Novel bispecific fusion protein and its uses

Abstract

The present invention relates to a novel bispecific fusion protein and its use, more specifically, to a novel bispecific fusion protein comprising a GLP-1 analog and a GLP-2 analog, which has improved effects on metabolic diseases such as obesity, diabetes, and metabolic disorder-related steatohepatitis by appropriately regulating the activities of GLP-1 and GLP-2.

Description

[Technical Field]

[0001] The present invention relates to a novel bispecific fusion protein and uses thereof, and more particularly to a bispecific fusion protein containing a GLP-1 analog and a GLP-2 analog, in which the activity of the GLP-1 analog and the activity of the GLP-2 analog are precisely regulated, and uses thereof. [Background technology]

[0002] GLP-1 and GLP-2 are peptide hormones that are expressed from a precursor called proglucagon and undergo a series of tissue-specific proteolytic cleavage processes. GLP-1 is produced and secreted by enteroendocrine L-cells in the small intestine and specific neurons in the solitary tract nucleus of the brainstem after food ingestion. The initial product, GLP-1(1-37), is easily amidated and cleaved into two equally biologically active cleaved forms (GLP-1(7-36)amide and GLP-1(7-37)). Active GLP-1 lowers blood glucose levels in a glucose-dependent manner and has been developed as a therapeutic agent for type 2 diabetes. Similarly to GLP-1, GLP-2 is a 33-amino acid peptide produced by a special post-translational cleavage process of proglucagon. It is produced by enteroendocrine L cells in the small intestine and various neurons in the central nervous system and is secreted together with GLP-1 in the small intestine upon ingestion of food. GLP-2 is known to improve the growth and function of the small intestine, reduce bone destruction, and exert neuroprotective effects upon administration, and is currently being developed as a treatment for diseases such as short bowel syndrome, Crohn's disease, and osteoporosis.

[0003] As previously mentioned, GLP-1 first attracted attention for its beneficial effects on glucose homeostasis in the treatment of type 2 diabetes patients (Gutniak et al., N. Engl. J. Med. 326:1316-1322, 1992). Similar to GLP-2, GLP-1 also reduces gastrointestinal secretion and motility, suggesting its potential role in the treatment of short bowel syndrome (Kunkel et al., Neurogastroenterol. Motil. 23:739-e328, 2011). Furthermore, activation of the GLP-1 receptor (GLP-1R) signaling pathway by GLP-1 promotes intestinal growth and crypt cell fusion (Koehler et al., Cell Metabol. 21(3):379-391, 2015). Based on this idea, Naia is currently conducting a Phase 1 clinical trial for short bowel syndrome using GLP-1-XTEN, which is GLP-1 linked to the half-life extending peptide XTEN. Furthermore, it has been reported that the combined administration of GLP-1 and GLP-2 showed an additive effect on small intestinal absorption compared to the administration of each peptide alone (Madsen et al., Regul. Pept. 184:30-39, 2013).

[0004] Metabolic dysfunction-associated fatty liver disease (MASLD) is not a single disease, but rather encompasses a variety of liver diseases, ranging from simple fatty liver without inflammation to chronic hepatitis and cirrhosis. It is known to be closely related to obesity and the resulting insulin resistance. Metabolic dysfunction-associated fatty liver disease (MASLD) ranges in severity from mild fatty liver with no hepatocellular damage, characterized by simple fatty liver with no hepatocellular damage, to persistent steatohepatitis with severe hepatocellular damage, and even liver fibrosis or cirrhosis accompanied by ascites and jaundice. Furthermore, MASLD is known to progress to liver cancer. Furthermore, MASLD is a Western-style disease whose incidence is rapidly increasing due to the spread of Western dietary habits. While most cases of metabolic dysfunction-associated fatty liver disease are mild, one in four patients with severe fatty liver will gradually progress to cirrhosis (or hepatic fibrosis), a serious liver disease, over time if left untreated. Therefore, MASLD is not a condition that can be ignored. The prevalence of metabolic disorder-related fatty liver disease varies depending on the characteristics of the population, but is reported to be 10-24% in the general population and 58-74% in obese people. However, to date, no drugs have been approved as treatments for metabolic disorder-related fatty liver disease or metabolic disorder-related steatohepatitis, and the development of a treatment for metabolic disorder-related fatty liver disease is considered an extremely urgent issue.

[0005] In this regard, the present inventors have developed a bispecific protein that simultaneously targets GLP-1R and GLP-2R (Korean Patent No. 10-2349718). Summary of the Invention [Problem to be solved by the invention]

[0006] However, although the bispecific fusion protein is widely used for various metabolic diseases such as short bowel syndrome, obesity, type 2 diabetes, and metabolic disorder-related steatohepatitis, there is no disclosure of a form of bispecific fusion protein that is more suitable for metabolic diseases.

[0007] Therefore, the present invention aims to solve various problems, including those mentioned above, and aims to provide a novel bispecific fusion protein that retains the functions of GLP-1 and GLP-2 and has activity more suitable for the treatment of metabolic diseases, but the scope of protection of the present invention is not limited to the above-mentioned object. [Means for solving the problem]

[0008] According to one aspect of the present invention, there is provided a bispecific fusion protein in which a GLP-1 analog and a GLP-2 analog are fused, wherein the activity of the GLP-1 analog is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analog is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).

[0009] Another aspect of the present invention provides a bispecific fusion protein comprising a first fusion protein in which a GLP-1 analog is linked to an antibody Fc region and a second fusion protein in which a GLP-2 analog is linked to an antibody Fc region, the bispecific fusion protein being produced by dimerization of the first and second fusion proteins, wherein the activity of the GLP-1 analog is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analog is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).

[0010] According to one aspect of the present invention, there is provided a composition comprising the bispecific fusion protein.

[0011] Another aspect of the present invention provides a pharmaceutical composition for treating metabolic diseases, comprising any one or more of the above bispecific fusion proteins as an active ingredient.

[0012] Another aspect of the present invention provides any one of the above bispecific fusion proteins for use in treating a metabolic disease.

[0013] According to another aspect of the present invention, there is provided a use of any one of the above bispecific fusion proteins in the manufacture of a therapeutic agent for a metabolic disease.

[0014] Another aspect of the present invention provides a method for treating a metabolic-related disorder in an individual, comprising administering to said individual a therapeutically effective amount of the bispecific fusion protein. [Effects of the Invention]

[0015] The bispecific fusion proteins of the present invention, when administered in vivo, can be used very effectively to treat metabolic diseases such as metabolic syndrome, obesity, type 1 diabetes, type 2 diabetes, metabolic disorder-related fatty liver disease, and liver fibrosis. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram showing the general structure of a bispecific fusion protein according to one embodiment of the present invention.

[0017] [Figure 2A] 1 is a series of gel photographs showing the results of SDS-PAGE analysis under non-reducing (NR) and reducing (R) conditions after purification of a GLP-2 homodimer (GLP-2-Fc homodimer, left) and MG12-5 containing a Knobs-into-Holes structure (right), which are comparative examples of the present invention:

[0018] M:size marker;

[0019] 1&2: GLP-2-Fc homo (10 μg); and

[0020] 3&4:MG12-5(10μg).

[0021] [Figure 2B] 1 is a series of gel photographs showing the results of SDS-PAGE analysis of MG12-6 according to one embodiment of the present invention under reducing and non-reducing conditions:

[0022] M:size marker;

[0023] 1:MG12-6; and

[0024] C: Ctrl (3 μg)-Herceptin.

[0025] [Figure 2C] 1 is a series of gel photographs showing the results of SDS-PAGE analysis of MG12-7 and MG12-8 under reducing and non-reducing conditions according to one embodiment of the present invention:

[0026] M:size marker;

[0027] 1:MG12-7;

[0028] 2:MG12-8; and

[0029] 3: Ctrl (3 μg)-Herceptin.

[0030] [Figure 2D] 1 is a gel photograph showing the results of SDS-PAGE analysis of MG12-9 according to an embodiment of the present invention under reducing and non-reducing conditions.

[0031] 1:Reducing MG12-9;

[0032] 2: Non-reducing MG12-9; and

[0033] 3: Ctrl (3 μg)-BSA.

[0034] [Figure 3A] FIG. 1 is a schematic diagram showing an experimental schedule, drug dosage, administration interval, etc. for analyzing the weight loss effect upon single administration of a bispecific fusion protein (MG12-8) according to one embodiment of the present invention. [Figure 3B]3B is a graph showing changes in body weight of experimental animals as a result of an experiment carried out according to the schedule of FIG. 3A. [Figure 3C] FIG. 1 is a schematic diagram illustrating an experimental schedule, drug dosage, and administration interval for analyzing the weight loss effect upon repeated administration of a bispecific fusion protein (MG12-8) according to one embodiment of the present invention. [Figure 3D] 3C. FIG. 3C is a graph showing the weight change of the experimental animals as a result of the experiment carried out according to the schedule shown in FIG. 3C. FIG. [Figure 3E] 3D shows the results of measuring the gallbladder volume of the experimental animals sacrificed after the experiment in FIG. NS indicates no statistical significance, ** indicates statistical significance of p<0.01, and error bars indicate standard error of the mean (SEM).

[0035] [Figure 4A] FIG. 1 is a schematic diagram showing an experimental schedule, drug dosage, administration interval, etc. for verifying the therapeutic effect of a bispecific fusion protein according to one embodiment of the present invention on fatty liver associated with metabolic disorders associated with leaky gut. [Figure 4B] 4B is a graph showing the weights of the experimental animals measured after the completion of the animal experiment carried out according to the schedule shown in FIG. 4A. [Figure 4C] FIG. 4C is a graph showing the results of measuring the level of serum fluorescence of orally administered FITC-Dextran 4 hours before the sacrifice of the experimental animals of FIG. 4B.

[0036] [Figure 5A] 1 is a graph showing the results of analyzing the in vitro GLP-1 activity of various bispecific fusion proteins prepared according to an embodiment of the present invention. [Figure 5B] FIG. 1 is a schematic diagram showing an intraperitoneal glucose tolerance test experiment schedule, dosages, and administration intervals of administered drugs for analyzing the in vivo GLP-1 activity of bispecific fusion protein samples having various in vitro GLP-1 activities according to one embodiment of the present invention. [Figure 5C]5B is a graph showing the results of an intraperitoneal glucose tolerance test performed according to the schedule shown in FIG. 5B. In the graph, * indicates statistical significance at the p<0.05 level, and ** indicates statistical significance at the p<0.01 level.

[0037] [Figure 6A] 1 is a series of graphs showing the results of measuring the percentage change in body weight (left) and the percentage change in fat relative to lean (right) following administration of a bispecific fusion protein according to an embodiment of the present invention in comparison with dapiglutide mimetic, a known GLP-1R / GLP-2R dual acting agent. [Figure 6B] 1 is a graph showing the change in weight change rate depending on the dose of a bispecific fusion protein according to an embodiment of the present invention. [Figure 6C] 1 is a graph showing the results of an analysis of the effect of a bispecific fusion protein according to one embodiment of the present invention on body weight change, compared with simple co-administration of GLP-1-Fc and GLP-2-Fc at the same doses. [Figure 6D] 1 is a graph showing the weight change rate in a diet-induced obesity model animal induced by a high-fat diet in a bispecific fusion protein according to an embodiment of the present invention compared with that in a GLP-1R / GIPR dual acting agent, tirzepatide mimetic. [Figure 6E] 6D is a graph showing the results of measuring the weight change rate of adipose tissue in the experimental animals in the experiment of FIG. 6D. [Figure 6F] 6D. FIG. 6C is a graph showing the results of measuring the weight change rate of non-adipose tissues of experimental animals in the experiment of FIG. 6D. [Figure 6G] 1 is a graph showing the results of measuring subcutaneous fat percentage (left) and visceral fat percentage (right) in a diet-induced obesity model animal in which obesity was induced by a high-fat diet, using a bispecific fusion protein according to an embodiment of the present invention. Tirzepatide mimetic was used as a positive control. In the graph, NS indicates no statistical significance, * indicates statistical significance at a p<0.05 level, and ** indicates statistical significance at a p<0.01 level.

