Human GLP-1 polypeptide variants and their applications

Abstract

The present application provides human GLP-1 polypeptide variants, and fusion proteins or immunoconjugates constructed using said human GLP-1 polypeptide variants can be used to prevent and / or treat GLP-1-related metabolic diseases or conditions.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to human GLP-1 polypeptide variants and their applications. [Background technology]

[0002] Glucagon-like peptide-1 (GLP-1) is an incretin secreted by L-cells of the small intestinal epithelium. In healthy individuals, GLP-1 can respond to the intake of nutrients in the body by promoting insulin release and inhibiting glucagon secretion. In patients with type 2 diabetes, infusion of hyperphysiological doses of GLP-1 can restore endogenous insulin secretion and lower blood glucose levels.

[0003] Enzymatic degradation and renal clearance by dipeptidyl peptidase IV (DPPIV) shorten the plasma half-life of natural GLP-1. Despite the development of several GLP-1R agonists (such as dulaglutide) modified to extend half-life for the treatment of type 2 diabetes, there is a need to develop effective long-term treatment plans that minimize side effects. [Overview of the project] [Means for solving the problem]

[0004] This application provides a human GLP-1 polypeptide variant, the amino acid sequence of which, compared to the amino acid sequence shown in Sequence ID No. 2, contains at least two amino acid mutations at amino acid positions selected from the group consisting of W31, K26, Q23, and Y19. This application further provides a fusion protein or immune complex comprising the human GLP-1 polypeptide variant, the fusion protein or immune complex having at least one of the following properties: (1) being able to bind to the GLP-1 receptor, (2) being able to activate the GLP-1 receptor and increase cAMP levels, (3) having a long blood half-life, (4) having high in vivo / in vitro stability, (5) having high blood drug concentration, and (6) being able to lower blood glucose.

[0005] In one embodiment, the present application provides a human GLP-1 polypeptide variant, the amino acid sequence of which, compared to the amino acid sequence shown in SEQ ID NO: 2, contains at least two amino acid mutations at amino acid positions selected from the group consisting of W31, K26, Q23, and Y19.

[0006] In one embodiment, the human GLP-1 polypeptide variant has at least some of the activity of human GLP-1.

[0007] In one embodiment, the amino acid sequence of the human GLP-1 polypeptide variant, compared to the amino acid sequence shown in SEQ ID NO: 2, contains at least two mutations at amino acid positions selected from the group consisting of W31, K26, and Y19.

[0008] In one embodiment, the amino acid sequence of the human GLP-1 polypeptide variant, compared to the amino acid sequence shown in SEQ ID NO: 2, contains at least two mutations at amino acid positions selected from the group consisting of W31, K26, and Q23.

[0009] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions W31 and K26.

[0010] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions W31 and Q23.

[0011] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions W31 and Y19.

[0012] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions K26 and Q23.

[0013] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions K26 and Y19.

[0014] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions W31, K26 and Y19.

[0015] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant contains mutations at the amino acid positions W31, K26 and Q23.

[0016] In one embodiment, at W31 of the human GLP-1 polypeptide variant, it contains amino acid substitutions of W31Y, W31K, W31R or W31A. For example, the amino acid substitution is W31Y, W31K or W31R. Also, for example, the amino acid substitution is W31Y.

[0017] In one embodiment, the Y19 of the human GLP-1 polypeptide variant contains any one amino acid substitution selected from the group consisting of Y19A, Y19L, Y19T, Y19F, Y19I, Y19V, and Y19S.

[0018] In one embodiment, the Q23 of the human GLP-1 polypeptide variant contains one amino acid substitution selected from the group consisting of Q23E, Q23T, Q23S, and Q23N.

[0019] In one embodiment, the K26 molecule of the human GLP-1 polypeptide variant includes the amino acid substitution K26R.

[0020] In one embodiment, the human GLP-1 polypeptide variant includes an amino acid sequence represented by any one of SEQ ID NOs: 29, 31-53, and 61-71. In one embodiment, the human GLP-1 polypeptide variant includes an amino acid sequence represented by any one of SEQ ID NOs: 29, 32-53, and 61-71. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by SEQ ID NO: 29. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by any one of SEQ ID NOs: 32-53. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by any one of SEQ ID NOs: 61-71.

[0021] In another embodiment, the present application provides a fusion protein or immune complex comprising the above-mentioned human GLP-1 polypeptide variant.

[0022] In one embodiment, the fusion protein or immune complex further comprises an immunoglobulin Fc region.

[0023] In one embodiment, the immunoglobulin Fc region of the fusion protein or immune complex is an IgG-derived Fc region.

[0024] In one embodiment, the immunoglobulin Fc region of the fusion protein or immune complex is an Fc region derived from IgG4.

[0025] In one embodiment, the immunoglobulin Fc region includes an amino acid sequence represented by any one of sequence numbers 139 to 143.

[0026] In one embodiment, the human GLP-1 polypeptide variant and the immunoglobulin Fc region are fused in frame.

[0027] In one embodiment, the fusion protein or immune complex includes a linker peptide.

[0028] In one embodiment, the human GLP-1 polypeptide variant and the immunoglobulin Fc region are linked by the linker peptide.

[0029] In one embodiment, the linker peptide includes the amino acid sequence shown in any one of SEQ ID NOs: 144 to 149.

[0030] In one embodiment, the human GLP-1 polypeptide variant, the linker peptide, and the immunoglobulin Fc region are fused in frame.

[0031] In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by any one of SEQ ID NOs: 96, 98-120, and 128-138. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by any one of SEQ ID NOs: 96, 97-120, and 128-138. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by SEQ ID NO: 96. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by any one of SEQ ID NOs: 99-122. In one embodiment, the fusion protein or immune complex includes an amino acid sequence represented by any one of SEQ ID NOs: 128-138.

[0032] In another embodiment, the present application provides isolated nucleic acid molecules encoding the human GLP-1 polypeptide variant or the fusion protein or immune complex described above.

[0033] In another embodiment, the present application provides a vector containing the isolated nucleic acid molecule described above.

[0034] In another embodiment, the present application provides cells that contain or express the human GLP-1 polypeptide variant, the fusion protein or immune complex, the isolated nucleic acid molecule, or the vector.

[0035] In another embodiment, the present application provides a pharmaceutical composition comprising the human GLP-1 polypeptide variant, the fusion protein or immune complex, the isolated nucleic acid molecule, the vector or cell, and an optional pharmaceutically acceptable adjuvant.

[0036] In another embodiment, the present invention provides uses for the human GLP-1 polypeptide variant or the fusion protein or immune complex in the preparation of agents for the prevention and / or treatment of metabolic diseases or conditions.

[0037] In another embodiment, the present application provides a method for preventing and / or treating a metabolic disease or condition, the method comprising administering the human GLP-1 polypeptide variant or the fusion protein or immune complex.

[0038] In another embodiment, the present application provides the above-mentioned human GLP-1 polypeptide variant or fusion protein or immune complex for the prevention and / or treatment of metabolic diseases or conditions.

[0039] In one embodiment, the metabolic disease or condition includes metabolic diseases related to GLP-1.

[0040] In one embodiment, the metabolic disease or condition includes diabetes mellitus.

[0041] In another embodiment, the present invention provides a method for increasing or promoting insulin expression in a subject in need thereof, the method comprising administering an effective amount of the human GLP-1 polypeptide variant or the fusion protein or immune complex to the subject.

[0042] In another embodiment, the present invention provides the human GLP-1 polypeptide variant or the fusion protein or immune complex described above for increasing or promoting insulin expression in subjects requiring it.

[0043] In another embodiment, the present invention provides the use of the human GLP-1 polypeptide variant or the fusion protein or immune complex in the preparation of agents for increasing or promoting insulin expression in subjects who require it.

