CN114163538B - 同时靶向gpc3和cd276的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用 - Google Patents

同时靶向gpc3和cd276的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用 Download PDF

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CN114163538B
CN114163538B CN202111501965.5A CN202111501965A CN114163538B CN 114163538 B CN114163538 B CN 114163538B CN 202111501965 A CN202111501965 A CN 202111501965A CN 114163538 B CN114163538 B CN 114163538B
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万晓春
曹国政
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

本发明公开了一种同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用。实验发现同时靶向GPC3和CD276的嵌合抗原受体T细胞对同时表达GPC3和CD276肿瘤细胞系有较强的杀伤活性，并且对GPC3阴性CD276阳性肿瘤细胞系有强的杀伤活性。因此所述嵌合抗原受体T细胞在肝细胞癌等的治疗中有较大的应用前景。

Description

同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞 及其制备方法和应用
技术领域
本发明涉及生物制品，具体涉及同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用。
背景技术
肝细胞癌是常见的恶性肿瘤，占所有原发性肝癌的90％，是全球第二大致死性癌症。大多数肝细胞癌病人接受局部或***性疗法，比如化疗、放疗、肝切除术、皮肤乙醇注射、射频消融术、各种栓塞疗法、抗体疗法如免疫检查点抑制剂、血管内皮生长因子受体抑制剂和靶向疗法如索拉菲尼，然而这些疗法的疗效都是不够的(Lee,Y.H.,et al.,Combinational Immunotherapy for Hepatocellular Carcinoma:Radiotherapy,ImmuneCheckpoint Blockade and Beyond.Frontiers in Immunology,2020.11)，对病人和病人的家庭造成了难以挽回的伤害。因此迫切需要新的治疗肝细胞癌的药物。
CAR-T(嵌合抗原受体T细胞)技术是近年来发展火热的细胞免疫疗法，它通过基因工程手段(例如基于重组病毒载体的转导)使嵌合抗原受体表达在T细胞表面，通过在体外制备和扩增嵌合抗原受体T细胞，然后回输至人体，激活自身免疫***，发挥对肿瘤细胞的高效杀伤(Rafiq,S.,C.S.Hackett,and R.J.Brentjens,Engineering strategies toovercome the current roadblocks in CAR T cell therapy.Nature Reviews ClinicalOncology,2020.17(3):p.147-167.Benmebarek,M.R.,et al.,Killing Mechanisms ofChimeric Antigen Receptor(CAR)T Cells.International Journal of MolecularSciences,2019.20(6).)。嵌合抗原受体T细胞技术被认为是最有可能攻克癌症的疗法，并且靶向CD19的CAR-T细胞在血液肿瘤中产生了良好的效果。目前全球已有6款CAR-T药物获批上市，展现了CAR-T细胞在治疗恶性肿瘤中的巨大应用前景。
然而CAR-T细胞治疗面临肿瘤抗原异质性表达、抗原丢失的难题。首先在急性淋巴细胞白血病中，CD19阴性细胞群可以逃避靶向CD19嵌合抗原受体T细胞的识别，导致细胞群的过度生长，产生疾病的复发。另外在胰腺癌、***癌和神经母细胞瘤的临床前研究中，单抗原靶向的嵌合抗原受体T细胞并不能根除肿瘤，这是因为嵌合抗原受体T细胞不能识别这些肿瘤细胞表达的低密度的靶抗原(Anurathapan,U.；Chan,R.C.；Hindi,H.F.；Mucharla,R.；Bajgain,P.；Hayes,B.C.；Fisher,W.E.；Heslop,H.E.；Rooney,C.M.；Brenner,M.K.；et al.Kinetics of tumor destruction by chimeric antigenreceptor-modified t cells.Mol.Ther.2014,22,623–633.O’Rourke,D.M.；Nasrallah,M.P.；Desai,A.；Melenhorst,J.J.；Mansfield,K.；Morrissette,J.J.D.；Martinez-Lage,M.；Brem,S.；Maloney,E.；Shen,A.；et al.A single dose of peripherally infusedegfrviii-directed car t cells mediates antigen loss and induces adaptiveresistance in patients with recurrent glioblastoma.Sci.Transl.Med.2017,9,eaaa0984)。在肝细胞癌中，血清中GPC3含量升高，抑制了靶向GPC3的嵌合抗原受体T细胞与细胞表面GPC3的结合，阻碍了嵌合抗原受体T细胞的疗效(Sun,L.,et al.,Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3in HepatocellularCarcinoma.Journal for Immunotherapy of Cancer,2021.9(4))。
CD276又称为B7-H3，是属于B7家族的一类跨膜蛋白。CD276在多种癌症中高表达，包括肺腺癌、胶质瘤、神经母细胞瘤、胰腺癌、卵巢癌，在正常组织中几乎不表达。特异性识别CD276的单克隆抗体在多种肿瘤模型中介导肿瘤的有效清除。CD276在肝细胞癌高表达并且与肝癌的进展和肿瘤病人的不良预后相关，靶向CD276的CAR-T细胞在体外能有效的杀伤肝细胞癌细胞系。但单靶点的CAR-T细胞治疗实体瘤(例如肝细胞癌)面临抗原异质性表达的障碍。
