Fatty-acid-chain-modified GLP-1-FGF21 fusion protein, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a fatty-acid-chain-modified GLP-1-FGF21 fusion protein, and a preparation method therefor and the use thereof. In the fusion protein, after a specific mutation site is introduced at the C-terminus of FGF21, the terminus of GLP-1-FGF21 is subjected to a fatty-acid-chain modification by using a Sortase-A enzyme-mediated molecular modification technique. The fatty-acid-chain modification can achieve: extended half-life of GLP-1-FGF21; slowed terminal degradation of FGF21 by fibroblast activation protease (FAP); and low-cost operation and rapid preparation of the GLP-1-FGF21 fusion protein in an aqueous solution at room temperature. The enzyme-mediated molecular modification technique is used on the GLP-1-FGF21 fusion protein of the present invention, thereby providing significant advantages.

Description

A GLP-1-FGF21 fusion protein modified by a fatty acid chain, and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to a GLP-1-FGF21 fusion protein modified by a fatty acid chain, and a preparation method and application thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Human fibroblast growth factor 21 (FGF21) is a polypeptide composed of 209 amino acids, wherein the N-terminal of the FGF21 protein contains a signal peptide composed of 28 amino acids, so that the mature FGF21 formed after signal peptide cleavage is composed of 181 amino acids.

[0003] FGF21 is expressed in various organs and tissues in humans, such as liver, pancreas and adipose tissue. In the human body, FGF21 can be secreted into the blood circulation and induce various signal pathways and functional activities in the liver, pancreas and adipose tissue, thereby achieving the physiological functions of regulating glucose and lipid metabolism and protecting pancreatic beta cells. Studies have shown that in obese mice induced by diet or genetically manipulated, injection of FGF21 protein can significantly reduce the body weight and blood glucose level of the mice, and the triglyceride content in the serum of the mice is also significantly reduced. Further studies have shown that FGF21 can improve the insulin sensitivity of the liver, thereby alleviating glucose intolerance. Studies on mammals closer to humans have shown that FGF21 can dose-dependently reduce the body weight and body fat of experimental animals, such as administration of FGF21 to diabetic rhesus monkeys, and it was found that the fasting plasma glucose and triglyceride levels were significantly reduced. In addition, FGF21 can also activate the signal pathways of exocrine pancreatic cells and liver cells, and inhibit the output of liver glycogen.

[0004] FGF21 belongs to the FGF (fibroblast growth factor) family, however, unlike most FGFs which have broad-spectrum mitogenic ability, FGF21 does not have obvious ability to promote cell proliferation. Studies have shown that FGF21 transgenic mice do not have tumors and tissue hyperplasia and other abnormal conditions in the whole life cycle. At the same time, pharmacokinetics and drug safety experiments have shown that FGF21 does not appear hypoglycemia beyond the pharmacological dose, which indicates that FGF21 is a potential ideal drug for treating diseases such as diabetes and obesity.

[0005] Unlike most FGF family members, FGF21 activates downstream signaling pathways through both FGF receptors (FGFRs) and the co-receptor β-klotho. Neither β-klotho nor FGFR alone can activate FGF21 signaling. Structural biology studies have shown that FGF21 can be divided into two parts, with the N-terminal domain primarily involved in FGFR binding and the C-terminal domain being a relatively flexible sequence involved in β-klotho binding. FGF21 can only activate downstream signaling pathways and exert its biological effects when it forms a ternary complex with β-klotho and FGFR.

[0006] The physiological functions of FGF21 in controlling blood glucose and reducing body weight have brought hope for the treatment of related diseases, but wild-type FGF21 has the disadvantages of being easily hydrolyzed by proteases and having a small molecular weight. Its half-life is only 0.5-2 hours, which is not suitable for direct use as a drug. At the same time, the flexible region at the C-terminal end of the FGF21 protein responsible for binding to the β-klotho receptor is easily degraded by fibroblast activation protein (FAP). The main degradation site is around P171. When P171 is cleaved by FAP protease, the FGF21 molecule can no longer bind to the β-klotho co-receptor, thereby causing the FGF21 molecule to be inactivated.

[0007] The main bioactive fragment of glucagon-like peptide-1 (GLP-1) is a 30 or 31 amino acid polypeptide fragment derived from the post-translational processing of proglucagon peptide (amino acids 7-36 or 7-37 of GLP-1). In humans, GLP-1 is a hormone produced mainly by cells in the gut and belongs to the incretin class. GLP-1 receptor agonists are a new type of hypoglycemic drug in recent years. By activating the GLP-1 receptor (GLP-1R), they enhance insulin secretion in a glucose concentration-dependent manner, inhibit glucagon secretion, and can delay gastric emptying, reduce food intake through central appetite suppression, and thus achieve the effect of lowering blood glucose. Endogenous GLP-1 has a half-life of only about 2 minutes, so diabetics need to supplement exogenous GLP-1 to achieve the purpose of lowering blood glucose.

