Fusion proteins containing improved GLP-1 receptor agonists and uses thereof

Abstract

The present invention relates to improved GLP-1 receptor agonists, fusion proteins containing the improved GLP-1 receptor agonists, nucleic acids encoding the fusion proteins, vectors and cells containing the nucleic acids, and uses thereof. Specifically, the present invention provides modified and improved GLP-1-IgG2 / Fc fusion proteins, nucleic acids encoding the fusion proteins, vectors and cells containing the nucleic acids, and compositions thereof. The present invention further relates to the use of the fusion proteins, nucleic acids, vectors, cells, and compositions thereof in the manufacture of medicaments for the treatment or prevention of metabolic diseases associated with disorders of glucose or lipid metabolism, as well as neurological disorders and other diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to improved GLP-1 receptor agonists, fusion proteins comprising the improved GLP-1 receptor agonists, nucleic acids encoding the fusion proteins, vectors and cells comprising the nucleic acids, and applications thereof. [Background technology]

[0002] Glucagon-like peptide-1 (GLP-1), also known as an incretin, is secreted by L-cells in the small intestine. GLP-1 exerts regulatory effects on multiple organs, including promoting insulin secretion, inhibiting glucagon release, delaying gastric emptying, and reducing appetite, and plays an important role in regulating nutrient intake and absorption. The biological effects of GLP-1 are primarily mediated by activation of the GLP-1 receptor (GLP-1R). GLP-1R is a G protein-coupled membrane protein primarily expressed in pancreatic β-cells, but is also expressed to varying degrees in other tissues and cells, including the lung, heart, kidney, gastrointestinal tract, and brain. Upon receptor binding, GLP-1 activates adenylate cyclase (AC), promoting the production of the second messenger cyclic adenosine monophosphate (cAMP), which interacts with protein kinase A (PKA) and the Epac family of cAMP-regulatory guanine nucleotide exchange factors (cAMP GEFs) [1].

[0003] The natural half-life of GLP-1 in the human body is only 1–2 minutes, primarily due to rapid enzymatic inactivation by dipeptidyl peptidase-IV (DPP-IV) and / or renal clearance [2]. Therefore, scientists have developed a variety of long-acting GLP-1 analogs that are resistant to degradation. For example, human GLP-1 analogs have been modified through amino acid substitutions [4,5] and N-terminal modifications (including fatty acid oxidation [6] and N-acetylation [7]) to extend their circulating half-lives. Albumin-bound GLP-1 (albiglutide) also exhibits an extended half-life [8]. In recent years, various GLP-1 receptor agonists and analogs have been widely used to treat metabolic diseases, particularly those related to glucose and lipid metabolism, especially type 2 diabetes mellitus (T2DM) and obesity. GLP-1 receptor agonists not only play an important role in diabetes treatment, but also have preventive and therapeutic effects on cardiovascular disease [9] and neurological disorders [10,11]. Furthermore, GLP-1 can bind to GLP-1 receptors in organs such as the kidney and skin, affecting tissue metabolism and related diseases

[12] .

[0004] The GLP-1 fusion protein disclosed in US Patent US8658174 is a GLP-1 peptide fused to an IgG / Fc domain and has the potential to be used in the treatment of diabetes.

[0005] Studies have shown that GLP1-Fc fusion proteins undergo post-translational modifications. In a 2019 study, Hou et al. reported that the addition of nicotinamide and cysteine during cell culture helped reduce the hydroxylation level of GLP1 analogs fused to IgG4 / Fc proteins (dalaglutide)

[13] .

[0006] Genetic engineering and recombinant protein technology are being utilized to produce therapeutic fusion proteins. This process involves cell engineering steps, including transcription, translation, and post-translational modifications. The manufacturing process directly affects the drug's physicochemical properties, conformation, half-life in the body, biological activity, and production yield. Therefore, there remains a demand for improved GLP-1 receptor agonists and their fusion proteins that are suitable for clinical treatment, suitable for large-scale manufacturing, and have other desirable properties such as high yield, high activity, and long half-life. Summary of the Invention

[0007] The objective of the present invention is to provide improved GLP-1 receptor agonists and their fusion proteins with high activity and yield. These fusion proteins can be obtained by various approaches or methods, including amino acid substitutions at specific positions in the protein sequence, or modifications such as hydroxylation or oxidation. For example, hydroxylation at the K34 position of the GLP-1 peptide or reduction of oxidation may be performed. Furthermore, the specific amino acid substitutions or modifications of the GLP-1 fusion proteins of the present invention may significantly extend the half-life of the improved fusion proteins and demonstrate superior prophylactic and therapeutic effects in human disease and animal disease models.

[0008] Therefore, one advantage of the present invention is that it provides improved GLP-1 fusion proteins with increased yield, activity, and / or half-life. Furthermore, the improved GLP-1 fusion proteins provided by the present invention exhibit significant benefits in lowering blood glucose levels, particularly in lowering glycated hemoglobin (HbA1c) and fasting plasma glucose (FPG). Furthermore, they are characterized by a low incidence of side effects such as hypoglycemia, nausea, diarrhea, and constipation.

[0009] One aspect of the present invention provides a fusion protein comprising a GLP-1 peptide and an immunoglobulin Fc domain, wherein the GLP-1 peptide is selected from human GLP-1(7-37), human GLP-1(7-36), and DPP-IV-resistant human GLP-1. The GLP-1 peptide contains one or more amino acid substitutions selected from the group consisting of A8G, G22E, and R36G relative to human GLP-1. The immunoglobulin Fc domain is also an IgG2-Fc domain, and the IgG2-Fc domain contains one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0010] In one embodiment, the GLP-1 peptide has a level of hydroxylation at lysine at position 34 (K34) relative to native human GLP-1, and in another embodiment, the GLP-1 peptide is substantially unoxidized at tryptophan at position 31 (W31) relative to native human GLP-1.

[0011] In one embodiment, the GLP-1 peptide has at least 90% sequence identity compared to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and contains one or more amino acid substitutions selected from the following group relative to human native GLP-1: A8G, G22E, and R36G.

[0012] In one embodiment, the IgG2-Fc domain is derived from human IgG2. In another embodiment, the IgG2-Fc domain has a level of oxidation at methionine 253 (M253), corresponding to SEQ ID NO:7.

[0013] In one embodiment, the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 and contains one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0014] In one embodiment, a fusion protein of the invention includes a GLP-1 peptide set forth in SEQ ID NO:3 and also includes an immunoglobulin Fc domain set forth in SEQ ID NO:6.

[0015] In some embodiments, the GLP-1 peptide is covalently linked to the immunoglobulin Fc domain via a linker. In some embodiments, the GLP-1 fusion protein of the invention also includes a GLP-1 peptide shown in SEQ ID NO:3, a linker shown in SEQ ID NO:9, and an immunoglobulin Fc domain shown in SEQ ID NO:6.

[0016] In some embodiments, the fusion protein also includes a signal peptide.

[0017] In another aspect, the invention provides a dimer comprising two identical peptide chains linked by a disulfide bond, each peptide chain comprising a fusion protein described herein.

[0018] In another aspect, the present invention provides a nucleic acid sequence comprising the coding sequence for a fusion protein described herein.

[0019] In another aspect, the present invention provides a vector comprising a nucleic acid sequence described herein.

[0020] In another aspect, the invention provides a cell comprising a nucleic acid sequence or vector described herein.

[0021] In another aspect, the invention provides a composition comprising a fusion protein, dimer, nucleic acid sequence, vector, or cell described herein.

[0022] In another aspect, the present invention provides a method of constructing a cell as described herein, the method comprising the steps of: a) A nucleic acid encoding the fusion protein is introduced into a vector to construct an expression vector. b) Transfer the expression vector into a transgenic recombinant or naturally expressing arginine hydroxylase cell to obtain a transgenic cell. Preferably, the level or activity of arginine hydroxylase expressed in the transcript or naturally-expressing cells is higher than the level or activity of arginine hydroxylase expressed in COS-7 cells. More preferably, the cells are CHO cells, particularly CHO-K1 cells. Furthermore, a preferred vector is the pKN012 vector. In another aspect, the present invention provides a method for constructing a transcriptome cell, comprising the steps of: c) Inserting the nucleic acid sequence shown (SEQ ID NO:26) into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GLP1-IgG2 / Fc expression vector. d) The pKN012-GLP1-IgG2 / Fc expression vector was transfected into CHO-K1 cells to obtain transfectant cells.

[0023] In another aspect, the present invention provides a method for producing a fusion protein, comprising the steps of: obtaining the fusion protein using a cell or cells prepared using the construction method described herein.

[0024] In another aspect, the present invention provides a method for assessing the quality of a fusion protein, which, when the fusion protein comprises a GLP-1 peptide and an immunoglobulin IgG2-Fc domain, comprises the steps of: detecting the hydroxylation level at position K34 of the fusion protein relative to native human GLP-1.

[0025] In another aspect, the present invention provides the use of a fusion protein, dimer, nucleic acid, vector, cell, or composition described herein in the formulation of a medicament for the treatment or prevention of a disease.

[0026] Other features and advantages of the present invention will become apparent in connection with the following detailed description and accompanying specific embodiments. The detailed description and specific examples provided herein are shown for the purpose of embodying preferred embodiments of the present invention. Various changes and modifications within the spirit and scope of the present invention will be readily apparent to those skilled in the art. [Brief explanation of the drawings]

[0027] [Figure 1] Schematic diagram of the pKN012-GLP1-IgG2 / Fc vector. [Figure 2-1] Mass spectra of peptide segment 27-34:EFIAWLVK(+16 Da) of the GLP-1 fusion protein YN-011 after Lys-C digestion are shown. A: Primary mass spectrum of EFIAWLVK, B: Primary mass spectrum of EFIAWLVK(+16 Da) modification, C: Secondary mass spectrum of EFIAWLVK, D: Secondary mass spectrum of EFIAWLVK(+16 Da) modification. [Figure 2-2] Mass spectra of peptide segment 27-34:EFIAWLVK(+16 Da) of the GLP-1 fusion protein YN-011 after Lys-C digestion are shown. A: Primary mass spectrum of EFIAWLVK, B: Primary mass spectrum of EFIAWLVK(+16 Da) modification, C: Secondary mass spectrum of EFIAWLVK, D: Secondary mass spectrum of EFIAWLVK(+16 Da) modification. [Figure 3A] Shown are UV detection data for modified peptide 27-34 (aa21-28) after Lys-C digestion:EFIAWLVK (+16 Da) modification, unmodified peptide 54-79 (aa48-73), and unmodified peptide 224-240 (aa218-234). [Figure 3B] This is an enlarged view of Figure 3A. [Figure 4]The mean blood concentration-time profiles after single and multiple subcutaneous injections of 1 mg, 2 mg, 3 mg, and 4 mg of YN-011 in patients with T2DM are shown in Figures 4A and 4B for single subcutaneous administration, and Figures 4C and 4D for multiple subcutaneous administration. [Figure 5] This shows a schematic diagram of a Phase IIa double-blind, placebo-controlled study conducted in patients with T2DM to evaluate the efficacy and safety of YN-011 administered subcutaneously at dose levels of 1 mg, 2 mg, 3 mg, and 4 mg. The filled triangles represent the administration of YN-011 at the designated dose levels, the filled circles represent the measurement of oral glucose tolerance test (OGTT), the open triangles represent the indicated dosing, and the five-pointed stars represent the safety evaluation. [Figure 6] The study demonstrates the effect of multiple doses of YN-011 on fasting blood glucose levels in patients with T2DM. (At each time point, YN-011 demonstrated significant differences compared to placebo at the 3 mg and 4 mg dose levels, with p values less than 0.05.) [Figure 7] The effect of multiple doses of YN-011 on HbA1c levels in patients with T2DM is shown. (At each time point, YN-011 showed significant differences compared to placebo at the dose levels of 1 mg, 3 mg, and 4 mg, with p values of <0.05.) [Figure 8] Changes in body weight (BW) in obese rhesus monkeys after repeated subcutaneous injections of YN-011 are shown. [Figure 9] The results are shown for SH-SY5Y neuronal cells after 48 hours of stimulation with TNF-α at different concentrations (*P<0.05 TNF-α (20 ng / ml) vs control; **P<0.01 TNF-α (40, 60, 80, 100 ng / ml) vs control). [Figure 10]The results show that YN-011 and GABA inhibited TNF-α-induced decrease in SH-SY5Y cell viability (**P<0.01 TNF-α vs. control; #P<0.05 YN-011 (10nM or 100nM) or GABA (100μM) + TNF-α vs. TNF-α; ##P<0.01 YN-011 (500nM) + TNF-α vs. TNF-α; n=6). [Figure 11] The combined use of YN-011 and GABA significantly increased SH-SY5Y cell viability (**P<0.01 TNF-α vs. control, #P<0.05 TNF-α+GABA+YN-011 vs. TNF-α+YN-011; n=6). [Figure 12] YN-011 reduced TNF-α-induced apoptosis in SH-SY5Y neuronal cells (**P<0.01 TNF-α vs. control, #P<0.05 10nM YN-011+TNF-α vs. TNF-α, ##P<0.01 100nM or 500nM YN-011+TNF-α vs. TNF-α, n=3). [Figure 13] GABA reduces TNF-α-induced apoptosis in SH-SY5Y neuronal cells (**P<0.01 TNF-α vs control, #P<0.05 10μM or 100μM GABA+TNF-α vs TNF-α, n=3). [Figure 14] The combined use of YN-011 and GABA reduced TNF-α-induced apoptosis in SH-SY5Y neuronal cells (**P<0.01 TNF-α vs control, #P<0.05 GABA or YN-011+TNF-α vs GABA+YN-011+TNF-α, n=3). [Figure 15] Live cell staining images showing TNF-α-promoted damage in SH-SY5Y neuronal cells (**: significant, ***: highly significant). [Figure 16]Live cell staining images showing the protective effect of YN-011 against TNF-α-induced injury in neuronal cells (**P<0.01 TNF-α vs. control; #P<0.05 10nM YN-011+TNF-α vs. TNF-α; ##P<0.01 100nM or 500nM YN-011+TNF-α vs. TNF-α; n=3). [Figure 17] Live cell staining images showing the protective effect of GABA against TNF-α-induced damage in neuronal cells (**P<0.01 TNF-α vs control; #P<0.05 10μM GABA+TNF-α vs TNF-α;##P<0.01 100μM GABA+TNF-α vs TNF-α; n=3). [Figure 18] Live cell staining images showing that the combination of YN-011 and GABA exerts a protective effect against TNF-α-induced damage in neurons (**P<0.01 TNF-α vs control; #P<0.05 GABA or YN-011+TNF-α vs GABA+YN-011+TNF-α; n=3). [Figure 19] The results show that YN-011 reduces TNF-α-induced neuronal apoptosis (**P<0.01 TNF-α vs control; #P<0.05 100nM or 500nM YN-011+TNF-α vs TNF-α; n=3). [Figure 20] GABA reduced apoptosis in TNF-α-induced neurons (**P<0.01 TNF-α vs control; #P<0.05 100μM GABA+TNF-α vs TNF-α; n=3). [Figure 21] The combined use of YN-011 and GABA reduced TNF-α-induced neuronal apoptosis (**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). [Figure 22]The figures show that YN-011 reduced TNF-α-induced neuronal apoptosis (**P<0.01 TNF-α vs control; #P<0.05 10nM, 100nM, or 500nM YN-011+TNF-α vs TNF-α; n=3). [Figure 23] GABA reduced TNF-α-induced neuronal apoptosis (**P<0.01 TNF-α vs control; #P<0.05 100μM GABA+TNF-α vs TNF-α; n=3). [Figure 24] The combined use of YN-011 and GABA reduced TNF-α-induced neuronal apoptosis (**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). [Figure 25] GABA reduced Aβ1-42 oligomer-induced proinflammatory cytokine mRNA expression in HMC3 microglial cells (*P<0.05, **P<0.01 Aβ1-42 oligomer vs control; #P<0.05 GABA+Aβ1-42 oligomer vs Aβ1-42 oligomer; n=3). [Figure 26] YN-011 reduced the mRNA expression of inflammatory cytokines induced by Aβ1-42 oligomers in HMC3 microglial cells (*P<0.05, **P<0.01 Aβ1-42 oligomers vs control; #P<0.05, ##P<0.01 YN-011+Aβ1-42 oligomers vs Aβ1-42 oligomers; n=3). [Figure 27]The combined use of YN-011 and GABA reduces the inflammatory cytokine mRNA expression induced by Aβ1-42 oligomers in HMC3 microglial cells (*P<0.05, **P<0.01 Aβ1-42 oligomers vs control; #P<0.05, GABA+YN-011+Aβ1-42 oligomers vs GABA+Aβ1-42 oligomers or YN-011+Aβ1-42 oligomers; n=3). [Figure 28] GABA reduced Aβ1-42 oligomer-induced proinflammatory cytokine expression in HMC3 microglial cells (**P<0.01 Aβ1-42 oligomer vs control; #P<0.05 GABA+Aβ1-42 oligomer vs Aβ1-42 oligomer; n=3). [Figure 29] YN-011 reduced the expression of inflammatory cytokines induced by Aβ1-42 oligomers in HMC3 microglial cells (**P<0.01 Aβ1-42 oligomers vs control; #P<0.05 YN-011+Aβ1-42 oligomers vs Aβ1-42 oligomers; n=3). [Figure 30] The results show that the combination of YN-011 and GABA reduced the inflammatory cytokine expression induced by Aβ1-42 oligomers in HMC3 microglial cells (**P<0.01 Aβ1-42 oligomers vs. control; #P<0.05 GABA+YN-011+Aβ1-42 oligomers vs. GABA+Aβ1-42 oligomers or YN-011+Aβ1-42 oligomers; n=3). DETAILED DESCRIPTION OF THE INVENTION

[0028] The following is a detailed description to help those skilled in the art to practice the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The terms used in the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention. All publications, patent applications, patents, figures, and other references mentioned herein are incorporated by reference in their entirety.

[0029] I. Definition Unless otherwise indicated, terms defined and used in this document should be understood as dictionary definitions, definitions in incorporated documents, and / or the commonly known meaning of the defined terms.

[0030] All references, patents, and patent applications cited in this document are incorporated by reference in their entirety for each subject matter cited, and in some cases may include the entire contents of the referenced documents.

[0031] All features disclosed in this specification may be combined in any manner. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a series of equivalent or similar features.

[0032] As used herein, the terms "peptide," "polypeptide," and "protein" refer to an amino acid chain containing two or more natural or unnatural amino acid residues, regardless of whether they are post-translationally modified (e.g., glycosylated or phosphorylated). The polypeptides disclosed herein can contain, for example, 3 to 3,500 natural or unnatural amino acid residues. The proteins referred to can be single peptide chains or multi-subunit proteins (e.g., composed of two or more polypeptides). The terms "peptide," "polypeptide," and "protein" described herein can be used interchangeably and can include not only natural amino acids but also unnatural amino acids or amino acid analogs or mimetics. The peptides, polypeptides, or proteins described in this application can be obtained by any method known in the art, including, but not limited to, natural isolation, recombinant expression, chemical synthesis, etc.

[0033] As used here, the term "amino acid" refers to an organic compound containing an amino group (-NH2), a carboxyl group (-COOH), and a side chain specific to each amino acid. In this application, the names of amino acids are also represented by standard one-letter or three-letter codes, summarized as follows:

[0034] [Table 1]

[0035] As used herein, the term "GLP-1 peptide" refers to a GLP-1 receptor agonist peptide in which the amino acid residue at or corresponding to position 34 is lysine. Examples of such peptides include SEQ ID NO: 1 or SEQ ID NO: 2. Specific examples include, but are not limited to, GLP-1(7-37), GLP-1(7-36-NH2) (also referred to as GLP-1(7-36)), DPP-IV-resistant GLP-1, and other GLP-1 analogs in which lysine is at or corresponding to position 34. For example, GLP-1 peptides include those derived from liraglutide (Novo Nordisk's VICTOZA®), semaglutide (Novo Nordisk's OZEMPIC®), albiglutide (GlaxoSmithKline's SYNCRIA®), taspoglutide (Roche), dulaglutide (Eli Lilly's TRULICITY®), or LY2428757 (Eli Lilly), as well as WO2021163972A1, CN111217915A, WO2011056713A2, and WO2000034332A1, which are incorporated by reference. For example, the referenced peptides can be GLP-1 analogs containing a "KG" amino acid motif sequence.

