WO2022117044A1

WO2022117044A1 - Glp-1/gcg dual receptor agonist polypeptide and fusion protein thereof

Info

Publication number: WO2022117044A1
Application number: PCT/CN2021/135120
Authority: WO
Inventors: Peng Jiang; Lin Xiao; Linjun Zhou; Hui Wang; Yi Ling; Xiaofeng Chen; Wenjia LI
Original assignee: Sunshine Lake Pharma Co., Ltd.
Priority date: 2020-12-03
Filing date: 2021-12-02
Publication date: 2022-06-09
Also published as: CN114591415B; CN114591415A

Abstract

Provided are a GLP-1/GCG dual receptor agonist polypeptide and a fusion protein containing the GLP-1/GCG dual receptor agonist polypeptide. The GLP-1/GCG dual receptor agonist polypeptide and fusion protein have different mutation sites from the existing published polypeptide or fusion protein. The polypeptide or fusion protein provided herein has good effect on reducing blood glucose and weight, and has a long half-life in vivo, which is a more effective weight-losing and glucose-controlling drug.

Description

GLP-1/GCG DUAL RECEPTOR AGONIST POLYPEPTIDE AND FUSION PROTEIN THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority and benefits of Chinese Patent Application No. 202011405942. X, filed with the State Intellectual Property Office of China on December 03, 2020, which is incorporated herein by reference in its entirety.

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

Obesity is closely related to type II diabetes, hyperlipidemia and hypertension. At present, the main effect of drugs for the tier-1 treatment of type II diabetes is to reduce blood glucose, but the effect of reducing weight is very limited. How to develop a drug with good effects of reducing blood glucose and weight 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 glucose is dependent on glucose concentration. While regulating blood glucose, it greatly reduces the risk of hypoglycemia. In recent years, GLP-1 based drugs, such as liraglutide, duraglutide and semaglutide, have gradually occupied a very important position in diabetes drugs. The GLP-1 drugs also have a weight loss effect when lowering blood glucose. 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 achieve the purpose of reducing food intake. Prior to this, liraglutide has been approved as a weight-loss drug, and its upgraded product semaglutide (trade name is wegovy) has a better weight loss effect and has been approved by the FDA in June 2021.

However, clinical trials have proved that the shortcomings of GLP-1 receptor agonists are also obvious, mainly in the following aspects: firstly, the short half-life leads to intensive injection frequency and inconvenience to patients; secondly, the pharmacokinetics and safety are both not clear, and how the introduced exogenous chemical group is metabolized and excreted, and what effects it has on the human body are still unclear, and further research is needed. The preclinical studies have shown that the glucagon-like peptide-1 and glucagon (GLP-1/GCG) dual receptor agonists have shown better weight loss and blood glucose lowering effects than single target GLP-1 agonists, and have a very large development potential.

Oxntomodulin (OXM) is a dual receptor agonist that activates glucagon-like peptide-1 (GLP-1) and glucagon (GCG) . Studies have shown that OXM has a good effect of reducing blood glucose and weight in the animal model, which is significantly better than existing GLP-1 drugs, such as liraglutide. However, some of the problems of oxyntomodulin (OXM) , such as poor stability and low receptor activity, lead to large doses and it is difficult to achieve the best effect of blood glucose control and weight loss.

SUMMARY OF THE INVENTION

In order to solve the above problems, the inventors optimized the OXM to improve its stability, and the half-life in vivo is prolonged. The receptor activity is increased, the balance between receptors is optimized, and achieved the best effect of blood glucose control and weight loss.

Based on the above optimization and transformation results, the technical solution provided by the present invention is:

In the first aspect, provided herein is a GLP-1/GCG dual receptor agonist polypeptide. According to the embodiments of the present invention, the polypeptide has an amino acid sequence as shown below: ^5’X ₁SQGT FTSDY SKYLD EKX ₁₈AK X ₂₁FX ₂₃EW LX ₂₇X ₂₈X ₂₉X ₃₀ ^3’, wherein, X ₁ is H or Y; X ₁₈ is R, A or K; X ₂₁ is E or D; X ₂₃ is V or I; X ₂₇ is L or I; X ₂₈ is A or E; X ₂₉ is A or G; X ₃₀ is GPSSGAPPPS or absent; when X ₂₇ is L, X ₂₈ is E.

