WO2024263005A1 - 신규 이중 특이성 융합단백질 및 그의 용도 - Google Patents

신규 이중 특이성 융합단백질 및 그의 용도 Download PDF

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WO2024263005A1
WO2024263005A1 PCT/KR2024/008655 KR2024008655W WO2024263005A1 WO 2024263005 A1 WO2024263005 A1 WO 2024263005A1 KR 2024008655 W KR2024008655 W KR 2024008655W WO 2024263005 A1 WO2024263005 A1 WO 2024263005A1
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glp
fusion protein
analogue
diabetes
dual
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French (fr)
Korean (ko)
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성영철
양상인
노재일
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Slbigen Inc
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Slbigen Inc
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Priority to EP24826320.4A priority Critical patent/EP4733314A1/en
Priority to JP2025549728A priority patent/JP2026507086A/ja
Priority to AU2024313859A priority patent/AU2024313859A1/en
Priority to CN202480041232.3A priority patent/CN121358758A/zh
Publication of WO2024263005A1 publication Critical patent/WO2024263005A1/ko
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to a novel dual-specificity fusion protein and its use, and more particularly, to a dual-specificity fusion protein comprising a GLP-1 analogue and a GLP-2 analogue, wherein the activity of the GLP-1 analogue and the activity of the GLP-2 analogue are precisely controlled, and its use.
  • GLP-1 and GLP-2 are peptide hormones that are produced through a series of tissue-specific protein cleavage processes that are expressed as a precursor called proglucagon.
  • GLP-1 is produced and secreted by specific nerve cells in the enteroendocrine L cells of the small intestine and the solitary tract nucleus of the brain stem when food is ingested.
  • the initial product, GLP-1(1-37) is easily amidated and converted into two truncated forms (GLP-1(7-36) amide and GLP-1(7-37)) with equivalent biological activities through cleavage. Since active GLP-1 plays a role in lowering blood sugar levels in a glucose-dependent manner, it has been developed and used as a treatment for type 2 diabetes.
  • GLP-2 is a 33 amino acid long peptide produced by a special post-translational cleavage process of proglucagon, produced in enteroendocrine L cells of the small intestine and various nerve cells of the central nervous system, and secreted together with GLP-1 in the small intestine when food is ingested.
  • GLP-2 is known to improve small intestine growth and function, reduce bone destruction, and have neuroprotective effects when administered, and is currently being developed as a treatment for diseases such as short bowel syndrome, Crohn's disease, and osteoporosis.
  • GLP-1 originally 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).
  • GLP-2 it has been suggested that it may play a role in the treatment of short bowel syndrome through research results showing that it reduces gastric secretion and motility (Kunkel et al ., Neurogastroenterol. Motil . 23: 739-e328, 2011).
  • GLP-1R GLP-1 receptor
  • metabolic dysfunction-associated steatotic liver disease is not a single disease, but rather includes various forms of liver disease ranging from simple fatty liver without inflammation to chronic hepatitis and cirrhosis, and is known to be closely related to obesity and resulting insulin resistance.
  • Metabolic dysfunction-associated fatty liver disease can vary greatly in severity from mild fatty liver disease with only fat accumulated without liver cell damage, to steatohepatitis with severe and persistent liver cell damage, and even liver fibrosis or cirrhosis (liver cirrhosis) accompanied by ascites or jaundice, and is known to progress further to liver cancer.
  • the incidence of MASLD is rapidly increasing as a Western-type disease due to the spread of Western eating habits.
  • metabolic-related fatty liver disease is a mild disease, but one in four patients with severe fatty liver disease will develop cirrhosis (or fibrosis), a serious liver disease, over time if left untreated. This is by no means a symptom that can be ignored.
  • the prevalence of metabolic-related fatty liver disease varies depending on the characteristics of the population, ranging from 10% to 24% in the general population to 58% to 74% in obese individuals.
  • drugs approved as treatments for metabolic-related fatty liver disease or metabolic-related steatohepatitis and the development of treatments for metabolic-related fatty liver disease is considered a very urgent task.
  • the present inventors have developed a dual specificity protein that simultaneously targets GLP-1R and GLP-2R (Korean Patent No. 10-2349718).
  • dual-specificity fusion protein can be used universally for various metabolic diseases such as short bowel syndrome, obesity, type 2 diabetes, and metabolic disorder-associated steatohepatitis, there is no disclosure at all about a form of dual-specificity fusion protein more suitable for metabolic diseases.
  • the present invention is intended to solve various problems including the above-described problems, and aims to provide a novel dual-specificity fusion protein having an activity more suitable for the treatment of metabolic diseases while maintaining the functions of GLP-1 and GLP-2.
  • the protection scope of the present invention is not limited to the above-described purpose.
  • a dual specificity fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused, wherein the activity of the GLP-1 analogue is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analogue is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).
  • a dual specificity fusion protein comprising a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a second fusion protein in which a GLP-2 analogue is linked to an antibody Fc region, wherein the dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein has an activity of 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analogue is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).
  • composition comprising the dual specificity fusion protein is provided.
  • a pharmaceutical composition for treating a metabolic disease comprising at least one of the above dual specificity fusion proteins as an active ingredient.
  • any one of the above dual specificity fusion proteins for use in the treatment of metabolic diseases.
  • a method of treating a metabolic disease in a subject comprising administering to the subject a therapeutically effective amount of the dual specificity fusion protein.
  • the dual-specificity fusion protein of the present invention can be very effectively used in the treatment of metabolic diseases such as metabolic syndrome, obesity, type 1 diabetes, type 2 diabetes, metabolic disorder-associated fatty liver disease, and liver fibrosis when administered in vivo.
  • metabolic diseases such as metabolic syndrome, obesity, type 1 diabetes, type 2 diabetes, metabolic disorder-associated fatty liver disease, and liver fibrosis when administered in vivo.
  • Figure 1 is a schematic diagram showing the schematic structure of a dual-specificity fusion protein according to one embodiment of the present invention.
  • Figure 2a 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 comparative example of the present invention, GLP-2 homodimer (GLP-2-Fc homodimer, left) and MG12-5 containing the Knobs-into-Holes structure (right):
  • Figure 2b is a series of gel photographs showing the results of SDS-PAGE analysis of MG12-6 under reducing and non-reducing conditions according to one embodiment of the present invention:
  • Figure 2c 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:
  • Figure 2d is a gel photograph showing the results of SDS-PAGE analysis of MG12-9 under reducing and non-reducing conditions according to one embodiment of the present invention:
  • FIG. 3a is a schematic diagram showing an experimental schedule, drug dosage, administration interval, etc. for analyzing the weight loss effect upon single administration of a dual-specificity fusion protein (MG12-8) according to an embodiment of the present invention.
  • FIG. 3b is a graph showing the change in body weight of experimental animals as a result of an experiment performed according to the schedule of FIG. 3a.
  • FIG. 3c is a schematic diagram showing an experimental schedule, drug dosage, administration interval, etc. for analyzing the weight loss effect upon repeated administration of a dual-specificity fusion protein (MG12-8) according to an embodiment of the present invention.
  • FIG. 3d is a graph showing the change in body weight of experimental animals as a result of an experiment performed according to the schedule illustrated in FIG. 3c.
  • 3e is a graph showing the results of measuring the volume of the gallbladder of experimental animals sacrificed after the end of the experiment of FIG. 3d.
  • NS indicates statistically not significant
  • ** indicates statistical significance of p ⁇ 0.01
  • the error bar indicates the standard error (SEM).
  • FIG. 4a is a schematic diagram showing an experimental schedule, drug dosage, administration interval, etc. for verifying the therapeutic effect of a dual-specificity fusion protein on fatty liver disease associated with metabolic abnormalities accompanied by intestinal leakage according to one embodiment of the present invention.
  • FIG. 4b is a graph showing the body weight of experimental animals measured after the end of the animal experiment performed according to the schedule shown in FIG. 4a.
  • FIG. 4c is a graph showing the results of measuring the serum fluorescence level of FITC-Dextran orally administered 4 hours before the sacrifice of the experimental animal of FIG. 4b.
  • FIG. 5a is a graph showing the results of analyzing the in vitro GLP-1 activity of various samples of dual-specific fusion proteins manufactured according to one embodiment of the present invention.
  • FIG. 5b is a schematic diagram showing an intraperitoneal administration glucose tolerance test experimental schedule, dosage of administered drugs, administration interval, etc. for analyzing the in vivo GLP-1 activity of various samples of dual-specific fusion proteins having in vitro GLP-1 activity according to one embodiment of the present invention.
  • FIG. 5c is a graph showing the results of the intraperitoneal administration glucose tolerance test performed according to the schedule shown in FIG. 5b.
  • * indicates statistical significance at the p ⁇ 0.05 level
  • ** indicates statistical significance at the p ⁇ 0.01 level.
