Short-Acting Factor VII Peptides

Abstract

The present invention discloses short-acting factor VII peptides. Shortened half-life is desirable for the treatment of acute bleeding and similar disorders. Modification of sialylation and / or glycosylation of factor VII and its variants produces peptides that can be used to treat acute bleeding conditions. Human coagulation factor VII variants and polynucleotides encoding such variants, vectors and host cells containing and expressing such variants, methods for obtaining such variants, methods for using such variants, compositions of the variants, and other inventive features related thereto are provided herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application Serial No. 61 / 745,674 filed on December 24, 2012 and U.S. Application Serial No. 61 / 787,026 filed on March 15, 2013, which are hereby incorporated by reference in their entirety. Technical Field

[0003] Provided herein are human coagulation factor VII variants and polynucleotides encoding such variants, vectors and host cells containing and expressing such variants, methods of obtaining such variants, methods of using such variants, compositions of the variants, and additional inventive features related thereto. Background Art

[0004] Blood coagulation is the process of being composed of the complex interaction of various blood components (or factors), and the complex interaction finally produces fibrin clot.Usually, the blood components involved in being referred to as coagulation "cascade" are enzymatically inactivated proteins (preenzymes or zymogens), which are converted into proteolytic enzymes by the action of activators (which themselves are activated coagulation factors).The coagulation factors that have undergone such conversion are generally referred to as "active factors", and are named (e.g., Factor VIIa) by adding the letter "a" to the name of the coagulation factor.

[0005] The beginning of the hemostatic process is mediated by the formation of a complex between tissue factor (which is exposed to circulating blood after vascular wall damage) and factor VIIa (which is present in the circulation in an amount corresponding to about 1% of the total factor VII protein amount). The complex is anchored to cells with tissue factor and converts factor IX and X into their active forms, factor IXa and factor Xa, on the cell surface. Factor Xa converts prothrombin into thrombin on cells with tissue factor, and the thrombin activates factor VIII, factor V, factor XI and factor XIII. In addition, the limited amount of thrombin formed in this initial step of hemostasis also activates platelets. After thrombin acts on platelets, platelets change shape and expose charged phospholipids on their surface. The activated platelet surface forms a template for other factor X activation and complete thrombin generation. Other factor X activations on the activated platelet surface occur via factor IXa and factor VIIIa complexes formed on the surface of activated platelets, and factor Xa then converts prothrombin into thrombin while still on the surface. Thrombin then converts fibrinogen into fibrin, which is insoluble and stabilizes the initial platelet plug. This process is localized to the tissue factor exposure site, thereby minimizing the risk of systemic activation of the coagulation system. In recent years, it has been found that factor VII and tissue factor are the main initiators of blood coagulation.

[0006] Factor VIIa is produced from its precursor factor VII, and the factor VII is synthesized in the liver and secreted into the blood, and it circulates in the blood as a single-chain glycoprotein (molecular weight of about 50,000 Da). The wild-type factor VII used herein has the amino acid sequence and nucleotide sequence disclosed in Figures 1 and 2. The term "factor VII" is intended to include the factor VII polypeptides in their uncut form (zymogen form) and those that have been proteolytically or otherwise processed to produce their respective biologically active forms (which can be referred to as factor VIIa). Wild-type factor VII is usually cut between residues 152 and 153 to produce factor VIIa.

[0007] Factor VII is converted into the double-chain form of Factor VIIa in vitro by Factor Xa, Factor XIIa, Factor IXa or thrombin. Like several other plasma proteins involved in hemostasis, the activity of Factor VII depends on vitamin K, which is necessary for the γ-carboxylation of multiple glutamic acid residues clustered near the amino terminus of the protein. These γ-carboxylated glutamic acids are necessary for the interaction of Factor VII with phospholipids induced by metal ions. In the presence of tissue factor, phospholipids and calcium ions, double-chain Factor VIIa rapidly activates Factor X or Factor IX by limited proteolysis. Factor VIIa is prone to proteolytic cleavage, thereby producing many degradation products that do not have coagulation activity.

[0008] Factor VII variants have been disclosed, which have an amino acid sequence derived from wild-type factor VII by substitution, deletion and / or insertion of one or more amino acids. For example, Dickinson et al. (Proc. Natl. Acad. Sci USA (1996) 93, 14379-14384) relate to factor VII variants, wherein Lys157, Val158, Glu296, Met298, Asp334, Ser336 or Lys227 have been replaced individually by Ala. Iwanaga et al. (Thromb. Haemost. (August 1999 supplement), 466, Abstract 1474) relate to factor VIIa variants, wherein residue 316-320 disappears or residue 311-322 is replaced by the corresponding residue derived from trypsin. Bolt et al., U.S. Patent Application Publication 2008 / 0058255 A1, relates to Factor VII variants having substitutions that disrupt glycosylation at N145 or N322, or at both N145 and N322. Toso et al. reported a series of Factor VII structure-function studies based on naturally occurring mutations. Mutant recombinant Factor VII proteins include T324M, E385K, and two mutant Factor VII proteins that lack glycosylation core sequences in the Factor VII heavy chain (N322Q) or the Factor VII light chain (N145Q). Toso et al., "Lack of Heavy Chain Glycosylation in Patient with Factor VII Deficiency Not Responsible for Mutant FVIIa Activity," Blood, Vol. 96, No. 11, Part 2 (November 16, 2000), p. 79b (American Society of Hematology 42nd Annual Meeting (42 nd Annual Meeting of the American Society of Hematology)).

[0009] Most naturally occurring peptides and proteins contain carbohydrate moieties that are connected to the peptide or protein via specific bonds with a selected number of amino acids along the length of the peptide or protein backbone. Thus, many naturally occurring peptides and proteins are referred to as "glycopeptides" or "glycoproteins", respectively. The variability of the glycosylation pattern on any given peptide or protein can affect the function of the peptide or protein. For example, the structure of the polysaccharide connected by N- on a peptide or protein can affect the various characteristics of the peptide or protein, including protease susceptibility, intracellular transport, secretion, tissue targeting, biological half-life, and the antigenicity of the peptide or protein in a cell or organism. One or more changes in these characteristics can affect the effectiveness of the peptide or protein in its natural setting, and can also affect the effectiveness of the peptide or protein as a therapeutic agent (when the peptide or protein has been prepared for this purpose).

[0010] The carbohydrate structure connected with peptide or protein chain is called " polysaccharide " molecule. The specific polysaccharide structure present in peptide or protein affects the solubility and aggregation characteristics of peptide or protein, the folding of peptide or protein backbone, and therefore affects its function or enzymatic activity, the resistance of peptide or protein to proteolytic attack and the control of proteolysis that causes the conversion of inactive form of peptide or protein to active form. For example, the terminal sialic acid residue present in the polysaccharide molecule affects the length of the half-life of peptide or protein in mammalian circulatory system. Peptides and proteins whose polysaccharides do not contain terminal sialic acid residues are usually removed from circulation more quickly by the liver.

[0011] The polysaccharide structures found in naturally occurring glycopeptides and glycoproteins are generally divided into two categories: N-connected polysaccharides and O-connected polysaccharides. Wild-type factor VIIa contains two N-connected glycosylation sites and two O-connected glycosylation sites. N-connected glycosylation is the most common covalent modification in eukaryotes. N-connected glycosylation occurs at the consensus sequence Asn-X-Ser / Thr, where polysaccharides are connected to the amine group of asparagine, and X represents any amino acid except proline. N-connected polysaccharides are based on the common core pentose Man3 (GlcNAc) 2, which can be further modified by adding monosaccharides such as N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fructose, mannose and fucose. Man3 (GlcNAc) 2 cores with different monosaccharides (including terminal sialic acid) can be connected to the Asn of the Asn-X-Ser / Thr consensus sequence via N-acetylglucosamine. This chemically complex co-translational modification serves multiple purposes and affects the biology of the protein in a variety of ways, including proper folding, functional group orientation, and clearance.

[0012] Various methods for tailoring the glycosylation pattern of peptides or proteins have been proposed in the art, including those described in US Pat. No. 8,008,252 to DeFrees et al.

[0013] It is often desirable to stimulate or enhance the coagulation cascade in a subject. Factor VIIa has been used to control bleeding disorders caused by coagulation factor deficiencies (e.g., hemophilia A and B, or deficiencies of coagulation factors XI or VII) or coagulation factor inhibitors. Recombinant Factor VIIa (developed by Novo Nordisk under the trade name NovoSeven ® Hedner, U.S. Pat. No. 5,180,583, discloses the use of Factor VIIa to control excessive bleeding in situations not caused by coagulation factor deficiency or coagulation factor inhibitors. Hedner discloses treatments for bleeding disorders caused, for example, by defective platelet function, thrombocytopenia, or von Wille's disease, and compositions for those uses.

[0014] Need to treat the bleeding of the obstacle that is not caused by congenital or developmental coagulation factor deficiency or coagulation factor inhibitor.Several clinical trials have confirmed the effectiveness of recombinant factor VIIa for controlling bleeding.But, there is the worry about the increase of undesirable thromboembolic events caused by using this molecule.Bleeding is a major problem in many obstacles, such as about surgical operation, complications after surgical operation, stem (stem) and organ transplant, intracranial hemorrhage, aortic aneurysm and trauma or overdose of some anticoagulants. Summary of the invention

[0015] One object is to treat bleeding disorders and seizures with short-acting Factor VII polypeptides. One object of this work is to provide compositions of short-acting Factor VII polypeptides (wild type or variants) characterized by one or more pharmacokinetic properties, such as a shortened half-life. One object is to provide such Factor VII molecules, which have a reduced chance of thrombotic events outside the target site and treatment time range. One object is to provide Factor VII polypeptides (wild type or variants) with enhanced clearance due to altered glycosylation patterns.

[0016] Described herein are compositions of variant Factor VII polypeptides, wherein the variant Factor VII polypeptides comprise an amino acid sequence having at least two sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the at least two sequence changes are (1) substitution of a glutamine residue for a proline residue at position 10, and (2) substitution of a glutamic acid residue for a lysine residue at position 32; and wherein the ratio of moles of conjugated sialic acid to moles of N-linked glycans in the composition is less than 0.05, less than 0.1, less than 1.0, less than 2.0, less than 3.0, less than 4.0, less than 5.0, or less than 6.0. Also described herein are compositions of variant Factor VII polypeptides, wherein the variant Factor VII polypeptide comprises an amino acid sequence having at least two sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the at least two sequence changes are (1) substitution of a glutamine residue for a proline residue at position 10, and (2) substitution of a glutamic acid residue for a lysine residue at position 32; and wherein the ratio of the number of moles of conjugated sialic acid to the number of moles of N-linked glycans is within a range selected from the group consisting of: (1) 0-5; (2) 0-4; (3) 0-3; (4) 0-2; (5) 0-1 and (6) 0-0.5.

[0017] Also described herein is an isolated variant Factor VII polypeptide comprising an amino acid sequence having at least two sequence alterations compared to the amino acid sequence of SEQ ID NO: 16, wherein the at least two sequence alterations are (1) substitution of a glutamine residue for a proline residue at position 10, and (2) substitution of a glutamic acid residue for a lysine residue at position 32, wherein the polypeptide has a ratio of moles of conjugated sialic acid to moles of N-linked glycans of less than 0.05, less than 0.1, less than 1.0, less than 2.0, less than 3.0, less than 4.0, less than 5.0 or less than 6.0. Also described herein are compositions of Factor VII polypeptides, wherein the Factor VII polypeptide comprises the amino acid sequence of SEQ ID NO: 16 (wild-type Factor VII) and the ratio of moles of conjugated sialic acid to moles of N-linked glycans in the composition is within a range selected from the group consisting of: (1) 1-5; (2) 1-4; (3) 1-3; (4) 1-2; and (5) 0.5-1; or the conjugated sialic acid is undetectable.

