Fusion protein molecule and use thereof

Abstract

Provided is a fusion protein, which binds to ACTRII, also can activate a GLP1 receptor and / or an FGF21 receptor, and has better muscle-building and fat-reducing effects in a weight-reducing use.

Description

Fusion protein molecules and their applications

[0001] This application claims priority to Chinese Patent Application No. CN2024107051421, filed on May 31, 2024, entitled "Fusion Protein Molecule and Its Application", and Chinese Patent Application No. CN202510637725X, 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, relates to fusion proteins that can activate GLP1 receptors and / or FGF21 receptors while binding to ACTRII, and their application 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 (GLP1 receptor agonist, Novo Nordisk), Tirzepatide (dual GLP1 and GIP receptor agonist, Eli Lilly), Mazdutide (dual GLP1 and GCG receptor agonist, Innovent + Eli Lilly), AMG133 (GIP receptor antagonist conjugated with GLP1, Amgen), and Retatrutide (GLP1, GIP, and GCG receptor triple agonist, 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, while also maintaining or even increasing muscle mass.

[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] ACTRII is a single-pass transmembrane protein expressed in various tissues. Its ligands include TGF-β family proteins such as activin, myostatin (or GDF8), and GDF11, all of which can inhibit muscle differentiation and induce myofibril atrophy. Myostatin primarily acts on muscle. ACTRII signaling is closely related to skeletal muscle growth and differentiation, as well as various diseases including obesity, pulmonary hypertension, heart failure, and osteoporosis. ACTRII acts on muscle and fat, exerting opposite effects: in muscle tissue, ACTRII binding to its ligands leads to the degradation of downstream proteins, inhibiting muscle differentiation and causing muscle atrophy; while in fat, through ligand binding, it can promote adipocyte growth and the differentiation and accumulation of preadipocytes. Therefore, developing antagonists targeting ACTRII could achieve the dual effects of fat reduction and muscle gain.

[0006] In non-muscle areas, Luspatercept can be used to treat transfusion-dependent β-thalassemia and anemia caused by low-risk ringed sideroblastic myelodysplastic syndrome; Sotatercept can be used to treat pulmonary hypertension, and its phase 3 clinical data are positive and its efficacy is significant, making it a blockbuster drug for pulmonary hypertension.

[0007] ACTRII antibodies, such as Bimagrumab, block the binding of ACTRIIs to ligands Activin and Myostatin, and promote brown adipose tissue increase and thermogenesis; clinical studies have shown that they can significantly improve body composition and insulin tolerance. ACTRII antibodies have a relatively unique mechanism of action that can achieve both fat reduction and muscle gain, and their safety has been validated in clinical trials, while also avoiding off-target effects. However, highly active ACTRII-blocking antibodies also block the binding of the ligand GDF11 to ACTRII, which involves osteoclast and osteoblast differentiation and function, thus potentially posing a risk of side effects. Furthermore, highly active ACTRII antibodies may also block the binding of the ligand Activin to ACTRII, thus creating a potential risk. Additionally, at higher doses, highly active ACTRII-blocking antibodies may also cause pancreatic and hepatotoxicity. These are some of the risks or problems associated with existing ACTRII antibodies.

[0008] Comparing only weight changes, Bimagrumab did not show a significant reduction in weight compared to other GLP-1 inhibitors, but it improved body composition. Subjects experienced a decrease in adipose tissue while increasing muscle tissue, with the adipose tissue reduction (20.5%) comparable to semaglutide (19.3%), but less than telpotide (33.9%). In mice, the ACTR1 antibody Bimagrumab, when used in combination with the GLP-1 drug Semaglutide, further reduced fat while maintaining or increasing muscle mass. Adding FGF21 to this combination further improved blood glucose, regulated lipid metabolism and insulin resistance, and even improved liver fibrosis.

[0009] Invention Overview

[0010] The purpose of this disclosure is to provide a fusion protein that, by introducing GLP1 and / or FGF21 polypeptide structures at the end of an ACTRII antibody, can activate GLP1 and / or FGF21 receptors while antagonizing ACTRII. Therefore, the fusion protein can enable subjects to achieve synergistic weight loss while maintaining muscle mass and improving the quality of weight loss. On the one hand, it can block the binding of ACTRII to its ligands, thereby exerting a fat-reducing and muscle-building effect. Furthermore, using the fusion protein form can appropriately reduce the activity of ACTRII antibodies in blocking their related ligands, thereby reducing side effects. On the other hand, the fusion protein can activate GLP1 receptors, producing weight-loss effects such as suppressing appetite and delaying gastric emptying; and / or, the fusion protein can also activate FGF21 receptors, which can exert effects such as weight loss, improving blood glucose, regulating lipid metabolism and insulin resistance, and improving liver fibrosis. In pulmonary arterial hypertension (PH or PAH), the fusion protein mainly inhibits ACTRII, thereby inhibiting the ACTRII / Smad2 / 3-mediated pro-proliferative effect and rebalancing anti-proliferative and pro-proliferative signals.

