DUAL-FUNCTION PROTEIN FOR LIPID AND BLOOD GLUCOSE REGULATION
20210371488 · 2021-12-02
Assignee
Inventors
- Zhao Dong (Shanghai, CN)
- Chi ZHOU (Shanghai, CN)
- Xiong FENG (Shanghai, CN)
- Jiyu ZHANG (Shanghai, CN)
- Shixiang Jia (Shanghai, CN)
- Qiang Li (Shanghai, CN)
Cpc classification
A01K2267/0362
HUMAN NECESSITIES
International classification
Abstract
The present disclosure relates to a dual-function protein for regulating blood glucose and lipid metabolism, wherein said dual-function protein comprises a human GLP-1 analog and human FGF21. In the present disclosure, provided is a method for preparing said dual function protein, and also provided is the use of said dual-function protein in the preparation of a biological substance for treating type 2 diabetes, obesity, dyslipidemia, fatty liver disease and/or metabolic syndrome. The dual-function protein provided in the present disclosure can synergistically regulate blood glucose and lipid levels in vivo, and satisfy multiple requirements for patients with type 2 diabetes such as lowering blood glucose, relieving hepatic steatosis, reducing body weight and improving metabolic disorders of circulating lipids.
Claims
1. A dual-function protein comprising sequentially human GLP-1 analog, linker peptide 1, human FGF21, linker peptide 2 and human immunoglobulin Fc fragment from the N to C-terminus; wherein: the linker peptide 1 comprises a flexible peptide; the linker peptide 2 comprises a flexible peptide and a rigid peptide; the rigid peptide comprises at least 1 rigid unit; and the rigid unit comprises a full-length or truncated sequence consisting of carboxyl terminal amino acids 113 to 145 of human chorionic gonadotropin β-subunit.
2. The dual-function protein of claim 1, wherein said dual-function protein is glycosylated.
3. The dual-function protein of claim 1, wherein said human GLP-1 analog is an analog, fusion peptide, or derivative thereof which is obtained by substituting, deleting or adding one or more amino acid residues on the amino acid sequence of SEQ ID NO: 1 and can maintain human GLP-1 activity.
4. The dual-function protein of claim 3, wherein the GLP-1 analog comprises an amino acid sequence of SEQ ID NO: 2, 3, 4 or 5.
5. The dual-function protein of claim 1, wherein said linker peptide 1 comprises a flexible peptide consisting of 2 or more amino acids.
6. The dual-function protein of claim 5, wherein the amino acids are selected from G, S, A and T.
7. The dual-function protein of claim 6, wherein the amino acid sequence of the flexible peptide is GGGGGGGSGGGGSGGGGS.
8. The dual-function protein of claim 1, wherein said human FGF21 comprises the sequence of SEQ ID NO: 6 wherein the leader peptide of amino acid position 1-28 is deleted.
9. The dual-function protein of claim 1, wherein said human FGF21 comprises the sequence of SEQ ID NO: 6 wherein the leader peptide of amino acid position 1-28 is deleted and which has G141S or L174P substitution.
10. The dual-function protein of claim 1, wherein the flexible peptide constituting said linker peptide 2 comprises 2 or more amino acids selected from G, S, A and T.
11. The dual-function protein of claim 10, wherein the general structural formula of the amino acid composition of said flexible peptide is (GS).sub.a(GGS).sub.b(GGGS).sub.c(GGGGS).sub.d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+c+d≥1.
12. The dual-function protein of claim 10, wherein the amino acid composition of said flexible peptide is selected from: TABLE-US-00006 (i) GGGGS; (ii) GSGGGSGGGGSGGGGS; (iii) GSGGGGSGGGGSGGGGSGGGGSGGGGS; (iv) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; (v) GGGSGGGSGGGSGGGSGGGS; and (vi) GGSGGSGGSGGS.
