Improved combination therapy of fusion protein of GLP-1 receptor agonist

Abstract

The present invention relates to a combination therapy of an improved GLP-1 receptor agonist or a fusion protein comprising the improved GLP-1 receptor agonist, e.g., a fusion protein comprising the improved GLP-1 receptor agonist. The compound is combined with gamma-aminobutyric acid (GABA) to be used for treating or preventing metabolic diseases related to glucose metabolism and / or lipid metabolism disorders, complications of the metabolic diseases, nervous system diseases and other related diseases.

Description

[0001] This application is a divisional application of a Chinese invention patent application with the application number 202380010196.X, the application date of June 21, 2023, and the invention title of "An improved fusion protein of GLP-1 receptor agonist and its application". The original application is the Chinese national phase application of a PCT patent application with the international application number PCT / CN2023 / 101978. Technical Field

[0002] The present invention relates to the field of biomedicine. Specifically, the present invention relates to an improved GLP-1 receptor agonist, a fusion protein comprising the improved GLP-1 receptor agonist, a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a cell, and their applications. Background Art

[0003] Glucagon-like peptide-1 (GLP-1), also known as incretin, is secreted by intestinal L cells and has multi-organ targeted regulatory effects, including promoting the secretion of pancreatic islet hormones, inhibiting the release of glucagon, slowing gastric emptying, reducing appetite, and playing an important regulatory role in nutrient uptake and promoting nutrient absorption. The biological action of GLP-1 is mainly through activating the GLP-1 receptor (GLP-1R). GLP-1R is a G protein-coupled membrane protein, mainly expressed in pancreatic β cells, and also has a certain degree of expression in other tissues and cells (lungs, heart, kidneys, gastrointestinal tract, and brain). After GLP-1 binds to the receptor, it activates adenylate cyclase (AC), thereby stimulating the generation of the second messenger cyclic adenosine monophosphate (cAMP), and binding to protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor (camp GEFs) Epac family. [1] .

[0004] The half-life of natural GLP-1 in the human body is only 1-2 minutes, mainly due to rapid enzymatic inactivation including dipeptidyl peptidase IV (DPP-IV) [2] and / or renal clearance rate [3] resulting therefrom. Therefore, scientists have developed a variety of long-acting anti-degradation GLP-1 analogs. For example, human GLP-1 analogs undergo amino acid substitution [4,5] and / or N-terminal modification, including fatty acylation [6] N-acetylation modification [7] to extend the circulating half-life. Albumin-conjugated GLP-1 (albiglutide) also has an extended half-life. [8]。In recent years, a variety of GLP-1 receptor agonists and their analogs have been widely used in the treatment of metabolic diseases related to glucose metabolism and / or lipid metabolism disorders, especially type 2 diabetes mellitus (T2DM) and obesity. GLP-1 receptor agonists play an important role in the treatment of diabetes and also have preventive and therapeutic effects on [9] cardiovascular diseases [10,11] and neurological diseases. In addition, GLP-1 can also bind to GLP-1 receptors in the kidneys, skin, etc., affecting tissue metabolism and related diseases

[12] 。

[0005] US Patent US8658174 discloses a GLP-1 fusion protein, which contains a GLP-1 polypeptide fused with an IgG / Fc domain and can be used for the treatment of diabetes.

[0006] Studies have shown that there are post-translational modifications in the GLP1-Fc fusion protein. Hou et al. reported in 2019 that increasing nicotinamide and cysteine during cell culture helps to reduce the hydroxylation level of a GLP analog fused with an IgG4 / Fc protein (dulaglutide)

[13] 。

[0007] Using genetic engineering recombinant protein technology to produce fusion proteins for therapeutic use aims to express the properties of natural polypeptides. The process involves cell engineering steps of transcription, translation, and post-translational modification, and its process directly affects the physical and chemical characteristics, conformation, in vivo half-life, biological activity, and production yield of the drug. Therefore, there is still a need for improved GLP-1 receptor agonists and their fusion proteins, such as those suitable for large-scale production, with higher yields and activities, and longer half-lives for clinical treatment. SUMMARY OF THE INVENTION

[0008] The object of the present invention is to provide an improved GLP-1 receptor agonist and its fusion protein with higher activity and yield. The fusion protein can be obtained through various ways or methods, such as replacing amino acids at specific positions in the protein sequence, or performing modifications such as hydroxylation and oxidation, such as hydroxylation at K34 of the GLP-1 polypeptide and / or reducing the oxidation of the GLP-1 polypeptide. In addition, the amino acid substitution or modification at specific positions of the GLP-1 fusion protein of the present invention significantly prolongs the half-life of the improved fusion protein, and at the same time has good preventive and therapeutic effects in human diseases and animal disease models.

[0009] Therefore, one of the advantages of the present invention is to provide an improved GLP-1 fusion protein, which has the characteristics of increased yield and activity, or extended half-life. In addition, the improved GLP-1 fusion protein provided by the present invention has outstanding advantages in reducing blood sugar, for example, reducing the level of glycated hemoglobin (HbA1c), reducing the level of fasting plasma glucose (FPG), etc., and has a low incidence of adverse reactions (such as hypoglycemia, nausea, diarrhea, constipation, etc.).

[0010] In one aspect, the present invention provides a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions selected from the group consisting of: A8G, G22E, and R36G relative to native human GLP-1; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

[0011] In one embodiment, the GLP-1 polypeptide has a certain level of hydroxylation at lysine 34 (K34) relative to native human GLP-1. In one embodiment, the GLP-1 polypeptide is substantially unoxidized at tryptophan 31 (W31) relative to native human GLP-1.

[0012] In one embodiment, the GLP-1 polypeptide has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and comprises one or more amino acid substitutions selected from the group consisting of: A8G, G22E, and R36G relative to native human GLP-1.

[0013] In one embodiment, the IgG2-Fc domain is the Fc domain from human IgG2. In one embodiment, the IgG2-Fc domain has a certain level of oxidation at methionine 253 (M253) corresponding to position 253 of SEQ ID NO:7.

[0014] In one embodiment, the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

[0015] In one embodiment, the fusion protein of the present invention comprises a GLP-1 polypeptide as shown in SEQ ID NO:3 and an immunoglobulin Fc domain as shown in SEQ ID NO:6.

[0016] In one embodiment, the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain via a linker. In one embodiment, the GLP-1 fusion protein of the present invention comprises a GLP-1 polypeptide as shown in SEQ ID NO:3, a linker as shown in SEQ ID NO:9, and an immunoglobulin Fc domain as shown in SEQ ID NO:6.

[0017] In one embodiment, the fusion protein further comprises a signal peptide.

[0018] In another aspect, the present invention provides a dimer comprising two identical peptide chains linked by a disulfide bond, wherein each peptide chain comprises the fusion protein described herein.

[0019] In another aspect, the present invention provides a polynucleotide comprising a polynucleotide encoding the fusion protein described herein.

[0020] In another aspect, the present invention provides a vector comprising the polynucleotide described herein.

[0021] In another aspect, the present invention provides a cell comprising the polynucleotide or vector described herein.

[0022] In another aspect, the present invention provides a composition comprising the fusion protein, dimer, polynucleotide, vector, or cell described herein.

[0023] In another aspect, the present invention provides a method for constructing the cell described herein, comprising:

[0024] a) introducing the polynucleotide encoding the fusion protein into a vector to construct an expression vector;

[0025] b) introducing the expression vector into a cell that recombinantly expresses or naturally expresses lysyl hydroxylase to obtain a recombinant cell;

[0026] Preferably, the level or activity of lysyl hydroxylase expressed by the cell that recombinantly expresses or naturally expresses lysyl hydroxylase is higher than the level or activity of lysyl hydroxylase expressed by COS-7 cells,

[0027] More preferably, the cell is a CHO cell, particularly a CHO-K1 cell,

[0028] Even more preferably, the vector is a pKN012 vector.

[0029] In another aspect, the present invention provides a method for constructing a recombinant cell, comprising:

[0030] c) Inserting the polynucleotide sequence shown in SEQ ID NO: 26 into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GLP1-IgG2 / Fc expression vector;

[0031] d) Introducing the pKN012-GLP1-IgG2 / Fc expression vector into CHO-K1 cells to obtain recombinant cells.

[0032] In another aspect, the present invention provides a method for producing a fusion protein, comprising the step of obtaining a fusion protein using the cells described herein or the cells prepared by the construction method.

[0033] In another aspect, the present invention provides a method for detecting the quality of a fusion protein, wherein the fusion protein comprises a GLP-1 polypeptide and an immunoglobulin IgG2-Fc domain, and the method comprises the following steps: detecting the hydroxylation level of the fusion protein at K34 relative to native human GLP-1.

[0034] In another aspect, the present invention provides the use of the fusion protein, dimer, polynucleotide, vector, cell, or composition described herein in a drug, and / or in the preparation of a drug for treating or preventing a disease.

[0035] Other features and advantages of the present invention will be apparent from the detailed description in the following embodiments. The detailed description and specific examples are given only by way of illustration while the preferred embodiments of the present invention are described, and various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art. Description of the Drawings

[0036] Figure 1 is a schematic diagram of the vector pKN012-GLP1-IgG2 / Fc.

[0037] Figure 2 is a mass spectrometry diagram of the enzymatically digested peptide segment 27-34: EFIAWLVK(+16Da) modified peptide segment of the GLP-1 fusion protein YN-011 sample after Lys-C digestion. Among them, A: primary mass spectrometry diagram of EFIAWLVK; B: primary mass spectrometry diagram of EFIAWLVK(+16Da) modification; C: secondary mass spectrometry of EFIAWLVK; D: secondary mass spectrometry diagram of EFIAWLVK(+16Da) modification.

[0038] Figure 3AAre the hydroxylation data detected by ultraviolet of the modified peptide 27-34 (aa21-28 in the figure) after digestion with Lys-C: the modified peptide EFIAWLVK(+16Da) (aa21-28(+16Da) in the figure), the unmodified peptide 54-79 without +16Da (aa48-73 in the figure), and the unmodified peptide 224-240 without +16Da (aa218-234 in the figure); Figure 3B Is Figure 3A An enlarged view of the small figure.

[0039] Figure 4 Are the mean plasma concentration-time curves after single and multiple subcutaneous injections of 1 mg, 2 mg, 3 mg, and 4 mg of YN-011 in T2DM subjects. Figure 4 A and Figure 4 B are the mean plasma concentration-time curves after single subcutaneous administration; Figure 4 C and Figure 4 D are the mean plasma concentration-time curves after multiple subcutaneous administrations.

[0040] Figure 5 Is a design chart showing a Phase IIa double-blind, placebo-controlled study in T2DM subjects, which evaluated the efficacy and safety of subcutaneous administration of YN-011 at dose levels of 1 mg, 2 mg, 3 mg, and 4 mg. Among them, solid triangles represent the administration of the official dose of YN-011, solid dots represent the detection of glucose tolerance test (OGTT), hollow triangles represent adaptive administration, and pentagrams represent safety evaluation tests.

[0041] Figure 6 Is a graph showing the effect of multiple doses of YN-011 on fasting blood glucose in T2DM subjects. (At each test time point, compared with placebo, P<0.05 at dose levels of 3 mg and 4 mg for YN-011.)

[0042] Figure 7 Is a graph showing the effect of multiple doses of YN-011 on HbA1c in T2DM subjects. (At each test time point, compared with placebo, P<0.05 at dose levels of 1 mg, 3 mg, and 4 mg for YN-011.)

[0043] Figure 8 Is a graph showing the change rate of body weight (BW) of obese rhesus monkeys after repeated subcutaneous injection of YN-011.

[0044] Figure 9It is the result graph after stimulating neuron cells SH-SY5Y with TNF-α at different concentrations for 48 hours (*P<0.05 TNF-α (20 ng / ml) vs control; **P<0.01 TNF-α (40, 60, 80, 100 ng / ml) vs control).

[0045] Figure 10 It shows the inhibitory effect of YN-011 and GABA on TNF-α reducing the viability of SH-SY5Y cells (**P<0.01 TNF-α vs control; #P<0.05 YN-011 (10 nM or 100 nM) or GABA (100 μM) + TNF-α vs TNF-α; ##P<0.01 YN-011 (500 nM) + TNF-α vs TNF-α; n = 6).

[0046] Figure 11 It shows that the combined use of YN-011 and GABA significantly increases the viability of SH-SY5Y cells (**P<0.01 TNF-α vs control, #P<0.05 TNF-α + GABA + YN-011 vs TNF-α + YN-011; n = 6).

[0047] Figure 12 It shows that YN-011 reduces the apoptosis of neuron cells SH-SY5Y induced by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 10 nM YN-011 + TNF-α vs TNF-α; ##P<0.01 100 nM or 500 nM YN-011 + TNF-α vs TNF-α; n = 3).

[0048] Figure 13 It shows that GABA reduces the apoptosis of neuron cells SH-SY5Y induced by TNF-α (**P<0.01 TNF-α vs control, #P<0.05 10 μM or 100 μM GABA + TNF-α vs TNF-α; n = 3).

[0049] Figure 14 It shows that the combined use of YN-011 and GABA reduces the apoptosis of neuron cells SH-SY5Y induced by TNF-α (**P<0.01 TNF-α vs control, #P<0.05 GABA or YN-011 + TNF-α vs GABA + YN-011 + TNF-α; n = 3).

[0050] Figure 15 It is the live cell staining graph, which shows that TNF-α promotes the damage of neuron cells SH-SY5Y (**: significant; ***: extremely significant).

[0051] Figure 16 It is a live cell staining image, which shows that YN-011 has a protective effect on neuron cells damaged by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 10 nM YN-011 + TNF-α vs TNF-α; ##P<0.01 100 nM or 500 nM YN-011 + TNF-α vs TNF-α; n = 3).

[0052] Figure 17 It is a live cell staining image, which shows that GABA has a protective effect on neuron cells damaged by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 10 μM GABA + TNF-α vs TNF-α; ##P<0.01 100 μM GABA + TNF-α vs TNF-α; n = 3).

[0053] Figure 18 It is a live cell staining image, which shows that the combined use of YN-011 and GABA has a protective effect on neuron cells damaged by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 GABA or YN-011 + TNF-α vs GABA + YN-011 + TNF-α; n = 3).

[0054] Figure 19 It shows that YN-011 reduces apoptosis of neuron cells induced by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 100 nM or 500 nM YN-011 + TNF-α vs TNF-α; n = 3).

[0055] Figure 20 It shows that GABA reduces apoptosis of neuron cells induced by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 100 μM GABA + TNF-α vs TNF-α; n = 3).

[0056] Figure 21 It shows that the combined use of YN-011 and GABA reduces apoptosis of neuron cells induced by TNF-α (**P<0.01 TNF-α vs control; #P<0.05 100 nM YN-011 + TNF-α vs 100 μM GABA + 100 nM YN-011 + TNF-α; ##P<0.01 100 μM GABA + TNF-α vs 100 μM GABA + 100 nM YN-011 + TNF-α; n = 3).

[0057] Figure 22It was shown that YN-011 reduced TNF-α-induced apoptosis of neuronal cells (**P < 0.01 TNF-α vs control; #P < 0.05 10 nM, 100 nM or 500 nM YN-011 + TNF-α vs TNF-α; n = 3).

[0058] Figure 23 It was shown that GABA reduced TNF-α-induced apoptosis of neuronal cells (**P < 0.01 TNF-α vs control; #P < 0.05 100 μM GABA + TNF-α vs TNF-α; n = 3).

[0059] Figure 24 It was shown that the combined use of YN-011 and GABA reduced TNF-α-induced apoptosis of neuronal cells (**P < 0.01 TNF-α vs control; #P < 0.05 TNF-α + 100 μM GABA + 100 nM YN-011 vs TNF-α + 100 μM GABA; ##P < 0.05 TNF-α + 100 μM GABA + 100 nM YN-011 vs TNF-α + 100 nM YN-011; n = 3).

[0060] Figure 25 It was shown that GABA decreased Aβ 1-42 oligomer-induced inflammatory factor mRNA expression (*P < 0.05, **P < 0.01 Aβ 1-42 oligomer vs control; #P < 0.05 GABA + Aβ 1-42 oligomer vs Aβ 1-42 oligomer; n = 3).

[0061] Figure 26 It was shown that YN-011 decreased Aβ 1-42 oligomer-induced inflammatory factor mRNA expression (*P < 0.05, **P < 0.01 Aβ 1-42 oligomer vs control; #P < 0.05, ##P < 0.01 YN-011 + Aβ 1-42 oligomer vs Aβ 1-42 oligomer; n = 3).

[0062] Figure 27 It was shown that the combined use of YN-011 and GABA decreased Aβ 1-42 oligomer-induced inflammatory factor mRNA expression (*P < 0.05, **P < 0.01 Aβ 1-42 oligomer vs control; #P < 0.05, GABA + YN-011 + Aβ 1-42Oligomers vs GABA + Aβ 1-42 Oligomers or YN-011 + Aβ 1-42 Oligomers; n = 3).

[0063] Figure 28 It shows that GABA reduces Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory factor expression (**P < 0.01 Aβ 1-42 Oligomers vs control; #P < 0.05 GABA + Aβ 1-42 Oligomers vs Aβ 1-42 Oligomers; n = 3).

[0064] Figure 29 It shows that YN-011 reduces Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory factor expression (**P < 0.01 Aβ 1-42 Oligomers vs control; #P < 0.05 YN-011 + Aβ 1-42 Oligomers vs Aβ 1-42 Oligomers; n = 3).

[0065] Figure 30 It shows that the combined use of YN-011 and GABA reduces Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory factor expression (**P < 0.01 Aβ 1-42 Oligomers vs control; #P < 0.05, GABA + YN-011 + Aβ 1-42 Oligomers vs GABA + Aβ 1-42 Oligomers or YN-011 + Aβ 1-42 Oligomers; n = 3). Detailed implementation mode

[0066] The following is a detailed description to help those skilled in the art practice the present 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 belongs. The terms used in this invention are only for describing specific embodiments and are not intended to limit the present invention. All publications, patent applications, patents, drawings and other references mentioned herein are incorporated herein by reference in their entirety.

[0067] I. Definitions or terms

[0068] Unless otherwise specified, the terms defined herein and used herein should be understood as having the meaning of a dictionary definition, or the definition incorporated in the document, and / or the well-known meaning of the defined term.

[0069] All references, patents, and patent applications referred to herein are incorporated by reference into the subject matter they each cite, and in some cases, may cover the entire content of the document.

[0070] All features disclosed in this specification can be combined in any manner. Each feature disclosed in this specification can be replaced by a replacement feature for the same, equivalent, or similar purpose. Thus, unless otherwise explicitly stated, each feature disclosed is merely an example of a series of equivalent or similar features.

[0071] 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 unnatural amino acid residues, whether or not there are post-translational modifications (e.g., glycosylation or phosphorylation). The polypeptides in the present invention can include, for example, 3 to 3500 natural or unnatural amino acid residues. The protein can be a single peptide chain or a multi-subunit protein (e.g., can be composed of 2 or more polypeptides). The "peptide", "polypeptide", and "protein" described herein can be used interchangeably, can contain natural amino acids, can also contain unnatural amino acids, or amino acid analogs and mimetics. The peptides, polypeptides, or proteins described in this application can be obtained by any method known in the art, such as but not limited to, natural isolation, recombinant expression, chemical synthesis, etc.

[0072] As used herein, the term "amino acid" refers to an organic compound containing an amino group (-NH 2 ) and a carboxyl group (-COOH) functional group, as well as a side chain unique to each amino acid. Amino acid names are also represented in this application by standard single-letter or three-letter codes, summarized as follows:

[0073] Name Three-letter code One-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0074] As used herein, the term "GLP-1 polypeptide" includes GLP-1 receptor agonist polypeptides having a lysine at amino acid residue position 34 or corresponding to amino acid residue position 34, such as the polypeptides shown in SEQ ID NO:1 or SEQ ID NO:2. Specifically, it includes but is not limited to GLP-1(7-37), GLP-1(7-36)amide (which can also be interchangeably referred to herein as GLP-1(7-36) amide, GLP-1(7-36)amide, DPP-IV resistant GLP-1, and other GLP-1 analogs having a lysine at amino acid residue position 34 or corresponding to amino acid residue position 34. For example, the GLP-1 polypeptide can contain components from Liraglutide (from Novo Nordisk ), Semaglutide (from Novo Nordisk )、Albiglutide (from GlaxoSmithKline), )、Taspoglutide (Roche), Dulaglutide (from Eli Lilly and Company), ) or the GLP-1 polypeptide of LY2428757 (Eli Lilly and Company), and may also include the GLP-1 polypeptides disclosed in WO2021163972A1, CN111217915A, WO2011056713A2 and WO2000034332A1, each of which is incorporated herein by reference. For example, the above polypeptide may be a GLP-1 analogue containing the "KG" amino acid motif sequence.

[0075] As used herein, the term "polynucleotide" or "oligonucleotide" refers to two or more covalently linked nucleotides. Unless otherwise clearly specified in the context, the term generally includes, but is not limited to, deoxyribopolynucleotides (DNA) and ribopolynucleotides (RNA), which may be single-stranded (ss) or double-stranded (ds). For example, the polynucleotide molecule or polynucleotide of the present invention may 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. A mixture of single-stranded and double-stranded regions, a hybrid molecule containing DNA and RNA, which may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, the polynucleotide molecule may consist of a triple-stranded region containing RNA or DNA or both RNA and DNA. As used herein, the term "oligonucleotide" generally refers to a polynucleotide having a length of no more than 200 base pairs and may be single-stranded or double-stranded. The sequences provided herein may be DNA sequences or RNA sequences, however, it should be understood that the sequences provided include DNA and RNA, as well as complementary RNA and DNA sequences, unless otherwise clearly specified in the context. For example, the sequence 5'-GAATCC-3' should be understood to include 5'-GAAUCC-3', 5'-GGATTC-3' and 5'-GGAUUC-3'.

