FGF21 fusion protein and use thereof

Abstract

Provided are an FGF21 fusion protein and use thereof in the treatment of metabolic diseases. The fusion protein has good activity in activating the reporter gene FGF21, a prolonged half-life, and excellent efficacy.

Description

An FGF21 fusion protein and its applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an FGF21 fusion protein and its applications. Background Technology

[0002] FGF21 is cleaved by the serine protease fibroblast activator protein (FAP) and dipeptidyl peptidase IV (DPP-IV). DPP-IV is responsible for cleavage at the N-terminus of residues 2 and 4, while FAP is responsible for cleavage at residue 171. Although cleavage at residues 2 and 4 does not significantly impair the function of FGF21, cleavage at residue 171 inactivates FGF21 because the ten C-terminal residues of FGF21 are crucial for its interaction with the receptor β-Klotho, which is also the main reason for the short half-life of FGF21 (approximately 0.5–1.5 hours).

[0003] Within the FGF21 polypeptide, the sites of mature polypeptides susceptible to protease degradation include positions 4-5, 20-21, 151-152, 171-172, and 178-181. After adding the signal peptide, the corresponding full-length amino acid positions of FGF21 are 1-209, and the numbered positions are the peptide bonds between amino acid residues at positions 32-33, 48-49, 179-180, 199-200, and 206-209.

[0004] Based on the natural sequence of human FGF21, this invention performs a series of sequence designs on FGF21 to improve the affinity of the C-terminal amino acid of FGF21 for KLB, increase the resistance of FGF21 to enzymatic degradation, and further prolong the half-life, thereby providing a fusion protein molecule with good FGF21 activation activity. Summary of the Invention

[0005] The present invention provides an FGF21 fusion protein comprising an FGF21 polypeptide, a linker, and an immunoglobulin Fc domain, wherein the FGF21 polypeptide and the immunoglobulin Fc domain are linked by the linker, the FGF21 polypeptide is wild-type FGF21 protein or a variant thereof, and the immunoglobulin Fc domain is the Fc of human IgG or a functional variant thereof.

[0006] In some embodiments, the N-terminus of the linker is connected to the C-terminus of the immunoglobulin Fc domain, and the C-terminus of the linker is connected to the N-terminus of the FGF21 polypeptide.

[0007] In some embodiments, the FGF21 peptide and the immunoglobulin Fc domain are linked via peptide linkers, such as short peptides, additional residues from the FGF21 translation sequence, or larger linkers to the entire protein. In some embodiments, the FGF21 peptide and the immunoglobulin Fc domain are linked via non-natural amino acid or non-peptide linkers.

[0008] In some embodiments, the linker is selected from short peptides (especially Ser / Gly repeat sequences), additional residues from the FGF21 translation sequence, larger linkers to many or complete proteins (such as Fc domains, human serum albumin (HSA)), or non-natural amino acids or non-peptide linkers (such as maleimide-Cys, NHS-Lys, simple amide bonds, lower alkyl groups (such as C1-C6), halogens (such as Cl, Br), CN, NH2, phenyl, etc.).

[0009] In some embodiments, the linker is selected from short peptides. In some embodiments, the linker consists of one or more amino acids. In some embodiments, the linker consists of 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. In some embodiments, the 1 to 20 amino acids are selected from glycine, serine, alanine, proline, asparagine, glutamine, or lysine. The linker can be a G-rich polypeptide, for example, it can be selected from several (G)-S (i.e., "GS"), (G)2-S (i.e., "GGS"), (G)3-S (i.e., "GGGS"), (G)4-S (i.e., "GGGGS"), and (G)5-S (i.e., "GGGGGS"). Other exemplary connectors may include, for example, GS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGSA, GGGGGSGGGSGGGGS, GGGKGGGG, GGGNGSGG, GGGCGGGG, or GPNGG. In some embodiments, the connector is selected from GS, GGGS, GGGGS, GGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGSGGGS, GGGGSGGGGSGGGSA, GGGGGSGGGSGGGGS, GGGKGGGG, GGGNGSGG, GGGCGGGG, or GPNGG.

[0010] In some embodiments, the linker comprises an amino acid sequence as shown in SEQ ID NO:29 (GS). In some embodiments, the linker is selected from the amino acid sequence shown in SEQ ID NO:29.

[0011] In some embodiments, the FGF21 polypeptide comprises an amino acid sequence selected from any of the following: SEQ ID NOs:3-24, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any of the amino acid sequences in SEQ ID NOs:3-24.