13. The dual-function protein of claim 1, wherein the rigid units constituting said linker peptide 2 are selected from SEQ ID NO: 7 and the truncated amino acid sequences thereof; wherein said truncated amino acid sequences comprise at least 2 glycosylation sites.
14. The dual-function protein of claim 13, wherein the rigid units comprise one of the following amino acid sequences: TABLE-US-00007 (i) SSSSKAPPPSLPSPSRLPGPSDTPILPQ; (ii) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ; (iii) SSSSKAPPPS; (iv) SRLPGPSDTPILPQ; or (v) SSSSKAPPPSLPSPSR.
15. The dual-function protein of claim 13, wherein said rigid units comprise an amino acid sequence that has at least 90% or 95% amino acid identity with SEQ ID NO: 7 or the truncated amino acid sequences of claim 13.
16. The dual-function protein of claim 1, wherein said rigid peptide comprises 1, 2, 3, 4 or 5 rigid units.
17. The dual-function protein of claim 1, wherein said human immunoglobulin Fc fragment is a variant having a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity with FcRn receptor.
18. The dual-function protein of claim 17, wherein said Fc variant is selected from: (i) hinge, CH2 and CH3 regions of human IgG1 containing Leu234Val, Leu235Ala and Pro331Ser mutations; (ii) hinge, CH2 and CH3 regions of human IgG2 containing Pro331Ser mutation; (iii) hinge, CH2 and CH3 regions of human IgG2 containing Thr250Gln and Met428Leu mutations; (iv) hinge, CH2 and CH3 regions of human IgG2 containing Pro331Ser, Thr250Gln and Met428Leu mutations; and (v) hinge, CH2 and CH3 regions of human IgG4 containing Ser228Pro and Leu235Ala mutations.
19. The dual-function protein of claim 1, wherein the amino acid sequence of said dual-function protein is of SEQ ID NO: 13 or 15.
20. A DNA molecule encoding the dual-function protein of claim 1.
21. The DNA molecule of claim 20, wherein said DNA molecule comprises the sequence as shown in SEQ ID NO: 14.
22. A vector comprising the DNA molecule of claim 20.
23. A host cell, wherein the host cell comprises the vector of claim 22.
24. A host cell, wherein the host cell is transfected with the vector comprising the DNA molecule of claim 20.
25. A pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent, and an effective dose of the dual-function protein of claim 1.
26. A method for preparing the dual-function protein, comprising: (a) introducing the DNA sequence encoding the dual-function protein of claim 20 into a mammalian cell; (b) screening a high-yield cell strain expressing more than 20 μg/10.sup.6 (million) cells within a period of every 24 hours in the growth medium thereof from step (a); (c) culturing the screened cell strain in step (b), and expressing the dual-function protein; (d) harvesting fermentation supernatant obtained from step (c), and purifying the dual-function protein; preferably, said mammalian cell in step (a) is a CHO cell; and more preferably, said mammalian cell is CHO-derived cell line DXB-11.
27. A method of treatment of one or more FGF21 related diseases and GLP-1 related diseases, and other metabolic, endocrine and cardiovascular diseases; comprising administering to a person suffering from at least one of said diseases an effective amount of the dual-function protein of claim 1.
28. The method of treatment of claim 27, wherein the disease is obesity, type 1 diabetes, type 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, nephropathy, diabetic complication, neuropathy, gastroparesis, or symptoms associated with the severe inactivation mutations of insulin receptor.
29. A method of treatment of one or more FGF21 related diseases and GLP-1 related diseases, and other metabolic, endocrine and cardiovascular diseases; comprising administering to a person suffering from at least one of said diseases an effective amount of the pharmaceutical composition of claim 24.
30. The method of treatment of claim 29, wherein the disease is obesity, type 1 diabetes, type 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, nephropathy, diabetic complication, neuropathy, gastroparesis, or symptoms associated with the severe inactivation mutations of insulin receptor.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0096] The present disclosure is further described below in combination with specific embodiments. It is to be understood that these embodiments serve only to illustrate the present disclosure and are not limiting the scope of the present disclosure. In the following embodiments, experimental methods without specifying specific conditions are generally performed under conventional conditions, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.
