WO2023030444A1 - Glp-1/gip双靶多肽、融合蛋白及其应用 - Google Patents

Glp-1/gip双靶多肽、融合蛋白及其应用 Download PDF

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WO2023030444A1
WO2023030444A1 PCT/CN2022/116513 CN2022116513W WO2023030444A1 WO 2023030444 A1 WO2023030444 A1 WO 2023030444A1 CN 2022116513 W CN2022116513 W CN 2022116513W WO 2023030444 A1 WO2023030444 A1 WO 2023030444A1
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seq
glp
ftsdy
gpssgappps
hec
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French (fr)
Chinese (zh)
Inventor
蒋鹏
肖�琳
周林俊
黄丽玫
杨敏
蒋燕
孙宁远
毛剑铭
李勇
李利佳
郭林峰
李静
李文佳
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Sunshine Lake Pharma Co Ltd
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Sunshine Lake Pharma Co Ltd
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Priority to US18/688,258 priority Critical patent/US20250223331A1/en
Priority to EP22863579.3A priority patent/EP4410827A4/en
Priority to JP2024514046A priority patent/JP2024532503A/ja
Publication of WO2023030444A1 publication Critical patent/WO2023030444A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • A61K38/2235—Secretins
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/645—Secretins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to the field of biomedicine, specifically to GLP-1/GIP dual-target polypeptides, fusion proteins and applications thereof, and more specifically to GLP-1/GIP dual-target polypeptides and GLP-1/GIP dual-target polypeptides Nucleic acid molecules, fusion proteins, nucleic acid molecules encoding fusion proteins, expression vectors, recombinant cells, GLP-1/GIP dual-target polypeptides, nucleic acid molecules, fusion proteins, nucleic acid molecules, expression vectors, and recombinant cells used in the preparation of medicines , pharmaceutical composition and method for preparing fusion protein.
  • Type 2 diabetes is a chronic metabolic disorder, which is closely related to obesity, hyperlipidemia and hypertension.
  • the current first-line drugs for the treatment of type 2 diabetes are mainly hypoglycemic, but the effect of reducing body weight is very limited.
  • Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by intestinal L-cells after eating, which can stimulate pancreatic ⁇ cells to secrete insulin, thereby stabilizing postprandial blood sugar fluctuations; its role in lowering blood sugar It is dependent on glucose concentration and greatly reduces the risk of hypoglycemia while regulating blood sugar.
  • GLP-1-based drugs such as liraglutide, dulaglutide and semaglutide
  • GLP-1 receptor agonist drugs are used for weight loss, the doses that need to be used are generally large, which are prone to gastrointestinal side effects, have poor tolerance, and have a narrow therapeutic window.
  • GIP Glucose-dependent incretin
  • intestinal K cells containing 42 amino acids, which mainly plays a role in glucose homeostasis and protects pancreatic ⁇ cells.
  • GIP and GLP-1 are incretins, both of which can promote the secretion of insulin and lower blood sugar in a blood sugar concentration-dependent manner, and the blood sugar lowering effect mediated by GIP is even stronger than that of GLP-1.
  • GIP receptor agonists alone in diabetic patients did not achieve the purpose of improving blood sugar.
  • single-molecule GLP-1/GIP dual receptor agonists can effectively solve the shortcomings of existing single-target drugs, and achieve better hypoglycemic and weight-loss effects by simultaneously activating multiple targets related to metabolism. Very big.
  • Single-molecule GLP-1/GIP dual receptor agonists can effectively solve the shortcomings of existing single-target drugs.
  • the inventors mutated wild-type GLP-1 and GIP through a large number of experiments, and obtained a variety of receptors that can simultaneously recognize GLP-1.
  • the GLP-1/GIP dual-target polypeptide of the GIP receptor and the GLP-1/GIP receptor and use the connecting peptide to fuse the GLP-1/GIP dual-target polypeptide with the Fc fragment of immunoglobulin to obtain a fusion protein.
