Fusion protein and use thereof

Abstract

The present invention relates to a fusion protein and the use thereof. Specifically, provided in the present invention are a GCGR-IgG2 / Fc fusion protein, a polynucleotide encoding the fusion protein, a vector and cell comprising the polynucleotide, and a composition thereof. The present invention also relates to the use of the fusion protein, polynucleotide, vector, cell and composition in a drug, and / or in the preparation of a drug for treating or preventing a glucose metabolism-related disease.

Description

A fusion protein and its applications Technical Field

[0001] This invention relates to the GCGR-IgG2 / Fc fusion protein, its preparation method, and its applications. Background Technology

[0002] Glucagon, a 29-amino acid peptide produced by pancreatic α cells, plays a crucial role in maintaining glucose homeostasis by activating the glucagon receptor (GCGR). GCGR is a G protein-coupled receptor expressed in various tissues, including the liver and brain. In cases of hypoglycemia, glucagon secretion increases to promote hepatic glucose production, primarily through glycolysis. Glucagon plays a vital role in preventing hypoglycemia during fasting. However, a lack of glucagon inhibition can lead to excessively high postprandial blood glucose in patients with type 2 diabetes.

[0003] Diabetes mellitus is considered a dual-hormonal disorder characterized by insulin deficiency and glucagon excess. Recent studies have shown that excessive glucagon secretion or activity, compared to insulin deficiency, is a major cause of diabetes development. Therefore, pharmacological interventions that reduce glucagon secretion or block glucagon receptor signaling are promising treatments for diabetes. For example, GCGR antagonists, antibodies, and antisense oligonucleotides have shown significant effects in alleviating hyperglycemia, with adverse reactions primarily including elevated blood pressure, elevated transaminases, and abnormal lipid metabolism. However, the adverse reactions related to blood pressure, transaminases, and lipid metabolism vary among different compounds or biologics.

[0004] Therefore, there is an urgent need to develop a GCGR-related drug with significant effects and low adverse reactions, higher yield and activity, and a more suitable half-life, which would be of great significance for the treatment of metabolic diseases such as diabetes. Summary of the Invention

[0005] The purpose of this invention is to provide an improved GCGR peptide fusion protein with higher activity and yield, which reduces the binding level of glucagon or its binding to cellular GCGR. This fusion protein can be obtained through various pathways or methods. Furthermore, the amino acid substitution at specific positions in the GCGR peptide fusion protein of this invention significantly prolongs the half-life of the improved fusion protein, while exhibiting good preventive and therapeutic effects in human and animal disease models. Compared to the direct inhibition of GCGR by antibodies and other methods to lower blood sugar, this invention has fewer side effects.

[0006] Therefore, the advantage of the present invention is that it provides an improved bioactive GCGR polypeptide fusion protein with improved yield and activity, or extended half-life.

[0007] In one aspect, the present invention provides a biologically active GCGR peptide fusion protein comprising a GCGR peptide and an immunoglobulin IgG2 / Fc domain, wherein the GCGR peptide is selected from the extracellular region of human GCGR, and the GCGR peptide is covalently linked to the immunoglobulin Fc domain.

[0008] In one embodiment, the IgG2 / Fc domain of the present invention includes a C222S substitution, and / or the IgG2 / Fc domain further includes one or both selected from A330S substitution and P331S substitution.

[0009] In one embodiment, the GCGR polypeptide fusion protein of the present invention has an amino acid sequence that is at least 90% identical to that of SEQ ID NO:1.

[0010] In one embodiment, in the GCGR polypeptide fusion protein of the present invention, the IgG2 / Fc domain has at least 90% sequence identity with SEQ ID NO:2 or SEQ ID NO:3 and contains A330S and / or P331S substitutions, and contains an amino acid sequence with C222S substitution.

[0011] In one embodiment, the GCGR polypeptide fusion protein of the present invention further comprises a linker peptide for connecting the GCGR polypeptide and the IgG2 / Fc domain.

[0012] In one embodiment, in the GCGR polypeptide fusion protein of the present invention, the linker peptide has an amino acid sequence selected from SEQ ID NO:8, 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, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, preferably SEQ ID NO:8. In one embodiment, the GCGR polypeptide fusion protein of the present invention further comprises a signal peptide. Preferably, the signal peptide is selected from one or more of human CD33 signal peptide, human GCGR signal peptide, and silkworm FH signal peptide, or the signal peptide comprises an amino acid sequence of SEQ ID NO:7, SEQ ID NO:23, or SEQ ID NO:24, or comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:23, or SEQ ID NO:24.

[0013] In one embodiment, the GCGR peptide fusion protein of the present invention has the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In one embodiment, the GCGR peptide fusion protein of the present invention is pharmaceutical grade.

[0014] In one embodiment, in the GCGR polypeptide fusion protein of the present invention, the amino acid sequence of the GCGR polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:1. In another embodiment, in the GCGR polypeptide fusion protein of the present invention, the GCGR polypeptide has the amino acid sequence of SEQ ID NO:1.

[0015] In one embodiment, in the GCGR polypeptide fusion protein of the present invention, the IgG2 / Fc domain has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% similarity to SEQ ID NO:2 or SEQ ID NO:3.