[0008] Physiologically, GLP-1 and FGF21 have strong complementarity, and the activation of GLP-1R and FGFR can more effectively reduce blood glucose, body weight and blood lipids, etc. Qi Pan et al. connected GLP-1 and FGF21 through Fc to obtain a GLP-1-Fc-FGF21 dual agonist (EBio Medicine. 2021 Jan; 63: 103202.). However, the operation method of the foregoing GLP-1-Fc-FGF21 dual agonist is complex, the molecular weight of the fusion protein constructed is large, which affects the molecular activity, and the mammalian cell expression is usually required, which is high in cost.

[0009] After directly expressing the GLP-1 and FGF21 fusion protein, the terminal of FGF21 can be modified by introducing appropriate mutation sites, for example, modified by a fatty acid chain or PEG to extend the half-life and shield the degradation of FAP protease on the terminal of FGF21. Although this method can prepare a GLP-1-FGF21 fusion protein with a smaller molecular weight, a chemical modification step needs to be introduced in the process, the process is relatively complex, and a large number of mutation sites need to be introduced in FGF21 or GLP-1 molecules to reduce non-specific modification.

[0010] Therefore, it is urgent to develop a GLP-1-FGF21 fusion protein and a preparation method thereof which are simple in process, low in cost, can extend the half-life of GLP-1-FGF21 and slow down the degradation of FAP on the terminal of FGF21. SUMMARY

[0011] The purpose of the present application is to introduce specific mutation sites at the terminal of FGF21, and then use Sortase-A enzyme-mediated coupling method to modify the terminal of GLP-1-FGF21 with a fatty acid chain to prepare a new GLP-1-FGF21 fusion protein. The fusion protein is easy to prepare, low in cost, high in coupling efficiency, can effectively extend the half-life of GLP-1-FGF21 and slow down the degradation of FAP on the terminal of FGF21.

[0012] In order to achieve the above purpose, in a first aspect, the present application provides a GLP-1-FGF21 fusion protein modified by a fatty acid chain, characterized in that the GLP-1-FGF21 fusion protein comprises GLP-1 or an active fragment thereof, a linker, an FGF21 variant or an active fragment thereof, the C-terminal of the FGF21 variant comprises a-LPXTG motif and the C-terminal is modified by a fatty acid chain, wherein X is any amino acid, and the linker is a connecting peptide.

[0013] In an embodiment of the present application, the FGF21 variant is obtained by mutation based on the sequence of wild-type human FGF21 protein, and the sequence of the wild-type human FGF21 is shown as SEQ ID NO: 1.

[0014] Further, in order to weaken the deamination reaction at position 121 and oxidation reaction at position 167, the N at position 121 and the M at position 168 of the sequence shown in SEQ ID NO: 1 are replaced by Q and L, respectively, and the sequence of the FGF21 variant is shown in SEQ ID NO: 4. The replacement of the aforementioned amino acids does not affect the activity or other functions of FGF21 per se.

[0015] Further, in order to enable Sortase-A enzyme to recognize FGF21, any 7 consecutive amino acids at positions 166-177 of the sequence shown in SEQ ID NO: 4 are replaced by LPXTGGG, wherein X represents any natural amino acid residue, and the sequence after the replacement is shown in SEQ ID NO: 5-8.

[0016] In a preferred embodiment of the present application, the sequence of the FGF21 variant is shown in SEQ ID NO: 8.

[0017] In a preferred embodiment of the present application, the GLP-1 polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11 or SEQ ID NO: 13.

[0018] In a preferred embodiment of the present application, the modification of the fatty acid chain is coupling a fatty acid chain to the side chain NH2- of the K at position 180 of the sequence shown in SEQ ID NO: 8.

[0019] In a preferred embodiment of the present application, the connecting peptide is GGGSGGGGS (SEQ ID NO: 14), (GGGGS)n, (GGGGS)n, SGGGGSGGGG (SEQ ID NO: 15), GGGGGSGGGGSSGGGGS (SEQ ID NO: 16), GGGGSGGGGSGGGG (SEQ ID NO: 17), GGGGSGGGGSGGGSGGGGS (SEQ ID NO: 18), GSPGSSSSGS (SEQ ID NO: 19), GSGSGSGS (SEQ ID NO: 20), GSGSGNGS (SEQ ID NO: 21), GGSGSGSG (SEQ ID NO: 22), GGSGSG (SEQ ID NO: 23), GGSG (SEQ ID NO: 24), GGSGNGSG (SEQ ID NO: 25), GGNGSGSG (SEQ ID NO: 26), GGNGSG (SEQ ID NO: 27) or (GGGGGS)n, n can be an integer between 1-5.

