CN116284336A - 一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒及其应用 - Google Patents
一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒及其应用 Download PDFInfo
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Abstract
本发明涉及肿瘤生物治疗领域,具体涉及一种特异性阻断关键促肿瘤炎症通路的重组复制型溶瘤腺病毒Ad5 mIL‑6R。本发明公开了该病毒的构建方法,成功获得了能够高表达可溶性白介素6受体突变体(mIL‑6R)的复制型溶瘤腺病毒(Ad5 mIL‑6R);该病毒能够感染肿瘤细胞,在肿瘤细胞内快速复制并产生溶瘤作用,所表达的mIL‑6R蛋白分子能够大量分泌到胞外,显著抑制IL‑6/STAT3炎症通路并促进抗肿瘤免疫作用;Ad5 mIL‑6R具有广谱的抗肿瘤作用,尤其对肝癌、肺癌和结直肠癌,具有预料不到的抑制肿瘤进展和延长荷瘤个体生存时间的作用;Ad5 mIL‑6R有极大的开发抗肿瘤药物的前景和价值。
Description
技术领域
本发明涉及肿瘤生物治疗领域,具体涉及一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒及其应用。
背景技术
癌症已经成为危害人类生命健康的疾病之首,在我国每年新增的癌症患者约400万人,每年有近300万人死于癌症。传统的手术、放疗、化疗等方案在一定程度上抑制了肿瘤的进展,但难以控制肿瘤的侵袭、转移和复发。
溶瘤病毒作为外来侵入颗粒,能够有效激活机体的天然免疫和适应性免疫。随着溶瘤病毒T-Vec在2015年底被FDA批准上市,溶瘤病毒介导的抗肿瘤免疫治疗受到越来越多的关注。溶瘤病毒本身能够在肿瘤细胞中复制并发挥溶瘤作用,同时能够激活免疫细胞,导致肿瘤局部的免疫细胞浸润,并诱发抗肿瘤免疫应答。另外,溶瘤病毒可以携带外源基因,通过在肿瘤局部高表达外源蛋白,直接发挥蛋白药物的抗肿瘤作用。
在溶瘤病毒药物开发领域,目前上市的产品有中国上海三维的V型重组溶瘤腺病毒注射液(安科瑞),通过选择性在肿瘤细胞内复制产生溶瘤作用,用于头颈部肿瘤的瘤内注射;美国安进公司的表达GM-CSF的重组单疱疹病毒(T-Vec),以激活抗肿瘤免疫达到抑癌,用于治疗复发不能手术的黑色素瘤患者。但这两者临床疗效均有限,应用受限。最近日本一三共获批上市的重组单疱疹病毒(ΔG47),在神经胶质瘤患者中的临床试验显示出好的疗效。
目前市场上溶瘤病毒产品研发,主要聚焦在通过重组病毒表达免疫活化因子、检查点抑制剂等,激活抗肿瘤免疫。但没有一种通过抑制促肿瘤炎症通路激活的重组溶瘤病毒产品。
据研究,85%的实体肿瘤都与炎症通路异常活化有关。IL-6/STAT3炎症通路的异常活化,与肿瘤的发生、发展和免疫逃逸均有密切的关系。IL-6存在两种形式的受体,其活化信号通路也分为两种:1)由跨膜受体介导的经典型信号通路;2)由可溶性白介素6(sIL-6R)介导的反式信号通路。经典型信号通路是IL-6直接与细胞膜上IL-6R结合,形成gp130二聚体活化下游信号通路。反式信号通路是IL-6先与胞外可溶性sIL-6R结合形成的复合物后,再结合gp130激活下游通路。由于IL-6R的存在局限于白细胞、肝细胞,而gp130的存在于几乎所有的细胞膜上,因此反式信号传导机制使IL-6能够在除上述细胞外更广泛的细胞上发挥作用,能增强IL-6的反应性和炎症反应,推动肿瘤细胞的增殖、存活、侵袭和转移,促进肿瘤微环境的血管生成、代谢与炎症反应。因此,反式信号传导机制被认为是一种潜在的危险信号。
目前有阻断IL-6的抗体药物,用于多种炎症相关疾病的治疗,市场上没有批准用于肿瘤治疗的阻断白介素6的抗体药物;目前也没有靶向IL-6反式激活通路的药物。
本发明首次公开了一种特异性阻断促肿瘤炎症活化通路重组溶瘤腺病毒,能够特异性阻断IL-6反式信号传导通路,显著活化抗肿瘤免疫反应,具有广谱的抗肿瘤作用,尤其对肺癌、肝癌及结直肠癌具有预料不到的显著疗效。
发明内容
针对现有技术的不足,本发明的目的在于提供一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒及其应用。本发明公开了该病毒的构建方法,成功获得了能够高表达可溶性白介素6受体突变体(mIL-6R)的复制型溶瘤腺病毒(Ad5 mIL-6R)。该病毒能够感染肿瘤细胞,在肿瘤细胞内快速复制并产生溶瘤作用,所表达的mIL-6R蛋白分子能够大量分泌到胞外,显著抑制IL-6/STAT3炎症通路;Ad5 mIL-6R在肝癌、肺癌和结直肠癌的小鼠模型中,具有显著的抑制肿瘤生长、进展和显著延长荷瘤小鼠生存时间的作用,有极大的开发抗肿瘤药物的前景和价值。
