EA044349B1 - LONG-ACTING BLOOD CLOTTING FACTORS AND METHODS OF OBTAINING THEM - Google Patents

Description

Field of invention

Disclosed are polypeptides comprising at least one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the carboxyl terminus of a coagulation factor, and polynucleotides encoding them. Also described are pharmaceutical compositions containing the polypeptides and polynucleotides according to the present invention, and methods for their use and preparation.

State of the art

The development of clotting factor replacement therapy has changed the lives of many people with hemophilia. Hemophilia is a group of inherited genetic diseases in which the body's ability to control blood clotting, or coagulation, is impaired. Patients with hemophilia do not produce adequate amounts of factor VIII or factor IX proteins, which are necessary for effective blood clotting. In severe cases of hemophilia, even minor damage can result in blood loss that lasts for days or weeks, and complete healing may never occur, creating the potential for permanent, disabling damage to joints and other organs and premature death.

One type of hemophilia, hemophilia B, is an X-linked bleeding disorder caused by a mutation in the factor IX (FIX) gene, which results in loss of the procoagulant activity of FIX. Patients with hemophilia B experience spontaneous soft tissue hemorrhages and recurrent hemarthrosis, which often lead to deforming arthropathy. Current treatment for these patients involves intravenous administration of recombinant FIX. However, the cost of treatment and the relatively rapid clearance of FIX from the circulation necessitate the development of long-acting FIX.

The commercial availability of FVIII and FIX has provided improved control of life-threatening bleeding episodes. Many patients receive prophylactic therapy, which reduces the risk of bleeding and associated complications. However, a significant proportion of patients (10-30%) develop inhibitory antibodies to administered exogenous FVIII and FIX. Administration of FVIIa, which is a bypass product, can enhance homeostasis and provide effective treatment for patients with inhibitory antibodies.

Recombinant FVIIa (NovoSeven®) is commercially available and was approved in 1996 for the treatment of bleeding episodes in hemophilia patients with inhibitory antibodies. However, clearance of rFVIIa occurs very quickly, with a terminal half-life of 2.5 hours. As a result, patients typically required multiple frequent infusions (2-3 doses given at 2-3 hour intervals) for achieving sufficient homeostasis after mild to moderate bleeding. Consequently, there is great interest in developing a long-acting form of FVIIa that would extend the duration of hemostatic activity after a single dose and allow for much less frequent administration. Long-acting FVIIa will also increase the feasibility of long-term preventive therapy.

Various methods are being developed to increase the half-life of FVIIa. However, the challenge is to provide an extended half-life of the protein while maintaining its biological activity and ensuring that modifications do not cause significant immunogenicity.

Brief description of the invention

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 pharmaceutical composition comprising 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 polynucleotide encoding a CTP-modified polypeptide consisting of a factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the FIX polypeptide.

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

In another embodiment, the present invention provides a cell containing an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FIX polypeptide.

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

In another embodiment, the present invention provides a method for increasing the biological half-life of a factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby extending the biological half-life of said FIX polypeptide.

In another embodiment, the present invention provides a method of increasing the area under the pharmacokinetic curve (AUC) of a factor IX (FIX) polypeptide, comprising the step of adding three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby increasing the AUC of the specified FIX polypeptide.

In another embodiment, the present invention provides a method of reducing the frequency of administration of a factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby reducing the frequency of administration of the specified FIX polypeptide.

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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby reducing the clearance rate of said FIX polypeptide.

In another embodiment, the present invention provides a method for producing a CTP-modified factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby producing a CTP-modified FIX polypeptide.

In another embodiment, the present invention provides a method of treating hemophilia in a subject, comprising administering to said subject a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of said FIX polypeptide, by what is the treatment for hemophilia in the specified subject.

In one embodiment, the invention provides a method of preventing a coagulation or coagulation disorder in a subject, the method comprising the step of administering to the subject a CTP-modified coagulation factor comprising three to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of said FVII polypeptide, such that provides prevention of hemophilia in the specified subject.

In another embodiment, the invention relates to a method of treating a blood clotting or coagulation disorder in a subject, the method comprising the step of administering to the subject a CTP-modified blood clotting factor containing three to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of the said clotting factor blood, which ensures the prevention of hemophilia in the specified subject.

Other features and advantages of the present invention will become apparent upon reading the following detailed description, examples and figures. It should be apparent, however, that the detailed description and specific examples, while preferred embodiments of the present invention are set forth herein, are given for purposes of illustration only, since various changes and modifications within the spirit and scope of the present invention will be apparent to experts in the field from this detailed description.

Brief description of the figures

Fig. 1A. A bar graph is shown showing harvested limited diluted, transfected, and selected FIX-CTP and FIX-CTP-CTP variant cells in the presence of 5 μg/ml vitamin K3. FIX levels were quantified using a human FIX enzyme-linked immunosorbent assay (ELISA) kit (Affinity Biologics; catalog number FIX-AG RUO), and the calculated protein concentration (μg/ml) was the average of two independent experiments. In fig. Figure 1B shows micrographs of gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining with anti-FIX antibody. Photomicrograph A shows Western blot staining with anti-FIX antibody; Photomicrograph B shows a Western blot stained with an antibody that recognizes gamma carboxylation. Lane 1 in AB was loaded with a sample containing recombinant FIX, and lane 2 in AB was loaded with a sample containing assembled FIX-CTP. In lane 3, AB, a sample containing assembled FIX-(CTP) ₂ was loaded.

Fig. 2. A graph is presented showing the comparative chromogenic activity of assembled FIX-CTP and FIX-(CTP) ₂ (measured using EC ₅₀ concentration) compared to recombinant human FIX (rhFIX) (American Diagnostics).

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Fig. 3. A chart is presented showing the PK profile of rhFIX, assembled FIX-CTP-CTP and assembled FIX-CTP.

Fig. 4. Shown is a bar graph showing the level of FIX antigen in assembled FIXCTP and assembled FIX-CTP-CTP and for purified FIX-CTP-CTP protein, as determined using a human FIX ELISA kit (Affinity Biologics; catalog number FIX-AG RUO ). Calculated protein concentration (μg/mL) was the average of two independent experiments.

Fig. 5. Micrographs of EP gels in PAGE/SDS recognition of FIX by the antibody are presented. Photomicrograph A shows Coomassie Brilliant Blue staining; Photomicrograph B shows Western blot staining with anti-FIX antibody; Photomicrograph C shows a Western blot stained with an antibody that recognizes gamma carboxylation. In lane 1, A-C, the sample containing FIX-(CTP)2 was loaded. Lane 2 in A-C was loaded with a sample containing free FIX(CTP)2. In lane 3, A-C, a sample containing concentrated FIX-(CTP)2 eluate was loaded.

Fig. 6. A graph is presented showing the chromogenic activity of FIX-(CTP) ₂ (sample concentration/optical density (OD)) compared to combined normal human plasma and rhFIX (American Diagnostics).

Fig. 7. A chart is presented showing the PK profile of purified FIX-CTP-CTP, rhFIX, pooled FIX-CTP-CTP, and pooled FIX-CTP.

Fig. 8. Anti-CTP and gamma-carboxylation antibody staining of Western blots of FIX fused to three, four, or five CTPs is shown. The assembled FIX-CTP3, FIX-CTP ₄ and FIX-CTP ₅ were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). EF analysis in PAGE/SDS was carried out using immunoblotting (Western blotting) using polyclonal antibodies (AT) to CTP (Adar Biotech Production) or AT to Gla (American Diagnostica).

Fig. 9. Detection of FIX-CTP3, FIX-CTP4 and FIX-CTP ₅ using Coomassie Brilliant Blue is presented. After the purification process using a Jacalin column (immunoaffinity purification of glycosylated proteins), FIX-CTP3, FIX-CTP4 and FIX-CTP ₅ were loaded onto a 12% trisglycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio -Rad). SDS-PAGE gels were stained with Coomassie Brilliant Blue for sample detection.

Fig. 10. The chromogenic activity of FIX is presented. Comparative in vitro activity assessment of fully purified (hydroxylapatite (HA) column) FIX-CTP3 FIX-CTP4 and FIX-CTP ₅ versus pooled normal human plasma was performed using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221802). Serial dilutions of all samples were prepared and activity was assessed by comparing the dose response curve of the samples with that of a reference drug consisting of normal human plasma.

Fig. 11. The comparative pharmacokinetic (PK) profile of FIX-CTP3, FIX-CTP4 and FIX-CTP ₅ is presented. FIX concentrations in plasma samples were quantified using human Elisa FIX kits (Affinity Biologics). The pharmacokinetic profile was calculated and was the average of 3 animals at each time point. The terminal half-life was calculated using PK Solutions 2.0 software.

Fig. 12. Analysis of FIX-CTP3 using SDS-PAGE followed by Coomassie staining of SDS-PAGE gels is presented. Gamma-carboxylated enriched protein FIX-CTP3, rhFIX, and rFIXa (activated FIX) were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Coomassie-PAGE/SDS-PAGE analysis was performed by staining the gel with Coomassie Brilliant Blue reagent (800 ng protein) (Figure 12A). Immunoblotting (Western blotting) was performed using 100 ng of protein and polyclonal AT to human FIX (Fig. 12B), monoclonal antibody recognizing human gammacarboxylation of protein (American Diagnostics, catalog number 499, 3570) (Fig. 12C), polyclonal AT to the FIX propeptide (Fig. 12D) and polyclonal AT to CTP (Fig. 12E).

Fig. 13. The chromogenic activity of FIX-CTP3 is presented. Comparative assessment of in vitro activity of assembled FIX-CTP3 and gamma-carboxylated enriched FIX-CTP3 protein versus pooled normal human plasma was performed using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221802). Serial dilutions of the collected FIX-CTP3 and protein were prepared and activity was assessed by comparing the dose response curve of the samples with that of a reference drug consisting of normal human plasma.

Fig. 14. Comparative blood clotting times are presented. An in vitro APTT (activated partial thromboplastin time) assay was performed comparing the coagulation activity of FIX-CTP3 with BeneFIX. Serial dilutions of the proteins were prepared and injected into human FIX-depleted plasma, and clotting times were assessed.

Fig. 15. Comparative PK profile FIX-CTP3 is presented. Performed quantitative analysis

- 3 044349 concentrations of FIX using human Elisa FIX kits (Affinity Biologics; catalog number FIX-AG

RUO). A pharmacokinetic profile was calculated for each protein and was the average of 3 animals at each time point.

Fig. 16. Activity profile parameters are presented. In parallel with PK sampling, the coagulation activity of citrated plasma samples from FIX-null animals treated with either BeneFIX® or FIX-CTP3 was assessed by aPTT assay, the results of which were converted to % activity. % activity at each collection time was calculated as current clotting time/clotting time of pooled normal mouse plasma * 100.

Fig. 17. The parameters of the first bleeding test are presented. FIX-null mice were given a single intravenous injection of 100 IU/kg BeneFIX® or rFIX-CTP ₃ . The tail vein was lightly dissected 48 hours after injection, and the tail vein bleeding time (TCVT) and bleeding intensity (optical density (OD) of hemoglobin) were assessed. A second bleeding test was performed 15 minutes after homeostasis was achieved and the same parameters were measured.

Fig. 18. The parameters of the second bleeding test are presented. As soon as the first bleeding described in the legend to Fig. 19, stopped spontaneously or was stopped manually, performed a second bleeding test 15 minutes after the first, and re-measured the time and intensity of bleeding.

Fig. 19. Shown is a diagram illustrating the rFVII-CTP construct (A), the rFVII-CTP-CTP construct (B), the rFIX-CTP construct (C), and the rFIX-CTP-CTP construct (D).

Fig. 20A. Shown is a bar graph showing harvested, limited dilution transfected, FVII-CTP variant clones and selected cells in the presence of 5 μg/ml vitamin K3. FVII levels were measured using an FVII ELISA (AssayPro).

Fig. 20B. Shown is a bar graph showing harvested, limited dilution-transfected clones with FVII-CTP variants and selected cells in the presence of 5 μg of vitamin K3. FVII activity was measured using the FVII chromogenic activity assay (AssayPro).

Fig. 20C. Shown is a bar graph showing harvested, limited dilution-transfected clones with FVII-CTP variants and selected cells in the presence of 5 μg of vitamin K3. Specific FVII activity was calculated for each treatment by dividing the activity value by the concentration of collected FVII.

Fig. 20D. A diagram showing the PK profile of collected FVII, FVII-CTP-CTP, and FVII-CTP is presented.

Fig. 21. Shown are Western blots of FVII fused to three, four, and five CTPs, detected with an anti-FVII antibody, an anti-CTP antibody, and a gamma carboxylation antibody. The collected FVII-CTP3, FVII-CTP4 and FVII-CTP ₅ were loaded onto a 12% Tris-glycine gel (Expedeon), which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). PAGE/SDS-PAGE analysis was performed by immunoblotting (Western blotting) using anti-FVII AT, polyclonal anti-CTP AT (Adar Biotech Production) or anti-Gla AT (American Diagnostica).

Fig. 22. The activity of FVII is presented - chromogenic activity. The in vitro activity of GA column-purified (highly gamma-carboxylated fraction) FVIICTP ₃ , FVII-CTP ₄ and FVII-CTP ₅ was compared with pooled normal human plasma using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221304 ). Serial dilutions of all samples were prepared and activity was assessed by comparing the dose response curve of the samples with that of a reference drug consisting of normal human plasma.

Fig. 23. The first comparative pharmacokinetic (PK) profile for FVII-3, 4 and 5 CTP is presented. FVII-CTP3, FVII-CTP4, and FVII-CTP ₅ (group A, B, and C, respectively) were administered as a single intravenous injection to Sprague-Dawle rats (six rats per treatment group) at a dose of 250 μg/kg body weight. Blood samples were collected from the retro-orbital sinus of 3 rats alternately at 0.083, 0.5, 2, 5, 8, 24, 48, 72 and 96 hours after administration. Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis. For FVII-CTP, ₅ showed a better profile compared to the other two variants.

Fig. 24. A second comparative PK profile is presented for FVII-3, 4 and 5 CTP. FVII-CTP3, FVIICTP4 and FVII-CTP ₅ after FVII selection and purification process on a GA column (group A, B and C, respectively) were administered by a single intravenous injection to Sprague-Dawle rats (three rats per substance) at a dose of 29. 45 mcg/kg body weight. Blood samples were collected from the retroorbital sinus at 0.083, 0.5 2, 8, 24, 48 and 72 hours after administration. Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis.

Fig. 25. A schematic diagram of the FVII-CTP3 purification process is presented. Batch 31 was received for PK/pharmacodynamics (PD) studies. Batch 38 was received for the survival study.

Fig. 26. Shown is EP/SDS-PAGE and Western blotting of final FVII and FVIIa. 10 mcg (batch

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31) or 5 μg (lot 38) was loaded onto each lane of a Coomassie-stained SDS-PAGE gel. 1 μg of protein was loaded into each lane of the Western blot gel. 1. Polypeptide FVIICTP3; 2. Heavy chain including 3x CTP; 3. Light chain. All three antibodies detected FVII. The FVIIa heavy chain was detected with an anti-CTP antibody and the light chain was detected with both FVII and Gla antibodies.

Fig. 27. The chromogenic activity of FVII-CTP3 was shown to be enhanced by purification on a ceramic hydroxyapatite (HA) column. The in vitro activity of pooled FVII-CTP3, purification fractions, and purified FVII-CTP3 was compared to pooled normal human plasma using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221304). Serial dilutions of the collected FVIICTP3 and protein were prepared and efficacy was assessed by comparing the dose response curve with that of a reference drug of normal human plasma.

Fig. 28. The PK profile of FVIIa-CTP ₃ compared with NovoSeven® in FVIII-null mice is presented. FVIIa-CTP ₃ was obtained after FVII selection, GA column purification process and activation. FVIIaCTP3 or NovoSeven® was administered by a single intravenous injection to hemophilic FVIII-/- mice. Blood samples were collected from the retroorbital sinus at 0.083, 0.5 2, 8, 24, 48 and 72 hours after administration. Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis, and the PK profile was determined based on FVIIa clotting activity using a commercially available STACLOT kit.

Fig. 29. It was shown that FVIIa-CTP ₃ was obtained after FVII selection, GA column purification process and activation. FVIIa-CTP ₃ or NovoSeven® was administered by a single intravenous injection to Haemophilus influenzae FVIII-/- mice. Blood samples were collected from the retroorbital sinus at 0.083, 0.5 2, 8, 24, 48 and 72 hours after administration. Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis. Parameters of thrombin generation were assessed during the PK experiment, and parameters including the maximum amount per peak, the amount of thrombin at each time point, and the rate of thrombin generation were evaluated.

Fig. 30. Survival curves of mice with hemophilia after tail vein transection (CVD) are presented. RCV was performed at (A) 15 min, (B) 24 h, or (C) 48 h after administration. The survival of mice was monitored for 24 hours after RCV and recorded hourly for the first 12 hours and then after 24 hours. FIG. 30D briefly describes the survival of mice recorded 24 hours after RCV. The results for the control group (medium) are the result of 3 experiments with 5 mice/experiment.

Fig. 31. Immunoblots of FVII - 3 CTP and FVII - 5 CTP are shown. A) GLA blotting, B) FVIIa blotting, C) CTP blotting.

Fig. 32. A comparative PK profile of FVII-3 and 5 CTP after selection and purification on a HA column (FVIIS versus HA-FVII) is presented.

Fig. 33. A comparative PK profile of FVII-3 and 5 CTP is presented. Second study (intravenous (IV) administration versus subcutaneous (SC) administration).

Fig. 34. Survival curves of mice with hemophilia after tail vein transection (CVD) are presented. RCV was performed 12 hours after subcutaneous administration. Mice survival was monitored for 24 h after RCV and recorded hourly for the first 12 h and then after 24 h.

Fig. 35. The PK profile of MOD-5014 compared with NovoSeven® after IV or SC administration is presented. A) presented with IV administration; B) subcutaneous administration is presented.

Fig. 36. Presents the PK profile of MOD-5014 (clone 61 #75, #81) compared to NovoSeven® after a single subcutaneous injection.

Detailed Description of the Invention

In one embodiment, the present invention provides long-acting blood clotting factors and methods for their preparation and use. In another embodiment, the long-acting blood clotting factors comprise a carboxy-terminal peptide (CTP, also referred to as a CTP molecule). In another embodiment, the long-acting polypeptides that contain a coagulation factor further comprise a carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). In another embodiment, CTP acts as a protective agent against clotting factor degradation. In another embodiment, CTP increases the value of the maximum concentration (C _max ) of the blood clotting factor. In another embodiment, CTP increases the time to reach maximum concentration (T _max ) of the clotting factor. In another embodiment, CTP prolongs the circulatory half-life of a coagulation factor. In some embodiments, CTP increases clotting factor activity.

In another embodiment, this application provides a method for increasing the biological half-life of a blood clotting factor, comprising the step of attaching one to ten CTPs to the carboxyl end of the blood clotting factor, thereby prolonging the biological half-life of a blood clotting factor.

- 5 044349 half-life of blood clotting factor. In another embodiment, the present application provides a method for increasing the biological half-life of a blood clotting factor, comprising the step of attaching one to five CTPs to the carboxyl end of the blood clotting factor, thereby extending the biological half-life of the blood clotting factor. In another embodiment, the present invention provides a method for increasing the circulatory half-life of a blood clotting factor. In another embodiment, the present invention provides a method for increasing the half-life of a blood clotting factor. In another embodiment, the present invention provides a method for increasing the half-life of a blood clotting factor. Blood coagulation factor VII (FVII) is a 444 amino acid (50 kDa) glycoprotein secreted by hepatocytes into the bloodstream as an inactive proenzyme. Upon tissue injury and upon contact with circulating blood, FVII forms a complex with tissue factor (TF), which is the true receptor protein for FVII and is expressed by various cells located in the deeper layers of the vessel wall. The formation of this FVII-TF complex leads to the activation of FVII. Activated FVII (FVIIa) initiates the extrinsic coagulation pathway by activating factor IX and factor X.

FVII belongs to a group of vitamin K-dependent glycoproteins associated with the blood coagulation system. In addition to FVII, this group consists of factor IX, factor X, protein C and prothrombin. These proteins have a similar domain organization and are synthesized as precursors with an N-terminal propeptide followed by a mature amino acid sequence. The propeptide contains an attachment site for gamma carboxylase, which converts glutamic acid (Glu) to gammacarboxyglutamic acid (Gla). This domain is followed by two epidermal growth factor (EGF)-like domains, a junction region (JR) and a C-terminal serine protease domain. Before secretion, the FVII propeptide is cleaved off to form the 406 amino acid single-chain zymogen of the FVII glycoprotein. Once secreted, the protein can be activated by cleavage at SU to produce a disulfide-linked double-chain heterodimer, FVIIa. The plasma concentration of FVII is 10 nM and approximately 1% circulates in active form in healthy individuals.

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

FIX is secreted as a single-chain molecule that undergoes complex post-transcriptional modifications, many of which are essential for its biochemical and pharmacokinetic properties. Among all posttranscriptional modifications, the 12 glutamic acid residues near the amino terminus of FIX, which are gamma-carboxylated by vitamin K-dependent gammacarboxylase, are the most important. Carboxylation is required for the interaction of FIX with phospholipid surfaces and for optimal FIX activity. The amino-terminal propeptide serves as a recognition site for gamma carboxylase and thus, after gamma carboxylation, it is cleaved by a serine protease from the Golgi apparatus, known as protein cleaving enzyme at the site of paired amino acids (PACE/furin). Four additional posttranscriptional modifications can occur in the Golgi apparatus: sulfation of tyrosine 155, phosphorylation of serine 158, O-glycosylation at serine Ser 63 and 61, and finally N-glycosylation at asparagine Asn 157 and 16, but they have not been shown to be required for proper activity FIX.

FIX circulates in plasma (average concentration 5 μg/ml) as a single-chain inactive zymogen. Upon proteolytic cleavage of the two peptide bonds Arg 145 and Arg 180 by one or two physiological activators, the FVIIa-TF complex or FIXa, the activation peptide is removed, converting FIX into a fully active enzyme consisting of a light and heavy chain held together by a single disulfide bond. The N-terminal light chain contains the non-catalytic gammacarboxyglutamic acid (Gla) and two epidermal growth factor-like domains, while the C-terminal heavy chain contains the molecule's trypsin-like catalytic domain. FIXa alone has weak catalytic activity. However, when it forms a complex with FVIII, its proteolytic activity increases by 4-5 orders of magnitude relative to its natural substrate FX.

In another embodiment, this application provides a method of increasing the biological half-life or a method of increasing the area under the pharmacokinetic curve (AUC) of a blood clotting factor, comprising the step of adding one to ten CTPs to the carboxyl end of the blood clotting factor, thereby extending the biological half-life or increasing AUC of blood clotting factor. In another embodiment, this application provides a method of increasing the biological half-life or a method of increasing the area under the pharmacokinetic curve (AUC) of a coagulation factor, comprising the step of adding one to five CTPs to the carboxyl end of the coagulation factor, thereby extending the biological half-life or increasing AUC of blood clotting factor. In another version

- 6 044349 lysis, the present application provides a method of increasing the biological half-life or a method of increasing the area under the pharmacokinetic curve (AUC) of FIX, which includes the step of attaching one to five CTPs to the carboxyl end of FIX, thereby extending the biological half-life or increasing the AUC of FIX. In another embodiment, this application provides a method of increasing the biological half-life or a method of increasing the area under the pharmacokinetic curve (AUC) of FVII or FVIIa, comprising the step of adding one to five CTPs to the carboxyl terminus of FVII or FVIIa, thereby extending the biological half-life or increasing AUC FVII or FVIIa. In another embodiment, the present invention provides a method for increasing the biological half-life of a factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby extending the biological half-life of said FIX polypeptide. In another embodiment, the present invention further provides a method of increasing the biological half-life of a factor VIIa (FVIIa) polypeptide, comprising the step of attaching up to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby extending the biological half-life of said FVIIa polypeptide. In one embodiment, three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin are attached to the carboxyl terminus of the specified FVIIa polypeptide. In another embodiment, four carboxy-terminal peptides (CTP) of human chorionic gonadotropin are attached to the carboxyl-terminus of the specified FVIIa polypeptide. In another embodiment, five carboxy-terminal peptides (CTP) of human chorionic gonadotropin are attached to the carboxyl terminus of the specified FVIIa polypeptide.

In another embodiment, the present invention provides a method of increasing the area under the pharmacokinetic curve (AUC) of a factor IX (FIX) polypeptide, comprising the step of adding three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby increasing the AUC of the specified FIX polypeptide. In another embodiment, the present invention provides a method of increasing the area under the pharmacokinetic curve (AUC) of a factor VIIa (FVIIa) polypeptide, comprising the step of adding up to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FVIIa polypeptide, thereby increasing the AUC of the specified FVIIa polypeptide . In one embodiment, three carboxy-terminal peptides (CTP) of human chorionic gonadotropin are attached to the carboxyl terminus of the specified FVIIa polypeptide. In another embodiment, four carboxy-terminal peptides (CTP) of human chorionic gonadotropin are attached to the carboxyl terminus of the specified FVIIa polypeptide. In another embodiment, five carboxy-terminal peptides (CTP) of human chorionic gonadotropin are attached to the carboxyl terminus of the specified FVIIa polypeptide.

In another embodiment, the blood clotting factor of the present invention is a protein. In another embodiment, the blood clotting factor of the present invention is a peptide. In another embodiment, the blood clotting factor of the present invention is a polypeptide. In another embodiment, the blood clotting factor is an enzyme. In another embodiment, the blood clotting factor is a serine protease. In another embodiment, the blood clotting factor is a glycoprotein. In another embodiment, the blood clotting factor is a transglutaminase. In another embodiment, the blood clotting factor is an inactive zymogen. In another embodiment, the blood clotting factor is any blood clotting factor known to one skilled in the art.

In another embodiment, the blood clotting factor is factor VIII (FVIII). In another embodiment, the clotting factor is factor V (FV). In another embodiment, the blood clotting factor is factor XIII (FXIII). In another embodiment, the blood clotting factor is factor X (FX). In another embodiment, the blood clotting factor is fibrin.

In another embodiment, the blood clotting factor is factor VIIa (FVIIa). In another embodiment, the blood clotting factor is factor VII (FVII). In another embodiment, the clotting factor is factor IX (FIX). In another embodiment, the blood clotting factor is factor X (FX). In another embodiment, the blood clotting factor is factor XIa (FXIa). In another embodiment, the blood clotting 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 blood clotting factor is prothrombin. In another embodiment, the blood clotting factor is thrombin. In another embodiment, the coagulation factor is factor XI (FXI). In another embodiment, the blood clotting factor is von Willebrand factor (vWF). In another embodiment, the blood clotting factor is

- 7 044349 factor Villa (FVIIIa). In another embodiment, the coagulation factor is FVIII with B domain deleted (FVIIIBDD). In another embodiment, the coagulation factor is FVIII with the B domain deleted (FVIIIBDD). In another embodiment, the coagulation factor is FVIII with beta domain deleted (FVIIIBDD). In another embodiment, the coagulation factor is factor IXa (FIXa). In another embodiment, the blood clotting factor is prekallikrein. In another embodiment, the blood clotting factor is kallikrein. In another embodiment, the blood clotting factor is factor XIIa (FXIIa). In another embodiment, the blood clotting factor is fibrinogen. In another embodiment, the blood clotting factor is thrombomodulin. In another embodiment, the coagulation factor is factor II (FII).

In another embodiment, the blood clotting factor is a glycoprotein. In another embodiment, the blood clotting factor is a vitamin K-dependent glycoprotein. In another embodiment, the blood clotting factor is a vitamin K independent glycoprotein.

In another embodiment, the blood clotting factor is a recombinant protein. In another embodiment, the blood clotting factor is a recombinant glycoprotein. In another embodiment, the blood clotting factor is a recombinant FV glycoprotein. In another embodiment, the blood clotting factor is recombinant FVI. In another embodiment, the blood clotting factor is recombinant FVII. In another embodiment, the blood clotting factor is recombinant FVIII. In another embodiment, the blood clotting factor is recombinant FIX. In another embodiment, the blood clotting factor is a recombinant FX. In another embodiment, the blood clotting factor is recombinant FXI. In another embodiment, the blood clotting factor is recombinant FXII. In another embodiment, the blood clotting factor is recombinant FvW. In another embodiment, the blood clotting factor is recombinant FII. In another embodiment, the blood clotting factor is recombinant FIXa. In another embodiment, the blood clotting factor is recombinant FXIa. In another embodiment, the blood clotting factor is recombinant fibrin. In another embodiment, the coagulation factor is recombinant FVIIa. In another embodiment, the blood clotting factor is recombinant FXa. In another embodiment, the blood clotting factor is recombinant FVa. In another embodiment, the blood clotting factor is recombinant prothrombin. In another embodiment, the blood clotting factor is recombinant thrombin. In another embodiment, the coagulation factor is recombinant FVIIIa. In another embodiment, the blood clotting factor is recombinant prekallikrein. In another embodiment, the coagulation factor is recombinant kallikrein. In another embodiment, the blood clotting factor is recombinant FXIIa. In another embodiment, the blood clotting factor is any known recombinant blood clotting factor. In another embodiment, the signal peptide-containing coagulation factor is any known recombinant coagulation factor.

In another embodiment, the clotting factor contains 1-10 CTP repeats attached to the C-terminus and no CTPs attached to the N-terminus. In another embodiment, the clotting factor contains at least one CTP attached to the C-terminus and no CTPs attached to the N-terminus. In another embodiment, a clotting factor having 1-10 CTP repeats attached to the C-terminus and no CTPs attached to the N-terminus is an engineered clotting factor. In another embodiment, a blood clotting factor containing at least one CTP attached to the C-terminus and no CTPs attached to the N-terminus is an engineered blood clotting factor. In another embodiment, a clotting factor having 1-10 CTP repeats attached to the C-terminus and no CTPs attached to the N-terminus is a conjugated clotting factor. In another embodiment, a blood clotting factor containing at least one CTP attached to the C-terminus and no CTPs attached to the N-terminus is a conjugated blood clotting factor.

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 further provides a CTP-modified factor VIIa (FVIIa) polypeptide consisting of an FVIIa polypeptide and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVIIa.

In another embodiment, the coagulation factor is a coagulation factor having a domain organization similar or identical to that of FIX, FVII, factor X, protein C, or prothrombin. In another embodiment, the coagulation factor is synthesized as a precursor with an N-terminal propeptide. In another embodiment, the coagulation factor described herein is in an inactive proenzyme form. In another embodiment, the clotting factor is produced in hepatocytes. In another embodiment, the blood clotting factor contains a gamma carboxylase attachment site that converts glutamic acid (Glu) to gamma-carboxyglutamic acid (Gla). In another embodiment, the blood clotting factor described herein is a commercially available blood clotting factor.

In one embodiment, the nucleic acid sequence encoding factor VII includes the following nucleic acid sequence:

ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggagga agcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaagg aggagcagtgctcct tcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggacc agtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccgg aactgtgagacgcacaaggatgaccagctgatctgt gtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaa gcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct attctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtg tgccccaaaggggagtgtccatggcaggtcct gttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaaga actggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggc gggtggcgcaggtc atcatccccagcacgtacgtcccggggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtg gtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctgg accgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtggg agactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccac atgccacccactaccggggcacgtggtacctgac gggcatcgtcagctggggccaggggctgcgcaaccgtgggccactttggggtgtac accagggtctcccagtacatcgagtggctgcaaaagctcatgcgctcagagccacgcccaggagtcctcctgcgagccccatttccctga ggatgcggccgc (SEQ ID NO: 11).

In another embodiment, the amino acid sequence of Factor VII includes the following amino acid sequence:

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEE QCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIP ILEKRNASKPQGRIVGGKVCPK GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKI KNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDH VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSR KVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVG HFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP (SEQ Ш NO: 9).

In another embodiment, the amino acid sequence of Factor VII includes the following amino acid sequence:

- 9 044349

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEE QCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIP ILEKRNASKPQGRIVGGKVCPK GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKI KNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDH VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSR KVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVG HFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP*GCGR (SEQ ID NO: 10).

In another embodiment, the nucleic acid sequence encoding factor VIICTP (attached to the carboxyl terminus) includes the following nucleic acid sequence:

ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggagga agcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaagg aggagcagtgctcct tcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggacc agtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccgg aactgtgagacgcacaaggatgaccagctgatctgt gtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaa gcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct attctgaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtg tgccccaaaggggagtgtccatggcaggtcct gttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaaga actggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggc gggtggcgcaggtc atcatccccagcacgtacgtcccggggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtg gtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctgg accgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtggg agactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccac atgccacccactaccggggcacgtggtacc tgacc ggcatcgtgagctggggccaggggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtggctg cagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccagcagcagctccaaggcccctccccctagcct gcccagccctagcagactgcctgggcccagc gacacccccatcctgccccagtgaggatccgcggccgc (SEQ TO NO: 12).

In another embodiment, the amino acid sequence of factor VII-CTP (attached to the carboxyl terminus) includes the following amino acid sequence:

MVSQALRLLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEE QCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIP ILEKRNASKPQGRIVGGKVCPK GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKI KNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDH VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSR KVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVG HFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQ * (SEQIDNO: 13).

In another embodiment, the nucleic acid sequence encoding factor VIICTP-CTP (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 10 044349 ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggagga agcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaa gg aggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggacc agtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccgg aactgtgagacgcacaag gatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaa gcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct attctagaaaaaagaaatgccagcaaaccccaaggccga attgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcct gttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaaga actggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgac ggggatgagcagagccggcgggtggcgcaggtc atcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtg gtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagc ggctggggccagctgctgg accgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtggg agactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccac atgccacccactaccgg ggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtac accagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccag cagcagctccaaggcccctccccctagcctgcccagccctag cagactgcctgggccctccgacacaccaatcctgccacagagcagct cctctaaggcccctcctccatccctgccatccccctcccggctgccaggcccctctgacacccctatcctgcctcagtgatgaaggtctggat ccgcggccgc (SEQ ID NO: 14).

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

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEE QCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIP ILEKRNASKPQGRIVGGKVCPK GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKI KNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDH VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSR KVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVG HFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQ SSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ ID NO: 15).

In another embodiment, the nucleic acid sequence encoding factor VIICTP-CTP-CTP (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 11 044349 ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggagga agcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaa gg aggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggacc agtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccgg aactgtgagacgcacaag gatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaa gcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct attctagaaaaaagaaatgccagcaaaccccaaggccga attgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcct gttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaaga actggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgac ggggatgagcagagccggcgggtggcgcaggtc atcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtg gtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagc ggctggggccagctgctgg accgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtggg agactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccac atgccacccactaccgg ggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtac accagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccag cagcagctccaaggcccctccccctagcctgcccagccctag cagactgcctgggcccagtgacacccctatcctgcctcagtccagctc cagcaaggccccaccccctagcctgccttctccttctcggctgcctggccccagcgatactccaattctgccccagtcctccagcagtaagg ctccccctccatctctgccatcccccagcagactgccaggcccttctgatacacccatcctcccacagtgat gaggatccgcggccgcttaa ttaa (SEQ ID NO: 24).

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

MVSQALRLLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEE QCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIP ILEKRNASKPQGRIVGGKVCPK GECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKI KNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDH VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSR KVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVG HFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQ SSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ Ш NO: 25).

In another embodiment, the nucleic acid sequence encoding factor VII(CTP) ₄ (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 12 044349 ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggagga agcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaa gg aggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggacc agtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccgg aactgtgagacgcacaag gatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaa gcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct attctagaaaaaagaaatgccagcaaaccccaaggccga attgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcct gttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaaga actggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgac ggggatgagcagagccggcgggtggcgcaggtc atcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtg gtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagc ggctggggccagctgctgg accgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtggg agactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccac atgccacccactaccgg ggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtac accagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccag cagcagctccaaggcccctccccctagcctgcccagccctag cagactgcctgggcccagtgacacccctatcctgcctcagtccagctc cagcaaggccccaccccctagcctgccttctccttctcggctgcctggccccagcgatactccaattctgccccagtcctccagcagtaagg ctccccctccatctctgccatcccccagcagactgccaggcccttctgatacacccatcctcccacagtgat gaggatccgc (SEQ ID NO: 26).

In another embodiment, the amino acid sequence of factor VII-(CTP) ₄ (attached to the carboxyl terminus) includes the following amino acid sequence:

LEDMVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLEREC KEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFE GRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPC GKIPILEKRNASKPQGRIVGGKVCP KGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHC FDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVL TDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQ QSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRG TWYLTGIVSWGQGCA TVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPI LPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**G (SEQ Ш NO: 27).

In another embodiment, the nucleic acid sequence encoding factor VII(CTP)5 (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 13 044349 ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggagga agcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaa gg aggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggacc agtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccgg aactgtgagacgcacaag gatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaa gcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct attctagaaaaaagaaatgccagcaaaccccaaggccga attgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcct gttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaaga actggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgac ggggatgagcagagccggcgggtggcgcaggtc atcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtg gtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagc ggctggggccagctgctgg accgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtggg agactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccac atgccacccactaccgg ggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtac accagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccag cagcagctccaaggcccctccccctagcctgcccagccctag cagactgcctgggccctctgacacccctatcctgcctcagtccagctcc tctaaggctccaccaccttccctgcctagcccttcaagactgccaggccctagcgatacaccaattctgccccagtcctccagcagcaaggc tcccccacctagcctgccttctccatcaaggctgcctggcccatccgataccccaattttgcctcagagcagctctag caaggcacctcccc ccagtctgccctctccaagcagactccctggcccttcagacactccaatcctcccacagtcctctagctctaaagctccacctcccagcctgc ccagccctagtagactccccggaccttctgatacccccatcttgccccagtgatgaggatccgc (SEQ ID NO: 28).

In another embodiment, the amino acid sequence of factor VII-(CTP) ₅ (attached to the carboxyl terminus) includes the following amino acid sequence:

LEDMVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLEREC KEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFE GRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPC GKIPILEKRNASKPQGRIVGGKVCP KGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHC FDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVL TDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQ QSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRG TWYLTGIVSWGQGCA TVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPI LPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKA PPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**GS (SEQ ID NO: 29 ).

In another embodiment, the nucleic acid sequence encoding factor IX includes the following nucleic acid sequence:

- 14 044349 gcgatcgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccattgccttttaggatatctactcagtgctgaatgta cagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaacct t gagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagta tgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggattt gaaggaa agaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtt tgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaa cttctaagctcaccc gtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagca cccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgat gcattctgtggaggctctatcgttaatga aaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtga acataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagta caaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgtt acacctatttgcattgctgacaaggaatacacgaa catcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttctccagtaccttagagttc cacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaag gaggtagagattca tgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaat gaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcacttgaacgcggccgc (SEQ ID NO: 16).

In another embodiment, the amino acid sequence of factor IX includes the following amino acid sequence:

MQRVNMIMAESPGLITICLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVP FPCGRVSVSQTSKLTRAETVFP DVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKP GQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQK RNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSG WGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIY NNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT* (SEQ Ш NO: Π)

In another embodiment, the nucleic acid sequence encoding the IXCTP factor (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 15 044349 gcgatcgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgt acagtttttctcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaacc ttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagt atgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggat ttgaagga aagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtgg tttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaa acttctaagctcacccg tgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagc acccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgat gcattctgtggaggctctatcgttaatgaa aaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtga acataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagta caaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttac acctatttgcattgctgacaaggaatacacgaa catcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttcc acttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaagga ggtagagattcat gtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatg aaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggccc ctcccccgagcctgccct ccccaagcaggctgcctgggccctccgacacaccaatcctgccacagtgatgaaggtctggatccgcggcc gc (SEQ ID NO: 18).

In another embodiment, the amino acid sequence of factor IX-CTP (attached to the carboxyl terminus) includes the following amino acid sequence:

MQRVNMIMAESPGLITICLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVP FPCGRVSVSQTSKLTRAETVFP DVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKP GQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQK RNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSG WGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIY NNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPS PSRLPGPSDTPILPQ** (SEQ ID NO: 19).

In another embodiment, the nucleic acid sequence encoding the factor IXCTP-CTP (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 16 044349 gcgatcgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgt acagtttttctcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaacc ttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagt atgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggat ttgaagga aagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtgg tttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaa acttctaagctcacccg tgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagc acccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgat gcattctgtggaggctctatcgttaatgaa aaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtga acataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagta caaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttac acctatttgcattgctacaaggaatacacgaac atcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttcca cttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaag gaggtagagattcatgt caaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaa aggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggcccct cccccgagcctgccctcc ccaagcaggctgcctgggccctccgacacaccaatcctgccacagagcagctcctctaaggcccctcctcca tccctgccatccccctcccggctgcctggcccctctgacacccctatcctgcctcagtgatgaaggtctggatccgcggccgc (SEQ ID NO: 20).

In another embodiment, the amino acid sequence of factor IX-CTP-CTP (attached to the carboxyl terminus) includes the following amino acid sequence:

MQRVNMIMAESPGLITICLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVP FPCGRVSVSQTSKLTRAETVFP DVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKP GQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQK RNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSG WGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIY NNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPS PSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ Ш NO: 21).

In another embodiment, the nucleic acid sequence encoding factor IX(CTP) ₃ (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 17 044349 tctagagtcgaccccgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgcctttaggatatctactcagt gctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttca agggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaatttt ggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgt ccctttggatt tgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgata acaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagttt ctgtttcacaaacttctaag ctcacccgtgctgaggcagtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacat cactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatg gtaaagttgatgcattctgtggaggctctatc gttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgt cgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagcta ttaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaac agctacgttacacctatttgcattgctgacaagga atacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtacc ttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctgg cttccatgaaggaggta gagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagag tgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcag caaggcccctcccccga gcctgccctccccaagcaggctgcctgggcccagtgacacccctatcctgcctcagtccagctccagcaagg ccccaccccctagcctgccttctccttctcggctgcctggccccagcgatactccaattctgccccagtcctccagcagtaaggctccccctc catctctgccatcccccagcagactgccaggcccttctgatacacccatcc tccccacagtgatgaggatccgcggccgc (SEQ ID NO: 30).

In another embodiment, the amino acid sequence of factor IX-(CTP) ₃ (attached to the carboxyl terminus) includes the following amino acid sequence:

MQRVNMIMAESPGLITICLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGN LERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVP FPCGRVSVSQTSKLTRAEAVFP DVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKP GQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQK RNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSG WGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIY NNMFCAGFHEGGRDSCQGDSGG PHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPS PSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSD TPILPQ** (SEQIDNO: 31).

In another embodiment, the nucleic acid sequence encoding factor IX(CTP) ₄ (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 18 044349 tctagagtcgaccccgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgcctttaggatatctactcagt gctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttca agggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaatttt ggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgt ccctttggatt tgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgata acaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagttt ctgtttcacaaacttctaag ctcacccgtgctgaggcagtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacat cactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatg gtaaagttgatgcattctgtggaggctctatc gttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgt cgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagcta ttaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaac agctacgttacacctatttgcattgctgacaagga atacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtacc ttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctgg cttccatgaaggaggta gagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagag tgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcag caaggcccctcccccga gcctgccctccccaagcaggctgcctgggccctctgacacccctatcctgcctcagtccagctcctctaaggcc ccaccaccttccctgcctagcccttcaagactgccaggccctagcgatacaccaattctgccccagtcctccagcagcaaggctcccccac ctagcctgccttctccatcaaggctgcctggcccatccgataccccaattttg cctcagagcagctctagcaaggcacctccccccagtctgc cctctccaagcagactccctggcccttcagacactcccattctgccacagtgatgaggatccgcggccgc (SEQ ID NO: 32).

In another embodiment, the amino acid sequence of factor IX-(CTP) ₄ (attached to the carboxyl terminus) includes the following amino acid sequence:

SRVDPAMQRVNMIMAESPGLITICILLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEE FVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDI NSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKWCSCTEGYRLAENQKSC EPAVPFPCGRVSVSQTSKLTRA EAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGG EDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETE HTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGS GYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTK FTIYNNMFCAGFHEGGRDSCQ GDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKA PPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSR LPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**GSAA (SEQ ID NO: 33).

In another embodiment, the nucleic acid sequence encoding factor IX(CTP)5 (attached to the carboxyl terminus) includes the following nucleic acid sequence:

- 19 044349 ctagagtcgaccccgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtg ctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaa gggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttg gaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtc cctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataa caaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttc tgtttcacaaacttctaagctcacccgtgctgaggcagtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatc actcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatgg taaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtc gcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctat taataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaa tacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtacct tagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggta gagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagag tgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcag caaggcccctcccccgagcctgccctccccaagcaggctgcctgggccctctgacacccctatcctgcctcagtccagctcctctaaggct ccaccaccttccctgcctagcccttcaagactgccaggccctagcgatacaccaattctgccccagtcctccagcagcaaggctcccccac ctagcctgccttctccatcaaggctgcctggcccatccgataccccaattttgcctcagagcagctctagcaaggcacctccccccagtctgc cctctccaagcagactccctggcccttcagacactccaatcctcccacagtcctctagctctaaagctccacctcccagcctgcccagcccta gtagactccccggaccttctgatacccccatcttgccccagtgatgaggatccgcggccgc (SEQ ID NO: 34).

In another embodiment, the amino acid sequence of factor IX-(CTP)5 (attached to the carboxyl terminus) includes the following amino acid sequence:

RVDPAMQRVNMIMAESPGLITICLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF VQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDIN SYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCE PAVPFPCGRVSVSQTSKLTRAEA VFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE DAKPGQFPWQWLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEH TEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSG YVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTK FTIYNNMFCAGFHEGGRDSCQG DSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPP PSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLP GPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILP Q**GS AA (SEQ Ш NO: 35).

In another embodiment, furin is added to a cell expressing the clotting factor-CTP of the present invention. In another embodiment, furin increases the efficiency of cellular production of clotting factor-CTP according to the present invention. In another embodiment, furin is cotransfected with a vector comprising a coding sequence for the coagulation factor-CTP of the present invention. In another embodiment, furin is encoded by a separate vector. In another embodiment, furin and coagulation factor-CTP are encoded by a single vector. In another embodiment, a furin coding sequence is inserted into pCI-DHFR. In another embodiment, the furin coding sequence is inserted into pCI-dhfr/smaI+NotI, Furin/AsisI F.I.+NotI.

