CN114591415A

CN114591415A - GLP-1/GCG dual-receptor agonist polypeptide and fusion protein thereof

Info

Publication number: CN114591415A
Application number: CN202111474768.9A
Authority: CN
Inventors: 蒋鹏; 肖�琳; 周林俊; 王慧; 凌伊; 陈小锋; 李文佳
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Sunshine Lake Pharma Co Ltd
Priority date: 2020-12-03
Filing date: 2021-12-03
Publication date: 2022-06-07
Anticipated expiration: 2041-12-03
Also published as: CN114591415B; WO2022117044A1

Abstract

The invention relates to a GLP-1/GCG dual-receptor agonist polypeptide, and further relates to a fusion protein containing the GLP-1/GCG dual-receptor agonist polypeptide. The GLP-1/GCG dual-receptor agonist polypeptide and the fusion protein have different mutation sites with the existing published polypeptide or fusion protein, have good effects of reducing blood sugar and weight and long half-life in vivo, and are more effective long-acting weight-reducing and sugar-controlling medicaments.

Description

GLP-1/GCG dual-receptor agonist polypeptide and fusion protein thereof

Technical Field

The invention relates to the field of biomedicine, in particular to GLP-1/GCG dual-receptor agonist polypeptides, fusion proteins, nucleic acids, constructs, recombinant cells, pharmaceutical compositions and pharmaceutical application.

Background

Obesity is closely related to type II diabetes, hyperlipidemia, hypertension and the like. At present, the primary effect of the first-line medicament for treating type II diabetes is blood sugar reduction, but the weight reduction effect is very limited, and how to develop the medicament with good blood sugar reduction and weight reduction effects is the current research fever point in the field.

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by intestinal L-cells after eating, and can stimulate pancreatic beta cells to secrete insulin, thereby stabilizing the fluctuation of postprandial blood sugar. The function of reducing blood sugar has glucose concentration dependency, and the risk of blood sugar is greatly reduced while blood sugar is regulated. GLP-1 based drugs, such as liraglutide, dolabrlutide and somagluteptide, have gradually taken a very important position in diabetes drugs in recent years. The GLP-1 medicines have the effect of losing weight when reducing blood sugar, and the mechanism is that GLP-1 acts on gastrointestinal tracts to delay gastric emptying and intestinal peristalsis, acts on a central nervous system to suppress appetite and the like, so that the purpose of reducing food intake is achieved. Liraglutide has been approved as an antiobesity drug before, and its upgraded product, namely somaglutide (trade name wegovy), has better weight-reducing effect and is approved by FDA to be marketed in 6 months of 2021.

However, clinical trials have demonstrated that the disadvantages of GLP-1 receptor agonists are also evident, mainly in the following areas: firstly, the short half-life results in intensive injection frequency and inconvenience for patients; secondly, the effects of weight loss and blood sugar reduction are still limited, and certain unmet clinical needs still exist. Preclinical research shows that the glucagon-like peptide 1 and glucagon (GLP-1/GCG) dual-receptor agonist shows better weight-reducing and blood-sugar-reducing effects compared with a single-target GLP-1 agonist, and has great development potential.

As a double-receptor agonist for activating glucagon-like peptide-1 (GLP-1) and glucagon (GCG), Oxyntomodulin (OXM) is proved to have good effects of reducing blood sugar and weight in an animal model by research, and is obviously superior to the existing GLP-1 medicaments, such as liraglutide. However, some problems of Oxyntomodulin (OXM) such as poor stability and low receptor activity lead to large dosage of oxyntomodulin, which makes it difficult to achieve optimal effect of sugar control and weight loss.

Disclosure of Invention

In order to solve the problems, the inventor optimizes and modifies OXM, improves the stability, prolongs the half-life period in vivo, improves the activity of receptors, optimizes the balance among the receptors and achieves the optimal effect of controlling sugar and losing weight.

Based on the optimization and transformation result, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, the invention features a GLP-1/GCG dual receptor agonist polypeptide. According to an embodiment of the invention, the polypeptide has an amino acid sequence as shown below:^5’X₁SQGT FTSDY SKYLD EKX₁₈AK X₂₁FX₂₃EW LX₂₇X₂₈X₂₉X₃₀ ^3’wherein, X₁Is H or Y; x₁₈Is R, A or K; x₂₁Is E or D; x₂₃Is V or I; x₂₇Is L or I; x₂₈Is A or E; x₂₉Is A or G; x₃₀Is GPSSGAPPPS or is absent; when X27 is L, X28Is E.

According to an embodiment of the present invention, the above-mentioned polypeptide may further have at least one of the following additional technical features:

according to an embodiment of the invention, the polypeptide has an amino acid sequence as shown below:^5’X₁SQGT FTSDY SKYLD EKX₁₈AK EFX₂₃EW LX₂₇X₂₈GX₃₀ ^3’wherein X is₁Is H or Y, X₁₈Is R, A or K, X₂₃Is V or I, X₂₇Is L or I, X₂₈Is A or E.

