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US-20260125444-A1 - FUSION PROTEIN HAVING TRIPLE ACTIVITY AND USE THEREOF

US20260125444A1US 20260125444 A1US20260125444 A1US 20260125444A1US-20260125444-A1

Abstract

A fusion protein having a triple activity and a use thereof are provided. More specifically, a fusion protein has a triple biological activity of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and epidermal growth factor 21 (FGF21). It can be used for treating type 2 diabetes (T2D), obesity, and other diseases related to glucose and lipid metabolic abnormalities.

Inventors

  • Weiyue Han
  • Guitao ZHANG
  • Dixiang LUO
  • Wenxin SONG
  • Shaodong ZHONG
  • Dan Liu
  • Jiangtao PENG

Assignees

  • SHANGHAI MINWEI BIOTECHNOLOGY CO., LTD

Dates

Publication Date
20260507
Application Date
20230118

Claims (20)

  1. 1 . A fusion protein, wherein the fusion protein has a structure as shown in formula I or formula II from N-terminus to C-terminus: wherein, Z0 is absent, or selected from: a signal peptide, a tag sequence, or a combination thereof; R1 is a mutant protein element of GLP-1 and GIP; L1 is absent or a linker peptide; Fc is a Fc element; L2 is absent or a linker peptide; R2 is a FGF21 element; “-” is a bond, wherein, the mutant protein element of GLP-1 and GIP comprises a GLP-1 and GIP mutant protein, wherein the amino acid sequence of the mutant protein is a polypeptide sequence based on the amino acid sequence shown in SEQ ID NO: 20 with amino acid residues at positions 1, 2, 13, 19, 21, 23, 24 selected from the group consisting of: His or Tyr at position 1, Gly or Ser at position 2, Tyr or Leu at position 13, Ala or Gln at position 19, Ala or Asp at position 21, Val or Ile at position 23, and Ala or Gln or Glu at position 24; and wherein the mutant protein has activity of binding and activating both a class B G protein-coupled receptor GLP-1R and a human glucose-dependent insulinotropic polypeptide (GIP) receptor.
  2. 2 . The fusion protein of claim 1 , wherein the amino acid sequence of the mutant protein is shown in any one of SEQ ID NOs: 8-19.
  3. 3 . The fusion protein of claim 1 , wherein the FGF21 element comprises FGF21 wild-type or a mutant thereof, wherein the amino acid sequence of the FGF21 mutant is a polypeptide sequence based on the amino acid sequence shown in SEQ ID NO: 1 with amino acid residues at positions 19, 98, 171 and/or 173 selected from the group consisting of: R19V, L98R, P171N, and Q173T.
  4. 4 . The fusion protein of claim 1 , wherein the fusion protein has an amino acid sequence selected from the group consisting of: (a) a sequence shown in any one of SEQ ID NOs: 4-7; (b) an amino acid sequence having at least 80%, preferably at least 85% or 90%, more preferably at least 95%, more preferably at least 98%, and more preferably at least 99% homology to a sequence shown in any one of SEQ ID NOs: 4-7; wherein the fusion protein has both activity of binding and activating a class B G protein-coupled receptor GLP-1R and a human glucose-dependent insulinotropic polypeptide (GIP) receptor, and activity of fibroblast growth factor 21 (FGF21).
  5. 5 . An isolated polynucleotide encoding the fusion protein of claim 1 .
  6. 6 . A vector comprising the polynucleotide of claim 5 .
  7. 7 . A host cell which comprises vector of claim 6 .
  8. 8 . A method for preparing a fusion protein, which comprises the following steps: (i) culturing the host cell of claim 7 under a suitable condition, thereby obtaining a mixture comprising the fusion protein; and (ii) purifying and/or separating the mixture obtained in step (i), thereby obtaining the fusion protein.
  9. 9 . A pharmaceutical composition comprising: (I) the fusion protein of claim 1 ; and (II) a pharmaceutically acceptable carrier.
  10. 10 . A method for: (i) controlling blood glucose content in the body of a subject in need thereof; (ii) reducing blood lipid content in the body of a subject in need thereof; (iii) reducing body fat rate, or suppressing weight gain of a subject in need thereof; (iv) improving liver function; and/or (v) preventing and/or treating a metabolic disease associated with diabetes or obesity, wherein the method comprises a step of administering an effective amount of the fusion protein of claim 1 to the subject.
  11. 11 . The fusion protein of claim 3 , wherein the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO: 2.
  12. 12 . The fusion protein of claim 1 , wherein the amino acid sequence of R2 is shown in SEQ ID NO: 1 or 2.
  13. 13 . The fusion protein of claim 1 , wherein the fusion protein has both activity of binding and activating a class B G protein-coupled receptor GLP-1R and a human glucose-dependent insulinotropic polypeptide (GIP) receptor, and activity of fibroblast growth factor 21 (FGF21).
  14. 14 . The fusion protein of claim 1 , wherein the sequence of the Fc element is shown in SEQ ID NO: 21.
  15. 15 . The fusion protein of claim 1 , wherein the L1s are each independently (G 4 S) n A, wherein n is a positive integer selected from 2 to 5.
  16. 16 . The fusion protein of claim 1 , wherein the L2s are each independently (G 4 S) n , wherein n is a positive integer selected from 2 to 5.
  17. 17 . The fusion protein of claim 1 , wherein the sequence of L1 is shown in SEQ ID NO: 22.
  18. 18 . The fusion protein of claim 1 , wherein the sequence of L2 is shown in SEQ ID NO: 23.
  19. 19 . The pharmaceutical composition of claim 9 , wherein the pharmaceutical composition further comprises other medicaments that can be used to prevent and/or treat a metabolic disease associated with diabetes or obesity.
  20. 20 . A method for preventing and/or treating a metabolic disease associated with diabetes or obesity, wherein the method comprises a step of administering an effective amount of the fusion protein of claim 1 to a subject in need thereof.

