A dual-target receptor agonist fusion protein and its application

Abstract

The present invention discloses a dual-target receptor agonist fusion protein and its application, and relates to the field of biomedical engineering. The fusion protein is a dual-target receptor agonist fusion protein (GLP-1 and GIP, GLP-1 and GCG) fused with a dual natural N-terminus of an antibody Fc fragment, which can be quickly prepared using a more efficient and low-cost bacterial expression system and has high in vitro agonist activity and in vivo activity. The fusion protein does not require purification and can be obtained in one step by SpyCatcher / SpyTag self-ligation, and is fused with a human antibody Fc fragment, which can improve mouse glucose tolerance well, and is superior to tirzepatide in reducing the weight of obese mice, inhibiting food intake, improving mouse lipid and liver metabolism, and improving liver cavitation. The fusion protein can be used to produce a new drug of an antibody Fc fragment-modified polypeptide / protein with a dual natural N-terminus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a dual-target receptor agonist fusion protein and applications thereof, specifically, to a GLP-1 and GIP dual-target receptor agonist fusion protein, a GLP-1 and GCG dual-target receptor agonist fusion protein and applications thereof, and more specifically, to the design of the GLP-1 and GIP dual-target receptor agonist fusion protein, a GLP-1 and GCG dual-target receptor agonist fusion protein, nucleic acid molecules encoding the GLP-1 and GIP dual-target receptor agonist fusion protein, a GLP-1 and GCG dual-target receptor agonist fusion protein, expression vectors, engineered bacteria, and applications of the GLP-1 and GIP dual-target receptor agonist fusion protein, the GLP-1 and GCG dual-target receptor agonist fusion protein, and nucleic acid molecules and engineered bacteria thereof in preparing drugs. Background Art

[0002] Glucagon-like peptide-1 receptor (GLP-1) is an incretin that can promote the synthesis and secretion of insulin by pancreatic β cells (Drucker et al., Nature Medicine, 1996, 2, 11), induce β cell proliferation and inhibit cell apoptosis (Kulkarni et al., Diabetologia, 2017, 60, 1442-1453), and inhibit the secretion of glucagon by α cells (Drucker et al., Cell Metabolism, 2018, 3, 714). It is currently one of the most effective drugs for the treatment of type 2 diabetes mellitus (T2DM) in the world. GLP-1 also has physiological functions such as slowing gastric emptying and inhibiting gastrointestinal motility (Iwasaki et al., Nature Communications, 2018, 9, 133), has a good weight loss effect, and has become a research hotspot for anti-obesity drugs. Since the half-life of natural GLP-1 in the body is only 2 to 3 minutes and it cannot be made into a drug, long-acting modification is one of the key research points for the drug development of GLP-1.

[0003] Glucose-dependent insulinotropic polypeptide receptor (GIP) is another incretin that has the function of promoting insulin secretion similar to GLP-1. In addition, the signal binding pathway activated by the interaction between GIP and GIPR can block vomiting and reduce other negative side effects of GLP-1 receptor (GLP-1R) activation (Hayes et al., Diabetes, 2021, 70, 2545-2553). Glucagon (GCG) is a pancreatic hormone secreted by pancreatic α cells that stimulates fat decomposition in adipose tissue, reduces food intake, and increases satiety and energy expenditure (Prato et al., 2021, Obesity Reviews. 2022, 23:e13372). In recent years, with the development and marketing of various glucagon-like peptide-1 receptor agonists (GLP-1RAs), a new generation of dual / multi-target receptor agonists have shown better administration effects, lower blood sugar levels, and less food intake, and have become the mainstream of GLP-1RAs research and development. Tirzepatide, a new generation of dual / multi-target receptor agonists, was approved for marketing by the US Food and Drug Administration (FDA) in May 2022. As a single-molecule dual-target GLP-1 / GIP (glucose-dependent insulinotropic polypeptide) agonist, Tirzepatide has shown superior hypoglycemic and weight loss effects than semaglutide (Coskun et al., Molecular metabolism, 2018, 18: 3-14). However, the design of such single-molecule agonists relies on the high homology of peptides, and it is difficult to accurately control the affinity of the molecule for different targets during the design process, making it difficult to predict the distribution of the molecule's efficacy for each target (Frias et al., New England Journal of Medicine, 2021, 385(6): 503-515). In addition, gastrointestinal peptides such as GLP-1, GIP, and GCG need to retain their natural N-termini to ensure activity (Zhang et al., Nature, 2017, 546(7657): 248-253), so the gene fusion multi-target receptor agonist expression strategy also has limitations.

[0004] Spy chemistry is a type of genetically encoded click chemistry based on two genetically encoded elements, SpyCatcher and SpyTag, which can spontaneously form polymers without complex post-translational modifications. This process does not require the use of chemical reagents, has a fast reaction, and is conducted under mild conditions, and has broad application prospects (Zakeri et al., Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(12): E690-E697).

[0005] In summary, there is still room for improvement in the design and preparation of dual (multi) target receptor agonists based on GLP-1. Therefore, this field still needs to develop long-acting multi-target receptor agonists with better blood sugar-lowering and weight-reducing effects and a wider range of indications. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a dual-target receptor agonist fusion protein and its application.

[0007] The present invention is based on the inventor's discovery and understanding of the following problems:

[0008] The dose-dependent side effects and short half-life of GLP-1 limit the clinical application of GLP-1. Combining with other targets such as GIP and GCG to form multi- / dual-target receptor agonists and prolonging half-life is currently the main research direction of GLP-1RAs. The multi- / dual-target receptor agonists currently in clinical research mainly design two polypeptides into a chimeric peptide that activates two / multiple targets at the same time, such as tirzepatide, a GLP-1 / GIP dual-target receptor agonist approved by the FDA in May 2022, and cotadutide and mazdutide, two GLP-1 / GCG dual-target receptor agonists in clinical phase III. This strategy is limited by the homologous sequence of the polypeptide and it is difficult to regulate its efficacy on each target. In addition, polypeptides such as GLP-1, GIP and GCG need to expose the natural N-terminus to retain their high activity on the receptor, which increases the difficulty of designing and preparing multiple targets.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The first aspect of the present invention provides a novel dual-target receptor agonist fusion protein, and more specifically, provides an antibody Fc fragment-modified dual-target receptor agonist fusion protein, the structure of which is shown in the following formula I:

[0011] abcdef (Ⅰ)

[0012] In the formula, each "-" is independently a connecting peptide or a peptide bond or an isopeptide bond;

[0013] a is a GLP-1 polypeptide, a GIP polypeptide or a GCG polypeptide; b is an Fc fragment; c is a SpyCatcher peptide; d is a SpyTag peptide; e is an ELP peptide, which serves as a third connecting peptide; f is a GLP-1 polypeptide, a GIP polypeptide or a GCG polypeptide; and a and f are not the same polypeptide at the same time.

