An NPQ-IgG2 / Fc fusion protein and its application

Abstract

This application provides an NPQ-IgG2 / Fc fusion protein and its applications. The fusion protein comprises an NPQ polypeptide and a human IgG2 / Fc region. The NPQ-IgG2 / Fc fusion protein has the ability to specifically bind to hepatocyte HEPG2 and insulin-secreting cells INS-1, and after binding to cells, it undergoes endocytosis, activating intracellular signaling pathways. This application also discloses that the NPQ-IgG2 / Fc fusion protein, through multi-target effects including promoting hepatocyte glucose and lipid metabolism and improving pancreatic β-cell function, can be used to treat liver diseases including fatty liver, diabetes, and other metabolic diseases related to glucose or lipid metabolism disorders.

Description

Technical Field

[0001] This invention relates to an NPQ-IgG2 / Fc fusion protein produced using recombinant genetic engineering technology and its applications. The fusion protein comprises an NPQ polypeptide and an IgG2 / Fc polypeptide. The NPQ-IgG2 / Fc fusion protein has the ability to specifically bind to HEPG2 hepatocytes and INS-1 insulin-secreting cells, and after binding, it undergoes endocytosis, activating intracellular signaling pathways. This application also discloses that the NPQ-IgG2 / Fc fusion protein, through multi-target effects including promoting hepatocyte glucose and lipid metabolism and improving pancreatic β-cell function, can be used to treat liver diseases including fatty liver, diabetes, and metabolic syndrome. Background Technology

[0002] Neuropeptide Q (NPQ), also known as Spexin, is a peptide hormone discovered in 2007 that is widely expressed in endocrine organs and epithelial tissues. The mature NPQ peptide consists of 14 amino acid residues and acts as a natural ligand, binding to galanin receptor 2 / 3 (GALR2 / 3) on the cell surface. It activates multiple downstream signaling pathways, stimulates insulin secretion, improves insulin resistance, regulates carbohydrate and lipid metabolism homeostasis, and affects various physiological functions, including food intake and energy balance. Furthermore, NPQ may participate in regulating the proliferation of adrenal cortical cells and influence the functions of the cardiovascular, renal, and endocrine systems. NPQ is associated with many diseases, including obesity, type 2 diabetes, fatty liver (Türkel, İbrahim et al. “Impact of spexin on metabolic diseases and inflammation: Anupdated minireview.” Experimental biology and medicine (Maywood, NJ) vol.247,7 (2022): 567-573.), and mental illnesses such as anxiety and depression (Lv, Shuang-Yu et al. “Emerging Roles of NPQ / Spexin in Physiology and Pathology.” Frontiers inpharmacology vol. 10 457. 7 May. 2019).

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

[0004] Therefore, NPQ may be a potential drug for treating diseases related to abnormal glucose and lipid metabolism. However, mature NPQ contains only 14 amino acids, is easily degraded by enzymes, and has a short half-life in vivo (about 1 hour), which limits its clinical application. Therefore, there is an urgent need to find NPQ analogs with better efficacy and metabolic properties. Summary of the Invention

[0005] On one hand, the present invention provides an NPQ fusion protein comprising an NPQ polypeptide and an immunoglobulin Fc domain, wherein the NPQ polypeptide is selected from human NPQ and mouse 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 the 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, and the IgG2 / Fc polypeptide has the 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 having an amino acid sequence selected from SEQ ID NO: 8.

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

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

[0008] In one embodiment, the linker peptide further has any amino acid sequence selected from 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 invention has the amino acid sequence of SEQ ID NO: 5.

[0010] In one embodiment, the NPQ fusion protein of the present invention further comprises a signal peptide. A signal peptide is a peptide chain of approximately 5-30 amino acids in length, following a start codon and encoding a hydrophobic amino acid sequence, responsible for guiding the protein into intracellular structures or guiding the transfer of newly synthesized proteins to secretory pathways. The present invention uses various forms of signal peptides. In one embodiment, the presented signal peptide includes, but is not limited to, the human CD33 signal peptide, or the signal peptide having the amino acid sequence SEQ ID NO: 6.

