A triple-target receptor agonist fusion protein and uses thereof

Abstract

The application discloses a three-target receptor agonist fusion protein and application thereof, and relates to the field of biological medicine engineering.The fusion protein is a three-target receptor agonist fusion protein (GLP-1, GIP and GCG) fused with an antibody Fc fragment at a natural N end, which can be rapidly prepared by using a more efficient and low-cost bacterial expression system and has high in-vitro agonist activity.The fusion protein is obtained in one step by self-connection of SpyCatcher / SpyTag and DogCatcher / DogTag without purification, and is fused with a human antibody Fc fragment, so that the fusion protein can well improve mouse sugar tolerance and greatly improve the yield.The fusion protein can be applied to production of a new type of drug of a polypeptide / protein with a natural N end and modified by an antibody Fc fragment.

Description

Technical Field

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

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized primarily by insulin resistance, accompanied by impaired pancreatic β-cell function, leading to elevated blood glucose levels. Glucagon-like peptide-1 receptor (GLP-1R) is one of the most effective targets for treating type 2 diabetes. GLP-1 is an incretin that improves and controls blood glucose levels by promoting insulin secretion and inhibiting glucagon (GCG) secretion, thus treating type 2 diabetes. Furthermore, GLP-1 can slow gastric emptying and inhibit gastrointestinal motility (Andersen, A. et al., Nature Reviews Endocrinology, 2018, 14, 390-403), achieving weight loss and therefore is also used to treat obesity and overweight. Natural GLP-1 has a half-life of only 2-3 minutes in vivo, which is insufficient for drug development. Long-acting modification is one of the challenges in developing glucagon-like peptide-1 receptor agonists (GLP-1RAs). Furthermore, these drugs also face adverse reactions such as nausea, vomiting, diarrhea, or constipation.

[0003] Another incretin-dependent glucose-dependent insulinotropic polypeptide receptor (GIP) also has a similar function in promoting insulin secretion (Lauritsen KB, Danish medical bulletin, 1983, 304, 205-14). Studies on its mechanism of action have found that GIP synergistically works with GLP-1 to reduce the gastrointestinal adverse reactions of GLP-1RAs (Hayes et al., Diabetes, 2021, 70, 2545-2553). GCG, secreted by pancreatic α cells, can mobilize lipolysis, inhibit food intake, prolong satiety, and increase energy expenditure (Prato et al., 2021, Obesity Reviews. 2022, 23: e13372). With in-depth research into the mechanisms of action of these hormones and receptors, the development of next-generation multi-target receptor agonists has become a mainstream research direction. Tirzepatide is a single-molecule dual-target GLP-1 / GIP agonist approved by the U.S. Food and Drug Administration (FDA) for improving glycemic control in adults with type 2 diabetes in combination with diet and exercise, and as a chronic weight management drug for weight loss and control in obese or overweight adults with weight-related diseases (FDA Approves New Medication for Chronic Weight Management, FDA News Release, 2023). Mazdutide is a single-molecule dual-target GLP-1 / GCG agonist, also approved for treating obesity or overweight and for glycemic control in type 2 diabetes (Ji L, New England Journal of Medicine, 2025, 392, 2215-2225). Retatrutide, a single-molecule triple-target GLP-1 / GIP / GCG agonist currently undergoing phase III clinical trials (Cell Metabolism, 2022, 34, 1234-1247). However, the design of these single-molecule agonists requires the high homology of the peptide sequence. During the design process, it is necessary to retain the natural N-terminus to maintain biological activity, and to precisely regulate the affinity of the molecule for different targets to adjust the efficacy distribution for each target. Therefore, 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 requires no chemical reagents, is rapid, and operates under mild conditions (Zakeri et al., Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(12): E690-E697). The modified SpyCatcher (ΔN) retains its activity while exhibiting lower immunogenicity (Zhida Liu et al., Scientific reports, 2014, 4, 7266), showing broad application prospects. Similar reaction systems include DogCatcher and DogTag (Keeble et al., Cell Chemical Biology, 2022, 29, 339-350), and these two reaction systems are orthogonal and do not interfere with each other.

[0005] Therefore, there is still a need for further optimization in the design and preparation of GLP-1-based dual / multi-target receptor agonists. There is an urgent need to develop novel multi-target agonists with longer duration of action, better hypoglycemic and weight loss efficacy, 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 three-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 ultra-short half-life of GLP-1 limit its clinical application. Combining it with other targets such as GIP and GCG to form dual / multi-target receptor agonists and prolonging its half-life are the main research directions for GLP-1 RAs. Currently, dual / multi-target receptor agonists in clinical trials mainly involve designing two / more peptides into a single chimeric peptide that simultaneously activates two / more targets. Examples include Tirzepatide, a GLP-1 / GIP dual-target receptor agonist approved by the FDA in May 2022; Mazdutide, a GLP-1 / GCG dual-target receptor agonist approved by the NMPA in June 2025; and Retatrutide, a GLP-1 / GIP / GCG triple-target receptor agonist currently undergoing 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. In addition, peptides such as GLP-1, GIP, and GCG require the exposure of their natural N-terminus to retain their high activity against receptors, 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 three-target receptor agonist fusion protein, and more specifically, provides a three-target receptor agonist fusion protein modified with an antibody Fc fragment, the structure of which is shown in Formula I or II below:

