WO2017116207A1 - Analogue de fgf21, conjugué de fgf21 et leur utilisation - Google Patents

Analogue de fgf21, conjugué de fgf21 et leur utilisation Download PDF

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WO2017116207A1
WO2017116207A1 PCT/KR2016/015561 KR2016015561W WO2017116207A1 WO 2017116207 A1 WO2017116207 A1 WO 2017116207A1 KR 2016015561 W KR2016015561 W KR 2016015561W WO 2017116207 A1 WO2017116207 A1 WO 2017116207A1
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fgf21
amino acid
acid sequence
analog
mature
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허용호
최승호
정성엽
권세창
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Hanmi Pharmaceutical Co Ltd
Hanmi Pharmaceutical Industries Co Ltd
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Hanmi Pharmaceutical Industries Co Ltd
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    • 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
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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
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    • A61K38/18—Growth factors; Growth regulators
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K47/54—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 organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to FGF21 analogs, FGF21 conjugates, and uses thereof.
  • Fibroblast growth factor 21 is a secretory polypeptide belonging to a subfamily of fibroblast growth factor (FGF), including FGF19, FGF21 and FGF23.
  • FGF21 is known to play an important role in several physiological functions including angiogenesis, mitosis, pattern formation, cell differentiation, metabolic regulation and tissue damage repair.
  • FGF21 has been reported to be expressed primarily in the liver and has been described as a therapeutic for ischemic vascular disease, wound healing, diseases associated with impairment of lung, bronchial or alveolar cell function, and numerous other disorders.
  • FGF21 stimulated glucose uptake in these cells when treated with mouse 3T3-L1 adipocytes in the presence and absence of insulin (72 hours), in ob / ob and db / db mice and 8 week old ZDF rats.
  • FGF21 stimulated glucose uptake in these cells when treated with mouse 3T3-L1 adipocytes in the presence and absence of insulin (72 hours), in ob / ob and db / db mice and 8 week old ZDF rats.
  • Fasting and postprandial blood glucose, triglycerides, and glucagon have been found to decrease in a dose-dependent manner.
  • the efficacy of the use of FGF21 as a therapy for the treatment of diabetes and obesity has been demonstrated (WO2003 / 011213).
  • human FGF21 is known to have a short half-life in vivo.
  • the half-life of human FGF21 in mice is only 1 to 2 hours, and the half-life of Cynomogus monkeys is 2.5 to 3 hours. Accordingly, in order to use FGF21 protein as a therapeutic agent in the treatment of type 2 diabetes, there is a need for an increase in half-life of FGF21 protein in vivo. Developing an improved half-life FGF21 protein can reduce the frequency of patients receiving protein injections and increase convenience.
  • FGF19 targets both adipocytes and hepatocytes and has an effect on them.
  • mice treated with recombinant human FGF19 show increased metabolic rate, increased lipid oxidation, lower respiratory index and weight loss despite being on a high fat diet.
  • such mice showed lower serum levels of leptin, insulin, cholesterol and triglycerides, and normal levels of blood sugar despite a high fat diet without appetite loss.
  • FGF19 showed weight loss, lowered cholesterol and triglycerides, and diabetes was not developed.
  • rhFGF19 was injected in db / db mice, its metabolic properties were reversed in the form of weight loss and decreased blood glucose.
  • FGF19 and FGF21 are achieved through their binding to the FGFR1c, FGFR2c and FGFR3c receptors, of which the binding to FGFR1c and FGFR2c is the most significant.
  • binding of FGF19 and FGF21 to these receptors requires co-receptor beta-klotho.
  • the metabolic effects of FGF19 and FGF21 are adipocyte specific due to the tissue-specific localized beta-cloto co-receptors.
  • FGF19 is known to regulate bile production by the liver through a liver-specific effect, distinct from FGF21. Specifically, in response to bile-production after meals, it inhibits the transcription of the cholesterol 7-alpha-hydroxylase gene (CYP7A1), a rate limiting enzyme in bile acid synthesis, whereby FGF19 negatively regulates bile production. In addition, FGF19 appears to have a hepatic scavenger effect not observed for FGF21. For example, hepatic adenocarcinoma develops due to increased proliferation and dysplasia of hepatocytes in FGF19 transgenic mice, and mice treated with rhFGF19 have shown hepatocyte proliferation of hepatocytes (Nicholes, K. et al., Am J). Pathol 160, 2295-2307 (2000).
  • CYP7A1 cholesterol 7-alpha-hydroxylase gene
  • FGF21 retains the activity of FGF19, it may be possible to retain useful activity of FGF19, such as bile control function in the liver.
  • Exendin-4 is also a 39 amino acid peptide produced by the salivary glands of Gila monster (Hloderma suspectum). Exendin-4 is an activator of glucagonlike peptide-1 (GLP-1) receptors, but does not significantly activate glucagon receptors.
  • GLP-1 glucagonlike peptide-1
  • Exendin-4 shares most of the glycemic action observed in GLP-1. Clinical and nonclinical studies show that exendin-4 enhances glucose-dependent insulin synthesis and secretion, inhibits glucose-dependent glucagon secretion, slows gastric emptying, food intake and weight loss, and increases beta cell mass and beta cell function. It has been shown to exhibit several beneficial antidiabetic properties, including an increase in markers.
  • Obese patients are at higher risk for diabetes, hypertension, hyperlipidemia, cardiovascular disease and musculoskeletal disease.
  • Exendin-4 exhibits resistance to cleavage by dipeptidyl peptidase (DPP4) compared to GLP-1, resulting in longer half-lives and duration of action in vivo.
  • DPP4 dipeptidyl peptidase
  • One object of the present invention is to provide an FGF21 analog, in which one or more amino acids in native fibroblast growth factor 21 (mutated) are removed, added, substituted, modified, or a combination thereof.
  • Another object of the present invention is to provide a polynucleotide encoding the FGF21 analog.
  • Another object of the present invention is to provide a vector comprising the polynucleotide.
  • Another object of the present invention is to provide a transformant containing the polynucleotide or a vector comprising the polynucleotide.
  • Another object of the present invention is (i) a native FGF21 or the FGF21 analog; And (ii) an FGF21 conjugate to which a carrier selected from the group consisting of an FcRn binder, a fatty acid, polyethylene glycol, an amino acid fragment, and albumin is bound.
  • a carrier selected from the group consisting of an FcRn binder, a fatty acid, polyethylene glycol, an amino acid fragment, and albumin is bound.
  • Another object of the present invention is the FGF21 analog; Or FGF21 analog or natural FGF21 to the carrier selected from the group consisting of FcRn binding material, fatty acid, polyethylene glycol, amino acid fragment, and albumin, FGF21 conjugate comprising an FGF21 conjugate as an active ingredient, for the prevention or treatment of metabolic syndrome It is to provide a pharmaceutical composition.
  • Another object of the present invention is to culture the transformant to express the FGF21 analog; And recovering the expressed FGF21 analog from a transformant or a culture thereof.
  • Another object of the present invention is to provide a method for preparing a FGF21 conjugate, comprising binding a native FGF21 or the FGF21 analog with a carrier selected from the group consisting of FcRn binders, fatty acids, polyethylene glycols, amino acid fragments, and albumin. will be.
  • One embodiment embodying the present invention is an FGF21 analog, wherein one or more amino acids in native fibroblast growth factor 21 (mutated) are removed, added, substituted, modified, or a combination thereof.
  • the FGF21 analog is characterized in that the FGF21 analog, having one or more of the following (i) to (iii).
  • the FGF21 analog is further characterized as being an FGF21 analog, having a variation of (iv), (v) or both:
  • the FGF21 analog is characterized in that the FGF21 analog, having the mutation of the following (a) and (b):
  • the FGF21 analog further comprises substitution of glutamic acid, glycine, amino acid 170 of naturally-occurring mature FGF21; Substitution of the serine of amino acid 172 for leucine; Or FGF21 analog, including both.
  • the FGF21 analog is characterized in that the FGF21 analog having an amino acid sequence, selected from the group consisting of SEQ ID NO: 18, 20, 22, 24, 26, and 86.
  • the other amino acid is alanine, characterized in that the FGF21 analog.
  • the amino acid sequence from No. 16 to No. 20 from the N-terminus of the native mature FGF19 is characterized in that the FGF21 analog, WGDPI (SEQ ID NO: 122).
  • the amino acid sequence comprising the amino acid sequence of 16 to 20 amino acids from the N-terminal end of the native mature FGF19 is characterized in that the FGF21 analog, consisting of 5 to 20 amino acids.
  • the amino acid sequence comprising the amino acid sequence 16 to 20 from the N-terminus of the native mature FGF19 is an amino acid sequence of 14 to 20 from the N-terminus of the native mature FGF19 or 15 to 20 It is characterized by being an FGF21 analog which is an amino acid sequence up to.
  • An FGF21 analog with an amino acid sequence comprising an amino acid sequence 16 to 20 from the N-terminus of a native mature FGF19 adds an amino acid sequence 16 to 20 from the N-terminus of a native mature FGF19. It is characterized in that the amino acid sequence is an FGF21 analog, added to the full length sequence of mature FGF21 or an active fragment thereof.
  • the amino acid sequence comprising the amino acid sequence 16 to 20 from the N-terminus of the native mature FGF19 is a form in which the C-terminus thereof is added to the full-length sequence of the mature FGF21 or the N-terminus of its active fragment, It is characterized by being an FGF21 analog.
  • amino acid sequence comprising the amino acid sequence 16 to 20 from the N-terminus of the native mature FGF19 is an FGF21 analog, directly or via a linker, to the full length sequence of the mature FGF21 or active fragment thereof do.
  • the FGF21 analog is a group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 119, and 121 It is characterized in that the FGF21 analog having an amino acid sequence selected from.
  • the FGF21 analog is the N-terminal amino acid sequence of the mature FGF21,
  • N-terminals 1 to 4 amino acids in the amino acid sequence of the mature FGF21 is characterized in that the analogue is the N-terminal amino acid sequence of FGF21 is excluded.
  • N-terminal amino acid sequence of the mature FGF21 is a contiguous amino acid sequence from the N-terminal to the amino acid sequence of the mature FGF21 from the fifth, sixth or ninth amino acid to any amino acid located in the C-terminal direction.
  • the contiguous amino acid sequence has 2 to 30 amino acids
  • the C-terminal amino acid sequence of the mature FGF21 is characterized in that the analog is a continuous amino acid sequence from any amino acid of the mature FGF21 up to the first, fourth, or eleventh amino acid from the C- terminal.