[0011] In one embodiment, the NPQ fusion protein of the present invention may further include an enzyme cleavage site.

[0012] In one embodiment, the NPQ fusion protein of the present invention may be further modified, including one or more modifications such as lysine hydroxylation, methionine oxidation, O-glycosylation (GalNAc), or aspartic acid isomerization.

[0013] In one embodiment, in the NPQ fusion protein of the present invention, the N-glycosylation modification has an N-glycoside type of GOF and G1F, preferably GOF.

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

[0015] On the other hand, the present invention provides a polynucleotide comprising a polynucleotide encoding the NPQ fusion protein of the present invention.

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

[0017] In another aspect, the present invention provides a carrier comprising the polynucleotide of the present invention.

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

[0019] In one embodiment, the composition further comprises a buffer solution, 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, poloxamer, lecithin, polyethylene glycol 15-hydroxystearate, cyclodextrins, etc.

[0021] In one embodiment, the composition further comprises excipients. In one embodiment, the excipients may include mannitol, sorbitol, maltitol, erythritol, arabinitol, xylitol, sucrose, lactose, trehalose, dextran, or mixtures thereof. In some embodiments, the excipients may be one or more selected from mannitol, sucrose, and sorbitol, preferably mannitol or sucrose.

[0022] In one embodiment, the composition comprises an NPQ fusion protein, formulated into a dosage form, and administered in doses 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 ... NPQ fusion protein in mg, approximately 17 mg, approximately 18 mg, approximately 19 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, and approximately 40 mg.

[0023] Furthermore, this invention provides the application of the NPQ fusion protein, the polynucleotide, the carrier, and the pharmaceutical composition of this invention in the preparation of a drug, which is a drug for treating or preventing metabolic diseases related to disorders of glucose or lipid metabolism. The pharmaceutical composition may include fusion proteins, polynucleotides, carriers, and cells, and is used for gene therapy.

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

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

[0026] In one embodiment, in the application of the present invention, the NPQ fusion protein, the polynucleotide, the carrier, or the pharmaceutical composition is used in combination with a drug for treating diabetes. The drug for treating diabetes may be a currently marketed drug, such as metformin, GLP-1 receptor agonists or GLP-1 analogs, and sulfonylureas, mainly including glimepiride, glibenclamide, gliclazide, gliquidone, etc., α-glucosidase inhibitors such as acarbose, and other marketed and under-development drugs for treating diabetes.

[0027] In one embodiment of the invention, the NPQ fusion protein or the pharmaceutical composition is administered via parenteral, intravenous, subcutaneous, or intramuscular routes.

[0028] In one embodiment, the fusion protein or its composition may be administered once a day, once every three days, once a week, or once every two weeks, etc.

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

[0030] In one embodiment, the recombinant cells of the present invention are obtained from Chinese hamster ovary cells (CHO), particularly CHO-S cells.

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

[0032] In one embodiment, the recombinant cell construction method of the present invention uses Chinese hamster ovary cells, particularly CHOS cells.

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

[0034] In one embodiment, the method for constructing the recombinant cells includes the following steps:

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

[0036] The expression vector was introduced into CHO-S cells to obtain recombinant cells.

[0037] The steps may also include screening cells in which the recombinant plasmid is stably integrated into the genome.

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

[0039] The NPQ(36-49)-IgG2 / Fc fusion protein of this invention has the ability to specifically bind to HEPG2 hepatocytes and INS-1 insulin-secreting cells, and after binding to cells, it undergoes endocytosis, activating intracellular signaling pathways. Simultaneously, the NPQ(36-49)-IgG2 / Fc fusion protein of this application, through multi-target effects including promoting hepatocyte glucose and lipid metabolism and improving pancreatic β-cell function, can be used to treat liver diseases including fatty liver, diabetes, and metabolic syndrome.

