CN110982841A - 重组腺相关病毒载体、重组腺相关病毒aav8-pd1及其应用 - Google Patents
重组腺相关病毒载体、重组腺相关病毒aav8-pd1及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,涉及一种重组腺相关病毒载体、重组腺相关病毒及其应用。该重组腺相关病毒载体为将PD‑1细胞外段核苷酸序列插入腺相关病毒载体而形成的载体;所述PD‑1细胞外段核苷酸序列如SEQ ID NO:1所示。本发明提供的重组腺相关病毒载体,将PD1的胞外段(共170个氨基酸)插入至腺相关病毒AAV8表达后分泌出可溶性PD1,这样就能够通过可溶性PD1与肿瘤细胞上的PDL1结合,有效阻断PD‑1/PD‑L1信号通路,以阻止肿瘤细胞上的PDL1与T细胞上PD1结合而避免T细胞耗竭,改善宿主的T细胞功能,提高免疫细胞对肿瘤细胞的杀伤作用,并且对正常的细胞、组织和器官几乎无损伤,具有极高的安全性和可靠性。
Description
技术领域
本发明属于生物技术领域,更具体地,涉及一种重组腺相关病毒载体、重组腺相关病毒及其应用。
背景技术
肝细胞癌(HCC)是世界上第六大最常见的恶性肿瘤和第二大常见癌症相关死亡原因。其主要病因是乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染以及肝硬化。GLOBOCAN2018数据估计每年将发生841,000例原发性肝癌新病例和782,000例死亡病例。原位肝移植(OLT)是治疗HCC和潜在肝硬化的有效方法,被认为是最佳治疗选择。HCC的早期治疗包括切除、消融和肝移植;不幸的是,大多数HCC病例处于晚期阶段或已经发生转移,适用全身治疗,不适合OLT。由于其高转移能力和复发率,晚期HCC的五年存活率很低。因此,寻求一种高效低毒的肝细胞癌治疗策略是非常迫切的。
近年来,一些新的治疗方法不断被应用于HCC治疗的临床实验中。如微小RNA靶向治疗,通过抑制miR-222/221,抑制PI3K/AKT途径而克服索拉非尼耐药;CDK4/6抑制剂通过诱导肿瘤抑制因子AMPK下调,促进HCC的凋亡和自噬;转化生长因子-β(TGFβ)阻断剂通过抑制SMAD蛋白磷酸化,抑制肿瘤的生长和迁移;Wnt信号通路拮抗剂抑制癌症干细胞(CSC)以及肿瘤细胞的增殖和迁移等。免疫疗法作为一种新的较为有效的治疗方法,也已经应用于临床HCC的治疗。
肿瘤细胞具有逃避免疫应答的能力,如何有效激活肿瘤微环境中T细胞的抗肿瘤活性,并保持其发现和攻击癌细胞的持久性能力是肿瘤免疫亟待探索的热点。新近有关免疫检测点及其抑制剂(ICI)在肿瘤治疗中的转化应用研究为解决这一瓶颈问题带来新的希望。程序性细胞死亡蛋白1(Programmed cell death protein1,PD1)是表达在T细胞表面的一种重要的免疫抑制跨膜蛋白,与其配体PDL1(主要表达于肿瘤细胞和肿瘤微环境中,长时间地暴露于抗原并介导T细胞抑制作用)相结合而对淋巴细胞的活化产生抑制作用,从而抑制肿瘤微环境中免疫细胞的免疫应答反应。目前最常见免疫检查点抑制剂包括nivolumab和pembrolizumab,它们已经在美国和欧盟等国家获得了的上市许可。这些抗体针对受体PD-1,PD-1由许多免疫细胞表达,如活化T细胞,B细胞,自然杀伤细胞,单核细胞和树突细胞(DC)等。其配体程序性细胞死亡蛋白-1配体1(PD-L1)也在多种细胞类型上表达,如活化T细胞和DC以及广泛的非造血组织如肺。其生理功能是在周围炎症期间维持体内的免疫学稳态以防止自身免疫。重点是,PD-L1在一些肿瘤细胞中高表达,它们在肿瘤微环境中的相互作用可以抑制局部抗肿瘤T细胞应答,从而促进癌细胞的免疫逃逸。因此,已设计抗体如nivolumab就是以阻断PD-1和PD-L1之间的相互作用,以恢复T细胞活化并防止癌细胞的免疫逃避。这种免疫检查点抑制剂(ICIs)已经在临床试验中观察到明显的疗效,癌症免疫疗法在2013年的时候就被认为是突破性技术,并被FDA批准用于治疗HCC。然而其价格昂贵,且不具有靶向性,静脉给药后会有全身反应。这种治疗通常与许多免疫相关的不良事件有关,包括结肠炎,皮炎以及肝炎以及Vogt-Koyanagi-Harada病(VKH)等。约7%-12%的接收单药αPD-1/αPD-L1单克隆抗体患者伴有3-4级免疫相关的不良事件。也有研究表明在进行小鼠实验时,会造成小鼠明显脱毛。因此需要探索肿瘤特异性递送ICI的创新策略。基因水平上的治疗性蛋白质的递送可以用病毒载体完成,包括衍生自腺相关病毒(AAV)的载体。
