EP4619423A1

EP4619423A1 - Dual function proteins and uses thereof

Info

Publication number: EP4619423A1
Application number: EP23892068.0A
Authority: EP
Inventors: Da-Eun Hwang; Do-Hoon Kim; Bora SIM; Yunji Lee; Mi Kyeong Ju; Seyoung LIM
Original assignee: Yuhan Corp
Current assignee: Yuhan Corp
Priority date: 2022-11-18
Filing date: 2023-11-17
Publication date: 2025-09-24
Also published as: WO2024107006A1; KR20240087545A; CN120530128A; AU2023381360A1; JP2025539794A

Abstract

One of the embodiment of the present invention relates to a fusion protein including a polypeptide represented by Formula (I) and a polypeptide represented by Formula (II) or a dimer thereof. The fusion protein according to the present invention or a dimer thereof is a material with improved in vivo persistence and protein productivity and stability, and has excellent effects of reducing body weight and controlling blood glucose levels.

Description

DUAL FUNCTION PROTEINS AND USES THEREOF

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

The present invention relates to a fusion protein including a bioactive protein or a dimer thereof; a method for treating or preventing metabolism-related diseases using the fusion protein or a dimer thereof and a medical use thereof; an isolated cell line producing the fusion protein or a dimer thereof; an isolated nucleic acid encoding the fusion protein or a dimer thereof; a recombinant expression vector including the nucleic acid; a host cell including the recombinant expression vector; and a method for producing the fusion protein or a dimer thereof.

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

Additionally, if two different types of bioactive protein can exert a synergistic effect with each other, these proteins may be fused to develop a dual function protein. For example, International Publication No. WO 2020/084496 discloses a GLP1-GDF15 fusion protein which includes a glucagon-like peptide-1 (GLP-1) peptide, a first linker peptide, a serum albumin protein, and GDF15 protein is described.

Meanwhile, it has been known that immunoglobulin Fc fusion increases the half-life of bioactive proteins in vivo and has fewer concerns about side effects such as toxicity and immune responses, and thus can be used in developing long-acting agents. However, even if immunoglobulin Fc fusion is applied, the activity of a bioactive protein may vary depending on the location of the fusion between a bioactive protein and an immunoglobulin Fc. In addition, even if the in vivo half-life is increased, it may be difficult to exhibit a sufficient pharmacokinetic profile to enable administration in humans at one-week intervals. Moreover, safety issues may arise due to immunogenicity due to the linker used during fusion or mutations, and there are various problems, such as the possibility of causing unintended immune responses depending on the type (isotype) of the immunoglobulin being introduced.

Further, since a dual function protein prepared using two different types of a bioactive protein is a structure that cannot exist naturally, its structure is inevitably more complex compared to those proteins which were fused with a single bioactive protein, and proteins for increasing the half-life (e.g., serum albumin protein, transferrin, etc.). Further, when developing a dual function protein, it is essential that individual bioactive proteins exist stably in the body while maintaining their physiological activity, and the difference in half-life between bioactive proteins be adjusted to an appropriate level. In order to achieve this object, introduction of a mutation and/or engineering by a linker may be required, in which due to the introduction of a mutation, the activity of a bioactive protein may vary, thus resulting in changes in the structure and stability of the bioactive protein. In addition, various problems (e.g., aggregation) may occur due to the linker and/or mutation of the generated dual function protein, thereby resulting in a decrease in protein stability.

The present inventors have found that in the process of developing dual function proteins, fusion proteins, which were prepared by fusing an Fc region of an immunoglobulin with GDF15 or a variant polypeptide thereof and then fusing with a linker and a bioactive protein different from GDF15, have excellent productivity, stability, pharmacokinetic profile, and pharmacological efficacy, thereby completing the present invention.

An object of the present invention is to provide a fusion protein, which is a dual function protein with excellent productivity, stability, pharmacokinetic profile, and pharmacological efficacy, and a dimer thereof; a method for treating or preventing metabolism-related diseases using the fusion protein or the dimer thereof and a medical use thereof; an isolated cell line producing the fusion protein or the dimer thereof; an isolated nucleic acid encoding the fusion protein or a dimer thereof; a recombinant expression vector including the nucleic acid; a host cell including the recombinant expression vector; and a method for producing the fusion protein or a dimer thereof.

In an embodiment of the present invention, the present invention provides a fusion protein including a polypeptide represented by Formula (I) and a polypeptide represented by Formula (II) or a dimer thereof.

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

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

In an embodiment of the present invention, the present invention provides a recombinant expression vector including the nucleic acid.

In another embodiment of the present invention, the present invention provides a host cell including the recombinant expression vector.

In an embodiment of the present invention, the present invention provides a method for producing a fusion protein or a dimer thereof, which includes culturing a cell line expressing a fusion protein under conditions in which the fusion protein is expressed; and recovering the fusion protein.

In another embodiment of the present invention, the present invention provides a pharmaceutical composition which includes a pharmaceutically acceptable carrier and the fusion protein, or a dimer thereof.

In a still another embodiment of the present invention, the present invention provides a method for treating or preventing a metabolism-related disease in an individual in need of treatment or prevention of the metabolism-related disease, which includes administering the fusion protein or a dimer thereof to the individual.

In an embodiment of the present invention, the present invention provides a use of the fusion protein in preparation of a drug for the treatment of metabolism-related diseases.

The fusion protein according to the present invention or a dimer thereof has improved pharmacological efficacy, in vivo persistence, and protein productivity and stability of a bioactive protein. Additionally, the fusion protein or the dimer thereof can be usefully used in methods for preventing and/or treating metabolism-related diseases.

FIG. 1 shows schematic diagrams of a fusion protein dimer including a GDF15 variant and a GLP-1 variant.

FIG. 2 shows the results of SDS-PAGE gel analysis of two-step purified HGK, HGH, and HGKH. It can be confirmed therefrom that the first and second polypeptides are not cleaved.

FIG. 3 shows graphs of the serum drug concentration of a GDF15 part over time for 240 hours after subcutaneous administration in mice of 13 types of long-acting fusion proteins. Data were expressed as means and standard deviations.

FIG. 4 shows graphs of the serum drug concentration of a GLP-1 part over time for 240 hours after subcutaneous administration in mice of 13 types of long-acting fusion proteins. Data were expressed as means and standard deviations.

FIG. 5 shows graphs of the serum drug concentration of a GDF15 part over time for 240 hours after subcutaneous administration in mice of 15 types of long-acting fusion proteins. Data were expressed as means and standard deviations.

FIG. 6 shows graphs of the serum drug concentration of a GLP-1 part over time for 240 hours after subcutaneous administration in mice of 15 types of long-acting fusion proteins. Data were expressed as means and standard deviations.

FIG. 7 shows graphs of the serum drug concentration of a GDF15 part and a GLP-1 part in rats over time for 504 hours after intravenous and subcutaneous administrations of the long-acting fusion protein HGKH-EKN-L4. Data were expressed as means and standard deviations.

FIG. 8 shows graphs comparing the GDF15 activity of 10 types of long-acting fusion proteins.

FIG. 9 shows graphs comparing the GLP-1 activity of 10 types of long-acting fusion proteins.

FIG. 10 shows graphs of the serum drug concentration of a GDF15 part in mice over time for 240 hours after subcutaneous administration of 10 types of long-acting fusion proteins. Data were expressed as means and standard deviations.

FIG. 11 shows graphs of the serum drug concentration of a GLP-1 part in mice over time for 240 hours after subcutaneous administration of 10 types of long-acting fusion proteins. Data were expressed as means and standard deviations.

FIG. 12 shows a graph comparing the body weight change (%) by repeated administration of GK-L3 and GH-L5 at different doses in diet-induced obesity (DIO) mice.

FIG. 13 shows a graph comparing the body weight change (%) in DIO mice by repeated administration of 5 types of long-acting fusion proteins (GK-L3-3, GH-L3, GH-L4, GKH-L2, and GKH-L4-L2).

FIG. 14 shows a graph comparing the body weight change (%) in DIO mice by repeated administration of HGH-EKN-L4 and HGKH-EKN-L4 at different doses.

FIG. 15 shows a graph comparing the body weight change (%) in DIO mice by repeated administration of 5 types of long-acting fusion proteins (HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGKH-EKN-L4, and HGKH-EKN-L4-49) at doses of 0.3 nmol/kg and 1 nmol/kg.

FIG. 16 shows the results of measuring non-fasting blood glucose levels on day 9 and day 21 from the initiation of administration in DIO mice repeatedly administered with 6 types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) (****p<0.0001 vs. DIO Vehicle (One-way ANOVA)).

FIG. 17 shows the results of measuring non-fasting blood glucose levels in DIO mice at the end of the administration of 6 types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) (**p<0.01, ***p<0.001, and ****p<0.0001 vs. DIO Vehicle (One-way ANOVA)).

FIG. 18 shows a graph comparing the cumulative food intake of 6 types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) measured in DIO mice from the initiation of repeated administration to day 24.

FIG. 19 shows a graph comparing the body weight change (%) in DIO mice caused by repeated administration of 6 types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4).

FIG. 20 shows a graph comparing the body weight change (%) in DIO mice caused by repeated administration of 9 types of long-acting fusion proteins (HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGK-EKN-L10-47, HGK-EKN-L10-49, HGKH-EKN-L4, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49).

FIG. 21 shows a graph comparing the cumulative food intake of HGKH-EKN-L4 measured in DIO mice from the initiation of administration to day 27 after repeated administration of HGKH-EKN-L4 at different doses.

FIG. 22 shows a graph comparing the body weight change (%) measured in DIO mice from the initiation of administration to day 27 after repeated administration of HGKH-EKN-L4 at different doses in DIO mice.

FIG. 23 shows a graph comparing the body weight change (%) measured in ob/ob mice from the initiation of administration to day 14 after single administration of HGK-EKN-L10 at different doses.

FIG. 24 shows a graph comparing the non-fasting blood glucose levels measured in ob/ob mice from the initiation of administration to day 14 after single administration of HGK-EKN-L10 at different doses.

FIG. 25 shows a graph comparing the body weight change (%) measured in ob/ob mice from the initiation of administration to day 14 after repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses.

FIG. 26 shows the results of measuring HbA1c levels in ob/ob mice at the end of the repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses (****p<0.0001 vs. Vehicle treated ob/ob control (One-way ANOVA)).

FIG. 27 shows the results of measuring the levels of total cholesterol, HDL, and LDL in ob/ob mice at the end of the repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses (**p<0.01, ***p<0.001, and ****p<0.0001 vs. ob/ob Vehicle (One-way ANOVA)).

FIG. 28 shows the results of measuring the levels of ALP, AST, ALT and relative liver weight in ob/ob mice at the end of the repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at different doses (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. ob/ob Vehicle (One-way ANOVA)).

FIG. 29 shows a graph comparing the non-fasting blood glucose levels measured in db/db mice after repeated administration of HGKH-EKN-L4 at different doses.

FIG. 30 shows the results of measuring HbA1c levels in db/db mice that were repeatedly administered HGKH-EKN-L4 at different doses on day 14 and day 26 from the initiation of administration (**p<0.01, ***p<0.001, and ****p<0.0001 vs. Vehicle treated db/db control (One-way ANOVA)).

FIG. 31 shows the results of an intraperitoneal insulin tolerance test performed in db/db mice at the end of the repeated administration of HGKH-EKN-L4 at different doses (**p<0.01, and ****p<0.0001 vs. db/db Vehicle (One-way ANOVA)).

FIG. 32 shows the results of measuring a body weight change (%) and a relative liver weight in a GAN-ob/ob NASH mouse model by repeated administration of HGKH-EKN-L4 at different doses (****p<0.0001 vs. GAN-ob/ob Vehicle (One-way ANOVA)).

FIG. 33 shows the results of measuring the levels of ALP, AST, and ALT in a GAN-ob/ob NASH mouse model at the end of the repeated administration of HGKH-EKN-L4 at different doses (^####p<0.0001 vs. Chow control (Unpaired t-test) and *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. GAN-ob/ob Vehicle (One-way ANOVA)).

FIG. 34 shows graphs comparing the degree of collagen deposition in the liver by repeated administration of HGKH-EKN-L4 at different doses in the GAN-ob/ob NASH mouse model using picro sirius red (PSR) stain (^####p<0.0001 vs. Chow Vehicle (Unpaired t-test) and ****p<0.0001 vs. GAN-ob/ob Vehicle (One-way ANOVA)).

FIG. 35 shows the amino acid sequence information of a human IgG1 Fc sequence used in the present invention.

In an aspect of the present invention, the present invention relates to a fusion protein including a polypeptide represented by Formula (I) below and a polypeptide represented by Formula (II) below or a dimer thereof:

A_p-B_q-C (I)

F_a-G_b-H-I-J (II)

wherein:

A and F are each independently a bioactive protein;

B, G, and I are each independently a linker;

C and H are each an immunoglobulin Fc or a variant thereof; and

J is a polypeptide consisting of an amino acid sequence represented by (Y₁Y₂)_t-N-(Y₃)_u-(Y₄)_v-(Y₅)_w-CPLGPGRCCRLHTV-Y_6-ASLEDLGWAD-Y₇-VLSPREVQVTMCIGACPSQFRAA-Y₈-MHA-Y₉-IKT-Y₁₀-LHRLKPDTVPAPCCVPASYNPMVLI-Y₁₁-KTDTGVSLQTYD-Y₁₂-LLAKDCHCI,

wherein:

Y₁, Y₃, and Y₇ to Y₉ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W);

Y₂, Y₆, and Y₁₁ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R);

Y₄ and Y₁₂ are a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid, which is aspartic acid (D) or glutamic acid (E);

Y₅ is a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R);

Y₁₀ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid which is aspartic acid (D) or glutamic acid (E); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R);

t, u, v, and w are each independently 0 or 1;

p and q in Formula (I) above and a and b in Formula (II) above are each independently 0 or 1, wherein when p is 0, q is 0, and when p is 1, q is 1; and

when a is 0, b is also 0, and when a is 1, b is 1, with the proviso that p and q in Formula (I) and a and b in Formula (II) are not 0 simultaneously.

In another aspect of the present invention, the present invention relates to a fusion protein or a dimer thereof, in which a bioactive protein and GDF15 or a variant thereof represented by the following Formula (III) are linked to the Fc of an immunoglobulin or a variant thereof, and optionally, a bioactive protein and/or GDF15 or a variant thereof represented by Formula (III) may be linked to the Fc of an immunoglobulin or a variant thereof by a linker:

(Y₁Y₂)_t-N-(Y₃)_u-(Y₄)_v-(Y₅)_w-CPLGPGRCCRLHTV-Y_6-ASLEDLGWAD-Y₇-VLSPREVQVTMCIGACPSQFRAA-Y₈-MHA-Y₉-IKT-Y₁₀-LHRLKPDTVPAPCCVPASYNPMVLI-Y₁₁-KTDTGVSLQTYD-Y₁₂-LLAKDCHCI (III).

GDF15 or a variant thereof represented by Formula (III) above is a polypeptide consisting of the amino acid sequence represented by (Y₁Y₂)_t-N-(Y₃)_u-(Y₄)_v-(Y₅)_w-CPLGPGRCCRLHTV-Y_6-ASLEDLGWAD-Y₇-VLSPREVQVTMCIGACPSQFRAA-Y₈-MHA-Y₉-IKT-Y₁₀-LHRLKPDTVPAPCCVPASYNPMVLI-Y₁₁-KTDTGVSLQTYD-Y₁₂-LLAKDCHCI,

wherein:

Y₅ is a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R); and

Y₁₀ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid which is aspartic acid (D) or glutamic acid (E); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R). In an embodiment of the present invention, GDF15 or a variant thereof represented by Formula (III) above may be a polypeptide corresponding to J of Formula (II) above.

As used herein, the term "bioactive protein" refers to a bioactive protein that has physiological activity in vivo. For example, a bioactive protein may be selected from the group consisting of insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor related protein (CTRP family), resistin, visfatin, omentin, retinol binding protein-4 (RBP4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, growth hormone, and variants thereof, but is not limited thereto.

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

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

As used herein, the term "GLP-1" is an incretin hormone consisting of 31 amino acids that is secreted from L cells in the intestinal tract upon stimulation by food, etc. GLP-1 exerts its activity by transmitting signals into cells through GLP-1 receptors, which are G protein-coupled protein receptors expressed in target tissues such as pancreatic beta cells and the brain. Human wild-type GLP-1 is a polypeptide represented by the amino acid sequence SEQ ID NO: 9 (HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR G).

