KR20220069610A - Novel fusion protein comprising FGF19 variant, and use thereof - Google Patents

Abstract

The present invention relates to a fusion protein comprising a novel fibroblast growth factor 19 (FGF 19) variant and uses thereof, and more specifically, to a fusion protein comprising a novel FGF19 variant; and a GLP-1 or an analogue thereof. The present invention relates to the fusion protein with increased persistence in the body and thus can prevent or treat liver disease.

Description

Novel fusion protein comprising FGF19 variant and use thereof {Novel fusion protein comprising FGF19 variant, and use thereof}

The present invention relates to a FGF19 variant, a novel fusion protein comprising the same, and uses thereof, and more particularly, to a novel fibroblast growth factor 19 (Fibroblast Growth Factor 19, FGF 19) variant; and to a fusion protein comprising GLP-1 or an analog thereof and uses thereof.

Non-alcoholic fatty liver disease (NAFLD) is divided into non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH). Simple fatty liver (NAFL) is a benign disease with a good clinical prognosis, although it shows fat deposition. In contrast, nonalcoholic steatohepatitis (NASH) is a state in which hepatocyte damage and inflammation are accompanied by fat deposition, the disease progresses slowly, and the prognosis is poor due to unclear pathological mechanisms, liver fibrosis, cirrhosis, and hepatocytes. It is known to cause cancer. Nonalcoholic steatohepatitis is a disease that gradually worsens into early NASH (F0/F1), fibrotic NASH (F2/F3), and cirrhosis NASH (F4) depending on the stage, and is an area with high unmet medical needs, F3 , stage F4 corresponds to 20-25% of all NASH patients.

According to the U.S. Food and Drug Administration (FDA), nonalcoholic steatohepatitis (NASH) is a disease that has a large burden on patients and eventually requires a liver transplant. It has been reported that there is a high unmet demand for existing therapeutic agents. There is still no FDA-approved treatment for NASH, and as a chemical-based drug, attempts are being made to treat NASH through various mechanisms of action, such as CCR2 and FXR agonist. However, there are many cases of failure in phase 2 or 3 clinical trials due to lack of efficacy among drugs, safety issues, and the like.

Therefore, NASH treatment is mainly in the development of biologics, and most biologic-based drugs are being developed mainly in phase 1 and 2 clinical trials. As the main drug group, GLP-1 class substances, FGF21 or FGF19, are used.

The GLP-1 family of substances is a substance used as a treatment for metabolic diseases such as diabetes due to its action as a glycemic control agent. FGF21 is an endocrine growth factor that acts directly on adiopse tissue to break down fat and regulate food preferences. FGF19 is an endocrine growth factor that directly acts on liver tissue to regulate lipid metabolism and deliver satiety. FGF21 or FGF19 is a therapeutic agent that can control metabolic problems that cause NASH, but it has been known that there is a risk of causing cancer when overdose, and that FGF19 is more likely to be a problem than FGF21. Therefore, a treatment using FGF21 is more preferred, but since FGF21 does not act directly on liver tissue, it is expected to have an indirect therapeutic effect rather than a direct therapeutic effect.

In this regard, U.S. Patent No. 10,413,590 discloses the use of a variant of FGF19 polypeptide for weight loss, and Republic of Korea Patent No. 10-2077721 discloses FGF19 variants and fusions, metabolic disorders containing FGF21 variants and fusions, and Compositions for the treatment of diseases have been disclosed. Korean Patent Application Laid-Open No. 10-2017-0080526 discloses a FGF21 analog in which one or more amino acids are removed, added, substituted, or modified from natural FGF21 and its use for metabolic syndrome, and U.S. Patent No. 10,588,880 discloses Ubenimax A NASH treatment composition using FGF19 and FGF19 has been disclosed. However, there has been no disclosure of clinical reports on sustainability studies using FGF19 and related drugs.

In addition, recently, co-administration of GLP-1 substances with other drugs is being tried as a new treatment method. For example, 'YH2574' developed by Yuhan Corporation is a fusion of GLP-1 and FGF21, which reduces the hassle of co-administration, and can be developed as a subcutaneous injection formulation administered once every two days to increase the half-life. It seems that the problem of single administration of each of the above substances can be solved. However, since FGF21 mediates the FGFR1c receptor of adipocytes, there is still a problem that a high concentration of administration is required to exhibit an effect similar to that of FGF19, which acts directly on the FGF4 receptor of liver cells.

Accordingly, the present inventors have newly prepared a fusion protein comprising a FGF19 mutant and GLP-1 or an analog thereof, which has excellent effects even when administered at a low dose and can reduce the hassle of co-administration. In addition, the present inventors confirmed that the fusion had a similar level of binding to the FGFR4 receptor compared to the case where the FGF19 single substance was used, and the persistence in vivo was increased, and thus the NASH, NAFLD treatment effect was excellent. The present invention was completed by finding the point.

The present invention has a high unmet need for nonalcoholic steatohepatitis or nonalcoholic fatty liver, and FGF19 can control metabolic problems by acting directly on liver tissue, but has a problem that it can cause cancer. In the absence of this, an object of the present invention is to provide a novel fusion protein comprising a FGF19 variant and GLP-1 or an analog thereof.

In order to achieve the above object, the present invention provides an FGF19 variant in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19).

In addition, the present invention provides a FGF19 variant wherein the FGF19 variant further comprises a persistent carrier.

In addition, the present invention provides an FGF19 variant in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19); and GLP-1 or an analog thereof.

In one embodiment of the present invention, the FGF19 variant may be selected from the group of FGF19 variants consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 7.

In one embodiment of the present invention, the long-acting carrier further comprising the FGF19 variant may be fused to the N-terminus or the C-terminus of the FGF19 variant.

In one embodiment of the present invention, the long-acting carrier is polyethylene glycol, fatty acid, albumin or fragment thereof, an albumin-binding substance, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or fragment thereof, a repeating unit polymer of a specific amino acid sequence, It may be selected from the group consisting of antibodies or fragments thereof, FcRn binding materials, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers.

In a specific embodiment of the present invention, the long-acting carrier may be alpha-1 antitrypsin or a variant thereof, or an immunoglobulin Fc or fragment thereof.

In a specific aspect of the present invention, in the alpha-1 antitrypsin variant, one or more amino acids are substituted with other amino acids, and the substitution comprises a substitution of at least one position from the 1st amino acid to the 25th amino acid from the N-terminus. can

In a specific embodiment of the present invention, the alpha-1 antitrypsin variant is substituted with asparagine at the ninth amino acid from the N-terminus, serine at the 232th amino acid, asparagine at the 37th amino acid, and threonine at the 359th amino acid It may include any one or more substitutions selected from the group consisting of substitutions.

In one embodiment of the present invention, the immunoglobulin Fc may be an animal, human, or modified immunoglobulin Fc thereof.

In a specific embodiment of the present invention, the immunoglobulin Fc may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, and combinations thereof.

In one embodiment of the present invention, the persistent carrier may be fused with the FGF19 variant directly or via a linker.

In a specific embodiment of the present invention, the linker may be a peptidic linker or a non-peptidyl linker.

In a more specific aspect of the present invention, the non-peptidyl linker is polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinylethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof.

In addition, in a more specific aspect of the present invention, the peptidic linker may be one in which two or more amino acids are linked.

FGF19 variant (FGF19 variant) in which one or more amino acids are substituted with other amino acids in the amino acid sequence of fibroblast growth factor 19 (FGF 19) according to an embodiment of the present invention; and in the fusion protein comprising GLP-1 or an analog thereof, the FGF19 variant is as defined above.

In one aspect of the present invention, the FGF19 variant; and GLP-1 or an analog thereof may be fused directly or via a long-acting carrier. Here, the long-acting carrier is as defined above.

In one specific aspect of the present invention, the long-acting carrier is a FGF19 variant; and to the N-terminus or C-terminus of GLP-1 or an analog thereof. In a more specific embodiment of the present invention, the persistent carrier is fused to the N-terminus of a FGF19 variant, and GLP-1 or an analog thereof. It may be fused to the C-terminus of the analog.

In one aspect of the present invention, the fusion protein may include any one of the amino acid sequences shown in SEQ ID NOs: 8 to 21.

