Pharmaceutical composition for preventing or treating hepatitis, hepatic fibrosis, and hepatic cirrhosis comprising fusion proteins

Abstract

To provide a composition for preventing or treating hepatitis, hepatic fibrosis, and hepatic cirrhosis, as well as uses and methods thereof.SOLUTION: A fusion protein comprising, as an active ingredient, a fibroblast growth factor 21 (FGF21) mutant protein; and a pharmaceutical composition for preventing or treating hepatitis, hepatic fibrosis, and hepatic cirrhosis, the composition comprising an Fc region of an immunoglobulin.SELECTED DRAWING: Figure 13A

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis. Specifically, the present invention relates to a fusion protein comprising a biologically active protein and an FGF21 mutant protein, and a pharmaceutical composition comprising the fusion protein, which is effective for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis. [Background technology]

[0002] There are various causes of liver disease, such as viral or bacterial infection, excessive accumulation of alcohol or toxic substances, fat or heavy metals, abnormal immune response, etc. These causes can include viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, toxic hepatitis, autoimmune liver disease, etc. In addition, some liver diseases can progress to cirrhosis, liver fibrosis, and liver cancer through chronic progression.

[0003] Acute liver diseases such as acute viral hepatitis or toxic hepatitis can cause severe fatigue, loss of appetite, and jaundice, and can rapidly progress to acute liver failure, which can lead to death if a liver transplant is not performed. On the other hand, slowly progressing chronic liver diseases such as chronic viral hepatitis and fatty liver disease are largely asymptomatic, and patients may not experience significant inconvenience in their daily lives. However, liver fibrosis is ongoing, and chronic liver disease can inadvertently progress to cirrhosis and liver cancer. According to the Korean Cirrhosis Incidence Statistics, the prevalence of cirrhosis was estimated at 0.5% in adults and approximately 1.0% in those aged 65 and older in 2012. Because this is based on a questionnaire survey conducted based on medical history, the actual prevalence of cirrhosis may be higher. The most common cause of cirrhosis in Korea is viral hepatitis, and the second most common cause is alcoholic liver disease.

[0004] When liver damage is repeatedly caused by some causes, regardless of whether symptoms exist or not, there is a high risk of developing cirrhosis, fibrosis, and liver cancer.When cirrhosis occurs, it is difficult to restore the hardened liver to its original state even with treatment.

[0005] Glucagon-like peptide-1 (GLP-1) is a 31-amino acid incretin hormone secreted by L-cells in the intestinal tract upon stimulation by food. Its biological effects occur via intracellular signaling via the GLP-1 receptor, a G protein-coupled receptor expressed in target tissues such as pancreatic β cells and the brain. GLP-1 secreted into the blood has a very short half-life of less than 2 minutes, caused by loss of activity due to cleavage of amino acids at its N-terminus by the enzyme dipeptidyl peptidase-4 (DPP-4). GLP-1 stimulates insulin secretion in pancreatic β cells based on blood glucose levels, thereby exerting a potent effect in lowering blood glucose without inducing hypoglycemia. Furthermore, administration of GLP-1 has been shown to result in weight loss in various animal models and humans, which is caused by reduced food intake due to its effect on appetite suppression. GLP-1 induces beta cell proliferation and enhances beta cell viability by inhibiting cell death caused by glucolipotoxicity via the GLP-1 receptor expressed in pancreatic beta cells. Excessive secretion of glucagon increases blood glucose and is known to be one of the causes of hyperglycemia in diabetes. In addition, GLP-1 is known to act on pancreatic alpha cells to inhibit fasting blood glucose elevation by inhibiting protein kinase A (PKA) protein-specific glucagon secretion.

[0006] Exendin-4 is a clinically important GLP-1 receptor agonist. It is a polypeptide with 39 amino acid residues and is normally produced in the salivary glands of the Gila monster lizard. It shares 52% amino acid sequence identity with GLP-1 and is known to interact with the GLP-1 receptor in mammals (Thorens et al. (1993) Diabetes 42:1678-1682). Exendin-4 has been shown to stimulate insulin secretion from insulin-producing cells in vitro, and its induction of insulin release from insulin-producing cells is stronger than that of GLP-1 under equimolar conditions. Exendin-4 potently stimulates insulin secretion and reduces blood glucose levels in both rodents and humans with a longer duration of action than GLP-1. However, exendin-4 is antigenic in mammals lacking GLP-1 due to the presence of an unidentified epitope.

[0007] The ability of GLP-1 and exendin-4 analogs (e.g., liraglutide and exenatide) to improve glucose control in humans has been clinically confirmed. It has been reported that GLP-1 increases beta cell mass through the inhibition of apoptosis and induction of proliferation. Furthermore, it has also been reported that GLP-1 acts as a gut hormone that inhibits gastric acid secretion and gastric emptying while enhancing satiety signals, thereby reducing appetite. This effect of GLP-1 may explain the weight loss observed when GLP-1 analogs are administered to patients with type 2 diabetes. Additionally, GLP-1 exhibits post-ischemic cardioprotective effects in rodents.

[0008] Various attempts have been made to develop long-acting GLP-1 analogs. Clinically confirmed long-acting GLP-1 analogs include dulaglutide (WO 2005 / 000892) and albiglutide (WO 2003 / 059934). Dulaglutide is an Fc-fused GLP-1 analog, and albiglutide is an albumin-fused GLP-1 analog, both of which have pharmacokinetic profiles that allow for once-weekly administration. Both drugs have excellent effects on lowering blood glucose and reducing body weight when administered once-weekly, and also provide significantly improved convenience in terms of treatment compared with exenatide and liraglutide.

[0009] Fibroblast growth factor 21 (FGF21), synthesized in the liver, is a hormone known to play an important role in glucose and lipid homeostasis. FGF21 exerts pharmacological effects in the liver, adipocytes, pancreatic β cells, hypothalamus, and muscle tissue, where both FGF21-specific receptors, i.e., FGF receptors and the β-Klotho complex, are expressed. It has been reported that FGF21 can reduce blood glucose levels, body weight, and blood triglyceride and low-density lipoprotein (LDL) concentrations in an insulin-independent manner in nonhuman primate and mouse models of various diabetes and metabolic diseases. Additionally, FGF21 is known to improve insulin sensitivity, making it a promising target for novel anti-diabetic or anti-obesity therapeutics (WO 2003 / 011213).

[0010] Therefore, in order to develop new antidiabetic drugs based on FGF21, attempts have been made to improve its biological activity and in vivo stability by constructing FGF21 mutants based on the wild-type FGF21 sequence through the substitution, insertion, and deletion of several amino acids (see WO2010 / 065439). However, FGF21 has a very short half-life, which has proven problematic when used directly as a biological drug (Kharitonenkov, A. et al., Journal of Clinical Investigation 115:1627-1635, 2005). The in vivo half-life of FGF21 is 1 to 2 hours in mice and 2.5 to 3 hours in monkeys. Therefore, for FGF21 to be used in its current form as a therapeutic agent for diabetes, daily administration is required.

[0011] Various approaches have been reported to increase the in vivo half-life of FGF21 recombinant protein. One such example is attaching polyethylene glycol (PEG), a polymeric material, to FGF21 to increase its molecular weight, thereby inhibiting renal excretion and increasing in vivo retention time (see WO 2012 / 066075). Another approach attempts to improve half-life by fusing it with a fatty acid that binds to human albumin (see WO 2012 / 010553). A further example attempts to increase half-life while maintaining pharmacological activity equivalent to that of wild-type FGF21 through the production of agonist antibodies that specifically bind to the human FGF receptor alone or in complex with β-Klotho (see WO 2012 / 170438). In another example, half-life was improved by preparing a long-acting fusion protein in which the Fc region of IgG binds to the FGF21 molecule (see WO 2013 / 188181).

[0012] Among the various available technologies for creating long-acting drugs, Fc fusion technology is widely used because it increases in vivo half-life while minimizing the drawbacks of other approaches, such as the induction of immune responses or toxicity. For the development of Fc-fused FGF21 proteins as long-acting therapeutic agents, the following conditions must be met:

[0013] First, the decrease in in vitro activity caused by the fusion should be minimized. Both the N- and C-termini of FGF21 contribute to FGF21 activity. In this regard, it is known that the activity of FGF21 fusion proteins varies significantly depending on the fusion position. Therefore, the activity of Fc-fused FGF21 fusion proteins in which mutations have been introduced into FGF21 may vary depending on the presence / absence or position of the fusion. Second, a pharmacokinetic profile that allows for weekly administration in humans should be achieved by increasing the in vivo half-life of the fusion. Third, given that immunogenicity can be expected in most patients after administration of biopharmaceuticals, the risk of immunogenicity due to the fusion linker or mutations should be minimized. Fourth, there should be no stability issues arising from the fusion position or the introduction of mutations. Fifth, because unwanted immune responses may occur depending on the isotype of the fusion immunoglobulin, solutions to prevent such responses are needed.

[0014] Attempts to develop long-acting fusion proteins by linking the Fc region of immunoglobulin G (IgG) to the FGF21 molecule have already been reported (see WO 2013 / 188181). Although the Fc region is fused to the N-terminus of wild-type FGF21, the Fc-FGF21 construct exhibits no clear difference in in vitro activity compared to wild-type FGF21. It is known that the half-life is extremely short due to in vivo protein degradation. To address this issue, attempts have been made to improve the in vivo half-life by introducing several mutations into specific sites in FGF21 to resist proteolysis. However, the risk of immunogenicity may increase with the introduction of multiple mutations. In contrast, the FGF21-Fc construct, in which the Fc region is fused to the C-terminus of the FGF21 molecule, exhibits a significant decrease in activity caused by fusion at this site compared to the Fc-FGF21 construct.

[0015] The combined administration of GLP-1 and FGF21 may have a synergistic effect compared to single administration depending on the mechanism of action and target tissue in the body, and is expected to potentially have superior anti-diabetic efficacy and additional benefits. The effects of combined administration of GLP-1 and FGF21 or GLP-1 / FGF21 fusion proteins have already been studied and reported (see WO 2010 / 142665 and WO 2011 / 020319).

[0016] Various issues must be resolved to develop a fusion protein containing GLP-1 and FGF21. Because wild-type GLP-1 and wild-type FGF21 have very short in vivo half-lives, even if developed as a therapeutic, they must be administered at least once daily. Therefore, long-acting technologies such as Fc fusion are needed to develop long-acting fusion proteins that improve patient convenience. For dual-function drugs targeting the two targets of GLP-1 and FGF21, it is important to introduce mutations that maintain the drug's activity and in vivo stability, and to address issues associated with changes in activity, structure, or stability caused by each mutation. The efficacy of the two targets, GLP-1 and FGF21, must be balanced. To achieve this, drug design is required that takes into account in vitro activity, pharmacokinetic profiles, pharmacological efficacy in animal models, and even clinical evaluation of efficacy in humans. Fusion proteins have structures that cannot exist in the human body and are structurally more complex than fusion proteins targeting a single target. In addition, because mutations or linker engineering are required to balance the two targets, the likelihood of forming aggregate complexes may increase, and further protein engineering may be required to prevent this. Furthermore, the potential for immunogenicity may increase due to novel mutant sequences or complex structures, which should be addressed or avoided.

[0017] The present inventors have made efforts to solve the above problems, and as a result have developed a fusion protein that is effective in treating hepatitis, liver fibrosis, and liver cirrhosis, and have completed the present invention. Summary of the Invention

[0018] technical issues An object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis.

