RU2794350C1 - Improved factor ix function protein and its conjugate and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to coagulation factor IX (FIX) conjugates, and can be used in medicine for the prevention or treatment of diseases associated with congenital or acquired FIX deficiency. A conjugate containing a FIX fusion protein and a 40 kDa polyethylene glycol attached to the C-terminus of the fusion protein is proposed. The fusion protein contains an active FIX group and a fusion partner (FP) and is represented by the structure FIX-L1-Fс-L2-P, where FIX is a full-length human coagulation factor IX, the fusion partner is an immunoglobulin Fc fragment from human IgG1 with point mutation N297A, L1 and L2 are peptide linkers, and P is an amino acid fragment containing the LPETGG sortase recognition site.

EFFECT: invention provides obtaining a FIX-based conjugate with an increased half-life in the bloodstream.

9 cl, 6 dwg, 2 tbl, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of Chinese Patent Application No. 201910591280.0 filed with the National Intellectual Property Office of China on July 02, 2019, the contents of which are incorporated by reference in their entirety.

FIELD OF TECHNOLOGY

[0002] This invention relates to a biologically active fusion protein with extended in vivo half-life and its polymer conjugate, and in particular relates to human coagulation factor IX with extended in vivo half-life, a pharmaceutical composition containing them, and their use in the treatment of hemorrhagic diseases (such as hemophilia B).

PRIOR ART

[0003] Hemophilia, a severe coagulopathy caused by a deficiency of certain blood clotting factors, is a group of hereditary hemorrhagic diseases including hemophilia A (deficiency of factor VIII, an antihemophilic globulin) and hemophilia B (deficiency of factor IX, a plasma component of thromboplastin).

[0004] Hemophilia B is a severe coagulopathy caused by deficiency of human coagulation factor IX (hFIX). The gene encoding hFIX (hFIX) is located in the Xq27.1 region at the far end of the long arm of the human X chromosome and is 34 kb in total length, including 8 exons and 7 introns. The total length of the hFIX cDNA is 2802 bp, of which 1383 bp is in the coding region (Genbank accession number: NM_000133). Patients with a severe form of the disease usually have less than 1% of normal factor IX activity, they often suffer from spontaneous bleeding, which causes hematomas in the muscles or joint deformities.

[0005] Recombinant compositions for the prevention and treatment of hemophilia are gaining acceptance in medicine and among patients due to their safety. In the case of hemophilia B, the administration of factor IX preparations (which is now usually a recombinant factor IX protein expressed in vitro) is the only effective treatment to maintain the required level of clotting factor IX in the patient's body. However, since peptide drugs generally have a short half-life, poor physical and chemical stability, and are easily degraded by various proteases in the body, these drugs require multiple injections throughout the day, resulting in frequent drug administration becoming a great physical, psychological, and financial burden for patients. limiting their adherence to treatment. Thus, there is an unmet need in the art for new drugs to increase the plasma half-life of the drug and increase its systemic exposure.

SUMMARY OF THE INVENTION

[0006] After many years of research, the inventors have created a fusion protein of clotting factor IX and Fc, which can be further modified to form a conjugate by a group including PEG, and has a significantly longer circulating half-life compared to FIX polypeptides.

[0007] In particular, in a first aspect of the invention, there is provided a clotting factor IX (FIX) fusion protein comprising a clotting factor IX active group and a fusion partner (FP) capable of increasing the half-life of the fusion protein, wherein the clotting factor IX active group and the fusion partner merge linked directly or through the first linker L1; preferably, the first linker L1 is a peptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids; more preferably, the first linker L1 contains a flexible peptide and/or a rigid element, and the flexible peptide is a peptide containing glycine (Gly, G), series (Ser, S), alanine (Ala, A) and/or threonine (Thr, T), for example, a flexible peptide is (GS) _m (GGS) _n (GGGS) _o (GGGGS) _p , where m, n, o and p are independently selected from integers from 0 to 50, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably not all of m, n, o and p are 0.

[0008] In one embodiment of the invention, the clotting factor IX active group is derived from human clotting factor IX, such as full-length or truncated clotting factor IX; a full-length or truncated human factor IX may contain one or more amino acid mutations, provided that it still retains factor IX activity, for example, the active group of factor IX contains the amino acid sequence shown in SEQ ID NO: 1, or is at least identical at least 80%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence given in SEQ ID NO: 1.

[0009] In another embodiment, the hard element is a peptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more prolines (Pro, P), preferably, the hard element contains the amino acid sequence selected from the group consisting of:

VAPPPALPAPVRLPGPA (SEQ ID NO: 3),

VAPPPALPAVAPPPALPA (SEQ ID NO: 4),

VAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPA (SEQ ID NO: 5),

VAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPA (SEQ ID NO: 6) and

VAPPPALPAVAPPPALPAGSVAPPPALPAVAPPPALPA (SEQ ID NO: 7).

[0010] Preferably, the first L1 linker is selected from the group consisting of:

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPA (SEQ ID NO: 8),

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPA (SEQ ID NO: 9),

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPA (SEQ ID NO 10),

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPA (SEQ ID NO: 11) and

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAGSVAPPPALPAVAPPPALPA (SEQ ID NO 12).

[0011] In yet another embodiment, the fusion partner is a full-length form, a truncated form, or a variant of an immunoglobulin, albumin, transferrin, or XTEN Fc fragment; for example, the fusion partner is derived from the Fc fragment of a human immunoglobulin; preferably, the Fc fragment consists of one to four domains selected from the group consisting of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; preferably, the Fc fragment is derived from an IgG Fc fragment of IgA, IgD, IgE or IgM, more preferably from an IgG Fc fragment; more preferably the Fc is derived from an IgG1, IgG2, IgG3 or IgG4 Fc, and more preferably the IgG Fc mediates less ADCC (antibody dependent cellular cytotoxicity) and/or CDC (complement dependent cytotoxicity) and/or has greater affinity binding to FcRn.

[0012] In yet another embodiment, when the fusion partner is also linked directly or via a second L2 linker to a P peptide containing a sortase recognition site, and the sortase is, for example, sortase A or sortase B; preferably, P contains a major LPXTG sortase A recognition site, where X is any amino acid and the P sequence is, for example, LPETG, LPETGG or LPETGGG; more preferably, the P amino acid sequence further comprises an affinity tag attached to a sortase recognition site, and the P amino acid sequence is, for example, LPETGGHHHHHH or LPETGGWSHPQFEK.

[0013] In another embodiment, the second L2 linker is a peptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids; preferably, the second linker L2 is a flexible peptide fragment containing glycine (Gly G), series (Ser, S), alanine (Ala, A) and/or threonine (Thr, T), such as (GS) _w (GGS) _x (GGGS) _y (GGGGS) _z where w, x, y, and z are independently selected from integers from 0 to 50, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably not all of w, x, y and z are 0; preferably, the second L2 linker is selected from the group consisting of GGGGS (SEQ ID NO: 14), GGGGSGGGGS (SEQ ID NO: 15) and GSGGSGGGGS (SEQ ID NO: 16).

[0014] In another embodiment, the fusion protein has the structure FLX-L1-FP-L2-P, where FIX, L1, L2, FP and P have the same definitions as in the above embodiments, and where one or both of L1 and L2 may be missing.

[0015] In another embodiment, the fusion protein contains the sequence shown in SEQ ID NO: 18, 20, 22, 24, 26, or 28, or is encoded by the nucleic acid sequence shown in SEQ ID NO: 19, 21, 23, 25, 27 or 29.

[0016] In addition, the present invention also provides a nucleic acid molecule encoding a fusion protein according to any of the above embodiments, and an expression vector containing the nucleic acid molecule.

[0017] In a second aspect of the present invention, there is provided a conjugate formed by attaching a hydrophilic polymer to the end of a fusion protein according to any of the embodiments of the first aspect. In the conjugate, one, two or more hydrophilic polymers are attached to the end of the fusion protein according to any of the embodiments of the first aspect, preferably, the hydrophilic polymer is attached to the sortase recognition site by a transpeptidation reaction; in particular, the fusion protein may be in the form of a monomer or a dimer.

[0018] In one embodiment, one, two or more hydrophilic polymers are independently selected from a polysaccharide and a polyalkylene glycol such as polypropylene glycol and polyethylene glycol; the polyalkylene glycol may be capped at the end, for example capped with an alkoxy group such as a methoxy group; and/or the polyalkylene glycol is linear or branched, for example the polyalkylene glycol is branched, such as a branched polyethylene glycol, in particular a branched polyethylene glycol copied with a methoxy group; the molecular weight of the polyalkylene glycol may be 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 or greater, or 160 or greater kDa, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kDa, or have a value between any two values; and preferably, the molecular weight of the hydrophilic polymer is 20 kDa or 40 kDa.

[0019] In a third aspect of the present invention, there is provided a method for preparing a conjugate of the second aspect, comprising contacting the fusion protein of the first aspect of the present invention with a sortase (such as sortase A or sortase B) and a hydrophilic polymer, wherein one end of the hydrophilic polymer has an amino group, which can be amidated by sortase.

[0020] In one embodiment, the N-terminus of the hydrophilic polymer contains poly-Gly such as GGGAA.

[0021] In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising an effective amount of the fusion protein described in the first aspect and/or the conjugate described in the second aspect, and optionally a pharmaceutically acceptable carrier.

[0022] In one embodiment, the pharmaceutical composition is used to prevent and/or treat hemorrhagic disease, where the hemorrhagic disease is preferably selected from hemorrhagic diseases in patients with congenital or acquired FIX deficiency and spontaneous or postoperative bleeding in patients with hemophilia B.

[0023] In yet another embodiment, the pharmaceutical composition is in the form of a liquid preparation or a lyophilized preparation.

[0024] In a fifth aspect of the present invention, there is provided the use of a fusion protein, conjugate or pharmaceutical composition according to the above aspects for the manufacture of a medicament for the prevention and/or treatment of a hemorrhagic disease, wherein the hemorrhagic disease is preferably selected from hemorrhagic diseases in patients with congenital or acquired FIX deficiency and spontaneous or postoperative bleeding in patients with hemophilia B.

[0025] In a sixth aspect of the present invention, there is provided a method for preventing and/or treating a hemorrhagic disease, comprising administering a fusion protein, conjugate, or pharmaceutical composition according to the above aspects to a subject in need thereof. The hemorrhagic disease is preferably selected from hemorrhagic disease in patients with congenital or acquired FIX deficiency and spontaneous or postoperative bleeding in patients with hemophilia B.

[0026] In a seventh aspect of the present invention, a kit is provided that includes a fusion protein, conjugate, or pharmaceutical composition according to the aspects set forth above, and optionally instructions for use.

[0027] In the eighth and ninth aspects of the present invention, respectively, a nucleic acid molecule encoding a fusion protein according to the first aspect and an expression vector containing the nucleic acid molecule are provided.

BRIEF DESCRIPTION OF GRAPHICS

[0028] In FIG. 1A shows the structure of the FIX/PACE (furin) bicistronic expression vector.

[0029] In FIG. 1B shows a map of the plasmid pcDNA3.1-Hyg-VKGC.

[0030] In FIG. 2 shows the results of purification of the FIX-C1-Fc-L2-P fusion protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (A) and size exclusion high performance liquid chromatography (SEC-HPLC) (B).

[0031] In FIG. 3 shows the results of the in vitro activity of the FIX-L1-FC-L2-P fusion protein and the FIX-C1-Fc-L2-P conjugate.

[0032] In FIG. 4 shows the results of the SEC-HPLC analysis of the 40 kDa conjugate after the PEG conjugation reaction.

[0033] In FIG. 5 shows the results of the SEC-HPLC analysis of the conjugate after purification and isolation.

[0034] In FIG. 6 shows the results of a study of the pharmacokinetics of the FIX-C1-Fc-L2-PEG conjugate in a model of hemophilia in mice, and the table below shows T1 / 2 (half-life in the final phase), Cmax, AUC0-inf (area under the curve), MRT ( mean retention time) and CL (clearance rate).

INFORMATION CONFIRMING THE POSSIBILITY OF IMPLEMENTING THE INVENTION

[0035] For a clearer understanding of the objectives, technical solutions and advantages of the present invention, a more detailed description of the present invention will be given below with reference to the drawings and embodiments. It is obvious that the described embodiments are only a part of all possible embodiments of the present invention. All other embodiments that a person skilled in the art will come to on the basis of embodiments of this invention without inventive activity, will be included in the scope of legal protection of this invention.

Definitions

[0036] Coagulation factor IX, also referred to as factor IX, FIX, is a vitamin K-dependent clotting factor with structural similarities to factor VII, prothrombin, factor X, and protein C.

[0037] As used herein, the term "coagulation factor IX active group" refers to a fusion protein group that exhibits factor IX clotting activity. The term "clotting factor IX" may refer to a natural wild-type protein (wild-type amino acid sequence is given in SEQ ID NO: 1), and the reference coding nucleic acid sequence can be found in the Genbank database under accession number: NM_000133, and in particular, is given in SEQ ID NO: 2), and also encompasses variant forms thereof, e.g., a protein variant resulting from one or more (e.g., 2, 3, 4, or 5) amino acid substitutions, deletions, or insertions, while retaining clotting factor IX activity. .

[0038] MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFDNPCGRVSVSQTSKLTRAETVFPNDRVDAFTVANSTE PGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECARYMKGKYGIK (NOWWITKIDVSFLTGIISWGEECARYMKGKYGIK1).

[0039] ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATCACCATCTGCCTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAATGTATGGAAGAAAAAGTGTAGTTTTGAAGATTTGAAAGAGA GAAGCAGTATGTTGATGGAGATCAGTGTGTGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGAATTTGAAGGAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTACTGCTCCTGTACTGAGGCGATATCAGACTTGCAGATCATG TGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGGATTTTGAATGGTAAAGTTGATGTGCATTCTGTGGAAGGCTCTAGTTACTACTACT ACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTTGAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTAGCATTGCTGACAAGGAATACACGAACATCTTCCTAGGCTATTGGATCTGAGCTAGGCTAACCTTGGAACT TTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCGATTAACTGGAATTATTAGCTGGGGTGAAGGAGTGATACTCAATGAAAGGCAAATAAGGATATATACCTAG AAACAAAGCTCACTTAA (SEQ ID NO: 2).

[0040] In one embodiment, the protein variant is at least 90% identical to the sequence of SEQ ID NO: 1. In another embodiment, the protein variant is at least 95%, e.g., 96%, 97%, 98%, or 99% identical to the sequence SEQ ID NO: 1. In this specification, unless otherwise indicated, reference to any particular position refers to the corresponding position in SEQ ID NO: 1.

[0041] This invention is not limited to the sequences described therein. Variants of FIX are, for example, those set forth in US5521070 (where the tyrosine in the first position is substituted with alanine) and WO2007/135182 (where one or more naturally occurring amino acid residues in FIX are substituted with cysteine residues). The cited documents are incorporated herein in their entirety by reference. Thus, FIX variants are well known in the art, and this invention embraces forms of variants that are known or that will be created or discovered in the future.

Fusion protein

[0042] The term "fusion partner", FP, refers to a polypeptide that is fused to a target polypeptide (a polypeptide in which it is desired to increase the circulating half-life). The fusion partner can influence the functional properties of the fusion protein through several different mechanisms, such as increasing the in vivo half-life of the target polypeptide.

[0043] In one embodiment, the fusion partner slows FIX clearance in vivo by interacting with the neonatal Fc receptor (FcRn). In another embodiment, the fusion partner is an immunoglobulin Fc fragment, albumin, transferrin, XTEN, or a portion thereof.

[0044] An immunoglobulin Fc fragment is safe for use as a pharmaceutical carrier because it is a biodegradable polypeptide that can be metabolized in the body. In addition, compared with the whole immunoglobulin molecule, the Fc region of the immunoglobulin has a relatively low molecular weight, which is favorable for the preparation, purification and production of the conjugate. Since the Fc fragment does not contain a Fab fragment (its amino acid sequence varies depending on the subclass of the antibody and is therefore very heterogeneous), it is expected that the Fc region of an immunoglobulin can significantly increase the homogeneity of the substance and have low antigenicity.

