WO2023061515A1 - Novel fgf analog for treating liver cancer and use thereof - Google Patents

Novel fgf analog for treating liver cancer and use thereof Download PDF

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WO2023061515A1
WO2023061515A1 PCT/CN2022/137532 CN2022137532W WO2023061515A1 WO 2023061515 A1 WO2023061515 A1 WO 2023061515A1 CN 2022137532 W CN2022137532 W CN 2022137532W WO 2023061515 A1 WO2023061515 A1 WO 2023061515A1
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fgf21
liver cancer
cancer
injection
tumor
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朱升龙
陈永泉
王振
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江南大学
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    • 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 factors [FGF]
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention relates to a novel FGF analogue for treating liver cancer and its application, in particular to the application of a human FGF21 analogue in the preparation of drugs for treating liver cancer, and belongs to the field of medical technology.
  • Liver cancer is one of the common malignant tumors in my country, with a high incidence in the southeast coastal areas.
  • the median age of patients with liver cancer in my country is 40 to 50 years old, and it is more common in men than women. In recent years, its incidence has been increasing.
  • the annual mortality rate of liver cancer accounts for the second place in the death rate of tumors, and ranks the third place among malignant tumors of the digestive system, second only to gastric and esophageal cancer.
  • Hepatitis B, C, liver cirrhosis, hepatocyte necrosis accompanied by regeneration of liver cells are prone to malignant transformation.
  • liver cancer is mainly based on radical surgery, chemotherapy, radiotherapy, traditional Chinese medicine treatment and biological therapy as its auxiliary treatment means.
  • radical surgery Approximately two-thirds of patients diagnosed for the first time underwent radical surgery, and more than half of these patients developed recurrence or distant metastasis.
  • radical surgery cannot be performed, and comprehensive medical treatment is often used as the main treatment.
  • liver cancer still has a high recurrence rate after surgery.
  • the commonly used therapeutic drugs are usually broad-spectrum chemical drugs, which have more side effects. Therefore, it is an ideal goal of current research and development to develop a safe drug for the treatment of liver cancer that can inhibit tumor growth and recurrence and protect organs, and has no obvious side effects.
  • liver cancer In recent years, a large number of clinical investigations have shown that the occurrence and development of liver cancer are closely related to metabolic abnormalities.
  • metabolic pathways including glycolysis, tricarboxylic acid cycle, fatty acid metabolism, and glutamine metabolism, have undergone reprogramming changes in tumor cells.
  • Tumor cells can provide the necessary for proliferation through the coordination of various metabolic pathways.
  • metabolic abnormalities are not only the result of tumor occurrence and development, but may also be directly involved in the initiation process of tumors.
  • the present invention finds that human fibroblast growth factor 21, an important member involved in multiple links of the glucose and lipid metabolism network, can significantly inhibit the development and metastasis of liver cancer. Based on this, the present invention provides a new application of FGF21 analogues in the preparation of drugs for preventing, alleviating and/or treating liver cancer.
  • the present invention provides FGF21 analogues, and the amino acid sequence of the FGF21 protein analogues is any one of SEQ ID NO.1-3.
  • the present invention provides the gene encoding the FGF21 protein analogue.
  • the present invention provides vectors and/or microbial cells carrying the genes.
  • the microbial cells comprise E. coli or mammalian cells.
  • the mammalian cells comprise 293T cells or CHO cells.
  • the invention provides the application of the gene in screening drugs for treating liver cancer.
  • the FGF21 gene is used as a drug target.
  • nucleotide sequence of the FGF21 gene is shown in any one of SEQ ID NO.4-6.
  • the use is for screening small molecule activators of FGF21 protein.
  • the use is for screening agents that inhibit the expression of alpha-fetoprotein.
  • the present invention provides a medicament or a pharmaceutical composition, which contains the FGF21 analogue.
  • the drug or pharmaceutical composition further includes human acceptable modifications, pharmaceutical carriers and/or excipients.
  • the modification includes PEG modification and FC modification.
  • the pharmaceutical excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers , Surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants.
  • the present invention provides the application of the FGF21 analog in the preparation of drugs or pharmaceutical compositions for preventing, alleviating and/or treating cancer.
  • the cancer includes but not limited to liver cancer, prostate cancer, breast cancer, esophageal cancer, colorectal adenocarcinoma, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, clear cell renal cancer Carcinoma, melanoma, multiple myeloma.
  • the prevention, alleviation and/or treatment of liver cancer includes but not limited to (a)-(d):
  • the liver cancer factor is alpha-fetoprotein.
  • the effective dose of the FGF21 analog is 0.1-100 mg/kg.
  • the administration route of the drug or pharmaceutical composition includes intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, arterial injection, intracavitary injection and/or oral administration.
