CN109608548A - 基于人源cd20抗体的嵌合抗原受体、慢病毒表达载体及其应用 - Google Patents

基于人源cd20抗体的嵌合抗原受体、慢病毒表达载体及其应用 Download PDF

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CN109608548A
CN109608548A CN201711467594.7A CN201711467594A CN109608548A CN 109608548 A CN109608548 A CN 109608548A CN 201711467594 A CN201711467594 A CN 201711467594A CN 109608548 A CN109608548 A CN 109608548A
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chimeric antigen
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张毅
李峰
张凯
张震
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First Affiliated Hospital of Zhengzhou University
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Abstract

本发明属于生物技术领域，具体涉及一种基于人源CD20抗体的嵌合抗原受体、慢病毒表达载体及其应用，所述嵌合抗原受体为CD20‑28z‑CAR嵌合抗原受体或为CD20‑BBz‑CAR嵌合抗原受体，所述CD20‑28z‑CAR嵌合抗原受体由人CD8a分子信号肽、人源CD20单链抗体、人CD8a分子柔性片段、人CD28分子跨膜区与胞内区、人CD3z分子胞内区依次串联构成；所述CD20‑BBz‑CAR嵌合抗原受体由人CD8a分子信号肽、人源CD20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。本发明制备了靶向CD20的CAR‑T细胞，治疗效果显著。

