Triple-active fusion protein and its applications

Abstract

The present invention provides a triple-active fusion protein and its applications. More specifically, the present invention relates to a triple-biologically active fusion protein of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and epidermal growth factor 21 (FGF21), which can be used to treat type 2 diabetes (T2D), obesity, and other disorders of glucose and lipid metabolism.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, specifically to a fusion protein with triple activity and its applications. [Background technology]

[0002] Diabetes and obesity are diseases caused by abnormalities in glucose and lipid metabolism, which are associated with various other diseases, including cardiovascular disease (CVD), peripheral arterial disease, microvascular complications, and osteoarthritis, etc. Therefore, the development of new treatments and drugs for obesity, diabetes, and their complications is of great importance for improving human health.

[0003] Fibroblast growth factor 21 (FGF21) is a member of the FGF family (fibroblast growth factors, FGFs) and contains 182 amino acids. FGF21 promotes glucose uptake by adipocytes, enhances insulin sensitivity, and lacks mitogenic activity, posing no potential tumor risk. Currently, there are no long-acting FGF21 protein products on the market, including Lilly's LY2405319, Pfizer's PF-05231023, and Bristol Myers Squibb's BMS986036, which are in clinical trials. However, these products cannot meet the clinical needs of type 2 diabetes, and their therapeutic effects in lowering body weight and blood lipids are not as good as those of GLP-1 drugs. Therefore, it is clear that FGF21 alone cannot meet clinical needs.

[0004] Bacterial studies have reported that the combined use of GLP-1 and FGF21 has a synergistic effect on blood sugar control and weight loss. Prior art has disclosed that the application of a GLP-1 and FGF21 composition can synergistically reduce blood sugar and body weight.

[0005] Currently, GLP-1 / GIP dual activity analogues (tirzepatide), FGF21 analogues and GLP-1 / FGF21 fusion proteins are used to treat diabetes and weight loss, but their effects are still insufficient, and there is no report on GLP-1 / GIP / FGF21 triple activity proteins in the art.

[0006] Therefore, there is a need in the art to develop a fusion protein with GLP-1 / GIP / FGF21 triple activity that can be effectively used for the treatment of diabetes and weight loss. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a fusion protein with triple activity and its applications.

[0008] Specifically, the present invention provides a triple biologically active fusion protein of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and epidermal growth factor 21 (FGF21) that can be used to treat type 2 diabetes (T2D), obesity, and other disorders of glucose and lipid metabolism. [Means for solving the problem]

[0009] A first aspect of the present invention provides a fusion protein, the fusion protein having, from N-terminus to C-terminus, the structure of Formula I or Formula II: Z0-R1-L1-Fc-L2-R2(I) Z0-R2-L1-Fc-L2-R1(II) In the formula: Z0 is absent or is selected from a signal peptide, a tag sequence, or a combination thereof; R1 is a GLP-1 and GIP mutein element; L1 is none or a linker, Fc is the Fc element, L2 is none or a linker, R2 is an FGF21 element, "-" is a bond, where: The GLP-1 and GIP mutein elements include GLP-1 and GIP muteins, wherein the amino acid sequence of the muteins is a polypeptide sequence having amino acid residues selected from the group consisting of: Position 1 is His or Tyr, Position 2 is Gly or Ser; position 13 is Tyr or Leu; position 19 is Ala or Gln; position 21 is Ala or Asp; Position 23 is Val or Ile, and position 24 is Ala or Gln or Glu; Furthermore, the mutein simultaneously has the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the human glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0010] In another preferred embodiment, the amino acid sequence of the mutein is as set forth in any one of SEQ ID NOs. 8-19. In another preferred embodiment, the FGF21 element comprises an FGF21 wild type or a mutant thereof. In another preferred embodiment, the amino acid sequence of the wild-type FGF21 is as set forth in SEQ ID NO.1.

[0011] In another preferred example, the amino acid sequence of the FGF21 mutant is a polypeptide sequence having amino acid residues selected from the group consisting of R19V, L98R, P171N, and Q173T at positions 19, 98, 171, and / or 173 based on the amino acid sequence shown in SEQ ID NO. 1.

[0012] In another preferred example, except for the mutations (such as amino acid residues at positions 19, 98, 171 and / or 173), the remaining amino acid sequence of the FGF21 mutant is identical or substantially identical to the sequence shown in SEQ ID NO.1.

[0013] In another preferred example, the substantially identical amino acid sequence differs by up to 10 amino acids (preferably 1 to 8, more preferably 1 to 10, more preferably 1 to 5), where the differences include amino acid substitutions, deletions, or additions, and the mutant still retains the activity of fibroblast growth factor 21 (FGF21).

