RU2822001C1 - Novel protein conjugate and use thereof for preventing or treating non-alcoholic steatohepatitis, obesity and diabetes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are protein conjugates containing polyubiquitin, a carrier linked through a linker to polyubiquitin, and four biomolecules linked through a linker to polyubiquitin and a carrier. In addition, present invention relates to a pharmaceutical composition containing a protein conjugate, and a method for preventing or treating non-alcoholic steatohepatitis, fatty liver disease, liver fibrosis, liver cirrhosis, liver cancer, obesity, or diabetes.

EFFECT: invention provides higher activity and improved therapeutic action of biomolecules.

16 cl, 55 dwg, 37 tbl, 9 ex

Description

Field of the present invention

The present invention relates to a protein conjugate comprising a polyubiquitin, a carrier linked to the polyubiquitin, and two or more biomolecules linked to the polyubiquitin or carrier. In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity and diabetes, containing a protein conjugate containing two or more biomolecules.

BACKGROUND OF THE INVENTION

Non-alcoholic fatty liver disease (NAFLD) is a type of disease whose histological manifestations are similar to alcoholic hepatitis, even with little or no alcohol consumption, and is a type of metabolic syndrome encompassing non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) , cirrhosis and liver cancer (hepatocellular carcinoma). Non-alcoholic fatty liver disease is on the rise as the population with obesity and diabetes increases, and the annual incidence rate in Korea is about 16%.

Non-alcoholic steatohepatitis (NASH) is mainly characterized by abnormal accumulation or deposition of fat in the liver (hepatic steatosis), liver inflammation and liver damage or damage to liver tissue (fibrosis). The worldwide prevalence of non-alcoholic steatohepatitis is 2-4% (3-12% of US adults). It is well known that non-alcoholic steatohepatitis has a faster histological progression and can progress to cirrhosis, while simple steatosis has a slower histological progression. Approximately 5-10% of people diagnosed with fatty liver develop steatohepatitis (Metabolism Clinical and Experimental 65 (2016) 1038-1048).

There is currently no commercially available therapeutic agent for the treatment of non-alcoholic steatohepatitis, and due to the lack of a therapeutic agent for it, other therapeutic agents are used to treat metabolic syndrome such as abdominal obesity, hyperlipidemia and diabetes, such as drugs that improve insulin resistance, antioxidants (for example, vitamins C and E), agents for the treatment of dyslipidemia, hepatoprotectors, etc., but they can hardly be considered as therapeutic agents for the direct treatment of non-alcoholic steatohepatitis.

Since nonalcoholic steatohepatitis is a complex disease in nature, licensing authorities in the United States, Europe, etc. established strict drug approval requirements for its therapeutic agents. For this reason, a number of therapeutic agents have recently failed to reach the clinical development stage, and accordingly, multiple agonist therapeutic agents have emerged to simultaneously improve various parameters. Multiple agonist therapeutics for nonalcoholic steatohepatitis currently in clinical trials include MEDI0382 from AstraZeneca, a dual GLP-1/glucagon (GCG) agonist, LY3298176 from Eli Lilly, a dual GIP/GLP-1 agonist, HM15211 from Hanmi Pharm.Co.,Ltd., glucagon/GIP/GLP-1 triple agonist, etc.

Obesity refers to the state of excess fat in the body and can be defined as a health risk condition due to excess fat in the body, which causes several diseases including heart disease. The World Health Organization (WHO) reported that there are more than 106 million adults worldwide who are overweight, at least 4 million are obese, and more than 2 in 3 Americans are overweight or obese (Low et al, 2009; Cooke and Bloom, 2006). Obese people have an increased risk of developing a number of diseases, including type 2 diabetes, hyperlipidemia, arthritis and apnea, compared to people of normal weight, and obesity is known to cause several types of cancer and heart disease.

Diabetes mellitus is a metabolic disease in which high blood glucose levels persist for a long time due to insufficient insulin secretion or insulin resistance. As blood glucose levels persist for a long time, glycation products enter the retina, kidneys, nerves, or large and small blood vessels throughout the body, causing chronic complications. Because complications of diabetes are more dangerous than diabetes itself, the most important goal of diabetes treatment today is to suppress the onset or progression of diabetes complications. Typical complications of diabetes include diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic osteoporosis, diabetic atherosclerosis, and the like.

In the development of multiple agonist-based therapeutics, due to the problem of reduced activity due to structural limitations, there are currently no studies of more than four-directional agonist-based therapeutics in non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, cancer liver, obesity or diabetes. Accordingly, the inventors of the present invention have worked tirelessly to develop a quadruple agonist/antagonist therapeutic agent for the treatment of non-alcoholic steatohepatitis, fatty liver disease, liver fibrosis, cirrhosis, liver cancer, obesity and diabetes. As a result, the present invention has been accomplished using a polyubiquitin structure.

State of the art document

Patent document

(Patent Document 0001) Korean Patent Application Publication No. 10-2016-0032699

(Patent Document 0002) Korea Patent No. 10-2034607

Detailed Disclosure of the Present Invention

Technical problem

The object of the present invention was to create a protein conjugate containing polyubiquitin, a carrier associated with polyubiquitin, and two or more biomolecules associated with polyubiquitin or carrier.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis (NASH), fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, comprising a protein conjugate containing two or more biomolecules. In particular, another object of the present invention was to provide a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, containing a quadridirectional GCG/GLP-1/FGF21 acceptor agonist/antagonist /GIP or GCG/GLP-1/FGF21/IL-1RA.

The solution of the problem

The present invention relates to a protein conjugate, characterized in that the protein conjugate contains: a polyubiquitin, a carrier linked directly or via a linker to the polyubiquitin, and a biomolecule linked directly or via a linker to the polyubiquitin or carrier, wherein the polyubiquitin consists of (i) a ubiquitin acceptor containing one or more unsubstituted lysines, wherein the ubiquitin lysines may be substituted with arginine or alanine, and (ii) a ubiquitin donor, wherein all of the ubiquitin lysines are substituted with arginine or alanine, and the biomolecule is two or more selected from the group consisting of GCG, GLP-1 , FGF21, GIP and IL-1RA, their analogues, GCG and GLP-1 dual acceptor agonist, and GLP-1 and GIP dual acceptor agonist.

In one embodiment, GCG and its analogue may be selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NO: 1 - 3, and preferably the GCG analogue may be a protein consisting of the amino acid sequence of SEQ ID NO: 2.

In one embodiment, GLP-1 and its analogue may be selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NO: 4 to 14, and preferably the GLP-1 analogue may be a protein consisting of the amino acid sequence of SEQ ID NO: : 12.

In one embodiment, the GCG GLP-1 dual acceptor agonist may be selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NOs: 15 and 16.

In one embodiment, FGF21 and an analogue thereof may be selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NO: 17 to 21. Preferably, the FGF21 analogue may be a protein consisting of the amino acid sequence SEQ ID NO: 20.

In one embodiment, the GIP and its analogue may be selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NO: 22 - 26. Preferably, the GIP and its analogue may be a protein consisting of the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the IL-1RA and its analogue may be selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NO: 27 and 28. Preferably, the IL-1RA may be a protein consisting of the amino acid sequence of SEQ ID NO: 27 .

In one embodiment, the polyubiquitin may consist of a ubiquitin acceptor in which the 6th, 11th, 27th, 29th, 33rd, and 48th lysines from the N-terminus of ubiquitin are replaced by arginine, and a ubiquitin donor, in which all ubiquitin lysines are replaced by arginine. Preferably, the polyubiquitin may consist of a ubiquitin acceptor consisting of the amino acid sequence of SEQ ID NO: 30, and a ubiquitin donor consisting of the amino acid sequence of SEQ ID NO: 31.

In one embodiment, the linker may be a polypeptide consisting of an amino acid sequence that combines from 1 to 30 GGGGS, EAAAK, or VPPPPP repeats. Preferably, the linker may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29.

In one embodiment, the carrier may be selected from the group consisting of albumin, antibody fragment, scFc (single chain Fc dimer), scFc dimer (single chain Fc dimer), transferrin, XTEN (genetic fusion of a loose repeat peptide sequence), CTP (carboxy terminal peptide) , PAS (proline-alanine-serine polymer), ELK (elastin-like peptide), HAP (homoamino acid polymer), GLK (gelatin-like protein), PEG (polyethylene glycol) and fatty acid. Preferably, the carrier may be albumin.

The present invention relates to a protein conjugate represented by the following formula:

in the above formula, W, X, Y and Z may each be a biomolecule selected from the group consisting of a GCG analogue consisting of the amino acid sequence SEQ ID NO: 2, a GLP-1 analogue consisting of the amino acid sequence SEQ ID NO: 12, an FGF21 analog consisting of the amino acid sequence SEQ ID NO: 20, a GIP analog consisting of the amino acid sequence SEQ ID NO: 24, and an IL-1RA consisting of the amino acid sequence SEQ ID NO: 27, Ub(A) may be a ubiquitin acceptor, consisting of the amino acid sequence of SEQ ID NO: 30, Ub(D) may be a ubiquitin donor consisting of the amino acid sequence of SEQ ID NO: 31, L may be independently absent or be a linker, A may be a carrier, and Ub(A) and Ub ( D) can be linked by a covalent bond.

In one embodiment, X may be a GCG analogue consisting of the amino acid sequence SEQ ID NO: 2, Y may be a GLP-1 analogue consisting of the amino acid sequence SEQ ID NO: 12, Z may be an FGF21 analogue consisting of the amino acid sequence SEQ ID NO: 12. NO: 20, and W may be a GIP analogue consisting of the amino acid sequence of SEQ ID NO: 24, or an IL-1RA analogue consisting of the amino acid sequence SEQ ID NO: 27.

In one embodiment, the linker may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29 and the carrier may be albumin.

The present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis (NASH), fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, containing a protein conjugate.

The present invention provides a method of preventing or treating non-alcoholic steatohepatitis (NASH), fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, comprising administering the composition to a non-human subject.

In addition, the present invention provides a method for producing a protein conjugate, characterized in that the method includes the steps of:

(i) obtaining an acceptor protein represented by the following formula;

X-L-Y-L-Ub(A)-L-A-L-Z

(ii) obtaining a donor protein represented by the following formula; And

W-L-Ub(D)

(iii) binding of Ub(A) in the acceptor protein and Ub(D) in the donor protein,

wherein W, X, Y and Z are each a biomolecule selected from the group consisting of a GCG analogue consisting of the amino acid sequence SEQ ID NO: 2, a GLP-1 analogue consisting of the amino acid sequence SEQ ID NO: 12, an FGF21 analogue consisting of the amino acid sequence SEQ ID NO: 20, a GIP analog consisting of the amino acid sequence SEQ ID NO: 24, and IL-1RA consisting of the amino acid sequence SEQ ID NO: 27, Ub(A) is a ubiquitin acceptor consisting of amino acid sequence SEQ ID NO: 30, Ub(D) is a ubiquitin donor consisting of the amino acid sequence SEQ ID NO: 31, each L is independently absent or a linker, and A is a carrier.