In another embodiment, the nucleic acid sequence encoding furin includes the following nucleic acid sequence:

- 20 044349 tctagagtcgaccccgccatggagctgaggccctggttgctatgggtggtagcagcaacaggaaccttggtcctgctagcagctgatgctc agggccagaaggtcttcaccaacacgtgggctgtgcgcatccctggaggcccagcggtggccaacagtgtggcacggaagcatgggtt cctca acctgggccagatcttcggggactattaccacttctggcatcgaggagtgacgaagcggtccctgtcgcctcaccgcccgcggca cagccggctgcagagggagcctcaagtacagtggctggaacagcaggtggcaaagcgacggactaaacgggacgtgtaccaggagcc cacagaccccaagtttcctcagcagtggtacctgtct ggtgtcactcagcgggacctgaatgtgaaggcggcctgggcgcagggctacac agggcacggcattgtggtctccattctggacgatggcatcgagaagaaccacccggacttggcaggcaattatgatcctggggccagtttt gatgtcaatgaccaggaccctgacccccagcctcggtacacacagatgaatgacaacaggcacggcacac ggtgtgcgggggaagtgg ctgcggtggccaacaacggtgtctgtggtgtaggtgtggcctacaacgcccgcattggaggggtgcgcatgctggatggcgaggtgaca gatgcagtggaggcacgctcgctgggcctgaaccccaaccacatccacatctacagtgccagctggggccccgaggatgacggcaaga cagtggatg ggccagcccgcctcgccgaggaggccttcttccgtggggttagccaggggccgaggggggctgggctccatctttgtctgg gcctcggggaacgggggccgggaacatgacagctgcaactgcgacggctacaccaacagtatctacacgctgtccatcagcagcgcca cgcagtttggcaacgtgccgtggtacagcgagg cctgctcgtccacactggccacgacctacagcagtggcaaccagaatgagaagcag atcgtgacgactgacttgcggcagaagtgcacggagtctcacacgggcacctcagcctctgcccccttagcagccggcatcattgctctca ccctggaggccaataagaacctcacatggcgggacatgcaacacctggtggtacagacctcgaagccagccc acctcaatgccaacgac tgggccaccaatggtgtggggccggaaagtgagccactcatatggctacgggcttttggacgcaggcgccatggtggccctggcccagaat tggaccacagtggccccccagcggaagtgcatcatcgacatcctcaccgagcccaaagacatcgggaaacggctcgaggtgcggaaga ccgtgaccgcgtgcct gggcgagcccaaccacatcactcggctggagcacgctcaggcgcggctcaccctgtcctataatcgccgtggc gacctggccatccacctggtcagccccatgggcacccgctccaccctgctggcagccaggccacatgactactccgcagatgggtttaat gactgggccttcatgacaactcattcctgggatgaggatccctctggcgagtgg gtcctagagatgaaaacaccagcgaagccaacaact atgggacgctgaccaagttcaccctcgtactctatggcaccgcccctgaggggctgcccgtacctccagaaagcagtggctgcaagaccc tcacgtccagtcaggcctgtgtggtgtgcgaggaaggcttctccctgcaccagaagagctgtgtccagcactgccctccagg cttcgcccc ccaagtcctcgatacgcactatagcaccgagaatgacgtggagaccatccgggccagcgtctgcgccccctgccacgcctcatgtgccac atgccaggggccggccctgacagactgcctcagctgccccagccacgcctccttggaccctgtggagcagacttgctcccggcaaagcc agagcagccgagagtcc ccgccacagcagcagccacctcggctgcccccggaggtggaggcggggcaacggctgcgggcagggct gctgccctcacacctgcctgaggtggtggccggcctcagctgcgccttcatcgtgctggtcttcgtcactgtcttcctggtcctgcagctgcg ctctggctttagttttcggggggtgaagg tgtacaccatggaccgtggcctcatctcctacaaggggctgccccctgaagcctggcaggag gagtgcccgtctgactcagaagaggacgagggccggggcgagaggaccgcctttatcaaagaccagagcgccctctgaacgcggccg c (SEQ ID NO: 22).

In another embodiment, the amino acid sequence of furin includes the following amino acid sequence:

MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLN LGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPT DPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGA SFDVNDQDP DPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRML DGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGL GSIF VWASGNGGREHD SCNCDGYTNSIYTLSIS S ATQFGNVPW YSEAC S STLATTYS SGN QNEKQIVTTDLRQKCTESHTGTSASAPLAAG IIALTLEANKNLTWRDMQHLVVQTSKPA HLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCiroiLTEPKDI GKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARP HDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGL PVPPESS GCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRA SVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEV EAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLIS YKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQSAL * (SEQ GO NO: 23).

- 21 044349

In one embodiment, the term coagulation factor further includes a homolog of a known coagulation factor. In one embodiment, the homologue has coagulating activity. In some embodiments, the term homology of the present invention also includes deletion, insertion or substitution variants, including amino acid substitutions, and biologically active polypeptide fragments. In one embodiment, the variant includes conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the coagulation factor. In another embodiment, the deletion, insertion, or substitution does not alter the function of interest of the coagulation factor that, in one embodiment, binds to a specific binding partner.

In another embodiment, the present invention includes a clotting factor homolog. In another embodiment, the present invention includes a clotting factor homologue having coagulating activity. In another embodiment, the present invention includes a coagulation factor homolog that performs functional binding. In another embodiment, the present invention includes homologs of a blood coagulation factor described herein having coagulating activity. In another embodiment, the present invention includes homologues of a blood coagulation factor described herein that perform functional binding. In another embodiment, homologs are, for example, polypeptides that are 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 at least 97%, at least 98%, or at least 99% coagulation factor homology as determined using National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

In another embodiment, the present invention includes homologs of furin. In another embodiment, the present invention includes furin homologs that retain the function of interest, which in one embodiment is the cleavage of a precursor protein. In another embodiment, homologs are, for example, polypeptides that are 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 at least 97%, at least 98%, or at least 99% homologous to furin as determined using National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

In another embodiment, the present application provides a polypeptide comprising a coagulation factor and one to ten carboxy-terminal peptides (CTPs) of a gonadotropin attached to the carboxyl terminus of the coagulation factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and one to three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a coagulation factor and one to five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the coagulation factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor containing at least one CTP at the carboxyl terminus.

In another embodiment, the present application provides 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.

In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and one to five CTPs attached to the carboxyl terminus of the clotting factor.

It should be apparent that the compositions and methods of the present invention including the components or steps described herein may, in another embodiment, consist of the same components or steps, or in another embodiment, consist of substantially the same components or steps. In some embodiments, the term include refers to the inclusion of said active agent, such as a CTP-modified coagulation factor, as well as the inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., known in the pharmaceutical industry. In some embodiments, the term consisting essentially of refers to a composition in which the only active ingredient is the specified active ingredient, however, other compounds that are useful for stabilization, preserving, etc. may also be included. dosage form, but do not directly participate in the therapeutic effect of the specified active ingredient. In some embodiments, a term consisting essentially of

- 22 044349 may refer to components that promote the release of the active ingredient. In some embodiments, the term comprised refers to a composition that contains an active ingredient and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the invention provides a polypeptide comprising a blood clotting factor and two gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to three CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to four CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to five CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to eight CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to nine CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and two to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the invention provides a polypeptide comprising a blood clotting factor and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to four CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to five CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to eight CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to nine CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and three to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide comprising a blood clotting factor and four gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and four to five CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and four to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and four to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and four to eight CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and four to nine CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and four to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide comprising a coagulation factor and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the coagulation factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and five to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and five to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment in

- 23 044349 This application provides a polypeptide comprising a blood clotting factor and five to eight CTPs attached to the carboxyl end of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and five to nine CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide comprising a blood clotting factor and five to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide consisting of a blood clotting factor and two gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and two to three CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and two to four CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and two to five CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and two to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and two to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and two to eight CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and two to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and two to ten CTPs attached to the carboxyl terminus of the clotting factor.

In one embodiment, the present invention provides a polypeptide consisting of a blood clotting factor and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and three to four CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and three to five CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and three to six CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and three to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and three to eight CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and three to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and three to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide consisting of a blood clotting factor and four gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and four to five CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and four to six CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and four to seven CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a blood clotting factor and four to eight CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and four to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and four to ten CTPs attached to the carboxyl terminus of the clotting factor.

In one embodiment, the present invention provides a polypeptide consisting of a blood clotting factor and five carboxy-terminal peptides (CTPs) of gonadotropin, attached

- 24 044349 blood clotting factors attached to the carboxyl end. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and five to six CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and five to seven CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and five to eight CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and five to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting of a clotting factor and five to ten CTPs attached to the carboxyl terminus of the clotting factor. In one embodiment, the present invention provides a polypeptide consisting essentially of a blood clotting factor and two gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and two to three CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and two to four CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and two to five CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and two to six CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and two to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and two to eight CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and two to nine CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and two to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide consisting essentially of a blood clotting factor and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and three to four CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and three to five CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and three to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and three to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and three to eight CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and three to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and three to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide consisting essentially of a blood clotting factor and four gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and four to five CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and four to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and four to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, this application provides a polypeptide consisting essentially of a blood clotting factor and four to eight

- 25 044349

CTP attached to the carboxyl end of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and four to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and four to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In one embodiment, the present invention provides a polypeptide consisting essentially of a blood clotting factor and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and five to six CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and five to seven CTPs attached to the carboxyl terminus of the blood clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and five to eight CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a clotting factor and five to nine CTPs attached to the carboxyl terminus of the clotting factor. In another embodiment, the present application provides a polypeptide consisting essentially of a blood clotting factor and five to ten CTPs attached to the carboxyl terminus of the blood clotting factor.

In another embodiment, the present application provides a polypeptide comprising, consisting essentially of, or consisting of a blood clotting factor that does not have a CTP at its amino terminus. In another embodiment, the present application provides a polypeptide comprising, consisting essentially of, or consisting of a blood clotting factor that lacks a CTP at the amino terminus. In another embodiment, the present application provides a polypeptide comprising, consisting essentially of, or consisting of a blood clotting factor containing at least one CTP at the carboxyl terminus and no CTP at the amino terminus. In another embodiment, the present application provides a polypeptide comprising, consisting essentially of, or consisting of a blood clotting factor containing multiple CTPs at the carboxyl terminus described herein and no CTP at the amino terminus.

In another embodiment, the present invention provides a polynucleotide encoding a polypeptide described above herein.

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

In another embodiment, the present invention further provides a polynucleotide encoding a CTP-modified polypeptide consisting of a factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVIIa polypeptide.

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

In one embodiment, the CTPs that are attached to the carboxyl end of the clotting factor are attached to the carboxyl end in series.

In one embodiment, the engineered coagulation factor described herein has equivalent or improved biological activity compared to an unmodified CTP coagulation factor. In another embodiment, the engineered coagulation factor described herein has equivalent or improved pharmacological properties compared to an unmodified CTP coagulation factor. In another embodiment, the engineered coagulation factor described herein has equivalent or improved pharmacokinetics compared to an unmodified CTP coagulation factor. In another embodiment, the engineered coagulation factor described herein has equivalent or improved pharmacodynamics compared to the unmodified CTP coagulation factor.

In one embodiment, the present invention provides a method for preventing or treating a clotting or coagulation disorder. 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 of treating hemophilia in a subject, comprising administering a CTP-modified factor VII according to the present invention.

In another embodiment, the present invention provides a method of treating hemophilia in a subject, comprising administering a CTP-modified factor IX according to the present invention. In one embodiment, the hemophilia is hemophilia B. In one embodiment, hemophilia B is known as factor IX deficiency or Christmas disease. In one embodiment, hemophilia is severe hemophilia, which in one embodiment is described as hemophilia in which clotting factor levels are 0-1%. In another embodiment, hemophilia is mild hemophilia, which in one embodiment is described as hemophilia in which clotting factor levels are 1-5%. In another embodiment, hemophilia is mild hemophilia, which in one embodiment is described as hemophilia in which clotting factor levels are 5-50%.

In another embodiment, the present invention provides a method of preventing or treating a clotting or coagulation disorder in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin linked to a carboxyl end of said FIX polypeptide, which prevents or treats a blood clotting or coagulation disorder in said subject. In another embodiment, the present invention provides a method of preventing or treating a clotting or coagulation disorder in a subject, comprising administering to the subject a CTP-modified factor VII (FVII) polypeptide comprising an FVII polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of the said subject. an FVII polypeptide that prevents or treats a blood clotting or coagulation disorder in the subject.

In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to said subject a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of said FIX polypeptide, such that provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to said subject a CTP-modified factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three carboxy-terminal human chorionic gonadotropin peptides (CTPs) attached to the carboxyl terminus of said FVIIa polypeptide. which prevents or treats hemophilia in said subject.

In another embodiment, the present invention provides a method of treating hemophilia in a subject, comprising administering to the subject one or more CTP-modified coagulation factors described herein. Thus, in one embodiment, the present invention provides a method of treating hemophilia in a subject, comprising administering to said subject a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three carboxy-terminal human chorionic gonadotropin peptides (CTPs) attached to the carboxyl terminus of said polypeptide FIX, and a CTP-modified factor VIIa (FVIIa) polypeptide comprising an FVIIa polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of the specified FVIIa polypeptide, thereby treating hemophilia in the specified subject. In one embodiment, CTP-modified FIX and CTP-modified FVIIa are administered in the same composition at the same time. In another embodiment, CTP-modified FIX and CTP-modified FVIIa are administered in separate compositions at the same time. In another embodiment, CTP-modified FIX and CTP-modified FVIIa are administered in separate compositions at different time points.

In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) or CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of a polypeptide of said FIX or said FVII, thereby preventing or treating hemophilia in said subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) or a CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and four carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of said FIX or said FVII polypeptide, thereby preventing or treating hemophilia in said subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) or CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin , attached to the carboxyl terminus of the polypeptide of the specified FIX or specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) or CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and three to five carboxy-terminal peptides (CTPs). ) human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX or specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) and a CTP-modified factor VII polypeptide comprising a FIX and FVII polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of a polypeptide of said FIX and said FVII, thereby preventing or treating hemophilia in said subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising administering to the subject a CTP-modified factor IX (FIX) and a CTP-modified factor VII polypeptide, comprising a FIX and FVII polypeptide and three to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX and specified FVII, which provides the prevention or treatment of hemophilia in the specified subject.

In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering to the subject a CTP-modified factor IX (FIX) or a CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and three carboxy-terminal peptides (CTPs). ) human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX or specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering to the subject a CTP-modified factor IX (FIX) or a CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and four carboxy-terminal peptides (CTPs). ) human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX or specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering to the subject a CTP-modified factor IX (FIX) or a CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and five carboxy-terminal peptides (CTPs). ) human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX or specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering to the subject a CTP-modified factor IX (FIX) or CTP-modified factor VII polypeptide comprising a FIX or FVII polypeptide and three to five carboxy-terminal peptides ( CTP) human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX or specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering to the subject a CTP-modified factor IX (FIX) and a CTP-modified factor VII polypeptide, comprising a FIX and FVII polypeptide and three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin, attached to the carboxyl terminus of the polypeptide of the specified FIX and specified FVII, which provides the prevention or treatment of hemophilia in the specified subject. In another embodiment, the present invention provides a method of preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering to said subject a CTP-modified factor IX (FIX) polypeptide and a CTP-modified factor VII polypeptide comprising a FIX and FVII polypeptide and three to five carboxy-terminal human chorionic gonadotropin peptides (CTPs) attached to the carboxyl terminus of a polypeptide of said FIX and said FVII, thereby preventing or treating hemophilia in said subject.

In some embodiments, this application provides a method of preventing or treating hemophilia in a subject, the method comprising the step of administering to the subject a CTP-modified clotting factor containing three to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to a carboxyl-terminal peptide (CTP) of human chorionic gonadotropin. end of said coagulation factor polypeptide, wherein the sequence of said CTP-modified coagulation factor is selected from the group consisting of SEQ ID NO: 25, 27 or 29, thereby preventing hemophilia in the specified subject.

In some embodiments, this application provides a method of preventing or treating hemophilia in a subject, the method comprising the step of subcutaneously administering to the subject a CTP-modified Factor VII comprising three to five carboxy-terminal peptides (CTPs) of human chorionic gonadotropin attached to the carboxyl terminus of said FVII polypeptide, wherein wherein the sequence of said CTP-modified FVII is selected from the group consisting of SEQ ID NO: 25, 27 or 29, thereby preventing hemophilia in said subject.

In other embodiments, the engineered coagulation factor is for the treatment of patients with hemophilia B. In one embodiment, a coagulation factor IX comprising 3 CTPs sequentially attached to its carboxyl terminus is for the treatment of patients with hemophilia B. In one embodiment, the factor A blood clotting factor IX comprising 4 CTPs sequentially attached to its carboxyl terminus is intended for the treatment of patients with hemophilia B. In one embodiment, a blood clotting factor IX comprising 5 CTPs sequentially attached to its carboxyl terminus is intended to treat patients with hemophilia B In another embodiment, coagulation factor IX comprising 2 CTPs sequentially attached to its carboxyl terminus is for the treatment of patients with hemophilia B. In another embodiment, coagulation factor IX including 1 CTP sequentially attached to its carboxyl terminus is for treating patients with hemophilia B. treating patients with hemophilia B. In other embodiments, the engineered clotting factor may reduce the number of infusions required for the patient, reduce the dosage required for the patient, or reduce both.

In one embodiment, coagulation factor IX comprising 3 CTPs sequentially attached to its carboxyl terminus exhibits an improved PK profile while maintaining its coagulating activity compared to assembled FIX-CTP-CTP, assembled FIX-CTP, or rhFIX. In one embodiment, the half-life of rFIX-CTP3 is 2.5 to 4 times greater than that of rFIX in rats and in mice lacking FIX. In one embodiment, administration of rFIX-CTP ₃ significantly prolongs the procoagulant effect in mice lacking FIX for at least 76 hours after administration. In one embodiment, administration of rFIX-CTP ₃ causes a higher peak of activity than rFIX in mice lacking FIX. In another embodiment, coagulation factor IX comprising 2 CTPs sequentially attached to its carboxyl terminus exhibits an improved PK profile while retaining its coagulating activity compared to assembled FIX-CTP or rhFIX. In another embodiment, coagulation factor IX, comprising 2 CTPs sequentially attached to its carboxyl terminus, has 3 times the half-life and 4.5 times the AUC compared to rhFIX.

In another embodiment, SC administration results in greater bioavailability of CTP-modified FVII compared to recombinant FVII. In another embodiment, the half-life is increased and bioavailability (AUC SC/AUC IV) is increased following SC administration of FVIIaCTP 3 and 5, compared to SC administration of NovoSeven®. In another embodiment, subcutaneously injected MOD-5014 and MOD-5019 showed improved survival in mice compared to recombinant FVII (NovoSeven®) (see Example 8 below).

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 different embodiment of the present invention.

In another embodiment, a signal peptide is attached to the N-terminus of the CTP, as described in US 7,553,940, which is incorporated herein by reference in its entirety.

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 clotting factor refers to a sequence of amino acids of a blood clotting factor including a signal sequence or signal peptide.

In another embodiment, the terms signal sequence and signal peptide are used interchangeably herein. In another embodiment, the term sequence, when describing a polynucleotide molecule, may refer to the coding portion. Each possibility represents a different embodiment of the present invention.

In another embodiment, an engineered coagulation factor containing at least one CTP as described herein has increased biological activity in vivo compared to the same coagulation factor without at least one CTP. IN

- 29 044349 In one embodiment, the increased biological activity is due to a longer half-life of the engineered coagulation factor, while at least some of the biological activity is retained. In another embodiment, the increased biological activity is due to the increased biological activity resulting from modification of the CTP. In another embodiment, the increased biological activity is due to both the longer half-life and increased functionality of the CTP-modified coagulation factor.

In some embodiments, at least one CTP sequence at the carboxyl terminus of a clotting factor provides enhanced protection against degradation of the clotting factor. In some embodiments, at least one CTP sequence at the carboxyl terminus of the clotting factor provides increased protection against clearance. In some embodiments, at least one CTP sequence at the carboxyl terminus of the clotting factor provides increased clearance time. In some embodiments, at least one CTP sequence at the carboxyl terminus of the clotting factor increases the _Cmax . In some embodiments, at least one CTP sequence at the carboxyl terminus of the clotting factor increases the _Tmax . In some embodiments, at least one CTP sequence at the carboxyl terminus of the clotting factor increases the half-life (T _1/2 ).

In another embodiment, the conjugated blood clotting factor of the present invention is administered in the same manner as the unmodified conjugated blood clotting factor. In another embodiment, the conjugated blood clotting factor of the present invention has a long circulation half-life and plasma retention time, reduced clearance, and increased clinical activity in vivo. In another embodiment, due to the improved properties of the conjugated coagulation factor described herein, the conjugate is administered at a lower frequency than the unmodified form of the same coagulation factor.

In another embodiment, reducing the frequency of administration will result in an improved treatment strategy, which, in one embodiment, will result in improved patient compliance, which will lead to improved treatment outcomes, as well as an improved patient's quality of life. In another embodiment, compared to conventional coagulation factor conjugates, conjugates having the molecular weight and linker structure of the conjugates of the present invention have been found to have increased potency, increased stability, increased AUC levels, and increased circulatory half-life.

In another embodiment, the present invention further provides a pharmaceutical composition comprising 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 said CTP-modified FIX polypeptide.

In another embodiment, the present invention further provides a pharmaceutical composition comprising a CTP-modified factor VIIa (FVIIa) polypeptide consisting of an FVIIa polypeptide and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVIIa.

In another embodiment, this application provides a composition containing a conjugated clotting factor described herein. In another embodiment, this application provides a pharmaceutical composition containing a conjugated blood clotting factor described herein. In another embodiment, this application provides a pharmaceutical composition containing a therapeutically effective amount of a conjugated blood clotting factor described herein. In one embodiment, the therapeutically effective amount of conjugated blood clotting factor is determined in accordance with factors such as the specific condition being treated, the general condition of the patient being treated, and other ingredients in the composition.

In another embodiment, the conjugated clotting factor described herein is useful for treating subjects suffering from hemophilia. In another embodiment, the conjugated clotting factor described herein is useful for the prophylactic treatment of hemophilia, thereby reducing the risk of bleeding and associated complications. In another embodiment, the conjugated coagulation factor described herein is useful for treating subjects suffering from hemophilia while reducing the risk of development of inhibitory antibodies to exogenously administered coagulation factors. In another embodiment, the conjugated coagulation factor described herein is useful for treating subjects suffering from hemophilia, thereby promoting homeostasis.

In one embodiment, the CTP-modified coagulation factor of the present invention is used for therapeutic purposes. In another embodiment, the CTP-modified blood clotting factor of the present invention is used in the prophylaxis

- 30 044349 for cultural purposes.

In another embodiment, the conjugated clotting factor described herein is useful for treating subjects susceptible to excessive bleeding or bruising, or having a prolonged prothrombin time (PTT) or partial thromboplastin time (PTT). In another embodiment, the conjugated clotting factor described herein is useful for treating subjects who have an acquired condition that causes bleeding, such as vitamin K deficiency or liver disease. In another embodiment, the conjugated clotting factor described herein is useful for treating subjects having clotting factor deficiencies that are acquired (from other diseases) or hereditary, mild or severe, permanent or temporary. In another embodiment, the conjugated blood clotting factor described herein is useful for treating subjects suffering from hemophilia A. In another embodiment, the conjugated blood clotting factor described herein is useful for treating subjects suffering from hemophilia B. In another embodiment in an embodiment, the conjugated clotting factor described herein is useful for treating subjects having acquired deficiency due to chronic diseases such as liver disease or cancer; due to an acute condition such as disseminated intravascular coagulation (DIC), in which clotting factors are consumed at a high rate; or due to vitamin K deficiency or treatment with a vitamin K antagonist such as warfarin (vitamin K is required for the production of factors II, VII, IX and X). In another embodiment, the conjugated blood clotting factor described herein is useful for treating subjects suffering from a disease that causes an imbalance in blood clotting, such as, but not limited to: liver disease, uremia, cancer, bone marrow disorder, snake oil exposure poison, vitamin K deficiency, anticoagulation therapy, accidental ingestion of the anticoagulant warfarin, multiple blood transfusions (blood units lose some clotting factors during storage), or a combination of these. In another embodiment, the present invention provides a method of 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 with 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 with hemophilia B (congenital factor IX deficiency).

In another embodiment, the compositions and methods of the present invention are for the treatment of bleeding episodes in patients with hemophilia A or B using FVIII or FIX inhibitors and in patients with acquired hemophilia; preventing bleeding during surgery or invasive procedures in patients with hemophilia A or B using FVIII or FIX inhibitors and in patients with acquired hemophilia; treating bleeding episodes in patients with congenital FVII deficiency and preventing bleeding during surgery or invasive procedures in patients with congenital FVII deficiency. In another embodiment, the compositions and methods of the present invention are for treating or preventing muscle bleeding. In another embodiment, the compositions and methods of the present invention are for treating or preventing joint bleeding. In another embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment for epistaxis and gum bleeding, mucosal bleeding, and bleeding within the central nervous system. In another embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment for gastrointestinal bleeding or cerebral bleeding. In another embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment for rare minor bleeding events. In another embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment for rare moderate bleeding events. In another embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment for frequent light bleeding. In another embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment for frequent moderate bleeding.

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

In one embodiment, the compositions and methods of the present invention provide therapeutic or prophylactic treatment of hemorrhage, which is in one embodiment intractable bleeding and in another embodiment intracerebral hemorrhage. In another embodiment, the compositions and methods of the present invention are used for the therapeutic or prophylactic treatment of neonatal coagulopathy; severe liver disease; for high-risk surgical procedures; traumatic blood loss; for bone marrow transplantation; thrombocytopenia and platelet dysfunction; for emergency inhibition of the action of oral anticoagulants; congenital deficiencies of factors V, VII, X and XI; or von Willebrand disease, in one embodiment for the therapeutic or prophylactic treatment of von Willebrand disease using von Willebrand factor inhibitors.

In one embodiment, the CTP-modified coagulation factor of the present invention is used to treat a subject with hemophilia or a related disorder described herein. In one embodiment, the subject is a human. 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 canid. In another embodiment, the subject is a feline. In another embodiment, the subject is a bovine, ovine, porcine, equine, mouse, or cervid animal. In one embodiment, the subject is a man. In another embodiment, the subject is a woman. 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 patient, in another embodiment, a geriatric patient.

In another embodiment (CTP), _{the n} > ₁ clotting factor described herein comprises a full-length clotting factor or an active fragment thereof linked via a peptide bond at the carboxyl terminus to at least one CTP molecule, wherein at the amino terminus thereof there are no CTPs. In another embodiment (CTP), _{the n} > ₁ clotting factor described herein comprises a clotting factor or an active fragment thereof linked via a peptide bond to at least one CTP molecule that is linked to an additional CTP molecule via a peptide bond , while there are no CTPs at its amino terminus. In another embodiment, a single nucleic acid molecule encodes an engineered coagulation factor having at least one CTP attached to its C terminus and no CTP at its amino terminus.

In another embodiment, CTP is attached to the clotting factor via a linker. In another embodiment, the linker that connects the CTP sequence to the clotting factor is a covalent bond. In another embodiment, the linker that connects the CTP sequence to the clotting factor is a peptide bond. In another embodiment, the linker that connects the CTP sequence to the clotting factor is a substituted peptide bond. In another embodiment, the CTP sequence includes: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 1). In another embodiment, the CTP sequence includes: SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 2). In another embodiment, the CTP sequence includes an amino acid sequence selected from the sequences described in SEQ ID NO: 1 and SEQ ID NO: 2.

In another embodiment, the carboxy-terminal peptide (CTP) of the present invention includes the amino acid sequence of human chorionic gonadotropin from amino acid position 112 to amino acid position 145 described in SEQ ID NO: 1. In another embodiment, the CTP sequence of the present invention includes the amino acid sequence of human chorionic gonadotropin from amino acid position 118 to position 145, described in the sequence SEQ ID NO: 2. In another embodiment, the CTP sequence also starts from any position between positions 112-118 and ends at position 145 of the amino acid sequence of human chorionic gonadotropin. In some embodiments, the CTP peptide sequence is 28, 29, 30, 31, 32, 33, or 34 amino acids in length and begins at position 112, 113, 114, 115, 116, 117, or 118 of the CTP amino acid sequence.

In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by 1-5 conservative amino acid substitutions, as described

- 32 044349 in US patent number 5712122, which is incorporated herein by reference. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by 1 conservative amino acid substitution. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by 2 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by 5 conservative amino acid substitutions.

In another embodiment, the amino acid sequence of the CTP peptide of the present invention is at least 70% homologous to the native amino acid sequence of the CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention is at least 80% homologous to the native amino acid sequence of the CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention is at least 90% homologous to the native amino acid sequence of the CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention is at least 95% homologous to the native amino acid sequence of the CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide of the present invention is at least 98% homologous to the native amino acid sequence of the CTP or the corresponding peptide.

In another embodiment, the polynucleotide encoding the CTP peptide of the present invention is at least 70% homologous to the native human CTP DNA sequence or the corresponding peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention is at least 80% homologous to the native human CTP DNA sequence or the corresponding peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention is at least 90% homologous to the native DNA sequence of the CTP or the corresponding peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention is at least 95% homologous to the native DNA sequence of the CTP or the corresponding peptide. In another embodiment, the polynucleotide encoding the CTP peptide of the present invention is at least 98% homologous to the native DNA sequence of the CTP or the corresponding peptide.

In one embodiment, at least one of the human chorionic gonadotropin CTP amino acid sequences is truncated. In another embodiment, both human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 2 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 3 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 4 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 5 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 2 or more of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, all human chorionic gonadotropin CTP amino acid sequences are truncated. In one embodiment, the truncated CTP includes the first 10 amino acids of SEQ ID NO: 3. In another embodiment, SEQ ID NO: 3 includes the following amino acid sequence (AA): SSSSKAPPPSLP.

In one embodiment, the truncated CTP includes the first 10 amino acids of SEQ ID NO: 4. In another embodiment, SEQ ID NO: 4 includes the following amino acid sequence (AA): SSSSKAPPPSLPSPSRLPGPSDTPILPQ.

In one embodiment, the truncated CTP includes the first 11 amino acids of SEQ ID NO: 4. In one embodiment, the truncated CTP includes the first 12 amino acids of SEQ ID NO: 4. In one embodiment, the truncated CTP includes the first 8 amino acids of SEQ ID NO: 4 or SEQ ID NO: 3. In one embodiment, the truncated CTP includes the first 13 amino acids of the sequence SEQ ID NO: 4. In one embodiment, the truncated CTP includes the first 14 amino acids of the sequence SEQ ID NO: 4. In one embodiment, the truncated CTP includes the first 6 amino acids of the sequence SEQ ID NO: 4 or SEQ ID NO: 3. In one embodiment, the truncated CTP includes the first 5 amino acids of the sequence SEQ ID NO: 4 or SEQ ID NO: 3.

In one embodiment, at least one of the human chorionic gonadotropin CTP amino acid sequences is glycosylated. In another embodiment, both amino acid sequences of the human chorionic gonadotropin CTP are glycosylated. In another embodiment, 2 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another va

- 33 044349 implementation 3 of the amino acid sequences CTP of human chorionic gonadotropin are glycosylated. In another embodiment, 4 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 5 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 2 or more of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, all human chorionic gonadotropin CTP amino acid sequences are glycosylated. In one embodiment, the CTP sequence of the present invention includes at least one glycosylation site. In one embodiment, the CTP sequence of the present invention includes 2 glycosylation sites. In one embodiment, the CTP sequence of the present invention includes 3 glycosylation sites. In one embodiment, the CTP sequence of the present invention includes 4 glycosylation sites. In one embodiment, one or more of the amino acid sequences of the human chorionic gonadotropin CTP is fully glycosylated. In another embodiment, one or more of the human chorionic gonadotropin CTP amino acid sequences is partially glycosylated. In one embodiment, partial glycosylation means that one of the glycosylation sites of the CTP is glycosylated. In another embodiment, two of the CTP glycosylation sites are glycosylated. In another embodiment, three of the CTP glycosylation sites are glycosylated.

In some embodiments, modification of the CTP sequence provides the advantage of allowing lower dosages to be used. In some embodiments, modification of the CTP sequences provides the advantage of allowing fewer dosages to be administered. In some embodiments, modification of CTP sequences provides the advantage of allowing safe, sustained action.

In some embodiments, as used herein, the term polypeptide, engineered coagulation factor, or protein includes native polypeptides (either degradation products, artificially synthesized polypeptides, or recombinant polypeptides) and peptidomimetics (typically, artificially synthesized polypeptides), as well as peptoids and hemeptoids, which are analogues of polypeptides that, in some embodiments, contain modifications that make the clotting factor polypeptides even more stable when in the body or better able to penetrate cells.

In some embodiments, modifications include, but are not limited to, C-terminal modification, polypeptide bond modification, 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 described in detail, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully described herein. Further details in this regard are described later in this text.

In some embodiments, the polypeptide bonds (-CO-NH-) within the polypeptide contain substituents. In some embodiments, the polypeptide bonds are replaced with N-methylated bonds (-N(CH3)-CO-). In some embodiments, the polypeptide bonds are replaced by ester bonds (-C(R)H-C-O-O-C(R)-N-). In some embodiments, the polypeptide bonds are replaced with ketomethylene bonds (-CO-CH2-). In some embodiments, the polypeptide bonds are replaced by alpha-azo bonds (-NH-N(R)-CO-), where R is any alkyl, such as methyl, with carbo bonds (-CH2-NH-). In some embodiments, the polypeptide bonds are replaced with hydroxyethylene bonds (-CH(OH)-CH2-). In some embodiments, the polypeptide bonds are replaced with thioamide bonds (-CS-NH-). In some embodiments, the polypeptide bonds are replaced by ethylene double bonds (-CH=CH-). In some embodiments, the polypeptide bonds are replaced with retroamide bonds (-NH-CO-). In some embodiments, the polypeptide bonds are replaced by polypeptide derivatives (-N(R)-CH2-CO-), wherein R is a normal side chain naturally present at a carbon atom. In some embodiments, these modifications can be made at any link along the peptide chain and, in one embodiment, at multiple (2-3 linkages) simultaneously.

In some embodiments, the natural aromatic amino acids of the polypeptide, such as Trp, Tyr, and Phe, are replaced with synthetic unnatural acids, such as phenylglycine, TIC, naphthylene (Nol), ring methylated derivatives of Phe, halogenated derivatives of Phe, or o-methyl-Tyr . In some embodiments, the polypeptides of the present invention include one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acid, complex carbohydrates, etc.).

In one embodiment, the term amino acid or amino acid sequence is intended to include the 20 naturally occurring amino acids; such amino acids are often post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine

- 34 044349 and phosphothreonine; and other uncommon amino acids include, but are not limited to: 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine and ornithine. In one embodiment, the amino acid includes both D- and L-amino acids.

In some embodiments, the polypeptides of the present invention are used to prepare a medicament, in which the polypeptides comprising the blood clotting factor are in soluble form. In some embodiments, the polypeptides of the present invention include one or more unnatural or naturally occurring polar amino acids, including, but not limited to, serine and threonine, which are capable of increasing the solubility of the polypeptide due to their hydroxyl-containing side chains.

In some embodiments, the engineered coagulation factor of the present invention is used in linear form, although one skilled in the art will appreciate that in cases where cyclization does not greatly interfere with the properties of the engineered coagulation factors, cyclic forms of the engineered coagulation factors may also be used. blood.

In some embodiments, the engineered coagulation factors of the present invention are synthesized biochemically, for example, using standard solid phase techniques. In some embodiments, these biochemical methods include purely solid-phase synthesis, partially solid-phase synthesis, fragment condensation, or classical solution synthesis.

In some embodiments, recombinant protein techniques are used to produce engineered coagulation factors of the present invention. In some embodiments, recombinant protein techniques are used to produce relatively long polypeptides (eg, greater than 18-25 amino acids). In some embodiments, recombinant protein techniques are used to produce large quantities of engineered coagulation factors of the present invention. In some embodiments, recombinant techniques are described in Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al (1990) Methods in Enzymol. 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) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, section VIII, pp. 421-463, which are incorporated herein by reference in their entirety.

In another embodiment, the present invention provides a polynucleotide molecule comprising a coding portion of a gene encoding a polypeptide comprising a coagulation factor and carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of the coagulation factor described above herein. In another embodiment, the present invention provides a polynucleotide molecule consisting of a coding portion of a gene encoding a polypeptide comprising a coagulation factor and carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of the coagulation factor described above herein. In another embodiment, the present invention provides a polynucleotide molecule consisting essentially of a coding portion of a gene encoding a polypeptide comprising a blood clotting factor and carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor described above herein.

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

In another embodiment, the present invention provides an expression vector comprising a polynucleotide molecule described herein. In another embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FIX polypeptide. 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 carboxy-terminal peptides (CTPs) linked to the carboxyl terminus of said FVIIa polypeptide.

In another embodiment, the present invention provides a cell containing an expression vector described herein. In another embodiment, the present invention provides a cell containing an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FIX polypeptide. In another embodiment, the present invention provides a cell containing an expression vector comprising a polynucleotide encoding a CTP-modified polypeptide consisting of a factor VIIa (FVIIa) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVIIa polypeptide.

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

In another embodiment, the present invention provides a method for producing a CTP-modified blood clotting factor, comprising the step of attaching one to ten carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified blood clotting factor, thereby obtaining a CTP-modified blood clotting factor. In another embodiment, the present invention provides a method for producing a CTP-modified blood clotting factor, comprising the step of attaching one to ten polynucleotide sequences encoding a carboxy-terminal peptide (CTP) of human chorionic gonadotropin to the carboxyl terminus of a polynucleotide sequence encoding said blood clotting factor, thereby allowing obtain CTP-modified blood clotting 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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby obtaining 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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby producing a CTP-modified FVIIa polypeptide.

In another embodiment, engineered coagulation factors of the present invention are synthesized using a polynucleotide molecule encoding a polypeptide of the present invention. In some embodiments, a polynucleotide molecule encoding engineered coagulation factors of the present invention is ligated into an expression vector including transcriptional control by a cis-regulatory sequence (eg, a promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for controlling the constitutive expression of the engineered coagulation factor of the present invention. In some embodiments, the cis-regulatory sequence is suitable for controlling the tissue-specific expression of the engineered coagulation factors of the present invention. In some embodiments, the cis-regulatory sequence is suitable for controlling the inducible expression of engineered coagulation factors of the invention.

In some embodiment, tissue-specific promoters suitable for use in the present invention include sequences that function in one or more specific cell populations. Examples include, but are not limited to, promoters such as the liver-specific albumin promoter (Pinkert et al., (1987) Genes Dev. 1:268277), lymphoid cell-specific promoters (Calame et al., (1988) Adv Immunol 43:235-275); in particular, T-cell receptor promoters (Winoto et al. (1989) EMBO J. 8:729-733) and immunoglobulin promoters (Banerji et al. (1983) Cell 33729-740), nerve cell-specific promoters such as a neurofilament promoter (Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlunch et al. (1985) Science 230:912-916) or mammary gland promoters such as whey promoter (US patent no.

- 36 044349

4873316 and European patent application, publication number 264166). Inducible promoters suitable for use in the present invention include, for example, the tetracycline inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase polynucleotide molecule refers to a single- or double-stranded nucleic acid sequence that is isolated and provided as an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and/or combined polynucleotide sequences (e.g., combinations of the sequences described above) .

In one embodiment, a complementary polynucleotide sequence refers to a sequence that is obtained by reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. In one embodiment, the sequence can subsequently be amplified in vivo or in vitro using DNA polymerase. In one embodiment, the genomic polynucleotide sequence refers to a sequence derived from a chromosome and thus represents a contiguous region of the chromosome.

In one embodiment, the combined polynucleotide sequence refers to a sequence that is at least partially complementary and at least partially genomic. In one embodiment, the combination sequence may include some of the exonic sequences required to encode the polypeptide of the present invention, as well as some of the intronic sequences inserted between them. In one embodiment, the intronic sequences can be obtained from any source, including other genes, and will typically include conserved splice signal sequences. In one embodiment, the intronic sequences include cis-acting expression regulatory components.

In one embodiment, after expression and secretion, the signal peptides are cleaved from the engineered coagulation factor precursors, resulting in mature engineered coagulation factors.

In some embodiments, the polynucleotides of the present invention are produced using PCR techniques or any other method or procedure known to one of ordinary skill in the art. In some embodiments, the procedure involves ligating two different DNA sequences (see, for example, Current Protocols in Molecular Biology, ed. Ausubel et al., John Wiley & Sons, 1992).

In one embodiment, polynucleotides of the present invention that encode engineered coagulation factors are inserted into expression vectors (ie, nucleic acid constructs) to allow expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences that make the vector suitable for replication and integration into prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences that make the vector suitable for replication and integration into eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector that makes the vector suitable for replication and insertion into both prokaryotes and eukaryotes. In some embodiments, cloning vectors include transcription and translation initiation sequences (eg, promoters, enhancers) and transcription and translation terminators (eg, polyadenylation signals).

In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as hosts for expression systems for expressing blood clotting factors according to the present invention. In some embodiments, these hosts include, but are not limited to, microorganisms such as bacteria transformed with a recombinant bacteriophage DNA expression vector, plasmid DNA, or cosmid DNA including a polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors comprising a polypeptide coding sequence; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as the Ti plasmid, including a polypeptide coding sequence.

In some embodiments, non-bacterial expression systems (eg, mammalian expression systems, such as CHO cells) are used to express the coagulation factors of the present invention. In one embodiment, the expression vector that is used to express the polynucleotides of the present invention in mammalian cells is a pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene. Construction of the pCI-dhfr vector is described, according to one embodiment, in Example 1.

In some embodiments, in the bacterial systems of the present invention,

- 37 044349 a variety of expression vectors can be successfully selected depending on the intended use of the polypeptide being expressed. In one embodiment, it is desirable to obtain large quantities of the polypeptide. In one embodiment, vectors are desired that direct the expression of high levels of a protein product, optionally coupled to a hydrophobic signal sequence that directs the expressed product to the bacterial periplasm or culture medium from which the protein product is readily purified. In one embodiment, certain fusion proteins are designed to contain a specific cleavage site to facilitate production of the polypeptide. In one embodiment, vectors amenable to such manipulation include, but are not limited to, the pET series E. coli expression vectors (Studier et al., Methods in Enzymol. 185:60-89 (1990)).

In one embodiment, yeast expression systems are used. In one embodiment, a variety of vectors containing constitutive or inducible promoters can be used in yeast, as described in US patent application number 5932447, which is incorporated herein by reference in its entirety. In another embodiment, vectors are used that facilitate the insertion of foreign DNA sequences into the yeast chromosome.

In one embodiment, the expression vector of the present invention may further comprise additional polynucleotide sequences that allow, for example, the translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for insertion into the genome of a chimeric promoter polypeptide.

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,l, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI, which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV, which are available from Strategene, pTRES, which is available from Clontech, and derivatives thereof.

In some embodiments, the present invention employs expression vectors comprising regulatory components from eukaryotic viruses, such as retroviruses. SV40 vectors include pSVT7 and pMT2. In some embodiments, the bovine papillomavirus-derived vectors include pBV-1MTHA and the Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other examples of vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the control of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mammary tumor virus promoter mouse, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that are effective for expression in eukaryotic cells.

In some embodiments, recombinant viral vectors are useful for in vivo expression of the coagulation factors of the present invention because they provide advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, a retrovirus, and is the process by which a single infected cell produces multiple generations of virions that bud off and infect neighboring cells. In one embodiment, this results in a large area quickly becoming infected, most of which was not initially infected by the original virus particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this property may be useful if the desired goal is to introduce a particular gene into only a limited number of target cells.

In one embodiment, various methods can be used to introduce the expression vector of the present invention into cells. Such methods are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. (Biotechniques 4 (6): 504-512, 1986), and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, for a description of positive-negative selection methods, see US Pat. Nos. 5,464,764 and 5,487,992, incorporated herein by reference. In some embodiments, introduction of nucleic acid by viral infection provides several advantages over other methods such as lipofection and electroporation because, due to the infectious nature of viruses, more efficient transfection can be achieved.

In one embodiment, it will be apparent that the engineered coagulation factors of the present invention can also be expressed from a nucleic acid construct that is administered to an individual using any suitable route of administration described above herein (ie, in vivo gene therapy). In one embodiment, the nucleic acid construct

- 38 044349 acids are introduced into a suitable cell using a suitable gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and expression system, if necessary, and then the modified cells are grown in culture and returned to the individual (i.e. e. ex vivo gene therapy).

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

It will be apparent that, in addition to the necessary components for transcription and translation of the introduced coding sequence (encoding the specified polypeptide), the expression construct of the present invention may also include sequences designed to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

In some embodiments, the transformed cells are cultured under effective conditions that allow the expression of large amounts of recombinant engineered coagulation factors. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH, and oxygen conditions that allow protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce a recombinant polypeptide of the present invention. In some embodiments, the medium typically includes an aqueous solution containing digestible sources of carbon, nitrogen and phosphate and suitable salts, minerals, metals and other nutrients such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri dishes. In some embodiments, culture is performed at a temperature, pH, and oxygen content appropriate for the recombinant cell. In some embodiments, determining culture conditions is within the skill of the average person skilled in the art.

In some embodiments, depending on the vector and host system used for production, the resulting engineered coagulation factors of the present invention are either retained in 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 are retained on the outer surface of the cell or viral membrane.

In one embodiment, after a predetermined culture time, the recombinant engineered clotting factor is produced.

In one embodiment, the formulation of obtaining a recombinant engineered coagulation factor as used herein refers to the collection of a whole fermentation medium containing the polypeptide and does not necessarily involve additional isolation or purification steps.

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, chromatography on concanavalin A, chromatofocusing and differential dissolution.

In one embodiment, to facilitate production, the expressed coding sequence can be designed to encode an engineered coagulation factor of the present invention fused to a cleavable molecule. In one embodiment, the fusion protein can be designed such that the polypeptide can be easily isolated using affinity chromatography; for example, by immobilization on a column that specifically binds the molecule being removed. In one embodiment, a cleavage site is designed between the engineered coagulation factor and the cleavable molecule, and the polypeptide can be released from the chromatography column by treatment with a suitable enzyme or agent that specifically cleaves the fusion protein at that site (see, for example, Booth and etc., Immunol.

Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854–15859 (1990)).

In one embodiment, the engineered coagulation factor of the present invention is obtained in substantially pure form.

In one embodiment, substantially pure refers to a purity that allows the protein to be used effectively in the applications described herein.

In one embodiment, the engineered coagulation factor of the present invention can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and components of the system are commercially available.

In some embodiments, recombinant engineered coagulation factors are synthesized and purified; their therapeutic efficacy can be analyzed either 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 one of ordinary skill in the art.

In another embodiment, the engineered blood clotting factor of the present invention may be administered to an individual by itself. In one embodiment, the engineered coagulation factor of the present invention may be administered to an individual in a pharmaceutical composition in which it is mixed with a pharmaceutically acceptable carrier.

