Homodimeric fusion protein with multiple regulation of blood glucose and blood lipid

Abstract

The application discloses a fusion protein, which comprises a structure shown in formula (I): Z1-L1-Z2-L2-Z3 (I), wherein Z1 is a first polypeptide, which has the agonistic activity of 28 peptides of GCGR and GLP-1R two receptors; L1 is a connecting peptide, Z2 is a second polypeptide, which is an IgG4 heavy chain constant region or a mutant thereof; L2 is a connecting peptide; and Z3 is a third polypeptide, which is an FGF-21 mutant. The homodimer fusion protein has the functions of regulating blood lipids, blood sugar, body weight and energy metabolism, and can be used for treating metabolic disorder related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology. Specifically, this invention relates to a homodimeric fusion protein with multiple regulatory functions on blood glucose and blood lipids. More specifically, this invention relates to a fusion protein, nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition, and their uses. Background Technology

[0002] Diabetes mellitus is the most common metabolic disease, mainly characterized by blood glucose dysregulation and long-term hyperglycemia leading to various complications. Drugs for treating diabetes include insulin, DPP-4 inhibitors, AMPK activators, SGLT-2 inhibitors, T2DM treatment drugs, and GLP-1 receptor agonists. Among these, DPP-4 inhibitors, AMPK activators, SGLT-2 inhibitors, and T2DM treatment drugs are small molecule drugs, with significant side effects and weight gain with long-term use, and their blood glucose control in the later stages of diabetes is not ideal (Kahn SE et al. N Engl J Med 2006; 355(23):2427-43). Insulin drugs can only supplement a certain amount of insulin, and due to individual dosage differences, they can also cause hypoglycemia. The ideal drug is a receptor agonist, which has a good effect on stimulating insulin production, protecting insulin cells, and protecting the cardiovascular system.

[0003] Obesity refers to the abnormal or excessive accumulation of fat that can impair health and easily induce type 2 diabetes, cardiovascular disease, respiratory diseases, musculoskeletal diseases, various cancers, and depression, leading to a greater need for healthcare services, surgery, and medications in obese individuals. Lifestyle changes based on diet and exercise are rarely able to sustainably reverse weight gain. While bariatric surgery can effectively reduce weight, it requires meeting certain criteria, carries high risks, and is expensive. Medications for treating obesity primarily work by inducing satiety or reducing nutrient absorption. This level of weight loss can effectively improve metabolic circulation and lipid management. However, these therapies are often accompanied by gastrointestinal side effects and cardiovascular effects that limit their use (Diets, drugs and surgery for weight loss[J].Treat Guidel Med Lett,2008,6(68):23-28).

[0004] Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by diffuse macrovesicular steatosis of hepatocellular carcinoma and lipid metabolism disorders, in addition to alcohol and other clearly defined hepatotoxic factors. The prevalence of NAFLD is increasing globally and has become a leading cause of chronic liver disease in many parts of the world. The pathogenesis of NAFLD is not fully understood, but its occurrence is related to the interaction and parallel effects of multiple metabolic disorders, such as abnormal lipid metabolism and obesity (J Hepatol. 2015 Apr; 62(1Suppl): S47-64.). Current treatment primarily involves liver transplantation, and to date, there is still a lack of definitive standardized drug treatment regimens in NAFLD treatment guidelines.

[0005] Therefore, there is an urgent need to develop a drug that can treat the above-mentioned metabolic diseases. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a fusion protein with multiple regulatory effects on blood glucose and lipids. This fusion protein possesses dual agonist biological activities of GCGR and GLP-1R and FGF21 biological activity. The GPGP repeat sequence of the L1 linker peptide of this invention is recognized and cleaved by FAP enzyme in vivo, thereby targeting and releasing the dual agonist 28-peptide of GCGR / GLP-1R, and a long-acting FGF-21 dimer fusion protein containing a second and third polypeptide. This more effectively synergistically targets cells with the dual agonist 28-peptide and the FC-FGF-21 dimer to exert corresponding biological effects. It achieves superior long-term regulation of blood glucose, blood lipids, and weight in vivo, and has the potential to treat diabetes, obesity, non-alcoholic fatty liver disease, and improve related diseases.

[0007] This invention is based on the following discoveries of the inventors:

[0008] GLP-1 is mainly secreted by L cells in the ileum and colon. It acts on G protein-coupled receptors (GLP-1R in GPCRs) to promote insulin secretion from pancreatic β cells in a glucose concentration-dependent manner, participating in the regulation of blood glucose homeostasis (Majaov S et al., J Biol Chem, 1986, 261: 11880-11889). The half-life of human-secreted GLP-1 is very short, only 1–2 minutes. After being secreted into the bloodstream, it is easily degraded by dipeptidyl peptidase 4 (DPP-4) and loses its insulin-promoting activity. To fully utilize the "natural" function of GLP-1, its structure has been modified, leading to the development of a series of GLP-1 receptor agonists. GLP-1 receptor agonists exert the same biological effects as natural GLP-1, while avoiding degradation and loss of activity, thus prolonging their duration of action and regulating blood glucose levels to treat diabetes (Arulmozhi DK et al., EurJPharmSci, 2006, 28: 96-108). Their mechanism of action differs from traditional insulin and oral secretagogues, overcoming many adverse reactions of oral hypoglycemic agents and traditional insulin. They can improve the control rate of type 2 diabetes mellitus (T2DM) and have been widely used in the treatment of T2DM. Recent studies have shown that GLP-1 receptor agonists have ameliorative effects on diabetes and related complications, NAFLD, cardiovascular disease, and other metabolic disorders, demonstrating broad application prospects.

[0009] Glucagon (GCG) is a polypeptide hormone secreted by pancreatic islet cells, whose main function is to maintain blood glucose homeostasis. It plays an important role in the development of metabolic diseases. Its receptor, GCGR, is mainly distributed in the liver. Signal transduction is initiated only after GCG and GCGR bind, converting liver glycogen into glucose. Studies have shown that exogenous administration of glucagon can reduce hepatic triglyceride (TG) synthesis and promote hepatic fat degradation. The catabolic effect and thermogenesis-enhancing effect of glucagon are beneficial to obese patients. In addition, acute administration of glucagon can moderately reduce food intake (Nat Rev Endocrinol, 2010, 6(12):689-697).

[0010] Glucose-dependent insulinotropic peptide (GIP) is secreted by K cells in the small intestine (duodenum and jejunum) and can promote fat storage in adipocytes and promote pancreatic β-cell function and glucose-dependent insulin secretion (Baggio LL et al. (2007) Gastroenterology 132(6), 2131-2157). Its receptor GIPR is highly expressed in many tissues. Food intake induces GIP secretion, but intact GIP is rapidly degraded into an inactive form by DPP-IV (Saxena R et al. (2010) Nature genetics 42(2), 142-148).

[0011] GLP-1R / GIPR / GCGR agonists of class B G protein-coupled receptors (GPCRs) are all intestinal hormones that can promote insulin secretion, moderately reduce food intake, and promote thermogenesis. Studies have shown that GLP-1 receptor agonists, when co-administered with other gastrointestinal hormones, exhibit promoting or synergistic effects, thereby improving glycemic control and reducing weight (Capozzi et al. Endocrine Reviews, October 2018, 39(5):719-738). Meanwhile, to address the issue of rapid drug metabolism in vivo, methods such as amino acid mutation, FC fusion, and PEG modification are mainly employed. Currently, several single-molecule multi-receptor agonists based on GLP-1, combined with other gastrointestinal hormones to exert promoting or synergistic effects, are in the clinical research stage (Bossart et al., 2022, Cell Metabolism 34, 59-74). Because the activities exhibited by the three receptors GLP-1R, GIPR, and GCGR under agonistic conditions are not entirely the same. GLP-1R agonists can lower blood glucose levels, suppress appetite, and reduce weight; GCGR agonists can reduce weight and promote lipid metabolism, but raise blood glucose; GIPR agonists can lower blood glucose levels, but their effect on weight requires further investigation. Eli Lilly's GLP-1R / GIPR dual agonist Tirzepatide has strong GIPR agonist activity, while Amgen's MariTide (AMG133) is a fusion protein of a GIPR antagonist monoclonal antibody and a GLP-1R agonist, both exhibiting excellent synergistic effects in vivo in lowering blood glucose, reducing weight, and regulating blood lipids. Therefore, whether GIPR agonist activity is retained in the design of GPCR multiple agonists needs further investigation. Furthermore, the key to designing GLP-1R / GIPR / GCGR multiple receptor agonists lies in the appropriate agonist ratio between the receptors to achieve synergistic effects in blood glucose control, weight loss, and lipid metabolism regulation.

[0012]

[0013] Human fibroblast 21 (FGF-21) is secreted by the liver, adipose tissue, membranous glands, and skeletal muscle. FGF21 requires the specific cofactor receptor β-Klotho (KLB) to help activate FGFR1, 2, and 3 receptors (IIIc) to exert its function, and is mainly involved in glucose and lipid metabolism (Lewis JE et al. (2019) Trends in Endocrinology & Metabolism 30(8), 491-504). The N-terminus of FGF21 interacts directly with the FGF receptor, while the C-terminus is important for the β-Klotho receptor; and the deletion of 1 to 6 amino acids at the N-terminus has no significant effect on receptor affinity, and the deletion of 1 to 4 amino acids at the N-terminus has no significant effect on activity (Micanovic R, Raches DW, Dunbar JD. [J]. J Cell Physiol, 2009).

[0014] Studies have found that FGF21 can inhibit glucagon secretion and increase insulin mRNA and protein expression, resulting in reduced body weight and fat mass. It also has protective effects on various tissues and cells, protecting pancreatic tissue in pancreatitis by limiting tissue fibrosis and the spread of inflammatory mediators; protecting vascular endothelial cells from hyperglycemic-induced cell damage; and protecting cardiac muscle cells from oxidative stress. Furthermore, FGF21 inhibits glucagon secretion and increases insulin mRNA and protein expression, resulting in reduced body weight and fat mass, and exhibits synergistic effects with GLP-1R / GIPR / GCGR agonists. However, natural FGF21 has poor in vivo stability and can be cleaved by FAP enzymes in serum, leading to its inactivation.