[0012] In some embodiments, the immunoglobulin Fc domain comprises an amino acid sequence selected from any of the following: SEQ ID NOs:25-28, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any of the amino acid sequences in SEQ ID NOs:25-28.

[0013] In some embodiments, the FGF21 fusion protein comprises an amino acid sequence selected from any of the following: SEQ ID NOs:32-55, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any of the amino acid sequences in SEQ ID NOs:32-55.

[0014] The present invention also provides a nucleic acid molecule encoding the FGF21 fusion protein.

[0015] The present invention also provides a recombinant vector comprising the nucleic acid molecule.

[0016] The present invention also provides a recombinant cell comprising the nucleic acid molecule and / or the recombinant vector, and capable of expressing the FGF21 fusion protein.

[0017] The present invention also provides the use of the FGF21 fusion protein in the preparation of drugs for metabolic diseases.

[0018] In some implementations, the metabolic disease is nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis or cirrhosis, type II diabetes, or obesity.

[0019] The present invention also provides a pharmaceutical composition comprising the FGF21 fusion protein and a pharmaceutically acceptable carrier, diluent or excipient.

[0020] Abbreviations and Terminology Definitions

[0021] The term "wild-type FGF21 protein" generally refers to a naturally occurring wild-type polypeptide expressed in the human body. For the purposes of this disclosure, the term "wild-type FGF21 protein" may be used interchangeably to refer to any full-length FGF21 polypeptide, such as any mature form of a polypeptide consisting of 209 amino acid residues with the amino acid sequence SEQ ID NO:1, or, for example, any mature form of a polypeptide consisting of 181 amino acid residues with the amino acid sequence SEQ ID NO:2, wherein 28 amino acid residues at the amino terminus of the full-length FGF21 polypeptide have been removed (i.e., it constitutes the signal peptide).

[0022] The term "FGF21 variant" refers to an FGF21 polypeptide variant in which the naturally occurring FGF21 amino acid sequence has been modified. Such modifications include, but are not limited to, substitutions of one or more amino acids, including substitutions by non-naturally occurring amino acid analogs and truncation. Therefore, FGF21 variants include, but are not limited to, the site-directed FGF21 variants described herein, truncated FGF21 polypeptides, protease-resistant FGF21 variants, aggregation-reducing FGF21 variants, FGF21 combinatorial variants, and FGF21 fusion proteins. To identify the specific truncation and amino acid substitutions of the FGF21 variants of the present invention, the truncated or mutated amino acid residue numbers correspond to the numbers of the mature 181-residue FGF21 polypeptide.

[0023] The term "FGF21 peptide" encompasses wild-type FGF21 protein or variants thereof. In some embodiments, FGF21 peptide refers to wild-type FGF21 protein. In some embodiments, FGF21 peptide refers to FGF21 variants.

[0024] The term "immunoglobulin Fc domain" generally refers to a domain that contains the CH2 and CH3 constant regions of an immunoglobulin (e.g., an antibody). For example, the immunoglobulin Fc domain can be a domain composed of the hinge region, CH2, and CH3 constant regions of an immunoglobulin (e.g., an antibody). For example, the immunoglobulin can be a human immunoglobulin. For example, the immunoglobulin can be human IgG1, wherein IgG1 can contain different allotypes.

[0025] The term "linker" is defined and described as follows: Linkers may be used, but are not required, when forming the fusion proteins of the present invention. When using linkers, their chemical structure may not be critical, as they primarily function as spacers. Linkers may consist of amino acids linked together by peptide bonds. In some embodiments of the invention, linkers consist of 1-20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. In many embodiments, the 1-20 amino acids are selected from the amino acids serine, glycine, proline, asparagine, alanine, glutamine, or lysine. In some embodiments, linkers consist of mostly sterically unobstructed amino acids, such as alanine and glycine. In some embodiments, linkers are combinations of polyglycine, polyalanine, glycine, and alanine (e.g., poly(Gly-Ala)), or combinations of glycine and serine, such as poly(Gly-Ser). Furthermore, the invention contemplates linkers of any length or composition. The linkers described herein are exemplary, and the invention also contemplates linkers that are much longer and include additional residues. The invention also contemplates non-peptide linkers. For example, alkyl linkers can be used. These alkyl linkers can be further substituted with any group that does not hinder stereostructure, including but not limited to lower alkyl groups (e.g., C1-C6), lower acyl groups, halogens (e.g., Cl, Br), CN, NH2, or phenyl groups. An exemplary non-peptide linker is a polyethylene glycol linker having a molecular weight of 100 to 5000 kD, approximately 100 to 500 kD. The terms "linker" and "linker" are used interchangeably.

