Novel protein binders and their use for the prevention or treatment of non-alcoholic steatohepatitis, obesity and diabetes

Abstract

The present application relates to a protein conjugate comprising polyubiquitin, a carrier bound to the polyubiquitin, and two or more biological molecules bound to the polyubiquitin or the carrier. Also, the present application relates to a pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, obesity, and diabetes, comprising the above protein conjugate comprising two or more biological molecules.

Description

Technical Field

[0001] This invention relates to a carrier comprising polyubiquitin and polyubiquitin bound to it.

[0002] Protein conjugates of two or more biomolecules that bind to the aforementioned polyubiquitin or carrier.

[0003] Furthermore, the present invention relates to protein conjugates comprising two or more biomolecules as described above.

[0004] It is used for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis,

[0005] Drug compositions for liver cancer, obesity, and diabetes. Background Technology

[0006] Non-alcoholic fatty liver disease (NAFLD) is a condition that presents with histological findings similar to alcoholic hepatitis even without or with minimal alcohol consumption. It is a metabolic syndrome associated with diseases ranging from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. The incidence of NAFLD is increasing with the rise in obesity and diabetes; in South Korea, the annual prevalence is approximately 16%.

[0007] Nonalcoholic steatohepatitis (NASH) is characterized by abnormal fat accumulation or deposition in the liver (hepatic steatosis), liver inflammation, and liver or stem tissue damage (fibrosis). The global incidence of NASH is 2%–4% (3%–12% in US adults). Compared to the slow histological progression of simple steatosis, NASH exhibits a faster histological progression and is known to develop into cirrhosis. Approximately 5%–10% of individuals diagnosed with fatty liver are diagnosed with steatohepatitis (Metabolism Clinical and Experimental 65 (2016) 1038–1048).

[0008] Currently, there are no commercially available treatments for non-alcoholic steatohepatitis (NAH). Because there are no treatments available, treatments for other metabolic syndromes such as abdominal obesity, hyperlipidemia, and diabetes are used. For example, insulin resistance improvers, antioxidants (such as vitamin C and vitamin E), dyslipidemia treatments, and liver protectants are used. However, these drugs are difficult to consider as direct treatments for NHA.

[0009] Nonalcoholic steatohepatitis (NASH) is a complex disease, and due to the stringent licensing requirements for treatments set by US and European authorities, many treatments have failed in clinical development recently. Consequently, the development of multi-agonist-based treatments that simultaneously improve multiple indicators is on the rise. Currently, clinically available multi-agonist-based NASH treatments include AstraZeneca's MEDI0382 (a GLP-1 / GCG dual agonist), Eli Lilly's LY3298176 (a GIP / GLP-1 dual agonist), and Hanmi Pharmaceutical's HM15211 (a GLP / GIP / GLP-1 triple agonist).

[0010] Obesity is a state of excessive body fat, which can be defined as a risk factor for a variety of diseases, including heart disease, due to excessive body fat. According to the World Health Organization (WHO), there are approximately 106 million overweight adults worldwide, with at least 4 million being obese. In the United States, more than two out of every three people are overweight or obese (Low et al., 2009; Cooke and Bloom, 2006). It is known that compared to people of normal weight, obese individuals have an increased risk of developing a variety of diseases, including type 2 diabetes, hyperlipidemia, arthritis, and sleep apnea, and are particularly at risk of developing various cancers and heart disease.

[0011] Diabetes mellitus is a metabolic disease characterized by persistently high blood sugar levels due to insufficient insulin secretion or insulin resistance. With prolonged hyperglycemia, glycated products damage the retina, kidneys, nerves, and blood vessels throughout the body, leading to chronic complications. These complications are more dangerous than diabetes itself; therefore, the primary goal of current diabetes treatment is to prevent the onset or progression of these complications. Representative diabetic complications include diabetic retinopathy, diabetic cataracts, diabetic nephropathy, diabetic neuropathy, diabetic cardiomyopathy, diabetic osteoporosis, and diabetic atherosclerosis.

[0012] In the development of therapeutic agents based on multiple agonists, the problem of reduced activity due to structural limitations has led to a lack of research on therapeutic agents for non-alcoholic steatohepatitis (NAH), fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity, or diabetes using four or more agonists. Therefore, the inventors, through continuous development of therapeutic agents for NHA, fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity, and diabetes based on four agonists / antagonists, have achieved this invention using a polyubiquitin structure.

[0013] Existing technical documents

[0014] Patent documents

[0015] Patent Document 0001: Korean Patent Publication No. 10-2016-0032699

[0016] Patent Document 0002: Korean Patent No. 10-2034607 Summary of the Invention

[0017] Technical issues

[0018] The purpose of this invention is to provide a protein conjugate comprising polyubiquitin, a carrier bound to polyubiquitin, and two or more biomolecules bound to the aforementioned polyubiquitin or carrier.

[0019] Furthermore, the present invention provides a pharmaceutical composition comprising a protein conjugate containing two or more biomolecules for the prevention or treatment of non-alcoholic steatohepatitis (NASH), fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity, or diabetes. Specifically, the object of the present invention is to provide a pharmaceutical composition comprising a GCG / GLP-1 / FGF21 / GIP or a GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity, or diabetes.

[0020] Technical solution

[0021] This invention provides a protein conjugate comprising: a polyubiquitin; a carrier that binds directly to or via a linker of the polyubiquitin; and a biomolecule that binds directly to or via a linker of the polyubiquitin or the carrier. The polyubiquitin comprises: (i) a receptor ubiquitin, wherein lysine residues of the ubiquitin can be replaced by arginine or alanine, but includes one or more unsubstituted lysine residues; and (ii) a donor ubiquitin, wherein all lysine residues of the ubiquitin are replaced by arginine or alanine. The biomolecule is selected from two or more of the group consisting of GCG, GLP-1, FGF21, GIP, and IL-1RA; analogues thereof; dual receptor agonists of GCG and GLP-1; and dual receptor agonists of GLP-1 and GIP.

[0022] In one embodiment, the GCG and its analogues may be selected from the group consisting of proteins formed by amino acid sequences of sequences 1 to 3. Preferably, the GCG analogues may be proteins formed by amino acid sequences of sequence 2.

[0023] In one embodiment, the GLP-1 and its analogues may be selected from the group consisting of proteins formed by amino acid sequences of sequences 4 to 14. Preferably, the GLP-1 analogues may be proteins formed by amino acid sequences of sequence 12.

[0024] In one embodiment, the aforementioned GCG and GLP-1 dual receptor agonist may be selected from the group consisting of proteins formed by the amino acid sequences of sequence 15 and sequence 16.

[0025] In one embodiment, the FGF21 and its analogues can be selected from the group consisting of proteins formed by amino acid sequences of sequences 17 to 21. Preferably, the FGF21 analogues can be proteins formed by amino acid sequences of sequence 20.

[0026] In one embodiment, the GIP and its analogues may be selected from the group consisting of proteins formed by the amino acid sequences of sequences 22 to 26. Preferably, the GIP and its analogues may be proteins formed by the amino acid sequence of sequence 24.

[0027] In one embodiment, the IL-1RA and its analogues may be selected from the group consisting of proteins formed by the amino acid sequences of sequences 27 and 28. Preferably, the IL-1RA may be a protein formed by the amino acid sequence of sequence 27.

[0028] In one embodiment, the aforementioned polyubiquitin may consist of a receptor ubiquitin in which lysine residues at positions 6, 11, 27, 29, 33, and 48 of the N-terminus of ubiquitin are replaced by arginine, and a donor ubiquitin in which all lysine residues of ubiquitin are replaced by arginine. Preferably, the aforementioned polyubiquitin may consist of a receptor ubiquitin formed by the amino acid sequence of sequence 30 and a donor ubiquitin formed by the amino acid sequence of sequence 31.

[0029] In one embodiment, the linker may be a polypeptide formed by repeating one to 30 GGGGS, EAAAK, or VPPPPP amino acid sequences. Preferably, the linker may be a polypeptide formed by the amino acid sequence of sequence 29.

[0030] In one embodiment, the carrier may be selected from the group consisting of albumin, antibody fragments, single-chain crystallizable fragments (scFc), single-chain crystallizable fragment dimers (single-chain Fc-dimer), transferrin, extended recombinant peptides (XTEN, genetic fusion of non-exact repeat peptide sequence), carboxy-terminal peptides (CTP), proline-alanine-serine polymers (PAS), elastin-like peptides (ELK), homo-amino acid polymers (HAP), gelatin-like proteins, polyethylene glycol (PEG), and fatty acids. Preferably, the carrier may be albumin.

[0031] The present invention provides protein conjugates represented by the following formula.