Description

基于人源CD20抗体的嵌合抗原受体、慢病毒表达载体及其 应用
技术领域
本发明属于生物技术领域，具体涉及一种基于人源CD20抗体的嵌合抗原受体、慢病毒表达载体及其应用。
背景技术
CD20在B细胞白血病或淋巴瘤恶性细胞上高表达，是一个较佳的血液系统肿瘤治疗靶点。嵌合抗原受体(CAR)是一种人工合成的结构。利用转基因技术，将CAR序列转导入T细胞中，能够产生识别特异靶点、杀伤肿瘤细胞的CAR-T细胞。但是，CAR-T细胞识别区的物种来源以及胞内共刺激信号组成严重影响该类细胞治疗效果。
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，但很多scFv序列是小鼠来源的，会造成较强的免疫排斥反应，导致CAR-T细胞治疗无效。
发明内容
本发明主要提供了一种基于人源CD20抗体的嵌合抗原受体、慢病毒表达载体及其应用，制备了靶向CD20的CAR-T细胞，治疗效果显著。其技术方案如下：
一种基于人源CD20抗体的嵌合抗原受体，所述嵌合抗原受体为CD20-28z-CAR嵌合抗原受体或为CD20-BBz-CAR嵌合抗原受体，所述CD20-28z-CAR嵌合抗原受体由人CD8a分子信号肽、人源CD20单链抗体、人CD8a分子柔性片段、人CD28分子跨膜区与胞内区、人CD3z分子胞内区依次串联构成；所述CD20-BBz-CAR嵌合抗原受体由人CD8a分子信号肽、人源CD20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，所述人源CD20单链抗体的氨基酸序列如SEQ ID NO.2所示。
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，所述人CD8a分子柔性片段的氨基酸序列如SEQ ID NO.3所示，所述人CD28分子跨膜区与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。
优选的，所述人CD8a分子柔性片段与跨膜区的氨基酸序列如SEQ ID NO.6所示，所述人41BB分子胞内区的氨基酸序列如SEQ ID NO.7所示。
一种核酸序列，所述核酸序列用于上述嵌合抗原受体，其中编码CD20-28z-CAR嵌合抗原受体的序列如SEQ ID NO.10所示，编码CD20-BBz-CAR嵌合抗原受体的序列如SEQ IDNO.11所示。
一种慢病毒表达载体，所述载体载有上述的嵌合抗原受体。
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-CD20-28z制粒或pCDH-EF1-CAR-CD20-BBz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CD20的CAR-T细胞中具有显著的医学应用。
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，但很多scFv序列是小鼠来源的，会造成较强的免疫排斥反应，导致CAR-T细胞治疗无效。而我们选择了高亲和性识别CD20的人源scFv序列(anti-CD20scFv)，既能够有效避免免疫排斥，又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，我们通过加入共刺激信号分子(CD28或41BB)，保证CAR-T细胞具备较强的杀伤功能与扩增能力。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铰链区保证胞外识别区与信号区之间有了很好的结合，保证了CAR-T细胞的功能。
(2)采用非人源scFv作为识别区时，CAR-T细胞往往受到机体排斥，导致治疗失败。本发明采用了完全人源的抗CD20scFv作为CAR-T细胞识别区，可以有效避免上述问题，有助于治疗效果改善。另外，通过添加胞内刺激信号(CD28或41BB)，能够改善T细胞功能。此类转基因修饰的CAR-T细胞具备更强、更持久的杀伤能力，有望提高肿瘤治疗效果。
附图说明
图1为CD20-28z-CAR结构示意图；
图2为CD20-BBz-CAR结构示意图；
图3为流式细胞术检测T细胞表达CAR表达情况图；
图4为CAR-T细胞增殖情况图；
图5为CAR-T细胞特异杀伤间皮素阳性细胞结果图；
图6为CAR-T细胞杀伤原代肿瘤细胞结果图；
图7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
1.CD20-28z-CAR结构
由人CD8a分子信号肽(Leading signal)、人源CD20单链抗体(scFv)、人CD8a分子柔性片段(CD8Hinge)、人CD28分子跨膜区与胞内区(CD28)，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CD20-28z-CAR结构的氨基酸序列如SEQ IDNO.8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CD20单链抗体(Human scFv)的氨基酸序列为：
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSGSTSGSGKPGSGEGSTKGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEI(SEQ ID NO.2)
人CD8a分子柔性片段(CD8Hinge)的氨基酸序列为：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.3)
人CD28分子跨膜区与胞内区(CD28)的氨基酸序列为：
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO.4)
人CD3z分子胞内区(CD3z)的氨基酸序列为：
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.5)
2.CD20-BBz-CAR结构
由人CD8a分子信号肽(Leading signal)、人源CD20单链抗体(scFv)、人CD8a分子柔性片段与跨膜区(CD8Hinge TM)、人41BB分子胞内区(41BB)，人CD3z分子胞内区(CD3z)依次串联构成，串联形成的结构如图2所示。所述CD20-BBz-CAR结构的氨基酸序列如SEQ IDNO.9所示。
其中，人CD8a分子信号肽(Leading signal)的氨基酸序列如SEQ ID NO.1所示。
人源CD20单链抗体(scFv)的氨基酸序列如SEQ ID NO.2所示。
人CD8a分子柔性片段与跨膜区(CD8Hinge TM)的氨基酸序列如SEQ ID NO.6所示。
人41BB分子胞内区(41BB)的氨基酸序列如SEQ ID NO.7所示。
人CD3z分子胞内区(CD3z)的氨基酸序列如SEQ ID NO.5所示。
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CD20-28z-CAR结构的DNA序列(SEQ IDNO.10所示)或CD20-BBz-CAR结构的DNA序列(SEQ ID NO.11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-CD20-28z或pCDH-EF1-CAR-CD20-BBz。
三、慢病毒包装
包装细胞293T铺板24小时后，将pCDH-EF1-CAR-CD20-28z或pCDH-EF1-CAR-CD20-BBz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3所示。图3显示CAR表达阳性率到达约50％，证明CAR表达质粒构建及病毒包装成功。
六、CAR-T细胞体外扩增
计数1×106个T细胞进行感染，并在感染后3，7，10和14天时进行计数，检测CAR-T细胞增殖情况。如图4所示，CAR-T细胞均能够高效增殖(14天内扩增30-70倍)，其中CD20-BBz-CAR细胞的增殖能力更好。
七、CAR-T细胞杀伤效果
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CD20高表达靶细胞(Raji)、CD20低表达靶细胞(Nalm6)或CD20阴性靶细胞(Jurkat)共孵育6小时。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。结果如图5所示，CAR-T细胞对表达CD20的肿瘤细胞具有很强的杀伤能力，而对CD20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细胞能够杀伤原代肿瘤细胞
将分离自B细胞白血病或淋巴瘤化患者的原代B细胞用细胞染料(eFluor670)标记后，与感染14天时的T细胞共孵育，效靶比(效应细胞:靶细胞，E:T)为1:1，1:5，1:20。6小时后，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原代肿瘤凋亡情况。结果如图6所示，CAR-T细胞对原代肿瘤细胞具有较强的杀伤能力。
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Raji细胞7天后，经尾静脉分别注射5×106个CAR-T细胞(CD20-28z-CAR或CD20-BBz-CAR)或T细胞(T cell)进行治疗。治疗0，3，7，14天时，利用小动物活体成像设备，观察肿瘤分布。如图7所示，在CAR-T细胞注射后7天(A)，肿瘤负荷明显降低，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。
序列表
<110> 郑州大学第一附属医院
<120> 基于人源CD20抗体的嵌合抗原受体、慢病毒表达载体及其应用
<160> 11
<170> SIPOSequenceListing 1.0
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<400> 1
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 2
<211> 245
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
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Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
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Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
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Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
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Gly Gln Gly Thr Thr Val Thr Val Ser Gly Ser Thr Ser Gly Ser Gly
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Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val Leu Thr
130 135 140
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
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Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln
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Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn
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Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
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Thr Arg Leu Glu Ile
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<210> 3
