AU2017201513B2

AU2017201513B2 - Long-acting coagulation factors and methods of producing same

Info

Publication number: AU2017201513B2
Application number: AU2017201513A
Authority: AU
Inventors: Udi Eyal Fima; Gili Hart
Original assignee: Opko Biologics Ltd
Current assignee: Opko Biologics Ltd
Priority date: 2009-07-09
Filing date: 2017-03-06
Publication date: 2019-07-18
Anticipated expiration: 2030-07-01
Also published as: AU2017201513A1; AU2015201906B2; AU2015201906A1

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 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 [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 carboxyterminal 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 [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.

[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 therapeutic levels of a drug unfeasible because of cost or frequency of the required dosing

2017201513 24 Jun 2019 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.

[005a] In a particular embodiment there is a CTP-modified polypeptide consisting of a coagulation factor and one to five chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or a coagulation factor and one to three chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

[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 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.

[007a] In a particular embodiment, there is provided a method of extending the biological half-life of a coagulation factor, comprising the step of attaching one to five chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or one to three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy

2017201513 24 Jun 2019 terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or

Factor IXa, thereby improving the biological half-life of said coagulation factor compared to an unmodified coagulation factor, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

[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.

[008a] In a particular embodiment, there is provided a method of improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to five chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or one to three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, thereby improving the AUC of said coagulation factor compared to an unmodified coagulation factor, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

[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.

[009a] In a particular embodiment, there is provided a method of reducing the dosing frequency of a coagulation factor, comprising the step of attaching one to five chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or

2a

2017201513 24 Jun 2019 one to three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or

Factor IXa, thereby reducing the dosing frequency of said coagulation factor compared to an unmodified coagulation factor, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

[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.

[010a] In another embodiment, there is a method of treating hemophilia, an acquired condition that causes bleeding or excessive bleeding or bruising, or any combination thereof, in a subject, the method comprising administering to said subject an effective amount of:

a CTP-modified polypeptide, or a pharmaceutical composition thereof, consisting of a coagulation factor and one to five chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or a coagulation factor and one to three chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

[010b] In yet another embodiment there is use of CTP-modified polypeptide or a pharmaceutical composition thereof in the preparation of a composition for treating hemophilia, an acquired condition that causes bleeding or excessive bleeding or bruising, or any combination thereof in a subject, said polypeptide consisting of:

a coagulation factor and one to five chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or

2b a coagulation factor and one to three chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

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2c

BRIEF DESCRIPTION OF THE DRAWINGS

2017201513 06 Mar 2017 [Oil] 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 5pg/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 5 pg 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 5 pg 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 15 plasma coagulation activity.

[016] Figure 2E is a graph showing PK profile of FVII, FVII-CTP-CTP, and FVII-CTP harvests.

[017] Figure 3 A 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 5pg/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 (pg/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-γ 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.

2017201513 06 Mar 2017 [019] Figure 4 is a graph showing FIX-CTP and FIX-(CTP)₂ harvests comparative chromogenic activity (measured by a the EC50. concentration) compared to rhFIX (American

Diagnostics).

[020] Figure 5 is a graph showing FIX-CTP and FIX-(CTP) harvests coagulation activity 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 FFX-CTP-CTP_MOD-3011 and MOD-3012, respectively harvests and MOD3012 purified protein FIX antigen level as determined using Human FIX ELISA kit (Affinity Biologicals; cat. No. FIX-AG RUO), the calculated protein concentration (gg/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-γ carboxylation antibody in Westernblot. Lane 1 in A-C was loaded with a sample containing FIX-(CTP)₂ (MOD-3012). Lane 2 in 20 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 OfFIX-(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 25 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 FIXCTP-CTP, and harvest of FIX-CTP.

[026a] Figure 12 shows a anti-CTP and anti-gamma carboxylation antibodies Western blots of

FIX fused to three, four or five CTPs. FIX-CTP3, FIX-CTP4, and FIX-CTP₅ harvests were 35 loaded on 12% Tris-Glycine gel using Precision plus dual color protein marker (Bio-Rad).

The SDS-PAGE analysis was performed by Western immuno-blot using anti-CTP polyclonal

Ab (Adar Biotech Production) or anti-Gla Ab (American Diagnostica). After a purification

2017201513 06 Mar 2017 process utilizing Jacalin column (immunoaffinity purification of glycosylated proteins), FIXCTP₃, FIX-CTP4, and FIX-CTP5 were loaded on 12% Tris-Glycine gel using Precision Plus

Dual Color Protein Marker (Bio-Rad). The SDS-PAGE was stained by Coomassie blue Dye for samples detection.

[026b] Figure 13 shows a coomassie blue detection of FIX-CTP3, FIX-CTP4, and FIX-CTP5. After a purification process utilizing Jacalin column (immunoaffinity purification of glycosylated proteins), FIX-CTP3, FIX-CTP4, and FIX-CTP₅ were loaded on 12% TrisGlycine gel using Precision Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGE was 10 stained by Coomassie blue dye for sample detection.

[026c] Figure 14 shows the results of a comparative assessment of the in vitro potency of fully purified (HA column) FIX-CTP3 FIX-CTP4 and FIX-CTP5 versus human pool normal plasma was performed using a commercially available chromogenic activity test kit, 15 BIOPHEN (Hyphen BioMed 221802). All samples were serially diluted and the potency was assessed by comparing a dose response curve to a reference preparation consisting of normal human plasma.

[026d] Figure 15 shows a comparative pharacokinetic (PK) profile of FIX-CTP3 FIX-CTP4 20 and FIX-CTP5. FIX concentration in plasma samples were quantified using human FIX Elisa kits (Affinity Biologicals). Pharmacokinetic profile was calculated and is the mean of 3 animals at each time point. Terminal half lives were calculated using PK Solutions 2.0 software.

[026e] Figure 16 shows western blots of FVII fused to three, four and five CTPs, detected using anti-FVII, anti-CTP, and anti-gamma carboxylation antibodies. FVII-CTP3, FVII-CTP4, and FVII-CTP5 harvests were loaded on 12% Tris -Glycine gel (expedeori) using Precision plus dual color protein marker (Bio-Rad). The SDS-PAGE analysis was performed by Western immunoblot using anti-FVII Ab, anti-CTP polyclonal Ab (Adar Biotech Production) 30 or anti-Gla Ab (American Diagnostica).

[026f] Figure 17 shows a comparative assessment of the in vitro potency of HA purified (highly gamma carboxylated fraction) FVII-CTP3, FVII-CTP4, and FVII-CTP₅ versus normal human pool plasma was performed using a commercially available chromogenic activity test kit, BIOPHEN (Hyphen BioMed 221304). All samples were serially diluted and the potency was assessed by comparing a dose response curve to a reference preparation consisting of normal human plasma.

4a

2017201513 06 Mar 2017 [026g] Figure 18 shows a first comparative pharmacokinetic (PK) profile-FVII 3, 4 and 5 CTPs. FVII-CTP3, FVII-CTP4, and FVII-CTP5 (Group A, B and C, respectively) were administered in a single intravenous injection to Sprague Dawley rats (six rats per treatment) 5 in a dose of 250 pg/kg body weight. Blood samples were drawn retro-orbitally from 3 rats alternately at 0.083, 0.5 2, 5, 8, 24, 48, 72 and 96 hours post dosing. Citrated plasma (0.38%) was prepared immediately after sampling and stored at -20°C until analysis. FVII-CTP₅demonstrated a superior profile as compared to the two other versions.

[026h] Figure 19 shows a second comparative PK profile-FVII 3, 4 and 5 CTPs. FVII-CTP3,

FVII-CTP4, and FVII-CTP₅ following FVII selection and the HA purification process (Group A, B and C, respectively) were administered in a single intravenous injection to Sprague Dawley rats (three rats per substance) in a dose of 29.45 pg/kg body weight. Blood samples were drawn retro-orbital at 0.083, 0.5 2, 8, 24, 48, and 72 hours post-dosing. Citrated plasma (0.38%) was prepared immediately after sampling and stored at -20°C until analysis.