[0019] Solutions to problems According to one object of the present invention, there is provided a pharmaceutical composition for preventing or treating hepatitis, liver fibrosis, and cirrhosis, comprising, as active ingredients, a fusion protein comprising a fibroblast growth factor 21 (FGF21) mutant protein; and an Fc region of an immunoglobulin, wherein the FGF21 mutant protein has one of the following mutations (1) to (7): (1) Substitution of amino acids at positions 98 to 101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 68); (2) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 69); (3) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 70); (4) substitution of the amino acid at position 170 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (5) substitution of the amino acid at position 174 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (6) a substitution of the amino acid at position 180 from the N-terminus of the wild-type FGF21 protein with the amino acid E, together with one or more of the mutations described in (1) to (5) above; and (7) Mutation of 1 to 10 amino acids to reduce the immunogenicity of wild-type FGF21 protein The present invention provides a pharmaceutical composition comprising at least one mutation selected from the group consisting of:

[0020] Furthermore, according to another object of the present invention, there is provided a pharmaceutical composition for preventing or treating hepatitis, liver fibrosis, and cirrhosis, comprising as active ingredients a fusion protein containing a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a mutant or fragment thereof; and an Fc region of an immunoglobulin, wherein the FGF21 mutant protein has one of the following mutations (1) to (7): (1) Substitution of amino acids at positions 98 to 101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 68); (2) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 69); (3) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 70); (4) substitution of the amino acid at position 170 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (5) substitution of the amino acid at position 174 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (6) a substitution of the amino acid at position 180 from the N-terminus of the wild-type FGF21 protein with the amino acid E, together with one or more of the mutations described in (1) to (5) above; and (7) Mutation of 1 to 10 amino acids to reduce the immunogenicity of wild-type FGF21 protein The present invention provides a pharmaceutical composition comprising at least one mutation selected from the group consisting of:

[0021] Furthermore, according to another object of the present invention, there is provided a use of the pharmaceutical composition of the present invention for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis.

[0022] Furthermore, according to another object of the present invention, there is provided use of the pharmaceutical composition of the present invention for producing a composition for preventing or treating hepatitis, liver fibrosis and liver cirrhosis.

[0023] Furthermore, in accordance with another object of the present invention, there is provided a method for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis, which comprises the step of administering the fusion protein of the present invention to a subject.

[0024] Advantageous Effects of the Invention The pharmaceutical composition of the present invention for preventing or treating hepatitis, liver fibrosis, and cirrhosis, which comprises as active ingredients a fusion protein containing a fibroblast growth factor 21 (FGF21) mutant protein and the Fc region of an immunoglobulin, has the effect of inhibiting the proliferation of inflammatory cells and fibroblasts, and can therefore be effectively used as a composition for preventing or treating hepatitis, liver fibrosis, and cirrhosis. [Brief explanation of the drawings]

[0025] [Figure 1A] 1A is a graph showing the in vitro activity of DFD4, DFD5, DFD6, DFD7, DFD9, and DFD13 as fusion proteins containing FGF21 mutant proteins (hereinafter, "FGF21 mutant fusion proteins") using an HEK293 cell line in which human β-Klotho is overexpressed. The FGF21 mutant fusion proteins did not show a significant decrease in activity due to the introduction of mutations.

[0026] [Figure 1B] Figure 1B is a graph showing the in vitro activity of DFD6, DFD13, DFD18, DFD72, DFD73, and DFD74 as fusion proteins, including FGF21 mutant fusion proteins, using a HEK293 cell line in which human β-Klotho is overexpressed. The FGF21 mutant fusion proteins did not show a significant decrease in activity due to the introduction of mutations.

[0027] [Figure 1C] Figure 1C is a graph showing the in vitro activity of DFD6 and DFD6 (E. coli) as fusion proteins containing FGF21 mutant fusion proteins using a HEK293 cell line in which human β-Klotho is overexpressed. The FGF21 mutant fusion proteins did not show a significant decrease in activity due to the introduction of mutations.

[0028] [Figure 2A]2A is a graph showing the in vitro activity of DFD3 and DFD4 as FGF21 mutant fusion proteins containing a linker linking the N-terminus and Fc region of FGF21, using a HEK293 cell line in which human β-Klotho is overexpressed. The FGF21 mutant fusion proteins showed slight differences in activity depending on the linker sequence, but did not show a significant decrease in activity.

[0029] [Figure 2B] Figure 2B is a graph showing the in vitro activity of DFD1 and DFD13 as FGF21 mutant fusion proteins containing a linker linking the N-terminus and Fc region of FGF21, using a HEK293 cell line in which human β-Klotho is overexpressed. The FGF21 mutant fusion proteins showed slight differences in activity depending on the linker sequence, but did not show a significant decrease in activity.

[0030] [Figure 3] Figure 3 is a graph showing the in vitro activity of RGE (Amgen), Fc-FGF21 (Lilly), and DFD1 using a HEK293 cell line in which human β-Klotho is overexpressed. DFD1 and RGE (Amgen) had similar activities, but Fc-FGF21 (Lilly) had two-fold higher in vitro activity than the other proteins.

[0031] [Figure 4A] FIG. 4A shows the results of size exclusion chromatography analysis of the FGF21 mutant fusion protein, DFD4.

[0032] [Figure 4B] FIG. 4B shows the results of size exclusion chromatography analysis of DFD13, an FGF21 mutant fusion protein into which the EIRP mutation has been introduced.

[0033] [Figure 4C]Figure 4C is a graph comparing the proposed content of high molecular weight aggregates (HMW%) of DFD4 and DFD13 using size exclusion chromatography to demonstrate the effect of the EIRP mutation of FGF21 on the stability of the fusion protein. DFD13 was associated with a lower percentage of high molecular weight aggregates (HMW%) compared to DFD4 at the initial stage and after 2 weeks or more, indicating that the introduction of the EIRP mutation improved the stability of the FGF21 mutant fusion protein, thereby significantly reducing the HMW%.

[0034] [Figure 5] Figure 5 shows the concentration of each protein in the blood over 96 hours after subcutaneous administration of FGF21 mutant fusion proteins. Data are presented as the mean and standard deviation.

[0035] [Figure 6A] Figure 6A is a graph showing the change in body weight in grams after a single administration of DFD18 in a diet-induced obese mouse model from the time of administration to 14 days. DFD18 showed a significant weight loss effect. Data are shown as the mean and standard error of the mean.

[0036] [Figure 6B] Figure 6B is a graph showing the change in body weight in % after a single administration of DFD18 in a diet-induced obese mouse model from the time of administration to day 14. DFD18 showed a significant weight loss effect. Data are shown as the mean and standard error of the mean.

[0037] [Figure 7]Figure 7 is a graph showing the in vitro GLP-1 activity of fusion proteins, depending on the GLP-1 variant and the hinge linking the C-terminus of GLP-1 to the Fc region, using a CHO cell line in which the human GLP-1 receptor is overexpressed. In general, the fusion protein (DFD23) containing the GLP-1(A2G) sequence exhibited 2- to 3-fold lower activity than other fusion proteins containing other GLP-1 variant sequences. No significant differences in GLP-1 activity were observed between fusion proteins containing variant sequences other than the GLP-1(A2G) sequence.

[0038] [Figure 8A] Figure 8A is a graph showing the GLP-1 activity of DFD59, DFD69, DFD112, and DFD114. The in vitro GLP-1 activity of the three fusion proteins (DFD69, DFD112, and DFD114) and FGF21-free Fc-fused GLP-1 variants was measured using a CHO cell line in which the human GLP-1 receptor is overexpressed. The three fusion proteins showed similar EC50 values, and the Fc-fused GLP-1 variant (DFD59) showed approximately two-fold higher activity than the fusion proteins.

[0039] [Figure 8B] Figure 8B is a graph showing the FGF21 activity of DFD69, DFD112, and DFD114. The in vitro activity of the fusion proteins dependent on the FGF21 mutants was measured using a HEK293 cell line in which human β-Klotho is overexpressed. It was demonstrated that the in vitro activity of the FGF21 moiety was similar in the three fusion proteins.

[0040] [Figure 9A] 9A is a graph showing serum drug concentrations after subcutaneous administration of the FGF21 portion of fusion proteins DFD69, DFD112, and DFD114. Data are presented as mean and standard deviation.

[0041] [Figure 9B]Figure 9B is a graph showing serum drug concentrations after subcutaneous administration of the GLP-1 portion of the fusion proteins DFD59, DFD69, DFD112, and DFD114. Data are presented as the mean and standard deviation.

[0042] [Figure 10A] Figure 10A is a graph showing changes in serum triglycerides (TG) after repeated subcutaneous administration of DFD114, DFD112, DFD74, or DFD72 in a diet-induced obese mouse model at 4-day intervals for 2 weeks. Administration of the fusion proteins and FGF21 mutant fusion proteins showed a serum lipid-lowering effect compared to the control group. Data are shown as the mean and standard error of the mean. Statistical analysis was performed by one-way analysis of variance followed by Dunnett's multiple comparison test (***: P<0.001 vs. vehicle control).

[0043] [Figure 10B] Figure 10B is a graph showing changes in serum total cholesterol (TC) after repeated subcutaneous administration of DFD114, DFD112, DFD74, or DFD72 in a diet-induced obese mouse model at 4-day intervals for 2 weeks. Administration of the fusion proteins and FGF21 mutant fusion proteins showed a serum lipid-lowering effect compared to the control group (***: P<0.001 vs. vehicle control).

[0044] [Figure 10C] Figure 10C is a graph showing changes in liver triglycerides (TG) after repeated subcutaneous administration of DFD114, DFD112, DFD74, or DFD72 in a diet-induced obese mouse model at 4-day intervals for 2 weeks. Administration of FGF21 mutant fusion proteins showed a lipid-lowering effect in the liver compared to the control group (*: P<0.05, ***: P<0.001 vs. vehicle control).

[0045] [Figure 11]Figure 11 shows histopathological images of the liver obtained in a diet-induced obese mouse model after repeated subcutaneous administration of DFD114 or DFD112 at 4-day intervals for 2 weeks. Administration of the fusion proteins demonstrated a reduced hepatic steatosis compared to the control group.

[0046] [Figure 12A] Figure 12A is a graph showing changes in ALT levels after repeated subcutaneous administration of DFD112 and DFD72 at 2-day intervals for 4 weeks in a methionine-choline-deficient (MCD) diet-induced nonalcoholic steatohepatitis mouse model. ALT levels were dose-dependently reduced in the fusion protein-treated groups compared with the control group, and ALT levels were also reduced in the FGF21 mutant fusion protein-treated groups. Data are shown as mean and standard error of the mean. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test (###: P<0.001 vs. MCS control, **: P<0.01, ***: P<0.001 vs. MCD control).

[0047] [Figure 12B] Figure 12B is a graph showing changes in AST levels after repeated subcutaneous administration of DFD112 and DFD72 at 2-day intervals for 4 weeks in a methionine choline-deficient (MCD) diet-induced nonalcoholic steatohepatitis mouse model. AST levels were dose-dependently reduced in the fusion protein-treated groups compared with the control group, and AST levels were also reduced in the FGF21 mutant fusion protein-treated groups (###: P<0.001 vs. MCS control, **: P<0.01 vs. MCD control).

[0048] [Figure 12C]Figure 12C is a graph showing changes in inflammation levels after repeated subcutaneous administration of DFD112 and DFD72 in a methionine choline deficient (MCD) diet-induced nonalcoholic steatohepatitis mouse model at 2-day intervals for 4 weeks. Inflammation levels were dose-dependently reduced in the fusion protein-treated group compared with the control group, and inflammation levels were also reduced in the FGF21 mutant fusion protein-treated group (###: P<0.001 vs. MCS control, ***: P<0.001 vs. MCD control).

[0049] [Figure 13A] Figure 13A is a graph showing changes in alpha-smooth muscle actin (α-SMA), a fibrosis-related indicator, in the liver after repeated subcutaneous administration of DFD112 and DFD72 at 2-day intervals for 4 weeks in an MCD diet-induced nonalcoholic steatohepatitis mouse model. α-SMA expression increased in the MCD control group compared with the methionine choline standard (MCS) control group. On the other hand, α-SMA levels in the fusion protein-treated group and the FGF21 mutant fusion protein-treated group decreased compared with the control group. Data are shown as mean and standard error of the mean. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test (###: P<0.001 vs. MCS control, ***: P<0.001 vs. MCD control).

