US20050164932A1 - FVII or FVIIa Gla domain variants - Google Patents

FVII or FVIIa Gla domain variants Download PDF

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US20050164932A1
US20050164932A1 US11/021,239 US2123904A US2005164932A1 US 20050164932 A1 US20050164932 A1 US 20050164932A1 US 2123904 A US2123904 A US 2123904A US 2005164932 A1 US2005164932 A1 US 2005164932A1
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variant
amino acid
polypeptide
substitution
fvii
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Jesper Haaning
Kim Andersen
Claus Bornaes
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Bayer Healthcare LLC
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Maxygen Holdings Ltd
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Priority to US11/021,239 priority Critical patent/US20050164932A1/en
Assigned to MAXYGEN HOLDINGS LTD. reassignment MAXYGEN HOLDINGS LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BORNAES, CLAUS, ANDERSEN, KIM VILBOUR, HAANING, JESPER MORTENSEN
Publication of US20050164932A1 publication Critical patent/US20050164932A1/en
Priority to US11/379,664 priority patent/US20060252128A1/en
Priority to US11/424,035 priority patent/US8987202B2/en
Priority to US11/424,030 priority patent/US7807638B2/en
Priority to US11/643,535 priority patent/US20070117756A1/en
Assigned to BAYER HEALTHCARE LLC reassignment BAYER HEALTHCARE LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MAXYGEN HOLDINGS LTD.
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
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    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/647—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)

Definitions

  • the present invention relates to novel Gla domain variants of Factor FVII (FVII) or Factor VIIa (FVIIa) polypeptides, as well as the use of such polypeptide variants in therapy, in particular for the treatment of a variety of coagulation-related disorders.
  • FVII Factor FVII
  • FVIIa Factor VIIa
  • Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually results in a fibrin clot.
  • the blood components participating in what has been referred to as the “coagulation cascade” are proenzymes or zymogens, i.e. enzymatically inactive proteins that are converted into an active form by the action of an activator.
  • proenzymes or zymogens i.e. enzymatically inactive proteins that are converted into an active form by the action of an activator.
  • FVII a zymogens
  • FVII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein with a molecular weight of 53 kDa (Broze & Majerus, J. Biol. Chem. 1980; 255:1242-1247).
  • the FVII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site, R152-1153, resulting in two chains linked by a single disulfide bridge.
  • FVIIa in complex with tissue factor is able to convert both factor IX (FIX) and factor X (FX) into their activated forms, followed by reactions leading to rapid thrombin production and fibrin formation (Osterud & Rapaport, Proc Natl Acad Sci USA 1977; 74:5260-5264).
  • FVII undergoes post-translational modifications, including vitamin K-dependent carboxylation resulting in ten ⁇ -carboxyglutamic acid residues in the N-terminal region of the molecule.
  • residues number 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO:1 are ⁇ -carboxyglutamic acid residues in the Gla domain important for FVII activity.
  • Other post-translational modifications include sugar moiety attachment at two naturally occurring N-glycosylation sites at position 145 and 322, respectively, and at two naturally occurring O-glycosylation sites at position 52 and 60, respectively.
  • hFVII human FVII
  • chromosome 13 The gene coding for human FVII (hFVII) has been mapped to chromosome 13 at q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). It contains nine exons and spans 12.8 Kb (O'Hara et al., Proc Natl Acad Sci USA 1987; 84:5158-5162).
  • FVII The gene organisation and protein structure of FVII are similar to those of other vitamin K-dependent procoagulant proteins, with exons 1a and 1b encoding for signal sequence; exon 2 the propeptide and Gla domain; exon 3 a short hydrophobic region; exons 4 and 5 the epidermal growth factor-like domains; and exon 6 through 8 the serine protease catalytic domain (Yoshitake et al., Biochemistry 1985; 24: 3736-3750).
  • NovoSeven® Commercial preparations of recombinant human FVIIa (rhFVIIa) are sold under the trademark NovoSeven®. NovoSeven® is indicated for the treatment of bleeding episodes in hemophilia A or B patients. NovoSeven® is the only rhFVIIa for effective and reliable treatment of bleeding episodes currently available on the market.
  • FVII inactive form of FVII in which arginine 152 and/or isoleucine 153 are modified has been reported in WO 91/11514. These amino acids are located at the activation site.
  • WO 96/12800 describes inactivation of FVIIa by a serine proteinase inhibitor. Inactivation by carbamylation of FVIIa at the ⁇ -amino acid group 1153 has been described by Petersen et al., Eur J Biochem, 1999;261:124-129. The inactivated form is capable of competing with wild-type FVII or FVIIa for binding to tissue factor and inhibiting clotting activity.
  • the inactivated form of FVIIa is suggested to be used for treatment of patients suffering from hypercoagulable states, such as patients with sepsis or at risk of myocardial infarction or thrombotic stroke.
  • FVIIa is capable of activating FX to FXa without binding to tissue factor, and this activation reaction is believed to occur primarily on activated blood platelets (Hedner et al. Blood Coagulation & Fibrinolysis, 2000; 11; 107-111).
  • hFVIIa or rhFVIIa has a low activity towards FX in the absence of tissue factor and, consequently, treatment of uncontrolled bleeding, for example in trauma patients, requires relatively high and multiple doses of hFVIIa or rhFVIIa.
  • FVIIa molecules which possess a high activity toward FX in the absence of tissue factor.
  • Such improved FVIIa molecules should exhibit a lowered clotting time (faster action/increased clotting activity) as compared to rhFVIIa when administered in connection with uncontrolled bleedings.
  • Gla domain variants of FVII/FVIIa have been disclosed in WO 99/20767, U.S. Pat. No. 6,017,882 and WO 00/66753, where some residues located in the Gla domain were identified as being important for phospholipid membrane binding and hence FX activation.
  • residues 10 and 32 were critical and that increased phospholipid membrane binding affinity, and hence increased FX activation, could be achieved by performing the mutations P10Q and K32E.
  • FX activation was enhanced as compared to rhFVIIa at marginal coagulation conditions, such as under conditions where a low level of tissue factor is present.
  • WO 01/58935 discloses a new strategy for developing FVII or FVIIa molecules having inter alia an increased half-life by means of directed glycosylation or PEGylation.
  • WO 03/093465 discloses FVII or FVIIa variants having certain modifications in the Gla domain and having one or more N-glycosylation sites introduced outside the Gla domain.
  • WO 2004/029091 discloses FVII or FVIIa variants having certain modifications in the tissue factor binding site.
  • the present inventors have now identified further residues in the Gla domain which further increase the phospholipid membrane binding affinity and hence further increase FX activation.
  • the FVII or FVIIa variants of the invention may also exhibit reduced tissue factor binding affinity.
  • the object of the present invention is 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.
  • 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.
  • the present invention relates to a Factor VII (FVII) or Factor VIIa (FVIIa) polypeptide variant having an amino acid sequence comprising 1-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, wherein a hydrophobic amino acid residue has been introduced by substitution in position 34.
  • the invention in a second aspect relates to a Factor VII (FVII) or Factor VIIa (FVIIa) polypeptide variant having an amino acid sequence comprising 1-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, wherein the amino acid sequence comprises an amino acid substitution in position 36.
  • FVII Factor VII
  • FVIIa Factor VIIa
  • FVIIa Factor VIIa polypeptide variant having an amino acid sequence comprising 1-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, wherein the amino acid sequence comprises an amino acid substitution in position 36.
  • the invention in a third aspect relates to a Factor VII (FVII) or Factor VIIa (FVIIa) polypeptide variant having an amino acid sequence comprising 3-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) having the amino acid sequence shown in SEQ ID NO:1, wherein amino acid sequence comprises an amino acid substitution in positions 10 and 32 and at least one further amino acid substitution in a position selected from the group consisting of positions 74, 77 and 116.
  • nucleotide sequence encoding the polypeptide variants of the invention
  • an expression vector comprising the nucleotide sequence
  • a host cell comprising the nucleotide sequence or expression vector.
  • Still further aspects of the invention relate to a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide variants of the invention, use of the polypeptide variants of the invention or the pharmaceutical composition of the invention as a medicament, as well as methods of treatment using the polypeptide variants or pharmaceutical compositions of the invention.
  • the polypeptide variants of the invention are used for the treatment of intracerebral haemorrhage or traumatic brain injury.
  • FIG. 1 shows the clotting time vs. concentration for variants of the invention when assayed in the “Whole Blood Assay”.
  • FIG. 2 shows the maximum tissue factor-dependent thrombin generation rate for variants of the invention determined in the “Thrombogram Assay”.
  • FIG. 3 shows the maximum phospholipid-dependent thrombin generation rate for variants of the invention determined in the “Thrombogram Assay”.
  • FIG. 4 is a thrombogram showing the phospholipid-dependent clotting activity of a variant of the invention (P10Q+K32E+A34E+R36E+T 106N+V253N) compared to rhFVIIa (NovoSeven®).
  • FIG. 5 is a thrombogram showing the tissue factor-dependent clotting activity of a variant of the invention (P10Q+K32E+A34E+R36E+T106N+V253N) compared to rhFVIIa (NovoSeven®).
  • FVII or “FVII polypeptide” refers to a FVII molecule provided in single chain form.
  • FVII polypeptide is the wild-type human FVII (hFVII) having the amino acid sequence shown in SEQ ID NO:1.
  • hFVII wild-type human FVII
  • FVII polypeptide also covers hFVII-like molecules, such as fragments or variants of SEQ ID NO:1, in particular variants where the sequence comprises at least one, such as up to 15, preferably up to 10, amino acid modifications as compared to SEQ ID NO:1.
  • FVIIa or “FVIIa polypeptide” refers to a FVIIa molecule provided in its activated two-chain form.
  • amino acid sequence of SEQ ID NO:1 is used to describe the amino acid sequence of FVIIa it will be understood that the peptide bond between R152 and I153 of the single-chain form has been cleaved, and that one of the chains comprises amino acid residues 1-152, the other chain comprises amino acid residues 153-406.
