Fusion protein molecule and use thereof

Abstract

Provided is a fusion protein, which can simultaneously target the GLP1 receptor or GLP1 / GCG receptors or GLP1 / GCG / GIP receptors, and the FGF21 receptor, can exhibit excellent effects of weight loss, improvement of NAS, etc., and can be used for treating related diseases.

Description

Fusion protein molecules and their applications

[0001] This application claims priority to Chinese Patent Application No. CN2024107067241, filed on May 31, 2024, entitled "Fusion Protein Molecule and Its Application", and Chinese Patent Application No. CN2025106376435, filed on May 16, 2025, entitled "Fusion Protein Molecule and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, it relates to fusion proteins that simultaneously bind to the GLP1 receptor or GLP1 / GCG or GLP1 / GCG / GIP receptor and the FGF21 receptor, and their use in the treatment of diseases. Background Technology

[0003] Obesity increases the risk of developing various diseases and dying from cancer. Currently, obese adults account for 13% of the global population, but less than 1% of patients receive drug treatment. Anti-obesity drugs mainly treat obesity by acting on the central nervous system, through incretin therapy, inducing thermogenesis, or through lipolysis. Drug development for obesity is shifting from single GLP1 receptor agonists to multi-receptor targets such as GLP1, GIP, and GCG. This has resulted in several drugs with good weight-loss effects, such as Semaglutide (a GLP1 receptor agonist, Novo Nordisk), Tirzepatide (a dual agonist of GLP1 and GIP receptors, Eli Lilly), Mazdutide (a dual agonist of GLP1 and GCG receptors, Innovent + Eli Lilly), AMG133 (a GIP receptor antagonist conjugated with GLP1, Amgen), and Retatrutide (a triple agonist of GLP1, GIP, and GCG receptors, Eli Lilly). However, these drugs still have drawbacks such as gastrointestinal side effects, weight rebound after discontinuation, and muscle loss. Therefore, next-generation drug development needs to focus more on better fat reduction and weight loss effects.

[0004] Fibroblast growth factor 21 (FGF21) is an endogenous metabolic hormone that regulates energy expenditure, glucose and lipid metabolism, and insulin resistance. FGF21 is an endocrine member of the FGF superfamily, and the liver is the primary site of its production. In addition, extrahepatic tissues such as white and brown adipose tissue and skeletal muscle also express FGF21. FGF21 reduces liver fat by increasing fatty acid oxidation, reducing the formation of new fat in the liver, and lowering systemic triglyceride levels. First, by improving metabolic regulation, FGF21 can reduce ongoing liver damage, allowing the liver time to heal. Second, FGF21 also has a targeted effect, increasing adiponectin, a hormone released from adipose tissue that inhibits the development and progression of liver fibrosis. In addition to its effects on the liver, FGF21 can also lower systemic lipid levels (triglycerides and LDL cholesterol) and improve glycemic control.

[0005] Although existing technologies have GLP1 single-target, GLP1 / GCG dual-target, GLP1 / GIP dual-target, and GLP1 / GCG / GIP triple-target peptide agonists, such as Semaglutide, Tirzepatide, Mazdutide, and Retatrutide, all of which have shown promising weight loss data, problems such as insufficient weight loss, muscle loss, severe gastrointestinal reactions, weight rebound, and inadequate lipid reduction and anti-fibrosis effects still exist. Therefore, adding FGF21 to GLP1 single-target, GLP1 / GCG dual-target, GLP1 / GIP dual-target, and GLP1 / GCG / GIP triple-target peptide agonists can further reduce weight, improve blood sugar, regulate lipid metabolism and insulin resistance, and even improve liver fibrosis.

[0006] Invention Overview

[0007] The purpose of this disclosure is to provide a fusion protein capable of activating single or multiple receptors for GLP1, GIP, and GCG, while also activating the FGF21 receptor. Therefore, this fusion protein, in addition to GLP1 stimulating insulin secretion, lowering blood glucose, suppressing appetite, increasing satiety, and delaying gastric emptying, can further stimulate insulin secretion, lower blood glucose, and suppress appetite with the addition of GIP. GCG further suppresses appetite, promotes energy metabolism and lipid breakdown, and FGF21 further reduces weight, improves blood glucose, regulates lipid metabolism and insulin resistance, and improves liver fibrosis. The multi-target combination fusion proteins provided in this disclosure, including GLP1 / FGF21, GLP1 / GCG / FGF21, and GLP1 / GIP / GCG / FGF21, can achieve better synergistic effects in lowering blood glucose and reducing weight than single-target proteins such as Semaglutide, dual-target proteins such as Tirzepatide, and tri-target proteins such as Retatrutide and Efruxifermin. Simultaneously, it can increase energy metabolism, maintain muscle mass, improve weight loss quality, and enhance resistance to NAFLD / MASH fibrosis.

[0008] This disclosure provides a fusion protein comprising: a GLP1 receptor agonist polypeptide domain, an FGF21 receptor agonist polypeptide domain, and a half-life extension polypeptide domain.