[0036] As used herein, the term "polynucleotide" or "oligonucleotide" refers to two or more covalently linked nucleotides. Unless otherwise specified by context, this term generally includes, but is not limited to, single-stranded (ss) or double-stranded (ds) deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). For example, polynucleotide molecules or oligonucleotides of the present invention may be composed of single- and double-stranded DNA, DNA containing mixtures of single- and double-stranded regions, single- and double-stranded RNA, and RNA. Mixtures of single- and double-stranded regions may include hybrid molecules containing both DNA and RNA, which may be either single-stranded or, more typically, a mixture of single- and double-stranded regions. Furthermore, polynucleotide molecules may be composed of RNA or DNA, or triple-stranded regions containing both RNA and DNA. As used herein, the term "oligonucleotide" generally refers to a polynucleotide that is 200 base pairs or less in length and can be single- or double-stranded. The sequences provided herein may be DNA or RNA sequences. However, unless the context indicates otherwise, the sequences provided should be understood to include both DNA and RNA, and complementary RNA and DNA sequences. For example, the sequence 5'-GAATCC-3' should be understood to include 5'-GAAUCC-3', 5'-GGATTC-3', and 5'-GGAUUC-3'.

[0037] As used in this document, the terms "sequence identity" or "sequence similarity" refer to the percentage of sequence similarity between two peptide sequences or two nucleotide sequences. To determine the percentage of sequence identity between two amino acid sequences or two nucleotide sequences, the sequences are aligned for optimal comparison (e.g., gaps are introduced in the first amino acid or nucleotide sequence to optimally align it with the second amino acid or nucleotide sequence) and the amino acid residues or nucleotides at corresponding 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 identical amino acid residues (or bases) in the reference sequence being compared by the total number of amino acid residues (or bases) in the candidate or reference sequence (whichever is shorter). If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the residue at that position is considered identical. The percentage of similarity between two sequences is a function of the number of shared identical positions in the sequences (i.e., percentage similarity = number of overlapping identical positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Mathematical algorithms can also be used to determine the percentage of sequence similarity between two sequences. One preferred, non-limiting example of a mathematical algorithm used to compare two sequences is the Karlin-Altschul algorithm

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

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

[16] and can be used with the NBLAST nucleotide program parameters set (e.g., score = 100, word length = 12) to obtain nucleotide sequences homologous to a particular polynucleotide molecule. BLAST protein searches can be performed with the XBLAST program parameters set (e.g., score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described in this document. Gapped BLAST can be used to obtain gapped alignments for comparison purposes

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

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

[0038] As used herein, "conservative amino acid substitution" refers to the substitution of one amino acid residue with another without impairing the essential properties of a protein. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R chain length. Examples of conservative substitutions include the substitution of one nonpolar (hydrophobic) residue with another nonpolar residue (e.g., alanine, isoleucine, valine, leucine, or methionine), one polar (hydrophilic) residue with another polar residue (e.g., between arginine and lysine), between glutamine and asparagine, between glycine and serine, the substitution of one basic residue with another basic residue (e.g., lysine, arginine, or histidine), or the substitution of one acidic residue with another acidic residue (e.g., aspartic acid or glutamic acid). The term "conservative substitution" also includes the use of chemically derivatized or unnatural amino acids in place of non-derivatized residues, as long as the peptide exhibits the required activity.

[0039] In the present invention, the term "fusion protein" refers to a protein containing two or more peptides that form distinct functional domains, for example, the GLP-1 fusion protein described herein contains a GLP-1 peptide and an immunoglobulin Fc domain.

[0040] In the present invention, the term "linker" refers to any chemical moiety that can covalently link one moiety to another. For example, a linker is a sequence of 1, 2, 3, 4, or 5 amino acid residues, or an artificial amino acid sequence having a length of 5 to 15, 20, 30, 50, or more amino acid residues, connected by peptide bonds, and used to link one or more peptides. A linker may or may not have a secondary structure. Linker sequences are known in the art; see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994).

[0041] In the present invention, the term "CH2" refers to constant domain 2 of an immunoglobulin heavy chain. Similarly, the term "CH3" refers to another structural domain of an immunoglobulin heavy chain, constant domain 3.

[0042] In the present invention, the term "hinge region" refers to the flexible region between the antigen binding fragment (Fab) and the fragment crystallizable (Fc) in the context of an immunoglobulin such as IgG.

[0043] As used in this document, the term "vector" refers to a vehicle into which a genetic element can be operatively inserted, expressing the genetic element, producing the protein, RNA, or DNA encoded by the genetic element, or replicating the genetic element. Vectors can be used to transform, transduce, or transfect host cells, thereby allowing the carried genetic element to be expressed within the host cell. Examples of vectors include plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Vectors may contain various regulatory elements for expression control, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication site. Vectors may also contain components that facilitate cell entry. These include, but are not limited to, viral particles, liposomes, or protein coats. Vectors may be expression vectors or cloning vectors.

[0044] The terms "DPPIV" and "DPP-IV" refer to dipeptidyl peptidase-IV, an enzyme that can inactivate native GLP-1.

[0045] The term "hydroxylation level" refers to the percentage of residues modified by hydroxylation at an amino acid position within a peptide sample. For example, a hydroxylation level of 20% means that 20% (molar fraction) of the peptide molecules are hydroxylated at a particular amino acid position. Modulators can be used to increase or decrease the hydroxylation level of an expressed protein. For example, minoxidil and Zn2+ (e.g., from ZnSO4) can inhibit hydroxylation and decrease the hydroxylation level when present in an expression system. Hydroxylation levels can be measured using the methods described in this embodiment or by mass spectrometry as described by Hou et al.

[13] , or as further described herein.

[0046] The term "oxidation level" refers to the percentage of residues at an amino acid position in a peptide sample that are modified by oxidation. For example, an oxidation level of 2% means that 2% (molar fraction) of the peptide molecules at a particular amino acid position are oxidized. The oxidation level can be measured by the method described in this embodiment, by the mass spectrometry method described in WO2002046227A2, or by the protein oxidation assay method by Bettinger et al.

[19] , or as further described in this document.

[0047] In the present invention, the term "pharmaceutical grade" refers to the chemical purity or proportion of a drug, biological molecule, or reagent that meets the requirements of pharmaceutical manufacturing.

[0048] In the present invention, the term "treatment" refers to the administration of an effective amount of a compound, composition, or formulation to a subject, which may consist of a single administration or may optionally include a series of procedures. As known in the art, "treatment" refers to a method used to achieve beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in disease severity, stabilization of the disease state (i.e., not worsening), prevention of disease progression, remission of the disease, improvement or alleviation of the disease, and alleviation (partial, complete, or temporary) of symptoms associated with the disease. Beneficial or desired clinical results include improvement in fasting blood glucose and / or HbA1c levels, weight loss, improvement in liver lipid content, as well as improvement in cognitive function, motor coordination, etc. In the present invention, the term "subject," also referred to as a "patient," as used herein, includes all animals, including mammals, and preferably refers to humans. The term "subject" also includes domestic animals such as cows, pigs, sheep, chickens, horses, etc., or rodents such as rats and mice, or primates such as apes, monkeys, chimpanzees, gorillas, orangutans, baboons, or domestic animals such as dogs and cats.

[0049] In the present invention, the term "pharmaceutically acceptable carrier" refers to any carrier, excipient, or formulation that is biologically or otherwise acceptable for a pharmaceutical product. As long as the carrier, excipient, or formulation is not incompatible with the active ingredient, its use in a therapeutic formulation is considered acceptable. The use of such pharmaceutically acceptable carriers is well known in the art, and various ingredients that can be included in pharmaceutical formulations are described in Reference

[20] .

[0050] In the present invention, the term "therapeutically effective amount" refers to any amount that produces a desired effect in a subject, such as alleviating symptoms, delaying the progression of a disease, or preventing the onset of a disease. This amount can be administered multiple times to achieve the desired effect over a period of time. In the context of this document, this term can refer to an amount that lowers the blood glucose level of a subject.

[0051] As used herein, the term "co-administration" refers to the administration of two or more substances (compounds, compositions, etc.) to a subject, which substances have biological activity. The specific administration regimen depends on the pharmacokinetics of the two or more substances in the presence of each other.

[0052] For purposes of understanding the scope of the present invention, the term "comprises" and its derivatives as used in this document are open-ended terms that specify the presence of stated features, elements, components, parts, integers, and / or steps, but do not exclude the presence of other features, elements, components, groups, integers, and / or steps that are not stated. The same applies to similar terms with similar meanings, such as "comprises" and "having" and their derivatives.

[0053] As used in this document, the terms "consisting of," "consisting of," and derivatives thereof, are closed-ended terms that identify the presence of stated features, elements, components, groups, integers, and / or steps, and further exclude the presence of other features, elements, components, groups, integers, and / or steps not stated.

[0054] Any numerical ranges enumerated in this document through endpoint lists include all numbers and fractions within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and fractions are deemed to be modified by the word "about."

[0055] Additionally, terms of degree such as "substantially," "approximately," and "approximately" used in this document indicate reasonable deviations from the qualifying term, without materially altering the final result. When such deviations do not negate the meaning of the qualifying term, these terms of degree should be interpreted as including deviations of at least ±5% from the qualifying term. More specifically, the term "about" refers to deviations from the reference value of approximately ±0.1-25%, ±1-20%, ±1-15%, ±1-10%, e.g., up to 10% or up to 5%.

[0056] As used in this specification and the appended claims, the singular forms "a," "an," and "one" include plural references unless otherwise indicated. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. Also, unless otherwise indicated, the term "or" is generally used in an inclusive sense, meaning "and / or."

[0057] Furthermore, it is intended that the definitions and embodiments described in particular sections are applicable to other embodiments described in this document, as would be understood by one of ordinary skill in the art. For example, in the following paragraphs, various aspects of the invention are defined in more detail. Each aspect so defined can be combined with any other aspect or aspects, unless expressly stated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0058] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are also described in this embodiment.

[0059] II. Proteins and Fusion Proteins The present invention provides stable GLP-1 peptides and fusion proteins, including, for example, GLP-1 peptides fused to an IgG / Fc domain.

[0060] One aspect of the present invention provides a GLP-1 fusion protein comprising a GLP-1 peptide and an immunoglobulin Fc domain. The fusion protein is covalently linked between the GLP-1 peptide and the immunoglobulin Fc domain. The GLP-1 peptide is selected from human GLP-1(7-37), human GLP-1(7-36), and DPP-IV-resistant human GLP-1. Compared to native human GLP-1, the GLP-1 peptide contains one or more amino acid substitutions selected from the following group: A8G, G22E, and R36G. The immunoglobulin Fc domain is an IgG2-Fc domain and contains one or more amino acid substitutions selected from the following group: C222S, A330S, and P331S.

[0061] GLP-1 peptides Surprisingly, the yield and activity of the GLP-1 fusion proteins of the present invention can be improved by increasing the hydroxylation level of the GLP-1 peptide at position 34 compared to native human GLP-1 (K34), decreasing oxidation at tryptophan position 31 (W31) compared to native human GLP-1, and / or introducing one or more site mutations in the GLP-1 peptide (e.g., A8G, G22E, R36G) or site mutations in the IgG2-Fc domain (e.g., C222S, A330S, P331S).

[0062] The native human GLP-1 peptide consists of 37 amino acids, and its amino acid sequence is set forth in SEQ ID NO:43, commonly referred to as "GLP-1(1-37)." The native human GLP-1 peptide is processed in the pancreas and small intestine to form GLP-1(7-37) or GLP-1(7-36). Unless otherwise specified, the amino acid positions of the GLP-1 peptides referred to herein correspond to the amino acid positions of SEQ ID NO:43. For example, the K34 position of the GLP-1 peptides referred to herein corresponds to the 34th position of SEQ ID NO:43. For example, GLP-1(7-36) refers to the GLP-1 peptide fragment formed by the amino acids between the 7th and 36th positions of SEQ ID NO:43, and GLP-1(7-37) refers to the GLP-1 peptide fragment formed by the amino acids between the 7th and 37th positions of SEQ ID NO:43. In certain embodiments, the amino acid sequence of GLP-1(7-36) is set forth as SEQ ID NO: 1. In certain embodiments, the amino acid sequence of GLP-1(7-37) is set forth as SEQ ID NO: 2.

[0063] Unless otherwise specified, the naming rules for amino acid mutations referred to in this application follow the naming rules for the amino acid sequence of SEQ ID NO: 43. The order of application is: name of amino acid before mutation - position of amino acid where mutation occurs - name of amino acid after mutation. For example, the A8G mutation in the GLP-1 peptide means that alanine (A) at position 8 corresponding to SEQ ID NO: 43 is replaced with glycine (G).

[0064] 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] can be used for measurement. Unless otherwise specified, the hydroxylation level referred to in this application is calculated based on the molecular percentage. For example, if, of 100 g of the GLP-1 fusion protein described in this invention, 10 g of the GLP-1 fusion protein is hydroxylated at the K34 position of the GLP-1 peptide and the remaining 90 g of the GLP-1 fusion protein is not hydroxylated at the K34 position, the hydroxylation level of the GLP-1 fusion protein is considered to be 10%.

[0065] In certain embodiments, the GLP-1 fusion proteins of the present invention have improved in vivo half-life and / or yield when produced by recombinant methods. Thus, the GLP-1 fusion proteins, as well as the reagents used to prepare the GLP-1 fusion proteins, can be used in the formulation of pharmaceuticals.

[0066] In one embodiment, the GLP-1 peptide has a certain level of hydroxylation at position 34, which corresponds to the lysine residue (K34) in native human GLP-1. K34 refers to the lysine residue at position 34 in native human GLP-1 peptide. Those skilled in the art will recognize that the same lysine may correspond to different positions in other GLP-1 peptide sequences.

[0067] The term "GLP-1 fusion protein" may refer to multiple molecules, which may or may not be hydroxylated at K34, and the level of hydroxylation may vary between these molecules.

[0068] The present invention also provides a composition comprising a GLP-1 fusion protein. In one embodiment, the hydroxylation level at K34 of the GLP-1 fusion protein is about 10% to 100%, for example, 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.

[0069] In one embodiment, the level of hydroxylation is between 10% and 100%, e.g., 10% or more, 15% or more, 20% or more, 26% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more.

[0070] As shown in the embodiments of the present application, the yield of hydroxylated GLP-1 fusion proteins is approximately 100-500 times higher compared to GLP-1 fusion proteins in which no hydroxylation is detected at the K34 position of GLP-1.

[0071] The present invention further discovered that GLP-1 peptides have a reduced oxidation level at the tryptophan residue at position 31 (W31) compared to native human GLP-1. A reduced oxidation level at W31 may be advantageous for the stability and / or activity of GLP-1 fusion proteins. In certain embodiments, the GLP-1 peptide is substantially unoxidized at the tryptophan residue at position 31 (W31) compared to native human GLP-1. In certain embodiments, the oxidation level at W31 of the GLP-1 peptide 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 is undetectable. W31 refers to the tryptophan residue at position 31 of native human GLP-1 peptides. Those skilled in the art will recognize that the same tryptophan residue may correspond to different positions in other GLP-1 peptides.

[0072] Oxidation detection can be performed using methods known in the present application or the prior art, such as the mass spectrometry method described in WO2002046227A2 or the protein oxidation assay method by Bettinger et al.

[19] . Unless otherwise specified, oxidation levels referred to in this application are expressed as a percentage based on the number of molecules. For example, if, in 100 g of a GLP-1 fusion protein disclosed in the present invention, 10 g of the GLP-1 fusion protein is oxidized at the tryptophan residue at position 31 (W31) of GLP-1, and the remaining 90 g of the GLP-1 fusion protein is not oxidized at W31, the oxidation level of the GLP-1 fusion protein is considered to be 10%.

[0073] The GLP-1 peptide of the fusion proteins disclosed herein may be of human origin. In certain embodiments, the GLP-1 peptide is selected from human GLP-1(7-37), human GLP-1(7-36), and DPP-IV-resistant human GLP-1, and contains the amino acid substitutions A8G and G22E compared to native human GLP-1.

[0074] In certain embodiments, the GLP-1 peptide is selected from human GLP-1(7-37), human GLP-1(7-36), and DPP-IV resistant human GLP-1, and contains the amino acid substitutions A8G, G22E, and R36G compared to native human GLP-1.

[0075] In some embodiments, the GLP-1 peptide is GLP-1(7-37). In some embodiments, the GLP-1 peptide is GLP-1(7-36). In some embodiments, the GLP-1 peptide is DPP-IV resistant GLP-1. In some embodiments, the GLP-1 peptide can include one, two, or three amino acid substitutions, such as mutations at A8G, G22E, and R36G.

[0076] In some embodiments, the GLP-1 peptides disclosed in the present application 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 compared to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. They contain one or more amino acid substitutions selected from the following group relative to native human GLP-1: A8G, G22E, and R36G. In some embodiments, the GLP-1 peptides disclosed in the present application 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 compared to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and stimulate insulin secretion in a glucose-dependent manner. In some embodiments, the GLP-1 peptides disclosed in the present application 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 compared to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. They contain one or more amino acid substitutions selected from the following group relative to native human GLP-1: A8G, G22E, and R36G, and stimulate insulin secretion in a glucose-dependent manner.

[0077] [Table 2]

[0078] In certain embodiments, the GLP-1 peptide is human GLP-1(7-37), which contains the following substitutions compared to native human GLP-1: A8G, G22E, and R36G.

[0079] In one embodiment, the amino acid sequence of the GLP-1 peptide comprises the amino acid sequence set forth in SEQ ID NO: 3. In a particular embodiment, the amino acid sequence of the GLP-1 peptide is set forth in SEQ ID NO: 3.

[0080] In one embodiment, the GLP-1 peptide is DPP-IV resistant human GLP-1.

[0081] Native GLP-1 has a short circulatory half-life (t<2 min) due to rapid enzymatic inactivation, including dipeptidyl peptidase IV (DPP-IV), and / or renal clearance. Therefore, when using native GLP-1, continuous subcutaneous infusion via a pump is required to maintain its efficacy in the body

[21] . As a result, long-acting GLP-1 analogs resistant to degradation have been developed for therapeutic purposes. 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] .

[0082] Fc domain In certain embodiments, the IgG2-Fc domain described in the present application is an Fc domain derived from human IgG2.

[0083] In this application, "IgG2-Fc" and "IgG2 / Fc" can be used interchangeably and both refer to the Fc domain of the immunoglobulin IgG2.

[0084] In some embodiments, the IgG2-Fc domains described in the present application have a level of oxidation at methionine at position 253 (M253), corresponding to SEQ ID NO: 7. In particular embodiments, the IgG2-Fc domains described in the present application have reduced oxidation at methionine at position 253 (M253), corresponding to SEQ ID NO: 7. In some embodiments, the IgG2-Fc domains described in the present application do not exhibit oxidation at methionine at position 253 (M253), corresponding to SEQ ID NO: 7. In the present application, M253 refers to the methionine residue at position 253 of IgG2, corresponding to SEQ ID NO: 7. Those skilled in the art will understand that the same methionine may correspond to different positions in other IgG peptides.

[0085] In some embodiments, the oxidation level of the IgG2-Fc domain at position M253 corresponding to SEQ ID NO: 7 is about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, or undetectable.

[0086] As mentioned above, a GLP-1 fusion protein may refer to multiple molecules, which may be oxidized or non-oxidized at one or two positions: M253 of the IgG2-Fc domain and / or W31 of the GLP-1 peptide. The oxidation level of the molecules may vary. Oxidation occurs at the boxed amino acid structures in the following formula:

[0087] [Table 3]

[0088] In certain embodiments, the GLP-1 fusion proteins of the invention exhibit no detectable oxidation at position W31 of the GLP-1 peptide and approximately 2% oxidation level at position M253 of the IgG2-Fc domain compared to native human GLP-1. Other GLP-1 analog fusion proteins exhibit 2-8% oxidation level at W31 (for the GLP-1 peptide) and / or M253 (for the IgG2-Fc domain).

[0089] The GLP-1 peptide in the fusion protein is covalently linked (directly or via a linker peptide) to the Fc portion of an immunoglobulin (Ig). In one embodiment, the immunoglobulin is an IgG. The fusion protein disclosed in this article can have an IgG-Fc, which is an IgG2-Fc.

[0090] In one embodiment, the IgG2-Fc domain contains a C222S substitution. The C222S substitution in IgG2-Fc increases the flexibility of the N-terminal hinge region by eliminating the disulfide bond between the two monomers of the dimer. Increased flexibility of the N-terminal hinge region reduces binding affinity to Fcγ receptors and reduces antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0091] In another embodiment, the IgG2-Fc domain contains A330S and P331S substitutions, which reduce affinity for Fcγ receptors and the C1q complement protein.

