AU2015201906A1 - Long-acting coagulation factors and methods of producing same - Google Patents

Abstract

Polypeptides and polynucleotides encoding same comprising at least one carboxy terminal peptide (CTP) of chorionic gonadotrophin attached to a carboxy terminus of a coagulation factor. Pharmaceutical compositions comprising the polypeptides 5 and polynucleotides of the invention and methods of using same are also disclosed.

Description

LONG-ACTING COAGULATION FACTORS AND METHODS OF PRODUCING SAME CROSS REFERENCE TO RELATED APPLICATIONS 5 [001] This Application claims priority from United States Provisional Application Serial Number 61/224,366. FIELD OF INVENTION [002] Polypeptides and polynucleotides encoding same comprising at least one carboxy terminal peptide (CTP) of chorionic gonadotrophin attached to a C-terminus (carboxy 10 terminus) of a coagulation factor are disclosed. Pharmaceutical compositions comprising the polypeptide and polynucleotides of the invention and methods of using same are also disclosed. BACKGROUND OF THE INVENTION [5 [003] Polypeptides are susceptible to denaturation or enzymatic degradation in the blood, liver or kidney. Accordingly, polypeptides typically have short circulatory half-lives of several hours. Because of their low stability, peptide drugs are usually delivered in a sustained frequency so as to maintain an effective plasma concentration of the active peptide. Moreover, since peptide drugs are usually administrated by infusion, frequent injection of 20 peptide drugs cause considerable discomfort to a subject. Thus, there is a need for technologies that will prolong the half-lives of therapeutic polypeptides while maintaining a high pharmacological efficacy thereof. Such desirous peptide drugs should also meet the requirements of enhanced serum stability, high activity and a low probability of inducing an undesired immune response when injected into a subject. 25 [004] Unfavorable pharmacokinetics, such as a short serum half-life, can prevent the pharmaceutical development of many otherwise promising drug candidates. Serum half-life is an empirical characteristic of a molecule, and must be determined experimentally for each new potential drug. For example, with lower molecular weight polypeptide drugs, physiological clearance mechanisms such as renal filtration can make the maintenance of 0 therapeutic levels of a drug unfeasible because of cost or frequency of the required dosing 1 regimen. Conversely, a long serum half-life is undesirable where a drug or its metabolites have toxic side effects. SUMMARY OF THE INVENTION [005] In one embodiment, the present invention provides a polypeptide consisting a coagulation factor and one to five gonadotrophin carboxy terminal (CTP) peptides attached to a carboxy terminus of the coagulation factor. [006] In another embodiment, the present invention further provides a polynucleotide molecule comprising a coding portion encoding a polypeptide consisting a coagulation factor and one to five gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor. [007] In another embodiment, the present invention further provides a A method of extending a biological half life of a coagulation factor, comprising the step of attaching one to five chorionic gonadotrophin carboxy terminal peptides to a carboxy terminus of the coagulation factor, thereby improving a biological half life of a coagulation factor. [008] In another embodiment, the present invention further provides a method of improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to five chorionic gonadotrophin carboxy terminal peptides to a carboxy terminus of the coagulation factor, thereby improving the area under the curve (AUC) of a coagulation factor. [009] In another embodiment, the present invention further provides a method of reducing a dosing frequency of a coagulation factor, comprising the step of attaching one to five chorionic gonadotrophin carboxy terminal peptides to a carboxy terminus of the coagulation factor, thereby reducing a dosing frequency of a coagulation factor. [010] In another embodiment, the present invention further provides a method of increasing compliance in the use of coagulation factor therapy, comprising providing to a subject in need thereof, a polypeptide comprising a coagulation factor, one to five chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. 2 BRIEF DESCRIPTION OF THE DRAWINGS [011] Figure 1 is a diagram illustrating rFVII-CTP construct (A), rFVII-CTP-CTP construct (B), rFIX-CTP construct (C), and rFIX-CTP-CTP construct (D). [012] Figure 2A is a bar graph showing harvests limited diluted clone transfected and selected cells with FVII-CTP variants in the presence of 5pLg/ml of Vitamin K3. The level of 5 FVII was quantified using FVII Elisa (AssayPro). [013] Figure 2B is a bar graph showing harvests of limited diluted transfected and selected cells with FVII-CTP variants in the presence of 5pLg of Vitamin K3.activity. FVII activity was quantified using FVII chromogenic activity assay (AssayPro). [014] Figure 2C is a bar graph showing harvests of limited diluted transfected and selected 10 cells with FVII-CTP variants in the presence of 5tg of Vitamin K3. The specific activity of FVII was calculated for each version by dividing the activity value by the harvest FVII concentration. [015] Figure 2D is a graph showing harvests of limited diluted transfected and selected cells with FVII, FVII-CTP and FVII-(CTP) 2 coagulation activity compared to normal poll human [5 plasma coagulation activity. [016] Figure 2E is a graph showing PK profile of FVII, FVII-CTP-CTP, and FVII-CTP harvests. [017] Figure 3A is a bar graph showing harvests of limited diluted transfected and selected cells with FIX-CTP and FIX-CTP-CTP variants in the presence of 5pLg/ml of Vitamin K3. 20 The level of FIX was quantified using Human FIX ELISA kit (Affinity Biologicals; cat. No. FIX-AG RUO), the calculated protein concentration (tg/ml) is the average of two independent runs. [018] Figure 3B depicts SDS-PAGE gel micrographs of FIX Ab recognition Micrograph A depicts recognition of anti-FIX antibody in Western-blot; Micrograph B depicts recognition 25 of anti-y carboxylation antibody in Western-blot. Lane 1 in A-B was loaded with a sample containing recombinant FIX Lane 2 in A-B was loaded with a sample containing FIX-CTP harvets. Lane 3 in A-B was loaded with a sample containing FIX-(CTP) 2 harvest. 3 [019] Figure 4 is a graph showing FIX-CTP and FIX-(CTP) 2 harvests comparative chromogenic activity (measured by a the EC 50 . concentration) compared to rhFIX ( American Diagnostics). [020] Figure 5 is a graph showing FIX-CTP and FIX-(CTP) harvests coagulation activity 5 compared to rhFIX( American Diagnostics) coagulation activity. [021] Figure 6 is a graph showing PK profile of rhFIX, harvest of FIX-CTP-CTP, and harvest of FIX-CTP. [022] Figure 7 is a bar graph showing harvests of FIX-CTP and FIX-CTP-CTPMOD-3011 and MOD-3012, respectively harvests and MOD3012 purified protein FIX antigen level as 0 detennined using Human FIX ELISA kit (Affinity Biologicals; cat. No. FIX-AG RUO), the calculated protein concentration (pg/ml) is the average of two independent runs. [023] Figure 8 depicts SDS-PAGE gel micrographs of FIX Ab recognition. Micrograph A depicts a coomassie blue staining; Micrograph B depicts recognition of anti-FIX antibody in Western-blot; Micrograph C depicts recognition of anti-y carboxylation antibody in Western 5 blot. Lane 1 in A-C was loaded with a sample containing FIX-(CTP) 2 (MOD-3012). Lane 2 in A-C was loaded with a sample containing unbound FIX-(CTP) 2 . Lane 3 in A-C was loaded with a sample containing a concentrated elution of FIX-(CTP) 2 (MOD-3012). [024] Figure 9 is a graph showing MOD3012 Comparative chromogenic activity (sample concentration/O.D.) compared to human normal pool plasma and rhFIX ( American 20 Diagnostics). [025] Figure 10 is a graph showing MOD3012 Comparative coagulation activity compared to human normal pool plasma and rhFIX. [026] Figure 11 is a graph showing PK profile of purified MOD3012, rhFIX, harvest of FIX-CTP-CTP, and harvest of FIX-CTP. 25 DETAILED DESCRIPTION OF THE INVENTION [027] In one embodiment, the present invention provides long-acting coagulation factors and methods of producing and using same. In another embodiment, long-acting coagulation factors comprise carboxy terminal peptide (CTP, also referred to as CTP unit). In another embodiment, long-acting pQlypeptides which comprise a coagulation factor further comprise 4 carboxy terminal peptide (CTP) of human Chorionic Gonadotropin (hCG). In another embodiment, CTP acts as a protectant against degradation of coagulation factors. In another embodiment, CTP extends the Cmax of coagulation factors. In another embodiment, CTP extends the Tmax of coagulation factors. In another embodiment, CTP extends circulatory 5 half-lives of coagulation factors. In some embodiments, CTP enhances the potency of coagulation factors. [028] In another embodiment, provided herein a method of extending a biological half life of a coagulation factor, comprising the step of attaching one to ten CTPs to a carboxy terminus of a coagulation factor, thereby improving a biological half life of a coagulation 10 factor. In another embodiment, provided herein a method of extending a biological half life of a coagulation factor, comprising the step of attaching one to five CTPs to a carboxy terminus of a coagulation factor, thereby improving a biological half life of a coagulation factor. [029] In another embodiment, provided herein a method of improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to ten CTPs to a ts carboxy terminus of a coagulation factor, thereby improving the area under the curve (AUC) of a coagulation factor. In another embodiment, provided herein a method of improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to five CTPs to a carboxy terminus of a coagulation factor, thereby improving the area under the curve (AUC) of a coagulation factor. :0 [030] In another embodiment, a coagulation factor of the invention is a protein. In another embodiment, a coagulation factor of the invention is a peptide. In another embodiment, a coagulation factor of the invention is a polypeptide. In another embodiment, the coagulation factor is an enzyme. In another embodiment, the coagulation factor is a serine protease. In another embodiment, the coagulation factor is a glycoprotein. In another embodiment, the 25 coagulation factor is a transglutaminase. In another embodiment, the coagulation factor is an inactive zymogen. In another embodiment, the coagulation factor is any coagulation factor known to one of skill in the art. In another embodiment, the coagulation factor is FVIII. In another embodiment, the coagulation factor is FV. In another embodiment, the coagulation factor is Factor XIII. In another embodiment, the coagulation factor is factor X. In another 10 embodiment, the coagulation factor is thrombin. In another embodiment, the coagulation factor is fibrin. In another embodiment, the coagulation factor is FVIIa. In another embodiment, the coagulation factor is FVII. In another embodiment, the coagulation factor is 5 FIX. In another embodiment, the coagulation factor is FX. In another embodiment, the coagulation factor is FXIa. In another embodiment, the coagulation factor is FXII. In another embodiment, the coagulation factor is FXa. In another embodiment, the coagulation factor is FVa. In another embodiment, the coagulation factor is prothrombin. In another embodiment, 5 the coagulation factor is thrombin. In another embodiment, the coagulation factor is FV. In another embodiment, the coagulation factor is FXI. In another embodiment, the coagulation factor is vWF. In another embodiment, the coagulation factor is FVIIIa. In another embodiment, the coagulation factor is B-deleted Domain FVIII (FVIIIBDD). In another embodiment, the coagulation factor is FIXa. In another embodiment, the coagulation factor is 10 prekallikrein. In another embodiment, the coagulation factor is kallikrein. In another embodiment, the coagulation factor is FXIIa. In another embodiment, the coagulation factor is fibrinogen. In another embodiment, the coagulation factor is thrombomodulin. In another embodiment, the coagulation factor is FII. [031] In another embodiment, the coagulation factor is a glycoprotein. In another [5 embodiment, the coagulation factor is a vitamin K dependent glycoprotein. In another embodiment, the coagulation factor is a vitamin K independent glycoprotein. In another embodiment, the coagulation factor is a recombinant protein. In another embodiment, the coagulation factor is a recombinant glycoprotein. In another embodiment, the coagulation factor is a recombinant glycoprotein FV. In another embodiment, the coagulation factor is a !0 recombinant FVI. In another embodiment, the coagulation factor is a recombinant FVII. In another embodiment, the coagulation factor is a recombinant FVIII. In another embodiment, the coagulation factor is a recombinant FIX. In another embodiment, the coagulation factor is a recombinant FX. In another embodiment, the coagulation factor is a recombinant FXI. In another embodiment, the coagulation factor is a recombinant FXII. In another embodiment, 25 the coagulation factor is a recombinant FvW. In another embodiment, the coagulation factor is a recombinant FII. In another embodiment, the coagulation factor is a recombinant FIXa. In another embodiment, the coagulation factor is a recombinant FXIa. In another embodiment, the coagulation factor is a recombinant fibrin. In another embodiment, the coagulation factor is a recombinant FVIIa. In another embodiment, the coagulation factor is a recombinant FXa. '0 In another embodiment, the coagulation factor is a recombinant FVa. In another embodiment, the coagulation factor is a recombinant prothrombin. In another embodiment, the coagulation factor is a recombinant thrombin. In another embodiment, the coagulation factor is a recombinant FVIIIa. In another embodiment, the coagulation factor is a recombinant 6 prekallikrein. In another embodiment, the coagulation factor is a recombinant kallikrein. In another embodiment, the coagulation factor is a recombinant FXIIa. In another embodiment, the coagulation factor is any known recombinant coagulation factor. In another embodiment, the coagulation factor comprising a signal peptide is any known recombinant coagulation 5 factor. In another embodiment, a coagulation factor comprises 1-10 CTP repeats attached to the C-terminus and no CTPs attached to the N-terminus. In another embodiment, the coagulation factor comprising a signal peptide is any known recombinant coagulation factor. In another embodiment, a coagulation factor comprises at least one CTP attached to the C terminus and no CTPs attached to the N-terminus. In another embodiment, a coagulation [0 factor comprising 1-10 CTP repeats attached to the C-terminus and no CTPs attached to the N-terminus is an engineered coagulation factor. In another embodiment, a coagulation factor comprising at least one CTP attached to the C-terminus and no CTPs attached to the N terminus is an engineered coagulation factor. In another embodiment, a coagulation factor comprising 1-10 CTP repeats attached to the C-terminus and no CTPs attached to the N [5 terminus is a conjugated coagulation factor. In another embodiment, a coagulation factor comprising at least one CTP attached to the C-terminus and no CTPs attached to the N terminus is a conjugated coagulation factor. [032] In another embodiment, the coagulation factor comprising a domain organization similar or identical to the domain organization of FIX, FVII, factor X, protein C and o prothrombin. In another embodiment, the coagulation factor is synthesized as precursors with N-terminal propeptide. In another embodiment, the coagulation factor as used herein is in an inactive pro-enzyme form. In another embodiment, the coagulation factor is produces in hepatocytes. In another embodiment, the coagulation factor comprises a docking site for gammacarboxylase which converts glutamic acids (Glu) into gamma carboxy glutamic acids 25 (Gla). In another embodiment, the coagulation factor as used herein is a commercially available coagulation factor. [033] In another embodiment, the amino acid sequence of factor VII comprises the following amino acid sequence: MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECK 30 EEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAF EGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVE YPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVV 7 SAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLR LHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP (SEQ ID NO: 5 9). In another embodiment, the amino acid sequence of factor VII comprises the following amino acid sequence: MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECK EEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAF EGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVE 0 YPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVV SAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLR LHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP*GCGR (SEQ 5 ID NO: 10). [034] In another embodiment, the nucleic acid sequence encoding factor VII comprises the following nucleic acid sequence: CTCGAGGACATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCTTGGGCTTCA GGGCTGCCTGGCTGCAGTCTTCGTAACCCAGGAGGAAGCCCACGGCGTCCTGCA 0o CCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCGGGCTCCCTGGA GAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGAGGCCCGGGAGATCTTCAA GGACGCGGAGAGGACGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAGTG TGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTCCAGTCCTAT ATCTGCTTCTGCCTCCCTGCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG 25 ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAG ACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATACCTATTCT AGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAAGGTGT GCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAATGGAGCTCAGTT 30 GTGTGGGGGGACCCTGATCAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTC GACAAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCACGACCTC AGCGAGCACGACGGGGATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCC AGCACGTACGTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACC 8 AGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGGACGTT CTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAG CTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCAACGTGCCCCGGC TGATGACCCAGGACTGCCTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA 5 TCACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTGCAA GGGGGACAGTGGAGGCCCACATGCCACCCACTACCGGGGCACGTGGTACCTGAC GGGCATCGTCAGCTGGGGCCAGGGCTGCGCAACCGTGGGCCACTTTGGGGTGTA CACCAGGGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCGCTCAGAGCC ACGCCCAGGAGTCCTCCTGCGAGCCCCATTTCCCTGAGGATGCGGCCGC (SEQ ID 0 NO: 11). [035] In another embodiment, the nucleic acid sequence encoding factor VII-CTP (attached to the carboxy terminus) comprises. the following nucleic acid sequence: CTCGAGGACATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCTTGGGCTTCA GGGCTGCCTGGCTGCAGTCTTCGTAACCCAGGAGGAAGCCCACGGCGTCCTGCA .5 CCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCGGGCTCCCTGGA GAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGAGGCCCGGGAGATCTTCAA GGACGCGGAGAGGACGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAGTG TGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTCCAGTCCTAT ATCTGCTTCTGCCTCCCTGCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG o ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAG ACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATACCTATTCT AGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAAGGTGT GCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAATGGAGCTCAGTT 25 GTGTGGGGGGACCCTGATCAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTC GACAAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCACGACCTC AGCGAGCACGACGGGGATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCC AGCACGTACGTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACC AGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGGACGTT 30 CTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAG CTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCAACGTGCCCCGGC TGATGACCCAGGACTGCCTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA TCACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTGCAA 9 GGGGGACAGTGGAGGCCCACATGCCACCCACTACCGGGGCACGTGGTACC TGACCGGCATCGTGAGCTGGGGCCAGGGCTGCGCCACCGTGGGCCACTTCGGCG TGTACACCAGGGTGTCCCAGTACATCGAGTGGCTGCAGAAACTGATGAGAAGCG AGCCCAGACCCGGCGTGCTGCTGAGAGCCCCCTTCCCCAGCAGCAGCTCCAAGG 5 CCCCTCCCCCTAGCCTGCCCAGCCCTAGCAGACTGCCTGGGCCCAGCGACACCCC CATCCTGCCCCAGTGAGGATCCGCGGCCGC (SEQ ID NO: 12). [036] In another embodiment, the amino acid sequence of factor VII-CTP (attached to the carboxy terminus) comprises the following amino acid sequence: MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECK 10 EEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAF EGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVE YPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVV SAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLR LHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR [5 LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSL PSPSRLPGPSDTPILPQ* (SEQ ID NO: 13). [037] In another embodiment, the nucleic acid sequence encoding factor VII-CTP-CTP (attached to the carboxy terminus) comprises the following nucleic acid sequence: 0 CTCGAGGACATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTGCTTGGGCTTCA GGGCTGCCTGGCTGCAGTCTTCGTAACCCAGGAGGAAGCCCACGGCGTCCTGCA CCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCGGGCTCCCTGGA GAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGAGGCCCGGGAGATCTTCAA GGACGCGGAGAGGACGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAGTG 25 TGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTCCAGTCCTAT ATCTGCTTCTGCCTCCCTGCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAG ACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATACCTATTCT 0 AGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAAGGTGT GCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAATGGAGCTCAGTT GTGTGGGGGGACCCTGATCAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTC 10 GACAAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCACGACCTC AGCGAGCACGACGGGGATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCC AGCACGTACGTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACC AGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGGACGTT 5 CTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAG CTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCAACGTGCCCCGGC TGATGACCCAGGACTGCCTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA TCACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTGCAA GGGGGACAGTGGAGGCCCACATGCCACCCACTACCGGGGCACGTGGTACCTGAC to CGGCATCGTGAGCTGGGGCCAGGGCTGCGCCACCGTGGGCCACTTCGGCGTGTA CACCAGGGTGTCCCAGTACATCGAGTGGCTGCAGAAACTGATGAGAAGCGAGCC CAGACCCGGCGTGCTGCTGAGAGCCCCCTTCCCCAGCAGCAGCTCCAAGGCCCCT CCCCCTAGCCTGCCCAGCCCTAGCAGACTGCCTGGGCCCTCCGACACACCAATCC TGCCACAGAGCAGCTCCTCTAAGGCCCCTCCTCCATCCCTGCCATCCCCCTCCCG [5 GCTGCCAGGCCCCTCTGACACCCCTATCCTGCCTCAGTGATGAAGGTCTGGATCC GCGGCCGC (SEQ ID NO: 14). [038] In another embodiment, the amino acid sequence of factor VII-CTP-CTP (attached to the carboxy terminus) comprises the following amino acid sequence: MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECK o EEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAF EGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVE YPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVV SAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLR LHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR 25 LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSL PSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ ID NO: 15). [039] In another embodiment, the nucleic acid sequence encoding factor IX comprises the following nucleic acid sequence: 30 GCGATCGCCATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATC ACCATTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCAT GAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTG 11 GAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGT TTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGG AAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCA GTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGG 5 . AAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCA GTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATAT CGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGA AGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGA TGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAA to AGCACCCAATCATTTAATGACTTCACTCGAGTTGTTGGTGGAGAAGATGCCAAAC CAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGG AGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACT GGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACAT ACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCA [5 GCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAG TGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACAT CTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAA GGGAGATCAGCTTTAGTTCTCCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCA CATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTC !o CATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACT GAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGT GCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGG ATTAAGGAAAAAACAAAGCTCACTTGAACGCGGCCGC (SEQ ID NO: 16). [040] In another embodiment, the amino acid sequence of factor IX comprises the following 25 amino acid sequence: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQ GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINS YECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSC EPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVV 30 GGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNI EETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNI FLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFH 12 EGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVRYVNWIKEK TKLT* (SEQ ID NO: 17). [041] In another embodiment, the nucleic acid sequence encoding factor IX-CTP (attached to the carboxy terminus) comprises the following nucleic acid sequence: 5 GCGATCGCCATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATC ACCATCTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCA TGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATT GGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAG TTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTG 10 GAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGC AGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGC AGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATA TCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGA 15 AGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGA TGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAA AGCACCCAATCATTTAATGACTTCACTCGAGTTGTTGGTGGAGAAGATGCCAAAC CAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGG AGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACT 2o GGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACAT ACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCA GCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAG TGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACAT CTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAA 25 GGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCA CATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTC CATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACT GAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGT GCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGG 30 ATTAAGGAAAAAACAAAGCTCACTAGCTCCAGCAGCAAGGCCCCTCCCCCGAGC CTGCCCTCCCCAAGCAGGCTGCCTGGGCCCTCCGACACACCAATCCTGCCACAGT GATGAAGGTCTGGATCCGCGGCCGC (SEQ ID NO: 18). 13 [042] In another embodiment, the amino acid sequence of factor IX-CTP (attached to the carboxy terminus) comprises the following amino acid sequence: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQ GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINS 5 YECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSC EPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVV GGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNI EETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNI FLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFH EGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEK TKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ ID NO: 19). [043] In another embodiment, the nucleic acid sequence encoding factor IX-CTP-CTP (attached to the carboxy terminus) comprises the following nucleic acid sequence: GCGATCGCCATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATC 5 ACCATCTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCA TGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATT GGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAG TTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTG GAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGC o AGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGC AGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATA TCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGA AGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGA 25 TGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAA AGCACCCAATCATTTAATGACTTCACTCGAGTTGTTGGTGGAGAAGATGCCAAAC CAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGG AGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACT GGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACAT 30 ACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCA GCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAG TGCTAAACAGCTACGTTACACCTATTTGCATTGCTACAAGGAATACACGAACATC TTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAG 14 GGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCAC ATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCC ATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTG AAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTG 5 CAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA TTAAGGAAAAAACAAAGCTCACTAGCTCCAGCAGCAAGGCCCCTCCCCCGAGCC TGCCCTCCCCAAGCAGGCTGCCTGGGCCCTCCGACACACCAATCCTGCCACAGAG CAGCTCCTCTAAGGCCCCTCCTCCATCCCTGCCATCCCCCTCCCGGCTGCCTGGCC CCTCTGACACCCCTATCCTGCCTCAGTGATGAAGGTCTGGATCCGCGGCCGC 0 (SEQ ID NO: 20). [044] In another embodiment, the amino acid sequence of factor IX-CTP-CTP (attached to the carboxy terminus) comprises the following amino acid sequence: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQ GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINS 5 YECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSC EPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVV GGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNI EETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNI FLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFH .0 EGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEK TKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ ID NO: 21). [045] In another embodiment, furin is added to a cell expressing the coagulation factor-CTP of the invention. In another embodiment, furin increases the production efficiency of a 25 coagulation factor-CTP of the invention in a cell. In another embodiment, furin is co transfected with the vector comprising the coding sequence of the coagulation factor-CTP of the invention. In another embodiment, furin is encoded by a separate vector. In another embodiment, furin and a coagulation factor-CTP are encoded by one vector. In another embodiment, the coding sequence of furin is inserted into pCI-DHFR. In another 30 embodiment, the coding sequence of furin is engineered in pCI-dhfr/smal+NotI, Furin/AsisI F.I.+NotI. 15 [046] In another embodiment, the nucleic acid sequence encoding furin comprises the following nucleic acid sequence: tetagagtcgacccCGCCATGGAGCTGAGGCCCTGGTTGCTATGGGTGGTAGCAGCAACA GGAACCTTGGTCCTGCTAGCAGCTGATGCTCAGGGCCAGAAGGTCTTCACCAACA 5 CGTGGGCTGTGCGCATCCCTGGAGGCCCAGCGGTGGCCAACAGTGTGGCACGGA AGCATGGGTTCCTCAACCTGGGCCAGATCTTCGGGGACTATTACCACTTCTGGCA TCGAGGAGTGACGAAGCGGTCCCTGTCGCCTCACCGCCCGCGGCACAGCCGGCT GCAGAGGGAGCCTCAAGTACAGTGGCTGGAACAGCAGGTGGCAAAGCGACGGA CTAAACGGGACGTGTACCAGGAGCCCACAGACCCCAAGTTTCCTCAGCAGTGGT 10 ACCTGTCTGGTGTCACTCAGCGGGACCTGAATGTGAAGGCGGCCTGGGCGCAGG GCTACACAGGGCACGGCATTGTGGTCTCCATTCTGGACGATGGCATCGAGAAGA ACCACCCGGACTTGGCAGGCAATTATGATCCTGGGGCCAGTTTTGATGTCAATGA CCAGGACCCTGACCCCCAGCCTCGGTACACACAGATGAATGACAACAGGCACGG CACACGGTGTGCGGGGGAAGTGGCTGCGGTGGCCAACAACGGTGTCTGTGGTGT 15 AGGTGTGGCCTACAACGCCCGCATTGGAGGGGTGCGCATGCTGGATGGCGAGGT GACAGATGCAGTGGAGGCACGCTCGCTGGGCCTGAACCCCAACCACATCCACAT CTACAGTGCCAGCTGGGGCCCCGAGGATGACGGCAAGACAGTGGATGGGCCAGC CCGCCTCGCCGAGGAGGCCTTCTTCCGTGGGGTTAGCCAGGGCCGAGGGGGGCT GGGCTCCATCTTTGTCTGGGCCTCGGGGAACGGGGGCCGGGAACATGACAGCTG 20 CAACTGCGACGGCTACACCAACAGTATCTACACGCTGTCCATCAGCAGCGCCAC GCAGTTTGGCAACGTGCCGTGGTACAGCGAGGCCTGCTCGTCCACACTGGCCACG ACCTACAGCAGTGGCAACCAGAATGAGAAGCAGATCGTGACGACTGACTTGCGG CAGAAGTGCACGGAGTCTCACACGGGCACCTCAGCCTCTGCCCCCTTAGCAGCCG GCATCATTGCTCTCACCCTGGAGGCCAATAAGAACCTCACATGGCGGGACATGC 25 AACACCTGGTGGTACAGACCTCGAAGCCAGCCCACCTCAATGCCAACGACTGGG CCACCAATGGTGTGGGCCGGAAAGTGAGCCACTCATATGGCTACGGGCTTTTGG ACGCAGGCGCCATGGTGGCCCTGGCCCAGAATTGGACCACAGTGGCCCCCCAGC GGAAGTGCATCATCGACATCCTCACCGAGCCCAAAGACATCGGGAAACGGCTCG AGGTGCGGAAGACCGTGACCGCGTGCCTGGGCGAGCCCAACCACATCACTCGGC 30 TGGAGCACGCTCAGGCGCGGCTCACCCTGTCCTATAATCGCCGTGGCGACCTGGC CATCCACCTGGTCAGCCCCATGGGCACCCGCTCCACCCTGCTGGCAGCCAGGCCA CATGACTACTCCGCAGATGGGTTTAATGACTGGGCCTTCATGACAACTCATTCCT GGGATGAGGATCCCTCTGGCGAGTGGGTCCTAGAGATTGAAAACACCAGCGAAG ,CCAACAACTATGGGACGCTGACCAAGTTCACCCTCGTACTCTATGGCACCGCCCC 16 TGAGGGGCTGCCCGTACCTCCAGAAAGCAGTGGCTGCAAGACCCTCACGTCCAG TCAGGCCTGTGTGGTGTGCGAGGAAGGCTTCTCCCTGCACCAGAAGAGCTGTGTC CAGCACTGCCCTCCAGGCTTCGCCCCCCAAGTCCTCGATACGCACTATAGCACCG AGAATGACGTGGAGACCATCCGGGCCAGCGTCTGCGCCCCCTGCCACGCCTCAT 5 GTGCCACATGCCAGGGGCCGGCCCTGACAGACTGCCTCAGCTGCCCCAGCCACG CCTCCTTGGACCCTGTGGAGCAGACTTGCTCCCGGCAAAGCCAGAGCAGCCGAG AGTCCCCGCCACAGCAGCAGCCACCTCGGCTGCCCCCGGAGGTGGAGGCGGGGC AACGGCTGCGGGCAGGGCTGCTGCCCTCACACCTGCCTGAGGTGGTGGCCGGCC TCAGCTGCGCCTTCATCGTGCTGGTCTTCGTCACTGTCTTCCTGGTCCTGCAGCTG 10 CGCTCTGGCTTTAGTTTTCGGGGGGTGAAGGTGTACACCATGGACCGTGGCCTCA TCTCCTACAAGGGGCTGCCCCCTGAAGCCTGGCAGGAGGAGTGCCCGTCTGACTC AGAAGAGGACGAGGGCCGGGGCGAGAGGACCGCCTTTATCAAAGACCAGAGCG CCCTCTGAACGCGGCCGC (SEQ ID NO: 22). [047] In another embodiment, the amino acid sequence of furin comprises the following 15 amino acid sequence: MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGF LNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDV YQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAG NYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNA !0 RIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFR GVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEAC SSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWR DMQHLVVQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTV APQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAI 25 HLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANN YGTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPG FAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTC SRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFVTVFL VLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQ 30 SAL* (SEQ ID NO: 23). [048] In some embodiments, the term coagulation factor further includes homologues of known coagulation factors which have a coagulating activity. In some embodiments, 17 homology according to the present invention also encompasses deletions, insertions, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. [049] In another embodiment, the invention includes homologues of a coagulation factor 5 having a coagulation activity. In another embodiment, the invention includes homologues of a coagulation factor as described herein having a coagulation activity. In another embodiment, homologues e.g., polypeptides which 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 % or more say 99 % homologous to a coagulation factor 10 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. [050] In another embodiment, the invention includes homologues of furin. In another embodiment, homologues e.g., polypeptides which 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 .5 least 89 %, at least 91 %, at least 93 %, at least 95 % or more say 99 % homologous to a furin as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. [051] In another embodiment, provided herein a polypeptide comprising a coagulation factor and one to ten gonadotrophin carboxy terminal (CTP) peptides attached to a carboxy o terminus of the coagulation factor. In another embodiment, provided herein a polypeptide comprising a coagulation factor and two to eight gonadotrophin carboxy terminal (CTP) peptides attached to a carboxy terminus of the coagulation factor. In another embodiment, provided herein a polypeptide comprising a coagulation factor and one to three gonadotrophin carboxy terminal (CTP) peptides attached to a carboxy terminus of the Z5 coagulation factor. In another embodiment, provided herein a polypeptide comprising a coagulation factor and one to five gonadotrophin carboxy terminal (CTP) peptides attached to a carboxy terminus of the coagulation factor. In another embodiment, provided herein a polypeptide consisting a coagulation factor and one to five gonadotrophin carboxy terminal (CTP) peptides attached to a carboxy terminus of the coagulation factor. In another o embodiment, provided herein a polypeptide comprising a coagulation factor and one to five CTPs attached to a carboxy terminus of the coagulation factor. In another embodiment, provided herein a polypeptide comprising a coagulation factor having no CTPs on its amino 18 terminus. In another embodiment, provided herein a polypeptide comprising a coagulation factor having at least one CTP on its carboxy terminus. In another embodiment, provided herein a polypeptide comprising a coagulation factor having at least one CTP on its carboxy terminus and no CTPs on its amino terminus. 5 [052] In other embodiments, engineered coagulation factor is a polypeptide comprising a coagulation factor having at least one CTP on its carboxy terminus. In other embodiments, engineered coagulation factor is a polypeptide comprising a coagulation factor having one CTP on its carboxy terminus. In other embodiments, engineered coagulation factor is a polypeptide consisting a coagulation factor having one CTP on its carboxy terminus. In other [0 embodiments, engineered coagulation factor comprises two CTP peptides attached in tandem to the carboxy terminus. In another embodiment, an engineered coagulation factor as described herein is equivalent to the non CTP modified coagulation factor in terms of biological activity. In another embodiment, an engineered coagulation factor as described herein is at least equivalent to the non CTP modified coagulation factor, in terms of [5 pharmacological measures such as pharmacokinetics and/or pharmacodynamics. [053] In other embodiments, engineered coagulation factor is intended to be used for the treatment of hemophilic B patients. In another embodiment, coagulation factor IX comprising 2 CTPs in tandem in its carboxy terminus (MOD-3012) is intended to be used for the treatment of hemophilic B patients. In another embodiment, coagulation factor IX comprising !o 1 CTP repeat in its carboxy terminus (MOD-3011) is intended to be used for the treatment of hemophilic B patients. In other embodiments, engineered coagulation factor can reduce the rate of infusions, reduce the required doses, or a combination thereof. [054] In another embodiment, coagulation factor IX comprising 2 CTPs in tandem in its carboxy terminus (MOD-3012) exhibits an improved PK profile while maintaining its 25 coagulation activity vs. FIX-CTP harvest or rhFIX. In another embodiment, coagulation factor IX comprising 2 CTPs in tandem in its carboxy terminus (MOD-3012) exhibits 3 fold increase in half life and 4.5 fold higher AUC compared to rhFIX. [055] 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. In another embodiment, the carboxy terminal peptide is a truncated CTP. Each possibility represents a separate embodiment of the present invention. 