CN101868249B - 治疗间皮瘤的药物和方法 - Google Patents
治疗间皮瘤的药物和方法 Download PDFInfo
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Abstract
本发明涉及使用至少一种减毒的麻疹病毒来制备旨在治疗个体的恶性间皮瘤的药物。
Description
技术领域
本发明涉及旨在治疗癌症(如恶性间皮瘤)的方法和药物。
背景技术
恶性间皮瘤(MM)是一种相对罕见并具有高侵染性的肿瘤，源于衬于浆膜腔的间皮细胞的失控增生，所述间皮细胞最常衬于胸膜(恶性胸膜间皮瘤或MPM)(Robinson等人，(2005)Lancet 366：397-408)。在工业化国家流行病学的研究已经确定暴露于石棉是引起MPM的一个最重要的病因(Gruber(2005)Lung Cancer 49S1：S21-S23；Bartrip(2004)Postgrad Med.J.80：72-76)。尽管在世界各地，石棉的使用已显著减少，但在未来20年中，间皮瘤的发病率仍预计上升，因为从暴露于石棉到表现临床症状之间有很长的潜伏期(20年～40年)。
今天，癌症诊断通常在疾病的晚期阶段得以确定，因为在疾病的早期阶段缺乏明显的症状，因此很难对间皮瘤患者进行预断。因此，事实上MPM被认为是一种用所有传统治疗方法都相对难以治疗的癌症。因此迫切需要研发新的治疗方法。
在过去的十年间，对病毒治疗法的兴趣日益增加，部分涉及在人类基因治疗中重组病毒载体生产上增加的认识。现在许多RNA复制病毒被认为是潜在的癌症治疗物。同样，已有人建议用改造的可复制型单纯疱疹病毒(HSV)治疗MPM，基于对MPM小鼠模型体外研究和所得结果(Adusumilli等人.(2006)J.Gene Med.8：603-615)。然而，这些改造的病毒载体在人体内的长期安全性仍未知，需要进行大量的临床实验来论证HSV这方面的应用。
因此，需要具有受认可的安全性(可靠地用于间皮瘤治疗)的病毒载体。
MV(麻疹病毒)是一种有包膜的，负链单链RNA病毒，属于副粘液病毒科、麻疹病毒属。已开发出用于生产抗麻疹疫苗的各种可复制的MV活减毒株。举例来说，施瓦茨(Schwartz)株、Moraten株或Zagreb株(这些源于从埃德蒙顿(Edmonston)患者分离的MV样品)是安全且充分验证的MV疫苗株。
最近已表明，体内施用可复制型埃德蒙顿MV株，可导致建立的淋巴瘤和骨髓瘤动物模型中肿瘤生长缓慢或有时消退(Grote等人.(2001)Blood 97：3746-3754；Peng等人.(2001)Blood 98：2002-2007)。另外，Anderson等人(2004)Cancer Res.64：4919-4926，已在体外实验中显示，肿瘤细胞大量表达CD46是用减毒的埃德蒙顿MV活株感染和杀死这些细胞所必需的。然而，已知人类癌症已不同程度表达CD46(Niehans等人.(1996)American J.Pathol.149：129-142)，因而令人怀疑用减毒的MV活株治疗癌症的普遍适用性。
发明概述
本发明源于本发明人的意外发现，即减毒的麻疹病毒可有效感染和杀死间皮瘤细胞。此外，本发明人已经证明，与来自减毒的麻疹病毒感染的间皮瘤细胞裂解物相接触的树突细胞可激活抗间皮瘤CD8T细胞。
因此，本发明涉及减毒的麻疹病毒，其用于治疗个体的恶性间皮瘤。
本发明也涉及至少一种减毒的麻疹病毒用于生产旨在治疗个体恶性间皮瘤的药物的用途。
本发明也涉及治疗个体恶性间皮瘤的方法，其中给所述个体施用治疗有效量的至少一种减毒的麻疹病毒。
本发明还涉及制备用于治疗个体癌症的疫苗性树突细胞的方法，包括下列步骤：
-用减毒的麻疹株体外感染优选取自所述个体的癌细胞，以产生细胞裂解物；
-使树突细胞与所述细胞裂解物接触，以产生疫苗性树突细胞。
本发明也涉及能根据上述制备方法得到的疫苗性树突细胞，涉及含所述疫苗性树突细胞作为活性成分及药学可接受载体的药物组合物，涉及所述用于治疗个体癌症的疫苗性树突细胞，并涉及所述疫苗性树突细胞用于生产旨在治疗个体癌症的药物的用途。
本发明还涉及治疗个体癌症的方法，其中将治疗有效量的根据上述制备方法获得疫苗性树突细胞施用于所述个体。
发明详述
正如本文所预期的，所述个体优选为哺乳动物，更优选为人。也优选已经暴露于石棉的个体。
正如本文所预期的，表述“减毒的麻疹病毒”是指任何源于麻疹致病病毒并且和所述麻疹致病病毒相比呈现毒性减弱的病毒。正如本文所预期，所述减毒的麻疹病毒可用本领域技术人员所知的任何技术由麻疹致病病毒得到，所述技术例如在培养的细胞上连续传代和/或基因工程。减毒的麻疹病毒具体可为重组病毒，任选表达其他基因。减毒的麻疹病毒更具体可为其中一种或多种蛋白(优选为辅助C蛋白)的表达被废除的麻疹病毒。当施用给人时，减毒的麻疹病毒优选不实质引起麻疹症状。此外，减毒的麻疹病毒优选为活的且可复制。
所述减毒的麻疹病毒优选为埃德蒙顿(Edmonston)株。减毒的麻疹病毒的埃德蒙顿株为本领域技术人员所熟知，并特别报道于Griffin(2001)Field′s Virology 4th Edition vol.2 Knipe和Howley(ed.)Lippincott-Raven Publishers，Philadelphia，1401-1441；Hilleman(2002)Vaccine 20：651-665)。所述减毒的麻疹病毒更优选选自：施瓦茨株和Moraten株。这些株(它们的基因组被证明相同)为本领域技术人员所熟知，并且广泛用于抗麻疹疫苗的生产。它们特别描述于在Schwarz(1962)Am.J.Dis.Child 103：216-219；Parks等人.(2001)J.Virol.75：921-933和Parks等人.(2001)J.Virol.75：910-920。所述减毒的麻疹病毒最优选用pTM-MVSchw质粒(SEQ ID NO：1)生产，报道于Combredet等人.(2003)J.Virol.77：11546-11554。
在本发明框架内待治疗的癌症优选为恶性间皮瘤，更优选为恶性胸膜间皮瘤或腹膜间皮瘤，最优选为恶性胸膜间皮瘤。这些癌症特别报道于Kazan-Allen(2005)Lung cancer 49S1：S3-S8和Robinson等人.(2005)Lancet 366：397-408。
将减毒的麻疹病毒施用给个体，可通过胸膜内腔或鼻内、肌肉、静脉或皮下途径施用。将减毒的麻疹病毒通过胸膜内腔施用给个体，优选施用于比邻待处理肿瘤处，或直接施用于待处理肿瘤。如有必要，减毒的麻疹病毒可与任何合适的药学可接受的载体组合。施用的减毒的麻疹病毒的治疗有效量优选在103～106的50％组织培养感染剂量(TCID50)。TCID50测定为本领域技术人员所熟知，并特别报道于Karber(1931)在Arch.Exp.Path.Pharmak.162：840-483。
从待用疫苗性树突细胞治疗的个体中提取癌症细胞的步骤优选不包括于上述制备疫苗性树突细胞的方法中。所述步骤可根据本领域技术人员知道的提取细胞的任何技术来进行，例如活组织检查和渗漏/积液术(effusions)(例如，胸膜积液)。在被提取后，癌细胞可根据经典技术培养在培养基中，或者例如冷冻(例如在-80℃)保存。当癌细胞不是来自待用疫苗性树突细胞处理的个体，它们可主要来自同种异体的人类间皮瘤细胞系。
在上述的制备方法中，癌细胞可通过和病毒直接接触(例如以1的感染复数(MOI)，在37℃温育2小时)而被减毒的麻疹病毒感染。在感染后，由于病毒作用感染的细胞自发死亡。在细胞裂解后通常首先形成合胞体。这一现象能通过直接显微镜观测感染的细胞证明。本文所预期的“细胞裂解物”包含整个(或总)细胞裂解物，或部分细胞裂解物，例如膜部分(例如胞浆包涵体或凋亡小体)。将被本领域技术人员所充分理解的是，在上述制备方法第一步中获得的细胞裂解物相当于病毒感染的癌细胞裂解物。
树突细胞可通过本领域技术人员熟知的许多方法得到。树突细胞优选来自待治疗个体。目前优选的树突细胞是单核细胞衍生的树突细胞。单核细胞衍生细胞的获取为本领域技术人员所特别熟知。单核细胞衍生的细胞优选用实施例4中报道的一般方法获得，或根据Spisek等人.(2001)Cancer Immunology Immunotherapy 50：417-427获得，或根据Royer等人.(2006)Scand.J.Immunol.63：401-409获得。单核细胞衍生的树突细胞源于待治疗个体，这些单核细胞可从所述个体的白细胞采集物中获得。
本领域技术人员所应明晰的是，应维持树突细胞和细胞裂解物接触足够长的时间，以确保所述树突细胞被细胞裂解物中存在的抗原有效装载。一旦装载(或负载的(pulsed))，便获得了本发明的疫苗性树突细胞。装载可用下文中实施例4描述的一般方法来进行。足以确保树突细胞有效装载的树突细胞和细胞裂解物之间的例示接触时间大约是24小时。接触时间具体要保持到所述树突细胞变为激活状态。激活状态通常在树突细胞装载完成后出现。树突细胞的激活状态(或成熟状态)可被本领域内技术人员熟知的许多标记物证实，例如膜或细胞因子标记物。这些激活的树突细胞的标记物特别列于实施例5中。
因此，根据本发明的制备方法获得的疫苗性树突细胞有着特别的优势，因为它们是抗癌CD8T细胞的有效刺激物。同样有优势的是，本发明的制备方法可允许制备处于激活状态的疫苗性树突细胞。
附图简述
图1、2、3、4和5：间皮瘤对减毒的麻疹病毒(MV)的易感性
图1：Schwarz MV疫苗株的选择性溶瘤活性。用非重组的MV(MOI 1.0)感染一组人上皮样间皮瘤细胞系(M11、M13、M47、M56和M61)和永生的正常间皮细胞系(Met5A)，并在72小时后进行感染的培养物形态的显微镜观测。
图2～3：和正常细胞相比，肿瘤细胞的CD46受体的更高表面表达。细胞用缀合FITC的CD46特异性抗体(灰色柱状图)或相关的同种异型Ig对照(白色柱状图)染色(图2)。数量表示平均荧光指数，柱状图表示得到的间皮细胞(白色柱)和间皮瘤细胞系(阴影线柱)的CD46表达的平均值(图3)。
图4～5：Schwarz MV疫苗株优先感染转化的肿瘤细胞。分别培养(图4)或共培养(图5)相同数量的M13和Met5A细胞过夜，使细胞粘附，并以1.0的MOI用eGFP-重组MV进行感染。在分别培养中，用流式细胞仪进行在感染后不同时间(24、48和72小时)的eGFP表达分析(图4)。在共培养模式中，用HLA-A2染色进行相同的实验，因为HLA等位基因的差异表达使可辨别两种细胞系。柱状图表示共培养中Met5A(白色柱)和M13(黑色柱)的％eGFP-阳性细胞(图5)。
图6：MV-感染的间皮瘤细胞系的免疫原性
图6：MV-和UV-处理诱导的细胞死亡。在指示的时刻(D1＝24h，D2＝48h，D3＝72h，及D4＝96h)由UV暴露(5kJ/cm2)或MV感染(MOI＝1.0)引起的M13肿瘤细胞凋亡(阴影线柱)对比未处理的对照细胞(白色条快)的流式细胞仪分析。
图7和8：单核细胞衍生的DC对抗体的吞噬作用
图7：UV-或MV-处理的M13肿瘤细胞用PKH-26标记，并和未成熟的DC共培养24小时。随后对获取的树突细胞用缀合FITC的抗HLA-DR抗体染色，并用流式细胞仪分析。显示了三个具有相似结果的实验中具有代表性的一个实验。双阳性DC数是基于HLA-DR表达选通的PKH-26阳性DC的百分率(缀合FITC的抗体，克隆B8.12.2，Immunotech)，表示凋亡细胞的吞噬效率。
图8：柱状图表示来自所测试各装载条件得到的吞噬作用产量的平均值。
图9、10和11：通过与MV-感染的间皮瘤细胞共培养诱导的DC
成熟
图9和10：以指定组合培养未成熟的DC和M13肿瘤细胞(1/1的比例)24小时。作为对照，将DC和TLR3配体、聚肌苷酸：聚胞苷酸(50μg/ml；Sigma)共温育，或将DC直接用MV感染(MOI＝1.0)。随后提取DC，并用特异于指定的细胞表面分子的缀合PE的抗体平板染色(图9-HLA分子；图10-成熟标记物)。根据DC的形态特征对它们选通，其中死细胞基于TOPRO-3染色(分子探针)被去除。用三色流式细胞仪分析DC表面显型。柱状图表示来自4种独立供体的平均值。
图11：通过CBA(针对IL-6、IL-1β、TNFα、IL-12和IL-10)和ELISA(针对IFNα)测定来研究24小时共培养上清上的DC细胞因子分泌模式。
图12：装载MV-感染的间皮瘤细胞的DC诱导MSLN特异性CD8
T细胞致敏
图12：MSLN特异性CD8 T细胞数通过流式细胞仪进行分析，所述CD8 T细胞源于和未负载或者负载UV-M13或MV-M13的DC致敏一周的共培养物。柱状图表示基于人CD8表达选通的T细胞中的PE-四聚物阳性细胞(缀合PE-Cy5的抗体，克隆RPA-T8，BDBiosciences)的百分率。显示的是一个有代表性的实验。
实施例
实施例1：间皮瘤对MV感染和溶瘤活性的易感性
为了比较MV-涉及的对肿瘤和非肿瘤细胞的细胞病变效应，一组五种上皮样间皮瘤细胞系(M11、M13、M47、M56、和M61)和一种间皮细胞(Met5A)用Schwarz疫苗株以1.0的感染复数(MOI)感染。
间皮瘤细胞系(M11、M13、M47、M56、和M61)来自对癌症患者进行胸腔穿刺术收集的胸膜积液。上皮样间皮瘤的诊断用活组织切片免疫组化染色来确定。对照间皮细胞系(Met5A)分离自无癌症的病人的胸膜液，并通过转染编码SV40 T-抗原的pRSV质粒(ATCC-LGC Promochem，Molsheim，France)使其永生。将细胞系维持在添加10％的热灭活的小牛胎儿血清(FCS来自Biowest，Nuaille，France)、1％的L-谷氨酰胺和1％青霉素/链霉素抗生素(都购自Sigma，St Quentin Fallavier，France)的RPMI-1640培养基中。用Hoechst33258染色(Sigma)常规检测细胞培养物的支原体污染。
减毒的MVSchwarz疫苗株获自F.Tangy(Pasteur Institut，France)。Schwarz MV从pTM-MVSchw(SEQ ID NO：1)cDNA，通过使用基于辅助细胞的营救系统(报道于Radecke等人(1995)EMBO J.14：5773-5784，并被Parks等人(1999)J.Virol.73：3560-3566改进)营救。简而言之，用5μg pTM-MVSchw和0.02μg表达SchwarzMV-L基因的pEMC-Lschw(Combredet等人.(2003)J.Virol.77：11546-11554)(SEQ ID NO：2)转然293-3-46辅助细胞。37℃过夜温育后，于43℃热激2小时，并将转染的细胞转移到Vero细胞单层上。将共培养15天后出现的合胞体转移到35mm孔中，然后在75cm2和150cm2 Vero细胞培养瓶中扩展，所述Vero细胞在5％FCS DMEM中培养。当合胞体达到80～90％汇合时，将细胞刮到小体积的OptiMEM中，并立即冻结。在低速离心成片状细胞残骸后，-80℃保存含有病毒的上清液。通过对Vero细胞的端点有限稀释测定来测定重组MV储存液的滴度。用方法计算出TCID50(Karber(1931)Arch.Exp.Path.Pharmak.162：480-483)。
以MOI＝1.0、37℃温育2小时来进行间皮瘤细胞系的病毒感染。MV感染三天后，观测到MV感染的细胞有典型的形态学变化，和非癌Met5A细胞相比，这是对大部分肿瘤MPM细胞系(4/5)的重要的细胞病变效应(CPE)的发生(图1)。通过发生或多或少重要的合胞体证实了CPE，这最终导致死肿瘤细胞的胞浆包含体流入培养上清液中(图1)。这些多核巨合胞体的发生是麻疹感染的特征，产生于HA+感染的细胞和邻近的CD46+培养细胞的融合。