GLP-1 secreted in the blood has a very short in vivo half-life of less than 2 minutes, which is due to loss of activity caused by cleavage of the N-terminal amino acid by the dipeptidyl peptidase-4 (DPP-4) enzyme in the body. Since GLP-1 promotes insulin secretion from pancreatic beta cells according to blood glucose concentration, it has a strong blood glucose lowering effect without causing hypoglycemia. Additionally, when GLP-1 is administered, weight loss occurs in various animal models and humans, and this is known to be resulted from a decrease in food intake due to the appetite-suppressing effect of GLP-1. GLP-1 induces an increase in viability and promotion of proliferation of beta cells by suppressing beta cell apoptosis caused by glycolipid toxicity through the GLP-1 receptors expressed in pancreatic beta cells. Excessive secretion of glucagon increases blood glucose levels and is known to be one of the causes of hyperglycemia appeared in diabetic patients. In addition, GLP-1 is known to suppress the rise in the fasting blood glucose levels by acting on pancreatic alpha cells and to suppress protein kinase A (PKA) protein-specific glucagon secretion.

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

Meanwhile, exenatide, liraglutide, dulaglutide, albiglutide, lixisenatide, semaglutide, tirzepatide, cotadutide, and taspoglutide described above are type of GLP-1 analogues (materials designed to mimic the effects of endogenous GLP-1, stimulating glucose-dependent insulin secretion and inhibiting glucagon secretion from the pancreas).

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

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

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

In still another embodiment of the present invention, the bioactive protein is a polypeptide consisting of an amino acid sequence represented by H-X₁-EGTFTSDVSSYLE-X₂-QAAKEFI-X₃-WL-X₄-X₅-G-X₆-G, in which X₁ may be (A) or glycine (G), and/or X₂ may be glycine (G) or glutamic acid (E), and/or X₃ may be alanine (A), and/or X₄ may be valine (V) or lysine (K), and/or X₅ may be lysine (K) or asparagine (N), and/or X₆ may be arginine (R) or glycine (G).

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

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

In another embodiment of the present invention, the bioactive protein is a polypeptide consisting of an amino acid sequence represented by H-X₁-EGTFTSDVSSYLE-X₂-QAAKEFI-X₃-WL-X₄-X₅-G-X₆-G, in which X₁ may be glycine (G), X₂ may be glutamic acid (E), and X₆ may be glycine (G).

In an embodiment of the present invention, the bioactive protein is a polypeptide consisting of an amino acid sequence represented by H-X₁-EGTFTSDVSSYLE-X₂-QAAKEFI-X₃-WL-X₄-X₅-G-X₆-G, in which X₃ may be alanine (A).

In another embodiment of the present invention, the bioactive protein is a polypeptide consisting of an amino acid sequence represented by H-X₁-EGTFTSDVSSYLE-X₂-QAAKEFI-X₃-WL-X₄-X₅-G-X₆-G, in which X₃ may be glutamic acid (E), and X₄ may be valine (V).

In still another embodiment of the present invention, the bioactive protein is a polypeptide consisting of an amino acid sequence represented by H-X₁-EGTFTSDVSSYLE-X₂-QAAKEFI-X₃-WL-X₄-X₅-G-X₆-G, in which X₃ may be glutamic acid (E), X₄ may be lysine (K), and X₅ may be asparagine (N).

In an embodiment of the present invention, A and F may each independently be selected from the group consisting of the amino acid sequences of SEQ ID NOS: 9 to 12.

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

In an embodiment of the present invention, t may be 0, Y₃ may be glycine (G) or serine (S), and/or Y₄ may be aspartic acid (D) or threonine (T), and/or Y₅ may be histidine (H), and/or Y₆ may be arginine (R) or asparagine (N), and/or Y₇may be tryptophan (W) or phenylalanine (F), and/or Y₈ may be asparagine (N), leucine (L), cysteine (C), or serine (S), and/or Y₉ may be glutamine (Q) or asparagine (N), and/or Y₁₀ may be serine (S), asparagine (N), aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), or leucine (L), Y₁₁ may be glutamine (Q) or histidine (H), and/or Y₁₂ may be aspartic acid (D), leucine (L), cysteine (C), or serine (S).

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

In still another embodiment of the present invention, w may be 0, Y₃ may be serine (S), and Y₄ may be threonine (T).

In another aspect of the present invention, J in Formula (II) above may be wild-type GDF15 or a variant thereof. Growth differentiation factor-15 (GDF15), which is also called macrophage inhibitory cytokine-1 (MIC-1), placental bone morphogenetic protein (PBMP) or nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), is a protein that is one of the members of the transforming growth factor-beta superfamily (TGF-β superfamily). Study results have been reported that GDF15 induces weight loss by suppressing dietary intake through binding to GDNF Family receptor alpha-like (GFRAL) and Ret proto-oncogene (RET), which are specifically expressed in brain tissue (Tsai VW, et al., PLoS One 2013; 8 (2): e55174; US 8,192,735). In addition, several studies have demonstrated excellent weight loss effects by administering GDF15 to various animal models with obesity; besides, additional metabolic benefits such as lowering of blood glucose levels, improvement of lipid levels, and improvement of insulin resistance were confirmed. The wild-type GDF15 protein may be derived from mammals such as humans, mice, pigs, and monkeys. Specifically, it may be a wild-type GDF15 protein derived from humans. More specifically, the wild-type GDF15 protein may be the human wild-type GDF15 protein represented by SEQ ID NO: 1, or the GDF15 protein whose NCBI reference sequence is NP_004855.2.

In another aspect of the present invention, J in Formula (II) above may be a GDF15 variant including one or more mutations selected from the group consisting of the mutations (1) to (12) below:

(1) The 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted (hereinafter described as "△N2");

(2) the 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and the 64th amino acid, serine, is substituted with arginine (hereinafter described as "△N2, S64R");

(3) the 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and the 32nd amino acid, tryptophan, is substituted with phenylalanine (hereinafter described as "△N2, W32F");

(4) the 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and the 90th amino acid, glutamine, is substituted with histamine (hereinafter described as "△N2, Q90H");

(5) the 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and the 60th amino acid, glutamine, is substituted with asparagine (hereinafter described as "△N2, Q60N");

(6) the 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and the 64th amino acid, serine, is substituted with asparagine (hereinafter described as "△N2, S64N");

(7) the 1st and 2nd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and the 64th amino acid, serine, is substituted with aspartic acid (hereinafter described as "△N2, S64D");

(8) the 1st to 3rd amino acids from the N terminus of the wild-type GDF15 protein (SEQ ID NO: 1) are deleted (hereinafter described as "△N3");

(9) the 1st to 3rd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and tryptophan and serine are inserted in their place (hereinafter described as "△N3, WS insertion");

(10) the 1st to 3rd amino acids from the N terminus of the wild-type GDF15 protein are deleted, and tryptophan and serine are inserted in their place; the 4th amino acid, glycine, is substituted with asparagine; the 5th amino acid, aspartic acid, is substituted with serine; and the 6th amino acid, histidine, is substituted with threonine (hereinafter described as "△N3, WS insertion, G4N, D5S, H6T");

(11) the 1st to 3rd amino acids from the N terminus of the wild-type GDF15 protein are deleted; the 4th amino acid, glycine, is substituted with asparagine; the 5th amino acid, aspartic acid, is substituted with serine; and the 6th amino acid, histidine, is substituted with threonine (hereinafter described as "△N3, G4N, D5S, H6T"); and

(12) the 1st to 3rd amino acids from the N terminus of the wild-type GDF15 protein are deleted; the 4th amino acid, glycine, is substituted with asparagine; the 5th amino acid, aspartic acid, is replaced with serine, the 6th amino acid, histidine, is substituted with threonine, and the 64th amino acid, serine, is substituted with arginine (hereinafter described as "△N3, NST, S64R").

In an embodiment of the present invention, the GDF15 variant may include a mutation, which is any one of S64R, W32F, Q90H, Q60N, S64N, and S64D in addition to △N2; NST and S64R in addition to △N3; or a combination of any one of S64R, S64N, and S64D with any one of W32F, Q90H, and Q60N.

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

In another embodiment of the present invention, J of Formula (II) above may be one in which the N-residue introduced by mutation is glycosylated or may include at least one N-linked glycan.

In one aspect of the present invention, J of Formula (II) above may include any one of the amino acid sequences of SEQ ID NOS: 2 to 8 and 93 to 108. In addition, the GDF15 variant protein may be in a form, in which, while the protein being represented by any one selected from the group consisting of amino acid sequences of SEQ ID NOS: 2 to 8 and 93 to 108 and 1 to 14 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type GDF15 protein.

In still another aspect of the present invention, B of Formula (I) above and G and I of Formula (II) above are linkers that can connect a bioactive protein and/or a GDF variant to any different protein. The linkers may be those known as a linker that can connect proteins and/or compounds to each other in the technical field to which the present invention belongs, but are not limited thereto.

In another aspect of the present invention, B of Formula (I) above and G and I of Formula (II) above may each independently be linkers selected from the group consisting of 1) a peptide consisting of 5 to 52 glycines (G) and/or serines (S); 2) a peptide consisting of 4 to 52 glutamic acids (E) and/or alanines (A); 3) a peptide consisting of 5 to 52 glutamic acids (E), lysines (K), and/or alanines (A); 4) a peptide consisting of 6 to 52 glycines (G), serines (S), glutamic acids (E), and/or alanines (A); and 5) a peptide consisting of 7 to 52 glycines (G), serines (S), glutamic acids (E), lysines (K), and/or alanines (A), but are not limited thereto.

In still another aspect of the present invention, the linker may be a linker selected from the group consisting of (G₄S)_n, GS(G₄S)_n, GS(EEEA)_n, (EEEA)_n, GS(EAAAK)_n, and (EAAAK)_n, or a polypeptide including the same. The n may be an integer of 1 to 10. In an embodiment of the present invention, a linker including (G₄S)₃ and a linker including GS(EEEA)₆and GS(EAAAK)₅ were used, but are not limited thereto.

In an embodiment of the present invention, the linker may each independently be selected from the group consisting of the amino acid sequences of SEQ ID NOS: 13 to 24, but is not limited thereto.

In still another aspect of the present invention, the linker may be GSEEEAEEEAEEEAEEEA (SEQ ID NO: 22), GSEEEAEEEAEEEAEEEAEEEAEEEA (SEQ ID NO: 23), GSEEEAEEEAEEEAEEEAEEEAEEEAEEEAEEEA (SEQ ID NO: 24), or GSEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 21). Preferably, the linker may be GSEEEAEEEAEEEAEEEAEEEAEEEA (SEQ ID NO: 23) or GSEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 21).

In an aspect of the present invention, C of Formula (I) above and H of Formula (II) above are each an Fc of each immunoglobulin or a variant thereof.

As used herein, the term "an Fc region", "an Fc fragment", or "an Fc" refers to a protein, which includes a heavy chain constant region 1 (CH1), a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include variable regions of heavy and light chains and light chain constant region 1 (CL1) of an immunoglobulin,

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

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

In another embodiment of the present invention, the immunoglobulin Fc or a variant thereof may be IgG1 Fc or a variant thereof, and a human IgG1 Fc may consist of the amino acid sequence represented by SEQ ID NO: 80 (see FIG. 35). The human IgG Fc or a variant thereof may have an adjacent amino acid sequence which is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80 or an Fc fragment including a CH3 domain. In an embodiment of the present invention, the human IgG Fc or a variant thereof may have an adjacent amino acid sequence which has an identity of 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 80 or an Fc fragment including a CH2 domain and a CH3 domain. In another embodiment of the present invention, the human IgG Fc or a variant thereof may have an adjacent amino acid sequence which is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80 or an Fc fragment including a partial hinge region, a CH2 domain, and a CH3 domain. In still another embodiment of the present invention, the human IgG Fc or a variant thereof may have an adjacent amino acid sequence which is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80.

In an aspect of the present invention, the Fc variant of an immunoglobulin may include at least one processed protuberance, or may include at least one processed cavity, and the protuberance and cavity may be located in a CH3 region. Specifically, an Fc variant of an immunoglobulin including a processed cavity may be processed to bind to an Fc variant of an immunoglobulin including a processed protuberance. The protuberance may be referred to as a "knob" and the cavity may be referred to as a "hole" When an Fc variant of an immunoglobulin includes a processed protuberance, it may be referred to as an Fc "knob", and if an Fc variant of an immunoglobulin includes a processed cavity, it may be referred to as an Fc "hole".

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

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

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

In still another aspect of the present invention, in a human IgG1 Fc fragment variant including a processed protuberance, the 146th amino acid, the 148th amino acid, and the 187th amino acid from the N-terminus in the amino acid sequences of SEQ ID NO: 80 may be substituted with serine (S), alanine (A), and valine (V), respectively.

In an aspect of the present invention, the immunoglobulin Fc or a variant thereof may be one in which the IgG effector function is reduced or eliminated. The immunoglobulin Fc or a variant thereof may include mutant(s) that eliminates (e.g., reduces or eliminates) the effector function. For example, the Fc partner sequence of the immunoglobulin Fc or a variant thereof may include mutant(s) that abolish(es) effector functions (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

In an embodiment of the present invention, the immunoglobulin Fc or a variant thereof may have E13A and L15A mutations introduced into the IgG1 amino acid sequence of SEQ ID NO: 80 to eliminate the IgG1 effect functionality. These mutations correspond to E233A and L235A when numbered according to Kabat's EU index.

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

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

In another aspect of the present invention, the Fc of the immunoglobulin or a variant thereof may be a polypeptide in which a hinge is added to the N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 81 to 86.

In still another aspect of the present invention, the Fc of the immunoglobulin or a variant thereof may be a polypeptide which includes a hinge consisting of one amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOS: 79, and 109 to 111.

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

In another aspect of the present invention, C of Formula (I) above may be a polypeptide consisting of any one amino acid sequence selected from SEQ ID NOS: 82, 84, 86, 88, 90, and 92; and H of Formula (II) above may be a polypeptide consisting of any one amino acid sequence selected from SEQ ID NOS: 81, 83, 85, 87, 89, and 91.

In another aspect of the present invention, the Fc of the immunoglobulin or a variant thereof may be a polypeptide consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: 81 to 92.

In an embodiment of the present invention, C of Formula (I) above may be an IgG Fc including a processed cavity or a variant thereof; H of Formula (II) above may be an IgG Fc including a processed protuberance; and through the arrangement of the protuberance and the cavity, the polypeptide represented by Formula (I) and the polypeptide represented by Formula (II) may be fused.

In still another embodiment of the present invention, the polypeptide represented by Formula (I) and the polypeptide represented by Formula (II) may be physically bound through a non-covalent interaction (e.g., a hydrophobic effect, such as a hydrophobic interaction between the Knob and Hole regions of an Fc), a covalent bond (e.g., a disulfide bond, such as 1 or 2 or more disulfide bonds between the hinge regions of an Fc), or through both a non-covalent interaction and a covalent bond.

As used herein, the term "dimer" means a protein complex including at least two polypeptides. Each of these polypeptides includes an N-terminus and a C-terminus. At least two polypeptides may be linked to each other through one or both of covalent and non-covalent interactions (e.g., an electrostatic, π-effect, a van der Waals force, and a hydrophobic effect). The two polypeptides may have the same amino acid sequence or may be different. When two types of polypeptides have two identical polypeptides, they are referred to as homodimers, whereas when the two types of polypeptides have two different polypeptides, they are referred to as heterodimers.

In an aspect of the present invention, a dimer of the fusion protein including a polypeptide represented by Formula (I) and a polypeptide represented by Formula (II) may be a homodimer in which the fusion proteins are identical to each other, or a heterodimer in which the fusion proteins are different from each other.

