WO2021083306A1 - Glp-1/gcg dual-acceptor agonist polypeptide - Google Patents

Abstract

A GLP-1/GCG dual-acceptor agonist polypeptide. The polypeptide comprises an amino acid sequence as follows: ^{5 '}X ₁SQGT FTSDX ₁₀ SKYLX ₁₅ X ₁₆X ₁₇X ₁₈AX ₂₀ X ₂₁FX ₂₃X ₂₄W L X ₂₇X ₂₈ X ₂₉X ₃₀ ^{3 '}, wherein X ₁ is H or Y, X ₁₀ is Y or L, X ₁₅ is D or E, X ₁₆ is M or E, X ₁₇ is Q, K, or R, X ₁₈ is R or A, X ₂₀ is H or K, X ₂₁ is E or D, X ₂₃ is V or I, X ₂₄ is Q, A, N, or E, X ₂₇ is L or M, X ₂₈ is A, N, or D, X ₂₉ is A or G, X ₃₀ is T, and PSSGAPPPS may not exist. The GLP-1/GCG dual-acceptor agonist polypeptide has a good blood sugar-lowering and weight-losing effect, and a long in vivo half-life period, and is a more effective long acting weight-losing and blood sugar control drug.

Description

GLP-1/GCG dual receptor agonist peptide Technical field

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

Background technique

Obesity is closely related to type 2 diabetes, hyperlipidemia and hypertension. At present, the main role of first-line drugs for the treatment of type 2 diabetes is to reduce blood sugar, but the effect of reducing weight is very limited. How to develop drugs with good blood sugar and weight reduction effects at the same time is the current research and development hotspot in this field.

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by the intestinal L-cells after eating, which can stimulate the pancreatic β-cells to secrete insulin, thereby stabilizing the fluctuation of blood glucose after a meal. Its effect of lowering blood sugar is glucose concentration-dependent. While regulating blood sugar, it greatly reduces the risk of hypoglycemia. In recent years, drugs based on GLP-1, such as liraglutide, duraglutide and semaglutide, have gradually occupied a very important position in diabetes drugs. GLP-1 drugs also have the effect of weight loss when lowering blood sugar. The mechanism is that GLP-1 acts on the gastrointestinal tract to delay gastric emptying and intestinal peristalsis, and acts on the central nervous system to suppress appetite, so as to reduce food intake. the goal of. At present, liraglutide has been approved as a weight-loss drug, and its second-generation product, semaglutide, has a better weight-loss effect. However, the main effect of this type of drugs is to control sugar and reduce weight, and the effect is limited, and it is still far from meeting clinical needs.

Glucagon (GCG) is a polypeptide hormone secreted by islet α cells, which can promote glycogen decomposition and gluconeogenesis, and significantly increase blood sugar; at the same time, it can also activate lipase, promote lipolysis, and increase fatty acid oxidation. Increase energy consumption, which has the effect of reducing fat and weight. Because of its physiological role in raising blood sugar, although it can be used in the treatment of hypoglycemia, it limits its application in obesity and weight loss, especially in obese patients with type 2 diabetes.

Oxyntomodulin (OXM) is a dual-receptor agonist that activates the glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR). The hypoglycemic effect of its GLP-1 receptor activity can balance the hypoglycemic effect caused by the activation of GCGR, and synergize the effects of the two in reducing weight, so as to achieve a better weight loss effect. Studies have shown that OXM has a good effect of reducing blood sugar and weight in animal models, which is significantly better than existing GLP-1 drugs, such as liraglutide.

Summary of the invention

This application is based on the inventor's discovery and understanding of the following facts and problems:

Some of the problems of oxyntomodulin (OXM), such as poor stability and low receptor activity, can lead to large doses. At the same time, the ratio of OXM to GLP-1R and GCGR activity is fixed, which makes it difficult to control glucose reduction. For best results. Therefore, the inventors optimized the OXM to improve its stability, prolong the half-life in vivo, increase the receptor activity, reduce the dose, and optimize the balance between the receptors to achieve the best glucose control. Weight loss effect.