[0076] As used herein, the terms "sequence identity" or "sequence homology" refer to the percentage of sequence identity between two polypeptide sequences or two polynucleotide sequences. To determine the percentage identity between two amino acid sequences or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid or polynucleotide sequence for optimal alignment with the second amino acid or polynucleotide sequence), and then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical to the reference sequence being compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). 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 molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = 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 identity between two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul

[14] , as modified by Karlin and Altschul

[15] . This algorithm is incorporated into the NBLAST and XBLAST programs

[16] , and BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameters set, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a particular polynucleotide molecule. BLAST protein searches can be performed using the XBLAST program parameters set, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain a gapped alignment for comparison purposes, Gapped BLAST can be used

[17] . Alternatively, PSI-BLAST can be used to perform an iterative search to detect distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm proposed by Myers and Miller

[18] , this algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percentage identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating the percentage identity, usually only exact matches are counted.

[0077] In the present invention, "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue without eliminating the required properties of the protein. Suitable conservative amino acid substitutions can be made by replacing amino acids with similar hydrophobicity, polarity, and R-chain length with each other. Examples of conservative substitutions include replacing one nonpolar (hydrophobic) residue with another nonpolar residue (e.g., alanine, isoleucine, valine, leucine, or methionine), replacing one polar (hydrophilic) residue with another polar residue (e.g., between arginine and lysine), between glutamine and asparagine, between glycine and serine, replacing one basic residue with another basic residue (e.g., lysine, arginine, or histidine), or replacing one acidic residue with another acidic residue (e.g., aspartic acid or glutamic acid). The phrase "conservative substitution" also includes using chemically derived residues or unnatural amino acids in place of non-derived residues, provided that such polypeptides exhibit the necessary activity.

[0078] In the present invention, the term "fusion protein" refers to a protein that contains two or more polypeptides that form different functional domains. For example, the GLP-1 fusion protein described herein contains a GLP-1 polypeptide and an immunoglobulin Fc domain.

[0079] In the present invention, the term "linker" refers to any chemical moiety capable of covalently linking one moiety to another. For example, a "linker" can be an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or a length between 5 and 15, 20, 30, 50, or more amino acid residues, linked by peptide bonds and used to link one or more polypeptides. The linker may or may not have a secondary structure. Linker sequences are known in the art, for example, see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994).

[0080] In the present invention, the term "CH2" refers to constant heavy chain 2, which is one of the domains of the immunoglobulin heavy chain. Similarly, the term "CH3" refers to constant heavy chain 3, which is another domain of the immunoglobulin heavy chain.

[0081] In the present invention, the term "hinge", when used in the context of immunoglobulins (e.g., IgG), refers to the flexible region between the antigen-binding fragment (Fab) and the crystallizable fragment (Fc).

[0082] As used herein, the term "vector" refers to a vehicle into which a genetic element can be operably inserted and which enables the genetic element to be expressed, such as to produce a protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce or transfect host cells so that the genetic element they carry is expressed within the host cells. By way of example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Vectors can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. Additionally, vectors can contain an origin of replication. Vectors can also include components that assist their entry into cells, including but not limited to, viral particles, liposomes or protein coats. Vectors can be expression vectors or cloning vectors.

[0083] The terms "DPPIV" and "DPP-IV" refer to dipeptidyl peptidase IV, which is an enzyme that can inactivate native GLP-1.

[0084] The term "hydroxylation level" refers to the percentage of residues at an amino acid position in a polypeptide sample that are modified by hydroxylation. For example, a hydroxylation level of 20% means that 20% (by number of molecules) of the polypeptide molecules are hydroxylated at a particular amino acid position, and the hydroxylation level of the expressed protein can be increased or decreased using a modulator. For example, when present in an expression system, minoxidil and Zn 2+ (e.g., from ZnSO 4 ) can inhibit hydroxylation and reduce the hydroxylation level. The hydroxylation level can be measured using the methods in the present embodiment or by mass spectrometry as described by Hou et al

[13] , or as further described herein.

[0085] The term "oxidation level" refers to the percentage of residues at an amino acid position in a polypeptide sample that are modified by oxidation. For example, an oxidation level of 2% means that 2% (by number of molecules) of the polypeptide molecules are oxidized at a particular amino acid position. The oxidation level can be measured using the methods in the present embodiment or by the mass spectrometry method described in WO2002046227A2 or according to the protein oxidation assay method of Bettinger et al

[19] , or as described herein.

[0086] In the present 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.

[0087] In the present invention, the term "treatment" refers to administering to a subject an effective amount of a compound, composition or formulation, 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 beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviating or improving one or more symptoms or disorders, reducing the severity of a disease, stabilizing (i.e., not worsening) the disease state, preventing the spread of a disease, reversing a disease, ameliorating or alleviating a disease, and remission (whether partial or complete, or temporary) of the disease state. Beneficial or desired clinical results include improved fasting blood glucose levels and / or HbA1c levels, weight loss, improved liver lipid content, and improved cognitive function, motor coordination, etc.

[0088] In the present invention, the term "subject" is also referred to as a patient and includes all animals, including mammals, as used herein, and preferably refers to humans. A "subject" can also be a domestic animal, such as cattle, pigs, sheep, poultry and horses; or a rodent, such as a rat, mouse; or a primate, such as an ape, monkey, chimpanzee, gorilla, orangutan, baboon; or a domestic pet, such as a dog and a cat.

[0089] In the present invention, the term "pharmaceutically acceptable carrier" refers to any carrier, reagent, excipient that is acceptable in a biological or other aspect. Its use in a therapeutic formulation is acceptable unless the carrier, reagent, excipient is incompatible with the active ingredient. The use of such pharmaceutically acceptable carriers is well known in the art. For example, the various components that can be included in a pharmaceutical formulation are described in Reference 20

[20] 。

[0090] In the present invention, the term "therapeutically effective amount" refers to any dose that causes a desired effect in a subject, where the desired effect refers to alleviating symptoms, slowing disease progression, preventing disease onset, etc. This amount can be effective with multiple administrations and / or achieve the desired effect over a period of time. As used herein, the term can refer to an amount that causes a reduction in blood glucose in a subject.

[0091] In the present invention, "co-administration" etc. means, for example, with respect to two or more substances (such as two or more compounds, two or more compositions, etc.), these two or more substances are administered to a subject and they are all biologically active simultaneously. The exact circumstances of administration will depend on the pharmacokinetics of the two or more substances in the presence of each other.

[0092] When understanding the scope of the present invention, the term "comprising" and its derivatives as used herein are open-ended terms that specify the recited features, elements, components, groups, integers, and / or steps, but do not preclude the presence of other unspecified features, elements, components, groups, integers, and / or steps. The foregoing also applies to words having similar meanings, such as the terms "including", "having", and their derivatives.

[0093] The terms "consisting of" and "consisting essentially of" as used herein are closed-ended terms that specify the presence of the recited features, elements, components, groups, integers, and / or steps and also preclude the presence of other unspecified features, elements, components, groups, integers, and / or steps.

[0094] Numerical ranges recited herein by endpoints include all numbers and fractions subsumed 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 fractions thereof are assumed to be modified by the term "about".

[0095] In addition, degree terms such as "substantially", "about", and "approximately" as used herein represent a reasonable deviation amount of the modified term such that the end result is not significantly changed. If such a deviation does not negate the meaning of its modifier, these degree terms should be construed to include a deviation of at least ±5% of the modified term. More specifically, the term "about" means ±0.1 - 25%, ±1 - 20%, ±1 - 15%, ±1 - 10%, e.g., up to 10%, up to 5% of the reference number.

[0096] As used in this specification and the appended claims, the singular forms "a", "an" include plural references unless the context clearly dictates otherwise. Thus, for example, a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally used herein in its meaning including "and / or" unless the context clearly dictates otherwise.

[0097] In addition, the definitions and embodiments described in a particular section are intended to apply to other embodiments described herein, which can be understood by those skilled in the art. For example, in the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other aspect or aspects, unless clearly indicated that they cannot be combined. In particular, any feature indicated as being preferred or advantageous can be combined with any other one or more features indicated as being preferred or advantageous.

[0098] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, specific methods and materials are also described in this embodiment.

[0099] II. Proteins and fusion proteins

[0100] The present invention provides stable GLP-1 polypeptides and fusion proteins comprising said GLP-1 polypeptides fused to, for example, an IgG / Fc domain.

[0101] In one aspect of the present invention, there is provided a GLP-1 fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions relative to native human GLP-1 selected from the group consisting of: A8G, G22E, and R36G; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

[0102] GLP-1 polypeptide

[0103] Surprisingly, the yield and activity of the GLP-1 fusion proteins of the present invention can be improved by increasing the level of hydroxylation at lysine 34 (K34) relative to native human GLP-1, or by reducing oxidation at tryptophan 31 (W31) relative to native human GLP-1 and / or introducing one or more site mutations in the GLP-1 polypeptide (e.g., one or more mutations among A8G, G22E, R36G) or site mutations in the IgG2-Fc domain (e.g., one or more mutations among C222S, A330S, P331S).

[0104] Native human GLP-1 polypeptide in its unprocessed form has 37 amino acids, and its amino acid sequence is shown in SEQ ID NO: 43, and is usually also referred to as "GLP-1(1-37)". The native human GLP-1 polypeptide in its unprocessed form is processed in the pancreas and small intestine to form GLP-1(7-37) or GLP-1(7-36) amide. Unless otherwise specified, the amino acid positions of the GLP-1 polypeptide mentioned in this application correspond to the amino acid positions of SEQ ID NO: 43. For example, the K34 of the GLP-1 polypeptide mentioned in this application corresponds to the 34th position of SEQ ID NO: 43. Another example, GLP-1(7-36) amide refers to the GLP-1 polypeptide fragment formed by the amino acids between the 7th and 36th positions of SEQ ID NO: 43; GLP-1(7-37) refers to the GLP-1 polypeptide fragment formed by the amino acids between the 7th and 37th positions of SEQ ID NO: 43. In some embodiments, the amino acid sequence of GLP-1(7-36) amide is shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO: 2.

[0105] Unless otherwise specified, the naming rule of the amino acid mutations mentioned in this application is: amino acid name before mutation – amino acid position where mutation occurs – amino acid name after mutation. For example, the A8G mutation in the GLP-1 polypeptide refers to the alanine (A) at the 8th position corresponding to SEQ ID NO: 43 being mutated to glycine (G).

[0106] The hydroxylation level of the GLP-1 fusion protein of the present invention can be measured by a variety of methods known in the prior art. For example, the mass spectrometry method described by Hou et al. is used for measurement

[13] . Unless otherwise specified, the hydroxylation level described in this application is calculated based on the proportion of the number of molecules. For example, in 100 g of the GLP-1 fusion protein of the present invention, if 10 g of the GLP-1 fusion protein has hydroxylation at K34 of the GLP-1 polypeptide, and the remaining 90 g of the GLP-1 fusion protein has no hydroxylation at K34 of the GLP-1 polypeptide, then the hydroxylation level of the GLP-1 fusion protein is considered to be 10%.

[0107] The GLP-1 fusion protein of the present invention has an increased in vivo half-life and / or yield during recombinant production in some embodiments. Therefore, the GLP-1 fusion protein and reagents for preparing the GLP-1 fusion protein can be used to prepare drugs.

[0108] In one embodiment, the GLP-1 polypeptide has a certain level of hydroxylation at lysine 34 (K34) relative to native human GLP-1. K34 refers to the lysine residue at position 34 of the native human GLP-1 polypeptide. Those skilled in the art will appreciate that the same lysine can correspond to different positions in other GLP-1 polypeptide sequences.

[0109] The GLP-1 fusion protein can refer to multiple molecules that can be hydroxylated or non-hydroxylated at K34, and the level of hydroxylation of these molecules can vary.

[0110] The present invention also provides a composition comprising the GLP-1 fusion protein. In one embodiment, the level of hydroxylation of the GLP-1 fusion protein at K34 is about 10% to 100%, such as at least about 20%, at least about 26%, at least about 30%, at least about 40%, or at least about 50%. Any percentage or range between 10% and 100% is contemplated.

[0111] In one embodiment, the level of hydroxylation is between 10% and 100%, such as greater than or equal to 10%, or greater than or equal to 15%, or greater than or equal to 20%, or greater than or equal to 26%, or greater than or equal to 30%, or greater than or equal to 35%, or greater than or equal to 40%, or greater than or equal to 45%, or greater than or equal to 50%, or greater than or equal to 55%, or greater than or equal to 60%, or greater than or equal to 65%, or greater than or equal to 70%, or greater than or equal to 75%, or greater than or equal to 80%, or greater than or equal to 85%, or greater than or equal to 90%, or greater than or equal to 95%.

[0112] As shown in the examples of the present application, the yield of the hydroxylated GLP-1 fusion protein is about 100-fold to about 500-fold higher than that of the GLP-1 fusion protein in which hydroxylation at K34 of GLP-1 is not detected.

[0113] The present invention also found that the GLP-1 polypeptide has a low level of oxidation at tryptophan 31 (W31) relative to native human GLP-1, and the low level of oxidation may be beneficial to the stability and / or activity of the GLP-1 fusion protein. In certain embodiments, the GLP-1 polypeptide is substantially unoxidized at tryptophan 31 (W31) relative to native human GLP-1. In certain embodiments, the level of oxidation of the GLP-1 polypeptide at W31 relative to native human GLP-1 is less than 0.5% (e.g., less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) or undetectable. W31 refers to the tryptophan residue at position 31 of the native human GLP-1 polypeptide. Those skilled in the art will appreciate that the same tryptophan can correspond to different positions in other GLP-1 polypeptides.

[0114] It can be detected by methods known in the embodiments of the present application or in the prior art (e.g., the mass spectrometry method described in WO2002046227A2 or Bettinger et al.)

[19] a protein oxidation assay method). Unless otherwise specified, the oxidation level described in the present application is calculated based on the proportion of the number of molecules. For example, in 100 g of the GLP-1 fusion protein of the present invention, if 10 g of the GLP-1 fusion protein is oxidized at W31 of GLP-1 and the remaining 90 g of the GLP-1 fusion protein is not oxidized at W31 of GLP-1, the oxidation level of the GLP-1 fusion protein is considered to be 10%.

[0115] The GLP-1 polypeptide of the fusion protein disclosed herein can be human GLP-1. In certain embodiments, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and comprises the A8G and G22E substitutions relative to native human GLP-1.

[0116] In certain embodiments, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and comprises the A8G, G22E, and R36G substitutions relative to native human GLP-1.

[0117] In some embodiments, the GLP-1 polypeptide is GLP-1(7-37). In some embodiments, the GLP-1 polypeptide is GLP-1(7-36) amide. In some embodiments, the GLP-1 polypeptide is DPP-IV resistant GLP-1. In some embodiments, the GLP-1 polypeptide can comprise amino acid substitutions, such as one, two, or three mutations among A8G, G22E, and R36G.

[0118] In certain embodiments, the GLP-1 polypeptide described in the present application 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 compared to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and comprises one or more amino acid substitutions selected from the group consisting of A8G, G22E and R36G relative to native human GLP-1. In certain embodiments, the GLP-1 polypeptide described in the present application 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 compared to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and stimulates insulin secretion by β-cells in a glucose-dependent manner. In certain embodiments, the GLP-1 polypeptide described in the present application 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 compared to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and comprises one or more amino acid substitutions selected from the group consisting of A8G, G22E and R36G relative to native human GLP-1, and stimulates insulin secretion by β-cells in a glucose-dependent manner.

[0119]

[0120] In certain embodiments, the GLP-1 polypeptide is human GLP-1(7-37) and comprises the A8G, G22E and R36G substitutions relative to native human GLP-1.

[0121] In one embodiment, the amino acid sequence of the GLP-1 polypeptide comprises the amino acid sequence shown in SEQ ID NO:3. In certain embodiments, the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3.

[0122] In one embodiment, the GLP-1 polypeptide is DPP-IV resistant human GLP-1.

[0123] Native GLP-1 has a short circulatory half-life (t1 / 2 < 2 minutes), which is mainly due to rapid enzymatic inactivation including dipeptidyl peptidase IV (DPP-IV) and / or renal clearance. Therefore, when using native GLP-1, continuous subcutaneous infusion by a pump must be used to maintain the action of GLP-1 in the body.

[21] Therefore, long-acting anti-degradation GLP-1 mimetic peptides for medicinal use have been developed. For example, dulaglutide is a DPP-IV protected GLP-1 analogue fused with an IgG4 / Fc fragment and has a half-life of 4.7 - 5.5 days.

[22] 。

[0124] Fc domain

[0125] In certain embodiments, the IgG2-Fc domain described in the present application is the Fc domain from human IgG2.

[0126] In the present application, "IgG2-Fc" and "IgG2 / Fc" can be used interchangeably and both refer to the Fc domain of immunoglobulin IgG2.

[0127] In certain embodiments, the IgG2-Fc domain described in the present application has a certain level of oxidation at methionine (M253) corresponding to position 253 of SEQ ID NO:7. In certain embodiments, the level of oxidation of the IgG2-Fc domain described in the present application at methionine (M253) corresponding to position 253 of SEQ ID NO:7 is reduced. In certain embodiments, the IgG2-Fc domain described in the present application is not oxidized at methionine (M253) corresponding to position 253 of SEQ ID NO:7. In the present application, M253 refers to the methionine residue at position 253 of IgG2 corresponding to SEQ ID NO:7. Those skilled in the art will know that the same methionine can correspond to different positions in other IgG polypeptides.

[0128] In certain embodiments, the level of oxidation of the IgG2-Fc domain at M253 corresponding to SEQ ID NO:7 is less than or equal to about 15%, less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, less than or equal to about 1%, less than or equal to about 0.5% or undetectable.

[0129] As mentioned above, the GLP-1 fusion protein can refer to multiple molecules, and these molecules can be oxidized or unoxidized at one or both of the positions of M253 in the IgG2-Fc domain and / or W31 in the GLP-1 polypeptide, and the levels of oxidation of these molecules can be different. The oxidation occurs at the rectangle in the following amino acid structural formula:

[0130]

[0131] In certain embodiments, no oxidation was detected at W31 of the GLP-1 polypeptide of the GLP-1 fusion protein of the present invention relative to native human GLP-1, and the oxidation level at M253 of the IgG2-Fc domain was approximately 2%. The oxidation levels of other GLP-1 analog fusion proteins at W31 (for the GLP-1 polypeptide) and / or M253 (for the IgG2-Fc domain) were both 2-8%.

[0132] The GLP-1 polypeptide in the fusion protein is covalently linked (directly or through a linker peptide) to the Fc portion of an immunoglobulin. In one embodiment, the immunoglobulin is IgG. The IgG-Fc of the fusion protein disclosed herein can be IgG2-Fc.

[0133] In one embodiment, the IgG2-Fc domain contains a C222S substitution. The C222S substitution of IgG2-Fc can increase 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 cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0134] In another embodiment, the IgG2-Fc domain contains A330S and P331S substitutions. The A330S and P331S substitutions reduce the affinity for the Fcγ receptor and C1q complement protein.

[0135] In one embodiment, the IgG2-Fc domain contains C222S, A330S, and P331S substitutions.

[0136] Except that the amino acid position of M253 corresponds to the amino acid position of SEQ ID NO:7, the other amino acid residue positions of the IgG2-Fc domain described in the present application, including for example A330, P331, and C222, correspond to the positions shown in human IgG2 such as Genbank accession number QRG33935.1. The IgG2-Fc portion can be 228 amino acids as shown in SEQ ID NO:5, corresponding to amino acids 219 to 445 of human IgG2 as shown in Genbank accession number QRG33935.1, for example. Those skilled in the art will easily identify the residue positions of amino acids, such as the residue positions of A330, P331, and C222 in the Fc fragment, shorter or longer, shown in the reference sequences SEQ ID NO:5 or SEQ ID NO:6.

[0137] In one embodiment, the IgG2-Fc domain of the present application 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 compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S. Wherein the fusion protein has an improved half-life compared to a GLP-1 polypeptide without an IgG / Fc domain fusion or fused with an IgG4 / Fc domain.

[0138] 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 compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0139] In one embodiment, the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and comprises A330S and P331S substitutions. Preferably, the IgG2-Fc domain further comprises a C222S substitution. More preferably, the amino acid sequence of the IgG2-Fc domain is as shown in SEQ ID NO:6.

[0140] In one embodiment, in the GLP-1 fusion protein of the present application, the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3, and the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO:6.

[0141]

[0142] The correspondence of the positions of the A8G, G22E, R36G substitutions and K34 hydroxylation of the GLP-1 polypeptide mentioned in the present application in SEQ ID NO:3 and in SEQ ID NO:7 is shown in the following table. Additionally, the correspondence of the positions of the C222S, A330S, P331S substitutions and M253 oxidation on the IgG2-Fc domain mentioned in the present application relative to QRG33935.1 and in SEQ ID NO:7 is also shown in the following table.

[0143]

[0144]

[0145] In one embodiment, the GLP-1 polypeptide is located at the N-terminus of the immunoglobulin Fc domain. In one embodiment, the GLP-1 polypeptide is located at the C-terminus of the immunoglobulin Fc domain.

[0146] Linker

[0147] In one embodiment, the GLP-1 polypeptide is directly covalently linked to the immunoglobulin Fc domain.

[0148] In one embodiment, the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain via a linker.

[0149] In one embodiment, the linker is selected from the group consisting of: a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0150] In one embodiment, the linker comprises a linking peptide.

[0151] The GLP-1 polypeptide and the IgG-Fc domain, such as the IgG2-Fc domain, of the fusion protein disclosed herein can be linked by a linking peptide. As used herein, the term "linking peptide" refers to any moiety that links different functional domains of a polypeptide together. The linking peptide can have any suitable length and structure.

[0152] In the present invention, the term "cleavable linker" refers to a linker that is sensitive to in vivo proteases, pH, or chemical factors and is readily cleaved in the presence of such factors.