  • FIG. 6a is a series of graphs showing the results of measuring the body weight change rate (left) and the change rate of fat to non-fat ratio (right) according to administration of a dual-specificity fusion protein according to an embodiment of the present invention, in comparison with Dapiglutide mimetic, a known GLP-1R/GLP-2R dual agonist.
  • FIG. 6b is a graph showing the change in body weight change rate according to the administration dose of a dual-specificity fusion protein according to an embodiment of the present invention.
  • FIG. 6c is a graph showing the results of analyzing the effect of a dual-specificity fusion protein according to an embodiment of the present invention on body weight change compared to simple co-administration of the same dose of GLP-1-Fc and GLP-2-Fc.
  • FIG. 6d is a graph showing the body weight change rate in a diet-induced obesity model animal induced by a high-fat diet of a dual-specificity fusion protein according to an embodiment of the present invention, in comparison with Tirzepatide mimetic, a GLP-1R/GIPR dual agonist.
  • FIG. 6e is a graph showing the results of measuring the weight change rate of the adipose tissue of the experimental animal in the experiment of FIG. 6d.
  • FIG. 6f is a graph showing the results of measuring the weight change rate of the non-adipose tissue of the experimental animal in the experiment of FIG. 6d.
  • 6g is a graph showing the results of measuring the subcutaneous fat percentage (left) and visceral fat percentage (right) of the dual specificity fusion protein according to one embodiment of the present invention in a diet-induced obesity model animal in which obesity is induced by a high-fat diet. Tirzepatide mimetic was used as a positive control.
  • NS indicates statistically not significant
  • * indicates statistical significance at the p ⁇ 0.05 level
  • ** indicates statistical significance at the p ⁇ 0.01 level.
  • FIG. 7a is a graph showing the results of measuring the serum LPS concentration for analyzing the endotoxemia reduction effect in a diet-induced obesity model animal induced by a high-fat diet of a dual-specificity protein according to one embodiment of the present invention.
  • FIG. 7b is a graph showing the results of measuring the serum ALT concentration for analyzing the liver damage inhibition effect in the experimental animal of FIG. 7a.
  • * indicates statistical significance at the p ⁇ 0.05 level
  • ** indicates statistical significance at the p ⁇ 0.01 level.
  • FIG. 8 is a series of graphs showing the results of insulin-stimulated glucose uptake analysis of a dual-specificity fusion protein according to one embodiment of the present invention, wherein (a) normalized mean fluorescence intensity measuring the degree of surface expression of GLP-1R and GLP-2R of differentiated 3T3-L1 adipocytes, (b, c) normalized mean fluorescence intensity measuring the degree of insulin-stimulated glucose uptake of differentiated 3T3-L1 adipocytes, (d) normalized mean fluorescence intensity measuring the degree of surface expression of GLP-1R and GLP-2R of differentiated L6-GLUT4myc myoblasts, and (e, f) normalized mean fluorescence intensity measuring the degree of surface expression of GLP-1R and GLP-2R of differentiated L6-GLUT4myc myoblasts.
  • * indicates statistical significance at the p ⁇ 0.05 level
  • ** indicates statistical significance at the p ⁇ 0.01 level.
  • FIG. 9 is a series of graphs showing the effects of administration of a dual-specificity fusion protein according to one embodiment of the present invention on glycemic control, changes in glycated hemoglobin, and insulin resistance, including (a) non-fasting blood sugar levels, (b) glycated hemoglobin (HbA1c) levels, (c) fasting blood sugar levels, (d) fasting insulin concentrations, (e) HOMA-IR scores, and (f) HOMA- ⁇ scores.
  • * indicates statistical significance at the p ⁇ 0.05 level
  • ** indicates statistical significance at the p ⁇ 0.01 level.
  • Fig. 10 shows the results of histological analysis on the pancreas removed after the experiment of Fig. 9 is completed, including (a-c) micrographs showing the results of immunohistochemical analysis on thin pancreatic tissue sections stained for (a) insulin, (b) insulin and glucagon, and (c) insulin and Ki-67 positive cells, and graphs showing (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 ⁇ m 2 , respectively.
  • * indicates statistical significance at the p ⁇ 0.05 level
  • ** indicates statistical significance at the p ⁇ 0.01 level.
  • GLP-1 as used in this document is an abbreviation for "glucagon-like peptide-1", a 30 or 31 amino acid long peptide hormone derived from the tissue-specific post-translational processing of the proglucagon peptide. GLP-1 is produced and secreted by enteroendocrine L cells of the small intestine and certain neurons in the nucleus of the solitary tract of the brain stem upon food ingestion. The primary product, GLP-1(1-37), is readily amidated and converted by cleavage into two truncated forms (GLP-1(7-36) amide and GLP-1(7-37)) with equally biological activity.
  • Active GLP-1 contains two alpha-helical regions at amino acid positions 13-20 and 24-35 and a linker region connecting the two alpha-helical regions. Since GLP-1 plays a role in lowering blood sugar levels in a glucose-dependent manner, it has been developed and used as a treatment for type 2 diabetes. However, since GLP-1 is rapidly degraded in vivo by dipeptidyl peptidase-4 (DPP-4), its in vivo half-life is only 2 minutes, and its effect as a natural peptide is extremely limited.
  • DPP-4 dipeptidyl peptidase-4
  • GLP-2 refers to a 33 amino acid long peptide produced by the post-translational cleavage process of proglucagon, similar to GLP-1, and produced by enteroendocrine L cells in the small intestine and various nerve cells in the central nervous system. GLP-2 is secreted together with GLP-1 when food is ingested. GLP-2 is known to improve small intestine growth and function, reduce bone destruction, and have neuroprotective effects when administered, and is currently being developed as a treatment for diseases such as short bowel syndrome, Crohn's disease, and osteoporosis.
  • GLP-1 analogue means a protein that performs the biological function of GLP-1 and can bind to the GLP-1/Exendin-4 receptor and mediate downstream signaling, and is also referred to as a "GLP-1 receptor agonist”.
  • GLP-2 analogue means a protein that performs the biological function of GLP-2 and can bind to the GLP-2 receptor and mediate downstream signaling, and is also referred to as a "GLP-2 receptor agonist”.
  • fusion protein used in the present invention means a recombinant protein in which two or more proteins or domains responsible for specific functions within a protein are linked so that each protein or domain takes on its original function.
  • half-life increasing moiety refers to a functional group linked to a recombinant protein to improve 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.
  • ELP elastine-like peptide
  • glycine-rich HAP homo-amino-acid polymer
  • GLK gelatine-like protein, Huang et al ., Eur. J. Pharm. Biopharm. 74(3): 435-441, 2010
  • serum albumin Sheffield et al ., Cell Physiol.
  • Biochem ., 45(2): 772-782, 2018 can be used, and the "half-life increasing moiety" added to such proteins is well known through review papers, etc. (Strohl, WR, BioDrugs , 29(4): 215-239, 2015). Accordingly, prior papers on the individual factors and the review papers are inserted into this document as references.
  • antibody Fc region refers to a crystallized fragment produced when an antibody is cleaved with papain, which interacts with cell surface receptors called Fc receptors and some proteins of the complement system.
  • the Fc region exhibits a homodimeric structure in which fragments containing the second and third constant domains (CH2 and CH3) of the heavy chain are linked by intermolecular disulfide bonds at the hinge region.
  • the Fc region of IgG has multiple N-glycan attachment sites, which are known to play an important role in Fc receptor-mediated actions.
  • hybrid Fc region refers to an Fc region peptide generated by combining portions of Ig Fc regions of various subtypes, and which can exhibit differences from the wild-type Fc region in binding ability to Fc receptors and complement due to the combination of these Fc region portions.
  • Exendin used in this document is 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, and is a member of the glucagon peptide family, known to perform a role equivalent to GLP-1 as an agonist of the GLP-1 receptor.
  • Exendin 4 is also called “extenatide”.
  • Exendin 3 is a mutant of Exendin 4 in which the second and third amino acids are substituted with serine and aspartic acid, respectively.
  • Lutisenatide used in this document is a GLP-1 receptor agonist, a drug manufactured by Sanofi and sold in Europe under the trade name Lyxumia and in the United States under the trade name Adlyxin, as a daily injection for the treatment of type 2 diabetes.
  • Albiglutide used in this document refers to one of the GLP-1 receptor agonists marketed by GlaxoSmithKline as a treatment for type 2 diabetes under the brand name Eperzan in Europe and Tanzeum in the United States.
  • Liraglutide refers to a subcutaneous GLP-1 receptor agonist marketed by Novo Nordisk under the brand name "Victoza” for the treatment of type 2 diabetes and obesity.
  • Taspoglutide used in this document is 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 of the GLP-1 (7-36) peptide, alanine, are methylated and the last amino acid is amidated. However, when manufactured in the form of a fusion protein with another peptide, the C-terminus may not be amidated but may be a general carboxyl group.
  • Semaglutide used in this document 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 is a drug developed by Novo Nordisk in 2012 and approved for sale in the United States in 2017.