According to an embodiment of the invention, the polypeptide has an amino acid sequence as shown below: HSQGT FTSDY SKYLD EKX₁₈AK EFX₂₃EW LX₂₇X₂₈G；

Wherein, X₁₈Is R, A or K, X₂₃Is V or I, X₂₇Is L or I, X₂₈Is A or E.

According to an embodiment of the invention, the polypeptide has the amino acid sequence as described above, and at least one of X23 or X27 in the amino acid sequence of the polypeptide is I.

According to an embodiment of the invention, the polypeptide has an amino acid sequence shown in any one of SEQ ID NOs 1-5.

HSQGT FTSDY SKYLD EKAAK EFIEW LLEG(SEQ ID NO:1)。

HSQGT FTSDY SKYLD EKRAK EFIEW LIAG(SEQ ID NO:2)。

HSQGT FTSDY SKYLD EKRAK EFVEW LIAG(SEQ ID NO:3)。

HSQGT FTSDY SKYLD EKKAK EFIEW LIAG(SEQ ID NO:4)。

HSQGT FTSDY SKYLD EKKAK EFVEW LIAG(SEQ ID NO:5)。

The inventor finds that the GLP-1/GCG dual-receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO. 1-5 has stronger in-vitro biological activity effect on GLP-1R and GCGR cells, and in-vivo experimental research results show that the GLP-1/GCG dual-receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO. 1-5 has obvious effects on controlling body weight and reducing blood sugar.

In a second aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, the fusion protein comprises the polypeptide as described above, a linking peptide and an IgG Fc fragment, the linking peptide being disposed between the head and the tail of the polypeptide and the IgG Fc fragment. The fusion protein provided by the embodiment of the invention has good effects of reducing blood sugar and weight and long half-life in vivo, and is an effective long-acting weight-reducing and sugar-controlling medicament.

According to an embodiment of the present invention, the above fusion protein may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the N-terminus of the linker peptide is linked to the C-terminus of the polypeptide, and the C-terminus of the linker peptide is linked to the N-terminus of the IgG Fc fragment.

According to an embodiment of the invention, the linker peptide has the amino acid sequence as shown in any one of SEQ ID NO 6-8.

GGGGSGGGGSGGGGS(SEQ ID NO:6)。

GGGGSGGGGSGGGGSA(SEQ ID NO:7)。

GGGGSGGGGS(SEQ ID NO:8)。

The IgG Fc fragment used in the present invention is derived from the Fc region of human IgG1, IgG2, or IgG4, or a mutant thereof. Preferably from the Fc region of human IgG4 or a mutant thereof.

According to an embodiment of the invention, the IgG Fc fragment is derived from a mutant Fc region of human IgG4 comprising the amino acid sequence:

ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:9)。

wherein the IgG4 Fc mutant has three mutations (EU numbering) at positions S228P, F234A and L235A, which correspond to positions 10, 16 and 17 of SEQ ID NO. 9, respectively, compared with the wild-type Fc of human IgG 4. The above mutations may further abolish the effector functions of IgG4 Fc, such as ADCC and CDC, to improve the safety of the fusion protein. And simultaneously, the K447 lysine amino acid at the C terminal is deleted, so that the heterogeneity condition of the fusion protein is avoided.

According to the embodiment of the invention, the fusion protein has an amino acid sequence shown in any one of SEQ ID NO 10-14.

HSQGTFTSDYSKYLDEKAAKEFIEWLLEGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:10)。

HSQGTFTSDYSKYLDEKRAKEFIEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:11)。

HSQGTFTSDYSKYLDEKRAKEFVEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:12)。

HSQGTFTSDYSKYLDEKKAKEFIEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:13)。

HSQGTFTSDYSKYLDEKKAKEFVEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:14)。

In a third aspect of the invention, the invention features a nucleic acid. According to an embodiment of the invention, the nucleic acid encodes a polypeptide as described above or a fusion protein as described above.

In a fourth aspect of the invention, the invention features a construct. According to an embodiment of the invention, the construct carries a nucleic acid as described above.

According to an embodiment of the present invention, the above-mentioned construct may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the vector of the construct is pxcc 17.4.

In a fifth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell expresses a polypeptide as described above or a fusion protein as described above.

In a sixth aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises the aforementioned construct or a nucleic acid integrated into its genome.

According to an embodiment of the present invention, the recombinant cell may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the recombinant cell is a CHO cell.

In a seventh aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises a polypeptide as described above or a fusion protein as described above.

According to an embodiment of the present invention, the pharmaceutical composition may further include at least one of the following additional technical features:

according to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

According to an embodiment of the invention, the pharmaceutical composition further comprises a further antidiabetic drug comprising at least one selected from the group consisting of insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha-glucosidase inhibitors and aminopeptidase IV inhibitors.

In an eighth aspect of the invention, a protein formulation is presented. According to an embodiment of the invention, a polypeptide as described above or a fusion protein as described above is included. The protein preparation provided by the embodiment of the invention is an effective long-acting weight loss and sugar control medicament.