[0040] Unless otherwise expressly stated, numerical ranges throughout the application include any subranges therein and any numerical values ​​incremented by the smallest subunit of a given value. Unless otherwise expressly stated, numerical values ​​throughout the application represent an approximate measure or limitation of the range of embodiments including minor deviations from a given value and having approximately the mentioned value as well as having the mentioned precise value. Except in the detailed description of the working embodiments provided at the end, all numerical values ​​of parameters (e.g., quantities or conditions) in this application (including the appended claims) should in all cases be understood to be modified by the term “approximately,” regardless of whether “approximately” actually precedes the numerical value. “Approximately” indicates that the stated numerical value allows for slight inaccuracies (some close to precision at that value; approximately or reasonably close to the value; approximate). If the inaccuracy provided by “approximately” is not understood in this common sense in the art, then “approximately” as used herein at least indicates a variation that can be produced by common methods of measuring and using these parameters. For example, “approximately” can include variations 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 present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments. Attached Figure Description

[0042] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

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

[0044] Figure 2A and Figure 2B This is an example of an NPQ(36-49)-IgG2 / Fc sequencing peak diagram. Note: Due to space limitations, only [the following is shown]. Figure 2A (1-80) and Figure 2B (81-210) Sequencing peak diagrams are used as examples.

[0045] Figure 3 This is a graph showing the SDS-PAGE electrophoresis results of SDS-PAGE and Western Blot detection of the physicochemical characteristics of the fusion protein. The corresponding bands for NPQ(36-49)-IgG2 / Fc reduced and non-reduced samples are shown. 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.

[0046] Figure 4 This is the deconvolutioned mass spectrum of the intact protein molecules from the NPQ sample after deglycosylation.

[0047] Figure 5 This is the mass spectrum of the NPQ sample after desugaring, molecular weight reduction, and deconvolution.

[0048] Figure 6 This is the deconvolutioned mass spectrum of the complete protein molecular weight of the NPQ sample.

[0049] Figure 7 This is the mass spectrum of the NPQ sample after deconvolution of molecular weight reduction.

[0050] Figure 8 This is a primary mass spectrum of the NPQ sample after Trypsin digestion.

[0051] Figure 9 This is a global FLD chromatogram of YN108 N-glycoside.

[0052] Figure 10 This is an enlarged FLD chromatogram of YN108 N-glycoside. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the various embodiments of the present invention will be described in detail below with reference to the embodiments. However, those skilled in the art will understand that many technical details have been presented in the various embodiments of the present invention to enable the reader to better understand this application. However, the technical solutions claimed in this application can be implemented even without these technical details and various changes and modifications based on the following embodiments.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] I. Definitions or Terms

[0056] Unless otherwise stated, the terms defined and used herein should be understood as dictionary definitions, or definitions in incorporated documents, and / or the well-known meanings of the defined terms.

[0057] All references, patents, and patent applications mentioned in this article are incorporated into their respective subjects by way of citation, and in some cases, the entire contents of the documents may be covered.

[0058] All features disclosed in this specification can be combined in any way. Each feature disclosed in this specification can be used to replace alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a series of equivalent or similar features.

[0059] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to an amino acid chain formed by the linkage of two or more natural or non-natural amino acid residues, regardless of the presence of post-translational modifications (e.g., glycosylation or phosphorylation). Polypeptides in this invention may comprise, for example, 3 to 3500 natural or non-natural amino acid residues. They include proteins with a single polypeptide chain and multi-subunit proteins (e.g., composed of two or more polypeptides).

[0060] In this invention, a “conservative amino acid substitution” is a substitution in which one amino acid residue is replaced by another amino acid residue without eliminating the desired properties of the protein. Suitable conservative amino acid substitutions can be performed by substituting amino acids with similar hydrophobicity, polarity, and R-chain length. Examples of conservative substitutions include replacing one nonpolar (hydrophobic) residue with another (e.g., alanine, isoleucine, valine, leucine, or methionine), replacing one polar (hydrophilic) residue with another (e.g., between arginine and lysine), between glutamine and asparagine, between glycine and serine, replacing one basic residue with another (e.g., lysine, arginine, or histidine), or replacing one acidic residue with another (e.g., aspartic acid or glutamic acid). The phrase “conservative substitution” also includes replacing non-derived residues with chemically derived residues or non-natural amino acids, provided that the polypeptide exhibits the necessary activity.