[0014] Furthermore, a is a GLP-1 polypeptide, and f is a GIP polypeptide or a GCG polypeptide; or, a is a GIP polypeptide or a GCG polypeptide, and f is a GLP-1 polypeptide.

[0015] Among them, a and b are connected by a first connecting peptide, b and c are connected by a second connecting peptide; c and d are connected by an isopeptide bond; d, e and f are connected in sequence by peptide bonds.

[0016] Furthermore, the C-terminus of a is connected to the N-terminus of the first connecting peptide; the C-terminus of the first connecting peptide is connected to the N-terminus of b; the C-terminus of b is connected to the N-terminus of the second connecting peptide; the C-terminus of the second connecting peptide is connected to the N-terminus of c; the N-terminus of d is connected to the C-terminus of e; and the N-terminus of e is connected to the C-terminus of f.

[0017] Furthermore, the amino acid sequence of the GLP-1 polypeptide is shown in SEQ ID NO: 1; the amino acid sequence of the GIP polypeptide is shown in SEQ ID NO: 2; and the amino acid sequence of the GCG polypeptide is shown in SEQ ID NO: 3.

[0018] In some embodiments, the GLP-1 polypeptide, GIP polypeptide and GCG polypeptide are amino acid sequences having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0019] In some embodiments, the GLP-1 polypeptide, GIP polypeptide and GCG polypeptide have no more than 6, 5, 4, 3, 2, or 1 mutations relative to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 while retaining the basic biological activity of the polypeptide before mutation.

[0020] In some embodiments, the GLP-1 polypeptide, GIP polypeptide and GCG polypeptide may be peptides having good binding activity to the GLP-1 receptor, GIP receptor or GCG receptor, such as Cotadutide (Henderson et al., Diabetes, Obesity and Metabolism, 2016, 18, 1176-1190), Retatrutide (Coskun et al., Cell Metabolism, 2022, 34, 1234-1247), and Tirzepatide.

[0021] Furthermore, the Fc fragment is selected from the Fc fragment of humanized IgG4.

[0022] In some embodiments, the amino acid sequence of the Fc fragment is shown in SEQ ID NO:4.

[0023] In some embodiments, the SpyCatcher peptide and SpyTag peptide are genetically editable Spy-chemistry peptides, and the SpyCatcher peptide and SpyTag peptide are original sequences, or are variants of the SpyCatcher peptide and SpyTag peptide.

[0024] In some embodiments, the SpyCatcher peptide is an N-terminal truncated version (SpyCatcher (ΔN)), and its amino acid sequence is shown in SEQ ID NO: 8; the SpyTag peptide is an original sequence, and its amino acid sequence is shown in SEQ ID NO: 9.

[0025] In some embodiments, the first connecting peptide and the second connecting peptide each independently comprise an amino acid sequence selected from the group consisting of: shown in SEQ ID NO: 5: GGGGSGGGGSGGGGSA and shown in SEQ ID NO: 6: GSGGSG.

[0026] In some embodiments, the amino acid sequence of the ELP peptide is shown in SEQ ID NO:7.

[0027] In the second aspect of the present invention, a nucleic acid molecule is proposed. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein required for forming the GLP-1 and GIP dual-target receptor agonist fusion protein, and the fusion protein required for the GLP-1 and GCG dual-target receptor agonist fusion protein. The nucleic acid molecule according to an embodiment of the present invention encodes the fusion protein required for forming the GLP-1 and GIP dual-target receptor agonist fusion protein, and the fusion protein required for the GLP-1 and GCG dual-target receptor agonist fusion protein has excellent binding activity with GLP-1 and / or GIP and / or GCG receptors, has the dual functions of weight loss and blood sugar reduction, and can effectively reduce the body weight and blood sugar level of mice.

[0028] Furthermore, the nucleic acid molecule includes the nucleic acid molecule shown at 589-1704 bp in SEQ ID NO: 14 and the nucleic acid molecule shown at 589-987 bp in SEQ ID NO: 17, or the nucleic acid molecule shown at 589-1737 bp in SEQ ID NO: 16 and the nucleic acid molecule shown at 589-954 bp in SEQ ID NO: 15; and is used to encode the fusion protein required to form a GLP-1 and GIP dual-target receptor agonist fusion protein.

[0029] The nucleic acid molecule includes the nucleic acid molecule shown at 589-1704 bp in SEQ ID NO: 14 and the nucleic acid molecule shown at 589-948 bp in SEQ ID NO: 19, or the nucleic acid molecule shown at 589-1698 bp in SEQ ID NO: 18 and the nucleic acid molecule shown at 589-954 bp in SEQ ID NO: 15; and is used to encode the fusion protein required to form a GLP-1 and GCG dual-target receptor agonist fusion protein.

[0030] In the third aspect of the present invention, the present invention provides an expression cassette or an expression vector. According to an embodiment of the present invention, the expression cassette comprises the nucleic acid molecule described in the second aspect; the expression vector comprises the nucleic acid molecule described in the second aspect or the above expression cassette. According to an embodiment of the present invention, the expression vector is a prokaryotic expression vector.

[0031] In the fourth aspect of the present invention, the present invention proposes an engineered bacterium. According to an embodiment of the present invention, the nucleic acid molecule described in the second aspect or the expression cassette or expression vector described in the third aspect is carried. The engineered bacterium according to the embodiment of the present invention can express fusion proteins containing GLP-1, GIP and GCG peptides respectively, and further form GLP-1 and GIP dual-target receptor agonist fusion proteins and GLP-1 and GCG dual-target receptor agonist fusion proteins through Spy-chemistry peptide biocoupling. The GLP-1 and GIP dual-target receptor agonist fusion proteins and GLP-1 and GCG dual-target receptor agonist fusion proteins have excellent binding activity with GLP-1 and / or GIP, GLP-1 / and / or GCG receptors, have the dual functions of weight loss and blood sugar reduction, and can effectively control or reduce body weight and blood sugar levels.