[0038] [Figure 7A] FIG. 1 is a graph showing the results of measuring serum LPS concentrations to analyze the effect of a bispecific protein according to an embodiment of the present invention on reducing endotoxemia in a diet-induced obesity model animal in which obesity was induced by a high-fat diet. [Figure 7B] 7B is a graph showing the results of measuring serum ALT levels to analyze the inhibitory effect on liver damage in the experimental animals of FIG. 7A. In the graph, * indicates statistical significance at the p<0.05 level, and ** indicates statistical significance at the p<0.01 level.

[0039] [Figure 8] 1 is a series of graphs showing the results of an analysis of insulin-stimulated glucose uptake of a bispecific fusion protein according to one embodiment of the present invention, including (a) normalized mean fluorescence intensity as a measure of surface expression of GLP-1R and GLP-2R in differentiated 3T3-L1 adipocytes, (b, c) the level of insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes, (d) normalized mean fluorescence intensity as a measure of surface expression of GLP-1R and GLP-2R in differentiated L6-GLUT4myc myoblasts, and (e, f) differentiated L6-GLUT4myc. In the graphs, * indicates statistical significance at the p<0.05 level, and ** indicates statistical significance at the p<0.01 level.

[0040] [Figure 9] 1 is a series of graphs showing the effects of administration of a bispecific fusion protein according to one embodiment of the present invention on blood glucose enhancement, changes in glycosylated hemoglobin, and insulin resistance, showing (a) non-fasting blood glucose levels, (b) glycosylated hemoglobin (HbA1c) levels, (c) fasting blood glucose levels, (d) fasting insulin levels, (e) HOMA-IR scores, and (f) HOMA-β scores. In the graphs, * indicates statistical significance at the p<0.05 level, and ** indicates statistical significance at the p<0.01 level.

[0041] [Figure 10]9 shows the results of histological analysis of the excised pancreas after the experiment in FIG. 9 , including (ac) immunohistochemical analysis of pancreatic tissue slices, including (a) micrographs staining for insulin, (b) insulin and glucagon, and (c) insulin and Ki-67-positive cells, and (d) islet area, (e) beta cell area, (f) alpha cell area, (g) beta / alpha cell ratio, and (h) the number of Ki-67-positive cells per 10,000 μm In the graphs, * indicates statistical significance at p<0.05, and ** indicates statistical significance at p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0042] Definitions of terms:

[0043] The term "GLP-1" as used herein is an abbreviation for "glucagon-like peptide-1," a 30- or 31-amino acid peptide hormone derived by tissue-specific post-translational processing of the proglucagon peptide. GLP-1 is produced and secreted by enteroendocrine L-cells in the small intestine and specific neurons in the nucleus tractus solitarius of the brainstem following food ingestion. The initial product, GLP-1(1-37), is readily amidated and cleaved to two equally biologically active truncated forms (GLP-1(7-36)amide and GLP-1(7-37)). Active GLP-1 contains two α-helical regions at amino acid positions 13-20 and 24-35, as well as a linker region connecting the two α-helical regions. GLP-1 has been developed and used as a therapeutic agent for type 2 diabetes due to its glucose-dependent role in lowering blood glucose levels. However, GLP-1 is rapidly degraded in vivo by dipeptidyl peptidase-4 (DPP-4), resulting in a half-life of only 2 minutes, making its efficacy as a natural peptide extremely limited.

[0044] As used herein, the term "GLP-2" refers to a 33-amino acid peptide produced by post-translational cleavage of proglucagon, similar to GLP-1, in the enteroendocrine L cells of the small intestine and various neurons in the central nervous system. GLP-2 is secreted along with GLP-1 upon ingestion of food. GLP-2 is known to improve small intestinal growth and function, reduce bone destruction, and exert neuroprotective effects upon administration, and is currently being developed as a therapeutic agent for diseases such as short bowel syndrome, Crohn's disease, and osteoporosis.

[0045] The term "GLP-1 analog" as used herein means a protein that biologically performs the function of GLP-1 and is capable of binding to the GLP-1 / Exendin-4 receptor and mediating downstream signal transduction, and is also referred to as a "GLP-1 receptor agonist."

[0046] As used herein, the term "GLP-2 analog" refers to a protein that performs the biological function of GLP-2 and is capable of binding to the GLP-2 receptor and mediating downstream signal transduction, and is also referred to as a "GLP-2 receptor agonist."

[0047] The term "fusion protein" as used herein refers to a recombinant protein in which two or more proteins or domains responsible for specific functions within a protein are linked together so that each protein or domain retains its original function.

[0048] As used herein, the term "half-life increasing moiety" refers to a functional group that is linked to a recombinant protein to increase the half-life of the recombinant protein in vivo. Such "half-life increasing moieties" include antibody Fc regions (Capon et al., Nature. 337:525-531, 1989), PEG (Caliceti and Veronese, Adv. Drug Delivery Rev. 55:1261-1277, 2003), XTEN (Schellenberger et al., Nat. Biotechnol. 27:1186-1190, 2009), PAS (Pro-Ala-Ser, Schlapschy et al., Protein Eng. Des. Sel. 26:489-501, 2013), ELP (elastine-like peptide, Floss et al., Trends Biotechnol. 28:37-45, 2010), and glycine-rich HAP (homo-amino-acid polymer, Schlapschy et al., Protein Eng. Des. Sel. 20:273-284, 2007), GLP (gelatin-like protein, Huang et al., Eur. J. Pharm. Biopharm. 74(3):435-441, 2010), and serum albumin (Sheffield et al., Cell Physiol. Biochem., 45(2):772-782, 2018) are used, and "half-life increasing moieties" added to these proteins are well known through review articles (Strohl, WR, BioDrugs, 29(4):215-239, 2015). Accordingly, prior articles on these individual factors and the review article are incorporated herein by reference.

[0049] As used herein, the term "antibody Fc region" refers to a crystallized fragment produced by papain cleavage of an antibody. This fragment interacts with cell surface receptors, also known as Fc receptors, and several proteins in the complement system. The Fc region exhibits a homotypic dimeric structure in which fragments containing the second and third constant regions (CH2 and CH3) of the heavy chain are linked by intermolecular disulfide bonds at the hinge region. The Fc region of IgG contains numerous N-glycan attachment sites, which are known to play an important role in Fc receptor-mediated actions.

[0050] As used herein, the term "hybrid Fc region" refers to an Fc region peptide generated by combining portions of Ig Fc regions of various subtypes, and such a combination of Fc region portions can exhibit differences in Fc receptor and complement binding ability compared to wild-type Fc regions.

[0051] The term "Exendin" as used herein refers to a peptide consisting of 39 amino acids isolated from the venom of the lizard Heloderma suspectum. Exendin 4 is 50% identical in amino acid sequence to GLP-1, is a member of the glucagon peptide family, and is known to play a role equivalent to GLP-1 as an agonist of the GLP-1 receptor. Exendin 4 is also known as "extenatide." Exendin 3 is a variant of Exendin 4 in which the second and third amino acids are substituted with serine and aspartic acid, respectively.

[0052] As used herein, the term "Lixisenatide" refers to a GLP-1 receptor agonist manufactured by Sanofi and sold as a daily injectable for the treatment of type 2 diabetes in Europe under the trade name Lyxumia and in the United States under the trade name Adlyxin.

[0053] As used herein, the term "Albiglutide" refers to a GLP-1 receptor agonist marketed by GSK under the trade name Eperzan in Europe and Tanzeum in the United States as a treatment for type 2 diabetes.

[0054] As used herein, the term "Liraglutide" is a subcutaneously injectable GLP-1 receptor agonist sold by Novo Nordisk under the trade name "Victoza" for the treatment of type 2 diabetes and obesity.

[0055] The term "Taspoglutide" as used herein refers to a GLP-1 receptor agonist jointly developed by Ipsen and Roche for the treatment of type 2 diabetes. It is a GLP-1 derivative in which the 8th and 35th amino acids, alanines, of the GLP-1(7-36) peptide are methylated and the final amino acid is amidated. However, when produced in the form of a fusion protein with another peptide, the C-terminus may be a general carboxyl group rather than amidated.

[0056] The term "Semaglutide" as used herein is a GLP-1R agonist, an oral hypoglycemic agent used to treat type 2 diabetes, and an anti-obesity agent used for long-term weight management. It was developed by Novo Nordisk in 2012 and approved for sale in the United States in 2017.

[0057] The term "tirzepatide" as used herein is a type of GLP-1R agonist currently used for the treatment of type 2 diabetes and weight loss. It is a drug developed by Eli Lilly and Company and approved for sale in the United States in 2022.

[0058] As used herein, the term "Dapiglutide" refers to a GLP-1R and GLP-2R dual agonist developed by Zealand Pharma, which is currently undergoing phase 1 clinical trials.

[0059] As used herein, the term "XTEN" refers to an unstructured immunogenic peptide containing six amino acids that is added to protein pharmaceuticals to improve their in vivo half-life, developed by Amunix, Inc., and is typically composed of 144 a.a. units or multiples thereof (US2010 / 0239554A1).

[0060] The term "teduglutide" as used herein refers to a mutant form of GLP-2 in which the second amino acid, alanine (A), is replaced with glycine (G), and is a GLP-2 analogue sold as a treatment for short bowel syndrome in the United States under the trade name Gattex and in Europe under the trade name Revestive.

[0061] The term "Glepaglutide" as used herein is a GLP-2 analogue with an improved half-life that has been developed as a therapeutic agent for short bowel syndrome and is currently undergoing phase 3 clinical trials for short bowel syndrome.

[0062] The term "GLP-2 analogue 10" as used herein refers to a GLP-2 analogue that has a stabilized structure by substituting the 11th and 18th amino acids of GLP-2 with cysteines and linking a lipidated intramolecular crosslinker through the thiol groups of the two substituted cysteines, and is characterized by the addition of the nine C-terminal amino acids of Exendin 4 to the C-terminus (Yang et al., J. Med. Chem. 61:3218-3223, 2018).

[0063] As used herein, the term "linker peptide" refers to an unstructured peptide used to link two or more proteins or peptides with other biological activities to form a fusion protein.

[0064] As used herein, the term "metabolic disorder" refers to a general term for diseases that occur due to abnormalities in the metabolic process, which is the process by which the body obtains energy from ingested food. Typical examples include obesity and diabetes, which are caused by the misregulation of carbohydrate metabolism. While various causes of metabolic disorders, including genetic and environmental factors, have been identified, carbohydrate-heavy diets have recently been identified as a major cause in addition to genetic factors. Examples of metabolic disorders include diabetes, including type 1 and type 2 diabetes, obesity, metabolic syndrome, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, and liver cirrhosis.

[0065] The term "metabolic syndrome" as used herein refers to a disease presumed to be caused by insulin resistance, characterized by abnormalities in two or more of the following: cholesterol, blood pressure, and blood glucose levels. It conceptualizes the phenomenon in which various risk factors for cardiovascular disease and type 2 diabetes cluster together as a single disease group. It is a useful concept that can comprehensively explain insulin resistance (IR) and the associated complex and diverse metabolic disorders and clinical manifestations. It is known that if metabolic syndrome is left untreated, the risk of developing cardiovascular diseases such as arteriosclerosis, myocardial infarction, and stroke, or type 2 diabetes, increases.