[0061] In this invention, the amino acid substitutions contained in IgG2 / Fc, such as C222S substitution, A330S substitution, and / or P331S substitution, are identified by the EU indexing system of Kabat et al. (1991) unless otherwise stated herein.

[0062] In this invention, 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 this invention, the term "linking peptide" refers to a preferred segment of amino acids that links different functional domains of a polypeptide together. Various linking peptides are considered, and linking peptides can have any suitable length and structure.

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

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

[0066] As used in this article, the term "vector" refers to a molecule used as a carrier to introduce foreign DNA into a cell.

[0067] In this invention, the term "pharmaceutically acceptable carrier" refers to any carrier, reagent, or excipient that is biologically or otherwise acceptable. Its use in therapeutic formulations is acceptable unless the carrier, reagent, or excipient is incompatible with the active ingredient. The use of such pharmaceutically acceptable carriers is well known in the art.

[0068] Furthermore, degree terms such as “basically,” “approximately,” and “roughly” as used herein indicate a reasonable amount of deviation from the modified terms such that the final result is not significantly altered. These degree terms should be interpreted as including at least ±5% deviation from the modifier if such deviation does not negate the meaning of the modifier.

[0069] More specifically, the term “about” refers to a range of plus or minus 0.1% to 25%, 1-20%, or 1-150%, 1-10%, such as a maximum of 10% or a maximum of 5%.

[0070] In understanding the scope of this invention, the term "comprising" and its derivatives as used herein are open-ended terms that specify the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The foregoing also applies to words with similar meanings, such as the terms "comprising," "having," and their derivatives.

[0071] Example 1: Preparation of NPQ fusion protein

[0072] Carrier construction:

[0073] A cDNA sequence (SEQ ID NO: 7) encoding a signal peptide, NPQ, and hIgG2 / Fc was inserted downstream of the transcription promoter of the eukaryotic expression vector pCDNA3.1 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) using chemical synthesis methods to construct the NPQ(36-49)-IgG2 / Fc expression vector pYN1.0 plasmid (e.g., ...). Figure 1 (As shown). In the cDNA sequence, the signal peptide is the secretion leader peptide sequence of the human CD33 gene; the NPQ portion contains the complete human NPQ sequence expressed and secreted by the cell; the hIgG2 / Fc region contains the hinge, CH2, and CH3 regions of the human IgG2 heavy chain. The NPQ(36-49)-IgG2 / Fc fusion protein synthesized under the guidance of the signal peptide is expressed and secreted into the extracellular culture medium. The characteristic of the NPQ(36-49)-IgG2 / Fc fusion protein is that it pre-forms a dimer on the cell surface, thus the binding of the polypeptide to its receptor will be more effective; 2) It is beneficial to the purification process in production, and can be purified in one step by protein A agarose gel.

[0074] Amplification of plasmid vectors:

[0075] The pYN1.0 plasmid containing the DNA sequence NPQ(36-49)-IgG2 / Fc was heat-transformed into DH5α competent cells and cultured on a plate containing kanamycin to form single colonies. Single colonies were picked and cultured stepwise to 100 mL, cultured overnight at 37°C and 220 rpm, and then centrifuged to extract plasmids to obtain high-concentration plasmids.

[0076] DNA sequence identification and verification

[0077] Sanger sequencing, a first-generation sequencing technology, was used to sequence the recombinant plasmid containing the target gene. The sequencing map is shown below. Figure 2A and Figure 2B As shown, the sequencing results are accurate and consistent with the NPQ(36-49)-IgG2 / Fc sequence.

[0078] Expression of the NPQ fusion protein:

[0079] To establish CHO-S cells stably expressing NPQ(36-49)-IgG2 / Fc, 10 7 CHO-S cells were electroporated 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 screen for cells that had stably integrated the recombinant plasmid into their genome. The culture medium was changed every 3 days until cell clones formed. Single-clonal cells were isolated and expanded into stable cell lines, and dot blot analysis of tissue cultures grown from these cell lines in 24-well plates was performed to detect the fusion protein. Cells that secreted the fusion protein were selected for subsequent characterization analysis.