Based on the above optimization and transformation results, in the first aspect of the present invention, the present invention proposes a GLP-1/GCG dual receptor agonist polypeptide. According to an embodiment of the present invention, the polypeptide having the amino acid sequence shown ^{_{below: 5 'X 1 SQGT FTSDX 10}} SKYLX 15 X 16 X 17 X 18 AX 20 X 21 FX 23 X 24 W L X 27 X 28 X 29 X 30 3 ^' , Where X ₁ is H or Y, X ₁₀ is Y or L, X ₁₅ is D or E, X ₁₆ is M or E, X ₁₇ is Q, K or R, X ₁₈ is R or A, X ₂₀ Is H or K, X ₂₁ is E or D, X ₂₃ is V or I, X ₂₄ is Q, A, N or E, X ₂₇ is L or M, X ₂₈ is A, N or D, X ₂₉ is A Or G, X ₃₀ is T, PSSGAPPPS or not present. The above-mentioned GLP-1/GCG dual receptor agonist polypeptide according to the embodiment of the present invention has a good effect of reducing blood sugar and weight, and has a long half-life in the body, and is a more effective long-acting weight-reducing and sugar-controlling drug.

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 present invention, the polypeptide having the amino acid sequence shown ^{_{below: 5 'X 1 SQGTFTSDX 10 SKYLX}} 15 EX 17 X 18 AK X 21 FX 23 X 24 W LLAGX 30 3', wherein, X ₃₀ is PSSGAPPPS or does not exist.

According to an embodiment of the present invention, the polypeptide having the amino acid sequence shown _{^{below: 5 'HSQGT FTSDY SKYLD X 16}} X 17 X 18 AX 20 X 21 FX 23 X 24 W LX 27 X 28 T 3', wherein, X ₂₄ It is Q, A or N, and X ₂₈ is N or D.

According to an embodiment of the present invention, the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 1-21, SEQ ID NO: 71.

According to an embodiment of the present invention, the polypeptide has an amino acid sequence shown in SEQ ID NO: 1 to 3.

The inventor found that the GLP-1/GCG dual receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO: 1 to 3 has significant advantages in stimulating the expression of GLP-1R or GCGR. At the same time, the results of in vivo experiments show The GLP-1/GCG dual receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO: 1 to 3 has significant effects in controlling body weight and reducing blood sugar.

According to an embodiment of the present invention, the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 22-34.

In the second aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein comprises a polypeptide linker peptide fragment and IgG ₄ -Fc previously described, the linker peptide disposed between the head and tail polypeptide fragments and IgG ₄ -Fc. The fusion protein according to the embodiment of the present invention has a good effect of reducing blood sugar and weight, and has a long half-life in the body, and is an effective long-acting weight-reducing and sugar-controlling drug.

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

According to an embodiment of the present invention, the N-terminus of the connecting peptide with the C-terminus of the polypeptide linked, a connecting peptide coupled to the N-terminus of the C-terminus of the IgG ₄ -Fc fragments.

According to an embodiment of the present invention, the connecting peptide has the amino acid sequence shown in SEQ ID NO:35.

According to an embodiment of the present invention, the IgG4-Fc fragment has the amino acid sequence shown in SEQ ID NO: 36.

According to an embodiment of the present invention, the fusion protein has an amino acid sequence shown in SEQ ID NO: 37-70, SEQ ID NO: 72.

In the third aspect of the present invention, the present invention proposes a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the aforementioned polypeptide or the aforementioned fusion protein.

In the fourth aspect of the present invention, the present invention proposes a construct. According to an embodiment of the present invention, the construct carries the aforementioned nucleic acid.

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

According to an embodiment of the present invention, the vector of the construct is pXC17.4.

In the fifth aspect of the present invention, the present invention proposes a recombinant cell. According to an embodiment of the present invention, the recombinant cell expresses the aforementioned polypeptide or the aforementioned fusion protein.

In the sixth aspect of the present invention, the present invention proposes a recombinant cell. According to an embodiment of the present invention, the recombinant cell contains the aforementioned construct or the genome integrates the aforementioned nucleic acid.