  • the inventors found that the fusion protein has Good ability to simultaneously activate GLP-1 receptor and GIP receptor, and different mutations can increase or decrease the agonistic activity of GLP-1 receptor and GIP receptor.
  • the present invention proposes a GLP-1/GIP dual target polypeptide.
  • the first polypeptide comprises the following amino acid sequence: X 1 X 2 X 3 GTFX 4 SDYSX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 X 19 , among them, X 1 is Y or H, X 2 is A or G or S, X 3 is E or Q, X 4 is I or T, X5 is I or K , X 6 is A or Y or L or I, X 7 is M or L, X 8 is D or E, X 9 is K or E, X 10 is I or Q or K or E or L, X 11 is H or A or R, X 12 is A or Q or V, X 13 is K or Q or R or H, X 14 is D or E or A or L
  • the above-mentioned GLP-1/GIP dual target polypeptide may further include at least one of the following additional technical features:
  • the first polypeptide comprises the following amino acid sequence: X 1 X 2 EGTFTSDYSIX 6 LDKX 10 AQX 13 X 14 FX 15 X 16 WLX 17 AX 19 , wherein X 1 is Y or H, X2 is A or G or S, X6 is A or Y or L, X10 is I or Q or K or L, X13 is Q or R, X14 is D or E or A, X 15 is V or I, X 16 is E or D or Q, X 17 is L or I or K, X 19 is Q or G; wherein, the first polypeptide does not comprise YAEGTFISDYSIAMDKIHQQDFVNWLLAQ amino acid sequence (SEQ IDNO: 122) .
  • the inventors carried out site-directed mutation of wild-type GLP-1 and GIP, and the obtained GLP-1/GIP dual-target polypeptide has good biological activity of binding to GLP-1 and/or GIP receptors, thereby simultaneously activating GLP-1 and GIP. 1 receptor and GIP receptor, the GLP-1/GIP dual target polypeptide has dual functions of weight loss and hypoglycemia.
  • the GLP-1/GIP dual target polypeptide further comprises a second polypeptide, and the second polypeptide has the amino acid sequence shown in SEQ ID NO:1.
  • the specific amino acid sequence of SEQ ID NO: 1 is: GPSSGAPPPS.
  • the C-terminal amino acid of the first polypeptide is linked to the N-terminal amino acid of the second polypeptide.
  • the GLP-1/GIP dual target polypeptide has at least one of the amino acid sequences shown in Table 1.
  • the present invention proposes a fusion protein.
  • it includes: (1) the GLP-1/GIP dual-target polypeptide described in the first aspect; (2) an Fc fragment, the C-terminal of the GLP-1/GIP dual-target polypeptide and the Fc The N-termini of the fragments are linked.
  • the fusion protein according to the embodiment of the present invention also has excellent binding activity to GLP-1 and/or GIP receptors, has dual functions of weight loss and hypoglycemia, and can effectively control or reduce body weight and blood sugar levels.
  • the above-mentioned fusion protein may further include at least one of the following additional technical features:
  • the Fc fragment is an Fc fragment of human IgG4 or a variant thereof.
  • the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 113.
  • a connecting peptide is further included, and the connecting peptide has the amino acid sequence shown in SEQ ID NO: 114.
  • GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla (SEQ ID NO: 114).
  • the N-terminal of the connecting peptide is connected to the C-terminal of the GLP-1/GIP dual target polypeptide, and the C-terminal of the connecting peptide is connected to the N-terminal of the Fc fragment.
  • the invention proposes a nucleic acid molecule.
  • the nucleic acid molecule encodes the GLP-1/GIP dual target polypeptide described in the first aspect or the fusion protein described in the second aspect.
  • the GLP-1/GIP dual-target polypeptides or fusion proteins encoded by the nucleic acid molecules according to the embodiments of the present invention also have excellent binding activity to GLP-1 and/or GIP receptors, and have dual functions of weight loss and hypoglycemia, Can effectively control or reduce body weight and blood sugar levels.
  • the present invention provides an expression vector. According to an embodiment of the present invention, it comprises the nucleic acid molecule described in the third aspect.