In the present invention, the fusion protein is characterized in that the half-life in the body is increased.

In addition, the present invention provides a pharmaceutical composition for preventing or treating liver disease comprising the FGF19 mutant or fusion protein.

In one embodiment of the present invention, the liver disease may be at least one selected from the group consisting of fatty liver disease, liver fibrosis, cirrhosis, and inflammatory liver disease.

In a specific embodiment of the present invention, the inflammatory liver disease is nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD).

The present invention relates to a FGF19 variant (FGF19 variant) in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19) and/or to a fusion protein comprising the same. The present invention is a fused form of protein, so it is possible to reduce the effort of co-administration, there is no side effect of liver cancer even when FGF19 is used, and it has advantages of excellent durability in the body even when a low dose is used.

In addition, there is an advantage in that it is possible to prevent or treat liver disease through the pharmaceutical composition comprising the FGF19 variant or fusion protein.

1 is a structural model of a fusion protein including a FGF19 mutant, and is a diagram illustrating a fusion protein in which GLP-1 and a FGF19 mutant are fused by NexP, a long-acting carrier.
2 is a structural model of a fusion protein including a FGF19 mutant, and is a diagram illustrating a fusion protein in which GLP-1 and FGF19 mutant are fused by immunoglobulin Fc as a long-acting carrier.
3 is a diagram showing the results of SDS-PAGE analysis of the final purified sample of the FGF19 mutant prepared according to an embodiment of the present invention.
4 is a diagram showing the results of SDS-PAGE analysis of the final purified sample of the fusion protein containing the FGF19 mutant prepared according to an embodiment of the present invention.
5 is a view showing the results of Western blot analysis of the FGF19 mutant and the fusion protein comprising the same prepared according to an embodiment of the present invention.
6a to 6c are diagrams confirming the FGFR4 receptor binding ability of the FGF19 mutant prepared according to an embodiment of the present invention.
7 is a view confirming the FGFR4 receptor binding ability of the fusion protein containing the FGF19 mutant prepared according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

Throughout the specification of the present invention, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Throughout the present specification, the term "analog" or "variant" includes any peptide that has some modifications of the corresponding native peptide and has some activity of the native peptide. Some modifications are optionally phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc. includes

In the present invention, "Fibroblast Growth Factor 19 (FGF 19)" refers to a protein encoded by the human FGF19 gene, and "FGF19 mutant" is compared with wild-type FGF19, one or more amino acids are substituted, It refers to a protein having an insertion or deletion.

In the present invention, "durable carrier" refers to a substance capable of increasing the half-life in vivo.

In the present invention, “GLP-1” refers to glucagon-like peptide-1, and “GLP-1 analogue” refers to a peptide having some modifications in the structure of GLP-1. It means a peptide having an effect similar to 1. GLP-1 analogues include, for example, Exenatide, Lixisenatide, Liraglutide, Dulaglutide, and the like. However, this is an example, and the GLP-1 analogue is not limited thereto.

The present invention relates to a FGF19 variant in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19).

In addition, the present invention provides an FGF19 variant in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19); and GLP-1 or an analog thereof.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating liver disease comprising the FGF19 variant or the fusion protein.

In a specific embodiment of the present invention, the FGF19 variant is selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 1 to 7 of Table 1 below.

division order SEQ ID NO: 1
(F2011-n1) RNSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPSFPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD SEQ ID NO: 2
(F2011-n2) RDTSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLFLSHFLPMLPMVPEEPEDLRGHLESDGLVTSSPLETDSMD SEQ ID NO: 3
(F2011-n4) MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLPGLPPALPEPPGDLRGHLESDMFSSPLETDSMDPF SEQ ID NO: 4
(F2011-n5) MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSTCHLRIRANGVVDCARGQSAHSLLEIKAVALRTIAIKGVHSVRYLCMGADGKIQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPLSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLESPLETDSMDPF SEQ ID NO: 5
(F2011-n6) RNESPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLFLSHFLPMLPMVPEEPEDLRGHLESDGLVTSSPLETDSMD SEQ ID NO: 6
(F2011-n7) RNASPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDSMD SEQ ID NO: 7
(F2011-n8) RNTSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPSFPLSHFLPMLPMVPEEPEDLRGHLESDGLVTSSPLETDSMD

In one aspect of the present invention, the FGF19 variant may further include a long-acting carrier.

In one embodiment of the present invention, the persistent carrier may be fused to the N-terminus or C-terminus of the FGF19 variant.

The long-acting carrier is a substance capable of increasing the half-life in vivo, and includes substances known in the art that can increase the half-life in vivo. In addition, as the long-acting carrier is used, the in vivo half-life is remarkably increased and the potency is improved.

In the present invention, specific long-acting carriers include polyethylene glycol, fatty acids, albumin or fragments thereof, albumin-binding substances, alpha-1 antitrypsin or variants thereof, immunoglobulin Fc or fragments thereof, repeating unit polymers of specific amino acid sequences, antibodies or fragments thereof. , FcRn binding material, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and may be selected from the group consisting of high molecular weight polymers. In addition, the long-acting carrier includes one or more.

In a specific embodiment of the present invention, the long-acting carrier is alpha-1 antitrypsin or a variant thereof, or an immunoglobulin Fc or fragment thereof.

In the present invention, the alpha-1 antitrypsin or a variant thereof has the characteristics as known in Prior Patent Publication Nos. 10-2013-0136883 and 10-2013-0029713.

Specifically, it is known that alpha-1 antitrypsin in the present invention has the following properties:

Alpha-1 antitrypsin is one of the proteins present in the blood of mammals with a molecular weight of about 50,000 Daltons, and is one of the main blood proteins with a concentration of about 2 mg/ml in the blood (Robin W.C. et al., Nature 298, 329-334, 1982), refers to a substance also called an alpha-1 protease inhibitor. This protein inhibits several types of proteolytic enzymes when tested with proteolytic enzymes. However, the main function in vivo related to currently known diseases is known as an inhibitor of neutrophil elastase (Beatty et al., J Biol Chem 255, 3931-3934, 1980). Alpha-1 antitrypsin deficiency leads to impaired lung function and serious genetic diseases. Therefore, alpha-1 antitrypsin extracted from blood has been approved by the FDA and sold under the trade name of Prolastin as a treatment for emphysema. Prolastin is usually administered to the human body by intravenous injection at a dose of 60 mg/kg at intervals of 1 week, and is a protein that has proven safety and harmfulness in the human body. In addition, the half-life of alpha-1 antitrypsin in the body is known to be about 5 to 6 days (Weweres, MD, et al., N. Engl J med 316, 1055-1062, 1987). Therefore, a logical rationale for creating a long-acting substance with an increased half-life in the body is provided by fusing alpha-1 antitrypsin, which has already been proven to be stable even when administered in excess to the human body, with a protein or peptide having physiological activity. The role and structure of alpha-1 antitrypsin as a protease inhibitor is already well known (Elliott, P. et al., JMB 275, 419-425, 1998). Although the amino acid P1 (position 358 from the N-terminus) of alpha-1 antitrypsin is a methionine group, this protein is known to inhibit the activities of various proteases such as trypsin, chymotrypsin, thrombin and elastase. have. In addition, more than 100 alleles of alpha-1 antitrypsin exist in nature, and phenotypes are classified from A to Z according to the type of isoelectric focusing (IEF) (Stoller et al., The Lancet). , 365, 2225 - 2236, 2005). Most of the M alleles are normal types and are again divided into various subtypes such as M1 (Val213), M1 (Ala213), M2, and M3 by amino acid sequence mutation. Therefore, the alpha-1 antitrypsin used in the present invention is a specific subtype that exists in nature, and the same effect can be obtained for other subtypes.

In the present invention, the alpha-1 antitrypsin variant may be prepared by mutating one or more amino acids using a site-directed mutagenesis method.

In a specific aspect of the present invention, in the alpha-1 antitrypsin variant, one or more amino acids are substituted with other amino acids, and the substitution comprises a substitution of at least one position from the 1st amino acid to the 25th amino acid from the N-terminus. .