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

According to the embodiments of the present invention, the polypeptide has an amino acid sequence as shown below: ^5’X ₁SQGT FTSDY SKYLD EKX ₁₈AK EFX ₂₃EW LX ₂₇X ₂₈GX ₃₀ ^3’, wherein, X ₁ is H or Y, X ₁₈ is R, A or K, X ₂₃ is V or I, X ₂₇ is L or I, X ₂₈ is A or E.

According to the embodiments of the present invention, the polypeptide has an amino acid sequence as shown below: HSQGT FTSDY SKYLD EKX ₁₈AK EFX ₂₃EW LX ₂₇X ₂₈G;

wherein, X ₁₈ is R, Aor K, X ₂₃ is V or I, X ₂₇ is L or I, X ₂₈ is A or E.

According to the embodiments of the present invention, the polypeptide has the aforementioned amino acid sequence, and in the amino acid sequence of the polypeptide, at least one of X ₂₃ or X ₂₇ is I.

According to the embodiments of the present invention, the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 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 inventors found that the GLP-1/GCG dual receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO: 1～5 has strong in vitro biological activity on GLP-1R and GCGR cells, and at the same time, the 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 significant effects in controlling body weight and reducing blood glucose.

In the second aspect, provided herein is a fusion protein. According to the embodiments of the present invention, the fusion protein comprises the aforementioned polypeptide, a connecting peptide, and an IgG Fc fragment, and the connecting peptide is arranged between the head and tail of the polypeptide and the IgG Fc fragment. The fusion protein according to the embodiments of the present invention has a good effect of reducing blood glucose and weight, and has a long half-life in the body, and it is an effective long-acting weight-losing and glucose-controlling drug.

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

According to the embodiments of the present invention, the N-terminus of the connecting peptide is connected with the C-terminus of the polypeptide, and the C-terminus of the connecting peptide is connected with the N-terminus of the IgG Fc fragment.

According to the embodiments of the present invention, the connecting peptide has an amino acid sequence 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 of the present invention is derived from the Fc region of human IgG1, IgG2, or IgG4 or a mutant thereof. Preferably, it is derived from the Fc region of human IgG4 or a mutant thereof.

According to the embodiments of the present invention, the IgG Fc fragment is derived from a mutant of the Fc region of human IgG4, which comprises the following amino acid sequence:

Wherein, the IgG4 Fc mutant has three site mutations (EU numbering) of S228P, F234A, and L235A compared with human wild-type IgG4-Fc, and these mutations correspond to the position 10, position 16 and position 17 in SEQ ID NO: 9 respectively. The above mutations can further eliminate the effector functions of IgG4-Fc, such as ADCC and CDC, to improve the safety of the fusion protein. At the same time, the lysine of K447 at the C-terminal is deleted to avoid the heterogeneity of the fusion protein.

According to the embodiments of the present invention, the fusion protein has an amino acid sequence shown in any one of SEQ ID NO: 10～14.

In the third aspect, provided herein is a nucleic acid. According to the embodiments of the present invention, the nucleic acid encodes the aforementioned polypeptide or the aforementioned fusion protein.

In the fourth aspect, provided herein is a construct. According to the embodiments of the present invention, the construct carries the aforementioned nucleic acid.

According to the embodiments of the present invention, the construct may further comprise at least one of the following additional technical features:

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

In the fifth aspect, provided herein is a recombinant cell. According to the embodiments of the present invention, the recombinant cell expresses the aforementioned polypeptide or the aforementioned fusion protein.

In the sixth aspect, provided herein is a recombinant cell. According to the embodiments of the present invention, the recombinant cell comprises the aforementioned construct or the genome of the recombinant cell integrates the aforementioned nucleic acid.

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

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

In the seventh aspect, provided herein is a pharmaceutical composition. According to the embodiments of the present invention, the pharmaceutical composition comprises the aforementioned polypeptide or the aforementioned fusion protein.

According to the embodiments of the present invention, the pharmaceutical composition may further comprise at least one of the following additional technical features:

According to the embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

According to the embodiments of the present invention, the pharmaceutical composition further comprises other anti-diabetic drugs, wherein the other anti-diabetic drugs comprise at least one selected from insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, α-glucosidase inhibitors, and aminodipeptidase IV inhibitors.