  • Tirzepatide used in this document is a type of GLP-1R agonist, currently used for the purpose of type 2 diabetes and weight loss, and is a drug developed by Eli Lilly and approved for sale in the United States in 2022.
  • Piglutide used in this document refers to a GLP-1R and GLP-2R dual agonist developed by Zealand Pharma, which is currently in phase 1 clinical trials.
  • XTEN refers to an unstructured, low-immunogenic peptide containing six amino acids added to improve the in vivo half-life of protein pharmaceuticals developed by Amunix, and is typically composed of amino acids in multiples of 144 a.a as a unit (US2010/0239554A1).
  • Teduglutide used in this document is a GLP-2 analogue that is a mutant in which the second amino acid of GLP-2, alanine (A), is replaced with glycine (G). It is sold under the trade name Gattex in the United States and under the trade name Revestive in Europe as a treatment for short bowel syndrome.
  • Glepaglutide used in this document is a GLP-2 analogue with an improved half-life that has been developed as a treatment for short bowel syndrome and is currently undergoing phase 3 clinical trials for short bowel syndrome.
  • GLP-2 analogue 10 refers to one of the GLP-2 analogues, which is characterized by having a stabilized structure in which the 11th and 18th amino acids of GLP-2 are substituted with cysteines, and a lipidated intramolecular cross-linker is linked through the thiol groups of the two substituted cysteines, and the C-terminal nine amino acids of Exendin 4 are added to the C-terminus (Yang et al ., J. Med. Chem . 61: 3218-3223, 2018).
  • linker peptide as used in this document is an unstructured peptide used when producing a fusion protein by linking two or more proteins or peptides with different biological activities.
  • metabolic disorder used in this document is a general term for diseases caused by abnormalities in the metabolic process, a series of processes that obtain energy from ingested food in the body.
  • diseases such as obesity and diabetes caused by failure to regulate carbohydrate metabolism are representative.
  • Various causes such as genetic and environmental factors have been suggested as the cause of these metabolic diseases, and recently, in addition to genetic factors, a carbohydrate-centered diet has been pointed out as the main cause.
  • These metabolic diseases include diabetes such as type 1 diabetes and type 2 diabetes, obesity, metabolic syndrome, metabolic disorder-associated fatty liver disease, metabolic disorder-associated steatohepatitis, and liver cirrhosis.
  • metabolic syndrome refers to a disease presumed to be caused by insulin resistance, and refers to symptoms in which two or more of the following values among cholesterol, blood pressure, and blood sugar levels are abnormal. It conceptualizes the phenomenon in which various cardiovascular diseases and risk factors for type 2 diabetes are clustered together as a single disease group. It is a useful concept that can comprehensively explain insulin resistance (IR) and various complex and diverse metabolic abnormalities and clinical aspects related to it. If metabolic syndrome is left untreated, it is known that the risk of developing cardiovascular diseases such as arteriosclerosis, myocardial infarction, and stroke or type 2 diabetes increases.
  • IR insulin resistance
  • a dual specificity fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused, wherein the activity of the GLP-1 analogue is 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analogue is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).
  • a dual specificity fusion protein comprising a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a second fusion protein in which a GLP-2 analogue is linked to an antibody Fc region, wherein the dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein has an activity of 40% or more compared to wild-type GLP-1 (human GLP-1 peptide), and the activity of the GLP-2 analogue is 50% or less compared to wild-type GLP-2 (human GLP-2 peptide).
  • the activity of the GLP-1 analogue 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, 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, or 70% or more, It means that it can have an activity of 71% or more, or 72% or more, or 73% or more, or 74% or more, or 75% or more, or 76% or more, or 77% or more, or 78% or more, or
  • the activity of the GLP-1 analogue 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, or 120%, and may have
  • the activity of the GLP-1 analogue can optionally exceed 120% compared to wild-type GLP-1 under in vitro conditions, and 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%, 192%, 19
  • the activity of the GLP-2 analogue under in vitro conditions may be 2% or more compared to wild-type GLP-2.
  • the activity of the GLP-2 analogue 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%, It can be 46%, 47%, 48%, 49%, or less than or equal to 50%, and can have a certain range with any of the above percentages as the lower limit and another greater value as the upper limit.
  • the above dual specificity fusion protein may have a half-life increasing moiety added thereto, and the half-life increasing moiety may be inserted between the GLP-1 analogue and the GLP-2 analogue or added to the N-terminus or C-terminus of the entire fusion protein, and preferably may be an antibody Fc region, PEG, XTEN, PAS (Pro-Ala-Ser), ELP (elastin-like peptide), glycine-rich HAP (homo-amino-acid polymer), GLP (gelatine-like protein), or serum albumin.
  • the half-life increasing moiety may be inserted between the GLP-1 analogue and the GLP-2 analogue or added to the N-terminus or C-terminus of the entire fusion protein, and preferably may be an antibody Fc region, PEG, XTEN, PAS (Pro-Ala-Ser), ELP (elastin-like peptide), glycine-rich HAP (homo-amino-a
  • the above dual specificity fusion protein may not be a proglucagon or an analogue thereof in which GLP-1 and GLP-2 naturally expressed in the body are linked via an intermediate peptide (intervention peptide), but may be a fusion protein in which a GLP-1 analogue and a GLP-2 analogue are directly linked or the two peptides are linked via a linker peptide of a different form than the intermediate peptide.
  • the GLP-1 analogue may be GLP-1, Exendin 3, Exendin 4, a GLP-1/Exendin 4 hybrid peptide, GLP-1-XTEN, Exendin 4-XTEN, Lixisenatide, Albiglutide, Liraglutide, or Taspoglutide, or a continuous repeat in which any one or more of these are linked.
  • the continuous repeat may increase GLP-1 activity or increase its length, thereby enabling quality control related to the production of the dual specificity protein (making it easy to distinguish whether the produced protein is a heterodimer or a homodimer), and may simplify the manufacturing process compared to PEGylation, which is well known as a conventional half-life increasing technology, thereby having an advantage in reducing the production cost.
  • the number of repeating units of the continuous repeat of the GLP-1 analogue may be adjusted according to the required GLP-1 activity.
  • the GLP-1 may include an amino acid sequence represented by SEQ ID NO: 1 or 2.
  • Exendin 3 may include an amino acid sequence represented by sequence number 3.
  • Exendin 4 may be composed of an amino acid sequence represented by sequence number 4.
  • the GLP-1/Exendin 4 hybrid may include an amino acid sequence represented by SEQ ID NO: 5.
  • the Lixisenatide may include an amino acid sequence represented by SEQ ID NO: 6.
  • the Exendin 4-XTEN may include an amino acid sequence represented by SEQ ID NO: 7.
  • the Albiglutide may include an amino acid sequence represented by SEQ ID NO: 8.
  • the Liraglutide may include an amino acid sequence represented by SEQ ID NO: 9.
  • the Taspoglutide may include an amino acid sequence represented by SEQ ID NO: 10.
  • the GLP-1 tandem repeats may include an amino acid sequence represented by SEQ ID NO: 11.
  • the antibody Fc region may be an antibody Fc region in which a heterodimerization moiety is formed, and the dimerization moiety may be Knobs-into-Holes (KiH), KiH SS . HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT SS , SEED or A107.
  • Knobs-into-Holes refers to a design strategy in antibody engineering used for heterodimerization of heavy chains in the production of recombinant proteins or IgG antibodies containing an Fc region.
  • the above KiH technology when dividing the two fusion proteins forming the heterodimer into the first fusion protein and the second fusion protein, may be a first fusion protein in which the 22nd amino acid of the CH3 domain in the first modified Fc region of the first fusion protein, threonine (T), is substituted with tryptophan (W) (T366W) (Knob), and the second fusion protein may be a second fusion protein in which the 22nd amino acid of the CH3 domain in the second modified Fc region, threonine (T), is substituted with serine (S) (T366S), the 24th amino acid, leucine (L), is substituted with alanine (A) (L368A), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V) (Y407V) (Hole structure), and conversely, the first fusion protein may be a first fusion protein in which the 22nd amino acid of the CH3 domain in the first
  • the second fusion protein may be a second variant Fc region in which the 22nd amino acid, threonine (T), of the CH3 domain is substituted with tryptophan (W) (T366W), 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 position of the amino acid where the mutation occurred is based on positions 224-330 (SEQ ID NO: 61) corresponding to the CH3 region of the amino acid sequence of human IgG1 of UniProt No. P01857, and the numbers related to specific amino acid substitutions in parentheses are according to the EU numbering rule related to the amino acid sequence of the antibody.
  • the amino acid corresponding to the corresponding position can be used as a mutated one based on the reference sequence.
  • the Knobs-into-Holes structure can be introduced through other amino acid mutations well known in the art.
  • Such selective mutations can generate a dual-specificity dimeric fusion protein through a combination of, for example, a Knob structure in which threonine (T), the 22nd amino acid of the CH3 domain of the first fusion protein, is substituted with tyrosine (Y) (T366Y) and a Hole structure in which tyrosine (Y), the 63rd amino acid of the CH3 domain of the second fusion protein, is substituted with threonine (T) (Y403T).