[0011] abcd(-fcg)-ce(-hci)(I)

[0012] ad(-fcg)-ce(-hci)-cb(II)

[0013] In the formula, each "-" independently represents a linking peptide, peptide bond, or isopeptide bond;

[0014] a is a GLP-1 peptide; b is an Fc fragment; c is an ELP peptide, serving as the second linker peptide; d is a SpyTag peptide; e is a DogTag peptide; f is a SpyCatcher peptide; g is a GIP peptide or a GCG peptide; h is a DogCatcher peptide; i is a GIP peptide or a GCG peptide; and g and i are not the same peptide at the same time.

[0015] Furthermore, g is a GIP peptide and i is a GCG peptide; or g is a GCG peptide and i is a GIP peptide.

[0016] Specifically, for structural formula I, a and b are connected by a first linker peptide, and b, c, d, c and e are connected sequentially by peptide bonds; g, c and f are connected sequentially by peptide bonds; i, c and h are connected sequentially by peptide bonds; d and f are connected by isopeptide bonds; and e and h are connected by isopeptide bonds.

[0017] Furthermore, regarding structural formula I, 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 d; the C-terminus of d is connected to the N-terminus of the second linker peptide; the N-terminus of e is connected to the C-terminus of the second linker peptide; the C-terminus of g 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 f; the C-terminus of i is connected to the N-terminus of the second linker peptide; and the C-terminus of the second linker peptide is connected to the N-terminus of h.

[0018] Specifically, for structure II, a and d are connected by a first linker peptide, and d, c, e, c and b are connected sequentially by peptide bonds; g, c and f are connected sequentially by peptide bonds; i, c and h are connected sequentially by peptide bonds; d and f are connected by isopeptide bonds; and e and h are connected by isopeptide bonds.

[0019] Furthermore, regarding structural formula II, 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 d; the C-terminus of d 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 e; the C-terminus of e 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 b; the C-terminus of g 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 f; the C-terminus of i is connected to the N-terminus of the second linker peptide; and the C-terminus of the second linker peptide is connected to the N-terminus of h.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] In some embodiments, the GLP-1 peptide, GIP peptide, and GCG peptide can 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.

[0024] Furthermore, the Fc fragment is selected from the Fc fragment of humanized IgG4.

[0025] In some embodiments, the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 4.

[0026] In some embodiments, the amino acid sequence of the first linker peptide is as shown in SEQ ID NO: 5: GGGGSGGGGSGGGSA.

[0027] In some embodiments, the amino acid sequence of the ELP peptide is shown in SEQ ID NO: 6.

[0028] 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.

[0029] In some embodiments, the SpyCatcher peptide is an N-terminal truncated form (SpyCatcher(ΔN)), whose amino acid sequence is shown in SEQ ID NO: 7; the SpyTag peptide is the original sequence, whose amino acid sequence is shown in SEQ ID NO: 8.

[0030] In some embodiments, the amino acid sequence of the DogCatcher peptide is shown in SEQ ID NO: 9; and the amino acid sequence of the DogTag peptide is shown in SEQ ID NO: 10.

[0031] A second aspect of 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, GIP, and GCG triple-target receptor agonist fusion protein. The fusion proteins encoded by the nucleic acid molecule according to embodiments of the present invention all possess excellent binding activity with GLP-1, GIP, and GCG receptors. The half-maximal effective concentration (EC50) of GLP-1(-Fc) / GIP / GCG for in vitro agonistic activity against the GLP-1 receptor is [not specified in the original text]. 50 The half-maximal effective concentration (EC50) for in vitro agonistic activity against the GIP receptor was 0.520 ± 0.040 nM. 50 The half-maximal effective concentration (EC50) for in vitro agonistic activity against the GCG receptor was 1.853 ± 0.335 nM. 50 The half-maximal effective concentration (CMC) of GLP-1 / GIP / GCG(-Fc) for in vitro agonistic activity against GLP-1 receptor was 0.894 ± 0.081 nM, and the half-maximal effective concentration (CMC) for in vitro agonistic activity against GIP receptor was 0.041 ± 0.007 nM. 50 The concentration was 0.831 ± 0.208 nM, and the half-maximal effective concentration (MCD) for in vitro agonistic activity against GCG receptors was 0.037 ± 0.005 nM; it has hypoglycemic activity and can effectively control or reduce blood glucose levels.

