GIP / GLP-1 receptor co-agonist and use thereof

Abstract

Provided in the present application are a GIP / GLP-1 receptor co-agonist and a GIP / GLP-1 fusion protein. By means of using a specific amino acid sequence, the GIP / GLP-1 receptor co-agonist provided in the present application achieves a superior agonistic activity against GIPR and / or GLP-1R and enhanced weight loss and hypoglycemic effects compared to the prior art. Further provided in the present application are a pharmaceutical composition comprising the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein; and the use of the pharmaceutical composition in the prevention or treatment of obesity and diabetes.

Description

A GIP / GLP-1 receptor co-agonist and its application Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to a novel GIP / GLP-1 receptor co-agonist and its application. Background Technology

[0002] Obesity is not only an independent disease but also a significant contributing factor to various chronic diseases such as cardiovascular and cerebrovascular diseases, multiple cancers (e.g., endometrial cancer, breast cancer), type 2 diabetes, and hypertension. Obesity leads to the deposition of excess lipids in adipose tissue and the liver, causing systemic chronic subclinical inflammation and insulin resistance, thereby resulting in organ damage. Therefore, developing new therapies and drugs for obesity, diabetes, and their complications is of great significance for improving human health.

[0003] Glucagon-like peptide-1 (GLP-1) is a naturally occurring incretin in the human body, composed of 37 amino acids. The GLP-1 polypeptide primarily functions to control blood sugar and reduce appetite by activating GLP-1 receptors distributed on the cell membrane surface.

[0004] However, the administration of GLP-1 analogs is subject to side effects such as nausea, vomiting, and vomiting, making it difficult to increase the dosage. Therefore, their clinical use cannot achieve comprehensive glycemic control and weight loss for patients.

[0005] Glucose-dependent insulinotropic peptide (GIP) receptor agonists have antiemetic properties in preclinical models, and therefore, their combination with GLP-1 agonists may be beneficial. GIP is primarily composed of 42 amino acid residues and is secreted by duodenal and adjacent jejunal K cells according to plasma glucose levels. GIP has a similar effect to GLP-1, being released upon food ingestion to promote insulin secretion. However, GIP exhibits a significant bidirectional regulatory effect on glucagon, inhibiting glucagon release during hyperglycemia and increasing glucagon secretion during hypoglycemia, thereby controlling glucose metabolism. Studies have found that co-agonism of GLP-1R / GIPR can exert a synergistic hypoglycemic effect, and dual receptor agonists of GIP and GLP-1 may produce superior hypoglycemic effects and insulin secretion stimulation.

[0006] Developing novel GIP / GLP-1 dual receptor agonists with lower dosing frequencies to provide safe and effective new treatments for obesity, diabetes and their complications is an important problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This application provides a GIP / GLP-1 receptor co-agonist and a GIP / GLP-1 fusion protein. The GIP / GLP-1 receptor co-agonist provided in this application, by employing a specific amino acid sequence, achieves superior agonistic activity against GIPR and / or GLP-1R compared to existing technologies, as well as superior weight loss and blood sugar lowering effects. This application also provides pharmaceutical compositions containing the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein; and the use of the GIP / GLP-1 receptor co-agonist, the GIP / GLP-1 fusion protein, or the pharmaceutical composition in the prevention or treatment of obesity and diabetes.

[0008] In a first aspect, this application provides a GIP / GLP-1 receptor co-agonist, wherein the GIP / GLP-1 receptor co-agonist comprises a structure of general formula Ia as shown below.

[0009] General formula Ia: X1[1-13]-R1-R2-R3-F-R4-R5-R6-X2[1-12]; where:

[0010] X1[1-13] is shown as SEQ ID NO:1, and X2[1-12] is shown as SEQ ID NO:2;

[0011] R1 is selected from any one of LDE, LDK, KEE, and KEK;

[0012] R2 is selected from IA, QA, and EA;

[0013] R3 is selected from any one of QKA, AQE, QKD, AQD, and QRA;

[0014] R4 is selected from VQ, IQ, IN, and VN;

[0015] R5 is selected from WL or YL; and

[0016] R6 is selected from I or L.

[0017] In one embodiment, the GIP / GLP-1 receptor co-agonist comprises the structure shown in general formula 1b below:

[0018] General formula 1b: X1[1-13]-R1-R2-R3-F-R4-WL-R5-X2[1-12]; where

[0019] X1[1-13] is shown as SEQ ID NO:1, and X2[1-12] is shown as SEQ ID NO:2;

[0020] R1 is selected from any one of LDE, LDK, KEE, and KEK;

[0021] R2 is selected from either IA or QA;

[0022] R3 is selected from any one of QKA, AQE, QKD, and AQD;

[0023] R4 is selected from any one of VQ, IQ, IN, and VN; and / or

[0024] R5 is selected from either I or L.

[0025] In one embodiment, in general formula 1a: R1 is LDE or KEK; R2 is IA; R3 is selected from QKA, AQE, and AQD; R4 is selected from VQ, IQ, and IN; R5 is selected from WL; and R6 is I; or

[0026] In the general formula 1b: R1 is LDE or KEK; R2 is IA; R3 is selected from any one of QKA, AQE and AQD; R4 is selected from any one of VQ, IQ and IN; and R5 is I.

[0027] In another embodiment, the GIP / GLP-1 receptor co-agonist comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:3-16.

[0028] In another embodiment, the GIP / GLP-1 receptor co-agonist comprises an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having a synonymous mutation only at position 24 and / or position 27 relative to the sequence shown in SEQ ID NO:9.

[0029] Furthermore, in one embodiment, the amino acids in the GIP / GLP-1 receptor co-agonist are all natural amino acids. In the embodiments of this application, "the amino acids in the GIP / GLP-1 receptor co-agonist are all natural amino acids" means that the amino acids in the GIP / GLP-1 receptor co-agonist do not contain side chain modifications, non-natural amino acid modifications, and / or amidation modifications of C-terminal amino acids.

[0030] In a second aspect, this application also provides a GIP / GLP-1 fusion protein comprising the aforementioned GIP / GLP-1 receptor co-agonist and an Fc region, wherein the GIP / GLP-1 receptor co-agonist and the Fc region are connected via a linker or directly. Optionally, the GIP / GLP-1 receptor co-agonist and the Fc region are connected via a linker.

[0031] In one embodiment, the Fc region comprises a natural Fc region sequence or a non-natural Fc region sequence; optionally, the Fc region is a human Fc region; further optionally, the Fc region is the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody or a variant thereof.

[0032] In one embodiment, compared to the Fc region of wild-type IgG4, the Fc region contains one or more of the mutations S228P, E233P, F234A, L235A, D265A, and R409K; or compared to the Fc region of wild-type IgG1, the Fc region contains one or more of the mutations L234A, L235A, P329A, and P331S.