  • T threonine
  • Y threonine
  • KiH SS refers to a structure in which cysteine is introduced into CH3 so that an intramolecular disulfide bond can be formed in the KiH structure.
  • the 10th amino acid of CH3 of Knob Fc, serine (S) is additionally substituted with cysteine (C) (S354C)
  • the 5th amino acid of Hole Fc, tyrosine (Y) is additionally substituted with cysteine (C) (Y349C) (Merchant et al ., Nat. Biotechnol . 16(7): 677-681, 1998).
  • HA-TF refers to a technology that induces heterodimerization by utilizing hydrophobic/steric complementarity by substituting serine (S), the 20th amino acid of CH3 of the first modified Fc region, with histidine (H) (S364H), phenylalanine (F), the 61st amino acid, with alanine (A) (F405A), tyrosine (Y), the 5th amino acid of CH3 of the second modified Fc region, with threonine (T) (Y349T), and threonine (T), the 50th amino acid, with phenylalanine (F) (T394F) (Moore et al ., MAbs 3(6): 546-557, 2011).
  • ZW1 refers to a first modified Fc region CH3 in which the 6th amino acid, threonine (T), is substituted with valine (V) (T350V), the 7th amino acid, leucine (L), is substituted with tyrosine (Y) (L351Y), the 61st amino acid, phenylalanine (F), is substituted with alanine (A) (F405A), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V) (Y407V), and the 6th amino acid, threonine (T), of the CH3 of the Fc region of the second modified Fc region is substituted with valine (V) (T350V), the 22nd amino acid, threonine (T), is substituted with tryptophan (W) (T366W), the 48th amino acid, lysine (K), is substituted with leucine (L) (K392L), and
  • 7.8.60 refers to a hydrophobic/stereotypic Fc region in which the 16th amino acid, lysine (K), of CH3 of the first modified Fc region 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), and the 1st amino acid, glutamic acid (E), of CH3 of the second modified Fc region is substituted with arginine (R) (E345R), the 3rd 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
  • DD-KK used in this document refers to a technology of inducing heterodimerization by utilizing electrostatic complementarity by substituting lysine (K), the 65th amino acid of CH3 of the first modified Fc region, to aspartic acid (D) (K409D), lysine (K), the 48th amino acid, to aspartic acid (D) (K392D), as well as substituting aspartic acid (D), the 55th amino acid of CH3 of the second modified Fc region, to lysine (K) (D399K), and glutamic acid (E), the 13th amino acid, to lysine (K) (E356K) (Gunasekaran et al ., J. Biol. Chem . 285(25): 19637-19646, 2010).
  • EW-RVT refers to a technique for inducing heterodimerization by utilizing hydrophobic/steric complementarity and long-range electrostatic interactions by substituting lysine (K), the 16th amino acid of CH3 of the first modified Fc region, with glutamic acid (E) (K360E), lysine (K), the 65th amino acid, with tryptophan (W) (K409W), glutamine (Q), the 3rd amino acid of CH3 of the Fc region of the second modified Fc region, with arginine (R) (Q347R), aspartic acid (D), the 55th amino acid, with valine (V) (D399V), and phenylalanine (F), the 61st amino acid, with threonine (T) (F405T) (Choi et al ., Mol. Cancer Ther. 12(12): 2748-2759, 2013).
  • EW-RVT SS refers to a structure in which cysteine is introduced into CH3 so that an intramolecular disulfide bond can be formed in the EW-RVT structure, and in which tyrosine (Y), the 5th amino acid of CH3 of the first modified Fc region of the EW-RVT, is additionally substituted with cysteine (C) (Y349C), and in which serine (S), the 10th amino acid of CH3 of the second modified Fc region, is additionally substituted with cysteine (C) (S354C) (Choi et al ., Mol. Immunol . 65(2): 377-383, 2015).
  • Y tyrosine
  • C cysteine
  • S354C serine
  • SEED refers to a technology that induces heterodimerization by utilizing hydrophobic/steric complementarity resulting from chain exchange between IgG and IgA by introducing 45 IgA-derived residues into CH3 of the first modified Fc region and 57 IgG1-derived residues into CH3 of the second modified Fc region (Davis et al ., Protein Eng, Des, Sel , 23(4): 195-202, 2010).
  • A107 refers to a technique of inducing heterodimerization by utilizing hydrophobic/steric complementarity and hydrogen bonding complementarity by substituting lysine (K), the 26th amino acid of CH3 of the first modified Fc region, with glutamic acid (E) (K370E), lysine (K), the 69th amino acid, with tryptophan (W) (K409W), glutamic acid (E), the 13th amino acid of CH3 of the second modified Fc region, with asparagine (N) (E357N), aspartic acid (D), the 55th amino acid, with valine (V) (D399V), and phenylalanine (F), the 61st amino acid, with threonine (T) (F405T) (Choi et al ., PLoS One 10(12): e0145349, 2015).
  • the above hybrid antibody Fc region may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 16.
  • the hybrid antibody Fc region may be additionally mutated so as not to cause undesirable side effects when administered into the body, such as antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell cytotoxicity
  • CDC complement-dependent cytotoxicity
  • 897938 may be a hybrid Fc region variant (NTIG Knob) comprising an amino acid sequence represented by SEQ ID NO: 15, wherein the 18th amino acid threonine (T) of the hybrid Fc region (Knob structure) consisting of an 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) comprising an amino acid sequence represented by SEQ ID NO: 16, wherein the 18th amino acid threonine (T) of the hybrid Fc region (Hole) consisting of an 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).
  • NTIG Knob comprising an amino acid sequence represented by SEQ ID NO: 15
  • the GLP-2 analogue can be GLP-2, a GLP-2 A2G mutant, a GLP-2 N16G_L17Q mutant, a GLP-2 A2G_N16G_L17Q mutant, Glepaglutilde, or a GLP-2 analogue 10, and mutations such as amino acid substitutions, additions, and deletions can be introduced for the purpose of lowering the binding affinity to the GLP-2 receptor in order to achieve lower activity compared to the activity of wild-type GLP-2.
  • an approach can be used to lower the binding affinity to the GLP-2 receptor by shortening the length compared to the GLP-1 analogue (for example, by inserting a longer linker peptide between the GLP-1 analogue and the Fc region).
  • the length of the linker of the first fusion protein may be about 5 to 40 aa longer than the linker of the second fusion protein, and more specifically, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38 aa, It can be about 39 aa or 40 aa long, and can have a certain range with any value among the above amino acid lengths as the lower limit and another value greater than that as the
  • the inventors of the present invention confirmed through Examples 2 to 4 of the present invention that when the length of a GLP-1 analogue is designed to be longer than that of a GLP-2 analogue (by using a slightly longer glycan linker peptide or forming a GLP-1 tandem repeat), the relative activity of GLP-2 is very low regardless of whether or not a mutation is introduced into the GLP-2 analogue.
  • the length of the fusion protein including the GLP-2 analogue is made longer than that of the fusion protein including the GLP-1 analogue by introducing a glycan linker into the GLP-2 analogue, it was confirmed that the relative activity of the GLP-2 analogue is significantly increased.
  • the GLP-2 analogue may include an amino acid sequence represented by any one of SEQ ID NOs: 17 to 22.
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 18 is a human GLP-2 wild-type peptide
  • the human GLP-2 mutant consisting of the amino acid sequence represented by SEQ ID NO: 17 is a human GLP-2 mutant in which the second amino acid, alanine, is substituted with glycine (A2G mutant or GLP-2 A2G , hereinafter, referred to as 'GLP-2-2G' for convenience) and is also called Teduglutide.
  • the GLP-2 variant consisting of the amino acid sequence represented by sequence number 19 is a GLP-2 variant (GLP-2 A2G_N16G_L17Q) in which not only the second amino acid, alanine (A), is mutated to glycine (G), but also the 16th amino acid, asparagine (N), is mutated to glycine (G) and the 17th amino acid, leucine (L), is mutated to glutamine (Q), and it is known to maintain the function of GLP-2 while inhibiting dimerization of GLP-2 or the formation of aggregates resulting from it during recombinant production (Baker et al ., J. Mol. Recognit . 25: 155-164, 2012).
  • GLP-2 A2G_N16G_L17Q GLP-2 variant in which not only the second amino acid, alanine (A), is mutated to glycine (G), but also the 16th amino acid, asparagine (N), is mutated to
  • a GLP-2 wild-type peptide in which the second amino acid, alanine, is substituted with glycine and the 17th amino acid, leucine, is substituted with glutamine can also exhibit the same function as a GLP-2 analogue composed of the amino acid sequence represented by SEQ ID NO: 19, and therefore can be used as a GLP-2 analogue in the present invention.
  • the Glepaglutide may include an amino acid sequence represented by SEQ ID NO: 21.
  • the GLP-2 analogue 10 may include an amino acid sequence represented by SEQ ID NO: 22.
