CN110157686A - 一种新型的免疫检查点激活免疫共刺激的复制型溶瘤腺病毒及其构建方法和应用 - Google Patents
一种新型的免疫检查点激活免疫共刺激的复制型溶瘤腺病毒及其构建方法和应用 Download PDFInfo
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- CN110157686A CN110157686A CN201910430993.9A CN201910430993A CN110157686A CN 110157686 A CN110157686 A CN 110157686A CN 201910430993 A CN201910430993 A CN 201910430993A CN 110157686 A CN110157686 A CN 110157686A
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Abstract
本发明公开了一种新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒及其构建方法和应用,所述的新型阻断免疫检查点并激活免疫共刺激信号通路的复制型重组溶瘤腺病毒,能够在肿瘤细胞内复制,并表达和分泌可溶性蛋白sPVR;所述的可溶性蛋白为特异性地结合TIGIT和CD226的PVR蛋白的胞外区,所述的sPVR蛋白具有同时阻断免疫检查点TIGIT介导的免疫负性调控,和激活共刺激通路CD226介导的抗肿瘤免疫功能。本发明的新型复制型溶瘤腺病毒AD5sPVR具有很强的活化抗肿瘤免疫作用,具有显著的抗肿瘤活性和安全性,有非常高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技术领域,具体涉及一种新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒及其构建方法和应用。
背景技术
癌症是对人类生命健康危害最大的疾病之一,每年都有数百万的人死于癌症。许多数病人在诊断时已经处于中晚期从而失去外科手术治疗的机会,传统的放疗化疗,并没有给肿瘤带来突破性的进展,即使小分子靶向药物,也面临着短期内复发的巨大挑战。
近十年来,抗肿瘤免疫治疗临床研究不断出现的令人振奋的结果,给肿瘤患者重新带来了希望。早在1090年,德国科学家(诺贝尔奖获得者)Paul Ehrlich就提出了免疫系统具有识别并清除异常肿瘤细胞的能力,并尝试免疫治疗,然而疗效不佳。随着科学认知的深入,人们发现肿瘤在发生发展过程中,不仅通过隐藏自己(缺乏被免疫细胞识别的抗原分子)逃避免疫攻击,还通过多种途径主动抑制抗肿瘤的免疫效应细胞,包括启动免疫检查点等诸多途径使免疫细胞失活,达到逃避免疫识别和免疫清除的目的。经典的PD-1及其配体PD-L1,还有最近被发现的另一个调控通路PVR及其受体TIGIT(T cell ITIM domain),都参与了抗肿瘤的负性免疫调控。这些分子通路显著抑制效应淋巴细胞的活化并使其进入失能状态,这一过程就是典型的启动免疫检查点的过程。同时阻断免疫负性调控和激活共刺激通路是被证明为有效的治疗肿瘤的方法,至今已有多个免疫检查点阻断剂被批准为用于临床肿瘤治疗的药物。
TIGIT(T cell immunoglobulin and immunoreceptor tyrosinebasedinhibitory motif[ITIM]domain)是近年来发现的另外一个表达在淋巴细胞上的免疫检查点调控分子,参与对活化的T细胞及NK细胞的负性调控。TIGIT作为免疫检查点分子对T细胞的调控主要通过以下几种方式实现:(1)T细胞上的TIGIT分子与肿瘤或者APC细胞膜上的PVR(CD155)分子结合,结合之后信号转导进入肿瘤细胞进而通过上调肿瘤或者APC细胞IL-10的表达,同时下调肿瘤细胞IL-12的表达等方式参与免疫抑制;(2)T细胞上的TIGIT分子可以直接与自身细胞上的免疫共刺激分子CD226结合,阻断CD226分子形成同源二聚体,从而抑制了CD226分子转导的免疫共刺激信号,间接起到免疫抑制的作用;(3)此外,PVR作为免疫共刺激分子CD226的配体,与TIGIT的亲和性远远大于CD226,因此,TIGIT分子能够通过竞争性地结合PVR,从而阻断PVR/CD226通路转导的免疫共刺激信号,参与免疫抑制调控。T细胞表达TIGIT所介导的负性调控,主要是通过阻断CD226共刺激通路,而后者在T细胞介导的抗肿瘤免疫中似乎不可或缺。因此,在阻断TIGIT的同时,恢复CD226的免疫共刺激通路,是一个巧妙而且有效的抗肿瘤免疫策略。
虽然阻断免疫检查点的抗肿瘤治疗已经被广泛证实为有效的治疗策略,但是也遇到了一些亟需解决的问题。首先,肿瘤患者对阻断免疫检查点的免疫治疗反应性低,普适性(有效率)有待提高。其次,临床研究发现,系统性阻断免疫检查点存在“脱靶”而“误伤”正常组织的情况,由此带来了自身性免疫损伤如心肌炎等副作用。进一步的临床试验数据表明,肿瘤浸润淋巴细胞(TILs)的多少、肿瘤局部的免疫活化状态等,是靶向免疫检查点治疗能否显效的重要预测指标。CD8+T细胞介导肿瘤清除过程中,I型干扰素(IFNα/β)通路的活化是靶向免疫检查点治疗的重要事件。因此,如何在肿瘤局部有效诱导I型干扰素介导的免疫活化、增强肿瘤微环境免疫细胞的浸润,可以使肿瘤对靶向免疫检查点的治疗更加敏感,这也许是解决免疫检查点治疗普适性(有效率)不高的有效手段之一。然而,如何解决免疫检查点治疗“脱靶”效应这一问题呢?