DETAILED DESCRIPTION OF THE INVENTION [027] In one embodiment, the present invention provides long-acting coagulation factors and 20 methods of producing and using same. In another embodiment, long-acting coagulation factors comprise carboxy terminal peptide (CTP, also referred to as CTP unit), hi another embodiment, long-acting polypeptides which comprise a coagulation factor further comprise

4b

2017201513 06 Mar 2017 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 C_max of coagulation factors. In another embodiment, CTP extends the T_max of coagulation factors. In another embodiment, CTP extends circulatory 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 [5 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.

Ό [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 30 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

2017201513 06 Mar 2017

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 FVilla. 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 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.

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

2017201513 06 Mar 2017 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 Cterminus and no CTPs attached to the N-terminus. In another embodiment, a coagulation 10 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 Nterminus 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 N15 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 Nterminus 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 >0 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 (Gia). 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

2017201513 06 Mar 2017

SAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLR LHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP (SEQ ID NO:

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 Ό CCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCGGGCTCCCTGGA

GAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGAGGCCCGGGAGATCTTCAA GGACGCGGAGAGGACGAAGCTGTTCTGGATTTCTTACAGTGATGGGGACCAGTG

TGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTCCAGTCCTAT ATCTGCTTCTGCCTCCCTGCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG

ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAG ACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATACCTATTCT AGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAAGGTGT GCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAATGGAGCTCAGTT

GTGTGGGGGGACCCTGATCAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTC GACAAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCACGACCTC AGCGAGCACGACGGGGATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCC AGCACGTACGTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACC

2017201513 06 Mar 2017

AGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGGACGTT

CTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAG CTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCAACGTGCCCCGGC

TGATGACCCAGGACTGCCTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA

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

CCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCGGGCTCCCTGGA

TGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTCCAGTCCTAT ATCTGCTTCTGCCTCCCTGCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG >0 ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC

ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAG ACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATACCTATTCT

AGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAAGGTGT

GCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAATGGAGCTCAGTT 25 GTGTGGGGGGACCCTGATCAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTC

GACAAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCACGACCTC AGCGAGCACGACGGGGATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCC

AGCACGTACGTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACC AGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGGACGTT

CTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAG

CTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCAACGTGCCCCGGC TGATGACCCAGGACTGCCTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA TCACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTGCAA

2017201513 06 Mar 2017

GGGGGACAGTGGAGGCCCACATGCCACCCACTACCGGGGCACGTGGTACC

TGACCGGCATCGTGAGCTGGGGCCAGGGCTGCGCCACCGTGGGCCACTTCGGCG

TGTACACCAGGGTGTCCCAGTACATCGAGTGGCTGCAGAAACTGATGAGAAGCG

AGCCCAGACCCGGCGTGCTGCTGAGAGCCCCCTTCCCCAGCAGCAGCTCCAAGG

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

EEQCSFEEAREIFKDAERTKLFWIS YSDGDQC ASSPCQNGGSCKDQLQSYICFCLP AF

SAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLR LHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPR

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

TGCCTCAAGTCCATGCCAGAATGGGGGCTCCTGCAAGGACCAGCTCCAGTCCTAT

ATCTGCTTCTGCCTCCCTGCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATG ACCAGCTGATCTGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACC ACACGGGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCAG

ACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATACCTATTCT

AGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTGGGGGGCAAGGTGT

GCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAATGGAGCTCAGTT GTGTGGGGGGACCCTGATCAACACCATCTGGGTGGTCTCCGCGGCCCACTGTTTC

2017201513 06 Mar 2017

GACAAAATCAAGAACTGGAGGAACCTGATCGCGGTGCTGGGCGAGCACGACCTC

AGCGAGCACGACGGGGATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCC AGCACGTACGTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACC

AGCCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGGACGTT

CTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGCGGCTGGGGCCAG

CTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATGGTCCTCAACGTGCCCCGGC

TGATGACCCAGGACTGCCTGCAGCAGTCACGGAAGGTGGGAGACTCCCCAAATA TCACGGAGTACATGTTCTGTGCCGGCTACTCGGATGGCAGCAAGGACTCCTGCAA

GGGGGACAGTGGAGGCCCACATGCCACCCACTACCGGGGCACGTGGTACCTGAC

CGGCATCGTGAGCTGGGGCCAGGGCTGCGCCACCGTGGGCCACTTCGGCGTGTA

CACCAGGGTGTCCCAGTACATCGAGTGGCTGCAGAAACTGATGAGAAGCGAGCC

CAGACCCGGCGTGCTGCTGAGAGCCCCCTTCCCCAGCAGCAGCTCCAAGGCCCCT CCCCCTAGCCTGCCCAGCCCTAGCAGACTGCCTGGGCCCTCCGACACACCAATCC

TGCCACAGAGCAGCTCCTCTAAGGCCCCTCCTCCATCCCTGCCATCCCCCTCCCG

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 10 EEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAF

LMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT

GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSL PSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ** (SEQ ID NO: 15).

[039] In another embodiment, the nucleic acid sequence encoding factor IX comprises the following nucleic acid sequence:

GCGATCGCCATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATC

ACCATTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCAT

GAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTG

2017201513 06 Mar 2017

GAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGT

TTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGG

AAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCA

GTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGG

AAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCA

GTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATAT

CGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGA

AGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGA

TGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAA

AGCACCCAATCATTTAATGACTTCACTCGAGTTGTTGGTGGAGAAGATGCCAAAC

CAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGG

AGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACT

GGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACAT

ACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCA ί5 GCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAG

TGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACAT

CTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAA

GGGAGATCAGCTTTAGTTCTCCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCA

CATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTC >0 CATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACT

GAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGT GCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGG ATTAAGGAAAAAACAAAGCTCACTTGAACGCGGCCGC (SEQ ID NO: 16).

[040] In another embodiment, the amino acid sequence of factor IX comprises the following amino acid sequence:

MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQ

GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCENGGSCKDDINS

YECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSC EPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVV 30 GGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNI

EETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNI

FLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFH

2017201513 06 Mar 2017

EGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEK

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

GAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGC

AGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG

GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGC

AGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATA TCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGA

AGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGA

TGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAA

AGCACCCAATCATTTAATGACTTCACTCGAGTTGTTGGTGGAGAAGATGCCAAAC CAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGG

AGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACT >0 GGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACAT

ACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCA

GCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAG TGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACAT

CTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAA

GGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCA

CATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTC CATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACT

GAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGT GCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGG

ATTAAGGAAAAAACAAAGCTCACTAGCTCCAGCAGCAAGGCCCCTCCCCCGAGC

CTGCCCTCCCCAAGCAGGCTGCCTGGGCCCTCCGACACACCAATCCTGCCACAGT GATGAAGGTCTGGATCCGCGGCCGC (SEQ ID NO: 18).

2017201513 06 Mar 2017 [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

ACCATCTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCA

TTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTG GAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGC 0 AGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG

GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGC AGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATA

TCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGA AGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGA

TGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAA

AGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACT GGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACAT

ACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCA

GCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAG TGCTAAACAGCTACGTTACACCTATTTGCATTGCTACAAGGAATACACGAACATC

TTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAG

2017201513 06 Mar 2017

GGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCAC

ATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCC

ATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTG

AAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTG

CAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA TTAAGGAAAAAACAAAGCTCACTAGCTCCAGCAGCAAGGCCCCTCCCCCGAGCC TGCCCTCCCCAAGCAGGCTGCCTGGGCCCTCCGACACACCAATCCTGCCACAGAG CAGCTCCTCTAAGGCCCCTCCTCCATCCCTGCCATCCCCCTCCCGGCTGCCTGGCC CCTCTGACACCCCTATCCTGCCTCAGTGATGAAGGTCTGGATCCGCGGCCGC (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

Ό 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 cotransfected 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+Notl, Furin/AsisI F.I.+Notl.