In a ninth aspect, the invention provides the use of a polypeptide as hereinbefore described or a fusion protein as hereinbefore described in the manufacture of a medicament for the treatment or prophylaxis of a metabolic disease.

According to an embodiment of the present invention, the metabolic disease includes at least one selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), diabetes, and obesity.

Drawings

FIG. 1 is a graph of blood glucose concentration versus time at various time points after administration of a sugar-loaded mouse according to an embodiment of the present invention;

FIG. 2 is a graph of blood glucose concentration versus time at various time points after administration of a sugar-loaded mouse according to an embodiment of the present invention;

FIG. 3 is a graph showing experimental results of the effect of a fusion protein according to an embodiment of the present invention on glucose tolerance in DIO mice;

FIG. 4 is a graph showing the results of experiments on the weight effect of fusion proteins according to the embodiments of the present invention on DIO mice;

fig. 5 is a graph showing experimental results of the fusion protein HEC-C70 on glucose tolerance in DIO mice according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of experiments on the effect of the fusion protein HEC-C70 on body weight of DIO mice according to an embodiment of the present invention;

FIG. 7 is a graph showing the results of experiments on random blood glucose in db/db mice using the fusion protein HEC-C70 according to an embodiment of the present invention;

FIG. 8 is a graph showing the results of experiments on the effect of the fusion protein HEC-C70 on the body weight of db/db mice according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Definition of terms

The terms "protein" and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound that is two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other linkages, such as ester, ether, amino, and the like. A protein or polypeptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. The term "amino acid" as used herein refers to natural and/or unnatural or synthetic amino acids, including D and L optical isomers of amino acids, e.g., glycine and D and L optical isomers, amino acid analogs, and peptidomimetics.

The "Fc fragment" described herein consists of the hinge, CH2 and CH3 constant region structures of an antibody. Antibodies comprise two functionally independent parts, a variable domain that binds antigen, called "Fab", and a constant domain, called "Fc", that is involved in effector functions (e.g., complement activation and attack by phagocytes). Fc has a long serum half-life, while Fab is short-lived (Capon et al, 1989, Nature 337: 525-31). When linked to a therapeutic protein, the Fc domain may provide a longer half-life or incorporate such functions as Fc receptor binding, protein a binding, complement binding and perhaps even placental transfer (Capon et al, 1989). The term "Fc" as used herein refers to a wild-type Fc sequence from a native antibody (e.g., human IgG1, IgG2, IgG3, or IgG4), and also includes variants thereof. Variants may include one or more amino acid substitutions, additions and/or deletions already disclosed. In some embodiments, the Fc variant has the activity of a wild-type Fc, such as binding to an Fc receptor.

The amino acid numbering of the IgG4 Fc fragment is according to EU numbering system, e.g., the "S228P" refers to a substitution of proline for serine at position 228, as numbered by EU numbering system; "K447" means that lysine at position 447 has been deleted or absent according to EU numbering system.

The term "fusion protein" generally refers to a protein resulting from the fusion of two or more proteins or polypeptides. Genes or nucleic acid molecules encoding the two or more proteins or polypeptides may be linked to each other to form a fusion gene or a fused nucleic acid molecule, which may encode the fusion protein. Translation of the fusion gene results in a single polypeptide having the properties of at least one, and even each, of the two or more proteins or polypeptides prior to fusion. Recombinant fusion proteins are created artificially by recombinant DNA techniques for biological research or therapy. Recombinant fusion proteins are proteins created by genetic engineering of fusion genes. The present invention relates to recombinant fusion proteins, and the terms fusion protein and recombinant fusion protein are used herein with the same meaning. The fusion proteins described herein typically comprise at least two domains (a and C), and optionally a third component, a linker between the two domains. The generation of recombinant fusion proteins is known in the art and typically involves removing a stop codon from a cDNA sequence encoding a first protein or polypeptide and then attaching the cDNA sequence of a second protein in frame by ligation or overlap extension PCR. The DNA sequence will then be expressed by the cell as a single protein. The protein may be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion of either.

When forming the fusion protein of the present invention, a linker or linker peptide may be, but need not be, used. The G-rich polypeptides of the invention may be selected from the group consisting of (G)3-S (i.e., "GGGS"), (G)4-S (i.e., "GGGGS"), and (G)5-S (i.e., "GGGGGS"). In some embodiments, the linker peptide comprises GGGGSGGGGS (SEQ ID NO:8), GGGGSGGGGSGGS (SEQ ID NO:6), or GGGGSGGGGSGGGGSA (SEQ ID NO: 7). The linkers described herein are exemplary and the linkers of the invention can be much longer linkers as well as linkers comprising other residues. The linker of the invention may also be a non-peptide linker.

As used herein, the term "comprising" or "includes" generally means including the stated elements, but not excluding others.