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

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

In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the aforementioned polypeptide or the aforementioned fusion protein.

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

According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

According to an embodiment of the present invention, the pharmaceutical composition further includes other anti-diabetic drugs, and the other anti-diabetic drugs include insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha -At least one of a glucosidase inhibitor and an aminodipeptidase IV inhibitor.

In the eighth aspect of the present invention, the present invention provides a protein preparation. According to an embodiment of the present invention, it includes the aforementioned polypeptide or the aforementioned fusion protein. The protein preparation according to the embodiment of the present invention is an effective long-acting weight-reducing and carbohydrate-controlling drug.

In the ninth aspect of the present invention, the present invention proposes the use of the aforementioned polypeptide or the aforementioned fusion protein in the preparation of medicines for the treatment or prevention of metabolic diseases.

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

Description of the drawings

Figure 1 is a blood glucose concentration-time curve at different time points after administration of a sugar-loaded mouse according to an embodiment of the present invention;

Figure 2 is a graph showing the experimental results of the effect of the fusion protein HEC-12063 on the glucose tolerance of DIO mice according to an embodiment of the present invention;

Figure 3 is a diagram showing the experimental results of the effect of the fusion protein HEC-12063 on the body weight of DIO mice according to an embodiment of the present invention;

Figure 4 is an experimental result diagram of the effect of the fusion protein HEC-C046 on the blood glucose of ob/ob mice according to an embodiment of the present invention;

Figure 5 is a diagram showing the experimental results of the effect of the fusion protein HEC-C046 on the body weight of ob/ob mice according to an embodiment of the present invention;

Fig. 6 is a graph showing the experimental results of the effect of the fusion protein HEC-C046 on the blood sugar of db/db mice according to an embodiment of the present invention;

Fig. 7 is a graph showing the experimental results of the effect of the fusion protein HEC-C046 on the body weight of db/db mice according to an embodiment of the present invention;

Fig. 8 is a blood glucose concentration-time curve at different time points after administration of a sugar-loaded mouse according to an embodiment of the present invention;

Figure 9 is a diagram showing the experimental results of the effect of the fusion protein HEC-C079 on the glucose tolerance of DIO mice according to an embodiment of the present invention;

Fig. 10 is a graph showing the experimental results of the effect of the fusion protein HEC-C079 on the body weight of DIO mice according to an embodiment of the present invention.

Detailed ways

The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention.

Example 1 Preparation method of GLP-1/GCG dual receptor agonist polypeptide

The fusion gene is formed by adding the gene sequence of the SUMO tag at the 5'end of the gene encoding the polypeptide. The fusion gene was cloned into a prokaryotic expression vector and induced expression in E. coli cells. After the cells are collected by centrifugation, they are ultrasonically broken and centrifuged to obtain the supernatant, and then purified by nickel column to obtain the fusion protein. Finally, after SUMO protease digestion treatment, the target polypeptide is obtained by reverse-phase purification. The specific process is as follows:

1. Vector construction and primer synthesis

Using pET-28a as the expression vector and BL21(DE3) as the host, the specific steps for preparing the polypeptide are as follows:

1) Design primers, which serve as templates for each other to amplify polypeptide gene fragments. And using polypeptide gene fragments and sumo gene fragments as templates, the fusion gene fragments are amplified by the fusion PCR method.

2) Construction of a recombinant expression vector: the fusion gene fragment was cloned into the prokaryotic expression vector pET-28a, E. coli BL21 (DE3) was transformed, the recombinant was selected, and the recombinant expression plasmid sample was sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing verification.

2. Expression purification

The resulting BL21(DE3) strain was constructed, cultured in LB, kanamycin (kanamycin) was added at a final concentration of 50μg/mL, and IPTG was used to induce expression for 5h after culture. Take the centrifuged cells, dissolve them with equilibration buffer (20mM Tris-HCl, pH8.0, 150mM NaCl), disrupt the cells with an ultrasonic instrument, and centrifuge the supernatant for purification of the fusion protein. The fusion protein was purified by Ni-NTA affinity chromatography column (GE Healthcare) and eluted with elution buffer (20mM Tris-HCl, pH8.0, 150mM NaCl, 200mM imidazole).