  • the above-mentioned expression vector may further include at least one of the following additional technical features:
  • the expression vector is a eukaryotic expression vector.
  • the present invention provides a recombinant cell. According to an embodiment of the present invention, it carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect.
  • the recombinant cells according to the embodiments of the present invention can express the GLP-1/GIP dual-target polypeptide or fusion protein, and the GLP-1/GIP dual-target polypeptide and fusion protein have excellent compatibility with GLP-1 and/or GIP receptors It has the dual functions of weight loss and hypoglycemia, and can effectively control or reduce body weight and blood sugar levels.
  • the present invention provides a pharmaceutical composition.
  • it comprises the GLP-1/GIP dual target polypeptide described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, Or the recombinant cell described in the fifth aspect.
  • the pharmaceutical composition may include: a pharmaceutically acceptable adjuvant, which includes at least one of a stabilizer, a wetting agent, an emulsifier, a binding agent, and an isotonic agent;
  • the composition is at least one of tablet, granule, powder, capsule, solution, suspension and freeze-dried preparation.
  • the pharmaceutical composition according to the embodiment of the present invention has a long-term function of reducing weight and/or lowering blood sugar, and can effectively control or reduce body weight and blood sugar level.
  • the present invention proposes the GLP-1/GIP dual-target polypeptide described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, and the nucleic acid molecule described in the fourth aspect.
  • the medicine is used to control or reduce blood sugar and body weight.
  • the present invention provides a method for preparing the fusion protein described in the second aspect.
  • the method includes: 1) constructing the expression vector described in the fourth aspect; 2) introducing the expression vector into a host cell to obtain a recombinant cell to express the fusion protein.
  • the fusion protein prepared according to the method of the embodiment of the present invention has excellent binding activity to GLP-1 and/or GIP receptors, has dual functions of weight loss and hypoglycemia, and can effectively control or reduce body weight and blood sugar levels.
  • the present invention provides a method for reducing blood sugar and/or body weight of a patient. According to an embodiment of the present invention, it includes administering to the patient at least one of the following: 1) the GLP-1/GIP dual target polypeptide described in the first aspect; 2) the fusion protein described in the second aspect; 3) the third aspect 4) the expression vector of the fourth aspect; 5) the recombinant cell of the fifth aspect; and 6) the pharmaceutical composition of the sixth aspect.
  • the method according to the embodiment of the present invention can effectively control or reduce the patient's body weight and/or blood sugar level in a long-term manner.
  • Fig. 1 has shown the blood glucose value of HEC-G123 group, HEC-G128 group, HEC-G131 group, HEC-G132 group of the embodiment of the present invention at different times;
  • Fig. 2 has shown the area under the curve of HEC-G123 group, HEC-G128 group, HEC-G131 group, HEC-G132 group of the embodiment of the present invention
  • Figure 3 shows the blood glucose values at different times in the HEC-G113 group, HEC-G122 group, HEC-G126 group, and HEC-G127 group of the embodiment of the present invention
  • Fig. 4 has shown the area under the curve of HEC-G113 group, HEC-G122 group, HEC-G126 group, HEC-G127 group of the embodiment of the present invention
  • Figure 5 shows the in vivo hypoglycemic effect of HEC-G20 in the db/db mouse model of the embodiment of the present invention
  • Fig. 6 shows the glycosylated hemoglobin value of HEC-G20 in the db/db mouse model of the embodiment of the present invention
  • Figure 7 shows the effect of long-term repeated administration of HEC-G115 and HEC-G124 of the embodiment of the present invention on the body weight of obese mice in the DIO model;
  • Figure 8 shows the effect of long-term repeated administration of HEC-G115 and HEC-G124 of the embodiment of the present invention on the cumulative food intake of obese mice in the DIO model
  • Fig. 9 has shown the structural diagram of the fusion protein of the embodiment of the present invention, wherein, fusion protein comprises three structural domains, from N-terminus to C-terminus are: GLP-1/GIP dual receptor agonist (GLP-1/GIP GIP dual target polypeptide), connecting peptide and Fc fragment.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • receptor agonist refers to a substance that acts on a receptor and can cause receptor activation, thereby causing a biological effect, which may be the enhancement or weakening of a specific expression of cell activity .