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。溶瘤病毒免疫疗法是否可以使肿瘤对靶向免疫检查点的治疗更加敏感,从而解决免疫检查点治疗普适性(有效率)不高这一问题(免疫检查点治疗遇到的第1个问题)。
另外,得益于溶瘤病毒在肿瘤细胞具有选择性复制的优势,利用表达免疫检查点阻断剂的溶瘤腺病毒在肿瘤局部复制的同时,使得免疫活化最大限度被局限在肿瘤的局部微环境,有效避免阻断免疫检查点“脱靶”而造成其它正常组织的“误伤”(免疫检查点治疗遇到的第2个问题)。
目前,缺乏一种同时具有阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒。
发明内容
本发明提供了一种同时具有阻断免疫检查点和激活免疫共刺激通路的的复制型溶瘤腺病毒及其构建方法和应用。
本发明的目的是通过下列技术方案实现的:本发明的一种新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒,所述复制型溶瘤腺病毒在肿瘤细胞内复制,并表达和分泌可溶性蛋白PVR的胞外区(sPVR),所述的可溶性蛋白为特异性地结合TIGIT和CD226的配体,所述的sPVR蛋白具有同时阻断TIGIT介导的免疫负性调控和激活CD226介导的免疫共刺激通路的功能。
进一步地,所述的可溶性蛋白为sPVR,sPVR的蛋白序列的氨基酸序列如序列表SEQID NO:1所示。
本发明所述的新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒,所述复制型溶瘤腺病毒能够溶瘤。
本发明所述的新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒在制备活化抗肿瘤免疫药物中的应用。
本发明所述的新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒在制备刺激IFN-γ表达药物中的应用。
本发明所述的新型的阻断免疫检查点并激活免疫共刺激信号通路的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
进一步地,所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
本发明一种新型的具有同时阻断TIGIT介导的免疫负性调控和激活CD226介导的共刺激通路的复制型溶瘤腺病毒AD5sPVR的构建方法,通过质粒构建、病毒拯救与病毒扩增三个步骤来实现。
有益效果:本发明的新型复制型溶瘤腺病毒AD5sPVR具有很强的活化抗肿瘤免疫作用,具有显著的抗肿瘤活性和安全性,有非常高的开发抗肿瘤药物的前景和价值。同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR在肿瘤细胞内和肿瘤部位选择性复制并且在局部表达和分泌sPVR,既提高了靶向性也保证了安全性;所表达的sPVR阻断免疫负性调控和激活共刺激通路,具有多靶点效应,从而确保药物的有效性。
与现有技术相比,本发明具有如下优点:
(1)不仅如此,从整体效果来看,同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR具有显著的活化抗肿瘤免疫作用,诱导IFN-γ的高表达,显著抑制肿瘤生长、延长生存期,具有极显著的抗肿瘤作用,是绝佳的用来制备抗肿瘤药物的原材料,也具有预料不到的效果。
(2)一种新型的同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR的设计和构建方法,成功获得了一株新型的可以同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR,该病毒可以在肿瘤细胞内和肿瘤部位选择性复制,并且能够高表达sPVR(CD155)的胞外区—sPVR,该可溶性蛋白能够分泌到细胞外,特异并高亲和地结合TIGIT,阻断TIGIT介导的许多负性免疫调控;同时,过量的sPVR能够直接特异性结合共刺激分子CD226,共同参与活化免疫的生物学和免疫学功能,起到活化抗肿瘤免疫的作用。
(3)本发明的新型同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR,能够有效活化抗肿瘤免疫反应,刺激IFN-γ的高表达。活体小动物肿瘤模型验证,新型复制型溶瘤腺病毒Ad5sPVR具有显著的抗肿瘤作用,能够用来制备抗肿瘤药物。