[0153] In the present invention, the term "non-cleavable linker" refers to a linker that is stable to in vivo proteases, pH, or chemical factors and is not readily cleaved.

[0154] In the present invention, the term "flexible linker" refers to a linker that can increase the spatial extensibility when linking different protein components, such that the spatial folding and conformation of the protein components are minimally affected by each other.

[0155] In the present invention, the term "rigid linker" refers to a linker that can maintain a fixed distance between protein components when linking different protein components.

[0156] In the present invention, the term "helical linker" refers to a linker in which the rigid units can form a helix (such as an α-helix) within themselves or with adjacent identical sequences, thereby enabling the resulting fusion protein to have a relatively stable three-dimensional conformation.

[0157] In the present invention, the term "non-helical linker" refers to a linker that cannot form a helical structure.

[0158] In one embodiment, the linker peptide comprises a linker containing glycine and serine. Preferably, the linker containing glycine and serine comprises one, two, three, four or more repeats as shown in SEQ ID NO:39 (GGGS), SEQ ID NO:40 (GGGGS), SEQ ID NO:41 (GGGGGS) or SEQ ID NO:42 (GGGGGGGGS).

[0159] In one embodiment, the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.

[0160] Name Amino acid sequence SEQ ID NO: Linker peptide SSGAPPPSGGGGS 9 Linker peptide GGGGSGGGGSGGGGS 10 Linker peptide GGGGSGGGGSGGGGSGGGGS 11 Linker peptide GSTGGGGSGKPGSGEGGGGS 12 Linker peptide ESGRSGGGGSGGGGS 13 Linker peptide EGKSSGSGSESKST 14 Linker peptide EGKSSGSGSESKSTQ 15 Linker peptide EGKSSGSGSESKVD 16 Linker peptide GSTSGSGKSSEGKG 17 Linker peptide KESGSVSSEQLAQFRSLD 18 Linker peptide ESGSVSSEELAFRSLD 19 Linker peptide GGGS 39 Linker peptide GGGGS 40 Linker peptide GGGGGS 41 Linker peptide GGGGGGGS 42

[0161] In one embodiment, the linker peptide linking the GLP-1 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 the amino acid sequence shown in SEQ ID NO:9. In one embodiment, the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9. In one embodiment, the amino acid sequence of the linker peptide is as shown in SEQ ID NO:9.

[0162] In one embodiment, in the GLP-1 fusion protein described in the present application, the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3, the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO:6, and the amino acid sequence of the linker is as shown in SEQ ID NO:9.

[0163] Fusion protein

[0164] In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:7. In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:7, and the GLP-1 polypeptide contains A8G, G22E, R36G substitutions, and the IgG2-Fc domain contains C222S, A330S and P331S substitutions. In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:7, and stimulates insulin secretion by β-cells in a glucose-dependent manner. In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:7, and the GLP-1 polypeptide contains A8G, G22E, R36G substitutions, the IgG2-Fc domain contains C222S, A330S and P331S substitutions, and stimulates insulin secretion by β-cells in a glucose-dependent manner.

[0165] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% (e.g., at least about 85%, at least about 90% or at least about 95%) sequence identity with SEQ ID NO:7.

[0166] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:7.

[0167] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:7.

[0168] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:7.

[0169]

[0170] As demonstrated in the examples, the GLP-1 fusion proteins disclosed herein, after sequence modification, have a longer half-life. For example, the GLP-1 fusion protein YN-011 has a half-life of approximately 8.6 days (207 h).

[0171] In one embodiment, the GLP-1 fusion protein of the present invention further comprises a signal peptide.

[0172] As used herein, the term "signal peptide" refers to a polypeptide that directs the secretion of a fusion protein into the extracellular medium. Such a polypeptide may also be referred to as a "leader peptide", "polypeptide precursor", "prepeptide", etc. The use of signal peptides to direct protein secretion is known in the art (e.g., U.S. Patent US8658174, the content of which is incorporated herein by reference in its entirety). Examples of signal peptides include, but are not limited to, the human CD33 signal peptide, the human growth hormone-releasing hormone (GHRH) signal peptide, the human alpha-1-microglobulin / bikunin precursor (AMBP) signal peptide, the Gaussia luciferase signal peptide, the murine immunoglobulin heavy chain signal peptide, and the murine immunoglobulin kappa light chain signal peptide. The signal peptide is cleaved during secretion. In some embodiments, cleavage of the signal peptide results in a reactive histidine residue at the N-terminus of the GLP-1 polypeptide.

[0173] In one embodiment, the signal peptide is the human CD33 signal peptide.

[0174] In one embodiment, the signal peptide 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 shown in SEQ ID NO:4 (MPLLLLLPLLWAGALA) and permits secretion of the fusion protein. In one embodiment, the signal peptide has the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:4. In one embodiment, the signal peptide has the amino acid sequence shown in SEQ ID NO:4.

[0175] In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:8. In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:8, and the GLP-1 polypeptide contains A8G, G22E, R36G substitutions, and the IgG2-Fc domain contains C222S, A330S and P331S substitutions. In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:8, and stimulates insulin secretion by β-cells in a glucose-dependent manner. In one embodiment, the fusion protein described in the present application 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 shown in SEQ ID NO:8, and the GLP-1 polypeptide contains A8G, G22E, R36G substitutions, the IgG2-Fc domain contains C222S, A330S and P331S substitutions, and stimulates insulin secretion by β-cells in a glucose-dependent manner.

[0176] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% (e.g., at least about 85%, at least about 90% or at least about 95%) sequence identity with SEQ ID NO:8.

[0177] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:8.

[0178] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:8.

[0179] In one embodiment, the fusion protein described in the present application has the amino acid sequence shown in SEQ ID NO:8.

[0180]

[0181] In one embodiment, the half-life of the fusion protein described in the present application in a subject (e.g., a human subject) is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0182] In another preferred embodiment, the GLP-1 fusion protein is of pharmaceutical grade.

[0183] Another aspect of the present invention provides a dimer comprising two identical peptide chains linked by a disulfide bond, wherein each peptide chain comprises the fusion protein as described herein.

[0184] III. Nucleic Acids

[0185] In another aspect, the present invention also provides a polynucleotide comprising a polynucleotide encoding the polypeptide or peptide described in the present invention (e.g., GLP-1 polypeptide, Fc fragment, fusion protein).

[0186] In another aspect, the present invention also provides a nucleic acid molecule comprising a polynucleotide encoding the polypeptide or peptide described herein (e.g., GLP-1 polypeptide, Fc fragment, fusion protein).

[0187] As used herein, the terms "nucleic acid" or "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless otherwise specified, a particular nucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixture of bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0188] In one embodiment, the polynucleotide described in the present application is codon-optimized, e.g., optimized for humans. The nucleic acid molecule can be used in the methods described herein.

[0189] Polypeptides and fusion proteins can be synthesized using standard protein chemistry techniques

[23] In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Mode1396; Milligen / Biosearch 9600). Alternatively, the peptides, polypeptides, or fragments or variants thereof described herein can be recombinantly produced using a variety of expression systems well known in the art.

[0190] In one embodiment, the polynucleotide described in the present application comprises a polynucleotide sequence as shown in SEQ ID NO:26 or as shown in SEQ ID NO:27 or a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO:26 or SEQ ID NO:27.

[0191] In one embodiment, the polynucleotide described in the present application has a polynucleotide 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:26. In another embodiment, the polynucleotide described in the present application is the polynucleotide sequence as shown in SEQ ID NO:26.

[0192] In one embodiment, the polynucleotide described in the present application has a polynucleotide 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:27. In another embodiment, the polynucleotide described in the present application is the polynucleotide sequence as shown in SEQ ID NO:27.

[0193] In certain embodiments, the polynucleotide described in the present application comprises a polynucleotide sequence as shown in any one of SEQ ID NO:20 - 22 or a polynucleotide sequence having at least 70% (e.g., 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:20 - 22.

[0194] In certain embodiments, the polynucleotide described in the present application comprises a polynucleotide sequence as shown in SEQ ID NO:24 or SEQ ID NO:25, or a polynucleotide sequence having at least 70% (e.g., 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:24 or SEQ ID NO:25.

[0195] In certain embodiments, the polynucleotide described in the present application comprises a polynucleotide sequence as shown in SEQ ID NO:23, or a polynucleotide sequence having at least 70% (e.g., 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.

[0196] In certain embodiments, the polynucleotide described in the present application comprises a polynucleotide sequence as shown in any one of SEQ ID NO:28 - 38, or a polynucleotide sequence having at least 70% (e.g., 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 the sequence shown in any one of SEQ ID NO:28 - 38.

[0197] The sequences of SEQ ID NO:20 - 38 are shown in the following table.

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] IV. Vectors and Cells

[0204] In another aspect of the present invention, there is also provided a vector comprising the polynucleotide described in the present application.

[0205] Any vector suitable for the intended purpose can be used. For example, some vectors can be introduced into an expression system, such as a mammalian, insect, or bacterial expression system, 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.

[0206] In some embodiments, the vector is used with an in vitro expression system to produce a fusion protein. The expression and purification of the fusion protein can be carried out by any suitable method known in the art.

[0207] 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, etc.). In one embodiment, the expression vector that can be used with the fusion protein of the present invention is pKN012, which can be obtained commercially (Beijing Kohnoor Science&Technology Co., Ltd).

[0208] The vector can contain suitable regulatory sequences and components. 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: transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, including translation initiation signals. In addition, 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 inducibility, 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. The recombinant expression vector can also contain a marker gene that facilitates the selection of host cells transformed, infected, or transfected with the vector for expressing the antibodies described herein. The recombinant expression vector can also contain other expression cassettes encoding, for example, fusion moieties (e.g., for generating antibody "fusion proteins") that can help with detection, including, for example, the tags and markers described herein.

[0209] In one embodiment, the vector contains one or more, optionally Figure 1 the components shown. For example, in one embodiment, the vector containing the polynucleotide encoding the GLP-1 fusion protein is pKN012-GLP1-IgG2.

[0210] A variety of methods for transducing cells can be used, 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 in vivo. Other vector systems that can be used to implement the present invention include vectors based on adenoviruses and adeno-associated viruses.

[0211] In another aspect of the present invention, there is provided a recombinant cell, which cell comprises a polynucleotide encoding the GLP-1 fusion protein, or comprises the vector described in the present application.

[0212] Stably expressing recombinant cells can be prepared, for example, by transforming, transfecting or transducing recombinant cells with a vector comprising a polynucleotide, preferably any polynucleotide described herein.

[0213] In one embodiment, in the cells of the present invention, the cells also recombinantly or naturally express lysyl hydroxylase.

[0214] In one embodiment, the level or activity of lysyl hydroxylase expressed by the cells of the present invention is higher than the level or activity of lysine hydroxylase expressed by COS-7 cells. In other words, the lysyl hydroxylase activity level of the cells is increased compared to COS-7 cells. For example, the cells can be cells modified to increase the expression of lysine hydroxylase (e.g., by preparing cells recombinantly expressing lysine hydroxylase), or can be cells having a naturally increased level of lysine hydroxylase compared to, for example, COS-7 cells. For example, the level or activity of lysyl hydroxylase expressed by the cells described in the present application is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% higher than the level or activity of lysine hydroxylase expressed by COS-7 cells.

[0215] In one embodiment, the cells described in the present application are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In one embodiment, the mammalian cells are cells derived from humans. For example, the mammalian cells are human embryonic kidney cells 293 (HEK293 cells), for example, HEK293T cells, HEK293S cells or HEK293F cells. In one embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells, for example, CHO-K1 cells, CHO-S cells or CHO-DG44 cells.

[0216] In one embodiment, the recombinant cells of the present invention are obtained by suspension domestication of Chinese hamster ovary cells, taking CHOK1 cells as an example.

[0217] V. Construction method of cells, production and detection methods of fusion proteins

[0218] In yet another aspect of the present invention, there is provided a method for constructing a recombinant cell, comprising: introducing a polynucleotide encoding the GLP-1 fusion protein of the present invention into a vector to construct an expression vector; and introducing the expression vector into a cell recombinantly or naturally expressing lysyl hydroxylase to obtain a recombinant cell.

[0219] In one embodiment, the lysyl hydroxylase expressed by the recombinant or native lysyl hydroxylase-expressing cells has an increased level or activity compared to the lysyl hydroxylase expressed by other cells (e.g., COS-7 cells).

[0220] In one embodiment, the cells are CHO cells, such as CHO-K1 cells, CHO-S cells or CHO-DG44 cells.

[0221] In one embodiment, the vector can be the pKN012 vector.

[0222] In one embodiment, the method for constructing the recombinant cells comprises the following steps:

[0223] a) Inserting the polynucleotide sequence shown in SEQ ID NO:26 into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GLP1-IgG2 / Fc expression vector;

[0224] b) Introducing the pKN012-GLP1-IgG2 / Fc expression vector into CHO-K1 cells to obtain recombinant cells.

[0225] In another embodiment, the method for constructing the recombinant cells comprises the following steps:

[0226] a) Inserting the polynucleotide sequence shown in SEQ ID NO:27 into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GLP-1-IgG2 / Fc expression vector;

[0227] b) Introducing the pKN012-GLP-1-IgG2 / Fc expression vector into CHO-K1 cells to obtain recombinant cells.

[0228] In yet another aspect of the present invention, there is provided a method for producing the GLP-1 fusion protein, comprising the step of obtaining the fusion protein using the recombinant cells described herein or the cells prepared by the construction method described herein.

[0229] As described herein, the GLP-1 fusion protein can be synthesized. As shown herein, the fusion protein can also be prepared using recombinant cells, including, for example, recombinant Chinese hamster ovary cells that recombinantly express the GLP-1 fusion protein. Accordingly, the present invention also provides a method for preparing the GLP-1 fusion protein, the method comprising culturing recombinant cells that express the GLP-1 fusion protein, wherein the culturing comprises one or more of the process steps or materials described in the examples. For example, the method may comprise one or more of the process steps described in Table 2 and / or one or more of the materials described in Table 3 or 4.

[0230] In some embodiments, the recombinant cells expressing the GLP-1 fusion protein are prepared using HEK293T, HEK293S, HEK293F, and / or CHO cells (such as CHO-K1 cells). For example, under conditions suitable for in vivo use, the recombinant cells can be used to produce recombinant polypeptides and / or fusion proteins.

[0231] In one embodiment, the method further comprises the step of: detecting the hydroxylation level of the fusion protein at K34 relative to native human GLP-1. Without being bound by any theory, it is believed that when the hydroxylation level of the GLP-1 fusion protein at K34 relative to native human GLP-1 is between 10% and 100% (e.g., greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 26%, greater than or equal to 27%, greater than or equal to 28%, greater than or equal to 29%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, or greater than or equal to 95%), the prepared fusion protein is considered qualified (e.g., meeting the drug factory standards).

[0232] In another aspect of the present invention, there is also provided a method for detecting the quality of a fusion protein, wherein the fusion protein comprises a GLP-1 polypeptide and an immunoglobulin IgG2-Fc domain, and the method comprises the following step: detecting the hydroxylation level of the fusion protein at K34 relative to native human GLP-1.

[0233] In one embodiment, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions selected from the group consisting of: A8G, G22E, and R36G; and / or the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S. In one embodiment, the GLP-1 polypeptide comprises the amino acid sequence shown in SEQ ID NO:3, and the IgG2-Fc domain comprises the amino acid sequence shown in SEQ ID NO:6. In one embodiment, the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3, and the amino acid sequence of the IgG2-Fc domain is as shown in SEQ ID NO:6.

[0234] In one embodiment, when the hydroxylation level of the fusion protein at lysine 34 (K34) relative to native human GLP-1 is between 10% and 100% (e.g., greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 26%, greater than or equal to 27%, greater than or equal to 28%, greater than or equal to 29%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, or greater than or equal to 95%), the quality of the fusion protein is confirmed as qualified. "Qualified" means meeting the drug standard system formulated by the national drug regulatory department or similar institutions, such as drug standards, drug registration standards, enterprise drug standards, pharmacopoeias, etc., so that the produced drugs meet the drug release standards.

[0235] VI. Composition

[0236] On the other hand, the present invention also provides a composition comprising the GLP-1 fusion protein, dimer, polynucleotide, vector or recombinant cell described in the present application.

[0237] In one embodiment, the composition may further comprise a suitable diluent or carrier. In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier.

[0238] In one embodiment, the composition is a pharmaceutical composition.

[0239] In one embodiment, the composition is a pharmaceutical composition comprising a GLP-1 fusion protein and a pharmaceutically acceptable carrier.

[0240] In one embodiment, the composition comprises a GLP-1 fusion protein, and the GLP-1 polypeptide of the fusion protein has a certain hydroxylation level at lysine 34 (K34) relative to native human GLP-1. In certain embodiments, in the fusion protein, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 26%, greater than or equal to 27%, greater than or equal to 28%, greater than or equal to 29%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, or greater than or equal to 95% of the GLP-1 polypeptides in the fusion protein are hydroxylated at K34 relative to native human GLP-1.

[0241] In one embodiment, the composition comprises a GLP-1 fusion protein, wherein the GLP-1 polypeptide of the fusion protein is substantially unoxidized at tryptophan 31 (W31) relative to native human GLP-1. In certain embodiments, the level of oxidation of the GLP-1 polypeptide at W31 relative to native human GLP-1 is less than 5% (e.g., less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%) or undetectable.

[0242] In one embodiment, the composition comprises a GLP-1 fusion protein, wherein the IgG2-Fc domain of the fusion protein has a certain level of oxidation at methionine 253 (M253) corresponding to SEQ ID NO:7. In certain embodiments, the level of oxidation of the IgG2-Fc domain at methionine 253 (M253) corresponding to SEQ ID NO:7 is less than or equal to 15%, such as less than or equal to 10%, less than or equal to 9%, less than or equal to 8%, less than or equal to 7%, less than or equal to 6%, 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% or undetectable.

[0243] In one embodiment, the composition further comprises buffered saline.

[0244] In one embodiment, the composition further comprises saccharides.

[0245] In one embodiment, the composition further comprises a surfactant. The pharmaceutical composition of the present invention can be prepared, packaged and / or sold as a unit dose and / or multiple unit doses. The composition can be prepared in various forms.

[0246] In one embodiment, the composition comprises a GLP-1 fusion protein formulated into a preparation with a dosage of about 0.25 mg to about 20 mg, for example, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg or about 20 mg of the GLP-1 fusion protein.

[0247] The GLP-1 fusion protein, polynucleotide, vector, recombinant cell or composition can be used for preparing a drug and / or for administration, for example, parenterally, intravenously, subcutaneously or intramuscularly.

[0248] In one embodiment, the GLP-1 fusion protein or its composition can be administered parenterally or formulated into a preparation for parenteral administration.

[0249] In one embodiment, the GLP-1 fusion protein or its composition can be administered subcutaneously or formulated into a preparation for subcutaneous administration.

[0250] In one embodiment, the GLP-1 fusion protein or its composition can be administered intravenously or formulated into a preparation for intravenous administration.

[0251] In one embodiment, the GLP-1 fusion protein or its composition can be administered intramuscularly or formulated into a preparation for intramuscular administration.

[0252] Diluents suitable for GLP-1 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 polypeptides and / or cells.

[0253] Diluents suitable for nucleic acids and / or vectors include, but are not limited to, saline solutions, pH buffer solutions and diluents described herein, as well as water and the like.

[0254] In another preferred embodiment, the diluent is sterile.

[0255] VII. Methods and Uses for Treating and Preventing Diseases

[0256] As shown herein, the GLP-1 polypeptide A8G, G22E, and / or R36G substitutions, and / or an increased level of hydroxylation at K34, a decreased level of oxidation at W31, and / or one or more substitutions of M253 in the IgG2 / Fc portion and / or C222S, A330S, and P331S in the IgG2 / Fc portion increase the yield, activity, and / or half-life of the GLP-1 fusion protein. The modified GLP-1 fusion proteins, nucleic acids, vectors, and recombinant cells described herein are suitable for preparing drugs and their compositions and for corresponding pharmaceutical therapeutic uses.

[0257] In another aspect of the invention, the fusion proteins, dimers, polynucleotides, vectors, cells, or compositions disclosed herein can be used in a subject for treating or preventing the onset of a disease or disorder or slowing its progression.

[0258] In another aspect of the invention, there is provided the use of a fusion protein, dimer, polynucleotide, vector, cell, or composition thereof as described herein as a GLP-1 receptor agonist.

[0259] In another aspect of the invention, there is provided a fusion protein, dimer, polynucleotide, vector, cell, or composition thereof for treating, preventing, or slowing the progression of a disease or disorder.

[0260] In another aspect of the invention, there is provided a fusion protein, dimer, polynucleotide, vector, cell, or composition thereof for preparing a drug for treating, preventing, or slowing the progression of a disease or disorder.

[0261] In another aspect of the invention, there is provided a method for treating, preventing, or slowing the progression of a disease or disorder by administering a therapeutically effective amount of the fusion protein, dimer, polynucleotide, vector, cell, or composition thereof to a subject in need thereof.

[0262] In another aspect of the invention, there is provided the use of a fusion protein, dimer, polynucleotide, vector, cell, or composition thereof as described herein in the preparation of a drug for treating or preventing a disease.

[0263] In another aspect of the invention, there is provided the use of a fusion protein, dimer, polynucleotide, vector, cell, or composition thereof as described herein for treating or preventing a disease.

[0264] In another aspect of the invention, there is provided a method for treating or preventing a disease, the method comprising: administering to a subject a therapeutically effective amount of a fusion protein, dimer, polynucleotide, vector, cell, or composition thereof as described herein.

[0265] In one embodiment, the drug comprising the fusion protein is administered in an amount of about 0.2 mg to about 20 mg (per person per administration) of the fusion protein. In one embodiment, the amount of the fusion protein is about 1 mg to about 10 mg. In one embodiment, the amount of the fusion protein is about 1 mg to about 5 mg. In one embodiment, the amount of the fusion protein is 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 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg or about 20 mg.

