Pharmaceutical compositions comprising fusion proteins and uses thereof

Abstract

The present application provides a pharmaceutical composition comprising a fusion protein comprising a human GLP-1 polypeptide variant and an immunoglobulin Fc region.

Description

Pharmaceutical composition containing fusion protein and use thereof

[0001] This application claims priority to Chinese patent application No. 2022117424922, filed on December 30, 2022. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present application relates to the field of biomedicine, and in particular to a pharmaceutical composition comprising a fusion protein and its use. Background Art

[0003] Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by L-cells in the small intestinal epithelium. In healthy individuals, GLP-1 responds to nutrient intake by promoting insulin release and inhibiting glucagon secretion. In patients with type 2 diabetes, infusion of supraphysiological doses of GLP-1 can restore endogenous insulin secretion and lower blood sugar.

[0004] Due to the enzymatic hydrolysis of dipeptidyl peptidase IV (DPPIV) and the clearance of the kidneys, the plasma half-life of natural GLP-1 is short. Although several modified GLP-1R agonists (e.g., dulaglutide) have been developed to extend the half-life for the treatment of type 2 diabetes, there is still a need to develop effective long-term treatments with minimal side effects. WO2022143515, WO2022143516 and international patent application PCT / CN2022 / 103777 disclose a class of mutated GLP-1 analogs and fusion proteins thereof, which have a more extended half-life and are expected to become an effective means for the long-term treatment of type 2 diabetes, and their entire contents are hereby incorporated by reference into this application.

[0005] Protein drugs are usually stored and used in the form of injections. Stable drug formulations are beneficial to the production, storage and transportation of drugs, and can improve the convenience and compliance of patients in taking drugs. Therefore, the field hopes to develop drug injections that can be stored stably for a long time.

[0006] Summary of the Invention

[0007] The present application provides a fusion protein comprising a human GLP-1 polypeptide variant and an immunoglobulin Fc region. The fusion protein has excellent pharmacokinetic properties, such as being difficult to be cleared, having a longer half-life (t1 / 2), and / or a higher exposure.

[0008] The present application also provides a pharmaceutical composition comprising the fusion protein, wherein the pH value of the pharmaceutical composition is 5.0-7.0. In certain embodiments, the pharmaceutical composition may further comprise a buffering component, a stabilizer, and / or a surfactant. The pharmaceutical composition of the present application has excellent stability. For example, after the pharmaceutical composition is stored at 25±2°C for 6 months, the monomer content of the fusion protein can still be maintained at above 96%.

[0009] The present application also provides a kit comprising the pharmaceutical composition and a container for holding the composition.

[0010] The present application further provides the use of the pharmaceutical composition in preventing and / or treating diseases, especially in preventing and / or treating metabolic diseases or disorders.

[0011] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0013] FIG1 shows the pharmacokinetic test results of the fusion proteins GM-RY-L1H2-Fc4, GM-RY-L2H2-Fc4 and GM-RY-L3H2-Fc4 described in the present application with different linkers.

[0014] FIG2 shows the pharmacokinetic test results of the fusion proteins GM-ARY-L2H2-Fc4 and GM-ARY-L2H1-Fc4 described in the present application with different hinge regions.

[0015] FIG3 shows the pharmacokinetic test results of the fusion protein GM-RY-L2H1-Fc4m described in the present application, wherein the Fc region contains a YTE mutation.

[0016] FIG4 shows the pharmacokinetic test results of the fusion proteins GM-ARY-L2H1-Fc4m and GM-RY-L2H1-Fc4m described in the present application, wherein the Fc region contains a YTE mutation.

[0017] Figure 5 shows the pharmacokinetic test results of the fusion proteins GM-ARY-L2H2-Fc4m, GM-RY-L2H2-Fc4m, GM-ARY-L2H1-Fc4m and GM-RY-L2H1-Fc4m described in the present application with different hinge regions and containing YTE mutations.

[0018] FIG6 shows an example of the numbering of the amino acid sequence of any GLP-1 polypeptide variant in the fusion protein of the present application.

[0019] FIG7 shows the test results showing that the fusion protein of the present application has the same level of GLP-1 receptor activation ability as dulaglutide. DETAILED DESCRIPTION

[0020] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0021] Definition of terms

[0022] In this application, the term "GLP-1" generally refers to glucagon-like peptide 1. Natural GLP-1 molecules are processed in vivo, with the first six amino acids cleaved off. Therefore, it is customary in the 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 by removing the C-terminal glycine residue and replacing it with an amide group. GLP-1 generally has two biologically active forms, namely GLP-1(7-37)OH and GLP-1(7-36)NH2. The "GLP-1" described in this application includes natural, synthetic, or modified GLP-1 proteins, as well as intact GLP-1 proteins or functional fragments thereof, and GLP-1 proteins in different biologically active forms. For example, wild-type human GLP-1 may comprise the amino acid sequence shown in SEQ ID NO:1, with the N-terminal amino acid residue H being designated as position 7. Therefore, the term "K26" in this application generally refers to the position of the 26th amino acid, calculated from the 7th H at the N-terminus of the GLP-1 protein, in which the amino acid at position 26 in the amino acid sequence set forth in SEQ ID NO: 1 is K; the term "W31" generally refers to the position of the 31st amino acid, calculated from the 7th H at the N-terminus of the GLP-1 protein, in which the amino acid at position 31 in the amino acid sequence set forth in SEQ ID NO: 1 is W. In this application, when describing amino acid mutations and / or substitutions, the numbering of amino acid residues in GLP-1 polypeptide variants is distinguished from the numbering of amino acid residues in the Fc region, in which the numbering of amino acid residues in the Fc region is based on the EU numbering system.

[0023] The human GLP-1 can be modified, and the modified GLP-1 variant can have at least some of the activity of the human GLP-1 before modification. For example, an exemplary modified amino acid sequence of wild-type human GLP-1 can be shown as SEQ ID NO: 2.

[0024] In this application, the term "at least a partial activity of human GLP-1" generally refers to a polypeptide having one or more activities of a human GLP-1 protein, or having at least 20% (e.g., at least 25%, 30%, 35%, 40%, 45%, or 50% or more) of the activity of a human GLP-1 protein. The human GLP-1 referred to as "at least a partial activity of human GLP-1" may be wild-type, such as the amino acid sequence set forth in SEQ ID NO: 1. The human GLP-1 referred to as "at least a partial activity of human GLP-1" may be a GLP-1 variant modified from wild-type human GLP-1, such as the amino acid sequence set forth in SEQ ID NO: 2. For example, the human GLP-1 polypeptide variant of the present application may have at least a partial activity of the human GLP-1 with an amino acid sequence such as that set forth in SEQ ID NO: 1. In other cases, the human GLP-1 polypeptide variant of the present application may have at least a partial activity of the human GLP-1 with an amino acid sequence such as that set forth in SEQ ID NO: 2.

[0025] The activity does not require the same level of activity as that of the human GLP-1 protein and can be higher, similar, or lower than that of the human GLP-1 protein. In certain cases, "at least a portion of the activity of human GLP-1" can refer to one or more selected from the following: activity binding to the GLP-1 receptor, activity activating the GLP-1 receptor, activity activating adenylate cyclase, activity promoting an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, activity positively regulating intracellular Ca2+ levels, activity stimulating insulin secretion, activity increasing liver glycogen storage, activity delaying gastric emptying, activity inhibiting gastric motility, activity reducing appetite, activity inhibiting β-cell apoptosis, activity inhibiting postprandial glucagon secretion, activity alleviating hypoglycemia, and activity reducing 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. For example, it can be detected by detecting the activation level of the cAMP / PKA signaling pathway using a luciferase assay. The “at least partial activity of human GLP-1” may be at least partial activity of a fusion protein comprising human GLP-1 (eg, a fusion protein with an Fc region).

[0026] In this application, the term "polypeptide" generally refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" can be any chain with two or more amino acids and does not refer to a product of a specific length. The polypeptides described herein include peptides, dipeptides, tripeptides, oligopeptides, proteins, amino acid chains, or any other term used to refer to chains with two or more amino acids, and the term "polypeptide" can replace any of these terms or be used interchangeably with them. The term "polypeptide" can also refer to products modified after expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, acylation, derivatization by known protective / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or generated by recombinant technology.

[0027] In this application, the term "variant" generally refers to a protein molecule with sequence homology to a natural biologically active protein. The polypeptide variants described herein include polypeptides having an altered amino acid sequence by adding (including insertions), deleting, modifying and / or replacing one or more amino acid residues, while retaining at least one therapeutic and / or biological activity of the parent sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2) and being different from the parent sequence. For example, the variant may have at least 0%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the parent protein. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants may be generated using techniques known in the art. The polypeptide variants of the present application may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0028] In this application, the term "mutation" generally includes any type of change or modification to a sequence (nucleic acid or amino acid sequence), including deletion, truncation, inactivation, destruction, substitution or translocation of an amino acid or nucleotide. In this application, an amino acid mutation can be an amino acid substitution, and the term "substitution" generally refers to replacing at least one amino acid residue in a predetermined parent amino acid sequence with a different "replacement" amino acid residue. The one or more replaced amino acid residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code).

[0029] In the present application, the term "amino acid substitution" refers to replacing an amino acid residue present in a parent sequence with another amino acid residue. The amino acid in the parent sequence can be substituted, for example, via chemical peptide synthesis or by recombinant methods known in the art.

[0030] In this application, the term "Fc region" generally refers to a domain derived from the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region, wherein "the immunoglobulin Fc region without the amino acid mutation" refers to the amino acid sequence shown in any one of SEQ ID NOs: 32-35, which correspond to the Fc sequences of human IgG1, IgG2, IgG3 and IgG4, respectively. The Fc region of the immunoglobulin described in this application generally comprises two constant domains (CH2 domain and CH3 domain), does not comprise a hinge region unless otherwise specified, and may optionally comprise a CH4 domain.