  • the contiguous amino acid sequence has 2 to 30 amino acids
  • C-terminal amino acid sequence of FGF19 is a contiguous amino acid sequence from any amino acid of FGF19 to C-terminal to the first amino acid
  • the contiguous amino acid sequence has 2 to 30 amino acids
  • N-terminal amino acid sequence of mature FGF21 and the C-terminal amino acid sequence of mature FGF21 or FGF19 are linked to each other via a linker
  • N-terminal amino acid sequence of mature FGF21 and C-terminal amino acid sequence of mature FGF21 or FGF19 are sequentially arranged in the N-terminal to C-terminal direction, or
  • the N-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 123 to 130,
  • the C-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 131 to 136,
  • the C-terminal amino acid sequence of the FGF19 is characterized in that the analog of SEQ ID NO: 137 or 138.
  • the FGF21 analog is characterized in that the FGF21 analog, having an amino acid sequence selected from the group consisting of SEQ ID NO: 90 to 101.
  • exendin-4 sequence is a sequence in which the alpha carbon of histidine, the first amino acid, is removed from the native exendin-4 sequence, and the 32nd to 39th amino acid sequence is excluded.
  • the N-terminal amino acid sequence of the mature FGF21 is characterized in that the FGF21 analog, which is the N-terminal amino acid sequence of FGF21, except for N-terminal 1 to 4 amino acids in the amino acid sequence of the mature FGF21.
  • the N-terminal amino acid sequence of the mature FGF21 is a contiguous amino acid sequence from the N-terminal to the fifth amino acid to any amino acid located in the C-terminal direction in the amino acid sequence of the mature FGF21, and the C-terminal of the mature FGF21.
  • the amino acid sequence is a contiguous amino acid sequence from any amino acid of mature FGF21 to the first or fourth amino acid from the C-terminus
  • the C-terminal amino acid sequence of FGF19 is the first amino acid from C-terminus to any amino acid of FGF19. It is characterized in that the FGF21 analog, which is a sequence of amino acids up to.
  • the contiguous amino acid sequence is FGF21 analogue having 2 to 30 amino acids.
  • the FGF21 analogue is an exendin-4 sequence, an N-terminal amino acid sequence of mature FGF21, and an amino acid sequence of C-terminal sequence of mature FGF21 or FGF19, which are sequentially connected from the N-terminal to the C-terminal direction. It is characterized in that the FGF21 analog.
  • the N-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 139 to 142, and the C-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 143 to 147, and the mature FGF19 C-terminal amino acid sequence of, is selected from the group consisting of SEQ ID NOs: 148 to 150,
  • the FGF21 analog is selected from the group consisting of SEQ ID NOs: 103 to 113, characterized in that the FGF21 analog.
  • Another embodiment embodying the present invention is a polynucleotide encoding said FGF21 analog.
  • Another embodiment of the invention is a vector comprising said polynucleotide.
  • Another embodiment of the invention is a transformant containing the polynucleotide or a vector comprising the polynucleotide.
  • the FcRn binding agent is an FGF21 conjugate, which is an immunoglobulin Fc region.
  • the carrier comprises (iii) a peptide linker or polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaka It is characterized in that it is connected via a non-peptidyl linker selected from the group consisting of a ride, dextran, polyvinylethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid and combinations thereof.
  • the FGF21 analog Or an FGF21 conjugate comprising an FGF21 conjugate, a carrier selected from the group consisting of an FcRn binding substance, a fatty acid, polyethylene glycol, an amino acid fragment, and albumin, bound to the FGF21 analog or natural FGF21,
  • compositions for the prevention or treatment of metabolic syndrome are provided.
  • the metabolic syndrome includes disorders of glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NAS), atherosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, and coronary heart disease ( Coronary heart disease), or liver metabolic disorders, characterized in that the pharmaceutical composition for the prevention or treatment of metabolic syndrome.
  • NAS nonalcoholic steatohepatitis
  • atherosclerosis caused by dyslipidemia
  • atherosclerosis atherosclerosis
  • arteriosclerosis arteriosclerosis
  • Coronary heart disease coronary heart disease
  • liver metabolic disorders characterized in that the pharmaceutical composition for the prevention or treatment of metabolic syndrome.
  • Another embodiment of the present invention comprises the steps of culturing the transformant to express the FGF21 analog; And recovering the expressed FGF21 analog from a transformant or a culture thereof.
  • Another embodiment embodying the present invention is a method for preparing a FGF21 conjugate, comprising binding a native FGF21 or FGF21 analog with a carrier selected from the group consisting of FcRn binders, fatty acids, polyethylene glycols, amino acid fragments, and albumin .
  • FGF21 analogue according to the present invention can be applied therapeutically useful in FGF21-related diseases.
  • Lane 1 shows the results of analysis of recombinant FGF21 purity by protein electrophoresis; Lane 1: size marker, lanes 2 and 3: purified FGF21, lanes 4 and 5: FGF21 containing impurities.
  • Figure 3 shows the results of confirming the binding capacity and in vitro glucose uptake activity of the FGF21 analog protein to beta-klotho (beta-klotho).
  • 4A to 4C are diagrams showing the results of reversal phase chromatography (RPC), ion exchange chromatography (EIX), and size exclusion chromatography (SEC) using HPLC to confirm purity after FGF21 analog purification, respectively.
  • 6A and 6B show the results of confirming the binding capacity and in vitro glucose uptake activity of beta-klotho of FGF19-FGF21 protein.
  • amino acids mentioned herein are abbreviated as follows according to the IUPAC-IUB nomenclature.
  • One embodiment embodying the present invention provides an FGF21 analog, wherein one or more amino acids in native fibroblast growth factor 21 (mutated) are removed, added, substituted, modified, or a combination thereof.
  • FGF21 analog refers to a modification of one or more amino acids in the amino acid sequence of native FGF21.
  • the FGF21 analog may be non-naturally occurring.
  • Naturally mature FGF21 can promote glucose uptake specifically for adipocytes.
  • Information about the native FGF21 protein can be easily obtained from known databases such as NCBI GenBank or UnitProt.
  • native FGF21 can be expressed in an immanture form with a total of 209 amino acids.
  • amino acids 1 to 28 from the N-terminus of the protein correspond to the signal sequence
  • amino acids 29 to 209 correspond to the sequence of mature FGF21 protein.
  • amino acid sequences of these immature forms of FGF21 are described in SEQ ID NOs: 87 and 88 (types L and P, respectively), but are not limited thereto.
  • amino acid sequence of this naturally occurring mature FGF21 examples include, but are not limited to, proteins represented by SEQ ID NOs: 4 (type L) and 151 (type P).
  • the FGF21 analog according to the present invention may have one or more of the following characteristics:
  • Modification of one or more amino acids in the native sequence means a modification selected from the group consisting of substitution, addition, removal, modification, and combinations thereof, of at least one amino acid in native FGF21. Can be.
  • the number of amino acids modified in the native FGF21 sequence is not particularly limited, but at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more , 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 At least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61,
  • amino acids commonly observed in human proteins can be used.
  • commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, Genzyme Pharmaceuticals, and the like.
  • Peptides containing these amino acids and formal peptide sequences can be synthesized and purchased through commercial peptide synthesis companies such as American Peptide Company or Bachem in the United States, or Anygen in Korea.
  • Amino acid derivatives can be obtained in the same manner, for example, desamino-histidine, beta-hydroxyimidazopropionic acid and 4-imidazoacetic acid. ), Beta-carboxyimidazopropionic acid and the like can be used.
  • the addition of the amino acid may be an addition of amino acid sequences derived from other peptides, such as FGF19 and / or insulin secreting peptides (eg exendin-3, exendin-4) other than FGF21.
  • the addition of such amino acids encompasses the addition of a large number of amino acids such as polypeptides or proteins.
  • the FGF21 analog may be a peptide having the same type of in vivo metabolic regulation as the native FGF21.
  • the FGF21 used in the examples of the present invention is an FGF21 analog made by genetic recombination technology, but the present invention is not limited to this, but encompasses various FGF21 analogs possessing the above-described characteristic activities.
  • the FGF21 analog may be prepared by a genetic recombination method according to its form, or may be prepared by a solid phase method, but is not limited thereto.
  • FGF21 analogs of the present invention include derivatives of native FGF21, fragments of native FGF21, and the like.
  • the term “derived from natural FGF21” refers to a peptide having one or more differences in amino acid sequence compared to a native FGF21, a peptide modified by modifying a native FGF21 sequence through modification, and the same kind as a native FGF21. Natural mimics of FGF21 that retain the physiological activity of
  • FGF21 may be homologous to a native FGF21 at least 80% amino acid sequence and / or some groups of one residue of FGF21 are chemically substituted (eg, alpha-methylation, alpha-hydroxylation), removed (eg; deamination) or modified form (eg, N-methylation), but is not limited thereto.
  • Combinations of the various methods for the preparation of derivatives can be used to prepare derivatives of the native FGF21 applied to the present invention.
  • modifications for the preparation of derivatives of native FGF21 include modifications with L- or D-type amino acids, and / or non-natural amino acids; And / or modifying by native or post-translational modification (eg, methylation, acylation, ubiquitination, intramolecular covalent bonds, etc.).
  • the substituted or added amino acids may use at least 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.
  • Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides and formal peptide sequences containing these amino acids can be synthesized and purchased through commercial peptide synthesis companies, for example, American peptide company or Bachem in the United States, or Anygen in Korea.
  • fragment of natural FGF21 or derivative of natural FGF21 refers to a form in which one or more amino acids are removed at the amino or carboxy terminus of a native FGF21 or a derivative of native FGF21. Such fragments may possess the same type of physiological activity as native FGF21.
  • the preparation methods used in the FGF21 derivatives and fragments of the present invention may be used independently and may be combined.
  • peptides having a glycemic control function in a body having one or more amino acid sequences different from each other and deaminated to amino residues at the N-terminus are included.
  • the FGF21 analog may have one or more or two or more of three of the following (i) to (iii):
  • glycine amino acid 170 of naturally-occurring mature FGF21, is substituted with an amino acid other than glycine
  • serine amino acid 172 of naturally occurring mature FGF21, is substituted with an amino acid other than serine
  • the removal of one or more amino acids of amino acid sequences 1 to 4 of the native mature FGF21 may include removal of amino acid 1 and amino acid 2 from the N-terminus of native mature FGF21.
  • removal of amino acids 1, 1 and 2, or amino acids 1 to 4 of the native mature FGF21 may be removed, but is not limited thereto.
  • the corresponding variation may be one in which 167th amino acid serine and / or 168th amino acid methionine is substituted with threonine, alanine, phenylalanine, or isoleucine, and more specifically, each in its natural form. It may correspond to, but is not limited to, substitution of threonine of amino acid 167 with threonine of mature FGF21 and substitution of alanine, phenylalanine, or isoleucine of 168 amino acid with methionine.