(4)本发明提供了一种可以同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR的设计和构建方法,成功获得了一株新型可以同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR,该病毒可以在肿瘤细胞内和肿瘤部位选择性复制、具有肿瘤靶向性,而且能够溶瘤,诱导免疫原性细胞死亡。与此同时,该病毒能够高表达可溶性蛋白sPVR,该蛋白能够分泌到细胞外,在肿瘤微环境特异性阻断TIGIT介导的免疫负性调控,同时特异性活化CD226共刺激通路,发挥活化免疫的生物学功能。
(5)药理学实验表明,本发明的同时阻断免疫负性调控和激活共刺激通路的新型复制型溶瘤腺病毒Ad5sPVR具有显著的活化抗肿瘤免疫作用、诱导淋巴细胞高表达IFN-γ,显著抑制肿瘤生长、延长生存期,具有极显著的抗肿瘤作用,可以用来制备抗肿瘤药物,是一个一石数鸟的绝佳设计和策略,具有预料不到的抗肿瘤效果。
附图说明
下面将结合附图进一步说明,附图中:
图1为本发明的表达可溶性融合蛋白sPVR的重组溶瘤腺病毒的构建(A)重组溶瘤腺病毒Ad5con、Ad5sPVR的基因结构原理图。(B-C)使用MOI=10的Ad5con、Ad5sPVR分别感染B16/F10细胞,使用MOI=20的Ad5con、Ad5sPVR分别感染H22细胞,48h后收集被感染细胞的上清,通过western blot的方法检测可溶性融合蛋白sPD1PVR的表达与分泌。数据代表三次独立性重复实验。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图2为本发明的重组溶瘤腺病毒Ad5sPVR的复制能力(A-F)分别用Ad5con和Ad5sPVR感染以下细胞:肝癌细胞株Hepa1-6(MOI=5)、H22(MOI=20)、LM3(MOI=2),黑色素瘤细胞株B16/F10(MOI=10)、乳腺癌细胞株4T1(MOI=10)和肺癌细胞株LLC1(MOI=10),在12h,24h,36h,48h,60h,72h收取细胞,提取病毒基因组DNA,通过Q-PCR检测Ad5的拷贝数。用MTT检测细胞活率。数据代表三次独立性重复实验数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图3为本发明的重组溶瘤腺病毒Ad5sPVR的溶瘤能力(A-F)分别用Ad5con和Ad5sPVR感染以下细胞:肝癌细胞株Hepa1-6(MOI=5)、H22(MOI=20)、LM3(MOI=2),黑色素瘤细胞株B16/F10(MOI=10)、乳腺癌细胞株4T1(MOI=10)和肺癌细胞株LLC1(MOI=10),在12h,24h,36h,48h,60h,72h收取细胞,用MTT检测细胞活率。数据代表三次独立性重复实验数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图4为本发明的重组溶瘤腺病毒Ad5sPVR所表达的可溶性蛋白sPVR与TIGIT和CD226间的相互作用。(A)PVR与TIGIT或CD226蛋白之间的亲和力动态监测图。
图5为本发明的重组溶瘤腺病毒Ad5sPVR在肝癌腹水瘤H22中活化免疫作用(A)在H22腹水瘤小鼠模型中评估Ad5sPVR的免疫活化作用,实验方案图如图所示。C57BL/6腹腔接种H22细胞5×106cells/只,待腹水出现后,随机分组,每组10只,分别在第8、12天抽取腹水后腹腔注射5×108pfu Ad5con或Ad5sPVR,第16天取腹水细胞,用ELISpot检测活化免疫细胞水平。(B)H22腹水瘤模型ELISpot检测结果。(C)H22腹水瘤模型ELISpot实验结果统计。数据代表三次独立性重复实验。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图6为本发明的重组溶瘤腺病毒Ad5sPVR在实体肿瘤黑色素瘤B16/F10中活化免疫作用(A)通过黑色素瘤B16/F10皮下瘤模型评估Ad5sPVR的免疫活化作用,实验方案图如图所示。C57BL/6右侧皮下接种B16/F10细胞2×106cells/只,出现肿瘤后,瘤内注射5×108pfuAD5con或Ad5sPVR,在第13天时,处死小鼠,分离肿瘤制成单细胞悬液,ELISpot检测免疫活化。(B)B16/F10实体瘤模型ELISpot检测结果。(C)B16/F10实体瘤模型ELISpot实验结果统计。数据代表三次独立性重复实验。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图7为本发明的重组溶瘤腺病毒Ad5sPVR的体内抗肿瘤作用(B16F10黑色素实体肿瘤模型)(A)B16/F10实体瘤体内实验方案图,选取6-8周龄C57/BL6雄性小鼠,皮下接种B16/F10细胞2×106cells/只,待第8天小鼠长出皮下瘤,随机分组,每组10只,分别在第8、9、10、11天皮下多位点注射Ad5con和Ad5sPVR5×108pfu/只,监测小鼠肿瘤生长。