WO2008089645A1 - Polypeptide de fusion inhibant la croissance cellulaire et son utilisation - Google Patents

Polypeptide de fusion inhibant la croissance cellulaire et son utilisation Download PDF

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WO2008089645A1
WO2008089645A1 PCT/CN2007/071381 CN2007071381W WO2008089645A1 WO 2008089645 A1 WO2008089645 A1 WO 2008089645A1 CN 2007071381 W CN2007071381 W CN 2007071381W WO 2008089645 A1 WO2008089645 A1 WO 2008089645A1
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polypeptide
fusion polypeptide
cancer
cell growth
arginine
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PCT/CN2007/071381
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French (fr)
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Xianmin Xia
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Xianmin Xia
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a fusion polypeptide comprising at least two polypeptides, one polypeptide being a polypeptide which inhibits cell growth and the other polypeptide being a polypeptide fragment which can cross the cell membrane. These two polypeptides can be directly linked by chemical covalent interaction or expressed in a cell as a fusion polypeptide.
  • the present invention also relates to the expression and purification process of this fusion polypeptide in Escherichia coli, its use in the preparation of antitumor drugs, and its application in the treatment of tumors and other proliferative diseases, and belongs to the field of medical bioengineering. Background technique
  • Tumor is a disease that seriously endangers human health. At present, its mortality rate has become the second leading cause of death in China after cardiovascular and cerebrovascular diseases. Chemotherapy, radiotherapy and surgical treatment are mainly used in the clinic. These methods have formed a relatively mature treatment system and achieved good results. However, due to the low selectivity of these methods, it is difficult to avoid the disadvantages of strong side effects and small therapeutic window between normal cells and tumors, so it is urgent to develop new effective treatment methods.
  • ErbB2 an epithelial growth factor receptor that promotes protein phosphorylation, is overexpressed in many breast cancer cells, and its protein kinase activity is also enhanced. It can be effectively inhibited by a monoclonal antibody specific for this protein. ErbB2 protein kinase activity blocks this protein-mediated signal transduction. This antibody is currently in clinical use.
  • phosphatidylinositol-3-kinase is an enzyme protein with lipophosphorylation kinase and protein phosphorylation kinase activity, and its kinase activity is affected by many signaling molecules. Due to the extremely important role of phosphatidylinositol-3-kinase in cell growth regulation, this protein is one of the important targets for research and development of new drugs for the treatment of tumors.
  • a regulatory subunit of the cell growth cycle regulatory protein a neuroblastoma protein (Rb), and a phosphatidylinositol-3-kinase.
  • Phosphatidylinositol-3-kinase is composed of a regulatory subunit that catalyzes the phosphorylation of serine and threonine on certain lipids and proteins.
  • the phosphatidylinositol 3-kinase protein is composed of a regulatory subunit and a catalytic subunit.
  • the core effect component however, the activity of the catalytic subunit is regulated by a regulatory subunit, which is achieved by two mechanisms: (1) the activity of the catalytic subunit itself is affected by the regulatory subunit; (2) the regulation subunit passage and The interaction of other proteins localizes the catalytic subunit to the target protein.
  • the object of the present invention is to provide a fusion polypeptide comprising: a polypeptide that inhibits cell growth and a polypeptide or molecule that penetrates a cell membrane, and a polypeptide or molecule that penetrates the cell membrane brings a polypeptide that inhibits cell growth into the cell, and has been experimentally proved This fusion peptide has inhibition
  • the fusion polypeptide is also effective in inhibiting tumor growth in a growth model of a plurality of tumor animals.
  • the fusion polypeptide comprises: a polypeptide that inhibits cell growth and a polypeptide or molecule that penetrates the cell membrane, a polypeptide that inhibits cell growth and a polypeptide or molecule that penetrates the cell membrane are chemically covalently linked, or a polypeptide that inhibits cell growth and a polypeptide that penetrates the cell membrane. Expressed as a fusion polypeptide in a cell.
  • the polypeptide that inhibits cell growth of the fusion polypeptide is linked to a tagged protein that purifies the fusion polypeptide.
  • the polypeptide that inhibits cell growth is a polypeptide consisting of 25 amino acid residues at the amino terminus of the regulatory subunit p55PIK of phosphatidylinositol-3-kinase, and its sequence is: methionine-aspartate-arginine- Aspartate-aspartate-alanine-aspartate-tryptophan-arginine-glutamate-valine-prosta-methionine-valine-proline-case -serine-threonine-glutamate-leucine-isoleucine-phenylalanine-tyrosine-isoleucine-glutamate-methionine (MDRDDADWREVMMP YSTELIFYIEM), or Is one of the polypeptides obtained by consecutively or intermittently deleting or substituting any number of amino acid residues in 1 to 12 from the amino terminus of the polypeptide, or having the sequence of the polypeptide obtained by addition of one or several amino acid residues
  • the polypeptide that penetrates the cell membrane is a polypeptide derived from the HIV virus tat protein, and its sequence is: tyrosine-glycine-arginine-lysine-lysine-arginine-arginine-glutamine-spermine Acid-arginine-arginine (YGRKKRRQRRR).
  • the tagged protein used to purify the fusion polypeptide is an oligohistidine tag in which the number of histidines is any number from 5-10.
  • the use of treatment in diseases associated with gold lymphoma, non-Hodgkin's lymphoma, breast cancer, gynecological tumors, prostate cancer, and genitourinary tumors and abnormal cell growth should be included.
  • the tat protein is one of the proteins with this ability, and its functional region that penetrates the cell membrane is one of the tat proteins.
  • the recombinant protein can be efficiently expressed by transforming a ribonucleic acid fragment encoding the corresponding polypeptide into E. coli.
  • a polypeptide consisting of 6 histidines (6xHis) is one of the commonly used tags to help affinity-purify recombinant proteins. This tag can effectively bind nickel ions or copper ions. This combination is stable, so it is combined with nickel ions. Or a copper ion agarose gel particle can effectively bind a recombinant protein containing a 6xHis tag.