In another embodiment, a pharmaceutical composition refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically appropriate carriers and excipients. The purpose of preparing a pharmaceutical composition is to facilitate the administration of the compound into the body.

In another embodiment, the active ingredient refers to the polypeptide sequence of interest that is responsible for the biological effect.

In another embodiment, any of the compositions of the present invention will contain at least one CTP sequence linked only to the carboxyl terminus of the engineered coagulation factor of interest, in any form. In one embodiment, the present invention provides combination medicinal products. In one embodiment, a combination drug means primarily a kit in the sense that the combination components described above can be dosed independently or by using different predetermined combinations with different amounts of the combination components, i.e. simultaneously, parallel, separately or sequentially. In some embodiments, parts of the set of parts can then, for example, be administered simultaneously or spaced out in time, namely, at different times and at equal or different time intervals for any part of the set of parts. The ratio of the total amounts of the combination components, in some embodiments, can be administered as a combination drug. In one embodiment, the combination drug may be modified, for example, to meet the needs of a subpopulation of patients to be treated, or the needs of a single patient who may require a different drug due to a particular disease, disease severity, age, gender, or body weight, which may easy to carry out by a person skilled in the art.

In another embodiment, physiologically acceptable carrier and pharmaceutically acceptable carrier, which are used interchangeably, refer to a carrier or diluent that does not cause significant irritation to the body and does not interfere with the biological activity and properties of the compound administered. These formulations include an adjuvant. In one embodiment, one of the ingredients included in the pharmaceutically acceptable carrier may be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979)).

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

Methods for the preparation and administration of drugs can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, the latest edition, which is incorporated herein by reference.

The present invention contemplates various embodiments of dosage ranges. The dosage of the engineered coagulation factor of the present invention, in one embodiment, 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

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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. 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 5565 mg/day.

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.

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.

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.

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. In another embodiment, the dosage of the clotting 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.

In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is included in an intranasal dosage form. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is included in an injectable dosage form. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 0.0001 mg to 0.6 mg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 0.001 mg to 0.005 mg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject at a dose ranging from

0.01 mg to 0.3 mg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 0.2 mg to 0.6 mg. In another embodiment, there are no CTPs at the N-terminus of the clotting factor.

In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 1-100 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 10-80 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 20-60 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 10-50 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 40-80 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 10-30 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject at a dose ranging from 30-60 μg.

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

In one embodiment, the dosage of CTP-modified FIX includes 50% of the amount of FIX administered to patients over the same period of time at the recommended dosage of recombinant FIX (eg, Benefix®, Wyeth or Mononine®, CSL Behring). In one embodiment, the dosage of CTP-modified FVIIa includes 50% of the amount of FVIIa administered to patients over the same period of time at the recommended dosage of recombinant FVIIa (eg, NovoSeven®). In one embodiment, the dosage of CTP-modified FVII includes 50% of the amount of FVII administered to patients over the same period of time at the recommended dosage of recombinant FVII. For example, if NovoSeven® is given to a patient before or after surgery at a dose of 90 mcg/kg every two hours (i.e., 7.65 mg every two hours or 45.9 mg in six doses over 12 hours, for a patient weighing 85 kg), the CTP-modified coagulation factor of the present invention can be given at a dose that is 50% of the 12-hour dose of recombinant FVIIa for a given patient (i.e., at a dose of 23 mg given once over 12 hours) .

In another embodiment, the dosage of the CTP-modified coagulation factor is such that it comprises 45% of the amount of coagulation factor that would be administered using non-CTP-modified coagulation factor. In another embodiment, the dosage of the CTP-modified coagulation factor is such that it comprises 10% of the amount of coagulation factor that would be administered using the non-CTP-modified coagulation factor. In another embodiment, the dosage of the CTP-modified coagulation factor is such that it comprises 25% of the amount of coagulation factor that would be administered using non-CTP-modified coagulation factor. In another embodiment, the dosage of the CTP-modified coagulation factor is such that it comprises 35% of the amount of coagulation factor that would be administered using non-CTP-modified coagulation factor. In another embodiment, the dosage of the CTP-modified coagulation factor is such that it comprises 75% of the amount of coagulation factor that would be administered using non-CTP-modified coagulation factor. In another embodiment, the dosage of the CTP-modified coagulation factor is such that it comprises 100% of the amount of coagulation factor that would be administered using non-CTP-modified coagulation factor. However, even if the dosage includes the same amount of clotting factor (eg, FIX) as the amount of unmodified CTP clotting factor, it is still preferable to subjects since it will be administered less frequently due to the increased half-life compared to recombinant blood clotting factors.

In another embodiment, the therapeutically effective amount of conjugated coagulation factor is in the range of 50-500 IU per kg body weight, and is administered once daily to once weekly for FIX or 10-500 μg/kg for FVIIa. In another embodiment, the therapeutically effective amount of conjugated clotting factor is 150-250 IU per kg body weight and is administered once daily. In another embodiment, the pharmaceutical composition,

- 42 044349 containing a conjugated blood clotting factor, formulated at a dose that is effective for administration to a human patient via various routes.

In one embodiment, FIX is administered in an amount effective to achieve 20-30 IU/dL of factor IX activity in the subject's bloodstream. In another embodiment, FIX is administered in an amount effective to achieve 25-50 IU/dL of factor IX activity in the subject's bloodstream. In another embodiment, FIX is administered in an amount effective to achieve 50-100 IU/dL of factor IX activity in the subject's bloodstream. In another embodiment, FIX is administered in an amount effective to achieve 100-200 IU/dL of factor IX activity in the subject's bloodstream. In another embodiment, FIX is administered in an amount effective to achieve 10-50 IU/dL of factor IX activity in the subject's bloodstream. In another embodiment, FIX is administered in an amount effective to achieve 20-100 IU/dL of factor IX activity in the subject's bloodstream.

In one embodiment, the CTP-modified clotting factor is administered to the subject weekly. In another embodiment, the CTP-modified clotting factor is administered to a subject twice a week. In another embodiment, the CTP-modified clotting factor is administered to the subject once every two weeks (once every two weeks). In another embodiment, the CTP-modified clotting factor is administered to a subject twice a month. In another embodiment, the CTP-modified clotting factor is administered to a subject once a month. In another embodiment, the CTP-modified clotting factor is administered to the subject daily. In another embodiment, the CTP-modified clotting factor is administered to the subject once every two days.

In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject once every three days. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject once every four days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject once every five days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP molecule is administered to a subject once every six days. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject once every 7-14 days. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject once every 10-20 days. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject once every 5-15 days. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is administered to a subject once every 15-30 days.

In another embodiment, the methods of the present invention include increasing a patient's compliance with clotting factor therapy, comprising administering to a subject in need thereof a polypeptide comprising a clotting factor and at least one carboxy-terminal peptide (CTP) of human chorionic gonadotropin linked to a carboxyl-terminal peptide (CTP) of human chorionic gonadotropin. end of the clotting factor, thereby increasing the patient's compliance with the use of clotting factor therapy.

In another embodiment, the methods of the present invention include improving the performance of patients suffering from chronic diseases that require clotting factor therapy. In another embodiment, the methods of the present invention reduce the frequency of clotting factor administration by modifying the clotting factor CTP described above herein.

In another embodiment, the present invention provides a method of reducing the frequency of administration of a Factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby reducing the frequency of administration of the specified FIX polypeptide. In another embodiment, the present invention provides a method of reducing the frequency of administration of a factor VIIa (FVIIa) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FVIIa polypeptide, thereby reducing the frequency of administration of the specified FVIIa polypeptide.

In another embodiment, the term compliance includes following directions. In another embodiment, the methods of the present invention include increasing the performance of patients requiring clotting factor therapy by reducing the frequency of clotting factor administration. In another embodiment, reducing the frequency of clotting factor administration is achieved by modifying the CTP, which makes the CTP-modified clotting factor more stable. In another embodiment, a reduction in the frequency of administration of the coagulation factor is achieved by increasing the T ¹ / ₂ of the coagulation factor. In another embodiment, reducing the frequency of administration of the clotting factor is achieved by increasing the clearance time or decreasing the rate of elimination of the clotting factor.

- 43 044349

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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby reducing the clearance rate of said 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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby reducing the clearance rate of said FVIIa polypeptide.

In another embodiment, a reduction in the frequency of clotting factor administration is achieved by increasing the clotting factor AUC measure.

In another embodiment, the present application provides a method for reducing the frequency of administration of a clotting factor, comprising the step of attaching one to ten CTPs to the carboxyl end of the clotting factor, thereby reducing the frequency of administration of the clotting factor. In another embodiment, the present application provides a method for reducing the frequency of administration of a clotting factor, comprising the step of attaching one to five CTPs to the carboxyl end of the clotting factor, thereby reducing the frequency of administration of the clotting factor. In another embodiment, the present application provides a method for reducing the frequency of administration of a clotting factor, comprising the step of attaching three CTPs to the carboxyl end of the clotting factor, thereby reducing the frequency of administration of the clotting factor. In another embodiment, the present application provides a method for reducing the frequency of administration of a clotting factor, comprising the step of attaching three to five CTPs to the carboxyl end of the clotting factor, thereby reducing the frequency of administration of the clotting factor.

In another embodiment, the present application provides a method of increasing compliance with the use of clotting factor therapy, comprising administering to a subject in need thereof a polypeptide comprising a clotting factor and one to ten carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the clotting factor, thereby thereby increasing compliance with the use of clotting factor therapy. In another embodiment, the present application provides a method of increasing compliance with the use of clotting factor therapy, comprising administering to a subject in need thereof a polypeptide comprising a clotting factor and one to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the clotting factor, thereby thereby increasing compliance with the use of clotting factor therapy. In another embodiment, the present application provides a method of increasing performance in relation to the use of clotting factor therapy, comprising administering to a subject in need thereof a polypeptide comprising a blood clotting factor and three carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the clotting factor, thereby increasing performance regarding the use of clotting factor therapy. In another embodiment, the present application provides a method of increasing compliance with the use of clotting factor therapy, comprising administering to a subject in need thereof a polypeptide comprising a clotting factor and three to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the clotting factor, thereby thereby increasing compliance with the use of clotting factor therapy.

In another embodiment, this application provides a method of preventing or treating a clotting or coagulation disorder in a subject, comprising administering to the subject a polypeptide comprising a blood clotting factor and one to ten carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby treating a bleeding or coagulation disorder in said subject. In another embodiment, the present application provides a method of preventing or treating a blood clotting or coagulation disorder in a subject, comprising administering to the subject in need thereof a polypeptide comprising a blood clotting factor and one to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, , which prevents or treats a blood clotting or coagulation disorder in the subject. In another embodiment, this application provides a method of preventing or treating a blood clotting or coagulation disorder in a subject, comprising administering to the subject in need thereof a polypeptide comprising a blood clotting factor and three carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby preventing or treating a bleeding or coagulation disorder in said subject. In another embodiment, this application provides a method of preventing or treating a blood clotting or coagulation disorder in a subject, comprising administering to the subject in need thereof a polypeptide comprising a blood clotting factor and three to five carboxy-terminal human chorionic gonadotropin peptides linked to

- 44 044349 to the carboxyl end of the blood clotting factor, which provides the prevention or treatment of a blood clotting or coagulation disorder in the specified subject.

In another embodiment, the present application provides a method of preventing hemophilia in a subject, comprising administering to the subject a polypeptide comprising a blood clotting factor and one to ten carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby preventing hemophilia in the subject. In another embodiment, the present application provides a method of preventing hemophilia in a subject, comprising administering to a subject in need thereof a polypeptide comprising a blood clotting factor and one to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby preventing hemophilia in the subject. subject. In another embodiment, the present application provides a method of preventing hemophilia in a subject, comprising administering to a subject in need thereof a polypeptide comprising a blood clotting factor and three carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby preventing hemophilia in the subject. In another embodiment, the present application provides a method of preventing hemophilia in a subject, comprising administering to a subject in need thereof a polypeptide comprising a blood clotting factor and three to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby preventing hemophilia in the subject. subject.

In another embodiment, the present invention showed that the compositions provided herein were unexpectedly more effectively absorbed into the bloodstream after subcutaneous administration (see Examples 7-9 herein). The ability to administer FVIIa subcutaneously is advantageous as it can be used for prophylactic applications. Subcutaneous injections are also much easier for patients to administer to themselves, which is an advantage when patients are very young and their veins are small and difficult to detect.

In another embodiment, the present application provides a method of treating hemophilia in a subject, comprising administering to the subject a polypeptide comprising a blood clotting factor and one to ten carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby treating hemophilia in the subject. In another embodiment, the present application provides a method of treating hemophilia in a subject, comprising administering to a subject in need thereof a polypeptide comprising a blood clotting factor and one to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby treating hemophilia in the subject. subject. In another embodiment, the present application provides a method of treating hemophilia in a subject, comprising administering to the subject in need thereof a polypeptide comprising a blood clotting factor and three carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby treating hemophilia in the subject. In another embodiment, the present application provides a method of treating hemophilia in a subject, comprising administering to a subject in need thereof a polypeptide comprising a blood clotting factor and three to five carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood clotting factor, thereby treating hemophilia in the subject. subject.

Oral administration in one embodiment includes unit dosage forms including tablets, capsules, lozenges, chewable tablets, suspensions, emulsions, and the like. Such unit dosage forms include a safe and effective amount of the desired clotting factor of the present invention, each of which is one embodiment from about 0.7 or 3.5 mg to about 280 mg/70 kg or, in another embodiment from approximately 0.5 or 10 mg to approximately 210 mg/70 kg. Pharmaceutically acceptable carriers suitable for the preparation of oral dosage forms 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; raising agents such as starch, alginic acid and croscarmellose; lubricants such as magnesium stearate, stearic acid and talc. In one embodiment, glidants, such as silica, can be used to improve the flow properties of the powder mixture. In one embodiment, dyes, such as FD&C dyes, can be added to enhance appearance. Sweeteners and flavoring agents such as aspartame, saccharin, menthol, peppermint, and fruit flavors are useful adjuvants for chewable tablets. Capsules typically contain one or more of the solid diluents described above. In some embodiments, the choice of carrier components depends on secondary factors such as taste, cost, and storage stability, which are not critical to the purposes of the present invention, and one skilled in the art

- 45 044349 region can easily make such a choice.

In one embodiment, the oral dosage form includes a predetermined release profile. In one embodiment, the oral dosage form of the present invention includes tablets, capsules, lozenges, or extended-release chewable tablets. In one embodiment, the oral dosage form of the present invention includes tablets, capsules, lozenges, or sustained release chewable tablets. In one embodiment, the oral dosage form of the present invention includes tablets, capsules, lozenges, or immediate release chewable tablets. In one embodiment, the oral dosage form is formulated to have a desired release profile of the pharmaceutically active ingredient as known to one of ordinary skill in the art.

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

In some embodiments, compositions for use in the methods of the present invention include solutions or emulsions, which, in some embodiments, are aqueous solutions or emulsions containing a safe and effective amount of the compounds of the present invention and possibly other compounds intended for topical administration. intranasal administration. In some embodiments, the compositions contain from about 0.001% to about 10.0% on a weight-to-volume basis of the subject compound, more preferably from about 00.1% to about 2.0%, which is used for systemic delivery of the compounds by the intranasal route.

In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is injected into a muscle (intramuscular injection). In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is injected under the skin (subcutaneous injection). In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is injected into a muscle. In another embodiment, a polypeptide comprising a blood clotting factor and at least one CTP molecule is injected into the skin. In another embodiment, the blood clotting factor described herein is administered by systemic administration. In another embodiment, the blood clotting factor described herein is administered by intravenous injection. In another embodiment, administration may be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, transnasal, intraocular, ophthalmic, epidural, buccal, rectal, transmucosal, enteric, or parenteral, including intramedullary injections, as well as by intrathecal or direct intraventricular injection.

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

In one embodiment, the route of administration may be enteral. In another embodiment, the route may be conjunctival, transdermal, intradermal, intraarterial, vaginal, rectal, intratumoral, paracanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, intranasal, sublingual, or a combination of these routes.

In another embodiment, the pharmaceutical compositions are administered by intravenous, intraarterial, or intramuscular injection of a liquid composition. In some embodiments, liquid dosage forms include solutions, suspensions, dispersions, emulsions, oils, and the like. In one embodiment, the pharmaceutical compositions are administered intravenously and are therefore formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially and are therefore formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly and are therefore formulated in a form suitable for intramuscular administration.

Moreover, in another embodiment, the pharmaceutical compositions are administered topically to the surface of the body and are therefore formulated in a form suitable for topical administration. Suitable topical dosage forms include gels, ointments, creams, lotions, drops and the like. For topical administration, the compounds of the present invention are combined with an additional suitable therapeutic agent or agents and are prepared and administered as solutions, suspensions or emulsions in a physiologically acceptable vehicle with or without the addition of a pharmaceutical carrier.

- 46 044349

In one embodiment, the pharmaceutical compositions of the present invention are prepared by processes well known in the art, such as conventional mixing, dissolving, granulating, drageeing, trituration, emulsification, encapsulation, entrapment, or lyophilization processes.

In one embodiment, pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and additives that facilitate the preparation of the active ingredients into pharmaceutically usable compositions. In one embodiment, the dosage form depends on the chosen route of administration.

In one embodiment, the injectable drugs of the present invention are formulated as aqueous solutions. In one embodiment, the injectable drugs of the present invention are formulated using physiologically compatible buffers, such as Hanks' solution, Ringer's solution, or physiological saline buffer. In some embodiments, penetrants suitable for the barrier to be penetrated are used for transmucosal administration of the dosage form. Such penetrants are well known in the art.

In one embodiment, the compositions described herein are formulated for parenteral administration, for example, by bolus injection or continuous infusion. In some embodiments, the injectable dosage forms are presented in a unit dosage form, such as ampoules or multi-dose containers, possibly with the addition of a preservative. In some embodiments, the compositions are suspensions, solutions or emulsions in oily or aqueous media and include auxiliary agents such as suspending, stabilizing and/or dispersing agents.

The compositions also include, in some embodiments, preservatives such as benzalkonium chloride and thimerosal and the like; chelating agents such as sodium edetate and other chelating agents; buffers such as phosphate, citrate and acetate; tonicity-regulating agents such as sodium chloride, potassium chloride, glycerin, mannitol and other tonicity-regulating agents; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfate and other antioxidants; aromatic agents; viscosity adjusting agents such as polymers, including cellulose and its derivatives; and polyvinyl alcohol and acid and base to adjust the pH of these aqueous compositions as necessary. The compositions also include, in some embodiments, local anesthetics or other active agents. The compositions can be applied in the form of sprays, aerosols, drops and the like.

In some embodiments, pharmaceutical compositions for parenteral administration include aqueous solutions of the active drug in water-soluble form. In addition, suspensions of the active ingredients, in some embodiments, are prepared as injection suspensions in a suitable oil or water base. Suitable lipophilic solvents or media include, in some embodiments, fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions include, in some embodiments, substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In another embodiment, the suspension also contains suitable stabilizers or agents that increase the solubility of the active ingredients to enable the preparation of highly concentrated solutions.

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

In another embodiment, the pharmaceutical composition delivered in a controlled release system is formulated for intravenous infusion, implantable osmotic pump, transdermal patch, liposomes, or other routes 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., N Engl J Med 321:574 (1989) In another embodiment, polymeric materials may be used. In yet another embodiment, the controlled release system may be placed in close proximity to the therapeutic target, i.e., the brain, thereby , only a fraction of the dose would be required for systemic administration (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in Langer's review (Science 249 :1527-1533 (1990).

In some embodiments, the active ingredient is in powder form for dilution with a suitable vehicle prior to use, such as a sterile pyrogen-free aqueous solution. The compositions, in some embodiments, are formulated for administration by atomization and inhalation. In another embodiment, the compositions are contained in a container with an atomizer attached to it.

In one embodiment, the medicament of the present invention is formulated into compositions for rectal administration, such as suppositories or retention microenemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

In some embodiments, pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate, or reduce the severity of symptoms of a disease, or to prolong the survival of a subject being treated.

In one embodiment, determination of a therapeutically effective amount is within the skill of those skilled in the art.

Some examples of substances that can serve 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, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween™ brand emulsifiers; wetting agents such as sodium lauryl sulfate; dyes; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer solutions. The selection of a pharmaceutically acceptable carrier to be used in conjunction with a compound is largely determined by the intended route of administration of the compound. If the claimed compound is intended for injection, in one embodiment, the pharmaceutically acceptable carrier is a sterile physiological saline solution with a blood-compatible suspending agent adjusted to a pH of approximately 7.4.

In addition, the compositions further include binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone), disintegrating agents (e.g., corn starch, potato starch, alginic acid, silica, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCl, acetate, phosphate) with varying pH and ionic strength, excipients such as albumin or gelatin to prevent absorption by surfaces, detergents (e.g. Tween 20 , Tween 80, Pluronic F68, bile salts), protease inhibitor, surfactants (e.g., sodium lauryl sulfate), penetration enhancers, solubilizing agents (e.g., glycerin, polyethylene glycerin), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropylcellulose, hydroxypropylmethylcellulose), viscosity increasing agents (e.g. carbomer, colloidal silica, ethylcellulose, 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), flow agent (e.g., colloidal silica), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate) , polymer coatings (eg, poloxamers or poloxamines), coating and film-forming agents (eg, ethylcellulose, acrylates, polymethacrylates) and/or adjuvants.

Common carrier components for syrups, elixirs, emulsions and suspensions include ethanol, glycerin, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For suspension, common suspending agents include methylcellulose, sodium carboxymethylcellulose, cellulose (eg, Avicel™, RC-591), tragacanth and sodium alginate; common wetting agents include lecithin and polyethylene oxide sorbitan (eg, polysorbate 80). Common preservatives include methylparaben and sodium benzoate. In another embodiment, the oral liquid compositions also include one or more of the sweeteners, flavoring agents, and colors described above.

The compositions also include incorporating or applying the active material to particulate polymer preparations such as polylactic acid, polyglycolic acid, hydrogels, etc., or to liposomes, microemulsions, micelles, monolamellar or multilamellar vesicles, red blood cell shadows or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

The present invention also includes particulate compositions coated with polymers

- 48 044349 (for example, poloxamers or poloxamines), and a compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens, or coupled to tissue-specific receptor ligands.

In some embodiments, the compounds are modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol-polypropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline. In another embodiment, the modified compounds have a substantially greater blood half-life after intravenous injection than that of the corresponding unmodified compounds. In one embodiment, the modifications also increase the solubility of the compound in aqueous solution, prevent aggregation, increase the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo biological activity is achieved by administering such polymer-compound abducts at lower frequencies or in lower doses than for the unmodified compound.

In some embodiments, the production of an effective amount or dose may first be assessed in in vitro assays. In one embodiment, the dose can be determined in animal models, and the information obtained can be used to more accurately determine useful doses in humans.

In one embodiment, the toxicity and therapeutic efficacy of the active ingredients described herein can be determined using standard pharmaceutical in vitro procedures, cell cultures, or experimental animals. In one embodiment, the results obtained from such in vitro assays and cell culture and animal studies can be used to determine dosage ranges for use in humans. In one embodiment, dosages vary depending on the dosage form used and the route of administration used. In one embodiment, the specific dosage form, route of administration, and dosage may be selected by the attending physician based on the patient's condition (see, for example, Fingl, et al., (1975) The Pharmacological Basis of Therapeutics, chapter 1, page 1).

In one embodiment, depending on the severity and responsiveness to treatment of the condition being treated, dosing may be administered in single or multiple doses, with treatment lasting from several days to several weeks or until cure is achieved or until will achieve a reduction in the symptoms of the disease state.

In one embodiment, the amount of the composition to be administered will, of course, depend on the subject being treated, the severity of the disease, the route of administration, the judgment of the attending physician, etc.

In one embodiment, compositions containing the drug of the present invention formulated in a dosage form together with a compatible pharmaceutical carrier are also prepared, placed in a suitable container and labeled as suitable for the treatment of the specified condition.

In another embodiment, the blood clotting factor described herein is a lyophilized (ie, freeze-dried) drug in combination with a combination of organic excipients and stabilizers, such as nonionic surfactants (ie, surfactants). active substances), various sugars, organic polyols and/or human serum albumin. In another embodiment, the pharmaceutical composition contains the described lyophilized blood clotting factor in sterile water for injection. In another embodiment, the pharmaceutical composition contains the described lyophilized blood clotting factor in sterile PBS for injection. In another embodiment, the pharmaceutical composition contains the described lyophilized blood clotting factor in sterile 0.9% NaCl for injection.

In another embodiment, the pharmaceutical composition contains a blood coagulation factor described herein and a combination of carriers, such as human serum albumin, polyols, sugars and anionic surfactant stabilizing agents. In another embodiment, the pharmaceutical composition contains a blood clotting factor described herein and lactobionic acid and an acetate/glycine buffer. In another embodiment, the pharmaceutical composition contains a blood clotting factor described herein and amino acids, such as arginine or glutamate, which increase the solubility of interferon compositions in water. In another embodiment, the pharmaceutical composition contains a lyophilized coagulation factor described herein and glycine or human serum albumin (HSA), a buffer (eg, acetate) and an isotonic agent (eg, NaCl). In another embodiment, the pharmaceutical composition contains a lyophilized blood clotting factor described herein and a phosphate buffer, glycine and HSA.

In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein is stabilized by placing it in buffer solutions having a pH between about 4 and 7.2. In another embodiment, a pharmaceutical composition containing a blood clotting factor is placed in a buffer solution having a pH between about 4 and 8.5. In another embodiment, a pharmaceutical composition containing a blood clotting factor is placed in a buffer solution having a pH between about 6 and 7. In another embodiment, a pharmaceutical composition containing a blood clotting factor is placed in a buffer solution having a pH of about 6.5 . In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein is stabilized with an amino acid as a stabilizing agent and, in some cases, with a salt (if the amino acid does not contain a charged side chain).

In another embodiment, the pharmaceutical composition containing the blood clotting factor described herein is a liquid composition containing a stabilizing agent in an amount between about 0.3 and 5 wt.%, which is an amino acid.

In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein ensures the accuracy of administration and the safety of the product. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein provides a biologically active, stable liquid dosage form for injection. In another embodiment, the pharmaceutical composition contains a non-lyophilized blood clotting factor described herein.

In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein provides a liquid dosage form capable of being stored for an extended period of time in a liquid state, thereby facilitating storage and transportation prior to administration.

In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein contains solid lipids as a matrix material. In another embodiment, an injectable pharmaceutical composition containing a blood clotting factor described herein contains solid lipids as a matrix material. In another embodiment, the preparation of lipid microparticles by spray crystallization is described by Speiser (Speiser et al., Pharm. Res. 8 (1991) 47-54), from which lipid nanobeads are then prepared for oral administration (Speiser EP 0167825 (1990)). In another embodiment, the lipids that are used are well tolerated by the body (eg, glycerides consisting of fatty acids that are present in the parenteral emulsion).

In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes polymer microparticles. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes nanoparticles. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes liposomes. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes a lipid emulsion. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes microspheres. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes lipid nanoparticles. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes lipid nanoparticles including amphiphilic lipids. In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein includes lipid nanoparticles containing a drug, a lipid matrix and a surfactant. In another embodiment, the lipid matrix contains at least 50% by weight monoglyceride.

In one embodiment, the compositions of the present invention are presented in a package or dispensing device, such as an FDA-approved kit, which includes one or more unit dosage forms containing the active ingredient. In one embodiment, the packaging, for example, includes a metal or polymer film, such as a blister pack. In one embodiment, the package or dispensing device is accompanied by instructions for administration. In one embodiment, the package or dispensing device is accompanied by a notice accompanying the container in the form required by the government agency regulating the manufacture, use, or marketing of the drug, and the notice reflects that agency's approval of the form of the compositions or administration to a person or animal. Such notice, in one embodiment, is a US Food and Drug Administration-approved prescription drug label or approved package insert.

In one embodiment, it will be apparent that the clotting factors of the present invention may be administered to a subject in conjunction with additional active agents,

- 50 044349 to achieve improved therapeutic effect compared to treatment with each agent separately. In another embodiment, measures are taken (eg, selection of dosage and additional agent) to avoid adverse side effects that are associated with combination therapies.

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

In another embodiment, the present invention provides a pharmaceutical composition comprising a CTP-modified factor VIIa (FVIIa) polypeptide consisting of an FVIIa polypeptide and five gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVIIa.

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

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

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

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

In another embodiment, the present invention provides a method of increasing the area under the pharmacokinetic curve (AUC) of a factor VIIa (FVIIa) polypeptide, comprising the step of adding three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FVIIa polypeptide, thereby increasing the AUC of the specified FVIIa polypeptide. In another embodiment, the present invention provides a method of reducing the frequency of administration of a factor VIIa (FVIIa) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FVIIa polypeptide, thereby reducing the frequency of administration of the specified FVIIa 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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby reducing the clearance rate of said FVIIa 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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby producing a CTP-modified FVIIa polypeptide.

In another embodiment, the present invention provides a method of treating hemophilia in a subject, comprising administering to said subject a CTP-modified factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three carboxy-terminal human chorionic gonadotropin peptides (CTPs) attached to the carboxyl terminus of said FVIIa polypeptide, by what is the treatment for hemophilia in the specified subject.

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 described in SEQ ID NO: 31. In another embodiment, the present invention provides a CTP-modified FIX polypeptide, in 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:

- 51 044349 at least one CTP is glycosylated. In another embodiment, the present invention provides a CTP-modified FIX polypeptide in which 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 said linker is a peptide bond.

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

In one embodiment, the present invention provides a polynucleotide encoding a CTP-modified polypeptide consisting of a factor IX (FIX) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FIX polypeptide. In another embodiment, the present invention provides a polynucleotide wherein the sequence of said polynucleotide is described 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 in which at least one CTP is truncated. In another embodiment, the present invention provides a polynucleotide wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a polynucleotide wherein said linker is a peptide bond. In another embodiment, the present invention provides an expression vector containing said polynucleotide.

In one embodiment, the present invention provides a cell containing an expression vector.

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

In one embodiment, the present invention provides a method of increasing the biological half-life of a factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby extending the biological half-life of said FIX polypeptide. In another embodiment, the present invention provides a method wherein the 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 in which at least one CTP is shortened. In another embodiment, the present invention provides a method wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a method wherein said linker is a peptide bond. In one embodiment, the present invention provides a method of increasing the area under the pharmacokinetic curve (AUC) of a factor IX (FIX) polypeptide, comprising the step of adding three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby increasing the AUC of the specified FIX polypeptide. In another embodiment, the present invention provides a method wherein the 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 in which at least one CTP is shortened. In another embodiment, the present invention provides a method wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a method wherein said linker is a peptide bond.

In one embodiment, the present invention provides a method of reducing the frequency of administration of a factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of the specified FIX polypeptide, thereby reducing the frequency of administration of the specified FIX polypeptide. In another embodiment, the present invention provides a method wherein the 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 in which at least one CTP is shortened. In another embodiment, the present invention provides a method wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a method wherein said linker is a peptide bond.

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 carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby reducing the clearance rate of said FIX polypeptide. In another embodiment, the present invention provides a method wherein the 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 in which at least one CTP is shortened. In another embodiment, the present invention provides a method wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a method wherein said linker is a peptide bond.

In one embodiment, the present invention provides a method for producing a CTP-modified factor IX (FIX) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FIX polypeptide, thereby producing a CTP-modified FIX polypeptide. In another embodiment, the present invention provides a method wherein the sequence of said CTP-modified FIX polypeptide is the sequence described in SEQ ID NO: 31. In another embodiment, the present invention provides a method wherein at least one CTP is encoded by the sequence amino acids 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 in which at least one CTP is shortened. In another embodiment, the present invention provides a method wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a method wherein said linker is a peptide bond.

In one embodiment, the present invention provides a method of treating hemophilia in a subject, comprising administering to said subject a CTP-modified factor IX (FIX) polypeptide comprising a FIX polypeptide and three carboxy-terminal human chorionic gonadotropin peptides (CTPs) attached to the carboxyl terminus of said FIX polypeptide, by what is the treatment for hemophilia in the specified subject. In another embodiment, the present invention provides a method wherein the sequence of said CTP-modified FIX polypeptide is the sequence described in SEQ ID NO: 31. In another embodiment, the present invention provides a method wherein at least one CTP is encoded by the sequence amino acids 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 in which at least one CTP is shortened. In another embodiment, the present invention provides a method wherein at least one CTP is attached to said FIX polypeptide via a linker. In another embodiment, the present invention provides a method wherein said linker is a peptide bond.

It is widely known in the art that the modified peptides and proteins of the present invention can be coupled to tags, drugs, targeting agents, carriers, solid supports, and the like, depending on the desired application. Tagged forms of modified biologics can be used to track their metabolic pathway; suitable labels for this purpose include, in particular, radioisotope labels such as iodine 131, technetium 99, indium 111 and the like. Tags can also be used as a means of detecting modified proteins or peptides in analysis systems; in this example, radioactive isotopes as well as enzyme tags, fluorescent tags, chromogenic tags and the like can also be used. The use of such labels is particularly useful if the peptide or protein is itself a targeting agent, such as an antibody or receptor ligand.

Similar coupling techniques, among others, can be used to couple the modified peptides and proteins of the present invention to solid supports. Once coupled, these modified peptides and proteins can then be used as affinity reagents to separate the desired components to which specific binding occurs.

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Finally, the modified peptides and proteins of the present invention can be used to produce antibodies, in particular immunoreactive with these new compounds. These antibodies are useful for a variety of diagnostic and therapeutic applications, depending on the nature of the biological activity of the unmodified peptide or protein. It should be obvious that the present invention provides antibodies that are immunoreactive with CTP-modified FIX, FVII or FVIIa described herein. In one embodiment, such antibodies can be used to distinguish or identify CTP-modified coagulation factors that have been administered from endogenous coagulation factors. In another embodiment, antibodies can be used to locate administered CTP-modified coagulation factors.

Additional aspects, advantages and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention described above in this text and claimed in the claims below is experimentally confirmed in the following examples.

Examples

In general, the terminology used in this text and laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are described in detail in the literature. See, for example, Molecular Cloning: A laboratory Manual Sambrook et al., (1989); Current Protocols in Molecular Biology, volumes I-III, Ausubel, R.M., ed. (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al (eds.) Genome Analysis: A Laboratory Manual Series, volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); techniques described in US patent numbers 4666828; 4683202; 4801531; 5192659 and 5272o57; Cell Biology: A Laboratory Handbook, Volumes I-III, Cellis, J.E., ed. (1994); Culture of Animal Cells - A Manual of Basic Technique by Freshney, Wiley-Liss, N.Y. (1994), third edition; Current Protocols in Immunology, volumes I-III, Coligan J.E., ed. (1994); Stites et al (eds.), Basic and Clinical Immunology (8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980); available immunoassays are described in detail in the patent and scientific literature, see, for example, US patent numbers 3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; Oligonucleotide Synthesis Gait, M.J., ed. (1984); Nucleic Acid Hybridization Hames, B.D., and Higgins S.J., eds. (1985); Transcription and Translation Hames, B. D., and Higgins S. J., eds. (1984); Animal Cell Culture Freshney, R.I., ed. (1986); Immobilized Cells and Enzymes IRL Press, (1986); A Practical Guide to Molecular Cloning Perbal, B., (1984) and Methods in Enzymology, volume 1-317, Academic Press; PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, California (1990); Marshak et al., Strategies for Protein Purification and Characterization - A Laboratory Course Manual CSHL Press (1996); each of which is incorporated by reference. Other major references are provided throughout this specification.

Example 1. Preparation and use of blood clotting factor IX.

Cloning and expression of the recombinant FIX molecule.

Factor IX clones were constructed in the eukaryotic expression vector pCI-neo (Promega, catalog no. E1841). An open reading frame (ORF) clone of Homo sapiens coagulation factor IX was ordered from OriGene (RC219065). Primers were ordered from Sigma-Genosys.

Construction of 301-1-pCI-neo-p200-11 (factor IX-ctp x2).

Primer 101:5' GTTTAGTGAACCGTCAGAAT 3' (SEQ ID NO: 36).

Primer 103 ^R: 5' TTGAGGAAGATGTTCGTGTA 3' (contains Factor IX SspI site) (SEQ ID NO: 37).

A polymerase chain reaction (PCR reaction) was carried out with primer 101 and reverse primer 103R and with plasmid DNA, factor IX cDNA clone (OriGene RC219065) as template; As a result of PCR amplification, a product of ~1085 bp in size was obtained. (PCR 10), which was purified from the gel (a fragment containing the N-terminus of the factor IX sequence).

Primer 98: 5' ATTACAGTTGTCGCAGTGA 3' (SEQ ID NO: 38).

Primer 99R: 5' GCTGGAGCTAGTGAGCTTTGTTTTTTCCTT 3' (SEQ ID NO: 39).

Primer 100: 5' GCTCACTAGCTCCAGCAGCAAGGCC 3' (SEQ ID NO: 40).

Primer 27R: 5' TTTTCACTGCATTCTAGTTGTGG 3' (SEQ ID NO: 41).

Three PCR reactions were performed. The first reaction was carried out with primer 98 and primer 99 ^R and plasmid DNA, factor IX cDNA clone (OriGene, RC219065) as template; As a result of PCR amplification, a product of ~540 bp was obtained.

The second reaction was carried out with primer 100 and primer 27 ^R and plasmid DNA 402-2-p72-3 (hGH-CTP-CTP) as template; As a result of PCR amplification, a product of ~258 bp was obtained.

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The last reaction (PCR 3) was carried out with primers 98 and 27R and a mixture of the products of the previous two reactions as a template; PCR amplification resulted in a product of ~790 bp, which was ligated into the TA cloning vector (Invitrogen, catalog number K2000-01). A fragment obtained by treatment with restriction enzymes SspI-EcoRI (TA 3-3) was isolated.

Another PCR reaction (PCR 12) was carried out with primer 101 and primer 27R, and with a mixture of PCR 10 products and the SspI-EcoRI fragment from PCR 3 as a template; As a result of PCR amplification, a product of ~1700 bp was obtained. (factor IX-ctp-ctp), which was ligated into the TA cloning vector (Invitrogen, catalog number K2000-01) (lig 180).

An error was detected in the Factor IX sequence, so the fragments were replaced to produce the Factor IX-ctp-ctp insert with the correct DNA sequence.

TA-PCR 3-3 was digested with restriction enzymes SspI and XbaI and a large fragment (vector) was isolated. TA 1804 was digested with restriction enzymes SspI and XbaI, and a small fragment (insert) was isolated, which was ligated with the isolated large fragment of TA-PCR-3-3, digested with SspI and XbaI. The new plasmid TA-183-2 was digested with SalI and NotI restriction enzymes, and the factor IX-ctp-ctp insert (~1575 bp) was isolated. This fragment was inserted into the eukaryotic expression vector pCI-neo (cleaved with SalI and Not I) to obtain clone 301-2-p200-11.

Construction of pCI-dhfr -factor 9-ctp x2 (p223-4). Vector pCI-dhfr (p6-1) was digested with restriction enzymes SmaI and NotI. Factor IX-CTP-CTP (p200-11) was digested with restriction enzymes ASisI F.I. and NotI. The resulting two fragments were ligated.

Construction of pCI-dhfr-factor 9-ctp x3 (p225-7). The pCI-dhfr OXM-CTPx3 (p216-4) vector was digested with XbaI and ApaI restriction enzymes. Factor IX-CTP-CTP (223-4) was digested with restriction enzymes XbaI and ApaI. The resulting two fragments were ligated.

Construction of pCI-dhfr-factor 9-ctp x3 T148A (p243-2). In plasmid p225-7, which contains threonine at position 148, Thr was replaced by Ala by site-directed mutagenesis, since the most common FIX variant contains alanine at this position.

Primer 75: ctcccagttcaattacagct (SEQ ID NO: 42).

Primer 122r: ggaaaaactgcctcagcacgggtgagc (SEQ ID NO: 43).

Primer 123: gtgctgaggcagtttttcctgatgtggactat (SEQ ID NO: 44).

Primer 124r: caacacagtgggcagcag (SEQ ID NO: 45).

Three PCR reactions were performed. The first reaction was carried out with primer 75 and primer 122r and plasmid DNA p225-7 as template; As a result of PCR amplification, a product of ~692 bp was obtained, which was purified from the gel. The second PCR reaction was carried out with primer 123 and primer 124r and p225-7 plasmid DNA as template; As a result of PCR amplification, a product of ~237 bp was obtained, which was purified from the gel. A third overlapping PCR reaction was carried out with primers 75 and 124r and a mixture of the products of the previous two reactions as a template; As a result of PCR amplification, a product of ~910 bp was obtained. The resulting overlap PCR product was digested with XbaI and NsiI and religated into plasmid p225-7 (digested with XbaI and NsiI) to yield factor IX-ctp x3 T148A plasmid designated p243-2.

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

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

Dg44 cells were seeded onto 100 mm cell culture dishes and grown to 50-60% confluency. A total of 2 μg (microgram) of FIX cDNA was used to transfect one 100 mm dish using FuGene reagent (Roche) in protein-free medium (Invitrogene CD Dg44). The media were removed 48 h after transfection and replaced with protein-free medium (Invitrogene CD Dg44) without nucleosides in the presence of 800 μg/ml G418 (neomycin). After 14 days, the transfected cell population was transferred to T25 cell culture flasks and selection continued for an additional 10–14 days until the cells grew as stable clones. Clones with high expression levels were selected. Approximately 2 x 10 ⁷ cells were used to inoculate 300 ml of growth medium in a 1700 cm ² roller bottle (Corning, Corning, NY) supplemented with 5 ng/ml vitamin K3 (menadione sodium bisulfate; Sigma). The conditioned medium was collected (harvested product) after cell viability rapidly decreased to approximately 70%. The conditioned medium was first clarified and then concentrated approximately 20-fold and dialyzed against PBS using a flow filtration cassette (10 kDa molecular weight cutoff; Millipore Corp.).

Determination of FIX antigen level. Antigen levels in the resulting FIX-CTP were determined using the AssayMax ELISA FIX Human ELISA Kit (AssayPro-EF1009-1). The calculated protein concentration was the average of three different dilutions in two independent experiments (Fig. 1A, Table 1).

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Table 1. Calculated protein concentration

FIX-CTP FIX-CTP-CTP AG level ΙΊΧ (µl ml) 41?) 19.2 Ciaiuapi noe οι cloppepe (CO) 8.7b 3.67 Variation coefficient ("<>CV) 20/)2 19.15

EF in PAGE/SDS - immunoblotting FIX. Assembled FIX-CTP or purified rhFIX (American Diagnostics), 100 ng of protein, were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Analysis of the gels after EF in PAGE/SDS was carried out by immunoblotting (Western blotting), using a polyclonal antibody to human FIX and a monoclonal antibody that recognizes human gamma-carboxylation of the protein (American Diagnostics). It was previously reported that rhFIX migrated in the gel at 55 kDa, whereas FIX fused to two CTPs migrated at 75 kDa. Both FIX-CTP protein variants were shown to be gammacarboxylated, a post-translational modification required for FIX activity and function (Fig. 1B).

Determination of chromogenic activity of FIX. The in vitro activity of assembled FIX-CTP was compared to rhFIX protein (American Diagnostics) using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221802). In the presence of thrombin, phospholipids, calcium, excess amounts of FXIa activate the assembled FIX into FIXa. FIXa forms an enzymatic complex with thrombin activated by FVIII:C (supplied in excess), phospholipids and calcium and activates Factor X present in the assay system into FXa. Activity directly correlates with the amount of FIX, which is the limiting factor. The specific activity of the resulting FXa towards the chromogenic substrate FXa (pNA) was then measured. The amount of pNA obtained is directly proportional to the activity of FIXa. Serial dilutions of rhFIX and pooled FIX-CTP were prepared and efficacy was assessed by comparing the dose response curve of pooled FIX to a reference formulation consisting of rhFIX or human plasma. The mean EC50 value for FIX was 21 ng/ml, whereas the calculated EC50 value for assembled FIX-(CTP)2 was 382 ng/ml, and the calculated EC50 value for assembled FIX-CTP was 1644 ng/ml. An approximately 15-fold reduction in the enzymatic activity of assembled FIX-(CTP)2 was observed (Fig. 2).

Coagulation activity FIX (aPTT). 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 intrinsic pathway activator, phospholipid, and calcium. The APTT reagent is called partial thromboplastin because tissue factor is not included in the phospholipid, unlike the prothrombin time (PT) reagent. The activator starts the system, and then, in the presence of a phospholipid, the remaining stages of the internal pathway are carried out. The reference aPTT range varies among laboratories, but is typically 27–34 s.

The primary objective of the assay was to quantify the ability of assembled FIX-CTP to restore the coagulation activity of FIX-depleted human plasma by adding rhFIX. 300 μl of FIX-depleted human plasma was mixed with 100 μl of rhFIX or collected FIX-CTP and serial dilutions were prepared. After incubation for 60 s at 37°C, thromboplastin, CaCl ₂ and phospholipids were added to the mixture, and the clotting time in seconds was determined (performed at American Medical Laboratories). Efficacy was assessed by comparing the dose response curve of the assembled FIX with a reference formulation consisting of rhFIX or human plasma. One unit of FIX activity corresponds to the concentration of FIX at which the activity is equal to that of one ml of normal human plasma. The aPTT results presented indicate that FIX-(CTP)2 demonstrated a 5.7-fold reduction in specific coagulating activity compared to rhFIX (Table 2). Moreover, the APTT results together with the results of the in vitro chromogenic activity assay suggest that assembled FIX-(CTP)2 has improved enzymatic activity compared to assembled FIX-CTP (Table 2). Improved activity of FIX-CTP proteins could be obtained after optimization of the expression system (i.e., cotransfection with furin and optimization of vitamin K3 concentration in the medium), which was enhanced after supertransfection with furin (results not shown).

Table 2. Coagulation activity of FIX

rhFIX(AD ) (µg/ml) PTT (sec.) FIX-CTP (µg/ml) PTT (sec.) FIX-CTP-CTP (µg/ml) PTT (sec.) 5 31.3 9 45.2 4 47.5 1.25 35.7 2.25 53.3 1 55.9 0.3125 43 0.5625 64.1 0.25 67 0.078125 52.1 0.140625 76.3 0.0625 77.4

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Pharmacokinetic study. RhFIX (American Diagnostic) and collected FIX-STP were administered as a single intravenous injection to Sprague-Dawle rats (six rats per substance) at a dose of μg/kg body weight (Table 3).

Table 3. PK study plan

Treatment groups Study drug Number of animals/group Route of administration Floor Dose level (µg/kg) Dose level (µg per animal) Injected volume (µl) Conc, (µg/ml) *Time points (hours after administration) 1 rFIX 6 IV m 75 15 500 thirty 0 (BEFORE administration) 0.083, 0.5, 1.5,4, 8, 24, 48, 72. 2 rFIX-PAGE 6 IV m 75 15 500 thirty 0 (before administration) 0.083, 0.5, 1.5,4, 8, 24, 48, 72. 3 rFIX-C TP PAGE 6 IV m 75 15 1000 15 0 (before administration) 0.083, 0.5, 1.5,4, 8, 24, 48, 72.