[0032] In the fifth aspect of the present invention, a pharmaceutical composition is proposed. According to an embodiment of the present invention, it comprises the GLP-1 and GIP dual-target receptor agonist fusion protein, the GLP-1 and GCG dual-target receptor agonist fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette or expression vector described in the third aspect, or the engineered bacteria described in the fourth aspect. The pharmaceutical composition may include: a pharmaceutically acceptable adjuvant, wherein the pharmaceutically acceptable adjuvant includes at least one of a stabilizer, a wetting agent, an emulsifier, a binder and an isotonic agent; the pharmaceutical composition is in the form of at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension and a dynamic preparation. The pharmaceutical composition according to an embodiment of the present invention has a long-lasting function of reducing weight and / or lowering blood sugar, and can effectively control or reduce body weight and blood sugar levels.

[0033] In the sixth aspect of the present invention, the present invention proposes the use of the GLP-1 and GIP dual-target receptor agonist fusion protein, the GLP-1 and GCG dual-target receptor agonist fusion protein, the nucleic acid molecule, the expression cassette or expression vector, or the engineered bacteria in the fourth aspect in the preparation of a drug. According to an embodiment of the present invention, the drug is used to control or reduce blood sugar and / or body weight; the drug is used to prevent and / or treat at least one of type 2 diabetes, obesity, metabolic dysfunction-related fatty hepatitis and dyslipidemia, and metabolic syndrome.

[0034] In the seventh aspect of the present invention, the present invention proposes a method for preparing the new dual-target receptor agonist fusion protein described in the first aspect. According to an embodiment of the present invention, the method includes: 1) constructing the nucleic acid molecule described in the second aspect; 2) introducing the expression vector into a host cell to obtain an engineered bacterium to express the fusion protein, and the fusion protein forms a GLP-1 and GIP dual-target receptor agonist fusion protein and a GLP-1 and GCG dual-target receptor agonist fusion protein according to the Spy-chemistry peptide biocoupling reaction. The fusion protein prepared by the method according to the embodiment of the present invention has excellent binding activity with GLP-1 and / or GIP, GLP-1 / and / or GCG receptors, has the dual functions of hypoglycemic and weight loss, and can effectively control or reduce body weight and blood sugar levels.

[0035] In the eighth aspect of the present invention, a method for reducing blood sugar and / or body weight of a patient is proposed. According to the embodiment, it includes using at least one of the following to the patient: 1) GLP-1 and GIP dual-target receptor agonist fusion protein, GLP-1 and GCG dual-target receptor agonist fusion protein described in the first aspect; 2) nucleic acid molecule described in the second aspect; 3) expression cassette or expression vector described in the third aspect; 4) engineered bacteria described in the fourth aspect; and 5) pharmaceutical composition described in the fifth aspect. The method according to the embodiment of the present invention can effectively control or reduce the patient's weight and / or blood sugar level for a long time.

[0036] Compared with the prior art, the present invention has the following advantages and effects:

[0037] The present invention provides a dual-target receptor agonist fusion protein, which is a dual-target receptor agonist fusion protein (GLP-1 and GIP, GLP-1 and GCG) fused with a dual natural N-terminus of an antibody Fc fragment, which can be rapidly prepared using a more efficient and low-cost bacterial expression system and has high in vitro agonist activity and in vivo activity. The fusion protein does not require purification and can be obtained in one step by SpyCatcher / SpyTag self-ligation (GLP-1 and GIP, GLP-1 and GCG), and is fused with a human antibody Fc fragment, which can well improve mouse glucose tolerance, and is superior to tirzepatide (Lilly) in reducing the weight of obese mice, inhibiting food intake, improving mouse lipid and liver metabolism, and improving liver cavitation. The fusion protein can be used to produce a new drug of an antibody Fc fragment-modified polypeptide / protein with dual natural N-termini. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0039] Figure 1These are the SDS-PAGE results of dual-target receptor agonist fusion proteins prepared based on icSAT purification and Spy-chemistry biocoupling and purified using Protein A; among them, A: GLP-1 / GIP dual-target receptor agonist fusion protein, B: GIP / GLP-1 dual-target receptor agonist fusion protein, C: GLP-1 / GCG dual-target receptor agonist fusion protein, D: GCG / GLP-1 dual-target receptor agonist fusion protein.

[0040] Figure 2 It is the molecular weight measurement result of GLP-1 / GIP dual-target receptor agonist fusion protein; wherein, A: GLP-1 / GIP dual-target receptor agonist fusion protein, B: GIP / GLP-1 dual-target receptor agonist fusion protein, C: GLP-1 / GCG dual-target receptor agonist fusion protein, D: GCG / GLP-1 dual-target receptor agonist fusion protein.

[0041] Figure 3 These are the affinity test results of the antibody Fc fragment of the dual-target receptor agonist fusion protein and human FcRn; wherein, A: GLP-1 / GIP dual-target receptor agonist fusion protein, B: GIP / GLP-1 dual-target receptor agonist fusion protein, C: GLP-1 / GCG dual-target receptor agonist fusion protein, D: GCG / GLP-1 dual-target receptor agonist fusion protein; E: affinity test results of the antibody Fc fragment of dulaglutide (Lilly) and human FcRn.

[0042] Figure 4 These are the results of in vitro agonist activity determination of dual-target receptor agonist fusion proteins on GLP-1R, GIPR or GCGR; among them, A and B: the results of in vitro agonist activity determination of GLP-1 / GIP dual-target receptor agonist fusion protein (i.e. GLP-1-Fc-Spy-GIP) and GIP / GLP-1 dual-target receptor agonist fusion protein (i.e. GIP-Fc-Spy-GLP-1) on GLP-1R and GIPR; C and D: the results of in vitro agonist activity determination of GLP-1 / GCG dual-target receptor agonist fusion protein (i.e. GLP-1-Fc-Spy-GCG) and GCG / GLP-1 (i.e. GCG-Fc-Spy-GLP-1) dual-target receptor agonist fusion protein on GLP-1R and GCGR.