[0097] Generally, the dual-function protein of the present disclosure is prepared synthetically. The nucleotide sequence according to the present disclosure, a person skilled in the art can conveniently use various known methods to prepare the encoding nucleic acid of the present disclosure. These methods are for example but not limited to: PCR, and DNA artificial synthesis etc., the specific methods can refer to J. Sambrook, “Molecular Cloning: A Laboratory Manual”. As an embodiment of the present disclosure, a method comprising fragment synthesis of nucleotide sequences, followed by overlap extension of PCR can be used for constructing the encoding nucleic acid sequence of the present disclosure.
[0098] Also provided in the present disclosure is an expression vector comprising a sequence encoding the dual-function protein of the present disclosure and a regulatory element transcriptionally linked thereto. Said “transcriptionally linked” or “transcriptionally linked to” refer to such a condition that some parts of a linear DNA sequence can regulate or control the activity of other parts in the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then the promoter is transcriptionally linked to the encoding sequence.
[0099] The expression vector can use commercially available ones, for example but not limited to: vectors pcDNA3, pIRES, pDR and pUC18 which can be used for expression in an eukaryotic system. A person skilled in the art can select a suitable expression vector according to the host cell.
[0100] According to the restriction map of the known expression vector, a person skilled in the art can insert the sequence encoding the dual-function protein of the present disclosure into a suitable restriction site to prepare the recombinant expression vector of the present disclosure following conventional methods via restriction digestion and ligation.
[0101] Also provided in the present disclosure is a host cell expressing the dual-function protein of the present disclosure, wherein said host cell comprises the sequence encoding the dual-function protein of the present disclosure. In at least one embodiment, said host cell is a eukaryotic cell, for example but not limited to CHO, a COS cell, a 293 cell and a RSF cell etc. As a at least one embodiment of the present disclosure, said cell is a CHO cell, which can well express the dual-function protein of the present disclosure, and the dual-function protein with a good binding activity and stability can be obtained.
[0102] Also provided in the present disclosure is a method for preparing the dual-function protein of the present disclosure using recombinant DNA, the steps thereof comprise: [0103] 1) Providing a nucleic acid sequence encoding the synergistic dual function protein; [0104] 2) Inserting the nucleic acid sequence of 1) into a suitable expression vector, and obtaining a recombinant expression plasmid; [0105] 3) Introducing the recombinant expression plasmid of 2) into a suitable host cell; [0106] 4) Culturing the transformed host cell under a condition suitable for expression; [0107] 5) Collecting the supernatant, and purifying the dual-function protein product.
[0108] To introduce said encoding sequence into the host cell one can use multiple known technologies in the art, for example but not limited to: calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, and alkali metal ion method.
[0109] With respect to the culture of and expression in the host cell can refer to Olander R M Dev Biol Stand, 1996, 86:338. Cells and debris in the suspension can be removed by centrifugation, and the supernatant is collected. Agarose gel electrophoresis technique can be used for identification.
[0110] The dual-function protein prepared as described herein can be purified to have a substantially homogeneous property, such as has a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed for secretion, a commercially available ultrafiltration membrane (such as products of Millipore and Pellicon etc.) can be used to separate said protein, wherein firstly, the expression supernatant is concentrated. The concentrate can be purified by the method of gel chromatography, or by the method of ion exchange chromatography, for example, by anion exchange chromatography (DEAE etc.) or cation exchange chromatography. The gel matrix can be common matrices for protein purification, such as agarose, glucan, and polyamide etc. Q- or SP-groups is a relatively ideal ion exchange group. Finally, the above-mentioned purified product can be further refined and purified by the methods of hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed high performance liquid chromatography (RP-HPLC). All the above-mentioned purification steps can be used in different combination in order to make the protein purity substantially homogeneous.