  • rFVII and rFVIIa refer to FVII and FVIIa polypeptides produced by recombinant techniques.
  • hFVII and hFVIIa refer to human wild-type FVII and FVIIa, respectively, having the amino acid sequence shown in SEQ ID NO:1
  • rhFVII and rhFVIIa refer to human wild-type FVII and FVIIa, having the amino acid sequence shown in SEQ ID NO:1, produced by recombinant means.
  • An example of rhFVIIa is NovoSeven®.
  • Ga domain is intended to cover amino acid residues 1 to 45 of SEQ ID NO:1.
  • position located outside the Gla domain covers amino acid residues 46-406 of SEQ ID NO:1.
  • FX Factor X
  • Tissue Factor Tissue Factor Pathway Inhibitor
  • proteease domain is used about residues 153-406 counted from the N-terminus.
  • catalytic site is used to mean the catalytic triad consisting of S344, D242 and H193 of the polypeptide variant.
  • parent is intended to indicate the molecule to be modified/improved in accordance with the present invention.
  • the parent polypeptide to be modified by the present invention may be any FVII or FVIIa polypeptide, and thus be derived from any origin, e.g. a non-human mammalian origin, it is preferred that the parent polypeptide is hFVII or hFVIIa.
  • a “variant” is a polypeptide which differs in one or more amino acid residues from its parent polypeptide, normally 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.
  • the parent polypeptide is hFVII or hFVIIa.
  • conjugate is intended to indicate a heterogeneous (in the sense of composite or chimeric) molecule formed by the covalent attachment of one or more polypeptides to one or more non-polypeptide moieties such as polymer molecules, lipophilic compounds, sugar moieties or organic derivatizing agents.
  • the conjugate is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood.
  • conjugated polypeptides of the invention include glycosylated and/or PEGylated polypeptides.
  • covalent attachment means that the polypeptide variant and the non-polypeptide moiety are either directly covalently joined to one another, or else are indirectly covalently joined to one another through at least one intervening moiety such as a bridge, spacer, or linkage moiety.
  • non-polypeptide moiety is intended to mean a molecule, different from a peptide polymer composed of amino acid monomers and linked together by peptide bonds, which molecule is capable of conjugating to an attachment group of the polypeptide variant of the invention.
  • Preferred examples of such molecules include polymer molecules, sugar moieties, lipophilic compounds or organic derivatizing agents.
  • the non-polypeptide moiety is linked to the polypeptide part of the conjugated variant through an attachment group of the polypeptide. As explained above, the non-polypeptide moiety can be directly or indirectly covalently joined to the attachment group.
  • a “polymer molecule” is a molecule formed by covalent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein.
  • the term “polymer” may be used interchangeably with the term “polymer molecule”.
  • the term is also intended to cover carbohydrate molecules attached by in vitro glycosylation, i.e. a synthetic glycosylation performed in vitro normally involving covalently linking a carbohydrate molecule to an attachment group of the polypeptide variant, optionally using a cross-linking agent.
  • sugar moiety is intended to indicate a carbohydrate-containing molecule comprising one or more monosaccharide residues, capable of being attached to the polypeptide variant (to produce a polypeptide variant conjugate in the form of a glycosylated polypeptide variant) by way of in vivo glycosylation.
  • in vivo glycosylation is intended to mean any attachment of a sugar moiety occurring in vivo, i.e. during posttranslational processing in a glycosylating cell used for expression of the polypeptide variant, e.g. by way of N-linked and O-linked glycosylation. The exact oligosaccharide structure depends, to a large extent, on the glycosylating organism in question.
  • 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 either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.
  • amino acid residue in position +3 relative to the asparagine residue is not a proline residue.
  • O-glycosylation site is the OH-group of a serine or threonine residue.
  • attachment group is intended to indicate a functional group of the polypeptide variant, in particular of an amino acid residue thereof or a carbohydrate moiety, capable of attaching a non-polypeptide moiety such as a polymer molecule, a lipophilic molecule, a sugar moiety or an organic derivatizing agent.
  • a non-polypeptide moiety such as a polymer molecule, a lipophilic molecule, a sugar moiety or an organic derivatizing agent.
  • Useful attachment groups and their matching non-polypeptide moieties are apparent from the table below.
  • Conjugation Attachment Examples of non- method/- group Amino acid polypeptide moiety Activated PEG Reference —NH 2 N-terminal, Polymer, e.g.
  • attachment group is used in an unconventional way to indicate the amino acid residues constituting a N-glycosylation site (with the sequence N-X-S/T/C as indicated above).
  • the asparagine residue of the N-glycosylation site is the one to which the sugar moiety is attached during glycosylation, such attachment cannot be achieved unless the other amino acid residues of the N-glycosylation site are present.
  • amino acid residue comprising an attachment group for a non-polypeptide moiety as used in connection with alterations of the lo amino acid sequence of the polypeptide is to be understood as meaning that one or more amino acid residues constituting an in vivo N-glycosylation site are to be altered in such a manner that a functional in vivo N-glycosylation site is introduced into the amino acid sequence.
  • amino acid names and atom names are used as defined by the Protein DataBank (PDB) (www. pdb .org) based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984) together with their corrections in Eur. J. Biochem., 152, 1 (1985)).
  • PDB Protein DataBank
  • amino acid residue is intended to include any natural or synthetic amino acid residue, and is primarily intended to indicate an amino acid residue contained in the group consisting of the 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 (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y)
  • G124 indicates that position 124 is occupied by a glycine residue in the amino acid sequence shown in SEQ ID NO:1.
  • G124R indicates that the glycine residue of position 124 has been substituted with an arginine residue.
  • Alternative substitutions are indicated with a “/”, e.g. N145S/T means an amino acid sequence in which asparagine in position 145 is substituted with either serine or threonine.
  • Multiple substitutions are indicated with a “+”, e.g.
  • K143N+N145S/T means an amino acid sequence which comprises a substitution of the lysine residue in position 143 with an asparagine residue and a substitution of the asparagine residue in position 145 with a serine or a threonine residue.
  • Insertion of an additional amino acid residue e.g. insertion of an alanine residue after G124, is indicated by G124GA. Insertion of two additional alanine residues after G124 is indicated by G124GAA, etc.
  • the term “inserted in position X” 1 or “inserted at position X” means that, the amino acid residue(s) is (are) inserted between amino acid residue X and X+1.
  • a deletion of an amino acid residue is indicated by an asterix. For example, deletion of the glycine residue in position 124 is indicated by G124*.
  • the term “differs from” as used in connection with specific mutations is intended to allow for additional differences being present apart from the specified amino acid difference.
  • the polypeptide may contain other modifications that are not necessarily related to this effect.
  • amino acid sequence of the polypeptide variant of the invention may, if desired, contain other alterations, i.e. other substitutions, insertions or deletions.
  • these may, for example, include truncation of the N- and/or C-terminus by one or more amino acid residues (e.g. by 1-10 amino acid residues), or addition of one or more extra residues at the N- and/or C-terminus, e.g. addition of a methionine residue at the N-terminus or introduction of a cysteine residue near or at the C-terminus, as well as “conservative amino acid substitutions”, i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids.
  • nucleotide sequence is intended to indicate a consecutive stretch of two or more nucleotide molecules.
  • the nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • vector refers to a plasmid or other nucleotide sequences that are capable of replicating within a host cell or being integrated into the host cell genome, and as such, are useful for performing different functions in conjunction with compatible host cells (a vector-host system) to facilitate the cloning of the nucleotide sequence, i.e. to produce useful quantities of the sequence, to direct the expression of the gene product encoded by the sequence and to integrate the nucleotide sequence into the genome of the host cell.
  • the vector will contain different components depending upon the function it is to perform.
  • Cell “Cell”, “host cell”, “cell line” and “cell culture” are used interchangeably herein and all such terms should be understood to include progeny resulting from growth or culturing of a cell.
  • Transformation and “transfection” are used interchangeably to refer to the process of introducing DNA into a cell.
  • “Operably linked” refers to the covalent joining of two or more nucleotide sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed.
  • “operably linked” means that the nucleotide sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.
  • modification or “amino acid modification” is intended to cover replacement of an amino acid side chain, substitution of an amino acid residue, deletion of an amino acid residue or insertion of an amino acid residue.
  • introduce refers to introduction of an amino acid residue, in particular by substitution of an existing amino acid residue, or alternatively by insertion of an additional amino acid residue.
  • removal refers to removal of an amino acid residue, in particular by substitution of the amino acid residue to be removed by another amino acid residue, or alternatively by deletion (without substitution) of the amino acid residue to be removed.
  • the term “activity” should be understood as the relevant activity associated with the assay in which the activity is actually measured.
  • a variant of the invention in its activated form, should have at least 10% of the amidolytic activity of rhFVIIa when assayed in the “Amidolytic Assay” described herein.
  • the variant, in its activated form has at least 20% of the amidolytic activity of rhFVIIa, such as at least 30%, e.g. at least 40%, more preferably at least 50%, such as at least 60%, e.g.
  • the variant in its activated form, has substantially the same amidolytic activity as rhFVIIa, such as an amidolytic activity of 75-125% of the amidolytic activity of rhFVIIa.
  • clotting activity refers to the activity measured in the “Whole Blood Assay” described herein, i.e. the time needed to obtain clot formation. Thus, a lower clotting time corresponds to a higher clotting activity.
  • the term “increased clotting activity” is used to indicate that the clotting time of the polypeptide variant is statistically significantly decreased relative to that generated by rhFVIIa or [P10Q+K32E]rhFVIIa as determined under comparable conditions and when measured in the “Whole Blood Assay” described herein.
  • the term “activity” is also used in connection with the variants' capability of activating FX to FXa. This activity is also denoted “FX activation activity” or “FXa generation activity” and may be determined in the “TF-independent Factor X Activation Assay” described herein.