  • the first fusion protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 25.
  • the second fusion protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 28.
  • the first fusion protein may be one in which the 10th amino acid, serine, of the CH3 domain in the hybrid Fc region is replaced with cysteine (C) and the 22nd amino acid, threonine (T), is replaced with tryptophan (W) (Knob structure)
  • the second fusion protein may be one in which the 5th amino acid, tyrosine (Y), of the CH3 domain in the Fc region is replaced with cysteine (C), the 22nd amino acid, threonine, is replaced with serine (S), the 24th amino acid, leucine (L), is replaced with alanine (A), and the 63rd amino acid, tyrosine (Y), is replaced with valine (V) (Hole structure), and conversely, the first fusion protein may be one in which the 5th amino acid, tyrosine (Y), of the CH3 domain in the Fc region is replaced with cysteine (C), the 22nd amino acid, threon
  • the first fusion protein may have threonine (T), which is the 22nd amino acid of the CH3 domain in the hybrid Fc region, substituted with tyrosine (Y)
  • the second fusion protein may have tyrosine (Y), which is the 63rd amino acid of the CH3 domain in the hybrid Fc region, substituted with threonine (T)
  • the second fusion protein may have tyrosine (Y), which is the 63rd amino acid of the CH3 domain in the hybrid Fc region, substituted with threonine (T)
  • the first fusion protein may have tyrosine (Y), which is the 63rd amino acid of the CH3 domain in the hybrid Fc region, substituted with threonine (T).
  • the mutation of the 63rd amino acid may be expressed as Y86T according to the numbering rules of IMGT (international ImMunoGeneTics information system) (Lefranc et al ., Dev. Comp. Immunol ., 27: 55-77, 2003), not based on the amino acid sequence of the CH3 domain of human IgG1 described in SEQ ID NO: 61.
  • IMGT international ImMunoGeneTics information system
  • various heterodimer-forming moieties that are introduced into the antibody Fc region to enable heterodimer formation may also be used.
  • one or more linker peptides may be inserted between the fusion partners of the fusion protein, that is, between the peptides or domains. That is, in the case of the dual specificity fusion protein in which the GLP-1 analogue and the GLP-2 analogue are fused, a linker peptide may be inserted between the GLP-1 analogue and the GLP-2 analogue, and in the case of the dual specificity fusion protein generated by dimerization of the first fusion protein and the second fusion protein, a linker peptide may be inserted between the GLP-1 analogue and the antibody Fc region within the first fusion protein, and similarly, a linker peptide may be inserted between the GLP-2 analogue and the antibody Fc region within the second fusion protein.
  • the linker peptide may or may not include an N-glycan attachment site.
  • the above N-glycan attachment site is intended to make it easier to confirm heterodimer formation by making the sizes of the first and second fusion proteins different, and it was confirmed that there was no significant effect on the binding affinity for GLP-1R or GLP-2R.
  • 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
  • AAGSGGGGGGGGSGGGGS SEQ ID NO: 36
  • GGGGSGGGGSGGGGS SEQ ID NO: 37
  • GGSGG SEQ ID NO: 38
  • GGSGGSGGS SEQ ID NO: 39
  • GGGSGG SEQ ID NO: 40
  • composition comprising the dual specificity fusion protein is provided.
  • a pharmaceutical composition for treating a metabolic disease comprising at least one of the above dual specificity fusion proteins as an active ingredient.
  • the metabolic disease may be metabolic syndrome, obesity, diabetes, metabolic disorder-associated fatty liver disease, metabolic disorder-associated steatohepatitis, liver fibrosis or liver cirrhosis, the diabetes may be type 1 diabetes or type 2 diabetes, and the liver fibrosis may be liver fibrosis caused by the progression of chronic metabolic disorder-associated steatohepatitis.
  • any one of the above dual specificity fusion proteins for use in the treatment of metabolic diseases.
  • the metabolic disease may be metabolic syndrome, obesity, diabetes, metabolic disorder-associated fatty liver disease, metabolic disorder-associated steatohepatitis, liver fibrosis or liver cirrhosis, the diabetes may be type 1 diabetes or type 2 diabetes, and the liver fibrosis may be liver fibrosis caused by the progression of chronic metabolic disorder-associated steatohepatitis.
  • the metabolic disease may be metabolic syndrome, obesity, diabetes, metabolic disorder-associated fatty liver disease, metabolic disorder-associated steatohepatitis, liver fibrosis or liver cirrhosis, the diabetes may be type 1 diabetes or type 2 diabetes, and the liver fibrosis may be liver fibrosis caused by the progression of chronic metabolic disorder-associated steatohepatitis.
  • a method for treating a metabolic disease in a subject comprising the step of administering a therapeutically effective amount of the dual specificity fusion protein to a patient suffering from the metabolic disease.
  • the metabolic disease may be metabolic syndrome, obesity, diabetes, metabolic disorder-associated fatty liver disease, metabolic disorder-associated steatohepatitis, liver fibrosis or liver cirrhosis
  • the diabetes may be type 1 diabetes or type 2 diabetes
  • the liver fibrosis may be liver fibrosis caused by the progression of chronic metabolic disorder-associated steatohepatitis.
  • Exendin 4 one of the GLP-1 receptor agonists, has already been proven to be effective in controlling blood sugar and weight in patients with type 2 diabetes in clinical trials (DeFronzo et al ., Diabetes Care 28: 1092-1100, 2005).
  • the dual specificity fusion protein according to one embodiment of the present invention can be used to treat metabolic diseases such as obesity, diabetes, and metabolic syndrome.
  • the composition may include a pharmaceutically acceptable carrier, and may additionally include a pharmaceutically acceptable auxiliary, excipient or diluent in addition to the carrier.
  • pharmaceutically acceptable refers to a composition that is physiologically tolerable and does not typically cause allergic reactions such as gastrointestinal upset, dizziness or the like when administered to a human.
  • carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives may be additionally included.
  • compositions according to one embodiment of the present invention can be formulated using methods known in the art to enable rapid release, or sustained or delayed release, of the active ingredient when administered to a mammal.
  • the formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.
  • composition according to one embodiment of the present invention may be administered by various routes, for example, orally, parenterally, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration, and may also be administered using an implantable device for sustained release or continuous or repeated release.
  • the number of administrations may be once a day or divided into several times within a desired range, and the administration period is not particularly limited.
  • composition according to one embodiment of the present invention can be formulated in a suitable form together with a pharmaceutically acceptable carrier commonly used.
  • pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline solution, aqueous glucose and glycol, and may further include stabilizers and preservatives.
  • stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
  • composition according to the present invention may appropriately include, depending on the administration method or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an antiadsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic, a buffer, an antioxidant, and the like, if necessary.
  • a suspending agent e.g., a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an antiadsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic, a buffer, an antioxidant, and the like.
  • the dosage of the composition to a patient will vary depending on many factors, including the patient's height, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the pharmaceutically active protein may be administered in an amount of 100 ng/kg body weight to 10 mg/kg body weight, more preferably 1 to 500 ⁇ g/kg body weight, and most preferably 5 to 50 ⁇ g/kg body weight, the dosage being adjusted taking into account the above factors.
  • therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.
  • the therapeutically effective amount of the composition of the present invention can be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage can be appropriately adjusted according to the age, sex and condition of the patient.
  • a linker peptide typically having a flexible structure, may be inserted between the two or more proteins or domains.
  • the above 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
  • the dual-specificity fusion protein can be produced by recombinantly transfecting a host cell with a recombinant expression vector including a first gene construct including a polynucleotide encoding the first fusion protein and a second gene construct including a polynucleotide encoding the second fusion protein, and then expressing the same.
  • the first gene construct and the second gene construct may be expressed by being inserted into one expression vector, or by being inserted into two separate expression vectors.
  • the vector may be designed so that each gene construct is operably linked to two separate regulatory sequences, or the two gene constructs may be operably linked to one regulatory sequence, and an internal ribosome entry site (IRES) may be used to connect the two gene constructs.
  • IRS internal ribosome entry site
  • 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 a regulatory sequence in such a way that its expression can occur.
  • regulatory sequence as described above is intended to include promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct the target nucleic acid to be constantly expressed in many host cells, those that direct the target nucleic acid to be expressed only in specific tissue cells (e.g., tissue-specific regulatory sequences), and those that direct expression to be induced by specific signals (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of an expression vector may vary depending on factors such as the choice of host cell to be transformed and the level of protein expression desired.
  • the expression vector of the present invention can be introduced into a host cell to express the fusion protein.
  • regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, these typically include regulatory sequences responsible for transcription initiation and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. Additional regulatory sequences may include, in addition to transcriptional regulators, translational enhancers and/or native-combination or heterologous promoter regions.
  • potential regulatory sequences that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rouse sarcoma virus), human elongation factor 1 ⁇ -promoter, glucocorticoid-inducible MMTV-promoter (Moloney mouse tumor virus), metallothionein-inducible or tetracycline-inducible promoters or enhancers such as the CMV enhancer or the SV40 enhancer.
