CN102844329B - 分离自猿腺病毒血清型19的六邻体、其高变区和使用其的嵌合型腺病毒 - Google Patents
分离自猿腺病毒血清型19的六邻体、其高变区和使用其的嵌合型腺病毒 Download PDFInfo
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Abstract
本发明涉及分离自由SEQ?ID?NO:3所示多核苷酸编码的猿腺病毒血清型19的新六邻体、其高变区、含有其的嵌合型腺病毒,及其治疗上的用途,提供了解决使用腺病毒开发基因治疗剂进行安全和有效地系统治疗的问题的方案。
Description
技术领域
本发明涉及分离自猿腺病毒血清型19(“SAd19”)的新六邻体,其高变区(“HVR”),使用其的嵌合型腺病毒及其治疗用途。
背景技术
腺病毒属于腺病毒科(Adenoviridae),其于1953年首次分离。根据基因组的相似性、致癌性和血液凝固特性,将人腺病毒分为六(6)个亚属(A至F)。腺病毒感染大多数非分裂细胞如肌肉细胞、肺细胞、脑细胞和心脏细胞,其分子生物学特征为本领域所熟知。腺病毒基因组由35kb的线性、双链DNA组成,其在宿主细胞中的复制依赖于病毒蛋白E1A。
通过使用将其E1A删除后的非复制性载体,可对上述腺病毒的特征加以利用。由于插入有腺病毒E1基因的HEK293细胞系的研制,腺病毒载体系统已经应用于许多研究中,其导致了利用宿主细胞的细胞毒性的病毒疗法的发展。2005年在中国商业化的融瘤病毒治疗剂安柯瑞是E1B55-缺失型腺病毒,其用于在p53-缺失型肿瘤中选择性诱导凋亡性细胞死亡。
在开发选择性复制的腺病毒治疗剂的过程中,最重要的是E1A蛋白的选择性表达,已经有很多案例表明使用肿瘤选择性表达启动子与可能的肿瘤-选择性腺病毒基因治疗剂有关。大多数肿瘤-选择性腺病毒是使用通常发现的人腺病毒血清型5(“HAd5”)制备的。据报道,由于HAd5占80%的普遍性,因此人具有高水平的腺病毒中和抗体(Appaiahgari,M.B.等人,(2007)临床及疫苗免疫学(ClinicalandVaccineImmunol.)14,1053-1055)。针对腺病毒衣壳蛋白的这些中和抗体影响腺病毒系统用药的药效和毒性(Chen,Y.等人,(2000)人类基因治疗(Hum.GeneTher.)11,1553-1567)。
病毒衣壳由三(3)种蛋白即六邻体、纤毛和五邻体组成,其包括具有由240个六邻体和12个五邻体组成的对称二十面体结构的壳粒(capsomere)。每个五邻体均结合突出的70~100nm的三聚体纤毛。当被腺病毒感染时,三聚体纤毛在腺病毒感染过程中附着于宿主细胞表面膜上的柯萨奇腺病毒受体(CAR)。五邻体的RGD区结合至整合蛋白,其导致病毒吸附并侵入至宿主细胞内。
据报道,六邻体蛋白的环1(L1)和环2(L2)暴露于病毒壳粒结构的外部。L1和L2分别含有六邻体蛋白的六(6)个高变区(HVRs),即在第132至320个氨基酸内的HVR-1至HVR-6,以及第408至459个氨基酸内的第7个HVR(HVR-7)。
腺病毒提供了优良且有效的将治疗基因转入细胞的方式。然而,使用腺病毒载体进行体内基因转移的一个问题是会产生针对腺病毒衣壳蛋白上抗原表位的抗体。
当腺病毒施用于人体时,形成了针对六邻体蛋白的中和抗体,该抗体主要靶向显性HVR区。也已知该抗体通过抑制宿主细胞感染而降低病毒复制效率(Wohlfart,C.(1988)J.Virol.62,2321-2328;Toogood,C.I.A.等人,(1992)J.Gen.Virol.73,1429-1435;Sumida,S.M.等人,(2005)J.Immunol.174,7179-7185)。
当使用病毒基因递送载体和病毒治疗剂而施用腺病毒时,必须解决由针对人HAd5的人中和抗体占多数导致的问题。除了上述与中和抗体相关的问题,也有报道说当全身接触时腺病毒感染肝脏。在这方面,有报道说腺病毒具有嗜肝性,当通过静脉途径施用腺病毒时,24小时内其90%转移至肝脏中(Worgall,S.等人,(1997)人类基因治疗(Hum.GeneTher.)8,37-44)。由于腺病毒的这种肝选择性,1999年年轻患者JessieGelsinger在施用腺病毒基因治疗剂的临床试验中发生了急性肝中毒。此后,腺病毒剂量一直被限制在不超过1×1013vp。因此,通常肝中毒被认为是使用腺病毒的许多基因治疗剂进行非临床/临床试验的剂量限制因素(Alemany,R.等人(2001)基因治疗(GeneTher.),8(17),1347-1353;Christ,M.等人,(2000)人类基因治疗(Hum.GeneTher.),11(3),415-427;Lieber,A.等人,(1997)J.Virol.,7(11),8798-8807)。这种肝选择性是腺病毒治疗剂进行系统用药时实现有效治愈的主要问题(Worgall,S.等人,(1997)人类基因治疗(Hum.GeneTher.),8,37-44)。
在这方面,Waddington等人近期报道,Gla域、凝血因子在血液中结合腺病毒六邻体蛋白,其促进了腺病毒转移至肝脏(Waddington,S.N.等人,(2008)细胞(Cell),132,397-409)。据推测六邻体的HVR-3、HVR-5或HVR-7可与凝血因子、Gla域结合(Kalyuzhniy,O.等人,(2008)Proc.Nat’lAcd.Sci.105,5483-5488)。HVR根据腺病毒的血清型而变化,目前还不清楚对于凝血因子的结合亲和力的关键因素是什么。据报道,通过插入特定的蛋白如RGD、RFP和BAP(生物素受体肽)以大大消弱对于凝血因子的结合亲和力从而降低嗜肝性,腺病毒的最大耐受剂量可提高10倍(Shashkova,E.V.等人,(2009)Mol.Ther.17,2121-2130)。
目前已知了六邻体蛋白的许多功能,当前进行了许多修饰六邻体蛋白的研究,以克服肝中毒和抗腺病毒免疫力的问题。有4种修饰六邻体蛋白的策略:1)用其他腺病毒血清型的对应六邻体基因代替所述六邻体基因,2)将肽插入至HVR中,3)用编码其他腺病毒血清型HVR的对应基因代替编码六邻体蛋白HVR的基因,以及4)将结合凝血因子与中和抗体的区域从HVR中去除。至今,对六邻体修饰使用的是将肽插入至HVR中的方法,以及用编码其他腺病毒血清型HVR的对应基因代替编码HVR的基因的方法。在上述4种策略中,完全的六邻体置换是改变病毒免疫原性的最显而易见的方法。然而,由于结合六邻体与五邻体和纤毛的微小结构差异会导致腺病毒衣壳结构不稳定,因此将完全的六邻体替换为修饰的六邻体蛋白的方法所带来的问题是产能退化(Roberts,D.M.等人,(2006)自然(Nature),441,p239-243;Youil,R.等人,(2002)人类基因治疗(Hum.GeneTher.)13,p311-320;Shashkova,E.等人,(2009)Mol.Ther.17,2121-2130)。