[0092] In one embodiment, the IgG2-Fc domain comprises C222S, A330S, and P331S substitutions.

[0093] With the exception of amino acid position M253, which corresponds to the amino acid position in SEQ ID NO:7, other amino acid residue positions in the IgG2-Fc domain described herein, such as A330, P331, and C222, correspond to positions in human IgG2 as set forth in GenBank Accession No. QRG33935.1. The IgG2-Fc portion may be composed of 228 amino acids, as set forth in SEQ ID NO:5, corresponding to amino acids 219 to 445 of human IgG2 as set forth in GenBank Accession No. QRG33935.1, for example. Those skilled in the art will readily recognize the residue positions of amino acids such as A330, P331, and C222 in the reference sequence SEQ ID NO:5 or SEQ ID NO:6, or shorter or longer Fc fragments. 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 to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. It contains one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S. The fusion protein has improved half-life compared to a GLP-1 peptide without an IgG / Fc domain or fused to an IgG4 / Fc domain.

[0094] 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 set forth in SEQ ID NO: 5 or SEQ ID NO: 6. It contains one or more amino acid substitutions selected from the following group: C222S, A330S, and P331S.

[0095] In one embodiment, the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6 and comprises an A330S and a P331S substitution. Preferably, the IgG2-Fc domain also comprises a C222S substitution. More preferably, the amino acid sequence of the IgG2-Fc domain is as set forth in SEQ ID NO: 6.

[0096] In one embodiment, the amino acid sequence of the GLP-1 peptide in the disclosed GLP-1 fusion protein is as set forth in SEQ ID NO:3, and the amino acid sequence of the immunoglobulin Fc domain is as set forth in SEQ ID NO:6.

[0097] [Table 4]

[0098] The corresponding positions of the A8G, G22E, R36G substitutions and K34 hydroxylation in the GLP-1 peptides referred to in this application are shown in the table below for SEQ ID NO:3 and SEQ ID NO:7. Furthermore, the corresponding positions of the C222S, A330S, P331S substitutions and M253 oxidation on the IgG2-Fc domain referred to in this application are shown in the table below for QRG33935.1 and SEQ ID NO:7.

[0099] [Table 5]

[0100] In one embodiment, the GLP-1 peptide is located at the N-terminus of the immunoglobulin Fc domain. In another embodiment, the GLP-1 peptide is located at the C-terminus of the immunoglobulin Fc domain.

[0101] Linker In one embodiment, the GLP-1 peptide is directly covalently attached to an immunoglobulin Fc domain.

[0102] In one embodiment, the GLP-1 peptide is covalently attached to the immunoglobulin Fc domain via a linker.

[0103] In one embodiment, the linker is selected from the group of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0104] In one embodiment, the linker comprises a connecting peptide.

[0105] The GLP-1 peptide and IgG-Fc domain (e.g., IgG2-Fc domain) of the fusion proteins disclosed in this article can be linked to each other via a connecting peptide. As used herein, the term "linking peptide" refers to any moiety that connects different functional domains of a peptide. The connecting peptide can have any suitable length and structure.

[0106] In the present invention, the term "connecting peptide" refers to a preferred segment of amino acids that connects different functional domains of a peptide. A variety of connecting peptides are contemplated, and the connecting peptide can have any suitable length and structure.

[0107] As used herein, the term "cleavable linker" refers to a linker that is sensitive to intracellular proteases, pH, or chemical agents and is readily cleaved in the presence of such agents.

[0108] In the present invention, the term "non-cleavable linker" refers to a linker that is stable and resistant to intracellular proteases, pH, or chemical agents, and is not easily cleaved.

[0109] In the present invention, the term "flexible linker" refers to a linker that increases spatial flexibility when linking different protein components, allowing the protein components to fold and assume a three-dimensional structure without significant interference with each other.

[0110] In the present invention, the term "rigid linker" refers to a linker that maintains a constant distance between different protein components when they are joined together.

[0111] In the present invention, the term "helical linker" refers to a linker in which the rigid units can form a helical structure (such as an alpha helix) internally or between identical adjacent sequences, thereby providing a linker that confers a relatively stable structure to the resulting fusion protein.

[0112] In the present invention, the term "non-helical linker" refers to a linker that is unable to form a helical structure.

[0113] In one embodiment, the linker comprises a sequence containing glycine and serine residues. Preferably, the linker containing glycine and serine residues 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).

[0114] In one embodiment, the linker comprises an amino acid sequence selected from the following group: 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.

[0115] [Table 6]

[0116] In one embodiment, the linker peptide connecting the GLP-1 peptide and the IgG2 / Fc domain can 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 to the amino acid sequence set forth in SEQ ID NO: 9. In one embodiment, the linker peptide comprises the amino acid sequence set forth in SEQ ID NO: 9. In one embodiment, the amino acid sequence of the linker peptide is the same as the sequence set forth in SEQ ID NO: 9.

[0117] In one embodiment, in the GLP-1 fusion protein described in the present application, the amino acid sequence of the GLP-1 peptide is as set forth in SEQ ID NO: 3, the amino acid sequence of the immunoglobulin Fc domain is as set forth in SEQ ID NO: 6, and the amino acid sequence of the linker peptide is as set forth in SEQ ID NO: 9.

[0118] fusion proteins In one embodiment, the fusion protein disclosed in the present application shares 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 set forth in SEQ ID NO: 7. In another embodiment, the fusion protein disclosed in the present application shares 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 set forth in SEQ ID NO: 7, wherein the GLP-1 peptide comprises A8G, G22E, and R36G substitutions, and the IgG2-Fc domain comprises C222S, A330S, and P331S substitutions. In yet another embodiment, the fusion protein disclosed in the present application shares 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 set forth in SEQ ID NO: 7, wherein the GLP-1 peptide comprises A8G, G22E, and R36G substitutions and the IgG2-Fc domain comprises C222S, A330S, and P331S substitutions, and stimulates insulin secretion from beta cells in a glucose-dependent manner. In a further embodiment, the fusion protein disclosed herein shares 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 set forth in SEQ ID NO:7, wherein the GLP-1 peptide comprises A8G, G22E, and R36G substitutions and the IgG2-Fc domain comprises C222S, A330S, and P331S substitutions, and stimulates insulin secretion from beta cells in a glucose-dependent manner. In one embodiment, the fusion protein disclosed herein has an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence that shares 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.

[0119] In one embodiment, the fusion protein disclosed in the present application has an amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence that shares at least about 90% sequence identity with SEQ ID NO:7.

[0120] In one embodiment, the fusion protein disclosed in the present application has an amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, the fusion protein also has an amino acid sequence set forth in SEQ ID NO: 7.

[0121] [Table 7]

[0122] As demonstrated in the exemplary embodiments, the GLP-1 fusion proteins disclosed herein have extended half-lives due to sequence modifications. For example, the GLP-1 fusion protein YN-011 exhibits a half-life of approximately 8.6 days (207 hours).

[0123] In one embodiment of the present invention, the GLP-1 fusion protein also includes a signal peptide.

[0124] The term "signal peptide" referred to herein refers to a peptide that directs the secretion of a fusion protein into the extracellular medium. This peptide is also referred to as a "leader peptide," "peptide precursor," "pre-peptide," etc. The use of signal peptides to direct protein secretion is known in the art (e.g., U.S. Patent US8658174, the contents of which are incorporated herein by reference in their entirety). Examples of signal peptides include, but are not limited to, the human CD33 signal peptide, human growth hormone-releasing hormone (GHRH) signal peptide, human alpha-1 microglobulin / bikunin precursor (AMBP) signal peptide, green fluorescent protein signal peptide, mouse immunoglobulin heavy chain signal peptide, and mouse immunoglobulin kappa light chain signal peptide. The signal peptide is cleaved during the secretion process. In some embodiments, cleavage of the signal peptide results in an active histidine residue at the N-terminus of the GLP-1 peptide.

[0125] In one embodiment, the signal peptide is the human CD33 signal peptide.

[0126] 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 set forth in SEQ ID NO:4 (MPLLLLLPLLWAGALA) and allows for secretion of the fusion protein. In one embodiment, the signal peptide has the amino acid sequence set forth in SEQ ID NO:4 or has at least 90% sequence identity to SEQ ID NO:4. In one embodiment, the signal peptide has the amino acid sequence set forth in SEQ ID NO:4.

[0127] 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 to the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO: 8, and the GLP-1 peptide contains substitutions such as A8G, G22E, and R36G, and the IgG2-Fc domain contains substitutions such as C222S, A330S, and P331S. 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 to the amino acid sequence set forth in SEQ ID NO: 8 and stimulates insulin secretion 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 to the amino acid sequence set forth in SEQ ID NO: 8, wherein the GLP-1 peptide contains substitutions such as A8G, G22E, and R36G, and the IgG2-Fc domain contains substitutions such as C222S, A330S, and P331S, and stimulates insulin secretion in a glucose-dependent manner.

[0128] In one embodiment, the fusion protein described in the present application has the amino acid sequence set forth in SEQ ID NO:8 or has at least 80% sequence identity (e.g., at least about 85%, at least about 90%, or at least about 95%) to the amino acid sequence set forth in SEQ ID NO:8.

[0129] In one embodiment, the fusion protein described in the present application has the amino acid sequence set forth in SEQ ID NO:8 or has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0130] In one embodiment, the fusion protein described in the present application has the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the fusion protein also has the amino acid sequence set forth in SEQ ID NO: 8.

[0131] [Table 8]

[0132] In one embodiment, the fusion proteins described in the present application have a half-life in a subject of 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.

[0133] In another preferred embodiment, the GLP-1 fusion protein is pharmaceutical grade.

[0134] Another aspect of the present invention provides a dimer comprising two identical peptide chains connected by a disulfide bond, each peptide chain comprising a fusion protein described in this disclosure.

[0135] III. Nucleic acids In another aspect, the present invention also provides polynucleotides comprising nucleotides encoding a peptide or polypeptide according to the present invention, such as a GLP-1 peptide, an Fc fragment, or a fusion protein.

[0136] In another aspect, the present invention also provides nucleic acid molecules comprising nucleotides encoding a peptide or polypeptide described herein, such as a GLP-1 peptide, Fc fragment, or fusion protein.

[0137] As used herein, the term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise specified, a particular nucleotide sequence also implicitly encompasses degenerate codon substitutions, alleles, orthologs, SNPs, complementary sequences, and conservative variants thereof, such as explicitly disclosed sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons at the third position are replaced with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0138] In one embodiment, the nucleic acids described herein are codon-optimized, e.g., optimized for human use. The nucleic acid molecules described herein can be used in the methods disclosed herein.

[0139] Peptides and fusion proteins can be synthesized using standard protein chemistry techniques.

[23] Additionally, automated peptide synthesizers, such as the Advanced ChemTech Model 1396 or Milligen / Biosearch 9600, are commercially available for peptide and fusion protein synthesis. Additionally, the peptides, polypeptides, or their fragments and variants described in this article can also be produced by recombinant expression using a variety of expression systems known in the art.

[0140] In one embodiment, the disclosed nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:26 or SEQ ID NO:27, or a nucleotide sequence that shares at least 70% sequence identity with SEQ ID NO:26 or SEQ ID NO:27.

[0141] In one embodiment, the disclosed nucleic acids have 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 disclosed nucleic acid is the nucleotide sequence set forth in SEQ ID NO: 26.

[0142] In one embodiment, the disclosed nucleic acids have 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 disclosed nucleic acid is the nucleotide sequence set forth in SEQ ID NO: 27.

[0143] In some embodiments, the disclosed plurality of nucleic acids comprises any one of the plurality of nucleic acid sequences set forth as SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, or a plurality of nucleic acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, or at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0144] In some embodiments, the disclosed nucleic acids include the nucleic acid sequences set forth as SEQ ID NO:24 or the nucleic acid sequences set forth as SEQ ID NO:25, or the nucleic acid sequences having at least about 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.

[0145] In certain embodiments, the disclosed nucleic acids include a plurality of nucleic acid sequences having at least about 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 plurality of nucleic acid sequences set forth as SEQ ID NO:23 or to SEQ ID NO:24 or SEQ ID NO:25. A nucleic acid sequence having at least about 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.

[0146] In certain embodiments, the disclosed plurality of nucleic acids includes any one of the plurality of nucleic acid sequences set forth as SEQ ID NO:28 through SEQ ID NO:38, or a plurality of nucleic acid sequences having at least about 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 or 99% sequence identity to any of the sequences set forth as SEQ ID NO:28 through SEQ ID NO:38.

[0147] The sequences of SEQ ID NOs: 20 to 38 are shown in the table below.

[0148] [Table 9-1]

[0149] [Table 9-2]

[0150] [Table 9-3]

[0151] IV. Vectors and Cells In another aspect of the present invention, there is provided a vector comprising a nucleic acid described in the present application.

[0152] Any suitable vector can be used for the intended purpose. For example, various vectors can be introduced into expression systems such as mammalian, insect, or bacterial expression systems for the expression and purification of expressed proteins. Some vectors can be used for virus production. These different vectors are well known in the art.

[0153] In some embodiments, the vectors are used in combination with in vitro expression systems to produce fusion proteins. Expression and purification of the fusion proteins can be carried out using any suitable method known in the art.

[0154] Possible expression vectors include, but are not limited to, plasmids or modified viruses (e.g., lentiviral vectors, adenoviruses, and replication-defective retroviruses, including adeno-associated viruses). In one embodiment, an expression vector that can be used in conjunction with the fusion proteins of the invention is pKN012, available from a commercial supplier (Beijing Konol Science and Technology Co., Ltd.).

[0155] The vector can include appropriate regulatory sequences and components. Suitable regulatory sequences can be selected from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Examples of such regulatory sequences include transcriptional promoters and enhancers, RNA polymerase binding sites, ribosome binding sites, and translation initiation signals. Additionally, depending on the cells to be transfected / infected / transduced and the vector used, other sequences, such as origins of replication, additional DNA restriction sites, enhancers, and transcription induction sequences, can be incorporated into the expression vector. In one embodiment, the regulatory sequences induce or enhance expression in neural tissues and / or cells. In one embodiment, the vector is a viral vector. The recombinant expression vector can also include a selectable marker gene to facilitate the selection of host cells transformed, infected, or transfected with the vector for expression of the antibodies described herein. The recombinant expression vector can further include other expression cassettes encoding fusion moieties (e.g., "fusion proteins") useful for, for example, detection, including the labels and tags described herein.

[0156] In one embodiment, the vector includes one or more optional components as shown in Figure 1. For example, in one embodiment, the vector containing the nucleic acid encoding the GLP-1 fusion protein is pKN012-GLP1-IgG2.

[0157] A variety of methods can be used to transduce cells, including viral vectors, "naked" DNA, DNA in lipid or other nanoparticles, DNA containing adjuvants, and gene guns. 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 adenoviral vectors and adeno-associated viral vectors.

[0158] Another aspect of the present invention provides a recombinant cell comprising a nucleic acid encoding a GLP-1 fusion protein or a vector described in this application.

[0159] Stably expressing recombinant cells can be prepared by transforming, transfecting, or transducing recombinant cells with a vector containing a nucleic acid, preferably a nucleic acid described in this application.

[0160] In one embodiment, the cells of the invention further express a recombinant or endogenous lysyl hydroxylase.

[0161] In one embodiment, the cells of the present invention express lysyl hydroxylase at a higher level or activity than COS-7 cells. In other words, the activity level of lysyl hydroxylase is increased compared to COS-7 cells. For example, the cells can be modified to increase expression of lysyl hydroxylase (e.g., by preparing cells recombinantly expressing lysyl hydroxylase), or can be cells that naturally have elevated levels of lysyl hydroxylase compared to COS-7 cells. For example, the level or activity of lysyl hydroxylase expressed by the cells of the present application can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the level or activity of lysyl hydroxylase expressed by COS-7 cells.

[0162] In one embodiment, the cell of the present application is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell. For example, the mammalian cell may be a human embryonic kidney 293 (HEK293 cell), such as a HEK293T cell, a HEK293S cell, or a HEK293F cell. In one embodiment, the mammalian cell is a Chinese hamster ovary (CHO) cell, such as a CHO-K1 cell, a CHO-S cell, or a CHO-DG44 cell.

[0163] In one embodiment, the recombinant cells of the invention are obtained by suspension adaptation of Chinese hamster ovary cells (CHO), with CHOK1 cells being provided as an example.

[0164] V. Cell Construction, Fusion Protein Production, and Detection Methods Another aspect of the invention provides a method for constructing a recombinant cell, comprising introducing a nucleic acid encoding a GLP-1 fusion protein of the invention into a vector to construct an expression vector, and transferring the expression vector into a cell that recombinantly or naturally expresses lysine hydroxylase to obtain the recombinant cell.

[0165] In one embodiment, the expression of lysine hydroxylase in recombinant or naturally expressing cells is increased compared to the level or activity of lysine hydroxylase expressed in other cells (eg, COS-7 cells).

[0166] In one embodiment, the cell is a CHO cell, such as a CHO-K1 cell, a CHO-S cell, or a CHO-DG44 cell.

[0167] In one embodiment, the vector may be a pKN012 vector.

[0168] In one embodiment, a method for constructing a recombinant cell comprises the steps of: a) The nucleotide sequence shown in SEQ ID NO: 26 is inserted into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GLP1-IgG2 / Fc expression vector. b) CHO-K1 cells are transfected with the pKN012-GLP1-IgG2 / Fc expression vector to obtain recombinant cells.

[0169] In another embodiment, a method of constructing a recombinant cell comprises the steps of: a) The nucleotide sequence shown in SEQ ID NO: 27 is inserted into the NcoI and HindIII sites of the pKN012 vector to generate the pKN012-GLP-1-IgG2 / Fc expression vector. b) CHO-K1 cells are transfected with the pKN012-GLP-1-IgG2 / Fc expression vector to obtain recombinant cells.

[0170] Another aspect of the invention provides a method for producing a GLP-1 fusion protein, comprising obtaining the fusion protein from a recombinant cell described herein or a cell prepared using the construction methods described herein.

[0171] GLP-1 fusion proteins can be synthesized as described herein. Additionally, as described herein, fusion proteins can also be prepared using recombinant cells, including recombinant Chinese hamster ovary (CHO) cells, that express the GLP-1 fusion protein. Accordingly, the present invention further provides a method for preparing a GLP-1 fusion protein, comprising culturing recombinant cells that express the GLP-1 fusion protein, wherein the culturing includes one or more process steps or materials described in the Examples. For example, the method can include one or more process steps listed in Table 2 and / or one or more materials listed in Tables 3 or 4.

[0172] In some embodiments, recombinant cells expressing GLP-1 fusion proteins can be prepared using HEK293T, HEK293S, HEK293F, and / or CHO cells (such as CHO-K1 cells). For example, the recombinant cells can be used to produce recombinant peptides and / or fusion proteins under conditions suitable for in vivo application.

[0173] In one embodiment, the method further comprises detecting the hydroxylation level of the fusion protein at residue K34 relative to native human GLP-1. Without being bound by any theory, it is believed that the prepared fusion protein is considered to be qualified (e.g., meets good manufacturing practice) if the hydroxylation level of the GLP-1 fusion protein at residue K34 relative to native human GLP-1 is 10% to 100% (e.g., 10%, 15%, 20%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater).

[0174] Another aspect of the present invention provides a method for detecting the quality of a fusion protein comprising a GLP-1 peptide and an immunoglobulin IgG2-Fc domain, the method comprising the step of detecting the hydroxylation level of the fusion protein on residue K34 compared to native human GLP-1.

[0175] In one embodiment, the GLP-1 peptide is selected from human GLP-1(7-37), human GLP-1(7-36), and DPP-IV-resistant human GLP-1. The GLP-1 peptide includes one or more amino acid substitutions selected from the following group compared to native human GLP-1: A8G, G22E, and R36G. The IgG2-Fc domain includes one or more amino acid substitutions selected from the following group: C222S, A330S, and P331S. In one embodiment, the GLP-1 peptide includes the amino acid sequence set forth in SEQ ID NO:3, and the IgG2-Fc domain includes the amino acid sequence set forth in SEQ ID NO:6. In another embodiment, the amino acid sequence of the GLP-1 peptide is as set forth in SEQ ID NO:3, and the amino acid sequence of the IgG2-Fc domain is as set forth in SEQ ID NO:6.

