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WO2024141054A1 - Pharmaceutical composition comprising fusion protein and use thereof - Google Patents

Pharmaceutical composition comprising fusion protein and use thereof Download PDF

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Publication number
WO2024141054A1
WO2024141054A1 PCT/CN2023/143414 CN2023143414W WO2024141054A1 WO 2024141054 A1 WO2024141054 A1 WO 2024141054A1 CN 2023143414 W CN2023143414 W CN 2023143414W WO 2024141054 A1 WO2024141054 A1 WO 2024141054A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
pharmaceutical composition
composition according
region
acid sequence
Prior art date
Application number
PCT/CN2023/143414
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French (fr)
Chinese (zh)
Inventor
徐霆
郭康平
恽丽红
吴圆丽
丁蒙
王成
金宇灏
Original Assignee
苏州康宁杰瑞生物科技有限公司
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Publication of WO2024141054A1 publication Critical patent/WO2024141054A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present application relates to the field of biomedicine, and in particular to a pharmaceutical composition comprising a fusion protein and uses thereof.
  • GLP-1 Glucagon-like peptide-1
  • GLP-1 is an incretin hormone secreted by the L-cells of the small intestinal epithelium. In healthy individuals, GLP-1 can respond to the body's nutrient intake, promote insulin release and inhibit glucagon secretion. In patients with type 2 diabetes, the infusion of supraphysiological doses of GLP-1 can restore endogenous insulin secretion and lower blood sugar.
  • the present application provides a fusion protein, which comprises a human GLP-1 polypeptide variant and an immunoglobulin Fc region.
  • the fusion protein has excellent pharmacokinetic properties, for example, not easily cleared, a longer half-life (t1/2), and/or a higher exposure amount.
  • 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.
  • FIG4 shows the pharmacokinetic test results of the fusion proteins GM-ARY-L2H1-Fc4m and GM-RY-L2H1-Fc4m described in the present application whose Fc region contains YTE mutation.
  • 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.
  • GLP-1 described in this application includes natural, synthetic or modified GLP-1 proteins, as well as complete GLP-1 proteins or functional fragments thereof, and GLP-1 proteins in different biologically active forms.
  • wild-type human GLP-1 may include an amino acid sequence as shown in SEQ ID NO:1, with the amino acid residue H at its N-terminus being marked as position 7.
  • the term "at least a portion of the activity of human GLP-1” generally refers to having one or more activities of 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 human GLP-1 protein.
  • the human GLP-1 of the "at least a portion of the activity of human GLP-1” may be wild-type, such as the amino acid sequence shown in SEQ ID NO:1.
  • the human GLP-1 in the "at least a portion of the activity of human GLP-1” may be a GLP-1 variant modified from wild-type human GLP-1, such as the amino acid sequence shown in SEQ ID NO:2.
  • the human GLP-1 polypeptide variant of the present application may have at least a portion of the activity of human GLP-1 with an amino acid sequence such as SEQ ID NO:1.
  • the human GLP-1 polypeptide variant of the present application may have at least a portion of the activity of human GLP-1 with an amino acid sequence such as SEQ ID NO:2.
  • the activity is not required to be the same level as that of human GLP-1 protein, but can be higher, similar or lower than that of human GLP- 1 protein.
  • "at least part of the activity of human GLP-1” may refer to one or more selected from the following groups: activity binding to GLP-1 receptor, activity activating GLP-1 receptor, activating adenylate cyclase, activity promoting elevated 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 ⁇ -cell apoptosis, activity inhibiting postprandial glucagon secretion, activity relieving hypoglycemia, and activity reducing body weight.
  • cAMP cyclic adenosine monophosphate
  • the "at least part of the activity of human GLP-1" may be at least part of the activity of a fusion protein (e.g., a fusion protein with an Fc region) comprising human GLP-1.
  • dulaglutide generally refers to a heterologous fusion protein comprising a GLP-1 analog (amino acid sequence as shown in SEQ ID NO: 2) and an Fc region derived from human IgG4 through a small Dulaglutide is also known as 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).
  • prevention and/or treatment includes not only the prevention and/or treatment of 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 any pharmacological effects that are generally beneficial to the treatment of patients.
  • the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid mutations at the amino acid positions W31 and Y19.
  • the amino acid sequence of the human GLP-1 polypeptide variant has two amino acid mutations, which may be located at the amino acid positions W31 and Y19.
  • the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NO: 12-18.
  • the amino acid mutation can make the fusion protein of the GLP-1 polypeptide variant and the Fc region still have at least partial activity of the fusion protein of human GLP-1 (for example, the human GLP-1 protein with an amino acid sequence as shown in any one of SEQ ID NO: 1 or 2) and the Fc region.
  • the amino acid sequence of the human GLP-1 polypeptide variant may contain amino acid substitutions W31Y and Y19A compared to the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the human GLP-1 polypeptide variant may include 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.
  • the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 25.
  • the fusion protein comprises an immunoglobulin Fc region.
  • the immunoglobulin Fc region may be located at the C-terminus or the N-terminus of the human GLP-1 polypeptide variant, preferably at the C-terminus.
  • the amino acid mutation at M252 may be M252Y.
  • the amino acid mutation at S254T may be S254T.
  • the amino acid mutation at T256 may be T256E.
  • the Fc region may be an Fc region derived from IgG1, and may include amino acid mutations of M252Y, S254T, and T256E.
  • the Fc region may be an Fc region derived from IgG2, and may include amino acid mutations of M252Y, S254T, and T256E.
  • the Fc region may be an Fc region derived from IgG3, and may include amino acid mutations of M252Y, S254T, and T256E.
  • the Fc region may be an Fc region derived from IgG4, and may include the amino acid sequence shown in SEQ ID NO:36.
  • 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.
  • the hinge region may be derived from IgG.
  • 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.
  • the hinge region in the fusion protein may have one or more additional amino acids at the N-terminus. One or more amino acids may be added at the C-terminus. In the present application, the hinge region in the fusion protein may reduce one or more 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 reduce one or more amino acids at the C-terminus compared to a naturally occurring IgG hinge region.
  • the hinge region in the fusion protein can increase one or more amino acids at the N-terminus, for example, increase Ala, compared to the naturally occurring IgG4 hinge region.
  • the hinge region in the fusion protein can include one or more amino acid mutations compared to the naturally occurring IgG4 hinge region to avoid undesirable protein formation. Such amino acid mutations are technologies known in the art.
  • the hinge region may comprise an amino acid sequence as shown in SEQ ID NO:30 or 31.
  • 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 with a length of less than 15 amino acids, their length may be increased to 15 amino acids by adding amino acids, for example, by adding Gly.
  • the linker described in the present application may comprise an amino acid sequence shown in any one of SEQ ID NO:27-29.
  • 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.
  • the human GLP polypeptide variant in the fusion protein described in the present application 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.
  • the immunoglobulin Fc region may be an Fc region of IgG comprising amino acid mutations of M252Y, S254T and T256E.
  • 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 the Fc region derived from IgG.
  • the immunoglobulin Fc region may be the Fc region of IgG comprising the amino acid mutations of M252Y, S254T and T256E.
  • the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 4, the linker may comprise the amino acid sequence shown 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 the Fc region derived from IgG4 and comprise the amino acid mutations of M252Y, S254T and T256E.
  • the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 43 and 45.
  • the fusion protein may be present in the form of an immunoconjugate.
  • the concentration of the buffer component described in the present application may be 1 nM-75 mM, and may preferably be 2 mM-50 mM; may more preferably be 5 mM-40 mM; and may even more preferably be 10 mM-30 mM.
  • the buffer component may include citrate, and the concentration of the citrate may 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.
  • the pharmaceutical composition of the present application may also include a stabilizer to protect the fusion protein from chemical and/or physical degradation, and the stabilizer may be selected from polyols, amino acids, sugars, salts and/or albumin.
  • the stabilizer described in the present application 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; even more preferably, the stabilizer comprises mannitol.
  • the concentration of the stabilizer can be 5mg/mL-270mg/mL, 5mg/mL-260mg/mL, 5mg/mL-250mg/mL, 5mg/mL-200mg/mL, 5mg/mL-170mg/mL, 10mg/mL-260mg/mL, 10mg/mL- or 30 mg/mL.
  • 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.
  • the concentration of 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.
  • the stabilizer comprises glycine
  • the concentration of the glycine can be 2mg/mL-50mg/mL, preferably 5mg/mL-40mg/mL, more preferably 10mg/mL-30mg/mL, and even more preferably 15mg/mL-25mg/mL.
  • the concentration of the glycine can be 1mg/mL-45mg/mL, 1mg/mL-40mg/mL, 2mg/mL-40mg/mL, 2mg/mL-35mg/mL, 5mg/mL-50mg/mL, 5mg/mL-40mg/mL, 5mg/mL-30mg/mL, 10mg/mL-35mg/mL, 10mg/mL-25mg/mL, 15mg/mL-40mg/mL, 15mg/mL-35mg/mL, 15mg/mL-25mg/mL, 18mg/mL-30mg/mL or 18mg/mL-20mg/mL.
  • the stabilizer comprises L-proline
  • the concentration of the L-proline may be 5 mg/mL-75 mg/mL, may be preferably 10 mg/mL-50 mg/mL, may be more preferably 15 mg/mL-40 mg/mL, and may even more preferably be 20 mg/mL-30 mg/mL.
  • the concentration of the L-proline can be 2mg/mL-100mg/mL, 2mg/mL-75mg/mL, 5mg/mL-75mg/mL, 5mg/mL-60mg/mL, 5mg/mL-50mg/mL, 10mg/mL-70mg/mL, 10mg/mL-50mg/mL, 10mg/mL-45mg/mL, 15mg/mL-80mg/mL, 15mg/mL-55mg/mL, 15mg/mL-45mg/mL, 20mg/mL-65mg/mL, 20mg/mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL, 25mg/mL-45mg/mL, 25mg/mL-40mg/mL or 25mg/mL-30mg/mL,
  • the stabilizer comprises L-arginine hydrochloride
  • 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.
  • 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, 30
  • 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.
  • 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.
  • 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.
  • 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.
  • the fusion protein can be stably present at a higher protein concentration, and the concentration of the fusion protein is 1mg/mL-30mg/mL, preferably 2mg/mL-20mg/mL, and more preferably 5mg/mL-10mg/mL.
  • the concentration of the fusion protein can be 0.5mg/mL-30mg/mL, 0.5mg/mL-25mg/mL, 1mg/mL-30mg/mL, 1mg/mL-25mg/mL, 1mg/mL-20mg/mL, 2mg/mL-20mg/mL, 2mg/mL-15mg/mL, 2mg/mL-10mg/mL, 5mg/mL-15mg/mL or 5mg/mL-10mg/mL.
  • 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 oil for injection, ethanol, and propylene glycol; more preferably, the solvent is water for injection.
  • the pharmaceutical composition of the present application has excellent stability.
  • the monomer content of the fusion protein is still above 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5%, and the monomer content can be measured by SE-HPLC purity.
  • the container may include a vial (e.g., a glass bottle), an ampoule, a syringe, an injection pen, and/or an IV bag.
  • the container can be sterile.
  • the container can include a glass bottle.
  • the kit may include a delivery device that can administer the composition described herein.
  • the delivery device may include a vial, an ampoule, a syringe, an injection pen, and/or an intravenous bag.
  • the pharmaceutical composition of the present application can be used to prevent and/or treat metabolic diseases or conditions.
  • 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.
  • 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.
  • the present application provides a method for increasing or promoting insulin expression in a subject in need thereof, the method comprising administering the pharmaceutical composition to the subject.
  • a subject in need may include a person suffering from a metabolic disease or condition.
  • a subject in need may include a person suffering from a GLP-1 related metabolic disease.
  • a subject in need may include a person suffering from diabetes.
  • a subject in need may include a person suffering from type II diabetes.
  • the insulin expression can include the activity of insulin secretion.
  • the insulin expression can include the amount and concentration of insulin.
  • the insulin expression can be detected by any method known in the art.
  • 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.
  • 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 is different from that of the amino acid sequence shown in SEQ ID NO: 2.
  • the row comprises at least 2 amino acid mutations, wherein the at least 2 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.
  • composition according to embodiment 1, wherein the immunoglobulin Fc region is located at the C-terminus of the human GLP-1 polypeptide variant.
  • 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.
  • 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.
  • immunoglobulin Fc region comprises an amino acid sequence as shown in SEQ ID NO:36.
  • 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 an amino acid sequence as shown in any one of SEQ ID NO: 30-31; preferably comprises an amino acid sequence as shown in SEQ ID NO: 30.
  • 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.
  • 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:
  • 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.
  • composition according to any one of embodiments 1-21, further comprising a stabilizer.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition according to any one of embodiments 1-28, further comprising a surfactant.
  • composition according to any one of embodiments 1-35 which is a liquid preparation.
  • 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%.
  • kits comprising the pharmaceutical composition of any one of embodiments 1-39 and a container for holding the composition.
  • kit of embodiment 40 wherein the container comprises a glass bottle.
  • kits 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.
  • a fusion protein of human GLP-1 polypeptide variant and Fc which comprises GLP-1 polypeptide variant (amino
  • the fusion protein is expressed in the form of a vector comprising a nucleic acid sequence encoding a fusion protein, wherein the ...
  • the in vivo pharmacokinetics of the fusion proteins GM-RY-L1H2-Fc4, GM-RY-L2H2-Fc4 and GM-RY-L3H2-Fc4 of the present application were investigated.
  • the experiment was 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 given free access to food and adapted for at least 3 days before the experiment.
  • SPF SD rats weighing 180-220 g were followed by The rats in each group were divided into groups.
  • the corresponding test sample was injected subcutaneously (SC) at a dose of 1 mg/kg, and dulaglutide (DULA) was used as a control.
  • SC subcutaneously
  • DULA dulaglutide
  • Example 2 the in vivo pharmacokinetics of the fusion proteins GM-ARY-L2H2-Fc4 and GM-ARY-L2H1-Fc4 of the present application were detected.
  • the samples were tested by ELISA.
  • the pharmacokinetic parameters were calculated using DAS (3.2.8) software.
  • the results are shown in Figure 2 and Table 3.
  • the in vivo half-life of GM-ARY-L2H2-Fc4 and GM-ARY-L2H1-Fc4 is comparable, among which the exposure of GM-ARY-L2H1-Fc4 using the hinge region of IgG1 (SEQ ID NO: 30) is better.
  • the in vivo pharmacokinetics of the fusion proteins GM-RY-L2H1-Fc4m and GM-ARY-L2H1-Fc4m of the present application were tested using cynomolgus monkeys. Two cynomolgus monkeys were included in each group, half male and half female. Each group was given a single subcutaneous injection of the corresponding test sample (1 mg/kg).
  • DULA duelaglutide without YTE mutation in the Fc region of IgG4
  • DULA-Fc4m duelaglutide with YTE mutation in the Fc region of IgG4 were used as controls.
  • the half-life or exposure of the fusion protein of the present application without YTE mutation in the Fc region is comparable to that of dulaglutide, while the half-life and exposure of the fusion protein of the present application after YTE mutation in the Fc region are significantly higher than those of dulaglutide.
  • the YTE mutation in the Fc region significantly improves the pharmacokinetic properties of the fusion protein of the present application, and the amino acid mutation of the human GLP-1 polypeptide variant of the fusion protein and the YTE mutation in the Fc region have a synergistic effect on the improvement of pharmacokinetic properties.
  • Example 4 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 containing the YTE mutation in the Fc region of the present application were detected.
  • ELISA was performed and the pharmacokinetic parameters were calculated using DAS (3.2.8) software.
  • Example 6 Luciferase assay to detect the activation effect of the fusion protein of the present application on the cAMP/PKA signaling pathway
  • HER293-GLP1R-CREB-D4 cells were digested and collected, counted, and the cell number was adjusted to 4 ⁇ 10 5 cells/ml with DMEM medium containing 10% FBS, and inoculated into 96-well plates, 50 ⁇ l per well.
  • the sample to be tested was diluted to 9 concentrations from 1000 ng/ml using DMEM medium containing 10% FBS; dulaglutide was used as a positive control. 50 ⁇ l of a series of concentrations of the sample to be tested 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.
  • the fusion protein of the present application has a GLP-1 receptor activation ability at a level comparable to that of dulaglutide.
  • Protein content Calculate the protein content based on the absorbance value of protein at 280 nm and the theoretical extinction coefficient of protein, with reference to Method 6 of Protein Content Determination Method 0731 in Part III of the 2020 edition of the Chinese Pharmacopoeia.

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Abstract

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

Description

包含融合蛋白的药物组合物及其用途Pharmaceutical composition containing fusion protein and use thereof
本申请要求申请日为2022/12/30的中国专利申请2022117424922的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of Chinese patent application No. 2022117424922 filed on December 30, 2022. This application cites the entire text of the above Chinese patent application.
技术领域Technical Field
本申请涉及生物医药领域，具体涉及一种包含融合蛋白的药物组合物及其用途。The present application relates to the field of biomedicine, and in particular to a pharmaceutical composition comprising a fusion protein and uses thereof.