[0011] This disclosure provides a fusion protein comprising: an anti-ACTRII antibody domain or an antigen-binding fragment domain thereof; a GLP1 receptor agonist polypeptide domain; and / or an FGF21 receptor agonist polypeptide domain.

[0012] This disclosure provides a fusion protein that can be any one of three structures:

[0013] 1) Anti-ACTRII antibody domain or its antigen-binding fragment domain, and GLP1 receptor agonist polypeptide domain;

[0014] 2) Anti-ACTRII antibody domain or its antigen-binding fragment domain, and FGF21 receptor agonist polypeptide domain;

[0015] 3) Anti-ACTRII antibody domain or its antigen-binding fragment domain, GLP1 receptor agonist peptide domain, and FGF21 receptor agonist peptide domain.

[0016] In some embodiments, the GLP1 receptor agonist polypeptide domain is linked to the N-terminus of the heavy or light chain of the antibody domain or its antigen-binding fragment domain of the anti-ACTRII; the FGF21 receptor agonist polypeptide domain is linked to the C-terminus of the heavy chain of the antibody domain or its antigen-binding fragment domain of the anti-ACTRII.

[0017] In some preferred embodiments, the GLP1 receptor agonist polypeptide domain is linked to the N-terminus of the light chain of the antibody domain or its antigen-binding fragment domain of the anti-ACTRII; the FGF21 receptor agonist polypeptide domain is linked to the C-terminus of the Fc of the antibody domain or its antigen-binding fragment domain of the anti-ACTRII.

[0018] In some embodiments, the GLP1 receptor agonist polypeptide domain is a GLP1R monoagonal, or a GLP1R / GIPR dual agonist, or a GLP1R / GCGR dual agonist, or a GLP1R / GIPR / GCGR triple agonist.

[0019] In some embodiments, the FGF21 receptor agonist polypeptide domain is a mutant of natural FGF21.

[0020] In some preferred embodiments, the fusion protein has the following structure 1):

[0021] Heavy chain from N-terminus to C-terminus: S0-Gp-L1-HC;

[0022] Light chain from N-terminus to C-terminus: S0-LC;

[0023] Wherein, S0 is absent, or selected from signal peptide, tag sequence or combination thereof; Gp is ​​GLP1 receptor agonist polypeptide domain; HC is antibody heavy chain against ACTRII; L1 is absent or linker; LC is antibody light chain against ACTRII.

[0024] In some preferred embodiments, the fusion protein has the following structure 2):

[0025] Heavy chain from N-terminus to C-terminus: S0-HC;

[0026] The light chain from the N-terminus to the C-terminus is: S0-Gp-L1-LC;

[0027] Wherein, S0 is absent, or selected from signal peptide, tag sequence or combination thereof; Gp is ​​GLP1 receptor agonist polypeptide domain; HC is antibody heavy chain against ACTRII; L1 is absent or linker; LC is antibody light chain against ACTRII.

[0028] In some preferred embodiments, the fusion protein has the following structure 3):

[0029] Heavy chain from N-terminus to C-terminus: S0-HC-L1-Fg;

[0030] Light chain from N-terminus to C-terminus: S0-LC;

[0031] Wherein, S0 is absent, or selected from signal peptide, tag sequence or combination thereof; Fg is FGF21 receptor agonist polypeptide domain; HC is antibody heavy chain against ACTRII; L1 is absent or linker; LC is antibody light chain against ACTRII.

[0032] In some preferred embodiments, the fusion protein has the following structure 4):

[0033] Heavy chain from N-terminus to C-terminus: S0-HC-L1-Fg;

[0034] The light chain from the N-terminus to the C-terminus is: S0-Gp-L1-LC;

[0035] Wherein, S0 is absent, or selected from signal peptide, tag sequence or combination thereof; Gp is ​​GLP1 receptor agonist polypeptide domain; Fg is FGF21 receptor agonist polypeptide domain; HC is antibody heavy chain against ACTRII; L1 is absent or linker; LC is antibody light chain against ACTRII.

[0036] In some preferred embodiments, L1 is (GGGGS)n, where n is any integer from 1 to 5, preferably 3.

[0037] In some preferred embodiments, the anti-ACTRII antibody is Bimagrumab, whose heavy chain sequence is shown in SEQ ID NO:1 and whose light chain sequence is shown in SEQ ID NO:2.

[0038] In some preferred embodiments, the sequence of the GLP1 receptor agonist polypeptide domain is any one of SEQ ID NO:3-14.

[0039] In some preferred embodiments, the sequence of the FGF21 receptor agonist polypeptide domain is any one of SEQ ID NO:15-16.