[0016] In one embodiment, in the GCGR polypeptide fusion protein of the present invention, the IgG2 / Fc domain has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3 and includes amino acid sequences with substitutions of A330S, P331S, and C222S. In another embodiment, in the GCGR polypeptide fusion protein of the present invention, the IgG2 / Fc domain has the amino acid sequence of SEQ ID NO:3.

[0017] In one embodiment, the GCGR polypeptide fusion protein of the present invention has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In another embodiment, the GCGR polypeptide fusion protein of the present invention has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0018] In one embodiment, the GCGR polypeptide fusion protein of the present invention further comprises a signal peptide.

[0019] In one embodiment, in the GCGR polypeptide fusion protein of the present invention, the signal peptide is selected from one or more of human CD33 signal peptide, human GCGR signal peptide, and silkworm FH signal peptide. In embodiments, the signal peptide has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:7, SEQ ID NO:23, or SEQ ID NO:24. In one embodiment, the signal peptide has the amino acid sequence of SEQ ID NO:7, SEQ ID NO:23, or SEQ ID NO:24.

[0020] In one embodiment, the signal peptide in the GCGR polypeptide fusion protein of the present invention is a CD33 signal peptide. In an embodiment, the signal peptide has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:7. In one embodiment, the signal peptide has the amino acid sequence of SEQ ID NO:7.

[0021] In another embodiment, in the GCGR polypeptide fusion protein of the present invention, the signal peptide comprises a CD33 signal peptide and a GCGR signal peptide. In embodiments, the signal peptide has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:23 and SEQ ID NO:24. In one embodiment, the signal peptide has the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24.

[0022] In one embodiment, the GCGR polypeptide fusion protein of the present invention may further include a tag protein, preferably a maltose-binding protein (MBP).

[0023] In another aspect, the present invention also provides a polynucleotide comprising a polynucleotide encoding the GCGR polypeptide fusion protein involved in the present invention.

[0024] In one embodiment, the polynucleotide encodes a GCGR polypeptide fusion protein of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In one embodiment, the polynucleotide has a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identical to SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. In another embodiment, the polynucleotide is a polynucleotide sequence of SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0025] In one embodiment, the polynucleotide has a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identical to SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. In another embodiment, the polynucleotide is a polynucleotide sequence of SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0026] In another aspect, the present invention provides a vector comprising the polynucleotide.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising the GCGR polypeptide fusion protein, polynucleotide, or carrier.

[0028] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0029] In one embodiment, the composition further comprises a buffered saline solution.

[0030] In one embodiment, the composition further comprises a surfactant.

[0031] In one embodiment, the composition comprises a GCGR peptide fusion protein, formulated into a dosage form, and administered at a dose of about 0.1 mg to about 20 mg, for example, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, 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... GCGR peptide fusion protein in doses of approximately 0.9 mg, 0.95 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0032] In another aspect, the present invention provides a GCGR polypeptide fusion protein, polynucleotide, carrier, or composition for preparing a drug.

[0033] In another aspect, the present invention provides the use of the GCGR polypeptide fusion protein, polynucleotide, carrier, or pharmaceutical composition in the preparation of a medicament.

[0034] In one embodiment of the invention, the drug is used to treat or prevent diseases related to glucose metabolism.

[0035] In one embodiment of the invention, the glucose metabolism-related disease is selected from diabetes.

[0036] In one embodiment of the invention, the metabolic disease associated with the glucose metabolism disorder is diabetes, preferably type 2 diabetes.

[0037] In one embodiment of the invention, the GCGR polypeptide fusion protein, polynucleotide, carrier, or composition is used in combination with a diabetes drug. In a preferred embodiment, the diabetes drug may be a currently marketed drug, such as insulin, metformin, sulfonylureas mainly including glimepiride, glibenclamide, gliclazide, gliquidone, etc., α-glucosidase inhibitors such as acarbose, etc., and other marketed and under-development drugs for treating diabetes. In one embodiment, the diabetes drug is metformin or insulin. In one embodiment, the composition may be used in combination with Alzheimer's disease drugs and / or non-pharmacological interventions such as cognitive therapy.

[0038] Another aspect of the present invention provides a method for treating or preventing glucose metabolism-related diseases in a subject in need, the method being to administer a therapeutically effective amount of the GCGR polypeptide fusion protein, polynucleotide, carrier, or composition to the subject.

[0039] In one embodiment, the glucose metabolism-related disease in the method of the present invention is diabetes mellitus. In another preferred embodiment, the diabetes mellitus is type 2 diabetes mellitus.

[0040] In one embodiment, the GCGR polypeptide fusion protein or its composition can also be used in combination with a drug for treating diabetes in the method of the present invention. In one embodiment, the drug for treating diabetes can be a currently marketed drug, such as insulin, metformin, sulfonylureas mainly including glimepiride, glibenclamide, gliclazide, gliquidone, etc., α-glucosidase inhibitors such as acarbose, etc., and also includes other marketed and under-development drugs for treating diabetes. In one embodiment, the diabetes drug is metformin or insulin.

[0041] In one embodiment, the GCGR polypeptide fusion protein or a composition thereof is administered via parenteral, intravenous, subcutaneous, or intramuscular routes.

[0042] In one embodiment, the dosage of the GCGR peptide fusion protein or the composition thereof is approximately 0.1 mg to 20 mg of the GCGR peptide fusion protein.