3. Fusion protease cleavage

After diluting the protein solution with equilibration buffer, add SUMO protease according to the ratio of protein amount: SUMO protease 50:1, and digest for 1.5h at room temperature.

4. Purification of peptides

Add 20% acetonitrile to the polypeptide aqueous solution obtained after digestion. The preparation and purification are carried out in the AKTA purification system through a 4.6*250mm C8 packed column with a particle size of 8μm. Start with 20% acetonitrile/H ₂ O (containing 20 mM Tris-HCl, pH 8.0), start with a gradient (increasing the proportion of acetonitrile at a rate of 1%/min) and a flow rate of 1 mL/min. The column is eluted by 30 Minutes, collect peptide-containing fractions. Analyze the separated products by liquid-mass spectrometry.

Nanomicro UniSil 8-120 C8 Ultra Plus 8um 4.6*250mm is from Suzhou Nanomicro Technology Co., Ltd.

Example 2 Determination of the in vitro activity of GLP-1/GCG dual receptor agonist polypeptide

The amino acid sequence of the GLP-1/GCG dual receptor agonist polypeptide obtained in Example 1 and the sample names of the corresponding polypeptide samples are shown in Table 1.

GLP-1/GCG dual receptor agonist polypeptide stimulates HEK293 cells expressing GLP-1R or GCGR, and detects cAMP produced by the recipient cells with a cAMP detection kit (Cisbio, 62AM6PEC), establishes a dose-effect curve, and calculates its EC ₅₀ , the results are shown in Table 2 and compared with each other.

Table 1:

Sample name	Amino acid sequence
HEC-1202	HSQGT FTSDY SEYLD SEAR DFVAW LEAGG
HEC-1204	YSQGTFTSDYSKYLDEQAAKEFVNWLLAGGPSSGAPPPS
HEC-1205	HSQGT FTSDY SKYLD MQRAH DFVQW LMNT
HEC-1206	HSQGT FTSDY SKYLD Ekrak EFVQW LMNT
HEC-12041	HSQGTFTSDYSKYLDEQAAKEFVNWLLAGGPSSGAPPPS

HEC-12042	HSQGT FTSDYSKYLDEQAAK EFVNW LLAG
HEC-12043	HSQGT FTSDYSKYLDEQAAK EFVNW LMNT
HEC-12045	HSQGT FTSDY SKYLD EQAAK EFVQW LLAG
HEC-12046	HSQGT FTSDY SKYLD EQAAK DFVQW LLAG
HEC-12047	HSQGT FTSDY SKYLD ERAAK EFVQW LLAG
HEC-12061	HSQGT FTSDY SKYLD Ekrak EFVQW LLAG
HEC-12062	HSQGT FTSDY SKYLD Ekrak EFVAW LLAG
HEC-12063	HSQGT FTSDY SKYLD Ekrak EFIAW LLAG
HEC-12064	HSQGT FTSDY SKYLD EKAAK EFVAW LLAG
HEC-12066	HSQGT FTSDY SKYLD EkrAK EFVAW LMNT
HEC-12067	HSQGT FTSDY SKYLD Ekrak EFIAW LMNT
HEC-12068	HSQGT FTSDY SKYLD Ekrak EFIAW LMDT
HEC-C007	HSQGT FTSDY SKYLD ERAAK DFVQW LMNT
HEC-C008	HSQGT FTSDY SKYLD ERAAK DFVQW LLDT
HEC-C009	HSQGT FTSDY SKYLD EQRAK DFVQW LLDT
HEC-C010	HSQGTFTSDYSKYLDERRAKEFVQWLLDT
HEC-C011	HSQGTFTSDYSKYLDERRAKDFIQW LLDT
HEC-C012	HSQGT FTSDY SKYLD ERRAK DFVAW LLDT
HEC-C044	HSQGT FTSDL SKYLD Ekrak EFIAW LLAG
HEC-C045	HSQGT FTSDY SKYLE EkrAK EFIAW LLAG
HEC-C046	HSQGT FTSDY SKYLD EkrAK EFIEW LLAG
HEC-C047	HSQGTFTSDYSKYLDEKRAKEFIAWLLAGGPSSGAPPPS
HEC-C048	HSQGT FTSDYSKYLDEKRAK EFIAW LLDT
HEC-C049	HSQGT FTSDYSRYLDEKRAK EFIAW LLAG
HEC-C050	HSQGT FTSDYSKYLD Ekrak EFIQW LLAG
HEC-C051	HSQGT FTSDYSKYLD Ekrak DFVQW LLAG
HEC-C052	HSQGT FTSDYSKYLD EQAAK EFIEW LLAG
HEC-C053	HSQGT FTSDYSKYLD EQAAK DFVEW LLAG
HEC-C054	HSQGT FTSDYSKYLD EKAAK EFIEW LLAG
HEC-C055	HSQGT FTSDY SKYLD EKAAK DFVEW LLAG
HEC-C079	HSQGT FTSDY SKYLD EKAAK EFVEW LLAG