可证实间皮瘤细胞上MV的减毒活株的受体CD46有显著的上调表达(图2～3)。
为了量化MV感染的敏感性，Met5A和M13细胞系被eGFP-重组的MV储存液感染(Combredet等人.(2003)J.Virol.77：11546-11554)。将该GFP-转基因的表达作为病毒感染的标记，因此允许用流式细胞仪检测感染细胞的百分率。两种培养细胞中的MV感染量都是剂量依赖的(MOI为0.01～5.0)，表示eGFP信号的特异性。然而Met5A被MV株感染(MOI为0.5～)，M13也明显被MV感染，但总是以最低的MOI(MOI为0.1～)。也观测到和正常细胞相比(MOI 1.0)，感染后48小时，在两种细胞分别培养(图4)和共培养(图5)系统(1∶3的比例)中，肿瘤细胞显著增加的感染量。此外，病毒感染也可通过观测到感染的细胞培养物中CD46表面表达的下调来证实。
因此，根据这些体外研究结果，和间皮组织相比，间皮瘤表现出对MV-介导的感染和MV-相关的细胞裂解活性有更明显的易感性。因此，MPM看起来是和基于麻疹病毒施用的病毒治疗法有关的候选物。
实施例2：MV和UV治疗诱导的肿瘤细胞死亡
在证明MV能够感染间皮瘤细胞之后，本发明人证明是否病毒感染也可导致凋亡介导的细胞死亡。
亚汇合单层M13细胞培养物或者经MV-感染(MOI 1.0)，或者用UV Stratalinker2400(Stratagene Europe，Amsterdam，Netherlands)进行UV-B-照射(312nm～5kJ/m2)，作为凋亡的阳性对照。在处理后的不同时间收集细胞，根据伴随物磷脂酰丝氨酸和膜联蛋白-V染色定量细胞的死亡，这报道于Ebstein等人.(2004)Am.J.Respir.Crit.Care Med.169：1322-1330。
如图6所示，暴露于UV-B照射24小时的M13细胞和经MV感染72小时的M13细胞表现出相等的肿瘤细胞死亡率(含有70％～80％之间的膜联蛋白-V阳性细胞)，这表示MV诱导感染的肿瘤细胞凋亡。由此定义的M13细胞死亡诱导的条件被用于下列实验中。
此外，通过观测到明显的细胞病变效应也证实了病毒涉及的细胞杀死，导致感染后72～96小时M13细胞层完全错位(图1)。
实施例3：MV感染的肿瘤细胞中随后的病毒复制循环
为了跟踪病毒在感染的M13细胞培养物中的生长动力学(MOI＝1.0)，进行特异于病毒dsRNA潜在受体(Mda-5、TLR-3、RIG-I和PKR)的RT-PCR。β-肌动蛋白基因的特异引物作为内部实验对照。
简而言之，M13细胞或者与聚肌苷酸：聚胞苷酸配体(10μg/ml)温育，或者与MV(MOI＝1.0)温育，并在不同时间收集细胞沉淀。然后用RNeasy试剂盒(Qiagen，Courtaboeuf，France)根据产品说明提取细胞的总细胞RNA，并用RTase(Invitrogen，Paisley，UK)进行逆转录。产生的cDNA作为PCR扩增的模版，该PCR使用特异于Mda-5、TLR-3、RIG-I、PKR、IFNβ，和β-激动蛋白的引物。PCR引物序列列在表1中。PCR产物通过琼脂糖凝胶电泳观察。
| 引物 | 序列 | 片段大小(bp) | SEQ IDNO： |
| β-激动蛋白 | 正向ATCTGGCACCACACCTTCTACAATGAGCTGCG反向CGTCATACTCCTGCTTGCTGATCCACATCTGC | 837 | 34 |
| TLR-3 | 正向ATTGGGTCTGGGAACATTTCTCTTC反向GTGAGATTTAAACATTCCTCTTCGG | 319 | 56 |
| Mda-5 | 正向GAGCAACTTCTTTCAACCAC反向GAACACCAGCATCTTCTCCA | 633 | 78 |
| RIG-I | 正向GAACGATTCCATCACTATCC反向GGCATCATTAIATTTCCGCA | 580 | 910 |
| PKR | 正向CTTCTCAGCAGATACATCAG反向GTTACAAGTCCAAAGTCTCC | 689 | 1112 |
表1：引物序列
因此这可证明病毒复制峰出现在感染间皮瘤M13细胞后的第一天到第四天之间。另外，也可证实相应于病毒dsRNA潜在受体(Mda-5、TLR-3、RIG-I和PKR)的PCR产物。
实施例4：未成熟DC对凋亡性间皮瘤细胞的有效摄入
然后研究树突细胞(DC)对MV-感染(72小时)所致的凋亡小体的摄入，并与其对UV-照射(24小时)的M13肿瘤细胞的摄入进行比较。
树突细胞来源于单核细胞，在获得知情同意后，从HLA-A0201健康供体(EFS，Nantes，France)的白细胞采集收获物产生所述单核细胞。富含单核细胞的级分(＞85％纯度)通过Ficoll密度梯度离心(PAA Laboratories，Les Mureaux，France)首先被分离。然后单核细胞通过用Beckman Avanti J20离心机(配备有JE5.0转子)和40ml淘洗室进行淘洗(逆流离心)来富集。如基于CD14标记物检测的流式细胞仪检测所评估，经淘洗的单核细胞的纯度通常超过80％。用500IU/ml GM-CSF和200IU/ml IL-4(Cell Genix Technology，Freiburg，Germany)以2×108细胞/ml培养单核细胞。然后使细胞分化6天。
在第6天，从培养物上清液中收集单核细胞衍生的DC，并接种于用于随后装载的培养物中。使未成熟的DC和2×108细胞/ml的凋亡物(来自UV-处理的或MV-感染的同种异体M13肿瘤细胞)温育共培养(比列1∶1)另外的24小时。DC吞噬量的分析通过用流式细胞仪检测和激光共聚焦显微镜检测来评估，如先前报道(Massé等人.(2002)Cancer Research 32：1050-1056)。简而言之，使用PKH-26膜染料着色剂，根据产品说明(Sigma，St Quentin Fallavier，France)标记经UV-或MV-处理的M13细胞。共温育24小时后，用缀合FITC的抗HLA-DR抗体(Immunotech，Marseilles，France)染色DC。用PBS洗涤后，收获细胞，并用FACSCalibur(BD Biosciences，Grenoble，France)或者用TCS NT显微镜(Leica Instruments，Heidelberg，Germany)分析。已摄入凋亡细胞的DC被鉴定为HLA-DR/PKH-26双阳性细胞(图7)。
如图8所示，能够证明DC以相同的速率有效吞入经UV-和MV-处理的间皮瘤细胞，如基于HLA-DR表达选通的PKH26-阳性DC的相似百分率所示(对于分别装载经UV-或MV-处理的M13细胞的DC分别为65％和74％)。
激光共聚焦扫描显微镜实验进一步证实凋亡的M13细胞在共培养24小时内被未成熟的DC有效摄入，无关于所使用的死亡诱导策略(MV-感染或UV-照射)。
实施例5：相比用UV照射诱导的凋亡性M13细胞，MV感染的
肿瘤细胞诱导DC自发成熟
接下来本发明人检验了是否来自MV感染的M13肿瘤细胞的细胞物质可有效刺激DC成熟。
在被暴露于照射或病毒介导的细胞裂解活性杀死的肿瘤细胞摄入后的24小时内评估DC成熟状态。
活DC(基于TOPRO-3阳性染色排除选通)的表型用Class I和II MHC分子(图9)和成熟标记物CD80、CD86、CD83和CD40(图10)的表面表达来研究，通过对共培养上清液进行细胞因子分泌模式分析来完成(图11)。作为对照，DC未经处理，或者组合TLR3配体和一种促炎症细胞因子(聚肌苷酸:聚胞苷酸/IFNα，作为病毒感染的模拟物)而成熟，或者直接通过接触麻疹病毒(MV)来致敏。
简而言之，用特异于HLA-ABC(克隆B9.12.1)、HLA-DR(克隆B8.12.2)、CD80(克隆MAB104)、CD83(克隆HB15a)、CD86(克隆HA5.2B7)、和CD40(克隆MAB89)的单克隆抗体(均购自Immunotech，Marseilles，France)进行免疫染色。在进行流式细胞仪检测前，DC与上述各抗体(1μg/ml)在4℃温育30分钟。测定吞入后24小时收集的上清液中的细胞因子的分泌模式。用可商购的Cytometric Beads Array试剂盒(BD Biosciences，Le Pont de Claix，France)根据产品说明测定了IL-10、IL-12、IL-6、IL-1β和TNFα浓度。IFNα的定量用ELISA检测(Biosource，Camarillo，USA)进行。
自发成熟程序只在装载有来自MV感染的间皮瘤细胞的凋亡小体的DC中观测到，以与实验中所用的阳性对照的成熟混合物(聚I:C/IFNα)基本相等的水平。通过共刺激分子(CD80、CD83、CD86、CD40和HLA-ABC)表达的显著上调和许多促炎细胞因子(IL-6、IL-1β、TNFα、和IFNα)的生成证实了自发成熟。
然而，与先前的报道一致，用经UV-照射的凋亡性肿瘤细胞负载DC，和用麻疹病毒(MV)直接感染DC不能诱发此效果。
所有这些数据都强烈证实，与UV-照射的肿瘤细胞相比，MV感染的肿瘤细胞的免疫原性升高。
实施例6：MSLN特异性CD8 T细胞应答的交叉致敏
最后，本发明人检测是否装载有来自MV感染的间皮瘤细胞的凋亡小体的DC可刺激特异于MPM-相关的肿瘤抗原(例如间皮素(MSLN))的效应子CD8应答。
为了评估这个问题，对CD8 T-淋巴细胞进行四聚物免疫染色，所述CD8T-淋巴细胞与装载来自经UV-或MV-处理的M13细胞的凋亡物质的自身DC致敏一周。作为对照，类似的实验用Jurkat淋巴瘤T细胞系进行，基于其对MV的易感性和其MSLN-阴性表达特征来选择(图12)。作为内部实验对照，完成了MelanA/Mart-1-特异性四聚物染色(MelanA26-35L)，以补充特异于两个选定的MSLN-衍生的CTL表位的那些。这些肽(MSLN 531～539和MSLN 541～550)通过使用两种计算算法BIMAS和SYFPEITHI扫描匹配对于HLA-A0201结合共有的基序的MSLN氨基酸序列(GenPept NP005814)来鉴定(表2)：
表2：四聚物特征
简而言之，通过使用CD8多重分选试剂盒(Miltenyi Biotec，Paris，France)用MACS柱系统阳性筛选来从HLA-A0201健康供体的PBMC制备CD8 T淋巴细胞。纯化的幼稚CD8 T细胞(＞90％纯度)用装载有各凋亡制剂的自身DC或未装载的DC(作为对照)刺激。在圆底的96孔平板(BD Falcon)中，通过将2×104成熟的DC与2×105应答T细胞在200μl 8％人血清RPMI 1640培养基(头三天补加10ng/ml IL-12(AbCys SA，Paris，France)，以后补加10U/ml IL-2(Proleukin，Chiron Therapeutics，USA))中混和(比列1∶10)来进行共培养。每三天加一次IL-2，允许常规的培养基更新。在培养7～8天后，收获T细胞，并如下用MSLN-特异性四聚物染色。
将所选定的CD8表位肽(由Eurogentec，Liege，Belgium合成)用于如先前报道于(Labarrière等人.(2002)Int.J.Cancer101：280-286)的单体生产(重组蛋白生产平台，U601-IFR26，Nantes，France)。HLA-A2 VLP9和HLA-A2 KLL10单体用PE-标记的链霉亲和素(BD Biosciences)寡聚化。染色和洗涤在0.1％BSA-PBS中进行。随后将T细胞在4℃下用10μg/ml的PE-标记的pMHC多聚体染色30分钟，然后再于4℃下用1μg/ml稀释的缀合PE-Cy5的抗-CD8抗体(克隆RPA-T8，BD Biosciences)染色30分钟。洗涤细胞，并立即在FACSCalibur上分析。
有趣的是，观测发现，与和装载有来自UV-处理的M13细胞的凋亡物质的DC的共培养物相比，与装载有来自MV-处理的M13细胞的凋亡物质的DC的共培养物中，CD8-阳性选通群中MSLN特异性T细胞的百分率显著增加。
序列表
<110>INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM)
INSTITUT PASTEUR
<120>治疗间皮瘤的药物和方法
<130>BET08P1093
<150>EP 07291232.2
<151>2007-10-10
<160>14
<170>PatentIn version 3.4
<210>1
<211>18967
<212>DNA
<213>人工序列
<220>
<223>质粒pTM-MVSchw
<400>1
gcggccgcta atacgactca ctatagggcc aactttgttt ggtctgatga gtccgtgagg 60
acgaaacccg gagtcccggg tcaccaaaca aagttgggta aggatagttc aatcaatgat 120
catcttctag tgcacttagg attcaagatc ctattatcag ggacaagagc aggattaggg 180
atatccgaga tggccacact tttaaggagc ttagcattgt tcaaaagaaa caaggacaaa 240
ccacccatta catcaggatc cggtggagcc atcagaggaa tcaaacacat tattatagta 300
ccaatccctg gagattcctc aattaccact cgatccagac ttctggaccg gttggtgagg 360
ttaattggaa acccggatgt gagcgggccc aaactaacag gggcactaat aggtatatta 420
tccttatttg tggagtctcc aggtcaattg attcagagga tcaccgatga ccctgacgtt 480
agcataaggc tgttagaggt tgtccagagt gaccagtcac aatctggcct taccttcgca 540
tcaagaggta ccaacatgga ggatgaggcg gaccaatact tttcacatga tgatccaatt 600
agtagtgatc aatccaggtt cggatggttc gggaacaagg aaatctcaga tattgaagtg 660
caagaccctg agggattcaa catgattctg ggtaccatcc tagcccaaat ttgggtcttg 720
ctcgcaaagg cggttacggc cccagacacg gcagctgatt cggagctaag aaggtggata 780
aagtacaccc aacaaagaag ggtagttggt gaatttagat tggagagaaa atggttggat 840
gtggtgagga acaggattgc cgaggacctc tccttacgcc gattcatggt cgctctaatc 900
ctggatatca agagaacacc cggaaacaaa cccaggattg ctgaaatgat atgtgacatt 960
gatacatata tcgtagaggc aggattagcc agttttatcc tgactattaa gtttgggata 1020