Example 1 preparation of GLP-1/GCG Dual receptor agonist Polypeptides

A fusion gene is formed by adding a gene sequence of SUMO tag to the 5' end of the gene encoding the polypeptide. The fusion gene is cloned to a prokaryotic expression vector and is induced and expressed in Escherichia coli cells. And after centrifugation, the thalli are crushed by ultrasound and centrifuged to take the supernatant, and then the fusion protein is obtained by nickel column purification. And finally, carrying out enzyme digestion treatment on the SUMO protease, and carrying out reversed-phase purification to obtain the target polypeptide. The specific process is as follows:

1. vector construction and primer Synthesis

The specific steps for preparing the polypeptide by taking pET-28a as an expression vector and BL21(DE3) as a host are as follows:

1) designing primers, and mutually using the primers as templates to amplify the polypeptide gene segment. And using the polypeptide gene fragment and sumo gene fragment as templates to amplify the fusion gene fragment by a fusion PCR method.

2) Constructing a recombinant expression vector: the fusion gene fragment is cloned into a prokaryotic expression vector pET-28a, escherichia coli BL21(DE3) is transformed, a recon is selected, and a recombinant expression plasmid sample is sent to Guangzhou Egyptian biotechnology limited company for sequencing verification.

2. Expression purification

The resulting BL21(DE3) strain was constructed, cultured in LB, added to kanamycin (kanamycin) at a final concentration of 50. mu.g/mL, and induced to express with IPTG for 5 hours after culture. The centrifuged cells were taken out, dissolved in an equilibrium buffer (20mM Tris-HCl, pH8.0, 150mM NaCl), disrupted by an ultrasonic instrument, and centrifuged to obtain a supernatant for purification of the fusion protein. Purification by Ni-NTA affinity chromatography (GE Healthcare) was performed, eluting with elution buffer (20mM Tris-HCl, pH8.0, 150mM NaCl, 200mM imidazole) to obtain the fusion protein.

3. Cleavage of fusion proteins

After diluting the protein solution with the balance buffer solution, the SUMO protease is added according to the ratio of the protein amount to the SUMO protease being 50:1, and the enzyme digestion is carried out for 1.5h at room temperature.

4. Purification of polypeptides

Adding acetonitrile with the final concentration of 20% into the polypeptide solution obtained after enzyme digestion. The column was packed with 4.6 x 250mm C8 (nanosil 8-120C8 Ultra Plus 8um 4.6 x 250mm, sozhou nanoscience gmbh) having a particle size of 8 μm. Preparative purification was performed in the AKTA purification system. With 20% acetonitrile/H₂Starting with O (20mM Tris-HCl, pH8.0), a gradient (1%/min increasing acetonitrile) was applied at a flow rate of 1mL/min for an elution time of 30 minutes, and fractions containing the peptide were collected. The isolated product was analyzed by LC-MS. The amino acid sequence of the obtained GLP-1/GCG dual receptor agonist polypeptide and the sample name of the corresponding polypeptide sample are shown in Table 1.

TABLE 1

Example 2 determination of in vitro Activity of Polypeptides

The polypeptide prepared by expression, human GLP-1(SEQ ID NO:17, TOCRIS, Cat. No. 5374(BATH:2A) and human GCG (SEQ ID NO:18, Novonide, Nuo Hesheng) were allowed to act on HEK293 cells expressing GLP-1R or GCGR, respectively, by the following specific operations:

1, genes GCGR and GLP-1R are optimized and synthesized conventionally in Jinwei Zhi company, the genes are cloned to a vector pUC57-Amp, and mini-scale recombinant plasmid DNA and a puncturing bacterium containing the recombinant plasmid are prepared;

2, performing double digestion on pUC57-GCGR recombinant plasmid DNA by Hind III and EcoR I, performing double digestion on pUC57-GLP-1R by Hind III and XhoI, performing electrophoresis on the digestion product by using 1% agarose gel, cutting a target band by using a clean blade, and recovering a target fragment by using a gel recovery kit;

3, connecting the enzyme digestion recovery product of the target fragment with a vector plasmid pcDNA3.1 fragment through T4 ligase, transforming DH5 alpha competent cells, coating a plate to separate a single colony, selecting a transformant for amplification culture, and performing enzyme digestion verification and sequencing verification.

4 inoculating 200mL of bacterial liquid which is verified to be correct and is used for large-scale extraction of plasmids, and using a kit comprising: PureLink HiPupure Plasmid Maxiprep Kit, as described. After the plasmid was verified by PCR and digestion, the plasmid was linearized with pvuI restriction enzyme. Finally, the plasmid is recovered by an ethanol precipitation method.

5 host cells are HEK293, the day before transfection, cells were plated at a density of 2X10^6 cells/well in 6-well plates, 1 mL/well. And (3) transfecting the recovered linearized plasmid into HEK293 cells by using a Lipofectamine 3000 liposome transfection method, adding G418 for screening to obtain a mixed strain, performing limited dilution separation to obtain a monoclonal, and performing activity detection and verification.

