WO2005042744A1 - Proteine de recombinaison anticancereuse, ses genes codant et ses utilisations - Google Patents

Proteine de recombinaison anticancereuse, ses genes codant et ses utilisations Download PDF

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WO2005042744A1
WO2005042744A1 PCT/CN2003/000928 CN0300928W WO2005042744A1 WO 2005042744 A1 WO2005042744 A1 WO 2005042744A1 CN 0300928 W CN0300928 W CN 0300928W WO 2005042744 A1 WO2005042744 A1 WO 2005042744A1
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seq
amino acid
protein
sequence
acid residue
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PCT/CN2003/000928
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English (en)
French (fr)
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Bing Zhu
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Beijing Sunbio Biotech Co., Ltd.
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Priority to AT03770858T priority Critical patent/ATE501256T1/de
Application filed by Beijing Sunbio Biotech Co., Ltd. filed Critical Beijing Sunbio Biotech Co., Ltd.
Priority to JP2005510086A priority patent/JP4688678B2/ja
Priority to CNB2003801106041A priority patent/CN100549175C/zh
Priority to PCT/CN2003/000928 priority patent/WO2005042744A1/zh
Priority to AU2003280921A priority patent/AU2003280921B2/en
Priority to DE60336353T priority patent/DE60336353D1/de
Priority to US10/577,535 priority patent/US7666989B2/en
Priority to EP03770858A priority patent/EP1688498B1/en
Priority to CA2544473A priority patent/CA2544473C/en
Priority to BRPI0318594A priority patent/BRPI0318594B8/pt
Priority to NZ547274A priority patent/NZ547274A/en
Publication of WO2005042744A1 publication Critical patent/WO2005042744A1/zh
Priority to HK06110111.7A priority patent/HK1089788A1/xx

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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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a recombinant protein in the field of genetic engineering and pharmacy, its coding gene and genetic engineering drug using it as an active ingredient, and in particular to a recombinant protein with anti-cancer effect, its coding gene and its It is an active ingredient for treating cancer.
  • mammals During evolution, mammals have gradually formed a set of apoptotic signaling mechanisms that can guide individual cells to undergo apoptosis.
  • the principle is that the ligand of a lethal cell interacts with a cell surface death receptor to achieve the purpose of apoptosis.
  • This beneficial apoptosis has important physiological effects on the removal of lymphocytes activated by the terminal of the immune response, as well as the removal of virus-infected cells and cancer-transducing cells.
  • TNF interacts with the receptor TNFR
  • Fas interacts with the FASFas / Apol / CD95 receptor.
  • TNF and Fasl can induce apoptosis in cancer cells, they have serious toxic side effects in anticancer treatment.
  • TNF injection can cause a fatal inflammatory response similar to septic shock, which is mainly mediated by TNF-B, a pre-transcription factor activated by TNF, located in vascular endothelial cells and macrophages.
  • Anti-Fas antibodies can cause liver Fas-dependent apoptosis in tissues, leading to fatal liver damage.
  • TRAIL TNF related apoptosis inducing ligand
  • the discovery of TRAIL is related to its identity with the sequences of TNF and FasL.
  • TRAIL causes apoptosis by interacting with DR5, the death receptor DR4.
  • DR5 the death receptor
  • TRAJL mRNA can be continuously expressed in many normal tissues of the human body, which means that TRAIL can protect normal cells while causing apoptosis.
  • the physiological mechanism of tissue include the expression of inhibitory receptors, which can compete with DR4 and DR5 for binding ligands, and TRAIL can interact with three receptors, such as DCR1, DCR2 and DPG. Therefore, TRAIL is less toxic than TNF and FasL.
  • An object of the present invention is to provide a recombinant protein having a tumor suppressing effect and a drug capable of effectively treating cancer using the protein as an active ingredient.
  • the recombinant protein with anti-cancer effect provided by the present invention is one of the following proteins-1) a protein having the sequence of SEQ ID No : 2 amino acid residues in the sequence listing; 2) a protein derived from SEQ ID No : 2 having a sequence homology of more than 90% with amino acid residues of SEQ ID N2: 2 and having the same activity as SEQ ID Na: 2;
  • amino acid residue sequence of SEQ ID Na: 2 has been replaced, deleted or added by one or more amino acid residues and has the same activity as the amino acid residue sequence of SEQ ID No : 2
  • the protein of SEQ ID No : 2 in the sequence listing is a recombinant human TRAIL circularly permuted form of TRAIL (CPT for Jane Herr), consisting of 166 amino acid residues.
  • the active ingredient of the medicament for treating cancer (CPT) provided by the present invention is one of the aforementioned proteins.
  • one or more pharmaceutically acceptable carriers may be added to the above medicine.
  • the carrier includes a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, etc., which are conventional in the pharmaceutical field, and a fragrance may be added if necessary.
  • the medicament of the present invention can be made into various forms such as injection solution, tablet, powder, granule, capsule, oral solution, ointment, cream and the like.
  • the aforementioned various dosage forms can be prepared according to a conventional method in the pharmaceutical field.
  • the coding sequence of the active ingredient (protein) of the cancer drug (CPT) provided by the present invention is one of the following nucleotide sequences:
  • a DNA sequence encoding a protein derived from SEQ ID No : 2 is a sequence in which the amino acid residue sequence of SEQ ID No : 2 is replaced by one or several amino acid residues, A polypeptide that is deleted or added and has the same activity as the amino acid residue sequence of SEQ ID Na : 2.
  • SEQ ID No : 1 in the Sequence Listing consists of 501 bases. Its coding frame is from nucleotide 1 to nucleotide 498 at the 5 'end.
  • Figure 1 is the tumor volume curve of human glioma U251 under different treatment conditions
  • Figure 2 shows the tumor size of human glioma U251 under different treatment conditions
  • Figure 3 is the tumor volume curve of human lung cancer NCI-H460 under different treatment conditions
  • Figure 4-1 to 4-6 are histopathological sections of human lung cancer NCI-H460 under different treatment conditions (HE staining X 100);
  • Figure 5 shows the tumor size of human lung cancer NCI-H460 under different treatment conditions
  • Figure 6 is the tumor volume curve of human colon cancer C0L0 205 under different treatment conditions
  • Example 1 Construction and expression of recombinant human-derived TRAIL circularized variant coding gene
  • the gene sequence encoding human TRAIL amino acid residues 121-281 is derived from the human spleen cDNA library.
  • the gene encoding human TRAIL amino acid residues 135-281 is obtained by conventional PCR using the human 7 / Z gene as a template.