而且,使用异源腺病毒和人腺病毒血清型对衣壳蛋白的修饰也在深入的研究中。据报道,针对黑猩猩腺病毒pan5、6、7和9(分别划分为猿腺病毒血清型22至25)的中和抗体的发生率小于6%,因此,包括黑猩猩腺病毒的猿腺病毒载体系统可用作基因治疗载体(Roy,S.等人(2004)人类基因治疗(Hum.GeneTher.)15,p519-530)。国际专利申请No.WO2006/040330和No.WO2002/083902教导了使用人血清型11、24、26、30、34、35、48、49和50的纤毛或六邻体蛋白抑制重组嵌合型腺病毒中由中和抗体引起的免疫应答,其中用其他血清型的对应区域代替结合至CAR或六邻体蛋白的腺病毒突起结构域。关于猿腺病毒血清型,国际专利申请No.WO2005/001103公开了使用猿腺病毒血清型18的嵌合型腺病毒。
然而,人们强烈需要开发一种具有较低免疫原性和较低毒性的腺病毒。因而,本发明人确定了一种新的分离自狒狒排泄物的SAd19六邻体基因,发现其具有很高的避免针对HAd5的中和抗体的能力且其具有低毒性。
发明概述
因而,本发明目的是提供一种用于制备嵌合型腺病毒的新六邻体蛋白和编码其的DNA。
本发明另一个目的是提供一种包括所述新六邻体蛋白的嵌合型腺病毒。
本发明还一个目的是提供一种包括所述嵌合型腺病毒的组合物。
本发明又一个目的是提供一种使用所述嵌合型腺病毒的基因治疗方法。
根据本发明的一个方面,提供分离自SAd19的一种六邻体和编码所述六邻体的DNA。
根据本发明另一个方面,提供分离自SAd19的六邻体的HVR和编码所述HVR的DNA。
根据本发明还一个方面,提供一种在六邻体中用分离自SAd19的六邻体蛋白或七(7)个或以上其中的连续的残基进行替换从而具有非天然氨基酸序列的嵌合型腺病毒。
根据本发明又一个方面,提供一种包括本发明嵌合型腺病毒的组合物。
根据本发明又一个方面,提供一种将治疗性转基因递送至哺乳动物细胞的方法,包括将本发明的嵌合型腺病毒引入所述细胞。
根据本发明又一个方面,提供一种治疗癌症的方法,包括将本发明的嵌合型腺病毒施用于个体。
根据本发明又一个方面,提供一种制备用于基因治疗的腺病毒载体的方法,包括将人腺病毒六邻体的七(7)个或以上氨基酸残基替换为本发明六邻体蛋白的七(7)个或以上氨基酸残基。
根据本发明又一个方面,提供包括本发明嵌合型腺病毒的分离的宿主细胞。
附图说明
参考附图并结合以下对本发明的描述,本发明上述及其他目的和特征将变得显而易见,附图所示分别为:
图1:人腺病毒(HAd)血清型5(A)、41(B)和SAd19的六邻体蛋白的氨基酸序列;
图2:pHex-SAd19六邻体载体(A)、pAdH5/S19_8DS载体(B)、pAd328H5/S19_DS载体(C)和pAd328H5/S19_DSΔ19k(D)的裂解图;
图3:蛋白质印迹分析(westernblotanalysis)结果表明,肿瘤特异性E1A的表达水平与AdH5_8DS和AdH5/S19_8DS的感染单位成正比;
图4:测量人凝血因子X和AdH5/S19_8DS(A)之间结合亲和力的SPR结果,以及人凝血因子IX或X与AdH5_8DS、AdH5/S19_8DS、Ad328H5/S19_DS或Ad328H5/S19_DSΔ19k(B-D)进行免疫共沉淀后的人凝血因子和腺病毒纤毛蛋白的蛋白质印迹法结果;
图5:琼脂糖凝胶电泳结果显示了静脉注射AdH5_8DS和AdH5/S19_8DS后各器官中的腺病毒纤毛基因的PCR扩增DNA条带;
图6:蛋白质印迹分析结果表明了通过使用针对HAd5的中和抗体,具有猿腺病毒血清型19、AdH5/S19_8DS(A)、Ad328H5/S19_DS和Ad328H5/S19_DSΔ19k(B)的六邻体的嵌合型腺病毒免于感染的能力;
图7:免疫染色图和柱形图表明了通过使用针对HAd5的中和抗体,具有SAd19、AdH5/S19_8DS(A)、Ad328H5/S19_DS和Ad328H5/S19_DSΔ19k(B)的六邻体的嵌合型腺病毒的免于感染能力;
图8:该图显示了施用于通过HAd5免疫的叙利亚仓鼠的AdH5/S19_8DS和AdH5_8DS所转移的血液中LK8基因的表达方式;
图9:对小鼠剂量依赖性地静脉施用AdH5_8DS和AdH5/S19_8DS后,从解剖的小鼠中切离的各器官照片;
图10:静脉施用AdH5_8DS的小鼠血液分析数据;
图11:静脉施用AdH5/S19_8DS的小鼠血液分析数据;
图12:静脉注射AdH5/S19_8DS后H460非小细胞肺癌的动物模型中的肿瘤生长曲线;
图13:根据针对HAd5的免疫,注射肿瘤特异性嵌合型腺病毒、AdH5/S19_8DS后的H2172原位肺癌解剖的动物模型的肺部照片;以及
图14:图13的肺部肿瘤的体积、重量、数目和大小的分析结果。
发明详述
除非另有定义,本文所用的技术和科学术语具有与本发明所属领域普通技术人员通常所理解的同样含义。而且,本文提到的所有文献均以全文引用的方式并入本文。
本文所用的术语“腺病毒”是指具有约36kb的线性基因组的无包膜二十面体双链DNA病毒。
本文所用的术语“嵌合型腺病毒”是指其核酸序列由至少2个腺病毒血清型核酸序列组成的腺病毒。
本文所用的“替换”是指一个或多个多核苷酸或氨基酸分别被不同的多核苷酸或氨基酸代替。
本文所用的术语“高变区”或“HVR”是指其序列为高度可变、形成结构限制环的可变域。
本文所用的术语“非天然氨基酸序列”是指腺病毒在给定血清型的天然六邻体蛋白中未被发现的任何氨基酸序列,其在基因表达水平上被引入至所述六邻体蛋白(即,产生编码所述非天然氨基酸序列的核酸序列)。
本文所用的术语“治疗性转基因”是指被转入至细胞中并且在合适条件下能被翻译和/或表达的多核苷酸,其赋予所引入的细胞所需的特性或带来所需的治疗结果。
本文所用的术语“载体”是指如本领域技术人员所知的用于基因转移的工具,包括病毒、质粒等。
本文所用的术语“中和抗体”是指能够抑制腺病毒感染(即,进入细胞)或由腺病毒控制基因表达的抗体。所述中和抗体可为从血清中纯化的或其中存在的抗体。
下面详细描述本发明。
本发明提供分离自SAd19的六邻体、其HVR和编码所述六邻体的DNA。
本发明的SAd19可通过从狒狒排泄物中分离提供,其被分类为F亚组。SAd19六邻体具有如SEQIDNO:16所示的氨基酸序列,其与人腺病毒血清型41(“HAd41”)六邻体的氨基酸序列具有85%的同源性,与HAd5六邻体的氨基酸序列具有76%的同源性。而且,编码SAd19六邻体的核苷酸序列与编码HAd41六邻体的核苷酸序列具有76%的同源性,并与编码HAd5六邻体的核苷酸序列具有70%的同源性。优选地,本发明所述SAd19六邻体具有如SEQIDNO:3所示的DNA序列。
所述SAd19六邻体或其七(7)个或以上氨基酸残基可通过替换而被整合至腺病毒的各种类型中以提供嵌合型腺病毒。相应地,提供在六邻体中具有非天然氨基酸序列的嵌合型腺病毒,其通过本发明的SAd19六邻体蛋白或其一个或多个残基的替换而实现。
优选地,所述嵌合型腺病毒可在六邻体中通过用具有如SEQIDNO:16所示氨基酸序列的六邻体的七(7)个或以上残基进行替换从而具有非天然氨基酸序列。所述SAd19六邻体的七(7)个或以上残基可为HVR。所述HVR可具有如SEQIDNO:21所示的氨基酸序列,并可由如SEQIDNO:20所示的核苷酸序列编码。更优选地,所述嵌合型腺病毒可在六邻体中具有非天然氨基酸序列,其通过替换为HVR片段而实现,即HVR-1至-7,分别对应于SEQIDNO:21的氨基酸残基11-41、46-52、69-78、106-119、126-138、160-173和275-303。