为达到上述目的,本发明采用了下列技术方案:
第一方面,本发明保护一种特异性阻断反式IL-6活化通路的可溶性IL-6R的突变体(mIL-6R),在如下a)或b)中的应用:
a)在制备促进溶瘤腺病毒溶瘤药物中的应用;
b)在制备抗肿瘤药物中的应用。
其中,所述可溶性IL-6R的突变体为将可溶性IL-6R的FN3结构域区的氨基酸序列的第228位丙氨酸突变为天冬氨酸;第230位天冬酰胺突变为天冬氨酸;第280位组氨酸突变为丝氨酸;第281位天冬氨酸突变为缬氨酸。该突变体与野生型可溶性IL-6R相比,包含有4个位点的氨基酸突变,能够极其显著阻断IL-6/STAT3信号转导通路,且具有显著的抗肿瘤作用。
其中,编码前述突变体mIL-6R的DNA分子的核苷酸序列如SEQ ID NO:1所示。
第二方面,本发明保护一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒,该溶瘤腺病毒该病毒能够在肿瘤中选择性复制、溶瘤、抑制肿瘤生长、促进抗肿瘤免疫并延长治疗个体生存时间;同时能够表达并分泌mIL-6R蛋白,该蛋白能够特异性阻断促肿瘤炎症。
优选的,所述重组复制型溶瘤腺病毒为Ad5 mIL-6R,即所用腺病毒载体为Ad5。
优选的,所述可溶性IL-6R的突变体为将可溶性IL-6R的FN3结构域区的氨基酸序列的第228位丙氨酸突变为天冬氨酸;第230位天冬酰胺突变为天冬氨酸;第280位组氨酸突变为丝氨酸;第281位天冬氨酸突变为缬氨酸。该突变体与野生型可溶性IL-6R相比,包含有4个位点的氨基酸突变,能够极其显著阻断IL-6/STAT3信号转导通路,且具有显著的抗肿瘤作用。
更优选的,编码前述突变体mIL-6R的DNA分子的核苷酸序列选自如下A)或B):
A)如SEQ ID NO:1所示;
B)与SEQ ID NO:1所述的序列具有80%以上同源性的核苷酸序列。
所述重组复制型溶瘤腺病毒的构建方法,包括如下步骤:
1)穿梭质粒构建:首先基因合成突变体mIL-6R的DNA序列,与pShuttle穿梭质粒酶切后连接;将连接产物转化DH5α感受态后,筛选阳性克隆,进行质粒提取;
2)病毒载体构建:分别用PCR技术克隆出CMV启动子基因片段、2A自剪切肽段基因片段、E1A基因片段、BGH PolyA加尾信号基因片段,再用PCR技术依顺序拼接接4个基因片段,最后用Exnase连接酶将拼接起来的片段连接到pShuttle;
3)病毒拯救、扩增及纯化,即得。
其中,编码突变体mIL-6R的DNA分子序列如SEQ ID NO:1所示;
所述CMV启动子的基因片段如SEQ ID NO:2所示;
所述2A自剪切肽段基因片段如SEQ ID NO:3所示;
所述E1A基因片段如SEQ ID NO:4所示;
所述BGH PolyA加尾信号基因片段如SEQ ID NO:5所示。
第三方面,本发明还提供上述复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
本发明还提供上述复制型溶瘤腺病毒与第二治疗药物联合在制备抗肿瘤药物中的应用。
联合用药时,采用同时给药、或不分次序先后单独给药。
优选的,所述肿瘤选自胶质瘤、食道癌、胃癌、乳腺癌、胰腺癌、肾癌、宫颈癌、卵巢癌、前列腺癌、头颈部鳞癌、黑色素瘤、骨肉瘤、纤维肉瘤、横纹肌肉瘤、多发性骨髓瘤、神经内分泌肿瘤,淋巴瘤中的其中一种;优选的,所述的肿瘤为肝癌、肺癌和结直肠癌等。
更优选的,所述肿瘤选自肝癌、肺癌或结直肠癌。本发明建立了多种实体瘤小鼠模型,包括肝癌、肺癌和结直肠癌,验证了Ad5 mIL-6R治疗肿瘤的显著效果和促进抗肿瘤免疫的活性。
优选的,所述药物为适于口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
第四方面,本发明请求保护一种药物组合物,该药物组合物包含上述重组复制型溶瘤腺病毒,以及药学上可接受的载体。
药学上可接受的载体包括填充剂、吸收剂、润湿剂、粘合剂、崩解剂、润滑剂等等。
第五方面,本发明还请求保护一种治疗肿瘤的方法,所述方法包括向适用对象施加有效量的前述重组复制型溶瘤腺病毒。
有益效果
本发明提供的一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒及其应用,与现有技术相比,具有如下有益效果:
(1)本发明构建了一种特异性阻断反式IL-6活化通路的可溶性IL-6R的突变体(mIL-6R),该突变体与野生型可溶性IL-6R相比,能够极其显著阻断IL-6/STAT3信号转导通路,且具有显著的抗肿瘤作用。