[0033] In one embodiment, the Fc region has an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences shown in SEQ ID NO:18-23. In some embodiments, the Fc region comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences shown in SEQ ID NO:18-23. In some embodiments, the Fc region is an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences shown in SEQ ID NO:18-23.

[0034] In one embodiment, the Fc region has an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:18. In some embodiments, the Fc region comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:18. In some embodiments, the Fc region is an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:18.

[0035] In one embodiment, the adapter comprises GGG(GGGGS)n, where n is an integer greater than or equal to 1, and n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; more preferably, the adapter comprises GGG(GGGGS)3; further preferably, the adapter comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:17. Even more preferably, the adapter is an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:17.

[0036] In a third aspect, this application provides a nucleic acid encoding any of the aforementioned GIP / GLP-1 receptor co-agonists, or any of the aforementioned GIP / GLP-1 fusion proteins.

[0037] In a fourth aspect, this application provides a vector comprising a nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or any of the GIP / GLP-1 fusion proteins. The vector may be a liposome, a virus, or a plasmid.

[0038] In a fifth aspect, this application provides a cell comprising any of the GIP / GLP-1 receptor co-agonists, any of the GIP / GLP-1 fusion proteins, any of the nucleic acids, or any of the vectors.

[0039] In a sixth aspect, this application provides a pharmaceutical composition comprising the GIP / GLP-1 receptor co-agonist of this application used as a medicament, the GIP / GLP-1 fusion protein, a nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or the GIP / GLP-1 fusion protein, or a carrier containing the nucleic acid; and optionally, the pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents or excipients, including but not limited to physiological saline, glucose injection, and / or physiologically acceptable buffer solutions.

[0040] In one specific embodiment, the pharmaceutical composition provided in this application comprises the GIP / GLP-1 receptor co-agonist of this application or the GIP / GLP-1 fusion protein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients; the pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, physiological saline, glucose injection, and / or physiologically acceptable buffer solutions.

[0041] The pharmaceutical compositions of this application may optionally be formulated for administration via a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or percutaneous). Such pharmaceutical compositions and methods of their preparation are well known in the art.

[0042] In one specific embodiment of this application, the pharmaceutical composition is an injectable formulation; optionally, the injectable formulation is an intravenous injection formulation or a subcutaneous injection formulation; further optionally, the pharmaceutical composition contains a therapeutically effective amount of the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein or a pharmaceutically acceptable salt thereof, nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or the GIP / GLP-1 fusion protein, or a carrier containing the nucleic acid, and one or more pharmaceutically acceptable carriers, diluents or excipients; the pharmaceutically acceptable carriers, diluents or excipients include, but are not limited to, physiological saline, glucose injection, and / or physiologically acceptable buffer solutions.

[0043] In another specific embodiment of this application, the pharmaceutical composition is a unit formulation, which is an injectable formulation; optionally, the injectable formulation is an intravenous injection formulation or a subcutaneous injection formulation; more preferably, the unit injectable formulation contains a therapeutically effective amount of the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein or a pharmaceutically acceptable salt thereof, nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or the GIP / GLP-1 fusion protein, or a carrier containing the nucleic acid, and one or more pharmaceutically acceptable carriers, diluents or excipients; the pharmaceutically acceptable carriers, diluents or excipients include, but are not limited to, physiological saline, glucose injection, and / or physiologically acceptable buffer solutions.

[0044] In a seventh aspect, this application provides the use of the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein or a pharmaceutically acceptable salt thereof, a nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or the GIP / GLP-1 fusion protein, a vector containing the nucleic acid, or the cell in the preparation of a weight loss and / or blood sugar lowering drug.

[0045] In an eighth aspect, this application provides the use of the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein or a pharmaceutically acceptable salt thereof, a nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or the GIP / GLP-1 fusion protein, a vector containing the nucleic acid, the cell, or the pharmaceutical composition for the prevention or treatment of obesity or diabetes.

[0046] In a ninth aspect, this application provides a method for preventing or treating obesity or diabetes, comprising administering to a subject in need an effective amount of the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein or a pharmaceutically acceptable salt thereof, a nucleic acid encoding either the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein, a carrier comprising the nucleic acid, the cell, or the pharmaceutical composition.

[0047] In a tenth aspect, this application provides a method for preparing the GIP / GLP-1 receptor co-agonist or the GIP / GLP-1 fusion protein or a pharmaceutically acceptable salt thereof, a nucleic acid encoding any of the GIP / GLP-1 receptor co-agonists or the GIP / GLP-1 fusion protein, a vector containing the nucleic acid, the cell, or the pharmaceutical composition.

[0048] The solution proposed in this application has the following advantages:

[0049] 1) The GIP / GLP-1 fusion protein provided in this application has good binding activity to GLP-1R and / or GIPR on the cell surface.

[0050] 2) The GIP / GLP-1 fusion protein provided in this application has good agonist activity against cells expressing GLP-1R and / or GIPR.

[0051] 3) The GIP / GLP-1 fusion protein provided in this application promotes the release of cAMP from cells expressing GLP-1R and / or GIPR, as well as the endocytosis of GLP-1R and / or GIPR on the cell surface.

[0052] 4) The GIP / GLP-1 fusion protein provided in this application can effectively prevent enzymatic cleavage by fusion proteases in human plasma and has good stability in plasma.

[0053] 5) The GIP / GLP-1 fusion protein provided in this application has good weight loss and blood glucose lowering effects on both normal-weight mice and obese mice: In normal-weight mice, in terms of blood glucose lowering, #2 has a blood glucose lowering effect comparable to or slightly better than Tirzepatide at 8h and 16h, and #7 has a blood glucose lowering effect slightly better than #2 at 8h, and it is expected that #7 will be slightly better than Tirzepatide at 8h; In terms of weight loss, #2, #7, #10 and Tirzepatide all have significant weight loss effects, and the weight loss of #7 is maintained for about 4 days, which is better than Tirzepatide, while the effects of #2 and #10 are comparable to Tirzepatide; In terms of weight loss, the weight curve of #7 is smoother, indicating that it can maintain the weight loss effect for a longer period of time during the dosing interval and the weight rebound is slower, and its effect is better than Tirzepatide.