19 [056] In other embodiments, the term engineered coagulation factor comprises the amino acid sequence of a matured coagulation factor. In other embodiments, the term engineered coagulation factor comprises the amino acid sequence of coagulation factor including its signal sequence or signal peptide. [057] In another embodiment, "signal sequence" and "signal peptide" are used interchangeably herein. In another embodiment, "sequence" when in reference to a polynucleotide molecule can refer to a coding portion. Each possibility represents a separate embodiment of the present invention. [058] In another embodiment, an engineered coagulation factor comprising at least one CTP as described herein has enhanced in-vivo biological activity compared the same coagulation factor without at least one CTP. [059] In some embodiments, at least one CTP sequence at the carboxy terminal end of the coagulation factor provides enhanced protection against degradation of a coagulation factor. In some embodiments, at least one CTP sequence at the carboxy terminal end of the coagulation factor provides enhanced protection against clearance. In some embodiments, at 5 least one CTP sequence at the carboxy terminal end of the coagulation factor provides prolonged clearance time. In some embodiments, at least one CTP sequence at the carboxy terminal end of the coagulation factor enhances its Cma. In some embodiments, at least one CTP sequence at the carboxy terminal end of the coagulation factor provides enhanced protection against enhances its Tma. In some embodiments, at least one CTP sequence at the o carboxy terminal end of the coagulation factor prolongs its T1/2. [060] In another embodiment, a conjugated coagulation factor of this invention is used in the same manner as an unmodified conjugated coagulation factor. In another embodiment, a conjugated coagulation factor of this invention have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. In another 15 embodiment, due to the improved properties of the conjugated coagulation factor as described herein, this conjugate is administered less frequently than the unmodified form of the same coagulation factor. [061] In another embodiment, decreased frequency of administration will result in improved patient compliance leading to improved treatment outcomes, as well as improved patient 0 quality of life. In another embodiment, compared to conventional conjugates of coagulation 20 factors it has been found that conjugates having the molecular weight and linker structure of the conjugates of this invention have an improved potency, improved stability, elevated AUC levels, and enhanced circulating half-life. [062] In another embodiment, provided herein a composition comprising the conjugated 5 coagulation factor as described herein. In another embodiment, provided herein a pharmaceutical composition comprising the conjugated coagulation factor as described herein. In another embodiment, a therapeutically effective amount of a conjugated coagulation factor is determined according to factors as the exact type of condition being treated, the condition of the patient being treated, as well as the other ingredients in the to composition. In another embodiment, a therapeutically effective amount of a conjugated coagulation factor is between 50-500 IU per kg body weight administered once a day to once a week. In another embodiment, a therapeutically effective amount of a conjugated coagulation factor is 150-250 IU per kg body weight, administered once a day. In another embodiment, a pharmaceutical composition comprising a conjugated coagulation factor is 15 formulated at a strength effective for administration by various means to a human patient. [063] In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects afflicted with Hemophilia. In another embodiment, a conjugated coagulation factor as described herein is useful in the prophylactic therapy of Hemophilia thus reducing the risk of bleeding and associated complications. In another embodiment, a conjugated coagulation factor as- described herein is useful in the treatment of subjects afflicted with Hemophilia while reducing the risk of developing inhibitory antibodies to exogenously administered coagulation factors. In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects afflicted with Hemophilia thus inducing homeostasis. [064] In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects experiencing excessive bleeding or bruising or having a prolonged Prothrombin Time (PT) or Partial Thromboplastin Time (PTT). In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects having an acquired condition that is causing bleeding, such as vitamin K deficiency or liver disease. In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects having deficiencies in coagulation factors that are acquired (due to other diseases) or inherited, mild or severe, permanent or temporary. In another embodiment, a 21 conjugated coagulation factor as described herein is useful in the treatment of subjects afflicted with hemophilia A. In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects afflicted with hemophilia B. In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects having acquired deficiencies due to chronic diseases, such as liver disease or cancer; to an acute condition such as disseminated intravascular coagulation (DIC), which uses up clotting factors at a rapid rate; or to a deficiency in vitamin K or treatment with a vitamin K antagonist like warfarin (the production of factors II, VII, IX, and X require vitamin K). In another embodiment, a conjugated coagulation factor as described herein is useful in the treatment of subjects afflicted with a disease in which causes clotting imbalances such as but not limited to: a liver disease, uremia, a cancer, a bone marrow disorder, an exposure to snake venom, a vitamin K deficiency, an anticoagulation therapy, an accidental ingestion of the anticoagulant warfarin, a multiple blood transfusions (stored units of blood lose some of their clotting factors). [065] In another embodiment, a subject as used herein is a human subject. In another embodiment, a subject is a pet. In another embodiment, a subject is a mammal. In another embodiment, a subject is a farm animal. In another embodiment, a subject is a monkey. In another embodiment, a subject is a horse. In another embodiment, a subject is a cow. In 5 another embodiment, a subject is a mouse. In another embodiment, a subject is a rat. [066] In another embodiment, a [(CTP)n>i-coagulation factor] as described herein comprises a full length coagulation factor or an active fragment thereof connected via a peptide bond on its carboxy terminus to at least one CTP unit with no CTPs on its amino terminus. In another embodiment, a [(CTP)n>i-coagulation factor] as described herein comprises a coagulation 10 factor or an active fragment thereof connected via a peptide bond to at least one CTP unit which is connected to an additional CTP unit via a peptide bond with no CTPs on its amino terminus. In another embodiment, one nucleic acid molecule encodes an engineered coagulation factor comprising at least one CTP attached to its C-terminus and no CTPs on its amino terminus. 15 [067] In another embodiment, the CTP is attached to the coagulation factor via a linker. In another embodiment, the linker which connects the CTP sequence to the coagulation factor is a covalent bond. In another embodiment, the linker which connects the CTP sequence to the coagulation factor is a peptide bond. In another embodiment, the linker which connects the 22 CTP sequence to the coagulation factor is a substituted peptide bond. In another embodiment, the CTP sequence comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2. [068] In another embodiment, SEQ ID NO: 1 comprises the following amino acid (AA) 5 sequence: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 1). In another embodiment, SEQ ID NO: 2 comprises the following amino acid (AA) sequence: SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 2). [069] In another embodiment, the carboxy terminal peptide (CTP) peptide of the present invention comprises the amino acid sequence from amino acid 112 to position 145 of human chorionic gonadotrophin, as set forth in SEQ ID NO: 1. In another embodiment, the CTP sequence of the present invention comprises the amino acid sequence from amino acid 118 to position 145 of human chorionic gonadotropin, as set forth in SEQ ID NO: 2. In another embodiment, the CTP sequence also commences from any position between positions 112 118 and terminates at position 145 of human chorionic gonadotrophin. In some embodiments, the CTP sequence peptide is 28, 29, 30, 31, 32, 33 or 34 amino acids long and commences at position 112, 113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence. [070] In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1-5 conservative amino acid substitutions as described [0 in U.S. Pat. No. 5,712,122 which is incorporated herein be reference. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1 conservative amino acid substitution. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 2 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic 15 gonadotrophin CTP which differs from the native CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 5 conservative amino acid substitutions. In another embodiment, the CTP 20 peptide amino acid sequence of the present invention is at least 70% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 80% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino 23 acid sequence of the present invention is at least 90% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 95% homologous to the native CTP amino acid sequence or a peptide thereof. 5 [071] In another embodiment, the CTP peptide DNA sequence of the present invention is at least 70% homologous to the native human CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequence of the present invention is at least 80% homologous to the .native human CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequence of the present invention is at least 90% 10 homologous to the native CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequence of the present invention is at least 95% homologous to the native CTP DNA sequence or a peptide thereof. [072] In one embodiment, at least one of the chorionic gonadotrophin CTP amino acid sequences is truncated. In another embodiment, both of the chorionic gonadotrophin CTP 15 amino acid sequences are truncated. In another embodiment, 2 of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, 2 or more of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, all of the chorionic gonadotrophin CTP amino acid sequences are truncated. In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 3. In another embodiment, 2o SEQ ID NO: 3 comprises the following amino acid (AA) sequence: SSSSKAPPPSLP. [073] In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 4. In another embodiment, SEQ ID NO: 4 comprises the following amino acid (AA) sequence: SSSSKAPPPSLPSPSRLPGPSDTPILPQ. [074] In one embodiment, the truncated CTP comprises the first 11 amino acids of SEQ ID 25 NO: 4. In one embodiment, the truncated CTP comprises the first 12 amino acids of SEQ ID NO: 4. In one embodiment, the truncated CTP comprises the first 8 amino acids of SEQ ID NO: 4 or SEQ ID NO: 3. In one embodiment, the truncated CTP comprises the first 13 amino acids of SEQ ID NO: 4. In one embodiment, the truncated CTP comprises the first 14 amino acids of SEQ ID NO: 4. In one embodiment, the truncated CTP comprises the first 6 amino 30 acids of SEQ ID NO: 4 or SEQ ID NO: 3. In one embodiment, the truncated CTP comprises the first 5 amino acids of SEQ ID NO: 4 or SEQ ID NO: 3. 24 [075] In one embodiment, at least one of the chorionic gonadotrophin CTP amino acid sequences is glycosylated. In another embodiment, both of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 2 of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In another embodiment, 2 or 5 more of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In another embodiment, all of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In one embodiment, the CTP sequence of the present invention comprises at least one glycosylation site. In one embodiment, the CTP sequence of the present invention comprises 2 glycosylation sites. In one embodiment, the CTP sequence of the present invention 10 comprises 3 glycosylation sites. In one embodiment, the CTP sequence of the present invention comprises 4 glycosylation sites. [076] In some embodiments, the CTP sequences modification is advantageous in permitting the usage of lower dosages. In some embodiments, the CTP sequences modification is advantageous in permitting fewer dosages. In some embodiments, the CTP sequences modification is advantageous in permitting a safe long acting effect. [077] In some embodiments, "polypeptide", "engineered coagulation factor", or "protein" as used herein encompasses native polypeptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, 15 synthetically synthesized polypeptides), as well as peptoids and semipeptoids which are polypeptide analogs, which have, in some embodiments, modifications rendering the polypeptides comprising a coagulation factor even more stable while in a body or more capable of penetrating into cells. [078] In some embodiments, modifications include, but are limited to C terminus 20 modification, polypeptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-0, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by 25 reference as if fully set forth herein. Further details in this respect are provided hereinunder. [079] In some embodiments, polypeptide bonds (-CO-NH-) within the polypeptide are substituted. In some embodiments, the polypeptide bonds are substituted by N-methylated 25 bonds (-N(CH3)-CO-). In some embodiments, the polypeptide bonds are substituted by ester bonds (-C(R)H-C-0-0-C(R)-N-). In some embodiments, the polypeptide bonds are substituted by ketomethylen bonds (-CO-CH2-). In some embodiments, the polypeptide bonds are substituted by a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, 5 carba bonds (-CH2-NH-). In some embodiments, the polypeptide bonds are substituted by hydroxyethylene bonds (-CH(OH)-CH2-). In some embodiments, the polypeptide bonds are substituted by thioamide bonds (-CS-NH-). In some embodiments, the polypeptide bonds are substituted by olefinic double bonds (-CH=CH-). In some embodiments, the polypeptide bonds are substituted by retro amide bonds (-NH-CO-). In some embodiments, the 10 polypeptide bonds are substituted by polypeptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. In some embodiments, these modifications occur at any of the bonds along the polypeptide chain and even at several (2-3 bonds) at the same time. [080] In some embodiments, natural aromatic amino acids of the polypeptide such as Trp, 15 Tyr and Phe, are substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (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 acid or one or more non-amino acid monomers (e.g. fatty acid, complex carbohydrates etc). 20 [081] In one embodiment, "amino acid" or "amino acid sequence" is understood to include the 20 naturally occurring amino acid; those amino acid often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acid including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment, "amino acid" 25 includes both D- and L-amino acid. [082] In some embodiments, the polypeptides of the present invention are utilized in therapeutics which requires the polypeptides comprising a coagulation factor to be in a soluble form. In some embodiments, the polypeptides of the present invention include one or more non-natural or natural polar amino acid, including but not limited to serine and 30 threonine which are capable of increasing polypeptide solubility due to their hydroxyl containing side chain. 26 [083] In some embodiments, the engineered coagulation factor of the present invention is utilized in a linear form, although it will be appreciated by one skilled in the art that in cases where cyclicization does not severely interfere with engineered coagulation factors characteristics, cyclic forms of the engineered coagulation factors can also be utilized. 5 [084] In some embodiments, the engineered coagulation factors of present invention are biochemically synthesized such as by using standard solid phase techniques. In some embodiments, these biochemical methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. [085] In some embodiments, recombinant protein techniques are used to generate the 10 engineered coagulation factors of the present invention. In some embodiments, recombinant protein techniques are used for the generation of relatively long polypeptides (e.g., longer than 18-25 amino acids). In some embodiments, recombinant protein techniques are used for the generation of large amounts of the engineered coagulation factors of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods 15 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. 20 [086] In another embodiment, the invention provides a polynucleotide molecule comprising a coding portion encoding a polypeptide comprising a coagulation factor and one to ten gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor. In another embodiment, the invention provides a polynucleotide molecule comprising a coding portion encoding a polypeptide consisting a coagulation factor and one to ten 25 gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor. In another embodiment, the invention provides a polynucleotide molecule comprising a coding portion encoding a polypeptide consisting a coagulation factor and one to seven gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor. In another embodiment, the invention provides a polynucleotide molecule comprising 30 a coding portion encoding a polypeptide consisting a coagulation factor and two to eight gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor. In another embodiment, the invention provides a polynucleotide molecule comprising 27 a coding portion encoding a polypeptide consisting a coagulation factor and one to five gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor. [087] In another embodiment, the invention provides an expression vector comprising a 5 polynucleotide molecule as described herein. In another embodiment, the invention provides a cell comprising the expression vector as described herein. In another embodiment, the invention provides a composition comprising the expression vector as described herein. In another embodiment, the invention provides a composition comprising the cell as described herein. In another embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is 10 a prokaryotic cell. [088] 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, the polynucleotide molecule encoding engineered coagulation factors of the present invention is ligated into an expression vector, comprising a transcriptional 15 control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis regulatory sequence is suitable for directing constitutive expression of the engineered coagulation factors of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue specific expression of the engineered coagulation factors of the present invention. In some embodiments, the cis-regulatory sequence is suitable 20 for directing inducible expression of the engineered coagulation factors of the present invention. [089] In some embodiment, tissue-specific promoters suitable for use with the present invention include sequences which are functional in specific cell population, example include, but are not limited to promoters such as albumin that is liver specific [Pinkert et al., (1987) 25 Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912 30 916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Inducible promoters suitable 28 for use with the present invention include for example the tetracycline-inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405). [090] In one embodiment, the phrase "a polynucleotide molecule" refers to a single or double stranded nucleic acid sequence which be isolated and provided in the form of an RNA 5 sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above). [091] In one embodiment, "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. In one embodiment, the sequence can be 10 subsequently amplified in-vivo or in-vitro using a DNA polymerase. [092] In one embodiment, "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome. [093] In one embodiment, "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a 15 composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements. 