In a specific embodiment of the present invention, the alpha-1 antitrypsin variant is substituted with asparagine at the ninth amino acid from the N-terminus, serine at the 232th amino acid, asparagine at the 37th amino acid, and threonine at the 359th amino acid Includes any one or more substitutions selected from the group consisting of substitutions.

In a specific embodiment of the present invention, the immunoglobulin Fc is an animal, human, or modified immunoglobulin Fc thereof.

In addition, in a specific embodiment of the present invention, the immunoglobulin Fc is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, and combinations thereof.

In the present invention, the persistent carrier and the FGF19 variant may be directly covalently or non-covalently bound, or may be linked through a suitable linker. Herein, the linker is a linker that does not cause functional modification of each part, and includes all linkers known in the art unless specifically defined herein.

In one embodiment of the present invention, the persistent carrier is fused with the FGF19 variant directly or via a linker.

In a specific embodiment of the present invention, the linker is a peptidic linker or a non-peptidyl linker.

Here, the peptide linker may be one in which two or more amino acids are linked, and the non-peptidyl linker is polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, poly saccharides, dextran, polyvinylethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof.

In addition, the present invention is a fibroblast growth factor 19 (Fibroblast Growth Factor 19, FGF 19) in the amino acid sequence of one or more amino acids substituted with other amino acids FGF19 variant (FGF19 variant); and GLP-1 or an analog thereof, comprising: the FGF19 variant; and GLP-1 or an analog thereof may be fused directly or via a long-acting carrier.

In the fusion protein of the present invention, the FGF19 variant, the persistent carrier, and the like are the same as defined above.

In one embodiment of the present invention, the long-acting carrier is a FGF19 variant; and to the N-terminus or C-terminus of GLP-1 or an analog thereof. In a specific embodiment of the present invention, the persistent carrier is fused to the N-terminus of the FGF19 variant and fused to the C-terminus of GLP-1 or an analog thereof.

In one aspect of the present invention, the fusion protein may include any one of the amino acid sequences shown in SEQ ID NO: 8 to SEQ ID NO: 21 of [Table 2] below.

In the fusion protein of the present invention, when the fusion protein includes the amino acid sequence of SEQ ID NOs: 15 to 21, the fusion protein may further include the amino acid sequence of SEQ ID NO: 22.

When it further comprises the amino acid sequence of SEQ ID NO: 22, it may be bound to the amino acid sequence of SEQ ID NOs: 15 to 21 through a disulfide bond.

Specifically, cysteines 48 and 51 of the amino acid sequence of SEQ ID NOs: 15 to 21 may form disulfide bonds with cysteines 2 and 5 of the amino acid sequence of SEQ ID NO: 22.

division order SEQ ID NO: 8
(F2012-n1) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSRNSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 9
(F2012-n2) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSRDTSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 10
(F2012-n4) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSMRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLPGLPPALPEPPGDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 11
(F2012-n5) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSMRDSSPLVHYGWGDPIRLRHLYTSGPHGLSTCHLRIRANGVVDCARGQSAHSLLEIKAVALRTIAIKGVHSVRYLCMGADGKIQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPLSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 12
(F2012-n6) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSRNESPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 13
(F2012-n7) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSRNASPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 14
(F2012-n8) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKGGGGSGGGGSRNTSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 15
(B8011-n1)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSRNSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 16
(B8011-n2)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSRDTSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 17
(B8011-n4)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSMRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLPGLPPALPEPPGDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 18
(B8011-n5)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSMRDSSPLVHYGWGDPIRLRHLYTSGPHGLSTCHLRIRANGVVDCARGQSAHSLLEIKAVALRTIAIKGVHSVRYLCMGADGKIQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPLSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 19
(B8011-n6)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSRNESPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 20
(B8011-n7)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSRNASPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 21
(B8011-n8)
(Chain A) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSRNTSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK SEQ ID NO: 22
(B8011)
(Chain B) TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLPPSRDELTKNQVSLPPSRDELTKNQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLPPSRDELTKNQVSLPPSRDELTKNQVSLSVKLSDKGFYPSDIVLSSRAVE

In one aspect of the present invention, the fusion protein is characterized in that the half-life in the body is increased. In a specific example of the present invention, the GLP-1 analogue and the FGF19 mutant may exist as a fusion protein in the form of binding to both ends of the immunoglobulin IgG1 or alpha-1 antitrypsin mutant. The fusion protein includes both a GLP-1 analogue and a FGF19 mutant, and has a remarkably increased in vivo half-life, and thus can exhibit effective therapeutic effects even when administered alone or at a low dose.

The present invention provides a pharmaceutical composition for preventing or treating liver disease comprising the FGF19 mutant or fusion protein.

In one embodiment of the present invention, the liver disease may be at least one selected from the group consisting of fatty liver disease, liver fibrosis, cirrhosis, and inflammatory liver disease.

In a specific embodiment of the present invention, the inflammatory liver disease may be nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD).

In addition, the pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.

In the present invention, the term 'pharmaceutically effective amount' means an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical prophylaxis or treatment, and the effective dose level depends on the severity of the disease, the drug of the activity, patient's weight, health, sex, patient's sensitivity to drug, administration time of the composition of the present application used, administration route and excretion rate, treatment period, the drug used in combination with or concurrently with the composition of the present application may be determined according to factors including and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may include other ingredients as needed. Other ingredients include, but are not particularly limited to, stabilizers, surfactants, plasticizers, lubricants, solubilizers, buffers, sweeteners, substrates, adsorbents, flavoring agents, binders, suspending agents, antioxidants, brightening agents, coating agents, flavoring agents, Perfume, wetting agent, wetting agent, antifoaming agent, chewing agent, refreshing agent, colorant, dragee, isotonic agent, pH adjusting agent, softening agent, emulsifying agent, adhesive, adhesion enhancing agent, thickening agent, thickening agent, foaming agent, excipient, dispersing agent, propellant, Pharmaceutical additives such as disintegrants, disintegrating aids, fragrances, desiccants, preservatives, preservatives, softening agents, solvents, solubilizers, solubilizers, and glidants are mentioned.

The dosage form of the pharmaceutical composition according to the present invention may be appropriately selected according to the formulation method, the age, weight, disease, symptom and degree of the patient, and the like, and is not particularly limited. For example, tablets (including sublingual tablets and orally disintegrating tablets), granules, powders, solutions, syrups (including dry syrups), jellies, capsules (including soft capsules and microcapsules), etc. do.

The dosage of the pharmaceutical composition according to the present invention is appropriately determined according to the patient's age, sex, weight, disease, symptoms and the degree thereof.

In the formulation of the pharmaceutical composition according to the present invention, additives known in the art may be blended. For example, when preparing a solid formulation for oral use, excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, odorants, etc. are added to the active ingredient, followed by molding, granulation, encapsulation, etc. according to a conventional method, Coated tablets, granules, powders, capsules, etc. can be manufactured. In addition, when preparing a liquid formulation for oral use, a solvent such as purified water or ethanol, a solubilizing agent, a suspending agent, an isotonic agent, a corrosive agent, a buffer, a stabilizer, an odorant, etc. are added to the active ingredient, and then according to a conventional method. By distributing the crude liquid or the like, it is possible to prepare an oral liquid preparation, a syrup preparation, and the like.

The dosage of the pharmaceutical composition according to the present invention may be administered in a dose of 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg when administered once to several times a day for adults. The dosage is not intended to limit the scope of the present invention in any way.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Preparation Example 1> Preparation of FGF19 mutant and fusion protein comprising the same

<1-1> Cloning

DNA encoding the amino acid sequence of wild-type FGF19 was produced by GenScript, amplified using polymerase chian reasction (hereinafter referred to as PCR), and inserted into pcDNA3.4-TOPO vector (vector). Then, the cDNA clone of the GLP-1-NexP-FGF19 mutant as shown in FIG. 1 or the GLP-1-Fc-FGF19 mutant as shown in FIG. It was prepared according to site-directed mutagenesis using a primer and a known cloning method. Thereafter, the mutation was confirmed using DNA sequencing. The plasmid whose base sequence was confirmed was mass-cultured and purified, and used for cell culture.