[0020] In a preferred embodiment of the present application, the connecting peptide is GGGGSGGGGS (SEQ ID NO: 28) or (GGGGS)4 (SEQ ID NO: 29).

[0021] In a preferred embodiment of the present application, the GLP-1-FGF21 fusion protein has a sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 12.

[0022] The second aspect of the present application provides a method for preparing a GLP-1-FGF21 fusion protein modified by a fatty acid chain, comprising: 1) obtaining the GLP-1-FGF21 fusion protein, 2) obtaining a conjugated polypeptide molecule modified by a fatty acid chain, and modifying the GLP-1-FGF21 fusion protein by a fatty acid chain using Sortase-A enzyme.

[0023] In an embodiment of the present application, the step of modifying by a fatty acid chain comprises: synthesizing a conjugated polypeptide molecule, mixing the conjugated polypeptide molecule with a fatty acid chain solution, stirring at room temperature to obtain a conjugated product A.

[0024] Further, the step of modifying by a fatty acid chain further comprises: mixing the conjugated product A with the GLP-1-FGF21 fusion protein, adding a Sortase-A enzyme solution, stirring at room temperature to obtain the GLP-1-FGF21 fusion protein modified by a fatty acid chain; and optionally, further purifying the GLP-1-FGF21 fusion protein modified by a fatty acid chain.

[0025] In a preferred embodiment of the present application, the conjugated polypeptide is as shown in SEQ ID NO: 10, and the modification by a fatty acid chain is coupling a fatty acid chain to the side chain NH2- of the 8th amino acid K of the sequence shown in SEQ ID NO: 10.

[0026] The third aspect of the present application provides a host cell comprising the nucleic acid molecule or the expression vector.

[0027] The fourth aspect of the present application provides a pharmaceutical composition comprising a safe and effective amount of the GLP-1-FGF21 fusion protein modified by a fatty acid chain or the GLP-1-FGF21 fusion protein modified by a fatty acid chain prepared by the method described above, and a pharmaceutically acceptable carrier.

[0028] The fifth aspect of the present application provides the use of the GLP-1-FGF21 fusion protein modified by a fatty acid chain, the preparation method, the nucleic acid molecule, the expression vector, and the host cell in the preparation of a medicament for treating or preventing hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome, and neurodegenerative diseases, in particular for delaying or preventing disease progression in type 2 diabetes, treating metabolic syndrome, treating obesity or preventing overweight, for reducing food intake, increasing energy consumption, reducing body weight, delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes; controlling blood glucose, blood lipids; delaying the progression from type 2 diabetes to insulin-requiring diabetes; regulating appetite; inducing satiety; preventing weight regain after successful weight loss; treating diseases or conditions associated with overweight or obesity; treating bulimia; treating binge eating; treating atherosclerosis, hypertension, IGT, dyslipidemia, coronary heart disease, fatty liver, treatment of beta-blocker poisoning, and in a medicament for inhibiting the movement of the gastrointestinal tract.

[0029] All the amino acid sequence numbers of FGF21 involved in the present application are numbered according to the wild-type FGF21 (as shown in SEQ ID NO: 1), and the first histidine residue site in SEQ ID NO: 1 is numbered as 1, the second as 2, and so on to the last amino acid as 181. Beneficial effects

[0030] The preparation method provided by the present application can prepare the GLP-1-FGF21 fusion protein modified by a fatty acid chain through a simple Sortase-A enzyme-mediated coupling technology, which is simple to operate, has no non-specific modification, and is operated in an aqueous solution at room temperature, thereby reducing the preparation cost and saving the preparation time; meanwhile, the GLP-1-FGF21 fusion protein modified by a fatty acid chain prepared by the preparation method prolongs the half-life of GLP-1-FGF21 and greatly slows down the degradation of FAP to the end of FGF21. BRIEF DESCRIPTION OF DRAWINGS

[0031] The concept, specific structure and technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application.

[0032] FIG. 1 is a comparison diagram of the C-terminal sequences of FGF21 proteins after four different Sortase-A couplings in Example 3, and the black bold italic part in the figure is the sequence after Sortase-A coupling.

[0033] FIG. 2 is a process schematic diagram of using Sortase-A enzyme to modify the GLP-1-FGF21 fusion protein by a fatty acid chain in Example 4.

[0034] Figure 3 is a plot of body weight change in mice after dosing in Example 7.

[0035] Figure 4 is a plot of blood glucose level change in mice after dosing in Example 7.

[0036] Figure 5 is a plot of liver pathology sections of mice at the end of dosing in Example 7, where A, B, C, D, E in the figure correspond to G1, G2, G3, G4 and G5 groups of mice, respectively, and white dots are fat tissues.