  • a DNA sequence encoding 3 ⁇ 4471 ⁇ ⁇ -? Amino acid residues was ligated at the 3 'end by a PCR method to a DNA sequence encoding TRAIL amino acid residues 122-135, and a DNA sequence encoding five glycines was inserted into the sequences encoding TRAIL135-281 and Between the sequences of TRAIL122-135 amino acid residues, the flexibility of glycine can ensure the correct folding of the protein. This way The obtained CPT-encoding gene was ligated to the vector plasmid pet 28b using Ncol and BamHI (other vector plasmids can also be used) to obtain an expression plasmid. The DNA sequence was tested to be correct.
  • the expression plasmid was transformed into E. coli strain BL21 (DE3), and the transformed E. coli was inoculated into 10 ml of LB liquid medium containing 20 ⁇ g / ml kanamycin and cultured on a 37 ° C shaker for 12 hours. Then, 10 ml of the culture was inoculated into 1 L of LB liquid culture medium containing 20 ⁇ g / ml kanamycin, and the culture was continued when it was 0D 6 . . When the value reached 0.6, 0.2 ml of 1M IPTG was added to the culture to induce protein expression. After 3 hours of induction, the cells were collected by centrifugation, and the pellet was suspended in 100 ml of a buffer containing 100 mM Tris (pH 7.9) and 150 mM NaCl.
  • the cells were centrifuged using a Beckman JA20 rotor at 15,000 rpm. Since the expressed protein can bind to a chelated metal resin, it can be purified by metal chelation chromatography. The supernatant after centrifugation was pumped into a solidified Ni chromatography column containing the resin, and washed with a buffer solution containing 50 mM Tris ( PH 7.7.9), 0.5 M NaCl, and 50 mM imidazole to remove foreign proteins. The bound protein was then eluted from a buffer containing 50 mM Tris (pH 7.9), 0.5 M NaCl and 200 mM imidazole, and the eluted protein was dialyzed against PBS buffer.
  • a buffer solution containing 50 mM Tris (pH 7.9), 0.5 M NaCl and 200 mM imidazole
  • the anti-cancer drug of the present invention is a white powder and is easily soluble in water.
  • Example 2 Determination of the killing effect of the drug of the present invention on cancer cells by the tetrazolium salt reduction method
  • RPMI1640 is a GIBC0 product
  • MTT is a Bebco product
  • calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.
  • C0L0 205 human colon cancer cells
  • NCI-H460 human lung cancer cells
  • RPMI 8226 human multiple myeloma cells
  • U251 human brain glioma cells
  • ATCC ATCC
  • HL-60 Human promyelocytic leukemia cells
  • MDA-MB-231 human breast cancer cells
  • MDA-MB-435 human breast cancer cells
  • SCLC human small cell lung cancer
  • H125 human lung cancer cells
  • PC-3 human pancreatic cancer cells
  • Collect well-grown tumor cells prepare IX 107ml cell suspension with RPMI 1640 medium containing 10% calf serum, and inoculate 100 ⁇ l (containing 1000 tumor cells) in a 96-well culture plate. After incubation at 37 ° C, 5% C0 2 incubator for 24 hours, add medicine.
  • a blank control, a fluorouracil, or a doxorubicin positive control was set, and the test sample was set at 5 concentrations. 3 parallel wells per concentration. Incubate at 37 ° C for 4 days in a 5% CO 2 incubator and discard the culture medium. Add MTT solution 100 ⁇ l (0.4mg / ml, prepared with RPMI 1640) to each well, and incubate at 37 ° C for 4 hours.
  • CPT has a strong killing activity on human tumor cells U251, C0L0205, NCI-H460. MDA-MB-435 cells, IC 5 . ⁇ 0. 01 g / ml. It also has a strong killing effect on HL-60, MM- Ying-231, PC-3, and H125 cells, IC 5 . ⁇ 0. Lg / ml. Good activity on RPMI 8226 cells, IC 5 . ⁇ g / ml. It also showed a certain inhibitory effect on SCLC cell growth. Compared with wild-type TRAIL, CPT is 5--10 times more potent in inducing apoptosis of cancer cells.
  • FU fluorouracil control
  • Adr doxorubicin control
  • Example 3 Inhibitory effect of the drug of the present invention on the growth of human cancer xenograft tumors in nude mice
  • the animals used were: BLAB / c-nu nude mice, 6-8 weeks in size, the same for each experiment gender.
  • Tumor strains human brain glioma U251, human lung cancer NCI-H460, human colon cancer COLO 205 nude mice
  • Transplanted tumors are inoculated subcutaneously in nude mice with in vitro cultured cells, subcultured and stored.
  • Experimental procedure Select tumor-bearing animals with good tumor growth and good general condition, and execute cervical dislocation.
  • the tumor mass was removed under aseptic conditions, cut with a scalpel into a tumor mass of 2-3 mm in diameter, and inoculated subcutaneously in the posterior axilla of a nude mouse with a trocar. About 7-10 days later, tumors can be seen in the experimental animals.
  • the vernier calipers were used to measure the length and width of tumors, and the tumors were grouped according to tumor volume. 7-8 animals per group.
  • tumor length, width and weight of the animal were measured twice a week. Calculate tumor volume (TV) and relative tumor volume (RTV) to draw a map of tumor volume growth.
  • RV tumor volume
  • RV relative tumor volume
  • the formula for calculating tumor volume (TV) is: length X width 2 ⁇ 2.
  • the formula for calculating the relative tumor volume (RTV) is: V t / V 0 (where V 0 is m ⁇ measured during divided-cage administration, and v t is ⁇ during each subsequent measurement.)
  • the t test was used to compare the statistical differences in tumor weight, tumor volume, RTV and other indicators of each group.
  • the evaluation index of antitumor activity is the relative tumor proliferation rate T / C (%), and the calculation formula is:
  • Carmustine (product of Tianjin Amino Acids People's Pharmaceutical Factory) was used as a positive control drug, and was administered by intraperitoneal injection at a dose of 40 mg / kg once.
  • Set 4 dose groups of 0.6 mg / kg, 1. 7 mg / kg, 5.0 mg / kg, and 15. 0 mg / kg.
  • Intraperitoneal injection was administered, and the experimental course was 10 times.
  • Cyclophosphamide (product of Shanghai Hualian Pharmaceutical Co., Ltd.) was used as a positive control drug, the applied dose was 100 mg / kg, intraperitoneal injection, and 80 mg / kg was added once after 2 weeks. Three dose groups of 1.7 mg / kg, 5.0 mg / kg, and 15. Omg / kg were set, which were administered by intraperitoneal injection once a day and 10 times.