[0031] In this application, the term "YTE mutation" refers to a group of mutations in the Fc region of IgG. YTE mutations can lead to increased binding of the Fc region to human FcRn and may alter the serum half-life of fusion proteins containing the Fc region. Fc regions containing YTE mutations include a combination of the following three amino acid mutations: M252Y, S254T, and T256E, where numbering is based on the EU index as in Kabat.

[0032] In this application, the term "hinge region" generally refers to a flexible amino acid sequence between 15 and 30 amino acids in length that allows the polypeptide portion at its N-terminus or C-terminus to move independently. The hinge region is typically derived from the region between the CH1 and CH2 functional domains of an immunoglobulin heavy chain. The hinge region is typically derived from IgG, for example, IgG1, IgG2, IgG3, or IgG4. Compared to the natural hinge region derived from IgG, the hinge region of this application may have one or more amino acid residues added to or deleted from the N-terminus or C-terminus, or one or more amino acid residues may be replaced, as long as the function of the hinge region is maintained.

[0033] In this application, the term "immunoglobulin" generally refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. In general, immunoglobulins can be heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). The NH2-terminus (about 110 amino acids) of the full-length immunoglobulin "light chain" (about 25KD and 214 amino acids) is encoded by the variable region gene, and the COOH-terminus is encoded by the kappa or lambda constant region gene. Natural immunoglobulins essentially consist of two Fab molecules and an Fc region connected via an immunoglobulin hinge region.

[0034] In this application, the term "linker" generally refers to an amino acid sequence that connects two heterologous polypeptides or fragments thereof. In this application, a linker can be an amino acid sequence that covalently links polypeptides to form a fusion polypeptide. The length of the linker can be 10-20 amino acids in length. The linker can be flexible or rigid.

[0035] In this application, the term "selective enhancement" generally refers to enhancement under specific conditions, which may be a specific pH range, a specific temperature range, a specific connection method, a specific sample source, or a specific reaction time. For example, an amino acid mutation in an Fc region that enhances the binding ability of the Fc region to an Fc receptor within a specific pH range but does not significantly alter the binding ability of the Fc region to an Fc receptor outside of the specific range can be referred to as "selective enhancement."

[0036] In this application, the term "fusion protein" generally refers to a longer polypeptide comprising or consisting of a polypeptide whose amino acid sequence can be fused directly or indirectly (via a linker, e.g., a connecting peptide) to the amino acid sequence of a heterologous polypeptide (e.g., the previous polypeptide or a polypeptide unrelated to its domain).

[0037] In this application, the term "nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.

[0038] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host cell, which transfers the inserted nucleic acid molecule to the host cell and / or between host cells. The vector may include a vector primarily for inserting DNA or RNA into a cell, a vector primarily for replicating DNA or RNA, and a vector primarily for expression of the transcription and / or translation of DNA or RNA. The vector may also include a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0039] In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that can express an antibody or its antigen-binding fragment as described herein. The host cell may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genome to the original parent cell, but may be able to express the antibody or its antigen-binding fragment as described herein. The host cell can be obtained by in vitro transfection of cells using the vectors described herein. The host cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). The recombinant host cell includes not only a specific cell, but also the offspring of these cells.

[0040] In this application, the term "metabolic disease or condition" generally refers to a disease or condition that disrupts the normal metabolic process in the body, making it impossible for the body to utilize and / or store energy normally. The metabolic disease or condition may refer to a disease of carbohydrate metabolism disorder, a disease of fat metabolism disorder and / or a disease of mitochondrial disorder. The metabolic disease or condition may be related to diet, toxins or infection, or may be a genetic disease. The metabolic disease or condition may include a disease or condition caused by a lack of metabolism-related enzymes, or abnormal metabolism-related enzyme function. The metabolic disease or condition described in this application may be selected from diabetes and other GLP-1 related metabolic disorders.

[0041] In this application, "dulaglutide" generally refers to a heterologous fusion protein containing a GLP-1 analog, which is formed by covalently linking a GLP-1 analog (amino acid sequence as shown in SEQ ID NO: 2) with the Fc region derived from human IgG4 through a small molecule peptide. Dulaglutide can also be called dulaglutide or dulaglutide, and its trade name is or Dulaglutide comprises the amino acid sequence shown in SEQ ID NO: 3 (see PCT patent WO2009009562 and US Patent US7452966).

[0042] In the present application, the term "composition" generally refers to a mixture comprising the fusion protein described in the present application, and the composition may comprise at least one pharmaceutically acceptable component, such as the buffer component, stabilizer and / or surfactant described in the present application.

[0043] In this application, the term "preparation" generally refers to a product comprising the fusion protein described herein in a predetermined amount or ratio, as well as any product produced directly or indirectly by combining predetermined amounts of the fusion proteins described herein. The term "preparation" as used herein may include pharmaceutical preparations, i.e., comprising the fusion protein described herein and other pharmaceutically acceptable ingredients, as well as any product produced directly or indirectly by combining, compounding, or aggregating any two or more ingredients. The preparation may be in liquid form.

[0044] In this application, the term "buffer component" generally refers to an agent that has the function of providing a buffering effect (e.g., resisting changes in pH). For example, the buffer component can adjust the change in pH due to the addition and / or release of acidic or basic substances. For example, the buffer component can include a weak acid and its conjugate base; it can also include a weak base and its conjugate acid. In this application, "buffer component" can be used interchangeably with descriptions such as "buffer system", "buffer salt", "buffer", and "buffer system".

[0045] In this application, the term "stabilizer" generally refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during preparation, storage and use. Commonly used protein drug stabilizers include, but are not limited to, amino acids, polyols, antioxidants, preservatives, cyclodextrins, polyethylene glycols, such as PEG 3000, 3350, 4000, 6000, albumins, such as human serum albumin (HSA), bovine serum albumin (BSA), salts, such as sodium chloride, magnesium chloride, calcium chloride, chelating agents, such as EDTA, etc.

[0046] In this application, the term "surfactant" generally refers to an agent that can protect a protein (e.g., a fusion protein described herein) from air / solution interface-induced stress, solution / surface-induced stress, to reduce aggregation of the protein or minimize the formation of particulate matter in the composition. The surfactant can contain both a hydrophobic group and a hydrophilic group. The surfactant can include a mixture or combination of one or more surfactants. The surfactant can include a nonionic surfactant.

[0047] In this application, the term "immunoconjugate" generally refers to a conjugate formed by directly or indirectly linking (e.g., covalently linking through a linker) a polypeptide or protein and an active substance of interest (e.g., protein, nucleic acid, drug or labeling molecule, etc.).

[0048] In this application, the term "subject in need" or "in need of treatment" includes those who already have a metabolic disease or condition as well as those in whom the condition is to be prevented. Subjects in need or "patients" include humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0049] In the present application, the term "prevention and / or treatment" includes not only preventing and / or treating a disease, but also generally includes preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and generally any pharmacological action that is beneficial to the treated patient.

[0050] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0051] In this application, the term "about" generally refers to variation within a range of 0.5%-10% above or below a specified value, for example, variation within a 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% above or below a specified value. Unless otherwise specified, the numerical values ​​mentioned in this application are considered to be modified by "about". If in doubt, or if the error range for a particular value or parameter is not commonly understood in the art, "about" means ±5% of that value or parameter.

[0052] Summary of the Invention

[0053] The present application relates to a pharmaceutical composition comprising a fusion protein comprising a human GLP-1 polypeptide variant and an immunoglobulin Fc region.

[0054] GLP-1 polypeptide variants

[0055] In the pharmaceutical composition of the present application, the fusion protein comprises a human GLP-1 polypeptide variant that has at least partial activity of human GLP-1. In certain instances, the human GLP-1 polypeptide variant may have one or more activities selected from the group consisting of: binding to the GLP-1 receptor, activating the GLP-1 receptor, activating adenylate cyclase, promoting an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, positively regulating intracellular Ca2+ levels, stimulating insulin secretion, increasing liver glycogen storage, delaying gastric emptying, inhibiting gastric motility, reducing appetite, inhibiting β-cell apoptosis, inhibiting postprandial glucagon secretion, alleviating hypoglycemia, and reducing body weight. For example, the human GLP-1 polypeptide variant may be detected by measuring its ability to bind to the GLP-1 receptor or the expression level of cAMP. The activity of the human GLP-1 polypeptide variant may be at least 20% (e.g., at least 25%, 30%, 35%, 40%, 45%, or 50% or more) of the activity of human GLP-1.

[0056] Compared to the amino acid sequence of SEQ ID NO: 2: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 2), the amino acid sequence of the human GLP-1 polypeptide variant may comprise at least two amino acid mutations, for example, two, three, or more amino acid mutations. For example, compared to the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two to four (e.g., 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, and Y19.

[0057] In this application, the amino acid position "Xn" refers to an amino acid substitution occurring at residue X at position n in the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, where n is a positive integer (for GLP-1, n starts at 7) and X is an abbreviation for any amino acid residue. For example, amino acid position "W31" refers to the position corresponding to amino acid position 31 in the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. For the amino acid sequence set forth in SEQ ID NO: 2, the correspondence between amino acid residue X and position n is shown in Figure 6.

[0058] 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 comprise at least two mutations at amino acid positions selected from the group consisting of W31, K26 and Y19.

[0059] For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid mutations at amino acid positions W31 and K26 compared to the amino acid sequence set forth in SEQ ID NO: 2. For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid mutations at amino acid positions W31 and K26. For example, the human GLP-1 polypeptide variant may comprise the amino acid sequence set forth in SEQ ID NO: 4.

[0060] For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid mutations at amino acid positions W31 and Y19 compared to the amino acid sequence set forth in SEQ ID NO: 2. For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid mutations at amino acid positions W31 and Y19. For example, the human GLP-1 polypeptide variant may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 12-18.

[0061] For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid mutations at amino acid positions K26 and Y19 compared to the amino acid sequence set forth in SEQ ID NO: 2. For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid mutations at amino acid positions K26 and Y19. For example, the human GLP-1 polypeptide variant may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5-11.