[0054] 6) The GIP / GLP-1 fusion protein provided in this application has good pharmacokinetic characteristics, among which #7 has a significantly longer half-life than Tirzepatide in cynomolgus monkeys. Attached Figure Description

[0055] Figure 1 shows the binding activity of the GIP / GLP-1 fusion protein to human GLP-1R expressed on the cell surface;

[0056] Figure 2 shows the binding activity of the GIP / GLP-1 fusion protein to monkey-derived GLP-1R expressed on the cell surface;

[0057] Figure 3 shows the binding activity of the GIP / GLP-1 fusion protein to murine GLP-1R expressed on the cell surface;

[0058] Figure 4 shows the binding activity of the GIP / GLP-1 fusion protein to human GIPR expressed on the cell surface;

[0059] Figure 5 shows the binding activity of the GIP / GLP-1 fusion protein to monkey-derived GIPR expressed on the cell surface;

[0060] Figure 6 shows the binding activity of the GIP / GLP-1 fusion protein to mouse GIPR expressed on the cell surface;

[0061] Figure 7 shows the reporter gene activity of the GIP / GLP-1 fusion protein in H_GLP-1R Reporter HEK-293 cells;

[0062] Figure 8 shows the reporter gene activity of the GIP / GLP-1 fusion protein in H_GIPR Reporter HEK-293 cells;

[0063] Figure 9 shows the reporter gene activity of the GIP / GLP-1 fusion protein in H_GCGR Reporter CHO-K1 cells;

[0064] Figure 10 shows that the GIP / GLP-1 fusion protein promotes cAMP release in H_GLP-1R Reporter HEK-293 cells;

[0065] Figure 11 shows that the GIP / GLP-1 fusion protein promotes cAMP release in H_GIPR Reporter HEK-293 cells;

[0066] Figure 12 shows that the GIP / GLP-1 fusion protein promotes the endocytosis of GLP-1R;

[0067] Figure 13 shows that the GIP / GLP-1 fusion protein promotes the endocytosis of GIPR;

[0068] Figure 14 shows the GLP-1R activity assay of the GIP / GLP-1 fusion protein in plasma after enzymatic digestion.

[0069] Figure 15 shows the GIPR activity assay of the GIP / GLP-1 fusion protein in plasma after enzymatic digestion.

[0070] Figure 16A shows the hypoglycemic effect of GIP / GLP-1 fusion protein on normal mice 16 h after administration of 3 g / kg glucose;

[0071] Figure 16B shows the hypoglycemic effect of GIP / GLP-1 fusion protein on normal mice 8 hours after administration of 3 g / kg glucose;

[0072] Figure 16C shows the hypoglycemic effect of GIP / GLP-1 fusion protein on normal mice 8 hours after administration of 3.5 g / kg glucose;

[0073] Figure 17A shows the weight loss effect of the GIP / GLP-1 fusion protein on normal mice;

[0074] Figure 17B shows the effect of the GIP / GLP-1 fusion protein on food intake in normal mice;

[0075] Figure 18A shows the weight loss effect of the GIP / GLP-1 fusion protein on DIO mice;

[0076] Figure 18B shows the effect of the GIP / GLP-1 fusion protein on food intake in DIO mice;

[0077] Figure 19 shows the pharmacokinetic properties of the GIP / GLP-1 fusion protein in cynomolgus monkeys. Detailed Implementation

[0078] The embodiments listed below are provided to better illustrate the content of this application, but are not intended to limit the scope of this application to the illustrated embodiments. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention are still within the protection scope of this application.

[0079] Terminology Explanation:

[0080] Unless otherwise stated, as used herein, the singular forms “a,” “an,” and “the” also include the plural forms. For example, the term “a cell” includes multiple cells and mixtures thereof.

[0081] As used herein, the terms "comprising" or "including" mean including the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this application, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0082] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to molecules comprising two or more amino acids linked together by peptide bonds. These terms encompass, for example, natural and artificial proteins and polypeptide analogs of protein sequences (e.g., mutant proteins, variants, and fusion proteins), as well as proteins that are post-transcriptionally or otherwise covalently or non-covalently modified. Peptides, polypeptides, or proteins can be monomers or polymers.

[0083] As used in this article, the term "treatment" refers to a clinical intervention aimed at altering the natural course of disease in the individual or cells receiving the treatment during the clinicopathological process. Ideal outcomes of treatment include slowing or reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0084] As used herein, the term "unit dosage form" refers to the smallest unit of packaging of each pharmaceutical preparation containing an appropriate amount of the active compound. For example, a unit dosage form in capsules refers to one capsule, a unit dosage form in tablets refers to one tablet, a unit dosage form in injections refers to one vial of injection, and so on for other types of preparations.

[0085] As used herein, the term “QD” means to be applied once a day, “Q2D” means to be applied once every two days (i.e., once every 1 day), “Q3D” means to be applied once every three days (i.e., once every 2 days), and “Q6D” means to be applied once every six days (i.e., once every 5 days).

[0086] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmaceutical salts or prodrugs, along with other chemical components, such as physiologically / pharmaceutical carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0087] As used herein, the term "therapeutic effective dose" refers to an amount of compound, when administered to a subject in single or multiple doses, sufficient to cure, alleviate, relieve, or partially resolve the clinical manifestations of a given disease or condition and its complications, exceeding the amount expected without such treatment. Therefore, the result may be a reduction and / or relief of the signs, symptoms, or cause of the disease, or any other desired biological alteration. It should be understood that "therapeutic effective dose" can vary from subject to subject, depending on the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the prescribing physician's judgment.

[0088] As used herein, the term "pharmaceutically acceptable salt" includes acid addition salts formed with organic or inorganic acids. Suitable pharmaceutically acceptable salts of the compounds of this application include acid addition salts, which may be salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc., or salts of organic acids such as, for example, acetic acid, benzenesulfonic acid, methanesulfonic acid, benzoic acid, citric acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, octanoic acid, azelaic acid, malic acid, tartaric acid, amino acids such as glutamic acid or aspartic acid, etc. Pharmaceutically acceptable acid addition salts of the compounds of this application include salts formed by adding one or more equivalent amounts of acid, such as monohydrochloride, dihydrochloride, etc. Salts can be prepared by any method within the knowledge of those skilled in the art.

[0089] As used herein, the terms "pharmaceutically acceptable carrier" or "pharmaceuticalally acceptable excipient" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Except where any conventional media or reagent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Furthermore, a variety of excipients commonly used in the art may be included.

[0090] GCGR (glucagon receptor): GCGR is a receptor belonging to the class B G protein-coupled receptor (GPCR) family, playing a crucial role in maintaining blood glucose homeostasis. GCGR is primarily expressed in the liver and kidneys. Its natural ligand is glucagon. It activates the Gs protein, inducing adenylate cyclase activation, producing cAMP, activating protein kinase A, promoting glycogenolysis and gluconeogenesis, thereby increasing blood glucose levels. GCGR holds significant therapeutic potential in diseases such as diabetes, obesity, and metabolic syndrome.

[0091] Tirzepatide: Tirzepatide is a novel drug primarily used to treat type 2 diabetes and obesity. It is a dual-mechanism drug that simultaneously activates glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors, improving glycemic control and weight by regulating blood glucose levels, promoting insulin secretion, reducing glucagon secretion, delaying gastric emptying, and increasing satiety.