[0018] Also described herein is an isolated variant Factor VII polypeptide selected from the group consisting of:

[0019] (1) A polypeptide comprising a Factor VII amino acid sequence having sequence alterations compared to the sequence of SEQ ID NO: 16, wherein the sequence alterations consist of: (1) a substitution of a glutamine residue for a proline residue at position 10, (2) a substitution of a glutamic acid residue for a lysine residue at position 32, and (3) a sequence alteration such that the N-linked glycosylation at position 145 is disrupted;

[0020] (2) a polypeptide comprising a Factor VII amino acid sequence having sequence alterations compared to the sequence of SEQ ID NO: 16, wherein the sequence alterations consist of: (1) a substitution of a glutamine residue for a proline residue at position 10, (2) a substitution of a glutamic acid residue for a lysine residue at position 32, and (3) a sequence alteration such that the N-linked glycosylation at position 322 is disrupted;

[0021] (3) a polypeptide comprising a Factor VII amino acid sequence having sequence alterations compared to the amino acid sequence of SEQ ID NO: 16, wherein the sequence alterations consist of: (1) a substitution of a glutamine residue for a proline residue at position 10, and (2) a substitution of a glutamic acid residue for a lysine residue at position 32, and (3) sequence alterations that disrupt N-linked glycosylation at positions 145 and 322;

[0022] (4) a polypeptide comprising a Factor VII amino acid sequence having a sequence alteration compared to the amino acid sequence of SEQ ID NO: 16, wherein the sequence alteration consists of: (1) a substitution of a proline residue at position 10 by a glutamine residue, and (2) a substitution of a lysine residue at position 32 by a glutamic acid residue, wherein positions 145 and 322 are asparagine and have linked N-linked glycosylation;

[0023] (5) a polypeptide comprising a Factor VII amino acid sequence having sequence alterations compared to the amino acid sequence of SEQ ID NO: 16, wherein the sequence alterations consist of: (1) a substitution of a proline residue at position 10 by a glutamine residue, (2) a substitution of a lysine residue at position 32 by a glutamic acid residue, (3) a substitution of an alanine residue at position 34 by a glutamic acid residue, (4) a substitution of an arginine residue at position 36 by a glutamic acid residue, and (5) a sequence alteration that disrupts N-linked glycosylation at position 145;

[0024] (6) A polypeptide comprising a Factor VII amino acid sequence having sequence alterations compared to the amino acid sequence of SEQ ID NO: 16, wherein the sequence alterations consist of: (1) a substitution of a proline residue at position 10 by a glutamine residue, (2) a substitution of a lysine residue at position 32 by a glutamic acid residue, (3) a substitution of an alanine residue at position 34 by a glutamic acid residue, (4) a substitution of an arginine residue at position 36 by a glutamic acid residue, and (5) a sequence alteration that disrupts N-linked glycosylation at position 322;

[0025] (7) A polypeptide comprising a Factor VII amino acid sequence having sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the sequence changes consist of: (1) a glutamine residue replacing the proline residue at position 10, (2) a glutamic acid residue replacing the lysine residue at position 32, (3) a glutamic acid residue replacing the alanine residue at position 34, (4) a glutamic acid residue replacing the arginine residue at position 36, and (5) a sequence change that disrupts N-linked glycosylation at positions 145 and 322; and

[0026] (8) A polypeptide comprising a Factor VII amino acid sequence having sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the sequence changes consist of: (1) a glutamine residue replacing the proline residue at position 10, (2) a glutamic acid residue replacing the lysine residue at position 32, (3) a glutamic acid residue replacing the alanine residue at position 34, and (4) a glutamic acid residue replacing the arginine residue at position 36, wherein positions 145 and 322 are asparagine and have linked N-linked glycosylation.

[0027] Also described herein are factor VII polypeptides having reduced sialic acid conjugated to factor VII polypeptides. In certain embodiments, the factor VII polypeptide is a variant polypeptide that produces an altered glycosylation pattern. In other embodiments, the factor VII polypeptide is a wild-type factor VII polypeptide conjugated with reduced sialic acid. In certain embodiments, reduced sialic acid conjugation can be achieved by treating the polypeptide with a sialidase. In other embodiments, reduced sialic acid conjugation can be achieved as follows: producing recombinant factor VII polypeptides in a cell line that partially or completely lacks sialylation of the peptide. In other embodiments, reduced sialic acid conjugation can be achieved as follows: co-expressing a recombinant factor VII polypeptide and a recombinant or exogenous sialidase in a cell line.

[0028] Also described is a method for treating a mammal having a disease or disorder requiring blood clot formation, the method comprising administering to a mammal in need thereof an effective amount of a Factor VII polypeptide having reduced sialic acid conjugation. In certain embodiments, the ratio of the number of moles of conjugated sialic acid to the number of moles of N-linked glycans is less than 0.05. In other embodiments, the Factor VII polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In other embodiments, the Factor VII polypeptide comprises wild-type Factor VII. In other embodiments, the disease or disorder to be treated is selected from the group consisting of: bleeding, gastrointestinal bleeding, uncontrolled bleeding, bleeding in a mammal undergoing a transplant or resection or surgery, bleeding caused by varicose veins, thrombocytopenia, hemophilia, intracranial hemorrhage, aortic aneurysm, and excessive administration of anticoagulants.

[0029] Other variations, compositions, methods, and related products and processes are disclosed in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1A-1H The nucleotide sequences of three kinds of factor VII molecules used in the present application are shown. "V1" is a human factor VII variant with 4 amino acid mutations compared to the wild-type human amino acid sequence of SEQ ID NO: 16: (P10Q, K32E, T106N and V253N). "V2" is a human factor VII variant with 6 amino acid mutations compared to the wild-type human amino acid sequence of SEQ ID NO: 16: (P10Q, K32E, A34E, R36E, T106N and V253N). Fig. 1 also shows the nucleotide sequences of the various constructs used in the embodiments.

[0031] Figure 2 Shown are the amino acid sequences of three factor VII molecules used in this application. The wild-type human factor VII used herein has the amino acid sequence of SEQ ID NO: 16. V1 has the amino acid sequence of SEQ ID NO: 17. V2 has the amino acid sequence of SEQ ID NO: 18. In V1 and V2, the changes in the wild-type factor VII of SEQ ID NO: 16 are shown in bold.

[0032] Figure 3 The diagram depicts three kinds of factor VII molecules used in the examples of the application.Show the connection of polysaccharides at N-glycosylation sites.In order to describe polysaccharides, the solid frame represents N-acetylglucosamine, the shaded oval represents mannose, the hollow oval represents galactose, the black diamond represents sialic acid (also known as N-acetylneuraminic acid), and the closed triangle represents fucose.Describe the polysaccharide structure with a possible variant of polysaccharides, and do not represent the polysaccharides actually measured.

[0033] Figure 4 The diagram depicts N-linked glycans showing the linkage at Asn in the Asn-X-Ser / Thr consensus sequence. A Man3(GlcNAc)2 core with different monosaccharides including a terminal sialic acid is shown.

[0034] Figure 5 The diagram depicts two approaches used in the present disclosure, exemplified with reference to V2, for reducing the half-life of Factor VII variants.

[0035] Figure 6 is a table of underglycosylated Factor VII molecules.

[0036] Figure 7 Shown are the results of an LC-MS method for identifying the remaining sialic acid on the heavy chain of V2 after desialylation according to experimental conditions.

[0037] Figure 8 Analysis of the sialic acid content of desialylated V2 is shown.

[0038] Fig. 9 Results of a phospholipid FX activation assay are shown.

[0039] Fig.10 Shown are the results of PL-TGA assays for desialylated proteins.

[0040] Fig.11 Expression of a hypoglycosylated Factor VII variant is shown.

[0041] Fig.12 The table shows the determination of the "specific activity" of the hypoglycosylated FVII variants using transfection supernatants.

[0042] Fig.13 The results of PL-TGA assays for the purified hypoglycosylated variant pMB121 are shown.

[0043] Fig.14 In vitro hepatocyte clearance of desialylated V2 relative to wild-type Factor VII is shown.

[0044] Fig.15 Results of in vitro hepatocyte clearance using Factor VII variants are shown. The hypoglycosylated variants did not show an increase in clearance in this model. This result suggests that the clearance mechanism of these molecules is different from that utilized by desialylated V2.

[0045] Fig.16Results of pharmacokinetic studies in rats are shown. The half-lives of desialylated V2 and V1 were significantly lower than their unmodified parent molecules in Sprague Dawley rats as measured by Factor VII ELISA.

[0046] Fig.17 Results of pharmacokinetic studies in HemA mice are shown.

[0047] Fig.18 Shown is a desialylated V2 efficacy study in HemA mice.

[0048] Fig.19 Shown is the desialylated V2 potency study in the TVT HemA model.

[0049] Fig. 20 Shown are the results of thrombin-antithrombin ("TAT") production in HemA mice using desialylated V2 compared to Factor VII.

[0050] Fig.21 Efficacy studies of desialylated V2 in coagulation-competent mice are shown.

[0051] Fig. 22 Shown is the in vitro hepatocyte clearance of desialylated wild-type Factor VII (dWT VIIa) compared to wild-type Factor VII with normal sialic acid conjugation.

[0052] Fig.23 Results of tail cleavage studies on dWT VIIa in human tissue factor knock-in (TFKI) mice compared to wild-type Factor VII are shown. Desialylated Factor VII was found to be significantly more potent than wild-type Factor VII.

[0053] Fig.24 Shown are the results of ELISA analysis of thrombin-antithrombin (TAT) complexes after administration of dWT VIIa or wild-type Factor VII.

[0054] Fig.25 Results of thrombus formation analysis in the FeCl3 thrombosis model are shown. A given dose of dWT VIIa produced greatly reduced thrombus formation compared to wild-type factor VII.

[0055] Fig.26 Shown are the apparent binding affinities of dWT VIIa and wild-type Factor VII to soluble tissue factor, measured using a fluorogenic substrate.

[0056] Fig. 27Soluble tissue factor in complex with dWT VIIa or wild-type factor VII is shown to convert factor X to factor Xa. DETAILED DESCRIPTION

[0057] Described herein is a method for regulating the pharmacokinetics of recombinant factor VII polypeptides (wild type or variant) to limit thrombotic complications in the treatment of acute bleeding. Also described is a factor VII polypeptide conjugated with reduced sialic acid. In addition, a variant of recombinant factor VII is described, which has an enhanced blood clearance rate and a decrease in the duration of efficacy. Due to the altered glycosylation pattern, such a variant has a shorter in vivo half-life than recombinant wild-type factor VII. The production and use of such short-acting factor VII polypeptides are also described.

[0058] To explain Factor VII and glycosylation, Figure 3 and 4 . Figure 3 Schematically show 3 embodiments of factor VII molecules and their domains.Factor VII is composed of Gla, EGF and catalytic domain and contains the protein of the polysaccharide (N145 and N322) that 2 N-connections are connected.V1 is a factor VII variant with 4 mutations (P10Q, K32E, T106N, V253N).V2 is a factor VII variant with 6 mutations (P10Q, K32E, A343, R36E, T106N, V253N).V1 and V2 all have the affinity to activated platelets increased and contain 2 extra N-glycosylation sites, thereby causing a longer half-life compared with wild-type factor VII.It is believed that 2 mutations (A34E, R36E) that are only present in V2 cause its tissue factor irrelevance.

[0059] Figure 4 An example of an N-linked glycan is shown schematically, showing linkage at Asn in the Asn-X-Ser / Thr consensus sequence. A Man3(GlcNac)2 core with different monosaccharides including a terminal sialic acid is shown.

[0060] Methods for preparing Factor VII polypeptides having a desired short half-life are provided herein. Two general methods are provided for preparing short-acting Factor VII polypeptides, which methods can be used alone or in combination. Figure 5 As schematically shown in one example using a factor VII variant, a glycosylated factor VII variant can be processed by desialylation or deglycosylation to change the glycosylation pattern of the variant and thereby change and preferably shorten its half-life. The method can also be used to desialylate a wild-type factor VII polypeptide.

[0061] Desialylation can be performed by any method known in the art. Examples of suitable methods include enzymatic desialylation by contact with any known enzyme that functions as a desialylation enzyme, including, but not limited to, sialidases including neuraminidase-agarose beads (Sigma N5254) and sialidases obtained from Clostridium perfringens ( ) identified in GI:40479 and in FEBS Lett. 238(1), 31-34 (1988). Clostridium perfringens ) of the neuraminidase. Such desialylation can be accomplished by contacting a partially purified recombinant Factor VII polypeptide with a sialidase in vitro under suitable conditions, or by co-expressing the sialidase in a host cell expressing the recombinant Factor VII polypeptide. The in vitro contact can be for a duration such that only partial desialylation occurs. For example, if a desired half-life can be obtained from the molecule at a ratio of 0.5-1 of moles of conjugated sialic acid to moles of N-linked polysaccharides in the composition of the Factor VII polypeptide, it is recommended to contact the sialidase for a limited period of time before complete desialylation occurs. Partial desialylation can also be obtained using a modified sialidase by contacting the Factor VII polypeptide with a sialidase under conditions that slow or impair the full function of the sialidase, or by other methods known to those skilled in the art for producing only partially desialylated polypeptides. Partial desialylation can be measured by comparison with the ratio of conjugated sialic acid / polysaccharides in a reference preparation that has been completely desialylated.

[0062] Desialylation can also be accomplished by expressing Factor VII polypeptides (wild type or variant) in a cell line lacking or lacking one or more cellular components required for the addition of sialic acid. Certain cell lines have been or can be modified to reduce or remove sialylation. For example, Lec2 cells with Chinese hamster ovary ("CHO") origin produce glycoproteins with about 10 times less sialic acid than wild-type cells. It is believed that desialylation of polysaccharides produces such molecules: it can be actively removed by liver receptors (including asialoglycoprotein receptors (ASGPR)), and its half-life is shortened for this reason.

[0063] The second approach is to deglycosylate the Factor VII variant and thereby obtain a molecule with a shortened half-life. Reduced glycosylation enhances clearance via the kidney (50-60 Kd cutoff, reviewed in Caliceti P and Veronese FM, “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates,” Adv Drug Deliv Rev. 2003;55(10):1261-77, Weinstein T et al., “Distribution of glycosaminoglycans in rat renal tubular epithelium,” J Am Soc Nephrol. 1997;8(4):586-95, Choi HS et al., “Renal clearance of quantum dots,” Nat Biotechnol. 2007;25(10):1165-70), surface charge, and changes in isoelectric point (pI) (which have been associated with increased glycoprotein circulation, reviewed in Byrne B. et al., “Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells,” Drug Discovery Today 2007;12(7-8):319) and clearance of Factor VII by less glycoprotein-mediated protection from any number of plasma proteases (Ton G., et al., 2005, Nie Y et al., 2006).

