Dual-target receptor agonist fusion protein and use thereof

Abstract

The present invention relates to the field of biomedical engineering. Disclosed are a dual-target receptor agonist fusion protein and a use thereof. The fusion protein is a dual-target receptor agonist fusion protein (GLP-1 and GIP, GLP-1 and GCG) having dual native N-termini and fused with an antibody Fc fragment. The fusion protein can be rapidly prepared by using a more efficient and low-cost bacterial expression system, and has high in vitro agonistic activity and in vivo activity. The fusion protein can be obtained in one step by means of self-ligation of SpyCatcher / SpyTag without the need for purification, and is fused with a human antibody Fc fragment. The fusion protein can be used in the production of a novel drug comprising a polypeptide / protein modified by an antibody Fc fragment and having dual native N-termini.

Description

A dual-target receptor agonist fusion protein and its applications Technical Field

[0001] This invention relates to the field of biomedical engineering, and particularly to a dual-target receptor agonist fusion protein and its applications. Specifically, it relates to a GLP-1 and GIP dual-target receptor agonist fusion protein, a GLP-1 and GCG dual-target receptor agonist fusion protein and their applications. More specifically, it relates to the design of the GLP-1 and GIP dual-target receptor agonist fusion protein, the nucleic acid molecules encoding the GLP-1 and GIP dual-target receptor agonist fusion protein, the expression vector, the engineered bacteria, and 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 and their nucleic acid molecules and engineered bacteria in the preparation of drugs. Background Technology

[0002] Glucagon-like peptide-1 receptor (GLP-1) is an incretin that promotes insulin synthesis and secretion by pancreatic β-cells (Drucker et al., Nature Medicine, 1996, 2, 11), induces β-cell proliferation and inhibits apoptosis (Kulkarni et al., Diabetologia, 2017, 60, 1442-1453), and inhibits glucagon secretion from α-cells (Drucker et al., Cell Metabolism, 2018, 3, 714). It is currently one of the most effective drugs for treating type 2 diabetes mellitus (T2DM) worldwide. GLP-1 also has physiological functions such as slowing gastric emptying and inhibiting gastrointestinal motility (Iwasaki et al., Nature Communications, 2018, 9, 133), exhibiting good weight-loss effects and becoming a research hotspot for anti-obesity drugs. Since the natural GLP-1 has a half-life of only 2 to 3 minutes in vivo, it cannot be used as a drug. Therefore, 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, similar to GLP-1, promotes insulin secretion. Furthermore, the signaling pathway activated by the interaction between GIP and GIPR can block vomiting and mitigate 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. It stimulates the breakdown of fat in adipose tissue, reduces food intake, increases satiety, and promotes energy expenditure (Prato et al., 2021, Obesity Reviews. 2022, 23:e13372). In recent years, with the development and market launch of various glucagon-like peptide-1 receptor agonists (GLP-1RAs), next-generation dual / multi-target receptor agonists have shown better drug delivery, lower blood glucose levels, and reduced food intake, becoming the mainstream of GLP-1RA research. The next-generation dual / multi-target receptor agonist tirzepatide was approved for marketing by the U.S. Food and Drug Administration (FDA) in May 2022. As a single-molecule dual-target GLP-1 / GIP (glucose-dependent insulinotropic peptide) agonist, tirzepatide has shown superior hypoglycemic and weight-loss efficacy compared to smegglutide (Coskun et al., Molecular Metabolism, 2018, 18:3-14). However, the design of such single-molecule agonists relies on the high homology of the peptides, and it is difficult to precisely control the affinity of the molecule for different targets during the design process, making it difficult to predict the distribution of the molecule's potency 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 native N-terminus 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 chemical reagents, is fast, and operates 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 GLP-1-based dual (multi)-target receptor agonists. Therefore, there is still a need in this field to develop multi-target receptor agonists with longer-acting effects, better blood sugar and weight loss 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 present invention aims to provide a dual-target receptor agonist fusion protein and its application.

[0007] This invention is based on the inventor's discoveries and understanding of the following problems:

[0008] The dose-dependent side effects and short half-life of GLP-1 limit its clinical application. The main research direction for GLP-1 receptor agonists is currently to combine them with other targets such as GIP and GCG to form multi / dual-target receptor agonists and extend their half-life. Currently, multi / dual-target receptor agonists in clinical trials mainly involve designing two peptides into a chimeric peptide that simultaneously activates two or more targets. Examples include tirzepatide, approved by the FDA in May 2022, a GLP-1 / GIP dual-target receptor agonist, and cotadutide and mazdutide, both GLP-1 / GCG dual-target receptor agonists currently in Phase III clinical trials. This strategy is limited by the homologous sequences of the peptides and makes it difficult to modulate their efficacy against each target. Furthermore, peptides such as GLP-1, GIP, and GCG require the exposure of their native N-terminus to retain their high receptor activity, which increases the difficulty of multi-target design and preparation.

