WO2012062078A1 - Variant de délétion au niveau de n-terminal du facteur de croissance humain des fibroblastes 21 et son conjugué - Google Patents

Variant de délétion au niveau de n-terminal du facteur de croissance humain des fibroblastes 21 et son conjugué Download PDF

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WO2012062078A1
WO2012062078A1 PCT/CN2011/071948 CN2011071948W WO2012062078A1 WO 2012062078 A1 WO2012062078 A1 WO 2012062078A1 CN 2011071948 W CN2011071948 W CN 2011071948W WO 2012062078 A1 WO2012062078 A1 WO 2012062078A1
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growth factor
fibroblast growth
human fibroblast
conjugate
terminal deletion
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Chinese (zh)
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范开
陈勇
陈海容
张淳
梅翔
张益�
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重庆富进生物医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of DNA recombination technology and medicine. More specifically, the present invention relates to a variant of N-terminally truncated human fibroblast growth factor 21 (FGF-21), a corresponding DNA molecule, an expression vector, and covalent attachment of the variant to polyethylene glycol And use.
  • FGF-21 N-terminally truncated human fibroblast growth factor 21
  • Fibroblast Growth Factor is a structurally related protein encoded by FGF gene family members. It has many mesodermal and neuroectodermal-derived cells such as endothelial cells, fibroblasts, and smooth muscle cells. It promotes DNA synthesis and cell division, and is mainly secreted by fibroblasts, endothelial cells, bone cells, and polymorphic cells. The relative molecular weight is generally 17 ⁇ 34kD. Amino acids in the central region of FGF family members have 30%-70% homology. FGF-21 is the latest member of a FGFs found to belong to the FGF-19 subfamily. The FGF-19 subfamily includes FGF-15, FGF-19, FGF-21, and FGF-23.
  • FGF-21 is mainly expressed in mature liver and thymus. It was first isolated from mouse embryos by Nishimura et al. (Nishimura T et al., Biochim Biophys Acta, 2000, 1492: 203-206). Its amino acid sequence and FGF were first obtained. The homology between -19 and FGF-23 was 35% and 24%, respectively. Subsequently, FGF-21 mRNA was also found in pancreas, adipose tissue and muscle tissue (Inagaki T et al, Cell Metab, 2007, 5(6): 415-425).
  • the human FGF-21 gene is located on chromosome 19, and the full-length FGF-21 protein consists of a 181 amino acid mature FGF-21 protein and a N-terminal 28 amino acid signal peptide.
  • the amino acid sequence is similar to the mouse FGF-21. 75% are the same.
  • FGF-21 plays a very important role in blood sugar, blood lipid metabolism and body weight control.
  • the N-terminal region and the C-terminal region play different biological functions, that is, the N-terminal amino acid participates in the interaction with the FGF receptor, and the C-terminal amino acid participates in a mutual interaction with a protein called ⁇ -Klotho.
  • Role Micanovic R et al, J Cell Phys, 2009, 219(2): 227-234). Kharitonenkov A et al.
  • FGF-21 glucose uptake by FGF-21 is non-insulin dependent and can increase glucose uptake when co-existing with insulin (Alexei Kharitonenkov et al, Biodrugs, 2008, 22(1): 37-44) .
  • FGF-21 can improve the function of islet beta cells. After 8 weeks of continuous treatment of FGF-21, db/db mice had normal blood glucose after meal, glucose clearance rate was significantly improved, insulin transcription, expression and secretion increased, but the number of islet cells did not change.
  • FGF-21 blocks glycolipid toxicity and cytokine-mediated apoptosis, protects the number and function of islet beta cells, and inhibits glucose-mediated glucagon release (Wente W et al, Diabetes, 2006) , 55: 2470-2478).
  • injection of recombinant human FGF-21 can lower LDL cholesterol, increase HDL cholesterol, and reduce the risk of cardiovascular disease (K aritonenkov A et al, Endocrinology, 2007, 148: 774- 781).
  • no side effects such as hypoglycemia, edema and obesity, which are often found in other diabetes, have shown good application prospects and safety.