In another aspect of the present invention, the fusion protein of the present invention may be selected from the group consisting of the followings:

(1) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(2) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(3) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(4) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 30 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(5) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(6) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 32 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(7) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(8) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(9) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(10) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(11) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 37 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(12) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 38 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(13) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 39 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(14) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(15) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked;

(16) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(17) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(18) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(19) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 68 are linked;

(20) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 69 are linked;

(21) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 70 are linked;

(22) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 67 are linked;

(23) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(24) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(25) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(26) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked;

(27) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(28) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(29) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(30) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 41 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(31) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(32) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(33) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 44 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(34) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(35) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(36) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(37) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(38) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(39) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(40) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked;

(41) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked;

(42) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked;

(43) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(44) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked;

(45) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked;

(46) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 71 are linked;

(47) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked;

(48) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked;

(49) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked;

(50) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(51) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked;

(52) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked;

(53) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 51 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(54) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 52 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(55) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 53 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(56) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 54 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(57) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 55 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(58) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 56 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(59) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 57 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(60) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 58 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(61) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 59 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(62) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 60 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(63) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 61 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; and

(64) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 62 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked.

In an aspect of the present invention, the present invention relates to an isolated cell line producing the fusion protein or a dimer thereof; an isolated nucleic acid encoding the fusion protein or a dimer thereof; a recombinant expression vector including the nucleic acid; and a host cell including the vector.

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

In another aspect of the present invention, the fusion protein or a dimer thereof may be expressed by a suitable cell expression system which is characterized by producing the same. As the cell expression system, a cell expression system, such as mammalian cells (e.g., CHO, COS, HEK 293, BHK, SK-Hip, etc.), E. coli, Saccharomyces cerevisiae, Pichia pastoris, and insect cells, which are known to be suitable for producing proteins in the technical field to which the present invention belongs, may be used.

The isolated host cell may be transformed or transfected with the DNA sequence of the present invention, and may be used to express and/or secrete a fusion protein or a dimer thereof. The host cell that may be used in the present invention include immortal hybridoma cells, NS/0 myeloma cells, HEK 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CAP cells (cells derived from human amniotic fluid), or COS cells.

As used herein, the term "isolated nucleic acid" refers to a nucleic acid molecule of the present invention, which is a part of a polynucleotide sequence that may have been isolated from about 50% or more of proteins, lipids, carbohydrates, or other materials that are discovered along with a nucleic acid in nature when the entire nucleic acid is isolated from a source cell, may be operably linked to a polynucleotide that is not linked in nature, or a part of a larger polynucleotide sequence that does not occur in nature. Specifically, the isolated nucleic acid molecule of the present invention is substantially free of any other contaminating nucleic acid molecules or other contaminants found in its natural environments that would impair the production of a polypeptide or its use in therapeutic, diagnostic, prophylactic, or research purposes do not exist. In particular, the isolated nucleic acid molecules encoding the fusion protein may have mutually-different sequences due to codon redundancy. Additionally, the isolated nucleic acid molecule may be appropriately modified depending on the purpose, or nucleotides may be added to the N-terminus or C-terminus, as long as the fusion protein can be produced.

As used herein, the term "recombinant expression vector" refers to a vector that is suitable for transformation of a host cell and includes a nucleic acid sequence that directs or controls the expression of an inserted heterologous nucleic acid sequence. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of the viral vector includeretroviruses, adenoviruses, and adeno-associated viruses, but are not limited thereto.

As used herein, the term "expression of a heterologous nucleic acid sequence" or "expression" of a target protein refers to transcription of an inserted DNA sequence, translation of an mRNA transcript, and production of an Fc fusion protein product or antibody or antibody fragment. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or variants thereof. The useful expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase a steady-state level of RNA after transcription. As an example of the present invention, the expression vector may be pAD15, a modified vector of RcCMV.

In another aspect, the present invention relates to a method for producing a fusion protein or a dimer thereof, which includes, under the conditions where the fusion protein is expressed, culturing a cell line expressing the fusion protein; and recovering the fusion protein. The conditions under which the fusion protein is expressed are those which allow the cell line to grow appropriately, and may be selected within the known technical scope of the technical field to which the present invention pertains.

Additionally, in an embodiment of the present invention, after recovering the fusion protein, a purification step using a purification method including column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, high performance liquid chromatography (HPLC), affinity chromatography, immunoprecipitation, etc., but not limited to, may be accomplished.

In still another aspect, the present invention relates to a composition which includes a pharmaceutically acceptable carrier and the fusion protein or a dimer thereof. Additionally, the present invention relates to a pharmaceutical composition for treating or preventing metabolism-related diseases which includes a pharmaceutically acceptable carrier and the fusion protein or a dimer thereof.

In an embodiment of the present invention, the pharmaceutically acceptable carrier may be any non-toxic material suitable for delivering a drug or a protein to an individual. Distilled water, alcohols, fats, waxes and inert solids may be included as the carrier. A pharmaceutically acceptable adjuvant (buffers, dispersants) may also be included in the pharmaceutical composition. The concentration of the fusion protein in such formulations may vary significantly.

In another embodiment of the present invention, the composition and the pharmaceutical composition may include formulation materials so as to modify, maintain, or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition. Suitable formulation materials may include amino acids (e.g., glycine, glutamine, asparagine, arginine, and lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, and sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrate, phosphates, and other organic acids), bulking agents (e.g., mannitol and glycine), chelating agents (e.g., ethylenediamine tetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (e.g., glucose, mannose, and dextrins), proteins (e.g., serum albumin, gelatin, and immunoglobulins), colorants, flavoring agents and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, and polyethylene glycol), sugar alcohols (e.g., mannitol and sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics; PEG; sorbitan ester; polysorbates (e.g., polysorbate 20 and polysorbate 80); triton; tromethamine; lecithin; cholesterol; and tyloxapal), stability enhancing agents (e.g., sucrose and sorbitol), tonicity enhancing agents (e.g., alkali metal halides; preferably sodium chloride or potassium chloride; and mannitol sorbitol), delivery Vehicles, diluents, excipients and/or pharmaceutical adjuvants, but are not limited thereto.

In still another embodiment of the present invention, the composition and the pharmaceutical composition may be administered by any route. For example, the composition and the pharmaceutical composition may be provided to an animal by any suitable means, either directly (e.g., topically, by injection, implantation, or local administration to a tissue site) or systemically (e.g., parenterally or orally). When the composition and the pharmaceutical composition are administered via parenteral administration (e.g., intravenous, subcutaneous, ophthalmic, intraperitoneal, intramuscular, oral, rectal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracistenal, intracapsular, intranasal, or aerosol administration), the compositions may be aqueous or may include a part of a body fluid suspension or solution that is physiologically applicable. Accordingly, since a carrier or Vehicle is physiologically acceptable, it may be added to the compositions and delivered to an individual. Accordingly, the composition and the pharmaceutical composition may generally include physiological saline as a carrier, such as a body fluid, for the formulation.

In an embodiment of the present invention, the frequency of administration of the composition and the pharmaceutical composition may vary depending on the pharmacokinetic parameters of the fusion protein in the formulation being used. Typically, a clinician will administer the pharmaceutical composition until the dose that achieves the desired effect is reached. Accordingly, the composition and the pharmaceutical composition may be administered as a single dose, in two or more doses separated by a time interval (which may or may not include an equal amount of the target fusion protein), or as continuous infusion through an implanted device or catheter. Further precision of appropriate doses is routinely made by those skilled in the art and it falls within the scope of their routine practice.

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

In an aspect of the present invention, the present invention relates to a method for treating or preventing metabolism-related diseases in an individual in need of the treatment or prevention of metabolism-related diseases, in which the method includes administering the fusion protein or a dimer thereof to the individual; and a use of the fusion protein or a dimer thereof in manufacturing a drug for the treatment of metabolism-related diseases.

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

As used herein, the term "metabolism-related diseases" refers to diabetes, obesity, hypercholesterolemia, hyperglycemia, insulin resistance, non-alcoholic steatoheaptitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver damage, hepatitis, liver fibrosis, liver cirrhosis, fatty liver, arteriosclerosis, dyslipidemia, cardiovascular disease, or metabolic syndrome, but is not limited thereto. The hepatitis may be non-viral hepatitis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E.

The fusion protein of the present invention or a dimer thereof has thermal stability. Preferably, the fusion protein of the present invention may have a melting temperature (Tm) and a temperature of aggregation (Tagg) of 40℃ or higher, 45℃ or higher, 46℃ or higher, 47℃ or higher, 50℃ or higher, 51℃ or higher, 52℃ or higher, 53℃ or higher, 54℃ or higher, 55℃ or higher, 56℃ or higher, 57℃ or higher, 58℃ or higher, 59℃ or higher, or 60℃ or higher, and the Tm and Tagg of the fusion protein may be 75℃, 74℃, 73℃, or 72℃ or below. Additionally, the fusion protein of the present invention may have the Tm and Tagg of 40℃ to 75℃, 40℃ to 74℃, 40℃ to 73℃, or 40℃ to 72℃.

The fusion protein of the present invention or a dimer thereof may reduce non-fasting blood glucose levels and/or fasting blood glucose levels, maintain the variation range in non-fasting blood glucsoe levels and/or fasting blood glucose levels at a low level, reduce blood glycated hemoglobin levels, blood total cholesterol, blood high-density lipoprotein (HDL) levels, blood low-density lipoprotein (LDL) levels, insulin resistance, blood liver damage parameters (e.g., ALT, AST, ALP), and/or the subject's body weight, inhibit the occurrence of liver damage and/or liver fibrosis or reduce the rate of progression thereof, but the effects are not limited thereto. Accordingly, in another embodiment of the present invention, the fusion protein of the present invention or a dimer thereof may treate diabetes, obesity, hypercholesterolemia, hyperglycemia, insulin resistance, NASH, NAFLD, liver damage, hepatitis, liver fibrosis, liver cirrhosis, fatty liver, arteriosclerosis, dyslipidemia, cardiovascular disease, or metabolic syndrome or improve the symptoms of these diseases.

In an embodiment of the present invention, the fusion protein of the present invention or a dimer thereof may be administered to a subject along with other biologically active molecules. The optimal combination between the fusion protein or a dimer thereof and other molecules, type of administration, and amount of dose may be determined through routine experimentation well known in the art.

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

Preparation Example 1. Preparation and purification of long-acting fusion proteins and dimers thereof (1)

Preparation Example 1-1. Gene cloning

A first polypeptide (corresponding to the polypeptide represented by Formula (II)), which is in the form of [IgG1 Fc_knob or IgG1 hFc_knob in which a hinge (SEQ ID NO: 79) is introduced into the IgG1 Fc_knob]-[linker]-[GDF15 or a variant thereof] or in the form of [GLP-1 or a variant thereof]-[linker]-[IgG1 Fc_knob or IgG1 hFc_knob in which a hinge (SEQ ID NO: 79) is introduced into the IgG1 Fc_knob]-[linker]-[GDF15 or a variant thereof], was designed (see Table 1).

GLP-1 Change in Sequence	GLP-1 Linker Sequence	IgG1 Fc	GDF15 Linker Sequence	GDF15 Change in Sequence	SEQ ID NO
-	-	IgG1 Fc knob (SEQ ID NO: 85)	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 25
-	-	IgG1 Fc_knob	GS(EEEA)6	△N2, S64R	SEQ ID NO: 26
A8G, G22E, R36G	(GGGGS)3	IgG1 Fc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 27
A8G, G22E, R36G	(EEEA)2	IgG1 Fc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 28
A8G, G22E, R36G	(EEEA)4	IgG1 Fc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 29
A8G, G22E, R36G	(EEEA)4	IgG1 Fc_knob	GS(EEEA)4	△N2, S64R	SEQ ID NO: 30
A8G, G22E, R36G	(EEEA)4	IgG1 Fc_knob	GS(EEEA)6	△N2, S64R	SEQ ID NO: 31
A8G, G22E, R36G	(EEEA)4	IgG1 Fc_knob	GS(EEEA)8	△N2, S64R	SEQ ID NO: 32
A8G, G22E, R36G	(EEEA)6	IgG1 Fc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 33
A8G, G22E, R36G	(EEEA)6	IgG1 Fc_knob	GS(EEEA)4	△N2, S64R	SEQ ID NO: 34
A8G, G22E, R36G	(EEEA)6	IgG1 Fc_knob	GS(EEEA)6	△N2, S64R	SEQ ID NO: 35
A8G, G22E, R36G	(EEEA)6	IgG1 Fc_knob	GS(EEEA)8	△N2, S64R	SEQ ID NO: 36
A8G, G22E, R36G	(EEEA)8	IgG1 Fc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 37
A8G, G22E, R36G	(EEEA)8	IgG1 Fc_knob	GS(EEEA)4	△N2, S64R	SEQ ID NO: 38
A8G, G22E, R36G	(EEEA)8	IgG1 Fc_knob	GS(EEEA)6	△N2, S64R	SEQ ID NO: 39
A8G, G22E, R36G	(EEEA)8	IgG1 Fc_knob	GS(EEEA)8	△N2, S64R	SEQ ID NO: 40
A8G, G22E, R36G	(EEEA)2	IgG1 Fc_knob	GS(EEEA)6	△N2, S64R	SEQ ID NO: 41
-	-	IgG1 hFc_knob (SEQ ID NO: 91)	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 42
A8G, G22E, R36G	(EAAAK)3	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 43
A8G, G22E, R36G	(EAAAK)5	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 44
A8G, G22E, A30E, R36G	(EAAAK)3	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 45
A8G, G22E, A30E, R36G	(EAAAK)5	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 46
A8G, G22E, A30E, R36G	(EAAAK)7	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 47
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)3	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 48
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)5	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 49
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)7	IgG1 hFc_knob	GS(EAAAK)5	△N2, S64R	SEQ ID NO: 50

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

GLP-1 Change in Sequence	GLP-1 linker sequence	IgG1 Fc	SEQ ID NO
-	-	IgG1 Fc_hole	SEQ ID NO: 86
-	-	IgG1 hFc_hole	SEQ ID NO: 92
A8G, G22E, R36G	(GGGGS)₃	IgG1 Fc_hole	SEQ ID NO: 63
A8G, G22E, R36G	(EEEA)₂	IgG1 Fc_hole	SEQ ID NO: 64
A8G, G22E, R36G	(EEEA)₄	IgG1 Fc_hole	SEQ ID NO: 65
A8G, G22E, R36G	(EEEA)₆	IgG1 Fc_hole	SEQ ID NO: 66
A8G, G22E, R36G	(EEEA)₈	IgG1 Fc_hole	SEQ ID NO: 67
A8G, G22E, R36G	(EAAAK)₃	IgG1 Fc_hole	SEQ ID NO: 68
A8G, G22E, R36G	(EAAAK)₅	IgG1 Fc_hole	SEQ ID NO: 69
A8G, G22E, R36G	(EAAAK)₇	IgG1 Fc_hole	SEQ ID NO: 70
A8G, G22E, R36G	(EAAAK)₃	IgG1 hFc_hole	SEQ ID NO: 71
A8G, G22E, A30E, R36G	(EAAAK)₃	IgG1 hFc_hole	SEQ ID NO: 72
A8G, G22E, A30E, R36G	(EAAAK)₅	IgG1 hFc_hole	SEQ ID NO: 73
A8G, G22E, A30E, R36G	(EAAAK)₇	IgG1 hFc_hole	SEQ ID NO: 74
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_hole	SEQ ID NO: 75
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_hole	SEQ ID NO: 76
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₇	IgG1 hFc_hole	SEQ ID NO: 77