背景技术Background technique
胰高血糖素样肽(glucagon-like peptide-1，GLP-1)是一种由小肠上皮L-细胞分泌的肠促胰素，在健康的个体中，GLP-1能响应机体营养物质的摄取，促进胰岛素释放并抑制胰高血糖素分泌。在患有II型糖尿病的患者中，输入超生理剂量的GLP-1可恢复内源性胰岛素分泌，降低血糖。Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the small intestinal epithelium. In healthy individuals, GLP-1 can respond to the body's nutrient intake, promote insulin release and inhibit glucagon secretion. In patients with type 2 diabetes, the infusion of supraphysiological doses of GLP-1 can restore endogenous insulin secretion and lower blood sugar.
由于二肽基肽酶IV(DPPIV)的酶解和肾脏的清除作用，天然GLP-1的血浆半衰期短。尽管已经开发了几种修饰以延长半衰期的GLP-1R激动剂(例如，度拉糖肽(dulaglutide))用于治疗II型糖尿病，但仍需开发有效的长期治疗并维持最低限度的副作用的方案。WO2022143515、WO2022143516及国际专利申请PCT/CN2022/103777中公开了一类突变的GLP-1类似物及其融合蛋白，具有更加延长的半衰期，有望成为II型糖尿病长期治疗的有效手段，在此将其整体内容引用加入到本申请中。Due to enzymatic hydrolysis by dipeptidyl peptidase IV (DPPIV) and clearance by 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.
蛋白类药物通常会以注射液的形式被贮藏及使用，稳定的药物制剂有利于药品的生产、保存及运输，并可以提高患者用药的便利性与依从性，因此，本领域期望开发可稳定长期保存的药物注射液。Protein drugs are usually stored and used in the form of injections. Stable drug preparations are beneficial to the production, storage and transportation of drugs, and can improve the convenience and compliance of patients in taking drugs. Therefore, the art hopes to develop drug injections that can be stably stored for a long time.
发明内容Summary of the invention
本申请提供了一种融合蛋白，所述融合蛋白包含人GLP-1多肽变体和免疫球蛋白Fc区。所述融合蛋白具有优异的药代动力学性质，例如，不易被清除、较长的半衰期(t1/2)，和/或较高的暴露量。The present application provides a fusion protein, which comprises a human GLP-1 polypeptide variant and an immunoglobulin Fc region. The fusion protein has excellent pharmacokinetic properties, for example, not easily cleared, a longer half-life (t1/2), and/or a higher exposure amount.
本申请还提供一种包含所述融合蛋白的药物组合物，所述药物组合物的pH值为5.0-7.0。在某些实施实施方案中，所述药物组合物还可以包含缓冲成分、稳定剂和/或表面活性剂。本 申请的药物组合物具有优良的稳定性，例如，药物组合物在25±2℃的条件下放置6个月后，其中融合蛋白的单体含量仍能保持在96％以上。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 buffer component, a stabilizer and/or a surfactant. The pharmaceutical composition of the application has excellent stability. For example, after the pharmaceutical composition is placed at 25±2° C. for 6 months, the monomer content of the fusion protein can still be maintained at more than 96%.
本申请还提供了一种试剂盒，其包含所述药物组合物以及盛放所述组合物的容器。The present application also provides a kit, which comprises the pharmaceutical composition and a container for containing the composition.
本申请另外还提供了药物组合物在预防和/或治疗疾病方面的应用，尤其是在预防和/或治疗代谢性疾病或病症方面的应用。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.
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。Those skilled in the art can easily perceive 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 the present 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 the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:
图1显示的是不同连接子的本申请所述融合蛋白GM-RY-L1H2-Fc4、GM-RY-L2H2-Fc4和GM-RY-L3H2-Fc4的药代动力学检测结果。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.
图2显示的是不同铰链区的本申请所述融合蛋白GM-ARY-L2H2-Fc4和GM-ARY-L2H1-Fc4的药代动力学检测结果。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.
图3显示的是Fc区包含YTE突变的本申请所述融合蛋白GM-RY-L2H1-Fc4m的药代动力学检测结果。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.
图4显示的是Fc区包含YTE突变的本申请所述融合蛋白GM-ARY-L2H1-Fc4m和GM-RY-L2H1-Fc4m的药代动力学检测结果。FIG4 shows the pharmacokinetic test results of the fusion proteins GM-ARY-L2H1-Fc4m and GM-RY-L2H1-Fc4m described in the present application whose Fc region contains YTE mutation.
图5显示的是不同铰链区且包含YTE突变的本申请所述融合蛋白GM-ARY-L2H2-Fc4m、GM-RY-L2H2-Fc4m、GM-ARY-L2H1-Fc4m和GM-RY-L2H1-Fc4m的药代动力学检测结果。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.
图6显示的是本申请融合蛋白中任GLP-1多肽变体的氨基酸序列的编号示例。FIG. 6 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.
图7显示的是本申请的融合蛋白具备与度拉糖肽同等水平的GLP-1受体激活能力的检测结果。 FIG. 7 shows the test results that the fusion protein of the present application has the same level of GLP-1 receptor activation ability as dulaglutide.
具体实施方式Detailed ways
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。The following is an explanation of the implementation of the present invention by means of 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.
术语定义Definition of Terms
在本申请中，术语“GLP-1”通常是指胰高血糖素样肽-1(glucagon like peptide 1)。天然GLP-1分子在体内经过加工，切割了前6个氨基酸，因此本领域习惯将GLP-1的氨基酸序列的N端第一个氨基酸定为第7位，C端最后一个氨基酸定为第37位。经过加工的肽可以在体内得到进一步修饰，去除C端的甘氨酸残基并以酰胺基替代。GLP-1通常有两种生物活性形式，分别为GLP-1(7-37)OH和GLP-1(7-36)NH2。本申请所述的“GLP-1”包含天然的、人工合成的或经修饰的GLP-1蛋白，还包含完整的GLP-1蛋白或其功能性片段，以及不同生物活性形式的GLP-1蛋白。例如，野生型的人GLP-1可包含如SEQ ID NO:1所示的氨基酸序列，其N末端的氨基酸残基H记为第7位。因此，本申请的术语“K26”通常是指从GLP-1蛋白N端第7位的H起算，第26位氨基酸的位置，在SEQ ID NO:1所示的氨基酸序列中，第26位的氨基酸为K；术语“W31”通常是指从GLP-1蛋白N端第7位的H起算，第31位氨基酸的位置，在SEQ ID NO:1所示的氨基酸序列中，第31位的氨基酸为W。在本申请中，在描述氨基酸突变和/或取代时，对于GLP-1多肽变体的氨基酸残基的编号描述区别于Fc区的氨基酸残基的编号描述，Fc区的氨基酸残基编号根据EU编码。In this application, the term "GLP-1" generally refers to glucagon-like peptide-1. The natural GLP-1 molecule is processed in vivo and the first 6 amino acids are cleaved. Therefore, it is customary in the art to define the first amino acid at the N-terminus of the amino acid sequence of GLP-1 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 glycine residue at the C-terminus 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 complete GLP-1 proteins or functional fragments thereof, and GLP-1 proteins in different biologically active forms. For example, wild-type human GLP-1 may include an amino acid sequence as shown in SEQ ID NO:1, with the amino acid residue H at its N-terminus being marked as position 7. Therefore, the term "K26" in the present application generally refers to the position of the 26th amino acid, starting from the 7th H at the N-terminus of the GLP-1 protein, and in the amino acid sequence shown in SEQ ID NO: 1, the 26th amino acid is K; the term "W31" generally refers to the position of the 31st amino acid, starting from the 7th H at the N-terminus of the GLP-1 protein, and in the amino acid sequence shown in SEQ ID NO: 1, the 31st amino acid is W. In the present application, when describing amino acid mutations and/or substitutions, the numbering description of the amino acid residues of the GLP-1 polypeptide variant is distinguished from the numbering description of the amino acid residues in the Fc region, and the amino acid residues in the Fc region are numbered according to the EU code.
所述人GLP-1可以被改造，改造后的GLP-1的变体可以具有改造前的人GLP-1的至少部分活性。例如，示例性的野生型人GLP-1改造后的氨基酸序列可以如SEQ ID NO:2所示。The human GLP-1 can be modified, and the modified GLP-1 variant can have at least some activity of the human GLP-1 before modification. For example, an exemplary amino acid sequence of wild-type human GLP-1 after modification can be shown as SEQ ID NO: 2.
在本申请中，术语“人GLP-1的至少部分活性”通常是指具有人GLP-1蛋白的一种或多种活性，或具有人GLP-1蛋白的至少20％(例如，至少25％、30％、35％、40％、45％、或50％或更高)的活性。所述“人GLP-1的至少部分活性”的人GLP-1可以是野生型的，如，SEQ ID NO:1所示的氨基酸序列。所述“人GLP-1的至少部分活性”中的人GLP-1可以是野生型人GLP-1改造后的GLP-1变体，如，SEQ ID NO:2所示的氨基酸序列。例如，本申请的人GLP-1多肽变体可以具有氨基酸序列如SEQ ID NO:1所示的人GLP-1的至少部分活性。在另一些情形中，本申请的人GLP-1多肽变体可以具有氨基酸序列如SEQ ID NO:2所示的人GLP-1的至少部分活性。In the present application, the term "at least a portion of the activity of human GLP-1" generally refers to having one or more activities of 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 human GLP-1 protein. The human GLP-1 of the "at least a portion of the activity of human GLP-1" may be wild-type, such as the amino acid sequence shown in SEQ ID NO:1. The human GLP-1 in the "at least a portion of the activity of human GLP-1" may be a GLP-1 variant modified from wild-type human GLP-1, such as the amino acid sequence shown in SEQ ID NO:2. For example, the human GLP-1 polypeptide variant of the present application may have at least a portion of the activity of human GLP-1 with an amino acid sequence such as SEQ ID NO:1. In other cases, the human GLP-1 polypeptide variant of the present application may have at least a portion of the activity of human GLP-1 with an amino acid sequence such as SEQ ID NO:2.
所述活性不要求与人GLP-1蛋白具有相同水平的活性，可以是高于、相近或低于人GLP- 1蛋白的活性。在某些情形中，“人GLP-1的至少部分活性”可以指选自下组中的一种或多种：与GLP-1受体结合的活性、激活GLP-1受体的活性、激活腺苷酸环化酶、促进细胞内环磷酸腺苷(cAMP)水平升高的活性、正性调节细胞内Ca2+水平的活性、刺激胰岛素分泌的活性、增加肝糖原储存量的活性、延缓胃排空的活性、抑制胃动力的活性、降低食欲的活性、抑制β细胞凋亡的活性、抑制餐后胰高血糖素的分泌的活性、缓解低血糖的活性和减轻体重的活性。例如，其可以通过检测与GLP-1受体结合的能力、cAMP的表达水平来检测。例如，其可以通过荧光素酶法检测对cAMP/PKA信号通路的激活水平来检测。所述“人GLP-1的至少部分活性”可以是包含人GLP-1的融合蛋白(例如，与Fc区的融合蛋白)的至少部分活性。The activity is not required to be the same level as that of human GLP-1 protein, but can be higher, similar or lower than that of human GLP- 1 protein. In some cases, "at least part of the activity of human GLP-1" may refer to one or more selected from the following groups: activity binding to GLP-1 receptor, activity activating GLP-1 receptor, activating adenylate cyclase, activity promoting elevated 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 relieving hypoglycemia, and activity reducing body weight. For example, it can be detected by detecting the ability to bind to GLP-1 receptor, the expression level of cAMP. For example, it can be detected by detecting the activation level of cAMP/PKA signaling pathway by luciferase method. The "at least part of the activity of human GLP-1" may be at least part of the activity of a fusion protein (e.g., a fusion protein with an Fc region) comprising human GLP-1.
在本申请中，术语“多肽”通常是指由通过酰胺键(也称为肽键)线性连接的单体(氨基酸)构成的分子。术语“多肽”可以是具有两个或更多个氨基酸的任意链，并且不指特定长度的产物。本申请所述多肽包括肽、二肽、三肽、寡肽、蛋白质、氨基酸链或任何其它用于指具有两个或更多个氨基酸的链，而且术语“多肽”可以代替这些术语中任一个或与其交换使用。术语“多肽”还可以指多肽的表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰化、通过已知的保护性/封闭性基团衍生化、蛋白水解分裂、或通过非天然存在的氨基酸修饰。多肽可以自天然的生物学来源衍生或通过重组技术生成。In the present application, the term "polypeptide" generally refers to a molecule consisting of monomers (amino acids) linearly connected 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 in the present application include peptides, dipeptides, tripeptides, oligopeptides, proteins, amino acid chains or any other chains used to refer to two or more amino acids, and the term "polypeptide" can replace any of these terms or be used interchangeably. The term "polypeptide" can also refer to a product modified after the 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-natural amino acids. Polypeptides can be derived from natural biological sources or generated by recombinant technology.
在本申请中，术语“变体”通常是指与天然生物活性蛋白质具有序列同源性的蛋白质分子。本申请所述的多肽变体包括通过添加(包含插入)、缺失、修饰和/或取代一个或多个氨基酸残基而具有改变的氨基酸序列的多肽，其同时保留亲本序列(例如，SEQ ID NO:2所示的氨基酸序列)的至少一种治疗和/或生物活性而与亲本序列不同。例如，变体可与亲本蛋白具有至少0％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性。变体可以天然存在或者是非天然存在的。可以使用本领域已知的技术来生成非天然存在的变体。本申请的多肽变体可以包含保守的或非保守的氨基酸替代、删除或添加。In the present application, the term "variant" generally refers to a protein molecule that has sequence homology with a natural biologically active protein. The polypeptide variants described in the present application include polypeptides having an altered amino acid sequence by adding (including insertion), deleting, modifying and/or replacing one or more amino acid residues, which retain at least one therapeutic and/or biological activity of the parent sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2) while 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 contain conservative or non-conservative amino acid substitutions, deletions or additions.
在本申请中，术语“突变”通常包括对序列(核酸或氨基酸序列)的任何类型的改变或修饰，包括氨基酸或核苷酸的缺失、截短、失活、破坏、取代或易位。在本申请中，氨基酸突变可以是氨基酸取代，术语“取代”通常是指用不同的“替换”氨基酸残基来替换预先确定的亲本氨基酸序列中的至少一个氨基酸残基。该一个或多个被替换的氨基酸残基可以是“天然存在的氨基酸残基”(即由遗传密码编码)。In the present application, the term "mutation" generally includes any type of change or modification to a sequence (nucleic acid or amino acid sequence), including the deletion, truncation, inactivation, destruction, substitution or translocation of an amino acid or nucleotide. In the present application, an amino acid mutation may 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 may be "naturally occurring amino acid residues" (i.e., encoded by a genetic code).
在本申请中，术语“氨基酸取代”是指用另一个氨基酸残基替代存在于亲本序列中的一个氨基酸残基。亲本序列中的氨基酸可以是例如经由化学肽合成或通过本领域已知的重组方 法来取代。In the present application, the term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent sequence with another amino acid residue. The amino acid in the parent sequence can be, for example, produced by chemical peptide synthesis or by recombinant methods known in the art. Method to replace.
在本申请中，术语“Fc区”通常是指源自免疫球蛋白重链C端区域的结构域，所述C端区域可以通过木瓜蛋白酶消化完整的抗体而产生。Fc区可以是天然序列Fc区或变体Fc区，其中，“未经所述氨基酸突变的免疫球蛋白Fc区”是指SEQ ID NO:32-35中任一项所示的氨基酸序列，其分别对应人IgG1、IgG2、IgG3和IgG4的Fc序列。本申请所述免疫球蛋白的Fc区通常包含2个恒定域(CH2结构域和CH3结构域)，如无特别说明，不包含铰链区，且任选地可以包含CH4结构域。In the present 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 NO:32-35, which corresponds to the Fc sequence of human IgG1, IgG2, IgG3 and IgG4, respectively. The Fc region of the immunoglobulin described in the present application generally comprises two constant domains (CH2 domain and CH3 domain), does not include a hinge region unless otherwise specified, and may optionally include a CH4 domain.
在本申请中，术语“YTE突变”是指IgG的Fc区中的一组突变。YTE突变可以导致Fc区与人类FcRn的结合增加，且可能改变包含该YTE突变的Fc区的融合蛋白的血清半衰期。包含YTE突变的Fc区包括下述三种氨基酸突变的组合：M252Y、S254T和T256E，其中根据在Kabat中的EU索引进行编号。In the present application, the term "YTE mutation" refers to a group of mutations in the Fc region of IgG. The YTE mutation can lead to increased binding of the Fc region to human FcRn and may change the serum half-life of the fusion protein of the Fc region containing the YTE mutation. The Fc region containing the YTE mutation includes a combination of the following three amino acid mutations: M252Y, S254T and T256E, which are numbered according to the EU index in Kabat.