  • the term “increased FX activation activity” or “increased FXa generation activity” is used to indicate that a variant of the invention, in its activated form, has a statistically significantly increased capability to activate FX to FXa as compared to a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa.
  • a variant of the invention in its activated form has an increased FX activation activity may conveniently be determined in the “TF-independent Factor X Activation Assay” described herein.
  • immunogenicity as used in connection with a given substance is intended to indicate the ability of the substance to induce a response from the immune system.
  • the immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (10 th Edition, Blackwell) for further definition of immunogenicity). Normally, reduced antibody reactivity will be an indication of reduced immunogenicity.
  • the immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.
  • the term “functional in vivo half-life” is used in its normal meaning, i.e. the time at which 50% of the biological activity of the polypeptide is still present in the body/target organ, or the time at which the activity of the polypeptide is 50% of the initial value.
  • serum half-life may be determined, i.e. the time at which 50% of the polypeptide circulates in the plasma or bloodstream prior to being cleared. Determination of serum half-life is often more simple than determining the functional in vivo half-life, and the magnitude of serum half-life is usually a good indication of the magnitude of functional in vivo half-life.
  • Alternative terms to 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 systems (RES), kidney, spleen or liver, by tissue factor, SEC receptor or other receptor mediated elimination, or by specific or unspecific proteolysis. Normally, clearance depends on size (relative to the cutoff for glomerular filtration), charge, attached carbohydrate chains, and the presence of cellular receptors for the protein.
  • the functionality to be retained is normally selected from procoagulant, proteolytic or receptor binding activity.
  • the functional in vivo half-life and the serum half-life may be determined by any suitable method known in the art.
  • the term “increased” as used about the functional in vivo half-life or serum half-life is used to indicate that the relevant half-life of the polypeptide variant is statistically significantly increased relative to that of as reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, as determined under comparable conditions (typically determined in an experimental animal, such as rats, rabbits, pigs or monkeys).
  • AUC iv or “Area Under the Curve when administered intravenously” is used in its normal meaning, i.e. as the area under the activity in serum-time curve, where the polypeptide variant has been administered intravenously, in particular when administered intravenously in rats.
  • the activity measured is the “clotting activity” as defined above.
  • the same amount of activity should be administered. Consequently, the AUC iv -values are typically normalized (i.e. corrected for differences in the injected dose) and expressed as AUC iv /dose administered.
  • the term “reduced sensitivity to proteolytic degradation” is primarily intended to mean that the polypeptide variant has reduced sensitivity to proteolytic degradation in comparison to hFVIIa, rhFVIIa or [P10Q+K32E]rhFVIIa as determined under comparable conditions.
  • the proteolytic degradation is reduced by at least 10% (e.g. by 10-25% or by 10-50%), such as at least 25% (e.g. by 25-50%, by 25-75% or by 25-100%), more preferably by at least 35%, such as at least 50%, (e.g. by 50-75% or by 50-100%) even more preferably by at least 60%, such as by at least 75% (e.g. by 75-100%) or even at least 90%.
  • Renal clearance is used in its normal meaning to indicate any clearance taking place by the kidneys, e.g. by glomerular filtration, tubular excretion or degradation in the tubular cells. Renal clearance depends on physical characteristics of the polypeptide, including size (diameter), hydrodynamic volume, symmetry, shape/rigidity, and charge. Normally, a molecular weight of about 67 kDa is considered to be a cut-off-value for renal clearance. Renal clearance may be established by any suitable assay, e.g. an established in vivo assay. Typically, renal clearance is determined by administering a labelled (e.g. radiolabelled or fluorescence labelled) polypeptide to a patient and measuring the label activity in urine collected from the patient.
  • a labelled e.g. radiolabelled or fluorescence labelled
  • Reduced renal clearance is determined relative to a corresponding reference polypeptide, e.g. rhFVIIa or [P10Q+K32E]rhFVIIa, under comparable conditions.
  • the renal clearance rate of the polypeptide variant is reduced by at least 50%, preferably by at least 75%, and most preferably by at least 90% compared to rhFVIIa or [P10Q+K32E]rhFVIIa.
  • tissue factor binding site tissue factor binding site
  • active site region active site region
  • ridge of the active site binding cleft are defined with reference to Example 1.
  • 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).
  • negatively charged amino acid residue includes the following amino acid residues: Aspartic acid (D) and glutamic acid (E).
  • positively charged amino acid residue includes the following amino acid residues: Lysine (K), arginine (R) and histidine (H).
  • the modifications performed in the Gla domain of the parent polypeptide preferably provide the resulting molecule with an increased phospholipid membrane binding affinity, an improved capability to activate FX to Fxa, and/or an increased clotting activity.
  • the variants of the invention may also have a reduced tissue factor binding affinity and a reduced activity when bound to tissue factor.
  • medical treatment with a polypeptide variant according to the invention may provide advantages over the currently available rhFVIIa compound (NovoSeven®), such as a lower dose, increased efficacy and/or faster action.
  • tissue factor-independent variants i.e. variants that have a reduced activity when bound to tissue factor compared to wild-type human Factor VIIa, may offer certain safety advantages in terms of reduced risk of undesired blood clot formation (e.g. thrombosis or thromboembolism), in particular when used for treatment of acute uncontrolled bleeding events such as trauma, including traumatic brain injury, or intracerebral haemorrhage.
  • the polypeptide variant, in its activated form and when compared to a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa has an increased FX activation activity, in particular when assayed in a tissue factor-independent assay, such as the “TF-independent Factor X Activation Assay” disclosed herein.
  • a tissue factor-independent assay such as the “TF-independent Factor X Activation Assay” disclosed herein.
  • the ratio between the FX activation activity of the polypeptide variant, in its activated form, and the FX activation activity of a reference molecule is at least 1.25 when assayed in the “TF-independent Factor X Activation Assay” disclosed herein. More preferably, this ratio is at least 1.5, such as at least 1.75, e.g. at least 2, even more preferably at least 3, such as at least 4, most preferably at least 5.
  • the ratio between the FX activation activity of the polypeptide variant, in its activated form, and the FX activation activity of rhFVIla is preferably at least about 5, typically at least about 10, when assayed in the “TF-independent Factor X Activation Assay” disclosed herein, such as at least about 15 or 20.
  • the variants of the invention possess an increased clotting activity (i.e. a reduced clotting time) as compared to rhFVIIa or [P10Q+K32E]rhFVIIa.
  • the ratio between the time to reach clot formation for the variant (t variant ) and the time to reach clot formation for rhFVIIa (t wt ) or [P10Q+K32E]rhFVIIa (tP10Q+K32E) is at the most 0.9 when assayed in the “Whole Blood Assay” described herein. More preferably this ratio is at the most 0.75, such as 0.7, even more preferably the ratio is at the most 0.6, and most preferably the ratio is at the most 0.5.
  • the present invention relates in a first aspect to a FVII or FVIIa polypeptide variant 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 has been introduced by substitution in position 34.
  • the hydrophobic amino acid residue to be introduced in 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.
  • 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.
  • the variant comprises substitutions in both of positions 10 and 32, such as P10Q+K32E.
  • the variant comprises the substitutions P10Q+K32E+A34L.
  • the variant further comprises an insertion of at least one (typically one) amino acid residue between position 3 and 4. It is preferred that the inserted amino acid residue is a hydrophobic amino acid residue. Most preferably the insertion is A3AY. Accordingly, in a particular interesting embodiment of the invention, the variant comprises the modifications A3AY+P10Q+K32E+A34L.
  • the variant may comprise a further substitution in position 33.
  • a hydrophobic amino acid residue is introduced by 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.
  • the Gla domain should not be modified to such an extent that the membrane binding properties are impaired. Accordingly, it is preferred that no modifications are made in the residues that become ⁇ -carboxylated, i.e. it is preferred that no modifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35.
  • non-polypeptide moieties such as sugar moieties and/or PEG groups, are introduced in the Gla domain. Consequently, it is preferred that no modifications are made in the Gla domain that create an in vivo N-glycosylation site.
  • the invention relates in a second aspect to a FVII or FVIIa polypeptide variant 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.
  • the amino acid residue top be introduced by substitution in position 36 is a negatively charged amino acid residue, e.g. R36E or R36D, in particular R36E.
  • 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.
  • the variant comprises substitutions in both of positions 10 and 32, such as P10Q+K32E.
  • the variant of the invention may further contain a substitution in position 38. It is preferred that a negatively charged amino acid residue is introduced by substitution in position 38, e.g. K38E or K38D, in particular K38E.
  • interesting variants are those that comprise the following substitutions P10Q+K32E+R36E or P10Q+K32E+R36E+K38E.
  • the variant further comprises an amino acid substitution in position 34 (i.e. the resulting variant comprises substitutions in the following residues 10+32+34+36 or 10+32+34+36+38).
  • a negatively charged amino acid residue is introduced by substitution in position 34, e.g. A34E or A34D.
  • preferred variants are those that comprise the following substitutions P10Q+K32E+A34E+R36E or P10Q+K32E+A34D+R36E+K38E.
  • the variant further comprises an insertion of at least one (typically one) amino acid residue between position 3 and 4. It is preferred that the inserted amino acid residue is a hydrophobic amino acid residue. Most preferably the insertion is A3AY.
  • the variant may comprise a further substitution in position 33.
  • a hydrophobic amino acid residue is introduced by substitution in position 33, in particular D33F.
  • the Gla domain may also contain modifications in other positions, in particular in positions 8, 1.1 and 28, such as R28F or R28E.
  • the Gla domain should not be modified to such an extent that the membrane binding properties are impaired. Accordingly, it is preferred that no modifications are made in the residues that become ⁇ -carboxylated, i.e. it is preferred that no modifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35.
  • non-polypeptide moieties such as sugar moieties and/or PEG groups, are introduced in the Gla domain. Consequently, it is preferred that no modifications are made in the Gla domain that create an in vivo N-glycosylation site.