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

Material Code	SEQ ID NOS	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Linker Sequence
GK-E1	27 and 86	IgG1 Fc_knob	(GGGGS)3	GS(EAAAK)5
GK-L1	28 and 86	IgG1 Fc_knob	(EEEA)2	GS(EAAAK)5
GK-L2	29 and 86	IgG1 Fc_knob	(EEEA)4	GS(EAAAK)5
GK-L2-2	30 and 86	IgG1 Fc_knob	(EEEA)4	GS(EEEA)4
GK-L2-3	31 and 86	IgG1 Fc_knob	(EEEA)4	GS(EEEA)6
GK-L2-4	32 and 86	IgG1 Fc_knob	(EEEA)4	GS(EEEA)8
GK-L3	33 and 86	IgG1 Fc_knob	(EEEA)6	GS(EAAAK)5
GK-L3-2	34 and 86	IgG1 Fc_knob	(EEEA)6	GS(EEEA)4
GK-L3-3	35 and 86	IgG1 Fc_knob	(EEEA)6	GS(EEEA)6
GK-L3-4	36 and 86	IgG1 Fc_knob	(EEEA)6	GS(EEEA)8
GK-L7	37 and 86	IgG1 Fc_knob	(EEEA)8	GS(EAAAK)5
GK-L7-2	38 and 86	IgG1 Fc_knob	(EEEA)8	GS(EEEA)4
GK-L7-3	39 and 86	IgG1 Fc_knob	(EEEA)8	GS(EEEA)6
GK-L7-4	40 and 86	IgG1 Fc_knob	(EEEA)8	GS(EEEA)8
GH-E1	25 and 63	IgG1 Fc_hole	(GGGGS)3	GS(EAAAK)5
GH-L1	25 and 64	IgG1 Fc_hole	(EEEA)2	GS(EAAAK)5
GH-L2	25 and 65	IgG1 Fc_hole	(EEEA)4	GS(EAAAK)5
GH-L3	25 and 66	IgG1 Fc_hole	(EEEA)6	GS(EAAAK)5
GH-L4	25 and 68	IgG1 Fc_hole	(EAAAK)3	GS(EAAAK)5
GH-L5	25 and 69	IgG1 Fc_hole	(EAAAK)5	GS(EAAAK)5
GH-L6	25 and 70	IgG1 Fc_hole	(EAAAK)7	GS(EAAAK)5
GH-L7	25 and 67	IgG1 Fc_hole	(EEEA)8	GS(EAAAK)5
GH-L4-L1	26 and 64	IgG1 Fc_hole	(EEEA)2	GS(EEEA)6
GH-L4-L2	26 and 65	IgG1 Fc_hole	(EEEA)4	GS(EEEA)6
GH-L4-L3	26 and 66	IgG1 Fc_hole	(EEEA)6	GS(EEEA)6
GKH-E1	27 and 63	IgG1 Fc_knob, IgG1 Fc_hole	(GGGGS)3	GS(EAAAK)5
GKH-L1	28 and 64	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)2	GS(EAAAK)5
GKH-L2	29 and 65	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)4	GS(EAAAK)5
GKH-L3	33 and 66	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)6	GS(EAAAK)5
GKH-L4-1	41 and 64	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)2	GS(EEEA)6
GKH-L4-2	31 and 65	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)4	GS(EEEA)6
GKH-L4-3	35 and 66	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)6	GS(EEEA)6
HGK-L10	44 and 92	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5
HGK-E-L8	45 and 92	IgG1 hFc_knob	(EAAAK)3	GS(EAAAK)5
HGK-E-L10	46 and 92	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5
HGK-E-L12	47 and 92	IgG1 hFc_knob	(EAAAK)7	GS(EAAAK)5
HGK-EKN-L8	48 and 92	IgG1 hFc_knob	(EAAAK)3	GS(EAAAK)5
HGK-EKN-L10	49 and 92	IgG1 hFc_knob	(EAAAK)5		GS(EAAAK)5
HGK-EKN-L12	50 and 92	IgG1 hFc_knob	(EAAAK)7	GS(EAAAK)5

(Continued from table 3)

Additionally, the two long-acting fusion proteins may be fused to each other through a GDF15-GDF15 interaction, and this is referred to as "a long-acting fusion protein dimer".

The long-acting fusion protein dimer in which the GLP-1 variant is bound to the first polypeptide is indicated by the material code "GK", the long-acting fusion protein dimer in which the GLP-1 variant is bound to the second polypeptide is indicated by the material code "GH", and the long-acting fusion protein dimer in which the GLP-1 variant is bound to the first and second polypeptides is indicated by the material code "GKH" (see FIGS. 1A to 1C). The HGK is one in which a hinge is added to both the IgG1 Fc_knob sequence and the IgG1 Fc_hole sequence compared to GK, HGH is one in which a hinge is added to both the IgG1 Fc_knob sequence and the IgG1 Fc_hole sequence compared to GH, and HGKH is one in which a hinge is added to both the IgG1 Fc_knob sequence and the IgG1 Fc_hole sequence compared to GKH (see FIGS. 1D to 1F).

Gene cloning was performed using the pcDNA3.3 (Invitrogen) expression vector, which includes the gene encoding the first polypeptide consisting of the amino acid sequence of SEQ ID NOS: 25 to 50 of Table 1 and the gene encoding the second polypeptide consisting of the amino acid sequences of SEQ ID NOS: 63 to 77, and 86 and 92 of Table 2. In particular, the nucleotide sequences encoding the amino acid sequences of SEQ ID NOS: 25 to 50, 63 to 77, 86, and 92 were synthesized by requesting to Macrogen Inc.

Preparation Example 1-2. Expression of long-acting fusion proteins

The pcDNA3.3 expression vector cloned in Preparation Example 1-1 above was transiently transfected into the ExpiCHO-S cell line (Invitrogen) as described in the Thermo Fisher manufacturer's protocol (the ExpiFectamine CHO transfection kit, ThermoFisher Scientific, Cat. No. A29129). Briefly, DNA and the ExpiFectamine complex were inoculated into ExpiCHO-S cells (6 x 10⁶ cells/mL) and an expression enhancer was added 24 hours thereafter. The cell culture was moved to 32°C on day 2 after transfection and harvested after further culturing for 6 days.

Preparation Example 1-3. Purification of long-acting fusion proteins

In order to purify the long-acting fusion protein comprised of the first and second polypeptides in the harvested culture medium, affinity purification using Protein A resin was performed in the first step. To obtain a high-purity long-acting fusion protein, in the second step of purification, ion exchange (IEX) purification using anion exchange (AEX) resin was performed on the pool in which the first step of purification has been completed.

Specifically, the culture medium was loaded and bound to MabSelect SuRe Protein A resin (GE Healthcare) in a state equilibrated with 1X PBS (pH 7.4). Upon completion of the binding of the first and second polypeptides, the MabSelect SuRe Protein A resin was washed with 1X PBS (pH 7.4), and then elution was performed using a 0.1 M Glycine (pH 3.5) solution so as to obtain the final material. All eluted proteins were pooled. The long-acting fusion protein in which the first and second polypeptides were accurately assembled was confirmed through SDS-PAGE gel (Invitrogen) analysis and size exclusion chromatography (Tosoh, Cat. No. 08541) analysis.

Then, in the case of anion exchange (AEX), the first step pooling, after adjusting the pH according to the isoelectric point, was loaded into POROS HQ Anion Exchange resin (Thermofisher) in a state equilibrated with a 50 mM Tris (pH 8.3) solution and bound thereto. Upon completion of the binding of the long-acting fusion protein comprised of the first and second polypeptides, the POROS HQ Anion Exchange resin was washed with a 50 mM Tris solution (pH 8.3), and then elution was performed by a concentration gradient using a 50 mM Tris solution (pH 8.3) containing 1 M NaCl so as to obtain the final material. Fractions satisfying the purity criteria of 95% or higher were pooled based on size exclusion chromatography (Tosoh, Cat. No. 08541) analysis, and then the buffer was exchanged with 1X PBS (pH 7.4). The concentration of the purified fusion protein was measured using a spectrophotometer (Biochrom, Libra UV/Vis Spectrophotometer).

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

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Linker Sequence	Purity after Protein A resin Purification (Intact %)
GK-E1	IgG1 Fc_knob	(GGGGS)3	GS(EAAAK)5	9.5
GK-L1	IgG1 Fc_knob	(EEEA)2	GS(EAAAK)5	77.7
GK-L2	IgG1 Fc_knob	(EEEA)4	GS(EAAAK)5	73.0
GK-L2-2	IgG1 Fc_knob	(EEEA)4	GS(EEEA)4	73.5
GK-L2-3	IgG1 Fc_knob	(EEEA)4	GS(EEEA)6	83.3
GK-L2-4	IgG1 Fc_knob	(EEEA)4	GS(EEEA)8	73.8
GK-L3	IgG1 Fc_knob	(EEEA)6	GS(EAAAK)5	67.1
GK-L3-2	IgG1 Fc_knob	(EEEA)6	GS(EEEA)4	76.9
GK-L3-3	IgG1 Fc_knob	(EEEA)6	GS(EEEA)6	77.7
GK-L3-4	IgG1 Fc_knob	(EEEA)6	GS(EEEA)8	72.6
GK-L7	IgG1 Fc_knob	(EEEA)8	GS(EAAAK)5	58.4
GK-L7-2	IgG1 Fc_knob	(EEEA)8	GS(EEEA)4	71.8
GK-L7-4	IgG1 Fc_knob	(EEEA)8	GS(EEEA)8	72.7
GH-E1	IgG1 Fc_hole	(GGGGS)3	GS(EAAAK)5	12.0
GH-L1	IgG1 Fc_hole	(EEEA)2	GS(EAAAK)5	72.6
GH-L2	IgG1 Fc_hole	(EEEA)4	GS(EAAAK)5	78.1
GH-L3	IgG1 Fc_hole	(EEEA)6	GS(EAAAK)5	60.6
GH-L4	IgG1 Fc_hole	(EAAAK)3	GS(EAAAK)5	62.5
GH-L5	IgG1 Fc_hole	(EAAAK)5	GS(EAAAK)5	79.3
GH-L6	IgG1 Fc_hole	(EAAAK)7	GS(EAAAK)5	69.1
GH-L4-L1	IgG1 Fc_hole	(EEEA)2	GS(EEEA)6	62.6
GH-L4-L2	IgG1 Fc_hole	(EEEA)4	GS(EEEA)6	52.1
GH-L4-L3	IgG1 Fc_hole	(EEEA)6	GS(EEEA)6	69.9
GKH-E1	IgG1 Fc_knob, IgG1 Fc_hole	(GGGGS)3	GS(EAAAK)5	2.0
GKH-L1	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)2	GS(EAAAK)5	63.0
GKH-L2	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)4	GS(EAAAK)5	62.7
GKH-L3	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)6	GS(EAAAK)5	64.1
HGK-L10	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5	80.64
HGK-E-L10	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5	83.39
HGK-EKN-L8	IgG1 hFc_knob	(EAAAK)3	GS(EAAAK)5	83.72
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5	82.00
HGK-EKN-L12	IgG1 hFc_knob	(EAAAK)7	GS(EAAAK)5	88.47
HGH-E-L6	IgG1 hFc_hole	(EAAAK)7	GS(EAAAK)5	69.03
HGH-EKN-L4	IgG1 hFc_hole	(EAAAK)3	GS(EAAAK)5	79.49
HGH-EKN-L5	IgG1 hFc_hole	(EAAAK)5	GS(EAAAK)5	81.58
HGH-EKN-L6	IgG1 hFc_hole	(EAAAK)7	GS(EAAAK)5	71.36
HGKH-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)3	GS(EAAAK)5	72.74
HGKH-E-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)3	GS(EAAAK)5	75.89
HGKH-E-L5	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)5	GS(EAAAK)5	74.04

(Continued from table 4)

Meanwhile, the region between Trp31 and Leu32 of the GLP-1 protein is a site cleaved by neutral endopeptidase (NEP) 24.11 in vivo, and it is known that when this site is cleaved, the original activity of GLP-1 disappears (Journal of Medicinal Chemistry 58 (2015): 1020 - 1037).

When HGK, HGH, and HGKH among the high purity sample proteins obtained through the two-step purification were analyzed using SDS-PAGE gel under reducing conditions, it was shown that the polypeptide fused with the GLP-1 variant was not cleaved at Trp31-Leu32 and thus maintained stability (see FIG. 2).

Experimental Example 1. Activity of long-acting fusion proteins

Experimental Example 1-1. Results of measurement of GDF15 activity of long-acting fusion proteins

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

The GDF15 activity was measured using the Bright-Glo^TM luciferase assay kit (Promega) and the HEK293 cell line (human embryonic kidney 293) in which GFRAL/RET/SRE-luc was overexpressed.

Specifically, 1 Х 10⁵ HEK293 cells, in which GFRAL/RET/SRE-luc was overexpressed, were seeded into each well of a 96-well plate comprising DMEM medium containing 10% FBS, and then cultured under 37℃ and 5% CO₂ conditions for 24 hours. After 24 hours, each medium in the 96-well plate was replaced with 50 mL of serum-free medium and cultured under 37℃ and 5% CO₂ conditions for 4 hours.

In addition, the long-acting fusion proteins prepared in Preparation Example 1 above were prepared by a 3-fold serial dilution from a concentration of 2,000 nM using a serum-free medium. Thereafter, 50 μL of a diluted solution of the long-acting GDF15 fusion proteins were added to each well containing 50 μL of a replaced serum-free medium and the GFRAL/RET/SRE-luc cell line such that the actual concentration was serially diluted 3-fold from 1,000 nM, and then reacted under 37℃ and 5% CO₂ conditions for 4 hours. After 4 hours, 100 μL of Bright-Glo^TM solution prepared by adding Bright-Glo^TM buffer to Bright-Glo^TM substrate was added to each well and reacted at room temperature for 1 minute.

Thereafter, the response value (relative light unit, RLU) was measured using a microplate reader (Perkin Elmer, Wallac Victor X5) capable of measuring luminescence. The results are shown in Table 5 below.

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Linker Sequence	EC₅₀ (nM)
Control (FM9-6 +Fc_hole)	-	-	GS(EAAAK)₅	5.0
GK-L1	IgG1 Fc_knob	(EEEA)₂	GS(EAAAK)₅	3.5
GK-L2	IgG1 Fc_knob	(EEEA)₄	GS(EAAAK)₅	4.5
GK-L3	IgG1 Fc_knob	(EEEA)₆	GS(EAAAK)₅	6.2
GH-L2	IgG1 Fc_hole	(EEEA)₄	GS(EAAAK)₅	3.1
GH-L4	IgG1 Fc_hole	(EAAAK)₃	GS(EAAAK)₅	5.3
GH-L5	IgG1 Fc_hole	(EAAAK)₅	GS(EAAAK)₅	8.3
GH-L6	IgG1 Fc_hole	(EAAAK)₇	GS(EAAAK)₅	8.6
GH-L4-L1	IgG1 Fc_hole	(EEEA)₂	GS(EEEA)₆	6.4
GH-L4-L2	IgG1 Fc_hole	(EEEA)₄	GS(EEEA)₆	3.6
GH-L4-L3	IgG1 Fc_hole	(EEEA)₆	GS(EEEA)₆	8.0
GKH-L1	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₂	GS(EAAAK)₅	6.7
GKH-L2	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₄	GS(EAAAK)₅	6.0
HGK-E-L10	IgG1 hFc_knob	(EAAAK)₅	GS(EAAAK)₅	8.4
HGK-EKN-L8	IgG1 hFc_knob	(EAAAK)₃	GS(EAAAK)₅	4.5
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)₅	GS(EAAAK)₅	4.6
HGH-E-L6	IgG1 hFc_hole	(EAAAK)₇	GS(EAAAK)₅	4.1
HGH-EKN-L4	IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	9.1
HGH-EKN-L5	IgG1 hFc_hole	(EAAAK)₅	GS(EAAAK)₅	7.0
HGKH-E-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	7.3
HGKH-E-L5	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₅	GS(EAAAK)₅	9.9
HGKH-EKN-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	6.6
HGKH-EKN-L5	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₅	GS(EAAAK)₅	7.0

Experimental Example 1-2. Results of measurement of GLP-1 activity of long-acting fusion proteins

The GLP-1 activity of the long-acting fusion proteins prepared in Preparation Example 1 above was measured.

The GLP-1 activity was measured using the cAMP Gs dynamic kit (Cisbio, Cat. No. 62AM4PEC) and the HEK293 cell line (human embryonic kidney 293) in which GLP-1R was overexpressed.

Specifically, 1 Х 10⁴ HEK293 cells, in which GLP-1R was overexpressed, were seeded into each well of a 96-well plate comprising DMEM medium containing 10% FBS and cultured under 37 ℃ and 5% CO₂ conditions for 16 hours. After 16 hours, the long-acting fusion proteins prepared in Preparation Example 1 above were prepared by a 5-fold serial dilution from a 20 nM concentration using DPBS containing 0.5% BSA (WELGENE, Cat. No. LS021-01) and 2 mM IBMX (Sigma Aldrich, Cat. No. I7018). Thereafter, 25 μL of the dilution of the long-acting fusion proteins was added to each well containing the GLP-1R overexpressing cell line such that the actual concentration was serially diluted by 5-fold from 10 nM and cultured under 37℃ and 5% CO₂ conditions for 30 minutes. After 30 minutes, 25 μL of the cAMP-d2 solution was added to each well. Then, 25 μL of an anti-cAMP-cryptate solution was added to each well and reacted at room temperature for 1 hour.

