GLP-1 / GIP dual targeting polypeptides and fusion proteins and their applications

Abstract

The amino acid sequence: X1X2X3GTFX4SDYSX5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 X 19 wherein X1 is Y or H, X2 is A or G or S, X3 is E or Q, X4 is I or T, X5 is I or K, X6 is A or Y or L or I, X7 is M or L, X8 is D or E, X9 is K or E, and X 10 is I or Q or K or E or L, and X 11 is H, A or R, and X 12 is A, Q, or V, and X 13 is K or Q or R or H, and X 14 is D or E or A or L, and X 15 is V or I, and X 16 is N or E or D or Q, and X 17 is L or I or K or V, and X 18 is A or E or K, and X 19 is Q or G, and X1 to X 13 X1 is Y, X2 is A, X3 is E, X4 is I, X5 is I, X6 is A, X7 is M, X8 is D, X9 is K, and X 10 is I and X 11 is H and X 12 is Q and X 13 is Q and X 14 is D and X 15 is V and X 16 is N and X 17 is L and X 18 is A and X 19 does not simultaneously satisfy Q. The GLP-1 / GIP dual targeting polypeptide can effectively reduce body weight and blood sugar level.

Description

[Technical field]

[0001] This application claims priority to and the benefit of Chinese Patent Application No. 202111026970.5, filed with the State Intellectual Property Office of China on September 2, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of biomedicine, in particular to GLP-1 / GIP dual target polypeptides, fusion proteins and their applications, more particularly to GLP-1 / GIP dual target polypeptides, nucleic acid molecules encoding GLP-1 / GIP dual target polypeptides, fusion proteins, nucleic acid molecules encoding fusion proteins, expression vectors, recombinant cells, the use of GLP-1 / GIP dual target polypeptides and their nucleic acid molecules, fusion proteins and their nucleic acid molecules, expression carriers and recombinant cells in the manufacture of medicines, pharmaceutical compositions and methods for preparing fusion proteins. [Background technology]

[0003] Type II diabetes is a chronic metabolic disorder closely related to obesity, hyperlipidemia, and hypertension. At present, the main effect of drugs for first-line therapy of type II diabetes is to reduce blood glucose, but the effect of weight loss is very limited. Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by intestinal L cells after a meal, which can stimulate pancreatic β cells to secrete insulin, thereby stabilizing the fluctuation of blood glucose after a meal. The blood glucose-lowering effect of the hormone depends on glucose concentration, and while regulating blood glucose, the hormone greatly reduces the risk of hypoglycemia. In recent years, GLP-1-based drugs, such as liraglutide, dulaglutide, and semaglutide, have gradually occupied a very important position of drugs for diabetes. GLP-1 drugs also have the effect of weight loss when lowering blood glucose, and 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 and achieve the purpose of reducing food intake.However, GLP-1 receptor agonist drugs are generally used at higher doses for weight loss, are prone to gastrointestinal side effects, have poor tolerability, and have a narrow therapeutic window.

[0004] Glucose-dependent incretin (GIP) is a peptide hormone secreted by intestinal K cells, which has 42 amino acids and plays a major role in glucose homeostasis and protects pancreatic β cells. Both GIP and GLP-1 are incretins, both of which can promote insulin secretion and lower blood glucose in a blood glucose concentration-dependent manner, and the effect of GIP-mediated blood glucose lowering is more potent than that of GLP-1. However, diabetic patients are insensitive to GIP, possibly due to receptor resistance induced by hyperglycemia, so the use of GIP receptor agonists alone in diabetic patients does not achieve the purpose of improving blood glucose. Summary of the Invention [Problem to be solved by the invention]

[0005] At present, in diseases with complex mechanisms, such as diabetes, obesity, etc., the effect of single-target drugs is limited, and some classical treatments, such as insulin injection, oral sulfonylurea and metformin, can easily cause hypoglycemia, so they still cannot meet clinical demands. By acting on different targets, multiple effects can be achieved to improve drug efficacy. Therefore, single molecule GLP-1 / GIP dual receptor agonists can effectively solve the shortcomings of existing single-target drugs, and can achieve better blood glucose lowering effect and weight loss effect by simultaneously activating multiple metabolism-related targets, and also have good development potential. [Means for solving the problem]

[0006] This application is based on the following discoveries and knowledge of the inventors.

[0007] Single molecule GLP-1 / GIP dual receptor agonist can effectively solve the shortcomings of existing single target drugs, the inventors have mutated wild type GLP-1 and GIP through many experiments to obtain various GLP-1 / GIP dual target polypeptides that can simultaneously recognize GLP-1 receptor and GIP receptor, and fused this GLP-1 / GIP dual target polypeptide with Fc fragment of immunoglobulin using a linker peptide to obtain fusion protein. The inventors have found that the fusion protein has good ability to simultaneously activate GLP-1 receptor and GIP receptor, and different mutations can increase or decrease the agonist activity of GLP-1 receptor and GIP receptor.

[0008] Thus, in a first aspect, the present invention provides a GLP-1 / GIP dual target polypeptide. According to an embodiment of the present invention, the dual target polypeptide comprises a first polypeptide, the first polypeptide having the following amino acid sequence:1 X 2 X 3 GTFX 4 SDYSX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 X 19 wherein X 1 is Y or H, and X 2 is A, G, or S, and X 3 is E or Q, and X 4 is I or T, and X 5 is I or K, and X 6 is A, Y, L, or I, and X 7 is M or L, and X 8 is D or E, and X 9 is K or E, and X 10 is I, Q, K, E, or L, and X 11 is H, A, or R, and X 12 is A, Q, or V, and X is 13 is K, Q, R, or H, and X 14 is D, E, A, or L, and X 15 is V or I, and X 16 is N, E, D, or Q, and X 17 is L, I, K, or V; X 18 is A, E, or K, and X 19 is Q or G, and X 1 ~X 13 X 1 is Y and X 2 is A and X 3 is E and X 4 is I and X 5 is I and X 6 is A and X 7 is M and X 8 is D and X 9 is K and X 10 is I and X11 is H and X 12 is Q and X 13 is Q and X 14 is D and X 15 is V and X 16 is N and X 17 is L and X 18 is A and X 19 and Q. The present inventors performed site-specific mutagenesis of wild-type GLP-1 and GIP, and the resulting GLP-1 / GIP dual target polypeptide has a certain degree of binding activity to the GLP-1 and / or GIP receptor.