Effects of the invention

The novel protein conjugate of the present invention contains two or more biomolecules and inhibits steatosis, inflammation and fibrosis in the liver, thereby having an excellent effect in the prevention or treatment of non-alcoholic steatohepatitis (NASH), fatty liver, liver fibrosis, liver cirrhosis, or cancer liver, and thus may also be usefully used for the prevention or treatment of obesity or diabetes. In addition, the protein conjugate can be stored in the body for a long time, exert its effect for a long time, and also has excellent safety through the use of polyubiquitin and a carrier harmless to the human body.

Brief description of drawings

Fig. 1 illustrates the results obtained by confirming the purification of the acceptor protein by SDS-PAGE.

Fig. 2 is a schematic diagram showing the donor protein purification process.

Fig. 3 and 4 illustrate the results obtained by confirming the purification of the donor protein through a Ni column using SDS-PAGE.

Fig. 5 and 6 illustrate the results obtained by confirming the purification of the donor protein by removing His-SUMO using SDS-PAGE.

Fig. 7 and 8 illustrate the results of polishing purification of the donor protein.

Fig. 9 is a schematic diagram showing a method for producing a protein conjugate by conjugating an acceptor protein and a donor protein.

Fig. 10 and 11 illustrate the results obtained by confirming the conjugation of the acceptor protein and the donor protein using SDS-PAGE.

Fig. 12 illustrates the results obtained by confirming the purification of a Ni protein conjugate by chromatography.

Fig. 13 and 14 illustrate the results obtained by confirming the purification of the protein conjugate by SDS-PAGE.

Fig. 15 illustrates the SDS-PAGE results of the final protein conjugate.

Figures 16-18 are graphs showing the results of the cAMP accumulation assay.

Fig. 19 is a graph showing the results of functional analysis of FGFR/KLB.

Fig. 20 is a graph showing the results of the NF-kB reporter assay.

Fig. 21 and 22 are graphs showing results obtained by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of mice in an in vivo efficacy test.

Fig. 23-26 illustrate the results obtained by observing steatosis and inflammation (lobular inflammation) in mouse liver tissue in an in vivo efficacy test and scoring according to the NAS evaluation criteria.

Fig. 27 and 28 are graphs showing results obtained by measuring the concentrations of TGT-β and triglycerides in mouse liver tissue in an in vivo efficacy test.

Fig. 29 and 30 are graphs showing results obtained by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of mice in an in vivo efficacy test.

Fig. 31-34 illustrate the results obtained by observing steatosis and inflammation (lobular inflammation) in mouse liver tissue in an in vivo efficacy test and scoring according to the NAS evaluation criteria.

Fig. 35 and 36 are graphs showing results obtained by measuring the concentrations of TGT-β and triglycerides in mouse liver tissue in an in vivo efficacy test.

Fig. 37-41 are graphs showing changes in triglycerides (TG), free fatty acids (NEFA), total cholesterol (T-Chol), low-density lipoprotein cholesterol (LDL cholesterol), and high-density lipoprotein cholesterol (HDL cholesterol) in mouse serum with diet-induced obesity in an in vivo efficacy test.

Fig. 42 and 43 are graphs showing changes in non-fasting blood glucose levels in type 2 diabetic mice in an in vivo efficacy test.

Fig. 44 and 45 are graphs showing body weight changes of type 2 diabetic mice in an in vivo efficacy test.

Fig. 46 and 47 are graphs showing changes in food intake of type 2 diabetic mice in an in vivo efficacy test.

Fig. 48 and 49 are graphs showing changes in water consumption of type 2 diabetic mice in an in vivo efficacy test.

In fig. 50 - 52 show the results of an in silico immunogenicity test for MHC class I.

In FIG. 53-55 show the results of an in silico MHC class II immunogenicity test.

Best Mode for Carrying Out the Present Invention

Embodiments and examples of the present invention will now be described in detail with reference to the accompanying drawings, so that those skilled in the art to which the present invention relates can easily put the present invention into practice. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described herein.

As used herein, when a certain part “includes” a certain component, it means that other components may be further included, and does not exclude other components, unless otherwise noted.

In the context of the present invention, the term "prevention" refers to any act of inhibiting or delaying the onset of a disease by administration of a composition, and "treatment" refers to any act in which the symptoms of a subject suspected of and suffering from a disease are ameliorated or beneficially modified by the administration of a composition .

In the context of the present invention, the term "subject" is a mammal, preferably a human, but may be an animal, including a domestic animal (eg, dog, cat, etc.), domesticated animal (eg, cow, sheep, pig, horse, etc. .) and laboratory animal (for example, rat, mouse, guinea pig, etc.).

The pharmaceutical composition of the present invention may be administered parenterally or orally depending on the desired route, and the dosage may vary depending on the patient's body weight, age, gender, health status, diet, time of administration, route of administration, excretion rate, severity of disease and the like . In addition, the therapeutically effective amount of the composition may vary depending on the route of administration, the target site and the condition of the patient, and when used in humans, the dose should be determined as appropriate taking into account both safety and effectiveness factors.

As used herein, the term "GCG" may refer to wild-type glucagon (native GCG), which is a protein consisting of the amino acid sequence SEQ ID NO: 1.

In the context of the present invention, the term "GCG analogue" means that some amino acids of the wild-type glucagon protein are substituted. Preferably, it may refer to a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 3. More preferably, it may refer to a protein consisting of the amino acid sequence of SEQ ID NO: 2, in which amino acids 16 to 20 The N termini of the wild-type glucagon protein are changed from SRRAQ to ERRAK, amino acids 23 to 24 are changed from VQ to IE, and amino acids 27 to 29 are changed from = MNT to LSA.

In the context of the present invention, the term "GLP-1" may refer to wild-type GLP-1 (native GLP-1), which is a protein consisting of the amino acid sequence SEQ ID NO: 4.

In the context of the present invention, the term "GLP-1 analog" means that some amino acids of the wild-type GLP-1 protein are substituted. Preferably, it may refer to a protein selected from the group consisting of a protein consisting of the amino acid sequences of SEQ ID NO: 5-14. More preferably, it may refer to a protein consisting of the amino acid sequence SEQ ID NO: 12 in which the 2nd amino acid from the N terminus of the wild type GLP-1 protein is changed from A to G, the 16th amino acid is changed from G to E , and the 30th amino acid is changed from R to GG.

In the context of the present invention, the term "FGF21" may refer to wild-type FGF21 (native FGF21), which is a protein consisting of the amino acid sequence SEQ ID NO: 17.

In the context of the present invention, the term "FGF21 analog" means that some amino acids of the wild-type FGF21 protein are substituted. Preferably, it may refer to a protein selected from the group consisting of a protein consisting of the amino acid sequences of SEQ ID NO: 18-21. More preferably, it may refer to a protein consisting of the amino acid sequence SEQ ID NO: 20 in which the 19th amino acid from the N terminus of the wild type FGF21 protein is changed from R to V, amino acids 98 to 100 are changed from LLL to DLK, amino acids 167 to 170 changed from SMVG to RLVE, amino acid 174 changed from G to L, and amino acids 179 to 181 changed from YAS to FE.

In the context of the present invention, the term "GIP" may refer to wild-type GIP (native GIP), which is a protein consisting of the amino acid sequence SEQ ID NO: 22.

In the context of the present invention, the term "GIP analog" means that some amino acids of the wild-type GIP protein are substituted. Preferably, it may refer to a protein selected from the group consisting of a protein consisting of the amino acid sequences of SEQ ID NO: 23-26. More preferably, it may refer to a protein consisting of the amino acid sequence of SEQ ID NO: 24 in which the 2nd amino acid from the N terminus of the wild type GIP protein is changed from A to S.

In the context of the present invention, the term "IL-1RA" may refer to wild-type IL-1RA (native IL-1RA), which is a protein comprised of the amino acid sequence SEQ ID NO: 27.

In the context of the present invention, the term “IL-1RA analog” means that some amino acids of the wild-type IL-1RA protein are substituted. Preferably, it may refer to a protein consisting of the amino acid sequence SEQ ID NO: 28.

The present invention will now be described in more detail by way of examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Acceptor protein

Hereinafter, the acceptor protein obtained in the examples is a polypeptide represented by the following formula:

X-L-Y-L-Ub(A)-L-A-L-Z

in the above formula

X is a GCG analogue consisting of the amino acid sequence SEQ ID NO: 2, Y is a GLP-1 analogue consisting of the amino acid sequence SEQ ID NO: 12, and Z is an FGF21 analogue consisting of the amino acid sequence SEQ ID NO: 20 ,

Ub(A) is a ubiquitin acceptor consisting of the amino acid sequence SEQ ID NO: 30,

L is a linker consisting of the amino acid sequence of SEQ ID NO: 29, and

A is albumin consisting of the amino acid sequence SEQ ID NO: 32.

The SEQ ID NOs of the amino acid sequences of each protein and signal peptide constituting the acceptor protein are shown in Table 1.

Table 1 Protein Amino acid sequence analogue of GCG SEQ ID NO: 2 GLP-1 analogue SEQ ID NO: 12 analogue of FGF21 SEQ ID NO: 20 Linker SEQ ID NO: 29 Ub(A) SEQ ID NO: 30 Albumen SEQ ID NO: 32 HAS SEQ ID NO: 33 IgGκ SEQ ID NO: 34

Donor protein

In the following, the donor protein obtained in the examples is a polypeptide represented by the following formula:

W-L-Ub(D)

in the above formula

W is a GIP analogue consisting of the amino acid sequence SEQ ID NO: 24, or IL-1RA consisting of the amino acid sequence SEQ ID NO: 27,

L is a linker consisting of the sequence SEQ ID NO: 29, and

Ub(D) is a ubiquitin donor consisting of the sequence SEQ ID NO: 31.

The SEQ ID NO of the amino acid sequence of each protein constituting the donor protein is shown in Table 2.

table 2 Protein Amino acid sequence GIP SEQ ID NO: 24 IL-1RA SEQ ID NO: 27 Linker SEQ ID NO: 29 Ub(D) SEQ ID NO: 31

In the context of the present invention, the terms "donor-191" and "D-191" refer to a donor protein containing a GIP analogue.

In the context of the present invention, the terms “donor-192” and “D-192” refer to a donor protein containing IL-1RA.

As used herein, the terms “RD-191” and “C-191” refer to a protein conjugate in which an acceptor protein and a GIP analog-containing donor protein are linked.

In the context of the present invention, the terms “RD-192” and “C-192” refer to a protein conjugate in which an acceptor protein and a donor protein containing IL-1RA are linked.

Example 1

Preparation of plasmid DNA for acceptor protein expression

An acceptor gene sequence, which is a gene encoding an acceptor protein, was synthesized and then cloned into pcDNA3.1(+), a simple structure expression vector containing a CMV promoter, an ampicillin resistance gene and the like.