  • Lilly Company discloses the amino acid sequence of human FGF-21 and its recombinant preparation, use (US7259248, WO/2009/020802), fusion protein of human FGF-21 protein with IgG4 or HSA (US20070237768), and a human FGF21
  • the biological effects are reduced to varying degrees, in which the simple N-terminal deletion of the 4 amino acid variants remains 90% (US 7622445) o
  • the researchers of the present invention found that the recombinant human wild-type FGF-21 has poor stability and exhibits an N-terminal break.
  • the N-terminal 9 amino acid sequence of the human FGF21 protein molecule was deleted (codenamed AN9FGF-21), which has higher stability and biological activity.
  • the above-mentioned characteristics of recombinant deletion human FGF21 were confirmed by using genetic engineering recombinant protein technology to prepare the deletion type AN9FGF21, including three major expression systems of E. coli, Pichia Pastoris and mammalian cells (CHO). .
  • the inventors of the present invention found that the conjugate obtained by modifying the polyethylene glycol of the deletion variant maintains the activity of the deletion variant in vivo and improves the deletion variant in vivo. Pharmacokinetic properties.
  • the deletion variant and its conjugate are more suitable as a pharmaceutical combination for the treatment or prevention of obesity, diabetes, high glucose or hyperlipidemia. Summary of the invention
  • One of the objects of the present invention is to provide a deletion type human fibroblast growth factor 21 ( ⁇ 9 FGF-21) variant. It has the amino acid sequence signature of SEQ ID No: 1, which is compared with the sequence of wild-type human fibroblast growth factor 21 (FGF-21), and has a total of 9 amino acids, 172 amino acids, truncated at the N-terminus of HPIPDSSPL. composition.
  • FGF-21 wild-type human fibroblast growth factor 21
  • the N-terminus may carry a methionine (Met) residue, which is expressed as Met-AN9FGF-21, and its amino acid sequence is SEQ ID NO: 3.
  • the N-terminal deletion of the N-terminal amino acid human fibroblast 21 (AN9FGF-21) of the present invention has Met at the N-terminus, and does not affect its biological properties.
  • the amino acid sequence of human AN9FGF-21 (SEQ ID NO: 1): Leu Gin Phe Gly Gly Gin 3 ⁇ 41 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp 1 5 1 0 15
  • Another object of the present invention is to provide a DNA molecule encoding the sequence of the deletion human human fibroblast growth factor 21 variant (SEQ ID NO: 1), and a recombinant expression vector containing the DNA molecule.
  • the expression vector may be derived from a prokaryotic or eukaryotic organism system, and in addition to the gene sequence encoding AN9FGF-21, further comprises a regulatory sequence for expression of the protein of interest such as a promoter and a screening marker such as ampicillin, tetracycline, neomycin, G418, Zeocin, DHFR, etc.
  • Another object of the present invention is to provide a host cell for use in the transformation of the above expression vector.
  • the host cell can be an E. coli, yeast or mammalian cell.
  • Another object of the present invention is to provide a method for culturing the above-described transformed host cells and isolating and purifying the recombinant deletion type human FGF-21 variant from the culture solution.
  • Another object of the present invention is to provide an analog of human fibroblast growth factor 21 which is improved in both stability and biological activity, i.e., human AN9FGF-21.
  • the polyethylene glycol derivative is covalently linked to the N-terminal amino group or the Lys amino group of the N-terminal deletion variant of human fibroblast growth factor 21.
  • the polyethylene glycol derivative is an aldehyde-activated monomethoxypolyethylene glycol molecule, and the aldehyde group is covalently linked to the Lys amino group.
  • the polyethylene glycol derivative is characterized by a linear or branched chain or a star shape and has a molecular weight of 5 KD to 40 KD.
  • the polyethylene glycol derivative has a molecular weight of 20 KD of linear monomethoxy polyethylene glycol propionaldehyde.
  • Another object of the present invention is to provide a conjugate of an N-terminal deletion variant of human fibroblast growth factor 21.
  • the conjugate is formed by covalent attachment of an activated polyethylene glycol molecule to the N-terminal amino group or Lys amino group of human AN9FGF-21, wherein the polyethylene glycol is mainly at the 47th position in the sequence of SEQ ID NO: 1.
  • the 50th, 60th and 113th amino groups of Lys are covalently linked, wherein the polyethylene glycol molecule may be linear or branched or star-shaped, and has a molecular weight of 5KD-40KD.