  • the present invention relates in a third aspect to a FVII or FVIIa polypeptide variant 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 an amino acid substitution in position 10, 32 and at least one further amino acid substitution in a position selected from the group consisting of position 74, 77 and 116.
  • amino acid substitution in position 10 is P10Q and the amino acid substitution in position 32 is K32E
  • substitution in position 74, 77 or 116 is selected from the group consisting of P74S, E77A and E116D.
  • the variant further comprises an amino acid substitution in position 34.
  • a negatively charged amino acid residue is introduced by substitution in position 34, e.g. A34E or A34D, in particular A34E.
  • the variant further comprises an insertion of at least one (typically one) amino acid residue between position 3 and 4. It is preferred that the inserted amino acid residue is a hydrophobic amino acid residue. Most preferably the insertion is A3AY.
  • variants comprising the following modifications A3AY+P10Q+K32E+E116D, A3AY+P10Q+K32E+E77A and P10Q+K32E+A34E+P74S.
  • the variant may comprise a further substitution in position 33.
  • a hydrophobic amino acid residue is introduced by 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.
  • the Gla domain should not be modified to such an extent that the membrane binding properties are impaired, i.e. preferably no modifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35, and it is preferred that an in vivo N-glycosylation site is not created in the Gla domain.
  • a molecule with a longer circulation half-life and/or increased bioavailability would decrease the number of necessary administrations.
  • a longer circulation half-life and/or increased bioavailability such as an increased Area Under the Curve as compared to rhFVIIa when administered intravenously
  • bioavailability such as an increased Area Under the Curve as compared to rhFVIIa when administered intravenously
  • a further object of the present invention is to provide improved FVII or FVII molecules (FVII or FVIIa variants) with an increased bioavailability (such as an increased Area Under the Curve as compared to a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, when administered intravenously) and which are capable of activating factor X to factor Xa (without binding to tissue factor) more efficiently than a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa (thereby being able to treat uncontrolled bleadings, such as a trauma, or chronic conditions such as hemophilia more efficiently).
  • an increased bioavailability such as an increased Area Under the Curve as compared to a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, when administered intravenously
  • a reference molecule such as rhFVIIa or
  • interesting variants of the invention are those which, in their activated forms and when compared to a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, generate an increased Area Under the Curve when administered intravenously (AUC iv ). This can conveniently be determined by intravenous administration in rats. More particularly, interesting variants of the present invention are those where the ratio between the AUC iv of said variant, in its actvated form, and the AUC iv of a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, is 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, even more preferably at least 4, such as at least 5, in particular when administered (intravenously) in rats.
  • the ratio between the functional in vivo half-life or the serum half-life for the variant, in its activated form, and the functional in vivo half-life or the serum half-life for a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa is at least 1.25.
  • the ratio between the relevant half-life for the variant, in its activated form, and the relevant half-life for the 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, even more preferably at least 3, such as at least 4, e.g. at least 5.
  • One way to increase the circulation half-life of a protein is to ensure that renal clearance of the protein is reduced. This may be achieved by conjugating the protein to a chemical moiety which is capable of conferring reduced renal clearance to the protein, e.g. polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • attachment of a chemical moiety to the protein or substitution of amino acids exposed to proteolysis may effectively block a proteolytic enzyme from contact that otherwise leads to proteolytic degradation of the protein.
  • proteolytic degradation is thus a major obstacle for obtaining a preparation in solution as opposed to a lyophilized product.
  • the advantage of obtaining a stable soluble preparation lies in easier handling for the patient, and, in the case of emergencies, quicker action, which potentially can become life saving.
  • Attempts to prevent proteolytic degradation by site directed mutagenesis at major proteolytic sites have been disclosed in WO 88/10295.
  • WO 01/58935 discloses a number of suitable modifications leading to an increase in AUC iv , functional in vivo half-life and/or serum half-life.
  • the variants disclosed in WO 01/58935 are the result of a generally new strategy for developing improved FVII or FVIIa molecules, which may also be used for the parent FVII or FVIIa polypeptide of the present invention.
  • the alteration embraces removal as well as introduction of amino acid residues comprising an attachment group for the non-polypeptide, moiety of choice.
  • the polypeptide variant may comprise other substitutions that are not related to introduction and/or removal of amino acid residues comprising an attachment group for the non-polypeptide moiety.
  • polypeptide variant may be attached to a serine proteinase inhibitor to inhibit the catalytic site of the polypeptide variant.
  • a serine proteinase inhibitor to inhibit the catalytic site of the polypeptide variant.
  • one or more of the amino acid residues present in the catalytic site may be mutated in order to render the resulting variant inactive.
  • S344A is an amino acid residue present in the catalytic site.
  • amino acid residue comprising an attachment group for a non-polypeptide moiety is selected on the basis of the nature of the non-polypeptide moiety of choice and, in most instances, on the basis of the method in which conjugation between the polypeptide variant and the non-polypeptide moiety is to be achieved.
  • amino acid residues comprising an attachment group may be selected from the group consisting of lysine, cysteine, aspartic acid, glutamic acid, histidine, and tyrosine, preferably lysine, cysteine, aspartic acid and glutamic acid, more preferably lysine and cysteine, in particular cysteine.
  • the position of the amino acid residue to be modified is preferably located at the surface of the parent FVII or FVIIa polypeptide, and more preferably occupied by an amino acid residue which has at least 25% of its side chain exposed to the surface (as defined in Example 1 herein), preferably at least 50% of its side chain exposed to the surface (as defined in Example 1 herein).
  • Such positions have been identified on the basis of an analysis of a 3D structure of the hFVII or hFVIIa molecule as described in WO 01/58935.
  • the position to be modified is preferably selected from a part of the FVII or FVIIa molecule that is located outside the tissue factor binding site, and/or outside the active site region, and/or outside the ridge of the active site binding cleft. These sites/regions are identified in Example 1 herein and in WO 01/58935.
  • the relevant amino acid residue comprising such group and occupying a position as defined above is preferably substituted with a different amino acid residue that does not comprise an attachment group for the non-polypeptide moiety in question.
  • the amino acid residue to be removed is one to which conjugation is disadvantageous, e.g. an amino acid residue located at or near a functional site of the polypeptide (since conjugation at such a site may result in inactivation or reduced activity of the resulting conjugate due to, e.g., impaired receptor recognition).
  • the term “functional site” is intended to indicate one or more amino acid residues which is/are essential for or otherwise involved in the function or performance of FVII or FVIIa.
  • the functional site may be determined by methods known in the art and is preferably identified by analysis of a structure of the FVIIa-tissue factor complex (See Banner et al., Nature 1996; 380:41-46).
  • an amino acid residue comprising such group is introduced into the relevant position, preferably by substitution of the amino acid residue occupying such position.
  • the exact number of attachment groups present and available for conjugation in the FVII or FVIIa polypeptide is dependent on the effect desired to be achieved by the conjugation.
  • the effect to be obtained is, e.g., dependent on the nature and degree of conjugation (e.g. the identity of the non-polypeptide moiety, the number of non-polypeptide moieties desirable or possible to conjugate to the polypeptide variant, where they should be conjugated or where conjugation should be avoided, etc.).
  • the FVII or FVIIa variant comprises an amino acid sequence which differs in 1-10 amino acid residues from amino acid residues 46-406 shown in SEQ ID NO:1, typically in 1-8 or in 2-8 amino acid residues, e.g. in 1-5 or in 2-5 amino acid residues, such as in 1-4 or in 1-3 amino acid residues, e.g. in 1, 2 or 3 amino acid residues from amino acid residues 46-406 shown in SEQ ID NO:1.
  • the polypeptide variant of the invention may contain 1-10 (additional) non-polypeptide moieties, typically 1-8 or 2-8 (additional) non-polypeptide moieties, preferably 1-5 or 2-5 (additional) non-polypeptide moieties, such as 1-4 or 1-3 (additional) non-polypeptide moieties, e.g. 1, 2 or 3 (additional) non-polypeptide moieties. It will be understood that such additional non-polypeptide moieties are covalently attached to an attachment group located outside the Gla domain.
  • Non-polypeptide Moiety is a Sugar Moiety
  • an attachment group for a 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 and/or removed, preferably introduced, in a position located outside the Gla domain.
  • naturally occurring glycosylation site covers the glycosylation sites at postions N145, N322, S52 and S60.
  • the term “naturally occurring in vivo O-glycosylation site” includes the positions S52 and S60, whereas the term “naturally occurring in vivo N-glycosylation site” includes positions N145 and N322.
  • the non-polypeptide moiety is a sugar moiety and the introduced attachment 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.
  • the introduced attachment 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.
  • 1-10 glycosylation sites, in particular in vivo N-glycosylation sites have been introduced, preferably 1-8, 1-6, 1-4 or 1-3 glycosylation sites, in particular in vivo N-glycosylation sites, have been introduced in one or more positionss located outside the Gla domain.
  • polypeptide variant wherein the polypeptide variant comprises one or more glycosylation sites, the polypeptide variant must be expressed in a host cell capable of attaching sugar (oligosaccharide) moieties at the glycosylation site(s) or alternatively subjected to in vitro glycosylation.
  • sugar oligosaccharide
  • Examples of glycosylating host cells are given in the section further below entitled “Coupling to a sugar moiety”.
  • positions wherein the glycosylation sites, in particular in vivo N-glycosylation sites, may be introduced include amino acid residues having at least 25% of their side chain exposed to the surface (as defined in Example 1 herein), such as at least 50% of the side chain exposed to the surface.
  • the position is preferably selected from a part of the molecule that is located outside the tissue factor binding site and/or the active site region and/or outside the ridge of the active site cleft, as defined in Example 1 herein.
  • substitutions creating an in vivo N-glycosylation site include a substitution selected from the group consisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, 1205S, 1205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T, R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N, D334N and combinations thereof.
  • 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.
  • 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, in particular one, two or three of T106N, I205T and V253N.
