Dual-function proteins and uses thereof

Abstract

One embodiment of the present invention relates to a fusion protein or a dimer thereof comprising a polypeptide represented by formula (I) and a polypeptide represented by formula (II). The fusion protein or a dimer thereof according to the present invention is a material with improved in vivo durability, protein productivity, and stability, and has excellent effects on weight loss and blood glucose regulation.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2022-0155543, filed on November 18, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a fusion protein or a dimer thereof comprising a biologically active protein; a method for treating or preventing a metabolic-related disease using the fusion protein or a dimer thereof, and medical uses thereof; an isolated cell line producing the fusion protein or a dimer thereof; an isolated nucleic acid encoding the fusion protein or a dimer thereof; a recombinant expression vector comprising the nucleic acid; a host cell comprising the recombinant expression vector; and a method for producing the fusion protein or a dimer thereof. [Background technology]

[0003] Various methods for treating and / or preventing diseases using bioactive proteins with in vivo physiological activity have been attempted in the pharmaceutical field. However, there are many cases where wild-type bioactive proteins have a short half-life. Therefore, to achieve the desired pharmacological effect by administering the bioactive protein, the frequency of administration must be increased. In this regard, various techniques (e.g., PEGylation, glycosylation, immunoglobulin Fc fusion, fusion with human serum albumin, fusion with human transferrin, etc.) are being actively researched to develop long-acting drugs that can increase the half-life of bioactive proteins.

[0004] Furthermore, when two different types of biologically active proteins can exert a synergistic effect on each other, these proteins can be fused to generate a dual-function protein. For example, International Publication No. WO2020 / 084496 discloses a GLP1-GDF15 fusion protein comprising a glucagon-like peptide-1 (GLP-1) peptide, a first linker peptide, a serum albumin protein, and a GDF15 protein.

[0005] On the other hand, immunoglobulin Fc fusion is known to increase the half-life of bioactive proteins in vivo and reduce the risk of adverse events such as toxicity and immune responses, so it can be used to develop long-acting drugs. However, even when immunoglobulin Fc fusion is applied, the activity of the bioactive protein may vary depending on the fusion position between the bioactive protein and the immunoglobulin Fc. Furthermore, even if the half-life in vivo is increased, it may be difficult to demonstrate a sufficient pharmacokinetic profile to allow administration in humans at weekly intervals. Furthermore, there are various issues, such as safety issues due to immunogenicity caused by the linker used during fusion or mutation, and the possibility of causing unintended immune responses depending on the type (isotype) of immunoglobulin introduced.

[0006] Furthermore, because bifunctional proteins prepared using two different types of bioactive proteins have a structure that does not exist in nature, their structure is inevitably more complex than that of proteins fused with a single bioactive protein and proteins for increasing half-life (e.g., serum albumin, tremensferin, etc.). Furthermore, when developing a bifunctional protein, it is essential that each bioactive protein exists stably in the body while maintaining its physiological activity, and the difference in half-life between the bioactive proteins is adjusted to an appropriate level. To achieve this goal, the introduction of mutations and / or linker manipulation may be required, whereby the activity of the bioactive proteins may be varied by the introduction of mutations, thereby changing the structure and stability of the bioactive proteins. Furthermore, various problems (e.g., aggregation) may occur due to the linker and / or mutations in the generated bifunctional protein, thereby reducing protein stability.

[0007] In the process of developing a dual-function protein, the present inventors found that a fusion protein prepared by fusing the Fc region of an immunoglobulin with GDF15 or a mutant polypeptide thereof, and then fusing it with a linker and a biologically active protein different from GDF15, has excellent productivity, stability, pharmacokinetic profile, and pharmacological efficacy, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to provide a fusion protein, which is a dual-function protein with excellent productivity, stability, pharmacokinetic profile, and pharmacological efficacy, and a dimer thereof; a method for treating or preventing a metabolic-related disease using the fusion protein or its dimer, and medical uses thereof; an isolated cell line producing the fusion protein or its dimer; an isolated nucleic acid encoding the fusion protein or its dimer; a recombinant expression vector comprising the nucleic acid; a host cell comprising the recombinant expression vector; and a method for producing the fusion protein or its dimer. [Means for solving the problem]

[0009] In an embodiment of the present invention, the present invention provides a fusion protein or a dimer thereof comprising a polypeptide represented by formula (I) and a polypeptide represented by formula (II).

[0010] In another embodiment of the present invention, the present invention provides a fusion protein or a dimer thereof, in which a biologically active protein represented by formula (III) and GDF15 or a variant thereof are linked to the Fc of an immunoglobulin or a variant thereof. In another embodiment of the present invention, the present invention provides an isolated cell line producing the fusion protein or a dimer thereof.

[0011] In yet another embodiment of the present invention, the present invention provides an isolated nucleic acid encoding a fusion protein or a dimer thereof.

[0012] In one embodiment of the present invention, the invention provides a recombinant expression vector comprising the nucleic acid.

[0013] In another embodiment, the present invention provides a host cell comprising the recombinant expression vector.

[0014] In one embodiment of the present invention, the present invention provides a method for producing a fusion protein or a dimer thereof, comprising culturing a cell line that expresses the fusion protein under conditions in which the fusion protein is expressed; and recovering the fusion protein.

[0015] In another embodiment of the present invention, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion protein, or a dimer thereof.

[0016] In yet another embodiment of the present invention, the present invention provides a method of treating or preventing a metabolic-related disease in an individual in need thereof, comprising administering to the individual a fusion protein or a dimer thereof.

[0017] In one embodiment of the present invention, the invention provides the use of the fusion protein in the preparation of a medicament for the treatment of a metabolic-related disease. [Effects of the Invention]

[0018] The fusion protein or its dimer according to the present invention has improved pharmacological efficacy, in vivo persistence, and protein productivity and stability of the biologically active protein. Furthermore, the fusion protein or its dimer can be useful in methods for preventing and / or treating metabolic diseases. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows a schematic diagram of a fusion protein dimer containing a GDF15 mutant and a GLP-1 mutant. [Figure 2] Figure 2 shows the results of SDS-PAGE gel analysis of the two-step purified HGK, HGH, and HGKH, confirming that the first and second polypeptides are not cleaved. [Figure 3] Figure 3 shows a graph of serum drug concentrations of the GDF15 moiety over time in mice 240 hours after subcutaneous administration of 13 types of long-acting fusion proteins. Data are expressed as the mean and standard deviation. [Figure 4] Figure 4 shows a graph of serum drug concentrations of the GLP1 moiety of 13 types of long-acting fusion proteins over time in mice 240 hours after subcutaneous administration. Data are expressed as the mean and standard deviation. [Figure 5] Figure 5 shows a graph of serum drug concentrations of the GDF15 moiety over time in mice 240 hours after subcutaneous administration of 15 types of long-acting fusion proteins. Data are expressed as the mean and standard deviation. [Figure 6] Figure 6 shows a graph of serum drug concentrations of the GLP1 moiety of 15 types of long-acting fusion proteins over time in mice 240 hours after subcutaneous administration. Data are expressed as the mean and standard deviation. [Figure 7] Figure 7 shows a graph of serum drug concentrations of the GDF15 and GLP-1 moieties in rats over a 504-hour period following intravenous and subcutaneous administration of the long-acting fusion protein HGKH-EKN-L4. Data are expressed as the mean and standard deviation. [Figure 8] FIG. 8 shows a graph comparing the GDF15 activity of 10 types of long-acting fusion proteins. [Figure 9] FIG. 9 shows a graph comparing the GLP-1 activity of 10 types of long-acting fusion proteins. [Figure 10] 10 shows a graph of serum drug concentrations of the GDF15 moiety in mice over a time course of 240 hours after subcutaneous administration of 10 types of long-acting fusion proteins. Data are expressed as the mean and standard deviation. [Figure 11]11 shows a graph of serum drug concentrations of the GLP-1 moiety in mice over time 240 hours after subcutaneous administration of 10 types of long-acting fusion proteins. Data are expressed as the mean and standard deviation. [Figure 12] FIG. 12 shows a graph comparing the % body weight change following repeated administration of different doses of GK-L3 and GH-L5 in diet-induced obese (DIO) mice. [Figure 13] FIG. 13 shows a graph comparing the percent weight change in DIO mice following repeated administration of five types of long-acting fusion proteins (GK-L3-3, GH-L3, GH-L4, GKH-L2, and GKH-L4-L2). [Figure 14] FIG. 14 shows a graph comparing the body weight change (%) in DIO mice following repeated administration of HGH-EKN-L4 and HGKH-EKN-L4 at different doses. [Figure 15] FIG. 15 shows a graph comparing the percent weight change in DIO mice following repeated administration of five types of long-acting fusion proteins (HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGKH-EKN-L4, and HGKH-EKN-L4-49) at doses of 0.3 nmol / kg and 1 nmol / kg. [Figure 16] FIG. 16 shows the results of non-fasting blood glucose level measurements on days 9 and 21 from the start of administration in DIO mice repeatedly administered six types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) [****p<0.0001 vs. DIO vehicle (one-way ANOVA)]. [Figure 17]FIG. 17 shows the results of measurement of non-fasting blood glucose levels in DIO mice at the end of administration of six types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) [**p<0.01, ***p<0.001, and ****p<0.0001 vs. DIO vehicle (one-way ANOVA)]. [Figure 18] FIG. 18 shows a graph comparing cumulative food intake of six types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) measured in DIO mice from the start of repeated dosing through 24 days. [Figure 19] FIG. 19 shows a graph comparing the percent weight change in DIO mice caused by repeated administration of six types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4). [Figure 20] FIG. 20 shows a graph comparing the % weight change in DIO mice caused by repeated administration of nine types of long-acting fusion proteins (HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGK-EKN-L10-47, HGK-EKN-L10-49, HGKH-EKN-L4, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49). [Figure 21] FIG. 21 shows a graph comparing the cumulative food intake of HGKH-EKN-L4 measured in DIO mice from the start of administration of HGKH-EKN-L4 at different doses until day 27 after repeated administration. [Figure 22] FIG. 22 shows a graph comparing the body weight change (%) measured in DIO mice from the start of administration of HGKH-EKN-L4 at different doses until day 27 after repeated administration. [Figure 23]FIG. 23 shows a graph comparing the % change in body weight measured in ob / ob mice from the start of administration of HGK-EKN-L10 at different doses until day 14 after a single administration. [Figure 24] FIG. 24 shows a graph comparing non-fasting blood glucose levels measured in ob / ob mice from the start of administration of HGK-EKN-L10 at different doses until day 14 after a single administration. [Figure 25] FIG. 25 shows a graph comparing the % change in body weight measured in ob / ob mice from the start of administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses until day 14 after repeated administration. [Figure 26] Figure 26 shows the results of measuring HbA1c levels in ob / ob mice at the end of repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses [****p<0.0001 vs. vehicle-treated ob / ob controls (one-way ANOVA)]. [Figure 27] Figure 27 shows the results of measuring total cholesterol, HDL, and LDL levels in ob / ob mice at the end of repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses [**p<0.01, ***p<0.001, and ****p<0.0001 vs. ob / ob vehicle (one-way ANOVA)]. [Figure 28] Figure 28 shows the results of measuring the values ​​of ALP, AST, ALT and relative liver weight in ob / ob mice at the end of repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses [*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. ob / ob vehicle (one-way ANOVA)]. [Figure 29] FIG. 29 shows a graph comparing non-fasting blood glucose levels measured in db / db mice after repeated administration of HGKH-EKN-L4 at different doses. [Figure 30]Figure 30 shows the results of measuring HbAlc levels in db / db mice repeatedly administered HGKH-EKN-L4 at different doses on days 14 and 26 from the start of administration [**p<0.01, ***p<0.001, and ****p<0.0001 vs. vehicle-treated db / db controls (one-way ANOVA)]. [Figure 31] FIG. 31 shows the results of an intraperitoneal insulin tolerance test performed in db / db mice at the end of repeated administration of HGKH-EKN-L4 at different doses [**p<0.01 and ****p<0.0001 vs. db / db vehicle (one-way ANOVA)]. [Figure 32] Figure 32 shows the results of measuring body weight change (%) and relative liver weight in the GAN-ob / ob NASH mouse model by repeated administration of HGKH-EKN-L4 at different doses [****p<0.0001 vs. GAN-ob / ob vehicle (one-way ANOVA)]. [Figure 33] Figure 33 shows the results of measuring the levels of ALP, AST, and ALT in the GAN-ob / ob NASH mouse model at the end of repeated administration of HGKH-EKN-L4 at different doses [####p<0.0001 vs. Chow control (unpaired t-test) and *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. GAN-ob / ob vehicle (one-way ANOVA)]. [Figure 34] Figure 34 shows a graph comparing the extent of hepatic collagen deposition following repeated administration of HGKH-EKN-L4 at different doses in a GAN-ob / ob NASH mouse model using picrosirius red (PSR) staining [####p<0.0001 vs. Chow vehicle (unpaired t-test) and ****p<0.0001 vs. GAN-ob / ob vehicle (one-way ANOVA)]. [Figure 35] FIG. 35 shows the amino acid sequence information of the human IgG1 Fc sequence used in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] In one aspect, the present invention relates to a fusion protein or a dimer thereof comprising a polypeptide represented by the following formula (I) and a polypeptide represented by the following formula (II): A p -B q -C(I) F a -G b -HIJ(II) [In formula: A and F are each independently a biologically active protein; B, G, and I are each independently a linker; C and H are immunoglobulin Fc or variants thereof, respectively; J is (Y1Y2) t -N-(Y3) u -(Y4) v -(Y5) w -CPLGPGRCCRLHTV-Y6-ASLEDLGWAD-Y7-VLSPREVQVTMCIGACPSQFRAA-Y8-MHA-Y9-IKT-Y 10 -LHRLKPDTVPAPCCVPASYNPMVLI-Y 11 -KTDTGVSLQTYD-Y 12 -LLAKDCHCI, During the ceremony: Y1, Y3, and Y7 to Y9 are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); Y2, Y6, and Y 11are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); Y4 and Y 12 is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or is an acidic amino acid that is aspartic acid (D) or glutamic acid (E); Y5 is a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); Y 10 is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid that is aspartic acid (D) or glutamic acid (E); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); t, u, v, and w are each independently 0 or 1; p and q in the above formula (I) and a and b in the above formula (II) are each independently 0 or 1, and when p is 0, q is 0, and when p is 1, q is 1; provided that p and q in formula (I) and a and b in formula (II) are not simultaneously 0, when a is 0, b is also 0, and when a is 1, b is 1.