  • the FGF21 fusion protein obtained in the present invention can be used in the treatment of liver cancer, and can significantly reduce the size of tumor lesions, so it can be used as an active ingredient of various dosage forms of drugs.
  • drugs similar to FGF21 are injected into the body by injection, which can effectively reduce the expression level of liver cancer factors, alleviate the occurrence and development of liver cancer, and inhibit the generation and metastasis of liver cancer, which is extremely important for the treatment of liver cancer. meaning.
  • Figure 1 is the effect of FGF21 on the growth rate of human liver cancer xenograft tumor; wherein, A is the change of tumor volume; B is the change of tumor mass;
  • Figure 2 is the effect of FGF21 on carbon tetrachloride-induced liver cancer in mice; wherein, A is the content of alpha-fetoprotein; B is liver cancer slices.
  • mice and C57BL/6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. They were raised in the Animal Center of Wuxi Medical College, Jiangnan University, under alternating lighting every 12 hours, and the temperature was 20 ⁇ 2°C.
  • liver cancer cell line HepG2 was provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences;
  • DMEM and 0.05% Trypsin were purchased from Boster Company; fetal bovine serum was purchased from Sijiqing Company. Other medicines were domestic analytically pure.
  • the hepatocarcinoma cell lines were grown adherently in DMEM medium containing 10% fetal bovine serum, cultured in a humidified incubator with 5% CO 2 at 37°C, and passaged once every other day.
  • mice were injected with 1 mg/kg, 5 mg/kg and 10 mg/kg of FGF21 protein, and the corresponding human doses were 0.1 mg/kg, 0.5 mg/kg and 1 mg/kg.
  • Example 1 Construction, expression and purification of recombinant proteins
  • the fusion FGF21 protein (amino acid sequence is shown in SEQ ID NO.1 ⁇ 3), according to the codon preference of Escherichia coli, the corresponding nucleotide sequence is designed, and its nucleotide sequence is respectively as FGF21-1 in the sequence list ( Nucleotide sequence as shown in SEQ ID NO.4), FGF21-2 (nucleotide sequence as shown in SEQ ID NO.5), FGF21-3 (nucleotide sequence as shown in SEQ ID NO.6) Show.
  • the three genes were sent to the company for synthesis, and the three genes were connected between the NdeI and BamHI restriction sites of the pET30a(+) vector, and the enzyme-ligated products were transformed into E.
  • coli DH5 ⁇ respectively.
  • the positive clones were picked and identified by restriction enzyme digestion, and three recombinant plasmids pET30a-FGF21-1, pET30a-FGF21-2 and pET30a-FGF21-3 were respectively constructed.
  • the recombinant plasmids containing the correct sequence obtained in step 1 were respectively transformed into competent cells of the expression strain Rosseta (DE3). Transformed single colonies were inoculated into 20 mL of LB medium containing Kan (50 ⁇ g/mL), cultured at 37 °C for 8 h, and inoculated into another 20 mL of LB medium containing Kan (50 ⁇ g/mL) at a volume ratio of 1:100 medium, cultured at 37°C, when A600 was around 0.35, IPTG was added to a final concentration of 0.25mmol/L for induction, the induction temperature was 30°C, and the cells were harvested after 5h, and Lysis buffer (20mmol/LTris, 150mmol/LNaCl, pH 8.0) to resuspend the bacteria, break the bacteria and centrifuge, take the supernatant and precipitate respectively for 12wt% SDS-PAGE electrophoresis analysis. And the bacteria were crushed, and the protein was purified to
  • the FGF21 protein whose amino acid sequence is shown in SEQ ID NO.1 is prepared according to the method of Example 1.
  • Human liver cancer cell HepG2 cells were inoculated under the skin of 6-week-old male nude mice according to 1 ⁇ 106 cells/only, and were randomly divided into four groups when the tumor grew to 200mm3 .
  • 1Normal saline group injected with an equal volume of normal saline;2 Low-dose group: inject 1 mg/kg FGF21; 3 medium-dose group: inject 5 mg/kg FGF21; 4 high-dose group: inject 10 mg/kg FGF21. Inject once a day for 15 consecutive days. Tumor volume was monitored daily, mice were sacrificed three weeks later, and tumor weight was measured. The results showed that three different doses of FGF21 could inhibit the transplanted tumor volume and final tumor weight in a dose-dependent manner.
  • FGF21 protein 10mg/kg
  • high-dose FGF21 protein 10mg/kg
  • it can completely inhibit tumor growth, keep the tumor at 250mm 3 , and the final volume is only 20% of that of the control group, and the tumor mass also decreased by about 70%.
  • 5mg/kg can reduce tumor mass by 45% and 53% respectively (as shown in Figure 1).