[0043] Figure 5 This is the scan result of liver tissue sections of ob / ob mice after long-term drug administration. DETAILED DESCRIPTION

[0044] The present invention is described in further detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be realized or understood by those skilled in the art with reference to the prior art. The reagents or instruments used that do not indicate the manufacturer are considered to be conventional products that can be purchased commercially. The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The primers used are all synthesized by Shanghai Shenggong Biotechnology.

[0045] As used herein, the terms "native," "wild type," or "WT" refer to a protein or polypeptide that occurs naturally or can be isolated from the environment without any genetically engineered mutations.

[0046] As used herein, the term "substantial biological activity" refers to a polypeptide that, after mutation, retains at least a portion (e.g., not less than about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) or all of its biological activity before mutation.

[0047] As used herein, the term "substitution" or "mutation" refers to replacing one amino acid in a polypeptide with another amino acid.

[0048] As used herein, amino acid substitutions are indicated by a first letter followed by a number followed by a second letter. The first letter indicates the amino acid in the wild-type protein or polypeptide; the number refers to the position of the substituted amino acid; and the second letter indicates the amino acid that replaces the wild-type amino acid.

[0049] Herein, a deletion at the amino terminus of a protein or polypeptide is indicated by ΔN followed by the name of the protein or polypeptide.

[0050] As used herein, "identity" or "homology" generally refers to the sequence similarity between two peptides or proteins or between two nucleic acid molecules. "Identity" or "homology" percentage refers to the percentage of identical residues between amino acids or nucleic acids in the compared molecules and is calculated based on the size of the smallest molecule in the molecules being compared.

[0051] Herein, "Fc" or "Fc fragment" is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The antibody Fc fragment may be a native Fc fragment or a variant Fc fragment.

[0052] In the fusion protein provided by the present invention, a natural antibody Fc fragment or an Fc fragment variant can be used. In a preferred embodiment, an Fc fragment variant is used. During the research process of the present invention, it was found that the antibody Fc fragment used can naturally form a dimer.

[0053] Herein, "dimer" refers to a protein dimer, which is composed of two protein (polypeptide) molecules and is a quaternary structure of a protein. The two proteins (polypeptides) that make up the dimer can be called monomer molecules or monomer proteins of the dimer. Dimers may include two cases of "homodimers" and "heterodimers". Among them, homodimers are composed of two identical monomer molecules; heterodimers are composed of two different monomer molecules. It is generally believed that homodimers have a simpler preparation method than heterodimers because only one peptide or protein needs to be synthesized. In the present invention, when the Fc fragment variant provided by the present invention is used, it has the ability to form homodimers; the fusion protein provided by the present invention has the ability to form homodimers.

[0054] Herein, "connector peptide" or "peptide linker" refers to a single amino acid or polypeptide sequence that connects two proteins (polypeptides) together. The length of the connecting peptide can be about 1-40 amino acids, including, for example, repeated proline and threonine, repeated alanine, glycine and serine. Common connecting peptides include flexible connecting peptides, such as a combination of alanine, glycine and serine, such as (GGGGS) 3A and GGGGS; rigid connecting peptides, PT-type connecting peptides with proline and threonine.

[0055] Herein, the GLP-1 and GIP dual-target receptor agonist fusion protein refers to a dual-target receptor agonist fusion protein containing two peptide components, GLP-1 and GIP, that is, a GLP-1 / GIP dual-target receptor agonist fusion protein in which the C-terminus of the GLP-1 peptide is connected to the N-terminus of the first connecting peptide and the C-terminus of the GIP peptide is connected to the N-terminus of the third connecting peptide, and a GIP / GLP-1 dual-target receptor agonist fusion protein in which the C-terminus of the GIP peptide is connected to the N-terminus of the first connecting peptide and the C-terminus of the GLP-1 peptide is connected to the N-terminus of the third connecting peptide. White; GLP-1 and GCG dual-target receptor agonist fusion protein refers to a dual-target receptor agonist fusion protein containing two peptide components, GLP-1 and GCG, that is, a GLP-1 / GCG dual-target receptor agonist fusion protein in which the C-terminus of the GLP-1 peptide is connected to the N-terminus of the first connecting peptide and the C-terminus of the GCG peptide is connected to the N-terminus of the third connecting peptide, and a GCG / GLP-1 dual-target receptor agonist fusion protein in which the C-terminus of the GCG peptide is connected to the N-terminus of the first connecting peptide and the C-terminus of the GLP-1 peptide is connected to the N-terminus of the third connecting peptide.

[0056] In the figure of Example 1, ES: cell lysate supernatant; EP: cell lysate precipitate; ES1, ES2: cell lysate supernatant 1, 2, Mix: a mixture of ES1 and ES2; ESS: supernatant after Mix was aggregated by adding salt; ESP: precipitate after Mix was aggregated by adding salt; CP: precipitate after ESP was cut; CS: supernatant after ESP was cut; among them, ES1~CP were all diluted 10 times, and 10*CS was not diluted; M: protein marker; BSA: bovine serum albumin standard.

[0057] The vectors used for expression constructs of the present invention include vectors that can replicate autonomously in host cells, such as plasmid vectors; and vectors that can be integrated into host cell DNA and replicated together with host cell DNA. In a specific embodiment, the expression construct of the present invention is derived from pET30a (+) of Novagen. Host cells used to express the fusion protein of the present invention include prokaryotes, yeast and higher eukaryotic cells. Exemplary prokaryotes include Escherichia coli ( Escherichia ), Bacillus ( Bacillus ), Pseudomonas ( Pseudomonas ) and Streptomyces ( Streptomyces ). In a preferred embodiment, the host cell is an Escherichia cell, preferably an Escherichia coli. In a specific embodiment of the present invention, the host cell used is an Escherichia coli BL21 (DE3) strain cell (Novagen).

[0058] Example 1: Preparation of GLP-1 and GIP dual-target receptor agonist fusion protein and GLP-1 and GCG dual-target receptor agonist fusion protein based on icSAT purification and Spy-chemistry coupling

[0059] This embodiment provides a method for biosynthesizing a dual-target receptor agonist fusion protein containing GLP-1.