[0040] In some preferred embodiments, when the fusion protein has structure 1), it comprises two homologous heavy chains and two homologous light chains, wherein the sequence of the homologous heavy chains is SEQ ID NO:17, 18, 21, 22 or 23, and the sequence of the homologous light chains is SEQ ID NO:2.

[0041] In some preferred embodiments, when the fusion protein has structure 2), it comprises two homologous heavy chains and two homologous light chains, wherein the sequence of the homologous heavy chains is SEQ ID NO:1, and the sequence of the homologous light chains is SEQ ID NO:19, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.

[0042] In some more preferred embodiments, the combinations of the homologous heavy chain and homologous light chain of the fusion protein are as follows: SEQ ID NO:17 and 2; SEQ ID NO:18 and 2; SEQ ID NO:1 and 19; SEQ ID NO:1 and 20; SEQ ID NO:21 and 2; SEQ ID NO:22 and 2; SEQ ID NO:23 and 2; SEQ ID NO:1 and 24; SEQ ID NO:1 and 25; SEQ ID NO:1 and 26; SEQ ID NO:1 and 27; SEQ ID NO:1 and 28; SEQ ID NO:1 and 29; SEQ ID NO:1 and 30; SEQ ID NO:1 and 31; SEQ ID NO:1 and 32; SEQ ID NO:1 and 33; SEQ ID NO:1 and 34.

[0043] In some preferred embodiments, when the fusion protein has structure 3), it comprises two homologous heavy chains and two homologous light chains, wherein the sequence of the homologous heavy chains is SEQ ID NO:35 or 36, and the sequence of the homologous light chains is SEQ ID NO:2.

[0044] In some preferred embodiments, when the fusion protein has structure 4), it comprises two homologous heavy chains and two homologous light chains, wherein the sequence of the homologous heavy chains is SEQ ID NO: 35 or 36, and the sequence of the homologous light chains is SEQ ID NO: 20, 24, 27, 28, 29, 30, 31, 32, 33 or 34.

[0045] In some more preferred embodiments, the combinations of the homologous heavy chain and homologous light chain of the fusion protein are as follows: SEQ ID NO:35 and 2; SEQ ID NO:36 and 2; SEQ ID NO:35 and 20; SEQ ID NO:35 and 24; SEQ ID NO:35 and 27; SEQ ID NO:35 and 28; SEQ ID NO:35 and 29; SEQ ID NO:35 and 30; SEQ ID NO:35 and 31; SEQ ID NO:35 and 32; SEQ ID NO:35 and 33; SEQ ID NO:35 and 34; SEQ ID NO:36 and 20; SEQ ID NO:36 and 24; SEQ ID NO:36 and 27; SEQ ID NO:36 and 33.

[0046] This disclosure also provides an isolated nucleic acid that encodes the fusion protein of this disclosure.

[0047] This disclosure also provides a vector containing the nucleic acid of this disclosure.

[0048] This disclosure also provides a host cell containing the vector or nucleic acid described in this disclosure.

[0049] 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.

[0050] This disclosure also provides a pharmaceutical composition comprising an effective amount of the fusion protein.

[0051] This disclosure also provides methods for using the fusion protein to treat diseases or its use in the preparation of medicaments for treating diseases.

[0052] 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.

[0053] This disclosure also provides the fusion proteins or pharmaceutical compositions described herein for the treatment of diseases.

[0054] 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.

[0055] 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:4-14.

[0056] This disclosure also provides a fusion protein comprising an FGF21 receptor agonist polypeptide domain, the sequence of which is either SEQ ID NO: 15 or 16. Attached Figure Description

[0057] Figure 1 shows the percentage change in body weight of DIO mice after multiple administrations of the fusion protein and the positive control. The numbers indicate the number of days of administration.

[0058] Figure 2 shows the percentage change in fat at the endpoint of the DIO mouse trial after multiple administrations of the fusion protein and the positive control.

[0059] Figure 3 shows the percentage change in muscle mass at the endpoint of the DIO mouse trial after multiple administrations of the fusion protein and the positive control.

[0060] Figure 4 shows the weight loss quality (WLQ) scores of the fusion protein and the positive control on days 7, 14, and 21.

[0061] Figure 5 shows the percentage change in body weight of DIO mice after multiple administrations of the fusion protein and the positive control. The numbers indicate the number of days of administration.

[0062] Figure 6 shows the percentage change in fat at the endpoint of the DIO mouse trial after multiple administrations of the fusion protein and the positive control.

[0063] Figure 7 shows the percentage change in muscle mass at the endpoint of the DIO mouse trial after multiple administrations of the fusion protein and the positive control.

[0064] Figure 8 shows the weight loss quality (WLQ) scores of the fusion protein and the positive control on days 7, 14, and 21. Detailed Implementation

[0065] the term

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0071] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain contains one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and one constant domain (CH).