Rapid cloning was performed to clone the signal peptide that allows the protein to be secreted from the cell into a plasmid into which the acceptor gene is inserted. Vector PCR was performed using plasmid DNA into which the acceptor gene was inserted as a template so that the signal peptide could be inserted at the 5' end of the acceptor protein, and insertion PCR was performed using human serum albumin (HSA) consisting of the sequence SEQ ID NO: 33, and an IgGκ signal peptide consisting of the sequence SEQ ID NO: 34 as a template so that each signal peptide can be inserted at the 5' end of the acceptor protein. The PCR reaction was performed using Phusion High-Fidelity DNA Polymerase (Thermo Fisher, Cat. No: F530). During primary denaturation at 98 °C for 3 minutes, secondary denaturation at 98 °C for 10 seconds, primer conjugation at 60 °C for 30 seconds and extension reaction at 72 °C for 3 minutes, processes from secondary denaturation until the extension reaction was repeated 18 times, and then the final extension reaction was carried out at 72 °C for 5 minutes.

After completion of the PCR reaction, it was confirmed whether the target gene was amplified by agarose gel electrophoresis. Next, 10 μl of each PCR vector product and PCR insert product were added at 1:1 to the PCR tube, followed by 0.5 μl of DpnI. The tube was treated at 37°C for 1 hour to remove template DNA and ligated. The ligated PCR product was added to competent DH5α cells and transformed by heat shock treatment at 42°C for 1 minute, plated on solid LB medium containing ampicillin, and then stationary cultured at 37°C for at least 16 hours to obtain colonies . One colony was selected and inoculated into 5 ml of LB medium containing ampicillin and then cultured at 37°C and 220 rpm for 16 hours. The culture solution was centrifuged at 3500 rpm for 20 minutes to obtain wet E. coli cells, and then solutions S1, S2 and S3 from DNA isolation kit (COSMO Genetech, Cat. No.: CMP0112) were added to disrupt the cell wall and obtained a cloudy solution of DNA in which proteins and DNA have been separated. Plasmid DNA was purified from the resulting turbid DNA solution using a purification column from a DNA isolation kit (COSMO Genetech, Cat. No.: CMP0112). COSMO Genetech was asked to perform gene sequencing of the plasmid DNA and two vectors were obtained in which the HSA consisting of the sequence SEQ ID NO: 33 or the IgGκ signal peptide consisting of the sequence SEQ ID NO: 34 were inserted at the 5' end acceptor protein.

The vector with the identified gene sequence was added to competent DH5α cells and transformed by heat shock treatment at 42°C for 1 minute, plated on solid LB medium containing ampicillin, and then stationary cultured at 37°C for at least 16 hours with obtaining colonies. One colony was picked and inoculated into 5 ml of LB medium and then cultured at 37°C and 220 rpm for 16 hours. A portion of this culture solution was again inoculated into 200 ml of LB medium containing ampicillin and then cultured at 37°C and 220 rpm for 16 hours. The culture solution was centrifuged at 3500 rpm for 30 minutes to obtain wet E. coli cells, and then solutions P1, P2 and P3 from the DNA extraction kit (QIAGEN, Cat. No.: 12263) were added to disrupt the cell wall, and a cloudy solution of DNA in which proteins and DNA have been separated. The plasmid DNA precipitate was obtained from the resulting turbid DNA solution using the purification column of the DNA Isolation Kit (QIAGEN, Cat. No.: 12263). The precipitate was dissolved in cell culture water (Sigma Aldrich, W3500) and then filtered through a 0.22 μm filter. The extracted plasmid DNA was used for protein expression after measuring the DNA concentration and purity using a nanodroplet device (IMPLEN, Nanodrop NP-80).

Example 2

Expression of acceptor protein in ExpiCHO-S cells

24 hours before the transfection process, ExpiCHO-S cells (Gibco, catalog number: A29127, lot: 1974423) were inoculated at 4 × 10 ⁶ cells/ml, prepared and placed on an orbital shaker in an incubator at 37 °C, 80% humidity or more, 8% CO _{2 ,} and cultured at 95 rpm (shaking diameter 50 mm). After 24 hours, cells were filtered through a 40-μm nylon filter (BD Falcon, Cat. No.: 352340) to remove clumps, and then cell viability and cell number were measured. The filtered cells were diluted with ExpiCHO-S expression medium (ExpiCHO Expression Media, Gibco, catalog number: A29100-01) to a final concentration of 6×10 ⁶ cells/ml and 200 ml of the final cells were added to a 1 L flask.

120 μg of DNA was diluted in 8 ml of OptiPRO SFM medium (Gibco, cat. no.: 12309-019), and 640 μl of ExpiFectamine CHO reagent (Gibco, cat. no. 100033022) was diluted in 7.4 ml of OptiPRO SFM medium (Gibco, cat. . No.: 12309-019), respectively, and then mixed. The mixed solution was reacted at room temperature for 3 minutes and then distributed into an inoculated flask to 6x10 ⁶ cells/ml and transfected. It was placed on an orbital shaker in an incubator at 37°C, 80% humidity or more, 8% CO ₂ and cultured at 95 rpm (shaking diameter 50 mm) for 18 hours. After 18 hours, 1200 µl ExpiFectamine CHO Enhancer (Gibco, part number: 100033019) and 48 ml ExpiCHO Feed (Gibco, part number: A29101-01) were added and placed on an orbital shaker in an incubator at 37 °C, 80% humidity or more, 8% CO _2, and cultured at 95 rpm for 7-8 days. After completion of culture, centrifugation was performed at 3500 rpm for at least 30 minutes to obtain only the acceptor protein expression culture solution without cell pellet. The resulting culture solution was filtered through two filters (Satorius stedim, cat. no.: DH-ST-29MDL20MC5FFV) to remove impurities.

80 μl of the resulting culture solution was taken, 20 μl of dye was added, which reduced the sample loading by 5 times, and mixed, and the mixture was left to stand at 95 °C for 5 minutes. To compare expression levels on the gel, 80 μl of bovine serum albumin, which was diluted to 62.5, 125 and 250 mg/ml, and 20 μl of dye reducing the sample loading by a factor of 5 were added and mixed and the mixture was kept at 95°C within 5 minutes. The prepared sample and marker protein were loaded onto a 10% Tris-glycine gel for size verification. The gel was stained with Coomassie Brilliant Blue R with gentle shaking, and the acceptor protein concentration was relatively quantified based on the bovine serum albumin protein band.

Example 3

Acceptor protein purification

The culture solution of the acceptor protein expressed in Example 2 was applied to a Blue sepharose HP resin column (GE, cat. no: 17-0413-01) equilibrated with equilibrium buffer (20 mM sodium phosphate, pH 7.0). Impurities were removed by pre-elution (20 mM sodium phosphate, pH 7.0, 0.15 M KCl), and acceptor protein was isolated using elution buffer (20 mM sodium phosphate, pH 7.0, 0.6 M KCl). The extracted acceptor protein was dialyzed with 20 mM sodium phosphate buffer, pH 7.0, to remove salts and ultrafiltrated so that the final sample was 5 mg/mL. The results of SDS-PAGE purification of the acceptor protein are shown in FIG. 1.

Example 4

Expression of donor protein in E. coli cells

The donor gene sequence encoding the donor protein was transformed into the pET21a vector with a His-SUMO tag, and then the plasmid into which the donor gene was inserted was added to BL21(DE3) competent cells and transformed by heat shock treatment at 42°C for 1 minutes and plated on solid LB medium containing ampicillin and then stationary cultured at 37°C for at least 16 hours to obtain colonies. One colony was selected and inoculated into 50 ml of LB medium, and then the inoculum was cultured at 37°C and 220 rpm for 16 hours.

In the case of the donor protein (Donor-191, D -191), containing GIP as a biomolecule, the main cultivation was carried out by sowing the seed culture solution in 1 liter of LB medium containing ampicillin in a ratio of 1:100. Cultivation was carried out at 37°C and 220 rpm for 2 hours. Thereafter, when OD600 nm reached 0.6, 0.25 M IPTG was added and cultured at 16 °C and 220 rpm for 20 hours. After completion of culture, centrifugation was performed at 3500 rpm for 30 minutes to obtain wet E. coli cells.

In the case of the donor protein (Donor-192, D -192), containing IL-1RA as a biomolecule, the main cultivation was carried out by sowing the seed culture solution in 1 liter of LB medium containing ampicillin in a ratio of 1:100. Autoinduction was carried out at 37°C and 220 rpm for 24 hours. After completion of culture, centrifugation was performed at 3500 rpm for 30 minutes to obtain wet E. coli cells.

Example 5

Purification of donor protein in E. coli cells

The cultured donor protein was purified by the following method, and the purification process is shown in FIG. 2.

Lysis/sonication

Wet cells obtained by culture were resuspended using lysis buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole, 0.1 mM PMSF). Lysis samples were placed on ice and sonicated under on/off conditions of Pules = 5 sec/3 sec and amplitude 45% for 15 min. The lysate was obtained by centrifugation at 14,000 rpm for 30 minutes to obtain only the supernatant.

Grip cleaning

The lysate was loaded onto Ni-Sepharose resin (QIAGEN, cat. no.: 30250). After sample loading was completed, nonspecific protein was sufficiently washed and removed using binding buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole). The Hig-SUMO-labeled donor protein was then recovered using elution buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.25 M imidazole). The extracted His-SUMO-tagged donor protein was dialyzed with 20 mM sodium phosphate buffer, pH 7.0, to remove salts and imidazole. The results obtained by confirming the purification of the donor protein through a nickel column using SDS-PAGE are shown in FIG. 3 and 4.

Enzymatic cleavage of SENP1

The His-SUMO-tagged donor protein and SENP1 were digested with SENP1 enzyme at a ratio of 100 mg: 1 mg. The protein concentration recovered by Ni purification was quantified, and SNEP1 (intrinsic) corresponding to 1/100 w/w. amounts (mg) of His-SUMO-tagged donor protein were mixed. The reaction mixture was allowed to stand at room temperature (15 to 25 °C) for 1 hour.

Removing His-SUMO

The reaction mixture was applied to Ni-Sepharose resin (QIAGEN, catalog no.: 30250) equilibrated with equilibrium buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole). Sample loading was performed and the donor protein in which the His-SUMO tag had been cleaved was allowed to flow. After sample loading was completed, the remaining donor protein was extracted using equilibrium buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole), and the extracted donor protein was dialyzed with 20 mM sodium phosphate buffer with pH 7.0, to remove salts and imidazole. Ultrafiltration was carried out so that the final donor product was 10 mg/ml. The results obtained by confirming His-SUMO removal purification by SDS-PAGE are shown in FIG. 5 and 6.

Cleaning

The donor recovered in the previous step was loaded onto a Capto Q ImpRes column (GE, cat. no: 17-5470-15) equilibrated with an equilibrium buffer (20 mM sodium phosphate, pH 7.0 buffer). The donor protein was allowed to pass through. The extracted proteins were concentrated to a high concentration. The results of polishing purification of the donor protein are shown in FIG. 7 and 8.

Example 6

Conjugation

As shown in FIG. 9, conjugation was carried out using the acceptor protein obtained in Example 3 and the donor protein obtained in Example 5. A mixture of the conditions shown in Table 3 below was prepared, and the conjugation reaction was carried out at 30°C for 4 hours.

