NPQ-igg2 / fc fusion protein and use thereof

Abstract

Provided are an NPQ-IgG2 / Fc fusion protein and the use thereof. The fusion protein comprises an NPQ polypeptide and a human IgG2 / Fc region, and can be used for treating glucose metabolism or lipid metabolism disorder-related metabolic diseases, such as liver diseases including fatty liver, and diabetes.

Description

NPQ-IgG2 / Fc fusion protein and application thereof TECHNICAL FIELD

[0001] The present application relates to a NPQ-IgG2 / Fc fusion protein prepared by genetic engineering recombination technology and application thereof, which comprises a NPQ polypeptide and an IgG2 / Fc polypeptide. The NPQ-IgG2 / Fc fusion protein has the ability to specifically bind to hepatocytes HEPG2 and insulin-secreting cells INS-1, and forms endocytosis after binding to cells, activates intracellular signaling pathways. At the same time, the present application also discloses the NPQ-IgG2 / Fc fusion protein, which can be used for treating liver diseases including fatty liver, diabetes and metabolic syndrome and the like by multi-targeting effect including promoting hepatocyte glycolipid metabolism and improving pancreatic beta cell function. BACKGROUND

[0002] Neuropeptide Q (NPQ), also known as Spexin, is a peptide hormone discovered in 2007 and widely expressed in endocrine organs and epithelial tissues. NPQ mature peptide is composed of 14 amino acid residues, which binds to cell surface galanin receptor 2 / 3 (GALR2 / 3) as a natural ligand, activates downstream multiple signaling pathways, stimulates insulin secretion, improves insulin resistance, regulates carbohydrate and fat metabolism homeostasis, and affects various physiological functions including food intake and energy balance. In addition, NPQ may be involved in the regulation of adrenal cortical cell proliferation, affecting the functions of cardiovascular, kidney and endocrine system. NPQ is related to many diseases, including obesity, type 2 diabetes, fatty liver (Turkel, Omer et al. “Impact of spexin on metabolic diseases and inflammation: An updated minireview.” Experimental biology and medicine (Maywood, N.J.) vol. 247, 7 (2022): 567-573.), and mental diseases such as anxiety and depression (Lv, Shuang-Yu et al. “Emerging Roles of NPQ / Spexin in Physiology and Pathology.” Frontiers in pharmacology vol. 10 457. 7 May. 2019).

[0003] ​Studies have shown that the decrease of NPQ level is associated with the severity of obesity, aging, and other metabolic diseases such as cardiovascular and cerebrovascular diseases; injection of exogenous NPQ (Spexin) in diet-induced obese (DIO) mice can reduce appetite, reduce energy intake, reduce body weight, increase exercise and fat consumption, etc. In addition, exogenous NPQ (Spexin) can significantly reduce the intracellular fat level of hepatocytes, reduce aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and reduce the level of non-alcoholic fatty liver and liver fibrosis in DIO mice; NPQ (Spexin) can reduce insulin resistance and glycosylated hemoglobin levels and enhance glucose tolerance in DIO mice.

[0004] Therefore, NPQ can become a potential drug for treating diseases related to abnormal glucose and lipid metabolism, but mature NPQ has only 14 amino acids, is easily degraded by enzymes, and has a short half-life in the body (about 1 hour), which limits its clinical application. Therefore, it is urgent to find NPQ analogs with good efficacy and metabolic properties. SUMMARY

[0005] In one aspect, the present application provides a NPQ fusion protein, the fusion protein comprising a NPQ polypeptide and an immunoglobulin Fc domain, wherein the NPQ polypeptide is selected from human NPQ and murine NPQ, the NPQ polypeptide is covalently linked to the immunoglobulin Fc domain, the immunoglobulin Fc domain comprises or is an IgG2 / Fc polypeptide, the NPQ polypeptide comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 19 or SEQ ID NO: 20 or an amino acid sequence having at least 90% sequence identity with the above sequences, the IgG2 / Fc polypeptide has an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; the NPQ fusion protein further comprises a linker peptide for linking the NPQ polypeptide and the immunoglobulin Fc domain, the linker peptide has an amino acid sequence selected from SEQ ID NO: 8.

[0006] In some embodiments, the NPQ fusion protein is further modified, and the modification comprises N-glycosylation or O-glycosylation or oxidation or succinimidation of asparagine or deamidation of asparagine or hydroxylation modification.

[0007] In one embodiment, in the NPQ fusion protein of the present application, the IgG2 / Fc polypeptide further comprises one or two or three selected from C222S substitution, A330S substitution and P331S substitution.

[0008] In one embodiment, the connecting peptide further has an amino acid sequence selected from any one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0009] In one embodiment, the NPQ fusion protein of the present application has an amino acid sequence of SEQ ID NO: 5.