  • an FGF21 analogue ( ⁇ H, M168A) with removal of histidine, amino acid 1 of native mature FGF21, and substitution of alanine for 168 amino acid methionine;
  • an FGF21 analogue ( ⁇ HP, S167T) having removal of histidine amino acid 1 and proline amino acid 2 of native-type mature FGF21 and substitution of threonine of amino acid 167;
  • an FGF21 analogue ( ⁇ HP, M168A) having the elimination of histidine, amino acid 1 and proline, amino acid 2 of the native mature FGF21, and substitution of alanine for methionine, amino acid 168;
  • FGF21 analogs ( ⁇ HP, M168F) having removal of histidine amino acid 1 and proline amino acid 2 of native-type mature FGF21 and substitution of 168 amino acid methionine with phenylalanine;
  • FGF21 analogs ( ⁇ HP, M168I) with removal of histidine amino acid 1 and proline amino acid 2 of native-type mature FGF21, and substitution of isoleucine with 168 amino acid methionine;
  • the FGF21 analog having the above-described mutations may include 1) a site cleaved upon expression of the analog and / or 2) a site cleaved in vivo, such as a site cleaved by DPPIV (Dipeptidyl peptidase-4) and And / or the site cleaved by the fibroblast activating protein (FAP) may have been removed, and / or 3) the binding to the receptor and / or co-receptor may be reduced compared to native mature FGF21, but is not limited thereto. .
  • DPPIV Dipeptidyl peptidase-4
  • FAP fibroblast activating protein
  • amino acids 1 to 4 eg HPIP
  • the amino acid located may be substituted with another amino acid, but is not limited thereto.
  • the FGF21 analogue according to the present invention has a long half-life compared to the native mature FGF21 due to the removal of the site cleaved at the time of expression and / or in vivo, while the removal by the receptor is significantly reduced by increasing the dissociation constant. It is characterized by an increased half-life.
  • the FGF21 analog may be an FGF21 analog having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, 26, and 86, but is not particularly limited thereto.
  • the FGF21 analog according to the present invention may include the following mutations together with or independently of the above-described mutations.
  • the FGF21 analogue according to the present invention is the fifth, sixth, seventh, eighth, ninth, 168, 172, 176, 177, and 178 amino acids of native FGF21.
  • One or more amino acids selected from the group consisting of the first amino acid are substituted with another amino acid, and / or
  • It may be an analog comprising a feature added with an amino acid sequence comprising the amino acid sequence 16 to 20 from the N-terminus of the native mature FGF19.
  • the other amino acid may be alanine, but is not particularly limited thereto.
  • any amino acid that can reduce the binding capacity to the FGF21 receptor and / or co-receptor compared to the unsubstituted FGF21 is included in the category of other amino acids without limitation.
  • FGF21 analogue ( ⁇ H, D5A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of apartin of aspartic acid, amino acid 5, with alanine, amino acid 168, ;
  • an FGF21 analogue ( ⁇ H, S6A, M168A) with the removal of histidine, amino acid 1 of amino acid FGF21, substitution of arineine for serine 6 amino acid, and alanine for amino acid 168;
  • FGF21 analogues ( ⁇ H, S7A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of arine for a serine of amino acid 7, and alanine for amino acid 168;
  • FGF21 analogs ( ⁇ H, P8A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of alanine for proline amino acid 8, and alanine for amino acid 168;
  • FGF21 analogs ( ⁇ H, L9A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of leucine of amino acid 9 with alanine, and substitution of alanine with methionine amino acid 168;
  • FGF21 analogs ( ⁇ H, S172A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of arineine for serine, amino acid 172, and alanine for amino acid 168;
  • FGF21 analogs ( ⁇ H, S176A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of arineine for serine, amino acid 176, and alanine for amino acid 168;
  • FGF21 analogs ( ⁇ H, P177A, M168A) with removal of histidine, amino acid 1 of amino acid of natural type FGF21, substitution of alanine for proline, amino acid 177, and alanine for amino acid 168;
  • FGF21 analogs ( ⁇ H, S178A, M168A) with removal of histidine, amino acid 1 of amino acid FGF21, substitution of arineine for serine, amino acid 178, and alanine for amino acid 168;
  • FGF21 analogue with removal of histidine amino acid 1 and proline amino acid 2 of amino acid FGF21, substitution of aspartic acid amino acid 5 with alanine, and substitution of 168 amino acid methionine with alanine ⁇ HP, D5A, M168A);
  • FGF21 analogue ( ⁇ HP) with the removal of histidine amino acid 1 and proline amino acid 2 of the native mature FGF21, substitution of a serine of serine 6 amino acid with alanine, and substitution of alanine with methionine amino acid 168 , S6A, M168A);
  • FGF21 analogue ( ⁇ HP) with removal of histidine amino acid 1 and proline amino acid 2 of amino acid FGF21, substitution of a serine of serine 7 amino acid, and alanine of amino acid 168 , S7A, M168A);
  • FGF21 analogue ( ⁇ HP) with the removal of histidine amino acid 1 and proline amino acid 2 of prophylactic mature FGF21, substitution of alanine for amino acid proline 8 with alanine, and substitution of alanine for amino acid 168 with alanine , P8A, M168A);
  • FGF21 analogue with the removal of histidine amino acid 1 and proline amino acid 2 of amino acid FGF21, substitution of leucine amino acid 9 with alanine, and substitution of alanine with methionine amino acid 168 , L9A, M168A);
  • FGF21 analogue ( ⁇ HP) with removal of histidine amino acid 1 and proline amino acid 2 of amino acid FGF21, substitution of arineine for serine amino acid 172, and alanine for amino acid 168 , S172A, M168A);
  • FGF21 analogue ( ⁇ HP) with the removal of histidine amino acid 1 and proline amino acid 2 of the native type FGF21, substitution of arineine for amino acid 176 with alanine, and alanine for amino acid 168 with alanine , S176A, M168A);
  • FGF21 analogues ( ⁇ HP) with removal of histidine amino acid 1 and proline amino acid 2 of the native mature FGF21, substitution of alanine for proline amino acid 177, and alanine for amino acid 168 with alanine , P177A, M168A);
  • FGF21 analogue ( ⁇ HP) with removal of histidine amino acid 1 and proline amino acid 2 of amino acid FGF21, substitution of arineine for serine amino acid 178, and alanine for amino acid 168 , S178A, M168A).
  • the above-described FGF21 analog has a cleavage site and / or has a reduced binding capacity with FGF21 receptors and / or co-receptors (beta-klotho) relative to native FGF21 and / or reduced receptor mediated rejection compared to native FGF21. -mediated clearance (RMC).
  • the FGF21 analog is selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 80 , May be an FGF21 analog having an amino acid sequence, but is not particularly limited thereto.
  • the FGF21 analogue according to the present invention may be a natural FGF21-derived amino acid sequence of FGF19 or a fragment thereof; Or is added to a derivative of native FGF21 or a fragment thereof.
  • an amino acid sequence comprising an amino acid sequence 16 to 20 from the N-terminus of native mature FGF19 may be added may be included independently or with the mutation (s) described above.
  • the "FGF19" described in the present invention includes a substance capable of expressing metabolic activity by targeting both adipocytes and hepatocytes.
  • the FGF19 may bind to FGFR4 and exhibit its metabolic activity, particularly metabolic activity to the liver.
  • An example is the ability to regulate bile acids.
  • the FGF19 is a concept including not only a naturally occurring mature FGF19 protein but also derivatives and fragments of native FGF19, and the detailed description of the derivatives and fragments also applies to the contents described above in FGF21.
  • the native mature FGF19 may exhibit metabolic activity against adipocytes and / or liver cells.
  • Information about the native FGF19 protein can be easily obtained from known databases such as NCBI GenBank or UnitProt.
  • the native FGF19 may have an amino acid sequence corresponding to NCBI Accession Number NP — 005108. That is, native FGF19 can be expressed as a protein of 216 amino acids in total. In this case, amino acids 1 to 22 from the N-terminus of the protein correspond to the signal sequence, and amino acids 23 to 216 correspond to the sequence of the mature FGF19 protein.
  • amino acid sequence of the native type FGF19 in this immature form examples include, but are not limited to, the protein represented by SEQ ID NO: 89, and amino acid sequences 23-216 in the sequence are mature FGF19 sequences. Corresponds to
  • amino acid sequence 16 to 20 from the N-terminus of the native mature FGF19 may be WGDPI (SEQ ID NO: 122).
  • amino acid sequence including the amino acid sequence of 16 to 20 amino acids from the N-terminal end of the native mature FGF19 may be composed of 5 to 20 amino acids.
  • amino acid sequence comprising the amino acid sequence 16 to 20 from the N- terminal of the native mature FGF19, the amino acid sequence of 14 to 20 from the N- terminal of the mature FGF19 of the native type ( SEQ ID NO: 152) or amino acid sequence 15 to 20 (SEQ ID NO: 153).
  • the FGF21 analog to which the amino acid sequence was added containing the amino acid sequence 16-16 of the N-terminus of a native mature FGF19 added the amino acid sequence 16-20 from the N-terminal of a native mature FGF19
  • the amino acid sequence, including the sequence may be added to the full length sequence of mature FGF21 or an active fragment thereof.
  • amino acid sequence comprising the amino acid sequence 16 to 20 from the N-terminus of the native mature FGF19, the C-terminus thereof is added to the full-length sequence of the mature FGF21 or the N-terminus of the active fragment thereof Form, FGF21 analog.
  • amino acid sequence comprising the amino acid sequence from 16 to 20 from the N-terminus of the native mature FGF19 may be linked directly or via a linker to the full length sequence of the mature FGF21 or an active fragment thereof.
  • the linker may be a flexible linker, and specifically, may be a peptide linker or a non-peptide linker.
  • non-peptide linker refers to a biocompatible linker in which two or more repeating units are linked, and the repeating units are connected to each other through any covalent bonds, not peptide bonds.
  • Non-limiting examples of such non-peptide linkers include polyethylene glycol (PEG) homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxy ethylated polyols, polyvinyl alcohols, polysaccharides, Dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid or combinations thereof, but is not limited thereto.
  • PEG polyethylene glycol
  • the conjugate may be in the form of a fusion protein including FGF19 and FGF21.
  • fusion protein examples include, but are not limited to, a fusion protein represented by the amino acid sequence of SEQ ID NO: 119 or 121. It is apparent to those skilled in the art that in the preparation of the fusion protein, there may be additional amino acid sequences for fusion as well as the original amino acid sequence of the fusion partner for recombinant linkage of each fusion partner. In this case, the scope of the fusion protein should not be construed as limited to the sequence numbers described.
  • the FGF21 analog according to the present invention may include the following mutations together with or independently of the above-described mutations, but is not particularly limited thereto.
  • amino acid sequence of (i) the N-terminal end of mature FGF21 may be added to (ii) the C-terminal amino acid sequence of mature FGF21 or FGF19.