腺相关病毒(Adeno-associated virus,AAV)是一种单链DNA细小病毒,以其安全性好、宿主范围广、免疫原性低、携带外源基因可长期表达等优点而备受关注,广泛应用于人类和动物相关疾病的基因治疗。由于衣壳蛋白序列之间存在差异,导致不同血清型的AAV与不同的细胞表面受体结合,这使得不同AAV血清型具有不同的组织嗜性,如AAV9对心脏、骨骼肌的高亲和力;AAV5对肺、中枢神经系统的高亲和力。其中AAV8在目前应用的AAV载体中嗜肝性最强,携带目的基因的载体在动物体内可以形成稳定的肝脏转染。重组AAV8可通过肝内注射或静脉注射的方法进入小鼠体内,可显示出显著的肝脏趋向性,且目的基因表达可以持续至少6个月,远比临床使用的PD-1抗体效果持久。
PD1作为一种跨膜蛋白,表达在细胞膜上,通过其细胞外段区域与配体PDL1相结合而发挥其作用。与单克隆抗体相比,可溶性PD1分子亦能够通过直接的受体/配体相互作用封闭肿瘤细胞表面PDL的协同刺激信号,且其剪切变异体片段较小,易于装载实现外源性表达分泌。
因此,亟需一种提高PD1在肿瘤细胞中表达的方法。
发明内容
本发明的目的是提供一种重组腺相关病毒载体、重组腺相关病毒及其应用,以阻止肿瘤细胞上的PDL1与T细胞上PD1结合而避免T细胞耗竭,提高抗肿瘤免疫活性。
腺病毒相关病毒(adenovirus associated virus,AAV)是一类单链线状DNA缺陷型病毒,其基因组DNA小于5kb,无包膜,外形为裸露的20面体颗粒。重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒,由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低,在体内表达外源基因时间长等特点,被视为最有前途的基因转移载体之一,在世界范围内的基因治疗和疫苗研究中得到广泛应用。AAV载体不需要腺病毒辅助,不插入宿主的基因组,而是游离于宿主细胞基因之外,呈卫星状稳定表达。并且腺相关病毒载体(AAV)体内的感染效率极高。
为了实现上述目的,本发明第一方面提供一种重组腺相关病毒载体。该重组腺相关病毒载体为将PD-1细胞外段核苷酸序列插入腺相关病毒载体而形成的载体;所述PD-1细胞外段核苷酸序列如SEQ ID NO:1所示。
SEQ ID NO:1为5'-ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG-3'。
具体地,所述PD-1细胞源自于人类,因而,PD-1细胞外段核苷酸序列为源自于人类的PD-1细胞外段核苷酸序列。
人PD1细胞外段的蛋白序列>sp|Q15116|1-170,如SEQ ID NO:2所示。
SEQ ID NO:2为MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV。
本发明第二方面一种重组腺相关病毒AAV8-PD1。所述重组腺相关病毒为重组8型腺相关病毒。
本发明第三方面提供上述的重组腺相关病毒载体在制备阻断PD-1/PD-L1信号通路或促进CD8阳性T细胞生长的制剂中的应用。上述的重组腺相关病毒载体或重组腺相关病毒可以通过静脉注射转染至宿主细胞中。
本发明第四方面提供上述的重组腺相关病毒载体在制备治疗肿瘤的制剂中的应用。
本发明第五方面提供上述的重组腺相关病毒载体在制备治疗肝癌的制剂中的应用。
本发明第六方面提供上述的重组腺相关病毒在制备阻断PD-1/PD-L1信号通路或促进CD8阳性T细胞生长的制剂中的应用。
本发明第七方面提供上述的重组腺相关病毒在制备治疗肿瘤的制剂中的应用。