  • Figures 4-1 to 4-6 The results of histopathological examination are shown in Figures 4-1 to 4-6, Figures 4-1, 4-3, and 4-5 are negative control groups, and Figure 4-1 shows that the tumor tissue is diffuse and solid. Large central liquefaction necrosis with a thin layer of connective tissue envelope at the edge, without obvious lymphocyte infiltration; Figure 4-13 shows that the tumor cells are nested and solid, with abundant capillaries in the tissue, and no obvious lymphocyte infiltration at the tumor edge; Figure 4-5 shows that the tumor cells are round, polygonal, with large nucleus, few cytoplasm, loose chromatin, obvious nucleoli, there are 2-3 nucleoli, and most mitotic images; Figure 4-2, 4- 4, 4-6 are CPT 15mg / kg group.
  • Figure 4-2 shows that the tumor tissue is solid, with central lamellar necrosis.
  • the tumors in the multi-connective group only proliferate, and there is a thin layer of connective tissue envelope on the edge. Lymphocytic infiltration;
  • Figures 4 to 4 show that tumor cells are arranged in a nest-like manner, with fewer capillaries in the tissue, central necrosis, a thin layer of connective tissue envelope at the edge, and a few lymphocytes infiltrating;
  • Figures 4-6 show that the tumor cells are round Shape, polygon, large nucleus, cell Small, loose chromatin, mitotic index, tumor tissue capillaries less, most point-like, special treatment 'f have evil Die.
  • the medicine of the present invention has a strong inhibitory effect on human lung cancer NCI-H460 cells.
  • Hydroxycamptothecin for injection (trade name: Xisu, Huangshi Li Shizhen Pharmaceutical Group Wuhan Li Shizhen Pharmaceutical Co., Ltd.) was used as a positive control drug at an application dose of 1 mg / kg. The drug was discontinued after 15 days of continuous administration. Due to observation, tumor growth in this group was more obvious. After 8 days of supplementary administration, the applied dose was 100 mg / kg, intraperitoneal injection, and 80 mg / kg was administered once every 2 weeks. Set three dose groups of 1. 7 mg / kg, 5.0 mg / kg, and 15. Omg / kg. Intraperitoneal injection is given once a day and 15 times.
  • Negative control 151 ⁇ 79 2532 ⁇ 1190 8.30 ⁇ 5.53
  • Tetrazolium salt (MTT) reduction method was used to determine the anti-cancer effect of the drug.
  • ⁇ The tumor cell inhibition rate was plotted with different concentrations of the drug to obtain a dose-response curve and the half effective concentration was calculated (IC 5 ).
  • CPT preparations have a very obvious inhibitory effect on tumor cell growth, including human lung cancer, colon cancer, breast cancer, gastric cancer, pancreatic cancer, glioma, blood cell cancer, bladder cancer, prostate Cancer, rectal colon cancer, cervical cancer, and brain tumor cells have more than 10 kinds of tumor cells have very significant in vitro killing activity, its U251 (human brain glioma cells), C0L0 205 (human colon cancer cells), NCI-H460 (human lung cancer cells), MDA-MB-435 (human L adenocarcinoma cells) half inhibitory concentration (IC 5 ) ⁇ 0. Ol g / ml.
  • IC 5 to HL-60 Human Promyelocytic Leukemia Slim Pack
  • MDA- MB- 231 human breast cancer cells
  • PC-3 human pancreatic cancer cells
  • H125 human lung cancer cells
  • IC 5 for RPMI 8226 human multiple myeloma cells
  • SCLC human small cell lung cancer
  • CPT can significantly slow down the tumor growth rate in animals, and high-dose medication in the early stage of transplantation tumor growth can reduce the tumor volume or even disappear.
  • the dose-effect relationship of the drug is obvious, and the pharmacodynamic effect shows a certain correlation with the course of administration.
  • mice were injected intraperitoneally with 15 mg / kg / day of CPT 15 times until the end of the experiment 4 weeks after the drug was stopped.
  • the observed parameters prove that CPT administration has a significant inhibitory effect on the growth of transplanted tumors of human cancer nude mice used in the experiment.
  • the tumor weight inhibition rates of CPT 15mg / kg for U251 were 87.5% and 87.7%, respectively, and the tumor weight inhibition rates of NCI-H460 were 87.0% and 88.
  • the tumor weight inhibition rates for COLO 205 were 97.4.0% and 97.8%, respectively, and the statistical difference was very significant; the present invention conducted experiments using 3 models, and the results showed that the CPT was 5.0 mg / kg Dose, administered continuously for 10-15 days, the relative tumor volume (RTV) of the three animal models showed statistically significant differences compared with the negative control group, and the 15 mg / kg dose showed very significant compared with the negative control group Statistical differences, T / C (%) are all below 25, even ⁇ 10, showing high antitumor activity and good experimental reproducibility.
  • CPT administration has no significant toxic and side effects on animals. Except for the tumor-bearing volume of mice in the administration group, the survival status such as body weight and survival number were not significantly different from those of the control group during the experimental observation period. It is proved that CPT has a significant selective inhibitory effect on tumor cells and does not cause apoptosis in normal tissue cells, so it can be used as a safe and effective drug for cancer treatment, which has important theoretical significance and application value, and has a broad market prospect.