Here, NexP is a mutant of alpha-1 antitrypsin (A1AT), a protein in the body that has an increased half-life in the body by maintaining the persistence in the body developed by the present inventors. A variant of alpha-1 anti-trypsin (A1AT) is a mutation of certain amino acids in order to abolish the intrinsic activity of alpha-1 anti-trypsin as a protease inhibitor and increase the half-life. The protein sequence and preparation method of alpha-1 anti-trypsin, which has eliminated its intrinsic activity as a proteolytic enzyme, are disclosed in Korean Patent Laid-Open No. 10-2010-0116558.

<1-2> Transformation and cell culture

The ExpiCHO-S ^™ cell culture medium was prepared in advance by shaking culture at 37° C., 8% CO ₂ in an incubator (humid incubator). DNA of the FGF19 mutant and the fusion protein (GLP-1-NexP-FGF19 mutant or GLP-1-Fc-FGF19 mutant) containing the same was dripped into the ExpiCHO-S ^™ cell culture medium to induce transformation (transfection). The transformation-induced cells were cultured with shaking in an incubator of 37° C., 8% CO ₂ . At 22 hours after transformation induction, a fed-batch culture was performed in which ExpiFectamine ^™ CHO enhancer and ExpiCHO ^™ feed were added to the culture medium once, and the culture temperature was changed to 32 °C after addition. and cultured with shaking for 6 days in an incubator of 8% CO ₂ (humid incubator). On the 6th day of culture, the culture medium was centrifuged at 4° C. to recover only the culture supernatant.

<1-3> Purification of FGF19 variants

For protein purification using a Q Sepharose FF column, buffer A (20 mM Tris, pH 8.5, 30 mM NaCl) and buffer B (20 mM Tris, pH 8.5, 1 M NaCl) were prepared. After binding the protein to the column, 5 column volume (CV) of buffer A was flowed to remove non-specifically bound protein, and after confirming that conductivity was maintained constant, 5% of buffer B 7 CV was flowed to remove non-target proteins. After that, buffer B was flowed with a concentration gradient of 5 to 15% to elute the target protein.

Buffer A (20 mM Sodium Phosphate, pH 7.0, 1.5 M NaCl) and Buffer B (20 mM Sodium Phosphate, pH 7.0) were prepared for protein purification using Butyl HP column. The protein recovered in the Q Sepharose FF purification process was bound to a Butyl HP column, and impurities were removed by flowing 5 CV of Buffer A, and 15 CV of Buffer B was flowed at a concentration gradient of 40-100% to elute the protein.

The eluted protein was dialyzed using a dialysis buffer (20 mM sodium phosphate, pH 7.0, 145 mM NaCl).

<1-4> Purification of GLP-1-NexP-FGF19 mutant with FGF19 mutant-containing fusion protein

For protein purification using a Q Sepharose FF column, buffer A (20 mM Tris, 30 mM NaCl, pH 8.0) and buffer B (20 mM Tris, 1 M NaCl, pH 8.0) were prepared. After binding the protein to the column, 5 CV of buffer A was flowed to remove the non-specifically bound protein, and after confirming that the conductivity was maintained constant, the buffer with a concentration gradient of 0-100% The target protein was eluted by flowing 5 CV of B.

Buffer A (20 mM Tris, 30 mM NaCl, pH 7.4) and Buffer B (20 mM Tris, 500 mM MgCl ₂ , pH7.4) were prepared for protein purification using an Alpha-1 anti-trypsin (a1AT) column. . After binding the protein to the column, 5 CV of buffer A was flowed to remove impurities, and 3 CV of buffer B was flowed at a concentration gradient of 0-100% to elute the target protein.

Buffer A (20 mM Tris, 1.5 M NaCl, pH 7.4) and buffer B (20 mM Tris, pH 7.4) were prepared for protein purification using Butyl HP column. The protein recovered in the Q Sepharose FF purification process was bound to a Butyl HP column, and impurities were removed by flowing 5 CV of Buffer A, and then 10 CV of Buffer B was flowed at a concentration gradient of 0-100% to elute the protein.

The eluted protein was dialyzed using a dialysis buffer (20 mM sodium phosphate, pH 7.0, 145 mM NaCl).

<1-5> Purification of GLP-1-Fc-FGF19 mutant with FGF19 mutant-containing fusion protein

For protein purification using a Q Sepharose FF column, buffer A (20 mM Tris, 30 mM NaCl, pH 8.0) and buffer B (20 mM Tris, 1 M NaCl, pH 8.0) were prepared. After binding the protein to the column, 5 CV of buffer A was flowed to remove the non-specifically bound protein, and after confirming that the conductivity was maintained constant, the buffer with a concentration gradient of 0-100% The target protein was eluted by flowing 5 CV of B.

Buffer A (20 mM NaPi, 150 mM NaCl, pH 7.0) and Buffer B (50 mM Citric acid, pH 7.0) were prepared for protein purification using a MabSelect Prism A column. After binding the protein to the column, 5 CV of buffer A was flowed to remove impurities, and 5 CV of 100% buffer B was flowed to elute the target protein.

Buffer A (20 mM Tris, 1.5 M NaCl, pH 7.4) and buffer B (20 mM Tris, pH 7.4) were prepared for protein purification using Butyl HP column. The protein recovered in the Q Sepharose FF purification process was bound to a Butyl HP column, and impurities were removed by flowing 5 CV of Buffer A, and then 10 CV of Buffer B was flowed at a concentration gradient of 0-100% to elute the protein.

The eluted protein was dialyzed using a dialysis buffer (20 mM sodium phosphate, pH 7.0, 145 mM NaCl).

<Example> Examples of FGF19 variants and fusion proteins comprising the same

FGF19 variants were prepared to improve the therapeutic effect of liver disease (Examples 1 to 7). In addition, the fusion protein GLP-1-NexP in which the FGF19 mutant and GLP-1 are fused through NexP (alpha-1 antitrypsin mutant), which is the applicant's long-acting technology, as a fusion protein for enhancing and providing durability for the treatment of liver disease -FGF19 mutant, GLP-1-Fc-FGF19 mutant fused via Fc as a long-acting carrier was prepared. Here NexP or Fc was fused to the C-terminus of GLP-1 and the N-terminus of the FGF19 variant. FGF19 mutant (Example 1-7), GLP-1-NexP-FGF19 mutant (Example 8-14) and GLP-1-Fc-FGF19 mutant (Example 15-21) are shown in Table 3, respectively. Specific amino acid sequences of the FGF19 mutant, GLP-1-NexP-FGF19 mutant and GLP-1-Fc-FGF19 mutant are as shown in Table 2 above.

Example FGF19 variant Mutations One F2011-n1
(SEQ ID NO: 1) Cut P2~S6, D7N, A8S, G9S, H11L 2 F2011-n2
(SEQ ID NO: 2) Cut P2~S6, A8T, G9S, H11L 3 F2011-n4
(SEQ ID NO: 3) Add N-terminal Met, cut P2~S6, cut A8S, G9S, H11L, S141P, H142~L144, cut P145G, M146L, L147P, M149A, V150L, E153P, E155G 4 F2011-n5
(SEQ ID NO: 4) Add N-terminal Met, cut P2~S6, A8S, G9S, H11L, S35T, F37H, D43N, V68I, M87I, V121L 5 F2011-n6
(SEQ ID NO: 5) Cut P2~S6, D7N, A8E, G9S, H11L, R105L, P106E 6 F2011-n7
(SEQ ID NO: 6) Cut P2~S6, D7N, G9S, H11L 7 F2011-n8
(SEQ ID NO: 7) Cut P2~S6, D7N, A8T, G9S, H11L Example fusion protein Included FGF19 variants 8 F2012-n1 (SEQ ID NO: 8) F2011-n1 9 F2012-n2 (SEQ ID NO: 9) F2011-n2 10 F2012-n4 (SEQ ID NO: 10) F2011-n4 11 F2012-n5 (SEQ ID NO: 11) F2011-n5 12 F2012-n6 (SEQ ID NO: 12) F2011-n6 13 F2012-n7 (SEQ ID NO: 13) F2011-n7 14 F2012-n8 (SEQ ID NO: 14) F2011-n8 15 B8011-n1 (SEQ ID NOs: 15 and 22) F2011-n1 16 B8011-n2 (SEQ ID NOs: 16 and 22) F2011-n2 17 B8011-n4 (SEQ ID NOs: 17 and 22) F2011-n4 18 B8011-n5 (SEQ ID NOs: 18 and 22) F2011-n5 19 B8011-n6 (SEQ ID NOs: 19 and 22) F2011-n6 20 B8011-n7 (SEQ ID NOs: 20 and 22) F2011-n7 21 B8011-n8 (SEQ ID NOs: 21 and 22) F2011-n8