2017201513 06 Mar 2017 [046] In another embodiment, the nucleic acid sequence encoding furin comprises the following nucleic acid sequence:

tctagagtcgacccCGCCATGGAGCTGAGGCCCTGGTTGCTATGGGTGGTAGCAGCAACA

GGAACCTTGGTCCTGCTAGCAGCTGATGCTCAGGGCCAGAAGGTCTTCACCAACA

CGTGGGCTGTGCGCATCCCTGGAGGCCCAGCGGTGGCCAACAGTGTGGCACGGA

AGCATGGGTTCCTCAACCTGGGCCAGATCTTCGGGGACTATTACCACTTCTGGCA

TCGAGGAGTGACGAAGCGGTCCCTGTCGCCTCACCGCCCGCGGCACAGCCGGCT GCAGAGGGAGCCTCAAGTACAGTGGCTGGAACAGCAGGTGGCAAAGCGACGGA

CTAAACGGGACGTGTACCAGGAGCCCACAGACCCCAAGTTTCCTCAGCAGTGGT

ACCTGTCTGGTGTCACTCAGCGGGACCTGAATGTGAAGGCGGCCTGGGCGCAGG

GCTACACAGGGCACGGCATTGTGGTCTCCATTCTGGACGATGGCATCGAGAAGA

ACCACCCGGACTTGGCAGGCAATTATGATCCTGGGGCCAGTTTTGATGTCAATGA CCAGGACCCTGACCCCCAGCCTCGGTACACACAGATGAATGACAACAGGCACGG CACACGGTGTGCGGGGGAAGTGGCTGCGGTGGCCAACAACGGTGTCTGTGGTGT

AGGTGTGGCCTACAACGCCCGCATTGGAGGGGTGCGCATGCTGGATGGCGAGGT

GACAGATGCAGTGGAGGCACGCTCGCTGGGCCTGAACCCCAACCACATCCACAT CTACAGTGCCAGCTGGGGCCCCGAGGATGACGGCAAGACAGTGGATGGGCCAGC CCGCCTCGCCGAGGAGGCCTTCTTCCGTGGGGTTAGCCAGGGCCGAGGGGGGCT GGGCTCCATCTTTGTCTGGGCCTCGGGGAACGGGGGCCGGGAACATGACAGCTG

CAACTGCGACGGCTACACCAACAGTATCTACACGCTGTCCATCAGCAGCGCCAC

GCAGTTTGGCAACGTGCCGTGGTACAGCGAGGCCTGCTCGTCCACACTGGCCACG

ACCTACAGCAGTGGCAACCAGAATGAGAAGCAGATCGTGACGACTGACTTGCGG CAGAAGTGCACGGAGTCTCACACGGGCACCTCAGCCTCTGCCCCCTTAGCAGCCG

GCATCATTGCTCTCACCCTGGAGGCCAATAAGAACCTCACATGGCGGGACATGC

AACACCTGGTGGTACAGACCTCGAAGCCAGCCCACCTCAATGCCAACGACTGGG

CCACCAATGGTGTGGGCCGGAAAGTGAGCCACTCATATGGCTACGGGCTTTTGG ACGCAGGCGCCATGGTGGCCCTGGCCCAGAATTGGACCACAGTGGCCCCCCAGC GGAAGTGCATCATCGACATCCTCACCGAGCCCAAAGACATCGGGAAACGGCTCG AGGTGCGGAAGACCGTGACCGCGTGCCTGGGCGAGCCCAACCACATCACTCGGC

TGGAGCACGCTCAGGCGCGGCTCACCCTGTCCTATAATCGCCGTGGCGACCTGGC CATCCACCTGGTCAGCCCCATGGGCACCCGCTCCACCCTGCTGGCAGCCAGGCCA CATGACTACTCCGCAGATGGGTTTAATGACTGGGCCTTCATGACAACTCATTCCT GGGATGAGGATCCCTCTGGCGAGTGGGTCCTAGAGATTGAAAACACCAGCGAAG -CCAACAACTATGGGACGCTGACCAAGTTCACCCTCGTACTCTATGGCACCGCCCC

2017201513 06 Mar 2017

TGAGGGGCTGCCCGTACCTCCAGAAAGCAGTGGCTGCAAGACCCTCACGTCCAG

TCAGGCCTGTGTGGTGTGCGAGGAAGGCTTCTCCCTGCACCAGAAGAGCTGTGTC

CAGCACTGCCCTCCAGGCTTCGCCCCCCAAGTCCTCGATACGCACTATAGCACCG

AGAATGACGTGGAGACCATCCGGGCCAGCGTCTGCGCCCCCTGCCACGCCTCAT

GTGCCACATGCCAGGGGCCGGCCCTGACAGACTGCCTCAGCTGCCCCAGCCACG

CCTCCTTGGACCCTGTGGAGCAGACTTGCTCCCGGCAAAGCCAGAGCAGCCGAG

AGTCCCCGCCACAGCAGCAGCCACCTCGGCTGCCCCCGGAGGTGGAGGCGGGGC AACGGCTGCGGGCAGGGCTGCTGCCCTCACACCTGCCTGAGGTGGTGGCCGGCC

TCAGCTGCGCCTTCATCGTGCTGGTCTTCGTCACTGTCTTCCTGGTCCTGCAGCTG

CGCTCTGGCTTTAGTTTTCGGGGGGTGAAGGTGTACACCATGGACCGTGGCCTCA

TCTCCTACAAGGGGCTGCCCCCTGAAGCCTGGCAGGAGGAGTGCCCGTCTGACTC AGAAGAGGACGAGGGCCGGGGCGAGAGGACCGCCTTTATCAAAGACCAGAGCG CCCTCTGAACGCGGCCGC (SEQ ID NO: 22).

[047] In another embodiment, the amino acid sequence of furin comprises the following amino acid sequence:

MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGF LNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDV YQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAG NYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEV AAV ANNGVCGVGV AYNA ’0 RIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFR

GVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEAC

SSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWR

DMQHLVVQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTV

APQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAI

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,

2017201513 06 Mar 2017 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 0 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 25 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 30 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

2017201513 06 Mar 2017 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.

[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 10 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 15 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 ’0 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.

2017201513 06 Mar 2017 [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 C_max. In some embodiments, at least one CTP sequence at the carboxy terminal end of the coagulation factor provides enhanced protection against enhances its T_max. In some embodiments, at least one CTP sequence at the 0 carboxy terminal end of the coagulation factor prolongs its Tl/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 20 quality of life. In another embodiment, compared to conventional conjugates of coagulation

2017201513 06 Mar 2017 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 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 L0 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

2017201513 06 Mar 2017 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. .

[067] In another embodiment, the CTP is attached to the coagulation factor via a linker, hi 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

2017201513 06 Mar 2017

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) sequence: DPREQDSSSSKAPPPSLPSPSRLPGPSDTPIL (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 112118 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 L0 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

2017201513 06 Mar 2017 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.

[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, 20 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.

2017201513 06 Mar 2017 [075] In one embodiment, at least one of the chorionic gonadotrophin CTP amino acid sequences is glycosylated. In another embodiment, both of the chorionic gonadotrophin 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-O, 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

2017201513 06 Mar 2017 bonds (~N(CH3)-CO-). In some embodiments, the polypeptide bonds are substituted by ester bonds (-C(R)H-C-O-O-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 oc-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).

[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 hydroxylcontaining side chain.

[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.

[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 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 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.

