CN109608547B - 表达Her2的嵌合抗原受体、慢病毒表达载体及其应用 - Google Patents
表达Her2的嵌合抗原受体、慢病毒表达载体及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种表达Her2的嵌合抗原受体、慢病毒表达载体及其应用,所述嵌合抗原受体由人CD8a分子信号肽、高亲和性Her2单链抗体、人CD8a分子柔性片段与跨膜区、人41BB分子胞内区、人CD3z分子胞内区依次串联构成。本发明制备的嵌合抗原受体能够靶向Her2,提高CAR‑T细胞治疗效果。
Description
技术领域
本发明属于生物技术领域,具体涉及一种表达Her2的嵌合抗原受体、慢病毒表达载体及其应用。
背景技术
表皮生长因子2(Her2)是重要的促癌基因,在多种肿瘤中高表达,是一种较好的肿瘤治疗靶点。利用转基因技术,将识别Her2的嵌合抗原受体(CAR)序列转导入T细胞中,能够产生识别特异靶点Her2、杀伤肿瘤细胞的CAR-T细胞。CAR-T细胞治疗在血液系统肿瘤中展示了良好效果,但在实体肿瘤中的效果却难以令人满意。CAR-T细胞在实体瘤治疗中出现的短板与以下4个问题有关:1、转基因序列能否在T细胞中高效表达;2、转基因CAR序列是否具有较强亲和力,能够高效识别靶抗原;3、CAR中的连接片段是否能够高效将胞外识别区信号传递给胞内活化区以启动T细胞杀伤;4、CAR活化后是否会导致T细胞迅速无能乃至耗竭。
发明内容
本发明主要提供了一种表达Her2的嵌合抗原受体、慢病毒表达载体及其应用,所述嵌合抗原受体能够靶向Her2,提高CAR-T细胞治疗效果。其技术方案如下:
一种表达Her2的嵌合抗原受体,所述嵌合抗原受体由人CD8a分子信号肽、高亲和性Her2单链抗体、人CD8a分子柔性片段与跨膜区、人41BB分子胞内区、人CD3z分子胞内区依次串联构成。
优选的,所述人CD8a分子信号肽的氨基酸序列如SEQ ID NO.1所示,所述高亲和性Her2单链抗体的氨基酸序列如SEQ ID NO.2所示,所述人CD8a分子柔性片段与跨膜区的氨基酸序列如SEQ ID NO.3所示,所述人41BB分子胞内区的氨基酸序列如SEQ ID NO.4所示,所述人CD3z分子胞内区的氨基酸序列如SEQ ID NO.5所示。
一种核酸序列,所述核酸序列用于编码上述嵌合抗原受体,所述核酸序列如SEQID NO.7所示。
一种慢病毒表达载体,所述载体载有上述嵌合抗原受体。
优选的,所述嵌合抗原受体为在pCDH-EF1-MSC质粒中表达,形成pCDH-EF1-CAR-her2BBz制粒。
一种慢病毒,所述慢病毒为将上述慢病毒表达载体与包装制粒psPAX2和pMD2.G共转染靶细胞得到。
上述嵌合抗原受体在制备识别特异靶点Her2的CAR-T细胞中具有医学上的应用。
采用上述方案,本发明具有以下优点:
(1)CAR主要由3部分构成:胞外识别区、信号转导区和胞内信号区。其中,胞外识别区决定了CAR-T细胞杀伤的特异性,往往由scFv序列构成,我们选择了高亲和性识别Her2的scFv序列(Her2scFv),能够高效识别靶细胞。胞内信号区也是决定CAR-T细胞治疗效果的重要结构,我们通过加入41BB信号域,显著提高了CAR-T细胞扩增与生存。此外,信号转导区的结构选择也影响CAR-T细胞功能。我们利用CD8铰链区与跨膜区序列保证胞外识别区与胞内信号区之间有了很好的结合,保证了CAR-T细胞的功能。
(2)在传统制备过程中,用于治疗的CAR-T细胞往往处于终末分化阶段,生存时间较短、难以发挥长效杀伤功能,因而治疗效果较差。而我们采取的41BB共刺激信号显著抑制T细胞终末分化,促进具备更长生存时间的记忆型T细胞生成。此类转基因修饰的CAR-T细胞具备更强、更持久的杀伤能力,有望提高实体瘤治疗效果。
附图说明
图1为Her2-CAR结构示意图;
图2为流式细胞术检测T细胞表达CAR表达情况图;
图3为流式细胞术检测CAR-T细胞分化表型图;
图4为CAR-T细胞特异杀伤Her2阳性细胞结果图;
图5为CAR-T细胞长效杀伤Her2阳性细胞结果图;
图6为CAR-T细胞控制小鼠肿瘤生长情况图。
具体实施方式
以下实施例中的实验方法如无特殊规定,均为常规方法,所涉及的实验试剂及材料如无特殊规定均为常规生化试剂和材料。
一、CAR结构
Her2-CAR基本结构由人CD8a分子信号肽(Leading signal)、高亲和性Her2单链抗体(scFv)、人CD8a分子柔性片段(CD8Hinge)与跨膜区(CD8TM)、人41BB分子胞内区(41BB)和人CD3z分子胞内区(CD3z)依次串联构成,串联形成的结构如图1所示。所述Her2-CAR结构的氨基酸序列如SEQ ID NO.6所示。
其中,人CD8a分子信号肽(Leading signal)的氨基酸序列为:
MALPVTALLLPLALLLHAARP(SEQ ID NO.1)
人源化Her2单链抗体(Humanized scFv)的氨基酸序列为:
DIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAWYQQKPGQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFGGGTKLEIKRGSTSGSGKPGSGEGSTKGEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSS(SEQ ID NO.2)
人CD8a分子柔性片段(CD8Hinge)与跨膜区(CD8TM)的氨基酸序列为:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT(SEQ ID NO.3)