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Description

一种具有抑癌作用的重组蛋白及其编码基因与应用
技术领域
本发明涉及基因工程及药学领域中的一种重组蛋白， 它的编码基因及以 其为活性成分的基因工程药物， 特别是涉及一种具有抑癌作用的重组蛋白， 它的编码基因及以其为活性成分的治疗癌症的药物。
背景技术
在进化过程中， 哺乳动物逐渐形成了一套凋亡信号机制， 能够引导个体 细胞发生凋亡， 其原理是致死细胞的配基与细胞表面死亡受体相互作用， 从 而达到使细胞凋亡的目的。 这种有益的凋亡对于去除免疫反应终端激活的淋 巴细胞， 以及去除病毒感染细胞和癌转细胞有着重要的生理作用。 例如 TNF 与受体 TNFR的相互作用， 以及 Fas 与 FASFas/Apol/CD95受体的相互作用。
死亡受体的激活直接参与细胞凋亡的级联反应。 它们可以诱导多种肿瘤 细胞的凋亡， 是潜在的抗癌因子。 虽然 TNF和 Fasl可以诱导癌细胞的凋亡， 但它们在抗癌治疗中有着严重的毒副作用。 TNF 的注射能引起类似于感染性 休克的致命的炎症反应， 这 反应主要是由位于血管内皮细胞及巨噬细胞内 的由 TNF激活的前转录因子 TNF- B介导， 抗 Fas抗体可导致肝组织内 Fas 依赖的细胞凋亡， 形成致命的肝损伤。
1995年 Wiley等发现了月中瘤坏死因子相关凋亡诱导配基 （TNF related apoptosis inducing ligand 简称 TRAIL) , TRAIL的发现与它和 TNF、 F asL 的序列具有同一性有关。 TRAIL通过与死亡受体 DR4的 DR5相互作用引起细胞凋 亡， 与 TNF和 FasL不同， TRAJL的 mRNA可以在人体许多正常组织中持续表达， 这就意味着 TRAIL在引起凋亡的同时存在着保护正常组织的生理机制。这些机 制包括表达抑制性的受体，可以与 DR4和 DR5竞争结合配基， TRAIL可以与 DCR1、 DCR2和 DPG等 3种受体相互作用， 因此 TRAIL的毒性要小于 TNF和 FasL。
发明公开
本发明的目的是提供一种具有抑癌作用的重组蛋白及其以该蛋白为活性 成分的能够有效治疗癌症的药物。
本发明所提供的具有抑癌作用的重组蛋白是下列蛋白质之一- 1 )具有序列表中 SEQ ID No : 2氨基酸残基序列的蛋白质； 2)与 SEQ ID N2 : 2的氨基酸残基序列同源性在 90%以上且具有与 SEQ ID Na: 2相同活性的由 SEQ ID No : 2衍生的蛋白质；
3) 在 SEQ ID No : 2的氨基酸残基序列的 N-端增加或删除 15个或 15个 以内氨基酸残基得到的具有与 SEQ ID No : 2相同活性的由 SEQ ID Ns: 2衍 生的蛋白质；
4) 在 SEQ ID No : 2的氨基酸残基序列的 C-端增加或删除 15个或 15个 以内氨基酸残基得到的具有与 SEQ ID No : 2相同活性的由 SEQ ID N2 : 2衍 生的蛋白质；
5) 将 SEQ ID Na: 2的氨基酸残基序列经过一个或几个氨基酸残基的取 代、 缺失或添加且具有与 SEQ ID No : 2的氨基酸残基序列相同活性的由 SEQ
ID No : 2衍生的蛋白质。
序列表中 SEQ ID No : 2 的蛋白质为重组人 TRAIL 环化变构体 (recombinant circularly permuted form of TRAIL, 简禾尔为 CPT), 由 166 个氨基酸残基组成。
本发明所提供的治疗癌症的药物（CPT)，其活性成分是上述蛋白质之一。 需要的时候， 在上述药物中还可以加入一种或多种药学上可接受的载 体。 所述载体包括药学领域常规的稀释剂、 赋形剂、 填充剂、 粘合剂、 湿 润剂、 崩解剂、 吸收促进剂、 表面活性剂、 吸附载体、 润滑剂等， 必要时 还可以加入香味剂、 甜味剂等。
本发明的药物可以制成注射液、 片剂、 粉剂、 粒剂、 胶囊、 口服液、 膏剂、 霜剂等多种形式。 上述各种剂型的药物均可以按照药学领域的常规 方法制备。
本发明所提供的治疗癌症药物 (CPT)的活性成分（蛋白）的编码序列是下 列核苷酸序列之一：
1) 序列表中的 SEQ ID Na: 1；
2) 编码序列表中 SEQ ID No : 2蛋白质序列的多核苷酸；
3) 与序列表中 SEQ ID No : 1限定的 DNA序列具有 90%以上同源性， 且 编码相同功能蛋白质的 DNA序列；
4) 编码由 SEQ ID No : 2衍生的蛋白质的 DNA序列， 所述由 SEQ ID Na: 2衍生的蛋白质为在 SEQ ID No : 2的氨基酸残基序列的 N-端增加或删除 15 个或 15个以内氨基酸残基得到的具有与 SEQ ID No : 2相同活性的多肽； 5) 编码由 SEQ ID No : 2衍生的蛋白质的 DNA序列， 所述由 SEQ ID Na: 2衍生的蛋白质为在 SEQ ID No : 2的氨基酸残基序列的 C-端增加或删除 15 个或 15个以内氨基酸残基得到的具有与 SEQ ID No : 2相同活性的多肽；
6) 编码由 SEQ ID No : 2衍生的蛋白质的 DNA序列， 所述由 SEQ ID Na: 2衍生的蛋白质为将 SEQ ID No : 2的氨基酸残基序列经过一个或几个氨基酸 残基的取代、 缺失或添加且具有与 SEQ ID Na: 2的氨基酸残基序列相同活性 的多肽。
序列表中的 SEQ ID No : 1由 501个碱基组成。 它的编码框是自 5' 端第 1位核苷酸到第 498位核苷酸。
含有本发明基因的表达载体及细胞系 (例如大肠杆菌， 酵母等表达体系） 均属于本发明的保护范围。 附图说明
图 1为不同处理条件下人神经胶质瘤 U251的肿瘤体积曲线；
图 2为不同处理条件下人神经胶质瘤 U251的瘤体大小；
图 3为不同处理条件下人肺癌 NCI-H460的肿瘤体积曲线；
图 4一 1至 4一 6为不同处理条件下人肺癌 NCI- H460的组织病理学切片 (HE染色 X 100);
图 5为不同处理条件下人肺癌 NCI-H460的瘤体大小；
图 6为不同处理条件下人结肠癌 C0L0 205的肿瘤体积曲线；
图 7— 1 至 7— 5 为接种了人结肠癌 C0L0 205 并经不同处理后的 BLAB/c-nu 裸小鼠。
实施发明的最佳方式
实施例 1、 重组人源 TRAIL环化变构体编码基因的构建及表达
编码人 TRAIL第 121- 281位氨基酸残基的基因序列来自于人脾脏 cDNA文 库， 编码人源 TRAIL第 135-281位氨基酸残基的基因用人源 7 /Z基因作模 板用常规 PCR方法获得， 然后将编码 ¾471 ·^- ? 位氨基酸残基的 DNA序 列通过 PCR方法在 3' 端连接上编码 TRAIL氨基酸残基 122-135的 DNA序列， 并将编码五个甘氨酸的 DNA序列插入编码 TRAIL135- 281和 TRAIL122- 135氨 基酸残基的序列之间， 甘氨酸的灵活性可以保证蛋白质的正确折叠。 这样得 到的编码 CPT基因用 Ncol和 BamHI连接到载体质粒 pet28b上 （也可以用其 它的载体质粒）， 得到表达质粒。 经检测 DNA序列正确。
