Long-acting coagulation factor and method for producing the same

Abstract

The present invention relates to a long-acting coagulation factor and a method for producing the same. A polypeptide and a polynucleotide encoding the same are disclosed, the polypeptide comprising at least one chorionic gonadotropin carboxyl terminal peptide (CTP) attached to the carboxyl terminus of the coagulation factor instead of the amino terminus. Pharmaceutical compositions and pharmaceutical preparations comprising the polypeptide and polynucleotide of the present disclosure and methods for using and producing the same are also disclosed.

Description

[0001] This application is a divisional application of the invention patent application with application date of June 19, 2016, application number 201680047897.0, and invention name “Long-acting coagulation factor and production method thereof”. Technical Field

[0002] Disclosed are polypeptides comprising at least one carboxy-terminal peptide (CTP) of chorionic gonadotropin attached to the carboxy terminus of a coagulation factor, and polynucleotides encoding the same. Also disclosed are pharmaceutical compositions and pharmaceutical formulations comprising the polypeptides and polynucleotides of the disclosure, and methods of using and producing the same. Background Art

[0003] The development of clotting factor replacement therapy has changed the lives of many individuals with hemophilia. Hemophilia is a group of inherited genetic disorders that impair the body's ability to control blood clotting, or coagulation. People with hemophilia do not produce adequate amounts of the Factor VIII protein or the Factor IX protein, which are necessary for effective blood clotting. In people with severe hemophilia, even minor injuries can result in blood loss lasting days or weeks, and full recovery may not occur, which can lead to debilitating, permanent damage to joints and other organs and premature death.

[0004] Hemophilia is an inherited, X-linked bleeding disorder caused by defects or deletions of key factors in the coagulation cascade. In patients with hemophilia, thrombin generation and fibrin clot formation are severely impaired, leading to spontaneous bleeding events, most commonly in joints and internal organs, as well as excessive bleeding during and after surgery or trauma. Frequent bleeding also leads to joint swelling, joint damage, severe deformities, frequent infections, and reduced mobility in patients with hemophilia (Mayo Clinic). Hemophilia A is caused by defective or missing expression of factor VIII, while hemophilia B is caused by defective or missing expression of factor IX.

[0005] Hemophilia B results in insufficient procoagulant activity of FIX. Patients with hemophilia B have spontaneous soft tissue bleeding and recurrent hemarthrosis, which often result in severe arthropathy. Current treatment for these patients involves intravenous administration of recombinant FIX. However, the cost and the relatively rapid clearance of FIX from the circulation make the development of long-acting FIX a challenging task. The commercial availability of FVIII and FIX has improved the control of life-threatening bleeding episodes. Many patients receive prophylactic treatment, which reduces the risk of bleeding and its associated complications. However, a significant proportion of patients (10% to 30%) develop inhibitory antibodies to exogenously administered FVIII and FIX. Administration of the byproduct FVIIa can induce homeostasis and provide effective treatment for patients with inhibitory Abs.

[0006] Recombinant FVIIa In the present invention, rFVIIa is commercially available and was approved in 1996 for the treatment of hemophiliacs with inhibitors. However, rFVIIa is quickly removed with a terminal half-life of 2.5 hours. As a result, patients usually need multiple frequent infusions (giving 2 to 3 dosages at intervals of 2 to 3 hours) to reach sufficient homeostasis after mild to moderate hemorrhage. Therefore, people are very interested in the long-acting form of developing FVIIa, which will extend the duration of the hemostatic activity after the single dose and allow to greatly reduce the frequency of administration. Long-acting FVIIa also can increase the feasibility of long-term preventive treatment.

[0007] Various technologies are being developed to extend the half-life of FVIIa. However, there remains a need to achieve an extended half-life of this protein while maintaining its biological activity and ensuring that the modification does not induce significant immunogenicity. The present invention addresses this need by attaching a gonadotropin carboxyl-terminal peptide (CTP) to FVIIa, thereby modifying it to extend its half-life and biological activity. Summary of the Invention

[0008] In one aspect, the invention provides a kind of polypeptide modified through CTP, it is made up of factor VII coagulation factor and three chorionic gonadotropin carboxyl terminal peptides (CTP) attached to the carboxyl terminus of the coagulation factor, wherein the polypeptide does not include a signal peptide. In another aspect, the invention provides a polypeptide modified through CTP, wherein the coagulation factor is activated FVII (FVIIa). In another aspect, the sequence of the coagulation factor modified through CTP is as listed in SEQ ID NO:46.

[0009] In another aspect, the CTP-modified polypeptide comprising activated FVIIa-CTP3 comprises a light chain and a heavy chain linked by a disulfide bond. In another aspect, separation of the light chain and the heavy chain on SDS-PAGE gel occurs under denaturing conditions, wherein the light chain migrates with a molecular weight of approximately 25 kDa, and the heavy chain migrates with a kDa molecular weight of approximately 60.

[0010] In one aspect, the present invention provides a pharmaceutical formulation comprising a buffer, an amino acid (which is glycine in another embodiment), a tonicity agent, and a CTP-modified polypeptide, wherein the CTP-modified polypeptide is composed of a factor VII coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the coagulation factor, wherein the polypeptide does not include a signal peptide. In another aspect, the buffer comprises 20 mM citrate. In another aspect, the tonicity agent is 150 mM sodium chloride. In another aspect, the formulation is a liquid formulation. In another aspect, the formulation is at a pH of about 6.4.

[0011] In another aspect, the invention provides a formulation comprising 20 mM citrate, 13.3 mM glycine, and 150 mM sodium chloride at a pH of about 6.4.

[0012] In another aspect, the invention provides a kind of pharmaceutical preparation, wherein coagulation factor is activated FVII (FVIIa).In another aspect, the sequence of the coagulation factor modified by CTP is as listed in SEQ ID NO:46.In another aspect, the pharmaceutical preparation comprising polypeptide is administered by daily, every other day, every three days, once a week, twice a week or once every other week or its any combination.In another aspect, the administration of pharmaceutical preparation is intravenous or subcutaneous administration.

[0013] In another aspect, the present invention provides a pharmaceutical composition comprising a CTP-modified polypeptide as described herein and a pharmaceutically acceptable carrier.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising the pharmaceutical formulation as described herein.

[0015] In one aspect, the invention provides a method for extending the biological half-life of factor VII (FVII) coagulation factor, comprising attaching three chorionic gonadotropin carboxyl terminal peptides (CTP) to the carboxyl terminal of the FVII coagulation factor, thereby extending the biological half-life of the coagulation factor, wherein the activated form of the coagulation factor does not include a signal peptide. In another aspect, the coagulation factor is activated FVII (FVIIa). In another aspect, the sequence of the activated CTP-modified coagulation factor is as listed in SEQ ID NO:46.

[0016] In one aspect, the invention provides a method for improving the area under the curve (AUC) of factor VII (FVII) coagulation factor, comprising attaching three chorionic gonadotropin carboxyl terminal peptides (CTP) to the carboxyl terminal of the FVII coagulation factor, thereby improving the AUC of the coagulation factor, wherein the activated form of the coagulation factor does not include a signal peptide. In another aspect, the coagulation factor is activated FVII (FVIIa). In another aspect, the sequence of the activated CTP-modified coagulation factor is as listed in SEQ ID NO:46.

[0017] In one aspect, the present invention provides a method for reducing the dosing frequency of a factor VIIa (FVII) coagulation factor, comprising attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII coagulation factor, thereby reducing the dosing frequency of the coagulation factor, wherein the activated form of the FVII coagulation factor does not include a signal peptide. In another aspect, the sequence of the activated CTP-modified coagulation factor is as set forth in SEQ ID NO: 46.

[0018] In one aspect, the present invention provides a method for preventing or treating blood coagulation or a coagulation disorder in a subject, the method comprising administering to the subject an activated CTP-modified coagulation factor polypeptide, wherein the coagulation factor is FVIIa-CTP3, thereby preventing or treating blood coagulation or a coagulation disorder in the subject. In another aspect, the disorder is hemophilia. In another aspect, hemophilia includes hemophilia A or hemophilia B with inhibitors. In another aspect, administration is performed subcutaneously. In another aspect, administration is performed intravenously.

[0019] In another aspect, the invention provides a method of reducing bleeding episodes.

[0020] Other features and advantages of the present invention will become apparent from the following detailed description, examples, and accompanying drawings. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] This patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Firm upon request and payment of the necessary fee.

[0022] Figure 1A Shown are bar graphs showing harvests of restricted, diluted, transfected, and selected cells with FIX-CTP and FIX-CTP-CTP variants in the presence of 5 μg / ml of vitamin K3. FIX levels were quantified using a human FIX ELISA kit (Affinity Biologicals; Catalog Number: FIX-AG RUO), and the calculated protein concentration (μg / ml) was the average of two independent runs.

[0023] Figure 1B Gel micrographs of SDS-PAGE showing FIX Ab recognition and Western blot depicting recognition of anti-FIX antibodies; Figure 1B Lane 1 in the panel was loaded with a sample containing recombinant FIX; Figure 1B Lane 2 in the Figure was loaded with a sample containing the FIX-CTP harvest. Figure 1B Lane 3 in the Figure was loaded with a sample containing the FIX-(CTP)2 harvest.

[0024] Figure 1C Micrographs of SDS-PAGE gels showing FIX Ab recognition. Figure 1C Recognition of anti-γ-carboxylation antibodies was depicted by Western blot. Figure 1C Lane 1 in the Figure is loaded with a sample containing recombinant FIX. Figure 1C Lane 2 in the Figure was loaded with a sample containing the FIX-CTP harvest. Figure 1C Lane 3 in the Figure was loaded with a sample containing the FIX-(CTP)2 harvest.

[0025] Figure 2 A graph is shown showing the comparative chromogenic activity of FIX-CTP and FIX-(CTP)2 harvests compared to rhFIX (American Diagnostics) (by EC 50 concentration was measured).

[0026] Figure 3 Shown are graphs showing the PK profiles of rhFIX, the harvest of FIX-CTP-CTP, and the harvest of FIX-CTP.

[0027] Figure 4 Shown are bar graphs showing the FIX antigen levels of the harvest of FIX-CTP, the harvest of FIX-CTP-CTP, and the FIX-CTP-CTP purified protein as determined using a human FIX ELISA kit (Affinity Biologicals; Catalog No.: FIX-AG RUO). The calculated protein concentration (μg / ml) is the average of two independent runs.

[0028] Figure 5A Gel micrographs of SDS-PAGE showing FIX Ab recognition and depicting Coomassie blue staining. Lane 1 is loaded with a sample containing FIX-(CTP)2. Lane 2 is loaded with a sample containing unbound FIX-(CTP)2. Lane 3 is loaded with a sample containing the concentrated eluate of FIX-(CTP)2.

[0029] Figure 5BGel micrographs of SDS-PAGE showing FIX Ab recognition and Western blot depicting recognition of anti-FIX antibodies. Lane 1 is loaded with a sample containing FIX-(CTP)2. Lane 2 is loaded with a sample containing unbound FIX-(CTP)2. Lane 3 is loaded with a sample containing the concentrated eluate of FIX-(CTP)2.

[0030] Figure 5C Gel micrograph of SDS-PAGE showing FIX Ab recognition and Western blot depicting recognition of anti-γ-carboxylation antibodies. Lane 1 is loaded with a sample containing FIX-(CTP)2. Lane 2 is loaded with a sample containing unbound FIX-(CTP)2. Lane 3 is loaded with a sample containing the concentrated eluate of FIX-(CTP)2.

[0031] Figure 6 Shown is a graph showing the chromogenic activity (sample concentration / OD) of FIX-(CTP)2 compared to human normal pooled plasma and rhFIX (American Diagnostics).

[0032] Figure 7 Shown are graphs showing the PK profiles of purified FIX-CTP-CTP, rhFIX, a harvest of FIX-CTP-CTP, and a harvest of FIX-CTP.

[0033] Figure 8A The Western blot of anti-CTP and anti-gamma carboxylation antibodies of the FIX fused to three, four or five CTPs is shown. The harvest of FIX-CTP3, FIX-CTP4 and FIX-CTP5 is loaded on 12% Tris-glycine gel using Precision plus dual-color protein markers (Bio-Rad). Anti-CTP polyclonal Ab (Adar Biotech Production) is used to perform SDS-PAGE analysis with Western blotting.

[0034] Figure 8B The Western blot of anti-CTP and anti-gamma carboxylation antibodies of the FIX fused to three, four or five CTPs is shown. The harvest of FIX-CTP3, FIX-CTP4 and FIX-CTP5 is loaded on 12% Tris-glycine gel using Precision plus dual-color protein markers (Bio-Rad). Anti-Gla Ab (American Diagnostica) is used to perform SDS-PAGE analysis with Western immunoblotting.

[0035] Figure 9The Coomassie blue detection of FIX-CTP3, FIX-CTP4 and FIX-CTP5 is shown. After the purification process (immunoaffinity purification of glycosylated proteins) utilizing a japonin column, FIX-CTP3, FIX-CTP4 and FIX-CTP5 were loaded onto a 12% Tris-glycine gel using Precision plus dual-color protein marker (Bio-Rad). SDS-PAGE was stained with Coomassie blue dye for sample detection.

[0036] Figure 10 The chromogenic activity of FIX is shown. Using commercially available chromogenic activity detection kit, BIOPHEN (HyphenBioMed 221802) performs a comparative assessment of the in vitro effectiveness of fully purified (HA column) FIX-CTP3 FIX-CTP4 and FIX-CTP5 with human pool normal plasma. All samples are serially diluted and effectiveness is assessed by comparing a dose-response curve with the benchmark configuration consisting of normal human plasma.

[0037] Figure 11 Comparative pharmacokinetic (PK) curves of FIX-CTP3, FIX-CTP4, and FIX-CTP5 are shown. The FIX concentration in plasma samples was quantified using a human FIX Elisa kit (Affinity Biologicals). Pharmacokinetic curves were calculated and are the average values ​​of 3 animals at each time point. Terminal half-life was calculated using PK Solutions 2.0 software.

[0038] Figure 12A FIG10 is a block diagram of the FIX-CTP3 SDS-PAGE analysis-Coomassie SDS-PAGE. Precision plus dual-color protein marker (Bio-Rad) was used to load the enriched protein of FIX-CTP3γ-carboxylation, rhFIX, and rFIXa (activated FIX) onto 12% Tris-glycine gels. SDS-PAGE Coomassie analysis was performed by staining the gel with Coomassie blue reagent (800 ng of protein).

[0039] Figure 12B Figure 2 shows FIX-CTP3 SDS-PAGE analysis - Coomassie SDS-PAGE. FIX-CTP3γ-carboxylated enriched protein, rhFIX and rFIXa (activated FIX) were loaded on a 12% Tris-glycine gel using a Precision plus dual-color protein marker (Bio-Rad). Western immunoblotting was performed using 100 ng of protein with an anti-human FIX polyclonal Ab.

[0040] Figure 12CFigure 29 shows FIX-CTP3 SDS-PAGE analysis-Coomassie SDS-PAGE. Precision plus dual color protein marker (Bio-Rad) is used to load the enriched protein of FIX-CTP3γ-carboxylation, rhFIX and rFIXa (activated FIX) on 12% Tris-glycine gel. Protein immunoblotting is performed using 100ng of protein (American Diagnostics, catalog #499,3570) with anti-human γ carboxylation monoclonal antibody.

[0041] Figure 12D Figure 2 shows FIX-CTP3 SDS-PAGE analysis - Coomassie SDS-PAGE. FIX-CTP3γ-carboxylated enriched protein, rhFIX and rFIXa (activated FIX) were loaded on a 12% Tris-glycine gel using Precision plus dual color protein marker (Bio-Rad). Western blotting was performed using 100 ng of protein with anti-FIX propeptide polyclonal Ab ( Figure 12D ).

[0042] Figure 12E Figure 24 shows FIX-CTP3 SDS-PAGE analysis - Coomassie SDS-PAGE. FIX-CTP3γ-carboxylated enriched protein, rhFIX and rFIXa (activated FIX) were loaded on a 12% Tris-glycine gel using a Precision plus dual color protein marker (Bio-Rad). Western immunoblotting was performed using 100 ng of protein with anti-CTP polyclonal Ab.

[0043] Figure 13 The chromogenic activity of FIX-CTP3 is shown. A comparative evaluation of the in vitro potency of FIX-CTP3 harvest and FIX-CTP3γ-carboxylated enriched protein with human pooled normal plasma was performed using a commercially available chromogenic activity assay kit, BIOPHEN (Hyphen BioMed 221802). The FIX-CTP3 harvest and protein were serially diluted and potency was assessed by comparing the dose-response curve to a benchmark formulation consisting of normal human plasma.

[0044] Figure 14 Comparative clotting times are shown. An in vitro aPTT (activated partial thromboplastin time assay) was performed to compare the clotting activity of FIX-CTP3 and BeneFIX. The proteins were serially diluted and spiked into human FIX-depleted plasma, and clotting times were assessed.

[0045] Figure 15Comparative PK curves for FIX-CTP3 are shown. FIX concentrations were quantified using a human FIX ELISA kit (Affinity Biologicals; catalog # FIX-AG RUO). Pharmacokinetic curves were calculated for each protein and are the average of three animals at each time point.

[0046] Figure 16A In parallel with PK sampling, clotting activity of citrated plasma samples from FIX-deficient animals administered FIX-CTP3 was assessed by aPTT assay, which was converted to % activity. % activity at each collection point was calculated as current clotting time / clotting time of normal pool mouse plasma*100.

[0047] Figure 16B In parallel with PK sampling, the performance was assessed by aPTT assay. The clotting activity of citrated plasma samples from dosed FIX-deficient animals was converted to % activity. The % activity at each collection point was calculated as current clotting time / clotting time of normal pooled mouse plasma*100.

[0048] Figure 17A Figure 2 shows the bleeding parameters of the first challenge. Or a single intravenous injection of rFIX-CTP3. 48 hours after dosing, the tail vein was slightly shortened and the tail vein bleeding time (TVBT) was assessed. A second bleeding challenge was performed 15 minutes after reaching homeostasis, and the same parameters were measured.

[0049] Figure 17B Figure 2 shows bleeding parameters for the first challenge. Or a single intravenous injection of rFIX-CTP3. 48 hours after administration, the tail vein was slightly shortened and the tail vein bleeding time (TVBT) was assessed. A second bleeding challenge was performed 15 minutes after reaching homeostasis, and the same parameters were measured.

[0050] Figure 17C Figure 2 shows the bleeding parameters of the first challenge. Or a single intravenous injection of rFIX-CTP3. At 48 hours after administration, the tail vein was slightly cut short and the bleeding intensity (hemoglobin OD) was assessed. A second bleeding challenge was performed 15 minutes after reaching homeostasis and the same parameters were measured.

[0051] Figure 17D Figure 2 shows bleeding parameters for the first challenge. Or a single intravenous injection of rFIX-CTP3. At 48 hours after administration, the tail vein was slightly cut short and the bleeding intensity (hemoglobin OD) was assessed. A second bleeding challenge was performed 15 minutes after reaching homeostasis and the same parameters were measured.

[0052] Figure 18A Bleeding parameters for the second challenge are shown. Once spontaneous or manual cessation of bleeding occurs 19A to 19D If the first bleeding challenge was not performed as described in the legend to the figure, a second bleeding challenge was performed 15 minutes after the first and the time was remeasured.

[0053] Figure 18B Bleeding parameters for the second challenge are shown. Once spontaneous or manual cessation of bleeding occurs 19A to 19D If the first bleeding challenge was not performed as described in the legend to the figure, a second bleeding challenge was performed 15 minutes after the first and the time was remeasured.

[0054] Figure 18C Bleeding parameters for the second challenge are shown. Once spontaneous or manual cessation of bleeding occurs 19A to 19D If the first bleeding challenge was not described in the legend to the figure, a second bleeding challenge was performed 15 minutes after the first one, and the bleeding intensity was re-measured.

[0055] Figure 18D Bleeding parameters for the second challenge are shown. Once spontaneous or manual cessation of bleeding occurs 19A to 19D If the first bleeding challenge was not described in the legend to the figure, a second bleeding challenge was performed 15 minutes after the first one, and the bleeding intensity was re-measured.

[0056] Figure 19A A diagram illustrating the rFVII-CTP construct is shown.

[0057] Figure 19B A diagram illustrating the rFVII-CTP-CTP construct is shown.

[0058] Figure 19C Shown is a diagram illustrating the rFIX-CTP construct.

[0059] Figure 19D Shown is a diagram illustrating the rFIX-CTP-CTP construct.

[0060] Figure 20A Shown are bar graphs showing harvests of restricted, diluted, clonally transfected and selected cells with FVII-CTP variants in the presence of 5 μg / ml of vitamin K3. Levels of FVII were quantified using a FVII ELISA (AssayPro).

[0061] Figure 20BShown are bar graphs showing harvests of restricted, diluted, transfected and selected cells with FVII-CTP variants in the presence of 5 μg / ml of vitamin K3 activity. FVII activity was quantified using a FVII chromogenic activity assay (AssayPro).

[0062] Figure 20C Shown are bar graphs showing harvests of restricted, diluted, transfected, and selected cells with FVII-CTP variants in the presence of 5 μg / ml of vitamin K3. The specific activity of FVII was calculated for each version by dividing the activity value by the FVII concentration of the harvest.

[0063] Figure 20D Shown are graphs showing the PK profiles of FVII, FVII-CTP-CTP, and the harvest of FVII-CTP.

[0064] Figure 21A The protein blot of the FVII that is fused to three, four and five CTPs that anti-FVII, anti-CTP and anti-gamma carboxylation antibody are used to detect is shown.The harvest of FVII-CTP3, FVII-CTP4 and FVII-CTP5 uses Precision plus two-color protein marker (Bio-Rad) to be loaded on 12% Tris-glycine gel (expedeon).Use anti-FVII to carry out SDS-PAGE analysis with protein immunoblotting.

[0065] Figure 21B The protein blot of the FVII that is fused to three, four and five CTPs that anti-FVII, anti-CTP and anti-gamma carboxylation antibody are used to detect is shown.The harvest of FVII-CTP3, FVII-CTP4 and FVII-CTP5 uses Precision plus two-color protein marker (Bio-Rad) to be loaded on 12% Tris-glycine gel (expedeon).Use anti-CTP polyclonal Ab (Adar Biotech Production) to carry out SDS-PAGE analysis by protein immunoblotting.

[0066] Figure 21CThe protein blot of the FVII that is fused to three, four and five CTPs that anti-FVII, anti-CTP and anti-gamma carboxylation antibody are used to detect is shown.The harvest of FVII-CTP3, FVII-CTP4 and FVII-CTP5 uses Precision plus two-color protein marker (Bio-Rad) to be loaded on 12% Tris-glycine gel (expedeon).Use anti-GlaAb (American Diagnostica) to carry out SDS-PAGE analysis by protein immunoblotting.

[0067] Figure 22 FVII activity-chromogenic activity is shown.Use commercially available chromogenic activity detection kit, BIOPHEN (HyphenBioMed 221304) carries out the comparative assessment of the in vitro effectiveness of (height gamma carboxylation component) FIX-CTP3, FIX-CTP4 and FIX-CTP5 of HA purification and normal human pool plasma.All samples are serially diluted and compared to assess effectiveness by the benchmark configuration product being composed of dose-response curve and normal human plasma.

[0068] Figure 23 Shown is the first comparative pharmacokinetic (PK) curve-FVII 3CTP, FVII 4CTP and FVII 5CTP.FVII-CTP3, FVII-CTP4 and FVII-CTP5 (being respectively group A, group B and group C) are pressed single intravenous injection with the dosage of 250 μ g / kg body weight to Sprague Dawley rats (each treatment six rats) are administered.At 0.083 hour, 0.5 hour, 2 hour, 5 hour, 8 hour, 24 hour, 48 hour, 72 hour and 96 hour after administration, alternately carry out the retroorbital extraction of blood sample from 3 rats.After sampling, prepare the plasma (0.38%) of citric acidization immediately and store it at-20 ℃ until being analyzed.Compared with other two versions, FVII-CTP5 has demonstrated outstanding curve.

[0069] Figure 24The second comparative PK curve-FVII 3CTP, FVII 4CTP and FVII 5CTP are shown.After FVII is selected and HA purification process, FVII-CTP3, FVII-CTP4 and FVII-CTP5 (being respectively group A, group B and group C) are administered to Sprague Dawley rats (three rats of every kind of material) by single intravenous injection with the dosage of 29.45 μ g / kg body weight.The back orbital extraction of blood sample was carried out in 0.083 hour, 0.5 hour, 2 hours, 8 hours, 24 hours, 48 ​​hours and 72 hours after administration.After sampling, prepare the plasma (0.38%) of citric acidization immediately and be stored in-20 ℃ until analyzing.

[0070] Figure 25A Schematic diagram showing the FVII-CTP3 purification process. Batch 31 was produced for PK / PD studies.

[0071] Figure 25B Schematic diagram showing the FVII-CTP3 purification process. Batch 38 was produced for survival studies.

[0072] Figure 26A Final SDS-PAGE and Western blot analysis of FVII and FVIIa are shown. 10 μg (batch 31) or 5 μg (batch 38) were loaded into each lane of a Coomassie-stained SDS-PAGE. 1. FVII-CTP3 polypeptide; 2. Heavy chain, including 3×CTP; 3. Light chain. All three antibodies detected FVII.

[0073] Figure 26B Final SDS-PAGE and Western blot of FVII and FVIIa are shown. 10 μg (batch 31) or 5 μg (batch 38) were loaded in each lane of Coomassie-stained SDS-PAGE. 1. FVII-CTP3 polypeptide; 2. Heavy chain, which includes 3×CTP; 3. Light chain.

[0074] Figure 26C Final SDS-PAGE and Western blot of FVII and FVIIa are shown. 10 μg (batch 31) or 5 μg (batch 38) were loaded in each lane of Coomassie-stained SDS-PAGE. 1. FVII-CTP3 polypeptide; 2. Heavy chain, which includes 3×CTP; 3. Light chain.

[0075] Figure 26DFinal SDS-PAGE and Western blot of FVII and FVIIa are shown. 10 μg (batch 31) or 5 μg (batch 38) were loaded in each lane of Coomassie-stained SDS-PAGE. 1. FVII-CTP3 polypeptide; 2. Heavy chain, which includes 3×CTP; 3. Light chain.