[0266] Different dosage forms can be used, and suitable dosage forms can include but are not limited to solutions, suspensions, pills, tablets.

[0267] In one embodiment, the treatment regimen can include multiple administrations.

[0268] In one embodiment, the GLP-1 fusion protein or its composition is administered once every 3 days, or once a week, or once every two weeks.

[0269] In one embodiment, the administration regimen of the GLP-1 fusion protein, dimer, polynucleotide, vector, cell or its composition is once a week followed by a drug withdrawal, and then once a week. In another preferred example, the drug withdrawal time is 2 weeks.

[0270] In one embodiment, the GLP-1 fusion protein, dimer, polynucleotide, vector, cell or its composition can be administered once followed by a two-week drug withdrawal, and then continuously administered 4 times a week. In another preferred embodiment, the treatment further includes administering an initial dose of 1 mg one week before the start of the administration regimen.

[0271] In one embodiment, the GLP-1 fusion protein, dimer, polynucleotide, vector, cell or its composition is administered by parenteral, intravenous, subcutaneous or intramuscular routes.

[0272] In one embodiment, the diseases include metabolic diseases related to glucose metabolism and / or lipid metabolism disorders, complications of metabolic diseases, neurological diseases or other related diseases.

[0273] In one embodiment, the disease or disorder is a metabolic disease associated with carbohydrate and / or lipid metabolism disorders.

[0274] In one embodiment, the metabolic disease associated with carbohydrate and / or lipid metabolism disorders is selected from the group consisting of: diabetes, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndrome.

[0275] In one embodiment, the metabolic disease associated with carbohydrate and / or lipid metabolism disorders is or includes diabetes. In one embodiment, the metabolic disease associated with carbohydrate and / or lipid metabolism disorders is type 2 diabetes. In another embodiment, the metabolic disease associated with carbohydrate and / or lipid metabolism disorders is or includes obesity. In another embodiment, the metabolic disease associated with carbohydrate and / or lipid metabolism disorders is or includes non-alcoholic fatty liver disease (NAFLD). In another embodiment, the metabolic disease associated with carbohydrate and / or lipid metabolism disorders is non-alcoholic steatohepatitis (NASH).

[0276] In one embodiment, the subject is newly diagnosed or previously diagnosed with diabetes (e.g., type 2 diabetes). Diabetes can be diagnosed in a variety of ways, such as fasting plasma glucose (FPG). According to the American Diabetes Association, diabetes is diagnosed when the fasting plasma glucose is greater than or equal to 126 mg / dl.

[0277] In one embodiment, the subject has an increased likelihood of developing diabetes (e.g., type 2 diabetes). For example, the subject may be predisposed to diabetes due to obesity or genetic susceptibility, such as when the subject has a family history of diabetes.

[0278] In one embodiment, the subject is an obese patient. Obesity can be defined by reference to the body mass index (BMI). For example, the World Health Organization (WHO) defines obesity as having a BMI equal to or greater than 30. In another embodiment, the subject has a BMI of at least about 20 kg / m 2 ². In another embodiment, the subject may have a blood glucose level higher than the average level of peers of comparable weight, but not high enough to be diagnosed with diabetes. In another embodiment, the subject may also be an individual with a family history of diabetes.

[0279] In one embodiment, the subject is newly diagnosed or previously diagnosed with NAFLD or NASH. In another embodiment, the subject has an increased likelihood of developing NAFLD or NASH. For example, the subject may have a genetic susceptibility to NAFLD or NASH.

[0280] In one embodiment, the complications of the metabolic disease include cardiovascular complications (e.g., coronary heart disease, sudden cardiac death, heart failure, etc.), kidney complications (e.g., acute kidney injury, diabetic nephropathy), or liver complications caused by the metabolic disease.

[0281] In one embodiment, the disease or disorder is a neurological disease. In one embodiment, the neurological disease is a neurodegenerative disease. In one embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), motor neuron disease, Huntington's disease, and Parkinson's disease (PD).

[0282] In one embodiment, the neurodegenerative disease is Alzheimer's disease. In another embodiment, the neurodegenerative disease is Parkinson's disease. In another embodiment, the neurodegenerative disease is motor neuron disease. In another embodiment, the neurodegenerative disease is Huntington's disease.

[0283] In one embodiment, the subject is newly diagnosed or previously diagnosed with Alzheimer's disease. In another embodiment, the subject has an increased likelihood of developing Alzheimer's disease. For example, the subject may be a subject who is predisposed to Alzheimer's disease due to the subject's genetics, such as when the subject has a family history of Alzheimer's disease or a Tau or APP mutation associated with Alzheimer's disease.

[0284] In one embodiment, the subject is newly diagnosed or previously diagnosed with Parkinson's disease. In another embodiment, the subject has an increased likelihood of developing Parkinson's disease. For example, the subject may be a subject who is predisposed to Parkinson's disease due to the subject's genetics, such as when the subject has a family history of Parkinson's disease.

[0285] The fusion protein, dimer, polynucleotide, vector, cell, or composition of the present invention can be combined with any other known drug or therapy for the treatment of diseases.

[0286] In one embodiment, the fusion proteins, dimers, polynucleotides, vectors, cells, or compositions disclosed herein can be used in combination with drugs for treating diabetes, which can be currently marketed drugs, such as insulin, metformin, sulfonylureas mainly including glimepiride, glibenclamide, gliclazide, gliquidone, etc., α-glucosidase inhibitors such as acarbose, etc., and also include other marketed and investigational drugs for treating diabetes. In one embodiment, the diabetes drug is metformin or insulin. In one embodiment, the diabetes drug is metformin or insulin. In one embodiment, the fusion proteins, dimers, polynucleotides, vectors, cells, or compositions disclosed herein can be used in combination with Alzheimer's drugs and / or non-drug interventions such as cognitive therapy. In one embodiment, the fusion proteins, dimers, polynucleotides, vectors, cells, or compositions disclosed herein are used in combination with γ-aminobutyric acid for treating neurodegenerative diseases. In one embodiment, the fusion proteins, dimers, polynucleotides, vectors, cells, or compositions disclosed herein are used in combination with γ-aminobutyric acid for treating Alzheimer's disease.

[0287] In certain embodiments, the GLP-1 fusion protein disclosed herein in combination with γ-aminobutyric acid can inhibit the effect of TNF-α on reducing the viability of SH-SY5Y cells.

[0288] In certain embodiments, the GLP-1 fusion protein disclosed herein in combination with γ-aminobutyric acid can reduce the apoptosis of neuronal cells SH-SY5Y induced by TNF-α.

[0289] In certain embodiments, the GLP-1 fusion protein disclosed herein in combination with γ-aminobutyric acid can protect neuronal cells damaged by TNF-α.

[0290] In certain embodiments, the GLP-1 fusion protein disclosed herein in combination with γ-aminobutyric acid can reduce the apoptosis of neuronal cells induced by TNF-α.

[0291] In certain embodiments, the GLP-1 fusion protein disclosed herein in combination with γ-aminobutyric acid can reduce the expression of inflammatory factors (such as TNF-α, IL-6) mRNA induced by Aβ oligomers in HMC3 microglial cells. 1-42 oligomers induced inflammatory factors (e.g., TNF-α, IL-6) mRNA expression.

[0292] In one embodiment, in the application of the present invention, the drug is a GLP-1 receptor agonist.

[0293] This application also provides the following embodiments.

[0294] Use of a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain in combination with an additional therapeutic agent in the preparation of a medicament for treating or preventing a disease, wherein,

[0295] the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions relative to native human GLP-1 selected from the group consisting of: A8G, G22E, and R36G;

[0296] the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

[0297] Use of a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain in the preparation of a medicament for treating or preventing a disease, wherein,

[0298] a) the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions relative to native human GLP-1 selected from the group consisting of: A8G, G22E, and R36G; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S; and

[0299] b) the medicament is used in combination with an additional therapeutic agent.

[0300] Embodiment 3: The use according to Embodiment 1 or 2, wherein the GLP-1 polypeptide has a certain level of hydroxylation at lysine 34 (K34) relative to native human GLP-1.

[0301] Embodiment 4: The use according to any one of Embodiments 1-3, wherein the level of hydroxylation is between 10% and 100%, such as greater than or equal to 10%, or greater than or equal to 15%, or greater than or equal to 20%, or greater than or equal to 26%, or greater than or equal to 30%, or greater than or equal to 40%, or greater than or equal to 50%, or greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%.

[0302] Embodiment 5: The use according to any one of Embodiments 1-4, wherein the GLP-1 polypeptide is substantially unoxidized at tryptophan (W31) at position 31 relative to native human GLP-1.

[0303] Embodiment 6: The use according to Embodiment 5, wherein the oxidation level of the GLP-1 polypeptide at W31 relative to native human GLP-1 is less than 0.5% or undetectable.

[0304] Embodiment 7: The use according to any one of Embodiments 1-6, wherein the GLP-1 polypeptide has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and comprises one or more amino acid substitutions selected from the group consisting of: A8G, G22E and R36G relative to native human GLP-1.

[0305] Embodiment 8: The use according to any one of Embodiments 1-7, wherein the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide and DPP-IV resistant human GLP-1, and comprises A8G and G22E substitutions relative to native human GLP-1.

[0306] Embodiment 9: The use according to Embodiment 8, wherein the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide and DPP-IV resistant human GLP-1, and comprises A8G, G22E and R36G substitutions relative to native human GLP-1.

[0307] Embodiment 10: The use according to Embodiment 9, wherein the GLP-1 polypeptide is human GLP-1(7-37), and comprises A8G, G22E and R36G substitutions relative to native human GLP-1.

[0308] Embodiment 11: The use according to any one of the foregoing embodiments, wherein the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3.

[0309] Embodiment 12: The use according to any one of Embodiments 1-11, wherein the IgG2-Fc domain is the Fc domain from human IgG2.

[0310] Embodiment 13: The use according to any one of the foregoing embodiments, wherein the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0311] Embodiment 14: The use according to any one of the foregoing embodiments, wherein the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6, and comprises A330S and P331S substitutions.

[0312] Embodiment 15: The use according to Embodiment 14, wherein the IgG2-Fc domain further comprises a C222S substitution.

[0313] Embodiment 16: The use according to Embodiment 15, wherein the amino acid sequence of the IgG2-Fc domain is as shown in SEQ ID NO:6.

[0314] Embodiment 17: The use according to any one of the foregoing embodiments, wherein the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3, and the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO:6.

[0315] Embodiment 18: The use according to any one of the foregoing embodiments, wherein the GLP-1 polypeptide is located at the N-terminus or C-terminus of the immunoglobulin Fc domain.

[0316] Embodiment 19: The use according to any one of the foregoing embodiments, wherein the GLP-1 polypeptide is directly covalently linked to the immunoglobulin Fc domain.

[0317] Embodiment 20: The use according to any one of Embodiments 1-19, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain through a linker.

[0318] Embodiment 21: The use according to Embodiment 20, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0319] Embodiment 22: The use according to Embodiment 21, wherein the linker comprises a linking peptide that links the GLP-1 polypeptide and the IgG2-Fc domain.

[0320] Embodiment 23: The use according to Embodiment 22, wherein the linker peptide comprises a linker containing glycine and serine.

[0321] Embodiment 24: The use according to Embodiment 23, wherein the linker containing glycine and serine comprises one, two, three, four or more repeats as shown in SEQ ID NO:39 (GGGS), SEQ ID NO:40 (GGGGGS), SEQ ID NO:41 (GGGGGS) or SEQ ID NO:42 (GGGGGGGS).

[0322] Embodiment 25: The use according to any one of Embodiments 20-24, wherein the linker comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.

[0323] Embodiment 26: The use according to Embodiment 25, wherein the linker comprises the amino acid sequence as shown in SEQ ID NO:9.

[0324] Embodiment 27: The use according to any one of Embodiments 20-26, wherein the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3, the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO:6, and the amino acid sequence of the linker is as shown in SEQ ID NO:9.

[0325] Embodiment 28: The use according to any one of the foregoing embodiments, wherein the fusion protein has the amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:7.

[0326] Embodiment 29: The use according to any one of the foregoing embodiments, wherein the IgG2-Fc domain has a certain level of oxidation at methionine (M253) corresponding to position 253 of SEQ ID NO:7.

[0327] Embodiment 30: The use according to Embodiment 29, wherein the oxidation level of the IgG2-Fc domain at M253 corresponding to SEQ ID NO:7 is less than or equal to 5%.

[0328] Embodiment 31: The use according to any one of the foregoing embodiments, wherein the fusion protein further comprises a signal peptide.

[0329] Embodiment 32: The use according to Embodiment 31, wherein the signal peptide is a human CD33 signal peptide.

[0330] Embodiment 33: The use according to Embodiment 31 or 32, wherein the signal peptide has the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4.

[0331] Embodiment 34: The use according to any one of Embodiments 31-33, wherein the fusion protein has the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8.

[0332] Embodiment 35: The use according to any one of the foregoing embodiments, wherein the half-life of the fusion protein in a human subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days.

[0333] Embodiment 36: The use according to any one of the foregoing embodiments, wherein in the fusion protein, greater than or equal to 10%, or greater than or equal to 15%, or at least greater than or equal to 20%, or greater than or equal to 26%, or greater than or equal to 30%, or greater than or equal to 40%, or greater than or equal to 50%, or greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90% of the GLP-1 polypeptide in the fusion protein is hydroxylated at K34 relative to native human GLP-1.

[0334] Embodiment 37: The use according to any one of the foregoing embodiments, wherein the disease is selected from the group consisting of: metabolic diseases related to glucose metabolism and / or lipid metabolism disorders, complications of metabolic diseases, central metabolic diseases (e.g., neurological diseases), and other related metabolic diseases.

[0335] Embodiment 38: The use according to Embodiment 37, wherein the metabolic diseases related to glucose metabolism and / or lipid metabolism disorders are selected from the group consisting of: diabetes, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndrome.

[0336] Embodiment 39: The use according to Embodiment 37 or 38, wherein the metabolic disease associated with sugar metabolism and / or lipid metabolism disorders is diabetes (e.g., type 2 diabetes, type 2 diabetes with poor blood glucose control after diet and exercise intervention).

[0337] Embodiment 40: The use according to Embodiment 37, wherein the complications of the metabolic disease include cardiovascular complications (such as heart failure), kidney complications (such as diabetic nephropathy), or liver complications (such as fatty liver, including NAFLD and NASH) caused by the metabolic disease.

[0338] Embodiment 41: The use according to Embodiment 37, wherein the neurological disease is a neurodegenerative disease.

[0339] Embodiment 42: The use according to Embodiment 41, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

[0340] Embodiment 43: The use according to any one of the foregoing embodiments, wherein the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea drugs (e.g., glimepiride, glibenclamide, gliclazide, gliquidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid.

[0341] Embodiment 44: The use according to Embodiment 43, wherein the additional therapeutic agent is γ-aminobutyric acid.

[0342] Embodiment 45: Use of a fusion protein comprising the amino acid sequence shown in SEQ ID NO:7 and γ-aminobutyric acid in the preparation of a medicament for the treatment of neurodegenerative diseases.

[0343] Embodiment 46: The use according to Embodiment 45, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

[0344] Embodiment 47: The use according to Embodiment 45 or 46, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:7.

[0345] Embodiment 48: A pharmaceutical combination comprising a fusion protein and an additional therapeutic agent, wherein the fusion protein comprises a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions relative to native human GLP-1 selected from the group consisting of: A8G, G22E, and R36G; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

[0346] Embodiment 49: The pharmaceutical combination according to Embodiment 48, wherein the additional therapeutic agent is selected from the group consisting of: insulin, metformin, sulfonylurea drugs (e.g., glimepiride, glibenclamide, gliclazide, gliquidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid.

[0347] Embodiment 50: The pharmaceutical combination according to Embodiment 49, wherein the additional therapeutic agent is γ-aminobutyric acid.

[0348] Embodiment 51: A pharmaceutical combination comprising a fusion protein and γ-aminobutyric acid, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:7.

[0349] Embodiment 52: A pharmaceutical combination comprising a fusion protein and γ-aminobutyric acid, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:7.

[0350] The above disclosure generally describes the present application. A more complete understanding can be obtained by referring to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the present application. Changes in form and substitution of equivalents may be considered when the circumstances may suggest or make it convenient. Although specific terms are used herein, these terms are for descriptive rather than limiting purposes.

[0351] The following non-limiting examples are used to describe the present invention.

[0352] Examples

[0353] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0354] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0355] Example 1: Expression and Activity Detection of Fusion Protein

[0356] 1.1 Plasmid construction

[0357] A vector encoding the GLP-1 fusion protein was constructed. This fusion protein contains human GLP-1(7-37) and human IgG2 / Fc (including the hinge, CH2, and CH3 regions in the human IgG2 heavy chain, i.e., Hinge-CH2-CH3). The prepropeptide sequence of human CD33 (hCD33) was fused with the GLP-1 sequence to direct the secretion of the synthesized peptide into the culture medium. A cDNA fragment encoding the fusion protein hCD33-GLP-1-IgG2 / Fc (hinge-ch2-ch3) was chemically synthesized (as shown in SEQ ID NO:27), and it was inserted into the NcoI and HindIII sites of the pKN012 vector to generate pKN012-GLP-1-IgG2 / Fc.

[0358] The pKN012-GLP-1-IgG2 / Fc stable expression vector was transformed into competent Escherichia coli DH5α cells. Plasmids were extracted from the strain containing pKN012-GLP-1-IgG2 / Fc and digested with PvuI. After electrophoresis, there was a target band with the correct molecular weight. The linearized plasmid was quantified after ethanol precipitation and used for stable transfection.

[0359] To establish CHO-K1 cells (Lot#58995535 / ATCC) stably expressing GLP-1-IgG2 / Fc, by electroporation (electrotransfection) technology, 2 μg of linearized pKN012-GLP-1-IgG2 / Fc was transfected into CHO-K1 cells growing in 6-well plates (2.5x 10 5 cells / well). 24 hours after transfection, the cells were dispersed and cultured in CD-CHO medium containing MSX (methionine sulfoximine, 100 μM / L) to select those cells that had stably integrated the recombinant plasmid into the genome. The medium was changed every 3 days until colonies formed. Single colonies were isolated and amplified into a stable cell line, and the GLP-1 fusion protein (YN-011) in the tissue culture supernatant of the cell line growing in 24-well plates was tested using a rat GLP-1 RIA kit. Cells capable of secreting the fusion protein were selected for further identification. The amino acid sequence of the prepared GLP-1 fusion protein is as shown in SEQ ID NO:7 (also referred to as "YN-011" in this application).

[0360] 1.2 Detection of YN-011 activity by inducing cAMP level

[0361] Natural GLP-1 stimulates insulin secretion from β-cells in a glucose-dependent manner. To evaluate whether purified YN-011 has the function of natural GLP-1, the effect on insulin secretion of cloned insulin-secreting INS-1 cells was determined. INS-1 cells were treated with serum and glucose starvation, and then treated with different amounts of purified YN-011 in the presence of 0, 5 or 20 mM glucose as shown. In the absence of glucose, YN-011 did not stimulate insulin secretion from β-cells. However, in the presence of 5 mM or 20 mM glucose, YN-011 stimulated insulin secretion from β-cells in a dose-dependent manner. The data indicate that the GLP-1-IgG2 / Fc fusion protein YN-011 has biological activity and can stimulate insulin secretion in INS-1 cells in a glucose-dependent manner.

[0362] In the absence of glucose, the cAMP of INS-1 cells treated with YN-011 (120 nM) was at the basal level. In the presence of 5 mM glucose, the cAMP level of YN-011-treated cells increased significantly, comparable to the dulaglutide level.

[0363] Example 2: Increasing the hydroxylation level of GLP-1 K34 can improve protein yield and activity

[0364] 2.1 Determination of modification level by LC-MS / MS-based peptide mapping

[0365] Identification was performed by mass spectrometry analysis using Lys-C digestion. The YN-011 sample was denatured, reduced and alkylated in sequence, and then hydrolyzed into peptides by Lys-C enzyme. Online LC-MS / MS analysis was performed using a Thermo LTQ velos Orbitrap instrument, and the data was processed and analyzed using mascot software. A modification with a molecular weight increase of 16 daltons (Da) was observed for the peptide segment 21-28: EFIAWLVK. The first-order and second-order mass spectra of the peptide without the +16 Da modification and the peptide with the +16 Da modification in the YN-011 sample are shown in Figure 2. The first-order molecular weight error was within 10 ppm. By analyzing the mass-to-charge ratio of the secondary fragment ions, the site of the +16 Da modification was K28. K28 is also known as K34 because the active GLP-1 (such as the 7-37 amino acids of GLP-1) contains a deletion of the first 6 residues. The 21-28 peptide can also be called the 27-34 peptide in natural human GLP-1.

[0366] The modification level at K34 in YN-011 was detected by UV280 and EIC (Extracted Ion Chromatography) methods, and the results are shown in Table 1 and Figure 3A and 3B as shown. The calculation method of UV280 is as follows:

[0367]

[0368] Wherein:

[0369] K: The proportion of the modified peptide containing +16 Da in the total amount of the amino acid 21-28 peptide segment (the sum of the unmodified peptide segment and the +16 Da modified segment content);

[0370] A1: The absorbance value of the amino acid 21-28 peptide segment without +16 Da modification at 280 nm;

[0371] A0: The absorbance value of the amino acid 21-28 peptide segment containing +16 Da modification at 280 nm

[0372] Table 1. Detection of the K34 modification level in YN-011 by UV280 and EIC methods

[0373]

[0374] * is the relative quantification of the mass spectrometry EIC

[0375] 2.2 Hydroxylation level of K34 in YN-011

[0376] After determining that +16 Da occurs at the 34th lysine (Lys) through mass spectrometry analysis, considering that 16 Da is the molecular weight of an oxygen atom, it is suggested that it may be the result of an oxidation reaction. In fact, there are two types of enzymes in cells that catalyze the modification of the lysine side chain. One is called lysyl oxidase (refer to https: / / en.wikipedia.org / wiki / Lysyl_oxidase), and the other is lysyl hydroxylase (refer to https: / / en.wikipedia.org / wiki / Lysyl_hydroxylase). Lysyl oxidase can oxidize the amino group on the 6th carbon of the lysine side chain into an aldehyde group, but the difference between this modification and the unmodified one is 1 Da, which does not match the result of an increase of 16 Da. Lysyl hydroxylase will add a hydroxyl group to the carbon (γ position) of the lysine side chain to form stable hydroxylysine. Hydroxylysine adds a hydroxyl group and reduces a hydrogen atom on the lysine side chain, so it just increases by 16 Da compared to the original molecular weight. The hydroxylation position at K34 is as follows:

[0377]

[0378] In summary, it is highly likely that the 16 Da increase on K34 in YN-011 is caused by lysine hydroxylation. Therefore, during cell culture, a hydroxylation reaction inhibitor was added. On the one hand, it was to verify that the +16 Da modification is lysine hydroxylation, and on the other hand, to harvest proteins with different +16 Da modification ratios and study the effect of the high or low +16 Da modification ratio on the biological activity of the protein.