[0021] In another aspect of the present invention, the present invention relates to a fusion protein or a dimer thereof, in which a biologically active protein represented by the following formula (III) and GDF15 or a variant thereof are linked to the Fc of an immunoglobulin or a variant thereof, and optionally the biologically active protein represented by formula (III) and / or GDF15 or a variant thereof may be linked to the Fc of an immunoglobulin or a variant thereof by a linker: (Y1Y2) t -N-(Y3) u -(Y4) v -(Y5) w -CPLGPGRCCRLHTV-Y6-ASLEDLGWAD-Y7-VLSPREVQVTMCIGACPSQFRAA-Y8-MHA-Y9-IKT-Y 10 -LHRLKPDTVPAPCCVPASYNPMVLI-Y 11 -KTDTGVSLQTYD-Y 12 -LLAKDCHCI (III).

[0022] GDF15 or a variant thereof represented by the above formula (III) is (Y1Y2) t -N-(Y3) u -(Y4) v -(Y5) w -CPLGPGRCCRLHTV-Y6-ASLEDLGWAD-Y7-VLSPREVQVTMCIGACPSQFRAA-Y8-MHA-Y9-IKT-Y 10 -LHRLKPDTVPAPCCVPASYNPMVLI-Y 11 -KTDTGVSLQTYD-Y 12 -LLAKDCHCI, During the ceremony: Y1, Y3, and Y7 to Y9 are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); Y2, Y6, and Y 11 are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); Y4 and Y 12 is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or is an acidic amino acid that is aspartic acid (D) or glutamic acid (E); Y5 is a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); Y 10is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid such as aspartic acid (D) or glutamic acid (E); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R). In one embodiment of the present invention, GDF15 represented by the above formula (III) or a variant thereof may be a polypeptide corresponding to J in the above formula (II).

[0023] As used herein, the term "bioactive protein" refers to a bioactive protein that has physiological activity in vivo. For example, the biologically active protein may be selected from the group consisting of, but is not limited to, 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 (RBP4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, growth hormone, and variants thereof.

[0024] In one embodiment of the present invention, the bioactive protein may be selected from the group consisting of GLP-1, exendin-4, or variants thereof.

[0025] Furthermore, in another embodiment of the present invention, the biologically active protein may be selected from the group consisting of a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, exenatide, liraglutide, dulaglutide, albiglutide, lixisenatide, semaglutide, tirzepatide, cotadutide, and taspoglutide, but is not limited to these.

[0026] As used herein, the term "GLP-1" refers to a 31-amino acid incretin hormone secreted from L cells in the intestinal tract upon stimulation by food or other substances. GLP-1 exerts its activity by transmitting signals to cells via the GLP-1 receptor, a G protein-coupled protein receptor expressed in target tissues such as pancreatic beta cells and the brain. Human wild-type GLP-1 is a polypeptide represented by the amino acid sequence of SEQ ID NO: 9 (HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR G).

[0027] GLP-1 secreted into the blood has a very short in vivo half-life of less than 2 minutes, due to the lack of activity caused by cleavage of the N-terminal amino acid by the dipeptidyl peptidase-4 (DPP-4) enzyme in the body. GLP-1 has a strong blood glucose-lowering effect without causing hypoglycemia because it stimulates insulin secretion from pancreatic beta cells in response to blood glucose concentrations. Furthermore, GLP-1 administration is known to induce weight loss in various animal models and humans, resulting from reduced food intake due to the appetite-suppressing effect of GLP-1. GLP-1 promotes beta cell proliferation by increasing survival rates and inhibiting beta cell apoptosis caused by glucolipotoxicity via the GLP-1 receptor expressed in pancreatic beta cells. Excessive glucagon secretion is known to increase blood glucose levels and is one of the causes of hyperglycemia observed in diabetic patients. Furthermore, GLP-1 is known to suppress the rise in fasting blood glucose levels by acting on pancreatic alpha cells and to inhibit protein kinase A (PKA) protein-specific glucagon secretion.

[0028] As used herein, the term "exendin-4" is known as a clinically important GLP-1 receptor agonist. Exendin-4 is a 39-residue polypeptide produced in the salivary glands of the Gila monster lizard. Exendin-4 shares 52% amino acid sequence homology with GLP-1 and is known to interact with the GLP-1 receptor in mammals (Thorens et al., Diabetes, 42: 1678-1682, 1993). Exendin-4 has been shown to promote insulin secretion by insulin-producing cells in vitro, and when administered in equimolar amounts, it is more potent at inducing insulin release from insulin-producing cells than GLP-1. Furthermore, exendin-4 potently stimulates insulin release in both rodents and humans, thereby lowering plasma glucose levels, and is longer acting than GLP-1; however, exendin-4 does not occur naturally in mammals and therefore has certain potential antigenic properties in GLP-1-deficient mammals.

[0029] On the other hand, the above-mentioned exenatide, liraglutide, dulaglutide, albiglutide, lixisenatide, semaglutide, tirzepatide, cotadutide, and taspoglutide are types of GLP-1 analogues (materials designed to mimic the effects of endogenous GLP-1, which stimulate glucose-dependent insulin secretion and inhibit glucagon secretion from the pancreas).

[0030] In one embodiment of the present invention, the biologically active protein may be a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); and X2 and X3 are each independently glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), X4, X5, and X6 are each independently a neutral amino acid selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid selected from the group consisting of aspartic acid (D) or glutamic acid (E); X4, X5, and X6 are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R).

[0031] In one embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 can be alanine (A) or glycine (G), and / or X2 can be glycine (G) or glutamic acid (E), and / or X3 can be alanine (A) or glutamic acid (E), and / or X4 can be valine (V) or lysine (K), and / or X5 can be lysine (K) or asparagine (N), and / or X6 can be arginine (R) or glycine (G).

[0032] In another embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 can be glycine (G), and / or X2 can be glutamic acid (E), and / or X3 can be alanine (A) or glutamic acid (E), and / or X4 can be valine (V) or lysine (K), and / or X5 can be lysine (K) or asparagine (N), and / or X6 can be glycine (G).

[0033] In yet another embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 can be (A) or glycine (G), and / or X2 can be glycine (G) or glutamic acid (E), and / or X3 can be alanine (A), and / or X4 can be valine (V) or lysine (K), and / or X5 can be lysine (K) or asparagine (N), and / or X6 can be arginine (R) or glycine (G).

[0034] In one embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 can be alanine (A) or glycine (G), and / or X2 can be glycine (G) or glutamic acid (E), and / or X3 can be glutamic acid (E), and / or X4 can be valine (V), and / or X5 can be lysine (K) or asparagine (N), and / or X6 can be arginine (R) or glycine (G).

[0035] In another embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 can be alanine (A) or glycine (G), and / or X2 can be glycine (G) or glutamic acid (E), and / or X3 can be glutamic acid (E), and / or X4 can be lysine (K), and / or X5 can be asparagine (N), and / or X6 can be arginine (R) or glycine (G).

[0036] In another embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X1 can be glycine (G), X2 can be glutamic acid (E), and X6 can be glycine (G).

[0037] In one embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, where X3 may be alanine (A).

[0038] In another embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, where X3 can be glutamic acid (E) and X4 can be valine (V).

[0039] In yet another embodiment of the present invention, the biologically active protein is a polypeptide consisting of an amino acid sequence represented by H-X1-EGTFTSDVSSYLE-X2-QAAKEFI-X3-WL-X4-X5-G-X6-G, wherein X3 can be glutamic acid (E), X4 can be lysine (K), and X5 can be asparagine (N).

[0040] In one embodiment of the present invention, A and F can each independently be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 9-12.

[0041] In another embodiment of the invention, t may be 1, Y1 may be alanine (A) or tryptophan (W), and Y2 may be arginine (R) or serine (S).

[0042] In one embodiment of the present invention, t may be 0, Y3 may be glycine (G) or serine (S), and / or Y4 may be aspartic acid (D) or threonine (T), and / or Y5 may be histidine (H), and / or Y6 may be arginine (R) or asparagine (N), and / or Y7 may be tryptophan (W) or phenylalanine (F), and / or Y8 may be asparagine (N), leucine (L), cysteine ​​(C), or serine (S), and / or Y9 may be glutamine (Q) or asparagine (N), and / or Y 10 may be serine (S), asparagine (N), aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), or leucine (L); Y 11 may be glutamine (Q) or histidine (H), and / or Y 12 may be aspartic acid (D), leucine (L), cysteine ​​(C), or serine (S).

[0043] In another embodiment of the invention, Y3 may be glycine (G), Y4 may be aspartic acid (D) and Y5 may be histidine (H).

[0044] In yet another embodiment of the invention, w may be 0, Y3 may be serine (S), and Y4 may be threonine (T).

[0045] In another embodiment of the present invention, J in the above formula (II) can be wild-type GDF15 or a mutant thereof. Growth differentiation factor-15 (GDF15), also known as macrophage inhibitory cytokine-1 (MIC-1), placental bone morphogenetic protein (PBMP), or nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), is a protein that is a member of the transforming growth factor beta superfamily (TGF-β superfamily). Research has reported that GDF15 induces weight loss by suppressing food intake through binding to GDNF family receptor alpha-like (GFRAL) and Ret proto-oncogene (RET), which are specifically expressed in brain tissue [Tsai VW, et al., PLoS One 2013; 8 (2): e55174; US 8,192,735]. Furthermore, some studies have demonstrated that administering GDF15 to various obese animal models results in significant weight loss, and additional metabolic benefits, such as lowering blood glucose levels, improving lipid levels, and improving insulin resistance, have been confirmed.Wild-type GDF15 protein can be derived from mammals such as humans, mice, pigs, and monkeys.Specifically, it can be the wild-type GDF15 protein derived from humans.More specifically, wild-type GDF15 protein can be the human wild-type GDF15 protein represented by SEQ ID NO: 1, or the GDF15 protein whose NCBI reference sequence is NP_004855.2.