  • mice On the 14th day after birth, the mice were intraperitoneally injected with 2 mg/kg of diethylnitrosamine (DEN), and 2 weeks after the administration, 5 mL/kg of 20% carbon tetrachloride (CCI4) was injected intraperitoneally, 2 times a week. A total of 16 weeks. After 16 weeks of induction, they were divided into normal saline group and FGF21 injection group. The injection dose of FGF21 injection group was 10 mg/kg, and the normal saline group was injected with the same amount of normal saline once a day for 6 consecutive weeks.
  • DEN diethylnitrosamine
  • CCI4 20% carbon tetrachloride
  • Figure 2A shows the alpha-fetoprotein levels of the FGF21 treatment group and the control group, and the alpha-fetoprotein content is a characteristic marker of liver cancer, indicating that the amino acid sequence of the FGF21 protein shown in SEQ ID NO.1 significantly After reducing the content of liver cancer markers
  • Figure 2B shows the pathological section of the tumor, further illustrating the inhibitory effect of FGF21 protein on liver cancer.
  • the inventor also tried to study the effect of the fusion protein of FGF21 protein shown in SEQ ID NO.
  • the FGF21 protein shown in SEQ ID NO.1 had comparable effects.

Abstract

Disclosed are a novel FGF analog for treating liver cancer and a use thereof, belonging to the technical field of medicine. The FGF21 protein provided by the present invention is a secreted protein composed of 428 amino acids. The present invention proves through cytological experiments and animal experiments that the prepared novel FGF21 analog can inhibit the proliferation, invasion and migration of liver cancer cell lines, and can significantly reduce the size of tumor lesions in a mouse model. A therapeutic drug for liver cancer prepared by using FGF21 as an active substance has the effects of good safety and long duration of drug effect, while protecting organs from damage and improving lipid metabolism disorders.

Description

一种新型治疗肝癌的FGF类似物及其应用A novel FGF analogue for treating liver cancer and its application 技术领域technical field
本发明涉及一种新型治疗肝癌的FGF类似物及其应用，特别涉及人FGF21类似物在制备用于治疗肝癌药物中的应用，属于医药技术领域。The invention relates to a novel FGF analogue for treating liver cancer and its application, in particular to the application of a human FGF21 analogue in the preparation of drugs for treating liver cancer, and belongs to the field of medical technology.
背景技术Background technique
肝癌为我国常见恶性肿瘤之一，高发于东南沿海地区。我国肝癌病人的中位年龄为40～50岁，男性比女性多见。近年来其发病率有增高趋势。我国肝癌年死亡率占肿瘤死亡率的第二位，在消化系统恶性肿瘤中列第三位，仅次于胃和食管癌。乙型、丙型肝炎、肝硬化，肝细胞坏死伴有肝细胞的再生过程中易于发生恶变。Liver cancer is one of the common malignant tumors in my country, with a high incidence in the southeast coastal areas. The median age of patients with liver cancer in my country is 40 to 50 years old, and it is more common in men than women. In recent years, its incidence has been increasing. In my country, the annual mortality rate of liver cancer accounts for the second place in the death rate of tumors, and ranks the third place among malignant tumors of the digestive system, second only to gastric and esophageal cancer. Hepatitis B, C, liver cirrhosis, hepatocyte necrosis accompanied by regeneration of liver cells are prone to malignant transformation.
目前，肝癌的治疗主要以外科根治手术为主，化疗、放疗、中医药治疗及生物治疗作为其辅助性治疗手段。首次诊断的病人中大约有三分之二接受了根治术，其中超过一半的患者出现复发或远处转移。对晚期癌肿或有广泛转移的癌肿，不能进行根治性手术，内科综合治疗常成为主要治疗手段，在可手术的患者中，肝癌术后仍有较高的复发率。而常用的治疗药物通常为广谱类化学药物，具有较多的副作用。因此，开发一种安全的、同时具有抑制肿瘤生长和复发且器官保护效应的、无相关明显副作用的、安全的治疗肝癌药物是目前研发的理想目标。At present, the treatment of liver cancer is mainly based on radical surgery, chemotherapy, radiotherapy, traditional Chinese medicine treatment and biological therapy as its auxiliary treatment means. Approximately two-thirds of patients diagnosed for the first time underwent radical surgery, and more than half of these patients developed recurrence or distant metastasis. For advanced cancer or cancer with extensive metastasis, radical surgery cannot be performed, and comprehensive medical treatment is often used as the main treatment. Among operable patients, liver cancer still has a high recurrence rate after surgery. The commonly used therapeutic drugs are usually broad-spectrum chemical drugs, which have more side effects. Therefore, it is an ideal goal of current research and development to develop a safe drug for the treatment of liver cancer that can inhibit tumor growth and recurrence and protect organs, and has no obvious side effects.