[0037] Figure 6 is a mass spectrometry identification plot of the intact GLP-1-FGF21 fusion protein after conjugation of fatty acid chains, which has a molecular weight of 24128 Da. DETAILED DESCRIPTION

[0038] DEFINITIONS

[0039] Unless otherwise indicated, the terms used in this application have the following meanings. A particular term should not be construed as indefinite or unclear if not specifically defined, but should be understood according to the ordinary meaning in the art.

[0040] Table 1 : List of abbreviations for 20 natural amino acids

[0041] The numerical range in the present application refers to each integer in the given range, for example, (GGGGS)n, n can be an integer between 1-5, i.e. n is 1, 2, 3, 4, 5.

[0042] As used herein, the term "fusion protein" refers to a protein resulting from the fusion of two or more proteins or polypeptides.

[0043] As used herein, the term "linker" refers to a commonly used linker known in the art or described in the present application, and the commonly used linker can be (GGGS)n, (GGGGS)n, (GGGGGS)n.

[0044] As used herein, the term "functional variant" refers to a protein or polypeptide that has one or more amino acids substituted, deleted or added based on the amino acid sequence of a parent protein and still substantially retains at least one biological property of the parent protein.

[0045] As used herein, the term "active fragment" refers to a protein or polypeptide that is truncated based on a parent protein but still substantially retains at least one biological property of the parent protein.

[0046] As used herein, the term "fatty acid chain" refers to a long aliphatic carbon-hydrogen chain organic compound with a carboxyl group at one end, with the general formula C(n)H(2n+1)COOH. It can be classified into four categories according to the length of the carbon chain: short-chain (containing 4-6 carbon atoms) fatty acids; medium-chain (containing 8-14 carbon atoms) fatty acids; long-chain (containing 16-20 carbon atoms) fatty acids; and very long-chain (containing 20 or more carbon atoms) fatty acids. The "fatty acid chain" coupled to the recombinant protein in the present application is preferably a fatty acid chain represented by Formula I:

[0047] -Linker-(CH2)x-COOH (Formula I)

[0048] The linker moiety includes, but is not limited to, a PEG (polyethylene glycol) segment, an amino acid such as glutamic acid, etc., and the number of (CH2) is between 10 and 20; preferably, the number of x is between 16 and 20; further preferably, the number of x is 16.

[0049] The wavy line indicates the site of connection to the FGF21 amino acid residue.

[0050] Further, the fatty acid chain described in the present application is coupled to a polypeptide sequence containing "GGGX1X2X3X4SPSYKS" through a chemical reaction, wherein X1, X2, X3, and X4 represent any amino acid or nothing, but not a lysine residue. The amino acid side chain is coupled to the side chain of the second lysine residue from the C-terminus through a chemical reaction.

[0051] Further preferably, the polypeptide sequence containing "GGGX1X2X3X4SPSYKS" is represented by SEQ ID NO: 10.

[0052] Currently, a variety of polypeptide drugs on the market are extended in half-life by increasing the fatty acid side chain to achieve non-covalent binding with plasma albumin, such as liraglutide and semaglutide of Novo Nordisk. The present application uses the purified FGF21 mutant (FGF21-P4, SEQ ID NO: 8) with GGGSPSYKS, and connects the polypeptide sequence containing "GGGX1X2X3X4SPSYKS" and modified with a fatty acid chain at the side chain of the second lysine residue from the C-terminus to FGF21-P4 through the transpeptidase Sortase-A enzyme-catalyzed ligation reaction, forming a fatty acid chain-modified FGF21 protein mutant.

[0053] As used herein, "treatment" of a subject having a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain unchanged, following treatment. Thus, treatment includes prevention, therapy, and / or cure. Prevention refers to preventing an underlying disease and / or preventing symptoms from worsening or disease from progressing. Treatment also includes any pharmaceutical use of any of the nucleic acid molecules provided herein and compositions provided herein.

[0054] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, after administration to the subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest symptoms of a disease or disorder.

[0055] As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to the subject, will have the intended prophylactic effect, e.g., prevent or delay the onset of a disease or symptoms, reduce the likelihood of a disease or symptoms from occurring. A fully prophylactically effective dose does not necessarily occur with the administration of one dose, and can only occur after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0056] As used herein, the term "subject" refers to a mammal, such as a human, a cow, and a dog.

[0057] As used herein and unless otherwise indicated, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, are generally understood either to be open- ended and non-limiting, e.g., not excluding additional unlisted elements or steps, or to be limiting, e.g., excluding additional unlisted elements or steps.

[0058] The concept, specific structure and generated technical effects of the present application will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present application.