[0064] Deglycosylation used herein includes, but is not limited to, the genetic modification of the factor VII polypeptides that produces an amino acid sequence that is changed compared to the reference factor VII polypeptide, and the change removes the glycosylation site connected by N-. For example, the change of the destruction of glycosylation at one or more amino acid residues required for the N-connected polysaccharide consensus sequence (i.e., Asn-X-Ser / Thr, wherein X represents any amino acid other than proline) can be used to produce factor VII variants. The "change that destroys glycosylation" of the factor VII amino acid sequence used herein means a change relative to wild-type factor VII: it causes the replacement, addition or deletion of one or more amino acid residues, and it causes the deletion of one or more sites of glycosylation connected by N-. For example, the glycosylation site connected by N- can be removed by replacing N145 and / or N322, which are both present in wild-type factor VII, with any amino acid (naturally occurring or non-naturally occurring). It should be identified that the glycosylation site that has the least effect on activity when it is changed to destroy glycosylation. In another embodiment, deglycosylation can occur by expressing the factor VII polypeptide (wild type or variant) in a cell line lacking a glycosylation mechanism. For example, it is expected that factor VII produced in bacterial cells is completely unglycosylated because bacterial cells lack the cellular machinery for glycosylation. In another embodiment, factor VII polypeptides are produced in such a cell line: it lacks a terminal glycosylase, or it has such an enzyme, but the activity of one or more enzymes is less than the activity found in a wild-type cell line. See, for example, Appa R. et al., 201, Narita M et al., 1998, Seested et al., 2010. In another embodiment, factor VII polypeptides are produced in such a cell line: it has a defect in an enzyme involved in the synthesis of glycans or the connection of glycans to factor VII, or has a defect in an enzyme involved in the synthesis of CMP-sialic acid transporter. In another embodiment, the factor VII polypeptide is treated with a deglycosylation enzyme or chemical substance to deglycosylate.

[0065] Treatment with sialidases, deglycosylases or chemicals to reduce or remove glycans from Factor VII polypeptides may occur at the expression, purification or post-purification stages.

[0066] In one embodiment, at least one N-connected glycosylation site in Factor VII variant V1 (N322, N145) or Factor VII variant V2 (N322, N145, N106, N253) is selectively removed, and activity is minimally affected. By destroying the N-polysaccharide consensus sequence, the N-polysaccharide site is deleted at the DNA level. This is achieved by removing the N (asparagine) codon and replacing it with a Q (glutamine) codon. Figure 6Table shows the embodiment of low glycosylated variant. Glycosylated variants are made on wild-type factor VII (referred to herein as " F7 "), V1 and V2 main chains. The engineered N-glycan sites (N106, N253) in V1 and V2 are restored to their wild-type sequences (T106, V253). Variant pMB113, pMB117 and pMB121 are wild-type factor VII, V1 and V2 constructs, which contain 2 endogenous N-glycosylation sites (N145, N322) respectively. Figure 6 All other variants in had one or both of their endogenous N-glycan sites removed by the introduction of N to Q mutations (N145Q, N322Q). This deglycosylation protocol resulted in faster clearance.

[0067] In one aspect of the present disclosure, deglycosylation and desialylation are combined to produce factor VII polypeptides with desirable shortened half-life. For example, factor VII molecules can be genetically modified to include glycosylation sites of additional N-connections in addition to the 2 present in wild-type factor VII. The variant can then be desialylated using one of the methods described herein. The resulting molecule can then retain the glycan structure at each N-connected glycosylation site, which does not have terminal sialic acid. In the experiments reported herein, the applicant reported such variants: its elimination time is faster than similar desialylated factor VII variants with less N-connected glycosylation sites. Similarly, factor VII polypeptides with only 2 N-connected glycosylation sites found in wild-type factor VII can be deglycosylated at one of these sites, and then desialylated. Based on the experimental evidence reported herein, the pharmacokinetics of the factor VII variants with an N-connected glycan (which lacks sialic acid) obtained are different from similar factor VII polypeptides with glycosylation sites that do not lack a second N-connected.

[0068] Definition and implementation plan

[0069] Unless otherwise defined, all technical and scientific terms used in this article generally have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Generally, the nomenclature used in this article and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are those well-known and commonly used in the art. Standard techniques are used for nucleic acid and polypeptide synthesis. The nomenclature used in this article and the laboratory procedures in analytical chemistry and organic synthesis described below are those well-known and commonly used in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. The procedures for genetic engineering are well-known and can be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY.

[0070] The term "sialic acid" or "sialyl" refers to any member of the nine-carbon carboxylated sugar family. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetylamino-3,5-dideoxy-D-glycero-D-galactononanopyran-1-osonic acid (often abbreviated as Neu5Ac, NeuAc or NANA)).

[0071] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers in which the monomers are amino acids and are linked together by amide bonds. In addition, non-natural amino acids are also included, for example, β-alanine, phenylglycine and homoarginine. Amino acids that are not genetically encoded can also be used with the technology disclosed herein. In addition, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules, etc. can also be used. All amino acids used herein can be D- or L-isomers. The L-isomer is generally preferred. "Polypeptide" and "protein" used herein refer to glycosylated and unglycosylated polypeptides and proteins, respectively.

[0072] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to chemical compounds whose structure is different from the general chemical structure of an amino acid, but which function in a manner similar to naturally occurring amino acids.

[0073] As used herein, the term "half-life" or "t1 / 2" in the context of administering a polypeptide or protein drug to a patient is defined as the time required for the plasma concentration of the drug in the patient to decrease by half.

[0074] The half-life can be determined in experimental animals, for example, by administering a dosage of about 25-250 micrograms / kg of the preparation; obtaining a plasma sample at a predetermined time after administration; and determining the content of the factor VII polypeptide in the sample using one or more of a coagulation assay (or any bioassay), an immunoassay, or an equivalent assay. The data can be displayed graphically, and the bioavailability is then determined as the area under the curve. In certain embodiments, a rat or mouse model is used for half-life measurements. The relative bioavailability of a factor VII polypeptide or a composition thereof represents the ratio of the area under the curve of a short-acting factor VII polypeptide to the area under the curve of a wild-type factor VII or another appropriate comparison polypeptide or protein. Any factor VII variant having the blood coagulation activity of factor VII can be used for the purposes and methods described herein. The factor VII variant used herein is a polypeptide. The terms "variant factor VII polypeptide" and "factor VII variant" are used interchangeably herein. In one embodiment, the factor VII variant has an amino acid sequence derived from wild-type factor VII (SEQ ID NO: 16) by substitution, deletion, and / or insertion of one or more amino acids. In naming an amino acid substitution, the first letter represents an amino acid present in wild-type human factor VII at a position. The numbers below represent the position in human wild-type factor FVII. The second letter represents the amino acid that replaces the amino acid found in the wild-type. For example, "P10Q" represents that glutamine (Q) replaces the proline (P) at amino acid position 10.

[0075] In certain embodiments, the factor VII variant comprises one or more amino acid substitutions selected from the group consisting of P10Q, K32E, R36E, A34E, T106N, and V253N. In other embodiments, the factor VII variant comprises at least 2, 3, 4, 5, or 6 of these substitutions. In other embodiments, the factor VII variant comprises an amino acid sequence having at least 2 sequence changes compared to the amino acid sequence of SEQ ID NO: 16 (wild-type human factor VII), wherein the at least 2 sequence changes are (1) a glutamine residue replacing a proline residue at position 10, and (2) a glutamic acid residue replacing a lysine residue at position 32. In another embodiment, the factor VII variant comprises an amino acid sequence having at least 3 sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the at least 3 sequence changes are: (1) a glutamine residue replacing a proline residue at position 10, (2) a glutamic acid residue replacing a lysine residue at position 32, and (3) a glutamic acid residue replacing an arginine residue at position 36. In another embodiment, the Factor VII variant comprises an amino acid sequence having at least 4 sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the at least 4 sequence changes are: (1) a glutamine residue replaces the proline residue at position 10, (2) a glutamic acid residue replaces the lysine residue at position 32, (3) a glutamic acid residue replaces the arginine residue at position 36, and (4) a glutamic acid residue replaces the alanine residue at position 34. In a specific embodiment, the factor VII variant comprises an amino acid sequence having at least 6 sequence changes compared to the amino acid sequence of SEQ ID NO: 16, wherein the at least 6 or 6 sequence changes are: (1) a glutamine residue replaces the proline residue at position 10, (2) a glutamic acid residue replaces the lysine residue at position 32, (3) a glutamic acid residue replaces the arginine residue at position 36, (4) a glutamic acid residue replaces the alanine residue at position 34, (5) an asparagine residue replaces the threonine residue at position 106, and (6) an asparagine residue replaces the valine residue at position 253. In another specific embodiment, the factor VII variant comprises only these 6 changes. For more details about these variants, see WO 200158935 of Maxygen and U.S. Patent No. 7,371,543 of Pedersen et al., both of which are incorporated herein by reference in their entirety.

[0076] Any functional factor VII polypeptide can be used as a starting polypeptide to design factor VII variants described herein. In certain embodiments, the factor VII polypeptide is a human factor VII polypeptide. In other embodiments, the factor VII polypeptide is a human factor VII polypeptide of SEQ ID NO: 16 or a modified form or allelic variant thereof. Useful starting polypeptides also include modified or variant factor VII polypeptides, which include and wild-type human factor VII (SEQ ID NO: 16) sequence with at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67% or 66% homogeneity amino acid sequence, and it also has factor VII activity. In addition, in certain embodiments, variant Factor VII polypeptides of the present disclosure include any polypeptide having at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67% or 66% identity to the sequence of SEQ ID NO: 16, which has Factor VII functionality and also contains one or more of the amino acid changes relative to SEQ ID NO: 16 discussed herein. In another embodiment, the Factor VII polypeptide comprises an amino acid sequence that is greater than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67% or 66% homologous to SEQ ID NO: 16 and has Factor VII activity and also has one or more amino acid changes as described herein.

[0077] The factor VII variant used herein also includes the glycosylation variant of wild-type factor VII. For example, partially desialylated wild-type factor VII variant and composition thereof can be useful because it has a shorter half-life than wild-type factor VII. Also useful herein are pharmaceutical preparations of partially or completely desialylated wild-type factor VII, and the purposes of such polypeptides and preparations in treating the diseases listed herein, which benefit from the short-acting polypeptides with factor VII activity. By the ratio of the moles of sialic acid conjugated to the moles of polysaccharides connected by N- in the composition of factor VII polypeptides as described herein, partial or complete desialylation can be measured.

[0078] The nucleotide sequence encoding the factor VII variant herein is also useful. In one embodiment, the factor VII polypeptide is encoded by such a nucleotide sequence: it has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67% or 66% homogeneity with the nucleotide sequence (SEQ ID NO: 1) of wild-type factor VII in total length, and its encoding function factor VII polypeptide. In certain embodiments, the nucleotide sequence also encodes one or more polypeptides containing the amino acid changes discussed herein relative to SEQ ID NO: 16. In another embodiment, the factor VII polypeptide is encoded by such a nucleotide sequence: it has more than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67% or 66% homology with the nucleotide sequence of wild-type factor VII (SEQ ID NO: 1), and its encoding function factor VII polypeptide. In certain embodiments, the nucleotide sequence also encodes a polypeptide containing one or more of the amino acid changes discussed herein relative to SEQ ID NO: 16.

[0079] The percent identity values ​​are calculated over the entire amino acid or nucleic acid sequence region. A range of programs based on a variety of algorithms are available to the skilled person for comparing different sequences. In at least one embodiment, the percent identity between two amino acid sequences is determined as follows: using the Needleman and Wunsch algorithm (Needleman 1970, J. Mol. Biol. (48): 444-453), which has been incorporated into the needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice, P., Longden, I., and Bleasby, A, Trends in Genetics 16(6), 276-277, 2000), using the BLOSUM 45 or PAM250 scoring matrix (for distantly related proteins) or the BLOSUM 62 or PAM160 scoring matrix (for more closely related proteins) and a gap opening penalty of 16, 14, 12, 10, 8, 6 or 4 and a gap extension penalty of 0.5, 1, 2, 3, 4, 5 or 6. In one embodiment, the identity percentage between 2 nucleotide sequences is as follows: use the pin program (EMBOSS:TheEuropean Molecular Biology Open Software Suite, Rice, P., Longden, I., and Bleasby, A, Trends in Genetics 16 (6), 276-277, 2000), use the EDNAFULL scoring matrix with 16,14,12,10,8,6 or 4 gap opening penalties and 0.5,1,2,3,4,5 or 6 gap extension penalties. A non-limiting example of parameters for aligning two amino acid sequences using the needle program is the default parameters, including the EDNAFULL scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5. Nucleic acid and protein sequences can further be used as "query sequences" to perform searches against public databases, for example, to identify other family members or related sequences.Such searches can be performed using the BLAST suite of programs (version 2.2) of Altschul et al. (Altschul 1990, J. Mol. Biol. 215:403-10). BLAST using the nucleic acid sequence of the present disclosure as the query sequence can be performed using the BLASTn, BLASTx or tBLASTx programs using default parameters to obtain nucleotide sequences (BLASTn, tBLASTx) or amino acid sequences (BLASTx) homologous to the sequences encoded by the nucleic acid sequences of the present disclosure. BLAST using the protein sequence encoded by the nucleic acid sequence of the present disclosure as the query sequence can be performed using the BLASTp or tBLASTn programs using default parameters to obtain amino acid sequences (BLASTp) or nucleic acid sequences (tBLASTn) homologous to the sequences of the present disclosure. In order to obtain gapped alignments for comparison purposes, Gapped BLAST using default parameters can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25(17):3389-3402.

