CN110305906B - 一种靶向pdl1的car嵌合受体的慢病毒载体及pdl1-car-t细胞 - Google Patents
一种靶向pdl1的car嵌合受体的慢病毒载体及pdl1-car-t细胞 Download PDFInfo
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- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
本发明公开了一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒,包含以下编码序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列;其编码序列为SEQ ID NO:1;本发明设计合成专门识别PD‑L1抗原的PD1胞外域,连带其他CAR共有的铰链区、共刺激区域等得到靶向PD‑L1的CAR嵌合受体的慢病毒,并将其感染T细胞,形成识别PD‑L1的CAR‑T细胞,而且PD‑1/PD‑L1结合后将会引起T细胞的激活和对肿瘤细胞的杀伤作用。
Description
技术领域
本发明涉及肿瘤的免疫治疗技术领域,具体说是一种靶向PDL1的CAR嵌合受体的慢病毒载体及PDL1-CAR-T细胞。
背景技术
CAR-T又被称为嵌合抗原受体T细胞,通过基因工程将抗原识别区、铰链区以及共刺激分子区域等全部整合到T细胞基因组内,使T细胞特异性的识别肿瘤细胞并激活T细胞对肿瘤的杀伤。目前CAR-T细胞治疗也仅仅在血液肿瘤细胞疗效显著,但存在一定的局限性,首先能完美表达在肿瘤细胞而在正常细胞无表达的靶标抗原几乎不存在,所以目前的CAR-T细胞治疗会存在脱靶效应。同时肿瘤细胞是个复杂的群体,外面有复杂的肿瘤微环境,当有靶标抗原的肿瘤细胞被清除后,在肿瘤微环境各种因子刺激下会诱导新的肿瘤细胞的形成,导致肿瘤的复发甚至转移。
随着对肿瘤免疫的深入研究发现肿瘤微环境可以保护肿瘤细胞不被机体的免疫系统识别和杀伤,肿瘤细胞的免疫逃逸在肿瘤的发生发展中起着至关重要的作用。有正性和负性信号调控机体免疫细胞的激活与抑制,其中程序性死亡分子(programmed death 1,PD-1)/PD-1配体(PD-1ligand,PD-L1)是负性免疫调节信号,抑制肿瘤特异性CD8+T细胞的免疫活性,介导免疫逃逸。越来越多的研究证实PD-1/PD-L1信号通路在肿瘤免疫中起到关键作用,也为肿瘤免疫治疗提供新的分子靶点,阻断PD-1/PD-L1信号通路的激活可以增强抗肿瘤免疫治疗效应,并且PD-L1在大多数癌症组织中过量表达,包括NSCLC、黑色素瘤、淋巴瘤、白血病、消化道肿瘤、肺癌等。
IFN-γ为γ干扰素,具有抗病毒、抗肿瘤和免疫调控的作用,研究发现在多发性骨髓瘤中,肿瘤微环境中IFN-γ可以促进骨髓瘤细胞表面PD-L1蛋白的表达,激活T细胞PD-1/PD-1信号通路从而抑制T细胞的激活、增殖,诱导特异性的CTL的凋亡,使CTL细胞毒杀伤作用下降,使肿瘤细胞发生免疫逃逸。
发明内容
为解决上述问题,本发明的目的是提供一种靶向PDL1的CAR嵌合受体的慢病毒载体及PDL1-CAR-T细胞。
本发明为实现上述目的,通过以下技术方案实现:
一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒,包含以下编码序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列;
其编码序列为SEQ ID NO:1。
一种PDL1-CAR-T细胞,包含权利要求1所述靶向PDL1的CAR嵌合受体的慢病毒载体质粒基因。
本发明还包括一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒的制备方法,包括以下步骤:
①设计合成以下多核苷酸序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列,并在多核苷酸序列的两端分别添加Xba I与BamH I,并将其连接在Blunt质粒载体中,得到Blunt-PD1质粒;
②用Xba I与BamH I酶酶切PLVX载体质粒与Blunt-PD1载体质粒,露出各自的粘性末端后,再用T4连接酶,将它们连接在一起,并转化至stbl3菌株中,挑取单克隆提质粒并用Xba I与BamH I双酶切,筛选质粒并测序验证,得到靶向PDL1的CAR嵌合受体的慢病毒载体质粒。
本发明还包括PDL1-CAR-T细胞的制备方法,包括以下步骤:
⑴对靶向PDL1的CAR嵌合受体的慢病毒载体质粒进行质粒大提,然后与293ft细胞进行质粒包装,转染,收集病毒上清液,得到PDL1-CAR病毒上清;
⑵分离人全血中的淋巴细胞,磁珠分选分离出T细胞,培养后加入PDL1-CAR病毒上清,培养,转染,得到PDL1-CAR-T细胞。
本发明还包括PDL1-CAR-T细胞的应用,在杀伤多发性骨髓瘤细胞方面的应用。
优选的PDL1-CAR-T细胞的应用,在应用过程中加入IFN-γ。
本发明相比现有技术具有以下优点:
本发明设计合成专门识别PD-L1抗原的PD1胞外域,连带其他CAR共有的铰链区、共刺激区域等得到靶向PD-L1的CAR嵌合受体的慢病毒,并将其感染T细胞,形成识别PD-L1的CAR-T细胞(PDL1-CAR-T细胞),而且PD-1/PD-L1结合后将会引起T细胞的激活和对肿瘤细胞的杀伤作用,而非抑制作用;并且体内外IFN-γ与PDL1-CAR-T细胞联用能明显进一步增强PDL1-CAR-T细胞对多发性骨髓瘤细胞的杀伤作用。
附图说明
图1为靶向PDL1的CAR嵌合受体的慢病毒载体质粒的多核苷酸序列连接示意图;
图2为病毒包装时PDL1-CAR不同剂量下观测到的荧光示意图;