[0015] Fibroblast activation protein (FAP) is a member of the DPP family of serine proteases, sharing 52% amino acid homology with DPP 4. FAP possesses dual proline dipeptidase and endopeptidase activity. Under normal physiological conditions, FAP expression levels in human tissues are low, primarily expressed in remodeled tissues and activated hepatic stellate cells. Clinical studies have reported increased FAP expression in activated hepatic stellate cells and plasma from patients with cirrhosis. Specific cleavage of the hFGF21 C-terminus at position 171 (Pro) inactivates FAP. Resisting FAP cleavage represents a novel strategy to enhance FGF21 activity.

[0016] The FGF-21 mutants P170G and G171P of this invention have been experimentally proven to be effectively resistant to FAP cleavage. This inversion mutation design reduces the risk of immunogenicity that may result from the mutation. The fusion protein of this invention is designed with a FAP cleavage site (GP) in the form GPGP(GGGGS)n. Specific cleavage of the fusion protein dimer using FAP enzyme gradually releases a 28-peptide with dual GCGR and GLP-1R agonist activity.

[0017] The FGF-FGFR signaling pathway is triggered by the binding of ligand-dependent receptor dimerization to FGF on the cell surface, leading to phosphorylation of the receptor kinase domain, intracellular signaling cascades, and activation of gene transcription in many intracellular survival and proliferation pathways. FGF21 and β-Klotho (KLB) act together on FGFR1, 2, and 3, causing FGFR receptor dimerization to activate extracellular signal transduction, thereby exerting their regulatory functions on life activities. The fusion protein of this invention is a dimer; the presence of two FGF21 molecules in the dimer makes it more conducive to FGFR binding, resulting in higher FGFR receptor dimerization efficiency and greater activation of extracellular signal transduction.

[0018] Based on this, in a first aspect of the present invention, a fusion protein is proposed. According to an embodiment of the present invention, the fusion protein comprises the structure shown in formula (I): Z1-L1-Z2-L2-Z3 (I), wherein Z1 is a 28-peptide with agonistic activity to both GCGR and GLP-1R receptors; the amino acid sequence of L1 is GPGP(GGGGS)n, where n is an integer from 1 to 3; Z2 is the constant region of the IgG4 heavy chain or a mutant thereof; the amino acid sequence of L2 is (GGGGS)n or (GGGG)n, where n is an integer from 1 to 3; Z3 is an FGF-21 mutant; wherein, compared with wild-type FGF-21, the mutation site of the FGF-21 mutant includes at least position 170 and / or position 171.

[0019] The fusion protein of this invention is a dimer. The FGF21 mutant, through mutations at positions 170 and 171, can resist FAP cleavage of FGF21, thereby obtaining a long-acting FGF-21 fusion protein containing the IgG4 heavy chain constant region or a mutant thereof. Furthermore, the GPGP sequence in the linker peptide of the fusion protein of this invention can be recognized and cleaved by FAP enzyme, thereby targeting and releasing the first polypeptide (a dual agonist of GCGR and GLP-1R), and the long-acting FGF-21 fusion protein containing the second and third polypeptides. This more effectively synergizes with the 28-peptide dual agonist and the FC-FGF-21 dimer to target cells and exert corresponding biological effects. It achieves superior long-term regulation of blood glucose, blood lipids, and weight in vivo, and has the potential to treat diabetes, obesity, non-alcoholic fatty liver disease, and improve related diseases.

[0020] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule according to embodiments of the present invention can encode the fusion protein described in the first aspect.

[0021] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0022] In a third aspect, the present invention provides an expression vector. According to embodiments of the invention, the expression vector comprises the nucleic acid molecule described in the second aspect. The expression vector according to embodiments of the invention carries the nucleic acid molecule described in the second aspect, thereby encoding the fusion protein described in the first aspect.

[0023] According to an embodiment of the present invention, the expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0024] According to an embodiment of the present invention, the expression vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0025] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. The recombinant cell according to embodiments of the present invention can obtain the fusion protein described in the first aspect.

[0026] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the expression vector into host cells.

[0027] According to embodiments of the present invention, the host cell includes a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

[0028] According to an embodiment of the present invention, the host cell is a mammalian cell.

[0029] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect; and optionally, pharmaceutically acceptable excipients or carriers. As is known prior, the fusion protein described in the first aspect can be cleaved by FAP enzymes to progressively release a 28-peptide dual agonist and an FGF-21 dimer containing a second and a third polypeptide, thereby enhancing tissue- and cell-specific biological effects; and the aforementioned nucleic acid molecule, expression vector, and recombinant cells carry or express the aforementioned fusion protein. Thus, the pharmaceutical composition comprising the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect can long-actingly regulate blood glucose, blood lipids, weight, etc., in vivo, and has the potential to treat metabolic disorders, cardiovascular diseases, or neurodegenerative diseases.

[0030] In a sixth aspect of the invention, the invention provides for the use of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for the prevention and / or treatment of at least one of metabolic disorder-related diseases, cardiovascular diseases, and neurodegenerative diseases.

[0031] According to embodiments of the present invention, the metabolic disorder-related diseases include at least one of obesity or overweight, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, diabetic retinopathy, hyperglycemia, dyslipidemia, atherosclerosis, non-alcoholic steatohepatitis, and non-alcoholic fatty liver disease.

[0032] According to embodiments of the present invention, the neurodegenerative disease includes at least one of Alzheimer's disease and Parkinson's disease.

[0033] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0034] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood in conjunction with the following description of the accompanying drawings and embodiments, wherein:

[0035] Figure 1 The results of non-reducing and reducing Western blot analysis of PJ-601, PJ-602, and PJ-603 in Example 2 of this invention;

[0036] Figure 2The results of non-reduction and reduction electrophoresis detection of PJ-601, PJ-602, and PJ-603 in Example 3 of this invention;

[0037] Figure 3 The GCGR bioactivity assay curves of Glucagon, PJ-601, PJ-602, and PJ-603 in Example 4 of this invention are shown.

[0038] Figure 4 The GLP-1R bioactivity assay curves of GLP-1, PJ-601, PJ-602, and PJ-603 in Example 4 of this invention are shown.

[0039] Figure 5 The FGF21 bioactivity assay curves of PJ-601, PJ-602, and PJ-603 in Example 5 of this invention are shown.

[0040] Figure 6 The results of HPLC-SEC determination of PJ-602 in Example 6 of this invention;

[0041] Figure 7 This is a graph showing the LC-MS results of the PJ-602 disulfide bond composition study in Example 6 of this invention;

[0042] Figure 8 The figure shows the LC-MS results of PJ-602 glycosylation in Example 6 of this invention;

[0043] Figure 9 This is a figure showing the LC-MS results of the PJ-602C terminal sequence in Example 6 of the present invention;

[0044] Figure 10 and 11 This is the blood glucose change curve of normal C57BL / 6 mice in the OGTT experiment in Example 9 of the present invention;

[0045] Figure 12 The relationship between body weight change (%) and time (days) in diet-induced obese (DIO) mice in Example 10 of the present invention;

[0046] Figure 13 The relationship between body weight change (%) and time (days) in diet-induced obese (DIO) mice in Example 11 of this invention;

[0047] Figure 14 The results of blood glucose and LDL-C detection in diet-induced obese (DIO) mice in Example 11 of this invention;

[0048] Figure 15 The results show the liver weight in diet-induced obese (DIO) mice in Example 11 of this invention;

[0049] Figure 16 The results of HE staining pathological analysis of liver tissue from diet-induced obese (DIO) mice in Example 11 of this invention;

[0050] Figure 17 The relationship between body weight change and time (days) in the HFD+CCL4-induced mouse NASH model of the present invention;

[0051] Figure 18 The liver-to-body weight ratio in the HFD+CCL4-induced mouse NASH model of this invention is shown in Example 12 of this invention.

[0052] Figure 19 The results of ALT / AST / TG / TC detection in the HFD+CCL4-induced mouse NASH model in Example 12 of this invention;

[0053] Figure 20 The results of HE staining pathological analysis of liver tissue in the HFD+CCL4-induced mouse NASH model in Example 12 of this invention;

[0054] Figure 21 The results of liver tissue fibrosis analysis in the HFD+CCL4-induced mouse NASH model in Example 12 of this invention;

[0055] Figure 22 The results of liver function indicators in the TAA-induced rat liver fibrosis model in Example 13 of this invention;

[0056] Figure 23 The results of liver tissue fibrosis analysis in the TAA-induced rat liver fibrosis model in Example 13 of this invention;

[0057] Figure 24 The relationship between body weight change and time (days) in the GAN-induced NASH mouse model in Example 14 of this invention;

[0058] Figure 25 The results of ALT / LDL-C detection in the GAN-induced NASH mouse model in Example 14 of this invention;

[0059] Figure 26 The results of HE staining pathological and fibrosis analysis of liver tissue in the GAN-induced NASH mouse model in Example 14 of this invention;

[0060] Figure 27 The results of weight change (%) and time (days) in the Foz / Foz mouse NASH model in Example 15 of this invention;

[0061] Figure 28The liver-to-body weight ratio of the Foz / Foz mouse NASH model in Example 15 of this invention is shown. Detailed Implementation

[0062] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0063] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0064] Detailed description of the invention

[0065] Definitions and General Terms

[0066] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0067] In this document, the terms “optional,” “optional,” “alternatively,” “optional,” or “optional” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0068] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0069] Without substantially affecting the protein or peptide activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of this invention to obtain variants of the anti-protein or peptide sequences. These are all considered to be included within the scope of protection of this invention. The variant sequences described in this invention can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences mentioned in this invention are shown in N-terminus to C-terminus format.