[0046] In another embodiment of the present invention, J in the above formula (II) may be a GDF15 mutant containing one or more mutations selected from the group consisting of the following mutations (1) to (12): (1) The first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted (hereafter referred to as “ΔN2”); (2) the first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted, and the 64th amino acid, serine, is replaced by arginine (hereafter referred to as “ΔN2, S64R”); (3) the first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted and the 32nd amino acid, tryptophan, is replaced by phenylalanine (hereafter referred to as “ΔN2, W32F”); (4) the first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted, and the 90th amino acid, glutamine, is replaced by histamine (hereafter referred to as “ΔN2, Q90H”); (5) the first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted, and the 60th amino acid, glutamine, is replaced by asparagine (hereafter referred to as “ΔN2, Q60N”); (6) the first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted, and the 64th amino acid, serine, is replaced by asparagine (hereafter referred to as “ΔN2, S64N”); (7) the first and second amino acids from the N-terminus of the wild-type GDF15 protein are deleted, and the 64th amino acid, serine, is replaced by aspartic acid (hereafter referred to as “ΔN2, S64D”); (8) The first to third amino acids from the N-terminus of the wild-type GDF15 protein (SEQ ID NO: 1) are deleted (hereinafter referred to as "ΔN3"); (9) The first to third amino acids from the N-terminus of the wild-type GDF15 protein are deleted, and a tryptophan and serine are inserted in their place (hereafter referred to as "ΔN3, WS insertion"); (10) The first to third amino acids from the N-terminus of the wild-type GDF15 protein are deleted, tryptophan and serine are inserted in their place, the fourth amino acid, glycine, is replaced by asparagine, the fifth amino acid, aspartic acid, is replaced by serine, and the sixth amino acid, histidine, is replaced by threonine (hereinafter referred to as "ΔN3, WS insertion, G4N, D5S, H6T"); (11) A variant in which the first to third amino acids from the N-terminus of the wild-type GDF15 protein are deleted, the fourth amino acid, glycine, is replaced by asparagine, the fifth amino acid, aspartic acid, is replaced by serine, and the sixth amino acid, histidine, is replaced by threonine (hereinafter referred to as "ΔN3, G4N, D5S, H6T"); (12) The first to third amino acids from the N-terminus of the wild-type GDF15 protein are deleted, the fourth amino acid, glycine, is replaced by asparagine, the fifth amino acid, aspartic acid, is replaced by serine, the sixth amino acid, histidine, is replaced by threonine, and the 64th amino acid, serine, is replaced by arginine (hereinafter referred to as "ΔN3, NST, S64R").

[0047] In one embodiment of the present invention, the GDF15 mutant may include mutations that are: ΔN2 plus any one of S64R, W32F, Q90H, Q60N, S64N, and S64D; ΔN3 plus NST and S64R; or a combination of any one of S64R, S64N, and S64D with any one of W32F, Q90H, and Q60N.

[0048] In another embodiment of the present invention, the GDF15 mutant may be in a form in which 1 to 14 amino acids are deleted at the N-terminus or C-terminus compared to the wild-type GDF15 protein represented by the amino acid sequence of SEQ ID NO:1.

[0049] In another embodiment of the present invention, J in the above formula (II) may be such that the N residue introduced by mutation is glycosylated or may comprise at least one N-linked glycan.

[0050] In one embodiment of the present invention, J in the above formula (II) may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 8 and 93 to 108. Furthermore, the GDF15 mutant protein may be a protein represented by any one selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 2 to 8 and 93 to 108, as well as a form in which 1 to 14 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type GDF15 protein.

[0051] In yet another embodiment of the present invention, B in formula (I) above and G and I in formula (II) above are linkers that can connect a biologically active protein and / or a GDF variant to any different protein. The linker may be, but is not limited to, any linker known in the art to which the present invention pertains that can connect proteins and / or compounds to each other.

[0052] In another embodiment of the present invention, B in the above formula (I) and G and I in the above formula (II) can each independently be a linker selected from the group consisting of, but not limited to: 1) a peptide consisting of 5 to 52 glycines (G) and / or serine (S); 2) a peptide consisting of 4 to 52 glutamic acids (E) and / or alanines (A); 3) a peptide consisting of 5 to 52 glutamic acids (E), lysines (K), and / or alanines (A); 4) a peptide consisting of 6 to 52 glycines (G), serine (S), glutamic acids (E), and / or alanines (A); and 5) a peptide consisting of 7 to 52 glycines (G), serine (S), glutamic acids (E), lysines (K), and / or alanines (A).

[0053] In yet another embodiment of the invention, the linker is (G4S) n , GS(G4S) n ,GS(EEEA) n , (EEEA) n , GS(EAAAK) n , and (EAAAK) nor a polypeptide comprising the same. n may be an integer of 1 to 10. In one embodiment of the present invention, a linker comprising (G4S)3 and linkers comprising GS(EEEA)6 and GS(EAAAK)5 are used, but are not limited to these.

[0054] In one embodiment of the present invention, each linker can be independently selected from the group consisting of the amino acid sequences of SEQ ID NOs: 13 to 24, but is not limited thereto.

[0055] In yet another aspect of the invention, the linker is GSEEEAEEEAEEEAEEEA (SEQ ID NO: 22), GSEEEAEEEAEEEAEEEAEEEAEEEAEEEA (SEQ ID NO: 23), GSEEEAEEEAEEEAEEEAEEEAEEEAEEEAEEEAEEEA (SEQ ID NO: 24), or GSEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 21) Preferably, the linker may be GSEEEAEEEAEEEAEEEAEEEAEEEAEE (SEQ ID NO: 23) or GSEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 21).

[0056] In one embodiment of the present invention, C of the above formula (I) and H of the above formula (II) are each Fc of each immunoglobulin or a variant thereof.

[0057] As used herein, the terms "Fc region," "Fc fragment," or "Fc" refer to a protein that includes immunoglobulin heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3), but does not include the variable regions of the immunoglobulin heavy and light chains and light chain constant region 1 (CL1).

[0058] Furthermore, as used herein, the term "immunoglobulin Fc or variant thereof" refers to an Fc region in which some amino acids are substituted or prepared by combining different types of Fc regions. The IgG Fc region or variant thereof may be an IgG1, IgG2, IgG3, or IgG4 Fc or variant thereof. Furthermore, the Fc region variant may be a variant that does not contain the hinge region of the heavy chain constant region, or may include the hinge region.

[0059] In another embodiment of the present invention, the immunoglobulin Fc or variant thereof may be an Fc consisting of any one of IgG1, IgG2, IgG3, IgG4, and IgD Fc, or a hybrid Fc consisting of a combination thereof.

[0060] In another embodiment of the invention, the immunoglobulin Fc or variant thereof may be an IgG1 Fc or variant thereof, and the human IgG1 Fc may consist of the amino acid sequence represented by SEQ ID NO: 80 (see Figure 35). The human IgG Fc or variant thereof may have a contiguous amino acid sequence or an Fc fragment comprising a CH3 domain that is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80. In one embodiment of the invention, the human IgG Fc or variant thereof may have a contiguous amino acid sequence or an Fc fragment comprising a CH2 domain and a CH3 domain that have 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 80. In another embodiment of the invention, the human IgG Fc or variant thereof may have a contiguous amino acid sequence or an Fc fragment comprising a partial hinge region, a CH2 domain, and a CH3 domain that is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80. In yet another embodiment of the invention, the human IgG Fc or variant thereof may have a contiguous amino acid sequence that is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80.

[0061] In one embodiment of the present invention, an Fc variant of an immunoglobulin may comprise at least one processed bulge and may comprise at least one processed cavity, and the bulge and cavity may be located in the CH3 region. Specifically, an Fc variant of an immunoglobulin comprising a processed cavity may be processed to bind to an Fc variant of an immunoglobulin comprising a processed bulge. The bulge may be referred to as a "knob," and the cavity may be referred to as a "hole." When an Fc variant of an immunoglobulin comprises a processed bulge, it may be referred to as an Fc "knob," and when an Fc variant of an immunoglobulin comprises a processed cavity, it may be referred to as an Fc "hole."

[0062] In yet another embodiment of the present invention, the processed protuberance may comprise at least one substitution in the amino acid sequence of human immunoglobulin Fc. Preferably, the processed protuberance may comprise at least one substitution in the IgG1 amino acid sequence of SEQ ID NO: 80, where the numbering of amino acid positions is according to EU numbering. The substitution may occur at a position selected from the group consisting of amino acid residues 347, 366, and 394. For example, the substitution may be, but is not limited to, any one selected from the group consisting of Q127W / Y, T146W / Y, and T174W / Y mutations (Q347W / Y, T366W / Y, and T394W / Y according to EU numbering), and combinations thereof.

[0063] In yet another embodiment of the present invention, the processed cavity may contain at least one substitution in the amino acid sequence of human immunoglobulin Fc. Preferably, the processed cavity may contain at least one substitution in the IgG1 amino acid sequence of SEQ ID NO: 80, where the numbering of amino acid positions is according to EU numbering. The substitution may occur at a position selected from the group consisting of amino acid residues 366, 368, 394, 405, and 407. For example, the substitution may be, but is not limited to, any one selected from the group consisting of T146S, L148A, T174S, F185T / V / A, and Y187T / V / A mutations (T366S, L368A, T394S, F405T / V / A, and Y407T / V / A according to EU numbering), and combinations thereof.

[0064] In another embodiment of the present invention, in a human IgG1 Fc fragment variant containing a processed bulge, the 146th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 80 may be substituted with tryptophan (W).

[0065] In yet another embodiment of the present invention, in a human IgG1 Fc fragment variant containing a processed bulge, the 146th, 148th, and 187th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 80 may be substituted with serine (S), alanine (A), and valine (V), respectively.

[0066] In one embodiment of the present invention, the immunoglobulin Fc or variant thereof may have reduced or eliminated IgG effector function. The immunoglobulin Fc or variant thereof may contain mutation(s) that eliminate (e.g., reduce or eliminate) the effector function. For example, the Fc partner sequence of the immunoglobulin Fc or variant thereof may contain mutation(s) that abolish the effector function [e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP)].

[0067] In one embodiment of the invention, the immunoglobulin Fc or variant thereof may have E13A and L15A mutations introduced into the IgG1 amino acid sequence of SEQ ID NO: 80, resulting in abolishing IgG1 functional action. These mutations correspond to E233A and L235A when numbered according to the EU index of Kabat.

[0068] In another embodiment of the invention, an N77A mutation (N297A according to EU numbering) may be introduced into the IgG1 amino acid sequence of SEQ ID NO: 80 for removal of N-linked glycans, or L14A, L15A, and N77A mutations (L234A, L235A, and N297A according to EU numbering) affect the functional properties of IgG1 and may be introduced into the IgG1 amino acid sequence of SEQ ID NO: 80 for removal of N-linked glycans.

[0069] In one embodiment of the present invention, the immunoglobulin Fc variant may be one in which the 14th, 15th, and 77th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 80 are substituted with alanine (A), respectively. These mutations correspond to amino acids 234, 235, and 297 according to EU numbering.

[0070] In another embodiment of the present invention, the immunoglobulin Fc or a variant thereof may be a polypeptide in which a hinge has been added to the N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NOs: 81 to 86.

[0071] In yet another embodiment of the present invention, the immunoglobulin Fc or variant thereof may be a polypeptide comprising a hinge consisting of one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 79 and 109 to 111.

[0072] In one embodiment of the present invention, the Fc of an immunoglobulin or a variant thereof may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 85 or 86, or a polypeptide in which the amino acid sequence of SEQ ID NO: 79 is added to the N-terminus of a polypeptide having the amino acid sequence of SEQ ID NO: 85 or 86.

[0073] In another embodiment of the present invention, C in the above formula (I) may be a polypeptide consisting of any one of the amino acid sequences selected from SEQ ID NOs: 82, 84, 86, 88, 90, and 92, and H in the above formula (II) may be a polypeptide consisting of any one of the amino acid sequences selected from SEQ ID NOs: 81, 83, 85, 87, 89, and 91.

[0074] In another embodiment of the present invention, the immunoglobulin Fc or a variant thereof can be a polypeptide consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 81 to 92.

[0075] In one embodiment of the present invention, C in the above formula (I) may be IgG Fc or a variant thereof comprising a processed cavity, and H in the above formula (II) may be IgG Fc comprising a processed protuberance, and the polypeptide represented by formula (I) and the polypeptide represented by formula (II) may be fused depending on the arrangement of the protuberance and the cavity.

[0076] In yet another embodiment of the invention, the polypeptide represented by formula (I) and the polypeptide represented by formula (II) can be physically associated by non-covalent interactions (e.g., hydrophobic effect, such as hydrophobic interactions between the knob and hole regions of Fc), covalent bonds (e.g., disulfide bonds, such as one or two or more disulfide bonds between the hinge regions of Fc), or by both non-covalent interactions and covalent bonds.

[0077] As used herein, the term "dimer" refers to a protein complex comprising at least two polypeptides. Each of these polypeptides comprises an N-terminus and a C-terminus. The at least two polypeptides may be linked to each other by one or both of covalent and non-covalent interactions (e.g., electrostatic, π effect, van der Waals force, and hydrophobic effect). The two polypeptides may have the same or different amino acid sequences. When two types of polypeptides have two identical polypeptides, they are called homodimers, while when two types of polypeptides have two different polypeptides, they are called heterodimers.

[0078] In one embodiment of the present invention, a dimer of a fusion protein comprising a polypeptide represented by formula (I) and a polypeptide represented by formula (II) may be a homodimer in which the fusion proteins are identical to each other, or a heterodimer in which the fusion proteins are different from each other.

