WO2023280144A1 - Fusion protein and medical use thereof - Google Patents

Fusion protein and medical use thereof Download PDF

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WO2023280144A1
WO2023280144A1 PCT/CN2022/103825 CN2022103825W WO2023280144A1 WO 2023280144 A1 WO2023280144 A1 WO 2023280144A1 CN 2022103825 W CN2022103825 W CN 2022103825W WO 2023280144 A1 WO2023280144 A1 WO 2023280144A1
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fusion protein
seq
fgf21
protein
protein according
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PCT/CN2022/103825
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French (fr)
Chinese (zh)
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毛东杰
谢岳峻
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上海翰森生物医药科技有限公司
常州恒邦药业有限公司
江苏豪森药业集团有限公司
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Priority to CN202280046816.0A priority Critical patent/CN117677640A/en
Publication of WO2023280144A1 publication Critical patent/WO2023280144A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a fusion protein of FGF21 and its medical application.
  • Fibroblast growth factor 21 (Fibroblast growth factor 21, FGF21) is a polypeptide (SEQ ID NO: 1) consisting of 209 amino acids, and its amino acid sequence has about 75% homology with mouse FGF21.
  • the N-terminus of FGF21 contains a signal peptide consisting of 28 amino acids, so mature FGF21 consists of 181 amino acids (SEQ ID NO: 3).
  • Mature FGF21 is the isoform or allelic form of native human FGF21 having Leu substituted for Pro at position 146 of SEQ ID NO: 3 herein. FGF21 is mainly expressed in the liver and pancreas, but also in fat and muscle tissue.
  • FGF21 can induce various signaling pathways and functional activities in the liver, pancreas and adipose tissue, thereby realizing the regulation of glucose and lipid metabolism and the protection of pancreatic ⁇ cells physiological function.
  • FGF21 regulates glucose uptake by adipocytes by activating insulin-independent glucose uptake.
  • studies have also shown that FGF21 can reduce body weight and body fat in a dose-dependent manner. For example, administering FGF21 to diabetic rhesus monkeys found that the levels of fasting plasma glucose, triglycerides, and glucagon were significantly reduced.
  • FGF21 may regulate lipid metabolism by promoting lipolysis and ketogenic effect.
  • FGF21 can inhibit glucose-mediated release of glucagon, stimulate insulin production, and prevent islet cell apoptosis, thereby improving pancreatic cell function.
  • FGF21 also activates signaling pathways in exocrine pancreatic cells and hepatocytes and inhibits hepatic glycogen export.
  • FGF21 is a member of the FGF gene family, and most FGFs have broad-spectrum mitogenic abilities. However, the results of the investigation show that FGF21 neither has the ability to promote cell proliferation nor antagonizes the functions of other members of the FGF family.
  • FGF21 neither has the ability to promote cell proliferation nor antagonizes the functions of other members of the FGF family.
  • Experiments have shown that in the whole life cycle of FGF21 transgenic mice (the amount of FGF21 in the body is about 150 times that of normal mice), no abnormalities such as tumors and tissue hyperplasia were found in the body. At the same time, its metabolic regulating effect is related to the metabolic level of the body, and the regulating effect only plays a role when the metabolism is abnormal. Even if the FGF21 exceeds the pharmacological dose, hypoglycemia will not occur.
  • the medicine (insulin, thiazolidinedione, etc.) of the main treatment diabetes on the market is easy to produce side effects when dosage is inappropriate, as the hypoglycemia that large doses of insulin cause, liver function damage and edema that thiazolidinedione causes, these None of them were found in animal experiments treated with FGF21, which is sufficient to prove that FGF21 is an ideal drug for treating diseases such as diabetes and obesity.
  • FGF21 The physiological function of FGF21 in controlling blood sugar and reducing body weight has brought hope for the treatment of related diseases.
  • wild-type FGF21 is easily hydrolyzed by proteases and can also be filtered by glomeruli, with a half-life of only 0.5-2h. It is difficult to guarantee the effective drug action time.
  • the pharmaceutical industry has improved the half-life of FGF21 by performing site-directed mutagenesis of amino acids at enzyme cleavage sites, preparing long-acting fusion proteins, or linking polyethylene glycol to the polypeptide backbone.
  • a major challenge in the development of FGF21 as a protein formulation also comes from the instability caused by its own aggregation.
  • the ideal effect of the target therapeutic protein is to increase the resistance to proteolysis and reduce the aggregation of the protein, thereby enhancing the half-life and stability of the FGF21 protein preparation, and realizing low-frequency administration to patients.
  • the present invention provides a kind of FGF21 protein or its variant, its general formula is as follows:
  • X 166 is selected from L or F;
  • X 175 is selected from R or W.
  • a preferred embodiment of the present invention is FGF21 protein described in general formula (II) or variant thereof, its sequence is as shown in SEQ ID NO:5 or SEQ ID NO:6.
  • the present invention also provides a fusion protein whose general formula is as follows:
  • F1 is selected from the FGF21 protein or variants thereof as described in general formula (II) ;
  • F 2 is connecting peptide
  • F3 is a domain composed of Fc or fragments of immunoglobulin.
  • F 1 is a sequence as shown in SEQ ID NO:5 or SEQ ID NO:6.
  • F 1 is a sequence as shown in SEQ ID NO:5.
  • F 2 is a sequence as shown in SEQ ID NO:7.
  • said F3 is represented by general formula ( III ):
  • F4 is the Fc fragment of immunoglobulin
  • n is an integer selected from 1-10;
  • n is an integer selected from 1-10.
  • F4 is SEQ ID NO: 8;
  • n 1;
  • n 9.
  • said F3 is SEQ ID NO: 9 .
  • the fusion protein is selected from SEQ ID NO: 12 or SEQ ID NO: 13.
  • the present invention also relates to a polynucleotide encoding the fusion protein described in general formula (I).
  • the coding of said nucleotide comprises FGF21 protein SEQ ID NO:5.
  • the coding of said nucleotides comprises FGF21 fusion protein SEQ ID NO: 12-13.
  • the present invention also relates to an expression vector comprising the polynucleotide as described above.
  • the present invention also relates to a host cell, which introduces or contains the above-mentioned expression vector, wherein the host cell is bacteria, preferably Escherichia coli; or the host cell is yeast, preferably Pichia Yeast; or the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
  • the host cell is bacteria, preferably Escherichia coli; or the host cell is yeast, preferably Pichia Yeast; or the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
  • the present invention also relates to a method for producing the fusion protein as described above, comprising the steps of:
  • the present invention further includes a pharmaceutical composition, which contains the fusion protein described in general formula (I) and pharmaceutically acceptable excipients, diluents or carriers.
  • the present invention also relates to the use of the fusion protein or the above-mentioned pharmaceutical composition in the preparation of medicines for the treatment or prevention of diabetes, obesity, dyslipidemia, metabolic syndrome, and non-alcoholic fatty liver Or related diseases such as non-alcoholic steatohepatitis.
  • the fusion protein provided by the present invention has the effect of significantly promoting fibroblast proliferation, good plasma stability, and can induce glucose uptake and promote phosphorylation of ERK1/2 protein. These characteristics are beneficial to the preparation and formulation of therapeutic proteins, and It has potential therapeutic effects on related diseases such as diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver or non-alcoholic steatohepatitis.
  • the amino acid position changes in the FGF21 mutants of the present invention are determined from the amino acid positions in the mature human wild-type FGF21 (SEQ ID NO: 3) polypeptide.
  • amino acid sequences of the present invention contain the standard one-letter or three-letter codes for the twenty amino acids.
  • FGF21 polypeptide refers to the naturally occurring wild-type polypeptide expressed in humans. Including SEQ ID NO: 1 consisting of the full-length form encoded by SEQ ID NO: 2 and SEQ ID NO: 3 consisting of the mature form encoded by SEQ ID NO: 4.
  • FGF21 mutant refers to a modified FGF21 polypeptide based on the amino acid sequence of naturally occurring FGF21 (SEQ ID NO: 4). Such modifications include, but are not limited to, one or more amino acid substitutions, including, but not limited to, protease-resistant FGF21 mutants, reduced aggregation FGF21 mutants, and FGF21 combination mutants described herein.
  • patient is a mammal, preferably a human.
  • treating refers to slowing, reducing or reversing the progression or severity of a symptom, disorder or disease.
  • Fc fragment refers to the constant region of the heavy chain of an immunoglobulin.
  • vector refers to any molecule (eg, nucleic acid, plasmid, or virus) used to deliver encoded information to a host cell.
  • expression vector refers to a vector suitable for transformation of host cells and containing nucleic acid sequences that direct and/or control the expression of an inserted heterologous nucleic acid sequence. Including but not limited to processes such as transcription, translation and RNA splicing.
  • host cell is used to refer to a cell transformed or capable of being transformed by a nucleic acid sequence and then capable of expressing a selected gene of interest.