[0009] According to an embodiment of the present invention, the GLP-1 / GIP dual target polypeptide may further comprise at least one of the following additional technical features:

[0010] According to an embodiment of the invention, the dual target polypeptide comprises a first polypeptide, the first polypeptide having the following amino acid sequence: 1 X 2 EGTFTSDYSIX 6 LDKX 10 AQX 13 X 14 FX 15 X 16 WLX 17 AX 19 wherein X 1 is Y or H, and X 2 is A, G, or S, and X 6 is A, Y, or L, and X 10 is I, Q, K, or L, and X 13 is Q or R, and X 14 is D, E, or A, and X 15 is V or I, and X 16 is E, D, or Q, and X 17 is L, I, or K, and X 19is Q or G, and the first polypeptide does not contain the amino acid sequence of YAEGTFISDYSIAMDKIHQQDFVNWLLAQ (SEQ ID NO: 122). The present inventors performed site-directed mutation of wild-type GLP-1 and GIP, and the resulting GLP-1 / GIP dual target polypeptide has good biological binding activity with GLP-1 and / or GIP receptor, therefore, GLP-1 receptor and GIP receptor can be activated simultaneously, and the GLP-1 / GIP dual target polypeptide has the dual functions of weight loss and blood glucose reduction.

[0011] According to an embodiment of the present invention, the GLP-1 / GIP dual target polypeptide further comprises a second polypeptide, and the second polypeptide has an amino acid sequence shown in SEQ ID NO: 1. The specific amino acid sequence of SEQ ID NO: 1 is GPSSGAPPPS.

[0012] According to an embodiment of the invention, the C-terminal amino acid of a first polypeptide is linked to the N-terminal amino acid of a second polypeptide.

[0013] According to an embodiment of the present invention, the GLP-1 / GIP dual target polypeptide has at least one of the amino acid sequences shown in Table 1.

[0014] In the second aspect, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises (1) the GLP-1 / GIP dual target polypeptide described in the first aspect, and (2) an Fc fragment, and the C-terminus of the GLP-1 / GIP dual target polypeptide is linked to the N-terminus of the Fc fragment. The fusion protein according to an embodiment of the present invention also has excellent binding activity with GLP-1 and / or GIP receptor, has dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0015] According to an embodiment of the present invention, the fusion protein may further comprise at least one of the following additional technical features:

[0016] According to an embodiment of the invention, the Fc fragment is an Fc fragment of human IgG4 or a variant thereof.

[0017] According to an embodiment of the invention, the Fc fragment comprises the amino acid sequence shown in SEQ ID NO:113.

[0018] [ka]

[0019] According to an embodiment of the invention, the fusion protein further comprises a linker peptide having the amino acid sequence set forth in SEQ ID NO:114.

[0020] [ka]

[0021] According to an embodiment of the invention, the N-terminus of the linker peptide is linked to the C-terminus of the GLP-1 / GIP dual target polypeptide, and the C-terminus of the linker peptide is linked to the N-terminus of the Fc fragment.

[0022] In the third aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the GLP-1 / GIP dual target polypeptide of the first aspect or the fusion protein of the second aspect. According to an embodiment of the present invention, both the GLP-1 / GIP dual target polypeptide or the fusion protein encoded by the nucleic acid molecule have excellent binding activity with GLP-1 and / or GIP receptor, have dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0023] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises a nucleic acid molecule as described in the third aspect.

[0024] According to an embodiment of the present invention, the expression vector may further include at least one of the following additional technical features:

[0025] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0026] In a fifth aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect. The recombinant cell according to the embodiment of the present invention can express GLP-1 / GIP dual target polypeptide or fusion protein, and the GLP-1 / GIP dual target polypeptide and fusion protein have excellent binding activity with GLP-1 and / or GIP receptor, have dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0027] In a sixth aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the GLP-1 / GIP dual target polypeptide described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect. The pharmaceutical composition may comprise a pharma- ceutically acceptable adjuvant, the pharma- ceutically acceptable adjuvant comprising at least one of a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent, and the pharmaceutical composition is at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, or a lyophilized preparation. According to an embodiment of the present invention, the pharmaceutical composition has the function of long-term weight loss and / or blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0028] In a seventh aspect, the present invention provides the use of a GLP-1 / GIP dual target polypeptide as described in the first aspect, a fusion protein as described in the second aspect, a nucleic acid molecule as described in the third aspect, an expression vector as described in the fourth aspect, or a recombinant cell as described in the fifth aspect in the manufacture of a medicament. According to an embodiment of the invention, the medicament is used for controlling or reducing glucose and body weight.

[0029] In an eighth aspect, the present invention provides a method for preparing the fusion protein described in the second aspect. According to an embodiment of the present invention, the method includes 1) constructing an expression vector described in the fourth aspect, and 2) introducing the expression vector into a host cell to obtain a recombinant cell for expressing the fusion protein. The fusion protein prepared by the method provided herein has excellent binding activity with GLP-1 and / or GIP receptors, has dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0030] In a ninth aspect, the present invention provides a method for reducing blood glucose and / or body weight in a patient. According to an embodiment of the present invention, the method comprises administering to a patient at least one of 1) a GLP-1 / GIP dual target polypeptide as described in the first aspect, 2) a fusion protein as described in the second aspect, 3) a nucleic acid molecule as described in the third aspect, 4) an expression vector as described in the fourth aspect, 5) a recombinant cell as described in the fifth aspect, and 6) a pharmaceutical composition as described in the sixth aspect. The method provided herein can effectively and long-term control or reduce the body weight and / or blood glucose level of the patient.

[0031] Additional aspects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention.

[0032] The above and / or further aspects and advantages of the present invention will become apparent and be readily understood from the following description of the embodiments in conjunction with the drawings, in which: [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows blood glucose levels of HEC-G123, HEC-G128, HEC-G131, and HEC-G132 groups at different time points in accordance with an embodiment of the present invention. [Diagram 2] FIG. 1 shows the areas under the curve for the HEC-G123 group, the HEC-G128 group, the HEC-G131 group, and the HEC-G132 group according to an embodiment of the present invention. [Diagram 3] FIG. 1 shows blood glucose levels of HEC-G113, HEC-G122, HEC-G126, and HEC-G127 groups at different time points according to an embodiment of the present invention. [Figure 4] FIG. 1 shows the areas under the curve for the HEC-G113 group, the HEC-G122 group, the HEC-G126 group, and the HEC-G127 group according to an embodiment of the present invention. [Diagram 5] FIG. 1 shows the in vivo blood glucose lowering effect of HEC-G20 in a db / db mouse model according to an embodiment of the present invention. [Figure 6] FIG. 13 shows glycosylated hemoglobin levels of HEC-G20 in a db / db mouse model according to an embodiment of the present invention. [Figure 7] FIG. 1 shows the effect of long-term repeated administration of HEC-G115 and HEC-G124 in an embodiment of the present invention on body weight in obese mice as a DIO model. [Figure 8] FIG. 13 shows the cumulative effect of long-term repeated administration of HEC-G115 and HEC-G124 in an embodiment of the present invention to obese mice as a DIO model. [Figure 9]1 is a structural diagram of a fusion protein according to an embodiment of the present invention, in which the fusion protein comprises three domains from N-terminus to C-terminus: a GLP-1 / GIP dual receptor agonist (GLP-1 / GIP dual target polypeptide), a linker peptide, and an Fc fragment. GLP-1 / GIP dual receptor agonist polypeptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0034] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 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 exemplary and are intended to explain the present invention and are not to be construed as limiting the present invention.