[0111] The expressed dual-function protein can be purified using an affinity column containing a specific antibody, receptor or ligand of said dual function protein. According to the properties of the affinity column, conventional methods, such as high salt buffer and changing pH etc. can used to elute the fusion polypeptide binding to the affinity column. Optionally, at the amino terminus or carboxyl terminus of said dual function protein, one or more polypeptide fragments also can be contained as protein tags. Any suitable tags can be used in the present disclosure. For example, said tags can be FLAG, HA, HAL c-Myc, 6-His or 8-His etc. These tags can be used for purifying the dual function protein.
Non-Limiting Exemplary Embodiments
[0112] 1. A dual-function protein comprising sequentially human GLP-1 analog, linker peptide 1, human FGF21, linker peptide 2 and human immunoglobulin Fc fragment from the N to C-terminus; wherein the linker peptide 1 comprises or consists of a flexible peptide; the linker peptide 2 comprises or consists of a flexible peptide and a rigid peptide, the rigid peptide comprises or consists of at least 1 rigid unit, and the rigid unit comprises a full-length or truncated sequence consisting of carboxyl terminal amino acids 113 to 145 of human chorionic gonadotropin β-subunit.
[0113] 2. The dual-function protein of embodiment 1, wherein said dual-function protein is glycosylated; preferably, said dual-function protein is glycosylated by being expressed in mammalian cells; and more preferably, said dual-function protein is glycosylated by being expressed in Chinese hamster ovary cells.
[0114] 3. The dual-function protein of embodiment 1, wherein said human GLP-1 analog is an analog, fusion peptide, or derivative thereof which is obtained by substituting, deleting or adding one or more amino acid residues on the amino acid sequence of SEQ ID NO: 1 and can maintain human GLP-1 activity.
[0115] 4. The dual-function protein of embodiment 3, wherein the GLP-1 analog comprises an amino acid sequence of SEQ ID NO: 2, 3, 4 or 5.
[0116] 5. The dual-function protein of embodiment 1, wherein said linker peptide 1 comprises a flexible peptide consisting of 2 or more amino acids; preferably, consisting of 5-30 amino acids.
[0117] 6. The dual-function protein of embodiment 5, wherein the amino acids of said linker peptide 1 are selected from the following amino acids: G, S, A and T; more preferably, said linker peptide 1 comprises G and S residues; and most preferably, the amino acid sequence of said linker peptide 1 is GGGGGGGSGGGGSGGGGS.
[0118] 7. The dual-function protein of embodiment 1, wherein said human FGF21 comprises the sequence as shown in SEQ ID NO: 6 in which the leader peptide of amino acid position 1-28 is deleted; or comprises the isoform sequence of SEQ ID NO: 6 in which the leader peptide of amino acid position 1-28 is deleted and which has G141S or L174P substitution.
[0119] 8. The dual-function protein of embodiment 1, wherein the flexible peptide constituting said linker peptide 2 comprises 2 or more amino acids selected from G, S, A and T; preferably, the general structural formula of the amino acid composition of said flexible peptide is (GS).sub.a(GGS).sub.b(GGGS).sub.c(GGGGS).sub.d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+c+d≥1; more preferably, the amino acid composition of said flexible peptide is selected from:
TABLE-US-00003 (i) GGGGS; (ii) GSGGGSGGGGSGGGGS; (iii) GSGGGGSGGGGSGGGGSGGGGSGGGGS; (iv) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; (v) GGGSGGGSGGGSGGGSGGGS; and (vi) GGSGGSGGSGGS.
[0120] 9. The dual-function protein of embodiment 1, wherein the rigid units constituting said linker peptide 2 are selected from SEQ ID NO: 7 and the truncated amino acid sequences thereof; wherein said truncated amino acid sequences comprise at least 2 glycosylation sites; preferably, said rigid units comprise one of the following amino acid sequences:
TABLE-US-00004 (i) SSSSKAPPPSLPSPSRLPGPSDTPILPQ; (ii) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ; (iii) SSSSKAPPPS; (iv) SRLPGPSDTPILPQ; or (v) SSSSKAPPPSLPSPSR.