[0045] The term “Immunoglobulin Fc region/fragment” refers to a protein that includes the 2nd heavy chain constant region (CH2) and the 3rd heavy chain constant region (CH3) of an immunoglobulin, but does not include the heavy chain and light chain variable regions. immunoglobulin. Also, the Fc fragment used in the present invention may further comprise the hinge region of the heavy chain constant region and/or all or part of the 1st heavy chain constant region (CH1) and/or the 1st light chain constant region (CL1), not including the heavy and light chain variable regions, provided that it has a physiological function substantially similar to or superior to that of the natural form.

[0046] In some embodiments, an Fc fragment of an immunoglobulin of the present invention may comprise 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) CH1 domain and CH2 domain; 3) CH1 domain and CH3 domain; 4) CH2 domain and CH3 domain; 5) a combination of one or more domains with (all or part of) the immunoglobulin hinge region; or 6) a dimer of any heavy chain constant region and light chain constant region domains.

[0047] Also, an immunoglobulin Fc fragment may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or be obtained by a combination or hybrid thereof. Preferably, it is derived from IgG or IgM (since they are the two most abundant proteins in human blood). In one embodiment, given that IgG antibodies have a longer half-life, the Fc domain of IgG is preferred.

[0048] The Fc fragment can also be modified to improve other functions, for example, to change through mutations its ability to induce immune responses, such as the ability to bind to complement and / or some Fc receptors, since this is not necessary when using Fc to increase half-life time. Mutations at positions 234, 235, and 237 of the IgG Fc region are known to generally result in reduced binding to FcγRI receptors and may also result in reduced binding to FcγRIIa and FcγRIII receptors, but these mutations do not alter its binding to FcRn receptors and therefore may still contribute to a long circulating half-life due to recycling by endocytosis. Preferably, the modified Fc fragment of an IgG fusion protein of the invention contains one or more of the following mutations: L234A, L235E and G237A mutations result in decreased affinity for certain Fc receptors, and A330S and P331S result in decreased Clq-mediated complement fixation.

[0049] In one embodiment, the clotting factor IX fusion protein is further linked to a fragment containing a sortase recognition site, ie. "peptide P", described in this invention.

[0050] For the first time, it was found that "sortase", including, in particular, sortase A and sortase B, has the function of attaching bacterial surface proteins to the cell wall. They found that the "sorting signal" of surface proteins in most cases has three key components: LPXTG sequences, a hydrophobic sequence, and a tail of positively charged residues. The sequence of LPXTG is very conserved and can be recognized by sortase, whereupon the cysteine (Cys) of sortase acts as a nucleophilic group, attacking the peptide bond between the threonine and glycine of the C-terminal motif of the LPXTG substrate (such as a surface protein), causing the peptide bond to be cleaved (acylation), which in turn leads to the formation of an acyl intermediate, and finally, the peptide covalently attaches to the cell wall or pili subunit to perform functions (deacylation).

[0051] In one embodiment of the invention, sortase A is used for site-specific PEG attachment. In a particular embodiment, the sortase A recognized LPETG LPETGG and LPETGGG sites are used. In another particular embodiment, various affinity tags can be added to the recognition site, such as LPETGGHHHHHH, LPETGGWSHPQFEK, etc.

[0052] In another embodiment, the clotting factor IX fusion protein of this invention is produced by recombinant methods, including, for example, expression of the protein in suitable prokaryotic or eukaryotic host cells, and the clotting factor IX fusion protein of this invention is isolated by standard methods. For example, a nucleotide sequence encoding a peptide can first be synthesized by chemical synthesis methods, and then the sequence can be cloned into a suitable expression vector for expression under the control of a suitable promoter. Alternatively, mutagenesis techniques can be used to obtain a nucleotide sequence encoding clotting factor IX from wild type clotting factor IX, and then the sequence of other elements to construct a fusion protein can be cloned into a suitable expression vector for expression under the control of a suitable promoter. These technologies are within the purview of a person skilled in the art, and many manuals exist in the art.

[0053] Suitable eukaryotic cells include mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hep and HepG2 cells. Preferably, the cells are grown under conditions suitable for expression of the clotting factor IX fusion protein of the invention. As for the reagents and compositions used to prepare or isolate the clotting factor IX fusion protein of this invention, there are no particular restrictions, and any system known in the art or commercially available can be used. In a preferred embodiment, the clotting factor IX fusion protein is produced by methods described in the art.

[0054] There are a variety of expression vectors that can be used to generate a coagulation factor IX fusion protein, which can be selected from vectors for expression in eukaryotic and prokaryotic cells. Vectors for expression in prokaryotic cells include, for example, plasmids such as pRSET, pET, and pBAD, which may use promoters including, for example, lac, trc, trp, recA, or araBAD. Vectors for expression in eukaryotic cells include: (1) vectors used for expression in yeast, such as pAO, pPIC, pYES, and pMET, which may use promoters such as AOX1, GAP, GAL1, AUG1, and so on; (2) vectors used for expression in insect cells, such as pMT, pAc[delta], plB, pMIB, pBAC, etc., in which promoters such as PH, p10, MT, Ac5, Op1E2, gp64 can be used, polh etc. and (3) vectors used for expression in mammalian cells such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV etc. and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus and the like, in which promoters such as CMV, SV40, EF-1, UbC, RSV, ADV promoters can be used. BPV and β-actin. In a preferred embodiment, the clotting factor IX fusion protein is expressed in prokaryotic or eukaryotic cell systems and codon-optimized sequences are used.

[0055] In a preferred embodiment, the sequence for expressing the clotting factor IX fusion protein comprises a propeptide and/or a signal peptide for improving secretion of the clotting factor IX fusion protein from the cell to the outside for isolation and purification; to improve the efficiency of propeptide cleavage, FIX yield and proper processing, the expression vector is optimized; in a particular embodiment, a FIX/PACE (furin) expression vector is constructed.

[0056] In another preferred embodiment, the sequence expressing the clotting factor IX fusion protein does not contain a propeptide and/or signal peptide, and instead of being secreted outside the cell for isolation and purification, the clotting factor IX fusion protein is obtained by cell lysis.

[0057] In one embodiment, in the fusion protein of the invention, the clotting factor IX protein and the Fc fragment are directly linked. In another embodiment of the present invention, the coagulation factor IX protein and the Fc fragment are linked through a first L1 linker. In another embodiment, the Fc fragment and the fragment containing the sortase recognition site are linked directly. In another embodiment, the Fc fragment and the fragment containing the sortase recognition site are linked through a second L2 linker.

[0058] In a preferred embodiment, the first L1 linker is a peptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids; more preferably, the first linker L1 contains a flexible peptide and/or a rigid element, and the flexible peptide is a peptide containing glycine (Gly, G), series (Ser, S), alanine (Ala, A) and/or threonine (Thr, T), for example, the flexible peptide is (GS)m(GGS)n(GGGS)o(GGGGS)p, where m, n, o, and p are independently selected from integers from 0 to 50, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably not all of m, n, o and p are 0.

[0059] A hard element is a peptide containing one or more prolines (Pro, P). Proline has a special cyclic structure, which imposes more restrictions on the structure of the peptide chain. The inventors have surprisingly found that the inclusion of a rigid unit in the linker can significantly improve the activity of the fusion protein. Preferably, the rigid element contains an amino acid sequence selected from the group consisting of:

VAPPPALPAPVRLPGPA (SEQ ID NO: 3),

VAPPPALPAVAPPPALPA (SEQ ID NO: 4),

VAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPA (SEQ ID NO: 5),

VAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPA (SEQ ID NO: 6) and

VAPPPALPAVAPPPALPAGSVAPPPALPAVAPPPALPA (SEQ ID NO: 7).

[0060] In another preferred embodiment, the sequence of the first linker

L1 is selected from the group consisting of:

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPA (SEQ ID NO: 8),

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPA (SEQ ID NO: 9),

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPA (SEQ ID NO 10),

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPA (SEQ ID NO: 11) and

GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAGSVAPPPALPAVAPPPALPA (SEQ ID NO 12).

[0061] The second linker L2 is a peptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, and is mainly used to separate Fc and sortase recognition site to facilitate recognition and action sortases; more preferably, the second linker L2 is a flexible peptide containing glycine (Gly, G), series (Ser, S), alanine (Ala, A) and/or threonine (Thr, T), such as (GS) _w (GGS ) _x (GGGS) _y (GGGGS) _z , where w, x, y, and z are independently selected from integers from 0 to 50, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably not all of w, x, y and z are 0.

[0062] In a preferred embodiment, the sequence of the second L2 linker is selected from the group consisting of GGGGS (SEQ ID NO: 14), GGGGSGGGGS (SEQ ID NO: 15) and GSGGSGGGGS (SEQ ID NO: 16). Coagulation factor IX fusion protein conjugate

[0063] The term "conjugate" refers to a product formed by a polypeptide or polypeptide variant covalently or non-covalently linked to a hydrophilic polymer modifying group described herein, wherein the hydrophilic polymer and polypeptide can be linked at any position, for example, the corresponding position on N -terminus, C-terminus or in the middle part of the polypeptide.

[0064] Generally, the term "polymer" as used herein has the conventional meaning known to those skilled in the art, and refers to both the polymer itself and its end-modified derivatives, unless expressly stated otherwise.

[0065] In addition, in the case of polymers such as polyethylene glycol, there are many ways to determine their molecular weight. Because polymers are made up of molecules with varying degrees of polymerization within a certain distribution range, the molecular weights of polymers are typically represented by their average molecular weight, in particular number average molecular weight or weight average molecular weight. The number average molecular weight refers to the sum of the products of the numerical fraction of each molecule with a different molecular weight and its corresponding molecular weight; weight average molecular weight refers to the sum of the products of the mass fraction of each molecule with a different molecular weight and its corresponding molecular weight. Although the number average molecular weight and the weight average molecular weight may diverge somewhat when the degree of polymerization of the polymers differs significantly, for polymers with a narrow distribution range, both indicators usually tend to the same value. In the case of polymers such as the polyethylene glycol mentioned here, its molecular weight may be indicated either as a number average molecular weight or as a weight average molecular weight.

[0066] The clotting factor IX fusion protein of the present invention may be conjugated with one or more hydrophilic polymers to form a clotting factor IX fusion protein conjugate. Preferably, the polymer is physiologically acceptable, which includes soluble in aqueous solution or suspension and does not adversely affect mammals, such as side effects after administration of the clotting factor IX fusion protein conjugate in a pharmaceutically acceptable amount. The polymers that can be used in this invention are not limited to any specific ones. Generally, polymers having 2 to about 3000 repeat units are preferred. The group of polymers may be selected from natural or synthetic polymers, examples of which include, without limitation, polysaccharides, polyalkylene glycols such as polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene oxide (PEO), ethylene glycol-propylene glycol copolymer, polyvinyl alcohol, and any combination thereof. . In a preferred embodiment, the clotting factor IX fusion protein conjugate of the invention is conjugated to one or more PEG moieties for modification.

[0067] In this invention, the polymer is not limited to a specific structure, it may be linear (such as alkoxyPEG or bifunctional PEG), branched or multi-arm (such as bifurcated PEG or PEG linked to a polyol core), arborescent, or may have degradable linkages. In addition, the internal structure of polymers can be organized into any number of different patterns, which can be selected from homopolymers, alternating copolymers, random copolymers, three alternating monomer copolymers, three monomer random polymers, three monomer block polymers, and the like. The polymer may also include polymers of poly(alkylene oxide), polymaleic acid, poly(D, L-alanine), and the like.

[0068] In some embodiments, the polymer is polyethylene glycol (PEG) or derivatives thereof, such as methoxypolyethylene glycol (mPEG). In this document, unless otherwise indicated, polyethylene glycols (PEGs) include having as an end group either hydroxyl groups or other groups. Other groups include alkoxy, cycloalkoxy, cycloalkylalkoxy, alkenyl, arylalkoxy, or aralkyloxy, without limitation. These PEG molecular species are known in the art and are routinely used in the modification of polypeptides. The PEG side chain can be linear, branched, bifurcated, or have multiple arms. Different polyethylene glycols may have different polymer chain lengths and different polymer structures.

[0069] In this invention, the molecular weight of PEG is not limited to specific values, and its molecular weight can vary from 0.1 to 200 kDa, for example, from 1 to 150 kDa, from 2 to 100 kDa, from 3 to 80 kDa, from 4 up to 50 kDa or from 5 to 40 kDa. Other useful PEGs include, for example, those described in WO 03/040211, US 6566506, US 6864350 and US 6455639. In particular, PEG has the general formula HO-CH ₂ CH ₂ O-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH, where n is in the range from about 5 to about 4000. As mentioned above, the PEG of this invention includes PEG with other end groups, such as methoxyPEG, branched PEG, bifurcated PEG and the like. Suitable branched PEGs can be prepared as described in US Pat. No. 5,932,462, the contents of which are hereby incorporated by reference in their entirety. Forked PEG refers to PEG that has a branch near one end of the polymer chain, and the backbone of the forked PEG may be linear or branched.

[0070] It is known to those skilled in the art that as the molecular weight of the polymeric group in the biologically active molecule conjugated to the polymeric group increases, the biological activity of the conjugate generally decreases gradually. It is also known to those skilled in the art that as the molecular weight of the polymeric group increases, the biological half-life of the conjugate and/or the plasma half-life of the conjugate increases accordingly. [0071] To ensure a stable therapeutic effect over a long period of time and to reduce the frequency of administration to improve patient compliance, it is desirable to increase the biological half-life of coagulation factor IX fusion protein conjugates as much as possible while maintaining significant coagulation factor IX receptor agonist activity. Thus, the present invention provides a clotting factor IX fusion protein conjugate with an extended biological half-life and significant clotting factor IX receptor agonist activity.

[0072] In a specific embodiment, the molecular weight of one or more polymeric groups (such as PEG) in the GC clotting factor fusion protein conjugate is 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more or 160 or more kDa e.g. 5, 10 , 20, 30, 40, 50, 60, 70, 80, 90, or 100 kDa, or a value between any two. It should be noted that if there are multiple conjugated polymeric groups in the conjugate, when describing the molecular weight of the polymeric group in the clotting factor IX fusion protein conjugate, the sum of the molecular weights of all conjugated polymeric groups in the conjugate is calculated unless otherwise noted.

[0073] The polymer used in this invention is known in the art and can be obtained by various methods, including, for example, commercial acquisition, or it can be obtained in-house according to methods known in the art. This invention is not limited to polymers obtained by any particular method.

[0074] After the conjugation reaction, the conjugate may be isolated by a suitable method, including, for example, ultrafiltration, dialysis or chromatography, and so on, all of which are within the skill of the art. pharmaceutical composition

[0075] The clotting factor IX fusion protein or factor IX fusion protein conjugate of the present invention may have various uses, including, for example, use in the prevention and/or treatment of hemorrhagic diseases. Thus, the present invention also provides a pharmaceutical composition for the prevention and/or treatment of hemorrhagic diseases, comprising a therapeutically effective amount of a fusion protein or conjugate of the present invention and optionally a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition may be used for the prevention and/or treatment of hemorrhagic diseases, more preferably for the prevention and/or treatment of hemorrhagic diseases in patients with congenital or acquired FIX deficiency and spontaneous or postoperative bleeding in patients with hemophilia B.

[0076] A therapeutically effective amount of a fusion protein or conjugate of this invention depends on the route of administration, the category of subjects, and the physical characteristics of the particular mammal in question. These factors and their relationship to quantification are well known to those skilled in the art of medicine. The amount and route of administration may be adjusted to achieve optimal efficacy for delivering the peptide to the subject, depending on factors well known to those skilled in the art such as body weight, diet, concomitant medications, and other factors.

[0077] The pharmaceutical composition of this invention can be administered as part of a combination therapy, that is, in combination with one or more other substances, while the pharmaceutical composition and other substances are administered together or sequentially. In other embodiments, other substances may be administered before, during, or after administration of one or more of: a fusion protein, a conjugate, or a pharmaceutical composition thereof of this invention.

[0078] As used herein, a "pharmaceutically acceptable carrier" or a "physiologically acceptable carrier" may be used interchangeably, including one or more of a variety of physiologically compatible salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. .d. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the mode of administration, the therapeutic agent may be coated with certain materials to protect the therapeutic agent from acid attack and other natural conditions that may lead to inactivation of the therapeutic agent.