将表达质粒转化进大肠杆菌菌株 BL21 (DE3)中， 转化后的大肠杆菌接种 到 10ml含有 20 μ g/ml 卡那霉素的 LB液体培养基中， 在 37 °C摇床上培养 12小时。然后将该 10ml培养物接种到 1L含 20 μ g/ml 卡那霉素的 LB液体培 养基中继续培养， 当其 0D6。。值达到 0. 6时， 将 0. 2 ml 1M的 IPTG加入 培 养物中来诱导蛋白质表达。 诱导 3小时后离心收集细胞， 将沉淀用 100ml含 lOOmM Tris (pH7. 9)、 150 mM NaCl的缓冲液悬浮。
4°C下超声波裂解细胞后， 用 Beckman JA20转头在 15， OOOrpm下离心。 由于表达的蛋白质可以结合螯合金属树脂，可通过金属螯合层析来进行纯化。 将离心后的上清泵入将含有树脂的固化 Ni 层析柱中， 经过含 50mM Tris (PH7. 9)、 0. 5M NaCl和 50mM咪唑的缓冲液冲洗来去除其中的杂蛋白。 然后 被结合上的蛋白质由含 50mM Tris (pH7. 9)、 0. 5M NaCl和 200mM咪唑的缓 冲液洗脱下来， 被洗脱下来的蛋白质用 PBS缓冲液进行透析。
最后， 蛋白质通过装在 AKTA HPLC系统 （Pharmacia) 中的离子交换柱和
Superdex 200 (Pharmacia)凝胶层析进行纯化。 分析表明得到的是氨基酸残 基序列为 SEQ ID No : 2的蛋白质。 即本发明的抑癌药物， 该产品呈白色粉末 状， 易溶于水。
实施例 2、采用四氮唑盐还原法（ΜΓΓ)测定本发明药物对癌细胞的杀伤作 用
试剂： RPMI1640 为 GIBC0产品； MTT为 Bebco产品； 小牛血清购自杭 州四季青生物工程材料有限公司。
细胞株： C0L0 205 (人结肠癌细胞）、 NCI-H460 (人肺癌细胞） RPMI 8226 (人多发性骨髓瘤细胞）、 U251 (人脑神经胶质瘤细胞）引自美国 ATCC， HL-60 (人早幼粒白血病细胞）、 MDA- MB- 231、 MDA- MB- 435 (人乳腺癌细胞、 SCLC (人小细胞肺癌）、 H125 (人肺癌细胞）、 PC- 3 (人胰腺癌细胞）。
收集生长良好的肿瘤细胞， 用含 10%小牛血清的 RPMI 1640培养基配制 成 I X 107ml细胞悬液， 接种于 96孔培养板内， 每孔 ΙΟΟμΙ (含 1000个肿瘤 细胞）。 置 37°C， 5% C02温箱内培养 24小时后加药。
实验设空白对照、 氟尿嘧啶或阿霉素阳性对照， 受试样品设 5个浓度， 每浓度 3个平行孔。 于 37°C， 5% C02温箱内培养 4天后弃培养液， 每孔加入 MTT溶液 ΙΟΟμΙ (0. 4mg/ml, RPMI 1640配制）， 37°C继续孵育 4小时。
弃去上清液， 每孔加入 DMS0 150μ1以溶解 Fomazan颗粒。 轻度振荡后， 用 BI0RAD 550型酶标仪在检测波长 540nm， 参考波长 450nm下测定 0D值。
以药物的不同浓度及对细胞的抑制率作图得到剂量反应曲线， 从中求出 半数抑制浓度（IC5Q)。 结果如表 1所示， 可以看出， CPT对人肿瘤细胞 U251, C0L0205, NCI-H460. MDA-MB-435细胞均有极强的杀伤活性， IC5。<0. 01 g/ml。 对 HL- 60， MM-應- 231， PC- 3, H125细胞也有很强的杀伤作用， IC5。<0. l g/ml。 对 RPMI 8226细胞有较好活性， IC5。< g/ml。 对 SCLC细胞生长也显示一定 抑制作用。 同野生型 TRAIL相比， CPT诱导癌细胞凋亡的活性要强 5- 10倍。
CPT对人肿瘤细胞株杀伤作用结果 （x±SD)
IC60 ( g/ral )
瘤株
CPT Fu Adr
U251 <0. 001 0. 981 ±0. 077
C0L0 205 0. 006±0 0. 826±0. 051
醒- MB- 435 0. 008 ±0. 002 0. 199±0. 138
HL-60 0. 014±0. 013 0. 006±0
NCI-H460 0. 002±0. 001 0. 566±0. 016
MDA-MB-231 0. 043±0. 0131 0. 318±0. 055
PC-3 0. 067 ±0. 002 4. 928±0. 753
H125 0. 085 ±0. 006 0. 138±0. 031
RPMI 8226 0. 824±0. 093 <0. 1
SCLC 10±0 0. 617 ±0. 0257
注： FU为氟尿嘧啶对照 Adr为阿霉素对照
实施例 3、 本发明药物对裸小鼠人癌异种移植瘤的生长抑制作用 在本实施例中， 所用动物为： BLAB/c- nu 裸小鼠， 6-8 周大小， 每次实 验选用同一性别。
瘤株： 人脑神经胶质瘤 U251、 人肺癌 NCI- H460、 人结肠癌 COLO 205裸小鼠 移植瘤， 由体外培养细胞接种于裸鼠皮下成活， 传代并保存。
实验过程： 选择肿瘤生长良好， 全身状况较好荷瘤动物， 颈椎脱臼处死。 无菌条件下取出瘤块， 用手术刀切割成直径 2-3mm的瘤块， 套管针接种于裸小 鼠腋后皮下。 约 7— 10天后实验动物腋后可见肿瘤。 用游标卡尺测量肿瘤长、 宽径， 按瘤体积大小分层分组。 每组 7-8只动物。
实验过程中，每周测量动物肿瘤长、宽径及体重 2次。计算肿瘤体积 (TV) 及相对肿瘤体积（RTV)， 绘制肿瘤体积生长变化图。 实验结束时将动物处死后 剥离肿瘤， 称瘤重， 计算药物对肿瘤生长抑制率。
肿瘤体积 （TV) 的计算公式为： 长 X宽 2÷2。
相对肿瘤体积 (RTV) 的计算公式为： V t / V 0 (其中 V 0为分笼给药时测 m τν, v t为以后每次测量时的 τν。 )
t检验法比较各组动物肿瘤重量、 肿瘤体积、 RTV等指标的统计学差别。 抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C (%)， 计算公式为：
治疗组 （T) RTV
T/C (%) = X 100
阴性对照组（C) RTV
疗效评价标准： T/C (%) >60为无效；
T/C (%) 60， 并经统计学处理 P<0. 05为有效。 一、 对人脑神经胶质瘤 U251的抑制作用
以卡莫斯汀 (天津氨基酸公司人民制药厂产品）为阳性对照药，以 40mg/kg 剂量腹腔注射给药 1次。 设 0. 6mg/kg、 1. 7 mg/kg、 5. 0 mg/kg、 15. Omg/kg 4 个剂量组， 腹腔注射给药， 实验疗程均为 10次给药。
实验结果如表 1、 表 2及图 1、 图 2所示， 表明， CPT对人脑神经胶质瘤 U251裸小鼠移植性肿瘤生长具有明显的抑制作用， 4剂量组肿瘤重量抑制率分 别为 30. 5 %、 44. 5 %、 64. 8 %和 87. 5 %。 各组肿瘤重量与对照组相比均显示 明显或非常明显统计学差别。 其抗肿瘤作用呈明显剂量依赖性。 5. 0mg/kg、 15mg/kg 组的相对肿瘤增殖率 T/C ( % ) 均 <60， 相对肿瘤体积 （RTV ) 在 15. Omg/kg剂量给药组与对照组相比统计学差异显著。 表 2. CPT对人神经胶质瘤 U251生长抑制作用 （x±SD) - 动物数 （只） 体重 (g)