  • substitutions selected from the group consisting of A51N+G58N, A51N+T106N, A51N+K109N, A51N+G124N, A51N+K143N+N145T, A51N+A175T, A51N+I205T, A51N+V253N, A51N+T267N+S269T, A51N+S314N+K316T, A51N+R315N+V317T, A51N+K316N+G318T, A51N+G318N, A51N+D334N, G58N+T106N, G58N+K109N, G58N+G124N, G58N+K143N+N
  • substitutions are selected from the group consisting of T106N+A175T, T106N+I205T, T106N+V253N, T106N+T267N+S269T, A175T+I205T, A175T+V253N, A175T+T267N+S269T, I205T+V253N, I205T+T267N+S269T and V253N+T267N+S269T, even more preferably from the group consisting of T106N+I205T, T106N+V253N and I205T+V253N.
  • three or more (such as three) in vivo N-glycosylation sites have been introduced by substitution.
  • preferred substitutions creating three in vivo N-glycosylation sites include substitutions selected from the group consisting of I205T+V253N+T267N+S269T and T106N+I205T+V253N.
  • the in vivo N-glycosylation site is introduced in a position which does not form part of the tissue factor binding site, the active site region or the ridge of the active site binding cleft as defined herein.
  • any of the modifications mentioned in the above sections may be combined with each other, in addition to being combined with the above-described substitutions in position 34 and/or 36, in particular A34E/L and/or R36E, and preferably in combination with the above-described substitutions in position 10 and/or 32, in particular P10Q and/or K32E.
  • preferred modifications include one, two or three of T106N, I205T and V253N, in particular two of these modifications, i.e. T106N+I205T, T106N+V253N or I205T+V253N.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34E+R36E+T106N+I205T.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34E+R36E+T106N+V253N.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34E+R36E+I205T+V253N.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+T106N+I205T.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+T106N+V253N.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+I205T+V253N.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+R36E+T106N+I205T.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+R36E+T106N+V253N.
  • the FVII or FVIIa variant comprises the modifications P10Q+K32E+A34L+R36E+I205T+V253N.
  • any one or more of these modifications may in addition be combined with insertion of at least one amino acid residue, typically a single amino acid residue, between position 3 and 4, where the inserted residue is preferably a hydrophobic amino acid residue. Most preferably the insertion is A3AY.
  • the FVII or FVIIa variant comprises modifications selected from:
  • the FVII or FVIIa variant may, in addition to the modifications described in the sections above, also contain mutations which are already known to increase the intrinsic activity of the polypeptide, for example those described in WO 02/22776.
  • the variant may comprise at least one modification in a position selected from the group consisting of 157, 158, 296, 298, 305, 334, 336, 337 and 374.
  • preferred substitutions include substitutions selected from the group consisting of V158D, E296D, M298Q, L305V and K337A.
  • substitutions are selected from the group consisting of V158D+E296D+M298Q+L305V+K337A, V158D+E296D+M298Q+K337A, V158D+E296D+M298Q+L305V, V158D+E296D+M298Q, M298Q, L305V+K337A, L305V and K337A.
  • the FVII or FVIIa variant may, in addition to the modifications described in the sections above, also contain other mutations, such as the substitution K341Q disclosed by Neuenschwander et al, Biochemistry, 1995; 34:8701-8707.
  • Other possible additional substitutions include D196K, D196N, G237L, G237GAA and combinations thereof.
  • a conjugated variant according to the invention may be produced by culturing an appropriate host cell under conditions conducive for the expression of the variant polypeptide, and recovering the variant polypeptide, wherein a) the variant polypeptide comprises at least one N- or O-glycosylation site and the host cell is an eukaryotic host cell capable of in vivo glycosylation, and/or b) the variant polypeptide is subjected to conjugation to a non-polypeptide moiety in vitro.
  • the polymer molecule to be coupled to the variant polypeptide may be any suitable polymer molecule, such as a natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300-100,000 Da, such as about 500-20,000 Da, more preferably in the range of about 500-15,000 Da, even more preferably in the range of about 2-12 kDa, such as in the range of about 3-10 kDa.
  • a suitable polymer molecule such as a natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300-100,000 Da, such as about 500-20,000 Da, more preferably in the range of about 500-15,000 Da, even more preferably in the range of about 2-12 kDa, such as in the range of about 3-10 kDa.
  • homo-polymers examples include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH 2 ) and a polycarboxylic acid (i.e. poly-COOH).
  • a hetero-polymer is a polymer comprising different coupling groups, such as a hydroxyl group and an amine group.
  • suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid 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.
  • PEO polyalkylene oxide
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PVA poly-vinyl alcohol
  • PVA poly-carboxylate
  • poly-(vinylpyrolidone) polyethylene-co-maleic acid anhydride
  • PEG is the preferred polymer molecule, since it has only few reactive groups capable of cross-linking compared to, e.g., polysaccharides such as dextran.
  • monofunctional PEG e.g. methoxypolyethylene glycol (mPEG)
  • mPEG methoxypolyethylene glycol
  • the hydroxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)).
  • Suitable activated polymer molecules are commercially available, e.g. from Nektar Therapeutics, Huntsville, Ala., USA, or from PolyMASC Pharmaceuticals plc, UK.
  • activated linear or branched polymer molecules for use in the present invention are described in the Nektar Molecule Engineering Catalog 2003 (Nektar Therapeutics), incorporated herein by reference.
  • 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 of which are incorporated herein by reference. Additional publications disclosing useful polymer molecules, PEGylation chemistries and conjugation methods are listed in WO 01/58935 and WO 03/093465.
  • activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG).
  • vinylsulfone-PEG VS-PEG
  • MAL-PEG maleimide-mPEG
  • OPSS-PEG orthopyridyl-disulfide-mPEG
  • PEG or mPEG polymers will have a size of about 5 kDa, about 10 kD, about 12 kDa or about 20 kDa.
  • the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the variant polypeptide (examples of which are given further above), as well as the functional groups of the polymer (e.g. being amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide, vinysulfone or haloacetate).
  • the PEGylation may be directed towards conjugation to all available attachment groups on the variant polypeptide (i.e. such attachment groups that are exposed at the surface of the polypeptide) or may be directed towards one or more specific attachment groups, e.g. the N-terminal amino group as described in U.S. Pat. No. 5,985,265 or to cysteine residues.
  • the conjugation may be achieved in one step or in a stepwise manner (e.g. as described in WO 99/55377).
  • the FVII or FVIIa variant is usually treated with a reducing agent, such as dithiothreitol (DDT) prior to PEGylation.
  • DDT dithiothreitol
  • the reducing agent is subsequently removed by any conventional method, such as by desalting. Conjugation of PEG to a cysteine residue typically takes place in a suitable buffer at pH 6-9 at temperatures varying from 4° C. to 25° C. for periods up to 16 hours.
  • the PEGylation is designed so as to produce the optimal molecule with respect to the number of PEG molecules attached, the size and form of such molecules (e.g. whether they are linear or branched), and the attachment site(s) in the variant polypeptide.
  • the molecular weight of the polymer to be used may e.g. be chosen on the basis of the desired effect to be achieved.
  • the polymer molecule which may be linear or branched, has a high molecular weight, preferably about 10-25 kDa, such as about 15-25 kDa, e.g. about 20 kDa.
  • the polymer conjugation is performed under conditions aimed at reacting as many of the available polymer attachment groups as possible with polymer molecules. This is achieved by means of a suitable molar excess of the polymer relative to the polypeptide.
  • the molar ratios of activated polymer molecules to polypeptide are up to about 1000-1, such as up to about 200-1, or up to about 100-1. In some cases the ration may be somewhat lower, however, such as up to about 50-1, 10-1, 5-1, 2-1 or 1-1 in order to obtain optimal reaction.
  • linker it is also contemplated according to the invention to couple the polymer molecules to the polypeptide through a linker. Suitable linkers are well known to the skilled person; see also WO 01/58935.
  • the amino acid sequence of the variant polypeptide e.g. the amino acid sequence of the variant polypeptide, the nature of the activated PEG compound being used and the specific PEGylation conditions, including the molar ratio of PEG to polypeptide, varying degrees of PEGylation may be obtained, with a higher degree of PEGylation generally being obtained with a higher ratio of PEG to variant polypeptide.
  • the PEGylated variant polypeptides resulting from any given PEGylation process will, however, normally comprise a stochastic distribution of conjugated polypeptide variants having slightly different degrees of PEGylation.
  • the expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells.
  • the host cell is a mammalian cell, such as a CHO cell, BHK or HEK, e.g. HEK 293, cell, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae or Pichia pastoris, or any of the host cells mentioned hereinafter.
  • Covalent in vitro coupling of sugar moieties such as dextran
  • amino acid residues of the variant polypeptide may also be used, e.g. as described, for example in WO 87/05330 and in Aplin et al., CRC Crit Rev. Biochem, pp.259-306, 1981. See also WO 03/093465 for further information on in vitro glycosylation of variants of FVII or FVIIa.
  • Attachment of a serine protease inhibitor can be performed in accordance with the method described in WO 96/12800.
  • the polypeptide variant of the present invention may be produced by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding the polypeptide variant and expressing the sequence in a suitable transformed or transfected host.
  • the host cell is a gamma-carboxylating host cell such as a mammalian cell.
  • polypeptide variants of the invention may be produced, albeit less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
  • a nucleotide sequence encoding a polypeptide of the invention may be constructed by isolating or synthesizing a nucleotide sequence encoding the parent FVII, such as hFVII with the amino acid sequence shown in SEQ ID NO:1 and then changing the nucleotide sequence so as to effect introduction (i.e. insertion or substitution) or removal (i.e. deletion or substitution) of the relevant amino acid residue(s).
  • nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with conventional methods.
  • nucleotide sequence is prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
  • oligonucleotides coding for portions of the desired polypeptide may 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).
  • the individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.