[0076] Figure 26E Final SDS-PAGE and Western blot of FVII and FVIIa are shown. 10 μg (batch 31) or 5 μg (batch 38) were loaded in each lane of Coomassie-stained SDS-PAGE. 1. FVII-CTP3 polypeptide; 2. Heavy chain, which includes 3×CTP; 3. Light chain.

[0077] Figure 26F Shown are SDS-PAGE and Western blots of final FVII and FVIIa. 1 μg of protein was loaded into each lane of the Western blot. 1. FVII-CTP3 polypeptide; 2. Heavy chain, including 3×CTP; 3. Light chain. All three antibodies detected FVII. The FVIIa light chain was detected using α-FVII.

[0078] Figure 26G Shown are the final SDS-PAGE and Western blots of FVII and FVIIa. 1 μg of protein was loaded into each lane of the Western blot. 1. FVII-CTP3 polypeptide; 2. Heavy chain, including 3×CTP; 3. Light chain. All three antibodies detected FVII. The FVIIa heavy chain was detected by α-CTP.

[0079] Figure 26H Shown are the final SDS-PAGE and Western blot of FVII and FVIIa. 1 μg of protein was loaded into each lane of the Western blot. 1. FVII-CTP3 polypeptide; 2. Heavy chain, including 3×CTP; 3. Light chain. All three antibodies detected FVII. The FVIIa heavy chain was detected by α-Gla.

[0080] Figure 27 Illustrate that due to the purification on ceramic hydroxyapatite (HA) post, the chromogenic activity of FVII-CTP3 is enhanced.Use commercially available chromogenic activity detection kit, BIOPHEN (Hyphen BioMed 221304) carries out the comparative assessment of the in vitro effectiveness of the component in FVII-CTP3 harvest, process and the FVII-CTP3 of purification and people's pool normal plasma.FVII-CTP3 harvest and protein are serially diluted and by comparing the benchmark configuration product of dose-response curve and normal human plasma to assess effectiveness.

[0081] Figure 28FVIIa-CTP3 and FVIII-deficient mice PK curve of FVIIa-CTP3. By single intravenous injection, FVIII- / -hemophilia mice are administered.After administration, 0.083 hour, 0.5 hour, 2 hour, 8 hour, 24 hour, 48 hour and 72 hour, blood sample is extracted retroorbitally.After sampling, prepare citrated plasma (0.38%) immediately and store it at-20 ℃ until analyzed, and use STACLOT commercial test kit to set up PK curve based on FVIIa coagulation activity.

[0082] Figure 29A The production of FVIIa-CTP3 after FVII selection, HA purification process and activation is shown. FVIII- / - hemophiliac mice were dosed by a single intravenous injection. Retroorbital blood samples were drawn at 0.083 hours, 0.5 hours, 2 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after dosing. Immediately after sampling, citrated plasma (0.38%) was prepared and stored at -20°C until analysis. Thrombin generation parameters were assessed during the PK experiment, including parameters for the maximum amount of the peak.

[0083] Figure 29B The production of FVIIa-CTP3 after FVII selection, HA purification process and activation is shown. FVIII- / - hemophiliac mice were dosed by a single intravenous injection. Retroorbital blood samples were drawn at 0.083 hours, 0.5 hours, 2 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after dosing. Immediately after sampling, citrated plasma (0.38%) was prepared and stored at -20°C until analysis. Thrombin generation parameters were assessed during the PK experiment, and the parameters including the amount of thrombin at each time point were assessed.

[0084] Figure 29C The production of FVIIa-CTP3 after FVII selection, HA purification process and activation is shown. FVIII- / - hemophiliac mice were dosed with a single intravenous injection. Retroorbital blood samples were drawn at 0.083 hours, 0.5 hours, 2 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after dosing. Immediately after sampling, citrated plasma (0.38%) was prepared and stored at -20°C until analysis. Thrombin generation parameters were assessed during the PK experiment, including parameters including thrombin generation rate.

[0085] Figure 30A Survival curves of hemophiliac mice after tail vein transection (TVT) are shown. TVT was performed 15 minutes after administration. After TVT, the survival of mice was observed for 24 hours and recorded every hour for the first 12 hours and 24 hours thereafter. The data for the control group (vehicle) are the sum of 3 experiments (5 mice / experiment).

[0086] Figure 30B Survival curves of hemophiliac mice after tail vein transection (TVT) are shown. TVT was performed 24 hours after administration. The survival of mice was observed for 24 hours after TVT and recorded every hour for the first 12 hours and 24 hours thereafter. The data for the control group (vehicle) are the sum of 3 experiments (5 mice / experiment).

[0087] Figure 30C Survival curves of hemophiliac mice after tail vein transection (TVT) are shown. TVT was performed 48 hours after administration. After TVT, the survival of the mice was observed for 24 hours and recorded every hour for the first 12 hours and 24 hours thereafter. The data for the control group (vehicle) are the sum of 3 experiments (5 mice / experiment).

[0088] Figure 30D Mouse survival recorded 24 hours after TVT is summarized.

[0089] Figure 31A Immunoblots of FVII-3-CTP and FVII-5CTP are shown as GLA blots.

[0090] Figure 31B Immunoblots of FVII-3-CTP and FVII-5CTP are shown as FVII blots.

[0091] Figure 31C Immunoblots of FVII-3-CTP and FVII-5CTP are shown as CTP blots.

[0092] Figure 32 Shown are comparative PK curves of FVII 3CTP and FVII 5CTP from selection and HA column purification (FVIIS vs. FVII HA).

[0093] Figure 33 Shown are comparative PK profiles of FVII 3CTP and FVII 5CTP - Second Study (IV vs. SC).

[0094] Figure 34 Shown is the survival curve of hemophilia mice after tail vein transection (TVT) after SC administration. TVT was performed 12 hours after administration. After TVT, the survival of mice was observed for 24 hours and recorded every hour after the first 12 hours and 24 hours.

[0095] Figure 35A MOD-5014 after IV administration is shown to be PK curve.

[0096] Figure 35B Showing that MOD-5014 after SC administration PK curve.

[0097] Figure 36 Shown are the effects of MOD-5014 (clones 61#75, #81) and PK curve.

[0098] Figure 37 Warfarin increases PT and aPTT values. SD rats were orally administered 10 mg / kg of warfarin, and blood samples were drawn at designated time points. Plasma was prepared and PT and aPTT values ​​were determined.

[0099] Figure 38 Inject MOD-5014 into the IV and Acute effects in warfarin-treated rats.

[0100] Figure 39 The results show that rats treated with warfarin responded to various MOD-5014 and Dose response.

[0101] Figure 40 MOD-5014 was shown to restore PT values ​​to normal values ​​up to 48 hours after administration. The effect disappears after 24 hours.

[0102] Figure 41 Shown with injection IV injection of MOD-5014 reduced bleeding time in warfarin-treated rats compared with 24 and 48 hours after administration.

[0103] Figure 42 MOD-5014 was able to restore PT values ​​to normal within 48 hours after administration. The effect disappears after 24 hours.

[0104] Figure 43 Shows benefit by keeping blood loss low for 48 hours after administration The superiority of.

[0105] Figure 44 Tissue factor pathway inhibitor (TFPI) was shown to inhibit MOD-5014 and

[0106] Figure 45 Antithrombin III was shown to inhibit MOD-5014 and

[0107] Figure 46 Shown by MOD-5014 and The results of the Factor X activation performed were almost identical.

[0108] Figure 47 The results show that the presence of TFPI (20 μg / ml to 0.002 ng / ml) was determined by MOD-5014 at 0.6 ng / ml and Factor X activation was performed.

[0109] Figure 48 The results show that the presence of TFPI (20 μg / ml to 0.002 ng / ml) was determined by MOD-5014 at 4.0 ng / ml and Factor X activation was performed.

[0110] Figure 49 Factor X activation in the presence of TFPI and heparin is shown, where MOD-5014 and showed similar activation.

[0111] Figure 50 Factor X activation in the presence of antithrombin III is shown, where MOD-5014 and showed similar activation.

[0112] Figure 51 In the presence of heparin, MOD-5014 and Factor X activation was performed with ELISA, where similar moderate inhibition was observed.

[0113] Figure 52 The results show that MOD-5014 and Similar Factor X activation was performed.

[0114] Figure 53Shown with commercially available high phospholipid (PL) concentration Compared with the thrombin generation curve of MOD-5014.

[0115] Figure 54 Shown with commercially available low phospholipid (PL) concentration Compared to the peak thrombin generation curve of MOD-5014.

[0116] Figure 55A and Figure 55B Shown for MOD-5014 and The thromboelastography results of and both reduced clotting time and increased clot formation rate.

[0117] Figure 56 Shows the recalcification Results of thrombin generation (TG). (Run #1)

[0118] Figure 57 Results of thrombin generation (TG) for MOD-5014 after recalcification are shown. (Run #1)

[0119] Figure 58 The AE provides a (NS) Results are overlaid with the results for MOD-5014(PRO)TG. A: Results at 1.25 μg / ml are provided. B: Results at 5 μg / ml are provided. C: Results at 15 μg / ml are provided. D: Results at 2.5 μg / ml are provided. E: Results at 10 μg / ml are provided. (Run #1)

[0120] Figure 59 AD provides the Overlay analysis of (NS) results relative to MOD-5014 (PRO) TG results. A: Shows results at 1.25 μg / ml of NS and 2.5 μg / ml of PRO. B: Shows results at 5 μg / ml of NS and 10 μg / ml of PRO. C: Shows results at 2.5 μg / ml of NS and 5 μg / ml of PRO. D: Shows results at 10 μg / ml of NS and 15 μg / ml of PRO. (Run #1)

[0121] Figure 60 The results show that the NS concentrations after recalcification are different. (NS) Thrombin generation (TG) results. (Run #2)

[0122] Figure 61Shown are the results of thrombin generation (TG) for MOD-5014 (PRO) after recalcification at different concentrations of PRO. (Run #2)

[0123] Figure 62 The AE provides a (NS) Results are overlaid with the results for MOD-5014(PRO)TG. A: Results at 1.25 μg / ml are provided. B: Results at 5 μg / ml are provided. C: Results at 15 μg / ml are provided. D: Results at 2.5 μg / ml are provided. E: Results at 10 μg / ml are provided. (Run #2)

[0124] Figure 63 AD provides the Overlay analysis of (NS) results relative to MOD-5014 (PRO) TG results. A: Shows results at 1.25 μg / ml of NS and 2.5 μg / ml of PRO. B: Shows results at 5 μg / ml of NS and 10 μg / ml of PRO. C: Shows results at 2.5 μg / ml of NS and 5 μg / ml of PRO. D: Shows results at 10 μg / ml of NS and 15 μg / ml of PRO. (Run #2)

[0125] Figure 64 Shown that after recalcification at low TF concentrations Data from the TG run. (Run #1)

[0126] Figure 65 Shown are data for TG performed with MOD-5014 after recalcification at low TF concentrations. (Run #1)

[0127] Figure 66 The AE provides a new perspective on the effect of recalcification at low TF at similar concentrations. (NS) Results are overlaid with those of MOD-5014 (PRO). A: Results at 1.25 μg / ml are provided. B: Results at 5 μg / ml are provided. C: Results at 15 μg / ml are provided. D: Results at 2.5 μg / ml are provided. E: Results at 10 μg / ml are provided. (Run #1)

[0128] Figure 67 Shown that after recalcification at low TF concentrations Data from the TG run. (Run #2)

[0129] Figure 68 Shown are data for TG performed with MOD-5014 after recalcification at low TF concentrations. (Run #2)

[0130] Figure 69 The AE provides a similar concentration of (NS) Results are overlaid with the results for MOD-5014(PRO)TG. A: Results at 1.25 μg / ml are provided. B: Results at 5 μg / ml are provided. C: Results at 15 μg / ml are provided. D: Results at 2.5 μg / ml are provided. E: Results at 10 μg / ml are provided. (Run #2)

[0131] Figure 70 AC provides the results of different concentrations of Overlay analysis of (NS) results relative to MOD-5014 (PRO) TG results. A: Shows results at 1.25 μg / ml of NS and 5 μg / ml of PRO. B: Shows results at 5 μg / ml of NS and 15 μg / ml of PRO. C: Shows results at 2.5 μg / ml of NS and 10 μg / ml of PRO. (Run #2)

[0132] Figure 71 Shown in the presence and absence of low TF as Total thrombin generation by FVIII compared to the results of MOD-5014.

[0133] Figure 72 Overlay analysis of thrombin generation by FVIII in the presence and absence of low TF is provided.

[0134] Figure 73 Provides the Overlay analysis of TG results for (NS; 1.25 μg / kg) versus TG results for MOD-5014 (PRO; 1.25 μg / kg). (Run #1)

[0135] Figure 74 The AE provides the Overlay analysis of TG results for (NS; 1.25 μg / kg) versus TG results for MOD-5014 (PRO; 1.25 μg / kg). A: Provides results at 0 pM TF. B: Provides results at 1 pM TF. C: Provides results at 5 pM TF. D: Provides results at 0.5 pM TF. E: Provides results at 2.5 pM TF. (Run #1)

[0136] Figure 75 Provides the Overlay analysis of TG results for (NS; 2.5 μg / kg) versus TG results for MOD-5014 (PRO; 2.5 μg / kg). (Run #1)

[0137] Figure 76 The AE provides the Overlay analysis of TG results (NS; 2.5 μg / kg) relative to MOD-5014 (PRO; 2.5 μg / kg). A: Provides results at 0 pM TF. B: Provides results at 1 pM TF. C: Provides results at 5 pM TF. D: Provides results at 0.5 pM TF. E: Provides results at 2.5 pM TF. (Run #1)

[0138] Figure 77 Provides the Overlay analysis of TG results (NS; 5 μg / kg) relative to MOD-5014 (PRO; 5 μg / kg). (Run #1)

[0139] Figure 78 The AE provides the Overlay analysis of TG results for (NS; 5 μg / kg) versus TG results for MOD-5014 (PRO; 5 μg / kg). A: Provides results at 0 pM TF. B: Provides results at 1 pM TF. C: Provides results at 5 pM TF. D: Provides results at 0.5 pM TF. E: Provides results at 2.5 pM TF. (Run #1)

[0140] Figure 79 AC shows the effect of increasing TF concentration on Dose-dependent TG responses in the presence of (NS). A: Provides results at 1.25 μg / kg of NS. B: Provides results at 5 μg / kg of NS. C: Provides results at 2.5 μg / kg of NS. (Run #1)

[0141] Figure 80 Figures AC show the dose-dependent TG response in the presence of MOD-5014 (PRO) at increasing TF concentrations. A: Results at 1.25 μg / kg PRO are provided. B: Results at 5 μg / kg PRO are provided. C: Results at 2.5 μg / kg PRO are provided. (Run #1)

[0142] Figure 81 Provides the Overlay analysis of TG results for (NS; 10 μg / kg) versus TG results for MOD-5014 (PRO; 10 μg / kg). (Run #2)

[0143] Figure 82 Provides the Overlay analysis of TG results for (NS; 2.5 μg / kg) versus TG results for MOD-5014 (PRO; 2.5 μg / kg). (Run #2)

[0144] Figure 83 Provides the Overlay analysis of TG results (NS; 5 μg / kg) relative to MOD-5014 (PRO; 5 μg / kg). (Run #2)

[0145] Figure 84 AC shows the effect of increasing TF concentration on Dose-dependent TG responses in the presence of (NS). A: Provides results at 2.5 μg / ml of NS. B: Provides results at 10 μg / ml of NS. C: Provides results at 5 μg / ml of NS. (Run #2)

[0146] Figure 85 Figures AC show the dose-dependent TG response in the presence of MOD-5014 (PRO) at increasing TF concentrations. A: Results at 2.5 μg / ml PRO are provided. B: Results at 10 μg / ml PRO are provided. C: Results at 5 μg / ml PRO are provided. (Run #2)

[0147] Figure 86 The AE provides a detailed description of the effect of TF on the Overlay analysis of TG results for (NS; 10 μg / kg) versus TG results for MOD-5014 (PRO; 10 μg / kg). A: Provides results at 0 pM TF. B: Provides results at 1 pM TF. C: Provides results at 5 pM TF. D: Provides results at 0.5 pM TF. E: Provides results at 2.5 pM TF. (Run #2)

[0148] Figure 87 The AE provides the Overlay analysis of TG results for (NS; 2.5 μg / kg) versus TG results for MOD-5014 (PRO; 2.5 μg / kg). A: Provides results at 0 pM TF. B: Provides results at 1 pM TF. C: Provides results at 5 pM TF. D: Provides results at 0.5 pM TF. E: Provides results at 2.5 pM TF. (Run #2)

[0149] Figure 88 The AE provides the Overlay analysis of TG results for (NS; 5 μg / kg) versus TG results for MOD-5014 (PRO; 5 μg / kg). A: Provides results at 0 pM TF. B: Provides results at 1 pM TF. C: Provides results at 5 pM TF. D: Provides results at 0.5 pM TF. E: Provides results at 2.5 pM TF. (Run #2)

[0150] Figure 89 Shown is a graph of WBCT after spiking MOD-5014 into dog blood.

[0151] Figure 90 Schematic diagram showing the two-compartment pharmacokinetic model.

[0152] Figure 91 Provided is a graph of mean plasma MOD-5014 concentration versus time following IV infusion in dogs.

[0153] Figure 92 Provided is a graph of mean plasma MOD-5014 activity versus time following IV infusion in dogs.

[0154] Figures 93A to 93B Comparative data showing MOD-5014 plasma concentration and activity following IV infusion of 50 μg / kg in dogs. Figure 93A Results for dog P14 are shown, and Figure 93B Results are shown for dog N06.

[0155] Figures 94A to 94D Comparative data showing MOD-5014 plasma concentration and activity following IV infusion of 200 μg / kg in dogs. Figure 94A Results for dog Blondie are shown; Figure 94B Results for the dog Josie; Figure 94C is the result for dog N06; and Figure 94D Results for dog P14.

[0156] Figures 95A to 95BComparative data showing MOD-5014 plasma concentration and activity following IV infusion of 400 μg / kg in dogs. Figure 95A Results for the dog Blondie are shown, and Figure 95B Results for the dog Josie are shown.

[0157] Figures 96A to 96B Comparative data showing MOD-5014 plasma concentration and activity following IV infusion of 600 μg / kg in dogs. Figure 96A Results for dog N05 are shown, and Figure 96B Results are shown for the dog Joanie.

[0158] Figures 97A to 97J Shown is a graph of plasma MOD-5014 concentration versus time. Points represent observed plasma concentrations, and lines represent the values ​​used to calculate T 1 / 2 The end slope. Figure 97A Results are shown for dog N06 over 30 hours; Figure 97B Results for dog P14 were obtained over 30 hours later; Figure 97C Results for dog Blondie approximately 50 hours later; Figure 97D Results for dog Josie approximately 50 hours later; Figure 97E Results for dog N06 after almost 100 hours; Figure 97F Results for dog P14 almost 100 hours later; Figure 97G The results for the dog Blondie almost 100 hours later; Figure 97H Results for Josie the dog almost 100 hours later; Figure 97I Results for Joanie the dog after almost 100 hours and Figure 97J Results for dog N05 after almost 100 hours.

[0159] Figures 98A to 98J Shown is a graph of plasma MOD-5014 activity versus time. Points represent observed plasma concentrations and lines represent the activity used to calculate T 1 / 2 The end slope. Figure 98A Results are shown for dog N06 over 30 hours; Figure 98B Results for dog P14 were obtained over 30 hours later; Figure 98C Results for dog Blondie almost 50 hours later; Figure 98D The results for Josie the dog almost 50 hours later; Figure 98E Results for dog N06 after almost 50 hours; Figure 98F Results for dog P14 were obtained almost 50 hours later; Figure 98G Results for dog Blondie almost 50 hours later; Figure 98HThe results for Josie the dog almost 50 hours later; Figure 98I Results for Joanie the dog after almost 50 hours and Figure 98J Results for dog N05 after almost 50 hours.

[0160] Figures 99A to 99J The modeling results - plasma concentrations are shown. The dots represent the observed plasma concentrations and the solid line represents the concentrations predicted by the model. Figure 99A Observed and predicted results for dog N06 are shown; Figure 99B Results for dog P14; Figure 99C Results for the dog Blondie; Figure 99D Results for the dog Josie; Figure 99E Results for dog N06 after almost 100 hours; Figure 99F Results for dog P14 almost 100 hours later; Figure 99G The results for the dog Blondie almost 100 hours later; Figure 99H Results for Josie the dog almost 100 hours later; Figure 99I Results for Joanie the dog after almost 100 hours and Figure 99J Results for dog N05 after almost 100 hours.

[0161] Figures 100A to 100J The modeling results - activity - are shown. The dots represent the observed plasma activity and the solid line represents the activity predicted by the model. Figure 100A Shown are the observed and predicted results for dog N06 after at least 32 hours; Figure 100B Results for dog P14 were obtained at least 32 hours later; Figure 100C Results for dogs Blondie at least 48 hours later; Figure 100D Results for dog Josie at least 48 hours later; Figure 100E Results for dog N06 were obtained at least 48 hours later; Figure 100F Results for dog P14 at least 48 hours later; Figure 100G Results for dogs Blondie at least 48 hours later; Figure 100H Results for dog Josie at least 48 hours later; Figure 100I Results for dog Joanie at least 48 hours later and Figure 100J Results for dog N05 are at least 48 hours later.

[0162] Figure 101Figures AD show dose-dependent changes in kaolin-induced TEG kinetics after administration of 50 μg / kg, 200 μg / kg, or 400 μg / kg of MOD-5014 to dog NO6. A: shows R time (reaction time); B: shows K time (time from the end of R to the clot reaching 20 mm, the speed of clot formation); C: shows angle (tangent to the curve when K is reached); and D: shows MA (maximum amplitude).

[0163] Figure 102 Figures AD show dose-dependent changes in kaolin-induced TEG kinetics after administration of 50 μg / kg and 200 μg / kg of MOD-5014 to dog P-14. A: shows R time (reaction time); B: shows K time (time from the end of R to the clot reaching 20 mm, the speed of clot formation); C: shows angle (tangent to the curve when K is reached); and D: shows MA (maximum amplitude).

[0164] Figure 103 Figures AD show dose-dependent changes in kaolin-induced TEG kinetics after administration of 200 μg / kg and 400 μg / kg of MOD-5014 to dog Blondie. A: shows R time (reaction time); B: shows K time (time from the end of R to the clot reaching 20 mm, the speed of clot formation); C: shows angle (tangent to the curve when K is reached); and D: shows MA (maximum amplitude).

[0165] Figure 104 Figures AD show dose-dependent changes in kaolin-induced TEG kinetics after administration of 200 μg / kg and 400 μg / kg of MOD-5014 to dog Josie. A: shows R time (reaction time); B: shows K time (time from the end of R to the clot reaching 20 mm, the speed of clot formation); C: shows angle (tangent to the curve when K is reached); and D: shows MA (maximum amplitude).

[0166] Figure 105 Figures AD show dose-dependent changes in kaolin-induced TEG kinetics after administration of 50 μg / kg, 200 μg / kg, or 400 μg / kg of MOD-5014 to dog Joanie. A: shows R time (reaction time); B: shows K time (time from the end of R to the clot reaching 20 mm, the speed of clot formation); C: shows angle (tangent to the curve when K is reached); and D: shows MA (maximum amplitude).

[0167] Figure 106Figures AD show the dynamics of TEG over time after MOD-5014 administration to dog NO5. A: shows R time (reaction time); B: shows K time (the time from the end of RT to the clot reaching 20 mm, the speed of clot formation); C: shows angle (the tangent of the curve when K is reached); and D: shows MA (maximum amplitude).

[0168] Figure 107 Figures AD show changes in TEG performance after administration of 270 μg / kg of rhFVIIa. A: R time (reaction time). B: Angle (tangent to the curve when K is reached). C: K time (the time from the end of RT to the clot reaching 20 mm, representing the speed of clot formation). D: MA ​​(maximum amplitude).

[0169] Figure 108 Figures A to D show the prolonged TEG effect of MOD-5014, representative data from an individual animal (Blondie). A: shows R time (reaction time). B: shows K time (the time from the end of RT to the clot reaching 20 mm, the speed of clot formation). C: shows the angle (the tangent to the plot when K is reached). D: shows MA (maximum amplitude). Arrows in B, C, and D point to the MOD-5014 TEG values ​​4 hours after dosing.

[0170] Figure 109 A map of the pCI-dhfr-MOD-5014 plasmid is shown.

[0171] Figure 110 Combined representative PK-PD curves from rat toxicology studies are shown, with the rate of change of plasma concentration (ng / ml) over time represented by open squares, and the rate of change of activity (mU / ml) over time represented by open circles.

[0172] Figure 111 Combined representative PK-PD curves from monkey toxicology studies are shown, with the rate of change of plasma concentration (ng / ml) over time represented by open circles, and the rate of change of activity (mU / ml) over time represented by open squares.

[0173] Figure 112 Shown are mean plasma MOD-5014 concentrations versus time following IV bolus administration to male cynomolgus monkeys.

[0174] Figure 113 Shown are mean MOD-5014 clotting activity versus time following IV bolus administration to male cynomolgus monkeys.

[0175] Figure 114To compare plasma concentrations and coagulation activity of MOD-5014 following a 1 mg / kg IV bolus injection in male cynomolgus monkeys.

[0176] Figure 115 To compare plasma concentrations and coagulation activity of MOD-5014 following a 7.5 mg / kg IV bolus injection in male cynomolgus monkeys.

[0177] Figure 116 To compare plasma concentrations and coagulation activity of MOD-5014 following a 15 mg / kg IV bolus injection in male cynomolgus monkeys.

[0178] Figures 117A to 117R The respective plasma MOD-5014 concentrations and clotting activity are plotted against time.

[0179] Figure 118 is the mean fluorescence intensity of platelets treated with MOD-5014 or FVIIa.

[0180] Figure 119 Thrombin generation in platelets treated with MOD-5014 or FVIIa.

[0181] Figure 120 Shown is a comparison of substrate (Pefachrome FVIIa) cleavage activity between FVIIa (NovoSeven) and CTP-modified Factor VIIa (MOD-5014).

[0182] Figure 121 Shown is a comparison of the activity of a substrate (Pefachrome FVIIa) between FVIIa (NovoSeven) and CTP-modified Factor VIIa (MOD-5014) when bound to tissue factor.