[0026] The term "amino acid" refers to twenty common, naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In some embodiments, the term "amino acid" also includes non-natural amino acids. Any suitable non-natural amino acid can be used. In some embodiments, the non-natural amino acid contains a reactive moiety for conjugating the agent with MIAC.

[0027] The term "mutation" refers to a mutation or change in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0028] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a control polypeptide sequence after sequence alignment and, where necessary, nicking to obtain the maximum percentage sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, like BLAST software or the FASTA package.

[0029] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence.

[0030] The term "positive control" refers to a natural or engineered cell or antibody that can bind to or express the target protein.

[0031] The term "vector" is used to refer to any molecule (such as nucleic acid, plasmid, or virus) used to deliver encoded information to a host cell. Attached Figure Description

[0032] Figure 1 shows the FGF21 activation activity of the fusion protein.

[0033] Figure 2 shows the FGF21 activation activity of the fusion protein.

[0034] Figure 3 shows the FGF21 activation activity of the fusion protein.

[0035] Figure 4 shows the FGF21 activation activity of the fusion protein.

[0036] Figure 5 shows the FGF21 activation activity of the fusion protein.

[0037] Figure 6 shows the FGF21 activation activity of the fusion protein.

[0038] Figure 7 shows the stability of the fusion protein.

[0039] Figure 8 shows the change in the rate of weight loss of the experimental mice.

[0040] Figure 9 shows the changes in the cumulative food intake of the experimental mice.

[0041] Figure 10 shows the changes in glucose tolerance levels in experimental mice.

[0042] Figure 11 shows the changes in ALT and AST levels in experimental mice.

[0043] Figure 12 shows the changes in TC, TG, HDL, and LDL levels in experimental mice.

[0044] Figure 13 is a histopathological image of liver stained with hematoxylin and eosin (HE). Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations and not for limiting the scope of protection of the present invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0046] Example 1: Humanized Molecular Design

[0047] Based on the wild-type human FGF21 polypeptide, a series of mutant combinations were designed to improve the affinity of the C-terminal amino acid of FGF21 for KLB, increase the resistance of FGF21 to enzymatic degradation, and further prolong its half-life. The wild-type FGF21 polypeptide sequence is SEQ ID NO:1 (here, SEQ ID NO:1 includes the first 1-28 signal peptides, and the amino acid positions are 1-209). Mutant combinations of the FGF21 polypeptide of this invention were designed according to Table 1. After removing the signal peptides at positions 1-28, the amino acid positions of the mature peptide are 1-181, and this invention uses this rule for numbering. It should be noted that in Table 1, FGF21 polypeptides 1-22 are truncated versions of the FGF21 polypeptide after removing the signal peptide and removing the first 1-4 amino acids (the first 1-4 amino acid sequences are HPIP). The N-terminal signal peptide sequence of the immunoglobulin Fc domain is SEQ ID NO:30.

[0048] The specific design schemes for positive controls and fusion proteins are shown in Table 2, where:

[0049] (a) The designed FGF21 polypeptide 1 (amino acid sequence SEQ ID NO:3) was linked to immunoglobulin Fc domain 1 (amino acid sequence SEQ ID NO:25) using a linker with amino acid sequence SEQ ID NO:29 to obtain positive control 1. The amino acid sequence of positive control 1 is SEQ ID NO:31.

[0050] (b) The designed FGF21 peptides 1-22 (amino acid sequences SEQ ID NO: 3-24) were linked to immunoglobulin Fc domain 2 (amino acid sequence SEQ ID NO: 26) using a linker with amino acid sequence SEQ ID NO: 29 to obtain fusion proteins 1-22. The amino acid sequences of fusion proteins 1-22 are SEQ ID NO: 32-53.

[0051] (c) The designed FGF21 polypeptide 19 (amino acid sequence SEQ ID NO:21) was linked to immunoglobulin Fc domains 3 and 4 (amino acid sequences SEQ ID NO:27 and 28, respectively) using a linker with amino acid sequence SEQ ID NO:29, to obtain fusion proteins 23 and 24, respectively. The amino acid sequences of fusion proteins 23 and 24 are SEQ ID NO:54 and 55, respectively.