[0070] In this paper, the term "at least 90% homology" means at least 90% homology with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0071] In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations. Exemplarily, it can be an amino acid sequence with a conserved modified form, wherein the amino acid modification in the "conserved modified form of the amino acid sequence" does not significantly affect or alter the activity of the amino acid sequence containing that amino acid; such modification includes amino acid substitution, addition, and deletion. Modifications can be introduced into the polypeptide sequences (e.g., a second or third polypeptide) of the present invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0072] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication by insertion into a suitable host (e.g., host cell or host bacterial cell), which transfers the inserted nucleic acid molecule into and / or between hosts. The expression vector may include expression vectors primarily for inserting DNA or RNA into cells, expression vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having a variety of the functions described above. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host. Typically, by culturing a suitable host containing the expression vector, the expression vector can produce the desired expression product.

[0073] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which an expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins or peptides. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0074] In this document, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active fusion protein with a liquid carrier, a finely fragmented solid carrier, or both.

[0075] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0076] In this document, the term "pharmaceuticalally acceptable amount" or "pharmaceuticalally acceptable dose" refers to a dose suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. For example, it may be "effective amount" or "effective dose," where "effective amount" or "effective dose" refers to an amount that is functional or active in humans and / or animals and is acceptable to them.

[0077] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients, except those incompatible with the fusion protein of the present invention, for example, any adverse biological effects they may produce or interactions that may occur in a harmful manner with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0078] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The fusion protein or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the fusion protein or pharmaceutical composition of the present invention is administered via intravenous or subcutaneous injection.

[0079] In this document, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) alleviation of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers a drug or fusion protein to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administering a drug containing the fusion protein described herein to an individual in need.

[0080] In this article, the term "EC" 50 "(concentration for 50% of maximal effect)" refers to the concentration of a drug or substance required to stimulate 50% of its corresponding biological response. EC 50 The lower the value, the stronger the stimulating or stimulating ability of the drug or substance. For example, more intuitively, it can be manifested as a stronger intracellular signal, thereby a better ability to induce the production of a hormone.

[0081] In this article, the term "G protein-coupled receptor" or "GPCR" refers to an important protein receptor in cell signaling, with a topological conformation of seven transmembrane segments. When an extracellular ligand acts on this receptor, the intracellular portion of the receptor binds to the G protein, activating the G protein. G proteins can transmit extracellular information through two pathways: the first is by opening transmembrane ion channels to allow external ions to enter; the second is by activating second messengers, such as cAMP and IP3 / DAG. Calcium ions are generally considered to be the third messenger downstream of cAMP and IP3 / DAG.

[0082] In this article, the terms "receptor agonist," "peptide agonist," or "agonist" are synonymous and refer to compounds, peptides, proteins, or other small molecule drugs that can bind to relevant ligands of certain endogenous receptors and exert biological effects through binding and signal transduction. Glucagons are gastrointestinal hormones that regulate blood glucose by enhancing glucose-stimulated insulin secretion (Drucker. DJ, Nauck, MA, Lancet 368:1696-705, 2006). To date, two known glucagons are: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). Preproglucagon is a 158-amino acid precursor peptide that undergoes differential processing in tissues to form various structurally related preproglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM).

[0083] This invention provides a detailed description of fusion proteins, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, and their uses.

[0084] This invention proposes a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, and their uses, which will be described in detail below.

[0085] Fusion protein

[0086] In a first aspect, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises the structure shown in formula (I): Z1-L1-Z2-L2-Z3 (I), wherein Z1 is a 28-peptide with agonistic activity to both GCGR and GLP-1R receptors; the amino acid sequence of L1 is GPGP(GGGGS)n, where n is an integer from 1 to 3; Z2 is the constant region of the IgG4 heavy chain or a mutant thereof; the amino acid sequence of L2 is (GGGGS)n or (GGGG)n, where n is an integer from 1 to 3; Z3 is an FGF-21 mutant; wherein, compared with wild-type FGF-21, the mutation site of the FGF-21 mutant includes at least position 170 and / or position 171 as shown in SEQ ID NO 10-13.

[0087] According to embodiments of the present invention, the FGF21 mutant, through mutations at positions 170 and 171, can effectively resist FAP cleavage, thereby obtaining a long-acting FGF-21 fusion protein containing the IgG4 heavy chain constant region or its mutant. Furthermore, the GPGP repeat sequence of the L1 linker peptide of the fusion protein of the present invention is recognized and cleaved by FAP enzyme in vivo, thereby targeting and releasing the GCGR / GLP-1R dual agonist 28-peptide, and the long-acting FGF-21 dimer fusion protein containing a second and a third polypeptide. This more effectively synergistically targets cells with the 28-peptide dual agonist and the FC-FGF-21 dimer to exert corresponding biological effects, achieving superior long-term regulation of blood glucose, blood lipids, and weight in vivo, and possessing the potential to treat diabetes, obesity, non-alcoholic fatty liver disease, and improve related diseases.

[0088] The first polypeptide in the fusion protein provided by this invention, namely the GCGR and GLP-1R dual agonist 28-peptide, can resist dipeptidyl peptidase-IV (DPP-IV) degradation, that is, the first polypeptide will not break near the second amino acid at the N-terminus (resistance to DPP-IV).

[0089] Furthermore, the inventors unexpectedly discovered during the experiment that the L1 linker peptide obtained by directly linking the GPGP sequence to (GGGGS)n, especially compared with linker peptides such as GGPSSGAPPPS(GGGGS)n, GPSSGAPPPS(GGGGS)n, GGPSSGAPPS(GGGGS)n, and GPSSGAPPS(GGGGS)n, not only ensures that it can be recognized by FAP enzyme and gradually and completely cleaved, but also does not affect the formation of homodimers. Moreover, it is beneficial to the biological activity of the dual agonist 28 peptide and FGF21, thereby ensuring that the fusion protein of the present invention can effectively regulate blood glucose, blood lipids, weight, etc. in vivo, and has a high therapeutic effect.

[0090] In this document, the term "agonistic activity" refers to the ability to activate a corresponding receptor, enabling the receptor to perform its corresponding function. In this document, the corresponding receptor is selected from at least one of GCGR and GLP-1R. For example, "having agonistic activity with the GCGR receptor" means being able to activate GCGR, enabling it to perform its corresponding function; for example, by binding to GCGR and initiating signal transduction, converting liver glycogen into glucose.

[0091] According to embodiments of the present invention, the fusion protein may further include at least one of the following technical features:

[0092] According to an embodiment of the present invention, the amino acid sequence of Z1 is as shown in the figure: X1GQGTFTSDY SKYLDEX2KAKX3FVX4WLLN;

[0093] Wherein, X1 is selected from H or Y;

[0094] X2 is selected from K or R;

[0095] X3 is selected from D or E;

[0096] X4 is selected from either Q or E.

[0097] According to an embodiment of the present invention, X1 is selected from H.

[0098] According to an embodiment of the present invention, X2 is selected from K.

[0099] According to an embodiment of the present invention, X3 is selected from E.

[0100] According to an embodiment of the present invention, X4 is selected from E.

[0101] According to an embodiment of the present invention, the amino acid sequence of Z1 is as shown in any one of SEQ ID NO:1 to 7.

[0102] HGQGTFTSDY SKYLDEKKAKEFVEWLLN(SEQ ID NO:1);

[0103] YGQGTFTSDY SKYLDEKKAK EFVQWLLN(SEQ ID NO:2);

[0104] HGQGTFTSDY SKYLDEKKAK EFVQWLLN(SEQ ID NO:3);

[0105] HGQGTFTSDY SKYLDEKKAK DFVQWLLN(SEQ ID NO:4);

[0106] HGQGTFTSDY SKYLDERKAKDFVEWLLN(SEQ ID NO:5);

[0107] HGQGTFTSDY SKYLDERAAQDFVQWLLD(SEQ ID NO:6);

[0108] HGEGTFTSDY SIALDKIAQK AFVQWLIA (SEQ ID NO: 7).

[0109] According to an embodiment of the present invention, the amino acid sequence of L1 has the following structure:

[0110] GPGP(GGGGS)n, where n is 1, 2 or 3.

[0111] According to an embodiment of the present invention, the amino acid sequence of L1 is shown in SEQ ID NO:8.

[0112] GPGPGGGGSGGGGSGGGGS (SEQ ID NO:8).

[0113] According to an embodiment of the present invention, Z2 is the constant region of human IgG4 heavy chain or a mutant thereof.

[0114] In this article, the heavy chain constant region includes the full-length heavy chain constant region or a fragment thereof. The full-length heavy chain constant region refers to the heavy chain constant region of a full-length antibody; for example, for human IgG, the full-length heavy chain constant region consists of CH1, the hinge region, CH2, and CH3 from the N-terminus to the C-terminus. A fragment of the full-length heavy chain constant region refers to a segment of a continuous amino acid sequence derived from the full-length heavy chain constant region, such as at least one of CH1, the hinge region, CH2, or CH3.

[0115] For example, the heavy chain constant region is an Fc fragment or CH2+CH3.

[0116] According to an embodiment of the present invention, Z2 has an amino acid sequence as shown in SEQ ID NO:9 or having at least 90% identity with it.

[0117] ESKYPPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:9).

[0118] According to an embodiment of the present invention, Z2 has an amino acid sequence as shown in SEQ ID NO:9 or having at least 90% identity with it.

[0119] According to an embodiment of the present invention, the amino acid sequence of L2 has the following structure:

[0120] (GGGGS)n or (GGGG)n, where n is 1, 2 or 3.

[0121] According to an embodiment of the present invention, the amino acid sequence of L2 is as follows:

[0122] GGGGS or GGGGSGGGGS or GGGGSGGGGSGGGGS;

[0123] Or GGGG or GGGGGGGG or GGGGGGGGGGGG.

[0124] According to an embodiment of the present invention, compared with wild-type FGF-21, the mutation sites of the FGF-21 mutant include G170P and P171G, as follows:

[0125] HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQIL GVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPA RFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYAS (SEQ ID NO: 10).

[0126] In some alternative embodiments of the present invention, the mutation site of the FGF-21 mutant further includes position 19 compared to wild-type FGF-21.

[0127] In some alternative embodiments of the present invention, the mutation site of the FGF-21 mutant further includes R19V compared to wild-type FGF-21.