具有SAd19的六邻体或其七(7)个或以上残基的嵌合型腺病毒显示出很小的由中和抗体引起的免疫抑制和肝中毒。
腺病毒的各种类型,优选地,人腺病毒血清型,更优选地,人腺病毒血清型5、11、24、26、30、34、35、48、49和50可用于制备本发明的嵌合型腺病毒。肿瘤特异性可复制的腺病毒或肿瘤特异性限制复制的腺病毒可用作腺病毒治疗剂。本发明的嵌合型腺病毒具有非天然氨基酸序列,以克服免疫应答和肝中毒的问题。
具体地,通过将野生型腺病毒的六邻体区替换为SAd19的六邻体区来制备本发明的非天然氨基酸序列。优选地,所得的非天然氨基酸序列为:使得用于中和直接针对相应野生型腺病毒六邻体蛋白抗体的七(7)个或以上的现有抗原表位没有免疫原性的非天然氨基酸序列。
根据本发明,嵌合型腺病毒包括七(7)个或以上氨基酸的六邻体修饰物,该六邻体修饰在七(7)个或以上的区进行。
最优选的嵌合型腺病毒可为AdH5/S19_8DS,其制备方法为将腺病毒载体pAdH5/S19_8DS转入至人肺腺癌上皮细胞系A549,从而将人腺病毒六邻体替换为SAd19的六邻体。
在本发明的一个优选实施方案中,本发明的嵌合型腺病毒还可含有治疗性转基因。所述治疗性转基因的非限制性实例包括肿瘤抑制基因、抗原基因、细胞毒素基因、细胞抑制基因、自杀基因、抗血管生成基因和免疫调节基因。
具体地,所述“肿瘤抑制基因”为其在靶细胞中的表达能够抑制肿瘤表现型和/或诱导细胞凋亡的核苷酸序列。所述肿瘤抑制基因的实例包括p53-基因、APC-基因、DPC-4/Smad4基因、BRCA-1基因、BRCA-2基因、WT-1基因、视网膜母细胞瘤基因(Lee等人,自然(Nature),1987,329,642)、MMAC-1基因、腺瘤性结肠息肉病蛋白(美国专利No.5,783,666)、DCC(在结肠直肠癌中除去的)基因、MMSC-2基因、位于染色体3p21.3上的鼻咽癌抑制基因(Cheng等人,Proc.Nat.Acad.Sci.,1998,95,3042-3047)、MTS1基因、CDK4基因、NF-1基因、NF-2基因、VHL基因等。
所述“抗原基因”为其在靶细胞中的表达导致产生能够被免疫系统识别的细胞表面抗原蛋白的核苷酸序列。抗原基因的实例包括癌胚抗原(CEA)、CD3、CD133、CD44和p53(国际申请No.WO94/02167)。为了易于被免疫系统识别,所述抗原基因可与MHC-I型抗原结合。
所述“细胞毒素基因”为其在细胞中表达时表现出毒性作用的核苷酸序列。细胞毒素基因的实例包括编码绿脓杆菌外毒素、蓖麻毒素、白喉毒素等的核苷酸序列。
所述“细胞抑制基因”为其在细胞中表达时诱导细胞周期终止的核苷酸序列。细胞抑制基因的非限制性实例包括p21、视网膜母细胞瘤基因、E2F-Rb融合蛋白基因、编码细胞周期蛋白依赖性激酶抑制剂的基因(例如,p16、p15、p18和p19)、生长终止特异性同源盒(GAX)基因(国际专利申请公开No.WO97/16459和No.WO96/30385)等。
所述“自杀基因”为其在细胞中表达时通过细胞凋亡诱导细胞死亡的核苷酸序列。自杀基因的非限制性实例包括编码单纯疱疹病毒胸苷激酶、水痘胸苷激酶、胞嘧啶脱氨酶、嘌呤核苷磷酸化酶、β-内酰胺酶、羧肽酶G2、细胞色素P450-2B1、硝基还原酶、β-葡糖醛酸酶、TRAIL(肿瘤坏死因子相关凋亡诱导配体)等的基因。
所述“抗血管生成基因”为其表达导致向细胞外分泌抗血管生成因子的核苷酸序列。抗血管生成因子包括血管生成抑制素、血管内皮生长因子(VEGF)和胎盘生长因子(PlGF)如可溶性VEGFR1(sFLT-1)的抑制剂(PNAS(美国),1998,95,8795-800)、内皮抑制素和载脂蛋白(a)三环结构域(LK8)。优选的抗血管生成基因为编码LK8的基因。LK8直接影响血管内皮细胞以诱导细胞凋亡并抑制上皮细胞迁移(KimJS等人,J.Biol.Chem.,(2003)278:29000)。具体地,据报道,腺病毒介导的LK8表达抑制肝细胞癌在小鼠中的生长(LeeK.等人,肝脏学(Hepatology)(2006)43:1063)。因此,预期腺病毒的溶瘤作用可通过引入LK8而改善。
所述“免疫调节基因”为其在细胞中表达时调节体液和细胞免疫应答的核苷酸序列。免疫调节基因的非限制性实例包括编码CD16、CTLA-4、IL24、GM-CSF等的基因。
所述治疗性转基因可通过本领域已知的各种DNA重组技术插入至本发明的嵌合型腺病毒中。
本发明还提供本发明嵌合型腺病毒在抑制肿瘤细胞生长中的应用,以及在制备腺病毒载体以递送用于治疗肿瘤和其他疾病的治疗性转基因中的应用。具体地,本发明提供一种将治疗性转基因递送至哺乳动物细胞的方法,其包括向所述细胞引入本发明的嵌合型腺病毒;一种治疗癌症的方法,其包括向个体施用所述腺病毒;以及一种制备用于基因治疗的腺病毒载体的方法,其包括将人腺病毒六邻体的七(7)个或以上氨基酸残基替换为SAd19六邻体蛋白的七(7)个或以上残基。
本发明还提供一种包括本发明的嵌合型腺病毒的组合物。本发明组合物用于基因治疗或病毒治疗,优选用于癌症治疗。
本发明组合物可进行配制以提供各种连同药用载体和/或赋形剂的制剂。因而,所述制剂的形式可为在油或水介质中的溶液、悬浮液或乳液、提取物、粉剂、颗粒剂、片剂或胶囊剂。
对于口服制剂,可使用各种制备方法包括特别为腺病毒释放设计的方法,例如,通过使用Eudragit或时间依赖型释药系统(timeclockreleasesystem)(Lubeck等人,Proc.Natl.Acad.Sci.USA,86(17),6763-6767(1989);以及Chourasia和Jain,J.Pharm.Pharm.Sci.,6(1),33-66(2003))。
如上所述,所述嵌合型腺病毒可通过本领域已知的任何基因转移系统进行转移。许多基因转移系统如[Rolland(1998)Crit.Rev.Therap.药物载体系统(DrugCarrierSystems)15:143-198]以及其中引用的参考文献中公开的系统,均为本领域所熟知。因此,本发明组合物可制备为适合于这些基因转移系统。
本发明的组合物可包括本领域已知的任何药用载体。合适的载体的实例为:水、盐水、酒精、脂、蜡、缓冲液,固体载体如甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、滑石粉、纤维素、葡萄糖、蔗糖和碳酸镁,或可生物降解的微球(例如,聚乳酸(polylactate)、聚甘醇酸(polyglycolate))。
本发明组合物的提供形式可以为单剂量或多剂量容器如密封安瓿或小瓶。优选地,所述容器密封以保持使用前药物制剂的无菌条件。通常,所述制剂可保存为悬浮液、流体和在油或水介质中的乳液。而且,所述药物制剂可在冻干条件下保存。