In the eighth aspect, provided herein is a protein formulation. According to the embodiments of the present invention, the protein formulation comprises the aforementioned polypeptide or the aforementioned fusion protein. The protein preparation according to the embodiments of the present invention is an effective long-acting weight-losing and glucose-controlling drug.

In the ninth aspect, provided herein is use of the aforementioned polypeptide or the aforementioned fusion protein or the aforementioned protein formulation in the manufacture of a medicament for treating or preventing metabolic diseases.

According to the embodiments of the present invention, the metabolic diseases comprise at least one selected from non-alcoholic fatty liver disease (NAFLD) , Non-alcoholic steatohepatitis (NASH) , diabetes, and obesity.

In another aspect, provided herein is a method of treating or preventing metabolic diseases in a subject comprising administering to the subject a therapeutically effective amount of the aforementioned polypeptide or the aforementioned fusion protein or the aforementioned protein formulation.

In another aspect, provided herein is the aforementioned polypeptide or the aforementioned fusion protein or the aforementioned protein formulation for use in treating or preventing metabolic diseases in a subject.

DESCRIPTION OF THE DRAWINGS

Figure 1 is a blood glucose concentration-time curve at different time points after administration of the glucose loaded mice according to the embodiments of the present invention;

Figure 2 is a blood glucose concentration-time curve at different time points after administration of the glucose loaded mice according to the embodiments of the present invention;

Figure 3 is a curve of the experimental results showing the effect of the fusion protein on the glucose tolerance of DIO mice according to the embodiments of the present invention;

Figure 4 is a curve of the experimental results showing the effect of the fusion protein on the body weight of DIO mice according to the embodiments of the present invention;

Figure 5 is a curve of the experimental results showing the fusion protein HEC-C70 on the glucose tolerance of DIO mice according to the embodiments of the present invention;

Figure 6 is a curve of the experimental results showing the effect of the fusion protein HEC-C70 on the body weight of DIO mice according to the embodiments of the present invention;

Figure 7 is a curve of the experimental results showing the fusion protein HEC-C70 on random blood glucose of db/db mice according to the embodiments of the present invention;

Figure 8 is a curve of the experimental results showing the effect of the fusion protein HEC-C70 on the body weight of db/db mice according to the embodiments of the present invention.

EXAMPLES

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

DEFINITION OF TERMS

The terms "protein" and "polypeptide" are interchangeable, and in their broadest sense, the terms refer to compounds of two or more subunit amino acids, amino acid analogue, or peptidomimetics. The subunits can be connected by peptide bonds. In another embodiment, the subunits can be connected by other bonds, such as esters, ethers, amino groups, and the like. The protein or polypeptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can constitute 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, such as glycine and D and L optical isomers, amino acid analogue, and peptidomimetics.

The "Fc fragment" described herein is composed of the hinge regions, CH2 and CH3 constant regions of an antibody. The antibody comprises two functionally independent parts, a variable domain called "Fab" that binds to an antigen and a constant domain called "Fc" that participates in effector functions (such as complement activation and attack by phagocytes) . Fc has a long serum half-life, while Fab has a short life (Capon et al., 1989, Nature 337: 525-31) . When linked to therapeutic proteins, the Fc domain can provide a longer half-life, or provide functions such as binding to Fc receptor, binding to protein A, binding to complement, or even placental transfer (Capon et al., 1989) . The term "Fc" used herein refers to the wild-type Fc sequence, which is derived from a natural antibody (for example, human IgG1, IgG2, IgG3 or IgG4) , and also comprises variants thereof. The variants may comprise one or more amino acid substitutions, additions, and/or deletions that have been disclosed. In some embodiments, the Fc variant has the activity of wild-type Fc, such as binding to Fc receptors.

The amino acid number of the IgG4 Fc fragment described herein is numbered according to the EU numbering system. For example, the “S228P” refers to that the serine at position 228 according to the EU numbering system is replaced by proline; the “K447” refers to that the lysine at position 447 according to the EU numbering system is deleted or absent.