[0092] Retatrutide (LY3437943) is a novel triple agonist peptide that simultaneously activates the glucagon receptor (GCGR), glucose-dependent insulinotropic peptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). It exhibits significant activity in both in vitro and in vivo studies, improving glucose tolerance, promoting weight loss, and demonstrating good safety and tolerability. Retatrutide shows significant potential in the treatment of obesity, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD).

[0093] cAMP-d2 is a fluorescence resonance energy transfer (FRET) receptor used to detect cAMP (cyclic adenosine monophosphate) levels. In HTRF (high-throughput time-resolved fluorescence) technology, cAMP-d2 binds to anti-cAMP antibodies, and cAMP levels are detected using FRET. This technology is widely used in drug screening and signaling pathway research.

[0094] Anti-cAMP-Eu is a reagent used in cAMP detection, its core principle being based on fluorescence resonance energy transfer (FRET) technology. In multiple studies, Anti-cAMP-Eu is described as an antibody labeled with europium (Eu) that binds to cAMP, thereby generating a signal in FRET experiments. This reagent plays a crucial role in cAMP detection, particularly in the quantitative analysis of intracellular cAMP levels. In this application, Anti-cAMP-Eu... 3+ -Cryptate refers to a cavitary compound (Eu) 3+ cAMP antibody labeled with α-Cryptate, wherein Eu 3+ Represents trivalent europium ions; Cryptote refers to a cavitary compound.

[0095] Example

[0096] Example 1. Preparation of GIP / GLP-1 fusion protein

[0097] Based on the amino acid sequences of the GIP / GLP-1 receptor co-agonists named #1 to #14 in Table 1 (see SEQ ID NO: 3-16 in Table 1), the corresponding gene sequences were chemically synthesized and coupled to the Fc region via a linker (amino acid sequences see SEQ ID NO: 17 in Table 1) (see SEQ ID NO: 18-23 in Table 1) and cloned into the pcDNA3.1 vector (Universal Bio). The GIP / GLP-1 fusion protein was expressed in mammalian CHO cells. After culturing for 7 days, the cell slurry was centrifuged to remove the precipitate, and the supernatant was collected. Protein A / G columns were pre-equilibrated with PB buffer, washed with 2-5 column volumes, and the supernatant sample was loaded onto the column. The target protein (i.e., the GIP / GLP-1 fusion protein) was eluted with 0.1M glycine solution at pH 2.7 into a container pre-filled with 1M Tris-HCl neutralization buffer at pH 9.0. The collected eluent (containing glycine, fusion protein, and Tris-HCl neutralization buffer) was exchanged into PBS buffer. The purity of the fusion protein was determined by HPLC-SEC, and the content of GIP / GLP-1 fusion protein was determined by Nanodrop (Thermo Scientific, NanoDrop 2000C).

[0098] Table 1. Amino acid sequences of GIP / GLP-1 receptor co-agonists, linkers, and Fc regions

[0099] Example 2. Detection of the binding ability of GIP / GLP-1 fusion protein to various receptor proteins expressed on the surface of HEK293 cell line.

[0100] Table 1-1. Sources of cell lines expressing each receptor protein used in the examples

[0101] The amino acid sequences of wild-type human GIP proteins in Table 1-1 above are shown in SEQ ID NO:24 below:

[0102] YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGGGPSSGAPPPSGGGGGGGSGGGGSGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:24). This protein can be obtained using conventional techniques in the art for use in this embodiment.

[0103] The binding activity of the GIP / GLP-1 fusion protein was determined by flow cytometry. The Reporter HEK293 cell line expressing human GLP-1 receptor (GLP-1R) or GIP receptor (GIPR) (i.e., the Reporter HEK293 cell line) and HEK293 cell lines overexpressing monkey or mouse GLP-1 receptor (GLP-1R) or GIP receptor (GIPR) were used to evaluate the binding ability of the GIP / GLP-1 fusion protein to human, monkey, or mouse GLP-1R or GIPR proteins expressed on the surface of HEK293 cells.

[0104] The GIP / GLP-1 fusion protein and control protein (wild-type GLP-1 (human) or wild-type GIP (human)) were diluted to 200 nM with PBS buffer, followed by a 3-fold serial dilution for a total of 9 dilutions (i.e., starting from 200 nM, 3-fold serial dilutions were performed for a total of 9 dilutions, resulting in a total of 11 different concentrations including 200 nM and 0 nM) (the dilution method for protein samples in Figures 1-3 and 6 follows this method); the protein sample dilution method in Figure 4 is: starting from 200 nM, 3-fold serial dilutions were performed for a total of 11 dilutions, including 12 different concentrations including 200 nM, excluding the 0 nM concentration; the protein sample dilution method in Figure 5 is: starting from 12.5 nM, 3-fold serial dilutions were performed for a total of 7 dilutions, including 9 different concentrations including 12.5 nM and 0 nM. HEK293 cells in logarithmic growth phase were digested with trypsin and counted. 1E5 cells were seeded per well in a V-bottom 96-well plate. After centrifugation and discarding the supernatant, diluted GIP / GLP-1 fusion protein or control protein sample was added, and the cells were incubated at 4°C for 1 h. Cells were washed three times with PBS buffer, and then labeled with Alexa Fluor 488-labeled goat anti-human IgG antibody on ice in the dark for 50 min. Cells were washed three times with PBS buffer, and the fluorescence intensity was read by flow cytometry. The results of the relatively preferred GIP / GLP-1 fusion protein (using the Fc of SEQ ID NO:18) are shown in Figures 1-6 and Tables 2-4.

[0105] Specifically, as shown in Table 2 and Figures 1 and 4, regarding the binding ability of human GLP-1R on the cell surface, using wild-type human GLP-1 protein as a positive control, fusion protein samples #2, #3, #6, #7, #8, #9, #10, #11, #12, #13, and #14 exhibited higher binding activity to GLP-1R on the HEK293 cell surface than wild-type GLP-1 protein. Regarding the binding ability of human GIPR on the cell surface, using wild-type GIP as a control, fusion protein samples #7, #9, and #10 exhibited higher binding activity to GIPR on the HEK293 cell surface than wild-type GIP protein.

[0106] Table 2. Binding ability of fusion proteins to human GLP-1R or GIPR on the surface of HEK293 cells.

[0107] As shown in Table 3 and Figures 2-3, fusion protein samples #7 and #10 can bind to both monkey-derived GLP-1R and mouse-derived GLP-1R on the cell surface. As shown in Table 4 and Figures 5-6, regarding the binding ability of monkey-derived and mouse-derived GIPR on the cell surface, using wild-type human GIP as a positive control, fusion protein samples #7 and #10 showed weaker binding to monkey-derived and mouse-derived GIPR on the cell surface compared to wild-type GIP.