[0009] In some embodiments, the GLP1 receptor agonist polypeptide domain is located at the N-terminus of the fusion protein, the half-life extension polypeptide domain is located in the middle of the fusion protein, and the FGF21 receptor agonist polypeptide domain is located at the C-terminus of the fusion protein.

[0010] In some embodiments, the GLP1 receptor agonist polypeptide domain is a GLP1R monoagonal, a GLP1R / GCGR dual agonist, or a GLP1R / GIPR / GCGR triple agonist.

[0011] In some embodiments, the half-life-extending polypeptide domain is Fc, wherein Fc is a heavy chain constant region fragment from human IgG1 or human IgG4 or a mutant derived therefrom.

[0012] In some embodiments, the FGF21 receptor agonist polypeptide domain is a mutant of natural FGF21.

[0013] In some preferred embodiments, the fusion protein has the following structure from the N-terminus to the C-terminus: S0-Gp-L1-Fc-L1-Fg; wherein S0 is absent or selected from a signal peptide, a tag sequence, or a combination thereof; Gp is ​​a GLP1 receptor agonist polypeptide domain; L1 is absent or an adapter; Fc is a heavy chain constant region fragment from human IgG1 or human IgG4 or a mutant derived from them; and Fg is an FGF21 receptor agonist polypeptide domain.

[0014] In some preferred embodiments, L1 is (GGGGS). n n is any integer from 1 to 5, preferably 3.

[0015] In some preferred embodiments, the sequence of Gp is ​​any one of SEQ ID NO:31-41.

[0016] In some preferred embodiments, the sequence of Fc is any one of SEQ ID NO:21-30.

[0017] In some preferred embodiments, the sequence of Fg is any one of SEQ ID NO:1-20, preferably SEQ ID NO:9 and 17.

[0018] In some preferred embodiments, the sequence of the fusion protein is any one of SEQ ID NO:42-62, preferably SEQ ID NO:42, 43, 44, 50, 51 and 52, and more preferably SEQ ID NO:51 and 52.

[0019] This disclosure also provides an isolated nucleic acid that encodes the fusion protein of this disclosure.

[0020] This disclosure also provides a vector comprising nucleic acid encoding the fusion protein of this disclosure.

[0021] This disclosure also provides a host cell containing the vector or nucleic acid described in this disclosure.

[0022] This disclosure also provides a method for preparing the fusion protein, comprising: culturing the host cell under suitable conditions, expressing the fusion protein, and further purifying and / or isolating it.

[0023] This disclosure also provides a pharmaceutical composition comprising an effective amount of the fusion protein.

[0024] This disclosure also provides methods for using the fusion protein to treat diseases or its use in the preparation of medicaments for treating diseases.

[0025] This disclosure also provides methods for treating diseases, including administering the fusion protein or pharmaceutical composition described herein to an individual, subject, or patient in need.

[0026] This disclosure also provides the fusion proteins or pharmaceutical compositions described herein for the treatment of diseases.

[0027] In some implementation schemes, the aforementioned diseases include type 1 diabetes, type 2 diabetes, obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), hyperlipidemia, hypertension, osteoarthritis, osteoporosis, obstructive sleep apnea-hypopnea syndrome (OSA), heart failure, pulmonary hypertension (PH or PAH), chronic kidney disease (CKD), and other diseases.

[0028] This disclosure also provides a fusion protein comprising a GLP1 receptor agonist polypeptide domain, the sequence of which is any one of SEQ ID NO:32-41.

[0029] This disclosure also provides a fusion protein comprising an FGF21 receptor agonist polypeptide domain, wherein the sequence of the polypeptide domain is any one of SEQ ID NO:2-20. Attached Figure Description

[0030] Figure 1: Body weight changes in DIO-NASH mice after multiple doses

[0031] Figure 2: Percentage change in body weight and fat in DIO-NASH mice after 28 days of multiple drug administrations

[0032] Figure 3: NAS scores of DIO-NASH mice after multiple doses

[0033] Figure 4: Body weight changes in DIO-NASH mice after multiple doses

[0034] Figure 5A: Changes in total cholesterol in the liver of DIO-NASH mice after multiple doses

[0035] Figure 5B: Changes in liver triglycerides in DIO-NASH mice after multiple doses

[0036] Figure 5C: Changes in low-density lipoprotein cholesterol in the liver of DIO-NASH mice after multiple doses

[0037] Figure 6A: NAS score of DIO-NASH mice after multiple doses

[0038] Figure 6B: Fatty degeneration score in DIO-NASH mice after multiple doses

[0039] Figure 6C: Ballooning score in DIO-NASH mice after multiple doses

[0040] Figure 7: Body weight changes in HFD+CCL4-induced NASH mice after multiple doses

[0041] Figure 8A: Changes in liver weight in HFD+CCL4-induced NASH mice after multiple doses

[0042] Figure 8B: Changes in liver weight to body weight in HFD+CCL4-induced NASH mice after multiple doses

[0043] Figure 9: NAS scores in HFD+CCL4-induced NASH mice after multiple doses Detailed Implementation Plan

[0044] the term

[0045] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated to be incorporated by reference.

