HUMAN FIBROBLAST GROWTH FACTOR 21 (HFGF21) FUSION PROTEIN, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

A fusion protein of hFGF21 or its analogs having improved pharmaceutical properties, and use of the fusion protein in preparing medicines for treating diseases, such as diabetes, obesity, non-alcoholic fatty liver disease, dyslipidemia, and/or metabolic syndrome.

Claims

1. A human fibroblast growth factor 21 fusion protein comprising, in order from N-terminus to C-terminus, wild-type hFGF21 or an analog thereof, a flexible peptide linker, at least one rigid unit comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit, and a fusion ligand, wherein the fusion ligand is selected from the group consisting of an immunoglobulin and an Fc fragment thereof, human serum albumin and transferrin.

2. (canceled)

3. The fusion protein of claim 1, wherein the wild-type hFGF21 comprises a sequence of SEQ ID NO: 1, wherein amino acids 1-28 (a leader peptide) are removed; or an isoform of the sequence of SEQ ID NO: 1 wherein amino acids 1-28 (a leader peptide) are removed and having G141S or L174P substitution.

4. The fusion protein of claim 1, wherein the hFGF21 analog has one or more amino acid deletions, insertions, additions or substitutions, and one or more amino acid deletions at the N-terminus or the C-terminus relative to the amino acid sequence of wild-type hFGF21.

5. The fusion protein of claim 4, wherein the hFGF21 analog has 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues deleted at the N-terminus.

6. The fusion protein of claim 5, wherein the hFGF21 analog has 4 amino acid residues, HPIP, deleted at the N-terminus.

7. The fusion protein of claim 4, wherein the hFGF21 analog has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues deleted at the C-terminus.

8. The fusion protein of claim 4, wherein the hFGF21 analog comprises one or more amino acid substitutions selected from the group consisting of Q55C, A109T, L126R, G148C, K150R, P158S, S195A, P199G and G202A.

9. The fusion protein of claim 1, wherein the flexible peptide linker comprises two or more amino acids selected from the group consisting of G, S, A and T.

10. The fusion protein of claim 9, wherein the flexible peptide linker has the structural formula of (GS).sub.a(GGS).sub.b(GGGS).sub.c(GGGGS).sub.d, wherein each a, b, c, and d is an integer equal to or greater than 0, and the sum of (a+b+c+d) is greater than 1.

11. The fusion protein of claim 10, wherein the flexible peptide linker has an amino acid sequence selected from the group consisting of: TABLE-US-00004 (i) GGGGS; (ii) GSGGGSGGGGSGGGGS; (iii) GSGGGGSGGGGSGGGGSGGGGSGGGGS; (iv) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; (v) GGGSGGGSGGGSGGGSGGGS; and (vi) GGSGGSGGSGGS.

12. The fusion protein of claim 1, wherein the rigid unit comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit comprises SEQ ID NO: 2 or a truncated sequence thereof; wherein the truncated sequence comprises at least 2 glycosylation sites.

13. The fusion protein of claim 12, wherein the rigid unit comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit comprises the following amino acid sequences: TABLE-US-00005 (i) SSSSKAPPPSLPSPSRLPGPSDTPILPQ; (ii) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ; (iii) SSSSKAPPPS; (iv) SRLPGPSDTPILPQ; and (v) SSSSKAPPPSLPSPSR.

14. The fusion protein of claim 13, wherein the amino acid sequence of the rigid unit comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit shares at least 70%, 80%, 90% or 95% identity to the corresponding sequence of the carboxyl terminal peptide.

15. The fusion protein of claim 1, wherein the fusion protein comprises 1, 2, 3, 4 or 5 rigid units comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit.

16. The fusion protein of claim 1, wherein the human immunoglobulin Fc fragment is a variant having one or more effects selected from the group consisting of a reduced ADCC effect, a reduced CDC effect, and an enhanced binding affinity to an FcRn receptor.

17. The fusion protein of claim 16, wherein the Fc fragment is selected from a human IgG Fc variant.

18. The fusion protein of claim 1, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 8.

19. A DNA molecule encoding the fusion protein of claim 1, wherein the DNA molecule comprises the sequence of SEQ ID NO:9.

20. (canceled)

21. (canceled)

22. (canceled)

23. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the fusion protein of claim 1.