在本申请中，术语“铰链区”通常是指一种长度在15-30个氨基酸之间的柔性氨基酸序列，其允许其N端或C端的多肽部分独立地移动。铰链区通常来自免疫球蛋白重链CHl和CH2功能区之间的区域。铰链区通常来源于IgG，例如，可来源于IgG1、IgG2、IgG3或IgG4。相对于天然的源自IgG的铰链区，本申请的铰链区的N端或C端可以增加一个或多个氨基酸残基，也可以删减一个或多个氨基酸残基，还可以替换一个或多个氨基酸残基，只要仍保持铰链区的功能即可。In the present application, the term "hinge region" generally refers to a flexible amino acid sequence of 15-30 amino acids in length, which allows the polypeptide portion at its N-terminus or C-terminus to move independently. The hinge region is generally derived from the region between the CH1 and CH2 functional regions of the immunoglobulin heavy chain. The hinge region is generally derived from IgG, for example, it can be derived from IgG1, IgG2, IgG3 or IgG4. Relative to the natural hinge region derived from IgG, the N-terminus or C-terminus of the hinge region of the present application can add one or more amino acid residues, can also delete one or more amino acid residues, and can also replace one or more amino acid residues, as long as the function of the hinge region is still maintained.
在本申请中，术语“免疫球蛋白”通常是指基本上由免疫球蛋白基因编码的一个或多个多肽组成的蛋白质。公认的人免疫球蛋白基因包括κ、λ、α(IgA1和IgA2)、γ(IgG1、IgG2、IgG3、IgG4)、δ、ε和μ恒定区基因，以及众多的免疫球蛋白可变区基因。一般来说，免疫球蛋白可以是约150,000道尔顿的异四聚体糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。全长免疫球蛋白“轻链”(约25KD和214个氨基酸)的NH2-末端(约110个氨基酸)由可变区基因编码，COOH-末端由κ或λ恒定区基因编码。天然的免疫球蛋白基本上由经由免疫球蛋白铰链区连接的两个Fab分子和Fc区组成。In the present 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 κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ constant region genes, as well as numerous immunoglobulin variable region genes. In general, immunoglobulins can be heterotetrameric glycoproteins of about 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 κ or λ constant region gene. Natural immunoglobulins are essentially composed of two Fab molecules and an Fc region connected via an immunoglobulin hinge region.
在本申请中，术语“连接子”通常是指连接两个异源性多肽或其片段的氨基酸序列。在本申请中，连接子可以是共价连接多肽以形成融合多肽的氨基酸序列。连接子的长度可以具有10-20个氨基酸长度。连接子可以是柔性的或刚性的。In the present application, the term "linker" generally refers to an amino acid sequence that connects two heterologous polypeptides or fragments thereof. In the present application, a linker can be an amino acid sequence that covalently connects polypeptides to form a fusion polypeptide. The length of the linker can have a length of 10-20 amino acids. The linker can be flexible or rigid.
在本申请中，术语“选择性增强”通常是指在特定条件下增强，所述特定条件可以是特定的pH范围、特定的温度范围、特定的连接方式、特定的样品来源或特定的反应时间等。例 如，Fc区的氨基酸突变使得Fc区在特定pH范围内与Fc受体的结合能力增强，但不显著改变Fc区在该特定范围内以外与Fc受体的结合能力，可以称为“选择性增强”。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, etc. For example, an amino acid mutation in the Fc region enhances the binding ability of the Fc region to Fc receptors within a specific pH range, but does not significantly change the binding ability of the Fc region to Fc receptors outside the specific range, which can be referred to as "selective enhancement".
在本申请中，术语“融合蛋白”通常是指包含或者由下述这样的多肽组成的更长的多肽，所述多肽的氨基酸序列可以直接或间接地(通过连接子，例如，连接肽)融合至一异源多肽(例如，前一多肽或其结构域无关的多肽)的氨基酸序列。In the present 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., a polypeptide unrelated to the previous polypeptide or its domain).
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。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.
在本申请中，术语“载体”通常是指能够在合适的宿主细胞中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还可以包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate 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 that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and/or translation of DNA or RNA. The vector may also include a vector with a variety of the above 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 may produce a desired expression product by cultivating a suitable host cell that contains the vector.
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞，细胞系或细胞培养物。所述宿主细胞可以包括单个宿主细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述宿主细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述宿主细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector including a nucleic acid molecule described in the present application, or that can express an antibody or its antigen-binding fragment described in the present application. 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 completely identical in morphology or genome to the original parent cell, but can express the antibody or its antigen-binding fragment described in the present application. The host cell can be obtained by transfecting cells in vitro using the vector described in the present application. The host cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell). The recombinant host cell includes not only a certain specific cell, but also the offspring of these cells.
在本申请中，术语“代谢性疾病或病症”通常是指破坏体内正常代谢过程，使得机体无法正常利用和/或贮存能量的疾病或病症。所述代谢性疾病或病症可以是指糖类代谢障碍的疾病、脂肪类代谢障碍的疾病和/或细胞线粒体障碍的疾病。所述代谢性疾病或病症可以是与饮食、毒素或感染相关的，也可以是遗传性疾病。所述代谢性疾病或病症可以包括由缺少代谢相关的酶、或代谢相关的酶功能异常引起的疾病或病症。本申请所述代谢性疾病或病症可以选自糖尿病和其他GLP-1相关代谢性病症。In the present 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 normally utilize and/or store energy. 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 the present application may be selected from diabetes and other GLP-1 related metabolic disorders.
在本申请中，“度拉糖肽(dulaglutide)”通常是指一种包含GLP-1类似物的异源融合蛋白质，由GLP-1类似物(氨基酸序列如SEQ ID NO:2所示)与源自人IgG4的Fc区通过小分 子肽共价连接形成。度拉糖肽也可称为杜拉鲁肽或度拉鲁肽，商品名为或 度拉糖肽包含如SEQ ID NO:3所示的氨基酸序列(参见PCT专利WO2009009562和美国专利US7452966)。In the present application, "dulaglutide" generally refers to a heterologous fusion protein comprising a GLP-1 analog (amino acid sequence as shown in SEQ ID NO: 2) and an Fc region derived from human IgG4 through a small Dulaglutide is also known as 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).
在本申请中，术语“组合物”通常是指包含本申请所述融合蛋白的混合物，所述组合物可以包含至少一种可药用的成分，例如本申请所述的缓冲成分、稳定剂和/或表面活性剂等。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 contain at least one pharmaceutically acceptable component, such as the buffer component, stabilizer and/or surfactant described in the present application.
在本申请中，术语“制剂”通常是指以预定量或比例包含本申请所述的融合蛋白的产品，以及任何直接或间接地通过将预定量的本申请所述的融合蛋白进行组合而产生的产品。本申请所述制剂的含义可以包含药物制剂，即包含了本申请所述的融合蛋白和和其他可药用成分，以及任何直接或间接地由任意两种或更多种成分组合、复合或聚集，而产生的产品。所述制剂可以以液态的形式存在。In the present application, the term "preparation" generally refers to a product containing the fusion protein described in the present application in a predetermined amount or ratio, and any product produced by combining a predetermined amount of the fusion protein described in the present application directly or indirectly. The meaning of the preparation described in the present application may include a pharmaceutical preparation, that is, a product containing the fusion protein described in the present application and other pharmaceutically acceptable ingredients, and any product produced by combining, compounding or aggregating any two or more ingredients directly or indirectly. The preparation may exist in a liquid form.
在本申请中，术语“缓冲成分”通常是指具有提供缓冲效应(例如抵抗pH值改变)的功能的试剂。例如，所述缓冲成分可以调节由于酸性或碱性物质的添加和/或释放所导致的pH的改变。例如，所述缓冲成分可以包括弱酸及其共轭碱；也可以包括弱碱及其共轭酸。在本申请中，“缓冲成分”可以与“缓冲体系”、“缓冲盐”、“缓冲液”、“缓冲系统”等描述相互替换使用。In the present application, the term "buffer component" generally refers to an agent having the function of providing a buffering effect (e.g., resisting pH changes). For example, the buffer component can adjust the change in pH due to the addition and/or release of acidic or alkaline 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 the present application, "buffer component" can be used interchangeably with descriptions such as "buffer system", "buffer salt", "buffer", and "buffer system".
在本申请中，术语“稳定剂”通常是指在制备、贮存和应用过程中保护活性药物成分和/或制剂免于化学和/或物理降解的药学上可接受的赋形剂。常用的蛋白药物稳定剂包括但不限于：氨基酸，多元醇，抗氧化剂，防腐剂，环糊精，聚乙二醇，例如PEG 3000、3350、4000、6000，白蛋白，例如人血清白蛋白(HSA)，牛血清白蛋白(BSA)，盐，例如氯化钠，氯化镁，氯化钙，螯合剂，例如EDTA等。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 application. 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.
在本申请中，术语“表面活性剂”通常是指可以保护蛋白质(例如本申请所述融合蛋白)免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少该蛋白质的聚集或使所述组合物中颗粒物的形成最小化的试剂。所述表面活性剂可以同时包含疏水性基团和亲水性基团。所述表面活性剂可以包括一种或多种表面活性剂的混合物或组合。所述表面活性剂可以包括非离子型表面活性剂。In the present application, the term "surfactant" generally refers to an agent that can protect proteins (e.g., fusion proteins 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 may contain a hydrophobic group and a hydrophilic group at the same time. The surfactant may include a mixture or combination of one or more surfactants. The surfactant may include a nonionic surfactant.
在本申请中，术语“免疫缀合物”通常是指多肽或蛋白质和感兴趣的活性物质(例如，蛋白质、核酸、药物或标记分子等)直接或间接连接(例如，通过连接子共价连接)而形成的缀合物。In the present application, the term "immunoconjugate" generally refers to a conjugate formed by directly or indirectly linking (eg, covalently linked via a linker) a polypeptide or protein and an active substance of interest (eg, protein, nucleic acid, drug or marker molecule, etc.).
在本申请中，术语“有需要的受试者”或“需要治疗的”包括已经患有代谢性疾病或病 症者以及待预防该病症者。有需要的受试者或“患者”包括接受预防性或治疗性治疗的人和其他哺乳动物受试者。In this application, the term "subject in need" or "in need of treatment" includes subjects who already have a metabolic disease or disease. Subjects in need thereof or "patients" include humans and other mammalian subjects receiving prophylactic or therapeutic treatment.
在本申请中，术语“预防和/或治疗”不但包括预防和/或治疗疾病，而且通常包括预防所述疾病的发作，减缓或逆转疾病进程，防止或减缓与所述疾病相关的一种或多种症状的发作，减少和/或减轻与所述疾病相关的一种或多种症状，减少所述疾病和/或与之相关的任何症状的严重性和/或持续时间，和/或防止所述疾病的严重性和/或与之相关的任何症状的进一步增加，防止、减少或逆转由所述疾病以及通常对治疗患者有益的任何药理作用而引起的任何生理损害。In the present application, the term "prevention and/or treatment" includes not only the prevention and/or treatment of 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 any pharmacological effects that are generally beneficial to the treatment of patients.
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。In this application, the term "comprising" generally means including the features explicitly stated, but not excluding other elements.
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。如无特别说明，本申请所提及的数值均视作经过“约”的修饰。如果有疑问，或者对于特定值或参数的误差范围没有被该领域承认的通常理解，则“约”意味着该值或参数的±5％。In this application, the term "about" generally refers to a change in the range of 0.5%-10% above or below the specified value, such as a change in the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value. Unless otherwise specified, the numerical values mentioned in this application are all considered to be modified by "about". If there is any doubt, or the error range for a particular value or parameter is not generally understood by the art, then "about" means ±5% of the value or parameter.
发明内容Summary of the invention
本申请涉及一种药物组合物，其包含融合蛋白，所述融合蛋白包含人GLP-1多肽变体和免疫球蛋白Fc区。The present application relates to a pharmaceutical composition comprising a fusion protein comprising a human GLP-1 polypeptide variant and an immunoglobulin Fc region.
GLP-1多肽变体GLP-1 polypeptide variants
本申请药物组合物中，所述融合蛋白包含人GLP-1多肽变体，其具有人GLP-1的至少部分活性。在某些情形中，人GLP-1多肽变体可以具有选自下组中的一种或多种活性：与GLP-1受体结合的活性、激活GLP-1受体的活性、激活腺苷酸环化酶、促进细胞内环磷酸腺苷(cAMP)水平升高的活性、正性调节细胞内Ca2+水平的活性、刺激胰岛素分泌的活性、增加肝糖原储存量的活性、延缓胃排空的活性、抑制胃动力的活性、降低食欲的活性、抑制β细胞凋亡的活性、抑制餐后胰高血糖素的分泌的活性、缓解低血糖的活性和减轻体重的活性。例如，其可以通过检测与GLP-1受体结合的能力、cAMP的表达水平来检测。所述人GLP-1多肽变体的活性可以为人GLP-1活性的至少20％(例如，至少25％、30％、35％、40％、45％、或50％或更高)。In the pharmaceutical composition of the present application, the fusion protein comprises a human GLP-1 polypeptide variant, which has at least part of the activity of human GLP-1. In some cases, the human GLP-1 polypeptide variant may have one or more activities selected from the following group: activity of binding to the GLP-1 receptor, activity of activating the GLP-1 receptor, activity of activating adenylate cyclase, activity of promoting the increase of intracellular cyclic adenosine monophosphate (cAMP) level, activity of positively regulating intracellular Ca2+ level, activity of stimulating insulin secretion, activity of increasing liver glycogen storage, activity of delaying gastric emptying, activity of inhibiting gastric motility, activity of reducing appetite, activity of inhibiting β-cell apoptosis, activity of inhibiting postprandial glucagon secretion, activity of alleviating hypoglycemia and activity of reducing body weight. For example, it can be detected by detecting the ability to bind to the GLP-1 receptor and 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.
与SEQ ID NO:2所示的氨基酸序列相比：HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO:2)，所述人GLP-1多肽变体的氨基酸序列可包含至少2个氨基酸突变，例如，可包含2个、3个或更多个氨基酸突变。例如，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含2-4个(例如，2-3个、3个或2个)氨基酸取代。所述至少2个氨基酸突变可以位于选自下组的氨基酸位置处：K26、W31和Y19。Compared with the amino acid sequence shown in SEQ ID NO: 2: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 2), the amino acid sequence of the human GLP-1 polypeptide variant may comprise at least 2 amino acid mutations, for example, 2, 3 or more amino acid mutations. For example, 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 2-4 (e.g., 2-3, 3 or 2) amino acid substitutions. The at least 2 amino acid mutations may be located at amino acid positions selected from the group consisting of K26, W31 and Y19.
在本申请中，所述氨基酸位置“Xn”是指在相应于SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列中第n位的残基X处发生氨基酸取代，其中n为正整数(对于GLP-1，n从7开始起算)，X为任意氨基酸残基的缩写。例如，氨基酸位置“W31”表示相应于SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列的第31位氨基酸的位置。对于SEQ ID NO:2所示的氨基酸序列，氨基酸残基X和位置n的对应关系如图6所示。In the present application, the amino acid position "Xn" refers to an amino acid substitution at the residue X at the nth position in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein n is a positive integer (for GLP-1, n starts from 7), and X is the abbreviation of any amino acid residue. For example, the amino acid position "W31" represents the position of the 31st amino acid in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. For the amino acid sequence shown in SEQ ID NO: 2, the correspondence between the amino acid residue X and the position n is shown in Figure 6.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含选自下组的氨基酸位置处的至少2个突变：W31、K26和Y19。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 contain at least 2 mutations at amino acid positions selected from the following group: W31, K26 and Y19.
例如，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含W31和K26氨基酸位置处的2个氨基酸突变。例如，所述人GLP-1多肽变体的氨基酸序列有2个氨基酸突变，可以位于W31和K26氨基酸位置处。例如，所述人GLP-1多肽变体可包含SEQ ID NO:4所示的氨基酸序列。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 include two amino acid mutations at the amino acid positions W31 and K26. For example, the amino acid sequence of the human GLP-1 polypeptide variant has two amino acid mutations, which may be located at the amino acid positions W31 and K26. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in SEQ ID NO: 4.
例如，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含W31和Y19氨基酸位置处的2个氨基酸突变。例如，所述人GLP-1多肽变体的氨基酸序列有2个氨基酸突变，可以位于W31和Y19氨基酸位置处。例如，所述人GLP-1多肽变体可包含SEQ ID NO:12-18中任一项所示的氨基酸序列。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 include two amino acid mutations at the amino acid positions W31 and Y19. For example, the amino acid sequence of the human GLP-1 polypeptide variant has two amino acid mutations, which may be located at the amino acid positions W31 and Y19. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NO: 12-18.
例如，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含K26和Y19氨基酸位置处的2个氨基酸突变。例如，所述人GLP-1多肽变体的氨基酸序列有2个氨基酸突变，可以位于K26和Y19氨基酸位置处。例如，所述人GLP-1多肽变体可包含SEQ ID NO:5-11中任一项所示的氨基酸序列。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 include two amino acid mutations at the amino acid positions of K26 and Y19. For example, the amino acid sequence of the human GLP-1 polypeptide variant has two amino acid mutations, which may be located at the amino acid positions of K26 and Y19. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NO: 5-11.