The term "fusion protein" generally refers to a protein obtained by fusing two or more proteins or polypeptides. The genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or a fusion nucleic acid molecule, and the fusion gene or fusion nucleic acid molecule can encode the fusion protein. The translation of the fusion gene produces a single polypeptide, which has the properties of at least one or even each of the two or more proteins or polypeptides before the fusion. The recombinant fusion protein is artificially created by recombinant DNA technology used in biological research or therapy. The recombinant fusion protein is a protein created by genetic engineering of fusion gene. The present invention relates to a recombinant fusion protein, and the terms "fusion protein" and "recombinant fusion protein" described herein have the same meaning. The fusion protein described herein generally comprises at least two domains (A and C) , and optionally it comprises a third component, a linker between the two domains. The production of recombinant fusion protein is known in the art, and usually involves removing the stop codon from the cDNA sequence encoding the first protein or polypeptide, and then attaching the cDNA sequence of the second protein in frame by ligation or overlap extension PCR. Then the DNA sequence will be expressed as a single protein by the cell. The protein can be engineered to include the complete sequence of the two original proteins or polypeptides, or only a portion of either.

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

As described herein, the term "comprise" , "include" or "contain" generally refers to the inclusion of the stated elements, but does not exclude other elements.

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

The fusion gene was 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. The cells were collected by centrifugation, and then ultrasonically broken and centrifuged to obtain the supernatant, then the supernatant was purified by a nickel column to obtain the fusion protein. Finally, after digestion with SUMO protease, the target polypeptide was obtained by reversed-phase purification. The specific processes are as follows:

1. Vector construction and primer synthesis

The pET-28a was used as an expression vector, and BL21 (DE3) was used as a host. The specific steps for preparing the polypeptides are as follows:

1) The primers were designed and served as templates for each other to amplify polypeptide gene fragments. The polypeptide gene fragments and SUMO gene fragments were used as templates. The fusion gene fragments were amplified by fusion PCR method.

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

2. Expression and purification

The constructed BL21 (DE3) bacterial strain was cultured in LB, then kanamycin at a final concentration of 50μg/mL was added. IPTG was used to induce expression for 5h after the culture. After centrifugation, the cells were taken and dissolved with equilibration buffer (20mM Tris-HCl, pH8.0, 150mM NaCl) , and the cells were broken by an ultrasonic instrument. The supernatant after centrifugation was used to purify the fusion protein. The supernatant was purified by Ni-NTA affinity chromatography column (GE Healthcare) and eluted with elution buffer (20mM Tris-HCl, pH8.0, 150mM NaCl, 200mM imidazole) to obtain the fusion protein.

3. Digestion of fusion protein

After the protein solution was diluted with equilibration buffer, SUMO protease was added according to the ratio of protein: SUMO protease = 50: 1, and the protein was digested for 1.5h at room temperature.

4. Purification of polypeptides

20%Acetonitrile was added to the polypeptide aqueous solution obtained after digestion. The mixture was passed through a 4.6*250mm C8 packed column with 8μm particle size (Nanomicro UniSil 8-120 C8 Ultra Plus 8 um 4.6*250mm, Suzhou Nanomicro Technology Co., Ltd. ) . Purification was carried out in the AKTA purification system. The purification system was started with 20%acetonitrile/H ₂O (containing 20mM Tris-HCl, pH 8.0) and the proportion of acetonitrile was increased at a gradient of 1%/min, the flow rate was 1mL/min. The column was eluted for 30 minutes, and the peptide-containing component was collected. Liquid-mass spectrometry was used to analyze the separated products. 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

SEQ ID	sample name	amino acid sequences
1	HEC-C080	HSQGT FTSDY SKYLD EKAAK EFIEW LLEG
2	HEC-C070	HSQGT FTSDY SKYLD EKRAK EFIEW LIAG
3	HEC-C085	HSQGT FTSDY SKYLD EKRAK EFVEW LIAG
4	HEC-C086	HSQGT FTSDY SKYLD EKKAK EFIEW LIAG
5	HEC-C087	HSQGT FTSDY SKYLD EKKAK EFVEW LIAG

Example 2 Determination of the in vitro activity of the polypeptide

The polypeptides prepared by expression, human GLP-1 (SEQ ID NO: 17, TOCRIS Company, catalog number 5374 (BATH: 2A) ) and GCG (SEQ ID NO: 18, Novo Nordisk, Novo Sang) were applied to the HEK293 cells expressing GLP-1R or GCGR respectively. The specific operations are:

1. The genes GCGR and GLP-1R were optimized and routinely synthesized in Jinweizhi Company, and the genes were cloned into the vector pUC57-Amp. The mini-scale recombinant plasmid DNA and puncture bacteria containing the recombinant plasmid were prepared;