[0032]

[0033] In the above formula, W, X, Y, and Z can be biomolecules in the group consisting of GCG analogs formed from the amino acid sequence of sequence 2, GLP-1 analogs formed from the amino acid sequence of sequence 12, FGF21 analogs formed from the amino acid sequence of sequence 20, GIP analogs formed from the amino acid sequence of sequence 24, and IL-1RA formed from the amino acid sequence of sequence 27, respectively. Ub(A) can be a receptor ubiquitin formed from the amino acid sequence of sequence 30, Ub(D) can be a donor ubiquitin formed from the amino acid sequence of sequence 31, L can be absent or a linker, A can be a carrier, and Ub(A) and Ub(D) can be linked by covalent bonds.

[0034] In one embodiment, X can be a GCG analog formed from the amino acid sequence of sequence 2, Y can be a GLP-1 analog formed from the amino acid sequence of sequence 12, Z can be an FGF21 analog formed from the amino acid sequence of sequence 20, and W can be a GIP analog formed from the amino acid sequence of sequence 24 or an IL-1RA formed from the sequence of sequence 27.

[0035] In one embodiment, the linker may be a polypeptide formed by the amino acid sequence of sequence 29, and the carrier may be albumin.

[0036] The present invention provides a pharmaceutical composition comprising the above-mentioned protein conjugates for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity or diabetes.

[0037] The present invention provides a method for preventing or treating non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity or diabetes, comprising the step of administering the above composition to an individual other than a human.

[0038] Furthermore, the present invention provides a method for preparing a protein conjugate, characterized by comprising: step (i), preparing a receptor protein represented by the following formula: XLYL-Ub(A)-LALZ; step (ii), preparing a donor protein represented by the following formula: WL-Ub(D); and step (iii), binding the Ub(A) of the above-mentioned receptor protein with the Ub(D) of the above-mentioned donor protein.

[0039] W, X, Y, and Z are biomolecules in the group consisting of GCG analogs formed from the amino acid sequence of sequence 2, GLP-1 analogs formed from the amino acid sequence of sequence 12, FGF21 analogs formed from the amino acid sequence of sequence 20, GIP analogs formed from the amino acid sequence of sequence 24, and IL-1RA formed from the amino acid sequence of sequence 27, respectively. Ub(A) is the receptor ubiquitin formed from the amino acid sequence of sequence 30, Ub(D) is the donor ubiquitin formed from the amino acid sequence of sequence 31, L is either absent or a linker, and A is a carrier.

[0040] The effects of the invention

[0041] The novel protein conjugates of this invention contain two or more biomolecules and exhibit excellent efficacy in the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, or liver cancer by inhibiting steatosis, inflammation, and fibrosis in the liver. They can also be useful in the prevention or treatment of obesity or diabetes. Furthermore, the protein conjugates maintain their effects in vivo for a long period and possess excellent safety due to the use of harmless polyubiquitin and carriers. Attached Figure Description

[0042] Figure 1 The results show the purification of the receptor protein confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0043] Figure 2 This is a schematic diagram illustrating the purification process of the donor protein.

[0044] Figure 3 and Figure 4 The results of purification of donor proteins via a nickel (Ni) column were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0045] Figure 5 and Figure 6 The purification results of His-SUMO by removing the donor protein were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0046] Figure 7 and Figure 8 The results of the fine purification of the donor protein are shown.

[0047] Figure 9 This is a schematic diagram illustrating the process of preparing protein conjugates through the conjugation of receptor and donor proteins.

[0048] Figure 10 and Figure 11The results show the conjugation of the receptor and donor proteins as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0049] Figure 12 The results show the nickel purification of the protein conjugate as confirmed by chromatography.

[0050] Figure 13 and Figure 14 The results show the purification of the protein conjugate as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0051] Figure 15 The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final protein conjugate are shown.

[0052] Figures 16 to 18 A graph showing the results of the cAMP accumulation assay.

[0053] Figure 19 A graph showing the results of the FGFR1 / KLB functional assay.

[0054] Figure 20 A graph showing the results of the NF-κB reporter assay.

[0055] Figure 21 and Figure 22 A graph showing the results of measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse blood during an in-vivo efficacy study.

[0056] Figures 23 to 26 The results of observing steatosis and inflammation (lobular inflammation) in mouse liver tissue in in vivo efficacy studies are shown, along with scores based on the Nonalcoholic Fatty Liver Disease Activity Score (NAS).

[0057] Figure 27 and Figure 28 A graph showing the results of measuring TGT-β and triglyceride concentrations in mouse liver tissue during in vivo efficacy studies.

[0058] Figure 29 and Figure 30 A graph showing the results of measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse blood during an in vivo efficacy study.

[0059] Figures 31 to 34The results of observing steatosis and inflammation (lobular inflammation) in mouse liver tissue in an in vivo efficacy study are shown, along with scores based on the non-alcoholic fatty liver disease activity score.

[0060] Figure 35 and Figure 36 A graph showing the results of measuring TGT-β and triglyceride concentrations in mouse liver tissue during in vivo efficacy studies.

[0061] Figures 37 to 41 To illustrate the changes in serum triglycerides (TG), free fatty acids (NEFA), total cholesterol (T-Chol), low-density lipoprotein cholesterol (LDL-Cholesterol), and high-density lipoprotein cholesterol (HDL-Cholesterol) in diet-induced obese mice in in vivo efficacy studies.

[0062] Figure 42 and Figure 43 A graph showing the changes in non-fasting blood glucose in type 2 diabetic mice during in vivo efficacy studies.

[0063] Figure 44 and Figure 45 A graph showing the change in body weight in type 2 diabetic mice during in vivo efficacy studies.

[0064] Figure 46 and Figure 47 A graph showing the changes in feed intake in type 2 diabetic mice during in vivo efficacy studies.

[0065] Figure 48 and Figure 49 A graph showing the changes in water intake in type 2 diabetic mice during in vivo efficacy studies.

[0066] Figures 50 to 52 The results of the in silico immunogenicity assay in major histocompatibility complex class I (MHC Class I) are shown.

[0067] Figures 53 to 55 The results of the in silico immunogenicity assay in major histocompatibility complex class II (MHC Class II) are shown. Detailed Implementation

[0068] The embodiments and examples of this application are described in detail below with reference to the accompanying drawings, so as to enable those skilled in the art to implement the present invention. However, this application can be implemented in many forms and is not limited to the embodiments and examples described herein.

[0069] Throughout this application specification, when a part is mentioned as "containing" a certain structural element, unless otherwise stated, it indicates that other structural elements may also be included, rather than excluding other structural elements.

[0070] As used in this application, the term "prevention" refers to all actions that suppress or delay the onset of a disease by administering the composition, and "treatment" refers to all actions that improve or cure the symptoms of an individual suspected of having a disease by administering the composition.

[0071] The term "individual" as used in this application can refer to mammals, although humans are preferred, but can also include pets (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.) and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0072] The pharmaceutical compositions of the present invention can be administered orally or parenterally according to the desired method. The dosage range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, metabolic rate, and disease severity. Furthermore, the therapeutically effective dosage of the above compositions varies depending on the administration method, target site, and patient condition. When used in humans, the dosage should be determined by considering both safety and efficacy.

[0073] The term “GCG” as used in this application may refer to wild-type glucagon (Native GCG) as a protein formed from the amino acid sequence of Sequence 1.

[0074] As used in this application, the term "GCG analogue" indicates that a portion of the amino acids in the wild-type glucagon protein have been substituted. Preferably, it may refer to a protein formed by the amino acid sequence of sequence 2 or sequence 3. More preferably, it may refer to a protein formed by the amino acid sequence of sequence 2, where amino acids 16 to 20, starting from the N-terminus of the wild-type glucagon protein, are substituted with SRRAQ for ERRAK, amino acids 23 to 24 are substituted with VQ for IE, and amino acids 27 to 29 are substituted with MNT for LSA.

[0075] The term “GLP-1” as used in this application may refer to wild-type GLP-1 (Native GLP-1) as a protein formed from the amino acid sequence of sequence 4.

[0076] As used in this application, the term "GLP-1 analog" can refer to a protein in which a portion of the amino acids of the wild-type GLP-1 protein have been substituted. Preferably, it can refer to a protein selected from the group consisting of proteins formed by amino acid sequences from sequence 5 to sequence 14. More preferably, it can refer to a protein formed by the amino acid sequence of sequence 12, where the second amino acid starting from the N-terminus of the wild-type GLP-1 protein is substituted with G instead of A, the 16th amino acid is substituted with E instead of G instead of G, and the 30th amino acid is substituted with GG instead of R.

[0077] The term "FGF21" as used in this application may refer to wild-type FGF21 (Native FGF21) as a protein formed from the amino acid sequence of sequence 17.

[0078] As used in this application, the term "FGF21 analogue" refers to a protein in which a portion of the amino acids of the wild-type FGF21 protein have been substituted. Preferably, it may refer to a protein selected from the group consisting of proteins formed by amino acid sequences of sequences 18 to 21. More preferably, it may refer to a protein formed by the amino acid sequence of sequence 20, where amino acid 19, starting from the N-terminus of the wild-type FGF21 protein, is substituted with R for V, amino acids 98 to 100 are substituted with LLL for DLK, amino acids 167 to 170 are substituted with SMVG for RLVE, amino acid 174 is substituted with G for L, and amino acids 179 to 181 are substituted with YAS for FE.