  • the term includes the progeny of a parental cell, whether or not the progeny is identical in morphology or genetic composition to the original parent, predominantly in the presence of selected genes.
  • the FGF21 mutant protein (SEQ ID NO:5-6) was expressed using the ExpiCHO system (Thermo Fisher #A29133).
  • the DNA sequence encoding the FGF21 mutant protein with a His tag at the C-terminal was cloned into the pCDNA3.1 vector, and after sequencing, it was confirmed that a plasmid expressing the fusion protein was obtained.
  • the method of preparing EQ buffer solution is to take a bag of PBS phosphate buffer solution powder, dissolve the powder in 2000ml ultrapure water, and filter it with a 0.22 ⁇ m filter membrane for later use.
  • the method to configure Elution buffer (500mM imidazole, pH7.4) is to weigh 34g of imidazole and add 450ml of EQ buffer, adjust the pH to 7.4, dilute to 500ml, and filter through a 0.22 ⁇ m filter membrane for later use.
  • the harvested supernatant was purified by AKTA Pure instrument. First, equilibrate the instrument with EQ buffer until the pH and conductivity of the effluent are consistent with the EQ buffer; then collect samples with Elution buffer.
  • the fusion protein SEQ ID NO: 10-16 was expressed by the same method, purified using Protein A affinity chromatography column, and the conditioned medium containing the expressed receptor was washed sequentially. The eluate was dialyzed against 10 mM tris-buffered saline, pH 7.2.
  • the concentration and purity of the prepared protein were determined by UV spectrophotometry.
  • 3T3-L1 mouse embryonic fibroblasts express glucose transporter 1 (GLUT1) on the surface of mature adipocytes, and FGF21 protein regulates the level of glucose uptake by adipocytes by regulating the expression level of GLUT1.
  • GLUT1 glucose transporter 1
  • Aspirate the original medium and add the induction medium that is, add 2 ⁇ g/ml human insulin (Sinobiologics , cat#:11038-HNAY) solution, 1 ⁇ M dexamethasone (Sigma, cat#:D4902-25MG) and 0.5mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma, cat#:I7018- 100MG), induced and cultured 3T3-L1 cells for 3 days, observed the number and size of fat granules in the cells under a microscope, and differentiated them into adipocytes, and then replaced the differentiation medium with complete medium containing only 2 ⁇ g/ml human insulin.
  • IBMX 3-isobutyl-1-methylxanthine
  • the adipocytes induced to differentiate and mature were digested to prepare a single-cell suspension, and the cell density was adjusted to 1x10 6 /mL with DMEM complete basal medium, 100uL/well, and seeded in a 96-well plate (Corning, cat#:3610). Adhere to the wall, add the mutant protein to be tested (SEQ ID NO:5) diluted with DMEM basal medium in the experimental group, so that the final concentration is 5000nM, 100uL/well is added to the plate, and the same volume of DMEM basal medium is added to the control group , and incubated overnight at 37°C, 5% carbon dioxide.
  • glucose uptake rate% (experimental group MFI-control group MFI)/control Group MFI*100%.
  • the experimental results showed that the FGF21 mutant (SEQ ID NO:6) significantly induced the uptake of the glucose analog 2-NBDG by adipocytes, and the induction efficiency was good.
  • FGF21 protein regulates the phosphorylation level of ERK1/2 protein through the Ras/Raf/MAPK signaling pathway to transduce cell signals and participate in energy metabolism in vivo. Evaluation Comparing FGF21 protein mutants to regulate the phosphorylation level of ERK1/2 protein can evaluate the level of cell signal transduction.
  • Seed HuH-7 (Chinese Academy of Sciences, cat#: SCSP-526) at 1 ⁇ 10 5 /mL in a 96-well plate, 100uL/well, incubate overnight at 37°C, 5% carbon dioxide, and the next day, the cells were placed in DMEM (gibco, cat #:11995-065) basal medium for 2 hours, add DMEM basal medium to the experimental group to dilute the protein to be tested, adjust the final concentration of the mutant protein to be tested (SEQ ID NO:5) to 50nM, add 100uL/well In the plate, add the same volume of DMEM basal medium to the control group, incubate at 37°C for 20 minutes, add fixative solution (BD Cytofix, cat#: 554655) and fix at 37°C for 30 minutes, centrifuge at 400g, 4°C for 5 minutes, wash twice all over.
  • DMEM gibco, cat #:11995-065
  • SEQ ID NO:5 final concentration of the mutant protein to
  • FGF21 mutants SEQ ID NO:5 and SEQ ID NO:6
  • SEQ ID NO:5 and SEQ ID NO:6 can up-regulate the phosphorylation level of ERK1/2 protein in HuH-7 cells, and the effect is good.
  • the FGF21 fusion protein is composed of functional FGF21 and Fc fragments, in which Fc binds to the extracellular domain of Fc ⁇ R through non-covalent bonding, activates the immunoreceptor tyrosine activation motif, and mediates immunity induced by ADCC (cytotoxicity) The activation effect is not good for disease treatment.
  • the combination of Fc and FcRn is beneficial for the fusion protein to have a longer circulation period in vivo.
  • fusion protein mutant 12 to Fc ⁇ R (Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIIa) is weak, and the risk of side effects caused by immune effects is small; the binding to FcRn is strong, which is conducive to its long-term circulation in the blood.
  • the human FcRn transgenic mouse model was used to evaluate the drug metabolism of FGF21 fusion protein mutants in mice.
  • Human FcRn transgenic mice with an average body weight of 18-22g and 18-22 weeks of age were randomly divided into groups, with 3 animals in each group, and the tested FGF21 fusion protein mutant 12 was administered at 4 mpk, s.c., in a single dose, PBS
  • the vehicle was used as a negative control group
  • blood was collected at 0.5, 2, 4, 6, 8, 24, 48, 72, 96, and 120 hours to separate plasma, frozen in a -20°C refrigerator, and then mutated using human KLB protein-FGF21 fusion protein
  • Body-Fc-HRP indirect ELISA method was used to detect the concentration of FGF21 fusion protein mutants in mouse plasma, and the PK solver software was used to analyze the PK parameters of the non-compartmental model and the formula of intravascular administration.
  • the experimental results are shown in the following table:
  • FGF21 fusion protein mutant 12 showed good metabolic activity in vivo.
  • the obesity model of C57BL/6 mice induced by high-fat diet was used to evaluate the effect of FGF21 fusion protein mutants on reducing body weight and liver weight.
  • the 12-week-old DIO male obesity model mice with a body weight of 30-50g were randomly divided into groups, with 8 animals in each group, and the tested FGF21 fusion protein mutants 12 and 13 were administered at 2mpk, s.c., q3d, in PBS vehicle As a negative control group, a total of 3 doses were administered. On the 0th day, the body weight was weighed and the drug was administered, and the body weight was measured every 3 days thereafter, and the food intake was accumulated. On the 9th day, we weighed and fasted overnight. On the 10th day, weighed and collected blood, took the liver, and weighed the liver.
  • test coefficient is p ⁇ 0.05, there is a statistical difference, using one-way ANOVA multi-group comparative analysis,
  • leptin-knockout mouse model of diabetes namely ob/ob model mice
  • FGF21 fusion protein mutant 12 in reducing body weight, blood glucose level, liver-to-weight ratio, and blood lipids: total cholesterol (TC), triglycerides Ester (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels.
  • TC total cholesterol
  • TG triglycerides Ester
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • 12-week-old ob/ob male obesity model mice with a body weight of 30-50g were randomly divided into groups, with 6 animals in each group, and the test FGF21 fusion protein mutant 12 was administered at 2mpk, s.c., q3d, PBS vehicle As a negative control group, a total of 3 doses were administered.
  • the body weight was weighed and the drug was administered, and the body weight was measured every 3 days thereafter, and the food intake was accumulated. Weighed on the 8th day and fasted overnight.
  • On the 9th day weighed and collected blood to detect blood sugar level and four items of blood sugar, separated the liver, weighed the liver and body weight respectively, and calculated the liver-to-body weight ratio.
  • the experimental results show that, compared with the candidate molecules of the vehicle group, they have obvious weight-lowering effects and blood-sugar-lowering effects.
  • CD57BL/6 mice were purchased from Nanjing Jicui Yaokang, and after a week of acclimatization in our laboratory, the NASH model was induced by diet. HFD was induced using a choline-deficient high-fat diet, ie, CDA.
  • the mice in the normal healthy group were fed with conventional feed, and the mice in the vehicle (pH7.4 PB solution) control group and the experimental group were fed with CDA/HFD feed containing 60% high fat and 2% cholesterol, and fed with 10% fructose in the drinking water , induced for 9 weeks. Subsequently, the experimental mice were randomly divided into 4 groups, with 5 animals in each group, and the administration started from the third week (ie, administration after 2 weeks of induction, and administration for 7 weeks).