(2)本发明构建的重组复制型溶瘤腺病毒Ad5 mIL-6R,能够感染多种真核细胞并复制,且能够表达并分泌mIL-6R蛋白。
(3)细胞实验中Ad5 mIL-6R对多种肿瘤细胞有溶瘤作用。
(4)细胞实验中Ad5 mIL-6R能够特异性地阻断IL-6/STAT3炎症通路的活化。
(5)动物实验中Ad5 mIL-6R对小鼠肝癌有显著治疗作用并显著延长小鼠生存时间。
(6)动物实验中Ad5 mIL-6R对小鼠肺癌有显著治疗作用并显著延长小鼠生存时间。
(7)动物实验中Ad5 mIL-6R对小鼠结直肠癌有治疗作用并延长小鼠生存时间。
(8)动物实验中Ad5 mIL-6R有显著促进免疫细胞肿瘤浸润和活化抗肿瘤免疫的作用。
附图说明
图1可溶性IL-6R(sIL-6R)的突变体(mIL-6R)示意图及其cDNA序列构建:
如图所示,在sIL-6R的FN3的结构域区插入四个突变氨基酸,具体为:228丙氨酸突变为天冬氨酸(A228D)、230位天冬酰胺突变为天冬氨酸(N230D)、280位组氨酸突变为丝氨酸(H280S)、281位天冬氨酸突变为缬氨酸(D281V)。
图2突变型可溶性IL-6受体阻断IL-6/STAT3信号通路并能抑制肿瘤生长:
如图A&B所示,表达突变型可溶性IL-6受体的质粒转染肺癌细胞株A549或LLC细胞后,分别于24、48、72小时加入IL-6刺激,与转染空白载体的对照相比,mIL-6R能够显著降低IL-6/STAT3的磷酸化水平,提示突变型可溶性IL-6受体可以结合IL-6并阻断其活化作用。
如图C-F所示在6-8周龄C57BL6小鼠肺癌皮下瘤模型中,给予对照空白质粒或表达突变型白介素-6受体质粒静脉高压水动力注射,如图D所示两组小鼠的体重均无明显的波动,说明突变型IL-6受体的治疗无明细副作用,且具有显著的抑制肺癌生长的作用(图E&F),两组有显著性差异。
图3为本发明的表达sIL-6R突变型(mIL-6R)的复制型腺病毒目的基因载体pShuttle Ad5-mIL-6R构建及目标蛋白的表达:
如图A所示,本发明的表达可溶性mIL-6R的重组溶瘤腺Ad5 mIL-6R病毒的构建基因序列结构示意图:首先将表达mIL-6R的基因片段克隆联接至腺病毒骨架质粒的CMV启动子上,该片段再通过表达可被剪切多肽T2A的基因序列链接腺病毒早期复制元件基因E1A;再将构建好的pShuttle Ad5-mIL-6R经PmeI酶切线性化之后,与骨架质粒pAdEasy-1同时转染大肠杆菌BJ5183,经卡那霉素抗性筛选阳性克隆菌落,扩增后提取质粒测序鉴定,再经PacI酶切后转染293T细胞,进行腺病毒的包装和拯救。图B&C为所构建的表达可溶性mIL-6R的重组溶瘤腺病毒(Ad5 mIL-6R)感染不同细胞并不同时间收取的细胞培养上清中,表达目标蛋白的情况。图B为293T细胞感染Ad5 mIL-6R后48h上清,图C为A549细胞感染Ad5 mIL-6R后72h的上清。结果显示重组溶瘤腺病毒Ad5 mIL-6R感染细胞后能够快速表达并大量分泌目标蛋白mIL-6R。
图4为本发明的重组溶瘤腺病毒Ad5 mIL-6R在肿瘤细胞中具有自我复制的能力:
进一步,在多种细胞株验证了重组溶瘤腺病毒Ad5 mIL-6R的复制能力。如图A所示,Ad5 mIL6R感染人或鼠的结肠癌细胞株后6小时、24小时、48小时、72小时、96小时的病毒复制情况,分别在相应时间点收取细胞,提取病毒基因组DNA,通过Q-PCR检测AD5的拷贝数;图B表明Ad5 mIL6R感染人或鼠的肺癌细胞株A549、LLC和H1299后不同时间点病毒复制的拷贝数。结果证实突变型可溶性IL-6受体的表达和分泌并不影响Ad5 mIL6R的复制能力。
图5表达突变型可溶性IL-6受体的重组溶瘤腺病毒Ad5 mIL-6R在细胞水平具有广谱的溶瘤作用:
在包括肺癌、肝癌、结直肠癌的多种细胞株中,Ad5 mIL6R具有显著的溶瘤作用,所表达的目的蛋白,没有影响腺病毒的溶瘤作用,甚至在A549、LLC、H1299、Hepa1-6、LM3及C3A细胞株中,表达突变型IL-6受体的复制性腺病毒具有稍强的溶瘤作用。
图6为本发明的重组溶瘤腺病毒Ad5 mIL-6R显著阻断IL-6/STAT3炎症活化通路的作用
Ad5 mIL6R抑制结直肠癌、肺癌细胞IL6/STAT3活化通路。Western blot检测重组溶瘤病毒Ad5 mIL6R分别感染结直肠癌、肺癌细胞24h后加IL6细胞因子刺激检测细胞中STAT3磷酸化水平。结果显示,Ad5 mIL6R感染的细胞中,STAT3磷酸化被抑制,证实Ad5mIL6R具有显著的抑制IL-6/STAT3炎症通路活化的作用。
图7为本发明的重组溶瘤腺病毒Ad5 mIL-6R具有显著抑制肝癌的作用