例如，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含W31、K26和Y19氨基酸位置处的3个氨基酸突变。例如，所述人GLP-1多肽变体的氨基酸序列有3个氨基酸突变，可以位于W31、K26和Y19氨基酸位置处。例如，所述人GLP-1多肽变体可包含SEQ ID NO:19-25中任一项所示的氨基酸序列。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 include three amino acid mutations at the amino acid positions of W31, K26 and Y19. For example, the amino acid sequence of the human GLP-1 polypeptide variant has three amino acid mutations, which may be located at the amino acid positions of W31, K26 and Y19. For example, the human GLP-1 polypeptide variant may include the amino acid sequence shown in any one of SEQ ID NO: 19-25.
在本申请中，所述氨基酸突变可以包含氨基酸取代。所述氨基酸突变使所述GLP-1多肽变体仍具有人GLP-1(例如，氨基酸序列如SEQ ID NO:1或2中任一项所示的人GLP-1蛋 白)的至少部分活性。所述氨基酸突变可以使所述GLP-1多肽变体的融合蛋白仍具有人GLP-1(例如，氨基酸序列如SEQ ID NO:1或2中任一项所示的人GLP-1蛋白)的融合蛋白的至少部分活性。所述氨基酸突变可以使所述GLP-1多肽变体与Fc区的融合蛋白仍具有人GLP-1(例如，氨基酸序列如SEQ ID NO:1或2中任一项所示的人GLP-1蛋白)与Fc区的融合蛋白的至少部分活性。In the present application, the amino acid mutation may include an amino acid substitution. The amino acid mutation allows the GLP-1 polypeptide variant to still have human GLP-1 (e.g., a human GLP-1 polypeptide with an amino acid sequence as shown in any one of SEQ ID NO: 1 or 2). The amino acid mutation can make the fusion protein of the GLP-1 polypeptide variant still have at least partial activity of the fusion protein of human GLP-1 (for example, the human GLP-1 protein with an amino acid sequence as shown in any one of SEQ ID NO: 1 or 2). The amino acid mutation can make the fusion protein of the GLP-1 polypeptide variant and the Fc region still have at least partial activity of the fusion protein of human GLP-1 (for example, the human GLP-1 protein with an amino acid sequence as shown in any one of SEQ ID NO: 1 or 2) and the Fc region.
在本申请中，所述K26处可以包含氨基酸取代，所述氨基酸取代可以为K26R。In the present application, the K26 may contain an amino acid substitution, and the amino acid substitution may be K26R.
在本申请中，所述W31处可以包含氨基酸取代，所述氨基酸取代可以为W31Y、W31A、W31R或W31K。在本申请中，所述W31处可以包含氨基酸取代，所述氨基酸取代可以为W31Y、W31K或W31R。例如，所述W31处可以包含氨基酸取代，所述氨基酸取代可以为W31Y。In the present application, the W31 may contain an amino acid substitution, and the amino acid substitution may be W31Y, W31A, W31R or W31K. In the present application, the W31 may contain an amino acid substitution, and the amino acid substitution may be W31Y, W31K or W31R. For example, the W31 may contain an amino acid substitution, and the amino acid substitution may be W31Y.
在本申请中，所述Y19处可以包含氨基酸取代，所述氨基酸取代可以为Y19A、Y19L、Y19T、Y19F、Y19I、Y19V或Y19S。例如，所述Y19处可以包含氨基酸取代，所述氨基酸取代可以为Y19A。In the present application, the Y19 may contain an amino acid substitution, and the amino acid substitution may be Y19A, Y19L, Y19T, Y19F, Y19I, Y19V or Y19S. For example, the Y19 may contain an amino acid substitution, and the amino acid substitution may be Y19A.
在本申请中，氨基酸取代“XnY”是指相应于SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列中第n位的残基X被取代为氨基酸残基Y，其中n为正整数(对于GLP-1，n从7开始起算)，X和Y分别独立地为任意氨基酸残基的缩写，且X不同于Y。例如，氨基酸取代“W31Y”是指相应于SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列中第31位的氨基酸残基W被取代为氨基酸残基Y。In the present application, the amino acid substitution "XnY" means that the residue X at position n in the amino acid sequence shown 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 from 7), X and Y are independently abbreviations of 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with the amino acid residue Y.
在本申请中，与SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含至少2个氨基酸取代，所述氨基酸取代可以选自下组：1)K26R，2)选自W31Y、W31R、W31K和W31A中的任意一个，和3)选自Y19A、Y19L、Y19T、Y19F、Y19I、Y19V和Y19S中的任意一个。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may contain at least 2 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 groups: 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.
例如，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含选自下组的至少2个氨基酸突变：1)K26R，2)选自W31Y、W31R和W31K和W31A中的任意一个，和3)选自Y19A、Y19L、Y19T、Y19F、Y19I、Y19V和Y19S中的任意一个。For example, compared with the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of the human GLP-1 polypeptide variant may contain at least 2 amino acid mutations selected from the following groups: 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.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含W31和K26氨基酸位置处的2个氨基酸取代，所述K26处的氨基酸取代可以为K26R，且所述W31处的氨基酸取代可以为W31Y、W31R和W31K和W31A。例如，与SEQ  ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含W31Y和K26R的氨基酸取代。In the present application, compared with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the human GLP-1 polypeptide variant may include two amino acid substitutions at the amino acid positions W31 and K26, the amino acid substitution at K26 may be K26R, and the amino acid substitution at W31 may be W31Y, W31R, W31K and W31A. For example, compared with SEQ ID NO: Compared with the amino acid sequence shown in ID NO:2, the amino acid sequence of the human GLP-1 polypeptide variant may contain amino acid substitutions of W31Y and K26R.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代W31Y。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may contain the amino acid substitution W31Y compared to the amino acid sequence shown in SEQ ID NO:2.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代K26R。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may contain the amino acid substitution K26R compared to the amino acid sequence shown in SEQ ID NO:2.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代Y19A。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may contain the amino acid substitution Y19A compared to the amino acid sequence shown in SEQ ID NO:2.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代W31Y和Y19A。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may contain amino acid substitutions W31Y and Y19A compared to the amino acid sequence shown in SEQ ID NO:2.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代W31Y和K26R。例如，所述人GLP-1多肽变体可包含SEQ ID NO：4和19-25中任一项所示的氨基酸序列。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 NO: 4 and 19-25.
例如，所述人GLP-1多肽变体的氨基酸序列的氨基酸取代可以为W31Y和K26R，例如，所述人GLP-1多肽变体可包含SEQ ID NO：4所示的氨基酸序列。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.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代K26R，氨基酸取代W31Y和Y19处的氨基酸取代。例如，所述GLP-1多肽变体可包含SEQ ID NO：19-25中任一项所示的氨基酸序列。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may include 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 include an amino acid sequence shown in any one of SEQ ID NO: 19-25.
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列可包含氨基酸取代K26R，氨基酸取代W31Y和氨基酸取代Y19A。例如，所述人GLP-1多肽变体可包括SEQ ID NO:25所示的氨基酸序列。In the present application, the amino acid sequence of the human GLP-1 polypeptide variant may include 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 include the amino acid sequence shown in SEQ ID NO: 25.
本申请中，所述人GLP-1多肽变体可以包含SEQ ID NO:26所示的氨基酸序列:HGEGTFTSDVSSX19LEEQAAREFIAX31LVKGGG；其中，X19＝Y、A、L、T、F、I、V或S；X31＝W、A、R、K或Y。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.
PCT国际专利申请WO2022143515、WO2022143516和PCT/CN2022/103777分别涉及人GLP-1多肽变体及其融合蛋白，在此将其作为整体并入本文作为参考。PCT international patent applications WO2022143515, WO2022143516 and PCT/CN2022/103777 respectively relate to human GLP-1 polypeptide variants and fusion proteins thereof, which are hereby incorporated by reference in their entirety.
Fc区Fc region
在本申请中，所述融合蛋白包含免疫球蛋白Fc区。所述免疫球蛋白Fc区可以位于人GLP-1多肽变体的C端，也可以位于人GLP-1多肽变体的N端，优选位于C端。 In the present application, the fusion protein comprises an immunoglobulin Fc region. The immunoglobulin Fc region may be located at the C-terminus or the N-terminus of the human GLP-1 polypeptide variant, preferably at the C-terminus.
免疫球蛋白Fc区可以源自IgG，例如，源自IgG1、IgG2、IgG3或IgG4。所述IgG1的Fc区可包含SEQ ID NO:32所示的氨基酸序列。所述IgG2的Fc区可包含SEQ ID NO:33所示的氨基酸序列。所述IgG3的Fc区可包含SEQ ID NO:34所示的氨基酸序列。所述IgG4的Fc区可包含SEQ ID NO:35所示的氨基酸序列。The immunoglobulin Fc region may be derived from IgG, for example, from IgG1, IgG2, IgG3 or IgG4. The Fc region of the IgG1 may comprise the amino acid sequence shown in SEQ ID NO:32. The Fc region of the IgG2 may comprise the amino acid sequence shown in SEQ ID NO:33. The Fc region of the IgG3 may comprise the amino acid sequence shown in SEQ ID NO:34. The Fc region of the IgG4 may comprise the amino acid sequence shown in SEQ ID NO:35.
在本申请中，所述Fc区可以包含氨基酸突变，与未经所述氨基酸突变的免疫球蛋白Fc区(例如，SEQ ID NO:32-35中任一项所示的氨基酸序列)相比，所述氨基酸突变可以使得所述免疫球蛋白Fc区与Fc受体的结合亲和力选择性增强。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 (for example, the amino acid sequence shown in any one of SEQ ID NO:32-35).
在本申请中，所述Fc区可以是源自IgG1、IgG2、IgG3或IgG4的Fc区，且在M252、S254和/或T256位置处包含一种或多种氨基酸突变(例如，所述氨基酸位置根据Kabat的EU编码)。例如，所述Fc区可以是源自IgG1、IgG2、IgG3或IgG4的Fc区，且在M252、S254和T256位置处包含氨基酸突变(例如，所述氨基酸位置根据Kabat的EU编码)。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).
例如，所述M252处的氨基酸突变可以为M252Y。例如，所述S254T处的氨基酸突变可以为S254T。例如，所述T256处的氨基酸突变可以为T256E。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.
在本申请中，所述Fc区可以是源自IgG1、IgG2、IgG3或IgG4的Fc区，且可包含M252Y、S254T和T256E的氨基酸突变。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.
例如，所述Fc区可以是源自IgG1的Fc区，且可包含M252Y、S254T和T256E的氨基酸突变。例如，所述Fc区可以是源自IgG2的Fc区，且可包含M252Y、S254T和T256E的氨基酸突变。例如，所述Fc区可以是源自IgG3的Fc区，且可包含M252Y、S254T和T256E的氨基酸突变。例如，所述Fc区可以是源自IgG4的Fc区，且可包含SEQ ID NO:36所示的氨基酸序列。For example, the Fc region may be an Fc region derived from IgG1, and may include amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG2, and may include amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG3, and may include amino acid mutations of M252Y, S254T, and T256E. For example, the Fc region may be an Fc region derived from IgG4, and may include the amino acid sequence shown in SEQ ID NO:36.
铰链区Hinge area
在本申请中，所述融合蛋白可包含铰链区，所述铰链区位于所述人GLP-1多肽变体和所述免疫球蛋白Fc区之间。在本申请中，所述铰链区可以源自IgG。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.
在本申请中，所述铰链区可以源自IgG1、IgG2、IgG3或IgG4。在本申请中，所述铰链区可以源自IgG1。在本申请中，所述铰链区可以源自IgG4。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.
在本申请中，与天然存在的IgG1铰链区相比，所述融合蛋白中的铰链区可以包含一个或多个氨基酸突变，以去除链间二硫键。这样的氨基酸突变是本领域已知的技术。In the present application, compared with the naturally occurring IgG1 hinge region, the hinge region in the fusion protein may contain one or more amino acid mutations to remove the interchain disulfide bonds. Such amino acid mutations are known in the art.
在本申请中，与天然存在的IgG铰链区相比，所述融合蛋白中的铰链区可以在N端增加一个或多个氨基酸。在本申请中，与天然存在的IgG铰链区相比，所述融合蛋白中的铰链区 可以在C端增加一个或多个氨基酸。在本申请中，与天然存在的IgG铰链区相比，所述融合蛋白中的铰链区可以在N端减少一个或多个氨基酸。在本申请中，与天然存在的IgG铰链区相比，所述融合蛋白中的铰链区可以在C端减少一个或多个氨基酸。In the present application, compared with the naturally occurring IgG hinge region, the hinge region in the fusion protein may have one or more additional amino acids at the N-terminus. One or more amino acids may be added at the C-terminus. In the present application, the hinge region in the fusion protein may reduce one or more 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 reduce one or more amino acids at the C-terminus compared to a naturally occurring IgG hinge region.
在本申请中，与天然存在的IgG4铰链区相比，所述融合蛋白中的铰链区可以在N端增加一个或多个氨基酸，例如，增加Ala。在本申请中，与天然存在的IgG4铰链区相比，所述融合蛋白中的铰链区可以包含一个或多个氨基酸突变，以避免不期望的蛋白形成。这样的氨基酸突变是本领域已知的技术。In the present application, the hinge region in the fusion protein can increase one or more amino acids at the N-terminus, for example, increase Ala, compared to the naturally occurring IgG4 hinge region. In the present application, the hinge region in the fusion protein can include one or more amino acid mutations compared to the naturally occurring IgG4 hinge region to avoid undesirable protein formation. Such amino acid mutations are technologies known in the art.
在本申请中，所述铰链区可以包含如SEQ ID NO:30或31所示的氨基酸序列。In the present application, the hinge region may comprise an amino acid sequence as shown in SEQ ID NO:30 or 31.
连接子Linker
在本申请中，所述融合蛋白可包含连接子。所述连接子位于所述人GLP-1多肽变体和所述免疫球蛋白Fc区之间，用于连接所述人GLP-1多肽变体和所述免疫球蛋白Fc区；优选位于所述人GLP-1多肽变体和所述铰链区之间。在本申请中，所述连接子可以是肽连接子。在本申请中，所述肽连接子可以是刚性的或柔性的。示例性的连接子可包括但不限于SEQ ID NO:27-29或47-70中任一项所示的氨基酸序列。In the present application, the fusion protein may include a linker. The linker is located 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 located 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 shown in any one of SEQ ID NO: 27-29 or 47-70.
在本申请中，所述连接子可以具有4-20个氨基酸长度，优选具有12-15个氨基酸长度。在本申请中，所述连接子可以具有15个氨基酸长度，对于上述列举的不足15个氨基酸长度的连接子，可以通过添加氨基酸使得其长度达到15个氨基酸，例如，添加Gly。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 with a length of less than 15 amino acids, their length may be increased to 15 amino acids by adding amino acids, for example, by adding Gly.
例如，本申请所述的连接子可包含SEQ ID NO:27-29中任一项所示的氨基酸序列。For example, the linker described in the present application may comprise an amino acid sequence shown in any one of SEQ ID NO:27-29.
融合蛋白Fusion Protein
在本申请中，所述融合蛋白可包含所述人GLP-1多肽变体和所述免疫球蛋白Fc区，优选地，可包含所述人GLP多肽变体、所述连接子、所述铰链区和所述免疫球蛋白Fc区。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.
在一些具体的实施方式中，本申请所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:4或25所示的氨基酸序列，所述免疫球蛋白Fc区可以是源自IgG的Fc区。例如，所述免疫球蛋白Fc区可以是包含M252Y、S254T和T256E的氨基酸突变的IgG的Fc区。In some specific embodiments, the human GLP polypeptide variant in the fusion protein described in the present application 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 amino acid mutations of M252Y, S254T and T256E.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:4或25所示的氨基酸序列，所述铰链区可以源自IgG1或IgG4的铰链区，所述免疫球蛋白Fc区可以是源自IgG的Fc区。例如，所述免疫球蛋白Fc区可以是包含M252Y、S254T和T256E的氨基酸突变的IgG的Fc区。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 the Fc region derived from IgG. For example, the immunoglobulin Fc region may be the Fc region of IgG comprising the amino acid mutations of M252Y, S254T and T256E.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:4或25所示 的氨基酸序列，所述连接子可以包含SEQ ID NO:27-29中任一项所示的氨基酸序列，所述铰链区可以源自IgG1或IgG4的铰链区，所述免疫球蛋白Fc区可以是源自IgG的Fc区。例如，所述免疫球蛋白Fc区可以是包含M252Y、S254T和T256E的氨基酸突变的IgG的Fc区。In the present application, the human GLP polypeptide variant in the fusion protein may comprise the human GLP polypeptide variant shown in SEQ ID NO: 4 or 25. The linker may comprise an amino acid sequence as shown 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 an Fc region derived from IgG. For example, the immunoglobulin Fc region may be an Fc region of IgG comprising the amino acid mutations of M252Y, S254T and T256E.