组别 瘤重 (g) 抑制率 （％) 开始 结束 开始 结束
阴性对照 8 8 21.9±1.1 22·3±2.6 1.28±0· 50
卡莫司汀对照 8 7 22.8±0.7 19.4±2· 1 0.09±0· 06** 93.0
CPT 0.6mg/kg 8 8 21.0±0.8 21.0±1.4 0.89±0· 49 30.5
1.7mg/kg 8 8 21.6±1·2 21.5±2.0 0.71±0· 22* 44.5
5. Omg/kg 8 8 22·0±1· 1 21.9±3.2 0.45+0.26** 64.8
15mg/kg 8 8 21.6±1.1 21.1±1.7 0.16±0· 15** 87.5 表 3. CPT对人神经胶质瘤 U251生长抑制作用 （x±SD)
Figure imgf000009_0001
二、 对人肺癌 NCI-H460的抑制作用
以环磷酰胺 （上海华联制药有限公司产品） 为阳性对照药， 应用剂量为 lOOmg/kg,腹腔注射， 2周后补加 80mg/kg给药 1次。设 1.7 mg/kg、 5.0 mg/kg、 15. Omg/kg 3个剂量组， 每日腹腔注射给药 1次， 给药 10次。
实验结果如表 4、 表 5及图 3、 图 5所示， 表明 CPT对人肺癌 NCI-H460 裸小鼠移植性肿瘤生长具有明显的抑制作用， 3剂量组肿瘤重量抑制率分别为 52.2%、 74.5%和 87.0%。 各组肿瘤重量与对照组相比均显示明显或非常明 显统计学差别。 中、 高剂量组的相对肿瘤增殖率 （％) 均〈 60、， 相对肿瘤体 积 （RTV) 与对照组相比统计学差异显著。 表 4. CPT对人肺癌 NCI-H460的生长抑制作用
Figure imgf000010_0001
表 5. CPT对人肺癌 NCI- H460的生长抑制作用
Figure imgf000010_0002
* Ρ<0. 05 ** Ρ〈0. 01 与阴性对照组比较
组织病理学检查结果如图 4—1至图 4一6所示， 图 4—1、 4一3、 4-5为阴 性对照组， 图 4一 1显示肿瘤组织呈弥漫性团实片状， 中央大片液化性坏死， 边缘有薄层结缔组织包膜， 无明显淋巴细胞浸润； 图 4一 3显示肿瘤细胞呈巢 索、 片状实性， 组织内毛细血管丰富， 肿瘤边缘无明显淋巴细胞浸润； 图 4 一 5 显示肿瘤细胞呈圆形、 多角形， 细胞核大、 胞质少， 染色质疏松， 核仁 明显， 有 2—3个核仁， 有多数核分裂像； 图 4—2、 4—4、 4一6为 CPT15mg/kg 组， 图 4一 2显示肿瘤组织呈团块实性， 中央片状坏死， 肿瘤内教多结缔组只 增生， 边缘有薄层结締组织包膜， 无明显淋巴细胞浸润； 图 4一 4显示肿瘤细 胞呈巢索状排列， 组织内毛细血管少， 中央坏死， 边缘有薄层结締组织包膜， 少数淋巴细胞浸润； 图 4— 6显示肿瘤细胞呈圆形、 多角形， 细胞核大、 胞质 少， 染色质疏松， 核分裂像， 肿瘤组织内毛细血管少， 多数点状、 小灶' f生坏 死。
可见本发明的药物对人肺癌 NCI-H460细胞具有较强的抑制作用。
三、 对人结肠癌 C0L0 205生长的抑制
以注射用羟基喜树碱（商品名： 喜素， 黄石李时珍药业集团武汉李时珍 药业有限公司产品）为阳性对照药， 应用剂量为 lmg/kg， 腹腔注射， 每日给 药 1次，于连续给药 15天后停药， 因观察发现该组肿瘤增长较明显， 于 8天 后补充给药 1周应用剂量为 100mg/kg， 腹腔注射， 2周后补加 80mg/kg给药 1次。 设 1. 7 mg/kg、 5. 0 mg/kg、 15. Omg/kg 3个剂量组， 每日腹腔注射给 药 1次， 给药 15次。
实验结果如表 6、 表 7、 图 6及图 7—1至 7— 5所示， 其中图 7—1为阴 性对照组； 图 7—2为 15. Omg/kg实验组； 图 7— 3为 5. 0mg/kg实验组； 图 7 一 4为 1. 7mg/kg实验组； 图 7— 5为羟基喜树碱阳性对照组； 结果表明， CPT 对人结肠癌 C0L0 205裸小鼠移植性肿瘤生长具有明显的抑制作用， 用药 5天 后可观察到高剂量组肿瘤体积较分组时明显缩小，随给药次数增多甚至消失， 停药后继续观察 4周， 1/3的动物未发现肿瘤生长。 大体解剖仅能于皮下见 到接种痕迹。 3剂量组肿瘤重量抑制率分别为 79. 6% , 90. 8 %和 97. 4%。 各 组肿瘤重量与对照组相比均显示明显或非常明显统计学差别。 各给药组的相 对肿瘤增殖率 T/C (%)均〈60， 相对肿瘤体积 （RTV)与对照组相比统计学差 异显著。 其中高剂量组的 T/C (%)〈10, 显示 CPT对该肿瘤的高度活性。 表 6. CPT对人结肠癌 C0L0 205生长抑制作用
Figure imgf000011_0001
表 7. CPT对人结肠癌 C0L0 205生长抑制作用 组别 肿瘤体积 （mm3) RTV T/C (%) 开始 结束
阴性对照 151 ±79 2532± 1190 8. 30±5. 53
喜树碱对照 150±34 845 ±66** 3. 92± 1. 91* 47. 2
CPT1. 7mg/kg 205 ±47 582 ±408** 1. 62±0. 62** 19. 5
5. Omg/kg 180 ±63 314± 175** 0. 91 ±0. 35** 11. 0
15mg/kg 192 ±81 52 ±53** 0. 33 ±0. 7** 4. 0
* P<0. 05 ** P<0. 01 与阴性对照组比较
工业应用
采用四氮唑盐（MTT)还原法测定药物的抗癌作用，·以不同浓度药品对肿 瘤细胞抑制率作图， 得到剂量效应曲线， 计算半数有效浓度 （IC5。）。 多种人 肿瘤细胞株观察证明： CPT制剂具有非常明显的抑制肿瘤细胞生长作用，对包 括人肺癌、 结肠癌、 乳腺癌、 胃癌、 胰腺癌、 神经胶质瘤、 血液细胞癌、 膀 胱癌、 前列腺癌、 直肠结肠癌、 子宫颈癌和脑瘤细胞等 10余种肿瘤细胞均有 非常明显的体外杀伤活性， 其对 U251 (人脑神经胶质瘤细胞）、 C0L0 205 (人 结肠癌细胞）， NCI-H460 (人肺癌细胞）， MDA-MB-435 (人 L腺癌细胞） 的半 数抑制浓度（IC5。）<0. Ol g/ml。对 HL- 60(人早幼粒白血病细月包）， MDA- MB- 231 (人乳腺癌细胞）， PC- 3 (人胰腺癌细胞）、 H125 (人肺癌细胞）的 IC5。< 0. 1 g/ml。 对 RPMI 8226 (人多发性骨髓瘤细胞） IC5。<l g/ml。 对 SCLC (人 小细胞肺癌）生长亦显示一定抑制作用。
体内试验结果显示： 用 15mg/kg/日剂量， 连续 10— 15 天给动物腹腔注 射 CPT， 对人结肠癌 C0L0 205、 人肺癌 NCI- H460、 人神经胶质瘤 U251等肿 瘤生长均显示非常明显的生长抑制作用。
与不给药对照组相比， CPT可明显减缓动物肿瘤生长速度， 在移植瘤生 长早期高剂量用药可使肿瘤体积缩小甚至消失。 药物剂量效应关系明显， 药 效作用与给药疗程显示一定相关。在人结肠癌 C0L0 205裸鼠移植瘤模型观察 到， 给小鼠腹腔注射 15mg/kg/日剂量的 CPT 15次至停药 4周后实验结束时， 该组动物所荷肿瘤仍小于给药前肿瘤体积， 其中 1/3的动物肿瘤接种部位无 法确认肿瘤存在， 仅在解剖后见到接种痕迹。