2. pUC57-GCGR recombinant plasmid DNA was taken and double digested with Hind III and EcoR I, pUC57-GLP-1R was taken and double digested with Hind III and Xho I. After the digested product was electrophoresed on 1%agarose gel, the target strip was cut with a clean blade, and the target fragment was recovered by a gel extraction kit;

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. the target fragment digested and recovered product and the vector plasmid pcDNA3.1 fragment were linked by T4 ligase and then transformed into DH5α competent cells. The plates were coated with the transformed DH5α competent cells to separate single colonies, and the transformants were picked for expanding culture, and verified by restriction enzyme digestion and sequencing.

4. 200 mL of the verified and correct bacterial solution was inoculated for large-scale plasmid extraction, the used kit was PureLink HiPure Plasmid Maxiprep Kit, and operated according to the instructions. After the plasmid was verified by PCR and restriction enzyme digestion, it was linearized with pvuI restriction enzyme. Finally, the plasmid was recovered by ethanol precipitation.

5. The host cell was HEK293. One day before transfection, the cells were plated in a 6-well plate at a density of 2×10 ⁶ cells/well, 1 mL/well. The recovered linearized plasmid was transfected into HEK293 cells by Lipofectamine 3000 liposome transfection method. G418 was added to screen to obtain a mixed strain, and then a single clone was obtained by limiting dilution and separation, and the activity test was carried out.

6. The cAMP detection kit (Cisbio, 62AM6PEC) was used to detect cAMP produced by the recipient cells according to the steps described in the operating instructions. The specific steps were as follows:

1) preparation of Assay buffer: complete culture solution (DMEM medium + 10%FBS) was taken and then 4/1000 of 500 mM IBMX stock solution was added. cAMP-d2 working solution and anti-cAMP-crytate working solution was prepared according to the kit instructions;

2) the test sample and the control sample human GLP-1 and GCG were diluted to a stock solution with an initial concentration of 500 nM, and then 20 μL of the stock solution was added to 80 μL Assay buffer (diluted 5 times) for gradient dilution. There were 8 compound concentration gradients including the stock solution;

3) preparation of cell suspension: The cells HEK293-GLP-1R and HEK293-GCGR were taken out from the liquid nitrogen tank, then added into a 37℃ water bath immediately. After the cells were completely thawed within 1.5 minutes, the cells were added dropwise to a 15mL centrifuge tube containing 8mL warm medium on the ultraclean table. The centrifuge tube was centrifuged at 900rpm for 5min, and the supernatant was discarded. The cells were resuspended in 1mL of complete culture solution (pipetted 15 times) , 20μL of the suspension was taken immediately and mixed with equal volume of Trypan Blue. 20μL of the mixed solution was taken to count the number of living cells, then the cells were diluted to 4 x 10 ⁵ cells/mL;

4) a 384-well plate was divided into GLP-1R cells area and GCGR cells area. A 12-lane change adjustable dispenser was used and the cell suspension was added to the wells in the corresponding area with 5μL/well. The test sample and the positive control sample gradient dilution were added to the 384-well plate corresponding to the cells by using the 12-lane change adjustable dispenser with 5μL/well (2 replicate wells for the same concentration sample) . Negative control: 10 μL assay buffer/well, each 384-well plate was set with 3 holes and covered with a white sealing film, then placed in a 37℃ constant temperature incubator. The plate was taken out after half an hour;

5) before use, the cAMP-d2 working solution and anti-cAMP-crytate working solution were diluted to 20 times with lysis buffer in the Hi-range kit, and mixed with 1: 1 to form a cAMP detection reagent mixed solution. In the sample group, cAMP detection reagent mixed solution was added with 10μL/well. In the negative control group, 5μL of lysis buffer and 5μL of diluted anti-cAMP-crytate working solution were added to each well. The plate was covered with a white lid, and stored at room temperature in the dark for 1 hour;

6) the fluorescence values of 665nm and 620nm were detected by the multifunctional enzyme marker;

7) based on this, a dose-effect curve was established, and the EC ₅₀ values were calculated and compared with each other. The specific results are shown in Table 2.

Table 2 Determination of in vitro EC ₅₀ values of polypeptides

It can be found from the experimental results in Table 2 that the GLP-1/GCG dual receptor agonist polypeptides provided herein can significantly activate GLP-1 receptor cells and GCG receptor cells, respectively.