[0128] In some alternative embodiments of the present invention, the mutation sites of the FGF-21 mutant further include positions 19 and 98 compared to wild-type FGF-21.

[0129] In some alternative embodiments of the present invention, the mutation sites of the FGF-21 mutant further include R19V and L98R compared to wild-type FGF-21.

[0130] In some alternative embodiments of the present invention, the mutation sites of the FGF-21 mutant further include positions 19, 98, and 180, compared to wild-type FGF-21.

[0131] In some alternative embodiments of the present invention, the mutation sites of the FGF-21 mutant further include R19V, L98R and A180E compared to wild-type FGF-21.

[0132] In some alternative embodiments of the present invention, the mutation site of the FGF-21 mutant further includes the deletion of the 4 amino-terminal amino acids.

[0133] In some alternative embodiments of the present invention, compared with wild-type FGF-21, the mutation sites of the FGF-21 mutant further include a 4-amino acid truncation at positions 19, 98, and 180 and at the N-terminus.

[0134] In some alternative embodiments of the present invention, compared with wild-type FGF-21, the mutation sites of the FGF-21 mutant further include R19V, L98R and A180E and a 4-amino acid truncation at the N-terminus.

[0135] According to an embodiment of the present invention, the amino acid sequence of Z3 is as shown in any one of SEQ ID NO: 11 to 13.

[0136] HPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQIL GVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPA RFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYAS(SEQ ID NO:11);

[0137] HPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQIL GVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPA RFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYES(SEQ ID NO:12).

[0138] DSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVK TSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLP LPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYES(SEQ ID NO:13).

[0139] According to an embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO: 14-21.

[0140] HGQGTFTSDYSKYLDEKKAKEFVEWLLNGPGPGGGGSGGGGSGGGGSESKYPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYAS(SEQ ID NO:14);

[0141] YGQGTFTSDYSKYLDEKKAKEFVQWLLNGPGPGGGGSGGGGSGGGGSESKYPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGSGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLGPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYES(SEQ ID NO:15);

[0142] HGQGTFTSDYSKYLDEKKAKEFVEWLLNGPGPGGGGSGGGGSGGGGSESKYPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGSGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLGPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYES(SEQ ID NO:16);

[0143] HGQGTFTSDYSKYLDEKKAKEFVEWLLNGGPGPGGGGSGGGGSGGGGSKYPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPPEPGILAPQPPDVGSSDPLSMVPGSQGRSPSYAS(SEQ ID NO:17);

[0144] HGQGTFTSDYSKYLDEKKAKEFVEWLLNGPGPGGGGSGGGGSGGGGSESKYPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYVNYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPPEPGILAPQPPDVGSSDPLSMVPGSQGRSPYES(SEQ ID NO:18);

[0145] HGQGTFTSDYSKYLDEKKAKEFVEWLLNGPGPGGGGSGGGGSGGGGSESKYPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGGGGSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLGPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYES(SEQ ID NO:19);

[0146] HGQGTFTSDYSKYLDERAAQDFVQWLLDGPGPGGGSGGGGSGGGGSESKYPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGSGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLGPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYES(SEQ ID NO:20);

[0147] HGEGTFTSDYSIALDKIAQKAFVQWLIAGPGPGGGGSGGGGSGGGGSESKYPPPCPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQIL GVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVPGSQGRSPSYAS(SEQ ID NO:21).

[0148] According to an embodiment of the present invention, the fusion protein is a homodimeric fusion protein, wherein the two fusion proteins in the homodimeric fusion protein are linked by disulfide bonds.

[0149] According to an embodiment of the present invention, the fusion protein is a homodimeric fusion protein, wherein the homodimeric fusion protein is linked by two pairs of disulfide bonds formed by two cysteine ​​residues located in the PPCPPCP sequence of the second polypeptide.

[0150] Nucleic acid molecules, expression vectors and recombinant cells

[0151] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule according to embodiments of the present invention can encode the fusion protein described in the first aspect.

[0152] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0153] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, if the amino acid sequence of the molecules in this invention includes either DNA or RNA form, disclosing one implies that the other is also disclosed.

[0154] In a third aspect, the present invention provides an expression vector. According to embodiments of the invention, the expression vector comprises the nucleic acid molecule described in the second aspect. The expression vector according to embodiments of the invention carries the nucleic acid molecule described in the second aspect, thereby encoding the fusion protein described in the first aspect.

[0155] By linking the aforementioned nucleic acid molecules to the expression vector, the nucleic acid molecules can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. The nucleic acid molecules and control elements simply need to be operatively linked.

[0156] According to embodiments of the present invention, the expression vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vectors used in this invention are not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0157] In this document, the term "operably ligated" refers to ligating a foreign gene to an expression vector such that the control elements within the expression vector, such as transcriptional control amino acid sequences and translational control amino acid sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable host cell, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the in vitro large-scale production of the fusion protein encoded by the nucleic acid molecule.

[0158] According to an embodiment of the present invention, the expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0159] According to an embodiment of the present invention, the expression vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0160] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell comprises: carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. The recombinant cell according to embodiments of the invention can efficiently express the fusion protein described in the first aspect under suitable conditions.

[0161] It should be noted that, in this document, "suitable conditions" refers to conditions suitable for the expression of the fusion protein described in this invention. Those skilled in the art will readily understand that suitable conditions for fusion protein expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the fusion protein according to the specific environment of their laboratory.

[0162] In some alternative embodiments of the present invention, the recombinant cells are obtained by introducing the expression vector into host cells.

[0163] According to embodiments of the present invention, the host cell includes a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

[0164] According to an embodiment of the present invention, the prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0165] According to an embodiment of the present invention, the eukaryotic cell is a fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0166] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces cerevisiae, or Trichoderma.

[0167] In some alternative embodiments of the present invention, the insect cells are grass armyworm cells.

[0168] In some alternative embodiments of the present invention, the plant cells are tobacco plant cells.

[0169] According to an embodiment of the present invention, the host cell is a mammalian cell.

[0170] According to embodiments of the present invention, the mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells, or human embryonic kidney 293 cells; and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0171] Pharmaceutical Composition

[0172] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect. As is known prior, the fusion protein described in the first aspect targets and releases a dual agonist and a long-acting FGF-21 fusion protein containing a second polypeptide and a third polypeptide, thereby enhancing the biological effects of the dual agonist and the long-acting FGF-21 fusion protein in the liver; and the aforementioned nucleic acid molecule, expression vector, and recombinant cells carry or express the aforementioned fusion protein. Therefore, the pharmaceutical composition containing the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect can long-actingly regulate blood glucose, blood lipids, weight, etc., in vivo, and has the potential to treat metabolic disorders, cardiovascular diseases, or neurodegenerative diseases.

[0173] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients or carriers.

[0174] It should be noted that the pharmaceutically acceptable excipients described in this invention include (but are not limited to): water, saline, liposomes, lipids, or combinations thereof. The choice of carrier should be related to the route of administration, which is well known to those skilled in the art.

[0175] According to embodiments of the present invention, the excipients include, but are not limited to, one or more of the following: one or more excipients, one or more diluents, one or more stabilizers, or one or more carriers.

[0176] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), etc., with injections being preferred. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0177] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0178] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the pharmaceutical composition may be administered to the subject as a whole or separately. When the components contained in the pharmaceutical composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0179] use

[0180] In a sixth aspect of the invention, the invention provides for the use of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for the prevention and / or treatment of at least one of metabolic disorder-related diseases, cardiovascular diseases, and neurodegenerative diseases.

[0181] According to embodiments of the present invention, the metabolic disorder-related diseases include at least one of obesity or overweight, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, diabetic retinopathy, hyperglycemia, dyslipidemia, atherosclerosis, non-alcoholic steatohepatitis, and non-alcoholic fatty liver disease.

[0182] In this article, the term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other symptoms that cause hyperglycemia. Type 1 diabetes, also known as insulin-dependent diabetes or juvenile-onset diabetes, is primarily caused by autoimmune disorders or pancreatic beta cell dysfunction, leading to insufficient insulin secretion. Type 2 diabetes, also known as non-insulin-dependent diabetes or adult-onset diabetes, is primarily caused by glucose intake exceeding insulin production, resulting in insufficient insulin production by the pancreas or insulin resistance.

[0183] In this article, the term "obesity" refers to excessive fat in organs such as the liver and subcutaneous fat. When energy intake exceeds energy expenditure, excess calories are stored in fat, leading to obesity and being overweight. In this article, individuals with a body mass index (BMI = weight (kg) divided by the square of height (m)) exceeding 25 are considered obese.

[0184] In this article, the term "NAFLD" refers to a clinicopathological syndrome characterized by diffuse macrovesicular steatosis of hepatocellular cells and lipid metabolism disorders, in addition to alcohol and other clearly defined hepatotoxic factors.

[0185] According to embodiments of the present invention, the neurodegenerative disease includes at least one of Alzheimer's disease and Parkinson's disease.

[0186] Methods of preventing and / or treating diseases

[0187] In a seventh aspect, the present invention provides a method for preventing and / or treating a disease. According to embodiments of the invention, the method comprises: administering to a subject a pharmaceutically acceptable dose of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cells of the fourth aspect, or the pharmaceutical composition of the fifth aspect; wherein the disease includes at least one of metabolic disorder-related diseases, cardiovascular diseases, and neurodegenerative diseases.

[0188] According to embodiments of the present invention, the metabolic disorder-related diseases include at least one of obesity or overweight, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, diabetic retinopathy, hyperglycemia, dyslipidemia, atherosclerosis, non-alcoholic steatohepatitis, and non-alcoholic fatty liver disease.

[0189] According to embodiments of the present invention, the neurodegenerative disease includes at least one of Alzheimer's disease and Parkinson's disease.