[0079] The term "GIP" as used in this application may refer to a wild-type GIP (Native GIP) as a protein formed from the amino acid sequence of sequence 22.

[0080] As used in this application, the term "GIP analogue" refers to a wild-type GIP protein in which a portion of amino acids have been substituted. Preferably, it may refer to a protein selected from the group consisting of proteins formed by amino acid sequences 23 to 26. More preferably, it may refer to a protein formed by an amino acid sequence 24 in which the second amino acid, starting from the N-terminus of the wild-type GIP protein, is substituted with S instead of A.

[0081] The term “IL-1RA” as used in this application may refer to wild-type IL-1RA (Native IL-1RA) as a protein formed from the amino acid sequence of sequence 27.

[0082] As used in this application, the term "IL-1RA analogue" indicates that a portion of the amino acids in the wild-type IL-1RA protein have been substituted. Preferably, it may refer to a protein formed by the amino acid sequence of sequence 28.

[0083] The present invention will be described in more detail below through embodiments, but the following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0084] receptor protein

[0085] The receptor protein prepared in the following examples is a polypeptide represented by the following formula.

[0086] XLYL-Ub(A)-LALZ

[0087] In the above formula, X is a GCG analog formed from the amino acid sequence of sequence 2, Y is a GLP-1 analog formed from the amino acid sequence of sequence 12, Z is an FGF21 analog formed from the amino acid sequence of sequence 20, Ub(A) is a receptor ubiquitin formed from the amino acid sequence of sequence 30, L is a linker formed from the amino acid sequence of sequence 29, and A is albumin formed from the amino acid sequence of sequence 32.

[0088] The amino acid sequences of the proteins and signal peptides that make up the above receptor proteins are shown in Table 1.

[0089] Table 1

[0090]

[0091]

[0092] donor protein

[0093] The donor proteins prepared in the following examples are polypeptides represented by the following formula.

[0094] WL-Ub(D)

[0095] In the above formula, W is a GIP analog formed from the amino acid sequence of sequence 24 or an IL-1RA formed from the amino acid sequence of sequence 27, L is a linker formed from the sequence of sequence 29, and Ub(D) is a donor ubiquitin formed from the sequence of sequence 31.

[0096] The amino acid sequences of the proteins that make up the above-mentioned donor proteins are shown in Table 2.

[0097] Table 2

[0098] protein amino acid sequence GIP Sequence 24 IL-1RA Sequence 27 connector Sequence 29 Ub(D) Sequence 31

[0099] As used in this application, the terms “Donor-191” and “D-191” refer to donor proteins containing GIP analogs.

[0100] The terms “Donor-192” and “D-192” used in this application refer to donor proteins containing IL-1RA.

[0101] As used in this application, the terms "RD-191" and "C-191" refer to protein conjugates containing donor proteins that include receptor proteins and GIP analogs.

[0102] As used in this application, the terms "RD-192" and "C-192" refer to protein conjugates containing a donor protein comprising a receptor protein and an IL-1RA.

[0103] Example 1

[0104] Preparation of plasmid deoxyribonucleic acid (DNA) for expressing receptor proteins.

[0105] After synthesizing the receptor gene sequence, which is the gene encoding the receptor protein, the gene was cloned into pcDNA3.1(+), an expression vector with a simple structure containing the CMV promoter, ampicillin resistance gene, etc.

[0106] To clone the signal peptide that enables the protein to be secreted extracellularly from a plasmid containing the receptor gene, fast cloning was performed. Vector polymerase chain reaction (PCR) was performed using the deoxyribonucleic acid of the plasmid containing the receptor gene as a template to insert the signal peptide at the 5' end of the receptor protein. Insertion PCR was performed using human serum albumin (HSA) formed from sequence 33 and IgGκ signal peptide formed from sequence 34 as templates to insert each signal peptide at the 5' end of the receptor protein. The PCR was performed using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Cat. No.: F530). The process involved a first denaturation at 98°C for 3 minutes, a second denaturation at 98°C for 10 seconds, primer binding at 60°C for 30 seconds, and an extension reaction at 72°C for 3 minutes. This process was repeated 18 times from the second denaturation to the extension reaction, with the final extension reaction performed at 72°C for 5 minutes.

[0107] After the polymerase chain reaction (PCR) was completed, the amplification of the target gene was confirmed by agarose gel electrophoresis. Then, 10 μL of the vector PCR product and the insertion PCR product were added to a PCR tube at a 1:1 ratio, followed by 0.5 μL of DpnI. The tube was treated at 37°C for 1 hour to remove template DNA before culturing. The cultured PCR product was then transferred to DH5α competent cells and heat-shocked at 42°C for 1 minute for transformation. The transformed cells were spread onto LB agar containing ampicillin and incubated at 37°C for 16 hours to obtain colonies. A single colony was inoculated into 5 mL of LB agar containing ampicillin and incubated at 37°C and 220 rpm for 16 hours. After centrifuging the culture medium at 3500 rpm for 20 minutes to obtain E. coli wet cells, the cell walls were disrupted by adding S1, S2, and S3 solutions from a deoxyribonucleic acid extraction kit (COSMO Genetech, Cat. No.: CMP0112), resulting in a turbid deoxyribonucleic acid solution from which proteins and deoxyribonucleic acid were separated. The plasmid deoxyribonucleic acid was purified from the obtained turbid deoxyribonucleic acid solution using a purification column from a deoxyribonucleic acid extraction kit (COSMO Genetech, Cat. No.: CMP0112). The gene sequence of the plasmid deoxyribonucleic acid was analyzed by COSMO Genetech to obtain two vectors: one containing human serum albumin (HSA) formed by sequence 33, and the other containing an IgGκ signal peptide formed by sequence 34, inserted at the 5' end of the receptor protein.

[0108] Transformation was achieved by placing the vector containing the confirmed gene sequence into DH5α competent cells and heat-shocking them at 42°C for 1 minute. The transformed cells were then spread onto LB agar containing ampicillin and incubated at 37°C for 16 hours to obtain colonies. A single colony was inoculated into 5 mL of LB agar and incubated at 37°C and 220 rpm for 16 hours. A portion of this culture was then re-inoculated into 200 mL of LB agar containing ampicillin and incubated at 37°C and 220 rpm for 16 hours. After centrifuging the culture at 3500 rpm for 30 minutes to obtain wet *E. coli* cells, the cell walls were disrupted by adding P1, P2, and P3 solutions from a deoxyribonucleic acid extraction kit (QIAGEN, Cat. No.: 12263), resulting in a turbid deoxyribonucleic acid solution from which proteins and deoxyribonucleic acid were separated. Plasmid DNA particles were extracted from the obtained DNA turbidity using a purification column of a DNA extraction kit (QIAGEN, Cat. No.: 12263). The particles were dissolved in cell culture water (Sigma Aldrich, W3500) and then filtered through a 0.22 μm filter. The concentration and purity of the extracted plasmid DNA were measured using a nanodrop instrument (IMPLEN, Nanodrop NP-80) before being used for protein expression.

[0109] Example 2

[0110] Receptor protein expression in ExpiCHO-S cells

[0111] 4×10 doses were administered 24 hours before the transfection process. 6 ExpiCHO-S cells (Gibco, Cat. No.: A29127, Lot: 1974423) were prepared and cultured in an orbital shaker at 95 rpm (50 mm shaking diameter) in a 37°C, 80%+ humidity, and 8% CO2 incubator. After 24 hours, to remove cell clumps, cells were filtered through a 40 μm nylon filter (BD Falcon, Cat. No.: 352340), and cell viability and cell number were measured. Cells were diluted with ExpiCHO-S expression medium (ExpiCHO Expression Media, Gibco, Cat. No.: A29100-01) to a final concentration of 6 × 10⁻⁶. 6 The cells / m were then transferred to a 1L flask, and 200mL of cells were placed into the flask.

[0112] 120 μg of deoxyribonucleic acid was diluted in 8 mL of OptiPRO SFM (Gibco, Cat. No.: 12309-019) medium, and 640 μL of ExpiFectamine CHO reagent (Gibco, Cat. No.: 100033022) was diluted in 7.4 mL of OptiPRO SFM (Gibco, Cat. No.: 12309-019) medium and mixed. After reacting the mixture at room temperature for 3 minutes, it was slowly dispensed into a solution containing 6 × 10⁻⁶ molecules. 6 Transfected cells were seeded in flasks at a concentration of [cells / mL]. The flasks were incubated at 95 rpm (50 mm shaking diameter) for 18 hours in a vortex mixer at 37°C, 80% humidity, and 8% CO2. After 18 hours, 1200 μL of ExpiFectamine CHO Enhancer (Gibco, Cat. No.: 100033019) and 48 mL of ExpiCHO Feed (Gibco, Cat. No.: A29101-01) were added, and the flasks were incubated at 95 rpm for 7-8 days in a vortex mixer at 37°C, 80% humidity, and 8% CO2. After incubation, the culture was centrifuged at 3500 rpm for at least 30 minutes to obtain only the receptor protein expression culture medium with cell granules removed. The culture medium was filtered using two filters (Satorius Stedim, Cat. No.: DH-ST-29MDL20MC5FFV; Satorius Stedim, Cat. No.: DH-ST-5441307G4OOB) to remove impurities.