[0183] Figure 122 Shown is a comparison of the production of activated factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) taking into account the concentration of factor VIIa.

[0184] Figure 123 Shown is a comparison of the production of activated factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) taking into account factor X concentration.

[0185] Figure 124A and 124BA comparison of the rates of production of activated factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) in the absence of tissue factor and taking into account lipid concentrations is shown (124A). A comparison of the rates of production of activated factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) in the absence of tissue factor and taking into account lipid concentrations is shown (124B).

[0186] Figure 125 Shown is a comparison of activated factor X production between FVIIa (NovoSeven) and MOD-5014 in the absence of tissue factor and taking into account factor X concentration.

[0187] Figure 126 Shown is a comparison of the inhibition of substrate (Pefachrome FVIIa) cleavage by FVIIa (NovoSeven) and CTP-modified FVIIa (MOD-5014) in view of polybrene.

[0188] Figures 127A to 127C It is shown that considering the TFPI concentration ( Figure 127A ) and against FVIIa ( Figure 127B ) and MOD-5014( Figure 127C ) of the inhibition of substrate (Pefachrome FXa) cleavage by FVIIa (NovoSeven) and CTP-modified FVIIa (MOD-5014) with varying durations of TFPI exposure. DETAILED DESCRIPTION

[0189] In one embodiment, the present invention provides a long-acting coagulation factor and its production and use methods. In another embodiment, the long-acting coagulation factor includes a carboxyl terminal peptide (CTP, also referred to as a CTP unit). In another embodiment, the long-acting polypeptide comprising the coagulation factor also includes the carboxyl terminal peptide (CTP) of human chorionic gonadotropin (hCG). In another embodiment, CTP acts as a protective agent to prevent the degradation of the coagulation factor. In another embodiment, CTP prolongs the CTP of the coagulation factor. max In another embodiment, CTP prolongs the T max In another embodiment, CTP extends the circulating half-life of a coagulation factor. In another embodiment, CTP enhances the potency of a coagulation factor.

[0190] In another embodiment, the present invention provides a method for extending the biological half-life of a coagulation factor, comprising the step of attaching one to ten CTPs to the carboxyl terminus of the coagulation factor, thereby extending the biological half-life of the coagulation factor. In another embodiment, the present invention provides a method for extending the biological half-life of a coagulation factor, comprising the step of attaching one to five CTPs to the carboxyl terminus of the coagulation factor, thereby extending the biological half-life of the coagulation factor. In another embodiment, the present invention provides a method for extending the circulatory half-life of a coagulation factor. In another embodiment, the present invention provides a method for increasing the half-life of a coagulation factor. In another embodiment, the present invention provides a method for extending the half-life of a coagulation factor.

[0191] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising a CTP-modified coagulation factor.

[0192] In one embodiment, the present disclosure relates to a pharmaceutical formulation comprising a buffer, a tonicity agent, and a CTP-modified polypeptide consisting of a coagulation factor and three chorionic gonadotropin CTPs attached to the carboxyl terminus of the coagulation factor.

[0193] In one embodiment, the present disclosure relates to a formulation for once-weekly administration to a subject with hemophilia A or B. In another embodiment, the subject is deficient in a coagulation factor. In another embodiment, the subject has acquired hemophilia. In another embodiment, the present disclosure relates to a process for manufacturing a pharmaceutical formulation for once-weekly administration to a subject deficient in a coagulation factor or with hemophilia, the process comprising the steps of:

[0194] a) modifying a coagulation factor by attaching three chorionic gonadotropin CTPs to the carboxyl terminus of the coagulation factor;

[0195] b) mixing the modified coagulation factor of step a with a buffer and a tonicity agent at a pH of about 6.4; and

[0196] c) Prefill a syringe with the formulation.

[0197] In one embodiment, the present disclosure relates to a process for filling a syringe with a formulation provided herein, comprising the steps of:

[0198] a) formulating a once-weekly dosage form of the CTP-modified coagulation factor having a predetermined amount of the CTP-modified coagulation factor; and

[0199] b) Fill the syringe with the formulation.

[0200] In one embodiment, a "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition or "pharmaceutical formulation" is to facilitate the administration of a compound to an organism. In certain embodiments, a "pharmaceutical composition" or "pharmaceutical formulation" provides a pharmaceutical dosage form of a drug. In certain embodiments, a "pharmaceutical composition" or "pharmaceutical formulation" includes sustained-release technology, a transdermal patch, or any dosage form known in the art.

[0201] Coagulation factor VII (FVII) is a glycoprotein (50KDa) of 444 amino acids as an inactive proenzyme secreted into the bloodstream by hepatocytes. When tissue damage and being exposed to circulating blood, FVII forms a complex with tissue factor (TF), which is the true receptor protein of FVII and is expressed by the various cells positioned at the deeper layers of the vascular wall. The formation of this FVII-TF complex has caused the activation of FVII. Activated FVII (FVIIa) initiates exogenous coagulation pathway by activating factors IX and factor X.

[0202] FVII belongs to a group of vitamin K dependent glycoproteins associated with the coagulation system. Except FVII, this group is made up of factor IX, factor X, protein C and prothrombin. These proteins have similar domain organization and are synthesized as precursors with N-terminal propeptides followed by mature amino acid sequences. The propeptide comprises a docking site for the gamma carboxylase that is used to convert glutamic acid (Glu) into gamma carboxylglutamate (Gla). The back of this domain is two epidermal growth factor-like (EGF) domains, a connecting region (CR) and a C-terminal serine protease domain. Before secretion, the FVII propeptide is cut to form a single-chain zymogen FVII glycoprotein of 406 amino acids. After secretion, the protein can be activated into a disulfide bond-connected double-chain heterodimer FVIIa by cutting in the CR. The plasma concentration of FVII is 10nM, and about 1% circulates in active form in healthy individuals. In other embodiments, the FVII modified through CTP comprises a glycosylation site that occupies at least one O-connection. In other embodiments, the CTP-modified FVIIa includes occupation of at least one O-linked glycosylation site.

[0203] Factor IX (FIX) is a 415 amino acid (55 kDa) glycoprotein that belongs to a group of vitamin K-dependent glycoproteins associated with the coagulation system. FIX has a similar domain organization to factor FVII, factor X, protein C, and prothrombin, which are synthesized as precursors with an N-terminal propeptide followed by the mature amino acid sequence.

[0204] FIX is secreted as a single-chain molecule, which has undergone complex post-transcriptional modifications, many of which are crucial for its biochemistry and pharmacokinetic properties. Among all post-transcriptional modifications, the 12 glutamic acid residues near the amino-terminal end of FIX that are γ-carboxylated by the γ-carboxylase of vitamin K dependence are the most critical. It is necessary to carry out carboxylation for the interaction of FIX with phospholipid surfaces and for realizing optimal FIX activity. The amino-terminal propeptide is used as the recognition site of γ-carboxylase, and therefore after γ-carboxylation, it is cut off by the Golgi serine protease that is referred to as paired alkaline amino acid cleavage enzyme (PACE / furin). Four additional post-transcriptional modifications may occur in the Golgi apparatus: sulfation of tyrosine 155, phosphorylation of serine 158, O-glycosylation on Ser63 and 61, and finally N-glycosylation on Asn 157 and 16, but they are shown to be not necessary for realizing the suitable activity of FIX.

[0205] FIX circulates in plasma as a single-chain, inactive zymogen (average concentration of 5 μg / ml). Following proteolytic cleavage at two peptide bonds: Arg145 and Arg180, by one or both of the physiological activators, the FVIIa-TF complex or FIXa, the activation peptide is removed, which converts FIX into a fully active enzyme consisting of a light chain and a heavy chain linked by a single disulfide bond. The N-terminal light chain contains a non-catalytic gamma-carboxyglutamate (Gla) and two epidermal growth factor-like domains, while the C-terminal heavy chain contains the trypsin-like catalytic domain of the molecule. FIXa alone is characterized by poor catalytic activity. However, when complexed with FVIII, its proteolytic activity towards its natural substrate, FX, is increased by 4-5 orders of magnitude.

[0206] In another embodiment, there is provided herein a method for extending the biological half-life or a method for improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to ten CTPs to the carboxyl terminus of a coagulation factor, thereby extending the biological half-life or improving the AUC of a coagulation factor. In another embodiment, there is provided herein a method for extending the biological half-life or a method for improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to five CTPs to the carboxyl terminus of a coagulation factor, thereby extending the biological half-life or improving the AUC of a coagulation factor. In another embodiment, there is provided herein a method for extending the biological half-life or a method for improving the area under the curve (AUC) of FIX, comprising the step of attaching one to five CTPs to the carboxyl terminus of FIX, thereby extending the biological half-life or improving the AUC of FIX. In another embodiment, provided herein is a method for extending the biological half-life or a method for improving the area under the curve (AUC) of FVII or FVIIa, comprising the step of attaching one to five CTPs to the carboxyl terminus of FVII or FVIIa, thereby extending the biological half-life or improving the AUC of FVII or FVIIa.

[0207] In another embodiment, the present invention provides a method for extending the biological half-life of a factor IX (FIX) polypeptide, comprising attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby extending the biological half-life of the FIX polypeptide. In another embodiment, the present invention provides a method for extending the biological half-life of a factor VIIa (FVIIa) polypeptide, comprising attaching one to a maximum of five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby extending the biological half-life of the FVIIa polypeptide. In one embodiment, three chorionic gonadotropin carboxyl-terminal peptides (CTPs) are attached to the carboxyl terminus of the FVIIa polypeptide. In another embodiment, four chorionic gonadotropin carboxyl-terminal peptides (CTPs) are attached to the carboxyl terminus of the FVIIa polypeptide. In another embodiment, five chorionic gonadotropin carboxyl-terminal peptides (CTPs) are attached to the carboxyl terminus of the FVIIa polypeptide.

[0208] In another embodiment, the present invention provides a method for improving the area under the curve (AUC) of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby improving the AUC of the FIX polypeptide. In another embodiment, the present invention provides a method for improving the area under the curve (AUC) of a factor VIIa (FVIIa) polypeptide, comprising the step of attaching up to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby improving the AUC of the FVIIa polypeptide. In one embodiment, three chorionic gonadotropin carboxyl-terminal peptides (CTPs) are attached to the carboxyl terminus of the FVIIa polypeptide. In another embodiment, four chorionic gonadotropin carboxyl-terminal peptides (CTPs) are attached to the carboxyl terminus of the FVIIa polypeptide. In another embodiment, five chorionic gonadotropin carboxyl-terminal peptides (CTPs) are attached to the carboxyl terminus of the FVIIa polypeptide.

[0209] In another embodiment, the coagulation factor of the present disclosure is a protein. In another embodiment, the coagulation factor of the present disclosure is a peptide. In another embodiment, the coagulation factor of the present disclosure is a polypeptide. In another embodiment, the coagulation factor is an enzyme. In another embodiment, the coagulation factor is a serine protease. In another embodiment, the coagulation factor is a glycoprotein. In another embodiment, the coagulation factor is a transglutaminase. In another embodiment, the coagulation factor is an inactive zymogen. In another embodiment, the coagulation factor is any coagulation factor known to those skilled in the art.

[0210] In another embodiment, the coagulation factor is factor VIII (FVIII). In another embodiment, the coagulation factor is factor V (FV). In another embodiment, the coagulation factor is factor XIII (FXIII). In another embodiment, the coagulation factor is factor X (FX). In another embodiment, the coagulation factor is fibrin.

[0211] In another embodiment, the coagulation factor is factor VIIa (FVIIa). In another embodiment, the coagulation factor is factor VII (FVII). In another embodiment, the coagulation factor is factor IX (FIX). In another embodiment, the coagulation factor is factor X (FX). In another embodiment, the coagulation factor is factor XIa (FXIa). In another embodiment, the coagulation factor is factor XII (FXII). In another embodiment, the coagulation factor is factor Xa (FXa). In another embodiment, the coagulation factor is factor Va (FVa). In another embodiment, the coagulation factor is prothrombin. In another embodiment, the coagulation factor is thrombin. In another embodiment, the coagulation factor is factor XI (FXI). In another embodiment, the coagulation factor is von Willebrand factor (vWF). In another embodiment, the coagulation factor is factor VIIIa (FVIIIa). In another embodiment, the coagulation factor is B-domain deleted FVIII (FVIIIBDD). In another embodiment, the coagulation factor is B-domain deleted FVIII (FVIIIBDD). In another embodiment, the coagulation factor is FVIII with a beta domain deletion (FVIIIBDD). In another embodiment, the coagulation factor is factor IXa (FIXa). In another embodiment, the coagulation factor is prekallikrein. In another embodiment, the coagulation factor is kallikrein. In another embodiment, the coagulation factor is factor XIIa (FXIIa). In another embodiment, the coagulation factor is fibrinogen. In another embodiment, the coagulation factor is thrombin modulin. In another embodiment, the coagulation factor is factor II (FII).

[0212] In another embodiment, the coagulation factor is a glycoprotein. In another embodiment, the coagulation factor is a vitamin K-dependent glycoprotein. In another embodiment, the coagulation factor is a vitamin K-independent glycoprotein.

[0213] In another embodiment, the coagulation factor is a recombinant protein. In another embodiment, the coagulation factor is a recombinant glycoprotein. In another embodiment, the coagulation factor is recombinant glycoprotein FV. In another embodiment, the coagulation factor is recombinant FVI. In another embodiment, the coagulation factor is recombinant FVII. In another embodiment, the coagulation factor is recombinant FVIII. In another embodiment, the coagulation factor is recombinant FIX. In another embodiment, the coagulation factor is recombinant FX. In another embodiment, the coagulation factor is recombinant FXI. In another embodiment, the coagulation factor is recombinant FXII. In another embodiment, the coagulation factor is recombinant FvW. In another embodiment, the coagulation factor is recombinant FII. In another embodiment, the coagulation factor is recombinant FIXa. In another embodiment, the coagulation factor is recombinant FXIa. In another embodiment, the coagulation factor is recombinant fibrin. In another embodiment, the coagulation factor is recombinant FVIIa. In another embodiment, the coagulation factor is recombinant FXa. In another embodiment, the coagulation factor is recombinant FVa. In another embodiment, the coagulation factor is recombinant prothrombin. In another embodiment, the coagulation factor is recombinant thrombin. In another embodiment, the coagulation factor is recombinant FVIIIa. In another embodiment, the coagulation factor is recombinant prekallikrein. In another embodiment, the coagulation factor is recombinant kallikrein. In another embodiment, the coagulation factor is recombinant FXIIa. In another embodiment, the coagulation factor is any known recombinant coagulation factor. In another embodiment, the coagulation factor comprising a signal peptide is any known recombinant coagulation factor. In another embodiment, the recombinant coagulation factor does not comprise a signal peptide. In another embodiment, the activated coagulation factor does not comprise a signal peptide.

[0214] In another embodiment, the coagulation factor comprises 1 to 10 CTP repeats attached to the C-terminus and does not include a CTP attached to the N-terminus. In another embodiment, the coagulation factor comprises at least one CTP attached to the C-terminus and does not include a CTP attached to the N-terminus. In another embodiment, the coagulation factor comprising 1 to 10 CTP repeats attached to the C-terminus and does not include a CTP attached to the N-terminus is an engineered coagulation factor. In another embodiment, the coagulation factor comprising at least one CTP attached to the C-terminus and does not include a CTP attached to the N-terminus is an engineered coagulation factor. In another embodiment, the coagulation factor comprising 1 to 10 CTP repeats attached to the C-terminus and does not include a CTP attached to the N-terminus is a conjugated coagulation factor. In another embodiment, the coagulation factor comprising at least one CTP attached to the C-terminus and does not include a CTP attached to the N-terminus is a conjugated coagulation factor.

[0215] In one embodiment, the present invention provides a CTP-modified Factor IX (FIX) polypeptide consisting of a FIX polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the CTP-modified FIX polypeptide.

[0216] In another embodiment, the present invention also provides a CTP-modified Factor VIIa (FVIIa) polypeptide, which consists of a FVIIa polypeptide and five gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa. In another embodiment, the present invention also provides a CTP-modified Factor VIIa (FVIIa) polypeptide, which consists of a FVIIa polypeptide and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa.

[0217] In another embodiment, coagulation factor is the coagulation factor that comprises the domain organization similar or identical with FIX, FVII, factor X, protein C or prothrombin.In another embodiment, coagulation factor is synthesized as the precursor with N-terminal propeptide.In another embodiment, coagulation factor as used herein is the precursor of inactive enzyme form.In another embodiment, coagulation factor is produced in hepatocyte.In another embodiment, coagulation factor comprises the docking site for the gamma carboxylase that is used to convert glutamic acid (Glu) into gamma carboxylglutamate (Gla).In another embodiment, coagulation factor as used herein is commercially available coagulation factor.

[0218]

[0219] In another embodiment, the amino acid sequence of Factor VII comprises the following amino acid sequence: MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCethKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLL VNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP(SEQ ID NO: 9).

[0220] In another embodiment, the amino acid sequence of Factor VII comprises the following amino acid sequence: MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCethKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVN GAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATA LELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP*GCGR(SEQ ID NO: 10).

[0221] In another embodiment, the nucleic acid sequence encoding Factor VII-CTP (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0222]

[0223] In another embodiment, the amino acid sequence of Factor VII-CTP (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0224] MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNC ETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKN WRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD SPNITYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQ*(SEQ ID NO: 13).

[0225] In another embodiment, the nucleic acid sequence encoding Factor VII-CTP-CTP (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0226]

[0227] In another embodiment, the amino acid sequence of Factor VII-CTP-CTP (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0228] MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQ LICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSE HDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**(SEQ ID NO: 15).

[0229] In another embodiment, the nucleic acid sequence encoding Factor VII-CTP-CTP-CTP (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0230]

[0231] In another embodiment, the amino acid sequence of Factor VII-CTP-CTP-CTP (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0232] (SEQ ID NO: 25) In another embodiment, amino acids 1 to 38 of SEQ ID NO: 25 include a signal sequence.

[0233] In another embodiment, the amino acid sequence of Factor VII-CTP-CTP-CTP (attached to the carboxyl terminus) lacking the signal peptide comprises the following amino acid sequence:

[0234] In another embodiment, the amino acid sequence of activated Factor VII-CTP-CTP-CTP (attached to the carboxyl terminus) (FVIIa-CTP3) lacks a signal peptide and includes the amino acid sequence set forth in SEQ ID NO: 46. In another embodiment, FVIIa-CTP3 lacks a signal peptide and includes a homolog of SEQ ID NO: 46. In another embodiment, FVIIa-CTP3 lacks a signal peptide and includes a variant of SEQ ID NO: 46. In another embodiment, the amino acid sequence of FVIIa-CTP3 is cleaved between the arginine (R) at residue 152 and the isoleucine (I) at residue 153. In another embodiment, the amino acid sequence of FVIIa-CTP3 structurally exists as a disulfide-linked two-chain heterodimer including a disulfide SS bridge between the cysteine ​​residues present on each chain. In another embodiment, the amino acid sequence of FVIIa-CTP3 exists structurally as a heterodimer comprising a light chain and a heavy chain linked by a disulfide-SS bond between a cysteine ​​residue present in the light chain and a cysteine ​​residue present in the heavy chain. In another embodiment, the light chain comprises the N-terminal fragment of the FVIIa-CTP3 amino acid sequence, and the heavy chain comprises the C-terminal fragment of the FVIIa-CTP3 amino acid sequence. In another embodiment, the cysteine ​​residue can be any cysteine ​​residue in either chain. In another embodiment, the amino acid sequence of FVIIa-CTP3 exists structurally as a disulfide-linked two-chain heterodimer comprising an SS bridge between cysteine ​​residues 135 and 262 of SEQ ID NO:46, wherein the two chains comprise a light chain comprising amino acids 1 to 152 and a heavy chain comprising amino acids 153 to 490.

[0235] In another embodiment, the light chain migrates at about 25 kDA in SDS-PAGE under denaturing conditions. In another embodiment, the heavy chain migrates at about 50 kDA in SDS-PAGE under denaturing conditions. In another embodiment, the heavy chain migrates at about 60 kDA in SDS-PAGE under denaturing conditions.

[0236] In another embodiment, the nucleic acid sequence encoding Factor VII-(CTP)4 (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0237]

[0238] In another embodiment, the amino acid sequence of Factor VII-(CTP)4 (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0239] (SEQ ID NO: 27).

[0240] In another embodiment, the nucleic acid sequence encoding Factor VII-(CTP)5 (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0241]

[0242] In another embodiment, the amino acid sequence of Factor VII-(CTP)5 (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0243] (SEQ ID NO: 29).

[0244] In another embodiment, the nucleic acid sequence encoding Factor IX comprises the following nucleic acid sequence:

[0245]

[0246] In another embodiment, the amino acid sequence of Factor IX comprises the following amino acid sequence: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQST QSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYT NIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT*(SEQ IDNO: 17).

[0247] In another embodiment, the nucleic acid sequence encoding Factor IX-CTP (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0248]

[0249] In another embodiment, the amino acid sequence of Factor IX-CTP (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0250] MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGF EGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNG KVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVL QYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO: 19).

[0251] In another embodiment, the nucleic acid sequence encoding Factor IX-CTP-CTP (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0252]

[0253] In another embodiment, the amino acid sequence of Factor IX-CTP-CTP (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0254] MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCEL DVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEK WIVTAAHHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTI YNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ IDNO: 21).

[0255] In another embodiment, the nucleic acid sequence encoding Factor IX-(CTP)3 (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0256]

[0257] In another embodiment, the amino acid sequence of Factor IX-(CTP)3 (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0258] (SEQ ID NO: 31).

[0259] In another embodiment, the nucleic acid sequence encoding Factor IX-(CTP)4 (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0260]

[0261] In another embodiment, the amino acid sequence of Factor IX-(CTP)4 (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0262] (SEQ ID NO: 33).

[0263] In another embodiment, the nucleic acid sequence encoding Factor IX-(CTP)5 (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

[0264]

[0265] In another embodiment, the amino acid sequence of Factor IX-(CTP)5 (attached to the carboxyl terminus) comprises the following amino acid sequence:

[0266] (SEQ ID NO: 35).

[0267] In another embodiment, furin is added to cells expressing the coagulation factor-CTP of the present disclosure. In another embodiment, furin increases the efficiency of production of the coagulation factor-CTP of the present disclosure in cells.

[0268] In another embodiment, furin is co-transfected with a vector comprising the coding sequence of coagulation factor-CTP of the present disclosure. In another embodiment, furin is encoded by an independent vector. In another embodiment, furin and coagulation factor-CTP are encoded by a vector. In another embodiment, the coding sequence of furin is inserted among the pCI-DHFR. In another embodiment, the coding sequence of furin is engineered in pCI-dhfr / smaI+NotI, Furin / AsisI FI+NotI.

[0269] In another embodiment, the nucleic acid sequence encoding furin comprises the following nucleic acid sequence:

[0270]

[0271] In another embodiment, the amino acid sequence of furin protease comprises the following amino acid sequence: MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTD average value ARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQSAL (SEQ ID NO: 23).

[0272] In one embodiment, the term coagulation factor also includes homologues of known coagulation factors. In one embodiment, the homologues have coagulation activity. In certain embodiments, homology according to the present invention also includes deletion variants, insertion variants or substitution variants, including amino acid substitutions and biologically active polypeptide fragments thereof. In one embodiment, the variant includes a conservative substitution or a deletion, insertion or substitution that does not significantly change the three-dimensional structure of the coagulation factor. In another embodiment, the deletion, insertion or substitution does not change the function of interest of the coagulation factor, which in one embodiment is bound to a specific binding complex.

[0273] In another embodiment, the present disclosure includes homologues of coagulation factors. In another embodiment, the present disclosure includes homologues of coagulation factors with coagulation activity. In another embodiment, the present disclosure includes homologues of coagulation factors with functional binding. In another embodiment, the present disclosure includes homologues of coagulation factors as described herein with coagulation activity. In another embodiment, the present disclosure includes homologues of coagulation factors as described herein with functional binding. In another embodiment, homologues with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to a coagulation factor, such as a polypeptide determined using BlastP software from the National Center for Biotechnology Information (NCBI) using default parameters.

[0274] In another embodiment, the disclosure includes a homologue of furin. In another embodiment, the disclosure includes a homologue of furin that keeps a function of interest, which function in one embodiment is for cutting a precursor protein. In another embodiment, a homologue having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with furin, for example polypeptide is determined using the default parameters of the BlastP software of National Center for Biotechnology Information (NCBI).

[0275] In another embodiment, a polypeptide is provided herein, which includes a coagulation factor and one to ten gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminal of the coagulation factor. In another embodiment, a polypeptide is provided herein, which includes a coagulation factor and one to three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminal of the coagulation factor. In another embodiment, a polypeptide is provided herein, which includes a coagulation factor and one to five gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminal of the coagulation factor. In another embodiment, a polypeptide is provided herein, which includes a coagulation factor with at least one CTP on its carboxyl-terminal.

[0276] In another embodiment, provided herein is a polypeptide consisting of a coagulation factor and one to five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the coagulation factor.

[0277] In another embodiment, provided herein is a polypeptide consisting essentially of a coagulation factor and one to five CTPs attached to the carboxyl terminus of the coagulation factor.

[0278] It is to be understood that the compositions, formulations and methods of the present invention that include elements or steps as described herein may consist of those elements or steps in another embodiment, or may consist essentially of those elements or steps in another embodiment. In some embodiments, the term "comprising" refers to including the specified active agent, such as a CTP-modified coagulation factor, as well as other active agents and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc. as known in the pharmaceutical industry. In some embodiments, the term "essentially consisting of" refers to a composition whose only active ingredient is the specified active ingredient, however, other compounds may also be included for stabilization, preservation, etc. of the formulation, but which are not directly related to the therapeutic effect of the specified active ingredient. In some embodiments, the term "essentially consisting of" may refer to components that facilitate the release of the active ingredient. In some embodiments, the term "consisting of" refers to a composition containing an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0279] In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and two gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and two to three CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and two to four CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and two to five CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and two to six CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and two to seven CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and two to eight CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, provided herein is a polypeptide comprising a coagulation factor and two to nine CTPs attached to the carboxyl terminus of the coagulation factor. In another embodiment, provided herein is a polypeptide comprising a coagulation factor and two to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0280] In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and three to four CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and three to five CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and three to six CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and three to seven CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and three to eight CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and three to nine CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, provided herein is a polypeptide comprising a coagulation factor and three to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0281] In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and four gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and four to five CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and four to six CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and four to seven CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and four to eight CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and four to nine CTPs attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein comprising a coagulation factor and four to ten CTPs attached to the carboxyl-terminus of the coagulation factor.