[0079] In another embodiment of the invention, the fusion protein of the invention comprises: (1) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (2) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (3) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (4) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 30 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (5) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (6) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 32 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (7) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (8) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (9) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (10) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (11) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 37 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (12) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 38 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (13) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 39 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (14) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (15) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked; (16) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (17) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (18) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (19) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 68 are linked; (20) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 69 are linked; (21) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 70 are linked; (22) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 67 are linked; (23) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (24) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (25) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (26) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked; (27) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (28) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (29) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (30) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 41 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (31) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (32) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (33) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 44 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (34) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (35) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (36) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (37) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (38) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (39) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (40) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked; (41) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked; (42) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked; (43) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (44) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked; (45) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked; (46) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 71 are linked; (47) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked; (48) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked; (49) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked; (50) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (51) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked; (52) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked; (53) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 51 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (54) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 52 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (55) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 53 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (56) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 54 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (57) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 55 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (58) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 56 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (59) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 57 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (60) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 58 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (61) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 59 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (62) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 60 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (63) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 61 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; and (64) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 62 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked may be selected from the group consisting of:

[0080] In one aspect, the invention relates to an isolated cell line producing the fusion protein or a dimer thereof; an isolated nucleic acid encoding the fusion protein or a dimer thereof; a recombinant expression vector comprising the nucleic acid; and a host cell comprising the vector.

[0081] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, the terms "transform" and "transfect" refer to the introduction of a nucleic acid (e.g., a vector) into a cell by any of a number of techniques known in the art.

[0082] In another embodiment of the present invention, the fusion protein or its dimer can be expressed in a suitable cell expression system characterized by producing the fusion protein or its dimer, such as mammalian cells (e.g., CHO, COS, HEK293, BHK, SK-Hip, etc.), E. coli, yeast (Saccharomyces cerevisiae), Pichia pastoris, and insect cells, which are known in the art to which the present invention pertains to be suitable for protein production.

[0083] Isolated host cells may be transformed or transfected with the DNA sequence of the present invention and used to express and / or secrete the fusion protein or dimer thereof. Host cells that may be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, HEK293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CAP cells (human amniotic fluid-derived cells), or COS cells.

[0084] As used herein, the term "isolated nucleic acid" refers to a nucleic acid molecule of the present invention that is part of a polynucleotide sequence that can be isolated from about 50% or more of the proteins, lipids, carbohydrates, or other materials that are naturally found with the nucleic acid when the entire nucleic acid is isolated from the source cell, can be operably linked to a polynucleotide with which it is not naturally linked, or is part of a larger polynucleotide sequence that does not occur in nature. Specifically, the isolated nucleic acid molecule of the present invention is substantially free of any other contaminating nucleic acid molecules or other contaminants found in its natural environment that would impair the production of the polypeptide or its use for therapeutic, diagnostic, preventive, or research purposes. In particular, isolated nucleic acid molecules encoding fusion proteins can have sequences that differ from each other due to codon redundancy. Furthermore, the isolated nucleic acid molecule can be appropriately modified or nucleotides can be added to the N- or C-terminus as long as a fusion protein can be produced.

[0085] As used herein, the term "recombinant expression vector" refers to a vector suitable for transformation of a host cell and containing a nucleic acid sequence that directs or controls the expression of an inserted heterologous nucleic acid sequence. 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.

[0086] 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, the translation of the mRNA transcript, and the production of an Fc fusion protein product or an antibody or antibody fragment. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. A useful expression vector may contain a human cytomegalovirus (CMV) promoter, which promotes continuous transcription of the target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence, which increases steady-state levels of RNA after transcription. As an example of the present invention, the expression vector may be pAD15, a modified vector of RcCMV.

[0087] In another aspect, the present invention relates to a method for producing a fusion protein or a dimer thereof, comprising culturing a cell line expressing the fusion protein under conditions for the expression of the fusion protein, and recovering the fusion protein. The conditions for the expression of the fusion protein are those that allow the cell line to grow appropriately and can be selected within the scope of known techniques in the technical field to which the present invention pertains.

[0088] Furthermore, in one embodiment of the present invention, after recovery of the fusion protein, a purification step may be completed using purification methods including, but not limited to, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, high performance liquid chromatography (HPLC), affinity chromatography, immunoprecipitation, and the like.

[0089] In yet another aspect, the present invention relates to a composition comprising a pharmaceutically acceptable carrier and the fusion protein or a dimer thereof. Furthermore, the present invention relates to a pharmaceutical composition for treating or preventing a metabolic-related disease, comprising a pharmaceutically acceptable carrier and the fusion protein or a dimer thereof.

[0090] In one embodiment of the present invention, the pharmaceutically acceptable carrier can be any non-toxic material suitable for delivering a drug or protein to an individual. Distilled water, alcohol, fat, wax, and intercalated solids can be included as carriers. Pharmaceutically acceptable auxiliary substances (buffers, dispersants) can also be included in the pharmaceutical composition. The concentration of the fusion protein in such a formulation can vary significantly.

[0091] In another embodiment of the present invention, compositions and pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, and lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, and sodium bisulfite), buffers (e.g., borate, bicarbonate, Tris-HCl, citric acid, phosphoric acid, and other organic acids), bulking agents (e.g., mannitol and glycine), chelating agents [e.g., ethylenediaminetetraacetic acid (EDTA)], complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, and hydroxyprolyl-beta-cyclodextrin), excipients, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, and dextrin), proteins (e.g., serum albumin, gelatin, and immunoglobulins), colorants, flavorings, and 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, and hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, and polyethylene glycol), sugar alcohols (e.g., mannitol and sorbitol), suspending agents, surfactants or wetting agents [e.g., pluronic; PEG; sorbitan esters; polysorbates (e.g., polysorbate 20 and polysorbate 80); Triton; tromethamine; lecithin; cholesterol; and tyloxapal], stability enhancers (e.g., sucrose and sorbitol), tonicity enhancers (e.g., alkali metal halides; preferably, sodium chloride or potassium chloride; and mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0092] In yet another embodiment of the present invention, the compositions and pharmaceutical compositions can be administered by any route. For example, the compositions and pharmaceutical compositions can be provided to animals by any suitable means, either directly (e.g., locally by injection, implantation, or local administration to a tissue site) or systemically (e.g., parenterally or orally). When the compositions and pharmaceutical compositions are administered parenterally (e.g., intravenously, subcutaneously, intraocularly, intraperitoneally, intramuscularly, orally, intrarectally, intraorbitally, intracranially, intraspinally, intraventricularly, intrathecally, intravesically, intraarticularly, intranasally, or by aerosol administration), the compositions can be aqueous solutions or can comprise part of a body fluid suspension or a physiologically acceptable solution. Thus, a physiologically acceptable carrier or vehicle can be added to the composition and delivered to an individual. Thus, the compositions and pharmaceutical compositions can typically comprise physiological saline, for example, a body fluid, as a carrier for formulation.

[0093] In one embodiment of the present invention, the frequency of administration of the composition and pharmaceutical composition can vary depending on the pharmacokinetic parameters of the fusion protein in the formulation used. Typically, a physician will administer the pharmaceutical composition until a dose that achieves the desired effect is reached. Thus, the composition and pharmaceutical composition can be administered as a single dose, in two or more doses (which may or may not contain the same amount of target fusion protein) separated by a period, or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dose can be routinely performed by those skilled in the art and is within the scope of their routine work.

[0094] In another embodiment of the present invention, the compositions and pharmaceutical compositions may be administered as a single dose or repeatedly administered two or more times, and may be administered once a day, twice a day, three times a day, every other day, once a week, once every two weeks, once every three weeks, once a month, or once every two months, but the number of administrations is not limited by such values.

[0095] In one aspect, the present invention relates to a method of treating or preventing a metabolic-related disease in an individual in need thereof, comprising administering to the individual a fusion protein or a dimer thereof, and to the use of the fusion protein or a dimer thereof in the manufacture of a medicament for the treatment of a metabolic-related disease.

[0096] As used herein, the term "individual" refers to a subject to which the fusion protein or dimer thereof is administered, and includes mammals such as livestock, primates, rabbits, and rodents, and the individual may be a human.

[0097] As used herein, the term " metabolic disease " refers to, but is not limited to, diabetes, obesity, hypercholesterolemia, hyperglycemia, insulin resistance, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver damage, hepatitis, liver fibrosis, cirrhosis, fatty liver, arteriosclerosis, dyslipidemia, cardiovascular disease or metabolic syndrome.Hepatitis can be non-viral hepatitis, hepatitis A, hepatitis B, hepatitis C, hepatitis D or hepatitis E.

[0098] The fusion proteins or dimers thereof of the present invention are thermostable. Preferably, the fusion proteins of the present invention may have a melting temperature (Tm) and aggregation temperature (Tag) of 40°C or higher, 45°C or higher, 46°C or higher, 47°C or higher, 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, 55°C or higher, 56°C or higher, 57°C or higher, 58°C or higher, 59°C or higher, or 60°C or higher. The Tm and Tagg of the fusion protein may be 75°C, 74°C, 73°C, or 72°C or lower. Furthermore, the fusion proteins of the present invention may have a Tm and Tagg of 40°C to 75°C, 40°C to 74°C, 40°C to 73°C, or 40°C to 72°C.

[0099] The fusion protein or dimer of the present invention can lower non-fasting blood glucose levels and / or fasting blood glucose levels, maintain the fluctuation range of non-fasting blood glucose levels and / or fasting blood glucose levels at a low level, reduce blood glycated hemoglobin levels, blood total cholesterol levels, blood high-density lipoprotein (HDL) levels, blood low-density lipoprotein (LDL) levels, insulin resistance, blood liver damage parameters (e.g., ALT, AST, ALP), and / or a subject's body weight, and inhibit the onset of liver damage and / or liver fibrosis or slow their progression rate, but its effects are not limited thereto. Therefore, in another embodiment of the present invention, the fusion protein or dimer of the present invention can treat diabetes, obesity, hypercholesterolemia, hyperglycemia, insulin resistance, NASH, NAFLD, liver damage, hepatitis, liver fibrosis, cirrhosis, fatty liver, arteriosclerosis, dyslipidemia, cardiovascular disease, or metabolic syndrome, or can ameliorate the symptoms of these diseases.

[0100] In one embodiment of the present invention, the fusion protein or dimer thereof of the present invention can be administered to a subject together with other biologically active molecules. The optimal combination between the fusion protein or dimer thereof and other molecules, the type of administration, and the amount of administration can be determined by routine experiments well known in the art.

[0101] Hereinafter, the present invention will be described in detail with reference to embodiments etc. in order to facilitate understanding of the present invention. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. [Example]

[0102] [Preparation Example 1] Preparation and purification of long-acting fusion protein or its dimer (1) [Preparation Example 1-1] Gene cloning A first polypeptide [corresponding to the polypeptide represented by formula (II)] is designed in the form of [IgG1 Fc_knob or IgG1 hFc_knob in which a hinge (SEQ ID NO: 79) has been introduced into the IgG1 Fc_knob]-[linker]-[GDF15 or a variant thereof], or in the form of [GLP-1 or a variant thereof]-[linker]-[IgG1 Fc_knob or IgG1 hFc_knob in which a hinge (SEQ ID NO: 79) has been introduced into the IgG1 Fc_knob]-[linker]-[GDF15 or a variant thereof] (see Table 1).

[0103] [Table 1] TIFF2025539794000003.tif107160

[0104] Furthermore, a second polypeptide [corresponding to the polypeptide represented by formula (I)] having the structure of [GLP-1 or a variant thereof]-[linker]-[IgG1 Fc_hole or IgG1 hFc_hole in which a hinge (SEQ ID NO: 79) has been introduced into the IgG1 Fc_hole] was designed (see Table 2).

[0105] [Table 2]

[0106] The first and second polypeptides form a fusion protein through a knob-in-hole interaction, and the protein formed by the knob-in-hole fusion is hereafter referred to as a "long-acting fusion protein" (see Table 3).

[0107] [Table 3-1] TIFF2025539794000006.tif92160 (Continued from Table 3)

[0108] [Table 3-2]

[0109] Furthermore, two long-acting fusion proteins may be fused together via GDF15-GDF15 interactions, which is referred to as a "long-acting fusion protein dimer."

[0110] Long-acting fusion protein dimers in which a GLP-1 variant is linked to a first polypeptide are designated by the material code "GK," long-acting fusion protein dimers in which a GLP-1 variant is linked to a second polypeptide are designated by the material code "GH," and long-acting fusion protein dimers in which a GLP-1 variant is linked to both a first and a second polypeptide are designated by the material code "GKH" (see Figures 1A-1C). Compared to GK, HGK has a hinge added to both the IgG1 Fc_knob sequence and the IgG1 Fc_hole sequence. Compared to GH, HGH has a hinge added to both the IgG1 Fc_knob sequence and the IgG1 Fc_hole sequence. Compared to GKH, HGKH has a hinge added to both the IgG1 Fc_knob sequence and the IgG1 Fc_hole sequence (see Figures 1D-1F).