20 [094] In one embodiment, following expression and secretion, the signal peptides are cleaved from the precursor engineered coagulation factors resulting in the mature engineered coagulation factors. [095] In some embodiments, polynucleotides of the present invention are prepared using PCR techniques, or any other method or procedure known to one skilled in the art. In some 25 embodiments, the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992). [096] In one embodiment, polynucleotides of the present invention which encode the engineered coagulation factors are inserted into expression vectors (i.e., a nucleic acid 29 construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this 5 vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation tenninators (e.g., polyadenylation 10 signals). [097] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host expression systems to express the coagulation factors of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA 15 expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence. 20 [098] In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems such as CHO cells) to express the coagulation factors of the present invention. In one embodiment, the expression vector used to express polynucleotides of the present invention in mammalian cells is pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene. Construction of the pCI-dhfr vector is described, according to one 25 embodiment, in Example 1. [099] In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly 30 as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage 30 site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)]. [0100] In one embodiment, yeast expression systems are used. In one embodiment, a number 5 of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. Application. No: 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used. [0101] In one embodiment, the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several 10 proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide. [0102] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which 15 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 their derivatives. [0103] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors 20 include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A*, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine 25 mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells. [0104] In some embodiments, recombinant viral vectors are useful for in-vivo expression of the coagulation factors of the present invention since they offer advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life 30 cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result 31 is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells. 5 [0105] In one embodiment, various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described 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, 10 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 at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative 15 selection methods. [0106] In some embodiments, introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses. [0107] In one embodiment, it will be appreciated that the engineered coagulation factors of Zo the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are 25 expanded in culture and returned to the individual (i.e., ex-vivo gene therapy). [0108] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu 30 et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment,, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671 32 1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques 5 well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention. [0109] It will be appreciated that other than containing the necessary elements for the 10 transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide. [0110] Various methods, in some embodiments, can be used to introduce the expression vector of the present invention into the host cell system. In some embodiments, such methods 15 are generally described 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, 20 Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods. [0111] In some embodiments, transformed cells are cultured under effective conditions, 25 which allow for the expression of high 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 permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some 30 embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in 33 conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art. 5 [0112] In some embodiments, depending on the vector and host system used for production, resultant engineered coagulation factors of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane. 10 [0113] In one embodiment, following a predetermined time in culture, recovery of the recombinant engineered coagulation factor is effected. [0114] In one embodiment, the phrase "recovering the recombinant engineered coagulation factor" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. .5 [0115] In one embodiment, engineered coagulation factors of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential 0 solubilization. [0116] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the engineered coagulation factor of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a Z5 column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the engineered coagulation factor and the cleavable moiety and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)]. 34 [0117] In one embodiment, the engineered coagulation factor of the present invention is retrieved in "substantially pure" fonr. [0118] In one embodiment, the phrase "substantially pure" refers to a purity that allows for the effective use of the protein in the applications described herein. 5 [0119] 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 the components of the system are commercially available. [0120] In some embodiments, the recombinant engineered coagulation factors are 0 synthesized and purified; their therapeutic efficacy can be assayed either in-vivo or in vitro. In one embodiment, the binding activities of the recombinant engineered coagulation factors of the present invention can be ascertained using various assays as known to one of skill in the art. [0121] In another embodiment, the engineered coagulation factor of the present invention 5 can be provided to the individual per se. In one embodiment, the engineered coagulation factor of the present invention can be provided to the individual as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. [0122] In another embodiment, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as 20 physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. [0123] In another embodiment, "active ingredient" refers to the polypeptide sequence of interest, which is accountable for the biological effect. [0124] In another embodiment, any of the compositions of this invention will comprise at 25 least one CTP sequence bound only to the carboxy terminus a engineered coagulation factor of interest, in any form. In one embodiment, the present invention provides combined preparations. In one embodiment, "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, 35 concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the 5 combined preparation. In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art. [0125] In another embodiment, the phrases "physiologically acceptable carrier" and 10 "pharmaceutically acceptable carrier" which be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. In one embodiment, one of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a 15 biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979). [0126] In another embodiment, "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and 20 types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. [0127] Techniques for formulation and administration of drugs are found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. [0128] In another embodiment, suitable routes of administration, for example, include oral, 25 rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. [0129] In another embodiment, the preparation is administered in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a 30 patient's body. 36 [0130] Various embodiments of dosage ranges are contemplated by this invention. 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 of 0.01-50 mg/day. In another 5 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 0.00 1-0.1 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 10 mg/day. In another embodiment, the dosage is in the range of 0.2-15mg/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 a range of 10-20mg/day. In another embodiment, the dosage is in the range of 20-40 15 mg/day. In another embodiment, the dosage is in a 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 a range of 50-100mg/day. In another embodiment, the dosage is in a 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 W mg/day. In another embodiment, the dosage is in the range of 55-65 mg/day. [0131] In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is formulated in an intranasal dosage form. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is formulated in an injectable dosage form. In another embodiment, a polypeptide comprising a coagulation factor and at 25 least one CTP unit is administered to a subject in 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 unit is administered to a subject in 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 unit is administered to a subject in a dose ranging from 0.005 mg to 0.01 mg. In another 30 embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in 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 unit is administered to a 37 subject in a dose in a dose ranging from 0.2 mg to 0.6 mg. In another embodiment, a coagulation factor is free of CTPs on its amino terminus. [0132] In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 1-100 micrograms. In another 5 embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 10-80 micrograms. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 20-60 micrograms. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a [o dose ranging from 10-50 micrograms. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 40-80 micrograms. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 10-30 micrograms. In another embodiment, a polypeptide comprising a coagulation factor and at .5 least one CTP unit is administered to a subject in a dose ranging from 30-60 micrograms. [0133] In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 0.2 mg to 2 mg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 2 mg to 6 mg. In another embodiment, a .O polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 4 mg to 10 mg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject in a dose ranging from 5 mg and 15 mg. [0134] In another embodiment, a polypeptide comprising a coagulation factor and at least one 25 CTP unit is injected into the muscle (intramuscular injection). In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is injected below the skin (subcutaneous injection). In another embodiment, a polypeptide comprising an IFN protein and CTP units is injected into the muscle. In another embodiment, a polypeptide comprising an IFN protein and CTP units is injected below the skin. [0135] In another embodiment, the methods of the invention include increasing the compliance in the use of coagulation factor therapy, comprising providing to a subject in need 38 thereof, a polypeptide comprising a coagulation factor, one chorionic gonadotrophin carboxy terminal peptide (CTP) attached to an amino terminus of the coagulation factor, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. [0136] In another embodiment, the methods of the invention include increasing the compliance of patients afflicted with chronic illnesses that are in need of a coagulation factor therapy. In another embodiment, the methods of the invention enable reduction in the dosing frequency of a coagulation factor by modifying the coagulation factor with CTPs as described 5 hereinabove. In another embodiment, the term compliance comprises adherence. In another embodiment, the methods of the invention include increasing the compliance of patients in need of a coagulation factor therapy by reducing the frequency of administration of the coagulation factor. In another embodiment, reduction in the frequency of administration of the coagulation factor is achieved due to the CTP modifications which render the CTP 10 modified coagulation factor more stable. In another embodiment, reduction in the frequency of administration of the coagulation factor is achieved as a result of increasing T1/2 of the coagulation factor. In another embodiment, reduction in the frequency of administration of the coagulation factor is achieved as a result of increasing the clearance time of the coagulation factor. In another embodiment, reduction in the frequency of administration of 15 the coagulation factor is achieved as a result of increasing the AUC measure of the coagulation factor. [0137] In another embodiment, provided a method of reducing a dosing frequency of a coagulation factor, comprising the step of attaching one to ten CTPs to a carboxy terminus of a coagulation factor, thereby reducing a dosing frequency of a coagulation factor. In another 20 embodiment, provided a method of reducing a dosing frequency of a coagulation factor, comprising the step of attaching one to five CTPs to a carboxy terminus of a coagulation factor, thereby reducing a dosing frequency of a coagulation factor. [0138] In another embodiment, provided a method of increasing compliance in the use of coagulation factor therapy, comprising providing to a subject in need thereof, a polypeptide 25 comprising a coagulation factor and one to ten chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of a coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. In another embodiment, provided a method of increasing compliance in the use of coagulation factor therapy, comprising 39 providing to a subject in need thereof, a polypeptide comprising a coagulation factor and one to five chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of a coagulation factor, thereby increasing compliance in the use of coagulation factor therapy. [0139] In another embodiment, a polypeptide comprising a coagulation factor and at least one 5 CTP unit is administered to a subject once a day. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every two days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every three days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a [0 subject once every four days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every five days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every six days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once 5 every week. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every 7-14 days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every 10-20 days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every 5-15 !0 days. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject once every 15-30 days. [0140] In another embodiment, the dosage is in a range of 50-500 mg/day. In another embodiment, the dosage is in a range of 50-150 mg/day. In another embodiment, the dosage is in a range of 100-200 mg/day. In another embodiment, the dosage is in a range of 150-250 mg/day. 25 In another embodiment, the dosage is in a range of 200-300 mg/day. In another embodiment, the dosage is in a range of 250-400 mg/day. In another embodiment, the dosage is in a range of 300 500 mg/day. In another embodiment, the dosage is in a range of 350-500 mg/day. [0141] 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 30 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 40 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. [0142] In another embodiment, the dosage is 1-90 mg/day. In another embodiment, the 5 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 10 mg/14 days. [0143] In another embodiment, the coagulation factor dosage 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 micrograms mg/5 days. In another embodiment, the dosage is 10-50 mg/6 days. In 15 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/1I days. In another embodiment, the dosage is 10-50 mg/14 days. [0144] Oral administration, in one embodiment, comprises a unit dosage form comprising tablets, capsules, lozenges, chewable tablets, suspensions, emulsions and the like. Such unit 20 dosage forms comprise a safe and effective amount of the desired coagulation factor of the invention, each of which is in one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or in another embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. The pharmaceutically acceptable carriers suitable for the preparation of unit dosage forms for peroral administration are well-known in the art. In some embodiments, tablets typically comprise conventional 25 pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. In one embodiment, glidants such as silicon dioxide can be used to improve flow characteristics of the powder-mixture. In one embodiment, coloring agents, such as 30 the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. In some 41 embodiments, the selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention, and can be readily made by a person skilled in the art. [0145] In one embodiment, the oral dosage form comprises predefined release profile. In one 5 embodiment, the oral dosage form of the present invention comprises an extended release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises a slow release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises an immediate release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage fonn is t0 formulated according to the desired release profile of the pharmaceutical active ingredient as known to one skilled in the art. [0146] Peroral compositions, in some embodiments, comprise liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. In some embodiments, liquid oral [5 compositions comprise from about 0.001% to about 0.933% of the desired compound or compounds, or in another embodiment, from about 0.0 1% to about 10 %. [0147] In some embodiments, compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of the compounds of the present invention and optionally, !0 other compounds, intended for topical intranasal administration. In some embodiments, h compositions comprise from about 0.001% to about 10.0% w/v of a 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. [0148] In another embodiment, the pharmaceutical compositions are administered by 25 intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially, and are thus 30 formulated in a form suitable for intra-arterial administration. In another embodiment, the 42 pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration. [0149] Further, in another embodiment, the pharmaceutical compositions are administered topically to body surfaces, and are thus formulated in a form suitable for topical administration. 