[0176] In one embodiment, the quality of a fusion protein is confirmed as "qualified" if the hydroxylation level of the fusion protein on K34 relative to native human GLP-1 is 10% to 100%. For example, the hydroxylation level may be 10%, 15%, 20%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher. "Qualified" means that the fusion protein meets pharmaceutical standards established by national drug regulatory authorities or similar organizations, such as pharmaceutical standards, drug registration standards, corporate drug standards, and pharmacopoeias, ensuring that the manufactured drug meets pharmaceutical manufacturing standards.

[0177] VI. Composition Another aspect of the present invention provides a composition comprising a GLP-1 fusion protein, dimer, nucleotide, vector, or recombinant cell described in this application.

[0178] In one embodiment, the composition may further comprise a suitable diluent or carrier. In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier.

[0179] In one embodiment, the composition is a pharmaceutical composition.

[0180] In one embodiment, the composition is a pharmaceutical composition comprising a GLP-1 fusion protein and a pharmaceutically acceptable carrier.

[0181] In one embodiment, the composition comprises a GLP-1 fusion protein, wherein the GLP-1 peptide of the fusion protein has a particular level of hydroxylation at the lysine 34 (K34) residue compared to native human GLP-1. In certain embodiments, the fusion protein has a hydroxylation level at the K34 residue of the GLP-1 peptide of 10%, 15%, 20%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater compared to native human GLP-1.

[0182] In one embodiment, the composition comprises a GLP-1 fusion protein, wherein the GLP-1 peptide of the fusion protein is substantially not oxidized at tryptophan residue 31 (W31) compared to native human GLP-1. In certain embodiments, the level of oxidation of the GLP-1 peptide at the W31 residue compared 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 is undetectable.

[0183] In one embodiment, the composition comprises a GLP-1 fusion protein, wherein the IgG2-Fc domain of the fusion protein has a particular level of oxidation at the methionine residue at position 253 (M253), corresponding to SEQ ID NO: 7. In certain embodiments, the oxidation level of the IgG2-Fc domain at the methionine residue at position 253 (M253), corresponding to SEQ ID NO: 7, is 15% or less, e.g., 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or undetectable.

[0184] In one embodiment, the composition further comprises buffered saline.

[0185] In one embodiment, the composition further comprises a carbohydrate.

[0186] In one embodiment, the composition further comprises a surfactant. Pharmaceutical compositions of the invention can be prepared, packaged, and / or sold as unit doses and / or multi-unit doses. The compositions can be prepared in a variety of forms.

[0187] In one embodiment, the composition comprises a GLP-1 fusion protein formulated as a dosage form in a dose range of about 0.25 mg to about 20 mg. For example, the dosage can be 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, or about 13 mg. 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.

[0188] The described GLP-1 fusion proteins, nucleotides, vectors, recombinant cells, or compositions can be used to prepare pharmaceuticals and / or administer them by various routes, such as parenteral (e.g., intravenous, subcutaneous, or intramuscular), enteral (e.g., oral), or other routes of administration.

[0189] In one embodiment, the GLP-1 fusion protein or combination thereof may be administered parenterally or formulated into a formulation for parenteral administration.

[0190] In one embodiment, the GLP-1 fusion protein or combination thereof may be administered by the subcutaneous route or formulated for subcutaneous administration.

[0191] In one embodiment, the GLP-1 fusion protein or combination thereof may be administered intravenously or formulated into a formulation for intravenous administration.

[0192] In one embodiment, the GLP-1 fusion protein or combination thereof may be administered intramuscularly or formulated into a formulation for intramuscular administration.

[0193] Suitable diluents for the GLP-1 fusion protein and / or cells include, but are not limited to, saline, a pH buffer solution, a diluent described herein, a glycerol solution, or other solution suitable for freezing the peptide and / or cells.

[0194] Suitable diluents for the nucleic acid and / or vector include, but are not limited to, saline, pH buffered solutions, diluents described herein, water, and the like.

[0195] In another preferred embodiment, the dilution solution is sterile.

[0196] VII. Methods and Uses for the Treatment and Prevention of Disease As demonstrated in this study, substitution of the GLP-1 peptide with A8G, G22E, and / or R36G, increasing the hydroxylation level at K34 and decreasing the oxidation level at W31, and / or substitution of C222S, A330S, and P331S in the IgG2 / Fc portion of the GLP-1 fusion protein enhances 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 in this study are suitable for the preparation of pharmaceuticals and combinations thereof for corresponding therapeutic purposes.

[0197] Another aspect of the present invention is that the fusion proteins, dimers, nucleic acids, vectors, cells, or compositions disclosed herein can be used to treat or prevent the onset or progression of a disease or condition in a subject.

[0198] Another aspect of the invention provides the use of the disclosed fusion proteins, dimers, nucleic acids, vectors, cells, or compositions as GLP-1 receptor agonists.

[0199] Another aspect of the present invention provides the use of the disclosed fusion proteins, dimers, nucleic acids, vectors, cells, or compositions for the treatment, prevention, or amelioration of the progression of a disease or condition.

[0200] Another aspect of the present invention provides methods for preparing a pharmaceutical composition comprising the fusion protein, dimer, nucleic acid, vector, cell, or composition for the treatment, prevention, or amelioration of the progression of a disease or condition.

[0201] Another aspect of the present invention provides methods for treating, preventing, or ameliorating the progression of a disease or condition by administering to a subject in need thereof a therapeutically effective amount of the fusion protein, dimer, nucleic acid, vector, cell, or composition.

[0202] Another aspect of the present invention provides the use of a fusion protein, dimer, nucleic acid, vector, cell, or composition described herein for the manufacture of a medicament for the treatment or prevention of disease.

[0203] Another aspect of the present invention provides for the use of a fusion protein, dimer, nucleic acid, vector, cell, or composition described herein for the treatment or prevention of disease.

[0204] Another aspect of the present invention provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of a fusion protein, dimer, nucleic acid, vector, cell, or composition described herein.

[0205] In one embodiment, the pharmaceutical composition containing the fusion protein is administered at a dose ranging from about 0.2 mg to about 20 mg per person per administration. In another embodiment, the amount of the fusion protein is from about 1 mg to about 10 mg. In yet another embodiment, the amount of the fusion protein is from about 1 mg to about 5 mg. In further embodiments, the amount of fusion protein is specifically selected from the range of about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, or about 10 mg. 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.

[0206] A variety of formulations can be used, including, but not limited to, solutions, suspensions, capsules, and tablets.

[0207] In one embodiment, the treatment regimen can include multiple administrations.

[0208] In one embodiment, the administration frequency of the GLP-1 fusion protein or combination thereof can be once every three days, once a week, or once every two weeks.

[0209] In one embodiment, the administration regimen of the GLP-1 fusion protein, dimer, nucleotide, vector, cell, or combination thereof can be weekly administration, followed by a rest period, followed by weekly administration. In another preferred example, the rest period is 2 weeks.

[0210] In one embodiment, the GLP-1 fusion protein, dimer, nucleotide, vector, cell, or combination thereof may be administered once, followed by a two-week rest, followed by four consecutive weekly doses. In another preferred embodiment, the treatment further includes an initial dose of 1 mg administered one week prior to the start of the dosing regimen.

[0211] In one embodiment, the GLP-1 fusion protein, dimer, nucleotide, vector, cell, or combination thereof can be administered via routes such as gastrointestinal, intravenous, subcutaneous, or intramuscular.

[0212] In one embodiment, the disease includes metabolic diseases, complications of metabolic diseases, or neurological disorders and diseases related to glucose or lipid metabolism.

[0213] In one embodiment, the disease or condition is a metabolic disease associated with glucose or lipid metabolism.

[0214] In one embodiment, the metabolic disease related to glucose 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.

[0215] In one embodiment, the metabolic disease associated with glucose or lipid metabolism is diabetes, including type 2 diabetes. In another embodiment, the metabolic disease associated with glucose or lipid metabolism is obesity. In another embodiment, the metabolic disease associated with glucose or lipid metabolism is NAFLD. In another embodiment, the metabolic disease associated with glucose or lipid metabolism is non-alcoholic liver fibrosis (NASH).

[0216] In one embodiment, the subject has newly diagnosed or previously diagnosed diabetes (e.g., type 2 diabetes). Diabetes can be diagnosed by various methods, such as fasting plasma glucose (FPG). According to American Diabetes Association guidelines, diabetes is diagnosed when fasting plasma glucose levels are 126 mg / dL or higher.

[0217] In one embodiment, the subject is more likely to have diabetes (e.g., type 2 diabetes). For example, the subject may be more susceptible to diabetes due to obesity or a genetic predisposition, such as having a family history of diabetes.

[0218] In one embodiment, the subject is obese. Obesity can be defined with reference to body mass index (BMI). For example, the World Health Organization (WHO) defines obesity as a BMI of 30 or greater. In another embodiment, the subject's BMI is at least about 20 kg / m2. In another embodiment, the subject's blood glucose levels are higher than average for their age-matched peers, but not high enough to diagnose diabetes. In another embodiment, the subject can be an individual with a family history of diabetes.

[0219] In one embodiment, the subject is newly diagnosed with NAFLD or NASH or has been previously diagnosed.In another embodiment, the subject is more likely to develop NAFLD or NASH.For example, the subject may have a genetic predisposition to NAFLD or NASH.

[0220] In one embodiment, the complications of a metabolic disease include cardiovascular complications (e.g., coronary heart disease, sudden cardiac death, heart failure), renal complications (e.g., acute kidney injury, diabetic nephropathy), or hepatic complications caused by a metabolic disease.

[0221] In one embodiment, the disease or condition is a neurological disease. In one embodiment, the neurological disease is a neurodegenerative disease. In one embodiment, neurodegenerative diseases include Alzheimer's disease (AD), motor neuron disease, Huntington's disease, and Parkinson's disease (PD).

[0222] 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 a motor neuron disease. In another embodiment, the neurodegenerative disease is Huntington's disease.

[0223] In one embodiment, the subject is newly diagnosed with Alzheimer's disease or has been previously diagnosed with Alzheimer's disease.In another embodiment, the possibility that the subject will suffer from Alzheimer's disease increases.For example, the subject may be susceptible to Alzheimer's disease due to a family history of Alzheimer's disease or due to genetic profile such as having the mutation related to tau or APP associated with Alzheimer's disease.

[0224] In one embodiment, the subject is newly diagnosed with Parkinson's disease or has previously been diagnosed with Parkinson's disease. In another embodiment, the subject is at an increased risk of developing Parkinson's disease. For example, the subject may be susceptible to Parkinson's disease due to a genetic profile, such as a family history of Parkinson's disease.

[0225] The fusion proteins, dimers, polynucleotides, vectors, cells, or compositions of the invention can be combined with other known drugs or therapies for the treatment of disease.

[0226] In one embodiment, the disclosed fusion proteins, dimers, polynucleotides, vectors, cells, or compositions can be used in combination with drugs for the treatment of diabetes, including currently marketed insulin, metformin, sulfonylureas (including glimepiride, glipizide, and glyburide), α-glucosidase inhibitors such as acarbose, and other drugs on the market and in development for the treatment of diabetes. In one embodiment, the diabetes drug is metformin or insulin. In another embodiment, the disclosed fusion proteins, dimers, polynucleotides, vectors, cells, or compositions can be used in combination with Alzheimer's disease drugs and / or non-pharmacological interventions, such as cognitive therapy. In one embodiment, the disclosed fusion proteins, dimers, polynucleotides, vectors, cells, or compositions are used in combination with gamma-aminobutyric acid (GABA) for the treatment of neurodegenerative diseases. In one embodiment, the disclosed fusion proteins, dimers, polynucleotides, vectors, cells, or compositions are used in combination with gamma-aminobutyric acid (GABA) for the treatment of Alzheimer's disease.

[0227] In some embodiments, the disclosed GLP-1 fusion proteins can be combined with gamma-aminobutyric acid (GABA) to inhibit TNF-α-induced loss of SH-SY5Y cell viability.

[0228] In some embodiments, the disclosed GLP-1 fusion proteins can be combined with gamma-aminobutyric acid (GABA) to attenuate TNF-α-induced apoptosis in neuronal SH-SY5Y cells.

[0229] In some embodiments, the disclosed GLP-1 fusion proteins can be combined with gamma-aminobutyric acid (GABA) to provide protection against TNF-α-induced damage in neuronal cells.

[0230] In some embodiments, the disclosed GLP-1 fusion proteins, when combined with gamma-aminobutyric acid (GABA), can reduce TNF-α-induced apoptosis in neuronal cells.

[0231] In some embodiments, the disclosed GLP-1 fusion proteins, when combined with gamma-aminobutyric acid (GABA), can reduce Aβ1-42 oligomer-induced inflammatory factor (e.g., TNF-α, IL-6) mRNA expression in HMC3 microglial cells.

[0232] In one embodiment of the application of the present invention, the drug is a GLP-1 receptor agonist.

[0233] The above disclosure summarizes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are provided for illustrative purposes and are not intended to limit the scope of the present application. Where appropriate or convenient, changes in form and substitution of equivalents may be considered. Although specific terms are used herein, they are used for purposes of description and not limitation.

[0234] The following non-limiting examples are provided to illustrate the present invention.

[0235] Example Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0236] Unless otherwise specified, materials and reagents used in the following examples are available from commercial sources.

[0237] Example 1: Fusion protein expression and activity assay 1.1 Plasmid construction A vector encoding a GLP-1 fusion protein was constructed. The fusion protein consisted of human GLP-1(7-37) and human IgG2 / Fc (including the hinge region, CH2, and CH3 of the human IgG2 heavy chain). The signal peptide sequence of human CD33 (hCD33) was fused to the GLP-1 sequence to direct secretion of the 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.

[0238] The pKN012-GLP-1-IgG2 / Fc stable expression vector was transformed into E. coli DH5α competent cells. Plasmid DNA was extracted from the bacterial strain containing pKN012-GLP-1-IgG2 / Fc and digested with PvuI. After electrophoresis, a target band of the correct molecular weight was observed. The linearized plasmid was quantified after ethanol precipitation and used for stable transfection.

[0239] To establish CHO-K1 cells stably expressing GLP-1-IgG2 / Fc (Lot No. 58995535 / ATCC), linearized pKN012-GLP-1-IgG2 / Fc (2 μg) was transfected into CHO-K1 cells grown in 6-well plates (2.5 x 10 cells / well) using electroporation (electroporation-mediated transfection). 24 h after transfection, the cells were dispersed and cultured in CD-CHO medium containing MSX (methionine sulfoximine, 100 μM / L) to select for cells that had stably integrated the recombinant plasmid into their genome. The culture medium was changed every 3 days until colonies were formed. Individual colonies were isolated and expanded into stable cell lines. The GLP-1 fusion protein in the culture supernatant of cell lines grown in 24-well plates was tested using a rat GLP-1 RIA kit (YN-011). Cells capable of secreting the fusion protein were selected and further identified. The amino acid sequence of the prepared GLP-1 fusion protein is shown in SEQ ID NO:7 (referred to as "YN-011" in this application).

[0240] 1.2 Evaluation of YN-011 activity by induction of cAMP levels Native GLP-1 stimulates insulin secretion from beta cells in a glucose-dependent manner. To assess whether purified YN-011 possesses the functions of native GLP-1, its effect on insulin secretion from clonal insulin-secreting INS-1 cells was measured. INS-1 cells were serum- and glucose-starved and then treated with different amounts of purified YN-011 in the presence of 0, 5, or 20 mM glucose, as indicated. In the absence of glucose, YN-011 did not stimulate insulin secretion from beta cells. However, in the presence of 5 or 20 mM glucose, YN-011 dose-dependently stimulated insulin secretion from beta cells. These data indicate 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.

[0241] In the absence of glucose, INS-1 cells treated with YN-011 (120 nM) maintained basal levels of cAMP. However, in the presence of 5 mM glucose, cAMP levels in YN-011-treated cells increased significantly, reaching levels comparable to those induced by exendin-4.

[0242] Example 2: Increasing GLP-1 K34 hydroxylation levels improves protein yield and activity 2.1 Determining modification levels by LC-MS / MS peptide mapping Mass spectrometry analysis was performed using Lys-C digestion for identification. The YN-011 sample was denatured, reduced, and alkylated, followed by Lys-C enzymatic hydrolysis to generate peptides. Online LC-MS / MS analysis was performed using a Thermo LTQ Velos Orbitrap instrument, and data were processed and analyzed using Mascot software. The peptide segment 21-28: EFIAWLVK was observed to have a molecular weight increase of 16 Da due to the modification. Figure 2 shows the primary and secondary mass spectra of the peptides from the YN-011 sample without and with the +16 Da modification. The mass measurement error for the primary spectrum was within 10 ppm. By analyzing the mass-to-charge ratio of the secondary fragment ions, the site of the +16 Da modification was determined to be K28. Because the first six residues are missing in active GLP-1 (e.g., amino acids 7-37 of GLP-1), K28 is also referred to as K34. Peptide 21-28 (also called the 27-34 peptide) is also found in native human GLP-1.

[0243] The modification level of the K34 site of YN-011 was determined using UV280 and EIC (extracted ion chromatography) methods, as shown in Table 1 and Figures 3A and 3B. The UV280 calculation method is as follows:

[0244]

number

[0245] [Table 10] * Note: UV280 and EIC methods were used to detect the K34 modification level of YN-011.

[0246] 2.K34 hydroxylation level of 2YN-011 Mass spectrometry confirmed the +16 Da modification at the 34th position of lysine (Lys), suggesting the possibility of an oxidation reaction, given that +16 Da corresponds to the molecular weight of an oxygen atom. In fact, there are two enzymes in cells that catalyze the modification of the lysine side chain. One is called lysyl oxidase (see https: / / en.wikipedia.org / wiki / Lysyl_oxidase) and the other is called lysyl hydroxylase (see https: / / en.wikipedia.org / wiki / Lysyl_hydroxylase). Lysyl oxidase can oxidize the amino group at the 6th carbon of the lysine side chain to form an aldehyde group. However, this modification only results in a 1 Da difference compared to the unmodified form, which is inconsistent with the observed +16 Da modification. On the other hand, lysyl hydroxylase adds a hydroxyl group to the γ carbon of the lysine side chain, forming a stable hydroxylated lysine (hydroxylysine). Lysine hydroxylation adds a hydroxyl group and reduces a side-chain hydrogen atom, resulting in a 16 Da increase in molecular weight. Therefore, the observed +16 Da modification is consistent with lysine hydroxylation. The hydroxylation site at position K34 is shown below.

[0247] [ka]

[0248] In conclusion, the +16 Da modification observed at K34 in YN-011 is most likely due to lysine hydroxylation. Therefore, adding a hydroxylation inhibitor during cell culture will enable us to verify that the +16 Da modification is lysine hydroxylation. Furthermore, by collecting proteins with different percentages of +16 Da modification, we can investigate the effect of the degree of +16 Da modification on the biological activity of proteins.

[0249] As described in Example 1, the cell culture process of stable CHOK1 cells capable of secreting the fusion protein YN-011 is shown in Table 2. The relevant materials and cell culture medium required for cell culture are listed in Tables 3 and 4, respectively. As mentioned above, mass spectrometry was used to detect the modification level of the fusion protein. The inventors found that the fusion protein was indeed hydroxylated at K34, with the hydroxylation level ranging from 15% to 40%.

[0250] To investigate the biological effects of K34 hydroxylation, we set up eight shake flasks (SF1-SF8) for culturing stable CHOK1 cells expressing the fusion protein YN-011. Using SF1-SF8, we evaluated various conditions. Hydroxylation inhibitors were added during the cell culture process to collect proteins with different hydroxylation ratios and examine the effect of the degree of hydroxylation on the protein's biological activity. Minoxidil is an inhibitor of lysyl hydroxylase, which suppresses lysyl hydroxylation. Zn2+ ions competitively inhibit the activity of the hydroxylation enzyme by competing with Fe2+ ions during the reaction. SF1 served as a control without added inhibitor. The minoxidil used in this experiment was dissolved in 0.1 N HCl solution. HCl was added to SF2 as an HCl control. SF3-SF5 contained different concentrations of minoxidil: 0.1 mM, 0.5 mM, and 1.0 mM, respectively. SF6 and SF7 were experimental groups with Zn2+ added. The Zn2+ reagent used was ZnSO4, and the concentrations of ZnSO4 used were 200 μM and 400 μM, respectively. SF8 contained both minoxidil and Zn2+ (provided by ZnSO4) as inhibitors, and the total concentrations of minoxidil and Zn2+ were 0.5 mM and 200 μM, respectively.