  • the present invention utilizes molecular biological techniques to bind the N25 polypeptide and a corresponding functional region of the HIV virus tat protein having a cell membrane effect and a specific binding nickel or copper ion.
  • the oligohistidine tag (6xHis) is linked by three different functional polypeptides to construct a fusion polypeptide that is easy to produce and purify and that can penetrate the cell membrane to perform biological functions.
  • fusion proteins including N25 and transmembrane polypeptides in several different cultured human and mouse tumor cell lines with growth and division ability. The DNA synthesis of these cells and several other observations indicating cell growth are significantly reduced. This fusion polypeptide has the ability to inhibit cell growth. In addition, the fusion polypeptide is also effective in inhibiting tumor growth in a growth model in a variety of tumor animals.
  • the present invention Compared with the existing methods for inhibiting cell growth, the present invention has the following characteristics:
  • the fusion polypeptide consisting of the transmembrane domain of N25 and tat protein can effectively inhibit tumor growth, and it is proved that N25 polypeptide still has the biological effect of inhibiting cell growth after penetrating cell membrane, and also indicates that the membrane of tat protein is transmissive.
  • other polypeptides or molecules with similar membrane-permeating functions may also be used to help the biologically active N25 penetrate the cell membrane.
  • N25 polypeptide exists in the protein sequence of human cells, with low toxic and side effects and weak antigenicity. The experimental results also show that it has no obvious effect on apoptosis or death, and has no obvious killing effect on normal cells.
  • a fusion polypeptide consisting of N25, a transmembrane functional polypeptide of tat protein and an oligohistidine was added to cultured cells and applied to animals, and no significant toxicity was observed.
  • the fusion polypeptide can effectively inhibit the growth of various human and murine tumor cells in the in vivo and in vitro culture systems, and prove that the fusion polypeptide has the advantages of high efficiency and broad spectrum in the treatment of abnormal cells such as tumors.
  • FIG. 1 Purified fusion polypeptide.
  • agarose gel particles chelated with nickel ions were used to bind the fusion polypeptide expressed by Escherichia coli; the imidazole was eluted, centrifuged, and the supernatant was collected to obtain a fusion polypeptide; The eluate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • the results show that the nickel ion agarose gel particles can specifically bind to the 6xHis tag and efficiently purify the fusion polypeptide containing this tag, and the purity of the purified polypeptide can be more than 90% after purification.
  • Example 1 A cDNA sequence encoding a fusion polypeptide consisting of an oligohistidine tag (6xHis), a TAT transmembrane domain and p55PIK ⁇ 25 was introduced into the plasmid.
  • the plasmid containing the oligohistidine tag (6xHis) and the TAT transmembrane domain cDNA was presented by Dr. Lu Shijiang from Chicago, USA.
  • the plasmid was digested with Ecom-BamHl (purchased from Promega, USA), and the digested plasmid was separated and purified in agarose gel for subsequent ligation.
  • the kit for recovering DNA fragments from the gel was obtained from Qiagen, Germany (catalog number: 28704).
  • the cDNA encoding the mouse p55PIK full-length protein was derived from a mouse testis tissue cDNA library (Stratagene, USA, catalog number: 937308), cloned into pcDNA3 (purchased from Invitrogen, catalog number: V79020), used to amplify N25
  • the PCR primer sequence of cDNA is:
  • Primer 1 TTTTTGGTACCATGGACCGCGATGACGCAGAC
  • Primer 2 TTTTTGAATTCTCAAGGATCCATTTCAATATAAAA
  • the PCR kit was obtained from Promega, USA (catalog number: M1861), and the PCR product was purified (purification kit from Qiagen, Germany, catalog number: 28704), digested with Ecom-BamHl; agarose The gel was purified and then ligated with the enzymatically purified vector (the kit was obtained from Promega, USA, catalog number: M1801). After transformation of the bacteria (competent bacteria from Promega, USA, catalog number: L2001), the clones were screened by conventional colony PCR.
  • a positive clone was selected, and the correctness of the cDNA sequence was confirmed by nucleic acid sequence determination, and then a large-scale purification preparation plasmid (large-scale purification kit from Promega, USA, catalog number: A7270) was used for subsequent experiments.
  • This plasmid contains an operon that is induced by IPTG to express recombinant proteins in bacteria, and the plasmid is transferred to BL21 bacteria (Novagen, USA, catalog number: A7270) After that, a 6xHis tag, a TAT transmembrane domain and a fusion protein of N25 can be expressed. This protein is called TAT-N25.
  • Example 2 Laboratory production process and purification process of fusion polypeptide TAT-N25.
  • the purified TAT-N25 fusion polypeptide was expressed in the laboratory, and the specific procedure is as follows:
  • Example 3 TAT-N25 fusion polypeptide affects cell growth and cell cycle experiments.
  • TAT-N25 fusion polypeptide The effect of TAT-N25 fusion polypeptide on cell growth was examined using a human cervical cancer cell line, Hela cells purchased from ATCC, USA, catalog number: HTB-22. Hela cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ° C, 5% CO 2 /95% air culture in 10 cm cell culture sub. After the cells were cultured to logarithmic growth phase, the polypeptide was added. The final concentration was 15 ⁇ g/ml, and the blank group and the urea group were cultured for control, and the cells were cultured for 24 hours, and the cell cycle distribution of these cells was analyzed by flow cytometry.
  • 6M urine It refers to 6 mol of urea per liter of water, and the following is called 6 M urea.
  • 6 M urea 6 M urine
  • mice were randomly divided into three groups. One day after the injection of the tumor cells, the mice in the group were separately injected with 0.1 ml of TAT-N25 at a concentration of 25 mg/ml, and TAT-N25 was dissolved in 6 M of urea, and the injection was repeated three times every other day.