[0282] In one embodiment, the present invention provides a kind of polypeptide, it includes coagulation factor and five gonadotropin carboxyl terminal peptides (CTP) attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it includes coagulation factor and five to six CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it includes coagulation factor and five to seven CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it includes coagulation factor and five to eight CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it includes coagulation factor and five to nine CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it includes coagulation factor and five to ten CTPs attached to the carboxyl terminal of coagulation factor.

[0283] In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and two gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide comprising a coagulation factor and two to three CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and two to four CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and two to five CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and two to six CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and two to seven CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and two to eight CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide is provided herein, which consists of a coagulation factor and two to nine CTPs attached to the carboxyl terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein, which consists of a coagulation factor and two to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0284] In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide comprising a coagulation factor and three to four CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and three to five CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and three to six CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and three to seven CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and three to eight CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and three to nine CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, provided herein is a polypeptide consisting of a coagulation factor and three to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0285] In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and four gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide comprising a coagulation factor and four to five CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and four to six CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and four to seven CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and four to eight CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and four to nine CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide comprising a coagulation factor and four to ten CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein.

[0286] In one embodiment, the present invention provides a kind of polypeptide, it is made up of coagulation factor and five gonadotropin carboxyl terminal peptides (CTP) attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it is made up of coagulation factor and five to six CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it is made up of coagulation factor and five to seven CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it is made up of coagulation factor and five to eight CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it is made up of coagulation factor and five to nine CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, there is provided herein a kind of polypeptide, it is made up of coagulation factor and five to ten CTPs attached to the carboxyl terminal of coagulation factor.

[0287] In one embodiment, the present invention provides a polypeptide consisting essentially of a coagulation factor and two gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide consisting essentially of a coagulation factor and two to three CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and two to four CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and two to five CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and two to six CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and two to seven CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and two to eight CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide is provided herein consisting essentially of a coagulation factor and two to nine CTPs attached to the carboxyl terminus of the coagulation factor. In another embodiment, a polypeptide is provided herein consisting essentially of a coagulation factor and two to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0288] In one embodiment, the present invention provides a polypeptide consisting essentially of a coagulation factor and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl-terminus of the coagulation factor. In another embodiment, a polypeptide consisting essentially of a coagulation factor and three to four CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and three to five CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and three to six CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and three to seven CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and three to eight CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, a polypeptide consisting essentially of a coagulation factor and three to nine CTPs attached to the carboxyl-terminus of the coagulation factor is provided herein. In another embodiment, provided herein is a polypeptide consisting essentially of a coagulation factor and three to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0289] In one embodiment, the invention provides a kind of polypeptide, it is essentially composed of coagulation factor and four gonadotropin carboxyl terminal peptides (CTP) attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and four to five CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and four to six CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and four to seven CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and four to eight CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and four to nine CTPs attached to the carboxyl terminal of coagulation factor. In another embodiment, provided herein is a polypeptide consisting essentially of a coagulation factor and four to ten CTPs attached to the carboxyl terminus of the coagulation factor.

[0290] In one embodiment, the invention provides a kind of polypeptide, it is essentially composed of coagulation factor and five gonadotropin carboxyl terminal peptides (CTP) attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and five to six CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and five to seven CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and five to eight CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and five to nine CTPs attached to the carboxyl terminal of coagulation factor.In another embodiment, herein provides a kind of polypeptide, it is essentially composed of coagulation factor and five to ten CTPs attached to the carboxyl terminal of coagulation factor.

[0291] In another embodiment, a polypeptide is provided herein, comprising, consisting essentially of, or consisting of a coagulation factor that does not have a CTP at its amino terminus. In another embodiment, a polypeptide is provided herein, comprising, consisting essentially of, or consisting of a coagulation factor that lacks a CTP at its amino terminus. In another embodiment, a polypeptide is provided herein, comprising, consisting essentially of, or consisting of a coagulation factor that has at least one CTP at its carboxyl terminus and does not have a CTP at its amino terminus. In another embodiment, a polypeptide is provided herein, comprising, consisting essentially of, or consisting of a coagulation factor that has at least one CTP at its carboxyl terminus and does not have a CTP at its amino terminus.

[0292] In another embodiment, the present invention provides a polynucleotide encoding the polypeptide as described above.

[0293] In another embodiment, the present invention also provides a composition comprising an expression vector comprising a polynucleotide encoding a Factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide and a CTP-modified polypeptide.

[0294] In another embodiment, the present invention also provides a polynucleotide encoding a factor VIIa (FVIIa) polypeptide and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the VIIa polypeptide. In another embodiment, the present invention also provides a composition comprising an expression vector comprising a polynucleotide encoding a factor VIIa (FVIIa) polypeptide and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide. In one embodiment, the CTP-modified FVIIa includes a signal peptide. In another embodiment, the CTP-modified FVIIa does not include a signal peptide.

[0295] In one embodiment, the present invention provides a recombinant coagulation factor as described above. In one embodiment, the present invention provides an engineered coagulation factor as described above. In one embodiment, the engineered coagulation factor as described above is referred to as a CTP-modified coagulation factor.

[0296] In one embodiment, the CTPs attached to the carboxyl terminus of the coagulation factor are attached in series to the carboxyl terminus.

[0297] In one embodiment, the engineered coagulation factors as described herein have equivalent or improved biological activity compared to coagulation factors not modified with CTP. In another embodiment, the engineered coagulation factors as described herein have equivalent or improved pharmacological measurements compared to coagulation factors not modified with CTP. In another embodiment, the engineered coagulation factors as described herein have equivalent or improved pharmacokinetics compared to coagulation factors not modified with CTP. In another embodiment, the engineered coagulation factors as described herein have equivalent or improved pharmacodynamics compared to coagulation factors not modified with CTP.

[0298] In one embodiment, the present invention provides a method for preventing or treating coagulation or coagulation disorders. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering a CTP-modified coagulation factor of the present invention. In another embodiment, the present invention provides a method for preventing and treating hemophilia in a subject, comprising administering a CTP-modified coagulation factor of the present invention. In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering a CTP-modified factor VII of the present invention.

[0299] In one embodiment, hemophilia is hemophilia A. In another embodiment, hemophilia is hemophilia B. In another embodiment, the method of the present invention for preventing or treating coagulation or coagulation disorders prevents or treats hemophilia in patients with hemophilia A or B containing inhibitors to FVIII or FIX, respectively. In another embodiment, the method of the present invention is for the prevention or treatment of patients with acquired hemophilia (hemophilia without inhibitors). In another embodiment, the method of the present invention for preventing or treating coagulation or coagulation disorders prevents or treats hemophilia A or B without inhibitors. In another embodiment, hemophilia is severe hemophilia. In another embodiment, hemophilia is moderate hemophilia. In another embodiment, hemophilia is moderate to severe hemophilia with or without inhibitors. It will be understood by those skilled in the art that the term "moderate to severe hemophilia" refers to a subject with less than or equal to 3% FVIII or FIX.

[0300] In another embodiment, the present invention provides a method for treating hemophilia in a subject comprising administering a CTP-modified Factor IX of the present invention. In one embodiment, the hemophilia is hemophilia B. In one embodiment, hemophilia B is also known as Factor IX deficiency or Krebsmacker's disease. In one embodiment, the hemophilia is severe hemophilia, which in one embodiment describes hemophilia in which the clotting factor level is 0-1%. In another embodiment, the hemophilia is moderate hemophilia, which in one embodiment describes hemophilia in which the clotting factor level is 1%-5%. In another embodiment, the hemophilia is mild hemophilia, which in one embodiment describes hemophilia in which the clotting factor level is 5%-50%.

[0301] In another embodiment, the present invention provides a method for preventing or treating a coagulation or clotting disorder in a subject, comprising administering to the subject a factor IX (FIX) polypeptide comprising a FIX polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, thereby preventing or treating the coagulation or clotting disorder in the subject. In another embodiment, the present invention provides a method for preventing or treating a coagulation or clotting disorder in a subject, comprising administering to the subject a factor VII (FVII) polypeptide comprising a FVII polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, thereby preventing or treating the coagulation or clotting disorder in the subject.

[0302] In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a factor IX (FIX) polypeptide comprising a FIX polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide, thereby preventing or treating hemophilia in the subject.

[0303] In another embodiment, the present invention provides a method for treating hemophilia for a subject, comprising administering to the subject one or more CTP-modified coagulation factors as described herein. Therefore, in one embodiment, the present invention provides a method for treating hemophilia for a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide and a CTP-modified factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide, thereby treating hemophilia for the subject. In one embodiment, CTP-modified FIX and CTP-modified FVIIa are administered simultaneously with the same composition. In another embodiment, CTP-modified FIX and CTP-modified FVIIa are administered simultaneously with separate compositions. In another embodiment, CTP-modified FIX and CTP-modified FVIIa are administered in separate compositions at separate times.

[0304] In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and four chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide and a CTP-modified factor VII polypeptide comprising a FIX polypeptide and a FVII polypeptide, and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl termini of the FIX polypeptide and the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide and a CTP-modified factor VII polypeptide comprising a FIX polypeptide and a FVII polypeptide, and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl termini of the FIX polypeptide and the FVII polypeptide, thereby preventing or treating hemophilia in the subject.

[0305] In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject subcutaneously or intravenously a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject subcutaneously or intravenously a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and four chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject subcutaneously or intravenously a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and five CTPs of chorionic gonadotropin attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject subcutaneously or intravenously a CTP-modified factor IX (FIX) polypeptide or a CTP-modified factor VII polypeptide comprising a FIX polypeptide or a FVII polypeptide and three to five CTPs of chorionic gonadotropin attached to the carboxyl terminus of the FIX polypeptide or the FVII polypeptide, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject subcutaneously or intravenously a CTP-modified factor IX (FIX) and a CTP-modified factor VII polypeptide comprising FIX and a FVII polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl termini of the FIX and FVII polypeptides, thereby preventing or treating hemophilia in the subject. In another embodiment, the present invention provides a method for preventing or treating hemophilia in a subject, comprising administering to the subject subcutaneously or intravenously a CTP-modified factor IX (FIX) and a CTP-modified factor VII polypeptide comprising FIX and a FVII polypeptide and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl termini of the FIX and FVII polypeptides, thereby preventing or treating hemophilia in the subject.

[0306] In some embodiments, provided herein is a method for preventing or treating hemophilia in a subject, the method comprising administering to the subject a CTP-modified coagulation factor comprising three to five CTPs of chorionic gonadotropin attached to the carboxyl terminus of a coagulation factor polypeptide, wherein the sequence of the CTP-modified coagulation factor is selected from the group consisting of SEQ ID NO: 25, 27, or 29, thereby preventing hemophilia in the subject. In another embodiment, the CTP-modified coagulation factor is selected from the group consisting of SEQ ID NO: 25, 27, 29, or 46. In another embodiment, the CTP-modified coagulation factor consists of SEQ ID NO: 46.

[0307] In one embodiment, the present invention provides a cell comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VII (FVII) polypeptide and three to five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, which is modified by a CTP. In another embodiment, the present invention provides a cell comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VII (FVII) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, which is modified by a CTP. In another embodiment, the present invention provides a cell comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VII (FVII) polypeptide and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, which is modified by a CTP.

[0308] In one embodiment, the term "three to five" when referring to gonadotropin carboxy-terminal peptides (CTPs) refers to the attachment of three, four, or five CTPs to the carboxy terminus of a coagulation factor provided herein.

[0309] In one embodiment, the present invention provides a composition comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VII (FVII) polypeptide and three to five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, which is modified by a CTP. In another embodiment, the present invention provides a composition comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VII (FVII) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, which is modified by a CTP. In another embodiment, the present invention provides a composition comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VII (FVII) polypeptide and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, which is modified by a CTP.

[0310] In one embodiment, the present invention provides a method for extending the biological half-life of a factor VII (FVII) polypeptide, comprising the step of attaching three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby extending the biological half-life of the FVII polypeptide. In another embodiment, the present invention provides a method for extending the biological half-life of a factor VII (FVII) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby extending the biological half-life of the FVII polypeptide. In another embodiment, the present invention provides a method for extending the biological half-life of a factor VII (FVII) polypeptide, comprising the step of attaching five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby extending the biological half-life of the FVII polypeptide.

[0311] In another embodiment, the present invention provides a method for improving the area under the curve (AUC) of a Factor VII (FVII) polypeptide, comprising the step of attaching three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby improving the AUC of the FVII polypeptide. In another embodiment, the present invention provides a method for improving the area under the curve (AUC) of a Factor VII (FVII) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby improving the AUC of the FVII polypeptide. In another embodiment, the present invention provides a method for improving the area under the curve (AUC) of a Factor VII (FVII) polypeptide, comprising the step of attaching five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby improving the AUC of the FVII polypeptide.

[0312] In one embodiment, the present invention provides a method for reducing the dosing frequency of a factor VII (FVII) polypeptide, comprising the step of attaching three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby reducing the dosing frequency of the FVII polypeptide. In another embodiment, the present invention provides a method for reducing the dosing frequency of a factor VII (FVII) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby reducing the dosing frequency of the FVII polypeptide. In another embodiment, the present invention provides a method for reducing the dosing frequency of a factor VII (FVII) polypeptide, comprising the step of attaching five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby reducing the dosing frequency of the FVII polypeptide.

[0313] In one embodiment, the present invention provides a method for reducing the clearance rate of a factor VII (FVII) polypeptide, comprising the step of attaching three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby reducing the clearance rate of the FVII polypeptide. In another embodiment, the present invention provides a method for reducing the clearance rate of a factor VII (FVII) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby reducing the clearance rate of the FVII polypeptide. In another embodiment, the present invention provides a method for reducing the clearance rate of a factor VII (FVII) polypeptide, comprising the step of attaching five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby reducing the clearance rate of the FVII polypeptide.

[0314] In one embodiment, the present invention provides a method for producing a CTP-modified Factor VII (FVII) polypeptide, comprising the step of attaching three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby producing a CTP-modified FVII polypeptide. In another embodiment, the present invention provides a method for producing a CTP-modified Factor VII (FVII) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby producing a CTP-modified FVII polypeptide. In another embodiment, the present invention provides a method for producing a CTP-modified Factor VII (FVII) polypeptide, comprising the step of attaching five chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide, thereby producing a CTP-modified FVII polypeptide.

[0315] In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a CTP-modified Factor VII (FVII) polypeptide comprising a FVII polypeptide and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, thereby treating hemophilia in the subject. In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a CTP-modified Factor VII (FVII) polypeptide comprising a FVII polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, thereby treating hemophilia in the subject. In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a CTP-modified Factor VII (FVII) polypeptide comprising a FVII polypeptide and five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVII polypeptide, thereby treating hemophilia in the subject.

[0316] In another embodiment, the methods provided herein further comprise the step of attaching four chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVII polypeptide.

[0317] In other embodiments, the engineered coagulation factors are used to treat patients with hemophilia B. In one embodiment, a coagulation factor IX comprising three CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In one embodiment, a coagulation factor IX comprising four CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In one embodiment, a coagulation factor IX comprising five CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In another embodiment, a coagulation factor IX comprising two CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In another embodiment, a coagulation factor IX comprising one CTP repeat at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In other embodiments, the engineered coagulation factors can reduce the number of infusions required for a patient, reduce the required dosage for a patient, or a combination of both.

[0318] In one embodiment, compared with the harvest of FIX-CTP-CTP, the harvest of FIX-CTP or rhFIX, the coagulation factors IX included in 3 CTPs in the series connection at coagulation factors IX carboxyl terminal shows an improved PK curve, and simultaneously maintained its coagulation activity. In one embodiment, the elimination half-life of rFIX-CTP3 is 2.5 to 4 times of rFIX in rats and the mice lacking FIX. In one embodiment, the administration of rFIX-CTP3 significantly prolongs the procoagulant effect in the mice lacking FIX and reaches at least 76 hours after administration. In one embodiment, the administration of rFIX-CTP3 has produced an activity peak higher than rFIX in the mice lacking FIX. In another embodiment, compared with the harvest of FIX-CTP or rhFIX, the coagulation factors IX included in 2 CTPs in the series connection at coagulation factors IX carboxyl terminal shows an improved PK curve, and simultaneously maintained its coagulation activity. In another embodiment, Factor IX comprising two CTPs in tandem at the carboxyl terminus of Factor IX exhibits a 3-fold increase in half-life and a 4.5-fold higher AUC compared to rhFIX.

[0319] In another embodiment, SC administration results in higher bioavailability of CTP-modified FVII compared to recombinant FVII. When compared to SC administration of FVIIa-CTP3 and CTP5, the half-life was longer and the bioavailability (AUC SC / AUC IV) was higher after SC administration of FVIIa-CTP3 and CTP5. In comparison, subcutaneously injected MOD-5014 and MOD-5019 showed improved mouse survival (see Example 8 below).

[0320] In one embodiment, MOD-5014 is FVIIa-CTP3 (having three CTP peptides attached to the C-terminus). In one embodiment, MOD-5014 provides a long-acting coagulation factor. In one embodiment, MOD-5014 provides a more durable and prolonged blood clotting reaction compared to recombinant human FVIIa. (See, for example, Example 14). It will be understood by those skilled in the art that the terms MOD-5014 or FVIIa-CTP3 can be used interchangeably, having all the same qualities and meanings, and in one embodiment refer to a disulfide-linked double-chain heterodimer structure comprising amino acid SEQ ID No: 46. In addition, it will be understood by those skilled in the art that when describing a CTP-modified coagulation factor, such as FVII-CTP3, the term FVII-CTP3 may in some cases refer to an inactive form of FVII-CTP3. Of course, those skilled in the art will recognize which form is being referred to based on associated details such as activity. Similarly, although the term MOD-5014 is interchangeable with the term FVIIa-CTP3, i.e., to refer to the active form of the coagulation factor modified with CTP, in certain circumstances the term MOD-5014 may be used to refer to the active form of FVII or the nucleotide sequence encoding FVII-CTP3, which is then expressed and secreted from cells and purified and activated in vitro, resulting in the active form of FVIIa present in the MOD-5014 molecule.

[0321] In one embodiment, the inactivation of MOD-5014 by tissue factor pathway inhibitor (TFPI) is dose-dependent. In one embodiment, the inactivation of MOD-5014 by TFPI is shown to be comparable to the inactivation of recombinant FVIIa by TFPI. In one embodiment, MOD-5014 is inhibited by antithrombin III. In one embodiment, the inhibition of MOD-5014 by antithrombin III is enhanced in the presence of heparin. In one embodiment, the inhibition of MOD-5014 by antithrombin III in the presence or absence of heparin is comparable to that of recombinant FVIIa. Similar inhibition pattern. (See Example 11 below).

[0322] In one embodiment, MOD-5014 generates thrombin in a dose-dependent manner. In one embodiment, MOD-5014 reduces the lag phase of thrombin generation. In one embodiment, MOD-5014 reduces blood clotting time. In one embodiment, MOD-5014 increases the efficiency of blood clot formation. In one embodiment, administration of MOD-5014 reduces blood clotting time in a subject. In one embodiment, administration of MOD-5014 increases the efficiency of blood clot formation in a subject. In one embodiment, thrombin generation achieved by MOD-5014 is similar to that achieved by recombinant FVIIa. In one embodiment, the reduction in the lag phase of thrombin generation caused by MOD-5014 is similar to that caused by recombinant FVIIa In one embodiment, the reduction in blood clotting time achieved by MOD-5014 is similar to that achieved by recombinant FVIIa In one embodiment, the increase in blood clot formation efficiency achieved by MOD-5014 is similar to that achieved by recombinant FVIIa. The resulting blood clot formation efficiency is increased (see Example 12 below).

[0323] As provided herein, the attachment of CTP to blood coagulation factors, for example, factor FVII, factor FVIIA and factor FX increases the half-life of blood coagulation factors. Example 11, 12 and 13 show the attachment of CTP, and three CTPs such as those attached to FVIIA do not seem to affect blood coagulation activity. In one embodiment, the attachment of CTP to FVII does not interfere with blood clot formation. In one embodiment, the attachment of CTP to FVII does not interfere with the increase of blood clot formation efficiency. In one embodiment, the attachment of CTP to FVII does not interfere with the reduction of blood clotting time. In one embodiment, after CTP is attached to blood coagulation factors, the combination of phospholipids to FVII is maintained. In one embodiment, the attachment of CTP to FVIIA does not interfere with blood clot formation. In one embodiment, the attachment of CTP to FVIIA does not interfere with the increase of blood clot formation efficiency. In one embodiment, the attachment of CTP to FVIIA does not interfere with the reduction of blood clot formation. In one embodiment, after CTP is attached to blood coagulation factors, the combination of phospholipids to FVIIA is maintained.

[0324] In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a factor IX (FIX) polypeptide comprising a FIX polypeptide and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, thereby treating hemophilia in the subject. In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a factor IX (FIX) polypeptide comprising a FIX polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, thereby treating hemophilia in the subject. In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a factor IX (FIX) polypeptide comprising a FIX polypeptide and five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, thereby treating hemophilia in the subject. In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a CTP-modified Factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide, thereby treating hemophilia in the subject.

[0325] In another embodiment, the present invention provides a method for treating hemophilia for a subject, comprising administering to the subject one or more CTP-modified coagulation factors as described herein. Therefore, in one embodiment, the present invention provides a method for treating hemophilia for a subject, comprising administering a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide and a CTP-modified factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three to five chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide, thereby treating hemophilia for the subject. In one embodiment, CTP-modified FIX and CTP-modified FVIIa are administered simultaneously with the same composition. In another embodiment, CTP-modified FIX and CTP-modified FVIIa are administered simultaneously with separate compositions. In another embodiment, CTP-modified FIX and CTP-modified FVIIa are administered at separate times in separate compositions.

[0326] In other embodiments, the engineered coagulation factors are used to treat patients with hemophilia B. In one embodiment, a coagulation factor IX comprising three CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In one embodiment, a coagulation factor IX comprising four CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In one embodiment, a coagulation factor IX comprising five CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In another embodiment, a coagulation factor IX comprising two CTPs in series at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In another embodiment, a coagulation factor IX comprising one CTP repeat at the carboxyl terminus of the coagulation factor IX is used to treat patients with hemophilia B. In other embodiments, the engineered coagulation factors can reduce the number of infusions required for a patient, reduce the required dosage for a patient, or a combination thereof.

[0327] Example 14 illustrates the result of MOD-5014 administration to large mammals (dogs). MOD-5014 administration provides effective and safe long-acting FVIIa for blood coagulation. Treatment using MOD-5014 can be preventive or on demand. In one embodiment, the invention provides a method for treating hemophilia at a subject, comprising administering MOD-5014 to the subject, thereby treating hemophilia at the subject. In one embodiment, the invention provides a method for preventing excessive bleeding at a subject, comprising administering MOD-5014 to the subject, thereby preventing excessive bleeding at the subject. In one embodiment, the invention provides a method for preventing hemophilia at a subject's prophylactic treatment, comprising administering MOD-5014 to the subject, thereby preventing hemophilia at the subject's prophylactic treatment.

[0328] In one embodiment, MOD-5014 is used to treat hemophilia in a subject comprising administering MOD-5014 to a subject with recombinant FVIIa. In one embodiment, the prophylactic treatment of hemophilia in a subject with MOD-5014 comprises administering recombinant FVIIa to the subject. In one embodiment, the use of MOD-5014 to prevent excessive bleeding in a subject comprises administering recombinant FVIIa Compared to the reduced dosing frequency of MOD-5014.

[0329] In one embodiment, compared with the harvest of FIX-CTP-CTP, the harvest of FIX-CTP or rhFIX, the coagulation factors IX included in 3 CTPs in the series connection at coagulation factors IX carboxyl terminal shows an improved PK curve, and simultaneously maintained its coagulation activity. In one embodiment, the elimination half-life of rFIX-CTP3 is 2.5 to 4 times of rFIX in rats and the mice lacking FIX. In one embodiment, the administration of rFIX-CTP3 significantly prolongs the procoagulant effect in the mice lacking FIX and reaches at least 76 hours after administration. In one embodiment, the administration of rFIX-CTP3 has produced an activity peak higher than rFIX in the mice lacking FIX. In another embodiment, compared with the harvest of FIX-CTP or rhFIX, the coagulation factors IX included in 2 CTPs in the series connection at coagulation factors IX carboxyl terminal shows an improved PK curve, and simultaneously maintained its coagulation activity. In another embodiment, Factor IX comprising two CTPs in tandem at the carboxyl terminus of Factor IX exhibits a 3-fold increase in half-life and a 4.5-fold higher AUC compared to rhFIX.

[0330] In one embodiment, In comparison, Factor VII comprising three CTPs in series at the carboxyl terminus of Factor VII exhibited an improved PK profile while maintaining its coagulation activity (see Tables 59 and Figure 36 ).

[0331] In another embodiment, the terms "CTP peptide," "carboxy-terminal peptide," and "CTP sequence" are used interchangeably herein. In another embodiment, the carboxy-terminal peptide is a full-length CTP. Each possibility represents a separate embodiment of the present disclosure.

[0332] In another embodiment, a signal peptide is attached to the amino terminus of the CTP as described in US Patent 7,553,940, which is incorporated herein by reference in its entirety. In another embodiment, a signal peptide is not attached to the amino terminus of the CTP.

[0333] In other embodiments, the term engineered coagulation factor refers to the amino acid sequence of a mature coagulation factor. In other embodiments, the term engineered coagulation factor refers to the amino acid sequence of a coagulation factor including its signal sequence or signal peptide.

[0334] In another embodiment, "signal sequence" and "signal peptide" are used interchangeably herein and have all the same properties and meanings. In another embodiment, when referring to a polynucleotide molecule, "sequence" may refer to the coding portion. In another embodiment, the engineered coagulation factor comprising at least one CTP as described herein has enhanced in vivo biological activity compared to the same coagulation factor without at least one CTP. In one embodiment, the enhanced in vivo biological activity is derived from the longer half-life of the engineered coagulation factor while maintaining at least some biological activity. In another embodiment, the enhanced biological activity is derived from the enhanced biological activity produced by the CTP modification. In another embodiment, the enhanced biological activity is derived from the longer half-life and enhanced function of the CTP-modified coagulation factor.