[0050] [Figure 13B]Figure 13B is a graph showing changes in transforming growth factor-beta (TGF-β), a fibrosis-related indicator, in the liver after repeated subcutaneous administration of DFD112 and DFD72 in an MCD diet-induced nonalcoholic steatohepatitis mouse model at 2-day intervals for 4 weeks. Expression of TGF-β increased in the MCD control group compared with the MCS control group. Meanwhile, TGF-β levels in the fusion protein-treated group and the FGF21 mutant fusion protein-treated group decreased compared with the control group (###: P<0.001 vs. MCS control, ***: P<0.001 vs. MCD control).

[0051] [Figure 13C] Figure 13C is a graph showing the results of Picrosirius Red staining, a fibrosis-related indicator, in the liver after repeated subcutaneous administration of DFD112 and DFD72 in an MCD diet-induced nonalcoholic steatohepatitis mouse model at 2-day intervals for 4 weeks. The amount of collagen increased in the MCD control group compared with the MCS control group. On the other hand, the Picrosirius Red levels in the fusion protein-treated group and the FGF21 mutant fusion protein-treated group decreased compared with the control group (###: P<0.001 vs. MCS control, **: P<0.01 vs. MCD control).

[0052] [Figure 14] Figure 14 shows histopathological photographs of the liver. Fat in the liver tissue was significantly reduced.

[0053] [Figure 15A]Figure 15A is a graph showing changes in ALT levels, a blood biochemical indicator, after repeated subcutaneous administration of DFD112 at 2-day intervals for 8 weeks in a mouse model of diet-induced obesity and nonalcoholic steatohepatitis. When the fusion protein was administered, a serum ALT-lowering effect was observed compared to the control group. Data are shown as the mean and standard error of the mean. Statistical analysis was performed by one-way analysis of variance followed by Dunnett's multiple comparison test (***: P<0.001).

[0054] [Figure 15B] Figure 15B is a graph showing the changes in AST levels after repeated subcutaneous administration of DFD112 in a mouse model of diet-induced obesity and non-alcoholic steatohepatitis at 2-day intervals for 8 weeks. When the fusion protein was administered, a serum AST lowering effect was observed compared to the control group (***: P<0.001).

[0055] [Figure 15C] Figure 15C is a graph showing the changes in TG after repeated subcutaneous administration of DFD112 in a mouse model of diet-induced obesity and non-alcoholic steatohepatitis at 2-day intervals for 8 weeks. When the fusion protein was administered, a serum TG lowering effect was observed compared to the control group (*: P<0.05, ***: P<0.001).

[0056] [Figure 15D] Figure 15D is a graph showing the changes in TC levels after repeated subcutaneous administration of DFD112 in a mouse model of diet-induced obesity and non-alcoholic steatohepatitis at 2-day intervals for 8 weeks. When the fusion protein was administered, a serum TC-lowering effect was observed compared with the control group (***: P<0.001).

[0057] [Figure 16A]Figure 16A provides a graph showing the change in NAFLD activity score (NAS) between before and after repeated subcutaneous administration of DFD112 in diet-induced obesity and non-alcoholic steatohepatitis mouse models for 8 weeks at 2-day intervals in the liver.When fusion protein is administered, the NAFLD activity score after administration is reduced compared with the score before administration.

[0058] [Figure 16B] Figure 16B provides a graph showing the change in liver fibrosis score between before and after repeated subcutaneous administration of DFD112 in diet-induced obesity and non-alcoholic fatty liver disease mouse model every 2 days for 8 weeks.When fusion protein is administered, the liver fibrosis score after administration is reduced compared with the score before administration.

[0059] [Figure 17A] Figure 17A is a graph showing changes in ALP levels, a blood biochemical indicator, after repeated subcutaneous administration of DFD112 at 2-day intervals for 8 weeks in a rat model of thioacetamide (TAA)-induced liver fibrosis. When the fusion protein was administered, a serum ALP-lowering effect was observed compared to the TAA control group. Data are shown as the mean and standard error of the mean. Statistical analysis was performed by one-way analysis of variance followed by Dunnett's multiple comparison test (###: P<0.001 vs. normal control, **: P<0.01 vs. TAA control).

[0060] [Figure 17B] Figure 17B is a graph showing changes in GGT levels, a blood biochemical indicator, after repeated subcutaneous administration of DFD112 in a rat model of thioacetamide (TAA)-induced liver fibrosis at 2-day intervals for 8 weeks. When the fusion protein was administered, a serum GGT lowering effect was observed compared to the TAA control group (###: P<0.001 vs. normal control, *: P<0.05 vs. TAA control).

[0061] [Figure 17C] Figure 17C is a graph showing changes in T-BIL levels, a blood biochemical indicator, after repeated subcutaneous administration of DFD112 in a rat model of thioacetamide (TAA)-induced liver fibrosis at 2-day intervals for 8 weeks. When the fusion protein was administered, a serum T-BIL reduction effect was observed compared to the control group (##: P<0.01 vs. normal control, *: P<0.05 vs. TAA control).

[0062] [Figure 17D] Figure 17D is a graph showing changes in fibrotic area in the liver after repeated subcutaneous administration of DFD112 in a rat model of thioacetamide (TAA)-induced liver fibrosis at 2-day intervals for 8 weeks. When the fusion protein was administered, a reduction in fibrotic area was observed compared to the control group (###: P<0.001 vs. normal control, ***: P<0.001 vs. TAA control). DETAILED DESCRIPTION OF THE INVENTION

[0063] In the following, the present invention will be described in detail.

[0064] The fusion protein contained as an active ingredient in the composition for preventing or treating hepatitis, liver fibrosis, and cirrhosis according to the present invention comprises a fibroblast growth factor 21 (FGF21) mutant protein; and an Fc region of an immunoglobulin, wherein the FGF21 mutant protein has the following mutations (1) to (7): (1) Substitution of amino acids at positions 98 to 101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 68); (2) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 69); (3) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 70); (4) substitution of the amino acid at position 170 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (5) substitution of the amino acid at position 174 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (6) a substitution of the amino acid at position 180 from the N-terminus of the wild-type FGF21 protein with the amino acid E, together with one or more of the mutations described in (1) to (5) above; and (7) Mutation of 1 to 10 amino acids to reduce the immunogenicity of wild-type FGF21 protein and at least one mutation selected from the group consisting of:

[0065] The fusion protein may further comprise a biologically active protein, or a variant or fragment thereof.

[0066] Specifically, the fusion protein contained as an active ingredient in the composition for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis according to the present invention comprises a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a mutant or fragment thereof; and an immunoglobulin Fc region, wherein the FGF21 mutant protein has the following mutations (1) to (7): (1) Substitution of amino acids at positions 98 to 101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 68); (2) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 69); (3) substitution of amino acids at positions 170 to 174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 70); (4) substitution of the amino acid at position 170 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (5) substitution of the amino acid at position 174 from the N-terminus of the wild-type FGF21 protein with the amino acid N; (6) a substitution of the amino acid at position 180 from the N-terminus of the wild-type FGF21 protein with the amino acid E, together with one or more of the mutations described in (1) to (5) above; and (7) Mutation of 1 to 10 amino acids to reduce the immunogenicity of wild-type FGF21 protein and at least one mutation selected from the group consisting of:

[0067] The wild-type FGF21 protein is a hormone known to play an important role in glucose and fat homeostasis, and may be derived from a mammal, such as a human, mouse, pig, monkey, etc., preferably a human. More preferably, the wild-type FGF21 protein may be a wild-type human FGF21 protein having the amino acid sequence shown in SEQ ID NO: 1.

[0068] The mutations contained in the FGF21 mutant protein may preferably be any one of the mutations EIRP, TGLEAV, TGLEAN, G170N, and G174N; a combination of any one of the mutations TGLEAV, TGLEAN, G170N, and G174N and the mutation EIRP; a combination of any one of the mutations EIRP, TGLEAV, TGLEAN, G170N, and G174N and the mutation A180E; or a combination of any one of the mutations TGLEAV, TGLEAN, G170N, and G174N, the mutation EIRP, and the mutation A180E. Furthermore, the FGF21 mutant protein may have a conformation in which 1 to 10 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type FGF21 protein. More preferably, the FGF21 mutant protein may comprise the amino acid sequence represented by any one of SEQ ID NOs: 6 to 23. Even more preferably, the FGF21 mutant protein may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 6 to 23, and may further have a conformation in which 1 to 10 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type FGF21 protein.

[0069] In the fusion protein, the asparagine (N) residue introduced by mutation of the FGF21 mutant protein may be glycosylated.

[0070] The biologically active protein may be one selected from the group consisting of insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor-related protein (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, and growth hormone. Preferably, the biologically active protein may be one selected from GLP-1, its variants, and exendin-4. Specifically, the fusion protein can simultaneously exhibit the effects of GLP-1 and FGF21 protein.

[0071] As used herein, the term "insulin" refers to a protein synthesized and secreted by beta cells in the pancreas and is a hormone that plays a role in maintaining a constant glucose level in the blood. Insulin is secreted when blood glucose levels are high, allowing blood glucose to enter cells where it is stored in the form of glycogen, and inhibiting glucose production in liver cells. It also aids in glucose oxidation and conversion to fatty acids in adipose tissue. In muscle, it promotes the absorption of amino acids and protein synthesis. Epinephrine and glucagon act as insulin antagonists by increasing blood glucose levels.

[0072] As used herein, the term "C-peptide" refers to the peptide that connects the A and B chains of proinsulin. C-peptide is secreted along with insulin by secretory granules of pancreatic cells but is not destroyed in the blood, and is therefore used as an indicator of the insulin secretory function of the pancreas.

[0073] As used herein, the term "leptin" refers to a hormone secreted by adipose tissue that maintains body fat at a constant level. Leptin secreted by adipose tissue acts on the brain to suppress appetite and activate the body's metabolism, thereby reducing body weight.

[0074] As used herein, the term "glucagon" refers to a protein synthesized and secreted by the pancreas, a hormone secreted in response to decreased blood glucose levels, and plays a role in increasing blood glucose levels. Glucagon consists of 29 amino acid residues and is secreted by the α cells of the islets of Langerhans.

[0075] As used herein, the term "gastrin" refers to a hormone secreted into the distal end of the stomach that induces gastric acid secretion and pancreatic juice production, and promotes motility of the stomach, small intestine, and large intestine.

[0076] As used herein, the term "gastrin-inhibitory polypeptide" refers to a linear polypeptide that inhibits all gastric secretions.

[0077] As used herein, the term "amylin" refers to a hormone synthesized and secreted by the beta cells of the pancreas, which, like insulin, regulates glucose metabolism.

[0078] As used herein, the term "calcitonin" refers to a thyroid hormone that regulates calcium levels in the blood. Calcitonin is a 32-amino acid polypeptide secreted by thyroid C cells.

[0079] As used herein, the term "cholecystokinin" refers to a 33-amino acid hormone produced by I cells in the duodenum and jejunum. Cholecystokinin accelerates splenic contraction and promotes pancreatic enzyme secretion, and inhibits gastric acid secretion.

[0080] As used herein, the term "peptide YY" is an abbreviation for peptide tyrosine tyrosine and refers to a 36 amino acid polypeptide secreted by cells of the large intestine and ileum in response to food.

[0081] As used herein, the term "neuropeptide Y" refers to a biologically active peptide consisting of 36 amino acids with an aminated carboxy terminus. Neuropeptide Y is widely distributed in the central and peripheral nervous systems of vertebrates, and is involved in blood pressure control in the sympathetic nervous system, endocrine or autonomic control in the central nervous system of vertebrates, feeding behavior, memory, and circadian rhythms.