[0251] [Table 11]

[0252] [Table 12]

[0253] [Table 13]

[0254] 2.3 Increasing the hydroxylation level of K34 in GLP-1 can increase protein yield and activity. The protein yield and activity results are shown in Table 5. SF1 was the blank control, and SF2 contained only HCl. The hydroxylation levels ranged from 15.7% to 16.0%. From SF3 to SF5, the hydroxylation rate gradually decreased as the concentration of minoxidil (a hydroxylation inhibitor) increased. When the minoxidil dosage reached 0.5 mM, the hydroxylation rate was 8.7%. When the minoxidil concentration increased to 1 mM, the rate was 9.4%. Cell growth and protein expression were significantly inhibited, with peak cell density decreasing by approximately 17% and protein yield decreasing by 57.0% to 67.9%. Furthermore, when 400 μM Zn2+ (another hydroxylation inhibitor) was added to the cell culture, the hydroxylation level also decreased to 10.7%. In an experiment in which a combination of inhibitors (0.5 mM minoxidil and 200 μM Zn2+) was added to SF8, the hydroxylation level decreased to 9.6%.

[0255] In the presence of a hydroxylation inhibitor such as minoxidil, the rate of hydroxylation gradually decreases as the concentration increases. Cell growth and protein expression are significantly inhibited, resulting in a decrease in peak cell density of approximately 17% and a gradual decrease in yield ranging from 57.0% to 67.9%. Similarly, in the presence of the hydroxylation inhibitor Zn2+, the hydroxylation level also decreases as the Zn2+ concentration increases, resulting in a decrease in protein yield of 22.9% to 34.2%. When the hydroxylation level reaches 15.7% to 16.0%, the yield increases, doubling compared to lower hydroxylation levels. Therefore, the higher the hydroxylation level, the higher the yield.

[0256] SUPA-1 is a GLP-1-IgG2 / Fc fusion protein disclosed in US Patent US8658174. No linking peptide is used between the GLP-1 peptide and the IgG2 / Fc. Other than the A8G substitution in the GLP-1 peptide, no other site mutations exist in the GLP-1 peptide or the IgG2 / Fc. SUPA-1 has been detected to be free of hydroxylation or other modifications, and under the same conditions, the protein yield is 22 mg / L. In contrast, when the K34 hydroxylation degree of YN-011 is 8.7%-9.6%, the yield reaches 0.77 g / L-1.03 g / L, more than 100-fold higher than that of SUPA-1. When the K34 hydroxylation degree exceeds 15%, the yield of YN-011 is more than 500-fold higher than that of SUPA-1.

[0257] Furthermore, the biological activity of YN-011 was tested using the method described in Example 1 for a blank control (SF1) and two samples with the lowest hydroxylation levels (SF4 and SF5). The results showed that the activity of SF1 was 88%, while the activities of SF4 and SF5 were 83% and 68%, respectively. Therefore, a higher hydroxylation level tends to increase biological activity.

[0258] In summary, it was confirmed that K34 in the fusion protein was indeed hydroxylated, and this modification significantly improved the yield of YN-011 and tended to increase its biological activity.

[0259] Furthermore, by scaling up the cell culture to a 15 L reactor and three 200 L reactors, YN-011 obtained from these batches showed 20%–30% hydroxylation at position 34, with a yield of approximately 2.7 g / L.

[0260] [Table 14]

[0261] Example 3 Measurement of oxidation levels by LC-MS / MS The oxidation level was measured according to the protein oxidation measurement method described in Example 2 or with reference to Bettinger et al.

[19] . The GLP-1 fusion protein YN-011 of the present invention was not oxidized at position W31, whereas the oxidation level of degludec at the same position was observed to be greater than 5%. Oxidation occurred at the position indicated by a box in the structure of W31.

[0262] [ka]

[0263] Furthermore, the GLP-1 fusion protein YN-011 of the present invention showed an oxidation level of about 2% to 4% at position M253, while degludec showed an oxidation level of over 5% at the same position.

[0264] Example 4: Preventive and therapeutic effects of YN-011 in db / db mice (type 2 diabetes model) The effect of multiple subcutaneous injections of YN-011 on lowering blood glucose levels was evaluated in db / db mice (Jackson Laboratories, 000642). Mice were housed under normal lighting conditions (12 hours light / 12 hours dark) and room temperature with free access to food (standard rodent chow) and water. Db / db mice lack leptin receptors and spontaneously develop obesity, hyperglycemia, and pancreatic beta cell atrophy by 4 weeks of age.

[0265] Sixty diseased db / db mice (weight 33-45 grams) were divided into six groups of 10 mice each (five males and five females) and administered YN-011 at doses of 0 (PBS buffer control), 0.15, 0.3, 0.6, or 1.2 mg / kg, or degludec at 0.3 mg / kg, subcutaneously. A separate group of wild-type mice (weight 17-22 grams) of the same genetic background served as normal controls. The dosing frequency was once every three days (Q3D), for a total of nine doses over 26 days.

[0266] Throughout the experiment, random plasma glucose (RPG) levels in the model control group of db / db mice were consistently high and significantly higher than those in normal control mice (P<0.001). After the first administration, YN-011 significantly reduced RPG concentrations in db / db mice, with therapeutic effects observed 3 hours after administration at a low dose of 0.15 mg / kg. The duration of the hypoglycemic effect ranged from 34 to 72 hours across the dose range of 0.15 to 1.2 mg / kg. YN-011 also significantly promoted insulin secretion, with an increase in serum insulin levels observed 3 hours after administration.

[0267] After the ninth dose of YN-011, RPG levels in mice treated with YN-011 were significantly lower than those in the control group at all doses and measurement time points, except for the 0.15 mg / kg group after the fourth dose (day 10). In this group, the difference was not significant (P=0.07). To better understand the reduction in blood glucose levels in each mouse group, we calculated the average reduction in random blood glucose levels at each measurement time point during the experiment. Results showed that the average reduction in random blood glucose levels 72 hours after the seventh dose was 31.8%, 46.2%, and 50.8% in the 0.15, 0.3, and 0.6 mg / kg YN-011 groups, respectively, and reached 54.3% in the 1.2 mg / kg YN-011 group. The hypoglycemic effect of YN-011 persisted for 72 hours after repeated administration.

[0268] Long-term administration of YN-011 also significantly reduced fasting blood glucose levels in db / db mice. Throughout the experiment, fasting blood glucose levels in db / db mice were consistently high and significantly higher than those in normal control mice (P<0.001). After subcutaneous injection of different doses of YN-011 every three days, fasting blood glucose levels in all YN-011 groups were significantly reduced compared to the model control group (P<0.001). At 54 hours after the seventh dose (day 21), the fasting blood glucose reduction rates were 46.8%, 55.6%, 59.5%, and 64.7% in the 0.15, 0.3, 0.6, and 1.2 mg / kg YN-011 groups, respectively. This suggests that the hypoglycemic effect of YN-011 at doses of 0.15, 0.3, 0.6, and 1.2 mg / kg administered every three days can be maintained for at least 54 hours (three days after administration). The positive control group receiving 0.3 mg / kg degludec also demonstrated significant reductions in fasting blood glucose levels at all time points.

[0269] To provide a more intuitive understanding of the reduction in blood glucose levels in each mouse group, the average percentage reduction in fasting blood glucose levels at each measurement time point throughout the experiment was calculated. The average percentage reductions in fasting blood glucose levels in the 0.15, 0.3, 0.6, and 1.2 mg / kg YN-011 groups were 55.0%, 61.8%, 63.7%, and 66.1%, respectively. The average percentage reduction in fasting blood glucose levels in the positive control group administered 0.3 mg / kg degludec was 63.6%.

[0270] Thus, multiple subcutaneous injections of YN-011 every 3 days significantly reduced fasting blood glucose levels in type 2 diabetic db / db mice. This effect was evident at a dose of 0.15 mg / kg, and the hypoglycemic effect of YN-011 at doses of 0.15, 0.3, 0.6, and 1.2 mg / kg was maintained for at least 54 hours (3 days after administration).

[0271] Subcutaneous injection of YN-011 at doses of 0.15 to 0.6 mg / kg into db / db mice tended to decrease serum fructosamine levels, but the 1.2 mg / kg YN-011 group showed a significant decrease in serum fructosamine levels.

[0272] In the model control group, the random and fasting body weights of db / db mice continued to increase throughout the experiment, while in the YN-011 treatment groups at doses of 0.3, 0.6, and 1.2 mg / kg, the random and fasting body weights were significantly reduced (P<0.05, P<0.01, P<0.001). In contrast, the SUPA-1 fusion protein disclosed in US Patent US8658174 did not significantly affect the body weight of db / db mice. This indicates that the GLP-1 fusion protein of this application is superior.

[0273] Compared with the vehicle control group, epididymal fat content and its ratio to body weight were significantly reduced in the YN-011-treated groups. Scapular fat content was significantly reduced in the 0.15, 0.3, and 0.6 mg / kg YN-011 groups. Subcutaneous fat mass, inguinal fat mass, and their ratio to body weight were significantly reduced in the 0.3, 0.6, and 1.2 mg / kg YN-011 groups. Perirenal fat mass was significantly reduced in the 1.2 mg / kg YN-011 group.

[0274] After the final administration (9th dose, day 26), YN-011 dose-dependently increased fasting serum insulin levels in db / db mice, accompanied by a significant increase in pancreatic β-cell mass.

[0275] Compared with the vehicle control group, multiple subcutaneous injections of YN-011 significantly reduced serum triglyceride levels, and 0.6 mg / kg YN-011 significantly reduced serum free fatty acid levels in db / db mice.

[0276] In summary, multiple subcutaneous injections of YN-011 demonstrated significant therapeutic effects in db / db mice, not only improving glucose metabolism but also suppressing abnormal lipid metabolism.

[0277] Example 5: Phase IIa clinical trial of YN-011 5.1 Study Design This Phase IIa clinical trial is a double-blind, placebo-controlled study to evaluate the efficacy and safety of subcutaneously administered YN-011 at doses of 1 mg, 2 mg, 3 mg, and 4 mg in subjects with type 2 diabetes mellitus (T2DM). The study design is shown in Figure 5.

[0278] Key inclusion criteria for Phase IIa trials: Patients with T2DM (WHO 1999) who have not taken metformin for at least one week and have not taken other oral antidiabetic drugs for at least two weeks. HbA1c value at screening: 7.0%≦HbA1c≦10.0%. Age 18 to 65 at screening. BMI ≥ 20kg / m2 and ≤ 40kg / m2.

[0279] Key exclusion criteria for Phase IIa trials: ·Type 1 diabetes. Fasting C-peptide <0.81ng / mL. Laboratory values meet any of the following criteria: alanine aminotransferase (ALT) level ≥ 2.5xULN, and / or aspartate aminotransferase (AST) level ≥ 2.5xULN, fasting triglycerides > 5.6mmol / L, estimated glomerular filtration rate (eGFR) < 45mL / min / 1.73m2 calculated by the CKD-EPI (EPI-(Scr)) formula, ·Familial (first-degree relative) or personal history of type 2 multiple endocrine neoplasia or medullary thyroid carcinoma. ·Uncontrolled high blood pressure. - History of pancreatitis, pancreatic cancer, serum amylase >1.2xULN at the time of screening, or high-risk factors for pancreatitis. Patients with uncontrolled hypothyroidism. - Suspected active infection. · Positive hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV-Ab), human immunodeficiency virus antibody (HIV-Ab), or Treponema pallidum antibody (TPAb). -Receipt of treatment with GLP-1 receptor agonists, DPP-4 inhibitors, or insulin in the 3 months prior to randomization. ·History of grade 3-4 allergy to protein drugs based on CTCAE. - Donated blood or lost more than 450 mL of blood in the 3 months prior to screening. - If you have a significant endocrine, immune, coagulation, genitourinary, or blood disorder. · Clinically significant gastric emptying disorder (e.g., gastric outlet obstruction), severe chronic gastrointestinal disease (e.g., active ulcer within 6 months), long-term use of medications that directly affect gastrointestinal motility, or previous gastrointestinal surgery. Any other condition that the investigator or attending physician deems inappropriate for participation in this study.

[0280] Subjects were randomly assigned in a 4:1 ratio to receive either YN-011 or placebo. YN-011 was administered at doses of 1 mg, 2 mg, or 3 mg. The administration regimen consisted of a single dose followed by a two-week washout period, followed by weekly administration for four consecutive weeks. Subjects in the 4 mg group received the same administration regimen, except that they received an initial dose of 1 mg one week prior to administration. All subjects received a total of five randomized doses.

[0281] The primary endpoint was the safety and tolerability of YN-011 in patients with type 2 diabetes. Secondary endpoints included weekly change from baseline in fasting plasma glucose, change from baseline in HbA1c at weeks 4 and 7, change from baseline in glycoalbumin at weeks 4 and 7, change in glucose tolerance, and change in pancreatic beta-cell function assessed by oral glucose tolerance test. Blood samples were collected from all subjects for pharmacokinetic studies. Safety assessments included adverse events, clinical tests, vital signs, 12-lead electrocardiograms, physical examinations, and anti-drug antibody (ADA) assessments.

[0282] 5.2 Pharmacokinetics of YN-011 after single or multiple doses YN-011 showed an increasing trend in efficacy across the 1.0 mg to 4.0 mg dose range. After a single dose, the half-life (T1 / 2) of YN-011 was approximately 207 hours (8.6 days), with a median Tmax of 60 to 84 hours (Figure 4). After the first dose of YN-011 after randomization, administration was once weekly, with the fourth dose administered consecutively. The plasma concentration of YN-011 reached a steady state after the fourth dose (Figure 4). The fusion protein in this application had a significantly longer half-life compared to SUPA-1 after amino acid substitution. Furthermore, compared to the commercially available drugs dulaglutide (elimination half-life 4.7 to 5.5 days

[22] ) and semaglutide (elimination half-life 5.7 to 6.7 days

[24] ), YN-011 exhibited a significantly longer half-life than dulaglutide and semaglutide.

[0283] 5.3 Efficacy of YN-011 in type 2 diabetes YN-011 was administered subcutaneously at formal doses of 1 mg, 2 mg, 3 mg, and 4 mg (according to the dosing schedule shown in Figure 5 ) for the treatment of type 2 diabetes.

[0284] A total of 40 subjects received at least one dose of YN-011 or placebo and were included in the safety analysis. The subjects' mean age was 51.7 ± 10.32 years, mean BMI was 25.80 ± 2.875 kg / m², and mean weight was 71.91 ± 12.360 kg. 40% of the subjects were female. Thirty-eight of the 40 subjects (95%) had comorbidities, including 32 (80%) with ultrasound-confirmed fatty liver, 25 (62.5%) with hyperlipidemia, 19 (47.5%) with hypertension, 10 (22.5%) with arteriosclerosis, and 10 (22.5%) with pulmonary involvement. None of the 40 subjects were taking any concomitant medications throughout the study. The subjects' baseline characteristics are summarized in the following table (Table 6).

[0285] [Table 15]

[0286] The effects of different doses of YN-011 on fasting blood glucose levels are shown in Figure 6. Multiple subcutaneous administration of 3 mg or 4 mg of YN-011 resulted in sustained, significant, and clinically and statistically significant improvements in fasting blood glucose levels compared to placebo.

[0287] The effects of different doses of YN-011 on HbA1c are shown in Figure 7. Multiple subcutaneous administration of YN-011 at 1 mg, 3 mg, or 4 mg resulted in sustained improvements in HbA1c levels, which were clinically and statistically significant compared to placebo. No serious side effects or safety risks were observed.

[0288] Example 6: Preventive and therapeutic effects of YN-011 on obesity 6.1 Effect of YN-011 on high-fat diet-induced obesity in mice The effects of multiple doses of YN-011 on glucose tolerance, insulin sensitivity, metabolism, and weight loss were evaluated in high-fat diet (HFD)-induced obese mice. Five-month-old male C57BL / 6 mice (obtained from commercial sources, such as Shanghai Slake Laboratory Animal Co., Ltd., under standard housing conditions) were fed an HFD (60% of total calories from fat and 20% from carbohydrates) for six months to establish a diet-induced obese (DIO) mouse model. DIO mice (weight >50g) were then divided into two groups (five per group). The experimental group received subcutaneous injections of 0.3mg / kg YN-011 twice weekly (BIW), while the control group received phosphate-buffered saline (PBS) injections. Treatment was continued for four weeks.

[0289] Experimental results showed that 4 weeks of BIW injection of YN-011 significantly reduced body weight in DIO mice. Compared to the PBS control group, mice treated with YN-011 showed a significant reduction in visceral fat, particularly epididymal fat mass. YN-011 did not affect the weight of other body weight-related tissues, such as brown adipose tissue (BAT), inguinal white adipose tissue (WAT), pancreas, or calf muscle.

[0290] Compared with the control group, 4 weeks of BIW injection of YN-011 significantly reduced ectopic lipid accumulation and liver triglycerides, as well as serum ALT levels (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 a 30% reduction in total cholesterol (TC), a 68% reduction in triglycerides (TG), and a 57% reduction in nonesterified fatty acids (NEFA) (P < 0.001). Compared with the control group, DIO mice receiving multiple doses of YN-011 showed a significant decrease in food intake. YN-011 administration showed a trend toward increased metabolic rate (VO2 and VCO2) and energy expenditure (EE), but the results were not statistically significant. When normalized to body weight, mice treated with YN-011 showed significant increases in VO2, VCO2, and EE during the night. YN-011 did not affect Ucp1 expression in BAT or epididymal WAT, but significantly upregulated Ucp1 expression in inguinal WAT. This indicates that YN-011 did not enhance BAT thermogenesis but promoted browning of inguinal white adipose tissue. Therefore, DIO mice treated with YN-011 exhibited higher core body temperatures at room temperature and maintained higher rectal temperatures even when exposed to a cold environment, indicating increased thermogenesis compared to the control group. These results suggest that YN-011 enhances adaptation to a cold environment in obese mice by generating more calories. Blood glucose fluctuation, intraperitoneal glucose tolerance test, and insulin tolerance experiment showed that YN-011 significantly reduced blood glucose levels (P<0.01) and improved insulin sensitivity (P<0.01) in DIO mice after 4 weeks of administration.

[0291] In conclusion, YN-011 effectively reduced body weight and ameliorated obesity-related metabolic disorders, including hyperglycemia, hyperlipidemia, and fatty liver, in obese mice. The beneficial metabolic effects of YN-011 were associated with the suppression of food intake and the browning and remodeling of WAT.

[0292] 6.2 Therapeutic effect of YN-011 on obese rhesus monkeys The obese rhesus monkeys used in this study were procured from Sichuan Primate Biotechnology Co., Ltd. Fifteen male obese rhesus monkeys were selected. They were aged 8–21 years (equivalent to 30–60 years in humans), weighed 9.25–15.70 kg, had fasting plasma glucose (FPG) levels of 5.50–8.58 mmol / L, and had HbA1c levels of 4.5–5.0%. These monkeys were within one year of onset of the disease and had not been receiving any medications. Liver and kidney function was normal. The animals were stratified based on FPG levels and randomly assigned to different groups. Selected animals underwent an intravenous glucose tolerance test (IVGTT) to measure blood glucose and insulin levels before administration of the test compound. The experimental groups included a placebo group and two YN-011 treatment groups (YN-011 50 μg / kg and YN-011 25 μg / kg), each with five animals. Treatment was administered by subcutaneous injection (SC) once weekly for 4 consecutive weeks on days 0 (D0), 7 (D7), 14 (D14), and 21 (D21).

[0293] The effects of YN-011 on body weight (BW) in obese rhesus monkeys are shown in Table 7 and Figure 8. In the placebo group, no significant changes in BW were observed throughout the study period, demonstrating the stability of the model. In the YN-011 25 μg / kg group, BW consistently decreased from D7 to D28 (2.60%-6.71%, P<0.05 or P<0.01) compared to baseline, with a significant decrease of 6.17% at D28. Compared to the placebo group, BW in the YN-011 25 μg / kg group significantly decreased on D14 and D21 (P<0.05). In the YN-011 50 μg / kg group, BW consistently decreased from D7 to D28 (4.06%-7.32%, P<0.05) compared to baseline, with a significant decrease of 7.32% at D28 (P<0.05). Compared with the placebo group, BW in the YN-011 50μg / kg group significantly or highly significantly decreased from D7 to D28 (P<0.05 or P<0.01).