[0072] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be assigned to one of two distinctly different types based on the amino acid sequence of its constant domain, termed κ and λ.

[0073] As used in this article, the broad types of "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0074] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] "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.

[0079] "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.

[0080] "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.

[0081] 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.

[0082] The term "GLP1 receptor agonist" as used in this disclosure encompasses four types: GLP1 monoreceptor agonists, GLP1 / GCG dual receptor agonists, GLP1 / GIP dual receptor agonists, and GLP1 / GCG / GIP triple receptor agonists. Specifically, a GLP1 monoreceptor agonist is a polypeptide that only binds to and activates GLP1R; a GLP1 / GCG dual receptor agonist is a polypeptide modified by amino acid mutation to simultaneously possess dual activities of binding to and activating both GLP1R and GCGR; a GLP1 / GIP dual receptor agonist is a polypeptide modified by amino acid mutation to simultaneously possess dual activities of binding to and activating both GLP1R and GIPR; and a GLP1 / GCG / GIP triple receptor agonist is a polypeptide modified by amino acid mutation to simultaneously possess triple activities of binding to and activating GLP1R, GIPR, and GCGR.

[0083] The FGF21 receptor agonists used in this article include various mutants of natural FGF21, such as Efruxifermin.

[0084] 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.

[0085] 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.

[0086] 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, and preferably comprises an amino acid sequence (GGGGS)3.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] Experimental Example

[0094] Preparation and expression of fusion proteins

[0095] 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.

[0096] 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), and 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 PBS phosphate buffer (pH 7.2). 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.

[0097] GLP1R / GCGR / GIPR / FGF21R ​​activation activity assay

[0098] 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 ratio of the 665 nm / 615 nm readings 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.

[0099] 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, anti-cAMP antibody-Eu was added. 3+ 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.

[0100] 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.

[0101] FGF21 receptor activation activity assay: Cell suspensions were prepared using the H_FGF21Reporter HEK-293luciferase reporter gene cell line at 5×10⁶ cells / mL. 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, add them to cells after removing the culture medium, incubate at 37°C for 6 h, and then detect fluorescence using a Bright-Glo luciferase assay system (Promega, Madison, MI).

[0102] Example

[0103] 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.

[0104] Example 1: Preparation of each domain of the fusion protein

[0105] The fusion protein disclosed herein comprises multiple domains: an anti-ACTRII antibody domain, and a GLP1 receptor agonist peptide domain, and / or an FGF21 receptor agonist peptide domain.

[0106] In the fusion protein, the anti-ACTRII antibody domain is an anti-ACTRII antibody or its antigen-binding fragment, and can be an antibody known in the prior art, such as Bimagrumab. In this disclosure, Bimagrumab is used as the anti-ACTRII antibody domain of the fusion protein, and the sequence of Bimagrumab is shown in Table 1.

[0107] The fusion protein contains four types of "GLP1 receptor agonists": GLP1 monoreceptor agonists, GLP1 / GCG dual receptor agonists, GLP1 / GIP dual receptor agonists, and GLP1 / GCG / GIP triple receptor agonists. This disclosure provides the sequences of several of these four types of GLP1 receptor agonists designed by the inventors, as shown in Table 2.

[0108] In the fusion protein, the FGF21 receptor agonist is a mutant of natural FGF21. This disclosure provides two mutant peptides of FGF21 designed by the inventors, as shown in Table 3.

[0109] Table 1 Anti-ACTRII antibody sequences

[0110] Table 2 GLP1 receptor agonist sequences

[0111] Table 3 FGF21 receptor agonist sequences

[0112] Example 2: Structural Exploration of Fusion Protein

[0113] I. The C-terminus of the GLP1 receptor agonist peptide domain is linked to the anti-ACTRII antibody domain.

[0114] Using the anti-ACTRII antibodies shown in Table 1 as the anti-ACTRII antibody domain of the fusion protein, and then linking the GLP1 receptor agonists shown in Table 2 to the C-terminus of the two heavy chains (IHG type) or light chains (ILG type) of the anti-ACTRII antibody domain via linkers, a fusion protein consisting of two homologous heavy chains and two homologous light chains is formed. An exemplary linker is (GGGGS)n (n can be a positive integer from 1 to 5). The structures of the heavy and light chains from the N-terminus to the C-terminus of the two configurations are illustrated below:

[0115] IHG type heavy chain: ACTRII antibody heavy chain + L1 + GLP1 IHG type light chain: ACTRII antibody light chain

[0116] ILG type heavy chain: ACTRII antibody heavy chain; ILG type light chain: ACTRII antibody light chain + L1 + GLP1

[0117] Table 4. Structural sequences of IHG or ILG fusion proteins

[0118] Table 5. Detection of activation activity of IHG or ILG type GLP1 receptor agonist peptide domains in fusion proteins.

