Fusion protein and use thereof

Abstract

A fusion protein and a use thereof. The fusion protein comprises an FGF21 mutant protein. The FGF21 mutant protein comprises modifications based on a wild-type human FGF21 sequence, the modifications being: deletion of a proline residue at position 171, or formation of a pair of disulfide bonds before and after the proline residue at position 171 by means of substitution, deletion, or addition of one or more amino acid residues. The FGF21 mutant protein is resistant to enzymatic degradation at the C-terminus thereof by a fibroblast activation protein (FAP), maintains C-terminus activity for binding to a β-klotho receptor, thereby extending an in vivo half-life and the pharmacological effect. The fusion protein enhances the therapeutic efficacy thereof in weight loss, blood glucose reduction, and non-alcoholic steatohepatitis (NASH) by fusing the FGF21 mutant protein with other molecules, such as GLP-1.

Description

A fusion protein and application thereof TECHNICAL FIELD

[0001] The present application relates to a fusion protein comprising FGF21 mutant protein and application thereof, and belongs to the field of biotechnology. BACKGROUND

[0002] Human fibroblast growth factor 21 (FGF21) is a polypeptide composed of 209 amino acids, wherein the N-terminal of FGF21 protein contains a signal peptide composed of 28 amino acids, and thus the mature FGF21 formed after signal peptide cleavage is composed of 181 amino acids (SEQ ID NO: 1).

[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 liver, pancreas and adipose tissue, thereby realizing 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 mice, and the triglyceride content in the serum of 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 hepatic glucose output.

[0004] FGF21 belongs to the FGF (fibroblast growth factor) family, however, unlike most FGFs which have broad 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, where the N-terminal domain is mainly related to the binding of FGFR, and the C-terminal domain is a relatively flexible sequence related to the binding of β-klotho. FGF21 can only activate downstream related signaling pathways and exert its biological effects after forming 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, too small molecular weight, and a half-life of 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 of FGF21 protein responsible for binding to the β-klotho receptor is easily degraded by fibroblast activation protein (FAP), and 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, resulting in inactivation of the FGF21 molecule. In the face of this difficulty, the pharmaceutical industry has prepared long-acting fusion proteins by performing site-directed mutagenesis of the amino acids at the cleavage site, or has connected polyethylene glycol, fatty acid chains, etc. to the polypeptide backbone to improve the half-life of FGF21. In US 8,034,770B2, some mutants based on the human wild-type FGF21 polypeptide sequence are described, in which P171G or P171A can effectively slow down the hydrolysis of the C-terminal of FGF21 protein by FAP protease, but whether according to the report of this patent or in our in vitro experiments, P171G still exists to some extent in the form of inactivation by FAP hydrolysis. Finding new and more effective mutants to weaken the hydrolysis of FAP protease is of great significance for further improving the drugability of FGF21 protein.

[0007] Further, FGF21 or its muteins can also be further enhanced in terms of weight loss, blood glucose reduction, blood lipid reduction, and NASH treatment by fusing or combining with other types of molecules. In 2021, Qi Pan et al. reported a GLP-1-FGF21 dual-target protein molecule using Fc domain fusion GLP-1 and FGF21 homodimer structure (https: / / doi.org / 10.1016 / j.ebiom.2020.103202), which showed stronger weight loss, blood glucose reduction, and blood lipid reduction than FGF21 alone or GLP-1 alone. Exploring better FGF21 muteins and better GLP-1 and FGF21 fusion methods is of great significance for the development of new blood glucose-lowering, weight-reducing, lipid-lowering, and NASH drugs. SUMMARY

[0008] The natural wild-type human FGF21 protein has an amino acid sequence as shown in SEQ ID NO: 1, and the proline residue (Pro) at position 171 is the enzyme cleavage site of FAP protease. The purpose of the present application is to reduce the hydrolysis rate of FGF21, prolong its in vivo half-life and drug efficacy by changing the enzyme cleavage site of wild-type FGF21 and / or the structure near the enzyme cleavage site, and to further enhance the therapeutic effect of FGF21 muteins in weight loss, blood glucose reduction, and non-alcoholic steatohepatitis (NASH) by fusing FGF21 muteins with other molecules such as GLP-1.

