Bifunctional fusion protein of GLP-1R agonist FGF21

Abstract

The present invention relates to fusion proteins comprising a GLP-1R (glucagon-like peptide-1 receptor) agonist and an FGF21 (fibroblast growth factor 21) compound, which are useful for the treatment of diabetes, obesity and other disorders of abnormal glycolipid metabolism.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a GLP-1R agonist-FGF21 bifunctional fusion protein, which is used for preventing and / or treating metabolic diseases such as obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH) and / or atherosclerosis, etc. BACKGROUND

[0002] Diabetes is a metabolic disease characterized by high blood sugar, which seriously endangers human health, is accompanied by sugar, fat and protein metabolic disorders caused by insulin resistance, and further causes various disabling or fatal complications. Diabetes has two types: type I diabetes and type II diabetes. Type I diabetes is an autoimmune disease, mainly caused by the destruction of pancreatic islet beta cells that produce insulin by the autoimmune system. Patients must be treated with insulin for life, and the main clinical symptoms are polydipsia, polyuria, polyphagia and weight loss. Type II diabetes accounts for about 90%-95% of diabetes cases, and the specific cause is not clear. It is mainly characterized by insulin resistance, accompanied by insufficient insulin secretion. The main clinical symptoms are polydipsia, polyuria, polyphagia, fatigue and obesity.

[0003] Non-alcoholic steatohepatitis (NASH) is a clinical syndrome with pathological changes similar to alcoholic hepatitis but without a history of excessive alcohol consumption, and is more common in middle-aged individuals, especially overweight and obese individuals. NASH is closely related to metabolic disorders such as obesity, insulin resistance, type II diabetes and hyperlipidemia. Its main features are hepatocyte macrovesicular steatosis with hepatocyte damage and inflammation, and severe cases can develop into cirrhosis. Therefore, selecting a treatment plan for diabetic patients with NASH has become a great challenge for many professional medical institutions.

[0004] Glucagon like peptide-1 (GLP-1) is a peptide hormone secreted by intestinal L cells. After specific binding with its receptor GLP-1R, GLP-1 promotes the secretion and release of insulin from pancreatic beta cells through signal transduction, thereby maintaining the balance of insulin in the body. The half-life of natural GLP-1 secreted in the blood is very short, only maintaining two minutes of activity, which is mainly due to the cleavage of the N-terminal amino acid by dipeptidyl peptidase-4 (DPP-4). At present, there are several long-acting GLP-1 receptor agonist drugs on the market worldwide, including semaglutide and liraglutide of Novo Nordisk, dulaglutide of Lilly, and GLP1 / GIP dual-target hypoglycemic drug tirzepatide (Tirzepatide). Tirzepatide has shown very outstanding hypoglycemic and weight loss effects in clinical trials, outperforming semaglutide in head-to-head competition, opening a new era of dual-target hypoglycemic drugs. The clinical advantage of multi-target agonists is that they can activate multiple signaling pathways in the human body at the same time, producing more excellent hypoglycemic effects while reducing side effects. Due to the complexity and heterogeneity of metabolic diseases such as diabetes, the therapeutic effect of a single target and a single molecule is limited, and the administration of multiple hypoglycemic drugs and clinical research is relatively complex, so a single molecule with multiple receptor agonists has become an important development direction in the field of diabetes.

[0005] Fibroblast growth factor-21 (FGF21) belongs to the FGF family and is a newly discovered endocrine hormone. It is mainly produced by the liver and also expressed in adipose tissue, pancreas and skeletal muscle. It can be secreted into the blood and participate in the regulation of the body's metabolism. Its C-terminal binds to the transmembrane protein β-klotho, and then binds to the N-terminal FGFR1c to form a stable FGF21 / β-klotho / FGFR complex, which activates the downstream tyrosine kinase phosphorylation signaling pathway. Studies have found that FGF21 has multiple physiological activities such as promoting glucose utilization, increasing insulin sensitivity, promoting fatty acid decomposition, and regulating cholesterol balance, and has the potential to treat chronic metabolic-related diseases such as diabetes, obesity, and NASH. Since FGF21 can control blood glucose while improving blood lipids, FGF21 has been highly expected by the medical community and has become a hot spot for the research and development of new drugs for type II diabetes. However, the in vivo half-life of FGF21 is very short, it is easy to form aggregates, and the bioavailability is low, which limits its drug development and clinical application.