[0059] The technical solutions of the present application will be further described below in conjunction with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[0060] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0061] Example 1: FGF21 protein and GLP-1-FGF21 fusion protein expression

[0062] 1.1, Expression plasmid construction

[0063] The Suzhou Jinyu Zhi Biological Technology Co., Ltd. was commissioned to synthesize genes capable of expressing proteins numbered FGF21-1414 (wild-type-like FGF21; mutant sites 121Q, 168L; as shown in SEQ ID NO: 4), FGF21-P1 (SEQ ID NO: 5), FGF21-P2 (SEQ ID NO: 6), FGF21-P3 (SEQ ID NO: 7), FGF21-P4 (SEQ ID NO: 8) and GLP-1-FGF21-P4 (SEQ ID NO: 9) as shown below, and cloned the genes into a pET21b vector, which was then transformed into a BL21 (DE3) (purchased from Merck) strain.

[0064] FGF21-1414 is a mutant of wild-type FGF21, referred to as "wild-type-like FGF21" (121Q, 168L), and its amino acid sequence is shown in SEQ ID NO: 4. The mutant sites 121Q and 168L are to weaken the deamination reaction at position 121 and the oxidation reaction at position 167, which do not affect its activity or other functions. Therefore, in the examples of the present application, the mutant FGF21-1414 is used as a control, and the experimental results thereof represent the results of wild-type FGF21. Therefore, all sequences in the present application have these two mutant sites. Table 2 shows the sequences and numbers of FGF21 and its variants, GLP-1 and its variants, GLP-1-FGF21-P4, and the coupled polypeptides involved in the present application.

[0065] Table 2. Sequences and numbers of FGF21 and its variants, GLP-1 and its variants, GLP-1-FGF21-P4, and the coupled polypeptides involved in the present application.

[0066] 1.2, Expression

[0067] The seeds of the overnight culture were transferred to a 500 mL TB medium containing Kana resistance at a volume ratio of 1:50, and the initial OD600 was about 0.1. The culture was incubated at 37°C and 220 rpm until the OD600 was 2.0. IPTG was added (the final concentration was 0.5 mmol / L), and the bacteria were collected after overnight culture at 37°C and 220 rpm. The protein was expressed in the form of inclusion bodies (IB).

[0068] Example 2: Renaturation and purification of FGF21 protein and GLP-1-FGF21 fusion protein

[0069] 2.1 Denaturation and dissolution of inclusion bodies

[0070] The FGF21-1414, FGF21-P1, FGF21-P2, FGF21-P3, FGF21-P4, GLP-1-FGF21-P4 inclusion bodies obtained after cell disruption and washing in Example 1 were dissolved using 8M urea and 10mM DTT. The dissolution of the inclusion bodies was carried out at room temperature for 4 hours.

[0071] 2.2 Refolding

[0072] The above dissolved inclusion body solution was added to a refolding solution containing 2M urea, 10mM cysteine and 20mM Tris-Cl buffer at pH 8.0, and incubated overnight at room temperature. Continuous stirring at 200 rpm was required during refolding.

[0073] 2.3 Hydrophobic interaction chromatography (Phenyl) purification of FGF21 proteins and GLP-1-FGF21 fusion proteins

[0074] The hydrophobic chromatography GE Capto Phenyl (purchased from GE) was eluted with a final concentration of 2M NaCl and 20mM Tris-Cl buffer, and then eluted with a gradient of 2M to 0M NaCl. The collected tubes containing the target proteins were selected according to SDS-PAGE and combined, which can capture the target proteins and remove some impurities.

[0075] 2.4 Ion exchange chromatography (Source 30Q) purification of FGF21 proteins and GLP-1-FGF21 fusion proteins

[0076] The combined proteins after hydrophobic interaction chromatography were diluted 10-fold and then subjected to ion exchange chromatography using Source 30Q, and eluted with a gradient of 0M to 1.0M NaCl, to further remove nucleic acids and some protein aggregates and impurities.

[0077] 2.5 Fine purification of FGF21 proteins and GLP-1-FGF21 fusion proteins by size exclusion chromatography

[0078] The combined proteins after ion exchange chromatography using Source 30Q were concentrated and then subjected to fine purification by size exclusion chromatography using Superdex 200, with PBS as the buffer. The purified proteins were used as the final samples, and were numbered as FGF21-1414, FGF21-P1, FGF21-P2, FGF21-P3, FGF21-P4, and GLP-1-FGF21-P4, respectively.

[0079] The purification information is shown in Table 3, and all the proteins reached electrophoretic purity.