[0046] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0047] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0048] The term “about” includes and describes the value or parameter itself. For example, “about x” includes and describes “x” itself. As used herein, when used in conjunction with a measurement or to modify a value, unit, constant, or range of values, the term “about” refers not only to the value or parameter itself, but also to variations within ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0049] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0050] GLP1 (glucagon-like peptide-1) is a 30- or 31-amino acid polypeptide incretin secreted by intestinal L-cells. It exists in two active forms: GLP1(7-36) and GLP1(7-37). GLP1 is released into circulation after meals and exerts its biological activity by activating GLP1 receptors. GLP1 has many biological functions, including stimulating insulin secretion, protecting pancreatic β-cells, inhibiting glucagon secretion, delaying gastric emptying, and suppressing appetite, thereby reducing food intake.

[0051] GCG (glucagon) is typically a 29-amino acid peptide, corresponding to amino acids 53-81 of proglucagon. Glucagon has numerous physiological effects, including raising blood glucose levels and maintaining blood glucose levels by binding to and activating glucagon receptors, regulating gluconeogenesis and glycogenolysis. It also increases blood glucose levels in hypoglycemic states by stimulating glycogenolysis and gluconeogenesis, regulates hepatic ketone production, regulates bile acid metabolism, and suppresses appetite through the vagus nerve's satiety effect, thereby reducing food intake, which in turn helps degrade fat and reduce weight.

[0052] GIP (glucose-dependent insulinotropic peptide) is usually a 42-amino acid peptide obtained by proteolysis of a 133-amino acid precursor. Its biological functions include glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and regulation of food intake.

[0053] "GLP1 receptor (GLP1R) agonists" are substances that can bind to GLP1R and elicit the same or similar characteristic responses as natural GLP1. These substances can be peptides, proteins, or small molecule compounds. GLP1R agonists activate GLP1R completely or partially, thereby triggering a series of downstream intracellular signaling pathway responses and producing corresponding cellular activity. Typical GLP1R agonists include natural GLP1 and its various mutants, derivatives, or analogs, such as Semaglutide, Mazdutide, Tirzepatide, and Retatrutide.

[0054] "GCG receptor (GCGR) agonists" are substances that can bind to GCGR and trigger a characteristic response similar to or the same as that of natural glucagon. These substances can be peptides, proteins, or small molecule compounds. GCGR agonists activate GCGR completely or partially, thereby inducing a series of downstream intracellular signaling pathway responses and producing corresponding cellular activity.

[0055] "GIP receptor (GIPR) agonists" are substances that can bind to GIPR and elicit the same or similar characteristic responses as natural GIP. These substances can be peptides, proteins, or small molecule compounds. GIPR agonists activate GIPR completely or partially, thereby triggering a series of downstream intracellular signaling pathway responses and producing corresponding cellular activity.

[0056] Fibroblast growth factor 21 (FGF21) is a member of the FGF family (fibroblast growth factors, FGFs) and contains 182 amino acids. FGFs have various functions, such as promoting fibroblast mitosis, mesodermal cell growth, and stimulating angiogenesis. FGF21 can promote glucose uptake by adipocytes, enhance insulin sensitivity, and reduce serum total cholesterol and low-density lipoprotein levels.

[0057] β-Klotho (KLB) is a member of the Klotho protein family, mainly distributed in organs and tissues such as the liver, adipose tissue, pancreas, and brain. KLB is a single-pass transmembrane protein and a key high-affinity co-receptor for fibroblast growth factor 19 / 21 (FGF19 / 21) to target and activate fibroblast growth factor receptors (FGFRs). KLB participates in the regulation of substances and energy such as blood glucose, lipids, body weight, and bile acid cycling in the FGF19 / 21-KLB-FGFRs pathway, and is involved in the regulation of cell proliferation in various tissues and organs.

[0058] The "GLP1 receptor agonist" used in this disclosure includes three types: GLP1 monoreceptor agonist, GLP1 / GCG dual receptor agonist, and GLP1 / GCG / GIP triple receptor agonist. Specifically, the GLP1 monoreceptor agonist is a polypeptide that only has the activity of binding to and activating GLP1R; the GLP1 / GCG dual receptor agonist is a polypeptide modified by amino acid mutation to simultaneously possess the dual activities of binding to and activating GLP1R and GCGR; and the GLP1 / GCG / GIP triple receptor agonist is a polypeptide modified by amino acid mutation to simultaneously possess the triple activities of binding to and activating GLP1R, GIPR, and GCGR.

[0059] The FGF21 receptor agonists used in this article include various mutants of natural FGF21, such as Efruxifermin.

[0060] The term "fusion protein" as used in this article typically refers to polypeptide domains, each with a specific function, that are combined together directly or via one or more linkers through peptide bonds.

[0061] As used herein, "connector" refers to any tool used to connect two polypeptide domains. The types of connectors include, but are not limited to, chemical connectors and polypeptide connectors. The sequence of polypeptide connectors is not limited. Polypeptide connectors are preferably non-immunogenic and flexible, such as those containing serine and glycine sequences. Depending on the specific construct, connectors can be long or short.