[0052] Table 1

[0053] Table 2

[0054] Example 2: Construction of gene synthesis and expression vector

[0055] The pcDNA3.1(+) vector was used as the plasmid vector for expressing the multifunctional antibody. The pcDNA3.1-G418 vector contains the promoter CMVPromoter, the eukaryotic selection tag G418, and the prokaryotic selection tag Ampicilline. The nucleotide sequences for the light and heavy chains of the constructed antibody were synthesized. The vector and target fragment were double-digested with HindIII and XhoI, recovered, and ligated using DNA ligase. The resulting fragments were then transformed into competent E. coli DH5α cells. Positive clones were selected, and plasmids were extracted and verified by enzyme digestion to obtain the plasmids.

[0056] Example 3 Plasmid Extraction

[0057] Recombinant plasmids containing the above-mentioned target genes were transformed into competent Escherichia coli DH5α cells. The transformed bacteria were plated on LB plates containing 100 μg / mL ampicillin and cultured. Plasmid clones were selected and cultured in liquid LB medium. The culture was shaken at 260 rpm for 14 hours. Plasmids were extracted using an endotoxin-free plasmid extraction kit, dissolved in sterile water, and their concentration was determined using a nucleic acid protein quantification instrument.

[0058] Example 4: Plasmid transfection, transient expression, and antibody purification

[0059] ExpiCHO cells were cultured at 37℃, 8% CO2, and 100 rpm until the cell density reached 6 × 10⁶ cells / year. 6The constructed plasmids were transfected into the above cells using liposomes according to the paired combinations. The plasmid concentration was 1 mg / mL, and the liposome volume was as per ExpiCHO. TM The Expression System kit specifies incubation at 32°C, 5% CO2, and 100 rpm for 7–10 days. Feedings are added 18–22 hours after transfection and again between day 5. The culture products are centrifuged at 4000g, filtered through a 0.22 μm filter, and the supernatant is collected. The resulting antibody protein is purified using Protein A ion exchange column, and the eluent is collected.

[0060] The specific steps for Protein A purification by ion exchange column are as follows: After high-speed centrifugation of cell culture medium, the supernatant is collected and subjected to affinity chromatography using a GE Protein A chromatography column. The chromatography uses 1×PBS (pH 7.4) as the equilibration buffer. After loading the cell supernatant, it is washed with PBS until the UV light returns to baseline. Then, the target protein is eluted with 0.1M glycine (pH 3.0) elution buffer, and the pH is adjusted to neutral using Tris for storage. The pH of the affinity chromatography product is adjusted to 1-2 pH units below or above the pI, and appropriately diluted to control the sample conductivity below 5 mS / cm. Using appropriate pH buffers such as phosphate buffer or acetate buffer, conventional ion exchange chromatography methods in the field, such as anion exchange or cation exchange, are used for NaCl gradient elution at the corresponding pH conditions. The collection tube containing the target protein is selected based on SDS-PAGE data and stored together.

[0061] The purified eluent was then ultrafiltered and transferred to buffer. Proteins were detected by SDS-polyacrylamide gel electrophoresis.

[0062] SDS-PAGE analysis confirmed the presence of the target band under non-reducing gel conditions and the target antibody under reducing gel conditions, corresponding to both the heavy and light chains of the desired antibody. Therefore, after plasmid transfection, transient expression, and purification, the correctly structured fusion protein was obtained.

[0063] Example 5: FGF21 reporter gene activation activity of the fusion protein

[0064] 293-FGFR1c-KLB-1G3 cells (the constructed KLB 293 cells) were transfected with plasmid pFA2-ELK1:pFR-luc at a ratio of 1:8. After 48 hours, the cells were resuspended in MEM complete medium and seeded at 3E4 cells / well in 96-well plates (50 μL / well) and incubated overnight at 37°C. The sample candidate antibody and irrelevant antibody IgG1 were diluted with MEM complete medium to an initial concentration of 80 μg / mL (final concentration 40 μg / mL), and then diluted 5-fold in 9 concentration gradients. The solutions were added to the cell culture plates at 50 μL / well and incubated at 37°C for 24 hours. 100 μL of luciferase reagent was added to each well, and the cells were incubated at room temperature for 10 minutes. Flow cytometry was used for analysis, and the results were analyzed using GraphPad Prism software. The amino acid sequence of positive control 1 is SEQ ID NO:31, and positive control 2 is fusion protein 1 with the amino acid sequence SEQ ID NO:32. The designed fusion proteins 2-24 all exhibited good FGF21 activation activity, as shown in Figures 1-6.