[0080] Table 3: Concentration of purified FGF21 protein and GLP-1-FGF21 fusion protein

[0081] Example 3: Screening of cell activity of different FGF21 mutants

[0082] In order to determine the suitable method for coupling using Sortase-A enzyme, the present application first screened the activity of different sequences after coupling. Sortase-A enzyme can connect the N- and C-termini of two polypeptides through enzyme-mediated method, and the specific principle is that when the C-terminus of a polypeptide has-LPXTG (X is any amino acid) sequence, Sortase-A enzyme can recognize the C-terminus of the polypeptide, and cut off the terminal G, and connect the N-terminus of another polypeptide with GGG- starting polypeptide to form-LPXTGGG- sequence, thereby realizing the enzyme-mediated coupling of two polypeptides. In the present application, four different C-terminal sequences of FGF21 protein after Sortase-A coupling were designed (as shown in Figure 1), and their cell activities were tested.

[0083] In order to further verify the activity of FGF21-1414 and its mutants in the present application, the activation of ERK signaling pathway of FGF21-1414 and its mutants was tested using HEK293 cells overexpressing β-Klotho (cells were constructed by Kanglong Huazheng). The specific experimental steps are as follows: the HEK293 cells overexpressing β-Klotho were cultured using DMEM + 10% FBS + 1* PS cell culture medium, and 20 μL of cell suspension was taken when the cells were cultured to the exponential phase and added to a white 384 assay plate, appropriate medium and appropriate cell density (5000 / well) were added, and the plate was incubated at 37°C under 5% CO2. FGF21-1414 and its mutants were diluted in the medium from 3 μM / 6 μM / 15 μM starting, 3-fold gradient, and 10 μl of compound was added to each well of the cell experiment plate. After adding the tested protein, the cells were incubated in a 37°C 5% CO2 incubator for 0.5 hours. After removing the cell supernatant, 16 μL of cell lysis buffer was added, and the plate was shaken at room temperature for 30-60 minutes. 4 μL of premixed antibody solution prepared in the detection buffer was added, the plate was sealed with a sealing plate, and the fluorescence emission at 665 nm and 620 nm was read after incubation at room temperature overnight. The results are shown in Table 4. Among the four different Sortase-A coupled FGF21 proteins designed in the present application, FGF21-P4 has a cell activity close to that of the wild-type FGF21, i.e. FGF21-1414, which can be used for further research.

[0084] Table 4: Cell activity test of four different Sortase-A coupled FGF21 proteins

[0085] Example 4: Fatty acid chain modification of GLP-1-FGF21 fusion protein using Sortase-A enzyme

[0086] In this example, we used Sortase-A enzyme to modify the fatty acid chain of GLP-1-FGF21-P4, and the specific operation steps are shown in Figure 2. The coupling polypeptide molecule GGGSPSYKS was synthesized by Nanjing Kingsray Company, and the specific modification steps are as follows: Take an appropriate amount of polypeptide molecule GGGSPSYKS solution (10 mg / ml, pH 9.5) and fatty acid chain solution (10 mg / ml) with a molar ratio of 1:1.2 and a volume ratio of 1:1 (the volume of the polypeptide molecule is supplemented with pH 9.5 carbonate buffer), mix quickly, and stir at room temperature for 15-30 min to obtain the coupling product A. Take an appropriate amount of GLP-1-FGF21-P4 recombinant protein solution (0.5-1.0 mg / ml, pH 8.0), add 10 times the molar amount of GLP-1-FGF21-P4 to the coupling product A (i.e. fatty acid chain modified coupling polypeptide molecule) solution, then add Sortase-A solution with a molar ratio of 50:1 (GLP-1-FGF21-P4:Sortase-A), and finally add 50 mM CaCl2 solution to make the final concentration of CaCl2 in the coupling reaction system 5 mM. After stirring at room temperature for 16-24 h, terminate the coupling reaction and perform anion chromatography (GE Q-FF) purification. The final product was analyzed by iodine staining SDS-PAGE, and the purity was more than 95%. The mass spectrometry identification results of GLP-1-FGF21 fusion protein after coupling fatty acid chain are shown in Figure 6.

[0087] Example 5: Cell activity experiment of GLP-1-FGF21 fusion protein modified by fatty acid chain

[0088] In order to further verify the cell activity of GLP-1-FGF21-P4-FA (FA = Fatty Acid) modified by fatty acid chain, we detected the cell activity of the molecule, and the specific method was consistent with the method described in Example 3. The specific results are shown in Table 5.

[0089] Table 5 Cell activity test of GLP-1-FGF21-P4-FA modified by fatty acid chain

[0090] From the above cell activity experiment, it can be known that the cell activity of GLP-1-FGF21-P4 fusion protein fused with GLP-1 is similar to that of FGF21-P4, but the activity of GLP-1-FGF21-P4-FA after modification of the fatty acid chain is greatly enhanced, and reaches the similar activity of wild type FGF21, i.e. FGF21-1414, which may be because the modification of the fatty acid chain changes the conformation of FGF21 and enhances its affinity to the receptor.