gaaactatgt atcctgctct tggactgcat gaatttgctg gtgagttatc cacacttgag 1080
tccttgatga acctttacca gcaaatgggg gaaactgcac cctacatggt aatcctggag 1140
aactcaattc agaacaagtt cagtgcagga tcataccctc tgctctggag ctatgccatg 1200
ggagtaggag tggaacttga aaactccatg ggaggtttga actttggccg atcttacttt 1260
gatccagcat attttagatt agggcaagag atggtaagga ggtcagctgg aaaggtcagt 1320
tccacattgg catctgaact cggtatcact gccgaggatg caaggcttgt ttcagagatt 1380
gcaatgcata ctactgagga caagatcagt agagcggttg gacccagaca agcccaagta 1440
tcatttctac acggtgatca aagtgagaat gagctaccga gattgggggg caaggaagat 1500
aggagggtca aacagagtcg aggagaagcc agggagagct acagagaaac cgggcccagc 1560
agagcaagtg atgcgagagc tgcccatctt ccaaccggca cacccctaga cattgacact 1620
gcaacggagt ccagccaaga tccgcaggac agtcgaaggt cagctgacgc cctgcttagg 1680
ctgcaagcca tggcaggaat ctcggaagaa caaggctcag acacggacac ccctatagtg 1740
tacaatgaca gaaatcttct agactaggtg cgagaggccg agggccagaa caacatccgc 1800
ctaccatcca tcattgttat aaaaaactta ggaaccaggt ccacacagcc gccagcccat 1860
caaccatcca ctcccacgat tggagccaat ggcagaagag caggcacgcc atgtcaaaaa 1920
cggactggaa tgcatccggg ctctcaaggc cgagcccatc ggctcactgg ccatcgagga 1980
agctatggca gcatggtcag aaatatcaga caacccagga caggagcgag ccacctgcag 2040
ggaagagaag gcaggcagtt cgggtctcag caaaccatgc ctctcagcaa ttggatcaac 2100
tgaaggcggt gcacctcgca tccgcggtca gggacctgga gagagcgatg acgacgctga 2160
aactttggga atccccccaa gaaatctcca ggcatcaagc actgggttac agtgttatta 2220
cgtttatgat cacagcggtg aagcggttaa gggaatccaa gatgctgact ctatcatggt 2280
tcaatcaggc cttgatggtg atagcaccct ctcaggagga gacaatgaat ctgaaaacag 2340
cgatgtggat attggcgaac ctgataccga gggatatgct atcactgacc ggggatctgc 2400
tcccatctct atggggttca gggcttctga tgttgaaact gcagaaggag gggagatcca 2460
cgagctcctg agactccaat ccagaggcaa caactttccg aagcttggga aaactctcaa 2520
tgttcctccg cccccggacc ccggtagggc cagcacttcc gggacaccca ttaaaaaggg 2580
cacagacgcg agattagcct catttggaac ggagatcgcg tctttattga caggtggtgc 2640
aacccaatgt gctcgaaagt caccctcgga accatcaggg ccaggtgcac ctgcggggaa 2700
tgtccccgag tgtgtgagca atgccgcact gatacaggag tggacacccg aatctggtac 2760
cacaatctcc ccgagatccc agaataatga agaaggggga gactattatg atgatgagct 2820
gttctctgat gtccaagata ttaaaacagc cttggccaaa atacacgagg ataatcagaa 2880
gataatctcc aagctagaat cactgctgtt attgaaggga gaagttgagt caattaagaa 2940
gcagatcaac aggcaaaata tcagcatatc caccctggaa ggacacctct caagcatcat 3000
gatcgccatt cctggacttg ggaaggatcc caacgacccc actgcagatg tcgaaatcaa 3060
tcccgacttg aaacccatca taggcagaga ttcaggccga gcactggccg aagttctcaa 3120
gaaacccgtt gccagccgac aactccaagg aatgacaaat ggacggacca gttccagagg 3180
acagctgctg aaggaatttc agctaaagcc gatcgggaaa aagatgagct cagccgtcgg 3240
gtttgttcct gacaccggcc ctgcatcacg cagtgtaatc cgctccatta taaaatccag 3300
ccggctagag gaggatcgga agcgttacct gatgactctc cttgatgata tcaaaggagc 3360
caatgatctt gccaagttcc accagatgct gatgaagata ataatgaagt agctacagct 3420
caacttacct gccaacccca tgccagtcga cccaactagt acaacctaaa tccattataa 3480
aaaacttagg agcaaagtga ttgcctccca aggtccacaa tgacagagac ctacgacttc 3540
gacaagtcgg catgggacat caaagggtcg atcgctccga tacaacccac cacctacagt 3600
gatggcaggc tggtgcccca ggtcagagtc atagatcctg gtctaggcga caggaaggat 3660
gaatgcttta tgtacatgtt tctgctgggg gttgttgagg acagcgattc cctagggcct 3720
ccaatcgggc gagcatttgg gttcctgccc ttaggtgttg gcagatccac agcaaagccc 3780
gaaaaactcc tcaaagaggc cactgagctt gacatagttg ttagacgtac agcagggctc 3840
aatgaaaaac tggtgttcta caacaacacc ccactaactc tcctcacacc ttggagaaag 3900
gtcctaacaa cagggagtgt cttcaacgca aaccaagtgt gcaatgcggt taatctgata 3960
ccgctcgata ccccgcagag gttccgtgtt gtttatatga gcatcacccg tctttcggat 4020
aacgggtatt acaccgttcc tagaagaatg ctggaattca gatcggtcaa tgcagtggcc 4080
ttcaacctgc tggtgaccct taggattgac aaggcgatag gccctgggaa gatcatcgac 4140
aatacagagc aacttcctga ggcaacattt atggtccaca tcgggaactt caggagaaag 4200
aagagtgaag tctactctgc cgattattgc aaaatgaaaa tcgaaaagat gggcctggtt 4260
tttgcacttg gtgggatagg gggcaccagt cttcacatta gaagcacagg caaaatgagc 4320
aagactctcc atgcacaact cgggttcaag aagaccttat gttacccgct gatggatatc 4380
aatgaagacc ttaatcgatt actctggagg agcagatgca agatagtaag aatccaggca 4440
gttttgcagc catcagttcc tcaagaattc cgcatttacg acgacgtgat cataaatgat 4500
gaccaaggac tattcaaagt tctgtagacc gtagtgccca gcaatgcccg aaaacgaccc 4560
ccctcacaat gacagccaga aggcccggac aaaaaagccc cctccgaaag actccacgga 4620
ccaagcgaga ggccagccag cagccgacgg caagcgcgaa caccaggcgg ccccagcaca 4680
gaacagccct gacacaaggc caccaccagc caccccaatc tgcatcctcc tcgtgggacc 4740
cccgaggacc aacccccaag gctgcccccg atccaaacca ccaaccgcat ccccaccacc 4800
cccgggaaag aaacccccag caattggaag gcccctcccc ctcttcctca acacaagaac 4860
tccacaaccg aaccgcacaa gcgaccgagg tgacccaacc gcaggcatcc gactccctag 4920
acagatcctc tctccccggc aaactaaaca aaacttaggg ccaaggaaca tacacaccca 4980
acagaaccca gaccccggcc cacggcgccg cgcccccaac ccccgacaac cagagggagc 5040
ccccaaccaa tcccgccggc tcccccggtg cccacaggca gggacaccaa cccccgaaca 5100
gacccagcac ccaaccatcg acaatccaag acgggggggc ccccccaaaa aaaggccccc 5160
aggggccgac agccagcacc gcgaggaagc ccacccaccc cacacacgac cacggcaacc 5220
aaaccagaac ccagaccacc ctgggccacc agctcccaga ctcggccatc accccgcaga 5280
aaggaaaggc cacaacccgc gcaccccagc cccgatccgg cggggagcca cccaacccga 5340
accagcaccc aagagcgatc cccgaaggac ccccgaaccg caaaggacat cagtatccca 5400
cagcctctcc aagtcccccg gtctcctcct cttctcgaag ggaccaaaag atcaatccac 5460
cacacccgac gacactcaac tccccacccc taaaggagac accgggaatc ccagaatcaa 5520
gactcatcca atgtccatca tgggtctcaa ggtgaacgtc tctgccatat tcatggcagt 5580
actgttaact ctccaaacac ccaccggtca aatccattgg ggcaatctct ctaagatagg 5640
ggtggtagga ataggaagtg caagctacaa agttatgact cgttccagcc atcaatcatt 5700
agtcataaaa ttaatgccca atataactct cctcaataac tgcacgaggg tagagattgc 5760
agaatacagg agactactga gaacagtttt ggaaccaatt agagatgcac ttaatgcaat 5820
gacccagaat ataagaccgg ttcagagtgt agcttcaagt aggagacaca agagatttgc 5880
gggagtagtc ctggcaggtg cggccctagg cgttgccaca gctgctcaga taacagccgg 5940
cattgcactt caccagtcca tgctgaactc tcaagccatc gacaatctga gagcgagcct 6000
ggaaactact aatcaggcaa ttgagacaat cagacaagca gggcaggaga tgatattggc 6060
tgttcagggt gtccaagact acatcaataa tgagctgata ccgtctatga accaactatc 6120
ttgtgattta atcggccaga agctcgggct caaattgctc agatactata cagaaatcct 6180
gtcattattt ggccccagtt tacgggaccc catatctgcg gagatatcta tccaggcttt 6240
gagctatgcg cttggaggag acatcaataa ggtgttagaa aagctcggat acagtggagg 6300
tgatttactg ggcatcttag agagcggagg aataaaggcc cggataactc acgtcgacac 6360
agagtcctac ttcattgtcc tcagtatagc ctatccgacg ctgtccgaga ttaagggggt 6420
gattgtccac cggctagagg gggtctcgta caacataggc tctcaagagt ggtataccac 6480
tgtgcccaag tatgttgcaa cccaagggta ccttatctcg aattttgatg agtcatcgtg 6540
tactttcatg ccagagggga ctgtgtgcag ccaaaatgcc ttgtacccga tgagtcctct 6600
gctccaagaa tgcctccggg ggtacaccaa gtcctgtgct cgtacactcg tatccgggtc 6660
ttttgggaac cggttcattt tatcacaagg gaacctaata gccaattgtg catcaatcct 6720