[0119] As can be seen from the data in Table 5, when the GLP1 receptor agonist polypeptide domain is linked to the C-terminus of the ACTRII antibody, whether it is linked to the C-terminus of the heavy chain or the light chain, the GLP1 receptor agonist polypeptide domain does not exhibit activation activity. This indicates that for the GLP1 receptor agonist polypeptide domain, linking it to the C-terminus causes the N-terminus of the polypeptide to be hidden, thus it does not have GLP1 receptor agonist activity.

[0120] II. The N-terminus of the GLP1 receptor agonist peptide domain is linked to the anti-ACTRII antibody domain.

[0121] Using the anti-ACTRII antibodies shown in Table 1 as the anti-ACTRII antibody domain of the fusion protein, and then linking the GLP1 receptor agonists shown in Table 2 to the N-terminus of the two heavy chains (II-GH type) or light chains (II-GL type) of the anti-ACTRII antibody domain via linkers, a fusion protein consisting of two homologous heavy chains and two homologous light chains is formed. An exemplary linker is (GGGGS)n. The structures of the heavy and light chains of the two conformations from the N-terminus to the C-terminus are illustrated below:

[0122] II-GH type heavy chain: GLP1+L1+ACTRII antibody heavy chain; II-GH type light chain: ACTRI antibody light chain

[0123] II-GL type heavy chain: ACTRII antibody heavy chain; II-GL type light chain: GLP1+L1+ACTRII antibody light chain

[0124] Table 6. Structural sequences of fusion proteins II-GH or II-GL.

[0125] Table 7. Detection of activation activity of the peptide domain of GLP1 receptor agonists in fusion proteins of type II-GH or type II-GL.

[0126] As shown in Table 7, when the GLP1 receptor agonist polypeptide domain is linked to the N-terminus of the ACTRII antibody, whether it is linked to the N-terminus of the heavy chain or the light chain, the GLP1 receptor agonist polypeptide domain exhibits high activation activity. This indicates that N-terminal exposure is a necessary condition for the activation activity of the GLP1 receptor agonist polypeptide domain. Therefore, the GLP1 receptor agonist polypeptide domain can only be linked to the N-terminus of the antibody, and linking the GLP1 receptor agonist polypeptide domain to the N-terminus of either the heavy chain or the light chain of the antibody is the preferred configuration.

[0127] III. The FGF21 receptor agonist polypeptide domain is linked to the C-terminus of the anti-ACTRII antibody domain.

[0128] Based on the aforementioned explorations in I. and II., it was found that linking the GLP1 receptor agonist polypeptide domain to the N-terminus of the anti-ACTRII antibody domain is the most ideal fusion protein conformation. If an FGF21 receptor agonist polypeptide domain is further introduced, this polypeptide domain is selectively linked to the C-terminus of the anti-ACTRII antibody domain. Considering molecular stability, it is preferable to link the FGF21 receptor agonist polypeptide domain to the C-terminus of the heavy chain of the anti-ACTRII antibody domain, and the GLP1 receptor agonist polypeptide domain to the N-terminus of the light chain of the anti-ACTRII antibody domain, thereby forming a fusion protein composed of two homologous heavy chains and two homologous light chains. The structural diagrams of the heavy and light chains from the N-terminus to the C-terminus of this conformation are shown below:

[0129] III-GLHF type heavy chain: ACTRII antibody heavy chain + L1 + FGF21

[0130] III-GLHF type light chain: GLP1+L1+ACTRII antibody light chain

[0131] Alternatively, the FGF21 receptor agonist polypeptide domain can be linked to the C-terminus of the anti-ACTRII antibody domain, thereby forming a fusion protein composed of two homologous heavy chains and two homologous light chains. The heavy and light chains of this conformation are illustrated below from the N-terminus to the C-terminus:

[0132] III-HF type heavy chain: ACTRII antibody heavy chain + L1 + FGF21

[0133] III-HF type light chain: ACTRII antibody light chain

[0134] Table 8. Structural sequences of fusion proteins III-GLHF and III-HF.

[0135] Example 3: Evaluation of the in vitro biological activity of the fusion protein GLP1 receptor agonist polypeptide domain and / or FGF21 receptor agonist polypeptide domain

[0136] The GLP1 receptor agonist polypeptide domain and / or FGF21 receptor agonist polypeptide domain of the fusion protein molecule of Example 2 were subjected to in vitro activity assays, including GLP1R activation activity assay, GCGR activation activity assay, GIPR activation activity assay, and FGF21R ​​activation activity assay.