[0066] Detailed description of the invention:

[0067] According to one aspect of the present invention, there is provided a bispecific fusion protein in which a GLP-1 analog and a GLP-2 analog are fused, wherein the activity of the GLP-1 analog is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analog is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).

[0068] Another aspect of the present invention provides a bispecific fusion protein comprising a first fusion protein in which a GLP-1 analog is linked to an antibody Fc region and a second fusion protein in which a GLP-2 analog is linked to an antibody Fc region, the bispecific fusion protein being produced by dimerization of the first and second fusion proteins, wherein the activity of the GLP-1 analog is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analog is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).

[0069] In the bispecific fusion protein, the activity of the GLP-1 analog is preferably 41% or more, or 42% or more, or 43% or more, or 44% or more, or 45% or more, or 46% or more, or 47% or more, or 48% or more, or 49% or more, or 50% or more, or 51% or more, or 52% or more, or 53% or more, or 54% or more, or 55% or more, or 56% or more, or 57% or more, or 58% or more, or 59% or more, or 60% or more, or 61% or more, or 62% or more, or 63% or more, or 64% or more, or 65% or more, or 66% or more, or 67% or more, or 68% or more, or 69% or more, compared to wild-type GLP-1 in vitro. or more, or 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more activity, and may have an activity of 100% or more but not more than 400%.

[0070] More specifically, the activity of the GLP-1 analogs is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, or 120%, and may have a range between any of the above percentages and any other higher value.

[0071] In addition, the activity of the GLP-1 analog may selectively exceed 120% compared to wild-type GLP-1 under in vitro conditions, more specifically, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%. 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 201%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%, 214%, 215%, 216%, 217%, 218%, 219%, 220%, 221%, 222%, 223%, 224%, 225%, 226%, 227%, 228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%, 240%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, 299%, 300%, 301%, 302%, 303%, 304%,305%, 306%, 307%, 308%, 309%, 310%, 311%, 312%, 313%, 314%, 315%, 316%, 317%, 318%, 319%, 320%, 321%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329% ,330%,331%,332%,333%,334%,335%,336%,337%,338%,339%,340%,341%,342%,343%,344%,345%,346%,347%,348%,349%,350%,351%,352%,353%,354% , 355%, 356%, 357%, 358%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%, 370%, 371%, 372%, 373%, 374%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%, 383%, 384%, 385%, 386%, 387%, 388%, 389%, 390%, 391%, 392%, 393%, 394%, 395%, 396%, 397%, 398%, 399%, or 400%, and may exceed 400%. Furthermore, the GLP-1 activity of the bispecific fusion protein according to an embodiment of the present invention under in vitro conditions may have a range with any of the above percentages as the lower limit and any other higher value as the upper limit.

[0072] In the bispecific fusion protein, the activity of the GLP-2 analogue in vitro is 2% or more compared to that of wild-type GLP-2.

[0073] In the bispecific fusion protein, the activity of the GLP-2 analog is 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% or less of that of wild-type GLP-2, and may have a range of any of the above percentages, with any lower limit and any other higher limit.

[0074] The bispecific fusion protein has a half-life-increasing moiety added thereto, which is inserted between the GLP-1 analog and the GLP-2 analog or added to the N-terminus or C-terminus of the entire fusion protein, and is preferably an antibody Fc region, PEG, XTEN, PAS, ELP, glycine-rich HAP, GLP, or serum albumin.

[0075] The bispecific fusion protein is not a proglucagon or analog thereof in which GLP-1 and GLP-2 naturally expressed in the body are linked by an intervention peptide, but a fusion protein in which a GLP-1 analog and a GLP-2 analog are directly linked, or the two peptides are linked by a linker peptide of another type other than the intervention peptide.

[0076] In the bispecific fusion protein, the GLP-1 analog may be GLP-1, exendin 3, exendin 4, GLP-1 / exendin 4 hybrid peptide, GLP-1-XTEN, exendin 4-XTEN, lixisenatide, albiglutide, liraglutide, or taspoglutide, or a contiguous repeat of any one or more of these. The contiguous repeat increases the activity or length of GLP-1, enabling quality control related to the production of bispecific proteins (easily distinguishing between heterodimers and homodimers). It also has the advantage of simplifying the manufacturing process compared to PEGylation, a well-known half-life-enhancing technology, thereby reducing production costs. The number of repeat units in the contiguous repeat of the GLP-1 analog can be adjusted depending on the desired GLP-1 activity.

[0077] In the bispecific fusion protein, the GLP-1 may comprise the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0078] In the bispecific fusion protein, the Exendin 3 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0079] In the bispecific fusion protein, the Exendin 4 can be composed of the amino acid sequence shown in SEQ ID NO:4.

[0080] In the bispecific fusion protein, the GLP-1 / Exendin 4 hybrid may comprise the amino acid sequence shown in SEQ ID NO:5.

[0081] In the bispecific fusion protein, the Lixisenatide may comprise the amino acid sequence shown in SEQ ID NO:6.

[0082] In the bispecific fusion protein, the Exendin 4-XTEN may comprise the amino acid sequence shown in SEQ ID NO:7.

[0083] In the bispecific fusion protein, the Albiglutide may comprise the amino acid sequence shown in SEQ ID NO:8.

[0084] In the bispecific fusion protein, the Liraglutide may comprise the amino acid sequence shown in SEQ ID NO:9.

[0085] In the bispecific fusion protein, the Taspoglutide may comprise the amino acid sequence shown in SEQ ID NO:10.

[0086] In the bispecific fusion protein, the GLP-1 contiguous repeat may comprise the amino acid sequence shown in SEQ ID NO:11.

[0087] In the bispecific fusion protein, the antibody Fc region is an antibody Fc region having a heterotypic dimerization moiety formed therein, and the dimerization moiety is also Knobs-into-Holes (KiH), KiHS-S.HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTS-S, SEED, or A107.

[0088] As used herein, the term "Knobs-into-Holes (KiH)" refers to a strategy in antibody engineering used for heavy chain heterodimerization in the production of recombinant proteins or IgG antibodies containing Fc regions. When two fusion proteins forming heterodimers are classified into a first fusion protein and a second fusion protein, the KiH technique involves substituting tryptophan (W) for threonine (T), the 22nd amino acid in the CH3 domain of the first fusion protein (Knob), (T366W). The second fusion protein involves substituting serine (S) for threonine (T), alanine (A) for leucine (L), the 24th amino acid in the CH3 domain (L368A), and valine (V) for tyrosine (Y), the 63rd amino acid in the CH3 domain of the second fusion protein (Knob). The first fusion protein is a first variant Fc region in which the 22nd amino acid, threonine (T), in the CH3 domain is substituted with serine (S) (T366S), the 24th amino acid, leucine (L) is substituted with alanine (A) (L368A), and the 62nd amino acid, tyrosine (T) is substituted with valine (V) (Y407V) (hole structure). The second fusion protein is a second variant Fc region in which the 22nd amino acid, threonine (T) in the CH3 domain is substituted with tryptophan (W) (T366W) (knob structure).

[0089] In this case, the positions of the mutated amino acids are based on positions 224-330 (SEQ ID NO: 61), which corresponds to the CH3 region of the amino acid sequence of human IgG1 in UniProt No. P01857, and the numbers in parentheses associated with specific amino acid substitutions are in accordance with the EU numbering convention for antibody amino acid sequences. Even if additional mutations such as addition, deletion, or substitution of amino acids occur at sites in the CH3 domain that are not related to the knobs-into-holes structure, the amino acids corresponding to those positions may be mutated based on the reference sequence. Alternatively, the knobs-into-holes structure may be introduced through other amino acid mutations well known to those skilled in the art. Such mutations have been well described in previous literature (Wei et al., Oncotarget 8(31):51037-51049, 2017; Ridgway et al., Protein Eng. 9(7):617-621, 1996; Carter, P., J. Immunol. Methods 48(1-2):7-15, 2001). For example, a bispecific dimeric fusion protein can be generated by combining a knob structure (T366Y) in which the 22nd amino acid, threonine (T), in the CH3 domain of the first fusion protein is substituted with tyrosine (Y) and a hole structure (Y403T) in which the 63rd amino acid, tyrosine (Y) in the CH3 domain of the second fusion protein is substituted with threonine (T). Conversely, the Knobs-into-Holes structure can be formed by introducing a Hole structure into the first fusion protein and a Knob structure into the second fusion protein.

[0090] As used herein, the term "KiH S-S" is a structure in which cysteine ​​is introduced into CH3 of the KiH structure so that an intermolecular disulfide bond (intramolecular) is formed, and in which serine (S), the tenth amino acid of CH3 of Knob Fc, is additionally substituted with cysteine ​​(C) (S354C), and tyrosine (Y), the fifth amino acid of Hole Fc, is additionally substituted with cysteine ​​(C) (Y349C) (Merchant et al., Nat. Biotechnol. 16(7):677-681, 1998).

[0091] As used herein, the term "HA-TF" refers to a technique for inducing heterodimerization using hydrophobic / steric complementarity by substituting the 20th amino acid, serine (S), with histidine (H) (S364H) and the 61st amino acid, phenylalanine (F), with alanine (A) (F405A) in the CH3 of the first variant Fc domain, and substituting the 5th amino acid, tyrosine (Y) with threonine (T) (Y349T) and the 50th amino acid, threonine (T) with phenylalanine (F) (T394F) in the CH3 of the second variant Fc domain (Moore et al., MAbs 3(6):546-557, 2011).

[0092] As used herein, the term "ZW1" refers to a variant of the first Fc region in which the sixth amino acid of CH3, threonine (T), is replaced with valine (V) (T350V), the seventh amino acid, leucine (L), is replaced with tyrosine (Y) (L351Y), the 61st amino acid, phenylalanine (F) is replaced with alanine (A) (F405A), and the 63rd amino acid, tyrosine (Y) is replaced with valine (V) (Y407V). This refers to a technique that induces heterodimerization using hydrophobic / steric complementarity by substituting the sixth amino acid of CH3, threonine (T), with valine (V) (T350V), the 22nd amino acid, threonine (T), with tryptophan (W) (T366W), the 48th amino acid, lysine (K), with leucine (L) (K392L), and the 50th amino acid, threonine (T), with tryptophan (W) (T394W) (von Kreudenstein et al., MAbs 5(5):646-654, 2013).

[0093] As used herein, the term "7.8.60" refers to a variant of the first Fc region in which the 16th amino acid, lysine (K), is substituted with aspartic acid (D) (K360D), the 55th amino acid, aspartic acid (D), is substituted with methionine (M) (D399M), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V) (Y407V). The second variant Fc region also includes the first amino acid, glutamic acid (E), is substituted with arginine (R) (E345R), the third amino acid, glutamine (Q), is substituted with arginine (R) (Q347R), the 22nd amino acid, threonine (T), is substituted with valine (V) (T366V), and the 63rd amino acid, lysine (K), is substituted with valine (V) (K409V). This variant exhibits hydrophobic / steric complementarity and electrostatic complementarity. This refers to a technique that induces heterodimerization using complementarity (Leaver-Fay et al., Structure 24(4):641-651, 2016).

[0094] As used herein, the term "DD-KK" refers to a technique for inducing heterodimerization using electrostatic complementarity by substituting the 65th amino acid, lysine (K), with aspartic acid (D) (K409D) and the 48th amino acid, lysine (K), with aspartic acid (D) (K392D) in the CH3 of the first variant Fc region, and by substituting the 55th amino acid, aspartic acid (D), with lysine (K) (D399K) and the 13th amino acid, glutamic acid (E), with lysine (K) (E356K) in the CH3 of the second variant Fc region (Gunasekaran et al., J. Biol. Chem. 285(25):19637-19646, 2010).