[0009] The objective of this invention is achieved through the following technical solution:

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

[0011] abcdef(Ⅰ)

[0012] In the formula, each "-" independently represents a linking peptide, peptide bond, or isopeptide bond;

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

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

[0015] Wherein, a and b are connected by a first linker peptide, b and c are connected by a second linker peptide; c and d are connected by isopeptide bonds; and d, e and f are connected sequentially by peptide bonds.

[0016] Furthermore, the C-terminus of a is connected to the N-terminus of the first linker peptide; the C-terminus of the first linker peptide is connected to the N-terminus of b; the C-terminus of b is connected to the N-terminus of the second linker peptide; the C-terminus of the second linker 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 peptide is shown in SEQ ID NO: 1; the amino acid sequence of the GIP peptide is shown in SEQ ID NO: 2; and the amino acid sequence of the GCG peptide 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 with respect to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0019] In some embodiments, the GLP-1 peptide, GIP peptide, and GCG peptide 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 peptide before the mutation.

[0020] In some embodiments, the GLP-1 peptide, GIP peptide, and GCG peptide may be peptides with 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 variants of the SpyCatcher peptide and SpyTag peptide.

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

[0025] In some embodiments, the first linker peptide and the second linker peptide each independently comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 5: GGGGSGGGGSGGGGSA and 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 a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes a fusion protein required for forming a GLP-1 and GIP dual-target receptor agonist fusion protein, and a fusion protein required for forming a GLP-1 and GCG dual-target receptor agonist fusion protein. Both the fusion proteins encoded by the nucleic acid molecule according to embodiments of the present invention exhibit excellent binding activity with GLP-1 and / or GIP and / or GCG receptors, and possess dual functions of weight reduction and blood glucose lowering, effectively reducing the weight and blood glucose levels of mice.

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

[0029] The nucleic acid molecules include those shown in SEQ ID NO: 14 (589-1704bp) and SEQ ID NO: 19 (589-948bp), or those shown in SEQ ID NO: 18 (589-1698bp) and SEQ ID NO: 15 (589-954bp); used to encode the fusion protein required for forming the GLP-1 and GCG dual-target receptor agonist fusion protein.

[0030] In a third aspect, the present invention provides an expression cassette or 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 expression cassette described above. According to an embodiment of the present invention, the expression vector is a prokaryotic expression vector.

[0031] In a fourth aspect, the present invention provides an engineered bacterium. According to embodiments of the invention, it carries the nucleic acid molecule described in the second aspect or the expression cassette or expression vector described in the third aspect. The engineered bacterium according to embodiments of the invention can express fusion proteins containing GLP-1, GIP, and GCG peptides respectively, and further form a GLP-1 and GIP dual-target receptor agonist fusion protein and a GLP-1 and GCG dual-target receptor agonist fusion protein through Spy-chemistry peptide bioconjugation. These GLP-1 and GIP dual-target receptor agonist fusion proteins and GLP-1 and GCG dual-target receptor agonist fusion proteins exhibit excellent binding activity with GLP-1 and / or GIP, GLP-1 and / or GCG receptors, and possess dual functions of weight loss and blood sugar reduction, effectively controlling or reducing weight and blood sugar levels.

[0032] A pharmaceutical composition is provided in the fifth aspect of the present invention. According to embodiments 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: pharmaceutically acceptable excipients, said pharmaceutically acceptable excipients including at least one of stabilizers, wetting agents, emulsifiers, binders, and isotonic agents; the pharmaceutical composition is in the form of at least one of tablets, granules, powders, capsules, solutions, suspensions, and dynamic formulations. The pharmaceutical composition according to embodiments of the present invention has a long-lasting effect in promoting weight loss and / or lowering blood sugar, and can effectively control or reduce weight and blood sugar levels.

[0033] In a sixth aspect, 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 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, in the preparation of a medicament. According to embodiments of the present invention, the medicament is used to control or reduce blood glucose and / or weight; the medicament is used to prevent and / or treat at least one of the following diseases: type 2 diabetes, obesity, metabolic dysfunction-related steatohepatitis, dyslipidemia, and metabolic syndrome.

[0034] In a seventh aspect, the present invention provides a method for preparing the novel dual-target receptor agonist fusion protein described in the first aspect. According to an embodiment of the present invention, the method comprises: 1) constructing the nucleic acid molecule described in the second aspect; 2) introducing the expression vector into a host cell to obtain engineered bacteria to express the fusion protein, wherein 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 a Spy-chemistry peptide bioconjugation 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, and GLP-1 and / or GCG receptors, and possesses dual functions of lowering blood sugar and reducing weight, effectively controlling or reducing weight and blood sugar levels.