[0062] According to this disclosure, the peptide linker preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises an amino acid sequence (GGGGS). n , where n is any integer choice from 1 to 5, preferably containing the amino acid sequence (GGGGS)3.

[0063] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, specifically the two polypeptide chains that form a dimer containing the self-binding C-terminal constant region of the immunoglobulin heavy chain. This term includes both native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of the IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain; for example, the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, antibodies generated by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Antibodies generated by host cells through the expression of specific nucleic acid molecules encoding the full-length heavy chain may include the full-length heavy chain, or they may include cleaved variants of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Therefore, the C-terminal lysine (K447) in the Fc region, or the C-terminal glycine (G446) and lysine (K447), may or may not be present.

[0064] The term "amino acid mutation" includes amino acid substitutions in a polypeptide sequence. As used herein, "amino acid substitution," "mutation," or "replacement" means replacing an amino acid at a specific position in the parental polypeptide sequence with another amino acid. For example, S32A indicates that serine at position 32 has been replaced by alanine.

[0065] The term "effective dose" refers to a dose of a pharmaceutical preparation containing the active ingredient of this disclosure that, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the mode of administration; the bioavailability characteristics of the administered preparation; the chosen dosing regimen; and the use of any concomitant therapies.

[0066] The term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in the clinicopathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and mitigating or improving prognosis.

[0067] The terms “individual,” “subject,” or “patient” refer to any animal, such as a mammal or marsupial. Individuals in this disclosure include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0068] The terms "disease," "symptom," or "disorder" refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. In this article, "diseases" include type 1 diabetes, type 2 diabetes, obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), hyperlipidemia, hypertension, osteoarthritis, osteoporosis, obstructive sleep apnea-hypopnea syndrome (OSA), heart failure, pulmonary hypertension (PH or PAH), and chronic kidney disease (CKD).

[0069] Experimental Example

[0070] Preparation and expression of fusion proteins

[0071] Primers were designed based on the amino acid sequence, and the various domains of the fusion protein were constructed by PCR. Homologous recombination was performed into the PTT5 expression vector to construct a full-length expression plasmid. After transformation and large-scale extraction of the plasmid, it was transfected into Expi293 cells for expression. The cells were then cultured in a shaker at 37°C, 5% humidity, and 120 rpm. Enhancer and feed were added 24 hours after transfection, and the cells were cultured for 7 days.

[0072] After culture, the cell culture solution was centrifuged (4500 rpm) for 10 min, and the supernatant was collected through a 0.45 μm filter. Affinity chromatography purification was performed using Protein A or its derivative (GE, MabSelect) that interacts with Fc. The MabSelect column was equilibrated with 5 column volumes of 1×PBS phosphate buffer (pH 7.4). The obtained cell supernatant was loaded onto the column. After loading, the column was washed with 5 column volumes of 1×PBS phosphate buffer until the A280 UV absorption peak returned to baseline. The column was then washed with 0.1 M HAc (pH 3.2). The eluted sample was collected based on the A280 UV absorption peak and neutralized with 1 M Tris-HCl (pH 8.0). Further, the obtained sample was concentrated by ultrafiltration and the buffer was changed to pH 7.4 PBS phosphate buffer. Protein content was measured at 280 nm using a UV spectrophotometer. The purity and integrity of the obtained protein were confirmed by SEC-HPLC and LC-MS, and the protein was stored in separate apparatus at -80℃ for long-term storage.

[0073] GLP1R / GCGR / GIPR / FGF21R ​​activation activity and KLB binding activity assay

[0074] GLP1R activation activity assay: The in vitro activity of the fusion protein GLP1 was detected using HEK293T cells overexpressing the human GLP1 receptor. The human GLP1R gene was cloned into pCDNA3.1 to construct the pCDNA3.1-hGLP1R expression vector, which was transfected into HEK293T cells. Stable high-expressing HEK293T / hGLP1R monoclonal cell lines were screened and cultured. HEK293T / hGLP1R overexpressing cells were resuspended in an experimental buffer (1×DPBS, Sigma, D8537), 500 μM IBMX (Sigma, I5879), 0.1% w / v BSA (Sigma, A1933-G5), 1000 cells / 5 μl / well, and mixed with an equal volume of serially diluted fusion protein. The mixture was then seeded in 384-well plates (Greiner, 784075) and cultured at 37°C and 5% CO2 for 30 min. Add 5 μl of anti-cAMP antibody-Eu to the plate after the above incubation. 3+ Cryptate and 5 μl cAMP-d2 were incubated at room temperature for 1 h. Optical density was read at 665 nm and 615 nm on an EnVision multi-plate reader, and the percentage of the 665 nm / 615 nm reading was calculated. Anti-cAMP antibody-Eu 3+ Cryptate binds to cAMP-d2 and they approach each other, then react with Eu under excitation light. 3+Cryptate transfers energy to receptor d2, causing d2 to emit fluorescence for an extended period. Meanwhile, the fusion protein activates hGLP1R on the cell surface, producing cAMP, which competes with the added cAMP-d2 for the anti-cAMP antibody-Eu. 3+ Cryptate binding reduces the energy received by receptor d2, resulting in a decrease in fluorescence readings.