[0121] 10. The dual-function protein of embodiment 9, wherein said rigid units have at least 90% or 95% identity to the amino acid sequences of the rigid units of embodiment 9.
[0122] 11. The dual-function protein of embodiment 1, wherein said rigid peptide comprises 1, 2, 3, 4 or 5 rigid units.
[0123] 12. The dual-function protein of embodiment 1, wherein said human immunoglobulin Fc fragment is a variant having a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity with FcRn receptor; preferably, said Fc variant is selected from:
[0124] (i) hinge, CH2 and CH3 regions of human IgG1 containing Leu234Val, Leu235Ala and Pro331Ser mutations;
[0125] (ii) hinge, CH2 and CH3 regions of human IgG2 containing Pro331Ser mutation;
[0126] (iii) hinge, CH2 and CH3 regions of human IgG2 containing Thr250Gln and Met428Leu mutations;
[0127] (iv) hinge, CH2 and CH3 regions of human IgG2 containing Pro331Ser, Thr250Gln and Met428Leu mutations; and
[0128] (v) hinge, CH2 and CH3 regions of human IgG4 containing Ser228Pro and Leu235Ala mutations.
[0129] 13. The dual-function protein of embodiment 1, wherein the amino acid sequence of said dual-function protein is shown in SEQ ID NO: 13 or 15.
[0130] 14. A DNA molecule encoding the dual-function protein of any of embodiments 1-13.
[0131] 15. The DNA molecule of embodiment 14, wherein said DNA molecule comprises the sequence as shown in SEQ ID NO: 14.
[0132] 16. A vector, wherein the vector comprises the DNA molecule of embodiment 14 or 15.
[0133] 17. A host cell, wherein the host cell comprises the vector of embodiment 16, or is transfected with the vector comprising the DNA molecule of embodiment 14 or 15.
[0134] 18. A pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent, and an effective dose of the dual-function protein of any of embodiments 1-13.
[0135] 19. A method for preparing the dual-function protein of any of embodiments 1-13, comprising: [0136] (a) introducing the DNA sequence encoding the dual-function protein of embodiment 14 or 15 into a mammalian cell; [0137] (b) screening a high-yield cell strain expressing more than 20 μg/10.sup.6 (million) cells within a period of every 24 hours in the growth medium thereof from step (a); [0138] (c) culturing the screened cell strain in step (b), and expressing the dual-function protein; [0139] (d) harvesting fermentation supernatant obtained from step (c), and purifying the dual-function protein; preferably, said mammalian cell in step (a) is a CHO cell; and more preferably, said mammalian cell is CHO-derived cell line DXB-11.
[0140] 20. The use of the dual-function protein of any of embodiments 1-13 in the preparation of a drug substance for treating FGF21 related diseases and GLP-1 related diseases, and other metabolic, endocrine and cardiovascular diseases; preferably, said diseases comprise obesity, types 1 and 2 diabetes, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, nephropathy, diabetic complication, neuropathy, gastroparesis, and symptoms associated with the severe inactivation mutations of insulin receptor; and more preferably, said diseases comprise obesity, types 1 and 2 diabetes, dyslipidemia, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis and metabolic syndrome.
EXAMPLES
Example 1: Construction of an Expression Plasmid of the Synergistic Dual Function Protein
[0141] All gene sequences encoding alpha 1 microglobulin secretion leader signal, GLP-1 analog, L1, FGF21 mature protein, L2 (comprising a flexible linker unit and rigid linker unit) and human IgG Fc variants were optimized using CHO preferred codons and the full-length gene sequences were synthesized. There are a SpeI at the 5′ and a EcoRI at the 3′ for subcloning the target gene encoding the fusion protein into the expression vector PXY1A1 modified from PCDNA3.1 (
[0142] Various dual-function proteins comprising GLP-1 and FGF21 were constructed. Here, three are exemplified: FP4I-1, FP4I-2 and FP4I-3. The amino acid composition is shown in Table 1 (L1 and L2 were underlined, and mutated amino acids in Fc variants were boxed).