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

[0036] The present invention has the following advantages and effects compared with the prior art:

[0037] This 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) with dual natural N-terminals fused with antibody Fc fragments. It can be rapidly prepared using a more efficient and cost-effective bacterial expression system and exhibits high in vitro and in vivo agonist activity. This fusion protein requires no purification and can be obtained in one step via SpyCatcher / SpyTag self-ligation (GLP-1 and GIP, GLP-1 and GCG), and incorporates a human antibody Fc fragment. It effectively improves glucose tolerance in mice and is superior to tirzepatide (Lilly) in reducing body weight in obese mice, inhibiting food intake, improving lipid and liver metabolism in mice, and improving liver vacuolation. This fusion protein can be used to produce novel drugs with dual natural N-terminal peptides / proteins modified with antibody Fc fragments. Attached Figure Description

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

[0039] Figure 1 shows the SDS-PAGE results of dual-target receptor agonist fusion proteins prepared by icSAT purification, Spy-chemistry bioconjugation, and Protein A purification; where 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, and D: GCG / GLP-1 dual-target receptor agonist fusion protein.

[0040] Figure 2 shows the molecular weight determination results of the GLP-1 / GIP dual-target receptor agonist fusion protein; where 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, and D: GCG / GLP-1 dual-target receptor agonist fusion protein.

[0041] Figure 3 shows the affinity assay results of the antibody Fc fragment of the dual-target receptor agonist fusion protein with human FcRn; where 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 assay results of the antibody Fc fragment of dulaglutide (Lilly) with human FcRn.

[0042] Figure 4 shows the in vitro agonistic activity assays of dual-target receptor agonist fusion proteins for GLP-1R, GIPR, or GCGR. A and B represent the in vitro agonistic activity assays of the GLP-1 / GIP dual-target receptor agonist fusion protein (GLP-1-Fc-Spy-GIP) and the GIP / GLP-1 dual-target receptor agonist fusion protein (GIP-Fc-Spy-GLP-1) for GLP-1R and GIPR. C and D represent the in vitro agonistic activity assays of the GLP-1 / GCG dual-target receptor agonist fusion protein (GLP-1-Fc-Spy-GCG) and the GCG / GLP-1 dual-target receptor agonist fusion protein (GCG-Fc-Spy-GLP-1) for GLP-1R and GCGR.

[0043] Figure 5 shows the results of liver tissue section scanning of ob / ob mice after long-term drug administration. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially. Unless otherwise specified, the methods used in the following embodiments are conventional methods, and specific steps can be found, for example, in *Molecular Cloning: A Laboratory Manual* (Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, 2001, NY, Cold Spring Harbor). All primers used were synthesized by Shanghai Sangon Biotech.

[0045] In this article, the terms “natural,” “wild-type,” or “WT” refer to proteins or peptides that are naturally occurring or can be isolated from the environment and do not contain any genetically engineered mutations.

[0046] In this article, the term "basic bioactivity" refers to the ability of a polypeptide to retain at least a portion (e.g., not less than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or all of its pre-mutation bioactivity after mutation.

[0047] In this article, the terms “substitution” or “mutation” refer to replacing one amino acid in a polypeptide with another amino acid.

[0048] In this article, the substitution of amino acids is indicated by a first letter followed by a number followed by a second letter. The first letter represents the amino acid in the wild-type protein or polypeptide; the number indicates the position of the amino acid that has been substituted; and the second letter indicates the amino acid that replaced the wild-type amino acid.

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

[0050] In this article, “identity” or “homology” generally refers to the sequence similarity between two peptides or proteins or between two nucleic acid molecules. The percentage of “identity” or “homology” refers to the percentage of identical residues among amino acids or nucleic acids in the molecules being compared, and is calculated based on the size of the smallest molecule being compared.

[0051] In this article, “Fc” or “Fc fragment” is used to define a C-terminal region in the antibody heavy chain that contains at least a portion of a constant region. Antibody Fc fragments can be native Fc fragments or variant Fc fragments.

[0052] The fusion protein provided by this invention can utilize a natural antibody Fc fragment or an Fc fragment variant; in a preferred embodiment, an Fc fragment variant is used. During the research process of this invention, it was discovered that the antibody Fc fragment used can naturally form a dimer.

[0053] In this article, "dimer" refers to a protein dimer, which is composed of two protein (peptide) molecules and represents a quaternary structure of a protein. The two proteins (peptides) that make up the dimer can be called the monomer molecules or monomeric proteins of the dimer. Dimers can include both "homodimers" and "heterodimers." 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 this invention, when the Fc fragment variant provided by this invention is used, it has the ability to form homodimers; the fusion protein provided by this invention also has the ability to form homodimers.

[0054] In this article, a "linker peptide" or "peptide linker" refers to a single amino acid or polypeptide sequence that links two proteins (peptides) together. Linker peptides can be approximately 1-40 amino acids long and contain, for example, repeated proline and threonine, or repeated alanine, glycine, and serine. Common linker peptides include flexible linker peptides, such as combinations of alanine, glycine, and serine, such as (GGGGS)3A and GGGGS; and rigid linker peptides, PT-type linker peptides containing proline and threonine.