[0080] The polynucleotide of the present disclosure is basically composed of the aforementioned nucleotide sequence, or comprises the aforementioned nucleotide sequence.Thereby, they can contain other nucleotide sequences equally.In certain embodiments, except open reading frame, the polynucleotide can also be included in other non-translated sequences at 3' end and / or 5' end of coding gene region, for example at least 10,20,30,40,50,60,70,80,90,100,200,300,400,500 or more nucleotides of the sequence at the upstream of 5' end of coding region and / or at least 10,20,30,40,50,60,70,80,90,100,200,300,400,500 or more nucleotides of the sequence at the downstream of 3' end of coding gene region.In addition, the polynucleotide can encode fusion protein, wherein a partner of the fusion protein is the polypeptide encoded by the nucleotide sequence listed above.Such fusion protein can include so-called "label", which can serve as a detectable marker or serve as an auxiliary measure for purification purposes. Tags used for different purposes are well known in the art and include FLAG-tag, 6-histidine-tag, MYC-tag, etc. In one embodiment, the polynucleotide further comprises an expression control sequence operably linked to the nucleotide sequence.

[0081] In certain embodiments, the nucleic acid sequence encoding the factor VII polypeptide is inserted into a suitable vector. Numerous vectors that can be used for different purposes are well known in the art, and those skilled in the art can easily select suitable vectors for their desired applications. In certain embodiments, the vector can be a cloning vector or an expression vector. In other embodiments, the vector can be a plasmid, a viral vector, a cosmid or an artificial chromosome. In certain embodiments, the nucleic acid encoding the factor VII polypeptide can be placed near a suitable promoter and / or under the control of a suitable promoter. Numerous promoters that can be used for different purposes are well known in the art, and those skilled in the art can easily select suitable promoters for their desired applications. In certain embodiments, the promoter can be a constitutive promoter, an inducible promoter or a tissue-specific promoter.

[0082] In certain embodiments, the Factor VII polypeptide is produced recombinantly in a cell, tissue or organism. In certain embodiments, such recombinant production is accomplished by transforming or transfecting a host cell with a nucleic acid molecule encoding a variant polypeptide or a vector containing such a nucleic acid. Numerous transformation and transfection methods are well known in the art, and those skilled in the art can easily select appropriate methods for their desired applications.

[0083] Such recombinant production can also be completed using any suitable host cell, tissue or organism. Suitable cells, tissues and organisms are well known in the art, and those skilled in the art can easily select suitable hosts for their desired applications. In certain embodiments, the host cell is a mammal. Examples of suitable mammalian cell lines are COS-1 (ATCC CRL 1650), baby hamster kidney (BHK), HEK293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), HEK293T (ATCC CRL 11268; DSM ACC 2494) and HEK293F (Invitrogen R79007) cell lines. A useful BHK cell line is tk 31ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, incorporated herein by reference), hereinafter referred to as BHK 570 cells. The BHK 570 cell line has been maintained at the American Type Culture Collection (12301 Parklawn Dr., Rockville, Md. 20852) under ATCC accession number CRL 10314. tk - The ts13 BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines can be used within the present disclosure, including Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61), CHO K1 (ATCC CCL 61), DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980), and CHO-DG44 cells (Urlaub et al. Cell 33:405-412, 1983).

[0084] Compositions of Factor VII polypeptides are useful: wherein the Factor VII polypeptide is as defined herein and the ratio of the number of moles of conjugated sialic acid to the number of moles of N-linked glycans in the composition is less than 0.05, less than 0.1, less than 1.0, less than 2.0, less than 3.0, less than 4.0, less than 5.0 or less than 6.0, or wherein the ratio of the number of moles of conjugated sialic acid to the number of moles of N-linked glycans is within a range selected from the group consisting of: (1) 0-8; (2) 0-7; (3) 0-6; (4) 0-5; (5) 0-4; (6) 0-3; (7) 0-2; (8) 0-1 and (9) The ratio of sialic acid to glycoprotein is 0-0.5, or 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5 and 0.1-1. The ratio is a measure of the number of moles of sialic acid bound to a glycoprotein relative to the number of glycans on the glycoprotein. The number of glycans represents the number of sugar moieties connected to the glycans connected to the N- in the glycoprotein, wherein the glycosylation sites connected to one N- can only support one glycan as defined herein for the purpose of the ratio. The ratio is determined using a sialic acid fluorescent labeling kit such as a kit sold by Takara Bio Inc. (Cat. No. 4400). Such sialic acid fluorescent labeling kits include a step of releasing sialic acid from bound glycoproteins, such as by partial acid hydrolysis or by using a sialidase, such as Arthrobacter ureagenes ( Arthrobacter ureafaciens ) sialidase. The free sialic acid is then labeled with a fluorophore such as 1,2-diamino-4,5-methyleneoxybenzene ("DMB"). The labeled sialic acid is then quantitatively measured using HPLC, and the peak height is compared to a calibration curve. Thus, the ratio measured is the ratio of the number of moles of sialic acid to the number of moles of glycans released from all Factor VII polypeptides of the composition.

[0085] In one series of embodiments, the composition of Factor VII polypeptides or the isolated polypeptide itself has a half-life as measured in human or mammalian plasma (e.g., mouse or rat plasma) of less than 2 hours, less than 1.5 hours, less than 1 hour, less than 75 hours, less than 5 hours, less than 25 hours, less than 0.1 hours, or so short that it cannot be properly measured.

[0086] The factor VII activity used herein is a biological activity that can be quantified as follows: as is well known in the art, the ability of the product to promote blood coagulation is measured using factor VII-deficient plasma and thromboplastin. In certain embodiments, the factor VII polypeptide having factor VII activity exhibits at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the activity of wild-type factor VII measured under the same conditions.

[0087] Pharmaceutical preparations of factor VII polypeptides and compositions comprising factor VII polypeptides and pharmaceutically acceptable excipients or carriers thereof are also useful. In certain embodiments, the pharmaceutical preparations are used for parenteral administration, such as by intravenous, subcutaneous or intramuscular administration, and administration may be as a single single dose, intermittent administration or as a continuous intravenous infusion. Topical preparations are also useful. One embodiment comprises such a pharmaceutical preparation: it contains the separated factor VII polypeptide as described herein, or contains the composition of the factor VII polypeptide as described herein, in the form of a lyophilized product reconstructed at the time of use. Alternatively, the pharmaceutical preparation may be a stable, ready-to-use liquid preparation, without the need for reconstruction. The pharmaceutical preparation may be a lyophilized powder of 1, 2, 5 or 8 mg of factor VII polypeptide in a disposable vial. After reconstitution with a specified volume of liquid (such as sterile water containing histidine), the final solution can contain any suitable amount of factor VII polypeptides that produce a therapeutic effect, for example, but not limited to, 1 mg / mL (1000 micrograms / mL), 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 1-2 mg / mL, 1-3 mg / mL, 1-5 mg / mL, 1-10 mg / mL, 0.5-1 mg / mL or 0.5-2 mg / mL of factor VII polypeptides. Those skilled in the art can easily determine the appropriate dosage for administration to the patient based on, for example, the weight of the patient, the type of bleeding disorder or attack to be treated and the activity of the specific factor VII polypeptides used. In certain embodiments, administration can be in the range of 70-110 micrograms / kg, 70-90 micrograms / kg or 80-100 micrograms / kg, and can be 90 micrograms / kg. Lyophilized powders can be reconstituted with aqueous carriers (such as water, buffered water, 0.4% saline, 0.3% glycine, etc.). The actual method for preparing the composition of parenteral administration is known or obvious to those skilled in the art, and is described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa. (1990). Topical application (such as being suitable in the case of trauma) can be achieved by means of sprays, perfusion methods, catheters, stents, vascular grafts or stents, ointments or other products known in the art. In certain embodiments, topical application can be by solid or semisolid matrix, such as surgical sponges or collagen matrix, which has been treated, impregnated, coated or soaked with a composition comprising factor VII variants.Methods for preparing such matrices are well known in the art (see, e.g., Thrombosis / Hemostasis 12:445, 2006), and the skilled artisan can readily determine appropriate dosages and methods for applying the composition to a given matrix.

[0088] In one embodiment, the disclosure relates to a kit comprising a factor VII polypeptide. In certain embodiments, the kit contains a bottle containing a ready-to-use liquid containing a factor VII polypeptide in a suitable pharmaceutical composition. In other embodiments, the kit contains a bottle containing a lyophilized factor VII polypeptide or a lyophilized formulation containing the polypeptide and a diluent for reconstruction. In other embodiments, the kit contains a topical preparation of a factor VII polypeptide, for example, an ointment, a spray or a liquid, and a substrate to which the topical preparation can be applied before being administered to a patient, such as a sponge or other medical substrate.

[0089] Compositions of factor VII polypeptides described herein are also useful. Factor VII is present in a mixture together with its natural degradation products. Therefore, compositions of factor VII polypeptides include polypeptides having one of the complete amino acid sequences as listed herein and degradation products having partial amino acid sequences of those described herein. In addition, because factor VII is a glycoprotein, it can be expected that compositions of factor VII contain a heterogeneous mixture of factor VII polypeptides, wherein each glycoprotein in the composition does not have the same glycosylation as other glycoproteins. Reference to compositions of factor VII polypeptides or separated factor VII polypeptides is intended to include mixtures of such polypeptides, wherein the various polypeptides have different glycosylation, and thus the term "composition" or "separated factor VII polypeptide" includes the heterogeneity of the glycosylation pattern within the polypeptide.

[0090] The Factor VII polypeptides and compositions described herein are useful for treating blood coagulation disorders, and those disorders that benefit from blood coagulation, and in particular for coagulation using drugs that have a shorter half-life than wild-type Factor VII. Thus, the Factor VII polypeptides and compositions herein can be used for penetrating traumatic injury; blunt traumatic injury; bleeding during elective surgery; bleeding during cardiac surgery; bleeding during spinal surgery; orthopedic surgery; neurosurgery; oncology surgery; postpartum surgery; menorrhagia; bleeding in stem cell transplantation; bleeding in liver transplantation; gastrointestinal bleeding; bleeding from active varicose veins in cirrhosis; bleeding from non-varicose veins in cirrhosis; diffuse alveolar hemorrhage; aortic aneurysm; intracerebral hemorrhage; traumatic brain injury; cerebral contusion; reversal of warfarin; reversal of heparin; reversal of anticoagulants; reversal of antithrombotics; Factor VII deficiency; burns; prophylaxis in hemophiliacs with inhibitors; partial hepatectomy in patients without cirrhosis and in patients with cirrhosis; acquired hemophilia; idiopathic thrombocytopenic purpura; Glanzmann's thrombasthenia; Glanzmann's thrombasthenia and Bernard-Soulier syndrome refractory to platelet transfusion.

[0091] Also disclosed herein is a useful assay for measuring the half-life of a coagulation factor (such as factor VII). There is a method for determining the half-life of a coagulation factor, the method comprising incubating living rat hepatocytes with the coagulation factor, taking out a sample at test time point 1, separating the supernatant in the sample from cells, and the activity or amount of the coagulation factor in the supernatant of the quantitative sample, wherein the activity or amount of the coagulation factor is determined using a double antibody sandwich ELISA assay. The method can be repeated at different time points to form a graph of the activity or amount of blood coagulation factor over time. Example

[0092] Method for obtaining desialylated factor VII polypeptide

[0093] A number of methods are used to prepare desialylated Factor VII polypeptides (wild-type and variants), including enzymatic desialylation of the polypeptide, production of Factor VII polypeptides in sialylation-deficient cell lines, and co-expression of Factor VII and sialidase in recombinant cells.

[0094] Preparation of sialic acid-deficient cell lines

[0095] Endogenous sialic acid is synthesized in mammalian cells in a complex pathway consisting of 32 enzymes (Wickramasinghe and Medrano 2011). The biosynthesis of sialic acid begins in the cytosol, which converts UDP-N-acetylglucosamine (UDP-GlcNAc) to Neu5Ac, involving several enzymes such as UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase (GNE), sialic acid 9-phosphate synthase (NANS) and sialic acid 9-phosphate phosphatase (NANP). Neu5Ac in the cytosol is imported into the nucleus through the nuclear pores and converted to CMP-Neu5Ac by an enzyme called CMP-Sia synthase (CMAS). The synthesized CMP-Neu5Ac is again transported back to the cytosol via the nuclear pores for further modification and conjugation in the Golgi apparatus. The conversion of Neu5Ac to Neu5Gc in the cytosol is catalyzed by the enzyme CMP-NeuAc-hydroxylase (CMAH). Then, CMP-Neu5Ac and CMP-Neu5Gc are transported into the Golgi compartment via the hydrophobic type 3 membrane transporter CMP-sialic acid transporter (SLC35A1) in the membrane of the middle trans-Golgi. CMP-sialic acid transporter is a key element in the sialylation pathway of cells (Hirschberg, et al. 1998). Homozygous mutations of this gene cause postnatal lethality in mice (MGI 4.32, homologous genes). In humans, mutations in SLC35A1 are associated with a reduction or complete lack of sialic acid conjugates. Some insertion and deletion mutations in SLC35A1 are associated with congenital disorders of glycosylation in humans, resulting in defects in nervous system development, coagulation and immunodeficiency (Martinez-Duncker, et al., 2005). Once CMP-Neu5Ac / CMP-Neu5Gc are transported into the Golgi apparatus, they are conjugated to carbohydrates, glycoproteins, and glycolipids by enzymes in the 20-member sialyltransferase (ST) family.