[0190] The effective amount of the fusion protein described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0191] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0192] Example 1: Preparation and in vitro activity of agonists with dual or multiple GPCR receptor agonistic activities

[0193] 1. Currently marketed GLP-1 receptor agonists (liraglutide, smegglutide, Trizepatide, etc.) all use a main chain amino acid sequence of 30-39. Studies have found that the N-terminal amino acid sequences of GLP-1 (natural), GCG (natural), and GIP (natural) of type B G protein-coupled receptors (GPCRs) have high homology, and positions 1-28 are the binding sites with affinity to the receptor and the sequences that exert biological effects. In order to reduce immunogenicity, the inventors have innovatively designed a variety of peptide agonists (hereinafter referred to as agonists) containing only 28 amino acids based on bioactivity, and synthesized the peptide agonist sequences in Table 1 using chemical synthesis methods.

[0194] Table 1: Amino acid sequences of each agonist

[0195] Name of agonist sequence agonist receptor targets SEQ ID NO: PJ-501 HGQGTFTSDY SKYLDEKKAK EFVEWLLN GCGR / GLP1 1 PJ-502 YGQGTFTSDY SKYLDEKKAK EFVQWLLN GCGR / GLP1 2 PJ-503 HGQGTFTSDY SKYLDEKKAK EFVQWLLN GCGR / GLP1 3 PJ-504 HGQGTFTSDY SKYLDEKKAK DFVQWLLN GCGR / GLP1 4 PJ-505 HGQGTFTSDY SKYLDERKAKDFVEWLLN GCGR / GLP1 5 PJ-506 HGQGTFTSDY SKYLDERAAQ DFVQWLLD GCGR / GLP1 / GIP 6 PJ-507 HGEGTFTSDY SIALDKIAQK AFVQWLIA GLP1 / GIP 7

[0196] 2. The in vitro bioactivity of the peptide agonists prepared in step 1 with their corresponding receptors (GLP-1R, GIPR, GCGR) was determined. The specific detection methods are shown below:

[0197] (1) In vitro activity assay method for GLP-1R agonists

[0198] The activating activity of GLP-1R was detected using the luciferase reporter gene assay (Jonathan W Day et al.: NatChem Biol. 2009 Oct; 5(10):749-57). The human GLP-1R gene (GKG gene) was cloned into the mammalian cell expression vector plasmid CMV hRluc-neo Vector to construct the recombinant expression plasmid CMV hRluc-neo Vector-rhGLP-1R. CMV hRluc-neo Vector-rhGLP-1R was transfected into CHO-K1-CRE cells (CHO-K1, Shanghai Cell Bank of Chinese Academy of Sciences). Stable transfected cells were selected by G418 and Hygromycin B to obtain the recombinant CHO-K1-rhGLP-1R-CRE stable transfected cell line.

[0199] In vitro activity assay: CHO-K1-rhGLP-1R-CRE cells in logarithmic growth phase were digested with trypsin and resuspended in complete culture medium to a concentration of 2.5-3.5 × 10⁻⁶. 5Cells / ml, 100 μl / well were seeded into 96-well cell plates (Shanghai Jing'an Biotechnology) and incubated overnight at 37℃ and 5% CO2. Purified recombinant dual or multiple regulatory peptide agonists for blood glucose and lipids (see Table 1 for details) were seeded at 100 μl / well into 96-well cell plates and incubated at 37℃ and 5% CO2 for 4-5 hours. The cell culture was discarded, and luciferase reagent (Promega E2550) was added. The plates were incubated at 20-25℃ for 40-50 minutes, and the chemiluminescence (RLU) value was read using a multi-functional microplate reader (MD M4). The RLU value and sample concentration were measured, and the EC50 of the purified recombinant protein's GLP-1R was calculated using a four-parameter curve fitting method with "Origin Pro 2017" parameter regression software. 50 .

[0200] (2) In vitro activity assay method for GCGR agonists:

[0201] The GCGR agonist activity assay also employed a similar luciferase reporter gene assay. The GCGR gene was cloned into the mammalian cell expression plasmid CMV hRluc-neo Vector to construct the recombinant expression plasmid CMV hRluc-neoVector-rhGCGR. HEK-293-CRE cells were transfected with this plasmid, and the stable cell line HEK-293-rhGCGR-CRE was selected. The construction and assay methods were the same as described above.

[0202] (3) Methods for detecting the in vitro activity of GIPR agonists:

[0203] The GIPR agonist activity assay was performed using a similar luciferase reporter gene assay. The GIPR gene was cloned into the mammalian cell expression plasmid CMV hRluc-neo Vector to construct the recombinant expression plasmid CMV hRluc-neoVector-rhGIPR. HEK-293-CRE cells were transfected with this plasmid, and the stable cell line HEK-293-rhGIPR-CRE was selected. The construction and detection methods were the same as described above.

[0204] The bioactivity results of PJ-501~PJ-507, (GLP-1)7-37, natural Glucagon, and natural GIP against GCGR, GLP-1R, and GIPR are shown in Table 2.

[0205] Table 2: Bioactivity of GLP-1R, GCGR, and GIPR in some peptide agonists

[0206]

[0207]

[0208] Example 2: Recombinant construction, transfection screening, and protein identification of fusion proteins

[0209] 1. Using the peptide agonist designed in Example 1, a fusion protein was designed. Its structure, from the N-terminus to the C-terminus, consists of the peptide agonist (SEQ ID NO: 1-7), the first linker L1, the heavy chain constant region of human IgG4, the second linker L2, and the human FGF21 mutant. The specific structure is shown in Table 3. Then, based on the amino acid sequence and codon table in Table 3, its nucleotide DNA sequence was designed.

[0210] Table 3: Amino acid sequences of each fusion protein

[0211]

[0212] 2. The company designed primers for PCR amplification, splicing the DNA fragments corresponding to each fusion protein to obtain the target gene. PCR splicing technology (including primer design, PCR-induced mutations, and enzyme digestion) is a well-known technique familiar to those skilled in the art. Those skilled in the art should understand that the PCR splicing process in this embodiment is not the only method; for example, the target gene can also be obtained through gene synthesis. The target genes corresponding to the fusion proteins with amino acid sequences as shown in SEQ ID NO:14-21 were successfully obtained. The vector plasmid pCHO1.0 was treated with the restriction enzymes AvrII and BstZ17 (TAKARA, Japan), and then the target gene was cloned and constructed into the mammalian cell expression vector pCHO1.0, which was then transformed into *E. coli* Top10. After positive clone identification, the plasmid was extracted using the Omega endotoxin-free plasmid kit and identified.

[0213] 3. Transfection of expression cell lines

[0214] CHO-S host cells (Thermo Fisher) were revived and cultured in Dynamis medium (Gibco A2617501), grown to the logarithmic growth phase, centrifuged, and resuspended at (1-2)×10⁻⁶. 6Cell suspension at 1 cell / ml; add 0.5 ml Dynamis medium to a transfection tube, and add the DNA obtained in step 2 above to PEI (linear PEI, 25 kDa) at a mass ratio of 1:2-6 to the transfection tube. Incubate at room temperature for 15-30 minutes, then slowly add the PEI / DNA mixture dropwise to the cell suspension. Place the cell shake flask in a large-capacity stacked CO2 shaking incubator and incubate at 37°C and 5% CO2 for 48 hours. On day 3, add culture medium to the cell shake flask, and increase the antibiotic resistance every two days until it reaches 15-50 μg / ml puromycin, 50-100 nm. After 14 days of screening, untransfected cells were removed to obtain mixed screening cell lines PJ-601, PJ-602, PJ-603, PJ-604, PJ-605, and PJ-606. After the cells grew, subclonal cell lines were selected to obtain the subclonal cell lines with the highest expression levels: PJ-601-subclone, PJ-602-subclone, PJ-603-subclone, PJ-604-subclone, PJ-605-subclone, and PJ-606-subclone.

[0215] 4. Expression and identification of recombinant fusion proteins

[0216] The supernatant of the fusion protein expression strains PJ-601, PJ-602, PJ-603, PJ-604, PJ-605, and PJ-606 from step 3 was separated by SDS-PAGE electrophoresis. After electrophoresis, Western blot detection was performed. A nitrocellulose membrane (PALL66485) was placed on the electrophoresis gel, and the protein in the gel was transferred to the nitrocellulose membrane under the action of an electric field. The membrane was blocked with TTBS buffer containing 5% skim milk powder for 1 hour. The solution was discarded, and the membrane was washed with TTBS and then incubated with polyclonal antibody containing 1:1000-1:2000 anti-FGF21 at RT for 2 hours with shaking. Discard the antibody solution; wash 3 times with TTBS buffer, then add 1:2000-1:4000 goat anti-rabbit IgG-HRP (ABCOM ab6721), and incubate at RT with shaking for 30 minutes; discard the solution, wash 4 times with TTBS buffer, discard the liquid, add substrate buffer and incubate at room temperature in the dark for color development. When the spots are clearly visible, wash with water to terminate the reaction.

[0217] The results showed that the fusion proteins in this embodiment were all homodimeric fusion proteins with a molecular weight of about 120 kDa, and the molecular weight of the monomers was about 60 kDa.

[0218] Western blot analysis of some fusion proteins in this embodiment is detailed in [link to sample]. Figure 1 . Figure 1The middle section shows the Western blot results for non-reduced and reduced PJ-601, PJ-602, and PJ-603. Lanes 1-7 (from left to right) represent PJ-601 non-reduced, PJ-601 reduced, M represents protein standards (240, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, 11 KD), PJ-602 non-reduced, PJ-602 reduced, PJ-603 non-reduced, and PJ-603 reduced, respectively.