[0137] In this embodiment, four GLP1 analogs—Semaglutide, Mazdutide, Tirzepatide, and Retatrutide—were used as positive controls representing different types of GLP1R agonists, and Efruxifermin (Fc-FGF21) was used as a positive control for FGF21R ​​agonists. As shown in Tables 9 and 10, most of the designed fusion proteins exhibited in vitro activity comparable to the positive controls, and some fusion proteins showed significantly stronger activity than the positive controls.

[0138] Table 9. Detection of activation activity of the peptide domain of GLP1 receptor agonists in fusion proteins of type II-GH or type II-GL.

[0139] Table 9 shows that the fusion proteins disclosed herein all exhibit GLP1R activation activity comparable to or better than Semaglutide. Furthermore, the fusion proteins with dual GLP1 / GCG receptor agonist activity comprehensively demonstrate stronger activity than the positive control Mazdutide; the fusion proteins with dual GLP1 / GIP receptor agonist activity have activity comparable to the positive control Tirzepatide; and the fusion proteins with triple GLP1 / GCG / GIP receptor agonist activity disclosed herein exhibit receptor activation activities with different receptor biases and imbalances, respectively demonstrating activity comparable to or stronger than the positive control Retatrutide.

[0140] Table 10 Detection of activation activity of the fusion protein III-GLHF or III-HF type GLP1 and FGF21 receptor agonist polypeptide domains

[0141] Table 10 shows that the III-GLHF fusion protein, while maintaining GLP1R and / or GCGR and GIPR activation activities, also exhibits FGF21R ​​activation activity comparable to the positive control Efruxifermin. The III-HF fusion protein also shows the same excellent performance. These data indicate that the fusion protein form disclosed herein can simultaneously possess strong GLP1, GCG, GIP, and / or FGF21 activities.

[0142] Example 4: In vitro evaluation of the affinity of the fusion protein for the anti-ACTRII antibody domain.

[0143] Antibodies were affinity-captured using a Protein A biosensor chip, and antigen molecules were then passed through the chip surface. Binding and dissociation curves were obtained in real-time using a Biacore 8K instrument. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). A 1:1 model was used for data fitting. The protein affinity level between the fusion protein and ACTRIIA was 10E-8 M, and the protein affinity level with ACTRIIB was 10E-10 to 10E-9 M.

[0144] Table 11. Biacore affinity test of fusion protein with human ACTRIIA.

[0145] Table 12. Biacore affinity assay of the fusion protein with human ACTRIIA.

[0146] Table 13 Biacore affinity assay of fusion protein and human ACTRIIB 1

[0147] Table 14. Biacore affinity test of fusion protein with human ACTRIIB 2

[0148] As shown in Tables 11-14, the affinity activity of ACTRII antibodies is as expected after linking multiple GLP1 receptor agonist polypeptide domains to the N-terminus and / or FGF21 receptor agonist polypeptide domains to the C-terminus.

[0149] Example 5: In vitro evaluation of the ACTRII blocking activity of the anti-ACTRII antibody domain of the fusion protein.

[0150] This embodiment uses the reporter gene cell line HEK-Blue™ TGF-β cells (Invivogen, hkb-tgfbv2) to evaluate the bioactivity of the fusion protein antagonizing ACTRII. HEK-Blue™ TGF-β cells stably express the Smad3 / 4-binding element (SBE)-inducible SEAP (secreted alkaline phosphatase) reporter gene. ACTRII-related cytokines bind to ACTRII on the cell surface, activating downstream signaling pathways including smad3 / 4, thereby inducing SEAP gene expression and secretion into the cell supernatant. The amount of secreted SEAP was measured using the QUANTIBlue™ reagent to assess the degree of ACTRII activation.

[0151] HEK-Blue™ TGF-β cells were cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (Gibco, 10091148), 50 U / mL penicillin, 50 μg / mL streptomycin (Gibco, 15140163), 100 μg / mL Normocin™ (Invivogen, ant-nr-1), 30 μg / mL blastcin (Blasticidin, Invivogen, ant-bl-1), 200 μg / mL HygroGold™ (Invivogen, ant-hg-1), and 100 μg / mL Zeocin™ (Invivogen, ant-zn-1). The day before the experiment, cells were seeded at 40,000 cells / well in complete medium in 96-well plates and cultured at 37°C and 5% CO2 for 16–18 h. The purified fusion protein disclosed herein was serially diluted with experimental reagents (DMEM + 0.1% inactivated bovine serum albumin, 50 μg / mL streptomycin, 50 U / mL penicillin, and 100 μg / mL Normocin™) and added together with Activin A at a final concentration of 5 ng / mL ACTRII activator to cells from which the culture medium had been discarded. After culturing at 37°C and 5% CO2 for 24 h, the cell supernatant was collected and mixed with QUANTIBlue™ (Invivogen, rep-qbs2) reagent at a 1:1 ratio, incubated for 2–4 h, and the optical density at 620 nm was measured using a colorimeter.