[0096] CMP-sialic acid transporter (SLC35A1) is a key molecule that supports sialic acid conjugation in the Golgi apparatus, and mutations in this transporter result in the synthesis of proteins lacking appropriate sialylation. In order to produce desialylated factor VII, a factor VII production cell line in which the CMP-sialic acid transporter gene has been knocked out is prepared. Alternatively, desialylation can be achieved by expressing factor VII variants in a cell line that produces protein therapeutic agents with very low sialylation levels or without sialylation on therapeutic molecules. This technology can be used to produce therapeutic proteins with short T1 / 2 in patients.

[0097] Lec2 cells of Chinese hamster ovary (CHO) origin were identified, which have the property of producing about 10 times less sialic acid in glycoproteins and glycolipids than wild-type cells (Stanley and Siminovitch, 1977, Stanley, 1980 and 1983). Later studies showed that Lec2 mutants were unable to translocate CMP-sialic acid across the membrane of Golgi vesicles in in vitro assays, while translocation of other nucleotide derivatives was relatively normal in mutant cells (Deutscher, et al., 1984). By using expression cloning, a gene encoding a CMP-sialic acid transporter from Lec2 cells was reported (Eckhardt et al., 1996). Further studies indicated that the deletion of nucleotides 575-751 in the CMP-sialic acid transporter gene caused the Lec2 phenotype (Eckhardt, et al., 1998). Other mutations in the CMP-sialic acid transporter gene, such as in the case of 1E3, 6B2, 8G8 and 9D3 cells, also result in the Lec2 phenotype (Eckhardt, et al., 1998).

[0098] Experiment 1

[0099] This experiment was designed to determine whether mutations in the gene for the CMP-sialic acid transporter (such as in the case of Lec2 cells) result in expressed recombinant proteins (eg, Factor VII) that are defective in sialylation compared to the same protein expressed from normal CHO cells.

[0100] (1) Test the expression of recombinant proteins (such as factor VII) obtained from Lec2 cells. Under normal transfection conditions, expression vectors (e.g., pMB117 and pMB121) containing factor VII variant genes are transfected into Lec2 cells and normal CHO cells. By factor VII activity assay, the expression level of factor VII obtained from cell cultures of these cells is monitored. The culture of transfected cells is amplified, and the culture conditioned medium is harvested for purification of factor VII.

[0101] (2) Test the sialic acid content of purified Factor VII expressed from Lec2 cells relative to the same protein expressed from normal CHO cells. According to the normal Factor VII purification method, Factor VII is purified from these conditioned media. Regarding the sialic acid content of purified Factor VII, purified Factor VII obtained from Lec2 cells or normal CHO cells is analyzed. As described herein, biological activity and pharmacokinetic (PK) parameters are analyzed.

[0102] Experiment 2

[0103] In order to produce a cell line to express Factor VII variants (which do not have sialic acid on the expressed recombinant protein), a gene deletion method targeting the CMP-sialic acid transporter gene is used to modify a cell line (e.g., a CHO cell line) expressing Factor VII. In order to completely inhibit sialylation in the cell, other targets may also be optionally deleted, such as UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase (GNE), sialic acid 9-phosphate synthase (NANS), sialic acid 9-phosphate phosphatase (NANP), and CMP-Sia synthase (CMAS) listed above in the introduction to enhance the inhibition of CMP-Neu5Ac biosynthesis, which provides substrates for the CMP-sialic acid transporter.

[0104] Two gene deletion technologies, TALE nucleases (TALENs) from Life Technologies and ZFP Nucleases (ZFNs) from Sangamo BioSciences / Sigma-Aldrich, can be used to knock out the CMP-sialic acid transporter gene or multiple genes in the sialic acid synthesis pathway.

[0105] The Factor VII expressing cell line in which the CMP-sialic acid transporter gene was knocked out was evaluated to confirm the deletion of the CMP-sialic acid transporter gene. The confirmed cell line was cultured to produce Factor VII. The Factor VII obtained from the cell line in which the CMP-sialic acid transporter gene was deleted was purified and evaluated as described herein, and compared with the Factor VII obtained from the parental Factor VII expressing cell line for the sialic acid content on the molecule.

[0106] Production of desialylated Factor VII by co-expression of Factor VII and bacterial sialidase

[0107] To prepare the desialylated form of FVII, FVII was reacted with a Arthrobacter ureagenesSialidase (AU sialidase) (N-acetylneuraminic acid glycohydrolase, EC 3.2.1.18) bacterial sialidase variants are co-expressed together. Using pJ608 expression vector, the CHO cells stably expressing FVII (e.g., SEQ ID NO 16 with P10Q, K32E, A34E and R36E mutations) are further transfected with AU sialidase. The expressed protein has a growth hormone signal sequence at the N-terminus to promote the secretion of the protein. Stable clones of the AU sialidase are selected for production, as detected by chromogenic assay for the sialidase in the culture medium. We also confirmed that the cells continue to express high levels of FVII protein, which is detected by ELISA assay, SDS PAGE and Western blotting using FVII specific antibodies as probes. Using lectin blotting assay, we use purified FVII to detect sialic acid on the FVII protein in the conditioned medium. On the contrary, similar levels of purified FVII derived from cells never transfected with sialidase show strong lectin binding signals. In summary, our data indicate that the level of AU sialidase expressed in CHO cell culture medium is sufficient to effectively desialylate FVII co-expressed by the cells.

[0108] Enzymatic preparation of desialylated Factor VII using soluble sialidase treatment

[0109] The following starting materials were used in this pilot test:

[0110] Factor VII: 20 mg wild-type factor VIIa, concentration approximately 1 mg / ml

[0111] Sialidase: 20ug, 0.25mg / ml, 50000U / ml, P0720L, purchased from New England BioLabs

[0112] Buffer A: 25 mM histidine, 50 mM NaCl, pH 6.4

[0113] Buffer B: 25 mM histidine, 1 M NaCl, pH 6.4

[0114] FVIIa preparation buffer: 2.3 mg / ml sodium chloride, 1.5 mg / ml dehydrated calcium chloride, 1.3 mg / ml glycylglycine, 0.1 mg / ml polysorbate 80, 25 mg / ml mannitol, 10 mg / ml sucrose, 0.5 mg / ml methionine, 1.6 mg / ml histidine, pH 6.0

[0115] Purification column: 5ml HiTrap Q Sepharose HP column.

[0116] Using these materials, follow the following procedure:

[0117] 1. Add 20ug sialidase (0.25mg / ml, 1:1000 mass ratio) to 20mg FVIIa (about 1mg / ml)

[0118] 2. The reaction was incubated at room temperature overnight (approximately 19 hours) and the desialylated FVIIa was then purified by chromatography as described below.

[0119] 3. Purify desialylated FVIIa on a 5 ml HiTrapQ Sepharose HP column as follows:

[0120] a) Equilibrate a Q-Sepharose column with 5 column volumes of buffer A (25 mM histidine, 50 mM NaCl, pH 6.4).

[0121] b) Before loading onto the column, the FVIIa and sialidase reactions were diluted with 200 ml of buffer A and the pH was adjusted to 6.4.

[0122] c) Load using AKTA Explorer system at a flow rate of 2.5 ml / min while monitoring A280. Collect flow-through fractions.

[0123] d) After loading is complete, wash the column with 10 column volumes of buffer A.

[0124] e) Elute the column with 20 column volumes of 0-50% buffer B (25 mM histidine, 1 M NaCl, pH 6.4) in 40 min. Collect the peak fractions (Desialylated NovoSeven)

[0125] f) Dialyze the peak fractions against FVIIa formulation buffer overnight at 4°C.

[0126] g) Freeze aliquots of the samples at -80°C.

[0127] The product was confirmed to be highly pure by SDS-PAGE, aSEC and active in biological assays for FVIIa. Assays for sialic acid content showed no residual sialic acid, and LC-MS analysis of the heavy chain showed no significant glycan structural changes other than the removal of sialic acid.

[0128] Enzymatic preparation of desialylated Factor VII using neuraminidase-agarose beads

[0129] The recombinant wild-type Factor VII used herein was NovoSeven® obtained from Novo Nordisk and is referred to herein as "F7". The other starting materials were V1 and V2 as described above.

[0130] The frozen starting material was thawed and combined quickly in a 37° C. water bath. The protein was concentrated 2.5-fold by centrifugation; the concentrate was gently mixed by pipetting to minimize any superconcentration (aggregation) at the protein-filter interface.

[0131] V2 was buffer exchanged from its V2 formulation buffer (containing histidine, CaCl2, trehalose, methionine and trace levels of Tween®-20 at pH 6.4 - 6.6) into MES buffer (containing 10 mM MES, 10 mM CaCl2, 50 mM NaCl, pH 6.0, sterile filtered). This was achieved in one of three ways. In the first option, V2 was buffer exchanged with a NAP-10 gravity flow column (GE, 17-0854-01), which was pre-washed 3-5 times with 3 column volumes of MES buffer at a time. V2 was then loaded onto the column and eluted with 1.5 times the loading volume of MES buffer. In the second option, V2 was buffer exchanged by dialyzing overnight in MES buffer. The dialysis cassette was pre-soaked in MES buffer and V2 was loaded into a 3.500 MWCO slide-a-lyzer cassette (Thermo Scientific, 66130) using a syringe in a 10 L pitcher containing sterile filtered MES buffer at 4° C. overnight. In a third option, V2 was buffer exchanged into MES buffer by passing through a Sephadex G-25 (Sigma, G-25-80) gel filtration column and equilibrated with MES buffer.

[0132] Buffer-exchanged V2 was desialylated with neuraminidase-agarose (Sigma N5254). The agarose bead product was provided in a 50% slurry mixture, stored in ammonium sulfate buffer; the beads were pre-washed 3-5 times in MES buffer; the bead / buffer mixture was separated by centrifugation at 1000 rcf for 3 min at 4°C, and the supernatant liquid was removed and discarded with a pipette. Buffer-exchanged V2 was added to the washed beads and gently mixed by rotation at room temperature for 16-22 hours. 2.08 mL of packed beads / mg protein were used for desialylation; for larger scale preparations, this was reduced 1:10 to 0.208 mL beads / mg protein. Thereafter, the desialylated V2 was recovered by centrifugation and removed with a pipette. The beads were gently washed again for 5 minutes by rotation in a 1:1 volume of fresh MES buffer; the wash mixture was centrifuged as before, and the supernatant was combined with V2. The beads were finally removed by sterile filtration through a 0.2 micron syringe filter or by vacuum filtration through a 0.45 micron filter.

[0133] EndoTrap HD resin (Hyglos) was used for several rounds of endotoxin removal. The resin was washed 3-5 times in MES buffer and the wash buffer was discarded. In two batches, 1-3 mL of washed resin was gently mixed with desialylated V2 at room temperature overnight. The resin was removed by centrifugation and then filtered through a syringe or vacuum filter.

[0134] Desialylated V2 was concentrated 4.75-fold to 2.1 mg / mL by 10-min cycles of centrifugation in Ultracels; the concentrate was gently mixed by pipetting to reduce any aggregation at the protein-filter interface.

[0135] With HiLoad 26 / 60 Superdex 200 size exclusion column, desialylated V2 is further separated from higher molecular weight substances (and aggregated endotoxin). The column and AKTA purifier system are pre-sterilized with 0.1N NaOH + 20%EtOH. The system is pH neutralized, rinsed with water, and balanced with reconstructed and merged V2 formulation buffer. Several batches of concentrate are manually injected into 12mL sample loops and loaded onto size exclusion columns at a flow rate of 3mL / min; the eluent is recovered and fractionated into polystyrene test tubes (17X100mm, Fisherbrand, 14-956-6D) with Frac-900. In the early stage, high molecular weight peaks are excluded, and the desired V2 fractions are merged, and Charles River EndoSafe PTS and NanoDrop ND-1000 are used to test endotoxin levels and concentrations. V2 desialylated V2 is eluted using V2 buffer.

[0136] 5 batches of size exclusion were performed and the collected eluates were combined into one batch which was concentrated to 1.0 mg / mL in Ultracels. The final preparation was sterile filtered through a 0.2 micron syringe filter and tested for endotoxin and concentration. 1 mL aliquots were transferred into labeled 2 mL tubes (Sarstedt, 72.694.006), flash frozen in ethanol / dry ice batches, and stored at -80°C in labeled boxes for later use.

[0137] Characterization - Protein Analysis and in vitro Determination

[0138] By using the protein gel analysis of 4-12%Bis-Tris NuPAGE (Novex NP0335BOX) in MES running buffer and by analyzing size exclusion (TSK3000 post; Running buffer: 200mM KH PO , 150mM KCl, pH 6.8, flow velocity: 0.15ml / min, fluorescence detection), characterize final preparation material and untreated starting material.With the sialic acid quantitative of DMB-labeled of total protein carried out by Takara Bio Inc. test kit discussed in this article, by LC-MS analysis the sialic acid content on the factor VII heavy chain in the small test sample.By the factor X activation of phospholipid dependency and thrombin generation determination, test activity.