[0079] When a pharmaceutical preparation of this invention is administered, it is administered in a pharmaceutically acceptable amount as part of a pharmaceutically acceptable composition. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the biologically active efficacy of the active ingredients. Such formulations typically contain salts, buffers, preservatives, compatible carriers, and possibly other therapeutic agents such as additional immunomodulators, including adjuvants, chemokines, and cytokines. When used in a medicinal product, the salt must be pharmaceutically acceptable. However, salts that are not pharmaceutically acceptable can be successfully used to obtain pharmaceutically acceptable salts, so they are not excluded from the scope of this invention.

[0080] If necessary, the coagulation factor IX fusion protein or coagulation factor IX fusion protein conjugate of the present invention may be combined with pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carriers" as used herein refers to one or more compatible solid or liquid excipients, diluents, or encapsulating agents that are suitable for administration to mammals, such as humans. The term "carrier" means organic or inorganic, natural or synthetic components, which are combined with active components to perform an auxiliary function. The components of the pharmaceutical composition can also be mixed in a form that does not interfere with the therapeutic effect of the desired drug.

[0081] Preferably, the pharmaceutical composition of this invention may contain a buffer system, and preferably, the buffer system is an acetate buffer solution with a pH of from about 3.0 to about 6.0 or a phosphate buffer solution with a pH of from about 5.0 to about 9.0. In some specific embodiments, suitable buffers include acetate, citrate, borate, and phosphate.

[0082] Optionally, the pharmaceutical composition may also contain suitable preservatives such as benzalkonium chloride, chloro-tert-butanol, parabens, and merthiolate.

[0100] the Pharmaceutical composition can be conveniently presented in the form of dosage forms and can be obtained by any method known in the field of pharmacology. The method includes the steps of bringing the active agent into association with the carrier which contains one or more accessory ingredients. In general, the composition is prepared by intimately mixing the active compound with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired shape.

[0083] Pharmaceutical compositions suitable for parenteral administration may be sterile aqueous or non-aqueous formulations containing one or more fusion proteins or conjugates. In some embodiments, the formulation is isotonic with respect to the subject's blood. The formulation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenteral acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this reason, any inert fixed oil can be used, including synthetic mono- or diglycerides. Also, fatty acids such as oleic acid can be used as injectable formulations. Formulations of carriers suitable for oral, subcutaneous, intravenous, intramuscular and the like administration can be prepared according to the guidelines in the art.

[0084] The fusion protein or conjugate of this invention can be formulated with carriers that protect it from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods are known in the art for preparing such compositions.

[0085] The pharmaceutical composition of this invention can be administered by any standard route, including injection or slow infusion over time. For example, administration may be oral, intravenous, intraperitoneal, intramuscular, intracavitary, intratumoral, or transdermal.

[0086] The pharmaceutical composition of this invention is administered in a pharmaceutically effective amount. An "effective amount" is the amount of any fusion protein or conjugate of the invention that provides the desired response alone or with additional doses and/or other therapeutic agents.

[0087] Undoubtedly, such an amount will depend on the specific disease being treated, the severity of the disease, the individual characteristics of the patient (including age, physical condition, height and body weight), the duration of treatment, the nature of concomitant treatment (if any), the specific route administration and similar factors known to medical and healthcare professionals. These factors are well known to those skilled in the art and can be ascertained through routine experimentation. It is generally preferred to use the maximum dose of each component, or combination thereof, which, based on medical judgment, can be called the maximum safe dose. However, those skilled in the art will appreciate that, for medical, psychological, or virtually any other reason, patients may require lower doses or allowable doses.

[0088] The pharmaceutical composition used in the method described above is preferably sterile and contains an effective amount of the fusion protein or conjugate alone or in combination with another drug in units of mass or units of volume suitable for administration to patients to obtain the desired response, such as reduction blood glucose.

[0089] The dosage of the fusion protein or conjugate administered to a subject may be selected based on various parameters, in particular depending on the route of administration and the condition of the subject. Other factors include the required duration of treatment. If the subject's response to the initially administered dose is insufficient, a higher dose (or a higher effective dose achievable by another, more local route of administration) tolerated by the patient may be used.

[0090] In some embodiments, the dosage range of the coagulation factor IX fusion protein or coagulation factor IX fusion protein conjugate may be from 30 mg/kg body weight per day to 0.00001 mg/kg body weight per day, or from 3 mg/kg per day. up to 0.0001 mg/kg per day or from 0.3 mg/kg per day to 0.01 mg/kg per day.

[0091] In some embodiments, the pharmaceutical composition of this invention contains 0.20-5 mg/ml clotting factor IX fusion protein and/or 4-40 mg/ml clotting factor IX fusion protein conjugate, preferably 0.20-5 mg/ml factor IX fusion protein and/or 4-40 mg/ml factor IX fusion protein conjugate, more preferably 0.5-2 mg/ml factor IX fusion protein and/or 10-20 mg/ml factor fusion protein conjugate coagulation IX. Typically, the dosage of the factor IX fusion protein or factor IX fusion protein conjugate of the present invention may range from about 10 µg/kg patient body weight to about 100,000 µg/kg patient body weight. In some embodiments, the dosage may vary from about 0.1 mg/kg to about 20 mg/kg. In some embodiments, the dosage may vary from about 0.1 mg/kg to 5 mg/kg, from 0.1 mg/kg to 10 mg/kg, or from 0.1 mg/kg to 15 mg/kg. In some embodiments the dosage may vary from about 1 mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 15 mg/kg, or 15 mg/kg to 20 mg/kg. In some embodiments, the dosage is about 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 17 mg/kg, 20 mg/kg, 25 mg/kg or 30 mg/kg. In some embodiments, the dosage is about 1 mg/kg, 3 mg/kg, 5 mg/kg, or 6 mg/kg. Depending on the properties of the composition, dosing can be carried out continuously (for example, by continuous administration) or at certain periods of time. In some embodiments, when administered intravenously, the dosage of the factor IX fusion protein or factor IX fusion protein conjugate of the present invention may be from 0.1 to 20 mg/kg, or any value within the indicated range. The ideal time span for multiple administrations of a particular composition can be determined by one skilled in the art without undue experimentation. Specialists in the field of technology are aware of other schemes for introducing the proposed composition, where the dosage, scheme of administration, site of administration, route of administration, and so on may vary depending on the following. In one embodiment, administration is intravenous. In another embodiment, the administration regimen is a single intravenous administration.

[0092] A kit containing a clotting factor IX fusion protein or a clotting factor IX fusion protein conjugate (eg, in a pharmaceutical composition) and instructions for use is also within the scope of this invention. The kit may additionally contain at least one of other agents, such as one or more other agents for the prevention and/or treatment of hemorrhagic diseases. In another embodiment, the kit may include a carrier that has compartments for firmly holding one or more containers or a series of containers (such as test tubes, test tubes, flasks, vials, syringes, and so on). The components of the kit may be packaged in an aqueous medium or in lyophilized form.

[0093] The composition may be in lyophilized form or in an aqueous medium.

[0094] Preferably, the subject is a vertebrate, more preferably a mammal, most preferably a human, however, the subject may also be other animals such as domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cattle , sheep and goats, pigs, horses, etc.) or laboratory animals (eg monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0095] The term "improved circulating half-life" or "increased circulating half-life" means that the molecule of the invention has an altered circulating half-life, preferably an increased circulating half-life compared to a wild-type factor IX molecule. The circulating half-life is preferably increased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65% , preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably at least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and more preferably at least 250 % or 300%. Even more preferably, the molecule has a circulating half-life increased by at least 400%, 500%, 600%, or even 700%.

[0096] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention in any way. All contents of documents cited in this application (including journal publications, issued patents, published patent applications, and pending patent applications) are expressly incorporated herein by reference. In the following examples, unless otherwise indicated, the reagents and materials used are commercially available products of at least analytical grade or equivalent.

DESCRIPTION OF EXAMPLES OF CARRYING OUT THE INVENTION

Example 1 Preparation of FIX-L1-Fc-L2-P Fusion Protein

1. Construction of an expression vector

[0097] The FIX fusion proteins constructed in this example have the general molecular structure FIX-L1-Fc-L2-P, where FIX is a natural coagulation factor IX of human origin, the sequence of which is shown in SEQ ID NO: 1.

[0098] L1, denoting the first linker between FLX and Fc, is a flexible peptide or linker peptide consisting of a flexible peptide and a rigid structure based on multiple prolines (Pro, P). In this example, a total of 6 L1 sequences were used, designated as C1, C2, C3, C4, C5 and GS, respectively, and their specific sequences are as follows:

C1: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPA (SEQ ID NO: 8);

C2: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPA (SEQ ID NO: 9);

C3: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPA (SEQ ID NO 10),

C4: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPA (SEQ ID NO 11)

C5: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAGSVAPPPALPAVAPPPALPA (SEQ ID NO: 12) and

GS: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO 13).

[0099] Fc is derived from human IgGl having point mutation N297A mediating reduced ADCC and CDC activity (see Lund J. et al. Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11. Mol Immunol. 1992 Jan; 29(1):53-9.).

[0100] L2 is the second linker, which is mainly used to separate the Fc and the sortase recognition site. In particular, the L2 sequence in this example is GSGGSGGGS (SEQ ID NO: 16).

[0101] P is an amino acid fragment containing a sortase recognition site.

[0102] Table 1 provides a summary of all FIX-L1-Fc-L2-P fusion proteins constructed in this Example.

[0103] Most of the recombinant FIX expressed in CHO cells exists as a precursor protein, and only about 30% of the propeptide is cleaved from the precursor protein to form mature FIX. Processing of mature FIX requires the enzyme PACE/furin, which is a subtilisin-like calcium-dependent serine propeptidase located in the trans Golgi complex and can cleave the C-terminus of R46 in the FIX precursor protein to catalyze the conversion of the FIX precursor protein to mature FIX. To improve the yield and ensure proper processing of FIX in this Example, a bicistronic FIX/PACE (furin) expression vector was further constructed, the structure of which is shown in FIG. 1 A.

Construction of a bicistronic expression vector FIX/PACE (furin)

A. Construction of the expression vector pFRL-rPS expressing PACE

[0104] The PACE/furin sequence has GenBank accession number NP 002560.1. For expression in the host, codon optimization of the sequence encoding amino acids 1-715 of PACE/furin (SEQ ID NO: 17) was performed. After the whole gene was synthesized, the sequence was cloned into the pcDNA3.1 (+) vector, and the resulting plasmid was designated pcDNA3.1-P-SOL (Shanghai Generay Biotech Co., Ltd.). The entire gene expression cassette, including the SV40 promoter, PACE/furin gene, and poly-A, was generated by PCR amplification with primers F1 and R1 (full sequences are shown below) using plasmid pcDNA3.1-P-SOL as template . After cleavage at the BglII restriction sites at both ends, the fragment was inserted into the pFRL-DHFR vector cleaved at one BamHI restriction site. Positive clones were selected, verified by sequencing, and vector pFRL-rPS was designated.

F1: 5'-TCAGGAAGATCTCGCGAATTAATTCTGTGGAATGTGT-3' (SEQ ID NO: 30);

R1: 5'-TATCCTAGATCTGCTGGCACGACAGGTTTCCCGACT-3' (SEQ ID NO: 31).

The amino acid sequence 1-715 of PACE/furin is shown below:

MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFDVNDQDPNDGPRYTQVNG NARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLCIWATNGVGRKVAGILSHAPQP KDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQPS (SEQ ID NO 17).

b. Construction of a bicistronic expression vector for the expression of FIX-L1-Fc-L2-P

b1) pFRL-rPS-FLX-C1-Fc-L2-P

[0105] Codon optimization was performed for the nucleotide sequence encoding FIX-C1-Fc-L2-P (i.e., the L1 sequence in FIX-L1-Fc-L2-P is C1, and so on) (the amino acid sequence is shown in SEQ ID NO: 18 and the codon-optimized coding nucleotide sequence is shown in SEQ ID NO: 19). After the whole gene was synthesized by Shanghai Generay Biotech Co., Ltd., the fragment was inserted into pFRL-rPS by cleavage at two HindIII/EcoRI restriction sites to construct the bicistronic expression vector pFRL-rPS-FIX-C1-Fc-L2-P, together expressing FIX-C1-Fc-L2-P/furin.

Amino acid sequence of FIX-C1-Fc-L2-P: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCVSCTEGYRLAENQKSCEPTSKEP ETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNGITKCAGFHEGGRDSCQGDSGGPHVTKEVKY NWIKEKTKLTGGGGSGGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPSEKTISKAKGQPREPDIQVYTLPPSRDELTKNQVSLNG PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGGSGGGGSLPETGG(SEQ ID NO 18).

Nucleotide sequence of FIX-C1-Fc-L2-P: TTGAGAATACTGAGCGCACTACTGAGTTTTGGAAGCAGTATGTGGATGGCGATCAGTGCGAGTCCAATCCATGCCTGAATGGCGGATCTTGCAAGGATGATATTAATTCTTACGAGTGTTGGTGCCCATTCGGCTTTGAGGGAAAGAATTGCGAACTGGATGTCACATGTAACATCAAGAACGGCAGATGCGAGCAGTTTTGTAAGAACAGAGCGCCGATAGCATAAGGTGGATGTGTAGTTGTCCTCACT TGAGCCCGCTGTGTCCATTTCCTTGTGGCCGGTCAGCGTCAGCCAGACTTCTAAGCTGACACGCGCCGAGACCGTCTTTCCAGATGTGGATTATGTCAACTCTACTGAGGCCGAGACCATTCTCGATAATATTACTCAGTCTACACAAGTCCTTTAACGACTTCACTCGGGTGGTGGGCGGAGAGGACGCAAAGCCTGGCCAGTTTCCTTGGCAGGTCGTGCTCAACGGAATTGGCTGGATCGA GATTGTGACCGCCTCTCACTGCGTCGAGACAGGCGTCAAGATTACCGTGGTCGCTGGAGAGCACAACATTGAGGAAACAGAGCACACCGAGCAGAAGCGCAATGTGATTCGCATCATCCCACACCACAACTACAACGCCGCTATTAATAAGTACAACCACGACATCGCCCTGTTAGAACTAGATGAGCCTCTGGTCCTGAATAGTTACGTCACTCCTATTTGTGTATCGCCGATAAAGACATTCTCCATCAT TGGGGACGGGTGTTTCACAAGGGACGGTCCGCACTCGTGCTACAATACCTCCGGGTGCCACTGGTGGATCGGGCAACATGCCTCCGGTCTACCAAGTTTACTATATACAATAACATGTTTTGCGCCGGCTTTCACGAGGGCGGACGGGATAGTTGCCAGGGCGATAGTGGAGGCCCTCATGTCACAGAGAGGTGGAGGGAACTTGCTTTCTGACAGGCATTATCATCG GCATCTATACTAAGGTCTCGCGTTACGTCAATTGGATCAAGGAAAAGACAAAGCTGACAGGCGGCGGAGGGAGCCGGCGGCGGAGGCTCTGGCGGCGGCGGATCTGGCGGAGGCGGCTCTGGCGGAGGCGGATCCGTCGCTCCACCACCTGCACTCCCCGCCCCTGTCAGACTGCCTGGACCCGCTGATAAGCCCACACATGTCCACCTTGCCCAGCACCCCTGGCCT CCAAAGGACACCCTCATGATTAGTAGAACCCCAAGGTGACTTGCGTCGTGGTCGATGTCAGTCACGAGGACCCCGAGGTGAAGTTTAATTGGTATGTAGATGGCGTCGAGTGCACAACGCTAAGACCAAGCCACGCGAAGGCAGTACGCATCTACTTACCGGGTCGTCTCTGTGCTGACTGTGCTGCACCATAGGATTGGCTCAATGGCCCCCTA AGGCAAAGGGCCAGCCTAGGGAGCCCCAGGTCTACACCCTGCCCCCTTCCCGCGATGAGCTGACTAAGAATCAGGTCTCTCTCACTTGCCTCGTGAAGGCTTTTACCCATCCGACATTGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAATAACTACAAGACAACTCCTCCCGTGCTCGATAGCGACGGTTCATTCTTCCTGTACTCCAAGCTCTGACCGTTGATAAGTCTCGGTCCTG GGCACTGCACAATCACTACACCCAGAAGTCCCTGTCCCTCAGCCCTGGCAAAGGCAGCGGCGGATCTGGAGGCGGCGGATCCCTCCCAGAGACCGGCGGATGA (SEQ ID NO 19).

b2) _P FRL-rPS-FIX-C2-Fc-L2-P

[0106] Using the FIX-C1-Fc-L2-P coding sequence as a template, the C2 sequence was added to the 5' end of the primer, and the Fc-L2-P sequence was amplified by PCR to obtain the C2-Fc-L2-P sequence. The sequences of primers F2 and R2 used for amplification are given below: Forward primer F2:

5'-TTCGCGGATCCGTCGCTCCACCACCTGCACTCCCCGCCGTGGCTCCTCCACCT

GCTTTGCCCGCTGATAAGACCCACACATGTCCAC-3' (SEQ ID NO: 32);

Reverse primer R2:

5'-TAACCGGAATTCATTATCCGCCGGTCTCTGGGAGAGATCCGCCGCCTCCAGATC

CGCCGCTGCCGCCAGGGCTGAGGGACAGGGACT-3' (SEQ ID NO: 33).