Blood samples were collected from the retro-orbital sinus of 3 rats alternately at 0.083, 0.5, 1.5, 4, 8, 24, 48 and 72 hours after administration. Plasma was obtained immediately after sample collection and stored at -20°C until analysis. The concentration of FIX was determined using a specific ELISA assay for FIX (AssayPro). A pharmacokinetic profile was calculated for each protein and was the average of 3 animals at each time point (Fig. 3). The terminal half-life was calculated using RK Solutions 2.0 software. In table Figure 4 summarizes the observed FIX concentrations at various sampling times.

Table 4. Observed FIX concentrations

Time (hours) FIX-AD (ng/ml) FIX-STR (ng/ml) FIX-CTP-CTP (ng/ml) 0.083 1506.7 1477.5 1914.8 0.5 1949.8 1150.1 1830.1 1.5 2189.4 1009.0 1264.3 4 733.90 709.33 1000.00 8 319.80 167.20 1234.67 24 NPKO 54.625 230 48 NPKO NPKO 120.9

The PK profile and summary of terminal half-lives are presented in Table. 5. The collected FIX-STP showed improved tV ₂ 3 values compared to rhFIX (2- and 5-fold increase, respectively). Because FIX concentrations in animal sera at 24 hours were below the limit of quantitation (LOQ) in the FIX dose series, additional PK parameters were not calculated.

Table 5. Brief description of PK parameters

Product Final half-life (hours) Ratio (FIX-(CTP)x/rhFIX) rhFIX (American Diagnostics) 2.62 - FIX-PAGE 5.55 2,I FIX-PAGE (FIX-CTP-CTP) 12.9 4.92

This study described a new approach to extend the half-life of FIX while maintaining its therapeutic efficacy. Attaching a CTP peptide to an active protein is potentially dangerous because it can reduce the activity of the protein. Therefore, the production of active recombinant FIX-CTP by adding a CTP sequence at the C-terminus of FIX is unexpected.

Immunoaffinity Purified FIX-CTP-CTP Study.

Cleaning FIX-CTP-CTP.

To evaluate a highly purified protein with increased activity whose PK profile mimics and can be extrapolated to clinical settings, FIX was modified with 2 CTP molecules sequentially attached to the carboxyl terminus to produce FIX-CTP-CTP. FIX-CTP-CTP was purified using a template-bound monoclonal antibody against the gammacarboxyglutamyl (Gla) residues present in the amino-terminal region of FIX (American Diagnostics, catalog number 3570MX). The monoclonal antibody was coupled to CL-4B Sepharose. The collected FIX-CTP-CTP at a concentration of 88 μg/mL was dialyzed against 20 mM Tris, 150 mM NaCl, and 10 mM EDTA at pH 7.4. The column loading rate was 0.5 ml/min, elution was carried out using 20 mM Tris-HCl, 350 mM NaCl and 50 mM CaCl, and the free fraction was purified again five more times. Finally, the eluted fraction was dialyzed against PBS, collected and concentrated.

Determination of FIX antigen level. Levels of assembled FIX-CTP, assembled FIX(CTP) ₂ , and purified FIX-(CTP)2 protein were determined using a human FIX ELISA kit (Affinity Biologics; catalog number FIX-AG RUO). The calculated protein concentration (μg/ml) was the average of two independent experiments (Fig. 4, Table 6).

Table 6. Calculated protein concentration FIX-CTP FIX-CTP-CTP FIX-CTP-CTP (purified) At the level of LH NH (μκιμ.ι) 125.78 88.53 172.0 SO G.28 21.3 1 2.03 o KV 1354 24.08 1.52

In addition, FIX-CTP-CTP quantitation was performed using the Bradford method. The calculated concentration was 202 μg/ml, which was similar to the concentration detected by the human FIX ELISA.

Blots of gels after EF in PAGE/SDS. The collected FIX-CTP-CTP, free fraction, and purified protein were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Coomassie-PAGE/SDS-PAGE analysis was performed by staining the gel with Coomassie Brilliant Blue reagent (800 ng protein). Immunoblotting (Western blotting) was performed with 100 ng of protein, polyclonal antibody (AT) to human FIX and monoclonal AT recognizing human gamma carboxylation of the protein (American Diagnostics, catalog no. 499 and #3570). The immunoaffinity purification procedure significantly enriched the FIX-CTP-CTP content while reducing the amount of impurities (Figure 5).

Amino-terminal sequencing. Purified FIX-CTP-CTP protein was separated by 12% Tris-glycine SDS-PAGE and then electroblotted onto a polyvinylidene fluoride (PVDF) membrane. The strip of interest was excised and placed on a cleaned Biobrene treated glass fiber filter. Amino-terminal sequence analysis was performed by Edman digestion using a pulsed liquid phase protein sequencer equipped with a 140 C microgradient HPLC system. Amino-terminal sequencing revealed that FIX-CTP-CTP was a mixture of proteins with incompletely and completely cleaved propeptide. Incomplete cleavage of the propeptide has been shown to reduce the coagulating activity of FIX. The propeptide cleavage process can be improved by cotransfection with furin.

Determination of chromogenic activity of FIX. The in vitro activity of purified FIX-CTP-CTP protein was compared to rhFIX (American Diagnostics) and pooled normal human plasma using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221802). In the presence of thrombin, phospholipids and calcium, excess amounts of FXIa activate FIX to FIXa. FIXa forms an enzymatic complex with thrombin (supplied in excess), phospholipids and calcium activate factor X present in the analytical system into FXa. Activity directly correlates with the amount of FIX, which is the limiting factor. The amount of FXa produced was measured by its specific activity towards the chromogenic substrate FXa (pNA). The amount of pNA formed is directly proportional to FIXa activity. Serial dilutions of rhFIX, human plasma, and FIXCTP-CTP were prepared and efficacy was assessed by comparing dose response curves (Fig. 6). The mean EC ₅₀ for rhFIX was 68.74 ng/mL, whereas the calculated EC ₅₀ for FIX-CTP-CTP was 505 ng/mL. An approximately 7-fold reduction in FIX-CTPCTP enzymatic activity was observed compared to recombinant FIX and a 16.5-fold reduction compared to collected normal human plasma. This reduced activity can be explained by incomplete cleavage of the amino-terminal propeptide, which was detected by amino-terminal analysis.

Coagulation activity FIX (aPTT). Activated partial thromboplastin time

- 58 044349 (APTT) is a measure of the integrity of the intrinsic and common pathways of the blood coagulation cascade. The aPTT is the time (measured in seconds) it takes plasma to clot after the addition of intrinsic pathway activator, phospholipid, and calcium.

This assay quantified the ability of the FIX-CTP-CTP protein to restore the coagulation activity of FIX-depleted human plasma by adding rhFIX. 300 μl of FIX-depleted human plasma was mixed with 100 μl of rhFIX, FIX-CTP-CTP (FIX-CTP-CTP (CTPs were sequentially attached to the C-terminus)), or pooled normal human plasma, and further diluted. After incubation for 60 s at 37°C, tissue factor (TF), CaCl2 and phospholipids were added to the mixture. Blood clotting time was determined in seconds. Efficacy was assessed by comparing the FIX-CTP-CTP dose response curve with that of the reference drug rhFIX or human plasma. One unit of FIX was taken to be the amount of FIX at which its activity is equal to the activity of 1 ml of normal human plasma.

The aPTT results presented indicate that the coagulating activity of FIX-CTP-CTP is only 1.4 times less than that of pooled normal human plasma and is similar to rhFIX. The APTT results, together with the results of the in vitro chromogenic activity assay, suggest that purification of FIX-CTP-CTP did not interfere with its activity.

Pharmacokinetic activity of FIX-CTP-CTP. Purified FIX-CTP-CTP, rhFIX (American Diagnostic), and collected FIX-CTP-CTP and FIX-CTP were administered as a single intravenous injection to Sprague-Dawle rats (eight rats per substance) at a dose of 100 μg/kg body weight (Table 7).

Table 7. PK study plan

Treatment groups Study drug Number of animals/group/time point and Dose level (µg/kg) Dose level (µg per animal) Injected volume (µl) Conc. (µg/ml) Time points (hours after administration) A rFIX 8 100 20 500 40 0 (before administration) 0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. IN rFIX-CTP (assembled) 8 100 20 500 40 0 (before administration) 0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. WITH rFIX-CTP PAGE (assembled) 6 100 20 500 40 0 (before administration) 0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. D rFIX-CTP PAGE (purified) 4 100 20 500 40 0.083, 0.5 1, 2, 4, 7, 10, 24, 4, 8, 72.

Blood samples were alternately collected from the retro-orbital sinus of 4 rats at 0.083, 0.5, 2, 4, 7, 10, 24, 48 and 72 hours after administration. Citrated plasma (0.32%) was obtained immediately after sample collection and stored at -20°C until analysis. FIX concentration was determined using a human FIX ELISA kit (Affinity Biologics). For each protein, the pharmacokinetic profile was calculated as the average of 4 animals at each time point (Fig. 7). The terminal half-life was calculated using PK Solutions 2.0 software. In table Figure 8 summarizes the observed FIX concentrations at various sampling times.

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Table 8. Observed FIX concentrations Time Assembled Assembled rhFIX, ng/ml Purified (h.) FIX-CTP, FIX-CTP2, FIX-CTP-CTP. ng/ml ng/ml ng/ml 0.085 1038.97 1123.62 325.05 886.48 0.5 939.12 956.80 274.58 670.92 1 791.97 843.85 222.90 674.17 2 304.98 673.31 186.00 503.91 4 315.37 525.50 109.69 357.36 7 171.45 384.36 67.62 257.02 10 50.34 250.73 40.20 158.66 24 10.07 78.50 NPKO 52.13 48 npko 23.40 npko 18.07

A brief description of the PK parameters is presented in Table. 9.

Table 9. Brief description of PK parameters TU ₂ (h.) AUC, ng/h/ml MRT (hours) Vd ml/kg C.L. ml/h/kg Assembled FIX-CTP 4.17 3622 4.5 155.1 27.6 Assembled FIX-STrg 10.44 9105.7 12 165.4 10.9 rhFIX 3.72 1416.8 5.1 373.8 70,183 Purified FIX-CTP-CTP 11.14 6314.2 12.3 254.5 15.83

MRT - mean retention time; Vd - volume of distribution; CL - ground clearance.

Assembled FIX-CTP showed an improved PK profile compared to assembled FIX-CTP. Moreover, purified FIX-CTP-CTP showed a 3-fold increase in T1/2e value and a 4.5-fold increase in AUC compared to rhFIX. The reduced amount of secreted FIX fused to sequential CTP molecules compared to FIX fused to CTP alone appears to be a consequence of the addition of additional CTP rather than impairment of ELISA detection, since the concentration of purified FIX-CTP-CTP calculated by the Bradford method , was similar to the concentration calculated by ELISA.

The clotting activity of FIX-CTP-CTP was similar to that of pooled human plasma; however, its in vitro chromogenic activity was significantly lower than that of rhFIX or pooled human plasma. The chromogenic activity assay is considered a very sensitive assay compared to the blood coagulation assay. The reason for decreased FIX-CTP-CTP activity may vary. The addition of CTP may reduce the affinity of FIX for FXIa or reduce posttranscriptional modifications (e.g., 12–10 GLA residues and propeptide cleavage). Amino-terminal analysis showed that proteolytic cleavage of the FIX-CTP-CTP propeptide was not completely completed before secretion. Since this posttranscriptional modification is important for the normal enzymatic activity of the protein, cotransfection with the furin-PACE plasmid is beneficial and can improve the activity of FIX-CTP-CTP.

Finally, a comparative pharmacokinetic study of FIX-CTP-CTP in rats demonstrated that fusion of two sequential CTPs to the C terminus of FIX produced FIX with an extended half-life.

FIX-deficient mouse model. In order to evaluate in vivo activity, FIX gene knockout mice were generated and a breeding line was created. 10 μg of either commercially available recombinant hFIX (BeneFIX®) or rFIX-(CTP) ₂ (FIX-CTP-CTP) was administered by tail vein injection into an anesthetized (22-28 g) FIX knockout mouse. The amount of protein injected was equal to the required concentration of FIX in normal plasma (5 μg/ml). Blood samples were collected from the dissected tail into heparinized capillary tubes at specific time points. FIX levels in plasma samples were assessed by ELISA and efficacy was measured using aPTT coagulation assay.

Increasing the efficiency of FIX propeptide cleavage. The CTP peptide cDNA was coupled to the 3' end of the human FIX cDNA. The corresponding constructs expressing rFIX and furin were cotransfected into Dg44 cells; Human rFIX cDNA was also cotransfected with a furin expression plasmid as a control. Secretion of high levels of FIX results in the secretion of a mixture of profactor and mature FIX factor, due to the limited amount of furin protease in the cell. Cotransfection of a furin-expressing vector with a profactor-expressing vector increases yield and results in secretion of fully processed FIX into the medium.

After cotransfection with FIX-(CTP)2 and furin, stable clones were obtained and collected for evaluation of propeptide cleavage. 100 ng of protein was loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). The gels were analyzed after EF in PAGE/SDS using immunoblotting (Western blotting), using polyclonal AT to human FIX (American Diagnostics) and a polyclonal antibody to propeptide. It was previously reported that rhFIX migrated at 55 kDa, whereas FIX fused to two CTPs migrated at 75 kDa. They showed that both variants of the FIX proteins underwent correct, complete propeptide cleavage.

To determine whether the enzymatic activity of FIX-(CTP)2 was improved by proper propeptide cleavage, the chromogenic and coagulating activities of assembled FIX-(CTP) ₂ cotransfected with furin were comparatively assessed. Significant improvements were observed in the specific activity of FIX-(CTP) ₂ , which was similar to that of rhFIX.

In conclusion, the results described herein suggest that FIX-CTPCTP can be effectively used to treat patients with hemophilia B. FIX fused to CTP constructs is beneficial due to improved in vivo pharmacological properties that compensate for the disadvantages of some in vitro properties. This proposed treatment has an advantage over known treatments in that it reduces the frequency of infusions and the number of doses required.

It is worth noting that when the strategy of attaching an albumin molecule was used to increase the half-life of FIX, the recombinant FIX became inactive. The proposed new approach resulted in the creation and purification of a new recombinant FIX fusion protein that exhibited improved long-term activity. Because the modifications themselves did not improve the pharmacokinetics of injected FIX, the finding that CTP fused to FIX improved pharmacokinetic parameters was unexpected. The presence of highly glycosylated sialic acid peptide stabilized the protein and protected it from interactions with vascular receptors without interfering with key factors determining FIX function.

FIX-CTP has therapeutic efficacy similar to that of rFIX in patients with hemophilia B and requires less frequent administration. A single injection of FIX-CTP is sufficient to control bleeding episodes and reduce the number of injections required during surgery in patients with hemophilia B.

CTP technology was used to develop long-acting FIX. Specifically, extension of the half-life of the recombinant rFIX molecule was achieved by fusing at least one human CTP with FIX. Recombinant FIX-CTP protein was expressed in mammalian cells and analyzed in vitro and in vivo. We demonstrated that the in vitro activity of rFIX-CTP was comparable to rFIX. Pharmacokinetic and efficacy studies in rats demonstrated improved properties of rFIX-CTP. The results of this study demonstrate that the development of an rFIX molecule with an extended half-life and hemostatic properties similar to those of the wild-type enzyme is feasible.

Example 2: Comparative evaluation of purified FIX-CTP3 against FIX-CTP ₄ and FIX-CTP ₅ .

2.1. Purpose of the study.

Comparative evaluation of pharmacokinetic parameters of FIX-CTP4 and FIX-CTP ₅ compared to FIXCTP3 after partial purification process.

2.2. Receiving assembled FIX-CTP4 and FIX-CTP ₅ .

FIX cDNA (OriGene RC219065) fused at the C terminus to four or five consecutive CTP sequences was expressed in Dg44 cells using the Excellgene expression system in the presence of 10 ng/L vitamin K3 (Sigma, Mennadion). The conditioned media were collected (300 ml), filtered and frozen.

2.3. Receiving assembled FIX-CTP3.

FIX-CTP3 was intrahost expressed in CHO cells using pCI-DFIFR vector, clone 196, BR-9 in the presence of 25 ng/L vitamin K3 (Sigma). The resulting cell suspensions were collected and filtered.

All FIX-CTP samples (3, 4 and 5 CTP) were purified using a Yacalin column only due to material shortage.

2.4. Determination of FIX antigen level.

FIX antigen levels were determined using a human FIX ELISA kit (Affinity Biologics;

- 61 044349 catalog number FIX-AG RUO). The calculated protein concentration was the average of four independent experiments. The concentration of FIX-CTP3 was slightly higher compared to the two additional variants (Table 10).

Table 10. FIX antigen level

3 PAGE Ultimate Yakalin40 4 PAGE Ultimate Yakalin40 5 PAGE Ultimate Yakalin40 Average (ng/ml) 1016.69 4644.11 1686.82 CO 225.41 925.63 160.07 %kv 22.17 19.93 9.49

2.5. Coomassie staining and FIX-CTP immunoblotting.

The assembled FIX-CTP3, FIX-CTP ₄ and FIX-CTP ₅ were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Analysis of gels after EF in PAGE/SDS was carried out by Western blot (immunoblotting), using polyclonal AT to CTP (Adar Biotech Production) or AT to Gla (American Diagnostica).

As previously reported, FIX fused to three CTPs migrated at 80 kDa, whereas FIX fused to four or five CTPs migrated at 85 kDa or 90 kDa, respectively. As expected, assembled FIX-CTP4 and FIX-CTP ₅ obtained from Excellgene showed very low levels of gammacarboxylation compared to assembled FIX-CTP3, which was obtained from Prolor (Fig. 8).

After the purification process using a jacalin column (immunoaffinity purification of glycosylated proteins), FIX-CTP3, FIX-CTP4 and FIX-CTP ₅ were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio -Rad). The gels after SDS-PAGE were stained with Coomassie Brilliant Blue to detect samples. All variants showed much purer band profiles (Fig. 9), suggesting an increase in protein purity.

2.6. Determination of chromogenic activity of FIX.

Comparative in vitro activity assessments of fully purified (GA column) FIX-CTP3, FIXCTP ₄ , and FIX-CTP ₅ versus pooled normal human plasma were performed using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221802). Serial dilutions of all samples were prepared and efficacy was assessed by comparing the dose response curve with that of a reference preparation of normal human plasma. The reduced chromogenic activity of FIX-CTP4 and FIX-CTP ₅ (Fig. 10) compared to plasma may be a consequence of inappropriate post-transcriptional modifications of the FIX proteins, such as inappropriate gamma-carboxylation and propeptide cleavage, or alternatively due to the addition of CTP cassettes. The change in the activity of FIX-CTP4 and FIX-CTP ₅ (Table 11) may be caused by inadequate ELISA quantitation capabilities of FIX due to masking of the CTP site of the antigen.

Table 11. EC50 sample/plasma ratio

Attitude Sample EU50 sample/plasma Plasma 3 STR, final, GA 1 2 4 STR, final, GA 5.35 5 STR, final, GA 2.73

2.7. Pharmacokinetic study.

FIX-CTP3, FIX-CTP4 and FIX-CTP ₅ purified on a Yacalin column (groups A, B and C, respectively) were administered by a single intravenous injection to Sprague-Dawle rats (six rats per treatment group) at a dose of 250 μg/kg body weight. Blood samples were taken from the retro-orbital sinus of 3 rats alternately at 0.083, 0.5, 2, 5, 8, 24, 48, 72 and 96 hours after administration (Table 12). Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis.

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Table 12. PK study plan

Group t 1 1 ί Level ! ί ί Moments in time ί (hours after administration) ! Quantity ! ANIMALS.x/y I group 1 Path introduced ' AND I i doses I (µg per Ϊ animal ί C) 1 Inscir 5 ! Volume (µl) ί Conc. (µg/ml) treatment । Treatment A FIX- i 6 ί i.v. 50 200 250 0.083, 0.5, 2, 5, 8, PAGE*3 24, 48, 72, 96 । Yakalin 1 40 FIX- PAGE *4 0.083, 0.5, 2, 5, 8, IN 6 ( i/v 50 200 250 t Yakalin 1 24, 48, 72, 96 ' 40 ί FIX- PAGE*5 0.083, 0.5, 2, 5, 8, WITH } 6 f i/v 50 200 250 j Yakalin 24, 48, 72, 96

FIX concentrations in plasma samples were quantified using human Elisa FIX kits (Affinity Biologics). The pharmacokinetic profile was calculated, which was the average of 3 animals at each time point. The terminal half-life was calculated using PK Solutions 2.0 software. In table 13 below summarizes the calculated FIX concentrations at various sampling times.

Table 13. Calculated FIX concentrations

Time (hours) Wed. ZTR ng/ml SO ZTR Wed. 4 STR ng/ml CO 4 PAGE Wed. 5 STR ng/ml CO 5 PAGE 0.083 1087.82 72.39 904.54 21.06 1097.23 82.24 0.5 774.18 86.31 736.82 66.93 998.79 70.43 2 562.23 3.70 627.09 32.47 747.85 14.02 5 357.44 8.63 431.23 29.41 576.49 27.36 8 239.20 7.82 327.46 30.26 394.96 36.48 24 77.08 4.26 107.38 5.18 142.42 16.13 48 27.73 2.02 39.83 1.85 53.66 3.33 72 12.55 1.48 21.53 1.55 23.54 3.32 96 6.66 1.23 10.63 0.13 18.54 3.39

The PK profile and a brief description of the PK parameters are presented below in Table. 14 and fig. 11. The complete PK profile at all time points suggested that addition of 4 or 5 CTP cassettes to FIX did not increase the half-life compared to FIX-CTP3. AUC after administration of FIX-CTP5 increased 1.4- to 1.6-fold compared with FIX-CTP3, but this increase was not statistically significant.

Table 14. PK profile and brief description of PK parameters

24 - 96 hours Half-life ('··)_ 20.43 22.02 23.96 AUC (ng/h/ml) 8218.38 10504.49 13329.41 Vd (ml/kg) 700.76 586.02 494.89 CL (ml/h/kg) 23.77 18.45 14.32

Because very low concentrations of FIX were found in samples obtained 96 hours after administration, which were at the lower limit of quantitative detection of the assay, the terminal half-life was recalculated, providing a more accurate and scientifically valid calculation (Table 15). According to this calculation, even the smallest differences were obtained between the half-lives of FIX-CTP3, FIX-CTP4 and FIX-CTP5.

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Table 15. Recalculated terminal half-life

Half-life 15.38 16.63 16.04 < ⁴ ·)

2.8. Conclusions.

This study evaluated the pharmacokinetic parameters and potential coagulation activities of FIX-CTP3, FIX-CTP4 and FIX-CTP ₅ . Fusion of CTP 4 and 5 to FIX did not provide improved or prolonged half-life compared to FIX-CTP3, and a decrease in chromogenic activity was observed. In table 16 below summarizes the percentage improvements in half-life for various FIX-CTP fusion variants (1 to 5 CTP). Fusion of CTP with FIX improved its pharmacokinetic properties, but, unexpectedly, the improvement was limited. Surprisingly, after sequential addition of 3, 4, or 5 CTPs to FIX, similar half-lives were calculated.

Table 16. Summary of percentage improvement in half-life

Option FIX 1U ₂ (8 - 72 hours) % increase rhHX versus KIP 112 KIP compared ..... with 2C 1 R 141 2(11’ vs. 3(11’ : 37 ZTR compared to 4TR b

4(TR compared to 5(11’ o

The results suggest that fusion of 3 CTP to FIX caused the greatest improvement in protein half-life, confirming that FIX-CTP3 represents an optimal option in terms of half-life, structure and potential coagulation activity for further clinical development.

Example 3 Treatment of FIX-CTP3 in the FIX-/- Haemophilus influenzae mouse model.

As described above, a study was conducted in which the PK profile and coagulating activity of the assembled FIX-CTP, FIX-CTP2 and FIX-CTP3 were tested in comparison with rhFIX. FIX-CTP3 exhibited an improved PK profile while maintaining coagulation activity compared to assembled FIX-CTP1 and FIX-CTP2 or rhFIX. In order to further evaluate the obtained result, the FIX-CTP3 protein containing gamma-carboxyglutamate residues was purified. Following a single IV administration of FIX-CTP3 to normal rats, a 3-fold increase in half-life and a 4.5-fold increase in AUC were observed compared with rhFIX. FIX-CTP ₃ showed reduced chromogenic and folding activity in vitro, most likely due to insufficient amino-terminal propeptide cleavage and inappropriate post-transcriptional modifications (PTMs), such as inappropriate gammacarboxylation.

The present study examined the pharmacokinetic and pharmacodynamic properties of recombinant human FIX fused to three sequential CTPs in FIX-null mice.

Purpose of the study.

To determine the pharmacokinetic and pharmacodynamic parameters of rFIX-(CTP) ₃ compared with commercially available rhFIX (BeneFIX®) in FIX-null mice following a single IV administration of FIX-(CTP)3 at a similar specific activity and dose (similar specific activity for FD and similar FIX parameters for FC).

Receiving assembled FIX-CTP3.

FIX cDNA (OriGene RC219065-Thr 148), with three CTP sequences sequentially appended to the C terminus, was expressed in Dg44 cells using the Excellgene expression system in the presence of 25 ng/ml vitamin K3 (Sigma, Mennadion). Five separate batches containing 5 liters of cell suspension were cultured (twenty-five liters total) and harvested after viability had decreased to 60-70%. The collected suspensions were filtered and frozen at -70°C.

Determination of the level of collected FIX antigen.

The level of collected FIX antigen was determined using a human FIX ELISA kit (Affinity Biologics; catalog number FIX-AG RUO). Antigen levels were calculated for each batch. The concentration of FIX was maintained among different batches (Table 17).

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Table 17. FIX antigen level

FIX antigen level The consignment 1 2 3 ( roll. (mkt ml) 28.81 3204 42.0 CO 2.69 4.0 % CV 8.84 8,38,2 9.4

FIX-CTP3 cleaning process.

After a short purification study, the purification process was carried out using the following 3 columns: diethylaminoethanol (DEAE)-Sepharose, heparin-Sepharose and Bio Rad type 1 ceramic hydroxyapatite (HA) (40 μm). The enriched gamma-carboxylated protein FIX-CTP3 was purified. Briefly:

Five liters of clarified collected cell suspension were thawed at 4°C for 4 days. For each purification batch, the clarified pooled cell suspension (2 liters) was concentrated 4-fold and dialyzed against 20 mM Tris-HCl pH 8.2 using a disposable hollow fiber cartridge with a nominal molecular weight cutoff of 10 kDa. This process (UF-DF1) was carried out twice, one liter of UV-DF1 was loaded onto a DEAE-Sepharose column, and factor IX was eluted with 20 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl ₂ , pH 8.2. The resulting product was diluted 1:1 with 20 mM Tris-HCl, 10 mM CaCl ₂ , pH 7.5, and the pH was adjusted to 7.5 before loading onto a heparin-Sepharose column. Elution was carried out with 20 mM Tris-HCl, 300 mM NaCl and 10 mM CaCl ₂ , pH 7.5. The eluted product was concentrated and dialyzed against 10 mM phosphate, pH 6.8, using a Pellicon XL cassette with a 10 kDa molecular weight cutoff membrane (UV-DP2). The resulting product was loaded onto a GA column and the activated factor IX fraction was eluted with 150 mM phosphate, pH 6.8. The purified product was concentrated to a target concentration of 2 mg/ml, dialyzed against Tris-buffered saline (TBS), pH 7.45, aliquoted and stored at -70°C.

The cleaning process was repeated five times weekly to clean the entire volume (25 liters). The purification processes were called GANo. 6-10. Each purified product was evaluated separately (Appendix No. 1-5). At the end of the purification process, the different batches were pooled and further concentrated to a target concentration of 4 mg/mL.

Analytical properties of FIX-CTP ₃ .

Determination of FIX antigen level.

The antigenic level of enriched gamma-carboxylated FIX-CTP3 protein was determined using a human FIX ELISA kit (Affinity Biologics; catalog number FIX-AG RUO). The calculated protein concentration was the average of two independent experiments (Table 18).

Table 18. FIX-CTP ₃ antigen level

ELISA No. 1 purified on a HA column FIX-STRz ELISA No. 2 purified on a HA-column FIXSTRz Final, avg. Razb. 1 2 Avg. Razb. 1 2 Avg. 130000 3412240 3781830 3597035 130000 3692260 3568240 3630250 3613643 260000 3915600 4158440 4037020 260000 3706820 3595540 3651180 3844100 520000 4158544 4334096 4246320 520000 3831464 3530748 3681106 3963713 1040000 4096352 4004104 4050228 1040000 3863392 3684304 3773848 3912038 Avg. (ng/ml) 3895684 4069618 3982651 Avg. (ng/ml) 3773484 3594708 3684096 3833373 CO 338367.5 234486.7 274313.5 CO 86576.66 65369.65 63369.86 154459.6 %kv 8.685703 5.761884 6.887712 %kv 2.294343 1.818497 1.720092 4.029338 Avg. (mg/ml) 3.895684 4.069618 3.982651 Avg. (mg/ml) 3.773484 3.594708 3.684096 3.833373

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ELISA No. 1 purified on a HA-column FIX-STRz ELISA No. 2 purified on a HA-column FIXSTRz Final, avg. Razb. 1 2 Avg. Razb. 1 2 Avg. 130000 3412240 3781830 3597035 130000 3692260 3568240 3630250 3613643 260000 3915600 4158440 4037020 260000 3706820 3595540 3651180 3844100 520000 4158544 4334096 4246320 520000 3831464 3530748 3681106 3963713 1040000 4096352 4004104 4050228 1040000 3863392 3684304 3773848 3912038 Avg. (ng/ml) 3895684 4069618 3982651 Avg. (ng/ml) 3773484 3594708 3684096 3833373 CO 338367.5 234486.7 274313.5 CO 86576.66 65369.65 63369.86 154459.6 %kv 8.685703 5.761884 6.887712 %kv 2.294343 1.818497 1.720092 4.029338 Avg. (mg/ml) 3.895684 4.069618 3.982651 Avg. (mg/ml) 3.773484 3.594708 3.684096 3.833373

Blots of gels after EF in PAGE/SDS.

Enriched gamma-carboxylated protein FIX-CTP3, rhFIX, and rFIXa (activated FIX) were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Coomassie staining analysis of the post-SDS-PAGE gel was performed by staining the gel with Coomassie Brilliant Blue reagent (800 ng protein) (Fig. 12). Immunoblotting (Western blotting) was performed using 100 ng of protein, using a polyclonal AT to human FIX (Fig. 12B), a monoclonal antibody that recognizes human gammacarboxylation of the protein (American Diagnostics, catalog number 499, 3570) (Fig. 12C), polyclonal AT to FIX propeptide (Fig. 12D) and polyclonal AT to CTP (Fig. 12E). FIX-CTP ₃ was previously reported to migrate at 75 kDa.

The purification procedure significantly enriched the FIX-CTP3 content while reducing the amount of impurities. The yield of the purification process was very low and was in the range of about 2-3% (results not shown) due to the need to collect only the gamma-carboxylated fractions of FIXCTP3, as demonstrated by an immunoblot stained with anti-Gla antibodies (Fig. 12B). Based on Coomassie staining and FIX immunoblotting, FIX-CTP3 was only approximately 60-70%, and additional bands of lower molecular weight, presumably less glycosylated forms, were also detected.

Coagulation activity of FIX-CTP ₃ .

Chromogenic activity of FIX-CTP3.

The in vitro activity of assembled FIX-CTP3 and enriched gamma-carboxylated FIX-CTP3 protein was comparatively assessed against pooled normal human plasma using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221802). Serial dilutions of assembled FIX-CTP3 and purified FIXCTP3 protein were prepared and activity was assessed by comparing the dose response curve of the samples with that of a reference drug consisting of normal human plasma. It was previously demonstrated that pooled FIX-CTP ₃ was 50 times less active than pooled human plasma (Table 19, Fig. 13). After purification of FIX-CTP3, the chromogenic activity was significantly improved and was only 4.72 times less than that of pooled human plasma (Table 19, Fig. 13). Reduced chromogenic activity of assembled FIX may be a consequence of inappropriate post-transcriptional modifications of FIX protein variants, such as inappropriate gamma-carboxylation and propeptide cleavage. After purification and enrichment of the gamma-carboxylated fraction of FIX-CTP3, activity improved, demonstrating the significant contribution of gamma-carboxylation to FIX activity.

Table 19. Chromogenic activity of FIX-CTP3

Sample ECso (ng/ml) Attitude ECso sample/plasma Assembled PH-STRz "41.3 54.4 ochpshch. HX( IPs M.b 4.X Pay 13.63 1

One-stage blood coagulation test (APTT).

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 intrinsic pathway activator, phospholipid, and calcium. Main goal

- 66 044349 of this assay is to quantify the ability of FIX-CTP3 to restore the coagulation activity of FIX-depleted human plasma upon addition of rhFIX. 200 μl of FIX-depleted human plasma was mixed with 25 μg/ml FIX-CTP3 and further diluted in TBS. After incubation for 60 seconds at 37°C, 50 μl of PTT activator (Actin FS) and 50 μl of 25 mM calcium were added to the mixture, and the clotting time in seconds was determined using a Sysmex® CA 1500 coagulometer (performed at Shiba Hospital, National Blood Coagulation Center using a validated APTT assay). Efficacy was assessed by comparing FIX-CTP3 to the reference drug pooled normal human plasma dose response curve. Results were expressed as percent activity interpolated from a standard curve covering FIX levels <1 to 110%. A 15-20-fold decrease in the coagulating activity of FIX-CTP3 was found compared to pooled normal human plasma, since the activity at a concentration of 5 μg/ml, which is the normal concentration of FIX in the body, was shown to be 6.5% (Table 20 ).

Table 20. Coagulation activity of FIX-CTP3

FIX-STRz FIX concentration from supplier (mg/ml) Concentration in the tested sample (µg/ml) % FIX activity (normalized to pooled normal human plasma activity) 3.83 25 34.7 5 6.5

FIX-CTP ₃ also showed increased clotting time compared to BeneFIX® (Table 21 and FIG. 14).

Table 21. Comparative blood clotting time (aPTT)

Clotting time

1 I.X-CTP; Beuel IX K 38 µt ml 77.6 19 µt ml 83.4 .b mkt Μ. 1 03.2 5(>.(ι 3.8 µt ml 104.8 57.6 1.9 µt m.ι 1 12.2 63.7 0.95 µt/ml 122.6 71.5 0.4^5 µt m. ι 83.7 0.238 µt ml 64.3

Additional blood coagulation analysis was performed independently on FIX-deficient mice by Dr. Paul Monahan at the University of North Carolina before the start of the PK-PD study. The results of the APTT assay suggested that the coagulating activity of FIX-CTP3 was 40 times less than that of pooled normal human plasma, as demonstrated by the requirement of a longer period (measured in seconds) and a higher concentration for sufficient coagulating activity (Table 22). .

Table 22. Comparative coagulation activity

FIX activity (units)

1 IX-C I r _; VspsNH^ 38 µt'm.· 13.0 19 µt ml 8.8 06 MKT M.I 4 1 16.8 3.8 µt'm.· . 1.6: 67.4 1.9 µt ml 0.0 41.7 0.95 µ1/ml • 0.4.'' 22.4 0.4^5 we ml 8.5 0.238 µt ml 3.7

The specific activity (U/ml), which was calculated based on the FIX antigen level as calculated by ELISA for FIX-CTP3 and BeneFIX®, was 4.46 and 198.9, respectively.

The discrepancy between the calculated FIX-CTP ₃ activity demonstrated in the chromogenic assay compared to the aPTT assay may be explained by the increased sensitivity of the aPTT assay and the relevance of the in vivo assay. In chromogenic activity assays, there are excess reagents and enzymes that may activate less effective FIX variants. The difference in specific activity values of FIX-CTP can be explained by the use of different reagents and automated devices. The activity value calculated at the University of North Carolina was used for the PK-PD study design.

Detection of FIXa protein.

To confirm that FIX (FIXa) was not activated after the cleaning process,

- 67 044349 FIXa detection assay using the FIXa Biophen chromogenic assay (cat. no. 221812). This assay measures the amount of FIXa present in a particular sample using the cascade of chromogenic activity described previously. FIX-CTP3 and rhFIX were diluted and FIXa levels were assessed. FIXCTP3 was not activated during purification or storage (Table 23).

Table 23. FIXa detection

Sample FIX- STRz rhFIX Original 1000 5.7 horses, (mgml) ι·|·ΊХа (mg ml) NICO 0.0048 % I IХа in NICO 0.085

image

FIX-CTP3 PK-PD study.

FIX-CTP3 and rhFIX (BeneFIX®) were administered by a single intravenous injection to FIX-null C57BI mice at a dose of 625 μg/kg body weight containing 100 IU FIX/kg body weight. Blood samples were collected from the retro-orbital sinus of 3 mice alternately at 0.25, 4, 24, 48, 72 and 96 hours after administration. Citrated plasma (0.32%) was obtained immediately after sample collection and stored at -20°C until analysis. hFIX antigen levels were assessed and a thorough PK analysis was performed. To evaluate the ability of FIX-CTP ₃ to prolong coagulation activity in FIX-null animals compared with BeneFIX®, FIX activity was calculated in citrated plasma samples collected from treated FIX'/' mice using an automated FIX activity assay (Table 24). ).

Table 24. Study design

Producing CT Introduction Dose Number of mice Collection points (hours after administration) Required amount **Group 1 FIXSTrz Single dose: i.v. 100 IU/kg 2.5 IU/mouse (553 µg/mouse) 12 mice 0.25, 1.4.8, 16, 24.48 6636 mcg Group 2 FIXSTrz Single dose: i.v. **472 mcg/kg 12.57 µg/mouse 18 mice *0,25,1*, 4*,8 *, 16*, 24*,48*, 72*, 96* 200 mcg 12.57 µg/mouse **Group 3 BeneFI X® Single dose: i.v. 100 IU/kg 2.5 IU/mouse 18 mice 0.25, 1,4,8, 16, 24, 48, *72, *96 226.3 µg 12.57 µg/mouse

* PK results collection points only ** Bleeding from the tail vein at T=48 hours after administration; groups 1 and 3

Pharmacokinetic profile of FIX-CTP3 in FIX’/’ mice.

FIX concentrations were calculated using human FIX ELISA kits (Affinity Biologics; catalog number FIX-AG RUO). A pharmacokinetic profile was calculated for each protein and was the average of three animals at each time point. Below in the table. 25 and in fig. Table 15 summarizes the calculated FIX concentrations at various sampling times for groups 1 and 3. The PK profile and a brief description of the PK parameters are presented below (Tables 26 and 27). PK analysis was also performed on group 2 to test the effect (results not shown).

- 68 044349

Table 25. Co: concentration FIX Moment FIX-STRz BeneFIX® time (hours) ng/ml ng/ml 0.25 3645.397 2823.023 1 241 1.00 2416.248 1703.205 1506.228 8 1 139.730 804.764 1I1® 415.32 dimm 24 238.37 158.7973 36 141.0105 94.40067 48 95.461 42.28833 alone 76.90953 96 24,955 npko

The two-compartment module (WinLin software) was used to determine the AUC _{of 0-6eCKOHe4} ., T _final . and clearance (CL). PK parameters are described below in table. 26.

Table 26. PK properties

Option FIX TU ₂ a (1/h.) 1½ β (1/h.) AUC ng/ml*h. CL ml/kg/h. MRT (hours) Vss (ml/kg) Benel lX k 3.4 12.7 22428 20 1 1.5 320.8 FIX-STRz 4 28.7 31770 19 22 425.2

Vss is the stationary volume of distribution.

Addition of three CTP cassettes to rhFIX extended the in vivo half-life of FIX by at least 2.5-fold. AUC after administration of FIX-CTP3 in vivo increased 2-fold compared to administration of rhFIX. Mice treated with FIX-CTP3 showed an improved PK profile compared to mice treated with BeneFIX®.

Pharmacodynamic profile of FIX-CTP ₃ in FIX-null mice.

In parallel with PK sampling, citrated plasma samples from FIX-null animals treated with either BeneFIX® or FIX-CTP3 were assessed for coagulation activity by APTT assay and converted to % activity. % activity at each collection time was calculated as current clotting time/clotting time of pooled normal mouse plasma*100. In table 27 summarizes activity values following administration of either BeneFIX® or FIX-CTP3.

Following administration of FIX-CTP ₃ , significant clotting activity was detected one hour post-administration, reaching 96% activity at 4 hours post-administration, whereas BeneFIX®'s highest activity value was 40% (Table 27, FIG. 16). The coagulation activity of FIX-CTP3 was maintained over a longer period of time, demonstrating prolonged activity. Coagulation activity in mice treated with BeneFIX® was not detected at time points after 36 hours, whereas mice treated with FIX-CTP3 continued to have measurable activity 72 hours after administration (Table 27, FIG. 16). Analysis of the pharmacokinetic profile of % clotting suggests that the clotting activity of FIX-CTP ₃ persists for a significantly longer period of time, and its half-life is almost 2 times higher than that of Benefix® (Table 28).

Table 27. % FIX activity

Hours after administration % BeneFIX® activity % FIX-STRz activity

0.25 39.9 1.0 1 33.4 15.5...... 8 18.8 65.2 16 24 1.7 11.9 36 48 <1 4.6 72 1 1.4

- 69 044349

Table 28. Clotting activity

Option FIX Τ'Λσ (1/h.) 1½ β (1/h.) BeneFIXk 5.7 — FIX-STRz 7.3 16

Bleeding test in FIX-null mice.

FIX-deficient mice were given a single intravenous injection of 100 IU/kg BeneFIX® or rFIX-CTP ₃ . The tail vein was slightly dissected 48 hours after injection, and the tail vein bleeding time (TCVT) and bleeding intensity (hemoglobin O.B.) were assessed. A second bleeding test was performed 15 min after homeostasis was achieved and the same parameters were measured. After the first bleeding test, FIX-CTP3-treated animals bled significantly less than BeneFIX®-treated animals, as demonstrated by O.P. values. hemoglobin (Fig. 17).

Because it has been previously described that during the first bleeding test in hemophiliac mice, bleeding time does not necessarily correlate with treatment response, it is recommended that homeostasis be assessed after additional bleeding. Once the first bleeding stopped spontaneously or was stopped manually, a second bleeding test was performed 15 minutes after the first and the time and intensity of bleeding were measured again. During the second episode of bleeding in animals that received FIX-CTP ₃ , the time and intensity of bleeding was less, demonstrating that FIX-CTP ₃ was effective at later time points (Fig. 18).

Finally, the animals were further monitored for 12 hours after the second bleeding test, and all repetitions of bleeding episodes were recorded. Animals treated with FIX-CTP3 were able to maintain blood homeostasis over the next 12 hours without recurrent bleeding episodes. In contrast, 50% of mice treated with BeneFIX® experienced spontaneous episodes of tail bleeding (Table 29).

Table 29. Result 12 hours after tail section

Group of mice Delayed rebleeding Death or severe impairment requiring euthanasia FIX-STRz (100 IU/kg) 0/5 (0%) 0/5 BeneFIX® (100 IU/kg) 3/6 (50%) 0/6 FIX-/- (not treated) 5/6 (100%) 1/6

Recombinant FIX-CTP3, a fusion protein consisting of a single FIX molecule fused to three CTP cassettes linked in series, was developed to address the short half-life of currently available FIX products used to treat patients with hemophilia B. We demonstrated that the half-life The half-life of rFIX-CTP ₃ was consistently 2.5-4 times greater than that of rFIX in rats (as previously reported) and in FIX-null mice. Without being bound to any particular theory, the fusion protein reduced the clearance of FIX and protected FIX from protease activity, degradation through masking, and reduced the affinity of FIX for liver receptors. Collectively, these properties of the CTP domain increase the half-life of FIX.

In addition to the pharmacokinetic analysis of rFIX-CTP ₃ , we examined the pharmacodynamic properties of FIX-CTP ₃ in FIX-null mice. rFIX-CTP ₃ and rFIX were administered at comparable doses (in units) to compensate for deficient coagulation levels in mice lacking FIX. However, the effects of rFIX-CTP3 in FIX-null mice were significantly increased until at least 76 h postadministration, reaching a higher peak of activity. The coagulation activity of FIX-CTP3 began to appear with a delay of 1 hour compared to BeneFIX®. Activation of FIX may be necessary because the addition of three consecutive CTPs could theoretically mask the activation site and delay the onset of the cascade. After administration of FIX-CTP3, a peak of 100% activity was observed, whereas BeneFIX® activity was only 40%. Higher initial activity is a very important parameter, and it has been demonstrated that the addition of 3 CTP can potentially improve yield.

The goal of prophylactic FIX replacement therapy in patients with hemophilia B is to maintain plasma levels of 1-2% of normal clotting activity. The Tail Vein Bleeding Assay is a sensitive in vivo test that measures the ability to maintain coagulation homeostasis at low levels of activity, simulating a homeostasis model

- 70 044349 blood coagulation in humans. In response to a tail vein bleeding test 48 hours after administration, animals administered rFIX-CTP ₃ maintained coagulation homeostasis with shorter and less severe bleeding episodes, demonstrating prolonged coagulation activity.

FIX is a complex protein that contains multiple functional domains that undergo a large number of post-translational modifications. One of the post-translational modifications required for FIX activity is gamma-carboxylation of the first 12 glutamic acids in the Gla domain by vitamin K-dependent gamma-glutamyl carboxylase. This modification facilitates the binding of FIX to phospholipid membranes and is thus important for its function. FIX that is not gamma-carboxylated is not functional, and therefore gamma-carboxylation is the rate-limiting step of the reaction.

This PK-PD study was performed using transiently transfected cells. Extensive analytical evaluation of post-translational modifications was performed on the stable FIXCTP ₃ protein produced and secreted by the stable optimized clone.

Based on the results presented, the coagulation factor FIX-CTP3 has the potential to reduce the frequency of injections in patients receiving routine prophylactic doses of FIX replacement therapy. It is expected that rFIX-CTP ₃ may provide prolonged protection against bleeding after each factor injection, reduce the total number of factor units required to treat bleeding episodes, and/or maintain adequate hemostasis during surgical procedures after fewer injections.

Example 4. Preparation and use of blood clotting factor FVII.

A long-acting version of activated coagulation factor VII (FVIIa) would be useful for the treatment of patients with hemophilia A and B. Recombinant FVIIa-CTP ₃ protein has clinical potential to improve the treatment of patients with hemophilia by reducing the frequency of infusions and even by reducing the drug load, making a possible preventive treatment approach that can significantly improve the patient's quality of life, avoid spontaneous bleeding episodes and accumulate damage to joints and other organs.