[0032] Furthermore, the nucleic acid molecule includes the nucleic acid molecule shown in SEQ ID NO: 11 (589-1989bp), the nucleic acid molecule shown in SEQ ID NO: 13 (589-1221bp), and the nucleic acid molecule shown in SEQ ID NO: 14 (589-1221bp), or the nucleic acid molecule shown in SEQ ID NO: 12 (589-1989bp), the nucleic acid molecule shown in SEQ ID NO: 13 (589-1221bp), and the nucleic acid molecule shown in SEQ ID NO: 14 (589-1221bp); used to encode the fusion protein required for forming the GLP-1, GIP, and GCG three-target receptor agonist fusion protein.

[0033] The nucleic acid molecules include those shown in SEQ ID NO: 11 (589-1989bp), SEQ ID NO: 15 (589-1182bp), and SEQ ID NO: 16 (589-1260bp), or those shown in SEQ ID NO: 12 (589-1989bp), SEQ ID NO: 15 (589-1182bp), and SEQ ID NO: 16 (589-1260bp); used to encode the fusion protein required for forming the GLP-1, GCG, and GIP tri-target receptor agonist fusion protein.

[0034] 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.

[0035] 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, GIP, and GCG three-target receptor agonist fusion protein and a GLP-1, GCG, and GIP three-target receptor agonist fusion protein through Spy-chemistry peptide and Dog-chemistry bioconjugation. The GLP-1, GIP, and GCG three-target receptor agonist fusion protein exhibits excellent binding activity to GLP-1, GIP, and GCG receptors, possesses hypoglycemic activity, and can effectively control or reduce blood glucose levels.

[0036] A pharmaceutical composition is provided in a fifth aspect of the present invention. According to embodiments of the invention, it comprises the three-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 hypoglycemic effect and can effectively control or reduce blood glucose levels.

[0037] In a sixth aspect, the present invention proposes the use of the three-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 in the preparation of a drug. According to embodiments of the present invention, the drug is used to control or lower blood glucose; the drug is used to prevent and / or treat at least one of diseases such as type 2 diabetes and obesity.

[0038] A method for preparing the three-target receptor agonist fusion protein described in the first aspect is provided in the seventh aspect of the present invention. 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 engineered bacteria to express the fusion protein, wherein the fusion protein forms a GLP-1, GIP, and GCG three-target receptor agonist fusion protein, or a GLP-1, GCG, and GIP three-target receptor agonist fusion protein, through a Spy-chemistry peptide and Dog-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, GIP, and GCG receptors, exhibits hypoglycemic activity, and can effectively control or reduce blood glucose levels.

[0039] In an eighth aspect of the invention, a method for lowering blood glucose levels in a patient is provided. According to embodiments, this includes administering to a patient at least one of the following: 1) the three-target receptor agonist fusion protein of the first aspect; 2) the nucleic acid molecule of the second aspect; 3) the expression cassette or expression vector of the third aspect; 4) the engineered bacteria of the fourth aspect; and 5) the pharmaceutical composition of the fifth aspect. The method according to embodiments of the invention can effectively and sustainably control or lower a patient's blood glucose levels.

[0040] The present invention has the following advantages and effects compared with the prior art:

[0041] This invention provides a three-target receptor agonist fusion protein, which is a natural N-terminal three-target receptor agonist fusion protein (GLP-1, GIP, and GCG) fused with an antibody Fc fragment. It can be rapidly prepared using a more efficient and cost-effective bacterial expression system and exhibits high in vitro and in vivo agonistic activity. This fusion protein requires no purification and can be obtained in one step via self-ligation of SpyCatcher / SpyTag and DogCatcher / DogTag (GLP-1, GIP, and GCG), and incorporates a human antibody Fc fragment. It exhibits nanomolar-level in vitro agonistic activity against different receptors, effectively improves glucose tolerance in mice, and significantly increases yield. This fusion protein can be used to produce novel drugs with naturally N-terminal peptides / proteins modified with antibody Fc fragments. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the process for preparing a three-target receptor agonist fusion protein based on icSAT purification and Spy-chemistry and Dog-chemistry bioconjugation.

[0043] Figure 2 The results are SDS-PAGE of the three-target receptor agonist fusion proteins prepared by icSAT purification and Spy-chemistry and Dog-chemistry bioconjugation. Among them, A: GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion protein, B: GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion protein, C: GLP-1(-Fc) / GCG / GIP three-target receptor agonist fusion protein, and D: GLP-1 / GCG / GIP(-Fc) three-target receptor agonist fusion protein.

[0044] Figure 3 The results are SDS-PAGE of the three-target receptor agonist fusion protein purified using Protein A; where A: GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion protein, and B: GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion protein.

[0045] Figure 4 These are the molecular weight determination results of the GLP-1, GIP, and GCG three-target receptor agonist fusion protein; where A: GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion protein, and B: GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion protein.