在本申请中，自N端至C端，所述融合蛋白可依次包含所述人GLP多肽变体、所述连接子、所述铰链区和所述免疫球蛋白Fc区。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.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:4所示的氨基酸序列，所述连接子可以包含SEQ ID NO:27-29中任一项所示的氨基酸序列，所述铰链区可以源自IgG1或IgG4的铰链区，所述免疫球蛋白Fc区可以是源自IgG4的Fc区。例如，所述融合蛋白可包含SEQ ID NO:37-39中任一项所示的氨基酸序列。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, 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 the Fc region derived from IgG4. For example, the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 37-39.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:25所示的氨基酸序列，所述连接子可以包含SEQ ID NO:28所示的氨基酸序列，所述铰链区可以源自IgG1或IgG4的铰链区，所述免疫球蛋白Fc区可以是源自IgG4的Fc区，且可选地包含M252Y、S254T和T256E的氨基酸突变。例如，所述融合蛋白可包含SEQ ID NO:40-42和44中任一项所示的氨基酸序列。In the present application, the human GLP polypeptide variant in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 25, the linker may comprise the amino acid sequence shown in SEQ ID NO: 28, the hinge region may be derived from the hinge region of IgG1 or IgG4, the immunoglobulin Fc region may be the Fc region derived from IgG4, and may optionally comprise the amino acid mutations of M252Y, S254T and T256E. For example, the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 40-42 and 44.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:4所示的氨基酸序列，所述连接子可以包含SEQ ID NO:28所示的氨基酸序列，所述铰链区可以源自IgG1或IgG4的铰链区，所述免疫球蛋白Fc区可以是源自IgG4的Fc区，且包含M252Y、S254T和T256E的氨基酸突变。例如，所述融合蛋白可包含SEQ ID NO:43和45中任一项所示的氨基酸序列。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, the linker may comprise the amino acid sequence shown 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 the Fc region derived from IgG4 and comprise the amino acid mutations of M252Y, S254T and T256E. For example, the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 43 and 45.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:4所示的氨基酸序列，所述连接子可以包含SEQ ID NO:28所示的氨基酸序列，所述铰链区可以源自IgG1的铰链区，所述免疫球蛋白Fc区可以是源自IgG4的Fc区，且包含M252Y、S254T和T256E的氨基酸突变。例如，所述融合蛋白可包含SEQ ID NO:43所示的氨基酸序列。In the present application, the human GLP polypeptide variant in the fusion protein may include the amino acid sequence shown in SEQ ID NO: 4, the linker may include the amino acid sequence shown 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 include the amino acid mutations of M252Y, S254T and T256E. For example, the fusion protein may include the amino acid sequence shown in SEQ ID NO: 43.
在本申请中，所述融合蛋白中的所述人GLP多肽变体可以包含SEQ ID NO:25所示的氨基酸序列，所述连接子可以包含SEQ ID NO:28所示的氨基酸序列，所述铰链区可以源自IgG1的铰链区，所述免疫球蛋白Fc区可以是源自IgG4的Fc区，且包含M252Y、S254T和T256E的氨基酸突变。例如，所述融合蛋白可包含SEQ ID NO:44所示的氨基酸序列。In the present application, the human GLP polypeptide variant in the fusion protein may include the amino acid sequence shown in SEQ ID NO: 25, the linker may include the amino acid sequence shown 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 include the amino acid mutations of M252Y, S254T and T256E. For example, the fusion protein may include the amino acid sequence shown in SEQ ID NO: 44.
在本申请中，所述融合蛋白可以以免疫缀合物的形式存在。In the present application, the fusion protein may be present in the form of an immunoconjugate.
药物组合物Pharmaceutical composition
本申请提供了一种药物组合物，其包含前述的融合蛋白，所述药物组合物的pH值可以为5.0-7.0。在本申请所指定的pH值条件下，本申请的药物组合物具备优良的稳定性。在一些实施方案中，所述药物组合物的pH值可以为5.5-6.5，可以优选为5.5-6.2，可以更优选为6.0-6.2，具体地，可以为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或7.0。The present application provides a pharmaceutical composition comprising the aforementioned fusion protein, and 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.
为抵抗pH值变化对组合物稳定性的影响，本申请的药物组合物还可以包含一种或多种的缓冲成分，例如，所述缓冲成分选自下组中的一种或多种：L-组氨酸-醋酸、柠檬酸盐、L-组氨酸-盐酸、醋酸-醋酸钠和磷酸盐，所述缓冲成分可以优选为L-组氨酸-醋酸或柠檬酸盐。In order to resist the influence of pH change 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-acetic acid, citrate, L-histidine-hydrochloric acid, acetic acid-sodium acetate and phosphate, and the buffer component may preferably be L-histidine-acetic acid or citrate.
本申请所述缓冲成分的浓度可以为1nM-75mM，可以优选为2mM-50mM；可以更优选为5mM-40mM；甚至可以更优选为10mM-30mM。具体地，所述缓冲成分的浓度可以为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或25mM-45mM。The concentration of the buffer component described in the present application may be 1 nM-75 mM, and may preferably be 2 mM-50 mM; may more preferably be 5 mM-40 mM; and may even more preferably be 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.
在一些实施方案中，所述缓冲成分可以包含L-组氨酸-醋酸，所述L-组氨酸-醋酸的浓度可以为2mM-50mM、5mM-45mM、10mM-40mM、10mM-30mM、15mM-35mM、15mM-30mM、15mM-25mM或20mM-25mM。In some embodiments, the buffer component may include 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.
在一些实施方案中，所述缓冲成分可以包含柠檬酸盐，所述柠檬酸盐的浓度可以为1mM-30mM、2mM-25mM、2mM-20mM、5mM-20mM、5mM-15mM或10mM-15mM。In some embodiments, the buffer component may include citrate, and the concentration of the citrate may 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.
本申请的药物组合物中还可以包含稳定剂，以保护融合蛋白免于化学和/或物理降解，所述稳定剂可以选自多元醇、氨基酸、糖类、盐和/或白蛋白。具体地，本申请所述的稳定剂选自下组中的一种或多种：甘露醇、L-精氨酸盐酸盐、甘氨酸、L-脯氨酸、山梨醇、甘油、麦芽糖、海藻糖和氯化钠；可以优选地，所述稳定剂选自下组中的一种或多种：甘露醇、L-精氨酸盐酸盐、甘氨酸和L-脯氨酸；可以更优选地，所述稳定剂选自下组中的一种或多种：甘露醇、甘氨酸和L-脯氨酸；甚至可以更优选的，所述稳定剂包含甘露醇。The pharmaceutical composition of the present application may also include a stabilizer to protect the fusion protein from chemical and/or physical degradation, and the stabilizer may be selected from polyols, amino acids, sugars, salts and/or albumin. Specifically, the stabilizer described in the present application 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; even more preferably, the stabilizer comprises mannitol.
本申请所述稳定剂的浓度可以为2mg/mL-300mg/mL，可以为5mg/mL-300mg/mL，可以优选为10mg/mL-150mg/mL，可以更优选为20mg/mL-100mg/mL，甚至可以更优选为25mg/mL-80mg/mL。具体地，所述稳定剂的浓度可以为5mg/mL-270mg/mL、5mg/mL-260mg/mL、5mg/mL-250mg/mL、5mg/mL-200mg/mL、5mg/mL-170mg/mL、10mg/mL-260mg/mL、10mg/mL- 240mg/mL、10mg/mL-220mg/mL、10mg/mL-190mg/mL、10mg/mL-160mg/mL、20mg/mL-240mg/mL、20mg/mL-230mg/mL、20mg/mL-180mg/mL、20mg/mL-150mg/mL、25mg/mL-210mg/mL、25mg/mL-200mg/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或30mg/mL-60mg/mL。The concentration of the stabilizer described in the present application can be 2mg/mL-300mg/mL, can be 5mg/mL-300mg/mL, can be preferably 10mg/mL-150mg/mL, can be more preferably 20mg/mL-100mg/mL, and can even be more preferably 25mg/mL-80mg/mL. Specifically, the concentration of the stabilizer can be 5mg/mL-270mg/mL, 5mg/mL-260mg/mL, 5mg/mL-250mg/mL, 5mg/mL-200mg/mL, 5mg/mL-170mg/mL, 10mg/mL-260mg/mL, 10mg/mL- or 30 mg/mL.
在一些实施方案中，所述稳定剂包含甘露醇，所述甘露醇的浓度可以为10mg/mL-150mg/mL，可以优选为20mg/mL-100mg/mL，可以更优选为30mg/mL-80mg/mL，甚至可以更优选为40mg/mL-60mg/mL。具体地，所述甘露醇的浓度可以为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或30mg/mL-70mg/mL。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 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.
在一些实施方案中，所述稳定剂包含甘氨酸，所述甘氨酸的浓度可以为2mg/mL-50mg/mL，可以优选为5mg/mL-40mg/mL，可以更优选为10mg/mL-30mg/mL，甚至可以更优选为15mg/mL-25mg/mL。具体地，所述甘氨酸的浓度可以为1mg/mL-45mg/mL、1mg/mL-40mg/mL、2mg/mL-40mg/mL、2mg/mL-35mg/mL、5mg/mL-50mg/mL、5mg/mL-40mg/mL、5mg/mL-30mg/mL、10mg/mL-35mg/mL、10mg/mL-25mg/mL、15mg/mL-40mg/mL、15mg/mL-35mg/mL、15mg/mL-25mg/mL、18mg/mL-30mg/mL或18mg/mL-20mg/mL。In some embodiments, the stabilizer comprises glycine, and the concentration of the glycine can be 2mg/mL-50mg/mL, preferably 5mg/mL-40mg/mL, more preferably 10mg/mL-30mg/mL, and even more preferably 15mg/mL-25mg/mL. Specifically, the concentration of the glycine can be 1mg/mL-45mg/mL, 1mg/mL-40mg/mL, 2mg/mL-40mg/mL, 2mg/mL-35mg/mL, 5mg/mL-50mg/mL, 5mg/mL-40mg/mL, 5mg/mL-30mg/mL, 10mg/mL-35mg/mL, 10mg/mL-25mg/mL, 15mg/mL-40mg/mL, 15mg/mL-35mg/mL, 15mg/mL-25mg/mL, 18mg/mL-30mg/mL or 18mg/mL-20mg/mL.
在一些实施方案中，所述稳定剂包含L-脯氨酸，所述L-脯氨酸的浓度可以为5mg/mL-75mg/mL，可以优选为10mg/mL-50mg/mL，可以更优选为15mg/mL-40mg/mL，甚至可以更优选为20mg/mL-30mg/mL。具体地，所述L-脯氨酸的浓度可以为2mg/mL-100mg/mL、2mg/mL-75mg/mL、5mg/mL-75mg/mL、5mg/mL-60mg/mL、5mg/mL-50mg/mL、10mg/mL-70mg/mL、10mg/mL-50mg/mL、10mg/mL-45mg/mL、15mg/mL-80mg/mL、15mg/mL-55mg/mL、15mg/mL-45mg/mL、20mg/mL-65mg/mL、20mg/mL-50mg/mL、20mg/mL-40mg/mL、20mg/mL-30mg/mL、25mg/mL-45mg/mL、25mg/mL-40mg/mL或25mg/mL-30mg/mL。In some embodiments, the stabilizer comprises L-proline, and the concentration of the L-proline may be 5 mg/mL-75 mg/mL, may be preferably 10 mg/mL-50 mg/mL, may be more preferably 15 mg/mL-40 mg/mL, and may even more preferably be 20 mg/mL-30 mg/mL. Specifically, the concentration of the L-proline can be 2mg/mL-100mg/mL, 2mg/mL-75mg/mL, 5mg/mL-75mg/mL, 5mg/mL-60mg/mL, 5mg/mL-50mg/mL, 10mg/mL-70mg/mL, 10mg/mL-50mg/mL, 10mg/mL-45mg/mL, 15mg/mL-80mg/mL, 15mg/mL-55mg/mL, 15mg/mL-45mg/mL, 20mg/mL-65mg/mL, 20mg/mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL, 25mg/mL-45mg/mL, 25mg/mL-40mg/mL or 25mg/mL-30mg/mL.
在一些实施方案中，所述稳定剂包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度可以为10mg/mL-100mg/mL，可以优选为20mg/mL-80mg/mL，可以更优选为25mg/mL-60mg/mL，甚至可以更优选为30mg/mL-50mg/mL。具体地，所述L-精氨酸盐酸盐的浓度可以为5mg/mL-100mg/mL、5mg/mL-80mg/mL、5mg/mL-70mg/mL、10mg/mL-100mg/mL、10mg/mL-60mg/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或35mg/mL-40mg/mL。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.
本申请的药物组合物还可以包括表面活性剂，所述表面活性剂选自聚山梨酯80、聚山梨酯20和泊洛沙姆188中的一种或多种；可以优选地，所述表面活性剂为聚山梨酯80或聚山梨酯20。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.
本申请所述表面活性剂的浓度可以为0.01mg/mL-1.0mg/mL，可以优选为0.05mg/mL-0.75mg/mL，可以更优选为0.1mg/mL-0.5mg/mL，甚至可以更优选为0.15mg/mL-0.25mg/mL。具体的，所述表面活性剂的浓度可以为0.01mg/mL-1.0mg/mL、0.01mg/mL-0.75mg/mL、0.05mg/mL-0.9mg/mL、0.05mg/mL-0.8mg/mL、0.05mg/mL-0.7mg/mL、0.1mg/mL-0.7mg/mL、0.1mg/mL-0.5mg/mL、0.15mg/mL-0.75mg/mL、0.15mg/mL-0.6mg/mL、0.15mg/mL-0.5mg/mL、0.15mg/mL-0.4mg/mL、0.15mg/mL-0.3mg/mL、0.15mg/mL-0.25mg/mL、0.2mg/mL-0.6mg/mL、0.2mg/mL-0.4mg/mL或0.2mg/mL-0.3mg/mL。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.
在本申请的药物组合物中，所述融合蛋白能够以较高的蛋白浓度稳定存在，所述融合蛋白的浓度为1mg/mL-30mg/mL，可以优选为2mg/mL-20mg/mL，可以更优选为5mg/mL-10mg/mL。具体的，所述融合蛋白的浓度可以为0.5mg/mL-30mg/mL、0.5mg/mL-25mg/mL、1mg/mL-30mg/mL、1mg/mL-25mg/mL、1mg/mL-20mg/mL、2mg/mL-20mg/mL、2mg/mL-15mg/mL、2mg/mL-10mg/mL、5mg/mL-15mg/mL或5mg/mL-10mg/mL。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 1mg/mL-30mg/mL, preferably 2mg/mL-20mg/mL, and more preferably 5mg/mL-10mg/mL. Specifically, the concentration of the fusion protein can be 0.5mg/mL-30mg/mL, 0.5mg/mL-25mg/mL, 1mg/mL-30mg/mL, 1mg/mL-25mg/mL, 1mg/mL-20mg/mL, 2mg/mL-20mg/mL, 2mg/mL-15mg/mL, 2mg/mL-10mg/mL, 5mg/mL-15mg/mL or 5mg/mL-10mg/mL.
本申请的药物组合物可以为液体制剂，例如，可以为注射液，可以优选为皮下注射液。所述液体制剂包括溶媒，所述溶媒为注射用水或注射用有机溶剂；可以优选地，所述注射用有机溶剂选自注射用油、乙醇、丙二醇中的一种或多种；可以更优选地，所述溶媒为注射用水。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 oil for injection, ethanol, and propylene glycol; more preferably, the solvent is water for injection.
本申请的药物组合物具备优良的稳定性，例如，在加速稳定性研究中，所述组合物在25±2℃的条件下放置6个月后，其中融合蛋白的单体含量仍在95％、96％、96.5％、97％、97.5％、98％、98.5％、99％或99.5％以上，所述单体含量可通过SE-HPLC纯度测得。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%, and the monomer content can be measured by SE-HPLC purity.
另一方面，本申请还提供一种试剂盒，其包括本申请所述的组合物和盛放本申请所述的组合物的容器。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.
例如，所述容器可以包括小瓶(例如，玻璃瓶)、安瓿、注射器、注射笔和/或静脉袋。在 本申请中，所述容器可以为无菌。例如，所述容器可以包括玻璃瓶。For example, the container may include a vial (e.g., a glass bottle), an ampoule, a syringe, an injection pen, and/or an IV bag. In the present application, the container can be sterile. For example, the container can include a glass bottle.
例如，所述试剂盒可以包括一个递送装置，所述递送装置可以施用本申请所述的组合物。例如，所述递送装置可以包括小瓶、安瓿、注射器、注射笔和/或静脉袋。For example, the kit may include a delivery device that can administer the composition described herein. For example, the delivery device may include a vial, an ampoule, a syringe, an injection pen, and/or an intravenous bag.
在本申请中，所述容器中所述组合物的体积可以为0.1mL-约5.0mL，可以优选为0.2mL-3.0mL，可以更优选为0.5mL-2.0mL。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.
治疗用途Therapeutic Uses
本申请的药物组合物可以用于预防和/或治疗代谢性疾病或病症。一方面，本申请提供了一种预防和/或治疗代谢性疾病或病症的方法，其包括以下步骤：向有需要的受试者施用所述的药物组合物。The pharmaceutical composition of the present application can be used to prevent and/or treat metabolic diseases or conditions. On the one hand, 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.
另一方面，本申请还提供了所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗代谢性疾病或病症。On the other hand, the present application also provides use of the pharmaceutical composition in preparing a drug for preventing and/or treating a metabolic disease or condition.
此外，本申请还提供了一种药物组合物，其可以用于预防和/或治疗代谢性疾病或病症。In addition, the present application also provides a pharmaceutical composition, which can be used to prevent and/or treat metabolic diseases or disorders.