[0107] Then, after cleavage at two BamHI/EcoRI restriction sites, the C1-Fc-L2-P sequence in the pFRL-rPS-FIX-C1-Fc-L2-P vector was replaced with the C2-Fc-L2-P amplified product, thus obtaining bicistronic expression vector pFRL-rPS-FIX-C2-Fc-L2-P co-expressing FIX-C2-Fc-L2-P/furin.

The amino acid sequence of FIX-C2-Fc-L2-P is: ETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAFNKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKEECAFTIYNNGITKVSWGVTGISFY VNWIKEKTKLTGGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPDIQVYTLPPSRDELVTKNQY TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSGGGGSLPETGG (SEQ ID NO: 20).

Nucleotide sequence of FIX-C2-Fc-L2-P: TTGAGAATACTGAGCGCACTACTGAGTTTTGGAAGCAGTATGTGGATGGCGATCAGTGCGAGTCCAATCCATGCCTGAATGGCGGATCTTGCAAGGATGATATTAATTCTTACGAGTGTTGGTGCCCATTCGGCTTTGAGGGAAAGAATTGCGAACTGGATGTCACATGTAACATCAAGAACGGCAGATGCGAGCAGTTTTGTAAGAACAGAGCGCCGATAGCATAAGGTGGATGTGTAGTTGTCCTCACT TGAGCCCGCTGTGTCCATTTCCTTGTGGCCGGTCAGCGTCAGCCAGACTTCTAAGCTGACACGCGCCGAGACCGTCTTTCCAGATGTGGATTATGTCAACTCTACTGAGGCCGAGACCATTCTCGATAATATTACTCAGTCTACACAAGTCCTTTAACGACTTCACTCGGGTGGTGGGCGGAGAGGACGCAAAGCCTGGCCAGTTTCCTTGGCAGGTCGTGCTCAACGGAATTGGCTGGATCGA GATTGTGACCGCCTCTCACTGCGTCGAGACAGGCGTCAAGATTACCGTGGTCGCTGGAGAGCACAACATTGAGGAAACAGAGCACACCGAGCAGAAGCGCAATGTGATTCGCATCATCCCACACCACAACTACAACGCCGCTATTAATAAGTACAACCACGACATCGCCCTGTTAGAACTAGATGAGCCTCTGGTCCTGAATAGTTACGTCACTCCTATTTGTGTATCGCCGATAAAGACATTCTCCATCAT TGGGGACGGGTGTTTCACAAGGGACGGTCCGCACTCGTGCTACAATACCTCCGGGTGCCACTGGTGGATCGGGCAACATGCCTCCGGTCTACCAAGTTTACTATATACAATAACATGTTTTGCGCCGGCTTTCACGAGGGCGGACGGGATAGTTGCCAGGGCGATAGTGGAGGCCCTCATGTCACAGAGAGGTGGAGGGAACTTGCTTTCTGACAGGCATTATCATCG GCATCTATACTAAGGTCTCGCGTTACGTCAATTGGATCAAGGAAAAGACAAAGCTGACAGGCGGCGGAGGGAGCCGGCGGCGGAGGCTCTGGCGGCGGCGGATCTGGCGGAGGCGGCTCTGGCGGAGGCGGATCCGTCGCTCCACCACCTGCACTCCCCGCCGTGGCTCCTCCCTGATAAGCTTTGCCCGCTGATAAGCCCACACATGGCCACCTTGCCCAGCCCCTGG AAAGCCAAAAGGACACCCTCATGATTAGTAGAACCCCAGAGGTGACTTGCGTCGTGGTCGATGTCAGTCACGAGGACCCCGAGGTGAAGTTTAATTGGTATGTAGATGGCGTCGAGTGCACAACGCTAAGACCAAGCCACGCGAAGAGCAGTACGCATCTACTTACCGGTCGTCTCTGTGCATGACTGTTTGCTGCACCAAGTCCACAAGGACCATATGTAAGTC TAGCAAGGCAAAGGGCCAGCCTAGGGAGCCCCAGGTCTACACCCTGCCCCCTTCCCGCGATGAGCTGACTAAGAATCAGGTCTCTCTCACTTGCCTCGTGAAGGCTTTTACCCATCCGACATTGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAATAACTACAAGACAACTCCTCCCGTGCTCGATAGCGACGGTTCATTCTTCCTGTACTCCAAGCTCCCCGTTGATAAGTCTCTGG GCACGAGGCACTGCCAATCACTACACCCCAGAAGTCCCTGTCCCTCAGCCCTGGCGGCAGCGGCGGAATTGGAGGCGGCGGATCTCTCCCAGAGACCGGCGGATAA (SEQ ID NO: 21).

63) Construction of pFRL-rPS-FIX-C3-Fc-L2-P

[0108] The vector construction method was the same as for pFRL-rPS-FIX-C2-Fc-L2-P. PCR primers to amplify the C3-Fc-L2-P coding sequence included forward primers F3: 5'-TGGCTCCTCCACCTGCTTTGCCCGCTGTCGCTCCACCACCTGCACTCC-3' (SEQ ID NO: 34) and F4: 5'-TAGATCTGTCGCTCCACCACCTGCACTCCCCGCCGTGGCTCCTCCACCTGCTTTGCC-3' (SEQ ID: 35) and reverse primer R3: 5'-TGAATTCTTATCATCCGCCGGTCTCTGGGAG-3' (SEQ ID NO: 36). The C3-Fc-L2-P sequence was obtained in two rounds of PCR amplification.

Amino acid sequence of FIX-C3-Fc-L2-P: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCVSCTEGYRLAENQKSCEPTSKEP ETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAArNKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNGITKVGITKEGGPMKISFY VNWIKEKTKLTGGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQDEEPQVY WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGGSGGGGSLPETGG (SEQ ID NO: 22).

Nucleotide sequence of FIX-C3-Fc-L2-P: TTGAGAATACTGAGCGCACTACTGAGTTTTGGAAGCAGTATGTGGATGGCGATCAGTGCGAGTCCAATCCATGCCTGAATGGCGGATCTTGCAAGGATGATATTAATTCTTACGAGTGTTGGTGCCCATTCGGCTTTGAGGGAAAGAATTGCGAACTGGATGTCACATGTAACATCAAGAACGGCAGATGCGAGCAGTTTTGTAAGAACAGAGCGCCGATAGCATAAGGTGGATGTGTAGTTGTCCTCACT TGAGCCCGCTGTGTCCATTTCCTTGTGGCCGGTCAGCGTCAGCCAGACTTCTAAGCTGACACGCGCCGAGACCGTCTTTCCAGATGTGGATTATGTCAACTCTACTGAGGCCGAGACCATTCTCGATAATATTACTCAGTCTACACAAGTCCTTTAACGACTTCACTCGGGTGGTGGGCGGAGAGGACGCAAAGCCTGGCCAGTTTCCTTGGCAGGTCGTGCTCAACGGAATTGGCTGGATCGA GATTGTGACCGCCTCTCACTGCGTCGAGACAGGCGTCAAGATTACCGTGGTCGCTGGAGAGCACAACATTGAGGAAACAGAGCACACCGAGCAGAAGCGCAATGTGATTCGCATCATCCCACACCACAACTACAACGCCGCTATTAATAAGTACAACCACGACATCGCCCTGTTAGAACTAGATGAGCCTCTGGTCCTGAATAGTTACGTCACTCCTATTTGTGTATCGCCGATAAAGACATTCTCCATCAT TGGGGACGGGTGTTTCACAAGGGACGGTCCGCACTCGTGCTACAATACCTCCGGGTGCCACTGGTGGATCGGGCAACATGCCTCCGGTCTACCAAGTTTACTATATACAATAACATGTTTTGCGCCGGCTTTCACGAGGGCGGACGGGATAGTTGCCAGGGCGATAGTGGAGGCCCTCATGTCACAGAGAGGTGGAGGGAACTTGCTTTCTGACAGGCATTATCATCG GCATCTATACTAAGGTCTCGCGTTACGTCAATTGGATCAAGGAAAAGACAAAGCTGACAGGCGGCGGAGGGAGCCGGCGGCGGAGGCTCTGGCGGCGGCGGATCTGGCGGAGGCGGCTCTGGCGGAGGCGGATCTGTCGCTCCACCAACCTGCACTCCCCGCCGTGGCTCCTCACCTGCTTTGCCCGCTGTCGCTCCACCACCTGCCAGACCACTCCCCGCCCCTGTCAG TTGCCCAGCACCTGAGCTGCTGGGCGGACCTAGCGTGTTTCTGTTCCCACCAAAGCCAAAAGGACACCCTCATGATTAGTAGAACCCCAGAGGTGACTTGCGTCGTGGTCGATGTCAGTCACGAGGACCCCGAGTGAAGTTTAATTGGTATGTAGATGGCGTCGAGTGCACAACGCTAAGACCAAGCCACGCGAAGAGCAGTACGCATCTTGGCACTTACCAGGGTCTCTCTCTG AGTACAAGTGTAAGGTCAGTAATAAGGCCCTCCCCGCACCTATTGAAAAGACTATTAGCAAGGCAAAGGGCCAGCCTAGGGAGCCCCAGGTCTACACCCTGCCCCCTTCCCGCGATGAGCTGACTAAGAATCAGGTCTCTCTCACTTGCCTCGTGAAGGCTTTTACCCATCCGACATTGCCGTGGGCTGGGAGTCCAACGGACAGCCAGAGAATAACTACAAGACACGCGATAGCCCTTGCT CGTTGATAAGTCTCGGTGGCAGCAGGGAAATGTGTTCTCTTGTTCCGTCATGCACGAGGCACTGCACAATCACTACACCCAGAAGTCCCTGTCCCTCAGCCCTGGCAAAGGCAGCGGCGGATCTGGAGGCGGCGGATCCCTCC WITH AG AG AC CGGCGG ATG A (SEQ ID NO: 23). 64) Construction of pFRL-rPS-FIX-C4-Fc-L2-P

[0109] The construction method was the same as for pFRL-rPS-FIX-C2-Fc-P. PCR primers for amplifying the C4-Fc-L2-P gene included forward primers F5: 5'-CCCTGT CAGACTGCCTGGACCCGCTGTCGCTCCACCACCTGCACTCC-3' (SEQ ID NO: 37) and F6:

5-TAGATCTGTCGCTCCACCACCTGCACTCCCCGCCCCTGTCAGACTGCCTGGACC-3' (SEQ ID NO: 38) and reverse primer R3. The C4-Fc-L2-P sequence was obtained in two rounds of PCR amplification.

Amino acid sequence of FIX-C4-Fc-L2-P: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCVSCTEGYRLAENQKSCEPTSK ETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNGITKCAGFHEGGRDSCQGDSGGPHVTKEVKY NWIKEKTKLTGGGGSGGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPS ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGS GGGGS LPETGG (SEQ ID NO: 24).

Nucleotide sequence of FIX-C4-Fc-L2-P: TTGAGAATACTGAGCGCACTACTGAGTTTTGGAAGCAGTATGTGGATGGCGATCAGTGCGAGTCCAATCCATGCCTGAATGGCGGATCTTGCAAGGATGATATTAATTCTTACGAGTGTTGGTGCCCATTCGGCTTTGAGGGAAAGAATTGCGAACTGGATGTCACATGTAACATCAAGAACGGCAGATGCGAGCAGTTTTGTAAGAACAGAGCGCCGATAGCATAAGGTGGATGTGTAGTTGTCCTCACT TGAGCCCGCTGTGTCCATTTCCTTGTGGCCGGTCAGCGTCAGCCAGACTTCTAAGCTGACACGCGCCGAGACCGTCTTTCCAGATGTGGATTATGTCAACTCTACTGAGGCCGAGACCATTCTCGATAATATTACTCAGTCTACACAAGTCCTTTAACGACTTCACTCGGGTGGTGGGCGGAGAGGACGCAAAGCCTGGCCAGTTTCCTTGGCAGGTCGTGCTCAACGGAATTGGCTGGATCGA GATTGTGACCGCCTCTCACTGCGTCGAGACAGGCGTCAAGATTACCGTGGTCGCTGGAGAGCACAACATTGAGGAAACAGAGCACACCGAGCAGAAGCGCAATGTGATTCGCATCATCCCACACCACAACTACAACGCCGCTATTAATAAGTACAACCACGACATCGCCCTGTTAGAACTAGATGAGCCTCTGGTCCTGAATAGTTACGTCACTCCTATTTGTGTATCGCCGATAAAGACATTCTCCATCAT TGGGGACGGGTGTTTCACAAGGGACGGTCCGCACTCGTGCTACAATACCTCCGGGTGCCACTGGTGGATCGGGCAACATGCCTCCGGTCTACCAAGTTTACTATATACAATAACATGTTTTGCGCCGGCTTTCACGAGGGCGGACGGGATAGTTGCCAGGGCGATAGTGGAGGCCCTCATGTCACAGAGAGGTGGAGGGAACTTGCTTTCTGACAGGCATTATCATCG GCATCTATACTAAGGTCTCGCGTTACGTCAATTGGATCAAGGAAAAGACAAAGCTGACAGGCGGCGGAGGGAGCCGGCGGCGGAGGCTCTGGCGGCGGCGGATCTGGCGGAGGCGTCTCTGGCGGAGGCGGATCTGTCGCTCCACCACCTGCACTCCCCGCCCCTGTCAGACTGCCTGGACCCGCTGTCGCATACACCACCTGCACTCCCCCCCGTGGCTCTCCCCCCACTAG CCAGCACCTGAGCTGCTGGGCGGACCTAGCGTGTTTCTGTTCCCACCAAAGCCAAAGGACACCCTCATGATTAGTAGAACCCCAGAGGTGACTTGCGTCGTGGTCGATGTCAGTCACGAGGACCCCGAGGTGAAGTTTAATTGGTATGTAGATGGCGTCGAGTGCACAACGCTAAGACCAAGCCGCGAAGAGCAGTACGCATCTACTGACCTGAGGCGTCAGTCTCCTTGCTGCTAG AGTGTAAGGTCAGTAATAAGGCCCTCCCCGCACCTATTGAAAAGACTATTAGCAAGGCAAAGGGCCAGCCTAGGGAGCCCCAGGTCTACACCCTGCCCCCTTCCCGCGATGAGCTGACTAAGAAATCAAGGTCTCTCTCACTTGCCTCGTGAAGGGCTTTTTACCCATCCGACATTGCCGTGGAGTGGGAGTCCAACGGAACCAGCCAGAGAATAACTACGCAAGACAACTCCTCCCGTGCTTCCATTTTCCG ATAAGTCTCGGTGGCAGCAGGGAAATGTGTTCTCTTGTTCCGTCATGCACGAGGCACTGCACAATCACTACACCCCAGAAGTCCCTGTCCCTCAGCCCTGGCGGCAGCGGCGGATCTGGAGGCGGCGGATCTCTCCCAGAGACCGGCGGATGA (SEQ ID NO: 25).

65) Construction of pFRL-rPS-FIX-C5-Fc-L2-P

[0110] Using FIX-C2-Fc-L2-P as a template, PCR amplification of the FIX-C2 fragment (F7 forward primer: 5'-ATATAAGCTTCCGCCACCATGCAGCGC-3' (SEQ ID NO: 39) and R4 reverse primer: 5'-ATATGGATCCAGCGGGCAAAGCAGGTGG AGGAGC-3' (SEQ ID NO: 40)). The resulting fragment was cloned into the pFRL-rPS-FIX-C2-Fc-L2-P vector using two HindIII/BamHI restriction sites to obtain a bicistronic expression vector pFRL-rPS-FIX-C5-Fc-L2-P co-expressing FIX-C5 -Fc-L2-P/fyrin (C5 is actually a tandem of two C2s).

