Application of improved fusion protein of GLP-1 receptor agonist in disease treatment

Abstract

The present invention relates to the use of an improved GLP-1 receptor agonist or a fusion protein comprising the improved GLP-1 receptor agonist in the treatment or prevention of nervous system diseases.

Description

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

[0002] The present invention relates to the field of biomedicine, and in particular, 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 applications thereof. Background Art

[0003] Glucagon-like peptide-1 (GLP-1), also known as incretin, is secreted by the L cells of the small intestine and has multi-organ targeted regulatory effects, including promoting pancreatic hormone secretion, inhibiting glucagon release, slowing gastric emptying, reducing appetite, and playing an important regulatory role in nutrient intake 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 that is mainly expressed in pancreatic β cells and is also expressed to a certain extent in other tissues and cells (lungs, heart, kidneys, gastrointestinal tract and brain). After binding to the receptor, GLP-1 activates adenylate cyclase (AC), thereby stimulating the production of the second messenger cyclic adenosine monophosphate (cAMP), and binds to protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (camp GEFs). [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 [3] Therefore, scientists have developed a variety of long-acting, resistant-to-degradation GLP-1 analogs. For example, human GLP-1 analogs have been replaced with amino acids. [4,5] and / or N-terminal modifications, including fatty acylation [6] N-acetylation [7] Prolonged circulation half-life. Albumin-conjugated GLP-1 (albiglutide) also has a prolonged half-life [8]In recent years, various GLP-1 receptor agonists and their analogs have been widely used to treat 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 have an important role in cardiovascular diseases. [9] and neurological diseases [10,11] It also plays a preventive and therapeutic role. 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 No. 8,658,174 discloses a GLP-1 fusion protein, which comprises a GLP-1 polypeptide fused to an IgG / Fc domain and can be used to treat diabetes.

[0006] Studies have shown that post-translational modifications exist in GLP1-Fc fusion proteins. Hou et al. reported in 2019 that the addition of nicotinamide and cysteine ​​during cell culture helped reduce the hydroxylation level of a GLP analog and IgG4 / Fc fusion protein (dulaglutide)

[13] .

[0007] The use of genetic engineering recombinant protein technology to produce fusion proteins for therapeutic purposes aims to express the properties of natural polypeptides. The process involves cell engineering steps of transcription, translation and post-translational modification, and its technology directly affects the drug's physicochemical characteristics, conformation, in vivo half-life, biological activity, and production yield, etc. Therefore, there is still a need for improved GLP-1 receptor agonists and fusion proteins thereof, 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 purpose of the present invention is to provide an improved GLP-1 receptor agonist and a fusion protein thereof with higher activity and yield, and the fusion protein can be obtained by a variety of ways or methods, such as replacing amino acids at specific positions of the protein sequence, or performing hydroxylation, oxidation and other modifications, such as by hydroxylating K34 of the GLP-1 polypeptide and / or reducing the oxidation of the GLP-1 polypeptide. In addition, the amino acid replacement 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 disease and animal disease models.

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

[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 following group relative to native human GLP-1: 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 following group: 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 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 following group: A8G, G22E and R36G relative to native human GLP-1.

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

[0014] In one embodiment, the IgG2-Fc domain has at least 90% sequence identity 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 includes 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 further comprises 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 lysine hydroxylase expressed by the recombinantly expressing or naturally expressing lysyl hydroxylase cell is higher than the level or activity of lysine hydroxylase expressed by COS-7 cells.

[0027] More preferably, the cell is a CHO cell, in particular a CHO-K1 cell.

[0028] Still 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 the fusion protein using the cell described herein or the cell 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 a use of the fusion protein, or dimer, or polynucleotide, or vector, or cell, or composition described herein in a medicine, and / or in the preparation of a medicine for treating or preventing a disease.

[0035] Other features and advantages of the present invention will be apparent in the detailed description of the embodiments below. Detailed description and specific examples are given by way of illustration only while the preferred embodiments of the present invention are provided, and various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0037] Figure 2 This is the mass spectrum of the 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: EFIAWLVK primary mass spectrum; B: EFIAWLVK (+16Da) modified primary mass spectrum; C: EFIAWLVK secondary mass spectrum; D: EFIAWLVK (+16Da) modified secondary mass spectrum.

[0038] Figure 3AThe UV-detected hydroxylation data of modified peptides 27-34 (aa21-28 in the figure) after Lys-C digestion: EFIAWLVK (+16Da) modified peptide (aa21-28 (+16Da) in the figure), unmodified peptides 54-79 without +16Da (aa48-73 in the figure), and unmodified peptides 224-240 without +16Da (aa218-234 in the figure); Figure 3B yes Figure 3A Enlarged images of small and medium images.

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

[0040] Figure 5 This is a design chart showing a Phase IIa double-blind, placebo-controlled study in T2DM subjects, which evaluates the efficacy and safety of subcutaneous administration of YN-011 at dose levels of 1mg, 2mg, 3mg, and 4mg. The solid triangle represents the formal dose administration of YN-011, the solid circle represents the glucose tolerance test (OGTT) test, the hollow triangle represents adaptive administration, and the five-pointed star represents the safety evaluation test.

[0041] Figure 6 The figure shows the effect of multiple doses of YN-011 on fasting blood glucose in T2DM subjects. (At each time point tested, YN-011 was compared with placebo at dose levels of 3 mg and 4 mg, P < 0.05.)

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

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

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

[0045] Fig.10 The results show that YN-011 and GABA inhibited the effect of TNF-α in reducing the viability of SH-SY5Y cells (**P<0.01TNF-α vs control; #P<0.05YN-011 (10nM or 100nM) or GABA (100μM) + TNF-α vs TNF-α; ##P<0.01YN-011 (500nM) + TNF-α vs TNF-α; n=6).

[0046] Fig.11 The results showed that the combined use of YN-011 and GABA significantly increased 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] Fig.12 It was shown that YN-011 reduced TNF-α-induced apoptosis in neuronal SH-SY5Y cells (**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] Fig.13 It was shown that GABA reduced TNF-α-induced apoptosis in neuronal SH-SY5Y cells (**P<0.01 TNF-α vs control, #P<0.05 10 μM or 100 μM GABA+TNF-α vs TNF-α; n=3).

[0049] Fig.14 It was shown that the combined use of YN-011 and GABA reduced TNF-α-induced apoptosis in SH-SY5Y neuronal cells (**P<0.01TNF-α vs control, #P<0.05GABA or YN-011+TNF-α vs GABA+YN-011+TNF-α; n=3).

[0050] Fig.15 The live cell staining images show that TNF-α promotes the damage of neuronal cells SH-SY5Y (**: significant; ***: extremely significant).

[0051] Fig.16 The live cell staining images show that YN-011 has a protective effect on TNF-α-injured neuronal cells (**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] Fig.17 The live cell staining images show that GABA has a protective effect on TNF-α-injured neuronal cells (**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] Fig.18 The live cell staining images show that the combined use of YN-011 and GABA has a protective effect on TNF-α-injured neuronal cells (**P<0.01TNF-α vs control; #P<0.05GABA or YN-011+TNF-α vs GABA+YN-011+TNF-α; n=3).

[0054] Fig.19 It is shown that YN-011 reduces TNF-α-induced apoptosis of neuronal cells (**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] Fig. 20 It is shown that GABA reduces 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).

[0056] Fig.21 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 100nM YN-011+TNF-α vs 100μM GABA+100nM YN-011+TNF-α; ##P<0.01 100μM GABA+TNF-α vs 100μM GABA+100nM YN-011+TNF-α; n=3).

[0057] Fig. 22It is shown that YN-011 reduces 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] Fig.23 It is shown that GABA reduces 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] Fig.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+100μM GABA+100nM YN-011 vs TNF-α+100μM GABA; ##P<0.05 TNF-α+100μM GABA+100nM YN-011 vs TNF-α+100nM YN-011; n=3).

[0060] Fig.25 GABA was shown to reduce Aβ in HMC3 microglia 1-42 Oligomer-induced mRNA expression of inflammatory factors (*P<0.05, **P<0.01Aβ 1-42 Oligomers vs. control; #P<0.05GABA+Aβ 1-42 Oligomers vs Aβ 1-42 oligomer; n=3).

[0061] Fig.26 YN-011 was shown to reduce Aβ in HMC3 microglia 1-42 Oligomer-induced mRNA expression of inflammatory factors (*P<0.05, **P<0.01Aβ 1-42 Oligomer vs control; #P<0.05, ##P<0.01YN-011+Aβ 1-42 Oligomers vs Aβ 1-42 oligomer; n=3).

[0062] Fig. 27 Combination of YN-011 and GABA reduces Aβ in HMC3 microglia 1-42 Oligomer-induced mRNA expression of inflammatory factors (*P<0.05, **P<0.01Aβ 1-42 Oligomers vs. control; #P<0.05, GABA+YN-011+Aβ 1-42Oligomers vs GABA+Aβ 1-42 Oligomer or YN-011+Aβ 1-42 oligomer; n=3).

[0063] Fig.28 GABA was shown to reduce Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory cytokine expression (**P<0.01Aβ 1-42 Oligomers vs. control; #P<0.05GABA+Aβ 1-42 Oligomers vs Aβ 1-42 oligomer; n=3).

[0064] Fig.29 YN-011 was shown to reduce Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory cytokine expression (**P<0.01Aβ 1-42 Oligomer vs control; #P<0.05YN-011+Aβ 1-42 Oligomers vs Aβ 1-42 oligomer; n=3).

[0065] Fig.30 Combination of YN-011 and GABA reduces Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory cytokine expression (**P<0.01Aβ 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 oligomer; n=3). DETAILED DESCRIPTION

[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 those generally understood by those of ordinary skill in the art to which the present invention belongs. The terms used in the present invention are only used to describe specific embodiments and are not intended to limit the present invention. All publications, patent applications, patents, drawings and other references mentioned herein are incorporated by reference in their entirety.

[0067] I. Definitions or Terms

[0068] Unless otherwise stated, the defined terms and terms used herein should be understood to refer to dictionary definitions, definitions in incorporated documents, and / or commonly understood meanings of the defined terms.

[0069] All references, patents, and patent applications referred to herein are incorporated by reference into the subject matter of each cited document, in some cases in their entirety.

[0070] All features disclosed in this specification may be combined in any manner. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly 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 connection of two or more natural or non-natural amino acid residues, regardless of whether there is post-translational modification (e.g., glycosylation or phosphorylation). The polypeptide of the present invention may include, for example, 3 to 3500 natural or non-natural amino acid residues. The protein may be a single peptide chain or a multi-subunit protein (e.g., it may be composed of 2 or more polypeptides). The "peptide", "polypeptide" and "protein" described herein may be used interchangeably and may contain natural amino acids, non-natural amino acids, or analogs and mimetics of amino acids. The peptides, polypeptides or proteins described in this application may be obtained by any method known in the art, such as, but not limited to, by natural separation, recombinant expression, chemical synthesis, etc.

[0072] As used herein, the term "amino acid" refers to a 2 ) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. The amino acid names are also represented in this application by standard single-letter or three-letter codes, which are summarized as follows:

[0073] name Three letter code Single letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate 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 lysine at amino acid residue 34 or corresponding to amino acid residue 34, such as 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 (also 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 lysine at amino acid residue 34 or corresponding to lysine at amino acid residue 34. For example, the GLP-1 polypeptide may include liraglutide (Liraglutide, from Novo Nordisk) ), Semaglutide (from Novo Nordisk ), Albiglutide (from GlaxoSmithKline ), tapoglutide (Roche), dulaglutide (from Eli Lilly ) or LY2428757 (Eli Lilly), and may also include 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 analog comprising a "KG" amino acid motif sequence.

[0075] As used herein, the term "polynucleotide" or "oligonucleotide" refers to two or more covalently linked nucleotides. Unless the context clearly states otherwise, the term generally includes, but is not limited to, deoxyribonucleotides (DNA) and ribonucleotides (RNA), which can be single-stranded (ss) or double-stranded (ds). For example, the polynucleotide molecules or polynucleotides of the present invention can be composed of single-stranded and double-stranded DNA, DNA, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions. A mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, the polynucleotide molecule can be composed of a triple-stranded region comprising 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 can be single-stranded or double-stranded. The sequence provided herein can be a DNA sequence or an RNA sequence, but it should be understood that the sequence provided includes DNA and RNA, as well as complementary RNA and DNA sequences, unless the context clearly states otherwise. For example, the sequence 5'-GAATCC-3' is understood to include 5'-GAAUCC-3', 5'-GGATTC-3', and 5'-GGAUUC-3'.

[0076] As used herein, the term "sequence identity" or "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two polynucleotide sequences. In order to determine the percentage identity of two amino acid sequences or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (for example, a gap can be introduced in the sequence of the first amino acid or polynucleotide sequence to optimally align the amino acid or polynucleotide sequence 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) identical to the reference sequence compared thereto by the total number of amino acid residues (or bases) in the candidate sequence or reference sequence (whichever is shorter). When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between the 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. A mathematical algorithm can also be used to determine the percentage identity between the two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the Karlin-Altschul algorithm.

[14] , which was later modified to the Karlin-Altschul algorithm

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

[16] BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameter 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 parameter set, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used.

[17] Alternatively, PSI-BLAST can be used to perform an iterated search to detect distant relationships (Id.) between molecules. When using 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 using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, only exact matches are usually calculated.

[0077] In the present invention, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue without eliminating the desired properties of the protein. Suitable conservative amino acid substitutions can be performed by replacing amino acids with similar hydrophobicity, polarity and R-chain length with each other. Examples of conservative substitutions include replacing one non-polar (hydrophobic) residue with another non-polar 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 replacing non-derivatized residues with chemically derived residues or non-natural amino acids, provided that the polypeptide exhibits the necessary activity.

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

[0079] In the present invention, the term "linker" refers to any chemical moiety that can covalently link one part to another part. 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, connected by peptide bonds, and used to connect 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 (eg, IgG) refers to the flexible region between the antigen binding fragment (Fab) and the crystallizable fragment (Fc).

[0082] The term "vector" as used herein refers to a vehicle into which a genetic element can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including but not limited to, viral particles, liposomes or protein shells. The vector may be an expression vector or a cloning vector.

[0083] The terms "DPPIV" and "DPP-IV" refer to dipeptidyl peptidase IV, an enzyme that inactivates 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. Modulators can be used to increase or decrease the hydroxylation level of the expressed protein. For example, when present in an expression system, minoxidil and Zn 2+ (e.g. from ZnSO 4 ) can inhibit hydroxylation and reduce the level of hydroxylation. The hydroxylation level can be measured using the method in this embodiment or 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 amino acid positions 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 determined using the method of the present embodiment or by mass spectrometry as described in WO2002046227A2 or according to Bettinger et al.

[19] Protein oxidation assay method, or as described herein.

[0086] In the present invention, the term "pharmaceutical grade" means that the chemical purity or proportion of drugs, biomacromolecules or reagents meets the requirements of drug production.

[0087] In the present invention, the term "treatment" refers to the administration of an effective amount of a compound or composition or formulation to a subject, which may consist of a single administration, or alternatively 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, alleviation or improvement of one or more symptoms or conditions, alleviation of the extent of the disease, stabilization (i.e., non-exacerbation) of the disease state, prevention of disease spread, reversal of the disease, improvement or alleviation of the disease, and alleviation of the disease state (whether partial or complete, or temporary). 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 as used herein, refers to all animals including mammals, and preferably refers to humans. "Subject" may also be livestock animals, such as cattle, pigs, sheep, poultry and horses; or rodents, such as rats and mice; or primates, such as apes, monkeys, chimpanzees, gorillas, orangutans, and baboons; or domestic animals, such as dogs and cats.