[0009] To achieve the above-mentioned purpose, one aspect of the present application provides a fusion protein comprising an FGF21 mutein linked to a heterologous protein. In a specific embodiment, the FGF21 mutein comprises an amino acid sequence having the following a or b change based on the wild-type human FGF21 sequence:

[0010] a. deleting the proline residue at position 171;

[0011] b. forming a pair of disulfide bonds at or adjacent to the proline residue at position 171 by replacing, deleting, or adding one or more amino acid residues.

[0012] One aspect of the present application is to directly delete the proline residue at position 171 (e.g., the sequences shown in SEQ ID NO: 4, 10, 16, and 22), so that the FGF21 protein loses the enzyme cleavage site of FAP and thus will not be hydrolyzed. Alternatively, the FGF21 mutein comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0013] Another aspect of the present application is to introduce two cysteine residues adjacent to the proline residue at position 171 by substitution and / or addition.

[0014] Optionally, a pair of disulfide bonds are formed before and after the Pro 171 by substitution, deletion or addition of amino acid residues adjacent to the Pro 171, which blocks the cleavage site of the Pro 171 and thus makes it difficult to be hydrolyzed.

[0015] Optionally, the disulfide bond can also be formed directly at the 171 site, i.e. the proline residue at the 171 site is substituted, and one or more amino acid residues adjacent to the 171 site are substituted, deleted or added to form a disulfide bond between the 171 site and the adjacent site.

[0016] Optionally, the adjacent site refers to within three sites before and after; further preferably within two sites before and after.

[0017] In another embodiment of the present application, two cysteine residues are introduced within three sites before and after the proline residue at the 171 site by substitution and / or addition.

[0018] Optionally, one cysteine residue is introduced within three sites before and after the proline residue at the 171 site by substitution and / or addition of one or two amino acid residues.

[0019] Optionally, the two cysteine residues forming the disulfide bond are separated by 1, 2 or 3 amino acids.

[0020] Optionally, the two sites where the cysteine residues are substituted or added are separated by 1, 2 or 3 amino acids, or the two sites where the cysteine residues are substituted or added are separated by 1, 2 or 3 amino acids by adding amino acid residues.

[0021] Optionally, one amino acid residue is substituted with a cysteine residue within the first three sites before the 171 site, or within the first two sites before and after the 171 site, and one amino acid residue is substituted with a cysteine residue within the last three sites after the 171 site, or within the first two sites before and after the 171 site; one or two amino acid residues are added between the 171 site and the site before the 171 site where the cysteine residue is substituted, and / or one or two amino acid residues are added between the 171 site and the site after the 171 site where the cysteine residue is substituted.

[0022] Optionally, the one or two amino acid residues added are selected from glycine, alanine and serine. Preferably, the added amino acid residue is glycine.

[0023] By spacing two cysteine residues forming disulfide bond by 2 or 3 amino acid residues, the introduction of the aforementioned spacer amino acid as glycine, alanine or serine (preferably glycine) can reduce the formation of mispaired dimers of FGF21 mutants.

[0024] Optionally, 170G is replaced by 170C, and 172S is replaced by 172C, and one or two amino acid residues are inserted between 171P and 172C.

[0025] Optionally, the FGF21 mutant protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 23, 24, 25, 26 and 27. Among them, SEQ ID NO: 4, 16, 22 are sequences formed by adding 1 alanine, serine, glycine residue respectively before the histidine at position 1 of SEQ ID NO: 10 on the basis of SEQ ID NO: 10, and SEQ ID NO: 5, 17, 23, SEQ ID NO: 6, 18, 24, SEQ ID NO: 7, 19, 25, SEQ ID NO: 8, 20, 26, SEQ ID NO: 9, 21, 27 and SEQ ID NO: 15 are similar to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, respectively, and will not be repeated here.

[0026] In another embodiment, the proline residue at position 171 is replaced, and one or more amino acid residues at its adjacent positions are replaced, deleted or added to form a disulfide bond between position 171 and its adjacent positions.

[0027] Optionally, 121N of the wild-type human FGF21 sequence is replaced by 121Q, and 168M is replaced by 168L, which can weaken the deamination reaction at position 121 and the oxidation reaction at position 168.

[0028] Optionally, 1 alanine, glycine or serine residue is added before the histidine residue at position 1 of the wild-type human FGF21 sequence, which can be used to remove the start codon methionine in prokaryotic expression (such as E. coli expression).