[0043] In one embodiment, the composition comprises a GCGR peptide fusion protein, formulated into a dosage form, and administered at a dose of about 0.1 mg to about 20 mg, for example, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, 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... GCGR peptide fusion protein in doses of approximately 0.9 mg, 0.95 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0044] In one embodiment, the GCGR polypeptide fusion protein or its composition is administered once every 3 days, once a week, or once every two weeks.

[0045] In another aspect, the present invention provides a recombinant cell comprising, or comprising, the polynucleotide encoding the GCGR polypeptide fusion protein, or comprising the polynucleotide, or comprising the vector.

[0046] In one embodiment, the recombinant cells of the present invention are obtained from Chinese hamster ovary cells through suspension domestication, with CHO cells as an example.

[0047] In another aspect, the present invention provides a method for constructing recombinant cells, comprising: introducing a polynucleotide encoding the GCGR polypeptide fusion protein of the present invention into a vector to construct an expression vector; and introducing the expression vector into recombinant or natural cells to obtain recombinant cells.

[0048] In one embodiment, the cells are human embryonic kidney cells 293 (HEK293 cells), or CHO-K1 cells, or CHO-S cells, or CHO-DG44, particularly CHO-S cells.

[0049] In one embodiment, the carrier may be a pKN012 carrier.

[0050] In one embodiment, the method for constructing the recombinant cells includes the following steps:

[0051] Insert the polynucleotide sequence shown in SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30 into the NcoI and HindIII sites of the pKN012 vector to generate pKN012-GCGR-IgG2 / Fc;

[0052] Recombinant cells were obtained by introducing the pKN012-GCGR-IgG2 / Fc expression vector into CHO-S cells.

[0053] In another aspect, the present invention provides a method for producing the GCGR peptide fusion protein, comprising the step of obtaining the GCGR peptide fusion protein using the recombinant cells.

[0054] Other features and advantages of the invention will become apparent from the detailed description below, as in the embodiments. The detailed description and specific examples are given by way of illustration only, while acknowledging preferred embodiments of the invention; various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art. Attached Figure Description

[0055] Figure 1 is a schematic diagram of the vector pKN012-GCGR-IgG2 / Fc.

[0056] Figure 2 shows the SDS-PAGE staining of the GCGR-IgG2 / Fc fusion protein. Where: R represents the reduced form (with DTT, single-chain molecule), and NR represents the non-reduced form (without DTT, double-chain molecule).

[0057] Figure 3 shows the inhibition of Glucagon activity by the GCGR-IgG2 / Fc fusion protein. Detailed Implementation

[0058] The following is a detailed description to assist those skilled in the art in practicing this invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, drawings, and other references mentioned herein are incorporated herein by reference in their entirety.

[0059] I. Definitions or Terms

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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. Proteins may be a single peptide chain or a multi-subunit protein (e.g., may consist of two or more polypeptides).

[0064] As used herein, the term "GCGR polypeptide" includes polypeptides of the GCGR extracellular domain (ECD), such as the polypeptide shown in SEQ ID NO:1.

[0065] As used herein, the term "polynucleotide" or "oligonucleotide" refers to two or more covalently linked nucleotides. Unless the context clearly indicates otherwise, the term generally includes, but is not limited to, deoxyribonucleotides (DNA) and ribonucleotides (RNA), which can be single-stranded (ss) or double-stranded (ds). For example, the polynucleotide molecules or polynucleotides of the present invention can consist of single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA. Mixtures of single-stranded and double-stranded regions, including hybrid molecules containing DNA and RNA, can be single-stranded or more typically double-stranded, or mixtures of single-stranded and double-stranded regions. Furthermore, polynucleotide molecules can consist of triple-stranded regions containing RNA or DNA, or both RNA and DNA. As used herein, the term "oligonucleotide" generally refers to a polynucleotide of no more than 200 base pairs in length and can be single-stranded or double-stranded. The sequences provided herein can be DNA sequences or RNA sequences; however, it should be understood that the provided sequences include DNA and RNA, as well as complementary RNA and DNA sequences, unless the context clearly indicates otherwise. For example, the sequence 5'-GAATCC-3' should be understood to include 5'-GAAUCC-3', 5'-GGATTC-3', and 5'-GGAUUC-3'.

[0066] As used herein, the term "sequence identity" refers to the percentage of sequence similarity between two polypeptide sequences or two polynucleotide sequences. To determine the percentage of similarity between two amino acid sequences or two polynucleotide sequences, sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first amino acid or polynucleotide sequence to best align with the second amino acid or polynucleotide sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of similarity between two sequences is a function of the number of shared identical positions (i.e., percentage of similarity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. Mathematical algorithms can also be used to determine the percentage of similarity between two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the Karlin-Altschul algorithm, later modified to Karlin-Altschul. This algorithm is incorporated into the NBLAST and XBLAST programs. BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameter set, for example, score = 100, word length = 12, to obtain nucleotide sequences homologous to a specific polynucleotide molecule. BLAST protein searches can be performed using the XBLAST program parameter set, for example, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecule described herein. Gapped BLAST can be used to obtain gap alignments for comparative purposes. Alternatively, PSI-BLAST can be used to perform iterative searches to detect long-distance relationships (Id.) between molecules. When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm proposed by Myers and Miller, which is incorporated into the ALIGN program (version 2.0), part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM120 weighted residue table, a 12-fold vacancy length penalty, and a 4-fold vacancy penalty can be used. The percentage of similarity between two sequences can be determined using techniques similar to those described above, with or without allowed gaps. When calculating the percentage of similarity, only perfect matches are typically counted.