[0089] In the present invention, the term "pharmaceutically acceptable carrier" refers to any carrier, agent, or excipient that is biologically or otherwise acceptable. Unless the carrier, agent, or excipient is incompatible with the active ingredient, its use in a therapeutic formulation is acceptable. The use of such pharmaceutically acceptable carriers is well known in the art. For example, various ingredients that may 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, and the desired effect refers to relieving symptoms, slowing disease progression, preventing disease onset, etc. This amount can be effectively administered multiple times and / or achieve the desired effect within a period of time. As used herein, the term can refer to an amount that causes a decrease in blood sugar in a subject.

[0091] In the present invention, "combined administration" and the like refer to, for example, two or more substances (e.g., two or more compounds, two or more compositions, etc.) that are administered to a subject and that both have biological activity at the same time. The exact circumstances of administration will depend on the pharmacokinetics of the two or more substances in the presence of each other.

[0092] In understanding the scope of the present invention, the term "comprise" and its derivatives as used herein are open terms that specify the features, elements, components, groups, integers and / or steps, but do not exclude the presence of other unspecified features, elements, components, groups, integers and / or steps. The above also applies to words with similar meanings, such as the terms "comprises", "having" and their derivatives.

[0093] As used herein, the terms "consisting of," "consisting of," and their derivatives are closed terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers, and / or steps.

[0094] Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (eg, 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, as used herein, terms of degree such as "substantially", "approximately" and "roughly" represent a reasonable amount of deviation of the modified term so that the end result is not significantly changed. These terms of degree should be interpreted as including a deviation of at least ±5% of the modifier if such deviation would not negate the meaning of its modifier. More specifically, the term "about" refers to ±0.1-25%, ±1-20%, ±1-15%, ±1-10%, for example, 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" and "the" include plural references unless the content 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 in its sense including "and / or" unless the content clearly dictates otherwise.

[0097] In addition, the definitions and embodiments described in the specific parts are intended to be applicable to other embodiments described herein, and those skilled in the art will understand. For example, in the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect defined in this way can be combined with any other aspect or aspects, unless it is clearly stated that it cannot be combined. In particular, any feature indicated as preferred or advantageous can be combined with any other one or more features indicated as 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 described in this embodiment.

[0099] II. Proteins and Fusion Proteins

[0100] The present invention provides stable GLP-1 polypeptides and fusion proteins comprising the GLP-1 polypeptides fused with, for example, IgG / Fc domains.

[0101] According to one aspect of the present invention, a GLP-1 fusion protein is provided, which comprises 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 following group relative to native human GLP-1: 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 following group: C222S, A330S and P331S.

[0102] GLP-1 peptide

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

[0104] The unprocessed natural human GLP-1 polypeptide has 37 amino acids, and its amino acid sequence is shown in SEQ ID NO:43, and is also commonly referred to as "GLP-1 (1-37)". The unprocessed natural human GLP-1 polypeptide 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 sites of the GLP-1 polypeptide mentioned in this application are the amino acid sites corresponding to SEQ ID NO:43. For example, K34 of the GLP-1 polypeptide mentioned in this application corresponds to the 34th site of SEQ ID NO:43. For another example, GLP-1 (7-36) amide refers to the GLP-1 polypeptide fragment formed by the amino acids between positions 7 and 36 of SEQ ID NO:43; GLP-1 (7-37) refers to the GLP-1 polypeptide fragment formed by the amino acids between positions 7 and 37 of SEQ ID NO:43. In certain embodiments, the amino acid sequence of GLP-1(7-36)amide is shown in SEQ ID NO: 1. In certain embodiments, the amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO: 2.

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

[0106] The hydroxylation level of the GLP-1 fusion protein of the present invention can be determined by various methods known in the art. For example, the mass spectrometry method described by Hou et al.

[13] Unless otherwise specified, the hydroxylation level described in this application is calculated as a percentage of the number of molecules. For example, among 100g of the GLP-1 fusion protein described in the present invention, if 10g of the GLP-1 fusion protein is hydroxylated at K34 of the GLP-1 polypeptide, and the remaining 90g of the GLP-1 fusion protein is not hydroxylated 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, in some embodiments, has an increased in vivo half-life and / or yield when recombinantly produced. 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 may correspond to different positions in other GLP-1 polypeptide sequences.

[0109] GLP-1 fusion protein may refer to a plurality of molecules, which may be hydroxylated at K34, or may not be hydroxylated, and the hydroxylation levels of the molecules may vary.

[0110] The present invention also provides compositions comprising a GLP-1 fusion protein. In one embodiment, the GLP-1 fusion protein has a hydroxylation level at K34 of 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 hydroxylation level is between 10% and 100%, for example, 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 is not detected at K34 of GLP-1.

[0113] The present invention also found that the GLP-1 polypeptide has a lower oxidation level at the 31st tryptophan (W31) relative to native human GLP-1, and the lower oxidation level 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 the 31st tryptophan (W31) relative to native human GLP-1. In certain embodiments, the oxidation level 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 may correspond to different positions in other GLP-1 polypeptides.

[0114] The chromatographic analysis can be performed by methods known in the examples of the present application or in the prior art (e.g., the mass spectrometry method described in WO2002046227A2 or the method described in Bettinger et al.

[19] Protein oxidation assay) is used for detection. Unless otherwise specified, the oxidation level described in this application is calculated based on the percentage of the number of molecules. For example, among 100g of the GLP-1 fusion protein described in the present invention, if 10g of the GLP-1 fusion protein is oxidized at W31 of GLP-1, and the remaining 90g of the GLP-1 fusion protein is not oxidized at W31 of GLP-1, then 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 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 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 may comprise amino acid substitutions, such as one, two or three mutations of A8G, G22E, R36G.

[0118] In certain embodiments, the GLP-1 polypeptide described herein 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: 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 herein 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: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and stimulates β cells to secrete insulin in a glucose-dependent manner. In certain embodiments, the GLP-1 polypeptide described herein 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: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and comprises one or more amino acid substitutions selected from the following group: A8G, G22E and R36G relative to native human GLP-1, and stimulates β cells to secrete insulin in a glucose-dependent manner.

[0119]

[0120] In certain embodiments, the GLP-1 polypeptide is human GLP-1(7-37) and comprises 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 shown in SEQ ID NO: 3.

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

[0123] Natural GLP-1 has a short circulation 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 natural GLP-1 is used, continuous subcutaneous infusion via a pump is necessary to maintain the effect of GLP-1 in vivo.

[21] Therefore, pharmaceutical long-acting degradation-resistant GLP-1 analog peptides have been developed. For example, dulaglutide is a DPP-IV-protected GLP-1 analog fused to an IgG4 / Fc fragment with a half-life of 4.7-5.5 days.

[22] .

[0124] Fc domain

[0125] In certain embodiments, the IgG2-Fc domain described herein is an Fc domain from human IgG2.

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

[0127] In certain embodiments, the IgG2-Fc domain described herein has a certain level of oxidation at the 253rd methionine (M253) corresponding to SEQ ID NO: 7. In certain embodiments, the IgG2-Fc domain described herein has a reduced level of oxidation at the 253rd methionine (M253) corresponding to SEQ ID NO: 7. In certain embodiments, the IgG2-Fc domain described herein has no oxidation at the 253rd methionine (M253) corresponding to SEQ ID NO: 7. In the present application, M253 refers to the methionine residue at the 253rd IgG2 corresponding to SEQ ID NO: 7. Those skilled in the art will appreciate that the same methionine may correspond to different positions in other IgG polypeptides.

[0128] In certain embodiments, the oxidation level 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 may refer to a plurality of molecules, which may be oxidized or unoxidized at one or both of the positions M253 of the IgG2-Fc domain and / or W31 of the GLP-1 polypeptide, and the oxidation levels of the molecules may be different. The oxidation occurs at the rectangular box in the following amino acid structure:

[0130]

[0131] In certain embodiments, the GLP-1 fusion protein of the present invention has no detected oxidation at W31 of the GLP-1 polypeptide relative to native human GLP-1, and has an oxidation level of about 2% at M253 of the IgG2-Fc domain. 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) are 2-8%.

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

[0133] In one embodiment, the IgG2-Fc domain comprises 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 cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

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

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

[0136] Except that the amino acid position of M253 is corresponding to the amino acid position of SEQ ID NO:7, 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 of human IgG2 as shown in Genbank Accession No. QRG33935.1. The IgG2-Fc portion can be 228 amino acids as shown in SEQ ID NO:5, corresponding to, for example, amino acids 219 to 445 of human IgG2 as shown in Genbank Accession No. QRG33935.1. Those skilled in the art will readily recognize the residue positions of amino acids, such as the residue positions of A330, P331 and C222 in the short or long Fc fragments shown in the reference sequences SEQ ID NO:5 or SEQ ID NO:6.

[0137] In one embodiment, the IgG2-Fc domain described herein 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. The fusion protein has an improved half-life compared to a GLP-1 polypeptide without an IgG / Fc domain fusion or with an IgG4 / Fc domain fusion.

[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 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 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 described in the present application, the amino acid sequence of the GLP-1 polypeptide is shown as SEQ ID NO:3, and the amino acid sequence of the immunoglobulin Fc domain is shown as SEQ ID NO:6.

[0141]

[0142] The positions of the sites where the A8G, G22E, R36G substitutions and K34 hydroxylation of the GLP-1 polypeptide mentioned in the present application occur in SEQ ID NO: 3 and in SEQ ID NO: 7 are shown in the following table. In addition, the positions of the sites where the C222S, A330S, P331S substitutions and M253 oxidation on the IgG2-Fc domain mentioned in the present application occur relative to the positions in QRG33935.1 and in SEQ ID NO: 7 are 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 covalently linked directly 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 linker peptide.

[0151] The GLP-1 polypeptide and IgG-Fc domain, e.g., IgG2-Fc domain, of the fusion protein disclosed herein can be connected by a connecting peptide. As used herein, the term "connecting peptide" refers to any portion that connects different functional domains of a polypeptide together. The connecting 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 factors such as proteases, pH or chemicals in vivo and is easily cleaved in the presence of the above 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 easily cleaved.

[0154] In the present invention, the term "flexible linker" refers to a linker that can increase spatial extensibility when connecting different protein components so that the spatial folding and conformation of the protein components are not affected by each other as much as possible.

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

[0156] In the present invention, the term "helical linker" refers to a linker in which a rigid unit can form a helix (eg, α-helix) within itself or with the same adjacent sequence, thereby allowing the formed 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 is unable to form a helical structure.

[0158] In one embodiment, the connecting peptide comprises a linker comprising glycine and serine. Preferably, the linker comprising 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 (GGGGGGGS).

[0159] In one embodiment, the connecting 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: Connector Peptide SSGAPPPSGGGGS 9 Connector Peptide GGGGSGGGGSGGGGS 10 Connector Peptide GGGGSGGGGSGGGGSGGGGS 11 Connector Peptide GSTGGGGSGKPGSGEGGGGS 12 Connector Peptide ESGRSGGGGSGGGGS 13 Connector Peptide EGKSSGSGSESKST 14 Connector Peptide EGKSSGSGSESKSTQ 15 Connector Peptide EGKSSGSGSESKVD 16 Connector Peptide GSTSGSGKSSEGKG 17 Connector Peptide KESGSVSSEQLAQFRSLD 18 Connector Peptide ESGSVSSEELAFRSLD 19 Connector Peptide GGGS 39 Connector Peptide GGGGS 40 Connector Peptide GGGGGS 41 Connector Peptide GGGGGGGS 42

[0161] In one embodiment, the connecting peptide connecting 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 as shown in SEQ ID NO: 9. In one embodiment, the connecting peptide comprises the amino acid sequence as shown in SEQ ID NO: 9. In one embodiment, the amino acid sequence of the connecting 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 shown as SEQ ID NO:3, the amino acid sequence of the immunoglobulin Fc domain is shown as SEQ ID NO:6, and the amino acid sequence of the linker is shown as SEQ ID NO:9.

[0163] Fusion Protein

[0164] In one embodiment, the fusion protein described herein 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 herein 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 comprises A8G, G22E, R36G substitutions, and the IgG2-Fc domain comprises C222S, A330S, and P331S substitutions. In one embodiment, the fusion protein described herein 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 beta cells to secrete insulin 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 β cells to secrete insulin in a glucose-dependent manner.

[0165] In one embodiment, the fusion protein described in the present application has an amino acid sequence as 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 an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID NO:7.

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

[0169]

[0170] As demonstrated in the examples, the GLP-1 fusion protein disclosed herein has a longer half-life after sequence modification. For example, the GLP-1 fusion protein YN-011 has a half-life of about 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 causes the fusion protein to be secreted into the extracellular medium. Such polypeptides may also be referred to as "leader peptides", "polypeptide precursors", "propeptides", and the like. The use of signal peptides to direct the secretion of proteins is known in the art (e.g., U.S. Pat. No. 8,658,174, the contents of which are incorporated herein by reference in their entirety). Examples of signal peptides include, but are not limited to, human CD33 signal peptide, human growth hormone-releasing hormone (GHRH) signal peptide, human α-1-microglobulin / bikunin precursor (AMBP) signal peptide, Gaussia luciferase signal peptide, mouse immunoglobulin heavy chain signal peptide, mouse immunoglobulin κ light chain signal peptide. The signal peptide is cleaved during the secretion process. In some embodiments, cleavage of the signal peptide produces an active histidine residue at the N-terminus of the GLP-1 polypeptide.

[0173] In one embodiment, the signal peptide is 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 to the amino acid sequence shown in SEQ ID NO:4 (MPLLLLLPLLWAGALA) and allows 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 with at least 90% sequence identity to 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 herein 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 herein 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 comprises A8G, G22E, R36G substitutions, and the IgG2-Fc domain comprises C222S, A330S and P331S substitutions. In one embodiment, the fusion protein described herein 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 beta cells to secrete insulin 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 β cells to secrete insulin in a glucose-dependent manner.

[0176] In one embodiment, the fusion protein described in the present application has an amino acid sequence as 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 an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID NO:8.

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

[0180]

[0181] In one embodiment, the half-life of the fusion protein described herein 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 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 (eg, GLP-1 polypeptide, Fc fragment, fusion protein) of the present invention.

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

[0187] The term "nucleic acid" or "nucleotide" used in this application refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a specific nucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. 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 by mixed 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 polynucleotides described herein are codon optimized, for example, optimized for humans. The nucleic acid molecules can be used in the methods described herein.

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

[23] In addition, automated peptide synthesizers are commercially available (eg, Advanced ChemTech Model 1396; Milligen / Biosearch 9600). Alternatively, the peptides, polypeptides, or fragments or variants thereof described herein can be recombinantly produced using various 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 herein has a polynucleotide sequence with 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 to SEQ ID NO: 26. In another embodiment, the polynucleotide described herein is a polynucleotide sequence as shown in SEQ ID NO: 26.

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

[0193] In certain embodiments, the polynucleotide described herein comprises a polynucleotide sequence as shown in any one of SEQ ID NOs: 20 to 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 to SEQ ID NOs: 20 to 22.

[0194] In certain embodiments, the polynucleotides described herein comprise a polynucleotide sequence as shown in SEQ ID NO:24 or as shown in 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 to SEQ ID NO:24 or SEQ ID NO:25.

[0195] In certain embodiments, the polynucleotides described herein comprise 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 to SEQ ID NO:23.

[0196] In certain embodiments, the polynucleotide described herein comprises a polynucleotide sequence as shown in any one of SEQ ID NOs: 28 to 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 to the sequence shown in any one of SEQ ID NOs: 28 to 38.

[0197] The sequences of SEQ ID NOs: 20 to 38 are shown in the following table.

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] IV. Vectors and Cells

[0204] In yet another aspect, the present invention provides a vector comprising the polynucleotide described in the present application.