[0010] In one embodiment, the NPQ fusion protein of the present application further comprises a signal peptide. A signal peptide is a peptide chain of about 5-30 amino acids in length that encodes a hydrophobic amino acid sequence responsible for directing a protein to an intracellular structure or for transferring a newly synthesized protein to a secretory pathway after the initiation codon. Various forms of signal peptides are used in the present application. In one embodiment, the presented signal peptide includes but is not limited to a human CD33 signal peptide, or the signal peptide has an amino acid sequence of SEQ ID NO: 6.

[0011] In one embodiment, the NPQ fusion protein of the present application can further comprise an enzyme cleavage site.

[0012] In one embodiment, the NPQ fusion protein of the present application can be further modified, comprising one or more of lysine hydroxylation, or methionine oxidation, or O-glycosylation (GalNAc), or aspartate isomerization, etc.

[0013] In one embodiment, the N-glycosylation modification of the NPQ fusion protein of the present application is of the N-glycoside type G0F and G1F, preferably G0F.

[0014] In one embodiment, in the NPQ fusion protein of the present application, the site of N-glycosylation modification can be, for example, asparagine (N) at position 105 of SEQ ID NO: 5; the site of O-glycosylation modification can be serine (S) at position 15, or 16, or 22, or 27 of SEQ ID NO: 5; the site of oxidation can be methionine (M) at position 60 of SEQ ID NO: 5, or methionine (M) at position 7 of SEQ ID NO: 5, or methionine (M) at position 205 of SEQ ID NO: 5, preferably methionine (M) at position 65 of SEQ ID NO: 5; the site of succinimide formation modification of asparagine can be asparagine (N) at position 123 of SEQ ID NO: 5; the site of deamidation modification of asparagine can be asparagine (N) at position 123 of SEQ ID NO: 5, or asparagine (N) at position 169 of SEQ ID NO: 5; the site of hydroxylation can be lysine (K) at position 11 of SEQ ID NO: 5, or tryptophan (W) at position 225 of SEQ ID NO: 5. The above modifications can also be at other suitable sites.

[0015] In another aspect, the present application provides a polynucleotide comprising a polynucleotide encoding the NPQ fusion protein of the present application.

[0016] In one embodiment, the polynucleotide of the present application has the polynucleotide sequence of SEQ ID NO: 7 or a polynucleotide sequence having at least about 70% sequence identity to SEQ ID NO: 7.

[0017] In another aspect, the present application provides a vector comprising the polynucleotide of the present application.

[0018] In yet another aspect, the present application provides a pharmaceutical composition comprising the NPQ fusion protein of the present application, or the polynucleotide of the present application, or the vector of the present application, and optionally a pharmaceutically acceptable carrier.

[0019] In one embodiment, the composition further comprises a buffer, including but not limited to phosphate buffer, citrate buffer, and borate buffer.

[0020] In one embodiment, the composition further comprises a surfactant. Suitable surfactants include, but are not limited to, Tween 80 (polysorbate 80), polyoxyethylene castor oil derivatives, poloxamers, lecithin, macrogol 15-hydroxystearate, cyclodextrins, and the like.

[0021] In one embodiment, the composition further comprises an excipient. In one embodiment, the excipient can include mannitol, sorbitol, maltitol, erythritol, arabinose, xylitol, sucrose, lactose, trehalose, dextran, or a mixture thereof. In some embodiments, the excipient can be one or more selected from mannitol, sucrose, sorbitol, preferably mannitol, sucrose.

[0022] In one embodiment, the composition comprises the NPQ fusion protein, formulated into a preparation, administered at a dosage of about 0.2 mg to about 40 mg, for example, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg of the NPQ fusion protein.

[0023] In still another aspect, the present application provides use of the NPQ fusion protein of the present application, the polynucleotide of the present application, the vector of the present application, the pharmaceutical composition of the present application in the manufacture of a medicament for treating or preventing a metabolic disease associated with glucose metabolism or lipid metabolism disorder. The pharmaceutical composition can include fusion proteins, polynucleotides, vectors, and cells, etc., and is used for treatment by gene therapy.

[0024] In one embodiment, in the use of the present application, the metabolic disease associated with glucose metabolism or lipid metabolism disorder is selected from diabetes, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and obesity.

[0025] In one embodiment, in the use of the present application, the metabolic disease associated with glucose metabolism or lipid metabolism disorder is diabetes, preferably type 2 diabetes.

[0026] In one embodiment, in the application of the present application, the NPQ fusion protein, the polynucleotide, the vector, or the pharmaceutical composition is used in combination with a drug for treating diabetes. The drug for treating diabetes can be a drug that has been marketed, such as metformin, a GLP-1 receptor agonist or a GLP-1 analogue, and a sulfonylurea, including glimepiride, glibenclamide, gliclazide, glipizide, etc., an alpha-glucosidase inhibitor such as acarbose, and other drugs for treating diabetes that have been marketed or are under development.

