3' overhangs for complementary sets.

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

One skilled in the art will be able to select the appropriate vector, expression control sequence, and host for expression of the polypeptide. The recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated, when introduced into the host cell.

The vector is preferably an expression vector in which the nucleotide sequence encoding the polypeptide variant of the invention is operably linked to additional segments required for transcription of the nucleotide sequence. The vector is typically derived from plasmid or viral DNA. Many expression vectors are commercially available or described in the literature for expression in the host cells described herein. For details of suitable vectors for expressing FVII see WO01/58935, incorporated herein by reference.

The term "control sequences" as used herein includes all components which are necessary or advantageous for the expression of the polypeptides of the invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide variant. These control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activation sequence, signal peptide sequence, and transcription terminator. The control sequences include at least a promoter.

A wide variety of expression control sequences may be used in the present invention, for example any of the control sequences disclosed in WO01/58935, which is incorporated herein by reference.

The nucleotide sequence of the present invention encoding a polypeptide variant, whether prepared by site-directed mutagenesis, synthesis, PCR, or other methods, optionally includes a nucleotide sequence encoding a signal peptide. A signal peptide is required if the polypeptide variant requires secretion from the cell in which it is expressed. Such a signal peptide, if present, should be recognized by a cell selected for expression of the polypeptide. The signal peptide may be homologous (e.g. typically linked to hFVII) or heterologous (e.g. originating from a source other than hFVII) to the polypeptide, or may be homologous or heterologous to the host cell, i.e. typically the signal peptide is a signal peptide expressed from the host cell or typically it is not a signal peptide expressed from the host cell.

Any suitable host may be used to produce polypeptide variants of the invention, including bacteria (but not particularly preferred), fungi (including yeast), plants, insects, mammals or other suitable animal cells or cell lines, and transgenic animals or plants. Examples of bacterial host cells include gram-positive bacteria such as strains of the genus bacillus, e.g. brevibacillus or bacillus subtilis, pseudomonas or streptomyces, or gram-negative bacteria such as e. Examples of suitable filamentous fungal host cells include Aspergillus strains, such as Aspergillus oryzae, Aspergillus niger or Aspergillus nidulans, Fusarium (Fusarium) or Trichoderma. Examples of suitable yeast host cells include strains of Saccharomyces, such as Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, such as Pichia pastoris or P.Methanolica, Hansenula, such as Hansenula polymorpha or Yarrowia. Examples of suitable insect host cells include lepidopteran cell lines, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusia ni cells (High Five) (US 5077214). Examples of suitable mammalian host cells include Chinese Hamster Ovary (CHO) cell lines (e.g., CHO-K1; ATCC CCL-61), green monkey cell lines (COS) (e.g., COS1(ATCC CRL-1650), COS7(ATCC CRL-1651)); mouse cells (e.g., NS/O), hamster kidney (BHK) cell lines (e.g., ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g., HEK293(ATCC CRL-1573)). Additional suitable cell lines are known in the art and are available from public collections such as the American type culture Collection, Rockville, Maryland. Furthermore, mammalian cells (such as CHO cells) may be modified to express sialyltransferases, e.g., 1, 6-sialyltransferase, as described in US5047335, in order to improve glycosylation of the variant polypeptide.

To increase secretion, it may be advantageous to prepare the polypeptides of the invention together with an endoprotease, in particular PACE (paired basic amino acid convertase), as described in US5986079, such as a Kex2 endoprotease as described in WO 00/28065.

Methods for introducing foreign DNA into the above cell types and additional information on expression, preparation and purification of FVII variants are described in WO01/58935, incorporated herein by reference.

The invention relates to a pharmaceutical composition and application thereof

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

The polypeptide variants or pharmaceutical compositions of the invention are useful as medicaments.

For the above improved properties, the polypeptide variant of the invention, or the pharmaceutical composition of the invention, is particularly useful for the treatment of uncontrolled bleeding events in trauma patients, thrombocytopenic patients, patients with anticoagulation therapy, and patients with cirrhosis of the liver, who suffer from bleeding due to varicose veins or other upper gastrointestinal bleeding, who receive orthotopic liver transplantation or hepatectomy (allowing for transfusions) or in hemophiliacs.

Trauma is defined as the damage of living tissue caused by an external factor. It is the fourth leading cause of death in the united states and places a large financial burden on the economy.

Trauma is classified as blunt or penetrating. Blunt trauma results in internal compression, organ damage and internal bleeding, while penetrating trauma (as a result of factors that penetrate the body and damage tissues, vessels and organs) results in external bleeding.