[0113] Take 80 μL of the prepared culture medium, add 20 μL of 5X reducing sample loading dye, mix, and incubate at 95°C for 5 minutes. To compare expression levels in the gel phase, add 80 μL of bovine serum albumin diluted to 62.5 mg / mL, 125 mg / mL, and 250 mg / mL, respectively, and 20 μL of 5X reducing sample loading dye, mix, and incubate at 95°C for 5 minutes. Load the prepared sample and the marker protein for size confirmation into a 10% Tris-Glycine gel. Stain with Coomassie brilliant blue R while gently shaking, and quantify the relative concentration of the receptor protein using the bovine serum albumin band as a reference.

[0114] Example 3

[0115] Purification of receptor proteins

[0116] The receptor protein culture medium expressed in Example 2 was loaded onto a Blue Sepharose HP resin (GE, Cat. No.: 17-0413-01) column equilibrated with equilibrium buffer (20 mM sodium phosphate, pH 7.0). After removing impurities with pre-elution (20 mM sodium phosphate, pH 7.0, 0.15 M KCl), the receptor protein was recovered using elution buffer (20 mM sodium phosphate, pH 7.0, 0.6 M KCl). The recovered receptor protein was dialyzed with 20 mM sodium phosphate buffer (pH 7.0) to remove salt, followed by ultrafiltration to achieve a final sample concentration of 5 mg / mL. The results of receptor protein purification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are shown below. Figure 1 As shown.

[0117] Example 4

[0118] Expression of donor proteins in Escherichia coli (E. coli) cells

[0119] After transforming the donor gene sequence encoding the donor protein into the pET21a vector with a His-SUMO tag, the plasmid containing the inserted donor gene was placed into BL21(DE3) competent cells. Transformation was performed by heat shock at 42°C for 1 minute. The cells were then spread onto LB agar containing ampicillin and incubated statically at 37°C for at least 16 hours to obtain colonies. Single colonies were inoculated into 50 mL of LB agar and seeded at 37°C and 220 rpm for 16 hours.

[0120] With GIP (Donor-191, D-191) as the biomolecule donor protein, the bacterial culture was inoculated at a ratio of 1:100 into 1L LB medium containing ampicillin for main culture. After culturing at 37°C and 220 rpm for 2 hours, when the OD600 nm reached 0.6, 0.25M IPTG was added, and the culture was incubated at 16°C and 220 rpm for 20 hours. After the culture was completed, E. coli wet cells were obtained by centrifugation at 3500 rpm for 30 minutes.

[0121] With IL-1RA as the biomolecule donor protein (Donor-192, D-192), the seed culture medium was inoculated at a ratio of 1:100 into 1L LB medium containing ampicillin for primary culture. Autoinduction was performed at 37°C and 220 rpm for 24 hours. After culture, E. coli wet cells were obtained by centrifugation at 3500 rpm for 30 minutes.

[0122] Example 5

[0123] Purification of donor proteins in E. coli cells

[0124] The cultured donor protein was purified using the following method, the purification process as follows: Figure 2 As shown.

[0125] Pyrolysis / Ultrasonic treatment

[0126] Wet cells obtained through 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). The lysed samples were loaded onto ice and sonicated for 15 minutes at 45% amplitude with pulls on / off = 5 sec / 3 sec. The lysate was then separated by centrifugation at 14,000 rpm for 30 minutes, and only the supernatant was collected.

[0127] Capture and Purification

[0128] Lysis products were loaded onto a Ni-sepharose resin (QIAGEN, Cat. No.: 30250). After sample loading, non-specific proteins were thoroughly washed away using binding buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole). Then, donor proteins with Hig-SUMO tags were recovered using elution buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.25 M imidazole). The recovered His-SUMO-tagged donor proteins were dialyzed against a 20 mM sodium phosphate buffer at pH 7.0 to remove salt and imidazole. The purification results of the donor proteins passing through the nickel column were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Figure 3 and Figure 4 As shown.

[0129] SENP1 enzyme decomposes

[0130] The His-SUMO-tagged donor protein and SENP1 were digested using SENP1 enzyme at a ratio of 100 mg:1 mg. The concentration of the protein recovered via Ni-purification was quantified, and 1 / 100 w / w of SNEP1 (in-house) equivalent to the amount (mg) of the His-SUMO-tagged donor protein was mixed in. The reaction mixture was incubated at room temperature (15°C–25°C) for 1 hour.

[0131] Remove His-SUMO

[0132] The reaction mixture was loaded onto a nickel-agarose gel resin (QIAGEN, Cat. No.: 30250) equilibrated with equilibration buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole). Sample loading was performed, and the His-SUMO-tagged donor protein was expelled via flow-through. After sample loading, the remaining donor protein was recovered using equilibration buffer (20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, 0.02 M imidazole). The recovered donor protein was dialyzed against 20 mM sodium phosphate buffer (pH 7.0) to remove salts and imidazole. Ultrafiltration was performed to achieve a final donor product concentration of 10 mg / mL. The His-SUMO removal purification results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Figure 5 and Figure 6 As shown.

[0133] purification

[0134] The donor protein was recovered in the step prior to loading onto a Capto Q ImpRes (GE, Cat. No.: 17-5470-15) column equilibrated with equilibration buffer (20 mM sodium phosphate, pH 7.0). The donor protein was expelled by overflow. The recovered protein was concentrated to a high concentration. The finely purified donor protein results are shown below. Figure 7 and Figure 8 As shown.

[0135] Example 6

[0136] Conjugate

[0137] like Figure 9 As shown, the receptor protein produced in Example 3 was conjugated with the donor protein produced in Example 5. A mixture was prepared under the conditions shown in Table 3 below, and the conjugation reaction was carried out at 30°C for 4 hours.

[0138] Table 3

[0139]

[0140]

[0141] The degree of conjugation was confirmed by loading 10 μg of the receptor onto an 8% sodium dodecyl sulfate-polyacrylamide gel. The results are as follows: Figure 10 and Figure 11 As shown.

[0142] Example 7

[0143] Enzyme removal using nickel agarose gel

[0144] To recover only the conjugate samples, the reactants were loaded onto nickel agarose gel resin (QIAGEN, Cat. No.: 30250) equilibrated with equilibration buffer (20 mM sodium phosphate, pH 8.0, 0.15 M NaCl). Impurities were then removed using equilibration buffer (20 mM sodium phosphate, pH 8.0, 0.5 M NaCl). The conjugates were then recovered using elution buffer (25 mM Tris, pH 8.0, 0.15 M NaCl, 0.01 M imidazole). The recovered protein conjugates (C-191 and C-192) were dialyzed against 20 mM sodium phosphate buffer (pH 7.0) to remove salts and imidazole. The nickel purification results were confirmed by spectroscopic methods. Figure 12 As shown.

[0145] Example 8

[0146] purification

[0147] The conjugates were recovered prior to loading onto a Capto Q ImpRes (GE, Cat. No.: 17-5470-15) column equilibrated with equilibration buffer (25 mM sodium phosphate, pH 7.0 buffer). The conjugates were recovered using elution buffer (25 mM sodium phosphate, pH 7.0, 158 mM NaCl buffer). The recovered protein conjugates were dialyzed against 20 mM sodium phosphate buffer (pH 7.0) to remove salts and imidazole. Ultrafiltration was performed to achieve a final conjugate product concentration of 5 mg / mL. Results are as follows. Figure 13 and Figure 14 As shown.

[0148] Example 9

[0149] Preparation

[0150] The protein conjugates recovered from AEX were dialyzed to remove salts and imidazoles using a formulation buffer (4.6 mM histidine, 5.7 mM Tris, pH 7.5, 10 mM arginine, 0.1 g / mL trehalose). Ultrafiltration was then performed to achieve a final product concentration of 5 mg / mL. Results are as follows: Figure 15 As shown.

[0151] Experimental Example 1

[0152] Cyclic adenosine monophosphate (cAMP) accumulation assay

[0153] To test the activity of GLP-1, GCG, and GIP agonists in protein conjugates C-191 and C-192, which are protein conjugates of GCG / GLP-1 / FGF21 / GIP or GCG / GLP-1 / FGF21 / IL-1RA receptor tetrad agonists / antagonists prepared by Examples 1 to 9, at the cellular level (in vitro), cyclic adenosine monophosphate (cAMP) accumulation was analyzed in cell lines that were transiently or stably overexpressing GLP-1 receptor, GIP receptor, and GCG receptor, respectively, using the Cisbio cAMP GsDynamic kit #62AM4PEC.