[0219] Example 3: Screening of stable expression lines for each fusion protein and preparation of fusion proteins

[0220] 1. Expression of each fusion protein:

[0221] Subclonal cell lines PJ-601 to PJ-603, which stably expressed cell lines in Example 2, were cultured in a 10L fermenter for expression. The subclonal cells were revived at 37°C, centrifuged at 1000 rpm for 5 minutes, and resuspended in Dynamis cell culture medium (0.5-1.0) × 10⁻⁶ cells / mL. 6 Cell suspension at 1-2 × 10⁶ cells / mL was aliquoted into 200-300 ml flasks. The flasks were then placed in a large-capacity stacked CO₂ shaker incubator at 37°C, 5% CO₂, and 125 rpm for scale-up culture. When the total cell count reached 1-2 × 10⁶ cells / mL... 10 When preparing to transfer cells to the jar, the cell density should be (1.0-2.0) × 10⁻⁶. 6 Cells were added to fermenters at a density of 1 cell / mL. Cell culture medium (Dynamis) was added to the fermenter and preheated to 37°C. Cells were then added, followed by the addition of 4-8% Glutamax, 0.5-1.0% P188, 0.5-1.0% tryptone, and 10-50 ng / mL insulin. The culture temperature was maintained at 37°C, and the rotation speed was 120-140 rpm / min. Air, oxygen, and carbon dioxide were introduced, and dissolved oxygen was maintained within the range of 40% ± 5%. Glucose was maintained at 3-7 mg / mL. Cells were cultured for 72 hours. At day 3, 2% 7A and 0.2% 7B feed (Hyclone) were added, and at day 5, 3% 7A and 0.3% 7B feed were added. After day 5, the temperature was lowered to 35-30°C and cultured further. When cell viability reached approximately 70%, the cell slurry was collected, with cell expression levels at 0.5-2 g / L. Cells were centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. The collected supernatant was stored at -4°C for later use.

[0222] 2. Purification of each fusion protein

[0223] The Protein A column was equilibrated with equilibration buffer (20 mM PBS, 0.5 M NaCl, pH 7.2). The pH of each centrifuged sample obtained in step 1 of this embodiment was adjusted to 7.2. The samples were loaded, washed with equilibration buffer, and eluted with 100% elution buffer (0.1 M Gly-HCl, pH 3.0). The eluted samples were collected and neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0) to a pH of 7.2-7.3.

[0224] The Q anion exchange chromatography column (Borglon) was regenerated using 2M sodium chloride and equilibrated with equilibration buffer (10mM Tris-HCl, pH 7.2). The neutralized sample was then loaded onto the column and washed with equilibration buffer. Elution was performed using 10mM Tris-HCl, 0.3M NaCl, pH 7.2. The eluted sample was collected, and the pH was adjusted to 7.2-7.3. The collected sample was then concentrated by ultrafiltration to obtain a fusion protein concentration of not less than 2 mg / ml and stored at 2-8℃.

[0225] The purified fusion proteins PJ-601 to PJ-606 were reduced with 10 mM TCEP and then subjected to SDS-PAGE electrophoresis. The SDS-PAGE results showed that the fusion proteins PJ-601 to PJ-606 were all homodimers with a molecular weight of approximately 120 kDa, while the monomers had a molecular weight of approximately 60 kDa.

[0226] For details of the SDS-PAGE electrophoresis results of some fusion proteins in this embodiment, please refer to [link to relevant documentation]. Figure 2 . Figure 2 The results of SDS-PAGE non-reducing electrophoresis for PJ-601, PJ-602, and PJ-603 are shown. Lanes 1 to 7 are used from left to right. Lane 1 is for protein standards (240, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, 11 KD). Lanes 2-3 are for PJ-601 reduced and non-reduced. Lanes 4-5 are for PJ-602 reduced and non-reduced. Lanes 6-7 are for PJ-603 reduced and non-reduced.

[0227] Example 4: Assay of GCG / GLP1 receptor cell activity of fusion proteins PJ601, PJ602, and J603

[0228] The purified fusion proteins PJ-601 to PJ-603 (derived from Example 3, see Table 3 for fusion proteins) were measured using the method in step 2 of Example 1, with (GLP-1)7-37 and Glucagon used as controls. The results showed that the fusion proteins of this invention all exhibited good agonistic activity. The statistical results of the in vitro biological activity assays for some of the fusion proteins are shown in Table 4. Figure 3 (GCGR activity assay results) Figure 4 (GLP-1R activity assay results are shown).

[0229] Table 4: GCGR and GLP-1R bioactivity of fusion proteins PJ-601, PJ-602, and PJ-603

[0230]

[0231] Example 5: Determination of FGF21 activity in fusion proteins PJ601, PJ602, J603, and PJ604

[0232] The in vitro activity of FGF21 was detected using the glucose consumption method. The β-klotho and FGFR1(IIIc) genes were cloned into the mammalian cell expression plasmid GV215 to construct recombinant expression plasmids GV215-β-klotho and GV215-FGFR1(IIIc) (β-klotho / FGFR1(IIIc) plasmids were purchased from Shanghai Jikai Gene Technology Co., Ltd.). The GV215-β-klotho and GV215-FGFR1(IIIc) recombinant plasmids were co-transfected into 3T3-L1 cells (Shanghai Cell Bank, Chinese Academy of Sciences), and 3T3-L1-β-klotho / FGFR1(IIIc) recipient cell lines were screened.

[0233] In vitro activity assay: 3T3-L1-β-klotho / FGFR1(IIIc) cells in logarithmic growth phase were digested with trypsin and resuspended in 5-7 × 10⁻⁶ mol / L solution. 5 200 μl / well was seeded into 48-well cell culture plates and cultured overnight at 37°C and 5% CO2. The cell culture medium was discarded, and FBS-free DMEM high-glucose medium was added for starvation. The cells were then cultured at 37°C and 5% CO2 for 48 hours.

[0234] Sample solution preparation: Using hFGF21 standard (Sinochem 10911-H07E) as the standard, purified fusion proteins PJ-601~PJ-604 (derived from Example 3, see Table 3 for fusion proteins) were tested. The cell starvation medium was discarded, and each fusion protein or standard was added to the cultured cell culture medium at 150 μL / well. The cells were incubated at 37℃ and 5% CO2 for 16-36 hours. 20 μL of supernatant was collected per well and added to a 96-well plate. POD / GOD glucose reagent (Shanghai Desay 26084) was added, and the plates were incubated at 37℃ and 5% CO2 for 10 minutes. The OD value was measured using a multi-functional microplate reader (MD M4). The EC50 of FGF21 for each purified fusion protein was calculated using a four-parameter curve fitting method with the OD value and the concentration of the sample in "Origin Pro 2017" parameter regression software. 50 The results showed that all fusion proteins exhibited good FGF21 biological activity. The in vitro biological activity assay results for some fusion proteins are as follows: Figure 5 As shown in Table 5.

[0235] Table 5: FGF21 bioactivity of each fusion protein

[0236] Variant number FGF21 in vitro biological activity EC 50 (nM) hFGF21 1.12 PJ-601 3.49 PJ-602 3.73 PJ-603 20.62 PJ-604 123.8

[0237] Example 6: Structural analysis of fusion proteins PJ-601 to PJ-603

[0238] (1) High-resolution mass spectrometry detection

[0239] 50 μg each of the fusion proteins PJ-601 to PJ-603 (derived from Example 3, see Table 3 for fusion proteins) were added to denaturing buffer, and 1 M DTT was added to a final concentration of 50 mM. Deglycosidases (including PNGase A, PNGase F, and O-glycosidase) were added, and the mixture was digested in a 65°C water bath for 30-60 min. Chromatographic separation was performed using ultra-high performance liquid chromatography (UHPLC), and molecular weight was detected using high-resolution mass spectrometry (HPLC). The detection time was 10 min, the detection mode was positive ion, and the scan range was 500-4000 m / z. The results showed that the molecular weight was 49.5–50.5 kDa, which was basically consistent with the theoretical molecular weight.

[0240] (2) SEC-HPLC analysis

[0241] The purified fusion proteins PJ-601–PJ-603 (derived from Example 3, see Table 3 for fusion proteins) were dissolved in 1000 μl of 20 mM Tris-HCl (pH 8.0) to a concentration of 1 mg / mL. Analysis was performed using HPLC-SEC (Agilent 1220 model) with a Cytiva Superdex™ 200 Increase 10 / 300GL column (10 × 300 mm); mobile phase: 0.05 mol / L Na₂CO₃-NaHCO₃, 0.15 mol / L NaCl, pH 10.3; elution gradient: isocratic elution for 50 min; flow rate: 0.5 mL / min; detection wavelength: 280 nm; column temperature: room temperature; injection volume: 50 μl. The results showed that the fusion protein exhibited a single main peak, with no monomers observed, and all polymers were less than 1%. In this embodiment, the HPLC-SEC analysis results of the fusion protein PJ-602 are exemplarily shown in [details omitted]. Figure 6 .

[0242] (3) Analysis of disulfide bond composition

[0243] The purified fusion proteins PJ-601–PJ-603 were diluted by volume with 100 mmol / L Tris-HCl at pH 8.0, and then urea solid at a final concentration of 8 mol / L was added. The samples were divided into two aliquots: one for non-reductive digestion and the other for TCEP reductive digestion. The digestion conditions were Lys-C digestion at 37°C for 16 h (mass ratio: 1:50). Mass spectrometry analysis was performed. The results showed that each fusion protein contained two pairs of interchain disulfide bonds (i.e., (T6+T7)—(T6+T7)), and the second polypeptide Fc had two pairs of intrachain disulfide bonds, while the third polypeptide FGF21 had one pair of intrachain disulfide bonds.

[0244] This embodiment exemplifies the results of the fusion protein PJ-602, as detailed in Table 6 and... Figure 7 Table 6 and Figure 7 The results showed that the peptide (T6+T7)—(T6+T7) was found in the non-reduced sample, and its molecular weight was about 4 Da less than that of the two reduced peptides, indicating the presence of two pairs of interchain disulfide bonds. Peptides T8—T12 and T18—T21 were also found, indicating that the second polypeptide FC has two pairs of intrachain disulfide bonds. Peptide T25 was found, and its molecular weight was about 2 Da less than that of the reduced polypeptide T25, indicating that the third polypeptide FGF21 has one pair of intrachain disulfide bonds. Furthermore, the corresponding peptides were found in the reductase-digested samples, confirming the accuracy of the analytical results.

[0245] Table 6: Disulfide bond composition analysis results of homodimer fusion protein PJ-602

[0246]

[0247] Note: In this step of the disulfide bond composition analysis, "—" indicates that two peptide segments are linked by a disulfide bond.