[0062] For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise three amino acid mutations at amino acid positions W31, K26, and Y19 compared to the amino acid sequence set forth in SEQ ID NO: 2. For example, the amino acid sequence of the human GLP-1 polypeptide variant may comprise three amino acid mutations at amino acid positions W31, K26, and Y19. For example, the human GLP-1 polypeptide variant may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 19-25.

[0063] In the present application, the amino acid mutation may comprise an amino acid substitution. The amino acid mutation allows the GLP-1 polypeptide variant to still have at least partial activity of human GLP-1 (e.g., a human GLP-1 protein with an amino acid sequence as shown in any one of SEQ ID NOs: 1 or 2). The amino acid mutation allows the fusion protein of the GLP-1 polypeptide variant to still have at least partial activity of a fusion protein of human GLP-1 (e.g., a human GLP-1 protein with an amino acid sequence as shown in any one of SEQ ID NOs: 1 or 2). The amino acid mutation allows the fusion protein of the GLP-1 polypeptide variant and the Fc region to still have at least partial activity of a fusion protein of human GLP-1 (e.g., a human GLP-1 protein with an amino acid sequence as shown in any one of SEQ ID NOs: 1 or 2) and the Fc region.

[0064] In the present application, the K26 may contain an amino acid substitution, and the amino acid substitution may be K26R.

[0065] In the present application, the W31 may comprise an amino acid substitution, and the amino acid substitution may be W31Y, W31A, W31R or W31K. In the present application, the W31 may comprise an amino acid substitution, and the amino acid substitution may be W31Y, W31K or W31R. For example, the W31 may comprise an amino acid substitution, and the amino acid substitution may be W31Y.

[0066] In the present application, the Y19 position may comprise an amino acid substitution, and the amino acid substitution may be Y19A, Y19L, Y19T, Y19F, Y19I, Y19V or Y19S. For example, the Y19 position may comprise an amino acid substitution, and the amino acid substitution may be Y19A.

[0067] In the present application, the amino acid substitution "XnY" means that the residue X at position n in the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with the amino acid residue Y, wherein n is a positive integer (for GLP-1, n starts at 7), X and Y are each independently an abbreviation for any amino acid residue, and X is different from Y. For example, the amino acid substitution "W31Y" means that the amino acid residue W at position 31 in the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with the amino acid residue Y.

[0068] In the present 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 SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid substitutions may be selected from the following group: 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.

[0069] For example, compared to the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant may comprise at least two amino acid mutations selected from the following group: 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.

[0070] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise two amino acid substitutions at amino acid positions W31 and K26 compared to the amino acid sequence set forth in SEQ ID NO: 2. 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, the amino acid sequence of the human GLP-1 polypeptide variant may comprise amino acid substitutions W31Y and K26R compared to the amino acid sequence set forth in SEQ ID NO: 2.

[0071] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise the amino acid substitution W31Y compared to the amino acid sequence shown in SEQ ID NO: 2.

[0072] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise the amino acid substitution K26R compared to the amino acid sequence shown in SEQ ID NO: 2.

[0073] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise the amino acid substitution Y19A compared to the amino acid sequence shown in SEQ ID NO: 2.

[0074] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise amino acid substitutions W31Y and Y19A compared to the amino acid sequence shown in SEQ ID NO: 2.

[0075] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise 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 comprise the amino acid sequence shown in any one of SEQ ID NOs: 4 and 19-25.

[0076] For example, the amino acid substitutions in the amino acid sequence of the human GLP-1 polypeptide variant may be W31Y and K26R. For example, the human GLP-1 polypeptide variant may comprise the amino acid sequence shown in SEQ ID NO:4.

[0077] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise an amino acid substitution K26R, an amino acid substitution W31Y, and an amino acid substitution at Y19 compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the GLP-1 polypeptide variant may comprise the amino acid sequence shown in any one of SEQ ID NOs: 19-25.

[0078] In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may comprise an amino acid substitution K26R, an amino acid substitution W31Y, and an amino acid substitution Y19A compared to the amino acid sequence shown in SEQ ID NO: 2. For example, the human GLP-1 polypeptide variant may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0079] In the present application, the human GLP-1 polypeptide variant may comprise the amino acid sequence shown in SEQ ID NO: 26: HGEGTFTSDVSSX19LEEQAAREFIAX31LVKGGG; wherein, X19 = Y, A, L, T, F, I, V or S; X31 = W, A, R, K or Y.

[0080] PCT international patent applications WO2022143515, WO2022143516 and PCT / CN2022 / 103777 respectively relate to human GLP-1 polypeptide variants and fusion proteins thereof, and are hereby incorporated by reference in their entirety.

[0081] Fc region

[0082] In the present application, the fusion protein comprises an immunoglobulin Fc region, which can be located at the C-terminus or the N-terminus of the human GLP-1 polypeptide variant, preferably at the C-terminus.

[0083] The immunoglobulin Fc region can be derived from IgG, for example, IgG1, IgG2, IgG3, or IgG4. The Fc region of IgG1 may comprise the amino acid sequence set forth in SEQ ID NO: 32. The Fc region of IgG2 may comprise the amino acid sequence set forth in SEQ ID NO: 33. The Fc region of IgG3 may comprise the amino acid sequence set forth in SEQ ID NO: 34. The Fc region of IgG4 may comprise the amino acid sequence set forth in SEQ ID NO: 35.

[0084] In the present application, the Fc region may contain amino acid mutations, and the amino acid mutations may selectively enhance the binding affinity of the immunoglobulin Fc region to the Fc receptor compared to an immunoglobulin Fc region without the amino acid mutations (e.g., the amino acid sequence shown in any one of SEQ ID NOs: 32-35).

[0085] In the present application, the Fc region may be an Fc region derived from IgG1, IgG2, IgG3 or IgG4, and comprises one or more amino acid mutations at positions M252, S254 and / or T256 (e.g., the amino acid positions are according to EU coding of Kabat). For example, the Fc region may be an Fc region derived from IgG1, IgG2, IgG3 or IgG4, and comprises amino acid mutations at positions M252, S254 and T256 (e.g., the amino acid positions are according to EU coding of Kabat).

[0086] For example, the amino acid mutation at M252 may be M252Y. For example, the amino acid mutation at S254T may be S254T. For example, the amino acid mutation at T256 may be T256E.

[0087] In the present application, the Fc region may be an Fc region derived from IgG1, IgG2, IgG3 or IgG4, and may contain amino acid mutations of M252Y, S254T and T256E.

[0088] For example, the Fc region may be an Fc region derived from IgG1 and may include amino acid mutations M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG2 and may include amino acid mutations M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG3 and may include amino acid mutations M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG4 and may include the amino acid sequence set forth in SEQ ID NO: 36.

[0089] Hinge region

[0090] In the present application, the fusion protein may comprise a hinge region, wherein the hinge region is located between the human GLP-1 polypeptide variant and the immunoglobulin Fc region. In the present application, the hinge region may be derived from IgG.

[0091] In the present application, the hinge region may be derived from IgG1, IgG2, IgG3 or IgG4. In the present application, the hinge region may be derived from IgG1. In the present application, the hinge region may be derived from IgG4.

[0092] In the present application, the hinge region in the fusion protein may comprise one or more amino acid mutations compared to the naturally occurring IgG1 hinge region to remove interchain disulfide bonds. Such amino acid mutations are well known in the art.

[0093] In the present application, the hinge region in the fusion protein may have one or more additional amino acids at the N-terminus compared to a naturally occurring IgG hinge region. In the present application, the hinge region in the fusion protein may have one or more additional amino acids at the C-terminus compared to a naturally occurring IgG hinge region. In the present application, the hinge region in the fusion protein may have one or more fewer amino acids at the N-terminus compared to a naturally occurring IgG hinge region. In the present application, the hinge region in the fusion protein may have one or more fewer amino acids at the C-terminus compared to a naturally occurring IgG hinge region.

[0094] In the present application, the hinge region in the fusion protein can increase one or more amino acids at the N-terminus compared to the naturally occurring IgG4 hinge region, for example, increase Ala. In the present application, the hinge region in the fusion protein can comprise one or more amino acid mutations compared to the naturally occurring IgG4 hinge region to avoid undesirable protein formation. Such amino acid mutations are known in the art.

[0095] In the present application, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 30 or 31.

[0096] Linker

[0097] In the present application, the fusion protein may comprise a linker. The linker is positioned between the human GLP-1 polypeptide variant and the immunoglobulin Fc region, and is used to connect the human GLP-1 polypeptide variant and the immunoglobulin Fc region; preferably, it is positioned between the human GLP-1 polypeptide variant and the hinge region. In the present application, the linker may be a peptide linker. In the present application, the peptide linker may be rigid or flexible. Exemplary linkers may include, but are not limited to, the amino acid sequence set forth in any one of SEQ ID NOs: 27-29 or 47-70.

[0098] In the present application, the linker may have a length of 4-20 amino acids, preferably a length of 12-15 amino acids. In the present application, the linker may have a length of 15 amino acids. For the linkers listed above that are less than 15 amino acids in length, their length can be increased to 15 amino acids by adding amino acids, for example, by adding Gly.

[0099] For example, the linker described herein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 27-29.

[0100] Fusion protein

[0101] In the present application, the fusion protein may comprise the human GLP-1 polypeptide variant and the immunoglobulin Fc region, preferably, may comprise the human GLP polypeptide variant, the linker, the hinge region and the immunoglobulin Fc region.

[0102] In some specific embodiments, the human GLP polypeptide variant in the fusion protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 4 or 25, and the immunoglobulin Fc region may be an Fc region derived from IgG. For example, the immunoglobulin Fc region may be an Fc region of IgG comprising the amino acid mutations M252Y, S254T, and T256E.