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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
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<210> 4
<211> 68
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<210> 5
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Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
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20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
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His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
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Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
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Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
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Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
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Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
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<211> 487
<212> PRT
<213> 人工序列(Artificial Sequence)
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His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
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Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr
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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
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Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Thr Thr Thr Pro Ala Pro
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Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
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Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
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Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
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Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly Arg
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Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
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Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
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Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
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Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
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Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210> 10
<211> 1485
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggaggtcc agctggtgga gagcgggggg gggctggttc agcccggcag gtccctgagg 120
ctgagctgcg ccgccagcgg cttcacattc aacgattacg ccatgcattg ggtgaggcag 180
gcccccggca aagggctgga gtgggtgagc acaatttcct ggaactccgg aagcatcggg 240
tacgctgact ctgtcaaggg ccgctttaca attagccggg acaacgccaa gaagagcctg 300
tacttgcaga tgaactccct gagggccgag gatacggccc tgtactactg cgccaaggat 360
atccagtacg ggaactacta ctacgggatg gatgtgtggg gccaggggac aactgtgacc 420
gtgagcgggg gcggagggtc cgggggaggc gggtccgggg ggggcggcag cggcggaggc 480
ggatcagaaa ttgtgcttac ccagtcacca gccacactct cactgtcccc tggggaacgg 540
gcaactttat cctgtagggc tagtcaatcc gtgagcagct acctcgcctg gtatcagcag 600
aagcctggac aggctcccag gctgctgatc tacgatgcca gcaacagggc cacaggcatt 660
cccgcccggt ttagcgggtc cggcagcggg accgacttca cactgaccat cagcagcctg 720
gagcccgagg acttcgccgt gtactactgc cagcagcggt ccaactggcc aatcacattc 780
ggccagggca ccaggctgga gatcaccacg acgccagcgc cgcgaccacc aacaccggcg 840
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900
ggcgcagtgc acacgagggg gctggacttc gcctgtgatt tttgggtgct ggtggtggtt 960
ggtggagtcc tggcttgcta tagcttgcta gtaacagtgg cctttattat tttctgggtg 1020
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1080
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1140
tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1200
ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1260
ggccgggacc ctgagatggg gggaaagccg cagagaagga agaaccctca ggaaggcctg 1320
tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380
gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440
gacacctacg acgcccttca catgcaggcc ctgccccctc gctag 1485
<210> 11
<211> 1470
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggccctgc ccgtgacagc cctgctgctg cccctggccc tgctgctcca cgccgcccgc 60
cccgaggtcc agctggtgga gagcgggggg gggctggttc agcccggcag gtccctgagg 120
ctgagctgcg ccgccagcgg cttcacattc aacgattacg ccatgcattg ggtgaggcag 180
gcccccggca aagggctgga gtgggtgagc acaatttcct ggaactccgg aagcatcggg 240
tacgctgact ctgtcaaggg ccgctttaca attagccggg acaacgccaa gaagagcctg 300
tacttgcaga tgaactccct gagggccgag gatacggccc tgtactactg cgccaaggat 360
atccagtacg ggaactacta ctacgggatg gatgtgtggg gccaggggac aactgtgacc 420
gtgagcgggg gcggagggtc cgggggaggc gggtccgggg ggggcggcag cggcggaggc 480
ggatcagaaa ttgtgcttac ccagtcacca gccacactct cactgtcccc tggggaacgg 540
gcaactttat cctgtagggc tagtcaatcc gtgagcagct acctcgcctg gtatcagcag 600
aagcctggac aggctcccag gctgctgatc tacgatgcca gcaacagggc cacaggcatt 660
cccgcccggt ttagcgggtc cggcagcggg accgacttca cactgaccat cagcagcctg 720
gagcccgagg acttcgccgt gtactactgc cagcagcggt ccaactggcc aatcacattc 780
ggccagggca ccaggctgga gatcaccacg acgccagcgc cgcgaccacc aacaccggcg 840
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960
gccgggactt gtggggtcct tctcctgtca ctggttatca ccaaacgggg cagaaagaaa 1020
ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1080
ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140
agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200
aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260
atggggggaa agccgcagag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgctag 1470

