CN104769431A - 以在癌干细胞中表达的分子为靶标的癌症诊断和治疗方法 - Google Patents
以在癌干细胞中表达的分子为靶标的癌症诊断和治疗方法 Download PDFInfo
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Abstract
本发明提供用于确定癌恶性程度的新方法、用于癌诊断的新方法、用于确定预后的新方法、用于癌治疗的新疫苗和用于遏制癌转移的新疫苗。具体地,本发明提供一种癌恶性程度评估方法,所述方法包括测量癌组织中DDX3X表达水平的步骤、以及通过使用DDX3X表达水平来评估癌组织恶性程度的步骤。
Description
技术领域
本发明涉及以在癌干细胞中表达的分子为靶标的癌症诊断和治疗方法,特别地,涉及用于确定癌恶性程度的方法,癌预后评估方法,癌抗原肽,用于制造用于过继性免疫的细胞组合物的方法,以及癌预防、癌治疗、癌转移遏制或癌复发遏制剂。
背景技术
日益增加的证据表明,大多数实体瘤由异质性的肿瘤细胞构成,相对小的细胞亚群显示出独特的特征,包括:高的致肿瘤性(tumorgenicity)、作为非粘附性球体生长、无限自更新、和非对称性分化。该独特的亚群的成员具有正常干细胞所具有的生物学、生物化学和分子学特征,因此被称为癌干细胞(cancer stem cells,CSC)。在经典的CSC模型中,CSC亚群与相对分化的、主体癌细胞群(bulkcancer population)之间的递进关系(hierarchy)是严格且单向的。但近来的数据显示,CSC和分化的癌细胞可在受调控的平衡下双向转化(非专利文献1)。
有报道称,在具有细胞毒性的化疗和分子靶向治疗后存活的极少量细胞展现出对CD133(可能的CSC标志物之一)的一致性表达(非专利文献2和3)。因为CSC具有抵抗细胞死亡的多种机制:例如改变的染色质状态、过量的多药排出转运蛋白(multidrug effluxtransporters)、抗凋亡因子、DNA修复基因产物和干细胞特异性生长信号机制,因此CSC可在致死性胁迫(例如具有细胞毒性的抗癌药物、分子靶向治疗剂和放疗)下存活。在此类致死性胁迫下存活的这些独特的癌细胞亚群可产生永久的、耐药的细胞群,所述细胞群具有遗传突变,并且可用作为母细胞(非专利文献2)。因此,CSC系统是大多数的治疗失败和癌复发的最可能的原因。除非开发出能够摧毁CSC亚群的有效治疗,否则极难实现持久性的治愈。因此,人们需要用于摧毁CSC亚群的有效治疗。
参考文献
非专利文献
非专利文献1:Li Y,Laterra J.Cancer stem cells:distinct entities ordynamically regulated phenotypes?Cancer research.2012;72:576-80
非专利文献2:Sharma SV,Lee DY,Li B,Quinlan MP,Takahashi F,Maheswaran S,et al.A chromatin-mediated reversible drug-tolerant state incancer cell subpopulations.Cell.2010;141:69-80
非专利文献3:Rappa G,Fodstad O,Lorico A.The stemcell-associated antigen CD133(Prominin-1)is a molecular therapeutic targetfor metastatic melanoma.Stem Cells.2008;26:3008-17
发明内容
本发明解决的技术问题
本发明的目的之一是提供用于确定癌恶性程度的新方法,新的癌预后评估方法,新的癌抗原肽,用于制造用于过继性免疫的细胞组合物的新方法,和新的癌预防、癌治疗、癌转移遏制或癌复发遏制剂。
解决技术问题的方案
本申请的发明人以前曾报道,在肿瘤引流淋巴结中经肿瘤抗原初免疫(primed)的效应T细胞在脑、肺和皮肤转移模型中具有抗肿瘤治疗效果(Kagamu H,Shu S.Purification of L-selectin(low)cellspromotes the generation of highly potent CD4 antitumor effector Tlymphocytes.J Immunol.1998;160:3444-52.、Fujita N,Kagamu H,Yoshizawa H,Itoh K,Kuriyama H,Matsumoto N,et al.CD40ligandpromotes priming of fully potent antitumor CD4(+)T cells in draininglymph nodes in the presence of apoptotic tumor cells.J Immunol.2001;167:5678-88)。再后来,本申请的发明人注目于CSC标志物之一CD133,并且成功地纯化出了CD133阳性黑色素瘤细胞,其在总的黑色素瘤细胞中占少于1%。该CD133阳性黑色素瘤细胞具有CSC性质。本发明的发明人发现,用黑色素瘤CSC进行接种诱导出了特异性CD8阳性T细胞,其包括辅助T细胞17(Th17细胞)和Th1细胞。特别地,黑色素瘤CSC特异性CD4阳性T细胞驱动效应器T细胞和具有高表达的MHC II类分子的活性树突状细胞在肿瘤细胞中的长期积累,展示出强的抗肿瘤效果。调控T细胞(Treg)(典型地被发现于肿瘤组织中)在注射了黑色素瘤CSC特异性CD4阳性T细胞的小鼠中没有被诱导。此外,该治疗摧毁了CD133阳性肿瘤细胞,由此使得亲本黑色素瘤(parental melanomas)治愈。这些结果显示,CD133阳性黑色素瘤细胞具有特异性的免疫原性抗原,以及,抗原特异性T细胞具有迄今未见的水平的、能摧毁CSC的抗肿瘤活性。
为了对优先表达于CD133阳性肿瘤细胞中的免疫原性蛋白质加以阐明,本申请的发明人使用二维电泳分析对蛋白质表达进行了比较,由此鉴定出了四种蛋白质。基于质谱(MS/MS)分析数据的Mascot检索将这些蛋白质之一鉴定为连接X的DEAD/H(Asp-Glu-Ala-Asp/His)盒多肽3(DDX3X)。
该蛋白质是ATP依赖性RNA解旋酶(helicase)DEAD盒家族(DEAD盒解旋酶)的成员,位于X染色体上。DEAD盒解旋酶具有多种功能,包括RNA剪切、从细胞核向胞质运出mRNA、转录和翻译调控、RNA分解和核糖体生物发生(Rocak S,Linder P.DEAD-boxproteins:the driving forces behind RNA metabolism.Nature reviewsMolecular cell biology.2004;5:232-41)。DDX3X在进化上从酵母到人高度保守,这暗示其是细胞存活所必需的。其具有位于Y染色体上的同源体DDX3Y,这两个基因均在胚胎发生中发挥作用。在人类中,DDX3X缺失或功能障碍导致生殖细胞形成受损(Matzuk MM,LambDJ.Genetic dissection of mammalian fertility pathways.Nat Cell Biol.2002;4Suppl:s41-9)。
在本发明中,本申请的发明人发现,DDX3X是CD133阳性黑色素瘤细胞的主要免疫原性靶标蛋白质。用合成的DDX3X进行接种在皮肤黑色素瘤治疗模型中展示出肿瘤消退(regression)效果。DDX3X在表达CSC标志物的人癌细胞系中强表达,但在正常的人上皮细胞和正常的人内皮细胞中仅略微表达。
本申请的发明人从上述结果设想:
癌组织中的DDX3X表达水平可用作为癌恶性程度的指标;
DDX3X特异性T细胞在癌患者血液中的存在与否可用作为癌预后评估的指标;
DDX3X的部分肽(片段)可用作为癌疫苗;以及
抗DDX3X免疫疗法可能是有望摧毁CSC、由此治愈癌症的有前途的策略。
本申请的发明人进行了进一步研究,并完成了本发明。
本发明包括下述方面。
项1、癌恶性程度评估方法,所述方法包括下述步骤:
测量癌组织中的DDX3X表达水平的步骤;和
通过使用所述DDX3X表达水平来评估癌组织的恶性程度的步骤。
项2、癌恶性程度评估试剂盒,所述试剂盒包含:
针对DDX3X的抗体、或者
与DDX3X mRNA或相应的cDNA特异性结合的多核苷酸,
其中,所述抗体或多核苷酸用于测量癌组织中DDX3X的表达水平。
项3、癌预后评估方法,所述方法包括下述步骤:
检测癌患者的血液中DDX3X特异性T细胞的步骤;和
通过使用检测结果评估癌预后的步骤。
项4、癌预后评估试剂盒,所述试剂盒包含:
DDX3X或其部分肽,
其中,所述DDX3X或其部分肽用于检测癌患者血液中的DDX3X特异性T细胞。
项5、一种肽,其由:
包含SEQ ID NO:1的氨基酸序列中SEQ ID NO:2至87中任一所示的氨基酸序列的9~20个连续氨基酸的序列,或
与上述氨基酸实质上相同的氨基酸序列
构成。
项6、根据项5所述的肽,其中,所述肽为癌抗原肽。
项7、癌疫苗,其包含项5所述的肽。
项7-2、根据项5所述的肽,其用于针对癌的疫苗。
项8、根据项7所述的癌疫苗,其中,所述疫苗用于预防或治疗癌,或用于遏制癌转移或癌复发。
项9、制造过继性免疫细胞的方法,所述方法包括下述步骤:
用DDX3X或其部分肽对具有抗原呈递能力的细胞进行脉冲的步骤。
项10、经DDX3X或其部分肽脉冲的抗原呈递细胞。
项11、DDX3X特异性细胞诱导剂,其包含项10所述的抗原呈递细胞作为活性组分。
项11-2、根据权利要求10所述的抗原呈递细胞,其中所述抗原呈递细胞用于诱导DDX3X特异性T细胞。
项12、制造过继性免疫细胞组合物的方法,所述方法包括下述步骤:
将具有抗原呈递能力的细胞暴露给DDX3X或其部分肽、以获得呈递源于DDX3X或其部分肽的抗原的细胞的步骤;和
用所述抗原呈递细胞诱导DDX3X特异性T细胞的步骤。
项13、根据项12所述的方法,其中所述DDX3X特异性T细胞是DDX3X特异性CD4阳性T细胞。
项14、癌预防、癌治疗、癌转移遏制或癌复发遏制剂,包含抑制DDX3X表达或活性的化合物。
项14-2、抑制DDX3X表达或活性的化合物,所述化合物用于预防或治疗癌、或用于遏制癌的转移或癌的复发。
本发明的有益效果
本发明的肽是癌抗原,其可用于提供用于确定癌恶性程度的方法,癌预后评估方法,癌抗原肽,用于制造用于过继性免疫的细胞组合物的方法,以及癌预防、癌治疗、癌转移遏制或癌复发遏制剂,等等。
附图简述
图1是人DDX3X的氨基酸序列。
图2是展示由CD8阳性T细胞进行的IFN-γ制造的图(实施例1)。
图3是展示由CD4阳性T细胞进行的IFN-γ和IL-17制造的图(实施例1)。
图4是展示由CD8阳性T细胞进行的IFN-γ制造的图(实施例1)。
图5是展示由CD4阳性T细胞进行的IFN-γ和IL-17制造的图(实施例1)。
图6显示了经接种的小鼠的肿瘤生长曲线(实施例2)。