[0027] In one embodiment, in the application of the present application, the NPQ fusion protein or the pharmaceutical composition is administered by parenteral, intravenous, subcutaneous, intramuscular, or the like.

[0028] In one embodiment, the NPQ fusion protein or the pharmaceutical composition is administered once every 1 day, once every 3 days, once a week, once every two weeks, or the like.

[0029] In another aspect, the present application provides a recombinant cell comprising a polynucleotide encoding the NPQ fusion protein of the present application, or comprising the polynucleotide of the present application, or comprising the vector of the present application.

[0030] In one embodiment, the recombinant cell of the present application is derived from a Chinese hamster ovary cell (CHO), in particular a CHO-S cell.

[0031] In another aspect, the present application provides a method for constructing a recombinant cell, comprising: introducing a polynucleotide encoding the NPQ fusion protein of the present application into a vector to construct an expression vector; and introducing the expression vector into a cell to obtain a recombinant cell.

[0032] In one embodiment, in the method for constructing a recombinant cell of the present application, the cell is a Chinese hamster ovary cell, in particular a CHO-S cell.

[0033] In one embodiment, the vector can be a pCDNA3.1 vector.

[0034] In one embodiment, the method for constructing a recombinant cell comprises the following steps:

[0035] inserting the polynucleotide sequence shown in SEQ ID NO: 7 into a pCDNA3.1 vector to generate a vector containing the polynucleotide sequence of the fusion protein;

[0036] introducing the expression vector into a CHO-S cell to obtain a recombinant cell.

[0037] The steps can further comprise screening cells in which the recombinant plasmid is stably integrated into the genome.

[0038] In still another aspect, the present application provides a method for producing the NPQ fusion protein of the present application, comprising the step of obtaining the NPQ fusion protein using the recombinant cell of the present application.

[0039] The NPQ(36-49)-IgG2 / Fc fusion protein of the present application has the ability to specifically bind to hepatocytes HEPG2 and insulin-secreting cells INS-1, and forms endocytosis after binding to the cells, and activates intracellular signaling pathways. At the same time, the NPQ(36-49)-IgG2 / Fc fusion protein of the present application, by multi-targeting effect including promoting hepatocyte glycolipid metabolism, improving pancreatic beta cell function, can be used for treating liver diseases including fatty liver, diabetes and metabolic syndrome and the like.

[0040] Unless otherwise expressly stated, ranges of values in the application file include any and all subranges of the values therein-stated and any and all values within the subranges. Unless otherwise expressly stated, values in the application file are presented only to the nearest 0.1 significant figure. Unless otherwise expressly stated, all numerical values in the application file (including the appended claims) are to be construed as modified by the term "about," even if the term "about" does not expressly appear before the numerical value. "About" means that the described value allows for slight imprecision (having a value that is approximately the same as that described; being within a range that is approximately the same as that described; being within a range that is slightly broader than that described; and / or being within a range that is slightly narrower than that described). If "about" is not expressly recited, it is not intended to be implied. "About" as used herein also includes a reasonable amount of deviation of the value as would be recognized by one of ordinary skill in the art. For example, "about" can include a deviation of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%.

[0041] The application has been described in detail by the foregoing embodiments, but the above-described embodiments are only illustrative in nature and are not intended to limit the application. Furthermore, the present application is not limited by any theory described in the foregoing prior art or summary or described in the following examples. BRIEF DESCRIPTION OF DRAWINGS

[0042] One or more embodiments are illustrated by way of example in the figures that form a part of this disclosure and which do not limit the scope of embodiments.

[0043] Figure 1 is a schematic diagram of pYN1.0 plasmid (NPQ-IgG2 / Fc-pCDNA3.1) construction.

[0044] FIG. 2A and FIG. 2B are NPQ(36-49)-IgG2 / Fc sequencing peak chart examples. Wherein: limited by the length, only show FIG. 2A(1-80) and FIG. 2B(81-210) sequencing peak chart as examples.

[0045] FIG. 3 is a chart showing SDS-PAGE electrophoresis results of detecting physicochemical characteristics of fusion protein by SDS-PAGE and Western Blot. The corresponding bands of NPQ(36-49)-IgG2 / Fc reduced and non-reduced samples, lane 1: protein marker; lane 2: ①NPQ(36-49)-IgG2 / Fc reduced sample; lane 3: ②NPQ(36-49)-IgG2 / Fc non-reduced sample.

[0046] FIG. 4 is a mass spectrum spectrum chart of NPQ sample desugared intact protein molecular weight deconvolution.

[0047] FIG. 5 is a mass spectrum spectrum chart of NPQ sample desugared reduced molecular weight deconvolution.

[0048] FIG. 6 is a mass spectrum spectrum chart of NPQ sample intact protein molecular weight deconvolution.

[0049] FIG. 7 is a mass spectrum spectrum chart of NPQ sample reduced molecular weight deconvolution.

[0050] FIG. 8 is a primary mass spectrum chart collected after Trypsin enzymolysis of NPQ sample.