[0014] In another preferred embodiment, the fusion protein may be modified or unmodified. In another preferred embodiment, the amino acid sequence of the FGF21 mutant is as set forth in SEQ ID NO.2. In another preferred embodiment, the amino acid sequence of R2 is as set forth in SEQ ID NO. 1 or 2.

[0015] In another preferred embodiment, the fusion protein simultaneously possesses the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the human glucose-dependent insulinotropic polypeptide (GIP) receptor, as well as the activity of fibroblast growth factor 21 (FGF21).

[0016] In another preferred embodiment, the Fc element is an Fc fragment of immunoglobulin IgG, wherein the IgG is selected from the group consisting of IgG1, IgG2, and IgG4, preferably IgG4. In another preferred embodiment, the sequence of the Fc element is as shown in SEQ ID NO.21. In another preferred example, the Fc fragment comprises the hinge region, CH2 and CH3 domains of IgG4.

[0017] In another preferred embodiment, L1 and / or L2 is a flexible linker. In another preferred embodiment, the amino acid sequence of L1 and / or L2 is (G4S) n or (G4S) n A or a variant thereof, where n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), preferably n=3.

[0018] In another preferred embodiment, the (G4S) n The variants of (G4S) can be obtained by substituting amino acids with similar or similar properties in the sequence, such as by mutating one or more S's to T, or by inserting 1 to 3 amino acids into the sequence (G4S). n A variant of the linker sequence.

[0019] In another preferred example, L1 and L2 may be the same or different. In another preferred example, each L1 is independently (GS) n A, where n is a positive integer selected from 2 to 5 (for example, 1, 2, 3, 4, 5, or 6), and preferably n is 3.

[0020] In another preferred example, each L2 is independently (GS) n and n is a positive integer selected from 2 to 5 (for example, 1, 2, 3, 4, 5, or 6), and preferably n is 3. In another preferred embodiment, the sequence of L1 is as shown in SEQ ID NO.22. In another preferred embodiment, the sequence of L2 is as shown in SEQ ID NO.23.

[0021] In another preferred embodiment, the fusion protein comprises: (a) a sequence as set forth in any one of SEQ ID NOs. 4 to 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%, more preferably at least 99% homology to the sequence set forth in any one of SEQ ID NOs. 4 to 7; Furthermore, the fusion protein simultaneously possesses the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the human glucose-dependent insulinotropic polypeptide (GIP) receptor, as well as the activity of fibroblast growth factor 21 (FGF21).

[0022] A second aspect of the invention provides an isolated polynucleotide, said polynucleotide encoding a fusion protein according to the first aspect of the invention.

[0023] A third aspect of the present invention provides a vector, said vector comprising a polynucleotide according to the second aspect of the invention. In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon, or a combination thereof.

[0024] A fourth aspect of the present invention provides a host cell, said host cell comprising a vector according to the third aspect of the invention or having integrated into its chromosome an exogenous polynucleotide according to the second aspect of the invention.

[0025] A fifth aspect of the present invention is (i) culturing under suitable conditions a host cell according to the fourth aspect of the invention to obtain a mixture comprising a fusion protein according to the first aspect of the invention; and (ii) purifying and / or separating the mixture obtained in step (i), thereby obtaining a fusion protein according to the first aspect of the invention.

[0026] A sixth aspect of the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising: (I) a fusion protein according to the first aspect of the invention, and (II) Contains a pharmaceutically acceptable carrier.

[0027] In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for preventing and / or treating metabolic diseases associated with diabetes or obesity, and the metabolic diseases associated with diabetes or obesity include type I diabetes, type II diabetes, gestational diabetes, obesity, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD).

[0028] In another preferred embodiment, the pharmaceutical composition further comprises other drugs used to prevent and / or treat metabolic diseases associated with diabetes or obesity.

[0029] A seventh aspect of the present invention provides the use of a fusion protein according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention for use in the preparation of a medicament, said medicament comprising: (i) controlling blood glucose content in the body of a subject in need thereof; (ii) reducing blood lipid content in a subject in need thereof; (iii) reducing body fat percentage or inhibiting weight gain in a subject in need thereof; (iv) improvement of liver function, and / or (v) For use in the prevention and / or treatment of diabetes or obesity-related metabolic diseases.

[0030] In another preferred embodiment, the blood lipids include total cholesterol and low-density lipoprotein cholesterol. In another preferred embodiment, the medicament is used to reduce body fat percentage or inhibit weight gain in a subject in need thereof.