图7显示了具有已建立的皮肤肿瘤的经接种小鼠的肿瘤生长曲线(实施例3)。
图8显示了经接种的小鼠的肿瘤生长曲线(实施例4)。
图9是展示通过用DD3C部分肽刺激进行的IFN-γ制造的图(实施例5)。
图10是展示通过用DD3C部分肽刺激进行的IFN-γ制造的图(实施例6)。
图11是展示经接种小鼠的肿瘤生长曲线的图(B)(实施例7)。
图12显示了CD133表达的流式细胞分析结果的图(实施例8)。
图13显示了使用针对DDX3X和β-肌动蛋白的抗体从肿瘤细胞获得的免疫印迹(实施例8)。
图14显示了在细胞损伤修复实验的损伤修复过程中拍摄的照片(实施例9)。
图15显示了拍摄来检查球状体形成的球状体(spheroid)照片(实施例11)。
具体实施方式
在本文中使用时,“癌(cancer)”指细胞在生物体中异常且不受控的增殖。例子包括实体瘤(例如,恶性肿瘤(carcinoma)和肉瘤(sarcoma))、淋巴瘤(lymphoma)和白血病(leukemia)。
更具体的例子包括:
儿童脑肿瘤,例如星形胶质细胞瘤(astroglioma)、恶性髓母细胞瘤(malignant medulloblastoma)、生殖细胞瘤(germ cell tumor)、颅咽管瘤(craniopharyngioma)、室管膜瘤(ependymoma);
成人脑肿瘤,例如神经胶质瘤(glioma)、神经胶质瘤(neuroglioma)、脑膜瘤(meningioma)、垂体腺瘤(pituitary adenoma)、神经鞘瘤(neurilemoma);
头颈部癌症,例如上颌窦癌(maxillary sinus cancer)、咽癌(pharyngeal cancer)(鼻咽癌(nasopharyngeal carcinoma)、中下咽癌(mesopharyngeal carcinoma)、下咽癌(hypopharyngeal carcinoma))、喉癌(laryngeal cancer)、口腔癌(oral cancer)、唇癌(lip cancer)、舌癌(tongue cancer)和腮腺癌(parotid cancer);
胸部癌症和肿瘤,例如小细胞肺癌(small cell lung cancer)、非小细胞肺癌(non-small cell lung cancer)、胸腺瘤(thymoma)和间皮瘤(mesothelioma);
胃肠癌和肿瘤,例如食道癌(esophageal cancer)、肝癌(livercancer)、原发性肝癌(primary hepatic cancer)、胆囊癌(gallbladdercancer)、胆管癌(bile duct cancer)、胃癌(stomach cancer)、大肠癌(large bowel cancer)、结肠癌(colonic cancer)、直肠癌(rectal cancer)、肛门癌(anal cancer)、胰腺癌(pancreatic cancer)和胰腺内分泌肿瘤(pancreatic endocrine tumor);
泌尿器官癌症和肿瘤,例如阴茎癌(penile cancer)、肾盂和输尿管癌(renal pelvis and ureteral cancer)、肾细胞癌(renal cell cancer)、睾丸肿瘤(testicular tumor)、前列腺癌(prostatic cancer)、膀胱癌(bladder cancer)、肾母细胞瘤(Wilms tumor)和尿路上皮癌(urothelialcancer);
妇科癌症和肿瘤,例如外阴癌(vulvar cancer)、子宫颈癌(uterinecervical cancer)、子宫体癌(corpus uteri cancer)、子宫内膜癌(endometrial cancer)、子宫肉瘤(uterine sarcoma)、绒毛膜癌(chorioniccancer)、阴道癌(vaginal cancer)、乳腺癌(breast cancer)、卵巢癌(ovarian cancer)和卵巢生殖细胞肿瘤(ovarian germ cell tumor);
成人和儿童软组织肉瘤;
骨肿瘤,例如骨肉瘤(osteosarcoma)和尤因氏瘤(Ewing’s tumor);
内分泌组织癌症和肿瘤,例如肾上腺皮质癌(adrenocorticalcancer)和甲状腺癌(thyroid cancer);
恶性淋巴瘤和白血病,例如恶性淋巴瘤(malignant lymphoma)、非霍奇金淋巴瘤(non-Hodgkin’s lymphoma)、霍奇金氏病(Hodgkin’sdisease)、多发性骨髓瘤(multiple myeloma)、浆细胞瘤(plasmacytictumor)、急性骨髓性白血病(acute myelogenous leukemia)、急性淋巴性白血病(acute lymphatic leukemia)、成人T细胞白血病淋巴瘤(adult T cell leukemia lymphoma)、慢性髓细胞性白血病(chronicmyelogenous leukemia)和慢性淋巴性白血病(chronic lymphaticleukemia);
皮肤癌症和肿瘤,如慢性骨髓增生性疾病(chronicmyeloproliferative disorders)、恶性黑色素瘤(malignant melanoma)(黑色素瘤)、鳞状细胞癌(squamous cell cancer)、基底细胞癌(basal cellcancer)和蕈样肉芽肿(mycosis fungoides);
以及这些肿瘤和癌症的转移灶。
本发明特别适合于在例如胸部癌症和肿瘤(例如小细胞肺癌和非小细胞肺癌)、皮肤癌症和肿瘤(例如恶性黑色素瘤(黑色素瘤))以及妇科癌症和肿瘤(例如乳腺癌)中的应用。
DDX3X是连接X的DEAD/H(Asp-Glu-Ala-Asp/His)盒多肽3。
如前文所述,该蛋白质是ATP依赖性RNA解旋酶DEAD盒家族(DEAD盒解旋酶)的成员,位于X染色体上。氨基酸序列已知。人DDX3X的序列(UniProtKB/Swiss-Prot:O00571.3)示于图1(SEQID NO:1)。
本文中使用的所有缩写,包括碱基序列(核苷酸序列)和核酸及氨基酸遵循IUPAC-IUB的规定(IUPAC-IUB communication onBiological Nomenclature,Eur.J.Biochem.,138;9(1984))、《包含碱基序列或氨基酸序列的说明书撰写指导(Guidelines for the Preparation ofSpecification which Contains Nucleotide and/or Amino Acid Sequence)》(JPO)和本领域的惯用标记法。
除非另有指明,在本申请文件中出现的碱基序列以从5’至3’末端表示。
除非另有指明,在本申请文件中出现的氨基酸序列以从N末端至C末端表示。
除非另有指明,在本文中使用时,“基因”包括双链DNA以及单链DNA(正义链)和具有与正义链互补的序列的单链DNA(反义链)、和它们的片段。此外,除非另有指明,本文中术语“基因”不区分调控区域、编码区域、外显子和内含子。
在本文中使用时,“核苷酸”(或“多核苷酸”)具有与核酸相同的含义,包括DNA和RNA二者。它们可以是双链或单链的。除非另有指明,某“核苷酸”(或“多核苷酸”)序列也包括具有互补序列的核苷酸(或多核苷酸)。
除非另有指明,在本文中使用时,“多核苷酸”包括寡核苷酸。
此外,除非另有指明,“核苷酸”(或“多核苷酸”)包括经修饰的核苷酸或核苷酸类似物(例如PNA和LNA)。
当“核苷酸”(或“多核苷酸”)是RNA时,序列表的碱基中的字母“T”应被理解为“U”。
除非另有指明,在本文中使用时,“cDNA”包括具有与mRNA互补的碱基序列的单链DNA(单链cDNA),以及单链cDNA和其互补序列的双链DNA(双链cDNA)。
在本文中使用时,“特异性杂交”表示:在普通杂交条件下、优选在严格杂交条件下(例如,在Sambrook,et al.,Molecular Cloning,Cold Spring Harbour Laboratory Press,New York,USA,2nd Ed.,1989中描述的条件下),以不与样品中其它多核苷酸显著杂交的方式进行杂交。在此类严格杂交条件的具体例子中,在6×SSC、0.5%SDS和50%甲酰胺溶液中于42℃进行加热,接着在0.1×SSC、0.5%SDS溶液中于68℃进行洗涤,之后观察到阳性杂交信号。
除非另有指明,在本文中使用时,“蛋白质/蛋白”包括经修饰的蛋白(例如,具有糖链)和未经修饰的蛋白。这也适用于不被特别指明为蛋白质的蛋白质。
在本文中使用时,来源于DDX3X或其部分肽的肽也被称为源于DDX3X的肽。
在本文中使用时,DDX3X的部分肽(或片段)表示含有DDX3X的部分氨基酸序列的肽。
癌恶性程度评估方法
本发明的癌恶性程度评估方法包括测量癌组织中的DDX3X表达水平的步骤、和通过使用所述DDX3X表达水平来评估癌组织的恶性程度的步骤。
在本文中使用时,“癌恶性程度”表示就临床意义而言癌杀死宿主的快速程度。具体地,其可被视为癌导致的死亡率的程度、转移可能性、癌预后程度、或癌治疗的困难程度。
在本文中使用时,“癌组织”可以例如是为了测试目的从患者收集的癌组织、通过手术摘除的癌组织、或此类癌组织的一部分。
DDX3X表达水平的测量可使用能将高恶性程度癌组织中DDX3X表达水平与低恶性程度癌组织中DDX3X表达水平区分开的任何手段来进行。
在本发明的一种实施方式中,可通过测量蛋白质DDX3X的量来进行对DDX3X表达水平的测量。具体而言,例如,根据需要使用常规方法从癌组织提取或制备蛋白质,通过使用下文例示的方法测量DDX3X表达水平。可通过使用例如市售试剂盒来提取或制备蛋白质。
对用于测量DDX3X的量的方法没有特别限定,只要可特异性地测量蛋白质的量即可。例子包括Western印迹、ELISA、荧光抗体方法、和蛋白质阵列(蛋白质芯片)方法。
在ELISA中,例如,将含有从癌组织提取或制备的蛋白质的溶液吸附至微多孔板的孔的固体表面,通过在应用针对DDX3X的抗体之后的酶反应来测量DDX3X的量。
在蛋白质阵列方法中,例如,制备具有针对DDX3X的抗体的蛋白质阵列(例如,抗体阵列(抗体芯片)),将从癌组织提取的蛋白质应用至蛋白质阵列。在抗体-抗原反应之后,通过使用例如ELISA等方法测量已与抗体结合的DDX3X的量。
抗体可以例如是多克隆抗体或单克隆抗体。
抗体可以例如是可以与抗原特异性结合的抗体片段,例如Fab片段和F(ab’)2片段。
可使用已知方法来制造抗体。
例如,当抗体是多克隆抗体时,可以用通过已知方法在大肠杆菌等中表达而制备的DDX3X或其部分肽、或用通过已知方法合成的DDX3X或其部分肽去免疫非人动物(例如兔),之后使用常用技术从经免疫的动物的血清来制备抗体。