  • the two control groups were injected with 0.1 ml of normal saline or 6 M of urea, respectively, and the number of injections and time were the same as those of the peptide test group.
  • mice Ten days after the tumor was injected, the mice were sacrificed and ascites was taken from the peritoneal cavity for volume measurement and BrdU incorporation into DNA. The results showed that the ascites in the mice treated with TAT-N25 was reduced by 85% compared with the control group. Correspondingly, cell DNA synthesis is also significantly reduced. See Table 3.
  • TAT-N25 fusion polypeptide inhibits the growth of ascites S180 in mice The average incorporation of DNA in BrdU into the intraperitoneal ascites volume (ml)
  • Example 5 Another experiment in which TAT-N25 fusion polypeptide inhibits the growth of ascites tumor in mice
  • TAT-N25 was dissolved in 6M urea, and 0.2 ml of 20 mg/ml TAT-N25 and 0.2 ml of fat emulsion nutrient solution were mixed.
  • the fat emulsion nutrient solution was from Huarui Pharmaceutical Co., Ltd.
  • the mice in the treatment group were injected through the tail vein separately, and the injection was repeated five times every other day.
  • the control group was injected with 0.2 ml of 6 M urea and 0.2 ml of fat emulsion nutrient solution, and the number and time of injection were the same as those of the peptide treatment group.
  • Ten days after the injection of the tumor the mice were sacrificed and ascites was taken from the peritoneal cavity for volume measurement and BrdU incorporation into DNA detection. The results showed that compared with the control group, the ascites in the mice treated with TAT-N25 was reduced by 70%; correspondingly, the DNA synthesis of the cells was reduced by 30%.
  • mice After 10 6 murine malignant sarcoma S180 cells were subcutaneously injected into mice, the mice were randomly divided into two groups. One day after the injection of tumor cells, TAT-N25 was dissolved in 6M urea, and 0.2 ml of 20 mg/ml TAT-N25 and 0.2 ml of fat emulsion nutrient solution were mixed. The fat emulsion nutrient solution was from Huarui Pharmaceutical Co., Ltd. For Intolit, the mice in the treatment group were injected through the tail vein separately, and the injection was repeated five times every other day.
  • the control group was injected with 0.2 ml of 6 M urea and 0.2 ml of fat emulsion nutrient solution, and the number and time of injection were the same as those of the peptide treatment group.
  • Example 7 Experiment of TAT-N25 fusion polypeptide inhibiting tumor growth in human rectal cancer in mice
  • mice After subcutaneous injection of 5 ⁇ 10 6 human rectal cancer HT29 cells into mice, the mice were randomly divided into two groups. One day after the injection of tumor cells, TAT-N25 was dissolved in 6M urea, and 0.2 ml of 20 mg/ml TAT-N25 and 0.2 ml of fat emulsion nutrient solution were mixed. The fat emulsion nutrient solution was from Huarui Pharmaceutical Co., Ltd. For Intolit, the mice in the treatment group were injected through the tail vein separately, and the injection was repeated five times every other day.
  • the control group was injected with 0.2 ml of 6 M urea and 0.2 ml of fat emulsion nutrient solution, and the number and time of injection were the same as the peptide treatment.
  • Treatment group Ten days after the tumor was injected, the tumor was sacrificed and the tumor was taken out from the mouse for weight measurement. The results showed that the tumor weight of the mice treated with TAT-N25 was reduced by 75% compared with the control group.
  • Example 8 TAT-N25 fusion polypeptide inhibits tumor growth of human squamous adenocarcinoma in mice
  • mice After subcutaneous injection of 5 ⁇ 10 6 human squamous cell carcinoma FTC236 cells in mice, the mice were randomly divided into two groups. One day after the injection of tumor cells, TAT-N25 was dissolved in 6M urea, and 0.2 ml of 20 mg/ml TAT-N25 and 0.2 ml of fat emulsion nutrient solution were mixed. The fat emulsion nutrient solution was from Huarui Pharmaceutical Co., Ltd. For Intolit, mice in the treatment group were injected through the tail vein, and the injection was repeated ten times every other day.
  • the control group was injected with 0.2 ml of 6 M urea and 0.2 ml of fat emulsion nutrient solution, and the number and time of injection were the same as those of the peptide treatment group. Twenty days after the tumor was injected, the tumor was sacrificed and the tumor was taken out from the mouse for weight measurement. The results showed that the tumor weight of the mice treated with TAT-N25 was reduced by 75% compared with the control group.
  • Example 9 Experiment of TAT-N25 fusion polypeptide inhibiting tumor growth in human leukemia in mice:
  • mice After subcutaneous injection of 5 ⁇ 10 6 human granulocyte leukemia HL60 cells in mice, the mice were randomly divided into two groups. One day after the injection of tumor cells, TAT-N25 was dissolved in 6M urea, and 0.2 ml of 20 mg/ml TAT-N25 and 0.2 ml of fat emulsion nutrient solution were mixed. The fat emulsion nutrient solution was from Huarui Pharmaceutical Co., Ltd. For Intolit, mice in the treatment group were injected through the tail vein, and the injection was repeated seven times every other day.
  • the control group was injected with 0.2 ml of 6 M urea and 0.2 ml of fat emulsion nutrient solution, and the number and time of injection were the same as those of the peptide treatment group. Fourteen days after the tumor was injected, the tumor was sacrificed and the tumor was taken out from the mouse for weight measurement. The results showed that the tumor weight of the mice treated with TAT-N25 was reduced by 50% compared with the control group.