  • it may comprise (i) the N-terminal amino acid sequence of mature FGF21 and (ii) the C-terminal amino acid sequence of mature FGF21 or FGF19, wherein (i) and (ii) may be directly or Indirectly linked, in the form of a FGF21 mock peptide derivative.
  • Such an FGF21 analog of the present invention may have a structure in which up to 30 amino acid sequences located at the N-terminal portion of the naturally occurring mature FGF21 and 30 or less amino acid sequences located at the C-terminal portion are combined.
  • some groups of amino acid residues here may be chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), removed (e.g., deamination) or modified (e.g., N-methylated), and metabolized in the body. It can have the ability to adjust.
  • the FGF21 analog has a portion of the N-terminus important for binding of the FGF21 receptor and a portion of the C-terminus of the FGF21 or FGF19 which directly binds to the FGF21 co-receptor beta-closo in the mature FGF21 sequence excluding the signal sequence. It may be combined.
  • the FGF21 analog may have a decreased binding capacity to the FGF21 receptor than the native FGF21, and may have an increase in blood half-life due to a significant decrease in receptor-mediated clearance caused by an increase in dissociation constant. have.
  • the FGF21 analog may include an N-terminal amino acid sequence of FGF21, in which all N-terminals 1 to 4 amino acids are excluded from an amino acid sequence of mature FGF21 that does not include a signal sequence. .
  • the N-terminal amino acid sequence of the mature FGF21 included in the FGF21 analog of the present invention is in the C-terminal direction from the fifth, sixth or ninth amino acid from the N-terminus in the amino acid sequence of the mature FGF21. It can be a contiguous amino acid sequence up to any amino acid located.
  • the consecutive amino acids are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, May have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, but is not limited thereto It doesn't work.
  • the N-terminal amino acid sequence of the mature FGF21 may be selected from the group consisting of SEQ ID NOs: 123 to 130, but is not limited thereto.
  • the FGF21 imitation peptide derivative exhibiting a lower titer than the natural FGF21 according to the present invention it is not limited to the sequence, 70% or more, specifically 80% or more, 81% or more with the sequence.
  • Amino acid sequences that exhibit at least%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of similarity are also included within the scope of the present invention.
  • sequence having such similarity it is obvious that the case where some sequences have an amino acid sequence deleted, modified, substituted or added is also included within the scope of the present invention. The same applies to the description of the C-terminal amino acid sequence of FGF21, the C-terminal amino acid sequence of FGF19, and the specific sequence of the FGF21 mock peptide derivative which will be described later.
  • similarity is intended to represent a similar degree to a wild type amino acid sequence or nucleic acid sequence, and the comparison of the homology may be performed by using a comparison program known in the art, which is easy to be seen with the naked eye.
  • Commercially available computer programs can calculate the similarity between two or more sequences as a percentage. In addition,% similarity can be calculated for adjacent sequences.
  • the C-terminal amino acid sequence of the mature FGF21 included in the FGF21 mock peptide derivative of the present invention is from any amino acid of mature FGF21 to the 1st, 4th, or 11th amino acid from the C-terminus. It may be a contiguous amino acid sequence.
  • the consecutive amino acids are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, May have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, but is not limited thereto It doesn't work.
  • the C-terminal amino acid sequence of the mature FGF21 may be selected from the group consisting of SEQ ID NOs: 131 to 136, but is not limited thereto.
  • the C-terminal amino acid sequence of FGF19 included in the FGF21 analog of the present invention may be a contiguous amino acid sequence from any amino acid of FGF19 to C-terminal to the first amino acid.
  • the consecutive amino acids are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, May have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, but is not limited thereto It doesn't work.
  • amino acid sequence of FGF19 is shown in SEQ ID NO: 89.
  • 1 to 24 is a signal sequence and 25 to 216 correspond to a mature FGF19 amino acid sequence.
  • the C-terminal amino acid sequence of the mature FGF19 may be SEQ ID NO: 137 or 138, but is not limited thereto.
  • the FGF21 mock peptide derivative is composed of several combinations of sequences starting from the N-terminal fourth, fifth and ninth amino acids of the native mature FGF21 and sequences starting from the first, fourth and eleventh amino acids of the C-terminal. Can be configured.
  • the present invention is not limited thereto, and all FGF21 analogs having reduced binding to the FGF21 receptor fall within the scope of the present invention.
  • the N-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 123 to 130
  • the C-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 131 to 136
  • the FGF19 The amino acid sequence of the C-terminus of may be an FGF21 analog, SEQ ID NO: 137 or 138. Various combinations thereof are included in the scope of the present invention.
  • the FGF21 analog may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 90 to 101. However, it is not limited thereto.
  • the FGF21 analog is not particularly limited in length as long as it can activate the FGF21 receptor and / or co-receptor (ie, beta-closo), but for example, may have 4 to 100 amino acids. However, it is not limited to what was described above.
  • amino acid sequence of the N- terminal of the mature FGF21 and (ii) the amino acid sequence of the C-terminal of the mature FGF21 or FGF19 may be linked to each other through a linker.
  • the linker is not particularly limited as long as the linker connects (i) and (ii) and brings the FGF21 imitation peptide derivative to the FGF21 receptor.
  • the linker may be a flexible linker.
  • the flexible linker may increase flexibility in binding the N-terminal amino acid sequence of FGF21 to the FGF21 receptor and the C-terminal amino acid sequence of FGF21 or FGF19 to the FGF21 co-receptor.
  • the linker may be a peptidic linker or a non-peptidyl linker, but is not limited thereto.
  • the linker is as described above.
  • the amino acid sequence of (i) the N-terminus of the mature FGF21 and (ii) the C-terminal amino acid sequence of the mature FGF21 or FGF19 are sequentially arranged from the N-terminal to the C-terminal direction, although not limited thereto. Can be.
  • the present invention is not limited thereto, and (ii) the amino acid sequence of the C-terminus of mature FGF21 or FGF19 and (i) the amino acid sequence of the N-terminus of mature FGF21 are sequentially arranged from the N-terminus to the C-terminal direction. Can be.
  • the FGF21 analog according to the present invention may include the following mutations together with or independently of the above-described mutations, but is not particularly limited thereto.
  • the N-terminal amino acid sequence of mature FGF21 is added to (ii) the C-terminal amino acid sequence of mature FGF21 or FGF19, and (iii) the exendin-4 sequence is added.
  • it may be FGF21 analog.
  • FGF21 analog which is a dual GLP-1 / FGF21 agonist, including an Excendin-4 sequence, a mature FGF21 N-terminal region and a mature FGF21 or FGF19 C-terminal region.
  • the FGF21 analog may be a reduced GLP-1 or FGF21 titer as compared to native GLP-1 (Glucagon-like peptide-1) or FGF21.
  • This double GLP-1 / FGF21 agonist, the FGF21 analog has a decreased binding capacity to the GLP-1 receptor, the FGF21 receptor, and / or the FGF21 co-receptor than the native GLP-1 and FGF21. This may be due to a significant decrease in clearance, resulting in increased blood half-life.
  • the "dual GLP-1 / FGF21 agonist” may comprise an exendin-4 peptide derived sequence, a mature FGF21 N-terminal sequence, and a C-terminal sequence of mature FGF21 or FGF19.
  • such structural properties may result in reduced GLP-1 and / or FGF21 titers, and any GLP-1 / FGF21 agonist possessing these properties may be included in the scope of the present invention without limitation.
  • exendin-4 peptide-derived sequence included in the double GLP-1 / FGF21 agonist is exendin-4, ie, imidazoacetyl-Exendin, from which the alpha carbon of histidine, the first amino acid, is removed from the exendin-4 sequence.
  • exendin-4 imidazoacetyl-exendin-4, CA-exendin-4 derived sequence.
  • the alpha carbon of the first amino acid histidine is removed from the native exendin-4 sequence, and may be a sequence from which the 32nd to 39th amino acid sequences are excluded.
  • the present invention is not limited thereto, and the exendin-4 sequence includes all CA-exedin-4 sequences having reduced GLP-1 titers.
  • the FGF21 sequence applied to the double GLP-1 / FGF21 agonist is directly linked to betacloso, an FGF21 co-receptor, and a portion of the N-terminus important for the binding of the FGF21 receptor in the mature FGF21 sequence except for the signal sequence.
  • -Part of the terminal may be a sequence consisting of a combination.
  • N-terminus or “C-terminus” may refer to the N-terminal region or C-terminal region of FGF21 or FGF19 which may constitute the dual GLP-1 / FGF21 agonist.
  • the dual GLP-1 / FGF21 agonists of the present invention are dual agents comprising an exendin-4 peptide, an N-terminal sequence of mature FGF21 amino acid sequence, and a C-terminal of mature FGF21 or FGF19 amino acid sequence, but the present invention is limited thereto. Rather, it includes all dual GLP-1 / FGF21 agonists with reduced in - vitro titers.
  • FGF19 binds to the FGF receptor like FGF21
  • FGF19 is preceded by betacloso, an FGF co-receptor, and it is known that the C-terminus of FGF19 binds to betacloso and its binding force is stronger than that of FGF21.
  • sequences consisting of the binding of the mature FGF21 N-terminus to the FGF19 C-terminus.
  • FGF21 N-terminal amino acid sequence used in the present invention is N-terminal except N-terminal 1st amino acid (Histidine) to 4th amino acid (Proline) does not affect activity in the mature FGF21 amino acid sequence that does not include a signal sequence
  • the amino acid sequence of the site can be used and specifically, in the amino acid sequence of mature FGF21, a contiguous amino acid sequence from the N-terminus to the fifth amino acid to any amino acid located in the C-terminus direction can be used.
  • the C-terminal amino acid sequence of mature FGF21 is a contiguous amino acid sequence from any amino acid of mature FGF21 to the first or fourth amino acid from the C-terminus
  • the C-terminal amino acid sequence of FGF19 is any of FGF19 Contiguous amino acid sequence from amino acid to C-terminus to first amino acid.
  • the contiguous amino acid sequence may be composed of 2 to 30 amino acid sequences.
  • exendin-4 sequence (ii) FGF21 N-terminal amino acid sequence, and (iii) FGF21 C-terminal amino acid sequence or FGF19 C-terminal amino acid sequence may be FGF21 analogs linked to each other.
  • N-terminal amino acid sequence of mature FGF21 and the C-terminal amino acid sequence of mature FGF21 or FGF19, or the N-terminal amino acid sequence and exendin-4 sequence of mature FGF21 are directly It may be connected or connected via a linker, the linker as described above.
  • the linker may increase flexibility in binding exendin-4, a GLP-1 analog, to the GLP-1 receptor.
  • the N-terminus of FGF21 binds to the FGF21 receptor and FGF21 or the C-terminus of FGF19 can increase flexibility in binding to the FGF21 co-receptor.