[0095] As used herein, the term "EW-RVT" refers to a technique for inducing heterodimerization using hydrophobic / steric complementarity and long-range electrostatic interactions by substituting the 16th amino acid, lysine (K), in the CH3 of the first variant Fc region with glutamic acid (E) (K360E) and the 65th amino acid, lysine (K), with tryptophan (W) (K409W), and the 3rd amino acid, glutamine (Q), in the CH3 of the Fc region of the second variant Fc region with arginine (R) (Q347R), the 55th amino acid, aspartic acid (D), with valine (V) (D399V), and the 61st amino acid, phenylalanine (F), with threonine (T) (F405T) (Choi et al., Mol. Cancer Ther. 12(12):2748-2759, 2013).

[0096] As used herein, the term "EW-RVT S-S" refers to a structure in which cysteine ​​has been introduced into CH3 of the EW-RVT structure so that an intermolecular disulfide bond is formed, in which the fifth amino acid, tyrosine (Y), in CH3 of the first variant Fc region of the EW-RVT has been additionally substituted with cysteine ​​(C) (Y349C), and the tenth amino acid, serine (S) in CH3 of the second variant Fc region has been additionally substituted with cysteine ​​(C) (S354C) (Choi et al., Mol. Immunol. 65(2):377-383, 2015).

[0097] As used herein, the term "SEED" refers to a technique that induces heterodimerization by utilizing hydrophobic / steric complementarity through chain exchange between IgG and IgA by introducing 45 IgA-derived residues into the CH3 of a first variant Fc region and 57 IgG1-derived residues into the CH3 of a second variant Fc region (Davis et al., Protein Eng. Des. Sel. 23(4):195-202, 2010).

[0098] As used herein, the term "A107" refers to a technique for inducing heterodimerization using hydrophobic / steric complementarity and hydrogen bonding complementarity by substituting the 26th amino acid, lysine (K), in CH3 of the first variant Fc domain with glutamic acid (E) (K370E), the 69th amino acid, lysine (K), in CH3 with tryptophan (W) (K409W), and the 13th amino acid, glutamic acid (E), in CH3 of the second variant Fc domain with asparagine (N) (E357N), the 55th amino acid, aspartic acid (D), in CH3 with valine (V) (D399V), and the 61st amino acid, phenylalanine (F), in CH3 with threonine (T) (F405T) (Choi et al., PLoS One 10(12):e0145349, 2015).

[0099] The hybrid antibody Fc region is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 16. The hybrid antibody Fc region is additionally mutated so as not to induce undesired side effects such as ADCC (antibody-dependent cell cytotoxicity) and CDC (complement-dependent cytotoxicity) upon administration to the body. Such hybrid antibody Fc regions are described in Korean Patent No. 897938, and include a hybrid Fc region variant (NTIG Knob) represented by SEQ ID NO: 15, in which the 18th amino acid, threonine (T), of a hybrid Fc region (knob structure) consisting of the amino acid sequence represented by SEQ ID NO: 14 is substituted with glutamine (Q) and the 196th amino acid, methionine (M), is substituted with leucine (L); or a hybrid Fc region variant (NTIG Hole) consisting of the amino acid sequence represented by SEQ ID NO: 16, in which the 18th amino acid, threonine (T), of a hybrid Fc region (hole) consisting of the amino acid sequence represented by SEQ ID NO: 13 is substituted with glutamine (Q) and the 196th amino acid, methionine (M), is substituted with leucine (L).

[0100] In the bispecific fusion protein, the GLP-2 analog is GLP-2, GLP-2 A2G Mutant, GLP-2 N16G_L17Q Mutant, GLP-2 A2G_N16G_L17QThe mutant, Glepaglulide, or GLP-2 analog 10, has amino acid substitutions, additions, deletions, or other mutations introduced to reduce binding affinity to the GLP-2 receptor, resulting in reduced activity compared to wild-type GLP-2. Alternatively, when wild-type GLP-2 is used, an approach can be used to reduce binding affinity to the GLP-2 receptor by shortening its length compared to GLP-1 analogs (e.g., by inserting a longer linker peptide between the GLP-1 analog and the Fc region). In this case, the length of the linker of the first fusion protein is about 5 to 40 a.a. longer than the linker of the second fusion protein, and more specifically, 5 a.a., 6 a.a., 7 a.a., 8 a.a., 9 a.a., 10 a.a., 11 a.a., 12 a.a., 13 a.a., 14 a.a., 15 a.a., 16 a.a., 17 a.a., 18 a.a., 19 a.a., 20 a.a., 21 a.a., 22 a.a. , 23 a.a., 24 a.a., 25 a.a., 26 a.a., 27 a.a., 28 a.a., 29 a.a., 30 a.a., 31 a.a., 32 a.a., 33 a.a., 34 a.a., 35 a.a., 36 a.a., 37 a.a., 38 a.a., 39 a.a., or 40 a.a., and may have a range with any of the above amino acid lengths as the lower limit and another larger value as the upper limit. In fact, the inventors confirmed through Examples 2 to 4 of the present invention that when the length of a GLP-1 analog is designed to be longer than that of a GLP-2 analog (by using a longer glycan linker peptide or forming a GLP-1 contiguous repeat), the GLP-2 activity is significantly reduced, whether or not a mutation is introduced into the GLP-2 analog. Conversely, when the length of the fusion protein containing a GLP-2 analog is increased by introducing a glycan linker into the GLP-2 analog compared to the fusion protein containing a GLP-1 analog, it was confirmed that the relative activity of the GLP-2 analog is significantly increased.

[0101] In the bispecific fusion protein, the GLP-2 analog may comprise any one of the amino acid sequences shown in SEQ ID NO: 17 to SEQ ID NO: 22. The peptide consisting of the amino acid sequence shown in SEQ ID NO: 18 is a human GLP-2 wild-type peptide, and the human GLP-2 mutant consisting of the amino acid sequence shown in SEQ ID NO: 17 is a human GLP-2 mutant in which the second amino acid, alanine, is replaced with glulisine (A2G mutant or GLP-2 A2G On the other hand, the GLP-2 variant consisting of the amino acid sequence shown in SEQ ID NO: 19 is a GLP-2 variant (GLP-2) in which not only the second amino acid, alanine (A), is substituted with glycine (G), but also the 16th amino acid, asparagine (N), is substituted with glycine (G), and the 17th amino acid, leucine (L), is substituted with glutamine (Q). A2G_N16G_L17Q ), which is known to suppress GLP-2 dimerization and the resulting aggregate formation during recombinant production while retaining the functionality of GLP-2 (Baker et al., J. Mol. Recognit. 25:155-164, 2012). Alternatively, a GLP-2 wild-type peptide in which the second amino acid, alanine, is substituted with glulisine and the 17th amino acid, leucine, is substituted with glutamine (A2G, L17Q, SEQ ID NO: 20) can also exhibit functions equivalent to those of the GLP-2 analog consisting of the amino acid sequence shown in SEQ ID NO: 19, and can therefore be used as a GLP-2 analog in the present invention.

[0102] In the bispecific fusion protein, the Glepaglutide may comprise the amino acid sequence shown in SEQ ID NO:21.

[0103] In the bispecific fusion protein, the GLP-2 analogue 10 may comprise the amino acid sequence shown in SEQ ID NO:22.

[0104] In the bispecific fusion protein, the first fusion protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:23 to SEQ ID NO:25.

[0105] In the bispecific fusion protein, the second fusion protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:26 to SEQ ID NO:28.

[0106] In the bispecific fusion proteins, the first fusion protein is a hybrid Fc region in which the 10th amino acid, serine, in the CH3 domain is substituted with cysteine ​​(C) and the 22nd amino acid, threonine (T), is substituted with tryptophan (W) (knob structure), and the second fusion protein is an Fc region in which the 5th amino acid, tyrosine (Y), in the CH3 domain is substituted with cysteine ​​(C), the 22nd amino acid, threonine, is substituted with serine (S), the 24th amino acid, leucine (L), is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V) (hole structure). The first fusion protein has an Fc region in which the fifth amino acid, tyrosine (Y), in the CH3 domain is substituted with cysteine ​​(C), the 22nd amino acid, threonine, is substituted with serine (S), the 24th amino acid, leucine (L), is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V) (hole structure). The second fusion protein has a hybrid Fc region in which the tenth amino acid, serine, in the CH3 domain is substituted with cysteine ​​(C) and the 22nd amino acid, threonine (T), is substituted with tryptophan (W) (knob structure).

[0107] Alternatively, the first fusion protein has a hybrid Fc region in which the 22nd amino acid, threonine (T), in the CH3 domain has been replaced with tyrosine (Y); the second fusion protein has a hybrid Fc region in which the 63rd amino acid, tyrosine (Y), in the CH3 domain has been replaced with threonine (T); the second fusion protein has a hybrid Fc region in which the 63rd amino acid, tyrosine (Y) in the CH3 domain has been replaced with threonine (T); and the first fusion protein has a hybrid Fc region in which the 63rd amino acid, tyrosine (Y) in the CH3 domain has been replaced with threonine (T). However, the mutation at amino acid 63 is represented as Y86T according to the numbering rules of the international ImMunoGeneTics information system (IMGT) (Lefranc et al., Dev. Comp. Immunol., 27:55-77, 2003), rather than based on the amino acid sequence of the CH3 domain of human IgG1 shown in SEQ ID NO: 61. Various heterodimer-forming moieties, as described above, that can be introduced into the antibody Fc region below the KiH structure to enable heterodimer formation can also be used.

[0108] In the bispecific fusion protein, one or more linker peptides are inserted between the fusion partners of the fusion protein, i.e., between the peptides or domains. That is, in the case of the bispecific fusion protein in which a GLP-1 analog and a GLP-2 analog are fused, a linker peptide is inserted between the GLP-1 analog and the GLP-2 analog. In the case of the bispecific fusion protein formed by dimerization of the first fusion protein and the second fusion protein, a linker peptide is inserted between the GLP-1 analog and the antibody Fc region in the first fusion protein, and similarly, a linker peptide is inserted between the GLP-2 analog and the antibody Fc region in the second fusion protein. In this case, the linker peptide may or may not contain an N-glycan attachment site. The N-glycan attachment site is used to facilitate the formation of heterodimers by differentiating the sizes of the first and second fusion proteins, and it has been confirmed that it does not significantly affect the binding affinity to GLP-1R or GLP-2R.

[0109] The linker peptides are EPKSSDKTHTCPPCP (SEQ ID NO: 29), EPKSCDKTHTCPPCP (SEQ ID NO: 30), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 31), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 32), AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 33), GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 34), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 35), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 36), GGGGSGGGGSGGGGS (SEQ ID NO: 37), GGSGG (SEQ ID NO: 38), GGSGGSGGS (SEQ ID NO: 39), GGGSGG (SEQ ID NO: 40), (GS) n (Unit: SEQ ID NO: 41, n is an integer from 1 to 10), (GGS) n (n is an integer from 1 to 10), (GS) n(n is an integer from 1 to 10), (GSSGGS) n (Unit: SEQ ID NO: 42, n is an integer from 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 43), EGKSSGSGSESKST (SEQ ID NO: 44), GSAGSAAGSGEF (SEQ ID NO: 45), (EAAAK) n (Unit: SEQ ID NO: 46, n is an integer from 1 to 10), CRRRRREAEAC (SEQ ID NO: 47), A(EAAAK)ALEA(EAAAK)A (SEQ ID NO: 48), GGGGGGGG (SEQ ID NO: 49), GGGGGG (SEQ ID NO: 50), AEAAAKEAAAAKA (SEQ ID NO: 51), PAPAP (SEQ ID NO: 52), (Ala-Pro) n (n is an integer between 1 and 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 53), PLGLWA (SEQ ID NO: 54), TRHRQPRGWE (SEQ ID NO: 55), AGNRVRRSVG (SEQ ID NO: 56), RRRRRRRR (SEQ ID NO: 57), GFLG (SEQ ID NO: 58), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 59), or GSTSGSGKPGSGEGS (SEQ ID NO: 60).