[0082] As used herein, the term "bone morphogenetic protein 6," also known as BMP-6, refers to a protein that is directly involved in bone formation.

[0083] As used herein, the term "bone morphogenetic protein 9," also known as BMP-9, refers to a protein that is directly involved in bone formation.

[0084] As used herein, the term "oxytomodulin" refers to a 37-amino acid polypeptide secreted by mucosal parietal cells. Oxytomodulin has a potent appetite suppressant effect.

[0085] As used herein, the term "oxytocin" refers to a hormone consisting of nine amino acids that promotes uterine contractions during labor and aids in milk production during lactation.

[0086] As used herein, the term "GLP-1" refers to an incretin hormone consisting of 31 amino acids that is secreted in intestinal L cells in response to stimulation by food, etc. For example, the GLP-1 protein may be represented by the amino acid sequence of SEQ ID NO:42.

[0087] A variant of GLP-1 may be represented, for example, by the amino acid sequence of any one of SEQ ID NOs: 43 to 46.

[0088] As used herein, the term "irisin" refers to a hormone secreted by muscles during exercise and delivered to adipocytes via the bloodstream to break down fat, such as by converting white adipocytes to brown adipocytes. Irisin is a 112-amino acid cleaved fragment of a membrane protein called FNDC5.

[0089] As used herein, the term "FNDC5" is an abbreviation for fibronectin type III domain-containing protein 5, which refers to the precursor substance of irisin.

[0090] As used herein, the term "apelin" refers to a peptide encoded by the APLN gene. Apelin is synthesized and secreted by adipose tissue and has the same function as insulin.

[0091] As used herein, the term "adiponectin" refers to a protein secreted by adipocytes that improves insulin resistance.

[0092] As used herein, the term "CTRP family" refers to C1q and tumor necrosis factor-related proteins, which are members of the adipokine family and act primarily on the liver and muscle tissue to regulate glucose and lipid metabolism, etc.

[0093] As used herein, the term "resistin" refers to a recently discovered adipokine secreted by adipocytes, a protein consisting of 108 amino acids, known to be increased during adipocyte differentiation and capable of inhibiting adipocyte differentiation.

[0094] As used herein, the term "visfatin" refers to one of the adipokines produced and secreted by adipose tissue, and is a 52 kDa protein.

[0095] As used herein, the term "omentin" refers to one of the adipokines, a protein produced and secreted by adipose tissue, that has anti-inflammatory properties.

[0096] As used herein, the term "retinol binding protein-4" refers to a protein secreted by adipocytes and contains vitamin A that improves insulin resistance.

[0097] As used herein, the term "glycetin" refers to the major enteroglucagon in the gastrointestinal tract, which consists of 69 amino acids, including all 29 amino acids of glucagon between amino acids at positions 33-66.

[0098] As used herein, the term "angiopoietin," also referred to as ANG, refers to a protein that acts during the growth of blood vessels or endothelial cells or during wound healing in the body.

[0099] As used herein, the term "IL-22," also known as IL-TIF, refers to the protein encoded by the IL-22 gene. IL-22 is secreted by natural killer cells or T cells activated in response to bacterial antigens on epithelial cells.

[0100] As used herein, the terms "Fc region," "Fc fragment," or "Fc" refer to a protein comprising immunoglobulin heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3), but excluding the heavy and light chain variable regions and immunoglobulin light chain constant region 1 (CL1). Furthermore, as used herein, the term "Fc region variant" refers to an Fc region prepared by substituting a portion of the amino acids in the Fc region or by combining different types of Fc regions.

[0101] The Fc region of an immunoglobulin may be the complete Fc region constituting an antibody, a fragment thereof, or an Fc region variant. Furthermore, the Fc region may include a molecule in the form of a monomer or a multimer, and may further include the hinge region of the heavy chain constant region. The Fc region variant may be modified to prevent cleavage at the hinge region. Furthermore, the hinge sequence of the Fc may have substitutions in several amino acids to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In addition, a portion of the amino acid sequence of the Fc hinge sequence may be substituted to inhibit rearrangement of the Fab region. The lysine (K) at the C-terminus of the Fc may be removed.

[0102] Preferably, the Fc region of the immunoglobulin may be any one of IgG1, IgG2, IgG3, IgG4, and IgD Fc regions, or a hybrid Fc region that is a combination thereof. Furthermore, the hybrid Fc region may contain an IgG4 region and an IgD region. Furthermore, the hybrid Fc region may contain a portion of the hinge sequence and CH2 of an IgD Fc and the CH2 and CH3 sequences of an IgG4 Fc.

[0103] In addition, the Fc fragment of the present invention may be in the form of a wild-type glycosylated chain, a more glycosylated chain than the wild-type, a less glycosylated chain than the wild-type, or a deglycosylated chain. Increased, decreased, or eliminated glycosylated chains can be prepared by conventional methods known in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

[0104] Preferably, the immunoglobulin Fc region may be represented by an amino acid sequence selected from SEQ ID NOs: 24 to 26, 47 and 48.

[0105] The fusion protein may comprise a biologically active protein, an immunoglobulin Fc region, and an FGF21 mutant protein linked in the following order from N-terminus to C-terminus: Furthermore, the fusion protein may comprise an FGF21 mutant protein, an immunoglobulin Fc region, and a biologically active protein linked in the following order from N-terminus to C-terminus: Preferably, the fusion protein may comprise a biologically active protein, an immunoglobulin Fc region, and an FGF21 mutant protein linked in the following order from N-terminus to C-terminus:

[0106] Furthermore, the fusion protein may comprise a GLP-1 variant protein, an Fc region of an immunoglobulin, and an FGF21 variant protein linked from N-terminus to C-terminus in the following order: Furthermore, the fusion protein may comprise an FGF21 variant protein, an Fc region of an immunoglobulin, and a GLP-1 variant protein linked from N-terminus to C-terminus in the following order: Preferably, the fusion protein may comprise a GLP-1 variant protein, an Fc region of an immunoglobulin, and an FGF21 variant protein linked from N-terminus to C-terminus in the following order:

[0107] Additionally, the fusion protein may further comprise a linker.

[0108] The fusion protein may be in a form in which the FGF21 mutant protein is directly linked to the N-terminus or C-terminus of the immunoglobulin Fc region, or in which the FGF21 mutant protein is linked to the immunoglobulin Fc region via a linker.

[0109] In such cases, the linker may be linked to the N-terminus, C-terminus, or free base of the Fc fragment, and may also be linked to the N-terminus, C-terminus, or free base of the FGF21 mutant protein. When the linker is a peptide linker, binding may occur at any region. For example, the linker may be linked to the C-terminus of the immunoglobulin Fc region and the N-terminus of the FGF21 mutant protein to form a fusion protein of the immunoglobulin Fc region and the FGF21 mutant protein.

[0110] Furthermore, the fusion protein of the present invention may be in a form in which a biologically active protein is linked to the N-terminus of the Fc region of an immunoglobulin of the fusion protein.

[0111] When the linker and Fc are expressed separately and then linked, the linker may be a cross-linking agent known in the art. Examples of cross-linking agents include, but are not limited to, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, imidoesters including N-hydroxysuccinimide esters such as 4-azidosalicylic acid and disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.

[0112] Furthermore, the linker may be a peptide, preferably a peptide consisting of 10 to 30 amino acid residues.

[0113] Furthermore, an alanine may be further attached to the end of the linker. Preferably, the linker may be a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 2 to 5.

[0114] The fusion protein may be in the form of a dimer or multimer of FGF21 mutant proteins, in which one or more FGF21 mutant proteins are linked to each other, linked to an immunoglobulin Fc region.Furthermore, the fusion protein may be in the form of a dimer or multimer in which two or more immunoglobulin Fc regions are linked, each having an FGF21 mutant protein linked thereto.

[0115] More specifically, the fusion protein may be represented by the amino acid sequence of any one of SEQ ID NOs: 36 to 39. Even more specifically, it may be represented by the amino acid sequence of SEQ ID NOs: 36, 37 or 39.

[0116] Furthermore, the fusion protein may preferably be a peptide having the amino acid sequence shown in any one of SEQ ID NOs: 58 to 67. More preferably, the fusion protein may be a peptide having the amino acid sequence shown in SEQ ID NO: 65, 66 or 67.

[0117] The FGF21 mutant protein may further comprise 1 to 10 amino acid mutations to reduce the immunogenicity of the wild-type FGF21 protein. Immunogenicity can be predicted by conventional methods known in the art. For example, the potential immunogenicity of a protein can be predicted by, for example, iTope TM and TCED TM The method can be used to screen.

[0118] Furthermore, mutations to minimize immunogenicity can be designed by routine methods known in the art, for example, EpiScreen™ to assess potential immunogenicity. TM As observed by performing analyses, amino acid sequences that induce immunogenicity can be identified through T cell epitope mapping, and variants with minimized immunogenicity can be designed through in silico prediction.

[0119] The fusion protein can be used to prevent or treat hepatitis, liver fibrosis, and cirrhosis.

[0120] Specifically, hepatitis can be acute viral hepatitis, chronic hepatitis, alcoholic hepatitis, autoimmune hepatitis, fulminant hepatitis, or non-alcoholic steatohepatitis (NASH). Specifically, cirrhosis can be alcoholic cirrhosis or primary biliary cirrhosis.

[0121] In addition, the pharmaceutical composition may further comprise a pharmaceutical carrier. The pharmaceutical carrier may be any carrier as long as it is a non-toxic material suitable for delivering the antibody to the patient. For example, distilled water, alcohol, fat, wax, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition. In these preparations, the concentration of the fusion protein may vary widely.

[0122] Specifically, pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. Suitable formulations may include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfate, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins ( serum albumin, gelatin, or immunoglobulin), coloring agents, flavoring agents, diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants, or wetting agents (e.g., pluronics; PEG; sorbitan esters; polysorbates, e.g., polysorbates 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), growth enhancers (e.g., alkyl metal halides, preferably sodium chloride or potassium chloride; or mannitol, sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants.

[0123] In addition, the present invention also provides a method for preventing or treating hepatitis, liver fibrosis, and liver cirrhosis, comprising administering a pharmaceutical composition of the present invention to a subject in need of treatment. Such a method may comprise administering an effective amount of a fusion protein of the present invention to a mammal with a hepatitis condition, such as acute viral hepatitis, chronic hepatitis, alcoholic hepatitis, autoimmune hepatitis, fulminant hepatitis, or nonalcoholic steatohepatitis (NASH). The method may also comprise administering an effective amount of a fusion protein of the present invention to a mammal with a cirrhosis condition, such as alcoholic cirrhosis, primary biliary cirrhosis, or liver fibrosis.

[0124] The pharmaceutical compositions of the present invention may be administered via any route. They may be provided to animals via any suitable means, either directly (e.g., locally, by injection into a tissue region, by implantation, or topical administration) or systemically (e.g., orally or parenterally). When the compositions of the present invention are provided parenterally via intravenous, subcutaneous, ophthalmic, intraperitoneal, intramuscular, oral, rectal, intraorbital, intracranial, intrathecal, intraventricular, intrathecal, intracisternal, intravesicular, intranasal, or aerosol administration, the compositions are preferably aqueous or may comprise part of a biologically compatible body fluid suspension or solution. Therefore, a carrier or vehicle, as long as it is biologically compatible, may be added to the composition and delivered to the patient. Thus, biologically compatible saline may generally be included as a carrier, such as a body fluid, for the formulation.

[0125] Furthermore, the frequency of administration may vary depending on the pharmacokinetic parameters of the fusion protein in the formulation used. Generally, the physician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a unit dose, in at least two doses (which may or may not contain the same amount of the target fusion protein) at time intervals, or by continuous injection via an implanted device or catheter. The accuracy of administering the appropriate dosage may be routinely performed by those skilled in the art and corresponds to the scope of research routinely performed by them.