  • the N-terminal deletion variant of the human fibroblast growth factor 21 and its conjugate protein are more useful for the treatment or prevention of obesity, diabetes, high fat and hyperglycemia.
  • a therapeutic or prophylactic drug or combination of drugs can be prepared by techniques known in the art at an effective dosage range.
  • injections, lyophilizates, sustained release agents, tablets or capsules can be prepared by known public formulation methods.
  • Figure 1 Recombinant human AN9FGF-21 protein expression and purification SDS-PAGE electropherogram. Wherein, M is a protein standard, 1 is uninduced, 2 is induced, 3 is purified by Phenyl, and 4 is purified by reverse phase.
  • Figure 2 PEG modification of recombinant human AN9FGF-21 and its purification SDS-PAGE electropherogram.
  • M is a protein standard
  • 1 is a pre-modification sample
  • 2 is a modified sample
  • 3 is a purified PEG-AN9FGF21.
  • Figure 3 Effect of recombinant human AN9FGF21 and PEG-AN9FGF21 on glucose uptake rate of 3T3-L1 cells (gross content is glucose content (mMol/L), abscissa is time (hour), where ⁇ is the control group, ⁇ ⁇ N9FGF21 , ⁇ is PEG-Z N9FGF21.
  • the present inventors conducted extensive and intensive research, using the preferred expression vector pET-3c and host cell BL21 (DE3) PlysS for expression and isolation of recombinant deletion-type human fibroblast growth factor 21 variant, using a molecular weight of 20KD.
  • the linear carboxy group-activated monomethoxypolyethylene glycol molecule modifies the deleted human fibroblast growth factor 21 variant.
  • the examples are for illustrative purposes only and are not to be construed as limiting the invention.
  • Example 1 Construction of recombinant human AN9FGF-21 protein expression engineering bacteria
  • the N-terminal 9-position His Pro lie Pro Asp Ser Ser Pro Leu sequence was deleted as the amino acid sequence of AN9FGF-21 (SEQ ID No: 1), and follows The codons preferred by E. coli were optimized to encode the cDNA encoding human AN9FGF-21 (SEQ ID NO: 2).
  • Entrusted Biotechnology (Dalian) Co., Ltd. for whole gene synthesis in which the Nde l restriction site (CAT ATG) was introduced at the 5' end of the cDNA, and the stop codon (TCA) and BamH I restriction site (GGA) were introduced at the 3' end. TCC), after inserting pMD-19-T simple vector, transferred to host DH5cx, the positive clone was confirmed by cDNA sequencing and the design sequence was identical, and named pMD-AN9FGF21.
  • the recombinant plasmid pMD-AN9FGF21 was extracted and digested with Nde I and BamH I.
  • the 540 bp fragment was recovered and ligated with the expression plasmid pET-3c (Invitrogen) which was also digested with Nde I and BamH I by T4 DNA ligase. .
  • CaThe CaC12 method was used to transform the E. coli host strain DH5a, and positive clones were selected.
  • the correct recombinant plasmid was identified by enzyme digestion and PCR as pET-3C- AN9FGF21 o
  • the plasmid was transformed into E. coli expression host strain by CaC12 method.
  • Example 2 Recombinant expression and preparation of recombinant human AN9FGF-21 in E. coli.
  • LA (Vg/ml Amp) agar plates were added to LB medium, and cultured overnight at 37 ° C. The lawns were picked from the overnight cultured LA plates and inoculated in LB-containing liquid culture.
  • Base tryptone 10g, yeast extract 5g, NaCl lOg, add water to lOOOmL, 121 °C, autoclave for 30min
  • lOOOmL 121 °C
  • autoclave for 30min a test tube, activate at 37 °C for 12 hours, then transfer to 1% In a 1000 mL flask containing 200 mL of LB medium, it was cultured overnight at 37 ° C to become the upper tank seed solution.
  • the upper tank seed solution was inoculated in a 5% ratio in a 30 L fermentor containing 20 L of M9+YT medium, 37 °. C culture, during the whole fermentation process, by adjusting the rotation speed, the amount of air, and the amount of pure oxygen to maintain dissolved oxygen>30%, adjust the pH with 28% ammonia water and keep at 7.0.