This text describes the production of a recombinant FVIIa-CTP molecule with an extended half-life based on the fusion of FVII with human CTP. Recombinant FVIIa-CTP was expressed in mammalian cells and characterized in vitro and in vivo. It was demonstrated that the activity of rFVII-CTP was comparable to rFVII. Pharmacokinetic and efficacy studies in rats demonstrated improved properties of rFVII-CTP. The results of this study demonstrated that the development of an rFVIIa molecule with an extended half-life and hemostatic properties very similar to the wild-type enzyme is feasible.

Cloning and expression of a recombinant FVII molecule.

Several factor VII clones were constructed in our eukaryotic expression vector (pCI-dhfrr) (Fig. 19). An MGC-verified human FL cDNA clone (IRCM) incorporating homo sapiens coagulation factor VII sequence was ordered from Open Biosystems (OB-MHS4426). The following primers were synthesized at Sigma-Genosys with the following sequences: primer 67: 5'CTCGAGGACATGGTCTCCCAGGCCC3' (includes 5' end of factor VII DNA and XhoI restriction site) (SEQ ID NO: 5); primer 68R: 5'TCTAGAATAGGTATTTTTCCACATG3' (includes XbaI restriction site) (SEQ ID NO: 6); primer 69: 5' TCTAGAAAAAAGAAATGCCAGC3' (includes XbaI restriction site) (SEQ ID NO: 7); and primer 70R: 5'GCGGCCGCATCCTCAGGGAAATGGGGCTCGCA3' (includes the 3' end of factor VII DNA and a NotI restriction site) (SEQ ID NO: 8).

Cloning was carried out in two series of PCR reactions. The first reaction was carried out with primer 67 and primer 68R using a cDNA plasmid with the factor VII sequence (OBMHS4426) as a template; As a result of PCR amplification, a product of ~534 bp was obtained, it was isolated and ligated into the TA cloning vector (Invitrogen, catalog number: K2000-01). The XhoI-XbaI fragment was isolated, including the sequence of the N-terminus of factor VII. A second reaction was carried out with primer 69 and primer 70R, and again the cDNA plasmid with the factor VII sequence (OBMHS4426) was used as a template. As a result of PCR amplification, a product of ~813 bp was obtained. and ligated it into a TA cloning vector (Invitrogen, catalog number: K2000-01). The XbaI-NotI fragment was isolated, including the sequence of the carboxyl terminus of factor VII. The resulting two fragments were inserted into our eukaryotic expression vector pCI-dhfr (triple ligation) to obtain clone 501-0-p136-1.

Plasmid 501-p136-1 (factor VII in the pCI-dhfr vector) was digested with the restriction enzymes XhoI and KpnI. A fragment of ~1186 bp was isolated. A partial factor VII clone (bp 1180-bp 1322) followed by a CTP sequence, a termination sequence, and a NotI sequence, which was synthesized at GeneArt (0721543), was digested with the restriction enzymes KpnI and NotI. A fragment of ~253 bp was isolated. The resulting two fragments were inserted into our eukaryotic expression vector pCI-dhfr (triple ligation) to obtain clone 501-1-p137-2. pCI-dhfr-701-2-p24-2 was digested with restriction enzymes XhoI and ApaI and a large fragment (vector) was isolated.

- 71 044349 pCI-dhfr-501-2-p137-2 (factor VII-ctp x1) was digested with restriction enzymes XhoI and ApaI and an insert of ~1200 bp was isolated. The vector and insert were ligated to produce 501-2-p139-2. Dg44 cells were seeded onto 100 mm cell culture dishes and grown to 50-60% confluency. A total of 2 μg of DNA was used to transfect one 100 mm dish using FuGene reagent (Roche) in protein-free medium (Invitrogen CD Dg44). The medium was removed 48 h after transfection and replaced with protein-free medium (Invitrogen CD Dg44) without nucleosides. After 14 days, the transfected cell population was transferred to T25 cell culture flasks, and selection was continued for 10-14 days until the cells began to grow well as stable clones. Clones with high expression levels were selected and approximately 2x10' cells were used to inoculate 300 ml of growth medium in a 1700 ^cc roller bottle (Corning, Corning, NY) supplemented with 5 ng/ml vitamin K3 (menadione sodium bisulfate; Sigma). Conditioned medium was collected after cell viability rapidly decreased to approximately 70%. The conditioned medium was first clarified and then concentrated approximately 20-fold and dialyzed against PBS using a flow filtration cassette (10 kDa molecular weight cutoff; Millipore Corp, Billerica, MA).

Determination of FVII antigen level.

The cDNA encoding the CTP peptide was fused to the 3' end of the cDNA encoding human FVII. Dg44 cells were transfected with the corresponding rFVII construct. Human rFVII cDNA was used as a control. The conditioned medium was collected, concentrated, and secreted recombinant FVII was further assessed. Levels of rFVII, rFVII-CTP and rFVII-CTP-CTP antigens were determined using the AssayMax human FVII ELISA kit (AssayPro) (Fig. 20A). No significant difference was observed in the level of secretion of rFVII-CTP and rFVII-(CTP)2 compared to native rFVII.

Blots of gels after EF in PAGE/SDS.

The EF/SDS-PAGE assay was performed by loading 50 ng of either assembled, purified, or activated rFVII protein. Samples were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Post-SDS-PAGE gel analysis was performed by immunoblotting (Western blotting) using anti-human FVII monoclonal antibody (AT) (R&D Systems) or rabbit polyclonal anti-CTP antibody.

The level of rFVII antigen correlated with the detected protein level on gels after EF in SDS-PAGE, subjected to immunoblotting with anti-FVII AT. rFVII-CTP migrated as a single band, whereas the corresponding molecular mass of the FVII control was approximately 52 kDa (results not shown). Both proteins reacted with FVII-specific antibodies on immunoblots. RFVII-CTP also reacted with CTP-specific antibodies. rFVII was secreted as a zymogen, with no trace of activated protein present.

Chromogenic activity of FVII.

The activities of the collected rFVII, rFVII-CTP and rFVII-(CTP)2 were determined using a commercially available chromogenic assay kit (AssaySense Human FVII Chromogenic Activity Assay Kit (AssayPro)). To functionally characterize rFVII-CTP and its ability to be further activated (FVIIa), concentrated collected rFVII-CTP was placed in a commercially available chromogenic assay kit, which measures the ability of TF/FVIIa to activate factor X to factor Xa, resulting in the presence of specific substrate, FXa releases a signal, the amount of which is determined (AssayPro). Addition of the CTP peptide to the C-terminus of the rFVII protein did not impair FVII activity as a serine protease (Fig. 20B, 20C).

Coagulation activity of FVII.

Prothrombin time (PT) is a measure of the extrinsic coagulation pathway. The PT is the time (measured in seconds) that plasma takes to clot after the addition of extrinsic pathway activator, phospholipid, and calcium. It is used to determine the propensity of blood to clot, particularly to measure warfarin dosage, liver damage, and vitamin K status. The reference range for prothrombin time is usually approximately 12-15 seconds. Specifically, this assay quantified the ability of collected FVII-CTP and FVII-(CTP)2 to restore the coagulation activity of FVII-depleted human plasma by adding rhFVII. 300 μl of FVII-depleted human plasma was mixed with 100 μl of pooled FVII, FVII-CTP, and FVII-(CTP)2 at specified concentrations, or pooled normal human plasma, and further diluted. After incubation for 60 s at 37°C, tissue factor (TF), CaCl ₂ and phospholipids were added to the mixture. Blood clotting time was determined in seconds. Efficacy was assessed by comparing the dose response curve of collected FVII-CTP and FVII-(CTP)2 with a reference formulation consisting of rhFVII or pooled human plasma. One unit of active FVII was defined as the amount of FVII whose activity was equal to the activity of one ml of normal human plasma. The PT of coagulation activity of rFVII and rFVII-CTP was measured using a coagulometer (Instrumentation Laboratory).

Previously, it was shown that the addition of the CTP peptide to the C-terminus of the rFVII protein did not disrupt its activity as a serine protease and led to stimulation and activation of native factor X and factor IX in plasma

- 72 044349 people. After addition of additional CTPs at the C terminus, a threefold decrease in serine protease activity was observed (results not shown).

Pharmacokinetic study.

The collected rFVII, rFVII-CTP and rFVII-(CTP)2 were administered intravenously to Sprague-Dawle rats (six rats per substance) at a dose of 100 μg/kg body weight. For all in vivo experiments, the corresponding protein was quantified using an FVII ELISA kit. For each FVII test substance, the injectable amount was calculated taking into account the differences in molecular weight between rFVII and rFVII-CTP, which lead to different molar concentrations.

Blood samples were collected from the retroorbital sinus using a modified sampling schedule to minimize the influence of the sampling procedure on the measured levels: alternately from 3 rats at 30 and 90 min and at 2, 6 and 48 h, and from the remaining three rats at 15 and 60 min and after 1.5, 4 and 24 hours. Plasma was obtained immediately after sample collection and stored at -20°C until analysis. FVII concentrations were quantified using an FVII-specific ELISA. Half-life and area under the pharmacokinetic curve (AUC) were calculated using the linear trapezoidal rule. Comparison of these clearance parameters showed that the in vivo half-life and AUC for rFVII-(CTP) ₂ were significantly higher than those for rFVII (Table 30).

Table 30. PK study parameters

Group Path Dose 1½ AUCo-t CL/F MRT MCT CT min 111 MIN Μ. 1 M.l MIN KT min FVII IV 60 4.07 3314.7 6,195 6.2 IVHf IP 13 51.06 3 1353.0 0.287 73.7 FVIISTRIST IV 60 β=13.66 7626.8 1.18 15.4

CL/F is the ratio of clearance to the degree of absorption (filtration).

Characterization of recombinant FVIIa-CTP.

During activation, FVII is cleaved at position R152, resulting in heavy and light chain domains that are linked by a single disulfide bond. rFVIIa-(CTP)2 was purified and activated using an ion exchange column purification process. In order to fully evaluate rFVIIa-(CTP) ₂ , the protein was loaded onto a reducing SDS-PAGE gel that was also loaded with commercially available FVIIa (NovoSeven®). The heavy and light chain domains separated and migrated as two distinct bands with molecular masses of 55 and 25 kDa. Both proteins reacted with FVII-specific antibodies, but the rFVIIa-CTP heavy chain reacted specifically with CTP-specific antibodies, suggesting that this band represents the FVII heavy chain fused to CTP. The light chain reacted specifically with AT to gammacarboxylase. FVIIa protein concentration was determined using an FVIIa-specific ELISA kit.

N-terminal sequencing of FVIIa.

Purified rFVII-CTP-CTP proteins in activated or zymogenic form were separated by SDS-PAGE (12% Tris-glycine gel) and then electroblotted onto a PVDF membrane. Bands of interest were excised and placed on a clean, Biobrene-treated glass fiber filter. Amino-terminal sequence analysis was performed by Edman digestion using a pulsed liquid-phase protein sequencer equipped with a 140 C microgradient HPLC system. The identity of the recombinant protein and correct propeptide cleavage were further verified by amino-terminal sequencing.

Coagulation activity of FVIIa.

To evaluate the coagulating activity of FVII-(CTP) ₂ , an activated partial thromboplastin time (aPTT) assay was performed. The FVII-depleted plasma sample was spiked with rFVIIa (NovoSeven®) or rFVIIa-(CTP)2. 300 μl of FVII-depleted human plasma was mixed with 100 μl of FVIIa or rFVIIa-(CTP) ₂ at specified concentrations, or normal pooled human plasma, and further diluted. After incubation for 60 s at 37°C, tissue factor (TF), CaCl2 and phospholipids were added to the mixture. Blood clotting time was determined in seconds. Efficacy was assessed by comparing the dose response curve for rFVIIa-(CTP) ₂ with that of a reference formulation consisting of rhFVIIa or pooled normal human plasma. One unit of FVIIa was defined as the amount of FVIIa whose activity was equal to the activity of 1 ml of normal human plasma. The aPTT coagulation activity of rFVII and rFVIIa-(CTP) ₂ was measured using a coagulometer (Instrumentation Laboratory). The aPTT coagulation activity of rFVIIa and rFVIIa-(CTP) ₂ was similar.

Pharmacokinetic studies in rats.

To characterize the effect of adding CTP to rFVIIa to increase its half-life, a comparative pharmacokinetic study was performed in rats. NovoSeven® (rFVIIa) and

- 73 044349 rFVIIa-(CTP)2 in TBS was administered by intravenous injection to 6 SD rats. FVIIa levels over time were detected using an FVIIa ELISA kit. Half-life and AUC were calculated for each protein. Comparison of the obtained clearance parameters showed that the in vivo half-life, yield and AUC values for rFVIIa-(CTP)2 were superior to those for NovoSeven®.

Model of FVIIa-CTP efficacy in vivo (model of hemophilia in mice lacking FVIII).

To evaluate the in vivo activity pattern, FVIII gene knockout mice were generated and a breeding line was established. 10 μg of either commercially available recombinant hFVIIa (NovoSeven®) or rFVIIa-(CTP)2 was administered by tail vein injection into an anesthetized FVIII gene knockout mouse (weighing 22–28 g). The amount of injected protein was equal to the required concentration of FVIII in normal plasma (5 μg/ml). Blood samples were collected from the dissected tail into heparinized capillary tubes at specific time points. FVIIa levels in plasma samples were assessed using ELISA and potency was measured using a PTT coagulation assay. In this study, a FVII fusion construct with CTP was generated. This recombinant protein was a core treatment component that provided an extended half-life and maintained therapeutic efficacy.

These results suggest that rFVIIa-(CTP) ₂ has therapeutic efficacy similar to that of rFVIIa in patients with hemophilia. Moreover, this technology requires less frequent administration. A single injection of rFVIIa-(CTP)2 appears to be sufficient to control bleeding episodes and reduce the number of injections required during surgery. This recombinant protein can be used for long-term preventive treatment.

Example 5 Comparative evaluation of purified FVII-CTP ₃ , FVII-CTP ₄ and FVII-CTP ₅ .

5.1. Purpose of the study.

Comparative assessment of the pharmacokinetic parameters and coagulation activity of FVII-CTP ₄ and FVII-CTP5 compared with FVII-CTP3.

5.2. Preparation of assembled FVII-CTP4 and FVII-CTP5.

FVII cDNA fused at the C terminus with four or five CTP sequences fused in series was expressed in Dg44 cells using the Excellgene expression system in the presence of 20 μg/L vitamin K3 (Sigma, Mennadion). The conditioned media were collected (300 ml), filtered and frozen.

5.3. Obtaining assembled FVII-CTP ₃ .

FVII-CTP3 was expressed intrahost in a mammalian expression system, in CHO cells, using the pCI-DHFR vector. Stably transfected pool #71 was grown in shake flasks in the presence of 25 ng/L vitamin K3 (Sigma). The resulting cell suspensions were collected and filtered.

All collected FVII-CTPs (3, 4, and 5 CTPs) were concentrated and dialyzed against TBS (50 mM Tris, 150 mM NaCl, pH 7.4) using a Pellicon XL cassette with a molecular weight cutoff of 10 kDa.

5.4. Determination of FVII antigen level.

FVII antigen levels were determined using a human FVII ELISA kit (Zymotest HyPhen) (Table 31). The calculated protein concentration was the average of two independent experiments.

Table 31. Level of FVII antigen (p. (Ill ml) 224^7 1 X”XS4 I W423 (O 44^ ^ι >ς ^4χ - o, cch

5.5. FVII-CTP immunoblotting.

The collected FVII-CTP3, FVII-CTP4, and FVII-CTP5 were loaded onto a 12% Tris-glycine gel (expedeon), which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Analysis of the gels after EF in SDS-PAGE was carried out using Western blotting (immunoblotting), using polyclonal AT to CTP (Adar Biotech Production) or AT to Gla (American Diagnostica). FVII fused to three, four, and five CTPs migrated at 80, 90, and 100 kDa, respectively. As expected, the assembled FVII-CTP4 and FVII-CTP5 obtained from Excellgene had low gammacarboxylation compared to the assembled FVII-CTP ₃ that was obtained from Prolor, since the preparation process was not optimized (Fig. 21).

5.6. Comparative assessment of FVII activity in vitro.

Comparative in vitro activity assessment of GA column purified (highly gamma-carboxylated fraction) FVII-CTP ₃ , FVII-CTP ₄ and FVII-CTP ₅ compared to pooled normal human plasma was performed using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221304). Serial dilutions of all samples were prepared and activity was assessed by comparing the dose response curve of the samples with that of a reference drug consisting of normal human plasma. FVII-CTP3 and FVII-CTP5 showed lower chromogenic activity than pooled normal plasma (FIG. 22).

FVII-CTP4 showed higher activity, as shown by EC50 ratios, compared to FVII-CTP3 and FVII-CTP5 (Table 32).

Table 32. Coagulation activity of FVII in vitro

ll.ia nia ().()> 1 VII 3(I P <>.I2 I VI1 4( I P 1 VII 5( I P O.O3 (1.(1(1

5.7. Coagulation activity of FVII in vitro.

Analysis of factor VII (FVII) activity, which was carried out at the Medical Center. Shiba, at the National Blood Coagulation Center of Israel, was a prothrombin (PT)-based assay using factor VII-depleted immunosorbed plasma (Siemens). The PV reagent was innovin and the analysis was performed using a CA 1500 Sysmex® device. The normal range for FVII was 55-145%.

Table 33. Chromogenic activity of FVII in vitro

l\ II 3(Ί P 36 0.5 224 2 ——.——— 0.25 6 ||j[^ 1\II 4( 1 P ——— 8" o 0.25 93 1\II 5(Ί P _____ YU 0.5 .............. 0.125 >8 o

Since the normal level of circulating FVII in the body is approximately 0.5 μg/ml, a 3-fold reduction in coagulating activity of pooled FVII-CTP3 and FVII-CTP5 was found compared to pooled normal human plasma; this result correlates with the obtained chromogenic activity (Table 33).

Pooled FVII-CTP4 exhibited a 3-fold increase in potential coagulating activity compared to pooled normal human plasma as observed in the chromogenic activity assay (Table 33). The percentage of activity of FVII-CTP ₄ was much higher than that of FVIICTP3 and FVII-CTP5. Methodological limitations of the ELISA method may limit the accuracy of FVII-CTP4 AT level calculations.

5.8. Pharmacokinetic study.

Two pharmacokinetic studies were conducted to determine the pharmacokinetic (PK) parameters of FVII-CTP3, FVII-CTP4 and FVII-CTP5. In the first study, FVII-CTP3, FVII-CTP4 and FVII-CTP5 (groups A, B and C, respectively) were administered as a single intravenous injection to Sprague-Dawle rats (six rats per treatment group) at a dose of 250 μg/kg body weight bodies. Blood samples were taken from the retro-orbital sinus of 3 rats alternately at 0.083, 0.5, 2, 5, 8, 24, 48, 72 and 96 hours after administration (Table 34). Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis.

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Table 34. Pharmacokinetic Study Design - Concentrated Composite Product

Group Explore Quantity Pu n> Level Inyenpro Conc. Moments of time . 1hsppya we are in lively ι puff/ vve, ie .uses bathroom (µg/m (hours after iipcnapai 1 ru inu/moment of time stump (mkt pa animal |1||D about whom (mkl) introduction) FVII- PAGE*3 V'V And 200 250 0 (before administration) 0.083. 0.5, 2, 5. 8, 24, 48, 72, 96 eid Ι·'VII- C'1P*4 V'V 111i 250 0 (before administration) 0.083. 0.5. 2, 5. 8, 24, 48, 72, FVIT- PAGE*5 V'V 200 250 0 (before administration) 0.083. 0.5.

Quantitative analysis of FVII concentrations in plasma samples was performed using human FVII ELISA kits (Zymutest FVII-Biophen). The pharmacokinetic profile was calculated and was the average of 3 animals at each time point. Terminal half-life values were calculated using PK Solutions 2.0 software. Below in the table. Figure 35 summarizes the calculated FVII concentrations at various sampling times. The PK profile (Figs. 23-24) and a brief description of the PK parameters (Table 36) are also presented below. FVII-CTP5 showed a better profile compared to FVII-CTP3 and FVII-CTP4 (Table 36).

Table 35. First pharmacokinetic study - FVII concentrations

Time nivmi SO (ed. ||||^^ 1\ 11-4- NNVNNNNNVV· (Ill ml) VNVVINVN· 0.083 4214 5X3 3600 42" 4888 504 0.5 3386 8<>2 5213 1682 5384 2>4 ¹ > 2 1138 2G> 3603 133Х 3082 28h 5 1390 3”4 226 112" 2480 561 8 333 16" 1349 44 2316 631 24 133 12 46 ¹ >8 88 U 48 38 3 165 24 384 2 12 2 91 62 16 ^1 96 IV|!^ 42 8 93 Table 36. Pharmacokinetic analysis 1\ PZ-( GR 1\ 11-4-( II’ l \ P-5( 11’ 1lepiyu i Ί.) but ι\ ,ι, and chin (0.083 - 8 2 5 6.6 1lepiyu i ।io.ι\ /КН ιιιιι (8 - 2 hours) 13.3 16.6 G." A1 C(n i h'ml) (8 - 2 hours) 18374.0 5 1224.4 72654.2 \d (ml hc) (8 - 2 hours) 2430 O| O 6" ( 1. (m.b h κι (8-72 h.) 1 i.o. 2 7

The addition of four or five CTPs significantly extended the half-life of FVII compared to the addition of 3 CTPs by a factor of 2 and 3, respectively (Table 36). This superiority was most important in the initial portion of the study (0.083-8 h), suggesting potentially improved protein yield and reduced extravascular clearance. The AUC following administration of FVII-CTP4 and FVII-CTP5 increased 3-fold and 4-fold, respectively, compared with FVII-CTP3. Clearance also decreased with the addition of 4 and 5 CTP to FVII (Table 36).

In this study, it was observed that the addition of four and five CTPs significantly prolonged the half-life of FVII compared with the addition of 3 CTPs, both the initial and the terminal half-life. The half-life values in the first and second studies were different due to different analytical approaches and were influenced by the dose and duration of the study, however, the general trend remained. AUC after administration of FVII-CTP4 and FVII-CTP5 increased

- 76 044349 was 2.5 and 7 times, respectively, compared with that for FVII-CTP3.

5.9. Conclusions.

This study assessed the PK parameters and potential coagulation activity of FVII-CTP3, FVII-CTP ₄ and FVII-CTP5. Coupling of 4 and 5 CTP to FVII provided the longest and improved half-life, improved exposure, and reduced clearance compared to coupling FVII-CTP3, while maintaining similar chromogenic and clotting activity in vitro. These results were observed at different protein concentrations and were similar between harvested and purified protein. When assessing the effect of CTP attachment to the C-terminus of FVII in general, attachment of 1-5 CTPs significantly increased the half-life and AUC of FVII in proportion to the attached CTP, suggesting that as the number of CTP molecules increased, the half-life and stability of FVII were significantly improved, while its original coagulation activity in vitro was preserved, as shown below in this text in table. 37.

Table 37

FVII versus FVII-CTP2 268 200 FVII-CTP2 versus FVII-CTP3 67 57.8 FVII-CTP versus FVII-CTP4 24 178 FVII-CTP4 versus FVII-CTPs 6 42

It has previously been reported that the half-life of FVII was correlated with the half-life of the activated form of FVII (FVIIa) in both humans and animals. Therefore, it is expected that a similar half-life improvement will be achieved for the activated variant after CTP attachment.

Example 6.

Utility studies of FVII-CTP3 in FVIII-deficient hemophiliac mice.

The studies described above in this text were conducted in which the PK profile and coagulating activity of the collected FVII-CTP, FVII-CTP ₂ and FVII-CTP3 were tested in comparison with those of commercially available FVII. FVII-CTP ₃ showed an improved PK profile, while maintaining its coagulating activity, compared with that of pooled FVII-CTP and FVII-CTP ₂ or rhFVII. To further characterize the properties of FVII-CTP3 in vitro and in vivo, a small stable pool of cells expressing and secreting the protein was obtained and purification and activation processes were developed.

The present study examined the pharmacokinetic and pharmacodynamic properties of FVIIa-CTP ₃ in FVIII-null mice. The PK profile of the protein was assessed. The PK profile was determined based on the specific activity of FVIIa and compared with that of the commercially available NovoSeven® product. In addition, the long-term hemostatic ability of FVIIa-CTP ₃ (the ability to induce blood clotting) was examined in vivo in FVIII-null mice after tail vein transection (survival study).

Objectives of the study.

To evaluate the pharmacokinetic and pharmacodynamic parameters of FVIIa-CTP ₃ compared with those of commercially available rhFVIIa (NovoSeven®) in FVIII-null mice following a single IV dose of similar activity.

To determine the in vivo ability of FVIIa-CTP3 to maintain homeostasis in FVIII-null mice following a single intravenous dose of FVIIa-CTP ₃ and NovoSeven® at a similar activity dose followed by a tail vein dissection test (survival study).

Obtaining assembled FVII-CTP ₃ .

FVII-CTP3 was intrahost expressed in Dg44 cells using the pCI-DHFR vector. Stable transfected pool No. 71 was grown in shake flasks in the presence of 25 ng/L vitamin K3 (Sigma). The cell suspension was cultured and harvested after viability decreased to 60-80%. The collected suspensions were filtered and frozen at -70°C.

Determination of antigen levels of collected FVIL.

FVII antigen levels were determined using a human FVII ELISA kit (Zymotest HyPhen) (Table 38). Antigen levels were calculated for each pooled batch collected.

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Table 38. FVII-CTP ₃ antigen level

Antigen IVI level 11 her. Yudovai ne FK-F, 1 1 1cc. yudova pps survival Assembled pairs 31L Assembled pairs 31B Collected couples 38 Avg. (µg/m. I) 16.0 15.9 16.6 CO 1.5 0.0 Oh % CV 9.1 0.1 4,у

FVII-CTP3 purification process (Fig. 25).

Process plan.

After a short purification study, the following purification process was carried out using 2 columns. The enriched gamma-carboxylated FVII-CTP3 protein was purified using a VII-Select affinity column (GE) and a ceramic hydroxyapatite type 1 (HA) 40 μm column (Bio Rad). Self-activation was induced by incubating purified FVII-CTP3 in the presence of CaCl ₂ overnight at 28°C. The purification process was in the final stages of development and was being optimized, so some of the purification steps were not identical for the two batches.

Ultrafiltration/diafiltration (UV-DF) using a hollow fiber cassette or Pellicon cassette with a molecular weight cut-off of 10 kDa.

The clarified collected cell suspension was thawed at 4°C over the weekend (2-3 days).

For batch 31, clarified pooled cell suspensions (12 L) were concentrated 4-fold (in two consecutive cycles) using a hollow fiber cartridge (GE Healthcare, catalog number UFP-10-C-4X2MA) with a molecular weight cutoff of 10 kDa. The concentrated cell suspension was diafiltered against 1-2 volumes of TBS (50 mM Tris, 150 mM NaCl, pH 7.4).

For batch 38, the clarified pooled cell suspension (8.5 L) was concentrated 4-fold using a Pellicon 2 cassette (Millipore) with a 10 kDa molecular weight cutoff. The concentrated cell suspension was directly loaded onto a VII-Select column.

Both ultrafiltration processes were performed on ice with ice-cold buffers. UV-DF samples were filtered through a 0.22 μm pore size filter before loading.

Capture on FVII-Select column.

UV-DF or concentrated collected cell suspension was loaded onto a VII-Select column (XK16/20, column volume 18 mL) pre-equilibrated with TBS, pH 7.4. The column was washed with 50 mM TrisHCl, 0.5 M NaCl, pH 7.5, and eluted FVII-CTP3 with 50 mM Tris-HCl, 1 M NaCl, 50% (v/v) propylene glycol, pH 7.5. The process was carried out in two successive cycles using the same column.

Separation on a ceramic hydroxyapatite column based on gamma-carboxylation.

The eluted product was diluted 1:10 with 10 mM sodium phosphate, pH 6.8, and loaded onto ceramic hydroxyapatite columns (XK16/20, column volume 24 ml). The column was washed with 59 mM sodium phosphate, pH 6.8, and the highly gamma-carboxylated factor VII fraction was eluted with 500 mM sodium phosphate, pH 6.8. This process was carried out in two successive cycles on the same column. For each batch, the eluates from two cycles were pooled and concentrated to 1.7-2 mg/ml and diafiltered with 20 mM Tris-HCl, 100 mM NaCl, pH 8.2 to reduce the volume and prepare the material for the activation step.

Activation of FVIL.

Purified FVII-CTP ₃ was diluted to 1 mg/ml and incubated with 20 mM Tris-HCl, 100 mM NaCl and 1 mM CaCl ₂ , pH 8.2, at 2-8°C for 24 hours. Activation was completed by exchanging the buffer ( UV-DF) to pre-formulation buffer (20 mM citrate, 240 mM NaCl, 13.3 mM glycine, pH 6.9).

Analytical properties of FVII-CTP3 and FVIIa-CTP3.

EF in PAGE/SDS and Western blotting.

Purified FVII-CTP ₃ and FVIIa-CTP ₃ were loaded onto a 12% Tris-glycine gel, which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Coomassie-PAGE/SDS-PAGE analysis was performed by staining the gel with Coomassie Brilliant Blue reagent (5 or 10 μg protein/lane). Western blot analysis was performed (1 μg protein/lane) using a polyclonal anti-human FVII antibody (R&D Systems; AF2338), a monoclonal antibody recognizing human gamma carboxylation (American Diagnostics, catalog no. 499, 3570), and polyclonal AT to CTP. Under reducing conditions, FVII-CTP3 migrated at 75 kDa and FVIIa-CTP ₃ migrated as two major bands: a heavy chain at 50 kDa and a light chain at 25 kDa, shown in FIG. 26 as lanes 2 and 3, respectively.

The purification procedure significantly increased the FVII-CTP ₃ content and reduced the amount of impurities. The yield of the purification process was 25-30% FVII (by ELISA). Most of the protein lost during the purification process had low or no FVII chromogenic activity. Based on Coomassie staining of the gel after EP using PAGE/SDS, it was revealed that the recovery

- 78 044349 local FVIIa-CTP ₃ migrated in more bands than predicted. The band that migrated at approximately ~75 kDa was non-activated FVII (FIG. 26, lane 1). This band consisted of two bands with slight differences in molecular weight, which may reflect different degrees of gamma-carboxylation. Additional bands with molecular weights below 20 kDa were also observed. They have previously been described as heavy chain degradation products.

Chromogenic activity of FVII-CTP3.

Comparative evaluation of in vitro activity of pooled FVII-CTP3, purification fractions, and purified FVII-CTP3 versus pooled normal human plasma was performed using the commercially available BIOPHEN Chromogenic Activity Assay Kit (Hyphen BioMed 221304). Serial dilutions of the collected FVII-CTP3 and purified protein were prepared and efficacy was assessed by comparing the dose response curve with that of a reference preparation of normal human plasma. After purification of FVII-CTP ₃ , the chromogenic activity was significantly improved and inactive fractions were preferentially separated on a GA column (FIG. 27). A strong correlation was observed between the chromogenic activity of FVII and the detection of FVII by anti-Gla monoclonal antibodies on Western blots. The EC50 value shows that the chromogenic activity of FVII in the collected material was impaired in both the carboxylated and non-carboxylated FVII fractions. After purification and enrichment of the gamma-carboxylated FVII-CTP ₃ fraction, activity improved, demonstrating the significant contribution of gamma-carboxylation to FVII activity (Figure 27). This parameter is important for adequate FVII activity in vivo and will be further developed in the clone development program.

Determination of protein quantity at wavelength A280.

The theoretical absorption coefficient of FVIIa-CTP ₃ and NovoSeven® was calculated using the ProtParam algorithm (http://web.expasy.org/protparam). The calculation was based on the amino acid sequence. The calculated absorption coefficients for FVII-CTP3 and NovoSeven® were 1.186 and 1.406, respectively. These values represent absorbance of 1 g/L at 280 nm.

The difference in absorption coefficient between the two proteins was solely due to the increased molecular weight of FVIIa-CTP ₃ compared to NovoSeven®, since CTP lacks aromatic and cysteine residues and therefore does not contribute to absorption.

Protein quantification at A280 was used for final FVII and for purified samples during the purification process, starting with elution from the VII-Select column.

Determination of FVIIa antigen level.

FVIIa antigen levels were determined using a human FVIIa ELISA kit (IMUBIND, American Diagnostica). Antigen levels were calculated for each batch. However, this method was not useful for determining the dose for injection because it did not reflect the amount of active product.

FVIIa-Staclot® VIIa-rTF blood clotting assay.

FVIIa was produced by intrachain cleavage of single-chain FVII. Native tissue factor (TF) is a cofactor of FVIIa. When bound to TF, FVII mediates the activation of factor X to Xa and is itself converted to FVIIa. Soluble tissue factor is the extracellular portion of native tissue factor. It can no longer activate FVII by self-activation, but tissue factor-bound FVIIa can activate FX to FXa.

The recombinant soluble tissue factor (rsTF) used in this assay is specific for FVIIa, which is used to create the FVIIa coagulation assay. RsTF in the presence of FVIIa, calcium and phospholipids leads to plasma clotting without activation of FVII to FVIIa.

The observed clotting time in this system is inversely proportional to the FVIIa content in the test sample, and does not depend on the presence of FVII in the sample.

The analysis was performed at Omri Laboratories (Ness Ziona, Israel). FVIIa activity was assessed in both NovoSeven® after rehydration and FVIIa-CTP ₃ before each study. The activity of NovoSeven® did not correlate with the predicted activity stated on the vial, but this discrepancy may have arisen due to different approaches to assessing activity. In table 39 briefly describes the clotting activity of FVIIa per volume without regard to protein concentration.

Table 39. FVIIa clotting activity in batch products

PK study Survival Study ΙΛ Na34 TR (GLNAZ!) NovoSeven 1\Ila- 34 IP (1\By 38) NovoSeven ΐΒίβϋβ· Activity (U/ml) 1.3*10 ⁶ 2.5*10 ⁵ 1.3*10 ⁶ 7.4*10 ⁵

Specific activity of FVIIa-CTP ₃ .

Specific activity (SA) of FVIIa (which was calculated as activity/ml divided by protein concentration) was calculated based on A280 and presented in Table. 40. When comparing the specific activities of two specified molecules that differ in molecular weight, an adjustment must be made in order to normalize the activity (i.e., due to the difference in molecular weight, the number of active sites in 1 mg of NovoSeven® is 1.185 times more than 1 mg FVIIa-CTP3). The calculation of the conversion factor is presented in the following equation:

Normalized_UA = (UA(FVΠa-CTP ₃ )/mol. mass(FVΠ-CTP ₃ )) x mol. mass of native FVII = (UA(FVIIa-CTP3)/53419.5 Da) x 45079.1 Da = (UA(FVIIa-CTP3) x 1.185

Table 40. Specific activity of FVIIa-CTP3 compared to NovoSeven®

Sample Wednesday CO (0=9) Cho Coeffpipen Conc, squirrel Units/m. । Specific activity The magnitude of the reduction in relation to TS.1Н1 |1И||||М^ИД NovoSeven R Λ2ΧΟ HF UA'U' ΤΥ U:.·. IIOI. boiling (mg/ml) Ε,ιΛιγ protein U/mg FVIIa NovoSeven® 1.274 0.03 1 2.39 8 1.406 0.906 8.36*10 5 9.23*10 5 9.23*10 5 1,o FVIIaSTrz 4,396 0.09 2 2.09 4 1,186 3,706 7.23*10 5 1.95*10 5 2.31*10 5 4.0

PK-PD study of FVIIa-CTP3.

Research plan.

FVIIa-CTP3 and rhFVIIa (NovoSeven®, NS) were administered as a single intravenous injection to FVIII-null C57B mice at a dose of 6.4*106 U/kg body weight (160,000 U/animal). Blood samples were taken from the retro-orbital sinus of 4 mice alternately at 0.166, 0.5, 2, 4, 8, 12, 24, 34, 48, 58 and 72 hours after administration (Table 41). Citrated plasma (0.32%) was obtained immediately after sample collection and stored at -20°C until analysis. The level of FVIIa coagulation activity was assessed and a thorough PK analysis was performed. The study was carried out at Omri Laboratories (Ness Ziona, Israel).

Table 41. Study design

1 ruins treated Research items Numbers ||I|1ID1MI animals/group point in time Pu। b entered Number of units livestock Pneumatic volume (µl) Moments in time (hours after entry) A rhFVIIa 4 IV 1.6*10 ⁵ 200 0 (up to introduction ) 0D66, 0.5, 2, 4, 8, 12, 24, 34, 48, 58, 72 IN FVIIa-STRz 4 IV 1.6*10 ⁵ 200 0 (up to administration) 0.166, 0.5, 2, 4, 8, 12, 24, 34, 48, 58, 72

PK profile of FVIIa-CTP3 in FVIII-null mice.

Quantitative analysis of FVIIa activity in blood samples was performed using the Staclot® VIIa-rTF kit (Stago, Parsippany, NJ). The pharmacokinetic profile was calculated for each protein and was the average of 4 animals at each time point. In fig. Figure 28 shows the PK profile of FVIIa during the experiment. The yield of FVIIa is presented in table. 42. A brief description of the PK parameters is presented in table. 43.

In table 41 summarizes the values of coagulation activity after administration of either NovoSeven® or FVIIa-CTP3. FVIIa-CTP3 and NovoSeven® reached maximum activity half an hour after administration. The highest activity value of NovoSeven® reached only 43% of the maximum activity value of FVIIa-CTP3. The coagulation activity of FVIIa-CTP3 was maintained over a longer period of time, demonstrating prolonged activity. Coagulation activity in mice treated with NovoSeven® was not detected at time points after 12 hours, whereas in mice treated with FVII-CTP3, measurable activity continued to persist for 48 hours after administration (Table 41 and FIG. 28). Addition of three consecutive copies of CTP to FVIIa increased yield by 100% (Table 42), as measured by peak activity after administration and compared with predicted activity based on in vitro assays, and increased the half-life and mean retention time (MRT) by 5 once. The exposure time (AUC) increased 3 times (Table 43).

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Table 41. Coagulation activity of FVIIa after a single IV injection

Time after administration (hours) Average coagulation activity 1 Vila (sd.ml) EVHa-STRz NovoSeven“ 0.16 6.84 o’ 3.24 0' O.5 9.7*1 o' 4.3*10' 2 2, RN ·' 3.94 0 4 7.7*10 ^{6 7.340s} 8 2.7 * 1 o 4.24 0 ¹ 12 3.74 0' 6.2* IG 24 2.4*1 o ¹ NPKO 34 4.64 o ^; nnko 48 1,540 ³ NPKO

Table 42. Output FVIIa-STRz

Treatment groups Study drug ii Quantities about units animal Fakgichssk n entered lot (e. ml) I am supposed to be Stakh (units. ml of blood) Stakh (ed. ml) exit a A rFVIIa 1.6040 ⁵ 1.2040 ⁶ 1.4040 ⁵ 4.25*10 4 thirty IN FVIIa-STRz 1.6040 ⁵ 1.2940 ⁶ 1.50*10 ⁵ 9.74*10 4 64.6

^The estimated _Cmax is the administered dose divided by the blood volume.

Table 43. PK parameters of FVIIa-CTP ₃ compared with NovoSeven®

FC parameters NovoSeven K EUPA-STrz 11 half-life (0.5 - 12 hours) 0.94 1.57 11srnol lie down sh-r (12-48 hours) nd 4.62 A1C (m1l.*chm.1) Vd-κτ (ml. kg) 5.80* |o' 1408 1.80* K G 2375 Cl.. KI ( M.I Ch. ΚΊ ) \1НТ (h) 1 < >34 1.3 356 6.7

Thrombin generation assay (TGA).

Thrombin generation is a major part of the coagulation cascade, and as such, assessing how well a particular individual can generate thrombin can correlate with the risk of bleeding or thrombosis. Variables that are commonly measured in thrombin generation assays include: lag time, time to peak thrombin generation, peak, endogenous thrombin potential (ETP) (ie, area under the pharmacokinetic curve and tail), thrombogram dynamics (TG). After the delay time, thrombin release was observed. However, blood clotting occurs after the lag time, when more than 95% of all thrombin has not yet been formed. Thrombin generation assays were performed at Omri Laboratories using Thrombinoscope reagents supplemented with human hemophilus influenzae plasma. TGA reflects the clotting ability of mouse plasma induced by injection of NovoSeven® and FVIIa-CTP ₃ . In fig. 29 shows the TGA parameters for mouse plasma after administration of FVIIa-CTP3 or NovoSeven®. After administration of FVIIa-CTP3, all three parameters (thrombin generation rate, maximum amount of thrombin generated and KPa) demonstrate an advantage of FVII-CTP3 treatment over NovoSeven® treatment. This further supports the potential long-acting benefit of FVII-CTP ₃ over NovoSeven®.

Study of treatment of FVIIa-CTP ₃ after tail vein dissection (TCV).

Research plan.

The results obtained from the PK/PD study of FVIIa-CTP ₃ provided insight into the functional properties of FVIIa-CTP ₃ and demonstrated that FVIIa-CTP ₃ has a pharmacokinetic advantage over NovoSeven®. However, the protein's ability to induce blood clotting in vivo after a traumatic event has not yet been demonstrated. To evaluate the ability of FVIIa-CTP ₃ to stop bleeding, the same FVIII knockout mouse model was used for the bleeding test.

FVIII-null mice were given a single intravenous injection of FVIIa-CTP ₃ or NovoSeven®. Mice were dosed with drug in amounts that provided equivalent FVIIa activity (1.6405 units, 200 μl) calculated according to the potency of each drug assessed in the FVIIa clotting activity assay (Table 44). The administered doses were 9 mg/kg NovoSeven® and 40 mg/kg FVII-CTP3 due to reduced activity

FVIIa-CTP ₃ . The control group was injected with 200 μl of medium. The tail vein was dissected at a distance of 2.7 cm from the tip of the tail at 15 min (injection 1), 24 h (injection 2), or 48 h (injection 3) after injection, and the survival of mice was recorded for 24 h.

Table 44. Evaluation of Injected Specimens

Xo>oSe>en “ 1 Vlla-CHL L Yinsky 11 Kopp, b. psa (mg/m.1) Akch IIBIIOC । b (units/ml) Specific aici and contribution ί ь (units/m·) Copp, protein (mg/m. 1) Aid II contribution • b (units/ml) Specific aid contribution । b (units/m) Ude. 1st aid contribution । b (normalized) 1 0.91 8.0*10 ⁵ 8.8*10 ⁵ 3.63 6.6*10 ⁵ 1.8*10 ⁵ 2.2*10 ⁵ 2 0.92 8.3*10 ⁵ 9.0*10 ⁵ 3.81 7.8*10 ⁵ 2.0*10 ⁵ 2.4*10 ⁵ 3 0.89 8.8*10 ⁵ 9.9*10 ⁵ 3.68 7.3*10 ⁵ 2.0*10 ⁵ 2.3*10 ⁵

Protein concentration was determined using A280.

Results.

The results for control groups that received medium by three injections (5 animals x 3 injections) are summarized and presented in FIG. 30. A 30% survival rate was observed 24 hours after tail vein transection.

Mice treated with NovoSeven® and FVIIa-CTP ₃ demonstrated adequate hemostatic activity after tail vein dissection, which was performed 15 minutes after FVIIa administration. Animals treated with FVIIa-CTP ₃ and NovoSeven® had a survival rate of 100% (FIG. 30).

The reduced rate of clearance of FVII-CTP3 demonstrated in the PK/PD study was most clearly seen when tail vein dissection was performed 24 hours after administration. A decrease in survival rate was observed for NovoSeven®. Similar to the control group, 50% of deaths were observed within 10 hours. Meanwhile, 90% of mice treated with FVIIaCTP3 survived (Fig. 30). The results highlight the long-term effectiveness of FVIIa-CTP3 treatment.

48 hours after administration showed a decrease in survival rate in groups treated with either FVIIa-CTP ₃ and NovoSeven® (Fig. 30C). A slight improvement was observed in mice treated with FVIIa-CTP, but the difference did not reach statistical significance.

Discussion.

Fusion of CTP to recombinant proteins extends the half-life of proteins from circulation while maintaining comparable activity. Although the mechanism causing reduced protein clearance above the 70 kDa size threshold is well understood in comparison to renal clearance, additional protection is achieved following CTP attachment. The addition of CTP is believed to unfold the protein shield and protect it from proteolytic degradation, increase its radial molecular weight due to its highly negative charge, and decrease its affinity for liver clearance receptors.

The purpose of this study was to provide some insight into the effect of CTP fusion with FVII on the half-life and clearance of the protein, and to examine its specific activity after this modification. FVIII-null mice were given a single IV injection of FVIIa-CTP ₃ or commercially available recombinant FVIIa (NovoSeven®) at similar unit doses and activity-based PK analysis was performed. FVIIa-CTP ₃ demonstrated a longer half-life, as evidenced by a 5- and 3.5-fold increase in half-life and AUC, respectively. The specific activity (U/mg) of FVIIa-CTP, calculated from the activity determined using the Staclot® kit divided by the protein concentration measured on the A280, was shown to be 4-5 times lower than the specific activity of NovoSeven®. To further our understanding of how CTP influences the hemostatic effects of FVIIa in vivo, the ability of FVIIa-CTP ₃ to reduce bleeding was examined. In the tail vein dissection hemorrhage model, a mouse model of hemophilia, administration of rFVIIa could improve the survival rate of transected animals and avoid fatal bleeding in them. In the study described in this text, animals were administered FVIIa-CTP ₃ or NovoSeven®. Both molecules were able to maintain homeostasis when dissection was performed 0.25 h after administration. A significantly longer duration of activity was demonstrated for the FVIIa-CTP ₃ treatment group when tail dissection was performed 24 hours after administration. The survival rate in the medium treatment group was higher than expected and higher than that found in previous studies (50% compared to 20% in previous studies, results not

- 82 044349 presented). The percentage of survival of treated animals was further assessed at earlier time points, for example 36 hours after administration.

In conclusion, FVIIa-CTP ₃ was demonstrated to have a longer duration of activity in hemophiliac mice, which resulted in a longer duration of hemostatic effect compared to NovoSeven®. The findings suggest that fusion of CTP with FVII represents a technology that has the potential to significantly improve preventive treatment for patients with hemophilia.

Example 7: Comparative evaluation of the profile of purified FVII-CTP3 versus that of FVII-CTP5 after a single IV or SC injection in SD rats.

Purpose of the study.

Two studies were conducted.

The purpose of the first study was to determine the pharmacokinetic parameters of rFVII-CTP ₃ compared to rFVII-CTP5 following FVII-Select (FVII-S) and GA column purification in male Sprague-Dawle rats following a single intravenous dose of 50 μg/animal.