[0029] Further, the heterologous protein comprises an Fc fragment of an immunoglobulin; preferably, the immunoglobulin is hlgG4. Alternatively, the Fc fragment of the immunoglobulin is a single-chain Fc (i.e. the C-terminal of one Fc monomer in a Fc homodimer is connected to the N-terminal of another Fc monomer by a linker of a specific length), preferably, the amino acid sequence of the scFc is shown as SEQ ID NO: 32-33.

[0030] Further preferably, the heterologous protein comprises an Fc mutant of an immunoglobulin. In a preferred embodiment of the present application, the Fc mutant of the immunoglobulin comprises an amino acid sequence shown as SEQ ID NO: 28.

[0031] Further, the FGF21 mutant protein is connected to the heterologous protein by a linker; preferably, the amino acid sequence of the linker is shown as SEQ ID NO: 34 or SEQ ID NO: 35.

[0032] Further, the fusion protein further comprises another fusion protein comprising GLP-1 or a mutant thereof, which forms a new fusion protein with the fusion protein comprising the FGF21 mutant protein described above. GLP-1 (glucagon-like peptide-1) is a hormone mainly produced by L cells in the intestine, which can reduce blood glucose by activating GLP-1 receptors, and the mutant GLP-1 described herein should also have the function of reducing blood glucose. The wild-type GLP-1 has an amino acid sequence shown as SEQ ID NO: 36, and possible GLP-1 mutants also include sequences shown as SEQ ID NO: 37-38. The new fusion protein comprising GLP-1, scFc and FGF21 has an amino acid sequence shown as SEQ ID NO: 29.

[0033] The third aspect of the present application provides a dimer comprising the fusion protein described above, and further the dimer is a homo- or hetero-dimer.

[0034] In a preferred embodiment of the present application, the dimer is a hetero-dimer forming a knob-into-hole (KIH) structure. Preferably, the knob-into-hole structure is located in the Fc mutant segment of the fusion protein.

[0035] Further, the dimer further comprises another fusion protein comprising GLP-1 or a mutant thereof, which forms a hetero-dimer with the fusion protein comprising the FGF21 mutant protein described above, and the specific amino acid sequences are shown as SEQ ID NO: 30-31.

[0036] The fourth aspect of the present application provides a vector containing a nucleic acid sequence capable of expressing the above molecule.

[0037] The fifth aspect of the present application provides a genetically engineered cell containing the above vector.

[0038] The sixth aspect of the present application provides a method for preparing a protein, comprising the steps of:

[0039] 1) constructing a vector as described above;

[0040] 2) transfecting the constructed vector into a host cell;

[0041] 3) culturing the host cell under conditions suitable for expression;

[0042] 4) isolating the FGF21 mutant protein and its fusion protein of the present application from the culture.

[0043] The seventh aspect of the present application provides a pharmaceutical composition containing the FGF21 mutant protein and its fusion protein as described above and a pharmaceutically acceptable excipient.

[0044] The eighth aspect of the present application provides the use of the FGF21 mutant protein and its fusion protein in controlling blood glucose, blood lipids, NASH and reducing body weight. Advantages

[0045] The FGF21 mutant protein provided by the present application can resist enzymatic degradation of the C-terminal by FAP enzyme and maintain the activity of the C-terminal, i.e. binding to the beta-klotho (KLB) receptor, thereby prolonging its in vivo half-life and drug efficacy. Compared with the mutant P171G, the FGF21 mutant in the embodiments of the present application has stronger ability to slow down the hydrolysis of FAP protease. The GLP-1-scFc-FGF21 fusion protein provided by the present application has a unique 1-to-1 molecular configuration, which solves the problem of short half-life of Fc homodimer form (i.e. 2-to-2 molecular form). At the same time, the C-terminal of FGF21 is introduced into a disulfide bond to avoid FAP protease hydrolysis. The production process of this molecule is based on E. coli refolding, which is low in cost and easy to scale up. In addition, the N-terminal of the GLP-1 mutant in the fusion protein is designed with HG, which weakens protease hydrolysis and prolongs the half-life.