[0108] Table 3. Binding ability of fusion proteins to monkey and mouse GLP-1R on the surface of HEK293 cells

[0109] Table 4. Binding ability of fusion proteins to monkey and mouse GIPR on the surface of HEK293 cells

[0110] Example 3. Detection of the agonist activity of the GIP / GLP-1 fusion protein against GLP-1R

[0111] The binding ability of the GIP / GLP-1 fusion protein to the GLP-1R protein on the surface of HEK293 cells was verified in Example 2. This example further tests the agonist activity of the GIP / GLP-1 fusion protein against GLP-1R. It should be understood that the GIP / GLP-1 fusion protein in this example and subsequent examples uses the relatively preferred SEQ ID NO:18 as the Fc amino acid sequence.

[0112] The assay was performed using H_GLP-1R Reporter HEK-293 cells expressing human GLP-1R. Cells were seeded in 96-well plates at 20,000 cells / well / 100 μL and cultured overnight at 37°C and 5% CO2. Each GIP / GLP-1 fusion protein and positive control protein (wild-type GLP-1, Tirzepatide (Selleck, P1206)) was serially diluted 4-fold starting from 500 nM in DMEM medium containing 1% serum. Each protein sample had 12 different concentrations (i.e., each protein sample was serially diluted 4-fold starting from 500 nM, for a total of 10 dilutions, resulting in 12 different concentrations including 500 nM and 0 nM). After removing the supernatant from H_GLP-1R Reporter HEK-293 cells, 100 μL of the diluted GIP / GLP-1 fusion protein or positive control protein sample was added to each well and incubated at 37°C, 5% CO2 for 16 h. Then, 50 μL of Bright-Glo Luciferase (Promega) was added to each well, and the mixture was incubated in the dark for 5 min. The chemiluminescence value was detected using a microplate reader. A curve was plotted with the logarithm of protein sample concentration on the x-axis and fluorescence value reading on the y-axis, and the half-maximal effect concentration (EC50) of each protein sample was calculated. The results are shown in Table 5 and Figure 7 below. The reporter gene activity of some of the screened fusion proteins #1, #2, #3, #5, #6, #7, #8, and #14 was much higher than that of the positive control protein Tirzepatide, while the reporter gene activity of sample #10 was similar to that of the positive control protein Tirzepatide.

[0113] Table 5. Reporter gene activity of the fusion protein in H_GLP-1R Reporter HEK-293 cells

[0114] Example 4. Detection of the agonist activity of the GIP / GLP-1 fusion protein against the GIP receptor

[0115] The binding ability of the GIP / GLP-1 fusion protein to the GIPR protein on the surface of HEK293 cells was verified in Example 2. This example further tests the agonist activity of the GIP / GLP-1 fusion protein to GIPR.

[0116] H_GIPR Reporter HEK-293 cells expressing GIPR were cultured to the logarithmic growth phase, triedpsinized, and resuspended in complete culture medium. 10,000 cells / well / 100 μL were added to white 96-well plates and incubated until adherent. Protein samples included fusion protein samples and positive control proteins (Tirzepatide (Selleck, P1206) and wild-type GIP). Protein samples were diluted to 5 nM with DMEM medium containing 1% serum, and then serially diluted 4-fold (10 times in total) to obtain 12 different concentrations, including 5 nM and 0 nM. After removing the supernatant from H_GIPR Reporter HEK-293 cells, 100 μL of each diluted protein sample was added to each well and incubated at 37°C, 5% CO2 for 16 h. 50 μL of Bright-Glo Luciferase (promega) was added to each well, and the plates were incubated in the dark for 5 min. Chemiluminescence values ​​were detected using a microplate reader. A curve was plotted with the logarithm of protein sample concentration on the x-axis and fluorescence value reading on the y-axis, and the half-maximal effect concentration (EC50) of each sample was calculated. The results are shown in Table 6 and Figure 8 below. The activity of some of the selected fusion proteins #6, #7, #8, #9, and #10 samples was much higher than that of the positive control protein Tirzepatide.

[0117] Table 6. Reporter gene activity of the fusion protein in H_GIPR Reporter HEK-293 cells

[0118] Example 5. Detection of the agonist activity of the GIP / GLP-1 fusion protein against GCGR

[0119] CHOK1-GCGR CRE-luc cells (GM-C09151, Jiman Biotechnology) were cultured to the logarithmic growth phase, triedpsin-digested, and resuspended in complete culture medium. 10,000 cells / well / 100 μL were added to white 96-well plates and incubated until adherence. Protein samples, including fusion proteins (#2, #6, #7, #8, #10) and control proteins (Tirzepatide (Selleck, P1206) and Retatrutide (MCE, HY-P3506A)), were diluted to 5 nM in DMEM medium containing 1% serum, and then serially diluted 4-fold (8 times in total) to obtain 10 different concentrations, including 5 nM and 0 nM. After removing the CHOK1-GCGR CRE-luc cell supernatant, 100 μL of the diluted protein sample was added to each well, and the plates were incubated at 37°C, 5% CO2 for 16 h. Add 50 μL of Bright-Glo Luciferase (promega) to each well and incubate in the dark for 5 min. Detect the chemiluminescence value using a microplate reader.

[0120] A curve was plotted with the logarithm of protein sample concentration on the x-axis and fluorescence value reading on the y-axis, and the half-maximal effect concentration (EC50) of each sample was calculated. The results are shown in Figure 9. The GIP / GLP-1 fusion proteins (#2, #6, #7, #8, #10) screened in this application did not activate GCGR, and the control Tirzepatide also did not activate GCGR. The control Retatrutide (a triple receptor agonist of GCGR, GIPR, and GLP-1R) showed agonist activity against GCGR, indicating that the dual-agonist fusion protein of this application can specifically activate the GLP-1 receptor (GLP-1R) and / or the GIP receptor (GIPR).

[0121] Example 6. GIP / GLP-1 fusion protein promotes cAMP release from GLP-1R cell line

[0122] In this embodiment, we examined whether the GIP / GLP-1 fusion protein promotes cAMP release in the GLP-1R cell line. In diabetic patients, GLP-1R signaling enhances glucose-dependent insulin secretion by upregulating cAMP, subsequently activating PKA and cAMP-activated exchange proteins.