[0031] In another preferred example, the metabolic disease associated with diabetes or obesity includes type I diabetes, type II diabetes, gestational diabetes, obesity, and non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD).

[0032] An eighth aspect of the present invention provides a method for preventing and / or treating metabolic diseases associated with diabetes or obesity, comprising administering to a subject in need thereof an effective amount of a fusion protein according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention. [Effects of the Invention]

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0034] [Figure 1] Random blood glucose after multiple doses in db / db diabetic mice. [Figure 2] 1 shows fasting blood glucose after multiple doses in db / db diabetic mice. DETAILED DESCRIPTION OF THE INVENTION

[0035] After extensive and detailed research and extensive screening, the inventors unexpectedly developed a fusion protein with triple activity of GLP-1, GIP, and FGF21. The resulting fusion protein significantly reduced body weight, plasma total cholesterol, and low-density lipoprotein cholesterol in DIO mice, improved liver function, and reduced blood glucose in db / db mice, with effects significantly superior to those of a single GLP-1 / GIP or FGF21 active protein. Based on this, the present invention was completed.

[0036] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] The term "about" can refer to a particular value or configuration within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on how the value or configuration is measured or determined.

[0038] As used herein, the terms "containing" or "comprising" can refer to open, semi-closed, and closed systems. In other words, the terms also include "consisting essentially of" or "consisting of."

[0039] Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-1 (GLP-1) is a 37-amino acid incretin that stimulates insulin secretion, protects pancreatic β cells, and inhibits glucagon secretion, gastric emptying, and food intake, leading to weight loss. Liraglutide and semaglutide are GLP-1 receptor agonists approved for the treatment of type 2 diabetes and obesity. However, many patients with type 2 diabetes and obesity remain inadequately controlled, and currently available incretin mimetics or dipeptidyl peptidase IV (DPP-IV) inhibitors utilize only a single established mechanism for glycemic and weight control.

[0040] Glucose-dependent insulinotropic polypeptide (GIP) Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β cells in the presence of glucose and protecting pancreatic β cells. The GLP-1 / GIP receptor dual agonist compound LY3298176 based on the native GIP polypeptide sequence has been described in the prior art and has good effects on lowering blood glucose and body weight. This product is currently approved by the FDA for the treatment of type 2 diabetes, and its generic name is tirzepatide.

[0041] Fibroblast growth factor 21 (FGF21) Fibroblast growth factor 21 (FGF21) is a member of the FGF family (fibroblast growth factors) and contains 182 amino acids. FGF21 can promote glucose uptake by adipocytes and enhance insulin sensitivity. It lacks mitogenic activity and therefore does not pose a potential tumor risk. Furthermore, FGF21 inhibits the synthesis of SREBP-2 in the liver, thereby lowering serum total cholesterol and low-density lipoprotein levels, thereby alleviating hypercholesterolemia. However, natural FGF21 has certain deficiencies, including easy aggregation in vitro, poor stability, susceptibility to protease hydrolysis, and a long in vivo half-life of only 2–3 hours in cynomolgus monkeys. These shortcomings limit the application of FGF21. Therefore, there are many ways to improve FGF21. For example, mutation of arginine (R) at position 19 to valine (V), increasing the number of N-glycosylation sites at the C-terminus, and binding of IgG Fc can inhibit the degradation of FGF21 in vitro and extend its half-life in cynomolgus monkeys; the three mutations L98R, P171G, and A180E extend the half-life of FGF21 in cynomolgus monkeys by approximately 70 hours.

[0042] Fusion proteins of the present invention The present invention provides a fusion protein having the triple biological activity of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and epidermal growth factor 21 (FGF21).

[0043] As used herein, the terms "fusion protein of the invention," "triple biologically active fusion protein of the invention," and "fusion protein with triple activity" are used interchangeably, and refer to a fusion protein having, from N-terminus to C-terminus, the structure of Formula I or Formula II, Z0-R1-L1-Fc-L2-R2(I) Z0-R2-L1-Fc-L2-R1(II) In the formula: Z0 is absent or is selected from a signal peptide, a tag sequence, or a combination thereof; R1 is a GLP-1 and GIP mutein element; L1 is none or a linker, Fc is the Fc element, L2 is none or a linker, R2 is an FGF21 element, "-" is a bond, where: The GLP-1 and GIP mutein elements include GLP-1 and GIP muteins, wherein the amino acid sequence of the muteins is a polypeptide sequence having amino acid residues selected from the group consisting of: Position 1 is His or Tyr, Position 2 is Gly or Ser; position 13 is Tyr or Leu; position 19 is Ala or Gln; position 21 is Ala or Asp; Position 23 is Val or Ile, and position 24 is Ala or Gln or Glu; Furthermore, the mutein simultaneously has the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the human glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0044] As used herein, the terms "GLP-1 and GIP mutein," "GLP-1 and GIP mutant," and "GLP-1 and GIP mutant polypeptide" are used interchangeably and refer to GLP-1 and GIP muteins that simultaneously have the activity of binding to and activating the class B G protein-coupled receptors GLP-1R and GIP receptors of the present invention.