[0046] All the amino acid sequence numbers involved in the present application are based on the wild-type FGF21 (SEQ ID NO: 1), and the first amino acid site number in SEQ ID NO: 1 is 1, the second is 2, and so on to the last amino acid.

[0047] The concept, specific structure and generated technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purposes, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0048] Fig. 1 is a schematic diagram of a molecular simulation structure forming a disulfide bond on both sides of a proline residue at position 171 (taking 170C / 173C as an example, SEQ ID NO: 5);

[0049] Fig. 2 is a SDS-PAGE electrophoresis diagram of the protein after expression and purification in Example 2, wherein M is Marker;

[0050] Fig. 3 is experimental data of GLP-1-scFc-FGF21 fusion protein 1300 in reducing body weight, reducing blood sugar and reducing blood lipid in DIO mice in Example 6. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be further described in detail below in combination 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 of the present application intended to be protected.

[0052] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0053] Example 1 Construction of Recombinant Expression Vector and Expression

[0054] 1.1 Construction of expression plasmid

[0055] Suzhou Jinyuzhi Biotechnology Co., Ltd. was commissioned to synthesize genes capable of expressing proteins numbered 1414, 1407, 1408, 1409, 1410, 1411, 1412, 1413 and 1300 as shown below, and clone the genes into pET21b vector (Merck), and then transform into BL21(DE3) strain (Merck).

[0056] FGF21 (121Q, 168L) mutant 1414, the amino acid sequence of which is shown as SEQ ID NO: 2, the mutation sites 121Q and 168L are to weaken the deamination reaction at position 121 and the oxidation reaction at position 168, which do not affect the activity or other functions thereof (as disclosed in WO2011154349A2, CN113728013A), therefore, in the examples, mutant 1414 is used as a control, and the experimental results thereof represent the results of wild-type FGF21, in addition, an alanine residue is added before the first histidine residue of FGF21 wild type for removing the start codon methionine in prokaryotic expression; FGF21 (P171G) mutant 1407, the amino acid sequence of which is shown as SEQ ID NO: 3; FGF21 (Del-P171) mutant 1408, the amino acid sequence of which is shown as SEQ ID NO: 4; FGF21 (170C / Ins-172G / 173C) mutant 1409, the amino acid sequence of which is shown as SEQ ID NO: 5; FGF21 (170C / 172C) mutant 1410, the amino acid sequence of which is shown as SEQ ID NO: 6; FGF21 (170C / 173C) mutant 1411, the amino acid sequence of which is shown as SEQ ID NO: 7; FGF21 (170C / Ins-172A / 173C) mutant 1412, the amino acid sequence of which is shown as SEQ ID NO: 8; FGF21 (170C / Ins-172S / 173C) mutant 1413, the amino acid sequence of which is shown as SEQ ID NO: 9; GLP-1-scFc-FGF21 fusion protein 1300, the amino acid sequence of which is shown as SEQ ID NO: 29, which is fused by GLP-1 protein and scFc and FGF21 (170C / Ins-172G / 173C) mutant 1409.

[0057] 1.2, Expression

[0058] The seeds of the overnight culture were transferred to a 500 mL TB medium containing Kana resistance at a volume ratio of 1:50, the initial OD600 was about 0.1, and the culture was incubated at 37°C, 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, 220 rpm. The protein was expressed in the form of inclusion body (IB).

[0059] Example 2 Renaturation and purification of FGF21 mutants and fusion proteins

[0060] 2.1 Denaturation and dissolution of inclusion body

[0061] The inclusion bodies obtained from cell disruption and washing in Example 1 were solubilized using 8 M urea and 10 mM DTT. The solubilization of the inclusion bodies was performed at room temperature for 4 hours.

[0062] 2.2 Refolding

[0063] The solubilized inclusion body solution was added to a refolding solution containing 2 M urea, 10 mM cysteine, and 20 mM Tris-Cl buffer, pH 8.0, and incubated overnight at room temperature. The refolding solution was continuously stirred at 200 rpm during the refolding.

[0064] 2.3 Hydrophobic interaction chromatography (Phenyl) purification of FGF21 and mutant proteins

[0065] The solubilized inclusion body solution was added to a refolding solution containing 2 M urea, 10 mM cysteine, and 20 mM Tris-Cl buffer, pH 8.0, and incubated overnight at room temperature. The refolding solution was continuously stirred at 200 rpm during the refolding.