[0123] H_GLP-1R Reporter HEK-293 cells were cultured to the logarithmic growth phase, triedpsinized, and resuspended in complete culture medium. 100,000 cells / well / 100 μL was added to 96-well plates and incubated until adherence. The fusion protein sample (#7) was diluted to 100 nM with DMEM medium containing 1% serum, and then serially diluted 5-fold (6 times) to obtain 8 different concentrations for each sample, including 100 nM and 0 nM. After removing the H_GLP-1R Reporter HEK-293 cell supernatant, 100 μL of the diluted fusion protein sample was added to each well and incubated at 37°C, 5% CO2 for 1 h. 40 μL was transferred from each well to a new white 96-well plate and analyzed using the Cisbio cAMP-Gs Dynamic Kit (Cisbio Cat#62AM4PEB). cAMP-d2 and Anti-cAMP-Eu were measured. 3+ Cryptate (from the Cisbio cAMP-Gs Dynamic kit) was diluted 20-fold with cAMP Lysis & Detection Buffer (from the Cisbio cAMP-Gs Dynamic kit) and mixed thoroughly. 20 μL of the diluted cAMP-d2 solution was added to each well, followed by 20 μL of the diluted Anti-cAMP-Eu. 3+ -Cryptate solution, shake for 30 seconds to mix, and incubate at room temperature in the dark for 1 hour. HTRF (homogeneous time-resolved fluorescence) signal readings were performed using a Biotek Synergy H1 microplate reader, with excitation wavelength at 320 nm and emission wavelengths at 620 nm and 665 nm. The signal ratio (665 nm / 620 nm * 10000) was calculated, and a four-parameter equation was used in GraphPad Prism (a medical graphing software) to perform nonlinear fitting between the signal ratio and sample concentration. The results are shown in Figure 10. Sample #7 promoted cAMP release from GLP-1R-expressing cells. At a certain concentration, the higher the sample concentration, the more cAMP was released. The EC50 value calculated using GraphPad was 5.8 nM.

[0124] Example 7. GIP / GLP-1 fusion protein promotes cAMP release in GIPR cell lines

[0125] H_GIPR Reporter HEK-293 cells (GM-C24030 from Jiman Biotechnology) were cultured to the logarithmic growth phase, triedpsin-digested, and resuspended in complete culture medium. 100 μL of 100,000 cells / well was added to 96-well plates and incubated until adherence. The fusion protein sample (#7) was diluted to 5 nM with DMEM medium containing 1% serum, and then serially diluted 5-fold (6 times) to obtain 8 different concentrations, including 5 nM and 0 nM. After removing the H_GIPR Reporter HEK-293 cell supernatant, 100 μL of the diluted fusion protein sample was added to each well and incubated at 37°C in a 5% CO2 incubator for 1 h. 40 μL of the diluted fusion protein sample was transferred from each well to a new white 96-well plate and analyzed using the Cisbio cAMP-Gs Dynamic Kit (Cisbio Cat#62AM4PEB). cAMP-d2 and Anti-cAMP-Eu were analyzed. 3+ -Cryptate (derived from the Cisbio cAMP-Gs Dynamic kit) was diluted 20-fold with cAMP Lysis & Detection Buffer and mixed thoroughly. 20 μL of the diluted cAMP-d2 solution was added to each well, followed by 20 μL of the diluted Anti-cAMP-Eu. 3+ -Cryptate solution, vortex for 30 seconds to mix, and incubate at room temperature in the dark for 1 hour. HTRF signals were read using a Biotek Synergy H1 microplate reader with excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm / 620 nm * 10,000) was calculated, and a four-parameter equation was used to perform nonlinear fitting between the signal ratio and sample concentration in GraphPad Prism. The results are shown in Figure 11. Sample #7 promoted cAMP release from GIPR-expressing cells. At a certain concentration, the higher the sample concentration, the more cAMP was released. The EC50 value calculated using GraphPad was 18 pM.

[0126] Example 8. GIP / GLP-1 fusion protein promotes GLP-1R endocytosis

[0127] Flow cytometry was used to assess the internalization of GLP-1R mediated by the fusion protein. H_GLP-1R Reporter HEK-293 cells were digested from T175 culture flasks with trypsin and washed with PBS. The cells were then suspended in serum-free DMEM medium, and fusion protein samples #7 and #10 were added to a final concentration of 50 nM, respectively, and incubated at 4°C for 1 h. After washing with FACS buffer, the cells were incubated at 4°C and 37°C for 2 h, respectively. Alexa Fluor 488-labeled goat anti-human secondary antibody was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, the fluorescence intensity of the cells was read by flow cytometry.

[0128] The results are shown in Table 7 and Figure 12. After incubating fusion protein samples #7 and #10 with cells for 2 hours, #7 induced approximately 28.1% of GLP-1R receptor internalization, and #10 induced approximately 76.8% of GLP-1R receptor internalization.

[0129] Table 7. Fusion protein promotes GLP-1R endocytosis

[0130] Example 9. GIP / GLP-1 fusion protein promotes GIPR endocytosis

[0131] Flow cytometry was used to assess the internalization of GIPR mediated by the fusion protein. H_GIPR Reporter HEK-293 cells expressing GIPR were digested from T175 culture flasks with trypsin and washed with PBS. The cells were then suspended in serum-free DMEM medium, and fusion protein samples #2, #6, #7, and #10 were added to a final concentration of 0.8 nM, and incubated at 4°C for 1 h. After washing with FACS buffer, the cells were incubated at 4°C and 37°C for 2 h, respectively. Alexa Fluor 488-labeled goat anti-human secondary antibody was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, the fluorescence intensity of the cells was read by flow cytometry.

[0132] The results are shown in Table 8 and Figure 13. After 2 hours of incubation, #2, #6, and #7 can induce more than 80% of GIPR receptor internalization, while #10 can induce about 56.6% of GIPR receptor internalization.

[0133] Table 8. Fusion proteins promote GIPR endocytosis

[0134] Example 10. Detection of GIP / GLP-1 fusion protease activity before and after digestion in human plasma

[0135] Samples #7, wild-type GIP, and wild-type GLP-1 were added to human plasma and digested using enzymes naturally present in human plasma. After digestion at 4°C for 36 hours, reporter gene activity was detected using the same method as in Examples 3 and 4. After the experiment, the values ​​were read, and a curve was plotted with the logarithm of sample concentration on the x-axis and the fluorescence value on the y-axis. The half-maximal effect concentration (EC50) for each sample was calculated. The results are shown in Table 9-1 and Figure 14 (GLP-1R), Table 9-2 and Figure 15 (GIPR). The reporter gene activities of the selected fusion proteins #7 and Tirzepatide in human GLP-1R cells and human GIPR cells after digestion were comparable to those before digestion. Both #7 and Tirzepatide effectively prevented digestion and exhibited good stability in plasma. However, the reporter gene activity of wild-type GLP-1R showed a significant decreasing trend.