Trauma can result from a variety of events, such as traffic accidents, gun injuries, falls, machine accidents, and punctures.

Cirrhosis can be caused by direct liver damage, including chronic alcohol abuse, chronic viral hepatitis (hepatitis b, c, and d) and autoimmune hepatitis, as well as by direct damage caused by biliary tract damage, including primary biliary cirrhosis, primary sclerosing cholangitis, and biliary atresia. Less common causes of liver cirrhosis include direct liver damage due to genetic diseases such as cystic fibrosis, alpha-1-antitrypsin deficiency, hemochromatosis, Wilson's disease, galactosemia, and glycogen storage disease. Transplantation is a key intervention in the treatment of patients with late stage cirrhosis.

Thus, another aspect of the invention relates to a polypeptide variant of the invention for use in the preparation of a medicament for the treatment of a disease or condition requiring blood clot formation. Another aspect of the invention relates to a method of treating a mammal having a disease or condition requiring blood clot formation comprising administering to a mammal in need thereof an effective amount of a polypeptide variant or pharmaceutical composition of the invention.

Examples of diseases/conditions that require increased clot formation include, but are not limited to, bleeding, including cerebral hemorrhage, and patients with severe uncontrolled bleeding, such as trauma. Other examples include patients receiving a living transplant, patients receiving a resection, and patients with bleeding due to varicose veins. Other broad diseases/disorders in which polypeptides of the invention may be used to increase blood clot formation are haemophilia, such as von Willebrand disease, haemophilia a, haemophilia B or haemophilia C.

The polypeptide variants of the invention are administered in a therapeutically effective dose which is approximately the same as that administered with rFVII such asThe dose used for treatment is the same or higher. As used herein, a "therapeutically effective dose" refers to a dose sufficient to produce the desired effect on the disease state being treated. The exact dosage to be administered depends on the particular circumstances and can be determined by methods well known to those skilled in the art. In general, the dose should be capable of preventing or reducing the severity or spread of the disease or condition being treated. It will be apparent to those skilled in the art that the effective amount of a polypeptide variant or composition of the invention will depend on the disease, dosage, schedule of administration, whether the polypeptide variant or composition is administered alone or in combination with other therapeutic agents, the serum half-life of the composition and the overall health of the patient.

The polypeptide variants of the invention are preferably administered in the form of a composition comprising a pharmaceutically acceptable carrier or excipient. By "pharmaceutically acceptable" is meant a carrier or excipient that does not cause any adverse effects in the patient to whom it is administered. Such pharmaceutically acceptable carriers and Excipients are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., Mack publishing company (1990); Pharmaceutical Formulation Development of Peptides and proteins, S.Frokjar and L.Hovgaard, Eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press (2000)).

The polypeptide variants of the invention can be used in "as is" and/or in the form of their salts. Suitable salts include, but are not limited to, salts with alkali or alkaline earth metals such as sodium, potassium, calcium and magnesium, and, for example, zinc salts. These salts or complexes may exist as crystalline and/or amorphous structures.

The pharmaceutical compositions of the present invention may be administered alone or in combination with other therapeutic agents. These agents may be administered as part of the same pharmaceutical composition, or separately, either simultaneously with the polypeptide variants of the invention or according to a therapeutic schedule. In addition, the polypeptide variants or pharmaceutical compositions of the invention may be used as adjuvants for other therapies.

In the present invention, "patient" includes humans and other mammals. Therefore, the method can be used for veterinary use.

Pharmaceutical compositions of the polypeptide variants of the invention may be formulated in a variety of forms, such as in liquid, gel, lyophilized or compressed solid form. The preferred form will vary according to the particular needs of the treatment, as will be apparent to those skilled in the art.

In particular, the pharmaceutical compositions of the polypeptide variants of the invention are formulated in lyophilized form or in stable solution form. The polypeptide variants can be lyophilized by a variety of well-known methods. Polypeptide variants can be prepared in a stable solution by removing or masking the proteolytic sites described herein. The advantage of obtaining a stable solution is that it is more convenient for the patient to use and, in case of emergency, acts faster, which may be life saving. The preferred form will vary according to the particular needs of the treatment, as will be apparent to those skilled in the art.

The formulations of the present invention may be administered in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebrally, intranasally, intradermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other suitable manner. The formulation may be administered by continuous infusion, although bolus injection (bolus injection) is acceptable, using techniques well known in the art, such as pumping or implantation. In some cases the formulation may be administered directly in solution or spray.