  • nucleotide sequence encoding the polypeptide is inserted into a recombinant vector and operably linked to control sequences necessary for expression of the FVII in the desired transformed host cell.
  • 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.
  • the vector is one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the 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.
  • suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature. Detailed information on suitable vectors for expressing FVII may be found in WO 01/58935, incorporated by reference.
  • control sequences is defined herein to include all components which are necessary or advantageous for the expression of the polypeptide variant of the invention.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide variant.
  • control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter.
  • control sequences may be used in the present invention, e.g. any of the control sequences disclosed in WO 01/58935, incorporated by reference.
  • the nucleotide sequence of the invention encoding a polypeptide variant may optionally include a nucleotide sequence that encode a signal peptide.
  • the signal peptide is present when the polypeptide variant is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide variant.
  • the signal peptide may be homologous (i.e. normally associated with hFVII) or heterologous (i.e. originating from another source than hFVII) to the polypeptide or may be homologous or heterologous to the host cell, i.e. a signal peptide normally expressed from the host cell or one which is not normally expressed from the host cell.
  • suitable signal peptides see WO 01/58935.
  • Any suitable host may be used to produce the polypeptide variant, including bacteria (although not particularly preferred), fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants.
  • Mammalian cells are preferred.
  • bacterial host cells include gram-positive bacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, such as strains of E. coli.
  • suitable filamentous fungal host cells include strains of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma.
  • yeast host cells examples include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. Polymorpha or Yarrowia.
  • suitable insect host cells include a Lepidoptora cell line, such as Spodoptera frugiperda (Sf9 or Sf2 1) or Trichoplusioa ni cells (High Five) (U.S. Pat. No. 5,077,214).
  • suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g.
  • CHO-K1 ATCC CCL-61
  • Green Monkey cell lines COS
  • COS Green Monkey cell lines
  • mouse cells e.g. NS/O
  • Baby Hamster Kidney (BHK) cell lines e.g. ATCC CRL-1632 or ATCC CCL-10
  • human cells e.g. HEK 293 (ATCC CRL-1573)
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md.
  • mammalian cells such as a CHO cell, may be modified to express sialyltransferase, e.g. 1,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335, in order to provide improved glycosylation of the polypeptide variant.
  • polypeptide variant of the invention in particular a PACE (paired basic amino acid converting enzyme) (e.g. as described in U.S. Pat. No. 5,986,079), such as a Kex2 endoprotease (e.g. as described in WO 00/28065).
  • PACE paired basic amino acid converting enzyme
  • Kex2 endoprotease e.g. as described in WO 00/28065
  • the present invention relates to a composition, in particular to a pharmaceutical composition, comprising a polypeptide variant of the invention and a pharmaceutically acceptable carrier or excipient.
  • polypeptide variant or the pharmaceutical composition according to the invention may be used as a medicament.
  • the polypeptide variants of the invention, or the pharmaceutical composition of the invention are particular useful for the treatment of uncontrollable bleeding events in trauma patients, thrombocytopenic patients, patients in anticoagulant treatment, and cirrhosis patients with variceal bleeding, or other upper gastrointestinal bleedings, in patients undergoing orthotopic liver transplantation or liver resection (allowing for transfusion free surgery), or in hemophilia patients.
  • Trauma is defined as an injury to living tissue caused by an extrinsic agent. It is the 4 th leading cause of death in the US and places a large financial burden on the economy.
  • Trauma may be caused by numerous events, e.g. traffic accidents, gunshot wounds, falls, machinery accidents, and stab wounds.
  • Cirrhosis of the liver may be caused by direct liver injury, including chronic alcoholism, chronic viral hepatitis (types B, C, and D), and autoimmune hepatitis as well as by indirect injury by way of bile duct damage, including primary biliary cirrhosis, primary sclerosing cholangitis and biliary atresia.
  • Less common causes of cirrhosis include direct liver injury from inherited disease such as cystic fibrosis, alpha-1-antitrypsin deficiency, hemochromatosis, Wilson's disease, galactosemia, and glycogen storage disease. Transplantation is the key intervention for treating late stage cirrhotic patients
  • the present invention relates to a poly eptide variant of the invention for the manufacture of a medicament for the treatment of diseases or disorder wherein. clot formation is desirable.
  • a still further aspect of the present invention relates to a method for treating a mammal having a disease or disorder wherein clot formation is desirable, comprising administering to a mammal in need thereof an effective amount of the polypeptide variant or the pharmaceutical composition of the invention.
  • diseases/disorders wherein increased clot formation is desirable include, but is not limited to, hemorrhages, including brain hemorrhages, as well as patient with severe uncontrolled bleedings, such as trauma. Further examples include patients undergoing living transplantations, patients undergoing resection and patients with variceal bleeding.
  • hemophilia e.g. von Willebrand disease, hemophilia A, hemophilia B or hemophilia C.
  • one particular aspect of the invention relates to use of the polypeptide variants of the invention for the treatment of intracerebral haemorrhage (ICH) or traumatic brain injury (TBI).
  • ICH intracerebral haemorrhage
  • TBI traumatic brain injury
  • ICH intracerebral haemorrhage
  • brain haemorrhage also known as brain haemorrhage, intracranial haemorrhage or haemorrhagic stroke
  • Asian populations is estimated to be 20-30%.
  • ICH is the most deadly form of stroke, for which there currently is no proven effective treatment.
  • ICH also results in very high rates of severe mental and physical disability among survivors.
  • ICH can be distinguished from other types of stroke using a CT scan or MRI, after which treatment may be initiated, although until now the available treatment options have only been symptomatic and largely ineffective. If initiated sufficiently early, however, e.g. within about 3-4 hours of the onset of the haemorrhagic stroke, it is contemplated that treatment with the polypeptide variants of the invention may result in significant improvements in terms of increase survival rates and/or decreased disability rates. In particular, it is contemplated that an increased TF-independent activity, optionally with a reduced TF-dependent activity, obtained by use of the polypeptide variants of the invention, may be advantageous over rhFVIIa (NovoSeven®) by reducing or eliminating the risk of thromboembolic events.
  • rhFVIIa NovoSeven®
  • One embodiment of this aspect of the invention thus relates to a method for treating ICH or TBI, comprising administering to a patient in need thereof an effective amount of a polypeptide variant of the invention as otherwise described above.
  • Another embodiment of this aspect of the invention relates to use of a polypeptide of the invention for the manufacture of a medicament for the treatment of ICH or TBI.
  • this aspect of the invention may generally be defined as a method for treating intracerebral haemorrhage or traumatic brain injury, comprising administering to a patient in need thereof an effective amount of a Factor VII (FVII). or Factor VIIa (FVIla) polypeptide variant having an amino acid sequence comprising 1-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, wherein the FX activation activity of the polypeptide variant, in its activated form, is greater than the FX activation activity of rhFVIIa when assayed in the “TF-independent Factor X Activation Assay” disclosed herein.
  • the ratio between the FX activation activity of the polypeptide variant, in its activated form, and the FX activation activity of rhFVIIa is preferably at least about 5, more preferably at least about 10, such as at least about 15.
  • variants have an amino acid sequence comprising 1-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, and comprise an amino acid substitution in position 10 and/or 32, and optionally at least one further substitution to introduce an in vivo N-glycosylation site.
  • the substitution in position 10 is P10Q and the substitution in position 32 is K32E.
  • the variant includes both substitutions P10Q and K32E, and optionally one or more additional substitutions to introduce at least one in vivo N-glycosylation site, e.g.
  • one, two or three glycosylation sites preferably two glycosylation sites, selected from A51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T, R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N and D334N.
  • the variant includes the substitutions P10Q and K32E as well as two substitutions selected from T106N, I205S/T and V253N, most preferably P10Q+K32E+T106N+V253N.
  • the polypeptide variants of the invention are administered to patients in a therapeutically effective dose, normally one approximately paralleling that employed in therapy with rFVII such as NovoSeven®.
  • therapeutically effective dose herein is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose will depend on the circumstances, and will be ascertainable by one skilled in the art using known techniques. Normally, the dose should be capable of preventing or lessening the severity or spread of the condition or indication being treated.
  • an effective amount of a polypeptide variant or composition of the invention depends, inter alia, upon the disease, the dose, the administration schedule, whether the polypeptide variant or composition is administered alone or in conjunction with other therapeutic agents, the plasma half-life of the compositions, and the general health of the patient.
  • a suitable dose of the variants of the invention for treatment of ICH, or TBI or other trauma will be in the range of about 20-300 ⁇ g protein per kg body weight, e.g. about 30-250 ⁇ g/kg, such as about 40-200 ⁇ g/kg, e.g. about 60-150 ⁇ g/kg.
  • ICH only a single dose of the of the variants of the invention will generally be indicated, while for TBI or other forms of trauma one or more additional doses may in certain cases be given as needed. Similar dose ranges are also applicable to hemophilia.
  • the polypeptide variant of the invention is preferably administered in a composition including a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient means a carrier or excipient that does not cause any untoward effects in patients to whom it is administered.
  • Such pharmaceutically acceptable carriers and excipients as well as suitable pharmaceutical formulation methods are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).
  • the polypeptide variant of the invention can be used “as is” and/or in a salt form thereof.
  • Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.
  • the pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the polypeptide variant of the invention, either concurrently or in accordance with another treatment schedule. In addition, the polypeptide variant or pharmaceutical composition of the invention may be used as an adjuvant to other therapies.
  • a “patient” for the purposes of the present invention includes both humans and other mammals. Thus, the methods are applicable to both human therapy and veterinary applications, in particular to human therapy.
  • composition comprising the polypeptide variant of the invention may be formulated in a variety of forms, e.g. as a liquid, gel, lyophilized, or as a compressed solid.
  • forms e.g. as a liquid, gel, lyophilized, or as a compressed solid.
  • the preferred form will depend upon the particular indication being treated and will be apparent to one skilled in the art.