(B)B16/F10肿瘤模型小鼠肿瘤生长曲线。(C)B16/F10肿瘤模型小鼠体重。(D)B16/F10肿瘤模型小鼠生存。数据代表三次独立性重复实验。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图8为本发明的Ad5sPVR病毒抗肿瘤的效果(4T1乳腺癌实体肿瘤模型)(A)4T1实体瘤体内实验方案图,选取6-8周龄BALB/c雌性小鼠,皮下接种4T1细胞5×104cells/只,待第8天小鼠长出皮下瘤,随机分组,每组10只,分别在第8、9、10、11天皮下多位点注射Ad5con和Ad5sPVR5×108pfu/只,监测小鼠肿瘤生长。(B)4T1肿瘤模型小鼠肿瘤生长曲线。(C)4T1肿瘤模型小鼠体重。(D)4T1肿瘤模型小鼠生存。数据代表三次独立性重复实验。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图9为本发明的Ad5sPVR病毒抗肿瘤的效果(H22肝癌腹水肿瘤模型)(A)H22腹水瘤体内实验方案图,选取6-8周龄C57BL/6雄性小鼠,腹腔注射H22细胞5×106cells/只,第8天分组,每组10只,第8、12、16分别腹腔注射Ad5con、Ad5sPVR5×108pfu/只,监测小鼠生存,直至小鼠死亡。(B)H22腹水瘤模型小鼠生存曲线。(C)H22腹水瘤模型三次独立性重复实验。(D)H22腹水瘤模型小鼠生存比例。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
图10为本发明的Ad5sPVR病毒在肿瘤局部的复制和融合蛋白的表达(A)H22腹水瘤模型实验方案图,选取6-8周龄C57BL/6雄性小鼠,腹腔注射H22细胞5×106cells/只,待第8天小鼠全部长出腹水,随机分组,分别在第8、12、16天先抽取腹水后腹腔注射Ad5con、Ad5sPVR5×108pfu/只,在第16天抽取腹水,流式检测淋巴细胞。(B)H22腹水瘤模型小鼠生存曲线。(C)第12天Ad5病毒拷贝数。(D)第16天Ad5病毒拷贝数。(E)第12天腹水中IFN-γ水平。(F)第16天腹水中IFN-γ水平。(G)第12天腹水中可溶性PVR表达量。(H)第16天腹水中可溶性PVR表达量。数据表示为Mean±SD,*P<0.05,**P<0.01,***P<0.001。
具体实施方式
通过以下实施例进一步详细说明本发明,但应注意本发明的范围并不受这些实施例的任何限制。
实施例1
新型复制型溶瘤腺病毒Ad5sPVR的构建、制备、抗肿瘤免疫活化评估和抗肿瘤作用评价
1实验材料和方法
1.实验材料和方法
1.1实验材料和仪器
1.1.1实验细胞系
人胚肾细胞株293T;人肝癌细胞株LM3;小鼠黑色素瘤细胞株B16/F10;小鼠肝癌细胞株H22和Hepa1-6;小鼠乳腺癌细胞株4T1。上述细胞均使用含10%胎牛血清,100U/I青霉素和1mg/ml链霉素的高糖DMEM培养基培养于37℃、5%CO2的培养箱中。
1.1.1.2实验仪器
生物安全柜(III advance,Class II Biological SafetyCabinet,The Baker Company),二氧化碳培养箱(FORMA SERIES II WATER JACKETCO2incubator,Thermo),低温离心机(HERAEUS MEGAFUGE1.0R,Thermo),垂直电泳槽(BIO-RAD),电泳仪(BIO-RAD),半干转-转膜仪(BIO-RAD),免疫印迹曝光系统(Alpha Innotech),PCR仪(PCR Thermal Cycler Dice,TaKaRa),实时定量PCR仪及分析软件(ABI384,SequenceDetection Software,Version1.3.1),酶标仪(VERSA max microplate reader),整套移液器(eppendorf和RAININ),细胞计数仪(Countstar Automated cell counter,Inno-Alliance Biotech Inc.,Wilmington,USA),流式细胞仪(FACSCalibur,Becton,Dickinsonand Company,USA),FlowJo software(Version7.6.5,Tree Star Inc,Ashland,Oregon),微孔板振荡器(QiLinBeiEr),核酸纯度浓度检测仪(Biophotometer plus,eppendorf),数显恒温水浴锅(国华电器)。