The FGF19 variants of Examples 1 to 7 were cloned by the method described in Preparation Example <1-1> using the primers of Table 4 below, and then described in Preparation Examples <1-2> and <1-3>. It was prepared by transformation, cell culture and purification by the method, and the state of the final purified protein was confirmed by SDS-PAGE (FIG. 3). The fusion proteins of Examples 8 to 14 were cloned by the method described in Preparation Example <1-1> using the primers of Table 4 below, and then described in Preparation Examples <1-2> and <1-4>. It was prepared by transformation, cell culture and purification by the method, and the state of the final purified protein was confirmed by SDS-PAGE (Fig. 4, left). The fusion proteins of Examples 15 to 21 were cloned by the method described in Preparation Example <1-1> using the primers of Table 4 below, and then described in Preparation Examples <1-2> and <1-5>. It was prepared by transformation, cell culture and purification by the method, and the state of the final purified protein was confirmed by SDS-PAGE (FIG. 4, right).

As a result, as shown in FIGS. 3 and 4 , it was confirmed that the fusion protein comprising the FGF19 variants of Examples 1 to 7 and the FGF19 variants of Examples 8 to 21 was purified with high purity.

No. Name Sequence (5′-3′) Usage One TOPO-seq-F CGC AAA TGG GCG GTA GGC GT (SEQ ID NO:23) Colony PCR
Sequencing 2 TOPO-seq-R CAA CAT AGT TAA GAA TAC CAG TC (SEQ ID NO: 24) Sequencing 3 F2-Xho CAT ACT TAC TCG AGG CCA CCA TGC GTA GCG GTT GCG TC (SEQ ID NO: 25) Insert PCR 4 F2-not CAT ACT TAG CGG CCG CTT ACT ATT TTT CAA ATG ATG GGG (SEQ ID NO: 26) Insert PCR
Colony PCR 5 F2-n1-R TCG CCC CAT CCG TAA TGC ACG AGA GGG CTA GAA TTA CGC CCT GCC ACT GCC AGC C (SEQ ID NO: 27) Insert PCR 6 F2-n2-R TCG CCC CAT CCG TAA TGC ACG AGA GGG CTA GTA TCA CGC CCT GCC ACT GCC AGC C (SEQ ID NO: 28) Insert PCR 7 F2-n3-R TCG CCC CAT CCG TAA TGC ACG AGA GGG CTA GAA TCA CGC ATC CCT GCC ACT GCC AGC CAC (SEQ ID NO: 29) Insert PCR 8 AT-B7H4-Xho GAA TAT CTC GAG GCC ACC ATG CCT AGT TCC GTG (SEQ ID NO: 30) Insert PCR 9 AT-F2-mega-R TCG GAG AAA GCC AGA GGA CGA GCC AGT GAG ACG GGG ACC AG (SEQ ID NO: 31) Insert PCR 10 ALB-SP-Xho GAA TAT CTC GAG GCC ACC ATG AAG TGG GTT ACA (SEQ ID NO: 32) Insert PCR 11 ALB-F2-mega-R TCG GAG AAA GCC AGA GGA CGA GAG TAA GCA GAG GAG
(SEQ ID NO: 33) Insert PCR 12 Xho-P1 CAA TAC CTC GAG GCC ACC ATG GCT ACA GGC TCC CG
(SEQ ID NO: 34) Insert PCR 13 GH-F2-R TCG GAG AAA GCC AGA GGA CGG GCA CTG CCC TCT TGA AGC C (SEQ ID NO: 35) Insert PCR 14 Q5-L-Xho CAG ATT CTC GAG GCC ACC ATG GGA TGG TCC TG (SEQ ID NO: 36) Insert PCR 15 Ig-F2-R TCG GAG AAA GCC AGA GGA CGT GAG TGG ACC CCA GTA GCA G (SEQ ID NO:37) Insert PCR 16 ALB-F2-n1-R TCG CCC CAT CCG TAA TGC ACG AGA GGG CTA GAA TTA CGA GAG TAA GCA GAG GAG (SEQ ID NO: 38) Insert PCR 17 ALB-F2-n2-R TCG CCC CAT CCG TAA TGC ACG AGA GGG CTA GTA TCA CGA GAG TAA GCA GAG GAG (SEQ ID NO:39) Insert PCR 18 ALB-F2-n3-R TCG CCC CAT CCG TAA TGC ACG AGA GGG CTA GAA TCA CGC ATA GAG TAA GCA GAG GAG (SEQ ID NO: 40) Insert PCR 19 F2-n4-F AGA ATC GCG GCT TCC TGC CAC TGC CTG GCC TGC CAC CTG CCC TGC CTG AGC CAC CCG GCG ACC TGA GGG GAC ATC TGG AGA GC (SEQ ID NO: 41) Insert PCR 20 F2-n3-inter1-R TCC CCT AGC ACA ATC CAC CAC GCC GTT AGC GCG GAT TCT CAG GTG GCA GGT GGA CAG GCC ATG GGG GCC
(SEQ ID NO: 42) Insert PCR 21 F2-n3-inter2-R TAT ACT GCA GCA GGC CCT GAA TCT TGC CAT CAG CGC CCA TGC ACA GGT ACC GCA CGG AAT GCA CGC CCT TGA TGG CGA TTG TCC TCA GAG CCA CGG CC (SEQ ID NO:43) Insert PCR 22 F2-V143L-F AGG TCT GAG AAG CAC AGG CTG CCC CTG TCC CTG TCT TCC GCT AAG CAG (SEQ ID NO: 44) Insert PCR 23 F19-S30E-n6I-R TCC GTA ATG CAC GAG AGG GCT CTC ATT ACG AGA GTA AGC AGA GGA G (SEQ ID NO: 45) Insert PCR 24 F19-R127L,P128E-n6 ACT GTG CCT TTG AGG AGG AGA TCC TAG AGG ATG GCT ACA ACG TGT ATA GGT C (SEQ ID NO:46) Insert PCR 25 F19-S30A-n7-R TCC GTA ATG CAC GAG AGG GCT AGC ATT ACG AGA GTA AGC AGA GGA G (SEQ ID NO:47) Insert PCR 26 F19-S30T-n8-R TCC GTA ATG CAC GAG AGG GCT AGT ATT ACG AGA GTA AGC AGA GGA G (SEQ ID NO:48) Insert PCR 27 BamH-F2-n3 CTT TAC TAG GAT CCA TGC GTG ATT CTA GCC CTC TCG TGC (SEQ ID NO: 49) Insert PCR
Colony PCR 28 Xho-SP-Dur-L CAA TAT CTC GAG GCC ACC ATG AAA TGG G (SEQ ID NO: 50) Insert PCR 29 Dur-L-NexP GCG GCG TCA CCC TGA GGA TCT TCT GAA CCG CCC CCT CCT GAC CCT CC (SEQ ID NO: 51) Insert PCR 30 Nex-seq-I CAG ATC AAT GAT TAT GTG G (SEQ ID NO: 52) Colony PCR
Sequencing 31 Dur-L-Fc CTG GTG CAG GAC AAG GGG GGC AAG TTG AAC CGC CCC CTC CTG ACC CTC C (SEQ ID NO: 53) Insert PCR 32 Fc-seq-I GCA AAG TGT CCA ATA AGG C (SEQ ID NO: 54) Colony PCR Sequencing 33 GLP1R-I-seq CTC TGC TAT CCT GCT GGG C (SEQ ID NO: 55) Colony PCR
Sequencing 34 AV2-R AAG GCA CAG TCG AGG CTG ATC (SEQ ID NO: 56) Colony PCR

In addition, the expression of the fusion protein comprising the FGF19 variants of Examples 1 to 7 and the FGF19 variants of Examples 8 to 21 prepared above was confirmed through Western blot analysis.