[0045] As used herein, a "linker" is preferably a flexible linker, typically a connecting peptide that may be rich in G, S, and / or A, e.g., may be composed of glycine G, serine S, and alanine A; a preferred linker is (G4S) n A or a variant thereof, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), preferably n=3, and the (G4S) n Mutants of nA were obtained by substituting amino acids with similar or similar properties within the sequence (G4S). n Variants of the A linker sequence are included, for example, by mutating one or more S's to T's, or by inserting 1-3 amino acids into the sequence.

[0046] In another preferred example, each L1 is independently (GS) n A, where n is a positive integer selected from 2 to 5 (for example, 1, 2, 3, 4, 5, or 6), and preferably n is 3. In another preferred example, each L2 is independently (GS) n and n is a positive integer selected from 2 to 5 (for example, 1, 2, 3, 4, 5, or 6), and preferably n is 3.

[0047] Preferably, in the fusion protein of the present invention, the sequence of R1 is selected from the amino acid sequences shown in any one of SEQ ID NOs. 8 to 19, the sequence of R2 is selected from the amino acid sequences shown in SEQ ID NO. 1 or 2 (preferably the FGF21 mutant sequence shown in SEQ ID NO. 2), the sequence of Fc is selected from the amino acid sequences shown in SEQ ID NO. 21, the sequence of L1 is as shown in SEQ ID NO. 22, and the sequence of L2 is as shown in SEQ ID NO. 23. More preferably, the fusion protein of the present invention has an amino acid sequence as shown in SEQ ID NOs. 4-7.

[0048] As used herein, the term "fusion protein of the present invention" further encompasses mutant forms that retain the above-mentioned activity. These mutant forms include, but are not limited to, deletion, insertion, and / or substitution of one to three (usually one to two, more preferably one) amino acids, and addition or deletion of one or several (usually up to three, preferably up to two, more preferably up to one) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or equivalent properties generally does not alter the function of the protein. As another example, addition or deletion of one or several amino acids at the C-terminus and / or N-terminus generally does not alter the structure and function of the protein. Furthermore, the term further encompasses monomeric and multimeric forms of the polypeptides of the present invention. The term further encompasses linear and nonlinear polypeptides (e.g., cyclic peptides).

[0049] The present invention further encompasses active fragments, derivatives, and analogs of the fusion proteins of the present invention. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the function or activity of the GLP-1 and GIP muteins or fusion proteins of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably, conservative amino acid residues) have been substituted, or (ii) a polypeptide having substitutions at one or more amino acid residues, or (iii) a polypeptide formed by fusing a polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a fusion protein formed by fusing a leader sequence, secretory sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the knowledge of those skilled in the art.

[0050] A preferred type of active derivative refers to a polypeptide in which up to three, preferably up to two, and more preferably up to one amino acid is replaced with an amino acid having the same or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably generated by replacing amino acids according to Table A. [Table A]

[0051] The present invention further provides analogs of the fusion proteins of the present invention. These analogs may differ from the polypeptides of the present invention in amino acid sequence, in modified forms that do not affect the sequence, or both. Analogs further include analogs with residues other than natural L-amino acids (e.g., D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (e.g., β-, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0052] Furthermore, the fusion proteins of the present invention can also be modified. Modified forms (which generally do not alter the primary structure) include polypeptides that have been chemically derivatized, such as by in vivo or in vitro acetylation or carboxylation. Modifications further include glycosylation, such as purified polypeptides, for example, by glycosylation modification of the polypeptide during synthesis and processing or in a further processing step. Such modifications can be achieved by exposing the polypeptide to a glycosylating enzyme (e.g., mammalian glycosylase or deglycosylase). Modified forms further include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Further included are polypeptides that have been modified to enhance their resistance to proteolysis or optimize their solubility.

[0053] The term "polynucleotide of the invention" can include polynucleotides that encode the GLP-1 and GIP muteins or fusion proteins of the invention, and can further include polynucleotides that add coding and / or non-coding sequences.