The second study examined the pharmacokinetic parameters of rFVII-CTP ₃ -GA compared with rFVII-CTP ₅ -GA in male Sprague-Dawle rats after a single intravenous or subcutaneous administration of 100 μg/animal.

Results.

Determination of FVII-CTP 3 and FVII-CTP 5 antigen levels.

FVII antigen levels were determined using a human FVII ELISA kit (Zymotest HyPhen) (see Table 45).

Table 45: Calculated protein concentration is summarized and represents the average of three independent experiments.

1 VII 3 PAGE 1 VII 5 PAGE FVITS46_ )!. END .ilia, ϊ. mi-ch 46).i. FVII-S m. FVH-I L 5 100% V horses, bottom 1. copy, dial. horses, lna.1. Average, (pgml) 3.78*10 ⁶ 1.594o 1.8840 7.92 4 CO 1.30^10 ⁶ 6.03 4 0 ⁵ 7.1540 ⁵ 3.5740 ⁵ CV (%) 3.43 4 0 ¹ 3.8040 ¹ 3.8040 ¹ 4.5140 ¹

Western blot analysis of the studied samples.

FVII-CTP ₃ , 5 samples were loaded onto a 4-12% Bis-Tris gel (NuPage, invitrogene), which was also loaded with a Precision plus dual color protein molecular weight marker (Bio-Rad). Analysis of the gels after EF in SDS-PAGE was carried out using Western blotting (immunoblotting), using polyclonal AT to FVII (R&D Systems), polyclonal AT to CTP (Adar Biotech Production) or AT to Gla (American Diagnostica). Briefly, FVII fused to three and five CTPs migrated at 80 and 100 kDa, respectively (see Fig. 31).

Comparative assessment of FVII activity in vitro.

Analysis of FVII activity, which was carried out at the Medical Center. Shiba, at the National Coagulation Center, was a PT-based assay using immunoadsorbed factor VII-depleted plasma (Siemens). The PV reagent was innovin and the analysis was carried out using a CA 1500 Sysmex device. The normal range for FVII was 55-145%. The activities of the samples are briefly described in Table. 46.

Table 46. Activity of samples

Obra sen Coppet early (mg/m. ι) according to XAXODROP Kopnen।early in psst. le.ioBainio.M sample (μt/m.) Rezu, ibiaibi (%) Average excise tax - ”/<” о । excise duty ii. ia >we FVII-5CTP FVII-S el. 2 87 2.19 1 thirty 16% conc. dial 0.5 10 FVII-5CTP-TA 5 100% 2 97 1 1 36 21% In conc. dial 0.5 13 FVII-S 46 el. conc. 3.17 2 100 1 0.5 35 18% dial 12 FVII-GA 46 el. conc. 1.5 2 92 20% dial (1) 1 33 0.5 10

The normal level of circulating FVII in the body is approximately 0.5 mcg/ml.

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Both FVII-CTP3 and FVII-CTP5 exhibited approximately a 5-fold reduction in coagulating activity compared to pooled normal human plasma.

Pharmacokinetic study.

Two pharmacokinetic studies were performed to determine the pharmacokinetic (PK) profile and parameters of FVII-CTP3 and FVII-CTP5 (after purification on the FVII-Select column and the FVII-GA column). In the first study, FVII-CTP3 and FVII-CTP5, after purification on a FVII-Select/GA column, were administered as a single intravenous injection to Sprague-Dawle rats (six rats per substance) at a dose of 50 μg/rat.

Blood samples were collected from the retro-orbital sinus of 3 rats alternately at 0.083, 0.5, 2, 5, 8, 24, 48, 72, 96 and 120 hours after administration. Citrated plasma (0.38%) was obtained immediately after sample collection and stored at -20°C until analysis.

In the second study, only samples after the GA column were studied. These samples were administered by a single intravenous or subcutaneous injection to Sprague-Dawle rats (six rats per substance), using a dose of 100 μg/rat. Blood samples were collected at the same time points and under the same conditions as in the first study described above.

Table 47. First study design (FVII-Select versus FVII-GA)

1 rune Ш>1 healed five II splenic blown steam ι Co. shches 1ALIVE! py x/ group 11pack> entered five Level D0>Y (mkt pa ZHIVO 1 pos.) Ipied obsm (“κι”) Copp, (m kt/m l) Moments of time (hours after administration) 0 (up to A FVII-CTP*3, batch 46, GA 6 IV 50 200 250 administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120 0 (up to IN FVII-CTP*3 batch 46, FVII-S 6 IV 50 200 250 administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120 0 (before administration) WITH FVII-CTP*5, batch 5, GA 6 IV 50 200 250 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120 0 (up to D FVII-CTP*5, batch 5, FVII-S 6 IV 50 200 250 administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120

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Table 4S. Study 2 (intravenous versus subcutaneous)

1 ruins. yuchennya Study preiaraι Co. shches 1 in the belly x/ ι ruin/ 1 |у ιь entered five DO level >Y (µt per animal) Inyenp rovappy yobnem (µl) Kopp, (μt/m. 1) Momep ιs of time (hours after administration) A FVII-CTP*3, batch 46, GA 6 IV 100 200 500 0 (before administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120 IN FVII-CTP*3, batch 46, GA 6 PC 100 200 500 0 (before administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120 WITH FVII-CTP*5, batch 5, GA 6 IV 100 200 500 0 (before administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120 D FVII-CTP*5, batch 5, GA 6 PC 100 200 500 0 (before administration) 0.083 0.5, 2, 5, 8, 24, 48, 72, 96, 120

The main differences between the two studies were the dosages and route of administration. In the first study, rats were administered a dose of 50 μg/rat by IV injection, whereas in the second study, rats were administered a dose of 100 μg/rat by IV or SC injection (total 500 μg/kg; rat weight 200 g). The increase in dosage occurred due to a change in the type of administration; SC administration requires large quantities to achieve an effect close to that of IV administration.

PK study analysis.

Quantitative analysis of FVII concentrations in plasma samples was performed using human FVII ELISA kits (zymutest FVII-Biophen). Pharmacokinetic profiles were calculated and reflect the average of 3 animals at each time point. The final half-life values were calculated using RK Solutions 2.0 software. The table below summarizes the calculated FVII concentrations at various sampling times. The PK profile and a brief description of the PK parameters are presented in the table below.

Table 49. First pharmacokinetic study (FVII-Select ______vs. FVII-GA) - FVII concentrations (ng/ml)______

Time watch) FVII PAGE*3. batch 46. GA 1 VII page ^h . batch 46. 1 VII-S FVII PAGE*5. batch 5, 1 A 1 VH PAGE*5. batch 5. 1 VII-S 0.083 1816.3 1633.9 2064.3 1853.5 0.5 1523.7 1409.9 1351.4 1418.0 2 1284.9 1041.7 1389.7 834.4 5 607.9 531.6 722.7 737.2 8 524.2 430.0 712.2 614.6 24 115.5 132.9 272.5 201.8 48 21.1 31.6 62.3 90.4 72 9.5 15.8 29.1 31.8 96 npko 5.8 7.0 16.9 120 npko NPKO 8.5 13.4

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Table 50. Second pharmacokinetic study (intravenous versus subcutaneous) - FVII concentrations (ng/ml)_____________

Time watch) FV II ( 1 R*3. batch 46, 1 A-in FVII ( 1 R ^y 5. nargia 5. GA-v/v FVII (1 R ^y 3, batch 46. GA-p'k* FVII ( | batch 5. GA-p-k 0.083 6452.6 6153.3 5.0 NPKO 0.5 3930.7 3660.6 14.5 14.6 2 1992.3 2176.2 113.6 96.2 5 1598.9 2087.3 106.6 70.5 8 781.6 1075.6 188.9 129.7 24 268.5 627.2 155.0 239.2 48 51.9 143.3 43.0 88.6 72 8.8 39.0 7.0 36.7 96 npko 10.8 NPKO 10.4 120 npko 8.2 npko 8.7

Table 51. PK analysis - first pharmacokinetic study (FVII-S versus GA)

IV II ( 1 R ^y 3. batch 46. 1 A IV II ( 1 R ^y 3. part five 46. FVII-S FVII (GR ^Y 5. nargia 5. 1 A IV II ( 1Р ^И 5. nargy 5. FVII-S Period iio.iyajBHii roZhZ - 8 hours) 4.3 4.0 5.51 5.59 Half-life (8 72\96\120 hours) P,1 12.1 16.46 20.29 Half-life (8 - 72) (hours) I,1 13.4 13.62 15.64 AUC(O-t) (observed area) (8 - 72/96/120 h) 14566.9 13686.4 21812.7 19307.9 Area AUC (co) (8 72/96/120 h) 14718.2 13788.1 22013.9 19701 Vd (area)/kg (ml/kg)(8 2/96/120 h.) 271.1 316.1 269.7 371.5 CL (area)/kg(ml/h/kg) (8 - 72/96/120 h.) 17.0 18.1 11,356 12.69

The addition of five CTPs prolonged the half-life of FVII compared with 3 CTPs. Both 5 CTP forms (i.e. FVII-S and FVII-GA) were detected at distant time points (96 and 120 h), whereas FVII-3 CTP-GA and FVII-3 CTP-S were detected before 72 h and 96 h, respectively. Based on this fact, the half-life of FVII-5 CTP was found to be longer than that of the 3CTP variants (see FIG. 32). Comparison of the half-lives of all materials tested (3 and 5 CTP) at the same time points (8-72 hours) showed that the half-lives were similar, although 5 CTP was much longer (Fig. 32).

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Table 52. PK analysis - second pharmacokinetic study (intravenous versus subcutaneous)

1 VII PAGE*3. batch 46. 1 A- in in 1 VII ( ΙΊ» ^ή 5. party 5. 1 Av in 1 VII ( ΊΊ»*3. nargnya 46. ΓΑ- ιι K’ ΙΛ II PAGE*5. Nargnya 5. 1 A- II k* 1>lifetime benefits STR*3 Knives nice feature (GR ^;: 5 Half-life (0.083 - 8 hours) h,o 3.9 -1.8 -3.18 Half-life (872\96\120 hours) 9.9 14.6 13.14 22.94 Half-life (8-72) (hours) 9.9 13.0 13.14 29.47 AUC(O-t) (observed area) (8 - 72/96/120 hours) 28866.8 43761.0 6600 9822.7 22.9 22.4 AUC (oo) area (8 72/96/120 h.) 28993.0 43934.4 6733 10110.8 23.22 23.01 Vd (area)/kg (ml/kg) (8 72/96/120 h.) 246.4 240.5 1407.6 1636.8 CL (area)/kg (ml/h/kg) (8 72/96/120 h) 17.2 I,4 74,261 49,452

Again, as observed in the first study, the addition of 5 CTP prolonged the half-life of FVII compared to the addition of 3 CTP, both the initial and terminal half-life and for both routes of administration (IV and SC, see Fig. 33 ). As expected, after SC administration, FVII was first detected in the blood at a later time point compared to IV administration.

Two PK studies were briefly described above. The main goal of the first study was to test the difference between FVII-3CTP and FVII-5CTP after purification on 2 different columns: FVII-Select and FVII-GA. In our previous studies, we compared assembled proteins with purified proteins and found that the difference between FVII variants with 3 and 5 CTP was greater when rats were injected with assembled proteins.

We did not observe a significant difference between the results of FVII 3/5 CTP after purification on both columns, therefore we decided to inject FVII 3/5 CTP-HA in the second study.

Example 8 Survival Study of FVIII Null Mice Following Subcutaneous Injection of FVIIA-CTP3 (MOD-5014).

Purpose of the study.

To evaluate the effectiveness of NovoSeven®, MOD-5014 (FVIIA-CTP3) and MOD-5019 (FVIIA-CTP5) in the tail vein dissection study, following subcutaneous administration.

Analytical properties of FVIIa-CTP ₃ (MOD-5014) and FVIIa-CTP ₅ (MOD 5019).

Determination of protein quantity on A280.

The theoretical absorption coefficient of NovoSeven® was calculated using the ProtParam algorithm (http://web.expasy.org/protparam). The calculation was based on the amino acid sequence. The calculated absorbance for NovoSeven® was 1.406 and for MOD-5019 was 1.075 (values represent absorbance of 1 g/L at 280 nm). The absorption coefficient of MOD-5014 was determined using the Mscan amino acid assay. The absorption coefficient for MOD-5014 was 1.27.

FVIIa blood clotting assay - STACLOT VIIa-rTF.

FVIIa was produced by intrachain cleavage of single-chain FVII. Native tissue factor (TF) is a cofactor of FVIIa, when bound to TF, FVII mediates the activation of factor X to Xa and is itself converted to FVIIa. Soluble tissue factor is the extracellular portion of native tissue factor. It can no longer activate FVII by self-activation, but tissue factor-bound FVIIa can activate FX to FXa.

The recombinant soluble tissue factor (rsTF) used in this assay is specific for FVIIa, which is used to create the FVIIa clotting test. Recombinant soluble tissue factor (rsTF) in the presence of FVIIa, calcium and phospholipids, causes plasma clotting without activating FVII to FVIIa. The observed clotting time in this system is inversely proportional to the FVIIa content in the test sample, and does not depend on the presence of FVII in the sample.

FVIIa activity was assessed for NovoSeven® rehydrated and for MOD5014 and MOD-5019 before each study.

Specific activity of FVIIa (which was calculated as activity/ml divided by protein concentration) was calculated based on A280 and presented in Table. 53. When comparing the specific activity of these two molecules that differ in molecular weight, an adjustment must be made in order to normalize the activity (i.e., due to the difference in molecular weight, the number of active sites in 1 mg of NovoSeven® is 1.185 times greater than in MOD-5014, and 1.307 times more than in MOD5019). Therefore, the calculation of the conversion factor is presented in the following equation: Normalized_UA = (UA(FVIIa-CTP ₃ )/mol. weight of native FVII) x mol. mass (FVII-CTP ₃ ) = (UA(FVIIaCTP3)/45079.1 Da) x 53419.5 Da = (UA(FVIIa-CTP3) x 1.185

Table 53. Specific activity of MOD-5014 compared to NovoSeven®

Obra yun Copp, protein at A280 (mg ml) 5 unit activity (e.g. mg FVIIa) Reduction ratio relative to ©.NovoSeven ©NovoSeven 0.93 52487 ι,ο MOD-5014, batch 73 1.4 25490 2.05 MOD-5019, batch 9 h,o 11698 4.48

Research plan.

The most relevant measure is the protein's ability to induce blood clotting in vivo after a traumatic event. To evaluate the ability of MOD-5014 to stop bleeding, the same FVIII knockout mouse model was used for the bleeding test.

FVIII-null mice were treated with a single subcutaneous injection of MOD-5014, MOD-5019, or

NovoSeven®. Groups A and B were dosed with NovoSeven® and MOD-5014, respectively, in equivalent amounts for FVIIa activity. Group C was dosed with MOD-5019 at an equivalent amount of FVIIa protein as MOD-5014 to assess the deciding factor (activity or amount of protein).

Doses administered were 4.2 mg/kg NovoSeven® and 8.6 mg/kg MOD-5014 and MOD-5019. The tail vein was dissected at a distance of 2.7 cm from the tip of the tail 12 hours after administration, and the survival of mice was recorded for 24 hours.

Table 54. Group designation

Group Date of injection Study drug Administration dose Injected volume (µl) Number of mice per group Bleeding time, hours after administration mg FVII/kg honey/kg A 13.1.13 ©NovoSeven 4.23 221876 100 10 12 IN 15.1.13 MOD-5014, batch 73 8.59 218750 160 10 12 WITH 27.1.13 MOD-5019, batch 9 8.59 100496 160 10 12

Results.

The results of the experiment are briefly described in table. 55 and in fig. 34.

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Table 55. Results of the RHV study

Time after RHV (h) Number of surviving mice % survival rate NovoSeven® MOD5014 MOD5019 NovoSeven® MOD5014 MOD5019 0 9 10 10 100 100 100 1 9 10 10 100 100 100 2 9 10 10 100 100 100 3 8 10 8 89 100 80 4 6 9 8 67 90 80 5 5 9 7 56 90 70 6 4 8 5 44 80 50 7 3 8 5 33 80 50 8 2 7 5 22 70 50 9 1 6 5 AND 60 50 10 1 5 5 AND 50 50 AND 1 3 5 AND thirty 50 12 1 3 5 And thirty 50 24 1 3 4 And thirty 40

At 24 hours post-RHV, only 11% of mice injected with NovoSeven® survived. 30% of mice injected with MOD-5014 and 40% of mice injected with MOD-5019 survived at this time point. Subcutaneous injection of MOD-5014 and MOD-5019 resulted in improved mouse survival compared to NovoSeven®. However, the results were not optimal as more than 50% of the animals died during the experiment.

Factor VIIa, like other clotting factors, is usually given by intravenous injection so that it goes directly into the bloodstream. However, the present invention has shown that the compositions provided herein are surprisingly more effective in entering the bloodstream after subcutaneous administration. The ability to administer FVIIa subcutaneously is advantageous because subcutaneous administration of FVIIa allows for prophylactic use. Subcutaneous injections are also much easier for patients to administer to themselves and offer an advantage when patients are very young and their veins are small and difficult to detect.

Therefore, subcutaneous administration can be used for prophylactic treatment.

Example 9 Comparative PK-PD study of recombinant MOD-5014 versus NovoSeven® after subcutaneous administration in SD rats.

Objectives of the study.

To determine the pharmacokinetic and pharmacodynamic parameters of MOD-5014 compared with commercially available rFVIIa in SD rats after a single subcutaneous administration.

Compare two independent experiments (05010 and 05034) with MOD-5014 products derived from two different clones (clone #28 versus #61) for their pharmacokinetic parameters.

Experimental methods.

Animals.

male SD rats arrived from Harlan Laboratories Israel, Ltd. at least 4 days before the start of the injections. The animals were healthy young adult rats weighing ~200 g at the start of the study. The deviation in body weight of animals at the start of treatment should not exceed ± 20% of the average weight of each sex. The health status of the animals used in this study was examined upon arrival. Only healthy animals were acclimated to laboratory conditions and used for the study.

FVIIa blood clotting assay - STACLOT VIIa-Rtf.

The recombinant soluble tissue factor (rsTF) used in this assay is specific for FVIIa, which is used to create the FVIIa clotting test. RsTF, in the presence of FVIIa, calcium and phospholipids, causes plasma clotting without activating FVII to FVIIa.

The observed clotting time in this system is inversely proportional to the FVIIa content in the test sample and does not depend on the presence of FVII in the sample.

FVIIa activity was assessed for both NovoSeven® after rehydration and MOD-5014 before each study. The specific activity of FVIIa was calculated based on A280. When comparing the specific activity of two molecules that differ in molecular weight, an adjustment must be made to normalize the activity (i.e., due to the difference in molecular weight, the number of active sites in 1 mg of NovoSeven® is 1.185 times greater than in MOD- 5014).

PK solver software.

Pharmacokinetic parameters were calculated using PK solver software. The IV administration curve was analyzed using two-compartment analysis (CA) of bolus dose administration, and then SC administration was analyzed using non-compartmental analysis (NCA) of extravascular administration - linear-log trapezoid method. Half-life, AUC, clearance and volume of distribution characteristics were calculated and output parameters were examined by comparison between experimental groups.

Experimental materials.

Experiment No. 05010.

A. NovoSeven® RT: (lot no. AU61553, received 7/31/12*). FVIIa concentration on A280: 0.86 mg/ml. FVIIa Staclot activity assay: 56867 units/mg. Injection dose: 946 mcg/kg. *Pool of NovoSeven® aliquots, all from the same lot no.

B. Clone 28. MOD-5014 RS12-001: 0.77 mg/ml** based on A280. FVIIa Staclot activity assay: 34162 units/mg. Injection dose: 850 mcg FVIIa/kg.

Experiment No. 05034.

A. NovoSeven® RT: (lot no. AU61347, received 1.1.13). FVIIa concentration on A280: 0.82 mg/ml, diluted to 0.4 mg/ml with sterile NS buffer. FVIIa Staclot Activity Assay: 55688 U/mg. Injection dose: 360 mcg/kg and 20047.7 units/kg.

B. Clone 61. MOD-5014 Lot 75: 1.9 mg/mL** based on A280, diluted to 0.89 mg/mL with formulation buffer. Injected dose: 20047.7 units/kg. FVIIa clotting activity: 25,002* units/mg based on FVIIa Staclot activity assay.

C. Clone 61. MOD-5014, lot 81A: 2.36 mg/ml based on A280 (filtered on the morning of the test and measured at 280 nm), diluted to 0.4 mg/ml with formulation buffer. Injection dose: 360 mcg FVIIa/kg. FVIIa clotting activity: 24,943 U/mg based on FVIIa Staclot activity assay.

D. Clone 61. MOD-5014 Lot 81A: 2.36 mg/mL based on A280, diluted to 0.89 mg/mL with formulation buffer. Injected dose: 20047.7 units/kg. FVIIa clotting activity: 24,943 U/mg based on FVIIa Staclot activity assay.

Research plan.

Experiment No. 05010.

MOD-5014 and NovoSeven® were administered as a single intravenous or subcutaneous injection to SD rats at a dose of 0.9 mg/kg body weight. Blood samples were collected from the retro-orbital sinus 3 of rats alternately at 0.5, 4, 8, 12, 24, 34, 48 and 58 hours after administration. Citrated plasma (0.32%) was obtained immediately after sample collection and stored at -20°C until analysis. This study was conducted at Science in Action Park, Hec-Ziona, Israel. The level of FVIIa coagulation activity was assessed and a thorough PK analysis was performed at Prolor-Biotech.

Table 55. Study design 05010

1 ruins. ichciiiisi Ike. yul hsmyy nrenar a1 Co. ι-βο ZIVO 1 II them /group a Co. । tow । in ALIVE 1 puff / group / XI0X101II time Nun, enter 1111" But. । Level LO1Y (ΜΚΊ 7 K 1) Pnyeinr ovappy volume (chm) Momen । and time (hours IIOC.IC introduction) A rFVIIa (Novo Seven®) 6 3 IV m 946 220 0, 0.5, 4, 8, 12, 24, 34, 48, 58 IN rFVIIa RS 12001 (clon 28) 6 3 IV m 850 220 0, 0.5, 4, 8, 12, 24, 34, 48, 58 11\Ila (\o\o Se\ ep M 6 11'k ιιιιιι^^ 940 220 O. (>.5.4. 8. 12. 24. 34. 48. 58 D ri Vila RS 12AXIS (bug 28) 6 3 PC m 850 220 0. 0.5. 4.8. 12. 24. 34. 48. 58

Experiment No. 05034.

MOD-5014 and NovoSeven® were administered as a single subcutaneous injection to SD rats at a dose of 0.9 mg/kg body weight. Blood samples were collected from the retro-orbital sinus 3 of rats alternately at 0.5, 2, 4, 6, 8, 12, 24, 34, 48 and 72 hours after administration. Citrated plasma (0.32%) was obtained immediately after sample collection and stored at -20°C until analysis. This study was conducted at Science in Action, Hec-Ziona.

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The level of FVIIa coagulation activity was assessed and a thorough PK analysis was performed at ProlorBiotech.

Table 56. Study design 05034

1 healing ruins lice. ie.i uem yp drug at Ko. mneciBO lively/puff/group/momesh of time * * * Path entered nya Floor Willow level pa ZHIVO 1II oe (μκί /kt) Level DO5Y PA ALIVE! BUT e (el./κι) 11 full volume (µl) Momep । s time (hours village ie introduction) A FVIIa (NovoS even®) 3 PC m 360 20047, 7 207 0, 0.5, 2, 4, 6, 8, 12, 24, 34, 48, 72 IN FVIIa 75 (clone 61) 3 PC m 801.8 4 20047, 7 207 0, 0.5, 2, 4, 6, 8, 12, 24, 34, 48, 72 WITH FVIIa 81A (clone 61) 3 PC m 360 8979.4 8 207 0, 0.5, 2, 4, 6, 8, 12, 24, 34, 48, 72 D FVIIa 81A (clone 61) 3 PC m 803.7 4 20047, 7 207 0, 0.5, 2, 4, 6, 8, 12, 24, 34, 48, 72

Results.

Quantitative analysis of FVIIa activity in blood samples was performed using the STACLOT VIIa-rTF kit (Stago). The pharmacokinetic profile for each protein was calculated and was the average of 3 animals at each time point.

Experiment No. 05010.

After background subtraction: 15 IU/ml.

In fig. Figure 35 shows the PK profile of FVIIa after IV and SC administration of either NovoSeven® or MOD5014. A brief description of FVIIa activity values at each time point is presented in Table. 57. PK patterns differ with IV and SC administration (see Fig. 35; after background subtraction: 15 mU/ml). Cmax after IV injection is higher than that after SC injection due to the drug entering the bloodstream immediately after administration (measured after 0.5 hours, Tables 57 and 58). However, after subcutaneous administration, drug molecules are transferred into the intracellular matrix and tissues, so Cmax can only be measured 2 hours after injection. The total drug recovery after SC administration was less than the Cmax value after IV injection.

At 8 hours post-injection, NovoSeven® was found to have the same PK pattern when administered by either IV or SC injection (after background subtraction: 15 mU/ml, FIG. 35). Moreover, coagulation activity in mice treated with NovoSeven® was not detected at time points after 12 hours, whereas mice treated with MOD-5014 continued to have measurable activity 58 hours after administration (Table 57; after background subtraction : 15 honey/ml; Fig. 35).

Table 57. Coagulation activity of FVIIa MOD-5014 compared with NovoSeven® after IV or SC administration

Time (hours) <>.5 4 NovoSevei (L) honey. ml 304051.7 40068.3 1 to . in in % CV 18.7 7.8 MOD-50I (B) chalk. ml 232818.3 62085.0 14. in in % CV 5.o 9.5 \ovoSci II k ( small ml 1 1401.7 21385.0 . ep k. About % q 2.4 22.6 MOD-: II to (med. m l 3001.7 12018, 3 '014. 1" % sq. 1 od I 15.8 8 5276.7 2.5 25931.7 6.1 5525.0 32.5 6445.0 2.2 12 255.0 13.8 5633.3 9.3 297.7 41.4 924.7 24.1 24 1.3 7.1 251.3 11.8 1.3 89.2 249.3 60.3 34 oh oh 78.3 4.5 oh oh 63.7 85.5 48 29.0 9.9 oh oh 35.0 47.2 58 10.3 4.6 oh oh 13.7 33.5

After background subtraction: 15 IU/ml.

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Table 58. PK parameters of MOD-5014 compared with NovoSeven® after IV or SC administration

A. i.v.

FC parameters NovoSeven'k: RT (M MOD-5014 (RS 12-001) (B) Half-life (0.5 0.24 1.04 - 4 hours) Half-life-β (4 58 hours) 1.31 3.17 AUC 0-infinite, honey/ml*h. 702467.95 820778.67 Vss (Units/kg/(mU/ml)) 0.13 0.13 CL ((U/kg)/(mU/ml)/hour) 0.08 0.04 MRT (hours) 1.74 3.62 B. s/c

FC parameters NovoSeven* RT (V) MOD-5014(RS 12-001)(() Half-life (hours) 1.40 7.78 Stach (med./ml) 21385.00 12018.33 AUC 0-infinite (med./ml*h.) 115099.72 84158.87 MRT 0-infinite (h.) 4.32 7.04 Vz/F (Unit/kg)/(mU/ml) 0.95 3.88 CL/F (Units/kg)/(mU/ml)/h. 0.47 0.35

Vz/F is the volume of distribution in the final phase.

Experiment No. 05034.

In fig. 36 shows the PK profile of FVII after subcutaneous administration of either NovoSeven® or MOD-5017. Two different batches of clone number 61 (#75 and 81) were tested at the same concentration or with the same units of activity compared to NovoSeven®. A brief description of FVIIa activity values at each time point is presented in Table. 59.

The results indicate a similar PK pattern after SC administration, consistent with previous experiments. Moreover, coagulation activity in mice treated with NovoSeven® was not detected at time points after 12 hours, whereas mice treated with MOD-5014 continued to have measurable activity 24 hours after administration (Table 59 and FIG. 36 ; and after subtracting the background: 56 honey/ml (8, 12 hours) or 32 honey/ml (0.5, 2, 6, 14 hours)).

The Cmax values of lot #81 (D) clone number 61 (1301 mU/ml) were lower than the Cmax values of lot #75 (B) clone number 61 and NovoSeven® (A) (3521 mU/ml and 5908 mU/ ml, respectively), although they were all administered by injection in the same number of activity units (Table 6). However, Lots No. 75 (B) and No. 81 (D) had almost identical activity (559 mU/ml and 478 mU/ml, respectively), which was measured 8 hours after injection (Fig. 36 and Table 59; and after subtracting the background: 56 honey/ml (8, 12 hours) or 32 honey/ml (0.5, 2, 6, 14 hours)).

Table 59. Coagulation activity of FVIIa MOD-5014 (clone 61, batches No. 75, 81) compared with NovoSeven® after a single subcutaneous injection

Time I (h.) NinoSe^en « (A) MOD-50I4. clone Y. nar।nya 75 (B) - equal units/kt MOD-5014. BUGS. guy 81A (C) equal to knight. 1A Na. ΜΚΊ /kt MOD-5014. clone 61. pairs 81A (1)) - equal units/ct chalk. /m. 1 % sq. ml./ml % CV m El./m % CV m El./m l kv <>.5 3271 40.5 350.3 26.6 101.3 24.1 208.7 51.2 2 5908.0 18.1 3521.3 70.9 1294.7 7.0 1301.3 31.6 6 1411.7 23.6 1349.7 45.6 425.3 27.6 663.0 13.4 8 1029.0 12.4 559.3 52.7 152.7 19.5 478.0 25.4 12 121, 3 9.9 563.0 17.4 148.7 36.3 712.7 16.2 24 1.0 25.0 117.0 41.9 21.3 36.4 99.0 36.7

After background subtraction: 56 honey/ml (8, 12 hours) or 32 honey/ml (0.5, 2, 6, 14 hours).

- 92 044349

Table 60. PK parameters of MOD-5014 (clone 61, batches No. 75, 81) compared with NovoSeven® after a single subcutaneous injection

FC naramera XovoSoen to RT(A) MOD-5014. k. yun 61. iiap i nya 75 (B) - equal e.i./ici MOD-50I4. kmop 61. nar। nya 81A (((equal to copy. FVIIa mkt/kt MOD-50I4. room 1011 61. frame 81A (D) - equal units/ct Half-life (hours) 1.67 5.70 4.62 6.41 Stach (med./ml) 5908.00 3521.33 1294.67 1301.33 AUC 0-infinite (med./ml*h.) 24688.18 20456.96 6260.23 13098.16 MRT 0-infinite (h.) 3.73 7.86 6.40 10.59 Vz/F (Units/kg)/(mU/ml) 1.96 8.06 9.55 14.15 CL/F (Units/kg)/(mU/ml)/h. 0.81 0.98 1.43 1.53

The present invention briefly describes two PK studies: 05010 and 05034. Our goal was to provide some insight into the effect of the addition of CTP to FVII on the half-life and clearance of the protein when administered subcutaneously, and to examine its specific activity after this modification. In these studies, SD rats were administered by a single subcutaneous injection of MOD-5014, prepared from two clones and two different batches, and compared with recombinant commercially available FVIIa (NovoSeven®). These components were administered by injection at similar concentrations of FVIIa (μg/kg) or at the same activity level (units/kg) and analyzed based on PK activity.

The main objective of the first study was to compare various PK parameters after IV and SC administration. Based on this study, we can conclude that there is a difference between the PK pattern measured after IV or SC administration. T1 ^/2 after subcutaneous injection of MOD-5014 was 7.78 hours, and only 4.2 hours after intravenous injection. AUC values were the same (Table 58).

The second study, however, focused on the differences between two batches of MOD-5014 clone number 61, which was injected at the same concentration of FVIIa or equal units of activity, and compared with NovoSeven®. In this study, we showed that clone 61 of lot #75 exhibited better PK parameters than lot #81. Lot #81, which was injected at the same activity level, had a lower Cmax for an unknown reason. Moreover, the same Cmax was measured when lot #81 clone 61 was injected at two different doses (by FVIIa concentration or by activity unit), rather than a 2.5-fold difference between the two activity values. After a joint analysis of both studies, we can come to the conclusion that clone 28 showed a greater t ^1/2 parameter than clone 61 No. 75 (best batch) after subcutaneous injection (7.78 and 5.7 hours, respectively, table 60). We can also conclude that samples at different time points produce different PK patterns, resulting in changes in the PK curves. The patterns of these curves can give us more insight into the behavior of the drug in the blood. Therefore, we decided to set time points similar to those found in Baxter (0, 0.5, 2, 6, 8, 12, 24, 34, 48, 72 h). Moreover, the FVIIa concentration in experiment 05010 was too high and was remeasured in the next experiment (05034) with SC administration. For future PK studies, we decided to administer the component by injection at a dose of 360 μg FVIIa/kg.

In summary, we can learn more about the MOD-5014 product after SC administration to identify the clone and lot with the best quality and to determine the best way to determine the amount of MOD-5014 injected - by concentration or FVIIa activity units.

Although the properties of the present invention have been illustrated and described herein, those of ordinary skill in the art will now be able to come up with many modifications, substitutions, variations and equivalents thereof. Therefore, it should be obvious that the scope of the appended claims should include all such modifications and changes as are expressly included within the scope of the present invention.

- 93 044349

List of sequences < 110> OPCO BIOLOGICS LTD.

< 12 0> LONG-ACTING BLOOD CLOTTING FACTORS AND METHODS FOR THEIR OBTAINING < 130> P-9520-PC5 < 150> 13/372,540 < 151> 2012-02-14 < 160>45 < 170> PatentIn version 3.5 < 210>1 < 211>32 <212>Protein <213>Homo sapiens <400>1

Asp Pro Arg 1 Phe Gln 5 Asp Ser Ser Ser Ser 10 Lys Ala Pro Pro Pro 15 Ser Leu Pro Ser Pro 20 Ser Arg Leu Pro Gly 25 Pro Ser Asp Thr Pro 30 Ile Leu <210> 2 <211> 28 <212> Protein <213> Homo sapiens <400> 2 Ser Ser Ser 1 Ser Lys 5 Ala Pro Pro Pro Ser 10 Leu Pro Ser Pro Ser 15 Arg Leu Pro Gly Pro 20 Ser Asp Thr Pro Ile 25 Leu Pro Gln

< 210>3 < 211>12 < 212> Protein < 213> Homo sapiens < 400>3

Ser Ser Ser

Ser Lys Ala Pro Pro Pro Ser Leu Pro

510 < 210>4 < 211>28 < 212> Protein < 213> Homo sapiens <400> 4

- 94 044349

Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg

5 10 15

Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220>

<223> PCR Primer for Factor VII <400> 5 ctcgaggaca tggtctccca ggccc <210> 6 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> PCR Primer for Factor VII <400> 6 tctagaatag gtatttttcc acat <210> 7 <211> 22 <212> DNA <213> Artificial sequence <220>

<223> PCR Primer for Factor VII <400> 7 tctagaaaaa agaaatgcca gc <210> 8 <211> 32 <212> DNA <213> Artificial sequence <220>

< 223> PCR Primer for Factor VII < 400>8 gcggccgcat cctcagggaa atggggctcg ca < 210>9 <211>444 <212> Protein <213> Homo sapiens <400> 9

- 95 044349

Met Val Ser Gln Ala 1 5

Leu Arg Leu Leu Cys

Leu Leu Leu Gly Leu

Gly Cys Leu Ala Ala 20

Val Phe Val Thr Gln

Glu Glu Ala His Gly 30

Leu His Arg Arg Arg 35

Arg Ala Asn Ala Phe 40

Leu Glu Glu Leu Arg 45

Gly Ser Leu Glu Arg 50

Glu Cys Lys Glu Glu 55

Gln Cys Ser Phe Glu 60

Ala Arg Glu Ile Phe 65

Lys Asp Ala Glu Arg 70

Thr Lys Leu Phe Trp 75

Ser Tyr Ser Asp Gly 85

Asp Gln Cys Ala Ser 90

Ser Pro Cys Gln Asn 95

Gly Ser Cys Lys Asp

100

Gln Leu Gln Ser Tyr

105

Ile Cys Phe Cys Leu

110

Ala Phe Glu Gly Arg 115

Asn Cys Glu Thr His 120

Lys Asp Asp Gln Leu

125

Cys Val Asn Glu Asn 130

Gly Gly Cys Glu Gln

135

Tyr Cys Ser Asp His 140

Gly Thr Lys Arg Ser 145

Cys Arg Cys His Glu 150

Gly Tyr Ser Leu Leu 155

Asp Gly Val Ser Cys

165

Thr Pro Thr Val Glu

170

Tyr Pro Cys Gly Lys

175

Pro Ile Leu Glu Lys

180

Arg Asn Ala Ser Lys

185

Pro Gln Gly Arg Ile

190

Gly Gly Lys Val Cys 195

Pro Lys Gly Glu Cys

200

Pro Trp Gln Val Leu

205

Leu Val Asn Gly Ala

210

Gln Leu Cys Gly Gly

215

Thr Leu Ile Asn Thr

220

Trp Val Val Ser Ala 225

Ala His Cys Phe Asp 230

Lys Ile Lys Asn Trp 235

Asn Leu Ile Ala Val

245

Leu Gly Glu His Asp

250

Leu Ser Glu His Asp

255

Gln

Val

Pro

Glu

Ile 80

Gly

Pro

Ile

Thr

Ala 160

Ile

Val

Leu

Ile

Arg 240

Gly

- 96 044349

Asp Glu Gln

Ser Arg Arg Val Ala Gln Val Ile Ile Pro 260 265

Val Pro Gly

275

Thr Thr Asn His Asp Ile Ala Leu Leu Arg

280 285

Ser Thr Tyr 270

Leu His Gln

Pro Val Val

290

Leu Thr Asp His Val Val Pro Leu Cys Leu

295 300

Pro Glu Arg

Thr Phe Ser 305

Glu Arg Thr Leu Ala Phe Val Arg Phe Ser

310 315

Leu Val Ser

320

Gly Trp Gly

Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu

325 330

Glu Leu Met

335

Val Leu Asn Val Pro Arg Leu Met

340

Thr Gln Asp Cys Leu Gln Gln Ser

345 350

Arg Lys Val Gly Asp Ser Pro Asn Ile

355 360

Gly Tyr Ser Asp Gly Ser Lys Asp Ser

370 375

Pro His Ala Thr His Tyr Arg Gly Thr 385 390

Ser Trp Gly Gln Gly Cys Ala Thr Val 405

Thr Glu Tyr Met 365 Phe Cys Ala Cys Lys Gly 380 Asp Ser Gly Gly Trp Tyr 395 Leu Thr Gly Ile Val 400 Gly 410 His Phe Gly Val Tyr 415 Thr

Arg Val Ser

Pro Arg Pro

435

Gln Tyr 420

Gly Val

Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu

425 430

Leu Leu Arg Ala Pro Phe Pro 440

<210> <211> <212> <213> <400> Met Val 1 10 448 Protein Leu Cys 10 Leu Leu Leu Gly Leu 15 Gln Homo 10 Ser sapiens Gln Ala 5 Leu Arg Leu Gly Cys Leu Ala 20 Ala Val Phe Val Thr 25 Gln Glu Glu Ala His 30 Gly Val

- 97 044349

Leu

Gly

Ala 65

Ser

Gly

Ala

Cys

Gly 145

Asp

Pro

Gly

Leu

Trp 225

Asn

Asp

Val

His Arg 35

Ser Leu 50

Arg Glu

Tyr Ser

Ser Cys

PheGlu

115

Val Asn 130

Thr Lys

Gly Val

Ile Leu

Gly Lys

195

Val Asn 210

Val Val

Leu Ile

Glu Gln

Pro Gly

Arg Arg

Glu Arg

Ile Phe

Asp Gly

Lys Asp 100

Gly Arg

Glu Asn

Arg Ser

Ser Cys

165

Glu Lys 180

Val Cys

Gly Ala

Ser Ala

Ala Val

245

Ser Arg 260

Thr Thr

Arg Ala

Glu Cys 55

Lys Asp 70

Asp Gln

Gln Leu

Asn Cys

Gly Gly

135

Cys Arg 150

Thr Pro

Arg Asn

Pro Lys

Gln Leu

215

Ala His 230

Leu Gly

Arg Val

Asn His

Asn Ala 40

Lys Glu

Ala Glu

Cys Ala

Gln Ser 105

Glu Thr 120

Cys Glu

Cys His

Thr Val

Ala Ser 185

Gly Glu 200

Cys Gly

CysPhe

Glu His

Ala Gln

265

Asp Ile

Phe Leu

Glu Gln

Arg Thr

Ser Ser 90

Tyr Ile

His Lys

Gln Tyr

Glu Gly

155

Glu Tyr 170

Lys Pro

Cys Pro

Gly Thr

Asp Lys

235

Asp Leu 250

Val Ile

Ala Leu

Glu Glu

Cys Ser 60

Lys Leu

Pro Cys

CysPhe

Asp Asp

125

Cys Ser 140

Tyr Ser

Pro Cys

Gln Gly

Trp Gln

205

Leu Ile

220

Ile Lys

Ser Glu

Ile Pro

Leu Arg

Leu Arg

PheGlu

Phe Trp

Gln Asn

Cys Leu 110

Gln Leu

Asp His

Leu Leu

Gly Lys

175

Arg Ile 190

Val Leu

Asn Thr

Asn Trp

His Asp

255

Ser Thr

270

Leu His

Pro

Glu

Ile 80

Gly

Pro

Ile

Thr

Ala 160

Ile

Val

Leu

Ile

Arg 240

Gly

Tyr

Gln

- 98 044349

275

280

285

Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro Glu Arg

290 295300

Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu Val Ser

305 310 315320

Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Met

325 330335

Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Ser

340 345350

Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe Cys Ala

355 360365

Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly

370 375380

Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val

385 390 395400

Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr

405 410415

Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu

420 425430

Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro Gly Cys Gly Arg

435 440445 <210>11 <211>1356 <212> DNA <213> Homo sapiens

<400> 11 ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc 60 tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg 120 cgcgccaacg cgttcctgga ggagctgcgg ccggggctccc tggagaggga gtgcaagggag 180 gagcagtgct ccttcgagga ggcccggggag atcttcaagg acgcggagag gacgaagctg 240 ttctggattt cttacagtga tggggaccag tgtgcctcaa gtccatgcca gaatggggggc 300 tcctgcaagg accagctcca gtcctatatc tgcttctgcc tccctgcctt cgaggggccgg 360 aactgtgaga cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag 420

- 99 044349

cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct 480 ctgctggcag acggggtgtc ctgcacacc acagttgaat atccatggtgg aaaaatacct 540 attctgaaa aaagaaatgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc 600 cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca gttgtgtggg 660 gggaccctga tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag 720 aactggagga acctgatcgc ggtgctggggc gagcacgacc tcagcgagca cgacggggat 780 gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc gggcaccacc 840 aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg tcctactga ccatgtggtg 900 cccctctgcc tgcccgaacg gacgttctct gagaggacgc tggccttcgt gcgcttctca 960 ttggtcagcg gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc 1020 ctcaacgtgc cccggctgat gaccagggac tgcctgcagc agtcacggaa ggtggggagac 1080 tccccaaata tcacggagta catgttctgt gccggctact cggatggcag caaggactcc 1140 tgcaaggggg acagtggagg cccacatgcc accactacc ggggcacgtg gtacctgacg 1200 ggcatcgtca gctggggcca gggctgcgca accgtgggcc actttggggt gtacaccagg 1260 gtctcccagt acatcgagtg gctgcaaaag ctcatgcgct cagagccacg cccaggagtc 1320 ctcctgcgag ccccatttcc ctgaggatgc ggccgc 1356

<210> 12 <211> 1442 <212> DNA <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400>12 ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc60 tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg120 cgcgccaacg cgttcct gga ggagctgcgg ccgggctccc tggagaggga gtgcaaggag180 gagcagtgct ccttcgagga ggcccgggag atcttcaagg acgcggagag gacgaagctg240 ttctggattt cttacagtga tggggaccag tgtgcctcaa gtccatgcca gaatgggggc300 tcctgcaagg a ccagctcca gtcctatatc tgcttctgcc tccctgcctt cgagggccgg360 aactgtgaga cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag420 cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct480 ctgctggcag acggggtgtc ctgcacaccc acagttgaat atccatgtgg aaaaatacct540 attctagaaa aaagaa atgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc600 cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca gttgtgtggg660

- 100 044349 gggaccctga aactggagga gagcagagcc aaccacgaca cccctctgcc ttggtcagcg ctcaacgtgc tccccaaata tgcaaggggg ggcatcgtga gtgtcccagt ctgctgagag cctagcagac gc tcaacaccat acctgatcgc ggcgggtggc tcgcg ctgct tgcccgaacg gctggggcca cccggctgat tcacggagta acagtggagg gctggggcca acatcgagtg cccccttccc tgcctgggcc ctgggtggtc ggtgctgggc gcaggtcatc ccgcctgcac gacgttctct gctgctggac gacccaggac catgttctgt cccacatgcc gggctg cgcc gctgcagaaa cagcagcagc cagcgacacc tccgcggccc gagcacgacc atccccagca cagcccgtgg gagaggacgc cgtggcgcca tgcctgcagc gccggctact acccactacc accgtgggcc ctgatgagaa tccaaggccc cccatcctgc actgtttcga tcagcgagca cgtacgtccc tcctcactga tggccttcgt cggccctgga agtcacggaa cggatggcag ggggcacgtg acttcggcgt gcgagcccag ctccccctag ccca gtgagg caaaatcaag cgacggggat gggcaccacc ccatgtggtg gcgcttctca gctcatggtc ggtgggagac caaggactcc gtacctgacc gtacaccagg acccggcgtg cctgcccagc atccgcggcc

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1442 <210> 13 <211> 472 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400>13