[0035] In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood to indicate or imply a relative importance or quantity of the technical features being shown. Thus, features defined as "first" and "second" may explicitly or implicitly include at least one of those features. In the present description, "plurality" means at least two, e.g., two, three, etc., unless expressly and specifically limited otherwise.

[0036] It should be noted that, in this specification, the term "receptor agonist" refers to a substance that can act on a receptor and cause the activation of the receptor, thus producing a biological effect. This effect can be an enhancement or a decrease in the specific occurrence of cellular activity.

[0037] It should be noted that in this specification, the terms "GLP-1 / GIP dual receptor agonist", "GLP-1 / GIP dual target polypeptide" and "recombinant polypeptide" all refer to polypeptides obtained by site-directed mutagenesis of wild-type GLP-1 and GIP protein molecules, and the amino acid sequences of the polypeptides obtained after site-directed mutagenesis of wild-type GLP-1 and GIP protein molecules, which can simultaneously activate the GLP-1 receptor and the GIP receptor, are shown in Table 1.

[0038] It should be noted that in this specification, the term "nucleic acid molecule" can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA and RNA, and is not subject to any particular length restriction. For constructs used in the construction of recombinant cells, the nucleic acid is preferably DNA, since DNA is more stable and easier to manipulate than RNA. The "nucleic acid" described in this application actually includes either or both of the complementary double strands. Those skilled in the art will also understand that one strand can be used to detect the other strand, and vice versa.

[0039] It should be noted that the "construct" described in this application refers to a gene vector that contains a specific nucleic acid sequence and can introduce a target nucleic acid sequence into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, the construct can be at least one of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus, and a plasmid is preferred. As a gene vector, a plasmid is simple to operate, can carry a larger fragment, and is easy to operate and handle. The form of the plasmid is not particularly limited, and the plasmid can be either a circular plasmid or a linear plasmid, that is, the plasmid can be single-stranded or double-stranded. Those skilled in the art can make a selection according to need.

[0040] The present invention relates to a compound having the following amino acid sequence:1 X 2 X 3 GTFX 4 SDYSX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 X 19 A GLP-1 / GIP dual targeting polypeptide comprising a first polypeptide having the formula: 1 is Y or H, and X 2 is A, G, or S, and X 3 is E or Q, and X 4 is I or T, and X 5 is I or K, and X 6 is A, Y, L, or I, and X 7 is M or L, and X 8 is D or E, and X 9 is K or E, and X 10 is I, Q, K, E, or L, and X 11 is H, A, or R, and X 12 is A, Q, or V, and X 13 is K, Q, R, or H, and X 14 is D, E, A, or L, and X 15 is V or I, and X 16 is N, E, D, or Q, and X 17 is L, I, K, or V, and X 18 is A, E, or K, and X 19 is Q or G, and X 1 ~X 19 But X 1 is Y and X 2 is A and X 3 is E and X 4 is I and X 5 is I and X 6 is A and X 7 is M and X 8 is D and X9 is K and X 10 is I and X 11 is H and X 12 is Q and X 13 is Q and X 14 is D and X 15 is V and X 16 is N and X 17 is L and X 18 is A and X 19 and Q. The present inventors have performed site-specific mutations of wild-type GLP-1 and GIP, and the resulting GLP-1 / GIP dual target polypeptide has a certain degree of binding activity to the GLP-1 and / or GIP receptor.

[0041] According to a specific embodiment of the present invention, the GLP-1 / GIP dual target polypeptide comprises a first polypeptide, the first polypeptide having the following amino acid sequence: 1 X 2 EGTFTSDYSIX 6 LDKX 10 AQX 13 X 14 FX 15 X 16 WLX 17 AX 19 wherein X 1 is Y or H, and X 2 is A or G or S, and X 6 is A, Y, or L, and X 10 is I, Q, K, or L, and X 13 is Q or R, and X 14 is D, E, or A, and X 15 is V or I, and X 16 is E, D, or Q, and X 17 is L, I, or K, and X 19is Q or G, and the first polypeptide does not contain the amino acid sequence YAEGTFISDYSIAMDKIHQQDFVNWLLAQ (SEQ ID NO: 122). The present inventors performed site-directed mutation of wild-type GLP-1 and GIP, and the resulting GLP-1 / GIP dual target polypeptide can activate GLP-1 receptor and GIP receptor, respectively, and the GLP-1 / GIP dual target polypeptide has the dual functions of weight loss and blood glucose reduction.

[0042] According to a specific embodiment of the present invention, the GLP-1 / GIP dual target polypeptide further comprises a second polypeptide, the second polypeptide having an amino acid sequence as shown in SEQ ID NO: 1. The specific amino acid sequence of SEQ ID NO: 1 is GPSSGAPPPS.

[0043] According to a specific embodiment of the invention, the C-terminal amino acid of the first polypeptide is linked to the N-terminal amino acid of the second polypeptide.

[0044] According to a specific embodiment of the present invention, the GLP-1 / GIP dual target polypeptide has at least one of the amino acid sequences shown in Table 1.

[0045] [Table 1A]

[0046] [Table 1B]

[0047] [Table 1C]

[0048] [Table 1D]

[0049] According to a specific embodiment of the present invention, the GLP-1 / GIP dual target polypeptide has the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:25-28, SEQ ID NO:37, SEQ ID NO:50-51, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:101-107, or SEQ ID NO:110-112.

[0050] The present invention provides a fusion protein comprising (1) the above GLP-1 / GIP dual target polypeptide and (2) an Fc fragment, in which the C-terminus of the GLP-1 / GIP dual target polypeptide is linked to the N-terminus of the Fc fragment. The fusion protein according to the embodiment of the present invention also has excellent binding activity to the GLP-1 and / or GIP receptor, has dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0051] According to a specific embodiment of the present invention, the Fc fragment is a human IgG4 Fc fragment or a variant thereof. Fusing an immunoglobulin Fc fragment to the GLP-1 / GIP dual target polypeptide can extend the half-life of the fusion protein in vivo.

[0052] According to a specific embodiment of the invention, the Fc fragment comprises the amino acid sequence shown in SEQ ID NO:113.

[0053] [ka]

[0054] According to a specific embodiment of the invention, the fusion protein further comprises a linker peptide, which has the amino acid sequence shown in SEQ ID NO:114.