[0006] Although GLP-1 receptor agonist drugs have strong hypoglycemic function, high-dose drugs are needed for weight loss effect, and gastrointestinal side effects often limit the use of high-dose drugs. GLP-1 / FGF21 dual agonists are expected to produce synergistic effects of hypoglycemia and weight loss, and the presence of FGF21 can also reduce liver fat and inflammation, reverse liver fibrosis, increase insulin sensitivity and improve lipoprotein, and treat NASH.

[0007] Since the in vivo half-lives of GLP-1 and FGF21 are very short, there are great challenges in their clinical application. In order to prolong the in vivo metabolic half-life of the drug, the methods commonly adopted include wrapping sodium hyaluronate slow-release microspheres, connecting chemical side chains, and constructing Fc fusion proteins, etc. Among them, the Fc fusion protein is to connect the Fc region (Hinge-CH2-CH3) of IgG antibody and the required protein to form a composition, which can prolong the half-life of the protein and improve the stability of the molecule. In order to increase the physiological activity of GLP-1 / FGF21 dual agonists in hypoglycemia, weight loss, and lipid reduction, it is very important to introduce mutations to increase their interaction with the target, enhance their in vivo biological activity, and maintain in vivo stability. Therefore, the present application aims to provide a GLP-1 / FGF21 dual functional fusion protein with excellent stability, obvious therapeutic effect, and reduced side effects, providing more medication options for patients. SUMMARY

[0008] The first aspect of the present application discloses a fusion protein comprising a first polypeptide, a second polypeptide and a third polypeptide, the first polypeptide being a GLP-1 analogue, the second polypeptide being an Fc part, and the third polypeptide being an FGF21 variant, the first polypeptide being connected to the second polypeptide through a first linker, and the third polypeptide being connected to the second polypeptide through a second linker; wherein,

[0009] The FGF21 variant is a variant of the natural FGF21 shown in SEQ ID NO: 6, and the FGF21 variant comprises an A180E mutation at the amino acid site of 180 and amino acid mutations at L98, S167 and P171 compared with the amino acid sequence shown in SEQ ID NO: 6:

[0010] The amino acid mutation at L98 is selected from any one of L98R and L98D; preferably, the amino acid mutation at L98 is L98R;

[0011] The amino acid mutation at S167 is selected from any one of S167R, S167H and S167C; preferably, the amino acid mutation at S167 is S167R;

[0012] the amino acid mutation at position P171 is selected from any one of P171A, P171C, P171E, P171G, P171H, P171Q, P171Y, P171S, P171T, and P171W; preferably, the amino acid mutation at position P171 is P171G;

[0013] and optionally further comprising a deletion of the S181 amino acid;

[0014] the amino acid sequence of the GLP-1 analogue is selected from the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2;

[0015] the amino acid sequence of the Fc moiety is selected from the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0016] In an embodiment according to the present application, the FGF21 variant comprises amino acid mutations of L98R, S167R, P171G, and A180E; or, comprises amino acid mutations of L98R, S167R, P171G, and A180E, and a deletion of the S181 amino acid.

[0017] In an embodiment according to the present application, wherein the first linker and the second linker are each independently selected from a polypeptide having an amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; preferably, the first linker and the second linker are each selected from a polypeptide having an amino acid sequence set forth in SEQ ID NO: 7, or the first linker and the second linker are each selected from a polypeptide having an amino acid sequence set forth in SEQ ID NO: 7 and SEQ ID NO: 9, respectively; preferably, the first linker and the second linker are each selected from a polypeptide having an amino acid sequence set forth in SEQ ID NO: 7.

[0018] In an embodiment according to the present application, the fusion protein further comprises an antibody light chain signal peptide having an amino acid sequence set forth in SEQ ID NO: 12.

[0019] In an embodiment according to the present application, the amino acid sequence of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; preferably, the amino acid sequence of the fusion protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; preferably, the amino acid sequence of the fusion protein is SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15; more preferably, the amino acid sequence of the fusion protein is SEQ ID NO: 13 or SEQ ID NO: 14.