  • the fermentation broth was centrifuged at 8000 rpm for 10 min at 4 ° C in a frozen high speed centrifuge, and the cells were collected and stored at -20 ° C for 24 hours.
  • 10 ml of sterilizing buffer 50 mmol/LTris, 5 mmol/L EDTA, pH 10.3 per lg of bacteria, stir for 1 hour in a water bath at 30 ° C, and use ATS pressure homogenizer (Model: AH-BASIC ) 500Pa Homogenize twice and observe with a microscope. The cells are completely broken and flocculated. Then, it was centrifuged at 1000 °C for 10 min at 4 ° C, and the supernatant was collected.
  • solution B was 10 mmol/L JL (pH 8.0) and 0.5 M NaCl.
  • the target protein peak containing AN9FGF-21 (about 50% gradient) was collected.
  • C18 reverse phase chromatography The separation medium was WelchXB-C18, solution A was 5% acetonitrile and 0.1% TFA, and solution B was 75% acetonitrile (containing 0.1% TFA).
  • the elution peak of the Q column was adjusted to pH 2.0 to 3.0 with TFA, and eluted with a gradient of solution A to solution B.
  • AN9FGF-21 elution peak (approximately 35% gradient) was collected. After lyophilization, a sample of recombinant human ⁇ N9FGF-21 was formed.
  • the sample is purified by the above steps, that is, salting out, hydrophobic chromatography, ion exchange chromatography, reverse phase chromatography, and the purity is over 95%.
  • recombinant human AN9FGF-21 is soluble, and each 100 g of bacteria can be purified to prepare recombinant human ⁇ N9FGF-21 protein about 100 mg.
  • Example 3 Modification and purification of AN9FGF-21 by mPEG-ButylALD-20KD
  • the ⁇ N9FGF-21 solution was prepared from Example 2, mPEG-ButylALD-20KD (relative molecular mass 20x10 3 ), and sodium cyanoborohydride (CH3BNNa) solution as the activation of ⁇ - ⁇ 2 .
  • the AN9FGF-21 solution was dialyzed against 100 mM PB pH 5.0 and added to a final concentration of CH3BNNa of 20 mM. Take AN9FGF-21 and mPEG-ButylALD-20KD at a mass ratio of 1:2, stir at 100 r/min at 4 °C for 24 hr, and sample for SDS-PAGE electrophoresis (Fig. 3) to determine the modification ratio of PEG-AN9FGF21.
  • the modification rate reaches 75%.
  • the modified sample PEG-20-Z ⁇ N9FGF21 was diluted 5-10 times with DDW and then adjusted to pH 8.0. Further separation and purification using Source 15-Q. After equilibration with an equilibration solution (50 mM Tris-HCl pH 8.0), the sample was re-equilibrated. The elution peak of PEG-AN9FGF21 containing no unmodified AN9FGF-21 was collected by linear elution with 0-500 mM NaCl.
  • the recombinantly prepared AN9FGF-21 protein was automatically analyzed by N-terminal amino acid sequence (Edman degradation method), and the N-terminal 15 amino acid sequence was: Met-Leu-Gln-Phe-Gly-Gly-Gln-Val-Arg-Gln-Arg- Tyr-Leu-Tyr-Thr, for
  • the 15% SDS-PAGE reduction electrophoresis showed that the prepared recombinant human AN9FGF-21 was a single-electrophoresis band with a molecular weight of 19 000 Daltons.
  • the HPLC (CHPLC) C18 reverse phase column showed that the purity was greater than 98.0%.
  • 3T3-L1 preadipocytes (Shanghai Cell Biology Cell Bank) were cultured in high glucose DMEM containing 10% FBS at 37 ° C, 5% CO 2 , and added 0.5 after 2 days.
  • Glucose transport assay 3T3-L1 adipocytes in 24-well plates were simultaneously added with recombinant human AN9FGF-21, PEG-AN9FGF21 and recombinant human FGF-21 (rhFGF-21) (Beijing Ai Dibo Biotechnology Co., Ltd.) The medium (0.2% BSA high glucose DMEM) was incubated at 37 ° C for 24 hours at final concentrations of 0, 0.032, 0.16, 0.8, 4, 20 and ⁇ , respectively.