[0067] 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.

[0068] In this invention, the term "fusion protein" refers to a protein comprising two or more polypeptides forming different functional domains. For example, the GCGR polypeptide fusion protein described herein comprises a GCGR polypeptide and an immunoglobulin IgG2 / Fc domain.

[0069] 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.

[0070] 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.

[0071] 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).

[0072] As used in this article, the term "vector" refers to a molecule that is used as a carrier to introduce foreign DNA into a cell.

[0073] In this invention, the term "pharmaceutical grade" refers to the chemical purity or proportion of a drug, biological macromolecule, or reagent that meets the requirements for drug production.

[0074] In this invention, the term "treatment" refers to administering an effective amount of a compound, composition, or formulation to a subject, which may consist of a single administration or optionally include a series of procedures. As is well known in the art, "treatment" is a method for obtaining a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions, reduction of disease severity, stabilization (i.e., non-exacerbation) of a disease state, prevention of disease spread, reversal of disease, improvement or relief of disease, and remission of a disease state (whether partial, total, or temporary).

[0075] Beneficial or desired results include improved blood sugar levels, weight loss, etc.

[0076] In this invention, the term "subject" is also referred to as a patient, as used herein to include all animals, including mammals, and preferably refers to humans.

[0077] 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.

[0078] In this invention, the term "therapeutic effective dose" refers to any dose that produces the desired effect in a subject, such as symptom relief, slowing disease progression, or prevention of disease flare-ups. This dose can be effective with multiple administrations and / or achieve the desired effect over a period of time. As used herein, this term can also refer to the amount that causes a decrease in blood glucose levels in a subject.

[0079] In this invention, "co-administration" and the like refer, for example, to two or more substances (e.g., two or more compounds, two or more compositions, etc.) that are administered to a subject, both of which are biologically active. The exact administration will depend on the pharmacokinetics of the two or more substances in the presence of each other.

[0080] 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.

[0081] The term “composition as” and its derivatives used herein are closed terms that specify the presence of the stated feature, element, component, group, integer and / or step, and exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0082] 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.

[0083] 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%.

[0084] As used in this specification and the appended claims, the singular form "a" includes plural references unless otherwise expressly specified. Thus, a composition comprising, for example, "a compound" includes a mixture of two or more compounds. It should also be noted that, unless otherwise expressly specified, the term "or" is generally used in its meaning including "and / or".

[0085] The definitions and embodiments described in certain sections are intended to apply simultaneously to other embodiments described herein, as will be understood by those skilled in the art.

[0086] The numerical ranges listed in this document by endpoints include all numbers and fractions contained within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and their fractions are assumed to be modified by the term “about”.

[0087] Furthermore, the definitions and embodiments described in certain sections are intended to apply to other embodiments described herein, as will be understood by those skilled in the art. For example, different aspects of the invention are defined in more detail in the following paragraphs. Each aspect thus defined may be combined with any other aspect or multiple aspects unless expressly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0088] Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, specific methods and materials are also described in this embodiment.

[0089] II. Proteins, fusion proteins, compositions, formulations, uses and methods

[0090] The present invention provides stable GCGR peptides and fusion proteins comprising said GCGR peptides fused with, for example, IgG2 / Fc domains.

[0091] The GCGR peptide of the fusion protein disclosed herein can be human GCGR. In some embodiments, the GCGR peptide is the extracellular amino acid sequence of GCGR.

[0092] In one embodiment, the GCGR polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with SEQ ID NO:1. In one embodiment, the GCGR polypeptide has the amino acid sequence of SEQ ID NO:1.

[0093] The GCGR peptide in the fusion protein is covalently linked (directly or via a linker peptide) to the IgG2 / Fc portion of the immunoglobulin. In one embodiment, the IgG2 / Fc domain contains a C222S substitution. The C222S substitution of IgG2 / Fc increases the flexibility of the N-terminal hinge region by removing the disulfide bond between the two monomers of the homodimer. The increased flexibility of the N-terminal hinge region can reduce the binding affinity of the Fcγ receptor, thereby reducing antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Unless otherwise stated herein, all amino acid residues referred to herein are identified using the EU indexing system of Kabat et al. (1991).

[0094] In another embodiment, the IgG2 / Fc domain includes an A330S / P331S substitution. The A330S / P331S substitution reduces affinity for the Fcγ receptor and C1q complement protein.

[0095] In one embodiment, the IgG2 / Fc domain includes C222S, A330S, and P331S substitutions.

[0096] In one embodiment, the IgG2 / Fc domain has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the IgG2 / Fc domain has the amino acid sequence of SEQ ID NO:3.

[0097] The GCGR peptide and IgG2 / Fc domain of the fusion protein disclosed herein, for example, the IgG2 / Fc domain, can be linked by a linker peptide. As used herein, the term "linker peptide" refers to any portion that links the different functional domains of the peptide together. Linker peptides can have any suitable length and structure. The linker peptide sequences that can be used are selected from any amino acid sequence of SEQ ID NO:8, 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, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0098] In one embodiment, the linker peptide connecting the GCGR polypeptide and the IgG2 / Fc domain may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with SEQ ID NO:8, 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, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. In one embodiment, the linker peptide has the amino acid sequence of SEQ ID NO:8.