在免疫健全Balbc小鼠中接种H22肝癌细胞株,形成小鼠皮下肝癌实体后,给予Ad5mIL6R瘤内注射,相比PBS治疗或Ad5 Ctrl治疗组,Ad5 mIL6R具有显著的抑制肝癌生长的作用,反应率达100%,其中约50%小鼠的肿瘤包块消失,且小鼠长期存活。结果提示Ad5mIL6R具有极其显著的治疗肝癌的作用。
图8为本发明的重组溶瘤腺病毒Ad5 mIL-6R具有显著抑制肺癌的作用
在免疫健全C57BL6小鼠中接种LLC肺癌细胞株,形成皮下实体瘤后,给予Ad5mIL6R瘤内注射,相比PBS治疗或Ad5 Ctrl治疗组,Ad5 mIL6R具有显著的抑制肺癌生长的作用,肿瘤应答反应率达100%,其中约20-30%小鼠的肿瘤包块完全消失,且小鼠长期存活。结果提示Ad5 mIL6R具有极其显著的治疗肺癌的作用。
图9为本发明的重组溶瘤腺病毒Ad5 mIL-6R具有显著抑制结直肠癌的作用
在免疫健全C57BL6小鼠中接种MC-38结直肠癌细胞株,形成皮下实体瘤后,给予Ad5 mIL6R瘤内注射,相比PBS治疗或Ad5 Ctrl治疗组,Ad5 mIL6R具有显著的抑制肿瘤生长的作用,且小鼠生存时间显著延长。结果提示Ad5mIL6R具有显著的治疗结直肠癌的作用。
图10为本发明的重组溶瘤腺病毒Ad5 mIL-6R显著提高CD8+T淋巴细胞瘤内浸润和抗肿瘤活性的作用
免疫健全的C57BL/6肺癌荷瘤小鼠,经瘤内注射溶瘤病毒Ad5 mIL-6R,隔天注射一次5*108PFU/只,共2次,第二次注射后48h,取肿瘤组织分离单细胞悬液,检测CD8+T淋巴细胞的数量及其抗肿瘤相关活化标志物如CD69、颗粒酶B(GzmB)和γ干扰素(IFNγ)的表达水平。结果显示,重组溶瘤腺病毒Ad5 mIL-6R治疗组肿瘤组织肿CD8+T淋巴细胞的比例较非治疗组显著升高,且表达活化标志CD69的CD8+T淋巴细胞比例也显著提高;且表达抗肿瘤活性标志物GzmB和IFNγ的CD8+T淋巴细胞比例也显著升高。说明Ad5 mIL-6R具有显著提高CD8+T淋巴细胞瘤内浸润和抗肿瘤活性的作用。
具体实施方式
下面结合具体实施例对本发明进行进一步的解释和说明,但应理解,所给出的实施例只作为举例说明,不以任何方式对本发明构成任何限制。
实施例1:人源突变型可溶性IL-6受体cDNA的构建:
IL-6R cDNA购于金斯瑞有限公司,在此基础上进行PCR改造,在IL-6R两端加上AGE1、XhoI酶切位点,并对目标位点成功突变。引物设计:
IL-6R AGE1 F:ATGACCGGTGCCACCATGCTGGCCGTC;
IL-6 225R:ACAGTCACTGCCGTGGACAGAGACCC;
IL-6 225F:CGTGGACAGAGACCCCCGCTGGCTCA;
IL-6 277R:CATCACTGTGTCATCTCCGTCGCCTG;
IL-6 277F:CATCTCCGTCGCCTGGAGCGGCCTGAG;
IL-6AD5 XhoI R:CATCATCACCACCATCACTAACTCGAGTCTAGAGGGC。
PCR扩增基因片段纯化:
a.进行柱平衡:收集管中放入吸附柱后,在吸附管中加入500μl平衡液BL,12000rpm,离心一分钟,将收集管中的废液倒掉,将吸附柱重新放入收集管中;
b.根据PCR反应液的体积,向其中加入五倍体积的结合液PB,混匀;
c.将b步骤中的液体加入到吸附柱CB2中,室温放置两分钟,12000rpm,离心一分钟,将收集管中的废液倒掉,吸附柱重新放入收集管中;
d.将600μl PW(使用前确认是否加入无水乙醇)加入到吸附柱CB2中,12000rpm,离心一分钟,将收集管中的废液倒掉,吸附柱重新放入收集管中;
e.将d步骤重复操作一次;
f.确认吸附柱放于收集管中后进行空转离心,12000rpm,离心一分钟,以确保去除多余的漂洗液,打开吸附柱盖子室温放置数分钟晾干;
g.将吸附柱CB2放于一个干净的离心管中,在吸附柱中央悬空滴加50μl TE,于室温中放置五分钟后,12000rpm离心两分钟收集DNA纯化后的片段;
h.对DNA浓度和纯度进行测定;
成功构建的突变型IL-6受体cDNA与pshuttle穿梭质粒酶切后连接。
DH5α感受态细胞转化:
a.将-80℃冰箱中将DH5α感受态细胞放置于冰上融化,解冻后立即使用;
b.取50μl感受态细胞加入1ng DNA,轻柔混匀,冰上静置5min;
c.42℃水浴,热激45s,立刻置于冰上,静置2min;
d.加入500μl SOC培养基
e.震荡培养箱37℃,150rpm培养1h;
f.2500g离心5min,倒掉适量培养基,剩余培养基重悬菌体,后将菌体涂于100μg/ml卡那霉素抗性的平板并轻轻涂匀,37℃倒置培养过夜。
单克隆的挑取:
在培养板上挑取十个单克隆菌点,分别放于十个含100μg/ml卡那霉素的五毫升LB培养基的无菌试管中,37℃,200rpm,摇床振荡培养12h。