5 Suitable topical formulations include gels, ointments, creams, lotions, drops and the like. For topical administration, the compounds of the present invention are combined with an additional appropriate therapeutic agent or agents, prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier. [0150] In one embodiment, pharmaceutical compositions of the present invention are tO manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. [0151] In one embodiment, pharmaceutical compositions for use in accordance with the present invention is formulated in conventional manner using one or more physiologically [5 acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. In one embodiment, formulation is dependent upon the route of administration chosen. [0152] In one embodiment, injectables, of the invention are formulated in aqueous solutions. In one embodiment, injectables, of the invention are formulated in physiologically compatible !o buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodinents, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0153] In one embodiment, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, 25 formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. [0154] The compositions also comprise, in some embodiments, preservatives, such as 30 benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and 43 others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases 5 to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like. [0155] In some embodiments, pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, 10 suspensions of the active ingredients, in some embodiments, are prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles 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 contain, in some embodiments, substances, which increase the viscosity of the suspension, such as sodium carboxymethyl 15 cellulose, sorbitol or dextran. In another embodiment,, the suspension also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. [0156] In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in 20 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; see generally ibid). [0157] 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 modes of administration. In one embodiment, a pump is used (see 25 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 can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, 30 pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990). 44 [0158] In some embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. Compositions are formulated, in some embodiments, for atomization and inhalation administration. In another embodiment, compositions are contained in a container with attached atomizing means. 5 [0159] In one embodiment, the preparation of the present invention is formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. [0160] In some embodiments, pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an to 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 ameliorate symptoms of disease or prolong the survival of the subject being treated. [0161] In one embodiment, determination of a therapeutically effective amount is well within the capability of those skilled in the art. [5 [0162] Some examples of substances which 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 carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, 0 cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TweenTM brand emulsifiers; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically-acceptable 25 carrier to be used in conjunction with the compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, in one embodiment, the pharmaceutically-acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4. [0163] In addition, the compositions further comprise binders (e.g. acacia, cornstarch, gelatin, 30 carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, 45 croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, 5 solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropyhnethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, 10 polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants. [0164] Typical components of carriers for syrups, elixirs, emulsions and suspensions include [5 ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, cellulose (e.g. AvicelTM, RC-591), tragacanth and sodium alginate; typical wetting agents include lecithin and polyethylene oxide sorbitan (e.g. polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. In another embodiment, peroral liquid compositions also .o contain one or more components such as sweeteners, flavoring agents and colorants disclosed above. [0165] The compositions also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar 25 vesicles, erythrocyte ghosts, or spheroplasts.) Such compositions will influence the physical state, solubility, stability, rate of in-vivo release, and rate of in-vivo clearance. [0166] Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific 0 receptors. 46 [0167] In some embodiments, compounds modified by the covalent attachment of water soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. In another embodiment, the modified compounds exhibit substantially longer 5 half-lives in blood following intravenous injection than do the corresponding umnodified compounds. In one embodiment, modifications also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance 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 the administration of such 10 polymer-compound abducts less frequently or in lower doses than with the unmodified compound. [0168] In some embodiments, preparation of effective amount or dose can be estimated initially from in-vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans. 15 [0169] In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in-vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form 20 employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.1]. [0170] In one embodiment, depending on the severity and responsiveness of the condition to 25 be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. [0171] In one embodiment, the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of 30 administration, the judgment of the prescribing physician, etc. 47 [0172] In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. [0173] In another embodiment, a coagulation factor as described herein is administered via 5 systemic administration. In another embodiment, a coagulation factor as described herein is administered by intravenous, intramuscular or subcutaneous injection. In another embodiment, a coagulation factor as described herein is lyophilized (i.e., freeze-dried) preparation in combination with complex organic excipients and stabilizers such as nonionic surface active agents (i.e., surfactants), various sugars, organic polyols and/or human serum .0 albumin. In another embodiment, a pharmaceutical composition comprises a lyophilized coagulation factor as described in sterile water for injection. In another embodiment, a pharmaceutical composition comprises a lyophilized coagulation factor as described in sterile PBS for injection. In another embodiment, a pharmaceutical composition comprises a lyophilized coagulation factor as described in sterile 0.9% NaCl for injection. .5 [0174] In another embodiment, the pharmaceutical composition comprises a coagulation factor as described herein and complex carriers such as human serum albumin, polyols, sugars, and anionic surface active stabilizing agents. See, for example, WO 89/10756 (Hara et al.- containing polyol and p-hydroxybenzoate). In another embodiment, the pharmaceutical composition comprises a coagulation factor as described herein and lactobionic acid and an !0 acetate/glycine buffer. In another embodiment, the pharmaceutical composition comprises a coagulation factor as described herein and amino acids, such as arginine or glutamate that increase the solubility of interferon compositions in water. In another embodiment, the pharmaceutical composition comprises a lyophilized coagulation factor as described herein and glycine or human serum albumin (HSA), a buffer (e g. acetate) and an isotonic agent (e.g 25 NaCl). In another embodiment, the pharmaceutical composition comprises a lyophilized coagulation factor as described herein and phosphate buffer, glycine and HSA. [0175] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein is stabilized when placed in buffered solutions having a pH between about 4 and 7.2. In another embodiment, the phannaceutical composition comprising 30 a coagulation factor as described herein is stabilized with an amino acid as a stabilizing agent and in some cases a salt (if the amino acid does not contain a charged side chain). 48 [0176] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein is a liquid composition comprising a stabilizing agent at between about 0.3% and 5% by weight which is an amino acid. [0177] In another embodiment, the pharmaceutical composition comprising a coagulation 5 factor as described herein provides dosing accuracy and product safety. In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein provides a biologically active, stable liquid formulation for use in injectable applications. In another embodiment, the pharmaceutical composition comprises a non lyophilized coagulation factor as described herein. to [0178] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein provides a liquid formulation permitting storage for a long period of time in a liquid state facilitating storage and shipping prior to administration. [0179] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises solid lipids as matrix material. In another embodiment, [5 the injectable pharmaceutical composition comprising a coagulation factor as described herein comprises solid lipids as matrix material. In another embodiment, the production of lipid microparticles by spray congealing was described by Speiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipid nanopellets for peroral administration (Speiser EP 0167825 (1990)). In another embodiment, lipids, which are used, are well tolerated by the Zo body (e. g. glycerides composed of fatty acids which are present in the emulsions for parenteral nutrition). [0180] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein is in the form of liposomes (J. E. Diederichs and al., Pharm./nd. 56 (1994) 267- 275). 25 [0181] In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises polymeric microparticles. In another embodiment, the injectable pharmaceutical composition comprising a coagulation factor as described herein comprises polymeric microparticles. In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises nanoparticles. In another 30 embodiment, the pharmaceutical composition comprising a coagulation factor as described herein, comprises liposomes. In another embodiment, the pharmaceutical composition 49 comprising a coagulation factor as described herein comprises lipid emulsion. In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises microspheres. In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises lipid nanoparticles. In another 5 embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises lipid nanoparticles comprising amphiphilic lipids. In another embodiment, the pharmaceutical composition comprising a coagulation factor as described herein comprises lipid nanoparticles comprising a drug, a lipid matrix and a surfactant. In another embodiment, the lipid matrix has a monoglyceride content which is at least 50% w/w. [0 [0182] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosage forms containing the active ingredient. In one embodiment, the pack , for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is [5 accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the fonn of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. o [0183] In one embodiment, it will be appreciated that the coagulation factors of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies. 25 [0184] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. 50 EXAMPLES [0185] Generally, the nomenclature used herein and the laboratory procedures utilized in the 5 present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained 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, Maryland (1989); Perbal, to "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", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III [5 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 0 immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" 25 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" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A 30 Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document. 51 EXAMPLE 1 Generation and utilization of Coagulation Factor IX [0186] Factor IX (FIX) is a 415 Amino acid (55KDa) glycoprotein; it belongs to a group of vitamin K dependent glycolproteins associated with the coagulation system. FIX has a similar 5 domain organization as factor FVII, factor X, protein C and prothrombin that are synthesized as precursors with N-terminal propeptide followed by a mature amino acid sequence. [0187] FIX is secreted as a single chain molecule that undergoes complex post transcriptional modifications many which are critical to its biochemical and pharmacokinetic properties. Among all the post transcriptional modifications ,12 glutamic acid residues near the amino 10 tenninus of FIX that are gamma carboxylated by the vitamin K dependent gamma carboxylase are the most crucial ones. Carboxylation is required for the interaction of FIX with the phospholipids surfaces and for optimal FIX activity. The amino terminus propeptide serves as a recognition site for the gamma carboxylase and thus following the gamma carboxylation it is cleaved off by the Golgi apparatus serine protease known as Paired basic 15 Amino acid Cleave Enzyme (PACE/Furin). Four additional post transcriptional modifications occur at the Golgi apparatus, sulfation of tyrosine 155 , phosphorylation of serine 158, 0 glycosylation on Ser 63 and on 61 and finally N- glycosylation on Asn 157 and 16. [0188] FIX circulates in the plasma (average concentration of 5ptg/ml) as a single chain inactive zymogen. Upon proteolytic cleavage at two peptide bonds: Arg 145 and Arg 180 by 20 either one or two physiological activators FVIIa- TF complex or FIXa , the activation peptide is removed converting FIX to a fully active enzyme consisting a light and heavy chain held together by a single disulfide bond. The N-terminal light chain contains the non catalytic gamma carboxglutamic acid (Gla) and two Epidrmal growth factor like domains while the C terminal heavy chain contains the trypsin like catalytic domain of the molecule. FIXa alone is 25 characterized by poor catalytic activity. However when complexed with FVIII, its proteolytic activity increase by 4-5 orders of magnitudes towards its natural substrate FX.. [0189] Hemophilia B is an X linked bleeding disorder caused by a mutation in the Factor IX (FIX) gene, resulting in a deficiency of the procoagulant activity of FIX. Hemophilia B patients have spontaneous soft tissue hemorrhages and recurrent hemarthroses that often lead 30 to a crippling Arthoathy. Current treatment for these patients includes an intravenous 52 administration of recombinant FIX. However issues of cost and relatively rapid clearance of FIX from the circulation makes developing a long acting FIX a challenging task. [0190] The CTP technology was utilized for the development of a long acting FIX. Specifically, extending half life of recombinant rFIX molecule was performed by fusion of at 5 least one human CTP to FIX. The recombinant FIX-CTP was expressed in mammalian cells and characterized in-vitro and in vivo. It was demonstrated that the in -vitro activity of rFIX CTP was comparable to rFIX. Phannacokinetics and efficacy studies in rats and demonstrated an improved properties of the rFIX-CTP. The results of this study demonstrate that it is feasible to develop a half life extended rFIX molecule having a similar haemostatic properties 10 to the wild type enzyme [0191] Cloning and expression of recombinant FIX molecule: Dg44 cells were plated in 100mm tissue culture dishes and grown to 50-60% confluence. A total of 2 pIg (microgram) of FIX cDNA was used for the transfection of one 100mm plate using the FuGene reagent (Roche) in protein free medium (Invitrogene CD Dg44). The media was removed 48 hours 15 after transfection and replaced with a protein free medium (Invitrogene CD Dg44) without nucleosides and in the presence of 800 pg/ml of G418 (Neomycin). After 14 days the transfected cell population was transferred into T25 tissue culture flasks and selection continued for additional 10-14 days until the cells began to grow as stable clones. High expressing clones were selected. Approximately 2x1 07 cells were used to inoculate 300ml of 2O growth medium in a 1700cm2 roller bottle (Coming, Coming NY) supplemented with 5ng/ml of Vitamin K3 (menadione sodium bisulfate; Sigma). The production medium (harvest) was collected after a rapid decrease in cells viability to about 70%. The production medium was first clarified and then concentrated approximately 20 fold and dialyzed with PBS using flow filtration cassette (1 OKDa MWCO; Millipore Corp.) 25 [0192] Determination of FIX antigen level:FIX-CTP harvests antigen levels were determined using AssayMax Human FIX ELISA kit (AssayPro- EF1009-1), the calculated protein concentration is the average of three different dilutions in two independent runs (Fig 3A). Table 1: Calculated protein concentration FIX-CTP FIX-CTP-CTP 53 FIX Ag level (ptg/ml) 41.9 19.2 SD 8.76 3.67 %CV 20.92 19.15 [0193] FIX SDS-PAGE - immune blot: FIX-CTP harvests or purified rhFIX (American Diagnostics), 100ng of protein, were loaded on 12% Tris -Glycine gel using Precision plus dual color protein marker (Bio-Rad). The SDS-PAGE analysis was performed by western 5 immuneblot using anti human FIX polyclonal Ab and anti human gamma carboxylation monoclonal antibody (American Diagnostics). As previously reported rhFIX migrated at 55KDa, while FIX fused to two CTPs migrated at 75KDa. Both variants of FIX-CTP proteins were shown to be gamma carboxylated (Fig 3B). [0194] Determination of FIX chromogenic activity: A comparative assessment of the in [0 vitro potency of FIX-CTP harvests versus rhFIX- protein (American Diagnostics) was performed using a commercially available chromogenic activity test kit, BIOPHEN (Hyphen BioMed 221802). In the present of thrombin, phospholipids, calcium, excess amounts of FXIa activates sampled FIX into FIXa. FIXa forms an enzymatic complex with thrombin, activated FVIII:C (supplied in an excess amounts) phospholipids and calcium and activates 5 factor X , present in the assay system, into FXa. The activity directly correlates to the amount of FIX, which is the limiting factor. The generated FXa is then measured by its specific activity on FXa chromogenic substrate (pNA). The amount of pNA generated is directly proportional to FIXa activity. rhFIX and FIX-CTP harvests were serially diluted and the potency was assessed by comparing a dose response curve of the FIX harvests to a reference 20 preparation consisting rhFIX or human plasma. The average EC50 of FIX was 21ng/ml while