  • the test drug was administered subcutaneously at q3d frequency at 5 mg/kg for 7 consecutive weeks. After reaching the end point of the experiment, the body weight was weighed, and the animal was euthanized. The liver was collected and weighed, and the liver was stored in 4% neutral formaldehyde. Tissue slices were used to detect indicators such as fat deposition, stem cell balloon-like lesions, and liver cell inflammation levels to evaluate the impact of the test drug on the development of NASH.
  • the experimental results show that compared with the healthy group, the tested drug can significantly reduce body weight by 24.92%, and at the same time significantly reduce the level of liver-to-body weight ratio, which is equivalent to the level of the healthy group; compared with the vehicle group, the candidate molecules can improve NASH fiber There is a statistically significant difference in levels.

Abstract

Provided are a fusion protein consisting of an FGF21 protein and immunoglobulin Fc or fragments thereof, and a method for treating diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver, non-alcoholic steatohepatitis, or other related diseases by using the FGF21-Fc fusion protein and pharmaceutical compositions thereof.

Description

一种融合蛋白及其医药用途A fusion protein and its medicinal use 技术领域technical field
本发明属于生物医药领域，具体涉及FGF21的融合蛋白及其医药用途。The invention belongs to the field of biomedicine, and in particular relates to a fusion protein of FGF21 and its medical application.
背景技术Background technique
成纤维细胞生长因21(Fibroblast growth factor 21,FGF21)是一种由209个氨基酸组成的多肽(SEQ ID NO:1)，其氨基酸序列与小鼠的FGF21具有约75％的同源性。FGF21N末端含有28个氨基酸构成的信号肽，因而成熟的FGF21由181个氨基酸组成(SEQ ID NO:3)。成熟的FGF21在本文SEQ ID NO:3的第146位上具有Leu替代Pro的天然人FGF21同等型(isoform)或等位形式(allelic form)。FGF21主要在肝脏和胰腺中表达，同时也存在于脂肪和肌肉组织中。通过FGFR的介导和跨膜蛋白βKlotho(KLB)的辅助，FGF21能诱导肝脏、胰腺和脂肪组织中的多种信号通路和功能活动，进而实现对糖脂代谢的调节和对胰岛β细胞进行保护的生理功能。FGF21通过激活非胰岛素依赖的葡萄糖吸收，调节脂肪细胞对葡萄糖的摄取。另外，研究还表明，FGF21可剂量依赖性的减少体重和全身体脂，如向患糖尿病的恒河猴施用FGF21，发现其空腹血浆葡萄糖、甘油三酯和胰高血糖素水平均有显著减少。同时，在FGF21转基因小鼠身上发现，其白色脂肪组织脂肪酶表达增加，血浆β-羟丁酸和游离脂肪酸水平升高。这意味着FGF21可能是通过促进脂肪分解以及生酮作用调控脂质代谢。在胰岛细胞和INS-1E细胞中，FGF21能够抑制葡萄糖介导的胰高血糖素的释放，刺激胰岛素的产生，并防止胰岛细胞凋亡，进而改善胰腺细胞功能。此外，FGF21还能激活外分泌胰腺细胞和肝细胞信号通路，抑制肝糖原输出。Fibroblast growth factor 21 (Fibroblast growth factor 21, FGF21) is a polypeptide (SEQ ID NO: 1) consisting of 209 amino acids, and its amino acid sequence has about 75% homology with mouse FGF21. The N-terminus of FGF21 contains a signal peptide consisting of 28 amino acids, so mature FGF21 consists of 181 amino acids (SEQ ID NO: 3). Mature FGF21 is the isoform or allelic form of native human FGF21 having Leu substituted for Pro at position 146 of SEQ ID NO: 3 herein. FGF21 is mainly expressed in the liver and pancreas, but also in fat and muscle tissue. Through the mediation of FGFR and the assistance of the transmembrane protein βKlotho (KLB), FGF21 can induce various signaling pathways and functional activities in the liver, pancreas and adipose tissue, thereby realizing the regulation of glucose and lipid metabolism and the protection of pancreatic β cells physiological function. FGF21 regulates glucose uptake by adipocytes by activating insulin-independent glucose uptake. In addition, studies have also shown that FGF21 can reduce body weight and body fat in a dose-dependent manner. For example, administering FGF21 to diabetic rhesus monkeys found that the levels of fasting plasma glucose, triglycerides, and glucagon were significantly reduced. Meanwhile, increased expression of lipase in white adipose tissue and increased levels of plasma β-hydroxybutyric acid and free fatty acids were found in FGF21 transgenic mice. This means that FGF21 may regulate lipid metabolism by promoting lipolysis and ketogenic effect. In islet cells and INS-1E cells, FGF21 can inhibit glucose-mediated release of glucagon, stimulate insulin production, and prevent islet cell apoptosis, thereby improving pancreatic cell function. In addition, FGF21 also activates signaling pathways in exocrine pancreatic cells and hepatocytes and inhibits hepatic glycogen export.
FGF21作为FGF基因家族的成员，大多数FGF具有广谱的促有丝***的能力，而探究结果表明，FGF21既没有促进细胞增殖的能力，也不会拮抗FGF家族其他成员的功能。实验证明，FGF21转基因鼠(体内FGF21量是正常鼠的150倍左右)在整个生命周期内，体内未发现肿瘤以及组织增生等异常状况。同时，其代谢调节作用与机体的代谢水平相关，调节作用仅在代谢异常时发挥作用，即使超过药理学剂量的FGF21也不会出现低血糖症。而目前市场上主要的治疗糖尿病的药物(胰岛素，噻唑烷二酮等)在剂量不当时易产生副作用，如大剂量的胰岛素导致的低血糖，噻唑烷二酮导致的肝功能损坏和水肿，这些在接受FGF21治疗的动物实验中均没有发现，这也足以证明FGF21是一种理想的治疗糖尿病以及肥胖等疾病的药物。FGF21 is a member of the FGF gene family, and most FGFs have broad-spectrum mitogenic abilities. However, the results of the investigation show that FGF21 neither has the ability to promote cell proliferation nor antagonizes the functions of other members of the FGF family. Experiments have shown that in the whole life cycle of FGF21 transgenic mice (the amount of FGF21 in the body is about 150 times that of normal mice), no abnormalities such as tumors and tissue hyperplasia were found in the body. At the same time, its metabolic regulating effect is related to the metabolic level of the body, and the regulating effect only plays a role when the metabolism is abnormal. Even if the FGF21 exceeds the pharmacological dose, hypoglycemia will not occur. And the medicine (insulin, thiazolidinedione, etc.) of the main treatment diabetes on the market is easy to produce side effects when dosage is inappropriate, as the hypoglycemia that large doses of insulin cause, liver function damage and edema that thiazolidinedione causes, these None of them were found in animal experiments treated with FGF21, which is sufficient to prove that FGF21 is an ideal drug for treating diseases such as diabetes and obesity.
FGF21在控制血糖和降低体重等方面的生理功能为治疗相关疾病带来了希望，但是野生型FGF21作为小分子蛋白易被蛋白酶水解，也可通过肾小球滤过，半衰期仅为0.5-2h，难以保证有效药物作用时间。面对这种困难，医药工业界通 过进行酶切位点氨基酸定点突变，制备长效融合蛋白或将聚乙二醇连接到多肽骨架等方法来提高FGF21的半衰期。此外，在研发FGF21作为蛋白质制剂中的重大挑战还来自其自身聚集造成的不稳定性。目标治疗蛋白的理想效果是增加对蛋白水解的耐受性以及降低蛋白的聚集性，进而增强FGF21蛋白制剂的半衰期和稳定性，实现对患者的低频给药。在WO2009/149171和WO2017/074117中已经描述了一些基于人野生型FGF21多肽序列的突变体，本领域亟需适合药用的FGF21多肽序列的突变体。The physiological function of FGF21 in controlling blood sugar and reducing body weight has brought hope for the treatment of related diseases. However, as a small molecular protein, wild-type FGF21 is easily hydrolyzed by proteases and can also be filtered by glomeruli, with a half-life of only 0.5-2h. It is difficult to guarantee the effective drug action time. Faced with this difficulty, the pharmaceutical industry has improved the half-life of FGF21 by performing site-directed mutagenesis of amino acids at enzyme cleavage sites, preparing long-acting fusion proteins, or linking polyethylene glycol to the polypeptide backbone. In addition, a major challenge in the development of FGF21 as a protein formulation also comes from the instability caused by its own aggregation. The ideal effect of the target therapeutic protein is to increase the resistance to proteolysis and reduce the aggregation of the protein, thereby enhancing the half-life and stability of the FGF21 protein preparation, and realizing low-frequency administration to patients. Some mutants based on the human wild-type FGF21 polypeptide sequence have been described in WO2009/149171 and WO2017/074117, and there is an urgent need in the art for mutants of the FGF21 polypeptide sequence suitable for pharmaceutical use.