所述嵌合型腺病毒和包括其的组合物可通过位点特异性注射或静脉注射而施用。位点特异性注射包括,例如,腹腔内注射,胸腔内注射,鞘内注射,动脉内注射,瘤内注射或局部应用。所述施用方法也可容易地应用于使用腺病毒载体的治疗和其他目标疾病的治疗的联用。优选的方法为静脉注射。
应当理解,实际施用的活性成分的合适用量应该根据各种相关因素而确定,包括治疗条件、患者个体的年龄和体重、饮食、施用时间、排泄率、患者症状的严重度和反应敏感性;因此,上述剂量并非旨在以任何形式限制本发明的范围。通常,本发明组合物含有1×107至1×1013pfu/ml本发明的嵌合型腺病毒,且本发明嵌合型腺病毒可以以每周一次1×1011pfu的用量注射3-5周。
本发明的组合物可用作单一治疗。但其可与其他抗肿瘤方案结合,如治疗癌症的常规化学疗法或放射疗法。可与本发明组合物联用的化学疗法药物包括:紫杉醇、顺铂、卡铂、丙卡巴肼、氮芥、环磷酰胺、异环磷酰胺、美法仑、苯丁酸氮芥、二甲磺酸-1,4-丁二醇(bisulfan)、亚硝基脲、放线菌素D、柔红霉素、阿霉素、博莱霉素、普卡霉素(plicomycin)、丝裂霉素、依托泊苷、三苯氧胺、紫杉酚、反铂(transplatinum)、5-氟尿嘧啶、长春新碱、长春碱和氨甲喋呤。可与本发明组合物联用的放射疗法可为X-射线辐射和γ-射线辐射等。
本发明的嵌合型腺病毒极少导致肝细胞转导,因为其不与凝血因子相互作用并表现出低的肝毒性。因此,由于其低风险的免疫应答和肝毒性,可将高剂量的本发明嵌合型腺病毒施用于个体,因而,本发明嵌合型腺病毒可用于安全有效的基因治疗和病毒治疗。
下列实施例旨在说明本发明而非限制其保护范围。
实施例1:SAd19六邻体基因的确认
用PCR从SAd19扩增六邻体基因,并克隆至pGEM-TEasy载体,用ABI自动DNA测序仪进行测序。
<1-1>六邻体基因的确认
用DNeasy组织试剂盒(QIAGEN,德国)分离SAd19的基因组,将其作为模板使用SEQIDNO:1和2引物组通过PCR扩增六邻体基因。用SV凝胶和PCR纯化系统(Promega公司,美国威斯康辛州)纯化扩增的六邻体基因,并借助T4DNA连接酶(Roche公司,瑞士)将其插入至pGEM-TEasy载体(Promega公司,美国威斯康辛州)。将所得的重组载体命名为pGEM-SAd19六邻体载体。用所述载体转化大肠杆菌(E.coli)细胞后,从10个转化子的克隆中提取载体DNA,用ABI自动DNA测序仪进行测序。在10个碱基序列中挑选出与其他序列同源性最高的序列,从而确定SAd19六邻体基因的核苷酸序列(SEQIDNO:3)。
<1-2>人腺病毒血清型的碱基和氨基酸序列比较
将SAd19六邻体基因的碱基和氨基酸序列与51份人血清型腺病毒的碱基和氨基酸序列进行比较。发现属于F亚组的SAd19六邻体与HAd41最相似,它们之间的氨基酸序列具有85%的同源性。其显示出与HAd5六邻体具有76%的氨基酸同源性。而且,发现SAd19六邻体与HAd41和HAd5的分别具有76%和70%的核苷酸序列同源性(图1)。
实施例2:其中由SAd19六邻体替换而制备嵌合型腺病毒
构建用于交换六邻体基因的穿梭载体并命名为pHex载体,其携带有用于同源重组的六邻体基因左和右延伸区。将SAd19六邻体基因克隆至pHex载体的唯一酶切位点,位于六邻体的左和右延伸臂之间以提供重组载体,命名为pHex-SAd19六邻体。将SphI-线性化的pHex-SAd19六邻体与AsiSI-线性化的pAdH5_8DS在BJ5183(Stratagene公司,美国加利福尼亚州)中进行同源重组以产生携带SAd19六邻体的重组载体,命名为pAdH5/S19_8DS。经PacI线性化后,将pAdH5/S19_8DS转染至A549细胞以产生其中固定了SAd19六邻体的新嵌合型腺病毒(AdH5/S19_8DS)。
<2-1>穿梭载体的构建
通过同源重组构建适合于替换六邻体基因的穿梭载体。为此,用SEQIDNO:4和5引物组通过PCR扩增HAd5六邻体基因5’端上游的约1kb-长区。将所得PCR产物命名为六邻体L,然后插入至pCR2.1Topo载体(Invitrogen公司,美国加利福尼亚州)。进行DNA序列分析以选出无突变的克隆,将其命名为pCR2.1-六邻体L。分别地,用SEQIDNO:6和7引物组通过PCR扩增HAd5六邻体基因3’端下游的约1kb-长区。将所得PCR产物命名为六邻体R,然后插入至pCR2.1Topo载体(Invitrogen公司,美国加利福尼亚州)。通过DNA测序分析选出无突变的克隆,并命名为pCR2.1-六邻体R。
使用XhoI和EcoRI对pCR2.1-六邻体L切离后,将六邻体L插入至pENTR2B(Invitrogen公司,美国加利福尼亚州)(其此前用SalI和EcoRI酶切)以产生重组载体pENTR2B-六邻体L。用HindIII消化pCR2.1-六邻体R,然后用Klenow片段处理以制成钝末端(bluntends)。用EcoRI消化以从钝末端的pCR2.1-六邻体R切割六邻体R。将该六邻体R插入至pENTR2B-六邻体L载体的钝化的XbaI和EcoRI位点。所得重组载体命名为pHex。在pfu聚合酶(Stratagene公司,美国加利福尼亚州)存在下,使用SEQIDNO:1和2引物组并以pGEM-SAd19六邻体为模板进行PCR。将SAd19的Mfe-1-限制性六邻体基因克隆至pHex载体的EcoRI位点。所得重组质粒经DNA测序分析发现其在恰当的位置具有SAd19六邻体,并命名为pHex-SAd19六邻体(图2A)。
<2-2>通过同源重组用SAd19六邻体替换
为了制备与SAd19六邻体重组的嵌合型腺病毒,首先用EcoRI处理pENTR2B载体(Invitrogen,CA)以去除其ccdB区。将此前由本发明人构建的pAAV-CMV_LK8_UN载体(参见PCT公开No.WO2009/102085)用KpnI/BglII处理以产生CMV_LK8片段(然后钝化末端),并将其插入至EcoRI-处理的pENTR2B的KpnI/XhoI位点(然后钝化末端)以产生重组质粒pENTR-CMV_LK8。
为了构建携带E1A基因的肿瘤特异性表达单位的质粒载体,从人基因组DNA中扩增DNMT-1(DNA(胞嘧啶-5)-甲基转移酶)基因(DS启动子)的近端启动子区,并克隆至pCR2.1-TOPO载体(Invitrogen,CA)以产生重组质粒pCR-DS。在Ex-Taq聚合酶(Takara公司,日本)存在下,以人基因组DNA为PCR模板,用SEQIDNO:8(5’-CTTCTCGCTGCTTTATCCCCATC-3’)和SEQIDNO:9(5’-CTCGGAGGCTTCAGCAGACGC-3’)引物组通过PCR进行基因扩增,所述引物组结合至DNMT-1基因的近端启动子区的两端。首先于94°C变性5分钟,再按如下进行30次PCR循环:94°C变性30秒,56°C退火30秒,72°C延伸1分钟,然后72°C再延伸3分钟。