[0044] Those skilled in the art will readily infer other aspects and advantages of the Application from the following detailed description. The following detailed description illustrates only exemplary embodiments of the Application. As will be apparent to those skilled in the art, by the nature of the Application, those skilled in the art can modify the specific embodiments disclosed without departing from the spirit and scope of the invention. Thus, the drawings and description in the specification are illustrative and not limiting.

[0045] The specific features of the invention described herein are as stated in the attached claims. The features and advantages of the invention described herein can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows. [Brief explanation of the drawing]

[0046] [Figure 1] The fusion proteins GM-WY and GM-FY demonstrate their binding activity to the GLP-1 receptor. [Figure 2] This demonstrates the binding activity of the fusion protein GM-WY to the GLP-1 receptor. [Figure 3] This demonstrates the binding activity of the fusion proteins GM-WR and GM-FH to the GLP-1 receptor. [Figure 4] The fusion proteins GM-WY, GM-QN, GM-QT, GM-QS, GM-YL, GM-YT, and GM-YF exhibit binding activity to the GLP-1 receptor. [Figure 5] The fusion proteins GM-WY, GM-YI, GM-YV, GM-YS, GM-QE, and GM-YA demonstrate their binding activity to the GLP-1 receptor. [Figure 6] This demonstrates the binding activity of the fusion proteins GM-FT, GM-FN, GM-FS, and GM-FQ to the GLP-1 receptor. [Figure 7] The fusion proteins GM-FE, GM-FV, GM-FK, and GM-FI demonstrate their binding activity to the GLP-1 receptor. [Figure 8]The binding activity of the fusion proteins GM-WY, GM-KRFY, GM-KRWY, and GM-KRWYFY to the GLP-1 receptor is demonstrated. [Figure 9] The fusion proteins GM-WY, GM-KRQN, GM-KRQT, GM-KRQS, GM-KRYL, GM-KRYT, and GM-KRYF demonstrate their binding activity to the GLP-1 receptor. [Figure 10] The fusion proteins GM-KRWY, GM-KRYI, GM-KRYV, GM-KRYS, GM-KRQE, and GM-KRYA demonstrate their binding activity to the GLP-1 receptor. [Figure 11] The fusion proteins GM-WY, GM-WYQN, GM-WYQS, and GM-WYQT demonstrate their binding activity to the GLP-1 receptor. [Figure 12] The fusion proteins GM-WY, GM-WYYL, GM-WYYT, GM-WYYF, GM-WYYI, GM-WYYV, and GM-WYYS exhibit binding activity to the GLP-1 receptor. [Figure 13] This demonstrates the binding activity of the fusion proteins GM-WYYA and GM-WYQE to the GLP-1 receptor. [Figure 14] The fusion protein GM-WYYA demonstrates the activating effect of GM-WYYA on the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 15] The fusion protein GM-WYQE demonstrates the activating effect of GM-WYQE on the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 16] This demonstrates the binding activity of the fusion proteins GM-WYFL and GM-WY to the GLP-1 receptor. [Figure 17] The fusion proteins GM-WIFL, GM-WA, GM-WL, and GM-WLFL demonstrate their binding activity to the GLP-1 receptor. [Figure 18] The fusion proteins GM-KRWYYF, GM-KRWYYL, GM-KRWYYT, GM-KRWYQN, GM-KRWYQT, and GM-KRWYQS demonstrate their binding activity to the GLP-1 receptor. [Figure 19]The fusion proteins GM-KRWYYI, GM-KRWYYV, GM-KRWYYS, GM-KRWYQE, and GM-KRWYYA demonstrate their binding activity to the GLP-1 receptor. [Figure 20] This demonstrates the binding activity of the fusion proteins GM-KRWYYA and GM-KRWYQN to the GLP-1 receptor. [Figure 21] The fusion proteins GM-KRWY and GM-KRWYQE demonstrate activation of the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 22] The fusion proteins GM-KRWYYA and GM-KRWYYL demonstrate activation of the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 23] The fusion proteins GM-KRWYYS and GM-KRWYYI demonstrate activation of the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 24] The fusion proteins GM-KRWYYT and GM-KRWYQN demonstrate activation of the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 25] The fusion proteins GM-KRWYQT and GM-KRWYQS demonstrate activation of the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 26] The fusion proteins GM-KRWYYA and GM-KRWYQN demonstrate activation of the cAMP / PKA signaling pathway at 6 h(A) and 24 h(B). [Figure 27] This shows the pharmacokinetic effects of the fusion protein GM-KRWY. [Figure 28] The pharmacokinetic effects of the fusion proteins GM-KRWYYL, GM-KRWYYI, and GM-KRWYYA are shown. [Figure 29] The pharmacokinetic effects of the fusion proteins GM-KRWYQN, GM-KRWYQE, and GM-KRWYYA are shown. [Figure 30]This demonstrates the blood glucose-lowering activity of the fusion protein GM-KRWY in oral glucose tolerance tests. [Figure 31] This shows the blood glucose-lowering activity of the fusion proteins GM-KRWYQE and GM-KRWYYA in oral glucose tolerance tests. [Figure 32] An example of amino acid sequence numbering in this application is shown. [Figure 33] The fusion proteins GM-KRWY, GM-KAWA, GM-KAWY, and GM-KRWA demonstrate their binding activity to the GLP-1 receptor. [Modes for carrying out the invention]

[0047] Embodiments of the present invention will be described below with reference to specific examples, and those skilled in the art will be able to easily understand other advantages and effects of the present invention from the contents disclosed herein.

[0048] Definition of Terms

[0049] In this application, the term "GLP-1" generally refers to glucagon-like peptide-1. Since the natural GLP-1 molecule has been processed in vivo to cleave the first six amino acids, it is customary in this art to define the first amino acid at the N-terminus of the GLP-1 amino acid sequence as position 7 and the last amino acid at the C-terminus as position 37. The processed peptide can be further modified in vivo, such as by removing the glycine residue at the C-terminus and substituting it with an amide group. GLP-1 generally has two bioactive forms: GLP-1(7-37)OH and GLP-1(7-36)NH2. As used in this application, "GLP-1" includes natural, artificially synthesized, or modified GLP-1 proteins, and further includes the complete GLP-1 protein or its functional fragments, and GLP-1 proteins of different bioactive forms. For example, wild-type human GLP-1 may contain the amino acid sequence shown in SEQ ID NO: 1, where the N-terminal amino acid residue H is designated as position 7. Therefore, the term "K26" in this application generally refers to the position of the 26th amino acid, counting from the H at position 7 of the N-terminus of the GLP-1 protein, where the amino acid at position 26 in the amino acid sequence shown in SEQ ID NO: 1 is K, and the term "W31" generally refers to the position of the 31st amino acid, counting from the H at position 7 of the N-terminus of the GLP-1 protein, where the amino acid at position 31 in the amino acid sequence shown in SEQ ID NO: 1 is W.

[0050] The above human GLP-1 can be modified, and variants of the modified GLP-1 may possess at least some of the activity of the original human GLP-1. For example, an exemplary modified amino acid sequence of wild-type human GLP-1 can be shown in SEQ ID NO: 2.

[0051] In this application, the term "activity of at least some of human GLP-1" generally refers to having the activity of one or more of the human GLP-1 protein, or having at least 20% (e.g., at least 25%, 30%, 35%, 40%, 45%, or 50%, or more) of the human GLP-1 protein. The human GLP-1 in "activity of at least some of human GLP-1" above may be the wild type, for example, the amino acid sequence shown in SEQ ID NO: 1. The human GLP-1 in "activity of at least some of human GLP-1" above may also be a GLP-1 mutant modified from wild-type human GLP-1, for example, the amino acid sequence shown in SEQ ID NO: 2. For example, the human GLP-1 polypeptide mutant of this application may have the activity of at least some of the human GLP-1 with the amino acid sequence shown in SEQ ID NO: 1. In other cases, the human GLP-1 polypeptide mutant of this application may have the activity of at least some of the human GLP-1 with the amino acid sequence shown in SEQ ID NO: 2.