[0103] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 4 or 25, the hinge region may be derived from the hinge region of IgG1 or IgG4, and the immunoglobulin Fc region may be derived from the Fc region of IgG. For example, the immunoglobulin Fc region may be the Fc region of IgG comprising the amino acid mutations M252Y, S254T, and T256E.

[0104] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 4 or 25, the linker may comprise the amino acid sequence shown in any one of SEQ ID NO: 27-29, the hinge region may be derived from the hinge region of IgG1 or IgG4, and the immunoglobulin Fc region may be derived from the Fc region of IgG. For example, the immunoglobulin Fc region may be the Fc region of IgG comprising the amino acid mutations M252Y, S254T, and T256E.

[0105] In the present application, from N-terminus to C-terminus, the fusion protein may sequentially comprise the human GLP polypeptide variant, the linker, the hinge region and the immunoglobulin Fc region.

[0106] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 4, the linker may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 27-29, the hinge region may be derived from the hinge region of IgG1 or IgG4, and the immunoglobulin Fc region may be the Fc region derived from IgG4. For example, the fusion protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 37-39.

[0107] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 25, the linker may comprise the amino acid sequence set forth in SEQ ID NO: 28, the hinge region may be derived from the hinge region of IgG1 or IgG4, and the immunoglobulin Fc region may be derived from the Fc region of IgG4 and optionally comprise the amino acid mutations M252Y, S254T, and T256E. For example, the fusion protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 40-42 and 44.

[0108] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:4, the linker may comprise the amino acid sequence set forth in SEQ ID NO:28, the hinge region may be derived from the hinge region of IgG1 or IgG4, and the immunoglobulin Fc region may be an Fc region derived from IgG4 and comprise the amino acid mutations M252Y, S254T, and T256E. For example, the fusion protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs:43 and 45.

[0109] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:4, the linker may comprise the amino acid sequence set forth in SEQ ID NO:28, the hinge region may be derived from the hinge region of IgG1, and the immunoglobulin Fc region may be derived from the Fc region of IgG4 and comprise the amino acid mutations M252Y, S254T, and T256E. For example, the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:43.

[0110] In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 25, the linker may comprise the amino acid sequence set forth in SEQ ID NO: 28, the hinge region may be derived from the hinge region of IgG1, and the immunoglobulin Fc region may be derived from the Fc region of IgG4 and comprise the amino acid mutations M252Y, S254T, and T256E. For example, the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 44.

[0111] In the present application, the fusion protein may be present in the form of an immunoconjugate.

[0112] Pharmaceutical composition

[0113] The present application provides a pharmaceutical composition comprising the aforementioned fusion protein, wherein the pH value of the pharmaceutical composition may be 5.0-7.0. Under the pH conditions specified in the present application, the pharmaceutical composition of the present application has excellent stability. In some embodiments, the pH value of the pharmaceutical composition may be 5.5-6.5, preferably 5.5-6.2, more preferably 6.0-6.2, specifically 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0.

[0114] To resist the effect of pH changes on the stability of the composition, the pharmaceutical composition of the present application may further comprise one or more buffer components, for example, the buffer component is selected from one or more of the following groups: L-histidine-acetate, citrate, L-histidine-hydrochloric acid, acetic acid-sodium acetate and phosphate, and the buffer component may preferably be L-histidine-acetate or citrate.

[0115] The concentration of the buffer component described in the present application may be 1 nM-75 mM, preferably 2 mM-50 mM, more preferably 5 mM-40 mM, and even more preferably 10 mM-30 mM. Specifically, the concentration of the buffer component can be 2mM-45mM, 2mM-40mM, 2mM-30mM, 5mM-50mM, 5mM-45mM, 5mM-35mM, 10mM-60mM, 10mM-55mM, 10mM-40mM, 10mM-35mM, 15mM-65mM, 15mM-60mM, 15mM-45mM, 15mM-35mM, 20mM-70mM, 20mM-55mM, 20mM-50mM, 20mM-40mM, 25mM-75mM, 25mM-60mM or 25mM-45mM.

[0116] In some embodiments, the buffer component may comprise L-histidine-acetate, and the concentration of the L-histidine-acetate may be 2mM-50mM, 5mM-45mM, 10mM-40mM, 10mM-30mM, 15mM-35mM, 15mM-30mM, 15mM-25mM, or 20mM-25mM.

[0117] In some embodiments, the buffer component can include citrate, and the concentration of the citrate can be 1 mM-30 mM, 2 mM-25 mM, 2 mM-20 mM, 5 mM-20 mM, 5 mM-15 mM, or 10 mM-15 mM.

[0118] The pharmaceutical composition of the present application may further comprise a stabilizer to protect the fusion protein from chemical and / or physical degradation. The stabilizer may be selected from polyols, amino acids, sugars, salts, and / or albumin. Specifically, the stabilizer described herein is selected from one or more of the following groups: mannitol, L-arginine hydrochloride, glycine, L-proline, sorbitol, glycerol, maltose, trehalose, and sodium chloride; preferably, the stabilizer is selected from one or more of the following groups: mannitol, L-arginine hydrochloride, glycine, and L-proline; more preferably, the stabilizer is selected from one or more of the following groups: mannitol, glycine, and L-proline; and even more preferably, the stabilizer comprises mannitol.

[0119] The concentration of the stabilizer described in the present application can be 2 mg / mL-300 mg / mL, can be 5 mg / mL-300 mg / mL, can be preferably 10 mg / mL-150 mg / mL, can be more preferably 20 mg / mL-100 mg / mL, and can even more preferably be 25 mg / mL-80 mg / mL. Specifically, the concentration of the stabilizer can be 5 mg / mL-270 mg / mL, 5 mg / mL-260 mg / mL, 5 mg / mL-250 mg / mL, 5 mg / mL-200 mg / mL, 5 mg / mL-170 mg / mL, 10 mg / mL-260 mg / mL, 10 mg / mL- 240mg / mL, 10mg / mL-220mg / mL, 10mg / mL-190mg / mL, 10mg / mL-160mg / mL, 20mg / mL-240mg / mL, 2 0mg / mL-230mg / mL, 20mg / mL-180mg / mL, 20mg / mL-150mg / mL, 25mg / mL-210mg / mL, 25mg / mL-200 mg / mL, 25mg / mL-150mg / mL, 25mg / mL-100mg / mL, 25mg / mL-80mg / mL, 30mg / mL-140mg / mL, 30mg / mL-130mg / mL, 30mg / mL-120mg / mL, 30mg / mL-110mg / mL, 30mg / mL-70mg / mL or 30mg / mL-60mg / mL.

[0120] In some embodiments, the stabilizer comprises mannitol, and the concentration of mannitol may be 10 mg / mL-150 mg / mL, preferably 20 mg / mL-100 mg / mL, more preferably 30 mg / mL-80 mg / mL, and even more preferably 40 mg / mL-60 mg / mL. Specifically, the concentration of the mannitol can be 10mg / mL-100mg / mL, 10mg / mL-80mg / mL, 15mg / mL-140mg / mL, 15mg / mL-120mg / mL, 15mg / mL-110mg / mL, 20mg / mL-130mg / mL, 20mg / mL-100mg / mL, 20mg / mL-90mg / mL, 20mg / mL-75mg / mL, 25mg / mL-120mg / mL, 25mg / mL-105mg / mL, 25mg / mL-85mg / mL, 25mg / mL-80mg / mL, 25mg / mL-65mg / mL, 30mg / mL-115mg / mL, 30mg / mL-95mg / mL or 30mg / mL-70mg / mL.

[0121] In some embodiments, the stabilizer comprises glycine, and the concentration of the glycine may be 2 mg / mL-50 mg / mL, preferably 5 mg / mL-40 mg / mL, more preferably 10 mg / mL-30 mg / mL, and even more preferably 15 mg / mL-25 mg / mL. Specifically, the concentration of the glycine may be 1 mg / mL-45 mg / mL, 1 mg / mL-40 mg / mL, 2 mg / mL-40 mg / mL, 2 mg / mL-35 mg / mL, 5 mg / mL-50 mg / mL, 5 mg / mL-40 mg / mL, 5 mg / mL-30 mg / mL, 10 mg / mL-35 mg / mL, 10 mg / mL-25 mg / mL, 15 mg / mL-40 mg / mL, 15 mg / mL-35 mg / mL, 15 mg / mL-25 mg / mL, 18 mg / mL-30 mg / mL, or 18 mg / mL-20 mg / mL.

[0122] In some embodiments, the stabilizer comprises L-proline, and the concentration of L-proline may be 5 mg / mL-75 mg / mL, preferably 10 mg / mL-50 mg / mL, more preferably 15 mg / mL-40 mg / mL, and even more preferably 20 mg / mL-30 mg / mL. Specifically, the concentration of L-proline can be 2 mg / mL-100 mg / mL, 2 mg / mL-75 mg / mL, 5 mg / mL-75 mg / mL, 5 mg / mL-60 mg / mL, 5 mg / mL-50 mg / mL, 10 mg / mL-70 mg / mL, 10 mg / mL-50 mg / mL, 10 mg / mL-45 mg / mL, 15 mg / mL-80 mg / mL, 15 mg / mL-55 mg / mL, 15 mg / mL-45 mg / mL, 20 mg / mL-65 mg / mL, 20 mg / mL-50 mg / mL, 20 mg / mL-40 mg / mL, 20 mg / mL-30 mg / mL, 25 mg / mL-45 mg / mL, 25 mg / mL-40 mg / mL or 25 mg / mL-30 mg / mL.