24. A method for preparing a human fibroblast growth factor 21 fusion protein comprising, in order from N-terminus to C-terminus, wild-type hFGF21 or an analog thereof, a flexible peptide linker, at least one rigid unit comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit, and a fusion ligand, wherein the fusion ligand is selected from the group consisting of an immunoglobulin and an Fc fragment thereof, human serum albumin and transferrin, the method comprising: (a) introducing a DNA sequence encoding the human fibroblast growth factor 21 fusion protein into a mammalian cell to obtain a high-yielding cell line; (b) screening the high-yielding cell line in step (a) which expresses more than 50 μg/10.sup.6 (million) cells per 24 hours in its growth medium; (c) growing the cell line obtained in step (b) to express the fusion protein to obtain a fermentation broth; and (d) harvesting the fermentation broth obtained in step (c) and purifying the fusion protein.

25. A method of treating a subject suffering from obesity, type 1 or type 2 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, and disorders associated with severe inactivation or mutation in the insulin receptor, comprising administering an effective amount of the fusion protein of claim 1 to the subject.

26. The fusion protein of claim 1, wherein the fusion ligand is an immunoglobulin Fc fragment.

27. The fusion protein of claim 4, wherein the amino acid sequence of the hFGF21 analog shares at least 70%, 80%, 90% or 95% identity to the corresponding sequence of wild-type hFGF21.

28. The fusion protein of claim 17, wherein the human IgG Fc variant is selected from the group consisting of: (i) hinge, CH2 and CH3 regions of human IgG1 containing mutations Leu234Val, Leu235Ala and Pro331Ser; (ii) hinge, CH2 and CH3 regions of human IgG2 containing mutation Pro331Ser; (iii) hinge, CH2 and CH3 regions of human IgG2 containing mutations Thr250Gln and Met428Leu; (iv) hinge, CH2 and CH3 regions of human IgG2 containing mutations Pro331Ser, Thr250Gln and Met428Leu; (v) hinge, CH2 and CH3 of human IgG4 regions containing mutations Ser228Pro and Leu235Ala.

29. The method of claim 24, wherein the mammalian cell used in step (a) is a CHO cell.

30. The method of claim 29, wherein the mammalian cell is CHO-derived cell line DXB-11.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0076] FIG. 1 shows the nucleotide sequence and deduced amino acid sequence of the hFGF21 fusion protein inserted into SpeI/EcoRI (restriction sites are marked with ) fragment in the PCDNA3.1 expression vector according to an embodiment of the present invention, consisting of a α1 microglobulin lead peptide (1-19, marked as ______), hFGF21 (20-200, having P instead of L), a flexible peptide linker (201-227, marked as ), CTP.sup.1 (228-255, marked as ), and vFc (256-478).

[0077] FIG. 2-a. Results of reducing and non-reducing SDS-PAGE electrophoresis for hFGF21 fusion protein FP4I, from left to right bands: NR (non-reducing); M (protein Marker); and R (reducing).

[0078] FIG. 2-b. SEC-HPLC results for hFGF21 fusion protein FP4I.

[0079] FIG. 3-a. Curve of the glucose tolerance test at 16 hours after a single injection of hFGF21 fusion protein FP4I (means±SEM, n=8).

[0080] FIG. 3-b. iAUC of the glucose tolerance test at 16 hours after a single injection of hFGF21 fusion protein FP4I (means±SEM, n=8). Notes on statistical difference markers: *P<0.05 compared to control group.

[0081] FIG. 3-c. Curve of the glucose tolerance test at 48 hours after a single injection of hFGF21 fusion protein FP4I (means±SEM, n=8).

[0082] FIG. 3-d. iAUC of the glucose tolerance test at 48 hours after a single injection of hFGF21 fusion protein FP4I (means±SEM, n=8). Notes on statistical difference markers: *P<0.05 compared to control group.

[0083] FIG. 4-a. iAUC of the glucose tolerance test at 16 hours after a single injection of several hFGF21 fusion proteins (means±SEM, n=8). Notes on statistical difference markers: *P<0.05 compared to control group.

[0084] FIG. 4-b. iAUC of the glucose tolerance test at 48 hours after a single injection of several hFGF21 fusion proteins (means±SEM, n=8). Notes on statistical difference markers: *P<0.05 compared to control group.