Table 3 Acceptor Donor ATP mUBA1 UBC13
/MMS2 Buffer Regenerating agent About. Condition 10 µM 15 µM 4 mM 1 µM 10 µM 50 mM Tris pH7.6,
2.5 mM MgCl2 0.05 mM TCEP 100 ml

10 μg of acceptor was loaded onto 8% SDS-PAGE and the degree of conjugation for the reaction was confirmed. The results are shown in Fig. 10 and 11.

Example 7

Enzyme Removal Using Ni-Sepharose

In order to recover only the conjugate sample, the reaction mixture was applied to Ni-Sepharose resin (QIAGEN, part number: 30250) equilibrated with equilibrium buffer (20 mM sodium phosphate, pH 8.0, 0.15 M NaCl). Once sample loading was complete, impurities were removed using equilibration buffer (20 mM sodium phosphate, pH 8.0, 0.5 M NaCl). The conjugate was then recovered using elution buffer (25 mM Tris, pH 8.0, 0.15 M NaCl, 0.01 M imidazole). The recovered protein conjugate (C-191 and C-192) was dialyzed with 20 mM sodium phosphate buffer, pH 7.0, to remove salts and imidazole. The results obtained by confirming the purification of Ni by chromatography are shown in FIG. 12.

Example 8

Cleaning

The conjugate obtained in the previous step was applied to a Capto Q ImpRes column (GE, cat. no: 17-5470-15) equilibrated with an equilibrium buffer (25 mM sodium phosphate, pH 7.0 buffer). The conjugate was recovered using elution buffer (25 mM sodium phosphate, pH 7.0, 158 mM NaCl buffer). The recovered protein conjugate was dialyzed with 20 mM sodium phosphate buffer, pH 7.0, to remove salts and imidazole. Ultrafiltration was carried out so that the final conjugate product was 5 mg/ml. The results are shown in Fig. 13 and 14.

Example 9

Compilation

The protein conjugate extracted from AEX was dialyzed with formulation buffer (4.6 mM histidine, 5.7 mM Tris, pH 7.5, 10 mM arginine, 0.1 g/mL trehalose) to remove salts and imidazole. Ultrafiltration was carried out so that the final conjugate product was 5 mg/ml. The results are shown in Fig. 15.

Test case

cAMP accumulation assay

To test the protein conjugates C-191 and C-192, which are tetra-acceptor agonists/antagonists of GCG/GLP-1/FGF21/GIP or GCG/GLP-1/FGF21/IL-1RA, prepared in the Examples 1 - 9, for the level of activity of GLP-1, GCG and GIP agonists at the cellular level (in vitro), a cAMP accumulation assay was performed in a cell line in which GLP-1 acceptor, GIP acceptor and GCG acceptor were overexpressed transiently or stably, respectively. , using the Cisbio cAMP Gs Dynamic #62AM4PEC kit as follows.

Receiving cells (temporary)

HEK293 cells were incubated for 2-3 days in an incubator at 37 °C and 5% CO ₂ so that the concentration of HEK293 cells in the T-75 flask was from 70 to 80%. The medium was removed and treated with 2 ml TryPLE Express, and then incubated in an incubator at 37°C and 5% CO ₂ for 3-5 minutes and the cells were detached. They were diluted by adding 6 ml of culture medium (MEM, 10% FBS, 1% Anti-anti) and transferred to a 15 ml tube and then centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded and poured into 5 ml of medium. The number of cells was counted and the concentration was adjusted to 3×10 ⁵ cells/ml. 2 ml was placed in a 6-well plate and incubated in an incubator at 37°C and 5% CO ₂ for 24 hours. The culture medium was removed and 1.7 ml of medium without antibiotic was added. FuGENE6 and each acceptor plasmid were added to Opti-MEM in the appropriate amount and cultured at room temperature for 5 min, respectively. Each mixture was mixed and cultured at room temperature for 15 minutes. The mixed solution was added to the appropriate wells and incubated in an incubator at 37°C and 5% CO ₂ for 24 hours. The medium was removed, and the cells were washed with 2 ml of prewarmed PBS. 0.5 ml of accutase was added per well and incubated in an incubator at 37°C and 5% CO ₂ for 5 minutes, after which the cells were separated. At this time, it was checked under a microscope whether the cells were completely detached, and the plate was struck to prevent the cells from detaching. Add 2.5 ml of prewarmed 37°C assay buffer (0.5% BSA, 2 mM IBMX in PBS) per well, transfer to a 15 ml tube and centrifuge at 1000 rpm for 3 minutes, and then released again into 2 ml of assay buffer. The number of cells was counted and the concentration was adjusted to 400,000 cells/ml in assay buffer.

Receiving cells (stable)

Cells in which GLP-1 acceptor (Genscript, cat. no. M00451), GIP acceptor (Genscript, cat. no. M00486), and GCG acceptor (Genscript, cat. no. M00345) were overexpressed were cultured such that the concentration of cells in the flask T-75 ranged from 70 to 80%. The medium was removed, and the cells were washed with 2 ml of PBS preheated to 37°C. 1 ml of accutase was added and incubated in an incubator at 37°C and 5% CO ₂ for 5 minutes, after which the cells were separated. At this time, it was checked under a microscope whether the cells were completely detached, and the plate was struck to prevent the cells from detaching. Add 3 ml of prewarmed 37°C assay buffer per well, transfer to a 15 ml tube and centrifuge at 1000 rpm for 3 minutes, then release back into 2 ml of assay buffer. The number of cells was counted and the concentration was adjusted to 400,000 cells/ml in assay buffer.

Procedure

5 μl of prepared cells were distributed into a small volume 96-well white plate. 5 µl each of standard and sample (2X), prepared by 4-fold serial dilution, were dispensed in duplicate and incubated in a 5% CO ₂ incubator for 30 minutes. At this time, 5 μl of assay buffer was added to the control and empty wells. 5 μl of 1 x cAMP-d2 solution was added. At this time, 5 μl of lysis and detection buffer was added to the empty well. 5 μL of 1X anti-cAMP cryptate solution was added and cultured for 1 hour at room temperature in a light-blocked state, and then fluorescence was measured using a plate reader.

Measurement and Analysis

Fluorescence of the sample in the plate was measured using a Synergy Neo2 instrument (excitation wavelength: 330 nm, emission wavelength: 665 nm and 620 nm).

The HTRF ratio was calculated as follows :

In addition, the delta ratio (ΔR) was calculated as follows.

ΔR = Ratio_Sample– Attitude_background = Signal - background fluorescence

EC50 values were obtained as HTRF ratio plot values using GraphPad Prism 8 (log(agonist) versus normalized response curve fitting - variable slope equation).

HTRF ratio plots for GLP-1R (GLP-1 acceptor), GIPR (GIP acceptor), and GCGR (GCG acceptor) are shown in FIG. 16-18 and EC50 values were calculated and shown in Table 4 below.

Table 4 EC50
(pM) GLP-1R GIPR GCGR 1 2 Average SD 1 2 Average SD 1 2 Average SD Liraglutide 40.7 36.7 38.7 2.83 - - - - - - - - Dulaglutide 1.23 0.9 1.07 0.23 - - - - - - - - GIP(1-39) - - - - 15.4 14.1 14.8 0.92 - - - - GCG - - - - - - - - 39.3 30.4 34.9 6.29 C-191 25.4 36.5 31.0 7.85 110 85 97.5 17.7 4487 3832 4160 463 C-192 13.2 15.9 14.6 1.91 - - - - 286 251 269 24.7

As shown in Table 4, C-191 was confirmed to have activity on each of the GLP-1 acceptor, GIP acceptor, and GCG acceptor. In addition, it was confirmed that C-192 also had activity against each of the GLP-1 acceptor and the GCG acceptor.

In particular, it was confirmed that, in terms of GLP-1 acceptor activity, C-191 and C-192 exhibited higher activity than liraglutide used as a control group. In addition, it was confirmed that C-192 had approximately 2-fold higher activity than C-191 in terms of GLP-1 acceptor activity, and had approximately 15-fold higher activity than C-191 , in terms of activity towards the GCG acceptor

Test case 2

Functional analysis of FGFR1/KLB

To test the protein conjugates C-191 and C-192, which are tetra-acceptor agonists/antagonists of GCG/GLP-1/FGF21/GIP or GCG/GLP-1/FGF21/IL-1RA, prepared in the Examples 1 - 9, For the level of FGF21 agonist activity at the cellular level (in vitro), functional analysis of FGFR1/KLB was performed on a cell line (Discover X, cat. no. 93-118C3), in which FGFR1/KLB was overexpressed, using a detection kit PathHunter (Discover X, part number 93-0001) as follows.

Cell seeding

Cells were cultured to 70-80% filling in a T-75 flask. The medium was removed and washed with 5 ml of prewarmed PBS. The PBS was removed and 2 ml of AssayComplete cell detachment reagent was added and then incubated in an incubator at 37°C and 5% CO ₂ for 3 minutes, after which the attached cells were detached. They were mixed with 6 ml of AssayComplete 0 cell culture reagent, the mixture was transferred to a 15 ml tube, centrifuged at 1000 rpm for 3 minutes and then released again into 5 ml of cell culture reagent. The number of cells was counted and the concentration was adjusted to 500,000 cells/ml. They were transferred to a reservoir and 40 μl per well were added to a white 96-well half-area cell culture plate using a multichannel pipette (20,000 cells/well). Only 40 μl of cell culture reagent was added to the empty well. Cells were incubated in an incubator at 37°C and 5% CO ₂ for 24 hours for attachment.

Procedure

10 μl each of rhFGF21 (5X) and test sample (5X), prepared by 4-fold serial dilution, were distributed in duplicate and cultured at room temperature for 4 hours. 10 µl of AssayComplete 0 cell culture reagent was added to the control and blank wells. After completion of culture, the medium was removed and 50 μl of AssayComplete 0 culture reagent was added. 30 μl of detection reagent was added and the reaction was carried out in a light-blocked state at room temperature for 1 hour, and then luminescence was measured using a plate reader.

Measurement and Analysis

The luminescence of the sample in the plate was measured on a Synergy Neo2 device.

Delta RLU (relative luminescence unit) (ΔRLU) was calculated as follows.

Δ RLU = RLU_sample - R.L.U._background = signal - background luminescence

In addition, the fold induction compared to the control group was calculated as follows.

EC50 values were derived from fold induction values for each concentration using GraphPad Prism 8 as follows (log (agonist) versus normalized response curve fitting - variable slope equation).

The graphs are shown in Fig. 19 and EC50 values were calculated and shown in Table 5 below.

Table 5 EC50 (nM) rhFGF21 C-191 C-192 1 0.75 92.2 42.2 2 0.88 45.1 40.5 Average 0.82 68.7 41.4 SD 0.09 33.3 1.20

As shown in Table 5 above, both C-191 and C-192 were confirmed to have FGFR1/KLB acceptor activity. In addition, in terms of FGFR1/KLB acceptor activity, C-192 was confirmed to have approximately 40% higher activity than C-191.