[0055] In this article, the GLP-1 and GIP dual-target receptor agonist fusion protein refers to a dual-target receptor agonist fusion protein containing both GLP-1 and GIP peptides. Specifically, it refers to the GLP-1 / GIP dual-target receptor agonist fusion protein where the C-terminus of the GLP-1 peptide is linked to the N-terminus of the first linker peptide, and the C-terminus of the GIP peptide is linked to the N-terminus of the third linker peptide; and the GIP / GLP-1 dual-target receptor agonist fusion protein where the C-terminus of the GIP peptide is linked to the N-terminus of the first linker peptide, and the C-terminus of the GLP-1 peptide is linked to the N-terminus of the third linker 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. Specifically, it is a GLP-1 / GCG dual-target receptor agonist fusion protein in which the C-terminus of the GLP-1 peptide is linked to the N-terminus of the first linker peptide and the C-terminus of the GCG peptide is linked to the N-terminus of the third linker peptide, and a GCG / GLP-1 dual-target receptor agonist fusion protein in which the C-terminus of the GCG peptide is linked to the N-terminus of the first linker peptide and the C-terminus of the GLP-1 peptide is linked to the N-terminus of the third linker peptide.

[0056] In the figure of Example 1, ES: cell lysate supernatant; EP: cell lysate precipitate; ES1, ES2: cell lysate supernatants 1 and 2; Mix: mixture of ES1 and ES2; ESS: supernatant after salting and aggregation of Mix; ESP: precipitate after salting and aggregation of Mix; CP: precipitate after cleavage of ESP; CS: supernatant after cleavage of ESP; wherein, ES1 to CP are all diluted 10-fold, and 10*CS is not diluted; M: protein marker; BSA: bovine serum albumin standard.

[0057] The vectors used for expressing the constructs of this invention include vectors capable of autonomous replication in host cells, such as plasmid vectors; and vectors capable of integrating into and replicating with the host cell DNA. In a specific embodiment, the expression constructs of this invention are derived from pET30a(+) of Novagen. Host cells used for expressing the fusion protein of this invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotes include bacteria of the genera *Escherichia*, *Bacillus*, *Pseudomonas*, and *Streptomyces*. In a preferred embodiment, the host cell is an *Escherichia* cell, preferably *Escherichia coli*. In one specific embodiment of this invention, the host cell used is *Escherichia coli* BL21(DE3) strain cells (Novagen).

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

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

[0060] In this embodiment, the expression vectors used to synthesize the GLP-1 and GIP dual-target receptor agonist fusion protein were pET30a-EFK8-MtuΔI-CM(m2)-GLP-1-Fc-SpyCatcher(ΔN), pET30a-EFK8-MtuΔI-CM(WT)-GIP-ELP15-SpyTag, pET30a-EFK8-MtuΔI-CM(WT)-GIP-Fc-SpyCatcher(ΔN), and pET30a-EFK8-MtuΔI-CM(m2)-GLP-1-ELP 15-SpyTag. In this embodiment, the expression vectors used to synthesize the GLP-1 and GCG dual-target receptor agonist fusion protein were pET30a-EFK8-MtuΔI-CM(m2)-GLP-1-Fc-SpyCatcher(ΔN) and pET30a-EFK8-MtuΔI-CM(m2)-GCG-ELP. 15 -SpyTag, pET30a-EFK8-MtuΔI-CM(m2)-GCG-Fc-SpyCatcher(ΔN) and pET30a-EFK8-MtuΔI-CM(m2)-GLP-1-ELP 15 -SpyTag. The target fragment is inserted between the NdeⅠ and XhoⅠ restriction sites of the vector pET30a(+) (purchased from Novagen). The encoding gene sequence of the fusion protein EFK8-MtuΔI-CM(m2)-GLP-1-Fc-SpyCatcher(ΔN) in this embodiment is shown in SEQ ID NO: 14. 15 The gene sequence encoding -SpyTag is shown in SEQ ID NO: 17. The gene sequence encoding the fusion protein EFK8-MtuΔI-CM(WT)-GIP-Fc-SpyCatcher(ΔN) is shown in SEQ ID NO: 16. The fusion protein EFK8-MtuΔI-CM(m2)-GLP-1-ELP 15 The gene sequence encoding -SpyTag is shown in SEQ ID NO: 15. The fusion protein EFK8-MtuΔI-CM(m2)-GCG-ELP 15 The gene sequence encoding -SpyTag is shown in SEQ ID NO: 19. The gene sequence encoding the fusion protein EFK8-MtuΔI-CM(m2)-GCG-Fc-SpyCatcher(ΔN) is shown in SEQ ID NO: 18. The initial ATG and the final TGA are the start and stop codons, respectively.