[0052] The above activities do not require the same level of activity as human GLP-1 protein, and may be higher, similar to, or lower than the activity of human GLP-1 protein. In some cases, "at least some of the activity of human GLP-1" may include activity that binds to the GLP-1 receptor, activity that activates the GLP-1 receptor, activity that activates adenylyl cyclase and promotes an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, and intracellular Ca 2+ The activity may be one or more selected from the group consisting of activities that positively regulate levels, activities that stimulate insulin secretion, activities that increase hepatic glycogen storage, activities that delay gastric emptying, activities that suppress gastric motility, activities that reduce appetite, activities that suppress β-cell apoptosis, activities that suppress postprandial glucagon secretion, activities that alleviate hypoglycemia, and activities that reduce body weight. For example, it can be detected by detecting the ability to bind to the GLP-1 receptor or the expression level of cAMP. The above "activity of at least a part of human GLP-1" may also be the activity of at least a part of a fusion protein containing human GLP-1 (for example, a fusion protein with an Fc region).

[0053] In this application, the term "GLP-1R" generally refers to the glucagon-like peptide-1 receptor. Binding of GLP-1R to GLP-1 can activate a signaling cascade, leading to activation of adenylyl cyclase and an increase in intracellular cAMP levels. The GLP-1R described herein may include full-length molecules, variants, fragments, or synthetic compounds of natural GLP-1R, provided that GLP-1R activity is preserved. For example, the GLP-1R described herein may include the amino acid sequence indicated by the NCBI database registration number NP_002053.3.

[0054] In this application, the term “polypeptide” generally refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term “polypeptide” may be any chain having two or more amino acids, rather than a product of a specific length. Polypeptides as described in this application include peptides, dipeptides, tripeptides, oligopeptides, proteins, amino acid chains, or any other chain having two or more amino acids, and the term “polypeptide” may be used by substituting or replacing any one of these terms. The term “polypeptide” may also refer to products obtained by expressing and modifying a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, acylation, derivatization with known protective / closing groups, hydrolysis of proteins, or modification with amino acids that do not exist in nature. Polypeptides may be derived from natural biosources or produced by recombinant technology.

[0055] In this application, the term “mutant” generally refers to a protein molecule having sequence homology to a naturally occurring biologically active protein. Polypeptide mutants described herein include polypeptides having an altered amino acid sequence by the addition (including insertion), deletion, modification, and / or substitution of one or more amino acid residues, while simultaneously retaining at least one therapeutic and / or biological activity of the parent sequence and being distinct from the parent sequence. For example, a mutant may have at least 0%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the parent protein. Mutants may or may not be naturally occurring. Techniques known in the art can be used to generate mutants that do not exist in nature. Polypeptide mutants may include conserved or non-conserved amino acid substitutions, deletions, or additions.

[0056] In this application, the term “mutation” generally includes any type of alteration or modification of a sequence (nucleic acid or amino acid sequence), including deletion, cleavage, inactivation, disruption, substitution or translocation of amino acids or nucleotides. In this application, an amino acid mutation may also be an amino acid substitution, and the term “substitution” generally refers to the substitution of at least one amino acid residue in a predetermined parent amino acid sequence with a different “substituted” amino acid residue. The one or more substituted amino acid residues may be “naturally occurring amino acid residues” (i.e., encoded by the genetic code).

[0057] In this application, the term "hinge region" generally refers to the region between the CH1 and CH2 functional regions of the heavy chain of an immunoglobulin. The hinge region is generally derived from IgG, and may be derived from, for example, IgG1, IgG2, IgG3, or IgG4.

[0058] In this application, the term "fusion protein" generally refers to a polypeptide that includes, or includes, a longer polypeptide consisting of, a polypeptide whose amino acid sequence can be directly or indirectly fused (via a linker such as a linker peptide) to a heterologous polypeptide (e.g., the previous polypeptide or a domain-unrelated polypeptide). In some cases, the fusion protein may include the polypeptide variant and an immunoglobulin Fc region that is directly or indirectly linked to the polypeptide variant (e.g., linked by a linker peptide). For example, the linker peptide may be GS, GAP, G4S, (G4S)2, (G4S)3, or (G4S)4.

[0059] In this application, the term "immune complex" generally refers to a complex formed by the direct or indirect linkage (e.g., covalent bond via a linker) of a polypeptide or protein to an active substance of interest (e.g., a protein, nucleic acid, drug, or marker molecule).

[0060] In this application, the term “immunoglobulin” generally refers to a protein consisting of one or more polypeptides that are essentially encoded by an immunoglobulin gene. Widely recognized human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ constant region genes, and many immunoglobulin variable region genes. The NH2-terminus (about 110 amino acids) of the full-length immunoglobulin “light chain” (about 25 kD and 214 amino acids) is encoded by a variable region gene, and the COOH-terminus is encoded by a κ or λ constant region gene. Generally, immunoglobulins may also be heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Natural immunoglobulins are essentially composed of two Fab molecules and an Fc region linked by an immunoglobulin hinge region.

[0061] In this application, the term "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain, which can be produced by complete antibody digestion with papain. The Fc region may be a native sequence Fc region or a mutant Fc region. The Fc region of an immunoglobulin generally includes two constant regions (CH2 domain and CH3 domain) and may optionally include a CH4 domain.

[0062] In this application, the term "in-frame fusion" generally refers to the process of ligating two or more open reading frames (ORFs) so as to preserve the precise reading frames of the original ORFs, thereby forming a continuous, longer ORF. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs.

[0063] In this application, the term "nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides, or ribonucleotides of any length, isolated from the natural environment or artificially synthesized.

[0064] In this application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate within a suitable host cell and transfers the inserted nucleic acid molecule within and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into a cell, vectors primarily for replicating DNA or RNA, and vectors primarily for expressing the transcription and / or translation of DNA or RNA. The vector may further include vectors having various of the above functions. The vector may also be a polynucleotide that can be transcribed and translated into polypeptides when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

[0065] In this application, the term “cell” generally refers to individual cells, cell lines, or cell cultures that include or contain plasmids or vectors of nucleic acid molecules described herein, or that are capable of expressing antibodies or antigen-binding fragments described herein. The host cells may include offspring of a single host cell. Due to natural, unexpected, or intentional mutations, the offspring cells may not necessarily be completely identical in morphology or genome to the original parent cells, but should be able to express antibodies or antigen-binding fragments described herein. The host cells can be obtained by transfecting cells in vitro using vectors described herein. The host cells may be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). The recombinant host cells may include not only certain cells but also their offspring.

[0066] In this application, the term “metabolic disorder or condition” generally refers to a disorder or condition that disrupts normal in vivo metabolic processes, preventing the body from properly using and / or storing energy. Such metabolic disorders or conditions may include disorders of carbohydrate metabolism, disorders of fat metabolism, and / or disorders of cellular mitochondrial dysfunction. Such metabolic disorders or conditions may be related to diet, toxins, or infections, or they may be genetic disorders. Such metabolic disorders or conditions may include disorders or conditions resulting from deficiencies of metabolic enzymes or abnormalities in the function of metabolic enzymes. The metabolic disorders or conditions described in this application may be selected from diabetes mellitus, obesity, and other GLP-1 related metabolic disorders.

[0067] In the application, "dulaglutide" generally refers to a heterofusion protein containing a GLP-1 analog, formed by covalently bonding a GLP-1 analog (amino acid sequence shown in SEQ ID NO: 2) and a human IgG4-derived Fc region (amino acid sequence shown in SEQ ID NO: 143) with a small peptide (amino acid sequence shown in SEQ ID NO: 148). The trade name for dulaglutide is TRULICITY® or Trulicity®. Dulaglutide contains the amino acid sequence shown in SEQ ID NO: 4 (see PCT Patent WO2009009562 and U.S. Patent US7452966).

[0068] In this application, the term “includes” generally means including the features that are clearly specified, but not excluding other elements.

[0069] In this application, the term "approximately" generally refers to a change within the range of 0.5% to 10% or more of the specified value, for example, a change within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% or more of the specified value.