将用SacI和XhoI从pCR-DS上切割的DNA片段插入至pΔE1Sp1B-E2F-1Rb7Δ19k载体(Kim,J.等人,(2007)人类基因治疗(Hum.GeneTher.)18,p773-786;mE1A,韩国专利No.746122;ΔE1B19K,韩国专利No.432953)中突变E1A基因前面的ClaI和SalI位点以构建重组质粒pSP72-DS_mE1A_ΔE1B19K。将用BamHI处理pSP72-DS_mE1A_ΔE1B19K所得的片段插入至pENTR-CMV_LK8的BglII位点以提供穿梭载体,命名为pENTR-CMV_LK8-DS_mE1A_ΔE1B19K。
向100ng的pAd-PLDest(Invitrogen公司,美国加利福尼亚州)和500ng的pENTR-CMV_LK8-DS_mE1A_ΔE1B19K的混合物中加入16μL的ClonaseI反应缓冲液(Invitrogen公司,美国加利福尼亚州)。将反应混合物于室温下用2μL的ClonaseI孵育1小时,然后于37°C用2μL蛋白酶K(2μg/μL)孵育10分钟。取10μL所得反应混合物用于转化大肠杆菌(E.coli)DH5α感受态细胞,再将其涂布至氨苄青霉素平板。将从限制酶图谱的阳性菌落中提取的质粒DNA通过DNA测序分析确认为目标腺病毒载体,并命名为pAdH5-8DS。
为了将pAdH5_8DS六邻体基因替换为SAd19六邻体,首先将500ng的pHex-SAd19六邻体和50ng的pAdH5_8DS分别用SphI和AsiSI进行线性化并混合,再通过电穿孔法转入至E.coliBJ5183。使用SEQIDNO:10(5’-ATGCGCAAGGTGTAGCCA-3’)和SEQIDNO:11(5’-AGCGTGCTGGCCAGCGTG-3’)引物组通过PCR筛选同源重组,所述引物组被设计用于检测同源重组。首先于94°C变性5分钟,再按如下进行30次PCR循环:94°C变性30秒,55°C退火40秒,72°C延伸1.5分钟,然后72°C再延伸3分钟。使用SEQIDNO:12(5’-CCCGTTACATAACTTACG-3’)(CMV正义引物)和SEQIDNO:13(5’-TTATGGCCTGGGGCGTTTACAG-3’)(E1A反义引物)引物组,通过PCR对筛选为阳性的克隆进行二次筛选,所述引物组被设计用于扩增含有LK8和E1A基因的区。首先于94°C变性5分钟,再按如下进行30次PCR循环:94°C变性30秒,55°C退火40秒,72°C延伸30秒,然后72°C再延伸5分钟。从通过两次PCR筛选为阳性的菌落中分离出DNA,并在大肠杆菌(E.coli)DH5α中扩增。与EcoRI、SpeI、XbaI和PshAI的酶切方式完全一致的克隆被确认并命名为pAdH5/S19_8DS(图2B)。DNA测序再次确认pAdH5/S19_8DS含有SAd19六邻体。
通过EcoRI消化将含有DS启动子的DNA片段从pCR-DS上切割,并克隆至phRL-null载体(Promega公司,美国威斯康辛州)的EcoRI位点以产生phRL-DS。将用SalI和PstI处理phRL-DS所得的片段插入至pE1.2(O.D.260Boise公司,美国爱达荷州)穿梭载体的SalI和PstI位点,产生pE1.2-DS。将用AlwNI消化pE1.2-DS切割的1.7kb大小DNA片段通过酶连接法使用大肠杆菌(E.coli)XL1-Blue电转感受态细胞克隆至pAd328(O.D.260Boise公司,美国爱达荷州)的SfiI位点。从生长在添加有氨苄青霉素和卡那霉素的LB平板上的菌落中提取的质粒DNA,通过与EcoRI、SpeI、XbaI和PshAI的切割方式完全一致而确认,并命名为pAd328-DS腺病毒载体(图2C)。从HAd5的基因组DNA扩增腺病毒E1基因并克隆至pGEM-TEasy载体(Promega公司,美国威斯康辛州),以产生重组质粒pGEMT-AdE1。将pGEMT-AdE1用BssHI消化后再用EcoNI短暂处理,以从中去除E1B19k区。将去除E1B19k的pGEMT-AdE1命名为pGEMT-AdE1Δ19k。为了将pAd328-DS的E1基因替换为去除E1BΔ19k的E1基因,首先将100ng的pAd328-DS和1μg的SphI-线性化的pGEMT-AdE1Δ19k混合,再通过电穿孔法转导至大肠杆菌(E.coli)BJ5183。通过用HindIII、SphI和EcoRV消化以筛选同源重组。确认与所有切割方式一致的克隆,并命名为pAd328-DSΔ19k。根据以上描述的步骤制备pAdH5/S198DS质粒,通过pHex-SAd19六邻体和pAd328_DS或pAd328-DSΔ19k(图2D)之间的同源重组产生pAd328H5/S19_DS和pAd328H5/S19_DSΔ19k。
<2-3>制备与SAd19六邻体重组的嵌合型腺病毒
质粒pAdH5/S19_8DS经PacI线性化后转染至A549细胞。转染14天后,观察到所述细胞的细胞病变,将其与培养基一起收集。为了完全将病毒从其中分离出来,首先将细胞冷冻和解冻3次,然后进行离心。将含有病毒的上清液进行两轮蚀斑纯化以得到纯病毒,并命名为AdH5/S19_8DS病毒。通过对其基因组DNA测序分析,AdH5/S19_8DS病毒被确认为具有SAd19六邻体基因的嵌合型腺病毒。按照上述步骤制备Ad328H5/S19_DS和Ad328H5/S19_DSΔ19k病毒以产生AdH5/S19_8DS。
<2-4>制备和纯化具有SAd19六邻体的嵌合型腺病毒
A549肺癌细胞在30个150mm大小的培养皿中生长到80%的融合度,然后在MOI为20时用AdH5/S19_8DS病毒感染。于37°C孵育2天后,以12,000×g离心10分钟收获细胞,然后悬浮于10mL裂解缓冲液(0.5MTris,pH8.0,1mMMgCl2)中。进行3轮冷冻和解冻以裂解细胞,然后以12,000×g冷冻离心10分钟去除细胞残余物。为了制备不连续CsCl密度梯度,将8mL比重为1.4的CsCl溶液放入超离心管(Beckman公司,美国加利福尼亚州),并用6mL比重为1.2的CsCl溶液覆盖,以这种方式保持二者之间的明确边界。将经0.22μm过滤器过滤的病毒样品加载至CsCl1.4/1.2密度梯度而不弄乱该边界,将该管用10mMTris-HCl(pH7.9)平衡重量。将该管放置到SW28离心机以23,000rpm在冷冻条件下超离心90分钟以形成病毒带。
将所分离的病毒用连续CsCl密度梯度超离心步骤再次纯化。为此,将8mL比重为1.4的CsCl溶液放入超离心管(Beckman公司,美国加利福尼亚州)并用比重为1.2的CsCl溶液覆盖,以这种方式保持二者之间的明确边界。使用密度梯度分离系统(Biocomp公司,加拿大)在该管中形成连续梯度。将通过所述不连续CsCl密度梯度超离心获得的病毒样品用一定体积的10mMTris-HCl(pH7.9)稀释并加载至CsCl1.4/1.2梯度管而不弄乱二者之间的边界。用10mMTris-HCl(pH7.9)平衡其重量后,将该管以23,000rpm在冷冻条件下超离心90分钟以形成病毒带。用PBSG缓冲液(含10%甘油的PBS)透析3次,每6小时更换新缓冲液。
实施例3:具有SAd19六邻体的嵌合型腺病毒的体外研究