[0123] In some embodiments, the stabilizer comprises L-arginine hydrochloride, and the concentration of the L-arginine hydrochloride may be 10 mg / mL-100 mg / mL, preferably 20 mg / mL-80 mg / mL, more preferably 25 mg / mL-60 mg / mL, and even more preferably 30 mg / mL-50 mg / mL. Specifically, the concentration of the L-arginine hydrochloride may be 5 mg / mL-100 mg / mL, 5 mg / mL-80 mg / mL, 5 mg / mL-70 mg / mL, 10 mg / mL-100 mg / mL, 10 mg / mL-60 mg / mL, 10mg / mL-50mg / mL, 15mg / mL-90mg / mL, 15mg / mL-60mg / mL, 15mg / mL-50mg / mL, 20mg / mL-80mg / mL, 20mg / mL-75mg / mL, 20mg / mL-70mg / mL, 25mg / mL- 70mg / mL, 25mg / mL-60mg / mL, 25mg / mL-45mg / mL, 30mg / mL-80mg / mL, 30mg / mL-70mg / mL, 30mg / mL-50mg / mL, 30mg / mL-40mg / mL or 35mg / mL-40mg / mL.

[0124] The pharmaceutical composition of the present application may further include a surfactant, which is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188; preferably, the surfactant is polysorbate 80 or polysorbate 20.

[0125] The concentration of the surfactant described in the present application may be 0.01 mg / mL-1.0 mg / mL, preferably 0.05 mg / mL-0.75 mg / mL, more preferably 0.1 mg / mL-0.5 mg / mL, and even more preferably 0.15 mg / mL-0.25 mg / mL. Specifically, the concentration of the surfactant can be 0.01 mg / mL-1.0 mg / mL, 0.01 mg / mL-0.75 mg / mL, 0.05 mg / mL-0.9 mg / mL, 0.05 mg / mL-0.8 mg / mL, 0.05 mg / mL-0.7 mg / mL, 0.1 mg / mL-0.7 mg / mL, 0.1 mg / mL-0.5 mg / mL, 0.15 mg / mL-0.75 mg / mL, 0.15 mg / mL-0.6 mg / mL, 0.15 mg / mL-0.5 mg / mL, 0.15 mg / mL-0.4 mg / mL, 0.15 mg / mL-0.3 mg / mL, 0.15 mg / mL-0.25 mg / mL, 0.2 mg / mL-0.6 mg / mL, 0.2 mg / mL-0.4 mg / mL or 0.2 mg / mL-0.3 mg / mL.

[0126] In the pharmaceutical composition of the present application, the fusion protein can be stably present at a higher protein concentration, and the concentration of the fusion protein is 1 mg / mL-30 mg / mL, preferably 2 mg / mL-20 mg / mL, and more preferably 5 mg / mL-10 mg / mL. Specifically, the concentration of the fusion protein can be 0.5 mg / mL-30 mg / mL, 0.5 mg / mL-25 mg / mL, 1 mg / mL-30 mg / mL, 1 mg / mL-25 mg / mL, 1 mg / mL-20 mg / mL, 2 mg / mL-20 mg / mL, 2 mg / mL-15 mg / mL, 2 mg / mL-10 mg / mL, 5 mg / mL-15 mg / mL or 5 mg / mL-10 mg / mL.

[0127] The pharmaceutical composition of the present application may be a liquid preparation, for example, an injection, preferably a subcutaneous injection. The liquid preparation includes a solvent, and the solvent is water for injection or an organic solvent for injection; preferably, the organic solvent for injection is selected from one or more of injection oil, ethanol, and propylene glycol; more preferably, the solvent is water for injection.

[0128] The pharmaceutical composition of the present application has excellent stability. For example, in an accelerated stability study, after the composition is placed at 25±2°C for 6 months, the monomer content of the fusion protein is still above 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5%. The monomer content can be measured by SE-HPLC purity.

[0129] On the other hand, the present application also provides a kit, which includes the composition described in the present application and a container for holding the composition described in the present application.

[0130] For example, the container can include a vial (e.g., a glass bottle), an ampoule, a syringe, an injection pen and / or an intravenous bag. In the present application, the container can be sterile. For example, the container can include a glass bottle.

[0131] For example, the kit can include a delivery device that can administer the compositions described herein. For example, the delivery device can include a vial, an ampoule, a syringe, an injection pen, and / or an intravenous bag.

[0132] In the present application, the volume of the composition in the container may be 0.1 mL to about 5.0 mL, preferably 0.2 mL to 3.0 mL, and more preferably 0.5 mL to 2.0 mL.

[0133] Therapeutic uses

[0134] The pharmaceutical composition of the present application can be used to prevent and / or treat metabolic diseases or conditions. In one aspect, the present application provides a method for preventing and / or treating metabolic diseases or conditions, comprising the following steps: administering the pharmaceutical composition to a subject in need thereof.

[0135] On the other hand, the present application also provides use of the pharmaceutical composition in preparing a medicament for preventing and / or treating metabolic diseases or disorders.

[0136] In addition, the present application also provides a pharmaceutical composition, which can be used to prevent and / or treat metabolic diseases or disorders.

[0137] The metabolic disease or disorder may comprise a GLP-1-related metabolic disease; preferably, the metabolic disease comprises diabetes; more preferably, the metabolic disease comprises type II diabetes.

[0138] In another aspect, the present application provides a method for increasing or promoting insulin expression in a subject in need thereof, comprising administering the pharmaceutical composition to the subject.

[0139] For example, a subject in need may include a person suffering from a metabolic disease or condition. For example, a subject in need may include a person suffering from a GLP-1 related metabolic disease. For example, a subject in need may include a person suffering from diabetes. For example, a subject in need may include a person suffering from type II diabetes.

[0140] For example, the expression of insulin can include the activity of insulin secretion. For example, the expression of insulin can include the amount and concentration of insulin. For example, the expression of insulin can be detected by any method known in the art.

[0141] For example, the increasing or promoting insulin expression in a subject in need thereof can comprise increasing or promoting the expression level of insulin in the subject by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300% or more relative to the level before administration of the drug combination.

[0142] This application specifically provides the following implementation schemes:

[0143] 1. A pharmaceutical composition comprising a fusion protein comprising a human GLP-1 polypeptide variant and an immunoglobulin Fc region; wherein the amino acid sequence of the human GLP-1 polypeptide variant comprises at least two amino acid mutations compared to the amino acid sequence of SEQ ID NO: 2, and the at least two amino acid mutations are located at amino acid positions selected from the group consisting of: W31, K26, and Y19;

[0144] The pH value of the pharmaceutical composition is 5.0-7.0.

[0145] 2. The pharmaceutical composition according to embodiment 1, wherein the immunoglobulin Fc region is located at the C-terminus of the human GLP-1 polypeptide variant.

[0146] 3. The pharmaceutical composition according to embodiment 1 or 2, wherein the immunoglobulin Fc region is an Fc region derived from IgG; preferably an Fc region derived from IgG1, IgG2, IgG3 or IgG4; more preferably an Fc region selected from IgG4.

[0147] 4. A pharmaceutical composition according to any one of embodiments 1-3, wherein the immunoglobulin Fc region comprises an amino acid mutation, and the amino acid mutation selectively enhances the binding affinity of the immunoglobulin Fc region to the Fc receptor compared to the immunoglobulin Fc region without the amino acid mutation.

[0148] 5. A pharmaceutical composition according to any one of embodiments 1-4, wherein the immunoglobulin Fc region comprises amino acid mutations at positions M252, S254 and T256 according to EU encoding; preferably comprises the following group of amino acid mutations: M252Y, S254T and T256E.

[0149] 6. A pharmaceutical composition according to any one of embodiments 1-5, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO: 36.

[0150] 7. A pharmaceutical composition according to any one of embodiments 1-6, wherein the fusion protein further comprises a hinge region, the hinge region is located between the human GLP-1 polypeptide variant and the immunoglobulin Fc region, and the hinge region comprises the amino acid sequence shown in any one of SEQ ID NOs: 30-31; preferably comprises the amino acid sequence shown in SEQ ID NO: 30.

[0151] 8. A pharmaceutical composition according to any one of embodiments 1-7, wherein the fusion protein further comprises a linker, and the linker is located between the human GLP-1 polypeptide variant and the immunoglobulin Fc region; preferably, it is located between the human GLP-1 polypeptide variant and the hinge region.

[0152] 9. According to the pharmaceutical composition of embodiment 8, the linker is a peptide having a length of 5-20 amino acids; preferably, the linker comprises an amino acid sequence as shown in any one of SEQ ID NOs: 27-29; more preferably, the linker comprises an amino acid sequence as shown in SEQ ID NO: 28.

[0153] 10. The pharmaceutical composition according to any one of embodiments 1 to 9, wherein the amino acid sequence of the human GLP-1 polypeptide variant comprises an amino acid mutation at a position selected from the group consisting of:

[0154] a) at amino acid positions W31 and K26;

[0155] b) at amino acid positions W31 and Y19;

[0156] c) at amino acid positions K26 and Y19;

[0157] and d) at amino acid positions W31, K26, and Y19

[0158] 11. The pharmaceutical composition according to any one of embodiments 1-10, wherein the mutation at amino acid position W31 is selected from W31Y, W31R, W31K and W31A; preferably W31Y.

[0159] 12. The pharmaceutical composition of any one of embodiments 1-11, wherein the mutation at amino acid position K26 is K26R.

[0160] 13. The pharmaceutical composition of any one of embodiments 1-12, wherein the mutation at amino acid position Y19 is selected from the group consisting of Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S; preferably Y19A.

[0161] 14. The pharmaceutical composition according to any one of embodiments 1 to 13, wherein the amino acid sequence of the human GLP-1 polypeptide variant comprises a combination of amino acid mutations of W31Y, K26R and Y19A; or W31Y and K26R, compared to the amino acid sequence shown in SEQ ID NO: 2.

[0162] 15. The pharmaceutical composition according to any one of embodiments 1-13, wherein the GLP-1 polypeptide variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-26.

[0163] 16. A pharmaceutical composition according to any one of embodiments 1-15, wherein the fusion protein comprises the human GLP-1 polypeptide variant, a linker, a hinge region and an immunoglobulin Fc region in sequence from N-terminus to C-terminus.

[0164] 17. A pharmaceutical composition according to any one of embodiments 1-16, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 37-45.