[0055] [ka]

[0056] According to a specific embodiment of the present invention, the N-terminus of the linker peptide is linked to the C-terminus of the GLP-1 / GIP dual target polypeptide, and the C-terminus of the linker peptide is linked to the N-terminus of the Fc fragment.

[0057] The present invention provides a nucleic acid molecule encoding the above-mentioned GLP-1 / GIP dual target polypeptide or fusion protein. The GLP-1 / GIP dual target polypeptide or fusion protein encoded by the nucleic acid molecule according to the embodiment of the present invention also has excellent binding activity with GLP-1 and / or GIP receptor, has dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0058] The present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the aforementioned nucleic acid molecule encoding a GLP-1 / GIP dual target polypeptide or fusion protein. When the aforementioned nucleic acid molecule is linked to a vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector as long as they can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or these control elements can be exogenous, i.e., these control elements do not have to be derived from the vector itself. Of course, if we dare to say, the nucleic acid molecule is operably linked to the control elements. In this specification, "operably linked" refers to linking an exogenous gene to a vector so that the control elements in the vector, such as the transcriptional control sequence and the translational control sequence, can perform their expected function of regulating the transcription and translation of the exogenous gene.

[0059] According to a specific embodiment of the invention, the expression vector is a eukaryotic expression vector.

[0060] The present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries a nucleic acid molecule encoding the above GLP-1 / GIP dual target polypeptide or fusion protein, or the above expression vector. According to a specific embodiment of the present invention, the recombinant cell can express the GLP-1 / GIP dual target polypeptide or fusion protein, and the GLP-1 / GIP dual target polypeptide and fusion protein have excellent binding activity to GLP-1 and / or GIP receptor, have dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0061] According to a specific embodiment of the invention, the recombinant cell is a mammalian cell, such as a CHO cell.

[0062] According to a specific embodiment of the present invention, recombinant cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0063] The present invention provides a pharmaceutical composition comprising the GLP-1 / GIP dual target polypeptide, the fusion protein, the nucleic acid molecule encoding the GLP-1 / GIP dual target polypeptide or the fusion protein, the expression vector, or the recombinant cell. The pharmaceutical composition may comprise a pharma- ceutical acceptable adjuvant, and the pharma-ceutical acceptable adjuvant comprises at least one of a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent, and the pharmaceutical composition is at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, or a lyophilized preparation. The pharmaceutical composition according to a specific embodiment of the present invention is used to treat diabetes, obesity, fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and other diseases, has the function of long-term weight loss and / or blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0064] The present invention provides the use of said GLP-1 / GIP dual target polypeptide, said fusion protein, said nucleic acid molecule encoding said GLP-1 / GIP dual target polypeptide or fusion protein, said expression vector, and said recombinant cell in the manufacture of a medicament. According to a specific embodiment of the present invention, the medicament is used for controlling or reducing blood glucose and body weight.

[0065] The present invention provides a method for preparing the above-mentioned fusion protein, comprising the steps of 1) constructing the above-mentioned expression vector, and 2) introducing the expression vector into a host cell to obtain a recombinant cell for expressing the above-mentioned fusion protein. The fusion protein prepared according to the method according to a specific embodiment of the present invention has excellent binding activity to GLP-1 and / or GIP receptor, has dual functions of weight loss and blood glucose reduction, and can effectively control or reduce body weight and blood glucose level.

[0066] Furthermore, the "GLP-1 / GIP dual target polypeptide" or "recombinant polypeptide" described in the present invention can be prepared not only by recombinant expression, but also by chemical synthesis. No matter which preparation method is used, as long as it has one of the blood glucose lowering or weight reducing activities described in the present invention, the method is within the protection scope of the present invention.

[0067] According to a specific embodiment of the present invention, recombinant cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0068] The present invention provides a method for reducing blood glucose and / or body weight of a patient, comprising administering to the patient at least one of 1) the GLP-1 / GIP dual target polypeptide, 2) the fusion protein, 3) the nucleic acid molecule encoding the GLP-1 / GIP dual target polypeptide or fusion protein, 4) the expression vector, 5) the recombinant cell, and 6) the pharmaceutical composition. The method according to a specific embodiment of the present invention can effectively and long-term control or reduce the body weight and / or blood glucose level of a patient, and can treat fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, and metabolic syndrome, and other diseases. The "Fc fragment" of the present invention can be a human IgG4 Fc fragment, or a variant of the IgG4 Fc fragment. The variant has one or more amino acid site mutations compared to wild-type IgG4 Fc.

[0069] The names of the GLP-1 / GIP dual target polypeptides of the present invention begin with "CG", e.g., "CG01", "CG02", etc., and the names of the GLP-1 / GIP dual target fusion proteins of the present invention begin with "HEC-G", e.g., "HEC-G01", "HEC-G02", etc.

[0070] The present invention will be described below with reference to specific examples. It should be noted that all experimental methods used in the following examples are conventional methods unless otherwise specified. All materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. These examples are merely illustrative and do not limit the present invention in any way.

[0071] Example 1 This example provides a method for synthesizing the polypeptides of the present invention.

[0072] 1. Solid phase peptide synthesis was carried out on a peptide synthesizer: the resin was added to a 150 ml reactor, then 50 ml of dichloromethane (DCM) was added and the resin was soaked for 2 hours. The resin was washed with N,N-dimethylformamide (DMF) and then drained, which was repeated four times, and then the resin was drained. The first amino acid at the C-terminus of Fmoc (protected form), DCM, and N,N-diisopropylethylamine (DIEA) were weighed and added to the reactor, and then the reactor was placed on a shaker at 30° C. for 2 hours. The mixture was blocked with a methanol solution (methanol:DIEA:DCM=1:1:2) for 0.5 hours, then washed four times with DMF, then drained. A 20% piperidine solution was added to the reactor to remove the Fmoc protecting group. After deprotection, the mixture was washed four times with DMF and then drained.

[0073] 2. The second amino acid at the C-terminus of Fmoc (protected), 1-hydroxybenzotriazole (HOBT), and N,N'-diisopropylcarbodiimide (DIC) were weighed and added to the reactor, then the reactor was placed on a shaker at 30°C for 1 hour. A small amount of resin was taken and tested by ninhydrin method. If the resin was colored, this means that the condensation was incomplete, and the reaction was continued. After the reaction was completed, the resin was washed with DMF four times and then drained. A certain amount of 20% piperidine (piperidine / DMF=1:4) was added to the reactor, then the reactor was placed on a decolorizing shaker and shaken for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, the resin was washed with DMF four times and then drained to check whether the protecting group was removed.

[0074] 3. Amino acids were linked in order according to step 2, and finally, a cleavage reagent was used to remove all the polypeptide protecting groups, and then the polypeptide was cleaved from the resin and sent for purification.