[0335] In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides enhanced protection against degradation of the coagulation factor. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides enhanced protection against clearance. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides an extended clearance time. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor enhances its Cmax. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor enhances its Tmax. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor extends its Tmax. 1 / 2.

[0336] In another embodiment, the conjugated coagulation factors of the present invention are used in the same manner as unmodified conjugated coagulation factors. In another embodiment, the conjugated coagulation factors of the present invention have increased circulation half-life and plasma residence time, reduced clearance and increased in vivo clinical activity. In another embodiment, due to the improved properties of the conjugated coagulation factors as described herein, the conjugates can be administered less frequently than unmodified forms of the same coagulation factors.

[0337] In another embodiment, reduced dosing frequency will lead to improved treatment strategies, which in one embodiment, will lead to improved patient compliance, which will lead to improved treatment outcomes and improved patient quality of life. In another embodiment, conjugates having the molecular weight and linker structure of the conjugates of the present invention have been found to have improved potency, improved stability, increased AUC levels, and enhanced circulation half-life compared to conventional coagulation factor conjugates.

[0338] In another embodiment, the present invention also provides a pharmaceutical composition or pharmaceutical formulation comprising a CTP-modified factor IX (FIX) polypeptide, wherein the CTP-modified factor IX (FIX) polypeptide consists of a FIX polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the CTP-modified FIX polypeptide.

[0339] In another embodiment, the present invention also provides a pharmaceutical composition or pharmaceutical formulation comprising a CTP-modified Factor VIIa (FVIIa) polypeptide, wherein the CTP-modified Factor VIIa (FVIIa) polypeptide consists of a FVIIa polypeptide and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa.

[0340] In another embodiment, the present invention also provides a pharmaceutical composition or pharmaceutical formulation comprising a CTP-modified Factor VIIa (FVIIa) polypeptide, wherein the CTP-modified Factor VIIa (FVIIa) polypeptide consists of a FVIIa polypeptide and four gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa.

[0341] In another embodiment, the present invention also provides a pharmaceutical composition or pharmaceutical formulation comprising a CTP-modified Factor VIIa (FVIIa) polypeptide, wherein the CTP-modified Factor VIIa (FVIIa) polypeptide consists of a FVIIa polypeptide and five gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa.

[0342] In another embodiment, provided herein is a composition comprising a conjugated coagulation factor as described herein. In another embodiment, provided herein is a pharmaceutical composition comprising a conjugated coagulation factor as described herein. In another embodiment, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a conjugated coagulation factor as described herein. In one embodiment, the therapeutically effective amount of the conjugated coagulation factor is determined based on factors such as the specific condition being treated, the condition of the patient being treated, and the other ingredients in the composition.

[0343] In one embodiment, the present disclosure provides a pharmaceutical preparation for the compositions, preparations and methods of the present disclosure. In another embodiment, the present disclosure provides a pharmaceutical preparation comprising a polypeptide consisting of a coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor. In another embodiment, the pharmaceutical preparation further comprises a buffer and a tonicity agent. In another embodiment, the buffer is 20mM citrate and 13.3mM glycine, and the tonicity agent is 150mM NaCl. In another embodiment, the preparation is at a pH of about 6.4. In another embodiment, the buffer is 20mM citrate and 13.3mM glycine, and the tonicity agent is 150mM NaCl and the pH is 6.4. In another embodiment, the preparation is a liquid preparation. In another embodiment, the preparation is a lyophilized preparation. In another embodiment, a lyophilized CTP-modified coagulation factor can be used to form a liquid preparation. In another embodiment, the CTP-modified coagulation factor is FVII-CTP-CTP-CTP. In another embodiment, the CTP-modified coagulation factor is FVIIa-CTP-CTP-CTP.

[0344] In another embodiment, a once-a-week dosage form comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a once-a-day dosage form comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a dosage form every other day comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a dosage form every two days comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a twice-a-week dosage form comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a twice-a-week dosage form comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a weekly dosage form comprising a pharmaceutical formulation provided herein is provided herein. In another embodiment, a biweekly (every two weeks) dosage form comprising a pharmaceutical formulation provided herein is provided herein.

[0345] In another embodiment, the present invention provides a formulation comprising a polypeptide consisting of a coagulation factor and three to five chorionic gonadotropin CTPs attached to the carboxyl terminus of the coagulation factor, and wherein the polypeptide optionally consists of a signal peptide, wherein the formulation has increased stability. In one embodiment, the formulation is stable for at least one year. In another embodiment, the formulation is stable for at least two years.

[0346] In one embodiment, the coagulation factor modified by CTP is formulated as a liquid formulation. In another embodiment, the factor VII modified by CTP is formulated as a liquid formulation. In another embodiment, the factor VIIa modified by CTP is formulated as a liquid formulation. In another embodiment, the factor IX modified by CTP is formulated as a liquid formulation. In one embodiment, the coagulation factor modified by CTP is formulated as an intranasal formulation. In another embodiment, the coagulation factor modified by CTP is formulated as an injectable formulation.

[0347] In another embodiment, the method of the present disclosure includes increasing compliance in the use of coagulation factor therapy, comprising providing a coagulation factor modified with a CTP to a subject in need thereof, thereby increasing compliance in the use of the coagulation factor therapy.

[0348] In another embodiment, the coagulation factor modified by CTP is administered to the subject once daily. In another embodiment, the polypeptide comprising the coagulation factor modified by CTP is administered to the subject once every two days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every three days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every four days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every five days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every six days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once a week. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every seven to fourteen days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every ten to twenty days. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every five to fifteen days. In another embodiment, the CTP-modified coagulation factor is administered to the subject once every 15 to 30 days.

[0349] In one embodiment, the formulations of the present invention are formulated as liquid formulations for injection via a syringe or pen device.

[0350] In one embodiment, the formulations provided herein further include preservatives such as benzalkonium chloride and thimerosal; chelating agents such as sodium edetate; buffers such as phosphates, citrates, and acetates; tonicity agents such as sodium chloride, potassium chloride, glycerol, mannitol, etc.; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfite, etc.; fragrances; viscosity modifiers such as polymers including cellulose and its derivatives; and polyvinyl alcohol and acids and bases to adjust the pH of these aqueous compositions as needed. The composition also includes a local anesthetic or other active ingredient. The composition can be used as a spray, mist, drops, etc.

[0351] In one embodiment, the coagulation factor as described herein is a human coagulation factor.

[0352] In another embodiment, the conjugated coagulation factors as described herein can be used to treat a subject with a coagulation or clotting disorder. In another embodiment, the coagulation or clotting disorder is hemophilia. In another embodiment, the conjugated coagulation factors as described herein can be used for the prophylactic treatment of hemophilia, thereby reducing the risk of bleeding and related complications. In another embodiment, reducing the risk of bleeding and related complications reduces the risk of spontaneous bleeding. In another embodiment, reducing the risk of bleeding and related complications reduces the risk of excessive bleeding. In another embodiment, the conjugated coagulation factors as described herein can be used to treat a subject with hemophilia while reducing the risk of developing inhibitory antibodies to the exogenously administered coagulation factors. In another embodiment, the conjugated coagulation factors as described herein can be used to treat a subject with hemophilia, thereby inducing homeostasis.

[0353] In one embodiment, the CTP-modified coagulation factor of the present invention has therapeutic applications. In one embodiment, the CTP-modified coagulation factor of the present invention has preventive applications.

[0354] In another embodiment, conjugated coagulation factors as described herein can be used to treat subjects experiencing excessive bleeding or bruising or with prolonged prothrombin time (PT) or partial thromboplastin time (PTT). In another embodiment, conjugated coagulation factors as described herein can be used to treat subjects with acquired conditions that cause bleeding, such as vitamin K deficiency or liver disease. In another embodiment, conjugated coagulation factors as described herein can be used to treat subjects with acquired (due to other diseases) or hereditary, mild or severe, permanent or temporary coagulation factor deficiency. In another embodiment, conjugated coagulation factors as described herein can be used to treat subjects with hemophilia A. In another embodiment, conjugated coagulation factors as described herein can be used to treat subjects with hemophilia B. In another embodiment, the conjugated coagulation factors as described herein can be used to treat subjects with acquired deficiencies due to chronic diseases such as liver disease or cancer; due to acute conditions that rapidly use up coagulation factors, such as disseminated intravascular coagulation (DIC); or due to vitamin K deficiency or treatment with vitamin K antagonists such as warfarin (the production of factors II, VII, IX, and X requires vitamin K). In another embodiment, the conjugated coagulation factors as described herein can be used to treat subjects with diseases that cause an imbalance in coagulation, such as, but not limited to, liver disease, uremia, cancer, bone marrow disorders, exposure to snake venom, vitamin K deficiency, anticoagulant therapy, accidental ingestion of the anticoagulant warfarin, multiple blood transfusions (stored blood units lose some of their coagulation factors), or a combination thereof. In another embodiment, the present invention provides a method for treating deep vein thrombosis in a subject, comprising administering a CTP-modified coagulation factor of the present invention. In another embodiment, the present invention provides a method for preventing uncontrolled bleeding in a subject with hemophilia, comprising administering a CTP-modified coagulation factor of the present invention. In another embodiment, the present invention provides a method for preventing bleeding episodes in a subject suffering from hemophilia, comprising administering a CTP-modified coagulation factor of the present invention. In another embodiment, the present invention provides a method for controlling bleeding episodes in a subject suffering from hemophilia B (congenital factor IX deficiency).

[0355] In one embodiment, the compositions of the present invention comprise a formulation as described herein. In another embodiment, the methods of the present invention comprise administering a formulation as described herein. In another embodiment, the methods of the present invention comprise administering a composition comprising a formulation as described herein.

[0356] In another embodiment, the compositions, formulations and methods of the present invention are used to treat bleeding episodes in patients with hemophilia A or B who have inhibitors to FVIII or FIX and in patients with acquired hemophilia; to prevent bleeding during surgical intervention or invasive treatment in patients with hemophilia A or B who have inhibitors to FVIII or FIX or in patients with acquired hemophilia; to treat bleeding episodes in patients with congenital FVII deficiency and to prevent bleeding during surgical intervention or invasive treatment in patients with congenital FVII deficiency. Acquired hemophilia is an idiopathic autoimmune disorder in which patients with previously normal hemostasis develop autoantibodies to clotting factors, most commonly FVIII. The production of autoantibodies to FVIII results in a deficiency of FVIII, which results in a deficiency of thrombin generated by the complex of factor IXa and factor VIIIa through the intrinsic pathway of the coagulation cascade. The following conditions may be associated with acquired hemophilia A: idiopathic, pregnancy, autoimmune diseases, inflammatory bowel disease, ulcerative colitis, skin diseases (e.g., psoriasis, pemphigus), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease), allergic drug reactions, diabetes, acute hepatitis B infection, acute hepatitis C infection, malignancies - solid tumors (prostate cancer, lung cancer, colon cancer, pancreatic cancer, gastric cancer, bile duct cancer, head and neck cancer, cervical cancer, breast cancer, melanoma, kidney cancer), hematological malignancies. It will be understood by those skilled in the art that autoimmune diseases may include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, temporal arteritis, Sjögren's syndrome, autoimmune hemolytic anemia, Goodpasture's syndrome, myasthenia gravis, Graves' disease, autoimmune hypothyroidism. It will be appreciated by those skilled in the art that allergic reactions may occur in subjects administered penicillin and its derivatives, sulfonamides, phenytoin, chloramphenicol, methyldopa, thioxanthene, interferon alpha, fludarabine, BCG (BCG) vaccination, desvenlafaxine. It will be appreciated by those skilled in the art that hematological malignancies may include chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome, myelofibrosis, and erythroleukemia. Therefore, in one embodiment, a method for treating acquired hemophilia is provided herein, comprising administering any one of the compositions provided herein to the subject.

[0357] In another embodiment, the compositions, formulations and methods of the present invention are used to treat or prevent muscle bleeding. In another embodiment, the compositions, formulations and methods of the present invention are used to treat or prevent joint bleeding. In another embodiment, the compositions, formulations and methods of the present invention provide treatment or prophylactic treatment for nose bleeding and gum bleeding, mucosal bleeding and bleeding into the central nervous system. In another embodiment, the compositions, formulations and methods of the present invention provide treatment or prophylactic treatment for gastrointestinal or cerebral bleeding. In another embodiment, the compositions, formulations and methods of the present invention provide treatment or prophylactic treatment for low-frequency mild bleeding. In another embodiment, the compositions, formulations and methods of the present invention provide treatment or prophylactic treatment for low-frequency moderate bleeding. In another embodiment, the compositions, formulations and methods of the present invention provide treatment or prophylactic treatment for high-frequency mild bleeding. In another embodiment, the compositions, formulations and methods of the present invention provide treatment or prophylactic treatment for high-frequency moderate bleeding.

[0358] In one embodiment, the compositions, formulations, and methods of the present invention provide for the treatment or prophylactic treatment of asymptomatic hemophilia. In another embodiment, the compositions, formulations, and methods of the present invention provide for the treatment or prophylactic treatment of mild to moderate hemophilia. In another embodiment, the compositions, formulations, and methods of the present invention provide for the treatment or prophylactic treatment of severe hemophilia.

[0359] In one embodiment, the compositions, formulations, and methods of the present invention provide therapeutic or prophylactic treatment for bleeding, which in one embodiment is uncontrollable bleeding and in another embodiment is intracerebral bleeding. In another embodiment, the compositions, formulations, and methods of the present invention provide therapeutic or prophylactic treatment for neonatal coagulopathy; severe liver disease; high-risk surgery; traumatic blood loss; bone marrow transplantation; thrombocytopenia and platelet dysfunction; urgent reversal of oral anticoagulation; congenital deficiency of factors V, VII, X, and XI; or von Willebrand disease, in one embodiment, von Willebrand disease with inhibitors to von Willebrand factor.

[0360] In one embodiment, the CTP-modified coagulation factors of the present invention are used to treat hemophilia or a related disease as described herein in a subject. In one embodiment, the subject is a human. In another embodiment, the subject is a human child. In another embodiment, the subject is a domesticated animal. In another embodiment, the subject is a mammal. In another embodiment, the subject is a farm animal. In another embodiment, the subject is a monkey. In another embodiment, the subject is a horse. In another embodiment, the subject is a cow. In another embodiment, the subject is a mouse. In another embodiment, the subject is a rat. In another embodiment, the subject is a canine. In another embodiment, the subject is a feline. In another embodiment, the subject is a bovine, ovine, porcine, equine, murine, or cervical. In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is a child, in another embodiment, an adolescent, in another embodiment, an adult, or in another embodiment, an elderly subject. In another embodiment, the subject is a pediatric subject, and in another embodiment, a geriatric subject.

[0361] In another embodiment, [(CTP)n>1-coagulation factor] as described herein comprises a full-length coagulation factor or an active fragment thereof, which is linked via a peptide bond at its carboxyl terminus to at least one CTP unit that does not have a CTP at its amino terminus. In another embodiment, [(CTP)n>1-coagulation factor] as described herein comprises a coagulation factor or an active fragment thereof, which is linked via a peptide bond to at least one CTP unit that is linked via a peptide bond to an additional CTP unit that does not have a CTP at its amino terminus. In another embodiment, a nucleic acid molecule encodes an engineered coagulation factor that includes at least one CTP attached to its C-terminus and does not have a CTP at its amino terminus.

[0362] In another embodiment, the CTP is attached to the coagulation factor via a linker. In another embodiment, the linker connecting the CTP sequence to the coagulation factor is a covalent bond. In another embodiment, the linker connecting the CTP sequence to the coagulation factor is a peptide bond. In another embodiment, the linker connecting the CTP sequence to the coagulation factor is a substituted peptide bond. In another embodiment, the CTP sequence comprises: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 1). In another embodiment, the CTP sequence comprises: SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 2). In another embodiment, the CTP sequence comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2.

[0363] In another embodiment, the carboxy-terminal peptide (CTP) sequence of the present invention comprises the amino acid sequence of amino acids 112 to 145 of human chorionic gonadotropin as set forth in SEQ ID NO: 1. In another embodiment, the CTP sequence of the present invention comprises the amino acid sequence of amino acids 118 to 145 of human chorionic gonadotropin as set forth in SEQ ID NO: 2. In another embodiment, the CTP sequence may also begin at any position between positions 112-118 and end at position 145 of human chorionic gonadotropin. In some embodiments, the CTP sequence peptide is 28, 29, 30, 31, 32, 33, or 34 amino acids long and begins at position 112, 113, 114, 115, 116, 117, or 118 of the CTP amino acid sequence.

[0364] In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by 1-5 conservative amino acid substitutions, as described in U.S. Patent No. 5,712,122, which is incorporated herein by reference. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by 1 conservative amino acid substitution. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by 2 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by 5 conservative amino acid substitutions.

[0365] In another embodiment, the amino acid sequence of the CTP peptide of the present invention has at least 70% homology to the amino acid sequence of a natural CTP or its peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention has at least 80% homology to the amino acid sequence of a natural CTP or its peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention has at least 90% homology to the amino acid sequence of a natural CTP or its peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention has at least 95% homology to the amino acid sequence of a natural CTP or its peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention has at least 98% homology to the amino acid sequence of a natural CTP or its peptide.

[0366] In another embodiment, the polynucleotide encoding the CTP peptide of the present invention has at least 70% homology to a natural human CTP DNA sequence or its peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention has at least 80% homology to a natural human CTP DNA sequence or its peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention has at least 90% homology to a natural CTP DNA sequence or its peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention has at least 95% homology to a natural CTP DNA sequence or its peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention has at least 98% homology to a natural CTP DNA sequence or its peptide.

[0367] In one embodiment, at least one of the chorionic gonadotropin CTP amino acid sequences is truncated. In another embodiment, both chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, two of the chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, three of the chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, four of the chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, five of the chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, two or more of the chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, all of the chorionic gonadotropin CTP amino acid sequences are truncated. In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 3. In another embodiment, SEQ ID NO: 3 comprises the following amino acid (AA) sequence: SSSSKAPPPSLP.

[0368] In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 2. In another embodiment, SEQ ID NO: 2 comprises the following amino acid (AA) sequence: SSSSKAPPPSLPSPSRLPGPSDTPILPQ.

[0369] In one embodiment, the truncated CTP comprises the first 11 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 12 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 8 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the truncated CTP comprises the first 13 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 14 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 6 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the truncated CTP comprises the first 5 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3.

[0370] In one embodiment, at least one of the chorionic gonadotropin CTP amino acid sequences is glycosylated. In another embodiment, both chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, two of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, three of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, four of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, five of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, two or more of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, all of the chorionic gonadotropin CTP amino acid sequences are glycosylated.

[0371] In one embodiment, the CTP sequence of the present invention comprises at least one glycosylation site. In one embodiment, the CTP sequence of the present invention comprises 2 glycosylation sites. In one embodiment, the CTP sequence of the present invention comprises 3 glycosylation sites. In one embodiment, the CTP sequence of the present invention comprises 4 glycosylation sites. In one embodiment, one or more of the chorionic gonadotropin CTP amino acid sequence is fully glycosylated. In another embodiment, one or more of the chorionic gonadotropin CTP amino acid sequence is partially glycosylated. In one embodiment, partially glycosylated means that one of the CTP glycosylation sites is glycosylated. In another embodiment, two of the CTP glycosylation sites are glycosylated. In another embodiment, three of the CTP glycosylation sites are glycosylated.

[0372] In certain embodiments, CTP sequence modification is advantageous in allowing the use of lower dosage. In certain embodiments, CTP sequence modification is advantageous in allowing less dosage. In certain embodiments, CTP sequence modification is advantageous in allowing safe long-lasting effects.

[0373] In some embodiments, "polypeptides," "engineered coagulation factors," or "proteins" as used herein include natural polypeptides (any of degradation products, synthetic polypeptides, or recombinant polypeptides) and peptidomimetics (typically synthetic polypeptides), as well as peptoids and hemi-peptoids that are polypeptide analogs that, in some embodiments, have modifications that render the polypeptides comprising the coagulation factors even more stable or more capable of penetrating cells in vivo.

[0374] In some embodiments, modifications include, but are not limited to, C-terminal modifications, polypeptide bond modifications, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated herein by reference as if fully set forth herein. Additional details in this regard are provided below.

[0375] In some embodiments, polypeptide bonds (-CO-NH-) within a polypeptide are substituted. In some embodiments, polypeptide bonds are substituted with N-methylated bonds (-N(CH3)-CO-). In some embodiments, polypeptide bonds are substituted with ester bonds (-C(R)HCOOC(R)-N-). In some embodiments, polypeptide bonds are substituted with ketomethylene bonds (-CO-CH2-). In some embodiments, polypeptide bonds are substituted with α-aza bonds (-NH-N(R)-CO-), where R is any alkyl group, such as methyl, or carbocyclic (carba) bonds (-CH2-NH-). In some embodiments, polypeptide bonds are substituted with hydroxyethylene bonds (-CH(OH)-CH2-). In some embodiments, polypeptide bonds are substituted with thioamide bonds (-CS-NH-). In some embodiments, polypeptide bonds are substituted with olefinic double bonds (-CH=CH-). In some embodiments, polypeptide bonds are substituted with reversed amide bonds (-NH-CO-). In some embodiments, polypeptide bonds are substituted with polypeptide derivatives (-N(R)-CH2-CO-), where R is a "normal" side chain naturally occurring on a carbon atom. In some embodiments, these modifications occur at any one of the linkages along the polypeptide chain, and in one embodiment at several (2 to 3 linkages) simultaneously.

[0376] In some embodiments, the natural aromatic amino acids of the polypeptide, such as Trp, Tyr and Phe, are substituted for synthetic non-natural acids such as phenylglycine, TIC, naphthylalanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In some embodiments, the polypeptides of the invention include one or more modified amino acids or one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, etc.).

[0377] In one embodiment, "amino acid" or "amino acid sequence" is understood to include the 20 naturally occurring amino acids; those amino acids that are commonly post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. In one embodiment, "amino acid" includes both D-amino acids and L-amino acids.

[0378] In some embodiments, the polypeptides of the present invention are used in treatments requiring a soluble form of a polypeptide comprising a coagulation factor. In some embodiments, the polypeptides of the present invention include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine, which can increase the solubility of the polypeptide due to their hydroxyl-containing side chains.

[0379] In some embodiments, the engineered coagulation factors of the present invention are used in linear form, but those skilled in the art will appreciate that cyclic forms of the engineered coagulation factors can also be used if cyclization does not severely interfere with the characteristics of the engineered coagulation factors.

[0380] In some embodiments, the engineered coagulation factors of the present invention are biochemically synthesized, such as by using standard solid phase techniques. In some embodiments, these biochemical methods include dedicated solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis.

[0381] In certain embodiments, recombinant protein technology is used to generate the coagulation factor of through engineering approaches of the present invention.In certain embodiments, recombinant protein technology is used to generate relatively long polypeptide (for example, longer than 18 to 25 amino acids).In certain embodiments, recombinant protein technology is used to generate a large amount of coagulation factors of through engineering approaches of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymology, 153:516-544; Studier et al., (1990) Methods in Enzymology, 185:60-89; Brisson et al., (1984) Nature, 310:511-514; Takamatsu et al., (1987) EMBO J., 6:307-311; Coruzzi et al., (1984) EMBO J. 3:1671-1680; and Brogli et al., (1984) Science, 224:838-843; Gurley et al., (1986) Acta Mol. Cell Biol., 6:559-565; and Weissbach & Weissbach, 1988, Methods in Plant Molecular Biology, Academic Press, New York, Chapter VIII, pp. 421-463, the entire contents of which are incorporated herein by reference.

[0382] In another embodiment, the present disclosure provides a polynucleotide molecule comprising a coding portion of a gene encoding a polypeptide comprising a coagulation factor and a gonadotropin carboxyl-terminal peptide attached to the carboxyl-terminus of the coagulation factor, as described above. In another embodiment, the present disclosure provides a polynucleotide molecule consisting of a coding portion of a gene encoding a polypeptide comprising a coagulation factor and a gonadotropin carboxyl-terminal peptide attached to the carboxyl-terminus of the coagulation factor, as described above. In another embodiment, the present disclosure provides a polynucleotide molecule consisting essentially of a coding portion of a gene encoding a polypeptide comprising a coagulation factor and a gonadotropin carboxyl-terminal peptide attached to the carboxyl-terminus of the coagulation factor, as described above.

[0383] In another embodiment, the present disclosure provides a polynucleotide encoding a polypeptide comprising a coagulation factor and three gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, as described above. In another embodiment, the present disclosure provides a polynucleotide encoding a polypeptide consisting of a coagulation factor and three gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, as described above. In another embodiment, the present disclosure provides a polynucleotide encoding a polypeptide consisting essentially of a coagulation factor and three gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, as described above. In one embodiment, the polynucleotide is a polynucleotide sequence. In one embodiment, the polynucleotide is a polynucleotide molecule.

[0384] In another embodiment, the present disclosure provides an expression vector comprising a polynucleotide molecule as described herein. In another embodiment, the present disclosure provides an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a Factor IX (FIX) polypeptide and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide. In another embodiment, the present disclosure provides an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a Factor VIIa (FVIIa) polypeptide and three to five gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide.

[0385] In another embodiment, the present disclosure provides a cell comprising an expression vector as described herein. In another embodiment, the present disclosure provides a cell comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, which is modified by a CTP. In another embodiment, the present disclosure provides a cell comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide, which is modified by a CTP.

[0386] In another embodiment, the present disclosure provides a composition comprising an expression vector as described herein. In another embodiment, the present invention provides a composition comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide and modified by a CTP. In another embodiment, the present invention provides a composition comprising an expression vector comprising a polynucleotide encoding a polypeptide consisting of a Factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide and modified by a CTP.

[0387] In another embodiment, the present disclosure provides a composition comprising a cell as described herein. In another embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a prokaryotic cell.