ttgcaagtgt tacacaacag gaacgatcat taatcaagac cctgacaaga tcctaacata 6780
cattgctgcc gatcactgcc cggtagtcga ggtgaacggc gtgaccatcc aagtcgggag 6840
caggaggtat ccagacgctg tgtacttgca cagaattgac ctcggtcctc ccatatcatt 6900
ggagaggttg gacgtaggga caaatctggg gaatgcaatt gctaagttgg aggatgccaa 6960
ggaattgttg gagtcatcgg accagatatt gaggagtatg aaaggtttat cgagcactag 7020
catagtctac atcctgattg cagtgtgtct tggagggttg atagggatcc ccgctttaat 7080
atgttgctgc agggggcgtt gtaacaaaaa gggagaacaa gttggtatgt caagaccagg 7140
cctaaagcct gatcttacgg gaacatcaaa atcctatgta aggtcgctct gatcctctac 7200
aactcttgaa acacaaatgt cccacaagtc tcctcttcgt catcaagcaa ccaccgcacc 7260
cagcatcaag cccacctgaa attatctccg gcttccctct ggccgaacaa tatcggtagt 7320
taatcaaaac ttagggtgca agatcatcca caatgtcacc acaacgagac cggataaatg 7380
ccttctacaa agataacccc catcccaagg gaagtaggat agtcattaac agagaacatc 7440
ttatgattga tagaccttat gttttgctgg ctgttctgtt tgtcatgttt ctgagcttga 7500
tcgggttgct agccattgca ggcattagac ttcatcgggc agccatctac accgcagaga 7560
tccataaaag cctcagcacc aatctagatg taactaactc aatcgagcat caggtcaagg 7620
acgtgctgac accactcttc aaaatcatcg gtgatgaagt gggcctgagg acacctcaga 7680
gattcactga cctagtgaaa ttaatctctg acaagattaa attccttaat ccggataggg 7740
agtacgactt cagagatctc acttggtgta tcaacccgcc agagagaatc aaattggatt 7800
atgatcaata ctgtgcagat gtggctgctg aagagctcat gaatgcattg gtgaactcaa 7860
ctctactgga gaccagaaca accaatcagt tcctagctgt ctcaaaggga aactgctcag 7920
ggcccactac aatcagaggt caattctcaa acatgtcgct gtccctgtta gacttgtatt 7980
taggtcgagg ttacaatgtg tcatctatag tcactatgac atcccaggga atgtatgggg 8040
gaacttacct agtggaaaag cctaatctga gcagcaaaag gtcagagttg tcacaactga 8100
gcatgtaccg agtgtttgaa gtaggtgtta tcagaaatcc gggtttgggg gctccggtgt 8160
tccatatgac aaactatctt gagcaaccag tcagtaatga tctcagcaac tgtatggtgg 8220
ctttggggga gctcaaactc gcagcccttt gtcacgggga agattctatc acaattccct 8280
atcagggatc agggaaaggt gtcagcttcc agctcgtcaa gctaggtgtc tggaaatccc 8340
caaccgacat gcaatcctgg gtccccttat caacggatga tccagtgata gacaggcttt 8400
acctctcatc tcacagaggt gttatcgctg acaatcaagc aaaatgggct gtcccgacaa 8460
cacgaacaga tgacaagttg cgaatggaga catgcttcca acaggcgtgt aagggtaaaa 8520
tccaagcact ctgcgagaat cccgagtggg caccattgaa ggataacagg attccttcat 8580
acggggtctt gtctgttgat ctgagtctga cagttgagct taaaatcaaa attgcttcgg 8640
gattcgggcc attgatcaca cacggttcag ggatggacct atacaaatcc aaccacaaca 8700
atgtgtattg gctgactatc ccgccaatga agaacctagc cttaggtgta atcaacacat 8760
tggagtggat accgagattc aaggttagtc cctacctctt cactgtccca attaaggaag 8820
caggcgaaga ctgccatgcc ccaacatacc tacctgcgga ggtggatggt gatgtcaaac 8880
tcagttccaa tctggtgatt ctacctggtc aagatctcca atatgttttg gcaacctacg 8940
atacttccag ggttgaacat gctgtggttt attacgttta cagcccaagc cgctcatttt 9000
cttactttta tccttttagg ttgcctataa agggggtccc catcgaatta caagtggaat 9060
gcttcacatg ggaccaaaaa ctctggtgcc gtcacttctg tgtgcttgcg gactcagaat 9120
ctggtggaca tatcactcac tctgggatgg tgggcatggg agtcagctgc acagtcaccc 9180
gggaagatgg aaccaatcgc agatagggct gctagtgaac caatcacatg atgtcaccca 9240
gacatcaggc atacccacta gtgtgaaata gacatcagaa ttaagaaaaa cgtagggtcc 9300
aagtggttcc ccgttatgga ctcgctatct gtcaaccaga tcttataccc tgaagttcac 9360
ctagatagcc cgatagttac caataagata gtagccatcc tggagtatgc tcgagtccct 9420
cacgcttaca gcctggagga ccctacactg tgtcagaaca tcaagcaccg cctaaaaaac 9480
ggattttcca accaaatgat tataaacaat gtggaagttg ggaatgtcat caagtccaag 9540
cttaggagtt atccggccca ctctcatatt ccatatccaa attgtaatca ggatttattt 9600
aacatagaag acaaagagtc aacgaggaag atccgtgaac tcctcaaaaa ggggaattcg 9660
ctgtactcca aagtcagtga taaggttttc caatgcttaa gggacactaa ctcacggctt 9720
ggcctaggct ccgaattgag ggaggacatc aaggagaaag ttattaactt gggagtttac 9780
atgcacagct cccagtggtt tgagcccttt ctgttttggt ttacagtcaa gactgagatg 9840
aggtcagtga ttaaatcaca aacccatact tgccatagga ggagacacac acctgtattc 9900
ttcactggta gttcagttga gttgctaatc tctcgtgacc ttgttgctat aatcagtaaa 9960
gagtctcaac atgtatatta cctgacattt gaactggttt tgatgtattg tgatgtcata 10020
gaggggaggt taatgacaga gaccgctatg actattgatg ctaggtatac agagcttcta 10080
ggaagagtca gatacatgtg gaaactgata gatggtttct tccctgcact cgggaatcca 10140
acttatcaaa ttgtagccat gctggagcct ctttcacttg cttacctgca gctgagggat 10200
ataacagtag aactcagagg tgctttcctt aaccactgct ttactgaaat acatgatgtt 10260
cttgaccaaa acgggttttc tgatgaaggt acttatcatg agttaactga agctctagat 10320
tacattttca taactgatga catacatctg acaggggaga ttttctcatt tttcagaagt 10380
ttcggccacc ccagacttga agcagtaacg gctgctgaaa atgttaggaa atacatgaat 10440
cagcctaaag tcattgtgta tgagactctg atgaaaggtc atgccatatt ttgtggaatc 10500
ataatcaacg gctatcgtga caggcacgga ggcagttggc caccgctgac cctccccctg L0560
catgctgcag acacaatccg gaatgctcaa gcttcaggtg aagggttaac acatgagcag 10620
tgcgttgata actggaaatc ttttgctgga gtgaaatttg gctgctttat gcctcttagc 10680
ctggatagtg atctgacaat gtacctaaag gacaaggcac ttgctgctct ccaaagggaa 10740
tgggattcag tttacccgaa agagttcctg cgttacgacc ctcccaaggg aaccgggtca 10800
cggaggcttg tagatgtttt ccttaatgat tcgagctttg acccatatga tgtgataatg 10860
tatgttgtaa gtggagctta cctccatgac cctgagttca acctgtctta cagcctgaaa 10920
gaaaaggaga tcaaggaaac aggtagactt tttgctaaaa tgacttacaa aatgagggca 10980
tgccaagtga ttgctgaaaa tctaatctca aacgggattg gcaaatattt taaggacaat 11040
gggatggcca aggatgagca cgatttgact aaggcactcc acactctagc tgtctcagga 11100
gtccccaaag atctcaaaga aagtcacagg ggggggccag tcttaaaaac ctactcccga 11160
agcccagtcc acacaagtac caggaacgtg agagcagcaa aagggtttat agggttccct 11220
caagtaattc ggcaggacca agacactgat catccggaga atatggaagc ttacgagaca 11280
gtcagtgcat ttatcacgac tgatctcaag aagtactgcc ttaattggag atatgagacc 11340
atcagcttgt ttgcacagag gctaaatgag atttacggat tgccctcatt tttccagtgg 11400
ctgcataaga ggcttgagac ctctgtcctg tatgtaagtg accctcattg cccccccgac 11460
cttgacgccc atatcccgtt atataaagtc cccaatgatc aaatcttcat taagtaccct 11520
atgggaggta tagaagggta ttgtcagaag ctgtggacca tcagcaccat tccctatcta 11580
tacctggctg cttatgagag cggagtaagg attgcttcgt tagtgcaagg ggacaatcag 11640
accatagccg taacaaaaag ggtacccagc acatggccct acaaccttaa gaaacgggaa 11700
gctgctagag taactagaga ttactttgta attcttaggc aaaggctaca tgatattggc 11760
catcacctca aggcaaatga gacaattgtt tcatcacatt tttttgtcta ttcaaaagga 11820
atatattatg atgggctact tgtgtcccaa tcactcaaga gcatcgcaag atgtgtattc 11880
tggtcagaga ctatagttga tgaaacaagg gcagcatgca gtaatattgc tacaacaatg 11940
gctaaaagca tcgagagagg ttatgaccgt taccttgcat attccctgaa cgtcctaaaa 12000
gtgatacagc aaattctgat ctctcttggc ttcacaatca attcaaccat gacccgggat 12060
gtagtcatac ccctcctcac aaacaacgac ctcttaataa ggatggcact gttgcccgct 12120
cctattgggg ggatgaatta tctgaatatg agcaggctgt ttgtcagaaa catcggtgat 12180
ccagtaacat catcaattgc tgatctcaag agaatgattc tcgcctcact aatgcctgaa 12240
gagaccctcc atcaagtaat gacacaacaa ccgggggact cttcattcct agactgggct 12300
agcgaccctt actcagcaaa tcttgtatgt gtccagagca tcactagact cctcaagaac 12360