Met Val Ser Gln Ala Leu Arg Leu 15

Leu Cys Leu Leu Leu Gly Leu Gln

1015

Gly Cys Leu Ala Ala Val Phe Val 20

Thr Gln Glu Glu Ala His Gly Val 25 30

Leu His Arg Arg Arg Arg Ala Asn

40

Ala Phe Leu Glu Glu Leu Arg Pro 45

Gly Ser Leu Glu Arg Glu Cys Lys

55

Glu Glu Gln Cys Ser Phe Glu Glu 60

Ala Arg Glu Ile Phe Lys Asp Ala 65 70

Glu Arg Thr Lys Leu Phe Trp Ile

80

Ser Tyr Ser Asp Gly Asp Gln Cys 85

Ala Ser Ser Pro Cys Gln Asn Gly

95

- 101 044349

Gly

Ala

Cys

Gly 145

Asp

Pro

Gly

Leu

Trp 225

Asn

Asp

Val

Pro

Thr 305

Gly

Val

Ser Cys

PheGlu

115

Val Asn 130

Thr Lys

Gly Val

Ile Leu

Gly Lys

195

Val Asn 210

Val Val

Leu Ile

Glu Gln

Pro Gly

275

Val Val 290

Phe Ser

Trp Gly

Leu Asn

Lys Asp 100

Gly Arg

Glu Asn

Arg Ser

Gln Leu

Asn Cys

Gly Gly

135

Cys Arg 150

Ser Cys

165

Glu Lys 180

Val Cys

Gly Ala

Ser Ala

Ala Val

245

Ser Arg 260

Thr Thr

Leu Thr

Glu Arg

Gln Leu

325

Val Pro 340

Thr Pro

Arg Asn

Pro Lys

Gln Leu

215

Ala His 230

Leu Gly

Arg Val

Asn His

Asp His

295

Thr Leu 310

Leu Asp

Arg Leu

Gln Ser 105

Glu Thr 120

Cys Glu

Cys His

Thr Val

Tyr Ile

CysPhe

His Lys

Gln Tyr

Glu Gly

155

Glu Tyr 170

Ala Ser

185

Gly Glu 200

Cys Gly

CysPhe

Glu His

Ala Gln

265

Asp Ile 280

Val Val

Ala Phe

Arg Gly

Met Thr

345

Lys Pro

Cys Pro

Gly Thr

Asp Lys

235

Asp Leu 250

Val Ile

Ala Leu

ProLeu

Val Arg

315

Ala Thr 330

Gln Asp

Asp Asp

125

Cys Ser 140

Tyr Ser

Pro Cys

Gln Gly

Trp Gln

205

Leu Ile

220

Ile Lys

Ser Glu

Ile Pro

Leu Arg

285

Cys Leu 300

Phe Ser

Ala Leu

Cys Leu

Cys Leu 110

Gln Leu

Asp His

Leu Leu

Gly Lys

175

Arg Ile 190

Val Leu

Asn Thr

Asn Trp

His Asp

255

Ser Thr

270

Leu His

Pro Glu

Leu Val

Glu Leu

335

Gln Gln 350

Pro

Ile

Thr

Ala 160

Ile

Val

Leu

Ile

Arg 240

Gly

Tyr

Gln

Arg

Ser 320

Met

Ser

- 102 044349

Arg Lys Val Gly Asp Ser Pro Asn

355 360

Ile Thr Glu Tyr Met Phe Cys Ala

365

Gly Tyr Ser Asp Gly Ser Lys Asp

370375

Pro His Ala Thr His Tyr Arg Gly

385390

Ser Cys Lys Gly Asp Ser Gly Gly 380

Thr Trp Tyr Leu Thr Gly Ile Val 395400

Ser Trp Gly Gln Gly Cys Ala Thr 405

Val Gly His Phe Gly Val Tyr Thr

410 415

Arg Val Ser Gln Tyr Ile Glu Trp 420

Leu Gln Lys Leu Met Arg Ser Glu

425 430

Pro Arg Pro Gly Val Leu Leu Arg

435 440

Ala Pro Phe Pro Ser Ser Ser Ser

445

Lys Ala Pro Pro Pro Ser Leu Pro

450 455

Ser Pro Ser Arg Leu Pro Gly Pro 460

Ser Asp Thr Pro Ile Leu Pro Gln

465470 <210>14 <211>1535 <212> DNA <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400> 14

ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc 60 tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg 120 cgcgccaacg cgttcctgga ggagctgcgg ccggggctccc tggagaggga gtgcaagggag 180 gagcagtgct ccttcgagga ggcccggggag atcttcaagg acgcggagag gacgaagctg 240 ttctggattt cttacagtga tggggaccag tgtgcctcaa gtccatgcca gaatggggggc 300 tcctgcaagg accagctcca gtcctatatc tgcttctgcc tccctgcctt cgaggggccgg 360 aactgtgaga cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag 420 cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct 480 ctgctggcag acggggtgtc ctgcacacc acagttgaat atccatggtgg aaaaatacct 540 attctgaaa aaagaaatgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc 600

- 103 044349

cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca gttgtgtggg 660 gggaccctga tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag 720 aactggagga acctgatcgc ggtgctggggc gagcacgacc tcagcgagca cgacggggat 780 gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc gggcaccacc 840 aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg tcctactga ccatgtggtg 900 cccctctgcc tgcccgaacg gacgttctct gagaggacgc tggccttcgt gcgcttctca 960 ttggtcagcg gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc 1020 ctcaacgtgc cccggctgat gaccagggac tgcctgcagc agtcacggaa ggtggggagac 1080 tccccaaata tcacggagta catgttctgt gccggctact cggatggcag caaggactcc 1140 tgcaaggggg acagtggagg cccacatgcc accactacc ggggcacgtg gtacctgacc 1200 ggcatcgtga gctggggcca gggctgcgcc accgtgggcc acttcggcgt gtacaccagg 1260 gtgtcccagt acatcgagtg gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg 1320 ctgctgagag cccccttccc cagcagcagc tccaaggccc ctccccctag cctgcccagc 1380 cctagcagac tgcctgggcc ctccgacaca ccaatcctgc cacagagcag ctcctctaag 1440 gcccctcctc catccctgcc atccccctcc cggctgccag gcccctctga cacccctatc 1500 ctgcctcagt gatgaaggtc tggatccgcg gccgc 1535

<210> 15 <211> 500 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400> 15

Met Val Ser Gln Ala Leu Arg Leu 1 5

Leu Cys Leu Leu Leu Gly Leu Gln

15

Gly Cys Leu Ala Ala Val Phe Val 20

Thr Gln Glu Glu Ala His Gly Val 25 30

Leu His Arg Arg Arg Arg Ala Asn

40

Ala Phe Leu Glu Glu Leu Arg Pro 45

Gly Ser Leu Glu Arg Glu Cys Lys

55

Glu Glu Gln Cys Ser Phe Glu Glu 60

Ala Arg Glu Ile Phe Lys Asp Ala 65 70

Glu Arg Thr Lys Leu Phe Trp Ile

80

- 104 044349

Ser

Gly

Ala

Cys

Gly 145

Asp

Pro

Gly

Leu

Trp 225

Asn

Asp

Val

Pro

Thr 305

Gly

Tyr Ser Asp

Gly Asp Gln 85

Cys Ala Ser

Ser Pro Cys

Gln Asn Gly 95

Ser Cys Lys

100

Phe Glu Gly 115

Val Asn Glu 130

Thr Lys Arg

Gly Val Ser

Ile Leu Glu

180

Gly Lys Val

195

Val Asn Gly 210

Val Val Ser

Leu Ile Ala

Glu Gln Ser

260

Pro Gly Thr

275

Val Val Leu 290

Phe Ser Glu

Trp Gly Gln

Asp Gln Leu

Arg Asn Cys

Asn Gly Gly

135

Ser Cys Arg

150

Cys Thr Pro 165

Lys Arg Asn

Cys Pro Lys

Ala Gln Leu

215

Ala Ala His

230

Val Leu Gly 245

Arg Arg Val

Thr Asn His

Thr Asp His

295

Arg Thr Leu

310

Leu Leu Asp 325

Gln Ser Tyr

105

Glu Thr His 120

Cys Glu Gln

Cys His Glu

Thr Val Glu

170

Ala Ser Lys

185

Gly Glu Cys 200

Cys Gly Gly

Cys Phe Asp

Glu His Asp

250

Ala Gln Val

265

Asp Ile Ala 280

Val Val Pro

Ala Phe Val

Arg Gly Ala

330

Ile Cys Phe

Lys Asp Asp

125

Tyr Cys Ser

140

Gly Tyr Ser 155

Tyr Pro Cys

Pro Gln Gly

Pro Trp Gln

205

Thr Leu Ile

220

Lys Ile Lys 235

Leu Ser Glu

Ile Ile Pro

Leu Leu Arg

285

Leu Cys Leu 300

Arg Phe Ser 315

Thr Ala Leu

Cys Leu Pro 110

Gln Leu Ile

Asp His Thr

Leu Leu Ala

160

Gly Lys Ile

175

Arg Ile Val 190

Val Leu Leu

Asn Thr Ile

Asn Trp Arg

240

His Asp Gly

255

Ser Thr Tyr 270

Leu His Gln

Pro Glu Arg

Leu Val Ser

320

Glu Leu Met

335

- 105 044349

Val Leu

Asn Val

340

Pro Arg

Leu Met

Arg Lys

Val Gly 355

Asp Ser

Pro Asn

360

Gly Tyr

370

Ser Asp

Gly Ser

Lys Asp 375

Pro His 385

Ala Thr

His Tyr

390

Arg Gly

Thr Gln 345

Ile Thr

Ser Cys

Thr Trp

Asp Cys

Glu Tyr

Lys Gly

380

Tyr Leu 395

Ser Trp

Gly Gln

Gly Cys 405

Ala Thr

Val Gly

410

His Phe

Arg Val

Ser Gln

420

Tyr Ile

Pro Arg

Pro Gly 435

Val Leu

Leu Gln

350

Gln Ser

Met Phe 365

Asp Ser

Thr Gly

Gly Val

Cys Ala

Gly Gly

Ile Val

400

Tyr Thr 415

Lys Ala

450

Pro Pro

Pro Ser

Ser Asp 465

Thr Pro

Ile Leu

470

Pro Ser

Leu Pro

Ser Pro

485

Glu Trp

Leu Arg 440

Leu Pro 455

Pro Gln

Ser Arg

Leu Gln 425

Ala Pro

Ser Pro

Ser Ser

Leu Pro

490

Ile Leu Pro Gln

500

Lys Leu

Phe Pro

Ser Arg

460

Ser Ser

475

Gly Pro

Met Arg

430

Ser Glu

Ser Ser 445

Ser Ser

Leu Pro

Gly Pro

Lys Ala

Pro Pro

480

Ser Asp

Thr Pro 495 <210>16 <211>1404 <212> DNA <213> Homo <400>16 gcgatcgcca gccttttagg acaaaattct ggaaccttga ttgaaaacac agtccaatcc sapiens tgcagcgcgt atatctactc gaatcggcca gagagaatgt tgaaagaaca atgtttaa at gaacatgatc agtgctgaat aagaggtata atggaagaaa actgaatttt ggcggcagtt atggcagaat gtacagtttt attcaggtaa agtgtagttt ggaagcagta gcaaggatga caccaggcct tcttgatcat attggaagag tgaagaagca tgttgatgga cattaattcc catcaccatt gaaaacgcca tttgttcaag cgagaagttt gatcagtgtg tatgaatgtt

120

180

240

300

360

- 106 044349 ggtgtccctt atggcagatg ctgagggata gtggaagagt atgtggacta cccaatcatt tcccttggca atgaaaaatg tcgcaggtga gaattattcc ttctggaact acaaggaata gagtcttcca accgagccac gcttccatga aagtggaagg aaggcaaata caaagctca c tggatttgaa cgagcagttt tcgacttgca ttctgtttca tgtaaattct taatgacttc ggttgttttg gattgtaact acataatatt tcaccacaac ggacgaaccc cacgaacatc caaagggaga atgtcttcga aggaggtaga gaccagtttc tggaatatat ttgaacgcgg ggaaagaact t gtaaaaata gaaaaccaga caaacttcta actgaagctg actcgagttg aatggtaaag gctgcccact gaggagacag tacaatgcag ttagtgctaa ttcctcaaat tcagctttag tctacaaagt gattcatgtc ttaactggaa accaaggtat ccgc gtgaattaga gtgctgataa agtcctgtga agctcacccg aaaccatttt ttggtggaga ttgatgcatt gtgttgaaac aacatacaga ctattaataa acagctacgt ttggatctgg ttct ccagta tcaccatcta aaggagatag ttattagctg cccggtatgt tgtaacatgt caaggtggtt accagcagtg tgctgagact ggataacatc agatgccaaa ctgtggaggc tggtgttaaa gcaaaagcga gtacaaccat tacacctatt ctatgtaagt ccttagagtt taacaacatg tggg ggaccc gggtgaagag caactggatt aacattaaga tgctcctgta ccatttccat gtttttcctg actcaaagca ccaggtcaat tctatcgtta attacagttg aatgtgattc gacattgccc tgcattgctg ggctggggaa ccacttgttg ttctgtgctg catgttactg tgtgcaatga aaggaaaaaa

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1404 <210>17 <211>461 <212> Protein <213> Homo sapiens <400>17

Met Gln 1

Arg Val

Asn Met 5

Ile Met

Ala Glu

Ser Pro

Gly Leu

Ile Cys

Leu Leu

Gly Tyr

Leu Leu

Ser Ala 25

Glu Cys

Thr Val

Asp His

Glu Asn 35

Ala Asn

Lys Ile 40

Leu Asn

Arg Pro

Lys Arg 45

Ser Gly 50

Lys Leu

Glu Glu

Phe Val 55

Gln Gly

Asn Leu

Glu Arg

Ile Thr 15

Phe Leu

Tyr Asn

Glu Cys

Met Glu Glu Lys Cys Ser Phe Glu 65 70

Glu Ala Arg Glu Val Phe Glu Asn

80

- 107 044349

Thr

Cys

Asn

Glu

Cys 145

Tyr

Pro

Glu

Thr

Thr 225

Gln

Val

Val

His

Tyr 305

Leu

Glu Arg Thr

Glu Ser Asn

100

Ser Tyr Glu

115

Leu Asp Val 130

Lys Asn Ser

Arg Leu Ala

Cys Gly Arg

180

Thr Val Phe

195

Ile Leu Asp 210

Arg Val Val

Val Val Leu

Asn Glu Lys

260

Lys Ile Thr

275

Thr Glu Gln 290

Asn Ala Ala

Asp Glu Pro

Thr Glu Phe 85

Pro CysLeu

Cys Trp Cys

Thr Cys Asn

135

Ala Asp Asn

150

Glu Asn Gln 165

Val Ser Val

Pro Asp Val

Asn Ile Thr

215

Gly Gly Glu

230

Asn Gly Lys 245

Trp Ile Val

Val Val Ala

Lys Arg Asn

295

Ile Asn Lys

310

Leu Val Leu 325

Trp Lys Gln 90

Tyr Val Asp

Gly Asp Gln 95

Asn Gly Gly

105

Pro Phe Gly 120

Ile Lys Asn

Lys Val Val

Lys Ser Cys

170

Ser Gln Thr

185

Asp Tyr Val 200

Gln Ser Thr

Asp Ala Lys

Val Asp Ala

250

Thr Ala Ala

265

Gly Glu His 280

Val Ile Arg

Tyr Asn His

Asn Ser Tyr

330

Ser Cys Lys

Phe Glu Gly

125

Gly Arg Cys

140

Cys Ser Cys 155

Glu Pro Ala

Ser Lys Leu

Asn Ser Thr

205

Gln Ser Phe

220

Pro Gly Gln 235

Phe Cys Gly

His Cys Val

Asn Ile Glu

285

Ile Ile Pro

300

Asp Ile Ala 315

Val Thr Pro

Asp Asp Ile 110

Lys Asn Cys

Glu Gln Phe

Thr Glu Gly

160

Val Pro Phe

175

Thr Arg Ala 190

Glu Ala Glu

Asn Asp Phe

Phe Pro Trp

240

Gly Ser Ile

255

Glu Thr Gly 270

Glu Thr Glu

His His Asn

Leu Leu Glu

320

Ile Cys Ile

335

- 108 044349

Ala Asp

Lys Glu

340

Tyr Thr

Asn Ile

Val Ser

Gly Trp 355

Gly Arg

Val Phe

360

Leu Gln

370

Tyr Leu

Arg Val

Pro Leu 375

Ser Thr

385

Lys Phe

Thr Ile

390

Tyr Asn

Glu Gly

Gly Arg

Asp Ser 405

Cys Gln

Thr Glu

Val Glu

420

Gly Thr

Ser Phe

Glu Glu

Cys Ala 435

Met Lys

Gly Lys

440

Arg Tyr

450

Val Asn

Trp Ile

Lys Glu 455

Phe Leu 345

His Lys

Val Asp

Asn Met

Gly Asp 410

Leu Thr 425

Tyr Gly

Lys Thr

Lys Phe

Gly Arg

Arg Ala

380

Phe Cys 395

Ser Gly

Gly Ile

Ile Tyr

Lys Leu

460

Gly Ser

350

Ser Ala

365

Thr Cys

Ala Gly

Gly Pro

Ile Ser

430

Thr Lys 445

Thr

Gly Tyr

Leu Val

Leu Arg

Phe His

400

His Val 415

Trp Gly

Val Ser <210> 18 <211> 1502 <212> DNA <213> Artificial sequence <220>

< 223> CTP-modified Factor IX <400> 18 gcgatcgcca tgccttttag aacaaaattc gggaaccttg tttgaaaaca gagtccaatc tggtgtccct aatggcagat actgagggat tgtggaagag tgcagcgcgt gatatctact tgaatcggcc agagagaatg ctgaa agaac catgtttaaa ttggatttga gcgagcagtt atcgacttgc tttctgtttc gaacatgatc cagtgctgaa aaagaggtat tatggaagaa aactgaattt tggcggcagt aggaaagaac ttgtaaaaat agaaaaccag acaaacttct atggcagaat tgtacagttt aattcaggta aag tgtagtt tggaagcagt tgcaaggatg tgtgaattag agtgctgata aagtcctgtg aagctcaccc caccaggcct ttcttgatca aattggaaga ttgaagaagc atgttgatgg acattaattc atgtaacatg acaaggtggt aaccagcagt gtgctgagac catcaccatc tgaaaacgcc gtttgttcaa acgagaagtt agatcagtgt ctatgaatgt taacatta ag ttgctcctgt gccatttcca tgtttttcct

120

180

240

300

360

420

480

540

600

- 109 044349

gatgtggact atgtaaattc tactgaagct gaaaccattt tggataacat cactcaaagc 660 acccaatcat ttaatgactt cactcgagtt gttggtggag aagatgccaa accaggtcaa 720 ttcccttggc aggttgtttt gaatggtaaa gttgatgcat tctgtggagg ctctatcgtt 780 aatgaaaaat ggattgtaac tgctgcccac tgtgttgaaa ctggtgttaa aattacagtt 840 gtcgcaggtg aacataatat tgaggagaca gaacatacag agcaaaagcg aaatgtgatt 900 cgaattattc ctcaccacaa ctacaatgca gctattaata agtacaacca tgacattgcc 960 cttctggaac tggacgaacc cttagtgcta aacagctacg ttacacctat ttgcattgct 1020 gacaaggaat acacgaacat cttcctcaaa tttggatctg gctatgtaag tggctgggga 1080 agagtcttcc acaaagggag atcagcttta gttcttcagt accttaggt tccacttgtt 1140 gaccgagcca catgtcttcg atctacaaag ttcaccactct aaacaacat gttctgtgct 1200 ggcttccatg aaggaggtag agattcatgt caaggagata gtggggggacc ccatgttact 1260 gaagtggaag ggaccagttt cttaactgga attatagct ggggtgaaga gtgtgcaatg 1320 aaaggcaaat atggaatata taccaaggta tcccggtatg tcaactggat taaggaaaaa 1380 acaaagctca ctagctccag cagcaaggcc cctcccccga gcctgccctc cccaagcagg 1440 ctgcctggggc cctccgacac accaatcctg ccacagtgat gaaggtctgg atccgcggcc 1500

gc

1502 <210> 19 <211> 489 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor IX <400>19

Met Gln Arg Val Asn Met Ile Met 15

Ala Glu Ser Pro Gly Leu Ile Thr

1015

Ile Cys Leu Leu Gly Tyr Leu Leu 20

Ser Ala Glu Cys Thr Val Phe Leu 25 30

Asp His Glu Asn Ala Asn Lys Ile

40

Leu Asn Arg Pro Lys Arg Tyr Asn 45

Ser Gly Lys Leu Glu Glu Phe Val

55

Gln Gly Asn Leu Glu Arg Glu Cys 60

Met Glu Glu Lys Cys Ser Phe Glu 65 70

Glu Ala Arg Glu Val Phe Glu Asn

80

- 110 044349

Thr

Cys

Asn

Glu

Cys 145

Tyr

Pro

Glu

Thr

Thr 225

Gln

Val

Val

His

Tyr 305

Leu

Glu Arg

Glu Ser

Ser Tyr

115

Leu Asp 130

Lys Asn

Arg Leu

Cys Gly

Thr Val

195

Ile Leu 210

Arg Val

Val Val

Asn Glu

Lys Ile

275

Thr Glu

290

Asn Ala

Asp Glu

Thr Thr

Glu Phe

Trp Lys

Asn Pro

100

Cys Leu

Asn Gly

105

Glu Cys

Trp Cys

Pro Phe 120

Val Thr

Cys Asn

135

Ile Lys

Gln Tyr 90

Gly Ser

Gly Phe

Asn Gly

Val Asp

Cys Lys

Glu Gly

125

Arg Cys 140

Ser Ala

Ala Glu

165

Arg Val 180

Phe Pro

Asp Asn

Val Gly

Leu Asn

245

Lys Trp 260

Thr Val

Gln Lys

Ala Ile

ProLeu

325

Asp Asn 150

Asn Gln

Ser Val

Asp Val

Ile Thr

215

Gly Glu 230

Gly Lys

Ile Val

Val Ala

Arg Asn

295

Asn Lys 310

Val Leu

Lys Val

Lys Ser

Ser Gln

185

Asp Tyr 200

Gln Ser

Asp Ala

Val Asp

Thr Ala

265

Gly Glu 280

Val Ile

Tyr Asn

Asn Ser

Gly Asp 95

Asp Asp 110

Lys Asn

Glu Gln

Val Cys

155

Cys Glu 170

Thr Ser

Val Asn

Thr Gln

Lys Pro

235

Ala Phe 250

Ala His

His Asn

Arg Ile

His Asp

315

Tyr Val 330

Ser Cys

Thr Glu

Pro Ala

Lys Leu

Ser Thr

205

Ser Phe 220

Gly Gln

Cys Gly

Cys Val

Ile Glu

285

Ile Pro 300

Ile Ala

Val Pro

175

Thr Arg 190

Glu Ala

Asn Asp

Phe Pro

Gly Ser

255

Glu Thr

270

Glu Thr

His His

Leu Leu

Gln

Ile

Cys

Phe

Gly 160

Phe

Ala

Glu

Phe

Trp 240

Ile

Gly

Glu

Asn

Glu 320

Ile

Thr Pro

Ile Cys

335

- 111 044349

Ala Asp

Lys Glu

340

Tyr Thr

Val Ser

Gly Trp 355

Gly Arg

Leu Gln

370

Tyr Leu

Arg Val

Ser Thr

385

Lys Phe

Thr Ile

390

Glu Gly

Gly Arg

Asp Ser 405

Thr Glu

Val Glu

420

Gly Thr

Glu Glu

Cys Ala 435

Met Lys

Arg Tyr

450

Val Asn

Trp Ile

Ser Lys 465

Ala Pro

Pro Pro

470

Pro Ser

Asp Thr

Pro Ile 485

Asn Ile

Val Phe

360

Pro Leu 375

Tyr Asn

Cys Gln

Ser Phe

Gly Lys

440

Lys Glu 455

Ser Leu

Leu Pro

Phe Leu 345

His Lys

Val Asp

Asn Met

Gly Asp 410

Leu Thr 425

Tyr Gly

Lys Thr

Pro Ser

Gln

Lys Phe

Gly Arg

Arg Ala

380

Phe Cys 395

Ser Gly

Gly Ile

Ile Tyr

Lys Leu

460

Pro Ser 475

Gly Ser

350

Ser Ala

365

Thr Cys

Ala Gly

Gly Pro

Ile Ser

430

Thr Lys 445

Thr Ser

Arg Leu

Gly Tyr

Leu Val

Leu Arg

Phe His

400

His Val 415

Trp Gly

Val Ser

Ser Ser

Pro Gly

480 <210> 20 <211> 1585 <212> DNA <213> Artificial sequence <220>

< 223> CTP-modified Factor IX < 400> 20 gcgatcgcca tgcagcgcgt gaacatgatc atggcagaat caccaggcct catcaccatc tgccttttag gatatctact cagtgctgaa tgtacagttt ttcttgatca tgaaaacgcc aacaaaattc tgaatcggcc aaagagg tat aattcaggta aattggaaga gtttgttcaa gggaaccttg agagagaatg tatggaagaa aagtgtagtt ttgaagaagc acgagaagtt tttgaaaaca ctgaaagaac aactgaattt tggaagcagt atgttgatgg agatcagtgt gagtccaatc catgtttaaa tggcggcagt tgca aggatg acattaattc ctatgaatgt tggtgtccct ttggatttga aggaaagaac tgtgaattag atgtaacatg taacattaag

120

180

240

300

360

420

- 112 044349

aatggcagat gcgagcagtt ttgtaaaaat agtgctgata acaaggtggt ttgctcctgt actgaggat atcgacttgc agaaaaccag aagtcctgtg aaccagcagt gccatttcca tgtggaagag tttctgtttc acaaacttct aagctcaccc gtgctgagac tgtttttcct gatgtggact atgtaaattc tactgaagct gaaaccattt tggataacat cactcaaagc acccaatcat ttaatgactt cactcgagtt gttggtggag aagatgccaa accaggtcaa ttcccttggc aggttgtttt gaatggtaaa gttgatgcat tctgtggagg ctctatcgtt aatgaaaaat ggattgtaac tgctgcccac tgtgttgaaa ctggtgttaa aattacagtt gtcgcaggtg aacataatat tgaggagaca gaacatacag agcaaaagcg aaatgtgatt cgaattattc ctcaccacaa ctacaatgca gctattaata agtacaacca tgacattgcc cttctggaac tggacgaacc cttagtgcta aacagctacg ttacacctat ttgcattgct acaaggaata cacgaacatc ttcctcaaat ttggatctgg ctatgtaagt ggctggggaa gagtcttcca caaagggaga tcagctttag ttcttcagta ccttagagtt ccacttgttg accgagccac atgtcttcga tctacaaagt tcaccatta taacaacatg ttctgtgctg gcttccatga aggaggtaga gattcatgtc aaggagatag tggggggaccc catgttactg aagtggaagg gaccagtttc ttaactggaa ttattagctg gggtgaagag tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt caactggatt aaggaaaaaa caaagctcac tagctccagc agcaaggccc ctccccccgag cctgccctcc ccaagcaggc tgcctgggcc ctccgacaca ccaatcctgc cacagagcag ctcctctaag gcccctcctc catccctgcc atccccctcc cggctgcctg gcccctctga cacccctatc ctgcctcagt gatgaaggtc tggatccgcg gccgc

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1585 <210> 21 <211> 517 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor IX <400> 21

Met Gln Arg Val 1

Asn Met Ile Met 5

Ala Glu Ser Pro

Gly Leu Ile Thr 15

Ile Cys Leu Leu 20

Gly Tyr Leu Leu

Ser Ala Glu Cys 25

Thr Val Phe Leu

Asp His Glu Asn 35

Ala Asn Lys Ile 40

Leu Asn Arg Pro

Lys Arg Tyr Asn 45

- 113 044349

Ser

Met 65

Thr

Cys

Asn

Glu

Cys 145

Tyr

Pro

Glu

Thr

Thr 225

Gln

Val

Val

His

Gly Lys 50

Glu Glu

Glu Arg

Glu Ser

Leu Glu

Lys Cys

Thr Thr

Asn Pro 100

Ser Tyr

115

Leu Asp 130

Lys Asn

Arg Leu

Cys Gly

Thr Val

195

Ile Leu 210

Arg Val

Val Val

Asn Glu

Lys Ile

275

Thr Glu

290

Glu Cys

Val Thr

Ser Ala

Ala Glu

165

Arg Val 180

Phe Pro

Asp Asn

Val Gly

Leu Asn

245

Lys Trp 260

Thr Val

Gln Lys

Glu Phe

Ser Phe 70

Glu Phe

Cys Leu

Trp Cys

Cys Asn

135

Asp Asn 150

Asn Gln

Ser Val

Asp Val

Ile Thr

215

Gly Glu 230

Gly Lys

Ile Val

Val Ala

Arg Asn

295

Val Gln

Glu Glu

Trp Lys

Asn Gly 105

Pro Phe 120

Ile Lys

Lys Val

Lys Ser

Ser Gln

185

Asp Tyr 200

Gln Ser

Asp Ala

Val Asp

Thr Ala

265

Gly Glu 280

Val Ile

Gly Asn

Ala Arg

Gln Tyr 90

Gly Ser

Gly Phe

Asn Gly

Val Cys

155

Cys Glu 170

Thr Ser

Val Asn

Thr Gln

Lys Pro

235

Ala Phe 250

Ala His

His Asn

Arg Ile

Leu Glu 60

Glu Val

Val Asp

Cys Lys

Arg Glu

PheGlu

Gly Asp 95

Asp Asp 110

Glu Gly

125

Lys Asn

Arg Cys 140

Glu Gln

Ser Cys

Thr Glu

Pro Ala

Lys Leu

Ser Thr

205

Ser Phe 220

Gly Gln

Cys Gly

Cys Val

Ile Glu

285

Ile Pro 300

Val Pro

175

Thr Arg 190

Glu Ala

Asn Asp

Phe Pro

Gly Ser

255

Glu Thr

270

Glu Thr

Cys

Asn 80

Gln

Ile

Cys

Phe

Gly 160

Phe

Ala

Glu

Phe

Trp 240

Ile

Gly

Glu

Asn

His His

- 114 044349

Tyr Asn Ala 305

Ala Ile Asn

310

Lys Tyr Asn

His Asp Ile

315

Ala Leu Leu

Leu Asp Glu

Pro Leu Val

325

Leu Asn Ser

Tyr Val Thr 330

Pro Ile Cys

335

Ala Asp Lys

Glu Tyr Thr 340

Asn Ile Phe

345

Leu Lys Phe

Gly Ser Gly

350

Val Ser Gly

355

Trp Gly Arg

Val Phe His

360

Lys Gly Arg

Ser Ala Leu 365

Leu Gln Tyr

370

Leu Arg Val

Pro Leu Val 375

Asp Arg Ala

380

Thr Cys Leu

Ser Thr Lys 385

Phe Thr Ile

390

Tyr Asn Asn

Met Phe Cys

395

Ala Gly Phe

Glu Gly Gly

Arg Asp Ser

405

Cys Gln Gly

Asp Ser Gly 410

Gly Pro His 415

Thr Glu Val

Glu Gly Thr 420

Ser Phe Leu

425

Thr Gly Ile

Ile Ser Trp

430

Glu Glu Cys

435

Ala Met Lys

Gly Lys Tyr

440

Gly Ile Tyr

Thr Lys Val 445

Arg Tyr Val

450

Asn Trp Ile

Lys Glu Lys 455

Thr Lys Leu

460

Thr Ser Ser

Ser Lys Ala 465

Pro Pro Pro

470

Ser Leu Pro

Ser Pro Ser

475

Arg Leu Pro

Pro Ser Asp

Thr Pro Ile

485

Leu Pro Gln

Ser Ser Ser 490

Ser Lys Ala

495

Pro Pro Ser

Leu Pro Ser 500

Pro Ser Arg

505

Leu Pro Gly

Pro Ser Asp

510

Glu 320

Ile

Tyr

Val

Arg

His 400

Val

Gly

Ser

Ser

Gly 480

Pro

Thr

Pro Ile Leu

515

Pro Gln <210> 22 <211> 2413 <212> DNA <213> Homo sapiens

- 115 044349

<400> 22 tctagagtcg accccgccat ggagctgagg ccctggttgc tatgggtggt agcagcaaca 60 ggaaccttgg tcctgctagc agctgatgct caggggccaga aggtcttcac caacacgtgg 120 gctgtgcgca tccctggagg cccagcggtg gccaacagtg tggcacggaa gcatgggttc 180 ctcaacctgg gccagatctt cggggactat taccacttct ggcatcgagg agtgacgaag 240 cggtccctgt cgcctcaccg cccgcggcac agccggctgc aggggagcc tcaagtacag 300 tggctggaac agcaggtggc aaagcgacgg actaaacgggg acgtgtacca ggagcccaca 360 gaccccaagt ttcctcagca gtggtacctg tctggtgtca ctcagcggga cctgaatgtg 420 aaggcggcct gggcgcaggg ctacacagg caggcattg tggtctccat tctggacgat 480 ggcatcgaga agaaccacc ggacttggca ggcaattatg atcctggggc cagttttgat 540 gtcaatgacc aggaccctga cccccagcct cggtacacac agatgaatga caacaggcac 600 ggcacacggt gtgcggggga agtggctgcg gtggccaaca acggtgtctg tggtgtaggt 660 gtggcctaca acgcccgcat tggaggggtg cgcatgctgg atggcgaggt gacagatgca 720 gtggaggcac gctcgctggg cctgaacccc aaccacatcc acatctacag tgccagctgg 780 ggccccgagg atgacggcaa gacagtggat gggccagccc gcctcgccga ggaggccttc 840 ttccgtgggg ttagccagg ccgagggggg ctggggctcca tctttgtctg ggcctcgggg 900 aacggggggcc gggaacatga cagctgcaac tgcgacggct acaccaacag tatctacacg 960 ctgtccatca gcagcgccac gcagtttggc aacgtgccgt ggtacagcga ggcctgctcg 1020 tccacactgg ccacgaccta cagcagtggc aaccagaatg agaagcagat cgtgacgact 1080 gacttgcggc agaagtgcac ggagtctcac acggggcacct cagcctctgc ccccttagca 1140 gccggcatca ttgctctcac cctggaggcc aataagaacc tcacatggcg ggacatgcaa 1200 cacctggtgg tacagacctc gaagccagcc cacctcaatg ccaacgactg ggccaccaat 1260 ggtgtgggcc ggaaagtgag ccactcatat ggctacggggc ttttggacgc aggcgccatg 1320 gtggccctgg cccagaattg gaccacagtg gccccccagc ggaagtgcat catcgacatc 1380 ctcaccgagc ccaaagacat cgggaaacgg ctcgaggtgc ggaagaccgt gaccgcgtgc 1440 ctggggcgagc ccaaccacat cactcggctg gagcacgctc aggcgcggct caccctgtcc 1500 tataatcgcc gtggcgacct ggccatccac ctggtcagcc ccatggggcac ccgctccacc 1560 ctgctggcag ccaggccaca tgactactcc gcagatgggt ttaatgactg ggccttcatg 1620 acaactcatt cctgggatga ggatccctct ggcgagtggg tcctagagat tgaaaacacc 1680 agcgaagcca acaactatgg gacgctgacc aagttcaccc tcgtactcta tggcaccgcc 1740 cctgaggggc tgcccgtacc tccagaaagc agtggctgca agaccctcac gtccagtcag 1800 gcctgtgtgg tgtgcgagga aggcttctcc ctgcaccaga agagctgtgt ccagcactgc 1860

- 116 044349 cctccaggct accatccggg gccctgacag tgctcccggc cccccggagg gaggtggtgg gtcctgcagc ggcctcatct tcagaagagg tgaacgcggc tcgcccccca ccagcgtctg actgcctcag aaagccagag tggaggcggg ccggcctcag tgcgctctgg cctacaaggg acgagggccg cgc agtcctcgat cgccccctgc ctgccccagc cagccgagag gcaacggctg ctgcgccttc ctttagtttt gctgccccct gggcgagagg acgcactata cacgcctcat cacgcctcct tccccgccac cgggcagggc atcgtgctgg cggggggtga ga agcctggc accgccttta gcaccgagaa gtgccacatg tggaccctgt agcagcagcc tgctgccctc tcttcgtcac aggtgtacac aggaggagtg tcaaagacca tgacgtggag ccaggggccg ggagcagact acctcggctg acacctgcct tgtcttcctg catggaccgt cccgtctgac gagcgccctc

1920

1980

2040

2100

2160

2220

2280

2340

2400

2413 <210>23 <211>794 <212> Protein <213> Homo sapiens <400>23

Met Glu 1

Leu Arg

Pro Trp 5

Leu Leu

Trp Val 10

Val Ala

Ala Thr

Leu Val

Leu Leu

Ala Ala

Asp Ala

Gln Gly 25

Gln Lys

Val Phe

Thr Trp

Ala Val 35

Arg Ile

Pro Gly 40

Gly Pro

Ala Val

Ala Asn 45

Ala Arg 50

Lys His

Gly Phe

Leu Asn 55

Leu Gly

Gln Ile

Phe Gly

Gly Thr 15

Thr Asn

Ser Val

Asp Tyr

Tyr His 65

Phe Trp

His Arg 70

Gly Val

Thr Lys

Arg Ser 75

Leu Ser

Pro His

Arg Pro

Arg His

Ser Arg 85

Leu Gln

Arg Glu 90

Pro Gln

Val Gln

TRp Leu 95

Glu Gln

Gln Val

100

Ala Lys

Arg Arg

Thr Lys 105

Arg Asp

Val Tyr

110

Gln Glu

Pro Thr

Asp Pro 115

Lys Phe

Pro Gln

120

Gln Trp

Tyr Leu

Ser Gly 125

Val Thr

Gln Arg

130

Asp Leu

Asn Val

Lys Ala 135

Ala Trp

Ala Gln

140

Gly Tyr

Thr Gly

- 117 044349

His Gly Ile Val Val 145

Ser Ile Leu Asp Asp 150

Gly Ile Glu Lys Asn 155

Pro Asp Leu Ala Gly

165

Asn Tyr Asp Pro Gly

170

Ala Ser Phe Asp Val

175

Asp Gln Asp Pro Asp

180

Pro Gln Pro Arg Tyr

185

Thr Gln Met Asn Asp

190

Arg His Gly Thr Arg 195

Cys Ala Gly Glu Val

200

Ala Ala Val Ala Asn

205

Gly Val Cys Gly Val 210

Gly Val Ala Tyr Asn

215

Ala Arg Ile Gly Gly

220

Arg Met Leu Asp Gly 225

Glu Val Thr Asp Ala 230

Val Glu Ala Arg Ser 235

Gly Leu Asn Pro Asn

245

His Ile His Ile Tyr

250

Ser Ala Ser Trp Gly

255

Glu Asp Asp Gly Lys

260

Thr Val Asp Gly Pro

265

Ala Arg Leu Ala Glu

270

Ala Phe Phe Arg Gly

275

Val Ser Gln Gly Arg

280

Gly Gly Leu Gly Ser

285

Phe Val Trp Ala Ser

290

Gly Asn Gly Gly Arg

295

Glu His Asp Ser Cys 300

Cys Asp Gly Tyr Thr 305

Asn Ser Ile Tyr Thr 310

Leu Ser Ile Ser Ser 315

Thr Gln Phe Gly Asn

325

Val Pro Trp Tyr Ser

330

Glu Ala Cys Ser Ser

335

Leu Ala Thr Thr Tyr

340

Ser Ser Gly Asn Gln

345

Asn Glu Lys Gln Ile

350

Thr Thr Asp Leu Arg

355

Gln Lys Cys Thr Glu 360

Ser His Thr Gly Thr

365

Ala Ser Ala Pro Leu

370

Ala Ala Gly Ile Ile

375

Ala Leu Thr Leu Glu

380

Asn Lys Asn Leu Thr 385

Trp Arg Asp Met Gln 390

His Leu Val Val Gln 395

His 160

Asn

Asn

Asn

Val

Leu 240

Pro

Glu

Ile

Asn

Ala 320

Thr

Val

Ser

Ala

Thr 400

- 118 044349

Ser

Gly

Ala

Lys

Leu 465

Ile

Arg

Ser

Asn

Gly 545

Gly

Gly

Ser

Ser

Thr 625

Cys

Lys Pro Ala

His Leu Asn 405

Ala Asn Asp

410

Trp Ala Thr

Asn Gly Val 415

Arg Lys Val

420

Met Val Ala

435

Cys Ile Ile 450

Glu Val Arg

Thr Arg Leu

Arg Gly Asp

500

Thr Leu Leu

515

Asp Trp Ala 530

Glu Trp Val

Thr Leu Thr

Leu Pro Val

580

Gln Ala Cys

595

Cys Val Gln 610

His Tyr Ser

Ala Pro Cys

Ser His Ser

Tyr Gly Tyr

425

Gly Leu Leu

Asp Ala Gly 430

Leu Ala Gln

Asp Ile Leu

455

Lys Thr Val

470

Glu His Ala 485

Leu Ala Ile

Ala Ala Arg

Phe Met Thr

535

Leu Glu Ile

550

Lys Phe Thr 565

Pro Pro Glu

Val Val Cys

His Cys Pro

615

Thr Glu Asn

630

His Ala Ser

Asn Trp Thr 440

Thr Val Ala

445

Pro Gln Arg

Thr Glu Pro

Lys Asp Ile

460

Gly Lys Arg

Thr Ala Cys

Leu Gly Glu 475

Pro Asn His

480

Gln Ala Arg

490

Leu Thr Leu

Ser Tyr Asn

495

His Leu Val

505

Pro His Asp 520

Thr His Ser

Glu Asn Thr

Leu Val Leu

570

Ser Ser Gly

585

Glu Glu Gly 600

Pro Gly Phe

Asp Val Glu

Cys Ala Thr

Ser Pro Met

Tyr Ser Ala

525

Trp Asp Glu

540

Ser Glu Ala 555

Tyr Gly Thr

Cys Lys Thr

Phe Ser Leu

605

Ala Pro Gln

620

Thr Ile Arg 635

Cys Gln Gly

Gly Thr Arg 510

Asp Gly Phe

Asp Pro Ser

Asn Asn Tyr

560

Ala Pro Glu

575

Leu Thr Ser 590

His Gln Lys

Val Leu Asp

Ala Ser Val

640

Pro Ala Leu

- 119 044349

655

Val Glu

645

650

Thr Asp

Cys Leu

660

Ser Cys

Pro Ser

Gln Thr

Cys Ser 675

Arg Gln

Ser Gln

680

Gln Gln

690

Pro Pro

Arg Leu

Pro Pro 695

Arg Ala 705

Gly Leu

Leu Pro

710

Ser His

Ser Cys

Ala Phe

Ile Val

725

Leu Val

Gln Leu

Arg Ser

740

Gly Phe

Ser Phe

Asp Arg

Gly Leu 755

Ile Ser

Tyr Lys

760

Glu Glu

770

Cys Pro

Ser Asp

Ser Glu

775

Thr Ala 785

Phe Ile

Lys Asp

790

Gln Ser

His Ala 665

Ser Ser

Glu Val

Leu Pro

Phe Val 730

Arg Gly 745

Gly Leu

Glu Asp

Ala Leu

Ser Leu

Arg Glu

Glu Ala

700

Glu Val 715

Thr Val

Val Lys

Pro Pro

Glu Gly

780

Asp Pro

670

Ser Pro 685

Gly Gln

Val Ala

Phe Leu

Val Tyr

750

Glu Ala 765

Arg Gly

Pro Gln

Arg Leu

Gly Leu

720

Val Leu 735

Thr Met

Trp Gln

Glu Arg <210> 24 <211> 1621 <212> DNA <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400> 24 ctcgaggaca tgcctggctg cgcgccaacg gagcagtgct ttctggattt tcctgcaagg aactgtgaga cagtactgca tggtctccca cagtcttcgt cgttcctgga ccttcgagga cttacagtga accagctcca cgcaca agga gtgaccacac ggccctcagg aacccaggag ggagctgcgg ggcccgggag tggggaccag gtcctatatc tgaccagctg gggcaccaag ctcctctgcc gaagcccacg ccgggctccc atcttcaagg tgtgcctcaa tgcttctgcc atctgtgtga cgctcctgtc ttctgcttgg gcg tcctgca tggagaggga acgcggagag gtccatgcca tccctgcctt acgagaacgg ggtgccacga gcttcagggc ccggcgccgg gtgcaaggag gacgaagctg gaatgggggc cgagggccgg cggctgtgag ggggtactct

120

180

240

300

360

420

480

- 120 044349

ctgctggcag acggggtgtc ctgcacacc acagttgaat atccatggtgg aaaaatacct attctgaaa aaagaaatgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca gttgtgtggg gggaccctga tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag aactggagga acctgatcgc ggtgctggggc gagcacgacc tcagcgagca cgacggggat gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc gggcaccacc aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg tcctactga ccatgtggtg cccctctgcc tgcccgaacg gacgttctct gagaggacgc tggccttcgt gcgcttctca ttggtcagcg gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc ctcaacgtgc cccggctgat gaccagggac tgcctgcagc agtcacggaa ggtggggagac tccccaaata tcacggagta catgttctgt gccggctact cggatggcag caaggactcc tgcaaggggg acagtggagg cccacatgcc accactacc ggggcacgtg gtacctgacc ggcatcgtga gctggggcca gggctgcgcc accgtgggcc acttcggcgt gtacaccagg gtgtcccagt acatcgagtg gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg ctgctgagag cccccttccc cagcagcagc tccaaggccc ctccccctag cctgcccagc cctagcagac tgcctgggcc cagtgacacc cctatcctgc ctcagtccag ctccagcaag gccccacccc ctagcctgcc ttctccttct cggctgcctg gccccagcga tactccaatt ctgccccagt cctccagcag taaggctccc cctccacatctc tgccatcccc cagcagactg ccaggccctt ctgatacacc catcctccca cagtgatgag gatccgcggc cgcttaatta

a

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1621 <210> 25 <211> 528 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400> 25