[0205] Any vector suitable for the intended use can be used. For example, some vectors can be introduced into expression systems, such as mammalian, insect or bacterial expression systems, for expression and purification of expressed proteins. 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 together with an in vitro expression system to produce a fusion protein. The expression and purification of the fusion protein can be performed 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, an 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 may include suitable regulatory sequences and components. Suitable regulatory sequences may be selected from a variety of sources, including bacteria, fungi, viruses, mammals 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, other sequences, such as replication origins, additional DNA restriction sites, enhancers and sequences that confer transcriptional induction ability, may be incorporated into the expression vector, depending on the cells to be transfected / infected / transduced and the vector used. In one embodiment, the regulatory sequence guides or increases expression in neural tissue and / or cells. In one embodiment, the vector is a viral vector. The recombinant expression vector may also include a marker gene, which helps select a host cell transformed, infected or transfected with a vector for expressing an antibody described herein. The recombinant expression vector may also include other expression cassettes encoding, for example, other fusion moieties (e.g., for producing antibody "fusion proteins") that can help detect, including, for example, tags and markers as described herein.

[0209] In one embodiment, the carrier comprises one or more, optionally Figure 1 For example, in one embodiment, the vector comprising 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 lipid 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 practice the present invention include vectors based on adenovirus and adeno-associated viruses.

[0211] Another aspect of the present invention provides a recombinant cell, wherein the 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 of the polynucleotides described herein.

[0213] In one embodiment, the cell of the present invention further recombinantly or naturally expresses lysyl hydroxylase.

[0214] In one embodiment, the lysyl hydroxylase level or activity of the cell expression of the present invention is higher than the lysine hydroxylase level or activity of COS-7 cell expression. In other words, the cell increases the lysyl hydroxylase activity level compared with COS-7 cells. For example, the cell can be a modified cell to increase the expression of lysine hydroxylase (e.g., by preparing a cell that recombinantly expresses lysine hydroxylase), or can be a cell with a naturally increased lysine hydroxylase level compared with, for example, COS-7 cells. For example, the lysyl hydroxylase level or activity of the cell expression 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 lysine hydroxylase level or activity expressed by COS-7 cells.

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

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

[0217] V. Cell construction method, fusion protein production and detection method

[0218] In another aspect of the present invention, a method for constructing a recombinant cell is provided, 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 that recombinantly or naturally expresses lysyl hydroxylase to obtain a recombinant cell.

[0219] In one embodiment, the recombinant or naturally-occurring cell expressing lysyl hydroxylase expresses an increased level or activity of lysine hydroxylase compared to lysine hydroxylase expressed by other cells (eg, COS-7 cells).

[0220] In one embodiment, the cell is a CHO cell, for example, a CHO-K1 cell, a CHO-S cell or a CHO-DG44 cell.

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

[0222] In one embodiment, the method for constructing the recombinant cell 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) The pKN012-GLP1-IgG2 / Fc expression vector was introduced into CHO-K1 cells to obtain recombinant cells.

[0225] In another embodiment, the method for constructing the recombinant cell 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) The pKN012-GLP-1-IgG2 / Fc expression vector was introduced into CHO-K1 cells to obtain recombinant cells.

[0228] In another aspect of the present invention, a method for producing the GLP-1 fusion protein is provided, 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, GLP-1 fusion proteins can be synthesized. As shown herein, the fusion proteins can also be prepared using recombinant cells, including, for example, recombinant Chinese hamster ovary cells that recombinantly express the GLP-1 fusion protein. Therefore, the present invention also provides a method for preparing a GLP-1 fusion protein, the method comprising culturing a recombinant cell expressing the GLP-1 fusion protein, wherein the culturing comprises one or more process steps or materials as described in the examples. For example, the method may include one or more process steps as described in Table 2 and / or one or more materials as described in Table 3 or 4.

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

[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., for example, 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., meets the drug factory standard).

[0232] In another aspect of the present invention, a method for detecting the quality of a fusion protein is provided, 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.

[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 relative to native human GLP-1; 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 as shown in SEQ ID NO:3, and the IgG2-Fc domain comprises the amino acid sequence as 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, the quality of the fusion protein is confirmed as qualified when the hydroxylation level of the 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%). "Qualified" means that it complies with the drug standard system formulated by the national drug supervision and administration department or similar institutions, such as drug standards, drug registration standards, enterprise drug standards, pharmacopoeia, etc., so that the produced drugs meet the drug factory standards.

[0235] VI. Composition

[0236] Another aspect of the present invention 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, wherein the GLP-1 polypeptide of the fusion protein has a certain level of hydroxylation 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 polypeptide in the fusion protein is 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 position 31 (W31) relative to native human GLP-1. In certain embodiments, the oxidation level 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 oxidation level at methionine 253 (M253) corresponding to SEQ ID NO: 7. In certain embodiments, the oxidation level 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 a carbohydrate.

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

[0246] In one embodiment, the composition comprises a GLP-1 fusion protein, formulated into a dosage form, and the dosage is 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 GLP-1 fusion protein.

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

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

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

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

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

[0252] Suitable diluents for GLP-1 fusion proteins and / or cells include, but are not limited to, saline solutions, pH buffered solutions, and the diluents described herein, as well as glycerol solutions or other solutions suitable for freezing polypeptides and / or cells.

[0253] Suitable diluents for nucleic acids and / or vectors include, but are not limited to, saline solutions, pH buffered solutions, and the 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, A8G, G22E and / or R36G substitutions in the GLP-1 polypeptide, and / or increased hydroxylation levels at K34, decreased oxidation levels at W31 and / or one or more substitutions of M253 of 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 compositions thereof, and for corresponding pharmaceutical therapeutic uses.

[0257] In another aspect of the present invention, the fusion proteins, dimers, polynucleotides, vectors, cells or compositions disclosed herein can be used to treat or prevent the onset of a disease or disorder or to slow its progression in a subject.

[0258] Another aspect of the present invention provides a use of the fusion protein, dimer, polynucleotide, vector, cell or a combination thereof as a GLP-1 receptor agonist.

[0259] Another aspect of the present invention provides a fusion protein, dimer, polynucleotide, vector, cell or a composition thereof for use in treating, preventing or slowing down the progression of a disease or condition.

[0260] Another aspect of the present invention provides a fusion protein, dimer, polynucleotide, vector, cell or a combination thereof for preparing a drug for treating, preventing or slowing down the progression of a disease or condition.

[0261] Another aspect of the present invention provides a method for treating, preventing or slowing down the progression of a disease or condition by administering a therapeutically effective amount of the fusion protein, dimer, polynucleotide, vector, cell or a composition thereof to a subject in need thereof.

[0262] Another aspect of the present invention provides use of the fusion protein, dimer, polynucleotide, vector, cell, or composition described herein in the preparation of a medicament for treating or preventing a disease.

[0263] Another aspect of the present invention provides use of the fusion protein, dimer, polynucleotide, vector, cell, or composition described herein for treating or preventing a disease.

[0264] Another aspect of the present invention provides a method for treating or preventing a disease, the method comprising: administering a therapeutically effective amount of the fusion protein, dimer, polynucleotide, vector, cell, or composition described herein to a subject.

[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 time) 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 g, 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 may be used, suitable dosage forms may include but are not limited to solutions, suspensions, pills, tablets.

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

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

[0269] In one embodiment, the GLP-1 fusion protein, dimer, polynucleotide, vector, cell or combination thereof is administered once a week followed by discontinuation of administration, followed by administration once a week. In another preferred embodiment, the discontinuation period is 2 weeks.

[0270] In one embodiment, the GLP-1 fusion protein, dimer, polynucleotide, vector, cell or composition thereof can be administered once for two weeks and then continuously administered four times a week. In another preferred embodiment, the treatment further comprises administering an initial dose of 1 mg one week before the start of the dosing regimen.

[0271] In one embodiment, the GLP-1 fusion protein, dimer, polynucleotide, vector, cell or a composition thereof is administered parenterally, intravenously, subcutaneously or intramuscularly.

[0272] In one embodiment, the disease includes metabolic diseases related to disorders of glucose metabolism and / or lipid metabolism, complications of metabolic diseases, nervous system diseases or other related diseases.

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

[0274] In one embodiment, the metabolic disease associated with disordered carbohydrate metabolism and / or lipid metabolism 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 related to the dysregulation of glucose metabolism and / or lipid metabolism is or includes diabetes. In one embodiment, the metabolic disease related to the dysregulation of glucose metabolism and / or lipid metabolism is type 2 diabetes. In another embodiment, the metabolic disease related to the dysregulation of glucose metabolism and / or lipid metabolism is or includes obesity. In another embodiment, the metabolic disease related to the dysregulation of glucose metabolism and / or lipid metabolism is or includes non-alcoholic fatty liver disease (NAFLD). In another embodiment, the metabolic disease related to the dysregulation of glucose metabolism and / or lipid metabolism is non-alcoholic liver fibrosis (NASH).

[0276] In one embodiment, the subject is newly diagnosed or has been previously diagnosed with diabetes (e.g., type 2 diabetes). Diabetes can be diagnosed in a variety of ways, such as fasting blood glucose (FPG). According to the American Diabetes Association, diabetes is diagnosed when fasting blood 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 susceptible to diabetes because the subject is obese or the subject has a 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 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 at least about 20 kg / m 2 In another embodiment, the subject may have a blood sugar level higher than the average level of peers of comparable weight, but not enough to be diagnosed as diabetes. In another embodiment, the subject may also be an individual with a family history of diabetes.

[0279] In one embodiment, the subject is a patient who 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 (eg, coronary heart disease, sudden cardiac death, heart failure, etc.), renal complications (eg, 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 has been 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 susceptible 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 has been 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 susceptible 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 drugs or therapies for treating diseases.

[0286] In one embodiment, the fusion protein, dimer, polynucleotide, vector, cell, or composition disclosed herein can be used in combination with a drug for treating diabetes, which can be a drug currently on the market, such as insulin, metformin, sulfonylureas mainly including glimepiride, glibenclamide, gliclazide, gliquidone, etc., α-glucosidase inhibitors such as acarbose, etc., and also include other drugs for treating diabetes that are already on the market and under development. 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 protein, dimer, polynucleotide, vector, cell, or composition disclosed herein can be used in combination with Alzheimer's disease drugs and / or non-drug interventions such as cognitive therapy. In one embodiment, the fusion protein, dimer, polynucleotide, vector, cell, or composition disclosed herein is used in combination with γ-aminobutyric acid for the treatment of neurodegenerative diseases. In one embodiment, the fusion protein, dimer, polynucleotide, vector, cell, or composition disclosed herein is used in combination with gamma-aminobutyric acid to treat Alzheimer's disease.

[0287] In certain embodiments, the GLP-1 fusion protein disclosed herein can be used in combination with γ-aminobutyric acid to inhibit the effect of TNF-α in 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 TNF-α-induced apoptosis of neuronal cells SH-SY5Y.

[0289] In certain embodiments, the GLP-1 fusion protein disclosed herein in combination with γ-aminobutyric acid can have a protective effect on 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 TNF-α-induced apoptosis of neuronal cells.

[0291] In certain embodiments, the GLP-1 fusion protein disclosed herein can be used in combination with γ-aminobutyric acid to reduce Aβ in HMC3 microglia. 1-42 Oligomers induce mRNA expression of inflammatory factors (e.g., TNF-α, IL-6).

[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 implementation schemes.

[0294] Embodiment 1. Use of a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain in the preparation of a drug 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 selected from the group consisting of A8G, G22E and R36G relative to native human GLP-1;

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

[0298] Embodiment 3. The use according to embodiment 1 or 2, wherein the hydroxylation level is between 10% and 100%, for example, 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%.

[0299] Embodiment 4. The use according to any one of embodiments 1-3, wherein the GLP-1 polypeptide is substantially not oxidized at tryptophan 31 (W31) relative to native human GLP-1.

[0300] Embodiment 5. The use according to embodiment 4, wherein the oxidation level of the GLP-1 polypeptide at W31 relative to native human GLP-1 is less than 0.5% or undetectable.

[0301] Embodiment 6. The use according to any one of embodiments 1-5, 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 following group: A8G, G22E and R36G relative to native human GLP-1.

[0302] Embodiment 7. The use according to any one of embodiments 1-6, 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.

[0303] Embodiment 8. The use according to embodiment 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, G22E and R36G substitutions relative to native human GLP-1.

[0304] Embodiment 9. The use according to embodiment 8, wherein the GLP-1 polypeptide is human GLP-1(7-37) and comprises A8G, G22E and R36G substitutions relative to native human GLP-1.

[0305] Embodiment 10. The use according to any one of the preceding embodiments, wherein the amino acid sequence of the GLP-1 polypeptide is as shown in SEQ ID NO:3.

[0306] Embodiment 11. The use according to any one of Embodiments 1-10, wherein the IgG2-Fc domain is an Fc domain from human IgG2.

[0307] Embodiment 12. The use according to any of the preceding 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.

[0308] Embodiment 13. The use according to any of the preceding 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.

[0309] Embodiment 14. The use according to Embodiment 13, wherein the IgG2-Fc domain further comprises a C222S substitution.

[0310] Embodiment 15. The use according to Embodiment 14, wherein the amino acid sequence of the IgG2-Fc domain is as shown in SEQ ID NO:6.

[0311] Embodiment 16. The use according to any one of the preceding 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.

[0312] Embodiment 17. The use according to any one of the preceding embodiments, wherein the GLP-1 polypeptide is located at the N-terminus or C-terminus of the immunoglobulin Fc domain.

[0313] Embodiment 18. The use according to any one of the preceding embodiments, wherein the GLP-1 polypeptide is covalently linked directly to the immunoglobulin Fc domain.

[0314] Embodiment 19. The use according to any one of embodiments 1-17, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain via a linker.

[0315] Embodiment 20. The use according to embodiment 19, 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.

[0316] Embodiment 21. The use according to Embodiment 20, wherein the linker comprises a connecting peptide connecting the GLP-1 polypeptide and the IgG2-Fc domain.

[0317] Embodiment 22. The use according to Embodiment 21, wherein the connecting peptide comprises a linker containing glycine and serine.

[0318] Embodiment 23. The use according to embodiment 22, 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 (GGGGS), SEQ ID NO:41 (GGGGGS) or SEQ ID NO:42 (GGGGGGGS).

[0319] Embodiment 24. The use according to any one of embodiments 19-23, 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.

[0320] Embodiment 25. The use according to embodiment 24, wherein the linker comprises an amino acid sequence as shown in SEQ ID NO:9.

[0321] Embodiment 26. The use according to any one of embodiments 19-25, 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.

[0322] Embodiment 27. The use according to any one of the preceding embodiments, wherein the fusion protein has an 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.

[0323] Embodiment 28. The use according to any one of the preceding embodiments, wherein the IgG2-Fc domain has a certain oxidation level at methionine 253 (M253) corresponding to SEQ ID NO:7.

[0324] Embodiment 29. The use according to Embodiment 28, wherein the oxidation level of the IgG2-Fc domain at M253 corresponding to SEQ ID NO: 7 is less than or equal to 5%.

[0325] Embodiment 30. The use according to any one of the preceding embodiments, wherein the fusion protein further comprises a signal peptide.

[0326] Embodiment 31. The use according to Embodiment 30, wherein the signal peptide is human CD33 signal peptide.

[0327] Embodiment 32. The use according to embodiment 30 or 31, wherein the signal peptide has an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:4.

[0328] Embodiment 33. The use according to any one of embodiments 30-32, wherein the fusion protein has an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:8.

[0329] Embodiment 34. The use according to any of the preceding 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.

[0330] Embodiment 35. The use according to any of the preceding 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 polypeptides in the fusion protein are hydroxylated at K34 relative to native human GLP-1.