FIX-(CTP)

2 harvest calculated EC50 was 382ng/ml, FIX-CTP harvest calculated EC50 was 1644ng/ml. Approximately 15 fold decrease in the enzymatic activity of FIX-(CTP) 2 harvest was observed (Fig 4). [0195] FIX Clotting activity (aPTT): The activated partial thromboplastin time (aPTT) is a 25 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 following the addition of an intrinsic pathway activator, phospholipid and calcium. The aPTT reagent is called a partial 54 thromboplastin because tissue factor is not included with the phospholipid as it is with the protime (PT) reagent. The activator initiates the system, then, the remaining steps of the intrinsic pathway take place in the presence of phospholipid. Reference aPTT range varies from laboratory to laboratory, but is usually in the range of 27 - 34 seconds. 5 [0196] The principal of the assay was to quantitate the ability of FIX-CTP harvests to restore the clotting activity of FIX depleted human plasma by the addition of rhFIX. 300d of FIX deficient human plasma was mixed with 100ptl of rhFIX or FIX-CTP harvests and serially diluted. Following 60 seconds incubation at 37C thromboplastin, CaCl2, and phospholipids were added to the mixture, clotting time in seconds was determined (performed by American 10 Medical Laboratories). The potency was assessed by comparing a dose response curve of the FIX harvests to a reference preparation consisting rhFIX or human plasma, one unit of FIX activity is the needed FIX concentration that equals the activity of one ml human normal plasma; The presented aPTT results indicate that FIX-(CTP) 2 exhibit a 5.7 fold reduction in its specific coagulation activity compared to rhFIX. Moreover, the aPTT results together with 15 the chromogenic activity in vitro assay suggest that FIX-(CTP) 2 harvest has an improved enzymatic activity vs. FIX-CTP harvest (Fig 5). An improved activity of FIX-CTP proteins will be obtained following optimization of the expression system (i.e. co-transfection with Furin and optimization of Vit K3 medium concentration). Table 2: FIX clotting activity rhFIX(A PTT(Sec) FIX-CTP PTT (Sec) FIX-CTP-CTP PTT (Sec) D) (pg/ml) (pg/ml) (Ig/ml) 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 0.07812 52.1 0.140625 76.3 0.0625 77.4 5 20 55 [0197] Pharmacokinetic study: rhFIX (American Diagnostic) , and FIX-CTP harvests were administered in a single intravenous injection to Spargue Dawley rats (six rats per substance) with a dose of 75ug/kg body weight. Table 3: PK study plan of operation No. of Dose Dose Dose Injected Con. *Time Treated. Test animals/ Route Gender Level Level Vol. ( g/ml) Points Groups Article group (pg/kg) (ptg per (ptl) (hours animal) post dose) 0 (Pre dose) 0.083, 1 rFIX 6 IV M 75 15 500 30 0.5, 1.5, 4, 8, 24, 1 48, 72. 0 (Pre dose) rFIX- 0.083, 2 CTP 6 IV M 75 15 500 30 0.5, 1.5,4, 8,24, 48, 72. 0 (Pre dose) rFIX- 0.083, 3 CTP- 6 IV M 75 15 1000 15 0.5, CTP 1.5,4, 8,24, 48, 72. 5 [0198] Blood samples were drawn retro-orbitaly from 3 Rats alternately at 0.083,0.5 1.5,4,8 ,24,48,72 hours post dosing. Plasma was prepared immediately after sampling and stored at 20C until analysis. FIX concentration was quantitated by FIX Elisa specific assay (AssayPro), Pharmacokinetic profile was calculated for each protein and is the mean of 3 animals at each 10 time point (Fig 6), terminal half lives was calculated using PK solutions 2.0 software. Table 4 summarizes the observed FIX concentrations at the different sampling time points. PK profile and summary of the terminal half-lives are summarized in table 5. 56 Table 4: Summary of PK profile Time FIX- AD (ng/ml) FIX-CTP (ng/ml) FIX-CTP-CTP (ng/ml) (Hr) 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 16720 1234.67 24 BLQ 54.625 230 48 BLQ BLQ 120.9 [0199] FIX-CTP harvests exhibit an improved T1/2p values compared to rhFIX (2 and 5 fold increase respectively). Since in FIX dosing collection animals serum concentrations at 5 24hr were below limit of quantitation (BLQ), additional PK parameters were not calculated. Table 5: Summary of PK parameters Product Terminal half life- (hr) Ratio (MOD-301X/rhFIX) rhFIX (American 2.62 Diagnostics) MOD-3011 (FIX-CTP) 5.55 2.11 MOD-3011 (FIX-CTP- 12.9 4.92 CTP) [0200] In this study a novel approach was described for prolonging FIX half life while retaining the therapeutic potency. Adding a CTP peptide to an active protein has a harmful 10 potential in interfering with the protein's activity, therefore, the generation of an active recombinant FIX-CTP by adding a CTP sequence at the C-terminus of the FIX is unexpected. 57 Characterization of an immunoaffinity purified FIX-CTP-CTP (MOD-3012) MOD3012 purification [0201] MOD3012 is a FIX modified with 2 CTP units in tandem in its carboxy-terminal. 5 MOD3012 was purified using matrix bound monoclonal antibody against y carboxyglutamyl (Gla) residues present in the N-terminal region of FIX (American Diagnostics Cat# 3570MX). The Monoclonal Ab was bound to Sepharose CL-4B. MOD3012 harvest in a concentration of 88pig/ml was dialyzed against 20mM Tris, 150Mm NaCI and 10mM EDTA at PH =7.4. The loading rate was 0.5ml/min, elution was perfonned using 20Mm Tris-HCl, 350mM NaCl and 10 50mM CaCl and the unbound fraction was recycled five times. Finally, the elution fraction was dialyzed with PBS, pulled and concentrated. Determination of FIX antigen level [0202] FIX-CTP and FIX-(CTP) 2 ; MOD-3011 and MOD-3012, respectively, harvests and MOD3012 purified protein levels were determined using the Human FIX ELISA kit (Affinity 15 Biologicals; cat# FIX-AG RUO), the calculated protein concentration (tg/ml) is the average of two independent runs (Fig 7). Table 1: Calculated protein concentration FIX-CTP FIX-CTP-CTP MOD3012 (purified) FIX Ag level (ptg/ml) 125.78 88.531 172.9 SD 17.28 21.31 2.63 %CV 13.74 24.08 1.52 [0203] Additionally, MOD-3012 was quantitated by Bradford assay. The calculated 20 concentration was 202pg/ml, which is similar to the concentration obtained by human FIX ELISA. SDS-PAGE blots [0204] MOD3012 harvest, unbound fraction and purified protein, were loaded on 12% Tris Glycine gel using Precision plus dual color protein marker (Bio-Rad). The SDS-PAGE 58 Coomassie analysis was performed by staining the gel with Commasie blue reagent (800ng of protein), western immunoblot was performed with (100ng of protein) anti human FIX polyclonal antibody (Ab) and anti human gamma carboxylation monoclonal Ab (American Diagnostics Cat #499, 3570). The immunoaffinity purification procedure significantly 5 enriched MOD3012 portion while reduced impurity (Fig. 8). N-terminal sequencing: [0205] MOD-3012 purified protein was separated by 12% Tris -Glycine SDS-PAGE and subsequently electro-blotted to PVDF membrane. The band of interest was cut out and put on a purified Biobrene treated glass fiber filter. The N-terminal sequence analysis was carried out 0 by Edmann degradation using a pulsed liquid protein sequencer equipped with a 140 C HPLC micro-gradient system. N-terminal sequencing revealed the MOD3012 is a mixture of un complete and complete pro peptide cleaved proteins. Inadequately pro peptide cleavage was shown to reduce FIX coagulation activity. By co-transfection with Furin an improved pro peptide cleavage process can be obtained. 5 Determination of FIX chromogenic activity [0206] A comparative assessment of the in-vitro potency of MOD-3012 purified protein versus rhFIX (American Diagnostics) and pool of human normal plasma was performed using a commercially available chromogenic activity test kit, BIOPHEN (Hyphen BioMed 221802). In the presence of thrombin, phospholipids and calcium; excess amounts of FXIa activated .0 FIX into FIXa. FIXa formed an enzymatic complex with thrombin, (supplied in an excess amounts) phospholipids and calcium and activates factor X, present in the assay system, into FXa. The activity directly correlated with the amount of FIX, which is the limiting factor. The generated FXa was measured by its specific activity on FXa chromogenic substrate (pNA). The amount of pNA generated was directly proportional to FIXa activity. rhFIX, human 25 plasma and MOD-3012 were serially diluted and potency was assessed by comparing a dose response curve (Fig. 9). The average EC 5 0 of rhFIX was 68.74ng/ml while MOD-3012 calculated EC 5 0 was 505ng/ml. Approximately 7 fold decrease in the enzymatic activity of MOD-3012 was observed vs. recombinant FIX and 16.5 fold decrease versus normal human pulled plasma. This reduced activity could be explained by inadequate cleavage of N-terminal 30 pro-peptide, which was identified by N-terminal analysis. FIX Clotting activity (aPTT) 59 [0207] The activated partial thromboplastin time (aPTT) is a measure of the integrity of the intrinsic and common pathways of the coagulation cascade. The aPTT is the time (measured in seconds) it takes plasma to clot following the addition of an intrinsic pathway activator, phospholipid and calcium. 5 [0208] The assay quantitated the ability of MOD-3012 protein to restore the clotting activity of FIX depleted human plasma by the addition of rhFIX. 300pl of FIX deficient human plasma was mixed with 100pl of rhFIX, MOD- 3012 (FIX-CTP-CTP (the CTP are in tandem in the C-terminal)), or normal pool human plasma which was further diluted. Following 60 seconds incubation at 37 0 C. Tissue Factor (TF), CaCl 2 , and phospholipids were added to the 10 mixture. Clotting time in seconds was determined. Potency was assessed by comparing a dose response curve of the MOD3012 to a reference preparation consisting rhFIX or human plasma. One unit of FIX was defined as the amount of FIX which equals to the activity of 1ml human normal plasma. [0209] The presented aPTT results (Fig. 10) indicate that MOD3012 coagulation activity is 15 only 1.4 less then normal pool human plasma and similar to the rhFIX. The aPTT results together with the chromogenic activity in-vitro assay suggest that MOD-3012 purification didn't damage its activity. Pharmacokinetic activity of MOD3012 [0210] Purified MOD3012, rhFIX (American Diagnostic) and harvests containing MOD3012 20 and MOD3011 (FIX-CTP) were administered in a single intravenous injection to Spargue Dawley rats (eight rats per substance) in a dose of 1 00pg/kg body weight. Table 2: PK study outline No.of Dose Dose Injected Con. Time-Points Treated. Test Article animals/ Level Level Vol. (pg/ml (hours post Groups group/ (pg/kg) (tg per (l) ) dose) time animal) point 0 (Pre-dose) A rFIX 8 100 20 500 40 0.083, 0.5,1, 2, 4, 7, 10, 24, 48, 72. 60 No.of Dose Dose Injected Con. Time-Points Treated. Test Article animals/ Level Level Vol. (ptg/ml (hours post Groups group/ (tg/kg) (tg per (tl) ) dose) time animal) point O (Pre-dose) B rFIX-CTP 8 100 20 500 40 0.083,0.5, 1, (harvest) 2, 4, 7, 10, 24, 48, 72. 0 (Pre-dose) C rFIX-CTP- 6 100 20 500 40 0.083, 0.5,1, CTP(harvest) 2, 4, 7, 10, 24, 48, 72. rFIX-CTP- 0.083, 0.5 1, 2, D CTP 4 100 20 500 40 4, 7, 10, 24, 4, (purified) 8,72. [0211] Blood samples were drawn retro-orbitally from 4 rats alternately at 0.083, 0.5, 2, 4, 7 10, 24, 48, and 72 hours post dosing. Citrated plasma (0.32%) was prepared immediately after sampling and stored at -20 0 C until analysis. FIX concentration was quantitated using human 5 FIX Elisa kit (Affinity Biologicals). Pharmacokinetic profile was calculated for each protein as the mean of 4 animals at each time point (Fig. 11). Terminal half live was calculated using PK solutions 2.0 software. Table 3 summarizes the observed FIX concentrations at different sampling time points. Summary of the PK parameters are also presented in table 4. Table 3: PK profile summary Time FIX-CTP FIX-(CTP) 2 rhFIX ng/ml Purified MOD harvest ng/ml harvest ng/ml 3012 ng/ml (hr) 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 61 7 171.45 384.36 67.62 257.02 10 50.34 250.73 40.20 158.66 24 10.07 78.50 BLQ 52.13 48 BLQ 23.40. BLQ 18.07 Table 4: Summary of PK parameters T1/2 (hr) AUC ng- MRT Vd CL hr/ml (hr) ml/Kg Mi/hr/Kg FIX-CTP harvest 4.17 3624.5r 155.1i 27.6