近年来，大量临床调查结果显示肝癌的发生发展与代谢异常存在十分紧密的联系。大量研究发现代谢通路包括糖酵解、三羧酸循环、脂肪酸代谢、谷氨酰胺代谢等在肿瘤细胞中均发生了重编程变化，肿瘤细胞可通过对各种代谢途径间的协调，提供增殖所必须的脂肪酸、核苷酸等，从而使生物大分子的合成过程更适合于肿瘤细胞的快速增殖。尤其需要指出的是，代谢异常并不仅仅是肿瘤发生发展的结果，还可能直接参与肿瘤的始发过程。目前，从代谢的角度开发抗肿瘤药物，已在诸多的肿瘤中获得了成功的应用，比如，最近的研究发现某些编码代谢酶类的基因本身就是癌基因，如延胡索酸羧化酶的突变与肾癌的发生、发展；琥珀酸羧化酶的突变与嗜铬细胞瘤的发生发展；异柠檬酸脱氢酶的突变产生代谢物小分子2HG一直HNF4α的表达从而诱导胆管癌等。基于以上分析表明靶向肿瘤细胞代谢可能是杀死肿瘤细胞的有效途径之一。然而，目前开发的药物都是一些小分子抑制剂，靶向性较弱，副作用较多，而生物蛋白类药物吸收快，靶向性强。因此，针对肝癌开发新型靶向生物蛋白类制剂是目前肿瘤研究的热点。In recent years, a large number of clinical investigations have shown that the occurrence and development of liver cancer are closely related to metabolic abnormalities. A large number of studies have found that metabolic pathways, including glycolysis, tricarboxylic acid cycle, fatty acid metabolism, and glutamine metabolism, have undergone reprogramming changes in tumor cells. Tumor cells can provide the necessary for proliferation through the coordination of various metabolic pathways. Essential fatty acids, nucleotides, etc., so that the biomacromolecule synthesis process is more suitable for the rapid proliferation of tumor cells. In particular, it should be pointed out that metabolic abnormalities are not only the result of tumor occurrence and development, but may also be directly involved in the initiation process of tumors. At present, the development of antitumor drugs from the perspective of metabolism has been successfully applied in many tumors. For example, recent studies have found that some genes encoding metabolic enzymes are themselves oncogenes, such as the mutation of fumarate carboxylase and The occurrence and development of kidney cancer; the mutation of succinate carboxylase and the occurrence and development of pheochromocytoma; the mutation of isocitrate dehydrogenase produces the metabolite 2HG and the expression of HNF4α to induce cholangiocarcinoma, etc. Based on the above analysis, targeting tumor cell metabolism may be one of the effective ways to kill tumor cells. However, the currently developed drugs are all small molecule inhibitors with weak targeting and many side effects, while bioprotein drugs are absorbed quickly and have strong targeting. Therefore, the development of new targeted biological protein preparations for liver cancer is currently a hot spot in tumor research.
发明内容Contents of the invention
本发明经过大量的研究发现，作为参与糖脂代谢网络多个环节的重要成员的人成纤维细胞生长因子21可以显著抑制肝癌的发生发展以及转移。基于此，本发明提供了FGF21类似物在制备预防、缓解和/或治疗肝癌的药物方面的新用途。After extensive research, the present invention finds that human fibroblast growth factor 21, an important member involved in multiple links of the glucose and lipid metabolism network, can significantly inhibit the development and metastasis of liver cancer. Based on this, the present invention provides a new application of FGF21 analogues in the preparation of drugs for preventing, alleviating and/or treating liver cancer.
本发明提供了FGF21类似物，所述FGF21蛋白类似物氨基酸序列如SEQ ID NO.1～3所示任一。The present invention provides FGF21 analogues, and the amino acid sequence of the FGF21 protein analogues is any one of SEQ ID NO.1-3.
本发明提供了编码所述FGF21蛋白类似物的基因。The present invention provides the gene encoding the FGF21 protein analogue.
本发明提供了携带所述基因的载体和/或微生物细胞。The present invention provides vectors and/or microbial cells carrying the genes.
在一种实施方式中，所述微生物细胞包括大肠杆菌或哺乳动物细胞。In one embodiment, the microbial cells comprise E. coli or mammalian cells.
在一种实施方式中，所述哺乳动物细胞包括293T细胞或CHO细胞。In one embodiment, the mammalian cells comprise 293T cells or CHO cells.
本发明提供了所述基因在筛选具有治疗肝癌的药物中的应用。The invention provides the application of the gene in screening drugs for treating liver cancer.
在一种实施方式中，所述FGF21基因作为药物靶标。In one embodiment, the FGF21 gene is used as a drug target.