[0110] According to one aspect of the present invention, there is provided a composition comprising the bispecific fusion protein.

[0111] Another aspect of the present invention provides a pharmaceutical composition for treating metabolic diseases, comprising any one or more of the above bispecific fusion proteins as an active ingredient.

[0112] In the pharmaceutical composition, the metabolic disease is metabolic syndrome, obesity, diabetes, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, liver fibrosis or liver cirrhosis, the diabetes is type 1 diabetes or type 2 diabetes, and the liver fibrosis is also liver fibrosis caused by the progression of chronic metabolic disorder-related steatohepatitis.

[0113] Another aspect of the present invention provides any one of the above bispecific fusion proteins for use in treating a metabolic disease.

[0114] In the dual isomer fusion protein, the metabolic disease is metabolic syndrome, obesity, diabetes, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, liver fibrosis or liver cirrhosis, the diabetes is type 1 diabetes or type 2 diabetes, and the liver fibrosis is also liver fibrosis caused by the progression of chronic metabolic disorder-related steatohepatitis.

[0115] According to another aspect of the present invention, there is provided a use of any one of the above bispecific fusion proteins in the manufacture of a therapeutic agent for a metabolic disease.

[0116] In the above use, the metabolic disease is metabolic syndrome, obesity, diabetes, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, liver fibrosis or liver cirrhosis, the diabetes is type 1 diabetes or type 2 diabetes, and the liver fibrosis is also liver fibrosis caused by the progression of chronic metabolic disorder-related steatohepatitis.

[0117] Another aspect of the present invention provides a method for treating a metabolic disorder in an individual, comprising administering to said patient a therapeutically effective amount of said bispecific fusion protein.

[0118] In the treatment method, the metabolic disease is metabolic syndrome, obesity, diabetes, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, liver fibrosis, or liver cirrhosis, the diabetes is type 1 diabetes or type 2 diabetes, and the liver fibrosis is also liver fibrosis caused by the progression of chronic metabolic disorder-related steatohepatitis.

[0119] Exendin 4, a GLP-1 receptor agonist, has already been shown in clinical trials to be effective in regulating blood glucose and body weight in patients with type 2 diabetes (DeFronzo et al., Diabetes Care 28:1092-1100, 2005). Therefore, the bispecific fusion protein according to one embodiment of the present invention can be used to treat metabolic diseases such as obesity, diabetes, and metabolic syndrome.

[0120] The composition may contain a pharmaceutically acceptable carrier, and may further contain a pharmaceutically acceptable adjuvant, excipient, or diluent in addition to the carrier.

[0121] As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause typical gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further contain fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

[0122] Furthermore, the pharmaceutical composition according to one embodiment of the present invention can be formulated using methods known to those skilled in the art to enable rapid, sustained, or delayed release of the active ingredient upon administration to a mammal, including powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, and sterile powders.

[0123] The pharmaceutical composition according to one embodiment of the present invention may be administered by various routes, for example, orally, parenterally, for example, by suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, or intraspinal administration, or by using an implantable device for sustained, continuous, or repeated release. The number of doses may be once a day or in divided doses within a desired range, and the administration period is not particularly limited.

[0124] The composition according to one embodiment of the present invention may be formulated into a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, parenteral carriers such as water, suitable oils, saline, aqueous glucose, and glycols, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Furthermore, the composition according to the present invention may appropriately contain suspending agents, solubilizers, stabilizers, isotonicity adjusting agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, antioxidants, and the like, as needed depending on the administration method and dosage form. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

[0125] The dosage of the composition administered to a patient will vary depending on many factors, including the patient's height, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered concomitantly. The pharmaceutically active protein is administered in an amount of 100 ng / kg body weight to 10 mg / kg body weight, more preferably 1 to 500 μg / kg body weight, and most preferably 5 to 50 μg / kg body weight, although dosages may be adjusted taking these factors into consideration.

[0126] The term "therapeutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined based on factors including the type and severity of the disease, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention is 0.1 mg / kg to 1 g / kg, more preferably 1 to 500 mg / kg, but the effective dose can be appropriately adjusted depending on the age, sex, and condition of the patient.

[0127] A linker peptide having a typical flexible structure is inserted between the two or more proteins or domains. The linker peptides are EPKSSDKTHTCPPCP (SEQ ID NO: 29), EPKSCDKTHTCPPCP (SEQ ID NO: 30), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 31), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 32), AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 33), GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 34), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 35), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 36), GGGGSGGGGSGGGGS (SEQ ID NO: 37), GGSGG (SEQ ID NO: 38), GGSGGSGGS (SEQ ID NO: 39), GGGSGG (SEQ ID NO: 40), (GS) n (Unit: SEQ ID NO: 41, n is an integer from 1 to 10), (GGS) n (n is an integer from 1 to 10), (GS) n (n is an integer from 1 to 10), (GSSGGS) n(Unit: SEQ ID NO: 42, n is an integer from 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 43), EGKSSGSGSESKST (SEQ ID NO: 44), GSAGSAAGSGEF (SEQ ID NO: 45), (EAAAK) n (Unit: SEQ ID NO: 46, n is an integer from 1 to 10), CRRRRREAEAC (SEQ ID NO: 47), A(EAAAK)ALEA(EAAAK)A (SEQ ID NO: 48), GGGGGGGG (SEQ ID NO: 49), GGGGGG (SEQ ID NO: 50), AEAAAKEAAAAKA (SEQ ID NO: 51), PAPAP (SEQ ID NO: 52), (Ala-Pro) n (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 53), PLGLWA (SEQ ID NO: 54), TRHRQPRGWE (SEQ ID NO: 55), AGNRVRRSVG (SEQ ID NO: 56), RRRRRRRR (SEQ ID NO: 57), GFLG (SEQ ID NO: 58), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 59), or GSTSGSGKPGSGEGS (SEQ ID NO: 60).

[0128] In another aspect of the present invention, the bispecific fusion protein can be produced by transfecting a host cell with a recombinant expression vector comprising a first gene construct comprising a polynucleotide encoding the first fusion protein and a second gene construct comprising a polynucleotide encoding the second fusion protein, followed by recombinant expression.

[0129] In this case, the first and second gene constructs are inserted into a single expression vector and expressed, or inserted into two separate expression vectors and expressed. In the former case, the vector can be designed so that each gene construct is operably linked to two separate regulatory sequences, or the two gene constructs can be operably linked to a single regulatory sequence and both gene constructs can be linked by an internal ribosome entry site (IRES).

[0130] As used herein, the term "operably linked to" means that a nucleic acid sequence of interest (e.g., in an in vitro transcription / translation system or in a host cell) is linked to said regulatory sequence in a manner that allows its expression to occur.

[0131] The term "regulatory sequence" is intended to encompass promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleic acid of interest in many host cells, those that direct expression of a nucleic acid of interest only in specific tissues (e.g., tissue-specific regulatory sequences), and those that direct expression in response to a specific signal (e.g., inducible regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector can vary depending on factors such as the choice of host cell to be transformed and the desired level of protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences enabling expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, these typically include a regulatory sequence responsible for transcription initiation and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. In addition to transcriptional regulatory elements, additional regulatory sequences may include translational enhancement elements and / or naturally occurring or heterologous promoter regions. For example, possible regulatory sequences enabling expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV promoter (Rous sarcoma virus), human height element 1α promoter, glucocorticoid-inducible MMTV promoter (Moloney murine tumor virus), metallothionein-inducible or tetracycline-inducible promoter, or amplifiers such as the CMV or SV40 amplifiers. For expression in neuronal cells, it is contemplated that the neurofilament promoter, PGDF promoter, NSE promoter, PrP promoter, or thy-1 promoter may be used. Such promoters are known in the art and described in Charron, J. Biol. Chem. 270:25739-25745, 1995. For expression in prokaryotic cells, numerous promoters have been described, including the lac promoter, tac promoter, or trp promoter.In addition to elements capable of initiating transcription, the regulatory sequence may also include a transcription termination signal, such as an SV40 poly-A site or a TK poly-A site, downstream of the polynucleotide according to one embodiment of the present invention. Suitable expression vectors for use herein are known in the art, and examples thereof include the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27 (Korean Patent No. 4142254), pX (Pagano et al., Science 255:1144-1147, 1992), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al., Cell 75:791-803, 1995), or prokaryotic expression vectors such as lambda gt11 or pGEX (Amersham Pharmacia). In addition to the nucleic acid molecule of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. The secretion signal is well known to those skilled in the art, and depending on the expression system used, a leader sequence capable of directing the fusion protein to a cellular compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the present invention, preferably a detoxified protein or a leader sequence capable of secreting the protein directly into the periplasm or extracellular medium.

[0132] The vectors of the present invention can be prepared by standard recombinant DNA techniques, including, for example, blunt-end and sticky-end ligation, restriction enzyme treatment to provide suitable termini, removal of phosphate groups by alkaline phosphatase treatment to prevent mismatching, and enzymatic ligation using T4 DNA ligase. The vectors of the present invention can be prepared by recombining DNA encoding a signal peptide obtained by chemical synthesis or recombinant DNA technology and DNA encoding the bispecific fusion protein of the present invention into a vector containing appropriate regulatory sequences. Vectors containing the regulatory sequences can be commercially purchased or prepared. In one embodiment of the present invention, the pBispecific backbone vector (Genexine, Inc., Republic of Korea) or pAD15 vector was used as the backbone vector.

[0133] The expression vector may further comprise a polynucleotide encoding a secretory signal sequence, which induces extracellular secretion of a recombinant protein expressed intracellularly, and may be a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus glycoprotein Ds (HSVgDs) signal sequence, or a growth hormone signal sequence.

[0134] The expression vector according to one embodiment of the present invention is an expression vector that expresses the protein in a host cell, and the expression vector may take any form, such as a plasmid vector, a virus vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.

[0135] The present invention will be described in more detail below through examples and experimental examples. However, the present invention is not limited to the examples and experimental examples disclosed below, and may be embodied in various different forms. The following examples and experimental examples are provided to fully disclose the present invention and to fully convey the scope of the invention to those skilled in the art.

[0136] Example: Design of a bispecific fusion protein

[0137] The present inventors have devised a variety of bispecific fusion proteins containing both GLP-1 and GLP-2 analogs, as shown in Figure 1 and Table 1 below.

[0138] In designing a bispecific fusion protein containing a GLP-1 analog and a GLP-2 analog, a GLP-1 / Exendin 4 hybrid (hereinafter referred to as "GLP-1 / Ex4 Hyb" for convenience) and its consecutive repeats and a GLP-2 analog (GLP-2-2G and a mutant at positions 16 and 17 (GLP-2-2G mutant)) were used to regulate the activity of the GLP-1 analog and the GLP-2 analog. For ease of purification, a glycan linker was used in either the first fusion protein containing the GLP-1 analog or the second fusion protein containing the GLP-2 analog, or neither of them used a glycan linker.