Thereafter, the response value (homogenous time-resolved fluorescence; HTRF) was measured using a microplate reader (Molecular Devices, FlexStation 3) capable of detecting time-resolved fluorescence. The results are shown in Table 6 below.

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Linker Sequence	EC50 (pM)
GK-L1	IgG1 Fc_knob	(EEEA)2	GS(EAAAK)5	277.3
GK-L2	IgG1 Fc_knob	(EEEA)4	GS(EAAAK)5	147.6
GK-L2-2	IgG1 Fc_knob	(EEEA)4	GS(EEEA)4	112.7
GK-L2-3	IgG1 Fc_knob	(EEEA)4	GS(EEEA)6	374.7
GK-L2-4	IgG1 Fc_knob	(EEEA)4	GS(EEEA)8	224.2
GK-L3	IgG1 Fc_knob	(EEEA)6	GS(EAAAK)5	17.6
GK-L3-2	IgG1 Fc_knob	(EEEA)6	GS(EEEA)4	88.1
GK-L3-3	IgG1 Fc_knob	(EEEA)6	GS(EEEA)6	105.4
GK-L3-4	IgG1 Fc_knob	(EEEA)6	GS(EEEA)8	84.7
GK-L7	IgG1 Fc_knob	(EEEA)8	GS(EAAAK)5	69.5
GK-L7-2	IgG1 Fc_knob	(EEEA)8	GS(EEEA)4	69.6
GK-L7-3	IgG1 Fc_knob	(EEEA)8	GS(EEEA)6	138.0
GK-L7-4	IgG1 Fc_knob	(EEEA)8	GS(EEEA)8	75.0
GH-L2	IgG1 Fc_hole	(EEEA)4	GS(EAAAK)5	92.9
GH-L3	IgG1 Fc_hole	(EEEA)6	GS(EAAAK)5	11.7
GH-L4	IgG1 Fc_hole	(EAAAK)3	GS(EAAAK)5	11.3
GH-L5	IgG1 Fc_hole	(EAAAK)5	GS(EAAAK)5	4.2
GH-L6	IgG1 Fc_hole	(EAAAK)7	GS(EAAAK)5	17.5
GH-L4-L1	IgG1 Fc_hole	(EEEA)2	GS(EEEA)6	722.2
GH-L4-L2	IgG1 Fc_hole	(EEEA)4	GS(EEEA)6	308.4
GH-L4-L3	IgG1 Fc_hole	(EEEA)6	GS(EEEA)6	48.9
GKH-L1	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)2	GS(EAAAK)5	11.7
GKH-L2	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)4	GS(EAAAK)5	10.2
GKH-L3	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)6	GS(EAAAK)5	12.9
GKH-L4-1	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)2	GS(EEEA)6	5.8
GKH-L4-2	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)4	GS(EEEA)6	44.3
GKH-L4-3	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)6	GS(EEEA)6	22.9
HGK-L10	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5	4.3
HGK-E-L8	IgG1 hFc_knob	(EAAAK)3	GS(EAAAK)5	4.8
HGK-E-L10	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5	2.5
HGK-E-L12	IgG1 hFc_knob	(EAAAK)7	GS(EAAAK)5	5.5
HGK-EKN-L8	IgG1 hFc_knob	(EAAAK)3	GS(EAAAK)5	6.8
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)5	GS(EAAAK)5	4.8
HGK-EKN-L12	IgG1 hFc_knob	(EAAAK)7	GS(EAAAK)5	5.5

(Continued from table 6)

As confirmed in Table 6 above, the IgG1 Fc type and/or linker type were shown to have some effects on the GLP-1 activity of the long-acting fusion proteins.

Experimental Example 2. Evaluation of thermal stability of long-acting fusion proteins

The thermal stability of the long-acting fusion proteins prepared in Preparation Example 1 above were evaluated using the UNcle equipment (Unchained Labs).

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

As shown in Table 7, the long-acting fusion proteins prepared in Preparation Example 1 above were confirmed to have Tm and Tagg values around 60°C.

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Linker Sequence	Tm (℃)	Tagg (℃)
GK-L2	IgG1 Fc_knob	(EEEA)₄	GS(EAAAK)₅	54.7	56.7
GK-L2-2	IgG1 Fc_knob	(EEEA)₄	GS(EEEA)₄	57.7	57.4
GK-L2-3	IgG1 Fc_knob	(EEEA)₄	GS(EEEA)₆	56.7	55.1
GK-L2-4	IgG1 Fc_knob	(EEEA)₄	GS(EEEA)₈	56.8	55.0
GK-L3	IgG1 Fc_knob	(EEEA)₆	GS(EAAAK)₅	55.7	58.0
GK-L3-2	IgG1 Fc_knob	(EEEA)₆	GS(EEEA)₄	57.0	51.3
GK-L3-3	IgG1 Fc_knob	(EEEA)₆	GS(EEEA)₆	57.0	52.8
GK-L3-4	IgG1 Fc_knob	(EEEA)₆	GS(EEEA)₈	57.0	52.7
GK-L7	IgG1 Fc_knob	(EEEA)₈	GS(EAAAK)₅	57.4	55.9
GK-L7-2	IgG1 Fc_knob	(EEEA)₈	GS(EEEA)₄	57.9	55.0
GK-L7-3	IgG1 Fc_knob	(EEEA)₈	GS(EEEA)₆	58.2	54.3
GK-L7-4	IgG1 Fc_knob	(EEEA)₈	GS(EEEA)₈	56.9	53.6
GH-L2	IgG1 Fc_hole	(EEEA)₄	GS(EAAAK)₅	53.7	55.9
GH-L3	IgG1 Fc_hole	(EEEA)₆	GS(EAAAK)₅	55.7	57.7
GH-L4	IgG1 Fc_hole	(EAAAK)₃	GS(EAAAK)₅	56.0	54.6
GH-L5	IgG1 Fc_hole	(EAAAK)₅	GS(EAAAK)₅	53.8	55.5
GH-L6	IgG1 Fc_hole	(EAAAK)₇	GS(EAAAK)₅	54.4	56.0
GKH-L2	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₄	GS(EAAAK)₅	56.0	52.5
GKH-L3	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₆	GS(EAAAK)₅	55.4	52.0
GKH-L4-1	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₂	GS(EEEA)₆	58.3	46.4
GKH-L4-2	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₄	GS(EEEA)₆	56.5	47.2
GKH-L4-3	IgG1 Fc_knob, IgG1 Fc_hole	(EEEA)₆	GS(EEEA)₆	57.1	54.0
HGK-L10	IgG1 hFc_knob	(EAAAK)₅	GS(EAAAK)₅	63.1	64.5
HGK-E-L8	IgG1 hFc_knob	(EAAAK)₃	GS(EAAAK)₅	62.5	62.4
HGK-E-L10	IgG1 hFc_knob	(EAAAK)₅	GS(EAAAK)₅	63.3	65.4
HGK-E-L12	IgG1 hFc_knob	(EAAAK)₇	GS(EAAAK)₅	64.3	61.9
HGK-EKN-L8	IgG1 hFc_knob	(EAAAK)₃	GS(EAAAK)₅	64.1	65.1
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)₅	GS(EAAAK)₅	65.1	69.5
HGK-EKN-L12	IgG1 hFc_knob	(EAAAK)₇	GS(EAAAK)₅	63.8	62.0
HGH-E-L4	IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	62.4	62.0
HGH-E-L6	IgG1 hFc_hole	(EAAAK)₇	GS(EAAAK)₅	50.9	66.9
HGH-EKN-L4	IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	62.7	63.1
HGH-EKN-L5	IgG1 hFc_hole	(EAAAK)₅	GS(EAAAK)₅	64.5	67.8
HGH-EKN-L6	IgG1 hFc_hole	(EAAAK)₇	GS(EAAAK)₅	50.5	63.1
HGKH-E-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	62.2	61.4
HGKH-E-L5	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₅	GS(EAAAK)₅	62.6	63.9
HGKH-EKN-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	GS(EAAAK)₅	64.9	67.0
HGKH-EKN-L5	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₅	GS(EAAAK)₅	64.5	63.4
HGKH-EKN-L6	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₇	GS(EAAAK)₅	65.1	63.0

Experimental Example 3. Pharmacokinetic evaluation of long-acting fusion proteins

Experimental Example 3-1. Experimental method for pharmacokinetic evaluation in mice

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

Specifically, in order to measure the serum concentration of the active GDF15-Fc part, ELISA analysis was performed using the GDNF family receptor alpha-like (GFRAL) protein, which is a receptor for GDF15 developed internally, and an antibody with immunoreactivity to Fc (Jackson ImmunoResearch, Cat. No. 109-035-098). In order to measure the serum levels of the active GLP1-Fc part, ELISA analysis was performed using an internally-developed antibody with immunoreactivity to the N-terminus of GLP-1 (Invitrogen, Cat. No. ABS033-10-02) and an antibody with immunoreactivity to Fc. After a single subcutaneous injection of each protein into mice, the serum concentrations of GDF15-Fc and GLP1-Fc parts for each material were measured for up to 240 hours so as to calculate each pharmacokinetic parameter.

Experimental Example 3-2. Pharmacokinetic evaluation results in mice

After a single subcutaneous administration of each protein prepared in Preparation Example 1 above to the mice, based on the serum concentration of each active material over time (see FIGS. 3 to 6), the pharmacokinetic parameters for the GDF15-IgG1 Fc part and the GLP1-IgG1 Fc part of the long-acting fusion protein were calculated and are shown in Tables 8 and 9 below. In addition, the pharmacokinetic profile of each long-acting fusion protein was compared and evaluated based on area under the curve (AUC), which indicates the degree of drug exposure.

GDF15-Fc Detection
Material Code	C_max(ng/mL)	T_max(hr)	AUC_last(ng·hr/mL)
GK-L2 (SEQ ID NOS: 29 and 86)	6008.31	24	426700.86
GK-L3 (SEQ ID NOS: 33 and 86)	5298.03	24	277759.86
GK-L3-3 (SEQ ID NOS: 35 and 86)	6097.27	4	258547.36
GK-L3-4 (SEQ ID NOS: 36 and 86)	2736.58	4	81828.26
GK-L7 (SEQ ID NOS: 37 and 86)	5407.21	4	236812.70
GH-L3 (SEQ ID NOS: 25 and 66)	6586.09	24	403636.42
GH-L4 (SEQ ID NOS: 25 and 68)	5664.67	24	467127.58
GH-L5 (SEQ ID NOS: 25 and 69)	7410.25	24	861227.02
GH-L6 (SEQ ID NOS: 25 and 70)	7388.62	48	897342.64
GH-L4-L3 (SEQ ID NOS: 26 and 66)	4814.14	24	274016.20
GKH-L2 (SEQ ID NOS: 29 and 65)	3377.58	24	192900.60
GKH-L3 (SEQ ID NOS: 33 and 66)	4415.16	4	123763.96
GKH-L4-2 (SEQ ID NOS: 31 and 65)	2548.91	4	85295.48
HGK-L10 (SEQ ID NOS: 44 and 92)	10176.70	48	1610291.80
HGK-E-L8 (SEQ ID NOS: 45 and 92)	14033.64	72	2430733.90
HGK-E-L10 (SEQ ID NOS: 46 and 92)	13066.94	48	2078272.70
HGK-E-L12 (SEQ ID NOS: 47 and 92)	14572.86	72	2188919.50
HGK-EKN-L8 (SEQ ID NOS: 48 and 92)	10515.86	48	1664240.80
HGK-EKN-L10 (SEQ ID NOS: 49 and 92)	11328.38	72	1930116.40
HGK-EKN-L12 (SEQ ID NOS: 50 and 92)	12002.98	48	1867846.30
HGH-E-L4 (SEQ ID NOS: 42 and 72)	29019.96	48	4433899.40
HGH-EKN-L4 (SEQ ID NOS: 42 and 75)	24064.09	48	3910492.40
HGH-EKN-L5 (SEQ ID NOS: 42 and 76)	11459.97	48	2160526.50
HGH-EKN-L6 (SEQ ID NOS: 42 and 77)	11664.84	48	2163979.20
HGKH-L4 (SEQ ID NOS: 43 and 71)	9165.70	48	1387246.60
HGKH-EKN-L4 (SEQ ID NOS: 48 and 75)	12909.92	48	1982712.40
HGKH-EKN-L5 (SEQ ID NOS: 49 and 76)	10158.31	48	1763416.50
HGKH-EKN-L6 (SEQ ID NOS: 50 and 77)	9424.28	48	1612796.80

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

GLP1-Fc Detection
Material Code	C_max(ng/mL)	T_max(hr)	AUC_last(ng·hr/mL)
GK-L2 (SEQ ID NOS: 29 and 86)	3753.28	4	167947.46
GK-L3 (SEQ ID NOS: 33 and 86)	6485.35	4	212192.34
GK-L3-3 (SEQ ID NOS: 35 and 86)	4983.43	4	89048.14
GK-L3-4 (SEQ ID NOS: 36 and 86)	1915.69	4	25225.08
GK-L7 (SEQ ID NOS: 37 and 86)	3583.66	4	70574.58
GH-L3 (SEQ ID NOS: 25 and 66)	3665.32	4	119461.34
GH-L4 (SEQ ID NOS: 25 and 68)	4123.73	24	155381.66
GH-L5 (SEQ ID NOS: 25 and 69)	2649.21	4	116782.60
GH-L6 (SEQ ID NOS: 25 and 70)	3667.66	4	101507.72
GH-L4-L3 (SEQ ID NOS: 26 and 66)	3265.86	4	69993.44
GKH-L2 (SEQ ID NOS: 29 and 65)	2061.60	4	48693.90
GKH-L3 (SEQ ID NOS: 33 and 66)	3658.94	4	50883.58
GKH-L4-2 (SEQ ID NOS: 31 and 65)	3802.44	4	47323.88
HGK-L10 (SEQ ID NOS: 44 and 92)	5576.44	24	316846.96
HGK-E-L8 (SEQ ID NOS: 45 and 92)	5416.43	24	505586.90
HGK-E-L10 (SEQ ID NOS: 46 and 92)	3349.47	48	344214.76
HGK-E-L12 (SEQ ID NOS: 47 and 92)	5295.37	4	453127.44
HGK-EKN-L8 (SEQ ID NOS: 48 and 92)	5703.09	24	636309.06
HGK-EKN-L10 (SEQ ID NOS: 49 and 92)	4519.23	48	523105.50
HGK-EKN-L12 (SEQ ID NOS: 50 and 92)	7624.32	24	717620.52
HGH-E-L4 (SEQ ID NOS: 42 and 72)	7138.04	24	670685.60
HGH-EKN-L4 (SEQ ID NOS: 42 and 75)	9890.47	24	1011232.90
HGH-EKN-L5 (SEQ ID NOS: 42 and 76)	4511.84	24	502577.72
HGH-EKN-L6 (SEQ ID NOS: 42 and 77)	4260.78	48	457281.58
HGKH-L4 (SEQ ID NOS: 43 and 71)	8194.99	24	576063.34
HGKH-EKN-L4 (SEQ ID NOS: 48 and 75)	12151.69	48	1444952.20
HGKH-EKN-L5 (SEQ ID NOS: 49 and 76)	8759.20	72	1240945.20
HGKH-EKN-L6 (SEQ ID NOS: 50 and 77)	9514.61	48	1101449.00

As shown in Table 9, when determining after comparing HGK-L10 with HGK-E-L10 and HGK-EKN-L10 and comparing HGKH-L4 with HGKH-EKN-L4, the AUC due to the introduction of the GLP-1 mutant sequence has increased. Additionally, when comparing HGKH-EKN-L4 with HGK-EKN-L8 and HGH-EKN-L4, HGKH-EKN-L5 with HGK-EKN-L10 and HGH-EKN-L5, and HGKH-EKN-L6 with HGK-EKN-L12 and HGH-EKN-L6, respectively, it was confirmed that the AUC of the GLP-1 part was increased about 1.4- to about 2.5-fold in the proteins where the GLP-1 variants were fused to both IgG1 hFc_knob and IgG1 hFc_hole compared to that of the proteins where the GLP-1 variants were fused to only IgG1 hFc_knob or IgG1 hFc_hole.