在一种实施方式中，所述FGF21基因的核苷酸序列如SEQ ID NO.4～6任一所示。In one embodiment, the nucleotide sequence of the FGF21 gene is shown in any one of SEQ ID NO.4-6.
在一种实施方式中，所述应用是用于筛选FGF21蛋白的小分子激活剂。In one embodiment, the use is for screening small molecule activators of FGF21 protein.
在一种实施方式中，所述应用是用于筛选抑制甲胎蛋白表达的试剂。In one embodiment, the use is for screening agents that inhibit the expression of alpha-fetoprotein.
本发明提供了一种药物或药物组合物，所述药物或药物组合物含有所述FGF21类似物。The present invention provides a medicament or a pharmaceutical composition, which contains the FGF21 analogue.
在一种实施方式中，所述药物或药物组合物还包括人体可接受的修饰、药用载体和/或辅料。In one embodiment, the drug or pharmaceutical composition further includes human acceptable modifications, pharmaceutical carriers and/or excipients.
在一种实施方式中，所述修饰包括PEG修饰、FC修饰。In one embodiment, the modification includes PEG modification and FC modification.
在一种实施方式中，所述药用辅料包括溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、粘合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗粘合剂、整合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂。In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers , Surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants.
本发明提供了所述FGF21类似物在制备预防、缓解和/或治疗癌症的药物或药物组合物中的应用。The present invention provides the application of the FGF21 analog in the preparation of drugs or pharmaceutical compositions for preventing, alleviating and/or treating cancer.
在一种实施方式中，所述癌症包括但不限于肝癌、前列腺癌、乳腺癌、食管癌、结肠直肠腺癌、宫颈癌、子宫内膜癌、卵巢癌、胰腺癌、胆囊癌、透明细胞肾癌、黑素瘤、多发性骨髓瘤。In one embodiment, the cancer includes but not limited to liver cancer, prostate cancer, breast cancer, esophageal cancer, colorectal adenocarcinoma, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, clear cell renal cancer Carcinoma, melanoma, multiple myeloma.
在一种实施方式中，所述预防、缓解和/或治疗肝癌包括但不限于(a)～(d)：In one embodiment, the prevention, alleviation and/or treatment of liver cancer includes but not limited to (a)-(d):
(a)降低肝癌因子的表达水平；(a) reducing the expression level of liver cancer factors;
(b)降低肿瘤质量、减少肿瘤体积；(b) reduce tumor mass and tumor volume;
(c)抑制肿瘤细胞的转移；(c) inhibiting the metastasis of tumor cells;
(d)抑制肿瘤的生成、生长和增殖。(d) Inhibition of tumor formation, growth and proliferation.
在一种实施方式中，所述肝癌因子为甲胎蛋白。In one embodiment, the liver cancer factor is alpha-fetoprotein.
在一种实施方式中，所述FGF21类似物的有效剂量为0.1～100mg/kg。In one embodiment, the effective dose of the FGF21 analog is 0.1-100 mg/kg.
在一种实施方式中，所述药物或药物组合物的给药途径包括皮内注射、皮下注射、静脉注射、肌肉注射、腹腔注射、静脉滴注、动脉注射、体腔内注射和/或口服。In one embodiment, the administration route of the drug or pharmaceutical composition includes intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, arterial injection, intracavitary injection and/or oral administration.
本发明的有益效果：Beneficial effects of the present invention:
(1)本发明获得FGF21融合蛋白能够作用与肝癌的治疗中，可显著降低肿瘤病灶的大小，因而可作为各种剂型药物的有效成分。(1) The FGF21 fusion protein obtained in the present invention can be used in the treatment of liver cancer, and can significantly reduce the size of tumor lesions, so it can be used as an active ingredient of various dosage forms of drugs.
(2)本发明将含有FGF21类似的药品通过注射的方式注射入体内，可有效降低肝癌症因子的表达水平、缓解肝癌的发生和发展、抑制肝癌的生成和转移，对于肝癌的治疗具有极其重大的意义。(2) In the present invention, drugs similar to FGF21 are injected into the body by injection, which can effectively reduce the expression level of liver cancer factors, alleviate the occurrence and development of liver cancer, and inhibit the generation and metastasis of liver cancer, which is extremely important for the treatment of liver cancer. meaning.
附图说明Description of drawings
图1为FGF21对人肝癌移植瘤生长速度的影响；其中，A为肿瘤体积的变化；B为肿瘤质量的变化；Figure 1 is the effect of FGF21 on the growth rate of human liver cancer xenograft tumor; wherein, A is the change of tumor volume; B is the change of tumor mass;
图2为FGF21对四氯化碳诱导的小鼠肝癌的影响；其中，A为甲胎蛋白含量；B为肝癌切片。Figure 2 is the effect of FGF21 on carbon tetrachloride-induced liver cancer in mice; wherein, A is the content of alpha-fetoprotein; B is liver cancer slices.