人41BB分子胞内区(41BB)的氨基酸序列为:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.4)
人CD3z分子胞内区(CD3z)的氨基酸序列为:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.5)
二、CAR序列合成及载体构建
CAR编码序列由上海生工公司合成,获得的Her2-CAR结构的DNA序列(SEQ ID NO.7所示)通过酶切连接插入到pCDH-EF1-MSC质粒中,构建慢病毒表达质粒pCDH-EF1-CAR-her2BBz。
三、慢病毒包装
包装细胞293T铺板24小时后,将pCDH-EF1-CAR-her2BBz表达质粒与包装质粒(psPAX2与pMD2.G)混合,利用磷酸钙转染试剂进行293T细胞转染。转染48小时后,收集上清,准备用于T细胞感染。
四、T细胞纯化与感染
人外周血经密度梯度离心后,分离外周血单个核细胞。利用德国美天旎公司的T细胞分离试剂盒获得纯化的CD3+T细胞,再按照2个细胞加入1个磁珠的比例,加入适量CD3/CD28磁珠活化2天。2天后,加入病毒上清与polybrene(6μg/mL)孵育过夜。次日,离心清洗T细胞3次后,加入含1000U IL-2与5%胎牛血清的RPMI1640培养基扩增T细胞。
五、T细胞CAR表达效率
T细胞感染3天后,利用流式细胞术对T细胞表面CAR的表达情况进行检测。结果如图2所示。图2显示CAR表达阳性率到达约60%,证明CAR表达质粒构建及病毒包装成功。
六、CAR-T细胞分化
T细胞感染14天后,利用流式细胞术对T细胞的分化情况进行分析。流式细胞术检测结果显示Her2BBz-CAR-T细胞主要向记忆性T细胞(CD45RO+CD62L+)亚型分化(图3),提示该类转基因CAR-T细胞有更加持久的杀伤功能。
七、CAR-T细胞短效杀伤
T细胞感染14天后,计数T细胞与靶细胞,并利用细胞染料(eFluor670)对靶细胞进行标记。然后按照效靶比(效应细胞:靶细胞,E:T)1:1,1:5,1:20的比例,将T细胞(效应细胞)与Her2阳性靶细胞(H322,KYSE70,TE-1,MCF-7,MDA-MD-231,A549)或Her2阴性靶细胞(16HBE与Raji)共孵育6小时(如图4所示)。共孵育结束后,离心收集细胞,利用凋亡染色试剂盒标记细胞,然后流式细胞术分析靶细胞凋亡情况。结果显示,CAR-T细胞对表达Her2的肿瘤细胞具有很强的杀伤能力,而对Her2阴性细胞的影响很小,说明该CAR-T细胞具有很强的特异性杀伤能力。
八、CAR-T细胞长效杀伤
为了进一步确定Her2BBz-CAR-T细胞具有更好的持续杀伤能力,我们利用细胞活性实时检测系统观察了CAR-T细胞对靶细胞(A549)的杀伤。CAR-T细胞按照E:T=0,1,2,5的比例加入到靶细胞中,然后对靶细胞的活性进行了96小时的连续观察(图5)。结果显示,Her2BBz-CAR-T细胞能够持续杀伤肿瘤细胞(图5),说明该CAR-T细胞具备发挥长效杀伤的能力。
九、动物实验验证CAR-T细胞功能
免疫缺陷小鼠经尾静脉接种5×106个A549细胞7天后,经尾静脉分别注射5×106个CAR-T细胞或T细胞进行治疗。治疗0,3,7,14天时,利用小动物活体成像设备,观察肿瘤大小。如图6所示,在CAR-T细胞注射后7天(A),肿瘤体积明显缩小,而且随着治疗时间的延长,CAR-T细胞治疗组的肿瘤体积越来越小(B),提示CAR-T细胞具有良好的肿瘤杀伤能力。
对本领域的技术人员来说,可根据以上描述的技术方案以及构思,做出其它各种相应的改变以及形变,而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
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Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro
130 135 140
Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Gln Gln Ser
145 150 155 160
Gly Pro Glu Val Val Lys Thr Gly Ala Ser Val Lys Ile Ser Cys Lys
165 170 175
Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Phe Ile Asn Trp Val Lys Lys
180 185 190
Asn Ser Gly Lys Ser Pro Glu Trp Ile Gly His Ile Ser Ser Ser Tyr
195 200 205
Ala Thr Ser Thr Tyr Asn Gln Lys Phe Lys Asn Lys Ala Ala Phe Thr
210 215 220
Val Asp Thr Ser Ser Ser Thr Ala Phe Met Gln Leu Asn Ser Leu Thr
225 230 235 240
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Tyr Glu
245 250 255
Glu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
260 265 270
Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