[0060] The expression vector used in this example to synthesize the GLP-1 and GIP dual-target receptor agonist fusion protein is pET30a-EFK8- Mt ΔI-CM(m2)-GLP-1-Fc-SpyCatcher(ΔN) and pET30a-EFK8- Mt ΔI-CM(WT)-GIP-ELP 15 -SpyTag, pET30a-EFK8- Mt ΔI-CM(WT)-GIP-Fc-SpyCatcher(ΔN) and pET30a-EFK8- Mt ΔI-CM(m2)-GLP-1-ELP 15-SpyTag. The expression vector used to synthesize the GLP-1 and GCG dual-target receptor agonist fusion protein in this example is pET30a-EFK8- Mt ΔI-CM(m2)-GLP-1-Fc-SpyCatcher(ΔN) and pET30a-EFK8- Mt ΔI-CM(m2)-GCG-ELP 15 -SpyTag, pET30a-EFK8- Mt ΔI-CM(m2)-GCG-Fc-SpyCatcher(ΔN) and pET30a-EFK8- Mt ΔI-CM(m2)-GLP-1-ELP 15 -SpyTag. The target fragment was inserted into the vector pET30a(+) (purchased from Novagen) N Ⅰ and XOt In this example, the fusion protein EFK8- Mt The coding gene sequence of ΔI-CM(m2)-GLP-1-Fc-SpyCatcher(ΔN) is shown in SEQ ID NO: 14. Fusion protein EFK8- Mt ΔI-CM(WT)-GIP-ELP 15 -The coding gene sequence of SpyTag is shown in SEQ ID NO: 17. Fusion protein EFK8- Mt The coding gene sequence of ΔI-CM (WT)-GIP-Fc-SpyCatcher (ΔN) is shown in SEQ ID NO: 16. Fusion protein EFK8- Mt ΔI-CM(m2)-GLP-1-ELP 15 -The coding gene sequence of SpyTag is shown in SEQ ID NO: 15. Fusion protein EFK8- Mt ΔI-CM(m2)-GCG-ELP 15 -The coding gene sequence of SpyTag is shown in SEQ ID NO: 19. Fusion protein EFK8- Mt The coding gene sequence of ΔI-CM(m2)-GCG-Fc-SpyCatcher(ΔN) is shown in SEQ ID NO: 18, wherein the initial ATG and the final TGA are the start codon and the stop codon, respectively.

[0061] The salinity-induced self-assembly peptide used in this example is EFK8, whose amino acid sequence is shown in SEQ ID NO: 11; the intein is Mt ΔI-CM(WT) or MtΔI-CM(m2), whose amino acid sequence is shown in SEQ ID NO: 12 and SEQ ID NO: 13; EFK8 is connected to the intein via a PT linker, whose amino acid sequence is shown in SEQ ID NO: 10. Mt The coding gene sequence of ΔI-CM (m2) is shown in SEQ ID NO: 14, bp 4-588. The amino acid sequence of GLP-1 is obtained from the literature (Glaesner et al., Diabetes / Metabolism Research and Reviews, 2010, 26(4):287-296). The amino acid sequence of GIP used in this example is obtained from the literature (Gault et al., Journal of Endocrinolody, 2003, 176, 133-141). The GCG amino acid sequence (SEQ ID NO: 3) used in this example is a humanized natural GCG sequence.

[0062] Preparation method: The expression vector used to synthesize the dual-target receptor agonist fusion protein was expressed in Escherichia coli BL21 (DE3), and the cells were collected. The cells were disrupted by heavy ultrasound using Buffer B1 (20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH8.0) (disruption conditions: power 200 W, ultrasound time 3 sec, interval time 3 sec, running time 15 min), and centrifuged at 15,000 g for 30 minutes at low temperature to separate the expression supernatant and cell fragments. The expression supernatant was quantified by SDS-PAGE protein, and the mixture was mixed at a molar ratio of SpyCatcher (ΔN) fusion protein: SpyTag fusion protein of 1:1, and reacted at 80 rpm for 2 hours at room temperature to form a dual-target receptor agonist fusion protein containing an icSAT tag. Then, an equal volume of Buffer B2 (1.4 M Na2SO4, 20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) was added to the mixture and placed at low temperature for 12 hours to precipitate the protein. The supernatant and protein precipitate were separated by centrifugation at 15,000 g for 30 minutes at low temperature. The precipitate was fully resuspended in cleavage Buffer B4 (PBS without NaCl, 20 mM Bis-Tris, 2 mM Na2·EDTA·2H2O, pH 6.2) without Na2SO4, placed on a rotary mixer, and reacted at 25 °C for 24 hours to allow the intein to be fully self-cleaved. The dual-target receptor agonist fusion protein without the icSAT tag in the supernatant was separated by centrifugation at 15,000 g for 30 minutes at low temperature. The purity and yield of the dual-target receptor agonist fusion protein in the supernatant were detected by SDS-PAGE.

[0063] The dual-target receptor agonist fusion protein was further finely purified using Protein A affinity purification. The specific steps were as follows: the supernatant of the dual-target receptor agonist fusion protein purified by icSAT was filtered through a 0.22 µm filter membrane, and at the same time, the column and purification system were rinsed with ultrapure water for 5-10 column volumes. The column was equilibrated with equilibration buffer (20 mM PB, 150 mM NaCl, pH7.4) for 5-10 column volumes. The filtered supernatant was passed through the column, and after the sample had completely passed through the column, it was rinsed with equilibration buffer until the baseline was "0". The sample value baseline was "0" when eluted with 50 mM acetic acid (50 mM HAc, pH3.6), and the sample was collected in a collection tube containing neutralization solution (1 M Tris-HCl, pH8.0). The column was rinsed with 1 M acetic acid solution and 0.1 M NaOH solution for 5-10 column volumes to remove some impurities and regenerate fillers in the column. Finally, the column was washed with ultrapure water for 5-10 column volumes, and the column and system were stored in 20% ethanol solution. SDS-PAGE was used to detect the purity and yield of the fusion protein. Figure 1 The SDS-PAGE results of the dual-target receptor agonist fusion protein prepared based on icSAT purification and Spy-chemistry biocoupling and purified using Protein A; the data involved in the preparation and purification of the dual-target receptor agonist fusion protein are shown in Table 1.