[0091] Example 6: FAP protease enzyme cutting experiment of GLP-1-FGF21 fusion protein modified by fatty acid chain

[0092] FAP is a protease existing in vivo, which can specifically cut the amino acids between 171-172 of FGF21, causing the C-terminal of FGF21 injected into the body or existing in the body to be incomplete, and thus unable to bind to the β-klotho receptor in vivo, losing activity and tissue specificity. This embodiment utilizes this property to perform an in vitro FAP (purchased from BPS, item number FAP-H5244) enzyme cutting experiment to test whether the GLP-1-FGF21-P4 molecule modified by the fatty acid chain has the property of slowing down the hydrolysis of FAP protease. The experiment was performed according to the literature (http: / / dx.doi.org / 10.1016 / j.molmet.2016.07.003), and the results are shown in Table 6. When the negative control FGF21-1414 is completely cut by FAP (i.e. 100% cut by the enzyme), GLP-1-FGF21-P4-FA is only about 20% to 30% cut by the enzyme, and this process is not affected by the presence or absence of HSA (human serum albumin).

[0093] Table 6 FAP protease enzyme cutting experiment of wild type GLP-1-FGF21-1414 and mutant GLP-1-FGF21-P4-FA

[0094] Example 7: pharmacodynamic experiment of GLP-1-FGF21-P4-FA modified by fatty acid chain in DIO mouse model

[0095] In order to further verify the activity of the GLP-FGF21 mutant fusion protein in the present application, we selected one of the FGF21 mutant fusion proteins, GLP-1-FGF21-P4-FA, and used 6-7 week old ob / ob mice (SPF level, Changzhou Cavens Experimental Animal Co., Ltd.). After the adaptation period, the mice were fed with high-fat feed for 3 weeks, and then entered the administration phase with a 4-week administration cycle. After the animals entered the experimental period, they were continuously fed with high-fat feed for 7 weeks to establish an obese DIO mouse model. The specific administration scheme is as follows:

[0096] G1 group is ordinary feed feeding animals (i.e. Lean Mice), as a control, a total of 10.

[0097] At the same time, the high-fat feed animals were grouped, Day 1 as the first day of administration, serum TC, LDL-C, fasting blood glucose, body weight data before administration as grouping basis, the animals were divided into 5 groups, 10 in each group, the specific grouping information is as follows: G1 group model (vehicle control group, i.e. ob / ob-Neg Ctr), G2 group model (Semaglutide control group, 30 nmol / kg, sc, q3d), G3 group model (GLP-1-FGF21-P4-FA experimental group, 10 nmol / kg, sc, q3d), G4 group model (GLP-1-FGF21-P4-FA experimental group, 30 nmol / kg, sc, q3d), G5 group model (GLP-1-FGF21-P4-FA experimental group, 60 nmol / kg, sc, q3d). And continue to monitor the changes of blood glucose and body weight, on the 30th day, the liver pathological changes of each group of mice were detected to test the effect of the molecules on liver and lipid metabolism, and the results are shown in Table 5 and Figures 3-5.

[0098] Table 7 Comparison of total cholesterol TC and low density lipoprotein LDL-C before and after administration of different administration groups on the 24th day

[0099] The above data analysis shows that GLP-1-FGF21-P4-FA is significantly better than the control molecule Semaglutide in blood glucose, body weight and lipid metabolism, and has obvious dose dependence.

[0100] The above, the preferred embodiments of the application are described in detail, and the embodiments of the application are described. However, the application is not limited to the above-mentioned embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A GLP-1-FGF21 fusion protein modified by a fatty acid chain, characterized in that, The GLP-1-FGF21 fusion protein comprises GLP-1 or an active fragment thereof, a linker, an FGF21 variant or an active fragment thereof, wherein the FGF21 variant or the active fragment thereof comprises a C-terminal -LPXTG motif or other motif that can be recognized by Sortase-A enzyme and a C-terminal fatty acid chain modification, wherein X is any amino acid, and the linker is a connecting peptide.

2. The fatty acid chain-modified GLP-1-FGF21 fusion protein according to claim 1, wherein, The FGF21 variant is mutated based on the sequence of wild-type human FGF21 protein, and the sequence of the wild-type human FGF21 is shown as SEQ ID NO:

1.

3. The fatty acid chain-modified GLP-1-FGF21 fusion protein according to claim 1 or 2, wherein, The mutation comprises replacing N at position 121 and M at position 168 of the sequence shown in SEQ ID NO: 1 with Q and L, respectively, and the sequence of the FGF21 variant is shown as SEQ ID NO:

4.