[0070] Polypeptide variants

[0071] In one embodiment, the present invention provides a human GLP-1 polypeptide variant having at least some of the activity of human GLP-1. In some cases, the human GLP-1 polypeptide variant has activity to bind to the GLP-1 receptor, activity to activate the GLP-1 receptor, activity to activate adenylyl cyclase and promote the increase of intracellular cyclic adenosine monophosphate (cAMP) levels, and intracellular Ca 2+The mutant may have one or more activities selected from the group consisting of activities that positively regulate levels, activities that stimulate insulin secretion, activities that increase hepatic glycogen storage, activities that delay gastric emptying, activities that suppress gastric motility, activities that reduce appetite, activities that suppress β-cell apoptosis, activities that suppress postprandial glucagon secretion, activities that alleviate hypoglycemia, and activities that reduce body weight. For example, this can be detected by detecting the ability to bind to the GLP-1 receptor or the expression level of cAMP. The activity of the above human GLP-1 polypeptide mutant may be at least 20% of the human GLP-1 activity (for example, at least 25%, 30%, 35%, 40%, 45%, or 50%, or more).

[0072] Compared to the amino acid sequence shown in Sequence ID No. 2, i.e., HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (Sequence ID No. 2), the amino acid sequence of the human GLP-1 polypeptide variant may contain at least two amino acid mutations, for example, two, three, four, or more amino acid mutations. For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may contain two to four (for example, two to three, three, or two) amino acid substitutions. The at least two amino acid mutations may be located at amino acid positions selected from the group consisting of K26, W31, Y19, and Q23.

[0073] In this application, the amino acid position "Xn" refers to an amino acid substitution that occurs at residue X corresponding to position n in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, where n is a positive integer (in the case of GLP-1, n starts from 7) and X is an abbreviation for any amino acid residue. For example, the amino acid position "W31" indicates the position corresponding to the 31st amino acid in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In the case of the amino acid sequence shown in SEQ ID NO: 2, the correspondence between amino acid residue X and position n is as shown in Figure 32.

[0074] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may contain at least two mutations at amino acid positions selected from the group consisting of W31, K26, and Y19, compared to the amino acid sequence shown in Sequence ID No. 2.

[0075] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may contain at least two mutations at amino acid positions selected from the group consisting of W31, K26, and Q23, compared to the amino acid sequence shown in Sequence ID No. 2.

[0076] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the W31 and K26 amino acid positions. For example, the amino acid sequence of the human GLP-1 polypeptide variant may have two amino acid mutations, which may be located at the W31 and K26 amino acid positions. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in Sequence ID No. 29.

[0077] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the W31 and Y19 amino acid positions. For example, the amino acid sequence of the human GLP-1 polypeptide variant may have two amino acid mutations, which may be at the W31 and Y26 amino acid positions. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of Sequence IDs No. 47 to 53.

[0078] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the W31 and Q23 amino acid positions. For example, the amino acid sequence of the human GLP-1 polypeptide variant may have two amino acid mutations, which may be located at the W31 and Q23 amino acid positions. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of Sequence IDs No. 43 to 46.

[0079] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the K26 and Y19 amino acid positions. For example, the amino acid sequence of the human GLP-1 polypeptide variant may have two amino acid mutations, which may be located at the K26 and Y19 amino acid positions. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of Sequence IDs No. 36 to 42.

[0080] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the K26 and Q23 amino acid positions. For example, the amino acid sequence of the human GLP-1 polypeptide variant may have two amino acid mutations, which may be located at the K26 and Q23 amino acid positions. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of Sequence IDs No. 32 to 35.

[0081] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the Y19 and Q23 amino acid positions.

[0082] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include three amino acid mutations at the W31, K26, and Y19 amino acid positions.

[0083] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include three amino acid mutations at the W31, K26, and Q23 amino acid positions.

[0084] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include three amino acid mutations at the Y19, K26, and Q23 amino acid positions.

[0085] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include three amino acid mutations at the W31, Y19, and Q23 amino acid positions.

[0086] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include four amino acid mutations at the W31, K26, Y19, and Q23 amino acid positions.

[0087] In this application, the above amino acid mutation may include amino acid substitution. Due to the above amino acid mutation, the GLP-1 polypeptide variant still possesses at least some of the activity of human GLP-1 (for example, human GLP-1 protein whose amino acid sequence is represented by either SEQ ID NO: 1 or 2). Due to the above amino acid mutation, the fusion protein of the GLP-1 polypeptide variant can still possess at least some of the activity of a fusion protein of human GLP-1 (for example, human GLP-1 protein whose amino acid sequence is represented by either SEQ ID NO: 1 or 2). Due to the above amino acid mutation, the fusion protein of the GLP-1 polypeptide variant and the Fc region can still possess at least some of the activity of a fusion protein of human GLP-1 (for example, human GLP-1 protein whose amino acid sequence is represented by either SEQ ID NO: 1 or 2) and the Fc region.

[0088] In this application, K26 may include an amino acid substitution, and the amino acid substitution may be K26R.

[0089] In this application, W31 may include an amino acid substitution, and the amino acid substitution may be W31Y, W31A, W31R, or W31K. In this application, W31 may include an amino acid substitution, and the amino acid substitution may be W31Y, W31K, or W31R. For example, W31 may include an amino acid substitution, and the amino acid substitution may be W31Y.

[0090] In this application, Y19 may include an amino acid substitution, and the amino acid substitution may be Y19A, Y19L, Y19T, Y19F, Y19I, Y19V, or Y19S.

[0091] In this application, Q23 may include an amino acid substitution, and the amino acid substitution may be Q23E, Q23T, Q23S, or Q23N.

[0092] In this application, the amino acid substitution "XnY" means that the residue X corresponding to position n in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is replaced by the amino acid residue Y, where n is a positive integer (in the case of GLP-1, n starts from 7), and X and Y are abbreviations for any amino acid residue independently, and X is different from Y. For example, the amino acid substitution "W31Y" means that the amino acid residue W corresponding to position 31 in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is replaced by the amino acid residue Y.

[0093] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may contain at least two amino acid substitutions compared to the amino acid sequence shown in Sequence ID No. 1 or Sequence ID No. 2, and these amino acid substitutions may be selected from the group consisting of 1) K26R, 2) any one selected from W31Y, W31R, W31K and W31A, 3) any one selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S, and 4) any one selected from Q23N, Q23T, Q23S and Q23E.

[0094] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the above human GLP-1 polypeptide variant may contain at least two amino acid mutations selected from the group consisting of 1) K26R, 2) any one selected from W31Y, W31R, W31K, and W31A, and 3) any one selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V, and Y19S.

[0095] For example, compared to the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the above human GLP-1 polypeptide variant may contain at least two amino acid mutations selected from the group consisting of 1) K26R, 2) any one selected from W31Y, W31R, W31K, and W31A, and 3) any one selected from Q23N, Q23T, Q23S, and Q23E.

[0096] In this application, compared with the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid substitutions at the W31 and K26 amino acid positions, the amino acid substitution at K26 may be K26R, and the amino acid substitution at W31 may be W31Y, W31R, W31K, and W31A. For example, compared with the amino acid sequence shown in Sequence ID No. 2, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitutions W31Y and K26R.

[0097] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitution W31Y compared to the amino acid sequence shown in Sequence ID No. 2.

[0098] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitution K26R compared to the amino acid sequence shown in Sequence ID No. 2.

[0099] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitution Q23E compared to the amino acid sequence shown in Sequence ID No. 2.

[0100] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitution Y19A compared to the amino acid sequence shown in Sequence ID No. 2.

[0101] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitutions W31Y and Q23E, compared to the amino acid sequence shown in Sequence ID No. 2.

[0102] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitutions W31Y and Y19A, compared to the amino acid sequence shown in Sequence ID No. 2.

[0103] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid substitutions W31Y and K26R compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NOs: 29, 31, and 61-71. Alternatively, for example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NOs: 29 and any one of 61-71.