[0139] Sialic acid content analysis

[0140] For untreated control and desialylated factor VII, sialic acid on the N-glycan of the heavy chain of factor VII was identified using LC-MS method. 10 µg of protein was reduced with 10 mM DTT mixture at 37 °C for 30 min and then analyzed on Agilent1200 Capillary LC system: Column: PLRP-S 8 µm 4000A, 0.3x150 mm, 75 °C. Buffer system: A: water with 0.2% formic acid + 0.01% TFA; B: ACN with 0.2% formic acid + 0.01% TFA. Gradient: 50 µL / min, reach 10% B in 2 min, reach 90% B in 25 min, wash at 90% B for 5 min, equilibrate at 10% B for 5 min.

[0141] Agilent 6520 Q-TOF system: DualEsi source, gas temperature: 350°C, drying gas: 7 psi, nebulizer: 10 psi, scan range: 500-3000 amu, 1 spectrum / s. Reference ions: 1221.990637 and 2421.91399 amu, 50 ppm window, Min 1000 counts.

[0142] The results are reported in Figure 7 middle.

[0143] Sialic acid quantification using the DMB labeling kit

[0144] The sialic acid fluorescent labeling kit (Takara Bio Inc., catalog number 4400) is used for quantitative and highly sensitive analysis of sialic acid glycoconjugates. The sialic acid fluorescent labeling technique based on HPLC using 1,2-diamino-4,5-methyleneoxybenzene (DMB) is a simple and highly sensitive quantitative method. In this method, after being labeled by DMB, free sialic acid is analyzed by reverse phase HPLC (GlycosepR, derived from Glyko, # 1-4727).

[0145] in conclusion

[0146] The V2 heavy chain has 2 N-glycosylation sites. The N-glycans are fucosylated, heavily sialylated di-, tri- and tetra-structures. No terminal sialic acid was found on the desialylated sample, suggesting that the sample was fully desialylated and had removed >99.9% of the sialic acid on the Factor VII N-glycan.

[0147] Half-life determination using rat hepatocytes

[0148] Preparation of hepatocytes

[0149] Primary rat hepatocytes of cryopreservation were obtained from CellzDirect (Invitrogen). Each bottle containing about 5 million cells was melted, and the cells were added to 10 ml of thawing medium, and then centrifuged at 60 g for 3 minutes. The cells were resuspended in incubation medium + 0.25% BSA (about 4 ml), and the cells were counted using a hemocytometer. After staining with trypan blue to identify dead cells, live cells were counted. Cell viability was 80-82%. Cells were used in the removal assay immediately after counting.

[0150] Thawing medium: Add Invitrogen CM3000 Thawing / Plating Supplement Pack to 500 ml Williams E medium. Incubation medium: Add Invitrogen CM4000 Cell Maintenance Supplement Pack to 500 ml Williams E medium.

[0151] In vitro hepatocyte clearance assay

[0152] Primary rat hepatocytes (1 million viable cells / ml) were incubated with 25 ng / ml different factor VII variants in CellzDirect incubation medium + 0.25% BSA in Eppendorf tubes at 37°C under gentle tumbling mixing, with a starting volume of 1.2 ml. At each specified time point, 0.25 ml of the mixture was removed and immediately centrifuged to precipitate the cells (1000rpm, 3 minutes, in an Eppendorf centrifuge). 0.18 ml of the clarified supernatant was taken out, quickly frozen, and stored overnight at -80°C. On the next day, the factor VII in the quantitative supernatant was determined using ELISA, with the corresponding purified mutant protein used as a standard. A cell-free control supernatant (wherein the factor VII variant was incubated at 37°C for 2 hours in a separate culture medium) was used as the zero time point value. Each incubation was performed in triplicate. Based on the method of Lu et al. (Lu reference), the equation CLint = 0.693 / was used. in vitro T 1 / 2 , calculated intrinsic clearance values, normalized for incubation volume and cell number. Using the program WinNonLin (Pharsight Corporation, Sunnyvale, CA), calculated in vitro Half-life (T 1 / 2). Supernatants from hepatocyte incubations were assayed using a double antibody sandwich ELISA format. 0.1 ml / well of anti-Factor VII monoclonal antibody (1.0 µg / ml in PBS) was added to a Greiner Microlon 655061 96-well plate. After incubation overnight at 4°C, the plates were blocked with 0.2 ml / well of 1% casein blocking buffer (50 mM TrisHCl, 100 mM NaCl, 0.05% Tween 20 pH 7.2) at 37°C for 1.5 hours. The plates were washed 4 times with 0.3 ml / well PBS + 0.05% Tween 20 (using a BioTek ELx405 plate washer) and the relevant Factor VII standards and unknown samples were then added to the plates. By adding 0.18 ml dilution buffer (50 mM TrisHCl, 100 mM NaCl, 0.1% casein, 0.05% Tween 20 pH7.2), 0.18 ml of each hepatocyte supernatant was diluted 2 times. 0.10 ml of each diluted supernatant was added to the ELISA plate in triplicate. Standards were prepared from the corresponding purified factor VII variants diluted in dilution buffer. 2 times serial dilutions of standards were prepared in dilution buffer to produce dilutions in the range of 50-0.8 ng / ml final concentration. Factor VII standards and samples (0.1 ml / well) were incubated at room temperature (21°C) for 2 hours. The plates were washed 4 times as above, followed by the addition of 1 µg / ml biotinylated detection antibody (0.1 ml / well) in dilution buffer (50 mM TrisHCl, 100 mM NaCl, 0.1% casein, 0.05% Tween 20 pH7.2), followed by incubation at room temperature for 1.5 hours. The plates were washed 4 times as above, followed by the addition of streptavidin-horseradish peroxidase (0.1 ml / well) diluted 1 / 1000 in dilution buffer, followed by incubation at room temperature for 1 hour. The plates were washed again, and 0.1 ml / well Ultra-TMB was added. After incubation at room temperature for 10-15 minutes, the reaction was stopped by the addition of 0.05 ml / well 2 M H2SO4. The absorbance was read at 450 nm using a Molecular Devices Spectramax M2 plate reader. Data analysis was performed using Softmax Pro5.4 (Molecular Devices).

[0153] Cryopreserved rat hepatocytes, thawing medium, and incubation medium (CellzDirect) were obtained from Invitrogen / Life Technologies (Grand Island, NY). 1-Step Ultra-TMB (One Step) substrate, catalog number 34028, was obtained from Thermo Scientific (Rockford, IL). Streptavidin-horseradish peroxidase (SA-HRP), catalog number DY998, was obtained from R&D Systems, Minneapolis, MN. Phosphate buffered saline (pH 7.2) was obtained from Invitrogen (Carlsbad, CA). Sprague-Dawley rat plasma (5% sodium citrate anticoagulant) was obtained from Bioreclamation (Westbury, NY). Greiner Microlon plates (catalog number 655061) were obtained by Fisher Scientific (Pittsburgh, PA).

[0154] Methods for obtaining deglycosylated variants: molecular variants

[0155] Wild-type factor VIIa has 2 N-glycans (N322 and N145), and V1 and V2 each have 4 N-glycans (N106, N145, N253, N322).Originally designed in V1 and V2 found in the other 2 N-glycans (N106, N253) to increase the half-life.For this work, by returning them to the endogenous amino acid sequence of wild-type factor VII (T106, V253) to remove these sites.Then by engineering N → Q mutations at these sites ( Figure 6 ), and the remaining two endogenous N-glycan sites (N145 and N322) were removed at the DNA level.

[0156] Wild-type factor VII is cloned in pmCMV to prepare pMB113.Synthesized the insertion sequence containing single N to Q mutation and double mutant (amino acid 145 and 322) at amino acid position 145 or 322, and cloned in pMB113 using XbaI and PmlI sites, produced clone pMB114-116.Then AscI and AfeI are used to clone the insertion sequence of the Gla domain encoding V1 and V2 in pMB113-116, and produce construct pMB117-120 (based on the variant of V1) and pMB121-124 (based on the variant of V2).Sequence verification (McLab) is carried out to all constructs.Via electroporation, with 6 hole forms, use every kind of construct transient transfection mammalian cells (CHO derived cell line).After transfection 4 days, collect supernatant, and express by western blotting assay, carry out hFVII ELISA (AssayPro) and FVII activity assay subsequently. A subset of variants were then single cell cloned.The V2 2-N glycan variant, designated pMB121, was purified and activated for further analysis.

[0157] Method for purifying / activating FVIIa from 10L WAVE expression

[0158] Method Introduction

[0159] The multi-step method that uses lasting several days to carry out, from the concentrated conditioned medium of diafiltration, purifies and activates FVII.First, the medium is melted and centrifuged to remove any aggregate that may have formed in the freezing / thawing process. Use pseudo-affinity capture step (it adopts the anion exchange column (Q-Sepharose) of eluting with CaCl) to further concentrate FVII protein and change buffer. Then, use hydroxyapatite column to further purify FVII protein. Then use smaller Q-Sepharose column to further purify FVII, then it is activated in the solution of pH 7.8 - 8.2 for 24 hours. By reducing pH to 4.0, stop activation reaction. Finally, FVIIa is dialyzed and frozen in preparation buffer (pH 6.5).

[0160] The final purified protein was characterized by SDS-PAGE, aSEC, ELISA, carbohydrate analysis, endotoxin and FVIIa activity assays.

[0161] FVII ELISA

[0162] Zymutest FVII enzyme-linked immunoassay (Aniara, West Chester, Ohio). The ELISA is a two-site immunoassay using rabbit anti-FVII polyclonal antibodies bound to the wells of a 96-well microplate. Samples are introduced followed by rabbit anti-FVII polyclonal antibodies conjugated to horseradish peroxidase (HRP). The assay is performed according to the manufacturer's instructions. Briefly, samples and calibrators are diluted in assay buffer in a 96-well round-bottom polypropylene dilution plate. A 50-µL aliquot of the diluted sample is transferred to the provided rabbit anti-FVII coated plate and incubated at room temperature for 15 minutes. 200 µL of HRP-conjugated rabbit anti-FVII is added and incubated at room temperature for 1 hour. The plate is washed five times with 300 µL of the provided wash buffer. TMB is added at 200 µL / well and incubated at room temperature for approximately 5 minutes. The reaction is stopped by the introduction of 50 µL of 0.45 M sulfuric acid. The absorbance is read at 450 nm. Factor VII levels were deduced by comparing sample values ​​to a V2 calibration curve generated using a 4-parameter curve fit.

[0163] Factor VII chromogenic assay

[0164] The Biophen FVII chromogenic assay (Aniara, West Chester, Ohio) was used. The chromogenic assay principle involves the formation of an enzyme complex consisting of Factor VII obtained from the sample and rabbit thromboplastin (tissue factor) supplied by the manufacturer. FX, added in excess, is activated to FXa, which in turn cleaves a chromogenic substrate (SXa-11) specific for FXa, thereby generating pNA. The amount of pNA released is directly proportional to the activity of FXa. The assay was performed according to the manufacturer's instructions. Briefly, samples and calibrators were diluted in Tris-BSA assay buffer in 96-well round-bottom polypropylene dilution plates (Fisher Scientific). Kit reagents R1, R2, and R3 and 96-well flat-bottom polystyrene assay plates (Costar) were warmed to 37°C prior to use. 30 µL of sample and calibrator were transferred from the dilution plate to the assay plate, followed by 30 µL of reagent R2 and then 60 µL of reagent R1. The assay plate was mixed and incubated for 7 minutes at 37°C on a rocking plate shaker (Boekel Scientific). 60 µL of R3 was added and the rate of change of absorbance (OD change at 405 nm / min) was measured at 37°C using a SpectraMax Plus microplate reader (Molecular Devices). Factor VII levels were deduced by comparing sample values ​​to the V2 calibration curve generated using a 4-parameter curve fit.

[0165] Phospholipid-dependent thrombin generation assay

[0166] Compared with wild-type FVIIa, as a result of additional γ-carboxylation, modification of the Gla-domain (P10Q / K32E) increases the potency of FX activation, thrombin generation and whole blood coagulation in the presence of anionic phospholipids or activated platelets. PL-dependent TGA is designed to measure rFVIIa activity in the presence of anionic phospholipids and is performed using thrombin calibrators and substrate reagents, FluCa-kit from Thrombinoscope, BV. Phospholipid (PL) vesicles composed of 20% phosphatidylserine (PS), 40% phosphatidylethanolamine (PE) and 40% phosphatidylcholine (PC) are obtained from Avanti Polar Lipids and prepared by sonication for 10 minutes in 100 mM NaCl, 50 mM Tris-HCl (pH 7.2).

[0167] 20 μL of PL-vesicles (500 μM) or thrombin calibrator were dispensed into 96-well plates. Different concentrations of rFVIIa were diluted in human HemA plasma and added to the PL mixture in triplicate and equilibrated at 37°C for 10 min. Thrombin generation was initiated by adding FluCa solution and thrombin was measured according to the Thrombinoscope. BV The reaction was monitored continuously for 60 min using the Calibrated Automated Thrombography (CAT) method described by . BV Data were acquired and analyzed using GraphPad (3.4.0) software, which was corrected for α2-macroglobulin activity using a thrombin calibrator. The analysis parameter 'peak height' represents the maximum level of thrombin generated, and 'endogenous thrombin potential' (ETP) corresponds to the total amount of thrombin generated. Thrombin generation parameters were analyzed using Prism 4.0 (GraphPad Inc) with a 4-parameter nonlinear curve fitting method.