[0020] In an embodiment according to the present application, the fusion protein is a homodimer fusion protein or a heterodimer fusion protein.

[0021] Another aspect of the present application provides a pharmaceutical composition comprising the fusion protein described above, and one or more pharmaceutical excipients.

[0022] The present application also provides a use of the fusion protein described above or the pharmaceutical composition described above in the preparation of a medicament, wherein the medicament is used for treating a metabolic disease selected from the group consisting of diabetes, obesity and fatty liver related diseases, preferably, the fatty liver related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

[0023] Still another aspect of the present application provides a method for treating a metabolic disease, comprising administering the fusion protein described above or the pharmaceutical composition described above to a patient in need thereof.

[0024] The present application further discloses a polynucleotide encoding the fusion protein described above. A vector comprising the polynucleotide described above. And a host cell comprising the vector described above. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1a The line graph shows the blood glucose concentration values of the bifunctional fusion protein of the present application, PBS control group, dulaglutide group and tirzepatide group in db / db mice during continuous administration. Figure 1b The line graph shows the blood glucose concentration values of the bifunctional fusion protein of the present application, PBS control group, dulaglutide group and tirzepatide group in db / db mice during continuous administration. Figure 1a The area under the curve AUC of the line graph.

[0026] Figure 2aThe blood glucose concentration values detected every day during the continuous administration of the bifunctional fusion protein of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group in ob / ob mice are shown. Figure 2b The area under the curve AUC of the line graph of Figure 2a

[0027] Figure 3a The body weight values detected every day during the continuous administration of the bifunctional fusion protein of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group in ob / ob mice are shown. Figure 3b The area under the curve AUC of the line graph of Figure 3a

[0028] Figure 4a The body weight values detected every day during the continuous administration of the bifunctional fusion protein of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group in DIO mice are shown. Figure 4b The change in body weight (%) is shown.

[0029] Figure 5a The body weight values detected every day during the continuous administration of the bifunctional fusion protein of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group in ob / ob mice are shown. Figure 5b The change in body weight (%) is shown.

[0030] Figure 6a The body weight values detected every day during the continuous administration of the bifunctional fusion protein of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group in DIO mice are shown. Figure 6b The change in body weight (%) is shown.

[0031] Figure 7 The triglyceride content in the body of the three bifunctional fusion proteins of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group after the end of the continuous administration in db / db mice is shown.

[0032] Figure 8 The total cholesterol content in the body of the three bifunctional fusion proteins of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group after the end of the continuous administration in ob / ob mice is shown.

[0033] Figure 9 The total cholesterol content in the body of the bifunctional fusion protein of the present application, the PBS control group, the dulaglutide group, and the tirzepatide group after the end of the continuous administration in DIO mice is shown.

[0034] Figure 10a and Figure 10b ​​The figure shows the content of glutamic transaminase and glutamic oxalate transaminase in vivo of the bifunctional fusion protein of the present application, PBS control group, dulaglutide group, tirzepatide group after the end of continuous administration in ob / ob mice.

[0035] Figure 11a and Figure 11b The figure shows the content of glutamic transaminase and glutamic oxalate transaminase in vivo of the bifunctional fusion protein of the present application, PBS control group, dulaglutide group, tirzepatide group after the end of continuous administration in DIO mice.

[0036] Figure 12a The figure shows the blood glucose concentration per day detected after single administration of the bifunctional fusion protein of the present application, dulaglutide group, tirzepatide group in db / db mice. Figure 12b is Figure 12a the area under the curve AUC of the line graph.

[0037] Definitions

[0038] For better understanding of the present application, the definitions and explanations of relevant terms are provided as follows.

[0039] The term "fusion protein" generally refers to a protein produced by linking (in particular, covalently linking) two or more different proteins (e.g., proteins and / or peptides), resulting in a single molecule with functional properties derived from each of the original proteins. Typically, the fusion proteins of the present application exhibit GLP-1R agonistic activity and FGF21 activity. Fusion proteins can be produced by genetic fusion (e.g., by recombinant DNA technology) or by chemical and / or enzymatic conjugation. In the fusion proteins according to the present application, the components of the fusion protein can be arranged in the order (from N-terminus to C-terminus) A-B-C or C-B-A, wherein A is a GLP-1R agonistic peptide, B is a linker molecule, and C is a human FGF21 functionally active variant.