[0104] For example, the amino acid substitutions in the amino acid sequence of the above human GLP-1 polypeptide variant may be W31Y and K26R, and the above human GLP-1 polypeptide variant may also contain the amino acid sequence shown in SEQ ID NO: 29.

[0105] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions Q23E and K26R compared to the amino acid sequence shown in Sequence ID No. 2.

[0106] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions Y19A and K26R, compared to the amino acid sequence shown in Sequence ID No. 2.

[0107] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Q23, compared to the amino acid sequence shown in SEQ ID NO: 2. In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Q23, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NOs: 61 to 64.

[0108] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions at amino acid substitution K26R, amino acid substitution W31Y, and Y19, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NOs: 65 to 71.

[0109] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Q23N, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 61.

[0110] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Q23T, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 62.

[0111] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Q23S, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 63.

[0112] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Q23E, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 64.

[0113] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Y19A, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 71.

[0114] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Y19L, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 66.

[0115] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Y19T, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 67.

[0116] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Y19F, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 65.

[0117] In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitutions K26R, W31Y, and Y19I, compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 68.

[0118] In the present application, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitution K26R, amino acid substitution W31Y, and amino acid substitution Y19V. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 69.

[0119] In the present application, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant may include amino acid substitution K26R, amino acid substitution W31Y, and amino acid substitution Y19S. For example, the GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 70.

[0120] In the present application, the human GLP-1 polypeptide variant has the amino acid sequence HGEGTFTSDVSSX shown in SEQ ID NO: 153 19 LEEX 23 AAX 26 EFIAX 31 and may include LVKGGG, wherein X 19 = Y, A, L, T, F, I, V or S, X 23 = Q, E, T, S or N, X 26 = K or R, X 31 = W, A, R, K or Y. Among them, X 19 = Y, X 23 = Q, X 26 = K and X <并 31 = W do not all exist simultaneously, or X 19 = Y, X 23 = Q, X 26 = K and X 31 = W do not exist simultaneously at least three of them.

[0121] In the present application, compared with the amino acid sequence shown in SEQ ID NO: 2, the human GLP-1 polypeptide variant has the amino acid sequence shown in SEQ ID NO: 153, that is, HGEGTFTSDVSSX 19 LEEX 23 AAX 26 EFIAX 31 There seems to be an issue with the tag "[[并0000014]]" in the original text. It's not clear what it's supposed to be. I've translated it as best as possible while keeping it as is in the translation. If this is a specific formatting or encoding issue, it might need further clarification.It may also contain LVKGGG, of which X is compared to the amino acid shown in SEQ ID NO: 2. 19 , X 23 , X 26 and / or X 31 This may include at least two amino acid substitutions, of which X 19 In this, amino acid Y can be substituted with A, L, T, F, I, V or S, and X 23 In this, amino acid Q can be substituted with E, T, S, or N, and X 26 In X, amino acid K can be substituted with R. 31 In this compound, amino acid W can be substituted with Y, K, R, or A.

[0122] In this application, the above human GLP-1 polypeptide variant is the amino acid sequence shown in SEQ ID NO: 150, namely HGEGTFTSDVSSX 19 LEEX 23 AAREFIAYLVKGGG may be included, of which X 19 =Y, A, L, T, F, I, V or S, X 23 = Q, E, T, S, or N. Of these, X 19 =Y and X 23 =Q does not exist simultaneously. In this application, compared to the amino acid sequence shown in SEQ ID NO: 2, the above human GLP-1 polypeptide variant has the amino acid sequence shown in SEQ ID NO: 150, i.e., HGEGTFTSDVSSX 19 LEEX 23 AAREFIAYLVKGGG may be included, of which the above human GLP-1 polypeptide variant is X 19 and X 23 This may include amino acid substitutions in X 19 In this, amino acid Y can be substituted with A, L, T, F, I, V or S, and X 23 In this compound, amino acid Q can be substituted with E, T, S, or N.

[0123] In this application, the amino acid sequence of the above human GLP-1 polypeptide variant is the amino acid sequence shown in SEQ ID NO: 151, i.e., HGEGTFTSDVSSYLEEX 23AAREFIAYLVKGGG may be included, of which X 23 =E, T, S, or N. In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 151, and the amino acid sequence of the human GLP-1 polypeptide variant is X 23 This may include amino acid substitutions in X 23 In this compound, amino acid Q can be substituted with E, T, S, or N.

[0124] In this application, the amino acid sequence of the above human GLP-1 polypeptide variant is the amino acid sequence shown in SEQ ID NO: 152, namely HGEGTFTSDVSSX 19 It may include LEEQAAREFIAYLVKGGG, of which X 19 =A, L, T, F, I, V, or S. In this application, the amino acid sequence of the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 151, and the amino acid sequence of the human GLP-1 polypeptide variant is X 19 This may include amino acid substitutions in X 19 In this compound, amino acid Y can be substituted with A, L, T, F, I, V, or S.

[0125] Fusion protein or immune complex

[0126] In another embodiment, the present application provides a fusion protein or immune complex which may contain the human GLP-1 polypeptide variants described herein. In the present application, the fusion protein or immune complex may contain one, two or more (e.g., three, four, or five) of the human GLP-1 polypeptide variants.

[0127] In this application, the fusion protein or immune complex may further include an immunoglobulin Fc region. The immunoglobulin Fc region described in this application may include the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of the immunoglobulin, and generally does not include the two antigen-binding regions (Fab fragments) from the antibody. Furthermore, the immunoglobulin Fc region of this application may include all or some of the Fc regions, provided that they have physiological activity similar to that of a natural protein. The Fc region of this application may include Fc regions derived from IgA, IgD, IgE, IgG, and IgM. For example, the above Fc region may be an Fc region derived from IgG. The above Fc region may be derived from human IgG. For example, the above Fc region may be an Fc region derived from IgG1, IgG2, IgG3, or IgG4. For example, the above Fc region may be derived from human IgG4.

[0128] In this application, the Fc region of the fusion protein or immune complex may further include an immunoglobulin hinge region. The hinge region may be derived from IgG. The hinge region may be derived from human IgG. For example, the hinge region may be a hinge region derived from IgG1, IgG2, IgG3, or IgG4. For example, the hinge region may be at the C-terminus of the GLP-1 polypeptide variant, or, for example, at the N-terminus of the Fc region CH2.

[0129] For example, the Fc region may include the amino acid sequence shown in SEQ ID NOs: 139-143. For example, the Fc region of the fusion protein or immune complex may include the amino acid sequence shown in SEQ ID NOs: 142 or 143. Also, for example, the Fc region may include the amino acid sequence shown in SEQ ID NO: 143.

[0130] In this application, one end of the GLP-1 polypeptide variant can be directly or indirectly ligated to one end of the Fc region, for example, by in-frame fusion. In some cases, the C-terminus of the GLP-1 polypeptide variant can be directly or indirectly ligated to the N-terminus of the Fc region.

[0131] In this application, the fusion protein or immune complex may include the GLP-1 polypeptide variant and the immunoglobulin Fc region. In some cases, the GLP-1 polypeptide variant and the Fc region can be linked via a linker (e.g., a linker peptide). For example, the linker peptide may be selected from GS, GAP, G4S, (G4S)2, (G4S)3, or (G4S)4. For example, the linker peptide may include the amino acid sequence shown in any one of SEQ ID NOs: 144 to 149.

[0132] In this application, the fusion protein or immune complex may, from the N-terminus to the C-terminus, sequentially include the human GLP-1 polypeptide variant described in this application, the linker peptide described in this application, and the Fc region described in this application. The fusion protein or immune complex may, from the N-terminus to the C-terminus, sequentially include the human GLP-1 polypeptide variant described in this application, the linker peptide (G4S)3 (whose amino acid sequence is shown in SEQ ID NO: 148), and the Fc region derived from IgG4 (whose amino acid sequence is shown in SEQ ID NO: 143). For example, the fusion protein or immune complex described in this application may include the amino acid sequence shown in any one of SEQ ID NOs: 96, 98-120, and 128-138.