[0248] (4) Glycosylation analysis:

[0249] Trypsin (TP) digestion and reduction: The peptides were digested with TP at a mass ratio of 1:50 in a water bath at 37°C for 20 h, and then reduced with 20 mmol / L TCEP. The molecular weight of the peptides was detected by mass spectrometry.

[0250] The peptides were digested with Trypsin at a mass ratio of 1:50 in a water bath at 37°C for 20 hours, followed by digestion with PNGase F-Fast glycosidase in a water bath at 65°C for 30 minutes. The molecular weight of the peptides was then determined by mass spectrometry.

[0251] Mass spectrometry analysis of glycosylation sites showed that the T10 peptide did not appear after TP enzyme cleavage. The T10 peptide appeared after PNGase F-Fast glycosidase cleavage, and glycosylation was present in the T10 peptide. This example exemplifies the analytical results of the fusion protein PJ-602, as detailed in Table 7. Figure 8 .

[0252] Table 7: Glycosylation Analysis Results of Homodimeric Fusion Protein PJ-602

[0253]

[0254] (5) C-terminal sequencing

[0255] The purified fusion protein PJ-602 (derived from Example 3, see Table 3 for fusion proteins) was digested with Lys-C at a ratio of 1:1000, and its molecular weight was determined by mass spectrometry. The mass spectrometry results showed that the C-terminal 59 amino acids of each fusion protein were consistent with the theoretical sequence. The LC-MS chromatogram of fusion protein PJ-602 is shown below. Figure 9 See Table 8.

[0256] Table 8: Results of sequence analysis of the C-terminal 59 amino acids of fusion protein PJ-602 compared with the theoretical sequence.

[0257]

[0258] Example 7: Formulation and preparation of homodimeric fusion proteins PJ-601 to PJ-603

[0259] Take fusion proteins PJ-601 to PJ-603, concentrate them by ultrafiltration, add 50-200 mmol / L glycine, 10-30 mmol / L histidine, 0.1-0.3% Tween-20 and 5-6% sucrose, sterilize by 0.22 μm filtration, aseptically aliquot into 1 ml vials and freeze-dry.

[0260]

[0261] The lyophilized fusion proteins PJ-601 to PJ-603 were analyzed by HPLC-SEC. The high and low molecular weights were less than 0.5%, and the biological activity was unchanged, which was basically consistent with the activity in Examples 4 and 5.

[0262] Example 8: FAP restriction enzyme digestion experiment of PJ602

[0263] The purified fusion protein PJ-602 was dissolved in 1000 μl of 20 mM Tris-HCl (pH 8.0) to a concentration of 1 mg / ml. 100 μl of the solution was then added to 5 μl of FAP enzyme (0.4 mg / ml), and the mixture was thoroughly mixed. The enzyme was digested in a water bath at 37 °C for 2 h, and then detected by high performance liquid chromatography. The chromatographic column was a Kromasil 100-3.5-C4; the mobile phase was: solution A was 0.1% formic acid aqueous solution, and solution B was 0.1% formic acid acetonitrile solution; the flow rate was 1.0 ml / min; the column temperature was 30℃; the detection wavelength was 214 nm; the elution gradient was: solution B 0-30 min, 5%-95%; the injection concentration was 1 mg / ml; the injection volume was 10 μl; the ion source was ESI+; the scanning mode was Scan, the scanning range was 300-2000 m / z, and the scanning time range was 4-30 min; hFGF21 standard (Yiqiao Shenzhou 10911-H07E) was used as a reference.

[0264] Samples of hFGF21 digested with FAP were detected using the aforementioned mass spectrometry conditions. The SQGRSPSYAS peptide at the C-terminus of hFGF21 was found, demonstrating that FAP enzyme specifically recognizes and cleaves the 172nd amino acid at the C-terminus of the hFGF21 protein. Under the same conditions, the SQGRSPSYAS peptide at the C-terminus of each fusion protein PJ-602 of the present invention was not detected under FAP enzyme action, but the first 30 amino acid sequences of the N-terminus of the fusion protein PJ-602 (HGQGTFTSDYSKYLDEKKAKEFVEWLLNGP) were detected. This indicates that FGF21 in each fusion protein PJ-602 of the present invention is resistant to FAP enzyme digestion, and that FAP enzyme digestion releases a peptide agonist and a long-acting FGF21 mutant.

[0265] Example 9: OGTT experiment of normal mice after single subcutaneous administration of PJ601, PJ602, and J603

[0266] 1. Sixty C57BL / 6 mice were randomly divided into six groups: Vehicle group, Tirzepatide group, Semaglutide group, PJ-601 group, PJ-602 group, and PJ-603 group, with 10 mice in each group. All treatment groups received a dose of 30 nmol / kg via subcutaneous administration, except for the Vehicle group, which received a corresponding volume of physiological saline subcutaneously. At 24 h and 72 h post-administration, five animals from each group underwent an oral glucose tolerance test (OGTT). Specific results are detailed in [link to results]. Figures 10-11 The results showed that PJ601, PJ602, and J603 all had hypoglycemic effects 24h and 72h after administration. Among them, the hypoglycemic effect of PJ602 lasted for a significantly longer time than that of Tirzepatide and Semaglutide.

[0267] 2. Using the above method, the inventors further conducted a single subcutaneous OGTT experiment on normal mice with other fusion proteins (PJ-604 to PJ-616) prepared in Example 3. The results showed that the fusion proteins of the present invention all had high hypoglycemic effects.

[0268] Example 10: Subcutaneous administration of PJ601, PJ602, and J603 to DIO model mice

[0269] Thirty male DIO model mice fed a high-fat diet for 16 weeks were randomly divided into five groups according to body weight: Vehicle group, Semaglutide group, PJ-601 group, PJ-602 group, and PJ-603 group, with six mice in each group. All treatment groups received a dose of 30 nmol / kg via subcutaneous administration. Semaglutide was administered twice weekly, while PJ-601, PJ-602, and PJ-603 mice received once weekly. The Vehicle group received the corresponding volume of physiological saline. All treatments were administered for two consecutive weeks. During the treatment period, the mice continued to be fed a high-fat diet, and their body weight was measured daily. At the end of the experiment, fasting blood glucose was measured, and plasma samples were collected for four lipid profiles. Detailed results are available in [link to results]. Figure 12 See Table 9.

[0270] Depend on Figure 12 As shown in Table 9, 30 nmol / kg PJ-601, PJ-602, and PJ-603 all have the effects of reducing body weight, regulating blood glucose, and regulating blood lipids in DIO mice. Among them, PJ-602 has the best effect on reducing body weight and regulating blood glucose and blood lipids.

[0271] Table 9: Results of the four regulatory effects of PJ601, PJ602, and J603 on blood glucose and blood lipids in DIO mice

[0272]

[0273]

[0274] Note: Compared with Vehicle, *P<0.05, **P<0.01.

[0275] Example 11: PJ-602 Repeated Subcutaneous Administration DIO Model Mouse Experiment

[0276] Twenty-four male DIO model mice fed a high-fat diet for 16 weeks were randomly divided into four groups according to body weight: Vehicle group, Tirzepatide group, and low- and high-dose PJ-602 groups, with six mice in each group. The low- and high-dose PJ-602 were administered subcutaneously twice weekly at doses of 30 nmol / kg and 60 nmol / kg, respectively. The Tirzepatide dose was 30 nmol / kg, administered subcutaneously twice weekly. The Vehicle group received the corresponding volume of physiological saline. Treatment continued for four weeks, with the mice fed a high-fat diet throughout the period. Body weight was measured daily. At the end of the experiment, fasting blood glucose was measured, plasma samples were collected for lipid profile analysis, and serum samples were collected for anti-FGF21 antibody detection. Liver samples were weighed, and a portion of the liver was fixed in formaldehyde for histopathological analysis (NAS score). Detailed results are detailed below. Figures 13-16 .in, Figures 14-16 In comparison with Vehicle, *P<0.05, **P<0.01.

[0277] Depend on Figures 13-16 PJ602 showed a good dose-response effect in reducing body weight, regulating blood glucose and blood lipids in DIO mice. Specifically, PJ-602's ability to regulate body weight, blood lipids, liver weight, hepatic steatosis, hepatocyte damage, and hepatic lobular inflammatory infiltration in DIO mice was significantly better than Tirzepatide at both 30 nmol / kg and 60 nmol / kg doses. At the end of the experiment, plasma from mice in the low- and high-dose PJ-602 groups was collected, and anti-drug antibodies were detected using a competitive ELISA assay after coating the fusion protein PJ-602. The results showed no detection of anti-peptide agonist (28-peptide) antibodies or anti-FGF21 antibodies.

[0278] Example 12: PJ-602 / PJ-603 repeated subcutaneous administration NASH model mouse experiment

[0279] Twenty-eight male model mice fed a high-fat diet (product number: D12492) for 12 weeks were randomly divided into four groups according to body weight: Vehicle group, MGL-3196 group (Resmetirom; THR-β agonist), PJ-602 group, and PJ-603 group, with seven mice in each group. A normal healthy mouse group of seven mice was also included. PJ-602 and PJ-603 were administered subcutaneously twice weekly at a dose of 60 nmol / kg, while MGL-3196 was administered orally once daily at a dose of 10 mg / kg. The Vehicle group and normal mice received the corresponding volume of physiological saline. All treatments were administered for four weeks. During the treatment period, the high-fat diet was maintained. Animals in the Vehicle, MGL-3196, PJ-602, and PJ-603 groups received intraperitoneal injections of 25% CCL4 at a dose of 0.5 mL / kg twice weekly. Body weight was measured daily. At the end of the experiment, plasma samples were collected to measure ALT / AST / TG / TC. Liver samples were weighed, and a portion of the liver was fixed in formaldehyde for histopathological analysis (NAS score) and Sirius staining for fibrosis assessment. See [link to results]. Figures 17-21 .

[0280] Depend on Figures 17-21 Both PJ-602 and PJ-603 can reduce the body weight, plasma ALT / AST / TG / TC of NASH mice, and have good regulatory effects on blood lipids, liver function, liver weight, hepatic steatosis, hepatic ballooning changes, and liver fibrosis in NASH mice.