ataactgcaa ggtttgtcct gatccatagt ccaaacccaa tgttaaaagg attattccat 12420
gatgacagta aagaagagga cgagggactg gcggcattcc tcatggacag gcatattata 12480
gtacctaggg cagctcatga aatcctggat catagtgtca caggggcaag agagtctatt 12540
gcaggcatgc tggataccac aaaaggcttg attcgagcca gcatgaggaa gggggggtta 12600
acctctcgag tgataaccag attgtccaat tatgactatg aacaattcag agcagggatg 12660
gtgctattga caggaagaaa gagaaatgtc ctcattgaca aagagtcatg ttcagtgcag 12720
ctggcgagag ctctaagaag ccatatgtgg gcgaggctag ctcgaggacg gcctatttac 12780
ggccttgagg tccctgatgt actagaatct atgcgaggcc accttattcg gcgtcatgag 12840
acatgtgtca tctgcgagtg tggatcagtc aactacggat ggttttttgt cccctcgggt 12900
tgccaactgg atgatattga caaggaaaca tcatccttga gagtcccata tattggttct 12960
accactgatg agagaacaga catgaagctt gccttcgtaa gagccccaag tcgatccttg 13020
cgatctgctg ttagaatagc aacagtgtac tcatgggctt acggtgatga tgatagctct 13080
tggaacgaag cctggttgtt ggctaggcaa agggccaatg tgagcctgga ggagctaagg 13140
gtgatcactc ccatctcaac ttcgactaat ttagcgcata ggttgaggga tcgtagcact 13200
caagtgaaat actcaggtac atcccttgtc cgagtggcga ggtataccac aatctccaac 13260
gacaatctct catttgtcat atcagataag aaggttgata ctaactttat ataccaacaa 13320
ggaatgcttc tagggttggg tgttttagaa acattgtttc gactcgagaa agataccgga 13380
tcatctaaca cggtattaca tcttcacgtc gaaacagatt gttgcgtgat cccgatgata 13440
gatcatccca ggatacccag ctcccgcaag ctagagctga gggcagagct atgtaccaac 13500
ccattgatat atgataatgc acctttaatt gacagagatg caacaaggct atacacccag 13560
agccatagga ggcaccttgt ggaatttgtt acatggtcca caccccaact atatcacatt 13620
ttagctaagt ccacagcact atctatgatt gacctggtaa caaaatttga gaaggaccat 13680
atgaatgaaa tttcagctct cataggggat gacgatatca atagtttcat aactgagttt 13740
ctgctcatag agccaagatt attcactatc tacttgggcc agtgtgcggc catcaattgg 13800
gcatttgatg tacattatca tagaccatca gggaaatatc agatgggtga gctgttgtca 13860
tcgttccttt ctagaatgag caaaggagtg tttaaggtgc ttgtcaatgc tctaagccac 13920
ccaaagatct acaagaaatt ctggcattgt ggtattatag agcctatcca tggtccttca 13980
cttgatgctc aaaacttgca cacaactgtg tgcaacatgg tttacacatg ctatatgacc 14040
tacctcgacc tgttgttgaa tgaagagtta gaagagttca catttctctt gtgtgaaagc 14100
gacgaggatg tagtaccgga cagattcgac aacatccagg caaaacactt atgtgttctg 14160
gcagatttgt actgtcaacc agggacctgc ccaccaattc gaggtctaag accggtagag 14220
aaatgtgcag ttctaaccga ccatatcaag gcagaggcta tgttatctcc agcaggatct 14280
tcgtggaaca taaatccaat tattgtagac cattactcat gctctctgac ttatctccgg 14340
cgaggatcga tcaaacagat aagattgaga gttgatccag gattcatttt cgacgccctc 14400
gctgaggtaa atgtcagtca gccaaagatc ggcagcaaca acatctcaaa tatgagcatc 14460
aaggctttca gacccccaca cgatgatgtt gcaaaattgc tcaaagatat caacacaagc 14520
aagcacaatc ttcccatttc agggggcaat ctcgccaatt atgaaatcca tgctttccgc 14580
agaatcgggt tgaactcatc tgcttgctac aaagctgttg agatatcaac attaattagg 14640
agatgccttg agccagggga ggacggcttg ttcttgggtg agggatcggg ttctatgttg 14700
atcacttata aagagatact taaactaaac aagtgcttct ataatagtgg ggtttccgcc 14760
aattctagat ctggtcaaag ggaattagca ccctatccct ccgaagttgg ccttgtcgaa 14820
cacagaatgg gagtaggtaa tattgtcaaa gtgctcttta acgggaggcc cgaagtcacg 14880
tgggtaggca gtgtagattg cttcaatttc atagttagta atatccctac ctctagtgtg 14940
gggtttatcc attcagatat agagaccttg cctgacaaag atactataga gaagctagag 15000
gaattggcag ccatcttatc gatggctctg ctcctgggca aaataggatc aatactggtg 15060
attaagctta tgcctttcag cggggatttt gttcagggat ttataagtta tgtagggtct 15120
cattatagag aagtgaacct tgtataccct agatacagca acttcatctc tactgaatct 15180
tatttggtta tgacagatct caaggctaac cggctaatga atcctgaaaa gattaagcag 15240
cagataattg aatcatctgt gaggacttca cctggactta taggtcacat cctatccatt 15300
aagcaactaa gctgcataca agcaattgtg ggagacgcag ttagtagagg tgatatcaat 15360
cctactctga aaaaacttac acctatagag caggtgctga tcaattgcgg gttggcaatt 15420
aacggaccta agctgtgcaa agaattgatc caccatgatg ttgcctcagg gcaagatgga 15480
ttgcttaatt ctatactcat cctctacagg gagttggcaa gattcaaaga caaccaaaga 15540
agtcaacaag ggatgttcca cgcttacccc gtattggtaa gtagcaggca acgagaactt 15600
atatctagga tcacccgcaa attctggggg cacattcttc tttactccgg gaacaaaaag 15660
ttgataaata agtttatcca gaatctcaag tccggctatc tgatactaga cttacaccag 15720
aatatcttcg ttaagaatct atccaagtca gagaaacaga ttattatgac ggggggtttg 15780
aaacgtgagt gggtttttaa ggtaacagtc aaggagacca aagaatggta taagttagtc 15840
ggatacagtg ccctgattaa ggactaattg gttgaactcc ggaaccctaa tcctgcccta 15900
ggtggttagg cattatttgc aatatattaa agaaaacttt gaaaatacga agtttctatt 15960
cccagctttg tctggtggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac 16020
attccgaggg gaccgtcccc tcggtaatgg cgaatgggac gcggccgatc cggctgctaa 16080
caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc 16140
ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg 16200
atgcggccgc gggccctatg gtacccagct tttgttccct ttagtgaggg ttaattccga 16260
gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 16320
cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgaggt 16380
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 16440
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 16500
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 16560
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 16620
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 16680
tccataggct cggcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 16740
gaaacccgac aggactataa agataccagg cgttcccccc tggaagctcc ctcgtgcgct 16800
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 16860
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 16920
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 16980
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 17040
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 17100
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 17160
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 17220
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 17280
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 17340
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 17400
caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 17460
cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactg cccgtcgtgt 17520
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 17580
acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 17640
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 17700
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 17760
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 17820
ggcgagttac atgatccccc atgttgtgaa aaaaagcggt tagctccttc ggtcctccga 17880
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat gcttatggca gcactgcata 17940
attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 18000
agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 18060
ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 18120
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 18180
cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 18240
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 18300
tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 18360
tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 18420
tgccacctga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 18480
tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 18540
agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 18600
tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 18660
catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 18720
aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 18780
ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 18840
taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca 18900
ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccac 18960
cgcggtg 18967
<210>2
<211>12082
<212>DNA
<213>人工序列
<220>
<223>质粒pEMC-LSchw
<400>2
aagcttttgc gatcaataaa tggatcacaa ccagtatctc ttaacgatgt tcttcgcaga 60
tgatgattca ttttttaagt atttggctag tcaagatgat gaatcttcat tatctgatat 120
attgcaaatc actcaatatc tagactttct gttattatta ttgatccaat caaaaaataa 180
attagaagcc gtgggtcatt gttatgaatc tctttcagag gaatacagac aattgacaaa 240
attcacagac tttcaagatt ttaaaaaact gtttaacaag gtccctattg ttacagatgg 300
aagggtcaaa cttaataaag gatatttgtt cgactttgtg attagtttga tgcgattcaa 360
aaaagaatcc tctctagcta ccaccgcaat agatcctgtt agatacatag atcctcgtcg 420
caatatcgca ttttctaacg tgatggatat attaaagtcg aataaagtga acaataatta 480
attctttatt gtcatcatga acggcggaca tattcagttg ataatcggcc ccatgttttc 540
aggtaaaagt acagaattaa ttagacgagt tagacgttat caaatagctc aatataaatg 600
cgtgactata aaatattcta acgataatag atacggaacg ggactatgga cgcatgataa 660
gaataatttt gaagcattgg aagcaactaa actatgtgat gtcttggaat caattacaga 720
tttctccgtg ataggtatcg atgaaggaca gttctttcca gacattgttg aattgatctc 780
gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagcggg 840
atcaattccg cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat 900
aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg 960
tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc 1020
tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt 1080
cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg 1140
acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac 1200
cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg 1260
tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg 1320
ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc 1380
gaaccacggg gacgtggttt tcctttgaaa aacacgataa taccatggac tcgctatctg 1440
tcaaccagat cttataccct gaagttcacc tagatagccc gatagttacc aataagatag 1500
tagccatcct ggagtatgct cgagtccctc acgcttacag cctggaggac cctacactgt 1560
gtcagaacat caagcaccgc ctaaaaaacg gattttccaa ccaaatgatt ataaacaatg 1620
tggaagttgg gaatgtcatc aagtccaagc ttaggagtta tccggcccac tctcatattc 1680
catatccaaa ttgtaatcag gatttattta acatagaaga caaagagtca acgaggaaga 1740
tccgtgaact cctcaaaaag gggaattcgc tgtactccaa agtcagtgat aaggttttcc 1800
aatgcttaag ggacactaac tcacggcttg gcctaggctc cgaattgagg gaggacatca 1860
aggagaaagt tattaacttg ggagtttaca tgcacagctc ccagtggttt gagccctttc 1920
tgttttggtt tacagtcaag actgagatga ggtcagtgat taaatcacaa acccatactt 1980
gccataggag gagacacaca cctgtattct tcactggtag ttcagttgag ttgctaatct 2040
ctcgtgacct tgttgctata atcagtaaag agtctcaaca tgtatattac ctgacatttg 2100
aactggtttt gatgtattgt gatgtcatag aggggaggtt aatgacagag accgctatga 2160
ctattgatgc taggtataca gagcttctag gaagagtcag atacatgtgg aaactgatag 2220
atggtttctt ccctgcactc gggaatccaa cttatcaaat tgtagccatg ctggagcctc 2280
tttcacttgc ttacctgcag ctgagggata taacagtaga actcagaggt gctttcctta 2340
accactgctt tactgaaata catgatgttc ttgaccaaaa cgggttttct gatgaaggta 2400
cttatcatga gttaactgaa gctctagatt acattttcat aactgatgac atacatctga 2460
caggggagat tttctcattt ttcagaagtt tcggccaccc cagacttgaa gcagtaacgg 2520
ctgctgaaaa tgttaggaaa tacatgaatc agcctaaagt cattgtgtat gagactctga 2580
tgaaaggtca tgccatattt tgtggaatca taatcaacgg ctatcgtgac aggcacggag 2640
gcagttggcc accgctgacc ctccccctgc atgctgcaga cacaatccgg aatgctcaag 2700
cttcaggtga agggttaaca catgagcagt gcgttgataa ctggaaatct tttgctggag 2760
tgaaatttgg ctgctttatg cctcttagcc tggatagtga tctgacaatg tacctaaagg 2820
acaaggcact tgctgctctc caaagggaat gggattcagt ttacccgaaa gagttcctgc 2880
gttacgaccc tcccaaggga accgggtcac ggaggcttgt agatgttttc cttaatgatt 2940
cgagctttga cccatatgat gtgataatgt atgttgtaag tggagcttac ctccatgacc 3000
ctgagttcaa cctgtcttac agcctgaaag aaaaggagat caaggaaaca ggtagacttt 3060
ttgctaaaat gacttacaaa atgagggcat gccaagtgat tgctgaaaat ctaatctcaa 3120
acgggattgg caaatatttt aaggacaatg ggatggccaa ggatgagcac gatttgacta 3180
aggcactcca cactctagct gtctcaggag tccccaaaga tctcaaagaa agtcacaggg 3240
gggggccagt cttaaaaacc tactcccgaa gcccagtcca cacaagtacc aggaacgtga 3300
gagcagcaaa agggtttata gggttccctc aagtaattcg gcaggaccaa gacactgatc 3360
atccggagaa tatggaagct tacgagacag tcagtgcatt tatcacgact gatctcaaga 3420
agtactgcct taattggaga tatgagacca tcagcttgtt tgcacagagg ctaaatgaga 3480
tttacggatt gccctcattt ttccagtggc tgcataagag gcttgagacc tctgtcctgt 3540
atgtaagtga ccctcattgc ccccccgacc ttgacgccca tatcccgtta tataaagtcc 3600
ccaatgatca aatcttcatt aagtacccta tgggaggtat agaagggtat tgtcagaagc 3660
tgtggaccat cagcaccatt ccctatctat acctggctgc ttatgagagc ggagtaagga 3720
ttgcttcgtt agtgcaaggg gacaatcaga ccatagccgt aacaaaaagg gtacccagca 3780
catggcccta caaccttaag aaacgggaag ctgctagagt aactagagat tactttgtaa 3840
ttcttaggca aaggctacat gatattggcc atcacctcaa ggcaaatgag acaattgttt 3900
catcacattt ttttgtctat tcaaaaggaa tatattatga tgggctactt gtgtcccaat 3960
cactcaagag catcgcaaga tgtgtattct ggtcagagac tatagttgat gaaacaaggg 4020
cagcatgcag taatattgct acaacaatgg ctaaaagcat cgagagaggt tatgaccgtt 4080
accttgcata ttccctgaac gtcctaaaag tgatacagca aattctgatc tctcttggct 4140
tcacaatcaa ttcaaccatg acccgggatg tagtcatacc cctcctcaca aacaacgacc 4200
tcttaataag gatggcactg ttgcccgctc ctattggggg gatgaattat ctgaatatga 4260
gcaggctgtt tgtcagaaac atcggtgatc cagtaacatc atcaattgct gatctcaaga 4320
gaatgattct cgcctcacta atgcctgaag agaccctcca tcaagtaatg acacaacaac 4380
cgggggactc ttcattccta gactgggcta gcgaccctta ctcagcaaat cttgtatgtg 4440
tccagagcat cactagactc ctcaagaaca taactgcaag gtttgtcctg atccatagtc 4500
caaacccaat gttaaaagga ttattccatg atgacagtaa agaagaggac gagggactgg 4560
cggcattcct catggacagg catattatag tacctagggc agctcatgaa atcctggatc 4620
atagtgtcac aggggcaaga gagtctattg caggcatgct ggataccaca aaaggcttga 4680
ttcgagccag catgaggaag ggggggttaa cctctcgagt gataaccaga ttgtccaatt 4740
atgactatga acaattcaga gcagggatgg tgctattgac aggaagaaag agaaatgtcc 4800
tcattgacaa agagtcatgt tcagtgcagc tggcgagagc tctaagaagc catatgtggg 4860
cgaggctagc tcgaggacgg cctatttacg gccttgaggt ccctgatgta ctagaatcta 4920
tgcgaggcca ccttattcgg cgtcatgaga catgtgtcat ctgcgagtgt ggatcagtca 4980