Table 2:

It can be seen from the experimental results in Table 2 that the GLP-1/GCG receptor dual agonist polypeptide sample obtained in the present invention can significantly activate GLP-1R or GCGR receptor cells.

Example 3 Construction of Fusion Protein Vector

Molecular cloning, the GLP-1 / GCG dual receptor agonist polypeptide and IgG4-Fc with linker peptide (GGGGSGGGGSGGGGSA) of (ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG) are fused sequence obtained by double digestion and inserted into the mammalian cell expression vector A series of mutant vectors were constructed between the same restriction sites of, and after being verified by sequencing, the plasmid vectors were extracted with endotoxin-free plasmid extraction kit (OMEGA) and stored at -20°C. The amino acid sequence of the GLP-1/GCG receptor dual agonist polypeptide in the vector and the name of the corresponding polypeptide sample are shown in Table 1. The sample name of the fusion protein containing the GLP-1/GCG receptor dual agonist polypeptide constructed in Example 3 is consistent with the sample name of the GLP-1/GCG receptor dual agonist polypeptide obtained in Example 1.

Example 4 Vector transfection and expression in cells

The Chinese hamster ovary cells (CHO) were resuscitated and subcultured. When the density was about 6*10 ⁶ cells/mL, the cells were collected and transfected. The ExpiCHOFectamineTM CHO Transfection Kit (ThermoFisher Scientific) was used. The final concentration of the vector constructed in Example 2 was 1μg/mL. On the second day of transfection, reagents such as enhancers were added to maintain the growth of transfected cells. The cell culture solution was harvested when the cell viability dropped to about 80%.

Example 5 Purification and identification of fusion protein

The cell culture solution was centrifuged to collect the supernatant, and filtered with a 0.22 μm filter to remove residual cell debris. The collected cell culture fluid was purified by Protein A chromatography column, the target peak was collected, and then further purified by anion exchange chromatography, and the protein was finally collected by elution with 0.02M PBS. The samples are quantified using a micro-nucleic acid protein analyzer (NanoDrop 2000/2000c Spectrophotometer). The sample was detected by 12% SDS-PAGE electrophoresis, and the electrophoresis pattern showed a single band. The precise molecular weight of the fusion protein determined by mass spectrometry is basically consistent with the theoretical molecular weight.

Example 6 Determination of the in vitro activity of the fusion protein

The HEK293 cells expressing GLP-1R or GCGR were stimulated by the fusion protein, cAMP detection kit (Cisbio, 62AM6PEC) was used to detect the cAMP produced by the recipient cells, the dose-response curve was established, and the EC _{50 was} calculated. The results are shown in Table 3. And compare them with each other.

table 3:

It can be seen from the experimental results in Table 3 that the GLP-1/GCG receptor dual agonist sample obtained in the present invention can significantly activate GLP-1R or GCGR receptor cells, among which HEC-12042, HEC-12063 and HEC-C046 are activating GLP-1R or GCGR receptor cells have significant advantages.