[0154] Cell preparation (brief)

[0155] HEK293 cells were cultured for 2-3 days in a 37°C, 5% CO2 incubator until they reached 70%-80% confluence in T-75 flasks. The culture medium was removed, and the cells were treated with 2 mL of TryPLE Express solution, then incubated for 3-5 minutes at 37°C, 5% CO2. The cells were then shaken off. After dilution with 6 mL of MEM medium (10% fetal bovine serum (FBS), 1% anti-antigen), the cells were transferred to 15 mL tubes and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed, and the cells were transferred to 5 mL of culture medium. After cell counting, the concentration was adjusted to 3 × 10⁻⁶ cells / mL. 5 Cells / mL. 2 mL of culture medium was aliquoted into each well of a 6-well plate and incubated at 37°C with 5% CO2 for 24 hours. After removing the culture medium, 1.7 mL of antibiotic-free medium was aliquoted into each well. FuGENE6 and each receptor plasmid were added to Opti-MEM and incubated separately at room temperature for 5 minutes. The cultures were then mixed and incubated at room temperature for 15 minutes. The mixture was aliquoted into the relevant wells and incubated at 37°C with 5% CO2 for 24 hours. After removing the culture medium, the cells were washed with 2 mL of pre-warmed phosphate buffer. 0.5 mL of accutase was aliquoted into each well and incubated at 37°C with 5% CO2 for 5 minutes to induce cell detachment. Under these conditions, complete cell detachment was confirmed using a microscope, and the plate was tapped to prevent further cell detachment. Add 2.5 mL of preheated (37°C) assay buffer (0.5% bovine serum albumin (BSA) in phosphate buffer containing 2 mM IBMX in PBS) to each well, transfer to a 15 mL tube, centrifuge at 1000 rpm for 3 minutes, and then add another 2 mL of assay buffer. After cell counting, adjust the concentration in the assay buffer to 400,000 cells / mL.

[0156] Cell preparation (stabilization)

[0157] Cells were cultured in T-25 flasks to achieve 70%–80% overexpression of GLP-1 receptor (Genscript, Cat. No. M00451), GIP receptor (Genscript, Cat. No. M00486), and GCG receptor (Genscript, Cat. No. M00345). After removing the culture medium, the cells were washed with 2 mL of phosphate buffer preheated to 37°C. 1 mL of cell digestion solution was added to each well, and the cells were incubated at 37°C with 5% CO2 for 5 minutes to induce cell detachment. Microscopic examination was used to confirm complete cell detachment, and the culture plate was tapped to prevent further detachment. 3 mL of analysis buffer preheated to 37°C was added to each well, and the cells were transferred to 15 mL tubes and centrifuged at 1000 rpm for 3 minutes. Then, 2 mL of analysis buffer was added. After cell counting, the concentration of cells in the analysis buffer was adjusted to 400,000 cells / mL.

[0158] Procedure

[0159] 5 μL of cells were aliquoted into each well of a 96-well low-volume white plate. Reference and sample (2X) solutions, prepared at 4-fold serial dilutions, were duplicated in 5 μL per well and incubated for 30 minutes in a 5% CO2 incubator. 5 μL of analysis buffer was then added to the control and blank wells. 5 μL of 1X cAMP-d2 solution was aliquoted into each well. 5 μL of lysis and detection buffer was added to the blank wells. 5 μL of 1X Anti-cAMP-Cryptate solution was aliquoted into each well and incubated at room temperature in the dark for 1 hour. Fluorescence was then measured using a plate reader.

[0160] Measurement and Analysis

[0161] Fluorescence of the sample in the culture plate was measured using a Synergy Neo2 instrument (excitation wavelength: 330 nm, emission wavelength: 665 nm and 620 nm).

[0162] The HTRF ratio is calculated as follows.

[0163]

[0164] Furthermore, the Delta ratio (ΔR) is calculated as follows.

[0165] △R=Ratio sample -Ratio blank =Signal - (background fluorescence)

[0166] Use GraphPad Prism 8 to calculate the EC50 value (curve-fitting of the log (agonist) vs. normalized response-variable slope equation) from the HTRF ratio plot.

[0167] Figures 16 to 18 The HTRF ratio plots for GLP-1R (GLP-1 receptor), GIPR (GIP receptor), and GCGR (GCG receptor) are shown below, and the calculated EC50 values ​​are shown in Table 4.

[0168] Table 4

[0169]

[0170]

[0171] As shown in Table 4, C-191 was confirmed to be active against each GLP-1 receptor, GIP receptor, and GCG receptor. Furthermore, C-192 was also confirmed to be active against each GLP-1 receptor and GCG receptor.

[0172] In particular, regarding activity against the GLP-1 receptor, C-191 and C-192 were found to exhibit superior activity compared to liraglutide, which was used as a control group. Furthermore, C-192 was found to be approximately twice as good as C-191 in terms of GLP-1 receptor activity, and approximately 15 times better in terms of GCG receptor activity.

[0173] Experimental Example 2

[0174] FGFR1 / KLB functional assay

[0175] To test the activity of FGF21 agonist in protein conjugates C-191 and C-192, which are GCG / GLP-1 / FGF21 / GIP or GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist prepared by Examples 1 to 9, at the cellular level (in vitro), FGFR1 / KLB functional analysis was performed using the PathHunter Detection Kit (Discover X, Cat. No. 93-0001) in an FGFR1 / KLB overexpressing cell line (Discover X, Cat. No. 93-118C3) as follows.

[0176] Cell seeding

[0177] The cells in the T-75 flask were cultured until they reached 70%–80% confluence. After removing the culture medium, the cells were washed with 5 mL of preheated phosphate buffer. After removing the phosphate buffer, 2 mL of AssayComplete cell detachment reagent was added, and the flask was incubated at 37°C with 5% CO2 for 3 minutes to allow attached cells to detach. The detached cells were then mixed with 6 mL of AssayComplete cell plating reagent and transferred to a 15 mL tube. After centrifugation at 1000 rpm for 3 minutes, the detached cells were re-added to 5 mL of cell plating reagent. After cell counting, the concentration was adjusted to 500,000 cells / mL. The flask was then transferred to a reservoir, and 40 μL (20,000 cells / well) was dispensed into each well of a white 96-well half-area cell culture plate using a multi-channel pipette. 40 μL of cell seeding reagent was dispensed into each empty well. The cells were then incubated for 24 hours at 37°C with 5% CO2 to allow cell attachment.

[0178] process

[0179] rhFGF21 (5X) prepared by serial 4-fold dilution and test sample (5X) were repeatedly aliquoted into each well at 10 μL and cultured at room temperature for 4 hours. 10 μL of AssayComplete cells were seeded into the control group and empty wells with reagent 0. After culture, the culture medium was removed, and 50 μL of AssayComplete cells were seeded into each well with reagent 0. 30 μL of detection reagent was aliquoted into each well, and the reaction was carried out at room temperature in the dark for 1 hour. The luminescence was then measured using a microplate reader.

[0180] Measurement and Analysis

[0181] The luminescence of the sample in the culture plate was measured using a Synergy Neo2 instrument.

[0182] Delta RLU (Relative luminescence unit) (ΔRLU) is calculated as follows.

[0183] △RLU=RLU sample -RLU blank =Signal - Background luminescence

[0184] Furthermore, the fold induction relative to the control group was calculated as follows.

[0185]

[0186] Based on different induction folds, the EC50 value was calculated using GraphPad Prism 8 in the following manner (curve-fitting of the log(agonist) vs. normalized response-variable slope equation).

[0187] Figure 19 The plot shows the calculated EC50 values, as shown in Table 5 below.

[0188] Table 5

[0189] EC50(nM) rhFGF21 C-191 C-192 1 0.75 92.2 42.2 2 0.88 45.1 40.5 average value 0.82 68.7 41.4 Standard deviation 0.09 33.3 1.20

[0190] As shown in Table 5 above, both C-191 and C-192 were confirmed to be active against the FGFR1 / KLB receptor. Furthermore, in terms of activity against the FGFR1 / KLB receptor, C-192 was confirmed to be approximately 40% superior to C-191.

[0191] Experimental Example 3

[0192] NF-κB reporter gene luminescent enzyme assay

[0193] To test the inhibitory effect of IL-1RA on NF-κB activity by IL-1β in the protein conjugate C-192, which is a protein conjugate of GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist prepared by Examples 1 to 9, at the cellular level (in vitro), the following luminescent enzyme analysis was performed in the NF-κB reporter (Luc) cell line (BPS Bioscience, Cat. No. 60650).

[0194] Cell inoculation

[0195] The cells in the T-75 flask were cultured until they reached 70%–80% confluence. After removing the culture medium, the cells were washed with 5 mL of preheated phosphate buffer. After removing the phosphate buffer, the cells were incubated in 1 mL of TryPLE Express at 37°C with 5% CO2 for 5 minutes to allow them to detach. The mixture was then combined with 4 mL of analytical medium (10% fetal bovine serum in Duchenne Modified Igor Medium (DMEM) containing 1% antibiotics), transferred to a 15 mL tube, centrifuged at 1000 rpm for 3 minutes, and then re-added to 5 mL of analytical medium. After cell counting, the concentration was adjusted to 500,000 cells / mL. The plates were then transferred to a water bath, and 40 μL (20,000 cells / well) was aliquoted into each well of a 96-well half-area cell culture plate using a multi-well pipette. Empty wells were aliquoted with only 40 μL of analytical medium. Cells were cultured for 24 hours in a constant temperature incubator at 37°C and 5% CO2 to allow them to attach.