[0075] GCGR activation activity assay: The human GCGR gene was cloned into pCDNA3.1 to construct the pCDNA3.1-hGCGR expression vector, which was transfected into HEK293T cells. Stable, high-expressing HEK293T / hGCGR monoclonal cell lines were screened and cultured. Similarly, GCGR activation activity was assessed using HEK293T / hGCGR cell lines. After activation with the fusion protein, the anti-cAMP antibody Eu3 was added. + Cryptate competes with cAMP-d2, and cAMP-d2 produced by human GCGR cells activated by a fusion protein with anti-cAMP antibody-Eu. 3+ Cryptate binding was used to assess the GCG activity of the fusion protein by changes in fluorescence readings.

[0076] GIPR activation activity assay: The human GIPR gene was cloned into pCDNA3.1 to construct the pCDNA3.1-hGIPR expression vector, which was transfected into HEK293T cells. Stable high-expressing HEK293T / hGIPR monoclonal cell lines were screened and cultured. The GIPR activity assay method was the same as above.

[0077] FGF21 receptor activation activity assay: Cell suspensions of 5 × 10⁵ cells were prepared using human / mouse / rat / monkey FGF21 Reporter HEK-293 luciferase reporter gene cell lines. 4 Cells / ml, DMEM + 5% FBS + 1 μg / ml puromycin, seeded in 96-well plates, 100 μl per well, and cultured overnight. Serially dilute the test samples and add them to cells after removing the culture medium. Incubate at 37°C for 6 h, then detect fluorescence using a Bright-Glo luciferase assay system (Promega, Madison, MI).

[0078] KLB binding assay: The human / mouse / rat / monkey KLB gene was cloned into pCDNA3.1 to construct the pCDNA3.1-KLB expression vector, which was transiently transfected into HEK293T cells. HEK293T / KLB transiently transfected cells were resuspended in experimental buffer (PBS + 2% FBS) and mixed with serially diluted fusion protein. After incubation at 4°C for 1 h, the cells were washed once with experimental buffer. Anti-human IgG (H+L) 488 fluorescent secondary antibody was added, and the cells were incubated at 4°C for 30 min, followed by washing twice with experimental buffer. Finally, the binding activity of the fusion protein to KLB from different species was detected using FACS.

[0079] Example

[0080] The following specific embodiments are provided to illustrate this disclosure. It should be understood that these examples are merely illustrative and not intended to limit the scope of this disclosure.

[0081] Example 1: Preparation and Screening of FGF21 Receptor Agonist Peptide Domains

[0082] The FGF21 receptor agonist polypeptide domain of the fusion protein provided in this disclosure consists of 19 mutant polypeptides of FGF21 designed by the inventors. All mutant polypeptides are based on F0 (FGF21 receptor agonist polypeptide domain of Efruxifermin) with multi-site amino acid combination mutations, as shown in Table 1. The mutation sites are marked with underlines.

[0083] Table 1. Sequence of FGF21 receptor agonist polypeptide domain

[0084] Various FGF21 receptor agonist polypeptide domains shown in Table 1 were linked to the C-terminus of Fc via linkers to form several Fc-FGF21 fusion proteins. Exemplary linkers include (GGGGS)n (n can be any positive integer from 1 to 5), and exemplary Fcs include IgG1 Fc or its mutants. The in vitro FGF21 receptor activation activity of the constructed Fc-FGF21 fusion proteins was tested, using Efruxifermin as a positive control. As shown in Table 2, most of the designed fusion proteins exhibited FGF21 receptor activation activity comparable to or better than the positive control.

[0085] Table 2. Detection of activation activity of the Fc-FGF21 receptor agonist polypeptide domain in the fusion protein.

[0086] As can be seen from the data in Table 2, the FGF21 receptor activation activity of Fc-F8, Fc-F11, Fc-F12, Fc-F14, Fc-F19 and Fc-F20 is significantly better than that of the positive control Efruxifermin. Therefore, the FGF21 receptor agonist polypeptide domain is preferred as the fusion protein of this disclosure.

[0087] Example 2: Pharmacokinetic assay of fusion protein Fc-FGF21

[0088] This embodiment evaluates the pharmacokinetics of the fusion protein Fc-FGF21 in mice. Six-week-old male C57BL / 6J mice were grouped (n=3) to have similar average body weights the day before drug treatment and administered a single subcutaneous dose of 4 mg / kg sample. Blood samples were then collected at 2, 8, 24, 48, 72, 96, 168, 240, 336, and 504 hours post-injection. The concentration of full-length FGF21 protein in blood was measured using the intact human FGF21 ELISA kit (F1231-K01, Eagle Biosciences, USA), which is immunoreactive to the N- and C-termini of the FGF21 protein. The concentrations of each fusion protein were measured in blood samples collected 504 hours after subcutaneous injection into mice, and pharmacokinetic parameters for each sample were calculated. The data are shown in Table 3.