FIX-(CTP)

2 10.44 9105.7 12 165.4 10.9 harvest rhFIX 3.72 1416.8 5.1 373.8 70.183 Purified MOD- 11.14 6314.2 12.3 254.5 15.83 3012 [0212] MOD-3012 harvest demonstrated an improved PK profile compared to MOD3011 harvest. Furthermore, Purified MOD-3012 exhibit 3-fold increase in T1/2p value and 4.5 fold 5 increase in AUC compared to rhFIX. [0213] The reduced amount of secreted FIX fused to tandem two CTP molecules versus fusion of a single CTP seems to be due to the addition of an extra CTP and not to reduced detection by ELISA. This assumption is based on the fact that Bradford purified MOD-3012 calculated concentration was similar to the obtained ELISA calculated concentration. 10 [0214] MOD3012 clotting activity was similar to pull human plasma; however, its in-vitro chromogenic activity was significantly lower when compared to rhFIX or pooled human plasma. The chromogenic activity assay was reported as a very sensitive assay compared to the coagulation assay. The reason for reduced activity of MOD3012 may vary. Decrease 62 affinity to FXIa by the addition of CTP or reduced post transcriptional modifications (e.g. 12 10 GLA residues and pro-peptide cleavage). N-terminal analysis revealed that the proteolytic cleavage of the MOD-3012 pro-peptide was not fully completed prior to secretion. Since this posttranscriptional modification is crucial for the normal enzymatic activity of the protein, co 5 transfection with Furine-PACE plasmid is favorable and may improves MOD3012 activity. [0215] Finally, MOD-3012 comparative PK study in rats demonstrated that fusion of two tandem CTPs to the C-terminal of FIX, generated a FIX with extended half life. Comparing the PK properties of MOD-3012 to FIX-FC or FIX-FP (competitive recombinant proteins; table 5 below) indicates that MOD3012 has an improved T1/2 compared to FIX-FP but 10 reduced T1/2 compared to FIX-FC. Table 5: PK properties of long lasting FIXs Product Company T1/2 AUC CL (Ratio) FIX-FP CSL- 2 Not Not Behring Indicated Indicated MOD-3012 Prolor 3 4.5 47 Ratio= long lasting / rhFIX or BeneFIX 15 FIX depleted mouse model [0216] In order to assess the in-vivo activity model, FIX knockout mice were obtained and a breeding colony was established. 10pg of either commercial recombinant hFIX (Benefix) or rFIX-(CTP) 2 (MOD-3012) are injected into the tail vein of an anaesthetized FIX knockout mouse (22-28g). The amount of injected protein equals to the required concentration of FIX 20 in normal plasma (5tg/ml). Blood samples are taken from the clipped tail into heparinized capillary tubes at specific time points. Plasma samples are assessed for FIX levels by ELISA and efficacy is measured by aPTT coagulation assay. [0217] Increasing FIX Propeptide cleavage efficacy: CTP peptide cDNA was fused to the 3' end of human FIX cDNA. The corresponding rFIX and Furin expressing constructs were 25 co-transfected into Dg44 cells; a human rFIX cDNA was also co-transfected with the Furin plasmid as a control. Secretion of high level of FIX leads to secretion of a mixture of pro 63 factor and a mature factor FIX, due to limited amount of the Furin protease in the cell. Co transfection, of a Furin expressing vector with a pro-factor expressing vector increases the recovery and result in the secretion of fully processed FIX in to the medium. [0218] Following FIX-(CTP) 2 and Furin co-transfection stable clones are generated and 5 harvest is collected for pro peptide cleavage evaluation. 1 OOng of protein, are loaded on 12% Tris -Glycine gel using Precision plus dual color protein marker (Bio-Rad). The SDS-PAGE analysis is performed by western immuneblot using anti human FIX polyclonal Ab (American Diagnostics) and anti pro peptide polyclonal antibody. As previously reported rhFIX migrated at 55KDa, while FIX fused to two CTPs migrated at 75KDa. Both variants of FIX- proteins .0 are shown to undergo a proper, full pro-peptide cleavage. [0219] To determine whether proper pro-peptide cleavage improve FIX-(CTP) 2 enzymatic activity, a comparative assessment of chromogenic and coagulation activity of FIX-(CTP) 2 harvest co transfecated with Furin is performed. A significant improvement in FIX-(CTP) 2 specific activity is observed which is similar to rhFIX was observed. 5 [0220] In conclusion, the results described herein suggest that MOD-3012 can be used efficiently for treating Hemophilia B patients. FIX fused to CTP constructs benefit from improved in-vivo pharmacologic performance that overcomes the drawback in certain in-vitro measures. This proposed treatment is advantageous over previous treatments as the rate of infusions and the amount of required doses are reduced. .0 [0221] It is important to notice that when an Albumin fused molecule strategy was used to improve the FIX half life the recombinant FIX became inactive. Using the present novel approach, lead to the design and purification of a novel recombinant FIX fused protein that presents an improved long lasting activity. Since mere size modifications didn't improve the pharmacokinetic of injected FIX. The finding that CTP fused to FIX facilitates 25 pharmacokinetic parameters was unexpected. The presence of highly glycosylated peptide sialic acid residues stabilized the protein and protected it from interactions with vascular receptors without abrogating key determinants of FIX function. [0222] FIX-CTP has a similar therapeutic efficacy to rFIX in hemophilia B patients and required less frequent dosing. It also may appear that a single injection of FIX-CTP is 30 sufficient to control bleeding episodes and reduce the number of injections that are needed ,during surgical intervention in hemophilic B patients. 64 EXAMPLE 2 Generation and utilization of Coagulation Factor VI [0223] Recombinant coagulation factor VIa (NovoSeven) (FVIIa) was commercially 5 available and was approved in 1996 for treatment of bleeding episodes in hemophilia patients with inhibitors. However, rFVIIa had a major disadvantage- rFVIIa was rapidly cleared with a terminal half life of 2.5 hours. As a result, patients generally required multiple, frequent infusions (2-3 doses given in 2-3 hours interval) to achieve adequate homeostasis following a mild to moderate bleed. 10 [0224] Here the generation of a recombinant FVIIa-CTP molecule with an extended half life based on fusion of FVII to a human CTP, as described. The recombinant FVIIa-CTP was expressed in mammalian cells and characterized In-vitro and In vivo. It was demonstrated that rFVII-CTP activity was comparable to rFVII, Pharmacokinetic and efficacy studies in rats demonstrated improved properties of the rFVII-CTP. The results of this study demonstrated 15 that it is feasible to develop a half life extended rFVIIa molecule with very similar haemostatic properties to the wild type enzyme. [0225] Cloning and expression of recombinant FV11 molecule: Several Factor VII clones were constructed in our eukaryotic expression vector (pCI-dhfrr) (Fig. 1). Human MGC verified FL cDNA clone (IRCM) containing the sequence of Homo sapiens coagulation factor 20 VII was ordered from "Open Biosystems" (OB-MHS4426). The following primers were synthesized by Sigma-Genosys in the following sequence: Primer 67: 5'CTCGAGGACATGGTCTCCCAGGCCC3' (contains the 5' end of Factor VII DNA and the restriction site of XhoI ) (SEQ ID NO: 5); Primer 6 8 R: 5' TCTAGAATAGGTATTTTTCCACATG3' (contains the restriction site of XbaI) (SEQ ID 25 NO: 6); Primer 69: 5' TCTAGAAAAAAGAAATGCCAGC3' (contains the restriction site of XbaI) (SEQ ID NO: 7); and Primer 70R. 5'GCGGCCGCATCCTCAGGGAAATGGGGCTCGCA3' (contains the 3' end of Factor VII DNA and the restriction site of NotI) (SEQ ID NO: 8). [0226] Cloning was performed in two sets of PCR reaction. The first reaction was conducted 30 with primer 67 and primer 6 8 R and cDNA plasmid with Factor VII sequence (OB-MHS4426) 65 was used as a template; as a result of the PCR amplification , a ~ 534 bp product was formed, isolated and ligated into TA cloning vector (Invitrogen, catalog K2000-01) . XhoI -XbaI fragment containing the amino terminus of factor VII sequence was isolated. The second reaction was conducted with primer 69 and primer 70R and again cDNA plasmid with Factor 5 VII sequence (OB-MHS4426) was used as a template; as a result of the PCR amplification, a ~ 813 bp product was formed and ligated into TA cloning vector (Invitrogen, catalog K2000 01) . XbaI-NotI fragment containing the Carboxy terminus of Factor VII sequence was isolated. The two fragments were inserted into our eukaryotic expression vector pCI-dhfr (triple ligation) to yield 501-0-pl36-1 clone. 10 [0227] Plasmid 501-pl36-1 (Factor VII in pCI-dhfr vector) was digested with restriction enzymes XhoI and KpnI. A fragment of ~1186 bp was isolated. A partial Factor VII clone (1180 bp-1322 bp) followed by CTP sequence, termination sequence and NotI sequence that was synthesized by GeneArt (0721543) was digested with restriction enzymes KpnI and NotI. A fragment of ~ 253 bp was isolated. The two fragments were inserted into our eukaryotic 15 expression vector pCI-dhfr (triple ligation) to yield 501-1-p137-2 clone. pCI-dhfr-701-2-p24 2 was digested with restriction enzymes XhoI and ApaI and the large fragment (vector) was isolated. [0228] pCI-dhfr-501-2-p137-2 (Factor VII-ctp xl) was digested with restriction enzymes XhoI and ApaI and ~ 1200 bp insert was isolated. The vector and insert were ligated to yield m0 501-2-pl139-2. Dg44 cells were plated in 100mm tissue culture dishes and grown to confluence of 50-60% .A total of 2ug of DNA was used for transfection of one 100mm plate using the FuGene reagent (Roche) in protein free medium (Invitrogen CD Dg44). The media was removed 48 hours post transfection and replaced with a protein free medium (Invitrogen CD Dg44) without nucleosides. After 14 days the transfected cells population were 25 transferred into T25 tissue culture flasks and the selection was continued for 10-14 days until the cells began to grow well as a stable clone. High expressing clones were selected and approximately 2x1 07 cells were used to inoculate 300ml of growth medium in a 1700cm 2 roller bottle (Coming , Corning NY) supplemented with 5ng/ml of Vitamin K3 ( menadione sodium bisulfate ;Sigma). The production medium (harvest) was collected after a rapid 30 decrease in the cells viability to around 70%. The production medium was first clarified using and then concentrated approximately 20 fold and dialyzed to PBS using flow filtration cassette (10KDaMWCO ; Millipore Corp , Billerica ,MA) 66 Determination of FVII antigen level [0229] The cDNA coding the CTP peptide was fused to the 3' end of the cDNA coding human FVII. The corresponding rFVII construct was transfected into Dg44 cells. As a control, a human rFVII cDNA was utilized. The production medium (harvest) was collected, 5 concentrated and the secreted recombinant FVII was further evaluated. rFVII , rFVII-CTP and rFVII- CTP-CTP antigen levels were determined by AssayMax Human FVII ELISA kit (AssayPro) (Fig. 2A). There was no significant difference in the secretion level of rFVII-CTP and rFVII-(CTP) 2 compared to native rFVII. SDS-PAGE blots o [0230] SDS-PAGE analysis was done by loading 50ng of either harvest, purified or activated rFVII protein. Samples were loaded on 12% Tris -Glycine gel using Precision plus dual color protein marker (Bio-Rad). The SDS-PAGE analysis was done by performing a western immunoblot using an anti human FVII monoclonal Ab (R&D systems) or anti-CTP polyclonal antibody generated in Rabbit. .5 [0231] The level of rFVII antigen correlated to the detected protein level in a SDS-PAGE immunoblotted with anti FVII Ab. rFVII- CTP migrated as a single band while the corresponding molecular weight of the FVII control was approximately 52KDa, both proteins reacted with antibodies specific for FVII on immunoblots. The rFVII-CTP also reacted with antibodies specific for CTP. rFVII was secreted in its zymogene form with no trace of .0 activated protein FVII Chromogenic activity: [0232] rFVII , rFVII-CTP and rFVII-(CTP) 2 harvests activity was determined using a commercially available chromogenic test kit (AssaySense Human FVII chromogenic Activity assay kit (AssayPro). For functional characterization of the rFVII-CTP and its ability to be 25 further activated (FVIIa), concentrated rFVII-CTP (harvests) were placed in a commercially available chromogenic test kit that measure the ability of TF/FVIIa to activate factor X to factor Xa that in the presence of FXa specific substrate releases a quantitated signal (AssayPro) . The addition of the CTP peptide at the C-terminal of the rFVII protein didn't impair its serine protease activity (Fig. 2B, C). 67 FV11 clotting activity: [0233] Prothrombin time (PT) measures the extrinsic pathway of coagulation. The PT is the time (measured in seconds) it takes plasma to clot following the addition of an extrinsic 5 pathway activator, phospholipid and calcium. It is used to detennine the clotting tendency of blood, specifically in the measure of warfarin dosage, liver damage, and vitamin K status. The reference range for prothrombin time is usually around 12-15 seconds. Specifically, the assay quantitated the ability of FVII-CTP and FVII-(CTP) 2 harvest to restore the clotting activity of FVII depleted human plasma by the addition of rhFVII. 300pl of FVII deficient human [0 plasma was mixed with 100pl of FVII, FVII-CTP and FVII-(CTP) 2 harvets at specific concentrations, or normal pool human plasma and were further diluted. Following 60 seconds incubation at 37 0 C, Tissue Factor (TF), CaCl 2 , and phospholipids were added to the mixture. Clotting time in seconds was detennined. Potency was assessed by comparing a dose response curve of FVII-CTP and FVII-(CTP) 2 harvests to a reference preparation consisting rhFVII or t5 human pool plasma. One unit of active FVII was defined as the amount of FVII which equals to the activity of one ml human normal plasma., The PT Clotting activity of rFVII and rFVII CTP was measured on a coagulometer (Instrumentation Laboratory). [0234] As previously shown the addition of the CTP peptide at the C-terminal of the rFVII protein didn't damage its serine protease activity and lead to initiation and activation of a !o native factor X and factor IX in human plasma. Following Additional CTP at the C terminal three fold reduction the serine protease activity was observed (Fig 2D) Pharmacokinetics study: rFVII, rFVII-CTP, and rFVII-(CTP) 2 harvests were administered intravenously to Spargue Dawley rats (six rats per substance) with a dose of 100ptg/kg body weight. For all the in-vivo experiments the amount of the respective protein was determined 25 on the basis of FVII Elisa kit. For each FVII test substance the injected amount was calculated by taking into account the differences in the molecular weight of rFVII verses rFVII-CTP which leads to different molar concentration. [0235] Blood samples were drawn retro-orbitally using an altering sampling scheme to minimize interference of the sampling procedure levels to be quantified: from 3 Rats at,30 30 min and 90, 2, 6, and 48 hrs, and from the remaining three rats at 15, 60 min and 1.5 , 4 , 24 hrs alternately . Plasma was prepared immediately after sampling and stored at -20 0 C until 68 analysis. FVII concentration was quantified by FVII Elisa specific assay. Half life and area under the curve (AUC) were calculated using linear trapezoidal rule. Comparison of these clearance parameters revealed that the in-vivo half life and rFVII-(CTP) 2 AUC are significantly higher than those of rFVII (Table 8). 5 Table 6: PK study parameters Group Route Dose Tv2 AUCo..t CL/F MRT pg/kg min ng/min/mL mL/min/kg min FV11 IV 60 4.07 3314.7 6.195 6.2 FVII- IV 60 b=51.06 31353.9 0.287 73.7 CTP FVII- IV 60 B=13.66 7626.8 1.18 15.4