[0064] Table 1 Data involved in the preparation and purification of dual-target receptor agonist fusion protein

[0065]

[0066] Note: a Aggregation efficiency (%) = the amount of fusion protein reduced in the supernatant after aggregation / the amount of fusion protein in the supernatant before aggregation × 100%, b Cutting efficiency (%) = the amount of fusion protein reduced after cutting / the amount of fusion protein before cutting × 100%, c Purity after icSAT (%) = amount of target protein / (amount of target protein + amount of impurity protein) × 100%.

[0067] Example 2: Molecular weight identification

[0068] The molecular weights of the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) prepared by fine purification in Example 1 were determined. The molecular weight of the samples was determined using an ultra-high performance liquid chromatography-time-of-flight mass spectrometry system UPLC-Q-TOF and a Biozen™ C4 chromatographic column. The conditions were: injection volume 10 μL, flow rate 0.5 mL / min, column temperature 75 °C, and detection wavelength 214 nm. Mobile phase A was an aqueous solution containing 0.1% (v / v) TFA, mobile phase B was an acetonitrile solution containing 0.1% (v / v) TFA, and the mobile phase gradient was set to: 5-60% B, elution 3 min. The results are as follows. Figure 2 The results showed that the actual molecular weight of the GLP-1 / GIP dual-target receptor agonist fusion protein was 105880.13 Da, which was consistent with the theoretical molecular weight (105878.64 Da) ( Figure 2 A in the figure); The actual molecular weight of the GIP / GLP-1 dual-target receptor agonist fusion protein is 105879.93 Da, which is consistent with the theoretical molecular weight (105878.64 Da) ( Figure 2 B); The actual molecular weight of the GLP-1 / GCG dual-target receptor agonist fusion protein is 102905.68 Da, which is consistent with the theoretical molecular weight (102905.10 Da) ( Figure 2 C in the figure); The actual molecular weight of the GCG / GLP-1 dual-target receptor agonist fusion protein is 102907.10 Da, which is consistent with the theoretical molecular weight (102905.10 Da) ( Figure 2 D in.

[0069] Example 3: Fc and FcRn affinity determination

[0070] In order to verify the affinity of the antibody Fc fragments of the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) finely purified in Example 1 to human FcRn, it is necessary to perform affinity measurement on them. The concentration of the purified dual-target receptor agonist fusion proteins was measured using a BCA Kit (Thermo Fisher, USA). Commercial dulaglutide (Lilly, USA) was used as a positive control. The entire BLI experiment was detected using a molecular interaction instrument OctetRED96 (Fortebio). Streptavidin biosensor (SA) (Sartorius, DE) was first equilibrated in a kinetic buffer (phosphate buffer containing 0.1% bovine serum albumin BSA and 0.02% Tween-20, pH 7.0). Then, 1 µg / mL of human FcRn (ACRO, UK) working solution was fixed to the SA biosensor in the kinetic buffer. The fixed SA biosensor was then equilibrated in the kinetic buffer. The equilibrated sensor was then incubated with the dual-target receptor agonist fusion protein samples to be tested (at a concentration gradient of 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM dual-target receptor agonist fusion protein sample) was affinity treated in kinetic buffer for 200 seconds, and then the affinity sensor was dissociated in kinetic buffer for 200 seconds. The kinetic results of binding and dissociation corresponding to some concentrations are shown in Figure 3 The calculation of the binding dissociation constant KD, the binding kinetic constant Kon and the dissociation kinetic constant Koff was performed according to the Octet kinetics operation manual, and the results are shown in Table 2.

[0071] Table 2 Correlation constants of dual-target receptor agonist fusion proteins

[0072]

[0073] The four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) have high affinity for FcRn, similar to the affinity between dulagultide and FcRn, to maintain the effect of antibody Fc fragment in extending the half-life of dual-target receptor agonist fusion proteins (Glaesner et al., Diabetes / Metabolism Research and Reviews, 2010, 26(4): 287-296).

[0074] Example 4: In vitro activity verification

[0075] In order to verify the binding activity of the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) finely purified in Example 1 with GLP-1R (GLP-1 receptor), GIPR (GIP receptor) and GCGR (GCG receptor), HEK293 / GLP-1R-Luciferase, HEK293 / GIPR-Luciferase and HEK293 / GCGR-Luciferase cells stably expressing GLP-1R (or GIPR or GCGR) and the reporter gene luciferase regulated by the NFAT response component were used. After GLP-1, GIP or GCG specifically binds to GLP-1R / GIPR / GCGR on the cell membrane, it can activate adenylate cyclase and transmit signals, so that the luciferase reporter gene can be expressed. After adding the luciferase substrate, the luciferase expression amount can be evaluated by detecting the chemiluminescence value, and then the biological activity of the four dual-target receptor agonist fusion proteins can be evaluated. The experimental results were expressed using the median effective concentration (EC 50 ) indicates that EC 50 The lower the value, the stronger the drug effect.

[0076] When determining the in vitro agonist activity of samples on HEK293 / GLP-1R-Luciferase cells, the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) and the reference substance GLP-1 need to be diluted to 2 or 4 nM with assay buffer (DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin) as the starting concentration, and then diluted stepwise in a 3-fold gradient. When determining the in vitro agonist activity of samples on HEK293 / GIPR-Luciferase cells, the GLP-1 / GIP dual-target receptor agonist fusion protein, the GIP / GLP-1 dual-target receptor agonist fusion protein and the reference substance GIP samples need to be diluted to 540 / 180 nM with assay buffer as the starting concentration, and then diluted stepwise in a 3-fold gradient. When determining the in vitro agonist activity of samples on HEK293 / GCGR-Luciferase cells, GLP-1 / GCG dual-target receptor agonist fusion protein, GCG / GLP-1 dual-target receptor agonist fusion protein and control GCG (SEQID NO: 3) need to be diluted to 3000 nM with assay buffer (DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin), which is used as the starting concentration, and then diluted step by step according to a 3-fold gradient concentration. HEK293 / GLP-1R-Luciferase (or HEK293 / GIPR-Luciferase or HEK293 / GCGR-Luciferase) cells were digested and the cells were diluted to a suspension of 1 million / mL with assay buffer. The cell suspension and the diluted samples were transferred to a 384-well plate (white bottom transparent), 20 μL / well, and 3 replicates were set. Finally, the 384-well plate was placed in a 37 ℃, 5% CO2 incubator for 6 h. After incubation, add Luciferase Assay Buffer, 30 μL / well. Wait for 3 min to allow cells to fully lyse, and then use an ELISA reader to read the data to establish a dose-effect curve and calculate the EC 50 value.