[0099] In one embodiment, the fusion protein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In one embodiment, the fusion protein has the amino acid sequence of SEQ ID NO:4.

[0100] As demonstrated in the examples, the GCGR peptide fusion protein disclosed herein has a longer half-life after sequence modification.

[0101] In some embodiments, the fusion proteins disclosed herein may include, or initially include, a signal peptide fused to GCGR. As used herein, the term "signal peptide" refers to a polypeptide that causes the fusion protein to be secreted into an extracellular medium. Such polypeptides may also be referred to as "lead peptides," "peptide precursors," "propeptides," etc. The use of signal peptides to direct protein secretion is known in the art (e.g., U.S. Patent US8,658,174, the contents of which are incorporated herein by reference in their entirety). Examples of signal peptides include, but are not limited to, human CD33 signal peptide, human growth hormone-releasing hormone (GHRH) signal peptide, human α-1-microglobulin / bikunin precursor (AMBP) signal peptide, Gaussian luciferase signal peptide, mouse immunoglobulin heavy chain signal peptide, mouse immunoglobulin κ light chain signal peptide, GCGR signal peptide, silkworm (Bombyx mori) FH (Fibroin heavy chain) signal peptide, etc. The signal peptide is cleaved during secretion.

[0102] In one embodiment, the signal peptide comprises having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with SEQ ID NO:7, SEQ ID NO:23, or SEQ ID NO:24, and allows for the secretion of the fusion protein. In one embodiment, the signal peptide is selected from one or more of SEQ ID NO:7, SEQ ID NO:23, or SEQ ID NO:24; preferably, the signal peptide is SEQ ID NO:7, or contains both SEQ ID NO:7 and SEQ ID NO:23.

[0103] In one embodiment, the GCGR polypeptide fusion protein of the present invention may further include a tag protein, preferably a maltose-binding protein (MBP).

[0104] In one embodiment, the fusion protein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with SEQ ID NO:4. In one embodiment, the fusion protein has the amino acid sequence of SEQ ID NO:4. In another preferred embodiment, the GCGR peptide fusion protein is pharmaceutical grade.

[0105] Peptides and fusion proteins can be synthesized using standard protein chemistry techniques. Furthermore, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Mode 1396; Milligen / Biosearch 9600). Alternatively, the peptides, polypeptides, or fragments or variants thereof described herein can be recombinantly generated using various expression systems well-known in the art.

[0106] In another aspect, the present invention provides a nucleic acid molecule comprising a polynucleotide sequence encoding the polypeptide or peptide described herein. In one embodiment, the polynucleotide is codon-optimized, for example, optimized for humans. The nucleic acid molecule can be used in the methods described herein.

[0107] In one embodiment, the nucleic acid may encode the GCGR polypeptide fusion protein disclosed herein. Any vector suitable for the intended use can be used. For example, some vectors can be introduced into expression systems, such as mammalian, insect, or bacterial expression systems, for the expression and purification of the expressed protein. Some vectors can be used to produce viruses. These different vectors are well known in the art. Suitable vectors include, but are not limited to, pMPGCR5, pAV0243, and pKN012.

[0108] In some embodiments, the vector, together with an in vitro expression system, is used to generate the fusion protein. Expression and purification of the fusion protein can be performed by any suitable method known in the art.

[0109] Possible expression vectors include, but are not limited to, plasmids or modified viruses (e.g., replication-defective retroviruses, including lentiviral vectors, adenoviruses, and adeno-associated viruses). In one embodiment, the expression vector that can be used with the fusion protein of the present invention is pKN012, which is commercially available (Beijing Kohnoor Science & Technology Co., Ltd.).

[0110] The vector can contain suitable regulatory sequences and components.

[0111] Suitable regulatory sequences can be selected from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase-binding sequences, ribosome-binding sequences, and translation initiation signals. Furthermore, depending on the cells to be transfected / infected / transduced and the vector used, other sequences, such as origins of replication, additional DNA restriction sites, enhancers, and sequences conferring transcriptional induction, can be incorporated into the expression vector. In one embodiment, the regulatory sequence directs or increases expression in neural tissue and / or cells. In one embodiment, the vector is a viral vector. Recombinant expression vectors may also contain marker genes that aid in the selection of host cells for vector transformation, infection, or transfection to express the antibodies described herein. Recombinant expression vectors may also contain additional expression cassettes encoding, for example, fusion moieties that can aid in detection (e.g., for generating antibody “fusion proteins”), including tags and markers as described herein.

[0112] In one embodiment, the vector comprises one or more, for example, in one embodiment, the vector comprising a polynucleotide encoding a GCGR polypeptide fusion protein is pKN012-GCGR-IgG2.

[0113] Various methods can be used to transduce cells, including viral vectors, "naked" DNA, DNA in lipids or other nanoparticles, adjuvant-assisted DNA, gene guns, etc. For example, retroviral vectors such as lentiviral vectors can also be used to transduce cells. Other vector systems that can be used to implement this invention include adenovirus and adeno-associated virus-based vectors.