[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 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 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

2017201513 06 Mar 2017 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 cisregulatory 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 230:91230 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

2017201513 06 Mar 2017 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.

[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

2017201513 06 Mar 2017 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 hostexpression 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.

[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

2017201513 06 Mar 2017 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 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, pBKRSV 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-ΙΜΤΗΑ, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. 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 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

2017201513 06 Mar 2017 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.

[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 >0 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., invivo 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. 3:167132

2017201513 06 Mar 2017

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 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

2017201513 06 Mar 2017 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.

[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.

[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 25 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 etal., J. Biol. Chem. 265:15854-15859 (1990)].

2017201513 06 Mar 2017 [0117] In one embodiment, the engineered coagulation factor of the present invention is retrieved in substantially pure form.

[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.

[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 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 least one CTP sequence bound only to the carboxy terminus a engineered coagulation factor of interest, in any form, hi 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,

2017201513 06 Mar 2017 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.

2017201513 06 Mar 2017 [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 embodiment, the dosage is in the range of 0.1-20 mg/day. In another embodiment, the dosage is in the range of 0.1-10 mg/day. In another embodiment, the dosage is in the range of 0.01-5 mg/day. In another embodiment, the dosage is in the range of 0.001-0.01 mg/day. In another embodiment, the dosage is in the range of 0.001-0.1 mg/day. In another embodiment, the dosage is in the range of 0.1-5 mg/day. In another embodiment, the dosage is in the range of 0.5-50 mg/day. hi another embodiment, the dosage is in the range of 0.2-15tng/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. hi 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 tire 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 ’0 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 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

2017201513 06 Mar 2017 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 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 0 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 Ό 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

2017201513 06 Mar 2017 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 CTP10 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 Tl/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

2017201513 06 Mar 2017 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

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 ίθ 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 300500 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

2017201513 06 Mar 2017 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 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/11 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 pharmaceuticallyacceptable 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

2017201513 06 Mar 2017 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 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 form is 10 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 15 compositions comprise from about 0.001% to about 0.933% of the desired compound or compounds, or in another embodiment, from about 0.01% to about 10 %.

[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, 10 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, hi 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

2017201513 06 Mar 2017 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.

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 [0 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 ί5 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 >0 buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodiments, 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

2017201513 06 Mar 2017 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).

2017201513 06 Mar 2017 [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.

[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 10 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.

[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 com 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, Ό cottonseed oil, sesame oil, olive oil, com oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the Tween™ 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,

2017201513 06 Mar 2017 croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., TrisHCI., 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, hyroxypropylmethyl 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 15 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. Avicel™, 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 Ό 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 30 receptors.

2017201513 06 Mar 2017 [0167] In some embodiments, compounds modified by the covalent attachment of watersoluble 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 unmodified 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 die 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.

[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.l].

[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.

2017201513 06 Mar 2017 [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 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 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 pharmaceutical 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).

2017201513 06 Mar 2017 [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 factor as described herein provides dosing accuracy and product safety. In another embodiment, the phannaceutical 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 nonlyophilized coagulation factor as described herein.

[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, 15 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 ’0 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).

[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 embodiment, the pharmaceutical composition comprising a coagulation factor as described herein^ comprises liposomes. In another embodiment, the pharmaceutical composition

2017201513 06 Mar 2017 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.

[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 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 form 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.

’0 [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.

[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.

EXAMPLES

2017201513 06 Mar 2017 [0185] Generally, the nomenclature used herein and the laboratory procedures utilized in the 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, ίθ 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 ί5 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.

EXAMPLE 1

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Generation and utilization of Coagulation Factor LX [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 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 terminus 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, Oglycosylation on Ser 63 and on 61 and finally N- glycosylation on Asn 157 and 16.

[0188] FIX circulates in the plasma (average concentration of 5pg/ml) as a single chain inactive zymogen. Upon proteolytic cleavage at two peptide bonds: Arg 145 and Arg 180 by ’0 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 (Gia) and two Epidrmal growth factor like domains while the Cterminal 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

2017201513 06 Mar 2017 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 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 rFIXCTP was comparable to rFIX. Pharmacokinetics 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 pg (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 2xl0⁷ cells were used to inoculate 300ml of 20 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 (lOKDa MWCO; Millipore Corp.) [0192] Determination of FIX antigen Ievel:FIX-CTP harvests antigen levels were determined using AssayMax Human FIX ELISA kit (AssayPro- EFl009-1), 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

2017201513 06 Mar 2017

FIX Ag level (fig/ml) 41.9 19.2

SD 8.76 3.67 %CV _? 20.92 1.9.15 [0193] FIX SDS-PAGE - immune blot: FIX-CTP harvests or purified rhFIX (American Diagnostics), lOOng 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 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 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

2017201513 06 Mar 2017 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.

[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. 300μ1 of FIX deficient human plasma was mixed with 100 pl of rhFIX or FIX-CTP harvests and serially diluted. Following 60 seconds incubation at 37C thromboplastin, CaC12, 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 D) (pg/ml)	PTT(Sec)	FIX-CTP (pg/ml)	PTT (Sec)	FIX-CTP-CTP (pg/ml)	PTT (Sec)
,5^/: > ;	31.3	9	45.2 .		< ' I:. X 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

[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.

2017201513 06 Mar 2017

Table 3: PK study plan of operation

Treated. Groups	Test Article	No. of animals/ group	Dose Route	Gender	Dose Level (fig/kg)	Dose Level (pg per animal)	Injected Vol. (μΐ)	Con. ( g/ml)	*TimePoints (hours postdose)
1	rFIX	6	IV	M	75	15	500	30	0 (Predose) 0.083, 0.5, 1.5, 4, 8, 24, 48, 72.
2	rFIX- CTP	6	IV	M	75	15	500	30	0 (Predose) 0.083, 0.5, 1.5, 4, 8,24, 48, 72.
3	rFIXCTPCTP	6	IV	M	75	15	1000	15	0 (Predose) 0.083, 0.5, 1.5, 4, 8,24, 48, 72.

[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.

2017201513 06 Mar 2017

Table 4: Summary of PK profile

Time	FIX- AD (ng/ml)	FIX-CTP (ng/ml)	FIX-CTP-CTP
(Hr)			(ng/ml)
0.083	1500: - A· ^:	\| J, 1477.5 u	A 1914.8
0.5	1949.8	1150.1	1830.1

4	733.90	709.33	1000.00
8	319.80	167.20	1234.67
24	BLQ	54.625	230
48	BLQ .......... < .	^BLQ	120.9

[0199] FIX-CTP harvests exhibit an improved Tl/2p values compared to rhFIX (2 and 5fold 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-30 lX/rhFIX)
rhTIX (American Diagnostics)	' 2.62
MOD-3011 (FIX-CTP)	5.55	2.11
MOD-3011 (FIX-CTP[ CTP)	. A )12.9	. 4M/^S 7

[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.

Characterization of an immunoaffinity purified FIX-CTP-CTP (MOD-3012)

2017201513 06 Mar 2017

MOD3012 purification [0201] MOD3012 is a FIX modified with 2 CTP units in tandem in its carboxy-terminal.

MOD3012 was purified using matrix bound monoclonal antibody against γ carboxyglutamyl (Gia) 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 88gg/ml was dialyzed against 20mM Tris, 150Mm NaCl and lOmM EDTA at PH =7.4. The 10 loading rate was 0.5ml/min, elution was performed using 20Mm Tris-HCl, 350mM NaCl and 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 Biologicals; cat# FIX-AG RUO), the calculated protein concentration (gg/ml) is the average of two independent runs (Fig 7).

Table 6: Calculated protein concentration

[0203] Additionally, MOD-3012 was quantitated by Bradford assay. The calculated concentration was 202gg/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

2017201513 06 Mar 2017

Coomassie analysis was performed by staining the gel with Commasie blue reagent (800ng of protein), western immunoblot was performed with (lOOng 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 uncomplete 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.