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Description

抑制细胞生长的融合多肽及其应用 技术领域
本发明涉及一种至少包括两个多肽的融合多肽， 一个多肽为抑制 细胞生长的多肽， 另一个多肽为能穿过细胞膜的多肽片段。 这两个多 肽可以通过化学共价健直接连接或在细胞中表达成融合多肽。 同时， 本发明还涉及这一融合多肽在大肠杆菌中的表达和纯化过程、 在制备 抗肿瘤药物中的用途以及在肿瘤及其它增殖性疾病治疗中的应用， 属 于医学生物工程领域。 背景技术
肿瘤是一种严重危害人类健康的疾病， 目前其死亡率在国内已成 为仅次于心脑血管疾病的第二位致死病因。 临床中主要应用化疗、 放 疗及手术治疗， 这些方法形成了比较成熟的治疗体系， 取得了较好的 效果。 然而， 由于这些方法选择性低， 难以避免毒副作用强、 正常细 胞和肿瘤之间治疗窗口小等缺点， 因此亟待研发新的有效治疗方法。
近来肿瘤细胞生物学的快速发展， 为满足这一社会需求提供了现 实的可能性， 其中， 随着细胞的信号转导途径 (Signaling transduction pathways)的阐明， 发现了越来越多可用作治疗肿瘤的药靶。 有一些已 经成功的应用于临床， 取得了非常好的效果。 比如在许多乳腺癌细胞 中 ErbB2 (一种能促进蛋白磷酸化的上皮生长因子受体 )过度表达， 并 且其蛋白激酶活性也增强， 应用一种特异性针对这一蛋白单克隆抗体 能有效的抑制 ErbB2蛋白激酶活性， 阻断这一蛋白介导的信号转导。 这一抗体目前已进入临床使用。
细胞生长的信号由细胞内外促进生长的因子经过一系列蛋白参与 传递到细胞核， 引起细胞周期的许多调节蛋白结构和功能变化而导致 细胞分裂。 在这些调节细胞生长周期的蛋白中， 磷脂酰肌醇 -3-激酶 ( PI3K )是一个具有脂磷酸化激酶和蛋白磷酸化激酶活性的酶蛋白， 其激酶活性受到许多信号传递分子的影响。 由于磷脂酰肌醇 -3-激酶在 细胞生长调节中的极重要的作用， 这一蛋白是目前研究开发治疗肿瘤 新药的重要靶标之一。 至今为止， 已发现多个磷脂酰肌醇 -3-激酶激酶 活性的抑制剂， 但由于磷脂酰肌醇 -3-激酶的酶活性也是体内一些重要 生理功能所必需的 （比如胰岛素调节人体内血糖和其它物质和能量代 谢过程） ， 而这些抑制剂在抑制磷脂酰肌醇 -3-激酶激酶活性的同时， 往往也阻断了这些生理功能， 具有^艮强的毒性， 所以在治疗疾病中的 应用受到较大限制。
2003年， 我们发现了细胞生长周期的调节蛋白一视神经母细胞瘤 蛋白 （Rb )和磷脂酰肌醇 -3-激酶的一个调节亚基的相互作用。 磷脂酰 肌醇 -3-激酶由调节亚基和催化亚基组成， 催化亚基催化某些脂类及蛋 白质上的丝氨酸和苏氨酸磷酸化的活性是磷脂酰肌醇 -3 -激酶蛋白的核 心效应组成部分， 然而， 催化亚基的活性受调节亚基的调控， 调控通 过以下两个机制实现： ( 1 ) 催化亚基的活性本身受调节亚基影响；（2 ) 调节亚基通过和其他蛋白的相互作用把催化亚基定位于目标蛋白。 我 们研究发现， 视神经母细胞瘤蛋白和磷脂酰肌醇 -3-激酶的相互作用是 由磷脂酰肌醇 -3-激酶中的 55千道尔顿的调节亚基（p55PIK )中氨基端 1-25号氨基酸（序列为： 曱硫氨酸 -天门冬氨酸 -精氨酸-天门冬氨酸-天 门冬氨酸 -丙氨酸-天门冬氨酸-色氨酸 -精氨酸-谷氨酸-缬氨酸 -曱硫氨酸 -曱硫氨酸-脯氨酸 -酪氨酸-丝氨酸-苏氨酸 -谷氨酸-亮氨酸-异亮氨酸-苯 丙氨酸-酪氨酸 -异亮氨酸-谷氨酸 -曱硫氨酸）参与的， 该多肽片段被称 为视神经母细胞瘤蛋白结合功能区 （以下称为 N25 ), 它把磷脂酰肌醇 -3-激酶和视神经母细胞瘤蛋白相互连接在一起， 磷脂酰肌醇 -3-激酶的 蛋白激酶活性导致了视神经母细胞瘤蛋白的磷酸化， 从而引起了细胞 分裂。我们进一步的实验结果证明在细胞中表达 p55PIK中 Ν25的多肽， 这一多肽能有效地结合于视神经母细胞瘤蛋白， 竟争性抑制细胞内磷 脂酰肌醇 -3-激酶和视神经母细胞瘤蛋白的结合， 由于这个多肽不具有 任何磷酸化蛋白激酶活性， 所以多肽结合视神经母细胞瘤蛋白将抑制 视神经母细胞瘤蛋白的磷酸化过程， 保持视神经母细胞瘤蛋白抑制细 胞生长的功能， 从而阻止肿瘤细胞分裂。
除视神经母细胞瘤蛋白外， 我们新的的实验结果也证实 Ν25多肽 在细胞内还能阻止 ρ55ΡΙΚ和其他细胞周期调节分子的结合，也能有效 地抑制细胞的分裂过程。
虽然试验结果证明在细胞中表达的 Ν25 多肽能有效抑制细胞生 长， 但由于 Ν25多肽自身不能穿过细胞膜， 导致直接应用 Ν25多肽作 为药物的疗效并不理想， 另外采用化学合成 Ν25多肽的成本较高， 所 以 Ν25多肽药物的临床应用受到了一定的限制。 发明内容