6 cAMP detection kit (Cisbio, 62AM6PEC) is used for detecting cAMP generated by receptor cells according to the steps described in the operating instruction, and the specific steps are as follows:

1) preparing an Assay buffer: taking complete culture solution (DMEM culture medium + 10% FBS), adding 4/1000 mM IBMX mother solution, cAMP-d2 working solution and anti-cAMP-crytate working solution, and preparing according to the kit specification;

2) diluting human GLP-1 and GCG of a sample to be detected and a control sample to a mother solution with an initial concentration of 500nM, and then gradually diluting the mother solution by adding 20 mu L of the mother solution to 80 mu L of Assay buffer (diluted by 5 times), wherein the mother solution comprises 8 compound gradients;

human GLP-1: HAEGT FTSDV SSYLE GQAAK EFIAW LVKGRG (SEQ ID NO:17)

Human GCG: HSQGT FTSDY SKYLD SRRAQ DFVQW LMNT (SEQ ID NO:18)

3) Preparing a cell suspension: taking out cells HEK293-GLP-1R and HEK293-GCGR from a liquid nitrogen tank, immediately carrying out water bath at 37 ℃, completely thawing within 1.5min, dropwise adding the cells into a 15mL centrifuge tube filled with 8mL warm culture medium in an ultraclean bench, centrifuging at 900rpm for 5min, discarding supernatant, resuspending the cells by 1mL complete culture solution (blowing and beating for 15 times), immediately mixing 20 mu L suspension with trypan equal volume of trypan blue, calculating the number of viable cells by taking 20 mu L, and diluting the cells to 4x10 cells/mL;

4) dividing the 384-well plate into GLP-1R cell and GCGR cell areas, adding cell suspension into the corresponding area of the 384-well plate by using a 12-channel lane-changing adjustable dispenser according to 5 mu L of each well, and adding the test sample and the positive control sample gradient diluent into the 384-well plate corresponding to the cells by using a 12-channel lane-changing adjustable dispenser, wherein each well is 5 mu L (the samples with the same concentration are in parallel with 2 multiple wells); negative control: setting 3 holes in each 384-hole plate at 10 mu L of assay buffer per hole, covering a white sealing plate film, putting the plate into a constant-temperature incubator at 37 ℃, and taking out the plate after half an hour;

5) just before use, lys buffer in Hi-range kit was used to mix cAMP-d2 working solution and anti-cAMP-cryptate

Diluting the working solution by 20 times, mixing 1:1 uniformly to prepare a cAMP detection reagent mixed solution, adding 10 mu L of LcAMP detection reagent mixed solution into each hole of a sample group, adding 5 mu L of lysine buffer and 5 mu L of diluted anti-cAMP-cryptate working solution into each hole of negative control, covering a white cover, and placing for 1h at room temperature in a dark place;

6) detecting the fluorescence values of 665nm and 620nm in a multifunctional microplate reader;

7) thus, dose-response curves were established, and EC50 values were calculated and compared with each other, and the specific results are shown in table 2.

TABLE 2 determination of the in vitro Activity of the polypeptides

The experimental results in Table 2 show that the GLP-1/GCG dual-receptor agonist polypeptide obtained by the invention can respectively and obviously activate GLP-1 receptor cells and GCG receptor cells.

EXAMPLE 3 construction of fusion protein vectors

A molecular cloning method is adopted, GLP-1/GCG double receptor agonist polypeptide and IgG4-Fc (SEQ ID NO:9) with connecting peptide (SEQ ID NO:6) are fused, the obtained sequence is inserted into the same enzyme cutting sites of a mammalian cell expression vector after double enzyme cutting, a series of mutant vectors are constructed, after sequencing verification is correct, a plasmid vector is extracted by an endotoxin-removing plasmid extraction kit (OMEGA), and the plasmid vector is preserved at the temperature of-20 ℃. Wherein the amino acid sequence of the GLP-1/GCG dual receptor agonist polypeptide in the carrier and the corresponding polypeptide sample name are shown in Table 1. The sample name of the fusion protein comprising the GLP-1/GCG dual receptor agonist polypeptide constructed in example 2 is identical to the sample name of the GLP-1/GCG dual receptor agonist polypeptide obtained in example 1.

Example 4 vector transfection and expression in cells

Chinese Hamster Ovary (CHO) cells were recovered and subcultured to a density of about 6 x10⁶Cells were harvested at cell/mL and transfected using the ExpichofectamineTM CHO Transfection Kit (ThermoFisher Scientific) at a final concentration of 1. mu.g/mL for the vectors constructed in example 2. The next day of transfection, agents such as enhancers are added to maintain the growth of the transfected cells. Cell culture fluid was harvested when the cell viability dropped to about 80%.

Example 5 purification and characterization of fusion proteins

The cell culture broth was centrifuged to collect the supernatant and filtered through a 0.22 μm filter to remove residual cell debris. Purifying the collected cell culture solution by adopting a Protein A chromatographic column, collecting a target peak, further purifying by using anion exchange chromatography, and finally eluting and collecting the Protein by using 0.02M PBS. The samples were quantified using a micro nucleic acid protein analyzer (NanoDrop 2000/2000 cSpectrophotometer). The sample is detected by 12% SDS-PAGE electrophoresis, and an electrophoretogram shows that the band is single. The exact molecular weight of the fusion protein was determined by mass spectrometry, essentially consistent with the theoretical molecular weight.