Parenteral administration of medicaments (Parentals)

A preferred example of a pharmaceutical composition is a solution designed for parenteral administration. The parenteral formulations may also be provided in frozen or lyophilized form, although in many cases, the pharmaceutical solution formulation may be provided in liquid form suitable for immediate use. In the former case, the composition must be thawed prior to use. The latter dosage form is typically used to enhance the stability of the active ingredients contained in the composition under various storage conditions, and as is known to those skilled in the art, lyophilized formulations are typically more stable than their liquid counterparts. The lyophilized formulation is reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

In the case of parenteral administration, a lyophilized formulation or an aqueous solution is prepared for use by appropriately mixing the polypeptide having the desired purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers (collectively, "excipients") commonly used in the art, such as buffers, stabilizers, preservatives, isotonic agents, nonionic surfactants or detergents, antioxidants, and/or various other additives such as bulking or bulking agents, chelating agents, antioxidants and solubilizing agents.

Details on parenteral formulations suitable for administration of FVII variants, as well as sustained release formulations, are set forth in WO01/58935 and WO 03/093465, which are incorporated herein by reference.

The invention is also described by the following non-limiting examples.

Materials and methods

Active site region

The active site region is defined as any residue of any atom within the catalytic triad (residues H193, D242, S344)With at least one atom within the distance.

Detection of reduced sensitivity to protease degradation

Proteolysis can be detected by the method described in example 5 of US5580560, wherein the proteolysis is an autonomous proteolysis.

In addition, reduced proteolysis can be detected in an in vivo model using radiolabeled samples, and the proteolysis of rhFVIIa and the polypeptide variants of the invention compared by taking blood samples and subjecting these blood samples to SDS-PAGE and autoradiography.

Regardless of which assay is used to determine proteolysis, "reduced proteolysis" refers to a significant reduction in cleavage compared to that obtained with rhFVIIa, as determined by gel scanning of coomassie blue stained SDS-PAGE gels, HPLC, or by conserved catalytic activity relative to wild-type using the tissue factor independent activity assay described below.

Determination of molecular weight of polypeptide variants

The molecular weight of the polypeptide variants is determined by SDS-PAGE, gel filtration, Western blotting, matrix assisted laser desorption, mass spectrometry or equilibrium centrifugation, e.g.SDS-PAGE as described by Laemmli, UK (Nature Vol 227(1970), pp.680-85).

Determination of phospholipid Membrane binding affinity

Phospholipid membrane binding affinities such as nelsesuen et al, Biochemistry, 1977; 30, of a nitrogen-containing gas; 10819-10824 or US 6,017,882.

TF-independent factor X activation assay

The assay is described in nelsesuen et al, J Biol Chem, 2001; 276: 39825 and 39831 are described on page 39826.

Briefly, the molecule to be targeted (hFVIIa, rhFVIIa or the activated form of the polypeptide variant of the invention) is mixed with a source of phospholipids (preferably a 8: 2 ratio of phosphatidylcholine to phosphatidylserine) and re-lipidated (relipidated) factor X in Tris buffer with BSA. After incubation for a specified time, the reaction was stopped by adding an excess of EDTA. The concentration of factor Xa was then determined by the change in absorbance at 405nm after addition of the chromogenic substrate (S-2222, Chromogenix). rhFVIIa (a) after correction against background_wt) The tissue factor-independent activity of (a) is determined as the change in absorbance after 10 minutes, the polypeptide variant of the invention (a)_Variants) The tissue factor-independent activity of (a) was determined as the change in absorbance after 10 minutes. The ratio of the activity of the activated form of the polypeptide variant to the activity of rhFVIIa is defined as a_Variants/a_wt。

Blood coagulation assay

The clotting activity of FVIIa and variants thereof was determined in a one-stage assay, and clotting times were recorded on a Thrombotrack IV coagulometer (Medinor). Factor VII-depleted human plasma (American Diagnostica) was lysed and equilibrated at room temperature for 15-20 min. Subsequently 50 microliters of plasma was transferred into the hemagglutination cup.

FVIIa and variants thereof were diluted in Glyoxaline buffer (5.7mM barbiturate, 4.3mM sodium citrate, 117mM NaCl, 1mg/ml BSA, pH 7.35). A50. mu.l sample was added to the hemagglutination cup and incubated at 37 ℃ for 2 minutes.

Thromboplastin (Medinor) is dissolved in water and CaCl is added₂To a final concentration of 4.5 mM. The reaction was initiated by the addition of 100. mu.l thromboplastin.

To determine clotting activity in the absence of TF, the same assay was used without addition of thromboplastin. Data were analyzed using PRISM software.