[0040] The term "GLP-1R agonistic peptide" refers to a peptide that binds to and activates the GLP-1 receptor, such as GLP-1 (as the primary GLP-1R agonist). GLP-1R agonistic peptides can also be referred to herein simply as "GLP-1R agonists".

[0041] The term "FGF21" refers to a member of the fibroblast growth factor (FGF) family of proteins. The amino acid sequence of FGF21 (GenBank Accession No. NP_061986.1) is set forth in SEQ ID NO: 6. "FGF21 variant", "FGF21 mutant", and similar terms describe modified versions of the FGF21 protein, e.g., in which component amino acid residues are deleted, added, modified, or replaced.

[0042] An "Fc portion" as described herein consists of the hinge region, the CH2 and CH3 constant region structure of an antibody. The term "Fc" includes wild-type Fc sequences, such as human IgGl, IgG2, IgG3 or IgG4, as well as variants thereof. Variants can include one or more substitutions, additions and / or deletions of amino acids.

[0043] The term "peptide" or "polypeptide" generally refers to a polymeric form of amino acids of any length, which can include from about 2 or more, or about 3 or more, or about 4 or more, or about 6 or more, or about 8 or more, or about 9 or more, or about 10 or more, or about 13 or more, or about 16 or more, or about 21 or more amino acids, covalently linked by peptide bonds. A peptide can consist, for example, of up to 100 amino acids. The terms "polypeptide" and "protein" are used interchangeably herein.

[0044] The term "antibody light chain signal peptide" or "signal peptide" (also known as "signal sequence" or "signal domain") refers to a peptide domain in a contiguous stretch of amino acid sequence located in the N-terminal region of a precursor protein, typically a membrane-bound or secreted protein, and is involved in post-translational protein trafficking. In many cases, the signal domain is removed from the full-length protein after completion of the sorting process by specialized signal peptidases.

[0045] The term "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be in the single- or double-stranded form and can be linear or covalently closed to form a circle.

[0046] The term "vector" refers to any molecule used to transfer coding information to a host cell, including nucleic acids, plasmids, cosmids, phages, viral vectors, artificial chromosomes, and the like.

[0047] The term "host cell" is used to refer to a cell that has been transformed with a nucleic acid sequence, or is capable of being transformed with a nucleic acid sequence, and to express a selected gene of interest, and includes the progeny of the parent cell, whether or not the progeny is identical in form or gene makeup to the parent cell, so long as the selected gene is still present.

[0048] The term "cell" includes prokaryotic cells, such as bacterial cells, as well as eukaryotic cells, such as yeast cells, fungal cells, or mammalian cells. DETAILED DESCRIPTION

[0049] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are conventional products that can be obtained by commercial purchase.

[0050] Examples

[0051] Example 1: Construction of vector

[0052] The present application first synthesizes the target gene (the target gene contains GLP-1, linker, Fc (WT / variant) of IgG, linker, FGF21 variant in turn from N-terminal to C-terminal), and constructs the vector of the fusion protein by the method of molecular cloning. First, the target gene is obtained by PCR amplification, and the PCR product is detected by 1% agarose gel electrophoresis, and the target gene is recovered by a Tian Gen gel recovery kit. Under the condition of 37℃, the target gene sequence and the vector pCGS3 are digested and treated by endonuclease HindIII and PaCI (NEB America), and the digestion products are purified and recovered by a Tian Gen kit according to the manufacturer's instructions. The purified and recovered target gene and the vector are connected by ClonExpress II OneStep Cloning Kit (Novagen, China) according to the manufacturer's instructions, 37℃ connection for 30 min, to obtain the recombinant expression plasmid.

[0053] The above recombinant expression plasmid is transformed into competent cells DH5α, and the bacterial body is coated on ampicillin plates. The single colony on the plate is picked and cultured in 1 mL LB medium to extract the plasmid, which is verified correct by sequencing, and then the Tian Gen plasmid large extraction kit is used to extract the verified correct expression plasmid, which is preserved at -20℃ for standby use.