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

[0081] Protein samples were purified using the AKTA Pure protein purifier. Transfected cell culture medium (typically 30 mL in a 125 mL flask) was loaded onto a 20 mL protein A affinity chromatography column pre-equilibrated with a buffer containing 150 mM NaCl, 25 mM Tris, and pH 7.0. The column was washed with a buffer containing 0.5 M NaCl, 50 mM citrate / sodium citrate, and pH 6.0, and then eluted with a buffer containing 0.1 M citrate / sodium citrate, pH 3.0. The fractions were neutralized with 1 M Tris, and the results were evaluated by SDS-PAGE followed by Coomassie Brilliant Blue staining to assess yield and purification efficiency. Results showed that 1 × 10⁶ cells per 30 mL of culture medium were optimal. 7After 14 days of cell culture, the yield of the fusion protein was approximately 2.8 mg / mL, indicating a high yield of the fusion protein, which is suitable for large-scale production.

[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. 10 μg of NPQ(36-49)-IgG2 / Fc protein was used to prepare reduced (containing 4% β-mercaptoethanol) and non-reduced samples, respectively, for SDS-PAGE electrophoresis. One gel was transferred using Coomassie Brilliant Blue. The gel was blocked with PBS containing 1% bovine serum albumin for 1 h to prevent non-specific binding. The gel was washed three times with PBS for 15 minutes each time. The gel was incubated with hFc antibody (Jacksonlab, 209-005-098) and NPQ antibody (G-bioscience; INT2475) for 1 h, followed by washing three times with PBS. The gel was then reacted with HRP-conjugated secondary antibody for 1 h, washed three times with PBS, and finally subjected to Western Blot development using ECL.

[0084] like Figure 3 As shown, the SDS-PAGE electrophoresis results show the 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 showed the presence of corresponding bands for NPQ(36-49)-IgG2 / Fc.

[0087] Example 3: Identification of NPQ fusion protein profiles

[0088] 3.1 Desugaring complete molecular weight determination: The measured desugaring complete molecular weight is consistent with the theoretical molecular weight.

[0089] After sample dilution, the samples were desaccharified using PNGase F enzyme. Data acquisition was performed using a Waters / ACQUITY Premier UPLC in series with Thermo / QE Plus, and the raw data were then analyzed using Biopharma Finder 4.1 software. The measured molecular weights obtained from the analysis were compared with the theoretical molecular weights to confirm their consistency.

[0090] The main components detected in the deglycosylated intact molecular weight of NPQ samples were deglycosylated protein molecules (see...). Figure 4 The measured desugar-free molecular weight was consistent with the theoretical molecular weight, with a difference of less than 100.0 ppm, as shown in Table 1.

[0091] Table 1. Measured and theoretical molecular weights of deglycosylated intact proteins from NPQ samples.

[0092]

[0093] 3.2 NPQ desugaring and reducing molecular weight analysis showed that the measured desugaring and reducing molecular weight was consistent with the theoretical molecular weight.

[0094] Samples were diluted and deglycosylated using PNGase F enzyme. The deglycosylated protein was then partially reduced by DTT under non-denaturing conditions. Data were acquired using a Waters / ACQUITY Premier UPLC in series with Thermo / QE Plus, and the raw data were analyzed using Biopharma Finder 4.1 software. The measured molecular weights were compared with the theoretical molecular weights to confirm their consistency.

[0095] The main component detected by the deglycosylation reduction molecular weight analysis of NPQ samples was deglycosylated single strands (see...). Figure 5 The measured desugar-reduced molecular weight was consistent with the theoretical molecular weight, with a difference of less than 100.0 ppm, as shown in Table 2.

[0096] Table 2. List of molecular weight information of deglycoreducing proteins in NPQ samples.

[0097]

[0098] 3.3 NPQ molecular weight analysis showed that the measured molecular weights were consistent with the theoretical molecular weights.

[0099] For the complete molecular weight of the NPQ sample, the main component detected was G0F glycosylation modification corresponding to both SC chains of the complete NPQ protein. Several other glycosylation modifications were also detected. The measured molecular weights of all sample components were consistent with the theoretical molecular weights, with differences within 100.0 ppm. The unconvolutioned complete molecular weight of the NPQ sample is shown below. Figure 6 The detailed molecular weight information is shown in Table 3.