  • the neurofilament-promoter For expression in neuronal cells, it is contemplated that the neurofilament-promoter, the PGDF-promoter, the NSE-promoter, the PrP-promoter or the thy-1-promoter may be used.
  • the above promoters are known in the art and are described in the literature (Charron, J. Biol. Chem. 270: 25739-25745, 1995).
  • a number of promoters have been disclosed, including the lac-promoter, the tac-promoter or the trp promoter.
  • the regulatory sequences may also include a transcription termination signal, such as the SV40-poly-A site or the TK-poly-A site, downstream of the polynucleotide according to one embodiment of the present invention.
  • Suitable expression vectors in the present invention are known in the art, including, but not limited to, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27 (Patent No.
  • the vector may additionally comprise a polynucleotide encoding a secretion signal.
  • secretion signals are well known to those skilled in the art.
  • a leader sequence capable of directing the fusion protein into a cellular compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the present invention, preferably a leader sequence capable of directly secreting the translated protein or its protein into the cytoplasmic periphery or the extracellular medium.
  • the vector of the present invention can be produced by, for example, standard recombinant DNA techniques, which include, for example, blunt-end and sticky-end ligation, restriction enzyme treatment to provide appropriate termini, removal of phosphate groups by alkaline phosphatase treatment to prevent inappropriate joining, and enzymatic ligation by T4 DNA ligase.
  • the vector of the present invention can be produced by recombining DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination techniques and DNA encoding the bispecific fusion protein of the present invention into a vector containing an appropriate regulatory sequence.
  • the vector containing the regulatory sequence can be purchased or produced commercially, and in one embodiment of the present invention, pBispecific backbone vector (Genexine, Inc., Korea) or pAD15 vector was used as a backbone vector.
  • the above expression vector may additionally include a polynucleotide encoding a secretion signal sequence, wherein the secretion signal sequence induces secretion of a recombinant protein expressed within a cell out of the cell, and may be a tPA (tissue plasminogen activator) signal sequence, a HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence, or a growth hormone signal sequence.
  • tPA tissue plasminogen activator
  • HSV gDs herpes simplex virus glycoprotein Ds
  • the expression vector according to one embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector may take any form, such as a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human chromosome, etc.
  • the present inventors designed various dual-specificity fusion proteins containing both GLP-1 analogues and GLP-2 analogues, as shown in Figure 1 and Table 1 below.
  • GLP-1/Exendin 4 hybrid hereinafter, conveniently referred to as 'GLP-1/Ex4 Hyb'
  • GLP-2-2G tandem repeats and GLP-2 analogue
  • a glycan linker was used in either the first fusion protein comprising the GLP-1 analogue or the second fusion protein comprising the GLP-2 analogue, or neither of them used a glycan linker.
  • Non-deformed (34) Knob hole First fusion protein (23)
  • Second fusion protein (26) 1 MG12-6 1.
  • GLP-1/Ex4 Hyb(5) Glycan (35)
  • Non-deformed (34) hole Knob Second fusion protein (27) First fusion protein (23) 2 MG12-7 1.
  • the first fusion protein may be one in which the 10th amino acid, serine (S), of the CH3 domain in the hybrid Fc region is replaced with cysteine (C) and the 22nd amino acid, threonine (T), is replaced with tryptophan (W) (Knob)
  • the second fusion protein may be one in which the 5th amino acid, tyrosine (Y), of the CH3 domain in the Fc region is replaced with cysteine (C), the 22nd amino acid, threonine (T), is replaced with serine (S), the 24th amino acid, leucine (L), is replaced with alanine (A), and the 63rd amino acid, tyrosine (Y), is replaced with valine (V) (Hole).
  • the position of the amino acid where the mutation occurred is based on the reference sequence (amino acid sequence of human IgG1 CH3 domain of SEQ ID NO: 61). Even if additional mutations such as addition, deletion or substitution of amino acids occur at a site unrelated to the Knobs-into-Holes structure on the CH3 domain, the amino acid corresponding to the corresponding position can be used based on the reference sequence.
  • the Knobs-into-Holes structure can be introduced through other amino acid mutations well known in the art. These mutations have been well described in the literature (Wei et al ., Oncotarget 2017, 8(31): 51037-51049; Ridgway et al ., Protein Eng .
  • Such selective mutations can generate a dual-specificity dimeric fusion protein through a combination of, for example, a Knob structure in which threonine, the 22nd amino acid of the CH3 domain of the first fusion protein, is substituted with tyrosine, and a Hole structure in which tyrosine, the 63rd amino acid of the CH3 domain of the second fusion protein, is substituted with threonine.
  • the above Knobs-into-Holes structure can be formed, conversely, by introducing a Hole structure into the first fusion protein and a Knob structure into the second fusion protein.
  • plasmid DNA transfection was performed on HEK293F cells using the N293F vector system (WiBiologics) for temporary expression in animal cells.
  • plasmid DNA was mixed in 3 ml of medium, and then 25 ⁇ g of 2 mg/ml PEI (Polyethylenimine, PolyPlus, USA) was added and mixed.
  • the reaction solution was allowed to stand at room temperature for 15 minutes and then added to 80–1000 ml of culture medium containing 1x106 cells/ml, and incubated for 24 hours under conditions of 120 rpm, 37°C, 8% CO2 .
  • 24 hours after DNA transfection nutrient supplement medium components (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 culture was continued for up to 7 days.
  • the supernatant obtained through the above culture was purified through the Protein A column and the secondary column, and the comparative dual-specificity fusion protein (named 'MG12-5') and the dual-specificity fusion proteins of Examples 1 to 4 (named 'MG12-6', 'MG12-7', 'MG12-8', and 'MG12-9', respectively) were appropriately diluted with 4X LDS sample buffer and water for injection to prepare a final concentration of 3-10 ⁇ g/20 ⁇ L.
  • 4X LDS sample buffer, 10X reducing agent, and water for injection were appropriately diluted to prepare a final concentration of 3-10 ⁇ g/20 ⁇ L, and heated in a 95°C heating block for 10 minutes.
  • the present inventors investigated the in vitro GLP-1 and GLP-2 activities of the dual-specificity fusion protein manufactured in the above examples. Specifically, the present inventors used CHO-K1 cells (DiscoverX) stably expressing the GLP-1 receptor to evaluate the activity of the GLP-1 receptor.
  • the GLP-1R expressing CHO-K1 cells were subcultured, dispensed 100 ⁇ L each at an amount of 5 x 10 5 cells/mL into a 96-well white plate, and cultured in a 5% CO 2 incubator at 37°C for 20-24 hours.
  • the drug was prepared at a concentration three times the final concentration, and then the culture medium in the plate was removed, and 30 ⁇ L of the experimental reagent and 15 ⁇ L of the GLP-1 series drug were treated. After DIRANF treatment, the reaction was performed at 37°C for 30 minutes, and then a total of 75 ⁇ L of cell lysis solution and activity evaluation reagent were added, reacted at room temperature for 1 hour, and fluorescence reaction was induced for 4-6 hours.
  • CHO-K1 cells stably expressing the GLP-2 receptor were used.
  • GLP-2 activity was evaluated using the same method as above, except that GLP-2R expressing CHO-K1 cells were used.
  • the activity of MG12-5, a comparative example, was referenced to the values described in Korean Patent No. 10-2349718.
  • the dual-specificity fusion proteins of Examples 1 to 3 of the present invention exhibited at least 40% of the activity compared to the wild-type GLP-1 peptide, confirming that they exhibit activities similar to or higher than the wild-type GLP-1, and in particular, Example 2 exhibited high activity of at least 150% compared to the wild-type GLP-1.
  • the present inventors evaluated the weight loss effect by a single administration of MG12-5 as a comparative group and MG12-8 according to one embodiment of the present invention. Specifically, normal 7-week-old male C57BL/6N mice were obtained and acclimatized through free food and water supply for 1 week. Thereafter, the weights of the mice were measured, and the experimental groups were randomly separated so that the average weights were similar. Then, MG12-5 (100 nmol/5 mL/kg), MG12-8 (100 nmol/5 mL/kg) prepared with MG12 formulation buffer, and a vehicle containing no drug were injected subcutaneously into the flanks of the mice. Three days after drug administration, the weights of the mice were measured to evaluate the weight loss due to the drugs (Fig. 3a).
  • the GLP-1 activity of the dual specificity fusion protein including a GLP-1 analogue and a GLP-2 analogue should be 40% or more in relative activity (%) compared to the wild-type GLP-1 peptide, or the GLP-2 activity of the fusion protein should be 50% or less in relative activity (%) compared to the wild-type GLP-2 peptide.
  • MG12-5 which is a comparative example
  • MG12-8 according to one embodiment of the present invention.
  • normal 7-week-old male C57BL/6J mice were obtained and acclimatized through free feeding and feeding for 1 week. Thereafter, the weights of the mice were measured, and groups were randomly divided so that the average weights were similar.