Amino acid sequence of FIX-C5-Fc-L2-P: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCVSCTEGYRLAENQKSCEPTSKEP ETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAFNKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKEECAFTIYNNGITKVSWGVTGISFY VNWIKEKTKLTGGGGSGGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAGSVAPPPALPAVAPPPALPADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQPSDWLNGKEYKCKVSNKALPPGFSRVETLVAKGQPREPQLT AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSGGGGSLPETGG (SEQ ID NO: 26).

Nucleotide sequence of FIX-C5-Fc-L2-P: TTGAGAATACTGAGCGCACTACTGAGTTTTGGAAGCAGTATGTGGATGGCGATCAGTGCGAGTCCAATCCATGCCTGAATGGCGGATCTTGCAAGGATGATATTAATTCTTACGAGTGTTGGTGCCCATTCGGCTTTGAGGGAAAGAATTGCGAACTGGATGTCACATGTAACATCAAGAACGGCAGATGCGAGCAGTTTTGTAAGAACAGAGCGCCGATAGCATAAGGTGGATGTGTAGTTGTCCTCACT TGAGCCCGCTGTGTCCATTTCCTTGTGGCCGGTCAGCGTCAGCCAGACTTCTAAGCTGACACGCGCCGAGACCGTCTTTCCAGATGTGGATTATGTCAACTCTACTGAGGCCGAGACCATTCTCGATAATATTACTCAGTCTACACAAGTCCTTTAACGACTTCACTCGGGTGGTGGGCGGAGAGGACGCAAAGCCTGGCCAGTTTCCTTGGCAGGTCGTGCTCAACGGAATTGGCTGGATCGA GATTGTGACCGCCTCTCACTGCGTCGAGACAGGCGTCAAGATTACCGTGGTCGCTGGAGAGCACAACATTGAGGAAACAGAGCACACCGAGCAGAAGCGCAATGTGATTCGCATCATCCCACACCACAACTACAACGCCGCTATTAATAAGTACAACCACGACATCGCCCTGTTAGAACTAGATGAGCCTCTGGTCCTGAATAGTTACGTCACTCCTATTTGTGTATCGCCGATAAAGACATTCTCCATCAT TGGGGACGGGTGTTTCACAAGGGACGGTCCGCACTCGTGCTACAATACCTCCGGGTGCCACTGGTGGATCGGGCAACATGCCTCCGGTCTACCAAGTTTACTATATACAATAACATGTTTTGCGCCGGCTTTCACGAGGGCGGACGGGATAGTTGCCAGGGCGATAGTGGAGGCCCTCATGTCACAGAGAGGTGGAGGGAACTTGCTTTCTGACAGGCATTATCATCG GCATCTATACTAAGGTCTCGCGTTACGTCAATTGGATCAAGGAAAAGACAAAGCTGACAGGCGGCGGAGGGAGCCGGCGGCGGAGGCTCTGGCGGCGGCGGATCTGGCGGAGGCGGCTCTGGCGGAGGCGGATCTGTCGCTCCACCAACCTGCACTCCCCGCCGTGGCTCCTCCACCTGCTTTGCCCACTGCATGGATCCGTCCCCCCGCTAG GTCCACCTTGCCCAGCACCTGAGCTGCTGGGCGGACCTAGCGTGTTTCTGTTCCCACCAAAGCCAAAGGACACCCTCATGATTAGTAGAACCCCAAGGTGACTTGCGTCGTGGTCGATGTCAGTCACGAGGACCCCGAGTGAAGTTTAATTGGTATGTAGATGGCGTCGAGGTGCACAACGCTAAGACCAAGCCACGCAACAGACAGTACGGCCATCTACTTACCGGTCGTCTCGTC GCAAAGAGTACAAGTGTAAGGTCAGTAATAAGGCCCTCCCCGCACCTATTGAAAAGACTATTAGCAAGGCAAAGGGCCAGCCTAGGGAGCCCCAGGTCTACACCCTGCCCCCTTCCCGCGATGAGCTGACTAAGAATCAGGTCTCTCTCACTTGCCTCGTGAAGGGCTTTTACCCATCCGACATTGCCGTGGAGTGGGAGTCCAAACGGACAGCCAGAGAATAACTACGACCTGGCATACCTCTC GCTCACCGTTGATAAGTCTCGGTGGCAGCAGGGAAATGTGTTCTCTTGTTCCGTCATGCACGAGGCACTGCACAATCACTACACCCAGAAGTCCCTGTCCCTCAGCCCTGGCGGCAGCGGCGGATCTGGAGGCGGCGGATCTCTCCCAGAGACCGGCGGATAA (SEQ ID NO: 27).

66) Construction of pFRL-rPS-FLX-GS-Fc-L2-P

[0111] The method for constructing the bicistronic expression vector pFRL-rPS-FIX-GS-Fc-L2-P co-expressing FIX-GS-Fc-L2-P/fypin was the same as for pFRL-rPS-FIX-C2 -Fc-L2-P. Primers for PCR amplification of the Fc-L2-P gene included the forward primer F8:

5'-GATTTGGATCCGATAAGACCCACACATGTCCACCTTG-3' (SEQ ID NO: 41) and reverse primer R5:

5'-TAACCGGAATTCATTATCCGCCGGTCTCTGGGAGAGATCCGCCGCCTCCAGAT

CCGCCGCTGCCGCCAGGGCTGAGGGACAGGGACT-3' (SEQ ID NO: 42).

The amino acid sequence of FIX-GS-Fc-L2-P is: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKTRANSADNKVVCVSCTEGYRLAENQKSCEPTSTENKVPLQVFL ILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEENTEQKRNVIRIIPHHNYNAAINКYNHDIALLELDEPLVLNSYVTPIСIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNGITKCAGFHEGGRDSCQGDSGGPMKISFIS VNWIKEKTKLTGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVSDFFKPPNGPSNG SKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSGGGGSLPETGG (SEQ ID NO: 28).

Nucleotide sequence of FIX-GS-Fc-L2-P: GAGAATACTGAGCGCACTACTGAGTTTTGGAAGCAGTATGTGGATGGCGATCAGTGCGAGTCCAATCCATGCCTGAATGGCGGATCTTGCAAGGATGATATTAATTCTTACGAGTGTTGGTGCCCATTCGGCTTTGAGGGAAAGAATTGCGAACTGGATGTCACATGTAACATCAAGAACGGCAGATGCGAGCAGTTTTGTAAGAACAGCGAGCCGATAGCATAAGGTGGTGTGTAGTTGTCATCCACTGACT AGCCCGCTGTGTCCATTTCCTTGTGGCCGGTCAGCGTCAGCCAGACTTCTAAGCTGACACGCGCCGAGACCGTCTTTCCAGATGTGGATTATGTCAACTCTACTGAGGCCGAGACCATTCTCGATAATATTACTCAGTCTACACAAGTCCTTTAACGACTTCACTCGGGTGGTGGGCGGAGAGGACGCAAAGCCTGGCCAGTTTCCTTGGCAGGTCGTGTGCTCAACGGAATGTGGCGATCGACATGATATGGCTGGATCGAATGATATG TTGTGACCGCCGCTCACTGCGTCGAGACAGGCGTCAAGATTACCGTGGTCGCTGGAGAGCACAACATTGAGGAAACAGAGCACACCGAGCAGAAGCGCAATGTGATTCGCATCATCCCACACCACAACTACAACGCCGCTATTAATAAGTACAACCACGACATCGCCCTGTTAGAACTAGATGAGCCTCTGGTCCTGAATAGTTACGTCACTCCTATTCGTGTCGCCGGATCTCTCAT GGGACGGGTGTTTCAAGGGACGGTCCGCACTCGTGCTACAATACCTCCGGGTGCCACTGGTGGATCGGGCAACATGCCTCCGGTCTACCAAGTTTACTATATACAATAACATGTTTTGCGCCGGCTTTCACGAGGGCGGACGGGATAGTTGCCAGGGCGATAGTGGAGGCCCTGGGTCACAGAGGTGGAGGAACTAGCTTTCTGACAGGCATTATCTCAGCGGA CATCTATACTAAGGTCTCGCGTTACGTCAATTGGATCAAGGAAAAGACAAAGCTGACAGGCGGCGGAGGGAGCCGGCGGCGGAGGCTCTGGCGGCGGCGGATCTGGCGGAGGCGGCTCTGGCGGAGGCGGATCCGATAAGACCCCACATGTCCACCTTGCCCAGCACCTGAGCTGCTGGGCGGACCTAGCGATGTTTCTGTGTTCCCACCAAAGCCAAAGGACACCACT GTCGATGTCAGTCACGAGGACCCCGAGGTGAAGTTTAATTGGTATGTAGATGGCGTCGAGTGCACAACGCTAAGACCAAGCCACGCGAAGAGCAGTACGCATCTACTTACCGGGTCGTCTCTGTGCTGACTGTGCTGCACCAGGATTGGCTCAATGGCAAAGTACAAGTGTGTAAGGTCAGTAATAAGGCCCTCCCCGCACCTATTGAAAAGACTGCATTAGCAAGCTCTAGGGCCCCA CCCGCGATGAGCTGACTAAGAATCAGGTCTCTCTCACTTGCCTCGTGAAGGCTTTTACCCATCCGACATTGCCGTGGAGTGGGAGTCCAAACGGACAGCCAGAGAATAACTACAAGACAACTCCTCCCGTGCTCGATAGCGACGGTTCATTCTTCCTGTACTCCAAGCTCACCGTTGATAAGTCTCGGTGGCAGCAGGGAAATGTGTTCTTGTTCCAGGCACCATCTCACTACTGCACCATTC GCAGCGGCGGATCTGGAGGCGGCGGATCTCTCCCAGAGACCGGCGGATAA (SEQ ID NO: 29).

[0112] A structural diagram of the above bicistronic expression vector FIX-Ll-Fc-L2-P/fusin is shown in FIG. 1 A.

Construction of a vector co-expressing GGCX and VKORC1 [0113] FIX is a vitamin K (VK)-dependent glycoprotein, and with the help of VK, the glutamate residue in its N-terminal G1a domain is carboxylated to form γ-carboxyglutamate, which provides the coagulation activity of FIX. During Gla modification, two key enzymes are γ-glutamyl carboxylase (GGCX) and subunit 1 of the vitamin K epoxide reductase complex (VKORC l). Thus, in this invention, the plasmid pcDNA3.1-Hyg-VKGC was constructed to increase the expression level of functional FLX by co-transfection with the plasmid with FIX. The structure of the plasmid pcDNA3.1-Hyg-VKGC is shown in FIG. 1B, and the construction method was as follows: gamma-glutamyl carboxylase (GGCX), 1 subunit of vitamin K-epoxide reductase complex (VKORCl), and internal ribosome entry site (IRES) DNA were synthesized by whole gene synthesis; The IRES was cloned into the pcDNA3.1(+)/hygro vector by cleavage at two NheI/XhoI restriction sites to construct the plasmid p3.1hyg-IRES; the VKORCl gene (Nhel/BamHI sites) and the GGCX gene (NotI/XhoI sites) were then cloned by restriction at the other two sites into p3.1hyg-IRES to construct a pcDNA3.1-Hyg-VKGC vector co-expressing GGCX and VKORCl.

2. Construction of stable cell lines

[0114] Each type of FIX fusion protein expression plasmid constructed as described above was transfected into CHO DG44 host cells by electroporation. For electroporation of 1×10 ⁷ cells, 40 μg of FIX-L1-Fc-L2-P/furin co-expressing plasmid and 20 μg of pcDNA3.1-Hyg-VKGC expression plasmid were mixed gently and added to electroporation cuvettes (Bio-Rad , 4 mm). A Bio-Rad electroporation device (GENE PULSER XCELL) was used and the following electroporation parameters were set:

[0115] After electroporation, the cells were transferred to recovery medium. After 24 hours of culture, cells were planted in fifteen 96-well plates and the medium was changed to screening medium containing Opti-MEM I+50 nM MTX. After culturing the cells for 4-5 days, medium containing 50 nM MTX was added 2-3 times. When cells reached a confluency of 50% or more, clones with high expression of the target protein were screened by dot blot, and a mouse anti-human IgG Fc fragment was used as an antibody. Selected clones with relatively high levels of expression were subsequently expanded into 24-well plates, 6-well plates, cell culture dishes, and cell culture shake flasks.

[0116] To increase the yield of the fusion protein, cells were cultured under pressure of increasing concentrations of MTX. Thus, by inhibiting the DHFR gene with MTX, co-amplification of the DHFR gene and the fusion protein gene was achieved.

3. Production of the fusion protein 3.1 Thawing and cell passage

[0117] Cells were thawed and cultured in culture flasks, and after density increase, cells were transferred to shake flasks containing CD DG44 + 5 mg/l VK medium for culture and passages. The passage was performed when the cells reached a density in the range of 2.5-3.5x10 ⁶ cells/ml, and planted at a density of 0.8-1.0x10 ⁶ cells/ml. When the cell culture reached 1.8-2 L and the cell density reached approximately 3.5x10 ⁶ cells/ml, the cells were cultured in the tanks.

3.2 Fermentation

[0118] A 15 L bioreactor (Applikon, Biobundle 15L) was used for fermentation, the inoculum volume was 8 L, and the cell density was 1.0×10 ⁶ cells/mL. The following cell culture parameters were set: 1) pH 6.8-7.4; 2) dissolved oxygen (DO) 40%; 3) oxygen supply mode: ventilation with large bubbles; 4) cooling method: cultivation at 37°C at an early stage and cooling to 33°C on the 5th day; 5) mixing speed: 80-140 rpm; 6) Nutrient medium supply method: 2%, 3%, 4%, 3%, 2%, 2%, 2%, 2% and 2% of the medium supplied on the fourth day, the total volume of the supplied nutrient medium was 24% , cells were counted with a cell counter daily; 7) collection: after culturing for about 13 days, the supernatant was collected by centrifugation at 3000 g for 10 minutes.

3.3 Sample analysis and purification

[0119] The conditioned supernatant was purified in three steps: affinity chromatography, mixed mode chromatography, and anion exchange chromatography. The purified fusion protein was analyzed by SDS-PAGE and SEC-HPLC. As shown in FIG. 2A, the SDS-PAGE results demonstrate that the size of the proteins of interest largely matched the calculated molecular weight.

размером частиц 2,7 мкм, внутренним диаметром 7,8 мм и длиной 300 мм, элюировали градиентом 50 мМ Tris+150 мМ NaCl, рН 7,2, при этом температура колонки составляла 30°С, скорость потока составляла 0,5 мл/мин, температура устройства для ввода образца составляла 4,0°С, а объем вводимого образца составлял 30 мкг.На Фиг. 2 В показаны результаты очистки на примере белка FIX-C1-Fc-L2-P, у которого время удерживания составляло 15,1 мин, а чистота по данным SEC после трехстадийной очистки составляла 95%.[0120] SEC-HPLC was run on an Agilent Advancebio SEC column with pore size

particle size 2.7 μm, inner diameter 7.8 mm and length 300 mm, eluted with a gradient of 50 mm Tris + 150 mm NaCl, pH 7.2, while the column temperature was 30°C, the flow rate was 0.5 ml/ min, the temperature of the sample injector was 4.0°C, and the volume of the injected sample was 30 µg. FIG. 2B shows purification results for the FIX-C1-Fc-L2-P protein, which had a retention time of 15.1 minutes and a SEC purity of 95% after three-step purification.

Example 2 Determination of FIX-Ll-Fc-L2-P activity in vitro and comparison

[0121] In this Example, the in vitro activity of FIX-C1-Fc-L2-P, FIX-C2-Fc-L2-P, FIX-C3-Fc-L2-P, FIX-C4-Fc-L2-P and FIX-C5-Fc-L2-P obtained in Example 1 and compared with the in vitro activity of FIX-GS-Fc-L2-P without a rigid element in the first L1 linker.

[0122] Human coagulation factor IX (hFIX) activity was measured in a one-step activated partial thromboplastin time (APTT) method using ellagic acid as an activator and using the 8th International Standard 2009 FIX concentrate (WHO standard for FIX activity) to determine APTT. . To construct a linear standard curve, the logarithms of the activity of the known standard and the measured APTT time were plotted. The test sample was diluted to fall within the range of the standard curve and mixed with FIX-deficient plasma to measure the APTT value. The biological activity of the test sample was calculated from the standard curve.