TABLE-US-00005 TABLE 1 Amino acid composition of each synergistic dual function protein FP4I-1
Example 2: Expression of the Dual-Function Protein in a Transfected Cell Line
[0143] A recombinant expression vector plasmid was transfected into a mammalian host cell line to express the synergistic dual function protein. In order to stabilize the high expression, a preferred host cell line was DHFR defective CHO-cell (U.S. Pat. No. 4,818,679). In the present example, the host cell was selected from CHO-derived cell line DXB11. A preferred transfection method was electroporation, but other methods such as calcium phosphate and liposome-induced transfection also can be used. A Gene Pulser electroporation apparatus (Bio-Rad Laboratories, Hercules, Calif.) setting at 300 V of electric field and 1500 μFd of capacitance was used in the present experiment and 50 μg pure expression plasmid was mixed with 5×10.sup.7 CHO cells in the cuvette. Two days after the transfection, a selection medium containing 0.6 mg/mL G418 was used. Quantitative ELISA using anti-human IgG Fc was applied to screen the transfectants with the resistance to G418. Anti-human FGF21 or anti-human GLP-1 by ELISA was used to quantify expression of the dual-function protein. A 96 well culture plate was subjected to the limiting dilution, the well generating a high level of the dual-function protein was subcloned.
[0144] In order to achieve a relatively high expression of the dual-function protein, it was appropriate to use the DHFR gene inhibited by MTX for co-amplification. In another selection medium containing incremental concentrations of MTX, the gene of the dual-function protein was co-amplified with the DHFR gene. The subclone with a positive DHFR expression was subjected to the limiting dilution, the selection pressure was gradually increased and the transfectant which can grow in a medium with up to 6 μM MTX was selected. The secreting rate of transfectant was determined and the cell line with a high expression of exogenous protein was screened out. The cell lines with secretory rate higher than about 10 (preferably about 20) μg/10.sup.6 (i.e. a million) cells/24 hours was subjected to an adaptive suspension culture in serum-free medium, then the dual-function protein was purified by a specified medium.
Example 3: Purification and Qualification of the Dual-Function Protein
[0145] This example describes the exemplary purification and qualification methods of FP4I-2. The cell culture supernatant was subjected to clarifying treatments, such as high speed refrigerated centrifugation and 0.22 μm sterile filtration etc., then purified by three chromatograph steps including protein A, anion exchange and hydrophobic chromatography, the specific method was as follows: In the first step, protein A was used for capture, wherein the equilibrium solution was PBS buffer, the eluant was a citrate buffer at pH 3.5, then the eluted protein was neutralized by 1 M Tris solution. In the intermediate purification process, high resolution anion exchange packing material Q Sepharose HP (GE company) was selected to remove residual impurity proteins. A combined mode was used, that is, 20 mM Tris-HCl, 0.2 M NaCl, pH 7.5 solution was used for rinsing, and 20 mM Tris-HCl, 0.3 M NaCl, pH 7.5 solution was used for elution. In the fine purification step, Butyl Sepharose FF (GE) was selected to remove polymers; due to different hydrophobic properties of FP4I-2 monomer and polymer, the monomer with weak hydrophobic property flowed through directly, but the polymer with high hydrophobic property bound to the medium; hydrophobic chromatography was selected as flow through mode, and the equilibrium solution was PBS buffer.