小提质粒:
a.取适量菌种保存于-80℃冰箱;
b.进行柱平衡:收集管中放入吸附柱后,在吸附管中加入500μl平衡液BL,12000rpm,离心1min,弃废液,将吸附柱重新放入收集管中;
c.12000rpm,离心1min,收集菌体;
d.将250μl的P1溶液加入到存有菌体的离心管中,彻底震荡悬浮菌体;
e.向离心管中加入250μl的P2溶液,上下温和翻转六到八次混匀,裂解菌体;
f.向离心管中加入250μl的P3溶液,立即上下翻转六到八次混匀,12000rpm离心10min;
g.收集f步骤中的上清液,用移液器将其转移至吸附柱CP3中,12000rpm离心1min,弃废液,吸附柱CP3重新放入收集管;
h.向吸附柱CP3中加入600μl漂洗液PW,12000rpm离心1min,弃废液,吸附柱CP3重新放入收集管中;
i.重复步骤h;
j.12000rpm,空转离心2min,弃残余漂洗液;
k.将吸附柱CB3置于离心管中,在吸附柱滴加100μl TE,于室温放置5min,12000rpm离心2min后收集质粒并测定DNA浓度及纯度。
获得的DNA进行测序鉴定,证实突变位点均正确。
实施例2突变型IL-6R有显著的抑制IL-6/STAT3炎症通路并抑制肿瘤生长的作用
表达突变型可溶性IL-6受体的Shuttle质粒(Mutant sIL-6R)转染A549细胞,分别在24、48、72小时后加入人白介素-6(hIL-6)15ng/ml刺激半小时后收集细胞提取蛋白,Western Blot检测P-STAT3和GAPDH,如图A所示。
表达突变型可溶性IL-6受体的Shuttle质粒(Mutant sIL-6R)转染LLC细胞,分别在24、48、72小时后加入鼠白介素-6(mIL-6)15ng/ml刺激半小时后收集细胞提取蛋白,western blot检测p-STAT3和GAPDH,如图B所示。
图C为小鼠体内治疗方案图,选用6-8周C57BL6雄性小鼠,皮下种植1x106LLC小鼠肺癌细胞,第七天根据肿瘤大小平均分为两组,分别给予尾静脉高压水动力注射空白质粒(对照组5只),或表达突变型可溶性IL-6受体质粒(治疗组,5只),共注射两次,间隔七天。小鼠体重四天测量一次,从第0天开始,如图D所示。E肿瘤接种日为-7天,第0天开始测量小鼠肿瘤体积,每四天一次,肿瘤体积为(长x宽2)/2,数据显示为均值+SD,*p<0.05,如图E所示。第12天所有小鼠肿瘤体积大小对比图,数据显示为均值+SD,*p<0.05,如图F所示。
实施例3表达sIL-6R突变型(mIL-6R)的复制型腺病毒载体Ad5-mIL-6R的构建及目标蛋白表达的检测:
本实验室使用的Ad5重组溶瘤腺病毒在AdEasy系统上进行改造,包括:构建连接目的基因的穿梭质粒pShuttle;穿梭质粒与病毒载体同源重组;拯救溶瘤腺病毒;扩增并纯化病毒;检测病毒滴度。
如图A所示重组腺病毒载体结构图,分别用PCR技术克隆出CMV启动子基因片段、2A自剪切肽段基因片段、E1A基因片段、BGH PolyA加尾信号基因片段,再用PCR技术依顺序拼接接4个基因片段,最后用Exnase连接酶将拼接起来的片段连接到pShuttle;图B为WB检测重组溶瘤病毒Ad5mIL6R感染293T细胞48h后上清中目标蛋白的表达(两个样本均为Ad5mIL6R感染);图C为重组复制型腺病毒Ad5 mIL6R感染A549细胞后72h胞培养上清中目标蛋白表达,对照病毒Ad5 Ctrl感染后无目标蛋白表达。
引物序列表
病毒拯救步骤:
将腺病毒的基因组转染入293T细胞中,在293T细胞内完成病毒各个原件的组装。主要包括5个过程:PacI酶线性化病毒基因组、293T细胞内包装病毒、病毒扩增、病毒纯化以及病毒滴度的测定。
重组腺病毒滴度测定:
a以密度2.5×105cells/孔,将293T细胞种在24孔板上;
b.依次十倍稀释病毒;
c.梯度稀释好的病毒放于24孔板中,每孔50μl,一个阴性对照;
d.于37℃,5%CO2培养箱培养48小时,弃掉培养基;
e.每孔加入1ml预冷的甲醇固定,置于-20℃,10分钟;
f.弃掉甲醇,每孔加入1ml PBS+1%BSA,轻柔地洗三次;
g.用PBS+1%BSA按照1:1000的比例稀释鼠源的Anti-Hexon抗体
h.弃掉上清,每孔加入500ul已稀释的Anti-Hexon抗体,置于37℃孵育1小时;
i.弃掉抗体,每孔加入1ml PBS+1%BSA,轻柔地洗三次;
j.用PBS+1%BSA按照1:500的比例稀释兔源的Anti-Mouse抗体;
k.弃掉上清,每孔加入500ul已稀释的兔源的Anti-Mouse抗体,置于37℃孵育1小时;