[0379] The cell culture process of the stable CHOK1 cells capable of secreting the fusion protein YN-011 used in Example 1 in this experiment is shown in Table 2. Tables 3 and 4 list the relevant materials and cell culture media required for cell culture. The modification level of the fusion protein was detected by the mass spectrometry analysis described above. The inventors found that the fusion protein was indeed hydroxylated at K34, with a hydroxylation level of 15% - 40%.

[0380] To detect the biological effect of K34 hydroxylation, 8 shake flasks (SF1 - SF8) were set up in the experiment for culturing CHOK1 cells stably expressing the fusion protein YN-011. SF1 - SF8 were used to evaluate various different conditions. During cell culture, a hydroxylation reaction inhibitor was added to harvest proteins with different hydroxylation modification ratios and study the effect of the high or low hydroxylation ratio on the biological activity of the protein. Minoxidil is an inhibitor of lysine hydroxylase and can inhibit lysine hydroxylation, while Zn 2+ ions can inhibit the action of hydroxylase by competing with Fe 2+ ions during the reaction process. SF1 was used as a control without adding any inhibitor. The Minoxidil used in this experiment was dissolved in 0.1 N HCl solution. HCl was added to SF2 as an HCl control. Different concentrations of Minoxidil, 0.1 mM, 0.5 mM, and 1.0 mM, were added to SF3 to SF5. SF6 and SF7 were Zn 2+ experimental groups, and the Zn 2+ reagent used was ZnSO 4 , and the ZnSO 4 concentrations used were 200 μM and 400 μM respectively. SF8 added both Minoxidil and Zn 2+ (provided by ZnSO 4 ) two inhibitors, and the total added concentrations of Minoxidil and Zn 2+ were 0.5 mM and 200 μM respectively.

[0381] Table 2. Cell culture process

[0382]

[0383] Table 3. Materials used in the cell culture process

[0384] Name Abbreviation Company Catalog number <![CDATA[Dynamis TM AGT medium]]> Dynamis Gibco A26175 CD Efficient Feed C AGT Feed C Gibco A1327501 Sheff-CHO PLUS PF ACF FM012 Sheffield 5X00483 L-Cystine Cys Applichem A3671 L-Tyrosine Tyr Applichem A3401 L-Glutamine Gln J.T.Baker 2078-06 Anhydrous glucose Glucose J.T.Baker 1919-09 Hydrochloric acid, 6.0N solution HCl J.T.Baker 0327-02 Sodium hydroxide, 10N solution NaOH J.T.Baker 5000-02 Minoxidil Minoxidil Sigma M4145 <![CDATA[ZnSO 4 > <![CDATA[Zn 2+ > Sigma Z0251 Anticoagulant ACA Gibco 0010057

[0385] Table 4. Cell culture medium and its components

[0386] Cell culture medium Composition Dynamis (0.5% ACA) IL Dynamis (added with 5 ml ACA) Feed C (containing 15 g / L FM012) Dissolve 15 g FM012 in 1 L Feed C FAA02 IL FAA02 (containing 7.95 g L-cystine and 4.75 g L-tyrosine)

[0387] 2.3 Increasing the hydroxylation level of K34 in GLP-1 can improve protein yield and activity

[0388] The results of protein yield and activity are shown in Table 5. The hydroxylation levels of the blank control SF1 and SF2 with only HCl added were 15.7% - 16.0%. From SF3 to SF5, as the concentration of minoxidil (one of the hydroxylation inhibitors) increased, the proportion of hydroxylation gradually decreased. When the dosage of minoxidil reached 0.5 mM, the hydroxylation proportion was 8.7%. When the minoxidil concentration increased to 1 mM, the proportion was 9.4%. Cell growth and protein expression were significantly inhibited: the peak density of live cells decreased by about 17%, and the protein yield decreased by 57.0% - 67.9%. In addition, when 400 μm of Zn 2+ (another hydroxylation inhibitor) was added to the cell culture, the hydroxylation level also decreased to 10.7%. In the experiment with the mixed addition of inhibitors (minoxidil, 0.5 mM; Zn 2+ , 200 μM), the hydroxylation level in SF8 also decreased to 9.6%.

[0389] When hydroxylation inhibitors such as minoxidil are present, the proportion of hydroxylation gradually decreases as the added concentration increases. Cell growth and protein expression are significantly inhibited: the peak density of live cells decreases by about 17%, and the yield gradually decreases, with a decrease of 57.0% - 67.9%; similarly, in the presence of the hydroxylation inhibitor Zn 2+ , the hydroxylation level also decreases as the concentration of Zn 2+ increases, and the protein recovery rate decreases by 22.9% - 34.2%; when the hydroxylation level reaches 15.7% - 16.0%, the recovery rate increases, and compared with the low hydroxylation level, the recovery rate doubles. Therefore, the higher the hydroxylation level, the higher the yield.

[0390] SUPA-1 is a GLP-1-IgG2 / Fc fusion protein disclosed in US Patent No. US8658174. There is no linker peptide used between the GLP-1 polypeptide and IgG2 / Fc. Except for the A8G substitution in the GLP-1 polypeptide, there are no other site mutations on the GLP-1 polypeptide and IgG2 / Fc. Hydroxylation was not detected in SUPA-1, no other modifications were detected, and the protein yield under the same conditions was 22 mg / L. When the hydroxylation level of K34 in YN-011 is 8.7% - 9.6%, the yield can reach 0.77 g / l - 1.03 g / l, which is more than 100 times that of SUPA-1. When the degree of hydroxylation of K34 is higher than 15%, the production rate of YN-011 can reach more than 500 times that of SUPA-1.

[0391] In addition, the blank control (SF1) and the two samples with the lowest hydroxylation levels (SF4 and SF5) were selected to detect the biological activity of YN-011 by the method described in 1.2 of Example 1. The results showed that SF1 was 88%, and SF4 and SF5 were 83% and 68% respectively. Therefore, the higher the hydroxylation ratio, the tendency of the biological activity to increase.

[0392] In summary, the modification on K34 in the fusion protein is indeed hydroxylation modification, and this modification greatly improves the yield of YN-011, and there is also a tendency for the biological activity to increase.

[0393] Further scaling up the cell culture scale, the YN-011 obtained from the 15 L pilot scale and three batches of 200 L reactors of YN-011 contains 20% - 30% lysine hydroxylation at position 34, and the yield of YN-011 can reach about 2.7 grams per liter.

[0394] Table 5. Summary of Yield and Protein Activity Results

[0395]

[0396] Example 3: Determination of Oxidation Level by LC-MS / MS

[0397] According to the protein oxidation assay method in Example 2 or Bettinger et al.

[19] The oxidation level was determined, and it was found that the GLP-1 fusion protein YN-011 of the present invention was not oxidized at W31, while the oxidation level of dulaglutide at the same position was greater than 5%. The oxidation occurred at the rectangle in the W31 structural formula:

[0398]

[0399] At the same time, the GLP-1 fusion protein YN-011 of the present invention has an oxidation level of about 2% - 4% at M253. While the oxidation level of dulaglutide at the same position is greater than 5%.

[0400] Example 4: Preventive and Therapeutic Effects of YN-011 in Diabetic db / db Mice (Type 2 Diabetes Model)

[0401] The effect of multiple subcutaneous injections of YN-011 on reducing blood glucose was evaluated in db / db mice (Jackson Laboratories, 000642). The mice were housed under normal light (12-hour light / 12-hour dark) and room temperature conditions and had free access to food (normal rodent chow) and water. db / db mice lack the leptin receptor and spontaneously develop obesity, hyperglycemia, and pancreatic β-cell atrophy at 4 weeks of age.

[0402] Sixty diseased db / db mice (33 - 45 g) were divided into 6 groups of 10 each (5 males and 5 females) and given 0 (solvent PBS buffer control), 0.15, 0.3, 0.6, 1.2 mg / kg of YN-011, or 0.3 mg / kg dulaglutide by subcutaneous injection. Another group of wild-type mice (17 - 22 g) with the same genetic background served as the normal control. The dosing frequency was once every 3 days (Q3D), and a total of 9 doses were administered over 26 days.

[0403] As Figure 6 shown, throughout the experimental period, the random blood glucose levels of the model control group of db / db mice remained at a relatively high level and were significantly higher than those of the normal control group mice (P < 0.001). After the first dose, YN-011 significantly reduced the random blood glucose (RPG) concentration in db / db mice, and the therapeutic effect could be observed at a dose as low as 0.15 mg / kg starting from 3 hours after dosing. In the dose range of 0.15 - 1.2 mg / kg, the duration of the hypoglycemic effect was 34 - 72 hours. YN-011 was also able to significantly promote insulin secretion and increase serum insulin levels starting from 3 hours after dosing.

[0404] After the 9th dose of YN-011, the RPG levels of YN-011-treated mice were significantly lower than those of the control group at all dose groups and measurement time points, except for no significant difference (P = 0.07) in the 0.15 mg / kg group after the 4th dose (day 10). To more intuitively understand the blood glucose decline in each group of mice, the average value of the random blood glucose decline rate at each measurement time point throughout the experiment was calculated. The results showed that the random blood glucose decline rates of the 0.15, 0.3, 0.6 YN-011 groups of mice 72 hours after the 7th dose were 31.8%, 46.2%, 50.8% respectively, and the blood glucose reduction rate of the 1.2 mg / kg YN-011 treatment group was as high as 54.3%. After repeated administration of YN-011, the effect of YN-011 in reducing RPG could be maintained for 72 hours.

[0405] Long-term administration of YN-011 can also significantly reduce the fasting blood glucose level of db / db mice. During the entire experimental period, the random blood glucose of db / db mice remained at a relatively high level, which was significantly higher than that of the normal control group mice (P<0.001). After db / db mice were continuously subcutaneously injected with different doses of YN-011 once every 3 days, the fasting blood glucose at all measured time points of the mice in each group decreased significantly compared with the model control group (P<0.001). The fasting blood glucose decline rates of the mice in the 0.15, 0.3, 0.6, and 1.2 mg / kg YN-011 groups at 54 hours (21 days) after the 7th administration were 46.8%, 55.6%, 59.5%, and 64.7% respectively. This indicates that the efficacy of reducing fasting blood glucose after continuous multiple administrations of 0.15, 0.3, 0.6, and 1.2 mg / kg YN-011 once every 3 days can be maintained for at least 54 hours (the 3rd day after administration). The fasting blood glucose at all measured time points of the mice in the positive control 0.3 mg / kg dulaglutide group also decreased significantly.

[0406] To intuitively understand the blood glucose decline of the mice in each group, the average value of the fasting blood glucose decline rate at each measured time point during the entire experiment was calculated. The average fasting blood glucose decline rates of the mice in the YN-011 groups of 0.15, 0.3, 0.6, and 1.2 mg / kg were 55.0%, 61.8%, 63.7%, and 66.1% respectively. The average fasting blood glucose decline rate of the mice in the positive control 0.3 mg / kg dulaglutide group was 63.6%.

[0407] Therefore, continuous multiple administrations of YN-011 once every 3 days can significantly reduce the fasting blood glucose of type 2 diabetic db / db mice. There is an obvious effect even at a dose of 0.15 mg / kg, and the effect of reducing fasting blood glucose of 0.15, 0.3, 0.6, and 1.2 mg / kg YN-011 can be maintained for at least 54 hours (the 3rd day after administration).

[0408] The serum fructosamine of db / db mice treated with subcutaneous injection of 9 times of 0.15 - 0.6 mg / kg YN-011 showed a decreasing trend, and the serum fructosamine level of db / db mice in the 1.2 mg / kg YN-011 treatment group decreased significantly.

[0409] The random and fasting body weights of the db / db mice in the solvent control group continued to increase during the experiment, while the random and fasting body weights of the animals in the YN-011 treatment groups of 0.3, 0.6, and 1.2 mg / kg decreased significantly (P<0.05, P<0.01, P<0.001). The SUPA-1 fusion protein disclosed in US Patent US8658174 had no obvious effect on the body weight of db / db mice. This shows that the GLP-1 fusion protein of this application has a better effect.

[0410] Compared with the solvent control group, the epididymal fat content and its ratio to body weight in the YN-011 treatment group were significantly reduced; the scapular fat content in the YN-011 groups at 0.15, 0.3, and 0.6 mg / kg was significantly reduced; the subcutaneous fat content, inguinal fat content, and their ratios to body weight in the YN-011 groups at 0.3, 0.6, and 1.2 mg / kg were significantly reduced; the perirenal fat content in the YN-011 group at 1.2 mg / kg was significantly reduced.

[0411] YN-011 increased the fasting serum insulin level in db / db mice in a dose-dependent manner 30 hours after the last administration (the 9th administration, day 26), accompanied by a significant increase in the pancreatic β-cell mass.

[0412] Compared with the solvent control group, multiple subcutaneous injections of YN-011 significantly reduced the serum triglyceride level, and YN-011 at 0.6 mg / kg significantly reduced the serum free fatty acid level in db / db mice.

[0413] In summary, multiple subcutaneous injections of YN-011 showed significant therapeutic effects in db / db mice, not only significantly improving glucose metabolism but also having obvious therapeutic effects on dyslipidemia.

[0414] Example 5: Phase IIa Clinical Trial of YN-011

[0415] 5.1 Experimental design

[0416] This Phase IIa clinical trial was a double-blind, placebo-controlled study conducted in subjects with T2DM to evaluate the efficacy and safety of subcutaneous administration of YN-011 at doses of 1 mg, 2 mg, 3 mg, and 4 mg. The design of this study was as Figure 5 shown.

[0417] Main inclusion criteria for the Phase IIa study:

[0418] · T2DM patients (WHO 1999) who had not taken metformin for at least 1 week and had not taken any other oral hypoglycemic drugs for at least 2 weeks.

[0419] · Glycated hemoglobin HbA1c at screening: 7.0% ≤ HbA1c ≤ 10.0%.

[0420] · Age at screening was 18 - 65 years old

[0421] · BMI ≥ 20 kg / m 2 and ≤ 40 kg / m 2

[0422] Main exclusion criteria for the Phase IIa study:

[0423] · Type 1 diabetes

[0424] · Fasting C-peptide < 0.81 ng / mL

[0425] · Laboratory test indicators meet one of the following criteria: alanine aminotransferase (ALT) level ≥ 2.5 x ULN, and / or aspartate aminotransferase (AST) ≥ 2.5 x ULN, fasting triglycerides > 5.6 mmol / L; glomerular filtration rate (eGFR) calculated by the CKD-EPI (EPI-(Scr)) equation < 45 mL / min / 1.73 m 2 ,

[0426] · Known susceptible family (first-degree relatives) or individual with a history of type 2 multiple endocrine neoplasia or medullary thyroid carcinoma

[0427] · Uncontrolled hypertension

[0428] · Known history of pancreatitis, pancreatic cancer or serum amylase > 1.2 x ULN, or high-risk factors for pancreatitis during screening

[0429] · Patients with uncontrolled thyroid insufficiency

[0430] · Suspected active infection

[0431] · Positive for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV-Ab), human immunodeficiency virus antibody (HIV-Ab) or treponema pallidum antibody (TPAb)

[0432] · Received GLP-1 receptor agonist or DPP-4 inhibitor or insulin treatment within 3 months before randomization

[0433] · History of CTCAE grade 3-4 allergy to any protein drug.

[0434] · Donated blood or lost more than 450 mL of blood within 3 months before screening

[0435] · Any obvious endocrine, immune, coagulation, urogenital tract abnormalities or blood diseases

[0436] · Clinically significant gastric emptying abnormalities (such as gastric outlet obstruction), severe chronic gastrointestinal diseases (such as active ulcers within 6 months), long-term use of drugs that directly affect gastrointestinal motility or underwent gastrointestinal surgery

[0437] · Any other conditions that the investigator or attending physician deems may be inappropriate for participation in the study.

[0438] Subjects were randomly assigned to receive YN-011 or placebo at a ratio of 4:1. The dosing regimens of YN-011 were 1, 2, and 3 mg, with a single-dose administration, followed by a two-week rest period, and then weekly administrations for 4 consecutive times. Subjects in the 4 mg group had the same dosing regimen, except for an initial dose of 1 mg administered one week before the first dose. All subjects received a total of 5 randomized administrations.

[0439] The primary endpoint was the safety and tolerability of YN-011 in T2DM subjects. The secondary endpoints included the change in fasting blood glucose relative to baseline each week, the change in HbA1c relative to baseline at week 4 and week 7, the change in glycated albumin relative to baseline at week 4 and week 7, the change in glucose tolerance, and the change in pancreatic β-cell function evaluated by an oral glucose tolerance test. Blood samples were collected from all subjects for pharmacokinetic studies. Safety assessments included adverse events, laboratory tests, vital signs, 12-lead electrocardiograms, physical examinations, and anti-drug antibody (ADA) evaluations.

[0440] 5.2 Pharmacokinetics of YN-011, administered either once or multiple times

[0441] In the dose range of 1.0 mg - 4.0 mg, YN-011 showed an increasing trend. After a single-dose administration, the half-life (T1 / 2) of YN-011 was approximately 207 hours (8.6 days), and the median Tmax was 60 - 84 hours ( Figure 4 ). YN-011 was administered for the first time after randomization, and then weekly after 2 weeks. After the fourth consecutive administration, the plasma concentration of YN-011 remained at a steady state ( Figure 4 ). After amino acid substitution of the fusion protein of this application, the half-life was significantly longer than that of SUPA-1. In addition, compared with the elimination half-lives of the already marketed dulaglutide and semaglutide, which are 4.7 - 5.5 days

[22] and 5.7 - 6.7 days

[24] respectively, the half-life of YN-011 was also significantly longer than that of dulaglutide and semaglutide.

[0442] 5.3 Therapeutic effect of YN-011 on type 2 diabetes

[0443] YN-011 was administered subcutaneously at formal dosing levels of 1 mg, 2 mg, 3 mg, and 4 mg (see the dosing regimen design in Figure 5 ).

[0444] A total of 40 subjects received at least one dose of YN-011 or placebo and were included in the safety analysis. Their average age was 51.7 ± 10.32 years, average BMI was 25.80 ± 2.875 kg / m2, average weight was 71.91 ± 12.360 kg, and 40% were female. Among the 40 subjects, 38 (95%) had comorbidities, including 32 (80%) with ultrasound-confirmed fatty liver, 25 (62.5%) with hyperlipidemia, 19 (47.5%) with hypertension, 10 (22.5%) with atherosclerosis, and 10 (22.5%) with lung masses. During the entire trial, none of the 40 subjects received combined drug therapy, and the basic conditions are as shown in the following table (Table 6).

[0445] Table 6. Basic characteristics of T2DM subjects

[0446]

[0447] The effects of different doses of YN-011 on fasting blood glucose are as Figure 6 shown. Compared with placebo, multiple subcutaneous administrations of 3 mg or 4 mg of YN-011 resulted in a persistent, clinically significant, and statistically significant improvement in fasting blood glucose.

[0448] The effects of different doses of YN-011 on HbA1c are as Figure 7 shown. Compared with placebo, multiple subcutaneous administrations of different doses of YN-011, 1 mg, 3 mg, or 4 mg, persistently improved HbA1c levels, with significant clinical significance and statistical differences. And no obvious side effects or safety risks were found.

[0449] Example 6: Preventive and therapeutic effects of YN-011 on obesity

[0450] 6.1 Effect of YN-011 in high-fat diet-induced obese mice

[0451] The effects of multiple administrations of YN-011 on glucose tolerance, insulin sensitivity, metabolism, and weight loss were evaluated in high-fat diet (HFD)-induced obese mice. Five-month-old male C57BL / 6 mice (commercially available, e.g., Shanghai SLAC Laboratory Animal Co., Ltd., under conventional breeding conditions) were continuously fed an HFD (60% total calories from fat, 20% from carbohydrates) for 6 months to establish obese model mice (diet-induced obesity, abbreviated as DIO). Then, the DIO mice (body weight > 50 g) were divided into 2 groups (5 animals per group). The experimental group was injected with 0.3 mg / kg of YN-011 twice a week (BIW), and the control group was injected with phosphate-buffered saline (PBS). The treatment continued for 4 weeks.

[0452] The experimental results showed that twice-weekly injection of YN-011 for 4 weeks had a significant weight loss effect on DIO mice. Compared with the PBS control group, significant reduction in visceral fat, especially epididymal fat content, was found in YN-011-treated mice. YN-011 had no effect on the weights of other weight-related tissues, including brown adipose tissue (BAT), white adipose tissue (WAT, inguinal), pancreas, or calf muscle.