另一方面,当抗体是单克隆抗体时,可通过下述方法制备抗体:将通过已知方法在大肠杆菌等中表达而制备的DDX3X或其部分肽用作为抗原,使用上文制备的抗原对非人哺乳动物(例如小鼠)进行免疫,将获得的抗体产生细胞与骨髓瘤融合,从由此制备的杂交瘤来制备抗体。
本发明中,优选用作为DDX3X部分肽的例如是下文描述的本发明的肽。
可根据可获得的氨基酸序列信息,使用常用的化学合成技术,来制造DDX3X或其部分肽。此类技术包括普通的液相或固相肽合成技术。此类肽合成技术的例子包括Peptide Synthesis(Maruzen,1975)和Peptide Synthesis,Interscience,New York,(1996)中描述的技术。已知的化学合成设备,例如,肽合成仪(Applied Biosystems),也可用于制造DDX3X或其部分肽。
还可使用表达载体、克隆载体等,使用常用的遗传工程技术(包括,例如基于编码基因的碱基序列信息进行的DNA克隆、质粒构建、转染进宿主、转染物培养和从培养的产物收集蛋白质等流程),来制造用作为抗原的DDX3X或由DDX3X的部分氨基酸序列构成的肽。
可通过将DDX3X或编码其部分肽的多核苷酸并入合适的载体DNA来获得重组载体。
可根据宿主的类型和期望的用途来合适地选择载体DNA。载体DNA可以是天然存在的DNA,或是除增殖所必需的区域之外的DNA部分被部分缺失的天然DNA。载体DNA的例子包括源于染色体、游离体或病毒的载体。具体例子包括:细菌质粒、噬菌体、转座子、酵母游离体、插入元件、酵母染色体元件、源于病毒(例如杆状病毒、乳头瘤病毒、SV40、痘病毒、腺病毒、禽痘病毒、假狂犬病毒和逆转录病毒)的载体和具有这些病毒的组合的载体、以及源于质粒和噬菌体的遗传元件的载体(例如粘粒(cosmids)和噬粒(phagemids))。
含有多核苷酸的重组载体可通过使用已知方法将多核苷酸插入进载体DNA来获得。具体而言,例如,使用合适的限制性酶在特异性位点对DNA和载体DNA加以切割,将它们混合并用连接酶重新连接。或者,可将合适的接头连接至多核苷酸,并插入进适用于所期望的目的的载体DNA的多克隆位点,以获得重组载体。
可通过使用已知方法,将含有多核苷酸的重组载体导入已知宿主,例如细菌,例如大肠杆菌(例如K12)和杆菌属细菌(例如MI114),酵母(例如AH22),昆虫细胞(例如Sf细胞)和动物细胞(例如COS-7细胞、Vero细胞和CHO细胞),来获得具有重组载体的转染体。
考虑到基因稳定性,优选使用染色体整合作为基因导入方法。为方便起见,可使用利用核外基因的自主复制系统。将载体DNA导入宿主细胞的过程可按照标准方法(例如Molecular Cloning:ALaboratory Manual(Sambrook et al.,Cold Spring Harbour Laboratory Press,Cold Spring Harbour,New York,1989)中描述的方法)来进行。具体例子包括磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、阳离子脂质介导的转染、电穿孔、转导、划痕负载(scrape loading)和生物弹轰击导入(ballistic introduction)。
DDX3X也可商业获得。
抗DDX3X抗体也可商业获得。
用于测量DDX3X的量的抗体可通过使用已知的标记方法(例如酶标记、放射性标记和荧光标记)来标记,或可用生物素等来修饰。
在本发明的另一实施方式中,可通过测量DDX3X的mRNA的量来进行对DDX3X表达水平的测量。具体而言,例如,根据需要,通过使用常规方法从癌组织提取或制备mRNA,测量DDX3X mRNA的量,这例如通过下文例示的方法来进行。mRNA的提取或制备可使用例如市售试剂盒来进行。
用于测量DDX3X mRNA的量的方法不受特别限制,只要其可测量特定mRNA的水平即可。例如,可以使用利用了与DDX3XmRNA或对应的cDNA特异性结合的多核苷酸探针或引物的已知方法,包括,例如southern印迹杂交、原位杂交、比较基因组杂交(CGH)、定量PCR(例如实时PCR)和Invader(来自HOLOGIC,USA的产品)方法。
在微阵列方法中,例如,制备具有将与DDX3X mRNA特异性结合的探针的核酸阵列(核酸芯片),将从癌组织提取、并用荧光或其它标记标记过的mRNA样品应用至核酸阵列。然后对来自已经与探针结合的、经标记的DDX3X mRNA的信号进行测量和分析。
在实时PCR中,例如,使用逆转录酶将从癌组织提取或制备的mRNA逆转录为cRNA。通过使用cDNA作为模板,使用与DDX3XcDNA特异性结合的引物,通过PCR扩增出预定区域,在扩增产物的产生期间对其实时监测。
用于测量mRNA的量的探针被设计为允许与DDX3X mRNA或cDNA特异性杂交。
作为探针的多核苷酸优选是选自下组的多核苷酸,所述组由DDX3X mRNA的完全碱基序列或部分碱基序列、或它们的互补序列,以及通过对所述多核苷酸的一个至数个碱基(例如1至10、1至5、1至3个)进行缺失、取代或添加而形成的那些多核苷酸构成。探针多核苷酸典型地为15至500个碱基长,优选为20至200个碱基长,更优选为20至50个碱基长。
探针多核苷酸可包括标记,例如荧光染料、酶、蛋白质、放射性同位素以及化学发光物质,它们被适当地加入,以使得对DDX3XmRNA量的测量得以实现。
用于通过定量PCR等进行mRNA的量的测量的引物被设计为允许与DDX3X mRNA或cDNA特异性杂交。用于设计引物的方法不受特别限制,例如,可以以用于引物设计引用的算法或软件使用已知的方法来进行。典型地,引物作为正向和反向引物组使用。
优选地,引物选自例如下组的多核苷酸,所述组由DDX3XmRNA的完全序列或部分序列、或它们的互补序列,以及通过对所述多核苷酸的一个至数个碱基(例如1至10、1至5、1至3个)进行缺失、取代或添加而形成的那些多核苷酸构成。引物多核苷酸典型地为15至30个碱基长。
引物多核苷酸可包括标记,例如荧光染料、酶、蛋白质、放射性同位素以及化学发光物质,它们被适当地加入,以使得对DDX3XmRNA量的测量得以实现。
可例如通过使用遗传工程技术(例如Enzymology,2005;392:24-35,73-96,173-185,405-419.;Nucleic Acids Res.1984;12:9441;NewBiochemical Experiment Course 1,Gene Research Technique II,TheJapanese Biochemical Society,p.105(1986)中描述的方法)、化学合成手段例如磷酸三酯法和亚磷酰胺法(J Am Chem Soc.1967;89(2):450-3.;J Am Chem Soc.1967;89(26):7146-7147.)和这些方法的组合来制造多核苷酸。RNA合成也可按照亚磷酰胺法进行,其中使用例如可商业获得的高通量DNA合成仪ABI3900(Applied Biosystems)与RNA合成试剂。
还可通过委托提供多核苷酸合成服务的公司或部门,来获得多核苷酸。
还可通过在癌组织中对表达DDX3X的细胞加以计数来进行DDX3X表达水平测量。对表达DDX3X的细胞的计数可通过在经免疫组织染色的癌组织中对表达DDX3X的细胞加以观察来进行,或通过使用例如流式细胞仪等技术,使用经例如荧光染料、酶、蛋白质或化学发光物质标记标记的抗DDX3X抗体对癌组织中表达DDX3X的细胞进行计数来进行。
在本发明的癌恶性程度评估方法中,当测量到的DDX3X表达水平高时,癌组织被评估为具有高恶性程度,当测量到的DDX3X表达水平低时,被评估为低恶性程度。
可使用统计学方法(例如学生t-检验和Kaplan-Meier方法),基于例如非癌组织中、通过传统评估鉴定为高恶性程度的癌组织和通过传统评估鉴定为低恶性程度的癌组织中的DDX3X表达水平,将DDX3X表达水平和癌组织恶性程度的水平关联起来。
本发明的癌恶性程度评估方法可与其它癌恶性程度评估方法一起进行。
癌恶性程度评估试剂盒
本发明的癌恶性程度评估试剂盒可用于本发明的癌恶性程度评估方法。
本发明的癌恶性程度评估试剂盒包含针对DDX3X的抗体、或能与DDX3X mRNA或对应的cDNA特异性结合的多核苷酸。
在本发明的一种实施方式中,癌恶性程度评估试剂盒包括针对DDX3X的抗体。
所述抗体用于测量蛋白质DDX3X的量。
与针对癌恶性程度评估方法描述的抗体相同的抗体可用作为癌恶性程度评估试剂盒中的抗体。
所述抗体可形成蛋白阵列(例如抗体阵列和抗体芯片)。蛋白阵列具有基底和抗体,抗体被配置于基底上。对基底没有特别限制,只要蛋白质可被配置在其上即可。例子包括玻璃板、尼龙膜、微珠粒、硅片和毛细管。蛋白阵列(抗体芯片)可通过使用常用于蛋白阵列制造的方法(例如使用喷墨技术的方法)将抗体固定到基底上来制造。
在本发明的另一实施方式中,本发明的癌恶性程度评估试剂盒包括能与DDX3X mRNA或对应的cDNA特异性结合的多核苷酸。
所述多核苷酸用于测量DDX3X mRNA的量。
与针对癌恶性程度评估方法描述的多核苷酸相同的多核苷酸可用作为癌恶性程度评估试剂盒中的多核苷酸。
所述多核苷酸可形成核酸阵列。核酸阵列具有基底和多核苷酸,多核苷酸被配置于基底上。多核苷酸可以是与针对癌恶性程度评估方法描述的多核苷酸相同的多核苷酸。对基底没有特别限制,只要蛋白质可被配置在其上即可。例子包括玻璃板、尼龙膜、微珠粒、硅片和毛细管。核酸阵列可通过使用常用于核酸阵列制造的方法(例如使用市售点样仪的方法和使用喷墨技术的方法)将多核苷酸固定到基底上来制造。
本发明的癌恶性程度评估试剂盒可包括酶、缓冲剂、试剂或手册,对它们可根据意欲的用途或形式来选择。
癌预后评估方法
本发明的癌预后评估方法包括:
检测癌患者的血液中DDX3X特异性T细胞的步骤;和
通过使用检测结果评估癌预后的步骤。
在本文中使用时,“癌预后”表示癌有可能的进展。
优选地,对癌患者血液中DDX3X特异性T细胞的检测通过对从癌患者获得的血液样品中的DDX3X特异性T细胞进行检测来进行。
血液样品可使用常用方法获得。
对血液样品中DDX3X特异性T细胞的检测可通过常用的抗原特异性T细胞检测方法来进行,所述方法包括例如,抗原依赖性增殖分析(例如3H-胸苷掺入检验)、细胞毒性测量(例如51Cr释放检验)、MHC肽四聚体染色、酶联免疫斑点(ELISPOT)检验和细胞外细胞因子检验。
用于这些检测方法中的抗原可以是与针对癌恶性程度评估方法中所描述的抗原相同的抗原。
当在癌患者的血液中检测到DDX3X特异性T细胞时,预后被评估为良好,当没有检测到DDX3X特异性T细胞时,则被评估为不良。
本发明的癌预后评估方法可以与其它癌预后评估方法一起进行。
癌预后评估试剂盒
本发明的癌预后评估试剂盒可用于本发明的癌预后评估方法。
本发明的癌预后评估试剂盒包含DDX3X或其部分肽。
DDX3X或其部分肽用于检测癌患者血液中的DDX3X特异性T细胞。