[0133] The fusion proteins or immune complexes described herein possess biological activity. Biological activity refers to the ability to bind to and activate GLP-1R in vivo or in vitro, thereby eliciting a response. The above responses include, but are not limited to, insulin secretion, glucagon inhibition, appetite suppression, weight loss, induction of satiety, inhibition of apoptosis, induction of pancreatic β-cell proliferation, and differentiation of pancreatic β-cells. The in vitro and in vivo activity of several representative fusion proteins will be tested.

[0134] The fusion protein or immune complex described in this application may reflect the ability to interact with human GLP-1R, as can be detected, for example, by ELISA, in Example 2. Example 3 reflects the in vitro activation ability of the fusion protein described in this application to GLP-1R, where activation of GLP-1R leads to activation of adenylyl cyclase, which in turn induces the expression of a reporter gene driven by cAMP response element binding protein (CREB), as indicated, for example, by the fluorescence intensity of the luciferase reporter gene or by the cAMP expression level. Example 4 reflects the long blood half-life of the fusion protein. Example 5 reflects the ability of the fusion protein described in this application to reduce elevated blood glucose levels.

[0135] In this application, the above-mentioned human GLP-1 polypeptide variants are named GLP-1-XX, where "XX" represents an amino acid substitution. For example, in the case of the human GLP-1 polypeptide variant GLP-WY, compared to the amino acid sequence shown in SEQ ID NO: 2, the amino acid mutation of the human GLP-1 polypeptide variant GLP-WY is that amino acid residue W at position 31 is replaced with amino acid residue Y. In the case of the human GLP-1 polypeptide variant GLP-KRWYYA, compared to the amino acid sequence shown in SEQ ID NO: 2, the amino acid mutation of the human GLP-1 polypeptide variant GLP-KRWYYA is that amino acid residue Y at position 19 is replaced with amino acid residue A, amino acid residue K at position 26 is replaced with amino acid residue R, and amino acid residue W at position 31 is replaced with amino acid residue Y.

[0136] In this application, the fusion protein of the human GLP-1 polypeptide variant and the Fc region is named GM-XX, where "XX" represents the corresponding amino acid substitution in the human GLP-1 polypeptide variant.

[0137] Nucleic acid molecules, vectors, cells, and methods for producing them

[0138] In another embodiment, the present application further provides one or more isolated nucleic acid molecules encoding the polypeptide variant or the fusion protein or immune complex described herein. For example, each of the one or more nucleic acid molecules may encode a complete polypeptide variant or the fusion protein or immune complex, or a part thereof.

[0139] The nucleic acid molecules described in this application may be isolated. For example, they can be produced or synthesized by (i) amplification in vitro, for example by polymerase chain reaction (PCR); (ii) cloning and recombination; (iii) purification, for example by enzymatic cleavage and gel electrophoresis; or (iv) synthesis, for example by chemical synthesis. In one embodiment, the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA technology. Recombinant DNA and molecular cloning techniques include those described in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis), T. Silhavy, MLBennan and L. WEnquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and Ausubel, FM et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Simply put, the above nucleic acids can be produced from genomic DNA fragments, cDNA, and RNA, all of which can be extracted directly from cells or recombinantly produced by various amplification methods (including, but not limited to, PCR and RT-PCR).

[0140] In another embodiment, the present application provides one or more vectors comprising the nucleic acid molecules described above. For example, the vector may comprise one or more of the nucleic acid molecules described above. The vector may further comprise other genes, such as marker genes, that enable selection of the vector in a suitable host cell and under suitable conditions. The vector may further comprise expression regulatory elements that enable precise expression of a coding region in a suitable host. Such regulatory elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. The one or more nucleic acid molecules described in the present application can be operably linked to the expression regulatory elements.

[0141] The above vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in genetic engineering. For example, the above vector is an expression vector.

[0142] In another embodiment, the present application provides cells that may contain one or more nucleic acid molecules and / or one or more vectors described herein. In some embodiments, each host cell may contain one or more nucleic acid molecules or vectors described herein. In some embodiments, each host cell may contain more (e.g., two or more) or more types (e.g., two or more) of nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the above cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS0 cells, HEK293T cells or HEK293A cells, or other eukaryotic cells such as plant-derived cells, fungal or yeast cells. The vectors described herein can be introduced into the above cells by methods known in the art, such as electroporation or lipofection.

[0143] In another embodiment, the present application provides a method for producing the polypeptide variant, the fusion protein, or the immune complex described herein. The method may include culturing the cells described herein under conditions that express the polypeptide variant, the fusion protein, or the immune complex. This may include, for example, using a suitable culture medium, a suitable temperature, and a suitable incubation time, methods that are well understood by those skilled in the art. In some cases, the above method may further include a step of collecting (e.g., isolating and / or purifying) the polypeptide variant, fusion protein, or immune complex described in this application. For example, affinity chromatography may be performed using protein G-agarose or protein A-agarose, and the polypeptide variant, fusion protein, or immune complex described in this application may be purified and isolated by gel electrophoresis and / or high-performance liquid chromatography.

[0144] Pharmaceutical compositions and uses

[0145] In another embodiment, the present application provides a pharmaceutical composition comprising the human GLP-1 polypeptide variant, the fusion protein or immune complex, the nucleic acid molecule, the vector or cell, and an optional pharmaceutically acceptable adjuvant.

[0146] For example, the above-mentioned pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants.

[0147] In this application, the above-mentioned pharmaceutical composition may be prepared for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or subcutaneous depot administration.

[0148] The pharmaceutical compositions described herein can be used to treat metabolic diseases or conditions. The pharmaceutical compositions can be used to treat diabetes (e.g., hyperglycemia, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, non-insulin-dependent diabetes, MODY (adult-onset diabetes mellitus), gestational diabetes, and / or decreased glycosylated hemoglobin (HbAlC)), prediabetes, hypertension, hyperglycemia, and / or dyslipidemia (or combinations of these metabolic risk factors). Acute symptoms of diabetes include excessive urination, resulting compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, drowsiness, and changes in energy metabolism. Chronic hyperglycemia of diabetes is associated with macrovascular and microvascular complications, which can lead to long-term damage, dysfunction, and, in some cases, ultimate failure of various organs (especially the eyes (particularly in the form of diabetic retinopathy), kidneys (in the form of diabetic nephropathy), nerves (in the form of diabetic neuropathy), heart, and blood vessels). The above pharmaceutical compositions may also be used to treat weight gain, promote weight loss, reduce excess weight and / or obesity (e.g., by controlling appetite, eating, food intake, calorie intake and / or energy expenditure) (including morbid obesity) and related diseases, abnormalities and health conditions (including, but not limited to, obesity-related inflammation, obesity-related gallbladder disease and obesity-related sleep apnea). For example, the above metabolic disease or condition may be selected from diabetes mellitus, obesity and other GLP-1 related metabolic diseases.

[0149] The frequency and dosage of the above-mentioned pharmaceutical composition can be determined by many relevant factors, including the type of disease being treated, the route of administration, the patient's age, sex, weight, and the severity of the disease, as well as the type of drug that constitutes the active ingredient. Because the above-mentioned pharmaceutical composition has excellent in vivo effects and sustained concentration, the frequency and dosage of the above-mentioned drug can be significantly reduced. Without being limited to any particular theory, the following examples are merely for the purpose of illustrating the fusion protein, manufacturing method, and uses related to the present invention, and do not limit the scope of the present invention. [Examples]

[0150] The positive control dulaglutide used in the examples of this application is an antidiabetic drug from Eli Lilly & Co., code name LY-2189265, the amino acid sequence of dulaglutide is shown in SEQ ID NO: 4, and is referred to in this application as GLP-1-Fc or KN042.