[0075] 4. After the target peptide was synthesized, it was separated from impurities via reverse phase liquid chromatography purification. The recovered target peptide was lyophilized into powder and sent to QC for quality inspection and purity and mass spectrometry identification. After HPLC test, the purity was above 95%. The molecular weight of the peptide identified by mass spectrometry was consistent with the theoretical molecular weight. Table 2 shows the synthesized polypeptide compounds.

[0076] [Table 2]

[0077] Example 2 Determination of in vitro activity of polypeptides HEK293 cells expressing GLP-1R or GIPR were treated with peptide samples, human GLP-1 (purchased) and human GIP (purchased), respectively, and the specific procedures are as follows. 1) Genes GIPR and GLP-1R were optimized and synthesized routinely by Genewiz, the genes were cloned into vector pUC57-Amp, mini-scale recombinant plasmid DNA was prepared, and bacteria harboring the recombinant plasmid were drilled. 2) pUC57-GIPR recombinant plasmid DNA was double-digested with HindIII and EcoRI, and pUC57-GLP-1R was double-digested with HindIII and XhoI. The digestion products were electrophoresed on a 1% agarose gel, the target bands were excised with a clean blade, and then the target fragments were recovered using a gel recovery kit. Specific experimental procedures were performed according to the kit's instructions. 3) The target fragment enzyme digestion recovery product and the vector plasmid pcDNA3.1 fragment were ligated via T4 ligase and transformed into DH5α competent cells, then single colonies were isolated by plating, transformants were selected, and grown for enzyme digestion verification and sequencing verification. 4) 200 mL of the bacterial solution obtained in inoculation step 3) and confirmed to have the fusion protein of the target product by sequencing was used for plasmid extraction. The kit used was the PureLink HiPure Plasmid Maxiprep Kit, and the instructions were followed. After confirming that the plasmid was correct by PCR and enzyme digestion, linearization was performed using pvuI restriction enzyme. Finally, the plasmid was recovered using ethanol precipitation. 5) The host cells were HEK293, and the day before transfection, the cells were cultured at 2 × 10 6 The cells were spread in a 6-well plate at a density of 1000 cells / well, and the volume added was 1 mL / well. The recovered linearized plasmids were transfected into HEK293 cells using Lipofectamine 3000 transfection method, then G418 was added to screen to obtain mixed strains, and single clones were obtained by limiting dilution and isolation, and the activity was tested and verified.

[0078] A cAMP detection kit (Cisbio, 62AM6PEC) was used to detect cAMP produced by the recipient cells according to the steps described in the instruction manual. The specific steps are as follows: 1) Preparation of assay buffer: 500 mM of 4 / 1000 IBMX stock solution was added to complete culture medium (DMEM medium + 10% FBS), and cAMP-d2 standard curve solution and anti-cAMP-cryptate standard curve solution were prepared according to the kit instructions. 2) Human GLP-1 (purchased) and GIP (purchased) test and control samples were diluted to a stock solution with an initial concentration of 500 nM, and then 20 μL was added to 80 μL of gradient serial dilution assay buffer (diluted 5 times) to obtain a total of 8 compound gradients including the stock solutions. 3) Preparation of cell suspension: Cells HEK293-GLP-1R and HEK293-GIPR were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath. If the mixture was not completely thawed within 1.5 min, the cells were added dropwise to a 15 mL centrifuge tube containing 8 mL of warm culture medium on a clean bench, then centrifuged at 900 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium (by pipetting 15 times). 20 μL of the suspension was immediately removed and mixed with an equal volume of trypan blue, and the cells were diluted to 4x10 5 The number of viable cells was counted after dilution to cells / mL. 4) 384-well plate was divided into GLP-1R cell area and GIPR cell area, cell suspension was added to the wells of corresponding area with 5μL per well using 12-channel adjustable dispenser, then serial dilutions of test substance and positive control substance were added to the 384-well plate corresponding to cells with 5μL per well using 12-channel adjustable dispenser (samples of the same concentration were replicated in two parallel wells). Negative control: Assay buffer 10μL / well, set up 3 wells in each 384-well plate, covered with white sealing film, placed in 37℃ constant temperature incubator, and removed after 0.5 hours. 5) cAMP-d2 calibration solution and anti-cAMP-cryptate calibration solution were diluted 20 times with the dissolution buffer of Hi-range kit before use, then mixed equally 1:1 to prepare cAMP detection reagent mixture, 10μL / well of sample group was added to cAMP detection reagent mixture, 5μL of dissolution buffer and 5μL of diluted anti-cAMP-cryptate calibration solution were added to each well of negative control, then the plate was covered with a white lid and placed in the dark at room temperature for 1 hour. 6) The fluorescence values ​​at 665 nm and 620 nm were detected by a multi-function microplate reader. 7) This was used to generate dose response curves, which were then used to calculate EC50 values ​​and compared to each other.

[0079] The specific results are shown in Table 3. All peptides showed strong GIPR agonist activity, but there were some differences in GLP-1R agonist activity. Among them, CG133 and CG134 had relatively weak GLP-1R agonist activity.

[0080] [Table 3]

[0081] Example 3 Construction of expression vectors In this embodiment, the GLP-1 / GIP dual receptor agonist polypeptide is fused with Fc fragment by a linking peptide using molecular cloning method, where the amino acid sequence of wild type GLP-1 is HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G (SEQ ID NO: 115), the amino acid sequence of wild type GIP is YAEGT FISDY SIAMD KIHQQ DFVNW LLAQ (SEQ ID NO: 116), and the GLP-1 / GIP dual receptor agonist polypeptide is obtained by mutating the amino acid sequences of wild type GLP-1 and GIP. The synthesized sequence is double digested and inserted into the same enzyme digestion site of mammalian cell expression vector. Alternatively, site-specific mutation primers were designed based on existing vectors, and a series of mutant vectors were constructed through polymerase chain reaction (PCR), then sent to a sequencing company to confirm that the sequencing was accurate, and the plasmid vector was extracted using OMEGA's Endotoxin Removal Plasmid Mini Kit, catalog number D6950-01, and the plasmid vector was stored at -20°C for later use. Table 4 shows the GLP-1 / GIP dual target polypeptide sequence in the GLP-1 / GIP dual target fusion protein.