[0061] In this embodiment, the salinity-induced self-assembly peptide used is EFK8, whose amino acid sequence is shown in SEQ ID NO: 11; the inteptide is MtuΔI-CM(WT) or MtuΔI-CM(m2), whose amino acid sequences are shown in SEQ ID NO: 12 and SEQ ID NO: 13; EFK8 and the inteptide are linked by a PT linker, the amino acid sequence of which is shown in SEQ ID NO: 10. The coding gene sequence of EFK8-MtuΔI-CM(m2) is shown in SEQ ID NO: 14, from 4 to 588 bp. The amino acid sequence of GLP-1 was obtained from the literature (Glaesner et al., Diabetes / Metabolism Research and Reviews, 2010, 26(4): 287-296). The GIP amino acid sequence used in this embodiment was 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 embodiment 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 *E. coli* BL21(DE3), and bacterial cells were collected. The bacterial cells were sonicated using Buffer B1 (20 mM Tris-base, 1 mM Na2·EDTA·2H2O, pH 8.0) (lysis conditions: power 200 W, sonication time 3 sec, interval time 3 sec, run time 15 min), and centrifuged at 15,000 g for 30 min at low temperature to separate the expression supernatant and bacterial fragments. SDS-PAGE was performed on the expression supernatant for protein quantification. The SpyCatcher(ΔN) fusion protein and SpyTag fusion protein were mixed at a molar ratio of 1:1 and reacted at 80 rpm for 2 hours at room temperature to form a dual-target receptor agonist fusion protein containing the icSAT tag. Then, an equal volume of Buffer B2 (1.4M Na2SO4, 20mM Tris-base, 1mM Na2·EDTA·2H2O, pH 8.0) was added to the mixture and incubated at low temperature for 12 hours to precipitate the protein. The supernatant and protein precipitate were separated by centrifugation at 15,000g for 30 minutes at low temperature. The precipitate was resuspended thoroughly in half the volume of Na2SO4-free cleavage Buffer B4 (NaCl-free PBS, 20mM Bis-Tris, 2mM Na2·EDTA·2H2O, pH 6.2), and incubated at 25°C for 24 hours to allow for complete self-cleavage of the integrins. Centrifugation at 15,000g for 30 minutes at low temperature yielded the supernatant containing the icSAT-tagged dual-target receptor agonist fusion protein. SDS-PAGE was used to analyze the purity and yield of the dual-target receptor agonist fusion protein in the supernatant.

[0063] The dual-target receptor agonist fusion protein was further purified using Protein A affinity purification. The specific steps were as follows: The supernatant of the icSAT-purified dual-target receptor agonist fusion protein was filtered through a 0.22 μm filter membrane, while simultaneously washing the column and purification system with ultrapure water for 5-10 column volumes. The column was then equilibrated with equilibration buffer (20 mM PB, 150 mM NaCl, pH 7.4) for 5-10 column volumes. The filtered supernatant was then flow-through the column until all the sample had passed through, followed by washing with equilibration buffer until the baseline was "0". The sample was eluted with 50 mM acetic acid (50 mM HAc, pH 3.6) until the baseline was "0", and the sample was collected in a collection tube containing neutralization buffer (1 M Tris-HCl, pH 8.0). The column was then washed successively with 1 M acetic acid solution and 0.1 M NaOH solution for 5-10 column volumes to remove impurities and regenerated packing material. Finally, the column was rinsed with ultrapure water for 5-10 column volumes, and the column and system were stored in 20% ethanol solution. SDS-PAGE was used to determine the purity and yield of the fusion protein. Figure 1 shows the SDS-PAGE results of the dual-target receptor agonist fusion protein prepared by icSAT purification, Spy-chemistry bioconjugation, and Protein A purification; data related to the preparation and purification of the dual-target receptor agonist fusion protein are shown in Table 1.

[0064] Table 1. Data related to the preparation and purification of the dual-target receptor agonist fusion protein.

[0065] Note: a Aggregation efficiency (%) = (Reduction in fusion protein in the supernatant after aggregation / Amount of fusion protein in the supernatant before aggregation) × 100%. b Cutting efficiency (%) = Reduction in fusion protein after cutting / Amount of fusion protein before cutting × 100% c Purity (%) after icSAT = Amount of target protein / (Amount of target protein + Amount of other proteins) × 100%.

[0066] Example 2: Molecular weight identification

[0067] The molecular weights of the four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, and GCG / GLP-1) obtained through fine purification in Example 1 were determined. Ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF) and Biozen were used. TMThe molecular weight of the sample was determined using a C4 column. The conditions were: injection volume 10 μL, flow rate 0.5 mL / min, column temperature 75℃, and detection wavelength 214 nm. Mobile phase A was an aqueous solution containing 0.1% (v / v) TFA, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) TFA. The mobile phase gradient was set to 5–60% B, and elution was performed for 3 min. The results are shown in 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, consistent with the theoretical molecular weight (105878.64 Da) (Figure 2, A); the actual molecular weight of the GIP / GLP-1 dual-target receptor agonist fusion protein was 105879.93 Da, 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 was 102905.68 Da, consistent with the theoretical molecular weight (102905.10 Da) (Figure 2, C); and the actual molecular weight of the GCG / GLP-1 dual-target receptor agonist fusion protein was 102907.10 Da, consistent with the theoretical molecular weight (102905.10 Da) (Figure 2, D).