[0168] Phospholipid-dependent FX activation assay

[0169] The ability of FVIIa to activate FX in the absence of tissue factor in the presence of phospholipid vesicles was measured using a PL-dependent FX activation assay. Factor VIIa or FVIIa variants were incubated with FX in the presence of phospholipid vesicles. FX activation was measured by the addition of S-2765, a chromogenic substrate for FXa. Briefly, calibrators and samples were diluted in Tris-HCl buffer in polypropylene round-bottom plates. 30 µL of 4 µg / mL FX (Haematologic Technologies Inc.) was added to all wells of a 96-well flat-bottom polystyrene plate, followed by 30 µL of phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine in a 20:40:40 weight % ratio. 30 µL of diluted samples and calibrators were transferred to the FX / phospholipid mixture. The plates were sealed, mixed gently, and incubated at 37°C for 20-23 hours. 40 µL of a 5 mM solution of S-2765 (DiaPharma) was added to all wells. The plates were sealed and incubated at 37°C for 6 hours. The absorbance was read at 405 nm in a microplate reader. The activity of the samples was determined by comparing their FX activation levels to the F7 calibration curve.

[0170] Rat PK study—animals, study protocol (injection of preparation, sampling of blood and preparation, ELISA, data analysis, sacrifice of animals).

[0171] 0.1 mg / kg of the proteins (F7, V2, V1, dV2 and dV1) were administered intravenously into Sprague Dawley rats. Plasma samples were taken starting 1 min after administration and analyzed by FVII ELISA.

[0172] HemA-PK study

[0173] 1.0 mg / Kg of protein (F7, dV1) was administered intravenously into HemA mice. Plasma samples were taken starting 5 min after administration and analyzed by FVII ELISA and sTF-PT assay.

[0174] FVII ELISA on plasma samples

[0175] Material

[0176] Monoclonal antibodies for FVIIa were used. In addition, a monoclonal antibody was biotinylated. Purified FVIIa variants (wild type or desialylated) were used as assay calibrants and assay controls. Blocking buffer was 1% (w / v) casein in 30mMTris pH 7.2, 60mM NaCl, 0.03% Tween-20. The assay dilution buffer (ADB) was 0.1% (w / v) casein, 50mM Tris pH 7.2, 0.1M NaCl, 0.05% Tween-20. The assay wash buffer was PBS + 0.05% Tween-20. The immunoassay plate was a Greiner Microlon high binding plate (#655061). Streptavidin-horseradish peroxidase (SA-HRP) was obtained from R&D Systems. HRP substrate Ultra-TMB was obtained from ThermoFisherPierce. Blank mouse plasma was obtained from CD1 or HemA mice either commercially (Bioreclamation) or by in-house sources. All other materials (casein, Tris, NaCl, Tween-20, PBS, sulfuric acid) were of reagent grade quality.

[0177] Method for FVIIa sandwich immunoassay

[0178] Coat 96-well assay plates with 0.1 ml / well of antibody to FVIIa (1 µg / ml in PBS) overnight at 4°C. Aspirate the plate and block with 0.2 ml / well of blocking buffer at room temperature for at least 2 hours under rotation (150 rpm). After blocking, wash the wells with 4 x 0.3 ml / well of wash buffer. Dilute FVIIa samples or standards 1:20 in ADB to a final concentration of 5% plasma and incubate 0.1 ml / well at room temperature under rotation for at least 1.5 hours. Measure all standards, controls, and samples in triplicate. After washing the plate as described above, add biotinylated antibody to FVIIa (42 ng / ml in ADB, 0.1 ml / well), and incubate the plate at room temperature under rotation for at least 1 hour. Wash the plate and then incubate with streptavidin-HRP (1:1000 in ADB) for at least 1 hour at room temperature under rotation. After the final plate wash, the wells were developed with 0.1 ml / well Ultra-TMB and the reaction was stopped with 0.05 ml / well 2M sulfuric acid. The stopped reaction was read at OD-450 nm and the data were analyzed and calibrated. The lower limit of quantitation (LLOQ) of the assay was typically 15-30 ng / ml FVIIa in 100% plasma.

[0179] Improved PT assay based on soluble tissue factor (sTF) for measuring rFVIIa activity

[0180] Prothrombin time (PT) assay was performed to measure the prothrombin time in HemA mice. In vitro Activity of human rFVIIa in plasma samples.

[0181] Briefly, 50 µL of a sample containing 10% HemA mouse plasma and 50% human FVII-deficient plasma (George King Inc) in aPTT buffer (0.15 M NaCl, 0.05 M Tris pH 7.5, 0.1% BSA) was mixed with 50 µL of sTF-PT reagent and incubated at 37°C for 30 seconds. sTF-PT reagent consisted of 1 volume of 2 µM recombinant human soluble TF (sTF 1-221 ) and 1 volume of 8µM phospholipid vesicles (PS 20 :PC 40 :PE 40 ). Clotting was initiated by the addition of 50 µL 25 mM CaCl2 and the clotting time was recorded on a STA coagulation analyzer (Diagnostica Stago Inc). Standards consisted of 2-fold serial dilutions of rFVIIa (wt-rFVII or modified rFVIIa variants) ranging from 200 to 0.78 ng / mL.

[0182] Efficacy of desialylated V2 in hemophilia A (HemA) mice

[0183] Acute Tail Cutting Efficacy Study

[0184] To determine blood loss, mice were anesthetized with isoflurane and the tail was placed in 0.9% saline warmed at 37-38°C in a 15 ml plastic tube for 10 min. The tail was cut with a scalpel 4 mm from the tip and immediately placed back in a separate 15 ml plastic tube containing 10 ml pre-warmed saline. The mice were allowed to bleed freely for 40 min. Desialylated V2 and F7 were administered intravenously 5 min after or 15- and 30-min before the tail cutting injury. Blood loss was quantified gravimetrically by weighing the tubes before and after blood collection.

[0185] Tail Vein Transection (TVT) Study

[0186] HemA mice were administered desialylated V2 or F7 by tail vein injection 1 hour before or 5 min after tail vein transection injury. Appropriate anesthesia was used. The tail vein was transected with a #11 scalpel straight blade and a timer was started. The mice were then returned to their individual clean cages with a white paper pad (Versi-Dri™) placed on top of a 4X8 inch heating pad. Animal activity was monitored hourly and at 24 hour time points over the next 9 hours. Any mice showing signs of reduced activity levels were recorded on the monitoring table, and any mice showing signs of excessive blood loss were euthanized immediately.

[0187] Thrombin-Antithrombin (TAT) Assay in HemA Mouse Plasma

[0188] Reagents:

[0189] (1) Capture antibody: anti-thrombin polyclonal antibody, obtained from Enzyme Research Labs, catalog number TAT-EIA-C.; (2) Detection antibody: HRP-conjugated anti-AT-III polyclonal antibody, obtained from Enzyme Research Labs, catalog number TAT-EIA-D, (3) Assay diluent: obtained from Enzyme Research Labs, catalog number TAT-EIA-D, (4) HRP substrate: Amplex Red, Invitrogen, catalog number A12216, (5) α-thrombin: obtained from Enzyme Research Labs, catalog number HT-1002a, stored at -80°C, (6) AT-III: obtained from Enzyme Research Labs, catalog number HAT, stored at -80°C, (7) BSA: obtained from Sigma, catalog number A-7030; (8) AT-III deficient plasma: purchased from EnzymeResearch Labs, catalog number: AT-DP, stored at -80°C.

[0190] Buffer

[0191] (1) TAT standard buffer: 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 0.05 U / mL heparin; (2) Coating buffer: 1 tablet of bicarbonate + 100 ml dH20, stored at 4°C; (3) Blocking buffer: 2% BSA-PBS; (4) Sample dilution buffer: add 0.1 M HEPES, pH 7.4, 0.15 M NaCl, 1% BSA, 0.05% Tween 20, filter and aliquot, store at -20°C; (5) Substrate buffer: add 50 µL 5 mg / mL Amplex Red and 20 µl 3% H2O2 to PBS buffer. Mix and prepare freshly before adding to the plate; (6) Preparation of 1µM TAT standard stock solution: add 100µL 1.36mg / ml human AT-III and 5.93µL 3.28 mg / mL human thrombin to 419µL TAT buffer, mix, and incubate at 37℃ for 10-20 min; (7) Preparation of 60 nM TAT standard stock solution: add 50µL 1µM TAT complex to 783µL AT-III deficient plasma, mix, aliquot into 50µL / vial, and store at -80℃.

[0192] Assay Procedure

[0193] 1. Dilute anti-thrombin pAb (capture antibody) in bicarbonate buffer (1:100 dilution: for one 96-well plate, add 110 µL antibody to 11 mL bicarbonate buffer).

[0194] 2. Add 100 µL of diluted coating antibody to each well of a 2HB Immulon 96-well plate. Tap the plate gently to ensure all liquid covers the bottom of the plate. Seal the plate and incubate overnight at 4°C.

[0195] 3. Wash 4 times with 300 µL of wash buffer in an automated plate washer. After the last wash, invert the plate and tap it on a clean paper towel.

[0196] 4. Add 150 µL of blocking buffer (2% BSA-PBS) to each well. Seal the plate and incubate at room temperature for 1.5 hours.

[0197] 5. Wash 4 times with 300 µL of wash buffer in an automated plate washer. After the last wash, invert the plate and tap it on a clean paper towel.

[0198] 6. Add 100 µL of standards, samples, and quality controls (QC) to each well in triplicate and incubate the plate at room temperature for 2 hours.

[0199] 7. Wash 4 times with 300 µL of wash buffer in an automated plate washer. After the last wash, invert the plate and tap it on a clean paper towel.

[0200] 8. Add 100 µL of HRP-detection antibody (1 / 100, 110 µL antibody in 11 mL conjugate diluent) to each well. Seal the plate and incubate at room temperature for 1 hour.

[0201] 9. Wash 4 times with 300 µL of wash buffer in an automated plate washer. After the last wash, invert the plate and tap it on a clean paper towel.

[0202] 10. Add 70 µL of Amplex Rd Substrate (freshly prepared) to each well.

[0203] 11. Incubate the plate in the dark at room temperature for 15-30 min.

[0204] 12. Read the plate at OD485nm / 595nm.

[0205] 13. Plot the standards using a 4-parameter curve fit; calculate the concentration from the controls and each sample from the standards in each ELISA plate.

[0206] Efficacy of desialylated V2 in coagulation-competent mice

[0207] Acute tail cleavage studies were performed to determine the efficacy of dV2 in coagulant mice. Coagulant mice were anesthetized with isoflurane and the tail was placed in 0.9% saline warmed at 37-38°C in a 15 ml plastic tube for 10 min. Following intravenous administration of 5 mg / kg tissue plasminogen activator (tPA), the tail was cut 50 mm from the tip with a scalpel and placed back into a separate pre-warmed 15 ml plastic tube containing 10 ml saline. Desialylated V2 and F7 were administered intravenously immediately after the tail cleavage injury. Mice were allowed to bleed freely for 45 min. Blood loss was quantified gravimetrically by weighing the tubes before and after blood collection.

[0208] result

[0209] In vitro characterization of desialylated or deglycosylated proteins

[0210] The heavy chain of dV2 was analyzed by LC-TOF MS. The analysis showed that the N-glycans on the heavy chain did not contain sialic acid after sialidase treatment. The presence of the Gla domain complicates such analysis of the light chain. In order to obtain an overall picture of the sialic acid content of the treated molecule, sialic acid fluorescent labeling was performed. This method showed that greater than 99.9% of the sialic acid on V2 was removed during the desialylation process. Figure 7 Analysis of the sialic acid content of desialylated V2 is shown. LC-TOFMS analysis and sialic acid fluorescence labeling were used. Similar results were obtained for the sialic acid content of dV1 (data not shown). The activity of the desialylated molecule was tested by phospholipid-dependent Xa activation and phospholipid-dependent TGA assays. PL-Xa and PL-TGA assays confirmed that the activity of the protein was not reduced after desialylation (see Fig. 9 and Fig.10 ). Fig. 9 Shown is the PL-FXa activation assay for desialylated proteins. Using the phospho-lipid FXa activation assay, the activity of desialylated V1 and V2 (dV1, dV2) was tested. Compared with their unmodified parent molecules, the two desialylated proteins have slightly higher activity in this assay. Fig.10 PL-TGA assays for desialylated proteins are shown. By PL-TGA, dV2 and dV1 showed slightly increased activity compared to their unmodified parent molecules. Results were normalized to F7.

[0211] PL-Xa assays consistently demonstrated measurable increases in activity of dV2 and dV1 compared to their unmodified parent molecules. Hypoglycosylated wtFVIIa, V2, and V1 molecules were expressed ( Fig.11 ) and were tested for expression and activity as crude expression extracts. Fig.11 The expression of hypoglycosylated FVII variants is shown. For FVII expression, culture medium samples 4 days after electroporation were analyzed. Western blot analysis using anti-Gla domain antibodies showed the expression of the variants. When the expression levels were normalized, the removal of the N-glycan sites did not seem to affect the activity. Fig.12The "specific activity" of hypoglycosylated FVII variants was determined using transfection supernatants. The activity of crude expression supernatants from 2 transient transfections of hypoglycosylated variants was determined by Xa activation assay. When the expression measured by ELISA was normalized, no decrease in activity caused by the removal of N-glycans was observed. As expected in this assay, V1 and F7 proteins had similar activities, while the V2 molecule had lower activity due to its TF-independence. This was further confirmed by a PL-TGA activity assay performed on purified hypoglycosylated V2 (having only 2 N-glycans (N322 and N145), referred to as pMB121). Fig.13 PL-TGA assays on purified hypoglycosylated variant pMB121 are shown. pMB1212 showed enhanced activity compared to F7 by PL-TGA assay, similar to unmodified V2. The in vitro clearance of these molecules was tested in a hepatocyte clearance model. dV2 showed significant clearance compared to unmodified V2 in this model ( Fig.14 ), whereas marginal or no increase in clearance was observed for the hypoglycosylated variant ( Fig.15 ).