[0331] Embodiment 36. The use according to any one of the preceding embodiments, wherein the disease is selected from the group consisting of metabolic diseases associated with disorders of carbohydrate metabolism and / or lipid metabolism, complications of metabolic diseases, central metabolic diseases (e.g., nervous system diseases), and other related metabolic diseases.

[0332] Embodiment 37. The use according to Embodiment 36, wherein the metabolic disease associated with dysregulation of carbohydrate metabolism and / or lipid metabolism is selected from the group consisting of diabetes, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity and metabolic syndrome.

[0333] Embodiment 38. The use according to embodiment 36 or 37, wherein the metabolic disease associated with dysregulation of carbohydrate metabolism and / or lipid metabolism is diabetes (e.g., type 2 diabetes, type 2 diabetes with poor glycemic control after diet and exercise intervention).

[0334] Embodiment 39. The use according to Embodiment 36, wherein the complications of the metabolic disease include cardiovascular complications (such as heart failure), renal complications (such as diabetic nephropathy) or liver complications (such as fatty liver, including NAFLD and NASH) caused by the metabolic disease.

[0335] Embodiment 40. The use according to embodiment 36, wherein the nervous system disease is a neurodegenerative disease.

[0336] Embodiment 41. The use according to embodiment 40, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease and Parkinson's disease.

[0337] Embodiment 42. The use according to any one of the preceding embodiments, wherein the disease is Alzheimer's disease.

[0338] Embodiment 43. Use of a fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 7 in the preparation of a medicament for treating neurodegenerative diseases.

[0339] Embodiment 44. The use according to embodiment 43, 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 45. The use according to embodiment 43 or 44, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:7.

[0341] Embodiment 46. Use of a fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 7 in the preparation of a medicament for treating Alzheimer's disease.

[0342] Embodiment 47. Use of a fusion protein in the preparation of a medicament for treating Alzheimer's disease, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:7.

[0343] The above disclosure generally describes the present application. A more complete understanding can be obtained with reference 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. When circumstances may suggest or become convenient, changes in form and replacement of equivalents may be considered. Although specific terms are used herein, these terms are for descriptive purposes rather than for limiting purposes.

[0344] The following non-limiting examples are provided to illustrate the present invention.

[0345] Example

[0346] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0347] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0348] Example 1: Fusion protein expression and activity detection

[0349] 1.1 Plasmid construction

[0350] A vector encoding a GLP-1 fusion protein was constructed, which contains human GLP-1 (7-37) and human IgG2 / Fc (including the hinge, CH2 and CH3 regions of the human IgG2 heavy chain, i.e., Hinge-CH2-CH3). The human CD33 (hCD33) secretory leader peptide sequence was fused to the GLP-1 sequence to guide the secretion of the synthetic 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 inserted into the NcoI and HindIII sites of the pKN012 vector to generate pKN012-GLP-1-IgG2 / Fc.

[0351] The pKN012-GLP-1-IgG2 / Fc stable expression vector was transformed into E. coli DH5α competent cells. The plasmid was 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.

[0352] To establish CHO-K1 cells (Lot#58995535 / ATCC) stably expressing GLP-1-IgG2 / Fc, 2 μg of linearized pKN012-GLP-1-IgG2 / Fc was transfected in 6-well plates (2.5 x 10 5 CHO-K1 cells grown in 24-well plates (cells / well). 24 hours after transfection, the cells were dispersed and cultured in CD-CHO medium containing MSX (methionine sulfoximine, 100 μM / L), and those cells that had stably integrated the recombinant plasmid into the genome were selected. The culture medium was replaced every 3 days until colonies were formed. Single colonies were isolated and expanded to stable cell lines, and the GLP-1 fusion protein (YN-011) in the tissue culture supernatant of the cell line grown in 24-well plates was tested using the rat GLP-1 RIA kit. Cells that can secrete the fusion protein are selected for further identification. The amino acid sequence of the prepared GLP-1 fusion protein is shown in SEQ ID NO:7 (also referred to as "YN-011" in this application).

[0353] 1.2 Detection of YN-011 activity by inducing cAMP levels

[0354] Natural GLP-1 stimulates insulin secretion from beta cells in a glucose-dependent manner. To evaluate whether purified YN-011 has the function of natural GLP-1, the effect on insulin secretion from cloned insulin-secreting INS-1 cells was determined. INS-1 cells were starved of serum and glucose 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 does not stimulate insulin secretion from beta cells. However, in the presence of 5 mM or 20 mM glucose, YN-011 stimulates insulin secretion from beta cells in a dose-dependent manner. The data show that the GLP-1-IgG2 / Fc fusion protein YN-011 is biologically active and can stimulate insulin secretion in INS-1 cells in a glucose-dependent manner.

[0355] In the absence of glucose, cAMP in INS-1 cells treated with YN-011 (120 nM) was at basal levels, and in the presence of 5 mM glucose, cAMP levels in YN-011-treated cells increased significantly, comparable to levels seen with dulaglutide.

[0356] Example 2: Increasing GLP-1K34 hydroxylation levels can improve protein production and activity

[0357] 2.1 Determination of modification levels by LC-MS / MS-based peptide mapping

[0358] Lys-C digestion was used for mass spectrometry analysis and identification. The YN-011 sample was denatured, reduced, and alkylated in sequence, and then hydrolyzed into peptides by Lys-C enzyme. The Thermo LTQ velos Orbitrap instrument was used for online LC-MS / MS analysis, and the mascot software was used to process and analyze the data. It was observed that the molecular weight of peptide 21-28: EFIAWLVK increased by 16 Daltons (Da). The primary and secondary mass spectra of the peptides without +16Da modification and the peptides with +16Da modification of the YN-011 sample are shown in Figure 2 Figure 2 As shown. The primary molecular weight error is within 10ppm. By analyzing the charge-to-mass ratio of the secondary fragment ions, the site of the 16Da modification is K28. K28 is also called K34 because the active GLP-1 (e.g., amino acids 7-37 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.

[0359] The modification level at K34 in YN-011 was detected by UV280 and EIC (Extracted Ion Chromatography) methods. The results are shown in Table 1 and Figure 3A and 3B The calculation method of UV280 is as follows:

[0360]

[0361] in:

[0362] K: The percentage of modified peptides containing +16Da in the total amount of peptides from amino acids 21 to 28 (the sum of the content of unmodified peptides and +16Da modified segments);

[0363] A1: absorbance at 280 nm of the peptide segment of amino acids 21-28 without the +16Da modification;

[0364] A0: Absorbance of the peptide containing amino acids 21-28 with +16Da modification at 280nm

[0365] Table 1. K34 modification levels in YN-011 detected by UV280 and EIC methods

[0366]

[0367] * Relative quantification by mass spectrometry EIC

[0368] 2.2 Hydroxylation level of K34 in YN-011

[0369] After mass spectrometry analysis confirmed that +16Da occurred at the 34th lysine (Lys), considering that 16Da is the molecular weight of an oxygen atom, it was suggested that it might be the result of an oxidation reaction. In fact, there are two types of enzymes in cells that catalyze the modification of lysine side chains, one is called lysine oxidase (Lysyl oxidase, see https: / / en.wikipedia.org / wiki / Lysyl_oxidase), and the other is lysine hydroxylase (Lysyl hydroxylase, see https: / / en.wikipedia.org / wiki / Lysyl_hydroxylase). Lysine oxidase can oxidize the amino group on the 6th carbon of the lysine side chain to an aldehyde group, but the difference between this modification and the unmodified one is 1Da, which is inconsistent with the result of an increase of 16Da. Lysine hydroxylase will add a hydroxyl group to the carbon (γ position) of the lysine side chain to form a stable hydroxylysine (Hydroxylysine). Hydroxylated lysine adds a hydroxyl group to the side chain of lysine and reduces a hydrogen atom, so it just increases by 16Da compared to the original molecular weight. The hydroxylation position of K34 is as follows:

[0370]

[0371] In summary, the increase of 16Da on K34 in YN-011 is most likely caused by lysine hydroxylation. Therefore, hydroxylation inhibitors were added during cell culture to verify that the +16Da modification was lysine hydroxylation, and to harvest proteins with different +16Da modification ratios to study the effect of the +16Da modification ratio on the biological activity of the protein.

[0372] The cell culture process of the stable CHOK1 cells capable of secreting the fusion protein YN-011 in Example 1 used in this experiment is shown in Table 2. Tables 3 and 4 list the relevant materials and cell culture medium required for cell culture. The modification level of the fusion protein was detected by mass spectrometry as described above. The inventors found that the fusion protein was indeed hydroxylated at K34, with a hydroxylation level of 15% to 40%.

[0373] In order to detect the biological effect of K34 hydroxylation, 8 shake flasks (SF1-SF8) were set up in the experiment to culture CHOK1 cells stably expressing the fusion protein YN-011. SF1-SF8 were used to evaluate various conditions. Hydroxylation inhibitors were added during cell culture, and proteins with different hydroxylation modification ratios were harvested to study the effect of high and low hydroxylation ratios on protein biological activity. Minoxidil is an inhibitor of lysine hydroxylase, which can inhibit lysine hydroxylation, while Zn 2+ ions can react with Fe 2+ ion competition, thereby inhibiting the action of hydroxylase. SF1 was used as a control without any inhibitor added. The minoxidil used in this experiment was dissolved in 0.1N HCl solution. HCl was added to SF2 as an HCl control. Different concentrations of minoxidil were added to SF3 to SF5, with concentrations of 0.1mM, 0.5mM, and 1.0mM, respectively. SF6 and SF7 were added with Zn 2+ Experimental group, using Zn 2+ The reagent is ZnSO 4 , the ZnSO used 4 The concentrations were 200 μM and 400 μM respectively. SF8 also added minoxidil and Zn 2+ (by ZnSO 4 Provides two inhibitors, minoxidil and Zn 2+ The total added concentrations were 0.5 mM and 200 μM, respectively.

[0374] Table 2. Cell culture process

[0375]

[0376] Table 3. Materials used in the cell culture process

[0377] name abbreviation company Part 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 JTBaker 2078-06 Anhydrous glucose Glucose JTBaker 1919-09 Hydrochloric acid, 6.0N solution HCl JTBaker 0327-02 Sodium hydroxide, 10N solution NaOH JTBaker 5000-02 Minoxidil Minoxidil Sigma M4145 <![CDATA[ZnSO 4 ]]> <![CDATA[Zn 2+ ]]> Sigma Z0251 Anticoagulants ACA Gibco 0010057

[0378] Table 4. Cell culture medium and its components

[0379] Cell culture medium Element Dynamis (0.5% ACA) IL Dynamis (added 5ml ACA) Feed C (containing 15g / L FM012) 1L Feed C dissolves 15g FM012 FAA02 IL FAA02 (containing 7.95g L-cystine and 4.75g L-tyrosine)

[0380] 2.3 Increasing the K34 hydroxylation level of GLP-1 can improve protein production and activity

[0381] The protein yield and activity results are shown in Table 5. The hydroxylation levels of the blank control SF1 and SF2 with only HCl added were 15.7% to 16.0%. From SF3 to SF5, the hydroxylation ratio gradually decreased with the increase in the concentration of minoxidil (one of the hydroxylation inhibitors). When the minoxidil dosage reached 0.5mM, the hydroxylation ratio was 8.7%. When the minoxidil concentration increased to 1mM, the ratio was 9.4%. Cell growth and protein expression were significantly inhibited: the peak density of viable cells decreased by about 17%, and protein production decreased by 57.0% to 67.9%. In addition, when 400μM Zn was added to the cell culture 2+ (another hydroxylation inhibitor), the hydroxylation level also dropped to 10.7%. 2+ , 200uM) experiment, the hydroxylation level in SF8 was also reduced to 9.6%.

[0382] When hydroxylation inhibitors such as minoxidil are present, the hydroxylation ratio gradually decreases with the increase of the added concentration. Cell growth and protein expression are significantly inhibited: the peak density of viable cells decreases by about 17%, and the yield gradually decreases by 57.0% to 67.9%. Similarly, in the presence of the hydroxylation inhibitor Zn 2+ In the presence of Zn 2+ The yield of protein decreased with the increase of hydroxylation concentration, and the yield decreased by 22.9% to 34.2%. When the hydroxylation level reached 15.7% to 16.0%, the yield increased, and compared with the low hydroxylation level, the yield was doubled. Therefore, the higher the hydroxylation level, the higher the yield.

[0383] SUPA-1 is a GLP-1-IgG2 / Fc fusion protein disclosed in US Patent No. 8658174. No connecting peptide is used between the GLP-1 polypeptide and IgG2 / Fc. Except for the replacement of GLP-1 polypeptide A8G, there are no other site mutations on the GLP-1 polypeptide and IgG2 / Fc. No hydroxylation was detected in SUPA-1, and no other modifications were detected. The protein yield under the same conditions was 22 mg / L. When the hydroxylation level of K34 in YN-011 is 8.7% to 9.6%, the yield can reach 0.77 g / l to 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 yield rate of YN-011 can reach more than 500 times that of SUPA-1.

[0384] 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 using the method in 1.2 of Example 1. The results showed that SF1 was 88%, SF4 and SF5 were 83% and 68%, respectively. Therefore, the higher the hydroxylation ratio, the higher the biological activity tends to be.

[0385] In summary, K34 in the fusion protein is indeed hydroxylated, and this modification greatly increases the yield of YN-011, and the biological activity also tends to increase.

[0386] The cell culture scale was further expanded, and the YN-011 obtained from the YN-011 pilot 15L and three batches of 200L reactors contained 20% to 30% lysine hydroxylation at position 34, and the yield of YN-011 could reach about 2.7 g / L.

[0387] Table 5. Summary of yield and protein activity results

[0388]

[0389] Example 3: Determination of oxidation levels by LC-MS / MS

[0390] According to Example 2 or Bettinger et al.

[19] Protein oxidation determination method, determination of oxidation level, found that the GLP-1 fusion protein YN-011 of the present invention was not oxidized on W31, while the oxidation level of dulaglutide at the same position was greater than 5%. The oxidation occurred at the rectangular box in the W31 structural formula:

[0391]

[0392] Meanwhile, 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%.

[0393] Example 4: Preventive and therapeutic effects of YN-011 in diabetic db / db mice (type 2 diabetes model)

[0394] The effect of multiple subcutaneous administration of YN-011 on lowering blood glucose was evaluated in db / db mice (Jackson Laboratories, 000642). Mice were housed under normal lighting (12 hours light / 12 hours dark) and room temperature with free access to food (normal rodent chow) and water. db / db mice lack leptin receptors and spontaneously develop obesity, hyperglycemia, and pancreatic β-cell atrophy at 4 weeks of age.

[0395] 60 diseased db / db mice (33-45g) were divided into 6 groups, 10 mice (5 males and 5 females) in each group, 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 of dulaglutide by subcutaneous injection. Another group of wild-type mice (17-22g) with the same genetic background served as normal controls. The frequency of administration was once every 3 days (Q3D), for a total of 9 administrations in 26 days.

[0396] like Figure 6 As shown in the figure, during the whole experiment, the random blood glucose of db / db mice in the model control group was maintained at a high level and was significantly higher than that of mice in the normal control group (P<0.001). After the first administration, YN-011 significantly reduced the random blood glucose (RPG) concentration of db / db mice. The therapeutic effect was observed from 3 hours after administration, at a dose as low as 0.15 mg / kg. Within the dose range of 0.15-1.2 mg / kg, the duration of the hypoglycemic effect was 34–72 hours. YN-011 can also significantly promote insulin secretion and increase serum insulin levels starting from 3 hours after administration.