Example 7 Effect of candidate substance on glucose tolerance of normal C57BL/6 mice

Experimental method: Normal C57BL/6 mice were divided into 6 groups according to blood sugar and body weight (Control, Dulaglutide-7.5nmol/kg, HEC-C046-7.5nmol/kg, HEC-C049-7.5nmol/kg, HEC-C050-7.5 nmol/kg, HEC-12063-7.5nmol/kg), each group of 8 animals, the corresponding vehicle or candidate was given by subcutaneous injection, the animals were fasted 56h after the administration, and the animals in each group were injected intraperitoneally with 2g/kg glucose 72h after the administration , And perform blood glucose testing before and 15, 30, 60, 90 min after sugar administration. Draw the blood glucose concentration-time curve according to the blood glucose values measured at different time points, as shown in Figure 1, calculate the AUC _{0～90min Glu of} each dose group and the hypoglycemic rate at the peak blood glucose.

Experimental results:

Table 4: Effects of GCG/GLP-1 series of proteins on blood glucose in mice with glucose load

Experimental conclusion: HEC-12063, HEC-C046, HEC-C049 and HEC-C050 can significantly reduce the blood glucose level of mice with glucose load.

Example 8 Effect of candidate HEC-12063 on glucose tolerance and body weight of DIO mice

Experimental method: 8-week-old C57BL/6 mice were fed with a high-fat diet for 4 months and divided into 5 groups according to their body weight (Model, semaglutide 10nmol/kg, HEC-C12063 3nmol/kg, HEC-C12063 10nmol/kg, HEC-C12063 30nmol/kg). The administration was started at the 5th month (the model group was given the corresponding vehicle), semaglutide was administered once a day, and each dose group of HEC-C12063 was administered twice a week for a total of 4 weeks. The animals were weighed before each administration. , The IPGTT experiment was performed on the 4th week of administration.

Experimental results: HEC-12063 decreased the blood glucose and body weight of DIO mice in a dose-dependent manner. At a dose of 3nmol/kg, it can significantly improve the glucose tolerance of DIO mice and have a certain weight reduction effect; HEC-12063 is at 3nmol/kg. The effect of improving glucose tolerance at the kg dose is better than that of semaglutide-10nmol/kg, and the weight reduction effect of HEC-12063 at the dose of 10nmol/kg is similar to that of semaglutide-10nmol/kg. See Table 5, Table 6 and Figure 2 and Figure 3 for specific data.

Table 5: The effect of long-term administration of HEC-12063 on the rate of blood glucose drop in glucose-loaded DIO mice

Table 6: Effect of long-term administration of HEC-12063 on body weight of DIO mice

Experimental conclusion: Long-term administration of HEC-12063 can significantly improve the glucose tolerance of DIO mice and significantly reduce the body weight of DIO mice.

Example 9 Effect of candidate HEC-C046 on blood glucose and body weight of ob/ob mice

Experimental method: 7-week-old ob/ob mice were fed with a high-fat diet for 3 weeks and divided into 3 groups (Model, semaglutide-10nmol/kg, HEC-C046-10nmol/kg group) according to body weight and blood sugar, and started subcutaneously at the 4th week Administration (the model group was given the corresponding vehicle), semaglutide was administered once a day, and HEC-C046 was administered twice a week for 4 weeks. During the administration, the blood glucose and body weight of each group were tested.

Experimental results: Long-term administration of HEC-C046 at a dose of 10 nmol/kg can significantly reduce blood glucose and body weight of ob/ob mice, and is superior to the effect of Semaglutide at a dose of 10 nmol/kg. See Table 7, Table 8, and Figure 4, Figure 5 for specific data.

Table 7: Effects of long-term administration of HEC-C046 on blood glucose in ob/ob mice

Table 8: Effects of long-term administration of HEC-C046 on body weight of ob/ob mice

Experimental conclusion: Long-term administration of HEC-C046 improves blood glucose and body weight of ob/ob mice better than Semaglutide at the same dose.