[0054] The present invention also relates to variants of the above polynucleotides that encode fragments, analogs, and derivatives of the polypeptides or fusion proteins of the present invention having the same amino acid sequences. These nucleotide variants include substitution, deletion, and insertion variants. As known in the art, allelic variants are alternative forms of polynucleotides, which may contain one or more nucleotide substitutions, deletions, or insertions, but which do not substantially alter the function of the GLP-1 and GIP muteins or fusion proteins that they encode.

[0055] The present invention further relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the above polynucleotides of the present invention under stringent conditions (or severe conditions). In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, and 60°C, or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, and 42°C, or (3) hybridization that occurs only when the identity between the two sequences is at least 90%, more preferably 95%.

[0056] The fusion proteins and polynucleotides of the present invention are preferably provided in an isolated form, more preferably in a homogeneously produced form.

[0057] The full-length polynucleotides of the present invention can be obtained typically by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the relevant sequences can be obtained by amplification using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. For long sequences, it is often necessary to perform two or more rounds of PCR amplification, followed by splicing the amplified fragments in the correct order.

[0058] Once the relevant sequence is obtained, recombinant methods can be used to obtain large quantities of the relevant sequence, typically by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the host cells grown by conventional methods.

[0059] Additionally, related sequences can be synthesized by artificial synthesis, especially when the fragments are relatively short in length, usually by first synthesizing several smaller fragments and then joining them together to obtain fragments of much longer sequences.

[0060] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis, and the DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art.

[0061] A method for amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used. Primers used in PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0062] Expression vector The present invention also relates to vectors comprising polynucleotides of the present invention and host cells genetically engineered with the vectors of the invention or the coding sequences of the fusion proteins of the invention, as well as methods for purifying the polypeptides according to the invention via recombinant techniques.

[0063] The polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins through conventional recombinant DNA techniques. (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the fusion protein of the invention, or with a recombinant expression vector comprising said polynucleotide; (2) culturing the host cells in an appropriate medium; (3) isolating and purifying the protein from the medium or cells.

[0064] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. Important features of an expression vector are that it usually contains a replication origin, a promoter, a marker gene, and a translation control element.

[0065] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include the E. coli lac or trp promoter, the lambda phage PL promoter, eukaryotic promoters such as the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, retroviral LTRs, and several other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector further contains a ribosome binding site for translation initiation and a transcription terminator.

[0066] Additionally, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as, for example, dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.

[0067] A vector containing the appropriate DNA sequence as described above and an appropriate promoter or control sequence can be transformed into an appropriate host cell to permit expression of the protein.

[0068] Host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, plant cells (e.g., carrot cells), and the like.

[0069] When expressing the polynucleotide of the present invention in higher eukaryotic cells, transcription can be enhanced by inserting an enhancer sequence into the vector. Enhancers are DNA cis-acting elements, typically consisting of approximately 10 to 300 base pairs, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer, which consists of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Those skilled in the art know how to select appropriate vectors, promoters, enhancers and host cells.

[0070] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be obtained by treating them with the CaCl2 method after the exponential growth phase, a process well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation, if necessary. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection and electroporation, liposome packaging, etc. can be selected.

[0071] The resulting transformant can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for culture can be selected from a variety of conventional media. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature change or chemical induction), and the cells are further cultured for a certain period of time.

[0072] The recombinant polypeptide in the method can be expressed intracellularly or at the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified using various isolation methods using physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0073] The main advantages of the present invention are: (1) The present invention provides, for the first time, a novel GLP-1 / GIP / FGF21 triple-activity fusion protein, which has many advantages over existing single-activity or dual-activity proteins in controlling blood glucose, body weight, and blood lipids. (2) The fusion protein of the present invention can significantly reduce mouse body weight, body fat percentage, plasma total cholesterol and low-density lipoprotein cholesterol, improve liver function, and lower blood glucose in mice, and its effects are significantly superior to those of a single GLP-1 / GIP active protein or FGF21 active protein, and have a synergistic effect. (3) The fusion proteins of the present invention can be used to treat type 2 diabetes (T2D), obesity, and other disorders of glucose and lipid metabolism.

[0074] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without detailed conditions are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0075] The structure of the fusion protein of the present invention is as shown in the following formula: R1-L1-Fc-L2-R2 Here, R1 is a polypeptide having GLP-1 and GIP dual activity, L1 and L2 are linkers, Fc is an IgG Fc fragment, and R2 is FGF21 or a mutant thereof.