[0397] After the 9th administration of YN-011, the RPG level of YN-011-treated mice was significantly lower than that of the control group in all dose groups and measurement time points, except for the 0.15 mg / kg group after the 4th administration (10th day), which showed no significant difference (P=0.07). In order to more intuitively understand the blood glucose reduction of each group of mice, the average value of the random blood glucose reduction rate at each measurement time point in the entire experiment was calculated. The results showed that the random blood glucose reduction rates of mice in the 0.15, 0.3, and 0.6 YN-011 groups were 31.8%, 46.2%, and 50.8% respectively 72 hours after the 7th administration, 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 RPG-reducing effect of YN-011 can be maintained for 72 hours.

[0398] Long-term administration of YN-011 can also significantly reduce the fasting blood glucose level of db / db mice. During the entire experiment, the random blood glucose of db / db mice was maintained at a high level and was significantly higher than that of normal control mice (P<0.001). After db / db mice were subcutaneously injected with different doses of YN-011 once every 3 days, the fasting blood glucose of each group of mice at all measured time points was significantly reduced compared with the model control group (P<0.001). The fasting blood glucose reduction rates of mice in the 0.15, 0.3, 0.6 and 1.2 mg / kg YN-011 groups were 46.8%, 55.6%, 59.5% and 64.7% respectively 54 hours after the 7th administration (21 days). This suggests that the efficacy of 0.15, 0.3, 0.6 and 1.2 mg / kg YN-011 in reducing fasting blood glucose after multiple administrations once every 3 days can be maintained for at least 54 hours after administration (3rd day after administration). The fasting blood glucose of mice in the positive control 0.3 mg / kg dulaglutide group also decreased significantly at all measured time points.

[0399] In order to more intuitively understand the blood sugar reduction of each group of mice, the average fasting blood sugar reduction rate at each measurement time point in the entire experiment was calculated. The average fasting blood sugar reduction rate of mice in the 0.15, 0.3, 0.6 and 1.2 mg / kg YN-011 groups was 55.0%, 61.8%, 63.7% and 66.1%, respectively. The average fasting blood sugar reduction rate of mice in the positive control 0.3 mg / kg dulaglutide group was 63.6%.

[0400] Therefore, YN-011 can significantly reduce the fasting blood glucose of type 2 diabetic db / db mice after multiple administrations every 3 days, with a significant effect at a dose of 0.15 mg / kg. The fasting blood glucose-lowering effect of 0.15, 0.3, 0.6 and 1.2 mg / kg YN-011 can be maintained for at least 54 hours after administration (3rd day after administration).

[0401] The serum fructosamine level of db / db mice treated with 9 subcutaneous injections 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 was significantly reduced.

[0402] The random and fasting body weights of db / db mice in the solvent control group continued to increase during the experiment, while the random and fasting body weights of animals in the 0.3, 0.6 and 1.2 mg / kg YN-011 treatment groups decreased significantly (P<0.05, P<0.01, P<0.001). The SUPA-1 fusion protein disclosed in U.S. Pat. No. 8,658,174 had no significant effect on the body weight of db / db mice, indicating that the GLP-1 fusion protein of the present application has a better effect.

[0403] 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 0.15, 0.3 and 0.6 mg / kg YN-011 groups was significantly reduced; the subcutaneous fat content, inguinal fat content and their ratio to body weight in the 0.3, 0.6 and 1.2 mg / kg YN-011 groups were significantly reduced; the perirenal fat content in the 1.2 mg / kg YN-011 group was significantly reduced.

[0404] YN-011 increased fasting serum insulin levels in db / db mice in a dose-dependent manner 30 hours after the last dose (9th dose, day 26), accompanied by a significant increase in pancreatic β-cell mass.

[0405] Multiple subcutaneous injections of YN-011 significantly reduced serum triglyceride levels compared with the vehicle control group, and 0.6 mg / kg of YN-011 significantly reduced serum free fatty acid levels in db / db mice.

[0406] In conclusion, multiple subcutaneous injections of YN-011 showed significant therapeutic effects in db / db mice, not only significantly improving glucose metabolism, but also having a significant therapeutic effect on abnormal lipid metabolism.

[0407] Example 5: Phase IIa clinical trial of YN-011

[0408] 5.1 Experimental design

[0409] The Phase IIa clinical trial is a double-blind, placebo-controlled study in subjects with T2DM evaluating the efficacy and safety of subcutaneous administration of YN-011 at doses of 1 mg, 2 mg, 3 mg, and 4 mg. The study is designed as follows: Figure 5 shown.

[0410] The main inclusion criteria for the Phase IIa study are:

[0411] Patients with T2DM who have not taken metformin for at least 1 week and have not taken any other oral glucose-lowering medications for at least 2 weeks (WHO 1999).

[0412] Glycated hemoglobin HbA1c at screening: 7.0% ≤ HbA1c ≤ 10.0%.

[0413] Age at screening: 18-65 years

[0414] BMI ≥ 20 kg / m 2 And ≤40kg / m 2

[0415] Major exclusion criteria for Phase IIa studies:

[0416] Type 1 diabetes

[0417] Fasting C-peptide <0.81 ng / mL

[0418] 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 >5.6mmol / L; glomerular filtration rate (eGFR) calculated by the CKD-EPI (EPI-(Scr)) equation <45mL / min / 1.73m 2 ,

[0419] A known susceptible family (first-degree relative) or personal history of multiple endocrine neoplasia type 2 or medullary thyroid cancer

[0420] Uncontrolled high blood pressure

[0421] Known history of pancreatitis, pancreatic cancer, or serum amylase >1.2x ULN, or high risk factors for pancreatitis at screening

[0422] Patients with uncontrolled thyroid dysfunction

[0423] Suspected active infection

[0424] Positive for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV-Ab), human immunodeficiency virus antibody (HIV-Ab), or Treponema pallidum antibody (TPAb)

[0425] · Received GLP-1 receptor agonists, DPP-4 inhibitors, or insulin within 3 months before randomization

[0426] · History of CTCAE grade 3-4 hypersensitivity to any protein-based drugs.

[0427] Donated blood or lost more than 450 mL of blood within 3 months before screening

[0428] Any significant endocrine, immune, coagulation, genitourinary or hematologic disorder

[0429] Clinically significant abnormal gastric emptying (such as gastric outlet obstruction), severe chronic gastrointestinal disease (such as active ulcer within 6 months), long-term use of drugs that directly affect gastrointestinal motility, or gastrointestinal surgery

[0430] Any other circumstances that the investigator or attending physician considers may not be suitable for participation in the study.

[0431] Subjects were randomized to receive YN-011 or placebo in a 4:1 ratio. YN-011 was administered at doses of 1, 2, and 3 mg, with a single dose followed by a two-week break and then once a week for four consecutive doses. Subjects in the 4 mg group had the same dosing regimen, except that an initial dose of 1 mg was given one week before dosing. All subjects received a total of five randomized doses.

[0432] The primary endpoint was the safety and tolerability of YN-011 in T2DM subjects, and secondary endpoints included weekly changes in fasting blood glucose from baseline, changes in HbA1c from baseline at weeks 4 and 7, changes in glycosylated albumin from baseline at weeks 4 and 7, changes in glucose tolerance, and changes in pancreatic β-cell function assessed by an oral glucose tolerance test. Blood samples were collected from all subjects for pharmacokinetic testing. Safety assessments included adverse events, laboratory tests, vital signs, 12-lead electrocardiograms, physical examinations, and anti-drug antibody (ADA) assessments. 5.2YN-011 Single or multiple generation Xie Dynamics

[0433] Within the dose range of 1.0mg-4.0mg, YN-011 showed an increasing trend. After a single dose, 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 once a week after 2 weeks. After the fourth consecutive administration, the plasma concentration of YN-011 remained at a steady state ( Figure 4 ). After amino acid replacement, the fusion protein of the present application has a significantly longer half-life than SUPA-1. In addition, the elimination half-life of dulaglutide and semaglutide, which are already on the market, is 4.7-5.5 days, respectively.

[22] and 5.7-6.7 days

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

[0434] 5.3 The therapeutic effect of YN-011 on type 2 diabetes

[0435] YN-011 was administered subcutaneously at the formal dosing levels of 1 mg, 2 mg, 3 mg, and 4 mg (dosing regimen design see Figure 5 ).

[0436] 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, the average BMI was 25.80±2.875kg / m2, the average weight was 71.91±12.360kg, and 40% were female. Of the 40 subjects, 38 (95%) had concurrent diseases, 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 tumors. During the entire trial, none of the 40 subjects received any other drug treatments. The basic information is shown in the following table (Table 6).

[0437] Table 6. Basic characteristics of T2DM subjects

[0438]

[0439] Effects of different doses of YN-011 on fasting blood glucose Figure 6 Multiple subcutaneous administration of 3 mg or 4 mg of YN-011 resulted in sustained, clinically meaningful, and statistically significant improvements in fasting glucose compared with placebo.

[0440] Effects of different doses of YN-011 on HbA1c Figure 7 As shown. Compared with placebo, multiple subcutaneous administration of YN-011 at different doses of 1 mg, 3 mg or 4 mg sustained improvement in HbA1c levels, with significant clinical significance and statistical differences. No obvious side effects or safety risks were found.

[0441] Example 6: Preventive and therapeutic effects of YN-011 on obesity

[0442] 6.1 Effects of YN-011 in high-fat diet-induced obese mice

[0443] 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. 5-month-old male C57BL / 6 mice (available from commercial sources, such as Shanghai Slake Laboratory Animal Co., Ltd., conventional feeding conditions) were given HFD (60% of total calories from fat and 20% from carbohydrates) for 6 months to establish an obese mouse model (diet-induced obesity, referred to as DIO). Then the DIO mice (weight>50g) were divided into 2 groups (5 animals in each group), and the experimental group was injected with 0.3mg / kg of YN-011 twice a week (BIW), and the control group was injected with phosphate buffered saline (PBS), and the treatment lasted for 4 weeks.

[0444] The results showed that YN-011 injected twice a week for 4 weeks had a significant weight loss effect on DIO mice. Compared with the PBS control group, visceral fat, especially epididymal fat content, was found to be significantly reduced in YN-011-treated mice. YN-011 had no effect on the weight of other body-related tissues, including brown adipose tissue (BAT), white adipose tissue (WAT, groin), pancreas or calf muscle.

[0445] Compared with the PBS control group, 4 weeks of BIW injection of YN-011 significantly reduced ectopic lipid accumulation and liver 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 BIW 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 free fatty acids (NEFA) reduced by 57% (P < 0.001).

[0446] DIO mice treated with multiple doses of YN-011 showed a significant reduction in food consumption compared with the control group. Metabolic rate (VO2 and VCO2) and energy expenditure (EE) in the YN-011-treated group showed an increasing trend, but it was not statistically significant. When normalized by body weight, YN-011-treated mice had a significant increase in VO2, VCO2, and EE 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 exhibited higher core body temperature at room temperature and maintained higher rectal temperature when exposed to cold environment, indicating more active thermogenesis compared with the control group. These results suggest that YN-011 enhances the adaptation ability of obese mice to cold environment exposure by generating more calories.

[0447] Through glucose excursion, 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.

[0448] In conclusion, YN-011 treatment effectively reduced body weight in obese mice and improved obesity-related metabolic disorders, including hyperglycemia, hyperlipidemia, and hepatic steatosis. The beneficial effects of YN-011 on metabolism were associated with the suppression of food consumption and WAT browning and remodeling.

[0449] 6.2 The therapeutic effect of YN-011 on obese rhesus monkeys

[0450] Obese rhesus monkeys were obtained from Sichuan Primi Biotechnology Group Co., Ltd. This experiment selected 15 male obese rhesus monkeys in the IGT / IFG and hyperglycemia stage who had not received drug treatment within 1 year of the initial onset. They were aged 8-21 years (equivalent to 30-60 years old in humans), weighed 9.25-15.70 kg, had FPG between 5.50-8.58 mmol / L, and HbA1c between 4.5-5.0%. Their liver and kidney functions were normal. They were randomly divided into groups according to FPG. All selected animals underwent IVGTT tests to detect blood glucose and insulin before administration. There were 5 cases in each group, including placebo group, YN-011 50 μg / kg group and YN-011 25 μg / kg group. The drugs were administered by subcutaneous injection (SC) once a week for 4 consecutive weeks, i.e., on day 0 (D0), day 7 (D7), day 14 (D14), and day 21 (D21).

[0451] The effects of YN-011 on body weight (BW) of obese rhesus monkeys are shown in Table 7. Figure 8 As shown. During the entire trial period, the body weight of the 5 animals in the placebo group did not change significantly, proving that the model was relatively stable. Compared with the baseline value, the body weight of the YN-011 25μg / kg group continued to decrease from D7 to D28 (2.60%-6.71%, P<0.05 or P<0.01) with a dosing frequency of once a week (4 times), and decreased by 6.17% on D28; compared with the placebo group, BW was significantly reduced on D14 and D21 (P<0.05). Compared with the baseline value, the body weight of the YN-011 50μg / kg group continued to decrease from D7 to D28 (4.06%-7.32%, P<0.05) with a dosing frequency of once a week (4 times), and was significantly reduced by 7.32% on D28 (P<0.05). Compared with the placebo group, BW was significantly or extremely significantly reduced on D7 to D28 (P<0.05 or P<0.01).

[0452] Table 7. Effects of repeated subcutaneous injections of YN-011 on BW in obese rhesus monkeys

[0453]

[0454] Note: “a” was measured 2 days before administration; # was compared with baseline, P<0.05; ## was compared with baseline, P<0.01. SC, subcutaneous injection.

[0455] Example 7: Preventive and therapeutic effects of YN-011 in the rhesus monkey non-alcoholic fatty liver disease (NASH) model 7.1 Practice Test Method

[0456] 7.1.1GE Ultrasound-Guided Liver Biopsy

[0457] Number of animals: 15

[0458] Specimen collection volume: ≤2 needles, each needle specimen length ≤1.5cm, the total length of the specimen collected each time ≤3cm

[0459] The removed tissue of about 2 cm was immediately placed in 4% paraformaldehyde buffer for fixation at room temperature for more than 24 hours, and then corrected, dehydrated, embedded, and sliced ​​for HE staining and Masson staining.

[0460] Instruments and equipment: GE Vivid S5 ultrasound machine was used for ultrasound, LEICA RM2135 slicer was used for slicing, OLYMPUSBX43 microscope was used for reading films, and OLYMPUSDP22-CU camera was used for microphotography.

[0461] Tissue sections were stained with H&E and Masson staining, and pathological sections were evaluated by pathologists according to the diagnosis and treatment guidelines for nonalcoholic fatty liver disease jointly drafted by the American Association of Liver Diseases (AASLD), the American College of Gastroenterology (ACG), and the American Gastroenterology Association (AGA). The diagnostic criteria are shown in Tables 2 and 3.

[0462] 7.1.2 Tissue processing, embedding and sectioning

[0463] After the liver biopsy specimens were well fixed, they were directly dehydrated, wax-impregnated, embedded, and sliced ​​(5 μm).

[0464] 7.1.3 HE staining

[0465] The sections were routinely dewaxed, and 100% anhydrous ethanol I, 100% anhydrous ethanol II, and 95%, 85%, and 75% ethanol were added for 3 minutes each, and then rinsed with tap water for 3 minutes, stained with hematoxylin solution for 12 minutes, rinsed with tap water for 5 minutes, and stained with eosin Y solution (water-soluble) for 3 minutes. The sections were rapidly dehydrated with 95% ethanol, dehydrated with anhydrous ethanol twice, each for 2-5 minutes, and transparentized with xylene twice, each for 10 minutes. The sections were sealed with neutral gum and observed under a microscope.

[0466] 7.1.4 Masson staining

[0467] Dewax the sections routinely, and stain with water-based red-fuchsin staining solution for 5-7 minutes. At the same time, prepare a weak acid working solution in a ratio of distilled water: weak acid solution = 2:1, and wash with the weak acid working solution for 1 minute. After washing with phosphomolybdic acid solution for 1-2 minutes, directly put it into aniline blue staining solution for 1-2 minutes, and wash with the prepared weak acid working solution for 1 minute. Dehydrate quickly with 95% ethanol, dehydrate with anhydrous ethanol twice, each time for 2-5 minutes, and transparentize with xylene twice, each time for 10 minutes. Seal with neutral gum and observe under a microscope.