Example 6 determination of the in vitro Activity of fusion proteins

By stimulating HEK293 cells expressing GLP-1R or GCGR with fusion protein, detecting cAMP generated by receptor cells with cAMP detection kit (Cisbio, 62AM6PEC), establishing dose-response curve, and calculating EC thereof₅₀The results are shown in Table 3 and compared with each other.

TABLE 3 in vitro Activity assay of fusion proteins

The experimental results in Table 3 show that the GLP-1/GCG dual receptor agonist sample obtained by the invention can obviously and respectively activate GLP-1 receptor cells and GCG receptor cells.

EXAMPLE 7 Effect of candidates on glucose tolerance in Normal C57BL/6 mice

The experimental method comprises the following steps: normal C57BL/6 mice were divided into 6 groups (Control, Dulaglutide-7.5nmol/kg, HEC-C80-7.5nmol/kg, HEC-C85-7.5nmol/kg, HEC-C86-7.5nmol/kg, HEC-C87-7.5nmol/kg) according to blood sugar and body weight, and 6 mice were administered with the corresponding vehicle or candidate by subcutaneous injection, animals were fasted 56h after administration, animals were injected 2g/kg of glucose intraperitoneally 72h after administration, and blood sugar measurements were performed 15, 30, 60, 90min before and after administration. The results are shown in Table 4. The blood glucose concentration-time curve is plotted from the blood glucose values measured at different time points, as shown in FIG. 1, and AUC is calculated for each dose group_{0～90min Glu}And the rate of blood glucose lowering at the peak of blood glucose.

The experimental results are as follows:

table 4: effect of fusion proteins on blood glucose in glucose-loaded mice

And (4) experimental conclusion: HEC-C80, HEC-C85, HEC-C86 and HEC-C87 all significantly reduced blood glucose levels in sugar-loaded mice.

EXAMPLE 8 Effect of candidates on glucose tolerance in Normal C57BL/6 mice

The experimental method comprises the following steps: normal C57BL/6 mice were divided into 3 groups (Control, Dulaglutide-7.5nmol/kg, HEC-C70-7.5nmol/kg) according to blood sugar and body weight, 8 mice per group were subcutaneously administered with the corresponding vehicle or candidate, animals were fasted 56h after administration, animals of each group were intraperitoneally injected 2g/kg after administration 72h, and blood sugar measurements were performed 15, 30, 60, 90min before and after administration. The results are shown in Table 5. The blood glucose concentration-time curve is plotted from the blood glucose values measured at different time points, as shown in FIG. 2, and AUC is calculated for each dose group_{0～90min Glu}And the rate of blood glucose lowering at the peak of blood glucose.

The experimental results are as follows:

table 5: effect of HEC-C70 on blood glucose in sugar-loaded mice

And (4) experimental conclusion: HEC-C70 significantly reduced blood glucose levels in glucose-loaded mice.

Example 9 Effect of candidates on glucose tolerance and body weight in DIO mice

The experimental method comprises the following steps: after 8-week-old C57BL/6 mice were fed with high-fat diet for 15 weeks, the mice were divided into 7 groups (Model, semaglutide-5nmol/kg, HEC-C70-5nmol/kg, HEC-C80-5nmol/kg, HEC-C85-5nmol/kg, HEC-C86-5nmol/kg, HEC-C87-5nmol/kg) according to body weight. Dosing began at week 16 (model group given the corresponding vehicle), semaglutide was administered once a day, the remaining groups were administered twice a week for 4 weeks, animal body weight was weighed prior to each administration, and IPGTT experiments were performed at week 4 of administration.

The experimental results are as follows: the effect of improving sugar tolerance of HEC-C70-5nmol/kg is similar to that of semaglutide-5 nmol/kg. HEC-C70-5nmol/kg, HEC-C80-5nmol/kg, HEC-C85-5nmol/kg, HEC-C86-5nmol/kg and HEC-C87-5nmol/kg have obvious effects of reducing the body weight of DIO mice, and the body weight reducing effects of HEC-C70-5nmol/kg and HEC-C85-5nmol/kg are superior to that of semaglutide-5 nmol/kg. The specific data are shown in table 6 and table 7, and fig. 3 and fig. 4.

Table 6: effect of Long-term administration on blood glucose lowering Rate in glucose-loaded DIO mice

Table 7: effect of Long-term administration on body weight of DIO mice

And (4) experimental conclusion: the long-term administration of HEC-C70, HEC-C80, HEC-C85, HEC-C86 and HEC-C87 can obviously improve the glucose tolerance of DIO mice and obviously reduce the body weight of the DIO mice.