[0151] Example 1: Production of a fusion protein

[0152] A fusion protein of the GLP-1 polypeptide variant GLP-1-XX and Fc was prepared, containing, from the N-terminus to the C-terminus, the GLP-1 polypeptide variant (whose amino acid sequence is shown in any one of SEQ ID NOs. 5 to 71), linker peptide (G4S) 3, and an Fc region derived from IgG4, in that order.

[0153] A vector containing the nucleic acid sequence encoding the fusion protein was transferred to cells, expressed, and purified to obtain the fusion protein GM-XX. The amino acid sequence is represented by one of sequence numbers 72 to 138. The amino acid mutations and sequences of the fusion protein and the corresponding human GLP-1 polypeptide variant are shown in Table 1.

[0154] [Table 1-1]

[0155] [Table 1-2]

[0156] Example 2 Binding affinity of the fusion protein according to the present invention to the GLP-1 receptor

[0157] The binding affinity of the fusion protein in Example 1 to the GLP-1 receptor was detected by ELISA. A fusion protein of the GLP-1 receptor and mouse Fc, called GLP1R-muFc (SEQ ID NO: 3), was expressed. GLP1R-muFc was diluted to 5 μg / mL, coated a 96-well plate, and fixed at room temperature for 30 minutes. After washing once with PBS, the plate was blocked with 1% BSA at 37°C for 30 minutes and washed three times with 0.05% PBST20. 50 μL of awaited sample, diluted with a 4-fold concentration gradient at 10 μg / mL, was added to each well, and KN042 was used as the positive control. The plates were incubated at 37°C for 1 hour and washed five times with PBST. 50 μL of mouse monoclonal HP6023 anti-human IgG4Fc-HRP (abcam, ab99817), diluted 2000-fold with 0.05% PBST20 containing 1% BSA, was added to each well, incubated at 37°C for 1 hour, and washed five times with PBST. 50 μL of TMB was added to each well and allowed to develop for 3 minutes. The reaction was stopped by adding 50 μL / well of 1M H2SO4, and the OD value was detected using a microplate reader (Molecular Devices, SpectraMax M3). KN042 was used as a positive control.

[0158] The results are shown in Figures 1-13, 16-20, and 33.

[0159] Figure 1 shows that the fusion protein with the amino acid mutation W31Y exhibits superior binding activity to the GLP-1 receptor compared to the F28Y mutation.

[0160] Figure 2 shows that the fusion protein after the amino acid mutation W31Y is similar to the positive control KN042.

[0161] Figure 3 shows that the fusion protein after amino acid mutation at W31 exhibits superior binding activity to the GLP-1 receptor compared to the fusion protein after amino acid mutation at F28.

[0162] Figures 4 and 5 show that the fusion proteins after multiple amino acid mutations in Q23 and Y19 both retain their binding activity to the GLP-1 receptor.

[0163] Figures 6 and 7 show that the binding activity of the fusion protein to the GLP-1 receptor is significantly reduced after amino acid mutation in F28.

[0164] Figures 1 to 7 show that the fusion proteins after amino acid mutations at W31, Q23, and Y19 still retain their GLP-1 receptor binding activity, but the amino acid mutation at F28 significantly reduces their GLP-1 receptor binding activity.

[0165] Figure 8 shows that the K26R-W31Y double mutation fusion protein exhibits superior GLP-1 receptor binding activity compared to the K26R-F28Y double mutation.

[0166] Figure 9 shows that the fusion proteins of the K26R-W31Y, K26R-Q23T, K26R-Q23S, K26R-Q23N, K26R-Y19L, K26R-Y19T, and K26R-Y19F double mutants exhibit superior binding activity to the GLP-1 receptor, similar to the positive control KN042.

[0167] Figure 10 shows that the fusion proteins of the K26R-W31Y, K26R-Q23E, K26R-Y19I, K26R-Y19V, K26R-Y19A, and K26R-Y19S double mutants exhibit superior binding activity to the GLP-1 receptor, similar to the positive control KN042.

[0168] Figure 11 shows that the fusion proteins of the K26R-W31Y, K26R-Q23N, K26R-Q23S, and K26R-Q23T double mutants exhibit good binding activity to the GLP-1 receptor.

[0169] Figure 12 shows that the fusion protein of the double mutant with amino acid mutations at W31Y and Y19 exhibits good binding activity to the GLP-1 receptor.

[0170] Figure 13 shows that the fusion proteins of the W31Y-Y19A and W31Y-Q23E amino acid double mutations exhibit good binding activity to the GLP-1 receptor.

[0171] Figures 8 to 13 show that fusion proteins containing amino acid mutations at two of the following positions—K26, W31, Y19, and Q23—have good binding activity to the GLP-1 receptor.

[0172] Figure 16 shows that both GM-WY and GM-WYFL can bind to the GLP-1 receptor.

[0173] Figure 17 shows that GM-WIFL, GM-WA, GM-WL, and GM-WLFL can all bind to the GLP-1 receptor.

[0174] Figures 16 and 17 show that the binding activity of fusion proteins of GLP-1 polypeptide variants that do not contain amino acid mutations at at least two amino acid positions selected from W31, Y19, Q23, and K26 is relatively weak.

[0175] Figure 18 shows that the triple mutant fusion protein with an amino acid mutation at W31Y-K26R-Q23, or the triple mutant fusion protein with an amino acid mutation at W31Y-K26R-Y19, exhibits good binding activity to the GLP-1 receptor.

[0176] Figure 19 shows that the triple mutant fusion protein with amino acid mutations at W31Y-K26R-Q23, or the triple mutant fusion protein with amino acid mutations at W31Y-K26R-Y19, exhibits good binding activity to the GLP-1 receptor.

[0177] Figure 20 shows that the fusion protein of the W31Y-K26R-Q23N triple mutation or the fusion protein of the W31Y-K26R-Y19A triple mutation exhibits good binding activity to the GLP-1 receptor.

[0178] Figures 18 to 20 show that fusion proteins containing amino acid mutations at three of the following positions—K26, W31, Y19, and Q23—have good binding activity to the GLP-1 receptor.

[0179] The test results of this invention further demonstrate that the in vitro biological activity (e.g., binding activity to the GLP-1 receptor, activity to activate the cAMP / PKA signaling pathway) of the fusion protein containing the K26R-W31Y double mutation is superior to that of the K26R-W31A and K26A-W31Y double mutations. For example, the results in Figure 33 show that the binding activity to the GLP-1 receptor of the fusion protein containing the K26R-W31Y double mutation is superior to that of the K26R-W31A, K26A-W31Y, and K26A-W31A double mutations.

[0180] Example 3: Detection of the activating effect of the fusion protein of the present invention on the cAMP / PKA signaling pathway using the luciferase method.

[0181] Based on the principle that a fusion protein of a GLP-1 polypeptide mutant binds to the GLP-1 receptor, activates adenylyl cyclase, and promotes an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, we detected the activating effect of the fusion protein on the cAMP / PKA signaling pathway based on the CREB (cAMP response element binding protein) reporter gene. HER293 cells were transfected using a human GLP-1R plasmid and a CREB-driven luciferase reporter plasmid. After transfection, HER293-GLP1R-CREB-D4 cells were digested, collected, counted, and the cell count was reduced to 4 × 10⁶ cells in DMEM medium containing 10% FBS. 5The cells / mL were adjusted and inoculated into 96-well plates at 50 μL per well. The awaited samples were diluted in DMEM medium containing 10% FBS to nine concentrations ranging from 1000 ng / mL, with KN042 used as the positive control. An additional 50 μL of the awaited samples at each of the following concentrations was added to each well of the 96-well plate, and incubated at 37°C for 6 or 24 hours. The supernatant was discarded, and luciferase substrate was added to each well. After standing at room temperature for 5 minutes, 50 μL was withdrawn, and luciferase activity was detected using the Bio-Glo® Luciferase Assay System (Promega, G7940), with values ​​read using a microplate reader (Molecular Devices, SpectraMax M3). Fluorescence intensity reflected the cAMP level and the degree of GLP-1 receptor activation.