[0082] [Table 4A]

[0083] [Table 4B]

[0084] [Table 4C]

[0085] [Table 4D]

[0086] Example 4 Transfection and expression of vectors in cells In this example, CHO-S cells were resuscitated and subcultured to a cell density of approximately 6×10 6 The cells were diluted to 1000 cells / mL. ExpiCHO Fectamine™ CHO Transfection Kit (ThermoFisher Scientific) was used for transfection. When a 50 mL expression system was used as an example, the specific experimental procedures were as follows. 1) Dilute the plasmid in 2mL of OptiPRO™ SFM Complex Formation Medium, the plasmid needed to be filtered and sterilized. The final concentration was 1μg / mL. 2) 1.84 mL of OptiPRO™ SFM Complex Formation Medium was added to 160 μL of ExpiFectamine™ CHO Reagent. 3) Mix the solutions from step 1 and step 2 equally, add them to the cells, mark them respectively, place the mixture on a shaker, and incubate at 37°C, 8% CO 2 The mixture was incubated at 140 rpm for 18 to 22 hours. 4) After culturing, 300 μL of ExpiFectamine™ CHO Enhancer and 12 mL of ExpiCHO™ Feed were added to the transfected cells, respectively. 5) The transfected cells obtained in step 4) were incubated at 37°C, 8% CO 2 After 6 days of cultivation, the cell fermentation broth was harvested for further purification.

[0087] Example 5 Purification and identification of the fusion protein The cell culture medium was centrifuged to collect the supernatant and filtered through a 0.22 μm filter to remove remaining cell debris. The collected cell culture medium was purified using a protein A chromatography column to collect the target peak, and then further purified using anion exchange chromatography. The protein was finally eluted and collected with 0.02 M PBS. The specific steps are as follows: 1) The cell fermentation culture was collected in a 50 mL centrifuge tube and centrifuged at low speed at 1000 rpm for 10 minutes. 2) At the same time, the column and purification system were rinsed with 5 to 10 column volumes of ultrapure water. 3) After centrifugation, the supernatant was filtered using a 0.45 μm ultrafiltration membrane to remove cell sediments. 4) The column was washed with 5 to 10 column volumes of 0.02 M PBS buffer to equilibrate the column. 5) The filtered supernatant was run through the column until all of the sample had passed through the column. 6) The column was then rinsed with 5-10 column volumes of 0.02M PBS buffer until the sample baseline reached "0". 7) The sample was then eluted using 0.1 M acetic acid-sodium acetate solution into a 50 mL centrifuge tube containing 2.5 mL of pH 8.0 Tris-HCl solution. 8) The column was rinsed with 0.1M NaOH solution (5-10 column volumes), then the packing was regenerated and some of the impurity proteins in the column were removed. 9) The column was rinsed with 5 to 10 column volumes of ultrapure water. 10) The above steps 4) to 9) were repeated until all samples were loaded. 11) The system was rinsed with 20% ethanol, and finally, the column was stored in ethanol and then removed. 12) The Protein A pre-packed column was removed from the purification apparatus and replaced with an anion exchange chromatography Q column. 13) The column and purification system were rinsed with 5 to 10 column volumes of ultrapure water. 14) The column was washed and equilibrated with 5-10 column volumes of 0.02M PB buffer. 15) The initially pure sample obtained in step (7) above was diluted until the conductivity was below 5 ms / cm and then the sample was loaded. 16) The column was then washed with 5-10 column volumes of 0.02M PB buffer. 17) The protein sample was eluted with 0.02M PBS buffer. 18) The sample was eluted with 1.5M NaCl solution to remove the dye in the column. 19) The column was rinsed with 0.1 M NaOH solution (5–10 column volumes) to remove some of the impurity proteins in the column. 20) The column was rinsed with 5 to 10 column volumes of ultrapure water. 21) Repeat steps 14) to 20) above until all samples have been loaded. 22) The system and column were rinsed with 20% ethanol until only ethanol remained in the column.

[0088] The samples were quantified using a micro nucleic acid protein analyzer (NanoDrop 2000 / 2000c spectrophotometer) and then detected by 12% SDS-PAGE electrophoresis, and the electrophoretic results showed a single band.

[0089] Example 6 Determination of in vitro activity of fusion proteins HEK293 cells expressing GLP-1R or GIPR were treated with fusion proteins prepared by expressing human GLP-1 (purchased) and GIP (purchased), respectively, and the specific procedures are as follows: 1) Genes GIPR and GLP-1R were optimized and synthesized routinely by Genewiz, the genes were cloned into vector pUC57-Amp, mini-scale recombinant plasmid DNA was prepared, and bacteria harboring the recombinant plasmid were drilled. 2) pUC57-GIPR recombinant plasmid DNA was double-digested with HindIII and EcoRI, and pUC57-GLP-1R was double-digested with HindIII and XhoI. The digestion products were electrophoresed on a 1% agarose gel, the target bands were excised with a clean blade, and then the target fragments were recovered using a gel recovery kit. Specific experimental procedures were performed according to the kit's instructions. 3) The target fragment enzyme digestion recovery product and the vector plasmid pcDNA3.1 fragment were ligated via T4 ligase and transformed into DH5α competent cells, then single colonies were isolated by plating, transformants were selected, and grown for enzyme digestion verification and sequencing verification. 4) 200 mL of the bacterial solution obtained in inoculation step 3) and confirmed to have the fusion protein of the target product by sequencing was used for plasmid extraction. The kit used was the PureLink HiPure Plasmid Maxiprep Kit, and the instructions were followed. After confirming that the plasmid was correct by PCR and enzyme digestion, linearization was performed using pvuI restriction enzyme. Finally, the plasmid was recovered using ethanol precipitation. 5) The host cells were HEK293, and the day before transfection, the cells were cultured at 2 × 10 6 The cells were spread in a 6-well plate at a density of 1000 cells / well, and the volume added was 1 mL / well. The recovered linearized plasmids were transfected into HEK293 cells using Lipofectamine 3000 transfection method, then G418 was added to screen to obtain mixed strains, and single clones were obtained by limiting dilution and isolation, and the activity was tested and verified.