[0089] Table 3. Pharmacokinetic analysis of the fusion protein Fc-FGF21

[0090] As shown in Table 3, the Tmax, Cmax, AUC, and half-life of F8 and F20 were significantly improved compared to Efruxifermin. In particular, the FGF21 fraction showed improvements in Tmax, Cmax, and half-life of at least two-fold, three-fold, and two-fold or more, respectively. F20 increased FGF21 activity while also reducing protein aggregation, thus improving drug-likeness and pharmacokinetics.

[0091] Example 3: Preparation of each polypeptide domain of the fusion protein GLP1-Fc-FGF21

[0092] The fusion protein disclosed herein contains multiple polypeptide domains: a half-life-extending polypeptide domain, such as Fc; a GLP1 receptor agonist polypeptide domain; and an FGF21 receptor agonist polypeptide domain.

[0093] Among them, Fc can be hIgG1 Fc and its mutants, or hIgG4 Fc and its mutants. The various Fc sequences used in this disclosure are shown in Table 4.

[0094] Furthermore, the "GLP1 receptor agonist polypeptide domain" includes three types: GLP1 monoreceptor agonist, GLP1 / GCG dual receptor agonist, and GLP1 / GCG / GIP triple receptor agonist. This disclosure provides sequences of several GLP1 receptor agonist polypeptide domains designed by the inventors, as shown in Table 5.

[0095] Furthermore, the FGF21 receptor agonist polypeptide domains selected according to Examples 1 and 2 are preferably F8 and F20.

[0096] Table 4 Fc domain sequence

[0097] Table 5. Sequences of GLP1 receptor agonist polypeptide domains

[0098] Example 4: Construction of the fusion protein GLP1-Fc-FGF21

[0099] The GLP1 receptor agonist polypeptide domain shown in Table 5 and the FGF21 receptor agonist polypeptide domain shown in Table 1 are connected to the N-terminus and C-terminus of the Fc domain shown in Table 4, respectively, via linkers to construct several GLP1-Fc-FGF21 fusion proteins of this disclosure. The sequences of the fusion proteins are shown in Table 6, and exemplary linkers are shown as (GGGGS). n (n can be a positive integer from 1 to 5).

[0100] Table 6 Construction of the fusion protein GLP1-Fc-FGF21

[0101] Example 5: In vitro biological activity evaluation and pharmacokinetic assay of the GLP1 receptor agonist polypeptide domain and FGF21 receptor agonist polypeptide domain of the fusion protein GLP1-Fc-FGF21

[0102] The GLP1 receptor agonist polypeptide domain and FGF21 receptor agonist polypeptide domain of the fusion protein molecule constructed in Example 4 were subjected to in vitro activity assays, including GLP1R activation activity assay, GCGR activation activity assay, GIPR activation activity assay, FGF21R ​​activation activity assay, and KLB binding activity assay.

[0103] This embodiment uses four GLP1 analogs—Semaglutide, Mazdutide, Tirzepatide, and Retatrutide—as positive controls representing different types of GLP1R agonists, Efruxifermin as a positive control for FGF21R ​​agonists, and 12C12-F (from CN116284441B) and C382L. 13 F8L 10 M2 and C495L 13 F8L 10 M2 (from CN109836504B) served as a positive control for the fusion protein GLP1-Fc-FGF21. As shown in Table 7, most of the fusion proteins designed in this disclosure exhibited in vitro activities comparable to the positive control, and some fusion proteins showed significantly stronger activities than the positive control.

[0104] Table 7. Detection of activation activity of the GLP1 and FGF21 receptor agonist peptide domains of the fusion protein.

[0105] Table 8. Detection of activation activity of the GLP1 and FGF21 receptor agonist peptide domains of the fusion protein.

[0106] Table 7 shows that most of the different types of GLP1-Fc-FGF21 fusion proteins designed in this disclosure, while maintaining the activation activity of GLP1 and / or GCG and GIP receptors, also exhibit FGF21R ​​activation activity comparable to the positive control Efruxifermin. Among them, T45-Fc1-F20 exhibits multiple agonist activities of GLP1R, GCGR, GIPR, and FGF21R, and the GLP1R activity is stronger than that of Retatrutide; the complete GCGR activation activity is 2.15 nM, which is consistent with the expected target GCGR activity at the nM level; the complete GIPR activation activity is 15.6 nM. It can be seen that this molecule exhibits relatively high and relatively balanced agonist activity on various receptors compared to other multiple agonist fusion proteins.

[0107] Table 8 shows that most of the different types of GLP1-Fc-FGF21 fusion proteins designed in this disclosure, while maintaining GLP1 and / or GCG and GIP receptor activation activities, also exhibit FGF21R ​​activation activity comparable to the positive control Efruxifermin. Among them, B3-Fc1-F8 and B3-Fc1-F20 exhibit multiple agonist activities of GLP1R, GCGR, and FGF21R. Taking B3-Fc1-F20 as an example, its GLP1R activation activity is 15 times that of Mazdutide and is significantly higher than that of C495L. 13 F8L 10 It is 4 times that of M2; its GCGR activation activity is 9 times that of Mazdutide, and it is comparable to C495L. 13 F8L 10 M2 is comparable; its FGF21R ​​activation activity is C495L. 13 F8L 10 It is 4 times that of M2 and comparable to Efruxifermin. This indicates that the molecule exhibits relatively high and balanced agonist activity at various receptors compared to other multi-agonist fusion proteins.