[0212] Rat PK and HemA PK

[0213] Pharmacokinetic studies in Sprague Dawley rats demonstrated that desialylated and hypoglycosylated proteins were cleared significantly faster than their unmodified counterparts as measured by FVII ELISA. Fig.16 Rat pharmacokinetic results are shown. As measured by FVII ELISA, the half-life of desialylated V2 and V1 in Sprague Dawley rats is significantly shorter than their unmodified parent molecules. This also applies to desialylated V2, desialylated V1, and pMB121 (hypoglycosylated V2). The t1 / 2 of the two desialylated molecules is less than 1 min, while the t1 / 2 of their parent proteins is approximately 2.5 hours. The clearance of the hypoglycosylated V2 molecule pMB121 is equivalent to that of F7, with a t1 / 2 of 1.6 hours. The PK study in HemA mice has similar results, with dV2 and F7 having a half-life of approximately 3 min and 2.6 hours, respectively ( Fig.17 (A)). The short half-life was confirmed by sTF-PTT clotting assay ( Fig.17 (B)). Fig.17 HemA PK results are shown. The half-life of desialylated V2 in HemA mice was significantly shorter than its unmodified parent molecule as measured by A) FVII ELISA and B) sTF-PT assay.

[0214] HemA potency model

[0215] The efficacy of dV2 was tested in HemA mice. Using the HemA tail cleavage model, a dose of 1 mg / kg (iv bolus) of dV2 was shown to be effective. By comparison, the effective dose of F7 in this model was 2.5 mg / kg (iv bolus). These results confirm that dV2 is more effective than F7 ( Fig.18 (A)). The model was also used to demonstrate that the potency of dV2 was cleared more rapidly than that of F7 ( Fig.18 (B)). Fig.18 Results of desialylated V2 potency studies in HemA mice are shown. Studies using dV2 demonstrated that this molecule is A) more potent than F7 and B) has a faster clearance of potency than F7 in the HemA tail cleavage model.

[0216] Using the more sensitive TVT model, the faster clearance of potency of dV2 compared to F7 was also confirmed and the effective dose was confirmed. Fig.19 Shown is a dV2 efficacy study in the TVT HemA model. TVT studies using a higher sensitivity efficacy model (TVT) confirmed that dV2 had a faster efficacy clearance than F7. Thrombin-antithrombin (TAT) measurements (as a marker of thrombogenicity) performed at 30 and 60 min after administration in HemA mice showed significantly lower levels of dV2. Fig. 20 TAT measurements are shown. In HemA mice, dV2 administered at its effective dose (1 mg / kg) produced less thrombin-antithrombin (TAT) compared to the effective dose (2.5 mg / kg) of F7. This data, together with the efficacy data, suggests that dV2 has a more favorable therapeutic index than F7.

[0217] Efficacy in coagulation-active mice

[0218] The efficacy of dV2 was tested in tPA-treated, coagulation-competent mice. Using the tail-cut model, dV2 was shown to be effective at doses of 0.3-1 mg / kg (iv bolus). By comparison, the effective dose of F7 in this model was 5 mg / kg (iv bolus). These results confirm that dV2 is more effective than F7 ( Fig.21 ). Fig.21 Shown are the results of desialylated V2 potency studies in tPA-treated, coagulant-competent mice.

[0219] Clearance and potency of desialylated wild-type factor VII (dWT VIIa)

[0220] Use NovoSeven® derived from Novo Nordisk as the starting factor VII material, and use soluble sialidase as described above to desialylate the starting polypeptide, produce desialylated wild-type factor VII (dWTVIIa) as described above. Found that dWT VIIa has> 99% purity, low endotoxin, and does not have detectable sialic acid. In addition, mass spectrometry analysis shows the selectivity of sialic acid removal.

[0221] The activity of this dWT VIIa material was analyzed using the Biophen FVII chromogenic assay and the modified PT assay as described above and compared to wild-type Factor VII. Each of these analyses demonstrated that dWT VIIa had nearly identical activity to the wild-type Factor VII polypeptide.

[0222] The clearance of dWT VIIa and wild-type factor VII (1 mg / kg) was also analyzed and compared using a human tissue factor knock-in (TFKI) mouse model. Fig. 22 As shown in , the half-life of dWT VIIa is significantly shorter than that of wild-type Factor VII, and the clearance (ml / h / kg) is more than 40-fold faster.

[0223] Using TFKI mice and the tail cutting method described above, the efficacy of dWT VIIa relative to wild-type factor VII was studied. Briefly, 5 mg / kg tPA was injected intravenously into mice, followed by tail cutting 50 mm from the tip. Wild-type factor VII (NovoSeven®) or dWT VIIa was then injected intravenously at doses ranging from 1-6 mg / kg. Blood was then collected from the tail for 45 minutes, with unstable clots disrupted every 6 minutes during the collection phase. As in Fig.23 In the present invention, it is surprisingly found that dWT VIIa is significantly more effective than wild-type factor VII. More specifically, the dWT VIIa at a dosage of 3 mg / kg causes reduced blood loss compared to the wild-type factor VII at a dosage of 6 mg / kg. In view of the results of this analysis, it is determined that 2 mg / kg dWT VIIa is the bioequivalent dose of 6 mg / kg wild-type factor VII.

[0224] The ability of dWT VIIa and wild-type factor VII (NovoSeven®) to cause systemic coagulation was also studied by the thrombin-antithrombin (TAT) method described above. Mice were treated with bioequivalent doses of dWT VIIa (2 mg / kg) and wild-type factor VII (6 mg / kg), and the formation of TAT complexes was measured by ELISA. Fig.24As shown in , wild-type NovoSeven® Factor VII produced significantly higher TAT levels than dWT VIIa. Given the fact that dWT VIIa only produced baseline TAT levels, this experiment suggests that this dose of dWT VIIa does not produce observable systemic coagulation, despite the fact that the polypeptide is as effective as wild-type Factor VII.

[0225] In addition, the ability of dWT VIIa and wild-type factor VII (NovoSeven®) to cause thrombosis was studied in the FeCl3 thrombosis model. Mice were treated with bioequivalent doses of dWT VIIa (2 mg / kg) and wild-type factor VII (6 mg / kg) 15 minutes before the start of the thrombosis study. Thrombosis was then initiated by administering a 3.25% FeCl3 solution, which was then measured using Doppler for 30 minutes. The resulting blood flow data were plotted on a graph of blood flow versus time, and the percentage of the area under the curve for the control sample was calculated to determine the reduction in blood flow caused by thrombosis in each factor VII treatment group. As in Fig.25 As shown in, wild-type NovoSeven® Factor VII produced a significant reduction in blood flow (average of about 40%), while dWT VIIa showed almost no reduction in blood flow (average>90%). This experiment confirmed that a given dose of dWT VIIa produced greatly reduced thrombosis compared to wild-type Factor VII.

[0226] The activity and potency of dWT VIIa relative to wild-type factor VII were further investigated by examining the apparent binding affinity of these peptides to soluble tissue factor (sTF) using the SN-17c tripeptide fluorogenic substrate (HTI). Fig.26 As shown in , this analysis demonstrated that dWT VIIa (dF7) and wild-type Factor VII (F7) had equivalent apparent binding affinities for sTF. Fig. 27 As shown in, in an experimental model that examines the ability of these peptides to activate factor X by titrating factor X concentration in the presence of sTF-Factor VII complex (0.5 nM Factor VII [dWT VIIa or wild-type], 125 nM sTF), the Michaelis Menten kinetics of dWT VIIa and wild-type Factor VII demonstrated that dWT VIIa (dF7) can activate factor X more efficiently (approximately 2-fold) than wild-type Factor VII (F7). This data suggests that dWT VIIa is able to convert more factor X to factor Xa per factor VII active site than its wild-type counterpart.

[0227] discuss

[0228] There is an unmet medical need for the development of therapeutic agents that are effective for the treatment of acute bleeding, but have reduced thrombogenicity. Potent Factor VII polypeptides with a short half-life could potentially yield molecules with a larger therapeutic window suitable for use in acute bleeding.

[0229] V2 and V1 are two kinds of factor VIIa variants (Fig. 1-3). These variants contain mutations to their Gla domains, which increase their affinity to activated platelets, and in the case of V2, cause tissue factor irrelevance. Two variants also have 2 other N-glycosylation sites, which result in a half-life extended compared with wild-type factor VIIa, i.e., a favorable characteristic for the treatment of hemophilia. However, their use as therapeutic agents for acute bleeding can benefit from the shortening of half-life. This modification can reduce the risk of off-target effects, and thus increase their therapeutic index. We have confirmed here that the removal of sialic acid present in the carbohydrate chains of V2 and V1 results in significantly faster removal of the molecules in the in vitro hepatocyte clearance model. Hypoglycosylated variants are not removed faster in this in vitro model, and this prompting removal mechanism between desialylated and hypoglycosylated molecules is different. In vivo studies carried out in Sprague Dawley rats confirm that desialylated molecules (dV2 and dV1) and hypoglycosylated variant pMB121 have significantly reduced half-life. Interestingly, compared with the clearance rate of the reported desialylated wild-type FVIIa (Appa et al., Thrombosis and Haemostasis 104.2 / 2010), desialylated V2 and V1 all have increased clearance rates, and this feature may be attributed to their 2 other N-polysaccharides. There is no limiting effect on the content claimed in the present invention, and a possible theory of this activity is that these additional N-polysaccharides can become the additional ligands of ASGPR or similar receptors after desialylation and mediate faster removal. As measured by in vitro activity assays, compared with their parent molecules, the activity of these molecules is retained or increased. Further in HemA mouse PK studies, the faster removal of dV2 was confirmed in vivo, and in HemA tail shearing and TVT studies, it was confirmed to be effective.

[0230] Furthermore, desialylation of wild-type Factor VII produces a Factor VII polypeptide that is cleared much faster than wild-type, while also providing the surprising result of increased potency, as demonstrated in numerous experimental models.

[0231] The removal of the N-glycans of Factor VIIa or Factor VIIa variants or the change of the monosaccharide composition of the N-glycans produces molecules that are cleared faster. These molecules retain activity and are effective in vivo. The development of these Factor VIIa molecules that are cleared faster will be beneficial for the treatment of acute bleeding indications, and may become an antidote for various anticoagulants on the market.

Claims

1. Use of a desialylated Factor VII polypeptide in the preparation of a medicament for treating a mammal suffering from a disease or disorder in which blood clot formation is required, wherein the ratio of the number of moles of conjugated sialic acid to the number of moles of N-linked glycans in the composition of the Factor VII polypeptide is less than 0.1; The desialylated Factor VII polypeptide consists of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO:

18.

2. The use according to claim 1, wherein the disease or disorder is selected from the group consisting of: bleeding, thrombocytopenia, hemophilia, aortic aneurysm and over-administration of anticoagulants.

3. The use according to claim 1, wherein the disease or disorder is selected from bleeding in mammals undergoing transplantation or resection or surgery, bleeding due to varicose veins, intracranial bleeding, gastrointestinal bleeding and uncontrolled bleeding.

4. The use according to claim 1, wherein the desialylated Factor VII polypeptide consists of the amino acid sequence of SEQ ID NO:

17.

5. The use according to claim 1, wherein the desialylated Factor VII polypeptide consists of the amino acid sequence of SEQ ID NO:

18.

6. The use according to claim 1, wherein the desialylated Factor VII polypeptide is glycosylated.

7. The use according to claim 1, wherein the ratio of the number of moles of conjugated sialic acid to the number of moles of N-linked glycans is less than 0.

05.

8. The use according to claim 1, wherein the desialylated Factor VII polypeptide is fully desialylated.

9. The use according to claim 1, wherein the disease or disorder is bleeding.

10. The use according to claim 1, wherein the disease or disorder is selected from the group consisting of: penetrating traumatic injury; blunt traumatic injury; bleeding in cardiac surgery; bleeding in spinal surgery; orthopedic surgery; neurosurgery; oncology surgery; postpartum surgery; menorrhagia; bleeding in stem cell transplantation; bleeding in liver transplantation; gastrointestinal bleeding; bleeding from active varicose veins in cirrhosis; bleeding from nonvaricose veins in cirrhosis; diffuse alveolar hemorrhage; aortic aneurysm; intracerebral hemorrhage; traumatic brain injury; cerebral contusion; reversal of warfarin; reversal of heparin; reversal of anticoagulants; reversal of antithrombotics; factor VII deficiency; burns; prophylaxis in hemophiliacs with inhibitors; partial hepatectomy in patients without cirrhosis and in patients with cirrhosis; acquired hemophilia; idiopathic thrombocytopenic purpura; Glanzmann's thrombasthenia; Glanzmann's thrombasthenia refractory to platelet transfusion and Bernard-Soulier syndrome.

Images