Example 3 Construction of fusion protein vector

Molecular cloning method was used to fuse the GLP-1/GCG dual receptor agonist polypeptide with IgG4-Fc (SEQ ID NO: 9) with a connecting peptide (SEQ ID NO: 6) . The resulting sequence was double digested and inserted between the same restriction site of the mammalian cell expression vector, and a series of mutant vectors were constructed. After being verified by sequencing, the plasmid vectors were extracted with endotoxin-free plasmid extraction kit (OMEGA) and stored at -20℃. The amino acid sequence of the GLP-1/GCG dual receptor 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 dual receptor agonist polypeptide constructed in Example 2 is consistent with the sample name of the GLP-1/GCG dual receptor 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*106 cells/mL, the cells were collected and transfected by using ExpiCHOFectamineTM CHO Transfection Kit (ThermoFisher Scientific) . The final concentration of the vector constructed in Example 2 was 1μg /mL. Reagents such as enhancers were added on the second day of the transfection to maintain the growth of the transfected cells. The cell culture solution was harvested when the cell viability lowered to about 80%.

Example 5 Purification and identification of fusion protein

The cell culture solution was centrifuged and the supernatant was collected, and the residual cell debris were removed by filtering with a 0.22 μm filter. The collected cell culture solution was purified by a Protein A chromatography column, the target peak was collected, and then further purified by anion exchange chromatography. The protein was finally eluted and collected with 0.02M PBS. The samples were quantified using a micro-nucleic acid protein analyzer (NanoDrop 2000/2000c Spectrophotometer) . The samples were detected by 12%SDS-PAGE electrophoresis, and the electrophoresis pattern showed as a single band. The precise molecular weight of the fusion protein was determined by mass spectrometry, and it was 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. A dose-effect curve was established, and the EC ₅₀ values were calculated and compared with each other. The results are shown in Table 3.

Table 3 Determination of in vitro EC ₅₀ values of fusion protein

It can be found from the experimental results in Table 3 that the GLP-1/GCG receptor dual agonist samples provided herein can significantly activate GLP-1 receptor cells and GCG receptor cells respectively.

Example 7 Effect of Candidates on Glucose Tolerance of Normal C57BL/6 Mice

Experimental method: Normal C57BL/6 mice were divided into 6 groups according to blood glucose and body weight (Control, Dulaglutide -7.5nmol/kg, HEC-C80-7.5nmol/kg, HEC-C85-7.5nmol/kg, HEC-C86-7.5 nmol/kg, HEC-C87-7.5nmol/kg) , 6 mice per group. The corresponding menstruum or candidate was administered by subcutaneous injection, the mice were fasted 56h after administration, and the mice in each group were injected intraperitoneally with 2g/kg glucose 72h after administration. The blood glucose test was performed before and 15, 30, 60, and 90 minutes after glucose administration. The results are shown in Table 4. The blood glucose concentration-time curve was plotted according to the blood glucose measured at different time points, as shown in Figure 1. The blood glucose drop rate of each dose group of AUC _{0～90min Glu} and at the peak blood glucose were calculated.

Results:

Table 4: Effects of different fusion proteins on blood glucose in mice with glucose load

Conclusion: HEC-C80, HEC-C85, HEC-C86 and HEC-C87 can significantly reduce the blood glucose level of glucose loaded mice.

Example 8 Effect of Candidates on Glucose Tolerance of Normal C57BL/6 Mice

Experimental method: Normal C57BL/6 mice were divided into 3 groups according to blood glucose and body weight (Control, Dulaglutide-7.5nmol/kg, HEC-C70-7.5nmol/kg) , 8 mice per group. The corresponding menstruum or candidate was administered by subcutaneous injection, the mice were fasted 56h after administration, and the mice in each group were injected intraperitoneally with 2g/kg glucose 72h after administration. The blood glucose test was performed before and 15, 30, 60, and 90 minutes after glucose administration. The results are shown in Table 5. The blood glucose concentration-time curve was plotted according to the blood glucose measured at different time points, as shown in Figure 2. The blood glucose drop rate of each dose group of AUC _{0～90min Glu} and at the peak blood glucose were calculated.

Results:

Table 5: The effect of HEC-C70 on blood glucose of glucose loaded mice

Conclusion: HEC-C70 can significantly reduce the blood glucose level of glucose loaded mice.