4. The fatty acid chain-modified GLP-1-FGF21 fusion protein according to any one of claims 1-3, wherein, The sequence of the FGF21 variant is shown as any one of SEQ ID NO: 5-8. Preferably, the sequence of the FGF21 variant is shown as SEQ ID NO:

8.

5. The fatty acid chain-modified GLP-1-FGF21 fusion protein of claim 4, wherein, The GLP-1 polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11 or SEQ ID NO:

13.

6. The fatty acid chain-modified GLP-1-FGF21 fusion protein according to claim 5, wherein, The fatty acid chain modification is coupling a fatty acid chain to the side chain NH2- of the amino acid K at position 180 of the sequence shown in SEQ ID NO:

8.

7. The fatty acid chain-modified GLP-l-FGF21 fusion protein according to any one of claims 1 to 6, wherein, The connecting peptide is GGGSGGGGS (SEQ ID NO: 14), (GGGGS)n, (GGGGS)n, SGGGGSGGGG (SEQ ID NO: 15), GGGGGSGGGGSSGGGGS (SEQ ID NO: 16), GGGGSGGGGSGGGG (SEQ ID NO: 17), GGGGSGGGGSGGGSGGGGS (SEQ ID NO: 18), GSPGSSSSGS (SEQ ID NO: 19), GSGSGSGS (SEQ ID NO: 20), GSGSGNGS (SEQ ID NO: 21), GGSGSGSG (SEQ ID NO: 22), GGSGSG (SEQ ID NO: 23), GGSG (SEQ ID NO: 24), GGSGNGSG (SEQ ID NO: 25), GGNGSGSG (SEQ ID NO: 26), GGNGSG (SEQ ID NO: 27), or (GGGGGS)n, n can be an integer between 1-5; preferably, the connecting peptide is GGGGSGGGGS (SEQ ID NO: 28) or (GGGGS)4 (SEQ ID NO: 29).

8. The fatty acid chain-modified GLP-1-FGF21 fusion protein according to any one of claims 1-7, wherein, The sequence of the GLP-1-FGF21 fusion protein is shown as SEQ ID NO: 9 or SEQ ID NO:

12.

9. The method of preparing a fatty acid chain-modified GLP-1-FGF21 fusion protein according to any one of claims 1 to 8, wherein, Comprise: 1) obtaining the GLP-1-FGF21 fusion protein, 2) obtaining a conjugated polypeptide molecule modified with a fatty acid chain, and modifying the GLP-1-FGF21 fusion protein with a fatty acid chain using Sortase-A enzyme.

10. The method of claim 9, wherein the GLP-1 -FGF21 fusion protein is prepared by, The step of fatty acid modification comprises: synthesizing a conjugated polypeptide molecule, mixing the conjugated polypeptide molecule with a fatty acid chain solution, stirring at room temperature to obtain a conjugated product A.

11. The method of claim 10, wherein the GLP-1 -FGF21 fusion protein is prepared by, The step of fatty acid modification further comprises: mixing the conjugated product A with the GLP-1-FGF21 fusion protein, adding a Sortase-A enzyme solution, stirring at room temperature to obtain the GLP-1-FGF21 fusion protein modified with a fatty acid chain; optionally, further purifying the GLP-1-FGF21 fusion protein modified with a fatty acid chain.

12. The production method according to any one of claims 10 to 11, wherein The conjugated polypeptide is shown in SEQ ID NO: 10, and the fatty acid chain modification is coupling a fatty acid chain to the side chain NH2- of the 8th amino acid K of the sequence shown in SEQ ID NO:

10.

13. A pharmaceutical composition, characterized by, A safe and effective amount of the GLP-1-FGF21 fusion protein modified with a fatty acid chain as claimed in any one of claims 1-8 or prepared by the method of any one of claims 9-12, and a pharmaceutically acceptable carrier.

14. The GLP-1-FGF21 fusion protein modified with a fatty acid chain as claimed in any one of claims 1-8, the method of preparation of any one of claims 9-12 for use in the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome and neurodegenerative diseases, in particular for delaying or preventing disease progression in type 2 diabetes, treating metabolic syndrome, treating obesity or preventing overweight, for reducing food intake, increasing energy expenditure, reducing body weight, delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes; controlling blood glucose, blood lipids; delaying the progression from type 2 diabetes to insulin-requiring diabetes; regulating appetite; inducing satiety; preventing weight regain after successful weight loss; treating diseases or conditions associated with overweight or obesity; treating bulimia; treating binge eating; treating atherosclerosis, hypertension, IGT, dyslipidemia, coronary heart disease, fatty liver, treatment of beta-blocker poisoning, for use in drugs for inhibiting the movement of the gastrointestinal tract.

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