[0136] Table 9-1. Reporter gene activity of the fusion protein before and after enzyme digestion in human GLP-1R cells

[0137] Table 9-2. Reporter gene activity of the fusion protein before and after enzyme digestion on human GIPR cells

[0138] Example 11. Hypoglycemic effect of GIP / GLP-1 fusion protein on normal mice

[0139] The experiment used 20-25g male ICR mice (purchased from Beijing Vital River Co., Ltd., 201) to detect the regulatory effects of a single subcutaneous administration of the fusion protein molecules #2, #7, and Tirzepatide on blood glucose in normal mice. Experimental design: Mice were divided into groups 1, 2, and 3; at hour 0, each group of mice was subcutaneously injected with 3 nmol / kg of #2, #7, Tirzepatide, and / or solvent (physiological saline, i.e., 0.9% sodium chloride injection), followed by fasting but free access to water; 8 hours after administration to group 1 mice, a 3g / kg glucose solution was injected intraperitoneally; 8 hours after administration to group 3 mice, a 3.5g / kg glucose solution was injected intraperitoneally; 16 hours after administration to group 2 mice, a 3g / kg glucose solution was injected intraperitoneally.

[0140] Blood glucose detection protocol: Based on the above experimental design, blood was collected from the tails of mice at time points of 0 min before intraperitoneal injection and 15 min, 30 min, 60 min, and 120 min after intraperitoneal injection, and blood glucose levels were measured using a glucometer. The area under the blood glucose curve (AUC) was calculated based on the results at each time point.

[0141] Experimental Results: In Group 2 mice, after administration of fusion protein molecule #2, solvent, or Tirzepatide, the changes in blood glucose levels after intraperitoneal injection of a 3 g / kg glucose solution 16 hours later are shown in Table 10 and Figure 16A. In Group 1 mice, after administration of fusion protein molecule #2, solvent, or Tirzepatide, the changes in blood glucose levels after intraperitoneal injection of a 3 g / kg glucose solution 8 hours later are shown in Table 11 and Figure 16B. In Group 3 mice, after administration of fusion protein molecules #2, #7, or solvent, the changes in blood glucose levels after intraperitoneal injection of a 3.5 g / kg glucose solution 8 hours later are shown in Table 12 and Figure 16C. Both #2 and #7 showed significant hypoglycemic effects.

[0142] Results Analysis: Tables 10-11 and Figures 16A-16B show that #2 has a blood glucose-lowering effect comparable to or slightly better than Tirzepatide at 8h and 16h. Table 12 and Figure 16C show that #7 has a slightly better blood glucose-lowering effect than #2 at 8h, and it is expected that #7 will be slightly better than Tirzepatide at 8h.

[0143] Table 10. Changes in blood glucose levels in ICR mice 16 hours after administration of #2, solvent, and Tirzepatide (corresponding to group 2 mice, injected with 3 g / kg glucose solution 16 hours after administration).

[0144] Table 11. Changes in blood glucose levels in ICR mice 8 hours after administration of #2, solvent, and Tirzepatide (corresponding to Group 1 mice, injected with 3 g / kg glucose solution 8 hours after administration).

[0145] Table 12. Changes in blood glucose levels in ICR mice 8 hours after administration of drugs #2, #7, and the solvent (corresponding to group 3 mice, injected with 3.5 g / kg glucose solution 8 hours after drug administration).

[0146] Example 12. Effects of GIP / GLP-1 fusion protein on weight loss and food intake in normal mice.

[0147] Male c57BL / 6J mice (20-25g, purchased from Beijing Vital River Co., Ltd.) were selected and administered subcutaneous injections of 10 nmol / kg of fusion protein molecules #2, #7, #10, Tirzepatide, or a solvent (physiological saline, i.e., 0.9% sodium chloride injection) on day 0. Body weight and food intake were monitored on day 0 after drug administration. The results of the mouse body weight gain rate are shown in Table 13 and Figure 17A. It can be seen that fusion protein molecules #2, #7, #10, and Tirzepatide all had significant weight loss effects: #7 maintained the weight loss for about 4 days, which was better than Tirzepatide; the effects of #2 and #10 were comparable to Tirzepatide. The results of the mouse food intake are shown in Table 14 and Figure 17B. It can be seen that #2, #7, #10, and Tirzepatide all significantly inhibited food intake.

[0148] Table 13. Effect on weight gain rate in normal mice (%)

[0149] Table 14. Effects on food intake (g) in normal mice

[0150] Example 13. Effects of GIP / GLP-1 fusion protein on weight loss and food intake in DIO mice

[0151] Male DIO mice (40-45g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., a high-fat diet-induced obese mouse model) were selected. On day 0, #7, #10, Tirzepatide, or a solvent (physiological saline, i.e., 0.9% sodium chloride injection) were administered subcutaneously. The dosage and frequency of administration are shown in Table 15. #10 was administered for 19 days and then discontinued; #7 and Tirzepatide were administered for 31 days and then discontinued. Daily body weight and food intake were recorded, and the results are shown in Figures 18A-18B. The comparison data of #7 administered at 10 nmol / kg Q3D and Tirzepatide administered at 10 nmol / kg Q3D showed that the weight loss curve of #7 was smoother, indicating that it maintained a longer weight loss effect during the dosing interval, and the weight recovery was slower, making it more effective than Tirzepatide. Comparative data between #7 (administered with 20 nmol / kg Q6D) and #10 (administered with 20 nmol / kg Q6D) showed that #7 had a smoother weight loss curve and was more effective than #10. The weight loss effect of #7 (administered with 20 nmol / kg Q6D) was comparable to that of Tirzepatide (administered with 10 nmol / kg Q3D), both reducing weight by less than 17% after administration (Figure 18A). Both #7 and #10 inhibited food intake (Figure 18B). The duration of food intake inhibition was longer after each administration of #7 and #10, and on the third day after administration, the inhibition was lower than that of Tirzepatide. Furthermore, under the same dosing cycle and dosage conditions, #7 showed superior food intake inhibition activity compared to #10.

[0152] Table 15. Experimental grouping of obese mice

[0153] Note: In Table 15, "Q3D" means once every 3 days (i.e., every 2 days); "Q6D" means once every 6 days (i.e., every 5 days).

[0154] Example 14. Pharmacokinetic properties of GIP / GLP-1 fusion protein in cynomolgus monkeys

[0155] Four male cynomolgus macaques, weighing 2–5 kg, were selected and divided into two groups. Group 1 (2 macaques) received a single subcutaneous injection of 30 nmol / kg of #7, while Group 2 (2 macaques) received a single subcutaneous injection of 30 nmol / kg of Tirzepatide. Whole blood (2 mL) was collected from both groups at 0 h before administration and at 1 h, 2 h, 6 h, 10 h (D1), 24 h (D2), 48 h (D3), 72 h (D4), 120 h (D6), 168 h (D8), 240 h (D11), 336 h (D15), 504 h (D22, #7 molecules only), and 672 h (D29, #7 molecules only). Serum was collected by centrifugation after blood collection. The concentration of #7 in serum was determined using ELISA, and the concentration of Tirzepatide in serum was determined using protein precipitation-LC-MS / MS. The results are shown in Table 16 and Figure 19. Table 16 shows data from four cynomolgus monkeys. It can be seen that molecule #7 of this application exhibited good pharmacokinetic characteristics after subcutaneous injection in two cynomolgus monkeys. Specifically, the Tg of #7 was [data missing]. 1 / 2 The exposure time was 174 hours for #7 and 46.3 hours for Tirzepatide. #7 was significantly longer than Tirzepatide, and the AUC (area under the curve of drug exposure) of #7 was also significantly better than that of Tirzepatide.