本发明第八方面提供上述的重组腺相关病毒在制备治疗肝癌的制剂中的应用。
本发明第一方面提供的重组腺相关病毒载体,将PD1的胞外段(共170个氨基酸)插入至腺相关病毒AAV8表达后分泌出可溶性PD1,这样就能够通过可溶性PD1与肿瘤细胞上的PDL1结合,有效阻断PD-1/PD-L1信号通路,以阻止肿瘤细胞上的PDL1与T细胞上PD1结合而避免T细胞耗竭,改善宿主的T细胞功能,提高免疫细胞对肿瘤细胞的杀伤作用,并且对正常的细胞、组织和器官几乎无损伤,具有极高的安全性和可靠性。
本发明第二方面提供的重组腺相关病毒(AAV8-PD1)能够靶向感染肝脏部位,在肝脏细胞内表达及分泌PD-1细胞外段蛋白,从而有效阻断PD-1/PD-L1信号通路,改善肿瘤患者的T细胞功能,提高免疫细胞对肿瘤细胞的杀伤作用,并且对正常的细胞、组织和器官几乎无损伤,具有极高的安全性和可靠性。
本发明提供的重组腺相关病毒AAV8-PD1能稳定感染肝组织并表达PD1细胞外段蛋白。
本发明提供的重组腺相关病毒AAV8-PD1明显抑制小鼠肿瘤生长。
本发明提供的重组腺相关病毒AAV8-PD1治疗后细胞毒性T淋巴细胞的数量增加。
本发明提供的重组腺相关病毒AAV8-PD1治疗后脾脏中CD8阳性记忆T细胞数量增加。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述,本发明的上述以及其它目的、特征和优势将变得更加明显。
图1示出了AAV、vGFP、以及AAV8-PD1的构建流程图。
图2A示出了不同滴度AAV9-PD1感染Hepa1-6细胞72小时后的荧光显微照片。
图2B示出了不同滴度AAV9-PD1感染Hepa1-6细胞72小时后的荧光统计结果。
图3示出了在对Hepa1-6细胞培养96小时后的PD1蛋白表达情况。
图4示出了实施例2中AAV8-PD1和AAV8-GFP在肝脏中的表达情况。
图5A示出了经重组腺相关病毒处理10天后小鼠肝脏的肉眼观察图。
图5B示出了经重组腺相关病毒处理10天后小鼠肝脏的数据统计分析结果图。其中,P>0.05;*P<0.05;**P<0.01;***P<0.001;****P<0.0001。
图6示出了经重组腺相关病毒处理7天后肿瘤组织中CD4+和CD8+CTL细胞的数量流式分析结果图。
图7示出了CD8+CTL细胞的数量占总活细胞的百分比统计结果图。其中,P>0.05;*P<0.05;**P<0.01;***P<0.001。
图8示出了经AAV8-PD1治疗后小鼠脾细胞中CD8+、CD44+、CD122+和CD4+、CD44+、CD122+的记忆性T细胞数量明显增多。
图9示出了CD8阳性记忆T细胞占总活细胞的百分比统计结果图。其中,P>0.05;*P<0.05;**P<0.01;***P<0.005。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式,然而应该理解,可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。
下面实施例中所使用的材料
小鼠肝癌细胞Hepa1-6来自南京医科大学陈云教授实验室细胞保种;6-8W雄性C57BL/6小鼠由湖南斯莱克景达实验动物有限公司提供(实验动物生产许可证:SCXK(湘)2016-0002);
大肠杆菌菌株DH5α来自Invitrogen;限制性内切酶来自Thermo FisherScientific;HB-infusionTM无缝克隆试剂盒来自Hanbio Biotechnology Co.,Ltd.;质粒DNA小,大量抽提试剂盒大来自Beijing ComWin Biotech;凝胶回收试剂盒来自ShanghaiGeneray Biotech;琼脂糖,琼脂粉来自Sangon Biotech;DNA ladder来自Thermo FisherScientific;KOD-Plus Kit来自TOYOBO CO.,LTD.。