通过比较用该病毒在不同MOI下感染的肺癌细胞系A549和正常细胞系MRC5中E1A和LK8的表达水平,对AdH5/S19_8DS病毒进行肺癌选择性体外检测。此外,测量AdH5/S19_8DS病毒与凝血因子的亲和力,该凝血因子介导血液循环传播病毒至肝脏。
<3-1>MOI-依赖性E1A表达
在MOI为100、25、10和1时,用AdH5/S19_8DS病毒感染在6-孔平板中生长至80%融合度的A549肺癌细胞和MRC5正常细胞。于37°C孵育24小时后,以3,000rpm离心5分钟收获所述细胞,并悬浮于1×SDS-PAGE缓冲液(50mMTris(pH6.8)、2%SDS、100mM二硫苏糖醇、0.1%溴酚蓝、10%甘油),于100°C水浴中加热5分钟,并以10,000rpm离心2分钟。所得上清液在4~12%的SDS-PAGE凝胶(Invitrogen公司,美国加利福尼亚州)上以20mA电泳2小时。用转移单元(Invitrogen公司,美国加利福尼亚州)在施加300mA电场的Tris-甘氨酸缓冲液(39mM甘氨酸、48mMTris、0.037%SDS、20%甲醇)中,将从凝胶分离的蛋白条带转移约90分钟至PVDF膜上。用TBS封闭溶液(ThermoScientific公司,美国伊利诺伊州)将所述膜于室温下封闭1小时。小鼠抗-E1A单克隆抗体(BDPharmingen公司,美国加利福尼亚州)作为一抗,用5%脱脂奶/1×TBST缓冲液以1:3,000稀释,于室温用探针探测1小时并用1×TBST缓冲液冲洗6次,每次5分钟。抗-小鼠HRP(KPL公司,美国马萨诸塞州)作为二抗,用5%脱脂奶/1×TBST缓冲液以1:5,000稀释,用探针探测30分钟并用1×TBST缓冲液冲洗6次,每次5分钟,并与显色剂(ECL,Amersham,英国)反应以显示E1A蛋白条带。E1A表达水平以MOI-依赖性方式增加,在A549癌症细胞中比MRC-5正常细胞高100倍(图3)。
<3-2>对凝血因子X的亲和力
使用SPR(表面等离子体共振)法分析AdH5/S19_8DS对凝血因子X的亲和力。将纯化的凝血因子X(HCX-0050,HaematologicalTechnologies公司,美国佛蒙特州)应用于CS5传感器芯片(Biacore公司,瑞典)。在含有5mMCaCl2的HBSP缓冲液(10mMHEPESpH7.4,150mMNaCl,0.005%吐温20)中分别以3.0×1011VP/mL、1.5×1011VP/mL和0.75×1011VP/mL的浓度稀释纯化的AdH5_8DS和AdH5/S19_8DS病毒。将HBSEP(10mMHEPESpH7.4,150mMNaCl,3mMEDTA,0.005%吐温20)作为再生缓冲液用于将病毒从所述芯片上分离。当病毒溶液以30μL/min的流速经过凝血因子X-固定的CM5传感器芯片时,记录RU值。在所述3个不同浓度下,腺病毒血清型5AdH5_8DS分别显示了300、150和70RU的固定化因子SPR信号,而具有SAd19六邻体的嵌合型腺病毒AdH5/S19_8DS显示出很弱或很小的对于凝血因子X的亲和力,因为在所有3个浓度下发现的SPR信号都在5RU以内(图4A)。还通过下述<3-3>中的描述的免疫沉淀法使用凝血因子X蛋白代替因子IX,检测了Ad328H5/S19_DS和Ad328H5/S19_DSΔ19k对凝血因子X的亲和力(图4D)。
<3-3>对凝血因子IX的亲和力
使用免疫沉淀法检测AdH5/S19_8DS对凝血因子IX的亲和力。向含有1mLPBS的管中加入1×1011VP的AdH5_8DS或AdH5/S19_8DS,以及10μg纯化的凝血因子IX(HCIX-0040,HaematologicalTechnologies公司,美国佛蒙特州)、5μg羊抗-凝血因子IX抗体和50μL琼脂糖-蛋白G(50%浆液),然后于回旋振荡器中在4°C孵育2小时。以5,000rpm离心5分钟后,将所获得的琼脂糖-蛋白G球团用PBS洗涤3次,悬浮于100μL的1×SDS-PAGE样品缓冲液(50mMTris(pH6.8),2%SDS,100mM二硫苏糖醇,0.1%溴酚蓝,10%甘油)中,加热5分钟。将悬浮液离心,所得上清液在4~12%SDS-PAGE凝胶上以20mA电泳约2小时。将分离的蛋白在含有Tris-甘氨酸缓冲液(39mM甘氨酸,48mMTris,0.037%SDS,20%甲醇)的转移单元中以300mA转移约90分钟至PVDF膜上。用TBS封闭溶液(ThermoScientific公司,美国伊利诺伊州)将所述膜于室温封闭1小时。将小鼠抗-HAd5纤毛单克隆抗体(NeoMarkers公司,美国加利福尼亚州)作为一抗,用5%脱脂奶/1×TBST缓冲液以1:3,000稀释,于室温用探针探测1小时,并用1×TBST缓冲液洗涤6次,每次5分钟。将抗-小鼠HRP(KPL公司,美国马萨诸塞州)作为二抗,用5%脱脂奶/1×TBST缓冲液以1:5,000稀释,用探针探测30分钟,用1×TBST缓冲液洗涤6次,每次5分钟,并与显色剂(ECL,Amersham,英国)反应以显示纤毛条带。将PVDF膜上的斑点浸没于RestoreTM蛋白质印迹膜再生液(ThermoScientific公司,美国伊利诺伊州)并于回旋振荡器中振荡1小时,用1×TBST缓冲液洗涤3次,每次10分钟,用TBS封闭溶液于室温封闭1小时。将羊抗-凝血因子IX抗体(AffinityBiologicals公司,加拿大)作为一抗,用5%脱脂奶/1×TBST缓冲液以1:3,000稀释,于室温用探针探测1小时,并用1×TBST缓冲液洗涤6次,每次5分钟。将抗-羊HRP(KPL公司,美国马萨诸塞州)作为二抗,用5%脱脂奶/1×TBST缓冲液以1:5,000稀释,用探针探测30分钟,用1×TBST缓冲液洗涤6次,每次5分钟,并与显色剂(ECL,Amersham,英国)反应以显示凝血因子IX条带(图4B)。还使用上述免疫沉淀法检测了Ad328H5/S19_DS和Ad328H5/S19_DSΔ19k对凝血因子IX的亲和力(图4C)。
实施例4:具有SAd19六邻体的嵌合型腺病毒的体内研究
<4-1>生物分布
将AdH5/S19_8DS以3×1010VP的剂量静脉注射至6周龄的Balb/c正常小鼠和裸鼠。注射2天后,用DNeasy血液和组织试剂盒(QIAGEN公司,德国)分离脑、肝、肺、心、胸腺、脾、卵巢、子宫和血液的DNA。用OD分光光度计定量分析分离的DNA,以200ng用量作为PCR模板。用SEQIDNO:14(5’-ACTCGAGCACGTTGTGCATTGTCA-3’)和SEQIDNO:15(5’-TGTCGACTAGTTTTCTTAAAATGG-3’)引物组通过PCR扩增腺病E4区毒。首先于94°C变性5分钟,再按如下进行30次PCR循环:94°C变性30秒,55°C退火40秒,72°C延伸1分钟,然后72°C再延伸3分钟。将PCR产物在电场存在下跑1%琼脂糖凝胶以按器官分析腺病毒的分布。注射后,HAd5即AdH5_8DS在肝和肺中观察到的最多,而在心和脾中部分地检出。相反,具有SAd19六邻体的嵌合型腺病毒,AdH5/S19_8DS病毒在心和脾中检出最多,而仅部分地在肝和肺中检出(图5)。这种分布方式非常不同于腺病毒血清型5的生物分布,已知腺病毒血清型5经静脉注射传导至肝中,大量注射则引起肝中毒。也即,AdH5/S19_8DS病毒极少量传导至肝,如同实施例<3-2>中观察到病毒显示出对凝血因子X很小的亲和力所推测的。