[0126] Furthermore, a preferred unit dose of the fusion protein in humans may be in the range of 0.01 μg / kg to 100 mg / kg body weight, and more preferably in the range of 1 μg / kg to 10 mg / kg body weight. While this is an optimal amount, the unit dose may vary depending on the disease being treated or the presence or absence of side effects. Nevertheless, the optimal administration dose may be determined by routine experimentation. Administration of the fusion protein may be by periodic bolus injections, an external reservoir (e.g., an intravenous bag), or continuous intravenous, subcutaneous, or intraperitoneal administration from an internal source (e.g., a bioerodable implant).

[0127] In addition, the fusion proteins of the present invention can be administered to a target recipient together with other biologically active molecules. The optimal combination of fusion protein and other molecule, mode of administration, and optimal dosage can be determined by routine experimentation, as is well known in the art.

[0128] The present invention provides the use of the pharmaceutical composition of the present invention, which comprises the fusion protein as an active ingredient, for preventing or treating hepatitis, liver fibrosis and liver cirrhosis.

[0129] The present invention provides use of the pharmaceutical composition of the present invention, which comprises the fusion protein as an active ingredient, for producing a composition for preventing or treating hepatitis, liver fibrosis and liver cirrhosis.

[0130] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding a fusion protein. The isolated nucleic acid molecule may be selected from the group consisting of DNA, RNA, and mRNA, and specifically, it may be DNA.

[0131] In such cases, isolated nucleic acid molecules encoding fusion proteins may have sequences that differ from each other due to codon redundancy. Furthermore, the isolated nucleic acid may be appropriately modified, or nucleotides may be added to the N- or C-terminus of the isolated nucleic acid, depending on the desired purpose, as long as the isolated nucleic acid is capable of producing the fusion protein.

[0132] The isolated nucleic acid molecule may, for example, comprise the nucleotide sequence set forth by any one of SEQ ID NOs:71-80.

[0133] In yet another aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule.

[0134] As used herein, the term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains a nucleic acid sequence that directs or controls the expression of an inserted heterologous nucleic acid sequence. Expression vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and their analogs. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0135] As used herein, the term "expression of a heterologous nucleic acid sequence" or "expression" of a target protein refers to the transcription of the inserted DNA sequence, translation of the mRNA transcript, and production of the Fc fusion protein product, antibody, or antibody fragment.

[0136] An effective expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. An effective expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence to enhance the level of post-transcriptional RNA stability. In an exemplary embodiment of the present invention, the expression vector is pAD15, a modified vector of RcCMV.

[0137] In yet another aspect, the present invention provides a host cell containing the expression vector.

[0138] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. As used herein, the terms "transformation" or "transfect" refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art.

[0139] Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and used for the expression and / or secretion of the target protein. Examples of suitable host cells that may be used in the present invention include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, CAP cells (human amniotic fluid-derived cells), and COS cells.

[0140] Form for invention Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to examples. However, these examples according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the examples described herein.

[0141] Preparation Example 1. Preparation and purification of fusion proteins containing FGF21 mutant proteins

[0142] Preparation Example 1-1. Preparation of expression vector for expression of FGF21 mutant protein Mutation studies of FGF21 were performed to improve the stability, activity, and pharmacokinetic profile of FGF21 in the Fc-FGF21 structure.

[0143] Specifically, protein variants were designed for the LLLE region (amino acids at positions 98 to 101 from the N-terminus of the FGF21 protein of SEQ ID NO: 1), the GPSQG region (amino acids at positions 170 to 174 from the N-terminus of the FGF21 protein of SEQ ID NO: 1), and the A180 region, which are expected to significantly affect protein activity based on three-dimensional structural analysis of the FGF21 protein.

[0144] The location, sequence information, target and expected effect of each mutation introduced into the FGF21 protein are listed in Table 1 below. N indicates glycosylated asparagine (N). [Table 1]

[0145] Additionally, FGF21 mutant proteins containing the mutations described in Table 1 are listed in Table 2 below. [Table 2]

[0146] The nucleotides encoding the amino acids were loaded into an expression vector in order from N-terminus to C-terminus so that the fused carrier, linker, and FGF21 mutant protein could be expressed. The material code of each FGF21 mutant fusion protein, the sequence of the mutation introduced into FGF21, the sequence of the fused carrier, and the linker sequence are listed in Table 3 below. In Table 3, N indicates glycosylated asparagine (N). [Table 3]

[0147] To produce FGF21 mutant fusion proteins, nucleotide sequences encoding each of the FGF21 mutant proteins were synthesized based on the amino acid sequence of each protein by consulting Bioneer Corporation (Korea). NheI and NotI restriction enzyme sequences were added to the 5' and 3' ends of the nucleotide sequences encoding each of the FGF21 mutant proteins, and an initiation codon for protein translation and a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) capable of secreting expressed proteins outside of cells were inserted at the 5' end following the restriction enzyme sequence. A stop codon was inserted next to the nucleotide sequence encoding each of the FGF21 mutant fusion proteins. The nucleotide sequences encoding each of the FGF21 mutant fusion proteins were cloned into the pTrans-empty expression vector using the two restriction enzymes NheI and NotI. The pTrans-empty expression vector, which has a simple structure containing a CMV promoter, a pUC-derived replication origin, an SV40-derived replication origin, and an ampicillin-resistance gene, was purchased from CEVEC Pharmaceuticals (Germany).

[0148] On the other hand, in the case of DFD6 (expressed in E. coli) and the Fc-FGF21 fusion protein RGE (Amgen), the nucleotide sequences encoding each fusion protein were inserted into a pET30a expression vector for expression in E. coli.

[0149] Preparative Example 1-2. Construction of plasmid DNA for expression of FGF21 mutant fusion protein To obtain large amounts of plasmid DNA for expression, E. coli was transformed with each of the expression vectors constructed in Preparation Example 1-1. Cell-wall-weakened E. coli cells were transformed with each expression vector via heat shock, and the transformants were plated on LB plates to obtain colonies. The resulting colonies were inoculated into LB medium and cultured at 37°C for 16 hours to obtain 100 mL of E. coli cultures containing each expression vector. The resulting E. coli was centrifuged to remove the culture medium, and then P1, P2, and P3 solutions (QIAGEN, Cat. No.: 12963) were added to disrupt the cell wall, thereby obtaining a DNA suspension from which proteins and DNA were separated. Plasmid DNA was purified from the resulting DNA suspension using a Qiagen DNA purification column. The eluted plasmid DNA was identified via agarose gel electrophoresis, and the concentration and purity were measured using a Nanodrop device (Thermo Scientific, Nanodrop Lite). The DNA obtained was used for expression.

[0150] Preparation Example 1-3. Expression of fusion protein in CAP-T cells Human cell lines were transfected with each of the plasmid DNA types obtained in Preparation Example 1-2. Each of the plasmid DNA types was transduced into CAP-T cells (CEVEC) cultured in PEM medium (Life Technologies) using a PEI solution (Polyplus, Cat. No.: 101-10N). The DNA and PEI solution mixture was mixed with the cell suspension using Freestyle293 expression medium (Invitrogen), and cultured at 37°C for 5 hours, after which PEM medium was added. After culturing at 37°C for 5-7 days, the culture was centrifuged to remove the cells, and the supernatant containing the FGF21 mutant fusion protein was obtained.

[0151] Preparation Example 1-4. Expression and purification of FGF21 mutant fusion proteins in E. coli E. coli strain BL21(DE3) was transformed with each plasmid DNA expressing the DFD6 (E. coli) and RGE (Amgen) fusion proteins. The transformed E. coli expressing each fusion protein was inoculated into 20 ml of LB medium and cultured at 37°C for 15 hours with shaking. A portion of the culture medium was then inoculated into 100 ml of LB medium and cultured at 37°C for 16 hours with shaking. After the culture was completed, the culture was centrifuged to obtain an E. coli pellet, and the cells were then disrupted using a high-pressure cell disrupter to obtain inclusion bodies.

[0152] The resulting inclusion bodies were purified by washing and elution, followed by a protein refolding process. Specifically, to remove bacterial proteins, the resulting inclusion bodies were washed 2-3 times with a buffer solution (pH 8.0) containing 0.5% Triton X-100, 50 mM Tris, 1 mM EDTA, and 0.1 M NaCl, and then resuspended in 8 M urea buffer containing 8 M urea, 50 mM Tris, and 1 mM DTT. To ensure complete denaturation of the protein in the 8 M urea buffer, the protein refolding process was carried out as follows.

[0153] First, urea was removed from the 8M urea buffer by stepwise dilution with 20mM glycine, pH 9.0 buffer. From 2M urea, copper sulfate (CuSO4) was added to a concentration of 80µM to induce stable structural folding of the protein. The refolded protein was suspended in PBS buffer solution (pH 7.4), filtered through a 0.22µm filter to remove impurities, and then loaded onto a Protein A affinity chromatography column. The column was washed with 1X PBS buffer solution (pH 7.4), and the protein was then eluted with 100mM glycine buffer solution (pH 3.0) to prepare the DFD6 (E. coli) fusion protein.

[0154] For the RGE (Amgen) fusion protein, the refolded protein was suspended in 50 mM Tris buffer solution (pH 8.0). The suspension was filtered through a 0.22 μm filter to remove impurities and then loaded onto an anion exchange resin column (POROS® HQ 50 μm, Thermo Fisher Scientific). The column was washed with 50 mM Tris buffer solution (pH 8.0), and then 50 mM Tris buffer solution (pH 8.0) was applied along a concentration gradient to elute the RGE (Amgen) fusion protein. The RGE (Amgen) fusion protein obtained by anion exchange resin was mixed with ammonium sulfate to a concentration of 1 M and then purified using a hydrophobic interaction chromatography column (Phenyl Sepharose FF, GE Healthcare). Specifically, the column was washed with 50 mM Tris buffer (pH 8.0) containing 1 M ammonium sulfate, and then a gradient of 50 mM Tris buffer (pH 8.0) was applied. The eluted fractions were analyzed by 10% Tris-glycine gel electrophoresis. The gel was stained with Coomassie Brilliant Blue R with gentle shaking. Fractions containing highly purified FGF21 mutant fusion proteins were collected and then dialyzed overnight at 4°C using a final buffer solution (1X PBS, 1 mM EDTA, pH 7.4). Upon completion of dialysis, the resulting protein stock solution was concentrated at 3,000 rpm at 4°C using a 30,000 MW cutoff centrifugal filter. The concentration of the FGF21 mutant fusion proteins was measured by BCA quantitative analysis.

[0155] Preparation Example 1-5. Purification of FGF21 mutant fusion protein A Protein A affinity chromatography column (GE Healthcare) was equilibrated with 1× PBS buffer solution (pH 7.4). The culture supernatant containing each FGF21 mutant fusion protein obtained in Preparative Examples 1-3 was filtered through a 0.2 μm filter and then loaded onto a Protein A affinity chromatography column. The column was washed with 1× PBS buffer solution (pH 7.4), and then the protein was eluted using 100 mM glycine buffer solution (pH 3.0). The fusion protein obtained by affinity chromatography was purified using an anion exchange resin column (POROS® HQ 50 μm, Thermo Fisher Scientific). Before eluting the FGF21 mutant fusion protein from the column, the anion exchange resin column was equilibrated with 50 mM Tris buffer solution (pH 8.0). Specifically, after washing the column with 50 mM Tris buffer solution (pH 8.0), 50 mM Tris buffer solution (pH 8.0) was distributed along a concentration gradient, and the eluted fractions were analyzed. Each eluted fraction was analyzed using size exclusion chromatography (SEC-HPLC), and fractions containing highly purified FGF21 mutant fusion proteins were collected. Concentration and quantitative analysis were performed according to the methods described in Preparation Examples 1-4.