[0146] The qualitative analysis result is shown in
Example 4: Effect of a Single Injection of the Dual-Function Protein on Glucose Utilization in C57BL/6 Mice
[0147] 8 weeks aged male C57BL/6J mice at SPF grade (purchased from Beijing HFK Bioscience Ltd.) were selected. Housing conditions: temperature 22-25° C., relative humidity 45-65%, and 12 h-light/dark cycle. After acclimation for 1 week, mice were randomly divided into control group, Dulaglutide 120 nmol/kg group, FP4I-2 120 nmol/kg group and FP4I-1 120 nmol/kg group (n=7) according to body weight. The mice in the treatment groups were injected subcutaneously with corresponding drug solutions, while the mice in the control group were injected subcutaneously with PBS buffer. After the injection, mice in each group were fasted for 16 h, and then glucose tolerance test was performed. The fasting blood glucose values of the mice were determined followed by an intraperitoneal injection of a 2 g/kg glucose solution, the blood glucose values were determined at 15 min, 30 min, 60 min, 90 min and 120 min after glucose injection, and the increased area below the curve and above the baseline (iAUC) was calculated by the trapezoidal method. The glucose tolerance test was further performed on the mice of each group at 96 h and 144 h after the administration, and the method was the same as above. The data were represented as means±SEM, and analyzed using SPSS18.0 statistical software. For the Gaussian distribution data, statistical comparison of the means among the groups was performed using one-way ANOVA, followed by LSD test for the homogeneity of variance or Dunnet T3 test for the heterogeneity of variance; non-parametric test was used for the Non-Gaussian distribution data. P<0.05 represented a significant statistical difference.
[0148] As shown in
[0149] In addition, the activity of Exendin4-FGF21 dual-function protein FP4I-3 on the glucose utilization was determined by above-mentioned method as well. C57BL/6 mice were divided into the control group, Dulaglutide group and FP4I-3 group. Corresponding drug solutions (120 nmol/kg) were administrated subcutaneously to the mice in Dulaglutide group and FP4I-3 group, respectively, and PBS buffer was administrated to the mice in the control group. The glucose tolerance test was performed at 16 h, 96 h, and 144 h after the injection. As shown in
[0150] The glucose tolerance test of FP4I-3 in animals demonstrated that Exendin-4 did not display a synergistic effect with FGF21. The hypoglycemic effect of FP4I-3 was significantly weaker than that of Dulaglutide indicated that the circulating half-life of FP4I-3 was shorter than Dulaglutide. In contrast, the preferred GLP-1-FGF21 dual-function proteins FP4I-2 and FP4I-1 had a relatively strong stability in vivo, and were not easily degraded and inactivated, and maintained a longer in vivo pharmacodynamic activity relative to Exendin4-FGF21 dual-function protein FP4I-3. Above results indicated that the combination modes of three functional components, GLP-1 analogs, FGF21 and Fc fragment in the dual-function protein were not random and arbitrarily, wherein the selection of GLP-1 analogs, the structure of linker peptide, the fusion sequence, even the difference of glycosylation pattern would affect accuracy and stability of the dual-function protein conformation to varying degrees, and it determined whether the active molecules were functionally synergetic and the half-life was prolonged or not.