[0100] Table 3. Measured molecular weight and theoretical molecular weight of intact protein in NPQ samples

[0101]

[0102] 3.4 NPQ reduction molecular weight analysis showed that the measured molecular weights were consistent with the theoretical molecular weights.

[0103] After sample dilution, the samples were partially reduced by DTT under non-denaturing conditions. Data were acquired using a Waters / ACQUITY Premier UPLC in series with Thermo / QE Plus, and the raw data were analyzed using Biopharma Finder 4.1 software. The measured molecular weights obtained from the analysis were compared with the theoretical molecular weights to confirm their consistency.

[0104] For the reduced molecular weight of NPQ, the detected major components corresponded to the molecular weights of single-chain G0F glycosylation modifications. The measured molecular weights of all components in the samples were consistent with the theoretical molecular weights, with differences within 100.0 ppm. The results of the reduced molecular weight detection for the NPQ samples are shown below. Figure 7 Detailed molecular weight information is shown in Table 4.

[0105] Table 4: Measured and Theoretical Molecular Weights of NPQ Samples

[0106]

[0107] 3.5 Amino acid sequence analysis showed that the NPQ amino acid sequence coverage was 91.7%.

[0108] After denaturation, reduction, and alkylation of NPQ-desugared and undesugared samples, they were digested with Trypsin. The digested peptides were collected using a Waters / ACQUITY Premier UPLC system in tandem with Thermo / QE Plus. Biopharma Finder 4.1 software was then used to analyze the precise molecular weight information from the primary mass spectra, as well as the fragment ion and post-translational modification site matching information from the secondary mass spectra, in conjunction with theoretical protein sequence information. These analyses were manually verified. The Trypsin digestion data were analyzed and assigned to determine the coverage of theoretical amino acid sequences and to confirm the consistency between the measured and theoretical amino acid sequences.

[0109] Enzymatic separation of peptide fragments from samples Figure 8 As shown in Table 5, the NPQ sequence coverage reached 91.7% by comparing the measured precise molecular weight information of the peptides using primary mass spectrometry and the fragment ion data using secondary mass spectrometry with the theoretical peptide information. Some short peptides, due to their strong hydrophilicity, were difficult to retain on reversed-phase chromatography and did not appear in the mass spectrometry results. The detected enzymatically digested peptide sequences were consistent with the theoretical sequences, with a molecular weight error within 10.0 ppm.

[0110] Table 5. Amino acid sequence coverage results of NPQ samples

[0111]

[0112] 3.6 Mass spectrometry analysis of amino acid post-translational modifications

[0113] After denaturation, reduction, and alkylation, the samples were digested with Trypsin. Data was acquired using a Waters / ACQUITY Premier UPLC tandem with Thermo / QE Plus. Combined with the theoretical amino acid sequence of YN-108, the precise molecular weight information of the primary mass spectra in the mass spectra was analyzed using BioPharma Finder software, as well as the matching information of fragment ions with theoretical sequences and post-translational modification sites in the secondary mass spectra. These were manually confirmed, and the proportion of post-translational modifications was quantified by extracting ion current maps (SIC).

[0114] The primary mass spectrum of the YN-108 sample after Trypsin digestion is shown below. Figure 8 As shown, the peptides were well separated. Table 6 shows the post-translational modification sites and their proportions above 1.0%. The YN-108 sample molecule mainly underwent N-glycosylation modification, along with minor modifications such as lysine hydroxylation, methionine oxidation, O-glycosylation, aspartic acid isomerization, asparagine deamidation, and asparagine succinimide modification. The N in the amino acid sequence SEQ ID NO:2 was converted to D after enzymatic digestion.

[0115] Table 6 shows the post-translational modification results of peptides from YN-108 samples after N-glycation by Trypsin digestion.