[0065] Example 6: Serum stability of the fusion protein

[0066] Candidate antibody positive control 1, fusion protein 23, and fusion protein 24 were diluted to a concentration of 1000 nM using fetal bovine serum FBS and dispensed into two tubes. One tube was placed at -20℃ as a 0h control, and the other tube was incubated at 37℃ for 24h. 293-FGFR1c-KLB-1G3 cells were transfected with plasmid pFA2-ELK1:pFR-luc = 1:8. After 48h, the cells were resuspended in MEM complete medium and seeded at 3E4 cells / well in 96-well plates, 50 μL / well, and incubated overnight at 37℃. Samples from the 0h control group and the 37℃-24h samples were diluted 10-fold with MEM complete medium in seven concentration gradients, 50 μL / well, and incubated at 37℃ for 24h. Add 100 μL of luciferase reagent per well, incubate at room temperature for 10 minutes, and detect by flow cytometry. Calculate the specific activity (specific activity = sample - 0 h / sample - 37℃ - 24 h), as shown in Table 3. Fusion proteins 23 and 24 remained stable after mixing with serum and incubating at 37℃ for 24 h, as shown in Figure 7.

[0067] Table 3

[0068] Example 7: PK of the fusion protein

[0069] SD rats (one female and one male) were used for rat pharmacokinetic (PK) assays of various candidate molecules. The drug was administered via tail vein bolus injection at a dose of 1 mpk. Blood samples were collected from the jugular / orbital veins of the experimental animals, and the actual blood collection time was recorded. After collection, the blood samples were left at room temperature for half an hour before centrifugation (centrifuge pre-cooled to 4°C; centrifugation conditions: 4°C, 3000 rpm, 10 min). Serum was separated after centrifugation and transferred to labeled centrifuge tubes. Blood drug concentrations were determined using ELISA.

[0070] Detection method: Coat 100 μL / well with 1.0 mg / mL monoclonal anti-human-IgG-Fc antibody and incubate overnight at 4°C. Discard the coating solution, wash 4 wells with 300 μL / well of 1×PBST (0.05%), block with 300 μL / well of 2% BSA, and incubate at 37°C for 1 h. Discard the blocking solution, wash 4 wells with 300 μL / well of 1×PBST (0.05%), dilute the sample 500-fold with 2% BSA, and add 100 μL / well to each well. Except for serum samples, perform parallel runs with 100 μL / well of standard curve solution. The initial concentration of the solution was 10 mg / mL, diluted 3-fold to obtain 10 concentrations. The diluent was 2% BSA in 0.2% serum, and a blank control (2% BSA in 0.2% serum) was included. The solution was incubated at 37°C for 1 hour. The liquid was discarded, and 300 μL of 1×PBST (0.05%) was added to each well. The plate was washed 4 times. Goat anti-human Fc-HRP was diluted 1:10000 and 100 μL was added to each well. The plate was incubated at 37°C for 45 minutes. The liquid was discarded again, and 300 μL of 1×PBST (0.05%) was added to each well. The plate was washed 6 times, and the liquid at the bottom of the wells was blotted dry on clean paper. Solarbiotin solution was added to each well at 100 mL / well, and the plate was wrapped in aluminum foil. The plate was incubated at 37°C in the dark for 3 minutes. Stop the colorimetric reaction by adding 100 μL of 1M hydrochloric acid to each well. Readings were taken at 450 nm using an ELISA reader, and the data were analyzed.

[0071] The plasma drug concentration data of the fusion protein of this invention were processed using pharmacokinetic software with a non-compartmental model. Relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method, as shown in Table 4. It can be seen that the half-life of fusion protein 23 is longer than that of other fusion proteins.

[0072] Table 4

[0073] Example 8: Effectiveness of the fusion protein

[0074] C57BL / 6 mice were fed a high-fat, high-sugar diet (XTHF60, reference D12492) to induce Diet-Induced Obesity (DIO) mice, and then administered the test drug. The efficacy of the test drug was evaluated based on various factors, including mouse body weight, food intake, and glucose tolerance level. Changes in mouse body weight are shown in Figure 8, changes in cumulative food intake are shown in Figure 9, and glucose tolerance level is shown in Figure 10. Experimental groups and administration methods are shown in Table 5.