DDX3X或其部分肽是与针对癌恶性程度评估方法描述的DDX3X或其部分肽相同的DDX3X或其部分肽。优选将本发明的肽(下文所述)用作为DDX3X或其部分肽。
肽
本发明的肽由SEQ ID NO:1的氨基酸序列中、包含SEQ ID NO:2至87中任一所示的氨基酸序列的9~20个连续氨基酸的序列,或与上述氨基酸实质上相同的氨基酸序列构成。
如上文所述,SEQ ID NO:1是DDX3X的氨基酸序列。
SEQ ID NO:2:FLLDLLNAT
SEQ ID NO:3:NITQKVVWV
SEQ ID NO:4:IQMLARDFL
SEQ ID NO:5:TFPKEIQML
SEQ ID NO:6:KYDDIPVEA
SEQ ID NO:7:RYIPPHLRN
SEQ ID NO:8:RNINITKDL
SEQ ID NO:9:KQYPISLVL
SEQ ID NO:10:IGLDFCKYL
SEQ ID NO:11:IELTRYTRP
SEQ ID NO:12:TRYTRPTPV
SEQ ID NO:13:MGNIELTRY
SEQ ID NO:14:LVLAPTREL
SEQ ID NO:15:YPISLVLAP
SEQ ID NO:16:QYPISLVLA
SEQ ID NO:17:LEDFLYHEGY
SEQ ID NO:18:FLDEYIFLA
SEQ ID NO:19:LLVEAKQEV
SEQ ID NO:20:FLLPILSQI
SEQ ID NO:21:DFLDEYIFL
SEQ ID NO:22:SHVAVENAL
SEQ ID NO:23:VAVENALGL
SEQ ID NO:24:ALGLDQQFA
SEQ ID NO:25:LGLDQQFAG
SEQ ID NO:26:GLDQQFAGL
SEQ ID NO:27:DQQFAGLDL
SEQ ID NO:28:NSSDNQSGG
SEQ ID NO:29:KGRYIPPHL
SEQ ID NO:30:PHLRNREAT
SEQ ID NO:31:RGRGDYDGI
SEQ ID NO:32:YDGIGSRGD
SEQ ID NO:33:RSGFGKFER
SEQ ID NO:34:KPLPPSERL
SEQ ID NO:35:LFSGGNTGI
SEQ ID NO:36:FSGGNTGIN
SEQ ID NO:37:INFEKYDDI
SEQ ID NO:38:YDDIPVEAT
SEQ ID NO:39:TGNNCPPHI
SEQ ID NO:40:EIIMGNIEL
SEQ ID NO:41:IIMGNIELT
SEQ ID NO:42:IPIIKEKRD
SEQ ID NO:43:GSGKTAAFL
SEQ ID NO:44:TAAFLLPIL
SEQ ID NO:45:AAFLLPILS
SEQ ID NO:46:IYADGPGEA
SEQ ID NO:47:LAVQIYEEA
SEQ ID NO:48:IYEEARKFS
SEQ ID NO:49:RPCVVYGGA
SEQ ID NO:50:CVVYGGADI
SEQ ID NO:51:LLVATPGRL
SEQ ID NO:52:ATPGRLVDM
SEQ ID NO:53:GLDFCKYLV
SEQ ID NO:54:LDFCKYLVL
SEQ ID NO:55:LVLDEADRM
SEQ ID NO:56:VLDEADRML
SEQ ID NO:57:GFEPQIRRI
SEQ ID NO:58:FSATFPKEI
SEQ ID NO:59:YIFLAVGRV
SEQ ID NO:60:RVGSTSENI
SEQ ID NO:61:ATGKDSLTL
SEQ ID NO:62:SLTLVFVET
SEQ ID NO:63:FLYHEGYAC
SEQ ID NO:64:LYHEGYACT
SEQ ID NO:65:LHQFRSGKS
SEQ ID NO:66:QFRSGKSPI
SEQ ID NO:67:ILVATAVAA
SEQ ID NO:68:TAVAARGLD
SEQ ID NO:69:ISNVKHVIN
SEQ ID NO:70:LPSDIEEYV
SEQ ID NO:71:EYVHRIGRT
SEQ ID NO:72:LGLATSFFN
SEQ ID NO:73:TSFFNERNI
SEQ ID NO:74:FFNERNINI
SEQ ID NO:75:NITKDLLDL
SEQ ID NO:76:DLLDLLVEA
SEQ ID NO:77:EVPSWLENM
SEQ ID NO:78:AYEHHYKGS
SEQ ID NO:79:EHHYKGSSR
SEQ ID NO:80:SRFSGGFGA
SEQ ID NO:81:FGARDYRQS
SEQ ID NO:82:GGGYGGFYN
SEQ ID NO:83:GGYGGFYNS
SEQ ID NO:84:GGFYNSDGY
SEQ ID NO:85:SDGYGGNYN
SEQ ID NO:86:GGNYNSQGV
SEQ ID NO:87:NYNSQGVDW
除了SEQ ID NO:2至87的氨基酸序列之外,例如,SEQ ID NO:88至92所示的氨基酸序列也可使用。
SEQ ID NO:88:KQYPISLVLAPTREL
SEQ ID NO:89:EIIMGNIELTRYTRPTPV
SEQ ID NO:90:KGADSLEDFLYHEGY
SEQ ID NO:91:FVETKKGADSLEDFLYHEGY
这些序列中的末端谷氨酰胺残基可被环化,形成焦谷氨酸。此类序列的例子是SEQ ID NO:92:pyroEYPISLVLA。
在本文中使用时,“由与包含SEQ ID NO:2至87中任一所示的氨基酸序列的9~20个连续氨基酸的序列实质上相同的氨基酸序列构成的肽”可以是由“包含SEQ ID NO:2至87中任一所示的氨基酸序列的9~20个连续氨基酸的序列”(其具有一个至数个,例如1至10、1至5、1至3、1至2、和1个,氨基酸残基的取代、缺失和/或添加)构成的肽。
在本文中使用时,“与……实质上相同的氨基酸序列”可以是具有80%或更高(优选地,85%或更高,更优选地,88%或更高)的氨基酸序列同一性的氨基酸序列。
取代可以是保守取代。
保守取代的例子包括天冬氨酸和谷氨酸之间的取代,精氨酸、赖氨酸和组氨酸之间的取代,色氨酸和苯丙氨酸之间的取代,苯丙氨酸和缬氨酸之间的取代,亮氨酸、异亮氨酸和丙氨酸之间的取代,以及甘氨酸和丙氨酸之间的取代。
例如,SEQ ID NO:2至87中任一所示的氨基酸序列中,优选SEQ ID NO:2至17及40和41中任一所示的氨基酸序列,更优选SEQ ID NO:9、11至17、40和41任一所示的氨基酸序列。
本发明的癌抗原肽由优选9至15个氨基酸残基、更优选9至12个氨基酸残基、进一步更优选9至11个氨基酸残基、特别优选10个氨基酸残基构成。
优选地,本发明的癌抗原肽由SEQ ID NO:1中包含SEQ ID NO:2至17和88至92中任一所示的氨基酸序列的9至20个连续氨基酸的序列构成。
特别优选地,本发明的癌抗原肽由SEQ ID NO:17、88或89所示的氨基酸序列构成。
本发明的癌抗原肽可作为使用已知方法分离的肽来制备,如前文针对癌组织恶性程度评估方法中的DDX3X或由DDX3X的部分氨基酸序列构成的肽所述。
在本文中使用时,“分离的”表示非天然存在的状态。
本发明的肽可以是盐的形式。此类盐的例子包括无机酸(例如盐酸和磷酸)的盐和有机酸(例如乙酸和酒石酸)的盐。
本发明的肽可以是附加有糖、聚乙二醇、脂质等的缀合物的形式使用,或者可以以例如放射性同位素等的衍生物、或聚合物的形式使用。
本发明的肽可以是癌抗原肽。
在本文中使用时,“癌抗原肽”可表示可被癌特异性细胞毒性T细胞(CTL)识别、并且可诱导和/或活化CTL的肽。
在本文中使用时,“被识别”表示被识别性物质鉴别为不同的,例如,识别性物质结合至被鉴别为不同的靶。在本文中使用时,“识别所述的肽”表示介由T细胞受体使得CTL与人白细胞抗原(HLA)及所述的肽结合。
在本文中使用时,“活化”可表示进一步增强或活化某些物质的活性或效果、或者此类物质的具有某些活性或效果的状态。具体而言,“活化CTL”表示CTL识别HLA呈递的肽并产生效应物(例如IFN-γ),或者,CTL展示出针对被识别的靶细胞的细胞毒性。
在本文中使用时,“诱导”可表示从基本不具有活性或效果的物质、或从此类物质的基本不具有活性或效果的状态产生活性或效果。具体而言,“诱导抗原特异性CTL”可表示在特异性识别一些抗原的CTL中导致分化和/或增殖,其可以是体外或体内的。
在本文中使用时,术语“特异性”在针对抗体或抗原使用时,表示免疫上特异性地结合抗体或抗原的能力。
癌疫苗
本发明的癌疫苗含有本发明的肽作为癌抗原。
本发明的肽可被单独或与各种载体一起制备成癌疫苗。
本发明的癌疫苗的剂型可以是经口施用的形式或肠胃外施用的形式。通常,优选肠胃外施用的形式。肠胃外施用的形式的例子包括皮下注射、肌内注射、静脉内注射和栓剂。
当本发明的癌疫苗是经口施用的形式时,本发明的肽可与可药用、并且不干扰作为癌抗原的本发明的肽的活性的赋形剂一起制备成癌疫苗。此类赋形剂的例子包括淀粉、甘露醇、乳糖、硬脂酸镁、纤维素、聚合的氨基酸和白蛋白。
当本发明的癌疫苗是肠胃外施用的形式时,本发明的肽可以与可药用、并且不干扰作为癌抗原的本发明的肽的活性的载体一起制备成癌疫苗。此类载体的例子包括水、常见的盐、右旋糖、乙醇、甘油和DMSO。
根据需要,本发明的癌疫苗还可含有白蛋白、润湿剂和/或乳化剂等材料。
本发明的肽还可与合适的佐剂一起使用,以活化细胞免疫性。本发明的癌疫苗可含有此类佐剂。
本发明的肽还可与增强细胞毒性T细胞(CTL)的肽识别的化合物一起使用,或例如与能免疫识别所述肽的抗体一起使用。本发明的癌疫苗可含有此类化合物和/或抗体。
本发明的癌疫苗可使用适合于所述剂型的常用方法来制造。
优选地,本发明的癌疫苗用于预防或治疗癌,或用于遏制癌转移或癌复发。
可通过使用适合于所述剂型的施用方法,将本发明的癌疫苗施用给人。
本发明的癌疫苗可以以例如大约0.01mg至100mg/天的剂量、优选大约0.1mg至30mg/天的剂量施用给成年人(按本发明的活性组分肽计)。给药间隔可根据症状和施用目的等因素合适地选择。
制造过继性免疫细胞的方法
本发明的制造过继性免疫细胞的方法包括下述步骤:
用DDX3X或其部分肽对具有抗原呈递能力的细胞进行脉冲的步骤。
具有抗原呈递能力的细胞的例子包括树突状细胞、巨噬细胞和B淋巴细胞。
脉冲可通过例如下述方式来进行:将具有抗原呈递能力的细胞在含有大约1至10μg/ml的DDX3X或其部分肽的培养基中于大约20至30℃的温度下孵育大约30分钟至大约1小时。以这种方式,可获得将癌抗原肽呈递到细胞表面以被DDX3X特异性CTL识别的细胞。所述细胞可以是分离的细胞。
优选地,DDX3X的部分肽是本发明的肽。
将癌抗原肽呈递到细胞表面以被DDX3X特异性CTL识别的细胞可以是呈递本发明的肽的抗原呈递细胞(APC)。