Whole blood assay

The clotting activity of FVIIa and variants thereof was determined in a one-stage assay, and clotting times were recorded on a Thrombotrack IV coagulometer (Medinor). 100. mu.l FVIIa or variants thereof in a medium containing 10mM Mglycydiglycine, 50mM NaCl, 37.5mM CaCl₂Diluted in buffer pH 7.35 and transferred to the reaction cup. The clotting reaction was initiated by adding 50 μ l of blood containing 10% 0.13M trisodium citrate as anticoagulant. Data were analyzed using Excel or PRISM software.

Deamidation assay

The ability of the variants to cleave small peptide substrates was determined using the chromogenic substrate S-2288 (D-Ile-Pro-Arg-p-nitroanilide). In assay buffer (50mM Na-Hepes pH 7.5, 150mM NaCl, 5mM CaCl₂0.1% BSA, 1U/ml heparin) to about 10-90 nM. In addition, soluble TF (sTF) was diluted to 50-450nM in assay buffer. Mu.l of assay buffer were mixed with 20. mu.l of FVIIa sample and 20. mu.l of sTF. After incubation at room temperature for 5 minutes with gentle shaking and then at 37 ℃ for 10 minutes, the reaction was initiated by adding S-2288 substrate to 1mM and absorbance at 405nm was measured at several time points.

ELISA assay

FVII/FVIIa (or variant) concentrations were determined by ELISA. The wells of the microtiter plate were coated with an antibody directed against the protease domain using a 2. mu.g/ml PBS solution (100. mu.l per well). After coating at R.T. (room temperature) overnight, wells were washed four times with THT buffer (100mM NaCl, 50mM Tris-HCl pH 7.20.05% Tween-20). Subsequently, 200. mu.l of 1% casein (prepared by diluting 2.5% stock solution with 100mM NaCl, 50mM Tris-HCl pH 7.2) was added per well for blocking. After 1hr of r.t. incubation, wells were emptied and 100 μ l of sample (optionally diluted in dilution buffer (THT + 0.1% casein)) was added. After further incubation at room temperature for 1hr, the wells were washed four times with THT buffer and 100. mu.l of a buffer directed against the EGF-like domain (1. mu.g)/ml) of biotin-labeled antibody. After 1h incubation at R.T., additional 4 washes with THT buffer, 100. mu.l streptavidin-horseradish peroxidase (DAKO A/S, Glostrup, Denmark, 1/10000 dilution) was added. After 1 hour incubation at R.T., additional 4 washes with THT buffer, 100. mu.l of TMB (3, 3 ', 5, 5' -tetramethylbenzidine, Kem-en-Tech A/S, Denmark) was added. Incubate R.T. for 30 min in the dark, add 100. mu.l of 1M H₂SO₄And determining the OD_450nm. Using rhFVIIa (f) ((r))) A standard curve was prepared.

Alternatively, FVII/FVIIa or variants may be quantified via the Gla domain rather than via the protease domain. In this ELISA format, wells were coated overnight with anti-EGF-like domain antibody and for detection, a calcium-dependent monoclonal anti-Gla domain antibody (2. mu.g/ml, 100. mu.l per well) was used. In this step, 5mM CaCl was added₂Add THT and dilution buffer.

Thrombogram assay

The effect of hFVIIa, rhFVIIa or FVIIa variants on human plasma thrombin generation is described in Hemker et al (2000) Thromb Haemost 83: 589-91 in page 589. Briefly, the molecule to be assayed (either hFVIIa, rhFVIIa or variants) is mixed with FVII-depleted platelet-poor plasma (PPP) containing re-lipidated tissue factor (such as Innovin by Dade Behring) or phospholipids (phosphatidylcholine and phosphatidylserine in a ratio of 8: 2, or phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine in a ratio of 4: 2: 4).

The reaction is initiated by the addition of a fluorogenic thrombin substrate and calcium chloride. Fluorescence was measured continuously and thrombin activity was determined by calculating the slope of the fluorescence curve (increase in fluorescence over time). In this method, a time (T) is obtained for maximum thrombin zymolytic amide activity_max) And thrombin generation rate (maximum increase in thrombin activity),and the total thrombin work (area under the curve (AUC)) can be calculated.

Frozen citrated FVII-depleted plasma was thawed in the presence of a cereal trypsin inhibitor (100. mu.g/ml serum) to inhibit the coagulation contact pathway. To each well of the 96-well plate, 80. mu.l of plasma and 20. mu.l of buffer containing the rhFVII or variant tested at a final concentration of 0.1-100nM were added. Recombinant human tissue factor (rTF) was added to 5. mu.l of assay buffer to a final concentration of 1 pM. The assay buffer was a distilled water solution containing 20mM hepes, 150mM NaCl and 60mg/ml BSA. The reaction was initiated by adding 20. mu.l of substrate solution containing 0.1M calcium chloride. The assay plate and reagents were preheated to 37 ℃ at which temperature the reaction was carried out. The fluorometer used was a BMG fluorometer with an excitation filter of 390nm and an emission filter of 460 nm. Fluorescence was measured in each well of a 96-well clean bottom plate (96-well clear bottom plates) at 20-40 second intervals over 30-180 minutes. Data were analyzed using PRISM software.