Example 5: Hypoglycemic Effect of Dual-Function Protein in Db/Db Mice
[0151] 8 weeks aged male db/db mice were purchased from Shanghai SLAC Laboratory Animal Ltd. Housing conditions: temperature 22-25° C., relative humidity 45-65%, and 12 h-light/dark cycle. After housed individually for 1 week as acclimation, the mice were divided into 4 groups according to body weight, blood glucose and food intake: control group, Dulaglutide group, FP4I-1 group and FP4I-2 group (n=7). Mice in the control group were injected subcutaneously with PBS buffer, and mice in other groups were injected subcutaneously with 120 nmol/kg corresponding drug solutions (twice per week, totally 8 times). Daily food intake of each mouse was recorded. At the end of the dosing period, mice were fasted for 16 hours, 5 μL whole blood sample was collected from the eye socket to measure glycosylated hemoglobin. The data were represented as means±standard error (
[0152] As shown in
Example 6. Therapeutic Effects of the Dual-Function Protein on Weight Loss, Hepatic Steatosis and Lipid Metabolism Disorder in Obese Mice Induced by High-Fat Diet
[0153] 8 weeks aged C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Ltd. Housing conditions: temperature 22-25° C., relative humidity 45-65%, and lighting time 12 h/d. After acclimation for 1 week, 7 mice were selected and fed with low-fat diet (D12450B, Research Diets), and other mice were fed with high-fat diet (D12451, Research Diets). 40 weeks later, obese mice were subjected to adaptive feeding with single animal/cage for 1 week, then the obese mice were divided into five groups according to body weight and weekly food intake: obese control group, Dulaglutide group, high fat diet pair-fed group, FP4I-1 group and FP4I-2 group (n=7). In the experiment, the amounts of daily diet per mouse in high fat diet pair-fed, FP4I-1 and FP4I-2 groups were consistent with daily food intake per mouse in Dulaglutide group. Mice in the obese control group and high fat diet pair-fed group were injected subcutaneously with PBS buffer solution, and mice in other groups were injected subcutaneously with 120 nmol/kg corresponding drug solutions, once every 6 days, and totally 2 times. The body weight of each mouse was recorded before and after the dosing period. At the end of the dosing period, mice in each group were fasted for 16 hours, whole blood was collected from the eye socket, and centrifuged at 2000×g for 15 min to obtain serum. Serum lipid profiles were determined by an automatic biochemical analyzer. Liver tissue was excised, washed with normal saline, then removed residual liquid with filter paper and weighed. About 50 mg liver tissue at the same part of each live was taken, and the triglyceride content was determined using the Folch method. The results were represented in the form of triglyceride content per mg liver tissue. The data were represented as means±SEM, and analyzed using SPSS18.0 statistical software. For the Gaussian distribution data, statistical comparison of the means among the groups was performed using one-way ANOVA, followed by LSD test for the homogeneity of variance or Dunnet T3 test for the heterogeneity of variance; non-parametric test was used for the Non-Gaussian distribution data. P<0.05 represented a significant statistical difference.
[0154] As shown in
[0155] Compared with Dulaglutide group, liver mass and liver triglyceride content of mice in FP4I-2 group were significantly decreased (P<0.01 or P<0.05), which demonstrated that FP4I-2 effectively reduced the excessive accumulation of triglyceride in liver, improved liver function. The results indicated that FP4I-2 could be used for treating various liver diseases induced by hepatic steatosis, such as nonalcoholic fatty liver, nonalcoholic steatohepatitis, liver fibrosis and liver cirrhosis.
[0156] Compared with Dulaglutide group, both total serum cholesterol and low density lipoprotein-cholesterol content of mice in FP4I-2 group were significantly reduced (P<0.01 or P<0.05), indicating that FP4I-2 can be used for treating hypercholesteremia and relevant cardiovascular and cerebrovascular diseases, such as hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis. Compared with Dulaglutide group, the body weight, liver mass, liver triglyceride content, serum triglycerides, total cholesterol and low density lipoprotein cholesterol levels in FP4I-1 group were mildly decreased but no significant differences were observed.
[0157] The present study demonstrated that FP4I-1 and FP4I-2 could treat obesity, fatty liver disease and lipid metabolic disorder via the physiological activity of FGF21, and was not completely dependent on the food intake regulation effect of GLP-1 analogs; the therapeutic effect of FP4I-2 in the obese mice was superior to Dulaglutide, which indicated that it could compensate for the deficiency of Dulaglutide in the clinic. In conclusion, the therapeutic mechanisms of FP4I-2 are more abundant than that of Dulaglutide, which is more suitable for the requirement of diversified clinical therapy.
[0158] This disclosure provides merely exemplary embodiments of the disclosure. One skilled in the art will readily recognize from the disclosure and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the disclosure as defined in the following claims.
[0159] All documents mentioned in this application are hereby incorporated by reference as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the invention, those skilled in the art can make various changes or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.