细胞培养瓶、冻存管、吸管、离心管、枪头、冻存盒、细胞培养板等耗材均购自Corning公司;DMEM培养基、胎牛血清(FCS)购自美国Gibico BRL公司;兔抗小鼠PCNA抗体购自Santa Cruz公司。PD1单克隆抗体购自Abcam公司;β-actin抗体购自CST公司;CD45、CD3、CD4、CD8、CD25、CD44、CD69、CD122、FOXP3等流式抗体来自BD公司。
PD-1细胞外段核苷酸序列如SEQ ID NO:1所示。
5'-ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG-3'。
人PD1细胞外段的蛋白序列>sp|Q15116|1-170,如SEQ ID NO:2所示。
5'-MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV-3'
8型腺相关病毒载体(AAV8病毒载体)来自Hanbio Biotechnology,其核苷酸序列如SEQ ID NO:3所示:
5'-ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGATCCGCCACCGGTACCTTAATTAACGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAATTAATTAACGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTACCGGTGCCACCATGGCCCAGTCCAAGCACGGCCTGACCAAAGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCACGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACC-3'。
统计处理
实施例1
(1)细胞培养
①细胞复苏培养
取出在液氮保种的小鼠肝癌Hepa1-6细胞迅速放入37℃的水浴锅中复温融化,在超净台中将冻存液转移置无菌15ml离心管,并加入3ml含有10%胎牛血清,100U/ml青霉素和100mg/ml链霉素的DMEM轻柔吹洗数次,500rpm离心5min,去上清,加入2ml培养基重悬,再转移置6孔板中,于37℃,5%CO2培养箱中培养。
②细胞传代培养
贴壁生长细胞传代:显微镜下观察细胞长满约90%后,弃除旧培养基,用PBS清洗2次,加入0.25%的胰酶,显微镜下观察细胞收缩变圆后,加入完全培养基终止消化,并轻柔吹打至细胞充分分散脱落,收集液体于无菌15ml离心管,800rpm离心5min,弃上清,用完全培养基重悬细胞沉淀,吹打均匀后分皿培养。
(2)重组8型腺相关病毒(AAV8-PD1)表达载体的构建
为了构建重组腺相关病毒8型载体(AAV8-PD1),用引物将人PD1细胞外段序列(来源于UniProtKB-Q15116(PDCD1_HUMAN);https://www.uniprot.org/uniprot/Q15116)扩增;载体37℃酶切,胶回收;片段PCR之后回收处理好的目的片段与载体连接;转化感受态细胞DH5α;抗性:Amp,37℃、230rpm,培养过夜;转化后的平板挑菌,37℃、230rpm摇菌14小时,用菌液进行PCR鉴定,将阳性克隆菌液送测序公司测序。其中,引物包括上游引物和下游引物,核苷酸序列如下所示。
上游引物h-PD1-F:ttttgacctccatagaagacaccgggatccgccaccatgcagatccca(SEQID NO:4)
下游引物h-PD1-R:tcatccttgtagtcgttaattaaggtacccaccagggtttggaactgg(SEQID NO:5)
(3)重组8型腺相关病毒(AAV8-PD1)表达载体的构建
AAV作为基因递送载体能将目的基因导入宿主细胞并稳定表达,并且在感染人类宿主细胞时,能够将位点特异性整合到人类19号染色体的特定区域。将人PD1蛋白的胞外域(氨基酸1-170),以及标记基因GFP,插入到AAV8的开放阅读框中,构建模式如图1。
(4)重组8型腺相关病毒AAV8-PD1表达载体的鉴定