发明内容
为了解决以上的问题，本发明提供一种同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用。
为达到上述目的，本发明采用了以下技术方案：
第一方面，本发明提供了一种同时靶向GPC3和CD276的嵌合抗原受体，该嵌合抗原受体命名为GPC3-CD276 TanCAR。
优选的，所述嵌合抗原受体包括从氨基端到羧基端依次排列的靶向GPC3的单链抗体、连接子(linker)、靶向CD276的单链抗体、胞外铰链区、跨膜区、共刺激信号区以及CD3ζ胞内区。
优选的，所述靶向GPC3的单链抗体的氨基酸序列如SEQ.ID.NO.3所示。
优选的，所述靶向GPC3的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.4所示。
优选的，所述连接子包括重复串联的G4S，例如氨基酸序列如SEQ.ID.NO.5所示的(G4S)3。
优选的，所述连接子，例如(G4S)3的编码基因的核苷酸序列如SEQ.ID.NO.6所示。
优选的，所述靶向CD276的单链抗体的氨基酸序列如SEQ.ID.NO.7所示。
优选的，所述靶向CD276的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.8所示。
优选的，所述胞外铰链区选自CD8α铰链区、CD28铰链区、IgG1铰链区或IgG4铰链区。
优选的，所述CD8α铰链区的氨基酸序列如SEQ.ID.NO.9所示。
优选的，所述CD8α铰链区的编码基因的核苷酸序列如SEQ.ID.NO.10所示。
优选的，所述跨膜区选自CD8α跨膜区、CD4跨膜区、CD28跨膜区、CD3ζ跨膜区或ICOS跨膜区。
优选的，所述CD8α跨膜区的氨基酸序列如SEQ.ID.NO.11所示。
优选的，所述CD8α跨膜区的编码基因的核苷酸序列如SEQ.ID.NO.12所示。
优选的，所述共刺激信号区选自4-1BB胞内区、CD28胞内区、OX40胞内区、ICOS胞内区或CD27胞内区。
优选的，所述4-1BB胞内区的氨基酸序列如SEQ.ID.NO.13所示。
优选的，所述4-1BB胞内区的编码基因的核苷酸序列如SEQ.ID.NO.14所示。
优选的，所述CD3ζ胞内区的氨基酸序列如SEQ.ID.NO.15所示。
优选的，所述CD3ζ胞内区的编码基因的核苷酸序列如SEQ.ID.NO.16所示。
优选的，所述嵌合抗原受体包括如SEQ.ID.NO.1所示的氨基酸序列。
优选的，所述嵌合抗原受体的基因序列包括如SEQ.ID.NO.2所示的核苷酸序列。
第二方面，本发明提供了一种同时靶向GPC3和CD276的嵌合抗原受体T细胞，该T细胞包括上述同时靶向GPC3和CD276的嵌合抗原受体(即T细胞表面具有同时靶向GPC3和CD276的嵌合抗原受体)。
第三方面，本发明提供了一种重组病毒载体及重组病毒，该重组病毒载体包括上述嵌合抗原受体的编码基因，该重组病毒采用所述重组病毒载体进行慢病毒包装而成。
优选的，所述嵌合抗原受体的编码基因包括从5’端到3’端依次连接的信号肽的编码基因、靶向GPC3的单链抗体的编码基因、连接子(例如(G4S)3)的编码基因、靶向CD276的单链抗体的编码基因、胞外铰链区(例如CD8α胞外铰链区)的编码基因、跨膜区(例如CD8α跨膜区)的编码基因、共刺激信号区(例如4-1BB胞内区)的编码基因以及CD3ζ胞内区的编码基因。
优选的，所述信号肽为CD8α信号肽，CD8α信号肽的编码基因的核苷酸序列如SEQ.ID.NO.19所示。
优选的，所述CD8α信号肽的氨基酸序列如SEQ.ID.NO.20所示。
优选的，所述重组病毒载体及重组病毒中，同时靶向GPC3和CD276的嵌合抗原受体的基因序列如SEQ.ID.NO.17所示，该基因序列包括所述嵌合抗原受体的编码基因的核苷酸序列。如SEQ.ID.NO.17所示的核苷酸序列与第一方面中如SEQ.ID.NO.2所示的核苷酸序列相比，增加了CD8α信号肽的编码基因。所述信号肽的编码基因可以较好地指导所述嵌合抗原受体表达到细胞表面。
优选的，所述嵌合抗原受体的氨基酸序列如SEQ.ID.NO.18所示，具体如下：MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
优选的，所述重组病毒载体为pWPXLd慢病毒载体。
第四方面，本发明提供了一种同时靶向GPC3和CD276的嵌合抗原受体T细胞的制备方法，包括以下步骤：
1)通过基因克隆构建或人工合成同时靶向GPC3和CD276的嵌合抗原受体GPC3-CD276TanCAR的基因序列，所述GPC3-CD276 TanCAR的基因序列包括如SEQ.ID.NO.2所示的核苷酸序列(例如GPC3-CD276 TanCAR的基因序列如SEQ.ID.NO.17所示)；
2)将所述GPC3-CD276 TanCAR的基因序列***到pWPXLd载体中，得到重组质粒pWPXLd-GPC3-CD276 TanCAR；
3)将所述重组质粒pWPXLd-GPC3-CD276 TanCAR与包膜质粒、包装质粒共转染宿主细胞，得到重组慢病毒；
4)将所述重组慢病毒转染CD3阳性T淋巴细胞(简称CD3阳性T细胞)，经分离获得同时靶向GPC3和CD276的嵌合抗原受体T细胞。
优选的，所述包膜质粒为PMD2G，包装质粒为psPAX2，宿主细胞为HEK293T细胞。
优选的，所述步骤4中，CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。
优选的，所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血或胎盘血等。
第五方面，本发明提供了一种治疗肝细胞癌的药物，该药物包括上述同时靶向GPC3和CD276的嵌合抗原受体T细胞。
本发明的有益效果体现在：
本发明所提出的同时靶向GPC3和CD276的嵌合抗原受体T细胞，不仅对表达GPC3和CD276的肿瘤细胞有杀伤作用，同时对GPC3阴性CD276阳性肿瘤细胞有杀伤活性，解决了抗原异质性表达或抗原丢失造成的嵌合抗原受体T细胞治疗肝细胞癌等实体瘤效果不佳的难题。
附图说明
图1为GPC3-CD276 TanCAR的编码基因的结构示意图。
图2为GPC3-CD276 TanCAR在T细胞上表达的流式分析结果；其中：UTD组为未转导病毒的CD3阳性T细胞，GPC3-CD276 TanCAR-T组为同时靶向GPC3和CD276的嵌合抗原受体T细胞。