主要实验试剂及耗材
引物均由金斯瑞公司合成。肿瘤细胞培养所需的DMEM高糖培养基,双抗,血清均购自Invitrogen(上海)公司。定量RT-PCR试剂Faststart Universal SYBR Green Master(Roche,04913914001)。Western Blot所需试剂耗材:蛋白酶抑制剂(Roche,11873580001),细胞裂解液(碧云天:P0013),PVDF膜(Roche,03010040001),WB Immobilon ECL发光液(Millipore,WBKLS0500),一抗稀释液(碧云天,P0023A),HRP标记的二抗(Multisciences,GAR007 and GAM007,1:5000稀释),其余所需试剂均为国产分析纯,购自南京大学化学化工学院。台盼蓝(碧云天,C0011),Opti-MEM购自Invitrogen(上海)公司。Western Blot抗体:anti-His(金斯瑞,MB001,1:5000稀释)。
1.1.2实验方法
1.1.2.1 Ad5sPVR病毒构建
可溶性蛋白sPVR的基因克隆以及携载sPVR基因的腺病毒穿梭质粒的构建小鼠PVR属于膜蛋白,其结构依次是:N端信号肽-胞外区-跨膜区-胞内区C端。PVR与TIGIT结合的功能单位是胞外区,保留PVR的N端信号肽区域(见图1);
可溶性蛋白sPVR的基因克隆:分别设计合成引物PVR-F、PVR-R、使用PVR-F和PVR-R引物,以小鼠肝癌细胞Hepa1-6的cDNA为模板扩增得到片段EXO-PVR。sPVR(EXO-PVR)、Linker和信号肽(CD33)的蛋白序列的氨基酸序列如序列表SEQ ID NO:1-3所示;sPVR(EXO-PVR)、Linker和信号肽(CD33)的DNA序列如序列表SEQ ID NO:4-6所示。基因模板构建相关引物如表1所示:
表1
携载可溶性蛋白基因的腺病毒穿梭质粒Ad5-pShuttle-sPVR载体的构建:使用Infusion技术将sPVR片段与Ad5-pShuttle(pZD55)连接。具体步骤:首先使用限制性内切酶BglII对Ad5-pShuttle(pZD55)线性化,纯化后片段按照sPVR:Ad5-pShuttle的2:1比例使用Infusion试剂盒(clontech lab.Inc.)进行连接,后经转化扩增验证获得携载sPVR基因的腺病毒穿梭质粒Ad5-pShuttle-sPVR。
1.2.2 Ad5sPVR病毒构建(质粒构建、病毒拯救与扩增)
A.Ad5sPVR全长质粒构建:将构建好的穿梭载体Ad5-pShuttle-sPVR用PmeI线性化后转入感受态pAdEasy-BJ5183中,使用含50ug/ml卡那霉素LB平板的进行筛选,挑取阳性克隆培养鉴定,鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定,鉴定正确后进行质粒大提获得Ad5sPVR全长质粒。
B.Ad5 sPVR病毒拯救:Ad5sPVR全长质粒使用PacI线性化,纯化后6孔板中1μg/well转染293T细胞,5%CO2、37℃培养,2天后将细胞消化后转入10cm平皿,2-3天换液,至80%细胞出现病变,使用10ml培养基将细胞吹下收集至15ml离心管,反复冻融2次,3000rpm/min离心15min,收集病毒上清-80℃保存做为毒种。
C.病毒扩增:取病毒种液50μl加入60%293T细胞10cm平皿中,5%CO2,37℃培养,细胞密度至90%以上,按照1传3比例传代,直至80%细胞出现病变,大约有10个平皿细胞,按上述方法收病毒,使用氯化铯密度梯度离心纯化病毒;使用TCID50方法进行滴度测定。
1.1.3 Ad5sPVR病毒功能评价
A.sPVR的表达和分泌功能:Ad5sPVR病毒感染肿瘤细胞72小时后,收细胞和上清,使用Western Blot检测sPVR的表达和分泌功能。
B.病毒复制能力:Ad5sPVR和Ad5con病毒相同MOI感染肿瘤细胞,72小时后收细胞,提取病毒DNA,通过Q-PCR检测病毒E1A,对病毒进行绝对定量,分析病毒复制能力变化。
C.溶瘤功能:分别使用Ad5sPVR和Ad5con病毒按照MOI1到100病毒量感染肿瘤细胞,48小时后使用MTT检测细胞活性,评价Ad5sPVR的杀瘤作用。
1.1.4体内研究AD5sPVR抗肿瘤机制
A.选用6-8周龄C57BL/6小鼠在右侧腋窝建立皮下瘤模型,每只小鼠一侧接种B16/F10细胞5×106个细胞,4-6天后测量肿瘤大小至200mm3,将小鼠随机分成3组,分别是:无处理组、对照Ad5病毒治疗组、Ad5sPVR病毒治疗组;a.按照分组使用相应病毒瘤内注射,每只注射病毒量5×108pfu,跟踪测量肿瘤体积,体重,至肿瘤体积大于2000mm3判定小鼠死亡,记录小鼠生存期。b.按照分组瘤内注射病毒,每只注射病毒量5×108pfu,注射两次,ELISpot检测免疫活化。