[0453] Compared with the PBS control group, twice-weekly injection of YN-011 for 4 weeks significantly reduced ectopic lipid accumulation and hepatic triglycerides, as well as serum ALT (YN-011 vs. Ctrl = 34.2 ± 7.7 vs. 153.4 ± 18.7, P < 0.01) and AST levels (YN-011 vs. Ctrl = 72.20 ± 19.29 VS. 145.6 ± 16.8, P < 0.05). After 4 weeks of twice-weekly treatment, YN-011 also significantly improved the lipid profile, with total cholesterol (TC) reduced by 30% (P < 0.001), triglycerides (TG) reduced by 68% (P < 0.001), and non-esterified fatty acids (NEFA) reduced by 57% (P < 0.001).

[0454] Compared with the control group, DIO mice administered YN-011 multiple times showed a significant reduction in food consumption. The metabolic rate (VO2 and VCO2) and energy expenditure (EE) trended upward in the YN-011 treatment group, but without statistical significance. When normalized by body weight, the VO2, VCO2, and EE of YN-011-treated mice were significantly increased at night. YN-011 had no effect on UcP1 expression in BAT and epididymal WAT, but significantly upregulated Ucp1 expression in inguinal WAT, indicating that YN-011 did not enhance BAT thermogenesis but promoted browning of inguinal white adipose tissue. Therefore, YN-011-treated DIO mice showed a higher core body temperature at room temperature and maintained a higher rectal temperature when exposed to a cold environment, indicating more active thermogenesis compared with the control group. These results suggest that YN-011 enhances the adaptability of obese mice to cold exposure by generating more calories.

[0455] Through glucose fluctuation, intraperitoneal glucose tolerance test, and insulin tolerance test, YN-011 significantly reduced blood glucose (P < 0.01) and improved insulin sensitivity (P < 0.01) in DIO mice after 4 weeks of treatment.

[0456] In summary, YN-011 treatment effectively reduced the body weight of obese mice and improved obesity-related metabolic disorders, including hyperglycemia, hyperlipidemia, and hepatic steatosis. The beneficial effects of YN-011 on metabolism were related to the inhibition of food consumption and browning and remodeling of WAT.

[0457] 6.2 Therapeutic effect of YN-011 on obese rhesus monkeys

[0458] The obese rhesus monkeys were sourced from Sichuan Prime Science & Technology Group Co., Ltd. In this experiment, 15 male obese rhesus monkeys in the IGT / IFG and hyperglycemia stages who had not received drug treatment within 1 year of the initial onset were selected. Their ages ranged from 8 to 21 years (equivalent to 30 to 60 years in humans), with body weights ranging from 9.25 to 15.70 kg, FPG between 5.50 and 8.58 mmol / L, and HbA1c between 4.5 and 5.0%; their liver and kidney functions were normal. They were randomly grouped according to FPG stratification. All selected animals underwent IVGTT tests to measure blood glucose and insulin before drug administration. A placebo group, a YN-011 50 μg / kg group, and a YN-011 25 μg / kg group were set up, with 5 animals in each group. They were administered subcutaneously (SC) once a week for 4 consecutive weeks, that is, subcutaneously administered on the 0th day (D0), 7th day (D7), 14th day (D14), and 21st day (D21).

[0459] The effects of YN-011 on the body weight (BW) of obese rhesus monkeys are shown in Table 7, Figure 8 as follows. During the entire test period, the body weights of the 5 animals in the placebo group did not show significant changes, proving that the model was relatively stable. Compared with the baseline value in the YN-011 25 μg / kg group, with a once-weekly dosing frequency (4 times), the body weight continuously decreased from D7 to D28 days (2.60% - 6.71%, P < 0.05 or P < 0.01), and decreased by 6.17% on D28; compared with the placebo group, the BW significantly decreased on D14 and D21 days (P < 0.05). Compared with the baseline value in the YN-011 50 μg / kg group, with a once-weekly dosing frequency (4 times), the body weight continuously decreased from D7 to D28 days (4.06% - 7.32%, P < 0.05), and significantly decreased by 7.32% on D28 (P < 0.05); compared with the placebo group, the BW significantly or extremely significantly decreased from D7 to D28 days (P < 0.05 or P < 0.01).

[0460] Table 7. Effects of repeated subcutaneous injection of YN-011 on the BW of obese rhesus monkeys

[0461]

[0462] Note: Detected 2 days before drug administration; # P < 0.05 compared with the baseline, ## P < 0.01 compared with the baseline. SC, subcutaneous injection administration.

[0463] Example 7: Preventive and therapeutic effects of YN-011 in a non-alcoholic steatohepatitis (NASH) model of rhesus monkeys

[0464] 7.1 Experimental methods

[0465] 7.1.1 Liver biopsy under GE ultrasound guidance

[0466] Number of animals: 15

[0467] Specimen collection volume: ≤ 2 needles, the length of each needle specimen ≤ 1.5 cm, and the total length of specimens collected each time ≤ 3 cm

[0468] The approximately 2-cm tissue taken out was immediately fixed in 4% paraformaldehyde buffer at room temperature for more than 24 h, and after being corrected, dehydrated, embedded, and sectioned, HE staining and Masson staining were performed.

[0469] Instrumentation: For ultrasound, GE Vivid S5 ultrasound machine was used; for sectioning, LEICA RM2135 microtome was used; for reading slides, OLYMPUS BX43 microscope was used; for microphotography, OLYMPUS DP22-CU camera was used.

[0470] The tissue sections were stained with H&E and Masson, and the pathological sections were evaluated by pathological experts according to the non-alcoholic fatty liver disease diagnosis and treatment guidelines jointly drafted by the American Association for the Study of Liver Diseases (AASLD), the American College of Gastroenterology (ACG), and the American Gastroenterological Association (AGA). The diagnostic criteria are shown in Tables 2 and 3.

[0471] 7.1.2 Tissue processing, embedding, and sectioning

[0472] After the liver biopsy specimens were fixed well, dehydration, wax infiltration, embedding, and sectioning (5 μm) were directly carried out.

[0473] 7.1.3 HE staining

[0474] The sections were routinely dewaxed, and 100% absolute ethanol I, 100% absolute ethanol II, 95%, 85%, and 75% ethanol were added respectively for 3 min each. Then they were rinsed with tap water for 3 min, stained with hematoxylin stain for 12 min, rinsed with tap water for 5 min, and stained with eosin Y solution (water-soluble) for 3 min. They were quickly dehydrated with 95% ethanol, dehydrated with absolute ethanol twice, 2 - 5 min each time, and cleared with xylene twice, 10 min each time. They were sealed with neutral gum and observed under a microscope.

[0475] 7.1.4 Masson staining

[0476] The sections were routinely dewaxed until reaching water. They were stained with ponceau magenta staining solution for 5 - 7 min. Meanwhile, a weak acid working solution was prepared according to the ratio of distilled water:weak acid solution = 2:1, and the sections were washed with the weak acid working solution for 1 min. After washing with phosphomolybdic acid solution for 1 - 2 min, they were directly placed into aniline blue staining solution and stained for 1 - 2 min, then washed with the prepared weak acid working solution for 1 min. They were rapidly dehydrated with 95% ethanol, dehydrated twice with absolute ethanol for 2 - 5 min each time, and cleared twice with xylene for 10 min each time. They were sealed with neutral balsam and observed under a microscope.

[0477] 7.1.5 Histopathological examination

[0478] The sections were observed, pathologically diagnosed, and photographed under a microscope (equipped with a digital imaging system) for analysis. According to the results of pathological histology diagnosis, the non - alcoholic fatty liver disease (NAFLD) activity score (NAS) (Table 8) and liver fibrosis staging (Table 9) were performed on the HE and Masson results according to the scoring criteria.

[0479] Table 8. NAS scoring criteria

[0480]

[0481] Note: NAS score = steatosis score + lobular inflammation score + ballooning degeneration score

[0482] Table 9. Liver fibrosis grading scoring criteria

[0483] Grade Description 0 No fibrosis observed 1a Mild perisinusoidal fibrosis in zone 3 1b Moderate perisinusoidal fibrosis in zone 3 1c Periportal fibrosis 2 Perisinusoidal and periportal fibrosis 3 Bridging fibrosis 4 Cirrhosis

[0484] 7.1.6 GE 3.0T MRI quantitative analysis of liver fat content

[0485] In this experiment, a GE 3.0T MRI scanner (750W 3T MRI, GE Healthcare) was used for liver scanning. The IDEAL - IQ sequence was used for scanning. The IDEAL - IQ technology is a technology based on IDEAL combined with fast three - dimensional multi - echo imaging. It includes multi - echo water - fat separation technology, regional growth technology, and also includes multiple imaging of tissue fat quantitative fraction value and R2* relaxation rate. Six echo signals with different TE times were acquired by a single breath - hold scan, and through computer reconstruction, in - phase, opposed - phase, water - phase, fat - phase, fat fraction map, and R2* Figure 6 kinds of contrast images can be obtained. The IDEAL - IQ hybrid water - fat separation algorithm is carried out in two steps. In the first step, the complex - domain reconstruction method is applied to obtain water image, fat image, and T2* map. In the second step, another set of estimated water and fat images are generated. Then the two groups of images are integrated by a hybrid algorithm to generate the final water and fat images.

[0486] Before performing this experiment, a Phantom experiment needs to be completed: For Clare P.

[11] After partial modification of the phantom preparation method used, a total of 5 standard fat solutions with known fat contents were prepared, using pure water as 0%. Weigh 60 mmol of sodium dodecyl sulfate and dissolve it in 1 L of deionized water. Heat the sodium dodecyl sulfate solution to 50 °C, add 40 g of carrageenan and mix well. Mix 3, 6, 12, 24, and 120 ml of soybean oil (Sinopharm Chemical Reagent Co., Ltd.) with 117, 114, 108, 96, and 0 ml of the above solution respectively to prepare solutions with fat contents of 0%, 2.5%, 5%, 10%, 20%, and 100%, and fill them into 6 100-ml EP tubes respectively for subsequent scanning operations.

[0487] Before each scan, a Phantom verification experiment is required. All animals were scanned twice, once at the baseline period and once after the end of drug administration. In the 3.0T MRI room, the scanning parameters are shown in Table 10. The anesthesia method is as follows: Intramuscular injection of 10 mg / kg ketamine hydrochloride, after anesthesia, endotracheal intubation, and maintenance of anesthesia with isoflurane accompanied by oxygen inhalation using a respiratory anesthesia machine. During the scanning process, a veterinarian monitors electrocardiogram, blood oxygen, respiratory rate, etc., and conducts veterinary supervision until the animal is fully awake and resumes spontaneous breathing.

[0488] Table 10. Liver MRI Scanning Parameters

[0489] Characteristics No. Sequence name Required grade Localizer 1 3-PI Loc SSFSE NA Water-fat separation 2 BH LAVA-Flex Liver cross-section Fat content 3 BH IDEAL IQ Liver cross-section

[0490] Image post-processing and analysis: After the MRI images of rhesus monkeys are acquired and saved, a 750W 3T MRI supporting analysis workstation is used. For the liver MRI images, the layers with the largest exposed area of the right lobe of the liver (layers 1-3) are mainly selected, avoiding large blood vessels, bile ducts, and gallbladders to avoid volume effects, and ROIs are selected. The area of each ROI is 90-110 mm 2 . When the MRI-PDFF% of intrahepatic fat content > 6%, it is moderate fatty liver (consistent with the clinical standard).

[0491] 7.2 Experimental results

[0492] The in vivo efficacy study of YN-011 was conducted in NASH rhesus monkeys. The clinical parameters evaluated included liver lipid content evaluated by magnetic resonance imaging proton density fat fraction (MRI-PDFF%), NAFLD activity score (NAS), and liver fibrosis stage evaluated by liver histology, body weight and body mass index (BMI), blood lipid profile, fructose metabolism, other biochemical parameters, and food consumption.

[0493] The rhesus monkeys selected in this study were 11 - 23 years old, equivalent to 30 - 70 years old in humans. All rhesus monkeys were male, with a body weight of 13.63 - 22.85 kg, had abnormal lipid metabolism for more than 2 years, an MRI - PDFF% of 7.8% - 11.9%, histological manifestations of NAS≥3, and a fibrosis score of 0 - 1c within 6 months. These animals clinically met the definition of NASH.

[0494] Fifteen rhesus monkeys were divided into 3 groups of 5 each and received subcutaneous injection of 0 (solvent control, placebo control group), 0.050 or 0.150 mg / kg YN - 011 once a week (QW) for 13 weeks. The 0.150 mg / kg YN - 011 group was given an adaptive dose (the first dose was 0.1 mg / kg, and the remaining 12 doses were 0.150 mg / kg).

[0495] During the study, all rhesus monkeys received the designated treatment, and none of the rhesus monkeys missed the end - of - treatment biopsy or withdrew from the treatment. No obvious adverse reactions were found in any treatment group during the study.

[0496] As shown in Table 11, compared with the placebo control group, the liver lipid content decreased by approximately 40% after 13 weeks of once - weekly YN - 011 treatment.

[0497] To study whether YN - 011 treatment led to improvement in liver histology, liver biopsy specimens were collected from all rhesus monkeys before the first injection and on the 89th day after the first injection. Liver sections were stained with HE and Masson for histological analysis, and the degrees of steatosis, inflammation, hepatocyte ballooning, and fibrosis were scored according to the criteria for NAFLD activity and NASH progression. The liver biopsy results showed that in rhesus monkeys treated with YN - 011, both the NAS and liver fibrosis scores decreased, and there was no obvious progression of NASH (Table 12).

[0498] Liver fat detected by MRI - PDFF decreased from 9.0% ± 0.9% to 5.0% ± 0.2% in the 50 μg / kg YN - 011 group and from 9.4% ± 1.5% to 5.6% ± 1.5% in the 150 μg / kg YN - 011 group. At the end of the drug administration, compared with the baseline, the percentage decreases were 43.8% and 39.7% respectively. There was no significant difference in the change of MRI - PDFF between the 50 μg / kg and 150 μg / kg YN - 011 groups.

[0499] Histological analysis showed that in the 50 μg / kg YN-011 group, the mean NAS decreased from 3.6 ± 0.5 to 1.6 ± 0.5 (P = 0.003) compared with the baseline at the end of dosing. In the 150 μg / kg YN-011 group, the mean NAS decreased from 3.6 ± 0.5 to 1.4 ± 0.5 (P < 0.001) compared with the baseline at the end of dosing. No significant difference in NAS was found between the 50 μg / kg and 150 μg / kg YN-011 groups.

[0500] Regarding metabolic measurements, including liver injury biomarkers, lipid profiles, and body weight, YN-011 treatment showed excellent improvement in both the 50 μg / kg and 150 μg / kg groups.

[0501] Table 11. Effects of YN-011 on the percentage change in liver fat content in NASH cynomolgus monkeys after repeated SC dosing

[0502]

[0503] Note: ROI: Region of interest; **P < 0.01 compared with the baseline; ##P < 0.01 compared with the placebo group; Rate of change = (Current value - Baseline value) / Baseline value × 100%.

[0504] Table 12. Effects of YN-011 on liver NAS and fibrosis scores in NASH cynomolgus monkeys after repeated SC dosing

[0505]

[0506]

[0507] In addition, the body weight and BMI of rhesus monkeys in the YN-011 treatment group (day 7-day 84) decreased significantly compared with baseline. YN-011 treatment also significantly reduced serum low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC) and total triglycerides (TG) at various measurement time points during treatment, and 0.15mg / kg YN-011 showed a more consistent lipid-lowering effect. Compared with baseline values, the level of high-density lipoprotein cholesterol (HDL-c) in the YN-011 treatment group showed a downward trend. These changes in serum lipid profiles are attributed to reduced food consumption in YN-011-treated animals. During the study, plasma FPG levels in all groups fluctuated within the normal range, and no significant inter-group differences were detected after administration of YN-011 and placebo. Plasma FRA, a validated biomarker that reflects the average level of blood glucose control over the past 2-3 weeks, showed a trend of decrease in the YN-011 group 1 week after the first injection, and a significant decrease of 4.3% after 12 weeks of treatment with 150μg / kg YN-011. These results indicate that YN-011 has blood glucose control efficacy in rhesus monkeys with NASH. There was a dose-dependent reduction in food consumption in YN-011-treated animals, which is a normal pharmacological effect of YN-011. In summary, these data indicate that multiple subcutaneous injections of YN-011 provide considerable therapeutic benefits for NASH rhesus monkeys.

[0508] Example 8: In vitro experiments on neurodegenerative diseases

[0509] 8.1 TNF-α concentration-dependently reduces the viability of neuronal cells SH-SY5Y

[0510] The neuronal cells SH-SY5Y were digested with 0.25% trypsin to prepare a single cell suspension. The single cell suspension was inoculated in a 96-well plate, with a cell density of 100 μl per well containing 10,000 cells. The culture plate was placed in an incubator for pre-culture overnight (37°C, 5% CO 2 ) to allow the cells to adhere to the wall. Replace the culture medium containing 10% FBS and 5% FBS respectively, and use 20, 40, 60, 80, and 100ng / ml TNF-α to stimulate the cells for 48 hours, and make 6 replicates for each drug treatment. After 48 hours of drug treatment, add 10μl CCK-8 solution to each well. After adding the reagent, gently shake the culture plate to mix. Also, pay attention to try not to generate bubbles during the addition process. Place the culture plate in the incubator and incubate for 1-4 hours. Use an enzyme reader to measure the absorbance value (OD) at 450nm, and use the following formula to calculate cell viability:

[0511] Cell viability (%) = [A (加药) -A (空白) ] / [A (未加药) -A (空白) ]×100

[0512] A (加药) : OD value of the wells with cells, CCK-8 solution and drug solution

[0513] A (未加药) : OD value of the wells with cells and CCK-8 solution but without drug solution

[0514] A (空白) : OD value of the wells without cells

[0515] The experimental results showed (as Figure 9 shown), the viability of neuron SH-SY5Y cells decreased significantly with the increase of TNF-α concentration.

[0516] 8.2 Effects of YN-011 and γ-aminobutyric acid (GABA) in inhibiting TNF-α from reducing the viability of SH-SY5Y cells

[0517] For the experimental procedures, please refer to Section 8.1, but the drug treatment procedures in this experiment are different from those in Section 8.1. Specifically, the drug treatment procedures in this experiment are as follows: Use the culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone, or simultaneously add 10, 100 or 500 nM YN-011 respectively, or simultaneously add 1, 10 or 100 μM GABA respectively to each well for 48 hours, and perform 6 replicates for each drug treatment.

[0518] The experimental results showed (as Figure 10 shown), the viability of SH-SY5Y cells decreased significantly in the presence of 60 ng / ml TNF-α, while the cell viability was significantly enhanced after treatment with 10, 100 and 500 nM YN-011, or after treatment with 100 μM GABA.

[0519] 8.3 Combining YN-011 and GABA significantly increases the viability of SH-SY5Y cells

[0520] For the experimental procedures, please refer to Section 8.1, but the drug treatment procedures in this experiment are different from those in Section 8.1. Specifically, the drug treatment procedures in this experiment are as follows: Use the culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone, or simultaneously add 100 nM YN-011 and / or 100 μM GABA to each well for 48 hours, and perform 6 replicates for each drug treatment.

[0521] The experimental results showed (as Figure 11 shown), the viability of neuron SH-SY5Y cells decreased significantly in the presence of 60 ng / ml TNF-α, while 100 nM YN-011 and 100 μM GABA significantly enhanced the cell viability. The combination of YN-011 and GABA showed stronger potency in enhancing cell viability compared with the individual use.

[0522] 8.4 YN-011 reduces apoptosis of neuronal cells SH-SY5Y induced by TNF-α

[0523] The steps of this experiment are as follows:

[0524] 1) Digest the neuron cells SH-SY5Y in a 10 cm culture dish with 0.25% trypsin and inoculate them into a 12-well plate.

[0525] 2) Place the culture plate in an incubator for pre-incubation overnight (37 °C, 5% CO 2 ) to allow the cells to adhere to the wall.

[0526] 3) Use a culture medium containing 5% FBS and treat the cells in a 37 °C incubator for 48 hours with 60 ng / ml TNF-α alone or with 10, 100 or 500 nM YN-011 added separately to each well. Do 3 replicates for each drug treatment.

[0527] 4) After 48 hours, discard the supernatant, add PBS and gently wash once. Place the cell culture plate on ice, add 120 μl of RIPA cell lysate (Beyotime P0013B) to each well. Add protease inhibitor and phosphorylated protease inhibitor to the lysate and lyse for 15 minutes.

[0528] 5) Transfer the cell lysate to a 1.5 ml EP tube and centrifuge at 14000 rpm for 30 minutes.

[0529] 6) It can be seen that there is cell precipitate at the bottom of the EP tube. Gently aspirate the supernatant into a new EP tube, add 5x loading buffer containing β-mercaptoethanol, and boil at 100 °C for 10 minutes to denature the protein.

[0530] 7) Use SDS-PAGE gel electrophoresis with a protein loading amount of 10 μg per well. Place the gel plate vertically against the power supply rack in the electrophoresis tank with the concave edge of the gel plate facing the power supply rack. Two gel plates share one power supply rack. Fix the gel plate and the power supply rack in the power supply tank as required. Add electrophoresis buffer as required so that the electrophoresis buffer in the power supply rack between the two gel plates does not communicate with the electrophoresis buffer in the electrophoresis tank. Gently pull out the comb in the gel plate.

[0531] 8) Electrophoresis: Connect the electrophoresis tank and the electrophoresis instrument with two wires, noting that the plugs and sockets of the red and black electrodes match. During electrophoresis, use low-voltage constant voltage electrophoresis for the upper gel. Turn on the power and adjust the voltage to 80 V (usually about 15 minutes). Use high-voltage constant voltage electrophoresis when the bromophenol blue enters the lower gel. Adjust the voltage to 120 V and stop electrophoresis when the bromophenol blue reaches near the bottom of the gel.