[0077] The results are as follows Figure 4 As shown in Table 3; the results of in vitro agonist activity assays of GLP-1R and GIPR by GLP-1 / GIP dual-target receptor agonist fusion protein and GIP / GLP-1 dual-target receptor agonist fusion protein were shown in Table 3. Figure 4 A and Figure 4 As shown in B; the results of the in vitro agonist activity assays of the GLP-1 / GCG dual-target receptor agonist fusion protein and the GCG / GLP-1 dual-target receptor agonist fusion protein on GLP-1R and GCGR are shown in Figure 4 C and Figure 4As shown in D.

[0078] Table 3 In vitro activity verification results of dual-target receptor agonist fusion proteins

[0079]

[0080] The four dual-target receptor agonist fusion proteins all have strong in vitro binding activity with GLP-1R, GIPR and GCGR. The fusion protein architecture does not affect the activity of polypeptides such as GLP-1, and may even increase the activity of polypeptides such as GLP-1 / GCG dual-target receptor agonist fusion protein and GCG / GLP-1 dual-target receptor agonist fusion protein.

[0081] Example 5: Glucose tolerance assessment

[0082] This example evaluates the effects of the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) finely purified in Example 1 on the glucose tolerance of normal C57BL / 6 mice.

[0083] Experimental methods: Normal C57BL / 6 mice, 5-6 weeks old, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. According to blood glucose and body weight, they were randomly divided into 7 groups (Vehicle group, Dulaglutide group, Tirzepatide group, GLP-1 / GIP group, GIP / GLP-1 group, GLP-1 / GCG group, GCG / GLP-1 group), with 6 mice in each group. For the Dulaglutide group, Tirzepatide group, GLP-1 / GIP group, GIP / GLP-1 group, GLP-1 / GCG group and GCG / GLP-1 group, each group of animals was subcutaneously injected with the corresponding drug at a dose of 10 nmol / kg; for the Vehicle group (i.e., Control group), the corresponding solvent was subcutaneously injected.

[0084] After a single dose of 8 hours, the animals were fasted for 16 hours and allowed to drink water freely. Blood was collected from the tail vein to determine the basal blood glucose value of each group of animals, followed by intraperitoneal injection of 2 g / kg glucose solution, and blood glucose was tested at 15, 30, 60, and 90 minutes after glucose administration. The blood glucose concentration-time curve was drawn according to the blood glucose values ​​measured at different time points, and the AUC of each dose group was calculated. 0~90 min ; The experimental results are shown in Table 4.

[0085] Table 4 Effects of a single administration of dual-target receptor agonist fusion protein on glucose tolerance in normal mice 24 hours after administration

[0086]

[0087] Note: In the same column, *** indicates P <0.001.

[0088] Experimental results: The four dual-target receptor agonist fusion proteins all have good effects on improving glucose tolerance in mice, which are similar to the commercially available dulaglutide and slightly lower than tirzepatide. The effects are ranked from large to small as tirzepatide>GLP-1 / GIP dual-target receptor agonist fusion protein>GIP / GLP-1 dual-target receptor agonist fusion protein>GCG / GLP-1 dual-target receptor agonist fusion protein>dulaglutide>GLP-1 / GCG dual-target receptor agonist fusion protein.

[0089] Example 6: In vivo drug efficacy evaluation in ob / ob mouse model

[0090] This example evaluates the effects of the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, and GCG / GLP-1) finely purified in Example 1 on the blood glucose level of ob / ob model mice.

[0091] Experimental methods: 56 C57BL / 6 ob / ob mice aged 5-6 weeks were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., weighing 35-40 g. They were randomly divided into 7 groups (Dulaglutide group, Tirzepatide group, GLP-1 / GIP group, GIP / GLP-1 group, GLP-1 / GCG group, GCG / GLP-1 group and Vehicle group) according to blood sugar and body weight, with 8 mice in each group. Each group of animals was given the corresponding drug subcutaneously, and each dose was 30 nmol / kg. For the Vehicle group, the solvent was injected subcutaneously. In addition, the Control group, i.e. normal C57BL / 6 mice, was set up and the solvent was injected subcutaneously. The injection frequency was once every 3 days, and a total of 10 times were administered; the blood sugar, body weight and food intake of the animals were measured before each administration. 72 hours after the last administration, an intraperitoneal glucose tolerance test was performed, samples were processed and collected, liver weight was recorded, and liver pathological conditions and blood biochemical indicators of mice in each group were tested. The above test results are shown in Tables 5-10 and Figure 5 .

[0092] Table 5: Effects of long-term administration of dual-target receptor agonist fusion protein on blood glucose in ob / ob mice

[0093]

[0094] Table 6: Effects of long-term administration of dual-target receptor agonist fusion protein on glucose tolerance in ob / ob mice

[0095]

[0096] Note: In the same column, * indicates P <0.05, ** indicates P <0.01; *** indicates P <0.001.

[0097] Table 7: Effects of long-term administration of dual-target receptor agonist fusion protein on body weight of ob / ob mice

[0098]

[0099] Note: Only the significant analysis results of D27 are added, with shoulders in the same column, * indicates P <0.05, ** indicates P <0.01; *** indicates P <0.001.

[0100] Table 8: Effects of long-term administration of dual-target receptor agonist fusion protein on food intake in ob / ob mice

[0101]

[0102] Table 9: Effects of long-term administration of dual-target receptor agonist fusion protein on fat and liver weight in ob / ob mice

[0103]

[0104] Note: In the same column, * indicates P <0.05, ** indicates P <0.01; *** indicates P <0.001.