所观察的各项指标证明， CPT给药对实验所选用的人癌裸鼠移植性肿瘤 生长抑制作用显著。 与对照组相比， CPT 15mg/kg给药对 U251的瘤重抑制率 分别为 87. 5 % %和 87. 7% , 对 NCI- H460的瘤重抑制率分别为 87. 0%和 88. 8 % , 对 COLO 205的瘤重抑制率分别为 97. 4. 0 %和 97. 8 % , 统计学差异非常 显著； 本发明利用 3个模型进行了实验， 结果显示， CPT以 5. 0mg/kg剂量， 连续 10— 15天给药， 3个动物模型的相对肿瘤体积 (RTV)与阴性对照组相比 均显示明显统计学差别， 15mg/kg剂量与阴'性对照组相比均显示非常明显统 计学差别， T/C ( % )均在 25以下， 甚至〈氐于 10， 显示高度的抗肿瘤活性 和良好的实验重现性。
CPT给药对动物无明显毒副作用， 在实验观察期内除给药组小鼠荷瘤体 积较小外， 体重、 存活数等生存状态与对照组比较无明显不同。 证明 CPT对 肿瘤细胞有明显的选择性抑制作用， 不会引起正常组织细胞的凋亡， 因此可 以用作安全有效的治疗癌症的药物， 具有重要的理论意义及应用价值， 市场 前景广阔。

Claims

权利要求书
1、 一种具有抑癌作用的重组蛋白， 是下列家族成员中的一种- 1)具有序列表中 SEQ ID No: 2氨基酸残基序列的蛋白质；
2)与 SEQ ID No: 2的氨基酸残基序列同源性在 90%以上且具有与 SEQ ID
No: 2相同活性的由 SEQ ID No: 2衍生的蛋白质；
3)在 SEQ ID No: 2的氨基酸残基序列的 N-端增加或删除 15个或 15个 以内氨基酸残基得到的具有与 SEQ ID No: 2相同活性的由 SEQ ID N2: 2衍 生的蛋白质；
4)在 SEQ ID No: 2的氨基酸残基序列的 C-端增加或删除 15个或 15个 以内氨基酸残基得到的具有与 SEQ ID Na: 2相同活性的由 SEQ ID N2: 2衍 生的蛋白质；
5)将 SEQ ID No: 2的氨基酸残基序列经过一个或几个氨基酸残基的取 代、 缺失或添加且具有与 SEQ ID N2: 2的氨基酸残基序列相同活性的由 SEQ ID a: 2衍生的蛋白质。
2、根据权利要求 1所述的蛋白质，其特征在于： 所述蛋白质为序列表中 的 SEQ ID No: 2。
3、一种具有抑癌作用的重组蛋白的编码基因，它是下列核苷酸序列之一: 1)序列表中的 SEQ ID No: 1；
2)编码序列表中 SEQ ID N2: 2蛋白质序列的多核苷酸；
3)与序列表中 SEQID No: 1限定的 DNA序列具有 90%以上同源性， 且 编码相同功能蛋白质的 DNA序列；
4)编码由 SEQ ID No: 2衍生的蛋白质的 DNA序列， 所述由 SEQ ID Na: 2衍生的蛋白质为在 SEQ ID No: 2的氨基酸残基序列的 N-端增加或删除 15 个或 15个以内氨基酸残基得到的具有与 SEQ ID No: 2相同活性的多肽；
5)编码由 SEQ ID No: 2衍生的蛋白质的 DNA序列， 所述由 SEQ ID Ns: 2衍生的蛋白质为在 SEQ ID a: 2的氨基酸残基序列的 C-端增加或删除 15 个或 15个以内氨基酸残基得到的具有与 SEQ ID No: 2相同活性的多肽；
6)编码由 SEQ ID No: 2衍生的蛋白质的 DNA序列， 所述由 SEQ ID Na: 2衍生的蛋白质为将 SEQ ID No: 2的氨基酸残基序列经过一个或几个氨基酸 残基的取代、 缺失或添加且具有与 SEQ ID No: 2的氨基酸残基序列相同活性 的多肽。
4、根据权利要求 3所述的编码基因， 其特征在于：所述基因是序列表中 的 SEQ ID No : 1。
5、 一种以权利要求 1所述蛋白质为活性成分的治疗癌症的药物。
6、 根据权利要求 5所述的药物， 其特征在于： 所述蛋白质是序列表中
SEQ ID No : 2。
7、根据权利要求 5或 6所述的药物，其特征在于：所述药物中还含有人 体可接受的药用载体。
8、 含有权利要求 4所述基因的表达载体。
9、 含有权利要求 4所述基因的细胞系。
10、 权利要求 1所述的重组蛋白在制备治疗癌症的药物中的应用。
11、 权利要求 2所述的重组蛋白在制备治疗癌症的药物中的应用。
PCT/CN2003/000928 2003-11-03 2003-11-03 Proteine de recombinaison anticancereuse, ses genes codant et ses utilisations WO2005042744A1 (fr)

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DE60336353T DE60336353D1 (de) 2003-11-03 2003-11-03 Rekombinantes protein mit krebsunterdrückender wirkung, sein codierendes gen und seine verwendung
JP2005510086A JP4688678B2 (ja) 2003-11-03 2003-11-03 抗癌作用を有する組換えタンパク質、それをコードする遺伝子、及びそれらの使用
CNB2003801106041A CN100549175C (zh) 2003-11-03 2003-11-03 一种具有抑癌作用的重组蛋白及其编码基因与应用
PCT/CN2003/000928 WO2005042744A1 (fr) 2003-11-03 2003-11-03 Proteine de recombinaison anticancereuse, ses genes codant et ses utilisations
AU2003280921A AU2003280921B2 (en) 2003-11-03 2003-11-03 A recombinant protein with cancer suppression action, its encoding gene and use
AT03770858T ATE501256T1 (de) 2003-11-03 2003-11-03 Rekombinantes protein mit krebsunterdrückender wirkung, sein codierendes gen und seine verwendung
US10/577,535 US7666989B2 (en) 2003-11-03 2003-11-03 Recombinant protein having an anti-cancer effect, its encoding gene and uses thereof
BRPI0318594A BRPI0318594B8 (pt) 2003-11-03 2003-11-03 proteína recombinante que possui um efeito anticâncer, seu gene de codificação, seu uso, medicamento para tratar cânceres, vetor de expressão e microrganismo transgênico