[0294] [Table 16] Note: "a" indicates pre-dose measurements. # indicates P<0.05 compared to baseline, ## indicates P<0.01 compared to baseline. SC indicates subcutaneous injection.

[0295] Example 7: Preventive and therapeutic effects of YN-011 in a rhesus monkey nonalcoholic steatohepatitis (NASH) model 7.1 Experimental method 7.1.1 GE Ultrasound-Guided Liver Biopsy Number of animals: 15 Number of specimens collected: no more than two needles, each specimen no longer than 1.5cm, total specimen length no more than 3cm.

[0296] Approximately 2 cm of the harvested tissue was immediately placed in 4% buffered formalin at room temperature and fixed for at least 24 hours. After appropriate modifications, the tissue was dehydrated, embedded, sectioned, HE stained, and Masson stained.

[0297] Equipment: A GE Vivid S5 ultrasound system was used for ultrasound, a LEICA RM2135 microtome for sectioning, an OLYMPUS BX43 microscope for slide examination, and an OLYMPUS DP22-CU camera for photomicrography.

[0298] Tissue sections were stained with H&E and Masson stains. Pathology slides were evaluated by a pathologist according to the diagnostic and treatment guidelines for nonalcoholic fatty liver disease (NAFLD) jointly developed by the American Association for the Study of Liver Diseases (AASLD), the American College of Gastroenterology (ACG), and the American Gastroenterological Association (AGA). The diagnostic criteria are listed in Tables 2 and 3.

[0299] 7.1.2 Tissue processing, embedding, and sectioning After appropriate fixation, liver biopsy specimens were dehydrated, infiltrated with paraffin, embedded, and directly sectioned (5 μm).

[0300] 7.1.3 H&E staining The sections were deparaffinized as per usual. They were immersed in 100% ethanol I, 100% ethanol II, 95%, 85%, and 75% ethanol for 3 minutes each. They were then rinsed in tap water for 3 minutes, stained in hematoxylin-eosin (HE) solution for 12 minutes, rinsed in tap water for 5 minutes, and stained in aqueous eosin Y solution for 3 minutes. Dehydration was performed quickly in 95% ethanol, followed by two cycles of absolute ethanol for 2–5 minutes each. The sections were then washed twice in xylene for 10 minutes each. A neutral mounting medium was applied and examined microscopically.

[0301] 7.1.4 Masson staining The sections were deparaffinized in the usual manner. They were stained with Weigert's iron hematoxylin for 5–7 minutes. At the same time, a weak acid working solution was prepared by mixing distilled water and weak acid solution in a 2:1 ratio. The sections were washed in the weak acid working solution for 1 minute. After washing in phosphomolybdic acid solution for 1–2 minutes, the sections were immersed directly in aniline blue staining solution for 1–2 minutes and then washed in the prepared weak acid working solution for 1 minute. The sections were quickly dehydrated in 95% ethanol, followed by two cycles of 2–5 minutes each in absolute ethanol. The sections were then washed twice in xylene for 10 minutes each. A neutral mounting medium was applied, and microscopic examination was performed.

[0302] 7.1.5 Histopathological examination of tissues Sections were examined under a microscope (using a digital imaging system), and pathological diagnoses and photographs were taken. Based on the histopathological diagnosis, HE and Masson results were scored according to the Nonalcoholic Fatty Liver Disease Activity Score (NAS, Table 8) and liver fibrosis stage scoring criteria (Table 9).

[0303] [Table 17] Note: NAS score = steatosis score + interlobular inflammation score + balloon degeneration score

[0304] [Table 18]

[0305] 7.1.6 Quantitative analysis of fatty liver using GE 3.0T MRI In this study, we used a GE 3.0T MRI scanner (750W3T MRI, GE Healthcare) for liver imaging. We used the IDEAL-IQ sequence, which combines IDEAL (iterative decomposition of water and fat by echo asymmetry and least-squares estimation) technology with fast 3D multi-echo imaging. This technology includes multi-echo water-fat separation, a region-growing algorithm, and tissue-fat quantification and R2 * Various imaging methods of relaxivity are included: in-phase, anti-phase, water only, fat only, fat fraction map, and R2 * Six contrast images, including T2 contrast maps, were acquired in a single breath-hold scan by collecting signals from six different echo time (TE) values. The images were then reconstructed using a computer algorithm to obtain the aforementioned contrast images. The IDEAL-IQ hybrid water-fat separation algorithm consists of two steps. The first step reconstructs the complex domain to separate the water and fat images, and the T2 contrast maps. * In the second step, we generate an additional set of estimated water and fat images. These two sets of images are then combined using a hybrid algorithm to generate the final water and fat images.

[0306] Prior to the experiment, a phantom test was performed. Five standard fat solutions with known fat contents were prepared using a modified body phantom preparation method used by Clare P. et al.

[11] . Pure water was used as 0% fat. Sodium dodecyl sulfate (60 mmol) was dissolved in 1 L of deionized water and heated to 50°C. 40 g of gelatin was added and mixed thoroughly. Soybean oil (96, 108, 114, 117, and 120 ml, obtained from China National Pharmaceutical Group Chemical Reagents Co., Ltd.) was mixed with 0, 24, 12, 6, and 3 ml of the above solution, respectively, to prepare solutions with fat contents of 0%, 2.5%, 5%, 10%, 20%, and 100%. These solutions were then filled into six 100 ml EP tubes for the subsequent scanning procedure.

[0307] A phantom validation experiment was performed before each scan. All animals underwent two scans: a baseline scan and one additional scan after drug administration was completed. Scans were performed in a 3.0T MRI room, and the scan parameters are shown in Table 10. The anesthesia procedure consisted of an intramuscular injection of 10 mg / kg ketamine hydrochloride, endotracheal intubation, and maintenance of anesthesia with isoflurane and oxygen via a ventilator. During the scanning process, veterinarians monitored the electrocardiogram, blood oxygenation, and respiratory rate until the animals fully regained consciousness and resumed spontaneous breathing.

[0308] [Table 19]

[0309] Image Postprocessing and Analysis: The collected and stored rhesus monkey fatty liver MRI images were analyzed using a 750W 3T MRI workstation. Liver MRI images were selected by selecting the maximally exposed surface of the right hepatic lobe (layers 1-3), avoiding large vessels, bile ducts, and the gallbladder to prevent volume effects. Regions of interest (ROIs) were selected, with each ROI measuring 90-110 mm2. Fatty liver was defined as an MRI-PDFF% (magnetic resonance imaging proton density fat fraction) >6% (in accordance with clinical criteria).

[0310] 7.2 Experimental results In vivo pharmacology studies of YN-011 were conducted in NASH rhesus monkeys. Clinical parameters evaluated included liver lipid content assessed by MRI-PDFF%, NAFLD activity score (NAS), liver fibrosis stage assessed by liver histology, body weight, body mass index (BMI), blood lipid profile, fructose metabolism, other biochemical parameters, and food intake.

[0311] The selected rhesus monkeys were aged 11–23 years, equivalent to a human age of 30–70 years. All monkeys were male and weighed 13.63–22.85 kg. They had abnormal lipid metabolism for more than two years, MRI-PDFF% of 7.8%–11.9%, NAS score of 3 or higher, and fibrosis score of 0–1c within 6 months. These animals met the clinical definition of NASH.

[0312] Fifteen rhesus monkeys were divided into three groups, five in each group. These monkeys received weekly subcutaneous injections of YN-011 at 0 (vehicle control, placebo control group), 0.050, or 0.150 mg / kg for 13 weeks (QW). The 0.150 mg / kg YN-011 group received adaptive dosing (0.1 mg / kg for the first dose, followed by 12 doses at 0.150 mg / kg).

[0313] All rhesus monkeys received their assigned treatment during the study, and no monkeys missed scheduled biopsies or discontinued treatment. No serious side effects were observed in any treatment group during the study.

[0314] As shown in Table ​ Table11, 13 weeks of once-weekly YN-011 treatment reduced liver lipid content by approximately 40% compared with the placebo control group.

[0315] To investigate whether YN-011 treatment resulted in histological improvement of the liver, liver biopsy specimens were collected from all rhesus monkeys before the first injection and 89 days after the first injection. Liver sections were stained with HE and Masson staining and subjected to histological analysis. The degree of steatosis, inflammation, hepatocyte swelling, and fibrosis was scored according to the criteria for NAFLD activity and NASH progression. Liver biopsy results showed that both NAS and liver fibrosis scores were reduced in rhesus monkeys treated with YN-011, and no significant progression of NASH was observed (Table 12).

[0316] Liver fat, as detected by MRI-PDFF, decreased from 9.0% ± 0.9% to 5.0% ± 0.2% in the 50 μg / kg YN-011 group and from 9.4% ± 1.5% to 5.6% ± 1.5% in the 150 μg / kg YN-011 group. Compared with baseline, the reduction rates at the end of treatment were 43.8% and 39.7%, respectively. There was no significant difference in the change in MRI-PDFF between the 50 μg / kg and 150 μg / kg YN-011 groups.

[0317] Histological analysis showed that in the 50 μg / kg YN-011 group, the mean NAS decreased from 3.6 ± 0.5 at baseline to 1.6 ± 0.5 (P = 0.003). In the 150 μg / kg YN-011 group, the mean NAS decreased from 3.6 ± 0.5 at baseline to 1.4 ± 0.5 (P < 0.001). There was no significant difference in NAS between the 50 μg / kg and 150 μg / kg YN-011 groups.

[0318] Regarding metabolic measures such as liver damage biomarkers, lipid profile, and body weight, YN-011 treatment showed significant improvements in both the 50μg / kg and 150μg / kg groups.

[0319] [Table 20] NOTE: ROI: Region of Interest; **Compared with baseline, P<0.01; ##Compared with placebo group, P<0.01; Percent change = (current value - baseline value) / baseline value × 100%.

[0320] [Table 21]

[0321] Furthermore, compared with baseline, the weight and BMI of NASH rhesus monkeys were significantly reduced in the YN-011-treated group (days 7-84). YN-011 treatment also significantly reduced serum low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC), and total triglycerides (TG) at various time points during the treatment period, with 0.15 mg / kg YN-011 demonstrating a more consistent lipid-lowering effect. High-density lipoprotein cholesterol (HDL-c) levels in the YN-011-treated group showed a trend toward a decrease compared with baseline. These changes in serum lipid profile were attributed to a decrease in food intake in animals treated with YN-011. Throughout the study, plasma fasting plasma glucose (FPG) levels fluctuated within the normal range, and no significant differences were detected between the YN-011 and placebo groups after administration of YN-011 or placebo. Plasma fructosamine (FRA), a validated biomarker reflecting average glycemic control over the past 2–3 weeks, showed a decreasing trend in the YN-011 group 1 week after the first injection and a significant decrease of 4.3% after 12 weeks of treatment with 150 μg / kg YN-011. These results demonstrate that YN-011 is effective in controlling blood glucose in NASH rhesus monkeys. The dose-dependent decrease in food intake observed in YN-011-treated animals is consistent with the normal pharmacological effect of YN-011. In conclusion, these data demonstrate that multiple subcutaneous injections of YN-011 have a significant therapeutic effect in NASH rhesus monkeys.

[0322] Example 8: In vitro experiments on neurodegenerative diseases 8.1 TNF-α concentration-dependent decrease in survival rate of SH-SY5Y neuronal cells SH-SY5Y neuronal cells were digested with 0.25% trypsin to prepare a single-cell suspension. The single-cell suspension was seeded into a 96-well plate at a density of 10,000 cells per well in 100 μl of medium. The plate was placed in a CO2 incubator (37°C, 5% CO2) overnight to allow cells to adhere. The medium was replaced with medium containing 10% or 5% FBS, and the cells were stimulated with 20, 40, 60, 80, or 100 ng / ml TNF-α for 48 hours. Each drug treatment was performed in six replicate wells. 48 hours after drug treatment, 10 μl of CCK-8 solution was added to each well, and the plate was gently shaken to mix the reagents. The plate was then incubated in a CO2 incubator for 1–4 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = [A(treated) - A(blank)] / [A(control) - A(blank)] x 100 A (treated): OD values of wells containing cells, CCK-8 solution, and drug solution A (Control): OD value of wells containing CCK-8 solution without cells or drug solution A (blank): OD value of wells without cells

[0323] The experimental results (shown in Figure 9 ) indicate that increasing concentrations of TNF-α significantly reduced the viability of SH-SY5Y neurons.

[0324] 8.2 Inhibitory effects of YN-011 and γ-aminobutyric acid (GABA) on TNF-α-induced decrease in SH-SY5Y cell viability See Section 8.1 for the experimental procedure. However, the drug treatment in this experiment differed from Section 8.1. Specifically, the drug treatment in this experiment was as follows: Using medium containing 5% FBS, cells were treated with 60 ng / ml TNF-α alone, or in combination with 10, 100, or 500 nM YN-011, or in combination with 1, 10, or 100 μM GABA for 48 hours. Each drug treatment was performed in six replicate wells.

[0325] The experimental results (see Figure 10) showed that the viability of SH-SY5Y cells was significantly reduced in the presence of 60 ng / ml TNF-α, but treatment with 10, 100, 500 nM YN-011 or 100 μM GABA significantly improved cell viability.

[0326] 8.3 Significant improvement in survival rate of SH-SY5Y cells by combined use of YN-011 and GABA See Section 8.1 for the experimental procedure. However, the drug treatment in this experiment differed from that in Section 8.1. Specifically, the drug treatment in this experiment was as follows: Using medium containing 5% FBS, cells were treated with 60 ng / ml TNF-α alone or in combination with 100 nM YN-011 and / or 100 μM GABA for 48 hours. Each drug treatment was performed in six replicate wells.

[0327] The experimental results (see Figure 11) showed that the viability of SH-SY5Y neurons was significantly reduced in the presence of 60ng / ml TNF-α, whereas the combined treatment of 100nM YN-011 and 100μM GABA significantly improved cell viability. The combined use of YN-011 and GABA showed a stronger effect on enhancing cell viability than either of them alone.

[0328] 8.4 YN-011 reduces TNF-α-induced apoptosis in SH-SY5Y neuronal cells The experimental procedure is as follows: 1) Digest SH-SY5Y neuronal cells using 0.25% trypsin in a 10 cm culture dish and seed them into a 12-well plate. 2) Place the culture plate in a CO2 incubator and pre-incubate overnight (37°C, 5% CO2) to promote cell attachment. 3) Using medium containing 5% FBS, cells in each well were treated with 60ng / ml TNF-α alone or in combination with 10, 100, or 500nM YN-011. Cells were incubated at 37°C for 48 hours. Each drug treatment was performed in triplicate wells. 4) After 48 hours, discard the supernatant, gently wash the cells once with PBS, and place the cell culture plate on ice. Add 120 μl of RIPA cell lysis buffer (Beyotime P0013B) containing protease and phosphatase inhibitors to each well and lyse the cells for 15 minutes. 5) Transfer the cell lysate to a 1.5 ml EP tube and centrifuge at 14,000 rpm for 30 minutes. 6) Carefully aspirate the supernatant into a new EP tube, add 5x loading buffer containing β-mercaptoethanol, and boil the sample at 100°C for 10 minutes to denature the proteins. 7) Load 10 μg of protein per lane and run SDS-PAGE gel electrophoresis. Place the gel perpendicular to the power rack of the electrophoresis tank, ensuring that the concave side of the gel faces the power rack. Two gels can share one power rack. If necessary, secure the gel and power rack to the electrophoresis tank. Add electrophoresis buffer to create separate chambers for the two gels and the buffer in the electrophoresis tank. Gently remove the comb from the gel. 8) Electrophoresis: Connect the electrophoresis tank with two electrodes to the power supply, ensuring the red and black electrode plugs match the corresponding ports. During electrophoresis, use low-voltage constant-voltage electrophoresis for the upper gel. Turn on the power supply and adjust the voltage to 80V (usually takes about 15 minutes). Once the bromophenol blue dye has entered the lower gel, switch to high-voltage constant-voltage electrophoresis and adjust the voltage to 120V until the bromophenol blue dye reaches the bottom of the gel. 9) Perform wet transfer. The sandwich arrangement for wet transfer is as follows: sponge / filter paper / gel / membrane / filter paper / sponge, firmly positioned. There should be no air bubbles between the gel and the membrane, and the sandwich should be correctly oriented, with the negative electrode facing the protein side of the gel and moving toward the positive electrode (membrane). After SDS-PAGE is complete, gently separate the two glass plates of the gel using a razor blade and place the gel on one of the glass plates. Use the blade to cut the gel at the boundary between the separating gel and stacking gel, and cut a small corner in the upper left corner of the separating gel to mark the sample order. Next, carefully transfer the gel to transfer buffer. Cut a piece of PVDF membrane the same size as the gel and soak it in methanol for 5 seconds. Cut six pieces of filter paper of the same size and equilibrate them in transfer buffer for 15 minutes simultaneously with the PVDF membrane and gel. Place the sponge pad, filter paper, gel, membrane, filter paper, and sponge pad (from bottom to top) on the transfer device, taking care not to trap air bubbles, especially between the membrane and filter paper, the gel and membrane, and the filter paper and gel. Set the transfer current to a constant 200 mA, and the transfer time is approximately 90 minutes. 10) Remove the transferred PVDF membrane and rinse it lightly with TBST. Discard the TBST and add 5% milk blocking solution to cover the PVDF membrane. Incubate at room temperature on a rocking shaker for 1 hour (30 rpm). 11) Incubate with primary antibodies. Remove blocking solution, wash membranes three times with TBST for 5 minutes each, and cut membranes according to molecular weight. Add primary antibodies at appropriate dilutions (Bcl-2, CST#3498S, 1:1000; Cleaved-caspase 3, CST#9661S, 1:1000; Caspase 3, CST#9662S, 1:1000; HSP90, Proteintech#13171-1-AP, 1:10000) and incubate overnight at 4°C on a rocking shaker. 12) Wash the membrane. Remove the PVDF membrane after overnight incubation and store the antibody at -20°C. Place the PVDF membrane in TBST solution and rinse quickly three times for 15 minutes each. 13) Incubate with secondary antibody: Add the corresponding species-specific secondary antibody (Jackson Lab, 1:10,000) prepared in blocking solution to the membrane and incubate at room temperature with gentle shaking for 1 hour. 14) Wash the membrane. Remove the secondary antibody and place the PVDF membrane in TBST solution. Rinse quickly three times for 15 minutes each. 15) Perform ECL chemiluminescence. In the dark, prepare fresh developer solution (solution A:solution B = 1:1) according to the instructions in the ECL chemiluminescence kit (Millipore #WBKLS0500). Add the developer solution evenly to the membrane and place it in the imaging device for image acquisition. 16) Protein expression analysis. The grayscale values of the Western blot bands were analyzed using Image J software and normalized to the grayscale value of the reference protein HSP90 to calculate the relative expression level of the target protein.

[0329] The experimental results (shown in Figure 12 ) indicate that YN-011 significantly reduces TNF-α-induced apoptosis in SH-SY5Y neuronal cells.

[0330] 8.5 GABA attenuates TNF-α-induced apoptosis in SH-SY5Y neuronal cells See Section 8.4 for the experimental procedure. However, the drug treatment in step 3) of this experiment differs from that in Section 8.4. In this experiment, the drug treatment in step 3) is as follows: Using medium containing 5% FBS, treat cells in each well with 60 ng / ml TNF-α alone or in combination with 1, 10, or 100 μM GABA for 48 hours at 37°C. Each drug treatment is performed in three replicate wells.

[0331] The experimental results (shown in Figure 13 ) indicate that GABA attenuates TNF-α-induced apoptosis in SH-SY5Y neuronal cells.

[0332] 8.6 Attenuation of TNF-α-induced apoptosis in SH-SY5Y neuronal cells by combined use of YN-011 and GABA See Section 8.4 for the experimental procedure. However, the drug treatment in step 3) of this experiment differs from that in section 8.4. In this experiment, the drug treatment in step 3) is as follows: Using medium containing 5% FBS, treat cells in each well with 60 ng / ml TNF-α alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours at 37°C. Each drug treatment is performed in triplicate wells.

[0333] The experimental results (shown in Figure 14 ) indicate that the combined use of YN-011 and GABA significantly reduced TNF-α-induced apoptosis in SH-SY5Y neuronal cells.