Example 10 Effect of candidates on glucose tolerance and body weight in DIO mice

The experimental method comprises the following steps: after being fed with high-fat diet for 15 weeks by C57BL/6N mice with 6 weeks of age, the mice are divided into 6 groups according to body weight (Model, LY3298176-10nmol/kg, semaglutide-10nmol/kg, MEDI0382-10nmol/kg, HEC-C70-2.5nmol/kg, HEC-C70-10 nmol/kg). Dosing began at week 16 (model group given vehicle), semaglutide and MEDI0382 were administered once daily, the remaining groups were administered twice weekly for 4 weeks, animals were weighed before each administration, and IPGTT experiments were performed at week 4.

Comparison group LY3298176 sequence:

YX₁EGTFTSDYSIX₂LDKIAQKAFVQWLIAGGPSSGAPPPS-NH₂(SEQ ID NO:15) in which X₁And X₂For Aib, K at position 20 is linked to ([2- (2-amino-ethoxy) ethoxy ] through its side chain epsilon-amino group]-acetyl group)₂-γGlu-CO-(CH₂)₁₈-CO₂H is connected.

Control MEDI0382 sequence: HSQGTFTSDKSEYLDSERARDFVAWLEAGG (SEQ ID NO:16) wherein K at position 10 is linked to gamma Glu-CO- (CH) through its side chain epsilon-amino group₂)₁₄-CO₂H is connected.

The experimental results are as follows: the effect of HEC-C70-10nmol/kg on improving sugar tolerance is similar to that of LY3298176-10nmol/kg, and is better than semaglutide-10nmol/kg and MEDI0382-10 nmol/kg. HEC-C70-10nmol/kg has the effect of remarkably reducing the body weight of a DIO mouse, and is superior to semaglutide-10 nmol/kg. The data are shown in tables 8 and 9 and FIGS. 5 and 6.

Table 8: effect of Long-term administration on blood glucose lowering Rate in glucose-loaded DIO mice

Table 9: effect of Long-term administration on body weight of DIO mice

And (4) experimental conclusion: the long-term administration of HEC-C70 can significantly improve the glucose tolerance and significantly reduce the body weight of DIO mice.

EXAMPLE 11 Effect of candidates on blood glucose and body weight in db/db mice

The experimental method comprises the following steps: the 6-7 week old ob/ob mice were divided into 3 groups (Model, Dulaglutide-30nmo/kg, HEC-C70-30nmol/kg) based on randomized blood glucose and body weight. For a total of 4 weeks (model group given the corresponding vehicle), Dulaglutide and HEC-C70 were administered twice weekly, during which time the animals of each group were monitored for random blood glucose and body weight.

The experimental results are as follows: the effect of improving sugar tolerance of HEC-C70-30nmol/kg is better than that of Dulaglutide-30 nmol/kg. HEC-C70-30nmol/kg has significant effect of reducing the body weight of db/db mice, and is superior to Dulaglutide-30 nmol/kg. The data are shown in tables 10 and 11 and in FIGS. 7 and 8.

Table 10: effect of Long-term administration on blood glucose in db/db mice

Table 11: effect of Long term administration on body weight of db/db mice

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Guangdong Dongyuang pharmaceutical Co., Ltd

<120> GLP-1/GCG dual-receptor agonist polypeptide and fusion protein thereof

<130> 2021-12-2

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of GLP-1/GCG dual receptor agonist polypeptide

<400> 1

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Glu Gly

20 25

<210> 2

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of GLP-1/GCG dual receptor agonist polypeptide

<400> 2

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Ile Glu Trp Leu Ile Ala Gly

20 25

<210> 3

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of GLP-1/GCG dual receptor agonist polypeptide

<400> 3

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Glu Trp Leu Ile Ala Gly

20 25

<210> 4

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of GLP-1/GCG dual receptor agonist polypeptide

<400> 4

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Lys Ala Lys Glu Phe Ile Glu Trp Leu Ile Ala Gly

20 25

<210> 5

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of GLP-1/GCG dual receptor agonist polypeptide

<400> 5

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Ile Ala Gly

20 25

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of linker peptide

<400> 6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 7

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of linker peptide

<400> 7

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of linker peptide

<400> 8

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 9

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of IgG4-Fc fragment

<400> 9

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly

225

<210> 10

<211> 273

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of fusion protein

<400> 10

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Glu Gly Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

35 40 45

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

50 55 60

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

65 70 75 80

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

85 90 95

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

100 105 110

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

115 120 125

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

130 135 140

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

145 150 155 160

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

165 170 175

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

180 185 190

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

195 200 205

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

210 215 220

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

225 230 235 240

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

245 250 255

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

260 265 270

Gly

<210> 11

<211> 273

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of fusion protein

<400> 11

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Ile Glu Trp Leu Ile Ala Gly Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

35 40 45

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

50 55 60

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

65 70 75 80

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

85 90 95

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

100 105 110

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

115 120 125

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

130 135 140

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

145 150 155 160

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

165 170 175

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

180 185 190

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

195 200 205

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

210 215 220

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

225 230 235 240

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

245 250 255

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

260 265 270

Gly

<210> 12

<211> 273

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of fusion protein

<400> 12

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Glu Trp Leu Ile Ala Gly Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