  • GLP-1/GIP dual receptor agonist refers to wild-type GLP-1, GIP protein molecule
  • GLP-1/GIP dual target polypeptide refers to wild-type GLP-1, GIP protein molecule
  • the polypeptide obtained after site-directed mutagenesis, the amino acid sequence of the polypeptide that can simultaneously activate GLP-1 receptor and GIP receptor obtained after site-directed mutagenesis of wild-type GLP-1 and GIP protein molecules in the present invention is shown in Table 1.
  • nucleic acid molecule can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any special restrictions.
  • the nucleic acid is preferably DNA, because DNA is more stable and easier to handle than RNA.
  • the "nucleic acid” mentioned in this application actually includes any one or both of the complementary double strands. It will also be understood by those skilled in the art that one strand can be used to detect the other, and vice versa.
  • the "construct" mentioned in this application refers to such a genetic carrier, which contains a specific nucleic acid sequence, and can transfer the target nucleic acid sequence into a host cell to obtain a recombinant cell.
  • the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a cosmid (Cosmid), and a virus, preferably a plasmid.
  • plasmids are easy to operate and can carry large fragments, which is convenient for operation and processing.
  • the form of the plasmid is also not particularly limited, and it can be either a circular plasmid or a linear plasmid, that is, it can be single-stranded or double-stranded. Those skilled in the art can make selections as needed.
  • the present invention provides a GLP-1/GIP dual-target polypeptide, comprising a first polypeptide having the following amino acid sequence: X 1 X 2 X 3 GTFX 4 SDYSX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 X 19 , among them, X 1 is Y or H, X 2 is A or G or S, X 3 is E or Q, X 4 is I or T, X5 is I or K, X6 is A or Y or L or I, X7 is M or L, X8 is D or E, X9 is K or E, X10 is I or Q or K or E or L, X 11 is H or A or R, X 12 is A or Q or V, X 13 is K or Q or R or H, X 14 is D or E or A or L, X 15 is V or I, X 16 is N or E or
  • the GLP-1/GIP dual target polypeptide comprises a first polypeptide, and the first polypeptide has the following amino acid sequence: X 1 X 2 EGTFTSDYSIX 6 LDKX 10 AQX 13 X 14 FX 15 X 16 WLX 17 AX 19 , wherein X 1 is Y or H, X 2 is A or G or S, X 6 is A or Y or L, X 10 is I or Q or K or L, X 13 is Q or R, X14 is D or E or A, X15 is V or I, X16 is E or D or Q, X17 is L or I or K, X19 is Q or G; wherein, the first poly The peptide does not contain the amino acid sequence YAEGTFISDYSIAMDKIHQQDFVNWLLAQ (SEQ ID NO: 122).
  • the inventors carried out site-directed mutation of wild-type GLP-1 and GIP, and the obtained GLP-1/GIP dual-target polypeptide can activate GLP-1 receptor and GIP receptor respectively, and the GLP-1/GIP dual-target polypeptide has weight loss and hypoglycemic dual functions.
  • the GLP-1/GIP dual target polypeptide further comprises a second polypeptide, and the second polypeptide has the amino acid sequence shown in SEQ ID NO:1.
  • the specific amino acid sequence of SEQ ID NO: 1 is: GPSSGAPPPS.
  • the C-terminal amino acid of the first polypeptide is linked to the N-terminal amino acid of the second polypeptide.
  • the GLP-1/GIP dual target polypeptide has at least one of the amino acid sequences shown in Table 1.
  • the GLP-1/GIP dual target polypeptide has the following amino acid sequences SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 25-28, SEQ ID NO: 37, SEQ ID NO: 50-51, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 101-107, SEQ ID NO: 110-112.