[0123] Aliquots of active FIX standard or sample were brought to room temperature, then pipetted at 10 µl of each and diluted to approximately 1 IU/ml with 5% FIX deficient plasma, and then diluted 10-fold and 20-fold (and further diluted 40-fold and 80-fold for the active standard) with 5% FIX deficient plasma and mixed thoroughly by vortexing. The diluted standard or sample was placed in the automatic coagulometer sample rack and the reagents used were placed in the appropriate reagent positions. The corresponding measurement program was then run on an automatic coagulation analyzer to determine the clotting time and measurements were repeated twice for each dilution. To construct a standard curve, the logarithm of the standard activity was plotted along the abscissa axis, and the logarithm of the clotting time was plotted along the ordinate axis. The activity corresponding to the measured clotting time of the diluted test product was calculated from the standard curve and then multiplied by the dilution factor to obtain the corresponding activity.

[0124] Unexpectedly found that, as shown in FIG. 3, specific activity of FIX-Cl-Fc-L2-P, FIX-C2-Fc-L2-P, FIX-C3-Fc-L2-P, FIX-C4-Fc-L2-P and FIX-C5-Fc molecules -L2-P, in which the first L1 linker contained a rigid element, was significantly higher than that of FIX-GS-Fc-L2-P, in which the first L1 linker did not have a rigid element, indicating that the first L1 linker, containing a rigid element can significantly increase the specific activity of the molecule.

[0125] In addition, unexpectedly, among all fusion proteins containing a rigid element in L1, FIX-C1-Fc-L2-P had a significantly higher activity, which was almost twice or more than the specific activity of other fusion proteins (Fig. 3). Thus, FIX-C1-Fc-L2-P was selected for further study in the following Examples. Example 3 Preparation of FIX-Cl-Fc-L2-PEG Fusion Protein Conjugate

[0126] The FIX-C1-Fc-L2-P protein contains LPETGG at the C-terminus, which can be specifically recognized by sortase A. The amide bond between T and G was cut by exposure to sortase A, and T interacted with the sulfhydryl group C at position 184 of sortase to form an intermediate thioether bond, which was then attacked by GGGAA-PEG with poly-Gly at the N-terminus. As a result, PEG of a certain molecular weight could bind to the C-terminus of the protein.

[0127] PEGylation reaction mixture: 40KD-GGGAA-PEG was dissolved in buffer and the pH was adjusted to 7.5. Protein and 40KD-GGGAA-PEG were then added to a 50 mM Tris 150 mM NaCl pH 7.5 buffer system at a ratio of 1:20, and then Sortase A and 10 mM CaCl ₂ were added. After 30 minutes, EDTA was added to stop the reaction.

[0128] The degree of conjugation of the conjugate (FIX-C1-Fc-L2-PEG) was checked using SEC-HPLC, which was carried out on a TOSOH TSKgel UItraSW Aggregate column, with a particle size of 3 μm, an internal diameter of 7.8 mm and a length of 300 mm, eluted with a gradient of 50 mM Tris + 150 mM NaCl elution buffer, pH 7.2, column temperature 25°C, flow rate 0.5 ml/min, sample injector temperature 4.0°C, injection volume sample was 50 μg, the retention time of the conjugated protein was 12-14.5 minutes, and the retention time of the substrate was 15 minutes. On FIG. 14 shows the results of a FIX-C1-Fc-L2-PEG assay where the PEG-conjugated product reached 70% after 30 minutes.

Purification and analysis of the PEG-conjugated product

[0129] Anion exchange chromatography of the PEG-conjugated products was performed to remove unreacted GGGAA-PEG sortase A and unconjugated substrate (FIX-C1-Fc-L2-P), and the residual amount of unconjugated substrate was less than 3%.

[0130] Conjugated samples were separated using anion exchange chromatography, and collected peak fractions at various stages. Samples taken at each peak were analyzed by SDS-HPLC and SEC, and then samples with less than 5% unconjugated substrate were pooled for purity by SEC. The results of the analysis of FIX-C1-Fc-L2-PEG by SEC are shown in FIG. 5 where the PEGylated product was 97.8% pure by SEC. Among them, the double conjugation product, whose retention time was 13.20 min, accounted for 15.7%, the single conjugation product, whose retention time was 13.70 min, accounted for 82.2%, and the unreacted substrate FIX-C1- Fc-L2-P, which had a retention time of 15.94 minutes, accounted for 1.24%.

Example 4 Maintaining excellent activity of FIX-Cl-Fc-L2-PEG conjugate in vitro

[0131] In this Example, the activity of the FIX-Cl-Fc-L2-PEG conjugate in vitro was further investigated. Human coagulation factor IX (hFIX) activity was measured in a one-step activated partial thromboplastin time (APTT) method using ellagic acid as an activator and the 8th International Standard 2009 for FIX Concentrate (WHO standard for FIX activity) to determine APTT. . To construct a linear standard curve, the logarithms of the activity of a known standard and the measured APTT time were used. The test sample was diluted to fall within the range of the standard curve and mixed with FIX-deficient plasma to measure the APTT value. The biological activity of the test sample was calculated from the standard curve.

[0132] Aliquots of active FIX standard or sample were brought to room temperature, then pipetted at 10 μl of each and diluted to approximately 1 IU/ml with 5% FIX deficient plasma, and then diluted 10-fold and 20-fold (and further diluted 40-fold and 80-fold for the active standard) with 5% FIX deficient plasma and mixed thoroughly by vortexing. The diluted standard or sample was placed in the automatic coagulometer sample rack and the reagents used were placed in the appropriate reagent positions. The corresponding measurement program was then run on an automatic coagulation analyzer to determine the clotting time and measurements were repeated twice for each dilution. To construct a standard curve, the logarithm of the standard activity was plotted along the abscissa axis, and the logarithm of the clotting time was plotted along the ordinate axis. The activity corresponding to the measured clotting time of the diluted test product was calculated from the standard curve and then multiplied by the dilution factor to obtain the corresponding activity.

[0133] As shown in FIG. 3, the specific activity of the FIX-Cl-Fc-L2-PEG conjugate was lower than that of FIX-C1-Fc-L2-P, reflecting that PEG conjugation reduces the specific activity of the molecule to some extent in vitro, and the above results were consistent with the general ideas in this area. However, it should be noted that the activity of FIX-C1-Fc-L2-PEG in vitro still remained at a relatively high level, not only much higher than that of non-PEG FIX-GS-Fc-L2-P, but was also comparable to the activity of other non-PEG FIX-L1-Fc-L2-P fusion proteins, with the exception of FIX-C1-Fc-L2-P

Example 5 FIX-C1-Fc-L2-PEG shows a significant increase in in vivo half-life

[0134] In this Example, the activity of the FIX-C1-Fc-L2-PEG conjugate in mice was further investigated. In particular, FIX-C1-Fc-L2-PEG was administered to HB mice and blood samples were taken from the retro-orbital space at various time points. Plasma was then separated to determine plasma hFIX activity in a one-step method, and pharmacokinetic parameters were calculated using WinNonlin 5.2.1 software.

[0135] The animals tested in this Example are described in the Table below.

[0136] In this Example, the following samples were used:

[0137] The experiment involved 2 groups. The BeneFIX reference drug and the FIX-C1-Fc-L2-PEG conjugate sample were administered via the tail vein at 300 IU/kg. Blood was taken at the following time points: 1) BeneFIX: 0.083, 0.5, 1, 4, 8, 24, 48 and 72 hours; 2) FIX-C1-Fc-L2-PEG: 0.083, 0.5, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 h. At each moment of blood sampling in each group, 5 HB animals (2 males, 3 females) were bled from the retroorbital space and hFIX activity was determined.

[0138] As shown in FIG. 6, BeneFIX maintained plasma hFIX levels above 5% for 2 days post-administration, but 3 days post-administration, plasma hFIX levels were only 1%-2%; while FIX-C1-Fc-L2-PEG still maintained the plasma hFIX level above 5% for 3 days after administration, and the plasma hFIX level was above 2% 6 days after administration, in particular, after 10 days after the introduction of hFIX in plasma was still maintained at the level of 1%-2%. The results of the experiment show that the terminal phase half-life (T1/2) of FIX-C1-Fc-L2-PEG reaches 70 h, which is significantly higher than the T1/2 of the reference drug BeneFIX (14 h; which is consistent with the values of the half-life described in literature) and T1/2 Alprolix (46 h) described in the literature (Peters et al. Prolonged activity of factor IX as a monomeric Fc fusion protein. Blood 2010; 115(10): 2057-64.). The maximum blood concentration (Cmax) was 119 IU/dL, the area under the drug concentration-time curve (AUClast) was 1475 h*IU/dL, the elimination rate (CL) was 0.2033 dd/h/kg, and the mean the retention time in the body (MRT) was 72 h, providing an excellent pharmacodynamic effect. It can be seen that the site-specific conjugation of hFIX fused with Fc and PEG significantly increases the half-life of the hFIX molecule and provides a significantly longer in vivo effect compared to existing drugs, which opens up good prospects for practical application.

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Sequence listing

SEQUENCE LISTING

<110> JIANGSU GENSCIENCES INC.

<120> IMPROVED FIX FUSION PROTEIN AND CONJUGATE AND USE THEREOF

<130> PCT/CN2020/097911

<150> CN 201910591280.0

<151> 2019-07-02

<150> PCT/CN2020/097911

<151> 2020-06-24

<160> 42

<170> PatentIn version 3.2

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Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

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His Gly Ile Val Val Ser Ile Leu Asp Asp Gly Ile Glu Lys Asn His

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Gly Val Cys Gly Val Gly Val Ala Tyr Asn Ala Arg Ile Gly Gly Val

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Arg Met Leu Asp Gly Glu Val Thr Asp Ala Val Glu Ala Arg Ser Leu

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Glu Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Arg Leu Ala Glu Glu

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Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Glu His Asp Ser Cys Asn

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Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Leu Ser Ile Ser Ser Ala

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Thr Gln Phe Gly Asn Val Pro Trp Tyr Ser Glu Ala Cys Ser Ser Thr

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Leu Ala Thr Thr Tyr Ser Ser Gly Asn Gln Asn Glu Lys Gln Ile Val

340 345 350

Thr Thr Asp Leu Arg Gln Lys Cys Thr Glu Ser His Thr Gly Thr Ser

355 360 365

Ala Ser Ala Pro Leu Ala Ala Gly Ile Ile Ala Leu Thr Leu Glu Ala

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Asn Lys Asn Leu Thr Trp Arg Asp Met Gln His Leu Val Val Gln Thr

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Ser Lys Pro Ala His Leu Asn Ala Asn Asp Trp Ala Thr Asn Gly Val

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Gly Arg Lys Val Ser His Ser Tyr Gly Tyr Gly Leu Leu Asp Ala Gly

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Ala Met Val Ala Leu Ala Gln Asn Trp Thr Thr Val Ala Pro Gln Arg

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Lys Cys Ile Ile Asp Ile Leu Thr Glu Pro Lys Asp Ile Gly Lys Arg

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Leu Glu Val Arg Lys Thr Val Thr Ala Cys Leu Gly Glu Pro Asn His

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Ile Thr Arg Leu Glu His Ala Gln Ala Arg Leu Thr Leu Ser Tyr Asn

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Arg Arg Gly Asp Leu Ala Ile His Leu Val Ser Pro Met Gly Thr Arg

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Ser Thr Leu Leu Ala Ala Arg Pro His Asp Tyr Ser Ala Asp Gly Phe

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Asn Asp Trp Ala Phe Met Thr Thr His Ser Trp Asp Glu Asp Pro Ser

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Gly Glu Trp Val Leu Glu Ile Glu Asn Thr Ser Glu Ala Asn Asn Tyr

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Gly Thr Leu Thr Lys Phe Thr Leu Val Leu Tyr Gly Thr Ala Pro Glu

565 570 575

Gly Leu Pro Val Pro Pro Glu Ser Ser Gly Cys Lys Thr Leu Thr Ser

580 585 590

Ser Gln Ala Cys Val Val Cys Glu Glu Gly Phe Ser Leu His Gln Lys

595 600 605

Ser Cys Val Gln His Cys Pro Pro Gly Phe Ala Pro Gln Val Leu Asp

610 615 620

Thr His Tyr Ser Thr Glu Asn Asp Val Glu Thr Ile Arg Ala Ser Val

625 630 635 640

Cys Ala Pro Cys His Ala Ser Cys Ala Thr Cys Gln Gly Pro Ala Leu

645 650 655

Thr Asp Cys Leu Ser Cys Pro Ser His Ala Ser Leu Asp Pro Val Glu

660 665 670

Gln Thr Cys Ser Arg Gln Ser Gln Ser Ser Arg Glu Ser Pro Pro Gln

675 680 685

Gln Gln Pro Pro Arg Leu Pro Pro Glu Val Glu Ala Gly Gln Arg Leu

690 695 700

Arg Ala Gly Leu Leu Pro Ser His Leu Pro Glu

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<223> FIX-C1-Fc-L2-P

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Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

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Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

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Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn

35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn

65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln

85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys

115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe

130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly

145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu

195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe

210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp

225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile

245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

275 280 285

His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn

290 295 300

Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu

305 310 315 320

Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile

325 330 335

Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr

340 345 350

Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val

355 360 365

Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg

370 375 380

Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His

385 390 395 400

Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val

405 410 415

Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly

420 425 430

Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser

435 440 445

Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Val Ala Pro Pro Pro Ala Leu Pro Ala Pro

485 490 495

Val Arg Leu Pro Gly Pro Ala Asp Lys Thr His Thr Cys Pro Pro Cys

500 505 510

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

515 520 525

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

530 535 540

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

545 550 555 560

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

565 570 575

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

580 585 590

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

595 600 605

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

610 615 620

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

625 630 635 640

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

645 650 655

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

660 665 670

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

675 680 685

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

690 695 700

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

705 710 715 720

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Ser Gly

725 730 735

Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

740 745

<210> 19

<211> 2241

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-C1-Fc-L2-P

<400> 19

atgcagcgcg tcaatatgat tatggccgag tctccaggcc tcattaccat ttgtctgctg 60

ggctacctgc tgtccgcaga gtgtaccgtc ttcctcgacc acgagaacgc aaataagatt 120

ctcaataggc ctaagcgcta caattccggga aagctcgaag agtttgtcca gggaaatctg 180

gagcgcgagt gtatggagga aaagtgtagc tttgaagaag ctagagaggt gtttgagaat 240

actgagcgca ctactgagtt ttggaagcag tatgtggatg gcgatcagtg cgagtccaat 300

ccatgcctga atggcggatc ttgcaaggat gatattaatt cttacgagtg ttggtgccca 360

ttcggctttg agggaaagaa ttgcgaactg gatgtcacat gtaacatcaa gaacggcaga 420

tgcgagcagt tttgtaagaa cagcgccgat aataaggtgg tgtgtagttg tactgaggga 480

taccgcctcg cagagaatca gaagtcctgt gagcccgctg tgccatttcc ttgtggccgg 540

gtcagcgtca gccagacttc taagctgaca cgcgccgaga ccgtctttcc agatgtggat 600

tatgtcaact ctactgaggc cgagaccatt ctcgataata ttactcagtc tacacagtcc 660

tttaacgact tcactcgggt ggtgggcgga gaggacgcaa agcctggcca gtttccttgg 720

caggtcgtgc tcaacggaaa ggtggacgca ttttgtggcg gatctatcgt gaatgagaag 780

tggattgtga ccgccgctca ctgcgtcgag acaggcgtca agattaccgt ggtcgctgga 840

gagcacaaca ttgaggaaac agagcacacc gagcagaagc gcaatgtgat tcgcatcatc 900

ccacaccaca actacaacgc cgctattaat aagtacaacc acgacatcgc cctgttagaa 960

ctagatgagc ctctggtcct gaatagttac gtcactccta tttgtatcgc cgataaagaa 1020

tacactaaca tctttctcaa gttcggctct ggctatgtat ctggatgggg acgggtgttt 1080

cacaagggac ggtccgcact cgtgctacaa tacctccggg tgccactggt ggatcgggca 1140

acatgcctcc ggtctaccaa gtttactata tacaataaca tgttttgcgc cggctttcac 1200

1260

ggaactagct ttctgacagg cattatctct tggggcgaag agtgcgctat gaagggaaag 1320

tacggcatct atactaaggt ctcgcgttac gtcaattgga tcaaggaaaa gacaaagctg 1380

acaggcggcg gagggagcgg cggcgggaggc tctggcggcg gcggatctgg cggaggcggc 1440

tctggcggag gcggatccgt cgctccacca cctgcactcc ccgcccctgt cagactgcct 1500

ggacccgctg ataagaccca cacatgtcca ccttgcccag cacctgagct gctgggcgga 1560

cctagcgtgt ttctgttccc accaaagcca aaggacaccc tcatgattag tagaacccca 1620

1680

tatgtagatg gcgtcgaggt gcacaacgct aagaccaagc cacgcgaaga gcagtacgca 1740

tctacttacc gggtcgtctc tgtgctgact gtgctgcacc aggattggct caatggcaaa 1800

gagtacaagt gtaaggtcag taataaggcc ctccccgcac ctattgaaaa gactattagc 1860

aaggcaaagg gccagcctag ggagccccag gtctacaccc tgcccccttc ccgcgatgag 1920

ctgactaaga atcaggtctc tctcacttgc ctcgtgaagg gcttttaccc atccgacatt 1980

gccgtggagt gggagtccaa cggacagcca gagaataact acaagacaac tcctcccgtg 2040

ctcgatagcg acggttcatt cttcctgtac tccaagctca ccgttgataa gtctcggtgg 2100

cagcagggaa atgtgttctc ttgttccgtc atgcacgagg cactgcacaa tcactacacc 2160

cagaagtccc tgtccctcag ccctggcaaa ggcagcggcg gatctggagg cggcggatcc 2220

ctcccagaga ccggcggatg a 2241

<210> 20

<211> 746

<212> PRT

<213> Artificial sequence

<220>

<223> FIX-C2-Fc-L2-P

<400> 20

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn

35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn

65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln

85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys

115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe

130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly

145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu

195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe

210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp

225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile

245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

275 280 285

His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn

290 295 300

Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu

305 310 315 320

Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile

325 330 335

Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr

340 345 350

Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val

355 360 365

Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg

370 375 380

Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His

385 390 395 400

Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val

405 410 415

Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly

420 425 430

Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser

435 440 445

Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Val Ala Pro Pro Pro Ala Leu Pro Ala Val