[0068] Example 3: Determination of the affinity between Fc and FcRn

[0069] To verify the affinity of the antibody Fc fragments of the four finely purified dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) obtained in Example 1 for human FcRn, affinity assays were performed. The concentrations of the purified dual-target receptor agonist fusion proteins were determined using a BCA Kit (Thermo Fisher, USA). Commercially available dulaglutide (Lilly, USA) was used as a positive control. The entire BLI experiment was performed using an Octet RED96 molecular interaction analyzer (Fortebio). Streptavidin was first... The biosensor (SA) (Sartorius, DE) was equilibrated in kinetic buffer (phosphate buffer containing 0.1% bovine serum albumin BSA and 0.02% Tween-20, pH 7.0). 1 μg / mL human FcRn (ACRO, UK) working solution was then fixed onto the SA biosensor in the kinetic buffer. The SA biosensor was then equilibrated in the kinetic buffer to immobilize it. The equilibrated sensor was then affinity-treated with a dual-target receptor agonist fusion protein sample (concentration gradients of 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, and 3.90625 nM) in the kinetic buffer for 200 seconds. The affinity-treated sensor was then dissociated in the kinetic buffer for 200 seconds. The binding and dissociation kinetics for some concentrations are shown in Figure 3. The binding dissociation constant KD, the binding kinetic constant Kon, and the dissociation kinetic constant Koff were calculated according to the Octet kinetic operation manual, and the results are shown in Table 2.

[0070] Table 2. Correlation constants of dual-target receptor agonist fusion proteins

[0071] 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, thus maintaining the antibody Fc fragment to prolong the half-life of the dual-target receptor agonist fusion protein (Glaesner et al., Diabetes / Metabolism Research and Reviews, 2010, 26(4):287-296).

[0072] Example 4: In vitro activity verification

[0073] 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) obtained by fine purification 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 NFAT response components-regulated reporter gene luciferase 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, enabling the expression of the luciferase reporter gene. After adding luciferase substrate, the expression level of luciferase can be assessed by detecting chemiluminescence value, thereby evaluating the biological activity of the four dual-target receptor agonist fusion proteins. The experimental results are based on the median effective concentration (EC50). 50 ) indicates that EC 50 The lower the value, the stronger the drug effect.

[0074] When determining the in vitro agonistic activity of the samples against 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 control GLP-1 should be diluted to 2 or 4 nM with analytical buffer (DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin) as the starting concentration, and then serially diluted in 3-fold increments. When determining the in vitro agonistic activity of the samples against 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 control GIP should be diluted to 540 / 180 nM with analytical buffer as the starting concentration, and then serially diluted in 3-fold increments. To determine the in vitro agonistic activity of the samples on HEK293 / GCGR-Luciferase cells, the GLP-1 / GCG dual-target receptor agonist fusion protein, the GCG / GLP-1 dual-target receptor agonist fusion protein, and the control GCG (SEQ ID NO: 3) were diluted to 3000 nM with analytical buffer (DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin) as the starting concentration, and then serially diluted 3-fold. HEK293 / GLP-1R-Luciferase (or HEK293 / GIPR-Luciferase or HEK293 / GCGR-Luciferase) cells were digested and diluted to a suspension of 1 million cells / mL with analytical buffer. The cell suspension and diluted samples were transferred to 384-well plates (white bottom permeable), 20 μL / well, with 3 replicates. Finally, the 384-well plates were incubated at 37°C in a 5% CO2 incubator for 6 h. After incubation, add 30 μL of Luciferase Assay Buffer to each well. Allow 3 minutes for cell lysis, then use a microplate reader to record the readings to establish a dose-response curve and calculate EC50. 50 value.

[0075] The results are shown in Figure 4 and Table 3; the in vitro agonistic activity assays of the GLP-1 / GIP dual-target receptor agonist fusion protein and the GIP / GLP-1 dual-target receptor agonist fusion protein on GLP-1R and GIPR are shown in Figure 4A and Figure 4B, respectively; the in vitro agonistic 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 4C and Figure 4D.

[0076] Table 3. Results of in vitro activity validation of the dual-target receptor agonist fusion protein

[0077] The four dual-target receptor agonist fusion proteins exhibit strong in vitro binding activity with GLP-1R, GIPR, and GCGR. This fusion protein architecture does not affect the activity of peptides such as GLP-1, and may even enhance the activity of peptides such as the GLP-1 / GCG dual-target receptor agonist fusion protein and the GCG / GLP-1 dual-target receptor agonist fusion protein.

[0078] Example 5: Glucose Tolerance Assessment

[0079] This embodiment evaluates the effects of four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) obtained by fine purification in Example 1 on glucose tolerance in normal C57BL / 6 mice.

[0080] Experimental Methods: Normal C57BL / 6 mice, 5–6 weeks old, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. 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, and GCG / GLP-1 group) according to blood glucose and body weight, 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 animal 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.