[0139] [Table 1]

[0140] Furthermore, in the case of the bispecific fusion proteins, the Knobs-into-holes technique was applied to preferentially generate heterodimers. Specifically, the first fusion protein (Knob) was constructed by substituting the 10th amino acid, serine (S), in the CH3 domain of the hybrid Fc region with cysteine ​​(C) and the 22nd amino acid, threonine (T), with tryptophan (W). The second fusion protein (Hole) was constructed by substituting the 5th amino acid, tyrosine (Y), in the CH3 domain of the Fc region with cysteine ​​(C), the 22nd amino acid, threonine (T), with serine (S), the 24th amino acid, leucine (L), with alanine (A), and the 63rd amino acid, tyrosine (Y), with valine (V). The positions of the mutated amino acids were determined based on the reference sequence (the amino acid sequence of the human IgG1 CH3 domain of SEQ ID NO: 61). If additional mutations, such as addition, deletion, or substitution of amino acids, occur in the CH3 domain at positions unrelated to the knobs-into-holes structure, the amino acids corresponding to those positions may be mutated relative to the reference sequence. Alternatively, the knobs-into-holes structure may be introduced through other amino acid mutations well known to those skilled in the art. Such mutations have been well described in previous literature (Wei et al., Oncotarget 2017, 8(31):51037-51049; Ridgway et al., Protein Eng. 1996, 9(7):617-621; Carter, P., J. Immunol. Methods 2001, 48(1-2):7-15; Merchant et al., Nat. Biotechnol. 1998, 16(7):677-681). For example, a bispecific dimeric fusion protein can be generated by combining a knob structure in which the 22nd amino acid, threonine, in the CH3 domain of the first fusion protein is replaced with tylosin and a hole structure in which the 63rd amino acid, tylosin, in the CH3 domain of the second fusion protein is replaced with threonine.The Knobs-into-Hole structure can also be formed by introducing a Hole structure into the first fusion protein and a Knob structure into the second fusion protein. Gene constructs encoding the first and second fusion proteins of the bispecific fusion proteins of the comparative example and the bispecific fusion proteins of Examples 1 to 4 were synthesized. For transient expression in animal cells, HEK293F cells were transfected with plasmid DNA using the N293F vector system (Ybiologics). To prepare the nucleic acid, 25 μg of plasmid DNA was added to 3 ml of medium and mixed, followed by the addition of 25 μg of 2 mg / ml PEI (Polyethylenimine, PolyPlus, USA). The reaction mixture was left at room temperature for 15 minutes and then diluted to 1 x 10. 6 The cells were cultured at 80-1000 ml of medium containing 1000 cells / ml and cultured for 24 hours at 120 rpm, 37°C, and 8% CO2. 24 hours after DNA transfection, nutrient supplements (Soytone, BD, USA) were added to a final concentration of 10 g / L. One day after transfection, the temperature was lowered to 32°C and the culture was continued until the seventh day.

[0141] The supernatant obtained from the culture was purified through a Protein A column and a secondary column to obtain a comparative bispecific fusion protein (designated "MG12-5") and the bispecific fusion proteins of Examples 1 to 4 (designated "MG12-6," "MG12-7," "MG12-8," and "MG12-9," respectively). These were then diluted appropriately with 4X LDS sample buffer and water for injection to prepare final concentrations of 3-10 μg / 20 μL. For reducing condition samples, each target substance was appropriately diluted with 4X LDS sample buffer, 10X reducing agent, and water for injection to prepare final concentrations of 3-10 μg / 20 μL. These samples were then heated in a 95°C heating block for 10 minutes. 20 μL of the prepared samples were loaded into each well of a gel installed in the pre-installed electrophoresis equipment. For size markers, 3-5 μL / well was loaded. Electrophoresis was performed after setting the power supply to 120 V and 90 minutes. After electrophoresis was completed, the gel was separated and stained using a staining solution and a destaining solution, and the results were analyzed.

[0142] As a result of the analysis, as can be seen from Figures 2A to 2D, all bispecific fusion proteins were observed between 50 and 75 kDa under non-reducing conditions (NR) and at 37 kDa under reducing conditions (R).

[0143] In conclusion, all bispecific fusion proteins according to the present invention can be successfully produced and purified by SDS-PAGE. In particular, as demonstrated by a comparison between MG12-5 and GLP-2-Fc homodimers, the formation of monomeric impurities was significantly reduced in bispecific fusion proteins incorporating the KiH structure compared to those without the KiH structure.

[0144] Experimental Example 1: Confirmation of in vitro activity of GLP-1 and GLP-2

[0145] The present inventors investigated the in vitro GLP-1 and GLP-2 activity of the bispecific fusion proteins prepared in the above examples. Specifically, the present inventors used CHO-K1 cells (DiscoverX) that stably express the GLP-1 receptor to evaluate GLP-1 receptor activity. GLP-1R-expressing CHO-K1 cells were subcultured and plated in a 96-well white plate at 5 x 10 5 The plates were then cultured in 100μL aliquots at 37°C for 20-24 hours in a 5% CO2 incubator. After confirming stable cell attachment, drugs were added at 3x the final concentration. The culture medium was then removed from the plate, and the plates were treated with 30μL of experimental reagent and 15μL of GLP-1 drugs. After DIRANF treatment, the plates were incubated at 37°C for 30 minutes. After incubation at 37°C, a total of 75μL of cell lysis solution and activity assessment reagent was added, and the incubation was continued at room temperature for 1 hour. A fluorescent reaction was then induced for 4-6 hours. The amount of cAMP increased by the GLP-1 receptor agonist binding to and stimulation of the CHO-K1 cells was measured by measuring the level of luciferase activity using the provided solution and a Spectramax M5 instrument. Drug activity was assessed by measuring the level of bioluminescence produced by luciferase activity.

[0146] To evaluate GLP-2 receptor activity, CHO-K1 cells (DiscoverX) stably expressing the GLP-2 receptor were used. Except for the use of GLP-2R-expressing CHO-K1 cells, GLP-2 activity was evaluated using the same method as described above. The activity of MG12-5, a comparative example, was measured using the values ​​described in Korean Patent No. 10-2349718.

[0147] As can be seen from Tables 2 and 3, the GLP-1 activity of Comparative Example MG12-5 was 35% of that of wild-type GLP-1, while the bispecific fusion proteins of Examples 1 to 3 of the present invention exhibited at least 40% or more activity compared to the wild-type GLP-1 peptide, demonstrating similar or even greater activity than wild-type GLP-1. In particular, Example 2 exhibited high activity of more than 150% compared to wild-type GLP-1. In addition, in terms of GLP-2 activity, while Comparative Example MG12-5 exhibited 59% of the activity of wild-type GLP-2, all of the bispecific fusion proteins of Examples 1 to 3 exhibited less than 50% activity. In particular, the bispecific fusion proteins of Examples 2 and 3 exhibited very low activity of approximately 2% and 7%, respectively, compared to wild-type GLP-2.

[0148] [Table 2]

[0149] [Table 3]

[0150] Experimental example 2: Analysis of weight loss effects

[0151] 2-1: Efficacy analysis of single-dose administration

[0152] The present inventors evaluated the efficacy of single administration of MG12-5 (a control group) and MG12-8 (an embodiment of the present invention) on weight loss. Specifically, normal 7-week-old male C57BL / 6N mice were obtained and acclimatized for one week with free access to food and water. The mice were then weighed and randomly assigned to experimental groups to ensure similar average weights. MG12-5 (100 nmol / 5 ml / kg), MG12-8 (100 nmol / 5 ml / kg), and drug-free vehicle, formulated in MG12 formulation buffer, were then subcutaneously injected into the flanks of the mice. Three days after drug administration, the mice were weighed to evaluate drug-induced weight loss (Figure 3A).

[0153] As a result, as can be seen in Figure 3B, in the vehicle-administered group, there was no change in body weight or an increase due to drug administration. Meanwhile, in the comparison group, the MG12-5-administered group, the body weight was maintained at a similar level to that before drug administration, while in the MG12-8-administered group according to one embodiment of the present invention, the body weight decreased by approximately 5%. This indicates that the GLP-1 activity of a bispecific fusion protein comprising a GLP-1 analog and a GLP-2 analog should be 40% or more relative to the wild-type GLP-1 peptide, and the GLP-2 activity of the fusion protein should be 50% or less relative to the wild-type GLP-2 peptide.

[0154] 2-2: Analysis of the effect of repeated administration

[0155] Next, the inventors evaluated the efficacy of repeated administration of MG12-5 (a comparative example) and MG12-8 (an embodiment of the present invention) on weight loss. Specifically, normal 7-week-old male C57BL / 6J mice were obtained and acclimatized for one week with free access to food and water. The mice were then weighed and randomly divided into groups to ensure similar average weights. MG12-5 (30 nmol / 5 ml / kg), MG12-8 (30 nmol / 5 ml / kg), and drug-free vehicle, formulated in MG12 formulation buffer, were then injected subcutaneously into the flank of the mice every other day for two weeks, for a total of seven injections. Body weights were then recorded every other day from the first day of drug administration, and the weight change compared to before drug administration was expressed as a percentage (%).

[0156] As a result, as can be seen from Figure 3D, unlike the vehicle-administered group, weight loss due to drug administration was observed in the experimental groups administered with the bispecific fusion protein according to the comparative example and one embodiment of the present invention. However, when the comparative example MG12-5 was repeatedly administered, it was confirmed that this weight loss effect gradually returned to normal values. Meanwhile, in the case of the MG12-8-administered group according to one embodiment of the present invention, an additional 5% weight loss effect was observed compared to the comparative example (MG12-5), and it was confirmed that weight loss due to repeated administration was maintained compared to MG12-5.

[0157] Meanwhile, GLP-2R agonists, which are primarily used to treat short bowel disease, are known to cause gallbladder hypertrophy as a side effect. Therefore, in the case of a bispecific fusion protein containing both a GLP-1R agonist and a GLP-2R agonist, as in the present invention, if the activity of the GLP-2R agonist is excessively strong, such a side effect may occur. Therefore, the inventors measured the volume of the gallbladder of the mice the day after the last drug administration by sacrificing the mice and then placing a scale around the gallbladder and taking photographs.

[0158] As a result, as can be seen in Figure 3E, when gallbladder hypertrophy due to repeated drug administration was observed, the gallbladder volume in the comparison group (MG12-5 administration group) increased 2.3 times compared to the vehicle administration group, while the gallbladder volume in the MG12-8 administration group according to one embodiment of the present invention was not statistically different from the vehicle administration group. This indicates that the GLP-2 activity of the bispecific fusion protein of the present invention is not toxic unless its relative activity (%) is less than 50% compared to the wild-type GLP-2 peptide.

[0159] Experimental Example 3: Analysis of the therapeutic effect of metabolic disorder-related steatohepatitis with leaky gut

[0160] Next, the inventors investigated the effects of Liraglutide (a control group) and MG12-8 (an embodiment of the present invention) on weight loss and the ability to regulate leaky gut in an animal model of metabolic disorder-associated steatohepatitis with leaky gut. Specifically, the inventors obtained 20-week-old male C57BL / 6J mice and induced overweight with a choline-deficient high-fat diet (CD-HFD). Furthermore, leaky gut was induced by treating the mice with a low concentration of 1% DSS (dextran sulfate sodium) for 7 days followed by a 10-day water cycle. Two weeks after the initial CD-HFD diet, Liraglutide and MG12-8 were subcutaneously injected at the given doses and intervals, and body weights were measured two weeks after the initial drug administration (Figure 4A). To confirm intestinal leakage, FITC (fluorescein isothiocyanate)-Dextran (4 kDa) solution was injected into the oral cavity of the mice 4 hours before sacrifice, and then feeding was discontinued. The amount of FITC-Dextran transferred to the blood was then assessed by measuring blood fluorescence.

[0161] As a result, as can be seen in Figure 4B, the final body weight of the vehicle-administered group was approximately 37g, which was confirmed to be 20% higher than the normal weight range of 30g. Meanwhile, the Liraglutide-administered group and the MG12-8-administered group according to an embodiment of the present invention both showed a decrease in body weight compared to the vehicle-administered group, and among them, MG12-8-administered according to an embodiment of the present invention was confirmed to have the most effective weight loss effect.