发明内容Contents of the invention
本发明提供了一种FGF21蛋白或其变体，其通式如下：The present invention provides a kind of FGF21 protein or its variant, its general formula is as follows:
Figure PCTCN2022103825-appb-000001
Figure PCTCN2022103825-appb-000001
其中：in:
X 166选自L或F； X 166 is selected from L or F;
X 175选自R或W。 X 175 is selected from R or W.
本发明的一个优选的实施方案是通式(II)所述的FGF21蛋白或其变体，其序列如SEQ ID NO:5或SEQ ID NO:6所示。A preferred embodiment of the present invention is FGF21 protein described in general formula (II) or variant thereof, its sequence is as shown in SEQ ID NO:5 or SEQ ID NO:6.
另一方面，本发明还提供一种融合蛋白，其通式如下：On the other hand, the present invention also provides a fusion protein whose general formula is as follows:
F 1-F 2-F 3 F 1 -F 2 -F 3
(I)(I)
其中：in:
F 1选自如通式(II)所述的FGF21蛋白或其变体； F1 is selected from the FGF21 protein or variants thereof as described in general formula (II) ;
F 2为连接肽； F 2 is connecting peptide;
F 3为免疫球蛋白的Fc或其片段组成的结构域。 F3 is a domain composed of Fc or fragments of immunoglobulin.
本发明的一个优选的实施方案中，F 1为序列如SEQ ID NO:5或SEQ ID NO:6所示。 In a preferred embodiment of the present invention, F 1 is a sequence as shown in SEQ ID NO:5 or SEQ ID NO:6.
本发明的一个优选的实施方案中，F 1为序列如SEQ ID NO:5所示。 In a preferred embodiment of the present invention, F 1 is a sequence as shown in SEQ ID NO:5.
本发明的一个优选的实施方案中，F 2为序列如SEQ ID NO:7所示。 In a preferred embodiment of the present invention, F 2 is a sequence as shown in SEQ ID NO:7.
在本发明的一个优选实施方案中，所述F 3如通式(III)所示： In a preferred embodiment of the present invention, said F3 is represented by general formula ( III ):
F 4(GGGGGS) m(GGGGS) nF 4 F 4 (GGGGGS) m (GGGGS) n F 4
(III)(III)
其中：in:
F 4为免疫球蛋白的Fc片段； F4 is the Fc fragment of immunoglobulin;
m选自1-10的整数；且m is an integer selected from 1-10; and
n选自1-10的整数。n is an integer selected from 1-10.
在本发明一个优选的实施方案中，对于通式(III)，其中：In a preferred embodiment of the present invention, for general formula (III), wherein:
F 4为SEQ ID NO：8； F4 is SEQ ID NO: 8;
m为1；且m is 1; and
n为9。n is 9.
在本发明一个优选的实施方案中，所述F 3为SEQ ID NO：9。 In a preferred embodiment of the present invention, said F3 is SEQ ID NO: 9 .
在本发明一个的优选方案中，所述融合蛋白选自SEQ ID NO：12或SEQ ID NO：13。In a preferred embodiment of the present invention, the fusion protein is selected from SEQ ID NO: 12 or SEQ ID NO: 13.
本发明还涉及一种多核苷酸，其编码包含通式(I)所述的融合蛋白。The present invention also relates to a polynucleotide encoding the fusion protein described in general formula (I).
在本发明的优选方案中，所述核苷酸的编码包含FGF21蛋白SEQ ID NO：5。In a preferred embodiment of the present invention, the coding of said nucleotide comprises FGF21 protein SEQ ID NO:5.
在本发明的优选方案中，所述核苷酸的编码包含FGF21融合蛋白SEQ ID NO：12-13。In a preferred embodiment of the present invention, the coding of said nucleotides comprises FGF21 fusion protein SEQ ID NO: 12-13.
本发明还涉及一种表达载体，其含有如上所述的多核苷酸。The present invention also relates to an expression vector comprising the polynucleotide as described above.
另外，本发明还涉及一种宿主细胞，其导入或含有如上所述的表达载体，其中所述的宿主细胞为细菌，优选为大肠杆菌；或者所述的宿主细胞为酵母菌，优选为毕赤酵母；或者所述的宿主细胞为哺乳动物细胞，优选为CHO细胞或HEK293细胞。In addition, the present invention also relates to a host cell, which introduces or contains the above-mentioned expression vector, wherein the host cell is bacteria, preferably Escherichia coli; or the host cell is yeast, preferably Pichia Yeast; or the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
本发明还涉及一种生产如上所述融合蛋白的方法，包括步骤：The present invention also relates to a method for producing the fusion protein as described above, comprising the steps of:
1)培养如上所述的宿主细胞；1) cultivating the host cell as described above;
2)从培养物中分离蛋白；2) Isolate the protein from the culture;
3)以及对所述的蛋白进行纯化。3) and purifying the protein.
本发明进一步包含一种药物组合物，其含有通式(I)所述的融合蛋白以及可药用的赋形剂、稀释剂或载体。The present invention further includes a pharmaceutical composition, which contains the fusion protein described in general formula (I) and pharmaceutically acceptable excipients, diluents or carriers.
本发明还涉及所述的融合蛋白，或如上所述的药物组合物在制备药物中的用途，所述药物用于治疗或预防治疗糖尿病、肥胖、血脂异常、代谢综合征、非酒精性脂肪肝或非酒精性脂肪肝炎等相关疾病。The present invention also relates to the use of the fusion protein or the above-mentioned pharmaceutical composition in the preparation of medicines for the treatment or prevention of diabetes, obesity, dyslipidemia, metabolic syndrome, and non-alcoholic fatty liver Or related diseases such as non-alcoholic steatohepatitis.
本发明提供的融合蛋白，具有显著促成纤维细胞增殖的作用，和良好的血浆稳定性，能够诱导葡萄糖摄取、促进ERK1/2蛋白的磷酸化，这些特征对于治疗性蛋白的制备和配制有利，并具有对糖尿病、肥胖、血脂异常、代谢综合征、非酒精性脂肪肝或非酒精性脂肪肝炎等相关疾病的潜在治疗效果。The fusion protein provided by the present invention has the effect of significantly promoting fibroblast proliferation, good plasma stability, and can induce glucose uptake and promote phosphorylation of ERK1/2 protein. These characteristics are beneficial to the preparation and formulation of therapeutic proteins, and It has potential therapeutic effects on related diseases such as diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver or non-alcoholic steatohepatitis.
发明的详细说明Detailed Description of the Invention
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。Unless stated to the contrary, the terms used in the specification and claims have the following meanings.
本发明的FGF21突变体中氨基酸的位置改变从成熟的人野生型FGF21(SEQ  ID NO:3)多肽中的氨基酸位置决定。The amino acid position changes in the FGF21 mutants of the present invention are determined from the amino acid positions in the mature human wild-type FGF21 (SEQ ID NO: 3) polypeptide.
本发明的氨基酸序列含有二十种氨基酸的标准单字母或三字母代码。The amino acid sequences of the present invention contain the standard one-letter or three-letter codes for the twenty amino acids.
术语“FGF21多肽”是指人体内表达的天然存在的野生型多肽。包括SEQ ID NO:1组成由SEQ ID NO:2编码的全长形式和SEQ ID NO:3组成由SEQ ID NO:4编码的成熟形式。The term "FGF21 polypeptide" refers to the naturally occurring wild-type polypeptide expressed in humans. Including SEQ ID NO: 1 consisting of the full-length form encoded by SEQ ID NO: 2 and SEQ ID NO: 3 consisting of the mature form encoded by SEQ ID NO: 4.
术语“FGF21突变体”是指基于天然存在的FGF21氨基酸(SEQ ID NO:4)序列而修饰的FGF21多肽。这类修饰包括但不仅限于一个或多个氨基酸被取代，包括且不仅限于本文所述的蛋白酶抗性FGF21突变体、聚集减少的FGF21突变体以及FGF21组合突变体。The term "FGF21 mutant" refers to a modified FGF21 polypeptide based on the amino acid sequence of naturally occurring FGF21 (SEQ ID NO: 4). Such modifications include, but are not limited to, one or more amino acid substitutions, including, but not limited to, protease-resistant FGF21 mutants, reduced aggregation FGF21 mutants, and FGF21 combination mutants described herein.
术语“患者”是哺乳动物，优选人。The term "patient" is a mammal, preferably a human.
术语“治疗”指减缓、降低或逆转症状、病症或疾病的进展或严重性。The term "treating" refers to slowing, reducing or reversing the progression or severity of a symptom, disorder or disease.
术语“Fc片段”指免疫球蛋白的重链的恒定区。The term "Fc fragment" refers to the constant region of the heavy chain of an immunoglobulin.
术语“载体”指用于向宿主细胞传递编码信息的任何分子(例如核酸、质粒或病毒)。The term "vector" refers to any molecule (eg, nucleic acid, plasmid, or virus) used to deliver encoded information to a host cell.