图3为四种多发性骨髓瘤细胞系在IFN-γ刺激24小时后,各组PDL1蛋白表达阳性的细胞占总细胞的百分比率示意图;
图4为空白T细胞与PDL1-CAR-T细胞分别与U266按照不同效靶比混合孵育24小时后,对U266的杀伤作用示意图;
图5为给药十五天后小鼠发光成像系统观察肿瘤细胞的增殖变化示意图。
具体实施方式
本发明的目的是提供一种靶向PDL1的CAR嵌合受体的慢病毒载体及PDL1-CAR-T细胞,通过以下技术方案实现:
一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒,包含以下编码序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列;
其编码序列为SEQ ID NO:1。
一种PDL1-CAR-T细胞,包含权利要求1所述靶向PDL1的CAR嵌合受体的慢病毒载体质粒基因。
本发明还包括一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒的制备方法,包括以下步骤:
①设计合成以下多核苷酸序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列,并在多核苷酸序列的两端分别添加Xba I与BamH I,并将其连接在Blunt质粒载体中,得到Blunt-PD1质粒;
②用Xba I与BamH I酶酶切PLVX载体质粒与Blunt-PD1载体质粒,露出各自的粘性末端后,再用T4连接酶,将它们连接在一起,并转化至stbl3菌株中,挑取单克隆提质粒并用Xba I与BamH I双酶切,筛选质粒并测序验证,得到靶向PDL1的CAR嵌合受体的慢病毒载体质粒。
本发明还包括PDL1-CAR-T细胞的制备方法,包括以下步骤:
⑴对靶向PDL1的CAR嵌合受体的慢病毒载体质粒进行质粒大提,然后与293ft细胞进行质粒包装,转染,收集病毒上清液,得到PDL1-CAR病毒上清;
⑵分离人全血中的淋巴细胞,磁珠分选分离出T细胞,培养后加入PDL1-CAR病毒上清,培养,转染,得到PDL1-CAR-T细胞。
本发明还包括PDL1-CAR-T细胞的应用,在杀伤多发性骨髓瘤细胞方面的应用。
优选的PDL1-CAR-T细胞的应用,在应用过程中加入IFN-γ。其中IFN-γ的添加量为IFN-γ:PDL1-CAR-T细胞个数=(1000IU/毫升:5*106~10*106细胞数)
本发明实施例中Xba I与BamH I酶购自于Thermofisher公司,PLVX载体质粒购自于上海信裕生物科技有限公司;DMEM完全培养基含10%胎牛血清,购自于Gibco公司;NCG小鼠购自于南京模式动物中心。
以下结合具体实施例来对本发明作进一步的描述。
实施例1
一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒,包含以下编码序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列;
其编码序列为SEQ ID NO:1。
一种靶向PDL1的CAR嵌合受体的慢病毒载体质粒的制备方法,包括以下步骤:
①设计合成多核苷酸序列,如图1所示,主要包括以下编码序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列;并且在核苷酸序列两端分别添加特定的酶切位点(Xba I与BamH I),并将其连接在Blunt质粒载体中得到Blunt-PD1质粒。
②用Xba I与BamH I酶酶切PLVX载体质粒与Blunt-PD1载体质粒,露出各自的粘性末端后,再用T4连接酶,将它们连接在一起,并转化至stbl3菌株中,挑取单克隆提质粒并用Xba I与BamH I双酶切,合成的多核苷酸序列在1.5kb左右,而PLVX质粒载体在9kb左右。筛选到合适的质粒后,进一步测序验证,质粒命名为PDL1-CAR。(质粒送往生工生物工程(上海)股份有限公司进行进一步测序验证)。
③将验证正确的质粒对应的菌株保存于冰箱,温度-80℃,同时对该菌进行质粒大提,按照天根质粒大提试剂盒的说明书进行操作。大提质粒测量浓度并进行Xba I与BamH I双酶切,结果正确进行下一步质粒包装。
实施例2
一种包含权利要求1所述靶向PDL1的CAR嵌合受体的慢病毒载体质粒基因的PDL1-CAR-T细胞,制备方法包括以下步骤:
培养状态良好的293ft细胞系,传代至10cm培养皿中,待细胞长至70-80%时,进行病毒包装,吸弃原有培养基,加入新的预热培养基8毫升,放置于培养箱中待转染;原有培养基以及新的预热培养基都是DMEM完全培养基。
取出两个已灭菌15毫升离心管,各加入3毫升的Opti-MEM培养基,其中一管加入转染试剂lipo300030微升,另外一管加入30微升p3000与适量的目的质粒和病毒包装质粒,将2个离心管液体吹打混匀,室温静置20分钟;
目的质粒是PDL1-CAR质粒;病毒包装质粒是PACK8与PACK2(质粒质量比为PDL1-CAR质粒:PACK8质粒:PACK2质粒=4:3:1)。
20分钟后,取出培养箱中的293ft细胞,将混合液用1毫升的小枪头轻轻滴入培养皿中,避免细胞在转染过程中漂起来,然后轻轻十字混匀,放置于培养箱中培养48-72h。293ft细胞是贴壁细胞,病毒包装时,293ft在培养皿中融合度在70%~80%左右,转染的目的质粒含有GFP,在倒置荧光显微镜下观测到绿色荧光即说明目的质粒已转入293ft细胞中,并成功表达,可进行病毒包装。观测绿色荧光覆盖率在60%及以上说明包装效果良好,可收集包装病毒上清,如图2中当PDL1-CAR高剂量时,可收集包装病毒上清。
选取二十四孔板,每孔加入4微升的Retronectin(TAKARA),再加入500微升的PBS,四度过夜;采用Ficoll分离人全血中的PBMC(人淋巴细胞),磁珠分选分离出T细胞,每孔1*105个,加入(100IU/孔)重组人IL-2与CD3/CD28beads(25微升每1*106个T细胞),培养箱过夜培养。