[0054] Example 2: Expression of fusion protein

[0055] The HEK293F host cells (ATCC, America) are resuscitated and cultured with resuscitation medium KOP293 (293 chemical limited high density serum-free cell culture solution), and the host cells are transfected. Taking 100 mL of cell suspension as an example, A: prepare two 15 mL sterile centrifuge tubes, add 5 mL KPM (serum-free cell transfection buffer) and 0.5 mL TA-293 (293 cell suspension chemical transfection reagent) in one of them; take another centrifuge tube, add 5 mL KPM and 100 μg plasmid, mix well, transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, mix well, and prepare the plasmid-carrier complex at room temperature for 10 minutes. Take the cells from the constant temperature shaker, add the prepared plasmid-carrier complex, and put it back into the CO2 constant temperature shaker for shaking culture. The product expression amount is determined on the sixth day after transfection.

[0056] Example 3: Purification of fusion protein

[0057] The cell culture solution of Example 2 is centrifuged at 8000 rpm for 15 min, and the supernatant is collected. The centrifuged cell culture supernatant is affinity purified by using a Protein A chromatography column (Protein A At Beads Lx, Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The equilibration buffer is 20 mM sodium phosphate dibasic-sodium phosphate monobasic buffer (PB), 150 mM NaCl, pH 7.0; the washing buffer is 20 mM PB, 0.5 M NaCl, pH 6.5; the elution buffer is 20 mM citric acid, and the pH is adjusted to 3.5 with 2 M NaOH; and the protein eluent under the target absorption peak is collected. After replacement with phosphate buffered saline (PBS), the sample is subjected to electrophoresis detection.

[0058] The present application prepares three fusion proteins by Examples 1, 2 and 3,

[0059]

[0060] Test example

[0061] Test example 1 Study on the hypoglycemic effect of bifunctional fusion protein in mice

[0062] This test example is carried out in db / db mice, ob / ob and DIO mice.

[0063] db / db mouse experiment

[0064] First, the purified fusion protein was diluted with PBS, and about 9-week-old, about 40-g ob / ob mice were randomly divided into 6 groups, i.e., a PBS control group, a dulaglutide group (purchased dulaglutide injection from Lilly Company, item number S20190022), a tirzepatide group (configuration: tirzepatide powder 15 mg, sodium chloride 4.1 mg, disodium hydrogen phosphate heptahydrate 0.7 mg, and water for injection, PH 7.0), a D1 group (GLP-1-IgG1-Fc-FGF21 group), a D2 group (GLP-1-IgG2-Fc-FGF21 group), and a D3 group (GLP-1-IgG4-Fc-FGF21 group).

[0065] Each ob / ob mouse was injected with 20 nmol / kg of protein, and the injection volume was 5 mL / kg. Four mice were injected in each group, and the injection was performed once a week for seven weeks. After the injection, the blood glucose change was observed to evaluate the hypoglycemic effect of the drug, and the experimental results are shown in FIGS. 5 and 6. Figure 1a and Figure 1b The experimental results of FIGS. 5 and 6 show that, in the ob / ob mice, D1 and D2 exhibit a better hypoglycemic effect than dulaglutide and tirzepatide, and D3 exhibits a better or equivalent hypoglycemic effect than dulaglutide and tirzepatide.

[0066] Figure 1a and Figure 1b The experimental results of FIGS. 5 and 6 show that, in the ob / ob mice, D1 and D2 exhibit a better hypoglycemic effect than dulaglutide and tirzepatide, and D3 exhibits a better or equivalent hypoglycemic effect than dulaglutide and tirzepatide.

[0067] ob / ob mouse experiment

[0068] First, the purified fusion protein was diluted with PBS, and about 9-week-old, about 40-g ob / ob mice were randomly divided into 6 groups, i.e., a PBS control group, a dulaglutide group (purchased dulaglutide injection from Lilly Company, item number S20190022), a tirzepatide group (configuration: tirzepatide powder 15 mg, sodium chloride 4.1 mg, disodium hydrogen phosphate heptahydrate 0.7 mg, and water for injection, PH 7.0), a D1 group (GLP-1-IgG1-Fc-FGF21 group), a D2 group (GLP-1-IgG2-Fc-FGF21 group), and a D3 group (GLP-1-IgG4-Fc-FGF21 group). Figure 2a and Figure 2b The experimental results of FIGS. 5 and 6 show that, in the ob / ob mice, D1 and D2 exhibit a better hypoglycemic effect than dulaglutide and tirzepatide, and D3 exhibits a better or equivalent hypoglycemic effect than dulaglutide and tirzepatide.