[0085] FIG. 5. Effect of hFGF21 fusion protein FP4I on body weight gain in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0086] FIG. 6. Effect of hFGF21 fusion protein FP4I on body composition of mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0087] FIG. 7. Effect of hFGF21 fusion protein FP4I on liver function in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0088] FIG. 8. Effect of hFGF21 fusion protein FP4I on liver weight of mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0089] FIG. 9. Effect of hFGF21 fusion protein FP4I on liver triglyceride content in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0090] FIG. 10-a. Blood glucose curve obtained from an experiment investigating the effect of hFGF21 fusion protein FP4I on insulin tolerance of mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0091] FIG. 10-b. AUC obtained from an experiment investigating the effect of hFGF21 fusion protein FP4I on insulin tolerance of mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0092] FIG. 11. Effect of hFGF21 fusion protein FP4I on fasting blood glucose levels in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0093] FIG. 12. Effect of hFGF21 fusion protein FP4I on fasting insulin levels in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0094] FIG. 13. Effect of hFGF21 fusion protein FP4I on insulin resistance index in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0095] FIG. 14. Effect of hFGF21 fusion protein FP4I on serum lipids in mice fed with high-fat diet (means±SEM, n=8). Notes on statistical difference markers: .sup.#P<0.05, .sup.##P<0.01 compared to the low-fat diet group; *P<0.05, **P<0.01 compared to the high-fat diet group.

[0096] FIG. 15. Effect of hFGF21 fusion protein FP4I on the morphopathology of liver tissue in mice fed with high-fat diet. Notes: A: the low-fat diet group; B: the high-fat diet group; C: the group with high-fat diet combined with hFGF21 fusion protein.

[0097] FIG. 16. Effect of hFGF21 fusion protein FP4I on the morphopathology of adipose tissue in mice fed with high-fat diet. Notes: A: the low-fat diet group; B: the high-fat diet group; C: the group with high-fat diet combined with hFGF21 fusion protein.

DETAILED DESCRIPTION

[0098] The present invention is further illustrated below in combination with specific embodiments. It should to be understood that the embodiments are only illustrative and are not intended to limit the scope of the present invention. In the following examples, experimental methods in which conditions are not specifically indicated are usually carried out according to conventional conditions, such as conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions suggested by the manufacturer.

[0099] The fusion proteins of the present invention are typically prepared by biosynthetic methods. Based on the nucleotide sequence of the present invention, one skilled in the art can conveniently prepare the nucleic acid of the present invention by various known methods, for example, but not limited to, PCR, DNA synthesis, etc. For specific methods, see J. Sambrook, Molecular Cloning: A Laboratory Manual. As an embodiment of the present invention, the nucleic acid sequence of the present invention can be constructed by segmentally synthesizing nucleotide sequences and then performing overlap extension PCR.

[0100] The present invention also provides an expression vector comprising a sequence encoding a fusion protein of the present invention and an expression regulatory sequence operably linked thereto. By “operably link” or “operably linked to” is meant a condition in which some portions of a linear DNA sequence are capable of regulating or controlling the activity of other portions of the same linear DNA sequence. For instance, a promoter is operably linked to a coding sequence if the promoter controls the transcription of the sequence.

[0101] The expression vector may be a commercially available vector such as, but not limited to, pcDNA3, pIRES, pDR, pUC18 or the like which can be used in a eukaryotic cell expression system. One skilled in the art can select a suitable expression vector based on the host cell.

[0102] The coding sequence of the fusion protein of the present invention may be introduced into suitable restriction sites by one skilled in the art by restriction enzyme cleavage and splicing according to a conventional method based on the restriction enzyme map of the known empty expression vector, to produce the recombinant expression vector of the present invention.

[0103] The invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence of a fusion protein of the present invention. The host cell is preferably a eukaryotic cell such as, but not limited to, CHO, COS cells, 293 cells, RSF cells and the like. In a preferred embodiment of the present invention, the cell is a CHO cell which can well express the fusion protein of the present invention to obtain a fusion protein having good binding activity and good stability.