[0156] Experimental Example 1. In vitro activity of fusion proteins

[0157] Experimental Example 1-1. Effect of FGF21 Mutations on Protein Activity The in vitro activities of the fusion proteins DFD4, DFD5, DFD6, DFD6 (E. coli), DFD7, DFD9, DFD13, DFD18, DFD72, DFD73 and DFD74 prepared in Preparation Example 1 were measured.

[0158] Specifically, the in vitro FGF21 activity of the fusion proteins was evaluated using a HEK293 cell line (Yuhan Corporation, Korea) modified to overexpress human β-Klotho, a coreceptor for FGF21. For activity evaluation, the concentrates containing the fusion proteins prepared in Preparation Examples 1-4 and 1-5 were subjected to three-fold serial dilutions at a concentration of 3 μM. After culturing under serum-starved conditions for 5 hours, the cell lines overexpressing human β-Klotho were treated with the diluted fusion proteins for 20 minutes and then lysed by adding cell lysis buffer (Cisbio / Cat# 64ERKPEG) while stirring at 60 rpm for 30 minutes at room temperature. The cell lysate solution was mixed with an antibody capable of detecting extracellular signal-regulated kinase (ERK) and phosphorylated ERK (Cisbio / Cat# 64ERKPEG), and the mixture was maintained at room temperature for 2 hours. Fluorescence was detected using a fluorescence detector (TECAN / GENiosPro). The activity of the fusion proteins was evaluated based on these EC 50 The results are shown in Figures 1A to 1C.

[0159] As shown in Figures 1A to 1C, the in vitro activity of fusion proteins prepared by introducing the mutant sequences into wild-type FGF21 protein was not inhibited, and the activities of each fusion protein were confirmed to be similar to each other. It was also confirmed that the in vitro activity of fusion proteins prepared by introducing N-glycosylation mutations into wild-type FGF21 protein was not inhibited through a DFD6 (E. coli) sample expressed in E. coli and a DFD6 sample expressed in animal cells.

[0160] Experimental Example 1-2. Effect of linker sequence on protein activity The in vitro activities of the fusion proteins DFD1, DFD3, DFD4 and DFD13 prepared in Preparation Example 1 were measured.

[0161] Specifically, the FGF21 activity of the fusion protein was measured using a concentrate containing the fusion protein prepared in Preparation Examples 1-5 according to the method described in Experimental Example 1-1. The results are shown in Figures 2A and 2B.

[0162] As shown in Figures 2A and 2B, slight differences in activity were observed depending on the linker sequence, but it was confirmed that the FGF21 mutant fusion proteins did not show a significant decrease in activity.

[0163] Experimental Examples 1-3. Experimental Results for DFD1, RGE (Amgen), and Fc-FGF21 (Lilly) The in vitro activities of the fusion protein DFD1 prepared in Preparation Example 1 and the control proteins RGE (Amgen) and Fc-FGF21 (Lilly) were measured.

[0164] Specifically, the FGF21 activity of the fusion proteins was measured using concentrates containing the fusion proteins prepared in Preparative Examples 1-5 and control proteins according to the method described in Experimental Example 1-1. The results are shown in Figure 3.

[0165] As shown in Figure 3, it was confirmed that DFD1 and RGE (Amgen) have similar in vitro activity, but Fc-FGF21 (Lilly) has two-fold higher in vitro activity than the other proteins.

[0166] Experimental Example 2. Evaluation of fusion protein stability Experimental Example 2-1. Experimental Method for Assessing Stability To measure the amount of protein aggregates during the initial sample preparation step, a size exclusion chromatography (SEC-HPLC) method was used to quantify high molecular weight aggregates (% HMW). The results are shown in Figures 4A to 4C.

[0167] Specifically, the TosoHaas model TSK-GEL G3000SWXL The column was used for the SEC-HPLC method. The column was equilibrated by flowing a buffer solution (1X PBS, 1 mM EDTA, pH 7.4) at a flow rate of 1 mL / min. The DFD4 and DFD13 protein stock solutions prepared in Preparative Examples 1-5 were concentrated to a target concentration of 20 mg / mL or higher using a 30,000 MW cutoff centrifugal filter at 3,000 rpm at 4°C. After measuring the concentration of each sample by BCA quantitative analysis, the sample was diluted with a buffer solution (1X PBS, 1 mM EDTA, pH 7.4) to a final concentration of 20 mg / mL. To measure the initial HMW% of DFD4 and DFD13, the 20 mg / mL sample was diluted to a concentration of 1 mg / mL with 1X PBS, 1 mM EDTA, pH 7.4. Next, 100 μL of each diluted sample was injected into the SEC-HPLC column and analyzed. To assess the stability of each sample, the %HMW of the samples was measured using the SEC-HPLC method on days 4, 8 and 14 while stored at 5°C, 25°C and 37°C for 2 weeks.

[0168] As shown in Figures 4A to 4C, DFD13 was confirmed to have a lower amount of high molecular weight aggregates (HMW%) compared to DFD4 at the initial stage and up to 2 weeks, indicating that the introduction of the EIRP mutation improved the stability of the FGF21 mutant fusion protein, thereby significantly reducing the HMW%.

[0169] Experimental Example 2-2. Stability Results To examine the effect of the EIRP mutation introduced into the original sequence LLLE(98-101) of FGF21 on stability, the stability of DFD4 (SEQ ID NO: 29) and DFD13 (SEQ ID NO: 35) was measured according to the method described in Experimental Example 2-1. The analytical results for the time 0 sample (initial stage; 0 days) and 4, 8, and 14-day storage samples of DFD4 and DFD13 are summarized in Table 4 below (in Table 4, ND means "not detected").

[0170] [Table 4]

[0171] As shown in Table 4, the %HMW content at the initial stage (day 0) was 0.91% for DFD4 and 0.56% for DFD13. After 2 weeks under storage conditions at 25°C, the %HMW content increased to 8.83% for DFD4, but was not observed in DFD13. DFD13 was shown to have a smaller %HMW ratio at the initial stage and 2 weeks compared to DFD4. It was found that when the EIRP mutation was introduced, the HMW% ratio of the FGF21 mutant fusion protein was greatly reduced.

[0172] Experimental Example 3: Pharmacokinetic measurements of fusion proteins Experimental Example 3-1. Experimental Method for Pharmacokinetic Measurements Six-week-old male ICR mice purchased from Orient BIO Co. (Korea) were divided into groups (n = 3 per blood collection time) so that their average body weights were similar one day before drug treatment. Each test substance was then administered subcutaneously at a single dose of 1 mg / kg (2 mg / kg in the case of RGE). Blood samples were then collected at 1, 4, 8, 12, 24, 48, 72, and 96 hours after injection. The concentration of intact, full-length FGF21 protein in the blood was measured using an Intact Human FGF21 ELISA kit (F1231-K01, Eagle Biosciences, USA), which immunoreacts with the N- and C-termini of FGF21 protein. The concentrations of blood samples collected up to 96 hours after subcutaneous injection of each fusion protein into mice were measured, and the pharmacokinetic parameters of each substance were calculated.

[0173] Experimental Example 3-2. Results of Pharmacokinetic Activity Measurements Pharmacokinetic parameters were calculated based on the graph showing the concentration of each protein in the blood versus time after subcutaneous administration of the fusion protein in mice (Figure 5), and the data are shown in Table 5 below. [Table 5]

[0174] The pharmacokinetic profiles of the fusion proteins were compared and evaluated based on the area under the curve (AUC), which indicates the drug exposure level.

[0175] As shown in Table 5, when comparing DFD4 with DFD13 and DFD6 with DFD73, it was determined that the introduction of the EIRP sequence resulted in an approximately 10 to 20% increase in the AUC value. When comparing DFD9 with DFD4, the introduction of TGLEAV resulted in an approximately 6-fold increase in the AUC value.

[0176] Additionally, we designed the TGLEAN, G170N, and G174N mutants to improve the durability of FGF21 via N-glycosylation at the C-terminus, which is known to be proteolytically degraded in vivo. We demonstrated an increase in AUC due to N-glycosylation over each control substance. Indeed, to demonstrate the AUC-improving effect of N-glycosylation, we compared the results with DFD6 (E. coli), a substance produced in E. coli without glycosylation. Here, we found that DFD6 produced in a human cell line exhibited an AUC level three times or more higher than that of DFD6 produced in E. coli, indicating that glycosylation improved the pharmacokinetic profile.

[0177] The A180E mutation, described in Amgen Co.'s patent (WO 2009 / 149171), was introduced into TGLEAV or G170N-introduced mutants such as DFD13 and DFD73 to obtain DFD18 and DFD74, where a further AUC increase of approximately 2- to 3-fold was observed.

[0178] To summarize the above results, the introduction of various mutations and their combinations resulted in improvements in pharmacokinetic parameters compared to the wild-type FGF21 fusion protein DFD9. The fusion protein with the highest AUC value was DFD74, which contained EIRP, G170N, and A180E, and showed an AUC approximately 45-fold higher than that of DFD9. Furthermore, considering the administration dose of RGE, i.e., 2 mg / kg, DFD74 was found to exhibit better drug exposure than Amgen's RGE. The overall pharmacokinetic improvement effects of the mutant sequences are summarized in Table 6. [Table 6]

[0179] Experimental Example 4. Activity of the fusion protein in diet-induced obese mice Experimental Example 4-1. Experimental Method for Evaluating Activity in Diet-Induced Obese Mice The weight-reducing effect of the FGF21 mutant fusion protein, DFD18, was evaluated in diet-induced obese mice. For the diet-induced obesity model, C57BL / 6J mice were purchased from Central Lab. Animal Inc. and fed a high-fat diet containing 60 kcal% fat (Research diet) for 8 to 12 weeks. Mice were divided into groups (n = 8 / group) so that they had similar mean body weights one day before drug treatment (day 0). Then, a single subcutaneous administration of 30 nmol / kg of the sample was performed. Changes in body weight were then observed compared to those observed with phosphate-buffered saline (PBS) as the vehicle.

[0180] Experimental Example 4-2. Protein activity in diet-induced obese mice The changes in body weight over time in a diet-induced obese mouse model after a single administration of 30 nmol / kg of DFD18 confirmed that the weight-reducing effect continued until day 10 after administration, with the maximum weight reduction (approximately 18%) occurring at day 11 after administration and maintained until day 14 (Figures 6A and 6B).

[0181] Preparation Example 2. Preparation and purification of fusion proteins Preparation Example 2-1. Preparation of expression vector for expression of fusion protein To identify the effects of the sequence of the GLP-1 variant protein and the sequence of the Fc hinge fused thereto on in vitro activity, pharmacokinetic profile, and pharmacological efficacy, various sequences for the Fc-fused GLP-1 variant protein were designed. The sequences of the GLP-1 variant proteins are listed in Table 7 below. [Table 7]

[0182] Additionally, the sequences of the Fc-fused GLP-1 variants are listed in Table 8. [Table 8]

[0183] In Table 8, HyFc5 refers to SEQ ID NO: 47, and HyFc40 refers to SEQ ID NO: 48. Various sequences for fusion proteins were designed to investigate the effects of the sequences of the GLP-1 mutant protein and the FGF21 mutant protein, the sequence of the Fc hinge fused to the GLP-1 mutant, and the sequence of the linker connecting the FGF21 mutant protein and the Fc on in vitro activity, pharmacokinetic profile, and pharmacological efficacy. The sequences of fusion proteins containing the GLP-1 mutant protein and the FGF21 mutant protein are listed in Table 9 below. Each fusion protein contains a GLP-1 mutant protein, an immunoglobulin Fc region, a linker, and an FGF21 mutant protein linked in the following order from N-terminus to C-terminus: [Table 9]

[0184] Specifically, the nucleotide sequences encoding each of the fusion proteins were synthesized based on the amino acid sequences of each protein after consulting with Bioneer Corporation (Korea). NheI and NotI restriction enzyme sequences were added to the 5' and 3' ends of the nucleotide sequences encoding each of the fusion proteins, and an initiation codon for protein translation and a leader sequence (MDAMLRGLCCVLLLCGAVFVSPSHA) capable of secreting the expressed protein outside the cell were inserted at the 5' end following the restriction enzyme sequence. A stop codon was inserted following the nucleotide sequence encoding each of the fusion proteins. The nucleotide sequences encoding each of the fusion proteins were cloned into the pTrans-empty expression vector using the two restriction enzymes NheI and NotI. The pTrans-empty expression vector, which has a simple structure containing a CMV promoter, a pUC-derived replication origin, an SV40-derived replication origin, and an ampicillin-resistance gene, was purchased from CEVEC Pharmaceuticals (Germany).