[0046] Figure 5 These are the affinity assay results of the antibody Fc fragment of the three-target receptor agonist fusion protein with human FcRn; where A: GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion protein, B: GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion protein, and C: Affinity assay results of the antibody Fc fragment of Dulaglutide (Lilly) with human FcRn.

[0047] Figure 6These are the results of in vitro agonistic activity assays of three-target receptor agonist fusion proteins for GLP-1R, GIPR, or GCGR; where A: results of in vitro agonistic activity assays of GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion proteins and GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion proteins for GLP-1R; B: results of in vitro agonistic activity assays of GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion proteins and GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion proteins for GIPR; C: results of in vitro agonistic activity assays of GLP-1(-Fc) / GIP / GCG three-target receptor agonist fusion proteins and GLP-1 / GIP / GCG(-Fc) three-target receptor agonist fusion proteins for GCGR.

[0048] Figure 7 This study examines the effect of a three-target receptor agonist fusion protein on glucose tolerance in normal mice; where A represents the blood glucose concentration-time curve plotted at different time points; and B represents the AUC. 0~90 min Values. Note: Compared to Control, **** P < 0.0001; compared to Retatrutide, ## P < 0.01, ### P < 0.001, #### P < 0.0001. Detailed Implementation

[0049] 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 Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.

[0050] 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.

[0051] 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.

[0052] In this article, the terms “substitution” or “mutation” refer to replacing one amino acid in a polypeptide with another amino acid.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The fusion protein provided by this invention can use a natural antibody Fc fragment or an Fc fragment variant. In a preferred embodiment, an Fc fragment variant is used.

[0058] 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 rigid linker peptides, specifically PT-type linker peptides containing proline and threonine.

[0059] In this article, the GLP-1, GIP, and GCG three-target receptor agonist fusion protein refers to a three-target receptor agonist fusion protein containing three peptide components: GLP-1, GIP, and GCG. Specifically, the C-terminus of the GIP peptide and the C-terminus of the GCG peptide are respectively linked to the N-terminus of an independent second linker peptide, and the C-terminus of the second linker peptide is linked to the N-terminus of SpyCatcher (ΔN) and DogCatcher, respectively. The GLP-1 / GIP / GCG three-target receptor agonist fusion protein is obtained by coupling via Spy-chemistry and Dog-chemistry.

[0060] A schematic diagram of the process for preparing a three-target receptor agonist fusion protein based on icSAT purification and Spy-chemistry / Dog-chemistry bioconjugation is shown below. Figure 1 As shown.

[0061] In the figure of Example 1, ES: cell lysate supernatant; EP: cell lysate precipitate; ES1, ES2, ES3: cell lysate supernatants 1, 2, and 3; Mix: mixture of ES1, ES2, and ES3; 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; M1 and M2: protein markers; BSA: bovine serum albumin standard.

[0062] 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).

[0063] Example 1: Preparation of a GLP-1, GIP, and GCG three-target receptor agonist fusion protein based on icSAT purification and Spy-chemistry and Dog-chemistry conjugation.

[0064] This embodiment provides a method for the biosynthesis of a GLP-1-containing three-target receptor agonist fusion protein.

[0065] In this embodiment, the expression vector used to synthesize the GLP-1, GIP, and GCG tri-target receptor agonist fusion protein was pET30a-EFK8-Mtu ΔI-CM(m2)-GLP-1-Fc-ELP. 15 -SpyTag-ELP 15 -DogTag, pET30a-EFK8-Mtu ΔI-CM(m2)-GLP-1-SpyTag-ELP 15 -DogTag-ELP 15 -Fc, pET30a-EFK8-Mtu ΔI-CM(WT)-GIP-ELP 15 -SpyCatcher(ΔN) and pET30a-EFK8-Mtu ΔI-CM(m2)-GCG-ELP 15 -DogCatcher. The expression vector used to synthesize the GLP-1, GCG, and GIP tri-target receptor agonist fusion protein in this embodiment was pET30a-EFK8-Mtu ΔI-CM(m2)-GLP-1-Fc-ELP. 15 -SpyTag-ELP 15 -DogTag, EFK8-Mtu ΔI-CM(m2)-GLP-1-SpyTag-ELP 15 -DogTag-ELP 15 -Fc, pET30a-EFK8-Mtu ΔI-CM(m2)-GCG-ELP 15 -SpyCatcher(ΔN) and pET30a-EFK8-Mtu ΔI-CM(WT)-GIP-ELP 15 -DogCatcher. The target fragment is inserted between the NdeⅠ and XhoⅠ restriction sites of the vector pET30a(+) (purchased from Novagen). In this example, the fusion protein EFK8-Mtu ΔI-CM(m2)-GLP-1-Fc-ELP 15 -SpyTag-ELP 15 The gene sequence encoding -DogTag is shown in SEQ ID NO: 11. Fusion protein EFK8-Mtu ΔI-CM(m2)-GLP-1-SpyTag-ELP 15 -DogTag-ELP 15 The gene sequence encoding -Fc is shown in SEQ ID NO: 12. Fusion protein EFK8-Mtu ΔI-CM(WT)-GIP-ELP15 The gene sequence encoding -SpyCatcher(ΔN) is shown in SEQ ID NO: 13. The fusion protein is EFK8-Mtu ΔI-CM(m2)-GCG-ELP. 15 The gene sequence encoding DogCatcher is shown in SEQ ID NO: 14. The fusion protein EFK8-Mtu ΔI-CM(m2)-GCG-ELP 15 The gene sequence encoding -SpyCatcher(ΔN) is shown in SEQ ID NO: 15. The fusion protein EFK8-Mtu ΔI-CM(WT)-GIP-ELP 15 The coding gene sequence for -DogCatcher is shown in SEQ ID NO: 16. The initial ATG and the final TGA, TAA, and TAG are the start and stop codons, respectively.