[0076] R1 has the following structural sequence and has dual activity of GLP-1 and GIP: X1X2EGTFTSDYSIX 13 LDKIAX 19 KX 21 FX 23 X 24 WLIAGGPSSGAPPPS (SEQ ID NO. 20) where X1 is His or Tyr, X2 is Gly or Ser, X 13 is Tyr or Leu, X 19 is Ala or Gln, X 21 is Ala or Asp, X 23 is Val or Ile, X 24 is Ala or Gln or Glu, L1 and / or L2 are linkers, Fc is an IgG Fc fragment and can be selected from IgG1, IgG2, and IgG4, and its main effect is to extend half-life; R2 is FGF21 or a mutant thereof, preferably an FGF21 mutant, and the amino acid sequence of the FGF21 mutant is a polypeptide sequence having amino acid residues selected from the group consisting of R19V, L98R, P171N, and Q173T at positions 19, 98, 171, and / or 173 based on the amino acid sequence shown in SEQ ID NO. 1. Here, L1 is (G4S)3A and L2 is (G4S)3.

[0077] Example 1. Preparation of fusion protein samples The corresponding genes were chemically synthesized based on the amino acid sequences of the 12C1-F, 12C2-F, 12C3-F, 12C4-F, 12C5-F, 12C6-F, 12C7-F, 12C8-F, 12C9-F, 12C10-F, 12C11-F, and 12C12-F fusion proteins and cloned into the pcDNA3.1 vector. After large-scale extraction of the plasmids, they were transfected into ExpiCHO-S cells (Life Technologies). Feed and enhancers were added 24 hours after transfection, and the cells were cultured in a thermostatic shaker at 5%, 120 rpm, and 32°C for 7 days. The cell suspension was then centrifuged at 10,000 × g for 30 minutes, and the supernatant was filtered through a 0.45 μm filter. The filtrate was loaded onto a MabSelect SuRe column equilibrated with phosphate buffer, and then the column was eluted with 10 column bed volumes of phosphate buffer. The bound fusion protein was eluted with 50 mmol / L citrate buffer (pH 3.0) and neutralized to pH 7.0 with 1 mol / L Tris-HCl solution. The purity of the fusion protein was detected using HPLC-SEC, and the absorbance value of the fusion protein at 280 nm was measured using a UV spectrophotometer. The content of the fusion protein was calculated based on the extinction coefficient and purity of the fusion protein.

[0078] Example 2. Measurement of in vitro biological activity of fusion proteins In vitro activity detection of the fusion protein GLP-1 was performed using CHO-K1 cells expressing the human GLP-1 receptor and CRE-luciferase. The cells were seeded into a 96-well plate at a density of 30,000 cells / well in 100 μL and incubated at 37°C and 5% CO2 for 1 hour. For various test protein samples, the protein solution was diluted 9 times in a 4-fold gradient starting from a fixed concentration with 2% HAS-containing PBS. Each sample had 10 dilutions with different concentrations. 50 μL of each dilution was added to the 96-well plate seeded with cells and incubated at 37°C and 5% CO2 for 5 hours. The 96-well plate was removed from the incubator and equilibrated at room temperature for 10 minutes. 100 μL of reaction solution was added to each well and shaken at 200 rpm for 10 minutes. The fluorescence readings were then measured using a multi-function microplate reader. A curve was plotted with sample concentration on the horizontal axis and fluorescence readings on the vertical axis, and the 50% effective concentration (EC50) of each sample was calculated. Tirzepatide was used as a positive control and the results are shown in the following table.

[0079] In vitro activity detection of the fusion protein GIP was performed using CHO-K1 cells expressing the human GIP receptor and CRE-luciferase. The cells were seeded into a 96-well plate at a density of 30,000 cells / well in 100 μL and incubated at 37°C and 5% CO2 for 1 hour. For various test protein samples, the protein solution was diluted 9 times in a 4-fold gradient starting from a fixed concentration with 2% HAS-containing PBS. Each sample had 10 dilutions with different concentrations. 50 μL of each dilution was added to the 96-well plate seeded with cells and incubated at 37°C and 5% CO2 for 5 hours. The 96-well plate was removed from the incubator and equilibrated at room temperature for 10 minutes. 100 μL of reaction solution was added to each well and shaken at 200 rpm for 10 minutes. The fluorescence readings were then measured using a multi-function microplate reader. A curve was plotted with sample concentration on the horizontal axis and fluorescence readings on the vertical axis, and the 50% effective concentration (EC50) of each sample was calculated. Tirzepatide was used as a positive control, and the results are shown in the following table, showing that the activity of some of the screened fusion proteins is much higher than that of the control tirzepatide. [Table 1] X1X2EGTFTSDYSIX 13 LDKIAX 19 KX 21 FX 23 X 24 WLIAGGPSSGAPPPS

[0080] The results show that 12C1-F, 12C3-F, 12C11-F, and 12C12-F are preferred fusion proteins based on the in vitro activity of GLP-1 and GIP, and the 12C12-F fusion protein with the best overall performance is selected for subsequent experiments.