[0090] The cAMP produced by the recipient cells was detected by a cAMP detection kit (Cisbio, 62AM6PEC) according to the steps described in the operating instructions. The specific steps are as follows: 1) Preparation of assay buffer: 500 mM of 4 / 1000 IBMX stock solution was added to complete culture medium (DMEM medium + 10% FBS), and cAMP-d2 standard curve solution and anti-cAMP-cryptate standard curve solution were prepared according to the kit instructions. 2) Human GLP-1 (purchased) and GIP (purchased) test and control samples were diluted to a stock solution with an initial concentration of 500 nM, and then 20 μL was added to 80 μL of gradient serial dilution assay buffer (diluted 5 times) to obtain a total of 8 compound gradients including the stock solutions. 3) Preparation of cell suspension: Cells HEK293-GLP-1R and HEK293-GIPR were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath. If the mixture was not completely thawed within 1.5 min, the cells were added dropwise to a 15 mL centrifuge tube containing 8 mL of warm culture medium on a clean bench, then centrifuged at 900 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium (by pipetting 15 times). 20 μL of the suspension was immediately removed and mixed with an equal volume of trypan blue, and the cells were diluted to 4x10 5 The number of viable cells was counted after dilution to cells / mL. 4) 384-well plate was divided into GLP-1R cell area and GIPR cell area, cell suspension was added to the wells of corresponding area with 5μL per well using 12-channel adjustable dispenser, then serial dilutions of test substance and positive control substance were added to the 384-well plate corresponding to cells with 5μL per well using 12-channel adjustable dispenser (samples of the same concentration were replicated in two parallel wells). Negative control: Assay buffer 10μL / well, set up 3 wells in each 384-well plate, covered with white sealing film, placed in 37℃ constant temperature incubator, and removed after 0.5 hours. 5) cAMP-d2 calibration solution and anti-cAMP-cryptate calibration solution were diluted 20 times with the dissolution buffer of Hi-range kit before use, then mixed equally 1:1 to prepare cAMP detection reagent mixture, 10μL / well of sample group was added to cAMP detection reagent mixture, 5μL of dissolution buffer and 5μL of diluted anti-cAMP-cryptate calibration solution were added to each well of negative control, then the plate was covered with a white lid and placed in the dark at room temperature for 1 hour. 6) The fluorescence values ​​at 665 nm and 620 nm were detected by a multi-function microplate reader. 7) This was used to generate dose response curves, which were then used to calculate EC50 values ​​and compared to each other.

[0091] The specific results are shown in Table 5. In this table, the fusion proteins represented by G04, G05, G12, G27, G28, G30, G42, G49-G51, G58, G62, and G108-G111 did not show agonistic activity, while the other fusion proteins showed agonistic activity. Among these, G06, G20, G23, G34, G35, G37, G38, G48, G66, G67, G72, G74, G99, G101, G113, G115, G121-G127, and G130-G132 had strong in vitro activity.

[0092] [Table 5A]

[0093] [Table 5B]

[0094] [Table 5C]

[0095] [Table 5D]

[0096] Example 7 Assessment of glucose tolerance This example evaluates the effects of HEC-G123, HEC-G128, HEC-G131, and HEC-G132 on glucose tolerance in normal C57BL / 6 mice.

[0097] Experimental Method: Normal C57BL / 6 mice were randomly divided into 6 groups (vehicle group, dulaglutide group, HEC-G123 group, HEC-G128 group, HEC-G131 group, HEC-G132 group) with 8 mice in each group according to blood glucose and body weight. In dulaglutide, HEC-G123, HEC-G128, HEC-G131, and HEC-G132 groups, the mice were subcutaneously injected with the corresponding drugs at a dose of 3 nmol / kg, and in the control group, the corresponding vehicle was subcutaneously injected.

[0098] After single administration for 60 hours, the mice were fasted for 12 hours and allowed to drink water ad libitum. Blood was collected from the tail vein to measure the basal blood glucose level of each group, and then 2 g / kg glucose solution was intraperitoneally injected, and blood glucose was measured 15 minutes, 30 minutes, 60 minutes, and 0 minutes after glucose administration. According to the blood glucose values ​​measured at different time points, blood glucose concentration-time curves were plotted, and the AUC 0~90分was calculated, and the experimental results are shown in Table 6 and Figures 1-2.

[0099] [Table 6]

[0100] Conclusion: HEC-G123, HEC-G128, HEC-G131, and HEC-G132 significantly reduced blood glucose levels in normal C57 mice after single administration for 72 hours. Compared with the positive control dulaglutide, HEC-G123 and HEC-G128 had more significant effects on improving glucose tolerance, while HEC-G131 and HEC-G132 had no significant difference in improving glucose tolerance.

[0101] Example 8 Assessment of glucose tolerance This example evaluates the effects of HEC-G113, HEC-G122, HEC-G126, and HEC-G127 on glucose tolerance in normal C57BL / 6 mice.

[0102] Experimental Method: Normal C57BL / 6 mice were randomly divided into 6 groups (vehicle group, dulaglutide group, HEC-G113 group, HEC-G122 group, HEC-G126 group, HEC-G127 group) with 8 mice in each group according to blood glucose and body weight. In dulaglutide (dulaglutide was purchased from Eli Lilly and Company, the lot number was D256504), HEC-G113, HEC-G122, HEC-G126, and HEC-G127 groups, the corresponding drugs were subcutaneously injected into the mice at a dose of 3 nmol / kg, and in the control group, the corresponding vehicle was subcutaneously injected into the mice.

[0103] After single administration for 60 hours, the mice were fasted for 12 hours and allowed to drink water ad libitum. Blood was collected from the tail vein to measure the basal blood glucose level of each group, and then 2 g / kg glucose solution was intraperitoneally injected, and blood glucose was measured 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 0 minutes after glucose administration. According to the blood glucose values ​​measured at different time points, blood glucose concentration-time curves were plotted, and the AUC 0~90分 was calculated, and the experimental results are shown in Table 7 and Figures 3-4.

[0104] [Table 7]

[0105] Conclusion: HEC-G113, HEC-G122, HEC-G126, and HEC-G127 significantly reduced blood glucose levels in normal C57 mice after single administration for 72 hours. The glucose tolerance improving effect of each group was better than that of the positive control dulaglutide.

[0106] Example 9 Evaluation of the in vivo efficacy of nine db / db mouse models This example evaluates the glucose effect of HEC-G20 on the db / db mouse model.

[0107] Methods: 7-8 week old db / db mice were randomly divided into 3 groups (model group, semaglutide group, HEC-G20 group) with 9 mice in each group according to blood glucose and body weight. In the semaglutide (semaglutide was purchased from Novo Nordisk, the lot number was JP52092) and HEC-G20 groups, the corresponding drugs were subcutaneously administered to the mice at a dose of 10 nmol / kg per group, and in the model group, the corresponding vehicle was subcutaneously injected. The semaglutide group was administered once a day, and the HEC-G20 group was administered twice a week for a total of 4 weeks, and the blood glucose of the animals was tested before each administration.

[0108] Experimental results: The HEC-G20 group could significantly reduce blood glucose after administration, and the blood glucose level reached the lowest level at 7 hours, and the effect was similar to that of the positive control semaglutide at the same dose. Compared with the model group, the blood glucose level of mice that underwent long-term repeated administration of the HEC-G20 group was significantly lower and remained stable for a long time, and its blood glucose lowering effect was similar to that of the semaglutide group, as shown in Table 8 and Figures 5 to 6.

[0109] [Table 8]

[0110] Conclusion: Long-term administration of HEC-G20 can significantly improve blood glucose levels in type II diabetic db / db mice.

[0111] Example 10 Evaluating the in vivo validity of the DIO model In this example, the effects of long-term repeated administration of HEC-G115 and HEC-G124 on body weight and food intake in obese mice as a DIO model were evaluated.