经DDX3X或其部分肽脉冲的APC可以是呈递源于DDX3X的肽的APC,其可用作为DDX3X特异性T细胞诱导剂。
APC可作为过继性免疫细胞被施用给需要过继性免疫治疗的人。
可使用已知的方法,在将APC在施用给人之前对其进行培养。
可通过将具有分化为CTL的潜能的前体细胞与上文所述经DDX3X或其部分肽脉冲的APC一起孵育,而离体诱导DDX3X特异性CTL。所述DDX3X特异性CTL可以是分离的细胞。
对前体细胞没有特别限制,只要它们能分化为CTL即可。例子包括外周血单核细胞(PBMC)、幼稚细胞(naive cells)和记忆型细胞(memory cells)。
如前文所述的DDX3X特异性CTL也可作为过继性免疫细胞施用给需要过继性免疫治疗的人。
可使用已知的方法,在将DDX3X特异性CTL在施用给人之前对其进行培养。
特别地,在本发明的另一实施方式中,本发明的过继性免疫细胞制造方法包括下述步骤:
将具有抗原呈递能力的细胞暴露给DDX3X或其部分肽、以获得呈递源于DDX3X或其部分肽的抗原的细胞的步骤;和
用所述抗原呈递细胞诱导DDX3X特异性T细胞的步骤。
优选地,DDX3X特异性T细胞是DDX3X特异性CD4阳性T细胞。
如上所述获得的过继性免疫细胞可被直接或与各种载体一起被制备成过继性免疫细胞组合物。
本发明的过继性免疫细胞组合物的剂型可以是经口施用的形式或肠胃外施用的形式。通常,优选肠胃外施用的形式。肠胃外施用的形式的例子包括皮下注射、肌内注射、静脉内注射和栓剂。
当本发明的过继性免疫细胞组合物是经口施用的形式时,本发明的过继性免疫细胞可与可药用、并且不干扰过继性免疫细胞的活性的赋形剂一起制备成过继性免疫细胞组合物。此类赋形剂的例子包括淀粉、甘露醇、乳糖、硬脂酸镁、纤维素、聚合的氨基酸和白蛋白。
当本发明的过继性免疫细胞组合物是肠胃外施用的形式时,本发明的过继性免疫细胞可以与可药用、并且不干扰过继性免疫细胞的活性的载体一起制备成过继性免疫细胞。此类载体的例子包括水、常见的盐、右旋糖、乙醇、甘油和DMSO。
癌预防、癌治疗、癌转移遏制或癌复发遏制剂
本发明的癌预防、癌治疗、癌转移遏制或癌复发遏制剂含有抑制DDX3X表达或活性的化合物。
抑制DDX3X表达的化合物的例子包括:包含与DDX3X基因正义链的全部区域或部分区域中的序列(下文中也简称为“靶序列”)互补的碱基序列(下文中也简称为“反义序列”)的多核苷酸。
此类多核苷酸的例子包括反义核苷酸、siRNA(小干扰RNA)和shRNA(小发卡RNA)。
靶序列可通过进行NCBI BLAST检索来确定。优选地,靶序列选自DDX3X基因的外显子区域。优选地,靶序列对靶DDX3X基因序列呈高度特异性。
靶序列例如为15至30个碱基长、优选18至25碱基长、更优选18至25个碱基长、进一步更优选19至23个碱基长、特别优选19至21个碱基长。
反义核苷酸可以是RNA或DNA。此外,反义核苷酸可具有在反义序列的至少一个末端连结一个至数个碱基(例如1至2个碱基、1至3个碱基、1至5个碱基)而成的序列,或在反义序列内一个至数个碱基被缺失、取代或添加而成的序列,只要反义核苷酸具有遏制DDX3X基因表达的作用即可。
例如,由包含靶序列(正义链)的多核苷酸和包含反义序列(反义链)的多核苷酸构成的双链多核苷酸可作为siRNA使用。
正义链和反义链可以比靶序列长一个或数个碱基(例如1至2个碱基、1至3个碱基、1至5个碱基),并且可具有例如,添加至末端(优选地,3’端)的两个尿嘧啶(U)碱基。此外,反义链和/或正义链可具有在反义链或靶序列的至少一个末端连结一个至数个碱基U、T、G、C或A(例如1至2个碱基、1至3个碱基、1至5个碱基)而成的序列,或在反义序列或靶序列内一个至数个此类碱基被缺失、取代或添加而成的序列,只要反义链和正义链具有遏制DDX3X基因表达的作用即可。
shRNA(小发卡RNA)的例子包括含有通过调控部分(环部分)(其可以是核苷酸序列、非核苷酸序列或它们的组合)互相连结的siRNA正义链和反义链的那些。
当调控部分是核苷酸序列时,核苷酸序列的例子包括至少一个碱基且小于10kb的核苷酸序列,优选为一个碱基至数百个碱基的核苷酸序列,更优选为一个碱基至数十个碱基的核苷酸序列,特别优选为1至20个碱基的核苷酸序列,以及由能通过剪接或其它细胞内机制在细胞质中产生前述长度的多核苷酸的序列构成的核苷酸序列。形成调控部分的核苷酸序列可包括正义序列和反义序列。此外,形成调控区域的核苷酸序列可以是下述序列之一、或下述序列中两条或多条的组合:
胞质定向的序列,例如多聚A、tRNA、Usn RNA、和源于逆转录病毒的CTE序列;
具有诱捕(decoy)活性的序列,例如NFκβ结合序列、E2F结合序列、SSRE和NF-AT;
干扰素诱导遏制序列,例如腺病毒VA1或VA2RNA;
具有RNase遏制活性、反义活性、核糖体活性等的序列;
用于特定出tRNA或表达位点的标志物序列;和
用于用大肠杆菌进行检测的选择标志物序列。
需要部分双链以用于诱捕活性等的功能性序列可以以互补核苷酸来生产。调控部分可被设计为包括剪接内含子供体序列和受体序列所需的序列,以使得调控部分序列的一部分能在具有剪接机制的细胞中被切掉并被再连结。如上所述的调控部分序列能更符合期望地改善RNA功能遏制作用,以及使得正义链和反义链稳定。
当调控部分是非核苷酸序列时,具体例子包括PNA(肽核酸),其是与核酸类似的具有聚酰胺主链的化学合成类似物。
在本发明中,还可将用于遏制DDX3X基因转录的、针对DDX3X基因的诱捕性核酸用于遏制DDX3X基因表达。
抑制DDX3X活性的已知化合物的例子包括WO2011/039735中描述的化合物,具体而言,下式所示的化合物:
[上式中,
Z表示CH2或S,
X和Y独立表示O或S,
n在0至4的范围内,
B不存在,或表示:
其中,q在0至4的范围内,R2'表示氢、-(CH2)w'-OH或-(CH2)w'-NH2(其中w'是1至3的整数),
或者,B是C=O,
R1、R2和R3每个独立选自由H、1至6个碳原子的直链或带支链的烷基、未取代或取代的苯基、未取代或取代的苯基烯基、未取代的或取代的苯基炔基、未取代或取代的二苯基烷基、未取代或取代的杂环基、未取代或取代的多环基、未取代或取代的脂环基、或(R1a-)m(L-)pR1b-(其中R1a和R1b可相同或不同,并表示未取代或取代的杂环基或未取代或取代的苯基,R1a还表示未取代或取代的多环基,L表示选自由-(CH2)q-、-HC=CH-、-C≡C-、-C(=O)-、-O-、-S-、-S(=O)-、-S(=O)2-、-NHCONH-和-NR1C-构成的组的二价连接基团,其中R1C是氢或烷基,m和p每个独立表示0或1,q是1至3的整数)构成的组;或
R2和R3可一起形成环烷基、环烯基、非芳香族杂环类环或稠合的或多环类环、或2-氧基吲哚(2-oxyindole)(环烷基、环烯基、稠合的或多环非芳香族杂环类环可被选自前述基团中的一个或多个取代基取代),
W不存在,或独立表示O、S、NH、NHCH2或N-R5(其中R5是1至6个碳原子的直链或带支链的烷基),
A不存在,或表示CONH、NHCO或NHCONH,
R4表示H、未取代或取代的1至6个碳原子的烷基、未取代或取代的烯基、未取代或取代的炔基、卤素、卤代烷基、COOH、OCH3、NO2、NH2、CN、OZ'或SZ'(其中Z'是H、未取代或取代的1至6个碳原子的烷基)。]
抑制DDX3X活性的已知化合物的其它例子包括Bioorganic&Medicinal Chemistry Letters,Volume 22,Issue 5,1March 2012,Pages 2094-2098中描述的化合物,具体而言,下式所示的化合物,
抑制DDX3X活性的已知化合物的其它例子包括下述化合物。
这些化合物可以是可药用盐的形式。
对解旋酶DDX3X的抑制使得下述四种miRNA减少:miRNA:hsa-mir-301a、hsa-mir-301b、hsa-mir-429和hsa-miR-3922。由此可以认为,抑制这些miRNA中的一种或多种(优选抑制全部)的活性与抑制DDX3X活性实质上相同。因此,抑制miRNA活性的化合物也被包括在根据本发明的抑制DDX3X活性的化合物的范围内。
这些miRNA的碱基序列如下所示。
hsa-mir-301a(miRBase登录号MI0000745):
ACUGCUAACGAAUGCUCUGACUUUAUUGCACUACUGUACUUUACAGCUAGCAGUGCAAUAGUAUUGUCAAAGCAUCUGAAAGCAGG(SEQ ID NO:93)
hsa-mir-301b(miRBase登录号MI0005568):
GCCGCAGGUGCUCUGACGAGGUUGCACUACUGUGCUCUGAGAAGCAGUGCAAUGAUAUUGUCAAAGCAUCUGGGACCA(SEQ IDNO:94)
hsa-mir-429(miRBase登录号MI0001641):
CGCCGGCCGAUGGGCGUCUUACCAGACAUGGUUAGACCUGGCCCUCUGUCUAAUACUGUCUGGUAAAACCGUCCAUCCGCUGC(SEQ ID NO:95)
hsa-miR-3922(miRBase登录号MI0016429):
GGAAGAGUCAAGUCAAGGCCAGAGGUCCCACAGCAGGGCUGGAAAGCACACCUGUGGGACUUCUGGCCUUGACUUGACUCUUUC(SEQ ID NO:96)
抑制miRNA活性的化合物的例子包括:包含与miRNA的整个或部分区域(下文中也简称为“靶序列”)互补的碱基序列(反义miRNA序列)的多核苷酸。
多核苷酸的例子包括反义核苷酸。
靶序列例如为10至30个碱基长、优选10至20个碱基长、更优选12至18个碱基长、更优选14至16个碱基长。
反义核苷酸例如是RNA、DNA或LNA。反义核苷酸可具有在反义miRNA序列的至少一个末端连结一个至数个碱基(例如1至2个碱基、1至3个碱基、1至5个碱基)而成的序列,或在反义miRNA序列内一个至数个碱基被缺失、取代或添加而成的序列,只要反义核苷酸具有遏制miRNA活性的作用即可。
实施例
下文中使用实施例对本发明进行更详细的描述。应当注意,本发明不以任何方式受这些描述所限。
实施例中使用的材料和方法如下所示。
材料和方法
小鼠
从CLEA Japan购买C57BL/6J(B6)雌性小鼠,将其保持在无病原体的环境中,在8-10周龄时用于实验。
所有动物实验都经Niigata University Ethics Committee for AnimalExperiments批准。
肿瘤细胞
B16F10是B6来源的黑色素瘤,其被体外保持。用缀合有藻红蛋白(PE)的抗CD133单克隆抗体(13A4)和抗PE微珠(Miltenyi Biotec)对亲本肿瘤细胞加以标记。使用autoMACS(商品名)(Miltenyi Biotec),按照厂商方案,分离CD133阳性和CD133阴性肿瘤细胞。细胞纯度高于90%。