Test case 3

NF-κ B reporter gene luciferase assay (reporter assay)

To test the protein conjugate C-192, which is a four-directional (tetra) GCG/GLP-1/FGF21/IL-1RA acceptor agonist/antagonist prepared in Examples 1-9, for the ability of IL-1RA to inhibit NF-κB activity using IL-1β at the cellular level (in vitro), luciferase assay was performed in the NF-κB reporter cell line (Luc) (BPS Bioscience, cat. no. 60650) as follows.

Cell seeding

Cells were cultured to 70-80% filling in a T-75 flask. The medium was removed and washed once with 5 ml of prewarmed PBS. PBS was removed, 1 ml TryPLE Express was added and incubated for 5 minutes in an incubator at 37°C and 5% CO ₂ , after which the cells were separated. They were mixed with 4 ml of assay medium (10% FBS, 1% antibiotic in DMEM) and the mixture was transferred to a 15 ml tube, centrifuged at 1000 rpm for 3 minutes, and then added back to 5 ml of assay medium. The number of cells was counted and the concentration was adjusted to 500,000 cells/ml. They were transferred to a reservoir and 40 μl per well were added to a white 96-well half-area cell culture plate using a multichannel pipette (20,000 cells/well). Only 40 μl of assay medium was added to an empty well. Cells were incubated in an incubator at 37°C and 5% CO ₂ for 24 hours for attachment.

Procedure

5 μl of each of the standard (10X) and sample (10X), prepared by 4-fold serial dilution, was distributed in duplicate. After this, 5 μl of 50 pM IL-1β (10X) was added and incubated in an incubator at 5% CO ₂ for 4 hours. 50 μl of ONE-Glo reagent (Promega, cat. no. E6120) was dispensed and cultured at room temperature for 5 minutes before luminescence was measured using a Synergy Neo2 instrument. At this time, the plate was removed from the incubator for 15 minutes and allowed to reach room temperature before treatment with ONE-Glo reagent.

Delta RLU (relative luminescence unit) (ΔRLU) was calculated as follows.

ΔRLU = _sample RLU - _background RLU = signal - background luminescence

IC50 values were obtained as ΔRLU for each concentration using GraphPad Prism 8 as follows (log(antagonist) versus normalized response curve fitting - variable slope equation).

The graphs are shown in Fig. 20 and IC50 values were calculated and shown in Table 6 below.

Table 6

IC50 (pM) rhIL-1RA C-192 1 39.9 561 2 51.5 594 3 46.6 708 4 51.9 568 Average 47.5 607 SD 4.85 68.4

As shown in Table 6 above, C-192 was confirmed to inhibit NF-κB activity by IL-1β.

Test case 4

Efficiency test in vivo: evaluation of effectiveness against non-alcoholic steatohepatitis

The in vivo efficacy of the GCG/GLP-1/FGF21/IL-1RA four-directional (tetra) receptor agonist/antagonist protein conjugate C-192 prepared in Examples 1-9 against non-alcoholic steatohepatitis was confirmed in C57BL mice /6J, in which fatty liver was caused by feeding MCD (methionine and choline deficient L-amino acid diet). The MCD mouse model is the most commonly used method for testing efficacy against nonalcoholic steatohepatitis, and the histological appearance of induced liver tissue from weeks 2 to 4 is known to be histologically similar to that of human nonalcoholic steatohepatitis. There are many precedents where a number of pharmaceutical companies and academia have conducted trials with various substances.

Fatty liver was induced by feeding MCD to male C57BL/6J mice for 8 weeks, and then the mice were divided into 6 groups shown in Table 7 below and administered the drug for 4 weeks. Two currently commercially available drugs were used as comparators: liraglutide (Saxenda) and dulaglutide (Trulycity). The administered composition was reintroduced subcutaneously using a disposable syringe in accordance with each administration cycle. During the 8-week diet period, body weight was measured once a week, and after 8 weeks, significant stratification into groups was performed based on body weight. The normal control group received MCS (L-amino acid diet with sufficient methionine and choline).

Table 7 No. Group Diet Input composition Administration dose Administration cycle Number of injections 1 Normal control group MCS - - - - 2 Negative control group MCD excipient - - - 3 Experimental group A MCD C-192 5 nmol/kg 1 time/2 days 14 times 4 Experimental group B MCD C-192 40 nmol/kg 1 time/2 days 14 times 5 Comparison group A MCD Liraglutide 50 nmol/kg 2 times/1 day 56 times 6 Comparative group B MCD Dulaglutide 2 nmol/kg 1 time/2 days 14 times

During the 4-week administration period, general symptoms and behavior were observed, and body weight was measured once a week after the start of administration and on the day of tissue extraction. Blood sampling and liver tissue isolation were carried out on the day of completion of observation, the blood was subjected to hematological and biochemical examination, and the extracted liver tissue was subjected to histopathological examination.

Alanine aminotransferase (ALT), which is used as a major indicator of liver disease, was measured using a hematology-biochemical test and the results are shown in Table 8 below and FIG. 21.

Table 8 No. Group ALT 1 Normal control group 18.3 2 Negative control group 230.6 3 Experimental group A 178.3 4 Experimental group B 126.3 5 Comparative group A (liraglutide administration group) 223.0 6 Comparative group B (Dulaglutide administration group) 220.5

As shown in Table 8 above, it was found that the ALT value was not significantly reduced in the liraglutide group and the dulaglutide group (comparative groups A and B), but the ALT value was significantly reduced in the C-192 group.

Since ALT indicates the degree of liver damage, it can be seen that when the protein conjugate of the present invention is administered, the degree of liver damage is reduced.

In addition, the ALT/AST values are shown in Table 9 below and FIG. 22.

Table 9 No. Group ALT/AST value 1 Normal control group 1.77 2 Negative control group 0.98 3 Experimental group A 1.35 4 Experimental group B 1.53 5 Comparative group A (liraglutide administration group) 0.99 6 Comparative group B (Dulaglutide administration group) 0.79

As shown in Table 9 above, it was confirmed that the ALT/AST value was less than 1.0 in the liraglutide group and the dulaglutide group (comparative groups A and B), but it was found that the ALT/AST value was above 1.0 in the group receiving C-192.

Since the ALT/AST value is often less than 1 in liver disease, it can be seen that when the protein conjugate of the present invention is administered, the degree of liver damage is reduced.

In addition, histopathological test was performed by observation through H&E and Masson's trichrome staining, and the results are shown in FIG. 23. In nonalcoholic steatohepatitis, steatosis and inflammation occurs in the lobules, and inflammatory cells can be identified by tissue staining. As shown in FIG. 23, it can be seen that the group that was administered the protein conjugate at a dose of 40 nmol/kg (experimental group B) showed improvement in fat and inflammatory cells to the values of the normal control group, compared with the negative control group.

In addition, based on the evaluation criteria shown in Table 10 below, the steatosis score, lobular inflammation score and NAS (NAFLD activity score) were evaluated.

Table 10 An object Definition Grade Steatosis Assessment of parenchymal damage from low to moderate steatosis <5% 0 5~33% 1 >33~66% 2 >66% 3 Lobular inflammation General assessment of all foci of inflammation No outbreaks 0 <2 lesions per 200 µm field 1 2~4 foci per 200 µm field 2 >4 foci per 200 µm field 3 Ballooning No 0 Several balloon cages 1 Ballooning multiple cells/bulges 2

The evaluation results are shown in Tables 11 - 13 below and in FIG. 24 - 26.

Table 11 No. Group Assessment of steatosis (steatosis) 1 Normal control group 0 2 Negative control group 2.1 3 Experimental group A 1.7 4 Experimental group B 0.9 5 Comparative group A (liraglutide administration group) 1.6 6 Comparative group B (Dulaglutide administration group) 1.9

Table 12 No. Group Evaluation of lobular inflammation
(Lobular inflammation) 1 Normal control group 0.3 2 Negative control group 1.3 3 Experimental group A 1.2 4 Experimental group B 0.2 5 Comparative group A (liraglutide administration group) 0.5 6 Comparative group B (Dulaglutide administration group) 1.5

Table 13 No. Group NAS (NAFLD Activity Score) 1 Normal control group 0.3 2 Negative control group 3.4 3 Experimental group A 2.9 4 Experimental group B 1.2 5 Comparative group A (liraglutide administration group) 2.2 6 Comparative group B (Dulaglutide administration group) 3.4

As shown in Tables 11-13 above, all groups administered the protein conjugate of the present invention showed excellent results, and in particular, the group administered the protein conjugate at a dose of 40 nmol/kg (experimental group B) showed significantly more different evaluation results than the liraglutide-administered group and the dulaglutide-administered group.

Liver tissue was analyzed by ELISA for TGF-β, a marker of liver fibrosis, and the results are shown in Table 14 below and FIG. 27.

Table 14 No. Group Hepatic TGF-β 1 Normal control group 74.40 2 Negative control group 164.10 3 Experimental group A 135.50 4 Experimental group B 98.70 5 Comparative group A (liraglutide administration group) 136.40 6 Comparative group B (Dulaglutide administration group) 159.60

As shown in Table 14 above, the group that was administered the protein conjugate according to the present invention showed a reduced TGF-β value, and in particular, the group that was administered the protein conjugate at a dose of 40 nmol/kg (experimental group B) showed almost the same value compared to the normal control group.

In addition, triglyceride accumulation in liver tissue was analyzed using a triglyceride assay kit, and the results are shown in Table 15 below and FIG. 28. The amount of triglycerides in liver tissue was almost not reduced in the liraglutide-treated group and the dulaglutide-treated group, but the group administered with the protein conjugate of the present invention showed a value almost the same as that of the normal control group.

Table 15 No. Group Hepatic triglyceride 1 Normal control group 17.76 2 Negative control group 40.31 3 Experimental group A 25.69 4 Experimental group B 24.43 5 Comparative group A (liraglutide administration group) 40.69 6 Comparative group B (Dulaglutide administration group) 40.78

As a result, it was found that the group administered the C-192 protein conjugate of the present invention showed a significantly superior effect in all experiments compared with the liraglutide administered group and the dulaglutide administered group.

Test Case 5

Efficiency test in vivo: evaluation of effectiveness against non-alcoholic steatohepatitis

The in vivo efficacy of the GCG/GLP-1/FGF21/GIP tetra-acceptor agonist/antagonist C-191 protein conjugate prepared in Examples 1-9 against nonalcoholic steatohepatitis was tested in C57BL/6J mice , in whom fatty liver disease was induced by feeding an MCD (an L-amino acid diet with sufficient methionine and choline).

The mice were divided into 4 groups as shown in Table 16 below, and the experiment was carried out in the same way as Test Example 4 above. Currently commercially available dulaglutide (Trulycity) was used as a reference substance.

Table 16 No. Group Diet Input composition Administration dose Administration cycle Number of injections 1 Normal control group MCS - - - - 2 Negative control group MCD Excipient - - - 3 Experimental group A MCD C-191 10 nmol/kg 1 time/2 days 14 times 4 Comparative group B MCD Dulaglutide 2 nmol/kg 1 time/2 days 14 times

During the 4-week administration period, general symptoms and behavior were observed, and body weight was measured once a week after the start of administration and on the day of tissue extraction. Blood sampling and liver tissue isolation were carried out on the day of completion of observation, the blood was subjected to hematological and biochemical examination, and the extracted liver tissue was subjected to histopathological examination.