Example 10 Effect of candidate HEC-C046 on blood glucose and body weight of db/db diabetic mice

Experimental method: 7-week-old db/db diabetic mice were divided into 4 groups (Model, semaglutide-10nmol/kg, HEC-C046-3nmol/kg, HEC-C046-10nmol/kg group) according to body weight and blood sugar, and were administered subcutaneously (The model group was given the corresponding vehicle), semaglutide was administered once a day, and HEC-C046 was administered twice a week, and the dose of HEC-C046-3nmol/kg was increased to 6nmol/kg on the 17th day of administration. All animals in all groups were given a total of The drug was administered for 4 weeks, and the blood glucose and body weight of each group of animals were tested during the administration period.

Experimental results: HEC-C046 can significantly reduce blood sugar and body weight of db/db mice after long-term administration at a dose of 10nmol/kg, and is superior to the effect of Semaglutide at a dose of 10nmol/kg. See Table 9, Table 10 and Figure 6, Figure 7 for specific data.

Table 9: Effect of long-term administration of HEC-C046 on blood sugar of db/db mice

Table 10: Effects of long-term administration of HEC-C046 on body weight of db/db mice

Experimental conclusion: Long-term administration of HEC-C046 has a significantly better effect on improving blood sugar and body weight of db/db mice than semaglutide.

Example 11 Effect of candidate HEC-C079 on glucose tolerance of normal C57BL/6 mice

Experimental method: Normal C57BL/6 mice were divided into 3 groups according to blood sugar and body weight (Control, Dulaglutide-7.5nmol/kg, HEC-C079-7.5nmol/kg), 8 mice in each group, and the corresponding vehicle or candidate was injected subcutaneously , The animals were fasted 56h after the administration. 72h after the administration, the animals in each group were intraperitoneally injected with 2g/kg glucose, and blood glucose was measured before and 15, 30, 60, and 90 minutes after the administration of the sugar. Draw the blood glucose concentration-time curve according to the blood glucose values measured at different time points, as shown in Figure 8, calculate the AUC _{0～90min Glu of} each dose group and the hypoglycemic rate at the peak blood glucose.

Experimental results:

Table 11: Effect of HEC-C079 on blood sugar of mice with glucose load

Experimental conclusion: HEC-C079 can significantly reduce the blood glucose level of mice with glucose load.

Example 12 Effect of candidate HEC-C079 on glucose tolerance and body weight of DIO mice

Experimental method: C57BL/6 mice aged 7-8 weeks were fed with a high-fat diet for 4 months and then divided into 3 groups (Model, semaglutide 5nmol/kg, HEC-C079 5nmol/kg) according to their body weight. The administration was started at the 5th month (the model group was given the corresponding vehicle), semaglutide was administered once a day, and HEC-C079 was administered twice a week for a total of 4 weeks. The animals were weighed before each administration. The IPGTT experiment was conducted in the 4th week.

Experimental results: HEC-C079 can significantly reduce the blood glucose and body weight of DIO mice at a dose of 5nmol/kg; HEC-C079 improves glucose tolerance better than semaglutide-5nmol/kg at a dose of 5nmol/kg, and HEC-C079 at 5nmol/kg The weight reduction effect of dose is similar to that of semaglutide-5nmol/kg. See Tables 12 and 13 and Figure 9 and Figure 10 for specific data.

Table 12: Effect of long-term administration of HEC-C079 on the rate of blood glucose drop in DIO mice with glucose load

Table 13: Effects of long-term administration of HEC-C079 on body weight of DIO mice

Experimental conclusion: Long-term administration of HEC-C079 can significantly improve the glucose tolerance of DIO mice and significantly reduce the body weight of DIO mice.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structure, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (22)

1. A GLP-1/GCG dual receptor agonist polypeptide, characterized in that the polypeptide has an amino acid sequence as shown below:

   ^{_{5 'X 1 SQGT FTSDX 10 SKYLX}} 15 X 16 X 17 X 18 AX 20 X 21 FX 23 X 24 W L X 27 X 28 X 29 X 30 3'