实施例5:具有SAd19六邻体的嵌合型腺病毒避免HAd5中和抗体免疫识别的能力的体外研究
<5-1>通过E1A表达方式检测抗-HAd5-阳性血浆对AdH5/S19_8DS和Ad328H5/S19_DS转导的影响
将野生型HAd5以1×1011VP的剂量肌肉注射至小鼠后腿,2周后再注射同样剂量的病毒以促进免疫应答。从所有注射病毒的小鼠血样中分离血清,测量抗-腺病毒抗体水平。为此,将1/25、1/100和1/1,000稀释的血清加入至涂布有腺病毒的ELISA平板,孵育1小时,用PBST(含0.1%吐温20的PBS)洗涤3次,然后用1/5,000稀释的HRP-标记的抗-小鼠IgG抗体孵育1小时。用PBST(含有0.1%吐温20的PBS)洗涤平板5次后,通过与TMB底物反应30分钟并用1M磷酸终止进行显色。测量平板的吸光度。与通过与以1/1000稀释的抗-腺病毒抗体(AbDSerotec公司,美国北卡罗来纳州)反应制备的阳性对照相比,当其吸光度为50%或更高时被认为是诱导阳性免疫应答。用AdH5/S19_8DS或Ad328H5/S19_DS在MOI为25时感染在6-孔平板上生长至90%融合度的A549细胞1小时,然后于室温与确定为阳性免疫应答的小鼠血浆一起孵育。于37°C孵育24小时后,以3,000rpm离心5分钟收获细胞,悬浮于1×SDS-PAGE样品缓冲液(50mMTris(pH6.8),2%SDS,100mM二硫苏糖醇,0.1%溴酚蓝,10%甘油),于100°C水浴中加热5分钟,以10,000rpm离心2分钟。用电泳试剂盒(Novex)在20mA下,将澄清的上清液在4~12%的SDS-PAGE凝胶上跑约2小时。在含有Tris-甘氨酸缓冲液(39mM甘氨酸、48mMTris、0.037%SDS、20%甲醇)转移单元(Invitrogen公司,美国加利福尼亚州)中施加300mA的电场,将分离的蛋白转移约90分钟至PVDF膜上。用TBS封闭溶液(ThermoScientific公司,美国伊利诺伊州)将所述膜于室温下封闭1小时。将小鼠抗-E1A单克隆抗体(BDPharmingen公司,美国加利福尼亚州)作为一抗,用5%脱脂奶/1×TBST缓冲液以1:3,000稀释,室温下以其孵育所述膜1小时,然后用1×TBST缓冲液冲洗6次,每次5分钟。将抗-小鼠HRP(KPL公司,美国马萨诸塞州)作为二抗,用5%脱脂奶/1×TBST缓冲液以1:5,000稀释,用其孵育所述膜30分钟,然后用1×TBST缓冲液冲洗6次,每次5分钟。用显色剂(ECL,Amersham,英国)显示E1A蛋白条带。而AdH5_8DS的转导被抗-HAd5抗体-阳性血浆抑制,抗-HAd5抗体-阳性血浆对嵌合型腺病毒AdH5/S19_8DS(图6A)、Ad328H5/S19_DS和Ad328H5/S19_DSΔ19k(图6B)的转导没有影响。
<5-2>通过免疫染色检测抗-HAd5-阳性血浆对H5/S19_8DS转导的影响
用确定为阳性免疫应答的小鼠血浆孵育1小时后,用AdH5/S19_8DS或AdH5/S19_DS在MOI为25时感染培养至90%融合度的A549细胞,然后于37°C孵育2天。用冷甲醇固定所述细胞,用无蛋白的T20(TBS)封闭缓冲液(ThermoScientific公司,美国伊利诺伊州)封闭1小时。在用PBST缓冲液稀释(1/3,000)的小鼠抗-E1A单克隆抗体(BDPharmingen公司,美国加利福尼亚州)中孵育所述细胞1小时,用1×TBST缓冲液冲洗6次,每次5分钟。然后,在用PBST缓冲液稀释(1/5,000)的HRP-标记的抗-小鼠二抗中再处理所述细胞1小时,用1×TBST缓冲液冲洗6次,每次5分钟,并与DAB溶液反应以显色。发现抗-HAd5抗体-阳性血浆抑制AdH5_8DS的转导,但对嵌合型腺病毒AdH5/S19_8DS(图7A)、Ad328H5/S19_DS和AdH5/S19_DSΔ19k(图7B)的转导无影响。
<5-3>具有SAd19六邻体的嵌合型腺病毒避免HAd5中和抗体免疫识别的能力的体内检测
将野生型HAd5以1×1011VP的剂量肌肉注射至仓鼠后腿,2周后再以同样剂量病毒注射以诱导免疫应答。从所有注射病毒的仓鼠血样中分离血清,测量其抗-腺病毒抗体水平。为此,将1/25、1/100和1/1,000稀释的血清加入至涂布有腺病毒的ELISA平板,孵育1小时,用PBST(含0.1%吐温20的PBS)洗涤3次,然后用1/5,000稀释的HRP-标记的抗-小鼠IgG抗体孵育1小时。用PBST(含有0.1%吐温20的PBS)洗涤平板5次后,通过与TMB底物反应30分钟并用1M磷酸终止进行显色。测量所述平板的吸光度。与通过与以1/1000稀释的抗-腺病毒抗体反应制备的阳性对照相比,当其吸光度为50%或更高时被认为是诱导阳性免疫应答。发现以1×1011VP剂量静脉注射了HAd5即AdH5_8DS的免疫仓鼠血液中LK8水平太低而无法检出。反之,以1×1011VP剂量静脉注射了AdH5/S19_8DS的仓鼠则确认血液LK8水平为200ng/mL或更高,其表达量维持在200ng/mL或更高水平达28天,这表明抗-人腺病毒血清型5中和抗体没有抑制具有SAd19六邻体的嵌合型腺病毒的转导(图8)。
实施例6:具有SAd19六邻体的嵌合型腺病毒的毒性
研究了SAd19六邻体的毒性。为此,将病毒以1×1011VP、5×1010VP、1×1010VP、5×109VP和1×109VP的剂量分别静脉注射至5组各5只Balb/c小鼠,小鼠隔日称重。注射后第3-6周,取小鼠血样和血清分析红细胞、白细胞、血小板、血红蛋白水平,红细胞比容,MCV(平均红细胞体积),MCH(平均红细胞血红蛋白浓度),MCHC(平均红细胞血红蛋白浓度)和白细胞分类计数。进行由白蛋白、总蛋白、SGPT(ALT)、SGOT(AST)和ALP水平的肝功能检测,肌酸酐和BUN水平的肾功能检测,肌酸酐激酶水平的肌肉检测,以及总胆固醇和葡萄糖水平的代谢检测所组成的检查。在5只以1×1011VP的剂量注射了人腺病毒血清型5病毒AdH5_8DS的小鼠组中,有4只小鼠在第5天死亡,而余下的1只为重病。经尸体解剖发现它们患有肝硬化,而其他组织无异常。经尸体解剖检查,观察到其他组别的小鼠均为正常(图9)。
<6-1>血液测试
分别以1×1011VP、5×1010VP、1×1010VP、5×109VP和1×109VP的剂量静脉注射HAd5即AdH5_8DS。在第5天,以1×1011VP的剂量注射所述病毒的所有小鼠均死亡。另一方面,其他组别的小鼠没有观察到异常的毒性。在血液学和血液生化测试方面,测试组和注射PBS的阴性对照组之间没有发现统计学上的差异(图10)。在以1×1011VP、5×1010VP、1×1010VP、5×109VP和1×109VP剂量注射了具有SAd19六邻体的嵌合型腺病毒AdH5/S19_8DS的所有组别小鼠均没有观察到异常的毒性。同样在血液学和血液生化测试方面,测试组和注射PBS的阴性对照组之间没有发现统计学上的差异(图11)。在以最大剂量注射HAd5的组中检测到的肝中毒没有在注射具有SAd19六邻体的嵌合型腺病毒的相应组中观察到。