[0334] 8.7 TNF-α damages SH-SY5Y neuronal cells SH-SY5Y neuronal cells in a 10 cm culture dish were digested with 0.25% trypsin to prepare a single-cell suspension. The single-cell suspension was then seeded into a 24-well plate. The culture plate was placed in a CO2 incubator at 37°C and 5% CO2 overnight to allow cells to adhere. Cells in each well were treated with 20, 40, 60, or 80 ng / ml TNF-α in medium containing 5% FBS at 37°C for 48 hours. Each drug treatment was performed in triplicate wells. The fluorescent dye Hoechst 33342 (Beyotime) was added to the wells at a dilution of 1:1000, and the plate was incubated at 37°C for 10 minutes. The culture medium was removed, the plate was gently washed twice with PBS, and the plate was immediately imaged under a fluorescence microscope (20x magnification). Ten fields of view were photographed for each cell well, and the number of blue-stained positive cells in each field was counted.

[0335] The experimental results (shown in Figure 15 ) indicate that TNF-α promotes damage to SH-SY5Y neuronal cells.

[0336] 8.8 Protective effects of YN-011 against TNF-α-induced injury in neuronal cells See Section 8.7 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.7. The drug treatment in this experiment is as follows: Using medium containing 5% FBS, cells in each well are treated with 60 ng / ml TNF-α alone or in combination with 10, 100, or 500 nM YN-011 for 48 hours at 37°C. Each drug treatment is performed in triplicate wells.

[0337] The experimental results (shown in Figure 16) indicate that YN-011 has a protective effect against TNF-α-induced damage in neuronal cells.

[0338] 8.9 Protective effects of GABA against TNF-α-induced damage in neuronal cells See Section 8.7 for experimental procedures. However, the drug treatment in this experiment will differ from that in Section 8.7. In this experiment, the drug treatment will be as follows: Using medium containing 5% FBS, cells in each well will be treated with 60 ng / ml TNF-α alone or in combination with 1, 10, or 100 μM GABA for 48 hours at 37°C. Each drug treatment will be performed in three replicate wells.

[0339] The experimental results (shown in Figure 17) indicate that GABA has a protective effect against TNF-α-induced damage in neuronal cells.

[0340] 8.10 Protective effect of combined use of YN-011 and GABA against TNF-α-induced damage in neurons See Section 8.7 for the experimental procedure. However, the drug treatment in this experiment will differ from that in Section 8.7. In this experiment, the drug treatment will be as follows: Using medium containing 5% FBS, cells in each well will be treated with 60 ng / ml TNF-α alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours at 37°C. Each drug treatment will be performed in triplicate wells.

[0341] The experimental results (shown in Figure 18) indicate that the combination of YN-011 and GABA has a protective effect against TNF-α-induced damage in neuronal cells.

[0342] 8.11 YN-011 reduces TNF-α-induced neuronal apoptosis The experimental procedure is as follows: 1) Digest SH-SY5Y neuronal cells in a 10 cm culture dish with 0.25% trypsin to prepare a single cell suspension. Seed the single cell suspension into a 24-well plate and place a sterile cover glass in each well. 2) Place the culture plate in a CO2 incubator and pre-incubate overnight at 37°C with 5% CO2 to allow cells to adhere. 3) Using medium containing 5% FBS, treat cells in each well with 60ng / ml TNF-α alone or in combination with 10, 100, or 500nM YN-011 for 48 hours at 37℃. Each drug treatment will be performed in triplicate wells. 4) After 48 hours, remove the culture medium and add 400 μl of 4% paraformaldehyde (PFA) to each well to fix the cells for 15 minutes at room temperature. 5) Wash twice with PBS for 5 minutes each. 6) To increase membrane permeability, add 0.1% Triton X-100 and wash for 10 minutes at room temperature. 7) Wash twice with PBS for 5 minutes each. 8) Add 10% goat serum (GS) and incubate at room temperature for 30 minutes to block. 9) Remove the 10% GS and add the primary antibody Cleaved-caspase 3 (CST 9661S) diluted 1:400 in 1% GS at 30 μl antibody solution per coverslip. 10) Place in the refrigerator at 4°C overnight. 11) The next day, remove the primary antibody and wash three times with PBS for 5 minutes each. 12) Add fluorescent secondary antibody (Alexa Fluor® 488 Conjugate) and incubate in the dark for 1 hour at room temperature. 13) Wash three times with PBS for 5 minutes each. 14) Stain with DAPI for 5 minutes and wash three times with PBS for 5 minutes each. 15) Apply mounting medium to a clean glass slide, gently place the cover glass with cells upside down on the mounting medium, and allow to dry overnight at room temperature in a cool, ventilated area. 16) Take images under a fluorescence microscope (40x magnification). 17) Photograph 10 fields for each treatment group and count the number of green-stained positive cells in each field.

[0343] The experimental results (shown in Figure 19) indicate that YN-011 significantly reduces TNF-α-induced neuronal apoptosis.

[0344] 8.12 GABA reduces TNF-α-induced neuronal apoptosis See Section 8.11 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.11. In this experiment, the drug treatment in step 3) is as follows: Using medium containing 5% FBS, treat cells in each well with 60 ng / ml TNF-α alone or in combination with 1, 10, or 100 μM GABA for 48 hours at 37°C. Each drug treatment is performed in three replicate wells.

[0345] The experimental results (shown in Figure 20) indicate that GABA reduces TNF-α-induced neuronal apoptosis.

[0346] 8.13 Combined use of YN-011 and GABA reduces TNF-α-induced neuronal apoptosis See Section 8.11 for the experimental procedure. However, the drug treatment in this experiment will be different from that in Section 8.11. In this experiment, the drug treatment in step 3) will be as follows: Using medium containing 5% FBS, treat cells in each well with 60 ng / ml TNF-α alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours at 37°C. Each drug treatment will be performed in triplicate wells.

[0347] The experimental results (shown in Figure 21) indicate that the combined use of YN-011 and GABA significantly reduced TNF-α-induced neuronal apoptosis.

[0348] 8.14 YN-011 reduces TNF-α-induced neuronal apoptosis SH-SY5Y neuronal cells in a 10 cm culture dish were digested with 0.25% trypsin to prepare a single-cell suspension. The single-cell suspension was seeded into a 12-well plate. The culture plate was placed in a CO2 incubator at 37°C and 5% CO2 overnight to allow cells to adhere. Using medium containing 5% FBS, cells in each well were treated with 60 ng / ml TNF-α alone or in combination with 10, 100, or 500 nM YN-011 for 48 hours at 37°C. Each drug treatment was performed in triplicate wells. The cells were collected and digested with 0.25% trypsin without EDTA for 1 minute. The cell suspension was transferred to a 1.5 ml EP tube and centrifuged at 1000 rpm for 5 minutes to settle the cells to the bottom of the tube. Resuspend 10 cells in 100 μl of binding buffer, add 5 μl of PI and 5 μl of Annexin V-FITC solution, and incubate for 15 minutes at room temperature in the dark. Add 400 μl of binding buffer to each tube and detect using a flow cytometer (Annexin V-FITC excitation wavelength: 488 nm, emission wavelength: 520 nm, PI excitation wavelength: 535 nm, emission wavelength: 617 nm). Count the number of Annexin V-FITC-positive and PI-negative cells in each sample.

[0349] The experimental results (shown in Figure 22) indicate that YN-011 significantly reduces TNF-α-induced neuronal apoptosis.

[0350] 8.15 GABA reduces TNF-α-induced neuronal apoptosis See Section 8.14 for experimental procedures. However, the drug treatments in this experiment will differ from those in Section 8.14. In this experiment, the drug treatments will be as follows: Using medium containing 5% FBS, cells in each well will be treated with 60 ng / ml TNF-α alone or in combination with 1, 10, or 100 μM GABA for 48 hours at 37°C. Each drug treatment will be performed in three replicate wells.

[0351] The experimental results (shown in Figure 23) indicate that GABA reduces TNF-α-induced neuronal apoptosis.

[0352] 8.16 Combined use of YN-011 and GABA reduces TNF-α-induced neuronal apoptosis See Section 8.14 for the experimental procedure. However, the drug treatment in this experiment differs from that in Section 8.14. The drug treatment in this experiment is as follows: Using medium containing 5% FBS, cells in each well are treated with 60 ng / ml TNF-α alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours at 37°C. Each drug treatment is performed in triplicate wells.

[0353] The experimental results (shown in Figure 24) indicate that the combined use of YN-011 and GABA significantly reduced TNF-α-induced neuronal apoptosis.

[0354] 8.17 GABA reduces the mRNA expression of inflammatory factors in HMC3 microglial cells induced by Aβ1-42 oligomers. Under sterile conditions, dissolve the synthesized Aβ1-42 peptide (AnaSpec #AS-20276) in hexafluoroisopropanol (Mecklin #H811026) to obtain a 1 mM solution. Divide the solution into 1.5 ml sterile centrifuge tubes and evaporate the hexafluoroisopropanol using a vacuum evaporator. The dried peptide forms a film in the tube and is stored at -20 °C for later use. Freshly prepare Aβ1-42 oligomers from the dried peptide film as follows: Dissolve the dried peptide film in anhydrous DMSO to obtain a 5 mM solution, then dilute it in cold basal cell culture medium to obtain a 100 µM stock solution. After incubating at 4 °C for 24 h, use the stock solution for cell treatment.

[0355] HMC3 human microglial cells were seeded in 24-well plates. After overnight incubation at 37°C and 5% CO2, cells in each well were treated with 10 μM Aβ1-42 oligomers alone or in combination with 1, 10, or 100 μM GABA for 48 hours. Each drug treatment was performed in triplicate wells. After 48 hours of treatment, total RNA was isolated from each well using 1 ml of Trizol (Invitrogen #15596026) according to the manufacturer's instructions. The RNA pellet was dissolved in 20 μl of DEPC-treated water. RNA concentration and purity were measured using a NanoDrop 2000. RNA was converted to cDNA using a reverse transcription kit (Yeasen #11141ES60). Prepare the PCR reaction 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. Run qPCR on an Applied Biosystems 7500 Real-Time PCR System using the following thermal program: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. Use the ΔΔCt method to compare the relative mRNA expression of TNF-α and IL-6 between treated and untreated cells, normalized to the expression of the housekeeping gene RPLP0. The experimental results (shown in Figure 25) indicate that in the presence of 10 μM Aβ1-42 oligomers, the mRNA expression of TNF-α and IL-6 in HMC3 cells significantly increased, whereas 100 μM GABA significantly decreased the mRNA expression of TNF-α, and both 10 μM and 100 μM GABA significantly decreased the mRNA expression of IL-6.

[0356] 8.18 YN-011 reduces the mRNA expression of inflammatory factors in HMC3 microglial cells induced by Aβ1-42 oligomers.

[0357] See Section 8.17 for the experimental procedure. However, the drug treatment in this experiment will differ from that in Section 8.17. In this experiment, the drug treatments will be as follows: Cells in each well will be treated with 10 μM Aβ1-42 oligomers alone or in combination with 10, 100, or 500 nM YN-011 for 48 hours. Each drug treatment will be performed in triplicate wells.

[0358] The experimental results (see Figure 26) show that the presence of 10 μM Aβ1-42 oligomers significantly increased the mRNA expression of TNF-α and IL-6 in HMC3 cells, whereas 100 nM and 500 nM YN-011 significantly decreased the mRNA expression of TNF-α and IL-6.

[0359] 8.19 The combination of YN-011 and GABA reduces the mRNA expression of inflammatory factors in HMC3 microglial cells induced by Aβ1-42 oligomers.

[0360] See Section 8.17 for the experimental procedure. However, the drug treatment in this experiment will differ from that in Section 8.17. In this experiment, the drug treatments will be as follows: Cells in each well will be treated with 10 μM Aβ1-42 oligomers alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours. Each drug treatment will be performed in triplicate wells.

[0361] The experimental results (shown in Figure 27) indicate that, compared with the individual treatment of GABA or YN-011, the combination of 100 μM GABA and 100 nM YN-011 exhibited a significantly stronger inhibitory effect on TNF-α mRNA expression induced by 10 μM Aβ1-42 oligomers.

[0362] 8.20 GABA reduces Aβ1-42 oligomer-induced expression of inflammatory factors in HMC3 microglial cells.

[0363] HMC3 human microglial cells were seeded in 12-well plates. After overnight incubation at 37°C and 5% CO2, cells in each well were treated with 10 μM Aβ1-42 oligomers alone or in combination with 1, 10, or 100 μM GABA for 48 hours. Each drug treatment was performed in triplicate. After 48 hours of treatment, cells were washed with PBS and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors for 15 minutes on ice. 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 120V. The gel was then transferred to a PVDF membrane (0.22 μm pore size) at 200 mA for 90 minutes. After unblocking, the membrane was incubated overnight at 4 °C with primary antibodies (TNF-α, Proteintech #60291-Ig; IL-6, Proteintech #21865-1-AP; HSP90, Proteintech #13171-1-AP) diluted in 5% milk solution. After washing, the membrane was incubated with the corresponding secondary antibody (Jackson Lab, 1:10,000) for 1 hour at room temperature. After washing, the antibody signals on the membrane were visualized using an ECL kit (Millipore #WBKLS0500) according to the manufacturer's instructions. ImageJ was used to calculate relative protein levels normalized to the endogenous protein HSP90.

[0364] The results (shown in Figure 28) show that in the presence of 10 μM Aβ1-42 oligomers, TNF-α and IL-6 expression in HMC3 cells was significantly increased, IL-6 expression was significantly decreased at 100 μM GABA, and TNF-α expression was significantly decreased at both 10 μM and 100 μM GABA.

[0365] 8.21 YN-011 reduces the expression of inflammatory factors in HMC3 microglial cells induced by Aβ1-42 oligomers.

[0366] See Section 8.20 for the experimental procedure. However, the drug treatments in this experiment differ from those in Section 8.20. The drug treatments in this experiment are as follows: Cells in each well are treated with 10 μM Aβ1-42 oligomers alone or in combination with 10, 100, or 500 nM YN-011 for 48 hours. Each drug treatment is performed in triplicate wells.

[0367] The experimental results (see Figure 29) show that the expression of TNF-α and IL-6 in HMC3 cells is significantly increased in the presence of 10 μM Aβ1-42 oligomers, whereas 100 nM and 500 nM YN-011 significantly decrease the expression of TNF-α and IL-6.

[0368] 8.22 The combination of YN-011 and GABA reduces the expression of inflammatory factors in HMC3 microglial cells induced by Aβ1-42 oligomers.

[0369] See Section 8.20 for the experimental procedure. However, the drug treatments in this experiment will differ from those in Section 8.20. In this experiment, the drug treatments will be as follows: Cells in each well will be treated with 10 μM Aβ1-42 oligomers alone or in combination with 100 μM GABA and / or 100 nM YN-011 for 48 hours. Each drug treatment will be performed in triplicate wells.

[0370] The experimental results (shown in Figure 30) indicate that, compared with the individual treatment of GABA or YN-011, the combination of 100 μM GABA and 100 nM YN-011 exhibits a significantly stronger inhibitory effect on the expression of TNF-α and IL-6 induced by 10 μM Aβ1-42 oligomers.

[0371] Example 9: Neurodegenerative diseases in vivo Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are central nervous system disorders characterized by the progressive loss of function of central neurons or their myelin sheaths, resulting in the gradual loss of neuronal structure and function, leading to symptoms such as dementia and movement disorders.

[0372] Among these, Alzheimer's disease (AD), also known as senile dementia, is characterized by progressive cognitive impairment and memory loss, and there is currently no effective treatment. Research has shown that the brains of AD patients accumulate neurotoxic plaques formed by the abnormal aggregation of beta-amyloid (Aβ). Aβ can cause the formation of senile plaques in the brain and neuronal apoptosis, making it an important factor in the development of AD. Aβ is a peptide consisting of 39–43 amino acids, and the most common Aβ subtypes in the human body are Aβ40 and Aβ42, which are prone to aggregation, leading to the formation of Aβ deposits and subsequent neurotoxic effects.

[0373] Studies have shown that GLP-1 has neuroprotective biological effects and can improve AD symptoms [10, 25, 26]. In animal models, GLP-1 improved cognitive function in AD mice, reduced Aβ deposition, attenuated Aβ-induced glial cell overactivation, and alleviated oxidative stress and inflammation in the brain, demonstrating the neuroprotective role of GLP-1 in AD. The present invention discloses a long-acting GLP-1 receptor agonist (GLP-1RA) that exerts biological effects to protect neurons and improve central nervous system function, thereby helping to improve AD symptoms.

[0374] The experiments involve Alzheimer's disease model mice and wild-type control adult mice of the same genetic background. The animals are housed individually in cages, maintained on a 12 / 12-hour light / dark cycle (lights on at 08:00, lights off at 20:00) and kept at 21.5°C. Food and water are available ad libitum. Prior to behavioral experiments, the mice are intraperitoneally (ip) injected with YN-011 or a placebo for 16 weeks.

[0375] Morris water maze setup The maze was made of white opaque plastic with a diameter of 120 cm and wall height of 40 cm. It was filled with water at 25°C to prevent excessive cooling of the body temperature. A small escape platform (10 × 6.5 × 21.5 cm) was placed in a fixed position within the quadrant, 25 cm away from the surrounding walls and hidden 1 cm below the water surface. Several fixed visual cues were present on the walls of the room.

[0376] spatial memory Four equidistant points (north, south, east, and west) along the circumference of the pool served as starting positions. Starting positions were randomly assigned across four trials per day. During training, mice were trained to find a fixed safety platform submerged in the pool. 24 hours after the end of the training period, the safety platform was removed from the pool, and the length and duration of each mouse's swim path were recorded, starting from the random starting position (n = 12 per group). The collected data were analyzed using one-way and two-way ANOVAs to assess spatial memory, represented by the time and path length required to reach the original location of the safety platform, and spatial cognition, represented by the time spent searching the area where the safety platform was located.

[0377] Detection of Aβ40 and Aβ42 levels Aβ40 and Aβ42 levels are measured using an Aβ detection kit. Briefly, brain hemispheres from YN-011-treated AD model mice and control mice are homogenized in Tris-buffered saline (25 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing proteinase inhibitors (Sigma, 250 ml / 5 ml buffer). The brain homogenates are then centrifuged at 100,000 g for 1 hour at 4°C. The supernatants are then diluted 1:10 and subjected to ELISA. ELISA measures only soluble beta-amyloid oligomer levels, not monomers. Protein quantification is performed using a Bradford protein assay. Final Aβ values are determined after normalization to total protein levels (n = 6 / group).

[0378] Example 10: Phase IIb and III clinical trials of YN-011 (monotherapy) 10.1 Test Design The Phase IIb and Phase III clinical trials are multicenter, randomized, double-blind, placebo-controlled studies to evaluate the efficacy and safety of YN-011 in patients with type 2 diabetes mellitus (T2DM) inadequately controlled despite diet and exercise therapy. Phase IIb is an exploratory clinical trial to investigate dose-response and safety (divided into four groups: YN-011 1 mg group, YN-011 2 mg group, YN-011 3 mg group, and placebo control group) and to obtain the recommended phase 3 dose (RP3D) of YN-011 for the Phase III phase. Phase III is a confirmatory clinical trial.