  • the pharmaceutical composition comprising the polypeptide variant of the invention may be formulated in lyophilised or stable soluble form.
  • the polypeptide variant may be lyophilised by a variety of procedures known in the art.
  • the polypeptide variant may be in a stable soluble form by the removal or shielding of proteolytic degradation sites as described herein.
  • the advantage of obtaining a stable soluble preparation lies in easier handling for the patient and, in the case of emergencies, quicker action, which potentially can become life saving.
  • the preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art.
  • the administration of the formulations of the present invention can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner.
  • the formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implantation. In some instances the formulations may be directly applied as a solution or spray.
  • a preferred example of a pharmaceutical composition is a solution, in particular an aqueous solution, designed for parenteral administration.
  • pharmaceutical solution formulations are provided in liquid form, appropriate for immediate use, such parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use.
  • the latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts.
  • Such lyophilized preparations are 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.
  • parenterals they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide variant having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are termed “excipients”), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic surfactants or detergents, afitioxidants, and/or other miscellaneous additives such as bulking agents or fillers, chelating agents, antioxidants and cosolvents.
  • excipients for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic surfactants or detergents, afitioxidants, and/or other miscellaneous additives such as bulking agents or fillers, chelating agents, antioxidants and cosolvents.
  • the active site region is defined as any residues having at least one atom within 10 ⁇ of any atom in the catalytic triad (residues H193, D242, S344).
  • Proteolytic degradation can be measured using the assay described in U.S. Pat. No. 5,580,560, Example 5, where proteolysis is autoproteolysis.
  • reduced proteolysis can be tested in an in vivo model using radiolabelled samples and comparing proteolysis of rhFVIIa and the polypeptide variant of the invention by withdrawing blood samples and subjecting these to SDS-PAGE and autoradiography.
  • reduced proteolytic degradation is intended to mean a measurable reduction in cleavage compared to that obtained by rhFVIIa as measured by gel scanning of Coomassie stained SDS-PAGE gels, HPLC or as measured by conserved catalytic activity in comparison to wild type using the tissue factor independent activity assay decribed below.
  • the molecular weight of polypeptide variants is determined by either SDS-PAGE, gel filtration, Western Blots, matrix assisted laser desorption mass spectrometry or equilibrium centrifugation, e.g. SDS-PAGE according to Laemmli, U. K., Nature Vol 227 (1970), pp. 680-85.
  • Phospholipid membrane binding affinity may be determined as described in Nelsestuen et al., Biochemistry, 1977; 30;10819-10824 or as described in Example 1 in U.S. Pat. No. 6,017,882.
  • the molecule to be assayed (either hFVIla, rhFVIIa or the polypeptide variant of the invention in its activated form) is mixed with a source of phospholipid (preferably phosphatidylcholine and phosphatidylserine in a ratio of 8:2) and relipidated Factor X in Tris buffer containing BSA. After a specified incubation time the reaction is stopped by addition of excess EDTA. The concentration of factor Xa is then measured from absorbance change at 405 nm after addition of a chromogenic substrate (S-2222, Chromogenix).
  • a source of phospholipid preferably phosphatidylcholine and phosphatidylserine in a ratio of 8:2
  • relipidated Factor X in Tris buffer containing BSA. After a specified incubation time the reaction is stopped by addition of excess EDTA. The concentration of factor Xa is then measured from absorbance change at 405 nm after addition
  • tissue factor independent activity of rhFVIIa (a wt ) is determined as the absorbance change after 10 minutes and the tissue factor independent activity of the polypeptide variant of the invention (a variant ) is also determined as the absorbance change after 10 minutes.
  • the ratio between the activity of the polypeptide variant, in its activated form, and the activity of rhFVIIa is defined as a variant /a wt .
  • the clotting activity of the FVIIa and variants thereof were measured in one-stage assays and the clotting times were recorded on a Thrombotrack IV coagulometer (Medinor).
  • Factor VII-depleted human plasma American Diagnostica
  • 50 microliters of plasma was then transferred to the coagulometer cups.
  • FVIIa and variants thereof were diluted in Glyoxaline Buffer (5.7 mM barbiturate, 4.3 mM sodium citrate, 117 mM NaCl, 1 mg/ml BSA, pH 7.35). The samples were added to the cup in 50 ul and incubated at 37° C. for 2 minutes.
  • Glyoxaline Buffer 5.7 mM barbiturate, 4.3 mM sodium citrate, 117 mM NaCl, 1 mg/ml BSA, pH 7.35.
  • Thromboplastin (Medinor) was reconstituted with water and CaCl 2 was added to a final concentration of 4.5 mM. The reaction was initiated by adding 100 ⁇ l thromboplastin.
  • the clotting activity of FVIIa and variants thereof were measured in one-stage assays and the clotting times were recorded on a Thrombotrack IV coagulometer (Medimnor). 100 ⁇ l of FVIIa or variants thereof were diluted in a buffer containing 10 mM glycylglycine, 50 mM NaCl, 37.5 mM CaCl 2 , pH 7.35 and transferred to the reaction cup. The clotting reaction was initiated by addition of 50 ⁇ l blood containing 10% 0.13 M tri-sodium citrate as anticoagulant. Data was analysed using Excel or PRISM software.
  • FVIIa is diluted to about 10-90 nM in assay buffer (50 mM Na-Hepes pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 0.1% BSA, 1 U/ml Heparin). Furthermore, soluble TF (sTF) is diluted to 50-450 nM in assay buffer. 120 ⁇ l of assay buffer is mixed with 20 ⁇ l of the FVIIa sample and 20 ⁇ l sTF. After 5 min incubation at room temperature with gentle shaking, followed by 10 min incubation at 37° C., the reaction is started by addition of the S-2288 substrate to 1 mM and the absorption at 405 nm is determined at several time points.
  • assay buffer 50 mM Na-Hepes pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 0.1% BSA, 1 U/ml Heparin.
  • soluble TF sTF
  • 120 ⁇ l of assay buffer is mixed with 20
  • FVII/FVIIa (or variant) concentrations are determined by ELISA.
  • Wells of a microtiter plate are coated with an antibody directed against the protease domain using a solution of 2 ⁇ g/ml in PBS (100 ⁇ l per well). After overnight coating at R.T. (room temperature), the wells are washed 4 times with THT buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.2 0.05% Tween-20). Subsequently, 200 ⁇ l of 1% Casein (diluted from 2.5% stock using 100 mM NaCl, 50 mM Tris-HCl pH 7.2) is added per well for blocking.
  • THT buffer 100 mM NaCl, 50 mM Tris-HCl pH 7.2
  • the wells are emptied, and 100 ⁇ l of sample (optionally diluted in dilution buffer (THT+0.1 % Casein)) is added. After another incubation of 1 hr at room temperature, the wells are washed 4 times with THT buffer, and 100 ⁇ l of a biotin-labelled antibody directed against the EGF-like domain (1 ⁇ g/ml) is added. After another 1 hr incubation at R.T., followed by 4 more washes with THT buffer, 100 ⁇ l of streptavidin-horse radish peroxidase (DAKO A/S, Glostrup, Denmark, 1/10000 diluted) is added.
  • DAKO A/S streptavidin-horse radish peroxidase
  • TMB 3,3′,5,5′-tetramethylbenzidine, Kem-en-Tech A/S, Denmark
  • 100 g of 1 M H 2 SO 4 is added and OD 450nm is determined.
  • a standard curve is prepared using rhFVIIa (NovoSeven®).
  • FVII/FVIIa or variants may be quantified through the Gla domain rather than through the protease domain.
  • wells are coated overnight with an antibody directed against the EGF-like domain and for detection, a calcium-dependent biotin-labelled monoclonal anti-Gla domain antibody is used (2 ⁇ g/ml, 100 ⁇ l per well).
  • a calcium-dependent biotin-labelled monoclonal anti-Gla domain antibody is used (2 ⁇ g/ml, 100 ⁇ l per well).
  • 5 mM CaCl 2 is added to the THT and dilution buffers.
  • the molecule to be assayed (either hFVIIa, rhFVIIa or a variant) is mixed with FVII-depleted platelet poor plasma (PPP) containing either relipidated recombinant tissue factor (such as Innovin from Dade Behring) or phospholipid (phosphatidylcholine and phosphatidylserine in a ratio of 8:2, or phosphatidylcholine, phosphatidylserine and phosphatidylethanol in a ratio of 4:2:4).
  • PPP FVII-depleted platelet poor plasma
  • the reaction is started by addition of a fluoregenic thrombin substrate and calcium chloride.
  • the fluorescence is measured continuously and the thrombin amidolytic activity is determined by calculating the slope of the fluorescence curve (the increase in fluorescence over time). In this way the time until maximum thrombin amidolytic activity is obtained (T max ), and the thrombin generation rate (maximal increase in thrombin activity) and the total thrombin work (area under the curve (AUC)) can be calculated.
  • Frozen citrated FVII-depeleted plasma is thawed in the presence of corn trypsin inhibitor (100 ⁇ g/ml serum) to inhibit the contact pathway of coagulation.
  • corn trypsin inhibitor 100 ⁇ g/ml serum
  • To each well of a 96-well microtiter plate is added 80 ⁇ l plasma and 20 ⁇ l buffer containing rhFVII or variant to be tested in final concentrations of between 0.1 and 100 nM.
  • Recombinant human tissue factor (rTF) is added in 5 ⁇ l assay buffer to a final concentration of 1 pM.
  • the assay buffer consists of 20 mM Hepes, 150 mM NaCl and 60 mg/ml BSA in distilled water. The reaction is started by adding 20 ⁇ l of the substrate solution containing 0.1 M calcium.
  • the assay plate and reagents are pre-warmed to 37° C. and the reaction takes place at this temperature.
  • the fluorimeter used is a BMG Fluormeter with an excitation filter at 390 nm and an emission filter at 460 nm. The fluorescence is measured in each well of 96-well clear bottom plates at 20-40 second intervals over 30-180 minutes. Data are analyzed using PRISM Software.