B.选用6-8周龄C57BL/6小鼠在腹腔建立腹水瘤模型,每只小鼠腹腔接种H22细胞5×106个细胞,第7-8天左右看到小鼠有腹水,将小鼠随机分成3组,分别是:无处理组、对照Ad5病毒治疗组、Ad5 sPVR病毒治疗组;a.按照分组使用相应病毒腹腔注射,每只注射病毒量5×108pfu,跟踪体重,直至小鼠死亡,记录小鼠生存期。b.按照分组腹腔注射病毒,每只注射病毒量5×108pfu,注射两次,ELISpot检测免疫活化。
1.1.5 Ad5sPVR病毒的滴度测定
293T细胞种于96孔板,每孔约1×104个细胞,待细胞贴壁后进行滴度测定。
病毒梯度的稀释:准备EP管,每个EP管加入1170μl含胎牛血清的DMEM;往第一个EP管中加入130μl病毒溶液,混匀,标记为10-1;从第一个EP管中吸取130μl于第二个EP管中,混匀,标记为10-2;依次类推,直至稀释到所需梯度为止。
每孔加入100μl相应梯度的病毒稀释液,每个梯度重复10个孔,37℃培养过夜。
5天后,将96孔板放于显微镜下观察GFP,记下每个梯度有GFP的孔数,用于病毒滴度的计算。
病毒滴度TCID50的计算公式:
Log10(TCID50)=L+d(s-0.5)+log10(1/v)
L=Log10最高稀释度(如最高稀释度为10倍稀释,L=1)
V=最初每孔细胞培养液的体积(ml/well);
d=Log10稀释度(如为10倍稀释,d=1);
s=各个梯度GFP比率之和。
1.2.3实时定量PCR
实时定量PCR的10μl体系组成:2.6μl PCR water,上下游引物各0.2μl,2μl的模板和5μl的SYBR Green荧光染料。样品混合后,于ABI 384 PCR仪上进行扩增。
1.2.4细胞总蛋白的提取及浓度测定
1)以六孔板为例,去掉细胞培养上清,用PBS洗涤2遍,去掉PBS,每孔加入200μl的胰酶,消化吹打细胞,并将细胞收入至EP管中,2500rpm离心5min。
2)去掉上清,加入PBS重悬细胞,1500rpm离心5min。
3)去掉PBS,每孔根据细胞量加入相应的含蛋白酶抑制剂的细胞裂解液,涡旋1min,置于冰上10min,重复操作三次。4℃,12000g离心10min。收集上清于另一干净的EP管中。
4)蛋白浓度的测定:根据BCA蛋白浓度测定盒说明书进行检测。取2μl蛋白样品于96孔板中,加入18μl的PBS稀释样品,最后在加入200μl的测定工作液(工作液由试剂A:试剂B=50:1),放置于60℃的烘箱中,30min后,用酶标仪在562nm测定吸光度,根据标准曲线,计算出蛋白样品的浓度。
5)每管加入1/4蛋白裂解液体积的5×loading buffer,混匀后,100℃金属浴5min,冷却后,-20℃保存备用。
1.2.5 Western blot实验
1)配胶和电泳:按照不同要求配制不同浓度的SDS-PAGE分离胶和浓缩胶根据蛋白定量的计算结果,每个样品上样量调为20μg。电泳条件:浓缩胶80V30min,分离胶120V,约60min,前提是将条带分开且不会跑出去。
2)转膜:准备滤纸和PVDF膜,先用甲醇浸泡PVDF膜,再和滤纸一同浸泡在转膜缓冲液中备用。从玻璃板中小心将胶取下,浸泡在转膜缓冲液中,按照负极-滤纸-PVDF膜-胶-滤纸-正极的三明治顺序放置,赶走气泡,根据所需条带大小不同,恒流110mA转膜60-70min。
3)封闭:转膜结束后,立即取出PVDF膜,放入5%脱脂奶粉中室温封闭1h。
4)一抗孵育:4℃孵育一抗过夜。
5)二抗孵育:用washing buffer洗涤条带,每次10min,共三次;再用相应的HPR标记的二抗室温孵育1h。
6)曝光:用washing buffer洗涤条带,每次10min,共三次;用化学发光液在WB曝光仪上曝光,并获取条带图像。
1.2.6台盼兰计数
以六孔板为例,去除细胞上清,用PBS洗涤2遍,去掉PBS,每孔加入200μl胰酶消化,轻轻吹打细胞并收集进入干净的EP管中,2500rpm,离心5min。去掉上清,加入PBS重悬细胞,2500rpm,离心5min。去掉PBS,根据细胞数量加入一定量的PBS重悬细胞,从中取出10μl细胞重悬液,加入10μl0.2%台盼蓝溶液混合,取混合液20μl于细胞计数板中,用细胞计数仪计数。
1.2.7流式细胞仪检测细胞表面分子
1)取实体瘤细胞(腹水)105cells,加PBS清洗一遍。
2)去掉PBS,每管加入100μl含相应量的流式抗体的PBS,重悬细胞,置于冰上30min,避光。期间拿出样品,轻轻吹打,防止其因沉淀而影响抗体结合效果。