本发明的目的提供一种融合多肽， 该融合多肽包括： 抑制细胞生 长的多肽和穿透细胞膜的多肽或分子， 穿透细胞膜的多肽或分子将抑 制细胞生长的多肽带入细胞中， 经过实验证明了这一融合多肽具有抑 制细胞生长的能力， 此外， 该融合多肽也能有效地抑制多种肿瘤动物 体内生长模型中的肿瘤生长。
融合多肽包括： 抑制细胞生长的多肽和穿透细胞膜的多肽或分子， 抑制细胞生长的多肽与穿透细胞膜的多肽或分子通过化学共价健连 接， 或抑制细胞生长的多肽与穿透细胞膜的多肽在细胞中表达成融合 多肽。
融合多肽的抑制细胞生长的多肽与纯化融合多肽的标签蛋白连 接。
抑制细胞生长的多肽是由磷脂酰肌醇 -3-激酶的调节亚基 p55PIK 中氨基端 25个氨基酸残基组成的多肽， 其序列为：曱硫氨酸-天门冬氨 酸 -精氨酸-天门冬氨酸-天门冬氨酸 -丙氨酸-天门冬氨酸-色氨酸-精氨酸 -谷氨酸-缬氨酸-曱硫氨酸-曱硫氨酸 -脯氨酸-酪氨酸-丝氨酸 -苏氨酸-谷 氨酸-亮氨酸-异亮氨酸-苯丙氨酸 -酪氨酸-异亮氨酸 -谷氨酸-曱硫氨酸 (MDRDDADWREVMMP YSTELIFYIEM) ,或是从该多肽氨基端连续或 间断地缺失或取代 1 - 12 中任意数值个氨基酸残基得到的多肽之一， 或是将所述多肽的序列经过一个或几个氨基酸残基的添加得到的具有 抑制细胞生长活性的多肽之一。
穿透细胞膜的多肽是衍生于 HIV病毒 tat蛋白的多肽， 其序列为： 酪氨酸-甘氨酸 -精氨酸-赖氨酸-赖氨酸 -精氨酸-精氨酸-谷酰胺 -精氨酸- 精氨酸-精氨酸 (YGRKKRRQRRR)。
用于纯化融合多肽的标签蛋白是寡聚组氨酸标签， 其中组氨酸数 目为 5-10中的任意数值个。
所述的融合多肽相应的核酸编码序列。
所述的融合或融合多肽相应的核酸编码序列多肽在治疗结肠癌、 食管癌、 胃癌、 胃肠道肿瘤、 耳鼻喉及头颈部肿瘤、 肺癌、 肝癌、 肉 瘤、 内分泌活化肿瘤、 白血病、 何杰金淋巴瘤、 非何杰金淋巴瘤、 乳 腺癌、 妇科肿瘤、 前列腺癌和泌尿生殖器官肿瘤及细胞生长异常有关 的疾病中治疗的用途。 应包括含有或应用该多肽或其片段以药学上可 接受的任何方式进行组合的内外用于治疗肿瘤及细胞生长异常有关的 疾病的用途。
大量实验证实， 自然界中存在一些具有穿透细胞膜能力的蛋白。 在这些蛋白中的一些片段是蛋白穿过细胞膜必需的， 被称为蛋白 （多 肽）传导功能区 （ protein/peptide transduction domain )或细胞穿透多肽 ( cell-penetrating peptides ) ,人类免疫缺陷病毒 (HIV)中的 tat蛋白便是 具备这种能力的蛋白之一， 其穿透细胞膜的功能区是 tat蛋白中的一个 多肽片段，序列为 YGRK RRQRRR。该片段能把其它自身不能穿透细 胞膜的生物大分子带进细胞。 在大肠杆菌中把编码相应多肽的核糖核 酸片断转化进去后， 能够有效地表达重组蛋白。 如果在重组蛋白中加 入能帮助亲合纯化重组蛋白的标签， 将极大地提高多肽产量， 降低生 产成本。 由 6个组氨酸组成的多肽 (6xHis)是常用的帮助亲合纯化重组 蛋白的标签之一， 该标签能有效结合镍离子或铜离子， 这种结合 4艮稳 定， 所以采用结合有镍离子或铜离子的琼脂糖凝胶颗粒能有效地结合 含有 6xHis标签的重组蛋白。
为了提高 N25多肽抑制细胞生长的效率， 本发明利用分子生物学 技术， 将 N25多肽和一种具有穿过细胞膜作用的 HIV病毒 tat蛋白中 相应的功能区及一种能特异性结合镍或铜离子的寡聚组氨酸标签 (6xHis)三个不同功能的多肽连接起来， 构建了一种易于生产纯化、 且 能够穿透细胞膜发挥生物学功能的融合多肽。
我们在几种不同的具有生长分裂能力的培养人类和老鼠肿瘤细胞 系中加入包括 N25和穿膜多肽的融合蛋白， 这些细胞的 DNA合成及 其他几个证明细胞生长的观察指标都显著降低， 证明了这一融合多肽 具有抑制细胞生长的能力。 此外， 该融合多肽也能有效地抑制多种肿 瘤动物体内生长模型中的肿瘤生长。
与现有抑制细胞生长的方法相比， 本发明有以下特点：
( 1 ) N25与 tat蛋白中的透膜功能区组成的融合多肽能有效地抑 制肿瘤生长， 证明 N25多肽在穿透细胞膜后仍具有抑制细胞生长的生 物学效应， 也说明除 tat蛋白的穿膜功能片段外， 其它有类似穿膜功能 的多肽或分子也可能用于帮助具有生物学活性 N25穿透细胞膜。
( 2 ) N25多肽的序列存在于人类细胞的蛋白质序列中， 毒副作用 低， 抗原性弱， 实验结果也表明其对细胞凋亡或死亡没有明显影响， 对正常细胞没有明显的杀伤作用。 由 N25、 tat蛋白的穿膜功能多肽和 寡聚组氨酸组成的融合多肽加入培养细胞和应用于动物体内， 也没有 观察到明显的毒性。
( 3 )融合多肽能有效抑制多种人类和鼠类肿瘤细胞在体内和体外 培养系统中的生长， 证明该融合多肽在肿瘤等细胞生长异常疾病的治 疗方面具有高效、 广谱等优点。