术语“表达载体”指适于宿主细胞转化并含有指导和/或控制***异源核酸序列表达的核酸序列的载体。包含但不限于诸如转录、翻译和RNA剪接等过程。The term "expression vector" refers to a vector suitable for transformation of host cells and containing nucleic acid sequences that direct and/or control the expression of an inserted heterologous nucleic acid sequence. Including but not limited to processes such as transcription, translation and RNA splicing.
术语“宿主细胞”用来指被核酸序列转化或能够被所述核酸序列转化然后能够表达选定目标基因的细胞。该术语包括亲代细胞的子代，无论子代在形态或遗传组成上是否与最初的亲代相同，主要存在选定的基因。The term "host cell" is used to refer to a cell transformed or capable of being transformed by a nucleic acid sequence and then capable of expressing a selected gene of interest. The term includes the progeny of a parental cell, whether or not the progeny is identical in morphology or genetic composition to the original parent, predominantly in the presence of selected genes.
具体实施方式detailed description
为了更详细的说明本发明，本说明书提供了下列具体实施方案，但本发明的方案并非仅限于此。In order to describe the present invention in more detail, the specification provides the following specific implementations, but the solutions of the present invention are not limited thereto.
1.主要实验试剂：1. Main experimental reagents:
Figure PCTCN2022103825-appb-000002
Figure PCTCN2022103825-appb-000002
2.主要实验仪器：2. Main experimental instruments:
名称name 品牌brand 型号model
蛋白纯化仪Protein Purifier GEGE AKTA-Pure150AKTA-Pure150
酶标仪Microplate reader MSMS 美谷分子American gluten molecules
实施例1 FGF21突变体蛋白的制备Example 1 Preparation of FGF21 mutant protein
采用ExpiCHO***(Thermo Fisher#A29133)表达FGF21突变体蛋白(SEQ ID NO:5-6)。The FGF21 mutant protein (SEQ ID NO:5-6) was expressed using the ExpiCHO system (Thermo Fisher #A29133).
具体的，将编码C端带有His标签的FGF21突变体蛋白的DNA序列克隆至pCDNA3.1载体中，经过测序，确认得到表达融合蛋白的质粒。使用ExpiFectamine试剂将质粒转染至ExpiCHO-S细胞中，在100毫升的ExpiCHO培养基中培养细胞7天后，收获上清液；采用离心过滤或深层过滤的方法进行发酵液的澄清。Specifically, the DNA sequence encoding the FGF21 mutant protein with a His tag at the C-terminal was cloned into the pCDNA3.1 vector, and after sequencing, it was confirmed that a plasmid expressing the fusion protein was obtained. Use ExpiFectamine reagent to transfect the plasmid into ExpiCHO-S cells. After culturing the cells in 100 ml of ExpiCHO medium for 7 days, harvest the supernatant; use centrifugal filtration or depth filtration to clarify the fermentation broth.
配置EQ缓冲液(PBS,pH7.4)的方法为取PBS磷酸缓冲液粉剂一袋，将该粉末用2000ml超纯水溶解，用0.22μm滤膜过滤备用。The method of preparing EQ buffer solution (PBS, pH7.4) is to take a bag of PBS phosphate buffer solution powder, dissolve the powder in 2000ml ultrapure water, and filter it with a 0.22μm filter membrane for later use.
配置Elution缓冲液(500mM咪唑,pH7.4)的方法为称取咪唑34g加EQ缓冲液450ml，调节ph至7.4，定容至500ml，0.22μm滤膜过滤备用。The method to configure Elution buffer (500mM imidazole, pH7.4) is to weigh 34g of imidazole and add 450ml of EQ buffer, adjust the pH to 7.4, dilute to 500ml, and filter through a 0.22μm filter membrane for later use.
将收获后的上清液通过AKTA Pure仪器进行纯化。首先用EQ缓冲液平衡仪器，直至流出液体的pH和电导值与EQ缓冲液一致；再用Elution缓冲液收集样品。The harvested supernatant was purified by AKTA Pure instrument. First, equilibrate the instrument with EQ buffer until the pH and conductivity of the effluent are consistent with the EQ buffer; then collect samples with Elution buffer.
采用同样的方法表达融合蛋白SEQ ID NO:10-16，使用Protein A亲和层析柱纯化，依次洗涤含有表达受体的条件培养基。将洗脱物在10mM tris缓冲盐水，pH 7.2的环境中透析。The fusion protein SEQ ID NO: 10-16 was expressed by the same method, purified using Protein A affinity chromatography column, and the conditioned medium containing the expressed receptor was washed sequentially. The eluate was dialyzed against 10 mM tris-buffered saline, pH 7.2.
用紫外分光光度法测定制备蛋白的浓度和纯度。The concentration and purity of the prepared protein were determined by UV spectrophotometry.
蛋白编号protein number 浓度(mg/L)Concentration (mg/L) 纯度(％)purity(%)
55 846846 9797
66 850850 9393
1010 710710 9696
1111 640640 8282
1212 350350 9191
1313 205205 9595
1414 460460 9494
1515 210210 8383
1616 310310 9191
实施例2 FGF21突变体诱导的葡萄糖摄取功能研究Example 2 Study on Glucose Uptake Function Induced by FGF21 Mutants
评价FGF21蛋白突变体对葡萄糖摄取水平的调节作用。3T3-L1小鼠胚胎成纤维细胞分化成熟的脂肪细胞表面表达葡萄糖转运蛋白1(GLUT1)，FGF21蛋白通过调节GLUT1的表达水平，进而调节脂肪细胞对葡萄糖摄取水平。The regulation of FGF21 protein mutants on glucose uptake levels was evaluated. 3T3-L1 mouse embryonic fibroblasts express glucose transporter 1 (GLUT1) on the surface of mature adipocytes, and FGF21 protein regulates the level of glucose uptake by adipocytes by regulating the expression level of GLUT1.
将培养的3T3-L1(南京科佰，cat#:CBP60758)小鼠胚胎成纤维细胞用胰蛋白酶(gibco，cat#:25200-056)消化，制备单细胞悬液，调整细胞密度至1x10 6/mL，接种于T75培养瓶，于37℃，5％二氧化碳培养箱内培养过夜。吸取原培养基，加入诱导培养基，即在含10％胎牛血清(gibco，cat#:1009141C)的DMEM(gibco，cat#:11995-065)完全培养基中加入2μg/ml人胰岛素(Sinobiologics，cat#:11038-HNAY)溶液，1μM***(Sigma，cat#:D4902-25MG)和0.5mM 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma，cat#:I7018-100MG)，诱导培养3T3-L1细胞3天，镜下观察细胞内脂肪粒的数量及大小，使其分化成脂肪细胞，之后将分化培养基换成只含有2μg/ml人胰岛素完全培养基。 Cultured 3T3-L1 (Nanjing Kebai, cat#:CBP60758) mouse embryonic fibroblasts were digested with trypsin (gibco, cat#:25200-056) to prepare a single cell suspension, and the cell density was adjusted to 1x10 6 / mL, inoculated into T75 culture flasks, and cultured overnight at 37°C in a 5% carbon dioxide incubator. Aspirate the original medium and add the induction medium, that is, add 2 μg/ml human insulin (Sinobiologics , cat#:11038-HNAY) solution, 1 μM dexamethasone (Sigma, cat#:D4902-25MG) and 0.5mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma, cat#:I7018- 100MG), induced and cultured 3T3-L1 cells for 3 days, observed the number and size of fat granules in the cells under a microscope, and differentiated them into adipocytes, and then replaced the differentiation medium with complete medium containing only 2 μg/ml human insulin.
将诱导分化成熟的脂肪细胞消化，制备单细胞悬液，用DMEM完全基础培养基调整细胞密度至1x10 6/mL，100uL/孔，接种于96孔板(Corning，cat#:3610)，待细胞贴壁，在实验组加入以DMEM基础培养基稀释待测突变体蛋白(SEQ ID NO:5)，使其终浓度为5000nM，100uL/孔加入板内，对照组加入相同体积的DMEM基础培养基，于37℃，5％二氧化碳孵育过夜。次日，将细胞在DMEM基础培养基中饥饿2小时，之后加入100uM 2-NBDG，于37℃孵育1小时，pH7.4PBS溶液洗细胞2次，胰蛋白酶消化，制备单细胞悬液，将细胞转移至V型底96孔板，利用ZE5流式细胞仪检测胞内2-NBDG信号，利用MFI计算葡萄糖的摄取率，计算公式：葡萄糖摄取率％＝(实验组MFI-对照组MFI)/对照组MFI*100％。 The adipocytes induced to differentiate and mature were digested to prepare a single-cell suspension, and the cell density was adjusted to 1x10 6 /mL with DMEM complete basal medium, 100uL/well, and seeded in a 96-well plate (Corning, cat#:3610). Adhere to the wall, add the mutant protein to be tested (SEQ ID NO:5) diluted with DMEM basal medium in the experimental group, so that the final concentration is 5000nM, 100uL/well is added to the plate, and the same volume of DMEM basal medium is added to the control group , and incubated overnight at 37°C, 5% carbon dioxide. The next day, starve the cells in DMEM basal medium for 2 hours, then add 100uM 2-NBDG, incubate at 37°C for 1 hour, wash the cells twice with pH 7.4 PBS solution, digest with trypsin, prepare a single cell suspension, and divide the cells Transfer to V-bottom 96-well plate, use ZE5 flow cytometer to detect intracellular 2-NBDG signal, use MFI to calculate glucose uptake rate, calculation formula: glucose uptake rate%=(experimental group MFI-control group MFI)/control Group MFI*100%.