Tissue factor binding surface plasmon resonance assay (Biacore assay)

Surface plasmon resonance analysis is used to determine the relative binding of wild-type factor VIIa and variants thereof to soluble tissue factor. Recombinant soluble tissue factor containing extracellular domain was coupled to a 270 reaction unit on a Biacore CM5 chip by NHS/EDC coupling. Soluble tissue factor was coupled at ph4.5 to enable reaction with the chip surface.

In this assay, tissue factor binding of factor VII proteins is compared at a single FVIIa or variant concentration to allow relative comparison of the variant to wild-type. The concentration was determined by flowing on the chip a standard curve of wild type FVIIa at concentrations of 75 and 0. mu.g/ml. FVIIa was removed by addition of 10mM EDTA. Determination of a concentration of 15. mu.g/ml in this manner yields binding in the linear range. The FVIIa variant was then flowed over the chip at 15. mu.g/ml to determine the relative binding strength of FVIIa or variant to tissue factor.

Examples

Example 1

Banner et al, J Mol Biol, 1996; 285: 2089X-ray structures of hFVIIa complexed with soluble tissue factor were used in this example. See WO01/58935 for additional information on the calculations in this example.

Surface exposure

A fractional ASA calculation was performed to determine that more than 25% of the side chains of the following residues were 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, A33, E33, R33, K33, L33, W33, I33, S33, S33, G33, D33, Q33, S33, K33, D33, D33, Q33, L33, Q33, S33, I33, F72, L33, P4, P7377, L5, N35122, N36142, N36122, N150, N175, N36122, N150, N36122, N33, N150, N33, N150, N175, N150, N33, N150, N175, N150, N175, N33, N175, N150, N33, N150, N33, N175, N150, N, 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, S394, K337, K341, G342, H351, R353, G354, Q367, G367, T372, T395, G19, P2, E325, G35398, G8521, G2, P2, N18, G21, P2, G398, G33, G18, G33, G21, G2, G18, G21, P2, G21, P2.

More than 50% of the side chains of the following residues were determined to be exposed on their surface: a1, A3, F4, L5, E6, E7L8, R9, P10, E14, E16, K18, E19, E20, Q21, S23, E25, E26, E29, K32, a34, E35R36, K38, L39, I42, S43, G47, D48, a51, S52, S53, Q56, G58, S60, K62, L65, Q66, S67, I69, F71, L73, P74, a75, E77, G77, R77, E77, H77, K77, D77, Q77, L77, I77, V149, N29, G121, N119, G175, G144, G175, N121, N175, G175, N121, N175, G175, N121, N175, N150, N121, N150, N121, N175, N150, N121, N175, N150, N121, N150, N175, N150, N175, N121, N175, N150, N175, N150, G121, N150, N175, N, k341, G354, G367, V371, E385, K389, R392, E394, R396, P397, G398, R402, P404 and P406(a1-S43 are located within the Gla domain, the rest being located outside the Gla domain).

Tissue factor binding sites

By ASA calculation, the following residues in FVII alter its ASA in the complex. These residues are defined as the residues that make up the receptor 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 R9.

Active site region

The active site region is one having at least one spacing from any atom in the catalytic triad (residues H193, D242, S344)Any residue of an atom within the range: 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, Q293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H, L378, T359, G360, I361, V362, S365, W363, W368, V368, L383, Y368, V383, V380, V383, V68.

Ridge of active site binding groove

The ridge of the active site binding groove is defined by visual observation of FVIIa structure 1fak. N173, a175, K199, N200, N203, D289, R290, G291, a292, P321 and T370.

Example 2

Expression cassette designed for expression of human factor VII in mammalian cells

The design and cloning of expression cassettes for the expression of rhFVII is described in example 2 of WO 01/58935.

Example 3

Construction of an expression cassette encoding a variant of the invention

Sequence Overhang Extension (SOE) PCR was used to generate constructs with variant FVII open reading frames containing substituted codons by standard methods.