[0066] In this embodiment, the salinity-induced self-assembly peptide used is EFK8, whose amino acid sequence is shown in SEQ ID NO: 17; EFK8 is linked to an inteptide via a PT linker, the amino acid sequence of which is shown in SEQ ID NO: 18; the inteptide is Mtu ΔI-CM(WT) or Mtu ΔI-CM(m2), whose amino acid sequences are shown in SEQ ID NO: 19 and SEQ ID NO: 20. The coding gene sequence of EFK8-Mtu ΔI-CM(m2) is shown in SEQ ID NO: 11, 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.

[0067] The preparation method was as follows: The expression vector used to synthesize the three-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 20 min), and centrifuged at 15,000 g for 30 min at 4 ℃ to separate the expression supernatant and bacterial fragments. SDS-PAGE was performed on the expression supernatant for protein quantification. Using the fusion protein containing SpyTag and DogTag as the backbone, the protein was mixed at a Catcher fusion protein: Tag fusion protein molar ratio of 1:1 and reacted at 80 rpm at 25 ℃ for 2 hours to form the three-target receptor agonist fusion protein containing the 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 incubated at 4 °C for 12 hours to precipitate the protein. The supernatant and protein precipitate were separated by centrifugation at 15,000 g for 30 minutes at 4 °C. The precipitate was resuspended thoroughly in half the volume of Na2SO4-free cleavage Buffer B4 (NaCl-free PBS, 20 mM Tris-base, 2 mM 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 15,000 g for 30 minutes at 4 °C yielded the icSAT-tagged three-target receptor agonist fusion protein in the supernatant. SDS-PAGE was used to determine the purity and yield of the three-target receptor agonist fusion protein in the supernatant.

[0068] Figure 2 The SDS-PAGE results are shown for the three-target receptor agonist fusion protein prepared by icSAT purification and Spy-chemistry and Dog-chemistry bioconjugation. The data involved in the preparation and purification of the three-target receptor agonist fusion protein are shown in Table 1.

[0069] Table 1. Data related to the preparation and purification of the tri-target receptor agonist fusion protein.

[0070]

[0071] Note: a Aggregation efficiency (%) = (Reduction in fusion protein in supernatant after aggregation / Amount of fusion protein in supernatant before aggregation) × 100%. b Cutting efficiency (%) = Reduction in fusion protein after cutting / Amount of fusion protein before cutting × 100% cPurity (%) after icSAT = Amount of target protein / (Amount of target protein + Amount of other proteins) × 100%.

[0072] Example 2: Protein Affinity Purification

[0073] Two GLP-1 / GIP / GCG tri-target receptor agonist fusion proteins from Example 1 were selected and further purified using Protein A affinity purification. The specific steps were as follows: The supernatant of the icSAT-purified tri-target receptor agonist fusion protein was filtered through a 0.22 µm filter membrane, while simultaneously washing the column and purification system with 5-10 column volumes of ultrapure water. The column was then equilibrated with 5-10 column volumes of equilibration buffer (20 mM PB, 150 mM NaCl, pH 7.4). The filtered supernatant was then flow-through the column until all the sample had flowed through, and then washed 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 washed with 1 M acetic acid solution and 0.5 M NaOH solution for 5-10 column volumes to remove some contaminating proteins and regenerated packing material. 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 determine the purity and yield of the fusion protein.

[0074] Figure 3 The SDS-PAGE results of the three-target receptor agonist fusion protein purified using Protein A are shown in Table 2.

[0075] Table 2. Data related to the fine purification of the tri-target receptor agonist fusion protein.

[0076]

[0077] Note: a Purity (%) of Protein A after purification = Amount of target protein / (Amount of target protein + Amount of other proteins) × 100%. b Recovery rate (%) = Amount of target protein after purification / Amount of target protein before purification × 100%.