[0388] In another embodiment, the present invention provides a method for producing a CTP-modified coagulation factor, comprising the step of attaching one to ten chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the coagulation factor, thereby producing a CTP-modified coagulation factor. In another embodiment, the present invention provides a method for producing a CTP-modified coagulation factor, comprising the step of attaching one to ten polynucleotide sequences encoding chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the polynucleotide sequence encoding the coagulation factor, thereby producing a CTP-modified coagulation factor. In another embodiment, the present invention provides a method for producing a CTP-modified factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby producing a CTP-modified FIX polypeptide. In another embodiment, the present invention provides a method for producing a CTP-modified Factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby producing a CTP-modified FVIIa polypeptide.

[0389] In another embodiment, the engineered coagulation factor of the present invention is synthesized using a polynucleotide molecule encoding a polypeptide of the present invention. In certain embodiments, the polynucleotide molecule encoding the engineered coagulation factor of the present invention is connected to an expression vector comprising a transcriptional control of a cis regulatory sequence (e.g., a promoter sequence). In certain embodiments, the cis regulatory sequence is suitable for instructing the constitutive expression of the engineered coagulation factor of the present invention. In certain embodiments, the cis regulatory sequence is suitable for instructing the tissue-specific expression of the engineered coagulation factor of the present invention. In certain embodiments, the cis regulatory sequence is suitable for instructing the inducible expression of the engineered coagulation factor of the present invention.

[0390] In some embodiments, tissue-specific promoters suitable for use with the present invention include sequences that are functional in one or more specific cell populations. Examples include, but are not limited to, liver-specific promoters such as albumin [Pinkert et al., (1987) Genes Dev. 1: 268-277], lymphoid-specific promoters [Calame et al., (1988) Adv. Immunol. 43: 235-275]; in particular, promoters for the T-cell receptor [Winoto et al., (1989) EMBO J. 8: 729-733] and immunoglobulins [Banerji et al., (1983) Cell 33: 729-740]. 33729-740)], a neuron-specific promoter, such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], a pancreas-specific promoter [Edlunch et al. (1985) Science 230:912-916], or a mammary gland-specific promoter, such as the cow's milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Inducible promoters suitable for use with the present invention include, for example, a tetracycline-inducible promoter (Srour, MA, et al., 2003. Thromb. Haemost. 90:398-405).

[0391] In one embodiment, the phrase "polynucleotide molecule" refers to a single-stranded or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and / or a composite polynucleotide sequence (e.g., a combination of the above).

[0392] In one embodiment, "complementary polynucleotide sequence" refers to a sequence produced by reverse transcription of a messenger RNA using a reverse transcriptase or any other RNA-dependent DNA polymerase. In one embodiment, the sequence can then be amplified in vivo or in vitro using a DNA polymerase.

[0393] In one embodiment, a "genomic polynucleotide sequence" refers to a sequence that is derived (isolated) from a chromosome and thus represents a contiguous portion of a chromosome.

[0394] In one embodiment, a "composite polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exon sequences required to encode a polypeptide of the present invention, as well as some intron sequences interposed therebetween. In one embodiment, intron sequences can be from any source, including other genes, and will typically include conserved splicing signal sequences. In one embodiment, intron sequences include cis-acting expression control elements.

[0395] In one embodiment, following expression and secretion, the signal peptide is cleaved from the precursor engineered coagulation factor, which generates a mature engineered coagulation factor lacking the signal peptide.

[0396] In some embodiments, the polynucleotides of the invention are prepared using PCR technology or any other method or process known to those skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (see, e.g., "Current Protocols in Molecular Biology," ed. Ausubel et al., John Wiley & Sons, 1992).

[0397] In one embodiment, the polynucleotide of the present invention that the coagulation factor of through engineering approaches is encoded is inserted into expression vector (that is, nucleic acid construct) to enable expression of recombinant polypeptide. In one embodiment, the expression vector of the present invention includes making this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes making this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes making this vector suitable for replication and integration in both prokaryotes and eukaryotes. In certain embodiments, cloning vector comprises transcription and translation initiation sequence (for example, promoter, enhancer) and transcription and translation terminator (for example, polyadenylation signal).

[0398] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host expression systems to express the coagulation factors of the present invention. In some embodiments, these include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with recombinant plasmid expression vectors containing the polypeptide coding sequence, such as Ti plasmids.

[0399] In some embodiments, a non-bacterial expression system (e.g., a mammalian expression system, such as CHO cells) is used to express the coagulation factors of the present invention. In one embodiment, the expression vector for expressing the polynucleotide of the present invention in mammalian cells is a pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene. The construction of a pCI-dhfr vector according to one embodiment is described in Example 1.

[0400] In some embodiments, in the bacterial system of the present invention, multiple expression vectors can be advantageously selected based on the intended use of the polypeptide being expressed. In one embodiment, a large amount of polypeptide is desired. In one embodiment, a vector is desired that directs high-level expression of a protein product, which may be fused to a hydrophobic signal sequence that introduces the expression product into the periplasm or culture medium of the bacteria for ease of purification of the protein product. In one embodiment, certain fusion proteins are engineered with specific cleavage sites to aid in the recovery of the polypeptide. In one embodiment, vectors suitable for such manipulations include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol., 185:60-89 (1990)].

[0401] In one embodiment, a yeast expression system is used. In one embodiment, a plurality of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Patent Application No. 5,932,447, the entire contents of which are incorporated herein by reference. In another embodiment, a vector that promotes integration of exogenous DNA sequences into yeast chromosomes is used.

[0402] In one embodiment, the expression vector of the present invention can also include additional polynucleotide sequences that allow, for example, translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for genomic integration of promoter-chimeric polypeptides.

[0403] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0404] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used in the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, the SV-40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or other promoters shown to be capable of efficient expression in eukaryotic cells.

[0405] In certain embodiments, recombinant viral vector can be used for the in vivo expression of coagulation factor of the present invention, and this is because it provides the advantage such as lateral infection and target specificity.In one embodiment, lateral infection is that for example, the cell that is intrinsic in the life cycle of retrovirus and is single infection produces the process of many buds and the daughter virus particle that infects adjacent cells.In one embodiment, the result is that large tracts of land is infected rapidly, and major part thereof is not infected by original virus particle at first.In one embodiment, produce the viral vector that can not lateral propagation.In one embodiment, if required purposes is to only introduce specific gene into the target cell of local quantity, this feature then may be useful.

[0406] In one embodiment, various methods can be used to introduce the expression vectors of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989, 1992); Ausubel et al., Modern Techniques in Molecular Biology, John Wiley & Sons, Baltimore, Maryland (1989); Chang et al., Somatic Cell Gene Therapy, CRC Press, Ann Arbor, Michigan (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor, Michigan (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworth, Boston, Massachusetts (1988); and Gilboa et al., [Biotechnology 4(6):504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, for positive and negative bidirectional selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992, which are incorporated herein by reference.

[0407] In some embodiments, introduction of nucleic acids by viral infection offers several advantages over other methods, such as lipofection and electroporation, because higher transfection efficiencies can be achieved due to the infectious nature of viruses.

[0408] In one embodiment, it will be understood that the engineered coagulation factors of the present invention can also be expressed from a nucleic acid construct administered to an individual using any suitable mode of administration as described above (i.e., in vivo gene therapy). In one embodiment, as needed, the nucleic acid construct is introduced into suitable cells via an appropriate gene delivery vector / method (transfection, transduction, homologous recombination, etc.) and expression system, and the modified cells are subsequently amplified in culture and returned to the individual (i.e., ex vivo gene therapy).

[0409] In one embodiment, a plant expression vector is used. In one embodiment, expression of the polypeptide coding sequence is driven by multiple promoters. In some embodiments, viral promoters are used, such as the 35S RNA promoter and the 19S RNA promoter of CaMV [Brisson et al., Nature, 310: 511-514 (1984)] or the coat protein promoter of TMV [Takamatsu et al., EMBO J. 6: 307-311 (1987)]. In another embodiment, a plant promoter is used, such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science, 224: 838-843 (1984)] or a heat shock promoter, for example, soybean hsp17.5-E or hsp17.3-B [Gurley et al., Acta Mol. Cell Biol., 6: 559-565 (1986)]. In one embodiment, the construct is introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and other techniques known to those skilled in the art. See, for example, Weissbach & Weissbach [Methods in Plant Molecular Biology, Academic Press, New York, Chapter VIII, pp. 421-463 (1988)]. The present invention can also use other expression systems known in the art, such as insect and mammalian host cell systems.

[0410] It will be appreciated that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (which encodes a polypeptide), the expression constructs of the invention can also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0411] In certain embodiments, under effective conditions, the transformed cells are cultivated, which allows the expression of a large amount of recombinant engineered coagulation factors. In certain embodiments, effective culture conditions include but are not limited to effective culture medium, bioreactor, temperature, pH and oxygen conditions that allow the production of protein. In one embodiment, effective culture medium refers to any culture medium in which cells are cultured to produce recombinant polypeptides of the present invention. In certain embodiments, culture medium generally includes an aqueous solution with assimilable carbon source, nitrogen source and phosphorus source, as well as suitable salts, minerals, metals and other nutrients, such as vitamins. In certain embodiments, cells of the present invention can be cultivated in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates and culture dishes. In certain embodiments, culture is carried out under temperature, pH and oxygen levels that are suitable for recombinant cells. In certain embodiments, culture conditions are determined within the scope of the professional knowledge of those of ordinary skill in the art.

[0412] In some embodiments, depending on the vector and host system used for production, the engineered coagulation factors of the present invention are either retained within the recombinant cell, secreted into the fermentation medium, secreted into the space between two cell membranes, such as the periplasmic space in E. coli; or retained on the outer surface of the cell or viral membrane.

[0413] In one embodiment, recovery of the recombinant engineered coagulation factor is performed after a predetermined period of incubation.

[0414] In one embodiment, the phrase "recovering the recombinant engineered coagulation factor" as used herein refers to collecting the whole fermentation medium containing the polypeptide and does not necessarily involve additional isolation or purification steps.

[0415] In one embodiment, the engineered coagulation factors of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubility.

[0416] In one embodiment, the expressed coding sequence can be engineered to encode the engineered coagulation factor of the present invention and the fused cleavable portion for ease of recovery. In one embodiment, the fusion protein can be designed so that the polypeptide can be easily separated by affinity chromatography; for example, by immobilization on a column dedicated to the cleavable portion. In one embodiment, the cleavage site is engineered between the engineered coagulation factor and the cleavable portion, and the polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or reagent that specifically cuts the fusion protein at that site [e.g., see Booth et al., Immunol. Lett., 19: 65-70 (1988); and Gardella et al., J. Biol. Chem., 265: 15854-15859 (1990)].

[0417] In one embodiment, the engineered coagulation factors of the present invention are recovered in a "substantially pure" form.

[0418] In one embodiment, the phrase "substantially pure" refers to a degree of purity that permits effective use of the protein in the applications described herein.

[0419] In one embodiment, the engineered coagulation factors of the present invention can also be synthesized using an in vitro expression system. In one embodiment, the in vitro synthesis method is well known in the art, and the components of the system are commercially available.

[0420] In some embodiments, recombinant engineered coagulation factors are synthesized and purified; their therapeutic efficacy can be determined in vivo or in vitro. In one embodiment, the binding activity of the recombinant engineered coagulation factors of the present invention can be determined using various assays known to those skilled in the art.

[0421] In another embodiment, the engineered coagulation factors of the present invention can be provided to the individual themselves. In one embodiment, the engineered coagulation factors of the present invention can be provided to the individual as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier.

[0422] In another embodiment, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0423] In another embodiment, "active ingredient" refers to a polypeptide sequence of interest that is responsible for a biological effect.

[0424] In another embodiment, any of the compositions of the present invention will include at least one CTP sequence that is bound in any form only to the carboxyl terminus of the engineered coagulation factor of interest. In one embodiment, the present invention provides a combined formulation. In one embodiment, a "combined formulation" specifically defines a "kit of parts," meaning that the combination complexes described above can be administered independently or by using different fixed combinations with different amounts of the combination complexes, i.e., simultaneously, concurrently, separately, or sequentially. In some embodiments, the various parts of the kit of parts can then be administered, for example, simultaneously or chronologically, i.e., at different time points and at equal or different time intervals for any part of the kit of parts. In some embodiments, the ratio of the total amount of the combination complexes can be managed in the combined formulation. In one embodiment, the combined formulation can be modified, for example, to address the needs of a subgroup of patients to be treated or the needs of a single patient who may have different needs due to a specific disease, disease severity, age, sex, or weight, as can be easily modified by one skilled in the art.

[0425] In another embodiment, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are included under these phrases. In one embodiment, one of the ingredients included in the pharmaceutically acceptable carrier can be, for example, polyethylene glycol (PEG), a biocompatible polymer with a broad range of solubility in organic and aqueous media (Mutter et al., (1979)).

[0426] In another embodiment, "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0427] Techniques for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0428] The present invention contemplates multiple embodiments of dosage ranges. In one embodiment, the dosage of the engineered coagulation factor of the present invention is in the range of 0.005-100 mg / day. In another embodiment, the dosage is in the range of 0.005-5 mg / day. In another embodiment, the dosage is in the range of 0.01-50 mg / day. In another embodiment, the dosage is in the range of 0.1-20 mg / day. In another embodiment, the dosage is in the range of 0.1-10 mg / day. In another embodiment, the dosage is in the range of 0.01-5 mg / day. In another embodiment, the dosage is in the range of 0.001-0.01 mg / day.

[0429] In another embodiment, the dosage is in the range of 0.001-0.1 mg / day. In another embodiment, the dosage is in the range of 0.1-5 mg / day. In another embodiment, the dosage is in the range of 0.5-50 mg / day. In another embodiment, the dosage is in the range of 0.2-15 mg / day. In another embodiment, the dosage is in the range of 0.8-65 mg / day. In another embodiment, the dosage is in the range of 1-50 mg / day. In another embodiment, the dosage is in the range of 5-10 mg / day. In another embodiment, the dosage is in the range of 8-15 mg / day. In another embodiment, the dosage is in the range of 10-20 mg / day. In another embodiment, the dosage is in the range of 20-40 mg / day. In another embodiment, the dosage is in the range of 60-120 mg / day. In another embodiment, the dosage is in the range of 12-40 mg / day. In another embodiment, the dosage is in the range of 40-60 mg / day. In another embodiment, the dosage is in the range of 50-100 mg / day. In another embodiment, the dosage is in the range of 1-60 mg / day. In another embodiment, the dosage is in the range of 15-25 mg / day. In another embodiment, the dosage is in the range of 5-10 mg / day. In another embodiment, the dosage is in the range of 55-65 mg / day.

[0430] In another embodiment, the dosage is in the range of 50-500 mg / day. In another embodiment, the dosage is in the range of 50-150 mg / day. In another embodiment, the dosage is in the range of 100-200 mg / day. In another embodiment, the dosage is in the range of 150-250 mg / day. In another embodiment, the dosage is in the range of 200-300 mg / day. In another embodiment, the dosage is in the range of 250-400 mg / day. In another embodiment, the dosage is in the range of 300-500 mg / day. In another embodiment, the dosage is in the range of 350-500 mg / day.

[0431] In one embodiment, the dosage is 20 mg / day. In one embodiment, the dosage is 30 mg / day. In one embodiment, the dosage is 40 mg / day. In one embodiment, the dosage is 50 mg / day. In one embodiment, the dosage is 0.01 mg / day. In another embodiment, the dosage is 0.1 mg / day. In another embodiment, the dosage is 1 mg / day. In another embodiment, the dosage is 0.530 mg / day. In another embodiment, the dosage is 0.05 mg / day. In another embodiment, the dosage is 50 mg / day. In another embodiment, the dosage is 10 mg / day. In another embodiment, the dosage is 20-70 mg / day. In another embodiment, the dosage is 5 mg / day.

[0432] In one embodiment, the dosage of the CTP-modified coagulation factor is 1-5 mg / day. In one embodiment, the dosage of the CTP-modified coagulation factor is 1-3 mg / day. In another embodiment, the dosage of the CTP-modified coagulation factor is 2 mg / day.

[0433] In another embodiment, the dosage is 1-90 mg / day. In another embodiment, the dosage is 1-90 mg / 2 days. In another embodiment, the dosage is 1-90 mg / 3 days. In another embodiment, the dosage is 1-90 mg / 4 days. In another embodiment, the dosage is 1-90 mg / 5 days. In another embodiment, the dosage is 1-90 mg / 6 days. In another embodiment, the dosage is 1-90 mg / week. In another embodiment, the dosage is 1-90 mg / 9 days. In another embodiment, the dosage is 1-90 mg / 11 days. In another embodiment, the dosage is 1-90 mg / 14 days.

[0434] In another embodiment, the dosage of the coagulation factor is 10-50 mg / day. In another embodiment, the dosage is 10-50 mg / 2 days. In another embodiment, the dosage is 10-50 mg / 3 days. In another embodiment, the dosage is 10-50 mg / 4 days. In another embodiment, the dosage is 10-50 mg / 5 days. In another embodiment, the dosage is 10-50 mg / 6 days. In another embodiment, the dosage is 10-50 mg / week. In another embodiment, the dosage is 10-50 mg / 9 days. In another embodiment, the dosage is 10-50 mg / 11 days. In another embodiment, the dosage is 10-50 mg / 14 days.

[0435] In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is formulated as an intranasal dosage form. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is formulated as an injectable dosage form. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 0.0001 mg to 0.6 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 0.001 mg to 0.005 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 0.01 mg to 0.3 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.2 mg to 0.6 mg. In another embodiment, the coagulation factor does not contain a CTP at its amino terminus.

[0436] In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 1-100 μg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 10-80 μg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 20-60 μg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 10-50 μg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 40-80 μg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 10-30 μg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 30-60 μg.

[0437] In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 0.2 mg to 2 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 2 mg to 6 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 4 mg to 10 mg. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose ranging from 5 mg to 15 mg.

[0438] In one embodiment, the coagulation factor modified by CTP is administered to a subject at a dose ranging from 10 μg / kg to 1000 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to a subject at a dose ranging from 25 μg / kg to 600 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to a subject at a dose ranging from 50 μg / kg to 400 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to a subject at a dose of approximately 25 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to a subject at a dose of approximately 50 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to a subject at a dose of approximately 100 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to a subject at a dose of approximately 200 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to the subject at a dose of about 300 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to the subject at a dose of about 400 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to the subject at a dose of about 500 μg / kg. In another embodiment, the coagulation factor modified by CTP is administered to the subject at a dose of about 600 μg / kg.

[0439] In one embodiment, the dosage of CTP-modified FIX includes the administration of recombinant FIX (e.g., Wyeth or CSL Behring) is administered to the patient at a dose of 50% of the amount of FIX. In one embodiment, the dosage of CTP-modified FVIIa includes the same time period as recombinant FVIIa (e.g., ). In one embodiment, the dose of CTP-modified FVII comprises 50% of the amount of FVII that would be administered to a patient at the recommended dose of recombinant FVII over the same time period. For example, if a patient is given 90 mcg / kg every 2 hours before or after surgery, (i.e., for an 85 kg patient, 7.65 mg every 2 hours or six doses over 12 hours totaling 45.9 mg), the CTP-modified coagulation factor of the present invention can be administered at a dose of 50% of the patient's 12-hour dose of recombinant FVIIa (i.e., a dose of 23 mg administered once every 12 hours).

[0440] In another embodiment, the dose of the CTP-modified coagulation factor is such that it contains 45% of the amount of coagulation factor administered using an unmodified coagulation factor. In another embodiment, the dose of the CTP-modified coagulation factor is such that it contains 10% of the amount of coagulation factor administered using an unmodified coagulation factor. In another embodiment, the dose of the CTP-modified coagulation factor is such that it contains 25% of the amount of coagulation factor administered using an unmodified coagulation factor. In another embodiment, the dose of the CTP-modified coagulation factor is such that it contains 35% of the amount of coagulation factor administered using an unmodified coagulation factor. In another embodiment, the dose of the CTP-modified coagulation factor is such that it contains 75% of the amount of coagulation factor administered using an unmodified coagulation factor. In another embodiment, the dose of the CTP-modified coagulation factor is such that it contains 100% of the amount of coagulation factor administered using an unmodified coagulation factor. However, even if the dose contains the same amount of coagulation factor (eg, FIX) as the non-CTP modified coagulation factor, it is still advantageous to the subject because less of it will need to be administered due to its extended half-life compared to recombinant coagulation factor.

[0441] In another embodiment, the therapeutically effective amount of the conjugated coagulation factor is 50-500 IU per kilogram of body weight, which is administered once a day to once a week for FIX or 10 μg / Kg-500 μg / Kg for FVIIa. In another embodiment, the therapeutically effective amount of the conjugated coagulation factor is 150-250 IU per kilogram of body weight, which is administered once a day. In another embodiment, the pharmaceutical composition comprising the conjugated coagulation factor is formulated at a strength that can be effectively administered to human patients by various routes.

[0442] In one embodiment, the administration of FIX is carried out for the experimenter with the amount that effectively makes the activity of circulating factor IX reach 20-30IU / dL. In another embodiment, the administration of FIX is carried out for the experimenter with the amount that effectively makes the activity of circulating factor IX reach 25-50IU / dL. In another embodiment, the administration of FIX is carried out for the experimenter with the amount that effectively makes the activity of circulating factor IX reach 50-100IU / dL. In another embodiment, the administration of FIX is carried out for the experimenter with the amount that effectively makes the activity of circulating factor IX reach 100-200IU / dL. In another embodiment, the administration of FIX is carried out for the experimenter with the amount that effectively makes the activity of circulating factor IX reach 10-50IU / dL. In another embodiment, the administration of FIX is carried out for the experimenter with the amount that effectively makes the activity of circulating factor IX reach 20-100IU / dL.

[0443] In one embodiment, the coagulation factor modified by CTP is administered to the subject once a week. In another embodiment, the coagulation factor modified by CTP is administered to the subject twice a week. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every two weeks. In another embodiment, the coagulation factor modified by CTP is administered to the subject twice a month. In another embodiment, the coagulation factor modified by CTP is administered to the subject once a month. In another embodiment, the coagulation factor modified by CTP is administered to the subject once a day. In another embodiment, the coagulation factor modified by CTP is administered to the subject once every two days.

[0444] In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every three days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every four days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every five days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every six days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every seven to fourteen days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every ten to twenty days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every five to fifteen days. In another embodiment, the polypeptide comprising a coagulation factor and at least one CTP unit is administered to the subject once every fifteen to thirty days.

[0445] In another embodiment, the method of the present disclosure includes increasing compliance in the use of coagulation factor therapy, which includes providing to a subject in need thereof a polypeptide comprising a coagulation factor and at least one chorionic gonadotropin carboxyl-terminal peptide (CTP) attached to the carboxyl terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy.

[0446] In another embodiment, the methods of the present disclosure include increasing compliance in patients with chronic diseases requiring coagulation factor therapy. In another embodiment, the methods of the present disclosure enable modification of coagulation factors with CTP as described above to reduce the frequency of coagulation factor dosing.

[0447] In another embodiment, the present invention provides a method for reducing the dosing frequency of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby reducing the dosing frequency of the FIX polypeptide. In another embodiment, the present invention provides a method for reducing the dosing frequency of a factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby reducing the dosing frequency of the FVIIa polypeptide.

[0448] In another embodiment, the term compliance includes compliance. In another embodiment, the method of the present disclosure includes increasing the compliance of patients requiring coagulation factor therapy by reducing the frequency of coagulation factor administration. In another embodiment, the reduction in the frequency of coagulation factor administration is achieved due to CTP modification that makes the CTP-modified coagulation factor more stable. In another embodiment, the reduction in the frequency of coagulation factor administration is achieved due to increasing the T1 / 2 of the coagulation factor. In another embodiment, the reduction in the frequency of coagulation factor administration is achieved due to increasing the clearance time or reducing the clearance rate of the coagulation factor.

[0449] In another embodiment, the present invention provides a method for reducing the clearance rate of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby reducing the clearance rate of the FIX polypeptide. In another embodiment, the present invention provides a method for reducing the clearance rate of a factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby reducing the clearance rate of the FVIIa polypeptide.

[0450] In another embodiment, a reduction in the dosing frequency of the coagulation factor is achieved due to an increased AUC measurement of the coagulation factor.

[0451] In another embodiment, provided herein is a method for reducing the dosing frequency of a coagulation factor, comprising the step of attaching one to ten CTPs to the carboxyl terminus of a coagulation factor, thereby reducing the dosing frequency of the coagulation factor. In another embodiment, provided herein is a method for reducing the dosing frequency of a coagulation factor, comprising the step of attaching one to five CTPs to the carboxyl terminus of a coagulation factor, thereby reducing the dosing frequency of the coagulation factor. In another embodiment, provided herein is a method for reducing the dosing frequency of a coagulation factor, comprising the step of attaching three CTPs to the carboxyl terminus of a coagulation factor, thereby reducing the dosing frequency of the coagulation factor. In another embodiment, provided herein is a method for reducing the dosing frequency of a coagulation factor, comprising the step of attaching three to five CTPs to the carboxyl terminus of a coagulation factor, thereby reducing the dosing frequency of the coagulation factor.

[0452] In another embodiment, provided herein is a method for increasing compliance in the use of coagulation factor therapy, comprising providing a subject in need thereof with a polypeptide comprising a coagulation factor and one to ten chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. In another embodiment, provided herein is a method for increasing compliance in the use of coagulation factor therapy, comprising providing a subject in need thereof with a polypeptide comprising a coagulation factor and one to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. In another embodiment, provided herein is a method for increasing compliance in the use of coagulation factor therapy, comprising providing a subject in need thereof with a polypeptide comprising a coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. In another embodiment, the present invention provides a method for increasing compliance in the use of coagulation factor therapy, which comprises providing to a subject in need thereof a polypeptide comprising a coagulation factor and three to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy.

[0453] In another embodiment, provided herein is a method for preventing or treating blood coagulation or a coagulation disorder for a subject, comprising providing to the subject a polypeptide comprising a coagulation factor and one to ten chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby treating blood coagulation or a coagulation disorder for the subject. In another embodiment, provided herein is a method for preventing or treating blood coagulation or a coagulation disorder for a subject, comprising providing to a subject in need a polypeptide comprising a coagulation factor and one to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby preventing or treating blood coagulation or a coagulation disorder for the subject. In another embodiment, provided herein is a method for preventing or treating blood coagulation or a coagulation disorder for a subject, comprising providing to a subject in need a polypeptide comprising a coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby preventing or treating blood coagulation or a coagulation disorder for the subject. In another embodiment, the present invention provides a method for preventing or treating blood coagulation or a coagulation disorder in a subject, comprising providing a subject in need thereof with a polypeptide comprising a coagulation factor and three to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, thereby preventing or treating blood coagulation or a coagulation disorder in the subject.