Example 4

Expression of polypeptide variants in CHO K1 cells

A50% confluent cell line CHO K1(ATCC # CCL-61) was inoculated into T-25 flasks containing MEM α, 10% FCS (Gibco/BRLCat #10091), P/S and 5 μ g/ml phytoquinone and the cells were grown to confluency. The full monolayer cells were transfected with 5. mu.g of the above-described plasmid of interest using Lipofectamine 2000 as transfection reagent (Life technologies) according to the manufacturer's protocol. The culture broth was sampled 24 hours after transfection and quantified using ELISA that recognizes EGF1 domain of hFVII. At this time point, the cells can be subjected to a relevant selection (e.g., hygromycin B) to generate a pool of stable transfectants. This is usually done within 1 week using CHO K1 cells and using the hygromycin B resistance gene as a selection marker on the plasmid.

Example 5

Preparation of CHO K1 cells stably expressing polypeptide variants

A vial of CHO-K1 transfectant colonies was thawed and cells were seeded in 175cm2 tissue culture flasks containing 25ml MEM α, 10% FCS, phytoquinone (5 μ g/ml), 100U/l penicillin, 100 μ g/l streptomycin for 24 hours. Collecting the cells, diluting them and mixing¹/₂Density of-1 cells/well 96-well microtiter plates. After one week of growth, colonies of about 20-100 cells appeared in the wells, and those wells containing only one colony were labeled. After 2 weeks of additional growth, the medium in all wells containing only one colony was replaced with 200 μ l of fresh medium. After 24 hours, media samples are removed and analyzed by, for example, ELISA. High yielding clones were selected and used for large scale production of FVII or variants.

Example 6

Purification and subsequent activation of polypeptide variants

FVII and FVII variants were purified as follows: the process is carried out at 4 ℃. Culture media harvested from large-scale production were ultrafiltered using a Millipore TFF system with a 30kDa cut-off Pellicon membrane. After concentrating the medium, citrate was added to 5mM, and the pH was adjusted to 8.6. The conductivity is reduced, if necessary, to 10 mS/cm. The samples were then loaded onto a Q-sepharose FF column equilibrated with 50mM NaCl, 10mM Tris pH 8.6. After washing with 100mM NaCl, 10mM Tris pH 8.6, then with 150mM NaCl, 10mM Tris pH 8.6, the mixture was washed with10mM Tris，25mM NaCl，35mM CaCl₂FVII eluted at pH 8.6.

For the second chromatography step, an affinity column was prepared by coupling a monoclonal calcium-dependent anti-Gla domain antibody to CNBr-activated sepharose ff. About 5.5mg of antibody was conjugated per ml of resin. The column was run with 10mM Tris, 100mM NaCl, 35mM CaCl₂pH 7.5. NaCl was added to the sample to a concentration of 100mM NaCl and the pH was adjusted to 7.4-7.6. After O/N application of the samples, 100mM NaCl, 35mM CaCl was used₂10mM Tris pH 7.5, eluting FVII protein with 100mM NaCl, 50mM citrate, 75mM Tris pH 7.5.

For the third chromatography, the conductivity of the sample is reduced to below 10mS/cm and the pH is adjusted to 8.6, if necessary. The sample was then applied to a Q-sepharose column (equilibrated with 50mM NaCl, 10mM Tris pH 8.6) at a density of about 3-5mg protein per ml gel to obtain efficient activation. After application, the column was washed with 50mM NaCl, 10mM Tris pH 8.6 for about 4 hours at a flow rate of 3-4 column volumes (cv) per hour. FVII proteins are eluted at 40cv using a gradient of 0-100% 500mM NaCl, 10mM Tris pH 8.6. FVII containing fractions were pooled.

For the final chromatography step, the conductivity is reduced to less than 10 mS/cm. Subsequently, the sample was applied to a Q-sepharose column (washed with 140mM NaCl, 10mM Mglycyclycene pH 8.6) at a concentration of 3-5mg protein per ml gel. The column is then washed with 140mM NaCl, 10mM gylcycenepH 8.6 and FVII with 140mM NaCl, 15mM CaCl₂10mM Glycylglycine, pH 8.6. The eluate was diluted to 10mM CaCl₂And the pH is adjusted to 6.8-7.2. Finally, Tween-80 to 0.01% was added, the pH was adjusted to 5.5 and the mixture was stored at-80 ℃.

Example 7

Test results-FX activating Activity

The variants of the invention were subjected to a "TF-independent factor X activation assay" giving the following results (expressed as a percentage of the activity of the reference P10Q + K32E variant):

TABLE 1

As shown by the above results, the variants of the present invention show substantial improvement in FX activation activity as compared to rhFVIIa and [ P10Q + K32E ] rhFVIIa.