[0078] Example 3: Molecular weight identification

[0079] The molecular weights of the two tri-target receptor agonist fusion proteins (GLP-1(-Fc) / GIP / GCG and GLP-1 / GIP / GCG(-Fc)) obtained by fine purification in Example 2 were determined. Ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF) and a Biozen™ C4 column were used to determine the molecular weights of the samples. The conditions were: injection volume 10 μL, flow rate 0.6 mL / min, column temperature 70 ℃, 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 15–40% B, and elution was performed for 7 min. The results are as follows: Figure 4 As shown. The results show that the actual molecular weight of the GLP-1(-Fc) / GIP / GCG tri-target receptor agonist fusion protein is 89672.26 Da, consistent with the theoretical molecular weight (89675.25 Da). Figure 4 The actual molecular weight of the GLP-1 / GIP / GCG(-Fc) tri-target receptor agonist fusion protein is 89675.93 Da, consistent with the theoretical molecular weight (89675.25 Da). Figure 4 (B in the middle).

[0080] Example 4: Determination of the affinity between Fc and FcRn

[0081] To verify the affinity of the antibody Fc fragments of the two finely purified three-target receptor agonist fusion proteins (GLP-1(-Fc) / GIP / GCG and GLP-1 / GIP / GCG(-Fc)) obtained in Example 2 for human FcRn, affinity assays were performed. The concentrations of the purified three-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). First, the Streptavidin 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). Then, 1 µg / mL of human FcRn (ACRO, UK) working solution was fixed onto the SA biosensor in the kinetic buffer. The SA biosensor was then equilibrated in the kinetic buffer. Finally, the equilibrated sensor was compared with the target tri-target receptor agonist fusion protein sample (concentration gradients of 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 15.625 nM). The three-target receptor agonist fusion protein samples (nM and 0nM) were affinity-treated in kinetic buffer for 200 seconds, and then the affinity-treated sensor was dissociated in kinetic buffer (phosphate buffer containing 0.1% bovine serum albumin BSA and 0.02% Tween-20, pH 7.4) for 200 seconds. Binding and dissociation kinetics at some concentrations are shown below. Figure 5 As shown in Table 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.

[0082] Table 3. Correlation constants of the tri-target receptor agonist fusion protein

[0083]

[0084] Two tri-target receptor agonist fusion proteins (GLP-1(-Fc) / GIP / GCG and GLP-1 / GIP / GCG(-Fc)) have a high affinity for FcRn, which is superior to the affinity between Dulagultide and FcRn. This can maintain the antibody Fc fragment and prolong the half-life of the tri-target receptor agonist fusion protein (Glaesner et al., Diabetes / Metabolism Research and Reviews, 2010, 26(4): 287-296).

[0085] Example 5: In vitro activity verification

[0086] To verify the binding activity of the two finely purified tri-target receptor agonist fusion proteins (GLP-1(-Fc) / GIP / GCG and GLP-1 / GIP / GCG(-Fc)) obtained in Example 2 with GLP-1R (GLP-1 receptor), GIPR (GIP receptor) and GCGR (GCG receptor), the cAMP Gs Dynamic Kit (Revvity, US) and HEK293 / GLP-1R, HEK293 / GIPR and HEK293 / GCGR cells stably expressing GLP-1R (or GIPR or GCGR) were used. The principle is that when GLP-1, GIP, and GCG specifically bind to GLP-1R / GIPR / GCGR on the cell membrane, adenylate cyclase is activated, promoting the conversion of intracellular ATP to cyclic adenosine monophosphate (cAMP) for signal transmission. The cAMP expression level can be assessed by detecting the FRET signal using HTRF (homogeneous time-resolved fluorescence) technology, thereby evaluating the biological activity of the two three-target receptor agonist fusion proteins. Experimental results are presented using the median effective concentration (EC50). 50 ) indicates that EC 50 The lower the value, the stronger the biological activity.

[0087] First, a standard curve for cAMP was established. The cAMP standard was diluted to 2848 nM with Stimulation Buffer 1 as the starting concentration, and then serially diluted 4-fold. The diluted standard was transferred to 384-well plates (white background) at 5 μL / well, with three replicates. 5 μL of Stimulation Buffer 1, 5 μL of cAMP-d2 (receptor marker) working solution, and 5 μL of europium-Cryptate-labeled anti-cAMP monoclonal antibody (donor) working solution were added to each well sequentially. The plates were sealed and incubated at room temperature in the dark for 1 hour. Fluorescence signals were detected at 620 nm and 665 nm using a microplate reader compatible with HTRF mode, and the HTRF ratio (665 nm / 620 nm × 10⁻¹⁰) was calculated. 4 This is used to establish a standard curve.