  • the present invention provides a fusion protein, comprising: (1) the aforementioned GLP-1/GIP dual-target polypeptide; (2) an Fc fragment, the C-terminal of the GLP-1/GIP dual-target polypeptide and the Fc fragment
  • the fusion protein according to the specific embodiment of the present invention also has excellent binding activity with GLP-1 and/or GIP receptors, has dual functions of weight loss and hypoglycemia, and can effectively control or reduce body weight and blood sugar levels.
  • the Fc fragment is an Fc fragment of human IgG 4 or a mutant thereof. Fusing the Fc fragment of immunoglobulin to the GLP-1/GIP dual target polypeptide can prolong the circulating half-life of the fusion protein in vivo.
  • the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 113.
  • the present invention further includes a connecting peptide, and the connecting peptide has the amino acid sequence shown in SEQ ID NO: 114.
  • GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla (SEQ ID NO: 114).
  • the N-terminal of the connecting peptide is connected to the C-terminal of the GLP-1/GIP dual target polypeptide, and the C-terminal of the connecting peptide is connected to the N-terminal of the Fc fragment.
  • the present invention provides a nucleic acid molecule, which encodes the aforementioned GLP-1/GIP dual-target polypeptide or fusion protein.
  • the GLP-1/GIP dual-target polypeptide or fusion protein encoded by the nucleic acid molecule according to specific embodiments of the present invention also has excellent binding activity to GLP-1 and/or GIP receptors, and has dual functions of weight loss and hypoglycemia, Can effectively control or reduce body weight and blood sugar levels.
  • the invention provides an expression vector. According to an embodiment of the present invention, it comprises the aforementioned nucleic acid molecule encoding the GLP-1/GIP dual-target polypeptide or fusion protein.
  • the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to the control element.
  • “Operably linked” herein refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function.
  • the expression vector is a eukaryotic expression vector.
  • the invention provides a recombinant cell.
  • a nucleic acid molecule carrying the aforementioned nucleic acid molecule encoding a GLP-1/GIP dual-target polypeptide or a fusion protein, or the aforementioned expression vector.
  • the recombinant cells according to specific embodiments of the present invention can express the GLP-1/GIP dual-target polypeptide or fusion protein, and the GLP-1/GIP dual-target polypeptide and fusion protein have excellent compatibility with GLP-1 and/or GIP It has the dual functions of weight loss and hypoglycemia, and can effectively control or reduce body weight and blood sugar levels.
  • the recombinant cells are mammalian cells, such as CHO cells.
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention provides a pharmaceutical composition, comprising the aforementioned GLP-1/GIP dual-target polypeptide, the aforementioned fusion protein, the aforementioned nucleic acid molecule encoding the GLP-1/GIP dual-target polypeptide or fusion protein, the aforementioned The expression vector, or the above-mentioned recombinant cells.
  • the pharmaceutical composition may include: a pharmaceutically acceptable adjuvant, which includes at least one of a stabilizer, a wetting agent, an emulsifier, a binding agent, and an isotonic agent;
  • the composition is at least one of tablet, granule, powder, capsule, solution, suspension and freeze-dried preparation.
  • the pharmaceutical composition according to specific embodiments of the present invention is used to treat diseases such as diabetes, obesity, fatty liver disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, dyslipidemia and metabolic syndrome, and has long-acting effects.
  • diseases such as diabetes, obesity, fatty liver disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, dyslipidemia and metabolic syndrome, and has long-acting effects.
  • the functions of weight loss and/or hypoglycemia can effectively control or reduce body weight and blood sugar levels.
  • the present invention provides the aforementioned GLP-1/GIP dual-target polypeptide, the aforementioned fusion protein, the aforementioned nucleic acid molecule encoding the GLP-1/GIP dual-target polypeptide or fusion protein, the aforementioned expression vector, the aforementioned The use of the recombinant cells in the preparation of medicines.
  • the medicament is used for controlling or reducing blood sugar and body weight.
  • the present invention provides a method for preparing the aforementioned fusion protein, the method comprising: 1) constructing the aforementioned expression vector; 2) introducing the expression vector into host cells to obtain recombinant cells to express the aforementioned fusion protein.