[0114] In another aspect, the present invention provides a recombinant cell that recombinantly expresses polypeptides or peptides or contains polynucleotide molecules or carriers as described herein.

[0115] Stably expressing recombinant cells can be prepared, for example, by transformation, transfection, or transduction of recombinant cells using a vector containing polynucleotides, preferably any polynucleotides described herein. In some embodiments, recombinant cells expressing the GCGR polypeptide fusion protein are prepared using HEK293T, HEK293S, HEK293F, and / or Chinese hamster ovary cells such as CHOK1 cells, which, for example, can be used to produce recombinant polypeptides and / or fusion proteins under conditions suitable for in vivo use.

[0116] As illustrated herein, substitutions of one or more of C222S, A330S, and P331S in the IgG2 / Fc moiety increase the yield, activity, and / or half-life of the GCGR peptide fusion protein. The modified GCGR peptide fusion proteins, nucleic acids, vectors, and recombinant cells described herein are suitable for the preparation of pharmaceuticals and compositions thereof, and for appropriate therapeutic uses.

[0117] As described herein, GCGR peptide fusion proteins can be synthesized. As also shown herein, the fusion protein can be prepared using recombinant cells, including, for example, recombinant Chinese hamster ovary cells expressing the GCGR peptide fusion protein. Therefore, the present invention also provides a method for preparing a GCGR peptide fusion protein, the method comprising culturing recombinant cells expressing the GCGR peptide fusion protein, wherein the culture comprises one or more of the process steps or materials described in the examples.

[0118] Another aspect of the present invention provides a composition comprising a GCGR polypeptide fusion protein, nucleic acid, vector or recombinant cells.

[0119] The composition may also contain a suitable diluent or a carrier. In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier.

[0120] In one embodiment, the composition is a pharmaceutical composition.

[0121] In one embodiment, the composition is a pharmaceutical composition comprising a GCGR peptide fusion protein and a pharmaceutically acceptable carrier.

[0122] In one embodiment, the composition comprises buffered saline solution.

[0123] In one embodiment, the composition comprises sugars.

[0124] In one embodiment, the composition comprises a surfactant. The pharmaceutical compositions of the present invention can be prepared, packaged, and / or sold in batches as a unit dose and / or in multiple unit doses. The compositions can be prepared in various forms.

[0125] The GCGR polypeptide fusion protein, polynucleotide, carrier, recombinant cell, or composition can be used to prepare a drug and / or for administration, for example, via parenteral, intravenous, subcutaneous, or intramuscular routes.

[0126] In one embodiment, the GCGR polypeptide fusion protein or a combination thereof may be administered parenterally or formulated into a preparation for parenteral administration.

[0127] In one embodiment, the GCGR polypeptide fusion protein or a combination thereof may be administered subcutaneously or formulated into a preparation for subcutaneous administration.

[0128] In one embodiment, the GCGR polypeptide fusion protein or a combination thereof may be administered intravenously or formulated into a preparation for intravenous administration.

[0129] In one embodiment, the GCGR polypeptide fusion protein or a combination thereof may be administered intramuscularly or formulated for intramuscular administration.

[0130] Diluents suitable for GCGR peptide fusion proteins and / or cells include, but are not limited to, saline solutions, pH buffer solutions and diluents described herein, as well as glycerol solutions or other solutions suitable for freezing peptides and / or cells.

[0131] Diluents suitable for nucleic acids and / or vectors include, but are not limited to, saline solutions, pH buffer solutions and the diluents described herein, as well as water.

[0132] In another preferred embodiment, the diluent is sterile.

[0133] In another aspect of the invention, the fusion proteins and compositions disclosed herein can be used in subjects to treat or prevent the onset of a disease or condition or to slow its progression.

[0134] In another aspect, the present invention provides the use of the fusion protein or a composition thereof as a GCGR receptor agonist.

[0135] In another aspect, the present invention provides the fusion protein or a composition thereof for treating, preventing or slowing the progression of a disease or condition.

[0136] In another aspect, the present invention provides a fusion protein or a composition thereof for use in the preparation of a medicament for treating, preventing or slowing the progression of a disease or condition.

[0137] In another aspect, the present invention provides a method for treating, preventing, or slowing the progression of a disease or condition by administering a therapeutically effective amount of the fusion protein or a composition thereof to a subject in need.

[0138] In one embodiment, the composition comprises a GCGR peptide fusion protein, formulated into a dosage form, and administered at a dose of about 0.1 mg to about 20 mg, for example, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, 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, or about 0. GCGR peptide fusion protein in doses of 9 mg, approximately 0.95 mg, approximately 1 mg, approximately 1.5 mg, approximately 2 mg, approximately 2.5 mg, approximately 3 mg, approximately 3.5 mg, approximately 4 mg, approximately 4.5 mg, approximately 5 mg, approximately 5.5 mg, approximately 6 mg, approximately 7 mg, approximately 7.5 mg, approximately 8 mg, approximately 8.5 mg, approximately 9 mg, approximately 9.5 mg, approximately 10 mg, approximately 11 mg, approximately 12 mg, approximately 13 mg, approximately 14 mg, approximately 15 mg, approximately 16 mg, approximately 17 mg, approximately 18 mg, approximately 19 mg, or approximately 20 mg.