Example 8

Test results-clotting Activity in "Whole blood assay

The variants of the invention, when subjected to the "whole blood assay", show a significantly increased clotting activity (i.e.a reduced clotting time) compared to rhFVIIa and [ P10Q + K32E ] rhFVIIa. The test results are shown in fig. 1 and table 2 below.

TABLE 2

Example 9

Test results-clotting Activity in the "clotting assay

When tested in a TF-dependent coagulation assay ("coagulation assay" as described in the materials and methods section above), it is clear that the coagulation activity of the variants of the invention having the R36E substitution is significantly reduced compared to rhFVII or other variants of the invention. See table 3 below. However, as described in example 7 above, the variant with the substitution R36E had significantly reduced clotting activity in the "TF-independent factor X activation assay".

TABLE 3

Example 10

Test results-Thrombin Generation in thrombogram assay

Maximum thrombin generation rates for FVIIa variants were determined at different variant protein concentrations using Phospholipid (PL) -dependent and Tissue Factor (TF) -dependent thrombograms (as described in the thrombogram assay above). By comparing the maximum thrombin generation rate (expressed as FU (fluorescence units) per second)²) The results shown in FIG. 2 (maximum tissue factor-dependent thrombin generation rate) and FIG. 3 (maximum phospholipid-dependent thrombin generation rate) were obtained plotted as a function of variant concentration in pM.

From these results, it is evident that the FVIIa variant P10Q K32E a34E R36E has different thrombin generating ability according to whether the reaction is PL-dependent or TF-dependent. The maximum TF-dependent thrombin generation rate of this variant was reduced by about 10 fold compared to FVIIa variants P10Q K32E or A3AY P10Q K32EA34L (dotted line in fig. 2). Lag time, peak arrival time, peak height and (to a lesser extent) AUC for P10Q K32E a34E R36E were reduced compared to the other variants (results are shown). In contrast to TF-dependent activity, the PL-dependent activity of the P10Q K32E a34E R36E variant was identical to the activity of the other variants tested in the examples (see fig. 3), i.e. the variant had full PL activity even in the case of a substantial reduction in TF-dependent activity.

In the same experiment, the variant P10Q K32E a34E R36E was directly compared with the variant P10Q K32EA34E P74S, which has the high TF-dependent thrombin generation rate shown in fig. 2. The difference in TF-binding of the two variants (i.e. reduced TF-binding of the variant P10Q K32E a34E R36E) is believed to be directly due to the presence of the R36E substitution, which may act synergistically with the a34E substitution.

Example 11

Test results-binding of FVIIa to tissue factor in Biacore assay

Using TF chips, the variants of the invention were tested by surface plasmon resonance on the Biacore system as described in the materials and methods section, with the following results:

TABLE 4^*n＝2

Consistent with the TF-dependent thrombin generation rate data from the thrombogram assay (example 10), the results in table 4 indicate that the R36E substitution binds weakly to tissue factor.

FVIIa variants having the same modifications as the variants shown in table 4, as well as two additional modifications introducing two glycosylation sites (T106N and V253N or I205T), were also tested for binding to tissue factor in the same assay. The results are shown in table 5 below.

TABLE 5

These results are consistent with those of table 4 and show that the presence of two new glycosylation sites in the variant of table 5 results in (further) reduced tissue factor binding compared to the same variant (or wild type) without other glycosylation sites in table 4. As with the variants of table 4, the presence of the T36E substitution in the glycosylation variant also results in a level of tissue factor binding that is substantially lower than that of other glycosylation variants without the substitution.

Claims

1. Factor VII or factor VIIa polypeptide variant having clotting activity comprising an amino acid sequence which is substantially identical to the amino acid sequence of SEQ ID NO: 1, wherein the negatively charged amino acid residue differs from the amino acid sequence of human factor VII or human factor VIIa by 1-15 amino acid modifications, wherein the negatively charged amino acid residue is represented by SEQ ID NO: 1 in position 36.

2. The variant of claim 1, wherein the substitution is R36D.

3. The variant of claim 1, wherein the substitution is R36E.

4. The variant of claim 1, further comprising an amino acid substitution in position 34.

5. The variant of claim 4, wherein the negatively charged amino acid residue is introduced by a substitution in position 34.

6. The variant of claim 5, wherein the substitution is a 34E.

7. The variant of claim 6, comprising the substitution A34E + R36E.

8. The variant of claim 4, wherein the hydrophobic amino acid residue is introduced by substitution in position 34.

9. The variant of claim 8, wherein the substitution is selected from the group consisting of: a34I, a34L, a34M, a34V, a34F, a34Y, and a 34W.