[0111] Gene cloning was performed using a pcDNA3.3 (Invitrogen) expression vector containing a gene encoding a first polypeptide consisting of the amino acid sequences of SEQ ID NOS: 25 to 50 in Table 1, and a gene encoding a second polypeptide consisting of the amino acid sequences of SEQ ID NOS: 63 to 77, 86, and 96 in Table 2. In particular, nucleotide sequences encoding the amino acid sequences of SEQ ID NOS: 25 to 50, 63 to 77, 86, and 92 were synthesized by request to Macrogen Inc. [Preparation Example 1-2] Expression of long-acting fusion protein

[0112] The pcDNA3.3 expression vector cloned in Preparation Example 1-1 above was transiently transfected into the ExpiCHO-S cell line (Invitrogen) as described in the Thermo Fisher protocol (ExpiFectamine CHO transfection kit, ThermoFisher Scientific, Cat. No. A29129). Briefly, the DNA and ExpiFectamine complex was transfected into ExpiCHO-S cells (6 x 10 6 The cells were seeded at 1000 x g (cells / mL) and expression enhancers were added 24 hours later. The cell culture was shifted to 32°C 2 days after transfection and harvested after 6 days of further culture. [Preparation Example 1-3] Purification of long-acting fusion protein

[0113] To purify the long-acting fusion protein containing the first and second polypeptides in the harvested culture medium, affinity purification using Protein A resin was performed in the first step. To obtain a highly purified long-acting fusion protein, ion exchange (IEX) purification using an anion exchange (AEX) resin was performed in the second step of purification on the pool after the first step of purification.

[0114] Specifically, the culture medium was loaded onto a MabSelect SuRe Protein A resin (GE Healthcare) equilibrated with 1x PBS (pH 7.4) and allowed to bind. Upon completion of binding of the first and second polypeptides, the MabSelect SuRe Protein A resin was washed with 1x PBS (pH 7.4), and then elution was performed using 0.1 M glycine (pH 3.5) solution to obtain the final material. All eluted proteins were pooled. Correct assembly of the first and second polypeptides into a long-acting fusion protein was confirmed by SDS-PAGE gel analysis (Invitrogen) and size exclusion chromatography (Tosoh, catalog no. 08541).

[0115] Next, in the case of anion exchange (AEX), after adjusting the pH according to the isoelectric point, the pool from the first step was loaded onto a POROS HQ anion exchange resin (Thermofisher) equilibrated with 50 mM Tris (pH 8.3) solution and allowed to bind. Upon completion of binding of the long-acting fusion protein containing the first and second polypeptides, the POROS HQ anion exchange resin was washed with 50 mM Tris (pH 8.3), and then eluted by a gradient using 50 mM Tris (pH 8.3) containing 1 M NaCl to obtain the final material. Fractions meeting the purity criteria of 95% or higher were pooled based on size exclusion chromatography (Tosoh, catalog number 08541) analysis, and then the buffer was exchanged with 1x PBS (pH 7.4). The concentration of the purified fusion protein was measured using a spectrophotometer (Biochrom, Libra UV / Vis Spectrophotometer).

[0116] The purity of the long-acting fusion protein of Preparation Example 1-1 above in the first purification step is shown in Table 4. The purity of the long-acting fusion protein varied after Protein A resin purification depending on the type of IgG1 Fc and linker sequence, but the overall purity was confirmed to be approximately 60% or higher.

[0117] [Table 4-1] TIFF2025539794000009.tif156160 (Continued from Table 4)

[0118] [Table 4-2]

[0119] On the other hand, the region between Trp31 and Leu32 of the GLP-1 protein is cleaved in vivo by neutral endopeptidase (NEP) 24.11, and it is known that cleavage at this site abolishes the original activity of GLP-1 [Journal of Medicinal Chemistry 58 (2015): 1020-1037].

[0120] When the highly purified sample proteins HGK, HGH, and HGKH obtained by the two-step purification were analyzed using SDS-PAGE gels under reducing conditions, it was shown that the polypeptides fused to the GLP-1 mutants were not cleaved at Trp31-Leu32 and therefore maintained stability (see Figure 2). [Experimental Example 1] Activity of long-acting fusion protein [Experimental Example 1-1] Measurement of GDF15 activity of long-acting fusion protein

[0121] The GDF15 activity of the long-acting fusion protein prepared in Preparation Example 1 above was compared using the long-acting GDF15 fusion protein FM9-6+Fc_hole (Korean Patent Application Publication No. 10-2021-0065057) consisting of the amino acids of SEQ ID NO: 25 and SEQ ID NO: 86 as a control substance.

[0122] GDF15 activity was measured using Bright-Glo (商標) It was measured using a luciferase assay kit (Promega) and the HEK293 cell line (human embryonic kidney 293) in which GFRAL / RET / SRE-luc is overexpressed.

[0123] Specifically, GFRAL / RET / SRE-luc was overexpressed in 1 × 10 5 HEK293 cells were seeded into each well of a 96-well plate containing DMEM medium containing 10% FBS and cultured for 24 hours at 37°C and 5% CO. After 24 hours, the medium in each well of the 96-well plate was replaced with 50 mL of serum-free medium and cultured for 4 hours at 37°C and 5% CO.

[0124] Furthermore, the long-acting fusion protein prepared in Preparation Example 1 above was prepared by 3-fold serial dilution from a concentration of 2000 nM using serum-free medium. Then, 50 μL of the diluted solution of the long-acting GDF15 fusion protein was serially diluted 3-fold from the actual concentration of 1000 nM, and then added to each well containing 50 μL of replaced serum-free medium and the GFRAL / RET / SRE-luc cell line, so that the reaction was carried out under the conditions of 37°C and 5% CO2 for 4 hours. After 4 hours, Bright-Glo (商標) Bright-Glo (商標) 100 μL of Bright-Glo prepared by adding buffer (商標) The solution was added to each well and allowed to react for 1 hour at room temperature.

[0125] The reaction values ​​(relative light units, RLU) were then measured using a microplate reader (Perkin Elmer, Wallac Victor X5) capable of measuring luminescence. The results are shown in Table 5 below.

[0126] [Table 5] [Experimental Example 1-2] Measurement results of GLP-1 activity of long-acting fusion protein

[0127] The GLP-1 activity of the long-acting fusion protein prepared in Preparation Example 1 above was measured.

[0128] GLP-1 activity was measured using a cAMP Gs dynamic kit (Cisbio, catalog number 62AM4PEC) and the HEK293 cell line (human embryonic kidney 293) in which GLP-1R is overexpressed.

[0129] Specifically, GLP-1R was overexpressed, 1 × 10 4HEK293 cells were seeded into each well of a 96-well plate containing DMEM medium containing 10% FBS and cultured for 16 hours at 37°C and 5% CO2. After 16 hours, the long-acting fusion protein prepared in Preparation Example 1 above was serially diluted 5-fold from a concentration of 20 nM using DPBS containing 0.5% BSA (WELGENE, Cat. No. LS021-01) and 2 mM IBMX (Sigma-Aldrich, Cat. No. I7018). 25 μL of the diluted solution of the long-acting fusion protein was then added to each well containing the GLP-1R-overexpressing cell line so that the actual concentration was serially diluted 5-fold from 10 nM. The cells were then cultured for 30 minutes at 37°C and 5% CO2. After 30 minutes, 25 μL of cAMP-d2 solution was added to each well. 25 μL of anti-cAMP-cryptate solution was then added to each well and allowed to react at room temperature for 1 hour.

[0130] The reaction values ​​(homogeneous time-resolved fluorescence energy transfer, HTRF) were then measured using a microplate reader (Molecular Devices, FlexStation 3) capable of detecting time-resolved fluorescence. The results are shown in Table 6 below.

[0131] [Table 6-1] TIFF2025539794000013.tif108160 (Continued from Table 6)

[0132] [Table 6-2]

[0133] As seen in Table 6 above, the IgG1 Fc type and / or linker type were shown to have some effect on the GLP-1 activity of the long-acting fusion proteins. [Experimental Example 2] Evaluation of thermal stability of long-acting fusion proteins

[0134] The thermal stability of the long-acting fusion protein prepared in Preparative Example 1 above was evaluated using an UNcle apparatus (Unchained Labs).

[0135] Specifically, the protein sample was diluted to 3 mg / mL with Dulbecco's phosphate buffered saline (DPBS, Gibco, USA), and fluorescence and static light scattering analyses were performed using an UNcle instrument. The melting temperature (Tm) and aggregation temperature (Tagg), which are indicators of thermal stability, are shown in Table 7 below.

[0136] As shown in Table 7, the long-acting fusion protein prepared in Preparation Example 1 above was confirmed to have a Tm and Tagg value of approximately 60°C.

[0137] [Table 7] TIFF2025539794000016.tif240160 TIFF2025539794000017.tif239160 [Experimental Example 3] Pharmacokinetic evaluation of long-acting fusion proteins [Experimental Example 3-1] Experimental method for evaluating pharmacokinetics in mice

[0138] After purchasing 7-week-old male C57BL / 6 mice from Orient BIO, Korea, the mice were acclimated for 7 days and then divided into groups with similar average body weights on the day of drug treatment (n=3 per blood collection time).The long-acting fusion proteins were then subcutaneously administered once at a dose of 1 mg / kg, and blood samples were collected 4, 24, 48, 72, 96, 120, 168, and 240 hours after administration.The blood concentration of each long-acting fusion protein was quantified by dividing it into the active protein portion, the GDF15-IgG1 Fc portion, and the GLP1-IgG1 Fc portion.

[0139] Specifically, to measure serum concentrations of the active GDF15-Fc portion, ELISA analysis was performed using an antibody immunoreactive against the endogenously generated GDF15 receptor, GDNF family receptor alpha-like (GFRAL) protein, and Fc (Jackson ImmunoResearch, catalog number 109-035-098). To measure serum levels of the active GLP1-Fc portion, ELISA analysis was performed using an endogenously generated antibody immunoreactive against the N-terminus of GLP-1 (Invitrogen, catalog number ABS033-10-02) and Fc (Invitrogen, catalog number ABS033-10-02). After a single subcutaneous injection of each protein into mice, serum concentrations of the GDF15-Fc and GLP1-Fc portions of each material were measured for up to 240 hours to calculate their respective pharmacokinetic parameters. [Experimental Example 3-2] Pharmacokinetic evaluation results in mice

[0140] Based on the serum concentration of each active ingredient over time (see Figures 3-6), the pharmacokinetic parameters of the GDF15-IgG1 Fc portion and GLP1-IgG1 Fc portion of the long-acting fusion proteins were calculated after a single subcutaneous administration of each protein prepared in Preparation Example 1 above to mice, and are shown in the following Tables 8 and 9. Furthermore, the pharmacokinetic profiles of each long-acting fusion protein were compared and evaluated based on the area under the curve (AUC), which indicates the degree of drug exposure.

[0141] [Table 8] TIFF2025539794000019.tif233160 TIFF2025539794000020.tif150160

[0142] As shown in Table 8, it was confirmed that HGH-E-L4 and HGH-EKN-L4 showed excellent AUC values ​​of the GDF15 portion. It was confirmed that other long-acting fusion proteins, except for HGH-E-L4 and HGH-EKN-L4, showed similar AUC values ​​of the GDF15 region within a maximum of about 1.8 times.

[0143] [Table 9] TIFF2025539794000022.tif233160 TIFF2025539794000023.tif150160

[0144] As shown in Table 9, the AUC increased with introduction of the GLP-1 mutant sequences as determined after comparing HGK-L10 to HGK-E-L10 and HGK-EKN-L10, and comparing HGKH-L4 to HGKH-EKN-L4. Furthermore, when HGKH-EKN-L4 was compared with HGK-EKN-L8 and HGH-EKN-L4, HGKH-EKN-L5 with HGK-EKN-L10 and HGH-EKN-L5, and HGKH-EKN-L6 with HGK-EKN-L12 and HGH-EKN-L6, respectively, it was confirmed that the AUC of the GLP-1 moiety was increased by approximately 1.4 to 2.5-fold in the protein in which the GLP-1 variant was fused to both the IgG1 hFc_knob and the IgG1 hFc_hole, compared to the protein in which the GLP-1 variant was fused to only the IgG1 hFc_knob or only the IgG1 hFc_hole.

[0145] The ratios of AUC of the GLP-1 portion to AUC of the GDF15 portion for the long-acting fusion proteins prepared in Preparation Example 1 above are shown in Table 10. A higher ratio of AUC of the GLP-1 portion to AUC of the GDF15 portion indicates a more balanced pharmacokinetic profile of the GLP-1 variant and the GDF15 variant.