485 490 495

Ala Pro Pro Pro Ala Leu Pro Ala Asp Lys Thr His Thr Cys Pro Pro

500 505 510

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

515 520 525

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

530 535 540

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

545 550 555 560

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

565 570 575

Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

580 585 590

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

595 600 605

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

610 615 620

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

625 630 635 640

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

645 650 655

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

660 665 670

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

675 680 685

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

690 695 700

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

705 710 715 720

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Ser Gly Gly Ser Gly

725 730 735

Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

740 745

<210> 21

<211> 2241

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-C2-Fc-L2-P

<400> 21

atgcagcgcg tcaatatgat tatggccgag tctccaggcc tcattaccat ttgtctgctg 60

ggctacctgc tgtccgcaga gtgtaccgtc ttcctcgacc acgagaacgc aaataagatt 120

ctcaataggc ctaagcgcta caattccggga aagctcgaag agtttgtcca gggaaatctg 180

gagcgcgagt gtatggagga aaagtgtagc tttgaagaag ctagagaggt gtttgagaat 240

actgagcgca ctactgagtt ttggaagcag tatgtggatg gcgatcagtg cgagtccaat 300

ccatgcctga atggcggatc ttgcaaggat gatattaatt cttacgagtg ttggtgccca 360

ttcggctttg agggaaagaa ttgcgaactg gatgtcacat gtaacatcaa gaacggcaga 420

tgcgagcagt tttgtaagaa cagcgccgat aataaggtgg tgtgtagttg tactgaggga 480

taccgcctcg cagagaatca gaagtcctgt gagcccgctg tgccatttcc ttgtggccgg 540

gtcagcgtca gccagacttc taagctgaca cgcgccgaga ccgtctttcc agatgtggat 600

tatgtcaact ctactgaggc cgagaccatt ctcgataata ttactcagtc tacacagtcc 660

tttaacgact tcactcgggt ggtgggcgga gaggacgcaa agcctggcca gtttccttgg 720

caggtcgtgc tcaacggaaa ggtggacgca ttttgtggcg gatctatcgt gaatgagaag 780

tggattgtga ccgccgctca ctgcgtcgag acaggcgtca agattaccgt ggtcgctgga 840

gagcacaaca ttgaggaaac agagcacacc gagcagaagc gcaatgtgat tcgcatcatc 900

ccacaccaca actacaacgc cgctattaat aagtacaacc acgacatcgc cctgttagaa 960

ctagatgagc ctctggtcct gaatagttac gtcactccta tttgtatcgc cgataaagaa 1020

tacactaaca tctttctcaa gttcggctct ggctatgtat ctggatgggg acgggtgttt 1080

cacaagggac ggtccgcact cgtgctacaa tacctccggg tgccactggt ggatcgggca 1140

acatgcctcc ggtctaccaa gtttactata tacaataaca tgttttgcgc cggctttcac 1200

1260

ggaactagct ttctgacagg cattatctct tggggcgaag agtgcgctat gaagggaaag 1320

tacggcatct atactaaggt ctcgcgttac gtcaattgga tcaaggaaaa gacaaagctg 1380

acaggcggcg gagggagcgg cggcgggaggc tctggcggcg gcggatctgg cggaggcggc 1440

tctggcggag gcggatccgt cgctccacca cctgcactcc ccgccgtggc tcctccacct 1500

gctttgcccg ctgataagac ccacacatgt ccaccttgcc cagcacctga gctgctgggc 1560

ggacctagcg tgtttctgtt cccaccaaag ccaaaggaca ccctcatgat tagtagaacc 1620

ccagaggtga cttgcgtcgt ggtcgatgtc agtcacgagg accccgaggt gaagtttaat 1680

tggtatgtag atggcgtcga ggtgcacaac gctaagacca agccacgcga agagcagtac 1740

gcatctactt accgggtcgt ctctgtgctg actgtgctgc accaggattg gctcaatggc 1800

aaagagtaca agtgtaaggt cagtaataag gccctccccg cacctattga aaagactatt 1860

agcaaggcaa agggccagcc tagggagccc caggtctaca ccctgccccc ttcccgcgat 1920

gagctgacta agaatcaggt ctctctcact tgcctcgtga agggctttta cccatccgac 1980

attgccgtgg agtgggagtc caacggacag ccagagaata actacaagac aactcctccc 2040

gtgctcgata gcgacggttc attcttcctg tactccaagc tcaccgttga taagtctcgg 2100

tggcagcagg gaaatgtgtt ctcttgttcc gtcatgcacg aggcactgca caatcactac 2160

acccagaagt ccctgtccct cagccctggc ggcagcggcg gatctggagg cggcggatct 2220

ctcccagaga ccggcggata a 2241

<210> 22

<211> 764

<212> PRT

<213> Artificial sequence

<220>

<223> FIX-C3-Fc-L2-P

<400> 22

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn

35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn

65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln

85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys

115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe

130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly

145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu

195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe

210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp

225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile

245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

275 280 285

His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn

290 295 300

Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu

305 310 315 320

Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile

325 330 335

Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr

340 345 350

Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val

355 360 365

Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg

370 375 380

Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His

385 390 395 400

Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val

405 410 415

Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly

420 425 430

Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser

435 440 445

Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Val Ala Pro Pro Pro Ala Leu Pro Ala Val

485 490 495

Ala Pro Pro Pro Ala Leu Pro Ala Val Ala Pro Pro Pro Ala Leu Pro

500 505 510

Ala Pro Val Arg Leu Pro Gly Pro Ala Asp Lys Thr His Thr Cys Pro

515 520 525

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

530 535 540

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

545 550 555 560

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

565 570 575

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

580 585 590

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

595 600 605

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

610 615 620

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

625 630 635 640

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

645 650 655

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

660 665 670

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

675 680 685

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

690 695 700

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

705 710 715 720

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

725 730 735

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly

740 745 750

Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

755 760

<210> 23

<211> 2295

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-C3-Fc-L2-P

<400> 23

atgcagcgcg tcaatatgat tatggccgag tctccaggcc tcattaccat ttgtctgctg 60

ggctacctgc tgtccgcaga gtgtaccgtc ttcctcgacc acgagaacgc aaataagatt 120

ctcaataggc ctaagcgcta caattccggga aagctcgaag agtttgtcca gggaaatctg 180

gagcgcgagt gtatggagga aaagtgtagc tttgaagaag ctagagaggt gtttgagaat 240

actgagcgca ctactgagtt ttggaagcag tatgtggatg gcgatcagtg cgagtccaat 300

ccatgcctga atggcggatc ttgcaaggat gatattaatt cttacgagtg ttggtgccca 360

ttcggctttg agggaaagaa ttgcgaactg gatgtcacat gtaacatcaa gaacggcaga 420

tgcgagcagt tttgtaagaa cagcgccgat aataaggtgg tgtgtagttg tactgaggga 480

taccgcctcg cagagaatca gaagtcctgt gagcccgctg tgccatttcc ttgtggccgg 540

gtcagcgtca gccagacttc taagctgaca cgcgccgaga ccgtctttcc agatgtggat 600

tatgtcaact ctactgaggc cgagaccatt ctcgataata ttactcagtc tacacagtcc 660

tttaacgact tcactcgggt ggtgggcgga gaggacgcaa agcctggcca gtttccttgg 720

caggtcgtgc tcaacggaaa ggtggacgca ttttgtggcg gatctatcgt gaatgagaag 780

tggattgtga ccgccgctca ctgcgtcgag acaggcgtca agattaccgt ggtcgctgga 840

gagcacaaca ttgaggaaac agagcacacc gagcagaagc gcaatgtgat tcgcatcatc 900

ccacaccaca actacaacgc cgctattaat aagtacaacc acgacatcgc cctgttagaa 960

ctagatgagc ctctggtcct gaatagttac gtcactccta tttgtatcgc cgataaagaa 1020

tacactaaca tctttctcaa gttcggctct ggctatgtat ctggatgggg acgggtgttt 1080

cacaagggac ggtccgcact cgtgctacaa tacctccggg tgccactggt ggatcgggca 1140

acatgcctcc ggtctaccaa gtttactata tacaataaca tgttttgcgc cggctttcac 1200

1260

ggaactagct ttctgacagg cattatctct tggggcgaag agtgcgctat gaagggaaag 1320

tacggcatct atactaaggt ctcgcgttac gtcaattgga tcaaggaaaa gacaaagctg 1380

acaggcggcg gagggagcgg cggcgggaggc tctggcggcg gcggatctgg cggaggcggc 1440

tctggcggag gcggatctgt cgctccacca cctgcactcc ccgccgtggc tcctccacct 1500

gctttgcccg ctgtcgctcc accacctgca ctccccgccc ctgtcagact gcctggaccc 1560

gctgataaga cccacacatg tccaccttgc ccagcacctg agctgctggg cggacctagc 1620

gtgtttctgt tcccaccaaa gccaaaggac accctcatga ttagtagaac cccagaggtg 1680

acttgcgtcg tggtcgatgt cagtcacgag gaccccgagg tgaagtttaa ttggtatgta 1740

gatggcgtcg aggtgcacaa cgctaagacc aagccacgcg aagagcagta cgcatctact 1800

taccgggtcg tctctgtgct gactgtgctg caccaggatt ggctcaatgg caaagagtac 1860

aagtgtaagg tcagtaataa ggccctcccc gcacctattg aaaagactat tagcaaggca 1920

aagggccagc ctagggagcc ccaggtctac accctgcccc cttcccgcga tgagctgact 1980

aagaatcagg tctctctcac ttgcctcgtg aagggctttt acccatccga cattgccgtg 2040

gagtgggagt ccaacggaca gccagagaat aactacaaga caactcctcc cgtgctcgat 2100

agcgacggtt cattcttcct gtactccaag ctcaccgttg ataagtctcg gtggcagcag 2160

ggaaatgtgt tctcttgttc cgtcatgcac gaggcactgc acaatcacta cacccagaag 2220

tccctgtccc tcagccctgg caaaaggcagc ggcggatctg gaggcggcgg atccctccca 2280

gagaccggcg gatga 2295

<210> 24

<211> 763

<212> PRT

<213> Artificial sequence

<220>

<223> FIX-C4-Fc-L2-P

<400> 24

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn

35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn

65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln

85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys

115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe

130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly

145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu

195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe

210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp

225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile

245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

275 280 285

His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn

290 295 300

Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu

305 310 315 320

Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile

325 330 335

Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr

340 345 350

Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val

355 360 365

Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg

370 375 380

Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His

385 390 395 400

Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val

405 410 415

Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly

420 425 430

Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser

435 440 445

Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Val Ala Pro Pro Pro Ala Leu Pro Ala Pro

485 490 495

Val Arg Leu Pro Gly Pro Ala Val Ala Pro Pro Pro Ala Leu Pro Ala

500 505 510

Val Ala Pro Pro Pro Ala Leu Pro Ala Asp Lys Thr His Thr Cys Pro

515 520 525

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

530 535 540

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

545 550 555 560

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

565 570 575

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

580 585 590

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

595 600 605

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

610 615 620

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

625 630 635 640

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

645 650 655

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

660 665 670

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

675 680 685

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

690 695 700

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

705 710 715 720

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

725 730 735

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Ser Gly Gly Ser

740 745 750

Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

755 760

<210> 25

<211> 2292

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-C4-Fc-L2-P

<400> 25

atgcagcgcg tcaatatgat tatggccgag tctccaggcc tcattaccat ttgtctgctg 60

ggctacctgc tgtccgcaga gtgtaccgtc ttcctcgacc acgagaacgc aaataagatt 120

ctcaataggc ctaagcgcta caattccggga aagctcgaag agtttgtcca gggaaatctg 180

gagcgcgagt gtatggagga aaagtgtagc tttgaagaag ctagagaggt gtttgagaat 240

actgagcgca ctactgagtt ttggaagcag tatgtggatg gcgatcagtg cgagtccaat 300

ccatgcctga atggcggatc ttgcaaggat gatattaatt cttacgagtg ttggtgccca 360

ttcggctttg agggaaagaa ttgcgaactg gatgtcacat gtaacatcaa gaacggcaga 420

tgcgagcagt tttgtaagaa cagcgccgat aataaggtgg tgtgtagttg tactgaggga 480

taccgcctcg cagagaatca gaagtcctgt gagcccgctg tgccatttcc ttgtggccgg 540

gtcagcgtca gccagacttc taagctgaca cgcgccgaga ccgtctttcc agatgtggat 600

tatgtcaact ctactgaggc cgagaccatt ctcgataata ttactcagtc tacacagtcc 660

tttaacgact tcactcgggt ggtgggcgga gaggacgcaa agcctggcca gtttccttgg 720

caggtcgtgc tcaacggaaa ggtggacgca ttttgtggcg gatctatcgt gaatgagaag 780

tggattgtga ccgccgctca ctgcgtcgag acaggcgtca agattaccgt ggtcgctgga 840

gagcacaaca ttgaggaaac agagcacacc gagcagaagc gcaatgtgat tcgcatcatc 900

ccacaccaca actacaacgc cgctattaat aagtacaacc acgacatcgc cctgttagaa 960

ctagatgagc ctctggtcct gaatagttac gtcactccta tttgtatcgc cgataaagaa 1020

tacactaaca tctttctcaa gttcggctct ggctatgtat ctggatgggg acgggtgttt 1080

cacaagggac ggtccgcact cgtgctacaa tacctccggg tgccactggt ggatcgggca 1140

acatgcctcc ggtctaccaa gtttactata tacaataaca tgttttgcgc cggctttcac 1200

1260

ggaactagct ttctgacagg cattatctct tggggcgaag agtgcgctat gaagggaaag 1320

tacggcatct atactaaggt ctcgcgttac gtcaattgga tcaaggaaaa gacaaagctg 1380

acaggcggcg gagggagcgg cggcgggaggc tctggcggcg gcggatctgg cggaggcggc 1440

tctggcggag gcggatctgt cgctccacca cctgcactcc ccgcccctgt cagactgcct 1500

ggacccgctg tcgctccacc acctgcactc cccgccgtgg ctcctccacc tgctttgccc 1560

1620

gtgtttctgt tcccaccaaa gccaaaggac accctcatga ttagtagaac cccagaggtg 1680

acttgcgtcg tggtcgatgt cagtcacgag gaccccgagg tgaagtttaa ttggtatgta 1740

gatggcgtcg aggtgcacaa cgctaagacc aagccacgcg aagagcagta cgcatctact 1800

taccgggtcg tctctgtgct gactgtgctg caccaggatt ggctcaatgg caaagagtac 1860

aagtgtaagg tcagtaataa ggccctcccc gcacctattg aaaagactat tagcaaggca 1920

aagggccagc ctagggagcc ccaggtctac accctgcccc cttcccgcga tgagctgact 1980

aagaatcagg tctctctcac ttgcctcgtg aagggctttt acccatccga cattgccgtg 2040

gagtgggagt ccaacggaca gccagagaat aactacaaga caactcctcc cgtgctcgat 2100

agcgacggtt cattcttcct gtactccaag ctcaccgttg ataagtctcg gtggcagcag 2160

ggaaatgtgt tctcttgttc cgtcatgcac gaggcactgc acaatcacta cacccagaag 2220

tccctgtccc tcagccctgg cggcagcggc ggatctggag gcggcggatc tctccgag 2280

accggcggat ga 2292

<210> 26

<211> 766

<212> PRT

<213> Artificial sequence

<220>

<223> FIX-C5-Fc-L2-P

<400> 26

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn

35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn

65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln

85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys

115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe

130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly

145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu

195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe

210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp

225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile

245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

275 280 285

His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn

290 295 300

Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu

305 310 315 320

Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile

325 330 335

Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr

340 345 350

Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val

355 360 365

Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg

370 375 380

Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His

385 390 395 400

Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val

405 410 415

Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly

420 425 430

Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser

435 440 445

Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Val Ala Pro Pro Pro Ala Leu Pro Ala Val