[0152] Table 15. Antagonistic activity of fusion proteins against ACTRII

[0153] As shown in Table 15, the fusion protein disclosed herein exhibits ACTRII receptor antagonistic activity of approximately 10-60 nM at the same concentration, which meets the original expectation of reducing ACTRII blocking activity to the nmol / L level. This approach minimizes the safety risks caused by excessive ACTRII blocking while satisfying the antagonistic activity requirements.

[0154] Example 6: Pharmacodynamic test of continuous subcutaneous administration of fusion protein in DIO mice

[0155] Construction of DIO mice: 5-6 week old male C57BL / 6J mice were fed a high-fat diet to establish the DIO model. Husbandry conditions included alternating 12h / 12h lighting, free access to food, a temperature of 20-26℃, and a relative humidity of 30-70%, with 4 mice per cage. Temperature and relative humidity were adjusted daily. After 12 weeks of modeling, mice were kept individually until 20-30 weeks of age. Once the mice reached a weight of approximately 50g, they were kept individually for another 20-30 weeks. One day before drug administration, mice were randomly assigned to groups of 6 mice each based on weight, food intake, and body composition. The groups included a normal control group, a positive control group, a solvent group (PBS), and a fusion protein group. Each group received subcutaneous injections of the drug: 20 nMol / kg for the positive control group, 20 nMol / kg for the low-dose D0-A-L3 group and 40 nMol / kg for the high-dose D0-A-L3 group, and 10 nMol / kg for the low-dose B3-A-L3 group and 20 nMol / kg for the high-dose B3-A-L3 group. Administration continued for 3 weeks. Food intake was monitored daily, and body weight was measured three times a week. Magnetic Resonance Imaging (MRI) was performed weekly to analyze changes in the percentage of fat and muscle mass in mice, calculating the weight loss quality (WLQ = fat loss / body weight loss × 100%). At the experimental endpoint (day 21), the percentage changes in fat and muscle mass were standardized according to the solvent group, and statistical analysis was performed using One-Way Anova (Dunnett's test).

[0156] D0-A-L3 exhibited dose-dependent efficacy in DIO mice. The low-dose group was comparable to semaglutide in terms of weight reduction, while the high-dose group showed significantly better weight and fat reduction than semaglutide (P<0.01) (Figures 1 and 2). D0-A-L3 significantly increased muscle mass while reducing weight (P<0.0001 compared to semaglutide), with the high-dose group showing comparable muscle-building effects to the low-dose group while demonstrating further fat reduction efficacy (Figure 3). With prolonged administration, both the low- and high-dose groups of D0-A-L3 showed increasingly better weight loss fractions (WLQ), reaching nearly 100% at the endpoint (day 21), significantly superior to semaglutide (P<0.0001) (Figure 4).

[0157] At the trial endpoint, B3-A-L3 demonstrated superior weight and fat reduction effects compared to Semaglutide, and comparable to Tirzepatide (Figures 5 and 6). Unlike Semaglutide and Tirzepatide, which resulted in significant muscle loss, B3-A-L3 effectively maintained muscle mass, with the low-dose group showing significantly better muscle retention than Semaglutide (P<0.05) and Tirzepatide (P<0.01) (Figure 7). B3-A-L3 showed a gradually increasing weight loss fraction (WLQ) compared to Semaglutide and Tirzepatide, with the high-dose group showing a significantly higher WLQ at the trial endpoint than Semaglutide (P<0.05). It is speculated that with prolonged administration, the WLQ of B3-A-L3 would continue to increase (Figure 8).

[0158] 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 comprising: an anti-ACTRII antibody domain or an antigen-binding fragment thereof domain; a GLP1 receptor agonist polypeptide domain, and / or, a FGF21 receptor agonist polypeptide domain; wherein the GLP1 receptor agonist polypeptide domain is linked to the N-terminus of the heavy chain or the light chain of the anti-ACTRII antibody domain or the antigen-binding fragment thereof domain, and / or, the FGF21 receptor agonist polypeptide domain is linked to the C-terminus of the heavy chain of the anti-ACTRII antibody domain or the antigen-binding fragment thereof domain; wherein the GLP1 receptor agonist polypeptide domain is a GLP1R single agonist, or a GLP1R / GIPR dual agonist, or a GLP1R / GCGR dual agonist, or a GLP1R / GIPR / GCGR triple agonist.

2. The fusion protein of claim 1, having the following structure: heavy chain from N-terminus to C-terminus: S0-Gp-L1-HC; light chain from N-terminus to C-terminus: S0-LC; wherein S0 is nothing, or is selected from a signal peptide, a tag sequence, or a combination thereof; Gp is the GLP1 receptor agonist polypeptide domain; HC is the antibody heavy chain against ACTRII; L1 is nothing or a linker; LC is the antibody light chain against ACTRII.