图3为同时靶向GPC3和CD276的嵌合抗原受体T细胞对Huh7细胞系的杀伤活性分析结果；其中：UTD组为未转导病毒的CD3阳性T细胞，GPC3-CD276 TanCAR-T组为同时靶向GPC3和CD276的嵌合抗原受体T细胞。
图4为同时靶向GPC3和CD276的嵌合抗原受体T细胞对SK-HEP-1-CD276细胞系杀伤活性分析结果；其中：UTD组为未转导病毒的CD3阳性T细胞，GPC3 CAR-T组为单靶点靶向GPC3的嵌合抗原受体T细胞，GPC3-CD276 TanCAR-T组为同时靶向GPC3和CD276的嵌合抗原受体T细胞；*表示差异显著。
具体实施方式
以下结合附图和实施例对本发明做进一步详细说明。所述实施例仅用于解释本发明，而非对本发明保护范围的限制。
(一)同时靶向GPC3和CD276的嵌合抗原受体T细胞的制备
(1)制备同时靶向GPC3和CD276的嵌合抗原受体GPC3-CD276 TanCAR的基因序列
参见图1，同时靶向GPC3和CD276的嵌合抗原受体GPC3-CD276 TanCAR的基因序列中，从5’端到3’端依次包括以下元件的编码基因：CD8α信号肽(CD8αSP)的编码基因、靶向GPC3的单链抗体(GPC3-scFv)的编码基因、连接子“(G4S)3”的编码基因、靶向CD276的单链抗体(CD276-scFv)的编码基因、CD8α铰链区(CD8αhinge)的编码基因、CD8α跨膜区(CD8αTM)的编码基因、4-1BB胞内区(4-1BB)的编码基因以及CD3ζ胞内区(CD3-zeta)的编码基因。
所述CD8α信号肽的编码基因的核苷酸序列如SEQ.ID.NO.19所示、靶向GPC3的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.4所示、(G4S)3的编码基因的核苷酸序列如SEQ.ID.NO.6所示、靶向CD276的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.8所示、CD8α铰链区的编码基因的核苷酸序列如SEQ.ID.NO.10所示、CD8α跨膜区的编码基因的核苷酸序列如SEQ.ID.NO.12所示、4-1BB胞内区的编码基因的核苷酸序列如SEQ.ID.NO.14所示、CD3ζ胞内区的编码基因的核苷酸序列如SEQ.ID.NO.16所示。通过在CD3ζ胞内区的编码基因的3’端连接终止密码子，即得到所述嵌合抗原受体GPC3-CD276 TanCAR的基因序列，具体如SEQ.ID.NO.17所示。
所述嵌合抗原受体GPC3-CD276 TanCAR的基因序列由江苏金唯智生物技术有限公司进行基因合成。
(2)构建重组质粒pWPXLd-GPC3-CD276 TanCAR
将合成的GPC3-CD276 TanCAR的基因序列***到pWPXLd载体的BamH1和EcoR1酶切位点之间，然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经验证正确的质粒标记为重组质粒pWPXLd-GPC3-CD276 TanCAR用于后续实验。
(3)重组慢病毒构建
将重组质粒pWPXLd-GPC3-CD276 TanCAR、包装质粒psPAX2及包膜质粒pMD2G使用lipofectamine3000转染试剂共转染入培养好的HEK293T细胞。第48h收集含病毒的上清：首先2000rpm室温离心培养体系5分钟，取上层清液，然后经0.45μm滤膜过滤，得到的重组慢病毒上清用于T细胞感染。
(4)嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC的来源：健康志愿者的自体静脉血。
分离PBMC的操作流程：抽取所述健康志愿者血液，使用Ficoll收集外周血单个核细胞，离心分离后取中间层细胞；经PBS洗涤、计数后得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入含10％血清和适量IL-2的KBM581培养基，配成细胞悬液；按磁珠与细胞的数目比例为1:1，加入CD3/CD28免疫磁珠，室温下于摇床以20rpm的转速孵育45分钟；采用磁铁对孵育磁珠的细胞进行筛选，去除未吸附的细胞悬液后，加入上述KBM581培养基重悬磁珠-细胞混合物，得到CD3阳性T淋巴细胞，继续培养24小时后用于慢病毒感染。
c)病毒转染法制备抗原特异性T淋巴细胞
取上述经过免疫磁珠法分离得到的CD3阳性T淋巴细胞，加入与CD3阳性T淋巴细胞数相应的所述重组慢病毒进行培养。
培养的第3天，收集一定数量的感染重组慢病毒的CD3阳性T淋巴细胞，流式细胞术分析该细胞表面CAR的表达，结果如图2所示，与未转导病毒的CD3阳性T淋巴细胞相比，感染重组慢病毒的T细胞中GPC3-CD276 TanCAR有约14％的表面表达，表明同时靶向GPC3和CD276的嵌合抗原受体T细胞制备成功。继续培养48小时，收集同时靶向GPC3和CD276的嵌合抗原受体T细胞用于杀伤实验分析，或保存在细胞冻存液中，并放置于程序降温盒中-80℃保存24小时后转移至液氮罐长期保存。
(二)使用RTCA***分析同时靶向GPC3和CD276的嵌合抗原受体T细胞对肝癌细胞系的杀伤活性
2.1实验1对照及效应细胞分组
按照针对靶细胞Huh7(同时表达GPC3和CD276的肝癌细胞系)所加入的效应细胞不同分为：UTD组(加入未转导病毒的CD3阳性T淋巴细胞)和GPC3-CD276 TanCAR-T组(同时靶向GPC3和CD276的嵌合抗原受体T细胞)，同时设置对照组(control，培养基组)。
2.2实验2对照及效应细胞分组
按照针对靶细胞SK-HEP-1-CD276(过表达CD276而GPC3不表达的肝癌细胞系)所加入的效应细胞不同分为：UTD组(加入未转导病毒的CD3阳性T淋巴细胞)、GPC3 CAR-T组(对应嵌合抗原受体GPC3 CAR的基因序列与GPC3-CD276Tan CAR相比缺少linker-CD276-scFv；CAR-T细胞生产方式一致，GPC3 CAR表达阳性率约为30％)、GPC3-CD276 TanCAR-T组(同时靶向GPC3和CD276的嵌合抗原受体T细胞)，同时设置对照组(control，培养基组)。
2.3实验操作
首先用50μL DMEM或RPMI1640培养基进行RTCA单板的平衡，然后收集培养好的靶细胞Huh7或SK-HEP-1-CD276，细胞计数后在每个孔中加入含有5000个靶细胞的50μL细胞悬液，37℃培养箱中放置15分钟，然后放置在RTCA电阻***中，24小时后按不同的效应细胞(例如收集的GPC3-CD276 TanCAR-T细胞)与靶细胞的比例(E:T)20:1、10:1、5:1，把相应细胞数目的效应细胞悬液100μL加入到靶细胞中，每组至少设置2个复孔，然后放入RTCA电阻***中，在一定时间后进行杀伤活性分析。