[0139] Different dosage forms can be used, and suitable dosage forms may include, but are not limited to, solutions, suspensions, pills, and tablets.

[0140] In one embodiment, the disease or condition is a glucose metabolism-related disease. In another preferred embodiment, the glucose metabolism-related disease is or includes diabetes, preferably type 2 diabetes.

[0141] In one embodiment, the subject of the embodiment is either newly diagnosed or previously diagnosed with diabetes. Diabetes can be diagnosed in several ways, such as by fasting plasma glucose (FPG). According to the American Diabetes Association, diabetes is diagnosed when fasting plasma glucose is greater than or equal to 126 mg / dL.

[0142] In one embodiment, the subject's likelihood of developing diabetes, such as type 2 diabetes, is increased. For example, the subject may be predisposed to diabetes due to obesity or a genetic predisposition, such as a family history of diabetes.

[0143] In one embodiment, the subject is an obese patient. Obesity can be defined by a reference body mass index (BMI). For example, the World Health Organization (WHO) defines obesity as having a BMI of 30 or greater. In another preferred embodiment, the subject has a BMI of at least about 20 kg / m². In another preferred embodiment, the subject may have blood glucose levels above the average for their age and weight, but not high enough to be diagnosed with diabetes. In another preferred embodiment, the subject may also be an individual with a family history of diabetes.

[0144] The compositions of the present invention can be combined with any other known drugs or therapies for the treatment of diseases.

[0145] In one embodiment, the compositions disclosed herein can be used in combination with a drug for treating diabetes, which may be a currently marketed drug, such as insulin, metformin, sulfonylureas mainly including glimepiride, glibenclamide, gliclazide, gliquidone, etc., alpha-glucosidase inhibitors such as acarbose, etc., and other marketed and under-development drugs for treating diabetes. In one embodiment, the diabetes drug is metformin or insulin.

[0146] The foregoing disclosure provides a general overview of this application. A more complete understanding can be obtained by referring to the specific examples below. These examples are described for illustrative purposes only and are not intended to limit the scope of this application. Changes in form and substitutions of equivalents may be considered where circumstances may suggest or become convenient. Although specific terminology is used herein, it is for descriptive purposes and not for limiting purposes.

[0147] The following non-limiting embodiments are used to describe the content of the present invention:

[0148] Example

[0149] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0150] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0151] Example 1: Plasmid Construction and Validation

[0152] As shown in Figure 1, the inventors constructed a secretory expression vector pKN012-GCGR-IgG2 / Fc encoding a GCGR-IgG2 / Fc fusion protein. This fusion protein comprises unmutated human IgG2 / Fc (containing the hinge, CH2, and CH3 regions of the human IgG2 heavy chain, i.e., Hinge-CH2-CH3, whose amino acid sequence is shown in SEQ ID NO:2), or mutant human IgG2 / Fc (mutated at C222S, A330S, and P331S sites, whose amino acid sequence is shown in SEQ ID NO:3), a linker peptide sequence (whose amino acid sequence is shown in SEQ ID NO:8), a signal peptide sequence (whose amino acid sequence is shown in SEQ ID NO:7, SEQ ID NO:23, and / or SEQ ID NO:24), and a human GCGR polypeptide (whose amino acid sequence is shown in SEQ ID NO:1). A cDNA fragment encoding the GCGR-IgG2 / Fc fusion protein (as shown in SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30) was chemically synthesized and inserted into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GCGR-IgG2 / Fc plasmid vector.

[0153] To establish CHO-S cells stably expressing the GCGR-IgG2 / Fc fusion protein, electroporation (electropy) was used to transfect 1x10 cells with 20 μg of linearized pKN012-GCGR-IgG2 / Fc. 7CHO-S cells were transfected. Twenty-four hours post-transfection, cells were cultured in CD-CHO medium containing MSX (methionine sulfoxide imide, 100 μM / L), with the medium replaced every 3 days until the recombinant plasmid was stably integrated into the genome. Single cells were seeded in 96-well plates using a limiting dilution method and gradually expanded to form stable monoclonal cell lines, which were then stored in liquid nitrogen. Selected cell lines were cultured in shake flasks, and the GCGR-IgG2 / Fc fusion protein was purified and isolated using a Protein A chromatography column. The purified GCGR-IgG2 / Fc fusion protein was detected and identified using SDS-PAGE protein electrophoresis and Western blotting with GCGR and Fc antibodies. The cell culture supernatant was purified to obtain the GCGR-IgG2 / Fc fusion protein (its amino acid sequence is shown in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6). After quantification by BCA method, 10 μg was taken and treated with or without DTT (to reduce disulfide bonds in the molecule). SDS-PAGE electrophoresis was followed by Coomassie brilliant blue staining. R represents the reduced form (with DTT, single-chain molecule), and NR represents the non-reduced form (without DTT, double-chain molecule). The gel staining results showed that the final GCGR-IgG2 / Fc fusion protein was of high purity. Figure 2 shows that the corresponding fusion protein band (SEQ ID NO:4) could be detected by Western blot analysis.

[0154] Example 2: HEK293 reporter gene assay for detecting GCGR peptide fusion protein activity and competitive inhibition.