  • MG12-5 (30 nmol/5 mL/kg), MG12-8 (30 nmol/5 mL/kg) prepared with MG12 formulation buffer, and a vehicle containing no drug were injected subcutaneously in the flanks of the mice once every two days for a total of 7 times for 2 weeks.
  • the body weights were recorded once every two days from the first drug administration date, and the change in body weight compared to before drug administration was expressed as a percentage (%).
  • the experimental group administered the dual-specificity fusion protein according to the comparative example and one embodiment of the present invention showed a decrease in body weight due to drug administration.
  • this weight loss effect gradually recovered to a normal value.
  • an additional weight loss effect of 5% was observed compared to the comparative example (MG12-5), and it was confirmed that the weight loss due to repeated administration was maintained compared to MG12-5.
  • the present inventors measured the volume of the gallbladder of the mice the day after the last drug administration by sacrificing the mice and placing a cutting board around the gallbladder and taking a picture.
  • the volume of the gallbladder in the comparative group increased 2.3 times compared to the vehicle administration group, but in the MG12-8 administration group according to one embodiment of the present invention, it was confirmed that there was no statistical difference in the volume of the gallbladder from the vehicle administration group.
  • the GLP-2 activity of the dual specificity fusion protein of the present invention does not exhibit toxicity when the relative activity (%) compared to the wild-type GLP-2 peptide is less than 50%.
  • the inventors of the present invention investigated the ability of Liraglutide, as a comparative group, and MG12-8 according to an embodiment of the present invention, to reduce body weight and control intestinal leakage in a metabolic disorder-associated steatohepatitis model animal accompanied by intestinal leakage.
  • the inventors obtained 20-week-old male C57BL/6J mice and induced overweight with a choline-deficient high-fat diet (CD-HFD).
  • CD-HFD choline-deficient high-fat diet
  • the leaky gut phenomenon was induced through a cycle of treating DSS (dextran sulfate sodium) at a low concentration of 1% for 7 days and then changing to drinking water for 10 days.
  • Liraglutide and MG12-8 were subcutaneously injected at given doses and administration intervals, respectively, and body weights were measured two weeks after the first drug administration date (Fig. 4a).
  • FITC fluorescein isothiocyanate
  • FITC-Dextran (4 kDa) solution was administered orally 4 hours before sacrifice of mice, after which feeding was stopped, and the amount of FITC-Dextran transferred to the blood was evaluated by measuring blood fluorescence.
  • the final body weight of the vehicle administration group was approximately 37 g, which was confirmed to be 20% higher than the normal weight range of 30 g.
  • a decrease in body weight was confirmed compared to the vehicle administration group, and among these, the most effective weight loss effect was confirmed by MG12-8 according to one embodiment of the present invention.
  • MG12-8 according to an embodiment of the present invention exhibited excellent intestinal leakage inhibition activity compared to the control group, as confirmed in FIG. 4c.
  • Liraglutide did not exhibit intestinal leakage inhibition activity compared to the control group.
  • MG12-8 according to an embodiment of the present invention used a relatively low dose of 15 nmol/kg, and the administration interval was also three times a week, which is more than four times longer than Liraglutide twice a day, so it can be seen that MG12-8 is a superior substance in terms of patient convenience.
  • 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 specifically secured four MG12-8 samples with different in vitro GLP-1 activities through RP-HPLC. Specifically, the four samples showed differences in the areas of peaks 1 and 3 as a result of RP-HPLC analysis. In the case of sample 1, the area of peak 1 was small, and as the sample number increased, the area of peak 1 increased, and conversely, the area of peak 3 decreased as the sample number increased (Table 4). In vitro GLP-1 activity was analyzed using HEK-GLP-1R cells (eEnzyme) stably expressing GLP-1R for the four samples with different ratios of the areas of peaks 1 and 3.
  • HEK-GLP-1R cells eEnzyme
  • the HEK-GLP-1R cells were sub-cultured, dispensed into 96-well black plates at a density of 3x105 cells/mL (100 ⁇ L each), and cultured in a 5% CO2 incubator at 37°C for 20-24 hours. After confirming that the cells were stably attached, the drug was prepared at a concentration 5 times the final concentration, and the culture medium in the plate was removed, followed by treatment with an appropriate amount of the experimental reagent and GLP-1 series drugs. After reacting with the drug for 2 hours, the activity of the drug was evaluated by measuring the degree of bioluminescence by measuring the increasing amount of cAMP using a solution provided in the kit and a Spectramax M5 device.
  • the relative GLP-1 activity was confirmed to be 100% for sample 1, about 200% for sample 2, about 300% for sample 3, and about 400% for sample 4.
  • the inventors of the present invention used the four samples with different in vitro GLP-1 activities as described above in an animal experiment to investigate the in vivo GLP-1 activity. Specifically, for this, the inventors performed an intraperitoneal glucose tolerance test (IPGTT). The drug was administered 18 hours before the start of the IPGTT, and then the feed and bedding were removed to start fasting. Then, 18 hours after the drug administration, a 20% glucose solution was administered intraperitoneally, and the changes in blood glucose over time before and after administration were observed (Fig. 5b).
  • IPGTT intraperitoneal glucose tolerance test
  • the present inventors then attempted to analyze the obesity treatment of a dual specificity fusion protein according to an embodiment of the present invention by comparing it with previously reported GLP-1R/GLP-2R dual agonists. Specifically, the present inventors investigated the body weight loss and body composition changes by treatment with 30 nmol/kg of Dapiglutide mimetic and 15 and 30 nmol/kg of MG12-8 (hereinafter referred to as 'MG12' for convenience) to compare its efficacy with that of a previously known GLP-1R/GLP-2R dual agonist peptide substance (Dapiglutide mimetic).
  • the fat mass of the whole body and the lean mass of muscles and bones were measured using a Minispec LF50 (Bruker biospin, BCA LF50) device, and in order to observe the body composition changes due to the drug, the rate of change in body composition was measured before drug administration and after drug administration for 2 weeks.
  • Minispec LF50 Bruker biospin, BCA LF50
  • DIO diet-induced obesity
  • the normal diet and high-fat diet groups showed similar positive fat weight increase rates, but the Dapiglutide mimetic administration group and the dual-specificity fusion protein administration group according to one embodiment of the present invention showed a decrease in fat among the body composition.
  • the Dapiglutide mimetic administration group showed a decrease of about 6%, while the 15 nmol/kg MG12 administration group showed a decrease of about 18%, and the 30 nmol/kg treatment group showed a decrease in the fat ratio of about 35%, indicating that the present invention is a substance with a very excellent effect in treating obesity compared to existing substances.
  • the present inventors analyzed the weight loss effect in a high-fat diet obese model animal using various concentrations of the dual-specificity protein. Specifically, in order to evaluate the weight loss effect according to the concentration of MG12, the present inventors obtained 5-week-old normal C57BL/6 male mice, and induced diet-induced obesity (DIO) by treating them with a 60% high-fat diet (HFD) after one week of acclimatization.
  • DIO induced diet-induced obesity
  • MG12 was prepared at 15, 30, and 60 nmol/5 mL/kg using MG12 formulation buffer, and administered subcutaneously to the abdomen of the mice once every two days.
  • the mice were randomly divided into groups so that the average body weights were similar, and the body weight change after drug administration was expressed as a percentage (%).
  • both the normal diet group and the HFD diet group showed a tendency to maintain or increase body weight for 2 weeks.
  • the group administered MG12 showed a weight loss effect of about 13 to 25% depending on the increase in concentration, and this weight loss effect appeared to be concentration-dependent.
  • the inventors of the present invention separately prepared GLP-1-Fc and GLP-2-Fc, which are fusion partners of the dual specificity fusion protein according to one embodiment of the present invention, and compared and analyzed the body weight change when simply combined administration (30 nmol/kg + 30 nmol/kg) was performed and when the dual specificity fusion protein (MG12) according to one embodiment of the present invention was administered at the same molar concentration (30 nmol/kg).
  • the present inventors compared and analyzed the fat-specific weight reduction effect of Trizpatide mimetic, a GLP-1R/GIPR peptide dual agonist conventionally used as an obesity treatment agent, and a dual specificity fusion protein according to an embodiment of the present invention.
  • the present inventors obtained 5-week-old normal C57BL/6 male mice, and after one week of acclimatization, 60% high-fat diet (HFD) was treated to induce diet-induced obesity (DIO). After 16 weeks from the first HFD, Tirzepatide mimetic and MG12 were prepared at 15 and 60 nmol/5 mL/kg, respectively, using MG12 formulation buffer and then administered subcutaneously.
  • HFD high-fat diet
  • MG12 diet-induced obesity
  • mice For body composition analysis, the total body fat mass and lean mass of the mice were measured using a Minispec LF50 (Bruker biospin, BCA LF50) device, and the body composition change rate before and after drug administration for 2 weeks was observed to observe the change in body composition due to the drug.