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 EC50 of rhFIX was 68.74ng/ml while MOD-3012 calculated EC50 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)

2017201513 06 Mar 2017 [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.

[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. 300μ1 of FIX deficient human plasma was mixed with ΙΟΟμΙ 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 10 seconds incubation at 37°C. Tissue Factor (TF), CaCf, and phospholipids were added to the 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 Iml human normal plasma.

[0209] The presented aPTT results (Fig. 10) indicate that MOD3012 coagulation activity is 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 and MOD3011 (FIX-CTP) were administered in a single intravenous injection to Spargue 25 Dawley rats (eight rats per substance) in a dose of 100pg/kg body weight.

Table 7: PK study outline

Trasted. Crnjapg	Artiste	atshn&ls/ point	Dsse Lewi WW)	Levs! skIwI)	&. > S \	Cess, (pg/ml )
A	fFIX	8	UM	20 .......	AM	40	0.5Λ X 4.7, Ki. 24, < 72,.

2017201513 06 Mar 2017

Tr&atsd, Ctoups		No.af entasis/ grmjp/ tee pata	Dass Level &Λ)	Dose Level (Hgpsr ssunial)	feleeted VOL. W)	Can. )	Ytaeteite (hsuss ptetee)
B	AIX-CTP (harvest)	8	w	20	590	48	0 uw, <72.................
C	rFKX-CIK CrKharvisij	6		20	500	40	0 ilteikte 0,083, ALL 48, 72.
D	τΗΧΑΤΡCT?	4	100	20	500	48	083,0 1,2, 4,7, 18,24, 4, 02.

[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 5 sampling and stored at -2O°C until analysis. FIX concentration was quantitated using human

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 8 summarizes the observed FIX concentrations at different sampling time points. Summary of the PK parameters are also presented in table 9.

Table 8: PK profile summary

0.085	1038.97	1123.62	325.05	886.48
0.5	939.12	956.80	274.58	670.92
	791.97	843.85	222.90	674.17
2	304.98	673.31	186.00	503.91
⁴	315.37	525.50	109.69	357.36
7	171.45	384.36	67.62	257.02
10	50.34	250.73	40.20	158.66
24	10.07	78.50	BLQ	52.13
48	BLQ	23.40	BLQ	18.07

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Table 9: Summary of PK parameters

I Purified MOD- 11.14 6314.2 12.3 254.5 15.83

[0212] MOD-3012 harvest demonstrated an improved PK profile compared to MOD3011 harvest. Furthermore, Purified MOD-3012 exhibit 3-fold increase in Tl/2p value and 4.5 fold 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 10 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.

[0214] MOD3012 clotting activity was similar to pull human plasma; however, its in- vitro chromogenic activity was significantly lower when compared to rhFLX or pooled human 15 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 affinity to FXIa by the addition of CTP or reduced post transcriptional modifications (e.g. 1210 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 20 posttranscriptional modification is crucial for the normal enzymatic activity of the protein, cotransfection 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 25 the PK properties of MOD-3012 to FIX-FC or FIX-FP (competitive recombinant proteins;

table 10 below) indicates that MOD3012 has an improved Tl/2 compared to FIX-FP but reduced Tl/2 compared to FIX-FC.

Table 10: PK properties of long lasting FIXs

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Product	Company	Tl/2 (Ratio)	AUC	CL
FIX-FP	CSL-Behring	2	Not	Not
			Indicated	Indicated
MOD-3012	Prolor	3	4.5	4.7

Ratio= long lasting / rhFIX or BeneFIX

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 10 mouse (22-28g). The amount of injected protein equals to the required concentration of FLX in normal plasma (5pg/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 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 pro63

2017201513 06 Mar 2017 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 harvest is collected for pro peptide cleavage evaluation. lOOng 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)₂specific activity is observed which is similar to rhFIX was observed.

[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 peptidesialic 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 appeal^- 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.

EXAMPLE 2

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Generation and utilization of Coagulation Factor FVII [0223] Recombinant coagulation factor Vila (NovoSeven) (FVIIa) was commercially 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.

[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 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 FVII 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 Xhol ) (SEQ ID NO: 5); Primer 68^R: 5'

TCTAGAATAGGTATTTTTCCACATG3' (contains the restriction site of Xbal) (SEQ ID 25 NO: 6); Primer 69: 5' TCTAGAAAAAAGAAATGCCAGC3' (contains the restriction site of

Xbal) (SEQ ID NO: 7); and Primer 70^R:

5'GCGGCCGCATCCTCAGGGAAATGGGGCTCGCA3' (contains the 3' end of Factor VII DNA and the restriction site of Notl) (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 68^R and cDNA plasmid with Factor VII sequence (OB-MHS4426)

2017201513 06 Mar 2017 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) . Xhol -Xbal fragment containing the amino terminus of factor VII sequence was isolated. The second reaction was conducted with primer 69 and primer 70^R 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 K200001) . Xbal-Notl fragment containing the Carboxy terminus of Factor VII sequence was isolated. The two fragments were inserted into our eukaryotic expression vector pCI-dhff (triple ligation) to yield 501-0-p 136-1 clone.

[0227] Plasmid 501-pl36-1 (Factor VII in pCI-dhfr vector) was digested with restriction enzymes Xhol 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 Notl sequence that was synthesized by GeneArt (0721543) was digested with restriction enzymes KpnI and Notl. A fragment of ~ 253 bp was isolated. The two fragments were inserted into our eukaryotic expression vector pCI-dhfr (triple ligation) to yield 501 -1 -p 137-2 clone. pCI-dhfr-701-2-p242 was digested with restriction enzymes Xhol and Apal and the large fragment (vector) was isolated.

[0228] pCI-dhff-501-2-pl37-2 (Factor VH-ctp xl) was digested with restriction enzymes Xhol and Apal and ~ 1200 bp insert was isolated. The vector and insert were ligated to yield >0 501-2-pl39-2. 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 2x10 cells were used to inoculate 300ml of growth medium in a 1700cm 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 (lOKDaMWCO ; Millipore Corp , Billerica ,MA)

2017201513 06 Mar 2017

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 .0 [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 Ό 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).

2017201513 06 Mar 2017

FVII 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 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 10 plasma was mixed with 100μ1 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°C, Tissue Factor (TF), CaCl₂, 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 L5 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 >0 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 100pg/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°C until 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 11).

2017201513 06 Mar 2017

Table 11: PK study parameters

Group	Route	Dose	Tl/2	AUC₀._t	CL/F	MRT
		Mg/kg	min	ng/min/mL	mL/min/kg	min
FVIICTP	IV	60	4.07	3314.7	6.195	6.2
FV LICIT	IV	60	β=51.06	31353.9	0.287	73.7
FVII-	IV	60	β=13.66	7626.8	1.18	15.4

CTPCTP

Characterization of recombinant FVIIa-CTP:

[0236] During activation, FVII is cleaved at RI 52 resulting in a heavy and a light chain domains that are held together by a single disulfide bridge. TFVIIa-(CTP)2 is purified and activated by ion exchange columns purification process. In order to fully evaluate rF Vlla(CTP)₂ , the protein is loaded on SDS-PAGE under reducing conditions to commercial FVIIa 15 (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 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 20 determined by FVIIa specific Elisa kit.