[0051] FIG. 9 is a global chart of FLD chromatogram of YN108 N-glycoside.

[0052] FIG. 10 is an enlarged chart of FLD chromatogram of YN108 N-glycoside. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the embodiments of the present application will be described in detail below. However, those skilled in the art can understand that in the embodiments of the present application, many technical details are proposed in order to make the readers better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed by the present application can be realized.

[0054] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0055] I. Definitions or Terms

[0056] Unless otherwise defined, the terms defined herein and the terms used in the present application shall be understood as dictionary definitions, or definitions in incorporated documents, and / or commonly understood meanings of the defined terms.

[0057] All references, patents and patent applications referred to in this document are incorporated by reference herein in their entirety on the subject matter for which each is cited.

[0058] All features disclosed in this specification may be combined in any way. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly excluded herein, every combination of features is within the scope of the application.

[0059] As used herein, the terms "peptide," "polypeptide," and "protein" refer to a chain of two or more natural or non-natural amino acid residues joined together, whether or not post-translational modifications (e.g., glycosylation or phosphorylation) are present. A polypeptide in the present application can include, for example, 3 to 3500 natural or non-natural amino acid residues. It includes proteins of a single polypeptide chain and multi-subunit proteins (e.g., composed of 2 or more polypeptides).

[0060] In the present application, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue without eliminating the required properties of the protein. Suitable conservative amino acid substitutions can be made by replacing an amino acid with another amino acid having similar hydrophobicity, polarity, and R-chain length. Examples of conservative substitutions include replacing one non-polar (hydrophobic) residue for another (e.g., alanine, isoleucine, valine, leucine, or methionine), one polar (hydrophilic) residue for another (e.g., between arginine and lysine), between glutamine and asparagine, between glycine and serine, replacing one basic residue (e.g., lysine, arginine, or histidine) for another, or replacing one acidic residue (e.g., aspartic acid or glutamic acid) for another. The phrase "conservative substitution" also includes replacing a non-derivatized residue with a chemically derivatized residue or a non-natural amino acid, provided that such polypeptide exhibits the necessary activity.

[0061] In the present application, IgG2 / Fc comprises amino acid substitutions such as C222S substitution, A330S substitution, and / or P331S substitution, and unless otherwise specified herein, the amino acid residue numbers referred to in the present application are identified in the EU index system of Kabat et al. (1991).

[0062] In the present application, the term "fusion protein" refers to a protein comprising two or more polypeptides forming different functional domains. For example, the NPQ fusion protein described herein comprises an NPQ polypeptide and an immunoglobulin Fc domain.

[0063] In the present application, the term "linker peptide" refers to a preferably stretch of amino acids that connects different functional domains of a polypeptide together. Various linker peptides are contemplated and the linker peptide can have any suitable length and structure.

[0064] In the present application, the term "CH2" refers to constant heavy chain 2, which is one of the domains of an immunoglobulin heavy chain. Similarly, the term "CH3" refers to constant heavy chain 3, which is another domain of an immunoglobulin heavy chain.

[0065] In the present application, the term "hinge" when used in the context of IgG refers to the flexible region between the antigen binding fragment (Fab) and the crystallizable fragment (Fc).

[0066] As used herein, the term "vector" refers to a molecule used as a vehicle to introduce foreign DNA into a cell.

[0067] In the present application, the term "pharmaceutically acceptable carrier" refers to any carrier, reagent, excipient that is biologically or otherwise acceptable for use in the treatment of a subject. The use of such pharmaceutically acceptable carriers in the treatment of a subject is well known in the art.

[0068] Furthermore, terms such as "substantially", "approximately", and "about" as used herein represent a reasonable deviation from the recited quantity so that the end result is essentially the same. These terms are to be interpreted to include at least ±5% deviation from the modifier that they modify, unless otherwise indicated.

[0069] More specifically, the term "about" refers to ±0.1 to 25%, 1-20%, or 1-150%, 1-10%, e.g. up to 10%, up to 5% of the referenced number.

[0070] The term "comprising", as used herein, and its derivatives, is an open term that is intended to mean the inclusion of one or more of the specified features, elements, components, groups, integers, and / or steps, but not excluding other unspecified features, elements, components, groups, integers, and / or steps. The above specification is to be construed in accordance with the following patent rules and regulations.