[0105] Table 10: Effects of long-term administration of dual-target receptor agonist fusion protein on liver function and blood lipids in ob / ob mice

[0106]

[0107] Note: In the same column, * indicates P <0.05, ** indicates P <0.01; *** indicates P <0.001; HbA1c%: glycated hemoglobin level; ALT: alanine aminotransferase; AST: aspartate aminotransferase; TG: triglyceride; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein.

[0108] Experimental results: Compared with Vehicle, long-term repeated administration of GLP-1 / GIP dual-target receptor agonist fusion protein, GIP / GLP-1 dual-target receptor agonist fusion protein, GLP-1 / GCG dual-target receptor agonist fusion protein and GCG / GLP-1 dual-target receptor agonist fusion protein can significantly reduce blood glucose in mice and maintain long-term stability. The effect of improving glucose tolerance in ob / ob mice is similar to that of tirzepatide and dulaglutide. GLP-1 / GCG dual-target receptor agonist fusion protein and GCG / GLP-1 dual-target receptor agonist fusion protein can significantly reduce the body weight of mice and inhibit their food intake. GLP-1 / GIP dual-target receptor agonist fusion protein, GIP / GLP-1 dual-target receptor agonist fusion protein, GLP-1 / GCG dual-target receptor agonist fusion protein and GCG / GLP-1 dual-target receptor agonist fusion protein can improve the liver weight and fat content of mice, and significantly improve liver function and blood biochemical indicators. In addition, GLP-1 / GCG dual-target receptor agonist fusion protein and GCG / GLP-1 dual-target receptor agonist fusion protein have the best effect in improving mouse liver cell cavitation ( Figure 5 ). The GLP-1 / GCG dual-target receptor agonist fusion protein has the best effect on improving all aspects of metabolism in mice, and its effect is better than tirzepatide.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An Fc fragment-modified dual-target receptor agonist fusion protein, characterized in that: The structure of the fusion protein is shown in the following formula I: abcdef (Ⅰ) In the formula, each "-" is independently a connecting peptide or a peptide bond or an isopeptide bond; a is a GLP-1 polypeptide and f is a GIP polypeptide or a GCG polypeptide; or, a is a GIP polypeptide or a GCG polypeptide and f is a GLP-1 polypeptide; b is an Fc fragment; c is a SpyCatcher peptide; d is a SpyTag peptide; e is an ELP peptide, which serves as a third connecting peptide; Wherein, a and b are connected by a first connecting peptide, b and c are connected by a second connecting peptide; c and d are connected by an isopeptide bond; d, e and f are connected in sequence by peptide bonds; The C-terminus of a is connected to the N-terminus of the first connecting peptide; the C-terminus of the first connecting peptide is connected to the N-terminus of b; the C-terminus of b is connected to the N-terminus of the second connecting peptide; the C-terminus of the second connecting peptide is connected to the N-terminus of c; the N-terminus of d is connected to the C-terminus of e; the N-terminus of e is connected to the C-terminus of f; The amino acid sequence of the GLP-1 polypeptide is shown in SEQ ID NO: 1; the amino acid sequence of the GIP polypeptide is shown in SEQ ID NO: 2; the amino acid sequence of the GCG polypeptide is shown in SEQ ID NO: 3; the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 4; the SpyCatcher peptide is SpyCatcher (ΔN), and its amino acid sequence is shown in SEQ ID NO: 8; the amino acid sequence of the SpyTag peptide is shown in SEQ ID NO: 9; the first connecting peptide and the second connecting peptide each independently comprise an amino acid sequence selected from the following group: shown in SEQ ID NO: 5 and shown in SEQ ID NO: 6; the amino acid sequence of the ELP peptide is shown in SEQ ID NO:

7.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the fusion protein required to form the Fc fragment-modified dual-target receptor agonist fusion protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule includes the nucleic acid molecule shown at 589-1704 bp in SEQ ID NO: 14 and the nucleic acid molecule shown at 589-987 bp in SEQ ID NO: 17, or the nucleic acid molecule shown at 589-1737 bp in SEQ ID NO: 16 and the nucleic acid molecule shown at 589-954 bp in SEQ ID NO: 15; and is used to encode the fusion protein required for forming the GLP-1 and GIP dual-target receptor agonist fusion protein; The nucleic acid molecule includes the nucleic acid molecule shown at 589-1704 bp in SEQ ID NO: 14 and the nucleic acid molecule shown at 589-948 bp in SEQ ID NO: 19, or the nucleic acid molecule shown at 589-1698 bp in SEQ ID NO: 18 and the nucleic acid molecule shown at 589-954 bp in SEQ ID NO: 15; and is used to encode the fusion protein required to form a GLP-1 and GCG dual-target receptor agonist fusion protein.

4. The Fc fragment-modified dual-target receptor agonist fusion protein-related biomaterial according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (1) An expression cassette comprising the nucleic acid molecule according to any one of claims 2 to 3; (2) An expression vector comprising the nucleic acid molecule according to any one of claims 2 to 3; (3) an expression vector comprising the expression cassette described in (1); (4) An engineered bacterium comprising the nucleic acid molecule according to any one of claims 2 to 3; (5) An engineered bacterium comprising the expression cassette described in (1); (6) An engineered bacterium comprising the expression vector described in (2) or (3).

5. A pharmaceutical composition, characterized in that: Comprising the Fc fragment modified dual-target receptor agonist fusion protein of claim 1, the nucleic acid molecule of any one of claims 2 to 3, or the biomaterial of claim 4.

6. Use of the Fc fragment-modified dual-target receptor agonist fusion protein of claim 1, the nucleic acid molecule of any one of claims 2 to 3, the biomaterial of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a drug, characterized in that: The drug is used to control or reduce blood sugar and / or body weight.

7. Use of the Fc fragment-modified dual-target receptor agonist fusion protein of claim 1, the nucleic acid molecule of any one of claims 2 to 3, the biomaterial of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a drug, characterized in that: The medicament is used for preventing and / or treating at least one of type 2 diabetes and obesity.

8. Use of the Fc fragment-modified dual-target receptor agonist fusion protein of claim 1, the nucleic acid molecule of any one of claims 2 to 3, the biomaterial of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a drug, characterized in that: The drug is used for preventing and / or treating at least one of metabolic dysfunction-related fatty hepatitis and dyslipidemia and metabolic syndrome.

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