[0185] Preparative Example 2-2. Construction of Plasmid DNA for Expression of Fc-Fused GLP-1 Variants and Fusion Proteins To obtain large amounts of plasmid DNA for expression, E. coli was transformed with each of the expression vectors constructed in Preparation Example 2-1. E. coli cells with cell walls weakened by heat shock were transformed with each expression vector, and the transformants were plated on LB plates to obtain colonies. The resulting colonies were inoculated into LB medium and cultured at 37°C for 16 hours to obtain 100 mL of E. coli cultures containing each expression vector. The resulting E. coli was then centrifuged to remove the culture medium, and P1, P2, and P3 solutions (QIAGEN, Cat. No.: 12963) were then added to disrupt the cell walls, thereby obtaining DNA suspensions from which proteins and DNA were separated. Plasmid DNA was purified from the resulting DNA suspension using a Qiagen DNA purification column. The eluted plasmid DNA was identified by agarose gel electrophoresis, and the concentration and purity were measured using a Nanodrop device (Thermo Scientific, Nanodrop Lite). The DNA obtained in this manner was used for expression.

[0186] Preparative Example 2-3. Expression of Fc-fused GLP-1 variants and fusion proteins in CAP-T cells Human cell lines were transformed with each of the plasmid DNAs obtained in Preparation Example 2-2. Each type of plasmid DNA was transduced into CAP-T cells (CEVEC) cultured in PEM medium (Life Technologies) using a PEI solution (Polyplus, Cat. No.: 101-10N). The DNA and PEI solution mixture was mixed with the cell suspension using Freestyle293 expression medium (Invitrogen), and the mixture was cultured at 37°C for 5 hours, after which PEM medium was added. After culturing at 37°C for 5-7 days, the culture was centrifuged to remove the cells, and the supernatant containing each protein was obtained.

[0187] Preparative Example 2-4. Purification of Fc-fused GLP-1 variants and fusion proteins A Protein A affinity chromatography column (GE Healthcare) was equilibrated with 1× PBS buffer solution (pH 7.4). The culture supernatant containing each Fc-fused GLP-1 variant and fusion protein obtained in Preparation Example 2-3 was filtered through a 0.2 μm filter and then loaded onto the Protein A affinity chromatography column. The column was washed with 1× PBS buffer solution (pH 7.4), and then the protein was eluted using 100 mM glycine buffer solution (pH 3.0). The protein obtained by affinity chromatography was purified using an anion exchange resin column (POROS® HQ 50 μm, Thermo Fisher Scientific). Before loading the protein eluted from affinity chromatography, the anion exchange resin column was equilibrated with 50 mM Tris buffer solution (pH 8.0).

[0188] After washing the column with 50 mM Tris buffer solution (pH 8.0), 50 mM Tris buffer solution (pH 8.0) was distributed along a concentration gradient, and the eluted fractions were analyzed. Each eluted fraction was analyzed by size exclusion chromatography (SEC-HPLC). Fractions containing Fc-fused GLP-1 variants and fusion proteins with high purity were collected and dialyzed overnight at 4°C using a final buffer solution (1X PBS, 1 mM EDTA, pH 7.4). Upon completion of dialysis, the resulting protein stock solution was concentrated at 3,000 rpm at 4°C using a 30,000 MW cutoff centrifugal filter. The concentration of each protein was measured via quantitative BCA analysis.

[0189] Experimental Example 5. In vitro activity of fusion proteins Experimental Example 5-1. Activities of DFD23, DFD24, DFD25, DFD26, DFD27, DFD28 and DFD29 The in vitro GLP-1 activity of the fusion proteins DFD23, DFD24, DFD25, DFD26, DFD27, DFD28, and DFD29 was measured. Specifically, a CHO cell line (Eurofins, HTS163C2) overexpressing the human GLP-1 receptor was purchased and used to evaluate the GLP-1 activity of the fusion proteins. To evaluate the activity, a sample containing the fusion protein (protein stock solution prepared in Preparation Example 2-4; hereinafter referred to as "sample") was subjected to 4-fold serial dilutions at a concentration of 25 nM. After treating the CHO cell line overexpressing the human GLP-1 receptor for 30 minutes, the intracellular cAMP produced was measured (Cisbio, 62AM4PEB). The activity of each protein was calculated using EC 50 The results were evaluated by comparing the values.

[0190] As shown in Figure 7, the fusion protein containing the GLP-1(A2G) sequence exhibited approximately 2- to 3-fold lower activity than the fusion proteins containing other GLP-1 mutant sequences. No significant difference in GLP-1 activity was observed among the fusion proteins containing mutant sequences other than the GLP-1(A2G) sequence.

[0191] Experimental Example 5-2. Activities of DFD59, DFD69, DFD112 and DFD114 The in vitro GLP-1 activity of the fusion proteins DFD69, DFD112, and DFD114 and DFD59 (Fc-fused GLP-1 variants) prepared in Preparation Example 2 was measured. Specifically, a CHO cell line (Eurofins, HTS163C2) overexpressing the human GLP-1 receptor was purchased and used to evaluate the GLP-1 activity of the fusion proteins. For activity evaluation, samples containing each of the fusion proteins were subjected to 4-fold serial dilutions at a concentration of 25 nM. After treating the CHO cell line overexpressing the human GLP-1 receptor for 30 minutes, the intracellular cAMP produced was measured (Cisbio, 62AM4PEB).

[0192] As shown in Figures 8A and 8B, the activity of each protein was measured using EC 50The three fusion proteins had similar EC 50 The values shown are those of the FGF21 mutant-free DFD59, which showed approximately two-fold higher activity than the fusion protein.

[0193] Next, the in vitro activity of the FGF21 moiety in DFD69, DFD112, and DFD114 was measured. Specifically, the in vitro activity of the FGF21 moiety in the fusion proteins was evaluated using a HEK293 cell line overexpressing human β-Klotho (a co-receptor for FGF21). For activity evaluation, samples containing each fusion protein were subjected to three-fold serial dilutions at a concentration of 3 μM. After culturing in a serum-starved state for 5 hours, the HEK293 cell line overexpressing human β-Klotho was treated for 20 minutes, and the cells were lysed by adding cell lysis buffer (Cisbio / Cat# 64ERKPEG) while stirring at 60 rpm for 30 minutes at room temperature. The cell lysate solution was mixed with antibodies capable of detecting ERK and phosphorylated ERK, and the mixture was maintained at room temperature for 2 hours. Fluorescence was detected using a fluorescence detector (TECAN / GENiosPro). Activity was assessed using these EC 50 The values were measured by comparing them.

[0194] As shown in Figures 8A and 8B, the in vitro activities of the FGF21 portions of the fusion proteins DFD69, DFD112 and DFD114 were confirmed to be similar.

[0195] Experimental Example 6. Pharmacokinetic evaluation of fusion proteins Experimental Example 6-1. Experimental Methods for Pharmacokinetic Evaluation Six-week-old male ICR mice purchased from Orient BIO (Korea) were divided into groups (n = 3 per blood collection time) so that their average body weights were similar one day before drug treatment. Each sample was subcutaneously administered once at a dose of 1 mg / kg. Blood samples were collected at 1, 4, 8, 12, 24, 48, 72, 96, 144, 192, and 240 hours after injection. The concentrations of each fusion protein in the blood were measured separately based on the FGF21 portion and the GLP-1-Fc portion. The concentration of the intact full-length FGF21 portion of the fusion protein in the blood was measured using an Intact Human FGF21 ELISA Kit (F1231-K01, Eagle Biosciences, USA), which immunoreacts with the N- and C-termini of the FGF21 protein. Additionally, the concentration of the active GLP-1-Fc portion of the fusion protein in blood was measured using antibodies immunoreactive with the N-terminus of GLP-1 and Fc, as determined via ELISA analysis. The concentrations of FGF21 and GLP-1-Fc portions of each protein were measured in blood samples collected up to 240 hours after a single subcutaneous injection of each protein into mice, and the pharmacokinetic parameters of each protein were calculated.

[0196] Experimental Example 6-2. Pharmacokinetic Activity Results Based on the concentration of each active substance in the blood over time after a single subcutaneous administration of each protein in mice (Figures 9A and 9B), pharmacokinetic parameters for the FGF21 and GLP-1-Fc portions of the fusion proteins were calculated, and the data are shown in Table 10 below. [Table 10]

[0197] The pharmacokinetic profiles of each fusion protein were compared and evaluated based on the area under the curve (AUC), which indicates the drug exposure level.

[0198] As shown in Table 10, for the pharmacokinetic parameters of the FGF21 moiety, DFD114 showed the highest degree of drug exposure (AUC) and half-life, followed by DFD112 and DFD69. DFD114 showed an AUC value that was approximately two-fold higher than that of DFD69. For the pharmacokinetics of the GLP-1-Fc moiety, four proteins (DFD59, DFD69, DFD112, and DFD114) containing the same GLP-1 variant sequence showed similar AUC values.

[0199] Experimental Example 7. Evaluation of fusion protein activity against non-alcoholic steatohepatitis in diet-induced obese mice Experimental Example 7-1. Method for evaluating activity against non-alcoholic steatohepatitis in diet-induced obese mice The steatohepatitis and lipid-ameliorating effects of the fusion proteins DFD114 and DFD112, and the FGF21 mutant fusion proteins DFD74 and DFD72, were evaluated in a diet-induced obese mouse model.

[0200] To induce diet-induced obesity, C57BL / 6J mice were fed a high-fat diet containing 60 kcal% fat (Research diet) for approximately 37 weeks to create an obese mouse model with steatohepatitis. Mice were divided into groups (n = 6 / group) so that their average body weights were similar to those before drug treatment. DFD114, DFD112, DFD74, and DFD72 were then administered three times at doses of 3 or 10 nmol / kg, 4 days apart, for 2 weeks.

[0201] The control group received subcutaneous administration of the same solvent (Dulbecco's Phosphate-Buffered Saline, DPBS, Gibco, USA) used for the preparation of the test drugs. Four days after the final administration, the animals were fasted overnight, blood was collected from the inferior vena cava, and liver tissue was excised after inhalation anesthesia and laparotomy. Blood biochemistry tests were performed on serum samples isolated from the collected blood samples, and fixed liver tissue was prepared as specimens through trimming, dehydration, paraffin embedding, and slicing. The prepared specimens were then stained with hematoxylin and eosin (H&E), and histopathological changes were observed using an optical microscope (Olympus, ECLIPSE E600).

[0202] Experimental Example 7-2. Evaluation of activity against non-alcoholic steatohepatitis in diet-induced obese mice To evaluate the steatohepatitis and lipid-improving effects of the fusion proteins and FGF21 mutant fusion proteins, DFD114, DFD112, DFD74, and DFD72 were administered repeatedly at doses of 3 or 10 nmol / kg every 4 days for 2 weeks in a diet-induced obese mouse model. Then, lipid changes in serum and liver tissue were analyzed, and histopathological changes were observed.