Description

TECHNICAL FIELD The present invention relates to the field of biomedicine, and specifically relates to a fusion protein having triple activity and use thereof. BACKGROUND Diabetes and obesity are diseases caused by abnormal glucose and lipid metabolism, which are associated with a variety of other diseases, including cardiovascular disease (CVD), peripheral arterial disease, microvascular complications, and osteoarthritis. Therefore, the development of new therapies and therapeutic drugs for obesity, diabetes and their complications are of great significance to improve human health. Fibroblast growth factor 21 (FGF21) is a member of fibroblast growth factors (FGFs) family with 182 amino acids. FGF21 can promote the absorption of glucose by adipocytes, enhance insulin sensitivity, and does not pose a potential tumor risk due to its lack of mitogenic activity. At present, no FGF21 long-acting protein product is available on the market. LY2405319 from Lilly, PF-05231023 from Pfizer and BMS 986036 from Bristol Squibb are now in clinical trials. However, they can not meet the clinical requirements in the treatment of type 2 diabetes, and they are not as effective as GLP-1 drugs in reducing weight and blood lipids. Therefore, it can be seen that FGF21 alone cannot meet clinical needs. Recently, research has reported that the combination of GLP-1 and FGF21 has a synergistic effect on blood glucose control and weight loss. Prior arts have disclosed that the combination of GLP-1 and FGF21 can synergistically reduce blood glucose and body weight, etc. At present, although the GLP-1/GIP dual active analog (Tirzepatide), FGF21 analog and GLP-1/FGF21 fusion protein are all used to treat diabetes and lose weight, their effects are still unsatisfactory, and there is no report on GLP-1/GIP/FGF21 triple active protein in this field. Therefore, there is an urgent need in this field to develop a fusion protein with GLP-1/GIP/FGF21 triple activity that can be effectively used to treat diabetes and lose weight. SUMMARY OF THE INVENTION The purpose of the present invention is to provide a fusion protein with triple activity and use thereof. Specifically, the present invention provides a fusion protein having a triple bioactivity of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and epidermal growth factor 21 (FGF21) for the treatment of type 2 diabetes (T2D), obesity, and other diseases related with glucose and lipid metabolic abnormality. In the first aspect of the present invention, it provides a fusion protein which has a structure as shown in formula I or formula II from N-terminus to C-terminus: wherein,Z0 is absent, or selected from: a signal peptide, a tag sequence, or a combination thereof;R1 is a mutant protein element of GLP-1 and GIP;L1 is absent or a linker peptide;Fc is an Fc element;L2 is absent or a linker peptide;R2 is a FGF21 element;“-” is a bond, wherein,the mutant protein element of GLP-1 and GIP comprises a GLP-1 and GIP mutant protein, wherein the amino acid sequence of the mutant protein is a polypeptide sequence based on the amino acid sequence shown in SEQ ID NO: 20 with amino acid residues at positions 1, 2, 13, 19, 21, 23, 24 selected from the group consisting of:His or Tyr at position 1,Gly or Ser at position 2,Tyr or Leu at position 13,Ala or Gln at position 19,Ala or Asp at position 21,Val or Ile at position 23, andAla or Gln or Glu at position 24; andwherein the mutant protein has activity of binding and activating both a class B G protein-coupled receptor GLP-1R and a human glucose-dependent insulinotropic polypeptide (GIP) receptor. In another preferred embodiment, the amino acid sequence of the mutant protein is shown in any one of SEQ ID NOs: 8-19. In another preferred embodiment, the FGF21 element comprises FGF21 wild-type or a mutant thereof. In another preferred embodiment, the amino acid sequence of the FGF21 wild-type is shown in SEQ ID NO: 1. In another preferred embodiment, the amino acid sequence of the FGF21 mutant is a polypeptide sequence based on the amino acid sequence shown in SEQ ID NO: 1 with amino acid residues at positions 19, 98, 171, and/or 173 selected from the group consisting of: R19V, L98R, P171N, Q173T. In another preferred embodiment, the amino acid sequence of the FGF21 mutant is identical or substantially identical to the sequence shown in SEQ ID NO: 1, except for the mutations (such as amino acid residues at positions 19, 98, 171, and/or 173). In another preferred embodiment, the substantially identical means that at most 10 amino acids (preferably 1-8, more preferably 1-10, more preferably 1-5) are different, wherein the differences comprise substitution, deletion, or addition of amino acids, and the mutant still retains the activity of fibroblast growth factor 21 (FGF21). In another preferred embodiment, the fusion protein may be modified or unmodified. In another preferred embodiment, the amino acid sequence of the FGF21 muta