485 490 495

Ala Pro Pro Pro Ala Leu Pro Ala Gly Ser Val Ala Pro Pro Pro Ala

500 505 510

Leu Pro Ala Val Ala Pro Pro Pro Ala Leu Pro Ala Asp Lys Thr His

515 520 525

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

530 535 540

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

545 550 555 560

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

565 570 575

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

580 585 590

Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser

595 600 605

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

610 615 620

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

625 630 635 640

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

645 650 655

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

660 665 670

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

675 680 685

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

690 695 700

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

705 710 715 720

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

725 730 735

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Ser

740 745 750

Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

755 760 765

<210> 27

<211> 2301

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-C5-Fc-L2-P

<400> 27

atgcagcgcg tcaatatgat tatggccgag tctccaggcc tcattaccat ttgtctgctg 60

ggctacctgc tgtccgcaga gtgtaccgtc ttcctcgacc acgagaacgc aaataagatt 120

ctcaataggc ctaagcgcta caattccggga aagctcgaag agtttgtcca gggaaatctg 180

gagcgcgagt gtatggagga aaagtgtagc tttgaagaag ctagagaggt gtttgagaat 240

actgagcgca ctactgagtt ttggaagcag tatgtggatg gcgatcagtg cgagtccaat 300

ccatgcctga atggcggatc ttgcaaggat gatattaatt cttacgagtg ttggtgccca 360

ttcggctttg agggaaagaa ttgcgaactg gatgtcacat gtaacatcaa gaacggcaga 420

tgcgagcagt tttgtaagaa cagcgccgat aataaggtgg tgtgtagttg tactgaggga 480

taccgcctcg cagagaatca gaagtcctgt gagcccgctg tgccatttcc ttgtggccgg 540

gtcagcgtca gccagacttc taagctgaca cgcgccgaga ccgtctttcc agatgtggat 600

tatgtcaact ctactgaggc cgagaccatt ctcgataata ttactcagtc tacacagtcc 660

tttaacgact tcactcgggt ggtgggcgga gaggacgcaa agcctggcca gtttccttgg 720

caggtcgtgc tcaacggaaa ggtggacgca ttttgtggcg gatctatcgt gaatgagaag 780

tggattgtga ccgccgctca ctgcgtcgag acaggcgtca agattaccgt ggtcgctgga 840

gagcacaaca ttgaggaaac agagcacacc gagcagaagc gcaatgtgat tcgcatcatc 900

ccacaccaca actacaacgc cgctattaat aagtacaacc acgacatcgc cctgttagaa 960

ctagatgagc ctctggtcct gaatagttac gtcactccta tttgtatcgc cgataaagaa 1020

tacactaaca tctttctcaa gttcggctct ggctatgtat ctggatgggg acgggtgttt 1080

cacaagggac ggtccgcact cgtgctacaa tacctccggg tgccactggt ggatcgggca 1140

acatgcctcc ggtctaccaa gtttactata tacaataaca tgttttgcgc cggctttcac 1200

1260

ggaactagct ttctgacagg cattatctct tggggcgaag agtgcgctat gaagggaaag 1320

tacggcatct atactaaggt ctcgcgttac gtcaattgga tcaaggaaaa gacaaagctg 1380

acaggcggcg gagggagcgg cggcgggaggc tctggcggcg gcggatctgg cggaggcggc 1440

tctggcggag gcggatctgt cgctccacca cctgcactcc ccgccgtggc tcctccacct 1500

1560

gctttgcccg ctgataagac ccacacatgt ccaccttgcc cagcacctga gctgctgggc 1620

ggacctagcg tgtttctgtt cccaccaaag ccaaaggaca ccctcatgat tagtagaacc 1680

ccagaggtga cttgcgtcgt ggtcgatgtc agtcacgagg accccgaggt gaagtttaat 1740

1800

gcatctactt accgggtcgt ctctgtgctg actgtgctgc accaggattg gctcaatggc 1860

aaagagtaca agtgtaaggt cagtaataag gccctccccg cacctattga aaagactatt 1920

agcaaggcaa agggccagcc tagggagccc caggtctaca ccctgccccc ttcccgcgat 1980

gagctgacta agaatcaggt ctctctcact tgcctcgtga agggctttta cccatccgac 2040

attgccgtgg agtgggagtc caacggacag ccagagaata actacaagac aactcctccc 2100

gtgctcgata gcgacggttc attcttcctg tactccaagc tcaccgttga taagtctcgg 2160

tggcagcagg gaaatgtgtt ctcttgttcc gtcatgcacg aggcactgca caatcactac 2220

acccagaagt ccctgtccct cagccctggc ggcagcggcg gatctggagg cggcggatct 2280

ctcccagaga ccggcggata a 2301

<210> 28

<211> 728

<212> PRT

<213> Artificial sequence

<220>

<223> FIX-GS-Fc-L2-P

<400> 28

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu

20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn

35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn

65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln

85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile

100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys

115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe

130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly

145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe

165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu

195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe

210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp

225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile

245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

275 280 285

His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn

290 295 300

Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu

305 310 315 320

Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile

325 330 335

Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr

340 345 350

Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val

355 360 365

Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg

370 375 380

Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His

385 390 395 400

Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val

405 410 415

Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly

420 425 430

Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser

435 440 445

Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

485 490 495

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

500 505 510

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

515 520 525

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

530 535 540

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

545 550 555 560

Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

565 570 575

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

580 585 590

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

595 600 605

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

610 615 620

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

625 630 635 640

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

645 650 655

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

660 665 670

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

675 680 685

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

690 695 700

Lys Ser Leu Ser Leu Ser Pro Gly Gly Ser Gly Gly Ser Gly Gly Gly

705 710 715 720

Gly Ser Leu Pro Glu Thr Gly Gly

725

<210> 29

<211> 2187

<212> DNA

<213> Artificial sequence

<220>

<223> FIX-GS-Fc-L2-P

<400> 29

atgcagcgcg tcaatatgat tatggccgag tctccaggcc tcattaccat ttgtctgctg 60

ggctacctgc tgtccgcaga gtgtaccgtc ttcctcgacc acgagaacgc aaataagatt 120

ctcaataggc ctaagcgcta caattccggga aagctcgaag agtttgtcca gggaaatctg 180

gagcgcgagt gtatggagga aaagtgtagc tttgaagaag ctagagaggt gtttgagaat 240

actgagcgca ctactgagtt ttggaagcag tatgtggatg gcgatcagtg cgagtccaat 300

ccatgcctga atggcggatc ttgcaaggat gatattaatt cttacgagtg ttggtgccca 360

ttcggctttg agggaaagaa ttgcgaactg gatgtcacat gtaacatcaa gaacggcaga 420

tgcgagcagt tttgtaagaa cagcgccgat aataaggtgg tgtgtagttg tactgaggga 480

taccgcctcg cagagaatca gaagtcctgt gagcccgctg tgccatttcc ttgtggccgg 540

gtcagcgtca gccagacttc taagctgaca cgcgccgaga ccgtctttcc agatgtggat 600

tatgtcaact ctactgaggc cgagaccatt ctcgataata ttactcagtc tacacagtcc 660

tttaacgact tcactcgggt ggtgggcgga gaggacgcaa agcctggcca gtttccttgg 720

caggtcgtgc tcaacggaaa ggtggacgca ttttgtggcg gatctatcgt gaatgagaag 780

tggattgtga ccgccgctca ctgcgtcgag acaggcgtca agattaccgt ggtcgctgga 840

gagcacaaca ttgaggaaac agagcacacc gagcagaagc gcaatgtgat tcgcatcatc 900

ccacaccaca actacaacgc cgctattaat aagtacaacc acgacatcgc cctgttagaa 960

ctagatgagc ctctggtcct gaatagttac gtcactccta tttgtatcgc cgataaagaa 1020

tacactaaca tctttctcaa gttcggctct ggctatgtat ctggatgggg acgggtgttt 1080

cacaagggac ggtccgcact cgtgctacaa tacctccggg tgccactggt ggatcgggca 1140

acatgcctcc ggtctaccaa gtttactata tacaataaca tgttttgcgc cggctttcac 1200

1260

ggaactagct ttctgacagg cattatctct tggggcgaag agtgcgctat gaagggaaag 1320

tacggcatct atactaaggt ctcgcgttac gtcaattgga tcaaggaaaa gacaaagctg 1380

acaggcggcg gagggagcgg cggcgggaggc tctggcggcg gcggatctgg cggaggcggc 1440

tctggcggag gcggatccga taagacccac acatgtccac cttgcccagc acctgagctg 1500

ctgggcggac ctagcgtgtt tctgttccca ccaaagccaa aggacaccct catgattagt 1560

agaaccccag aggtgacttg cgtcgtggtc gatgtcagtc acgaggaccc cgaggtgaag 1620

tttaattggt atgtagatgg cgtcgaggtg cacaacgcta agaccaagcc acgcgaagag 1680

cagtacgcat ctacttaccg ggtcgtctct gtgctgactg tgctgcacca ggattggctc 1740

aatggcaaag agtacaagtg taaggtcagt aataaggccc tccccgcacc tattgaaaag 1800

actattagca aggcaaaggg ccagcctagg gagccccagg tctacaccct gcccccttcc 1860

cgcgatgagc tgactaagaa tcaggtctct ctcacttgcc tcgtgaaggg cttttaccca 1920

tccgacattg ccgtggagtg ggagtccaac ggacagccag agaataacta caagacaact 1980

cctcccgtgc tcgatagcga cggttcattc ttcctgtact ccaagctcac cgttgataag 2040

tctcggtggc agcagggaaa tgtgttctct tgttccgtca tgcacgaggc actgcacaat 2100

cactacaccc agaagtccct gtccctcagc cctggcggca gcggcggatc tggaggcggc 2160

ggatctctcc cagagaccgg cggataa 2187

<210> 30

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> F1

<400> 30

tcaggaagat ctcgcgaatt aattctgtgg aatgtgt 37

<210> 31

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> R1

<400> 31

tatcctagat ctgctggcac gacaggtttc ccgact 36

<210> 32

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> F2

<400> 32

ttcgcggatc cgtcgctcca ccacctgcac tccccgccgt ggctcctcca cctgctttgc 60

ccgctgataa gacccacaca tgtccac 87

<210> 33

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> R2

<400> 33

taaccggaat tcattatccg ccggtctctg ggagagatcc gccgcctcca gatccgccgc 60

tgccgccagg gctgagggac aggact 87

<210> 34

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> F3

<400> 34

tggctcctcc acctgctttg cccgctgtcg ctccaccacc tgcactcc 48

<210> 35

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> F4

<400> 35

tagatctgtc gctccaccac ctgcactccc cgccgtggct cctccacctg ctttgcc 57

<210> 36

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> R3

<400> 36

tgaattctta tcatccgccg gtctctggga g 31

<210> 37

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> F5

<400> 37

ccctgtcaga ctgcctggac ccgctgtcgc tccaccacct gcactcc 47

<210> 38

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> F6

<400> 38

tagatctgtc gctccaccac ctgcactccc cgcccctgtc agactgcctg gacc 54

<210> 39

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> F7

<400> 39

atataagctt ccgccaccat gcagcgc 27

<210> 40

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> R4

<400> 40

atatggatcc agcgggcaaa gcaggtggag gagc 34

<210> 41

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> F8

<400> 41

gatttggatc cgataagacc cacacatgtc caccttg 37

<210> 42

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> R5

<400> 42

taaccggaat tcattatccg ccggtctctg ggagagatcc gccgcctcca gatccgccgc 60

tgccgccagg gctgagggac aggact 87

<---

Claims (22)

1. A conjugate for the prevention or treatment of diseases associated with congenital or acquired clotting factor IX (FIX) protein deficiency, containing a clotting factor IX (FIX) fusion protein and a hydrophilic polymer, where the hydrophilic polymer is a polyethylene glycol attached to the C-terminus of the fusion protein , and the specified polyethylene glycol has a molecular weight of 40 kDa; and the fusion protein contains an active group of coagulation factor IX and a fusion partner (FP) represented by the structure FIX-L1-Fc-L2-P, where the active group of clotting factor IX is a full length human clotting factor IX containing the sequence shown in SEQ ID NO: 1; And the fusion partner is an immunoglobulin Fc fragment from human IgG1 with the point mutation N297A, L1 is the first linker between FIX and Fc, L1 is selected from the group consisting of: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPA (SEQ ID NO: 8), GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPA (SEQ ID NO: 9), GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAVAPPPALPAPV RLPGPA (SEQ ID NO: 10), GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPAVAPPPALPAVAP PPALPA (SEQ ID NO: 11) and GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAGSVAPPPALPA VAPPPALPA (SEQ ID NO: 12); L2 is the second linker between Fc and the sortase P recognition site, L2 contains the sequence GSGGSGGGGS (SEQ ID NO: 16); And P is an amino acid fragment containing the LPETGG sortase recognition site. 2. The conjugate of claim 1 wherein the first L1 linker is GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPA (SEQ ID NO: 8). 3. The conjugate of claim 1 wherein the fusion protein is in monomer or dimer form. 4. The method of obtaining a conjugate according to any one of paragraphs. 1-3, including expression of the coagulation factor IX (FIX) fusion protein in the host cell; purification of the coagulation factor IX fusion protein (FIX); bringing the fusion factor IX protein (FIX) into contact with sortase, preferably sortase A or sortase B, and a hydrophilic polymer, where one end of the hydrophilic polymer has an amino group that can be amidated by sortase, where the hydrophilic polymer is polyethylene glycol (PEG) with a molecular weighing 40 kDa. 5. The method of claim 4, wherein the N-terminus of the hydrophilic polymer contains poly-Gly, preferably GGGAA. 6. Pharmaceutical composition for the prevention or treatment of diseases associated with congenital or acquired FIX deficiency, containing an effective amount of the conjugate according to any one of paragraphs. 1-3 and a pharmaceutically acceptable carrier. 7. The pharmaceutical composition according to claim 6, which is in the form of a liquid preparation or a lyophilized preparation. 8. The use of the conjugate according to any one of paragraphs. 1-3 for the manufacture of a medicament for the prevention or treatment of a hemorrhagic disease, where the hemorrhagic disease is selected from hemorrhagic diseases in patients with congenital or acquired FIX deficiency and spontaneous or postoperative bleeding in patients with hemophilia B. 9. Kit for the prevention or treatment of diseases associated with congenital or acquired FIX deficiency, containing a conjugate according to any one of claims. 1-3 and instructions for use.

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