3)30min后,每管加入1ml PBS混合细胞,2500rpm,离心5min。去掉上清,再加入PBS重悬细胞,2500rpm,离心5min。去掉PBS,每管加入300μl PBS重悬,避光。
将准备好的样品,用流式细胞仪检测。用FlowJo软件分析实验结果。
1.2.8 Mouse IFN-γELISpot检测
1)ELISpot板子在使用前每孔加入200μl含10%血清的培养基孵育30min以上,放入细胞培养箱中。
2)去掉培养基,每孔加入200μl含细胞的培养体系。细胞体系组成:100μl的肿瘤细胞和100μl的脾脏细胞。混合均匀之后,加入孔板中,放入细胞培养箱中。且在实验结束取出之前,不要随意挪动板子。12h后,取出板子检测。
3)去掉培养基,每孔加入200μl的PBS清洗,需要清洗五遍以上。
4)去掉PBS,每孔加入100μl含一抗的稀释液。一抗稀释液:含0.5%FBS的PBS;一抗稀释比例1:1000。室温放置2h。
5)去掉一抗稀释液,用PBS洗五遍以上;每孔加入100μl二抗稀释液。二抗稀释液:含0.5%FBS的PBS;二抗稀释比例1:1000。室温放置1h。
6)去掉二抗稀释液,用PBS洗五遍以上。每孔加入200μl显色剂显色。待有蓝色斑点出现,且又不显色过头的情况下,甩掉显色液,用自来水洗多遍。
7)将自来水甩掉,室温晾干。注意:不要在室温晾的过久,且晾干过程保持避光。最后用避光保存于封口袋中。扫描读板。
1.2.9肿瘤组织及脾细胞的mouse IFN-γ的ELISpot检测
1)肿瘤组织单细胞悬液制备及mouse IFN-γ的ELISpot检测:处死小鼠,取下一小块的肿瘤组织,用PBS清洗,再放入培养皿中,加入1ml的胶原酶溶液,用剪刀剪碎,将肿瘤组织浑浊液吸入干净的离心管中,再加入1ml胶原酶溶液,放入37℃培养箱1小时,使得肿瘤组织得以消化完全。期间,每隔10min要取出,用枪头吹打混匀。2h后,取出装有肿瘤组织浑浊液的离心管,确认肿瘤组织消化完全。将浑浊液离心,取沉淀即肿瘤组织细胞。用含血清的DMEM重悬,细胞计数,将细胞浓度调为2×106个/ml,取100μl细胞混合液做鼠IFN-γ的ELISpot检测,方法同1.2.8。
2)脾单细胞悬液的制备及鼠IFN-γ的ELISpot检测:处死小鼠,取出脾脏,用PBS清洗,剪下一小块脾脏组织,放于70μl的细胞够滤网中,用5ml的注射器研磨,边研磨边加入适量的PBS冲洗。用Ficoll法去掉红细胞,离心重悬获得脾脏单细胞混合液,细胞计数,将细胞浓度调为2×106个/ml,取100μl与肿瘤细胞混合做鼠IFN-γ的ELISpot检测,方法同1.2.8。
1.2.10游离PVR的表达纯化和亲和力分析
293T细胞经Ad5sPVR感染,将细胞培养基上清经过镍柱。加入洗涤液将未结合的蛋白洗去,然后加入洗脱液并收集蛋白。收集的蛋白通过超滤的方式将溶液换为PBS。将收集的样品通过PAGE胶和考马斯亮蓝染色鉴定其纯度大于85。纯化的PVR蛋白用于Octet RED96蛋白互作分析系统进行亲和力分析。将够买的小鼠TIGIT和CD226蛋白配制为200nM的PBS溶液。Octet RED96使用的感受器具有抗his活性,因而能够加载纯化的PVR蛋白。通过动态监测PVR与TIGIT或CD226蛋白的结合过程计算出蛋白之间的解离常数。
2.实验结果与结论
图1的结果显示我们已经成功构建可以同时阻断免疫负性调控和激活共刺激通路TIGIT的复制型溶瘤腺病毒Ad5sPVR,图中对照组对应位置没有条带、是空白的,而Ad5sPVR重组溶瘤腺病毒对应的条带深黑,而且从蛋白大小判断,是我们拟表达的目标蛋白:可溶性的sPVR。证明Ad5sPVR重组溶瘤腺病毒能够在感染的细胞中复制并表达目标蛋白,且能够被分泌到细胞外。
图2的结果显示我们所构建的重组溶瘤腺病毒Ad5sPVR在Hepa1-6、H22、LM3、4T1、B16/F10、LLC1肿瘤细胞内保留了与对照病毒相当的复制能力。
图3的结果显示我们所构建的重组溶瘤腺病毒Ad5sPVR在Hepa1-6、H22、LM3、4T1、B16/F10、LLC1肿瘤细胞内保留了与对照病毒相当的溶瘤能力。
图4蛋白与蛋白间相互作用的结果表明,病毒所表达分泌的sPVR与其特异性的受体蛋白TIGIT(免疫负性调控分子)和CD226(免疫共刺激分子)有极高的亲和力,且与TIGIT的亲和力显著大于CD226。
图5在H22肝癌腹水瘤中腹腔注射重组溶瘤腺病毒Ad5sPVR,通过ELISpot实验结果看出肿瘤微环境中抗肿瘤免疫活性显著增强。
图6在B16/F10黑色素瘤实体肿瘤中瘤内注射重组溶瘤腺病毒Ad5sPVR,通过ELISpot实验结果看出肿瘤微环境中抗肿瘤免疫活性显著增强。