[0071] Example 1 Preparation of NPQ fusion proteins

[0072] Construction of vectors:

[0073] The cDNA nucleic acid sequence (SEQ ID NO: 7) encoding the fusion protein comprising a signal peptide part, NPQ and hIgG2 / Fc was inserted into the transcription promoter of the eukaryotic expression vector pCDNA3.1 (purchased from Shengong Bioengineering (Shanghai) Co., Ltd.) by using the chemical synthesis method to construct the NPQ(36-49)-IgG2 / Fc expression vector pYN1.0 plasmid (as shown in FIG. 1). In the cDNA sequence, the signal peptide is the secretion leader peptide sequence of the human CD33 gene; the NPQ part comprises the complete human NPQ sequence expressed and secreted by cells; and the hIgG2 / Fc region comprises the hinge, CH2 and CH3 regions in the human IgG2 heavy chain. The signal peptide guides the expression and secretion of the synthesized NPQ(36-49)-IgG2 / Fc fusion protein into the extracellular culture medium. The NPQ(36-49)-IgG2 / Fc fusion protein has the characteristics of forming dimers on the cell surface in advance, so that the binding of the polypeptide to its receptor will be more effective; and it is beneficial to the purification process in production, and can be purified by protein A agarose gel in one step.

[0074] Amplification of the plasmid vector:

[0075] The pYN1.0 plasmid containing the DNA sequence of NPQ(36-49)-IgG2 / Fc was heat-shocked in DH5a competent cells, cultured on a plate containing kanamycin to form single colonies; single colonies were picked and gradually expanded to 100 mL. After overnight culture at 37°C on a shaker at 220 rpm, the cells were centrifuged and the plasmid was extracted to obtain high-concentration plasmid.

[0076] DNA sequence identification and verification

[0077] The recombinant plasmid containing the target gene was sequenced using the first-generation sequencing technology Sanger sequencing, and the sequencing map is shown in FIGS. 2A and 2B. The sequencing results were accurately matched with the NPQ(36-49)-IgG2 / Fc sequence.

[0078] Expression of NPQ fusion protein:

[0079] To establish CHO-S cells stably expressing NPQ(36-49)-IgG2 / Fc, 10 7A CHO-S cell was electrotransformed with 30 μg of linearized NPQ(36-49)-IgG2 / Fc and cultured in Dynamis medium containing MTX (0.5 μg / ml) and purimycin (500 nmol / L) to select cells in which the recombinant plasmid had stably integrated into the genome. During the culture, the medium was changed every 3 days until cell clones were formed. Single clones were isolated and expanded into stable cell lines and these cell lines were grown in tissue culture dot blots to detect the fusion protein. Cells that secreted the fusion protein were selected for further characterization.

[0080] Purification of NPQ(36-49)-IgG2 / Fc fusion protein:

[0081] Protein samples were purified using an AKTA pure protein purification system. Culture medium from transfected cells (typically 30 mL from a 125 mL flask) was loaded onto a 20 mL protein A affinity column pre-equilibrated with a buffer containing 150 mM NaCl, 25 mM Tris pH 7.0, washed with a buffer containing 0.5 M NaCl, 50 mM citric acid / sodium citrate pH 6.0 and then the protein was eluted from the affinity column using a buffer containing 0.1 M citric acid / sodium citrate, pH 3.0. The components of the sample were neutralized with 1 M Tris and assessed for yield and purity by SDS PAGE and visualized by Coomassie Brilliant Blue staining. The results showed that 1 x 10 7 After 14 days of cell culture, the yield of the fusion protein was about 2.8 mg / mL, indicating that the production of the fusion protein was high and could be produced on a large scale.

[0082] Example 2 Identification of the physicochemical characteristics of the NPQ fusion protein

[0083] The physicochemical characteristics of the NPQ fusion protein were identified using SDS-PAGE and Western Blot methods. 10 μg of NPQ(36-49)-IgG2 / Fc protein was used to prepare a reduced (containing 4% β mercaptoethanol) sample and a non-reduced sample for SDS-PAGE electrophoresis. One of the gels was transferred to a membrane using Coomassie Brilliant Blue. The membrane was blocked with PBS containing 1% bovine serum albumin for 1 h to prevent non-specific binding. The membrane was washed with PBS 3 times for 15 minutes each time. An hFc antibody (Jackson lab, 209-005-098) and an NPQ antibody (G-bioscience; INT2475) were incubated for 1 h. After the primary antibody reaction, the membrane was washed with PBS 3 times. The membrane was reacted with a secondary antibody conjugated with HRP for 1 h, washed with PBS 3 times and developed using ECL developing solution for Western Blot development.

[0084] As shown in Figure 3, SDS-PAGE electrophoresis results show corresponding bands of NPQ(36-49)-IgG2 / Fc reduced and non-reduced samples.

[0085] Figure legend: Lane 1: protein marker; Lane 2: ① NPQ(36-49)-IgG2 / Fc reduced sample; Lane 3: ② NPQ(36-49)-IgG2 / Fc non-reduced sample.

[0086] Western Blot results show corresponding bands of NPQ(36-49)-IgG2 / Fc.