2.4结果分析
使用指定时间点的细胞指数(cell index)进行效应细胞杀伤率分析。
所述UTD组杀伤率计算公式：
UTD杀伤率＝(controlcell index-UTDcell index)/controlcell index×100
所述GPC3-CD276 TanCAR-T组、GPC3 CAR-T组杀伤率计算公式：
CAR-T杀伤率＝(controlcell index-CAR-Tcell index)/controlcell index×100
实验结果表明(图3)，GPC3-CD276 TanCAR-T与UTD相比(杀伤24小时)，同时靶向GPC3和CD276的嵌合抗原受体T细胞对GPC3阳性CD276阳性肝癌细胞系Huh7有更强的杀伤活性，杀伤效应在效靶比(E:T)为10:1、20:1时较优。
另外实验结果还表明(图4)，在效靶比20:1时，GPC3-CD276 TanCAR-T与GPC3 CAR-T相比(杀伤20小时)，同时靶向GPC3和CD276的嵌合抗原受体T细胞对GPC3阴性CD276阳性肝癌细胞系SK-HEP-1-CD276有更强的杀伤效果。
总之，本发明构建的同时靶向GPC3和CD276的嵌合抗原受体T细胞对GPC3和CD276阳性肿瘤细胞系有较强的杀伤活性，并且与GPC3 CAR-T相比，对GPC3阴性CD276阳性肿瘤细胞系有更强的杀伤效果，克服了GPC3异质性表达导致的肿瘤抗原逃逸的问题。因此所述同时靶向GPC3和CD276的嵌合抗原受体T细胞在肝细胞癌等实体瘤的治疗中有较大的应用前景。
<110> 深圳先进技术研究院；中国科学院深圳理工大学（筹）
<120> 同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用
<160> 20
<210> 1
<211> 725
<212> PRT
<213> 嵌合抗原受体（不含CD8α SP）
<400> 1
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
260 265 270
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
275 280 285
Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
290 295 300
Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp
305 310 315 320
Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
325 330 335
Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr
340 345 350
Tyr Cys Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp
355 360 365
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
370 375 380
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr
385 390 395 400
Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
405 410 415
Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln
420 425 430
Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr
435 440 445
Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
450 455 460
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
465 470 475 480
Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly
485 490 495
Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
500 505 510
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
515 520 525
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
530 535 540
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
545 550 555 560
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
565 570 575
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
580 585 590
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
595 600 605
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
610 615 620
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
625 630 635 640
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
645 650 655
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
660 665 670
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
675 680 685
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
690 695 700