[0116]

[0117] Table 6 (continued)

[0118]

[0119] 3.7 N-glycoside analysis

[0120] Glycosylation modification affects the stability, biological activity, solubility, in vivo half-life, and immunogenicity of therapeutic proteins to some extent. N-glycoside analysis can characterize the major N-glycoside types on proteins and provide guidance for the safety and efficacy of therapeutic proteins.

[0121] N-glycoside analysis of YN108 involved separating N-glycosides from the protein using PNGase F enzyme. The protein was precipitated with ice-cold ethanol, and the supernatant containing N-glycosides was collected after centrifugation. After drying, the sample was labeled with the fluorescent reagent 2-AB. The labeled sample was then separated by hydrophilic interaction chromatography, and peaks were detected on a fluorescence detector. Finally, relative quantification of each glycoform was performed using the area normalization method. The FLD chromatograms of N-glycosides in the YN108 sample are shown in Figures 9 and 10, and detailed analytical results are shown in Table 7. The results indicate that the main N-glycoside types in the YN108 sample are G0F and G1F.

[0122] Table 7. Relative content of N-glycosides in YN108 sample

[0123]

[0124] Example 4: Determination of the affinity between the NPQ fusion protein and the receptor

[0125] The affinity of the NPQ fusion protein for its receptor was determined using ELISA. INS-1 and HEPG2 cells grown in 96-well plates (BDBiosciences) were washed with PBS and fixed with 4% paraformaldehyde (ThermoScientific) for 10 min at room temperature, followed by quenching in PBS solution containing 2% glycine (pH 7.5) for 5 min. In the binding capacity assay, logarithmically diluted NPQ(36-49)-IgG2 / Fc fusion protein (1×10⁻⁶) was used. -5 Up to 1×10 -12 Cells were incubated alone with 10 mM NPQ (Abcam, USA) or co-incubated with 10 mM NPQ to detect specific binding and non-specific binding, respectively. In the competitive binding assay, a fixed concentration of 10 mM NPQ(36-49)-IgG2 / Fc fusion protein was used, and different concentrations of NPQ (1×10⁻⁶) were used. -5 Up to 1×10 -12 M) Competitive binding. After culturing for 4 hours at 4°C in a final volume of 100 mL, excess NPQ and NPQ(36-49)-IgG2 / Fc fusion protein were washed off, and cells were blocked with 5% BSA (BD Biosciences). The binding of NPQ(36-49)-IgG2 / Fc was detected using goat anti-human IgG Fc antibody (1:4000, Southern Biotech) and HRP-conjugated donkey anti-goat IgG (1:5000, Jackson Immuno Research).

[0126] An enzymatic reaction was initiated with o-phenylenediamine (OPD) (Fisher Scientific), and colorimetric changes were analyzed by reading the absorbance at 490 nm using a Beckman microplate reader. The results showed that the NPQ fusion protein binds well to receptors in INS-1 and HEPG2 cells.

[0127] Example 5: Effects of NPQ fusion protein on the INS-1 cell signaling pathway

[0128] INS-1 cells were washed with PBS and dissolved in lysis buffer containing 0.1% protease inhibitor. The lysis buffer was then incubated on ice for 30 minutes and centrifuged at 8000 g for 10 minutes. Cell samples and an equal volume of protein GAPDH were subjected to SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride membrane. Immunoblotting was performed with the following primary antibodies: anti-CREB antibody (#9197); anti-phospho-CREB antibody (Ser133) (#9198); anti-Akt antibody (#2920); and anti-phospho-Akt (Ser473) (#4060), all purchased from Cell Signaling Technology. The membrane was washed with PBS-Tween-20 and incubated with peroxidase-conjugated secondary antibody. Protein bands were detected using the ECL Plus kit.

[0129] Example 6: Effect of NPQ fusion protein on pancreatic islet cell secretory function

[0130] INS-1 cells were seeded in 24-well plates at a density of 2.5 × 10⁻⁶ cells / well. 5 Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) per well. The next day, cells were cultured in fresh KRB (128.8 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄) without glucose. S Cells were cultured for 120 minutes in a buffer solution of O4, 1.0 mM CaCl2, 5.0 mM HEPES, 0.1% fetal bovine serum, and 2.8 mM glucose instead of the original culture medium. Cells were then treated for 2 hours with 2.8 mM or 16.8 mM glucose and various concentrations of purified NPQ(36-49)-IgG2 / Fc fusion protein. Insulin levels in the culture medium were detected using a rat insulin RIA kit (Linco, St. Charles, MO, USA), following the manufacturer's instructions.