具体实施方式Detailed ways
实验动物及饲养：裸鼠及C57BL/6小鼠购于江苏集萃药康生物科技有限公司。饲养于江南大无锡医学院动物中心，每12小时交替照明，温度20±2℃。Experimental animals and breeding: Nude mice and C57BL/6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. They were raised in the Animal Center of Wuxi Medical College, Jiangnan University, under alternating lighting every 12 hours, and the temperature was 20±2°C.
细胞培养：肝癌细胞系HepG2由中国科学院生物化学与细胞生物学研究所提供；Cell culture: the liver cancer cell line HepG2 was provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences;
DMEM、0.05％Trypsin，购于博士德公司；胎牛血清购于四季青公司。其他药品为国产分析纯。肝癌细胞系贴壁生长于含10％胎牛血清的DMEM培养液中，于37℃，5％CO 2湿化培养箱中培养，隔天传代一次。 DMEM and 0.05% Trypsin were purchased from Boster Company; fetal bovine serum was purchased from Sijiqing Company. Other medicines were domestic analytically pure. The hepatocarcinoma cell lines were grown adherently in DMEM medium containing 10% fetal bovine serum, cultured in a humidified incubator with 5% CO 2 at 37°C, and passaged once every other day.
下述实施例中对小鼠注射1mg/kg、5mg/kg和10mg/kg的FGF21蛋白，分别对应的人体剂量为0.1mg/kg、0.5mg/kg和1mg/kg。In the following examples, mice were injected with 1 mg/kg, 5 mg/kg and 10 mg/kg of FGF21 protein, and the corresponding human doses were 0.1 mg/kg, 0.5 mg/kg and 1 mg/kg.
实施例1：重组蛋白的构建、表达及纯化Example 1: Construction, expression and purification of recombinant proteins
(1)FGF21蛋白表达载体的构建(1) Construction of FGF21 protein expression vector
将融合FGF21蛋白(氨基酸序列如SEQ ID NO.1～3所示)，根据大肠杆菌密码子偏好性，设计出对应的核苷酸序列，其核苷酸序列分别如序列表中FGF21-1(核苷酸序列如SEQ ID  NO.4所示)、FGF21-2(核苷酸序列如SEQ ID NO.5所示)、FGF21-3(核苷酸序列如SEQ ID NO.6所示)所示。将这3种基因送至公司合成，将3个基因连接至pET30a(+)载体的NdeI与BamHI酶切位点间，将酶连产物分别转化至大肠杆菌DH5α中。挑取阳性克隆，经过酶切鉴定后，即分别构建得到3种重组质粒pET30a-FGF21-1、pET30a-FGF21-2和pET30a-FGF21-3。The fusion FGF21 protein (amino acid sequence is shown in SEQ ID NO.1～3), according to the codon preference of Escherichia coli, the corresponding nucleotide sequence is designed, and its nucleotide sequence is respectively as FGF21-1 in the sequence list ( Nucleotide sequence as shown in SEQ ID NO.4), FGF21-2 (nucleotide sequence as shown in SEQ ID NO.5), FGF21-3 (nucleotide sequence as shown in SEQ ID NO.6) Show. The three genes were sent to the company for synthesis, and the three genes were connected between the NdeI and BamHI restriction sites of the pET30a(+) vector, and the enzyme-ligated products were transformed into E. coli DH5α respectively. The positive clones were picked and identified by restriction enzyme digestion, and three recombinant plasmids pET30a-FGF21-1, pET30a-FGF21-2 and pET30a-FGF21-3 were respectively constructed.
(2)蛋白的表达及纯化(2) Protein expression and purification
将步骤1得到的含有正确序列的重组质粒分别转化至表达菌株Rosseta(DE3)感受态细胞中。转化后的单菌落分别接种至20mL含Kan(50μg/mL)的LB培养基中，37℃培养8h，以体积比为1:100接种于另一20mL含Kan(50μg/mL)的LB培养基中，37℃培养，当A600在0.35左右时，加入IPTG至终浓度为0.25mmol/L进行诱导，诱导温度为30℃，5h后收获菌体，用Lysis buffer(20mmol/LTris，150mmol/LNaCl，pH8.0)重悬菌体，破碎菌体后离心，分别取上清和沉淀进行12wt％SDS-PAGE电泳分析。并对菌体进行破碎、并纯化蛋白得到纯化后的FGF21-1、FGF21-2和FGF21-3，其对应的氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示。The recombinant plasmids containing the correct sequence obtained in step 1 were respectively transformed into competent cells of the expression strain Rosseta (DE3). Transformed single colonies were inoculated into 20 mL of LB medium containing Kan (50 μg/mL), cultured at 37 °C for 8 h, and inoculated into another 20 mL of LB medium containing Kan (50 μg/mL) at a volume ratio of 1:100 medium, cultured at 37°C, when A600 was around 0.35, IPTG was added to a final concentration of 0.25mmol/L for induction, the induction temperature was 30°C, and the cells were harvested after 5h, and Lysis buffer (20mmol/LTris, 150mmol/LNaCl, pH 8.0) to resuspend the bacteria, break the bacteria and centrifuge, take the supernatant and precipitate respectively for 12wt% SDS-PAGE electrophoresis analysis. And the bacteria were crushed, and the protein was purified to obtain purified FGF21-1, FGF21-2 and FGF21-3, and the corresponding amino acid sequences were as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 shown.