  • the FGF21 analog may be one in which the exendin-4 sequence, the amino acid sequence of the N-terminus of mature FGF21, and the amino acid sequence of the C-terminus of mature FGF21 or FGF19 are sequentially connected from the N-terminal to the C-terminal direction. .
  • the N-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 139 to 142
  • the C-terminal amino acid sequence of the mature FGF21 is selected from the group consisting of SEQ ID NOs: 143 to 147
  • the amino acid sequence of the C-terminus of mature FGF19 may be selected from the group consisting of SEQ ID NOs: 148 to 150, but is not limited thereto.
  • the FGF21 analog may be selected from the group consisting of SEQ ID NOs: 103 to 113.
  • the FGF21 analog of the present invention can be produced by standard synthetic methods, recombinant expression systems, or any other method of the art.
  • FGF21 analogs according to the invention can be synthesized in a number of ways, including for example, including:
  • Another embodiment embodying the present invention provides a transformant comprising a polynucleotide encoding the FGF21 analog, a vector comprising the polynucleotide, or a vector comprising the polynucleotide or the polynucleotide.
  • the FGF21 analog is as described above.
  • vector refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding said FGF21 analog operably linked to a suitable regulatory sequence such that the target protein, such as said FGF21 analog, can be expressed in a suitable host. do.
  • the regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.
  • the recombinant vector can be transformed into a suitable host cell and then replicated or functioned independently of the host genome and integrated into the genome itself.
  • the recombinant vector to be used in the present invention is not particularly limited as long as it can be replicated in a host cell, and may be produced using any vector known in the art.
  • Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc.
  • the vector usable in the present invention is not particularly limited and known expression vectors can be used.
  • the recombinant vector can be used to transform host cells to produce the FGF21 analog of the present invention.
  • Such transformed cells which are also part of the present invention, may be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors of the invention or used for recombinant production of the conjugates of the invention.
  • transformation in the present invention means introducing a recombinant vector comprising a polynucleotide encoding a target protein into a host cell to allow the protein encoded by the polynucleotide to be expressed in the host cell.
  • the transformed polynucleotide it includes all of them regardless of whether they are inserted into or located outside the chromosome of the host cell.
  • the polynucleotide also includes DNA and RNA encoding the target protein.
  • the polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression.
  • the expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal, which are typically operably linked to the polynucleotide.
  • the expression cassette may be in the form of an expression vector capable of self replication.
  • the polynucleotide may be introduced into a host cell in its own form, and may be operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • operably linked means that the gene sequence is functionally linked with a promoter sequence for initiating and mediating the transcription of a polynucleotide encoding the FGF21 analog of the present invention.
  • Suitable hosts for the present invention are not particularly limited as long as they allow expression of the polynucleotide of the present invention.
  • Specific examples of hosts that can be used in the present invention include bacteria of the genus Escherichia , such as E. coli ; Bacillus subtilis (Bacillus subtilis) and Bacillus (Bacillus), such as bacteria belonging to the genus; Bacteria of the genus Pseudomonas , such as Pseudomonas putida ; Pichia pastoris , Saccharomyces cerevisiae ), Schizosaccharomyces yeast such as pombe ); Insect cells such as Spodoptera luciferda (Sf9); And animal cells such as CHO, COS, BSC and the like.
  • FGF21 native FGF21 or said FGF21 analog
  • FGF21 conjugate to which a carrier selected from the group consisting of FcRn binder, fatty acid, polyethylene glycol, amino acid fragment, and albumin is bound.
  • the FGF21 and the FGF21 analog are as described above.
  • the FcRn binding agent may be an immunoglobulin Fc region.
  • the FGF21 conjugate may include (i) a native FGF21 or the FGF21 analog and (ii) a carrier (iii) a peptide linker, or polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, Polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, and combinations thereof.
  • each end of the non-peptidyl polymer may be bound to an immunoglobulin Fc region and an amine group or thiol group of FGF21, respectively.
  • the immunoglobulin Fc region, or immunoglobulin constant region, of the present invention is a biodegradable polypeptide that is metabolized in vivo, it is safe to use as a carrier for drugs.
  • the immunoglobulin Fc region is advantageous in terms of the manufacture, purification and yield of the conjugate because of its relatively low molecular weight compared to the whole immunoglobulin molecule, as well as the removal of Fab moieties that exhibit high heterogeneity because the amino acid sequence varies from antibody to antibody. It is also expected that the homogeneity will be greatly increased and the likelihood of inducing blood antigens will be lowered.
  • an "immunoglobulin Fc region” or an “immunoglobulin constant region” is a site including a heavy chain constant region 2 (CH2) and / or a heavy chain constant region 3 (CH3) moiety except for the heavy and light chain variable regions of an immunoglobulin. Means.
  • the immunoglobulin Fc region may be one component of a moiety of the FGF21 conjugate of the present invention.
  • the immunoglobulin Fc region may include a hinge portion in the heavy chain constant region, but is not limited thereto.
  • some or all heavy chain constant region 1 (CH1) and / or light chain constant region It may be an extended Fc region including 1 (CL1). It may also be a region from which some fairly long amino acid sequences corresponding to CH2 and / or CH3 have been removed.
  • the immunoglobulin Fc regions of the present invention may comprise 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) Combination of one or two or more of the CH1 domain, CH2 domain, CH3 domain and CH4 domain with an immunoglobulin hinge region (or a portion of the hinge region), 6) heavy chain constant region may be a dimer of each domain and light chain constant region .
  • an immunoglobulin hinge region or a portion of the hinge region
  • the immunoglobulin Fc region may be in a dimeric form, and one molecule of FGF21 or an analog thereof may be covalently linked to one Fc region in a dimeric form, wherein the immune Globulin Fc and FGF21 or analogs thereof may be linked to each other by non-peptidyl polymers.
  • the immunoglobulin Fc and FGF21 or an analog thereof may be connected to each other by a non-peptidyl polymer.
  • immunoglobulin Fc regions of the present invention include naturally occurring amino acid sequences as well as sequence derivatives thereof.
  • Amino acid sequence derivatives mean that one or more amino acid residues in a natural amino acid sequence have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • IgG Fc amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331 which are known to be important for binding can be used as suitable sites for modification.
  • various kinds of derivatives are possible, such as a site capable of forming disulfide bonds, a few amino acids at the N-terminus in the native Fc, or a methionine residue may be added at the N-terminus of the native Fc.
  • the complement binding site such as C1q binding site may be removed, ADCC (antibody dependent cell mediated cytotoxicity) site may be removed in order to eliminate the effector function.
  • ADCC antibody dependent cell mediated cytotoxicity
  • Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979).
  • the most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.
  • it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation and amidation. may be modified.
  • Fc derivatives may exhibit biological activities equivalent to those of the Fc region of the present invention and may increase structural stability to heat, pH, etc. of the Fc region.
  • the Fc region may be obtained from a natural type separated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
  • the method of obtaining from the natural form may be a method of separating the whole immunoglobulin from a human or animal living body, and then processing the protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F (ab) 2 . Fc or pF'c may be separated by size-exclusion chromatography or the like.
  • the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.
  • the immunoglobulin Fc region may be in a natural sugar chain, an increased sugar chain compared to the natural form, a reduced sugar chain or a sugar chain removed from the natural form.
  • Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease such immunoglobulin Fc sugar chains.
  • the immunoglobulin Fc region in which the sugar chain is removed from the Fc is significantly reduced in binding strength with the complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, thereby not causing an unnecessary immune response in vivo. Do not.
  • a form more consistent with the original purpose as a carrier of the drug would be the immunoglobulin Fc region from which the sugar chains have been removed or unglycosylated.
  • “Deglycosylation” refers to an Fc region in which sugar is removed by an enzyme
  • Aglycosylation refers to an Fc region which is not produced and glycosylated in prokaryotes, and more specifically, in Escherichia coli. .
  • the immunoglobulin Fc region may be of human origin or animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and in more specific embodiments is human origin.
  • the immunoglobulin Fc region may be an Fc region by IgG, IgA, IgD, IgE, IgM derived or combinations thereof or hybrids thereof. In more specific embodiments it is derived from IgG or IgM, which is most abundant in human blood, and in more specific embodiments it is of IgG known to enhance the half-life of ligand binding proteins. In even more specific embodiments the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.
  • “combination” in the present invention means that, when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of different origin. That is, it is possible to prepare dimers or multimers from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE.
  • non-peptidyl polymer refers to a biocompatible polymer having two or more repeating units bonded thereto, and may be one component forming a moiety of the FGF21 conjugate of the present invention.
  • the repeating units are linked to each other through any covalent bond, not a peptide bond.
  • the non-peptidyl polymer may be used in combination with the non-peptidyl linker.
  • Non-peptidyl polymers usable in the present invention include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxy ethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA ( Biodegradable polymers such as polylactic acid, polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Glycol (PEG). Analogs thereof known in the art and analogs which can be easily manufactured at the technical level in the art are included in the scope of the present invention.
  • the non-peptidyl polymer of the present invention may have a reactor capable of binding to the carrier of the present invention, for example, an immunoglobulin Fc region and FGF21. That is, it can be linked to a carrier such as an immunoglobulin Fc region and FGF21 or these analogues through the reactor of the non-peptidyl polymer.
  • Both terminal reactors of the non-peptidyl polymer are preferably selected from the group consisting of reaction aldehyde groups, propion aldehyde groups, butyl aldehyde groups, maleimide groups and succinimide analogs.
  • the functional group containing the aldehyde group may be an alkyl aldehyde such as propion aldehyde, butyl aldehyde. Therefore, specifically, the non-peptidyl polymer may be PEG in which a propion aldehyde group is substituted at the sock end.
  • succinimid analog succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate can be used.
  • the non-peptidyl polymer has a reactor of reactive aldehyde groups at both ends, it is effective to minimize nonspecific reactions and to bind FGF21 and immunoglobulins at both ends of the non-peptidyl polymer, respectively.
  • the final product, resulting from reductive alkylation by aldehyde bonds, is much more stable than linked by amide bonds.
  • the aldehyde reactor selectively reacts to the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, for example pH9.0 conditions.
  • Both terminal reactors of the linker which is the non-peptidyl polymer may be the same or different from each other.
  • one end may have a maleimide group and the other end may have an aldehyde group, a propion aldehyde group, or a butyl aldehyde group.
  • the hydroxy group may be activated into the various reactors by a known chemical reaction, or a polyethylene glycol having a commercially available modified reactor may be used. Sustained conjugates of the invention can be prepared.
  • the non-peptidyl polymer of the present invention may each have a reactor selected from the group consisting of aldehyde, maleimide, vinylsulfone, thiol, disulfide orthopyridine, iodide acetamide, succinimide and their derivatives each independently And, it may be covalently linked to each of the FGF21 and the carrier, for example immunoglobulin Fc region through the reactor of the both ends.