Specifically, SDS-PAGE (150 volt, 60 min) was performed on the fusion protein comprising the FGF19 variants of Examples 1 to 7 and the FGF19 variants of Examples 8 to 21, and then the gel was removed. Transfer (25 volt, 70 min) was performed on the back NC membrane. After the transfer process was completed, the membrane was separated and immersed in 5% skim milk to perform blocking. After the blocking process, as the primary antibody, anti-FGF19 antibody (Abcam) was mixed with skim milk at a ratio of 1:2000 and added to the membrane, followed by reaction in a low temperature room (4°C) for one day. After the reaction was finished, it was washed 3 times (15 minutes) with TBST buffer, and then the secondary antibody Goat anti-Rabbit IgG (Jackson) was reacted on the membrane at a ratio of 1:3000 (60 min). After terminating the reaction by washing with TBST buffer 3 times, the ECL solutions A and B were mixed 1:1 and evenly sprayed on the membrane. Detection was performed with Chemi-Doc.

As a result, as shown in FIG. 5 , it was confirmed that fusion proteins including the FGF19 variants of Examples 1 to 7 and the FGF19 variants of Examples 8 to 21 were properly prepared.

<Experimental Example 1> FGFR4 receptor binding ability of FGF19 variants and fusion proteins including FGF19 variants

Competitive enzyme-linked immunosorbent assay (competitive enzyme-linked immunosorbent assay, competitive ELISA) was performed to determine the presence or absence of binding between the FGFR4 receptor and the fusion protein comprising the FGF19 variants of Examples 1 to 7 and the FGF19 variants of Examples 8 to 21 did

Specifically, after adsorbing 2 μg/ml anti-hFc specific antibody to a 96 well plate, blocking was performed using 3% BSA to prevent non-specific binding, and 1 μg/ml FGFR4-hFc was added It was aliquoted and reacted with an anti-hFc specific antibody. Then, 0.5 μg/mL of biotinylated human FGF19 and FGF19 variants of Examples 1 to 7, and each of the fusion proteins comprising the FGF19 variants of Examples 8 to 21 were mixed and dispensed, followed by reaction for 1 hour and 30 minutes. Then, streptavidin-HRP (horseradish peroxidase) was added and reacted for 30 minutes. In order to check the amount of bound HRP, it was reacted for 10 minutes after treatment with TMB, and the reaction was completed with 1 M sulfuric acid solution. Absorbance was measured at 450 nm wavelength.

As a result, as shown in FIGS. 6a to 6c, it was confirmed that all of the FGF19 variants of Examples 1 to 7 bind to the FGFR4 receptor.

In addition, as shown in FIG. 7 , it was confirmed that all of the fusion proteins including the FGF19 variants of Examples 8 to 21 bind to the FGFR4 receptor. In particular, it was confirmed that these fusion proteins exhibited superior binding ability compared to wild-type FGF19.

<Experimental Example 2> Confirmation of in vivo efficacy of FGF19 variant and fusion protein including FGF19 variant

<2-1> Animal

db/db mice (The Jackson Laboratory) were exposed to controlled light (12 h light/12 h light off, 6:30 pm - 6:30 am), temperature (22±4 ℃), and humidity (50%±20%) Stored according to welfare guidelines under conditions. Ad libitum access to water (sterile water) was provided, and a commercial diet containing 17 kcal % fat, 23 kcal % protein and 60 kcal % carbohydrate was provided ad libitum. For diet-induced obesity, C57BL6/J mice (Jackson Laboratory) were fed a high-fat diet (D12492, Research Diet, New Brunswick, JJ USA) containing 60 kCal % fat, 20 kcal % protein, and 20 kcal % carbohydrate (D12492, Research Diet, New Brunswick, JJ USA). It was maintained for 20 weeks. All animal studies were conducted with the approval of the Animal Ethics Committee.

<2-2> Blood glucose analysis

Blood collected from the tail of the mouse was analyzed using an ACCU-CHEK blood glucose meter from Roche Diagnostics, a manufacturer.

<2-3> Lipid profile analysis (Ripid profie analyis)

Whole blood collected from the tail of a mouse was collected in a general capillary tube (BD Clay Adams SurePrep), and then serum and blood cells were separated using a centrifuge. Lipid profiles of serum samples were analyzed using an Integra 400 Clinical analyzer according to the manufacturer's instructions, Roche Diagnostics.

<2-4> Hepatocelluar Carcinoma (HCC) analysis

Liver specimens were collected from db/db mice 6 months after drug injection. The HCC score was recorded as the number of HCC nodules on the total liver surface of mice injected with the strain divided by the number of HCC nodules of mice injected with wild-type FGF19.