[0066] 2.4 Ion exchange chromatography (Source 30Q) purification of FGF21 and mutant proteins

[0067] The solubilized inclusion body solution was added to a refolding solution containing 2 M urea, 10 mM cysteine, and 20 mM Tris-Cl buffer, pH 8.0, and incubated overnight at room temperature. The refolding solution was continuously stirred at 200 rpm during the refolding.

[0068] 2.5 Size exclusion chromatography (Superdex 200 pg) purification of FGF21 and mutant proteins

[0069] The solubilized inclusion body solution was added to a refolding solution containing 2 M urea, 10 mM cysteine, and 20 mM Tris-Cl buffer, pH 8.0, and incubated overnight at room temperature. The refolding solution was continuously stirred at 200 rpm during the refolding.

[0070] The purification information is shown in Table 1. All proteins reached the purity of electrophoretic purity.

[0071] Table 1. Concentration of purified FGF21 mutants and fusion proteins

[0072] The SDS-PAGE analysis of the proteins purified from the final FGF21 mutant 1414 and other mutants (Figure 2) shows that FGF21 mutant 1414 and mutants 1407, 1408 and 1409 all exist as monomers, among which the additional disulfide bond in FGF21 mutant 1409 has completely formed intramolecular disulfide bond and has not incorrectly formed intermolecular disulfide bond to cause the formation of FGF21 dimer. FGF21 mutant 1410 has formed a large amount of dimer, indicating that the additional disulfide bond in the molecule cannot form the correct intramolecular disulfide bond, but incorrectly forms a large amount of intermolecular disulfide bond. Comparing the molecular design of 1409 and 1410, 1409 has inserted a glycine (Ins-G172) after P171, and the insertion of G172 can make more space between C170-C173 to form intramolecular disulfide bond. Replacing the inserted amino acid G172 with A172 (FGF21 mutant 1412) or S172 (FGF21 mutant 1413) can have a similar technical effect, but still a small amount of incorrect dimer can be formed. FGF21 mutant 1411 is to move the mutation after P171 to 173 (SEQ ID NO: 7), and the mutant only produces a small amount of incorrect dimer.

[0073] Example 3 FAP Enzymatic Cleavage Test in Vitro

[0074] FAP is a protease existing in vivo, which can specifically cleave the amino acids between 171-172 of FGF21, causing the C-terminal of FGF21 injected into the body or existing in vivo to be incomplete, and thus unable to bind to the β-klotho receptor in vivo, losing activity and tissue specificity. This embodiment uses this property to perform an in vitro FAP (purchased from Peprotech, item number FAP-H5244) enzymatic cleavage experiment to select FGF21 mutants that are not cleaved by the enzyme. The experiment is performed according to the literature (http: / / dx.doi.org / 10.1016 / j.molmet.2016.07.003), and the results are shown in Table 2. When the negative control FGF21 mutant 1414 is completely cleaved by FAP, about half of FGF21 mutant P171G (1407) is also cleaved under the experimental conditions, while mutants 1408-1413 (1410 not detected) and fusion protein 1300 designed in the application have only a small amount of enzymatic cleavage or are not cleaved.

[0075] Table 2 FGF21 mutant and fusion protein cleavage experiment by FAP

[0076] Example 4 FGF21 Mutant and β-Klotho Receptor Binding Test

[0077] The extracellular domain of human β-Klotho receptor protein (C-terminal fusion with Poly-His tag) was expressed and purified by our company, and the sequence was from Uniprot database. The affinity assay was performed using Biacore-8K, specifically the human β-Klotho protein was immobilized on the NTA chip, the immobilization amount was 1000 RU, and the flow buffer used was PBS (containing 0.05% Tween-20). During the experiment, the binding of β-Klotho to different concentrations of FGF21 mutants was detected by flowing through. The final data was fitted using kinetics and the dissociation constant KD was calculated. The specific affinity results are shown in Table 3. It can be seen that the 1407-1413 mutants (1410 not tested) all showed similar affinity to β-Klotho as the FGF21 control mutant 1414. This indicates that the affinity activity of each mutant is not inhibited, and the activity of each mutant is similar to each other.