l.用1x Stable Peroxidase Buffer按照1:10稀释的比例稀释10x DAB;
m.弃掉抗体,每孔加入1ml PBS+1%BSA,轻柔地洗三次;
n.弃掉上清,每孔加入500ul 1x DAB,室温孵育10分钟;
o.弃掉DAB,每孔加入1ml PBS;
p.在20倍物镜下,最少计算三个视野内棕色阳性细胞的数量,然后计算每孔内棕色阳性细胞的数量平均值;
q.病毒滴度的计算:病毒滴度=(每个视野内阳性细胞数x视野内总细胞数)/(每孔加入病毒体积x稀释倍数)。
实施例4表达sIL-6R突变型的复制型腺病毒Ad5 mIL-6R在肿瘤细胞内能够大量复制:
Ad5 mIL-6R分别以不同的MOI感染不同的细胞株,包括人或鼠的结直肠癌细胞株(图A)、肺癌细胞株(图B),并在相应的时间点收取细胞并提取DNA,用Q-PCR标准曲线法测定病毒的拷贝数,以6小时病毒拷贝数为1,根据病毒拷贝数相对增加倍数进行呈现。数据表示为Mean+SD。如图所示,结果证实突变型可溶性IL-6受体的表达和分泌并不影响Ad5 mIL6R的复制能力。
实施例5表达sIL-6R突变型的复制型腺病毒Ad5 mIL-6R具有显著的溶瘤作用:
Ad5 mIL-6R分别以不同的MOI(0.1、1、5、10、20、40、80)感染A549、LLC、H1299、Hepa1-6、LM3、SK-Hep-1、MC-38、HT29、CT-26及SW620细胞种细胞株,72小时后,用MTT法检测肿瘤细胞活率。数据表示为均值+SD。如图所示,在包括肺癌、肝癌、结直肠癌的多种细胞株中,Ad5 mIL6R具有显著的溶瘤作用,所表达的目的蛋白,没有影响腺病毒的溶瘤作用,甚至在A549、LLC、H1299、Hepa1-6、LM3及C3A细胞株中,表达突变型IL-6受体的复制性腺病毒具有稍强的溶瘤作用。
实施例6表达sIL-6R突变型的复制型腺病毒Ad5 mIL-6R显著阻断IL-6/STAT3炎症活化通路
Ad5 mIL-6R感染不同的肿瘤细胞株24h后,在培养基中加入15ng/ml人或鼠细胞因子IL-6,半小时后收取细胞蛋白,用WB检测磷酸化STAT3的表达水平。结果显示,Ad5 mIL6R感染的细胞中,STAT3磷酸化被抑制,证实Ad5mIL6R具有显著的抑制IL-6/STAT3炎症通路活化的作用。
实施例7重组溶瘤腺病毒Ad5 mIL-6R具有显著抑制肝癌的作用
6-8周龄的Balbc雄性小鼠皮下接种肝癌细胞H22,当肿瘤体积>100mm3时,给予小鼠瘤内注射Ad5 mIL-6R,5x108PFU/次/只,隔天重复注射一次共3次,每两天监测肿瘤大小、小鼠体重及生存状况。NS,无统计学意义,**P<0.01,****P<0.0001。如图所示,相比PBS治疗或Ad5 Ctrl治疗组,Ad5mIL6R具有显著的抑制肝癌生长的作用,反应率达100%,其中约50%小鼠的肿瘤包块消失,且小鼠长期存活。提示Ad5 mIL6R具有极其显著的治疗肝癌的作用。
实施例8重组溶瘤腺病毒Ad5 mIL-6R具有显著抑制肺癌的作用
6-8周龄C57BL6雄性小鼠,皮下接种小鼠肺癌细胞株LLC,当肿瘤体积>100mm3时,给予小鼠瘤内注射溶瘤病毒Ad5 mIL6R,5x108PFU/次/只,隔天一次共3次,每两天监测肿瘤大小、小鼠体重及生存状况。**P<0.01,****P<0.0001。如图所示,相比PBS治疗或Ad5 Ctrl治疗组,Ad5 mIL6R具有显著的抑制肺癌生长的作用,肿瘤应答反应率达100%,其中约20-30%小鼠的肿瘤包块完全消失,且小鼠长期存活。结果提示Ad5 mIL6R具有极其显著的治疗肺癌的作用。
实施例9重组溶瘤腺病毒Ad5 mIL-6R具有显著抑制结直肠癌的作用
6-8周龄C57BL6雄性小鼠,皮下接种小鼠结直肠癌细胞株MC38,当肿瘤体积>100mm3时,给予小鼠瘤内注射溶瘤病毒Ad5 mIL6R,5x108PFU/次/只,隔天一次共3次,每两天监测肿瘤大小、小鼠体重及生存状况。**P<0.01,NS,无统计学意义。如图所示,相比PBS治疗或Ad5 Ctrl治疗组,Ad5 mIL6R具有显著的抑制肿瘤生长的作用,且小鼠生存时间显著延长。结果提示Ad5mIL6R具有显著的治疗结直肠癌的作用。
实施例10重组溶瘤腺病毒Ad5 mIL-6R显著提高CD8+T淋巴细胞瘤内浸润和抗肿瘤活性