  • the conjugates of the present invention are those wherein the reactors at both ends of the non-peptidyl polymer are bound to the carrier and the amine group or thiol group of FGF21, respectively, or the reactors at both ends of the non-peptide polymer are respectively FGF21 and It may be bound to the N-terminal amine group of the carrier or the amine group of the Lys residue side chain.
  • the non-peptidic polymer includes a reactor capable of binding to a carrier, for example, an immunoglobulin Fc region and FGF21 at both ends, and the reactor is not particularly limited thereto, but is not particularly limited to an aldehyde group, propionaldehyde.
  • a carrier for example, an immunoglobulin Fc region and FGF21 at both ends
  • the reactor is not particularly limited thereto, but is not particularly limited to an aldehyde group, propionaldehyde.
  • a succinimide analog succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate
  • non-peptidyl polymer that can be used in the present invention can be used without limitation so long as it is a polymer resistant to proteolytic enzymes in vivo.
  • Non-peptidyl polymers preferably have a molecular weight in the range of 1 to 100 kDa, specifically 1 to 20 kDa.
  • the non-peptidyl polymer of the present invention which is combined with FGF21 or a carrier, may be used not only with one kind of polymer but also with a combination of different kinds of polymers.
  • the non-peptidyl polymer used in the present invention may have a two- or three-terminal reactor that can be combined with FGF21 and a carrier. Specifically, the reactor may be an aldehyde group.
  • the present invention provides a composition comprising the FGF21 analog or FGF21 conjugate.
  • the FGF21 conjugate is as described above.
  • a persistent formulation for FGF21 comprising an FGF21 analog or FGF21 conjugate.
  • the long-acting formulation may be an FGF21 conjugated persistent formulation or an FGF21 analog persistent formulation having increased in vivo persistence and stability compared to native FGF21.
  • the long-acting formulation may be a pharmaceutical composition for preventing or treating metabolic syndrome to be described later.
  • long-acting formulations or pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound.
  • the carrier may include a kind of non-naturally occuring carrier.
  • excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavorings can be used.
  • buffers, preservatives, analgesics, solubilizers, isotonic and stabilizing agents can be used in combination.
  • bases, excipients, lubricants and preservatives may be used.
  • the formulations of the long-acting formulations or pharmaceutical compositions of the present invention can be prepared in a variety of mixtures with the pharmaceutically acceptable carriers described above.
  • FGF21 analog or FGF21 analog or natural FGF21 to the carrier selected from the group consisting of FcRn binding material, fatty acid, polyethylene glycol, amino acid fragment, and albumin, FGF21 conjugate comprising an FGF21 conjugate as an active ingredient, for the prevention or treatment of metabolic syndrome It provides a pharmaceutical composition.
  • prevention refers to any action that inhibits or delays the onset of metabolic syndrome disease by administration of the FGF21 analog, FGF21 conjugate, or a composition comprising the same
  • treatment refers to the FGF21 analog, FGF21 conjugate, Or it refers to any action that improves or benefits the symptoms of metabolic syndrome disease by administration of a composition comprising the same.
  • the metabolic syndrome includes disorders of glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NAS), atherosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, and coronary heart disease (Coronary heart disease) may be selected from the group consisting of, but is not limited thereto.
  • NAS nonalcoholic steatohepatitis
  • atherosclerosis caused by dyslipidemia atherosclerosis
  • arteriosclerosis arteriosclerosis
  • coronary heart disease Coronary heart disease
  • the term "administration" means introducing a predetermined substance into a patient in any suitable manner, and the route of administration of the composition is not particularly limited, but any general route by which the composition can reach a target in vivo.
  • Can be administered for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. have.
  • intraperitoneal administration intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. have.
  • the oral composition since the peptide is digested, it is desirable to formulate the oral composition to coat the active agent or to protect it from degradation in the stomach. It may preferably be administered in the form of an injection.
  • the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
  • the pharmaceutical composition of the present invention may include excipients or diluents as well as pharmaceutically acceptable carriers.
  • pharmaceutically acceptable means a sufficient amount and side effects that do not cause a therapeutic effect, the type of disease, the age, weight, health, sex, sensitivity of the patient to the drug It can be easily determined by those skilled in the art according to well-known factors in the medical field, such as the route of administration, the method of administration, the frequency of administration, the duration of treatment, the combination or the drug used simultaneously.
  • the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as oral administration binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors, etc.
  • buffers, preservatives, analgesic A topical agent, a solubilizer, an isotonicity agent, a stabilizer, etc. can be mixed and used, and in case of topical administration, a base, an excipient, a lubricant, a preservative, etc. can be used.
  • the formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above.
  • oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups and wafers, and in the case of injections, they may be prepared in unit dosage ampoules or multiple dosage forms. Others may be formulated into solutions, suspensions, tablets, pills, capsules and sustained release preparations.
  • suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
  • fillers may be further included.
  • the invention comprises administering to a subject in need thereof a selected from the group consisting of the FGF21 analogs, FGF21 conjugates thereof, persistent agents thereof, pharmaceutical compositions thereof, and combinations thereof.
  • a selected from the group consisting of the FGF21 analogs, FGF21 conjugates thereof, persistent agents thereof, pharmaceutical compositions thereof, and combinations thereof are provided.
  • FGF21 analogs FGF21 conjugates, their sustained preparations, and pharmaceutical compositions thereof are as described above.
  • the FGF21-related diseases may be metabolic syndrome, as described above.
  • the dosage of the FGF21 analog, FGF21 conjugate, and the like of the present invention depends on the type of active ingredient, FGF21 analog, FGF21 conjugate, etc., along with various related factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease. Is determined. Since the FGF21 analogs, FGF21 conjugates, and the like of the present invention have excellent in vivo persistence and titer, the frequency and frequency of administration of the FGF21 analogs, FGF21 conjugates, their sustained preparations, or pharmaceutical compositions thereof of the present invention can be significantly reduced. have.
  • Another embodiment of the present invention comprises the steps of culturing the transformant to express the FGF21 analog; And recovering the expressed FGF21 analog from a transformant or a culture thereof.
  • the FGF21 analog and transformant are as described above.
  • it may be a method for producing an FGF21 analog including the following steps:
  • step (c) obtaining a supernatant containing FGF21 analog from the precipitate of step (b), applying it to anion binding chromatography, and obtaining an eluate comprising FGF21 analog;
  • Another embodiment embodying the present invention provides a method of making a FGF21 conjugate comprising binding a native FGF21 or the FGF21 analog with a carrier selected from the group consisting of FcRn binding agents, fatty acids, polyethylene glycols, amino acid fragments, and albumin to provide.
  • a carrier selected from the group consisting of FcRn binding agents, fatty acids, polyethylene glycols, amino acid fragments, and albumin to provide.
  • the FGF21, its analog, FGF21 conjugate, and transformants are as described above.
  • the present invention provides a method for increasing the half-life in vivo using the FGF21 and analog or FGF21 analog conjugate, combined with FGF21, analog and carrier.
  • the FGF21, its analog, FGF21 conjugate, and carrier are as described above.
  • the FGF21 gene was amplified from FGF21 cDNA (OriGene, RC204538) to construct FGF21 expression vector.
  • FGF21 cDNA OriGene, RC204538
  • the following primers SEQ ID NO: 1, 2 was prepared and PCR was performed.
  • FGF21 amplification primer order SEQ ID NO: FGF21 5'-GTCAAGCTCACCCCATCCCTGACTCCAGTC-3 '
  • FGF-21 expression vector was constructed. The correct sequence was confirmed by sequence analysis. Nucleotide and protein sequences are as follows. The following FGF21 sequence corresponds to the mature FGF21 sequence.
  • the backbone vector of the FGF21 analog was produced to prepare the FGF21 analog using the native FGF21 expression vector as a template.
  • Naturally mature FGF21 is found in about 20% of histidine's first amino acid histidine (Histidine) in the protein expression process, and cut between amino acids 167 and 168 near the C terminus. To prevent this, the first histidine or the second proline at the N-terminus is removed, and serine, the 167th amino acid, threonine, and alanine, the 168th amino acid, Methionine.
  • FGF21 muteins substituted with Alanine, Phenylalanine and Isoleucine were prepared and used as backbone vectors for the FGF21 analog fabrication described above.
  • the histidine removal form was first prepared using SEQ ID NO: 5 and SEQ ID NO: 6, and then the FGF21 M168A substituted FGF21 analog backbone vector was completed.
  • FGF21 analog backbone vector DNA protein sequence order SEQ ID NO: ⁇ H, M168A DNA ATGAAAAAGA CAGCTATCGC GATTGCAGTG GCACTGGCTG GTTTCGCTAC CGTTGCGCAA GCTCCCATCC CTGACTCCAG TCCTCTCCTG CAATTCGGGG GCCAAGTCCG GCAGCGGTAC CTCTACACAG ATGATGCCCA GCAGACAGAA GCCCACCTGG AGATCAGGGA GGATGGGACG GTGGGGGGCG CTGCTGACCA GAGCCCCGAA AGTCTCCTGC AGCTGAAAGC CTTGAAGCCG GGAGTTATTC AAATCTTGGG AGTCAAGACA TCCAGGTTCC TGTGCCAGCG GCCAGATGGG GCCCTGTATG GATCGCTCCA CTTTGACCCT GAGGCCTGCA GCTTCCGGGA GCTGCTTCTT GAGGACGGAT ACAATGTTTA CCAGTCCGAA GCCCACGGCC TCCCTCCT GAGGACGGAT ACAATG
  • FGF21 analogs in which the N- and C-terminal amino acids of FGF21 were modified one by one were prepared.
  • forward and reverse oligonucleotides were synthesized (Table 5), followed by PCR to amplify each analog gene.
  • amino acid numbers indicated in the analog names below are numbered based on the sequence of the native mature FGF21, ⁇ H is the deletion of the first amino acid histidine in the native mature FGF21 sequence, ⁇ HP in the native mature FGF21 sequence The first and second amino acids histidine and proline are deleted.