CTP

CTP Characterization of recombinant FVIIa-CTP: [0236] During activation, FVII is cleaved at R152 resulting in a heavy and a light chain domains that are held together by a single disulfide bridge. rFVIla-(CTP) 2 is purified and 10 activated by ion exchange columns purification process. In order to fully evaluate rFVIIa

(CTP)

2 , the protein is loaded on SDS-PAGE under reducing conditions to commercial FVIIa (Novoseven@). The heavy and the light chain domains are separated and migrate as separated bands of molecular weights 55 and 25 KDa. Both proteins react with antibodies specific for FVII but the heavy chain of the rFVIIa-CTP specifically reacts with anti-CTP specific 15 antibodies indicating that this band represents the FVII heavy chain fused to CTP. The light chain reacts specifically with anti gamma caroxylase Ab. FVIIa protein concentration is determined by FVIIa specific Elisa kit. FVIa N-terminal sequencing: [0237] rFVII -CTP-CTP in activated or zymogene purified proteins is separated by SDS 20 PAGE (on 12% Tris -Glycine) and subsequently electroblotted to PVDF membrane. The bands of interest are cut out and put on a purified Biobrene treated glass fiber filter. The N terminal sequence analysis is carried out by Edmann degradation using a pulsed liquid protein sequencer equipped with a 140 C HPLC microgradient system. The identity of the 69 recombinant protein and proper pro peptide cleavage is further verified by N-terminal sequencing. FVIIa clotting activity: [0238] In order to evaluate FVII-(CTP) 2 coagulation activity, activated partial thromboplastin 5 time assay (aPTT) is performed,. FVIII deficient plasma sample is substituted with rFVIIa (NovoSeven) or rFVIla-(CTP) 2 , 300d of FVII deficient human plasma is mixed with 100pl of FVIIa or rFVIa-(CTP) 2 at specific concentrations, or normal pull human plasma which is further diluted. Following 60 seconds incubation at 37 0 C. Tissue Factor (TF), CaCl 2 , and phospholipids are added to the mixture. Clotting time in seconds is determined. Potency is 10 assessed by comparing a dose response curve of rFVIIa-(CTP) 2 to a reference preparation consisting rhFVIIa or human pool nonnal plasma. One unit of FVIa is defined as the amount of FVIIa which equals to the activity of 1ml human normal plasma., The aPTT Clotting activity of rFVII and rFVIa-(CTP) 2 is measured on a coagulometer '(Instrumentation Laboratory). The aPTT Clotting activity of rFVIIa and rFVIla-(CTP) 2 is similar. [5 Pharmacokinetics studies in rats: [0239] In order to Characterize the influence of the CTP addition to the rFVIIa on its longevity potential a comparative pharmacokinetic study in rats is performed. NovoSeven (rFVIla) and rFVIIa-(CTP) 2 in TBS are injected IV to 6 SD rats. The time course levels of FVIIa are detected using FVIIa Elisa kit. Half life and AUC are calculated for each protein. 0 Comparison of these clearance parameters reveals that the in-vivo measures of half life, recovery, and AUC of the rFVIIa-(CTP) 2 are superior to those of NovoSeven. FVIIa-CTP In-vivo efficacy model: [0240] In order to evaluate the in-vivo activity of the rFVIIa-(CTP) 2 , 6 SD rats are treated with phenprocoumn in order to inhibit vitamin K depended gamma carboxylation of the 5 coagulation factors Gla domain. Due to the short half life of FVIII, the native FVIII is depleted faster than the other vitamin K dependent coagulation factors. It is shown that after 16 hours the FVIII activity is almost completely depleted. At this time point externally adding FVIIa corrected-reduced clotting time in rats. In order to compare NovoSeven and rFVIIa

(CTP)

2 , equal dose of both proteins are injected into rats 16 hours post phenprocounm 70 treatment. Clotting time of rat blood is corrected to normal value by both recombinant proteins. Thus, both proteins display a comparable effect in this model. [0241] In a separate experiment NovoSeven and rFVIla-(CTP) 2 are injected immediately after phenprocoumn treatment but coagulation parameters are determined after 16 hours. 5 NovoSeven does not correct the clotting time under these conditions due to a short half life. In contrast the clotting time of animals treated with rFVIIa-(CTP) 2 is corrected to values close to the values of the healthy controls. This indicates that rFVIIa-(CTP) 2 is still present and retains biology activity after a longer period. This data further confirms the great advantage in using CTP modified rFVIIa. FIX depleted mouse model (wasn't conducted yet): 0 FVIII hemophilic mice model: [0242] In order to assess the in-vivo activity model, FVII knockout mice are obtained and a breeding colony is established. 10ptg of either commercial recombinant hFVIIa (Novoseven) or rFVIIa-(CTP) 2 are injected into the tail vein of an anaesthetized FVIII knockout mouse (22-28g). The amount of injected protein equals to the required concentration of FVIII in 5 normal plasma (5p.g/ml). Blood samples are taken from the clipped tail into heparinized capillary tubes at specific time points. Plasma samples are assessed for FVIIa levels by ELISA and efficacy is measured by a PTT coagulation assay. [0243] In this study a fusion construct of FVII with CTP is generated. This recombinant protein is the basis for a treatment that provides a prolonged half life and retention of 'o adequate favorable therapeutic potency. [0244] These results suggest that rFVIIa-(CTP) 2 has a similar therapeutic efficacy to rFVIIa in hemophilia patients. Moreover, this technology requires less frequent dosing. It appears that a single injection of rFVIIa-(CTP) 2 is sufficient to control bleeding episodes and reduce the number of injections that are needed during surgical intervention. This recombinant 25 protein may be used as a long term prophylactic treatment. 71

Claims (4)

1. A method of increasing patient compliance in the use of a coagulation factor therapy, comprising providing to a subject in need thereof a CTP-modified coagulation factor polypeptide comprising a least three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII, Factor VIla, or Factor IX, thereby increasing patient compliance.

2. The method of claim 1, comprising the step of attaching three CTPs to the carboxy terminus of said Factor IX.

3. The method of claim 1, comprising the step of attaching three to five CTPs to the carboxy terminus of said Factor VII, or said Factor VIla.

4. The method of any one of claims 1-3, wherein said method comprises the step of administering a once a weekly dose of said coagulation factor polypeptide. OPKO BIOLOGICS LTD WATERMARK PATENT AND TRADE MARKS ATTORNEYS P36646AU01