  • Glucose uptake assay using heated (37 ° C) KRP buffer (NaCl 131.2 mmol / Lol / L, KC1 4.7 mmol / Lol / L, MgS0 4 1.2 mmol / Lol / L, CaCl 2 2.5 mmol / Lol / L , NaH 2 P0 4 2.5 mmoVLol/L, pH 7.4) After washing twice; using KRP containing 1% BSA, 0.2 ⁇ ( ⁇ /hole 2-deoxy-[14C]-glucose (Beijing Institute of Atomic Energy) ( ⁇ ) The buffer was incubated for 1 hr, and lOumol/1 cytochalasin B was added to stop the glucose uptake.
  • KRP buffer NaCl 131.2 mmol / Lol / L, KC1 4.7 mmol / Lol / L, MgS0 4 1.2 mmol / Lol / L, CaCl 2 2.5 m
  • ⁇ /L cytochalasin B Another set of ⁇ /L cytochalasin B was used as the non-specific uptake rate of 2-deoxy-(14C)-glucose. All values were subtracted from this value as the glucose uptake rate (CPM, liquid scintillation counter count per minute) for each group of cells. Results showed that recombinant human AN9FGF-21 was more than human The activity of FGF-21 for extracting glucose from fat cells in vitro was about 45% higher under the same experimental conditions. The activity of PEG-AN9FGF21 to promote glucose extraction from fat cells in vitro under the same experimental conditions was about 70%.
  • Glucose utilization analysis After 3T3-L1 was induced into adipocytes (the method is the same as above), PEG-AN9FGF21 and human ⁇ N9FGF-21 samples (using low-sugar DMEM + 5% FCS) were added to prepare a final concentration of 4nM at 37 °C. The culture supernatant was taken at 12 hr, 24 hr and 48 hr in 5% C0 2 , and the glucose content in the supernatant was determined by a glucose assay kit and plotted (Fig. 3). As shown in the figure, recombinant human AN9FGF-21 and PEG-AN9FGF21 significantly promoted the utilization of glucose by 3T3-L1 cells.
  • Example 6 Pharmacokinetics of recombinant human AN9FGF-21 and PEG-AN9FGF21 in CD-I mice
  • mice Twenty-four CD-I mice were randomly divided into two groups (AN9FGF-21 group and PEG-AN9FGF21 group). Each group was injected subcutaneously at 0.4 mg/kg, and blood was taken at intervals of 0 to 144 hrs after the injection. The blood concentration of FGF-21 in the blood was measured using a FGF-21 ELISA Kit (Kang Peptide Bios) to calculate the half-life.
  • Example 7 Stability of recombinant human AN9FGF-21
  • the recombinant human AN9FGF-21 protein solution was replaced by 10 mmol/L Mol/L phosphate buffer (pH 7.0) and B 0.15 Mol/L sodium chloride by dialysis treatment to adjust the protein concentration (measured by Lowry method) to 1.0. Mg/ml. Sterile filtration was carried out in a 4 °C refrigerator and a 25 °C constant temperature chamber for stability testing. Samples were taken at different time points for in vitro activity, SDS-PAGE electrophoresis and reverse HPLC analysis.
  • Example 8 Recombinant human AN9FGF-21 expressed in Pichia pastoris
  • the cDNA sequence encoded by AN9FGF-21 was inserted into the pIC9K (AOX promoter) or pPICZ ci (GAP promoter) plasmid according to Invitrogen's instructions to construct a corresponding secretion-inducible (methanol-induced) or constitutive recombinant expression vector.
  • pIC9K AOX promoter
  • pPICZ ci GAP promoter
  • the Pichia pastoris containing the foreign gene of AN9FGF-21 is cultured to express the target protein, and recombinant AN9FGF-21 can be obtained by a common separation technique such as centrifugation, ultrafiltration, concentration, precipitation, column chromatography and the like.
  • the AN9FGF-21 protein (N-terminal no Met) can also be recombinantly expressed in the S. cerevisiae expression plasmid containing the GAL-1 gene promoter.
  • the obtained recombinant human AN9FGF-21 protein was analyzed by the method of Example 3 to prove that it also had the same biological characteristics.