[0081] Eight hours after a single dose, animals were fasted for 16 hours but allowed free access to water. Baseline blood glucose levels were measured by blood collection from the tail vein. Subsequently, animals were administered a 2 g / kg glucose solution via intraperitoneal injection, with blood glucose levels measured at 15, 30, 60, and 90 minutes post-glucose administration. Blood glucose concentration-time curves were plotted based on the blood glucose values ​​measured at different time points, and the AUC for each dose group was calculated. 0～90min The experimental results are shown in Table 4.

[0082] Table 4. Effects of a single dose of the dual-target receptor agonist fusion protein on glucose tolerance in normal mice 24 hours after administration. Note: In the same column, *** indicates P<0.001.

[0083] Experimental results: All four dual-target receptor agonist fusion proteins showed good effects in improving glucose tolerance in mice, similar to commercially available dulaglutide, but slightly lower than tirzepatide. The effects were ranked from greatest to least: 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.

[0084] Example 6: In vivo efficacy evaluation in ob / ob mouse model

[0085] This embodiment evaluates the effects of four dual-target receptor agonist fusion proteins (GLP-1 / GIP, GIP / GLP-1, GLP-1 / GCG, GCG / GLP-1) obtained by fine purification in Example 1 on blood glucose in ob / ob model mice.

[0086] Experimental Methods: Fifty-six 5-6 week old C57BL / 6ob / ob mice, weighing 35-40g, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. They were randomly divided into seven groups (Dulaglutide group, Tirzepatide group, GLP-1 / GIP group, GIP / GLP-1 group, GLP-1 / GCG group, GCG / GLP-1 group, and Vehicle group) with eight mice in each group, based on blood glucose and body weight. Each group received a subcutaneous injection of the corresponding drug at a dose of 30 nmol / kg. For the Vehicle group, a solvent was injected subcutaneously. A Control group (normal C57BL / 6 mice) was also included, receiving a solvent injection subcutaneously. Injections were administered every three days for a total of 10 doses. Blood glucose, body weight, and food intake were measured before each administration. A glucose tolerance test was performed 72 hours after the last administration. Samples were processed and collected, liver weight was recorded, and liver pathology and blood biochemical indicators were measured in each group. The test results are shown in Table 5-10 and Figure 5.

[0087] Table 5: Effects of long-term administration of dual-target receptor agonist fusion protein on blood glucose levels in ob / ob mice

[0088] Table 6: Effects of long-term administration of dual-target receptor agonist fusion protein on glucose tolerance in ob / ob mice Note: In the same column, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0089] Table 7: Effects of long-term administration of dual-target receptor agonist fusion protein on body weight in ob / ob mice Note: Only the significance analysis results for D27 have been added. The same column headings indicate that * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0090] Table 8: Effects of long-term administration of dual-target receptor agonist fusion protein on food intake in ob / ob mice

[0091] Table 9: Effects of long-term administration of dual-target receptor agonist fusion protein on fat and liver weight in ob / ob mice Note: In the same column, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0092] Table 10: Effects of long-term administration of dual-target receptor agonist fusion protein on liver function and blood lipids in ob / ob mice Note: In the same column, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; HbA1c%: Glycated hemoglobin level; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TG: Triglycerides; TC: Total cholesterol; LDL: Low-density lipoprotein; HDL: High-density lipoprotein.

[0093] Experimental results: Compared with the 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 significantly reduced blood glucose in mice and maintained long-term stability. The effect on improving glucose tolerance in ob / ob mice was 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 significantly reduced mouse body weight and inhibited food intake in mice. The 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 all improved liver weight and fat content in mice, and significantly improved liver function and blood biochemical indicators. Furthermore, the GLP-1 / GCG and GCG / GLP-1 dual-target receptor agonist fusion proteins showed the best effect in improving hepatocyte vacuolation in mice (Figure 5). The GLP-1 / GCG dual-target receptor agonist fusion protein showed the best improvement in all aspects of metabolic mice, and its effect was superior to tirzepatide.

[0094] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dual-target receptor agonist fusion protein modified with an Fc fragment, characterized in that: The structure of the fusion protein is shown in Formula I: abcdef(Ⅰ) In the formula, each "-" independently represents a linking peptide, peptide bond, or isopeptide bond; a is a GLP-1 peptide, GIP peptide, or GCG peptide; b is an Fc fragment; c is a SpyCatcher peptide; d is a SpyTag peptide; e is an ELP peptide, serving as the third linker peptide; f is a GLP-1 peptide, GIP peptide, or GCG peptide; and a and f are not the same peptide at the same time. Wherein, a and b are connected by a first linker peptide, b and c are connected by a second linker peptide; c and d are connected by isopeptide bonds; and d, e and f are connected sequentially by peptide bonds. The C-terminus of a is connected to the N-terminus of the first linker peptide; the C-terminus of the first linker peptide is connected to the N-terminus of b; the C-terminus of b is connected to the N-terminus of the second linker peptide; the C-terminus of the second linker 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.