Alanine aminotransferase (ALT), which is used as a major indicator of liver disease, was measured using a hematology-biochemical test and the results are shown in Table 17 below and FIG. 29.

Table 17 No. Group ALT 1 Normal control group 19.1 2 Negative control group 312.4 3 Experimental group A 151.9 4 Comparative group A (Dulaglutide administration group) 246.8

As shown in Table 17 above, it was confirmed that the ALT value was almost not reduced in the dulaglutide administered group, but the ALT value was significantly reduced in the C-191 administered group.

Since ALT indicates the degree of liver damage, it can be seen that when the protein conjugate of the present invention is administered, the degree of liver damage is reduced.

In addition, the AST/ALT values are shown in Table 18 below and FIG. thirty.

Table 18 No. Group AST/ALT value 1 Normal control group 1.84 2 Negative control group 0.82 3 Experimental group A 0.98 4 Comparative group A (Dulaglutide administration group) 0.76

As shown in Table 18 above, the ALT/AST value was confirmed to be 0.76 in the dulaglutide group, but the ALT/AST value was confirmed to be 0.98 in the C-191 group, which is higher than in the group receiving dulaglutide.

Since the ALT/AST value is often less than 1 in liver disease, it can be seen that when the protein conjugate of the present invention is administered, the degree of liver damage is reduced.

In addition, histopathological test was performed by observation through H&E and Masson's trichrome staining, and the results are shown in FIG. 31. In nonalcoholic steatohepatitis, steatosis and inflammation occurs in the lobules, and inflammatory cells can be identified by tissue staining. As shown in FIG. 31, it can be seen that the group administered the protein conjugate at a dose of 10 nmol/kg showed improvement in fat and inflammatory cells to the values of the normal control group, compared to the negative control group.

In addition, based on the evaluation criteria shown in Table 10 above, the steatosis score, lobular inflammation score and NAS (NAFLD activity score) were evaluated.

The evaluation results are shown in Tables 19 - 21 below and in FIG. 32 - 34.

Table 19 No. Group Assessment of steatosis (steatosis) 1 Normal control group 0 2 Negative control group 2.4 3 Experimental group A 1.4 4 Comparative group A (Dulaglutide administration group) 1.8

Table 20 No. Group Evaluation of lobular inflammation
(Lobular inflammation) 1 Normal control group 0.2 2 Negative control group 1.6 3 Experimental group A 1.0 4 Comparative group A (Dulaglutide administration group) 1.6

Table 21 No. Group NAS (NAFLD Activity Score) 1 normal control group 0.2 2 negative control group 4.0 3 Experimental group A 2.4 6 Comparative group A (Dulaglutide administration group) 3.4

As shown in Tables 19-21 above, all groups administered the protein conjugate of the present invention showed excellent results and showed significantly better evaluation results than the group administered dulaglutide.

Liver tissue was analyzed by ELISA for TGF-β, a marker of liver fibrosis, and the results are shown in Table 22 below and FIG. 35.

Table 22 No. Group Hepatic TGF-β 1 Normal control group 77.3 2 Negative control group 177.9 3 Experimental group A 117.4 4 Comparative group A (Dulaglutide administration group) 165.3

As shown in Table 22 above, it was confirmed that the group administered with the protein conjugate of the present invention exhibited a reduced TGF-β value, and this value was significantly reduced compared with the group administered dulaglutide.

In addition, triglyceride accumulation in liver tissue was analyzed using a triglyceride assay kit, and the results are shown in Table 23 below and FIG. 36. The amount of triglycerides in the liver tissue was practically not reduced in the dulaglutide-treated group, but the group administered with the protein conjugate of the present invention showed a value almost similar to the normal control group.

Table 23 No. Group Hepatic triglyceride 1 Normal control group 17.13 2 Negative control group 40.09 3 Experimental group A 24.02 6 Comparative group A (Dulaglutide administration group) 36.08

As a result, it can be seen that the group administered the C-191 protein conjugate of the present invention showed a significantly superior effect in all experiments compared with the group administered dulaglutide.

Test Case 6

Efficiency test in vivo: evaluation of effectiveness against obesity

To confirm the effect of the GCG/GLP-1/FGF21/IL-1RA tetra-acceptor agonist/antagonist protein conjugate C-192 prepared in Examples 1-9 on reducing blood lipids , induced obesity mice were used for evaluation. To induce obesity by dietary method, 5-week-old C57BL/6J mice were purchased from Central Lab. Animal Inc., and after an acclimation period of about 7 days was completed, were fed a Western diet for 16 weeks to induce obesity in the mice. As shown in Table 24 below, animals without abnormalities were selected based on the average body weight and change in body weight, and the groups were divided into 6 animals per group so that the average body weight in each group was the same. Regarding the route of administration, subcutaneous administration was carried out in the same way as the planned clinical route of administration, and the frequency of administration was 1 time/2 days for 8 weeks, i.e. only 28 times.

Table 24 No. Group Average body weight (g) Diet Input composition Administration dose Administration cycle Number of injections 1 Normal control group 27.1 ± 1.3 Solid feed for experimental animals excipient - 1 time/2 days 28 times 2 Negative control group 44.7 ± 2.3 RD western diet excipient - 1 time/2 days 28 times 3 Experimental group A 43.9 ± 1.1 RD western diet C-192 10 nmol/kg 1 time/2 days 28 times 4 Experimental group B 42.1 ± 4.1 RD western diet C-192 30 nmol/kg 1 time/2 days 28 times 5 Experimental group C 41.7 ± 4.4 RD western diet C-192 60 nmol/kg 1 time/2 days 28 times

For the administered composition, the excipient was administered to the normal control group and the negative control group, and C-192 was administered at a dose of 10, 30 and 60 nmol/kg to the experimental group. During the 8-week administration period, general symptoms such as appearance, behavior and excretion were observed once daily. On the day of completion of administration, blood was collected from the abdominal vena cava, placed in an SST tube and centrifuged at 3000 rpm for 15 minutes to separate the serum, and then a hematology-biochemical analyzer (7180, HTTACHI, Japan) was used to measure the biomarkers shown in Table 25 below.

Table 25 Lipid biomarker Units Measuring method Triglyceride
(TG) mg/dl GPO-HMMPS glycerol blanking Free fatty acid
(non-esterified fatty acid, NEFA) µeq/l ACS ACOD Total cholesterol
(T-Chol) mg/dl Cholesterol oxidase -HMMPS Low-density lipoprotein cholesterol
(LDL-cholesterol) mg/dl Selective protective enzymatic High-density lipoprotein cholesterol
(HDL-cholesterol) mg/dl Straight

To evaluate the effect of C-192 on lipid improvement, serum triglycerides (TG), free fatty acids (NEFA), total cholesterol (T-Chol), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HD) were measured, and the results are shown in Table 26 below and FIG. 37-41.

Table 26 No. group TG
(mg/dl) NEFA
(µequiv./l) T-Chol
(mg/dl) LDL
(mg/dl) HDL
(mg/dl) 1 Normal control group 14.7 1.2 105.3 2.9 62.8 2 Negative control group 26.0 1.5 241.8 21.7 99.2 3 Experimental group A 24.2 1.3 227.0 19.9 102.8 4 Experimental group B 18.0 1.1 162.0 13.6 74.7 5 Experimental group C 16.0 1.2 167.2 12.7 79.8

As shown in Table 26 above and FIG. 37–41, it was confirmed that blood levels of lipid-related biomarkers were reduced in the C-192-treated group. Specifically, it was confirmed that in the case of the group receiving C-192 at a dose of 60 nmol/kg (experimental group C), triglycerides (TG) decreased by 38.5%, free fatty acids (NEFA) decreased by 20%, total cholesterol (T-Chol) was reduced by 30.9%, low-density lipoprotein cholesterol (LDL-Chol) was reduced by 41.5%, and high-density lipoprotein cholesterol (HDL-Chol) was reduced by 19.6% compared with the negative control group. In addition, there was a statistically significant difference in all biomarkers except high-density lipoprotein cholesterol compared to the negative control group. Therefore, it can be seen that the protein conjugate of the present invention reduces blood lipid levels in diet-induced obese mice.

Test Case 7

Efficiency test in vivo: diabetes effectiveness assessment

To confirm the antidiabetic efficacy of the C-192 protein conjugate, which is a four-directional (tetra) GCG/GLP-1/FGF21/IL-1RA acceptor agonist/antagonist prepared in Examples 1 to 9, db/db mice, which are the model type 2 diabetic mice were used for the evaluation. db/db mice are an animal model in which obesity and type 2 diabetes are induced because db/db mice have a mutation in Lepr, a leptin acceptor gene on chromosome 4, resulting in a lack of signaling of leptin, a hormone secreted by adipocytes . C57BL/6J mice were used as normal animals, and 7-week-old male mice were purchased from Japan SLC and quarantined and acclimated for 7 days to obtain 8-week-old mice, which were used as experimental animals.

As shown in Table 27 below, the composition of the experimental group was divided into a normal control group, a negative control group, a test substance administered group, and a positive control group. After completion of quarantine and acclimatization, groups of 5 animals per group were divided so that body weight and blood glucose levels were equal. Regarding the route of administration, subcutaneous administration was carried out in the same way as the planned clinical application route, and the administration was carried out for 8 days. The administration frequency was 1 time/1 day for the normal control group, negative control group and experimental group and 1 time/2 days for the positive control group.

Table 27 No. Origin and species Group Input composition Administration dose Administration cycle Number of injections 1 C57BL/6J mouse Normal control group excipient - 1 time/1 day 8 times 2 db/db mouse Negative control group excipient - 1 time/1 day 8 times 3 Experimental group A C-192 10 nmol/kg 1 time/1 day 8 times 4 Experimental group B C-192 60 nmol/kg 1 time/1 day 8 times 5 Positive control group Semaglutide 10 nmol/kg 1 time/2 days 4 times

For the administered composition, the excipient was administered to the normal control group and the negative control group, and C-192 was administered at a dose of 10 and 60 nmol/kg to the experimental group. During the 8-day administration period, general symptoms were observed once daily. To confirm the antidiabetic effect, blood glucose levels, body weight, food, and water were measured in db/db mice. Regarding the blood glucose level, non-fasting blood glucose level was measured, and blood was collected from the tail vein 1 time/2 days before administration, and the blood glucose level was measured using a glucometer (G-Doctor). To confirm changes in body weight, food and water, measurements were taken on the 1st day of administration, the 4th day of administration and the 8th day of administration, and the results are shown in Table 28 below and FIG. 42-49.