<6-2>活组织检查
将从注射小鼠中切除的脑、心、肝、肺、肾、脾、子宫和卵巢用3.7%的中性福尔马林固定,包埋于石蜡中,切片后用H&E染色。除了以1×1011VP剂量注射人腺病毒血清型的组以外,所有测试组均没有发现器官异常。肝活组织检查结果表明以1×1011VP剂量注射的组存在大量肝坏死,其被认为是诱导了死亡小鼠的急性肝功能障碍。这就是通常观察到的腺病毒肝中毒。对于具有SAd19六邻体的嵌合型腺病毒,即便以1×1011VP的剂量注射也没有导致肝中毒,这表明SAd19六邻体有希望成为解决使用HAd5所导致的肝中毒问题的方案。
实施例7:具有SAd19六邻体的嵌合型腺病毒在动物肿瘤模型中的抗肿瘤活性
<7-1>嵌合型腺病毒在人肺癌异种移植动物模型中的肿瘤选择性、抗肿瘤活性
将非小细胞肺癌(NSCLC)细胞系NCI-H460以5×106个细胞的剂量皮下注射至免疫缺陷型Balb/c裸鼠右侧以形成50~100mm3大小的肿瘤。将小鼠随机分为4组,每组5只。将对照组小鼠以1×109pfu剂量静脉注射携带LK8基因的Ad-LK8复制缺陷型腺病毒或静脉注射PBS,以常规两天的间隔注射3次。对于测试组,以1×109pfu和2×108pfu的剂量静脉注射AdH5/S19_8DS,以常规间隔注射3次。每隔2或3天测量肿瘤大小以绘制肿瘤生长曲线。首次注射病毒后第24天,以1×109pfu剂量注射AdH5/S19_8DS的组,相比施用PBS的组表现出74%以上的肿瘤生长抑制率,而相比施用Ad-LK8的组表现出64%以上的肿瘤生长抑制率。另一方面,以2×108pfu剂量注射AdH5/S19_8DS的组,相比施用PBS的组表现出61%以上的肿瘤生长抑制率,而相比施用Ad-LK8的组表现出48%以上的肿瘤生长抑制率。自注射后第17天,双向重复测量方差分析(2-wayRMANOVA)检测揭示了注射AdH5/S19_8DS的两组相对于施用PBS组的统计学意义(注射后第17天,P<0.05;注射后第21天和第24天,P<0.01)(图12)。
<7-2>嵌合型腺病毒在用HAd5免疫的人肺癌原位动物模型中的肿瘤选择性、抗肿瘤活性
将Balb/c裸鼠以1×1010VP的剂量用HAd5肌肉注射至其后腿进行两轮免疫,每轮常规间隔2周。从取自所述小鼠的血样中发现其含有抗-HAd5抗体。将NSCLC细胞系NCI-H2172以1×106细胞的剂量接种至尾部血管。在以1×109pfu注射剂量接种肿瘤后的第7、9和11天,空白对照和免疫的小鼠接受三次静脉注射Ad-LK8、AdH5/S19_8DS或AdH5_8DS。在第6周,从所述小鼠切离肺(图13)。对肺中产生的肿瘤计数,并按照大小分组:x≤0.5cm,0.5cm<x≤0.7cm和0.7cm<x≤1cm,并将其分别记为5、7和10分。对肺产生的肿瘤进行计数,在施用PBS的阴性对照组中平均为18.6,而在施用Ad-LK8的组中平均为11.2。对于施用AdH5/S19_8DS的组,发现分别在用和未用HAd5进行免疫的肺中平均肿瘤数为5.6和5.4。另一方面,对于施用AdH5_8DS的组,发现分别在免疫或空白对照的小鼠肺中平均肿瘤数为10.8和6.2。因此,AdH5/S19_8DS显示出与免疫无关的几乎相同的滴度,但是,由于抗-HAd5抗体AdH5_8DS显示出明显下降的效价。测量平均肺体积,正常小鼠为274mm3,施用PBS的组为570mm3,施用Ad-LK8的组为480mm3。然而,发现注射AdH5/S19_8DS的免疫或未免疫小鼠组的肺体积分别为370mm3和360mm3。它们之间的肺体积没有显著性差异。在施用AdH5_8DS的组中,免疫或未免疫小鼠平均肺体积分别为410mm3和350mm3。测量平均肺重量,正常小鼠为170mg,施用PBS的组为376mg,施用Ad-LK8的组为278mg。当注射AdH5/S19_8DS时,免疫或未免疫小鼠组所测量的平均肺重量均为218mg,而对于施用AdH5_8DS的组的平均肺重量,免疫小鼠为240mg,未免疫小鼠为230。对于根据肿瘤大小所得的分数,测量的平均数为:施用PBS的组为107.8,而施用Ad-LK8的组为60.8。当注射AdH5/S19_8DS时,所述免疫组和未免疫组得到的分数分别为28和27。另一方面,当注射AdH5_8DS时,所述免疫组和未免疫组分别得到的分数为57.2和32.6(图14)。总的来说,这些数据表明,不考虑HAd5的免疫,AdH5/S19_8DS甚至进行静脉注射也能具有抗肿瘤活性。
尽管已经针对上述具体实施方式描述了本发明,应该理解,本领域技术人员可对本发明做出各种修改和变化,其同样落入所附权利要求书所限定的本发明保护范围内。
Claims (11)
1.分离自由SEQIDNO:16所示氨基酸序列组成的猿腺病毒血清型19的六邻体。
2.编码如权利要求1所述六邻体的DNA。
3.如权利要求2所述的DNA,其由SEQIDNO:3所示的核苷酸序列组成。
4.嵌合型腺病毒,其六邻体由SEQIDNO:16所示的氨基酸序列组成,所述嵌合型腺病毒为人腺病毒血清型5。
5.如权利要求4所述的嵌合型腺病毒,其中所述嵌合型腺病毒还包括治疗性转基因。
6.如权利要求5所述的嵌合型腺病毒,其中所述治疗性转基因选自下组:肿瘤抑制基因、抗原基因、细胞毒素基因、细胞抑制基因、自杀基因、抗血管生成基因和免疫调节基因。
7.如权利要求6所述的嵌合型腺病毒,其中所述肿瘤抑制基因选自下组:p53基因、APC基因、DPC-4/Smad-4基因、BRCA-1基因、BRCA-2基因、WT-1基因、视网膜母细胞瘤基因、MMAC-1基因、腺瘤性结肠息肉病蛋白、DCC(在大肠癌中去除的)基因、MMSC-2基因、NF-1基因、NF-2基因、MTS1基因、CDK4基因和VHL基因;
所述抗原基因为癌胚抗原(CEA)、CD3、CD133、CD44或p53;
所述细胞毒素基因选自编码绿脓杆菌外毒素、蓖麻毒素和白喉毒素的基因;
所述细胞抑制基因选自下组:p21、视网膜母细胞瘤基因、E2F-Rb融合蛋白基因、编码细胞周期蛋白依赖性激酶抑制剂的基因和生长终止特异性同源盒(GAX)基因;
所述自杀基因选自编码单纯疱疹病毒胸苷激酶、水痘胸苷激酶、胞嘧啶脱氨酶、嘌呤核苷磷酸化酶、β-内酰胺酶、羧肽酶G2、细胞色素P450-2B1、硝基还原酶、β-葡糖醛酸酶和TRAIL(肿瘤坏死因子相关的凋亡诱导配体)的基因;
所述抗血管生成基因选自编码血管内皮生长因子(VEGF)、可溶性血管内皮生长因子受体、血管生成抑制素、内皮抑制素和载脂蛋白(a)三环结构域(LK8)的基因;以及
所述免疫调节基因选自编码CD16、CTLA-4、IL24和GM-CSF的基因。
8.包括如权利要求4所述嵌合型腺病毒的组合物。
9.一种制备用于基因治疗的权利要求4所述的嵌合型腺病毒的方法。
10.包括如权利要求4所述嵌合型腺病毒的分离的宿主细胞。
11.如权利要求10所述的分离的宿主细胞,其中所述分离的宿主细胞为人细胞。
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