[0379] Key inclusion criteria for Phase IIb and III studies: Ages 18 to 75 at the time of screening -Diagnosed type 2 diabetes (according to WHO 2000 criteria) without antihyperglycemic drug treatment in the 8 weeks prior to screening Glycated hemoglobin HbA1c at screening: 7.5%≦HbA1c≦11% Glycated hemoglobin HbA1c before randomization: 7.5%≦HbA1c≦10.5% Fasting plasma glucose (FPG) <13.9mmol / L at screening and before randomization BMI ≥ 18.5 kg / m² and ≤ 40 kg / m²

[0380] Key exclusion criteria for Phase IIb and III studies: ·Type 1 diabetes Use of any medications: taking DPP-4 inhibitors and / or GLP-1 analogues in the 3 months prior to screening -Continued insulin treatment for 14 days or more in the year prior to screening (excluding gestational diabetes requiring insulin treatment) Fasting C-peptide <0.3nmol / mL -Having had diabetic ketoacidosis, diabetic lactic acidosis, or hyperosmolar nonketotic diabetic coma within 6 months prior to screening Have had or require treatment for proliferative retinopathy or maculopathy, severe diabetic neuropathy, intermittent claudication, or diabetic foot disease in the 6 months prior to screening Severe hypoglycemic events without a clear cause (grade 3 hypoglycemia) in the 6 months prior to screening, or 3 or more hypoglycemic events (blood glucose <3.9 mmol / L) in the month prior to screening, or recurrent hypoglycemic symptoms · Have had a serious injury, serious infection, or surgery within the month prior to screening · Have a medical condition prior to screening that may affect blood sugar control Donated blood or received significant blood loss (>400 mL) or transfusion within the past 3 months Uncontrolled high blood pressure Patients with a history of acute or chronic pancreatitis, symptomatic gallbladder disease, pancreatic injury, or other high-risk factors for pancreatitis, or patients with amylase and / or lipase levels ≥ 1.5 times the upper limit of normal (ULN) at screening History of medullary thyroid cancer, multiple endocrine neoplasia (MEN) 2A or 2B syndrome, or a related family history or personal history of other malignancies Clinically significant abnormal gastric emptying, severe chronic gastrointestinal disease, long-term use of medications that directly affect gastrointestinal motility, or gastrointestinal surgery within 6 months prior to screening (as determined by the investigator) Blood disorders or any condition that causes hemolysis or red blood cell instability Uncontrolled hyperthyroidism or hypothyroidism Hepatitis B surface antigen (HBsAg) positive, with hepatitis B viral load (HBV-DNA) above the local laboratory detection limit, hepatitis C antibody (HCV-Ab), human immunodeficiency virus antibody (HIV-Ab), Treponema pallidum antibody (TP-Ab) positive, or novel coronavirus pneumonia (COVID-19) nucleic acid test positive Acute or chronic hepatitis, or laboratory findings meeting any of the following criteria: alanine aminotransferase (ALT) level ≥ 2.5xULN and / or aspartate aminotransferase (AST) ≥ 2.5xULN, fasting triglycerides > 5.7mmol / L, estimated glomerular filtration rate (eGFR) < 60mL / min / 1.73m2 calculated by the CKD-EPI (EPI-(Scr)) formula Any other condition that the investigator or attending physician determines to be inappropriate for participation in the clinical trial.

[0381] The dose-confirming treatment regimen for the Phase IIb stage for enrolled subjects is as follows: Subjects enrolled in the Phase IIb validation phase:

[0382] [Table 22]

[0383] Subjects enrolled in the Phase 3 efficacy phase:

[0384] [Table 23]

[0385] The primary efficacy endpoint was a comparison of YN-011 and placebo in the change from baseline in HbA1c levels after 12 weeks (Phase IIb) or 24 weeks (Phase III) of double-blind treatment in patients with type 2 diabetes. Secondary efficacy endpoints primarily included changes from baseline in FPG, fasting insulin, fasting C-peptide, fasting glucagon, fasting lipid profile, and fasting body weight (at 12 weeks in Phase IIb and 24 weeks in Phase III). Other secondary endpoints included HbA1c goal achievement rates (proportions of subjects achieving HbA1c <7.0% and <6.5%), area under the glucose curve during a mixed meal tolerance test (MMTT), and area under the insulin or C-peptide curve during the MMTT. Safety evaluations included adverse events, clinical laboratory tests, vital signs, and 12-lead electrocardiogram assessments.

[0386] 10.2 Therapeutic Effect of YN-011 on Type 2 Diabetes Clinical trials have shown that 1 mg of YN-011 reduced HbA1c levels by 1.73% after 24 weeks of treatment. 1 mg of semaglutide reduced HbA1c levels by 1.55% after 30 weeks of treatment (Sorli et al., Lancet Diabetes Endocrinol 2017;5:251-60), and 1.5 mg of dulaglutide reduced HbA1c levels by 1.46% after 26 weeks of treatment (Shi et al., J Diabetes Investig 2020;11:142-150).

[0387] Furthermore, clinical trial results showed that the incidence of hypoglycemia (<3.9 mmol / L) after 24 weeks of treatment with YN-011 1 mg and 3 mg was 0.8% and 1.7%, respectively. The incidence of hypoglycemia after 26 weeks of treatment with dulaglutide 0.75 mg and 1.5 mg was reported to be 4.1% and 6.3%, respectively (Shi et al., J Diabetes Investig 2020;11:142-150).

[0388] Furthermore, the incidence of nausea after 24 weeks of treatment with YN-011 1 mg and 3 mg was 3.4% and 6.0%, respectively. The incidence of nausea after 30 weeks of treatment with semaglutide 0.5 mg and 1 mg doses was reported to be 20% and 24%, respectively (Sorli et al., Lancet Diabetes Endocrinol 2017;5:251-60). The incidence of nausea after 26 weeks of treatment with dulaglutide 1.5 mg dose was 9.5% (Shi et al., J Diabetes Investig 2020;11:142-150). For liraglutide, the incidence of nausea after 24 weeks of treatment with liraglutide 1 mg and 3 mg doses was 5.6% and 10%, respectively (Shuai et al., Diabetes Obes Metab. 2021;23(1):116-124).

[0389] Example 11: Phase IIb and Phase III clinical trials of YN-011 in combination with metformin 11.1 Test Design The Phase IIb and Phase III clinical trials were multicenter, randomized, double-blind, placebo-controlled studies to evaluate the efficacy and safety of YN-011 in patients with T2DM whose glycemic control was inadequate with metformin therapy. The Phase IIb study aimed to investigate dose-response, evaluate safety (divided into three groups: YN-011 1 mg + metformin, YN-011 3 mg + metformin, and placebo + metformin), and determine the recommended Phase 3 dose (RP3D) for the Phase III study to confirm efficacy.

[0390] The inclusion criteria for subjects in the Phase IIb and Phase III trials of YN-011 in combination with metformin were essentially the same as those in Implementation Example 10, with the following exceptions: · Have been diagnosed with type 2 diabetes for at least 8 weeks (WHO 2000) and meet any of the following criteria: a) receiving metformin monotherapy for ≥8 weeks at a dose of ≥1500 mg per day or the maximum tolerated dose (<1500 mg per day, ≥1000 mg per day) (eligible subjects could enter a direct run-in period). b) received metformin monotherapy for less than 8 weeks at a dose of ≥1500 mg / day or the maximum tolerated dose (<1500 mg / day but ≥1000 mg / day) (eligible subjects were required to enter a metformin dose stabilization period); c) received metformin monotherapy at a dose of less than 1500 mg / day and had not reached the maximum tolerated dose (eligible subjects were required to enter a metformin dose-titration and dose-stabilization period).

[0391] For key exclusion criteria for subjects in the Phase IIb and Phase III studies of YN-011 in combination with metformin, see Exclusion Criteria in Implementation Example 10.

[0392] The treatment regimen for subjects enrolled in the Phase IIb dose-confirmation phase was as follows:

[0393] Subjects enrolled in the Phase IIb dose-confirmation phase:

[0394] [Table 24]

[0395] Subjects enrolled in the Phase 3 dose-confirmation phase:

[0396] [Table 25]

[0397] The primary efficacy endpoint included a comparison of YN-011 in combination with metformin versus placebo in combination with metformin in terms of change from baseline in HbA1c levels after 12 weeks (Phase IIb) or 24 weeks (Phase III) of double-blind treatment in patients with type 2 diabetes who were inadequately controlled with metformin therapy. For secondary efficacy endpoints and safety evaluations, please see Implementation Example 10.

[0398] 11.2 Therapeutic Effect of YN-011 in Combination with Metformin in Type 2 Diabetes Clinical trials have shown that after 24 weeks of treatment with 3 mg of YN-011 in combination with metformin, HbA1c levels decreased by 1.8% and FPG levels decreased by 2.42%. Administration of 1.5 mg of dulaglutide in combination with metformin has also been reported to reduce HbA1c levels by 1.42% and FPG levels by 1.93% after 40 weeks of treatment (Dungan et al., Lancet 2014;384:1349-57). Furthermore, clinical trials have shown that the incidence of hypoglycemia (<3.9 mmol / L) after 24 weeks of treatment with 3 mg of YN-011 in combination with metformin was 1.8%, comparable to the incidence of hypoglycemia in the placebo group (1.7%). The incidence of hypoglycemia after 40 weeks of dulaglutide 1.5 mg in combination with metformin was reported to be 9% (Dungan et al., Lancet 2014;384:1349-57). Furthermore, the incidence of nausea after 24 weeks of YN-011 3 mg in combination with metformin was reported to be 7.0%. The incidence of nausea after 40 weeks of dulaglutide 1.5 mg in combination with metformin was reported to be 20% (Dungan et al., Lancet 2014;384:1349-57), and the incidence of nausea after 30 weeks of semaglutide 1 mg in combination with metformin was reported to be 13.4% (Ji et al., Diabetes Obes Metab. 2021;23:404-414).

[0399] The above examples represent preferred embodiments disclosed in this application. However, it should be understood that this application is not limited to the disclosed examples. Instead, this application is intended to cover various modifications and equivalents within the spirit and scope of the appended claims.

[0400] All publications, patents, and patent applications are incorporated by reference in their entirety into this disclosure. Specifically, the sequences associated with each accession number provided in this disclosure (including, for example, protein and / or nucleotide sequences provided in tables or elsewhere) are incorporated by reference.

[0401] The scope of the claims should not be limited by the preferred examples and embodiments, but should be understood in the broadest possible interpretation consistent with the specification.

[0402] References 1.Leech, CA, et al., Expression of cAMP-regulated guanine nucleotide exchange factors in pancreatic beta-cells.Biochem Biophys Res Commun, 2000.278(1):p.44-7. 2. Drucker, DJ, Glucagon-like peptides. Diabetes, 1998.47(2):p.159-69. 3. Montrose-Rafizadeh, C., et al., Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells. Endocrinology, 1999.140(3):p.1132-40. 4.Ahren,B.and O.Schmitz,GLP-1 receptor agonists and DPP-4 inhibitors in the treatment of type 2 diabetes.Horm Metab Res,2004.36(11-12):p.867-76. 5.Green,B.D.,et al.,Structurally modified analogues of glucagon-like peptide-1(GLP-1)and glucose-dependent insulinotropic polypeptide(GIP)as future antidiabetic agents.Curr Pharm Des,2004.10(29):p.3651-62. 6.Chang,A.M.,et al.,The GLP-1 derivative NN2211 restores beta-cell sensitivity to glucose in type 2 diabetic patients after a single dose.Diabetes,2003.52(7):p.1786-91. 7.Liu,H.K.,et al.,N-acetyl-GLP-1:a DPP IV-resistant analogue of glucagon-like peptide-1(GLP-1)with improved effects on pancreatic beta-cell-associated gene expression.Cell Biol Int,2004.28(1):p.69-73. 8.Kim,J.G.,et al.,Development and characterization of a glucagon-like peptide 1-albumin conjugate:the ability to activate the glucagon-like peptide 1 receptor in vivo.Diabetes,2003.52(3):p.751-9. 9.Nielsen,R.,et al.,Effect of liraglutide on myocardial glucose uptake and blood flow in stable chronic heart failure patients:A double-blind,randomized,placebo-controlled LIVE sub-study.J Nucl Cardiol,2019.26(2):p.585-597. 10.Duarte,A.I.,et al.,Liraglutide Protects Against Brain Amyloid-beta1-42 Accumulation in Female Mice with Early Alzheimer’s Disease-Like Pathology by Partially Rescuing Oxidative / Nitrosative Stress and Inflammation.Int J Mol Sci,2020.21(5). 11.Elbassuoni,E.A.and R.F.Ahmed,Mechanism of the neuroprotective effect of GLP-1 in a rat model of Parkinson’s with pre-existing diabetes.Neurochem Int,2019.131:p.104583. 12.Muskiet,M.H.A.,et al.,GLP-1 and the kidney:from physiology to pharmacology and outcomes in diabetes.Nat Rev Nephrol,2017.13(10):p.605-628. 13.Hou,Y.,et al.,Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc-Fusion Protein in a CHO Fed-Batch Process.Biotechnol J,2019.14(3):p.e1700706. 14.Karlin,S.and S.F.Altschul,Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes.Proc Natl Acad Sci U S A,1990.87(6):p.2264-8. 15.Karlin,S.and S.F.Altschul,Applications and statistics for multiple high-scoring segments in molecular sequences.Proc Natl Acad Sci U S A,1993.90(12):p.5873-7. 16.Altschul,S.F.,et al.,Basic local alignment search tool.J Mol Biol,1990.215(3):p.403-10. 17.Altschul,S.F.,et al.,Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Res,1997.25(17):p.3389-402. 18.Myers,E.W.and W.Miller,Optimal alignments in linear space.Comput Appl Biosci,1988.4(1):p.11-7. 19.Bettinger,J.Q.,et al.,Quantitative Analysis of in Vivo Methionine Oxidation of the Human Proteome.J Proteome Res,2020.19(2):p.624-633. 20.Allen,L.V.,Jr.,Remington:The Science and Practice of Pharmacy:from the past into the future.Int J Pharm Compd,2012.16(5):p.358-62. 21.Toft-Nielsen,M.B.,S.Madsbad,and J.J.Holst,Continuous subcutaneous infusion of glucagon-like peptide 1 lowers plasma glucose and reduces appetite in type 2 diabetic patients.Diabetes Care,1999.22(7):p.1137-43. 22.Geiser,J.S.,et al.,Clinical Pharmacokinetics of Dulaglutide in Patients with Type 2 Diabetes:Analyses of Data from Clinical Trials.Clin Pharmacokinet,2016.55(5):p.625-34. 23.Bodanszky,M.,Principles of peptide synthesis.2nd rev.ed.Springer laboratory.1993,Berlin ;New York:Springer-Verlag.xii,329 p. 24.Pratley,R.E.,et al.,Semaglutide versus dulaglutide once weekly in patients with type 2 diabetes(SUSTAIN 7):a randomised,open-label,phase 3b trial.Lancet Diabetes Endocrinol,2018.6(4):p.275-286. 25.Zheng,J.,et al.,GLP-1 improves the supportive ability of astrocytes to neurons by promoting aerobic glycolysis in Alzheimer’s disease.Mol Metab,2021.47:p.101180. 26.Park,J.S.,et al.,Blocking microglial activation of reactive astrocytes is neuroprotective in models of Alzheimer’s disease.Acta Neuropathol Commun,2021.9(1):p.78.

Claims

1. A fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently bonded to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from the amino acid sequences shown in SEQ ID NO: 1 Human GLP-1 (7-37), SEQ ID NO: 2 (Human GLP-1 (7-36)), or SEQ ID NO: 3 (DPP-IV resistant human GLP-1), the GLP-1 polypeptide contains one or more amino acid substitutions A8G, G22E, and R36G relative to natural human GLP-1, and the immunoglobulin Fc domain contains an IgG2-Fc domain or is an IgG2-Fc domain, the IgG2-Fc domain has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and contains the amino acid substitution C222S, and optionally the GLP-1 polypeptide is directly covalently bonded to the Fc domain or covalently bonded via a linker.

2. The fusion protein according to claim 1, wherein the GLP-1 polypeptide has a hydroxylation level of lysine 34 (K34) of 10% or more, 15% or more, or 20% or more compared to natural human GLP-1, and the GLP-1 polypeptide preferably has an Fc domain comprising one or more additional amino acid substitutions selected from the group consisting of A330S and P331S as described in SEQ ID NO: 6, and / or the amino acid sequence of the GLP-1 polypeptide is shown in SEQ ID NO: 3, and the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO:

6.

3. The fusion protein according to claim 1 or 2, wherein the GLP-1 polypeptide is substantially unoxidized with tryptophan 31 (W31) relative to natural human GLP-1, or the fusion protein according to claim 2, wherein the hydroxylation level is 26% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

4. The fusion protein according to claim 3, wherein the oxidation level of the GLP-1 polypeptide at W31 is less than 0.5% or undetectable relative to natural human GLP-1.

5. The fusion protein according to claim 1 or 2, wherein the linker comprises a connecting peptide, the connecting peptide comprises glycine and serine residues, the connecting peptide comprises one, two, three, four or more repetitions of SEQ ID NO: 39 (GGGS), SEQ ID NO: 40 (GGGGGS), SEQ ID NO: 41 (GGGGGGGS), or SEQ ID NO: 42 (GGGGGGGGGS), and the linker is 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, and preferably the linker comprises the amino acid sequence described in SEQ ID NO:

9.

6. The fusion protein according to claim 1 or 2, having the amino acid sequence described in SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7, and / or the IgG2-Fc domain having an oxidation level at a methionine residue corresponding to the methionine residue (M253) of SEQ ID NO:

7.

7. The fusion protein according to claim 6, wherein the oxidation level of the M253 residue is 5% or less.

8. The fusion protein according to claim 1 or 2, further comprising a signal peptide and / or having a half-life in the body of a subject of 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.

9. The fusion protein according to claim 8, wherein the signal peptide is a human CD33 signal peptide, wherein the signal peptide has the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4, or the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

8.

10. A dimer comprising two identical peptide chains linked by disulfide bonds, each peptide chain comprising the fusion protein described in claim 1 or 2.

11. A nucleic acid molecule comprising a vector containing i) a nucleotide sequence encoding the fusion protein described in claim 1 or 2, or ii) a nucleotide sequence described in SEQ ID NO: 26 or SEQ ID NO: 27, or a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 26 or SEQ ID NO: 27, or i) or ii).

12. A cell comprising the nucleic acid molecule or vector described in claim 11.

13. The cell according to claim 12, further expressing lysine hydroxylase recombinantly or naturally, preferably the level of expression or activity of lysine hydroxylase expressed in the cell is higher than the level or activity of lysine hydroxylase in COS-7 cells, and / or the cell is a prokaryotic or eukaryotic cell, preferably the eukaryotic cell is a mammalian cell, more preferably the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell, or the mammalian cell is a human embryonic kidney cell 293 (HEK293), or a CHO-K1 cell, or a CHO-S cell, or a CHO-DG44 cell.

14. A composition comprising a fusion protein according to any one of claims 1 to 9, or a dimer according to claim 10, or a nucleic acid molecule or vector according to claim 11, or a cell according to claim 12 or 13.

15. The composition according to claim 14, wherein in the fusion protein, at least 10%, or at least 15%, or at least 20%, or at least 26%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the GLP-1 polypeptide is hydroxylated at the K34 position relative to human GLP-1.

16. A fusion protein according to any one of claims 1 to 9, a dimer according to claim 10, a nucleotide molecule or vector according to claim 11, a cell according to claim 12 or 13, a composition according to claim 14 or 15, for use in the treatment or prevention of a disease, or in the manufacture of a pharmaceutical for the treatment or prevention of a disease, or an additional therapeutic agent.

17. The fusion protein, or dimer, or nucleic acid molecule or vector, or cell or composition according to claim 16, wherein the disease is selected from the group consisting of metabolic diseases related to glucose or lipid metabolism disorders, complications of metabolic diseases, and neurological diseases.

18. The fusion protein, or dimer, or nucleic acid molecule or vector, or cell or composition according to claim 17, wherein the metabolic disorder associated with glucose or lipid metabolism disorder is selected from diabetes mellitus, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), obesity, and metabolic syndromes, or the metabolic disorder associated with glucose or lipid metabolism disorder is diabetes mellitus (e.g., type 2 diabetes mellitus).

19. A fusion protein or dimer or nucleic acid molecule or vector or cell or composition for use according to claim 18, wherein the neurodegenerative disease is selected from the group selected from Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

20. The fusion protein, dimer, nucleic acid molecule, vector, cell, or composition according to claim 16, wherein the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea drugs (e.g., glimepiride, glibenclamide, gliclazide, glikidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid, and is preferably metformin or γ-aminobutyric acid.

21. The fusion protein according to claim 16, wherein the fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the additional therapeutic agent is metformin used in the manufacture of pharmaceuticals for the treatment of diabetes (preferably type 2 diabetes), or the fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the additional therapeutic agent is γ-aminobutyric acid used in the manufacture of pharmaceuticals for the treatment of neurodegenerative diseases, preferably the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

22. A combination of a pharmaceutical product comprising the fusion protein according to claim 1 or 2, or the dimer according to claim 10, or the polynucleotide or vector according to claim 11, or the cell according to claim 12 or 13, or the composition according to claim 14, and the additional therapeutic agent.

23. A combination of pharmaceuticals wherein the additional therapeutic agent is a drug for treating diabetes or a drug for treating neurodegenerative diseases. Or, the combination of pharmaceuticals according to claim 22, wherein the additional therapeutic agent is selected from the group consisting of insulin, metformin, sulfonylurea (e.g., glimepiride, glibenclamide, gliclazide, glacidone), α-glucosidase inhibitors (e.g., acarbose), and γ-aminobutyric acid, preferably metformin or γ-aminobutyric acid.

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