Claims (9)

1.一种基于人源CD20抗体的嵌合抗原受体，其特征在于：所述嵌合抗原受体为CD20-28z-CAR嵌合抗原受体或为CD20-BBz-CAR嵌合抗原受体，所述CD20-28z-CAR嵌合抗原受体由人CD8a分子信号肽、人源CD20单链抗体、人CD8a分子柔性片段、人CD28分子跨膜区与胞内区、人CD3z分子胞内区依次串联构成；所述CD20-BBz-CAR嵌合抗原受体由人CD8a分子信号肽、人源CD20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所述的基于人源CD20抗体的嵌合抗原受体，其特征在于：所述人源CD20单链抗体的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的基于人源CD20抗体的嵌合抗原受体，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，所述人CD8a分子柔性片段的氨基酸序列如SEQ ID NO.3所示，所述人CD28分子跨膜区与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。
4.根据权利要求1所述的基于人源CD20抗体的嵌合抗原受体，其特征在于：所述人CD8a分子柔性片段与跨膜区的氨基酸序列如SEQ ID NO.6所示，所述人41BB分子胞内区的氨基酸序列如SEQ ID NO.7所示。
5.一种核酸序列，其特征在于：所述核酸序列用于编码权利要求1所述的嵌合抗原受体，其中编码CD20-28z-CAR嵌合抗原受体的序列如SEQ ID NO.10所示，编码CD20-BBz-CAR嵌合抗原受体的序列如SEQ ID NO.11所示。
6.一种慢病毒表达载体，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。
7.根据权利要求6所述的慢病毒表达载体，其特征在于：所述嵌合抗原受体为在pCDH-EF1-MSC质粒中表达，形成pCDH-EF1-CAR-CD20-28z制粒或pCDH-EF1-CAR-CD20-BBz制粒。
8.一种慢病毒，其特征在于：所述慢病毒为将权利要求6或7中的慢病毒表达载体与包装制粒psPAX2和pMD2.G共转染靶细胞得到。
9.权利要求1-4任一项所述的嵌合抗原受体在制备识别特异靶点CD20的CAR-T细胞中的应用。
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WO2016164731A2 (en) * 2015-04-08 2016-10-13 Novartis Ag Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car) - expressing cell
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CN112390891A (zh) * 2019-08-14 2021-02-23 苏州方德门达新药开发有限公司 嵌合抗原受体及其构建方法和应用
CN112390891B (zh) * 2019-08-14 2022-06-03 苏州方德门达新药开发有限公司 嵌合抗原受体及其构建方法和应用

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