FVIIa N-terminal sequencing:

[0237] rFVII -CTP-CTP in activated or zymogene purified proteins is separated by SDS25 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 Nterminal 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

2017201513 06 Mar 2017 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 time assay (aPTT) is performed,. FVIII deficient plasma sample is substituted with rFVIIa (NovoSeven) or rFVIIa-(CTP)2, 300μ1 of FVII deficient human plasma is mixed with 100μ1 of FVIIa or rFVIIa-(CTP)2 at specific concentrations, or normal pull human plasma which is further diluted. Following 60 seconds incubation at 37°C. Tissue Factor (TF), CaCl₂, and phospholipids are added to the mixture. Clotting time in seconds is determined. Potency is assessed by comparing a dose response curve of rFVIIa-(CTP)₂ to a reference preparation consisting rhFVIIa or human pool normal plasma. One unit of FVIIa is defined as the amount of FVIIa which equals to the activity of 1ml human normal plasma., The aPTT Clotting activity of rFVII and rFVIIa-(CTP)₂ is measured on a coagulometer (Instrumentation Laboratory). The aPTT Clotting activity of rFVIIa and rFVIIa-(CTP)₂ is similar.

L5 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 (rFVIIa) 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.

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 coagulation factors Gia 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 phenprocoumn

2017201513 06 Mar 2017 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 No voSeven and rFVIIa-(CTP)2 are injected immediately 5 after phenprocoumn treatment but coagulation parameters are determined after 16 hours.

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 10 CTP modified rFVIIa.

FVII hemophilic mice model:

[0242] In order to assess the in-vivo activity model, FVII knockout mice are obtained and a 15 breeding colony is established. 10gg of either commercial recombinant hFVIIa (Novoseven) or rF VIIa-(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 normal plasma (5gg/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 20 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 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 rF VIIa-(CTP)2 is sufficient to control bleeding episodes and reduce the number of injections that are needed during surgical intervention. This recombinant protein 30 may be used as a long term prophylactic treatment.

Comparative Assessment of Purified FIX-CTP3 vs. FIX-CTP4 and FIX-CTP5

Study objective [0245] A comparative assessment of the pharmacokinetic parameters of FIX-CTP4 and FIXCTP5 versus FIX-CTP3 following a partial purification process.

2017201513 06 Mar 2017

Production of FIX-CTP₄ and FIX-CTP5 harvests [0246] FIX cDNA (OriGene RC219065) fused at the C-terminal to four or five tandem CTP sequences was expressed in Dg44 cells using Excellgene expression system in the presence of ng/L of vitamin K3 (Sigma, Mennadion). The harvests were collected (300ml), filtered and frozen.

Production of FIX-CTP3 harvest [0247] FIX-CTP3 was expressed in-house in CHO cells using pCI-DHFR vector, clone 196, BR-9 in the presence of 25 ng/L of vitamin K3 (Sigma). The harvests were collected and filtered. All FIX-CTP samples (3, 4 and 5 CTP) were purified only by Jacalin column because of a lack of material.

Determination of FIX antigen level [0248] FIX antigen level was determined using Human FIX ELISA kit (Affinity Biologicals; Cat. # FIX-AG RUO). The calculated protein concentration is the average of four independent 20 runs. FIX-CTP3 concentration was slightly higher as compared to the two additional versions (Table 12).

Table 12: FIX antigen level

1686.82	4644.11	1016.69	Av. (ng/ml)
160.07	925.63	225.41	SI)
9.49	19.93	22.17	%CV

FIX-CTP Coomassie stain and immune-blot [0249] FIX-CTP3, FIX-CTP4, and FIX-CTP5 harvests were loaded on 12% Tris-Glycine gel using Precision Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGE analysis was performed by Western immuno-blot using anti-CTP polyclonal Ab (Adar Biotech Production) or anti-Gla Ab (American Diagnostica).

2017201513 06 Mar 2017 [0250] As previously reported, FIX fused to three CTPs migrated at 80 kDa while FIX fused to four or five CTPs migrated at 85 KDa or 90 KDa, respectively. As expected, FIX-CTP4 and

FIX-CTP5 harvests from Excellgene showed very low levels of gamma carboxylation compared to FIX-CTP3 harvest, which was produced at Prolor (Figure 12). All variants showed much cleaner band profiles (Figure 13), suggesting an improved purity.

Determination of FIX chromogenic activity [0251] A comparative assessment of the in vitro potency of fully purified (HA column) FIX10 CTP3, FIX-CTP4, and FIX-CTP5 versus human pool normal plasma was performed using a commercially available chromogenic activity test kit, BIOPHEN (Hyphen BioMed 221802). All samples were serially diluted, and the potency was assessed by comparing a doseresponse curve to a reference preparation of normal human plasma. The reduced chromogenic activity of FIX-CTP4 and FIX-CTP₅ (Figure 14) as compared to plasma can be a consequence 15 of improper post-transcriptional modifications of FIX proteins, e.g. inappropriate gamma carboxylation and pro-peptide cleavage or, alternatively, due to the addition of CTP cassettes.

[0252] The fluctuation in the FIX-CTP4 and FIX-CTP5 activity (Table 13) might be caused by inappropriate quantitation capabilities of the FIX ELISA due to CTP masking of the antigen 20 site.

Table 13: Sample/plasma EC50 ratio

Plasma

3 CTP Final HA ί

5.35 4 CTP Final HA

2.73 5 CTP Final HA |

Pharmacokinetic study [0253] Jacalin-purified FIX-CTP3, FIX-CTP4, and FIX-CTP₅ (Group A, B and C, respectively) were administered in a single intravenous injection to Sprague-Dawley rats (six rats per treatment group) at a dose of 250 pg/kg body weight. Blood samples were drawn retro-orbitally from 3 rats alternately at 0.083, 0.5 2, 5, 8, 24, 48, and 72 hours post-dosing (Table 14). Citrated plasma (0.38%) was prepared immediately after sampling and stored at 2017201513 06 Mar 2017

20°C until analysis.

Table 14: PK study plan of operation

Dose

Treatm	i s No. of \| \|	\| Injected
i s \| Dose	Level
ent	i Treatment i animals/		\| Vol.
	ί i Route	\| (pg per	5
Group	s i group	5	i (pl)

Cone.

(pg/ml) <

<

J

FIXTime-Points (hr post-dose)

CTP*3

IV

200

250

0.083, 0.5, 2, 5, 8, i Jacalin 40

Thx-..............

24, 48, 72,

CTP*4

IV

200

250

0.083, 0.5, 2, 5, 8,

Jacalin 40

24, 48, 72,

FIXCTP*5

IV

200

250

0.083, 0.5, 2, 5, 8,

Jacalin 40

24, 48, 72 [0254] FIX concentration in plasma samples were quantified using human FIX ELISA kits (Affinity Biologicals). The pharmacokinetic profile was calculated and is the mean of 3 animals at each time point. Terminal half-lives were calculated using PK Solutions Software.

Table 15 below summarizes the calculated FIX concentrations at the different sampling time points.

Table 15: Calculated FIX concentrations

2017201513 06 Mar 2017

Time (hr)

0.083

0.5

Γ48

Av. 3 CTP | SD

Αν. 4 CTP SD Αν. 5 CTP | SD ng/ml j 3 CTP j ng/ml ΐ 4 CTP j ng/ml

1087.82

T774JL8 j 72.39

ΓδόΑΪ i 904.54

1'736782

06

66.93

562.23

357.44 ΐ 3.70 γ—

627.09

431.23

32.47

29.41

1097.23

998.79

576.49

5CTP

24

70.43 j 14.02 ΐ'27'36 j 239.20 f77O8™ Jiiill Tn®

327.46

26

394.96

48 j 4.26 f'2O2 T L48

107.38

T39®' j 5.18 'Π'®

Tl55 fl42A2 | 53?66 | 16.13 'Ρ'?33“ summary of the PK parameters are presented in Table 16 below [0255] The PK profile and and in Figure 15. A full PK analysis profile at all time points suggested that addition of 4 or 5 CTP cassettes to FIX did not increase its half-life as compared to FIX-CTP3. The AUC values were not statistically significant.