[0069] Figure 2a and Figure 2b The experimental results of FIGS. 5 and 6 show that, in the ob / ob mice, D1 and D2 exhibit a better hypoglycemic effect than dulaglutide and tirzepatide, and D3 exhibits a better or equivalent hypoglycemic effect than dulaglutide and tirzepatide.

[0070] DIO mouse experiment

[0071] First, the purified fusion protein was diluted with PBS, and DIO mice of about 9 weeks of age and about 40 g in weight were randomly divided into 4 groups, including a PBS group, a dulaglutide group (purchased from Eli Lilly dulaglutide injection, item number S20190022), a tirzepatide group (configuration: tirzepatide powder 15 mg, sodium chloride 4.1 mg, disodium hydrogen phosphate heptahydrate 0.7 mg, and water for injection, PH 7.0), and a D2 group (GLP-1-IgG2-Fc-FGF21 group).

[0072] The corresponding original drug or fusion protein was injected at a dose of 10 nmol / kg per DIO mouse, and the injection volume was 5 mL / kg. Four mice were injected with each original drug or fusion protein, and the injection was performed every 3 days for 25 days. The blood glucose changes after administration were observed to evaluate the hypoglycemic effect of the drug, and the experimental results are shown in Figure 3a and Figure 3b .

[0073] Figure 3a and Figure 3b The experimental results show that D2 has a better hypoglycemic effect than dulaglutide and tirzepatide in DIO mice.

[0074] Test Example 2 Weight loss effect of bifunctional fusion protein in mice

[0075] This test example was tested in db / db mice, ob / ob and DIO mice. The experimental steps were the same as in Example 1, and the body weight changes were observed after administration. The experimental results are shown in Figures 4a to 4b , Figures 5a to 5b and Figures 6a to 6b .

[0076] Figure 4a and Figure 4b The experimental results show that D1, D2, D3, dulaglutide, and tirzepatide all exhibit a certain weight loss effect compared with the PBS control group in db / db mice, and D1 and D2 have the best weight loss effect.

[0077] Figure 5a and Figure 5b The experimental results show that D1, D2, D3, dulaglutide, and tirzepatide all exhibit a significant weight loss effect compared with the PBS control group in ob / ob mice, and D1 and D2 have the best weight loss effect.

[0078] Figure 6a and Figure 6b The experimental results show that D2, dulaglutide, and tirzepatide have a significant weight loss effect compared with the PBS control group in DIO mice, and D2 has the best weight loss effect.

[0079] Test Example 3 Study on the effect of the bifunctional fusion protein on improving blood lipid in mice

[0080] This test example was carried out in db / db mice and ob / ob mice respectively. The experimental steps were the same as in Example 1. After administration, the mice were sacrificed, and the blood triglyceride or total cholesterol (TC) level was detected to evaluate the effect of the drug on improving blood lipid, and the experimental results are shown in Figure 7 , Figure 8 and Figure 9 .

[0081] Figure 7 The experimental results of D1 and D2 showed that, in db / db mice, they had significantly better effect on reducing triglyceride than dulaglutide and tirzepatide; D3 had better or equivalent effect on reducing triglyceride than dulaglutide and tirzepatide.

[0082] Figure 8 The experimental results of D1 and D2 showed that, in ob / ob mice, they had significantly better effect on reducing total cholesterol than dulaglutide and tirzepatide; D3 had better or equivalent effect on reducing total cholesterol than dulaglutide and tirzepatide.

[0083] Figure 9 The experimental results of D2 showed that, in DIO mice, it had significantly better effect on reducing serum total cholesterol than dulaglutide and tirzepatide.

[0084] Test Example 4 Study on the protective effect of the bifunctional fusion protein on liver function in mice

[0085] This test example was carried out in ob / ob and DIO mice. The experimental steps were the same as in Example 1. After administration, the mice were sacrificed, and the blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected to evaluate the protective effect of the drug on liver function, and the experimental results are shown in Figures 10a to 10b and Figures 11a to 11b .