[0468] 7.1.5 Histopathological examination

[0469] Slice microscope (with digital imaging system) observation, pathological diagnosis, photography and analysis. According to the results of pathological histology, HE and Masson results were scored according to the scoring criteria for non-alcoholic fatty liver disease (NAFLD) activity score (NAS) (Table 8) and liver fibrosis stage (Table 9).

[0470] Table 8. NAS scoring criteria

[0471]

[0472] Note: NAS score = fatty degeneration score + interlobular inflammation score + ballooning degeneration score

[0473] Table 9. Scoring criteria for liver fibrosis grading

[0474] grade describe 0 No fibrosis observed 1a Zone 3 mild perisinusoidal fibrosis 1b Zone 3 moderate sinus fibrosis 1c Periportal fibrosis 2 Perisinusoidal and periportal fibrosis 3 Bridging fibrosis 4 Cirrhosis

[0475] 7.1.6 Quantitative analysis of liver fat content using GE 3.0T MRI

[0476] This study used a GE3.0T MRI scanner (750W3T MRI, GE Healthcare) for liver scans. The IDEAL-IQ sequence scan was used. The IDEAL-IQ technology is based on IDEAL and combines fast three-dimensional multi-echo imaging. It includes multi-echo water-fat separation technology, regional growth technology, and multiple imaging of tissue fat quantitative score and R2* relaxation rate. Six echo signals with different TE times were collected through a breath-hold scan. After computer reconstruction, the same-phase, anti-phase, water phase, lipid phase and fat fraction maps, R2* Figure 6 The IDEAL-IQ hybrid water-fat separation algorithm is performed in two steps. The first step is to use the complex domain reconstruction method to obtain water images, fat images and T2* images. The second step is to generate another set of estimated water and fat images. Then the two sets of images are integrated by the hybrid algorithm to generate the final water and fat images.

[0477] Before performing this test, the Phantom test must be completed: After the Clare P.

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

[0478] Phantom validation experiments were performed before each scan. All animals underwent baseline scans and one scan after the end of drug administration, for a total of 2 times, in a 3.0T MRI room. The scanning parameters are shown in Table 10. The anesthesia method was as follows: intramuscular injection of 10 mg / kg ketamine hydrochloride, endotracheal intubation after anesthesia, and anesthesia was maintained by isoflurane and oxygen inhalation connected to a respiratory anesthesia machine. During the scan, the veterinarian monitored the electrocardiogram, blood oxygen, respiratory rate, etc., and veterinary supervision was performed until the animal was fully awake and resumed spontaneous breathing.

[0479] Table 10. Liver MRI scan parameters

[0480] feature No. sequence name Required level Positioning image 1 3-PI Loc SSFSE NA Water and fat separation 2 BH LAVA-Flex Liver cross section Fat Content 3 BH IDEAL IQ Liver cross section

[0481] Image post-processing and analysis: After the rhesus monkey MRI images were collected and saved, they were analyzed using a 750W 3T MRI supporting workstation. The liver MRI images were selected mainly from the layer with the largest exposure of the right lobe of the liver (1-3 layers), avoiding large blood vessels, bile ducts and gallbladder to avoid volume effects, and ROI selection was performed. The area of ​​each ROI was 90-110mm 2 Intrahepatic fat content MRI-PDFF%>6% is considered moderate fatty liver (consistent with clinical standards).

[0482] 7.2 Experimental Results

[0483] The in vivo efficacy study of YN-011 was conducted in NASH rhesus monkeys. Clinical parameters evaluated included liver lipid content assessed by magnetic resonance imaging proton density fat fraction (MRI-PDFF%), NAFLD activity score (NAS), and liver fibrosis stage assessed by liver histology, body weight and body mass index (BMI), lipid profile, fructose metabolism, other biochemical parameters, and food consumption.

[0484] The rhesus monkeys selected for this study were 11-23 years old, equivalent to 30-70 years old in humans. All rhesus monkeys were male, weighed 13.63-22.85 kg, had abnormal lipid metabolism for more than 2 years, MRI-PDFF% was 7.8%-11.9%, histologically showed NAS ≥ 3, and fibrosis score 0-1c within 6 months. These animals met the clinical definition of NASH.

[0485] Fifteen rhesus monkeys were divided into three groups of 5 monkeys each and received 0 (solvent control, placebo control group), 0.050 or 0.150 mg / kg YN-011 subcutaneous injection once a week (QW) for 13 weeks. The 0.150 mg / kg YN-011 group was given adaptive dosing (0.1 mg / kg for the first dose and 0.150 mg / kg for the remaining 12 doses).

[0486] During the study, all rhesus monkeys received their assigned treatment, and no rhesus monkey missed the end-of-treatment biopsy or withdrew from treatment. No significant adverse reactions were found in any treatment group during the study.

[0487] As shown in Table 11, once-weekly YN-011 treatment for 13 weeks reduced liver lipid content by approximately 40% compared to the placebo control group.

[0488] To investigate whether YN-011 treatment leads to improvement in liver histology, liver biopsy specimens were collected from all rhesus monkeys before the first injection and on day 89 after the first injection. Liver sections were histologically analyzed using HE and Masson staining, and the degree of steatosis, inflammation, hepatocyte ballooning, and fibrosis were scored according to the criteria for NAFLD activity and NASH progression. Liver biopsy results showed that in rhesus monkeys treated with YN-011, NAS and liver fibrosis scores were reduced, while NASH did not progress significantly (Table 12).

[0489] Hepatic fat detected by MRI-PDFF decreased from 9.0%±0.9% to 5.0%±0.2% in the 50μg / kg YN-011 group and from 9.4%±1.5% to 5.6%±1.5% in the 150μg / kg YN-011 group, with a reduction of 43.8% and 39.7% at the end of administration compared with baseline, respectively. There was no significant difference in MRI-PDFF changes between the 50μg / kg and 150μg / kg YN-011 groups.

[0490] Histological analysis showed that in the 50 μg / kg YN-011 group, the average NAS decreased from 3.6±0.5 to 1.6±0.5 compared with the baseline at the end of dosing (P=0.003). In the 150 μg / kg YN-011 group, the average NAS decreased from 3.6±0.5 to 1.4±0.5 compared with the baseline at the end of dosing (P<0.001). No significant difference in NAS was found between the 50 μg / kg and 150 μg / kg YN-011 groups.

[0491] Regarding metabolic measures, including liver injury biomarkers, lipid profiles, and body weight, YN-011 treatment demonstrated superior improvements in both the 50 μg / kg and 150 μg / kg groups.

[0492] Table 11. Effect of YN-011 on the change in liver fat content percentage in NASH rhesus monkeys after repeated SC administration

[0493]

[0494] Note: ROI: region of interest; ** compared with baseline, P < 0.01; ## compared with placebo group, P < 0.01; change rate = (current value - baseline value) / baseline value × 100%.

[0495] Table 12. Effects of repeated SC administration of YN-011 on liver NAS and fibrosis scores in NASH rhesus monkeys

[0496]

[0497]

[0498] 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.

[0499] Example 8: In vitro experiments on neurodegenerative diseases

[0500] 8.1 TNF-α reduces the viability of SH-SY5Y neuronal cells in a concentration-dependent manner

[0501] 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:

[0502] Cell viability (%) = [A (加药) -A (空白) ] / [A (未加药) -A (空白) ]×100

[0503] A (加药) : OD values ​​of wells with cells, CCK-8 solution and drug solution

[0504] A (未加药) : OD value of the wells with cells, CCK-8 solution and no drug solution

[0505] A (空白) : OD value of the well without cells

[0506] The experimental results show (such as Fig. 9 As shown in Figure 3, the viability of neuronal SH-SY5Y cells decreased significantly with the increase of TNF-α concentration.

[0507] 8.2 YN-011 and γ-aminobutyric acid (GABA) inhibit the effect of TNF-α on reducing the viability of SH-SY5Y cells

[0508] Please refer to Section 8.1 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.1. That is, the drug treatment steps of this experiment are as follows: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone, or add 10, 100 or 500nM YN-011, or add 1, 10 or 100μM GABA at the same time for 48 hours in each well, and make 6 replicate wells for each drug treatment.

[0509] The experimental results show (such as Fig.10 As shown in Figure 3, the viability of SH-SY5Y cells was significantly decreased in the presence of 60 ng / ml TNF-α, while after treatment with 10, 100 and 500 nM YN-011, or after treatment with 100 μM GABA, the cell viability was significantly enhanced.

[0510] 8.3 Combined use of YN-011 and GABA significantly increased SH-SY5Y cell viability

[0511] Please refer to Section 8.1 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.1. That is, the drug treatment steps of this experiment are: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone, or add 100nM YN-011 and / or 100μM GABA at the same time for 48 hours in each well, and make 6 replicate wells for each drug treatment.

[0512] The experimental results show (such as Fig.11 As shown in Figure 2, the viability of neuronal SH-SY5Y cells was significantly decreased in the presence of 60ng / ml TNF-α, while 100nM YN-011 and 100μM GABA significantly enhanced cell viability. The combination of YN-011 and GABA showed a stronger effect in enhancing cell viability than either alone.

[0513] 8.4 YN-011 reduces TNF-α-induced apoptosis in SH-SY5Y neuronal cells

[0514] The steps of this experiment are as follows:

[0515] 1) The neuronal cells SH-SY5Y in a 10 cm culture dish were digested with 0.25% trypsin and seeded in a 12-well plate.

[0516] 2) Place the culture plate in an incubator for pre-culture overnight (37°C, 5% CO 2 ) to allow cells to adhere.

[0517] 3) Using culture medium containing 5% FBS, treat cells with 60 ng / ml TNF-α alone or with 10, 100 or 500 nM YN-011 in a 37°C incubator for 48 hours. Perform 3 replicate wells for each drug treatment.

[0518] 4) After 48 hours, discard the supernatant, add PBS to wash gently, place the cell culture plate on ice, add 120ul RIPA cell lysis buffer (Biyuntian P0013B) to each well, add protease inhibitors and phosphorylated protease inhibitors to the lysis buffer, and lyse for 15 minutes.

[0519] 5) Place the cell lysate in a 1.5 ml EP tube and centrifuge at 14,000 rpm for 30 minutes.

[0520] 6) Cells can be seen precipitating 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℃ for 10 minutes to denature the protein.

[0521] 7) Use SDS-PAGE gel electrophoresis, with 10 μg of protein sample per well. Place the gel plate vertically against the power rack in the electrophoresis tank, with the concave edge of the gel plate against the power rack. Two gel plates share one power rack. Fix the gel plate and the power rack in the power tank as required. Add electrophoresis buffer as required, so that the electrophoresis buffer added to the power rack between the two gel plates and the electrophoresis buffer added to the electrophoresis tank are not connected to each other. Gently pull out the comb in the gel plate.

[0522] 8) Electrophoresis: Use two wires to connect the electrophoresis tank and the electrophoresis instrument, and make sure that the plugs and sockets of the red and black electrodes match. Use low-voltage constant-voltage electrophoresis for the upper gel, turn on the power and adjust the voltage to 80V (usually about 15 minutes), and use high-voltage constant-voltage electrophoresis when bromophenol blue enters the lower gel, adjust the voltage to 120V until the bromophenol blue reaches the bottom of the gel, then stop the electrophoresis.

[0523] 9) Use wet transfer for membrane transfer. The wet transfer sandwich is arranged as follows: sponge / filter paper / gel / membrane / filter paper / sponge, which are arranged tightly. No bubbles can be left between the gel / membrane. The direction of the sandwich is confirmed to be correct. The negative electrode is the negatively charged protein in the gel, which migrates to the positive electrode (membrane). After the SDS-PAGE electrophoresis is completed, use a thin plate to gently pry open the two glass plates of the gel plate so that the gel is tilted on one of the glass plates. Use a blade to cut the separation gel along the junction of the separation gel and the concentrated gel on the gel, and cut off a small corner in the upper left corner of the separation gel to mark the sample order. Then carefully transfer the gel into the transfer buffer. Cut a 0.22μm PVDF membrane of the same size as the separation gel and soak it in methanol for 5 seconds. Cut 6 pieces of filter paper of the same size and equilibrate them with the transfer buffer for 15 minutes at the same time as the PVDF membrane and gel. Place the sponge gasket, filter paper, glue, membrane, filter paper, and sponge gasket (from bottom to top) on the transfer device from the negative electrode (black bottom) to the positive electrode. Be sure to remove bubbles when placing them, especially between the membrane and the filter paper, the glue and the membrane, and the filter paper and the glue. Set the transfer current to a constant current of 200mA, and the time takes about 90 minutes.

[0524] 10) Take out the PVDF membrane after transfer, wash it in TBST, pour off 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).

[0525] 11) Primary antibody incubation, remove the blocking solution, wash the membrane three times with TBST, 5 minutes each time, cut the membrane according to the molecular weight, add the primary antibody of the corresponding dilution multiple (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 in a shaker at 4°C overnight.

[0526] 12) Wash the membrane, take out the PVDF membrane that has been incubated overnight, and recover the antibody in a -20°C refrigerator. Place the taken out PVDF membrane in TBST solution and quickly rinse it three times, each time for 15 minutes.

[0527] 13) Secondary antibody incubation: Add secondary antibodies of the corresponding species (Jackson Lab, 1:10000) prepared in blocking solution and slowly block at room temperature for 1 hour.

[0528] 14) Wash the membrane, remove the secondary antibody, place the PVDF membrane in TBST solution, and quickly rinse three times, each time for 15 minutes.

[0529] 15) ECL chemiluminescence: prepare fresh developing solution (solution A: solution B = 1:1) according to the instructions of the ECL luminescence kit (Millipore #WBKLS0500) under light-proof conditions, add it evenly on the membrane, and place it in an imager to develop and collect images.

[0530] 16) Protein expression analysis: Image J software was used to statistically analyze the gray value of western blot bands, and the gray value of the internal reference protein HSP90 was used for correction to calculate the relative expression of the target protein.

[0531] The experimental results show (such as Fig.12 YN-011 significantly reduced TNF-α-induced apoptosis in SH-SY5Y neuronal cells.

[0532] 8.5 GABA reduces TNF-α-induced apoptosis in SH-SY5Y neuronal cells

[0533] Please refer to Section 8.4 for the experimental steps, but the drug treatment step 3) of this experiment is different from that of Section 8.4. That is, the drug treatment step 3) of this experiment is: using a culture medium containing 5% FBS, treat the cells with 60ng / ml TNF-α alone or with 1, 10, or 100μM GABA in each well in a 37°C incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0534] The experimental results show (such as Fig.13 As shown), GABA reduced the apoptosis of SH-SY5Y neuronal cells induced by TNF-α.

[0535] 8.6 Combined use of YN-011 and GABA reduces TNF-α-induced apoptosis in SH-SY5Y neuronal cells

[0536] Please refer to Section 8.4 for the experimental steps, but the drug treatment step 3) of this experiment is different from that of Section 8.4. That is, the drug treatment step 3) of this experiment is: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 100μM GABA and / or 100nM YN-011 in each well in a 37°C incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0537] The experimental results show (such as Fig.14 As shown in Figure 3, the combined use of YN-011 and GABA significantly reduced the apoptosis of SH-SY5Y neuronal cells induced by TNF-α.