[0131] Experimental results show that the NPQ fusion protein can promote insulin secretion.

[0132] Example 7: Effect of NPQ fusion protein on triglyceride levels

[0133] The effect of NPQ fusion protein on triglyceride levels was investigated in HepG2 cells. HepG2 cells were grown in 24-well plates and incubated with 1–100 µg / mL (final concentration) NPQ(36–49)-IgG2 / Fc for 1 h. Then, palmitic acid (50–300 µM) was added for further incubation. Cells were washed three times with phosphate-buffered saline and fixed with 4% paraformaldehyde at room temperature for 30 min. Fixed cells were washed with deionized water, soaked in 60% isopropanol for 3 min, stained with 2 mg / mL Oil Red O for 60 min, and washed three times with deionized water to remove unbound dye. Cell nuclei were stained with hematoxylin for 3 min and rinsed with deionized water. After microscopic examination, the triglyceride content in each well was quantitatively determined based on Oil Red O.

[0134] Experimental results show that the NPQ fusion protein can effectively reduce triglyceride levels.

[0135] Table 8 Replacement Sites

[0136]

[0137] Sequence List:

[0138]

[0139]

[0140]

[0141] The foregoing examples list what are currently considered to represent preferred embodiments of this application; however, it should be understood that this application is not limited to the disclosed examples. Rather, this application is intended to cover various modifications and equivalent examples that fall within the spirit and scope of the appended claims.

[0142] All publications, patents, and patent applications are incorporated herein by reference in their entirety. Specifically, sequences associated with each accession number provided herein, including accession numbers and / or biomarker sequences (e.g., proteins and / or polynucleotides) provided, for example, in tables or elsewhere, are incorporated herein by reference in their entirety.

[0143] The scope of the claims should not be limited to the preferred embodiments and examples, but should be understood as the broadest interpretation consistent with the specification.

Claims

1. An 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 has the amino acid sequence shown in SEQ ID NO: 2, and the IgG2 / Fc polypeptide has the amino acid sequence shown 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 has the amino acid sequence shown in SEQ ID NO: 8, and the NPQ fusion protein comprises the amino acid sequence shown in SEQ ID NO: 5; the NPQ fusion protein further comprises N-glycosylation modification.

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

6.

3. A polynucleotide, characterized in that, The polynucleotide comprises a polynucleotide encoding the NPQ fusion protein of claim 1 or 2.

4. A carrier, characterized in that, The vector comprises the polynucleotide of claim 3.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the NPQ fusion protein of claim 1 or 2, or the polynucleotide of claim 3, or the carrier of claim 4, and optionally a pharmaceutically acceptable carrier.

6. The use of an NPQ fusion protein as described in claim 1 or 2, a polynucleotide as described in claim 3, a carrier as described in claim 4, and a pharmaceutical composition as described in claim 5 in the preparation of a medicament, wherein the medicament is a medicament for treating or preventing metabolic diseases related to disorders of glucose or lipid metabolism, wherein... The metabolic diseases related to disorders of glucose or lipid metabolism are selected from diabetes, non-alcoholic fatty liver disease, and obesity.

7. The use of an NPQ fusion protein as described in claim 1 or 2, a polynucleotide as described in claim 3, a carrier as described in claim 4, and a pharmaceutical composition as described in claim 5 in the preparation of a medicament, wherein the medicament is a medicament for treating or preventing metabolic diseases related to disorders of glucose or lipid metabolism, wherein... The metabolic disease associated with disorders of glucose or lipid metabolism is non-alcoholic steatohepatitis.

8. A recombinant cell, characterized in that, The cell contains the polynucleotide as described in claim 3, or the carrier as described in claim 4.

9. The recombinant cell according to claim 8, characterized in that, The recombinant cells were obtained from CHO cells.

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