Table 16: PK profile and a summary of the PK parameters

Half-life (hr)

22.02

23.96

Vd (ml/kg)

700.76

586.02

494.89

CL (ml/hr/kg)

23.77

18.45

14.32 shown to have very low FIX concentrations, [0256] Since 96 hr post-dosing samples were which were at the lower limit of quantification of the assay, the terminal half-life was recalculated providing a more precise and scientifically appropriate calculation (Table 17). According to this calculation, even smaller differences were obtained between the half-life of FIX-CTP3, FIX-CTP4, and FIX-CTP₅.

Table 17: Recalculated terminal half-life

Half-life (hr) 15.38

16.63

16.04

2017201513 06 Mar 2017

Conclusions [257] In this study, the pharmacokinetic parameters and potential clotting activity of FIX5 CTP3, FIX-CTP4, and FIX-CTP5 were assessed. Fusion of 4 and 5 CTPs to FIX did not provide a superior or improved half-life extension, as compared to FIX-CTP3, and reduced chromogenic activity was observed. Table 18 below summarizes the percent improvement of half-life for the different FIX-CTP fused variants (1 to 5 CTPs). Fusion of CTP to FIX improved its pharmacokinetic behavior, but, unpredictably, this improvement was limited.

Surprisingly, following fusion of 3, 4 or 5 CTPs in tandem to FIX, a similar half-life value was calculated.

Table 18: Summary of the percent improvement of half-life

FIX Version

T'A (8-72hr)l % increase s rhFIX vs. 1 CTP M12 ]

1CTP vs.

2CTP

141

J

2( I P vs.

3CTP

4( I P vs

5( TP [0258] These data demonstrate that fusion of 3 CTPs to FIX produces a maximal improvement in protein half-life, confirming that FIX-CTP3 is the optimal variant in terms of half-life, structure and potential clotting activity for further clinical development.

Comparative assessment of Purified FVII-CTP3, FVII-CTP4, and FVII-CTP5

Study objective [0259] Comparative assessment of pharmacokinetic parameters and clotting activity of FVII35 CTP₄ and FVII-CTP₅ versus FVII-CTP3.

Production of FVII-CTP4 and FVII-CTP5 harvests [0260] FVII cDNA fused at the C-terminal to four or five tandem CTP sequences was expressed in Dg44 cells using the Excellgene expressing system in the presence of 20 pg/L of vitamin K3 (Sigma, Mennadion). The harvest was collected (300 ml), filtered and frozen.

2017201513 06 Mar 2017

Production of FVII-CTP₃ harvest [0261] FVII-CTP3 was expressed in-house in mammalian expressing system, CHO cells, using pCI-DHFR vector. Stable transfected pool #71 was grown in shake flasks, in the 5 presence of 25 ng/L of vitamin K3 (Sigma). The harvests were collected and filtered. All

FVII-CTP harvests (3, 4 and 5 CTPs) were concentrated and dialyzed against TBS (50 mM Tris, 150mM NaCl, pH 7.4) using Pellicon XL MWCO lOkDa.

Determination of FVII antigen level [0262] FVII antigen level was determined using Human FVII ELISA kit (Zymotest HyPhen) (Table 19). The calculated protein concentration is the average of two independent runs.

Table 19: FVII antigen level

SD	44789.5	3248.7	5309
	—..........................

FVII-CTP immune-blot [0263] FVII-CTP3, FVII-CTP4, and FVII-CTP5 harvests were loaded on 12% Tris-Glycine gel (expedeori) using Precision plus dual color protein marker (Bio-Rad). The SDS-PAGE 20 analysis was performed by Western immune-blot using anti-CTP polyclonal Ab (Adar

Biotech Production) or anti-Gla Ab (American Diagnostica).

[0264] FVII fused to three, four and five CTP migrated at 80, 90 and lOOkDa, respectively.

As expected, FVII-CTP4 and FVII-CTP5 harvests from Excellgene contain low gamma 25 carboxylation content as compared to FVII-CTP3 harvest which was produced at Prolor since the production process wasn't optimized (Figure 16).

Comparative assessment of FVII in vitro potency [0265] A comparative assessment of the in vitro potency of HA purified (highly gamma carboxylated fraction) FVII-CTP3, FVII-CTP4, and FVII-CTP5 versus normal human pool plasma was performed using a commercially available chromogenic activity test kit, BIOPHEN (Hyphen BioMed 221304). All samples were serially diluted, and the potency was assessed by comparing a dose-response curve to a reference preparation consisting of normal human plasma. FVII-CTP3 and FVII-CTP5 demonstrated chromogenic activity lower than pooled normal plasma (Figure 17). FVII-CTP4 demonstrated higher activity as reflected by

EC50 ratios, compared to FVII-CTP3 and FVII-CTP₅ (Table 20).

2017201513 06 Mar 2017

Table 20: FVII In Vitro Clotting Activity

05

Plasma

FVII 3CTP

0.12

2.72

03

71

FVII 5CTP

0.06

1.35

FVII In Vitro Clotting Activity [0266] Factor VII (FVII) activity assay, which was performed in Sheba Medical Center, the

Israel National Coagulation Center, is a prothrombin (PT)-based assay using immunoadsorbed plasma deficient in Factor VII (Siemens). The PT reagent is innovin, and the assay is performed in the Sysmex® CA 1500 instrument. FVII normal range is within 55-145%.

[0267] Since the normal level of circulating FVII in the body is around 0.5 pg/ml, FVII-CTP3 and FVII-CTP₅ harvests exhibit 3-fold reductions in their coagulation activity versus normal human pool plasma; this result correlates with the obtained chromogenic activity (Table 21).

Table 21: FVII In Vitro Chromogenic Activity

2017201513 06 Mar 2017

[0268] The FVII-CTP4 harvest exhibits a 3-fold increase in its potential coagulation activity 5 versus normal human pool plasma as observed in the chromogenic activity assay (Table 21).

The activity percentage of FVII-CTP4 is much higher compared to activity percentage of FVII-CTP3 and FVII-CTP5. Methodological limitations of the EEISA method may limit the accuracy of Ag level calculations of FVII-CTP4.

Pharmacokinetic study [0269] Two pharmacokinetic studies were performed in order to determine the FVII-CTP3, FVII-CTP4, and FVII-CTP5 pharmacokinetics (PK) parameters. During the first study, FVIICTP₃, FVII-CTP4, and FVII-CTP₅ (Group A, B and C, respectively) were administered in a single intravenous injection to Sprague Dawley rats (six rats per treatment) in a dose of 250 15 pg/kg body weight. Blood samples were drawn retro-orbitally from 3 rats alternately at 0.083,

0.5 2, 5, 8, 24, 48, 72 and 96 hours post-dosing (Table 22). Citrated plasma (0.38%) was prepared immediately after sampling and stored at -20°C until analysis.

2017201513 06 Mar 2017

Table 22: Pharmacokinetic Study Design - Concentrated Harvest

Treat m ent Group	Test Article	No. of animals/ group/ point	Dose Rout IIIQII	Dose Level (pg per animal)	Injected Vol. (pl)	Cone, (pg/ml)	Time-Foints (hours post-dose)
A	FVII iiilii·	6	lliiiil	50	200	250	0 (Pre-dose) 0.083,
B	CTPM	ϊΒΐϊΐϊΐϊΐϊόΐ	ijilll	50	iiSiii	250	0 (Pre-dose) 0.083, iiiiiiiiiiiiiiiiiiiiii 2,5,8,24,4872,96
C	FVII-	6	iv	50	200	250	0 (Pre-dose) 0.083, 0.5,

[0270] FVII concentration in plasma samples were quantified using human FVII Elisa kits (Zymutest FVII-Biophen). The pharmacokinetic profile was calculated and is the mean of 3 animals at each time point. Terminal half-life values were calculated using PK Solutions 2.0 Software. Table 23 below summarizes the calculated FVII concentrations at the different sampling time points. The PK profile (Figures 18-19) and a summary of the PK parameters 10 (Table 22) are also presented below. FVII-CTP5 demonstrated a superior profile as compared to FVII-CTP3 and FVII-CTP4 (Table 24).