[0078] Table 3 FGF21 and mutant binding activity test with β-Klotho

[0079] Example 5 FGF21 mutant and fusion protein in β-Klotho-ERK cell activity experiment

[0080] To further verify the activity of the FGF21 mutants and fusion proteins in the present application, the FGF21 and mutants were tested for their activation of the ERK signaling pathway using HEK293 cells overexpressing β-Klotho (cells were constructed by Kanglong Huazheng). The specific experimental procedures were 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 from the cells cultured to the exponential phase and added to a white 384 assay plate, with appropriate medium and appropriate cell density (5000 / well) added, and the plate was incubated at 37°C under 5% CO2. The FGF21 and mutants were diluted in the medium starting from 3 uM / 6 uM / 15 uM with a 3-fold gradient, and 10 ul of the compound was added to each well of the cell experiment plate. After the addition of the test protein, the cells were incubated at 37°C in a 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 the prepared antibody solution in the detection buffer was added, the plate was sealed with a plate sealing module, and the plate was incubated at room temperature overnight. The fluorescence emission at 665 nm and 620 nm was then read, and the activity of the mutants was measured by comparing their EC50 values. The results are shown in Table 4, and the 1407-1413 mutants (1410 was not tested) had very similar biological activity relative to the FGF21 control mutant 1414. The cell activity of FGF21 in the GLP-1-scFc-FGF21 fusion protein 1300 molecule was about 20 times lower than that of the FGF21 molecule 1414, which was related to the steric hindrance formed by the scFc protein fused to the N-terminus of the FGF21 molecule. We also tested the cell activity of FGF21 in the GLP-1-scFc-FGF21 (1414) protein, and the activity was 21 nM. The cell activity of FGF21 in the GLP-1-scFc-FGF21 fusion protein 1300 molecule was slightly higher than that of FGF21 in the GLP-1-scFc-FGF21 (1414) protein.

[0081] Table 4 Cell activity test of FGF21 mutants and fusion proteins

[0082] Example 6 Activity experiment of FGF21 mutant fusion protein in ob / ob mice

[0083] To further verify the activity of the FGF21 mutant fusion protein in the application, we selected one of the FGF21 mutant fusion proteins, GLP-1-scFc-FGF21 1300, and used 6-7 week old ob / ob mice (SPF level, Changzhou Cavens Experimental Animal Co., Ltd.). After the adaptation period, the animals 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 fed with high-fat feed for 7 weeks to establish an obese DIO mouse model. The specific administration scheme is as follows:

[0084] G1 group is the animal fed with ordinary feed (i.e. Lean Mice), as a control, a total of 10.

[0085] At the same time, the animals fed with high-fat feed were grouped. Day 1 was the first day of administration, and the serum TC, LDL-C, fasting blood glucose, and body weight data before administration were used as grouping criteria. The animals were divided into 5 groups, 10 in each group, and the specific grouping information is as follows: G2 group model (vehicle control group, i.e. ob / ob-Neg Ctr), G3 group model (Semaglutide control group, 30 nmol / kg, sc, q3d), G4 group model (1300 experimental group, 10 nmol / kg, sc, q3d), G5 group model (1300 experimental group, 30 nmol / kg, sc, q3d), and G6 group model (1300 experimental group, 60 nmol / kg, sc, q3d). The changes in blood glucose, body weight, TC, and LDL-C were continuously monitored, and the results are shown in Figure 3.

[0086] The above data analysis shows that the use of scFc fusion GLP-1 and FGF21 mutant 1300 is significantly better than the control molecule Semaglutide in blood glucose, body weight, TC, and LDL-C, and has obvious dose dependence.

[0087] The above detailed description of the preferred embodiments of the application has described the implementation of the application. It should be understood that the application is not limited to the above-described embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the application should be included in the protection scope of the application.

Claims

1. A fusion protein, characterized in that, This includes FGF21 mutant proteins linked to heterologous proteins, wherein the FGF21 mutant protein comprises an amino acid sequence modified based on the wild-type human FGF21 sequence by either of the following a or b: a. Delete the proline residue at position 171; b. A disulfide bond is formed at or before and after the proline residue at position 171 by substitution, deletion or insertion of one or more amino acid residues.

2. The fusion protein as described in claim 1, characterized in that, The FGF21 mutant protein comprises an amino acid sequence selected from SEQ ID NO:4, 10, 16 and 22.