单克隆抗体和流式细胞分析
从American Type Culture Collection获得产生针对鼠CD4(GK1.5,L3T4)、CD8(2.43,Lyt-2)、CD3(2C11)和鼠CD62L(MEL14)的单克隆抗体的杂交瘤。抗CD4单克隆抗体、抗CD8单克隆抗体和抗CD62L单克隆抗体从经亚致死量照射的(500cGy)DBA/2小鼠作为腹水产生。缀合PE的抗CD80(16-10A)、抗CD86(GL1)、抗CD62L(MEL14)、抗CD8(2.43)和抗CD25(PC61)单克隆抗体、缀合有荧光素异硫氰酸酯(FITC)的抗Thy1.2(30-H12)单克隆抗体和抗CD4(GK1.5)单克隆抗体购自BD PharMingen。对细胞表面表性的分析通过用缀合的抗体对0.5至1×106个细胞进行直接免疫染色来实现。使用FACScan(商品名)流动显微荧光计(Becton Dickinson)对每份样品中总共10,000个细胞进行了分析。缀合有PE的匹配各亚类的抗体被用作为同位素对照,它们也购自BD PharMingen。使用CellQuest(商品名)软件(Becton Dickinson)对样品进行分析。
T细胞分选
通过经过尼龙毛柱(Wako Pure Chemical Industries)对淋巴结(LN)细胞悬浮液中的T细胞进行了浓缩。为获得具有被下调控的CD62L表达(CD62Llow)的经高度纯化的(>90%)细胞,通过使用预先涂布有山羊抗大鼠免疫球蛋白抗体(Ig Ab)(Jackson ImmunoResearchLaboratories)/抗CD62L(MEL14)单克隆抗体的T-25瓶进行的盘选(panning)技术,以及通过使用经山羊抗大鼠Ig Ab/抗CD62L单克隆抗体涂布的DynaBeads M-450(Dynal)的磁性珠粒技术,对LN T细胞进行进一步分离。在一些实验中,按照Hiura T,Kagamu H,Miura S,IshidaA,Tanaka H,Tanaka J,et al.,Both regulatory T cells and antitumor effectorT cells are primed in the same draining lymph nodes during tumorprogression.J Immunol.2005;175:5058-66所述,通过使用磁性珠粒进行消耗(depletion),将细胞进一步分离为CD4阴性和CD8阴性细胞。出于纯化目的,使用经抗CD4单克隆抗体涂布的Dynabeads和Detachabeads(Invitrogen)进行阳性选择,获得经高度纯化的CD4阳性细胞。
源于骨髓的树突状细胞
按照Fujita N,Kagamu H,Yoshizawa H,Itoh K,Kuriyama H,Matsumoto N,et al.CD40ligand promotes priming of fully potent antitumorCD4(+)T cells in draining lymph nodes in the presence of apoptotic tumorcells,J Immunol.2001;167:5678-88中描述的方法,从骨髓细胞(BM)产生树突状细胞(DC)。简言之,将从小鼠股骨和胫骨获得的BM于37℃在T-75瓶中于含有10ng/ml重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF,由KIRIN馈赠)完全培养基(CM)中放置2小时。分离非粘附细胞,并在新的瓶中培养。在第6天,通过轻柔吸取收获非粘附细胞。CM由其中补充有10%经脂多糖(LPS)检验(无内毒素)的失活胎牛血清、0.1mM非必需氨基酸、1μM丙酮酸钠、100U/mL青霉素、100μg/mL链霉素硫酸酯(全部均来自Life Technologies,Inc.)和5×10-5M 2-巯基乙醇(Sigma Chemical)的RPMI 1640培养基构成。
引流DC/肿瘤疫苗的LN细胞
将BM和DC与相同数量的经照射肿瘤细胞(5,000cGy)在CM中共培养过夜。在两侧均用1×106BM-DC和肿瘤细胞对B6小鼠进行s.c.接种。收获引流BM-DC和肿瘤疫苗的腹股沟LN。按照Watanabe S,Kagamu H,Yoshizawa H,Fujita N,Tanaka H,Tanaka J,et al.,The durationof signaling through CD40directs biological ability of dendritic cells toinduce antitumor immunity.J Immunol.2003;171:5828-36中所述的方法制备单细胞悬浮液。
过继性免疫疗法
用悬浮于100μl Hanks’平衡盐溶液(HBSS)中的B16-F10肿瘤细胞,对B6小鼠在正中线进行s.c.注射,以建立皮下肿瘤模型。接种后两或三天,对小鼠进行亚致死量照射(500cGy),然后i.v.输注从引流BM-DC/肿瘤疫苗的淋巴结分离的T细胞。用抗CD3单克隆抗体(2C11)刺激这些LN细胞,并将其在含有40U/ml IL-2的CM中培养3天,以获得足够量的细胞,如Fujita N,Kagamu H,Yoshizawa H,Itoh K,Kuriyama H,Matsumoto N,et al.CD40ligand promotes priming of fullypotent antitumor CD4(+)T cells in draining lymph nodes in the presence ofapoptotic tumor cells.J Immunol.2001;167:5678-88所述。使用卡钳测量皮下肿瘤的垂直直径。
细胞因子ELISA
在CM中被固定的抗CD3单克隆抗体或经抗原脉冲的BM-DC对T细胞进行刺激。收获上清液,并使用小鼠IFN-γ、IL-4和IL-17ELISA试剂盒(Genzyme),按照厂商方案,通过定量“夹心”酶免疫检验,检测其IFN-γ、IL-4和IL-17含量。
体外增殖检验
用HBSS中的5μM 5-(6)-羧基荧光素二乙酸琥珀酰亚胺二酯(CFSE;Molecular Probes)于37℃对黑色素瘤细胞标记15分钟,在CD3刺激前洗两次。被CFSE标记的肿瘤细胞与未被标记的肿瘤细胞的比例为1:10。在CM中对肿瘤细胞以1×105/mL进行培养,并使用流动显微荧光计进行计数和分析,以确定被CFSE标记的细胞。
免疫杂交印迹检验
在含有蛋白酶抑制剂混合物(Sigma)的Nonidet P-40缓冲液中收获并裂解细胞。对相等微克量的蛋白质进行SDS-7.5%PAGE,并转移至聚偏氟乙烯膜(Millipore)上。用针对DDX3X(Sigma)和β-肌动蛋白(Sigma)的抗体探测来自肿瘤细胞的免疫印迹。二抗由与辣根过氧化物酶(HRP;Bio Rad,Dako)缀合的抗小鼠Ig和抗兔Ig构成。使用ECL试剂盒(Pierce)观察免疫反应性蛋白质条带。针对所有分析至少进行三次独立实验。
通过shRNA敲除DDX3X
DDX3X的敲除使用shRNA慢病毒(pLKO.1-puro)质粒(SigmaAldrich)来获得。使用的含有DDX3X靶序列的寡核苷酸为CCGGACGTTCTAAGAGCAGTCGATTCTCGAGAATCGACTGCTCTTAGAACGTTTTTTG(SEQ ID NO:97)。在新培养基中添加B16CD133阳性细胞,向每个孔中添加溴化己二甲胺(8μg/ml)。按照厂商方案,将细胞与pLKO.1-puro质粒加包装载体共转染。转染后大约16小时更换培养基,对细胞再培养48-72小时。用含有嘌呤霉素(2.0μg/ml)的新鲜培养基孵育实验细胞,每3-4天用新鲜的含嘌呤霉素(2.0μg/ml)的培养基更换培养基,直到可鉴定到抗性集落。拣出最少五个嘌呤霉素抗性集落,每个克隆被扩展以用于检验。DDX3X敲除的效率通过免疫杂交印迹来测定。
统计分析
使用学生t检验来进行组间比较。通过多因变量一般线性模型分析动态肿瘤生长数据。针对P<0.05考虑差异为显著。使用SPSS统计软件(SPSS)或GraphPad Prism 5.0软件(GraphPad Software)进行统计分析。
DDX3X和其部分肽
通过化学合成制备DDX3X和其部分肽。
DDX3X具有SEQ ID NO:1所示的氨基酸序列。
肽J具有SEQ ID NO:89所示的氨基酸序列。
肽K具有SEQ ID NO:88所示的氨基酸序列。
DDX3X-10元寡聚体(DDX3X-10mer)具有SEQ ID NO:17所示的氨基酸序列。
DDX3X-15元寡聚体(DDX3X-15mer)具有SEQ ID NO:90所示的氨基酸序列。
DDX3X-20元寡聚体(DDX3X-20mer)具有SEQ ID NO:91所示的氨基酸序列。
实施例1
从DDX3X特异性CD4阳性T细胞释放CD133阳性肿瘤抗原特异
性细胞因子
本申请的发明人研究了用合成的DDX3X抗原初免疫的T细胞是否能识别CD133阳性黑色素瘤细胞。为对此加以测试,从经合成的DDX3X脉冲的引流DC的淋巴结(LN)分离具有表达被下调控的CD62L的T细胞(CD62Llow)。
用1×104个树突状细胞,在96孔板中于200μl完全培养基(CM)中对从淋巴结分离的CD62Llow CD4阳性或CD8阳性T细胞(1×105个细胞)进行48小时的刺激。用于进行刺激的树突状细胞用相同数量的经照射CD133阳性肿瘤细胞或CD133阴性肿瘤细胞(5,000cGy)或合成的DDX3X(5μg/ml)刺激过夜。在共培养之前,用CD11c微珠粒对树突状细胞进行纯化。
本申请的发明人发现,由此获得的DDX3X特异性CD4阳性T细胞以黑色素瘤CSC特异性方式分泌IFN-γ和IL-17。但是,DDX3X特异性CD8阳性T细胞应答于CD133阴性和CD133阳性黑色素瘤细胞两者(图2和3)。
接着,本申请的发明人黑色素瘤CSC特异性T细胞是否识别DDX3X和产生细胞因子。发现,用经免疫的CD133阳性黑色素瘤细胞初免疫的黑色素瘤CSC特异性CD4阳性T细胞以DDX3X特异性方式产生细胞因子(图4和5)。令人吃惊地,黑色素瘤CSC特异性CD4阳性T细胞在DDX3X刺激的情况下产生的细胞因子比用黑色素瘤CSC自身刺激时更多。
由此发现黑色素瘤CSC特异性CD4阳性T细胞具有针对表达DDX3X的肿瘤细胞的抗肿瘤活性,并可用于过继性免疫疗法。
实施例2
DDX3X免疫接种诱导针对黑色素瘤细胞的保护性免疫