[0146] [Table 10]

[0147] As shown in Table 10, proteins in which GLP-1 variants were fused to IgG1 hFc_knob (HGK-L10, HGK-E-L8, HGK-E-L10, HGK-E-L12, HGK-EKN-L8, HGK-EKN-L10, and HGK-EKN-L12) were shown to have GLP-1 AUC / GDF15 AUC ratios of 0.17 to 0.38. Furthermore, proteins in which GLP-1 variants were fused to IgG1 hFc_hole (HGH-E-L4, HGH-EKN-L4, HGH-EKN-L5, and HGH-EKN-L6) were shown to have GLP-1 AUC / GDF15 AUC ratios of 0.15 to 0.27. On the other hand, proteins in which GLP-1 mutants were fused to IgG1 hFc_knob and IgG1 hFc-hole (HGKH-EKN-L4, HGKH-EKN-L5, and HGKH-EKN-L6) were shown to have GLP-1 AUC / GDF15 AUC ratios of 0.68–0.73. [Experimental Example 3-3] Experimental method for evaluating pharmacokinetics in rats

[0148] Six-week-old male Sprague-Dawley [SD(Crl:CD)] rats were purchased from Orient BIO, Korea. The purchased rats were acclimated for 7 days and then divided into groups with similar average body weights on the day of drug treatment (n=3 per group). The long-acting fusion protein HGKH-EKN-L4 prepared in Preparation Example 1 above was then administered intravenously at a dose of 1 mg / kg, and subcutaneously at doses of 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg. Blood samples were collected at 0.05, 1, 4, 8, 24, 48, 72, 96, 120, 168, 240, 336, 408, and 504 hours after intravenous administration and at 4, 8, 24, 48, 48, 72, 96, 120, 168, 240, 336, 408, and 504 hours after subcutaneous administration, respectively. The serum concentration of the long-acting fusion protein was quantified by dividing it into active protein moieties, the GDF15-IgG1 Fc moiety and the GLP1-IgG1 Fc moiety. The serum concentrations of the GDF15-Fc moiety and the GLP1 Fc moiety of each material were measured in rats up to 504 hours after injection of the long-acting fusion protein to calculate each pharmacokinetic parameter. [Experimental Example 3-4] Pharmacokinetics evaluation results in rats

[0149] After a single administration of HGKH-EKN-L4 to rats, the pharmacokinetic parameters of the GDF15-IgG1 Fc portion and GLP1-IgG1 Fc portion of HGKH-EKN-L4 were calculated based on the serum concentrations according to administration time and dose (see Figure 7), and are shown in Tables 11 and 12 below.

[0150] [Table 11]

[0151] [Table 12] [Preparation Example 2] Preparation and purification of long-acting fusion protein (2) [Preparation Example 2-1] Gene cloning and expression of long-acting fusion protein

[0152] To determine whether the stability, in vitro activity, and pharmacokinetic profile of the long-acting fusion protein may be altered when a GDF15 mutant with reduced GDF15 activity is used, a first polypeptide in Table 13 below and a second polypeptide in Table 14 below were designed where the GDF15 mutant had structural features identical to those of the long-acting fusion protein prepared in Preparative Example 1 above.

[0153] [Table 13] TIFF2025539794000028.tif59160

[0154] [Table 14]

[0155] Gene cloning was performed using a pcDNA3.3 (Invitrogen) expression vector containing a gene encoding a first polypeptide consisting of the amino acid sequences of SEQ ID NOs: 42, 48, 49, and 51 to 62, and a gene encoding a second polypeptide consisting of the amino acid sequences of SEQ ID NOs: 75 and 92. In particular, nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 42, 48, 49, and 51 to 62 and SEQ ID NOs: 75 and 92 were synthesized by request to Macrogen Inc.

[0156] The cloned pcDNA3.3 expression vector was transiently transfected into the ExpiCHO-S cell line (Invitrogen) as described in the Thermo Fisher protocol (ExpiFectamine CHO transfection kit, ThermoFisher Scientific, Cat. No. A29129). Briefly, the DNA and ExpiFectamine complex was transfected into ExpiCHO-S cells (6 × 10 6The cells were seeded at 1000 x g (cells / mL) and expression enhancers were added 24 hours later. The cell culture was shifted to 32°C 2 days after transfection and harvested after 6 days of further culture. [Preparation Example 2-2] Purification of long-acting fusion protein

[0157] To purify the long-acting fusion protein containing the first and second polypeptides in the culture medium, Protein A resin purification was performed as the first step, followed by AEX purification in the second step, as in Preparative Examples 1-3. The long-acting fusion protein, in which the first and second polypeptides were correctly assembled, was confirmed by SDS-PAGE gel (Invitrogen) analysis and SE-HPLC (Tosoh, Cat. No. 08541) analysis. [Experimental Example 4] Measurement results of activity of long-acting fusion protein [Experimental Example 4-1] Measurement results of GDF15 activity of long-acting fusion protein

[0158] The GDF15 activity of the long-acting fusion proteins prepared in Preparation Example 2 above was compared. The GDF15 activity was measured in the same manner as in Experimental Example 1-1 above. The results are shown in Table 15 below and Figure 8. In particular, the GDF15 activity of the long-acting fusion proteins was significantly higher than the in vitro GDF15 activity (EC max Comparisons were made based on a 100% accuracy (see Table 15).

[0159] [Table 15] [Experimental Example 4-2] Measurement results of GLP-1 activity of long-acting fusion protein

[0160] The GLP-1 activity of the long-acting fusion proteins prepared in Preparation Example 2 above was compared. The GLP-1 activity was measured in the same manner as in Experimental Examples 1-2 above. The results are shown in Table 16 below and Figure 9.

[0161] [Table 16]

[0162] As shown in Table 16, when HGK-EKN-L10 was compared with HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-47, and HGK-EKN-L10-49, it was confirmed that the GLP-1 activity was similar regardless of the GDF15 mutant sequence. Similarly, when HGKH-EKN-L4 was compared with HGKH-EKN-L4-23, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49, it was confirmed that the GLP-1 activity was similar regardless of the GDF15 mutant sequence. [Experimental Example 5] Evaluation of the thermal stability of long-acting fusion proteins

[0163] The thermal stability of the long-acting fusion protein prepared in Preparative Example 2 above was assessed using an UNcle apparatus (Unchained Labs).

[0164] Specifically, the protein sample was diluted to 3 mg / mL with Dulbecco's phosphate buffered saline (DPBS, Gibco, USA), and fluorescence and static light scattering analyses were performed using an UNcle instrument. The melting temperature (Tm) and aggregation temperature (Tagg), which are indicators of thermal stability, are shown in Table 17 below.

[0165] [Table 17]

[0166] As shown in Table 17, when HGK-EKN-L10, HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-38, HGK-EKN-L10-47, HGK-EKN-L10-49, and HGK-EKN-L10-484 were compared, it was confirmed that they had similar Tm and Tagg values ​​higher than 60°C regardless of the mutant sequence of GDF15. Similarly, when HGKH-EKN-L4, HGKH-EKN-L4-23, HGKH-EKN-L4-30, HGKH-EKN-L4-38, HGKH-EKN-L4-47, HGKH-EKN-L4-49, and HGKH-EKN-L4-484 were compared, it was confirmed that they had similar Tm and Tagg values ​​higher than 60°C, regardless of the mutant sequence of GDF15. [Experimental Example 6] Pharmacokinetic evaluation results of long-acting fusion protein in mice

[0167] The pharmacokinetics of the long-acting fusion proteins were evaluated using the method described in Experimental Example 3-1 above. After a single subcutaneous administration of each protein prepared in Preparation Example 2 above to mice, the pharmacokinetic parameters of the GDF15-IgG1 Fc portion and GLP1-IgG1 Fc portion of the long-acting fusion proteins were calculated based on the serum concentration of each active ingredient over time (see Figures 10 and 11), and are shown in Tables 18 and 19 below. The pharmacokinetic profiles of each long-acting fusion protein were compared and evaluated based on the area under the curve (AUC), which indicates the degree of drug exposure.

[0168] [Table 18]

[0169] As shown in Table 18, it was confirmed that the other long-acting fusion proteins, except for HGK-EKN-L10-L484, exhibited AUC values ​​similar to that of the GDF15 moiety within a maximum of about 1.4-fold. In the case of HGK-EKN-L10-484, the AUC value of the GDF15 moiety was reduced by about 2-fold.

[0170] [Table 19]

[0171] As shown in Table 19, it was confirmed that HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-38, HGK-EKN-L10-47, and HGK-EKN-L10-49 showed an increase in the AUC value of the GLP-1 moiety of approximately 3.7 to 4.9 times compared to HGK-EKN-L10-484.

[0172] Furthermore, the AUC of the GLP-1 moiety was increased by about 2.0- to about 2.5-fold in the protein in which the GLP-1 variant was fused to both the IgG1 hFc_knob and the IgG1 hFc_hole compared to that in the protein in which the GLP-1 variant was fused to only the IgG1 hFc_knob, as determined by comparing HGK-EKN-L10-23 with HGKH-EKN-L4-23, HGK-EKN-L10-30 with HGKH-EKN-L4-30, HGK-EKN-L10-47 with HGKH-EKN-L4-47, and HGK-EKN-L10-49 with HGKH-EKN-L4-49, respectively.

[0173] The ratios of AUC of the GLP-1 portion to AUC of the GDF15 portion for the long-acting fusion proteins prepared in Preparation Example 2 above are shown in Table 20. A higher ratio of AUC of the GLP-1 portion to AUC of the GDF15 portion indicates a more balanced pharmacokinetic profile of the GLP-1 variant and the GDF15 variant.

[0174] [Table 20]

[0175] As shown in Table 20, proteins in which GLP-1 variants were fused to IgG1 hFc_knob (HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-38, HGK-EKN-L10-47, HGK-EKN-L10-49, and HGK-EKN-L10-484) were shown to have GLP-1 AUC / GDF15 AUC ratios of 0.18 to 0.46. On the other hand, proteins in which GLP-1 mutants were fused to IgG1 hFc_knob and IgG1 hFc_hole (HGKH-EKN-L4-23, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49) were shown to have GLP-1 AUC / GDF15 AUC ratios of 0.66 to 0.853. In other words, proteins in which GLP-1 mutants were fused to IgG1 hFc_knob and IgG1 hFc_hole were confirmed to have pharmacokinetic profiles comparable to those of GLP-1 and GDF15 mutants.

[0176] A comprehensive review of the above experimental examples 1-6 reveals that even when the activity of the GDF15 variant is reduced, it does not affect the stability of the long-acting fusion protein and has a pharmacokinetic profile that is equilibrated with that of the GLP-1 variant; therefore, altering the activity of the GDF15 variant is not expected to significantly affect the stability and pharmacokinetic profile of the long-acting fusion protein. [Experimental Example 7] Evaluation of the efficacy of long-acting fusion protein in diet-induced obese (DIO) mice

[0177] DIO mice are C57BL / 6N mice fed a 60% high-fat diet (Research Diets, Cat. No. D12492), and these mice exhibit obesity, hyperglycemia, and insulin resistance.

[0178] DIO mice (Taconic, USA) were purchased from Raonbio (Korea) and fed a 60% high-fat diet for 8 weeks. The purchased DIO mice were further fed a 60% high-fat diet for 5 weeks and used in this study. [Experimental Example 7-1] Effect of weight loss in DIO mice (1)

[0179] The DIO mice were divided into groups (n=6 per group) with similar body weights on the day before the start of administration. The long-acting fusion protein prepared in Preparation Example 1 above was then subcutaneously administered every 3 days for a total of 4 weeks at a dose of 1 nmol / kg, 3 nmol / kg, or 10 nmol / kg, and Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as a vehicle. Body weight was measured daily or every 3 days from the start of administration until day 26, and the results are shown in Table 21 below and Figure 12.

[0180] As shown in Table 21 and FIG. 12, it was confirmed that GK-L3 and GH-L5 exhibited a dose-dependent effect of reducing body weight.

[0181] [Table 21] [Experimental Example 7-2] Effect of weight loss in DIO mice (2)

[0182] The day before the start of administration, DIO mice were divided into groups (n=6 per group) with similar average body weights. The long-acting fusion proteins (GK-L3-3, GH-L3, GH-L4, GKH-L2, and GKH-L4-L2) prepared in Preparation Example 1 above were then subcutaneously administered at a dose of 3 nmol / kg every 3 days for a total of 4 weeks, with Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) administered as a vehicle. Body weight was measured daily or every 3 days from the start of administration until day 26, and the results are shown in Table 22 and Figure 13 below.

[0183] As shown in Table 22 and FIG. 13, it was confirmed that GK-L3-3, GH-L3, GH-L4, GKH-L2, and GKH-L4-L2 exhibited superior effects on weight loss compared to the control.