所述代谢性疾病或病症可以包含GLP-1相关代谢性疾病；可以优选地，所述代谢性疾病包含糖尿病；可以更优选地，所述代谢性疾病包含II型糖尿病。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.
另一方面，本申请提供了一种增加或促进有需要的受试者中胰岛素表达的方法，所述方法包括向所述受试者施用所述药物组合物。In another aspect, the present application provides a method for increasing or promoting insulin expression in a subject in need thereof, the method comprising administering the pharmaceutical composition to the subject.
例如，有需要的受试者可以包含患有代谢性疾病或病症的人。例如，有需要的受试者可以包含患有GLP-1相关代谢性疾病的人。例如，有需要的受试者可以包含患有糖尿病的人。例如，有需要的受试者包含患有II型糖尿病的人。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.
例如，所述胰岛素表达可以包含胰岛素分泌的活性。例如，所述胰岛素表达可以包含胰岛素的量和浓度。例如，所述胰岛素的表达可以通过本领域已知的任何方法进行检测。For example, the insulin expression can include the activity of insulin secretion. For example, the insulin expression can include the amount and concentration of insulin. For example, the insulin expression can be detected by any method known in the art.
例如，所述增加或促进有需要的受试者中胰岛素表达可以包含相对于施用药物组合之前，受试者胰岛素的表达水平增加或促进至少约5％、至少约10％、至少约15％，至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、至少约90％、至少约100％、至少约150％、至少约200％、至少约300％或更高的水平。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.
本申请具体提供了以下的实施方案：This application specifically provides the following implementation scheme:
1.一种药物组合物，其包含融合蛋白，所述融合蛋白包含人GLP-1多肽变体和免疫球蛋白Fc区；其中与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序 列包含至少2个氨基酸突变，所述至少2个氨基酸突变位于选自下组的氨基酸位置处：W31、K26和Y19；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 is different from that of the amino acid sequence shown in SEQ ID NO: 2. The row comprises at least 2 amino acid mutations, wherein the at least 2 amino acid mutations are located at amino acid positions selected from the group consisting of: W31, K26, and Y19;
所述药物组合物的pH值为5.0-7.0。The pH value of the pharmaceutical composition is 5.0-7.0.
2.根据实施方案1所述的药物组合物，其中所述免疫球蛋白Fc区位于所述人GLP-1多肽变体的C端。2. A pharmaceutical composition according to embodiment 1, wherein the immunoglobulin Fc region is located at the C-terminus of the human GLP-1 polypeptide variant.
3.根据实施方案1或2所述的药物组合物，其中所述免疫球蛋白Fc区为源自IgG的Fc区；优选为源自IgG1、IgG2、IgG3或IgG4的Fc区；更优选为选自IgG4的Fc区。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.
4.根据实施方案1-3中任一项所述的药物组合物，其中所述免疫球蛋白Fc区包含氨基酸突变，与未经所述氨基酸突变的免疫球蛋白Fc区相比，所述氨基酸突变使得所述免疫球蛋白Fc区与Fc受体的结合亲和力选择性增强。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.
5.根据实施方案1-4中任一项所述的药物组合物，其中所述免疫球蛋白Fc区在根据EU编码的M252、S254和T256位置处包含氨基酸突变；优选包含下组的氨基酸突变：M252Y、S254T和T256E。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 encoded according to EU; preferably comprises the following group of amino acid mutations: M252Y, S254T and T256E.
6.根据实施方案1-5中任一项所述的药物组合物，其中所述免疫球蛋白Fc区包含如SEQ ID NO:36所示的氨基酸序列。6. A pharmaceutical composition according to any one of embodiments 1-5, wherein the immunoglobulin Fc region comprises an amino acid sequence as shown in SEQ ID NO:36.
7.根据实施方案1-6中任一项所述的药物组合物，其中所述融合蛋白还包含铰链区，铰链区位于所述人GLP-1多肽变体和所述免疫球蛋白Fc区之间，所述铰链区包含如SEQ ID NO:30-31中任一项所示的氨基酸序列；优选包含如SEQ ID NO:30所示的氨基酸序列。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 an amino acid sequence as shown in any one of SEQ ID NO: 30-31; preferably comprises an amino acid sequence as shown in SEQ ID NO: 30.
8.根据实施方案1-7中任一项所述的药物组合物，其中所述融合蛋白还包含连接子，所述连接子位于所述人GLP-1多肽变体和所述免疫球蛋白Fc区之间；优选位于所述人GLP-1多肽变体和所述铰链区之间。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 between the human GLP-1 polypeptide variant and the hinge region.
9.根据实施方案8所述的药物组合物，所述连接子为具有5-20个氨基酸长度的肽；优选地，所述连接子包含如SEQ ID NO:27-29中任一项所示的氨基酸序列；更优选地，所述连接子包含如SEQ ID NO:28所示的氨基酸序列。9. According to the pharmaceutical composition described in 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 NO: 27-29; more preferably, the linker comprises an amino acid sequence as shown in SEQ ID NO: 28.
10.根据实施方案1-9中任一项所述的药物组合物，其中与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列包含位于选自以下位置处的氨基酸突变：10. A 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 following positions compared to the amino acid sequence shown in SEQ ID NO: 2:
a)在W31和K26氨基酸位置处；a) at amino acid positions W31 and K26;
b)在W31和Y19氨基酸位置处；b) at amino acid positions W31 and Y19;
c)在K26和Y19氨基酸位置处； c) at amino acid positions K26 and Y19;
以及d)在W31、K26和Y19氨基酸位置处and d) at amino acid positions W31, K26 and Y19
11.根据实施方案1-10中任一项所述的药物组合物，其中在W31氨基酸位置处的突变选自W31Y、W31R、W31K和W31A；优选为W31Y。11. A pharmaceutical composition according to any one of embodiments 1-10, wherein the mutation at the amino acid position W31 is selected from W31Y, W31R, W31K and W31A; preferably W31Y.
12.根据实施方案1-11中任一项所述的药物组合物，其中在K26氨基酸位置处的突变为K26R。12. The pharmaceutical composition of any one of embodiments 1-11, wherein the mutation at amino acid position K26 is K26R.
13.根据实施方案1-12中任一项所述的药物组合物，其中在Y19氨基酸位置处的突变选自Y19A、Y19L、Y19T、Y19F、Y19I、Y19V和Y19S；优选为Y19A。13. A pharmaceutical composition according to any one of embodiments 1-12, wherein the mutation at the Y19 amino acid position is selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S; preferably Y19A.
14.根据实施方案1-13中任一项所述的药物组合物，其中与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列包含的氨基酸突变组合为W31Y、K26R和Y19A；或W31Y和K26R。14. A pharmaceutical composition according to any one of embodiments 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 an amino acid mutation combination of W31Y, K26R and Y19A; or W31Y and K26R.
15.根据实施方案1-13中任一项所述的药物组合物，其中所述GLP-1多肽变体包含SEQ ID NO:4-26中任一项所示的氨基酸序列。15. A 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 NO:4-26.
16.根据实施方案1-15中任一项所述的药物组合物，其中所述融合蛋白自N端至C端，依次包含所述人GLP-1多肽变体、连接子、铰链区以及免疫球蛋白Fc区。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.
17.根据实施方案1-16中任一项所述的药物组合物，其中所述融合蛋白包含如SEQ ID NO:37-45中任一项所示的氨基酸序列。17. A pharmaceutical composition according to any one of embodiments 1-16, wherein the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO:37-45.
18.根据实施方案1-17中任一项所述的药物组合物，所述药物组合物的pH值为5.5-6.5；优选pH值为5.5-6.2；更优选pH值为6.0-6.2。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.
19.根据实施方案1-18中任一项所述的药物组合物，所述药物组合物包含缓冲成分。19. The pharmaceutical composition according to any one of embodiments 1-18, comprising a buffer component.
20.根据实施方案19所述的药物组合物，其中所述缓冲成分选自下组中的一种或多种：L-组氨酸-醋酸、柠檬酸盐、L-组氨酸-盐酸、醋酸-醋酸钠和磷酸盐；所述缓冲成分优选为L-组氨酸-醋酸或柠檬酸盐。20. A 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.
21.根据实施方案19或20所述的药物组合物，其中所述缓冲成分的浓度为2mM-50mM；优选为5mM-40mM；更优选为10mM-30mM；甚至更优选为20mM。21. A pharmaceutical composition according to embodiment 19 or 20, wherein the concentration of the buffer component is 2mM-50mM; preferably 5mM-40mM; more preferably 10mM-30mM; even more preferably 20mM.
22.根据实施方案1-21中任一项所述的药物组合物，所述药物组合物还包含稳定剂。22. The pharmaceutical composition according to any one of embodiments 1-21, further comprising a stabilizer.
23.根据实施方案22所述的药物组合物，其中所述稳定剂选自下组中的一种或多种：甘露醇、L-精氨酸盐酸盐、甘氨酸、L-脯氨酸、山梨醇、甘油、麦芽糖、海藻糖和氯化钠；优选地，所述稳定剂选自下组中的一种或多种：甘露醇、L-精氨酸盐酸盐、甘氨酸和L-脯氨酸；更优选地，所述稳定剂选自下组中的一种或多种：甘露醇、甘氨酸和L-脯氨酸。 23. A pharmaceutical composition according to embodiment 22, wherein the stabilizer is selected from one or more of the following group: 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 group: mannitol, L-arginine hydrochloride, glycine and L-proline; more preferably, the stabilizer is selected from one or more of the following group: mannitol, glycine and L-proline.
24.根据实施方案23所述的药物组合物，所述稳定剂的浓度为2mg/mL-150mg/mL，优选为10mg/mL-150mg/mL；优选为20mg/mL-100mg/mL。24. According to the pharmaceutical composition of embodiment 23, 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.根据实施方案23所述的药物组合物，所述稳定剂包含甘露醇，所述甘露醇的浓度为10mg/mL-150mg/mL；优选为20mg/mL-100mg/mL；更优选为30mg/mL-80mg/mL；甚至更优选为40mg/mL-60mg/mL。25. According to the pharmaceutical composition of embodiment 23, 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.
26.根据实施方案23所述的药物组合物，所述稳定剂包含甘氨酸，所述甘氨酸的浓度为2mg/mL-50mg/mL；优选为5mg/mL-40mg/mL；更优选为10mg/mL-30mg/mL；甚至更优选为15mg/mL-25mg/mL。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.
27.根据实施方案23所述的药物组合物，所述稳定剂包含L-脯氨酸，所述L-脯氨酸的浓度为5mg/mL-75mg/mL；优选为10mg/mL-50mg/mL；更优选为15mg/mL-40mg/mL；甚至更优选为20mg/mL-30mg/mL。27. According to the pharmaceutical composition of embodiment 23, 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.
28.根据实施方案23所述的药物组合物，所述稳定剂包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度为10mg/mL-100mg/mL；优选为20mg/mL-80mg/mL；更优选为25mg/mL-60mg/mL；甚至更优选为30mg/mL-50mg/mL。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.
29.根据实施方案1-28中任一项所述的药物组合物，所述药物组合物还包含表面活性剂。29. The pharmaceutical composition according to any one of embodiments 1-28, further comprising a surfactant.
30.根据实施方案29所述的药物组合物，其中所述表面活性剂选自聚山梨酯80、聚山梨酯20和泊洛沙姆188中的一种或多种；优选地，所述表面活性剂为聚山梨酯80或聚山梨酯20。30. A 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.
31.根据实施方案30所述的药物组合物，其中所述表面活性剂的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。31. A 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.
32.根据实施方案30所述的药物组合物，其中所述表面活性剂包含聚山梨酯80，所述聚山梨酯80的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。32. A pharmaceutical composition according to 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.
33.根据实施方案30所述的药物组合物，其中所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。33. A pharmaceutical composition according to 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.
34.根据实施方案30所述的药物组合物，其中所述表面活性剂包含泊洛沙姆188，所述泊洛沙姆188的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为 0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。34. The pharmaceutical composition according to 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.
35.根据实施方案1-34中任一项所述的药物组合物，其中所述融合蛋白的浓度为1mg/mL-30mg/mL；优选为2mg/mL-14mg/mL；更优选为5mg/mL-10mg/mL。35. A pharmaceutical composition according to 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.
36.根据实施方案1-35中任一项所述的药物组合物，所述药物组合物为液体制剂。36. The pharmaceutical composition according to any one of embodiments 1-35, which is a liquid preparation.
37.根据实施方案36所述的药物组合物，其中所述液体制剂为注射液，优选为皮下注射液。37. A pharmaceutical composition according to embodiment 36, wherein the liquid preparation is an injection solution, preferably a subcutaneous injection solution.
38.根据实施方案36或37所述的药物组合物，其中所述液体制剂包含溶媒，所述溶媒为注射用水或注射用有机溶剂；优选地，所述注射用有机溶剂选自注射用油、乙醇、丙二醇中的一种或多种。38. A 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 injection oil, ethanol, and propylene glycol.
39.根据实施方案1-38中任一项所述的药物组合物，所述药物组合物在25±2℃的条件下放置6个月后，其中融合蛋白的单体含量仍在96％以上；优选单体含量在97％以上；更优选单体含量在98％以上，甚至更优选单体含量在99％以上。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%.
40.试剂盒，其包含实施方案1-39中任一项所述的药物组合物以及盛放所述组合物的容器。40. A kit comprising the pharmaceutical composition of any one of embodiments 1-39 and a container for holding the composition.
41.根据实施方案40所述的试剂盒，其中所述容器包括玻璃瓶。41. The kit of embodiment 40, wherein the container comprises a glass bottle.
42.根据实施方案40或41所述的试剂盒，其中所述容器中所述药物组合物的体积为0.1mL-5.0mL；优选为0.2mL-3.0mL；更优选为0.5mL-2.0mL。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.
43.实施方案1-39中任一项所述的药物组合物，和/或实施方案40-42任一所述的试剂盒在制备预防和/或治疗代谢性疾病或病症药物中的用途。43. Use of the pharmaceutical composition described in any one of embodiments 1-39 and/or the kit described in any one of embodiments 40-42 in the preparation of a drug for preventing and/or treating a metabolic disease or disorder.
44.根据实施方案43所述的用途，其中所述代谢性疾病或病症包含GLP-1相关代谢性疾病或病症；优选地，所述代谢性疾病或病症包含糖尿病；更优选地，所述代谢性疾病或病症包含II型糖尿病。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.
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。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.
实施例Example
实施例1融合蛋白的制备Example 1 Preparation of fusion protein
制备人GLP-1多肽变体和Fc的融合蛋白，从N端至C端依次包含GLP-1多肽变体(氨 基酸序列如SEQ ID NO:4-25中任一项所示)、连接肽(氨基酸序列如SEQ ID NO:27-29中任一项所示)、铰链区(氨基酸序列如SEQ ID NO:30-31中任一项所示)和源自IgG的Fc区(氨基酸序列如SEQ ID NO:32-36中任一项所示)。将包含编码融合蛋白的核酸序列的载体转入细胞，表达纯化得融合蛋白。示例性的融合蛋白全长序列和各部分序列如下表1所示。Prepare a fusion protein of human GLP-1 polypeptide variant and Fc, which comprises GLP-1 polypeptide variant (amino The fusion protein is expressed in the form of a vector comprising a nucleic acid sequence encoding a fusion protein, wherein the ...
表1示例性融合蛋白的结构和氨基酸序列
Table 1 Structure and amino acid sequence of exemplary fusion proteins
实施例2使用不同连接子的融合蛋白的药代动力学检测Example 2 Pharmacokinetics of fusion proteins using different linkers
考察本申请融合蛋白GM-RY-L1H2-Fc4、GM-RY-L2H2-Fc4和GM-RY-L3H2-Fc4的体内药代动力学。实验在SPF动物房进行，动物房环境温度23±2℃，相对湿度40-70％，12小时明暗交替。实验动物自由饮食，实验前适应至少3天。体重为180-220g的SPF级SD大鼠随 机分组。皮下注射(SC)各组大鼠相对应供试品，剂量为1mg/kg，度拉糖肽(DULA)作为对照。于给药前、给药后6h、24h、48h、72h、96h和120h经颈静脉采血约100ul。将采集的血液加入离心管中，室温静置30-60min。之后4℃，两小时内4000rpm离心10分钟，迅速分离血清。-80℃保存。样本进行ELISA检测。使用DAS(3.2.8)软件计算药代动力学参数。The in vivo pharmacokinetics of the fusion proteins GM-RY-L1H2-Fc4, GM-RY-L2H2-Fc4 and GM-RY-L3H2-Fc4 of the present application were investigated. The experiment was 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 given free access to food and adapted for at least 3 days before the experiment. SPF SD rats weighing 180-220 g were followed by The rats in each group were divided into groups. The corresponding test sample was injected subcutaneously (SC) at a dose of 1 mg/kg, and dulaglutide (DULA) was used as a control. About 100ul of blood was collected from the jugular vein before administration and 6h, 24h, 48h, 72h, 96h and 120h after administration. The collected blood was added to a centrifuge tube and allowed to stand at room temperature for 30-60min. After that, the blood was centrifuged at 4000rpm for 10 minutes at 4℃ within two hours to quickly separate the serum. Store at -80℃. The samples were tested by ELISA. The pharmacokinetic parameters were calculated using DAS (3.2.8) software.