[0081] Example 3. Testing blood glucose reduction by multiple subcutaneous injections in db / db diabetic mice The purpose of this example was to study the effect of subcutaneous injection of 12C12-F fusion protein on indices such as body weight in DIO mice, and at the same time to compare its efficacy with that of tirzepatide and Fc-FGF21.

[0082] After the animals were adapted to the breeding environment, their fasting blood glucose levels were measured once a week after a 4-hour fast. With the exception of six wild-type control mice, the db / db mice were divided into groups once their 4-hour fasting blood glucose levels reached the hyperglycemic threshold (≥16.7 mmol / L). The 24 male db / db mice that reached the hyperglycemic threshold were divided equally into four groups based on blood glucose levels and body weight. Each group consisted of six mice: the tirzepatide group, the Fc-FGF21 group, the 12C12-F group, and the vehicle (PBS) group. A control group of wild-type mice was also established. After the experimental animals were divided into groups, treatment with the corresponding drug was initiated via subcutaneous injection (twice a week) at a dose of 30 nmol / kg. Random blood glucose and fasting blood glucose (4-hour fasting) were measured twice a week during the treatment period. Random blood glucose levels were as shown in Figure 1, and fasting blood glucose levels were as shown in Figure 2.

[0083] The results showed that the blood glucose lowering effect of 12C12-F was significantly superior to that of tirzepatide (GLP-1 / GIP dual activity) and Fc-FGF21 (FGF21 monoactivity), and was essentially consistent with that of the normal control group, indicating that the blood glucose of diabetic mice was restored to normal.

[0084] Example 4. Weight loss test in DIO mice following multiple subcutaneous injections The purpose of this example is to study the effect of subcutaneous injection of the fusion protein on indexes such as body weight of DIO mice, and at the same time to compare its efficacy with that of tirzepatide.

[0085] Seven-week-old C57BL / 6Nju mice were fed a high-fat diet (approximately 20% protein and 60% fat) for 12 weeks to model the disease. The conditions were: 12 / 12 hr daily light / dark cycle, free access, temperature 20-25°C, relative humidity 40-70%, and ventilation 10-15 times / hour. The day before administration, mice were randomly assigned to groups based on body weight, water intake, and food intake. Each group consisted of six mice: tirzepatide, Fc-FGF21, 12C12-F, vehicle (PBS), and normal control. The mice were administered subcutaneous injections twice a week for five consecutive weeks, a total of 10 times. After the administration, the mice were examined for body weight, OGTT, body fat percentage, blood lipids, and liver function. The data were expressed as mean ± standard deviation, and statistical analysis of the data was performed using one-way ANOVA.

[0086] 12C12-F significantly reduced body weight and body fat percentage in DIO obese mice, with the magnitude of reduction being significantly greater than that of the controls tirzepatide and Fc-FGF21. At the same time, it significantly reduced blood glucose, plasma total cholesterol, and low-density lipoprotein cholesterol, and improved liver function, essentially restoring it to normal.

[0087] [Table 2]

[0088] [Table 3]

[0089] The sequence of the present invention is as follows: [Table 4]

[0090] SEQ. ID NO. 1 (wild-type FGF21) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

[0091] SEQ.ID NO.2(FGF21 sudden mutation) HPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGNSTGRSPSYAS

[0092] SEQ.ID NO.3(Fc-FGF21 sudden mutation) ESKYGPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGNSTGRSPSYAS*

[0093] SEQ.ID NO.4 (12C1-F fusion protein) YGEGTFTSDYSILLDKIAQKAFIEWLIAGGPSSGAPPPS GGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPPEPGILAPQPPDVGSSDPLSMVGNSTGRSPSYAS

[0094] SEQ.ID NO.5 (12C3-F fusion protein) YGEGTFTSDYSIYLDKIAQKAFIEWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPPEPGILAPQPPDVGSSDPLSMVGNSTGRSPSYAS

[0095] SEQ.ID NO.6 (12C11-F fusion protein) HGEGTFTSDYSIYLDKIAAKDFIEWLIAGGPSSGAPPPS GGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPPEPGILAPQPPDVGSSDPLSMVGNSTGRSPSYAS

[0096] SEQ. ID NO. 7 (12C12-F fusion protein) HSEGFTSDYSILLDKIAQKAFIEWLIAGGPSSGAPPPS GGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTS RFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGNSTGRSPSYAS

[0097] SEQ ID NO. 20 (polypeptide template) X1X2EGTFTSDYSIX 13 LDKIAX 19 KX 21 FX 23 X 24 WLIAGGPSSGAPPPS where X1 is H or Y, X2 is G or S, and X 13 is Y or L, and X 19 is A or Q, and X 21 is A or D, and X 23 is V or I, and X 24 is A, Q, or E.