[0184] [Table 22] [Experimental Example 7-3] Effect of weight loss in DIO mice (3)

[0185] The day before the start of administration, DIO mice were divided into groups (n=6 per group) with similar average body weights. The long-acting fusion proteins prepared in Preparation Examples 1 and 2 above were then subcutaneously administered every 3 days for a total of 4 weeks at doses of 1 nmol / kg, 0.3 nmol / kg, or 0.1 nmol / kg, with Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) administered as a vehicle. Body weight was measured daily or every 3 days from the start of administration until the 26th or 30th day.

[0186] As shown in Table 23 and FIG. 14, it was confirmed that HGH-EKN-L4 and HGKH-EKN-L4 exhibited a dose-dependent effect of reducing body weight.

[0187] [Table 23]

[0188] Furthermore, as shown in Table 24 below and Figure 15, it was confirmed that HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGKH-EKN-L4, and HGKH-EKN-L4-49 exhibited a dose-dependent effect of reducing body weight.

[0189] [Table 24] [Experimental Example 7-4] Hypoglycemic effect in DIO mice

[0190] The day before the start of administration, the mice were divided into groups (n=6 per group) with similar non-fasting blood glucose levels and body weights. Then, the six types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) prepared in Preparation Examples 1 and 2 above were subcutaneously administered at a dose of 1 nmol / kg every 3 days for a total of 4 weeks, and Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was administered as a vehicle.

[0191] Non-fasting blood glucose levels were measured on days 9 and 21 from the start of administration, and fasting blood glucose levels were measured at the end of the study. Blood glucose concentrations were measured using a GlucoDr blood glucose meter (Allmedicus, Korea), and the blood glucose measurement results are shown in Figures 16 and 17.

[0192] Figure 16 shows that non-fasting blood glucose levels were measured on days 9 and 21 after the start of administration, confirming that all six long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) maintained lower glucose levels than the vehicle-treated group at a dose of 1 nmol / kg. Figure 17 shows that all six long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) showed excellent effects in improving blood glucose levels at a dose of 1 nmol / kg. [Experimental Example 7-5] Effect of weight loss in DIO mice (4)

[0193] The day before the start of administration, the mice were divided into groups (n=6 per group) with similar non-fasting blood glucose levels and body weights. Then, the six types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) prepared in Preparation Examples 1 and 2 above were subcutaneously administered at a dose of 1 nmol / kg every 3 days for a total of 4 weeks, and Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was administered as a vehicle.

[0194] Body weight and cumulative food intake were measured daily or every three days from the start of dosing until day 24 or 27. Cumulative food intake is shown in Figure 18, and the effects of weight loss are shown in Table 25 and Figure 19.

[0195] [Table 25]

[0196] The cumulative food intake of all six long-acting fusion proteins was reduced compared to the vehicle-treated group (see Figure 18), demonstrating their superior effect on weight loss compared to the vehicle-treated group (see Figure 19). Furthermore, it was confirmed that all six long-acting fusion proteins showed continuous weight loss from the time of administration until day 27 (see Figure 19). [Experimental Example 7-6] Effect of weight loss in DIO mice (5)

[0197] The day before the start of administration, the mice were divided into groups (n=6 per group) with similar mean body weights, and nine types of long-acting fusion proteins (HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGK-EKN-L10-47, HGK-EKN-L10-49, HGKH-EKN-L4, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49) prepared in Preparation Examples 1 and 2 above were subcutaneously administered at a dose of 1 nmol / kg every 3 days for a total of 4 weeks, and Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as a vehicle.

[0198] Body weight was measured daily or every three days from the start of dosing until day 26. The effects of weight loss are shown in Table 26 and Figure 20.

[0199] [Table 26]

[0200] As shown in Table 26 above, it was confirmed that the nine long-acting fusion proteins exhibited excellent weight loss effects ranging from approximately 18% to approximately 30%. Furthermore, it was confirmed that all nine long-acting fusion proteins exhibited continuous weight loss up to day 26 (see Figure 20). [Experimental Example 7-7] Effect of weight loss in DIO mice (6)

[0201] The day before the start of administration, the mice were divided into groups (n=6 per group) with similar mean body weights, and the long-acting fusion protein HGKH-EKN-L4 prepared in Preparation Examples 1 and 2 above was subcutaneously administered every three days for a total of four weeks at doses of 0.3 nmol / kg, 1 nmol / kg, 3 nmol / kg, 10 nmol / kg, or 30 nmol / kg. Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as a vehicle. Cumulative food intake and body weight were measured daily or every three days from the start of administration until day 27. Cumulative food intake is shown in Figure 21, and the effects of weight loss are shown in Table 27 and Figure 22.

[0202] [Table 27]

[0203] As a result, it was confirmed that HGKH-EKN-L4 reduced cumulative food intake and body weight in a dose-dependent manner. [Experimental Example 8] Evaluation of the efficacy of long-acting fusion proteins in ob / ob mice

[0204] Ob / ob mice, in which excessive obesity is induced by hyperphagia due to a mutation in the ob gene responsible for leptin production, are characterized by hyperglycemia and insulin resistance. Five- to seven-week-old male ob / ob mice (Jackson Laboratory, USA) were purchased from Raonbio (Korea) and allowed to acclimate for 4 weeks before initiating treatment. [Experimental Example 8-1] Evaluation of efficacy of single administration in ob / ob mice

[0205] The day before the start of administration, mice were divided into groups (n = 5 per group) with similar mean non-fasting blood glucose levels and body weights. HGK-EKN-L10 was administered subcutaneously once at doses of 0.1 nmol / kg, 0.3 nmol / kg, 1 nmol / kg, 3 nmol / kg, or 10 nmol / kg. Specifically, Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as a vehicle, and blood glucose levels were measured using a GlucoDr blood glucose meter (Allmedicus, Korea). Body weight and non-fasting blood glucose levels were measured daily from the start of administration until day 14.

[0206] The results of body weight measurements are shown in Table 28 below. The dose-dependence of HGK-EKN-L10 in the effect of weight loss was confirmed, and it was confirmed that weight loss continued to occur for approximately 14 days after a single administration of HGK-EKN-L10 at a dose of 1 nmol / kg or more (see Figure 23). Furthermore, HGK-EKN-L10 showed the highest effect at a dose of 3 nmol / kg or more.

[0207] [Table 28]

[0208] The results of the non-fasting blood glucose level measurements are shown in Figure 24. HGK-EKN-L10 was confirmed to dose-dependently lower blood glucose levels, and blood glucose levels were maintained at low levels immediately after administration. Furthermore, the blood glucose lowering effect was confirmed to be maintained for approximately 14 days after a single administration of HGK-EKN-L10 at doses of 1 nmol / kg or higher. [Experimental Example 8-2] Evaluation of the effects of repeated drug administration in ob / ob mice

[0209] The day before the start of treatment, mice were divided into groups (n = 6 per group) with similar non-fasting blood glucose levels and mean body weights. HGK-EKN-L10 and HGKH-EKN-L4 were subcutaneously administered at doses of 0.3 nmol / kg, 1 nmol / kg, or 3 nmol / kg every three days for a total of eight doses. Specifically, Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA) was administered as vehicle, and body weight was measured daily or every three days from the start of treatment until day 24. Upon completion of the study, glycated hemoglobin (HbAlc) levels were quantified using a DCA2000 HbAlc kit (Siemens, 5035C), and lipid, metabolic, and liver injury indices were analyzed.

[0210] It was demonstrated that after repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 every 3 days for 8 times, a dose-dependent effect on body weight reduction was observed (Table 29 and Figure 25).

[0211] [Table 29]

[0212] At the completion of the study, measurements of glycated hemoglobin levels, which reflect average blood glucose levels, confirmed that HGK-EKN-L10 and HGKH-EKN-L4 had reduced glycated hemoglobin levels compared to the vehicle-treated group at all doses (see Figure 26).

[0213] At the completion of the study, lipid and metabolic parameters were measured by serum chemistry analysis, and HGK-EKN-L10 and HGKH-EKN-L4 showed improved effects on total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels compared to the vehicle-treated group (see Figure 27).

[0214] Furthermore, serum liver damage indicators confirmed that all doses of the long-acting fusion protein showed statistically significant decreases in alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels compared to the vehicle-treated group (see Figures 28a-28c). Furthermore, when evaluating relative liver weight (%), taking body weight into account, statistically significant decreases in relative liver weight were confirmed in all doses of HGK-EKN-L10 and HGKH-EKN-L4-treated groups compared to the vehicle-treated group (see Figure 28d). [Experimental Example 9] Evaluation of the efficacy of administration of long-acting fusion protein in db / db mice

[0215] The db / db mouse is a model of type 2 diabetes and obesity characterized by hyperglycemia, insulin resistance, and hyperphagia due to a genetic defect in the leptin receptor gene. Five- to six-week-old male db / db mice (Janvier, France) were purchased from Raonbio (Korea) and allowed to acclimate for 4 weeks before initiating treatment.

[0216] The day before the start of treatment, mice were divided into groups (n = 8 per group) with similar body weights, non-fasting blood glucose levels, and glycated hemoglobin levels. HGKH-EKN-L4 was then administered subcutaneously every three days at doses of 0.3 nmol / kg, 1 nmol / kg, 3 nmol / kg, or 10 nmol / kg for a total of eight doses. Specifically, vehicle treatment was performed using Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA), and blood glucose levels were measured using an Accu-Check instant blood glucose meter (Roche, Switzerland). Non-fasting blood glucose levels were measured once daily until day 27 after the start of treatment, and glycated hemoglobin levels were measured on days 14 and 26 after the start of treatment. Furthermore, to evaluate the improvement of insulin resistance, mice were fasted for 6 hours starting the morning of day 3 after the last treatment. The insulin administration level for each mouse was determined based on its fasting body weight. Mice were given an intraperitoneal injection of insulin (1 U / kg, Humulin R, Eli Lilly). Blood samples were collected serially from the initial tail cut and measured before insulin injection (0) and at 15, 30, 60, 90, and 120 minutes, respectively.

[0217] As a result of repeated administration of HGKH-EKN-L4 eight times every three days, non-fasting blood glucose levels were maintained at low levels at all doses compared to the vehicle-treated group, confirming that HGKH-EKN-L4 exhibited a dose-dependent effect of reducing blood glucose levels (see Figure 29). Specifically, it was confirmed that the group administered HGKH-EKN-L4 at a dose of 10 nmol / kg maintained blood glucose levels at normal control levels until the end of the study.

[0218] The results of measuring glycated hemoglobin levels on days 14 and 26 after the start of administration are shown in Table 30 below and Figure 30. Before the start of administration, the glycated hemoglobin levels of the vehicle-treated group and the HGKH-EKN-L4-administered group were similar, but after administration, the glycated hemoglobin levels improved compared to the vehicle-treated group at all doses, confirming that the glycated hemoglobin levels decreased in a dose-dependent manner with HGKH-EKN-L4.

[0219] [Table 30]

[0220] The results of the insulin tolerance test (ITT) performed after the final administration are shown in Figure 31. The results of the insulin tolerance test confirmed that the groups receiving HGKH-EKN-L4 at doses of 1 nmol / kg, 3 nmol / kg, and 10 nmol / kg showed a dose-dependent decrease in blood glucose levels after insulin administration, which was similar to the improvement in insulin sensitivity of the normal control group (see Figure 31a). Analysis based on AUC glucose of the vehicle-treated group confirmed that the AUC glucose values ​​were significantly decreased in the groups receiving HGKH-EKN-L4 at doses of 1 nmol / kg, 3 nmol / kg, and 10 nmol / kg (see Figure 31b). [Experimental Example 10] Evaluation of the efficacy of long-acting fusion protein in Gubra Amylin NASH (GAN) ob / ob mice [Experimental Example 10-1] GAN-ob / ob nonalcoholic steatohepatitis (NASH) mouse model

[0221] Animal models of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) can be divided into four types: diet-induced models, chemically induced models, genetically modified models, and combined models. Among these, the GAN-ob / ob model, a double combined model in which ob / ob mice, a mutant model of the ob gene, are fed a GAN diet, is a relevant model that resembles the human NASH phenotype.

[0222] Male ob / ob mice (Jackson Lab, USA) aged 5-7 weeks were purchased from Raonbio (Korea). The ob / ob mice were fed a GAN diet (Research Diets, Catalog No. D09100310; 40 kcal% fat, 22 kcal% fructose, 10% sucrose, and 2% cholesterol) for 12 weeks, after which drug treatment began. The day before drug treatment, mice were randomized into groups (n = 8 per group) based on body weight and AST, ALT, triglyceride (TG), and total cholesterol levels.