第二天将铺好retronectin的24孔板加入500微升2%BSA封闭半个小时,吸弃上清,加入2毫升PDL1-CAR病毒上清,4℃,3000rpm离心2h。吸弃上清,将活化的T细胞分别加入各孔,4℃,3000rpm离心2h,培养箱孵育48h。48h后离心收集各组细胞,PBS清洗一遍,加入适量PBS,混匀,过筛网,将过滤液上流式检测空白T以及转染PDL1-CAR-T细胞内GFP荧光强度,间接显示PDL1-CAR的转染效率。我们经过多次试验显示PDL1-CAR的转染效率能达到70-80%。
实施例3
一、选取多发性骨髓瘤细胞系H929、L363、RPMI8226以及U266作为体外研究对象,IFN-γ刺激下,流式细胞仪检测各细胞表面PDL1蛋白表达变化,具体步骤如下:
1)12孔板,分别接入四种骨髓瘤细胞系,每个细胞系各3孔(分为三组:Negative对照组、Control组、IFN-γ刺激组),每孔细胞数>5*105,孵育过夜;
2)在每个细胞系的IFN-γ组的孔内,加入适量IFN-γ干扰素,使其终浓度为1000IU/毫升,5%CO2,37℃孵育24小时;
3)24小时后,选取12个1.5毫升EP管,分别收集12孔板中12个孔内的四种骨髓瘤细胞(1000rpm,离心10min),弃掉上清,预冷的PBS清洗细胞一次;
4)四种细胞系的Control组与IFN-γ组,分别加入100微升预冷PBS,重悬细胞,各加入3微升PE-PDL1抗体,混匀后室温孵育15分钟;
5)1000rpm,离心10min,弃掉上清,预冷PBS清洗细胞一次;
6)所有12个1.5毫升EP管内的细胞,用500微升预冷PBS重悬,加入流式管内,上机检测各组PE荧光强度。
如图3所示,四种多发性骨髓瘤细胞系(H929、L363、RPMI8226、U266)在IFN-γ(1000IU/mL)刺激24小时后,各组PDL1蛋白表达阳性的细胞占总细胞的百分比率,由图中可以看出,U266与RPMI8226细胞系在IFN-γ作用下,细胞内PDL1蛋白的表达显著高于空白对照组,U266细胞的表达升高高于RPMI8226细胞系。
二、以U266作为后续的研究对象,观察IFN-γ干扰素刺激24小时后U266细胞的凋亡情况,具体步骤如下:
1)U266细胞传代至12孔板其中的9个孔内,每孔细胞数>5*105,37℃,5%CO2孵育过夜;
2)将其分为三组(Control组、IFN-γ组、顺铂阳性对照组,3孔/组),FIN-γ组加入适量IFN-γ干扰素,使其终浓度为1000IU/mL,顺铂阳性对照组加入25μM的顺铂(化疗药物,可以引起U266细胞的凋亡,作为阳性对照),Control组加入与前两孔等量的培养基(2微升)作为空白对照组,37℃,5%CO2孵育24小时;
3)选取9个1.5毫升EP管,分别对应收集各孔细胞至EP管内,1000rpm,离心10min,弃掉上清,预冷PBS洗一遍;
4)BD试剂公司凋亡试剂盒(货号556570)内的1×binding buffer稀释细胞至1*106/mL,吸取100微升至流式管内,加入5μl FITC Annexin V和5μl PI,轻轻震荡混匀,并室温避光孵育15min;
5)向各管内加入400微升预冷1×binding buffer,轻轻震荡混匀,流式细胞仪检测FITC和PI荧光变化。
图4为流式细胞仪检测IFN-γ对U266细胞凋亡的影响示意图,可以看出,25μM顺铂刺激24小时后,U266细胞有明显的凋亡(约18%的细胞凋亡与坏死,空白对照组为0.2%),而1000IU/mL的IFN-γ刺激24小时对U266细胞的凋亡无明显影响(凋亡与坏死不到1%,空白对照组约0.2%)。
三、将IFN-γ与PDL1-CAR-T细胞联合应用,研究其对多发性骨髓瘤细胞系U266的杀伤性,将空白T细胞与PDL1-CAR-T细胞分别与U266按照不同效靶比(1:1、2:1、5:1、10:1)混合孵育24小时后,如图4所示,PDL1-CAR-T细胞对U266的杀伤明显高于空白对照组,并且有显著性差异。同时,我们还发现,将IFN-γ与PDL1-CAR-T细胞联合应用,能进一步增强PDL1-CAR-T细胞对肿瘤细胞U266的杀伤,并且与PDL1-CAR-T单独应用组相比有显著性差异。
四、在体内动物实验中进一步验证IFN-γ对PDL1-CAR-T细胞的增效作用,现实构建了可以表达luciferase的U266-luc细胞系,以便于我们对U266细胞进行示踪。选取适龄的NCG小鼠,随机分为三组,分别尾静脉注射2*106个U266-luc细胞系,两天后,三组小鼠分别做以下处理:PDL1-CAR-T组:尾静脉注射1*107PDL1-CAR-T细胞;PDL1-CAR-T+IFN-γ组:尾静脉注射1*107PDL1-CAR-T细胞+IFN-γ干扰素;control组:尾静脉注射等量的生理盐水。给药后十五天,小鼠发光成像系统观察肿瘤细胞的增殖变化,如图5结果显示,PDL1-CAR-T细胞单独应用组能明显减少多发性骨髓瘤细胞系U266在NCG小鼠体内的增殖,而且当IFN-γ与PDL1-CAR-T细胞联合应用时,能进一步减少多发性骨髓瘤细胞系U266在体内的增殖。
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<110> 山东大学第二医院
<120> 一种靶向PDL1的CAR嵌合受体的慢病毒载体及PDL1-CAR-T细胞
<141> 2019-07-18
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 368