Table 23: First Pharmacokinetic Study - FVII Concentrations

0.083	4214		3600		4888	504
						____
0.5	3386	892	5213	1682	5384
	........1138...........	............219......	......3603...........	Illi··		...........289...............
5	'1390		2726	............_™	2480	_..............
8	'333		'Ϊ349	..........................	23Ϊ6	......................™.............................
		............................	...............................	...............................	V»W»W»W»W»W»W»W»W»W»W·	W»W»W»W»W»W»W»W»W»W»W»W»
24	133	12	476	98	788	34
48			165		384
72			91
						™___
96	26	1	42	8

2017201513 06 Mar 2017

Table 24: Pharmacokinetic Analysis

half-life (8-72hr) (hr)	13.3	16.6	17.7
AUC(ng/\|₁r/mi)(8-72hr)	.............18374.6..............	..............51'224.4	72954.2
Vd (ml/kg)(8-72hr)	-----	----	----
	—	............................................

[0271] The addition of four or five CTPs significantly elongated FVII half-life as compared to

CTPs by 2- and 3-fold, respectively (Table 24). This superiority was more significant in the initial part of the study (0.083-8 hr), suggesting a potential improved protein recovery and reduced extra vascular clearance. AUC following FVII-CTP₄ and FVII-CTP₅ administration increased by 3- and 4-fold, respectively, versus FVII-CTP3. Clearance was also reduced while 10 adding 4 and 5 CTPs to FVII (Table 24).

[0272] As observed in the study, the addition of four and five CTPs significantly elongated FVII half-life as compared to 3 CTPs, both in the initial and terminal half-life. The half-life values in the first and second study are different due to a different analysis approach which 15 was effected by the dose and study duration, nevertheless the overall trend was maintained.

The AUC following FVII-CTP4 and FVII-CTP5 administration increased by 2.5- and 7-fold, respectively, versus FVII-CTP3.

Conclusions [0273] In this study, the PK parameters and potential clotting activity of FVII-CTP3, FVIICTP₄, and FVII-CTP5 were assessed. Fusion of 4 and 5 CTPs to FVII provided a superior and improved half-life, exposure and reduced clearance as compared to FVII-CTP3 while maintaining a similar chromogenic and in vitro clotting activity. These results were observed 25 at different concentrations of protein and were consistent for both harvest and purified protein. While evaluating the overall effect of fusion of CTP at the C terminus to FVII, fusion of 1-5 CTPs considerably increased the half-life and AUC of FVII in a CTP proportional manner, suggesting that as the CTP portion of the molecule increases, FVII longevity and stability is significantly improved while maintaining its initial in vitro clotting activity, as

2017201513 06 Mar 2017 summarized in Table 25 herein below.

Table 25:

FVII vs. FV11-CTP₂	268	200
I VII-CTIb vs. 1 VII-CTPj	67	-----

pyjj^Yp—	ό

[0274] As previously reported, FVII half-life correlates with the half-life of the activated form of FVII (FVIIa) both in humans and animals. Therefore, it is likely that a similar improvement in half-life will be obtained for the activated versions following CTP fusion.

Claims

2017201513 24 Jun 2019

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A CTP-modified polypeptide consisting of a coagulation factor and one to five chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or a coagulation factor and one to three chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.
2. The CTP-modified polypeptide of claim 1, wherein the amino acid sequence of said Factor VII or said Factor Vila coagulation factor is set forth in amino acids 39-444 of SEQ ID NO: 9 or a sequence at least 95% homologous thereto, or wherein the amino acid sequence of said Factor IX or said Factor IXa coagulation factor is set forth in amino acids 47-461 of SEQ ID NO: 17 or a sequence at least 95% homologous thereto.
3. The polypeptide of any one of claims 1-2, wherein the sequence of at least one CTP comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.
4. The polypeptide of any one of claims 1-3, wherein at least one CTP comprises at least one glycosylation site.
5. The polypeptide of any one of claims 1-4, wherein at least one CTP is attached to said coagulation factor via a linker.
6. The polypeptide of claim 5, wherein said linker is a peptide bond.
7. A pharmaceutical composition comprising the polypeptide of any one of claims 1-6.
8. A method of extending the biological half-life of a coagulation factor, comprising the step of attaching one to five chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or one to three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, thereby improving the biological half-life of said coagulation factor compared to an unmodified

2017201513 24 Jun 2019 coagulation factor, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.
9. A method of improving the area under the curve (AUC) of a coagulation factor, comprising the step of attaching one to five chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or one to three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, thereby improving the AUC of said coagulation factor compared to an unmodified coagulation factor, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.
10. A method of reducing the dosing frequency of a coagulation factor, comprising the step of attaching one to five chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or one to three chorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, thereby reducing the dosing frequency of said coagulation factor compared to an unmodified coagulation factor, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.
11. The method of any one of claims 8-10, wherein the amino acid sequence of said Factor VII or said Factor Vila coagulation factor is set forth in amino acids 39-444 of SEQ ID NO: 9 or a sequence at least 95% homologous thereto, or wherein the amino acid sequence of said Factor IX or said Factor IXa coagulation factor is set forth in amino acids 47-461 of SEQ ID NO: 17 or a sequence at least 95% homologous thereto.
12. The method of any one of claims 8-11, wherein the sequence of at least one CTP comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

2017201513 24 Jun 2019
13. A method of treating hemophilia, an acquired condition that causes bleeding or excessive bleeding or bruising, or any combination thereof, in a subject, the method comprising administering to said subject an effective amount of:

a CTP-modified polypeptide, or a pharmaceutical composition thereof, consisting of a coagulation factor and one to five chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or a coagulation factor and one to three chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.
14. The method of claim 13, wherein the amino acid sequence of said Factor VII or said Factor Vila coagulation factor is set forth in amino acids 39-444 of SEQ ID NO: 9 or a sequence at least 95% homologous thereto, or wherein the amino acid sequence of said Factor IX or said Factor IXa coagulation factor is set forth in amino acids 47-461 of SEQ ID NO: 17 or a sequence at least 95% homologous thereto.
15. The method of any one of claims 13-14, wherein the sequence of at least one CTP comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.
16. Use of CTP-modified polypeptide or a pharmaceutical composition thereof in the preparation of a composition for treating hemophilia, an acquired condition that causes bleeding or excessive bleeding or bruising, or any combination thereof in a subject, said polypeptide consisting of:

a coagulation factor and one to five chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor VII or Factor Vila, or a coagulation factor and one to three chorionic gonadotropin carboxy terminal peptides (CTPs) attached to the carboxy terminus of said coagulation factor, wherein said coagulation factor is Factor IX, or Factor IXa, wherein the attachment of any CTPs to the amino terminus of said coagulation Factor VII, Factor Vila, Factor IX, or Factor IXa is excluded.

2017201513 24 Jun 2019
17. The use of claim 16, wherein the amino acid sequence of said Factor VII or said Factor Vila coagulation factor is set forth in amino acids 39-444 of SEQ ID NO: 9 or a sequence at least 95% homologous thereto, or wherein the amino acid sequence of said Factor IX or said Factor IXa coagulation factor is set forth in amino acids 47-461 ofSEQIDNO: 17 orasequence at least 95% homologous thereto.
18. The use of any one of claims 16-17, wherein the sequence of at least one CTP comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.
19. A method of increasing patient compliance in the use of a coagulation factor therapy, comprising providing to a subject in need thereof the CTP-modified coagulation factor polypeptide of any one of claims 1-6, thereby increasing patient compliance.
20. The method of claim 19, wherein said method comprises the step of administering a once a week dose of said coagulation factor polypeptide.