[0538] 8.7 TNF-α promotes the damage of SH-SY5Y neuronal cells

[0539] The neuronal SH-SY5Y cells in the 10 cm culture dish were digested with 0.25% trypsin to prepare a single cell suspension. The single cell suspension was inoculated in a 24-well plate. The culture plate was placed in an incubator for pre-culture overnight (37°C, 5% CO 2 ) to allow the cells to adhere. Using a culture medium containing 5% FBS, treat the cells with 20, 40, 60 or 80ng / ml TNF-α in a 37°C incubator for 48 hours, and perform 3 replicates for each drug treatment. Add the fluorescent dye Hoechst 33342 (Biyuntian) to the well plate at a dilution concentration of 1:1000, incubate in a 37°C incubator for 10 minutes, and then remove the cell culture plate. Wash off the culture medium, gently wash twice with PBS, and immediately take a picture under a fluorescence microscope (20 times). Take 10 fields of view for each cell well, and count the number of blue positive cells in each field of view.

[0540] The experimental results show (such as Fig.15 As shown in Figure 3, TNF-α promoted the damage of neuronal cells SH-SY5Y.

[0541] 8.8 Protective effect of YN-011 on neuronal cells damaged by TNF-α

[0542] Please refer to Section 8.7 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.7. That is, the drug treatment steps of this experiment are as follows: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 10, 100 or 500nM YN-011 in each well in a 37°C incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0543] The experimental results show (such as Fig.16 YN-011 has a protective effect on TNF-α-damaged neuronal cells.

[0544] 8.9 Protective effect of GABA on neurons damaged by TNF-α

[0545] Please refer to Section 8.7 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.7. That is, the drug treatment steps of this experiment are as follows: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 1, 10 or 100μM GABA in a 37℃ incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0546] The experimental results show (such as Fig.17 As shown in Figure 3, GABA has a protective effect on TNF-α-damaged neuronal cells.

[0547] 8.10 Protective effect of combined use of YN-011 and GABA on TNF-α-damaged neurons

[0548] Please refer to Section 8.7 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.7. That is, the drug treatment steps of this experiment are: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 100μM GABA and / or 100nM YN-011 in a 37℃ incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0549] The experimental results show (such as Fig.18 The combined use of YN-011 and GABA has a protective effect on TNF-α-damaged neuronal cells.

[0550] 8.11 YN-011 reduces TNF-α-induced apoptosis of neuronal cells

[0551] The steps of this experiment are as follows:

[0552] 1) Use 0.25% trypsin to digest the neuronal cells SH-SY5Y in a 10 cm culture dish to prepare a single cell suspension. The single cell suspension is inoculated in a 24-well plate, and a sterile slide is placed in the 24-well plate.

[0553] 2) Place the culture plate in an incubator for pre-culture overnight (37°C, 5% CO 2 ) to allow cells to adhere.

[0554] 3) Using culture medium containing 5% FBS, treat cells with 60 ng / ml TNF-α alone or with 10, 100 or 500 nM YN-011 in a 37°C incubator for 48 hours. Perform 3 replicate wells for each drug treatment.

[0555] 4) After 48 hours, the culture medium was removed and 400 μl of 4% paraformaldehyde (PFA) was added to each well for fixation at room temperature for 15 minutes.

[0556] 5) Wash twice with PBS, 5 minutes each time.

[0557] 6) Add 0.1% Triton X-100 to permeabilize the membrane and incubate at room temperature for 10 minutes.

[0558] 7) Wash twice with PBS, 5 minutes each time.

[0559] 8) Add 10% goat serum (GS) and block at room temperature for 30 minutes.

[0560] 9) Aspirate 10% GS and add primary antibody Cleaved-caspase3 (CST 9661S) using 1% GS antibody diluent at an antibody dilution ratio of 1:400, 30 μl per slide.

[0561] 10) Place in a 4°C refrigerator overnight.

[0562] 11) The next day, remove the primary antibody and wash with PBS three times, 5 minutes each time.

[0563] 12) Add fluorescent secondary antibody (Alexa 488Conjugate) and incubate at room temperature in the dark for 1 hour.

[0564] 13) Wash with PBS three times, 5 minutes each time.

[0565] 14) DAPI staining for 5 minutes, then wash 3 times with PBS, 5 minutes each time.

[0566] 15) Drop the mounting medium onto a clean glass slide, drain the cell slide and place it upside down on the mounting medium, and place it in a cool and ventilated place at room temperature overnight.

[0567] 16) Take photos under a fluorescence microscope (40 times).

[0568] 17) Ten fields of view were photographed for each treatment group, and the number of green positive cells in each field of view was counted.

[0569] The experimental results show (such as Fig.19 YN-011 significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0570] 8.12 GABA reduces TNF-α-induced neuronal apoptosis

[0571] Please refer to Section 8.11 for the experimental steps, but the drug treatment step 3) of this experiment is different from that of Section 8.11. That is, the drug treatment step 3) of this experiment is: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 1, 10 or 100μM GABA in each well in a 37℃ incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0572] The experimental results show (such as Fig. 20 GABA reduced TNF-α-induced apoptosis of neuronal cells.

[0573] 8.13 Combination of YN-011 and GABA reduces TNF-α-induced neuronal apoptosis

[0574] Please refer to Section 8.11 for the experimental steps, but the drug treatment step 3) of this experiment is different from that of Section 8.11. That is, the drug treatment step 3) of this experiment is: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 100μM GABA and / or 100nM YN-011 in each well in a 37°C incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0575] The experimental results show (such as Fig.21 The combined use of YN-011 and GABA significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0576] 8.14 YN-011 reduces TNF-α-induced apoptosis of neuronal cells

[0577] Use 0.25% trypsin to digest the neuronal SH-SY5Y cells in a 10 cm culture dish to prepare a single cell suspension. The single cell suspension was inoculated in a 12-well plate. 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. Using a culture medium containing 5% FBS, treat the cells with 60ng / ml TNF-α alone or with 10, 100, or 500nM YN-011 in a 37°C incubator for 48 hours. Perform 3 replicates for each drug treatment. Collect the cells and digest them with 0.25% EDTA-free trypsin for 1 minute. Pipette the cell suspension into a 1.5ml EP tube and centrifuge at 1000rpm for 5 minutes. The cells will settle at the bottom of the tube. Place 10 5 Resuspend each cell in 100μl Binding Buffer, add 5μl PI and 5μl Annexin V-FITC solution, and incubate at room temperature in the dark for 15 minutes. Add 400μl Binding Buffer to each tube and detect by flow cytometry (Annexin V-FITC excitation wavelength is 488nm, emission wavelength is 520nm; PI excitation wavelength is 535nm, emission wavelength is 617nm). Count the number of Annexin V-FITC positive cells and PI negative cells in each sample.

[0578] The experimental results show (such as Fig. 22 YN-011 significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0579] 8.15 GABA reduces TNF-α-induced neuronal apoptosis

[0580] Please refer to Section 8.14 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.14. That is, the drug treatment steps of this experiment are as follows: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 1, 10 or 100μM GABA in a 37℃ incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0581] The experimental results show (such as Fig.23 GABA reduced TNF-α-induced apoptosis of neuronal cells.

[0582] 8.16 Combination of YN-011 and GABA reduces TNF-α-induced neuronal apoptosis

[0583] Please refer to Section 8.14 for the experimental steps, but the drug treatment steps of this experiment are different from those of Section 8.14. That is, the drug treatment steps of this experiment are as follows: using culture medium containing 5% FBS, treat cells with 60ng / ml TNF-α alone or with 100μM GABA and / or 100nM YN-011 in a 37℃ incubator for 48 hours, and perform 3 replicates for each drug treatment.

[0584] The experimental results show (such as Fig.24 The combined use of YN-011 and GABA significantly reduced TNF-α-induced apoptosis of neuronal cells.

[0585] 8.17 GABA reduces the mRNA expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglia.

[0586] Under sterile conditions, the synthesized Aβ 1-42 The peptide (AnaSpec #AS-20276) was dissolved in hexafluoroisopropanol (Mecklin #H811026) to obtain a 1 mM solution. The solution was aliquoted into 1.5 ml sterile centrifuge tubes and the hexafluoroisopropanol was evaporated in a vacuum desiccator. The dried peptide was formed into a film in the tube and stored at -20°C until use. Aβ was freshly prepared from the dried peptide film as shown below. 1-42 Oligomers. The dried peptide film was first dissolved in anhydrous DMSO to obtain a 5 mM solution, and then diluted with cold cell culture basal medium to obtain a 100 μM stock solution. After incubation at 4°C for 24 hours, the stock solution was used for cell culture.

[0587] Human microglial HMC3 cells were seeded into 24-well plates. Incubate at 37°C and 5% CO 2 After overnight incubation in an incubator, 10 μM Aβ 1-42Oligos were treated with 1, 10 or 100 μM GABA alone or in combination for 48 hours. Three replicates were made for each drug treatment. After 48 hours of treatment, total RNA was isolated from the cells using 1 ml Trizol (Invitrogen #15596026) per well according to the manufacturer's instructions. The RNA pellet was dissolved in 20 μl DEPC-treated water. RNA concentration and purity were measured by NanoDrop 2000. RNA was converted to cDNA using a reverse transcription kit (Yeasen #11141ES60). PCR reactions were prepared as follows: 5 μl PCR mix (Yeasen #10108ES03), 0.2 μl forward primer, 0.2 μl reverse primer, 1 μl cDNA and 3.6 μl water. qPCR was performed on an Applied Biosystems 7500 Real-Time PCR System using the following thermal program: 95°C pre-denaturation for 3 min and 40 cycles of denaturation (95°C, 30 s), 60°C annealing for 30 s, and extension for 1 min at 72°C. The ΔΔCt method was used to compare the relative mRNA expressions of TNF-α and IL-6 between treated and untreated cells, which were normalized to the expression of the housekeeping gene RPLP0.

[0588] The experimental results show (such as Fig.25 As shown), at 10 μM Aβ 1-42 In the presence of oligomers, the mRNA expressions of TNF-α and IL-6 in HMC3 cells were significantly increased, 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.

[0589] 8.18 YN-011 reduces the mRNA expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglia cells.

[0590] Please refer to Section 8.17 for the experimental steps, but the drug treatment steps in this experiment are different from those in Section 8.17. That is, the drug treatment steps in this experiment are: 10 μM Aβ 1-42 The oligomers were treated with 10, 100 or 500 nM YN-011 alone or simultaneously for 48 hours. Three replicate wells were prepared for each drug treatment.

[0591] The experimental results show (such as Fig.26 As shown), at 10 μM Aβ 1-42 In the presence of oligomers, the mRNA expressions of TNF-α and IL-6 in HMC3 cells were significantly increased, while 100 nM and 500 nM YN-011 significantly decreased the mRNA expressions of TNF-α and IL-6.

[0592] 8.19 Combination of YN-011 and GABA reduces Aβ in HMC3 microglia 1-42 Oligomer-induced inflammatory factors mRNA expression.

[0593] Please refer to Section 8.17 for the experimental steps, but the drug treatment steps in this experiment are different from those in Section 8.17. That is, the drug treatment steps in this experiment are: 10 μM Aβ 1-42 The oligomers were treated with 100 μM GABA and / or 100 nM YN-011 alone or simultaneously for 48 hours. Three replicates were performed for each drug treatment.

[0594] The experimental results show (such as Fig. 27 Compared with GABA or YN-011 alone, the combined treatment of 100 μM GABA and 100 nM YN-011 had an effect on the expression of 10 μM Aβ 1-42 The oligomers had a significantly stronger inhibitory effect on TNF-α mRNA expression induced by TNF-α.

[0595] 8.20 GABA reduces the expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglia.

[0596] Human microglial HMC3 cells were seeded into 12-well plates. Incubate at 37°C and 5% CO 2 After culturing overnight in an incubator, the cells were treated with 10 μM Aβ 1-42Oligos were treated with cells alone or with 1, 10, or 100 μM GABA for 48 hours. Three replicates were performed for each drug treatment. After 48 hours of treatment, cells were rinsed with PBS and lysed for 15 minutes on ice with RIPA buffer (Beyotime #P0013B) supplemented with protease inhibitors and phosphatase inhibitors. The cell lysate was 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. 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 unwinding the sandwich, the membrane was blocked in 5% milk solution for 1 hour at room temperature, and then incubated overnight at 4°C with primary antibody diluent (TNF-α, Proteintech#60291-Ig; IL-6, Proteintech#21865-1-AP; HSP90, Proteintech#13171-1-AP). After washing, the membrane was incubated at room temperature for 1 hour with the corresponding secondary antibody diluent (JacksonLab, 1:10000). After washing, the secondary antibody signal on the membrane was visualized using an ECL kit (Millipore#WBKLS0500) according to the manufacturer's instructions. ImageJ was used to calculate the amount of protein normalized to the endogenous protein HSP90.

[0597] The results show (such as Fig.28 As shown), at 10 μM Aβ 1-42 In the presence of oligomers, the expression of TNF-α and IL-6 in HMC3 cells was significantly increased, while 100 μM GABA significantly reduced the expression of IL-6, and 10 μM and 100 μM GABA significantly reduced the expression of TNF-α.

[0598] 8.21 YN-011 reduces the expression of inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglia.

[0599] Please refer to Section 8.20 for the experimental steps, but the drug treatment steps in this experiment are different from those in Section 8.20. That is, the drug treatment steps in this experiment are: use 10μM Aβ 1-42 The oligomers were treated with 10, 100 or 500 nM YN-011 alone or simultaneously for 48 hours. Three replicate wells were prepared for each drug treatment.

[0600] The experimental results show (such as Fig.29 As shown), at 10 μM Aβ 1-42In the presence of oligomers, the expression of TNF-α and IL-6 in HMC3 cells was significantly increased, while 100nM and 500nM YN-011 significantly decreased the expression of TNF-α and IL-6.

[0601] 8.22 Combined use of YN-011 and GABA reduces inflammatory factors induced by Aβ1-42 oligomers in HMC3 microglia Sub-expression.

[0602] Please refer to Section 8.20 for the experimental steps, but the drug treatment steps in this experiment are different from those in Section 8.20. That is, the drug treatment steps in this experiment are: use 10μM Aβ 1-42 The oligomers were treated with 100 μM GABA and / or 100 nM YN-011 alone or simultaneously for 48 hours. Three replicates were performed for each drug treatment.

[0603] The experimental results show (such as Fig.30 Compared with GABA or YN-011 alone, the combined treatment of 100 μM GABA and 100 nM YN-011 had an effect on the expression of 10 μM Aβ 1-42 The oligomers had a significantly stronger inhibitory effect on TNF-α and IL-6 expression induced by the oligomers.

[0604] Example 9: Neurodegenerative Diseases in Vivo

[0605] Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), are central nervous system diseases in which central neurons or their myelin sheaths are damaged and gradually lose function. They are characterized by the gradual loss of neuronal cell structure and function, and manifest as dementia, movement disorders, and other disorders.

[0606] Among them, Alzheimer's disease (AD), also known as senile dementia, is characterized by progressive cognitive dysfunction and memory impairment, and there is currently no effective treatment. Studies have shown that neurotoxic plaques accumulate in the brains of AD patients. These plaques are formed by 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 to 43 amino acids. The most common subtypes of Aβ in the human body are Aβ40 and Aβ42, which are easy to aggregate, thus forming the core of Aβ precipitation, triggering neurotoxic effects.

[0607] Studies have shown that GLP-1 has a neuroprotective biological effect and can improve AD symptoms [10 ,25 ,26]. In animal models, GLP-1 improves the cognitive function of AD model mice while reducing Aβ deposition, alleviating Aβ-induced glial cell overactivation, and reducing oxidative stress and inflammation in the brain, indicating that GLP-1 plays a protective role in the central nervous system in AD. The present invention discloses a long-acting GLP-1 receptor agonist (GLP-1RA), which exerts the biological effect of GLP-1 in protecting neurons and can improve central nervous system function, thus helping to improve AD symptoms.