6至8周龄的雄性C57BL/6小鼠,皮下接种小鼠肺癌细胞LLC 4x106/只,当肿瘤体积达到500mm3时,将小鼠随机分组,瘤内(I.T.)注射溶瘤病毒Ad5mIL-6R,隔天注射一次5*108PFU/只,共2次,第二次注射后48h,麻醉后处死小鼠,剥取肿瘤组织并将其放入六孔板中。加入200μl 0.2%的胶原酶IV溶液,用眼科剪将组织剪成1-2mm的组织块,然后补充胶原酶溶液至1ml并转移至2ml Eppendorf管中,经胶原酶消化2h收取细胞悬液过筛获得单细胞悬液,计数后细胞进行荧光染色。具体步骤如下:2×106的单细胞悬液中加入相应的单克隆抗体,室温下孵育15min。用4%(w/v)多聚甲醛(PFA;Cat#1004965000,Sigma-Aldrich)固定细胞,并且用FACS Caliber流式细胞仪进行检测。数据分析使用FlowJo软件(Version10.4.0,Tree Star Inc)。
进行细胞内蛋白染色时,取2×106细胞按照流式表面染色方法标记细胞表面抗原,再加入4%PFA固定细胞,经1X Permeabilization buffer透膜后离心,1xPermeabilization buffer重悬细胞,避光放置30min,分别加入PE anti-mouse IFN-γAntibody、PE anti-human/mouse Granzyme B Recombinant Antibody,避光孵育30min。PBS洗涤细胞、离心后重悬,上流式细胞仪检测。抗体如下:大鼠抗小鼠CD45(APC,Clone 30-F11,Cat#559864,BD或PerCP-Cy5.5,Clone30-F11,Cat#103132,Biolegend);CD8(PerCP-Cy5.5,Clone 53-6.7,Cat#551162,BD);Granzyme B(PE,Clone QA16A02,Cat#372207,Biolegend);IFN-γ(PE,Clone XMG1.2,Cat#505807,Biolegend);CD69(FITC,CloneH1.2F3,Cat#104505,Biolegend)。结果如图显示,重组溶瘤腺病毒Ad5 mIL-6R治疗组肿瘤组织肿CD8+T淋巴细胞的比例较非治疗组显著升高,且表达活化标志CD69的CD8+T淋巴细胞比例也显著提高;且表达抗肿瘤活性标志物GzmB和IFNγ的CD8+T淋巴细胞比例也显著升高。说明Ad5 mIL-6R具有显著提高CD8+T淋巴细胞瘤内浸润和抗肿瘤活性的作用。
由以上结果可知,本发明提供了一种对多种实体恶性肿瘤具有显著治疗作用的复制型溶瘤腺病毒Ad5 mIL-6R,该病毒在肿瘤细胞内能够高表达并大量分泌可溶性mIL-6R,该蛋白能够显著阻断IL-6/STAT3炎症通路,有效促进抗肿瘤疗效。一个重组溶瘤腺病毒,同时整合多种独特的抗肿瘤机制,对多种炎症相关性的恶性肿瘤具有广谱的治疗作用,尤其是肝癌、肺癌和结直肠癌等,具有预料不到的抗肿瘤效果,可以用来制备抗肿瘤药物。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。
序列表
<110> 南京惟亚德生物医药有限公司
<120> 一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒及其应用
<160> 5
<170> SIPOSequenceListing 1.0
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gacagcgtga ctctgacctg cccgggggta gagccggaag acaatgccac tgttcactgg 180
gtgctcagga agccggctgc aggctcccac cccagcagat gggctggcat gggaaggagg 240
ctgctgctga ggtcggtgca gctccacgac tctggaaact attcatgcta ccgggccggc 300
cgcccagctg ggactgtgca cttgctggtg gatgttcccc ccgaggagcc ccagctctcc 360
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ccggagggag acagctcttt ctacatagtg tccatgtgcg tcgccagtag tgtcgggagc 600
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aacatcacag tcactgccgt ggacagagac ccccgctggc tcagtgtcac ctggcaagac 720
ccccactcct ggaactcatc tttctacaga ctacggtttg agctcagata tcgggctgaa 780
cggtcaaaga cattcacaac atggatggtc aaggacctcc agcatcactg tgtcatctcc 840