FGF21蛋白突变体对葡萄糖摄取水平的调节作用Regulatory Effect of FGF21 Protein Mutant on Glucose Uptake Level
蛋白编号/SEQ ID NO:Protein number/SEQ ID NO: 葡萄糖摄取率5000nM(％)Glucose uptake rate 5000nM(%)
66 42.7642.76
实验结果表明，FGF21突变体(SEQ ID NO:6)显著诱导脂肪细胞对葡糖糖类似物2-NBDG摄取，诱导效率良好。The experimental results showed that the FGF21 mutant (SEQ ID NO:6) significantly induced the uptake of the glucose analog 2-NBDG by adipocytes, and the induction efficiency was good.
实施例3 FGF21突变体诱导ERK1/2蛋白磷酸化的细胞功能研究Example 3 Cellular Functional Study of ERK1/2 Protein Phosphorylation Induced by FGF21 Mutants
FGF21蛋白通过Ras/Raf/MAPK信号通路途径调节ERK1/2蛋白的磷酸化水平转导细胞信号参与体内能量代谢。评价比较FGF21蛋白突变体对ERK1/2蛋白的磷酸水平调节差异可评价其转导细胞信号的水平。FGF21 protein regulates the phosphorylation level of ERK1/2 protein through the Ras/Raf/MAPK signaling pathway to transduce cell signals and participate in energy metabolism in vivo. Evaluation Comparing FGF21 protein mutants to regulate the phosphorylation level of ERK1/2 protein can evaluate the level of cell signal transduction.
将HuH-7(中科院，cat#:SCSP-526)以1x10 5/mL接种于96孔板，100uL/孔，于37℃，5％二氧化碳孵育过夜，次日，将细胞在DMEM(gibco，cat#:11995-065)基础培养基中饥饿2小时，在实验组加入以DMEM基础培养基稀释待测蛋白，调整待测突变体蛋白(SEQ ID NO:5)终浓度为50nM，100uL/孔加入板内，对照组加入相同体积的DMEM基础培养基，37℃孵育20分钟，加入固定液(BD Cytofix，cat#:554655)于37℃固定30分钟，以400g，4℃离心5分钟，洗两遍。 加入通透液(BD Phosflow，cat#:558050)4℃通透1小时之后加入Alexa
Figure PCTCN2022103825-appb-000003
647标记小鼠抗人pERK1/2蛋白抗体(Biolegend，cat#:369504)，4℃孵育1小时，以400g，4℃离心5分钟，洗两遍，100uL/孔加入2％FBS溶液重悬细胞于ZE5(Bio-Rad)流式细胞仪检测Alexa
Figure PCTCN2022103825-appb-000004
647通道信号，利用平均荧光强度(MFI)计算pERK1/2增长效率，计算公式：pERK1/2增长率％＝(实验组MFI-对照组MFI)/对照组MFI*100％。 Seed HuH-7 (Chinese Academy of Sciences, cat#: SCSP-526) at 1×10 5 /mL in a 96-well plate, 100uL/well, incubate overnight at 37°C, 5% carbon dioxide, and the next day, the cells were placed in DMEM (gibco, cat #:11995-065) basal medium for 2 hours, add DMEM basal medium to the experimental group to dilute the protein to be tested, adjust the final concentration of the mutant protein to be tested (SEQ ID NO:5) to 50nM, add 100uL/well In the plate, add the same volume of DMEM basal medium to the control group, incubate at 37°C for 20 minutes, add fixative solution (BD Cytofix, cat#: 554655) and fix at 37°C for 30 minutes, centrifuge at 400g, 4°C for 5 minutes, wash twice all over. Add permeation solution (BD Phosflow, cat#:558050) to permeate for 1 hour at 4°C and then add Alexa
Figure PCTCN2022103825-appb-000003
647-labeled mouse anti-human pERK1/2 protein antibody (Biolegend, cat#:369504), incubate at 4°C for 1 hour, centrifuge at 400g, 4°C for 5 minutes, wash twice, add 100uL/well in 2% FBS solution to resuspend the cells Detection of Alexa on ZE5 (Bio-Rad) flow cytometer
Figure PCTCN2022103825-appb-000004
647 channel signals, using the mean fluorescence intensity (MFI) to calculate the growth efficiency of pERK1/2, the calculation formula: pERK1/2 growth rate%=(MFI of the experimental group-MFI of the control group)/MFI of the control group*100%.
蛋白编号protein number pERK1/2增长率％pERK1/2 Growth %
SEQ ID NO:5SEQ ID NO:5 13.1313.13
SEQ ID NO:6SEQ ID NO:6 16.2516.25
实验结果表明，FGF21突变体(SEQ ID NO:5和SEQ ID NO:6)可上调HuH-7细胞内ERK1/2蛋白的磷酸水平，效果良好。The experimental results show that FGF21 mutants (SEQ ID NO:5 and SEQ ID NO:6) can up-regulate the phosphorylation level of ERK1/2 protein in HuH-7 cells, and the effect is good.
实施例4 融合蛋白与Fc受体结合的BLI检测Example 4 BLI detection of fusion protein binding to Fc receptors
FGF21融合蛋白由发挥功能的FGF21和Fc片段组成，其中Fc结合FcγR的胞外域通过非共价键结合，激活免疫受体酪氨酸激活基序，介导的ADCC(细胞毒性)所引发的免疫激活效应对于疾病治疗不利。而Fc与FcRn(新生儿Fc受体)的结合，则有利于融合蛋白在体内具有更长的循环周期。The FGF21 fusion protein is composed of functional FGF21 and Fc fragments, in which Fc binds to the extracellular domain of FcγR through non-covalent bonding, activates the immunoreceptor tyrosine activation motif, and mediates immunity induced by ADCC (cytotoxicity) The activation effect is not good for disease treatment. The combination of Fc and FcRn (neonatal Fc receptor) is beneficial for the fusion protein to have a longer circulation period in vivo.
通过融合蛋白与FcγRI、FcγRIIa、FcγRIIIa、FcRn之间的亲和力分析来评估Fc片段介导的潜在免疫效应及体内循环特性。使用PBST(pH＝7.4)溶液将融合蛋白12稀释到1108.0nM，553.8nM，276.9nM，138.4nM和69.2nM；将FcγRI(Sino,#10256-H27H-B)、FcγRIIa(Sino,#10374-H27H-B)、FcγRIIIa(Sino,10389-H27H1-B)和FcRn(Sino,#CT009-H08H-B)分别稀释到10μg/mL，3μg/mL，5μg/mL和3μg/mL浓度。The potential immune effects mediated by Fc fragments and the in vivo circulation characteristics were evaluated by the affinity analysis between the fusion protein and FcγRI, FcγRIIa, FcγRIIIa, and FcRn. Fusion protein 12 was diluted to 1108.0nM, 553.8nM, 276.9nM, 138.4nM and 69.2nM using PBST (pH=7.4) solution; -B), FcγRIIIa (Sino, 10389-H27H1-B) and FcRn (Sino, #CT009-H08H-B) were diluted to concentrations of 10 μg/mL, 3 μg/mL, 5 μg/mL and 3 μg/mL, respectively.
在PBST(pH＝7.4)溶液中用蛋白相互作用仪(PALL,#Red)的SA探针捕获FcγRI、FcγRIIa、FcγRIIIa；随后与融合蛋白12混合进行反应，充分反应后的固相结合物在PBST缓冲液中进行解离分析，以Data Analysis软件对结果分析。同样的方法，使用PBST(pH＝6.0)溶液用于融合蛋白与FcRn的结合检测。获得亲和力常数如下表所示：In PBST (pH=7.4) solution, FcγRI, FcγRIIa, and FcγRIIIa were captured with the SA probe of protein interaction instrument (PALL, #Red); then mixed with fusion protein 12 for reaction, and the fully reacted solid-phase conjugate was released in PBST Dissociation analysis was performed in the buffer solution, and the results were analyzed with Data Analysis software. In the same way, PBST (pH=6.0) solution was used to detect the binding of the fusion protein to FcRn. The obtained affinity constants are shown in the table below:
Figure PCTCN2022103825-appb-000005
Figure PCTCN2022103825-appb-000005
融合蛋白突变体12与FcγR(FcγRI、FcγRIIa、FcγRIIIa)的结合均较弱，免疫效应带来的副作用风险较小；与FcRn之间结合较强，有利于其在血液中长效循环。The binding of fusion protein mutant 12 to FcγR (FcγRI, FcγRIIa, FcγRIIIa) is weak, and the risk of side effects caused by immune effects is small; the binding to FcRn is strong, which is conducive to its long-term circulation in the blood.