  • the fusion protein prepared according to the method of specific embodiments of the present invention has excellent binding activity to GLP-1 and/or GIP receptors, has dual functions of weight loss and hypoglycemia, and can effectively control or reduce body weight and blood sugar levels.
  • GLP-1/GIP dual-target polypeptide or "recombinant polypeptide” described in the present invention can be prepared not only through recombinant expression, but also through chemical synthesis, regardless of the preparation method, As long as it has one of the hypoglycemic or weight-reducing activities described in the present invention, it is within the protection scope of the present invention.
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention provides a method for reducing blood sugar and/or body weight of a patient, comprising administering to the patient at least one of the following: 1) the aforementioned GLP-1/GIP dual-target polypeptide; 2) the aforementioned fusion protein; 3) the aforementioned nucleic acid molecule encoding the GLP-1/GIP dual target polypeptide or fusion protein; 4) the aforementioned expression vector; 5) the aforementioned recombinant cell; and 6) the aforementioned pharmaceutical composition.
  • the method according to specific embodiments of the present invention can effectively and long-term control or reduce the patient's body weight and/or blood sugar level, treat fatty liver disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, dyslipidemia and metabolic syndrome symptoms and other diseases.
  • the "Fc fragment" of the present invention may be a human IgG4 Fc fragment, or a variant of an IgG4 Fc fragment, and the mutant has one or more amino acid mutations compared to the wild-type IgG4 Fc.
  • GLP-1/GIP dual target polypeptide of the present invention starts with "CG”, such as “CG01”, “CG02”, etc.
  • HEC-G such as " HEC-G01”, “HEC-G02”, etc.
  • This example provides a method for synthesizing the polypeptide of the present invention.
  • step 3 Connect amino acids in sequence according to step 2, and finally cut off all the protective groups of the polypeptide with a cleavage reagent, cut it off from the resin, and send it for purification.
  • the target peptide is synthesized, the target peptide is separated from impurities by reversed-phase liquid chromatography purification, and the collected target peptide is freeze-dried into powder, and sent to QC quality inspection for purity and mass spectrometry identification. Detected by HPLC, the purity was greater than 95%. The molecular weight of the peptide identified by mass spectrometry was consistent with the theoretical molecular weight. Table 2 is the synthesized polypeptide compound.
  • Embodiment 2 measures the in vitro activity of polypeptide
  • Inoculation step 3 After sequencing, 200mL of the bacterial liquid containing the fusion protein of the target product was obtained and used for plasmid extraction.
  • the kit used was: PureLink HiPure Plasmid Maxiprep Kit, and the operation was performed according to the instructions. After the plasmid was verified by PCR and enzyme digestion, it was linearized with pvuI restriction endonuclease. Finally, the plasmid was recovered by ethanol precipitation.
  • the host cell is HEK293.
  • One day before transfection spread the cells on a 6-well plate at a density of 2x10 ⁇ 6 cells/well, and add 1 mL/well.
  • the recovered linearized plasmid was transfected into HEK293 cells using Lipofectamine 3000 liposome transfection method. After adding G418 to screen to obtain mixed strains, limited dilution was performed to isolate single clones for activity detection and verification.
  • cAMP detection kit (Cisbio, 62AM6PEC) to detect the cAMP produced by the recipient cells according to the steps described in the operation manual. The specific steps are as follows:
  • the method of molecular cloning is used to fuse the GLP-1/GIP dual receptor agonist polypeptide with the Fc fragment with the connecting peptide.
  • the GLP-1 wild-type amino acid sequence is: HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G (SEQ ID NO: 115)
  • the GIP wild-type amino acid sequence is: YAEGT FISDY SIAMD KIHQQ DFVNW LLAQ (SEQ ID NO: 116), the GLP- 1.