   Where X ₁ is H or Y,

   X ₁₀ is Y or L,

   X ₁₅ is D or E,

   X ₁₆ is M or E,

   X ₁₇ is Q, K or R,

   X ₁₈ is R or A,

   X ₂₀ is H or K,

   X ₂₁ is E or D,

   X ₂₃ is V or I,

   X ₂₄ is Q, A, N or E,

   X ₂₇ is L or M,

   X ₂₈ is A, N or D,

   X ₂₉ is A or G,

   X ₃₀ is T, PSSGAPPPS or does not exist.
2. The polypeptide of claim 1, wherein the polypeptide has an amino acid sequence as shown below:

   ^{_{5 'X 1 SQGT FTSDX 10 SKYLX}} 15 EX 17 X 18 AK X 21 FX 23 X 24 W LLAGX 30 3'

   Among them, X ₃₀ is PSSGAPPPS or does not exist.
3. The polypeptide of claim 1, wherein the polypeptide has an amino acid sequence as shown below:

   _{^{5 'HSQGT FTSDY SKYLD X 16 X}} 17 X 18 AX 20 X 21 FX 23 X 24 W LX 27 X 28 T 3'

   Among them, X ₂₄ is Q, A or N, and X ₂₈ is N or D.
4. The polypeptide according to claim 2, wherein the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 1-21, SEQ ID NO: 71, and preferably, the polypeptide has SEQ ID NO: 1 ~3 shown in the amino acid sequence.
5. The polypeptide according to claim 3, wherein the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 22 to 34.
6. A fusion protein characterized by comprising: the polypeptide according to any one of claims 1 to 5, a connecting peptide and an IgG ₄ -Fc fragment, the connecting peptide being arranged at the head and tail of _{the polypeptide and the IgG 4 -Fc fragment} between.
7. Fusion protein according to claim 6, characterized in that, connected to the N-terminus of the peptide to the C-terminus of said polypeptide is connected to the N-terminus of the connection with the C-terminal peptide fragment of IgG ₄ -Fc connected.
8. The fusion protein according to claim 6, wherein the connecting peptide has the amino acid sequence shown in SEQ ID NO:35.
9. The fusion protein of claim 6, wherein the IgG4-Fc fragment has the amino acid sequence shown in SEQ ID NO: 36.
10. The fusion protein according to claim 6, wherein the fusion protein has an amino acid sequence shown in SEQ ID NO: 37-70, SEQ ID NO: 72.
11. A nucleic acid, characterized in that the nucleic acid encodes the polypeptide according to any one of claims 1 to 5 or the fusion protein according to any one of claims 6 to 10.
12. A construct characterized by carrying the nucleic acid of claim 11.
13. The construct according to claim 12, wherein the vector of the construct is pXC17.4.
14. A recombinant cell characterized by expressing the polypeptide according to any one of claims 1 to 5 or the fusion protein according to any one of claims 6 to 10.
15. A recombinant cell, characterized in that the recombinant cell contains the construct according to claim 12 or 13 or the nucleic acid according to claim 11 is integrated into the genome.
16. The recombinant cell according to claim 14 or 15, wherein the recombinant cell is a CHO cell.
17. A pharmaceutical composition characterized by comprising the polypeptide of any one of claims 1 to 5 or the fusion protein of any one of claims 6-10.
18. The pharmaceutical composition of claim 17, further comprising a pharmaceutically acceptable carrier.
19. The pharmaceutical composition according to claim 17, characterized in that it further comprises other anti-diabetic drugs, and the other anti-diabetic drugs are selected from insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone At least one of a ketone, an α-glucosidase inhibitor, and an aminodipeptidase IV inhibitor.
20. A protein preparation characterized by comprising the polypeptide of any one of claims 1 to 5 or the fusion protein of any one of claims 6-10.
21. Use of the polypeptide according to any one of claims 1 to 5 or the fusion protein according to any one of claims 6 to 10 or the protein preparation according to claim 20 in the preparation of medicines for treatment or prevention Metabolic disease.
22. The use according to claim 21, wherein the metabolic disease comprises at least one selected from the group consisting of non-alcoholic fatty liver disease, diabetes, and obesity.

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