[0108] Table 9. Detection of activation activity and KLB binding activity of the FGF21 receptor agonist peptide domain of the fusion protein GLP1-Fc-FGF21.

[0109] Table 10. Detection of activation activity of the FGF21 receptor agonist polypeptide domain in the fusion protein GLP1-Fc-FGF21

[0110] As shown in Table 9, T45-Fc1-F20 exhibits improved FGF21 activity. Specifically, its activation activity against human FGF21R ​​is 2.5 times that of 12C12-F, against mouse FGF21R ​​is 2 times that of 12C12-F, against rat FGF21R ​​is 8 times that of 12C12-F, and against monkey FGF21R ​​is 2.5 times that of 12C12-F. Furthermore, T45-Fc1-F20 also possesses strong KLB binding activity. Specifically, its binding activity against human KLB is 2 times that of 12C12-F, against mouse KLB is 5 times that of 12C12-F, against rat KLB is 3 times that of 12C12-F, and against monkey KLB is 2 times that of 12C12-F.

[0111] As shown in Table 10, B3-Fc1-F20 exhibits improved FGF21 activity. Specifically, the activation activity of B3-Fc1-F20 on human FGF21R ​​is C495L. 13 F8L10 Four times that of M2.

[0112] Example 6: Pharmacodynamic study of multiple subcutaneous injections in a DIO-NASH model mouse model of diet-induced obesity (DIO) and non-alcoholic steatohepatitis (NASH).

[0113] The purpose of this embodiment is to investigate the effects of subcutaneous injection of the disclosed fusion protein on the body weight and NAFLD activity score (NAS score, including steatosis, inflammation and ballooning) of diet-induced obese (DIO) mice.

[0114] Male C57BL / 6J mice aged 5–6 weeks were fed a high-fat diet to establish the model. Husbandry conditions included alternating 12h / 12h lighting, free access to food, a temperature of 20–24℃, and a relative humidity of 30–70%. One day before administration (Day 0), mice were randomly assigned to groups based on body weight, food intake, and body composition: B3-Fc1-F8, B3-Fc1-F20, T45-Fc1-F20, Tirzepatide, Retatrutide, Efruxifermin, 12C12-F, solvent group (PBS), and a normal control group, with 6 mice in each group. Each group received subcutaneous injections twice weekly for 4 weeks, with body weight and food intake monitored three times weekly. Animals were assessed for lean meat and fat mass in vivo using a Minispec (Bruker) MRI scanner on Days 0, 7, 14, 21, and 28, and underwent an oral glucose tolerance test (OGTT) on Day 22. At the end of the experiment on Day 29, all animals had their body weight, food intake, and fasting blood glucose measured. Serum, plasma, and liver tissue were collected after euthanasia for liver function, blood lipids, liver fat, and liver pathology tests. Data are expressed as mean ± standard deviation and statistically analyzed using one-way ANOVA.

[0115] As shown in Figure 1, after multiple administrations to DIO-NASH model mice, the same dose of T45-Fc1-F20 showed the best weight loss effect. As shown in Figure 2, after multiple administrations to DIO-NASH model mice, T45-Fc1-F20's fat-reducing effect was significantly better than Tirzepatide (p<0.001 compared to Tirzepatide) and also better than Retatrutide (p<0.0001 compared to Retatrutide). As shown in Figure 3, T45-Fc1-F20 significantly improved NAS scores (p<0.0001 compared to the solvent group), especially in improving fatty degeneration. As shown in Figure 4, after multiple administrations to DIO-NASH model mice, the same doses of B3-Fc1-F8 and B3-Fc1-F20 showed significantly better weight loss effects than Tirzepatide. As shown in Figures 5A-5C, administration of B3-Fc1-F8 significantly improved total cholesterol (*p<0.05 compared to Tirzepatide), triglycerides (****p<0.0001 compared to Tirzepatide), and low-density lipoprotein cholesterol (*p<0.05 compared to Tirzepatide) in mouse liver compared to Tirzepatide. Administration of B3-Fc1-F20 also significantly improved total cholesterol (**p<0.01 compared to Tirzepatide), triglycerides (***p<0.001 compared to Tirzepatide), and low-density lipoprotein cholesterol (**p<0.01 compared to Tirzepatide) in mouse liver compared to Tirzepatide. As shown in Figures 6A-6C, administration of B3-Fc1-F8 and B3-Fc1-F20 significantly improved the NAS score in mice. Compared to the solvent group, both B3-Fc1-F8 and B3-Fc1-F20 showed highly significant changes in fatty degeneration and ballooning degeneration (both p < 0.0001 compared to the solvent group). Furthermore, compared to Tirzepatide, B3-Fc1-F8 and B3-Fc1-F20 demonstrated greater benefits in improving fatty degeneration (both p < 0.05 compared to Tirzepatide).