3. The fusion protein as described in claim 1, characterized in that, The FGF21 mutant protein is obtained by introducing two cysteine ​​residues within three positions before and after the proline residue at position 171 of the wild-type human FGF21 sequence through substitution and / or addition.

4. The fusion protein as described in claim 3, characterized in that, The two cysteine ​​residues that form a disulfide bond are separated by one, two, or three amino acids.

5. The fusion protein as described in claim 4, characterized in that, The two sites that are replaced with cysteine ​​residues or added cysteine ​​residues are separated by one, two or three amino acids, or the two sites that are replaced with cysteine ​​residues or added cysteine ​​residues are separated by one, two or three amino acids by adding amino acid residues.

6. The fusion protein as described in claim 3, characterized in that: a. Replace one amino acid residue with a cysteine ​​residue within the first three positions of position 171; b. Replace one amino acid residue with a cysteine ​​residue within three positions after position 171; c. Add one or two amino acid residues between the position 171 and the site before 171 where a cysteine ​​residue is replaced, and / or add one or two amino acid residues between the position 171 and the site after 171 where a cysteine ​​residue is replaced.

7. The fusion protein as described in claim 6, characterized in that, The one or two amino acid residues added are selected from glycine, alanine, or serine.

8. The fusion protein as described in claim 6, characterized in that... : a. Replace 170G with 170C; b. Replace 172S with 172C; c. Insert one or two amino acid residues between 171P and 172C.

9. The fusion protein as described in claim 3, characterized in that, The FGF21 mutant protein comprises an amino acid sequence selected from SEQ ID NO:5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 23, 24, 25, 26 and 27.

10. The fusion protein according to claim 1, characterized in that, A disulfide bond is formed between position 171 and its neighboring sites by replacing the proline residue at position 171 and by replacing, deleting or adding one or more amino acid residues at its neighboring sites.

11. The fusion protein according to any one of claims 1, 3-8, and 10, characterized in that, Replace 121N with 121Q and 168M with 168L in the wild-type human FGF21 sequence; and / or add one alanine, glycine, or serine residue before the first histidine residue in the wild-type human FGF21 sequence.

12. The fusion protein according to any one of claims 1-11, characterized in that, The heterologous protein includes an Fc fragment of an immunoglobulin, or the heterologous protein includes an Fc fragment of an immunoglobulin and GLP-1 or a mutant thereof; preferably, the immunoglobulin is hIgG4; preferably, the amino acid sequence of the GLP-1 or a mutant thereof is shown in any one of SEQ ID NO:35-39.

13. The fusion protein as described in claim 12, characterized in that, The heterologous protein includes an Fc fragment of an immunoglobulin and GLP-1 or a mutant thereof, wherein the Fc fragment of the immunoglobulin is a single-chain Fc with an amino acid sequence as shown in SEQ ID NO:32 or SEQ ID NO:

33.

14. The fusion protein according to any one of claims 12-13, characterized in that, The heterologous proteins include Fc mutants of immunoglobulins.

15. The fusion protein as described in claim 14, characterized in that, The Fc mutant of the immunoglobulin contains the amino acid sequence shown in SEQ ID NO:

28.

16. The fusion protein according to any one of claims 12-15, characterized in that, The FGF21 mutant protein is linked to the heterologous protein via a linker.

17. The fusion protein of claim 13, characterized in that, The fusion protein contains the amino acid sequence shown in SEQ ID NO:

29.

18. A dimer, characterized in that, Includes the fusion protein as described in any one of claims 12, 14-16.

19. The dimer as claimed in claim 18, characterized in that, It is a heterodimer that forms a pestle-and-mortar structure. The heteroprotein in the fusion protein is an Fc fragment of an immunoglobulin. The heterodimer contains the amino acid sequences shown in SEQ ID NO:30 and SEQ ID NO:

31.

20. A pharmaceutical composition, characterized in that, It contains a fusion protein as described in any one of claims 1-17, or a dimer as described in any one of claims 18-19, and a pharmaceutically acceptable excipient.

21. The use of the fusion protein of any one of claims 1-17, the dimer of any one of claims 18-19, or the pharmaceutical composition of claim 20 in controlling blood glucose, blood lipids, NASH, and reducing weight.

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