实施列5 FGF21融合蛋白突变体PK研究Implementation column 5 FGF21 fusion protein mutant PK research
利用人FcRn转基因小鼠模型评价FGF21融合蛋白突变体在小鼠体内的药物代谢情况。The human FcRn transgenic mouse model was used to evaluate the drug metabolism of FGF21 fusion protein mutants in mice.
将平均体重为18-22g，18-22周龄的人FcRn转基因小鼠随机分组，每组3只动物，受试FGF21融合蛋白突变体12均以4mpk，s.c.，单次的方式给药，PBS溶媒作为阴性对照组，分别在0.5、2、4、6、8、24、48、72、96、120小时采血分离血浆，冻存于-20℃冰箱，之后利用人KLB蛋白-FGF21融合蛋白突变体-Fc-HRP间接ELISA法检测小鼠血浆中FGF21融合蛋白突变体的浓度，并利用PK solver软件非房室模型，血管内给药的公式分析其PK参数，实验结果如下表所示：Human FcRn transgenic mice with an average body weight of 18-22g and 18-22 weeks of age were randomly divided into groups, with 3 animals in each group, and the tested FGF21 fusion protein mutant 12 was administered at 4 mpk, s.c., in a single dose, PBS The vehicle was used as a negative control group, blood was collected at 0.5, 2, 4, 6, 8, 24, 48, 72, 96, and 120 hours to separate plasma, frozen in a -20°C refrigerator, and then mutated using human KLB protein-FGF21 fusion protein Body-Fc-HRP indirect ELISA method was used to detect the concentration of FGF21 fusion protein mutants in mouse plasma, and the PK solver software was used to analyze the PK parameters of the non-compartmental model and the formula of intravascular administration. The experimental results are shown in the following table:
PK参数PK parameters 单位unit 突变体12mutant 12
t1/2t1/2 hh 71.371.3
CmaxCmax ng/mlng/ml 1.49E+051.49E+05
AUC 0-tAUC 0-t ng/ml*hng/ml*h 5.43E+065.43E+06
AUC 0-inf_obsAUC 0-inf_obs ng/ml*hng/ml*h 6.95E+066.95E+06
MRT 0-inf_obsMRT 0-inf_obs hh 93.493.4
Cl_obsCl_obs (mg/kg)/(ng/ml)/h(mg/kg)/(ng/ml)/h 5.8E-075.8E-07
综合半衰期t 1/2、暴露量、血药最高浓度达峰时间等参数，FGF21融合蛋白突变体12显示出良好的体内代谢活性。 Considering parameters such as half-life t 1/2 , exposure dose, and time to peak plasma drug concentration, FGF21 fusion protein mutant 12 showed good metabolic activity in vivo.
实施例6 FGF21融合蛋白在DIO小鼠体内药效研究Example 6 Study on the efficacy of FGF21 fusion protein in DIO mice
利用高脂饮食诱导C57BL/6小鼠肥胖模型，即DIO模型小鼠，评价FGF21融合蛋白突变体在降低体重、肝脏重量的效果。The obesity model of C57BL/6 mice induced by high-fat diet, that is, DIO model mice, was used to evaluate the effect of FGF21 fusion protein mutants on reducing body weight and liver weight.
将体重为30-50g，12周龄的DIO雄性肥胖模型小鼠随机分组，每组8只动物，受试FGF21融合蛋白突变体12、13，以2mpk,s.c.，q3d的方式给药，PBS溶媒作为阴性对照组，共给药3次。第0天称量体重并给药，以后每3天给药并测量体重，累计进食量。第9天称重并禁食过夜于第10天称重并采血，取肝脏，并称量肝脏。The 12-week-old DIO male obesity model mice with a body weight of 30-50g were randomly divided into groups, with 8 animals in each group, and the tested FGF21 fusion protein mutants 12 and 13 were administered at 2mpk, s.c., q3d, in PBS vehicle As a negative control group, a total of 3 doses were administered. On the 0th day, the body weight was weighed and the drug was administered, and the body weight was measured every 3 days thereafter, and the food intake was accumulated. On the 9th day, we weighed and fasted overnight. On the 10th day, weighed and collected blood, took the liver, and weighed the liver.
DIO小鼠体重变化(％)(Mean±SD)Body weight change of DIO mice (%) (Mean±SD)
Figure PCTCN2022103825-appb-000006
Figure PCTCN2022103825-appb-000006
Figure PCTCN2022103825-appb-000007
Figure PCTCN2022103825-appb-000007
DIO小鼠实验终点(第10天)血糖水平(Mean±SD)Blood glucose level (Mean±SD) at the experimental end point (day 10) of DIO mice
Figure PCTCN2022103825-appb-000008
Figure PCTCN2022103825-appb-000008
*检验系数为p<0.05有统计学差异，采用one-way ANOVA多组比较分析，*The test coefficient is p<0.05, there is a statistical difference, using one-way ANOVA multi-group comparative analysis,
DIO小鼠实验终点(第10天)肝重变化Changes in liver weight at the end point (day 10) of DIO mice
Figure PCTCN2022103825-appb-000009
Figure PCTCN2022103825-appb-000009
*检验系数为p<0.05有统计学差异，采用one-way ANOVA多组比较分析。* There is a statistical difference when the test coefficient is p<0.05, and one-way ANOVA is used for multi-group comparative analysis.
综合实验结果，2mpk,s.c.，q3d的方式给药方式下，随着小鼠进食量增加，FGF21融合蛋白突变体12、13显示出显著且持久的降体重作用；对于降低肝脏重量及血糖水平，与溶媒组PBS相比均具有明显的统计学差异，结果表明在减少肝脏脂肪蓄积，降低血糖水平，均显示出较好的代谢调节效果。According to the comprehensive experimental results, under the administration of 2mpk, s.c., q3d, FGF21 fusion protein mutants 12 and 13 showed a significant and lasting effect on reducing body weight as the food intake of mice increased; for reducing liver weight and blood sugar levels, Compared with the PBS of the vehicle group, there are significant statistical differences, and the results show that it can reduce liver fat accumulation and lower blood sugar levels, and both show better metabolic regulation effects.
实施例7 FGF21融合蛋白在ob/ob小鼠体内药效研究Example 7 Study on the efficacy of FGF21 fusion protein in ob/ob mice
利用瘦素敲除的小鼠糖尿模型，即ob/ob模型小鼠，评价FGF21融合蛋白突变体12在降低体重、血糖水平、肝-体重比、血脂四项：总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)水平效果。Using leptin-knockout mouse model of diabetes, namely ob/ob model mice, to evaluate FGF21 fusion protein mutant 12 in reducing body weight, blood glucose level, liver-to-weight ratio, and blood lipids: total cholesterol (TC), triglycerides Ester (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels.
将体重为30-50g，12周龄的ob/ob雄性肥胖模型小鼠随机分组，每组6只动物，受试FGF21融合蛋白突变体12，以2mpk,s.c.，q3d的方式给药，PBS溶媒作为阴性对照组，共给药3次。第0天称量体重并给药，以后每3天给药并测量体重，累计进食量。第8天称重并禁食过夜于第9天称重并采血检测血糖水平及 血糖四项，分离肝脏，并分别称量肝脏、体重，计算肝脏-体重比。12-week-old ob/ob male obesity model mice with a body weight of 30-50g were randomly divided into groups, with 6 animals in each group, and the test FGF21 fusion protein mutant 12 was administered at 2mpk, s.c., q3d, PBS vehicle As a negative control group, a total of 3 doses were administered. On the 0th day, the body weight was weighed and the drug was administered, and the body weight was measured every 3 days thereafter, and the food intake was accumulated. Weighed on the 8th day and fasted overnight. On the 9th day, weighed and collected blood to detect blood sugar level and four items of blood sugar, separated the liver, weighed the liver and body weight respectively, and calculated the liver-to-body weight ratio.
实验结果表明，相对于溶媒组候选分子具有明显的降体重效果和降血糖效果。The experimental results show that, compared with the candidate molecules of the vehicle group, they have obvious weight-lowering effects and blood-sugar-lowering effects.
ob/ob小鼠体重变化(％)(Mean±SD)Ob/ob mouse body weight change (%) (Mean±SD)
Figure PCTCN2022103825-appb-000010
Figure PCTCN2022103825-appb-000010
*检验系数为p<0.05有统计学差异，采用one-way ANOVA多组比较分析。* There is a statistical difference when the test coefficient is p<0.05, and one-way ANOVA is used for multi-group comparative analysis.
ob/ob小鼠实验终点(第9天)血糖水平变化(mmoL/L)(Mean±SD)Ob/ob mice experimental endpoint (day 9) changes in blood glucose levels (mmoL/L) (Mean±SD)
Figure PCTCN2022103825-appb-000011
Figure PCTCN2022103825-appb-000011
*检验系数为p<0.05有统计学差异，采用one-way ANOVA多组比较分析。* There is a statistical difference when the test coefficient is p<0.05, and one-way ANOVA is used for multi-group comparative analysis.