Table 28 No. Group Glucose
(mg/dl) Weight
(gram) Food
(gram) Water
(ml) 1 Normal control group 161.0 23.6 15.4 33.1 2 Negative control group 561.9 33.2 26.6 97.2 3 Experimental group A 409.6 33.7 20.4 61.5 4 Experimental group B 216.8 30.6 16.7 48.6 5 Positive control group 444.5 32.3 21.9 41.3

As shown in Table 28 above and FIG. 42–49, it was confirmed that blood glucose levels, food intake and water intake were reduced in the C-192 administered group. In particular, in the case of the group receiving C-192 at a dose of 60 nmol/kg (experimental group B), it was confirmed that blood glucose levels on the 9th day were significantly reduced by 61.4%, body weight on the 8th day day was significantly reduced by 7.8%, food intake was significantly reduced by 37.1%, and water intake was significantly reduced by 50.0% compared to the negative control group. Thus, it can be seen that the protein conjugate of the present invention exhibits an antidiabetic effect.

Test Case 8

Immunogenicity assay in silico

In silico immunogenicity analysis was performed for each acceptor and donor included in the C-191 and C-192 protein conjugates, which are quaternary agonist/antagonist (tetra) acceptor GCG/GLP-1/FGF21/GIP or GCG/GLP -1/FGF21/IL-1RA obtained in examples 1-9. Immunogenicity refers to the property that triggers an immune response when a drug is introduced into the human body, and is a factor influencing not only the effectiveness of the drug but also its safety. In silico analysis was performed using an immunogenicity-inducing sequence database and a computer analysis program, and in vitro immunogenicity assessment was performed using human-derived PBMCs.

As a result, as shown in FIG. 50-55, the level of induction of immunogenicity was predicted for the 27 HLA types shown in Table 29 below, which are the most common worldwide.

Table 29 Sequence no. HLA type Sequence no. HLA type 1 HLA-DRB1*01:01 15 HLA-DRB5*01:01 2 HLA-DRB1*03:01 16 HLA-DQA1*05:01/DQB1*02:01 3 HLA-DRB1*04:01 17 HLA-DQA1*05:01/DQB1*03:01 4 HLA-DRB1*04:05 18 HLA-DQA1*03:01/DQB1*03:02 5 HLA-DRB1*07:01 19 HLA-DQA1*04:01/DQB1*04:02 6 HLA-DRB1*08:02 20 HLA-DQA1*01:01/DQB1*05:01 7 HLA-DRB1*09:01 21 HLA-DQA1*01:02/DQB1*06:02 8 HLA-DRB1*11:01 22 HLA-DPA1*02:01/DPB1*01:01 9 HLA-DRB1*12:01 23 HLA-DPA1*01:03/DPB1*02:01 10 HLA-DRB1*13:02 24 HLA-DPA1*01/DPB1*04:01 eleven HLA-DRB1*15:01 25 HLA-DPA1*03:01/DPB1*04:02 12 HLA-DRB3*01:01 26 HLA-DPA1*02:01/DPB1*05:01 13 HLA-DRB3*02:02 27 HLA-DPA1*02:01/DPB1*14:01 14 HLA-DRB4*01:01

Each acceptor and donor protein sequence was predicted to have a low probability of binding to the MHC I and MHC II antigen-binding sites for each of the 27 HLA types. Safety was predicted to be high because each acceptor and donor had low immunogenicity.

Test case 9

API Screening

Screening for GLP-1 acceptor agonists

To test the activity levels of native GLP-1 and GLP-1 analogue at the cellular level (in vitro), a cAMP accumulation assay was performed in the same manner as Test Example 1.

EC50 values for single GLP-1 acceptor agonists were calculated and shown in Table 30 below.

Table 30 An object Cell line EC50 (pM) a comment SEQ ID NO Liraglutide Temporary 43.0 ± 10.6 n=11 - Stable 50.3 ± 21.9 n=8 Dulaglutide Stable 1.45 ± 0.45 n=6 - GLP-1(7-36)_native Temporary 4.00 n=1 SEQ ID NO: 4 GLP#1 Temporary 159 n=1 SEQ ID NO: 5 GLP#3 Temporary 2.25 ± 0.19 n=2 SEQ ID NO: 6 GLP#4 Temporary 3.19 ± 0.74 n=2 SEQ ID NO: 7 GLP#5 Temporary 7.52 n=1 SEQ ID NO: 8 GLP#6 Temporary 1.50 ± 0.06 n=2 SEQ ID NO: 9 GLP#7 Temporary 23.0 n=1 SEQ ID NO: 10 GLP#8 Temporary 1.95 ± 0.03 n=2 SEQ ID NO: 11 GLP#9 Temporary 0.71 ± 0.37 n=3 SEQ ID NO: 12 Stable 2.08 ± 1.60 n=4 GLP#10 Temporary 8.26 ± 4.02 n=2 SEQ ID NO: 13 GLP#11 Temporary 6.05 n=1 SEQ ID NO: 14 GLP#12 Temporary 559 n=1 - GLP#13 Temporary 31.9 ± 7.28 n=2 - GLP#14 Temporary >5000 n=1 - GLP#15 Temporary >5000 n=1 -

EC50 values for dual GLP-1/GCG acceptor agonists were calculated and shown in Table 31 below.

Table 31 An object Cell line EC50 (pM) a comment SEQ ID NO GCG#2 Temporary 52.0 ± 27.4 n=2 SEQ ID NO: 2 Stable 10.1 ± 1.23 n=2 GCG#3 Temporary 43.9 ± 28.3 n=2 SEQ ID NO: 3 Stable 25.6 ± 26.9 n=3 GLP/GCG#1 Temporary 588 n=1 - GLP/GCG#2 Temporary 201 n=1 - GLP/GCG#3 Temporary 13 n=1 SEQ ID NO: 15 GLP/GCG#4 Temporary 15.0 ± 7.00 n=2 - GLP/GCG#5 Temporary 165 n=1 - GLP/GCG#6 Temporary 129 n=1 - GLP/GCG#7 Temporary 3860 n=1 - GLP/GCG#8 Temporary 12.5 n=1 - GLP/GCG#9 Temporary 1589 n=1 - GLP/GCG#10 Temporary 9742 n=1 - GLP/GCG#11 Temporary >5000 n=1 - GLP/GCG#12 Temporary 80.1 n=1 - GLP/GCG#13 Temporary 604 n=1 - GLP/GCG#14 Temporary 221 n=1 - GLP/GCG#15 Temporary 16 n=1 SEQ ID NO: 16

EC50 values for dual GLP-1/GIP acceptor agonists were calculated and shown in Table 32 below.

Table 32 An object Cell line EC50 (pM) a comment SEQ ID NO GLP/GIP#2 Temporary 57.8 n=1 - GLP/GIP#3 Temporary >5000 n=1 - GLP/GIP#4 Temporary >5000 n=1 -

Screening for GIP acceptor agonists

To test the activity levels of native GIP and GIP analogue at the cellular level (in vitro), a cAMP accumulation assay was performed in the same manner as Test Example 1.

EC50 values for single GIP acceptor agonists and dual GLP-1/GIP acceptor agonists were calculated and are shown in Table 33 below.

Table 33 An object Cell line EC50 (pM) A comment SEQ ID NO GIP native-Ub Temporary 8.93 ± 5.77 n=4 SEQ ID NO: 22 Stable 35.0 n=1 GIP#1 Temporary 17.5 ± 10.6 n=3 SEQ ID NO: 23 GIP#2 Temporary 6.63 ± 3.87 n=3 SEQ ID NO: 24 Stable 12.0 ± 5.73 n=2 GIP#3 Temporary 171 n=1 - GIP#4 Temporary >5000 n=1 - GIP#5 Temporary 81.9 n=1 SEQ ID NO: 25 GIP#6 Temporary 398 n=1 - GIP#7 Temporary 76.9 n=1 SEQ ID NO: 26 GIP#8 Temporary 560 n=1 - GLP/GIP#2 Temporary 128 n=1 - GLP/GIP#3 Temporary 566 n=1 - GLP/GIP#4 Temporary 1283 n=1 -

Screening for GCG acceptor agonists

To test the activity levels of native GCG and GCG analogue at the cellular level (in vitro), a cAMP accumulation assay was performed in the same manner as Test Example 1.

The EC50 values of single GCG acceptor agonists were calculated and shown in Table 34 below.

Table 34 An object Cell line EC50 (pM) A comment SEQ ID NO GCG native-Ub Temporary 41.7 ± 29.9 n=3 SEQ ID NO: 1 Stable 35.3 ± 32.7 n=2 GCG#1 Temporary >5000 n=1 - GCG#2 Temporary 41.7 ± 5.51 n=3 SEQ ID NO: 2 Stable 68.5 ± 41.8 n=2 GCG#3 Temporary 47.8 ± 21.9 n=3 SEQ ID NO: 3 Stable 21.1 ± 12.3 n=3 GCG#4 Temporary >5000 n=1 - GCG#5 Temporary 661 ± 193 n=3 - GCG#6 Temporary 8400 n=1 - GCG#7 Temporary 956 n=1 - GCG#8 Temporary 761 ± 73.5 n=2 - GCG#9 Temporary 1073 n=1 - GCG#10 Temporary >5000 n=1 - GCG#11 Temporary >5000 n=1 -

EC50 values for dual GLP-1/GCG acceptor agonists were calculated and shown in Table 35 below.

Table 35 An object Cell line EC50 (pM) a comment SEQ ID NO GLP/GCG#1 Temporary >5000 n=1 - GLP/GCG#2 Temporary >5000 n=1 - GLP/GCG#3 Temporary >5000 n=1 SEQ ID NO: 15 GLP/GCG#4 Temporary >5000 n=1 - GLP/GCG#5 Temporary >5000 n=1 - GLP/GCG#6 Temporary >6250 n=1 - GLP/GCG#7 Temporary >5000 n=1 - GLP/GCG#8 Temporary >5000 n=1 - GLP/GCG#9 Temporary >5000 n=1 - GLP/GCG#10 Temporary >5000 n=1 - GLP/GCG#11 Temporary >5000 n=1 - GLP/GCG#12 Temporary 321 n=1 - GLP/GCG#13 Temporary >5000 n=1 - GLP/GCG#14 Temporary >5000 n=1 - GLP/GCG#15 Temporary 424 n=1 SEQ ID NO: 16

Screening for FGF21 acceptor agonists

To test the activity levels of native FGF21 and the FGF21 analog at the cellular level (in vitro), a functional FGFR1/KLB assay was performed in the same manner as Test Example 2.

EC50 values for single FGF21 acceptor agonists were calculated and shown in Table 36 below.

Table 36 An object EC50 (nM) A comment SEQ ID NO rhFGF21 (native) 1.17 ± 0.56 n=4 SEQ ID NO: 17 FGF#1 21.0 ± 0.49 n=2 SEQ ID NO: 18 FGF#5 22.3 ± 0.71 n=2 SEQ ID NO: 19 FGF#7 17.7 ± 2.55 n=2 SEQ ID NO: 20 FGF#9 19.1 ± 0.49 n=2 SEQ ID NO: 21 FGF#11 > 5000 n=1 - FGF#12 > 5000 n=1 - FGF#13 > 5000 n=1 - FGF#14 > 5000 n=1 - FGF#15 > 5000 n=1 -

Screening for IL-1RA acceptor antagonists

To test the ability of native IL-1RA and IL-1RA analog to inhibit NF-κB activity by IL-1β at the cellular level (in vitro), an NF-κB reporter gene luciferase assay was performed in the same manner as Test Example 3.