[0104] The present invention also provides a method for producing a fusion protein of the present invention by using recombinant DNA, including the steps of:

[0105] 1) providing a nucleic acid sequence encoding a fusion protein;

[0106] 2) inserting the nucleic acid sequence of 1) into a suitable expression vector to obtain a recombinant expression vector;

[0107] 3) introducing the recombinant expression vector of 2) into a suitable host cell;

[0108] 4) growing the transformed host cell under conditions suitable for expression;

[0109] 5) collecting the supernatant and purifying the fusion protein product.

[0110] The coding sequence can be introduced into a host cell by various techniques known in the art such as, but not limited to, calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage transduction and method using alkali metal ions.

[0111] For the culture and expression of host cells, see Olander R M Dev Biol Stand, 1996, 86:338. The cells and debris in the suspension can be removed by centrifugation and the supernatant is collected. Identification can be performed by agarose gel electrophoresis.

[0112] The fusion protein obtained as described above can be purified to a substantially uniform nature, for example, showing a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed by secretion, a commercially available ultrafiltration membrane such as products from Millipore, Pellicon, etc. can be used to separate the protein.

[0113] The supernatant is firstly to be concentrated. The concentrated supernatant may be further purified by gel chromatography or by ion exchange chromatography, such as anion exchange chromatography (DEAE, etc.) or cation exchange chromatography. The gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like. The Q- or SP-group is a preferred ion exchange group. Finally, the purified product may be further finely purified by methods such as hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the like. All of the above purification steps can be used in different combinations to ultimately obtain proteins with a substantially uniform purity.

[0114] The expressed fusion protein can be purified by using an affinity chromatography column containing an antibody, receptor or ligand specific for the fusion protein. Depending on the nature of the affinity column used, the fusion polypeptide bound to the affinity column can be eluted by using conventional methods such as high salt buffer, pH change, and the like. Alternatively, the fusion protein may also contain one or more polypeptide fragments as a protein tag at the amino terminus or carboxyl terminus. Any suitable label can be used in the present invention. For example, the label may be FLAG, HA, HAL c-Myc, 6-His or 8-His, and the like. These tags can be used to purify the fusion protein.

Example 1. Construction of an Expression Plasmid Encoding the hFGF21 Fc Fusion Protein

[0115] Gene sequences encoding the α1 microglobulin leader peptide and mature hFGF21 or its analog, flexible peptide linker, CTP rigid unit and human IgG Fc variant were artificially-optimized, CHO cell-biased codons. The full-length sequence was obtained by chemical synthesis. A restriction site, SpeI or EcoRI respectively, were present at each of the 5′-end and 3′-end of the synthesized fragment to facilitate insertion of the target fragment into a specific site of the expression vector. The verified gene of the fusion protein of hFGF21 or its analog was digested with SpeI and EcoRI, and then inserted into corresponding restriction sites in PCDNA3.1-modified plasmid PXY1A1 to obtain an expression plasmid of the fusion gene. This plasmid contains a cytomegalovirus early promoter which is an enhancer required for mammalian cells to express foreign genes at high levels. This plasmid also contains selectable markers which may confer kanamycin resistance to bacteria and G418 resistance to mammalian cells. In addition, the PXY1A1 expression vector contains the mouse dihydrofolate reductase (DHFR) gene, thus the fusion gene and DHFR gene may be co-amplified in the presence of methotrexate (MTX) in host cells deficient in expressing a DHFR gene (See U.S. Pat. No. 4,399,216).

[0116] As shown in Table 1, the present invention constructed a series of fusion proteins of hFGF21, which comprised wild-type hFGF21 and its analogs, flexible peptide linkers (Linkers) with different lengths and Fc variant (vFc) elements of different IgG subtypes. In addition, the position and length of the CTP rigid unit were also different. In order to verify that fusion proteins comprising at least one CTP rigid unit with different lengths have a high biological activity, we constructed fusion proteins FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H and FP4I; as well as FP4J comprising no CTP rigid unit. The nucleotide sequence and translated amino acid sequence of FP4I are shown in FIG. 1.