The ratios of AUC of the GLP-1 part/AUC of the GDF15 part of the long-acting fusion proteins prepared in Preparation Example 1 above are shown in Table 10 below. It is determined that the higher the ratio of AUC of the GLP-1 part/AUC of the GDF15 part, the more balanced the pharmacokinetic profiles of the GLP-1 variants and GDF15 variants.

Material Code	HGK-L10	HGK-E-L8	HGK-E-L10	HGK-E-L12	HGK-EKN-L8	HGK-EKN-L10	HGK-EKN-L12
GLP-1 to GDF15 Ratio (AUC_last)	0.20	0.22	0.17	0.21	0.38	0.27	0.38
Material Code	HGH-E-L4	HGH-EKN-L4	HGH-EKN-L5	HGH-EKN-L6	HGKH-EKN-L4	HGKH-EKN-L5	HGKH-EKN-L6
GLP-1 to GDF15 Ratio (AUC_last)	0.15	0.27	0.23	0.21	0.73	0.70	0.68

As shown in Table 10, the proteins (HGK-L10, HGK-E-L8, HGK-E-L10, HGK-E-L12, HGK-EKN-L8, HGK-EKN-L10, and HGK-EKN-L12) in which GLP-1 variants are fused to IgG1 hFc_knob were shown to have a GLP-1 AUC/GDF15 AUC ratio of 0.17 to 0.38. In addition, the proteins (HGH-E-L4, HGH-EKN-L4, HGH-EKN-L5, and HGH-EKN-L6) in which the GLP-1 variants are fused to the IgG1 hFc_hole were shown to have a GLP-1 AUC/GDF15 AUC ratio of 0.15 to 0.27. Meanwhile, the proteins (HGKH-EKN-L4, HGKH-EKN-L5, and HGKH-EKN-L6) in which the GLP-1 variants are fused to IgG1 hFc_knob and IgG1 hFc_hole were shown to have a GLP-1 AUC/GDF15 AUC ratio of 0.68 to 0.73.

Experimental Example 3-3. Experimental method for evaluation of pharmacokinetics in rats

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

Experimental Example 3-4. Evaluation results of rat pharmacokinetics

Pharmacokinetic parameters for the GDF15-IgG1 Fc part and the GLP1-IgG1 Fc part of HGKH-EKN-L4 based on the serum concentration by administration time and dose (see FIG. 7) after a single administration of HGKH-EKN-L4 to rats were calculated and shown in Tables 11 and 12 below.

GDF15-Fc Detection
Parameter	1 mg/kg (i.v.)	0.1 mg/kg (s.c.)	0.3 mg/kg (s.c.)	1 mg/kg (s.c.)
AUC_last (ng·hr/mL)	2591621.53	128779.01	502945.08	2716726.90
C₀(ng/mL)	24704.35	-	-	-
C_max (ng/mL)	-	537.77	2159.89	9493.08
T_max (hr)	-	48	48	96
Bioavailability (%)	-	49.69	64.69	104.83

GLP1-Fc Detection
Parameter	1 mg/kg (i.v.)	0.1 mg/kg (s.c.)	0.3 mg/kg (s.c.)	1 mg/kg (s.c.)
AUC_last (ng·hr/mL)	1364699.13	66636.17	247059.49	981687.85
C₀(ng/mL)	23108.22	-	-	-
C_max (ng/mL)	-	595.30	1794.17	6881.07
T_max (hr)	-	48	48	48
Bioavailability (%)	-	48.83	60.35	71.83

Preparation Example 2. Preparation and purification of long-acting fusion proteins (2)

Preparation Example 2-1. Gene cloning and expression of long-acting fusion proteins

In order to confirm whether the stability, in vitro activity, and pharmacokinetic profiles of long-acting fusion proteins may vary when a GDF15 variant with reduced GDF15 activity is used, in a case where the GDF15 variant has structural characteristics identical to those of the long-acting fusion proteins prepared in Preparation Example 1 above, the first polypeptide in Table 13 below and the second polypeptide in Table 14 below were designed.

GLP-1 Change in Sequence	GLP-1 Linker Sequence	IgG1 Fc	GDF15 Linker Sequence	GDF15 Change in Sequence	SEQ ID NO of Fusion Protein
-	-	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64R	SEQ ID NO: 42
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64R	SEQ ID NO: 49
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N2, W32F	SEQ ID NO: 51
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N2, Q90H	SEQ ID NO: 52
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N2, Q60N	SEQ ID NO: 53
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64N	SEQ ID NO: 54
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64D	SEQ ID NO: 55
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₅	IgG1 hFc_knob	GS(EAAAK)₅	△N3, NST, S64R	SEQ ID NO: 56
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64R	SEQ ID NO: 48
GEGEKN (A8G, G22E, A30E, V33K, K34N, R36G)	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N2, W32F	SEQ ID NO: 57
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N2, Q90H	SEQ ID NO: 58
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N2, Q60N	SEQ ID NO: 59
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64N	SEQ ID NO: 60
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N2, S64D	SEQ ID NO: 61
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_knob	GS(EAAAK)₅	△N3, NST, S64R	SEQ ID NO: 62

GLP-1 Change in Sequence	GLP-1 Linker Sequence	IgG1 Fc	SEQ ID NO of Fusion Protein
-	-	IgG1 hFc_hole	SEQ ID NO: 92
A8G, G22E, A30E, V33K, K34N, R36G	(EAAAK)₃	IgG1 hFc_hole	SEQ ID NO: 75

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

The cloned pcDNA3.3 expression vector was transiently transfected into the ExpiCHO-S cell line (Invitrogen) as described in the Thermo Fisher manufacturer's protocol (ExpiFectamine CHO transfection kit, ThermoFisher Scientific, Cat. No. A29129). Briefly, DNA and the ExpiFectamine complex 6 x 10⁶ cells/mL were inoculated into ExpiCHO-S cells (6 x 10⁶ cells/mL), and an expression enhancer was added 24 hours thereafter. The cell culture was moved to 32°C on day 2 after transfection and harvested after further culturing for 6 days.

Preparation Example 2-2. Purification of long-acting fusion proteins

In order to purify the long-acting fusion proteins comprised of the first and second polypeptides in the culture medium, protein A resin purification was performed as the first step, and then AEX purification was performed in the second step as in Preparation Example 1-3. The long-acting fusion proteins in which the first and second polypeptides were accurately assembled were confirmed through SDS-PAGE gel (Invitrogen) analysis and SE-HPLC (Tosoh, Cat. No. 08541) analysis.

Experimental Example 4. Measurement results of activity of long-acting fusion proteins

Experimental Example 4-1. Measurement results of GDF15 activity measurement of long-acting fusion proteins

The GDF15 activity of the long-acting fusion proteins prepared in Preparation Example 2 above was compared. GDF15 activity was measured in the same manner as in Experimental Example 1-1 above. The results are shown in Table 15 and FIG. 8 below. In particular, the GDF15 activity of the long-acting fusion proteins was compared based on the in vitro GDF15 activity of HGK-EKN-L10 and HGKH-EKN-L4 (EC_max 100%) (see Table 15).

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Mutation Sequence	EC_max (%)
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)₅	△N2, S64R	100
HGK-EKN-L10-23	IgG1 hFc_knob	(EAAAK)₅	△N2, W32F	36
HGK-EKN-L10-30	IgG1 hFc_knob	(EAAAK)₅	△N2, Q90H	49
HGK-EKN-L10-47	IgG1 hFc_knob	(EAAAK)₅	△N2, S64N	84
HGK-EKN-L10-49	IgG1 hFc_knob	(EAAAK)₅	△N2, S64D	61
HGKH-EKN-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64R	100
HGKH-EKN-L4-23	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, W32F	43
HGKH-EKN-L4-30	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, Q90H	38
HGKH-EKN-L4-47	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64N	84
HGKH-EKN-L4-49	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64D	50

Experimental Example 4-2. Measurement results of GLP-1 activity of long-acting fusion proteins

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

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Mutation Sequence	EC₅₀ (pM)
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)₅	△N2, S64R	6.933
HGK-EKN-L10-23	IgG1 hFc_knob	(EAAAK)₅	△N2, W32F	6.174
HGK-EKN-L10-30	IgG1 hFc_knob	(EAAAK)₅	△N2, Q90H	7.610
HGK-EKN-L10-47	IgG1 hFc_knob	(EAAAK)₅	△N2, S64N	8.153
HGK-EKN-L10-49	IgG1 hFc_knob	(EAAAK)₅	△N2, S64D	7.343
HGKH-EKN-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64R	3.000
HGKH-EKN-L4-23	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, W32F	3.278
HGKH-EKN-L4-30	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, Q90H	3.359
HGKH-EKN-L4-47	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64N	3.391
HGKH-EKN-L4-49	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64D	3.588

As shown in Table 16, when HGK-EKN-L10 was compared with and HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-47, and HGK-EKN-L10-49, it was confirmed that GLP-1 activity was similar regardless of the GDF15 mutant sequences. Likewise, when HGKH-EKN-L4 was compared with HGKH-EKN-L4-23, HGKH-EKN-L4-30, HGKH-EKN-L4-47 and HGKH-EKN-L4-49, it was confirmed that GLP-1 activity was similar regardless of the GDF15 mutant sequences.

Experimental Example 5. Evaluation results of thermal stability of long-acting fusion proteins

The thermal stability of the long-acting fusion proteins prepared in Preparation Example 2 above was evaluated using the UNcle equipment (Unchained Labs).

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

Material Code	IgG1 Fc	GLP-1 Linker Sequence	GDF15 Mutation Sequence	Tm (℃)	Tagg (℃)
HGK-EKN-L10	IgG1 hFc_knob	(EAAAK)₅	△N2, S64R	65.1	69.5
HGK-EKN-L10-23	IgG1 hFc_knob	(EAAAK)₅	△N2, W32F	66.5	71.8
HGK-EKN-L10-30	IgG1 hFc_knob	(EAAAK)₅	△N2, Q90H	65.3	71.0
HGK-EKN-L10-38	IgG1 hFc_knob	(EAAAK)₅	△N2, Q60N	66.0	71.9
HGK-EKN-L10-47	IgG1 hFc_knob	(EAAAK)₅	△N2, S64N	65.3	71.3
HGK-EKN-L10-49	IgG1 hFc_knob	(EAAAK)₅	△N2, S64D	65.6	69.9
HGK-EKN-L10-484	IgG1 hFc_knob	(EAAAK)₅	△N3, NST, S64R	64.1	71.9
HGKH-EKN-L4	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64R	64.9	67.0
HGKH-EKN-L4-23	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, W32F	65.5	66.5
HGKH-EKN-L4-30	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, Q90H	64.5	66.8
HGKH-EKN-L4-38	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, Q60N	65.0	66.6
HGKH-EKN-L4-47	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64N	64.8	66.5
HGKH-EKN-L4-49	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N2, S64D	64.8	66.1
HGKH-EKN-L4-484	IgG1 hFc_knob, IgG1 hFc_hole	(EAAAK)₃	△N3, NST, S64R	63.6	67.3

As shown in Table 17, when HGK-EKN-L10, HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-38, HGK-EKN-L10-47, HGK-EKN-L10-49, and HGK-EKN-L10-484 were compared, it was confirmed that these had similar Tm and Tagg values of higher than 60 °C regardless of the mutant sequences of GDF15. Likewise, when HGKH-EKN-L4, HGKH-EKN-L4-23, HGKH-EKN-L4-30, HGKH-EKN-L4-38, HGKH-EKN-L4-47, HGKH-EKN-L4-49, and HGKH-EKN-L4-484 were compared, it was confirmed that these had similar Tm and Tagg values of higher than 60 °C regardless of the mutant sequences of GDF15.

Experimental Example 6. Pharmacokinetic evaluation results of long-acting fusion proteins in mice

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

GDF15-Fc Detection
Material Code	C_max (ng/mL)	T_max(hr)	AUC_last(ng·hr/mL)
HGK-EKN-L10-23 (SEQ ID NOS: 51 and 92)	8998.87	48	1638440.40
HGK-EKN-L10-30 (SEQ ID NOS: 52 and 92)	10064.69	72	1780192.60
HGK-EKN-L10-38 (SEQ ID NOS: 53 and 92)	9272.04	72	1533821.80
HGK-EKN-L10-47 (SEQ ID NOS: 54 and 92)	11147.21	48	1970426.40
HGK-EKN-L10-49 (SEQ ID NOS: 55 and 92)	10620.50	120	2032097.20
HGK-EKN-L10-484 (SEQ ID NOS: 56 and 92)	5528.63	48	873395.16
HGKH-EKN-L4-23 (SEQ ID NOS: 57 and 75)	10504.12	48	1786175.80
HGKH-EKN-L4-30 (SEQ ID NOS: 58 and 75)	11055.33	48	1913355.60
HGKH-EKN-L4-47 (SEQ ID NOS: 60 and 75)	12688.87	48	2212259.90
HGKH-EKN-L4-49 (SEQ ID NOS: 61 and 75)	9226.26	24	1767434.00

As shown in Table 18, it was confirmed that other long-acting fusion proteins, excluding HGK-EKN-L10-L484, showed AUC values similar to that of the GDF15 part within a maximum of about 1.4-fold. In HGK-EKN-L10-484, the AUC value of the GDF15 part decreased by about 2-fold.

GLP-1 Fc Detection
Material Code	C_max (ng/mL)	T_max(hr)	AUC_last(ng·hr/mL)
HGK-EKN-L10-23 (SEQ ID NOS: 51 and 92)	6972.79	24	754438.66
HGK-EKN-L10-30 (SEQ ID NOS: 52 and 92)	5254.10	24	586812.32
HGK-EKN-L10-38 (SEQ ID NOS: 53 and 92)	5952.38	48	606108.72
HGK-EKN-L10-47 (SEQ ID NOS: 54 and 92)	6881.02	24	650361.52
HGK-EKN-L10-49 (SEQ ID NOS: 55 and 92)	7212.20	24	637269.68
HGK-EKN-L10-484 (SEQ ID NOS: 56 and 92)	2440.36	24	154970.08
HGKH-EKN-L4-23 (SEQ ID NOS: 57 and 75)	11862.17	48	1524991.90
HGKH-EKN-L4-30 (SEQ ID NOS: 58 and 75)	11256.77	48	1460510.60
HGKH-EKN-L4-47 (SEQ ID NOS: 60 and 75)	10432.60	48	1463371.70
HGKH-EKN-L4-49 (SEQ ID NOS: 61 and 75)	9703.09	48	1392072.20

As shown in Table 19, it was confirmed that HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-38, HGK-EKN-L10-47, and HGK-EKN-L10-49 showed an increased AUC value of the GLP-1 part by about 3.7-fold to 4.9-fold compared to HGK-EKN-L10-484.

Additionally, when determining by comparing HGK-EKN-L10-23 with HGKH-EKN-L4-23, HGK-EKN-L10-30 with HGKH-EKN-L4-30, HGK-EKN-L10-47 with HGKH-EKN-L4-47, and HGK-EKN-L10-49 with HGKH-EKN-L4-49, respectively, the AUC of the GLP-1 part was increased about 2.0-fold to about 2.5-fold in the proteins where the GLP-1 variants were fused to both IgG1 hFc_knob and IgG1 hFc_hole compared to that of the proteins where the GLP-1 variants were fused to only IgG1 hFc_knob.

The ratios of AUC of the GLP-1 part/AUC of the GDF15 part of the long-acting fusion proteins prepared in Preparation Example 2 above are shown in Table 20 below. It is determined that the higher the ratio of AUC of the GLP-1 part/AUC of the GDF15 part, the more balanced the pharmacokinetic profiles of the GLP-1 variants and GDF15 variants.

Material Code	HGK-EKN-L10-23	HGK-EKN-L10-30	HGK-EKN-L10-38	HGK-EKN-L10-47	HGK-EKN-L10-49
GLP-1 to GDF15 ratio (AUC_last)	0.46	0.33	0.40	0.33	0.31
Material Code	HGK-EKN-L10-484	HGKH-EKN-L4-23	HGKH-EKN-L4-30	HGKH-EKN-L4-47	HGKH-EKN-L4-49
GLP-1 to GDF15 Ratio (AUC_last)	0.18	0.85	0.76	0.66	0.79

As shown in Table 20, the proteins (HGK-EKN-L10-23, HGK-EKN-L10-30, HGK-EKN-L10-38, HGK-EKN-L10-47, HGK-EKN-L10-49, and HGK-EKN-L10-484), in which GLP-1 variants are fused to IgG1 hFc_knob, were shown to have a GLP-1 AUC/GDF15 AUC ratio of 0.18 to 0.46. Meanwhile, the proteins (HGKH-EKN-L4-23, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49) in which the GLP-1 variants are fused to IgG1 hFc_knob and IgG1 hFc_hole, were shown to have a GLP-1 AUC/GDF15 AUC ratio of 0.66 to 0.853. In other words, it was confirmed that the proteins fused to the IgG1 hFc_knob and IgG1 hFc_hole of the GLP-1 variants had balanced pharmacokinetic profiles of the GLP-1 variants and the GDF15 variants.