为检测是否可通过合成的DDX3X进行免疫接种是否能诱导针对B16黑色素瘤细胞的保护性免疫,将经DDX3X或卵白蛋白(OVA)以5μg/ml脉冲的树突状细胞或与经照射的CD133阳性肿瘤细胞(5,000cGy)共培养了8小时的树突状细胞皮下(s.c.)注射于小鼠的右侧。14天后,用2×106个黑色素瘤细胞在腹部正中线对小鼠进行接种。每组含有5只小鼠。如图6所示,较之未经处理或用经OVA脉冲的DC处理的小鼠而言,在获得经DDX3X脉冲的树突状细胞免疫接种的小鼠中,肿瘤生长被更显著地抑制。此外,较之用与经照射的CD133阳性肿瘤细胞共培养的树突状细胞注射的小鼠而言,用经DDX3X脉冲的DC免疫接种的小鼠显著出显著更强的保护性免疫作用。
实施例3
用DDX3X免疫接种展示出针对已建立的皮肤肿瘤的疗效
本申请的发明人进一步测试了用DDX3X进行的免疫接种针对建立的肿瘤是否具有疗效。在腹部正中线s.c.接种1×106个B16黑色素瘤细胞后的第2、9和6天,将1×106个树突状细胞注射于右侧。在右侧以5μg/ml皮下(s.c.)注射经DDX3X或OVA脉冲的1×106个树突状细胞。每组含有12只小鼠。图7表示每只小鼠的肿瘤生长曲线。发现,用经DDX3X脉冲的树突状细胞免疫接种的12只小鼠中,有6只被永久性治愈。在用经DDX3X脉冲的树突状细胞免疫接种的其它小鼠中,皮肤肿瘤生长被显著遏制。而没有被处理或用经OVA脉冲的DC免疫接种的所有小鼠均死于肿瘤。
实施例4
DDX3X对于CD133阳性黑色素瘤的免疫原性的意义
以前已显示,B16黑色素瘤细胞具有多种免疫原性蛋白。
为阐明DDX3X在DDX3X免疫原性中的意义,本申请的发明人通过使用shRNA敲除DDX3X而建立了CD133阳性黑色素瘤细胞(缺乏DDX3X的CD133阳性B16细胞)。将总共5,000cGy照射的空白转染-shRNA导入的CD133阳性B16细胞和DDX3X敲除CD133阳性B16细胞与树突状细胞(DC)共培养8小时。将用CD11c微珠粒和autoMACS(商品名)纯化的106个CD11c阳性细胞皮下施予B6小鼠。免疫后2周,使用2×106个黑色素瘤细胞沿着腹部正中线对小鼠进行皮下接种。每组含有5只小鼠。如图8所示,用CD133阳性亲本细胞或CD133阳性空白转染肿瘤细胞(对照)免疫接种的小鼠具有有效的保护性免疫。而相反,用缺乏DDX3X的CD133阳性肿瘤细胞进行的免疫接种不能诱导出抗肿瘤保护性免疫。换言之,缺乏DDX3X的CD133阳性黑色素瘤失去了疫苗作用。
实施例5
从小细胞肺癌患者收集外周血(15ml)。使用Lymphoprep(商品名)(Cosmo Bio)通过密度梯度离心收集单核细胞级分,用CD14微珠粒和autoMACS分离CD14+细胞。将CD14+细胞与rhGM-CSF(1ng/ml,由Kirin馈赠)和IL-4(10ng/ml,R&D systems)一起培养,在被分化并成熟成为树突状细胞之后的第5天之前使用。将树突状细胞在含有合成的DDX3X蛋白质(3.3μg/ml)或相同浓度的肽(肽J、肽K)的培养基中培养过夜,经CD11c微珠粒和autoMACS纯化的CD11c阳性细胞被用作为抗原呈递细胞。为了从CD14-级分细胞中除去幼稚T细胞和调控T细胞,通过使用缀合有抗人CD62L抗体(1H3)的Dynabeads除去CD62Lhigh细胞。
在BD BioCoat T细胞活化板(Becton Dickinson)对CD62Llow CD14-细胞培养48小时,在含有20U/ml的rhIL-2(Shionogi的馈赠)的培养基中培养4天。FACS验证了:增加了大约10倍的细胞中95%或更多均为CD3+T细胞,CD3+T细胞被用作应答细胞。在圆底96孔板中在200μl培养基中对应答细胞(1×105)和抗原呈递细胞(1×104)培养24小时,通过ELISA测量收集的上清液中的IFN-γ浓度。在固定有抗CD3抗体的96孔板中孵育的1×105个应答细胞的培养物的上清液被用作为阳性对照。结果示于表9。
实施例6
使用源于DDX3X的肽进行的CTL诱导和IFN-γ产生
对通过用源于DDX3X的肽进行的刺激实现的DDX3X特异性CTL诱导和IFN-γ产生进行了评估。
表1列出了使用的试剂。表2是用于诱导的肽的列表。
表1
表2
培养基制备
将人AB血清在56℃失活30分钟,并滤经0.22-μm过滤器(SerumAcrodisc,Pall)。添加失活的人AB血清(50mL),并在干净的操作台上与500mL AIM-V混合,以制备培养基。通过添加10mL肝素钠注射液(10000U/mL)和500mL Hank’s平衡盐溶液(20U/mL肝素)并混合,来制备含肝素的HBSS。使用前将它们贮藏于4℃。
对血液供体的筛选
对健康志愿者加以筛选,选出具有通过软件计算预计对DDX3X肽显示出高亲和性的HLA-A*2601或HCT116HLA-A0201的个体(选择十聚体)。这些志愿者中,六名具有HLA-A*0201,五名具有HLA-A*2601。
源于外周血的单核细胞(PBMC)的制备
用含肝素的HBSS(每20mL血,13mL HBSS)稀释从健康志愿者获得的外周血(40mL),并放置于装有15mL Lymphoprep的淋巴细胞分离管(Leucosep(trade name);Greiner)中。离心(2,000rpm,20℃,20min)后,将中间层(PBMC)收集进50mL离心管,在用含肝素HBSS稀释两倍后再进行离心(1,800rpm,20℃,5min)。将得到的沉淀团悬浮于10mL含肝素HBSS中并离心(1,200rpm,4℃,5min)。重复该过程。将得到的PBMS沉淀团悬浮于培养基(1mL)中,使用1.5×107个细胞进行DDX3X特异性CTL诱导,而剩下的细胞则在再刺激中用于抗原呈递细胞。将在再刺激中用于抗原呈递细胞的PBMC悬浮于CellBanker中,并冻存于-80℃,在使用前解冻。
DDX3X特异性CTL的诱导
将PBMC(1.5×107个细胞)以1.5×106个细胞/孔接种于24孔板中(第0天)。然后,向每个孔中加入三种类型的源于DDX3X的肽(每种的终浓度为20μg/mL)和IL-7(终浓度10ng/mL),于37℃在5%CO2中对细胞加以培养。
1周后(第7天)对细胞细胞进行再刺激。为进行再刺激,对在第0天作为抗原呈递细胞冻存的PBMC解冻,然后,在将细胞调节为3×106cells/mL或更低之后,添加三种类型的源于DDX3X的肽(每种的终浓度为20μg/mL),并在37℃缀合2小时。这之后是添加丝裂霉素(KyowaHakko Kirin)溶液,至终浓度为50μg/mL,于37℃对细胞进行45分钟的处理。用AIM-V对细胞洗两次,重悬浮于培养基中,以获得抗原呈递细胞悬浮液。对培养了1周的细胞进行收获之后,将细胞以1.2×106个细胞/孔接种进24孔板,并用相同数量的细胞接种抗原呈递细胞悬浮液。最终,以10ng/ml添加IL-7,于37℃在5%CO2中对细胞加以培养。2天后(第9天),从每个孔轻柔移除一半培养基,并更换为含40U/mL的IL-2的培养基,再进行培养。每隔一天(第11天、第13天)以相同的方式用含20U/ml IL-2的培养基重复进行培养基的半体积更换。使用相同的流程在第14天和第21天重复进行再刺激。在20U/mL的IL-2存在下对细胞进行共培养,细胞在每隔一天(第16天、第18天、第20天和第23天、第25天、第27天)用含20U/mL的IL-2的培养基半量替换的培养基中生长直至第28天。
对源于DDX3X的肽刺激导致的IFN-γ生产的评估
将在第21天和第28天收获的细胞合适地用培养基稀释,并接种进96孔圆底板(各100μL)。然后,将被制备为含有40μg/mL源于DDX3X的肽的培养基添加进96孔圆底板(各100μL)中,将细胞在5%CO2/37℃中培养(肽的终浓度=20μg/mL)。针对各刺激进行三次重复ELISA。作为阴性对照,刺激中使用溶剂DMSO。
肽刺激之后24小时,从每个孔轻柔收集培养物上清液。在适宜修正后,按照厂商方案,使用ELISA试剂套装(BD OptEIA ELISA set(人IFN-γ)(商品名)),通过ELISA检测每份培养物上清液中的IFN-γ浓度。具体而言,涂布抗体、检测抗体和经HRP标记的抗体在稀释500倍后使用。使用可见波长吸光度微孔板读数仪(VERSAmax(商品名);Molecular Device)进行测量。
结果
结果示于图10。在图中,各供体(A至J)在棒状图中的柱从左至右表示DMSO、10聚体、15聚体和20聚体。
在第21天的评估中,在A*0201的三份样品中的任何刺激中均未观察到IFN-γ产生。另一方面,A*2601的五份样品中的两份仅在DDX3X-10元寡聚体刺激时显示出IFN-γ产生。
在第28天的评估中,A*0201的六份样品之一在DDX3X-10元寡聚体刺激中显示出IFN-γ产生。另一方面,除了在第21天的DDX3X-10元寡聚体刺激中显示出IFN-γ产生的两份样品之外,在A*2601的五份样品之一的DDX3X-20元寡聚体刺激中也观察到IFN-γ产生。
上述结果表明,通过用源于DDX3X的肽刺激健康PBMC来对IFN-γ产生细胞进行刺激特异性诱导是有可能的。
实施例7
经DDX3X/DC免疫刺激的CD4阳性T细胞具有抗肿瘤作用
用合成的DDX3X以5μg/mL对树突状细胞脉冲8小时,并使用CD11c微珠粒和autoMACS(商品名)将其作为CD11c阳性细胞(DDX3X/DC)分离。从引流DDX3X/DC疫苗的淋巴结分离CD62Llow T细胞。按照“材料和方法”所述,将CD62Llow T细胞(其为淋巴结T细胞)培养5天。将培养的CD62Llow T细胞静脉输注进在亚致死全身照射(500cGy)之后具有建立了2天的皮肤黑色素瘤的小鼠。DDX3X特异性细胞被发现具有抗肿瘤活性,并且显著地遏制皮肤肿瘤生长(图11A)。
因此,发现引流DDX3X/DC疫苗的淋巴结T细胞具有抗肿瘤疗效。
还研究了CD4阳性T细胞和CD8阳性T细胞中何者负责抗肿瘤活性。用磁性珠粒对培养5天后的淋巴结T细胞进行进一步纯化,以获得CD4阳性T细胞和CD8阳性T细胞。静脉输注10x106个CD4阳性淋巴结T细胞或CD8阳性T细胞。将10x106个CD8阳性T细胞输注进亚致死全身照射(500cGy)之后具有建立了2天的皮肤黑色素瘤的小鼠。但是,没有发现显著的抗肿瘤活性。而另一方面,DDX3X特异性CD4阳性T细胞却显示出高抗肿瘤活性,并且治愈了肿瘤(图11B)。
实施例8
DDX3X的特异性表达
使用可能的CSC标志物CD133、CD44和CD24,针对DDX3X在人肿瘤细胞中的表达,对87.5和S2(人小细胞肺癌)、HCT116(人结肠癌)、A549(人非小细胞肺癌)、WM115(人黑色素瘤)和MCF7(人乳腺癌)细胞进行分析。
如图12所示,87.5和HCT116细胞表达CD133,而其它癌细胞却不。87.5细胞作为漂浮性聚积物增殖,其容易形成肿瘤球体。发现MCF7细胞展示出CD44+和CD24-/low表型,这在传统上被认为是乳腺癌干细胞表型(下文的参考文献1至3)。