2. The Fc fragment-modified dual-target receptor agonist fusion protein according to claim 1, characterized in that: a is a GLP-1 peptide, f is a GIP peptide or a GCG peptide; or a is a GIP peptide or a GCG peptide, f is a GLP-1 peptide.

3. The Fc fragment-modified dual-target receptor agonist fusion protein according to claim 1 or 2, characterized in that: The amino acid sequence of the GLP-1 peptide is as shown in SEQ ID NO: 1; or, the amino acid sequence of the GLP-1 peptide is an amino acid sequence with at least 85%, 90%, 95%, or 99% sequence identity relative to SEQ ID NO: 1; or, the GLP-1 peptide has no more than 6, 5, 4, 3, 2, or 1 mutations relative to SEQ ID NO: 1 while retaining the basic biological activity of the peptide before mutation; or, the GLP-1 peptide is a peptide with good binding activity to the GLP-1 receptor: Cotadutide, Retatrutide, or Tirzepatide. The amino acid sequence of the GIP peptide is as shown in SEQ ID NO: 2; or, the amino acid sequence of the GIP peptide is an amino acid sequence with at least 85%, 90%, 95% or 99% sequence identity relative to SEQ ID NO: 2; or, the GIP peptide has no more than 6, 5, 4, 3, 2 or 1 mutations relative to SEQ ID NO: 2 while retaining the basic biological activity of the peptide before the mutation; or, the GIP peptide is a peptide with good binding activity to the GIP receptor: Retatrutide or Tirzepatide. The amino acid sequence of the GCG polypeptide is as shown in SEQ ID NO: 3; or, the amino acid sequence of the GCG polypeptide is an amino acid sequence with at least 85%, 90%, 95% or 99% sequence identity relative to SEQ ID NO: 3; or, the GCG polypeptide has no more than 6, 5, 4, 3, 2 or 1 mutations relative to SEQ ID NO: 3 while retaining the basic biological activity of the polypeptide before the mutation; or, the GCG polypeptide is a peptide with good binding activity to the GCG receptor: Cotadutide or Retatrutide. The Fc fragment is selected from the Fc fragment of humanized IgG4; The SpyCatcher peptide is SpyCatcher(ΔN) or a variant thereof, and the amino acid sequence of SpyCatcher(ΔN) is shown in SEQ ID NO: 8; The amino acid sequence of the SpyTag peptide shown is as shown in SEQ ID NO: 9, or a variant thereof; The first linker peptide and the second linker peptide each independently comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 5 and SEQ ID NO: 6; The amino acid sequence of the ELP peptide is shown in SEQ ID NO:

7.

4. The Fc fragment-modified dual-target receptor agonist fusion protein according to claim 3, characterized in that: The amino acid sequence of the Fc fragment is shown in SEQ ID NO:

4.

5. 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 according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleic acid molecule includes the nucleic acid molecule shown in SEQ ID NO: 14 (589-1704bp) and the nucleic acid molecule shown in SEQ ID NO: 17 (589-987bp), or the nucleic acid molecule shown in SEQ ID NO: 16 (589-1737bp) and the nucleic acid molecule shown in SEQ ID NO: 15 (589-954bp); used to encode the fusion protein required for forming the GLP-1 and GIP dual-target receptor agonist fusion protein; The nucleic acid molecules include those shown in SEQ ID NO: 14 (589-1704bp) and SEQ ID NO: 19 (589-948bp), or those shown in SEQ ID NO: 18 (589-1698bp) and SEQ ID NO: 15 (589-954bp); used to encode the fusion protein required for forming the GLP-1 and GCG dual-target receptor agonist fusion protein.

7. The Fc fragment modified bi-target receptor agonist fusion protein related biomaterial according to any one of claims 1 to 4, characterized by: It can be 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 5 to 6; (2) An expression vector comprising the nucleic acid molecule according to any one of claims 5 to 6; (3) An expression carrier comprising the expression box described in (1); (4) Engineered bacteria comprising the nucleic acid molecules according to any one of claims 5 to 6; (5) Engineered bacteria containing the expression cassette described in (1); (6) Engineered bacteria containing the expression vector described in (2) or (3).

8. A pharmaceutical composition, characterized by: The invention comprises a dual-target receptor agonist fusion protein modified with the Fc fragment as described in any one of claims 1 to 4, a nucleic acid molecule as described in any one of claims 5 to 6, or a biological material as described in claim 7.

9. Use of the Fc fragment modified dual target receptor agonist fusion protein according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5 to 6, the biomaterial according to claim 7 or the pharmaceutical composition according to claim 8 for the manufacture of a medicament, characterized in that: The medication is used to control or reduce blood sugar and / or weight.

10. Use of the Fc fragment modified dual target receptor agonist fusion protein according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5 to 6, the biomaterial according to claim 7 or the pharmaceutical composition according to claim 8 for the manufacture of a medicament, characterized in that: The drug is used for the prevention and / or treatment of at least one of type 2 diabetes, obesity, metabolic dysfunction-related steatohepatitis, dyslipidemia, and metabolic syndrome.

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