CA2544473A CA2544473C (en) 2003-11-03 2003-11-03 A recombinant protein having an anti-cancer effect, its encoding gene and uses thereof
EP03770858A EP1688498B1 (en) 2003-11-03 2003-11-03 A recombinant protein with cancer suppression action, its encoding gene and use
NZ547274A NZ547274A (en) 2003-11-03 2003-11-03 A recombinant protein with cancer suppression action, its encoding gene and use
HK06110111.7A HK1089788A1 (en) 2003-11-03 2006-09-12 A recombinant protein with cancer suppression action, its encoding gene and use

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CA (1) CA2544473C (zh)
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US7960348B2 (en) 2005-12-22 2011-06-14 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US8361968B2 (en) 2002-12-26 2013-01-29 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
WO2013037090A1 (zh) 2011-09-16 2013-03-21 北京沙东生物技术有限公司 包含trail/apo2l环化变构体的融合蛋白及其编码基因与应用
US8404643B2 (en) 2005-12-22 2013-03-26 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US8765909B2 (en) 2006-10-25 2014-07-01 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US8778871B2 (en) 2004-06-25 2014-07-15 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof

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JPWO2009131191A1 (ja) * 2008-04-24 2011-08-25 武田薬品工業株式会社 メタスチン誘導体およびその用途
HUE027068T2 (en) 2010-12-03 2016-08-29 Adamed Sp Zoo Anti-cancer fusion protein
PL219845B1 (pl) 2011-01-05 2015-07-31 Adamed Spółka Z Ograniczoną Odpowiedzialnością Przeciwnowotworowe białko fuzyjne
PL394618A1 (pl) 2011-04-19 2012-10-22 Adamed Spólka Z Ograniczona Odpowiedzialnoscia Przeciwnowotworowe bialko fuzyjne
PL397167A1 (pl) 2011-11-28 2013-06-10 Adamed Spólka Z Ograniczona Odpowiedzialnoscia Przeciwnowotworowe bialko fuzyjne
PL223487B1 (pl) 2011-12-28 2016-10-31 Adamed Spółka Z Ograniczoną Odpowiedzialnością Przeciwnowotworowe białko fuzyjne

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US8361968B2 (en) 2002-12-26 2013-01-29 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US8778871B2 (en) 2004-06-25 2014-07-15 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US7960348B2 (en) 2005-12-22 2011-06-14 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US8404643B2 (en) 2005-12-22 2013-03-26 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
US8765909B2 (en) 2006-10-25 2014-07-01 Takeda Pharmaceutical Company Limited Metastin derivatives and use thereof
WO2013037090A1 (zh) 2011-09-16 2013-03-21 北京沙东生物技术有限公司 包含trail/apo2l环化变构体的融合蛋白及其编码基因与应用
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CN1860229A (zh) 2006-11-08
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AU2003280921B2 (en) 2007-09-13
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CA2544473C (en) 2013-08-13
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US20080280821A1 (en) 2008-11-13
DE60336353D1 (de) 2011-04-21
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EP1688498B1 (en) 2011-03-09
ATE501256T1 (de) 2011-03-15
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