[0086] Figure 10a and Figure 10b The experimental results of D1, D2 and D3 showed that, in ob / ob mice, they had more effective effect on reducing alanine aminotransferase and aspartate aminotransferase than dulaglutide and tirzepatide.

[0087] Figure 11a and Figure 11b The experimental results of D2 showed that, in DIO mice, it had more effective effect on reducing alanine aminotransferase and aspartate aminotransferase than dulaglutide and tirzepatide.

[0088] Test Example 5 Study on the hypoglycemic effect of the bifunctional fusion protein in mice after single administration

[0089] The test example was tested in db / db mice. The experimental procedure was the same as that of Example 1, and the blood glucose change after administration was observed clinically to evaluate the long-term hypoglycemic effect of the drug. The experimental results are shown in Table 1. Figure 12a and Figure 12b

[0090] Figure 12a and Figure 12b The experimental results of D1, D2, dulaglutide and taspoglutide showed that the blood glucose was rapidly reduced on the first day after administration; dulaglutide and taspoglutide were maintained for 1 day and rebounded rapidly; D1 was maintained for 4 days and then rebounded; D2 was maintained for 5 days and then rebounded. It can be seen that D1 and D2 showed stronger and more persistent hypoglycemic effect than dulaglutide and taspoglutide.

[0091] Sequence Listing

[0092]

[0093] ​

Claims

1. A fusion protein comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide is a GLP-1 analog, the second polypeptide is an Fc moiety, the third polypeptide is an FGF21 variant, the first polypeptide is linked to the second polypeptide via a first linker, and the third polypeptide is linked to the second polypeptide via a second linker; wherein... The FGF21 variant is a variant of the natural FGF21 shown in SEQ ID NO:

6. Compared with the amino acid sequence shown in SEQ ID NO:6, the FGF21 variant contains an A180E mutation at amino acid position 180, and amino acid mutations at positions L98, S167, and P171. The amino acid mutation at position L98 is selected from any one of the following: L98R and L98D; preferably L98R. The amino acid mutation at position S167 is selected from any one of the following: S167R, S167H, and S167C; preferably, it is S167R. The amino acid mutation at position P171 is selected from any one of the following: P171A, P171C, P171E, P171G, P171H, P171Q, P171Y, P171S, P171T, and P171W; preferably P171G; Optionally, it may further include the deletion of the S181 amino acid; The amino acid sequence of the GLP-1 analog is selected from the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2; The amino acid sequence of the Fc portion is selected from the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:

5.

2. The fusion protein of claim 1, wherein, The FGF21 variant contains amino acid mutations in L98R, S167R, P171G, and A180E; or, contains amino acid mutations in L98R, S167R, P171G, and A180E, and the deletion of the S181 amino acid.

3. The fusion protein according to claim 1 or 2, wherein, The first and second adapters are each independently selected from polypeptides with amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; preferably, both the first and second adapters are selected from polypeptides with amino acid sequences shown in SEQ ID NO:7, or the first and second adapters are selected from polypeptides with amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:9, respectively; preferably, both the first and second adapters are selected from polypeptides with amino acid sequences shown in SEQ ID NO:

7.

4. The fusion protein according to any one of claims 1-3, further comprising an antibody light chain signal peptide with the amino acid sequence SEQ ID NO:

12.

5. The fusion protein according to any one of claims 1-4, wherein the amino acid sequence of the fusion protein comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; preferably, the amino acid sequence of the fusion protein is composed of an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; preferably, the amino acid sequence of the fusion protein is SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; more preferably, the amino acid sequence of the fusion protein is SEQ ID NO:13 or SEQ ID NO:

14.

6. The fusion protein according to any one of claims 1-5, characterized in that, The fusion protein is a homodimeric fusion protein or a non-homodimeric fusion protein.

7. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 6, and one or more pharmaceutical excipients.

8. Use of a fusion protein according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament, wherein, The drug is used to treat metabolic diseases, which are selected from diseases related to diabetes, obesity, and fatty liver; preferably, the fatty liver-related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, and cirrhosis.

9. A polynucleotide encoding the fusion protein of any one of claims 1 to 6.

10. A carrier comprising the polynucleotide of claim 9.

11. A host cell comprising the vector of claim 10.

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