[0087] Example 3 NPQ fusion protein mass spectrometry identification

[0088] 3.1 Detection of deglycosylated intact molecular weight The measured deglycosylated intact molecular weight is consistent with the theoretical molecular weight

[0089] After sample dilution, PNGaseF enzyme was used for deglycosylation treatment. Data acquisition was performed by Waters / ACQUITY Premier UPLC connected with Thermo / QE Plus, and then the original data was analyzed by Biopharma Finder 4.1 software. The measured molecular weight obtained by analysis was compared with the theoretical molecular weight and the consistency was confirmed.

[0090] The main component detected in the deglycosylated intact molecular weight detection of NPQ sample is the deglycosylated protein molecule (see Figure 4), and the measured deglycosylated intact molecular weight is consistent with the theoretical molecular weight, with a difference within 100.0 ppm, as shown in Table 1.

[0091] Table 1 Measured deglycosylated intact protein molecular weight and theoretical molecular weight of NPQ sample

[0092] 3.2 NPQ deglycosylated reduced molecular weight detection shows that the measured deglycosylated reduced molecular weight is consistent with the theoretical molecular weight

[0093] After sample dilution, PNGaseF enzyme was used for deglycosylation treatment. The deglycosylated protein was partially reduced by DTT under non-denaturing conditions. Data acquisition was performed by Waters / ACQUITY Premier UPLC connected with Thermo / QE Plus, and then the original data was analyzed by Biopharma Finder 4.1 software. The measured molecular weight obtained by analysis was compared with the theoretical molecular weight and the consistency was confirmed.

[0094] The main component detected in the deglycosylated reduced molecular weight detection of NPQ sample is the deglycosylated single chain (see Figure 5), and the measured deglycosylated reduced molecular weight is consistent with the theoretical molecular weight, with a difference within 100.0 ppm, as shown in Table 2.

[0095] Table 2 List of de-glycosylated reduced protein molecular weight information of NPQ sample

[0096] 3.3 NPQ intact molecular weight detection shows that the measured molecular weights are consistent with the theoretical molecular weights

[0097] For the intact molecular weight of NPQ sample, the main component detected corresponds to NPQ intact protein with G0F glycosylation modification on both SC chains, in addition to other various glycosylation modifications. The measured molecular weights of each component of the sample are consistent with the theoretical molecular weights, with a difference of within 100.0 ppm. The deconvoluted intact molecular weight of NPQ sample is shown in Figure 6. The detailed molecular weight information measured is shown in Table 3.

[0098] Table 3 Measured intact protein molecular weight and theoretical molecular weight of NPQ sample

[0099] 3.4 NPQ reduced molecular weight detection shows that the measured molecular weights are consistent with the theoretical molecular weights

[0100] After sample dilution, partial reduction under non-denaturing conditions by DTT, data acquisition by Waters / ACQUITY Premier UPLC coupled with Thermo / QE Plus, and then analysis of raw data by Biopharma Finder 4.1 software. The measured molecular weights obtained by analysis are compared with the theoretical molecular weights and confirmed to be consistent.

[0101] For the reduced molecular weight of NPQ, the main component detected corresponds to the molecular weight of single chain with G0F glycosylation modification. The measured molecular weights of each component of the sample are consistent with the theoretical molecular weights, with a difference of within 100.0 ppm. The results of reduced molecular weight detection of NPQ sample are shown in Figure 7. The detailed molecular weight information measured is shown in Table 4.

[0102] Table 4: Information table of measured reduced molecular weight and theoretical molecular weight of NPQ sample

[0103] 3.5 Amino acid sequence analysis results show that the NPQ amino acid sequence coverage is 91.7%

[0104] The NPQ deglycosylated and non-deglycosylated samples were denatured, reduced and alkylated, then digested by Trypsin, and the digested peptides were subjected to data acquisition by Waters / ACQUITY Premier UPLC coupled with Thermo / QE Plus. Then, by using Biopharma Finder 4.1 software in combination with the theoretical protein sequence information, the mass spectrometry spectrum was analyzed for the accurate molecular weight information of the primary mass spectrometry, the secondary mass spectrometry fragment ions and the matching information of the post-translational modification sites, and manual confirmation was performed. The data of the Trypsin digestion experiment sample was analyzed and attributed to determine the theoretical sequence coverage of the amino acids, and to confirm whether the measured amino acid sequence of the sample is consistent with the theoretical sequence.

[0105] The separation of the peptide segments of the digested sample is shown in FIG. 8. By comparing the accurate molecular weight information of the primary mass spectrometry and the secondary mass spectrometry fragment ion data of the measured peptide segments with the theoretical peptide segment information, the sequence coverage of NPQ reached 91.7%. Some short peptides were difficult to retain on the reversed-phase chromatography due to strong hydrophilicity, and were not present in the mass spectrometry detection results. The results are shown in Table 5, the detected digested peptide sequences are consistent with the theoretical sequences, and the molecular weight error is within 10.0 ppm.