( 4 )利用大肠杆菌表达融合多肽并采用亲合纯化融合蛋白， 不仅 筒化了生产过程， 提高了生产效率， 而且降低了生产成本， 便于大规 模生产并直接应用于临床。 附图说明
附图 1 : 纯化后的融合多肽。 按实施实例 2所述的工艺流程， 采用 螯合有镍离子的琼脂糖凝胶颗粒， 结合大肠杆菌表达出来的融合多肽； 加入咪唑洗脱、 离心、 收集上清液得到融和多肽； 取 5微升洗脱液进 行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测。 结果表明， 镍离子的 琼脂糖凝胶颗粒能够特异性结合 6xHis 标签并有效地纯化含有这一标 签的融合多肽， 纯化后融合多肽的纯度可达 90%以上。 具体实施方式
例 1: 在质粒中引入编码寡聚组氨酸标签 (6xHis), TAT穿膜功能 区和 p55PIK Ν 2 5组成的融合多肽的 cDNA序列。
含有编码寡聚组氨酸标签 (6xHis) , TAT穿膜功能区 cDNA序列的 质粒由美国芝加哥的卢世江博士惠赠。 质粒用 Ecom-BamHl (购于美 国 Promega公司）酶切质粒， 酶切后的质粒在琼脂糖胶中分离、 纯化， 用于以后的连接反应。从胶中回收 DNA片段的试剂盒来自德国 Qiagen 公司 （产品目录号为： 28704 )。 编码小鼠 p55PIK全长蛋白的 cDNA来 源于小鼠睾丸组织 cDNA 文库 （美国 Stratagene公司， 目录号为： 937308 ),克隆到 pcDNA3 (购于 Invitrogen公司， 目录号为： V79020 ), 用于扩增 N25 cDNA的 PCR引物序列为：
引物 1： TTTTTGGTACCATGGACCGCGATGACGCAGAC
引物 2： TTTTTGAATTCTCAAGGATCCATTTCAATATAAAA
PCR条件：
94 °C 2分钟；
94 °C 1分钟、 55 °C 1分钟、 72 °C 2分钟共 25个循环；
72 °C 延伸 5分钟。
PCR试剂盒来自美国 Promega公司 （目录号为： M1861 ), PCR产 物经过纯化后（纯化用的试剂盒来自德国 Qiagen公司，产品目录号为： 28704 ), 用 Ecom-BamHl酶切; 再用琼脂糖胶纯化， 然后和酶切纯化 后的载体进行连接反应 (连接试剂盒来自美国 Promega公司， 目录号 为： M1801 )。 转化细菌后 （感受态细菌来自美国 Promega公司， 目录 号为： L2001 ),克隆用常规菌落 PCR进行筛选。选出阳性克隆，其 cDNA 序列的正确性通过核酸序列测定得到证实后，大规模纯化制备质粒（大 规模纯化试剂盒来自美国 Promega公司， 目录号为： A7270 ), 用于以 后的实验。 这种质粒含有在细菌中受 IPTG诱导表达重组蛋白的操纵 子，把质粒转移到 BL21细菌中（美国 Novagen公司，目录号为： A7270 ) 后， 即可表达一个 6xHis标签， TAT穿膜功能区和 N25的融合蛋白。 该蛋白称为 TAT-N25。
例 2: 融合多肽 TAT-N25的实验室生产流程及纯化工艺。
用含有表达融合多肽质粒的 BL21大肠杆菌细胞培养后，在实验室 开始表达纯化 TAT-N25融合多肽， 具体流程如下：
(1)、 在超净工作台上接种 50微升菌种于 100毫升的浓度为 100微 克 /毫升含氨苄青霉素的 LB细菌培养基中， 然后在 37°C摇床上以 220 转 /分钟的速度进行培养过夜； （2)、 加入 700毫升含氨苄青霉素的培养 基继续培养 2小时； （3)、 加入 300微升浓度 0.2毫摩尔 /升异丙基 -β-D- 硫代半乳糖苷（IPTG )诱导细菌表达融合多肽， 培养 16 小时； （4)、 离心收集细菌， 离心是在转速为 3600转 /分钟、 4°C的条件下离心 5分 钟， 倒去上清液， 然后先加入 20毫升 6摩尔 /升的尿素到收集的细菌， 并将细菌抽提液转移倒 50毫升离心管中； （5)、 在超声波细胞破碎仪上 超声破碎细菌， 超声波细胞破碎仪功率 300瓦， 工作 3秒， 间隔 3秒、 冰浴条件下， 破碎 6分钟， 细菌破碎后在摇床上混匀 1小时； （6)、 离 心分离上清液， 离心是在转速为 10000转 /分钟、 4°C的条件下离心 15 分钟， 将上清液转移到另一支 50毫升离心管中； （7)、 在上清液中加入 600微升螯合有镍离子的琼脂糖凝胶颗粒，该螯合有镍离子的琼脂糖凝 胶颗粒由美国 Promega公司提供， 混匀并在摇床上混合 2小时， 然后 在 3600转 /分钟、 4°C条件下离心 5分钟， 除去上清液将琼脂糖凝胶颗 粒转移到 1.5毫升离心管中； （8)、 加入 1毫升 6摩尔 /升的尿素于 1.5 毫升离心管中在 5000转 /分钟、常温条件下离心 1分钟来离心清洗琼脂 糖凝胶颗粒， 重复 3遍； （9)、 加入 10ml浓度为 200毫摩尔 /升咪唑洗 脱多肽， 在摇床上混匀 1 小时， 然后在 5000 转 /分钟、 室温条件下离 心 1分钟， 收集含有洗脱的融和多肽上清液， 重复 2次； （10)、 浓缩纯 化将多肽转入截流分子量为 3500道尔顿的透析袋，再于 6摩尔 /升的尿 素中透析纯化； （11)、取 5微升洗脱液进行十二烷基硫酸钠-聚丙烯酰 胺（SDS - PAGE )凝胶电泳检测， 提取的多肽保存于冰箱 4°C保存。