结果如图1和表2显示，GM-RY-L1H2-Fc4、GM-RY-L2H2-Fc4和GM-RY-L3H2-Fc4的体内代谢性质与对照的度拉糖肽没有显著差异，其中使用L2连接子(SEQ ID NO:28)的GM-RY-L2H2-Fc4具有最好的半衰期，暴露量与对照相当。The results are shown in Figure 1 and Table 2. There was no significant difference in the in vivo metabolic properties of GM-RY-L1H2-Fc4, GM-RY-L2H2-Fc4 and GM-RY-L3H2-Fc4 compared with the control dulaglutide, among which GM-RY-L2H2-Fc4 using L2 linker (SEQ ID NO:28) had the best half-life and the exposure was comparable to that of the control.
表2药代动力学检测结果
Table 2 Pharmacokinetic test results
实施例3使用不同铰链区的融合蛋白的药代动力学检测Example 3 Pharmacokinetic testing of fusion proteins using different hinge regions
根据实施例2的方法，检测本申请融合蛋白GM-ARY-L2H2-Fc4和GM-ARY-L2H1-Fc4的体内药代动力学。样本进行ELISA检测。使用DAS(3.2.8)软件计算药代动力学参数。结果如图2和表3显示，GM-ARY-L2H2-Fc4和GM-ARY-L2H1-Fc4的体内半衰期相当，其中使用IgG1的铰链区(SEQ ID NO:30)的GM-ARY-L2H1-Fc4的暴露量较好。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 detected. The samples were tested by ELISA. The pharmacokinetic parameters were calculated using DAS (3.2.8) software. The results are shown in Figure 2 and Table 3. The in vivo half-life of GM-ARY-L2H2-Fc4 and GM-ARY-L2H1-Fc4 is comparable, among which the exposure of GM-ARY-L2H1-Fc4 using the hinge region of IgG1 (SEQ ID NO: 30) is better.
表3药代动力学检测结果
Table 3 Pharmacokinetic test results
实施例4使用Fc区包含YTE突变的融合蛋白的药代动力学检测Example 4 Pharmacokinetic testing of fusion proteins containing YTE mutations in the Fc region
使用食蟹猴检测本申请融合蛋白GM-RY-L2H1-Fc4m和GM-ARY-L2H1-Fc4m的体内药代动力学。实验每组2只食蟹猴，雌雄各半，每组分别单次皮下注射给予对应供试品(1mg/kg)， DULA(IgG4的Fc区未进行YTE突变的度拉糖肽)和DULA-Fc4m(IgG4的Fc区进行YTE突变的度拉糖肽)作为对照。分别于给药前和给药后4h、8h、24h、48h、72h、120h、168h、264h、336h、504h、672h、840h和1008h采集血样。分离血清，ELISA法测定给予受试物后食蟹猴血清中受试物的浓度。使用DAS(3.2.8)软件计算药代动力学参数。The in vivo pharmacokinetics of the fusion proteins GM-RY-L2H1-Fc4m and GM-ARY-L2H1-Fc4m of the present application were tested using cynomolgus monkeys. Two cynomolgus monkeys were included in each group, half male and half female. Each group was given a single subcutaneous injection of the corresponding test sample (1 mg/kg). DULA (dulaglutide without YTE mutation in the Fc region of IgG4) and DULA-Fc4m (dulaglutide with YTE mutation in the Fc region of IgG4) were used as controls. Blood samples were collected before and 4h, 8h, 24h, 48h, 72h, 120h, 168h, 264h, 336h, 504h, 672h, 840h and 1008h after administration. Serum was separated and the concentration of the test substance in the serum of cynomolgus monkeys after administration of the test substance was determined by ELISA. Pharmacokinetic parameters were calculated using DAS (3.2.8) software.
结果如图3、图4、表4和表5显示，其中表4的数据来自图3的曲线，表5的数据来自图4的曲线。与同样包含YTE突变的度拉糖肽相比，本申请的GM-RY-L2H1-Fc4m(图3和图4)和GM-ARY-L2H1-Fc4m(图4)具有更好的体内半衰期和暴露量；而度拉糖肽的Fc区进行YTE突变的效果较弱，与未突变的度拉糖肽相比，Fc区的YTE突变并没有提升度拉糖肽的体内半衰期和暴露量(图4)。The results are shown in Figures 3, 4, Table 4 and 5, where the data in Table 4 are from the curve in Figure 3, and the data in Table 5 are from the curve in Figure 4. Compared with dulaglutide also containing the YTE mutation, 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; while the effect of YTE mutation in the Fc region of dulaglutide is weak, and compared with the unmutated dulaglutide, the YTE mutation in the Fc region does not improve the in vivo half-life and exposure of dulaglutide (Figure 4).
结合图1的数据，未进行Fc区YTE突变的本申请融合蛋白的半衰期或暴露量与度拉糖肽相当，而Fc区进行YTE突变后的本申请融合蛋白的半衰期和暴露量均显著高于度拉糖肽。这说明Fc区的YTE突变显著提升了本申请融合蛋白的药代动力学性质，且融合蛋白的人GLP-1多肽变体的氨基酸突变和Fc区的YTE突变对药代动力学性质的提升具有协同效果。Combined with the data in Figure 1, the half-life or exposure of the fusion protein of the present application without YTE mutation in the Fc region is comparable to that of dulaglutide, while the half-life and exposure of the fusion protein of the present application after 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 the amino acid mutation of the human GLP-1 polypeptide variant of the fusion protein and the YTE mutation in the Fc region have a synergistic effect on the improvement of pharmacokinetic properties.
表4药代动力学检测结果
Table 4 Pharmacokinetic test results
表5药代动力学检测结果
Table 5 Pharmacokinetic test results
实施例5使用不同铰链区的Fc区YTE突变的融合蛋白的药代动力学检测Example 5 Pharmacokinetic testing of fusion proteins with YTE mutations in the Fc region using different hinge regions
根据实施例4的方法，检测本申请包含Fc区YTE突变的融合蛋白GM-ARY-L2H2-Fc4m、GM-RY-L2H2-Fc4m、GM-ARY-L2H1-Fc4m和GM-RY-L2H1-Fc4m的体内药代动力学。样本 进行ELISA检测。使用DAS(3.2.8)软件计算药代动力学参数。According to the method of Example 4, 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 containing the YTE mutation in the Fc region of the present application were detected. ELISA was performed and the pharmacokinetic parameters were calculated using DAS (3.2.8) software.
结果如图5和表6显示，四种融合蛋白的体内半衰期或暴露量均较好，其中使用IgG1的铰链区(SEQ ID NO:30)的融合蛋白的综合效果较好。The results are shown in Figure 5 and Table 6. The in vivo half-life or exposure of the four fusion proteins are all good, among which the fusion protein using the hinge region of IgG1 (SEQ ID NO: 30) has the best overall effect.
表6药代动力学检测结果
Table 6 Pharmacokinetic test results
实施例6荧光素酶法检测本申请融合蛋白对cAMP/PKA信号通路的激活作用Example 6 Luciferase assay to detect the activation effect of the fusion protein of the present application on the cAMP/PKA signaling pathway
利用GLP-1多肽变体的融合蛋白结合GLP-1受体，激活腺苷酸环化酶，促进细胞内环磷酸腺苷(cAMP)水平升高的原理，基于CREB(cAMP反应元件结合蛋白)报告基因检测本申请融合蛋白对cAMP/PKA信号通路的激活作用。利用人GLP-1R质粒和CREB驱使的荧光素酶报告质粒转染HER293细胞。转染后，消化收集HER293-GLP1R-CREB-D4细胞，计数，用含有10％FBS的DMEM培养基调整细胞数至4×105细胞/ml，接种至96孔板，每孔50μl。使用含有10％FBS的DMEM培养基将待测样品从1000ng/ml起被稀释为9个浓度；度拉糖肽作为阳性对照。上述96孔板每孔再加入50μl一系列浓度的待测样品，37℃孵育数小时。弃去上清，每孔加入荧光素酶底物。室温放置5min后，取出50μl，使用荧光素酶分析系统Bio-GloTMLuciferase Assay System(Promega,G7940)检测荧光素酶的活性，并且用酶标仪(Molecular Devices,SpectraMax M3)进行读数。荧光强度体现cAMP的水平，从而反应GLP-1受体激活的程度。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. Based on the CREB (cAMP response element binding protein) reporter gene, the activation effect of the fusion protein of the present application on the cAMP/PKA signaling pathway is detected. HER293 cells were transfected with human GLP-1R plasmid and CREB-driven luciferase reporter plasmid. After transfection, HER293-GLP1R-CREB-D4 cells were digested and collected, counted, and the cell number was adjusted to 4×10 5 cells/ml with DMEM medium containing 10% FBS, and inoculated into 96-well plates, 50 μl per well. The sample to be tested was diluted to 9 concentrations 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 sample to be tested 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 activity of luciferase was detected using the luciferase analysis system Bio-Glo TM Luciferase Assay System (Promega, G7940), and the reading was performed using a microplate reader (Molecular Devices, SpectraMax M3). The fluorescence intensity reflects the level of cAMP, thereby reflecting the degree of GLP-1 receptor activation.
结果如图7所示，本申请的融合蛋白具备与度拉糖肽相当水平的GLP-1受体激活能力。The results are shown in FIG7 , and the fusion protein of the present application has a GLP-1 receptor activation ability at a level comparable to that of dulaglutide.
实施例7配制并考察融合蛋白的不同配方制剂Example 7 Preparation and investigation of different formulations of fusion protein
将制备的融合蛋白GM-ARY-L2H1-Fc4m采用不同缓冲溶液超滤换液，然后采用10kD截留分子量的超滤离心管浓缩到考察的蛋白质浓度要求，添加不同配方中的稳定剂，采用0.2μm无菌过滤后，分装到相应包装材料中，进行稳定性考察实验。 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 molecular weight cutoff of 10kD. Stabilizers in different formulas were added and the product was sterile filtered at 0.2μm and then packaged into corresponding packaging materials for stability testing.
检测项目及方法Testing items and methods
(1)SE-HPLC纯度：参考《中国药典》2020年版三部通则0514分子排阻色谱法，用于检测产品的纯度。其中，色谱柱为TOSOH TSKgel UP-SW3000 4.6*150mm，2μm；流动相为50mM Na2HPO4，250mM L-Arg-HCl；检测波长280nm，按面积归一化法计算。(1) SE-HPLC purity: Refer to the molecular exclusion chromatography method in Part III of the 2020 edition of the Chinese Pharmacopoeia, which is used to detect the purity of the product. The chromatographic column is TOSOH TSKgel UP-SW3000 4.6*150mm, 2μm; the mobile phase is 50mM Na2HPO4, 250mM L-Arg-HCl; the detection wavelength is 280nm, and the calculation is based on the area normalization method.
(2)RP-HPLC纯度：参考《中国药典》2020年版三部通则0512高效液相色谱法，用于检测产品的降解。其中，色谱柱为Waters ACQUITY UPLC Protein BEH C4 300A 2.1×100mm，1.7μm；检测波长为214nm，按面积归一化法计算。(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 is Waters ACQUITY UPLC Protein BEH C4 300A 2.1×100 mm, 1.7 μm; the detection wavelength is 214 nm, calculated by the area normalization method.
(3)CE-SDS纯度：参考参考《中国药典》2020年版三部通则3127单抗分子大小变异体测定法，用于检测产品的纯度。供试品经过变性(非还原条件)或变性还原(还原条件)后，放入毛细管电泳仪，电泳分析，在220nm波长下，按面积归一化法计算。(3) CE-SDS purity: Refer to the 3127 monoclonal antibody molecular size variant determination method in the 2020 edition of the Chinese Pharmacopoeia. The test 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 and calculated by the area normalization method.
(4)蛋白质含量：参考《中国药典》2020年版三部通则0731蛋白质含量测定法第六法，根据蛋白质在280nm波长下的吸光值和蛋白质的理论消光系数，计算蛋白质含量。(4) Protein content: Calculate the protein content based on the absorbance value of protein at 280 nm and the theoretical extinction coefficient of protein, with reference to Method 6 of Protein Content Determination Method 0731 in Part III of the 2020 edition of the Chinese Pharmacopoeia.
(5)不溶性微粒：在AccuSizer 780SIS不溶性微粒分析仪上测量≥10μm和≥25μm颗粒的数量。(5) Insoluble particles: The number of particles ≥10 μm and ≥25 μm were measured on the AccuSizer 780SIS insoluble particle analyzer.
(6)动态光散色(DLS)：在PUNK型粒度仪上测量样品平均粒子直径(Z.average.Diameter)和分布系数(PDI)。(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.
(7)相对结合活性：采用酶联免疫吸附测定法(ELISA法)。(7) Relative binding activity: Enzyme-linked immunosorbent assay (ELISA) was used.
实施例8缓冲液pH的筛选Example 8 Screening of buffer pH
蛋白质含量在10mg/ml条件下，考察pH 5.5-6.5范围30±2℃高温条件下的稳定性。通过SE-HPLC和RP-HPLC检测蛋白的纯度差异，通过不溶性微粒检测形成的颗粒差异。具体配方组成及检测结果汇总见表7。The protein content was 10 mg/ml, and the stability was investigated at pH 5.5-6.5 and 30±2℃. The protein purity was tested by SE-HPLC and RP-HPLC, and the particle size was tested by insoluble particles. The specific formula composition and test results are summarized in Table 7.
表7各pH条件下高温加速14天和28天检测结果汇总


注：不溶性微粒为高温加速14天样品的结果Table 7 Summary of the test results of high temperature acceleration 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
总体上，配方在该pH范围内较为稳定。SE-HPLC检测结果显示，随着pH降低，单体含量有降低的趋势，但pH 6.0-6.5的纯度差异不明显；RP-HPLC检测显示不同pH之间的差异不明显；pH 6.0-6.2时不溶性微粒水平相对较低，整体上均在可接受的范围内。In general, the formula is relatively stable within this pH range. SE-HPLC test results show that as the pH decreases, the monomer content tends to decrease, but the purity difference between pH 6.0-6.5 is not obvious; RP-HPLC test shows that the difference between different pH values is not obvious; the level of insoluble particles is relatively low at pH 6.0-6.2, which is generally within an acceptable range.
实施例9缓冲系统的筛选Example 9 Screening of buffer systems
蛋白质含量在10mg/ml条件下，在两个稳定剂种类中，选择pH 6.0条件考察了两种缓冲体系：L-组氨酸-醋酸和柠檬酸盐在30±2℃高温条件下的稳定性。通过SE-HPLC和RP-HPLC检测蛋白的纯度差异，具体配方组成及检测结果汇总见表8。When the protein content was 10 mg/ml, the stability of two buffer systems, L-histidine-acetic acid and citrate, at a high temperature of 30±2°C was investigated under the condition of pH 6.0 among the two stabilizer types. The purity of the protein was tested by SE-HPLC and RP-HPLC. The specific formula composition and test results are summarized in Table 8.
表8不同缓冲系统高温加速14天和28天检测结果汇总
Table 8 Summary of the test results of different buffer systems at high temperature for 14 days and 28 days
各组的SE-HPLC检测结果无明显差异，稳定性均较好。SE-HPLC检测结果显示两种稳定剂的配方在L-组氨酸-醋酸缓冲系统中的降解速度略低于柠檬酸盐。There was no significant difference in the SE-HPLC test results of each group, and the stability was good. 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.
实施例10稳定剂种类的选择Example 10 Selection of stabilizer type
蛋白质含量在10mg/ml条件下，在L-组氨酸-醋酸缓冲液中，考察不同稳定剂在30±2℃高温条件下对蛋白稳定性的影响。通过SE-HPLC和RP-HPLC检测蛋白的纯度差异，通过DLS检测蛋白均一性差异。具体配方组成及检测结果见表9。When the protein content was 10 mg/ml, the effects of different stabilizers on protein stability at 30±2°C were investigated in L-histidine-acetate buffer. The protein purity was tested by SE-HPLC and RP-HPLC, and the protein homogeneity was tested by DLS. The specific formula composition and test results are shown in Table 9.
表9含不同稳定剂的配方高温加速14天和28天检测结果汇总


注：DLS是未加速2-8℃保存9个月样品检测结果。Table 9 Summary of the test results of the formulations containing different stabilizers at high temperature for 14 days and 28 days


Note: DLS is the test result of samples stored at 2-8℃ for 9 months without acceleration.
SE-HPLC和RP-HPLC检测结果显示，含不同稳定剂的配方差异不明显，蛋白纯度都较高。DLS结果显示，各组的均一性都较高。综合来说，稳定剂甘露醇、甘氨酸和L-脯氨酸对蛋白的稳定性较好。The results of SE-HPLC and RP-HPLC showed that there was no significant difference between the formulations containing different stabilizers, and the protein purity was high. The DLS results showed that the uniformity of each group was high. In general, the stabilizers mannitol, glycine and L-proline had good stability for the protein.