The IC50 values of the single IL-1RA acceptor antagonists were calculated and are shown in Table 37 below.

Table 37 An object IC50 (pM) Comments SEQ ID NO rhIL-1RA (native) 68.6 ± 7.71 n=2 SEQ ID NO: 27 IL-1RA-Ub (donor) 236 ± 43.8 n=2 SEQ ID NO: 28

As a result of screening the above various agonists and antagonists to obtain the protein conjugate of the present invention, biomolecules having excellent effects were selected. As a result, it was confirmed that the protein conjugate of the present invention has an excellent effect in the prevention and treatment of non-alcoholic steatohepatitis (NASH), fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity or diabetes, and also has excellent stability.

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LIST OF SEQUENCES

<110> Wanjin Biotechnology Inc.

<120> NEW PROTEIN CONJUGATE AND ITS USE FOR PREVENTION

OR TREATMENT OF NON-ALCOHOLIC STEATHOEPATITIS, OBESITY AND DIABETES

<130> OGB20P-0001-KR

<160> 34

<170> KoPatentIn 3.0

<210> 1

<211> 29

<212>PRT

<213> Artificial sequence

<220>

<223> GCG native

<400> 1

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

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Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

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<210> 2

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<212>PRT

<213> Artificial sequence

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<223> GCG#2

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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

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Arg Arg Ala Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala

20 25

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<223> GCG#3

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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

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Arg Arg Ala Lys Asp Phe Val Glu Trp Leu Leu Ser Ala

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<210> 4

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His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

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Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 5

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<212>PRT

<213> Artificial sequence

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<223> GLP-1№1

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His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

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Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asn Gly Arg

20 25 30

<210> 6

<211> 30

<212>PRT

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<220>

<223> GLP-1№3

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His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Lys

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Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Asn Gly Arg

20 25 30

<210> 7

<211> 31

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№4

<400> 7

His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<210> 8

<211> 31

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№5

<400> 8

His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly

20 25 30

<210> 9

<211> 30

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№6

<400> 9

His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Lys

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 10

<211> 31

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№7

<400> 10

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Lys

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<210> 11

<211> 31

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№8

<400> 11

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<210> 12

<211> 31

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№9

<400> 12

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly

20 25 30

<210> 13

<211> 30

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№10

<400> 13

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Glu Tyr Leu Glu Lys

1 5 10 15

Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 14

<211> 30

<212>PRT

<213> Artificial sequence

<220>

<223> GLP-1№11

<400> 14

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 15

<211> 29

<212>PRT

<213> Artificial sequence

<220>

<223> GLP/GCG№3

<400> 15

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Met Asn Thr

20 25

<210> 16

<211> 29

<212>PRT

<213> Artificial sequence

<220>

<223> GLP/GCG№15

<400> 16

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Met Asn Thr

20 25

<210> 17

<211> 181

<212>PRT

<213> Artificial sequence

<220>

<223> FGF21 native

<400> 17

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 18

<211> 182

<212>PRT

<213> Artificial sequence

<220>

<223> FGF21№1

<400> 18

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg

165 170 175

Ser Pro Ser Tyr Glu Ser

180

<210> 19

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<212>PRT

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<220>

<223> FGF21№5

<400> 19

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Val Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Asp Leu Lys Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Glu Gly Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 20

<211> 180

<212>PRT

<213> Artificial sequence

<220>

<223> FGF21№7

<400> 20

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Val Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Asp Leu Lys Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Arg Leu Val Glu Pro Ser Gln Leu Arg Ser

165 170 175

Pro Ser Phe Glu

180

<210> 21

<211> 181

<212>PRT

<213> Artificial sequence

<220>

<223> FGF21№9

<400> 21

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Val Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Arg Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Glu Gly Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 22

<211> 42

<212>PRT

<213> Artificial sequence

<220>

<223> GIP native

<400> 22

Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210> 23

<211> 42

<212>PRT

<213> Artificial sequence

<220>

<223> GIP№1

<400> 23

Tyr Gly Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210> 24

<211> 42

<212>PRT

<213> Artificial sequence

<220>

<223> GIP№2

<400> 24

Tyr Ser Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210> 25

<211> 42

<212>PRT

<213> Artificial sequence

<220>

<223> GIP№5

<400> 25

Phe Ser Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210> 26

<211> 42

<212>PRT

<213> Artificial sequence

<220>

<223> GIP№7

<400> 26

Tyr Gly Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile Arg Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210> 27

<211> 152

<212>PRT

<213> Artificial sequence

<220>

<223> IL-1RA native

<400> 27

Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp

1 5 10 15

Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala

20 25 30

Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp Val

35 40 45

Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly Lys

50 55 60

Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu

65 70 75 80

Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys

85 90 95

Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu

100 105 110

Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala Asp

115 120 125

Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val Thr

130 135 140

Lys Phe Tyr Phe Gln Glu Asp Glu

145 150

<210> 28

<211> 153

<212>PRT

<213> Artificial sequence

<220>

<223> IL-1RA

<400> 28

Met Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile

1 5 10 15

Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val

20 25 30

Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp

35 40 45

Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly

50 55 60

Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln

65 70 75 80

Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp

85 90 95

Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe

100 105 110

Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala

115 120 125

Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val

130 135 140

Thr Lys Phe Tyr Phe Gln Glu Asp Glu

145 150

<210> 29

<211> 15

<212>PRT

<213> Artificial sequence

<220>

<223> Linker

<400> 29

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 30

<211> 77

<212>PRT

<213> Artificial sequence

<220>

<223>Ub(A)

<400> 30

Met Gln Ile Phe Val Arg Thr Leu Thr Asp Arg Thr Ile Thr Leu Glu

1 5 10 15

Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp

20 25 30

Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg

35 40 45

Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu

50 55 60

Ser Thr Leu His Leu Val Leu Arg Pro Arg Val Val Asp

65 70 75

<210> 31

<211> 76

<212>PRT

<213> Artificial sequence

<220>

<223>Ub(D)

<400> 31

Met Gln Ile Phe Val Arg Thr Leu Thr Asp Arg Thr Ile Thr Leu Glu

1 5 10 15

Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp

20 25 30

Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg

35 40 45

Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu

50 55 60

Ser Thr Leu His Leu Val Leu Arg Pro Arg Gly Gly

65 70 75

<210> 32

<211> 585

<212>PRT

<213> Artificial sequence

<220>

<223>Albumin

<400> 32

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu

580 585

<210> 33

<211> 18

<212>PRT

<213> Artificial sequence

<220>

<223>HSA

<400> 33

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser

<210> 34

<211> 20

<212>PRT

<213> Artificial sequence

<220>

<223> IgGk

<400> 34

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<---

Claims (41)

1. A protein conjugate for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, characterized in that the protein conjugate contains: polyubiquitin, a carrier linked via a linker to polyubiquitin, and four biomolecules connected via a linker to polyubiquitin and a carrier, wherein the polyubiquitin consists of a ubiquitin acceptor consisting of the amino acid sequence of SEQ ID NO: 30, and a ubiquitin donor consisting of the amino acid sequence of SEQ ID NO: 31, and the biomolecule is selected from the group consisting of GCG, GLP-1, FGF21, GIP and IL-1RA, their analogues. 2. The protein conjugate according to claim 1, characterized in that the GCG analogue is a protein consisting of the amino acid sequence SEQ ID NO: 2. 3. Protein conjugate according to claim 1, characterized in that the GLP-1 analogue is a protein consisting of the amino acid sequence SEQ ID NO: 12. 4. Protein conjugate according to claim 1, characterized in that the FGF21 analogue is a protein consisting of the amino acid sequence SEQ ID NO: 20. 5. The protein conjugate according to claim 1, characterized in that GIP and its analogue are a protein selected from the amino acid sequence SEQ ID NO: 24. 6. Protein conjugate according to claim 1, characterized in that IL-1RA is a protein consisting of the amino acid sequence SEQ ID NO: 27. 7. Protein conjugate according to claim 1, characterized in that the linker is a polypeptide consisting of an amino acid sequence in which from 1 to 30 repeats of GGGGS, EAAAK or VPPPPP are combined. 8. Protein conjugate according to claim 7, characterized in that the linker is a polypeptide consisting of the amino acid sequence SEQ ID NO: 29. 9. Protein conjugate according to claim 1, characterized in that the carrier is selected from the group consisting of albumin, antibody fragment, scFc (single chain Fc), scFc dimer (single chain Fc dimer), transferrin, XTEN (genetic fusion of loose repeating peptide sequences ), CTP (carboxy-terminal peptide), PAS (proline-alanine-serine polymer), ELK (elastin-like peptide), HAP (homoamino acid polymer), GLK (gelatin-like protein), PEG (polyethylene glycol) and fatty acid. 10. Protein conjugate according to claim 9, characterized in that the carrier is albumin. 11. A protein conjugate for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, represented by the following formula: in the above formula W is a GIP analogue consisting of the amino acid sequence SEQ ID NO: 24, or IL-1RA consisting of the amino acid sequence SEQ ID NO: 27, X is a GCG analogue consisting of the amino acid sequence SEQ ID NO: 2, Y is a GLP-1 analogue consisting of the amino acid sequence SEQ ID NO: 12, Z is an FGF21 analogue consisting of the amino acid sequence SEQ ID NO: 20, Ub(A) is a ubiquitin acceptor consisting of the amino acid sequence SEQ ID NO: 30, Ub(D) is a ubiquitin donor consisting of the amino acid sequence SEQ ID NO: 31, L each independently represents a linker, A represents a carrier, and Ub(A) and Ub(D) are connected by a covalent bond. 12. The protein conjugate according to claim 11, characterized in that the linker is a polypeptide consisting of the amino acid sequence SEQ ID NO: 29. 13. Protein conjugate according to claim 11, characterized in that the carrier is albumin. 14. Pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, containing a protein conjugate according to any one of paragraphs. 1-13 and one or more excipients. 15. A method of preventing or treating non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity or diabetes, comprising administering the composition of claim 14 to a non-human subject. 16. A method for producing a protein conjugate, characterized in that the method includes the stages: (i) obtaining an acceptor protein represented by the following formula X-L-Y-L-Ub(A)-L-A-L-Z (ii) obtaining a donor protein represented by the following formula W-L-Ub(D), and (iii) binding of Ub(A) in the acceptor protein and Ub(D) in the donor protein, W is a GIP analogue consisting of the amino acid sequence SEQ ID NO: 24, or IL-1RA consisting of the amino acid sequence SEQ ID NO: 27, X is a GCG analogue consisting of the amino acid sequence SEQ ID NO: 2, Y is a GLP-1 analogue consisting of the amino acid sequence SEQ ID NO: 12, Z is an FGF21 analogue consisting of the amino acid sequence SEQ ID NO: 20, Ub(A) is a ubiquitin acceptor consisting of the amino acid sequence SEQ ID NO: 30, Ub(D) is a ubiquitin donor consisting of the amino acid sequence SEQ ID NO: 31, L is each independently a linker, and A represents the carrier.