  • the GLP-1/GIP dual-receptor agonist polypeptide is obtained by mutating the wild-type amino acid sequence of GIP; the synthesized sequence is double-digested and inserted between the same restriction sites of the mammalian cell expression vector; or Design primers for site-directed mutagenesis on the basis of existing vectors, construct a series of mutant vectors by polymerase chain reaction (PCR), and send them to the sequencing company to verify correctness by sequencing, then use OMEGA’s endotoxin-free plasmid extraction kit ( Endo-free Plasmid Mini kit II) to extract the plasmid vector, Cat. No. D6950-01, and store the plasmid vector at -20°C for future use.
  • Table 4 shows the GLP-1/GIP dual target polypeptide sequence in the GLP-1/GIP dual target fusion protein.
  • CHO-S cells were subcultured after resuscitation, and the cell density was diluted to about 6*10 6 cells/mL.
  • ExpiCHO FectamineTM CHO Transfection Kit (ThermoFisher Scientific) was used for transfection. Taking the 50mL expression system as an example, the specific experiment The operation is as follows:
  • step 3 Add the uniformly mixed reagents of the solutions of step 1 and 2 into the cells, mark them respectively, place them in a shaker, and incubate at 37°C, 8% CO 2 , 140rpm for 18-22h;
  • step 5) The transfected cells obtained in step 4) were placed in a shaker at 37° C., 8% CO 2 , and cultured at 140 rpm for six days, and then the cell fermentation broth was collected for subsequent purification.
  • Embodiment 5 Fusion protein purification and identification
  • the cell culture solution was centrifuged to collect the supernatant, and filtered through a 0.22 ⁇ m filter membrane to remove residual cell debris.
  • the collected cell culture fluid was purified by Protein A chromatography column, the target peak was collected, and further purified by anion exchange chromatography, and the protein was finally collected by elution with 0.02M PBS.
  • the specific operation steps were as follows:
  • the sample was quantified by a micronucleic acid protein analyzer (NanoDrop 2000/2000c Spectrophotometer), and then detected by 12% SDS-PAGE electrophoresis, and the electrophoresis result showed a single band.
  • Embodiment 6 measures the in vitro activity of fusion protein
  • Inoculation step 3 After sequencing, 200mL of the bacterial liquid containing the fusion protein of the target product was obtained and used for plasmid extraction.
  • the kit used was: PureLink HiPure Plasmid Maxiprep Kit, and the operation was performed according to the instructions. After the plasmid was verified by PCR and enzyme digestion, it was linearized with pvuI restriction endonuclease. Finally, the plasmid was recovered by ethanol precipitation.
  • the host cell is HEK293.
  • One day before transfection spread the cells on a 6-well plate at a density of 2x10 ⁇ 6 cells/well, and add 1 mL/well.
  • the recovered linearized plasmid was transfected into HEK293 cells using Lipofectamine 3000 liposome transfection method. After adding G418 to screen to obtain mixed strains, limited dilution was performed to isolate single clones for activity detection and verification.
  • cAMP detection kit (Cisbio, 62AM6PEC) to detect the cAMP produced by the recipient cells according to the steps described in the operation manual. The specific steps are as follows:
  • This example evaluates the effects of HEC-G123, HEC-G128, HEC-G131, and HEC-G132 on the glucose tolerance of normal C57BL/6 mice.
  • mice Normal C57BL/6 mice were randomly divided into 6 groups according to blood glucose and body weight (Vehicle group, Dulaglutide group, HEC-G123 group, HEC-G128 group, HEC-G131 group, HEC-G132 group), 8 mice in each group .
  • the animals in each group were subcutaneously injected with the corresponding drug, and the dosage was 3nmol/kg; for the control group, the corresponding drug was injected subcutaneously. the solvent.
  • HEC-G123, HEC-G128, HEC-G131, and HEC-G132 can all significantly reduce blood glucose levels in normal C57 mice. Compared with the positive control dulaglutide, the effects of HEC-G123 and HEC-G128 on improving glucose tolerance were more significant, while the effects of HEC-G131 and HEC-G132 on improving glucose tolerance had no significant difference.
  • This example evaluates the effects of HEC-G113, HEC-G122, HEC-G126, and HEC-G127 on the glucose tolerance of normal C57BL/6 mice.