[0532] 9) Wet transfer is used for membrane transfer. The wet transfer sandwich arrangement is: sponge / filter paper / gel / membrane / filter paper / sponge, tightly arranged. There should be no air bubbles between the gel and the membrane. Confirm the correct placement direction of the sandwich. The negative electrode side is the protein in the negatively charged gel, which migrates electrophoretically towards the positive electrode side (membrane). After SDS-PAGE electrophoresis is completed, gently pry open the two pieces of glass of the gel plate with a thin plate to make the gel lie on one of the glass plates. Use a blade to cut the separating gel along the junction of the separating gel and the stacking gel on the gel, and cut off a small corner at the upper left corner of the separating gel to mark the sample order. Then carefully transfer the gel into the transfer buffer. Cut a 0.22 μm PVDF membrane of the same size as the separating gel, and soak it in methanol for 5 seconds. Cut 6 pieces of filter paper of the same size, and equilibrate them with the PVDF membrane and the gel in the transfer buffer for 15 minutes. On the transfer device, place the sponge gasket, filter paper, gel, membrane, filter paper, sponge gasket (from bottom to top) from the negative electrode (black bottom) to the positive electrode. When placing, be sure to remove air bubbles, especially between the membrane and the filter paper, the gel and the membrane, and the filter paper and the gel. Set the transfer current to constant current, 200 mA, and the time required is about 90 minutes.

[0533] 10) Take out the PVDF membrane after membrane transfer, wash it slightly in TBST, pour out the TBST, add 5% milk blocking solution, cover the PVDF membrane, and slowly block it on a shaker at room temperature for 1 hour (rotation speed 30 rpm).

[0534] 11) Primary antibody incubation. Remove the blocking solution, wash the membrane 3 times with TBST, 5 minutes each time. Then cut the membrane according to the molecular weight size, add the corresponding dilution of the primary antibody (Bcl-2, CST#3498S, 1:1000; Cleaved-caspase3, CST#9661S, 1:1000; Caspase3, CST#9662S, 1:1000; HSP90, Proteintech#13171-1-AP, 1:10000), and incubate it overnight on a shaker at 4°C.

[0535] 12) Wash the membrane. Take out the PVDF membrane incubated overnight, recover the antibody and store it in a -20°C refrigerator. Place the taken-out PVDF membrane in the TBST solution and quickly rinse it 3 times, 15 minutes each time.

[0536] 13) Secondary antibody incubation. Add the corresponding species-specific secondary antibody (Jackson Lab, 1:10000) prepared by mixing with the blocking solution, and slowly block it at room temperature for 1 hour.

[0537] 14) Wash the membrane. Remove the secondary antibody, place the PVDF membrane in the TBST solution, and quickly rinse it 3 times, 15 minutes each time.

[0538] 15) ECL chemiluminescence. Under light-proof conditions, prepare a fresh developing working solution (Solution A: Solution B = 1:1) according to the instructions in the ECL chemiluminescence kit (Millipore #WBKLS0500), uniformly drop it on the membrane, and place it in an imager for developing and image acquisition.

[0539] 16) Protein expression level analysis. Use Image J software to statistically analyze the gray value of the western blot bands, correct it with the gray value of the internal reference protein HSP90, and calculate the relative expression level of the target protein.

[0540] The experimental results showed (as Figure 12 shown) that YN-011 significantly reduced the apoptosis of TNF-α-induced neuronal cells SH-SY5Y.

[0541] 8.5 GABA reduces apoptosis of neuronal cells SH-SY5Y induced by TNF-α

[0542] For the experimental procedures, please refer to Section 8.4, but the drug treatment step 3) in this experiment is different from that in Section 8.4. Specifically, the drug treatment step 3) in this experiment is as follows: Use a culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone per well, or add 1, 10, 100 μM GABA respectively at the same time in a 37 °C incubator for 48 hours. Make 3 replicates for each drug treatment.

[0543] The experimental results showed (as Figure 13 shown) that GABA reduced the apoptosis of TNF-α-induced neuronal cells SH-SY5Y.

[0544] 8.6 Combining YN-011 and GABA reduces apoptosis of neuronal cells SH-SY5Y induced by TNF-α

[0545] For the experimental procedures, please refer to Section 8.4, but the drug treatment step 3) in this experiment is different from that in Section 8.4. Specifically, the drug treatment step 3) in this experiment is as follows: Use a culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone per well, or add 100 μM GABA and / or 100 nM YN-011 respectively at the same time in a 37 °C incubator for 48 hours. Make 3 replicates for each drug treatment.

[0546] The experimental results showed (as Figure 14 shown) that the combined use of YN-011 and GABA significantly reduced the apoptosis of TNF-α-induced neuronal cells SH-SY5Y.

[0547] 8.7 TNF-α promotes damage of neuronal cells SH-SY5Y

[0548] Digest the neuron cells SH-SY5Y in a 10 cm culture dish with 0.25% trypsin to prepare a single cell suspension. Inoculate the single cell suspension into a 24-well plate. Place the culture plate in an incubator for pre-incubation overnight (37 °C, 5% CO 2 2), to allow the cells to adhere. Use a culture medium containing 5% FBS, and treat the cells in each well with 20, 40, 60 or 80 ng / ml TNF-α in a 37 °C incubator for 48 hours, with 3 replicates for each drug treatment. Add the fluorescent dye Hoechst 33342 (Beyotime) to the well plate at a dilution concentration of 1:1000. After incubating in a 37 °C incubator for 10 minutes, take out the cell culture plate. Wash off the culture medium, gently wash 2 times with PBS, and immediately take it to a fluorescence microscope for photographing (20 times magnification). Take 10 fields of view for each cell well, and count the number of blue positive cells in each field of view.

[0549] The experimental results showed (as Figure 15 shown), TNF-α promoted the damage of neuron cells SH-SY5Y.

[0550] 8.8 Protective effect of YN-011 on neuronal cells damaged by TNF-α

[0551] Please refer to Section 8.7 for the experimental procedures, but the drug treatment procedures in this experiment are different from those in Section 8.7. That is, the drug treatment procedures in this experiment are as follows: Use a culture medium containing 5% FBS, and treat the cells in each well with 60 ng / ml TNF-α alone, or simultaneously add 10, 100 or 500 nM YN-011 respectively in a 37 °C incubator for 48 hours, with 3 replicates for each drug treatment.

[0552] The experimental results showed (as Figure 16 shown), YN-011 had a protective effect on neuron cells damaged by TNF-α.

[0553] 8.9 Protective effect of GABA on neuronal cells damaged by TNF-α

[0554] Please refer to Section 8.7 for the experimental procedures, but the drug treatment procedures in this experiment are different from those in Section 8.7. That is, the drug treatment procedures in this experiment are as follows: Use a culture medium containing 5% FBS, and treat the cells in each well with 60 ng / ml TNF-α alone, or simultaneously add 1, 10 or 100 μM GABA respectively in a 37 °C incubator for 48 hours, with 3 replicates for each drug treatment.

[0555] The experimental results showed (as Figure 17 shown), GABA had a protective effect on neuron cells damaged by TNF-α.

[0556] 8.10 Protective effect of combining YN-011 and GABA on neuronal cells damaged by TNF-α

[0557] Please refer to Section 8.7 for the experimental procedures, but the drug treatment procedures in this experiment are different from those in Section 8.7. Specifically, the drug treatment procedures in this experiment are as follows: Use a culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone or add 100 μM GABA and / or 100 nM YN-011 separately to each well, and incubate the cells in a 37°C incubator for 48 hours. Three replicate wells are set up for each drug treatment.

[0558] The experimental results showed (as Figure 18 shown), that the combined use of YN-011 and GABA had a protective effect on TNF-α-damaged neuronal cells.

[0559] 8.11 YN-011 reduces apoptosis of neuronal cells induced by TNF-α

[0560] The experimental procedures are as follows:

[0561] 1) Digest the neuronal cells SH-SY5Y in a 10-cm culture dish with 0.25% trypsin to prepare a single-cell suspension. Inoculate the single-cell suspension into a 24-well plate, and place a sterile coverslip in the 24-well plate.

[0562] 2) Place the culture plate in an incubator for pre-incubation overnight (37°C, 5% CO 2 2) to allow the cells to adhere to the plate.

[0563] 3) Use a culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone or add 10, 100, or 500 nM YN-011 separately to each well, and incubate the cells in a 37°C incubator for 48 hours. Three replicate wells are set up for each drug treatment.

[0564] 4) After 48 hours, aspirate the culture medium, and add 400 μl of 4% paraformaldehyde (PFA) to each well to fix the cells at room temperature for 15 minutes.

[0565] 5) Wash the cells twice with PBS, 5 minutes each time.

[0566] 6) Add 0.1% Triton X-100 to permeabilize the cells, and incubate at room temperature for 10 minutes.

[0567] 7) Wash the cells twice with PBS, 5 minutes each time.

[0568] 8) Add 10% goat serum (GS) and incubate at room temperature for 30 minutes to block non-specific binding.

[0569] 9) Aspirate the 10% GS, and add the primary antibody Cleaved-caspase3 (CST 9661S). Use an antibody dilution solution containing 1% GS, with an antibody dilution ratio of 1:400, and add 30 μl of the antibody solution to each coverslip.

[0570] 10) Place it in a 4°C refrigerator overnight.

[0571] 11) The next day, aspirate the primary antibody, add PBS and wash 3 times, 5 minutes each time.

[0572] 12) Add the fluorescent secondary antibody (Alexa 488 Conjugate), and incubate in the dark at room temperature for 1 hour.

[0573] 13) Wash with PBS 3 times, 5 minutes each time.

[0574] 14) Stain with DAPI for 5 minutes, then wash with PBS 3 times, 5 minutes each time.

[0575] 15) Drop the mounting medium on a clean glass slide, drain the cell culture slide and invert it onto the mounting medium, and leave it overnight at room temperature in a cool and well-ventilated place.

[0576] 16) Take pictures under a fluorescence microscope (40× magnification).

[0577] 17) Take pictures of 10 fields of view for each treatment group, and count the number of green positive cells in each field of view.

[0578] The experimental results showed (as Figure 19 shown) that YN-011 significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0579] 8.12 GABA reduces apoptosis of neuronal cells induced by TNF-α

[0580] Please refer to Section 8.11 for the experimental procedures, but the drug treatment step 3) in this experiment is different from that in Section 8.11. That is, the drug treatment step 3) in this experiment is as follows: Use the culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone or add 1, 10, or 100 μM GABA respectively in each well at 37°C in an incubator for 48 hours, and make 3 replicates for each drug treatment.

[0581] The experimental results showed (as Figure 20 shown) that GABA reduced TNF-α-induced apoptosis of neuronal cells.

[0582] 8.13 Combining YN-011 and GABA reduces apoptosis of neuronal cells induced by TNF-α

[0583] For the experimental procedures, please refer to Section 8.11. However, step 3) of the drug treatment in this experiment is different from that in Section 8.11. Specifically, step 3) of the drug treatment in this experiment is as follows: Use the culture medium containing 5% FBS, and treat the cells in a 37°C incubator for 48 hours with 60 ng / ml TNF-α alone in each well, or add 100 μM GABA and / or 100 nM YN-011 separately at the same time. For each drug treatment, three replicate wells are prepared.

[0584] The experimental results showed (as Figure 21 shown), that the combined use of YN-011 and GABA significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0585] 8.14 YN-011 reduces apoptosis of neuronal cells induced by TNF-α

[0586] Digest the neuronal cells SH-SY5Y in a 10-cm culture dish with 0.25% trypsin to prepare a single-cell suspension. Inoculate the single-cell suspension into a 12-well plate. Place the culture plate in an incubator for pre-incubation overnight (37°C, 5% CO 2 2) to allow the cells to adhere. Use the culture medium containing 5% FBS, and treat the cells in a 37°C incubator for 48 hours with 60 ng / ml TNF-α alone in each well, or add 10, 100, or 500 nM YN-011 separately at the same time. For each drug treatment, three replicate wells are prepared. Collect the cells, and digest the cells with 0.25% trypsin without EDTA for 1 minute. Aspirate the cell suspension into a 1.5-ml EP tube, centrifuge at 1000 rpm for 5 minutes, and the cell pellet settles at the bottom of the tube. Resuspend 10 5 cells in 100 μl of Binding Buffer, add 5 μl of PI and 5 μl of Annexin V-FITC solution, and incubate in the dark at room temperature for 15 minutes. Add 400 μl of Binding Buffer to each tube and detect with a flow cytometer (the excitation wavelength of Annexin V-FITC is 488 nm, and the emission wavelength is 520 nm; the excitation wavelength of PI is 535 nm, and the emission wavelength is 617 nm). Count the number of Annexin V-FICT-positive cells and PI-negative cells in each sample.

[0587] The experimental results showed (as Figure 22 shown), that YN-011 significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0588] 8.15 GABA reduces apoptosis of neuronal cells induced by TNF-α

[0589] The experimental procedures are referred to Section 8.14, but the drug treatment procedures in this experiment are different from those in Section 8.14. Specifically, the drug treatment procedures in this experiment are as follows: Use the culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone or add 1, 10, or 100 μM GABA respectively at the same time in each well, and incubate the cells in a 37°C incubator for 48 hours. Three replicates are made for each drug treatment.

[0590] The experimental results showed (as Figure 23 shown), GABA reduced the apoptosis of neuron cells induced by TNF-α.

[0591] 8.16 Combining YN-011 and GABA reduces apoptosis of neuronal cells induced by TNF-α

[0592] The experimental procedures are referred to Section 8.14, but the drug treatment procedures in this experiment are different from those in Section 8.14. Specifically, the drug treatment procedures in this experiment are as follows: Use the culture medium containing 5% FBS, and treat the cells with 60 ng / ml TNF-α alone or add 100 μM GABA and / or 100 nM YN-011 respectively at the same time in each well, and incubate the cells in a 37°C incubator for 48 hours. Three replicates are made for each drug treatment.

[0593] The experimental results showed (as Figure 24 shown), the combined use of YN-011 and GABA significantly reduced the apoptosis of neuron cells induced by TNF-α.

[0594] 8.17 GABA reduces the mRNA expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglial cells

[0595] Under sterile conditions, dissolve the synthesized Aβ 1-42 peptide (AnaSpec#AS-20276) in hexafluoroisopropanol (Mecklin#H811026) to obtain a 1 mM solution. Aliquot the solution into 1.5 ml sterile centrifuge tubes, and then evaporate the hexafluoroisopropanol in a vacuum dryer. The dried polypeptide forms a film in the tube and is stored at -20°C for later use. As shown below, freshly prepare Aβ 1-42 oligomers from the dried peptide film. First dissolve the dried peptide film in anhydrous DMSO to obtain a 5 mM solution, and then dilute it with cold cell culture basal medium to obtain a 100 μM stock solution. After incubating at 4°C for 24 hours, the stock solution is used for cell culture.

[0596] Seed human microglial HMC3 cells into a 24-well plate. After incubating overnight in an incubator at 37°C and 5% CO 2 , use 10 μM Aβ 1-42Oligomers alone, or 1, 10, or 100 μM GABA were added separately to the cells for 48 hours. Three replicates were performed for each drug treatment. After 48 hours of treatment, 1 ml of Trizol (Invitrogen #15596026) was used per well to isolate total RNA from the cells according to the manufacturer's instructions. The RNA pellet was dissolved in 20 μl of DEPC-treated water. The RNA concentration and purity were measured by NanoDrop 2000. RNA was reverse transcribed into cDNA using a reverse transcription kit (Yeasen #11141ES60). The PCR reaction was prepared as follows: 5 μl of PCR mixture (Yeasen #10108ES03), 0.2 μl of forward primer, 0.2 μl of reverse primer, 1 μl of cDNA, and 3.6 μl of water. qPCR was performed on an Applied Biosystems 7500 real-time PCR system using the following thermal program: pre-denaturation at 95 °C for 3 minutes and 40 denaturation cycles (95 °C, 30 seconds), annealing at 60 °C for 30 seconds, and extension at 72 °C for 1 minute. The relative mRNA expression of TNF-α and IL-6 between treated and untreated cells was compared using the ΔΔCt method, and these mRNA expressions were normalized to the expression of the housekeeping gene RPLP0.

[0597] The experimental results showed (as Figure 25 shown), in the presence of 10 μM Aβ 1-42 oligomers, the mRNA expression of TNF-α and IL-6 in HMC3 cells increased significantly, while 100 μM GABA significantly decreased the mRNA expression of TNF-α, and 10 μM and 100 μM GABA significantly decreased the mRNA expression of IL-6.

[0598] 8.18 YN-011 reduces the mRNA expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglial cells

[0599] Please refer to Section 8.17 for the experimental procedure, but the drug treatment procedure in this experiment is different from that in Section 8.17. That is, the drug treatment procedure in this experiment is as follows: Cells were treated with 10 μM Aβ 1-42 oligomers alone, or 10, 100, or 500 nM YN-011 were added separately for 48 hours. Three replicates were performed for each drug treatment.

[0600] The experimental results showed (as Figure 26 shown), in the presence of 10 μM Aβ 1-42 oligomers, the mRNA expression of TNF-α and IL-6 in HMC3 cells increased significantly, while 100 nM and 500 nM YN-011 significantly decreased the mRNA expression of TNF-α and IL-6.

[0601] 8.19 Combined use of YN-011 and GABA to reduce Aβ 1-42 oligomer-induced inflammatory factors mRNA expression

[0602] Please refer to Section 8.17 for the experimental procedures, but the drug treatment procedures in this experiment are different from those in Section 8.17. Specifically, the drug treatment procedures in this experiment are as follows: Treat the cells with 10 μM Aβ 1-42 oligomers alone, or simultaneously add 100 μM GABA and / or 100 nM YN-011 respectively and treat the cells for 48 hours. Three replicates are made for each drug treatment.

[0603] The experimental results show (as Figure 27 shown), compared with the treatment with GABA or YN-011 alone, the combined treatment with 100 μM GABA and 100 nM YN-011 has a significantly stronger inhibitory effect on the TNF-α mRNA expression induced by 10 μM Aβ 1-42 oligomers.

[0604] 8.20 GABA reduces the expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglial cells

[0605] Seed human microglial HMC3 cells into a 12-well plate. After culturing overnight in an incubator at 37 °C and 5% CO 2 2, treat with 10 μM Aβ 1-42Oligomers alone, or 1, 10, or 100 μM GABA were added separately to treat the cells for 48 hours. Three replicates were set up for each drug treatment. After 48 hours of treatment, the cells were rinsed with PBS and lysed on ice for 15 minutes with RIPA buffer (Beyotime #P0013B) supplemented with protease inhibitors and phosphatase inhibitors. The cell lysates were centrifuged at 14,000 rpm for 30 minutes. The supernatant was collected, mixed with 5x loading buffer supplemented with β-mercaptoethanol, and boiled at 100 °C for 10 minutes. The denatured proteins were separated by SDS-PAGE. Briefly, 10 μg of protein was loaded onto a precast mini polyacrylamide gel (SurePAGE, GenScript #M00657) and run at 120 V. Thereafter, the gel was electroblotted onto a 0.22-μm PVDF membrane at 200 mA for 90 minutes. After removing the sandwich, the membrane was blocked in a 5% milk solution at room temperature for 1 hour and then incubated overnight at 4 °C with primary antibody diluents (TNF-α, Proteintech #60291-Ig; IL-6, Proteintech #21865-1-AP; HSP90, Proteintech #13171-1-AP). After washing, the membrane was incubated with the corresponding secondary antibody diluent (JacksonLab, 1:10000) at room temperature for 1 hour. After washing, the secondary antibody signal on the membrane was visualized using an ECL kit (Millipore #WBKLS0500) according to the manufacturer's instructions. The protein amount normalized to the endogenous protein HSP90 was calculated using ImageJ.

[0606] The results showed (as Figure 28 shown), in the presence of 10 μM Aβ 1-42 oligomers, the expression of TNF-α and IL-6 in HMC3 cells increased significantly, while 100 μM GABA significantly decreased the expression of IL-6, and 10 μM and 100 μM GABA significantly decreased the expression of TNF-α.

[0607] 8.21 YN-011 reduces the expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglial cells

[0608] Please refer to Section 8.20 for the experimental procedures, but the drug treatment procedures in this experiment are different from those in Section 8.20. Specifically, the drug treatment procedures in this experiment are as follows: Cells were treated with 10 μM Aβ 1-42 oligomers alone, or 10, 100, or 500 nM YN-011 were added separately and simultaneously to treat the cells for 48 hours. Three replicates were set up for each drug treatment.

[0609] The experimental results showed (as Figure 29 shown), in the presence of 10 μM Aβ 1-42In the presence of oligomers, the expression of TNF-α and IL-6 in HMC3 cells was significantly increased, while 100 nM and 500 nM YN-011 significantly decreased the expression of TNF-α and IL-6.

[0610] 8.22 Combined use of YN-011 and GABA reduces the expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglia Factor expression.

[0611] For the experimental procedures, please refer to Section 8.20. However, the drug treatment procedures in this experiment are different from those in Section 8.20. Specifically, the drug treatment procedures in this experiment are as follows: Treat the cells with 10 μM Aβ 1-42 oligomers alone, or add 100 μM GABA and / or 100 nM YN-011 separately and simultaneously to treat the cells for 48 hours. Three replicate wells were set up for each drug treatment.

[0612] The experimental results showed (as Figure 30 shown), compared with the treatment with GABA or YN-011 alone, the combined treatment with 100 μM GABA and 100 nM YN-011 had a significantly stronger inhibitory effect on the TNF-α and IL-6 expression induced by 10 μM Aβ 1-42 oligomers.

[0613] Example 9: Neurodegenerative Diseases in Vivo

[0614] Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), etc., are central nervous system diseases in which central neurons or their myelin sheaths are damaged and gradually lose their functions, characterized by the gradual loss of the structure and function of neuronal cells, manifested as dementia, movement disorders, etc.

[0615] Among them, Alzheimer's disease (AD), also known as senile dementia, is manifested as progressive cognitive impairment and memory impairment, and there is currently no effective treatment method. Research has shown that neurotoxic plaques accumulate in the brains of AD patients, and these plaques are formed by the abnormal aggregation of beta-amyloid (Aβ). Aβ can lead to the formation of senile plaques in the brain and apoptosis of nerve cells, and is an important factor leading to AD. Aβ is a polypeptide containing 39-43 amino acids. The most common subtypes of Aβ in the human body are Aβ40 and Aβ42, which are prone to aggregation, thus forming the core of Aβ precipitation and triggering neurotoxic effects.