例 3： TAT-N25融合多肽影响细胞生长及细胞周期的实验。
用人宫颈癌细胞系， 购自美国 ATCC, 目录号为： HTB-22的 Hela 细胞检测 TAT-N25融合多肽对细胞生长的影响。 Hela细胞在含 10% 胎 牛血清的 DMEM培养液， 37°C , 5%C02/95%空气培养条件下培养于 10厘米细胞培养亚， 将细胞培养至对数生长期后， 加入多肽至最终浓 度为 15微克 /毫升， 同时培养空白组和尿素组用以对照， 培养 24小时 收取细胞， 利用流式细胞仪分析分析这些细胞的细胞周期分布。 6M尿 素指的是每升水中含有 6摩尔尿素， 以下都筒称为 6M尿素。 实验结 果表明： 在这一剂量下表达 TAT-N25融合多肽对细胞凋亡没有明显影 响， 增加 G0/G1期细胞数 ，其作用见下表 1。
表 1： TAT-N25对细胞周期的影响
Figure imgf000009_0001
DNA合成是细胞增殖的标志。 下一个实验中， 我们检测 TAT-N25对 细胞 DNA合成的影响。 因为 BrdU为胸腺嘧啶类似物， 在 DNA合成 的 S期可掺入到 DNA中， 掺入到 DNA的 BrdU经过免疫染色后， 利 用流式细胞仪通过检测荧光强度的变化来分析 DNA的合成情况，空白 组和尿素组用以对照。 结果显示： 加入 TAT-N25多肽后， 肿瘤细胞中 有 DNA合成的 S期的细胞数目变化不大， 但平均 BrdU的掺入量较空 白组有明显减小， 见表 2, 这说明融合多肽抑制了 DNA的合成， 减少 了肿瘤细胞的增殖。 表 2: TAT-N25抑制细胞 DNA合成
Figure imgf000009_0002
例 4: TAT-N25融合多肽抑制小鼠体内腹水瘤生长的实验 在小鼠的腹膜内注射鼠恶性肉瘤 S180细胞后，将小鼠随机分成三 组。 注射肿瘤细胞一天之后， 向组中的小鼠分别注射 0.1 毫升浓度为 25 mg/ml TAT-N25 , TAT-N25溶于 6M的尿素中， 隔天注射重复三次。 两个对照组分别注射 0.1毫升生理盐水或 6M的尿素，注射次数和时间 同多肽试验组。 注射肿瘤十天后杀死小鼠并从其腹膜腔内取出腹水进 行体积测量和 BrdU掺入到 DNA的检测。 结果显示， 与对照组相比， 加入 TAT-N25 治疗的小鼠体内的腹水减少了 85%。 与之对应， 细胞 DNA合成也明显减少。 见表 3。
表 3 TAT-N25融合多肽抑制小鼠体内腹水瘤 S180的生长 DNA的 BrdU的平均掺入强 腹膜腔内腹水体积 (毫升）
度
空白对照 7.5 42.6
6M尿素 7.1 41.1
TAT-N25 1.3 26.5
例 5: TAT-N25融合多肽抑制小鼠体内腹水瘤生长的另一实验 在小鼠的腹膜内注射鼠恶性肉瘤 S180细胞后，将小鼠随机分成二 组。 注射肿瘤细胞一天之后， TAT-N25溶于 6M的尿素中， 将 0.2毫 升浓度为 20 mg/ml TAT-N25和 0.2毫升脂肪乳营养液混合后， 脂肪乳 营养液来自华瑞制药公司， 商品名为英托利特， 通过尾静脉向治疗组 中的小鼠分别注射， 隔天注射重复五次。 对照组分别注射 0.2毫升 6M 的尿素和 0.2 毫升脂肪乳营养液混合后， 注射次数和时间同多肽治疗 组。 注射肿瘤十天后杀死小鼠并从其腹膜腔内取出腹水进行体积测量 和 BrdU掺入到 DNA的检测。结果显示，与对照组相比，加入 TAT-N25 治疗的小鼠体内的腹水减少了 70%； 与之对应， 细胞 DNA合成减少 30%。
例 6: TAT-N25融合多肽抑制小鼠体内肉瘤生长的实验
在小鼠的皮下注射 106个鼠恶性肉瘤 S180细胞后， 将小鼠随机分 成二组。 注射肿瘤细胞一天之后， TAT-N25 溶于 6M 的尿素中， 将 0.2毫升浓度为 20 mg/ml TAT-N25和 0.2毫升脂肪乳营养液混合后，脂 肪乳营养液来自华瑞制药公司， 商品名为英托利特， 通过尾静脉向治 疗组中的小鼠分别注射， 隔天注射重复五次。对照组分别注射 0.2毫升 6M的尿素和 0.2毫升脂肪乳营养液混合后， 注射次数和时间同多肽治 疗组。 注射肿瘤十天后杀死并从小鼠体内取出肿瘤进行重量测量。 结 果显示， 与对照组相比， 加入 TAT-N25治疗的小鼠体内的肿瘤重量减 少了 75%。
例 7: TAT-N25融合多肽抑制小鼠体内人类直肠癌肿瘤生长的实 验