实施例2：FGF21对人肝癌移植瘤生长速度的影响Example 2: Effect of FGF21 on growth rate of human liver cancer xenografts
按照实施例1的方法制备得到氨基酸序列如SEQ ID NO.1所示的FGF21蛋白。The FGF21 protein whose amino acid sequence is shown in SEQ ID NO.1 is prepared according to the method of Example 1.
将人肝癌细胞HepG2细胞按照1×10 6个细胞/只接种6周龄的雄性裸鼠的皮下，待肿瘤长至200mm 3随机分为四组，①生理盐水组：注射等体积生理盐水；②低剂量组：注射1mg/kg FGF21；③中剂量组：注射5mg/kg FGF21；④高剂量组：注射10mg/kg FGF21。每天注射一次，连续注射15天。每天监测肿瘤体积，三周后处死小鼠，称量肿瘤重量。结果显示：三种不同剂量的FGF21均能抑制移植瘤体积和最终肿瘤重量，且呈现剂量依赖性。高剂量FGF21蛋白(10mg/kg)注射后，可完全抑制肿瘤生长，使肿瘤维持在250mm 3，最终体积仅为对照组的20％，同样肿瘤质量也下降了约70％，注射1mg/kg和5mg/kg分别能使肿瘤质量下降45％和53％(如图1所示)。 Human liver cancer cell HepG2 cells were inoculated under the skin of 6-week-old male nude mice according to 1× 106 cells/only, and were randomly divided into four groups when the tumor grew to 200mm3 . ①Normal saline group: injected with an equal volume of normal saline;② Low-dose group: inject 1 mg/kg FGF21; ③ medium-dose group: inject 5 mg/kg FGF21; ④ high-dose group: inject 10 mg/kg FGF21. Inject once a day for 15 consecutive days. Tumor volume was monitored daily, mice were sacrificed three weeks later, and tumor weight was measured. The results showed that three different doses of FGF21 could inhibit the transplanted tumor volume and final tumor weight in a dose-dependent manner. After injection of high-dose FGF21 protein (10mg/kg), it can completely inhibit tumor growth, keep the tumor at 250mm 3 , and the final volume is only 20% of that of the control group, and the tumor mass also decreased by about 70%. 5mg/kg can reduce tumor mass by 45% and 53% respectively (as shown in Figure 1).
实施例3：FGF21对四氯化碳诱导的小鼠肝癌的影响Example 3: Effect of FGF21 on mouse liver cancer induced by carbon tetrachloride
按照实施例1的方法制备氨基酸序列如SEQ ID NO.1所示的FGF21蛋白。Prepare the FGF21 protein whose amino acid sequence is shown in SEQ ID NO.1 according to the method of Example 1.
小鼠在出生后第14天，腹腔注射二乙基亚硝胺(DEN)2mg/kg，并在给药2周后，腹腔注射20％四氯化碳(CCI4)5mL/kg，每周2次共16周。诱导16周后开始分组为生理盐水组及FGF21注射组，FGF21注射组注射剂量为10mg/kg，生理盐水组注射同样量的生理盐水，每天注射一次，连续注射6周。On the 14th day after birth, the mice were intraperitoneally injected with 2 mg/kg of diethylnitrosamine (DEN), and 2 weeks after the administration, 5 mL/kg of 20% carbon tetrachloride (CCI4) was injected intraperitoneally, 2 times a week. A total of 16 weeks. After 16 weeks of induction, they were divided into normal saline group and FGF21 injection group. The injection dose of FGF21 injection group was 10 mg/kg, and the normal saline group was injected with the same amount of normal saline once a day for 6 consecutive weeks.