[0454] In another embodiment, the present invention provides a method for preventing hemophilia for a subject, comprising providing to the subject a polypeptide comprising a coagulation factor and one to ten chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby preventing hemophilia for the subject. In another embodiment, the present invention provides a method for preventing hemophilia for a subject, comprising providing to a subject in need a polypeptide comprising a coagulation factor and one to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby preventing hemophilia for the subject. In another embodiment, the present invention provides a method for preventing hemophilia for a subject, comprising providing to a subject in need a polypeptide comprising a coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby preventing hemophilia for the subject. In another embodiment, the present invention provides a method for preventing hemophilia in a subject, comprising providing a subject in need thereof with a polypeptide comprising a coagulation factor and three to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, thereby preventing hemophilia in the subject.

[0455] In another embodiment, the present invention is presented in that the compositions provided herein are surprisingly more effectively absorbed into the bloodstream after SC administration (see Examples 7 to 9 herein). Because it can be used for prophylactic applications, it is advantageous to be able to administer FVIIa subcutaneously. For patients, subcutaneous injection is also easier to self-inject, and is advantageous when the patient is very young and their vein is very small and difficult to find.

[0456] In another embodiment, provided herein is a method for treating hemophilia in a subject, comprising providing to the subject a polypeptide comprising a coagulation factor and one to ten chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby treating hemophilia in the subject. In another embodiment, provided herein is a method for treating hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a coagulation factor and one to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby treating hemophilia in the subject. In another embodiment, provided herein is a method for treating hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl terminus of the coagulation factor, thereby treating hemophilia in the subject. In another embodiment, provided herein is a method for treating hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a coagulation factor and three to five chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, thereby treating hemophilia in the subject.

[0457] In one embodiment, oral administration includes unit dosage forms, which include tablets, capsules, lozenges, chewable tablets, suspensions, emulsions, and the like. Such unit dosage forms include a safe and effective amount of the desired coagulation factor of the present disclosure, each of which is about 0.7 mg or 3.5 mg to about 280 mg / 70 kg in one embodiment, or about 0.5 mg or 10 mg to about 210 mg / 70 kg in another embodiment. Pharmaceutically acceptable carriers suitable for preparing unit dosage forms for oral administration are well known in the art. In some embodiments, tablets typically include conventional pharmaceutically compatible adjuvants such as, inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; binders such as starch, gelatin, and sucrose; disintegrants such as starch, alginic acid, and cross-linked carboxymethyl cellulose; lubricants such as magnesium stearate, stearic acid, and talc. In one embodiment, a glidant such as silicon dioxide can be used to improve the flow characteristics of the powder mixture. In one embodiment, a colorant such as FD&C dye can be added for appearance. For chewable tablets, sweeteners and flavorings, such as aspartame, saccharin, menthol, mint and fruit flavorings are useful adjuvants. Capsules typically include one or more solid diluents disclosed above. In some embodiments, the choice of carrier component depends on secondary considerations that are not critical for the purposes of the present invention, such as taste, cost and storage stability, and can be easily made by those skilled in the art.

[0458] In one embodiment, the oral dosage form comprises a predetermined release profile. In one embodiment, the oral dosage form of the present invention comprises a controlled release tablet, capsule, lozenge, or chewable tablet. In one embodiment, the oral dosage form of the present invention comprises a sustained release tablet, capsule, lozenge, or chewable tablet. In one embodiment, the oral dosage form of the present invention comprises an immediate release tablet, capsule, lozenge, or chewable tablet. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutical active ingredient known to those skilled in the art.

[0459] In some embodiments, oral compositions include liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically acceptable carriers suitable for the preparation of such compositions are well known in the art. In some embodiments, liquid oral compositions include from about 0.001% to about 0.933% of the desired compound, or in another embodiment, from about 0.01% to about 10% of the desired compound.

[0460] In some embodiments, the compositions used in the methods of the present invention include solutions or emulsions, which in some embodiments are aqueous solutions or emulsions, which include a safe and effective amount of a compound of the present invention and, optionally, other compounds for topical intranasal administration. In some embodiments, the compositions include about 0.001% to about 10.0% w / v of the subject compound, more preferably about 0.01% to about 2.0% w / v of the subject compound, for systemic delivery of the compound via the intranasal route.

[0461] In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is injected into a muscle (intramuscular injection). In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is injected under the skin (subcutaneous injection). In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is injected into a muscle. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is injected into the skin. In another embodiment, a coagulation factor as described herein is administered systemically. In another embodiment, a coagulation factor as described herein is administered by intravenous injection. In another embodiment, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, nasal, intraocular, ocular, epidural, oral, rectal, transmucosal, enteral, or parenteral delivery, including intramedullary injection and intrathecal or direct intracerebroventricular administration.

[0462] In another embodiment, the formulation is administered locally rather than systemically, for example, by injecting the formulation directly into a specific area of ​​the patient's body.

[0463] In one embodiment, the route of administration can be enteral. In another embodiment, the route can be conjunctival, transdermal, intradermal, intraarterial, vaginal, rectal, intratumoral, parenteral (parcanceral), transmucosal, intramuscular, intravascular, intraventricular, intracranial, intranasal, sublingual or a combination thereof.

[0464] In another embodiment, pharmaceutical compositions and pharmaceutical preparations are administered by intravenous, intraarterial or intramuscular injection of liquid preparations. In certain embodiments, liquid preparations include solutions, suspensions, dispersants, emulsions, oils, etc. In one embodiment, pharmaceutical compositions and pharmaceutical preparations are administered intravenously, and thus are prepared in a form suitable for intravenous administration. In another embodiment, pharmaceutical compositions and pharmaceutical preparations are administered intraarterially, and thus are prepared in a form suitable for intraarterial administration. In another embodiment, pharmaceutical compositions and pharmaceutical preparations are administered intramuscularly, and thus are prepared in a form suitable for intramuscular administration.

[0465] In addition, in another embodiment, the pharmaceutical compositions and pharmaceutical preparations are administered topically to the body surface and are thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops, etc. For topical administration, the compounds of the present invention are combined with additional appropriate therapeutic agents and prepared and applied as solutions, suspensions, or emulsions in physiologically acceptable diluents with or without a pharmaceutical carrier.

[0466] In one embodiment, the pharmaceutical compositions and pharmaceutical preparations of the present invention are manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, embedding or lyophilizing processes.

[0467] In one embodiment, the pharmaceutical compositions and pharmaceutical preparations for use according to the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and adjuvants that facilitate processing of the active ingredient into pharmaceutically acceptable preparations. In one embodiment, the formulation depends on the selected route of administration.

[0468] In one embodiment, the injection of the present disclosure is prepared in an aqueous solution. In one embodiment, the injection of the present disclosure is prepared in a physiologically compatible buffer, such as Hank's solution, Ringer's solution or physiological saline buffer. In certain embodiments, for transmucosal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art.

[0469] In one embodiment, the formulations described herein are formulated for parenteral administration, for example, by bolus injection or continuous infusion. In some embodiments, the formulations for injection are presented in unit dosage form, for example, in ampoules or multidose containers with optional added preservatives. In some embodiments, the composition is a suspension, solution, or emulsion in an oily or aqueous vehicle and contains formulating agents such as suspending agents, stabilizers, and / or dispersants.

[0470] In some embodiments, the composition further includes preservatives such as benzalkonium chloride and thimerosal; chelating agents such as sodium edetate; buffers such as phosphates, citrates, and acetates; tonicity agents such as sodium chloride, potassium chloride, glycerol, mannitol; antioxidants such as ascorbic acid, acetylcysteine, sodium metabisulfite; fragrances; viscosity modifiers such as polymers including cellulose and its derivatives; and polyvinyl alcohol and acids and bases to adjust the pH of these aqueous compositions as needed. In some embodiments, the composition further includes a local anesthetic or other active ingredient. The composition can be used as a spray, mist, drops, etc.

[0471] In certain embodiments, pharmaceutical compositions and pharmaceutical preparations for parenteral administration include aqueous solutions of active agents in water-soluble form. In addition, in certain embodiments, the suspensoid of active component is prepared into suitable oil base or water-based injection suspension. In certain embodiments, suitable lipophilic solvent or carrier include fatty oil, such as sesame oil or synthetic fatty acid ester, such as ethyl oleate, triglyceride or liposome. In certain embodiments, aqueous injection suspension contains the material that increases the viscosity of suspensoid, such as sodium carboxymethyl cellulose, sorbitol or dextran. In another embodiment, suspensoid also contains suitable stabilizer or increases the solubility reagent of active component to allow preparation of highly concentrated solution.

[0472] In another embodiment, the active compound can be delivered in vesicles, particularly liposomes (see, Langer, Science, 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Beresteint and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; JE Diederichs et al., Pharm. / nd. 56, (1994) 267-275).

[0473] In another embodiment, the pharmaceutical composition delivered by a controlled-release system is formulated for intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507 (1980); Saudek et al., New England Journal of Medicine, 321:574 (1989)). In another embodiment, a polymeric material can be used. In yet another embodiment, a controlled-release system can be placed near the therapeutic target, i.e., the brain, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Other controlled-release systems are discussed in the review by Langer (Science, 249:1527-1533 (1990)).

[0474] In some embodiments, the active ingredient is in powder form to be combined with a suitable carrier, e.g., a sterile, pyrogen-free aqueous solution, prior to use. In some embodiments, the composition is formulated for aerosolization and inhalation administration. In another embodiment, the composition is contained in a container with an attached aerosolizing tool.

[0475] In one embodiment, the preparations of the invention are formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0476] In some embodiments, pharmaceutical compositions and pharmaceutical formulations used in the context of the present invention include compositions comprising an amount of an active ingredient effective to achieve its intended purpose. In some embodiments, a therapeutically effective amount refers to an amount of the active ingredient effective to prevent, alleviate, or ameliorate symptoms of a disease or to prolong the survival of the subject being treated.

[0477] In one embodiment, determination of a therapeutically effective amount is well within the capabilities of those skilled in the art.

[0478] Some examples of substances that can be used as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween™ brand emulsifiers; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffered saline. The choice of a pharmaceutically acceptable carrier to be used in conjunction with a compound is essentially determined by the manner in which the compound is to be administered. If the subject compound is to be injected, in one embodiment the pharmaceutically acceptable carrier is sterile saline with a blood compatible suspending agent, the pH of which has been adjusted to about 7.4.

[0479] In addition, the composition may include a binder (e.g., gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), a disintegrant (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), a buffer with various pH and ionic strength (e.g., Tris-HCl, acetate, phosphate), an additive to prevent absorption to surfaces such as albumin or gelatin, a detergent (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), a protease inhibitor, a surfactant (e.g., sodium lauryl sulfate), a penetration enhancer, a solubilizer (e.g., glycerol, polyethylene glycol), an antioxidant (e.g., Ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), glidants (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and / or adjuvants.

[0480] Typical components of carriers for syrups, elixirs, emulsions, and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. For suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, cellulose (e.g., Avicel TM , RC-591), tragacanth and sodium alginate; Typical wetting agents include lecithin and polyethylene oxide dehydrated sorbitan (e.g., polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. In another embodiment, the oral liquid composition further comprises one or more components, such as the sweeteners, flavorings, and coloring agents disclosed above.

[0481] The compositions also include incorporating the active material into or onto a polymeric compound, such as polylactic acid, polyglycolic acid, hydrogel, or granular formulation, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0482] The present disclosure also encompasses particulate compositions coated with a polymer (eg, poloxamer or poloxamine) and a compound coupled to an antibody directed against a tissue-specific receptor, ligand, or antigen, or coupled to a ligand of a tissue-specific receptor.

[0483] In some embodiments, the compound is modified by covalent attachment of a water-soluble polymer, such as polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, or polyproline. In another embodiment, the modified compound exhibits a substantially longer half-life in the blood following intravenous injection compared to the corresponding unmodified compound. In one embodiment, the modification also increases the solubility of the compound in aqueous solution, eliminates aggregation, enhances the physical and chemical stability of the compound, and substantially reduces the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo biological activity is achieved by administering the polymer-compound adduct less frequently or at a lower dose than the unmodified compound.

[0484] In some embodiments, the preparation of effective amounts or doses can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models, and this information can be used to more accurately determine useful doses in humans.

[0485] In one embodiment, the toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro and in cell culture or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for use in humans. In one embodiment, the dosage varies depending on the dosage form employed and the route of administration used. In one embodiment, the exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition. [See, for example, Fingl et al. (1975) "Pharmacological Basis of Therapeutics," Chapter 1, page 1].

[0486] In one embodiment, administration can be single or multiple administrations, depending on the severity and responsiveness of the condition being treated, with the course of treatment lasting from several days to several weeks or until a cure is effected or a diminution of the disease state is achieved.

[0487] In one embodiment, the amount of composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0488] In one embodiment, compositions comprising a formulation of the invention formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0489] In another embodiment, the coagulation factor as described herein is a lyophilized (i.e., freeze-dried) formulation in combination with a complex organic excipient and stabilizer, such as a nonionic surfactant (i.e., surfactant), various sugars, organic polyols, and / or human serum albumin. In another embodiment, the pharmaceutical composition comprises the lyophilized coagulation factor in sterile water for injection. In another embodiment, the pharmaceutical composition comprises the lyophilized coagulation factor in sterile PBS for injection. In another embodiment, the pharmaceutical composition comprises the lyophilized coagulation factor in sterile 0.9% NaCl for injection.

[0490] In another embodiment, the pharmaceutical composition includes coagulation factor as described herein and a composite carrier, such as human serum albumin, polyols, sugar and anionic surfactant stabilizers. In another embodiment, the pharmaceutical composition includes coagulation factor as described herein and lactobionic acid and acetate / glycine buffer. In another embodiment, the pharmaceutical composition includes coagulation factor as described herein and an amino acid that increases the solubility of interferon compositions in water, such as arginine or glutamate. In another embodiment, the pharmaceutical composition includes lyophilized coagulation factor as described herein and glycine or human serum albumin (HSA), buffer (e.g., acetate) and isotonic agent (e.g., NaCl). In another embodiment, the pharmaceutical composition includes lyophilized coagulation factor as described herein and phosphate buffer, glycine and HSA.

[0491] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein is stabilized when placed in a buffer solution with a pH of about 4 to 7.2. In another embodiment, the pharmaceutical composition comprising a coagulation factor is in a buffer solution with a pH of about 4 to 8.5. In another embodiment, the pharmaceutical composition comprising a coagulation factor is in a buffer solution with a pH of about 6 to 7. In another embodiment, the pharmaceutical composition comprising a coagulation factor is in a buffer solution with a pH of about 6.5. In another embodiment, the pharmaceutical composition comprising a coagulation factor is in a buffer solution with a pH of about 6.4. In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein is stabilized by an amino acid as a stabilizer and in some cases by a salt (if the amino acid does not contain a charged side chain).

[0492] In another embodiment, the pharmaceutical composition comprising a coagulation factor described herein is a liquid composition comprising about 0.3% to 5% by weight of a stabilizer that is an amino acid.

[0493] In another embodiment, the pharmaceutical composition comprising the coagulation factors described herein provides dosing accuracy and product safety. In another embodiment, the pharmaceutical composition comprising the coagulation factors described herein provides a biologically active and stable liquid formulation for injectable applications. In another embodiment, the pharmaceutical composition comprises a non-lyophilized coagulation factor described herein.

[0494] In another embodiment, pharmaceutical compositions comprising the coagulation factors described herein provide liquid formulations that allow for prolonged storage in a liquid state that facilitates storage and transportation prior to administration.

[0495] In another embodiment, the pharmaceutical composition comprising coagulation factor as herein described comprises a solid lipid as a matrix material. In another embodiment, the injectable pharmaceutical composition comprising coagulation factor as herein described comprises a solid lipid as a matrix material. In another embodiment, Speiser describes producing lipid microparticles (Speiser et al., Chemistry Research, 8 (1991) 47-54) by spray condensation, followed by oral administration (Speiser, EP0167825 (1990)) by lipid nanoparticles. In another embodiment, the lipid used is well tolerated by the body (for example, a glyceride consisting of fatty acids present in an emulsion for parenteral nutrition).

[0496] In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises polymer microparticles. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises nanoparticles. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises liposomes. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises a lipid emulsion. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises microspheres. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises lipid nanoparticles. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises lipid nanoparticles comprising amphiphilic lipids. In another embodiment, a pharmaceutical composition comprising a coagulation factor as described herein comprises lipid nanoparticles comprising a drug, a lipid matrix, and a surfactant. In another embodiment, the lipid matrix has a monoglyceride content of at least 50% w / w.

[0497] In one embodiment, the compositions of the present invention are presented in a package or dispensing device, such as a kit approved by the FDA, which contains one or more unit dosage forms comprising an active ingredient. In one embodiment, the packaging, for example, comprises metal or plastic foil, such as a blister pack. In one embodiment, the packaging or dispensing device is accompanied by instructions for administration. In one embodiment, the packaging or dispenser is provided with precautions associated with the container that are prescribed in the form by a government agency that manages the manufacture, use or sale of the drug, and the precautions reflect the approval of the agency to the form of the composition or the approval of a human or veterinary administration department. Such precautions are labeled in one embodiment on a product insert approved for prescription drugs or approved by the U.S. Food and Drug Administration.

[0498] In one embodiment, it will be appreciated that the coagulation factors of the present invention can be provided to an individual together with additional active agents to achieve an improved therapeutic effect compared to the treatment performed with each agent alone. In another embodiment, measures are taken (e.g., administration and selection of supplements) to avoid the adverse side effects associated with the combination therapy.

[0499] In another embodiment, the present invention provides a CTP-modified Factor VIIa (FVIIa) polypeptide consisting of a FVIIa polypeptide and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa.

[0500] In another embodiment, the present invention provides a pharmaceutical composition comprising a CTP-modified Factor VIIa (FVIIa) polypeptide, which consists of a FVIIa polypeptide and five gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa.

[0501] In another embodiment, the present invention provides a polynucleotide encoding a Factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the VIIa polypeptide and modified with a CTP.

[0502] In another embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a Factor VIIa (FVIIa) polypeptide and three gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide.

[0503] In another embodiment, the present invention provides a cell comprising an expression vector comprising a polynucleotide encoding a Factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide and a CTP-modified polypeptide.

[0504] In another embodiment, the present invention provides a composition comprising an expression vector comprising a polynucleotide encoding a Factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide and a CTP-modified polypeptide.

[0505] In another embodiment, the present invention provides a method for extending the biological half-life of a Factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby extending the biological half-life of the FVIIa polypeptide.

[0506] In another embodiment, the present invention provides a method for improving the area under the curve (AUC) of a Factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby improving the AUC of the FVIIa polypeptide.

[0507] In another embodiment, the present invention provides a method for reducing the dosing frequency of a Factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby reducing the dosing frequency of the FVIIa polypeptide.

[0508] In another embodiment, the present invention provides a method for reducing the clearance rate of a Factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby reducing the clearance rate of the FVIIa polypeptide.

[0509] In another embodiment, the present invention provides a method for producing a CTP-modified Factor VIIa (FVIIa) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FVIIa polypeptide, thereby producing a CTP-modified FVIIa polypeptide.

[0510] In another embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a CTP-modified Factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three chorionic gonadotropin carboxyl-terminal peptides (CTPs) attached to the carboxyl terminus of the FVIIa polypeptide, thereby treating hemophilia in the subject.

[0511] In one embodiment, the present invention provides a CTP-modified Factor IX (FIX) polypeptide consisting of a FIX polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the CTP-modified FIX polypeptide. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, wherein the sequence of the CTP-modified FIX polypeptide is the sequence set forth in SEQ ID NO: 31. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, wherein at least one CTP is truncated. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, wherein the linker is a peptide bond.

[0512] In one embodiment, the present invention provides a pharmaceutical composition comprising a CTP-modified FIX polypeptide.

[0513] In one embodiment, the present invention provides a polynucleotide encoding a factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, modified with a CTP. In another embodiment, the present invention provides a polynucleotide, wherein the sequence of the polynucleotide is set forth in SEQ ID NO: 30. In another embodiment, the present invention provides a polynucleotide, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a polynucleotide, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a polynucleotide, wherein at least one CTP is truncated. In another embodiment, the present invention provides a polynucleotide, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a polynucleotide, wherein the linker is a peptide bond. An expression vector comprises the polynucleotide.

[0514] In one embodiment, the present invention provides a cell comprising an expression vector.

[0515] In one embodiment, the present invention provides a composition comprising an expression vector.

[0516] In one embodiment, the present invention provides a method for extending the biological half-life of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby extending the biological half-life of the FIX polypeptide. In another embodiment, the present invention provides a method, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a method, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a method, wherein at least one CTP is truncated. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a method, wherein the linker is a peptide bond.

[0517] In one embodiment, the present invention provides a method for improving the area under the curve (AUC) of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby improving the AUC of the FIX polypeptide. In another embodiment, the present invention provides a method, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a method, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a method, wherein at least one CTP is truncated. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a method, wherein the linker is a peptide bond.

[0518] In one embodiment, the present invention provides a method for reducing the frequency of administration of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby reducing the frequency of administration of the FIX polypeptide. In another embodiment, the present invention provides a method, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a method, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a method, wherein at least one CTP is truncated. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a method, wherein the linker is a peptide bond.

[0519] In one embodiment, the present invention provides a method for reducing the clearance rate of a factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby reducing the clearance rate of the FIX polypeptide. In another embodiment, the present invention provides a method, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a method, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a method, wherein at least one CTP is truncated. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FVII polypeptide via a linker. In another embodiment, the present invention provides a method, wherein the linker is a peptide bond.

[0520] In one embodiment, the present invention provides a method for producing a CTP-modified Factor IX (FIX) polypeptide, comprising the step of attaching three chorionic gonadotropin carboxyl-terminal peptides (CTPs) to the carboxyl terminus of the FIX polypeptide, thereby producing a CTP-modified FIX polypeptide. In another embodiment, the present invention provides a method, wherein the sequence of the CTP-modified FIX polypeptide is the sequence set forth in SEQ ID NO: 31. In another embodiment, the present invention provides a method, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a method, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a method, wherein at least one CTP is truncated. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a method, wherein the linker is a peptide bond.

[0521] In one embodiment, the present invention provides a method for treating hemophilia in a subject, comprising administering to the subject a CTP-modified Factor IX (FIX) polypeptide comprising a FIX polypeptide and three chorionic gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide, thereby treating hemophilia in the subject. In another embodiment, the present invention provides a method, wherein the sequence of the CTP-modified FIX polypeptide is the sequence set forth in SEQ ID NO: 31. In another embodiment, the present invention provides a method, wherein at least one CTP is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the present invention provides a method, wherein at least one CTP is glycosylated. In another embodiment, the present invention provides a method, wherein at least one CTP is truncated. In another embodiment, the present invention provides a method, wherein at least one CTP is attached to the FIX polypeptide via a linker. In another embodiment, the present invention provides a method, wherein the linker is a peptide bond.

[0522] As is generally known in the art, the modified peptides and proteins of the present disclosure can be coupled to labels, drugs, targeting agents, carriers, solid supports, and the like, depending on the desired application. Labeled forms of modified biological products can be used to track their metabolic fate; in particular, suitable labels for this purpose include radioisotope labels, such as iodine-131, technetium-99, indium-111, and the like. Labels can also be used to mediate the detection of modified proteins or peptides in assay systems; in this case, radioisotopes as well as enzyme labels, fluorescent labels, chromogenic labels, and the like can also be used. The use of such labels is particularly useful if the peptide or protein itself is a targeting agent, such as an antibody or receptor ligand.

[0523] Similar linking techniques can be employed to couple the modified peptides and proteins of the present disclosure to solid supports. Once coupled, these modified peptides and proteins can then be used as affinity reagents to isolate desired components exhibiting a specific reaction.

[0524] Finally, the modified peptides and proteins of the present disclosure can be used to produce antibodies with specific immunoreactivity with these new compounds. These antibodies can be used in multiple diagnostic and therapeutic applications, depending on the properties of the biological activity of the unmodified peptide or protein. It should be understood that the present disclosure provides antibodies with immunoreactivity with FIX, FVII or FVIIa modified through CTP as described herein. In one embodiment, this antibody can be used to distinguish or identify a coagulation factor modified through CTP and an endogenous coagulation factor administered. In another embodiment, an antibody can be used to locate a coagulation factor modified through CTP administered.

[0525] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon review of the following examples, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the present invention as delineated above and as claimed in the claims section finds experimental support in the following examples.

[0526] Examples

[0527] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Modern Molecular Biology Techniques," Vols. I-III, Ausubel, RM, ed., (1994); Ausubel et al., "Modern Molecular Biology Techniques," John Wiley & Sons, Baltimore, MD (1989); Perbal, "Molecular Cloning: A Practical Guide," John Wiley & Sons, Baltimore, MD (1989); and Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Book Review, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual System", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methods as described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Manual", Vols. I-III, Cellis, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Techniques", Freshney, Wiley-Liss, New York (1994), 3rd ed.; "Modern Immunological Techniques", Vols. I-III, Coligan JE, ed. (1994); Stites et al. (eds.), Basic and Clinical Immunology (8th ed.), Appleton & Lange, New York, CT (1994); Mishell and Shiigi (eds.), Selected Methods in Cellular Immunology, WH Freeman and Co., New York (1980); available immunoassays are described extensively in the patent and scientific literature, see, for example, U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; "Oligonucleotide Synthesis," Gait, MJ, ed. (1984); "Nucleic Acid Hybridization," Hames, BD, and Higgins, ed. SJ, eds. (1985); Transcription and Translation, Hames, BD, and Higgins SJ, eds. (1984); Animal Cell Culture, Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes," IRL Press, (1986); "Molecular Cloning: A Practical Guide," Perbal, B., (1984) and "Methods in Enzymology," Vols. 1-317, Academic Press; "PCR Protocols: A Guide to Methods and Applications," Academic Press, San Diego, CA (1990); Marshak et al., "Protein Purification and Characterization Strategies: A Guide to Experimental Disciplines," CSHL Press, (1996); all of which are incorporated herein by reference. Other general references are provided throughout this document.