[0117] In the present example, the wild-type hFGF21 had the amino acid sequence as shown in SEQ ID NO: 1 (expressed as hFGF21), and it had an equivalent form, .sup.L174PhFGF21, wherein the Leu at position 174 of SEQ ID NO: 1 was replaced by Pro. The hFGF21 analog preferably comprises a truncated wild-type hFGF21 polypeptide, hFGF21(HPIP.sup.−), wherein the HPIP at the N-terminus of the wild-type hFGF21 mature protein has been deleted (having an amino acid sequence of amino acids 33-209 of SEQ ID NO: 1). The hFGF21 analog also preferably comprises variants in which an amino acid has been substituted/replaced, such as variant Q55C, which is obtained by replacing Gln at position 55 of SEQ ID NO: 1 with Cys, and several other variants including A109T, L126R K150R, P199G and G202A which are obtained by replacing the amino acid at the corresponding site of SEQ ID NO: 1.

TABLE-US-00003 TABLE 1 Composition of the constructed hFGF21 fusion protein Elemental composition of a series of fusion proteins of hFGF21 Code and its analogs (from N-terminus to C-terminus) FP4A .sup.P199GhFGF21-L1-CTP.sup.1-vFcγ.sub.2-3 FP4B hFGF21(HPIP.sup.−)-L2-CTP.sup.1-vFcγ.sub.2-3 FP4C .sup.K150RhFGF21-L3-CTP.sup.5-vFcγ.sub.1 FP4D .sup.G202AhFGF21-L4-CTP.sup.4-vFcγ.sub.4 FP4E .sup.A109ThFGF21-L1-CTP.sup.3-CTP.sup.3-CTP.sup.3-vFcγ.sub.2-2 FP4F hFGF21-L5-CTP.sup.5-CTP.sup.5-vFcγ.sub.2-3 FP4G .sup.Q55ChFGF21-L4-CTP.sup.3-vFcγ.sub.2-2 FP4H .sup.L126RhFGF21-L6-CTP.sup.2-vFcγ.sub.4 FP4I .sup.L174PhFGF21-L3-CTP.sup.1-vFcγ.sub.2-3 FP4J .sup.L174PhFGF21-L3-vFcγ.sub.2-3

Example 2. Expression of Fusion Protein in Transfected Cell Lines

[0118] The recombinant expression vector plasmid was transfected into a mammalian host cell line to express the hFGF21 fusion protein. DHFR enzyme-deficient CHO cells are preferred host cell line for stable expression at high levels (See U.S. Pat. No. 4,818,679). CHO-derived cell line DXB11 was used as a host cell in this example. A preferred method of transfection was electroporation, and other methods including calcium phosphate co-deposition and liposome transfection might also be used. In electroporation, Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, Calif.) was used at a voltage of 300 V and a capacitance of 1500 μFd, and 50 μg of high-purity expression plasmid was added to 5×10.sup.7 cells in the cuvette. Two days after the transfection, the medium was replaced with a growth medium containing 0.6 mg/mL of G418. Transfectants resistant to the selected drug were screened by an ELISA assay against human IgG Fc. Quantification of the expression of the fusion protein can also be performed by using an ELISA assay against hFGF21. The wells producing high levels of Fc fusion protein were subcloned by limiting dilutions on 96-well culture plates.

[0119] To achieve higher levels of fusion protein expression, co-amplification utilizing the DHFR gene that can be inhibited by an MTX drug is preferred. The transfected gene of the fusion protein was co-amplified with the DHFR gene in growth media containing increasing concentrations of MTX. Subclones with positive DHFR expression were subjected to limiting dilution, and transfectants capable of growing in media containing up to 6 μM of MTX were screened by increasing the selection pressure gradually. The transfectants were measured for secretion rates and cell lines yielding high levels of exogenous protein were screened. Cell lines with a secretion rate of more than about 30, preferably about 50 μg/10.sup.6 [i.e. million] cells/24 h, were adapted to suspension culture by using serum-free growth media. Conditioned media was then used to purify the fusion protein.

Example 3. Purification and Characterization of the Fusion Protein

[0120] The conditioned media containing the fusion protein was titrated to pH 7˜8 with 1 N NaOH and then filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a Protein A column that had been equilibrated with phosphate buffered saline (PBS). After the fusion protein was bound onto the Protein A column, the effluent fractions were discarded. The column was washed with PBS until the OD value at 280 nm was less than 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer (pH 3.75). The eluate was neutralized with 0.4 volume of 1 M K.sub.2HPO.sub.4, and fractions containing the purified protein were combined, dialyzed against PBS, and then filtered through a 0.22 micron nitrocellulose filter and stored at 4° C. The protein product was identified and analyzed by SDS-PAGE under non-reducing and reducing conditions. The results of SDS-PAGE electrophoresis for purified FP4I protein were shown in FIG. 2-a. Under non-reducing conditions, only one clear band (at approximately 130 KD) was present, without polymer. Under reducing conditions, only one band (at approximately 65 KD) was present, without degradation products. In addition, the purity of the fusion protein FP4I sample was examined by SEC-HPLC. As a result, as shown in FIG. 2-b, the percentage of the main peak area reached 98.87%, without polymer. The above results indicated that the fusion protein constructed by the present invention had a good stability and was not susceptible to degradation and aggregation.