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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Ala Thr Phe Thr Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp
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Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Phe Pro Glu
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Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu
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His Met Ser Val Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile
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Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Arg Ala Glu Leu Arg
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Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser
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Pro Arg Pro Ala Gly Phe Gln Lys Arg Gly Arg Lys Lys Leu Leu Tyr
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Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
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Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
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Leu Leu Ala Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile
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Leu Thr Ala Leu Phe Leu Arg Val Arg Ser Lys Arg Ser Arg Leu Leu
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Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
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Ser Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly Gly Gly
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Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr
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Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly Leu
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Pro Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu
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Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala
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Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala
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Claims (2)
1.一种PDL1-CAR-T细胞,其特征在于:包含下述靶向PDL1的CAR嵌合受体的慢病毒载体质粒基因;其中靶向PDL1的CAR嵌合受体的慢病毒载体质粒基因包含以下编码序列:专门识别PDL1抗原的PD1蛋白的胞外域编码序列,人CD8铰链区编码序列、CD28跨膜区编码序列、人CD28胞内域编码序列和人CD3ζ编码序列;
靶向PDL1的CAR嵌合受体的慢病毒载体质粒基因的编码序列为SEQ ID NO:1;
在应用过程中加入IFN-γ,其中IFN-γ的添加量为IFN-γ:PDL1-CAR-T细胞个数=(1000IU/毫升:5*106~10*106细胞数)。
2.权利要求1所述PDL1-CAR-T细胞的应用,其特征在于:在制备治疗杀伤多发性骨髓瘤细胞药物方面的应用。
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