  • mice Normal C57BL/6 mice were randomly divided into 6 groups according to blood glucose and body weight (Vehicle group, Dulaglutide group, HEC-G113 group, HEC-G122 group, HEC-G126 group, HEC-G127 group), 8 mice in each group .
  • Dulaglutide group Dulaglutide was purchased from Eli Lilly, batch number: D256504
  • HEC-G113 group, HEC-G122 group, HEC-G126 group, and HEC-G127 group each group of animals was subcutaneously injected with the corresponding drug, and the dosage was 3 nmol/kg; for the control group, the corresponding vehicle was injected subcutaneously.
  • HEC-G113, HEC-G122, HEC-G126, and HEC-G127 can all significantly reduce blood glucose levels in normal C57 mice.
  • the effect of each group of drugs on improving glucose tolerance was better than that of the positive control dulaglutide.
  • This example evaluates the effect of HEC-G20 on blood glucose in the db/db mouse model.
  • mice aged 7-8 weeks were randomly divided into 3 groups (Model group, Semaglutide group, HEC-G20 group) according to blood glucose and body weight, 9 mice in each group.
  • Semaglutide group Semaglutide was purchased from Novo Nordisk, batch number: JP52092
  • HEC-G20 group the animals in each group were subcutaneously injected with the corresponding drug, and each dose was 10 nmol/kg; for the Model group, the corresponding drug was injected subcutaneously. solvent.
  • the semaglutide group was administered once a day, and the HEC-G20 group was administered twice a week for a total of 4 weeks; the blood glucose of the animals was tested before each administration.
  • the HEC-G20 group can significantly reduce blood sugar after administration, and the blood sugar level reaches the lowest level at 7 hours, and the effect is similar to that of the positive control Semaglutide at the same dose. Compared with the Model, the blood glucose level of the mice in the long-term repeated administration of HEC-G20 group was significantly lower and remained stable for a long time.
  • HEC-G20 long-term administration of HEC-G20 can significantly improve the blood glucose level of type II diabetes model db/db mice.
  • This example evaluates the effects of long-term repeated administration of HEC-G115 and HEC-G124 on body weight and food intake of obese mice in the DIO model.
  • mice were randomly divided into normal group NFD and model group HFD at the fifth week of age.
  • the normal group was fed with normal maintenance feed, while the other groups were fed with high-fat feed D12492. Changes in body weight and food intake of mice were monitored every 3 weeks. After feeding for 16 weeks, the body weights of the mice in the model group and the normal group were (47.9 ⁇ 3.4) g and (29.6 ⁇ 1.5) g, respectively, and the difference between the two groups was statistically significant.
  • the normal group was divided into Control group, and the model group mice were divided into Vehicle group, Semaglutide group, HEC-G115 group and HEC-G124 group, with 10 mice in each group.
  • the corresponding drugs were injected subcutaneously to the animals in each group; for the Vehicle group, PBS was injected subcutaneously. Observed for 7 days after the first administration, and then each group was administered once every 3 days, and the dose of each group was 10nmol/kg. Body weight and food intake were measured before each administration. Three weeks after administration, the intraperitoneal glucose tolerance test was performed. The samples were processed and collected 72 hours after the last administration, the liver weight was recorded, and the liver pathological conditions and blood biochemical indicators of each group were detected. The test results are shown in Table 9-12 and Figure 7-8.
  • Table 11 Effects of long-term administration of HEC-G115 and HEC-G124 on body weight and liver index of DIO mice
  • Table 12 Effects of long-term administration of HEC-G115 and HEC-G124 on liver function and blood lipids in DIO mice
  • HEC-G115 and HEC-G124 were comparable to those of the control group Semaglutide administered once a day at the same dose.
  • HEC-G115 and HEC-G124 also significantly improved the liver function and blood lipids of DIO mice, and the effect was similar to that of Semaglutide.

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WO2025180387A1 (en) * 2024-02-26 2025-09-04 Serpentide Inc. Super long-lasting glp1 or glp1/gip analogue drugfor type-2 diabetes and obesity

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