Example 4. Effect of a Single Injection of hFGF21 Fusion Protein on Glucose Utilization Rate in Obese Mice Induced by High-Fat Diet

[0121] 32 male C57BL/6J mice aged 7 weeks (purchased from SLAC Laboratory Animal Co., Ltd, Shanghai, SPF-grade) were housed in a controlled environment: temperature of 22-25° C., relative humidity of 45-65%, and 12 h light/dark cycle. After a week of adaption, the mice were fed with high-fat diet (D12492 diet, a product from Research Diets, USA) for 12 weeks. The mice were divided into 4 groups according to the body weight: control group, FP4I-2.5 mg/kg (low-dose group), FP4I-5 mg/kg (middle-dose group) and FP4I-10 mg/kg (high-dose group). The mice in the drug-administration groups were subcutaneously injected with the corresponding FP4I solutions, and the mice in the control group were subcutaneously injected with PBS buffer. Thereafter, all the mice in each group were fasted for 16 hours followed by a glucose tolerance test. The fasting blood glucose level was measured. The mice were then intraperitoneally injected with 2 g/kg of glucose solution and the blood glucose levels at 15 min, 30 min, 60 min, 90 min and 120 min after injection were measured. The incremental area under the curve and above the baseline (iAUC) was calculated by trapezoidal method. 48 hours after drug administration, glucose tolerance test was carried out once more as described above. Data were expressed as mean±standard error of mean (mean±SEM) and analyzed using SPSS 18.0 statistical software. In normal distribution, differences between the means of multiple groups were analyzed by one-way ANOVA, followed by LSD test for homogeneous variances or Dunnett's T3 test for heterogeneous variances. Abnormal distribution was examined by non-parametric test. P values of less than 0.05 were considered statistically significant.

[0122] As show in FIG. 3a and FIG. 3b, middle- and high-dose of FP4I significantly improved the glucose utilization in obese mice at 16 h after administration when compared with the control group (P<0.05). As shown in FIG. 3c and FIG. 3d, the high dose of FP4I could significantly improve the glucose utilization level in obese mice at 48 h after administration as well (P<0.05). The data demonstrated that the hFGF21 fusion protein provided by the present invention had a long-lasting effect on improving glucose utilization.

[0123] The metabolic activities of other hFGF21 fusion proteins in vivo were evaluated by glucose tolerance test as described above. The obese mice induced by high-fat diet were randomly divided into 10 groups (n=6 each group): FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H, FP4J, and control group. Mice in drug-administration groups were subcutaneously injected with 10 mg/kg of corresponding protein solutions while the mice in the control group were subcutaneously injected with PBS buffer. Glucose tolerance tests were performed at 16 h and 48 h after injection, respectively. The results showed that FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H and FP4J significantly improved glucose utilization 16 h after drug-administration (P<0.05) (FIG. 4a), and FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G and FP4H significantly improved glucose utilization at 48 h after drug-administration (P<0.05). However, there was no significant difference in blood glucose level between the FP4J group and the control group (FIG. 4b). Additionally, the data indicated that the hFGF21 fusion protein provided by the present invention had a strong anti-proteolytic ability and was not easily degraded in vivo, thereby maintaining a long physiological activity. Furthermore, compared to the FP4J, a CTP-free fusion protein, the FP4I which contained a CTP structure showed a longer half-life in vivo, demonstrating that the presence of CTP can prolong the half-life in vivo.