[0155] Human GCGR(Luc)HEK293 reporter gene cells not only express the CREB signal transduction response element but also the full-length human GCGR receptor. This receptor can drive the luciferase expression system through stimulation by GCGR agonists or glucagon. In the absence of agonists or glucagon, the GCGR receptor is not activated, resulting in low luminescence signal. In the presence of agonists or glucagon, luminescence activating the GCGR pathway can be detected in a dose-dependent manner.

[0156] After diluting the required samples, add them to the cultured 96-well plates at gradient concentrations according to their corresponding concentrations, and then incubate at 37°C. After incubation, aspirate the supernatant from each well, add 100 μL of reporter gene cell lysis buffer, and lyse at room temperature for 10 min. Then, gently pipette to mix 15 times, avoiding the generation of air bubbles during the pipetting process. Transfer 75 μL of the lysed cell suspension from each well to the corresponding 96-well plate, and centrifuge at 2000g for 30 s to remove air bubbles. Add 50 μL of firefly luciferase assay reagent to each well, and read the values ​​of the 96-well plate within 10 min using a microplate reader.

[0157] The concentration gradients of GCGR-IgG2 / Fc fusion protein (928-01 in Figure 3) were 50 nM, 250 nM, and 1000 nM, respectively.

[0158] The glucagon concentration gradients were 0.000128 nM, 0.00064 nM, 0.0032 nM, 0.016 nM, 0.08 nM, 0.4 nM, 2 nM, and 10 nM.

[0159] Experimental results show that the GCGR-IgG2 / Fc fusion protein can bind well to the GCGR receptor.

[0160] As shown in Figure 3, the 1000 nM GCGR-IgG2 / Fc fusion protein can effectively inhibit Glucagon activity.

[0161] The fusion protein of this invention can reduce hepatic gluconeogenesis, promote pancreatic β-cell proliferation, and increase insulin synthesis and secretion, and can be used in the preparation of drugs for the prevention or treatment of diabetes.

[0162] Table 1 Amino acid sequence list

[0163] Table 2 Nucleic Acid Sequence List

[0164] The foregoing examples list what is 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 modifications and equivalent examples that are included within the spirit and scope of the appended claims.

[0165] 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.

[0166] 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. A biologically active GCGR peptide fusion protein, the fusion protein comprising a GCGR peptide and an IgG2 / Fc domain, wherein the GCGR peptide comprises extracellular amino acids of GCGR, the GCGR peptide is covalently linked to an immunoglobulin Fc domain, and the IgG2 / Fc domain is a mutated or non-mutated IgG2 / Fc.

2. The GCGR polypeptide fusion protein according to claim 1, wherein, The GCGR polypeptide has an amino acid sequence that is at least 90% identical to that of SEQ ID NO:1; and / or The IgG2 / Fc domain has a sequence of SEQ ID NO:2 or SEQ ID NO:

3.

3. The GCGR polypeptide fusion protein according to any one of claims 1-2, wherein, The fusion protein further comprises a linker peptide for connecting the GCGR polypeptide and the IgG2 / Fc domain. Preferably, the linker peptide has any amino acid sequence selected from SEQ ID NO:8, 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, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, with SEQ ID NO:8 being the most preferred.

4. The GCGR polypeptide fusion protein according to any one of claims 1-3, wherein, The fusion protein further comprises a signal peptide, preferably selected from one or more of human CD33 signal peptide, human GCGR signal peptide and silkworm FH signal peptide, or the signal peptide comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:23 or SEQ ID NO:24, or comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7 or SEQ ID NO:23 or SEQ ID NO:

24.

5. The GCGR polypeptide fusion protein according to any one of claims 1-4, wherein the fusion protein has an amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

6. A polynucleotide comprising a polynucleotide encoding the GCGR polypeptide fusion protein of any one of claims 1-5.

7. The polynucleotide of claim 6, having a polynucleotide sequence of SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, or having a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:

30.

8. A vector comprising the polynucleotide of claim 6 or 7.

9. A pharmaceutical composition comprising the GCGR polypeptide fusion protein of any one of claims 1-5, or the polynucleotide of claim 6 or 7, or the carrier of claim 8, and optionally a pharmaceutically acceptable carrier.

10. The use of a GCGR polypeptide fusion protein as described in any one of claims 1-5, a polynucleotide as described in claim 6 or 7, a carrier as described in claim 8, or a pharmaceutical composition as described in claim 9 in the preparation of a medicament. Preferably, the drug is a drug for treating or preventing diseases related to glucose metabolism. Preferably, the glucose metabolism-related disease is selected from diabetes, and more preferably, the diabetes is type 2 diabetes.

11. A recombinant cell comprising the polynucleotide as described in claim 6 or 7 and / or the vector as described in claim 8.

12. The recombinant cells according to claim 11, wherein the cells are derived from human embryonic kidney cells 293 (HEK293 cells), or CHO-K1 cells, or CHO-S cells, or CHO-DG44 cells.

13. A method for constructing recombinant cells as described in claim 11 or 12, comprising: A polynucleotide encoding the GCGR polypeptide fusion protein was introduced into a vector to construct an expression vector; the expression vector was then introduced into cells to obtain recombinant cells. Preferably, the carrier is a pKN012 carrier.

14. A method for producing a biologically active GCGR peptide fusion protein, comprising the step of obtaining the GCGR peptide fusion protein using recombinant cells as described in claim 11 or 12 or recombinant cells prepared by the construction method as described in claim 13.

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