实施例8 FGF21融合蛋白对NASH模型药效研究Example 8 Study on the efficacy of FGF21 fusion protein on NASH model
CD57BL/6小鼠采购于南京集萃药康，在本实验室适应一周后即进行饮食诱导NASH模型。使用胆碱缺乏的高脂肪含量饲料即，CDA HFD诱导。正常健康组小鼠常规饲料饲喂，溶媒(pH7.4PB溶液)对照组和实验组小鼠使用含60％高脂肪，2％胆固醇的CDA/HFD饲料，并在饮水中添加10％果糖饲喂，诱导9周。随后将实验小鼠随机分为4组，每组5动物，从第3周开始给药(即诱导2周后给药，给药7周)。受试药物以5mg/kg,通过皮下以q3d频次给药，连续给药7周，到达实验终点后，称量体重，安乐死处理动物，收集肝脏称重后将肝脏保存于4％中性甲醛，供组织切片以检测脂肪沉积、干细胞气球样病变、肝细胞炎症水平等指标评估受试药物对NASH发生进程的影响。CD57BL/6 mice were purchased from Nanjing Jicui Yaokang, and after a week of acclimatization in our laboratory, the NASH model was induced by diet. HFD was induced using a choline-deficient high-fat diet, ie, CDA. The mice in the normal healthy group were fed with conventional feed, and the mice in the vehicle (pH7.4 PB solution) control group and the experimental group were fed with CDA/HFD feed containing 60% high fat and 2% cholesterol, and fed with 10% fructose in the drinking water , induced for 9 weeks. Subsequently, the experimental mice were randomly divided into 4 groups, with 5 animals in each group, and the administration started from the third week (ie, administration after 2 weeks of induction, and administration for 7 weeks). The test drug was administered subcutaneously at q3d frequency at 5 mg/kg for 7 consecutive weeks. After reaching the end point of the experiment, the body weight was weighed, and the animal was euthanized. The liver was collected and weighed, and the liver was stored in 4% neutral formaldehyde. Tissue slices were used to detect indicators such as fat deposition, stem cell balloon-like lesions, and liver cell inflammation levels to evaluate the impact of the test drug on the development of NASH.
实验结果表明，与健康组对照比，受试药物在减轻体重方面效果明显可达24.92％，同时明显在降低肝脏-体重比水平，与健康组水平相当；相对于溶媒组候选分子在改善NASH纤维化水平有统计学差异。The experimental results show that compared with the healthy group, the tested drug can significantly reduce body weight by 24.92%, and at the same time significantly reduce the level of liver-to-body weight ratio, which is equivalent to the level of the healthy group; compared with the vehicle group, the candidate molecules can improve NASH fiber There is a statistically significant difference in levels.
NASH小鼠体重变化(％)(Mean±SEM)Body weight change of NASH mice (%) (Mean±SEM)
Figure PCTCN2022103825-appb-000012
Figure PCTCN2022103825-appb-000012
受试药物对纤维化水平及NAS评分的改善Improvement of fibrosis level and NAS score by test drugs
Figure PCTCN2022103825-appb-000013
Figure PCTCN2022103825-appb-000013
受试药物对肝脏-体重比的影响Effects of Tested Drugs on Liver-to-Body Weight Ratio
Figure PCTCN2022103825-appb-000014
Figure PCTCN2022103825-appb-000014
Figure PCTCN2022103825-appb-000015
Figure PCTCN2022103825-appb-000015
Figure PCTCN2022103825-appb-000016
Figure PCTCN2022103825-appb-000016
Figure PCTCN2022103825-appb-000017
Figure PCTCN2022103825-appb-000017
Figure PCTCN2022103825-appb-000018
Figure PCTCN2022103825-appb-000018
Figure PCTCN2022103825-appb-000019
Figure PCTCN2022103825-appb-000019

Claims (13)

  1. 一种融合蛋白，其通式如下：A fusion protein, its general formula is as follows:
    F 1-F 2-F 3 F 1 -F 2 -F 3
    (I)(I)
    其中：in:
    F 1为FGF21蛋白或其变体，其结构如下通式(II)所示： F 1 is FGF21 protein or its variant, its structure is shown in the following general formula (II):
    HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSWDPX 166SMVGGSQGX 175SPSYES HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARPLPGLPPALPEPPGILAPQPPDVGSWDPX 166 SMVGGSQGX 175 SPSY
    (II)(II)
    X 166选自L或F； X 166 is selected from L or F;
    X 175选自R或W； X 175 is selected from R or W;
    F 2为连接肽； F 2 is connecting peptide;
    F 3为免疫球蛋白的Fc或其片段组成的结构域。 F3 is a domain composed of Fc or fragments of immunoglobulin.
  2. 根据权利要求2所述的融合蛋白，其特征在于，F 1为序列如SEQ ID NO:5或SEQ ID NO:6所示。 The fusion protein according to claim 2, wherein F1 is a sequence as shown in SEQ ID NO:5 or SEQ ID NO:6.
  3. 根据权利要求2所述的融合蛋白，其特征在于，F 2为序列如SEQ ID NO:7所示。 The fusion protein according to claim 2 , wherein F2 is a sequence as shown in SEQ ID NO:7.
  4. 根据权利要求1-3任一项所述的融合蛋白，其特征在于，所述F 3如通式(III)所示： The fusion protein according to any one of claims 1-3, wherein said F 3 is shown in general formula (III):
    F 4(GGGGGS) m(GGGGS) nF 4 F 4 (GGGGGS) m (GGGGS) n F 4
    (III)(III)
    其中：in:
    F 4为免疫球蛋白的Fc片段； F4 is the Fc fragment of immunoglobulin;
    m选自1-10的整数；且m is an integer selected from 1-10; and
    n选自1-10的整数。n is an integer selected from 1-10.
  5. 根据权利要求4所述的融合蛋白，其特征在于，The fusion protein according to claim 4, wherein,
    F 4为SEQ ID NO：8； F4 is SEQ ID NO: 8;
    m为1；且m is 1; and
    n为9。n is 9.
  6. 根据权利要求1-3任一项所述的融合蛋白，其特征在于，所述F 3为SEQ ID NO：9。 The fusion protein according to any one of claims 1-3 , wherein the F3 is SEQ ID NO:9.
  7. 根据权利要求1-6任一项所述的融合蛋白，其特征在于，所述融合蛋白选自SEQ ID NO：12或SEQ ID NO：13。The fusion protein according to any one of claims 1-6, wherein the fusion protein is selected from SEQ ID NO: 12 or SEQ ID NO: 13.
  8. 一种多核苷酸，其编码包含权利要求1-7任一项所述的融合蛋白。A polynucleotide encoding the fusion protein according to any one of claims 1-7.
  9. 一种表达载体，其含有权利要求8所述的多核苷酸。An expression vector comprising the polynucleotide according to claim 8.
  10. 一种宿主细胞，其导入或含有权利要求9所述的表达载体，其中所述宿主细胞选自细菌、酵母菌或哺乳动物细胞，所述细菌优选为大肠杆菌；所述酵母菌，优选为毕赤酵母；所述哺乳动物细胞，优选为CHO细胞或HEK293细胞。A host cell, which introduces or contains the expression vector according to claim 9, wherein the host cell is selected from bacteria, yeast or mammalian cells, and the bacteria is preferably Escherichia coli; the yeast is preferably Bi red yeast; the mammalian cells are preferably CHO cells or HEK293 cells.
  11. 一种生产蛋白的方法，包括步骤：A method for producing protein, comprising the steps of:
    培养权利要求10所述的宿主细胞；cultivating the host cell of claim 10;
    从培养物中分离蛋白；Isolate protein from culture;
    以及对所述的蛋白进行纯化。and purifying the protein.
  12. 一种药物组合物，其含有权利要求1-7任一项所述的融合蛋白，以及可药用的赋形剂、稀释剂或载体。A pharmaceutical composition, which contains the fusion protein according to any one of claims 1-7, and pharmaceutically acceptable excipients, diluents or carriers.
  13. 根据权利要求1-7任一项所述的融合蛋白，或权利要求12所述的药物组合物在制备药物中的用途，所述药物用于治疗或预防治疗糖尿病、肥胖、血脂异常、代谢综合征、非酒精性脂肪肝或非酒精性脂肪肝炎相关疾病。According to the fusion protein according to any one of claims 1-7, or the pharmaceutical composition according to claim 12 in the preparation of medicines, the medicine is used for the treatment or prevention of diabetes, obesity, dyslipidemia, metabolic syndrome syndrome, non-alcoholic fatty liver or non-alcoholic steatohepatitis-related diseases.
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