[0203] As shown in Figures 10A to 10C, serum triglyceride (TG) and total cholesterol (TC) were measured after repeated subcutaneous administration of the fusion proteins DFD114 and DFD112, and the FGF21 mutant fusion proteins DFD74 and DFD72. As a result, serum triglyceride and total cholesterol levels were reduced, and triglyceride levels in liver tissue were also reduced compared to the vehicle control group.

[0204] As shown in Figure 11, histopathological experiments performed after repeated subcutaneous administration of the fusion proteins DFD114 and DFD112 showed that lipids in liver tissue were significantly reduced compared with the vehicle control group.

[0205] Experimental Example 8. Evaluation of the activity of the fusion protein in MCD-induced non-alcoholic steatohepatitis mice Experimental Example 8-1. Method for evaluating activity in mice with MCD-induced non-alcoholic steatohepatitis To evaluate the inflammation- and fibrosis-reducing effects of the fusion protein and FGF21 mutant fusion proteins in a nonalcoholic steatohepatitis model, we evaluated the effects of DFD112 and DFD72 in an MCD model.

[0206] The methionine-choline-deficient (MCD) diet-induced nonalcoholic liver disease animal model is one of the widely used models for evaluating nonalcoholic steatohepatitis. Steatohepatitis with liver fibrosis was induced by feeding a diet lacking methionine and choline, which play important roles in beta-oxidation and very low-density lipoprotein (VLDL) synthesis. This is known to be similar to the pathological model of human steatohepatitis. To induce the steatohepatitis model, C57BL / 6 mice were alternately fed the MCD diet and the methionine-choline standard (MCS) diet ad libitum for 17 weeks, with 10 days of feeding the MCD diet followed by 4 days of feeding the MCS diet.

[0207] Animals were weighed before administration of the test substances and randomly assigned to groups so that the average body weight of each group was as evenly distributed as possible. After assigning 10 mice to each group, 3, 10, and 30 nmol / kg of DFD112 and 10 nmol / kg of DFD72 were administered subcutaneously every 2 days for 4 weeks. In the MSC control group and MCD diet control group, mice were administered subcutaneously with the solvent used to prepare the test substances (Dulbecco's Phosphate-Buffered Saline, DPBS, Gibco, USA) every 2 days for 4 weeks in the same manner.

[0208] After repeated administration of the test substance for 4 weeks, mice were fasted overnight, blood was collected from the inferior vena cava, and liver tissue was excised after inhalation anesthesia and laparotomy. Blood biochemistry tests were performed on serum samples isolated from the collected blood samples, and fixed liver tissue was prepared as specimens using a standard tissue processing process. Hematoxylin and eosin (H&E) and immunohistochemical staining were then performed, and histopathological changes were observed using a light microscope (Olympus, BX53).

[0209] Experimental Example 8-2. Activity evaluation results in mice with MCD-induced non-alcoholic steatohepatitis Mice with MCD-induced NASH were repeatedly administered 3, 10, or 30 nmol / kg of the fusion protein DFD112 or 10 nmol / kg of the FGF21 mutant fusion protein DFD72 every 2 days for 4 weeks. Blood biochemistry and histopathological tests were then performed to evaluate the effects on NASH.

[0210] As shown in Figures 12A to 12C, the MCD control group, in which nonalcoholic steatohepatitis was induced, showed significantly higher alanine aminotransferase (ALT) and aspartate transaminase (AST), indicators of liver damage, compared with the normal-diet MCS control group (p<0.001). Serum AST and ALT levels were dose-dependently reduced in the DFD112-treated group compared with the MCD control group. In addition, the MCD control group showed significantly higher inflammation levels in histopathological examination compared with the MCS control group (p<0.01). When DFD112 was administered, inflammation levels were dose-dependently reduced compared with the MCD control group.

[0211] As shown in Figures 13A to 13C and 14, to evaluate the effect of the fusion protein on liver fibrosis, alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β) in liver tissue were stained using immunohistochemical staining and quantified by image analysis as indicators of liver fibrosis. The expression of α-SMA and TGF-β was increased in the MCD control group compared to the MCS control group (p<0.001). When DFD112 and DFD72 were administered, the expression of α-SMA and TGF-β was decreased compared to the MCD control group. In addition, collagen in liver tissue was stained using Picrosirius red staining and quantified by image analysis. As a result, the amount of collagen was higher in the MCD control group than in the MCS control group, and a tendency for decreased collagen in liver tissue was observed when DFD112 and DFD72 were administered compared to the MCD control group (p<0.001).

[0212] Experimental Example 9. Evaluation of the activity of the fusion protein in diet-induced obesity and non-alcoholic steatohepatitis mice Experimental Example 9-1. Method for assessing the activity of fusion proteins in diet-induced obese and non-alcoholic steatohepatitis mice To evaluate the inflammation- and fibrosis-reducing effects of the fusion protein in a nonalcoholic steatohepatitis model, the effects of DFD112 were evaluated in a diet-induced obesity and nonalcoholic steatohepatitis mouse model.

[0213] A mouse model with obesity and nonalcoholic steatohepatitis was prepared by feeding C57BL / 6 mice a high-fat diet (Research diet) containing 40% fat, 40% carbohydrate, and 2% cholesterol for approximately 30 weeks. Histopathological examination of liver tissue was performed approximately 3 weeks before drug treatment, and ALT and AST levels and body weight were measured before administration. Experimental animals were selected and divided into groups to ensure equal distribution of NASH induction levels and body weights. Twelve mice were assigned to each group and repeatedly administered DFD112 subcutaneously at doses of 3 or 10 nmol / kg every 2 days for 8 weeks. A control group was injected with the vehicle used to prepare the test substances (Dulbecco's Phosphate-Buffered Saline, DPBS, Gibco, USA) in the same manner.

[0214] After the final administration, mice were fasted overnight, blood was collected from the inferior vena cava, and liver tissue was excised after inhalation anesthesia and laparotomy. Blood biochemistry tests were performed on serum samples isolated from the collected blood samples. Fixed liver tissue samples were prepared and stained with hematoxylin and eosin (H&E) or Picrosirius red to observe histopathological changes.

[0215] Experimental Example 9-2. Evaluation of fusion protein activity in diet-induced obese and non-alcoholic steatohepatitis mice DFD112 was administered at doses of 3 or 10 nmol / kg every 2 days for 8 weeks to mice with diet-induced obesity and NASH. Blood biochemistry and histopathological tests were then performed to evaluate the effects on NASH.

[0216] As shown in Figures 15A to 15D, serum ALT and AST levels, as indicators of liver damage, were reduced to normal levels in the DFD112-treated group (p<0.001). Serum triglyceride (TG) and total cholesterol (TC) levels were also significantly reduced compared to the vehicle control group (p<0.05 or p<0.001).

[0217] As shown in Figures 16A and 16B, histopathological examination results from before and after administration showed that the NAFLD activity score (NAS), an index for classifying nonalcoholic steatohepatitis grade, increased or maintained in the vehicle control group after 8 weeks of repeated administration compared with the pre-administration score. On the other hand, the NAFLD activity score significantly decreased in all subjects in the DFD112-administered group compared with the pre-administration score. In addition, when fibrosis severity was assessed, the severity of fibrosis was maintained or worsened in the control group compared with the severity before administration, while a significant improvement was observed in the DFD112-administered group (p<0.05).

[0218] Experimental Example 10. Evaluation of fusion protein activity in rats with TAA-induced liver fibrosis Experimental Example 10-1. Method for evaluating fusion protein activity in rats with TAA-induced liver fibrosis To assess the liver fibrosis-reducing effect of the fusion protein, the effect of DFD112 was evaluated in a rat model in which liver fibrosis was induced by drinking water.

[0219] A liver fibrosis model was prepared in Wistar rats by providing drinking water containing thioacetamide (TAA) for approximately 14 weeks. Animals were weighed before administration of the test substance and randomly assigned to groups so that the average body weight of each group was as evenly distributed as possible. After assigning eight rats to each group, 30 nmol / kg of DFD112 was administered subcutaneously every two days for 8 weeks. TAA drinking water was continuously provided until the end of administration. The normal control group received standard drinking water from the start of the study until the end of administration, while the TAA control group received TAA drinking water from the start of the study until the end of administration.

[0220] After the end of the treatment, the mice were fasted overnight, blood was collected from the inferior vena cava, and liver tissue was excised after inhalation anesthesia and laparotomy. Blood biochemistry tests were performed on serum samples isolated from the collected blood samples, and fixed liver tissue was prepared as specimens and stained with Picrosirius red to examine histopathological changes to compare the degree of liver fibrosis.

[0221] Experimental Example 10-2. Method for evaluation of fusion protein activity in TAA-induced liver fibrosis rats The fusion protein DFD112 was administered at 30 nmol / kg repeatedly to rats with TAA-induced liver fibrosis every 2 days for 8 weeks, and the effect on liver fibrosis was evaluated by blood biochemistry and histopathological examination.

[0222] As shown in Figures 17a to 17d, the TAA control group in which liver fibrosis was induced showed significantly higher levels of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), and total bilirubin (T-bil), as indicators of liver damage, compared with the control group provided with standard drinking water (p<0.01 or p<0.001).

[0223] ALT, GGT, and T-Bil levels were significantly decreased in the DFD112-treated group compared with the TAA control group. Histopathological examination showed that the percentage of liver fibrosis areas stained positive with Picrosirius red staining was significantly increased in the TAA control group compared with the normal control group. In addition, the percentage of liver fibrosis areas was significantly decreased in the DFD112-treated group compared with the TAA control group (p<0.001).

Claims

1. A pharmaceutical composition for preventing or treating hepatitis, liver fibrosis, and / or liver cirrhosis, comprising as an active ingredient a fusion protein comprising a fibroblast growth factor 21 (FGF21) mutant protein, an Fc region of an immunoglobulin, a biologically active protein, and a linker, The FGF21 mutant protein contains the mutation (1): (1) Substitution of amino acids at positions 98 to 101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 68). Including, Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the linker connects the FGF21 mutant protein to the Fc region of an immunoglobulin.

3. The pharmaceutical composition of claim 2, wherein the linker is linked to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the FGF21 mutant protein.

4. The pharmaceutical composition according to claim 2 , wherein the linker is a peptide consisting of 10 to 30 amino acid residues.

5. The pharmaceutical composition of claim 4, wherein the linker has an amino acid sequence represented by any one of SEQ ID NOs: 2 to 5.

6. 2. The pharmaceutical composition of claim 1, wherein the Fc region of the immunoglobulin is a hybrid Fc comprising any one of the Fc regions of IgG1, IgG2, IgG3, IgG4, and IgD, or a combination thereof.

7. The pharmaceutical composition of claim 6 , wherein the hybrid Fc comprises an IgG4 region and an IgD region.

8. 2. The pharmaceutical composition of claim 1, wherein the fusion protein comprises a biologically active protein, an Fc region of an immunoglobulin, a linker, and an FGF21 mutant protein linked in the following order from N-terminus to C-terminus.

9. The pharmaceutical composition of claim 8, wherein the linker is linked to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the FGF21 mutant protein.

10. 10. Use of the pharmaceutical composition of claim 1 for the manufacture of a composition for preventing or treating hepatitis, liver fibrosis, and / or liver cirrhosis, wherein the hepatitis is selected from the group consisting of acute viral hepatitis, chronic hepatitis, alcoholic hepatitis, autoimmune hepatitis, and fulminant hepatitis.

11. The pharmaceutical composition of claim 1 for preventing or treating liver fibrosis or cirrhosis.

12. The pharmaceutical composition of claim 1 for preventing or treating liver fibrosis.

13. 11. The use according to claim 10 for the manufacture of a composition for preventing or treating liver fibrosis or cirrhosis.

14. 11. The use according to claim 10 for the manufacture of a composition for preventing or treating liver fibrosis.

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