结果如图2A所示，图2A显示了FGF21治疗组和对照组的甲胎蛋白水平，甲胎蛋白含 量是肝癌的特征性标志物，表明氨基酸序列如SEQ ID NO.1所示的FGF21蛋白显著降低肝癌标志物含量，图2B显示了肿瘤的病理切片，进一步说明FGF21蛋白对肝癌的抑制效果。The results are shown in Figure 2A, and Figure 2A shows the alpha-fetoprotein levels of the FGF21 treatment group and the control group, and the alpha-fetoprotein content is a characteristic marker of liver cancer, indicating that the amino acid sequence of the FGF21 protein shown in SEQ ID NO.1 significantly After reducing the content of liver cancer markers, Figure 2B shows the pathological section of the tumor, further illustrating the inhibitory effect of FGF21 protein on liver cancer.
发明人还尝试对SEQ ID NO.2～3所示的FGF21蛋白的融合蛋白在治疗肝癌方面的效果进行了研究，结果显示，如SEQ ID NO.2～3任一所示的FGF21融合蛋白与SEQ ID NO.1所示的FGF21蛋白具有相当的效果。The inventor also tried to study the effect of the fusion protein of FGF21 protein shown in SEQ ID NO. The FGF21 protein shown in SEQ ID NO.1 had comparable effects.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (13)

  1. FGF21类似物，其特征在于，所述FGF21蛋白类似物氨基酸序列如SEQ ID NO.1～3所示任一。FGF21 analogs, characterized in that the amino acid sequence of the FGF21 protein analogs is any one of SEQ ID NO.1-3.
  2. 编码权利要求1所述FGF21类似物的基因。A gene encoding the FGF21 analogue of claim 1.
  3. 携带权利要求2所述基因的载体和/或微生物细胞。Carriers and/or microbial cells carrying the genes of claim 2.
  4. 权利要求2所述基因在筛选具有治疗肝癌的药物中的应用。The application of the gene described in claim 2 in screening drugs for treating liver cancer.
  5. 根据权利要求4所述的应用，其特征在于，用于筛选FGF21蛋白的小分子激活剂。The application according to claim 4, characterized in that it is used for screening small molecule activators of FGF21 protein.
  6. 一种药物或药物组合物，其特征在于，含有权利要求1所述FGF21类似物。A medicine or pharmaceutical composition, characterized in that it contains the FGF21 analogue of claim 1.
  7. 根据权利要求6所述的药物或药物组合物，其特征在于，所述药物或药物组合物还包括人体可接受的修饰、药用载体和/或辅料。The medicament or pharmaceutical composition according to claim 6, characterized in that, the medicament or pharmaceutical composition further comprises human acceptable modifications, pharmaceutical carriers and/or adjuvants.
  8. 权利要求1所述FGF21类似物在制备预防、缓解和/或治疗癌症的药物或药物组合物中的应用。The use of the FGF21 analog according to claim 1 in the preparation of medicines or pharmaceutical compositions for preventing, alleviating and/or treating cancer.
  9. 根据权利要求8所述的应用，其特征在于，所述癌症包括但不限于肝癌、前列腺癌、乳腺癌、食管癌、结肠直肠腺癌、宫颈癌、子宫内膜癌、卵巢癌、胰腺癌、胆囊癌、透明细胞肾癌、黑素瘤、多发性骨髓瘤。The application according to claim 8, wherein the cancers include but not limited to liver cancer, prostate cancer, breast cancer, esophageal cancer, colorectal adenocarcinoma, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, Gallbladder carcinoma, clear cell renal carcinoma, melanoma, multiple myeloma.
  10. 根据权利要求8所述的应用，其特征在于，所述预防、缓解和/或治疗肝癌包括但不限于(a)～(d)：The application according to claim 8, wherein the prevention, alleviation and/or treatment of liver cancer includes but not limited to (a)-(d):
    (a)降低肝癌因子的表达水平；(a) reducing the expression level of liver cancer factors;
    (b)降低肿瘤质量、减少肿瘤体积；(b) reduce tumor mass and tumor volume;
    (c)抑制肿瘤细胞的转移；(c) inhibiting the metastasis of tumor cells;
    (d)抑制肿瘤的生成、生长和增殖。(d) Inhibition of tumor formation, growth and proliferation.
  11. 根据权利要求10所述的应用，其特征在于，所述肝癌因子为甲胎蛋白。The use according to claim 10, characterized in that the liver cancer factor is alpha-fetoprotein.
  12. 根据权利要求10所述的应用，其特征在于，所述FGF21类似物的有效剂量为0.1～100mg/kg。The use according to claim 10, characterized in that the effective dose of the FGF21 analog is 0.1-100 mg/kg.
  13. 根据权利要求10所述的应用，其特征在于，所述药物的给药途径包括皮内注射、皮下注射、静脉注射、肌肉注射、腹腔注射、静脉滴注、动脉注射、体腔内注射和/或口服。The application according to claim 10, wherein the route of administration of the drug comprises intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, arterial injection, intrabody injection and/or oral.
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