[0608] The experiment used Alzheimer's disease model mice and wild-type control adult mice with the same genetic background. The animals were housed individually in cages, maintained with 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 degrees Celsius. Food and water were available ad libitum. Before the behavioral experiments, the mice were injected intraperitoneally (ip.) with YN-011 or placebo every week for 16 weeks.

[0609] Morris water maze setup

[0610] The maze was made of white opaque plastic with a diameter of 120 cm and a wall height of 40 cm, and was filled with water at 25°C to prevent hypothermia. A small escape platform (10 × 6.5 × 21.5 cm) was placed in 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.

[0611] Spatial Memory

[0612] Four points (north, south, east, and west) equidistantly distributed along the circumference of the pool were used as the starting positions. The starting positions were randomly assigned in four experiments every day for training. The mice were trained to find a safety platform fixed at a certain position in the pool and half-submerged in the water. 24 hours after the end of the training period, the safety platform in the pool was removed, and the length and time of the mouse's swimming path were recorded from the random starting position (n = 12 per group). The collected data were analyzed by one-way and two-way ANOVA, respectively, to evaluate spatial memory as the time required to reach the original position of the safety platform and the length of the path, and spatial acuity as the time to stay and search in the area where the safety platform is located.

[0613] Detection of Aβ40 and Aβ42 levels

[0614] Aβ40 and Aβ42 levels were measured using an Aβ detection kit. In brief, the hemispheres of the AD model mouse brains treated with control and YN-011 and the control hemispheres were homogenized in Tris-buffered saline (25mM Tris HCl, pH 7.4; 150mM NaCl) with the addition of protease inhibitors (Sigma, 250ml / 5ml buffer). The brain homogenate was centrifuged at 100,000g and 4°C for 1 hour. The supernatant was then diluted 1:10 and then subjected to ELISA, which only measures soluble amyloid beta oligomer levels, but not amyloid monomers. Protein was quantified using the Bradford protein assay. Final Aβ values ​​(n=6 per group) were determined after normalization to total protein levels.

[0615] Example 10: Phase IIb and Phase III clinical trials of YN-011 (monotherapy)

[0616] 10.1 Experimental Design

[0617] The Phase IIb and Phase III clinical trial is a multicenter, randomized, double-blind, placebo-controlled efficacy and safety clinical study in patients with T2DM who have poor blood sugar control after diet and exercise intervention. The Phase IIb clinical trial is a clinical trial to explore the dose effect and safety evaluation (divided into four groups: YN-011 (1mg) group, YN-011 (2mg) group, YN-011 (3mg) group and placebo control group), and obtains the Phase III clinical recommended dose (Recommended Phase 3 Dose, RP3D) of YN-011 for the Phase III clinical trial, namely RP3D low dose and RP3D high dose. The Phase III clinical trial is a clinical trial to confirm the efficacy.

[0618] Key inclusion criteria for Phase IIb and III studies:

[0619] Age at screening: 18-75 years old

[0620] ·Confirmed with type 2 diabetes for at least 8 weeks (WHO 2000) and not receiving any glucose-lowering medication within 8 weeks before screening.

[0621] Glycated hemoglobin HbA1c at screening: 7.5% ≤ HbA1c ≤ 11%.

[0622] Glycated hemoglobin (HbA1c) before randomization: 7.5% ≤ HbA1c ≤ 10.5%

[0623] Fasting plasma glucose (FPG) at screening and before randomization: <13.9mmol / L.

[0624] BMI ≥ 18.5 kg / m 2 And ≤40kg / m 2

[0625] Major exclusion criteria for Phase IIb and III studies:

[0626] Type 1 diabetes

[0627] Use of any DPP-4 inhibitors and / or GLP-1 analogs within 3 months before screening

[0628] ·Continuous use of insulin for more than 14 days within one year before screening (the duration of insulin treatment for gestational diabetes is not within this limit),

[0629] Fasting C-peptide <0.3nmol / mL

[0630] · Diabetic ketoacidosis, diabetic lactic acidosis or hyperosmolar non-ketotic diabetic coma within 6 months before screening;

[0631] ·Proliferative retinopathy or maculopathy that is unstable or requires treatment, severe diabetic neuropathy, intermittent claudication, or diabetic foot within 6 months of the screening period;

[0632] Severe hypoglycemia (grade III hypoglycemia) of unknown cause occurred within 6 months before screening, or 3 or more hypoglycemia events (blood sugar <3.9mmol / L) occurred within 1 month before screening, or symptoms related to hypoglycemia occurred repeatedly,

[0633] · Severe trauma, infection or surgery that may affect blood sugar control occurred within 1 month before screening,

[0634] · Blood donation or massive blood loss (>400mL) or blood transfusion within 3 months before screening;

[0635] Uncontrolled high blood pressure

[0636] Patients with a history of acute or chronic pancreatitis, symptomatic gallbladder disease, pancreatic injury, and other high-risk factors that may lead to pancreatitis, or patients with blood amylase and / or blood lipase ≥ 1.5 times the upper limit of normal (ULN) at screening;

[0637] Patients with a history of medullary thyroid cancer, multiple endocrine neoplasia (MEN) 2A or 2B syndrome, or a related family history; or a history of other malignancies;

[0638] Patients with clinically significant gastric emptying abnormalities, severe chronic gastrointestinal diseases, long-term use of drugs that have a direct effect on gastrointestinal motility, or gastrointestinal surgery within 6 months before screening, who are assessed by the researchers as unsuitable to participate in this clinical study;

[0639] · Suffering from blood system diseases or any diseases causing hemolysis or red blood cell instability;

[0640] Uncontrolled hyperthyroidism or hypothyroidism;

[0641] · 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;

[0642] 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; estimated glomerular filtration rate (eGFR) calculated by the CKD-EPI (EPI-(Scr)) equation <60mL / min / 1.73m 2 ,

[0643] Any other circumstances that the investigator or attending physician considers may not be suitable for participation in the study.

[0644] The study drug treatment regimen is as follows:

[0645] Subjects enrolled in the dose confirmation phase of Phase IIb:

[0646]

[0647] Subjects enrolled in the Phase III efficacy confirmation phase:

[0648]

[0649] 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.

[0650] 10.2 The therapeutic effect of YN-011 on type 2 diabetes

[0651] Clinical trial results showed that after 24 weeks of treatment with 1 mg YN-011, HbA1c levels were reduced by 1.73%. It was reported that after 30 weeks of treatment with 1 mg semaglutide, HbA1c levels were reduced by 1.55% (Sorli et al., Lancet Diabetes Endocrinol 2017; 5: 251–60); after 26 weeks of treatment with 1.5 mg dulaglutide, HbA1c levels were reduced by 1.46% (Shi et al., J Diabetes Investig 2020; 11: 142–150).

[0652] In addition, clinical trial results also showed that after 24 weeks of treatment with 1mg and 3mg YN-011, the incidence of adverse reactions of hypoglycemia (<3.9mmol / L) was 0.8% and 1.7%, respectively. It was reported that after 26 weeks of treatment with dulaglutide at a dose of 0.75mg and 1.5mg, the incidence of adverse reactions of hypoglycemia was 4.1% and 6.3%, respectively (Shi et al., J Diabetes Investig 2020; 11: 142–150).

[0653] In addition, clinical trial results also showed that the incidence of adverse reactions of nausea was 3.4% and 6.0% after 24 weeks of treatment with 1mg and 3mg YN-011, respectively. It is reported that the incidence of adverse reactions of nausea after 30 weeks of treatment with semaglutide at a dose of 0.5mg and 1mg was 20% and 24%, respectively (Sorli et al., Lancet Diabetes Endocrinol 2017; 5: 251–60); the incidence of adverse reactions of nausea after 26 weeks of treatment with dulaglutide at a dose of 1.5mg was 9.5% (Shi et al., J Diabetes Investig 2020; 11: 142–150); the incidence of adverse reactions of nausea after 24 weeks of treatment with loxenatide at a dose of 1mg and 3mg was 5.6% and 10%, respectively (Shuai et al., Diabetes Obes Metab. 2021; 23(1): 116-124).

[0654] Example 11: Phase IIb and III clinical trials of YN-011 combined with metformin

[0655] 11.1 Experimental Design

[0656] The Phase IIb and Phase III clinical trial is a multicenter, randomized, double-blind, placebo-controlled efficacy and safety clinical study conducted in T2DM patients with poor blood sugar control after metformin treatment. The Phase IIb clinical trial is a clinical trial to explore the dose effect and safety evaluation (divided into three groups: 1mg YN-011 combined with metformin group, 3mg YN-011 combined with metformin group and placebo combined with metformin control group), and it is a Phase III clinical trial to obtain the Phase III clinical recommended dose (Recommended Phase 3 Dose, RP3D). The Phase III clinical trial is a clinical trial to confirm the efficacy.

[0657] The main inclusion criteria for subjects in the Phase IIb and III studies of YN-011 in combination with metformin are the same as those in Example 10 except for the following:

[0658] Diagnosed with type 2 diabetes for at least 8 weeks (WHO 2000) and meets any of the following conditions:

[0659] a) Patients who have received metformin monotherapy for ≥8 weeks and metformin dose ≥1500 mg / day or maximum tolerated dose (<1500 mg / day but ≥1000 mg / day) (those who pass the screening can directly enter the lead-in period);

[0660] b) Patients who have received metformin monotherapy for less than 8 weeks and whose metformin dose is ≥1500 mg / day or the maximum tolerated dose (<1500 mg / day but ≥1000 mg / day) (patients who are qualified need to enter the metformin dose stabilization period);

[0661] c) Patients receiving metformin monotherapy at a dose of <1500 mg / day and who have not yet reached the maximum tolerated dose (after passing the screening, they need to enter the metformin dose titration period and dose stabilization period).

[0662] Please refer to the exclusion criteria section in Example 10 for the main exclusion criteria for subjects in the Phase IIb and III studies of YN-011 in combination with metformin.

[0663] The study drug treatment regimen is as follows:

[0664] Subjects enrolled in the dose confirmation phase of Phase IIb:

[0665]

[0666] Subjects enrolled in the Phase III efficacy confirmation phase:

[0667]

[0668] 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 combined with metformin and placebo combined with metformin in T2DM patients with poor glycemic control after metformin treatment. For secondary efficacy endpoints and safety assessments, please refer to the corresponding parts of Example 10.

[0669] 11.2 The therapeutic effect of YN-011 combined with metformin on type 2 diabetes

[0670] The results of this clinical trial showed that after 24 weeks of treatment with 3 mg YN-011 combined with metformin, HbA1c levels decreased by 1.8% and FPG levels decreased by 2.42%. It was reported that after 40 weeks of treatment with 1.5 mg dulaglutide combined with metformin, HbA1c levels decreased by 1.42% and FPG levels decreased by 1.93% (Dungan et al., Lancet 2014; 384: 1349–57).

[0671] The results of this clinical trial also showed that after 24 weeks of treatment with 3mg YN-011 combined with metformin, the incidence of adverse reactions of hypoglycemia (<3.9mmol / L) was 1.8%, which was comparable to the incidence of hypoglycemia in the control group of placebo combined with metformin (1.7%). It was reported that after 40 weeks of treatment with dulaglutide at a dose of 1.5mg combined with metformin, the incidence of adverse reactions of hypoglycemia was 9% (Dungan et al., Lancet 2014; 384: 1349–57).

[0672] In addition, the results of this clinical trial also showed that the incidence of adverse reactions of nausea was 7.0% after 24 weeks of treatment with 3mg YN-011 combined with metformin. It is reported that the incidence of adverse reactions of nausea was 20% after 40 weeks of treatment with dulaglutide at a dose of 1.5mg combined with metformin (Dungan et al., Lancet 2014; 384: 1349–57); the incidence of adverse reactions of nausea was 13.4% after 30 weeks of treatment with 1mg semaglutide combined with metformin (Ji et al., Diabetes Obes Metab. 2021; 23: 404–414).

[0673] The above examples list the contents currently considered to be the preferred examples of the present application, but it should be understood that the present application is not limited to the disclosed examples. On the contrary, the present application is intended to cover various modifications and equivalent examples included in the spirit and scope of the appended claims.

[0674] All publications, patents and patent applications are incorporated herein by reference in their entirety. Specifically, the sequences associated with each accession number provided herein, including, for example, accession numbers and / or biomarker sequences (e.g., proteins and / or polynucleotides) provided in the tables or elsewhere, are incorporated by reference in their entirety.

[0675] The scope of the claims should not be limited by the preferred embodiments and examples, but should be construed to be given the broadest interpretation consistent with the description.

[0676] References

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[0682] 6.Chang,A.M.,et al.,The GLP-1 derivative NN2211 restores beta-cellsensitivity to glucose in type 2 diabetic patients after a singledose.Diabetes,2003.52(7):p.1786-91.

[0683] 7.Liu,H.K.,et al.,N-acetyl-GLP-1:a DPP IV-resistant analogue ofglucagon-like peptide-1(GLP-1)with improved effects on pancreatic beta-cell-associated gene expression.Cell Biol Int,2004.28(1):p.69-73.

[0684] 8.Kim,J.G.,et al.,Development and characterization of a glucagon-likepeptide 1-albumin conjugate:the ability to activate the glucagon-like peptide1 receptor in vivo.Diabetes,2003.52(3):p.751-9.

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[0686] 10.Duarte,A.I.,et al.,Liraglutide Protects Against Brain Amyloid-beta1-42 Accumulation in Female Mice with Early Alzheimer's Disease-LikePathology by Partially Rescuing Oxidative / Nitrosative Stress andInflammation.Int J Mol Sci,2020.21(5).

[0687] 11.Elbassuoni,E.A.and R.F.Ahmed,Mechanism of the neuroprotectiveeffect of GLP-1 in a rat model of Parkinson's with pre-existingdiabetes.Neurochem Int,2019.131:p.104583.

[0688] 12.Muskiet,M.H.A.,et al.,GLP-1 and the kidney:from physiology topharmacology and outcomes in diabetes.Nat Rev Nephrol,2017.13(10):p.605-628.

[0689] 13.Hou,Y.,et al.,Nutrient Optimization Reduces Phosphorylation andHydroxylation Level on an Fc-Fusion Protein in a CHO Fed-BatchProcess.Biotechnol J,2019.14(3):p.e1700706.

[0690] 14.Karlin,S.and S.F.Altschul,Methods for assessing the statisticalsignificance of molecular sequence features by using general scoringschemes.Proc Natl Acad Sci U S A,1990.87(6):p.2264-8.

[0691] 15.Karlin,S.and S.F.Altschul,Applications and statistics for multiplehigh-scoring segments in molecular sequences.Proc Natl Acad Sci U S A,1993.90(12):p.5873-7.

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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 drug 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 following group relative to native human GLP-1: 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 following group: C222S, A330S and P331S.

2. The use according to claim 1, wherein The amino acid sequence of the GLP-1 polypeptide is shown in SEQ ID NO:

3.

3. The use according to claim 1 or 2, wherein The amino acid sequence of the IgG2-Fc domain is shown in SEQ ID NO:

6.

4. The use according to any one of the preceding claims, wherein The fusion protein has an 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.

5. according to any one of the preceding claims purposes, wherein the disease is selected from the group consisting of: metabolic diseases associated with dyslipidemia, complication of metabolic diseases, central metabolic diseases (for example, nervous system diseases) and other related metabolic diseases. The use according to claim 5 , wherein the nervous system disease is a neurodegenerative disease.

7. Use according to any one of the preceding claims, wherein the disease is Alzheimer's disease.

8. Use of a fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 7 in the preparation of a drug for treating neurodegenerative diseases.

9. Use of a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 7 in the preparation of a medicament for treating Alzheimer's disease.

10. Use of a fusion protein in the preparation of a drug for treating Alzheimer's disease, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 7.

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