为了检验构建病毒是否成功,分别用不同滴度的重组腺相关病毒AAV8-PD1感染Hepa1-6细胞,72小时后观察到重组腺相关病毒GFP绿色荧光能够在细胞内表达,结果如图2A和图2B所示。上述结果表明,随着病毒感染滴度的增高,GFP绿色荧光蛋白表达增高,病毒感染效率增高;其中5×105vg病毒的感染效果最好。用MOI值为5×105的病毒感染Hepa1-6细胞,在37℃,5%CO2培养箱中培养96小时后提取蛋白质,并进行western blot分析,结果详见图3。由图3可知,Hepa1-6细胞感染AAV8-PD1后可分泌PD1蛋白,而对照组未见PD1蛋白分泌。
实施例2
本实施例用于确定重组腺相关病毒AAV8-PD1对肝脏具有较强的亲和力。
将重组腺相关病毒AAV8-PD1治疗组(AAV8-PD1)、AAV8-GFP治疗组(vGFP)、以及对照组(Mock)用1×1010vg病毒/鼠对小鼠进行尾静脉注射,10天后小动物成像可见光照射可以观察到GFP荧光在肝脏区域表达明显,说明重组腺相关病毒AAV8-PD1对肝脏组织具有靶向作用,详见图4。
实施例3
本实施例构建小鼠肝癌模型。
将6-8周龄雄性C57BL/6小鼠用1×107Hepa1-6细胞在100μl无菌PBS中注射至小鼠右侧腋窝皮下。实验小鼠随机分成四组:重组腺相关病毒AAV8-PD1治疗组(vPD1)、AAV8-GFP治疗组(vGFP)、PD1单克隆抗体治疗组(aPD1)和对照组(Mock)。在Hepa1-6细胞注射后皮下肿瘤大小约为150mm3时,将2.5×1010vg病毒用PBS稀释到100μl肿瘤内注射一次,空白对照仅给PBS。每天监测小鼠的体况和体重,使用游标卡尺测量肿瘤直径大小,并且当肿瘤在任何方向上达到20mm时使小鼠安乐死,结果请参见图5A和图5B。肿瘤体积按照公式计算:肿瘤体积(mm3)=0.52×长×宽2。
由图5A和图5B可知,与对照组比较,AAV8-PD1治疗组肿瘤体积明显减小,肿瘤体重明显减轻(请参见图5A)。AAV8和PD1单抗单独治疗组均有不同程度的抗肿瘤作用,但AAV8-PD1治疗组效果更显著(P<0.0001,请参见图5B)。
实施例4
本实施例对经重组相关病毒AAV8-PD1处理7天后的小鼠,取肿瘤组织流式检测。实验分为四组:重组腺相关病毒AAV8-PD1治疗组(vPD1)、AAV8-GFP治疗组(vGFP)、PD1单克隆抗体治疗组(aPD1)和对照组(Mock)。
取新鲜肿瘤组织放于含少量PBS培养皿中,用剪刀将组织剪成匀浆状;加入10mlPBS,用吸管吸取组织匀浆,以300目尼龙网过滤到试管内;1000rpm离心5min,再用PBS洗3次,每次以800rpm、5min去除细胞碎片;70%冰乙醇固定细胞4℃过夜;1000rpm,3min离心去除上清后分别加CD4、CD8、CD44、CD122流式抗体孵育30min;1000rpm,3min离心去除上清;PBS洗3次,每次1000rpm,3min离心去除上清;最后一次清洗后加入200μl PBS重悬细胞,上流式细胞仪检测。结果请参见图6和图7。
如图6所示,vPD1治疗组的细胞毒性T细胞(CD8阳性T细胞)数量较对照组明显增多。如图7所示,各组细胞毒性T细胞数量分别为对照组为0.16%、vGFP治疗组为0.33%、aPD1治疗组为1.22%、vPD1治疗组为6.08%,组间比较有显著差异(P<0.001)。
在进行上述流式细胞检测的过程中,检测不同处理对脾脏中CD8阳性记忆T细胞(TCM)数量的改变。请参见图8和图9,Mock对照组为13.4%、vGFP治疗组为20.4%、aPD1治疗组为19.7%、vPD1治疗组为25.7%,组间比较有显著差异(P<0.001)。
由上述实施例可知,使用本发明的重组腺相关病毒载体或重组腺相关病毒,与直接使用抗体作为蛋白质相比,治疗性抗体的遗传递送就有了许多优点。如使用重组腺相关病毒载体进行递送可导致单次注射后长期抗体产生,因此可以降低制造成本和治疗周期。在这种情况下,天然可用的AAV血清型将抗体基因递送到肝脏或肌肉组织中,从那里把ICI释放到血流中,导致持久的和高抗体血清水平。