[0088] To determine the in vitro agonistic activity of samples on HEK293 / GLP-1R cells (or HEK293 / GIPR or HEK293 / GCGR), two tri-target receptor agonist fusion proteins (GLP-1(-Fc) / GIP / GCG and GLP-1 / GIP / GCG(-Fc)) and control GLP-1 (or GIP or GCG (SEQ ID NO: 3)) need to be diluted to appropriate concentrations with Stimulation Buffer 1 as the starting concentration (2× working concentration), followed by step-by-step dilutions at 5-fold concentrations. HEK293 / GLP-1R (or HEK293 / GIPR or HEK293 / GCGR) cells were digested and diluted with Stimulation Buffer 1 to a concentration of 4×10⁻⁶. 6 Cell suspensions of 5 μL / well were transferred to 384-well plates, with three replicates. A negative control group, a basal cell control group, and a sample group were set up. 5 μL of Stimulation Buffer 1, Stimulation Buffer 1, and 5 μL of sample were added to each well, respectively. The 384-well plates were sealed and protected from light, and incubated at 37 °C for 30 min to induce receptor activation and promote intracellular cAMP accumulation. After incubation, 5 μL of Lysis & Detection Buffer, 5 μL of cAMP-d2 (receptor marker) working solution, and 5 μL of cAMP-d2 (receptor marker) working solution were added to each well, respectively. Finally, 5 μL of europium (Europium) Cryptoate-labeled anti-cAMP monoclonal antibody (donor) working solution was added to each well, allowing intracellularly generated endogenous cAMP to competitively bind to cAMP-d2. The microplate was incubated at room temperature in the dark for 1 hour. Fluorescence signals were detected at 620 nm and 665 nm wavelengths using a microplate reader compatible with HTRF mode, and the HTRF ratio (665 nm / 620 nm × 10⁻¹⁰) was calculated. 4 This allows for the establishment of a dose-response curve and the calculation of EC. 50 value.

[0089] The results are as follows Figure 6 As shown in Table 4; the in vitro agonistic activity assays of the GLP-1(-Fc) / GIP / GCG tri-target receptor agonist fusion protein and the GLP-1 / GIP / GCG(-Fc) tri-target receptor agonist fusion protein against GLP-1R, GIPR, and GCGR are as follows. Figure 6 As shown.

[0090] Table 4. Results of in vitro activity verification of the three-target receptor agonist fusion protein

[0091]

[0092] The two three-target receptor agonist fusion proteins exhibit strong in vitro binding activity with GLP-1R, GIPR, and GCGR, and the fusion protein architecture does not affect the activity of peptides such as GLP-1.

[0093] Example 6: Glucose Tolerance Assessment

[0094] This embodiment evaluates the effects of two finely purified tri-target receptor agonist fusion proteins, GLP-1(-Fc) / GIP / GCG and GLP-1 / GIP / GCG(-Fc), obtained in Example 2, on glucose tolerance in normal C57BL / 6 mice.

[0095] Experimental Methods: Normal C57BL / 6 mice, 5–6 weeks old, were purchased from Zhuhai Beston Biotechnology Co., Ltd. They were randomly divided into 5 groups (Control group, Retatrutide group, Dulaglutide group, GLP-1(-Fc) / GIP / GCG group, and GLP-1 / GIP / GCG(-Fc) group) according to blood glucose and body weight, with 6 mice in each group. For the Retatrutide group, Dulaglutide group, GLP-1(-Fc) / GIP / GCG group, and GLP-1 / GIP / GCG(-Fc) group, each animal was subcutaneously injected with the corresponding drug at a dose of 10 nmol / kg; for the Control group, the corresponding solvent was injected subcutaneously.

[0096] 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. 0~90 min The experimental results are shown in Table 5 and Figure 7 As shown.

[0097] Table 5. Effects of a single dose of the tri-target receptor agonist fusion protein on glucose tolerance in normal mice 24 hours after administration.

[0098]

[0099] Note: In the same column, **** indicates P < 0.0001.

[0100] Experimental Results: Both tri-target receptor agonist fusion proteins showed good effects in improving glucose tolerance in mice, similar to commercially available dulaglutide, but slightly less effective than tirzepatide. The effects, ranked from greatest to least, were: Retatrutide > Dulaglutide > GLP-1 / GIP / GCG(-Fc) > GLP-1(-Fc) / GIP / GCG. Therefore, both tri-target receptor agonist fusion proteins could significantly reduce blood glucose levels in mice.