gtcgcctgga gcggcctgag gcacgtggtg cagcttcgtg cccaggagga gttcgggcaa 900
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cctccagctg agaacgaggt gtccaccccc atgcaggcac ttactactaa taaagacgat 1020
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cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180
gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240
ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300
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gcaagaccta cccgccgtcc taaaatggcg cctgctatcc tgagacgccc gacatcacct 660
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cctgagatac acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt 780
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gacttgagct gtaaacgccc caggccataa 870
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tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180
gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225
Claims (10)
1.一种可溶性IL-6R的突变体mIL-6R在如下a)或b)中的应用:
在制备促进溶瘤腺病毒溶瘤药物中的应用;
在制备抗肿瘤药物中的应用。
2.根据权利要求1所述的应用,所述的突变体mIL-6R为将可溶性IL-6R的FN3结构域区的氨基酸序列的第228位丙氨酸突变为天冬氨酸;第230位天冬酰胺突变为天冬氨酸;第280位组氨酸突变为丝氨酸;第281位天冬氨酸突变为缬氨酸。
3.一种特异性阻断促肿瘤炎症通路的重组复制型溶瘤腺病毒,其特征在于:该病毒能够在肿瘤中选择性复制、溶瘤、抑制肿瘤生长、促进抗肿瘤免疫并延长治疗个体生存时间;同时能够表达并分泌mIL-6R蛋白,该蛋白能够特异性阻断促肿瘤炎症;优选的,所述重组复制型溶瘤腺病毒为Ad5 mIL-6R,即所用腺病毒载体为Ad5。
4.根据权利要求3所述的重组复制型溶瘤腺病毒,其特征在于,所述的突变体mIL-6R为将可溶性IL-6R的FN3结构域区的氨基酸序列的第228位丙氨酸突变为天冬氨酸;第230位天冬酰胺突变为天冬氨酸;第280位组氨酸突变为丝氨酸;第281位天冬氨酸突变为缬氨酸;
优选的,编码前述突变体mIL-6R的DNA分子的核苷酸序列选自如下A)或B):
如SEQ ID NO:1所示;
与SEQ ID NO:1所述的序列具有80%以上同源性的核苷酸序列。
5.权利要求3或4任一所述的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
6.权利要求3或4任一所述的复制型溶瘤腺病毒与第二治疗药物联合在制备抗肿瘤药物中的应用。
7.根据权利要求6所述的应用,其特征在于,联合用药时,采用同时给药、或不分次序先后单独给药。
8.根据权利要求1、2、5或6所述的应用,其特征在于,所述肿瘤选自胶质瘤、食道癌、胃癌、乳腺癌、胰腺癌、肾癌、宫颈癌、卵巢癌、前列腺癌、头颈部鳞癌、黑色素瘤、骨肉瘤、纤维肉瘤、横纹肌肉瘤、多发性骨髓瘤、神经内分泌肿瘤,淋巴瘤中的其中一种;优选的,所述的肿瘤为肝癌、肺癌或结直肠癌。
9.根据权利要求1、2、5或6所述的应用,其特征在于,所述药物为适于口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
10.一种药物组合物,其特征在于,包含权利要求3-5所述的重组复制型溶瘤腺病毒,以及药学上可接受的载体;优选的,所述药学上可接受的载体选自填充剂、吸收剂、润湿剂、粘合剂、崩解剂或润滑剂。
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