在此之前,溶瘤麻疹病毒和腺病毒已被改造以编码αPD-L1的scFv-Fc和αCLTA-4的scFv-Fc或αCTLA-4抗体。这两种溶瘤病毒都是具有复制能力的颗粒,可裂解人类肿瘤细胞,必须在生物安全水平2级条件下处理。虽然这种特性可能会增强肿瘤浸润淋巴细胞(TILs)的侵袭,但它们不能或只能在感染和复制中消失。而这里应用的AAV载体可以在安全水平1级条件处理。此外,现在对于AAV载体的应用已经有足够的体内临床经验,已经有两种产品现已在欧盟和美国获得上市许可。这使得AAV载体成为遗传递送的理想工具,因为它们比溶瘤病毒更容易应用于研究和治疗。
在本发明中,AAV8-PD1重组腺病毒明显抑制肿瘤生长,肿瘤体积明显减小,肿瘤体重明显减轻。AAV8和PD1单抗单独治疗组均有不同程度的抗肿瘤作用,但AAV8-PD1治疗组效果更显著。在本发明中,AAV8本身的抗肿瘤作用与其表达的Rep蛋白及基因组特殊的ITR结构有关。Rep主要通过与原癌基因或病毒基因的异常启动子结合影响这些基因的活性,从而发挥抗肿瘤作用,且具有细胞特异性。ITR主要通过激活DNA损伤反应,从而引起细胞凋亡、死亡或细胞周期阻滞。同时,AAV还能增加肿瘤细胞对多种抗肿瘤因子的敏感性。此外,AAV感染基因整合至细胞后,可能激发机体抗病毒蛋白免疫的同时,杀伤被AAV感染的肿瘤细胞。
在本发明中,重组的腺相关病毒AAV8-PD1能稳定感染肝组织,并且导致原位种植瘤ICI优先于其他部位表达,因此感染后的肿瘤细胞产生的AAV8-PD1不断释放到肿瘤微环境中,与肿瘤细胞表面的PDL1特异性结合进而活化T细胞免疫应答,发挥抗肿瘤免疫效应。
由上述实施例的实验结果可知,AAV8-PD1治疗组的细胞毒性T细胞(CD8阳性T细胞)数量较对照组明显增多,这表明病毒感染肿瘤细胞后,释放可溶性PD1,与肿瘤细胞上的PDL1结合,阻断T细胞与肿瘤细胞的PD1-PDL1结合。
由上述实施例的实验结果还可知,AAV8-PD1治疗组的脾脏中CD8阳性记忆T细胞数量较对照组明显增多,表示存在全身免疫效应,这可能这对肿瘤转移和复发有重要意义。
以上已经描述了本发明的各实施例,上述说明是示例性的,并非穷尽性的,并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下,对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。
序列表
<110> 海南医学院第一附属医院
<120> 重组腺相关病毒载体、重组腺相关病毒及其应用
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Claims (10)
1.一种重组腺相关病毒载体,其特征在于,所述重组腺相关病毒载体为将PD-1细胞外段核苷酸序列插入腺相关病毒载体而形成的载体;所述PD-1细胞外段核苷酸序列如SEQ IDNO:1所示。
2.根据权利要求1所述的重组腺相关病毒载体,其特征在于,所述PD-1细胞源自于人类。
3.根据权利要求1或2所述的重组腺相关病毒载体,其特征在于,所述腺相关病毒载体为8型腺相关病毒载体。
4.一种重组腺相关病毒AAV8-PD1,其特征在于,所述重组腺相关病毒具有权利要求1-3任一项所述的重组腺相关病毒载体。
5.根据权利要求4所述的重组腺相关病毒,其特征在于,所述重组腺相关病毒为重组8型腺相关病毒。
6.权利要求1-3任一项所述的重组腺相关病毒载体在制备阻断PD-1/PD-L1信号通路或促进CD8阳性T细胞生长的制剂中的应用。
7.权利要求1-3任一项所述的重组腺相关病毒载体在制备治疗肿瘤的制剂中的应用。
8.权利要求1-3任一项所述的重组腺相关病毒载体在制备治疗肝癌的制剂中的应用。
9.权利要求4或5所述的重组腺相关病毒在制备阻断PD-1/PD-L1信号通路或促进CD8阳性T细胞生长的制剂中的应用。
10.权利要求4或5所述的重组腺相关病毒在制备治疗肿瘤的制剂中的应用,优选为在制备治疗肝癌的制剂中的应用。
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