Upon comprehensive review of Experimental Examples 1 to 6 above, even when the activity of the GDF15 variants was lowered, it did not affect the stability of the long-acting fusion proteins and had a balanced pharmacokinetic profile with GLP-1 variants; therefore, it is expected that changing the activity of GDF15 variants will not significantly affect the stability and pharmacokinetic profile of the long-acting fusion proteins.

Experimental Example 7. Evaluation of drug efficacy of long-acting fusion proteins in diet-induced obese (DIO) mice

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

DIO mice (Taconic, USA) fed with 60% high-fat diet for 8 weeks were purchased from Raonbio (Korea). The DIO mice purchased were additionally fed with 60% high-fat diet for 5 weeks and used in this study.

Experimental Example 7-1. Effect of reducing body weight in DIO mice (1)

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

As shown in Table 21 and FIG. 12, it was confirmed that GK-L3 and GH-L5 showed the effect of reducing body weight in a dose-dependent manner.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Maximal efficacy	Efficacy on Day 26
Control	-	-	2.3
GK-L3	1	-8.9	-2.5
GK-L3	3	-11.2	-10.4
GK-L3	10	-21.0	-21.0
GH-L5	1	-14.6	-14.0
GH-L5	3	-26.9	-26.9
GH-L5	10	-29.6	-29.6

Experimental Example 7-2. Effect of reducing body weight in DIO mice (2)

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

As shown in Table 22 and FIG. 13, it was confirmed that GK-L3-3, GH-L3, GH-L4, GKH-L2, and GKH-L4-L2 showed an excellent effect of reducing body weight compared to the control.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Maximal efficacy	Efficacy on Day 26
Vehicle	-	-	-1.9
GK-L3-3	3	-7.6	-2.7
GH-L3	3	-13.8	-13.2
GH-L4	3	-27.2	-27.2
GKH-L2	3	-9.9	-4.9
GKH-L4-L2	3	-7.5	-3.8

Experimental Example 7-3. Effect of reducing body weight in DIO mice (3)

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

As shown in Table 23 and FIG. 14, it was confirmed that HGH-EKN-L4 and HGKH-EKN-L4 showed the effect of reducing body weight in a dose-dependent manner.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Maximal efficacy	Efficacy on Day 26
Vehicle	-	-	2.14
HGH-EKN-L4	0.1	-10.3	-10.3
HGH-EKN-L4	0.3	-14.2	-14.2
HGH-EKN-L4	1	-18.0	-18.0
HGKH-EKN-L4	0.1	-3.8	-3.2
HGKH-EKN-L4	0.3	-15.9	-15.9

Additionally, as shown in Table 24 and FIG. 15 below, it was confirmed that HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGKH-EKN-L4, and HGKH-EKN-L4-49 showed the effect of reducing body weight in a dose-dependent manner.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Maximal efficacy	Efficacy on Day 30
Vehicle	-	-	2.55
HGK-L10	0.3	-11.59	-11.59
HGK-L10	1	-23.09	-23.09
HGK-EKN-L10	0.3	-7.68 (on Day 24)	-7.37
HGK-EKN-L10	1	-18.57	-18.57
HGK-EKN-L10-30	0.3	-7.07	-7.07
HGK-EKN-L10-30	1	-22.21	-22.21
HGKH-EKN-L4	0.3	-8.68 (on Day 27)	-8.48
HGKH-EKN-L4	1	-20.86 (on Day 27)	-20.42
HGKH-EKN-L4-49	0.3	-6.16	-6.16
HGKH-EKN-L4-49	1	-21.27	-21.27

Experimental Example 7-4. Hypoglycemic effect in DIO mice

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

The non-fasting blood glucose levels were measured on day 9 and day 21 from the initiation of administration, and the fasting blood glucose levels were measured at the termination. Glucose concentration in the blood was measured using a GlucoDr blood glucose meter (Allmedicus, Korea), and the measurement results of blood glucose levels are shown in FIGS. 16 and 17.

In FIG. 16, as a result of measuring the non-fasting blood glucose levels on day 9 and day 21 from the initiation of administration, it was confirmed that all of the 6 types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) were maintained at a lower glucose level than the Vehicle-treated group at a dose of 1 nmol/kg. In FIG. 17, it was confirmed that all of the 6 types of long-acting fusion proteins (HGK-EKN-L8, HGK-EKN-L10, HGK-EKN-L12, HGH-EKN-L5, HGH-EKN-L6, and HGKH-EKN-L4) showed an excellent effect of improving the blood glucose levels at a dose of 1 nmol/kg.

Experimental Example 7-5. Effect of reducing body weight in DIO mice (4)

The body weight and the cumulative food intake was measured daily or every 3 days from the initiation of administration until day 24 or day 27. The cumulative food intake are shown in FIG. 18, and the effect of reducing body weight is shown in Table 25 and FIG. 19.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Maximal efficacy	Efficacy on Day 27
Vehicle	-	-	3.0
HGK-EKN-L8	1	-17.85	-17.85
HGK-EKN-L10	1	-22.51	-22.51
HGK-EKN-L12	1	-10.66 (on Day 12)	-6.52
HGH-EKN-L5	1	-14.99	-14.99
HGH-EKN-L6	1	-18.79	-18.79
HGKH-EKN-L4	1	-23.07	-23.07

The cumulative food intake of all of the 6 long-acting fusion proteins decreased compared to the Vehicle-treated group (see FIG. 18) and were shown to have an excellent effect of reducing body weight compared to the Vehicle-treated group (see FIG. 19). In addition, it was confirmed that all of the 6 types of long-acting fusion proteins showed continuous weight loss from the time of administration until day 27 (see FIG. 19).

Experimental Example 7-6. Effect of reducing body weight in DIO mice (5)

The mice were divided into groups (n = 6 per group) having similar average values of body weight on the day before the start of administration, and the 9 types of long-acting fusion proteins prepared in Preparation Examples 1 and 2 above (HGK-L10, HGK-EKN-L10, HGK-EKN-L10-30, HGK-EKN-L10-47, HGK-EKN-L10-49, HGKH-EKN-L4, HGKH-EKN-L4-30, HGKH-EKN-L4-47, and HGKH-EKN-L4-49) were administered subcutaneously every 3 days for a total 4 weeks at a dose of 1 nmol/kg, and Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was administered as a Vehicle.

The body weight was measured daily or every 3 days from the initiation of administration until day 26. The effect of reducing body weight is shown in Table 26 and FIG. 20.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Efficacy on Day 26
Vehicle	-	1.96
HGK-L10	1	-28.28
HGK-EKN-L10	1	-29.69
HGK-EKN-L10-30	1	-22.54
HGK-EKN-L10-47	1	-23.95
HGK-EKN-L10-49	1	-18.11
HGKH-EKN-L4	1	-25.14
HGKH-EKN-L4-30	1	-26.63
HGKH-EKN-L4-47	1	-22.32
HGKH-EKN-L4-49	1	-26.96

As shown in Table 26 above, it was confirmed that 9 types of long-acting fusion proteins showed an excellent body weight loss effects ranging from approximately 18% to about 30%. In addition, it was confirmed that all 9 long-acting fusion proteins showed continuous weight loss until day 26 (see FIG. 20).

Experimental Example 7-7. Effect of reducing body weight in DIO mice (6)

The mice were divided into groups (n = 6 per group) having similar average values of body weight on the day before the start of administration, and the long-acting fusion protein HGKH-EKN-L4 prepared in Preparation Examples 1 and 2 above was administered subcutaneously every 3 days for a total 4 weeks at doses of 0.3 nmol/kg, 1 nmol/kg, 3 nmol/kg, 10 nmol/kg, or 30 nmol/kg, and Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was administered as a Vehicle. The cumulative food intake and body weight were measured daily or every 3 days from the initiation of administration until day 27. The cumulative food intake are shown in FIG. 21, and the effect of reducing body weight is shown in Table 27 and FIG. 22.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Efficacy on Day 27
Vehicle	-	4.13
HGKH-EKN-L4	0.3	-12.05
HGKH-EKN-L4	1	-19.33
HGKH-EKN-L4	3	-28.02
HGKH-EKN-L4	10	-29.93
HGKH-EKN-L4	30	-38.06

As a result, it was confirmed that HGKH-EKN-L4 reduced cumulative food intake and body weight in a dose-dependent manner.

Experimental Example 8. Evaluation of drug efficacy of long-acting fusion proteins in ob/ob mice

Ob/ob mice, in which excessive obesity is induced by hyperphagia due to a mutation in the ob gene, which is responsible for producing leptin, are characterized by hyperglycemia and insulin resistance. 5- to 7-week-old male ob/ob mice (Jackson Laboratory, USA) were purchased from Raonbio (Korea), acclimatized for 4 weeks, and then administration was initiated.

Experimental Example 8-1. Evaluation results of single-dose drug efficacy in ob/ob mice

The mice were divided into groups (n = 5 per group) having similar average values of non-fasting blood glucose levels and body weight on the day before the start of administration, and HGK-EKN-L10 was administered subcutaneously once at doses of 0.1 nmol/kg, 0.3 nmol/kg, 1 nmol/kg, 3 nmol/kg, or 10 nmol/kg. In particular, Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was administered as a Vehicle, and glucose concentration in blood was measured using a GlucoDr blood glucose meter (Allmedicus, Korea). Body weight and non-fasting blood glucose levels were measured every day from the initiation of administration until day 14.

The measurement results of body weight are shown in Table 28 below. The dose dependence of HGK-EKN-L10 in the effect of reducing body weight was confirmed, and after single administration of HGK-EKN-L10 at a dose of 1 nmol/kg or higher, it was confirmed that weight loss continued to occur for about 14 days (see FIG. 23). In addition, HGK-EKN-L10 showed the highest efficacy at a dose of 3 nmol/kg or higher.

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. ob/ob Control
Group	Dose (nmol/kg)	Maximal Efficacy	Efficacy on Day 14
Vehicle	-	-	-
HGK-EKN-L10	0.1	-3.05 (on Day 2)	-1.26
HGK-EKN-L10	0.3	-3.08 (on Day 3)	-1.31
HGK-EKN-L10	1	-13.42 (on Day 14)	-13.42
HGK-EKN-L10	3	-18.88 (on Day 14)	-18.88
HGK-EKN-L10	10	-18.56 (on Day 14)	-18.56

The measurement results of the non-fasting blood glucose levels are shown in FIG. 24. It was confirmed that HGK-EKN-L10 lowered the blood glucose levels in a dose-dependent manner and it was confirmed that the blood glucose levels were maintained at a low level immediately after administration. In addition, it was confirmed that the effect of lowering the blood glucose levels was maintained for about 14 days after single administration of HGK-EKN-L10 at a dose of 1 nmol/kg or higher.

Experimental Example 8-2. Evaluation results of efficacy of repeated administration of drugs in ob/ob mice

The mice were divided into groups (n = 6 per group) having similar average values of non-fasting blood glucose levels and body weight on the day before the start of administration, and HGK-EKN-L10 and HGKH-EKN-L4 were administered subcutaneously every 3 days for a total 8 times at doses of 0.3 nmol/kg, 1 nmol/kg, or 3 nmol/kg. In particular, Dulbecco's phosphate buffered saline (DPBS, Gibco, USA) was administered as a Vehicle, and body weight was measured daily or every 3 days from the initiation of administration until day 24. Upon completion of the study, glycated hemoglobin (HbA1c) levels were quantified using the DCA 2000 HbA1c kit (Siemens, 5035C), and and analysis of lipid, metabolic parameters, and liver damage indicators was performed.

Following the repeated administration of HGK-EKN-L10 and HGKH-EKN-L4 at 8 times of every 3 days, it was substantiated that a dose-dependent effect in reducing body weight was confirmed. (Table 29 and FIG. 25).

Group	Dose (nmol/kg)	Change of Body Weight (%) vs. Day 0
Group	Dose (nmol/kg)	Maximal Efficacy	Efficacy on Day 24
Vehicle	-	-	19.67
HGK-EKN-L10	0.3	-9.73 (on Day 24)	-9.73
HGK-EKN-L10	1	-19.66 (on Day 24)	-19.66
HGK-EKN-L10	3	-17.52 (on Day 24)	-17.52
HGKH-EKN-L4	0.3	-7.03 (on Day 24)	-7.03
HGKH-EKN-L4	1	-14.77 (on Day 21)	-14.72
HGKH-EKN-L4	3	-14.29 (on Day 24)	-14.29

Upon completion of the study, as a result of measuring the glycated hemoglobin level, which reflects the average blood glucose level, it was confirmed that the glycated hemoglobin levels of HGK-EKN-L10 and HGKH-EKN-L4 decreased compared to the Vehicle-treated group at all doses (see FIG. 26).

Upon completion of the study, as a result of measuring the lipid and metabolism parameters through serum chemistry analysis, HGK-EKN-L10 and HGKH-EKN-L4 showed the effect of improving the total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels compared to the Vehicle-treated group (see FIG. 27).

Additionally, upon observation of liver damage indicators in serum, it was confirmed that all doses of the long-acting fusion proteins showed statistically significant reductions in the levels of alkaline phosphatases (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) compared to the Vehicle-treated group (see FIGS. 28a to 28c). Additionally, upon evaluation of the relative liver weight (%) considering body weight, a statistically significant reduction in relative liver weight was confirmed in administration groups at all doses of HGK-EKN-L10 and HGKH-EKN-L4 compared to the Vehicle-treated group (see FIG. 28d).

Experimental Example 9. Evaluation results of efficacy of administration of long-acting fusion proteins in db/db mice

db/db mice are genetically deficient in the leptin receptor gene and model type 2 diabetes and obesity, and are characterized by hyperglycemia, insulin resistance, and hyperphagia. 5- to 6-week-old male db/db mice (Janvier, France) were purchased from Raonbio (Korea), acclimatized for 4 weeks, and then administration was initiated.

The mice were divided into groups (n = 8 per group) having similar average values of body weight, non-fasting blood glucose level, and glycated hemoglobin levels on the day before the start of administration, and then, HGKH-EKN-L4 was administered subcutaneously every 3 days for a total 8 times at doses of 0.3 nmol/kg, 1 nmol/kg, 3 nmol/kg, or 10 nmol/kg. In particular, Vehicle treatment was performed using Dulbecco's phosphate buffered saline (DPBS, Gibco, USA), and the blood glucose concentration was measured using an Accu-Chek instant blood glucose meter (Roche, Switzerland). Non-fasting blood glucose levels were measured once daily until day 27 after the start of administration, and glycated hemoglobin levels were measured on day 14 and day 26 from the initiation of administration. Additionally, in order to evaluate improvement in insulin resistance, the mice were fased for 6 hours from the morning on the third day after the last administration. The insulin dose levels for each mouse was determined based on body weight after fasting. Given the mouse an intraperitoneal injection of insulin (1 U/kg, Humulin R, Eli Lilly). Blood samples were continuously taken from the initial tail cut and measured before the insulin injection (0) and at 15, 30 60, 90, and 120 minutes, respectively.

As a result of repeated administrations of HGKH-EKN-L4at 8 times every 3 days, it was confirmed that non-fasting blood glucose levels were maintained at a lower level compared to the Vehicle-treated group at all doses, and that HGKH-EKN-L4 exhibited the effect of reducing blood glucose levels in a dose-dependent manner (see FIG. 29). In particular, it was confirmed that the group administered with HGKH-EKN-L4 at a dose of 10 nmol/kg maintained blood glucose levels at the normal control level until the end of the study.

The results of measuring glycated hemoglobin levels on day 14 and day 26 from the initiation of administration isshown in Table 30 and FIG. 30 below. It was confirmed that before the start of administration, the glycated hemoglobin levels of the Vehicle-treated group and the group administered with HGKH-EKN-L4 were similar, but after administration, the glycated hemoglobin levels were improved compared to the Vehicle-treated group at all doses, and glycated hemoglobin levels were decreased in a dose-dependent manner of HGKH-EKN-L4.