Example 5. Pharmacokinetic Studies on a Single Dose of hFGF21 Fusion Protein in Rats

[0124] Six male SD rats weighted 180±10 g (purchased from SLAC Laboratory Animal Co., Ltd, Shanghai, SPF-grade) were housed in the following environment: temperature of 22-25° C., relative humidity of 45-65%, and 12 h light/dark cycle. After one week of adaption, 3 mg/kg of FP4I and FP4J were injected into the tail vein. 0.3 ml of blood was collected from the orbit respectively before administration, and at 1 h, 4 h, 7 h, 24 h, 48 h, 72 h, 96 h, 120 h, 149 h, 168 h, 192 h, 216 h and 240 h after administration. The whole blood was allowed to stand, and then centrifuged at 2000×g for 15 minutes to obtain serum. The sandwich ELISA assay was performed to detect drug concentrations in serum. The mouse anti-hFGF21 antibody (R&D, Cat. No. MAB25373-100) was used as capturing antibody while HRP-labeled goat anti-human IgG-Fc mAb (Jackson, Inc., Cat. No. 109-035-098) was used as detecting antibody. The software PKSOLVER was applied to calculate the pharmacokinetic parameters: T.sub.1/2 and AUC.sub.(0-t).

[0125] The results showed that the circulating half-life T.sub.1/2 of FP4I at the dosage of 3 mg/kg in SD rats was 29.81±1.56 h, and the AUC.sub.(0-t) thereof was 1716711±201507 ng/ml.Math.h, the circulating half-life T.sub.1/2 of FP4J at the dosage of 3 mg/kg in SD rats was 22.43±1.45 h, and the AUC.sub.(0-t) thereof was 1210604±191426 ng/ml.Math.h, while the circulating half-life of native FGF-21 in rats was 1-1.5 h. In summary, FP4I had a significantly increased half-life in vivo, and it had a longer circulating half-life in vivo and higher bioavailability than FP4J.

Example 6. Preventive Effect of hFGF21 Fusion Protein on Obesity, Nonalcoholic Hepatic Steatosis, Insulin Resistance and Hypercholesterolemia in Obese Mie Induced by High-Fat Diet

1. Model Establishment and Drug Administration

[0126] Twenty-four 7-week-old C57BL/6J male mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were housed in a controlled environment: temperature of 22-25° C., relative humidity of 45-65%, and 12 h light/dark cycle. After a week of adaption, the mice were divided into 3 groups according to their body weight: low-fat diet (LFD) group, high-fat diet (HFD) group, and high-fat diet plus 3.6 mg/kg hFGF21 fusion protein FP4I (HFD+FP4I 3.6 mg/kg) group. The mice in the LFD group and HFD groups were fed with D12450B diet and D12492 diet (Research diets, USA) respectively. Mice in the HFD+FP4I 3.6 mg/kg group were subcutaneously injected with 3.6 mg/kg of FP4I every four days, and mice in LFD and HFD groups were subcutaneously injected with PBS solution. The study was conducted for 116 days. At the end of the experiment, all the mice were fasted for 16 hours, and the fasting blood glucose levels and body weights were measured. Whole blood samples were collected by removing eyeballs, and then centrifuged at 2000×g for 15 min to separate and obtain the serum. The liver tissue was excised, washed with physiological saline, cleared the remaining fluid with filter paper and then weighed. Two parts of liver tissue were taken from the same site, wherein one was fixed in 10% formalin solution for HE staining, and the other was rapidly frozen in liquid nitrogen and stored at −80° C. for liver index analysis. The adipose tissue around the epididymis on one side was collected and fixed in a 10% formalin solution for HE staining.

2. Index Detection

2.1. Body Weight Observation

[0127] The mice were weighed every 4 days, and muscle, fat and body fluid contents of the mice were analyzed by time-domain nuclear magnetic resonance on day 104 after the start of the experiment. Changes between body weight before and after the experiment were calculated according to the following formulae: body weight gain=the body weight of the mouse at the end of the experiment—the body weight of the mouse at the beginning of the experiment.

2.2. Serum Biochemical Analysis

[0128] ALT, AST, HDL-c, LDL-c, TG and TC concentrations in serum were measured with automatic biochemical analyzer (Erba XL-200). The specific procedure was performed according to the instrument manual.