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
705 710 715 720
Ala Leu Pro Pro Arg
725
<210> 2
<211> 2178
<212> DNA
<213> 嵌合抗原受体（不含CD8α SP）
<400> 2
gatgtggtta tgacccaaag ccccctgagc ctgcctgtga ctcctgggga gcctgctagc 60
atcagctgca gaagctctca gagcctggtg cacagcaatg ccaacaccta cctgcactgg 120
tacctgcaga agcctgggca gagccctcag ctgctgatct acaaggtgag caatagattt 180
tctggggtgc ctgacagatt ttctggctct ggctctggca cagacttcac cctgaagatc 240
agcagagtgg aggctgagga tgtgggggtg tactactgct ctcagaacac ccatgtgccc 300
cccacctttg gccaaggcac aaagctggag atcaagagag gtggcggtgg ctcgggcggt 360
ggtgggtcgg gtggcggcgg atctcaagtg cagctggtgc agtctggggc tgaggtgaag 420
aagcctgggg cctctgtgaa ggtgagctgc aaggcctctg gctacacctt cacagactat 480
gagatgcact gggtcagaca agcccctggc caaggcctag aatggatggg ggccctggac 540
cccaagactg gggacacagc ctactctcag aagttcaagg gcagagtgac cctgacagct 600
gatgagagca caagcacagc ctacatggag ctgagcagcc tgagatctga ggacacagct 660
gtgtactact gcactagatt ctacagctac acctactggg gccaaggcac cctggtgaca 720
gtgagcagcg gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaggta 780
caactggtgg agtctggggg gggcctggtt cagcctgggg gctctctgag actgagctgt 840
gctgcctctg gcttcacctt cagcagcttt ggcatgcact gggtgagaca agcccctggc 900
aagggcctgg agtgggtggc ctacatcagc tctgacagct ctgccatcta ctatgctgac 960
acagtgaagg gcagattcac catcagcaga gacaatgcca agaacagcct gtacctgcag 1020
atgaacagcc tgagagatga ggacacagct gtgtactact gtggcagagg cagagagaac 1080
atctactatg gcagcagact ggactattgg ggccaaggca caacagtgac agtcagctct 1140
gggggtggag gatctggagg tgggggctct gggggtgggg gatctgacat tcagctgaca 1200
cagagcccta gcttcctgtc tgcctctgtg ggggacagag tgaccatcac ctgcaaggct 1260
tctcagaatg tggacaccaa tgtggcctgg tatcagcaga agcctggcaa ggcccccaag 1320
gccctgatct actctgctag ctacagatac tctggggtgc ctagcagatt ctctggctct 1380
ggctctggca cagacttcac cctgaccatc agcagcctgc agcctgagga ctttgccacc 1440
tactactgtc agcagtacaa caactacccc ttcacctttg gccaaggcac taagctggaa 1500
atcaagacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 1560
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 1620
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 1680
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1740
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1800
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1860
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1920
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1980
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2040
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2100
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 2160
gccctgcccc ctcgctga 2178
<210> 3
<211> 243
<212> PRT
<213> GPC3-scFv
<400> 3
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
243
<210> 4
<211> 729
<212> DNA
<213> GPC3-scFv
<400> 4
gatgtggtta tgacccaaag ccccctgagc ctgcctgtga ctcctgggga gcctgctagc 60
atcagctgca gaagctctca gagcctggtg cacagcaatg ccaacaccta cctgcactgg 120