实施例11考察蛋白质含量的影响Example 11 Investigating the influence of protein content
在10mM柠檬酸盐，50mg/ml甘露醇，0.02％聚山梨酯80，pH 6.0制剂组成中考察不同蛋白质含量的影响，进行30±2℃高温加速实验，通过SE-HPLC和RP-HPLC检测蛋白的纯度差异，不溶性微粒检测蛋白的颗粒差异。具体配方组成及检测结果见表10。The effects of different protein contents were investigated in the formulation composition of 10 mM citrate, 50 mg/ml mannitol, 0.02% polysorbate 80, pH 6.0. A high temperature accelerated test at 30 ± 2 °C was conducted. The purity of the protein was detected by SE-HPLC and RP-HPLC, and the particle size of the protein was detected by insoluble particles. The specific formulation composition and test results are shown in Table 10.
表10不同蛋白质含量配方高温加速14天和28天检测结果汇总

注：不溶性微粒检测高温加速14天样品的结果Table 10 Summary of test results of different protein content formulas accelerated at high temperature for 14 days and 28 days

Note: The results of insoluble particles detection were obtained by accelerating the test for 14 days at high temperature.
结果显示，两种不同浓度配方经高温加速后的蛋白纯度和微粒数均无显著差异，稳定性良好。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.
实施例12表面活性剂种类的筛选Example 12 Screening of surfactant types
在10mM柠檬酸盐，50mg/ml甘露醇，pH 6.0制剂组成中考察不同表面活性剂对稳定性的影响，样品室温(25±2℃)150rpm震荡14天后，通过SE-HPLC和RP-HPLC检测蛋白的纯度差异，通过DLS检测蛋白均一性差异。具体配方组成及检测结果见表11。The effects of different surfactants on stability were investigated in a formulation of 10 mM citrate, 50 mg/ml mannitol, pH 6.0. After the samples were shaken at 150 rpm at room temperature (25 ± 2 ° C) for 14 days, the protein purity was detected by SE-HPLC and RP-HPLC, and the protein homogeneity was detected by DLS. The specific formulation composition and test results are shown in Table 11.
表11不同表面活性剂种类筛选考察检测结果汇总


注：DLS是未加速2-8℃保存9个月样品的检测结果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
SE-HPLC和RP-HPLC检测结果显示不同表面活性剂对蛋白纯度的影响无显著差异；DLS检测结果中，含有泊洛沙姆188的配方PDI较高，提示其均一性较低，含聚山梨酯80和聚山梨酯20的配方的均一性都较高。The results of SE-HPLC and RP-HPLC 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.
实施例13加速稳定性研究Example 13 Accelerated Stability Study
样品采用配方10mg/ml蛋白，20mM L-组氨酸-醋酸，50mg/ml甘露醇，0.02％(w/v)聚山梨酯80，pH 6.2±0.1，分装在西林瓶中，在25±2℃的条件下放置6个月，分别于第0、1、2、3、6个月末取样。按照稳定性重点考察项目进行检测，考察样品单体纯度(SE-HPLC)、降解含量(RP-HPLC)、还原和非还原毛细管凝胶电泳(CE-SDS)、蛋白质含量、相对结合活性等，结果见表12。The sample was formulated with 10 mg/ml protein, 20 mM L-histidine-acetate, 50 mg/ml mannitol, 0.02% (w/v) polysorbate 80, pH 6.2±0.1, and was packaged in vials and placed at 25±2°C for 6 months. Samples were taken at the end of the 0th, 1st, 2nd, 3rd, and 6th months. The samples were tested according to the key stability inspection items, including the monomer purity (SE-HPLC), degradation content (RP-HPLC), reduced and non-reduced capillary gel electrophoresis (CE-SDS), protein content, relative binding activity, etc. The results are shown in Table 12.
表12加速(25±2℃，6个月)稳定性研究数据
Table 12 Accelerated (25±2℃, 6 months) stability study data
结果显示，在25±2℃条件下放置6个月，同第0天比较，样品各项指标都在可接受范围内，表明本申请的制剂配方在室温6个月仍能保持稳定性。 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.
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Claims (44)

  1. 一种药物组合物，其包含融合蛋白，所述融合蛋白包含人GLP-1多肽变体和免疫球蛋白Fc区；其中与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列包含至少2个氨基酸突变，所述至少2个氨基酸突变位于选自下组的氨基酸位置处：W31、K26和Y19；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 2 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 2, and the at least 2 amino acid mutations are located at amino acid positions selected from the group consisting of: W31, K26 and Y19;
    所述药物组合物的pH值为5.0-7.0。The pH value of the pharmaceutical composition is 5.0-7.0.
  2. 根据权利要求1所述的药物组合物，其中所述免疫球蛋白Fc区位于所述人GLP-1多肽变体的C端。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. 根据权利要求1或2所述的药物组合物，其中所述免疫球蛋白Fc区为源自IgG的Fc区；优选为源自IgG1、IgG2、IgG3或IgG4的Fc区；更优选为选自IgG4的Fc区。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. 根据权利要求1-3中任一项所述的药物组合物，其中所述免疫球蛋白Fc区包含氨基酸突变，与未经所述氨基酸突变的免疫球蛋白Fc区相比，所述氨基酸突变使得所述免疫球蛋白Fc区与Fc受体的结合亲和力选择性增强。The pharmaceutical composition according to any one of claims 1 to 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.
  5. 根据权利要求1-4中任一项所述的药物组合物，其中所述免疫球蛋白Fc区在根据EU编码的M252、S254和T256位置处包含氨基酸突变；优选包含下组的氨基酸突变：M252Y、S254T和T256E。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. 根据权利要求1-5中任一项所述的药物组合物，其中所述免疫球蛋白Fc区包含如SEQ ID NO:36所示的氨基酸序列。A pharmaceutical composition according to any one of claims 1-5, wherein the immunoglobulin Fc region comprises an amino acid sequence as shown in SEQ ID NO:36.
  7. 根据权利要求1-6中任一项所述的药物组合物，其中所述融合蛋白还包含铰链区，铰链区位于所述人GLP-1多肽变体和所述免疫球蛋白Fc区之间，所述铰链区包含如SEQ ID NO:30-31中任一项所示的氨基酸序列；优选包含如SEQ ID NO:30所示的氨基酸序列。A pharmaceutical composition according to any one of claims 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 an amino acid sequence as shown in any one of SEQ ID NO:30-31; preferably comprises an amino acid sequence as shown in SEQ ID NO:30.
  8. 根据权利要求1-7中任一项所述的药物组合物，其中所述融合蛋白还包含连接子，所述连接子位于所述人GLP-1多肽变体和所述免疫球蛋白Fc区之间；优选位于所述人GLP-1多肽变体和所述铰链区之间。The pharmaceutical composition according to any one of claims 1 to 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. 根据权利要求8所述的药物组合物，所述连接子为具有5-20个氨基酸长度的肽；优选地，所述连接子包含如SEQ ID NO:27-29中任一项所示的氨基酸序列；更优选地，所述连接子包含如SEQ ID NO:28所示的氨基酸序列。According to the pharmaceutical composition of claim 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 NO: 27-29; more preferably, the linker comprises an amino acid sequence as shown in SEQ ID NO: 28.
  10. 根据权利要求1-9中任一项所述的药物组合物，其中与SEQ ID NO:2所示的氨基酸 序列相比，所述人GLP-1多肽变体的氨基酸序列包含位于选自以下位置处的氨基酸突变：The pharmaceutical composition according to any one of claims 1 to 9, wherein the amino acid shown in SEQ ID NO: 2 Compared with the sequence, the amino acid sequence of the human GLP-1 polypeptide variant comprises an amino acid mutation at a position selected from the following positions:
    a)在W31和K26氨基酸位置处；a) at amino acid positions W31 and K26;
    b)在W31和Y19氨基酸位置处；b) at amino acid positions W31 and Y19;
    c)在K26和Y19氨基酸位置处；c) at amino acid positions K26 and Y19;
    以及d)在W31、K26和Y19氨基酸位置处and d) at amino acid positions W31, K26 and Y19
  11. 根据权利要求1-10中任一项所述的药物组合物，其中在W31氨基酸位置处的突变选自W31Y、W31R、W31K和W31A；优选为W31Y。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. 根据权利要求1-11中任一项所述的药物组合物，其中在K26氨基酸位置处的突变为K26R。The pharmaceutical composition according to any one of claims 1-11, wherein the mutation at the amino acid position K26 is K26R.
  13. 根据权利要求1-12中任一项所述的药物组合物，其中在Y19氨基酸位置处的突变选自Y19A、Y19L、Y19T、Y19F、Y19I、Y19V和Y19S；优选为Y19A。The pharmaceutical composition according to any one of claims 1 to 12, wherein the mutation at the Y19 amino acid position is selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S; preferably Y19A.
  14. 根据权利要求1-13中任一项所述的药物组合物，其中与SEQ ID NO:2所示的氨基酸序列相比，所述人GLP-1多肽变体的氨基酸序列包含的氨基酸突变组合为W31Y、K26R和Y19A；或W31Y和K26R。A pharmaceutical composition according to any one of claims 1-13, 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 an amino acid mutation combination of W31Y, K26R and Y19A; or W31Y and K26R.
  15. 根据权利要求1-13中任一项所述的药物组合物，其中所述GLP-1多肽变体包含SEQID NO:4-26中任一项所示的氨基酸序列。A pharmaceutical composition according to any one of claims 1-13, wherein the GLP-1 polypeptide variant comprises an amino acid sequence shown in any one of SEQ ID NO:4-26.
  16. 根据权利要求1-15中任一项所述的药物组合物，其中所述融合蛋白自N端至C端，依次包含所述人GLP-1多肽变体、连接子、铰链区以及免疫球蛋白Fc区。The pharmaceutical composition according to any one of claims 1 to 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. 根据权利要求1-16中任一项所述的药物组合物，其中所述融合蛋白包含如SEQ ID NO:37-45中任一项所示的氨基酸序列。A 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 NO:37-45.
  18. 根据权利要求1-17中任一项所述的药物组合物，所述药物组合物的pH值为5.5-6.5；优选pH值为5.5-6.2；更优选pH值为6.0-6.2。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. 根据权利要求1-18中任一项所述的药物组合物，所述药物组合物包含缓冲成分。The pharmaceutical composition according to any one of claims 1 to 18, comprising a buffer component.
  20. 根据权利要求19所述的药物组合物，其中所述缓冲成分选自下组中的一种或多种：L-组氨酸-醋酸、柠檬酸盐、L-组氨酸-盐酸、醋酸-醋酸钠和磷酸盐；所述缓冲成分优选为L-组氨酸-醋酸或柠檬酸盐。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. 根据权利要求19或20所述的药物组合物，其中所述缓冲成分的浓度为2mM-50mM；优选为5mM-40mM；更优选为10mM-30mM；甚至更优选为20mM。The pharmaceutical composition according to claim 19 or 20, wherein the concentration of the buffer component is 2mM-50mM; preferably 5mM-40mM; more preferably 10mM-30mM; even more preferably 20mM.
  22. 根据权利要求1-21中任一项所述的药物组合物，所述药物组合物还包含稳定剂。 The pharmaceutical composition according to any one of claims 1-21, further comprising a stabilizer.
  23. 根据权利要求22所述的药物组合物，其中所述稳定剂选自下组中的一种或多种：甘露醇、L-精氨酸盐酸盐、甘氨酸、L-脯氨酸、山梨醇、甘油、麦芽糖、海藻糖和氯化钠；优选地，所述稳定剂选自下组中的一种或多种：甘露醇、L-精氨酸盐酸盐、甘氨酸和L-脯氨酸；更优选地，所述稳定剂选自下组中的一种或多种：甘露醇、甘氨酸和L-脯氨酸。The pharmaceutical composition according to claim 22, wherein the stabilizer is selected from one or more of the following group: 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 group: mannitol, L-arginine hydrochloride, glycine and L-proline; more preferably, the stabilizer is selected from one or more of the following group: mannitol, glycine and L-proline.
  24. 根据权利要求23所述的药物组合物，所述稳定剂的浓度为2mg/mL-150mg/mL，优选为10mg/mL-150mg/mL；优选为20mg/mL-100mg/mL。According to the pharmaceutical composition of claim 23, 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. 根据权利要求23所述的药物组合物，所述稳定剂包含甘露醇，所述甘露醇的浓度为10mg/mL-150mg/mL；优选为20mg/mL-100mg/mL；更优选为30mg/mL-80mg/mL；甚至更优选为40mg/mL-60mg/mL。According to the pharmaceutical composition of claim 23, 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.
  26. 根据权利要求23所述的药物组合物，所述稳定剂包含甘氨酸，所述甘氨酸的浓度为2mg/mL-50mg/mL；优选为5mg/mL-40mg/mL；更优选为10mg/mL-30mg/mL；甚至更优选为15mg/mL-25mg/mL。The pharmaceutical composition according to claim 23, wherein 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.
  27. 根据权利要求23所述的药物组合物，所述稳定剂包含L-脯氨酸，所述L-脯氨酸的浓度为5mg/mL-75mg/mL；优选为10mg/mL-50mg/mL；更优选为15mg/mL-40mg/mL；甚至更优选为20mg/mL-30mg/mL。The pharmaceutical composition according to claim 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.
  28. 根据权利要求23所述的药物组合物，所述稳定剂包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度为10mg/mL-100mg/mL；优选为20mg/mL-80mg/mL；更优选为25mg/mL-60mg/mL；甚至更优选为30mg/mL-50mg/mL。According to the pharmaceutical composition of claim 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.
  29. 根据权利要求1-28中任一项所述的药物组合物，所述药物组合物还包含表面活性剂。The pharmaceutical composition according to any one of claims 1-28, further comprising a surfactant.
  30. 根据权利要求29所述的药物组合物，其中所述表面活性剂选自聚山梨酯80、聚山梨酯20和泊洛沙姆188中的一种或多种；优选地，所述表面活性剂为聚山梨酯80或聚山梨酯20。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. 根据权利要求30所述的药物组合物，其中所述表面活性剂的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。The pharmaceutical composition according to 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. 根据权利要求30所述的药物组合物，其中所述表面活性剂包含聚山梨酯80，所述聚山梨酯80的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。The pharmaceutical composition according to claim 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.
  33. 根据权利要求30所述的药物组合物，其中所述表面活性剂包含聚山梨酯20，所述聚 山梨酯20的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。The pharmaceutical composition according to claim 30, wherein the surfactant comprises polysorbate 20, The concentration of sorbitan 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. 根据权利要求30所述的药物组合物，其中所述表面活性剂包含泊洛沙姆188，所述泊洛沙姆188的浓度为0.01mg/mL-1.0mg/mL；优选为0.05mg/mL-0.75mg/mL；更优选为0.1mg/mL-0.5mg/mL；甚至更优选为0.15mg/mL-0.25mg/mL。The pharmaceutical composition according to 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. 根据权利要求1-34中任一项所述的药物组合物，其中所述融合蛋白的浓度为1mg/mL-30mg/mL；优选为2mg/mL-14mg/mL；更优选为5mg/mL-10mg/mL。The pharmaceutical composition according to any one of claims 1 to 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. 根据权利要求1-35中任一项所述的药物组合物，所述药物组合物为液体制剂。The pharmaceutical composition according to any one of claims 1 to 35, wherein the pharmaceutical composition is a liquid preparation.
  37. 根据权利要求36所述的药物组合物，其中所述液体制剂为注射液，优选为皮下注射液。The pharmaceutical composition according to claim 36, wherein the liquid preparation is an injection, preferably a subcutaneous injection.
  38. 根据权利要求36或37所述的药物组合物，其中所述液体制剂包含溶媒，所述溶媒为注射用水或注射用有机溶剂；优选地，所述注射用有机溶剂选自注射用油、乙醇、丙二醇中的一种或多种。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. 根据权利要求1-38中任一项所述的药物组合物，所述药物组合物在25±2℃的条件下放置6个月后，其中融合蛋白的单体含量仍在96％以上；优选单体含量在97％以上；更优选单体含量在98％以上，甚至更优选单体含量在99％以上。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. 试剂盒，其包含权利要求1-39中任一项所述的药物组合物以及盛放所述组合物的容器。A kit comprising the pharmaceutical composition according to any one of claims 1 to 39 and a container for containing the composition.
  41. 根据权利要求40所述的试剂盒，其中所述容器包括玻璃瓶。The kit of claim 40, wherein the container comprises a glass bottle.
  42. 根据权利要求40或41所述的试剂盒，其中所述容器中所述药物组合物的体积为0.1mL-5.0mL；优选为0.2mL-3.0mL；更优选为0.5mL-2.0mL。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. 权利要求1-39中任一项所述的药物组合物，和/或权利要求40-42任一所述的试剂盒在制备预防和/或治疗代谢性疾病或病症药物中的用途。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 drug for preventing and/or treating a metabolic disease or condition.
  44. 根据权利要求43所述的用途，其中所述代谢性疾病或病症包含GLP-1相关代谢性疾病或病症；优选地，所述代谢性疾病或病症包含糖尿病；更优选地，所述代谢性疾病或病症包含II型糖尿病。 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.
PCT/CN2023/143414 2022-12-30 2023-12-29 Pharmaceutical composition comprising fusion protein and use thereof WO2024141054A1 (en)

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