图7显示,在黑色素瘤实体瘤小鼠模型中,注射重组溶瘤腺病毒Ad5sPVR能够显著抑制肿瘤的生长,延长荷瘤小鼠生存时间;并且注射重组溶瘤腺病毒Ad5sPVR和对照病毒Ad5组与模型组相比,小鼠体重未收到影响,证明重组溶瘤腺病毒Ad5sPVR和对照病毒Ad5具有一定的安全性。
图8显示,在乳腺癌实体瘤小鼠模型中,注射重组溶瘤腺病毒Ad5sPVR能够显著抑制肿瘤的生长,延长荷瘤小鼠生存时间;并且注射重组溶瘤腺病毒Ad5sPVR和对照病毒Ad5组与模型组相比,小鼠体重未收到影响,证明重组溶瘤腺病毒Ad5sPVR和对照病毒Ad5具有一定的安全性。
图9肝癌腹水瘤模型中,给与重组溶瘤腺病毒Ad5sPVR治疗的小鼠,其生存时间显著延长,约有20%小鼠长期无瘤生存,治愈的小鼠能够完全排斥腹腔再次注射同种肿瘤细胞,说明能够产生长期免疫记忆。
图10肝癌腹水瘤模型中显示,注射重组溶瘤腺病毒Ad5sPVR后,病毒在肿瘤局部能够复制、表达融合蛋白。
由以上结果显示,本发明提供了一种可以同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR的设计和构建方法,成功获得了一株新型可以同时阻断免疫负性调控和激活共刺激通路的复制型溶瘤腺病毒Ad5sPVR,该病毒可以在肿瘤细胞内和肿瘤部位选择性复制、具有肿瘤靶向性,而且能够溶瘤,诱导免疫原性细胞死亡。与此同时,该病毒能够高表达可溶性蛋白sPVR,该蛋白能够分泌到细胞外,在肿瘤微环境特异性阻断TIGIT介导的免疫负性调控,同时特异性活化CD226共刺激通路,发挥活化免疫的生物学功能。本发明可以阻断TIGIT免疫检查点的复制型溶瘤腺病毒Ad5sPVR,具有显著的活化抗肿瘤免疫作用,诱导IFN-γ的高表达,显著抑制肿瘤生长、延长生存期,具有极显著的抗肿瘤作用,可以用来制备抗肿瘤药物,是一个一石数鸟的绝佳设计和策略,具有预料不到的抗肿瘤效果。
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> 南京大学
<120> 一种新型的免疫检查点激活免疫共刺激的复制型溶瘤腺病毒及其构建方法和应用
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Claims (8)
1.一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒,其特征在于:所述的复制型溶瘤腺病毒在肿瘤细胞内复制;并使肿瘤细胞表达和分泌可溶性蛋白,所述的可溶性蛋白为特异性地结合TIGIT和CD226的配体PVR蛋白的胞外区,所述的sPVR蛋白具有同时阻断TIGIT介导的免疫负性调控和激活CD226介导的共刺激通路的功能。
2.如权利要求1所述的一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒,其特征在于:所述的可溶性蛋白为sPVR,sPVR的蛋白序列的氨基酸序列如序列表SEQ ID NO:1所示。
3.如权利要求1或2所述的一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒,其特征在于:所述复制型溶瘤腺病毒能够溶瘤。
4.权利要求1或2所述的一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒在制备活化抗肿瘤免疫药物中的应用。
5.权利要求1或2所述的一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒在制备刺激IFN-γ表达药物中的应用。
6.权利要求1或2所述的一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
7.如权利要求6所述的一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用,其特征在于:所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
8.一种新型的阻断免疫检查点和激活免疫共刺激通路的复制型溶瘤腺病毒AD5sPVR的构建方法,其特征在于:通过质粒构建、病毒拯救与病毒扩增三个步骤来实现;
可溶性蛋白sPVR的基因克隆:分别设计合成引物PVR-F、PVR-R,使用PVR-F和PVR-R引物,以小鼠肝癌细胞Hepa1-6的cDNA为模板扩增得到片段EXO-PVR;sPVR即EXO-PVR和信号肽CD33的蛋白序列的氨基酸序列如序列表SEQ ID NO:1-3所示;sPVR即EXO-PVR和信号肽CD33的DNA序列如序列表SEQ ID NO:4-6所示。
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