275 280 285
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
290 295 300
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
305 310 315 320
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
325 330 335
Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 7
<211> 1491
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacattg tgctgaccca aactccatcc tccctacctg tgtcagttgg agagaaggtt 120
actatgacct gcaagtccag tcagaccctt ttatatagta acaatcaaaa gaactacttg 180
gcctggtacc agcagaaacc agggcagtct cctaaactgc tgatttcctg ggcattcact 240
aggaaatctg gggtccctga tcgcttcaca ggcagtggat ctgggacaga tttcactctc 300
accatcggca gtgtgaaggc tgaagacctg gcagtttatt actgtcagca atattctaac 360
tatccgtgga cgttcggtgg aggcaccaag ctggaaatca aacggggtgg tggtggttct 420
ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg gatccgaggt ccagctgcag 480
cagtctggac ctgaggtagt gaagactggg gcttcagtga agatatcctg caaggcttct 540
ggttactcat tcactggtta cttcataaac tgggtcaaga agaactctgg aaagagccct 600
gagtggattg gacacattag ttcttcctat gctacctcta cctacaacca gaagtttaaa 660
aacaaggccg catttactgt agacacatcc tccagcacag ccttcatgca gcttaacagc 720
ctgacatctg aggactctgc agtctattat tgtgttagaa gtggtaacta cgaagaatat 780
gctatggact attggggtca aggaacctca gtcaccgtct cgtcaaccac gacgccagcg 840
ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 900
gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 960
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020
accaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1080
caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1140
tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1200
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1260
agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa 1320
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta g 1491
Claims (6)
1.一种靶向Her2的嵌合抗原受体,其特征在于:所述编码嵌合抗原受体的核酸序列如SEQ ID NO.7所示,由人CD8a分子信号肽、高亲和性Her2单链抗体、人CD8a分子柔性片段与跨膜区、人41BB分子胞内区、人CD3z分子胞内区依次串联构成。
2.根据权利要求1所述的靶向Her2的嵌合抗原受体,其特征在于:所述人CD8a分子信号肽的氨基酸序列如SEQ ID NO.1所示,所述高亲和性Her2单链抗体的氨基酸序列如SEQ IDNO.2所示,所述人CD8a分子柔性片段与跨膜区的氨基酸序列如SEQ ID NO.3所示,所述人41BB分子胞内区的氨基酸序列如SEQ ID NO.4所示,所述人CD3z分子胞内区的氨基酸序列如SEQ ID NO.5所示。
3.一种慢病毒表达载体,其特征在于:所述载体载有权利要求1所述的嵌合抗原受体的编码基因。
4.根据权利要求3所述的慢病毒表达载体,其特征在于:所述嵌合抗原受体的编码基因为在pCDH-EF1-MSC质粒中表达,形成pCDH-EF1-CAR-her2BBz质粒。
5.一种慢病毒,其特征在于:所述慢病毒为将权利要求3或4中的慢病毒表达载体与包装质粒psPAX2和pMD2.G共转染靶细胞得到。
6.权利要求1或2所述的嵌合抗原受体在制备识别特异靶点Her2的 CAR-T细胞中的应用。
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