[0075] Table 5

[0076] The mice were administered the drug for 4 weeks. After the last administration, mice in each group underwent an oral glucose tolerance test (OGTT). Blood glucose levels were collected at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after administration to assess glucose tolerance. Blood samples were then collected to measure total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Biochemical results are shown in Table 6. The mice were then dissected, and liver weight, inguinal fat weight, and epididymal fat weight were measured. Figures 8-10 and Table 6 show that fusion protein 23 effectively reduced weight without affecting food intake, and glucose tolerance returned to normal after administration. Liver weight and fat weight were measured after dissection, and the results are shown in Table 7. The results indicate that fusion protein 23 can effectively reduce liver fat and total body fat weight. As shown in Figures 11 and 12, after administration, fusion protein 23 effectively reduced ALT, AST, TC, and LDL in mice, improving liver function. The liver histopathological image stained with hematoxylin and eosin (HE) is shown in Figure 13. The pathological results after administration indicate that fusion protein 23 can effectively improve hepatocellular steatosis.

[0077] Table 6

[0078] Table 7

Claims

1. An FGF21 fusion protein, characterized in that, The FGF21 fusion protein comprises an FGF21 polypeptide, a linker, and an immunoglobulin Fc domain, wherein the FGF21 polypeptide and the immunoglobulin Fc domain are linked by the linker, the FGF21 polypeptide is wild-type FGF21 protein or a variant thereof, and the immunoglobulin Fc domain is the Fc of human IgG or a functional variant thereof.

2. The FGF21 fusion protein according to claim 1, characterized in that, The N-terminus of the linker is connected to the C-terminus of the immunoglobulin Fc domain, and the C-terminus of the linker is connected to the N-terminus of the FGF21 polypeptide.

3. The FGF21 fusion protein according to claim 1, characterized in that, The linkers are selected from short peptides (especially Ser / Gly repeat sequences), additional residues from the FGF21 translation sequence, larger linkers from multiple to complete proteins (such as Fc domains, HSA), or non-natural amino acids or non-peptide linkers (such as maleimide-Cys, NHS-Lys, simple amide bonds, lower alkyl groups (such as C1-C6), halogens (such as Cl, Br), CN, NH2, phenyl, etc.).

4. The FGF21 fusion protein according to claim 3, characterized in that, The linker is a short peptide.

5. The FGF21 fusion protein according to claim 4, characterized in that, The linker consists of 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids.

6. The FGF21 fusion protein according to claim 5, characterized in that, The 1 to 20 amino acids are selected from glycine, serine, alanine, proline, asparagine, glutamine, or lysine.

7. The FGF21 fusion protein according to claim 5, characterized in that, The linker is selected from GS, GGGS, GGGGS, GGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGSGGGGSA, GGGGSGGGSGGGGGS, GGGKGGGG, GGGNGSGG, GGGCGGGG or GPNGG.

8. The FGF21 fusion protein according to claim 1, characterized in that, The FGF21 polypeptide comprises an amino acid sequence selected from any of the following: SEQ ID NOs:3-24, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any of the amino acid sequences in SEQ ID NOs:3-24.

9. The FGF21 fusion protein according to claim 1, characterized in that, The immunoglobulin Fc domain comprises an amino acid sequence selected from any of the following: SEQ ID NOs:25-28, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any of the amino acid sequences in SEQ ID NOs:25-28.

10. The FGF21 fusion protein according to claim 1, characterized in that, The FGF21 fusion protein comprises an amino acid sequence selected from any of the following: SEQ ID NOs:32-55, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any of the amino acid sequences in SEQ ID NOs:32-55.

11. A nucleic acid molecule encoding the FGF21 fusion protein according to any one of claims 1-10.

12. A recombinant vector comprising the nucleic acid molecule according to claim 11.

13. A recombinant cell comprising the nucleic acid molecule of claim 11 and / or the recombinant vector of claim 12, and capable of expressing the FGF21 fusion protein.

14. Use of the FGF21 fusion protein according to any one of claims 1-10 in the preparation of a medicament for metabolic diseases.

15. The application according to claim 14, characterized in that, The metabolic diseases mentioned are non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis or cirrhosis, type II diabetes, or obesity.

16. A pharmaceutical composition comprising the FGF21 fusion protein according to any one of claims 1-10 and a pharmaceutically acceptable carrier, diluent or excipient.

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