actacggatg gttttttgtc ccctcgggtt gccaactgga tgatattgac aaggaaacat 5040
catccttgag agtcccatat attggttcta ccactgatga gagaacagac atgaagcttg 5100
ccttcgtaag agccccaagt cgatccttgc gatctgctgt tagaatagca acagtgtact 5160
catgggctta cggtgatgat gatagctctt ggaacgaagc ctggttgttg gctaggcaaa 5220
gggccaatgt gagcctggag gagctaaggg tgatcactcc catctcaact tcgactaatt 5280
tagcgcatag gttgagggat cgtagcactc aagtgaaata ctcaggtaca tcccttgtcc 5340
gagtggcgag gtataccaca atctccaacg acaatctctc atttgtcata tcagataaga 5400
aggttgatac taactttata taccaacaag gaatgcttct agggttgggt gttttagaaa 5460
cattgtttcg actcgagaaa gataccggat catctaacac ggtattacat cttcacgtcg 5520
aaacagattg ttgcgtgatc ccgatgatag atcatcccag gatacccagc tcccgcaagc 5580
tagagctgag ggcagagcta tgtaccaacc cattgatata tgataatgca cctttaattg 5640
acagagatgc aacaaggcta tacacccaga gccataggag gcaccttgtg gaatttgtta 5700
catggtccac accccaacta tatcacattt tagctaagtc cacagcacta tctatgattg 5760
acctggtaac aaaatttgag aaggaccata tgaatgaaat ttcagctctc ataggggatg 5820
acgatatcaa tagtttcata actgagtttc tgctcataga gccaagatta ttcactatct 5880
acttgggcca gtgtgcggcc atcaattggg catttgatgt acattatcat agaccatcag 5940
ggaaatatca gatgggtgag ctgttgtcat cgttcctttc tagaatgagc aaaggagtgt 6000
ttaaggtgct tgtcaatgct ctaagccacc caaagatcta caagaaattc tggcattgtg 6060
gtattataga gcctatccat ggtccttcac ttgatgctca aaacttgcac acaactgtgt 6120
gcaacatggt ttacacatgc tatatgacct acctcgacct gttgttgaat gaagagttag 6180
aagagttcac atttctcttg tgtgaaagcg acgaggatgt agtaccggac agattcgaca 6240
acatccaggc aaaacactta tgtgttctgg cagatttgta ctgtcaacca gggacctgcc 6300
caccaattcg aggtctaaga ccggtagaga aatgtgcagt tctaaccgac catatcaagg 6360
cagaggctat gttatctcca gcaggatctt cgtggaacat aaatccaatt attgtagacc 6420
attactcatg ctctctgact tatctccggc gaggatcgat caaacagata agattgagag 6480
ttgatccagg attcattttc gacgccctcg ctgaggtaaa tgtcagtcag ccaaagatcg 6540
gcagcaacaa catctcaaat atgagcatca aggctttcag acccccacac gatgatgttg 6600
caaaattgct caaagatatc aacacaagca agcacaatct tcccatttca gggggcaatc 6660
tcgccaatta tgaaatccat gctttccgca gaatcgggtt gaactcatct gcttgctaca 6720
aagctgttga gatatcaaca ttaattagga gatgccttga gccaggggag gacggcttgt 6780
tcttgggtga gggatcgggt tctatgttga tcacttataa agagatactt aaactaaaca 6840
agtgcttcta taatagtggg gtttccgcca attctagatc tggtcaaagg gaattagcac 6900
cctatccctc cgaagttggc cttgtcgaac acagaatggg agtaggtaat attgtcaaag 6960
tgctctttaa cgggaggccc gaagtcacgt gggtaggcag tgtagattgc ttcaatttca 7020
tagttagtaa tatccctacc tctagtgtgg ggtttatcca ttcagatata gagaccttgc 7080
ctgacaaaga tactatagag aagctagagg aattggcagc catcttatcg atggctctgc 7140
tcctgggcaa aataggatca atactggtga ttaagcttat gcctttcagc ggggattttg 7200
ttcagggatt tataagttat gtagggtctc attatagaga agtgaacctt gtatacccta 7260
gatacagcaa cttcatctct actgaatctt atttggttat gacagatctc aaggctaacc 7320
ggctaatgaa tcctgaaaag attaagcagc agataattga atcatctgtg aggacttcac 7380
ctggacttat aggtcacatc ctatccatta agcaactaag ctgcatacaa gcaattgtgg 7440
gagacgcagt tagtagaggt gatatcaatc ctactctgaa aaaacttaca cctatagagc 7500
aggtgctgat caattgcggg ttggcaatta acggacctaa gctgtgcaaa gaattgatcc 7560
accatgatgt tgcctcaggg caagatggat tgcttaattc tatactcatc ctctacaggg 7620
agttggcaag attcaaagac aaccaaagaa gtcaacaagg gatgttccac gcttaccccg 7680
tattggtaag tagcaggcaa cgagaactta tatctaggat cacccgcaaa ttctgggggc 7740
acattcttct ttactccggg aacaaaaagt tgataaataa gtttatccag aatctcaagt 7800
ccggctatct gatactagac ttacaccaga atatcttcgt taagaatcta tccaagtcag 7860
agaaacagat tattatgacg gggggtttga aacgtgagtg ggtttttaag gtaacagtca 7920
aggagaccaa agaatggtat aagttagtcg gatacagtgc cctgattaag gactaattgg 7980
ttgaactccg gaaccctaat cctgccctag gtggttaggc attatttacc tcgagggggc 8040
cggatccact agttctagaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 8100
aaaaaaaaaa aaaaaaaaaa acgtcgcgca ggtgacaatg tcgagctagc tatgaattcc 8160
ccggggagct cactagtgga tccctgcagc tcgagaggcc taattaatta agtcgacgat 8220
ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa 8280
ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga 8340
actatatccg gatcgagatc aattctgtga gcgtatggca aacgaaggaa aaatagttat 8400
agtagccgca ctcgatggga catttcaacg taaaccgttt aataatattt tgaatcttat 8460
tccattatct gaaatggtgg taaaactaac tgctgtgtgt atgaaatgct ttaaggaggc 8520
ttccttttct aaacgattgg gtgaggaaac cgagatagaa ataataggag gtaatgatat 8580
gtatcaatcg gtgtgtagaa agtgttacat cgactcataa tattatattt tttatctaaa 8640
aaactaaaaa taaacattga ttaaatttta atataatact taaaaatgga tgttgtgtcg 8700
ttagataaac cgtttatgta ttttgaggaa attgataatg agttagatta cgaaccagaa 8760
agtgcaaatg aggtcgcaaa aaaactgccg tatcaaggac agttaaaact attactagga 8820
gaattatttt ttcttagtaa gttacagcga cacggtatat tagatggtgc caccgtagtg 8880
tatataggat ctgctcccgg tacacatata cgttatttga gagatcattt ctataattta 8940
ggagtgatca tcaaatggat gctaattgac ggccgccatc atgatcctat tttaaatgga 9000
ttgcgtgatg tgactctagt gactcggttc gttgatgagg aatatctacg atccatcaaa 9060
aaacaactgc atccttctaa gattatttta atttctgatg tgagatccaa acgaggagga 9120
aatgaaccta gtacggcgga tttactaagt aattacgctc tacaaaatgt catgattagt 9180
attttaaacc ccgtggcgtc tagtcttaaa tggagatgcc cgtttccaga tcaatggatc 9240
aaggactttt atatcccaca cggtaataaa atgttacaac cttttgctcc ttcatattca 9300
gggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 9360
ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 9420
cttcccaaca gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt agcggcgcat 9480
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 9540
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 9600
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 9660
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 9720
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 9780
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 9840
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 9900
taacgtttac aatttcccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 9960
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 10020
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 10080
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 10140
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 10200
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 10260
agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 10320
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 10380
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 10440
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 10500
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 10560
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 10620
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 10680
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 10740
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 10800
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 10860
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 10920