[0156] Table 16. Key pharmacokinetic parameters of a single dose in cynomolgus monkeys

Claims

1. A GIP / GLP-1 receptor co-agonist, wherein the GIP / GLP-1 receptor co-agonist comprises the structure of general formula Ia as shown below. General formula Ia: X1[1-13]-R1-R2-R3-F-R4-R5-R6-X2[1-12]; where: X1[1-13] is shown as SEQ ID NO:1, and X2[1-12] is shown as SEQ ID NO:2; R1 is selected from any one of LDE, LDK, KEE, and KEK; R2 is selected from IA, QA, and EA; R3 is selected from any one of QKA, AQE, QKD, AQD, and QRA; R4 is selected from VQ, IQ, IN, and VN; R5 is selected from WL or YL; and R6 is selected from I or L.

2. A GIP / GLP-1 receptor co-agonist, wherein, The GIP / GLP-1 receptor co-agonist comprises the structure shown in general formula Ib below: General formula Ib: X1[1-13]-R1-R2-R3-F-R4-WL-R5-X2[1-12]; in X1[1-13] is shown as SEQ ID NO:1, and X2[1-12] is shown as SEQ ID NO:2; R1 is selected from any one of LDE, LDK, KEE, and KEK; R2 is selected from either IA or QA; R3 is selected from any one of QKA, AQE, QKD, and AQD; R4 is selected from any one of VQ, IQ, IN, and VN; and / or R5 is selected from either I or L.

3. The GIP / GLP-1 receptor co-agonist according to claim 1 or 2, wherein: (1) In the general formula 1a, R1 is LDE or KEK; R2 is IA; R3 is selected from any one of QKA, AQE, and AQD; R4 is selected from any one of VQ, IQ, and IN; R5 is selected from WL; and R6 is I; or (2) In the general formula 1b, R1 is LDE or KEK; R2 is IA; R3 is selected from any one of QKA, AQE and AQD; R4 is selected from any one of VQ, IQ and IN; and R5 is I.

4. The GIP / GLP-1 receptor co-agonist according to claim 1, comprising an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:3-16.

5. The GIP / GLP-1 receptor co-agonist according to claim 4, comprising the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having a synonymous mutation only at position 24 and / or position 27 relative to the sequence shown in SEQ ID NO:

9.

6. The GIP / GLP-1 receptor co-agonist according to any one of claims 1-5, wherein the amino acids in the GIP / GLP-1 receptor co-agonist are all natural amino acids.

7. A GIP / GLP-1 fusion protein comprising a GIP / GLP-1 receptor co-agonist according to any one of claims 1-6 and an Fc region, wherein the GIP / GLP-1 receptor co-agonist is connected to the Fc region via a linker or directly; optionally, the GIP / GLP-1 receptor co-agonist is connected to the Fc region via a linker.

8. The GIP / GLP-1 fusion protein according to claim 7, wherein, The Fc region contains a natural Fc region sequence or a non-natural Fc region sequence; optionally, the Fc region is a human Fc region; further optionally, the Fc region is the Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody or a variant thereof.

9. The GIP / GLP-1 fusion protein according to claim 8, wherein, Compared to the Fc region of wild-type IgG4, the Fc region contains one or more of the mutations S228P, E233P, F234A, L235A, D265A, and R409K; or compared to the Fc region of wild-type IgG1, the Fc region contains one or more of the mutations L234A, L235A, P329A, and P331S.

10. The GIP / GLP-1 fusion protein according to claim 8, wherein the Fc region has an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences in SEQ ID NO:18-23; optionally, the Fc region has an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

18.

11. The fusion protein according to any one of claims 7-10, wherein the linker comprises GGG(GGGGS)n, where n is an integer greater than or equal to 1; optionally, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; more preferably, the linker comprises GGG(GGGGS)3; further optionally, the linker comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:

17.

12. A nucleic acid encoding a GIP / GLP-1 receptor co-agonist according to any one of claims 1-6, or a GIP / GLP-1 fusion protein according to any one of claims 7-11.

13. A vector comprising the nucleic acid of claim 12; Optionally, the vector is selected from liposomes, viruses, or plasmids.

14. A cell comprising the GIP / GLP-1 receptor co-agonist of any one of claims 1-6, the GIP / GLP-1 fusion protein of any one of claims 7-11, the nucleic acid of claim 12, or the vector of claim 13.

15. A pharmaceutical composition comprising the GIP / GLP-1 receptor co-agonist of any one of claims 1-6, the GIP / GLP-1 fusion protein of any one of claims 7-11, the nucleic acid of claim 12, or the carrier of claim 13, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

16. The pharmaceutical composition according to claim 15, wherein it is an injectable formulation; optionally, the injectable formulation is an intravenous injection formulation or a subcutaneous injection formulation; further optionally, the pharmaceutical composition contains a therapeutically effective amount of any one of claims 1-6, any one of claims 7-11, the GIP / GLP-1 fusion protein, the nucleic acid according to claim 12, or the carrier according to claim 13; even further optionally, the pharmaceutical composition is a unit injection formulation.

17. Use of the GIP / GLP-1 receptor co-agonist of any one of claims 1-6, the GIP / GLP-1 fusion protein of any one of claims 7-11, the nucleic acid of claim 12, the vector of claim 13, or the cell of claim 14 in the preparation of weight loss and / or hypoglycemic drugs.

18. The GIP / GLP-1 receptor co-agonist of any one of claims 1-6, the GIP / GLP-1 fusion protein of any one of claims 7-11, the nucleic acid of claim 12, the vector of claim 13, the cell of claim 14, or the pharmaceutical composition of claim 15 or 16, for use in the prevention or treatment of obesity or diabetes.

19. A method for preventing or treating obesity or diabetes, comprising administering to a subject in need an effective amount of any one of claims 1-6 of the GIP / GLP-1 receptor co-agonist, any one of claims 7-11 of the GIP / GLP-1 fusion protein, any one of claims 12 of the nucleic acid, any one of claims 13 of the vector, any one of claims 14 of the cell, or any one of claims 15 or 16 of the pharmaceutical composition.

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