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

Experimental method: 8-week-old C57BL/6 mice were fed with high-fat diet for 15 weeks and divided into 7 groups according to body weight (Model, semaglutide-5nmol/kg, HEC-C70-5nmol/kg, HEC-C80-5nmol/kg, HEC-C85-5nmol/kg, HEC-C86-5nmol/kg, HEC-C87-5nmol/kg) . From the 16th week, the mice were administered (the model group was administered the corresponding menstruum) . The semaglutide group was administered once a day, and the other groups were administered twice a week, and a total of 4 weeks of administration. The mice were weighed before each administration and the IPGTT experiment was carried out in 4 weeks.

Experimental results: the effect of HEC-C70-5nmol/kg on improving glucose tolerance is similar to semaglutide-10nmol/kg. HEC-C70-5nmol/kg, HEC-C80-5nmol/kg, HEC-C85-5nmol/kg, HEC-C86-5nmol/kg and HEC-C87-5nmol/kg all have a significant effect on reducing the body weight of DIO mice, and the effects of HEC-C70-5nmol/kg and HEC-C85-5nmol/kg are better than semaglutide-5nmol/kg. The specific data are shown in Table 6, Table 7, Figure 3 and Figure 4.

Table 6: Effects of long-term administration of different fusion proteins on the rate of blood glucose drop in glucose-loaded DIO mice

Table 7: Effects of long-term administration of different fusion proteins on body weight of DIO mice

Conclusion: Long-term administration of HEC-C70, HEC-C80, HEC-C85, HEC-C86, HEC-C87 can significantly improve the glucose tolerance and significantly reduce the body weight of DIO mice.

Example 10 Effect of Candidates on Glucose Tolerance and Body Weight of DIO Mice

Experimental method: 6-week-old C57BL/6N mice were fed with high-fat diet for 15 weeks and divided into 6 groups according to body weight (Model, LY3298176-10nmo/kg, semaglutide-10nmol/kg, MEDI0382-10nmol/kg, HEC-C70-2.5nmol/kg, HEC-C70-10nmol/kg) . From the 16th week, the mice were administered (the model group was administered the corresponding menstruum) . The semaglutide group and MEDI0382 group were administered once a day, and the other groups were administered twice a week, and a total of 4 weeks of administration. The mice were weighed before each administration and the IPGTT experiment was carried out in 4 weeks.

Control group LY3298176 sequence: YX ₁EGTFTSDYSIX ₂LDKIAQKAFVQWLIAGGPSSGAPPPS-NH ₂ (SEQ ID NO: 15) , wherein the X ₁ and X ₂ are Aib, and K at position 20 is connected with ( [2- (2-amino-ethoxy) -ethoxy] -acetyl) ₂-γGlu-CO- (CH ₂) ₁₈-CO ₂H through the side chain ε-amino.

Control group MEDI0382 sequence: HSQGTFTSDKSEYLDSERARDFVAWLEAGG (SEQ ID NO: 16) , wherein, K at position 10 is connected with γGlu-CO- (CH ₂) ₁₄-CO ₂H through the side chain ε-amino.

Experimental results: the effect of HEC-C70-10nmol/kg on improving glucose tolerance is similar to that of LY3298176-10nmol/kg, and better than semaglutide-10nmol/kg and MEDI0382-10nmol/kg. HEC-C70-10nmol/kg has a significant effect on reducing the body weight of DIO mice, and it is better than semaglutide-10nmol/kg. The specific data are shown in Table 8, Table 9, Figure 5 and Figure 6.

Table 8: The effect of long-term administration on the rate of blood glucose drop of DIO mice with glucose load

Table 9: The effect of long-term administration on the body weight of DIO mice

Conclusion: Long-term administration of HEC-C70 can significantly improve the glucose tolerance and significantly reduce the body weight of DIO mice.

Example 11 Effects of candidates on blood glucose and body weight of db/db mice

Experimental method: Ob/ob mice aged 6-7 weeks were divided into 3 groups (Model, Dulaglutide-30nmo/kg, HEC-C70-30nmol/kg) according to random blood glucose and body weight. Ob/ob mice were administered for a total of 4 weeks (the model group was administered the corresponding menstruum) , Dulaglutide and HEC-C70 were administered twice a week. During the administration, the random blood glucose and body weight of the mice in each group were monitored.