[0116] Example 7: Pharmacodynamic test of the fusion protein by multiple subcutaneous injections in NASH model mice induced by a high-fat diet (HFD) and 5% carbon tetrachloride (CCL4).

[0117] The purpose of this embodiment is to study the effect of subcutaneous injection of the disclosed fusion protein on the NAS score of HFD+CCL4-induced NASH model mice, while simultaneously performing liver function, blood lipids, liver lipids, and liver pathology tests.

[0118] The model was established using 19-week-old male DIO-C57BL / 6J mice fed a high-fat diet. Husbandry conditions included: alternating 12-hour / 12-hour lighting, free access to food, a temperature of 20–24℃, and a relative humidity of 30–70%. One day before drug administration (Day 0), mice were randomly assigned to groups based on body weight and food intake: B3-Fc1-F8, B3-Fc1-F20, and C495L. 13 F8L 10 M2, Mazdutide, Efruxifermin, MGL-3196, solvent group (PBS), and normal control group, with 9–10 mice in each group. Mice were continued to be fed a high-fat diet and received intraperitoneal injections of 5% (v / v) CCL4 twice weekly to establish the model. Simultaneously, they received subcutaneous injections of the drug once weekly at a dose of 10 nmol / kg. Body weight and food intake were monitored three times weekly for 4 weeks. At the end of the experiment on Day 29, body weight and food intake of all animals were measured. Serum, plasma, and liver tissue were collected after euthanasia for liver function, blood lipids, liver lipids, and liver pathology. Data are expressed as mean ± standard deviation and statistically analyzed using one-way ANOVA.

[0119] As shown in Figure 7, after multiple administrations of B3-Fc1-F8 and B3-Fc1-F20 to HFD+CCL4-induced NASH model mice, the same doses significantly reduced mouse body weight, and the weight reduction effect was superior to that of Mazdutide. As shown in Figures 8A-8B, after multiple administrations of B3-Fc1-F8 and B3-Fc1-F20 to HFD+CCL4-induced NASH model mice, compared with the solvent group, liver weight was significantly reduced by both B3-Fc1-F8 and B3-Fc1-F20 (both ****p<0.0001 compared to the solvent group). Furthermore, B3-Fc1-F8 and B3-Fc1-F20 also significantly improved the liver-to-body ratio (***p<0.001 and ****p<0.0001, respectively, compared to the solvent group). As shown in Figure 9, B3-Fc1-F8 and B3-Fc1-F20 significantly improved NAS scores (both ****p<0.0001 compared to the solvent group), especially in improving steatosis.

[0120] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for extraordinary experimentation. All such equivalents are within the scope of this disclosure and are encompassed by the claims.

Claims

1. A fusion protein having the following structure from N-terminus to C-terminus: So-Gp-L1-Fc-L1-Fg; wherein, S0 is nothing or selected from a signal peptide, a tag sequence or a combination thereof; Gp is a GLP1 receptor agonist polypeptide domain; L1 is nothing or a linker; Fc is a heavy chain constant region fragment from or derived from human IgG1 or human IgG4; Fg is a FGF21 receptor agonist polypeptide domain; the sequence of the Gp is any one of SEQ ID NOs: 31-41; the sequence of the Fc is any one of SEQ ID NOs: 21-30; the sequence of the Fg is any one of SEQ ID NOs: 1-20.

2. The fusion protein of claim 1, having a full-length sequence of any one of SEQ ID NOs: 42-62.

3. The fusion protein of claim 1, having a full-length sequence of SEQ ID NOs: 42, 43, 44, 50, 51 and 52.

4. The fusion protein of claim 1, having a full-length sequence of SEQ ID NOs: 51 and 52.

5. An isolated nucleic acid encoding the fusion protein of any one of claims 1-4.

6. A vector comprising the nucleic acid of claim 5.

7. A host cell comprising the vector of claim 6 or the nucleic acid of claim 5.

8. A method of making the fusion protein of any one of claims 1-4, comprising: culturing the host cell of claim 7 under suitable conditions to express the fusion protein, and further purifying and / or isolating the fusion protein.

9. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1-4.

10. Use of the fusion protein of any one of claims 1-4 in the preparation of a medicament for treating a disease.

11. The use of claim 10, wherein the disease comprises type I diabetes, type II diabetes, obesity, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), hyperlipidemia, hypertension, osteoarthritis, osteoporosis, obstructive sleep apnea hypopnea syndrome (OSA), heart failure, pulmonary arterial hypertension (PH or PAH), chronic kidney disease (CKD).

12. A fusion protein comprising a GLP1 receptor agonist polypeptide domain, the sequence of the polypeptide domain being any one of SEQ ID NOs: 32-41.

13. A fusion protein comprising a FGF21 receptor agonist polypeptide domain, the sequence of the polypeptide domain being any one of SEQ ID NOs: 2-20.

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