[0616] Research has shown that GLP-1 has a neuroprotective biological effect and can improve the symptoms of AD [10,25,26]。In animal models, while GLP-1 improves the cognitive function of AD model mice, it is accompanied by a reduction in Aβ deposition, alleviates Aβ-induced overactivation of glial cells, and reduces oxidative stress and inflammation in the brain, indicating that GLP-1 plays a central nervous protective role in AD. The present invention discloses a long-acting GLP-1 receptor agonist (GLP-1RA) that exerts the biological function of GLP-1 in protecting neurons and can improve central nervous function, contributing to the improvement of AD symptoms.

[0617] Model mice with Alzheimer's disease and wild-type control adult mice of the same genetic background were used in the experiment. The animals were individually housed in cages, maintained on a 12 / 12-hour light / dark cycle (lights on at 08:00 and off at 20:00), and the temperature was controlled at 21.5 °C. Food and water were available ad libitum. Before the behavioral experiments, YN-011 or placebo was intraperitoneally (ip.) injected into the mice once a week for 16 weeks.

[0618] Morris water maze setup

[0619] The maze was made of white opaque plastic with a diameter of 120 cm and a wall height of 40 cm, and filled with water at 25 °C to avoid hypothermia. A small escape platform (10 × 6.5 × 21.5 cm) was placed at a fixed position in one quadrant, 25 cm from the perimeter, and hidden 1 cm below the water surface. There were many fixed visual cues on the walls of the room.

[0620] Spatial memory

[0621] Four points (north, south, east, west) evenly distributed along the circumference of the pool were used as starting positions. The starting positions were randomly assigned in four experiments every day to train the mice to find the safe platform fixed at a certain position in the pool and half-submerged in the water. Twenty-four hours after the end of the training period, the safe platform in the pool was removed, and starting from a random starting position, the length and time of the mice's swimming paths were recorded (n = 12 per group). The collected data were analyzed by one-way ANOVA and two-way ANOVA respectively to evaluate spatial memory as the time and path length required to reach the original position of the safe platform, and spatial acuity as the time spent swimming to the area where the safe platform is located and staying to search.

[0622] Detect Aβ40 and Aβ42 levels

[0623] Measure Aβ40 and Aβ42 levels using an Aβ detection kit. Briefly, homogenize the hemispheres of the brains of control and YN-011-treated AD model mice and the control hemispheres in Tris-buffered saline (25 mM Tris HCl, pH 7.4; 150 mM NaCl) supplemented with protease inhibitor (Sigma, 250 μl / 5 ml buffer). Centrifuge the brain homogenate at 100,000 g and 4 °C for 1 hour. Then dilute the supernatant 1:10 and perform ELISA, which measures only the soluble β-amyloid oligomer level but not the amyloid monomer. Quantify the protein using the Bradford protein assay. Determine the final Aβ value after normalization to the total protein level (n = 6 per group).

[0624] Example 10: Phase IIb and III Clinical Trials of YN-011 (Monotherapy)

[0625] 10.1 Experimental design

[0626] The Phase IIb and III clinical trials were a multicenter, randomized, double-blind, placebo-controlled clinical study of efficacy and safety in patients with T2DM with poor glycemic control after dietary and exercise interventions. The Phase IIb clinical trial was a clinical trial to explore dose-effect and safety assessment (divided into four groups: YN-011 (1 mg) group, YN-011 (2 mg) group, YN-011 (3 mg) group, and placebo control group), and to obtain the Recommended Phase 3 Dose (RP3D) of YN-011 for the Phase III clinical trial, namely the low-dose RP3D and high-dose RP3D. The Phase III clinical trial was a clinical trial to confirm efficacy.

[0627] Main inclusion criteria for the Phase IIb and III studies:

[0628] · Aged 18 - 75 years at screening

[0629] · Diagnosed with type 2 diabetes for at least 8 weeks (WHO 2000) and not received any hypoglycemic drug treatment within 8 weeks before screening.

[0630] · Glycated hemoglobin HbA1c at screening: 7.5% ≤ HbA1c ≤ 11%.

[0631] · Glycated hemoglobin (HbA1c) before randomization: 7.5% ≤ HbA1c ≤ 10.5%

[0632] · Fasting plasma glucose (FPG) at screening and before randomization: < 13.9 mmol / L.

[0633] ·BMI ≥ 18.5 kg / m 2 and ≤ 40 kg / m 2

[0634] Major exclusion criteria for Phase IIb and III studies:

[0635] · Type 1 diabetes

[0636] · Use of any DPP-4 inhibitor and / or GLP-1 analogue within 3 months prior to screening

[0637] · Continuous insulin treatment for more than 14 days within one year prior to screening (treatment time for gestational diabetes with insulin is not within this limit),

[0638] · Fasting C-peptide < 0.3 nmol / mL

[0639] · Diabetic ketoacidosis, diabetic lactic acidosis, or hyperosmolar non-ketotic diabetic coma within 6 months prior to screening

[0640] · Proliferative retinopathy or macular lesions, severe diabetic neuropathy, intermittent claudication, or diabetic foot that is unstable or requires treatment within 6 months during the screening period

[0641] · Severe hypoglycemia (Grade III hypoglycemia) events of unknown cause within 6 months prior to screening, or 3 or more hypoglycemia events (blood glucose < 3.9 mmol / L) within 1 month prior to screening, or recurrent hypoglycemia-related symptoms

[0642] · Severe trauma, severe infection, or surgery that may affect blood glucose control within 1 month prior to screening

[0643] · Blood donation, significant blood loss (> 400 mL), or blood transfusion within 3 months prior to screening;

[0644] · Uncontrolled hypertension

[0645] · Patients with a history of acute or chronic pancreatitis, symptomatic cholecystitis, pancreatic injury history, or other high-risk factors that may lead to pancreatitis, or patients with serum amylase and / or serum lipase ≥ 1.5 times the upper limit of normal value (ULN) at screening;

[0646] · Patients with a history of medullary thyroid carcinoma, multiple endocrine neoplasia (MEN) 2A or 2B syndrome, or a related family history; or patients with a history of other malignancies;

[0647] · Patients with clinically significant gastric emptying abnormalities, severe chronic gastrointestinal diseases, long-term use of drugs that directly affect gastrointestinal motility, or gastrointestinal surgery within 6 months prior to screening, and are considered by the investigator to be unsuitable for participating in this clinical study;

[0648] · Suffering from blood system diseases or any diseases causing hemolysis or red blood cell instability;

[0649] Uncontrolled hyperthyroidism or hypothyroidism;

[0650] · Those who are positive for hepatitis B surface antigen (HBsAg) and hepatitis B virus load (HBV-DNA) higher than the lower limit of local laboratory detection, hepatitis C antibody (HCV-Ab), immunodeficiency virus antibody (HIV-Ab), syphilis treponema pallidum antibody (TP-Ab), or novel coronavirus (COVID-19) nucleic acid test;

[0651] Acute or chronic hepatitis, or laboratory test indicators meet one of the following criteria: alanine aminotransferase (ALT) level ≥2.5x ULN, and / or aspartate aminotransferase (AST) ≥2.5x ULN, fasting triglycerides >5.7mmol / L; glomerular filtration rate (eGFR) calculated by the CKD-EPI (EPI-(Scr)) equation <60mL / min / 1.73m 2 ,

[0652] Any other circumstances that the investigator or attending physician considers may not be suitable for participation in the study.

[0653] The study drug treatment regimen is as follows:

[0654] Subjects enrolled in the dose confirmation phase of Phase IIb:

[0655]

[0656] Subjects enrolled in the Phase III efficacy confirmation phase:

[0657]

[0658] The primary efficacy endpoint is to compare the change in HbA1c level relative to baseline after 12 weeks (Phase IIb) or 24 weeks (Phase III) of double-blind administration of YN-011 and placebo in patients with T2DM. Secondary efficacy endpoints mainly include: changes in FPG, fasting insulin, fasting C-peptide, fasting glucagon, fasting lipid profile and fasting weight relative to baseline (for 12 weeks of treatment in Phase IIb and 24 weeks of treatment in Phase III) and 24 weeks (for 28 weeks of open treatment in Phase III); HbA1c target rate (proportion of subjects with HbA1c <7.0% and HbA1c <6.5%); blood glucose area under the curve during the mixed meal tolerance test (MMTT) and insulin or C-peptide area under the curve during the MMTT. Safety assessment includes adverse events, laboratory tests, vital signs, and 12-lead electrocardiogram evaluation.

[0659] 10.2 Therapeutic effect of YN-011 on type 2 diabetes

[0660] Clinical trial results showed that after 24 weeks of treatment with 1 mg of YN-011, the HbA1c level decreased by 1.73%. It was reported that after 30 weeks of treatment with 1 mg of semaglutide, the HbA1c level decreased by 1.55% (Sorli et al., Lancet Diabetes Endocrinol 2017; 5:251–60); after 26 weeks of treatment with 1.5 mg of dulaglutide, the HbA1c level decreased by 1.46% (Shi et al., J Diabetes Investig 2020; 11:142–150).

[0661] In addition, clinical trial results also showed that after 24 weeks of treatment with 1 mg and 3 mg of YN-011, the incidences of adverse reactions of hypoglycemia (<3.9 mmol / L) were 0.8% and 1.7%, respectively. It was reported that after 26 weeks of treatment with 0.75 mg and 1.5 mg of dulaglutide, the incidences of adverse reactions of hypoglycemia were 4.1% and 6.3%, respectively (Shi et al., J Diabetes Investig 2020; 11:142–150).

[0662] In addition, clinical trial results also showed that after 24 weeks of treatment with 1 mg and 3 mg of YN-011, the incidences of adverse reactions of nausea were 3.4% and 6.0%, respectively. It was reported that after 30 weeks of treatment with 0.5 mg and 1 mg of semaglutide, the incidences of adverse reactions of nausea were 20% and 24%, respectively (Sorli et al., Lancet Diabetes Endocrinol 2017; 5:251–60); after 26 weeks of treatment with 1.5 mg of dulaglutide, the incidence of adverse reaction of nausea was 9.5% (Shi et al., J Diabetes Investig 2020; 11:142–150); after 24 weeks of treatment with 1 mg and 3 mg of lixisenatide, the incidences of adverse reactions of nausea were 5.6% and 10%, respectively (Shuai et al., Diabetes Obes Metab. 2021; 23(1):116-124).

[0663] Example 11: Phase IIb and Phase III Clinical Trials of YN-011 in Combination with Metformin

[0664] 11.1 Experimental design

[0665] The Phase IIb and III clinical trials were multicenter, randomized, double-blind, placebo-controlled clinical studies on the efficacy and safety in patients with type 2 diabetes mellitus (T2DM) who had poor glycemic control after metformin treatment. The Phase IIb clinical trial was a clinical trial for exploring dose-effect and safety assessment (divided into three groups: 1 mg YN-011 combined with metformin group, 3 mg YN-011 combined with metformin group, and placebo combined with metformin control group), to obtain the Recommended Phase 3 Dose (RP3D) for the Phase III clinical trial. The Phase III clinical trial was a clinical trial for efficacy confirmation.

[0666] The major inclusion criteria for the subjects in the Phase IIb and III studies of YN-011 combined with metformin were the same as those in Example 10, except for the following point:

[0667] · Diagnosed with type 2 diabetes for at least 8 weeks (WHO 2000), and meeting any of the following conditions:

[0668] a) Those who had received metformin monotherapy for ≥8 weeks and the metformin dose was ≥1500 mg / day or the maximum tolerated dose (<1500 mg / day but ≥1000 mg / day) (could directly enter the lead-in period after being screened qualified);

[0669] b) Those who had received metformin monotherapy for <8 weeks and the metformin dose was ≥1500 mg / day or the maximum tolerated dose (<1500 mg / day but ≥1000 mg / day) (needed to enter the metformin dose stabilization period after being screened qualified);

[0670] c) Those who had received metformin monotherapy with a dose <1500 mg / day and had not reached the maximum tolerated dose (needed to enter the metformin dose titration period and the dose stabilization period after being screened qualified).

[0671] For the major exclusion criteria of the subjects in the Phase IIb and III studies of YN-011 combined with metformin, please refer to the exclusion criteria section in Example 10.

[0672] The treatment regimen of the study drug is as follows:

[0673] Subjects enrolled in the dose confirmation stage of the Phase IIb:

[0674]

[0675] Subjects enrolled in the efficacy confirmation stage of the Phase III:

[0676]

[0677] The primary efficacy endpoint was to compare the change in HbA1c levels relative to baseline after 12 weeks (Phase IIb) or 24 weeks (Phase III) of double-blind administration of YN-011 combined with metformin versus placebo combined with metformin in T2DM patients with poor glycemic control after metformin treatment. For the secondary efficacy endpoints and safety assessments, please refer to the corresponding parts of Example 10.

[0678] 11.2 Therapeutic effect of the combination of YN-011 and metformin on type 2 diabetes

[0679] The results of this clinical trial showed that after 24 weeks of treatment with 3 mg of YN-011 combined with metformin, the HbA1c level decreased by 1.8% and the FPG level decreased by 2.42%. It was reported that after 40 weeks of treatment with dulaglutide at 1.5 mg combined with metformin, the HbA1c level decreased by 1.42% and the FPG level decreased by 1.93% (Dungan et al., Lancet 2014; 384:1349–57).

[0680] The results of this clinical trial also showed that after 24 weeks of treatment with 3 mg of YN-011 combined with metformin, the incidence of adverse reactions of hypoglycemia (<3.9 mmol / L) was 1.8%, which was comparable to the incidence of hypoglycemia (1.7%) in the control group of placebo combined with metformin. It was reported that after 40 weeks of treatment with dulaglutide at 1.5 mg combined with metformin, the incidence of adverse reactions of hypoglycemia was 9% (Dungan et al., Lancet 2014; 384:1349–57).

[0681] In addition, the results of this clinical trial also showed that after 24 weeks of treatment with 3 mg of YN-011 combined with metformin, the incidence of adverse reactions of nausea was 7.0%. It was reported that after 40 weeks of treatment with dulaglutide at 1.5 mg combined with metformin, the incidence of adverse reactions of nausea was 20% (Dungan et al., Lancet 2014; 384:1349–57); after 30 weeks of treatment with semaglutide at 1 mg combined with metformin, the incidence of adverse reactions of nausea was 13.4% (Ji et al., Diabetes Obes Metab. 2021; 23:404–414).

[0682] The above examples list the content that is currently considered to present the preferred examples of this application. However, it should be understood that this application is not limited to the disclosed examples. On the contrary, this application aims to cover various modifications and equivalent examples included within the spirit and scope of the appended claims.

[0683] All publications, patents, and patent applications are hereby incorporated by reference in their entirety. Specifically, the sequences associated with each accession number provided herein, including, for example, accession numbers provided in tables or elsewhere and / or biomarker sequences (such as proteins and / or polynucleotides), are incorporated by reference in their entirety.

[0684] The scope of the claims should not be limited by the preferred examples and embodiments, but should be construed in the broadest manner consistent with the specification.

[0685] References

[0686] 1. Leech, C.A., et al., Expression of cAMP-regulated guanine nucleotide exchange factors in pancreatic beta-cells. Biochem Biophys Res Commun, 2000. 278(1): p. 44-7.

[0687] 2. Drucker, D.J., Glucagon-like peptides. Diabetes, 1998. 47(2): p. 159-69.

[0688] 3. Montrose-Rafizadeh, C., et al., Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells. Endocrinology, 1999. 140(3): p. 1132-40.

[0689] 4. Ahren, B. and O. Schmitz, GLP-1 receptor agonists and DPP-4 inhibitors in the treatment of type 2 diabetes. Horm Metab Res, 2004. 36(11-12): p. 867-76.

[0690] 5. Green, B. D., et al., Structurally modified analogues of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) as future antidiabetic agents. Curr Pharm Des, 2004. 10(29): p. 3651-62.

[0691] 6. Chang, A. M., et al., The GLP-1 derivative NN2211 restores beta-cell sensitivity to glucose in type 2 diabetic patients after a single dose. Diabetes, 2003. 52(7): p. 1786-91.

[0692] 7. Liu, H. K., et al., N-acetyl-GLP-1: a DPP IV-resistant analogue of glucagon-like peptide-1 (GLP-1) with improved effects on pancreatic beta-cell-associated gene expression. Cell Biol Int, 2004. 28(1): p. 69-73.

[0693] 8. Kim, J. G., et al., Development and characterization of a glucagon-like peptide 1-albumin conjugate: the ability to activate the glucagon-like peptide 1 receptor in vivo. Diabetes, 2003. 52(3): p. 751-9.

[0694] 9. Nielsen, R., et al., Effect of liraglutide on myocardial glucose uptake and blood flow in stable chronic heart failure patients: A double-blind, randomized, placebo-controlled LIVE sub-study. J Nucl Cardiol, 2019. 26(2): p. 585-597.

[0695] 10. Duarte, A. I., et al., Liraglutide Protects Against Brain Amyloid-beta 1-42 Accumulation in Female Mice with Early Alzheimer's Disease-Like Pathology by Partially Rescuing Oxidative / Nitrosative Stress and Inflammation. Int J Mol Sci, 2020. 21(5).

[0696] 11. Elbassuoni, E. A. and R. F. Ahmed, Mechanism of the neuroprotective effect of GLP-1 in a rat model of Parkinson's with pre-existing diabetes. Neurochem Int, 2019. 131: p. 104583.

[0697] 12. Muskiet, M. H. A., et al., GLP-1 and the kidney: from physiology to pharmacology and outcomes in diabetes. Nat Rev Nephrol, 2017. 13(10): p. 605-628.

[0698] 13. Hou, Y., et al., Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc-Fusion Protein in a CHO Fed-Batch Process. Biotechnol J, 2019. 14(3): p. e1700706.

[0699] 14. Karlin, S. and S. F. Altschul, Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA, 1990. 87(6): p. 2264-8.

[0700] 15. Karlin, S. and S. F. Altschul, Applications and statistics for multiple high-scoring segments in molecular sequences. Proc Natl Acad Sci U S A, 1993. 90(12): p. 5873-7.

[0701] 16. Altschul, S. F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-10.

[0702] 17. Altschul, S. F., et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997. 25(17): p. 3389-402.

[0703] 18. Myers, E. W. and W. Miller, Optimal alignments in linear space. Comput Appl Biosci, 1988. 4(1): p. 11-7.

[0704] 19. Bettinger, J.Q., et al., Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome. J Proteome Res, 2020. 19(2): p. 624 - 633.

[0705] 20. Allen, L.V., Jr., Remington: The Science and Practice of Pharmacy: from the past into the future. Int J Pharm Compd, 2012. 16(5): p. 358 - 62.

[0706] 21. Toft - Nielsen, M.B., S. Madsbad, and J.J. Holst, Continuous subcutaneous infusion of glucagon - like peptide 1 lowers plasma glucose and reduces appetite in type 2 diabetic patients. Diabetes Care, 1999. 22(7): p. 1137 - 43.

[0707] 22. Geiser, J.S., et al., Clinical Pharmacokinetics of Dulaglutide in Patients with Type 2 Diabetes: Analyses of Data from Clinical Trials. Clin Pharmacokinet, 2016. 55(5): p. 625 - 34.

[0708] 23. Bodanszky, M., Principles of peptide synthesis. 2nd rev. ed. Springer laboratory. 1993, Berlin;New York: Springer - Verlag. xii, 329 p.

[0709] 24. Pratley, R.E., et al., Semaglutide versus dulaglutide once weekly in patients with type 2 diabetes (SUSTAIN 7): a randomised, open-label, phase 3b trial. Lancet Diabetes Endocrinol, 2018. 6(4): p. 275-286.

[0710] 25. Zheng, J., et al., GLP-1 improves the supportive ability of astrocytes to neurons by promoting aerobic glycolysis in Alzheimer's disease. Mol Metab, 2021. 47: p. 101180.

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Claims

1. Use of a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain in the preparation of a medicament for treating or preventing a disease, wherein, the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions selected from the group consisting of: A8G, G22E, and R36G relative to native human GLP-1; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

2. Use of a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain in the preparation of a medicament for treating or preventing a disease, wherein, a) the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions selected from the group consisting of: A8G, G22E, and R36G relative to native human GLP-1; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S; and b) the medicament is used in combination with an additional therapeutic agent.

3. Use according to any one of the preceding claims, wherein, the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3, and the amino acid sequence of the immunoglobulin Fc domain is as shown in SEQ ID NO:

6.

4. Use according to any one of the preceding claims, wherein the disease is selected from the group consisting of: metabolic diseases related to disorders of glucose and / or lipid metabolism, complications of metabolic diseases, central metabolic diseases (e.g., neurological diseases), and other related metabolic diseases.

5. Use according to claim 4, wherein the neurological disease is a neurodegenerative disease.

6. Use according to claim 5, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

7. Use of a fusion protein comprising the amino acid sequence as shown in SEQ ID NO:7 and gamma-aminobutyric acid in the preparation of a medicament for treating a neurodegenerative disease.

8. A pharmaceutical combination comprising a fusion protein and an additional therapeutic agent, wherein the fusion protein comprises a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(7-37), human GLP-1(7-36) amide, and DPP-IV resistant human GLP-1, and the GLP-1 polypeptide comprises one or more amino acid substitutions relative to native human GLP-1 selected from the group consisting of: A8G, G22E, and R36G; the immunoglobulin Fc domain comprises or is an IgG2-Fc domain, and the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of: C222S, A330S, and P331S.

9. A pharmaceutical combination comprising a fusion protein and gamma-aminobutyric acid, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

7.

10. A pharmaceutical combination comprising a fusion protein and gamma-aminobutyric acid, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:7.

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