[0162] Meanwhile, analysis of the effect on intestinal leakage showed that MG12-8 according to an embodiment of the present invention exhibited superior inhibitory activity against intestinal leakage compared to the control group, as confirmed in Figure 4C. On the other hand, Liraglutide did not exhibit inhibitory activity against intestinal leakage compared to the control group. Furthermore, while the comparative Liraglutide was administered at a high dose of 55 nmol / kg twice daily, MG12-8 according to an embodiment of the present invention was administered at a relatively low dose of 15 nmol / kg, and the administration interval was also more than four times longer than that of Liraglutide, at twice daily, three times a week. Considering this, it can be seen that MG12-8 according to an embodiment of the present invention is also an even superior substance from the patient's perspective.

[0163] Experimental Example 4: Analysis of GLP-1 activity in vivo

[0164] The present inventors analyzed the in vivo GLP-1 activity of MG12-8 according to one embodiment of the present invention. To this end, the present inventors obtained four MG12-8 samples with different in vitro GLP-1 activity through RP-HPLC. Specifically, the four samples showed differences in the areas of peaks 1 and 3 in the RP-HPLC analysis. In sample 1, the area of ​​peak 1 was small, and the area of ​​peak 1 increased with increasing sample number. Conversely, the area of ​​peak 3 decreased with increasing sample number (Table 4). The in vitro GLP-1 activity of the four samples with different ratios of the areas of peak 1 and peak 3 was analyzed using HEK-GLP-1R cells (eEnzyme) stably expressing GLP-1R. Specifically, the HEK-GLP-1R cells were subcultured and placed in a 96-well assay plate at 3 x 105 The plates were then cultured in 100μL aliquots at 37℃ in a 5% CO2 incubator for 20-24 hours. After confirming stable cell attachment, drugs were added at 5x the final concentration. The culture medium was then removed from the plate, and appropriate amounts of experimental reagents and GLP-1 drugs were added. After 2 hours of drug reaction, the increased cAMP levels were measured using the provided solution and a Spectramax M5 instrument to assess drug activity.

[0165] [Table 4]

[0166] As shown in Figure 5A, the relative GLP-1 activity was 100% for Sample 1, approximately 200% for Sample 2, approximately 300% for Sample 3, and approximately 400% for Sample 4. Next, the inventors used the four samples with different in vitro GLP-1 activity in animal experiments to investigate in vivo GLP-1 activity. Specifically, to do this, the inventors performed an intraperitoneal glucose tolerance test (IPGTT). After administering the drug 18 hours before the IPGTT, the animals were fasted by removing food and bedding. Then, 18 hours after drug administration, blood glucose changes were observed over time before and after intraperitoneal administration of a 20% glucose solution (Figure 5B).

[0167] As a result, as can be seen from Figure 5C, samples that exhibited 100-300% in vitro GLP-1 activity exhibited similar in vivo blood glucose-lowering efficacy. Meanwhile, samples that exhibited 400% in vitro GLP-1 activity exhibited even higher in vivo blood glucose-lowering efficacy than samples with 100% GLP-1 activity. This demonstrates that a bispecific fusion protein according to an embodiment of the present invention, which exhibits 100-300% GLP-1 activity in vitro compared to a wild-type GLP-1 peptide, exhibits similar efficacy in vivo.

[0168] Experimental Example 5: Analysis of the effect of obesity treatment in a high-fat diet model

[0169] 5-1: Comparative analysis of efficacy compared with conventional GLP-1R / GLP-2R dual acting agents

[0170] The present inventors further analyzed the efficacy of a bispecific fusion protein according to one embodiment of the present invention in treating obesity, in comparison with previously reported GLP-1R / GLP-2R dual agonists. Specifically, the present inventors investigated the effects of 30 nmol / kg dapiglutide mimetic and MG12-8 (hereinafter referred to as "MG12" for convenience) at 15 and 30 nmol / kg to compare its efficacy with that of a known GLP-1R / GLP-2R dual agonist peptide substance (dapiglutide mimetic). To analyze body composition, mice were measured for total body fat mass and lean mass (muscle and bone mass) using a Minispec LF50 (Bruker Biospin, BCA LF50) instrument. To observe drug-induced changes in body composition, the percentage change in body composition was measured before and after two weeks of drug administration. To this end, 5-week-old normal C57BL / 6N male mice were obtained and, after one week of acclimation, administered a 60% high-fat diet (HFD) to induce diet-induced obesity (DIO). Sixteen weeks after the initial HFD, appropriately formulated dapiglutide mimetics and a bispecific fusion protein (MG12) according to one embodiment of the present invention were injected subcutaneously into the flank of the mice every two days.

[0171] The changes in body weight and body composition before and after two weeks of drug administration were analyzed. As can be seen from the graph on the left side of Figure 6A, administration of 30 nmol / kg of dapiglutide mimetic resulted in a weight loss of approximately 5%, while administration of 15 nmol / kg of the bispecific fusion protein (MG12) according to one embodiment of the present invention resulted in a weight loss of 13%, and administration of 30 nmol / kg resulted in a weight loss of approximately 20%.

[0172] Furthermore, after the experiment was completed and the experimental animals were sacrificed, the weight ratio of adipose tissue to non-adipose tissue was measured. As can be seen from the graph on the right side of Figure 6A, the normal diet and high-fat diet groups showed similar increases in fat weight, but the dapiglutide mimetic and bispecific fusion protein according to an embodiment of the present invention groups showed decreases in fat among body components. In particular, the dapiglutide mimetic group showed a decrease of about 6%, while the 15 nmol / kg MG12 group and the 30 nmol / kg MG12 group showed decreases of about 18% and about 35%, respectively, demonstrating that this substance has a significantly superior effect in treating obesity compared to existing substances.

[0173] 5-2: Concentration-dependent effect analysis

[0174] To determine whether the in vivo effects of a bispecific fusion protein (MG12) according to one embodiment of the present invention are concentration-dependent, the inventors analyzed the weight loss effect in a high-fat diet-induced obesity model animal using various concentrations of the bispecific protein. Specifically, to evaluate the weight loss efficacy of MG12 at different concentrations, 5-week-old normal C57BL / 6 male mice were obtained and, after one week of acclimation, administered a 60% high-fat diet (HFD) to induce diet-induced obesity (DIO). Starting 16 weeks after the initial HFD, MG12 was formulated in MG12 formulation buffer at 15, 30, and 60 nmol / 5 ml / kg and administered subcutaneously to the abdominal region of the mice every other day. Mice were randomly divided into groups based on their weight to ensure similar mean body weights, and weight changes after drug administration were expressed as a percentage (%).

[0175] As shown in Figure 6B, both the normal diet group and the HFD diet group tended to maintain or increase their body weight over the two-week period. However, in the MG12-treated group, increasing the concentration of MG12 resulted in a weight loss of approximately 13-25%, and this weight loss effect was concentration-dependent.

[0176] Next, to confirm whether the effect of the bispecific fusion protein according to one embodiment of the present invention is similar to that of the simple co-administration of GLP-1 peptide and GLP-2 peptide, the inventors separately prepared GLP-1-Fc and GLP-2-Fc, which are the fusion partners of the bispecific fusion protein according to one embodiment of the present invention, and performed a comparative analysis of the weight changes observed when the fusion partners were simply co-administered (30 nmol / kg + 30 nmol / kg) with the bispecific fusion protein according to one embodiment of the present invention (MG12) at the same molar concentration (30 nmol / kg).

[0177] As a result, as can be seen from Figure 6C, it was confirmed that the same dose of MG12 further improved weight loss by approximately 5% compared to the simple combination of GLP-1-Fc and GLP-2-Fc alone.

[0178] 5-3: Analysis of the effect of fat-specific weight loss

[0179] The present inventors compared the fat-specific weight loss effects of tirzepatide mimetic, a GLP-1R / GIPR peptide dual-acting agent used as a conventional anti-obesity agent, with a bispecific fusion protein according to one embodiment of the present invention. Specifically, the present inventors obtained 5-week-old normal C57BL / 6 male mice and, after one week of acclimation, induced diet-induced obesity (DIO) by feeding them a 60% high-fat diet (HFD). Sixteen weeks after the initial HFD, tirzepatide mimetic and MG12 were formulated in MG12 formulation buffer at 15 and 60 nmol / 5 ml / kg, respectively, and then subcutaneously administered. For body composition analysis, mice were measured for total body fat, muscle, and bone weights using a Minispec LF50 (Bruker Biospin, BCA LF50) instrument. To observe drug-induced changes in body composition, the rate of change in body composition was observed before and after two weeks of drug administration.

[0180] As a result, as shown in Figure 6D, two weeks of treatment with 15 nmol / kg tirzepatide mimetic and 60 nmol / kg MG12 resulted in a similar 23% weight loss compared to the control group (vehicle-treated group). Also, as shown in Figure 6E, MG12 treatment resulted in a 52% reduction in body fat, compared to a 44% reduction in fat mass with the tirzepatide mimetic. Meanwhile, as shown in Figure 6F, muscle loss was measured and found to be only 7% in the MG12-treated group, compared to a 14% reduction in muscle mass with the tirzepatide mimetic-treated group.

[0181] 5-4: Analysis of efficacy of visceral fat suppression

[0182] Next, the inventors investigated whether the fat-reducing effect of the bispecific fusion protein according to one embodiment of the present invention was due to a reduction in subcutaneous fat or visceral fat. To this end, the inventors sacrificed the experimental animals after completing the experiment in Experimental Example 5-3, excised the subcutaneous fat and visceral fat, measured their respective weights, and divided the weights by body weight to convert them into the body fat percentage of each adipose tissue.

[0183] As a result, as can be seen from the left graph in Figure 6G, in the case of subcutaneous fat, both the tirzepatide mimetic and MG12 showed a tendency for subcutaneous fat percentage to be similar or decreased compared to the control group (vehicle administration group), but the results were not statistically significant. Meanwhile, when examining the body fat percentage relative to visceral fat, as can be seen from the right graph in Figure 6G, the tirzepatide mimetic administration group showed a decrease to the level of the normal control group compared to the vehicle administration group. The visceral fat content in the group administered with the bispecific fusion protein according to one embodiment of the present invention (MG12) was even lower than that in the tirzepatide mimetic administration group, demonstrating that the compound is a highly effective substance for reducing visceral fat in obese animals.

[0184] Experimental Example 6: Analysis of the therapeutic effects of endotoxin and liver injury

[0185] Left untreated, obesity can progress to metabolically impaired fatty liver disease (NAFLD), metabolically impaired steatohepatitis (NASH), and even irreversible liver damage, liver cirrhosis. To investigate whether metabolic endotoxemia and liver damage, which are common symptoms in obese animal models, could be suppressed, the inventors administered the GLP-1 / GIPR dual-acting agent tirzepatide mimetic and a bispecific fusion protein (MG12) according to one embodiment of the present invention at the same potency concentration for two weeks, and then collected serum samples and analyzed for their components. Endotoxemia was analyzed using a mouse LPS ELISA kit (CSB-E13066m, Cusabio), and liver damage was analyzed by measuring ALT levels using dry-chem equipment (DRI-CHEM NX-500, FUJI).

[0186] As a result, as can be seen in Figure 7A, diet-induced obesity (DIO) increased serum lipopolysaccharide (LPS) levels, inducing endotoxemia. This level did not decrease with administration of the tirzepatide mimetic, but decreased to the control level with administration of the bispecific fusion protein according to one embodiment of the present invention. Furthermore, analysis of the extent of liver damage confirmed, as can be seen in Figure 7B, that diet-induced obesity caused liver damage and a significant increase in ALT levels. In the tirzepatide mimetic-administered group, ALT levels were reduced to half the level of the vehicle-administered group, and in the bispecific fusion protein (MG12)-administered group according to one embodiment of the present invention, a statistically significant improvement in ALT levels was observed compared to the tirzepatide mimetic-administered group.