Experimental results: the effect of HEC-C70-30nmol/kg on improving glucose tolerance is better than Dulaglutide-30nmol/kg. HEC-C70-30nmol/kg has a significant effect on reducing the body weight of db/db mice, and it is better than Dulaglutide-30nmol/kg. The specific data are shown in Table 10, Table 11, Figure 7 and Figure 8.

Table 10: The effect of long-term administration on blood sugar of db/db mice

Table 11: The effect of long-term administration on the body weight of db/db mice

Reference throughout this specification to "an embodiment, " "some embodiments, " "one embodiment" , "another example, " "an example, " "a specific example, " or "some examples, " means 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 present disclosure. Thus, the appearances of the above terms throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can integrate and combine different embodiments, examples or the features of them as long as they are not contradictory to one another.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

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

^5’X ₁SQGT FTSDY SKYLD EKX ₁₈AK X ₂₁FX ₂₃EW LX ₂₇X ₂₈X ₂₉X ₃₀ ^3’

wherein, X ₁ is H or Y; X ₁₈ is R, A or K; X ₂₁ is E or D; X ₂₃ is V or I; X ₂₇ is L or I; X ₂₈ is A or E; X ₂₉ is A or G; X ₃₀ is GPSSGAPPPS or absent;

when X ₂₇ is L, X ₂₈ is E.
The polypeptide of claim 1, wherein the polypeptide has an amino acid sequence as shown below:

HSQGT FTSDY SKYLD EKX ₁₈AK EFX ₂₃EW LX ₂₇X ₂₈G;

wherein, X ₁₈ is R, A or K, X ₂₃ is V or I, X ₂₇ is L or I, X ₂₈ is A or E.
The polypeptide of any one of claims 1 to 2, wherein in the amino acid sequence of the polypeptide, at least one of X ₂₃ or X ₂₇ is I.
The polypeptide of any one of claims 1～3, wherein the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 1～5.
A fusion protein comprising the polypeptide of any one of claims 1～4, a connecting peptide and an IgG-Fc fragment, wherein the connecting peptide is arranged between the head and tail of the polypeptide and the IgG-Fc fragment.
The fusion protein of claim 5, wherein the N-terminus of the connecting peptide is connected with the C-terminus of the polypeptide, and the C-terminus of the connecting peptide is connected with the N-terminus of the IgG-Fc fragment.
The fusion protein of any one of claims 5～6, wherein the connecting peptide has an amino acid sequence shown in any one of SEQ ID NO: 6～8.
The fusion protein of any one of claims 5～7, wherein the IgG-Fc fragment is an IgG4-Fc mutant and has an amino acid sequence shown in SEQ ID NO: 9.
The fusion protein of any one of claims 5～8, wherein the fusion protein has an amino acid sequence shown in any one of SEQ ID NO: 10～14.
A nucleic acid encoding the polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9.
A construct carrying the nucleic acid of claim 10.
The construct of claim 11, wherein the vector of the construct is pXC17.4.
A recombinant cell expressing the polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9, or comprising the construct of claim 11 or 12, or the genome of which integrates the nucleic acid of claim 10.
The recombinant cell of claim 13, wherein the recombinant cell is a CHO cell.
A pharmaceutical composition comprising the polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9.
The pharmaceutical composition of claim 15, further comprising a pharmaceutically acceptable carrier.
The pharmaceutical composition of claim 15, further comprising other anti-diabetic drugs, and the other anti-diabetic drugs comprising at least one selected from insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, α-glucosidase inhibitors, and aminodipeptidase IV inhibitors.
A protein formulation comprising the polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9.
Use of the polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9 or the protein formulation of claim 18 in the manufacture of a medicament for treating or preventing metabolic diseases, wherein the metabolic diseases comprise at least one selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, diabetes, and obesity.
A method of treating or preventing metabolic diseases in a subject comprising administering to the subject a therapeutically effective amount of the polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9 or the protein formulation of claim 18, wherein the metabolic diseases comprise at least one selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, diabetes, and obesity.
The polypeptide of any one of claims 1～4 or the fusion protein of any one of claims 5～9 or the protein formulation of claim 18 for use in treating or preventing metabolic diseases, wherein the metabolic diseases comprise at least one selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, diabetes, and obesity.