[0182] As a result, as shown in Figures 14-15 and 21-26, all of the fusion proteins related to this invention can activate the cAMP / PKA signaling pathway by activating the GLP-1 receptor. The fusion proteins GM-WYYA, GM-WYQE, GM-KRWY, GM-KRWYQE, GM-KRWYYA, GM-KRWYYL, GM-KRWYYS, GM-KRWYYI, GM-KRWYYT, GM-KRWYQN, GM-KRWYQT, and GM-KRWYQS related to this invention demonstrate excellent in vitro stability.

[0183] Figure 14 shows that the fusion protein of the W31Y-Y19A double mutant GLP-1 polypeptide variant and its Fc region can increase cAMP levels by activating the GLP-1 receptor, and that the incubation time can be extended from 6 hours (14A) to 24 hours (14B), indicating a stronger activation capacity. The increased activation capacity compared to the positive control demonstrates that the fusion protein GM-WYYA of this application exhibits superior in vitro stability.

[0184] Figure 15 shows that the fusion protein of the W31Y-Q23E double mutant GLP-1 polypeptide variant and its Fc region can increase cAMP levels by activating the GLP-1 receptor, and that the incubation time can be extended from 6 hours (15A) to 24 hours (15B), indicating a stronger activation capacity. The increased activation capacity compared to the positive control indicates that the fusion protein GM-WYQE of the application showed superior in vitro stability.

[0185] Figure 21 shows that both the K26R-W31Y double mutation and the K26R-W31Y-Q23 triple mutation fusion proteins can activate the GLP-1 receptor.

[0186] Figures 22 and 23 show that the triple mutant fusion proteins with amino acid mutations at K26R-W31Y-Y19 can all activate the GLP-1 receptor.

[0187] Figures 24 and 26 show that both the triple mutant fusion protein with amino acid mutations at K26R-W31Y-Q23 and the triple mutant fusion protein with amino acid mutations at K26R-W31Y-Y19 can activate the GLP-1 receptor.

[0188] Figure 25 shows that a triple mutant fusion protein with amino acid mutations at K26R-W31Y-Q23 can activate the GLP-1 receptor.

[0189] Example 4: Pharmacokinetic study

[0190] The in vivo pharmacokinetics of the GLP-1-Fc fusion protein were investigated. Experiments were conducted in an SPF animal room with an ambient temperature of 23±2°C, relative humidity of 40-70%, and a 12-hour light-dark cycle. Experimental animals were allowed free feeding and acclimatized for at least 3 days prior to the experiment. SPF-grade SD rats weighing 180-220 g were randomly assigned to groups. Each group of rats received a subcutaneous injection of the corresponding test product, with dulaglutide used as a control. Approximately 100 μL of blood was collected from the jugular vein before administration and at 6, 24, 48, 72, 96, and 120 hours after administration. The collected blood was added to a centrifuge tube and allowed to stand at room temperature for 30-60 minutes. Then, serum was rapidly separated by centrifugation at 4000 rpm for 10 minutes within 2 hours at 4°C. The samples were stored at -80°C. Samples were detected by ELISA. Pharmacokinetic parameters were calculated using DAS(3.2.8) software.

[0191] As a result, the fusion proteins according to the present invention were shown to have better pharmacokinetic properties compared to the control. Figure 27 shows that the half-life of the fusion protein GM-KRWY according to the present invention is longer than that of dulaglutide, at 16.199 h, and that the drug exposure is higher than that of dulaglutide, while the blood drug concentration is similar. Figure 28 shows that the half-lives of the fusion proteins GM-KRWYYL, GM-KRWYYA, and GM-KRWYYI according to the present invention are longer than that of dulaglutide, at approximately 13-16.5 h, and that the drug exposure and blood drug concentration are higher than that of dulaglutide. Figure 29 shows that the half-lives of the fusion proteins GM-KRWYQN, GM-KRWYQE, and GM-KRWYYA according to the present invention are longer than that of dulaglutide, at approximately 22-25 h, and that the drug exposure and blood drug concentration are higher than that of dulaglutide.

[0192] Example 5: Oral glucose tolerance test (OGTT)

[0193] The in vivo hypoglycemic activity of GLP-1-Fc fusion protein was investigated. Mice were fasted overnight, and their blood glucose and body weight were measured before the experiment. They were then randomly assigned to groups. Each group of mice received the corresponding test substance intraperitoneally, with dulaglutide as the control. 0.5 hours after administration, each mouse was orally administered 1.5 g / kg of glucose. Blood glucose levels were measured at the tip of the mouse's tail using a handheld Roche blood glucose meter (Accu-chek) before glucose loading and at 10, 20, 30, 60, and 120 minutes after loading. Approximately 1 mm of the tip of the mouse's tail was removed, and venous blood was collected by pressing the tail from the base to the tip to measure blood glucose levels.

[0194] As a result, compared to the control, the fusion proteins GM-KRWY (Figures 30A and 30B), GM-KRWYYA, and GM-KRWYQE (Figures 31A and 31B) related to this application demonstrated the same level of in vivo hypoglycemic activity.

Claims

1. A human GLP-1 polypeptide variant containing the amino acid sequence represented by any one of sequence numbers 29, 61-64, 66, 68, and 71.

2. A fusion protein or immune complex comprising the human GLP-1 polypeptide variant described in claim 1.

3. The fusion protein or immune complex according to claim 2, further comprising an immunoglobulin Fc region.

4. The fusion protein or immune complex according to claim 3, wherein the immunoglobulin Fc region is an IgG-derived Fc region.

5. The fusion protein or immune complex according to claim 3, wherein the immunoglobulin Fc region is an Fc region derived from IgG4.

6. The fusion protein or immune complex according to claim 3, wherein the immunoglobulin Fc region comprises an amino acid sequence represented by any one of SEQ ID NOs: 139 to 143.

7. The fusion protein or immune complex according to claim 3, wherein the human GLP-1 polypeptide variant and the immunoglobulin Fc region are in-frame fused.

8. A fusion protein or immune complex according to claim 3, comprising a linker peptide.

9. The fusion protein or immune complex according to claim 8, wherein the human GLP-1 polypeptide variant and the immunoglobulin Fc region are linked by the linker peptide.

10. The fusion protein or immune complex according to claim 8, wherein the linker peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 144 to 149.

11. The fusion protein or immune complex according to claim 2, comprising the amino acid sequence represented by any one of SEQ ID NOs: 96, 128, 131, 133, 135, and 138.

12. Human GLP-1 polypeptide variant according to claim 1 Alternatively, an isolated nucleic acid molecule encoding a fusion protein or immune complex as described in any one of claims 2 to 11.

13. A vector comprising the isolated nucleic acid molecule described in claim 12.

14. A cell containing or expressing a human GLP-1 polypeptide variant according to claim 1, a fusion protein or immune complex according to any one of claims 2 to 11, or a vector according to claim 13.

15. A pharmaceutical composition comprising a human GLP-1 polypeptide variant according to claim 1, a fusion protein or immune complex according to any one of claims 2 to 11, or a vector according to claim 13, and an optionally pharmaceutically acceptable adjuvant.

16. A human GLP-1 polypeptide variant according to claim 1, for use in the prevention and / or treatment of metabolic diseases or conditions. Alternatively, the fusion protein or immune complex according to any one of claims 2 to 11.

17. The use of human GLP-1 polypeptide variants or fusion proteins or immune complexes according to claim 16, wherein the metabolic disease or condition includes metabolic diseases related to GLP-1.

18. The metabolic disease or condition described above includes diabetes mellitus, and the human GLP-1 polypeptide variant or fusion protein or immune complex for use according to claim 16.

19. A human GLP-1 polypeptide variant according to claim 1, for use in increasing or promoting insulin expression in subjects who require increased or promoted insulin expression. Alternatively, the fusion protein or immune complex according to any one of claims 2 to 11.

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