参考文献1:Al-Hajj M,Wicha MS,Benito-Hernandez A,Morrison SJ,Clarke MF.Prospective identification of tumorigenic thoracic cancer cells.Proceedings of the National Academy of Sciences of the United States ofAmerica.2003;100:3983-8。
参考文献2:Ponti D,Costa A,Zaffaroni N,Pratesi G,Petrangolini G,Coradini D,et al.Isolation and in vitro propagation of tumorigenic thoraciccancer cells with stem/progenitor cell properties.Cancer research.2005;65:5506-11。
参考文献3:Kai K,Arima Y,Kamiya T,Saya H.Breast cancer stemcells.Breast cancer.2010;17:80-5。
从正常人细胞(人表皮角化细胞(NHEK)、人微血管内皮细胞(HMEC)、正常人支气管上皮细胞(NHBE))和癌细胞(87.5、S2、HCT116、A549、WM115、MCF7)提取全细胞裂解物。使用针对DDX3X和β-肌动蛋白的抗体进行肿瘤细胞的免疫杂交印迹检验。此外,可能的CSC标志物阳性细胞,例如87.5、HCT116和MCF7强烈表达DDX3X(图13)。因此,有可能DDX3X不仅在小鼠黑色素瘤干细胞中表达,而是在各种人肿瘤中也表达。
实施例9
人结肠癌细胞系HCT116是占优势的CD133阳性细胞,其高度表达DDX3X。使用通过shRNA(其是用慢病毒载体导入的)敲除DDX3X获得的1-4细胞和空白转染1-6细胞进行细胞损伤重复实验。已证实,1-4细胞和1-6细胞的生长速率不同。使用移液吸头,将在24孔板中同时接种之后生长至亚会合的1-4细胞和1-6细胞以直线状剥离,观察损伤恢复的时间进程。具有DDX3X敲除的1-4细胞中,组织修复延迟(图14)。
实施例10
在小细胞肺癌细胞系中DDX3X高表达,因此针对在小细胞肺癌患者的外周血中是否存在能识别DDX3X并产生细胞因子的T淋巴细胞进行了研究。该实验经Niigata University,School of Medicine,EthicsCommittee批准。
在知情许可后收集外周血(15mL)。使用Lymphoprep(商品名)(Cosmo Bio)通过密度梯度离心收集单核细胞级分,用CD14微珠粒和autoMACS分离CD14+细胞。将CD14+细胞与rhGM-CSF(1ng/ml,由Kirin馈赠)和IL-4(10ng/ml,R&D systems)一起培养,并在第5天前分化为树突状细胞。将树突状细胞在含有合成的DDX3X蛋白质(3.3μg/ml)或相同浓度的OVA的培养基中培养过夜,经CD11c微珠粒和autoMACS纯化的CD11c阳性细胞被用作为抗原呈递细胞。为了从CD14-级分细胞中除去幼稚T细胞和调控T细胞,通过使用缀合有抗人CD62L抗体(1H3)的Dynabeads除去CD62Lhigh细胞。在BD BioCoatT细胞活化板(Becton Dickinson)对CD62Llow CD14-细胞培养48小时,在含有20U/ml的rhIL-2(Shionogi的馈赠)的培养基中培养4天。FACS验证了:细胞中95%或更多为CD3+T细胞,CD3+T细胞被用作应答细胞。在圆底96孔板中在200μl培养基中对应答细胞(1×105)和抗原呈递细胞(1×104)共培养24小时,使用共培养后收集的上清液,通过ELISA测量IFN-γ浓度。未经脉冲的树突状细胞、经DDX3X脉冲的树突状细胞和经OVA脉冲的树突状细胞被用作为抗原呈递细胞。在表3,“有”表示仅在与经DDX3X脉冲的树突状细胞的共培养物中以显著差异证实了来自T细胞的IFN-γ生产。在该表中,“%Treg”和“%Teff”表示调控T细胞和效应T细胞分别相对于CD4+T细胞总数的比例(在从外周血分离之后立即对单核细胞级分进行FACS分析而测定的)。CD62Lhigh CD25+CD4+T细胞和CD62Llow CD4+T细胞在FACS分析中分别被用作为调控T细胞(Treg)和效应T细胞(Teff),FACS分析如Koyama K,Kagamu H,et al.Reciprocal CD4+T-cell balance of effectorCD62Llow CD4+and CD62Lhigh CD25+CD4+regulatory T cells in small celllung cancer reflects disease stage.Clin Cancer Res.2008;14:6770-9所报道的那样进行。在健康个体(HV)、具有远端转移的小细胞肺癌患者(SCLC-ED)和治愈的小细胞肺癌患者中,均无法检测到应答于DDX3X的T细胞。但是,实验发现,在12名没有远端转移的小细胞肺癌患者(SCLC-LD)中,有5名存在应答于DDX3X的特异性地产生IFN-γ的T细胞。该结果表明,具有DDX3X特异性T细胞的小细胞肺癌患者具有令人期待的预后。
在表中,“有”表示血液中检测到DDX3X特异性T细胞,“无”表示在血液中没有检测到DDX3X特异性T细胞。
表3
实施例11
针对形成漂浮性的细胞聚集体(球状体)的能力,对通过从人结肠癌细胞系HCT116敲除DDX3X获得的1-4细胞进行了分析。与具有在非粘附培养物中形成球状体的能力的亲本株HCT116(CDD133+)相反,DDX3X敲除1-4细胞不具有球状体形成能力(图15)。
实施例12
已知DDX3X具有RNA解旋酶活性,还已知其在C.elegans和果蝇中参与miRNA的核-胞质转运、加工和成熟。但是,关于DDX3X涉及人细胞的miRNA没有任何报道。因此进行了研究,通过在具有上调的DDX3X表达的人肿瘤细胞和HCT116中敲除DDX3X,来研究是否存在miRNA变动。
使用第6代miRCURY LNAnaration(商品名)microRNA阵列(Filgen),使用通过从HCT116细胞敲除DDX3X获得的1-4细胞和空白转染1-6细胞来进行实验。使用miRBase Release 17作为注释信息。使用GenePix 4000B(Molecular Devices)进行阵列扫描,使用Array-ProAnalyzer Ver4.5(Media Cybemetics)来产生图像数据以及校正图像。使用局部回归进行归一化。在分析的2,684个miRNA中,均没有呈现在1-4细胞中比1-6细胞中表达增加。另一方面,发现有下述4个miRNA,即hsa-miR-301a、hsa-miR-429、hsa-miR-301b和hsa-miR-3922-3p,满足归一化强度≥10,归一化强度(之和)≥阴性对照平均值(=85),并且较之在1-6细胞中而言在1-4细胞中降低的归一化强度比例≤0.5。
Claims (14)
1.癌恶性程度评估方法,所述方法包括下述步骤:
测量癌组织中的DDX3X表达水平的步骤;和
通过使用所述DDX3X表达水平来评估癌组织的恶性程度的步骤。
2.癌恶性程度评估试剂盒,所述试剂盒包含:
针对DDX3X的抗体、或者
与DDX3X mRNA或相应的cDNA特异性结合的多核苷酸,
其中,所述抗体或多核苷酸用于测量癌组织中DDX3X的表达水平。
3.癌预后评估方法,所述方法包括下述步骤:
检测癌患者的血液中DDX3X特异性T细胞的步骤;和
通过使用检测结果评估癌预后的步骤。
4.癌预后评估试剂盒,所述试剂盒包含:
DDX3X或其部分肽,
其中,所述DDX3X或其部分肽用于检测癌患者血液中的DDX3X特异性T细胞。
5.一种肽,其由:
SEQ ID NO:1的氨基酸序列中、包含SEQ ID NO:2至87中任一所示的氨基酸序列的9~20个连续氨基酸的序列,或
与上述氨基酸实质上相同的氨基酸序列
构成。
6.根据权利要求5所述的肽,其中,所述肽为癌抗原肽。
7.癌疫苗,其包含权利要求5所述的肽。
8.根据权利要求7所述的癌疫苗,其中,所述疫苗用于预防或治疗癌,或用于遏制癌转移或癌复发。
9.制造过继性免疫细胞的方法,所述方法包括下述步骤:
用DDX3X或其部分肽对具有抗原呈递能力的细胞进行脉冲的步骤。
10.经DDX3X或其部分肽脉冲的抗原呈递细胞。
11.DDX3X特异性细胞诱导剂,其包含权利要求10所述的抗原呈递细胞作为活性组分。
12.制造过继性免疫细胞组合物的方法,所述方法包括下述步骤:
将具有抗原呈递能力的细胞暴露给DDX3X或其部分肽、以获得呈递源于DDX3X或其部分肽的抗原的细胞的步骤;和
用所述抗原呈递细胞诱导DDX3X特异性T细胞的步骤。
13.根据权利要求12所述的方法,其中所述DDX3X特异性T细胞是DDX3X特异性CD4阳性T细胞。
14.抑制DDX3X表达或活性的化合物,所述化合物用于预防或治疗癌、或用于遏制癌的转移或癌的复发。
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CN110446928A (zh) * | 2017-02-07 | 2019-11-12 | 学校法人埼玉医科大学 | 用于预测癌症免疫疗法临床效果的免疫学生物标志物 |
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CA2883577A1 (en) | 2014-03-13 |
BR112015004653A2 (pt) | 2017-07-04 |
SG11201501394SA (en) | 2015-03-30 |
IN2015DN01646A (zh) | 2015-07-03 |
IL237411A0 (en) | 2015-04-30 |
KR20150046787A (ko) | 2015-04-30 |
JP2015529825A (ja) | 2015-10-08 |
WO2014038682A2 (en) | 2014-03-13 |
PH12015500388A1 (en) | 2015-04-27 |
US20150297694A1 (en) | 2015-10-22 |
EP2893352A2 (en) | 2015-07-15 |
EA201590495A1 (ru) | 2015-07-30 |
TW201413245A (zh) | 2014-04-01 |
AU2013313974A1 (en) | 2015-03-12 |
MX2015002749A (es) | 2015-09-25 |
WO2014038682A3 (en) | 2014-05-15 |
AR092423A1 (es) | 2015-04-22 |
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