[0528] Example 1

[0529] Production and use of coagulation factor IX

[0530] Cloning and expression of recombinant FIX molecules:

[0531] Factor IX clones were constructed into our eukaryotic expression vector pCI-neo (Promega, catalog number E1841). Homo sapiens coagulation factor IX ORF clones were ordered from "OriGene" (RC219065). Primers were ordered from Sigma-Genosys.

[0532] Construction of 301-1-pCI-neo-p200-11 (Factor IX-ctp x2):

[0533] Primer 101: 5'GTTTAGTGAACCGTCAGAAT 3' (SEQ ID NO: 36)

[0534] Primer 103 R : 5'TTGAGGAAGATGTTCGTGTA 3' (containing the SspI site of Factor IX) (SEQ ID NO: 37)

[0535] Primer 101 and primer 103 R PCR reactions were performed using plasmid DNA and cDNA clones of Factor IX (OriGene" RC219065) as templates; as a result of PCR amplification, a product of ∼1085 bp (PCR 10) was formed and purified from the gel (a fragment containing the amino terminus of the Factor IX sequence).

[0536] Primer 98: 5'ATTACAGTTGTCGCAGGTGA 3' (SEQ ID NO: 38)

[0537] Primer 99 R :5'GCTGGAGCTAGTGAGCTTTGTTTTTTCCTT 3'(SEQ ID NO:39)

[0538] Primer 100: 5'GCTCACTAGCTCCAGCAGCAAGGCC 3' (SEQ ID NO: 40)

[0539] Primer 27 R :5'TTTTCACTGCATTCTAGTTGTGG 3'(SEQ ID NO:41)

[0540] Three PCR reactions were performed using primers 98 and 99. R The first reaction was performed using plasmid DNA and cDNA clone of Factor IX (OriGene" RC219065) as templates; as a result of PCR amplification, a product of ∼540 bp was formed.

[0541] Primer 100 and primer 27 R A second reaction was performed using plasmid DNA of 402-2-p72-3 (hGH-CTP-CTP) as a template; as a result of PCR amplification, a product of ∼258 bp was formed.

[0542] Primers 98 and 27 R A final reaction (pcr3) was performed with a mixture of the products of the first two reactions and PCR amplification of 790 bp, which was ligated into a TA cloning vector (Invitrogen, catalog K2000-01). The SspI-EcoRI fragment was isolated (TA 3-3).

[0543] Primers 101 and 27 R Another PCR reaction was performed with a mixture of the products of pcr 10 and the SspI-EcoRI fragment from pcr 3 as template; as a result of PCR amplification, a ∼1700 bp product (Factor IX-ctp-ctp) was formed and ligated into a TA cloning vector (Invitrogen, catalog K2000-01) (ligation 180).

[0544] An error was discovered in the Factor IX sequence, so the fragment was replaced to form an insert of Factor IX-ctp-ctp with the correct DNA sequence.

[0545] Digest TA-pcr 3-3 with SspI and XbaI and separate the large fragment (vector). Digest TA 180-4 with SspI and XbaI and separate the small fragment (insert) and connect it to the large fragment of the separation of TA-pcr-3-3 digested with SspI and XbaI. Digest new plasmid TA-183-2 with Sal I and NotI, and separate Factor IX-CTP-CTP inset (~1575bp). This fragment is inserted into the eukaryotic expression vector pCI-neo (digested with Sal I and Not I) to obtain 301-2-p200-11 clone.

[0546] Construction of pCI-dhfr-Factor IX-ctpx2 (p223-4): Digest the vector pCI-dhfr (p6-1) with SmaI and NotI. Digest Factor IX-CTP-CTP (p200-11) with ASisI FI and NotI. Ligate the two fragments.

[0547] Construction of pCI-dhfr Factor IX-CTP x3 (p225-7): Digest the vector pCI-dhfr OXM-CTP x3 (p216-4) with XbaI and ApaI. Digest Factor IX-CTP-CTP (223-4) with XbaI and ApaI. Ligate the two fragments.

[0548] Construction of pCI-dhfr factor 9-ctp x3 T148A (p243-2): Plasmid p225-7 contains a threonine at position 148, and since the more common version of FIX contains an alanine at this position, site-directed mutagenesis was used to replace Ala with Thr.

[0549] Primer 75: ctcccagttcaattacagct (SEQ ID NO: 42)

[0550] Primer 122r: ggaaaaactgcctcagcacgggtgagc (SEQ ID NO: 43)

[0551] Primer 123: gtgctgaggcagtttttcctgatgtggactat (SEQ ID NO: 44)

[0552] Primer 124r: caacacagtgggcagcag (SEQ ID NO: 45)

[0553] Three PCR reactions were performed. The first reaction was performed with primer 75 and primer 122r and plasmid DNA p225-7 as a template; as a result of PCR amplification, a ~692 bp product was formed and purified from the gel. The second PCR reaction was performed with primer 123 and primer 124r and plasmid DNA p225-7 as a template; as a result of PCR amplification, a ~237 bp product was formed and purified from the gel. A third overlapping PCR reaction was performed with primer 75 and 124r and a mixture of the products of the first two reactions as a template; as a result of PCR amplification, a ~910 bp product was formed. This overlapping PCR product was digested with XbaI and NsiI and religated into the p225-7 plasmid (digested with XbaI and NsiI) to generate Factor IX-ctpx3T148A, designated p243-2.

[0554] Construction of FIX-4CTP (p259-4): A 3.5CTP fragment was isolated from oxym-4CTP (p254-3) using restriction enzymes Apa1 and Xba1. A FIX + 0.5CTP fragment was isolated from FIX-3CTP (p243-2) using restriction enzymes Apa1 and Xba1. The two fragments were ligated.

[0555] Construction of FIX-5CTP (p260-18): A 4.5CTP fragment was isolated from oxym-5CTP (255-1) using restriction enzymes Apa1 and Xba1. A FIX + 0.5CTP fragment was isolated from FIX-3CTP (p243-2) using enzymes Apa1 and Xba1. The two fragments were ligated.

[0556] Dg44 cells are layered in 100mm tissue culture dishes and grown to a confluence of 50-60%. Use 2 μ g (microgram) of FIX cDNA to be transfected in protein-free culture medium (Invitrogene CD Dg44) using FuGene reagent (Roche) to a 100mm plate. This culture medium is removed after transfection 48 hours and replaced with the protein-free culture medium (Invitrogene CD Dg44) without nucleosides in the presence of 800 μ g / ml of G418 (neomycin). After 14 days, the transfected cell colony is transferred to a T25 tissue culture flask, and selection is continued for another 10-14 days until cells begin to grow as stable clones. Select the clones of high expression. Use about 2x10 7 Cells were grown at 1700 cm in a 5-well plate supplemented with 5 ng / ml vitamin K3 (menadione sodium bisulfate; Sigma). 2300 ml of growth medium was inoculated into roller bottles (Corning, Corning NY). Production medium (harvest) was collected after cell viability rapidly decreased to about 70%. The production medium was first clarified and then concentrated approximately 20-fold and dialyzed against PBS using a flow filtration cassette (10 kDa MWCO; Millipore Corp.).

[0557] Determination of FIX antigen levels: Antigen levels of FIX-CTP harvests were determined using the AssayMax Human FIX ELISA Kit (AssayPro-EF1009-1). The calculated protein concentration was the average of three different dilutions from two independent runs ( Figure 1A , Table 1).

[0558] Table 1: Calculated protein concentrations

[0559]

[0560] FIX SDS-PAGE-immunoblotting: FIX-CTP harvests or purified rhFIX (American Diagnostics) and 100 ng of protein were loaded onto 12% Tris-glycine gels using Precision plus dual-color protein markers (Bio-Rad). SDS-PAGE analysis was performed by protein immunoblotting using anti-human FIX polyclonal antibodies and anti-human gamma carboxylation monoclonal antibodies (American Diagnostics). As previously reported, rhFIX migrates at 55 kDa, while FIX fused to two CTPs migrates at 75 kDa. Both variants of the FIX-CTP protein were shown to be gamma carboxylated, an essential post-translational modification for FIX activity and function ( Figure 1B ).

[0561] Determination of FIX chromogenic activity: Using a commercially available chromogenic activity detection kit, BIOPHEN (HyphenBioMed 221802) performs a comparative assessment of the in vitro efficacy of FIX-CTP harvests and rhFIX protein (American Diagnostics). In the presence of thrombin, phospholipids, and calcium, excess FXIa activates the sampled FIX into FIXa. FIXa forms an enzyme complex with thrombin, activated FVIII:C (supplied in excess), phospholipids, and calcium, and activates the factor X present in the assay system into FXa. Activity is directly associated with the amount of FIX as a limiting factor. The generated FXa is then measured by its specific activity on a FXa chromogenic substrate (pNA). The amount of pNA produced is directly proportional to the FIXa activity. Serial dilutions of rhFIX and FIX-CTP harvests are performed and efficacy is assessed by comparing the dose-response curve of the FIX harvest with a benchmark configuration consisting of rhFIX or human plasma. The average EC50 for FIX was 21 ng / ml, while the calculated EC50 for FIX-(CTP)2 harvest was 382 ng / ml and the calculated EC50 for FIX-CTP harvest was 1644 ng / ml. An approximately 15-fold decrease in the enzyme activity of the FIX-(CTP)2 harvest was observed ( Figure 2 ).

[0562] FIX Clotting Activity (aPTT): The activated partial thromboplastin time (aPTT) is a measure of the integrity of the intrinsic and common pathways of the coagulation cascade. The aPTT is the time in seconds for plasma to clot after the addition of an intrinsic pathway activator, phospholipids, and calcium. The aPTT reagent is called a partial thromboplastin because, like the prothrombin time (PT) reagent, tissue factor does not include phospholipids. The activator primes the system, and the remaining steps of the intrinsic pathway then proceed in the presence of phospholipids. The range of baseline aPTT varies by laboratory, but is generally in the range of 27-34 seconds.

[0563] The principle of this assay is to quantify the ability of the coagulation activity of the human plasma of FIX removed by adding rhFIX to the recovery of the FIX-CTP harvest. The human plasma lacking FIX of 300 μ l is mixed with the rhFIX or FIX-CTP harvest of 100 μ l and serially diluted. After incubation for 60 seconds at 37 ℃, thromboplastin, CaCl 2 and phospholipid are added to the mixture, and the coagulation time (performed by the American Medical Laboratory) in seconds is determined. Effectiveness is assessed by comparing the dose-response curve of the FIX harvest with the benchmark configuration consisting of rhFIX or human plasma. The FIX activity of one unit corresponds to the active FIX concentration equal to 1 ml of normal human plasma. The aPTT result shown shows that, compared with rhFIX, FIX-(CTP) 2 shows that its specific coagulation activity has reduced by 5.7 times (table 2). In addition, the aPTT result shows that the FIX-(CTP) 2 harvest has the improved enzymatic activity (table 2) relative to the FIX-CTP harvest together with the chromogenic activity of in vitro assay. After optimization of the enhanced expression system following supertransfection with furin (ie, co-transfection with furin and optimization of vitamin K3 medium concentration), improved activity of the FIX-CTP protein could be obtained (data not shown).

[0564] Table 2: FIX coagulation activity

[0565]

[0566] Pharmacokinetic Studies: rhFIX (American Diagnostic) and FIX-CTP harvest were administered as a single intravenous injection to Sprague-Dawley rats (six rats for each substance) at a dose of 75 μg / kg body weight (Table 3).

[0567] Table 3: Operational PK Study Plan

[0568]

[0569]

[0570] Blood samples were drawn from three rats at 0.083 hours, 0.5 hours, 1.5 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after administration. Plasma was immediately prepared and stored at -20°C until analysis. FIX concentrations were quantified by a FIX ELISA specific assay (AssayPro). Pharmacokinetic curves were calculated for each protein, representing the mean of three animals at each time point ( Figure 3The terminal half-life was calculated using PKSolutions 2.0 software. Table 4 summarizes the FIX concentrations observed at different sampling time points.

[0571] Table 4: Observed FIX concentrations

[0572]

[0573] A summary of the PK profiles and terminal half-life is summarized in Table 5. FIX-CTP harvest exhibited improved T1 / 2 compared to rhFIX β Since the 24-hour serum concentrations of FIX in the FIX dosing collection were below the limit of quantification (BLQ), no additional PK parameters were calculated.

[0574] Table 5: Summary of PK parameters

[0575]

[0576] In this study, a novel method for extending the FIX half-life and maintaining therapeutic efficacy was described. Adding the CTP peptide to the active protein has the harmful possibility of interfering with protein activity. Therefore, it is unexpected to produce active recombinant FIX-CTP by adding the CTP sequence at the C-terminus of FIX.

[0577] Characterization of immunoaffinity purified FIX-CTP-CTP

[0578] FIX-CTP-CTP purification

[0579] In order to assess its PK curve, simulate and can be extrapolated to the protein of the active high content with improvement of clinical environment, FIX-CTP-CTP is the FIX modified with 2 CTP units in series at its carboxyl terminus.Use the monoclonal antibody of the matrix binding for the gamma carboxyl glutamyl (Gla) residue present in the N-terminal region of FIX (American Diagnostics, catalog #3570MX) to purify FIX-CTP-CTP.Monoclonal antibody is bound to sepharose CL-4B.The FIX-CTP-CTP harvest that is 88 μ g / ml is dialyzed with 20mM Tris, 150mM NaCl and 10mM EDTA at pH=7.4 by concentration.Loading rate is 0.5ml / minute, uses 20mM Tris-HCl, 350mM NaCl and 50mM CaCl to perform elution and unbound part is reclaimed five times.Finally, the eluted portion is dialyzed, pulled and concentrated with PBS.

[0580] Determination of FIX antigen levels: FIX-CTP harvest, FIX-(CTP)2 harvest, and FIX-(CTP)2 purified protein levels were determined using a human FIX ELISA kit (Affinity Biologicals; catalog #FIX-AG RUO). The calculated protein concentration (μg / ml) is the average of two independent runs ( Figure 4 , Table 6).

[0581] Table 6: Calculated protein concentration

[0582]

[0583] In addition, FIX-CTP-CTP was quantified by Bradford assay. The calculated concentration was 202 μg / ml, which is similar to the concentration obtained by human FIX ELISA.

[0584] SDS-PAGE blotting: The FIX-CTP-CTP harvest, unbound fraction, and purified protein were loaded onto a 12% Tris-glycine gel using Precision plus dual color protein marker (Bio-Rad). SDS-PAGE Coomassie analysis was performed by staining the gel with Coomassie blue reagent (800 ng of protein). Western blotting was performed using 100 ng of protein, anti-human FIX polyclonal antibody (Ab), and anti-human gamma carboxylate monoclonal Ab (American Diagnostics, catalog #499 and #3570). The immunoaffinity purification process significantly enriched the FIX-CTP-CTP fraction while reducing impurities ( Figures 5A to 5C ).

[0585] N-terminal sequencing: The FIX-CTP-CTP purified protein was separated by 12% Tris-glycine SDS-PAGE and subsequently electroblotted onto a PVDF membrane. The band of interest was excised and placed on a purified Biobrene-treated glass fiber filter. N-terminal sequence analysis was performed by Edmann degradation using a pulsed liquid protein sequencer equipped with a 140C HPLC microgradient system. N-terminal sequencing showed that FIX-CTP-CTP was a mixture of incomplete and complete propeptide cleavage proteins. This showed that insufficient propeptide cleavage reduced the coagulant activity of FIX. The propeptide cleavage process could be improved by co-transfection with furin.

[0586] Determination of FIX chromogenic activity: Using a commercially available chromogenic activity detection kit, BIOPHEN (HyphenBioMed 221802) performs a comparative assessment of the in vitro effectiveness of FIX-CTP-CTP purified protein and rhFIX (American Diagnostics) and a pool of normal human plasma. In the presence of thrombin, phospholipids, and calcium, excess FXIa activates FIX to FIXa. FIXa forms an enzyme complex with thrombin (in excess), phospholipids, and calcium, and activates the factor X present in the assay system to FXa. Activity is directly associated with the amount of FIX as a limiting factor. The FXa produced is measured by its specific activity on FXa chromogenic substrate (pNA). The amount of pNA produced is directly proportional to FIXa activity. rhFIX, human plasma, and FIX-CTP-CTP are serially diluted, and effectiveness is assessed by comparing dose-response curves ( Figure 6 The average EC of rhFIX 50 was 68.74 ng / ml, while the calculated EC 50 The enzyme activity of FIX-CTP-CTP was observed to be approximately 7-fold lower than that of recombinant FIX and 16.5-fold lower than that of normal human plasma. This reduced activity may be explained by insufficient cleavage of the N-terminal propeptide as identified by N-terminal analysis.

[0587] FIX clotting activity (aPTT): The activated partial thromboplastin time (aPTT) is an integrated measure of the intrinsic and common pathways of the coagulation cascade. The aPTT is the time (measured in seconds) for plasma to clot following the addition of an intrinsic pathway activator, phospholipids, and calcium.

[0588] This assay quantifies the ability of the FIX-CTP-CTP protein to restore the clotting activity of FIX-depleted human plasma by adding rhFIX. 300 μl of FIX-deficient human plasma was mixed with 100 μl of rhFIX, FIX-CTP-CTP (CTP in series at the C-terminus) or further diluted normal pool human plasma. After incubation at 37°C for 60 seconds, tissue factor (TF), CaCl2 and phospholipids were added to the mixture. The clotting time in seconds was determined. Efficacy was assessed by comparing the dose-response curve of FIX-CTP-CTP with the benchmark configuration of rhFIX or human plasma. One unit of FIX was defined as the amount of FIX equivalent to the activity of 1 ml of normal human plasma.

[0589] The aPTT results showed that the clotting activity of FIX-CTP-CTP was only 1.4-fold lower than that of normal pooled human plasma and similar to that of rhFIX. The aPTT results, together with the chromogenic activity determined in vitro, indicated that the purification of FIX-CTP-CTP did not impair its activity.

[0590] Pharmacokinetic activity of FIX-CTP-CTP: Purified FIX-CTP-CTP, rhFIX (American Diagnostic), and harvests containing FIX-CTP-CTP and FIX-CTP were administered as a single intravenous injection to Sprague-Dawley rats (eight rats for each substance) at a dose of 100 g / kg body weight (Table 7).

[0591] Table 7: Overview of PK studies

[0592]

[0593] Blood samples were drawn retro-orbitally from four rats at 0.083, 0.5, 2, 4, 7, 10, 24, 48, and 72 hours after dosing. Citrated plasma (0.32%) was immediately prepared after sampling and stored at -20°C until analysis. FIX concentrations were quantified using a human FIX ELISA kit (Affinity Biologicals). Pharmacokinetic curves ( p < 0.05) were calculated for each protein based on the mean of the four animals at each time point. Figure 7 The terminal half-life was calculated using PK Solutions 2.0 software. Table 8 summarizes the FIX concentrations observed at different sampling time points.

[0594] Table 8: Observed FIX concentrations

[0595]

[0596] A summary of the...

Claims

1. Use of a pharmaceutical preparation in the preparation of a medicament for reducing the frequency of administration of factor VII coagulation factor, wherein: The pharmaceutical preparation comprises a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl-terminal peptide, wherein the polypeptide modified with a chorionic gonadotropin carboxyl-terminal peptide is composed of a factor VII coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, and wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the preparation is at a pH of 6.4 to 6.

5.

2. Use of a pharmaceutical preparation in the preparation of a drug for prolonging the biological half-life of factor VII coagulation factor, wherein: The pharmaceutical preparation comprises a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl-terminal peptide, wherein the polypeptide modified with a chorionic gonadotropin carboxyl-terminal peptide is composed of a factor VII coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, and wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the preparation is at a pH of 6.4 to 6.

5.

3. Use of a pharmaceutical preparation in the preparation of a medicament for improving the area under the curve of factor VII coagulation factor, wherein: The pharmaceutical preparation comprises a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl-terminal peptide, wherein the polypeptide modified with a chorionic gonadotropin carboxyl-terminal peptide is composed of a factor VII coagulation factor and three chorionic gonadotropin carboxyl-terminal peptides attached to the carboxyl-terminus of the coagulation factor, and wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the preparation is at a pH of 6.4 to 6.

5.

4. The use according to any one of claims 1 to 3, wherein The carboxy-terminal peptide-modified polypeptide does not include a signal peptide.

5. The use according to any one of claims 1 to 3, wherein The pharmaceutical formulation was at a pH of 6.

4.

6. The use according to any one of claims 1 to 3, wherein The buffer also included 13.3 mM glycine.

7. The use according to any one of claims 1 to 3, wherein The pharmaceutical formulation included 20 mM citrate, 13.3 mM glycine, 150 mM sodium chloride, and was at a pH of 6.

4.

8. The use according to any one of claims 1 to 3, wherein The pharmaceutical preparation is a liquid preparation.

9. The use according to any one of claims 1 to 3, wherein The amino acid sequence of at least one carboxy-terminal peptide is set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

10. The use according to any one of claims 1 to 3, wherein At least one of the carboxy-terminal peptides is truncated.

11. The use according to any one of claims 1 to 3, wherein At least one of the carboxy-terminal peptides is glycosylated.

12. The use according to any one of claims 1 to 3, wherein At least one carboxy-terminal peptide is attached to the coagulation factor, optionally via a linker.

13. The use according to any one of claims 1 to 3, wherein The sequence of the carboxyl-terminal peptide-modified polypeptide is listed in SEQ ID NO:

46.

14. The use according to any one of claims 1 to 3, wherein The Factor VII coagulation factor includes an activated Factor VII coagulation factor (Factor VIIa coagulation factor).

15. The use according to claim 14, wherein The carboxyl-terminal peptide-modified polypeptide includes a light chain and a heavy chain linked by a disulfide bond.

16. The use according to claim 15, wherein The light chain comprises amino acids 1-152 of SEQ ID NO: 46 and the heavy chain comprises amino acids 153-490 of SEQ ID NO:

46.

17. The use according to any one of claims 15-16, wherein Separation of the light chain and the heavy chain on the SDS-PAGE gel occurs under denaturing conditions, and wherein the light chain migrates with a molecular weight of 25 kDa and the heavy chain migrates with a molecular weight of 60 kDa.

18. The use according to any one of claims 15-16, wherein The disulfide bond occurs between cysteine ​​residue 135 and cysteine ​​residue 262 of SEQ ID NO:

46.

19. The use according to any one of claims 1 to 3, wherein The pharmaceutical formulation is administered to a subject.

20. The use according to claim 19, wherein The subject is a human adult or a human child.

21. The use according to claim 19, wherein The Factor VII coagulation factor includes an activated Factor VII coagulation factor (Factor VIIa coagulation factor).

22. The use according to claim 21, wherein The carboxyl-terminal peptide-modified polypeptide includes a light chain and a heavy chain linked by a disulfide bond.

23. The use according to claim 22, wherein Separation of the light chain and the heavy chain on the SDS-PAGE gel occurs under denaturing conditions, and wherein the light chain migrates with a molecular weight of 25 kDa and the heavy chain migrates with a molecular weight of 60 kDa.

24. The use according to claim 22, wherein The light chain comprises amino acids 1-152 of SEQ ID NO: 46 and the heavy chain comprises amino acids 153-490 of SEQ ID NO:

46.

25. The use according to claim 24, wherein The disulfide bond occurs between cysteine ​​residue 135 and cysteine ​​residue 262 of SEQ ID NO:

46.

26. The use according to claim 19, wherein The subject suffers from a blood coagulation or clotting disorder.

27. The use according to claim 26, wherein The disorder is hemophilia.

28. The use according to claim 27, wherein The hemophilia includes hemophilia A or hemophilia B.

29. The use according to claim 27, wherein The hemophilia includes hemophilia A or hemophilia B with inhibitors.

30. The use according to any one of claims 20 to 29, wherein The administration is performed subcutaneously or intravenously.

31. The use according to claim 12, wherein The linker is a peptide bond.

32. Use of a pharmaceutical preparation in the preparation of a medicament for treating blood coagulation or coagulation disorders, wherein: The pharmaceutical preparation comprises a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide, wherein the polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide consists of a factor VII coagulation factor and three chorionic gonadotropin carboxyl terminal peptides attached to the carboxyl terminal of the coagulation factor, Wherein, the pharmaceutical preparation is used to reduce the dosing frequency of Factor VII coagulation factor, prolong the biological half-life of Factor VII coagulation factor, and / or improve the area under the curve of Factor VII coagulation factor, wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the preparation is at a pH of 6.4 to 6.

5.

33. Use of a pharmaceutical preparation in the preparation of a medicament for treating blood coagulation or coagulation disorders, wherein: The pharmaceutical preparation comprises a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide, wherein the polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide is composed of an activated factor VII coagulation factor (factor VIIa coagulation factor) and three chorionic gonadotropin carboxyl terminal peptides attached to the carboxyl terminal of the coagulation factor, wherein the pharmaceutical preparation is used to reduce the administration frequency of the factor VIIa coagulation factor, prolong the biological half-life of the factor VIIa coagulation factor, and / or improve the area under the curve of the factor VIIa coagulation factor, wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the formulation is at a pH of 6.4 to 6.

5.

34. Use of a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide in the preparation of a pharmaceutical preparation for treating blood coagulation or coagulation disorders, wherein the polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide consists of a factor VII coagulation factor and three chorionic gonadotropin carboxyl terminal peptides attached to the carboxyl terminus of the coagulation factor, wherein: The pharmaceutical preparation is used to reduce the administration frequency of the factor VII coagulation factor, prolong the biological half-life of the factor VII coagulation factor, and / or improve the area under the curve of the factor VII coagulation factor. wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the formulation is at a pH of 6.4 to 6.

5.

35. Use of a buffer, a tonicity agent and a polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide in the preparation of a pharmaceutical preparation for treating blood coagulation or coagulation disorders, wherein the polypeptide modified with a chorionic gonadotropin carboxyl terminal peptide consists of an activated factor VII coagulation factor (factor VIIa coagulation factor) and three chorionic gonadotropin carboxyl terminal peptides attached to the carboxyl terminus of the coagulation factor, wherein: The pharmaceutical preparation is used to reduce the administration frequency of the factor VIIa coagulation factor, prolong the biological half-life of the factor VIIa coagulation factor, and / or improve the area under the curve of the factor VIIa coagulation factor. wherein the buffer comprises 20 mM citrate, the tonicity agent is 150 mM sodium chloride, and the formulation is at a pH of 6.4 to 6.5.

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