35 40 45

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

50 55 60

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

65 70 75 80

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

85 90 95

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

100 105 110

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

115 120 125

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

130 135 140

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

145 150 155 160

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

165 170 175

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

180 185 190

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

195 200 205

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

210 215 220

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

225 230 235 240

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

245 250 255

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

260 265 270

Gly

<210> 13

<211> 273

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of fusion protein

<400> 13

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Lys Ala Lys Glu Phe Ile Glu Trp Leu Ile Ala Gly Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

35 40 45

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

50 55 60

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

65 70 75 80

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

85 90 95

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

100 105 110

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

115 120 125

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

130 135 140

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

145 150 155 160

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

165 170 175

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

180 185 190

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

195 200 205

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

210 215 220

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

225 230 235 240

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

245 250 255

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

260 265 270

Gly

<210> 14

<211> 273

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of fusion protein

<400> 14

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Ile Ala Gly Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

35 40 45

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

50 55 60

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

65 70 75 80

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

85 90 95

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

100 105 110

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

115 120 125

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

130 135 140

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

145 150 155 160

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

165 170 175

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

180 185 190

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

195 200 205

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

210 215 220

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

225 230 235 240

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

245 250 255

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

260 265 270

Gly

<210> 15

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> control LY3298176 sequence, Xaa is Aib

<400> 15

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys

1 5 10 15

Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 16

<211> 30

<212> PRT

<213> native sequence

<220>

<223> control MEDI0382 sequences

<400> 16

His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Ser

1 5 10 15

Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly

20 25 30

<210> 17

<211> 31

<212> PRT

<213> native sequence

<220>

<223> human GLP-1 sequences

<400> 17

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210> 18

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> human GCG sequence

<400> 18

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

Claims

1. A GLP-1/GCG dual receptor agonist polypeptide, wherein said polypeptide has an amino acid sequence as shown below:

^5’X₁SQGT FTSDY SKYLD EKX₁₈AK X₂₁FX₂₃EW LX₂₇X₂₈X₂₉X₃₀ ^3’

wherein, X₁Is H or Y; x₁₈Is R, A or K; x₂₁Is E or D; x₂₃Is V or I; x₂₇Is L or I; x₂₈Is A or E; x₂₉Is A or G; x₃₀Is GPSSGAPPPS or is absent;

when X is present₂₇When is L, X₂₈Is E.

2. The polypeptide of claim 1, wherein the polypeptide has an amino acid sequence as shown in seq id no:

HSQGT FTSDY SKYLD EKX₁₈AK EFX₂₃EW LX₂₇X₂₈G；

3. The polypeptide of any one of claims 1-2, wherein in the amino acid sequence of the polypeptide, X is₂₃Or X₂₇At least one of which is I.

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide has an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 5.

5. A fusion protein, comprising: the polypeptide of any one of claims 1 to 4, a linker peptide and an IgG-Fc fragment, wherein the linker peptide is disposed between the polypeptide and the IgG-Fc fragment.

6. The fusion protein of claim 5, wherein the N-terminus of the linker peptide is linked to the C-terminus of the polypeptide, and the C-terminus of the linker peptide is linked to the N-terminus of the IgG-Fc fragment.

7. The fusion protein of any one of claims 5 to 6, wherein the linker peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 6 to 8.

8. The fusion protein of any one of claims 5 to 7, wherein the IgG-Fc fragment is an IgG4-Fc variant having the amino acid sequence set forth in SEQ ID NO. 9.

9. The fusion protein of any one of claims 5 to 8, wherein the fusion protein has an amino acid sequence of any one of SEQ ID NOs 10 to 14.

10. A nucleic acid encoding the polypeptide of any one of claims 1 to 4 or the fusion protein of any one of claims 5 to 9.

11. A construct carrying the nucleic acid of claim 10.

12. The construct of claim 11, wherein the vector of said construct is pxcc 17.4.

13. A recombinant cell expressing a polypeptide according to any one of claims 1 to 4 or a fusion protein according to any one of claims 5 to 9 or comprising a construct or genome according to claim 11 or 12 having integrated therein a nucleic acid according to claim 10.

14. The recombinant cell of claim 13, wherein the recombinant cell is a CHO cell.

15. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 4 or the fusion protein of any one of claims 5 to 9.

16. The pharmaceutical composition of claim 15, further comprising a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 15, further comprising an additional antidiabetic agent including at least one selected from the group consisting of insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha-glucosidase inhibitors and aminopeptidase IV inhibitors.

18. A protein preparation comprising the polypeptide of any one of claims 1 to 4 or the fusion protein of any one of claims 5 to 9.

19. Use of the polypeptide of any one of claims 1 to 4 or the fusion protein of any one of claims 5 to 9 or the protein formulation of claim 18 in the manufacture of a medicament for treating or preventing a metabolic disease comprising at least one member selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, diabetes, obesity.