tacctgcaga agcctgggca gagccctcag ctgctgatct acaaggtgag caatagattt 180
tctggggtgc ctgacagatt ttctggctct ggctctggca cagacttcac cctgaagatc 240
agcagagtgg aggctgagga tgtgggggtg tactactgct ctcagaacac ccatgtgccc 300
cccacctttg gccaaggcac aaagctggag atcaagagag gtggcggtgg ctcgggcggt 360
ggtgggtcgg gtggcggcgg atctcaagtg cagctggtgc agtctggggc tgaggtgaag 420
aagcctgggg cctctgtgaa ggtgagctgc aaggcctctg gctacacctt cacagactat 480
gagatgcact gggtcagaca agcccctggc caaggcctag aatggatggg ggccctggac 540
cccaagactg gggacacagc ctactctcag aagttcaagg gcagagtgac cctgacagct 600
gatgagagca caagcacagc ctacatggag ctgagcagcc tgagatctga ggacacagct 660
gtgtactact gcactagatt ctacagctac acctactggg gccaaggcac cctggtgaca 720
gtgagcagc 729
<210> 5
<211> 15
<212> PRT
<213> （G4S）3
<400> 5
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 6
<211> 45
<212> DNA
<213> （G4S）3
<400> 6
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45
<210> 7
<211> 244
<212> PRT
<213> CD276-scFv
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser
130 135 140
Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
145 150 155 160
Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys
165 170 175
Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr
180 185 190
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
210 215 220
Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys
<210> 8
<211> 732
<212> DNA
<213> CD276-scFv
<400> 8
gaggtacaac tggtggagtc tggggggggc ctggttcagc ctgggggctc tctgagactg 60
agctgtgctg cctctggctt caccttcagc agctttggca tgcactgggt gagacaagcc 120
cctggcaagg gcctggagtg ggtggcctac atcagctctg acagctctgc catctactat 180
gctgacacag tgaagggcag attcaccatc agcagagaca atgccaagaa cagcctgtac 240
ctgcagatga acagcctgag agatgaggac acagctgtgt actactgtgg cagaggcaga 300
gagaacatct actatggcag cagactggac tattggggcc aaggcacaac agtgacagtc 360
agctctgggg gtggaggatc tggaggtggg ggctctgggg gtgggggatc tgacattcag 420
ctgacacaga gccctagctt cctgtctgcc tctgtggggg acagagtgac catcacctgc 480
aaggcttctc agaatgtgga caccaatgtg gcctggtatc agcagaagcc tggcaaggcc 540
cccaaggccc tgatctactc tgctagctac agatactctg gggtgcctag cagattctct 600
ggctctggct ctggcacaga cttcaccctg accatcagca gcctgcagcc tgaggacttt 660
gccacctact actgtcagca gtacaacaac taccccttca cctttggcca aggcactaag 720
ctggaaatca ag 732
<210> 9
<211> 45
<212> PRT
<213> CD8α hinge
<400> 9
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 10
<211> 135
<212> DNA
<213> CD8α hinge
<400> 10
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 11
<211> 24
<212> PRT
<213> CD8α TM
<400> 11
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 12
<211> 72
<212> DNA
<213> CD8α TM
<400> 12
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 13
<211> 42
<212> PRT
<213> 4-1BB
<400> 13
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 14
<211> 126
<212> DNA
<213> 4-1BB
<400> 14
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 15