[0165] 18. The pharmaceutical composition according to any one of embodiments 1-17, wherein the pH value of the pharmaceutical composition is 5.5-6.5; preferably the pH value is 5.5-6.2; more preferably the pH value is 6.0-6.2.

[0166] 19. The pharmaceutical composition of any one of embodiments 1-18, comprising a buffer component.

[0167] 20. The pharmaceutical composition according to embodiment 19, wherein the buffer component is selected from one or more of the following groups: L-histidine-acetate, citrate, L-histidine-hydrochloric acid, acetic acid-sodium acetate and phosphate; the buffer component is preferably L-histidine-acetate or citrate.

[0168] 21. The pharmaceutical composition according to embodiment 19 or 20, wherein the concentration of the buffer component is 2 mM-50 mM; preferably 5 mM-40 mM; more preferably 10 mM-30 mM; even more preferably 20 mM.

[0169] 22. The pharmaceutical composition of any one of embodiments 1-21, further comprising a stabilizer.

[0170] 23. The pharmaceutical composition according to embodiment 22, wherein the stabilizer is selected from one or more of the following groups: mannitol, L-arginine hydrochloride, glycine, L-proline, sorbitol, glycerol, maltose, trehalose and sodium chloride; preferably, the stabilizer is selected from one or more of the following groups: mannitol, L-arginine hydrochloride, glycine and L-proline; more preferably, the stabilizer is selected from one or more of the following groups: mannitol, glycine and L-proline.

[0171] 24. The pharmaceutical composition according to embodiment 23, wherein the concentration of the stabilizer is 2 mg / mL-150 mg / mL, preferably 10 mg / mL-150 mg / mL; preferably 20 mg / mL-100 mg / mL.

[0172] 25. The pharmaceutical composition according to embodiment 23, wherein the stabilizer comprises mannitol, and the concentration of mannitol is 10 mg / mL-150 mg / mL; preferably 20 mg / mL-100 mg / mL; more preferably 30 mg / mL-80 mg / mL; even more preferably 40 mg / mL-60 mg / mL.

[0173] 26. According to the pharmaceutical composition of embodiment 23, the stabilizer comprises glycine, and the concentration of glycine is 2 mg / mL-50 mg / mL; preferably 5 mg / mL-40 mg / mL; more preferably 10 mg / mL-30 mg / mL; even more preferably 15 mg / mL-25 mg / mL.

[0174] 27. The pharmaceutical composition according to embodiment 23, wherein the stabilizer comprises L-proline, and the concentration of L-proline is 5 mg / mL-75 mg / mL; preferably 10 mg / mL-50 mg / mL; more preferably 15 mg / mL-40 mg / mL; even more preferably 20 mg / mL-30 mg / mL.

[0175] 28. According to the pharmaceutical composition of embodiment 23, the stabilizer comprises L-arginine hydrochloride, and the concentration of L-arginine hydrochloride is 10 mg / mL-100 mg / mL; preferably 20 mg / mL-80 mg / mL; more preferably 25 mg / mL-60 mg / mL; even more preferably 30 mg / mL-50 mg / mL.

[0176] 29. The pharmaceutical composition of any one of embodiments 1-28, further comprising a surfactant.

[0177] 30. The pharmaceutical composition according to embodiment 29, wherein the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188; preferably, the surfactant is polysorbate 80 or polysorbate 20.

[0178] 31. The pharmaceutical composition according to embodiment 30, wherein the concentration of the surfactant is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

[0179] 32. The pharmaceutical composition of embodiment 30, wherein the surfactant comprises polysorbate 80, and the concentration of polysorbate 80 is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

[0180] 33. The pharmaceutical composition of embodiment 30, wherein the surfactant comprises polysorbate 20, and the concentration of polysorbate 20 is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

[0181] 34. The pharmaceutical composition of embodiment 30, wherein the surfactant comprises poloxamer 188, and the concentration of poloxamer 188 is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

[0182] 35. The pharmaceutical composition of any one of embodiments 1-34, wherein the concentration of the fusion protein is 1 mg / mL-30 mg / mL; preferably 2 mg / mL-14 mg / mL; more preferably 5 mg / mL-10 mg / mL.

[0183] 36. The pharmaceutical composition according to any one of embodiments 1-35, which is a liquid preparation.

[0184] 37. The pharmaceutical composition according to embodiment 36, wherein the liquid preparation is an injection solution, preferably a subcutaneous injection solution.

[0185] 38. The pharmaceutical composition according to embodiment 36 or 37, wherein the liquid preparation comprises a solvent, and the solvent is water for injection or an organic solvent for injection; preferably, the organic solvent for injection is selected from one or more of oil for injection, ethanol, and propylene glycol.

[0186] 39. According to any one of embodiments 1-38, after the pharmaceutical composition is placed at 25±2°C for 6 months, the monomer content of the fusion protein is still above 96%; preferably, the monomer content is above 97%; more preferably, the monomer content is above 98%, and even more preferably, the monomer content is above 99%.

[0187] 40. A kit comprising the pharmaceutical composition of any one of embodiments 1-39 and a container for holding the composition.

[0188] 41. The kit of embodiment 40, wherein the container comprises a glass bottle.

[0189] 42. The kit according to embodiment 40 or 41, wherein the volume of the pharmaceutical composition in the container is 0.1 mL-5.0 mL; preferably 0.2 mL-3.0 mL; more preferably 0.5 mL-2.0 mL.

[0190] 43. Use of the pharmaceutical composition of any one of embodiments 1-39, and / or the kit of any one of embodiments 40-42, in the preparation of a medicament for preventing and / or treating a metabolic disease or disorder.

[0191] 44. The use according to embodiment 43, wherein the metabolic disease or disorder comprises a GLP-1 related metabolic disease or disorder; preferably, the metabolic disease or disorder comprises diabetes; more preferably, the metabolic disease or disorder comprises type II diabetes.

[0192] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0193] Example

[0194] Example 1 Preparation of fusion protein

[0195] A fusion protein of a human GLP-1 polypeptide variant and Fc is prepared, comprising, from N-terminus to C-terminus, a GLP-1 polypeptide variant (amino acid sequence as shown in any one of SEQ ID NOs: 4-25), a connecting peptide (amino acid sequence as shown in any one of SEQ ID NOs: 27-29), a hinge region (amino acid sequence as shown in any one of SEQ ID NOs: 30-31), and an IgG-derived Fc region (amino acid sequence as shown in any one of SEQ ID NOs: 32-36). A vector containing a nucleic acid sequence encoding the fusion protein is introduced into cells, and the fusion protein is expressed and purified. Exemplary full-length and partial sequences of the fusion protein are shown in Table 1 below.

[0196] Table 1 Structure and amino acid sequence of exemplary fusion proteins

[0197] Example 2 Pharmacokinetics of fusion proteins using different linkers

[0198] The in vivo pharmacokinetics of the present invention's fusion proteins, GM-RY-L1H2-Fc4, GM-RY-L2H2-Fc4, and GM-RY-L3H2-Fc4, were investigated. The experiments were conducted in an SPF animal room with an ambient temperature of 23±2°C, a relative humidity of 40-70%, and a 12-hour light-dark cycle. The experimental animals were allowed to eat freely and acclimate for at least 3 days before the experiment. SPF-grade Sprague-Dawley rats weighing 180-220g were randomly divided into groups. Each group of rats received a subcutaneous (SC) injection of the corresponding test article at a dose of 1 mg / kg, with dulaglutide (DULA) serving as a control. Approximately 100 μl of blood was collected from the jugular vein before, and 6, 24, 48, 72, 96, and 120 hours after administration. The collected blood was placed in a centrifuge tube and allowed to stand at room temperature for 30-60 minutes. The serum was then rapidly separated by centrifugation at 4000 rpm for 10 minutes within two hours at 4°C. The samples were stored at -80°C. The samples were tested by ELISA. The pharmacokinetic parameters were calculated using DAS (3.2.8) software.

[0199] The results are shown in Figure 1 and Table 2. The in vivo metabolic properties of GM-RY-L1H2-Fc4, GM-RY-L2H2-Fc4, and GM-RY-L3H2-Fc4 were not significantly different from those of the control dulaglutide. Among them, GM-RY-L2H2-Fc4 using the L2 linker (SEQ ID NO: 28) had the best half-life and the exposure was comparable to that of the control.

[0200] Table 2 Pharmacokinetic test results

[0201] Example 3 Pharmacokinetics of Fusion Proteins with Different Hinge Regions

[0202] According to the method of Example 2, the in vivo pharmacokinetics of the fusion proteins GM-ARY-L2H2-Fc4 and GM-ARY-L2H1-Fc4 of the present application were tested. Samples were tested by ELISA. Pharmacokinetic parameters were calculated using DAS (3.2.8) software. The results, as shown in Figure 2 and Table 3, show that the in vivo half-lives of GM-ARY-L2H2-Fc4 and GM-ARY-L2H1-Fc4 are comparable, with GM-ARY-L2H1-Fc4 using the hinge region of IgG1 (SEQ ID NO: 30) showing better exposure.

[0203] Table 3 Pharmacokinetic test results

[0204] Example 4 Pharmacokinetics of Fusion Proteins Containing YTE Mutations in the Fc Region

[0205] The in vivo pharmacokinetics of the fusion proteins GM-RY-L2H1-Fc4m and GM-ARY-L2H1-Fc4m of the present application were detected using cynomolgus monkeys. The experiment consisted of two cynomolgus monkeys in each group, half male and half female. Each group was given a single subcutaneous injection of the corresponding test article (1 mg / kg), DULA (dulaglutide with no YTE mutation in the Fc region of IgG4) and DULA-Fc4m (dulaglutide with YTE mutation in the Fc region of IgG4) as controls. Blood samples were collected before and 4h, 8h, 24h, 48h, 72h, 120h, 168h, 264h, 336h, 504h, 672h, 840h and 1008h after administration. The serum was separated and the concentration of the test substance in the serum of the cynomolgus monkey after administration was determined by ELISA. The pharmacokinetic parameters were calculated using DAS (3.2.8) software.