Met Val Ser Gln 1

Ala Leu Arg Leu 5

Leu Cys Leu Leu 10

Leu Gly Leu Gln 15

Gly Cys Leu Ala 20

Ala Val Phe Val

Thr Gln Glu Glu 25

Ala His Gly Val 30

Leu His Arg Arg 35

Arg Arg Ala Asn

Ala Phe Leu Glu

Glu Leu Arg Pro 45

- 121 044349

Gly

Ala 65

Ser

Gly

Ala

Cys

Gly 145

Asp

Pro

Gly

Leu

Trp 225

Asn

Asp

Val

Pro

Ser Leu Glu 50

Arg Glu Ile

Tyr Ser Asp

Ser Cys Lys

100

Phe Glu Gly 115

Val Asn Glu 130

Thr Lys Arg

Gly Val Ser

Ile Leu Glu

180

Gly Lys Val

195

Val Asn Gly 210

Val Val Ser

Leu Ile Ala

Glu Gln Ser

260

Pro Gly Thr

275

Val Val Leu 290

Arg Glu Cys 55

Phe Lys Asp 70

Gly Asp Gln 85

Asp Gln Leu

Arg Asn Cys

Asn Gly Gly

135

Ser Cys Arg

150

Cys Thr Pro 165

Lys Arg Asn

Cys Pro Lys

Ala Gln Leu

215

Ala Ala His

230

Val Leu Gly 245

Arg Arg Val

Thr Asn His

Thr Asp His

295

Lys Glu Glu

Ala Glu Arg

Cys Ala Ser

Gln Ser Tyr 105

Glu Thr His 120

Cys Glu Gln

Cys His Glu

Thr Val Glu

170

Ala Ser Lys

185

Gly Glu Cys 200

Cys Gly Gly

Cys Phe Asp

Glu His Asp

250

Ala Gln Val

265

Asp Ile Ala 280

Val Val Pro

Gln Cys Ser 60

Thr Lys Leu 75

Ser Pro Cys

Ile Cys Phe

Lys Asp Asp

125

Tyr Cys Ser

140

Gly Tyr Ser 155

Tyr Pro Cys

Pro Gln Gly

Pro Trp Gln

205

Thr Leu Ile

220

Lys Ile Lys 235

Leu Ser Glu

Ile Ile Pro

Leu Leu Arg

285

Leu Cys Leu 300

Phe Glu Glu

Phe Trp Ile 80

Gln Asn Gly 95

Cys Leu Pro 110

Gln Leu Ile

Asp His Thr

Leu Leu Ala

160

Gly Lys Ile

175

Arg Ile Val 190

Val Leu Leu

Asn Thr Ile

Asn Trp Arg

240

His Asp Gly

255

Ser Thr Tyr 270

Leu His Gln

Pro Glu Arg

- 122 044349

Thr Phe Ser Glu Arg 305

Thr Leu Ala Phe Val 310

Arg Phe Ser Leu Val 315

Gly Trp Gly Gln Leu

325

Leu Asp Arg Gly Ala

330

Thr Ala Leu Glu Leu

335

Val Leu Asn Val Pro

340

Arg Leu Met Thr Gln

345

Asp Cys Leu Gln Gln

350

Arg Lys Val Gly Asp

355

Ser Pro Asn Ile Thr

360

Glu Tyr Met Phe Cys

365

Gly Tyr Ser Asp Gly

370

Ser Lys Asp Ser Cys

375

Lys Gly Asp Ser Gly

380

Pro His Ala Thr His 385

Tyr Arg Gly Thr Trp 390

Tyr Leu Thr Gly Ile 395

Ser Trp Gly Gln Gly

405

Cys Ala Thr Val Gly

410

His Phe Gly Val Tyr

415

Arg Val Ser Gln Tyr

420

Ile Glu Trp Leu Gln

425

Lys Leu Met Arg Ser

430

Pro Arg Pro Gly Val

435

Leu Leu Arg Ala Pro

440

Phe Pro Ser Ser Ser

445

Lys Ala Pro Pro Pro

450

Ser Leu Pro Ser Pro

455

Ser Arg Leu Pro Gly

460

Ser Asp Thr Pro Ile 465

Leu Pro Gln Ser Ser 470

Ser Ser Lys Ala Pro 475

Pro Ser Leu Pro Ser

485

Pro Ser Arg Leu Pro

490

Gly Pro Ser Asp Thr

495

Ile Leu Pro Gln Ser

500

Ser Ser Ser Lys Ala

505

Pro Pro Pro Ser Leu

510

Ser Pro Ser Arg Leu

515

Pro Gly Pro Ser Asp

520

Thr Pro Ile Leu Pro

525

Ser 320

Met

Ser

Ala

Gly

Val 400

Thr

Glu

Ser

Pro

Pro 480

Pro

Pro

Gln <210> 26 <211> 1607 <212> DNA <213> Artificial sequence

- 123 044349 <220>

<223> CTP-modified Factor VII <400>26 ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc60 tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg120 cgcgccaacg cgttcct gga ggagctgcgg ccgggctccc tggagaggga gtgcaaggag180 gagcagtgct ccttcgagga ggcccgggag atcttcaagg acgcggagag gacgaagctg240 ttctggattt cttacagtga tggggaccag tgtgcctcaa gtccatgcca gaatgggggc300 tcctgcaagg a ccagctcca gtcctatatc tgcttctgcc tccctgcctt cgagggccgg360 aactgtgaga cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag420 cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct480 ctgctggcag acggggtgtc ctgcacaccc acagttgaat atccatgtgg aaaaatacct540 attctagaaa aaagaa atgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc600 cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca gttgtgtggg660 gggaccctga tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag720 aactgg agga acctgatcgc ggtgctgggc gagcacgacc tcagcgagca cgacggggat780 gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc gggcaccacc840 aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg tcctcactga ccatgtggtg900 cccctctgcc tgcccgaacg gacgttctct gagaggacgc tggccttcgt gcgcttctca960 ttggtcagcg gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc1020 ctcaacgtgc cccggctgat gacccaggac t gcctgcagc agtcacggaa ggtgggagac1080 tccccaaata tcacggagta catgttctgt gccggctact cggatggcag caaggactcc1140 tgcaaggggg acagtggagg cccacatgcc acccactacc ggggcacgtg gtacctgacc1200 ggcatcgtga gctggggcca gggctgcgcc accgtgggcc acttcggcgt gtacaccagg1260 gtgtcccagt acatcgagtg gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg1320 ctgctgagag cccccttccc cagcagcagc tccaaggccc ctccccctag cctgcccagc1380 cctagcagac tgcctgggcc cagtgacacc cctatcctgc ctcagtccag ctccagcaag1440 gccccacccc ctagcctgcc ttctccttct cggctgcctg gccccagcga tactccaatt1500 ctgccccagt cctccagcag taaggctccc cctccatctc tgccatcccc cagcagactg15 60 ccaggccctt ctgatacacc catcctccca cagtgatgag gatccgc1607 <210>27 <211>532 <212> Protein <213> Artificial sequence

- 124 044349 <220>

<223> CTP-modified Factor VII <400> 27

Leu Glu Asp Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu 1 5 10 15

Gly Leu Gln Gly Cys Leu Ala Ala Val Phe Val Thr Gln Glu Glu

25 30

His Gly Val Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu 35 40 45

Leu Arg Pro Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys 50 55 60

Phe Glu Glu Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys 65 70 75

Phe Trp Ile Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro 85 90 95

Gln Asn Gly Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys

100 105 110

Cys Leu Pro Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp

115 120 125

Gln Leu Ile Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr Cys

130 135 140

Asp His Thr Gly Thr Lys Arg Ser Cys Arg Cys His Glu Gly Tyr

145 150 155

Leu Leu Ala Asp Gly Val Ser Cys Thr Pro Thr Val Glu Tyr Pro

165 170 175

Gly Lys Ile Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pro Gln

180 185 190

Arg Ile Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp

195 200 205

Val Leu Leu Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu

210 215 220

Asn Thr Ile Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Ile

Leu

Ala

Glu

Ser

Leu 80

Cys

Phe

Asp

Ser

Ser 160

Cys

Gly

Gln

Ile

Lys

- 125 044349

230

235

240

225

Asn

His

Ser

Leu

Pro 305

Leu

Glu

Gln

Phe

Ser 385

Gly

Val

Arg

Ser

Pro 465

Trp Arg Asn

Leu Ile Ala 245

Val Leu Gly

250

Glu His Asp

Leu Ser Glu

255

Asp Gly Asp

260

Thr Tyr Val

275

His Gln Pro 290

Glu Arg Thr

Val Ser Gly

Leu Met Val

340

Gln Ser Arg

355

Cys Ala Gly 370

Gly Gly Pro

Ile Val Ser

Tyr Thr Arg

420

Ser Glu Pro

435

Ser Ser Lys 450

Gly Pro Ser

Glu Gln Ser

Pro Gly Thr

Val Val Leu

295

Phe Ser Glu

310

Trp Gly Gln 325

Leu Asn Val

Lys Val Gly

Tyr Ser Asp

375

His Ala Thr

390

Trp Gly Gln 405

Val Ser Gln

Arg Pro Gly

Ala Pro Pro

455

Asp Thr Pro

470

Arg Arg Val

265

Ala Gln Val

Ile Ile Pro 270

Thr Asn His 280

Thr Asp His

Arg Thr Leu

Leu Leu Asp

330

Pro Arg Leu

345

Asp Ser Pro 360

Gly Ser Lys

His Tyr Arg

Gly Cys Ala

410

Tyr Ile Glu

425

Val Leu Leu 440

Pro Ser Leu

Ile Leu Pro

Asp Ile Ala

285

Val Val Pro

300

Ala Phe Val 315

Arg Gly Ala

Met Thr Gln

Asn Ile Thr

365

Asp Ser Cys 380

Gly Thr Trp 395

Thr Val Gly

Trp Leu Gln

Arg Ala Pro

445

Pro Ser Pro

460

Gln Ser Ser 475

Leu Leu Arg

Leu Cys Leu

Arg Phe Ser

320

Thr Ala Leu

335

Asp Cys Leu 350

Glu Tyr Met

Lys Gly Asp

Tyr Leu Thr

400

His Phe Gly 415

Lys Leu Met 430

Phe Pro Ser

Ser Arg Leu

Ser Ser Lys

480

- 126 044349

Ala Pro Pro Pro Ser Leu Pro Ser Pro

485

Asp Thr Pro Ile Leu Pro Gln Ser Ser

500505

Ser Leu Pro Ser Pro Ser Arg Leu Pro

515520

Ser Arg Leu Pro Gly Pro Ser

490 495

Ser Ser Lys Ala Pro Pro Pro 510

Gly Pro Ser Asp Thr Pro Ile 525

Leu Pro Gln Gly

530 <210>28 <211>1775 <212> DNA <213> Artificial sequence <220>

<223> CTP-modified Factor VII

<400> 28 ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc 60 tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg 120 cgcgccaacg cgttcctgga ggagctgcgg ccggggctccc tggagaggga gtgcaagggag 180 gagcagtgct ccttcgagga ggcccggggag atcttcaagg acgcggagag gacgaagctg 240 ttctggattt cttacagtga tggggaccag tgtgcctcaa gtccatgcca gaatggggggc 300 tcctgcaagg accagctcca gtcctatatc tgcttctgcc tccctgcctt cgaggggccgg 360 aactgtgaga cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag 420 cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct 480 ctgctggcag acggggtgtc ctgcacacc acagttgaat atccatggtgg aaaaatacct 540 attctgaaa aaagaaatgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc 600 cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca gttgtgtggg 660 gggaccctga tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag 720 aactggagga acctgatcgc ggtgctggggc gagcacgacc tcagcgagca cgacggggat 780 gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc gggcaccacc 840 aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg tcctactga ccatgtggtg 900 cccctctgcc tgcccgaacg gacgttctct gagaggacgc tggccttcgt gcgcttctca 960 ttggtcagcg gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc 1020 ctcaacgtgc cccggctgat gaccagggac tgcctgcagc agtcacggaa ggtggggagac 1080 tccccaaata tcacggagta catgttctgt gccggctact cggatggcag caaggactcc 1140

- 127 044349

tgcaaggggg acagtggagg cccacatgcc accactacc ggggcacgtg gtacctgacc 1200 ggcatcgtga gctggggcca gggctgcgcc accgtgggcc acttcggcgt gtacaccagg 1260 gtgtcccagt acatcgagtg gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg 1320 ctgctgagag cccccttccc cagcagcagc tccaaggccc ctccccctag cctgcccagc 1380 cctagcagac tgcctgggcc ctctgacacc cctatcctgc ctcagtccag ctcctctaag 1440 gctccaccac cttccctgcc tagcccttca agactgccag gcctagcga tacaccatt 1500 ctgccccagt cctccagcag caaggctccc ccacctagcc tgccttctcc atcaaggctg 1560 cctggcccat ccgatacccc aattttgcct cagagcagct ctagcaaggc acctcccccc 1620 agtctgccct ctccaagcag actccctggc ccttcagaca ctccaatcct cccacagtcc 1680 tctagctcta aagctccacc tccagcctg cccagcccta gtagactccc cggaccttct 1740 gatacccca tcttgcccca gtgatgagga tccgc 1775

<210> 29 <211> 589 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor VII <400>29

Leu Glu Asp Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu

5 1015

Gly Leu Gln Gly Cys Leu Ala Ala Val Phe Val Thr Gln Glu Glu Ala 20 2530

His Gly Val Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu Glu 35 4045

Leu Arg Pro Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser 50 5560

Phe Glu Glu Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys Leu 65 70 7580

Phe Trp Ile Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro Cys 85 9095

Gln Asn Gly Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys Phe

100 105110

Cys Leu Pro Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp Asp

115 120125

- 128 044349

Gln

Asp 145

Leu

Gly

Arg

Val

Asn 225

Asn

His

Ser

Leu

Pro 305

Leu

Glu

Gln

Phe

Leu Ile Cys 130

His Thr Gly

Leu Ala Asp

Lys Ile Pro

180

Ile Val Gly

195

Leu Leu Leu 210

Thr Ile Trp

Trp Arg Asn

Asp Gly Asp

260

Thr Tyr Val

275

His Gln Pro 290

Glu Arg Thr

Val Ser Gly

Leu Met Val

340

Gln Ser Arg

355

Cys Ala Gly

Val Asn Glu

135

Asn Gly Gly

Cys Glu Gln

140

Tyr Cys Ser

Thr Lys Arg

150

Gly Val Ser 165

Ile Leu Glu

Gly Lys Val

Val Asn Gly

215

Val Val Ser

230

Leu Ile Ala 245

Glu Gln Ser

Pro Gly Thr

Val Val Leu

295

Phe Ser Glu

310

Trp Gly Gln 325

Leu Asn Val

Lys Val Gly

Tyr Ser Asp

Ser Cys Arg

Cys Thr Pro

170

Lys Arg Asn

185

Cys Pro Lys 200

Ala Gln Leu

Ala Ala His

Val Leu Gly

250

Arg Arg Val

265

Thr Asn His 280

Thr Asp His

Arg Thr Leu

Leu Leu Asp

330

Pro Arg Leu

345

Asp Ser Pro 360

Gly Ser Lys

Cys His Glu 155

Gly Tyr Ser

160

Thr Val Glu

Tyr Pro Cys

175

Ala Ser Lys

Pro Gln Gly 190

Gly Glu Cys

205

Cys Gly Gly

220

Cys Phe Asp 235

Glu His Asp

Ala Gln Val

Asp Ile Ala

285

Val Val Pro

300

Ala Phe Val 315

Arg Gly Ala

Met Thr Gln

Asn Ile Thr

365

Asp Ser Cys

Pro Trp Gln

Thr Leu Ile

Lys Ile Lys

240

Leu Ser Glu

255

Ile Ile Pro 270

Leu Leu Arg

Leu Cys Leu

Arg Phe Ser

320

Thr Ala Leu

335

Asp Cys Leu 350

Glu Tyr Met

Lys Gly Asp

- 129 044349

370

375

380

Ser Gly Gly Pro His 385

Ala Thr His Tyr Arg 390

Gly Thr Trp Tyr Leu 395

Gly Ile Val Ser Trp

405

Gly Gln Gly Cys Ala

410

Thr Val Gly His Phe

415

Val Tyr Thr Arg Val

420

Ser Gln Tyr Ile Glu

425

Trp Leu Gln Lys Leu

430

Arg Ser Glu Pro Arg 435

Pro Gly Val Leu Leu

440

Arg Ala Pro Phe Pro 445

Ser Ser Ser Lys Ala

450

Pro Pro Pro Ser Leu

455

Pro Ser Pro Ser Arg

460

Pro Gly Pro Ser Asp 465

Thr Pro Ile Leu Pro 470

Gln Ser Ser Ser Ser 475

Ala Pro Pro Pro Ser

485

Leu Pro Ser Pro Ser

490

Arg Leu Pro Gly Pro

495

Asp Thr Pro Ile Leu

500

Pro Gln Ser Ser Ser

505

Ser Lys Ala Pro Pro

510

Ser Leu Pro Ser Pro

515

Ser Arg Leu Pro Gly 520

Pro Ser Asp Thr Pro

525

Leu Pro Gln Ser

530

Ser Ser Lys Ala Pro

535

Pro Pro Ser Leu Pro

540

Pro Ser Arg Leu Pro 545

Gly Pro Ser Asp Thr 550

Pro Ile Leu Pro Gln 555

Ser Ser Ser Lys Ala

565

Pro Pro Pro Ser Leu

570

Pro Ser Pro Ser Arg

575

Thr

400

Gly

Met

Ser

Leu

Lys 480

Ser

Pro

Ile

Ser

Ser 560

Leu

Pro Gly Pro Ser Asp

580

Thr Pro Ile Leu Pro

585

Gln Gly Ser <210> 30 <211> 1673 <212> DNA <213> Artificial sequence <220>

<223> CTP-modified Factor IX

- 130 044349 <400> 30 tctagagtcg accccgccat gcagcgcgtg aacatgatca tggcagaatc accaggcctc 60 atcaccatct gccttttagg atatctactc agtgctgaat gtacagtttt tcttgatcat120 gaaaacgcca acaaaattct gaatcgg cca aagaggtata attcaggtaa attggaagag180 tttgttcaag ggaaccttga gagagaatgt atggaagaaa agtgtagttt tgaagaagca240 cgagaagttt ttgaaaacac tgaaagaaca actgaatttt ggaagcagta tgttgatgga300 gatcagtgtg agtccaatcc atgtt taaat ggcggcagtt gcaaggatga cattaattcc360 tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact gtgaattaga tgtaacatgt420 aacattaaga atggcagatg cgagcagttt tgtaaaaata gtgctgataa caaggtggtt480 tgctcctgta ctgagggata tcgacttgca gaaaaccaga agtcctgtga accagcagtg540 ccatttccat gtggaagagt ttctgtttca caaacttcta agctcacccg t gctgaggca600 gtttttcctg atgtggacta tgtaaattct actgaagctg aaaccatttt ggataacatc660 actcaaagca cccaatcatt taatgacttc actcgagttg ttggtggaga agatgccaaa720 ccaggtcaat tcccttggca ggttgttttg aatggtaaag ttgatgcat t ctgtggaggc780 tctatcgtta atgaaaaatg gattgtaact gctgcccact gtgttgaaac tggtgttaaa840 attacagttg tcgcaggtga acataatatt gaggagacag aacatacaga gcaaaagcga900 aatgtgattc gaattattcc tcaccacaac tacaatgcag ctattaataa gtacaaccat960 gacattgccc ttctggaact ggacgaaccc ttagtgctaa acagctacgt tacacctatt1020 tgcattgctg acaaggaata cacgaacatc ttcctcaaat ttggatctgg ctatgtaagt1080 ggctggggaa gagtcttcca ca aagggaga tcagctttag ttcttcagta ccttagagtt1140 ccacttgttg accgagccac atgtcttcga tctacaaagt tcaccatcta taacaacatg1200 ttctgtgctg gcttccatga aggaggtaga gattcatgtc aaggagatag tgggggaccc1260 catgttactg aagtgg aagg gaccagtttc ttaactggaa ttattagctg gggtgaagag1320 tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt caactggatt1380 aaggaaaaaa caaagctcac tagctccagc agcaaggccc ctcccccgag cctgccctcc1440 ccaagcaggc tgcctgggcc cagtgacacc cctatcctgc ctcagtccag ctccagcaag1500 gccccacccc ctagcctgcc ttctccttct cggctgcctg gccccagcga tactccaatt1560 ctgccccagt cctccagcag taaggctccc cctcca tctc tgccatcccc cagcagactg1620 ccaggccctt ctgatacacc catcctccca cagtgatgag gatccgcggc cgc1673 <210>31 <211>545 <212> Protein <213> Artificial sequence

- 131 044349 <220>

<223> CTP-modified Factor IX <400> 31

Met Gln Arg Val Asn 1 5

Met Ile Met Ala Glu

Ser Pro Gly Leu Ile

Ile Cys Leu Leu Gly 20

Tyr Leu Leu Ser Ala

Glu Cys Thr Val Phe

Asp His Glu Asn Ala 35

Asn Lys Ile Leu Asn 40

Arg Pro Lys Arg Tyr 45

Ser Gly Lys Leu Glu 50

Glu Phe Val Gln Gly 55

Asn Leu Glu Arg Glu 60

Met Glu Glu Lys Cys 65

Ser Phe Glu Glu Ala 70

Arg Glu Val Phe Glu 75

Thr Glu Arg Thr Thr

Glu Phe Trp Lys Gln 90

Tyr Val Asp Gly Asp 95

Cys Glu Ser Asn Pro

100

Cys Leu Asn Gly Gly

105

Ser Cys Lys Asp Asp

110

Asn Ser Tyr Glu Cys 115

Trp Cys Pro Phe Gly 120

Phe Glu Gly Lys Asn 125

Glu Leu Asp Val Thr 130

Cys Asn Ile Lys Asn

135

Gly Arg Cys Glu Gln 140

Cys Lys Asn Ser Ala 145

Asp Asn Lys Val Val 150

Cys Ser Cys Thr Glu 155

Tyr Arg Leu Ala Glu

165

Asn Gln Lys Ser Cys

170

Glu Pro Ala Val Pro

175

Pro Cys Gly Arg Val

180

Ser Val Ser Gln Thr

185

Ser Lys Leu Thr Arg

190

Glu Ala Val Phe Pro

195

Asp Val Asp Tyr Val 200

Asn Ser Thr Glu Ala

205

Thr Ile Leu Asp Asn

210

Ile Thr Gln Ser Thr

215

Gln Ser Phe Asn Asp

220

Thr Arg Val Val Gly 225

Gly Glu Asp Ala Lys 230

Pro Gly Gln Phe Pro 235

Thr

Leu

Asn

Cys

Asn 80

Gln

Ile

Cys

Phe

Gly 160

Phe

Ala

Glu

Phe

Trp 240

- 132 044349

Gln

Val

Val

His

Tyr 305

Leu

Ala

Val

Leu

Ser 385

Glu

Thr

Glu

Arg

Ser 465

Pro

Val Val Leu

Asn Glu Lys

260

Lys Ile Thr

275

Thr Glu Gln 290

Asn Ala Ala

Asp Glu Pro

Asp Lys Glu

340

Ser Gly Trp

355

Gln Tyr Leu 370

Thr Lys Phe

Gly Gly Arg

Glu Val Glu

420

Glu Cys Ala

435

Tyr Val Asn 450

Lys Ala Pro

Ser Asp Thr

Asn Gly Lys 245

Trp Ile Val

Val Val Ala

Lys Arg Asn

295

Ile Asn Lys

310

Leu Val Leu 325

Tyr Thr Asn

Gly Arg Val

Arg Val Pro

375

Thr Ile Tyr

390

Asp Ser Cys 405

Gly Thr Ser

Met Lys Gly

Trp Ile Lys

455

Pro Pro Ser

470

Pro Ile Leu

Val Asp Ala

250

Phe Cys Gly

Gly Ser Ile

255

Thr Ala Ala

265

Gly Glu His 280

Val Ile Arg

Tyr Asn His

Asn Ser Tyr

330

Ile Phe Leu

345

Phe His Lys 360

Leu Val Asp

Asn Asn Met

Gln Gly Asp

410

Phe Leu Thr

425

Lys Tyr Gly 440

Glu Lys Thr

Leu Pro Ser

Pro Gln Ser

His Cys Val

Glu Thr Gly 270

Asn Ile Glu

285

Glu Thr Glu

Ile Ile Pro

300

His His Asn

Asp Ile Ala 315

Val Thr Pro

Lys Phe Gly

Gly Arg Ser

365

Arg Ala Thr

380

Phe Cys Ala 395

Ser Gly Gly

Gly Ile Ile

Ile Tyr Thr

445

Lys Leu Thr

460

Pro Ser Arg 475

Ser Ser Ser

Leu Leu Glu

320

Ile Cys Ile

335

Ser Gly Tyr 350

Ala Leu Val

Cys Leu Arg

Gly Phe His

400

Pro His Val

415

Ser Trp Gly 430

Lys Val Ser

Ser Ser Ser

Leu Pro Gly

480

Lys Ala Pro

- 133 044349

Asp Thr

485 490 495

Pro Pro

Ser Leu

500

Pro Ser

Pro Ser

Pro Ile

Leu Pro 515

Gln Ser

Ser Ser

520

Pro

Ser Pro Ser 530

Arg Leu

Pro Gly 535

Arg Leu 505

Ser Lys

Pro Ser

Pro Gly

Ala Pro

Asp Thr

540

Pro Ser

510

Pro Pro 525

Pro Ile

Ser Leu

Leu Pro

Gln

545 <210> 32 <211> 1757 <212> DNA <213> Artificial sequence <220>

<223> CTP-modified Factor IX

<400> 32 tctagagtcg accccgccat gcagcgcgtg aacatgatca tggcagaatc accaggcctc 60 atcaccact gcctttagg atatctactc agtgctgaat gtacagtttt tcttgatcat 120 gaaaacgcca acaaaattct gaatcggcca aagaggtata attcaggtaa attggaagag 180 tttgttcaag ggaaccttga gagagaatgt atggaagaaa agtgtagttt tgaagaagca 240 cgagaagttt ttgaaaacac tgaaagaaca actgaatttt ggaagcagta tgttgatgga 300 gatcagtgtg agtccaatcc atgtttaaat ggcggcagtt gcaaggatga cattaattcc 360 tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact gtgaattaga tgtaacatgt 420 aacattaaga atggcagatg cgagcagttt tgtaaaaata gtgctgataa caaggtggtt 480 tgctcctgta ctgaggata tcgacttgca gaaaaccaga agtcctgtga accagcagtg 540 ccattccat gtggaagagt ttctgtttca caaacttcta agctcacccg tgctgaggca 600 gtttttcctg atgtggacta tgtaaattct actgaagctg aaaccatttt ggataacatc 660 actcaaagca cccaatcatt taatgacttc actcgagttg ttggtggaga agatgccaaa 720 ccaggtcaat tcccttggca ggttgttttg aatggtaaag ttgatgcatt ctgtggaggc 780 tctatcgtta atgaaaaatg gattgtaact gctgccact gtgttgaaac tggtgttaaa 840 attacagttg tcgcaggtga acataatatt gaggacag aacatacaga gcaaaagcga 900 aatgtgattc gaattattcc tcaccacaac tacaatgcag ctattaataa gtacaaccat 960 gacattgccc ttctggaact ggacgaaccc ttagtgctaa acagctacgt tacacctatt 1020 tgcattgctg acaaggaata cacgaacatc ttcctcaaat ttggatctgg ctatgtaagt 1080

- 134 044349

ggctggggaa gagtcttcca caaagggaga tcagctttag ttcttcagta ccttagagtt 1140 ccacttgttg accgagccac atgtcttcga tctacaaagt tcaccatta taacaacatg 1200 ttctgtgctg gcttccatga aggaggtaga gattcatgtc aaggagatag tggggggaccc 1260 catgttactg aagtggaagg gaccagtttc ttaactggaa ttattagctg gggtgaagag 1320 tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt caactggatt 1380 aaggaaaaaa caaagctcac tagctccagc agcaaggccc ctccccccgag cctgccctcc 1440 ccaagcaggc tgcctgggcc ctctgacacc cctatcctgc ctcagtccag ctcctctaag 1500 gccccaccac cttccctgcc tagcccttca agactgccag gcctagcga tacaccatt 1560 ctgccccagt cctccagcag caaggctccc ccacctagcc tgccttctcc atcaaggctg 1620 cctggcccat ccgatacccc aattttgcct cagagcagct ctagcaaggc acctcccccc 1680 agtctgccct ctccaagcag actccctggc ccttcagaca ctcccattct gccacagtga 1740 tgaggatccg cggccgc 1757

<210> 33 <211> 583 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor IX <400>33

Ser Arg Val Asp Pro Ala Met Gln Arg Val Asn Met Ile Met Ala Glu

5 1015

Ser Pro Gly Leu Ile Thr Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala 20 2530

Glu Cys Thr Val Phe Leu Asp His Glu Asn Ala Asn Lys Ile Leu Asn 35 4045

Arg Pro Lys Arg Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly 50 5560

Asn Leu Glu Arg Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala 65 70 7580

Arg Glu Val Phe Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln 85 9095

Tyr Val Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly

100 105110

- 135 044349

Ser

Phe

Gly 145

Cys

Glu

Ser

Asn

Gln 225

Pro

Phe

His

Asn

Ile 305

Asp

Val

Lys

Cys Lys

115

Glu Gly 130

Arg Cys

Ser Cys

Pro Ala

Lys Leu

195

Ser Thr

210

Ser Phe

Gly Gln

Cys Gly

Cys Val

275

Ile Glu

290

Ile Pro

Ile Ala

Thr Pro

Phe Gly

355

Asp Asp

Lys Asn

Glu Gln

Thr Glu

165

Val Pro 180

Thr Arg

Glu Ala

Asn Asp

Phe Pro

245

Gly Ser 260

Glu Thr

Glu Thr

His His

Leu Leu

325

Ile Cys 340

Ser Gly

Ile Asn

Cys Glu

135

Phe Cys 150

Gly Tyr

Phe Pro

Ala Glu

Glu Thr

215

Ph Thr 230

Trp Gln

Ile Val

Gly Val

Glu His

295

Asn Tyr 310

Glu Leu

Ile Ala

Tyr Val

Ser Tyr 120

Leu Asp

Lys Asn

Arg Leu

Cys Gly 185

Ala Val 200

Ile Leu

Arg Val

Val Val

Asn Glu

265

Lys Ile 280

Thr Glu

Asn Ala

Asp Glu

Asp Lys

345

Ser Gly 360

Glu Cys

Val Thr

Ser Ala

155

Ala Glu 170

Arg Val

Phe Pro

Asp Asn

Val Gly

235

Leu Asn

250

LysTrp

Thr Val

Gln Lys

Ala Ile

315

Pro Leu 330

Glu Tyr

Trp Gly

Trp Cys

125

Cys Asn 140

Asp Asn

Asn Gln

Ser Val

Asp Val

205

Ile Thr

220

Gly Glu

Gly Lys

Ile Val

Val Ala

285

Arg Asn 300

Asn Lys

Val Leu

Thr Asn

Arg Val

365

Pro Phe

Ile Lys

Lys Val

Lys Ser

175

Ser Gln 190

Asp Tyr

Gln Ser

Asp Ala

Val Asp

255

Thr Ala

270

Gly Glu

Val Ile

Tyr Asn

Asn Ser

335

Ile Phe

350

Phe His

Gly

Asn

Val 160

Cys

Thr

Val

Thr

Lys 240

Ala

Ala

His

Arg

His 320

Tyr

Leu

Lys

- 136 044349

Gly Arg Ser

370

Ala Leu Val

Arg Ala Thr 385

Cys Leu Arg

390

Phe Cys Ala

Gly Phe His

405

Ser Gly Gly

Pro His Val 420

Gly Ile Ile

435

Ser Trp Gly

Ile Tyr Thr

450

Lys Val Ser

Lys Leu Thr 465

Ser Ser Ser

470

Pro Ser Arg

Leu Pro Gly

485

Ser Ser Ser

Lys Ala Pro 500

Pro Gly Pro

515

Ser Asp Thr

Ala Pro Pro

530

Pro Ser Leu

Asp Thr Pro 545

Ile Leu Pro

550

Ser Leu Pro

Ser Pro Ser

565

Leu Pro Gln

Gly Ser Ala 580

Leu Gln Tyr 375

Ser Thr Lys

Glu Gly Gly

Thr Glu Val

425

Glu Glu Cys 440

Arg Tyr Val 455

Ser Lys Ala

Pro Ser Asp

Pro Pro Ser

505

Pro Ile Leu 520

Pro Ser Pro 535

Gln Ser Ser

Arg Leu Pro

Ala

Leu Arg Val

380

Phe Thr Ile

395

Arg Asp Ser 410

Glu Gly Thr

Ala Met Lys

Asn Trp Ile

460

Pro Pro Pro

475

Thr Pro Ile 490

Leu Pro Ser

Pro Gln Ser

Ser Arg Leu

540

Ser Ser Lys

555

Gly Pro Ser 570 <210> 34 <211> 1840 <212> DNA <213> Artificial sequence

Pro Leu Val

Tyr Asn Asn

Cys Gln Gly

415

Ser Phe Leu

430

Gly Lys Tyr 445

Lys Glu Lys

Ser Leu Pro

Leu Pro Gln

495

Pro Ser Arg

510

Ser Ser Ser 525

Pro Gly Pro

Ala Pro Pro

Asp Thr Pro

575

Asp

Met 400

Asp

Thr

Gly

Thr

Ser 480

Ser

Leu

Lys

Ser

Pro 560

Ile

- 137 044349 <220>

<223> CTP-modified Factor IX <400>34 ctagagtcga ccccgccatg cagcgcgtga acatgatcat ggcagaatca ccaggcctca60 tcaccatctg ccttttagga tatctactca gtgctgaatg tacagttttt cttgatcatg120 aaaacgccaa caaaattctg aatcgg ccaa agaggtataa ttcaggtaaa ttggaagagt180 ttgttcaagg gaaccttgag agagaatgta tggaagaaaa gtgtagtttt gaagaagcac240 gagaagtttt tgaaaacact gaaagaacaa ctgaattttg gaagcagtat gttgatggag300 atcagtgtga gt ccaatcca tgtttaaatg gcggcagttg caaggatgac attaattcct360 atgaatgttg gtgtcccttt ggatttgaag gaaagaactg tgaattagat gtaacatgta420 acattaagaa tggcagatgc gagcagtttt gtaaaaatag tgctgataac aaggtggttt480 gctcctgtac tgagggatat cgacttgcag aaaaccagaa gtcctgtgaa ccagcagtgc540 catttccatg t ggaagagtt tctgtttcac aaacttctaa gctcacccgt gctgaggcag600 tttttcctga tgtggactat gtaaattcta ctgaagctga aaccattttg gataacatca660 ctcaaagcac ccaatcattt aatgacttca ctcgagttgt tggtggagaa gatgccaaac720 caggt caatt cccttggcag gttgttttga atggtaaagt tgatgcattc tgtggaggct780 ctatcgttaa tgaaaaatgg attgtaactg ctgcccactg tgttgaaact ggtgttaaaa840 ttacagttgt cgcaggtgaa cataatattg aggagacaga acatacagag caaaagcgaa900 atgtgattcg aattattcct caccacaact acaatgcagc tattaataag tacaaccatg960 acattgccct tctggaactg gacgaaccct tagtgctaaa cagctacgtt acacctattt1020 gcattgctga caaggaatac acgaacatct tcctcaaatt tggatctggc tatg taagtg1080 gctggggaag agtcttccac aaagggagat cagctttagt tcttcagtac cttagagttc1140 cacttgttga ccgagccaca tgtcttcgat ctacaaagtt caccatctat aacaacatgt1200 tctgtgctgg cttccatgaa ggaggtagag attcatgtca aggaga tagt gggggacccc1260 atgttactga agtggaaggg accagtttct taactggaat tattagctgg ggtgaagagt1320 gtgcaatgaa aggcaaatat ggaatatata ccaaggtatc ccggtatgtc aactggatta1380 aggaaaaaac aaagctcact agctccagca gcaaggcccc tcccccgagc ctgccctccc1440 caagcaggct gcctgggccc tctgacaccc ctatcctgcc tcagtccagc tcctctaagg1500 ctccaccacc ttccctgcct agcccttcaa gactgccagg ccctagcgat acaccaattc 1560 tgccccagtc ctccagcagc aaggctcccc cacctagcct gccttctcca tcaaggctgc1620 ctggcccatc cgatacccca attttgcctc agagcagctc tagcaaggca cctcccccca1680 gtctgccctc tccaagcaga ctccctggcc cttcagacac tccaatcctc ccacagtcc t1740 ctagctctaa agctccacct cccagcctgc ccagccctag tagactcccc ggaccttctg1800

- 138 044349 atacccccat cttgccccag tgatgaggat ccgcggccgc

1840 <210> 35 <211> 610 <212> Protein <213> Artificial sequence <220>

<223> CTP-modified Factor IX <400> 35

Arg Val 1

Asp Pro

Ala Met 5

Gln Arg

Val Asn

Met Ile

Met Ala

Pro Gly

Leu Ile

Thr Ile

Cys Leu

Leu Gly 25

Tyr Leu

Leu Ser

Cys Thr

Val Phe 35

Leu Asp

His Glu

Asn Ala

Asn Lys

Ile Leu 45

Pro Lys 50

Arg Tyr

Asn Ser

Gly Lys 55

Leu Glu

Glu Phe

Val Gln

Glu Ser 15

Ala Glu

Asn Arg

Gly Asn

Leu Glu 65

Arg Glu

Cys Met 70

Glu Glu

Lys Cys

Ser Phe 75

Glu Glu

Ala Arg 80

Glu Val

PheGlu

Asn Thr 85

Glu Arg

Thr Thr

Glu Phe

Trp Lys

Gln Tyr 95

Val Asp

Gly Asp

100

Gln Cys

Glu Ser

Asn Pro 105

Cys Leu

Asn Gly

110

Gly Ser

Cys Lys

Asp Asp 115

Ile Asn

Ser Tyr

120

Glu Cys

Trp Cys

Pro Phe 125

Gly Phe

Glu Gly

130

Lys Asn

Cys Glu

Leu Asp 135

Val Thr

Cys Asn

140

Ile Lys

Asn Gly

Arg Cys 145

Ser Cys

Pro Ala

Lys Leu

Glu Gln

Thr Glu

Val Pro

180

Thr Arg

Phe Cys

150

Gly Tyr 165

Phe Pro

Ala Glu

Lys Asn

Arg Leu

Cys Gly

Ala Val

Ser Ala

Ala Glu

170

Arg Val 185

Phe Pro

Asp Asn 155

Asn Gln

Ser Val

Asp Val

Lys Val

Lys Ser

Ser Gln

190

Asp Tyr

Val Cys

160

Cys Glu 175

Thr Ser

Val Asn

- 139 044349

195

200

205

Ser Thr Glu Ala Glu

210

Thr Ile Leu Asp Asn

215

Ile Thr Gln Ser Thr

220

Ser Phe Asn Asp Phe 225

Thr Arg Val Val Gly 230

Gly Glu Asp Ala Lys 235

Gly Gln Phe Pro Trp

245

Gln Val Val Leu Asn

250

Gly Lys Val Asp Ala

255

Cys Gly Gly Ser Ile

260

Val Asn Glu Lys Trp

265

Ile Val Thr Ala Ala

270

Cys Val Glu Thr Gly

275

Val Lys Ile Thr Val

280

Val Ala Gly Glu His

285

Ile Glu Glu Thr Glu

290

His Thr Glu Gln Lys

295

Arg Asn Val Ile Arg 300

Ile Pro His His Asn 305

Tyr Asn Ala Ala Ile 310

Asn Lys Tyr Asn His 315

Ile Ala Leu Leu Glu

325

Leu Asp Glu Pro Leu

330

Val Leu Asn Ser Tyr

335

Thr Pro Ile Cys Ile

340

Ala Asp Lys Glu Tyr

345

Thr Asn Ile Phe Leu

350

Phe Gly Ser Gly Tyr

355

Val Ser Gly Trp Gly

360

Arg Val Phe His Lys 365

Arg Ser Ala Leu Val

370

Leu Gln Tyr Leu Arg

375

Val Pro Leu Val Asp

380

Ala Thr Cys Leu Arg 385

Ser Thr Lys Phe Thr 390

Ile Tyr Asn Asn Met 395

Cys Ala Gly Phe His

405

Glu Gly Gly Arg Asp

410

Ser Cys Gln Gly Asp

415

Gly Gly Pro His Val

420

Thr Glu Val Glu Gly

425

Thr Ser Phe Leu Thr

430

Ile Ile Ser Trp Gly

435

Glu Glu Cys Ala Met

440

Lys Gly Lys Tyr Gly 445

Gln

Pro 240

Phe

His

Asn

Ile

Asp 320

Val

Lys

Gly

Arg

Phe 400

Ser

Gly

Ile

- 140 044349

Tyr

Thr

450

Lys

Val

Ser

Arg

Tyr

455

Val

Asn

Trp

Ile

Lys 460

Glu

Lys

Thr

Lys

Leu

465

Thr

Ser

Ser

Ser

Ser 470

Lys

Ala

Pro

Pro

Pro

475

Ser

Leu

Pro

Ser

Pro

480

Ser

Arg

Leu

Pro

Gly 485

Pro

Ser

Asp

Thr

Pro

490

Ile

Leu

Pro

Gln

Ser

495

Ser

Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro

500 505 510

Gly Pro Ser Asp Thr Pro

515

Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala

520 525

Pro Pro Pro Ser Leu Pro Ser Pro

530 535

Ser Arg Leu Pro Gly Pro Ser Asp 540

Thr Pro Ile Leu Pro Gln Ser Ser

545 550

Ser Ser Lys Ala Pro Pro Pro Ser

555 560

Leu Pro Ser Pro Ser Arg Leu Pro 565

Gly Pro Ser Asp Thr Pro Ile Leu

570 575

Pro Gln Ser Ser

580

Ser Ser

Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro

585 590

Ser Arg Leu Pro

595

Gly Pro

Ser Asp Thr Pro Ile Leu Pro Gln Gly Ser

600 605

Ala Ala

610 <210> 36 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> Primer 101 for FIX-(CTP)2 <400> 36 gtttagtgaa ccgtcagaat <210> 37 <211> 20 <212> DNA <213> Artificial sequence <220>

<223>Primer 103-R for FIX-(CTP)2

- 141 044349 <400> 37 ttgaggaaga tgttcgtgta <210> 38 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> Primer 98 for FIX-(CTP)2 <400> 38 attacagttg tcgcaggtga <210> 39 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Primer 99-R for FIX-(CTP)2 <400> 39 gctggagcta gtgagctttg ttttttcctt <210> 40 <211> 25 <212> DNA <213> Artificial sequence <220>

<223> Primer 100 for FIX-(CTP)2 <400> 40 gctcactagc tccagcagca aggcc <210> 41 <211> 23 <212> DNA <213> Artificial sequence <220>

<223> Primer 27-R for FIX-(CTP)2 <400> 41 ttttcactgc attctagttg tgg <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220>

< 223 > Primer 75 < 400 > 42 ctcccagttc aattacagct 20

- 142 044349 <210> 43 <211> 27 <212> DNA <213> Artificial sequence <220>

<223> Primer 122r <400>43 ggaaaaactg cctcagcacg ggtgagc27 <210>44 <211>32 <212> DNA <213> Artificial sequence <220>

<223> Primer 123 <400>44 gtgctgaggc agtttttcct gatgtggact at32 <210>45 <211>18 <212> DNA <213> Artificial sequence <220>

<223> Primer 124r <400> 45 caacacagtg ggcagcag 18

Claims (19)

1. The use of a modified carboxy-terminal peptide (CTP) clotting factor of human chorionic gonadotropin, consisting of a clotting factor and three CTPs attached to the carboxyl terminus of said clotting factor, in the preparation of a medicament for reducing excessive bleeding or bruising in a subject or for maintaining hemostasis in during surgical procedures or during surgical procedures in a subject, wherein said coagulation factor contains either a precursor with an N-terminal propeptide or a mature coagulation factor without an N-terminal propeptide, wherein said N-terminal propeptide contains a signal peptide, wherein said CTP-modified the blood clotting factor is selected from the group consisting of: (a) a CTP-modified Factor VII (FVII) polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 25, wherein said blood clotting factor contains a blood clotting factor precursor, and amino acids 39-528 of SEQ ID NO: 25, wherein said coagulation factor comprises a mature coagulation factor, and (b) a CTP-modified activated Factor VII polypeptide (FVIIa) having an amino acid sequence selected from the group consisting of SEQ ID NO: 25, wherein said coagulation factor comprises a precursor coagulation factor; and amino acids 39-528 of SEQ ID NO: 25, wherein said coagulation factor comprises a mature coagulation factor. 2. Use according to claim 1, wherein said subject has reduced bleeding time and intensity after said use. 3. Use according to claim 1, wherein maintaining hemostasis includes maintaining blood hemostasis without recurrence of bleeding episodes. 4. Application according to claim 3, where maintaining blood hemostasis includes maintaining coagulation activity. 5. Use according to any one of claims 1 to 4, wherein at least one CTP is glycosylated. - 143 044349 6. Use according to any one of claims 1 to 5, where the use of said medicinal product is carried out by subcutaneous administration. 7. Use according to any one of claims 1 to 6, wherein the subject is a child. 8. Use according to any one of claims 1 to 7, wherein the CTP-modified FVII polypeptide or the CTP-modified FVIIa polypeptide contains a light chain and a heavy chain linked by a disulfide bond. 9. Use according to claim 8, where the separation of said light chain and said heavy chain in a PAGE/SDS gel occurs under denaturing conditions. 10. Use according to claim 9, wherein said light chain migrates at a molecular weight level of 25 kDa and said heavy chain migrates at a molecular weight level of 50 kDa. 11. Use according to any one of claims 8 to 10, wherein the disulfide bond is present at residue 152 of said blood clotting factor FVII or blood clotting factor FVIIa. 12. Use according to claim 11, wherein said light chain contains amino acids 39-190 of the sequence SEQ ID NO: 25 and said heavy chain contains amino acids 191-528 of the sequence SEQ ID NO: 25. 13. A modified carboxy-terminal peptide (CTP) coagulation factor of human chorionic gonadotropin, consisting of a coagulation factor and three CTPs attached to the carboxyl terminus of said coagulation factor, wherein said coagulation factor contains either a precursor with an N-terminal propeptide or a mature coagulation factor blood without an N-terminal propeptide, wherein said CTP-modified coagulation factor comprises a disulfide-linked double-chain heterodimeric structure, wherein said N-terminal propeptide comprises a signal peptide, wherein said CTP-modified coagulation factor is selected from the group consisting of: (a) CTP -modified Factor VII (FVII) polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 25, where the specified blood coagulation factor contains a precursor of the blood coagulation factor, and amino acids 39-528 of the sequence SEQ ID NO: 25, where the specified the blood coagulation factor comprises a mature coagulation factor, and (b) a CTP-modified activated Factor VII polypeptide (FVIIa) having an amino acid sequence selected from the group consisting of SEQ ID NO: 25, wherein said coagulation factor comprises a coagulation factor precursor , and amino acids 39-528 of the sequence SEQ ID NO: 25, wherein said coagulation factor comprises a mature coagulation factor. 14. The CTP-modified coagulation factor of claim 13, wherein the CTP-modified FVII polypeptide or the CTP-modified FVIIa polypeptide comprises a light chain and a heavy chain linked by a disulfide bond. 15. The CTP-modified coagulation factor of claim 14, wherein separation of said light chain and said heavy chain in a PAGE/SDS gel occurs under denaturing conditions. 16. The CTP-modified coagulation factor of claim 15, wherein said light chain migrates at a molecular weight level of 25 kDa and said heavy chain migrates at a molecular weight level of 50 kDa. 17. The CTP-modified coagulation factor according to any one of claims 13 to 16, wherein a disulfide bond is present at residue 152 of said coagulation factor FVII or coagulation factor FVIIa. 18. The CTP-modified coagulation factor of claim 17, wherein said light chain comprises amino acids 39-190 of SEQ ID NO: 25 and said heavy chain comprises amino acids 191-528 of SEQ ID NO: 25. 19. A pharmaceutical composition comprising a CTP-modified blood coagulation factor as defined in any one of claims 13 to 18 and a pharmaceutically acceptable carrier.