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 10980
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 11040
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 11100
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 11160
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 11220
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 11280
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 11340
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 11400
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 11460
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 11520
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 11580
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 11640
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 11700
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 11760
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 11820
cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 11880
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 11940
tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 12000
tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 12060
cagctatgac catgattacg cc 12082
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cgtcatactc ctgcttgctg atccacatct gc 32
<210>5
<211>25
<212>DNA
<213>人工序列
<220>
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<400>5
attgggtctg ggaacatttc tcttc 25
<210>6
<211>25
<212>DNA
<213>人工序列
<220>
<223>引物
<400>6
gtgagattta aacattcctc ttcgg 25
<210>7
<211>20
<212>DNA
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<220>
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<400>7
gagcaacttc tttcaaccac 20
<210>8
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<400>8
gaacaccagc atcttctcca 20
<210>9
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<400>9
gaacgattcc atcactatcc 20
<210>10
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<400>10
ggcatcatta tatttccgca 20
<210>11
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cttctcagca gatacatcag 20
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<400>12
gttacaagtc caaagtctcc 20
<210>13
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<223>MSLN 531-539表位
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Val Leu Pro Leu Thr Val Ala Glu Val
1 5
<210>14
<211>10
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<220>
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Lys Leu Leu Gly Pro His Val Glu Gly Leu
1 5 10
Claims (15)
1.减毒的麻疹病毒用于制备治疗个体的恶性间皮瘤的药物的用途。
2.权利要求1的用途，其中所述减毒的麻疹病毒是埃德蒙顿(Edmonston)株。
3.权利要求1的用途，其中所述减毒的麻疹病毒选自：施瓦茨(Schwartz)株和Moraten株。
4.权利要求1的用途，其中所述恶性间皮瘤是恶性胸膜间皮瘤。
5.权利要求1的用途，其中所述个体已暴露于石棉。
6.制备用于治疗个体的恶性间皮瘤的疫苗性树突细胞的方法，包括下列步骤：
●用减毒的麻疹株体外感染取自所述个体的恶性间皮瘤细胞，以产生细胞裂解物；
●使树突细胞与所述细胞裂解物接触，以产生疫苗性树突细胞。
7.权利要求6的方法，其中所述树突细胞源于所述个体。
8.权利要求6或7的方法，其中所述树突细胞是单核细胞衍生的树突细胞。
9.权利要求6的方法，其中所述恶性间皮瘤是恶性胸膜间皮瘤。
10.权利要求6的方法，其中所述减毒的麻疹病毒是埃德蒙顿株。
11.权利要求6的方法，其中所述减毒的麻疹病毒选自：施瓦茨株和Moraten株。
12.疫苗性树突细胞，其根据权利要求6～11中任一项的方法得到。
13.药物组合物，其包含：
●根据权利要求6～11中任一项的方法得到的疫苗性树突细胞作为活性成分；及
●药学可接受的载体。
14.疫苗性树突细胞用于制备治疗个体的恶性间皮瘤的药物的用途，所述疫苗性树突细胞根据权利要求6～11中任一项的方法得到。
15.权利要求14的用途，其中所述恶性间皮瘤是恶性胸膜间皮瘤。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP07291232A EP2058003A1 (en) | 2007-10-10 | 2007-10-10 | Medicaments and methods for treating mesothelioma |
| EP07291232.2 | 2007-10-10 | ||
| PCT/EP2008/063626 WO2009047331A1 (en) | 2007-10-10 | 2008-10-10 | Medicaments and methods for treating mesothelioma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101868249A CN101868249A (zh) | 2010-10-20 |
| CN101868249B true CN101868249B (zh) | 2013-05-29 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200880116771XA Active CN101868249B (zh) | 2007-10-10 | 2008-10-10 | 治疗间皮瘤的药物和方法 |
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| Country | Link |
|---|---|
| US (2) | US9023643B2 (zh) |
| EP (2) | EP2058003A1 (zh) |
| CN (1) | CN101868249B (zh) |
| CA (1) | CA2702186C (zh) |
| DK (1) | DK2203183T3 (zh) |
| ES (1) | ES2550938T3 (zh) |
| WO (1) | WO2009047331A1 (zh) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2432738A1 (en) * | 2003-02-26 | 2004-08-26 | Philippe Despres | New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications |
| EP2058003A1 (en) | 2007-10-10 | 2009-05-13 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Medicaments and methods for treating mesothelioma |
| EP2759301A1 (en) * | 2013-01-24 | 2014-07-30 | Institut Pasteur | Use of a genetically modified infectious measles virus with enhanced pro-apoptotic properties (MV-DeltaC virus) |
| CN107085095A (zh) * | 2017-03-02 | 2017-08-22 | 江苏华冠生物技术股份有限公司 | 用作酶联试剂盒包被抗原的麻疹病毒裂解液的制备方法 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7118740B1 (en) * | 2000-09-22 | 2006-10-10 | Mayo Foundation For Medical Education And Research | Method for limiting the growth of cancer cells using an attenuated measles virus |
| EP2058003A1 (en) | 2007-10-10 | 2009-05-13 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Medicaments and methods for treating mesothelioma |
-
2007
- 2007-10-10 EP EP07291232A patent/EP2058003A1/en not_active Withdrawn
-
2008
- 2008-10-10 WO PCT/EP2008/063626 patent/WO2009047331A1/en active Application Filing
- 2008-10-10 CA CA2702186A patent/CA2702186C/en active Active
- 2008-10-10 US US12/682,457 patent/US9023643B2/en active Active
- 2008-10-10 DK DK08805222.0T patent/DK2203183T3/en active
- 2008-10-10 EP EP08805222.0A patent/EP2203183B1/en active Active
- 2008-10-10 ES ES08805222.0T patent/ES2550938T3/es active Active
- 2008-10-10 CN CN200880116771XA patent/CN101868249B/zh active Active
-
2015
- 2015-04-08 US US14/681,362 patent/US9636395B2/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Anne Gauvrit et al.measles virus induces oncolysis of mesothelioma cells and allows dendritic cells to cross-prime tumor specific cd8 response.《Cancer Research》.2008,第68卷(第12期),全文. * |
| Hegmans JP et al.Immunotherapy of murine malignant mesothelioma using tumor lysate-pulsed dendritic cells.《American Journal of Respiratory and Critical Care Medicine》.2005,第171卷(第10期),1167-1177. * |
| Hilleman MR et al.Development and Evaluation of the Moraten Measles Virus Vaccine.《JAMA》.1968,第206卷(第3期),摘要. * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2550938T3 (es) | 2015-11-13 |
| CA2702186A1 (en) | 2009-04-16 |
| EP2203183A1 (en) | 2010-07-07 |
| US20150209423A1 (en) | 2015-07-30 |
| CN101868249A (zh) | 2010-10-20 |
| US9636395B2 (en) | 2017-05-02 |
| DK2203183T3 (en) | 2015-11-09 |
| US9023643B2 (en) | 2015-05-05 |
| EP2058003A1 (en) | 2009-05-13 |
| WO2009047331A1 (en) | 2009-04-16 |
| US20100278872A1 (en) | 2010-11-04 |
| CA2702186C (en) | 2019-05-28 |
| EP2203183B1 (en) | 2015-08-19 |
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