[0196] process

[0197] Reference (10X) and sample (10X) prepared by serial 4-fold dilution were aliquoted into each well in 5 μL increments. Next, 5 μL of 50 pM IL-1β (10X) was aliquoted into each well and incubated in a 5% CO2 incubator for 4 hours. Then, 50 μL of ONE-Glo reagent (Promega, Cat. No. E6120) was aliquoted into each well and incubated at room temperature for 5 minutes before measuring luminescence using a SynergyNeo2 instrument. In this case, the culture plates were removed from the incubator 15 minutes before ONE-Glo reagent treatment to allow them to reach room temperature.

[0198] Calculate Delta RLU (relative light-emitting unit) (ΔRLU) as follows.

[0199] △RLU=RLU sample -RLU blank =Signal - Background luminescence

[0200] Using GraphPad Prism 8 with different concentrations of ΔRLU, the IC50 value (curve-fitting of the log (antagonist) vs. normalized response - variable slope equation) is calculated as follows.

[0201] Figure 20 The plot shows the calculated IC50 values, as shown in Table 6 below.

[0202] Table 6

[0203] IC50(pM) rhIL-1RA C-192 1 39.9 561 2 51.5 594 3 46.6 708 4 51.9 568 average value 47.5 607 Standard deviation 4.85 68.4

[0204] As shown in Table 6 above, C-192 was confirmed to inhibit the activity of NF-κB via IL-1β.

[0205] Test Example 4

[0206] In vivo efficacy trial: evaluating efficacy against non-alcoholic steatohepatitis.

[0207] The in vivo efficacy of C-192, a protein conjugate of a GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist prepared in Examples 1 to 9, against non-alcoholic steatohepatitis (NAH) was tested in C57BL / 6J mice with fatty liver induced by feeding them a diet deficient in methionine and choline (MCD, Methionine and Choline-Deficient L-Amino Acid Diet). The MCD mouse model is the most typical method used in efficacy studies for NHA, as the histological morphology of liver tissue induced for 2–4 weeks is known to resemble that of human NHA, and there are numerous precedents in pharmaceutical companies and academia using multiple substances in these studies.

[0208] Fatty liver was induced in male C57BL / 6J mice by feeding them a diet containing monoclonal antibodies (MCS) for 8 weeks. The mice were then divided into six groups as shown in Table 7 below and administered the drug for 4 weeks. Commercially available liraglutide (Saxenda) and dulaglutide (Trulycity) were used as two comparative substances. The drug combination was administered subcutaneously using a disposable syringe for each dosing cycle. Body weight was measured weekly during the 8-week feeding period, and groups were intentionally formed based on body weight after 8 weeks. The normal control group received a diet containing recombinant methionine and choline (MCS, Methionine and Choline Sufficient L-Amino Acid Diet).

[0209] Table 7

[0210] No Group diet Dosing Composition Dosage Dosage cycle Dosage frequency 1 normal control group MCS - - - - 2 negative control group MCD excipient - - - 3 Experimental Group A MCD C-192 5nmol / 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 / day 56 times 6 Comparison group B MCD Dulaglutide 2nmol / kg 1 time / 2 days 14 times

[0211] During the 4-week drug administration period, general symptoms and behavior were observed. Weight was measured once a week from the start of drug administration, and also on the day of tissue removal. On the last day of observation, blood was drawn and liver tissue was removed for blood biochemistry analysis and histopathological examination of the removed liver tissue.

[0212] Alanine aminotransferase (ALT), a fundamental indicator of liver disease, is measured through blood biochemistry tests, as shown in Table 8 below. Figure 21 As shown.

[0213] Table 8

[0214]

[0215]

[0216] As shown in Table 8 above, it can be seen that in the groups treated with liraglutide and duraglutide (comparison group A and comparison group B), the alanine aminotransferase (ALT) value was hardly reduced, but in the group treated with C-192, the ALT value was significantly reduced.

[0217] Since alanine aminotransferase (ALT) indicates the degree of liver damage, it is known that the degree of liver damage is reduced when the protein conjugate of the present invention is administered.

[0218] Furthermore, Table 9 below and Figure 22 The alanine aminotransferase (ALT) / aspartate aminotransferase (AST) values ​​are shown.

[0219] Table 9

[0220] No Group ALT / AST levels 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 (duraglutide administration group) 0.79

[0221] As shown in Table 9 above, it was confirmed that in the groups treated with liraglutide and duraglutide (comparison group A and comparison group B), the alanine aminotransferase (ALT) / aspartate aminotransferase (AST) ratio was less than 1.0, while in the group treated with C-192, the ALT / AST ratio was greater than 1.0.

[0222] In liver diseases, since the alanine aminotransferase (ALT) / aspartate aminotransferase (AST) ratio is often less than 1, it is known that the degree of liver damage is reduced when the protein conjugate of the present invention is administered.

[0223] Furthermore, histopathological examination was conducted using hematoxylin and eosin (H&E) staining and Masson's trichrome staining, and the results were as follows: Figure 23 As shown. In cases of non-alcoholic steatohepatitis, due to fatty degeneration and lobular inflammation, inflammatory cells can be identified during tissue staining. For example... Figure 23 As shown, compared with the negative control group, the fat and inflammatory cells in the group treated with 40 nmol / kg of protein conjugate (experimental group B) were improved to the extent that of the normal control group.

[0224] Furthermore, the steatosis score, lobular inflammation score, and non-alcoholic fatty liver disease activity score (NAS, NAFLD activity score) were assessed according to the evaluation criteria shown in Table 10 below.

[0225] Table 10

[0226]

[0227] The above assessment results are shown in Tables 11 to 13 below. Figures 24 to 26 As shown.

[0228] Table 11

[0229] No Group Fatty degeneration fraction 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 (duraglutide administration group) 1.9

[0230] Table 12

[0231] No Group Lobular inflammation score 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 (duraglutide administration group) 1.5

[0232] Table 13

[0233]

[0234] As shown in Tables 11 to 13 above, the groups administered the protein conjugate of the present invention all showed excellent results, especially the group administered 40 nmol / kg of the protein conjugate (experimental group B), which showed much better evaluation results than the liraglutide and duraglutide groups.

[0235] TGF-β, a marker of liver fibrosis, was analyzed in liver tissue by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 14 below. Figure 27 As shown.

[0236] Table 14

[0237]

[0238] As shown in Table 14 above, the TGF-β values ​​of the groups administered the protein conjugate of the present invention decreased, especially the group administered 40 nmol / kg of the protein conjugate (experimental group B), which showed values ​​almost similar to those of the normal control group.

[0239] Furthermore, the accumulation of triglycerides in liver tissue was analyzed using a triglyceride assay kit, and the results are shown in Table 15 below. Figure 28 As shown, the amount of triglycerides in the liver tissue of the liraglutide and duraglutide administration groups was hardly reduced, while the protein conjugate administration group of the present invention showed values ​​almost similar to the normal control group.

[0240] Table 15

[0241]

[0242] Ultimately, it can be seen that compared with the liraglutide and duraglutide administration groups, the protein conjugate C-192 administration group of the present invention showed excellent results in all experiments.

[0243] Experimental Example 5

[0244] In vivo efficacy study: evaluating efficacy against non-alcoholic steatohepatitis.

[0245] The in vivo efficacy of C-191, a protein conjugate of a GCG / GLP-1 / FGF21 / GIP receptor quadruple agonist / antagonist prepared in Examples 1 to 9, against non-alcoholic steatohepatitis was tested in C57BL / 6J mice with fatty liver induced by ingestion of a diet lacking methionine and choline (MCD, Methionine and Choline Deficient L-Amino Acid Diet).

[0246] As shown in Table 16 below, the groups were divided into four groups and were conducted in the same manner as in Test Example 4 above, with commercially available duraglutide used as the comparative substance.

[0247] Table 16

[0248] No Group diet Dosing Composition Dosage Dosage cycle Dosage frequency 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 Comparison group B MCD Dulaglutide 2nmol / kg 1 time / 2 days 14 times

[0249] During the 4-week drug administration period, general symptoms and behavior were observed. Weight was measured once a week from the start of drug administration, and also on the day of tissue removal. On the last day of observation, blood was drawn and liver tissue was removed for blood biochemistry analysis and histopathological examination of the removed liver tissue.

[0250] Alanine aminotransferase (ALT), a fundamental indicator of liver disease, was measured using blood biochemistry tests. The results are shown in Table 17 below. Figure 29 As shown.

[0251] Table 17

[0252]

[0253]

[0254] As shown in Table 17 above, it was confirmed that in the group treated with duraglutide, the alanine aminotransferase (ALT) level was almost not reduced, but in the group treated with C-191, the ALT level was significantly reduced.