[0187] Experimental Example 7: Analysis of the effect of glucose absorption

[0188] To confirm whether a bispecific fusion protein according to an embodiment of the present invention can be used to treat diabetes, the inventors performed a 2-NBDG glucose absorption assay using mouse pre-adipocyte 3T3-L1 cells and rat myoblast L6-GLUT4myc cells (provided by: From the laboratory of Amira Klip, PhD, Hospital for Sick Children).

[0189] Specifically, the culture medium of saturated 3T3-L1 preadipocytes cultured in a 24-well plate was replaced with 24-differentiation induction medium [DMEM medium containing 10% FBS (Cytiva, USA), 1% penicillin / streptomycin (P / S, Cytiva, USA), 1 μM dexamethasone (DEXA, Sigma, USA), 0.5 mM 3-isobutly-1-methylxanthine (IBMX, Sigma, USA), 2 μM rosiglitazone (Cayman, Germany), and 1 μg / mL insulin (Sigma, USA)], and the cells were cultured for 11 days under 5% CO2 conditions to induce differentiation into adipocytes. The differentiated cells were then insulin-starved and treated with MG12 (300 nM), a bispecific fusion protein according to one embodiment of the present invention, and 10 μg / mL insulin. They were then cultured in glucose-free DMEM containing 100 μg / mL 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-Deoxyglucose (2-NDBG, Cayman, USA). Semaglutide and dapiglutide were used as positive controls at the same dose (300 nM). After 30 minutes of incubation, the cells were washed with glucose-free DMEM and then cultured in lysis buffer (RIPA buffer) for 20 minutes. Fluorescence signals were measured at 485 nM excitation and 535 nM emission wavelengths using a microplate reader (SpectraMax M3, USA).

[0190] Similarly, 2 × 10 rat L6-GLUTmyc muscle cells were plated in a 24-well plate. 4After two days, the medium was replaced with α-MEM media (Gibco, USA) containing 1% penicillin streptomycin and 2% FBS (Cytiva, USA), and muscle cell differentiation was performed for five days. After differentiation, the cells were treated with MG12 (300 nM), a bispecific fusion protein according to one embodiment of the present invention, and 10 μg / mL insulin. Subsequently, the cells were treated with 2-NDBG at a concentration of 100 μg / mL for 30 minutes. After washing with α-MEM media and incubation in lysis buffer, the intracellular 2-NBDG content was measured using a microplate reader (SpectraMax M3, USA) at 485 nm (excitation wavelength) and 535 nm (emission wavelength).

[0191] As a result, as shown in Figure 8, it was confirmed that the bispecific fusion protein (MG12) according to one embodiment of the present invention has a glucose uptake efficiency in differentiated adipocytes as well as muscle fibers that is superior to that of GLP-1-Fc, GLP-2-Fc, the existing GLP-1 analog (Semaglutide), and the GLP-1R / GLP-2R dual agonist (Dapiglutide).

[0192] Experimental Example 8: Animal experiment to verify the effectiveness of diabetes treatment

[0193] 8-1: Fasting blood glucose, blood insulin concentration, HOMA-IR and HOMA-β score analysis

[0194] The present inventors conducted animal experiments using diabetic model mice to confirm whether the bispecific fusion protein according to one embodiment of the present invention improves insulin resistance as well as blood glucose control.

[0195] Specifically, the inventors administered designated doses of semaglutide, tirzepatide, or the bispecific fusion protein MG12-8 according to one embodiment of the present invention subcutaneously to 14-week-old male db / db mice (Janvier Labs, France) once every three days for 12 weeks.

[0196] Thereafter, non-fasting blood glucose levels were measured at 25-day intervals on days 25, 50, and 75 of the study, and blood glycosylated hemoglobin (HbA1c) levels were measured at monthly intervals until month 3. After the study was completed, fasting blood glucose and blood insulin levels, as well as HOMA-IR and HOMA-β scores were calculated.

[0197] As a result, as shown in Figure 9, the bispecific fusion protein (MG12) according to one embodiment of the present invention was confirmed to have significantly superior non-fasting blood glucose lowering ability and blood glycosylated hemoglobin (HbA1c) levels at a dose of 30 nmol / kg compared to 30 nmol / kg semaglutide and 15 nmol / kg tirzepatide. It also significantly reduced fasting blood glucose and significantly increased fasting blood insulin levels. Furthermore, while 30 nmol / kg semaglutide and 15 nmol / kg tirzepatide both exceeded 40 and did not eliminate insulin resistance, the bispecific fusion protein according to one embodiment of the present invention lowered the HOMA-IR score to the control group level and also showed significantly higher HOMA-β scores, which indicate β-cell function, compared to 30 nmol / kg semaglutide and 15 nmol / kg tirzepatide, suggesting its potential application in the treatment of not only type 2 diabetes but also type 1 diabetes.

[0198] 8-2:Histological analysis

[0199] After the experiment was completed, the inventors sacrificed the experimental animals and then performed histological analysis of the pancreatic tissues.

[0200] Specifically, after the animal experiments, the experimental animals were sacrificed under general anesthesia, and the pancreas was removed and fixed in 4% paraformaldehyde and embedded in paraffin. The tissue was then cut into cross sections (5 μm) using a microtome, and stained for insulin alone, double stained for insulin and glucagon, or double stained for insulin and Ki-67. Double staining was performed using fluorescently labeled antibodies, and images were taken using a confocal laser microscope.

[0201] As a result, as can be seen from FIG. 10, the bispecific protein according to an embodiment of the present invention not only significantly increased the area of ​​pancreatic islets, unlike the conventional diabetes treatments semaglutide and tirzepatide, but also significantly increased the area of ​​β cells among the total islets, while decreasing the area of ​​α cells, resulting in a reduction of 10,000 μm 2 The positive control, semaglutide, showed a significant increase, but its effect was weaker than that of MG12 at the same dose, and tirzepatide showed no improvement compared to the control group.

[0202] These results suggest that the bispecific fusion protein according to one embodiment of the present invention is a substance that has a very high protective effect on pancreatic β cells, unlike conventional diabetes treatments.

[0203] As described above, the bispecific fusion protein according to one embodiment of the present invention is safe and highly effective in reducing body weight and visceral fat content in obese animals, and has been confirmed to be a highly effective substance for treating metabolic diseases such as liver damage caused by prolonged conditions such as diabetes, including type 1 diabetes as well as type 2 diabetes, and obesity.

[0204] The present invention has been described with reference to the above-mentioned embodiments and experimental examples, but these are merely illustrative, and those skilled in the art will recognize that various modifications and equivalent embodiments are possible. Therefore, the true technical scope of the present invention should be determined by the technical spirit of the claims. [Industrial Applicability]

[0205] The bispecific fusion proteins according to one embodiment of the present invention can be used as medicines, particularly for the treatment of metabolic diseases such as obesity, diabetes, and metabolic disorders related to steatohepatitis.

Claims

1. In a bispecific fusion protein formed by the fusion of a GLP-1 analog and a GLP-2 analog, A bispecific fusion protein in which the activity of the GLP-1 analog is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analog is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).

2. A bispecific fusion protein comprising a first fusion protein in which a GLP-1 analog is linked to the antibody Fc region and a second fusion protein in which a GLP-2 analog is linked to the antibody Fc region, wherein the bispecific fusion protein produced by dimerization of the first and second fusion proteins, A bispecific fusion protein in which the activity of the GLP-1 analog is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analog is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).

3. The bispecific fusion protein according to claim 1 or 2, wherein the GLP-1 analog exhibits at least 80% of the activity of wild-type GLP-1 under in vitro conditions.

4. The bispecific fusion protein according to claim 1 or 2, wherein the GLP-2 analog exhibits at least 2% of the activity of wild-type GLP-2 under in vitro conditions.

5. A bispecific fusion protein according to claim 1 or 2, wherein a half-life-enhancing moiety is added.

6. The bispecific fusion protein according to claim 5, wherein the half-life increasing moiety is inserted between the GLP-1 analog and the GLP-2 analog, or is attached to the N-terminus or C-terminus of the overall fusion protein.

7. The bispecific fusion protein according to claim 5, wherein the half-life increasing moiety is an antibody Fc region, PEG, XTEN, PAS, ELP, glycine-rich HAP, GLP, or serum albumin.

8. The bispecific fusion protein according to claim 1, wherein GLP-1 and GLP-2 are not proglucagon or analogs linked by an intermediate peptide, but rather the GLP-1 analog and GLP-2 analog are directly linked, or the two peptides are linked by another form of linker peptide that is not the intermediate peptide.

9. The bispecific fusion protein according to claim 1 or 2, wherein the GLP-1 analog is a continuous repeat of GLP-1, exendin 3, exendin 4, GLP-1 / Exendin 4 hybrid peptide, GLP-1-XTEN, Exendin 4-XTEN, Lixisenate, Albiglutide, Liraglutide, or Taspoglutide, or one or more of these linked together.

10. The aforementioned GLP-2 analogs are GLP-2, GLP-2 Ａ２Ｇ Mutant, GLP- ２Ｎ１６Ｇ＿Ｌ１７Ｑ Mutant, GLP-2 Ａ２Ｇ＿Ｎ１６Ｇ＿Ｌ１７Ｑ The bispecific fusion protein according to claim 1 or 2, wherein the protein is a mutant, Glepaglutilde, or GLP-2 analogue 10.

11. The bispecific fusion protein according to claim 2, wherein the antibody Fc region is a hybrid Fc region in which two or more isotype antibody Fc regions are mixed.

12. The bispecific fusion protein according to claim 11, wherein the hybrid Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 16.

13. The bispecific fusion protein according to claim 2, wherein the antibody Fc region is an antibody Fc region on which heterodimerized moiety has been formed.

14. The aforementioned heterodimerized moiety is Knobs-into-Holes (KiH), KiH Ｓ－Ｓ .. HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT Ｓ－Ｓ The bispecific fusion protein according to claim 13, which is SEED or A107.

15. A composition comprising the bispecific fusion protein described in claim 1 or 2.

16. A pharmaceutical composition for the treatment of metabolic diseases, comprising the bispecific fusion protein described in claim 1 or 2 as an active ingredient.

17. The pharmaceutical composition according to claim 16, wherein the metabolic disease is obesity, diabetes, metabolic syndrome, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, hepatic fibrosis, or cirrhosis.

18. The pharmaceutical composition according to claim 17, wherein the diabetes is type 1 diabetes or type 2 diabetes.

19. A bispecific fusion protein according to claim 1 or 2, used for the treatment of metabolic diseases.

20. The bispecific fusion protein according to claim 19, wherein the metabolic disease is obesity, diabetes, metabolic syndrome, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, hepatic fibrosis, or cirrhosis.

21. The bispecific fusion protein according to claim 20, wherein the diabetes is type 1 diabetes or type 2 diabetes.

22. Use of the bispecific fusion protein according to claim 1 or 2 for the manufacture of a therapeutic agent for metabolic diseases.

23. The use according to claim 22, wherein the metabolic disease is obesity, diabetes, metabolic syndrome, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, hepatic fibrosis, or cirrhosis.

24. The use according to claim 23, wherein the diabetes is type 1 diabetes or type 2 diabetes.

25. A method for treating a metabolic disorder in an individual, comprising the step of administering a therapeutically effective amount of the bispecific fusion protein according to claim 1 or 2 to a patient suffering from a metabolic disorder.

26. The method according to claim 25, wherein the metabolic disease is obesity, diabetes mellitus, metabolic syndrome, metabolic disorder-related fatty liver disease, metabolic disorder-related steatohepatitis, hepatic fibrosis, or cirrhosis.

27. The method according to claim 26, wherein the diabetes is type 1 diabetes or type 2 diabetes.

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