  • Minispec LF50 Bruker biospin, BCA LF50
  • the inventors of the present invention attempted to investigate whether the fat-reducing effect of the dual-specificity fusion protein according to one embodiment of the present invention was due to the reduction of subcutaneous fat or visceral fat.
  • the inventors specifically sacrificed the experimental animals after the end of the experiment of Experimental Example 6-3, extracted the subcutaneous fat and visceral fat, measured the weight of each, and converted it into the body fat percentage of each adipose tissue by dividing it by the body weight.
  • both Tirzepatide mimetic and MG12 showed a tendency for the subcutaneous fat ratio to be similar or decreasing compared to the control group (vehicle administration group), but it was confirmed that there was no statistical significance.
  • the Tirzepatide mimetic administration group decreased to the level of the control group (normal mice) compared to the vehicle administration group.
  • the visceral fat content was lower than that of the Tirzepatide mimetic administration group, and it was found that it is a very effective substance for reducing visceral fat in obese animals.
  • liver cirrhosis which is an irreversible liver damage disease. Therefore, the inventors of the present invention investigated whether the present inventors could suppress the increase in metabolic endotoxemia (blood endotoxemia) and liver damage, which are symptoms that often occur in obese animal models. After administering Tirzepatide mimetic, a GLP-1/GIPR dual agonist at the same efficacy concentration, and a dual specificity fusion protein (MG12) according to an embodiment of the present invention for 2 weeks, the serum was obtained and component analysis was performed.
  • NASH non-metabolic fatty liver disease
  • NASH metabolic syndrome-associated steatohepatitis
  • a mouse LPS ELISA kit (CSB-E13066m, Cusabio) was used for analysis, and for liver damage analysis, ALT levels were measured using dry-chem equipment (DRI-CHEM NX-500, FUJI).
  • Fig. 7a As a result, as confirmed in Fig. 7a, it was confirmed that the amount of serum LPS (lipopolysaccharide) increased due to diet-induced obesity (DIO), which induced endotoxemia. This value was not decreased when Tirzepatide mimetic was administered, but it was confirmed that it was decreased to the control level when the dual specificity fusion protein according to an embodiment of the present invention was administered. In addition, as a result of analyzing the degree of liver damage, it was confirmed that the ALT level was significantly increased due to liver damage caused by diet-induced obesity, as confirmed in Fig. 7b.
  • DIO diet-induced obesity
  • the ALT level was decreased to half that of the vehicle administration group, and in the case of the dual specificity fusion protein (MG12) administration group according to an embodiment of the present invention, it was confirmed that the ALT level reduction effect was improved to a statistically significant level compared to the Tirzepatide mimetic administration group.
  • a bispecific fusion protein according to one embodiment of the present invention can be used for the treatment of diabetes
  • the inventors of the present invention performed a 2-NBDG glucose uptake assay on 3T3-L1 cells, which are mouse pre-adipocytes, and L6-GLUT4myc cells, which are rat myoblasts (provided by: From the laboratory of Amira Klip, PhD, Hospital For Sick Children).
  • the culture medium of 3T3-L1 adipocyte precursor cells cultured in a 24-well plate at a confluent state was replaced with 24-differentiation-inducing medium [DMEM medium containing 10% FBS (Cytiva, USA), 1% Penicillin/Streptomycin (P/S, Cytiva, USA), 1 ⁇ M dexamethasone (DEXA, Sigma, USA), 0.5 mM 3-isobutly-1-methylxanthine (IBMX, Sigma, USA), 2 ⁇ M Rosiglitazone (Cayman, Germany), and 1 ⁇ g/mL insulin (Sigma, USA)], and then cultured under 5% CO2 conditions for 11 days to induce differentiation into adipocytes.
  • DMEM medium containing 10% FBS (Cytiva, USA), 1% Penicillin/Streptomycin (P/S, Cytiva, USA), 1 ⁇ M dexamethasone (DEXA, Sigma, USA), 0.5 mM 3-isobutly-1-methylx
  • the bispecific fusion protein MG12 (300 nM) according to one embodiment of the present invention and 10 ⁇ g/mL of insulin were treated, and then cultured in glucose-free DMEM containing 100 ⁇ g/mL of 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-Deoxyglucose (2-NDBG, Cayman, USA). Semaglutide, Tirzepatide, and Retatrutide at the same dose (300 nM) were used as positive controls.
  • the cells were washed with glucose-free DMEM medium and incubated with lysis buffer (RIPA buffer) for 20 minutes.
  • the fluorescence signals were measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, respectively, using a microplate reader (SpectraMax M3, USA).
  • rat L6-GLUTmyc muscle cells were seeded in 24-well plates at a density of 2 ⁇ 10 4 cells/well, and after 2 days, the medium was changed to ⁇ -MEM media (Gibco, USA) containing 1% penicillin streptomycin and 2% FBS (Cytiva, USA), and the muscle cells were differentiated for 5 days.
  • ⁇ -MEM media Gibco, USA
  • FBS FBS
  • the muscle cells were differentiated for 5 days.
  • the dual-specificity fusion protein MG12 300 nM
  • 10 ⁇ g/mL insulin were treated.
  • 2-NDBG was treated at a concentration of 100 ⁇ g/mL for 30 minutes.
  • the intracellular 2-NBDG content was measured at 485 nm (excitation wavelength) and 535 nm (emission wavelength) using a Microplate reader (SpectraMax M3, USA).
  • the dual-specificity fusion protein (MG12) according to one embodiment of the present invention has a superior glucose uptake efficiency in differentiated adipocytes as well as muscle fibers than GLP-1-Fc or GLP-2-Fc, as well as the existing GLP-1 analog (Semaglutide) and GLP-1R/GLP-2R dual agonist (Dapiglutide).
  • the inventors of the present invention conducted an animal experiment using diabetic model mice to confirm whether the dual-specificity fusion protein according to one embodiment of the present invention not only improves blood sugar levels but also improves insulin resistance.
  • the present inventors administered Semaglutide, Tirzepatide, or the dual specificity fusion protein MG12-8 according to one embodiment of the present invention at the indicated doses to 14-week-old male db/db mice (Janvier Labs, France) by subcutaneous injection once every three days for 12 weeks.
  • non-fasting blood glucose levels were measured on days 25, 50, and 75 from the start of the experiment at 25-day intervals, and blood glycated hemoglobin (HbA1c) levels were measured at monthly intervals until month 3.
  • HbA1c blood glycated hemoglobin
  • the dual specificity fusion protein (MG12) according to one embodiment of the present invention exhibited significantly better non-fasting blood glucose lowering ability and blood glycated hemoglobin (HbA1c) level lowering effect at a dose of 30 nmol/kg than Semaglutide at 30 nmol/kg or Tirzepatide at 15 nmol/kg, and not only significantly lowered fasting blood glucose level but also significantly increased fasting blood insulin concentration.
  • HbA1c blood glycated hemoglobin
  • the dual specificity fusion protein according to one embodiment of the present invention lowered the HOMA-IR score to the control level, and the HOMA- ⁇ score indicating beta-cell function also showed a significantly higher value compared to Semaglutide at 30 nmol/kg or Tirzepatide at 15 nmol/kg, thereby exploring the possibility of application to the treatment of type 1 diabetes as well as type 2 diabetes.
  • the inventors sacrificed the experimental animals and performed a histological analysis on the pancreatic tissue.
  • the experimental animals were sacrificed by general anesthesia, the pancreas was removed, fixed in 4% paraformaldehyde, embedded in paraffin, and the tissue was cut into transverse sections (5 ⁇ m) using a microtome. The sections were stained with insulin alone, double stained with insulin and glucagon, or double stained with insulin and Ki-67. All double stainings were performed using fluorescently labeled antibodies, and photographs were taken using a confocal laser microscope.
  • the dual specificity protein according to one embodiment of the present invention not only significantly increased the pancreatic islet area, unlike the conventional diabetes treatments Semaglutide and Tirzepatide, but also very significantly increased the area of beta cells among the entire pancreatic islets, rather decreased the area of alpha cells, and also significantly increased the number of Ki-67 positive cells per 10,000 ⁇ m 2.
  • Semaglutide, the positive control showed a significant increase, but its effect was lower than that of MG12 at the same dose, and Tirzepatide showed no improvement at all compared to the control group.
  • the dual-specificity fusion protein according to one embodiment of the present invention is a substance with a very high protective effect on pancreatic beta cells, unlike conventional diabetes treatment agents.
  • the dual-specificity fusion protein according to one embodiment of the present invention safely and very effectively showed the effect of reducing body weight and visceral fat content in obese animals, and was confirmed to be a very effective substance for the treatment of metabolic diseases such as diabetes, including type 1 diabetes as well as type 2 diabetes, and liver damage caused by the persistence of obesity.
  • the dual specificity fusion protein can be used as a medicine, particularly, as a medicine for treating metabolic diseases such as obesity, diabetes, and metabolic disorder-associated steatohepatitis.

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