[0206] The results are shown in Figures 3, 4, Tables 4, and 5, where the data in Table 4 are derived from the curve in Figure 3, and the data in Table 5 are derived from the curve in Figure 4. Compared to dulaglutide, which also contains the YTE mutation, the GM-RY-L2H1-Fc4m (Figures 3 and 4) and GM-ARY-L2H1-Fc4m (Figure 4) of the present application have better in vivo half-life and exposure; however, the effect of the YTE mutation in the Fc region of dulaglutide is weak, and compared with unmutated dulaglutide, the YTE mutation in the Fc region does not increase the in vivo half-life and exposure of dulaglutide (Figure 4).

[0207] Combined with the data in Figure 1, the half-life and exposure of the fusion protein of the present application without the YTE mutation in the Fc region are comparable to those of dulaglutide, while the half-life and exposure of the fusion protein of the present application after the YTE mutation in the Fc region are significantly higher than those of dulaglutide. This indicates that the YTE mutation in the Fc region significantly improves the pharmacokinetic properties of the fusion protein of the present application, and that the amino acid mutations in the human GLP-1 polypeptide variant of the fusion protein and the YTE mutation in the Fc region have a synergistic effect on improving pharmacokinetic properties.

[0208] Table 4 Pharmacokinetic test results

[0209] Table 5 Pharmacokinetic test results

[0210] Example 5 Pharmacokinetic testing of fusion proteins using Fc region YTE mutations with different hinge regions

[0211] The in vivo pharmacokinetics of the fusion proteins GM-ARY-L2H2-Fc4m, GM-RY-L2H2-Fc4m, GM-ARY-L2H1-Fc4m, and GM-RY-L2H1-Fc4m comprising the YTE mutation in the Fc region of the present application were tested according to the method of Example 4. Samples were tested by ELISA. Pharmacokinetic parameters were calculated using DAS (3.2.8) software.

[0212] The results are shown in FIG5 and Table 6. The in vivo half-life or exposure of the four fusion proteins were all good, among which the fusion protein using the hinge region of IgG1 (SEQ ID NO: 30) had the best overall effect.

[0213] Table 6 Pharmacokinetic test results

[0214] Example 6 Luciferase assay to detect the activation effect of the fusion protein of the present application on the cAMP / PKA signaling pathway

[0215] The fusion protein of the GLP-1 polypeptide variant binds to the GLP-1 receptor, activates adenylate cyclase, and promotes the increase of intracellular cyclic adenosine monophosphate (cAMP) levels. The activation effect of the fusion protein of this application on the cAMP / PKA signaling pathway is detected based on the CREB (cAMP response element binding protein) reporter gene. HER293 cells were transfected with a human GLP-1R plasmid and a CREB-driven luciferase reporter plasmid. After transfection, the HER293-GLP1R-CREB-D4 cells were digested and collected, counted, and the cell number was adjusted to 4×10 cells using DMEM medium containing 10% FBS. 5cells / ml, inoculated into a 96-well plate, 50 μl per well. The test sample was diluted into 9 concentrations starting from 1000 ng / ml using DMEM medium containing 10% FBS; dulaglutide was used as a positive control. 50 μl of a series of concentrations of the test sample were added to each well of the above 96-well plate and incubated at 37°C for several hours. The supernatant was discarded and luciferase substrate was added to each well. After standing at room temperature for 5 minutes, 50 μl was taken out and the luciferase analysis system Bio-Glo was used. TM Luciferase activity was detected using the Luciferase Assay System (Promega, G7940) and read using a microplate reader (Molecular Devices, SpectraMax M3). Fluorescence intensity reflects the level of cAMP, which in turn reflects the degree of GLP-1 receptor activation.

[0216] The results are shown in FIG7 , which show that the fusion protein of the present application has a GLP-1 receptor activation ability comparable to that of dulaglutide.

[0217] Example 7 Preparation and Investigation of Different Formulas of Fusion Protein

[0218] The prepared fusion protein GM-ARY-L2H1-Fc4m was ultrafiltrated with different buffer solutions and then concentrated to the required protein concentration using an ultrafiltration centrifuge tube with a 10kD molecular weight cutoff. Stabilizers in different formulations were added and the product was sterile filtered at 0.2μm before being packaged into corresponding packaging materials for stability testing.

[0219] Testing items and methods

[0220] (1) SE-HPLC purity: Refer to the 0514 size exclusion chromatography method in the 2020 edition of the Chinese Pharmacopoeia, Part III, for product purity testing. The chromatographic column was TOSOH TSKgel UP-SW3000 4.6*150 mm, 2 μm; the mobile phase was 50 mM Na2HPO4, 250 mM L-Arg-HCl; the detection wavelength was 280 nm, and the area normalization method was used for calculation.

[0221] (2) RP-HPLC purity: Refer to the 2020 edition of the Chinese Pharmacopoeia, Part III, General Chapter 0512, High Performance Liquid Chromatography, for detecting product degradation. The chromatographic column was a Waters ACQUITY UPLC Protein BEH C4 300A 2.1×100 mm, 1.7 μm; the detection wavelength was 214 nm, and the purity was calculated by the area normalization method.

[0222] (3) CE-SDS purity: Refer to the 3127 Monoclonal Antibody Size Variant Determination Method in the 2020 edition of the Chinese Pharmacopoeia. The sample is denatured (non-reducing conditions) or denatured and reduced (reducing conditions) and placed in a capillary electrophoresis instrument for electrophoresis analysis at a wavelength of 220 nm, calculated by the area normalization method.

[0223] (4) Protein content: refer to the sixth method of protein content determination in Part III of the 2020 edition of the Chinese Pharmacopoeia, Part 0731, and calculate the protein content based on the absorbance value of protein at a wavelength of 280 nm and the theoretical extinction coefficient of protein.

[0224] (5) Insoluble particles: The number of particles ≥10 μm and ≥25 μm was measured on an AccuSizer 780SIS insoluble particle analyzer.

[0225] (6) Dynamic light scattering (DLS): The average particle diameter (Z.average.Diameter) and distribution index (PDI) of the sample were measured on a PUNK type particle size analyzer.

[0226] (7) Relative binding activity: enzyme-linked immunosorbent assay (ELISA) was used.

[0227] Example 8 Screening of buffer pH

[0228] At a protein content of 10 mg / ml, stability was tested at 30 ± 2°C in the pH range of 5.5-6.5. Protein purity was determined by SE-HPLC and RP-HPLC, and differences in particle size were determined by insoluble particulate matter analysis. The specific formulation composition and test results are summarized in Table 7.

[0229] Table 7 Summary of test results of high temperature accelerated test for 14 days and 28 days under various pH conditions Note: Insoluble particles are the result of samples accelerated at high temperature for 14 days

[0230] Overall, the formulation is relatively stable within this pH range. SE-HPLC analysis revealed a decreasing trend in monomer content with decreasing pH, but no significant purity differences were observed between pH 6.0 and 6.5. RP-HPLC analysis also revealed no significant differences between pH values. Insoluble particulate levels were relatively low at pH 6.0-6.2, generally within an acceptable range.

[0231] Example 9 Screening of buffer systems

[0232] At a protein content of 10 mg / ml, the stability of two buffer systems, L-histidine-acetate and citrate, was investigated at pH 6.0 at 30 ± 2°C. Protein purity was determined by SE-HPLC and RP-HPLC. The specific formulations and test results are summarized in Table 8.

[0233] Table 8 Summary of the test results of different buffer systems at high temperature for 14 days and 28 days

[0234] There was no significant difference in the SE-HPLC test results among the groups, indicating good stability. The SE-HPLC test results showed that the degradation rate of the two stabilizer formulas in the L-histidine-acetate buffer system was slightly lower than that of citrate.

[0235] Example 10 Selection of stabilizer types

[0236] The effects of different stabilizers on protein stability at 30±2°C were investigated in L-histidine-acetate buffer at a protein content of 10 mg / ml. Protein purity was determined by SE-HPLC and RP-HPLC, and protein homogeneity was determined by DLS. The specific formulation composition and test results are shown in Table 9.

[0237] Table 9 Summary of the results of accelerated high temperature testing for 14 and 28 days for formulations containing different stabilizers Note: DLS is the test result of the sample stored at 2-8℃ for 9 months without acceleration.

[0238] SE-HPLC and RP-HPLC analysis revealed no significant differences between formulations containing different stabilizers, with high protein purity across all groups. DLS analysis demonstrated high homogeneity across all groups. Overall, the stabilizers mannitol, glycine, and L-proline demonstrated excellent protein stability.

[0239] Example 11 Investigating the influence of protein content

[0240] The effects of varying protein content were investigated in a formulation containing 10 mM citrate, 50 mg / ml mannitol, and 0.02% polysorbate 80 at pH 6.0. Accelerated high-temperature testing at 30 ± 2°C was performed. Protein purity was determined by SE-HPLC and RP-HPLC, and protein particle size was determined by insoluble particulate matter analysis. The specific formulation composition and test results are shown in Table 10.

[0241] Table 10 Summary of test results of different protein content formulas at high temperature for 14 days and 28 days Note: The results of insoluble particulate matter test were obtained by high temperature accelerated 14-day sample testing.

[0242] The results showed that there was no significant difference in protein purity and particle number between the two formulations with different concentrations after high-temperature acceleration, and the stability was good.

[0243] Example 12 Screening of surfactant types

[0244] The effects of different surfactants on stability were investigated in a formulation containing 10 mM citrate, 50 mg / ml mannitol, pH 6.0. After shaking at 150 rpm for 14 days at room temperature (25 ± 2°C), protein purity was determined by SE-HPLC and RP-HPLC, and protein homogeneity was determined by DLS. The specific formulation composition and test results are shown in Table 11.