[0112] Experimental method: C57 / BL6 mice were randomly divided into normal group NFD and model group HFD at the age of 5 weeks, the normal group was fed with normal maintenance diet, while the model group was fed with high fat diet D12492. The changes in body weight and food intake of the mice were monitored every 3 weeks. After 16 weeks of feeding, the body weights of the mice in the model group and normal group were (47.9±3.4)g and (29.6±1.5)g, respectively, and the difference between the two groups was statistically significant. The normal group was divided into the control group, and the successfully modeled model group mice were divided into the vehicle group, semaglutide group, HEC-G115 group, and HEC-G124 group, with 10 mice in each group. In the semaglutide, HEC-G115, and HEC-G124 groups, the corresponding drugs were subcutaneously injected into each group, and PBS was subcutaneously injected into the vehicle group. The first administration was for 7 days at a dose of 10 nmol / kg in each group, and then observed every 3 days. Body weight and food intake were measured before each administration. After 3 weeks of administration, intraperitoneal glucose tolerance test was performed. After the last administration for 72 hours, samples were collected, liver mass was recorded, and liver pathological status and blood biochemical index were detected in each group. The test results are shown in Table 9 to Table 12 and Figure 7 to Figure 8.

[0113] [Table 9]

[0114] [Table 10]

[0115] [Table 11]

[0116] [Table 12]

[0117] Results: After 4 weeks, the weight loss and feeding inhibition effects of HEC-G115 and HEC-G124 administered once every 3 days were comparable to those of control semaglutide administered once daily at the same dose.HEC-G115 and HEC-G124 also significantly improved liver function and blood lipids in DIO mice, with effects similar to those of semaglutide.

[0118] Throughout this specification, reference to "one embodiment," "some embodiments," "one example," "particular example," or "some examples" means that the particular feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the disclosure. As used herein, graphic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples. In addition, one of ordinary skill in the art may incorporate and combine different embodiments, examples, or characteristics thereof, as long as they are not mutually inconsistent.

[0119] While embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that the above embodiments should not be construed as limiting the present disclosure, and that changes, substitutions, and modifications may be made in the embodiments without departing from the spirit, principle, and scope of the present disclosure.

Claims

1. The amino acid sequence: X 1 X 2 X 3 GTFX 4 SDYSX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 X 19 A GLP-1 / GIP dual target polypeptide comprising a first polypeptide having the formula: 1 is Y or H, and X 2 is A, G, or S, and X 3 is E or Q, and X 4 is I or T, and X 5 is I or K, and X 6 is A, Y, L, or I, and X 7 is M or L, and X 8 is D or E, and X 9 is K or E, and X 10 is I, Q, K, E, or L, and X 11 is H, A, or R, and X 12 is A, Q, or V, and X 13 is K, Q, R, or H, and X 14 is D, E, A, or L, and X 15 is V or I, and X 16 is N, E, D, or Q, and X 17 is L, I, K, or V, and X 18 is A, E, or K, and X 19 is Q or G, X 1 ~X 19 But X 1 is Y and X 2 is A and X 3 is E and X 4 is I and X 5 is I and X 6 is A and X 7 is M and X 8 is D and X 9 is K and X 10 is I and X 11 is H and X 12 is Q and X 13 is Q and X 14 is D and X 15 is V and X 16 is N and X 17 is L and X 18 is A and X 19 does not simultaneously satisfy that GLP-1 / GIP dual target polypeptide.

2. 1. The GLP-1 / GIP dual target polypeptide, comprising the amino acid sequence: X 1 X 2 EGTFTSDYSIX 6 LDKX 10 AQX 13 X 14 FX 15 X 16 WLX 17 AX 19 wherein X 1 is Y or H, and X 2 is A, G, or S, and X 6 is A, Y, or L, and X 10 is I, Q, K, or L, and X 13 is Q or R, and X 14 is D, E, or A, and X 15 is V or I, and X 16 is E, D, or Q, and X 17 is L, I, or K, and X 19 is Q or G, the first polypeptide does not contain the amino acid sequence YAEGTFISDYSIAMDKIHQQDFVNWLLAQ (SEQ ID NO: 122) The GLP-1 / GIP dual target polypeptide according to claim 1,

3. 3. The GLP-1 / GIP dual target polypeptide according to claim 1 or 2, further comprising a second polypeptide having the amino acid sequence shown in SEQ ID NO:

1.

4. 4. The GLP-1 / GIP dual target polypeptide according to claim 1, wherein the C-terminus of the first polypeptide is linked to the N-terminus of the second polypeptide.

5. A GLP-1 / GIP dual target polypeptide according to any one of claims 1 to 4, characterized in that it has at least one of the following amino acid sequences: SEQ ID NOs: 2 to 112, SEQ ID NOs: 117 to 121.

6. The GLP-1 / GIP dual target polypeptide according to claim 5, characterized in that it has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NOs: 25 to 28, SEQ ID NO: 37, SEQ ID NOs: 50 to 51, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NOs: 101 to 107, or SEQ ID NOs: 110 to 112.

7. (1) a GLP-1 / GIP dual target polypeptide according to any one of claims 1 to 6, and (2)Fc fragment wherein the C-terminus of the GLP-1 / GIP dual target polypeptide is linked to the N-terminus of the Fc fragment.

8. 8. The fusion protein according to claim 7, wherein the Fc fragment is an Fc fragment of human IgG4 or a variant thereof.

9. The fusion protein according to claim 7 or 8, characterized in that the Fc fragment comprises the amino acid sequence shown in SEQ ID NO:

113.

10. A fusion protein according to any one of claims 7 to 9, characterized in that it further comprises a linker peptide having the amino acid sequence shown in SEQ ID NO:

114.

11. The fusion protein according to any one of claims 7 to 10, characterized in that the N-terminus of the linker peptide is linked to the C-terminus of the GLP-1 / GIP dual target polypeptide, and the C-terminus of the linker peptide is linked to the N-terminus of the Fc fragment.

12. A nucleic acid molecule encoding a GLP-1 / GIP dual target polypeptide according to any one of claims 1 to 6 or a fusion protein according to any one of claims 7 to 11.

13. An expression vector comprising the nucleic acid molecule of claim 12, characterized in that it is a eukaryotic expression vector.

14. A pharmaceutical composition comprising a GLP-1 / GIP dual target polypeptide described in any one of claims 1 to 6, a fusion protein described in any one of claims 7 to 11, a nucleic acid molecule described in claim 12, or an expression vector described in claim 13.

15. A GLP-1 / GIP dual target polypeptide according to any one of claims 1 to 6, a fusion protein according to any one of claims 7 to 11, a nucleic acid molecule according to claim 12, or an expression vector according to claim 13, for use in treating diabetes, obesity, fatty liver disease, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, dyslipidemia, and metabolic syndrome in a subject.

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