<110> Alteogen, Inc <120> Novel fusion protein comprising FGF19 variant, and use thereof <130> PB2020-225 <160> 56 <170> KoPatentIn 3.0 <210> 1 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fgf19 variant <400> 1 Arg Asn Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg 1 5 10 15 Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25 30 Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser 35 40 45 Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala 50 55 60 Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly 65 70 75 80 Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu 85 90 95 Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His 100 105 110 Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125 Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140 Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met 145 150 155 160 Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170 175 Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 <210> 2 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fgf19 variant <400> 2 Arg Asp Thr Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg 1 5 10 15 Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25 30 Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser 35 40 45 Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala 50 55 60 Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly 65 70 75 80 Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu 85 90 95 Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His 100 105 110 Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125 Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140 Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met 145 150 155 160 Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170 175 Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 <210> 3 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fgf19 variant <400> 3 Met Arg Asp Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile 1 5 10 15 Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys 20 25 30 Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln 35 40 45 Ser Ala His Ser Leu Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val 50 55 60 Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp 65 70 75 80 Gly Lys Met Gln Gly Leu Leu Gln T yr Ser Glu Glu Asp Cys Ala Phe 85 90 95 Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys 100 105 110 His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr 115 120 125 Lys Asn Arg Gly Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro 130 135 140 Glu Pro Pro Gly Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser 145 150 155 160 Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly 165 170 175 Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 <210> 4 <211> 190 <212> PRT <213> Artificial Sequence <220> <223> fgf19 variant <400> 4 Met Arg Asp Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile 1 5 10 15 Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Thr Cys 20 25 30 His Leu Arg Ile Arg Ala Asn Gly Val Val Asp Cys Ala Arg Gly Gln 35 40 45 Ser Ala Hi s Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Ile 50 55 60 Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp 65 70 75 80 Gly Lys Ile Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe 85 90 95 Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys 100 105 110 His Arg Leu Pro Leu Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr 115 120 125 Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro 130 135 140 Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp 145 150 155 160 Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu 165 170 175 Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 190 <210> 5 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fgf19 variant <400> 5 Arg Asn Glu Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg 1 5 10 15 Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25 30 Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser 35 40 45 Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala 50 55 60 Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly 65 70 75 80 Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu 85 90 95 Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His 100 105 110 Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125 Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140 Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met 145 150 155 160 Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170 175 Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 <210> 6 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fgf19 variant <400> 6 Arg Asn Ala Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg 1 5 10 15 Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25 30 Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser 35 40 45 Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala 50 55 60 Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly 65 70 75 80 Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu 85 90 95 Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His 100 105 110 Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125 Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140 Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met 145 150 155 160 Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170 175 Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 <210> 7 <211> 189 <212> PRT < 213> Artificial Sequence <220> <223> fgf19 variant <400> 7 Arg Asn Thr Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg 1 5 10 15 Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe 20 25 30 Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser 35 40 45 Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala 50 55 60 Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly 65 70 75 80 Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu 85 90 95 Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His 100 105 110 Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys 115 120 125 Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met 130 135 140 Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met 145 150 155 160 Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 165 170 175 Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 180 185 <210> 8 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 8 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gin Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Arg Asn Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro 450 455 460 Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 465 470 475 480 Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 485 490 495 Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 500 505 510 Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala 515 520 525 Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 530 535 540 Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 545 550 555 560 Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 565 570 575 Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 580 585 590 Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser 595 600 605 Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 610 615 620 Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 <210> 9 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 9 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly G ly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gin Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Il e Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr As p Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Arg Asp Thr Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro 450 455 460 Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 465 470 475 480 Cys Phe Leu Arg Il e Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 485 490 495 Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 500 505 510 Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala 515 520 525 Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 530 535 540 Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 545 550 555 560 Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 565 570 575 Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 580 585 590 Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser 595 600 605 Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 610 615 620 Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 <210> 10 <211> 637 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 10 His Gly Glu Gly Thr Phe Thr S er Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gln Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp V al Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 P he Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Met Arg Asp Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp 450 455 460 Pro Ile A rg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 465 470 475 480 Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg 485 490 495 Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 500 505 510 Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly 515 520 525 Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 530 535 540 Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 545 550 555 560 Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln 565 570 575 Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 580 585 590 Leu Pro Glu Pro Pro Gly Asp Leu Arg Gly His Leu Glu Ser Asp Met 595 600 6 05 Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val 610 615 620 Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 <210> 11 <211> 640 <212> PRT <213 > Artificial Sequence <220> <223> fusion protein <400> 11 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gln Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Al a Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln Hi s Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Ph e Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Met Arg Asp Ser Ser Pro Leu Val His Tyr Gly Trp Gly Asp 450 455 460 Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser 465 470 475 480 Thr Cys His Leu Arg Ile Arg Ala Asn Gly Val Val Asp Cys Ala Arg 485 490 495 Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg 500 505 510 Thr Ile Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly 515 520 525 Ala Asp Gly Lys Ile Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys 530 535 540 Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser 545 550 555 560 Glu Lys His Arg Leu Pro Leu Ser Leu Ser Ser Ala Lys Gln Arg Gln 565 570 575 Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met 580 585 590 Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu 595 600 605 Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe 610 615 620 Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 640 <210> 12 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 12 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gln Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Arg Asn Glu Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro 450 455 460 Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 465 470 475 480 Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 485 490 495 Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 500 505 510 Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala 515 520 525 Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 530 535 540 Phe Glu Glu Glu Ile Leu Glu Asp Gly Tyr Asn Val Tyr Arg Ser Glu 545 550 555 560 Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 565 570 575 Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 580 585 590 Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser 595 600 605 Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 610 615 620 Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 <210> 13 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 13 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gln Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Arg Asn Ala Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro 450 455 460 Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 465 470 475 480 Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 485 490 495 Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 500 505 510 Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala 515 520 525 Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 530 535 540 Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 545 550 555 560 Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 565 570 575 Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 580 585 590 Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser 595 600 605 Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 610 615 620 Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 <210> 14 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 14 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp 35 40 45 Pro Gln Gly Asp Ala Ala Asn Lys Thr Asp Thr Ser His His Asp Gln 50 55 60 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 65 70 75 80 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 85 90 95 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 100 105 110 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 115 120 125 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 130 135 140 Leu Leu His Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 145 150 155 160 G ly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 165 170 175 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 180 185 190 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 195 200 205 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 210 215 220 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 225 230 235 240 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 245 250 255 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 260 265 270 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 275 280 285 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 290 295 300 Lys Leu G ln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 305 310 315 320 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 325 330 335 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 340 345 350 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 355 360 365 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 370 375 380 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 385 390 395 400 Ala Ile Asn Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe 405 410 415 Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 420 425 430 Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 4 45 Gly Ser Arg Asn Thr Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro 450 455 460 Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser 465 470 475 480 Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly 485 490 495 Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr 500 505 510 Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala 515 520 525 Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala 530 535 540 Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu 545 550 555 560 Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu 565 570 575 Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu 580 585 590 Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser 595 600 605 Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly 610 615 620 Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 625 630 635 <210> 15 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 15 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 45 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Gl u Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 25 5 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Arg Asn Ser Ser 275 280 285 Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu 290 295 300 Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg 305 310 315 320 Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu 325 330 335 Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val 340 345 350 His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly 355 360 365 Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg 370 375 380 Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val 385 390 395 400 Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe 405 410 415 Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu 420 425 430 Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro 435 440 445 Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu 450 455 460 Ala Val Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 16 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 16 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 45 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asp Thr Ser 275 280 285 Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu 290 295 300 Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg 305 310 315 320 Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu 325 330 335 Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val 340 345 350 His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly 355 360 365 Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg 370 375 380 Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val 385 390 395 400 Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe 405 410 415 Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu 420 425 430 Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro 435 440 445 Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu 450 455 460 Ala Val Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 17 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 17 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 4 5 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Arg Asp Ser 275 280 285 Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His 290 295 300 Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile 305 310 315 320 Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser 325 330 335 Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly 340 345 350 Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln 355 360 365 Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile 370 375 380 Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro 385 390 395 400 Val Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly 405 410 415 Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Gly 420 425 430 Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu 435 440 445 Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val 450 455 460 Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 18 <211 > 474 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 18 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 45 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Arg Asp Ser 275 280 285 Ser Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His 290 295 300 Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Thr Cys His Leu Arg Ile 305 310 315 320 Arg Ala Asn Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser 325 330 335 Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Ile Ala Ile Lys Gly 340 345 350 Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Ile Gln 355 360 365 Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile 370 375 380 Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro 385 390 395 400 Leu Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly 405 410 415 Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu 420 425 430 Glu Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser 435 440 445 Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu 450 455 460 Glu Ala Val Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 19 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 19 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 45 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Glu Ser 275 280 285 Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu 290 295 300 Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg 305 310 315 320 Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu 325 330 335 Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val 340 345 350 His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly 355 360 365 Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Leu 370 375 380 Glu Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val 385 390 395 400 Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe 405 410 415 Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu 420 425 430 Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro 435 440 445 Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu 450 455 460 Ala Val Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 20 <211> 473 <212> PRT <213> Artificial Sequen ce <220> <223> fusion protein <400> 20 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 45 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu P ro Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Ala Ser 275 280 285 Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu 290 295 300 Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile A rg 305 310 315 320 Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu 325 330 335 Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val 340 345 350 His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly 355 360 365 Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg 370 375 380 Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val 385 390 395 400 Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe 405 410 415 Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu 420 425 430 Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro 435 440 445 Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val T hr Gly Leu Glu 450 455 460 Ala Val Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 21 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 21 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Cys 35 40 45 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 50 55 60 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 65 70 75 80 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 85 90 95 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 100 105 110 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 115 120 125 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 130 135 140 Val Ser As n Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys 145 150 155 160 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 165 170 175 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 180 185 190 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 195 200 205 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Lys Asp Gly 210 215 220 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 225 230 235 240 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Thr Ser 275 280 28 5 Pro Leu Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu 290 295 300 Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg 305 310 315 320 Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu 325 330 335 Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val 340 345 350 His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly 355 360 365 Leu Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg 370 375 380 Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val 385 390 395 400 Ser Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe 405 410 415 Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu 420 425 430 Pro Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Pro 435 440 445 Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu 450 455 460 Ala Val Arg Ser Pro Ser Phe Glu Lys 465 470 <210> 22 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> fusion protein_chain B <400> 22 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1 5 10 15 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 35 40 45 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55 60 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95 Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile 100 105 110 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 130 135 140 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 145 150 155 160 Gly Gln Pro Glu Asn Asp Tyr Lys Thr Thr Pro Val Leu Asp Ser 165 170 175 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 180 185 190 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 195 200 205 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TOPO-seq-F < 400> 23 cgcaaatggg cggtaggcgt 20 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TOPO-seq-R <400> 24 caacatagtt aagaatacca gtc 23 <210> 25 <211> 38 <212> DNA <213> Artificial Sequ ence <220> <223> F2-Xho <400> 25 catacttact cgaggccacc atgcgtagcg gttgcgtc 38 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> F2-not <400> 26 catacttagc ggccgcttac tatttttcaa atgatgggg 39 <210> 27 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> F2-n1-R <400> 27 tcgccccatc cgtaatgcac gagagggcta gaattacgcc ctgccactgc cagcc 55 <210> 28 < 55 <212> DNA <213> Artificial Sequence <220> <223> F2-n2-R <400> 28 tcgccccatc cgtaatgcac gagagggcta gtatcacgcc ctgccactgc cagcc 55 <210> 29 <211> 60 <212> DNA <213> Artificial Sequence < 220> <223> F2-n3-R <400> 29 tcgccccatc cgtaatgcac gagagggcta gaatcacgca tccctgccac tgccagccac 60 60 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> AT-B7H4-Xho <400> 30 gaatatctcg aggccaccat gcctagttcc gtg 33 <210> 31 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> AT-F2-mega-R <400> 31 tcggagaaag ccagaggacg agccagtgag acggggacca g 41 < 210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> ALB-SP-Xho <400 > 32 gaatatctcg aggccaccat gaagtgggtt aca 33 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> ALB-F2-mega-R <400> 33 tcggagaaag ccagaggacg agagtaagca gaggag 36 <210> 34 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Xho-P1 <400> 34 caatacctcg aggccaccat ggctacaggc tcccg 35 <210> 35 <211> 40 <212> DNA <213> Artificial Sequence <220 > <223> GH-F2-R <400> 35 tcggagaaag ccagaggacg ggcactgccc tcttgaagcc 40 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Q5-L-Xho <400> 36 cagattctcg aggccaccat gggatggtcc tg 32 <210> 37 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Ig-F2-R <400> 37 tcggagaaag ccagaggacg tgagtggacc ccagtagcag 40 <210> 38 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> ALB-F2-n1-R <400> 38 tcgccccatc cgtaatgcac gagagggcta gaattacgag agtaagcaga ggag 54 <210> 39 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> ALB-F2-n2-R <400> 39 tcgccccatc cgtaatgcac gagagggcta gtatcacgag agtaagcaga ggag 54 <210> 40 <211> 5 7 <212> DNA <213> Artificial Sequence <220> <223> ALB-F2-n3-R <400> 40 tcgccccatc cgtaatgcac gagagggcta gaatcacgca tagagtaagc agaggag 57 <210> 41 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> F2-n4-F <400> 41 agaatcgcgg cttcctgcca ctgcctggcc tgccacctgc cctgcctgag ccacccggcg 60 acctgagggg acatctggag agc 83 <210> 42 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> F2-n3-inter1-R <400> 42 tcccctagca caatccacca cgccgttagc gcggattctc aggtggcagg tggacaggcc 60 atggggggcc 69 <210> 43 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> F2-n3-inter2-R <400> 43 tatactgcag caggccctga atcttgccat cagcgcccat gcacaggtac cgcacggaat 60 gcacgccctt gatggcgatt gtcctcaga < ccacggcc 98 48 <212> DNA <213> Artificial Sequence <220> <223> F2-V143L-F <400> 44 aggtctgaga agcacaggct gcccctgtcc ctgtcttccg ctaagcag 48 <210> 45 <211> 46 <212> DNA <213> Artificial Sequence <220 > <223> F19-S30E-n6I-R <400> 45 tccgtaatgc acgagagggc tctcattacg agagtaagca gaggag 46 <210> 46 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> F19-R127L,P128E- n6 <400> 46 actgtgcctt tgaggaggag atcctagagg atggctacaa cgtgtatagg tc 52 <210> 47 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> F19-S30A-n7-R <400> 47 tccgtaatgc acgagagggc tagcattacca gaggag 46 <210> 48 <2 11> 46 <212> DNA <213> Artificial Sequence <220> <223> F19-S30T-n8-R <400> 48 tccgtaatgc acgagagggc tagtattacg agagtaagca gaggag 46 <210> 49 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> BamH-F2-n3 <400> 49 ctttactagg atccatgcgt gattctagcc ctctcgtgc 39 <210> 50 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Xho-SP-Dur -L <400> 50 caatatctcg aggccaccat gaaatggg 28 <210> 51 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Dur-L-NexP <400> 51 gcggcgtcac cctgaggatc ttctgaaccg ccccctcctg accctcc 47 <210 > 52 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nex-seq-I <400> 52 cagatcaatg attatgtgg 19 <210> 53 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Dur-L-Fc <400> 53 ctggtgcagg acaagggggg caagttgaac cgccccctcc tgaccctcc 49 <210> 54 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fc-seq-I < 400> 54 gcaaagtgtc caataaggc 19 <210> 55 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GLP1R-I-seq <400> 55 ctctgctatc ctgctgggc 19 <210 > 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AV2-R<400> 56 aaggcacagt cgaggctgat c 21