[0255] Since alanine aminotransferase (ALT) indicates the degree of liver damage, it is known that the degree of liver damage is reduced when the protein conjugate of the present invention is administered.

[0256] Furthermore, the AST / ALT ratios are shown in Table 18 below. Figure 30 As shown.

[0257] Table 18

[0258] No Group AST / ALT levels 1 normal control group 1.84 2 negative control group 0.82 3 Experimental Group A 0.98 4 Comparative group A (duraglutide administration group) 0.76

[0259] As shown in Table 18 above, the ALT / AST ratio in the group treated with duraglutide was confirmed to be 0.76, while the ALT / AST ratio in the group treated with C-191 was 0.98, which was higher than that in the duraglutide group.

[0260] In liver diseases, since the alanine aminotransferase (ALT) / aspartate aminotransferase (AST) ratio is often less than 1, it is known that the degree of liver damage is reduced when the protein conjugate of the present invention is administered.

[0261] Furthermore, histopathological examination was conducted using hematoxylin-eosin staining and Masson's trichrome staining, and the results were as follows: Figure 31 As shown. In cases of non-alcoholic steatohepatitis, due to fatty degeneration and lobular inflammation, inflammatory cells can be identified during tissue staining. For example... Figure 31 As shown, compared with the negative control group, the group that received 10 nmol / kg of protein conjugate showed improvement in fat and inflammatory cells to the extent that of the normal control group.

[0262] Furthermore, the steatosis score, lobular inflammation score, and non-alcoholic fatty liver disease activity score were assessed according to the assessment criteria shown in Table 10 above.

[0263] The above assessment results are shown in Tables 19 to 21 below. Figures 32 to 34 As shown.

[0264] Table 19

[0265] No Group Fatty degeneration fraction 1 normal control group 0 2 negative control group 2.4 3 Experimental Group A 1.4 4 Comparative group A (duraglutide administration group) 1.8

[0266] Table 20

[0267] No Group Hepatic lobule inflammation score 1 normal control group 0.2 2 negative control group 1.6 3 Experimental Group A 1.0 4 Comparative group A (duraglutide administration group) 1.6

[0268] Table 21

[0269]

[0270] As shown in Tables 19 to 21 above, the groups administered the protein conjugate of the present invention all showed excellent results, demonstrating significantly better evaluation results than the duraglutide group.

[0271] TGF-β, a marker of liver fibrosis, was analyzed in liver tissue by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 22 below. Figure 35 As shown.

[0272] Table 22

[0273] No Group liver TGF-β 1 normal control group 77.3 2 negative control group 177.9 3 Experimental Group A 117.4 4 Comparative group A (duraglutide administration group) 165.3

[0274] As shown in Table 22 above, it was confirmed that the TGF-β value was reduced in the group that received the protein conjugate of the present invention, which was significantly reduced compared to the group that received duraglutide.

[0275] Furthermore, triglyceride accumulation in liver tissue was analyzed using a triglyceride assay kit, and the results are shown in Table 23 below. Figure 36 As shown, the amount of triglycerides in the liver tissue of the duraglutide-treated group was almost not reduced, while the protein conjugate-treated group of the present invention showed values ​​almost similar to those of the normal control group.

[0276] Table 23

[0277] No Group Liver triglycerides 1 normal control group 17.13 2 negative control group 40.09 3 Experimental Group A 24.02 6 Comparative group A (duraglutide administration group) 36.08

[0278] Ultimately, it can be seen that the protein conjugate C-191 administration group of the present invention showed far superior effects compared with the duraglutide administration group in all experiments.

[0279] Experimental Example 6

[0280] In vivo efficacy studies: evaluating efficacy against obesity.

[0281] To confirm the lipid-lowering effect of C-192, a protein conjugate of the GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist prepared in Examples 1 to 9, an evaluation was conducted using diet-induced obese mice. Five-week-old C57BL / 6J mice were purchased from Chuo Laboratory Animal Co., Ltd. After approximately seven days of acclimatization, they were fed a western diet for 16 weeks to induce obesity. Animals without abnormalities were selected based on average body weight and weight change, and were divided into groups of six to ensure similar average body weight across groups, as shown in Table 24 below. Subcutaneous administration was administered via the same route as the intended clinical administration, once every two days, repeated 28 times over eight weeks.

[0282] Table 24

[0283]

[0284] The excipients were used as the dosing composition in the normal and negative control groups, while the experimental groups were administered C-192 at doses of 10 nmol / kg, 30 nmol / kg, and 60 nmol / kg. During the 8-week dosing period, general symptoms such as appearance, mobility, and excretion were observed once daily. On the last day of dosing, blood was collected from the abdominal vein and placed in an SST tube. Serum was separated by centrifugation at 3000 rpm for 15 minutes, and the biomarkers shown in Table 25 below were measured using a blood biochemistry analyzer (7180, HTTACHI Corporation, Japan).

[0285] Table 25

[0286]

[0287] To evaluate the effect of C-192 on lipid improvement, serum levels of triglycerides (TG), non-tert-form fatty acids (NEFA), total cholesterol (T-Chol), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) were measured. The results are shown in Table 26 below. Figures 37 to 41 As shown.

[0288] Table 26

[0289]

[0290] As shown in Table 26 above and Figures 37 to 41As shown, a reduction in blood lipid-related biomarkers was confirmed in the C-192-treated groups. Specifically, in the group treated with 60 nmol / kg C-192 (experimental group C), compared to the negative control group, a 38.5% reduction in triglycerides, a 20% reduction in free fatty acids, a 30.9% reduction in total cholesterol, a 41.5% reduction in low-density lipoprotein cholesterol, and a 19.6% reduction in high-density lipoprotein cholesterol were observed. Furthermore, compared to the negative control group, all biomarkers except high-density lipoprotein cholesterol showed statistically significant differences. Therefore, it is evident that the protein conjugate of the present invention reduces blood lipids in diet-induced obese mice.

[0291] Experimental Example 7

[0292] In vivo efficacy studies: evaluating efficacy in treating diabetes.

[0293] To confirm the antidiabetic efficacy of C-192, a protein conjugate of the GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist prepared in Examples 1 to 9, evaluation was conducted using db / db mice, a type 2 diabetes model. db / db mice are animal models of obesity and type 2 diabetes caused by a mutation in the Leptin receptor gene on chromosome 4, which prevents signal transduction of leptin, a hormone secreted from adipocytes. C57BL / 6J mice were used as normal animals, and 8-week-old male mice purchased from SLC Corporation of Japan, after one week of quarantine and acclimatization, were used as experimental animals.

[0294] As shown in Table 27 below, the experimental group consisted of a normal control group, a negative control group, an experimental substance administration group, and a positive control group. After quarantine and acclimatization, the animals were divided into groups of 5 each, with equal body weight and blood glucose levels. Subcutaneous administration was administered via the same route as expected for clinical application, and the administration lasted for 8 days. The administration frequency was once a day for the normal control group, negative control group, and experimental group, and once every two days for the positive control group.

[0295] Table 27

[0296]

[0297] The excipients were administered as the drug composition to the normal and negative control groups, while the experimental groups were administered C-192 at doses of 10 nmol / kg and 60 nmol / kg. General symptoms were observed once daily during the 8-day administration period. To confirm the anti-diabetic effect in db / db mice, blood glucose, body weight, feed intake, and water intake were measured. Non-fasting blood glucose was measured using a G-Doctor glucometer, collected from the tail vein once every two days before administration. Changes in body weight, feed intake, and water intake were measured on days 1, 4, and 8 of administration. The results are shown in Table 28 below. Figures 42 to 49 As shown.

[0298] Table 28

[0299]

[0300] As shown in Table 28 above and Figures 42 to 49 As shown, it was confirmed that blood glucose, feed, and water intake were reduced in the group treated with C-192. In particular, it was confirmed that in the group treated with 60 nmol / kg C-192 (experimental group B), compared with the negative control group, blood glucose was significantly reduced by 61.4% on day nine, body weight was significantly reduced by 7.8% on day eight, feed intake was significantly reduced by 37.1%, and water intake was significantly reduced by 50.0%. Therefore, it can be seen that the protein conjugate of the present invention exhibits an antidiabetic effect.

[0301] Experimental Example 8

[0302] Immunogenicity analysis in silico

[0303] In silico immunogenicity analysis was performed on the various receptors and donors of protein conjugates C-191 and C-192, which are GCG / GLP-1 / FGF21 / GIP or GCG / GLP-1 / FGF21 / IL-1RA receptor quadruple agonist / antagonist prepared by Examples 1 to 9. Immunogenicity refers to the nature of the immune response induced when a drug is administered to the human body, and it is a factor that affects not only efficacy but also safety. In silico analysis using an immunogenicity-inducing sequence database and computer analysis software, and in vitro immunogenicity assessment using human PBMCs were performed.