[0223] Mice were then subcutaneously administered HGKH-EKN-L4 at doses of 3 nmol / kg, 10 nmol / kg, and 30 nmol / kg every 3 days for a total of 8 weeks. Specifically, vehicle treatment was performed using Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA). The test substance was administered for 8 weeks during the total 20-week GAN dietary induction period. [Experimental Example 10-2] Improvement of body weight, liver weight, and liver damage-related blood indices in the GAN-ob / ob NASH mouse model

[0224] Repeated administration of HGKH-EKN-L4 for 8 weeks resulted in a weight loss effect of approximately -12.36% in the HGKH-EKN-L4 (3 nmol / kg)-treated group, approximately -18.14% in the HGKH-EKN-L4 (10 nmol / kg)-treated group, and approximately -36.51% in the HGKH-EKN-L4 (30 nmol / kg)-treated group compared to the GAN-ob / ob control group (GAN-ob / ob vehicle) (4.79%) (see Figure 32a). Furthermore, when evaluating relative liver weight (%) taking into account fasting body weight, a statistically significant decrease in relative liver weight was confirmed in the HGKH-EKN-L4-treated group (see Figure 32b).

[0225] When liver damage indicators in the blood were observed, the values ​​of ALT, AST, and ALP were significantly increased in the GAN-ob / ob NASH control group (GAN-ob / ob vehicle) compared with the normal control group (Chow vehicle). Administration of HGKH-EKN-L4 demonstrated a dose-dependent improvement in liver damage indicators, as verified by the statistically significant decrease in ALT, AST, and ALP values ​​observed in the HGKH-EKN-L4-treated group (see Figure 33). [Experimental Example 10-3] Histological improvement effect in GAN-ob / ob NASH mouse model

[0226] Picrosirius red staining (PSR) analysis is an analytical method for staining collagen deposition in liver tissue. Taking into account the liver weight of each individual, the total PSR content (mg) is shown in Figure 34. A significant increase in PSR was observed in the GAN-ob / ob control group (GAN-ob / ob vehicle) compared to the normal control group (Chow vehicle). The total PSR content was reduced by HGKH-EKN-L4 administration, indicating that the progression of liver fibrosis was inhibited by HGKH-EKN-L4 administration. Statistical significance was observed in the groups administered HGKH-EKN-L4 at doses of 3 nmol / kg, 10 nmol / kg, and 30 nmol / kg (see Figure 34).

[0227] In conclusion, when HGKH-EKN-L4 was administered to the GAN-ob / ob NASH mouse model, it not only reduced body weight but also inhibited the progression of liver injury and liver fibrosis, indicating an improvement in nonalcoholic steatohepatitis.

Claims

1. A fusion protein or a dimer thereof comprising a polypeptide represented by the following formula (I) and a polypeptide represented by the following formula (II): A ｐ -B ｑ -C(I) F ａ -G ｂ -H-I-J(II) [In the formula: A and F are each independently a biologically active protein; B, G, and I are each independently a linker; C and H are each an immunoglobulin Fc or a variant thereof; J is (Y １ Y ２ ) ｔ -N-(Y ３ ) ｕ -(Y ４ ) ｖ -(Y ５ ) ｗ -CPLGPGRCCRLHTV-Y ６ -ASLEDLGWAD-Y ７ -VLSPREVQVTMCIGACPSQFRAA-Y ８ -MHA-Y ９ -IKT-Y １０ -LHRLKPDTVPAPCCVPASYNPMVLI-Y １１ -KTDTGVSLQTYD-Y １２ -LLAKDCHCI, During the ceremony: Y １ , Y ３ , and Y ７ ~Y ９ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); Y ２ , Y ６ , and Y １１ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W), or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); Y ４ and Y １２ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid that is aspartic acid (D) or glutamic acid (E); Y ５ is a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); Y １０ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid that is aspartic acid (D) or glutamic acid (E); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); t, u, v, and w are each independently 0 or 1; p and q in the above formula (I) and a and b in the above formula (II) are each independently 0 or 1, and when p is 0, q is 0, and when p is 1, q is 1; provided that p and q in formula (I) and a and b in formula (II) are not simultaneously 0, when a is 0, b is also 0, and when a is 1, b is 1.

2. 2. The fusion protein or dimer thereof according to claim 1, wherein the biologically active protein is 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 (RBP4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, growth hormone, and variants thereof.

3. The fusion protein or dimer thereof according to claim 2, wherein the biologically active protein is selected from the group consisting of GLP-1, exendin-4, or a variant thereof.

4. The biologically active protein is H-X １ -EGTFTSDVSSYLE-X ２ -QAKEFI-X ３ -WL-X ４ -X ５ -G-X ６ -G, exenatide, liraglutide, dulaglutide, albiglutide, lixisenatide, semaglutide, tirzepatide, cotadutide, and taspoglutide, wherein X １ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); X ２ and X ３ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid that is aspartic acid (D) or glutamic acid (E); X ４ , X ５ and X ６ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), glutamine (Q), asparagine (N), and tryptophan (W); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R).

5. X １ is alanine (A) or glycine (G), and / or X ２ is glycine (G) or glutamic acid (E), and / or X ３ is alanine (A) or glutamic acid (E), and / or X ４ is valine (V) or lysine (K), and / or X ５ is lysine (K) or asparagine (N), and / or X ６ The fusion protein or a dimer thereof according to claim 4, wherein is arginine (R) or glycine (G).

6. X １ is glycine (G), and X ２ is glutamic acid (E), and X ３ The fusion protein or a dimer thereof according to claim 5, wherein is glycine (G).

7. X ３ The fusion protein or dimer thereof according to claim 5, wherein is alanine (A).

8. X ３ is glutamic acid (E), and X ４ The fusion protein or dimer thereof according to claim 5, wherein is valine (V).

9. X ３ is glutamic acid (E), and X ４ is lysine (K) and X ５ The fusion protein or dimer thereof according to claim 5, wherein is asparagine (N).

10. 2. The fusion protein or a dimer thereof according to claim 1, wherein A and F are each independently selected from the group consisting of the amino acid sequences of SEQ ID NOs: 9 to 12.

11. t is 1, and Y １ is alanine (A) or tryptophan (W), and Y ２ The fusion protein or a dimer thereof according to claim 1, wherein is arginine (R) or serine (S).

12. t is 0, and Y ３ is glycine (G) or serine (S), and / or Y ４ is aspartic acid (D) or threonine (T), and / or Y ５ is histidine (H), and / or Y ６ is arginine (R) or asparagine (N), and / or Y ７ is tryptophan (W) or phenylalanine (F), and / or Y ８ is asparagine (N), leucine (L), cysteine ​​(C), or serine (S), and / or Y ９ is glutamine (Q) or asparagine (N), and / or Y １０ is serine (S), asparagine (N), aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), or leucine (L); Y １１ is glutamine (Q) or histidine (H), and / or Y １２ is aspartic acid (D), leucine (L), cysteine ​​(C), or serine (S), or a dimer thereof according to claim 1 .

13. Y ３ is glycine (G), and Y ４ is aspartic acid (D), and Y ５ The fusion protein or dimer thereof according to claim 12, wherein is histidine (H).

14. w is 0 and Y ３ is serine (S), and Y ４ The fusion protein or dimer thereof according to claim 12, wherein is threonine (T).

15. The fusion protein or a dimer thereof according to claim 1, wherein J is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2 to 8 and 93 to 108.

16. B, G and I are each independently: 1) A peptide consisting of 5 to 52 glycines (G) and / or serine (S); 2) Peptides consisting of 4 to 52 glutamic acids (E) and / or alanines (A); 3) peptides consisting of 5 to 52 glutamic acid (E), lysine (K), and / or alanine (A); 4) peptides consisting of 6 to 52 glycines (G), serine (S), glutamic acid (E), and / or alanines (A); and 5) Peptides consisting of 7 to 52 glycine (G), serine (S), glutamic acid (E), lysine (K), and / or alanine (A) The fusion protein or dimer thereof according to claim 1, wherein the linker is selected from the group consisting of:

17. B, G, and I are each independently ４ S) ｎ , GS (G ４ S) ｎ , GS (EEEA) ｎ , (EEEA) ｎ , GS (EAAAK) ｎ , and (EAAAK) ｎ The fusion protein or a dimer thereof according to claim 16, wherein n is an integer of 1 to 10.

18. 2. The fusion protein or a dimer thereof according to claim 1, wherein B, G, and I are each independently selected from the group consisting of the amino acid sequences of SEQ ID NOs: 13 to 24.

19. The fusion protein or dimer thereof according to claim 1 , wherein the immunoglobulin Fc or variant thereof comprises a processed protuberance or a processed cavity.

20. The fusion protein or dimer thereof according to claim 1, wherein the immunoglobulin Fc or variant thereof is a hybrid Fc containing any one of the Fc regions of IgG1, IgG2, IgG3, IgG4, and IgD, or a combination thereof.

21. The fusion protein or dimer thereof according to claim 19, wherein the human IgG1 Fc fragment variant containing a processed protuberance has the 146th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 80 substituted with tryptophan (W).

22. The fusion protein or dimer thereof according to claim 19, wherein the human IgG1 Fc fragment variant containing the processed cavity is one in which the 146th, 148th, and 187th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 80 are substituted with serine (S), alanine (A), and valine (V), respectively.

23. The fusion protein or dimer thereof according to claim 1, wherein the immunoglobulin Fc variant has reduced or eliminated Fc effector functions.

24. The fusion protein or dimer thereof according to claim 23, wherein the immunoglobulin Fc variant is one in which the 14th, 15th, and 77th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 80 are each substituted with alanine (A).

25. The fusion protein or a dimer thereof according to claim 1, wherein the immunoglobulin Fc or a variant thereof is a polypeptide comprising an amino acid sequence of SEQ ID NO: 81 to 86 and a hinge added to the N-terminus of the polypeptide.

26. The fusion protein or a dimer thereof according to claim 20, wherein the immunoglobulin Fc or variant thereof is a polypeptide comprising a hinge consisting of one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 79 and 109 to 111.

27. The fusion protein or dimer thereof according to claim 1, wherein the immunoglobulin Fc or variant thereof is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 85 or 86, or a polypeptide in which the amino acid sequence of SEQ ID NO: 79 is added to the N-terminus of a polypeptide having the amino acid sequence of SEQ ID NO: 85 or 86.

28. The fusion protein or a dimer thereof according to claim 1, wherein the immunoglobulin Fc or variant thereof is a polypeptide consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 81 to 92.

29. The fusion protein: (1) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (2) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (3) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (4) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 30 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (5) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (6) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 32 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (7) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (8) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (9) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (10) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (11) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 37 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (12) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 38 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (13) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 39 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (14) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked; (15) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked; (16) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (17) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (18) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (19) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 68 are linked; (20) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 69 are linked; (21) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 70 are linked; (22) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 67 are linked; (23) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (24) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (25) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (26) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked; (27) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (28) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (29) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (30) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 41 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked; (31) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked; (32) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked; (33) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 44 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (34) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (35) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (36) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (37) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (38) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (39) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (40) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked; (41) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked; (42) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked; (43) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (44) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked; (45) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked; (46) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 71 are linked; (47) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked; (48) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked; (49) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked; (50) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (51) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked; (52) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked; (53) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 51 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (54) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 52 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (55) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 53 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (56) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 54 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (57) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 55 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (58) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 56 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked; (59) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 57 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (60) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 58 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (61) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 59 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (62) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 60 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; (63) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 61 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; and (64) A protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 62 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked. The fusion protein or a dimer thereof according to claim 1,

30. An isolated cell line producing the fusion protein or a dimer thereof according to any one of claims 1 to 29.

31. An isolated nucleic acid encoding the fusion protein or a dimer thereof according to any one of claims 1 to 29.

32. 32. A recombinant expression vector comprising the nucleic acid of claim 31.

33. A host cell comprising the vector of claim 32.

34. Culturing a cell line expressing the fusion protein under conditions in which the fusion protein of any one of claims 1 to 29 is expressed; and Recovering the fusion protein A method for producing a fusion protein or a dimer thereof, comprising:

35. A composition comprising a pharmaceutically acceptable carrier and the fusion protein or dimer thereof according to any one of claims 1 to 29.

36. 30. A method for treating or preventing a metabolic-related disease in an individual in need thereof, comprising administering to the individual the fusion protein or dimer thereof according to any one of claims 1 to 29.

37. 37. The method of claim 36, wherein the metabolic-related disease is diabetes, obesity, hypercholesterolemia, hyperglycemia, insulin resistance, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), liver damage, hepatitis, liver fibrosis, cirrhosis, fatty liver, arteriosclerosis, dyslipidemia, cardiovascular disease, or metabolic syndrome.

38. A pharmaceutical composition for treating or preventing a metabolic disease, comprising a pharmaceutically acceptable carrier and the fusion protein or dimer thereof according to any one of claims 1 to 29.

39. 30. Use of the fusion protein or dimer thereof according to any one of claims 1 to 29 in the preparation of a medicament for the treatment of a metabolic-related disease.

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