[0245] Table 11 Summary of screening and testing results of different surfactant types Note: DLS is the test result of the sample stored at 2-8℃ for 9 months without acceleration

[0246] The SE-HPLC and RP-HPLC test results showed that there was no significant difference in the effects of different surfactants on protein purity; in the DLS test results, the PDI of the formula containing poloxamer 188 was higher, indicating that its uniformity was lower, while the uniformity of the formulas containing polysorbate 80 and polysorbate 20 was higher.

[0247] Example 13 Accelerated stability study

[0248] Samples were formulated with 10 mg / ml protein, 20 mM L-histidine-acetate, 50 mg / ml mannitol, and 0.02% (w / v) polysorbate 80, pH 6.2±0.1. The samples were stored in vials at 25±2°C for 6 months, with samples collected at the end of months 0, 1, 2, 3, and 6. Stability testing was performed according to key criteria, including monomer purity (SE-HPLC), degradation content (RP-HPLC), reducing and non-reducing capillary gel electrophoresis (CE-SDS), protein content, and relative binding activity. The results are shown in Table 12.

[0249] Table 12 Accelerated (25±2°C, 6 months) stability study data

[0250] The results showed that after being placed at 25±2°C for 6 months, compared with the 0th day, all indicators of the samples were within the acceptable range, indicating that the formulation of the present application can still maintain stability at room temperature for 6 months.

[0251] Sequence information

Claims

1. A pharmaceutical composition comprising a fusion protein, wherein the fusion protein comprises a human GLP-1 polypeptide variant and an immunoglobulin Fc region; wherein the amino acid sequence of the human GLP-1 polypeptide variant comprises at least two amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 2, and the at least two amino acid mutations are located at amino acid positions selected from the group consisting of: W31, K26 and Y19; The pH value of the pharmaceutical composition is 5.0-7.

0. 2 . The pharmaceutical composition according to claim 1 , wherein the immunoglobulin Fc region is located at the C-terminus of the human GLP-1 polypeptide variant.

3. The pharmaceutical composition according to claim 1 or 2, wherein the immunoglobulin Fc region is an Fc region derived from IgG; preferably an Fc region derived from IgG1, IgG2, IgG3 or IgG4; more preferably an Fc region selected from IgG4.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the immunoglobulin Fc region comprises an amino acid mutation, and compared with the immunoglobulin Fc region without the amino acid mutation, the amino acid mutation selectively enhances the binding affinity of the immunoglobulin Fc region to the Fc receptor.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the immunoglobulin Fc region comprises amino acid mutations at positions M252, S254 and T256 encoded according to EU; preferably comprises the following group of amino acid mutations: M252Y, S254T and T256E.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO:

36.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein the fusion protein further comprises a hinge region, the hinge region is located between the human GLP-1 polypeptide variant and the immunoglobulin Fc region, and the hinge region comprises the amino acid sequence shown in any one of SEQ ID NOs: 30-31; preferably comprises the amino acid sequence shown in SEQ ID NO:

30.

8. The pharmaceutical composition according to any one of claims 1-7, wherein the fusion protein further comprises a linker, and the linker is located between the human GLP-1 polypeptide variant and the immunoglobulin Fc region; preferably between the human GLP-1 polypeptide variant and the hinge region.

9. The pharmaceutical composition according to claim 8, wherein the linker is a peptide having a length of 5-20 amino acids; preferably, the linker comprises an amino acid sequence as shown in any one of SEQ ID NOs: 27-29; more preferably, the linker comprises an amino acid sequence as shown in SEQ ID NO:

28.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the amino acid sequence of the human GLP-1 polypeptide variant comprises an amino acid mutation at a position selected from the following positions compared to the amino acid sequence shown in SEQ ID NO: 2: a) at amino acid positions W31 and K26; b) at amino acid positions W31 and Y19; c) at amino acid positions K26 and Y19; and d) at amino acid positions W31, K26 and Y19 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the mutation at the amino acid position W31 is selected from W31Y, W31R, W31K and W31A; preferably W31Y.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the mutation at the amino acid position K26 is K26R.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the mutation at the amino acid position Y19 is selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S; preferably Y19A.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant comprises a combination of amino acid mutations of W31Y, K26R and Y19A; or W31Y and K26R.

15. The pharmaceutical composition according to any one of claims 1-13, wherein the GLP-1 polypeptide variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-26.

16. The pharmaceutical composition according to any one of claims 1-15, wherein the fusion protein comprises, from N-terminus to C-terminus, the human GLP-1 polypeptide variant, a linker, a hinge region and an immunoglobulin Fc region.

17. The pharmaceutical composition according to any one of claims 1-16, wherein the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 37-45.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the pH value of the pharmaceutical composition is 5.5-6.5; preferably the pH value is 5.5-6.2; more preferably the pH value is 6.0-6.

2.

19. The pharmaceutical composition according to any one of claims 1 to 18, comprising a buffer component.

20. The pharmaceutical composition according to claim 19, wherein the buffer component is selected from one or more of the following groups: L-histidine-acetate, citrate, L-histidine-hydrochloric acid, acetic acid-sodium acetate and phosphate; the buffer component is preferably L-histidine-acetate or citrate.

21. The pharmaceutical composition according to claim 19 or 20, wherein the concentration of the buffer component is 2 mM-50 mM; preferably 5 mM-40 mM; more preferably 10 mM-30 mM; even more preferably 20 mM.

22. The pharmaceutical composition according to any one of claims 1-21, further comprising a stabilizer.

23. The pharmaceutical composition of claim 22, wherein the stabilizer is selected from one or more of the group consisting of mannitol, L-arginine hydrochloride, glycine, L-proline, sorbitol, glycerol, maltose, trehalose and sodium chloride; preferably, the stabilizer is selected from one or more of the group consisting of mannitol, L-arginine hydrochloride, glycine and L-proline; more preferably, the stabilizer is selected from one or more of the group consisting of mannitol, glycine and L-proline.

24. The pharmaceutical composition according to claim 23, wherein the concentration of the stabilizer is 2 mg / mL-150 mg / mL, preferably 10 mg / mL-150 mg / mL; preferably 20 mg / mL-100 mg / mL.

25. The pharmaceutical composition according to claim 23, wherein the stabilizer comprises mannitol, and the concentration of the mannitol is 10 mg / mL-150 mg / mL; preferably 20 mg / mL-100 mg / mL; more preferably 30 mg / mL-80 mg / mL; even more preferably 40 mg / mL-60 mg / mL.

26. The pharmaceutical composition according to claim 23, wherein the stabilizer comprises glycine, and the concentration of the glycine is 2 mg / mL-50 mg / mL; preferably 5 mg / mL-40 mg / mL; more preferably 10 mg / mL-30 mg / mL; even more preferably 15 mg / mL-25 mg / mL.

27. The pharmaceutical composition according to claim 23, wherein the stabilizer comprises L-proline, and the concentration of the L-proline is 5 mg / mL-75 mg / mL; preferably 10 mg / mL-50 mg / mL; more preferably 15 mg / mL-40 mg / mL; even more preferably 20 mg / mL-30 mg / mL.

28. The pharmaceutical composition of claim 23, wherein the stabilizer comprises L-arginine hydrochloride, and the concentration of the L-arginine hydrochloride is 10 mg / mL-100 mg / mL; preferably 20 mg / mL-80 mg / mL; more preferably 25 mg / mL-60 mg / mL; even more preferably 30 mg / mL-50 mg / mL.

29. The pharmaceutical composition according to any one of claims 1-28, further comprising a surfactant.

30. The pharmaceutical composition according to claim 29, wherein the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188; preferably, the surfactant is polysorbate 80 or polysorbate 20.

31. The pharmaceutical composition of claim 30, wherein the concentration of the surfactant is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

32. The pharmaceutical composition of claim 30, wherein the surfactant comprises polysorbate 80 at a concentration of 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

33. The pharmaceutical composition of claim 30, wherein the surfactant comprises polysorbate 20, wherein the concentration of polysorbate 20 is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

34. The pharmaceutical composition of claim 30, wherein the surfactant comprises poloxamer 188, and the concentration of poloxamer 188 is 0.01 mg / mL-1.0 mg / mL; preferably 0.05 mg / mL-0.75 mg / mL; more preferably 0.1 mg / mL-0.5 mg / mL; even more preferably 0.15 mg / mL-0.25 mg / mL.

35. The pharmaceutical composition according to any one of claims 1-34, wherein the concentration of the fusion protein is 1 mg / mL-30 mg / mL; preferably 2 mg / mL-14 mg / mL; more preferably 5 mg / mL-10 mg / mL.

36. The pharmaceutical composition according to any one of claims 1-35, which is a liquid preparation.

37. The pharmaceutical composition according to claim 36, wherein the liquid preparation is an injection, preferably a subcutaneous injection.

38. The pharmaceutical composition according to claim 36 or 37, wherein the liquid preparation comprises a solvent, and the solvent is water for injection or an organic solvent for injection; preferably, the organic solvent for injection is selected from one or more of oil for injection, ethanol, and propylene glycol.

39. According to any one of claims 1-38, after the pharmaceutical composition is placed at 25±2°C for 6 months, the monomer content of the fusion protein is still above 96%; preferably the monomer content is above 97%; more preferably the monomer content is above 98%, and even more preferably the monomer content is above 99%.

40. A kit comprising the pharmaceutical composition of any one of claims 1 to 39 and a container for holding the composition.

41. The kit of claim 40, wherein the container comprises a glass bottle.

42. The kit according to claim 40 or 41, wherein the volume of the pharmaceutical composition in the container is 0.1 mL-5.0 mL; preferably 0.2 mL-3.0 mL; more preferably 0.5 mL-2.0 mL.

43. Use of the pharmaceutical composition according to any one of claims 1 to 39, and / or the kit according to any one of claims 40 to 42, in the preparation of a medicament for preventing and / or treating a metabolic disease or condition.

44. The use according to claim 43, wherein the metabolic disease or disorder comprises a GLP-1 related metabolic disease or disorder; preferably, the metabolic disease or disorder comprises diabetes; more preferably, the metabolic disease or disorder comprises type II diabetes.