  • FGF21 analog backbone vector DNA protein sequence analog order SEQ ID NO: ⁇ H, D5A, M168A DNA ATGAAAAAGA CAGCTATCGC GATTGCAGTG GCACTGGCTG GTTTCGCTAC CGTTGCGCAA GCTCCCATCC CTGCATCCAG TCCTCTCCTG CAATTCGGGG GCCAAGTCCG GCAGCGGTAC CTCTACACAG ATGATGCCCA GCAGACAGAA GCCCACCTGG AGATCAGGGA GGATGGGACG GTGGGGGGCG CTGCTGACCA GAGCCCCGAA AGTCTCCTGC AGCTGAAAGC CTTGAAGCCG GGAGTTATTC AAATCTTGGG AGTCAAGACA TCCAGGTTCC TGTGCCAGCG GCCAGATGGG GCCCTGTATG GATCGCTCCA CTTTGACCCT GAGGCCTGCA GCTTCCGGGA GCTGCTTCTT GAGGACGGAT ACAATGTTTA CCAGTCCGAA GCCCACGGCC TCCCTCCCCCCCCC
  • E. coli BL21 E. coli B F-dcm ompT hsdS (rB-mB-) gal, Agilent
  • E. coli B F-dcm ompT hsdS (rB-mB-) gal, Agilent was transformed using the recombinant FGF21 expression vector.
  • Fermentation proceeded when the nutrients in the culture was limited, feeding medium was added to the fed value culture.
  • the growth of the strains was monitored by OD values and introduced IPTG with a final concentration of 0.5 mM at OD values above 80. After the introduction, the culture was further carried out until about 23 to 25 hours, and after the incubation, the recombinant strain was harvested using a centrifuge and stored at -80 ° C until use.
  • Histidine is added to the supernatant obtained in the above example to 15 mM. Then, 60% HCl was added slowly to pH 5.2, centrifuged at 7,000 rpm to remove the pellet, and the supernatant containing the recombinant FGF21 analog was collected.
  • 1M Tris pH 8.0 buffer was added to the supernatant obtained in the above example so that the pH was 6 or more, and then diluted with 10 mM Tris 8.0 buffer so that the conductivity was 5 or less.
  • the protein was conjugated to a DEAE (GE, USA) column equilibrated with 10 mM Tris pH 8.0 buffer, followed by concentration using a 10 mM Tris pH 8.0 buffer containing 0.5 M sodium chloride.
  • the recombinant FGF21 analog was eluted with a linear concentration gradient of 10 column volumes from% to 100%.
  • the eluate obtained in the above example was diluted 10-fold with 10 mM Tris pH 8.0 buffer, and then slowly added 3 M ammonium sulfate (1.2 M) buffer to 1.2 M.
  • Bound to Source Iso (GE, USA) column equilibrated with 10 mM Tris pH 8.0 buffer containing 1.2 M Ammonium Sulfate and then concentrated using 10 mM Tris pH 8.0 buffer.
  • the recombinant FGF21 protein was eluted with a linear gradient of 10 column volumes from 0% to 100%.
  • the present inventors confirmed that about 50% of the cleavage form of FGF21 was expressed in the course of expressing native FGF21 in E. coli, and the cleaved FGF21 in the full sequencing result was serine at position 167. It was confirmed that the cleavage between (167S) and methionine (168M) located at 168 by an unknown route.
  • the FGF21 analog was produced using the native FGF21 expression vector as a template.
  • the native FGF21 forms a truncated form between amino acids 167 and 168 near the C-terminus during protein expression.
  • FGF21 was replaced with Ser, the 167th amino acid, and Thr, with Met, the 168th amino acid.
  • amino acids 1-4 were removed at the gene level.
  • the 170th amino acid Gly was replaced with Glu and the 172th amino acid Ser was replaced with Leu to prevent the formation of the cleaved form between the 171th and 172th amino acids by the fibroblast activating protein (FAP) in vivo.
  • FAP fibroblast activating protein
  • Oligonucleotides used to prepare the FGF21 analog were synthesized as shown in Table 8, followed by site-directed mutagenesis PCR to amplify the FGF21 analog gene.
  • the oligonucleotides of SEQ ID NOs: 81 and 82 were first prepared to form a form in which 1-4 amino acids (HPIP) were removed, and then 167, 168, 170, and 172 using oligonucleotides of SEQ ID NOs: 83 and 84. Th amino acid was substituted to complete the FGF21 analog vector.
  • FGF21 Analog Fabrication Primer order SEQ ID NO: HPIP 5'-CGCTACCGTTGCGCAAGCTGACTCCAGTCCTCTCCTGCA-3 ' 81 5'-TGCAGGAGAGGACTGGAGTCAGCTTGCGCAACGGTAGCGA-3 ' 82 S167T, M168I, G170ES172L 5'-GGCTCCTCGGACCCTCTGACCATCGTGGAACCTCTCCAGGG-3 ' 83 5'-CCCTGGAGAGGTTCCACGATGGTCAGAGGGTCCGAGGAGCC-3 ' 84
  • FGF21 analog backbone vector DNA protein sequence order SEQ ID NO: FGF21 (del-HPIP, S167T, M168I, G170E, S172L) DNA
  • E. coli BL21 E. coli BF - dcm ompT hsdS (r B -m B- ) gal, Agilent
  • E. coli BL21 E. coli BF - dcm ompT hsdS (r B -m B- ) gal, Agilent
  • the transformation method was recommended by Agilent.
  • Single colonies transformed with the recombinant FGF-21 analog expression vector were taken and inoculated in 1X Luria Broth (LB) medium containing ampicillin (50 ⁇ g / ml) and incubated at 37 ° C. for 15 hours.
  • LB 1X Luria Broth
  • Recombinant FGF21 strain cultures and 1X LB medium containing 30% glycerol were mixed at a ratio of 1: 1 (v / v), and each 1 ml was dispensed into cryo-tubes and stored at -140 ° C. This was used as a cell stock for the production of recombinant fusion proteins.
  • Fermentation proceeded when the nutrients in the culture was limited, supplemented with additional feeding (feeding solution) to proceed with the oil value culture.
  • the growth of the strain was monitored by OD values, and introduced with an IPTG of 0.5 mM final concentration at an OD value of 80 or higher. After the introduction, the culture was further performed until about 23 to 25 hours, and after the incubation, the recombinant strain was harvested using a centrifuge and stored at -80 ° C until use.
  • Example 2 (2) 200 g of the E. coli cells obtained in Example 2 (2) were dissolved in 3 L of a buffer solution (20% sucrose, 1 mM EDTA, 30 mM Tris pH 7.5) at room temperature, stirred for 2 hours, and centrifuged at 7,000 rpm. Collected. The pellet was again dissolved in 3 L of distilled water at 4 ° C., stirred at 4 ° C. for 2 hours, centrifuged at 7,000 rpm to remove the pellet, and the supernatant containing the surrounding cytoplasmic protein was collected.
  • a buffer solution 20% sucrose, 1 mM EDTA, 30 mM Tris pH 7.5
  • Example 2 To the supernatant obtained in Example 2 (3) was added histidine to a final concentration of 15 Mm. Then, 60% HCl was added slowly to pH 5.2, centrifuged at 7,000 rpm to remove the pellet, and the supernatant containing the recombinant FGF21 analog protein was collected.
  • 1M Tris pH 8.0 buffer was added to the supernatant obtained in Example 2 (4) to have a pH of 6 or more, and then diluted with 10 mM Tris 8.0 buffer to have a conductivity of 5 or less. Conjugate the protein to a DEAE (GE, USA) column equilibrated with 10 mM Tris pH 8.0 buffer, then use a 10 mM Tris pH 8.0 buffer containing 0.5 M sodium chloride to give a concentration of 0 to 100%. The recombinant FGF21 analog protein was eluted with a linear concentration gradient of.
  • Example 2 (5) The eluate obtained in Example 2 (5) was diluted 10-fold with 10 mM Tris pH 8.0 buffer, and then slowly added 3M ammonium sulfate buffer solution to 1.2M.
  • the protein was bound to a Source Iso (GE, USA) column equilibrated with 10 mM Tris pH 8.0 buffer containing 1.2 M Ammonium Sulfate, followed by zero concentration using 10 mM Tris pH 8.0 buffer.
  • Recombinant FGF21 analog was eluted with a linear gradient of 10 column volumes from% to 100%.
  • FIGS. 4A-4C Purity of the purified FGF21 analog protein was analyzed using electrophoresis (SDS-PAGE) and high pressure chromatography (HPLC) and the results are shown in FIGS. 4A-4C (FIGS. 4A-4C).
  • FGF21 analog FGF21 mock peptide, having the following sequence was synthesized (Table 10).
  • the FGF21 analog may have a high production efficiency when mass-produced compared to the natural FGF21, and may have a reduced titer compared to the natural FGF21.
  • the FGF21 analog a dual GLP-1 / FGF21 agonist, capable of simultaneously acting on the GLP-1 receptor and the FGF21 receptor, was prepared and its sequence is shown in Table 11 below.
  • the first amino acid (Xaa) in the table means that the alpha carbon is removed from histidine, ie imidazoacetyl.
  • the novel dual GLP-1 / FGF21 agonist prepared above has a binding capacity to the GLP-1 receptor, FGF21 receptor, FGF21 co-receptor, its binding capacity is reduced compared to the natural type has a reduced titer, diabetes, It can be used as a therapeutic substance in obese or nonalcoholic steatohepatitis patients.
  • the amino acids 15-20 of FGF19 refer to amino acids 15-20, based on the mature FGF19 sequence excluding amino acids 1-22, which are signal sequences in the native immature FGF19 sequence.
  • FGF19 (15 ⁇ 20) Insertion Primer order SEQ ID NO: FGF-19 (15-20) 5'-GAAGGAGATATACATATGGGCTGGGGTGATCCGATTCACCCCATCCCTGACTCCAG-3 ' 114 5'-CTGGAGTCAGGGATGGGGTGAATCGGATCACCCCAGCCCATATGTATATCTCCTTC-3 ' 115
  • the site-directed mutagenesis method was used to insert the FGF19 (15-20) sequence, which is the FGFR4 binding site, between the start codon of the pET22b vector and the FGF21 N-terminus.
  • PCR amplification conditions were repeated 18 times at 95 °C 30 seconds, 55 °C 30 seconds, 68 °C 6 minutes and confirmed the exact sequence through the sequence analysis.
  • FGF19 (15-20) -FGF21 sequence present in pET22b in the surrounding cytoplasm of the host cell, it was intended to be inserted into the pFlage-CTS vector.
  • the following primers SEQ ID NOs: 116 and 117
  • PCR was performed. It was.
  • FGF19 (14 ⁇ 20) Insertion Primer order SEQ ID NO: FGF-19 (14-20) 5'-GTAAGCTTATGGCTGGGGTGATCCGATTC-3 ' 116 5'-CACTCGAGTCAGGAAGCGTAGCTGGGGCTTC-3 ' 117
  • PCR amplification conditions were repeated 25 times at 95 ° C., 30 sec, 55 ° C., 30 sec, and 68 ° C. at 40 sec.
  • the amplified gene and vector were digested with Hind III and Xho I restriction enzymes, followed by pFlag- with T4 ligase.
  • An expression vector was prepared by inserting into a CTS vector. Sequence analysis confirmed the correct sequence.