  • Example 9 Hypoglycemia and hypolipidemic effects of AN9FGF-21 in a db/db mouse model
  • mice of 8 weeks old were randomly divided into three groups (AN9FGF-21 group, Met-AN9FGF-21 group and model control group), subcutaneously injected at 2 mg/kg daily for 10 days. .
  • the model control group was injected with normal saline in the same manner and volume, and the animals were routinely fed in SPF.
  • the animals were treated for blood and blood lipids (triglycerides and cholesterol).
  • the results showed that Met-AN9FGF-21 and AN9FGF-21 had the same hypoglycemic and blood lipid effects in the diabetic model animals as compared with the model group.

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Abstract

L'invention concerne un variant du facteur de croissance humain des fibroblastes 21 qui présente une délétion de 9 aminoacides N-terminal du facteur de croissance humain des fibroblastes 21 de type sauvage naturel. L'invention concerne également un conjugué du variant qui est obtenu par modification chimique du variant avec des dérivés de polyéthylène glycol. L'invention concerne aussi l'utilisation du variant et du conjugué dans la fabrication d'un médicament destiné au traitement ou à la prévention de l'obésité, du diabète, de l'hyperglycémie ou de l'hyperlipidémie.
PCT/CN2011/071948 2010-11-11 2011-03-18 Variant de délétion au niveau de n-terminal du facteur de croissance humain des fibroblastes 21 et son conjugué WO2012062078A1 (fr)

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US10517929B2 (en) 2014-10-23 2019-12-31 Ngm Biopharmaceuticals, Inc. Pharmaceutical compositions comprising FGF19 variants
US10695404B2 (en) 2015-10-30 2020-06-30 Salk Institute For Biological Studies Treatment of steroid-induced hyperglycemia with fibroblast growth factor (FGF) 1 analogs
US10744185B2 (en) 2015-11-09 2020-08-18 Ngm Biopharmaceuticals, Inc. Methods of using variants of FGF19 polypeptides for the treatment of pruritus
US10800843B2 (en) 2015-07-29 2020-10-13 Ngm Biopharmaceuticals, Inc. Beta klotho-binding proteins
US11370841B2 (en) 2016-08-26 2022-06-28 Ngm Biopharmaceuticals, Inc. Methods of treating fibroblast growth factor 19-mediated cancers and tumors
US11542309B2 (en) 2019-07-31 2023-01-03 Salk Institute For Biological Studies Fibroblast growth factor 1 (FGF1) mutant proteins that selectively activate FGFR1B to reduce blood glucose

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106220724B (zh) * 2016-09-13 2019-10-11 河南师范大学 人成纤维细胞生长因子21重组蛋白及其制备方法和应用
CN106432509B (zh) * 2016-09-13 2019-05-21 河南师范大学 一种治疗代谢疾病的重组人成纤维细胞生长因子21融合蛋白及其制备方法和应用
CN106397607A (zh) * 2016-09-13 2017-02-15 河南师范大学 重组人成纤维细胞生长因子21融合蛋白及其在制备治疗代谢疾病药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009149171A2 (fr) * 2008-06-04 2009-12-10 Amgen Inc. Mutants fgf21 et leurs utilisations
CN101663046A (zh) * 2007-03-30 2010-03-03 Ambrx公司 经修饰fgf-21多肽和其用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1789443A1 (fr) * 2004-09-02 2007-05-30 Eli Lilly And Company Muteines du facteur de croissance 21 du fibroblaste

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101663046A (zh) * 2007-03-30 2010-03-03 Ambrx公司 经修饰fgf-21多肽和其用途
WO2009149171A2 (fr) * 2008-06-04 2009-12-10 Amgen Inc. Mutants fgf21 et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MICANOVIC, R. ET AL.: "Different Roles of N- and C-Termini in the Functional Activity of FGF21.", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 219, no. 2, 30 December 2008 (2008-12-30), pages 227 - 234 *
YIE, J. ET AL.: "FGF21 N- and C-termini play different roles in receptor interaction and activation.", FEBS LETTERS., vol. 583, no. 1, 5 January 2009 (2009-01-05), pages 19 - 24 *

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