2.3. Insulin Tolerance Test, Fasting Insulin Level and Insulin Resistance Index

[0129] At day 118, the mice in each group were fasted for 6 h (10:00 am-4:00 pm) and then the basal blood glucose levels and body weight were measured. 0.75 IU/kg of insulin solution was injected intraperitoneally to the mice. The blood glucose levels were measured at 15, 30, 60, 90, 120 min after injection to draw an insulin tolerance curve. The AUC was calculated by trapezoidal method. The fasting serum insulin content in mice was measured by ELISA assay, and the insulin resistance index was calculated according to the following formula: insulin resistance index=fasting blood glucose level (mmol/L)×fasting insulin content (mIU/L)/22.5.

2.4. Liver Triglyceride Content Measurement

[0130] 50 mg of liver tissue was accurately excised for each mouse. The liver TG contents were measured following the method described by Floch and the results were expressed as the TG content per mg liver tissue.

2.5. Histopathology Examination

[0131] Mouse liver and adipose tissues which were collected from the same site and preserved in 10% formalin solution were stained with HE for morphopathological observation.

3. Statistics and Analysis

[0132] Data were expressed as mean±standard error of mean (mean±SEM) and analyzed by using SPSS 18.0 statistical software. In normal distribution, differences between the means of multiple groups were analyzed by one-way ANOVA, followed by LSD test for homogeneous variances or Dunnett's T3 test for heterogeneous variances. Abnormal distribution was examined by non-parametric test. P<0.05 indicated significant statistical difference.

4. Results

[0133] 4.1. Effect of the hFGF21 Fusion Protein on Body Weight and Fat Content in Mice Fed with High-Fat Diet

[0134] Compared with the low-fat diet group, mice in the high-fat diet group had significantly increased body weight, body weight gain and body fat content, showing obvious obesity symptoms. The hFGF21 fusion protein significantly reduced the body weight gain and body fat content induced under high-fat diet feeding conditions. The results are shown in FIGS. 5-6.

4.2. Effect of the hFGF21 Fusion Protein on Liver Function and Hepatic Steatosis in Mice Fed with High-Fat Diet

[0135] As shown in FIG. 7, compared with the low-fat diet group, serum ALT levels in mice of the high-fat diet group significantly increased, showing significant liver damage and lipid metabolism disorder. Compared to the high-fat diet group, the hFGF21 fusion protein significantly reduced serum ALT levels in mice. As shown in FIG. 8 and FIG. 9, the liver weight and liver triglyceride content in mice fed with high-fat diet were significantly increased, while the hFGF21 fusion protein significantly reduced the liver weight and liver triglyceride content in mice fed with high-fat diet.

4.3. Effect of hFGF21 Fusion Protein on Insulin Resistance in Mice Fed with High-Fat Diet.

[0136] The insulin tolerance test showed that mice fed high-fat diet showed significant symptoms of insulin resistance. Results of the serum insulin test further indicated that mice developed hyperinsulinemia and insulin resistance. The hFGF21 fusion protein significantly alleviated the insulin resistance symptoms and the increase in fasting blood glucose in mice fed with high-fat diet. The results are shown in FIGS. 10-13.

4.4 Effect of hFGF21 Fusion Protein on Hypercholesterolemia in Mice Fed with High-Fat Diet

[0137] As shown in FIG. 14, compared with the low-fat diet group, serum TC and LDL-c levels in mice of the high-fat diet group significantly increased, showing lipid metabolism disorder. Compared to the high-fat diet group, the hFGF21 fusion protein significantly reduced serum TC and LDL-c levels in mice.

4.5. Pathological Morphology Test

[0138] As shown in FIGS. 15 and 16, compared with the low-fat diet group, significant steatosis occurred in the liver tissue, the lipid droplet vacuoles were clearly visible and fused into a sheet-like structure, the morphology of liver cells was severely damaged, and the cross-sectional area of fat cells was significantly increased in mice of the high-fat diet group. Compared with the high-fat diet group, the hFGF21 fusion protein significantly attenuated the steatosis of liver tissue and reduced the cross-sectional area of fat cells.

[0139] Based on the above results, it was confirmed that hFGF21 fusion protein can effectively control the body weight, improve insulin resistance, reverse liver steatosis and hypercholesterolemia in obese mice induced by high-fat diet.

[0140] All documents mentioned in the present invention are hereby incorporated by reference to the same extent as if each of the documents is individually recited for reference. It is to be understood that various changes and modifications may be made by those skilled in the art upon reading the above teachings of the present invention, which also fall within the scope of the claims appended hereto.