[0304] The results are as follows Figures 50 to 55 As shown, the degree of immunogenicity induced is predicted to be associated with the 27 most frequently occurring human leukocyte antigen (HLA) types listed in Table 29 below.

[0305] Table 29

[0306]

[0307]

[0308] The protein sequences of the various receptors and donors were predicted to have a low probability of binding to the antigen-binding sites of major histocompatibility complex I (MHC I) and major histocompatibility complex II (MHC II) for 27 different human leukocyte antigen types. This result indicates that the receptors and donors have low immunogenicity and therefore predict high safety.

[0309] Experimental Example 9

[0310] API Screening

[0311] Screening for GLP-1 receptor agonists

[0312] To test the activity of wild-type GLP-1 and GLP-1 analogs at the cellular level (in vitro), cyclic adenosine monophosphate (cAMP) accumulation analysis was performed using the same method as in Experimental Example 1.

[0313] The EC50 values ​​of GLP-1 receptor monoagonals were calculated as shown in Table 30 below.

[0314] Table 30

[0315]

[0316]

[0317] The EC50 values ​​of the GLP-1 / GCG receptor dual agonists were calculated as shown in Table 31 below.

[0318] Table 31

[0319]

[0320]

[0321] The EC50 values ​​of the GLP-1 / GIP receptor dual agonists were calculated as shown in Table 32 below.

[0322] Table 32

[0323] distinguish cell lines EC50(pM) Remark Serial Number GLP / GIP#2 Briefly 57.8 n＝1 - GLP / GIP#3 Briefly >5000 n＝1 - GLP / GIP#4 Briefly >5000 n＝1 -

[0324] Screening for GIP receptor agonists

[0325] To test the activity of wild-type GIP and GIP analogs at the cellular level (in vitro), cyclic adenosine monophosphate (cAMP) accumulation analysis was performed using the same method as in Experimental Example 1.

[0326] The EC50 values ​​of GIP receptor monoagonal agents and GLP-1 / GIP receptor dual agonists are calculated as shown in Table 33 below.

[0327] Table 33

[0328]

[0329] Screening for GCG receptor agonists

[0330] To test the activity of wild-type GCG and GCG analogs at the cellular level (in vitro), cyclic adenosine monophosphate (cAMP) accumulation analysis was performed using the same method as in Experimental Example 1.

[0331] The EC50 values ​​of the GCG receptor monoagonal were calculated as shown in Table 34 below.

[0332] Table 34

[0333]

[0334] The EC50 values ​​of the GLP-1 / GCG receptor dual agonists were calculated as shown in Table 35 below.

[0335] Table 35

[0336]

[0337]

[0338] Screening for FGF21 receptor agonists

[0339] To test the activity of wild-type FGF21 and FGF21 analogs at the cellular level (in vitro), FGFR1 / KLB functional analysis was performed using the same method as in Experimental Example 2.

[0340] The EC50 values ​​of the FGF21 receptor monoagonal were calculated as shown in Table 36 below.

[0341] Table 36

[0342] distinguish EC50(nM) Remark Serial Number rhFGF21(wild type) 1.17±0.56 n＝4 Sequence 17 FGF#1 21.0±0.49 n＝2 Sequence 18 FGF#5 22.3±0.71 n＝2 Sequence 19 FGF#7 17.7±2.55 n＝2 Sequence 20 FGF#9 19.1±0.49 n＝2 Sequence 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 -

[0343] Screening for IL-1RA receptor antagonists

[0344] To test the inhibitory capacity of wild-type IL-1RA and IL-1RA analogs on NF-κB activity via IL-1β at the cellular level (in vitro), NF-κB reporter gene luminescent enzyme analysis was performed using the same method as in Experimental Example 3.

[0345] The IC50 values ​​of IL-1RA receptor monoantagonists were calculated as shown in Table 37 below.

[0346] Table 37

[0347] distinguish IC50(pM) Remark Serial Number rhIL-1RA (wild type) 68.6±7.71 n＝2 Sequence 27 IL-1RA-Ub (donor) 236±43.8 n＝2 Sequence 28

[0348] Based on the screening results of various agonists and antagonists, biomolecules with excellent effects were selected, thereby preparing the protein conjugate of the present invention. The results confirmed that the protein conjugate of the present invention has excellent preventive and therapeutic effects on non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity, or diabetes, and also exhibits excellent stability. <110> Genjin Biotechnology Co., Ltd. <120> Novel protein conjugates and their applications in the prevention or treatment of non-alcoholic steatohepatitis, 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 Wild Type <400> 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 2 <211> 29 <212> PRT <213> Artificial sequence <220> <223> GCG#2 <400> 2 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Ile Glu Trp Leu Leu Ser Ala 20 25 <210> 3 <211> 29 <212> PRT <213> Artificial sequence <220> <223> GCG#3 <400> 3 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Val Glu Trp Leu Leu Ser Ala 20 25 <210> 4 <211> 30 <212> PRT <213> Artificial sequence <220> <223> GLP-1 Wild Type <400> 4 His Ala 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> 5 <211> 30 <212> PRT <213> Artificial sequence <220> <223> GLP-1#1 <400> 5 His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asn Gly Arg 20 25 30 <210> 6 <211> 30 <212> PRT <213> Artificial sequence <220> <223> GLP-1#3 <400> 6 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 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 <4​​​​​​​​​​​​​​​​​​​​​​​​​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> Synthetic 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> Synthetic 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> Synthetic 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 wild type <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 ​​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 <211> 181 <212> PRT <213> Artificial Sequence <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 wild type <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> Synthetic 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> Synthetic 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 wild type <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 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 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 The Ser Al of Lys Gln Arg Leu Lys Cys Al 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 Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys His Gly Asp Leads To 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 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 With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 405 410 415 Gln Will Be Thr Pro Thr Leu Val Glu Will Be Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Only 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 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 With Cys Thr Leu Ser Glu Lys Glu Arg Gln and Lys Gln Thr Ala 515,520,525 Leu Val Glu Leu Val Lys Lys 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 Ser Ser Gln 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

1. A protein conjugate, characterized in that, Include: Polyubiquitin; The carrier binds directly to or through a linker to the aforementioned polyubiquitin; and Biomolecules bind directly to or through linkers of the aforementioned polyubiquitins or carriers. The above-mentioned polyubiquitin is composed of the following substances: (i) Receptor ubiquitin, wherein the lysine residue of ubiquitin can be replaced by arginine or alanine, but contains more than one unsubstituted lysine residue; and (ii) Donor ubiquitin, in which all lysine residues are replaced by arginine or alanine. The above protein conjugate is represented by the following formula: In the above formula, W, X, Y, and Z are different biomolecules selected from the following group: Glucagon, i.e., GCG or its analogues, is selected from proteins formed by amino acid sequences 1 to 3. Glucagon-like peptide-1 (GLP-1) or its analogues are selected from the group of proteins composed of amino acid sequences 4 to 14. Fibroblast growth factor 21 (FGF21) or its analogues are selected from proteins formed by amino acid sequences 17 to 21. Glucose-dependent insulinotropic peptides (GIPs) or their analogues, selected from the group consisting of proteins formed by amino acid sequences 22 to 26, and Interleukin-1 receptor antagonists, namely IL-1RA or its analogues, are selected from the group consisting of proteins formed by the amino acid sequences of sequences 27 and 28. Ub(A) is the receptor ubiquitin. Ub(D) is the donor ubiquitin. L can be either non-existent or a connector, each independently. A is the carrier albumin. Ub(A) and Ub(D) are connected by covalent bonds.

2. The protein conjugate according to claim 1, characterized in that, The aforementioned polyubiquitin consists of receptor ubiquitin in which lysine residues at positions 6, 11, 27, 29, 33 and 48 of the N-terminus of ubiquitin are replaced by arginine, and donor ubiquitin in which all lysine residues of ubiquitin are replaced by arginine.

3. The protein conjugate according to claim 1, characterized in that, The aforementioned polyubiquitin consists of a receptor ubiquitin formed by the amino acid sequence of sequence 30 and a donor ubiquitin formed by the amino acid sequence of sequence 31.

4. The protein conjugate according to claim 1, characterized in that, The linker described above is a polypeptide formed by repeating amino acid sequences of 1 to 30 GGGGS, EAAAK or VPPPPP.

5. The protein conjugate according to claim 4, characterized in that, The linker described above is a polypeptide formed from the amino acid sequence of sequence 29.

6. The protein conjugate according to claim 1, characterized in that, X is a GCG analog formed from the amino acid sequence of sequence 2, Y is a GLP-1 analog formed from the amino acid sequence of sequence 12, Z is an FGF21 analog formed from the amino acid sequence of sequence 20, and W is a GIP analog formed from the amino acid sequence of sequence 24 or an IL-1RA formed from the sequence of sequence 27.

7. The protein conjugate according to claim 1, characterized in that, The linker described above is a polypeptide formed from the amino acid sequence of sequence 29.

8. A pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, fatty liver, liver fibrosis, cirrhosis, liver cancer, obesity, or diabetes, characterized in that, It comprises the protein conjugate of claim 1.

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