[0281] Example 13: Efficacy experiment of repeated subcutaneous administration of PJ-602 on thioacetamide (TAA)-induced liver fibrosis in rats

[0282] Eighteen male SD rats were randomly divided into three groups according to body weight: a normal control group, a model control group, and a PJ-602 group, with six rats in each group. Liver fibrosis was induced by intraperitoneal injection of 300 mg / kg TAA twice weekly for 8 weeks. The normal control group received an equivalent volume of physiological saline intraperitoneally. Two weeks after induction, the model animals began receiving PJ-602 at a dose of 25 nmol / kg, administered subcutaneously twice weekly. The normal and model control groups received an equivalent volume of physiological saline subcutaneously for 6 consecutive weeks. At the end of the experiment, blood samples were collected to detect platelet counts and plasma levels to assess liver function. Liver samples were weighed, and a portion of the liver was fixed in formaldehyde and stained with Sirius to evaluate fibrosis. Detailed results are shown below. Figures 22-23 .

[0283] Depend on Figures 22-23PJ-602 can significantly improve liver function indicators (ALT / AST / GGT / TBIL / DBIL) in model animals and significantly improve the degree of liver fibrosis in model animals, indicating that PJ-602 has a good therapeutic effect on TAA-induced rat liver fibrosis model.

[0284] Example 14: Experiment on NASH model mice induced by repeated subcutaneous administration of PJ-602 and GAN diet

[0285] Thirty-two male model mice fed a high-fat GAN diet (catalog number: D09100310) for 18 weeks were randomly divided into four groups according to body weight: Vehicle group, MGL-3196 group (Resmetirom; THR-β agonist), Efruxifermin group, and PJ-602 group, with eight mice in each group. A normal healthy mouse group of eight was also included. PJ-602 was administered at a dose of 50 nmol / kg, and Efruxifermin at a dose of 50 nmol / kg, subcutaneously twice weekly. MGL-3196 was administered orally at a dose of 3 mg / kg once daily. The Vehicle group and normal mice received the corresponding volume of physiological saline. All treatments were administered for 8 weeks. The mice were continued to be fed a high-fat diet during the treatment period. Body weight was measured daily. At the end of the experiment, plasma ALT / LDL-C was measured, liver weight was collected, and a portion of the liver was fixed in formaldehyde for histopathological analysis (NAS score) and Sirius staining for fibrosis assessment. Detailed results are detailed below. Figures 24-26 .

[0286] Depend on Figures 24-26 PJ-602 can reduce the body weight and plasma ALT / LDL-C of NASH mice, and also has a good regulatory effect on blood lipids, liver function, liver weight, hepatic steatosis, hepatic ballooning changes, and liver fibrosis in NASH mice.

[0287] Example 15: Experiment on the NASH model of Foz / Foz mice with repeated subcutaneous administration of PJ-602

[0288] Eighteen male Foz / Foz mice fed a high-fat diet (catalog number: D12492) for 8 weeks were randomly divided into three groups according to body weight: Vehicle group, Tirzepatide group, and PJ-602 group, with 6 mice in each group. PJ-602 and Tirzepatide were administered subcutaneously twice weekly at a dose of 30 nmol / kg, while the Vehicle group received the corresponding volume of physiological saline for 4 consecutive weeks. The high-fat diet was maintained throughout the treatment period. Body weight was measured daily. At the end of the experiment, plasma samples were collected to measure ALT, AST, and / or LDL-C. Liver samples were weighed, and partial liver tissue was fixed in formaldehyde for histopathological analysis (NAS score) and Sirius staining for fibrosis assessment. Detailed results are detailed below. Figures 24-26 .

[0289] Depend on Figures 27-28 As shown in Tables 10-11, PJ-602 can reduce body weight, plasma ALT, AST, and LDL-C in Foz / Foz mouse NASH models, and is superior to Tirzepatide in improving ALT, AST, and LDL-C. Regarding the regulatory effects of PJ-602 on blood lipids, liver function, liver weight, hepatic steatosis, and ballooning changes in the liver in Foz / Foz mouse NASH models, the improvement effect of PJ-602 at a dose of 30 nmol / kg is superior to that of Tirzepatide at 30 nmol / kg.

[0290] Table 10: Effects of PJ602 on the regulation of blood lipids and liver function in Foz / Foz mouse NASH model

[0291] Group ALT (IU / L) AST (IU / L) LDL-C (mmol / L) Vehicle 871.37±82.3 867.09±68.3 1.05±0.15 Tirzepatide 427.20±109.6 368.60±59.7 0.63±0.17 PJ-602 64.79±26.3 168.05±32.6 0.14±0.10

[0292] Table 11: Results of PJ602 in improving hepatic steatosis and fibrosis in Foz / Foz mouse NASH model

[0293] Group Fatty degeneration Hepatic lobular inflammation balloon-like changes NAS rating Fibrosis score Vehicle 2.4±1.2 1.8±0.6 0.9±0.2 5.1±1.4 1.9±0.3 Tirzepatide 1.2±0.2 0.9±0.4 0.2±0.1 2.3±0.4 1.0±0.2 PJ-602 0.0±0.0 0.6±0.3 0.0±0.0 0.6±0.2 0.9±0.4

[0294] Example 16: Pharmacokinetics of a single subcutaneous administration of PJ-602 to cynomolgus monkeys

[0295] The pharmacokinetics of PJ-602 after a single subcutaneous dose in cynomolgus monkeys were evaluated. The specific steps were as follows: Four cynomolgus monkeys (half male and half female) were administered a subcutaneous dose of 1 mg / kg. Approximately 1 ml of blood was collected before administration and at 2, 8, 24, 48, 72, 96, 120, 144, 168, 216, and 288 hours after administration. Plasma was collected by centrifugation. Serum PJ-602 concentration was detected using a double-antibody sandwich ELISA method. An ELISA plate was coated with rabbit anti-human FGF21 antibody (from Yiqiao Shenzhou) and blocked with BSA. Different dilutions of PJ-602 and the plasma sample were added and incubated. After washing three times with PBST, Biotin anti-human IgG Fc Antibody (Abcam) was added, followed by Avidin-HRP+TMB chromogenic development. The absorbance (OD value) was read at dual wavelengths of 450 nm and 630 nm. Specific test results are shown in Table 12. The results showed that a single subcutaneous administration of PJ-602 to cynomolgus monkeys resulted in T... max It takes approximately 36 hours, with a half-life of approximately 72 hours, supporting a clinical dosing frequency of once weekly or once every two weeks.

[0296] Table 12: Pharmacokinetic parameters after a single dose

[0297] Pharmacokinetic parameters Mean±SD T max (h)]]> 36.0±13.9 C max (ng / ml) 945.2±38.2 <![CDATA[T 1 / 2 (h)]]> 72.4±8.4 <![CDATA[AUC 0-last (ng / ml*h)]]> 123374.5±21424.6

[0298] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0299] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises the structure shown in formula (I): Z1-L1-Z2-L2-Z3(I), Z1 is a 28-peptide with agonistic activity against both GCGR and GLP-1R receptors. The amino acid sequence of L1 is shown as GPGP(GGGGS)n, where n is 1 to 3; Z2 is the constant region of the IgG4 heavy chain or a mutant thereof; The amino acid sequence of L2 is shown as (GGGGS)n or (GGGG)n, where n is 1 to 3; Z3 is an FGF-21 mutant that contains G170P and P171G mutations compared to wild-type FGF-21.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of Z1 is shown as X1GQGTFTSDYSKYLDEX2KAK X3FVX4WLLN; Wherein, X1 is selected from H or Y, preferably H; X2 is selected from K or R, preferably K; X3 is selected from D or E, with E being preferred; X4 is selected from Q or E, with E being preferred; Optionally, the amino acid sequence of Z1 is shown in any one of SEQ ID NO:1 to 7.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of L1 has the following structure: GPGP(GGGGS)n, where n is 1, 2 or 3; Optionally, the amino acid sequence of L1 is as shown in SEQ ID NO:8; Optionally, the amino acid sequence of L2 has the following structure: (GGGGS)n, where n is 1, 2 or 3; Optionally, the Z2 is the human IgG4 heavy chain constant region or a mutant thereof; Optionally, the Z2 has an amino acid sequence as shown in SEQ ID NO:9 or having at least 90% identity with it.

4. The fusion protein according to claim 1, characterized in that, The mutation site of the FGF-21 mutant further includes R19V; Optionally, the mutation sites of the FGF-21 mutant further include L98R, or L98R and A180E; Optionally, the mutation site of the FGF-21 mutant further includes the deletion of the 4 amino acids at the N-terminus.

5. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 14-21; Optionally, the fusion protein is a homodimeric fusion protein, wherein the homodimeric fusion protein is linked by two pairs of disulfide bonds formed by two cysteine ​​residues located in the PPCPP sequence of the second polypeptide.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 5; Optionally, the nucleic acid molecule is DNA.

7. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 6; Optionally, the expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors; Optionally, the expression vector includes at least one of plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

8. A recombinant cell, characterized in that, The recombinant cells include: Carrying the nucleic acid molecule of claim 6 or the expression vector of claim 7; or Express the fusion protein according to any one of claims 1 to 5; Optionally, the recombinant cells are obtained by introducing the expression vector into host cells; Optionally, the host cell includes eukaryotic cells and prokaryotic cells; Optionally, the host cell is a mammalian cell.

9. A pharmaceutical composition, characterized in that, include: The fusion protein of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, or the recombinant cell of claim 8; and optionally pharmaceutically acceptable excipients or carriers.

10. Use of the fusion protein of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the recombinant cell of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament, wherein the medicament is used for the prevention and / or treatment of at least one of metabolic disorder-related diseases, cardiovascular diseases, and neurodegenerative diseases; Optionally, the metabolic disorder-related diseases include at least one of obesity or overweight, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, diabetic retinopathy, hyperglycemia, dyslipidemia, atherosclerosis, non-alcoholic steatohepatitis, and non-alcoholic fatty liver disease. Optionally, the neurodegenerative disease includes at least one of Alzheimer's disease and Parkinson's disease.

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