在小鼠的皮下注射 5xl06个人类直肠癌 HT29细胞后，将小鼠随机 分成二组。 注射肿瘤细胞一天之后， TAT-N25溶于 6M的尿素中， 将 0.2毫升浓度为 20 mg/ml TAT-N25和 0.2毫升脂肪乳营养液混合后，脂 肪乳营养液来自华瑞制药公司， 商品名为英托利特， 通过尾静脉向治 疗组中的小鼠分别注射， 隔天注射重复五次。对照组分别注射 0.2毫升 6M的尿素和 0.2毫升脂肪乳营养液混合后， 注射次数和时间同多肽治 疗组。 注射肿瘤十天后杀死并从小鼠体内取出肿瘤进行重量测量。 结 果显示， 与对照组相比， 加入 TAT-N25治疗的小鼠体内的肿瘤重量减 少了 75%。
例 8: TAT-N25融合多肽抑制小鼠体内人类曱状腺癌肿瘤生长的 实验
在小鼠的皮下注射 5χ106个人类曱状腺癌 FTC236细胞后，将小鼠 随机分成二组。 注射肿瘤细胞一天之后， TAT-N25溶于 6M的尿素中， 将 0.2毫升浓度为 20 mg/ml TAT-N25和 0.2毫升脂肪乳营养液混合后， 脂肪乳营养液来自华瑞制药公司， 商品名为英托利特， 通过尾静脉向 治疗组中的小鼠分别注射， 隔天注射重复十次。 对照组分别注射 0.2 毫升 6M的尿素和 0.2毫升脂肪乳营养液混合后，注射次数和时间同多 肽治疗组。 注射肿瘤二十天后杀死并从小鼠体内取出肿瘤进行重量测 量。 结果显示， 与对照组相比， 加入 TAT-N25治疗的小鼠体内的肿瘤 重量减少了 75%。
例 9: TAT-N25融合多肽抑制小鼠体内人类白血病肿瘤生长的实 验：
在小鼠的皮下注射 5χ106个人类粒细胞白血病 HL60细胞后，将小 鼠随机分成二组。 注射肿瘤细胞一天之后， TAT-N25溶于 6M的尿素 中， 将 0.2毫升浓度为 20 mg/ml TAT-N25和 0.2毫升脂肪乳营养液混 合后， 脂肪乳营养液来自华瑞制药公司， 商品名为英托利特， 通过尾 静脉向治疗组中的小鼠分别注射， 隔天注射重复七次。 对照组分别注 射 0.2毫升 6M的尿素和 0.2毫升脂肪乳营养液混合后， 注射次数和时 间同多肽治疗组。 注射肿瘤十四天后杀死并从小鼠体内取出肿瘤进行 重量测量。 结果显示， 与对照组相比， 加入 TAT-N25治疗的小鼠体内 的肿瘤重量减少了 50%。

Claims

权 利 要 求 书
1、 融合多肽， 包括： 抑制细胞生长的多肽和穿透细胞膜的多肽或 分子， 抑制细胞生长的多肽与穿透细胞膜的多肽或分子通过化学共价 健连接， 或抑制细胞生长的多肽与穿透细胞膜的多肽在细胞中表达成 融合多肽。
2、 根据权利要求 1所述的融合多肽， 其特征在于： 融合多肽的抑 制细胞生长的多肽与纯化融合多肽的标签蛋白连接。
3、 根据权利要求 1或 2所述的融合多肽， 其特征在于： 抑制细胞 生长的多肽是由磷脂酰肌醇 -3-激酶的调节亚基 p55PIK中氨基端 25个 氨基酸残基组成的多肽， 其序列为：甲硫氨酸-天门冬氨酸 -精氨酸-天门 冬氨酸-天门冬氨酸 -丙氨酸-天门冬氨酸-色氨酸 -精氨酸-谷氨酸-缬氨酸 -甲硫氨酸-甲石克氨酸-脯氨酸-酪氨酸 -丝氨酸-苏氨酸-谷氨酸 -亮氨酸-异 亮氨酸-苯丙氨酸 -酪氨酸-异亮氨酸 -谷氨酸-甲硫氨酸 (MDRDDADWREVMMPYSTELIFYIEM) ,或是从该多肽氨基端连续或 间断地缺失或取代 1 - 12 中任意数值个氨基酸残基得到的多肽之一， 或是将所述多肽的序列经过一个或几个氨基酸残基的添加得到的具有 抑制细胞生长活性的多肽之一。
4、 根据权利要求 1或 2所述的融合多肽， 其特征在于： 穿透细胞 膜的多肽是衍生于 HIV病毒 tat蛋白的多肽， 其序列为：酪氨酸-甘氨酸 -精氨酸-赖氨酸-赖氨酸 -精氨酸-精氨酸-谷酰胺 -精氨酸-精氨酸-精氨酸 (YGRKKRRQRRR)0
5、 根据权利要求 1或 2所述的融合多肽， 其特征在于： 用于纯化 融合多肽的标签蛋白是寡聚组氨酸标签， 其中组氨酸数目为 5-10中的 任意数值个。
6、 根据权利要求 3所述的融合多肽相应的核酸编码序列。
7、根据权利要求 3所述的融合多肽在治疗结肠癌、食管癌、 胃癌、 胃肠道肿瘤、 耳鼻喉及头颈部肿瘤、 肺癌、 肝癌、 肉瘤、 内分泌活化 肿瘤、 白血病、 何杰金淋巴瘤、 非何杰金淋巴瘤、 乳腺癌、 妇科肿瘤、 前列腺癌和泌尿生殖器官肿瘤及细胞生长异常有关的疾病中治疗的用 途。
8、 根据权利要求 3中所述的融合多肽， 应包括含有或应用该多肽 或其片段以药学上可接受的任何方式进行组合的内外用于治疗肿瘤及 细胞生长异常有关的疾病的用途。
9、 根据权利要求 6所述的融合多肽相应的核酸编码序列在治疗结 肠癌、 食管癌、 胃癌、 胃肠道肿瘤、 耳鼻喉及头颈部肿瘤、 肺癌、 肝 癌、 肉瘤、 内分泌活化肿瘤、 白血病、 何杰金淋巴瘤、 非何杰金淋巴 瘤、 乳腺癌、 妇科肿瘤、 前列腺癌和泌尿生殖器官肿瘤及细胞生长异 常有关的疾病中治疗的用途。
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