<211> 112
<212> PRT
<213> CD3-zeta
<400> 15
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 16
<211> 336
<212> DNA
<213> CD3-zeta
<400> 16
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 17
<211> 2241
<212> DNA
<213> 嵌合抗原受体（含CD8α SP）
<400> 17
atggccctgc ctgtgacagc cctgttactt cccctggccc tgttactgca tgctgctaga 60
cctgatgtgg ttatgaccca aagccccctg agcctgcctg tgactcctgg ggagcctgct 120
agcatcagct gcagaagctc tcagagcctg gtgcacagca atgccaacac ctacctgcac 180
tggtacctgc agaagcctgg gcagagccct cagctgctga tctacaaggt gagcaataga 240
ttttctgggg tgcctgacag attttctggc tctggctctg gcacagactt caccctgaag 300
atcagcagag tggaggctga ggatgtgggg gtgtactact gctctcagaa cacccatgtg 360
ccccccacct ttggccaagg cacaaagctg gagatcaaga gaggtggcgg tggctcgggc 420
ggtggtgggt cgggtggcgg cggatctcaa gtgcagctgg tgcagtctgg ggctgaggtg 480
aagaagcctg gggcctctgt gaaggtgagc tgcaaggcct ctggctacac cttcacagac 540
tatgagatgc actgggtcag acaagcccct ggccaaggcc tagaatggat gggggccctg 600
gaccccaaga ctggggacac agcctactct cagaagttca agggcagagt gaccctgaca 660
gctgatgaga gcacaagcac agcctacatg gagctgagca gcctgagatc tgaggacaca 720
gctgtgtact actgcactag attctacagc tacacctact ggggccaagg caccctggtg 780
acagtgagca gcggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctgag 840
gtacaactgg tggagtctgg ggggggcctg gttcagcctg ggggctctct gagactgagc 900
tgtgctgcct ctggcttcac cttcagcagc tttggcatgc actgggtgag acaagcccct 960
ggcaagggcc tggagtgggt ggcctacatc agctctgaca gctctgccat ctactatgct 1020
gacacagtga agggcagatt caccatcagc agagacaatg ccaagaacag cctgtacctg 1080
cagatgaaca gcctgagaga tgaggacaca gctgtgtact actgtggcag aggcagagag 1140
aacatctact atggcagcag actggactat tggggccaag gcacaacagt gacagtcagc 1200
tctgggggtg gaggatctgg aggtgggggc tctgggggtg ggggatctga cattcagctg 1260
acacagagcc ctagcttcct gtctgcctct gtgggggaca gagtgaccat cacctgcaag 1320
gcttctcaga atgtggacac caatgtggcc tggtatcagc agaagcctgg caaggccccc 1380
aaggccctga tctactctgc tagctacaga tactctgggg tgcctagcag attctctggc 1440
tctggctctg gcacagactt caccctgacc atcagcagcc tgcagcctga ggactttgcc 1500
acctactact gtcagcagta caacaactac cccttcacct ttggccaagg cactaagctg 1560
gaaatcaaga ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1620
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 1680
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 1740
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1800
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1860
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1920
agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1980
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 2040
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 2100
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 2160
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 2220
caggccctgc cccctcgctg a 2241
<210> 18
<211> 746
<212> PRT
<213> 嵌合抗原受体（含CD8α SP）