Claims (28)

FGF19 variant in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19).
The FGF19 variant according to claim 1, wherein the FGF19 variant is selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 7.
The FGF19 variant according to claim 1, wherein the FGF19 variant further comprises a long-acting carrier.
4. The FGF19 variant according to claim 3, wherein the persistent carrier is fused to the N-terminus or C-terminus of the FGF19 variant.
According to claim 3, wherein the long-acting carrier is polyethylene glycol, fatty acid, albumin or fragment thereof, albumin-binding substance, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or fragment thereof, repeating unit polymer of a specific amino acid sequence, antibody Or fragments thereof, FcRn binding material, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and FGF19 variants that are selected from the group consisting of polymers.
The FGF19 variant according to claim 5, wherein the long-acting carrier is alpha-1 antitrypsin or a variant thereof, or an immunoglobulin Fc or fragment thereof.
7. The method of claim 6, wherein in the alpha-1 antitrypsin variant, one or more amino acids are substituted with other amino acids, wherein the substitution comprises a substitution of at least one position from the 1st amino acid to the 25th amino acid from the N-terminus. , FGF19 variants.
7. The method of claim 6, wherein the alpha-1 antitrypsin variant is substituted with asparagine at the ninth amino acid from the N-terminus, serine at the 232th amino acid, asparagine at the 37th amino acid, and threonine at the 359th amino acid The FGF19 variant comprising any one or more substitutions selected from the group consisting of.
7. The FGF19 variant of claim 6, wherein the immunoglobulin Fc is an animal, human, or modified immunoglobulin Fc thereof.
7. The FGF19 variant of claim 6, wherein the immunoglobulin Fc is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, and combinations thereof.
5. The FGF19 variant according to claim 4, wherein the persistent carrier is fused with the FGF19 variant directly or via a linker.
The FGF19 variant according to claim 11, wherein the linker is a peptidic linker or a non-peptidyl linker.
13. The method of claim 12, wherein the non-peptidyl linker is polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinylethyl ether , polylactic acid (PLA), polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof, FGF19 variants.
The FGF19 variant according to claim 12, wherein the peptidic linker is two or more amino acids linked.
FGF19 variant in which one or more amino acids are substituted with other amino acids in the amino acid sequence of Fibroblast Growth Factor 19 (FGF 19); and GLP-1 or an analog thereof.
The fusion protein according to claim 15, wherein the FGF19 variant is selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 7
16. The method of claim 15, wherein said FGF19 variant; and GLP-1 or an analog thereof is fused directly or via a long-acting carrier.
18. The method of claim 17, wherein the long-acting carrier is an FGF19 variant; and fused to the N-terminus or C-terminus of GLP-1 or an analog thereof.
The fusion protein of claim 18 , wherein the persistent carrier is fused to the N-terminus of the FGF19 variant and fused to the C-terminus of GLP-1 or an analog thereof.
18. The method of claim 17, wherein the long-acting carrier is polyethylene glycol, fatty acid, albumin or fragment thereof, albumin-binding substance, alpha-1 antitrypsin or variant thereof, immunoglobulin Fc or fragment thereof, repeat unit polymer of a specific amino acid sequence, antibody Or fragments thereof, FcRn binding material, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharide, and a fusion protein that is selected from the group consisting of high molecular weight polymers.
The fusion protein according to claim 17, wherein the durable carrier is alpha-1 antitrypsin or a variant thereof, or an immunoglobulin Fc or fragment thereof.
18. The fusion protein of claim 17, wherein the persistent carrier further comprises a linker.
The fusion protein according to claim 22, wherein the linker is a peptidic linker or a non-peptidyl linker.
The fusion protein according to claim 15, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NOs: 8 to 21.
The fusion protein according to claim 15, wherein the fusion protein has an increased half-life in the body.
A pharmaceutical composition for preventing or treating liver disease comprising the FGF19 variant of any one of claims 1 to 14, or the fusion protein of any one of claims 15 to 25.
The pharmaceutical composition for preventing or treating liver disease according to claim 26, wherein the liver disease is at least one selected from the group consisting of fatty liver disease, liver fibrosis, cirrhosis, and inflammatory liver disease.
The pharmaceutical composition for preventing or treating liver disease according to claim 27, wherein the inflammatory liver disease is nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD).

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