[0098] SEQ ID NO. 21 (Fc segment) ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0099] SEQ ID NO. 22 (Linker 1) GGGGSGGGGSGGGGSA SEQ ID NO. 23 (Linker 2) GGGGSGGGGSGGGGS

[0100] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. A fusion protein comprising: The fusion protein has, from N-terminus to C-terminus, the structure of Formula I or Formula II, Z0-R1-L1-Fc-L2-R2(I) Z0-R2-L1-Fc-L2-R1 (II) In the formula: Z0 is absent or is selected from a signal peptide, a tag sequence, or a combination thereof; R1 is a GLP-1 and GIP mutein element; L1 is none or a linker; Fc is the Fc element, L2 is none or a linker; R2 is an FGF21 element, "-" is a bond, where: The GLP-1 and GIP mutein elements include GLP-1 and GIP muteins, and the amino acid sequences of the muteins are as follows, based on positions 1, 2, 13, 19, 21, 23, and 24 of the amino acid sequence shown in SEQ ID NO. 20: position 1 is His or Tyr; position 2 is Gly or Ser; position 13 is Tyr or Leu; position 19 is Ala or Gln; position 21 is Ala or Asp; Position 23 is Val or He, and position 24 is Ala or Gln or Glu; a polypeptide sequence having amino acid residues selected from the group consisting of: The fusion protein is characterized in that the mutein simultaneously has the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the human glucose-dependent insulinotropic polypeptide (GIP) receptor.

2. The amino acid sequence of the mutein is as set forth in any one of SEQ ID NOs. 8 to 19. The fusion protein of claim 1.

3. The FGF21 element includes a wild-type FGF21 or a mutant thereof, and the amino acid sequence of the FGF21 mutant is a polypeptide sequence having amino acid residues selected from the group consisting of R19V, L98R, P171N, and Q173T at positions 19, 98, 171, and / or 173 based on the amino acid sequence shown in SEQ ID NO.

1. The fusion protein of claim 1.

4. The fusion protein may comprise: (a) a sequence as set forth in any one of SEQ ID NOs. 4 to 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%, more preferably at least 99% homology with the sequence set forth in any one of SEQ ID NOs. 4 to 7; having an amino acid sequence selected from the group consisting of The fusion protein is characterized by simultaneously having the activity of binding to and activating the class B G protein-coupled receptor GLP-1R and the human glucose-dependent insulinotropic polypeptide (GIP) receptor, as well as the activity of fibroblast growth factor 21 (FGF21). The fusion protein of claim 1.

5. 1. An isolated polynucleotide comprising: The isolated polynucleotide, characterized in that the polynucleotide encodes a fusion protein according to any one of claims 1 to 4.

6. A vector comprising: The vector, characterized in that it comprises the polynucleotide of claim 5 .

7. A host cell comprising: The host cell is characterized in that it contains the vector of claim 6 or has the exogenous polynucleotide of claim 5 integrated into its chromosome.

8. 10. A method for preparing the fusion protein of claim 1, comprising: (i) culturing the host cell of claim 7 under suitable conditions to obtain a mixture containing the fusion protein of claim 1; and (ii) purifying and / or separating the mixture obtained in step (i), thereby obtaining the fusion protein of claim 1. The method, characterized in that it comprises:

9. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (I) a fusion protein according to claim 1, and (II) A pharmaceutically acceptable carrier The pharmaceutical composition characterized by comprising:

10. Use of the fusion protein of claim 1 or the pharmaceutical composition of claim 9, used in the preparation of a drug, said drug comprising: (i) controlling blood glucose content in the body of a subject in need thereof; (ii) reducing blood lipid content in a subject in need thereof; (iii) reducing body fat percentage or inhibiting weight gain in a subject in need thereof; (iv) improving liver function, and / or (v) The use as described above, characterized in that it is used for the prevention and / or treatment of diabetes or obesity-related metabolic diseases.

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