[0101] 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 three-target receptor agonist fusion protein, characterized in that, The structure of the fusion protein is shown in formula I or II below: I: abcdcehci, and d is connected to f in fcg via an isopeptide bond, and e is connected to h via an isopeptide bond; II: adcecb, and d is connected to f in fcg via an isopeptide bond, and e is connected to h in hci via an isopeptide bond; In the formula, each "-" independently represents a linking peptide or peptide bond; a is a GLP-1 peptide; b is an Fc fragment; c is an ELP peptide, serving as the second linker peptide; d is a SpyTag peptide; e is a DogTag peptide; f is a SpyCatcher peptide; g is a GIP peptide or a GCG peptide; h is a DogCatcher peptide; i is a GIP peptide or a GCG peptide; and g and i are not the same peptide at the same time. In Formula I, a and b are connected by a first linker peptide, and b, c, d, c and e are connected sequentially by peptide bonds; g, c and f are connected sequentially by peptide bonds; i, c and h 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 d; the C-terminus of d is connected to the N-terminus of the second linker peptide; the N-terminus of e is connected to the C-terminus of the second linker peptide; the C-terminus of g 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 f; the C-terminus of i 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 h. In Formula II, a and d are connected by a first linker peptide, and d, c, e, c and b are connected in sequence by peptide bonds; g, c and f are connected in sequence by peptide bonds; i, c and h are connected in sequence 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 d; the C-terminus of d 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 e; the C-terminus of e 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 b; the C-terminus of g 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 f; the C-terminus of i 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 h.

2. The three-target receptor agonist fusion protein according to claim 1, characterized in that: The amino acid sequence of the GLP-1 peptide is as shown in SEQ ID NO: 1; or, the GLP-1 peptide is an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity with respect 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 the mutation; or, the GLP-1 peptide is a peptide with good binding activity to the GLP-1 receptor: Cotadutide, Retatrutide, or Tirzepatide. And / or, the amino acid sequence of the GIP peptide is as shown in SEQ ID NO: 2; or, the GIP peptide is an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with respect 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. And / or, the amino acid sequence of the GCG peptide is as shown in SEQ ID NO: 3; or, the GCG peptide is an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with respect to SEQ ID NO: 3; or, the GCG peptide 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 peptide before the mutation; or, the GCG peptide is a peptide with good binding activity to the GCG receptor: Cotadutide or Retatrutide. And / or, the SpyCatcher peptide is SpyCatcher(ΔN) or a variant thereof, the amino acid sequence of which is shown in SEQ ID NO: 7; And / or, the amino acid sequence of the SpyTag peptide is as shown in SEQ ID NO: 8; or a variant thereof; And / or, the amino acid sequence of the DogCatcher peptide is shown in SEQ ID NO: 9; the amino acid sequence of the DogTag peptide is shown in SEQ ID NO: 10; And / or, the Fc fragment is selected from the Fc fragment of humanized IgG4; And / or, the amino acid sequence of the first linker peptide is as shown in SEQ ID NO: 5; And / or, the amino acid sequence of the ELP peptide is shown in SEQ ID NO:

6.

3. The three-target receptor agonist fusion protein according to claim 2, characterized in that: The amino acid sequence of the Fc fragment is shown in SEQ ID NO:

4.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the fusion protein required to form the three-target receptor agonist fusion protein according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that: The nucleic acid molecules include those shown in SEQ ID NO: 11 (589-1989bp), SEQ ID NO: 13 (589-1221bp), and SEQ ID NO: 14 (589-1221bp), or those shown in SEQ ID NO: 12 (589-1989bp), SEQ ID NO: 13 (589-1221bp), and SEQ ID NO: 14 (589-1221bp); used to encode the fusion protein required for forming the GLP-1, GIP, and GCG three-target receptor agonist fusion protein.

6. The nucleic acid molecule according to claim 4, characterized in that: The nucleic acid molecules include those shown in SEQ ID NO: 11 (589-1989bp), SEQ ID NO: 15 (589-1182bp), and SEQ ID NO: 16 (589-1260bp), or those shown in SEQ ID NO: 12 (589-1989bp), SEQ ID NO: 15 (589-1182bp), and SEQ ID NO: 16 (589-1260bp); used to encode the fusion protein required for forming the GLP-1, GCG, and GIP tri-target receptor agonist fusion protein.

7. The biomaterial related to the three-target receptor agonist fusion protein according to any one of claims 1 to 3, characterized in that: 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 4 to 6; (2) An expression vector comprising the nucleic acid molecule according to any one of claims 4 to 6; (3) An expression carrier comprising the expression box described in (1); (4) Engineered bacteria comprising the nucleic acid molecules described in any one of claims 4 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 in that: It comprises the three-target receptor agonist fusion protein according to any one of claims 1 to 3, the nucleic acid molecule according to any one of claims 4 to 6, or the biological material according to claim 7.

9. The use of the three-target receptor agonist fusion protein according to any one of claims 1 to 3, the nucleic acid molecule according to any one of claims 4 to 6, the biomaterial according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for controlling or lowering blood glucose.

10. The use of the three-target receptor agonist fusion protein according to any one of claims 1 to 3, the nucleic acid molecule according to any one of claims 4 to 6, the biomaterial according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a drug, characterized in that, The drug is used for the prevention and / or treatment of at least one of type 2 diabetes and obesity.

Images