3. The fusion protein of claim 1, having the following structure: heavy chain from N-terminus to C-terminus: S0-HC; light chain from N-terminus to C-terminus: S0-Gp-L1-LC; wherein S0 is nothing, or is selected from a signal peptide, a tag sequence, or a combination thereof; Gp is the GLP1 receptor agonist polypeptide domain; HC is the antibody heavy chain against ACTRII; L1 is nothing or a linker; LC is the antibody light chain against ACTRII.

4. The fusion protein of claim 1, having the following structure: heavy chain from N-terminus to C-terminus: S0-HC-L1-Fg; light chain from N-terminus to C-terminus: S0-LC; wherein S0 is nothing, or is selected from a signal peptide, a tag sequence, or a combination thereof; Fg is the FGF21 receptor agonist polypeptide domain; HC is the antibody heavy chain against ACTRII; L1 is nothing or a linker; LC is the antibody light chain against ACTRII.

5. The fusion protein of claim 1, having the following structure: heavy chain from N-terminus to C-terminus: S0-HC-L1-Fg; light chain from N-terminus to C-terminus: S0-Gp-L1-LC; wherein S0 is nothing, or is selected from a signal peptide, a tag sequence, or a combination thereof; Gp is the GLP1 receptor agonist polypeptide domain; Fg is the FGF21 receptor agonist polypeptide domain; HC is the antibody heavy chain against ACTRII; L1 is nothing or a linker; LC is the antibody light chain against ACTRII.

6. The fusion protein of any one of claims 2-5, wherein L1 is (GGGGS) n , and n is any integer from 1-5, preferably 3.

7. The fusion protein of any one of claims 1-5, wherein, the anti-ACTRII antibody is Bimagrumab; preferably, the heavy chain sequence of the anti-ACTRII antibody is SEQ ID NO: 1, and the light chain sequence is SEQ ID NO:

2.

8. The fusion protein of any one of claims 1-5, wherein, the sequence of the GLP1 receptor agonist polypeptide domain is any one of SEQ ID NOs: 3-14.

9. The fusion protein of any one of claims 1-5, wherein, the sequence of the FGF21 receptor agonist polypeptide domain is any one of SEQ ID NOs: 15-16.

10. The fusion protein of any one of claims 1-5, comprising two cognate heavy chains and two cognate light chains, the sequence of the cognate heavy chain is SEQ ID NO: 1, 17, 18, 21, 22, 23, 35 or 36, and the sequence of the cognate light chain is SEQ ID NO: 2, 19, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.

11. The fusion protein of claim 10, comprising the combination of cognate heavy chain and cognate light chain as follows: SEQ ID NO: 17 and 2; SEQ ID NO: 18 and 2; SEQ ID NO: 1 and 19; SEQ ID NO: 1 and 20; SEQ ID NO: 21 and 2; SEQ ID NO: 22 and 2; SEQ ID NO: 23 and 2; SEQ ID NO: 1 and 24; SEQ ID NO: 1 and 25; SEQ ID NO: 1 and 26; SEQ ID NO: 1 and 27; SEQ ID NO: 1 and 28; SEQ ID NO: 1 and 29; SEQ ID NO: 1 and 30; SEQ ID NO: 1 and 31; SEQ ID NO: 1 and 32; SEQ ID NO: 1 and 33; SEQ ID NO: 1 and 34; SEQ ID NO: 35 and 2; SEQ ID NO: 36 and 2; SEQ ID NO: 35 and 20; SEQ ID NO: 35 and 24; SEQ ID NO: 35 and 27; SEQ ID NO: 35 and 28; SEQ ID NO: 35 and 29; SEQ ID NO: 35 and 30; SEQ ID NO: 35 and 31; SEQ ID NO: 35 and 32; SEQ ID NO: 35 and 33; SEQ ID NO: 35 and 34; SEQ ID NO: 36 and 20; SEQ ID NO: 36 and 24; SEQ ID NO: 36 and 27; SEQ ID NO: 36 and 33, respectively.

12. An isolated nucleic acid encoding the fusion protein of any one of claims 1-11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the vector of claim 13 or the nucleic acid of claim 12.

15. A method of making the fusion protein of any one of claims 1-11, comprising:

15. Culturing the host cell of claim 14 under suitable conditions to express the fusion protein, and further purifying and / or isolating the fusion protein.

16. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1-11.

17. Use of the fusion protein of any one of claims 1-11 in the manufacture of a medicament for treating a disease.

18. The use of claim 17, 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).

19. A fusion protein comprising a GLP1 receptor agonist polypeptide domain, the sequence of the polypeptide domain being any one of SEQ ID NOs: 4-14.

20. A fusion protein comprising a FGF21 receptor agonist polypeptide domain, the sequence of the polypeptide domain being any one of SEQ ID NOs: 15 and 16.

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