[0106] Table 5 NPQ sample amino acid sequence coverage results

[0107] 3.6 Mass spectrometry analysis of post-translational modification of amino acids

[0108] After denaturation, reduction and alkylation of the sample, the sample was digested by Trypsin, and the sample was subjected to data acquisition by Waters / ACQUITY Premier UPLC coupled with Thermo / QE Plus in combination with the theoretical amino acid sequence of YN-108. By using BioPharma Finder software, the mass spectrometry spectrum was analyzed for the accurate molecular weight information of the primary mass spectrometry, the secondary mass spectrometry fragment ions and the matching information of the theoretical sequence and the post-translational modification sites, and manual confirmation was performed. The proportion of post-translational modification was quantified by extracting the ion chromatogram (SIC).

[0109] The primary mass spectrometry spectrum of the YN-108 sample digested by Trypsin is shown in FIG. 8, and the peptide segments are well separated. Table 6 shows the post-translational modification sites and proportion information with a proportion higher than 1.0%. The YN-108 sample molecules mainly undergo N-glycosylation modification, and also undergo a small amount of lysine hydroxylation, methionine oxidation, O-glycosylation, aspartic acid isomerization, deamidation of asparagine and succinimidation of asparagine modification, etc. The N in the contained amino acid sequence SEQ ID NO: 2 becomes D after digestion.

[0110] Table 6. Post-translational modification results of YN-108 sample tryptic peptides digested with deglycosylating trypsin

[0111] Table 6 (continued)

[0112] 3.7 N-glycan analysis

[0113] Glycosylation modification affects the stability, bioactivity, solubility, in vivo half-life and immunogenicity of therapeutic proteins to some extent. N-glycan analysis can characterize the main N-glycan types on the protein, which has certain guiding significance for the safety and efficacy of therapeutic proteins.

[0114] N-glycan analysis of YN108 separates N-glycan on glycoprotein from protein by PNGase F enzyme, adds ice alcohol to precipitate protein, takes the supernatant containing N-glycan after centrifugation, labels it with fluorescent reagent 2-AB after drying, separates the labeled sample by hydrophilic interaction chromatography, detects the peaks on the fluorescence detector, and finally uses area normalization method for relative quantification of each glycan type. The FLD chromatogram of N-glycan in YN108 sample is shown in Figure 9 and Figure 10, and the detailed analysis results are shown in Table 7. The results show that the main N-glycan types in YN108 sample are G0F and G1F.

[0115] Table 7. Relative content of N-glycan in YN108 sample

[0116] Example 4. Determination of affinity of NPQ fusion protein to receptor

[0117] ELISA technology was used to determine the affinity of NPQ fusion protein to receptor. INS-1 and HEPG2 cells growing in 96-well plates (BD Biosciences) were washed with PBS and fixed with 4% paraformaldehyde (Thermo Scientific) at room temperature for 10 minutes, and then quenched in a PBS solution containing 2% glycine (pH 7.5) for 5 minutes. In the binding capacity experiment, cells were incubated with logarithmically diluted NPQ(36-49)-IgG2 / Fc fusion protein (1 x 10 -5 to 1 x 10 -12 M) alone or with 10 mM NPQ (Abcam, USA) to detect total and non-specific binding, respectively. In the competitive binding experiment, 10 mM fixed concentration of NPQ(36-49)-IgG2 / Fc fusion protein was used, and different concentrations of NPQ (1 x 10 -5 to 1 x 10 -12M) Competitive binding. After incubation at 4°C for 4 hours in a final volume of 100 μl, cells were washed to remove excess NPQ and NPQ(36-49)-IgG2 / Fc fusion protein, and blocked with 5% BSA (BD Biosciences). Bound NPQ(36-49)-IgG2 / Fc was detected with goat anti-human IgG Fc antibody (1 :4000, Southern Biotech) and HRP-conjugated donkey anti-goat IgG (1 :5000, Jackson ImmunoResearch).

[0118] The enzymatic reaction was initiated by the addition of o-phenylenediamine (OPD) (Fisher Scientific) and the colorimetric change was analyzed by reading the absorbance at 490 nm on a Beckman microplate reader. The results of the experiment showed that the NPQ fusion protein bound well to the receptor in both INS-1 and HEPG2 cells.

[0119] Example 5 Effect of NPQ fusion protein on signaling pathways in INS-1 cells

[0120] INS-1 cells were washed with PBS and lysed in lysis buffer containing 0.1% protease inhibitors. The lysate was then incubated on ice for 30 minutes and centrifuged at 8000 g for 10 minutes. Cell samples and an equal amount of protein GAPDH were run on SDS-PAGE and transferred to a polyvinylidene difluoride membrane, which was immunoblotted with the following primary antibodies: anti-CREB antibody (#9197); anti-phospho-CREB antibody (Ser 133) (#9198); anti-Akt antibody (#2920); anti-phospho-Akt (Ser 473) antibody (#4060), all purchased from Cell Signaling Technology; the membrane was washed with PBS-Tween-20 and incubated with a peroxidase-conjugated secondary antibody. Protein bands were detected using an ECL Plus kit.