  • tissue factor binding of factor VII protein was compared at a single concentration of FVIIa or variant to allow a relative comparison of the variants to wild-type.
  • This concentration was determined by means of a standard curve of wild type FVIIa that was flowed over the chip in concentrations between 75 and 0 ⁇ g/ml.
  • FVIIa was removed by addition of 10 mM EDTA. It was determined in this manner that a concentration of 15 ⁇ g/ml gave binding in the linear range.
  • Variants of FVIIa were then flowed over the chip at 15 ⁇ g/ml to determine the relative binding strength of FVIIa or variants to tissue factor.
  • residues in hFVII change their ASA in the complex. These residues were defined as constituting 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 R379.
  • the active site region is defined as any residue having at least one atom within a distance of 10 ⁇ from any atom in the catalytic triad (residues H193, D242, S344): I153, Q167, V168, L169, L170, L171, Q176, L177, C178, G179, G180, T181, V188, V189, S190, A191, A192, H193, C194, F195, D196, K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234, V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246, V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328, D338, S339, C340, K341, G342, D343, S344, G345, G346, P3
  • the ridge of the active site binding cleft region was defined by visual inspection of the FVIIa structure 1FAK.pdb as: N173, A175, K199, N200, N203, D289, R290, G291, A292, P321 and T370.
  • the expression cassette for expression of rhFVII was designed and cloned as described in Example 2 of WO 01/58935.
  • Sequence overhang extension (SOE) PCR was used for generating constructs having variant FVII open reading frames with substituted codons by using standard methods.
  • the cell line CHO K1 (ATCC #CCL-61) is seeded at 50% confluence in T-25 flasks using MEMO, 10% FCS (Gibco/BRL Cat #10091), P/S and 5 ⁇ g/ml phylloquinone and allowed to grow until confluent.
  • the confluent mono cell layer is transfected with 5 ⁇ g of the relevant plasmid described above using the Lipofectamine 2000 transfection agent (Life Technologies) according to the manufacturer's instructions. Twenty four hours post transfection a sample is drawn and quantified using e.g. an ELISA recognizing the EGF1 domain of hFVII. At this time point relevant selection (e.g. Hygromycin B) may be applied to the cells with the purpose of generating a pool of stable transfectants. When using CHO K1 cells and the Hygromycin B resistance gene as selectable marker on the plasmid, this is usually achieved within one week.
  • a vial of CHO-K1 transfectant pool is thawed and the cells seeded in a 175 cm 2 tissue flask containing 25 ml of MEM(X, 10% FCS, phylloquinone (5 ⁇ g/ml), 100 U/l penicillin, 100 ⁇ g/l streptomycin and grown for 24 hours.
  • the cells are harvested, diluted and plated in 96-well microtiter plates at a cell density of 1 ⁇ 2-1 cell/well. After a week of growth, colonies of 20-100 cells are present in the wells and those wells containing only one colony are labelled. After a further two weeks, the media in all wells containing only one colony is substituted with 200 ⁇ l fresh medium. After 24 hours, a medium sample is withdrawn and analysed by e.g. ELISA. High producing clones are selected and used for large scale production of FVII or variants.
  • FVII and FVII variants are purified as follows: The procedure is performed at 4° C. The harvested culture media from large-scale production is ultrafiltered and subsequently diafiltered against 10 mM Tris pH 8.6, using a Millipore TFF system with 30 kDa cut-off Pellicon membranes. After concentration of the medium, citrate is added to 5 mM and the pH is adjusted to 8.6. If necessary, the conductivity is lowered to below 10 mS/cm. Alternatively, the media may be diluted and pH- and citrate-adjusted without prior ultra- and diafiltration.
  • the sample is applied to a Q-sepharose FF column, equilibrated with 50 mM NaCl, 10 mM Tris pH 8.6. After washing the column with 100 mM NaCl, 10 mM Tris pH 8.6, and for some variants 150 mM NaCl, 10 mM Tris pH 8.6, FVII is eluted using 10 mM Tris, 25 mM NaCl, 35 mM CaCl 2 , pH 8.6. In the elution step, the concentration of CaCl 2 or NaCl can be increased if necessary to improve the yield.
  • an affinity column is prepared by coupling of a monoclonal Calcium-dependent antiGla-domain antibody to CNBr-activated Sepharose FF. About 5.5 mg antibody is coupled per ml resin. The column is equilibrated with 10 mM Tris, up to 100 mM NaCl, 35 mM CaCl 2 ,pH 7.5. NaCl is for some variants added to the sample to a concentration of 100 mM NaCl and the pH is adjusted to 7.4-7.6.
  • the column is washed with up to 100 mM NaCl, 35 mM CaCl 2 , 10 mM Tris pH 7.5, and the FVII protein is eluted with 100 mM NaCl, 50 mM citrate, 75 mM Tris pH 7.5, or alternatively 10 mM Tris, 25 mM NaCl, 5 mM EDTA pH 8.6.
  • the latter elution buffer allows for a direct load onto the third chromatographic column without any adjustment of the eluate.
  • the conductivity of the sample is lowered to below 10 mS/cm, if necessary, and the pH is adjusted to 8.6.
  • the sample is then applied to an anion exchange column, typically a Q-sepharose column at a density around 3-10 mg protein per ml gel or a Poros 50 HQ column (Applied BioSciences) at a density of 5-40 mg protein per ml gel to obtain efficient activation, both columns previously equilibrated with 25-50 mM NaCl, 10 mM Tris pH 8.6.
  • an anion exchange column typically a Q-sepharose column at a density around 3-10 mg protein per ml gel or a Poros 50 HQ column (Applied BioSciences) at a density of 5-40 mg protein per ml gel to obtain efficient activation, both columns previously equilibrated with 25-50 mM NaCl, 10 mM Tris pH 8.6.
  • the column is washed with up to 50 mM NaCl, 0.25 mM CaCl 2 , 10 mM Tris pH 8.6 for 2-4 hours with a flow of 3-4 column volumes (cv) per hour.
  • the FVII protein is eluted using a gradient of 0-100% of 500 mM NaCl, 10 mM Tris pH 8.6 over 40 cv. FVII containing fractions are pooled.
  • an anion exchange or a gelfiltration step isused.
  • the conductivity is lowered to below 10 mS/cm.
  • the sample is-applied to a Q-sepharose column (equilibrated with 140 mM NaCl, 10 mM glycylglycine pH 8.6) at a concentration of 3-5 mg protein per ml gel.
  • the column is then washed with 140 mM NaCl, 10 mM glycylglycine pH 8.6 and FVII is eluted with 140 mM NaCl, 15-35 mM CaCl 2 , 10 mM glycylglycine pH 8.6.
  • the eluate is diluted to 10 mM CaCl 2 and the pH is adjusted 6.8-7.2. Finally, Tween-80 is added to 0.01% and the pH is adjusted to 5.5 for storage at ⁇ 80° C.
  • a G25 column HiPrep, Amersham Biosciences
  • 10 mM glycylglycine 10 mM CaCl 2 , 140 mM NaCl, 0.01% Tween-80, pH 5.5.
  • the variants of the invention showed a substantial improvement in FX activation activity as compared to rhFVIIa and also as compared to [P10Q+K32E]rhFVIIa.
  • variants of the invention having the R36E substitution have a significantly reduced clotting activity when compared to rhFVII or to other variants of the invention. See Table 3 below. Nevertheless, as illustrated in Example 7 above, variants having the R36E substitution have an increased Factor X activation activity in the “TF-independent Factor X Activation Assay”.
  • the FVIIa variant P10Q K32E A34E R36E has a differentiated thrombin generation ability depending on the whether the reaction is PL-dependent or TF-dependent.
  • the maximum TF-dependent thrombin generation rate of this variant is decreased by approximately 10-fold (punctuated line in FIG. 2 ) when compared to the FVIIa variants P10Q K32E or A3AY P10Q K32E A34L.
  • lag time, time to peak, peak height and (to a lesser extent) AUC are reduced for P10Q K32E A34E R36E compared to the other variants (results not shown).
  • the PL-dependent activity of the P10Q K32E A34E R36E variant is equivalent to that of the other variants tested in this example (see FIG. 3 ), i.e. this variant has full PL-dependent activity even though the TF-dependent activity is substantially reduced.
  • the variant P10Q K32E A34E R36E was compared directly to the variant P10Q K32E A34E P74S, which has a high TF-dependent thrombin generation rate as shown in FIG. 2 .
  • the differences in TF-binding between these two variants i.e. the reduced TF-binding of the variant P10Q K32E A34E R36E is believed to be directly attributable to the presence of the R36E substitution, possibly in synergy with the A34E substitution.
  • FIG. 4 shows that the PL-dependent activity of the variant of the invention, compared to rhFVIIa, has a reduced lag time, a reduced time to peak, an increased peak height, and an increased maximum rate of thrombin generation.
  • FIG. 5 shows that the TF-dependent activity of the variant of the invention, compared to rhFVIIa, has an increased lag time, an increased time to peak, a reduced peak height, and a reduced maximum rate of thrombin generation.
  • FIGS. 4 and 5 show that the variant of the invention, P10Q+K32E+A34E+R36E+T106N+V253N, has an enhanced phospholipid-dependent activity and at the same time a reduced tissue factor-dependent activity compared to rhFVIIa. Both of these properties are contemplated to be advantageous in a clinical setting, e.g. in the case of trauma or intracerebral haemorrhage, the enhanced PL-dependent activity for obtaining faster and more effective blood clotting, and the reduced TF-dependent activity for minimizing the risk of undesired blood clot formation.

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US11/424,035 US8987202B2 (en) 2003-06-19 2006-06-14 Factor VII or Factor VIIa Gla domain variants
US11/424,030 US7807638B2 (en) 2003-06-19 2006-06-14 Factor V11 or factor V11A GLA domain variants
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