Group	Dose (nmol/kg)	HbA1c (%)
Group	Dose (nmol/kg)	On Day 14	On Day 24
Normal control	-	4.2	-
Vehicle	-	7.3	8.6
HGKH-EKN-L4	0.3	6.3	6.2
HGKH-EKN-L4	1	6.4	5.5
HGKH-EKN-L4	3	5.7	5.4
HGKH-EKN-L4	10	5.2	4.6

The results of the insulin tolerance test (ITT) performed after the last administration are shown in FIG. 31. As a result of insulin tolerance test, it was confirmed that the groups receiving HGKH-EKN-L4 at doses of 1 nmol/kg, 3 nmol/kg, and 10 nmol/kgshowed improved insulin sensitivity similar to normal control group, with a dose-dependent reduction in blood glucose levels following insulin administration. (see FIG. 31a). As a result of analysis based on the AUCglucose of the Vehicle-treated group, it was confirmed that the value of AUCglucose was significantly reduced in the groups administered with HGKH-EKN-L4 at doses of 1 nmol/kg, 3 nmol/kg, and 10 nmol/kg (see FIG. 31b).

Experimental Example 10. Evaluation of drug efficacy of long-acting fusion proteins in Gubra Amylin NASH (GAN) ob/ob mice

Experimental Example 10-1. GAN-ob/ob non-alcoholic steatohepatitis (NASH) mouse model

Animal models of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) may be divided into four types: a diet-induced model, a chemical-induced model, a genetic modification model, and a complex model. Among these, the GAN-ob/ob model, which is a double complex model in which a GAN diet is fed to ob/ob mice that is a mutation model of the ob gene, is a relevant model similar to the human NASH phenotype.

5- to 7-week-old male ob/ob mice (Jackson lab, USA) were purchased from Raonbio (Korea). The ob/ob mice were fed with a GAN diet (Research Diets, Cat. No. D09100310; 40 kcal% Fat, 22 kcal% Fructose, 10% sucrose, and 2% Cholesterol) for 12 weeks and then drug treatment was initiated. On the day before the drug treatment, mice were randomized into groups (n = 8 per group) according to their body weight and the levels of AST, ALT, triglycerides (TG), and total cholesterol.

Then, the mice were subcutaneously administered with HGKH-EKN-L4 at doses of 3 nmol/kg, 10 nmol/kg, and 30 nmol/kg every 3 days for a total of 8 weeks. In particular, Vehicle treatment was performed using Dulbecco's phosphate buffered saline (DPBS, Gibco, USA). The test articles were administered for 8 weeks during GAN diet induction for a total 20 weeks.

Experimental Example 10-2. Effects of improvement on body weight, liver weight, and liver damage-related blood indicators in the GAN-ob/ob NASH mouse model

As a result of repeated administration of HGKH-EKN-L4 for 8 weeks, compared to the GAN-ob/ob control group (GAN-ob/ob Vehicle) (4.79%), the effect of reducing body weight of about -12.36% was shown in the group administered with HGKH-EKN-L4 (3 nmol/kg), about -18.14% in the group administered with HGKH-EKN-L4 (10 nmol/kg), and about -36.51% in the group administered with HGKH-EKN-L4 (30 nmol/kg) (see FIG. 32a). Additionally, when evaluating relative liver weight (%) considering fasting body weight, it was confirmed that there was a statistically significant decrease in relative liver weight in the group administered with HGKH-EKN-L4 treated group (see FIG. 32b).

When the liver damage indicators in the blood were observed, in the GAN-ob/ob NASH control group (GAN-ob/ob Vehicle), the levels of ALT, AST, and ALP were significantly increased compared to the normal control group (Chow Vehicle). Upon administration of HGKH-EKN-L4, a dose-dependnet improvedment in liver damage indicators was validated with a statistically significant reduction in ALT, AST, and ALP levels observed in the HGKH-EKN-L4 administered group(see FIG. 33).

Experimental Example 10-3. Effect of histological improvement in GAN-ob/ob NASH mouse model

Picrosirius Red stain (PSR) analysis is an analysis method for staining collagen deposited in liver tissue, and considering the liver weight of each individual, the total PSR contents (mg) are shown in FIG. 34. A significant increase in PSR was confirmed in the GAN-ob/ob control group (GAN-ob/ob Vehicle) compared to the normal control group (Chow Vehicle), and the total PSR content was decreased by administration of HGKH-EKN-L4, which indicates that the progression of liver fibrosis is inhibited through HGKH-EKN-L4 administration. Statistical significance was shown in the groups administered with HGKH-EKN-L4 at does of 3 nmol/kg, 10 nmol/kg, and 30 nmol/kg (see FIG. 34).

In conclusion, when HGKH-EKN-L4 was administered in the GAN-ob/ob NASH mouse model, it not only reduced body weight, but also inhibited the progression of liver damage and liver fibrosis, which indicates that non-alcoholic steatohepatitis has improved.

Claims

A fusion protein comprising a polypeptide represented by Formula (I) below and a polypeptide represented by Formula (II) below or a dimer thereof:

A_p-B_q-C (I)

F_a-G_b-H-I-J (II)

wherein:

A and F are each independently a bioactive protein;

B, G, and I are each independently a linker;

C and H are each an immunoglobulin Fc or a variant thereof; and

J is a polypeptide consisting of an amino acid sequence represented by (Y₁Y₂)_t-N-(Y₃)_u-(Y₄)_v-(Y₅)_w-CPLGPGRCCRLHTV-Y_6-ASLEDLGWAD-Y₇-VLSPREVQVTMCIGACPSQFRAA-Y₈-MHA-Y₉-IKT-Y₁₀-LHRLKPDTVPAPCCVPASYNPMVLI-Y₁₁-KTDTGVSLQTYD-Y₁₂-LLAKDCHCI,

wherein:

Y₁, Y₃, and Y₇ to Y₉ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W);

Y₂, Y₆, and Y₁₁ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W) or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R);

Y₄ and Y₁₂ are a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid, which is aspartic acid (D) or glutamic acid (E);

Y₅ is a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R);

Y₁₀ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid which is aspartic acid (D) or glutamic acid (E); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R);

t, u, v, and w are each independently 0 or 1;

p and q in Formula (I) above and a and b in Formula (II) above are each independently 0 or 1, wherein when p is 0, q is 0, and when p is 1, q is 1; and

when a is 0, b is also 0, and when a is 1, b is 1, with the proviso that p and q in Formula (I) and a and b in Formula (II) are not 0 simultaneously.
The fusion protein or the dimer thereof according to claim 1, wherein the bioactive protein is selected from the group consisting of insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor related protein (CTRP) family, resistin, visfatin, omentin, retinol binding protein-4 (RBP4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, growth hormone, and variants thereof.
The fusion protein or the dimer thereof according to claim 2, wherein the bioactive protein is selected from the group consisting of GLP-1, exendin-4, and variants thereof.
The fusion protein or the dimer thereof according to claim 3, wherein the bioactive protein is selected from the group consisting of a polypeptide consisting of an amino acid sequence represented by H-X₁-EGTFTSDVSSYLE-X₂-QAAKEFI-X₃-WL-X₄-X₅-G-X₆-G, exenatide, liraglutide, dulaglutide, albiglutide, lixisenatide, semaglutide, tirzepatide, cotadutide, and taspoglutide, wherein:

X₁ is a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W);

X₂ and X₃ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or an acidic amino acid which is aspartic acid (D) or glutamic acid (E); and

X₄, X₅, and X₆ are each independently a neutral amino acid selected from the group consisting of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), phenylalanine (F), proline (P), methionine (M), serine (S), threonine (T), tyrosine (Y), cysteine (C), glutamine (Q), asparagine (N), and tryptophan (W); or a basic amino acid selected from the group consisting of lysine (K), histidine (H), and arginine (R).
The fusion protein or the dimer thereof according to claim 4, wherein X₁ is alanine (A) or glycine (G), and/or X₂ is glycine (G) or glutamic acid (E), and/or X₃ is alanine (A) or glutamic acid (E), and/or X₄ is valine (V) or lysine (K), and/or X₅ is lysine (K) or asparagine (N), and/or X₆ is arginine (R) or glycine (G).
The fusion protein or the dimer thereof according to claim 5, wherein X₁ is glycine (G), X₂ is glutamic acid (E), and X₃ is glycine (G).
The fusion protein or the dimer thereof according to claim 5, wherein X₃ is alanine (A).
The fusion protein or the dimer thereof according to claim 5, wherein X₃ is glutamic acid (E), and X₄ is valine (V).
The fusion protein or the dimer thereof according to claim 5, wherein X₃ is glutamic acid (E), X₄ is lysine (K), and X₅ is asparagine (N).
The fusion protein or the dimer thereof according to claim 1, wherein A and F are each independently selected from the group consisting of amino acid sequences of SEQ ID NOS: 9 to 12.
The fusion protein or the dimer thereof according to claim 1, wherein t is 1, Y₁ is alanine (A) or tryptophan (W), and Y₂ is arginine (R) or serine (S).
The fusion protein or the dimer thereof according to claim 1, wherein t is 0, Y₃ is glycine (G) or serine (S), and/or Y₄ is aspartic acid (D) or threonine (T), and/or Y₅ is histidine (H), and/or Y₆ is arginine (R) or asparagine (N), and/or Y₇is tryptophan (W) or phenylalanine (F), and/or Y₈ is asparagine (N), leucine (L), cysteine (C), or serine (S), and/or Y₉ is glutamine (Q) or asparagine (N), and/or Y₁₀ is serine (S), asparagine (N), aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), or leucine (L), Y₁₁ is glutamine (Q) or histidine (H), and/or Y₁₂ is aspartic acid (D), leucine (L), cysteine (C), or serine (S).
The fusion protein or the dimer thereof according to claim 12, wherein Y₃ is glycine (G), Y₄ is aspartic acid (D), and Y₅ is histidine (H).
The fusion protein or the dimer thereof according to claim 12, wherein w is 0, Y₃ is serine (S), and Y₄ is threonine (T).
The fusion protein or the dimer thereof according to claim 1, wherein J is selected from the group consisting of amino acid sequences of SEQ ID NOS: 2 to 8 and 93 to 108.
The fusion protein or the dimer thereof according to claim 1, wherein B, G, and I are each independently a linker selected from the group consisting of:

1) a peptide consisting of 5 to 52 glycines (G) and/or serines (S);

2) a peptide consisting of 4 to 52 glutamic acids (E), and/or alanines (A);

3) a peptide consisting of 5 to 52 glutamic acids (E), lysines (K), and/or alanines (A);

4) a peptide consisting of 6 to 52 glycines (G), serines (S), glutamic acids (E), and/or alanines (A); and

5) a peptide consisting of 7 to 52 glycines (G), serines (S), glutamic acids (E), lysines (K) and/or alanines (A).
The fusion protein or the dimer thereof according to claim 16, wherein B, G, and I are each independently a linker selected from the group consisting of (G₄S)_n, GS(G₄S)_n, GS(EEEA)_n, (EEEA)_n, GS(EAAAK)_n, and (EAAAK)_n, and n is an integer from 1 to 10.
The fusion protein or the dimer thereof according to claim 1, wherein B, G, and I are each independently selected from the group consisting of amino acid sequences of SEQ ID NOS: 13 to 24.
The fusion protein or the dimer thereof according to claim 1, wherein the immunoglobulin Fc or a variant thereof comprises a processed protuberance or a processed cavity.
The fusion protein or the dimer thereof according to claim 1, wherein the immunoglobulin Fc or a variant thereof is any one of the Fc regions of IgG1, IgG2, IgG3, IgG4, and IgD, or a hybrid Fc containing a combination thereof.
The fusion protein or the dimer thereof according to claim 19, wherein the human IgG1 Fc fragment variant comprising the processed protuberance is one in which the 146th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 80 is substituted with tryptophan (W).
The fusion protein or the dimer thereof according to claim 19, wherein the human IgG1 Fc fragment variant comprising the processed cavity is one in which the 146th amino acid, the 148th amino acid, and the 187th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 80 are substituted with serine (S), alanine (A), and valine (V), respectively.
The fusion protein or the dimer thereof according to claim 1, wherein the immunoglobulin Fc variant is one in which the effector function of Fc is reduced or eliminated.
The fusion protein or the dimer thereof according to claim 23, wherein the immunoglobulin Fc variant is one in which the 14th amino acid, 15th amino acid, and 77th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 80 are each substituted with alanine (A).
The fusion protein or the dimer thereof according to claim 1, wherein the immunoglobulin Fc or a variant thereof is a polypeptide in which a hinge is added to the N-terminus of the polypeptide consisting of the amino acid sequences of SEQ ID NOS: 81 to 86.
The fusion protein or the dimer thereof according to claim 20, wherein the immunoglobulin Fc or a variant thereof is a polypeptide which comprises a hinge consisting of one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: 79 and 109 to 111.
The fusion protein or the dimer thereof according to claim 1, wherein the immunoglobulin Fc or a variant thereof is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 85 or 86, or a polypeptide in which the amino acid sequence of SEQ ID NO: 79 is added to the N-terminus of the polypeptide having the amino acid sequence of SEQ ID NO: 85 or 86.
The fusion protein or the dimer thereof according to claim 1, wherein the immunoglobulin Fc or a variant thereof is a polypeptide which consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: 81 to 92.
The fusion protein or the dimer thereof according to claim 1, wherein the fusion protein is:

(1) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(2) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(3) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(4) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 30 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(5) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(6) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 32 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(7) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(8) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(9) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(10) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(11) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 37 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(12) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 38 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(13) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 39 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(14) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 86 are linked;

(15) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked;

(16) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(17) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(18) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(19) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 68 are linked;

(20) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 69 are linked;

(21) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 70 are linked;

(22) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 25 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 67 are linked;

(23) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(24) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(25) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(26) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63 are linked;

(27) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(28) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(29) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(30) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 41 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 64 are linked;

(31) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 31 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 65 are linked;

(32) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 66 are linked;

(33) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 44 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(34) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(35) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(36) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(37) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(38) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(39) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(40) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked;

(41) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked;

(42) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked;

(43) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(44) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked;

(45) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked;

(46) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 71 are linked;

(47) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 45 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 72 are linked;

(48) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 46 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 73 are linked;

(49) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 47 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 74 are linked;

(50) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(51) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 49 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 76 are linked;

(52) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 77 are linked;

(53) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 51 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(54) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 52 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(55) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 53 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(56) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 54 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(57) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 55 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(58) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 56 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 92 are linked;

(59) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 57 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(60) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 58 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(61) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 59 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(62) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 60 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked;

(63) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 61 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked; and

(64) a protein in which a polypeptide consisting of the amino acid sequence of SEQ ID NO: 62 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 75 are linked.
An isolated cell line producing the fusion protein or the dimer thereof according to any one of claims 1 to 29.
An isolated nucleic acid encoding the fusion protein or the dimer thereof according to any one of claims 1 to 29.
A recombinant expression vector comprising the nucleic acid according to claim 31.
A host cell comprising the vector according to claim 32.
A method for producing a fusion protein or a dimer thereof, comprising:

culturing a cell line expressing a fusion protein under conditions in which the fusion protein of any one of claims 1 to 29 is expressed; and

recovering the fusion protein.
A composition comprising a pharmaceutically acceptable carrier and the fusion protein or the dimer thereof according to any one of claims 1 to 29.
A method for treating or preventing a metabolism-related disease in an individual in need of treatment or prevention of a metabolism-related disease, comprising administering the fusion protein or the dimer thereof according to any one of claims 1 to 29 to the individual.
The method according to claim 36, wherein the metabolism-related disease is diabetes, obesity, hypercholesterolemia, hyperglycemia, insulin resistance, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver damage, hepatitis, liver fibrosis, liver cirrhosis, fatty liver, arteriosclerosis, dyslipidemia, cardiovascular disease, or metabolic syndrome.
A pharmaceutical composition for treating or preventing metabolic diseases, comprising a pharmaceutically acceptable carrier and the fusion protein or the dimer thereof according to any one of claims 1 to 29.
Use of the fusion protein or the dimer thereof according to any one of claims 1 to 29 in preparation of a drug for the treatment of metabolism-related diseases.