  • FGF21, FGF19 (15-20) -FGF21 recombinant fusion protein, and FGF19 (14-20) -FGF21 recombinant fusion protein are as follows.
  • E. coli BL21 E. coli BF - dcm ompT hsdS (r B -m B- ) gal, Agilent
  • E. coli BL21 E. coli BF - dcm ompT hsdS (r B -m B- ) gal, Agilent
  • the transformation was followed by the method recommended by Agilent.
  • Single colonies transformed with the recombinant FGF19-FGF21 expression vector were taken and inoculated in 1X Luria Broth (LB) medium containing ampicillin (50 ⁇ g / ml) and incubated at 37 ° C. for 15 hours.
  • LB 1X Luria Broth
  • Recombinant FGF19-FGF21 strain cultures and 1X LB medium containing 30% glycerol were mixed at a ratio of 1: 1 (v / v), and each 1 ml was dispensed into cryo-tubes and stored at -140 ° C. This was used as a cell stock for the production of recombinant fusion proteins.
  • FGF19-FGF21 For expression of recombinant FGF19-FGF21, 1 vial of each cell stock was dissolved, inoculated into 1 L of 1 ⁇ Luria broth, and cultured at 37 ° C. for 14-15 hours. When the value of the OD600 was 1.0 or more, the culture was terminated, and this was used as the seed culture solution. Seed cultures were inoculated into 9.2 L of fermentation medium using a 30 L fermenter (BIOSTAT C-plsu 30L, Sartorius, Germany) and initial bath fermentation was started. Culture conditions were maintained at pH 6.70 using a temperature of 30 °C, air volume 13 L / min (1 vvm), agitation speed 500 rpm and 30% ammonia water.
  • a 30 L fermenter BIOSTAT C-plsu 30L, Sartorius, Germany
  • Fermentation proceeded when the nutrients in the culture was limited, supplemented with additional feeding (feeding solution) to proceed with the oil value culture.
  • the growth of the strains was monitored by OD values and introduced with IPTG with a final concentration of 0.5 mM at an OD value of 80 or higher.
  • the culture proceeds further to about 23-25 hours after introduction, and after the incubation, the recombinant strain is harvested using a centrifuge and stored at -80 ° C until use.
  • Histidine was added to the supernatant obtained in the above (3) to 15 mM. Then, 60% HCL was slowly added to pH 5.2, centrifuged at 7,000 rpm to remove the pellet, and the supernatant containing the recombinant FGF19-FGF21 protein was collected.
  • 1M Tris pH 8.0 buffer was added to the supernatant obtained in (4) so that the pH was 6 or more, and then diluted with 10 mM Tris 8.0 buffer so that the conductivity was 5 or less. Conjugate the protein to a DEAE (GE, USA) column equilibrated with 10 mM Tris pH 8.0 buffer, then use a 10 mM Tris pH 8.0 buffer containing 0.5 M sodium chloride to provide a linear concentration of 10 column volumes so that the concentration is from 0% to 100%. The gradient eluted the recombinant FGF19-FGF21 protein.
  • the eluate obtained in (5) was diluted 10-fold with 10 mM Tris pH 8.0 buffer, and then slowly added 3M ammonium sulfate buffer to 1.2M.
  • the protein was bound to a Source Iso (GE, USA) column equilibrated with 10 mM Tris pH 8.0 buffer containing 1.2 M ammonium sulfate, and then the concentration was 0% to 100% using 10 mM Tris pH 8.0 buffer.
  • Recombinant FGF19-FGF21 protein was eluted at a linear concentration gradient of 10 columns.
  • reaction solution was mono-pegylated by application to Source15Q (GE, USA) using a buffer containing 10 mM Tris pH 7.0 and a 0.25 M Sodium Chloride gradient. Monopegylated) native FGF21 was purified.
  • the molar ratio of the purified mono-pegylated natural FGF21 and immunoglobulin Fc was 1: 3 and the protein concentration was 50 mg / mL, and the reaction was carried out at 4 ° C. for 16 hours.
  • the reaction solution was performed in an environment in which 20 mM sodium cyanoborohydride (NaCHBH 3 ) as a reducing agent was added to 100 mM potassium phosphate pH 6.0 buffer. After the reaction was completed, the reaction solution was applied to Source15Q (GE, USA) using a 20 mM Bis Tris pH 6.0 and 0.25 M sodium chloride concentration gradient, and 1.2 M ammonium sulfate (Ammonium Sulfate). ) And a concentration gradient of 20 mM Tris pH 7.5 was applied to Source ISO (GE, USA) to purify the conjugate covalently linked with PEG by native FGF21 to immunoglobulin Fc.
  • Source15Q GE, USA
  • Ammonium Sulfate ammonium Sulf
  • the binding force of the representative FGF21 analogs of the present invention with the receptor and the co-receptor is measured.
  • the FGF21 analog of the present invention confirms that the binding force between the FGF21 receptor and the co-receptor (beta-klotho) is reduced compared to the native type FGF21.
  • Receptor-mediated clearance (RMC) of the FGF21 analog is measured.
  • the FGF21 analog of the present invention confirms that the receptor-mediated elimination is reduced than that of the native FGF21.
  • the SPR test was conducted to confirm whether the recombinant FGF19-FGF21 protein, which is the FGF21 analog obtained in Example 5, has a binding capacity to FGFR4. As a result, it was confirmed that the FGF19-FGF21 protein had a binding capacity of about 1.7% of FGFR4 compared to the native type FGF19 (FIG. 7).
  • FGF19-FGF21 protein has the activity of FGF19.
  • the concentration of FGF21-immunoglobulin Fc conjugate was increased (1.2, 2.4, 4.8 nmol / kg / Q2D) in diabetic model mice (db / db mouse). Blood glucose was measured for 4 weeks after subcutaneous administration once. As a result, it was confirmed that the blood glucose of the diabetic model mice was significantly reduced (FIG. 9).

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Abstract

La présente invention concerne un analogue de FGF21, un conjugué de FGF21, et leur utilisation.
PCT/KR2016/015561 2015-12-31 2016-12-30 Analogue de fgf21, conjugué de fgf21 et leur utilisation Ceased WO2017116207A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836486A (zh) * 2019-01-30 2019-06-04 北京双因生物科技有限公司 成纤维生长因子21变体、其融合蛋白及其用途
CN111187845A (zh) * 2020-02-05 2020-05-22 内蒙古农业大学 Fgf21基因在产毛动物选育和/或调节毛发长短中的应用
CN111662373A (zh) * 2019-03-05 2020-09-15 广东东阳光药业有限公司 一种多肽分子及其应用
WO2022227707A1 (fr) * 2021-04-30 2022-11-03 江南大学 Procédé de préparation et utilisation d'une protéine de fusion double cible
US11510990B2 (en) 2020-01-11 2022-11-29 Beijing Ql Biopharmaceutical Co., Ltd. Conjugates of fusion proteins of GLP-1 and FGF21
EP4410817A4 (fr) * 2021-09-29 2025-01-29 Caregen Co., Ltd. Peptide ayant une activité anti-vieillissement, et son utilisation
US12331093B2 (en) 2021-07-14 2025-06-17 Beijing Ql Biopharmaceutical Co., Lt Fusion polypeptides for metabolic disorders

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10360948B2 (en) 2017-06-26 2019-07-23 Samsung Electronics Co., Ltd. Memory device and operating method of memory device

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010065439A1 (fr) * 2008-12-05 2010-06-10 Eli Lilly And Company Variants de facteurde croissance 21 du fibroblaste
US20100216715A1 (en) * 2009-01-23 2010-08-26 Novo Nordisk A/S FGF21 Derivatives With Albumin Binder A-B-C-D-E- And Their Use
US20140148388A1 (en) * 2009-10-15 2014-05-29 Genentech, Inc. Chimeric fibroblast growth factors with altered receptor specificity
US8809499B2 (en) * 2009-08-20 2014-08-19 Chongqing Fagen Biomedical Inc. Fusion protein of human fibroblast growth factor-21 and exendin-4
US20140243503A1 (en) * 2008-06-04 2014-08-28 Amgen Inc. Fgf21 mutants and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140243503A1 (en) * 2008-06-04 2014-08-28 Amgen Inc. Fgf21 mutants and uses thereof
WO2010065439A1 (fr) * 2008-12-05 2010-06-10 Eli Lilly And Company Variants de facteurde croissance 21 du fibroblaste
US20100216715A1 (en) * 2009-01-23 2010-08-26 Novo Nordisk A/S FGF21 Derivatives With Albumin Binder A-B-C-D-E- And Their Use
US8809499B2 (en) * 2009-08-20 2014-08-19 Chongqing Fagen Biomedical Inc. Fusion protein of human fibroblast growth factor-21 and exendin-4
US20140148388A1 (en) * 2009-10-15 2014-05-29 Genentech, Inc. Chimeric fibroblast growth factors with altered receptor specificity

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KHARITONENKOV ET AL.: "Rational Design of a Fibroblast Growth Factor 21-Based Clinical Aandidate, LY2405319", PLOS ONE, vol. 8, no. e58575, 11 March 2013 (2013-03-11), pages 1 - 10, XP055268887 *
WU ET AL.: "Separating Mitogenic and Metabolic Activities of Fibroblast Growth Factor 19 (FGF19", PNAS, vol. 107, no. 32, 10 August 2010 (2010-08-10), pages 14158 - 14163, XP055198815 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836486A (zh) * 2019-01-30 2019-06-04 北京双因生物科技有限公司 成纤维生长因子21变体、其融合蛋白及其用途
CN111662373A (zh) * 2019-03-05 2020-09-15 广东东阳光药业有限公司 一种多肽分子及其应用
CN111662373B (zh) * 2019-03-05 2024-05-14 广东东阳光药业股份有限公司 一种多肽分子及其应用
US11510990B2 (en) 2020-01-11 2022-11-29 Beijing Ql Biopharmaceutical Co., Ltd. Conjugates of fusion proteins of GLP-1 and FGF21
CN111187845A (zh) * 2020-02-05 2020-05-22 内蒙古农业大学 Fgf21基因在产毛动物选育和/或调节毛发长短中的应用
CN111187845B (zh) * 2020-02-05 2023-03-31 内蒙古农业大学 Fgf21基因在产毛动物选育和/或调节毛发长短中的应用
WO2022227707A1 (fr) * 2021-04-30 2022-11-03 江南大学 Procédé de préparation et utilisation d'une protéine de fusion double cible
US12331093B2 (en) 2021-07-14 2025-06-17 Beijing Ql Biopharmaceutical Co., Lt Fusion polypeptides for metabolic disorders
EP4410817A4 (fr) * 2021-09-29 2025-01-29 Caregen Co., Ltd. Peptide ayant une activité anti-vieillissement, et son utilisation

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