[0121] Example 6 Effect of NPQ fusion protein on the secretory function of pancreatic islet cells

[0122] INS-1 cells were seeded in 24-well plates at a density of 2.5 x 10 5 cells per well and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). The next day, fresh KRB (128.8 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, 0.1% BSA, 0.01% sodium azide, pH 7.4) without glucose was used. SO4, 1.0 mM CaCl2, 5.0 mM HEPES, 0.1% fetal bovine serum, 2.8 mM glucose) for 120 minutes, and then treated with 2.8 mM or 16.8 mM glucose and various concentrations of purified NPQ(36-49)-IgG2 / Fc fusion protein for 2 hours. Insulin levels in the culture medium were measured using a rat insulin RIA kit (Linco, St Charles, MO, USA) according to the manufacturer's instructions.

[0123] The results of the experiment show that the NPQ fusion protein promotes insulin secretion.

[0124] Example 7 Effect of NPQ fusion protein on triglyceride levels

[0125] The effect of NPQ fusion protein on triglyceride levels was tested on HepG2 cells. HepG2 cells were grown in 24-well plates and incubated with 1-100 μg / mL (final concentration) of NPQ(36-49)-IgG2 / Fc for 1 hour, then incubated with a final concentration of 50-300 μM palmitic acid, washed 3 times with phosphate buffered saline, and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The fixed cells were washed with deionized water, soaked in 60% isopropanol for 3 minutes, and stained with 2 mg / mL Oil Red O stain for 60 minutes, then washed 3 times with deionized water to remove unbound stain. The nuclei were stained with hematoxylin for 3 minutes and washed with deionized water. After microscopic examination, the Oil Red-based triglyceride content in each well was quantified.

[0126] The results of the experiment show that the NPQ fusion protein effectively reduces triglyceride levels.

[0127] Table 8 Substitution sites

[0128] SEQUENCE LISTING:

[0129] The foregoing illustration, while indicating presently preferred examples of the application, should not be construed to limit the application to the examples disclosed. Rather, the application is intended to cover any modifications and equivalents within the spirit and scope of the claims appended hereto.

[0130] All publications, patents, and patent applications are herein incorporated by reference in their entirety. In particular, the sequences associated with each accession number provided herein, including, for example, the accession numbers and / or biomarker sequences (e.g., proteins and / or polynucleotides) provided in the tables or elsewhere, are incorporated by reference in their entirety.

[0131] The scope of the claims should not be limited to the preferred embodiments and examples described herein but should be given the broadest interpretation consistent with the specification as a whole.

Claims

1. A NPQ fusion protein, characterized in that, The NPQ fusion protein comprises an NPQ polypeptide and an immunoglobulin Fc domain, wherein the NPQ polypeptide is human NPQ, the NPQ polypeptide is covalently linked to the immunoglobulin Fc domain, the immunoglobulin Fc domain is an IgG2 / Fc polypeptide, the NPQ polypeptide is an amino acid sequence set forth in SEQ ID NO: 2, and the IgG2 / Fc polypeptide is an amino acid sequence set forth in SEQ ID NO: 4; the NPQ fusion protein further comprises a linker peptide for linking the NPQ polypeptide and the immunoglobulin Fc domain, the linker peptide is an amino acid sequence set forth in SEQ ID NO: 8, and the NPQ fusion protein comprises an amino acid sequence set forth in SEQ ID NO:

5.

2. The NPQ fusion protein according to claim 1, characterized in that, The NPQ fusion protein is further modified, and the modification comprises N-glycosylation or O-glycosylation or oxidation or succinimidation of asparagine or deamidation of asparagine or hydroxylation modification.

3. The NPQ fusion protein according to claim 1 or 2, characterized in that, The NPQ fusion protein further comprises a signal peptide, and the signal peptide is a human CD33 signal peptide or the signal peptide has an amino acid sequence of SEQ ID NO:

6.

4. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1 or 2. The polynucleotide comprises a polynucleotide encoding the NPQ fusion protein of any one of claims 1-3.

5. A vector, characterized in that, The vector comprises the polynucleotide of claim 4.

6. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the NPQ fusion protein of any one of claims 1-3, or the polynucleotide of claim 4, or the vector of claim 5, and optionally a pharmaceutically acceptable carrier.

7. Use of the NPQ fusion protein of any one of claims 1-3, the polynucleotide of claim 4, the vector of claim 5, or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating or preventing a metabolic disease associated with glucose metabolism or lipid metabolism disorder.

8. Use according to claim 7, characterized in that, The metabolic disease associated with glucose metabolism or lipid metabolism disorder is selected from diabetes, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and obesity.

9. A recombinant cell, characterized in that, The cell comprises the polynucleotide of claim 4 or comprises the vector of claim 5.

10. The recombinant cell of claim 9, wherein, The recombinant cell is obtained from a CHO cell.

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