10. The variant of claim 9, wherein the substitution is selected from the group consisting of: a34I, a34L and a 34V.

11. The variant of claim 10, wherein the substitution is a 34L.

12. The variant of claim 11, comprising the substitution a34L + R36E.

13. The variant of claim 1, further comprising an amino acid substitution in position 10 and/or 32.

14. The variant of claim 13 which comprises the substitution K32E.

15. The variant of claim 13 which comprises the substitution P10Q.

16. The variant of claim 13, which comprises the substitutions P10Q + K32E.

17. The variant of claim 16, which comprises the substitutions P10Q + K32E + a34E + R36E.

18. The variant of claim 16, which comprises the substitutions P10Q + K32E + a34L + R36E.

19. The variant according to claim 1, wherein at least one amino acid residue comprising a linking group for the non-polypeptide moiety has been introduced at a position outside the Gla domain.

20. The variant of claim 19, wherein the attachment group is a glycosylation site.

21. The variant of claim 20, wherein the glycosylation site is an in vivo N-glycosylation site introduced by substitution.

22. The variant of claim 21, wherein the N-glycosylation site is introduced by a substitution selected from the group consisting of: a51N, G58N, T106N, K109N, G124N, K143N + N145T, a175T, I205S, I205T, V253N, T267N, T267N + S269T, S314N + K316S, S314S + K316S, R315S + V317S, K316S + G318S, D334S, and combinations thereof.

23. The variant of claim 22, comprising at least one substitution selected from the group consisting of: T106N, I205T and V253N.

24. The variant according to claim 23, comprising the substitution P10Q + K32E + a34E + R36E + T106N + I205T.

25. The variant according to claim 23, comprising the substitution P10Q + K32E + a34E + R36E + T106N + V253N.

26. The variant according to claim 23, comprising the substitution P10Q + K32E + a34E + R36E + I205T + V253N.

27. The variant according to claim 23, comprising the substitution P10Q + K32E + a34L + R36E + T106N + I205T.

28. The variant according to claim 23, comprising the substitution P10Q + K32E + a34L + R36E + T106N + V253N.

29. The variant according to claim 23, comprising the substitution P10Q + K32E + a34L + R36E + I205T + V253N.

30. The variant of claim 1, further comprising an insertion of at least one amino acid residue between positions 3 and 4.

31. The variant of claim 30, comprising an insertion of one amino acid residue between positions 3 and 4.

32. The variant of claim 30, wherein the hydrophobic amino acid residue is inserted between positions 3 and 4.

33. The variant of claim 32, wherein the insertion is A3 AY.

34. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34E + R36E.

35. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34L + R36E.

36. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34E + R36E + T106N + I205T.

37. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34E + R36E + T106N + V253N.

38. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34E + R36E + I205T + V253N.

39. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34L + R36E + T106N + I205T.

40. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34L + R36E + T106N + V253N.

41. The variant of claim 33, comprising the insertion A3AY and the substitution P10Q + K32E + a34L + R36E + I205T + V253N.

42. The variant of claim 1, wherein the amino acid sequence in SEQ ID NO: 1 in positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35.

43. The variant of claims 1-42, wherein the variant is an activated form thereof.

44. The variant of claims 1-42, wherein the ratio of FX activation activity of the activated form of said variant to that of rhFVIIa is at least 5, when determined in the "TF-independent factor X activation assay" as described herein.

45. A nucleotide sequence encoding the variant of any of claims 1-42.

46. An expression vector comprising the nucleotide sequence of claim 45.

47. A host cell comprising the nucleotide sequence of claim 45 or the expression vector of claim 46.

48. The host cell of claim 47, wherein the host cell is a gamma-carboxylated cell capable of glycosylation in vivo.

49. A composition comprising the variant of any of claims 1-44 and at least one pharmaceutically acceptable carrier or excipient.

50. Use of a variant of any of claims 1-44 in the manufacture of a medicament for treating a disease or condition in need of clot formation.

51. The use according to claim 50, wherein the disease or condition is selected from the group consisting of: severe uncontrolled bleeding, bleeding in mammals undergoing transplants or resections, bleeding due to varicose veins, upper gastrointestinal bleeding in patients with cirrhosis, thrombocytopenia and hemophilia.

52. The use according to claim 50, wherein the disease or condition is trauma.

53. The use according to claim 51, wherein the disease or condition is hemophilia.

54. The use according to claim 52, wherein the trauma is blunt trauma.

55. The use according to claim 52, wherein the trauma is penetrating trauma.

56. The use according to claim 50, wherein the disease or condition is bleeding.

57. The use according to claim 56, wherein said bleeding is cerebral bleeding.