<400> 18
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 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
20 25 30
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
100 105 110
Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
145 150 155 160
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
180 185 190
Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser
210 215 220
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
275 280 285
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
290 295 300
Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro
305 310 315 320
Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala
325 330 335
Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
340 345 350
Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu
355 360 365
Asp Thr Ala Val Tyr Tyr Cys Gly Arg Gly Arg Glu Asn Ile Tyr Tyr
370 375 380
Gly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
385 390 395 400
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
405 410 415
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
420 425 430
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn
435 440 445
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile
450 455 460
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
465 470 475 480
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
485 490 495
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe
500 505 510
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala
515 520 525
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
530 535 540
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
545 550 555 560
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
565 570 575
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
580 585 590
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
595 600 605
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
610 615 620
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
625 630 635 640
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
645 650 655
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
660 665 670
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
675 680 685
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
690 695 700
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
705 710 715 720
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
725 730 735
Ala Leu His Met Gln Ala Leu Pro Pro Arg
740 745
<210> 19
<211> 63
<212> DNA
<213> CD8α SP
<400> 19
atggccctgc ctgtgacagc cctgttactt cccctggccc tgttactgca tgctgctaga 60
cct 63
<210> 20
<211> 21
<212> PRT
<213> CD8α SP
<400> 20
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

Claims (6)

1.一种同时靶向GPC3和CD276的嵌合抗原受体，其特征在于：该嵌合抗原受体包括靶向GPC3的单链抗体、连接子、靶向CD276的单链抗体、胞外铰链区、跨膜区、共刺激信号区以及CD3ζ胞内区；
所述靶向GPC3的单链抗体的氨基酸序列如SEQ.ID.NO.3所示，所述靶向CD276的单链抗体的氨基酸序列如SEQ.ID.NO.7所示；
所述胞外铰链区选自CD8α铰链区；
所述跨膜区选自CD8α跨膜区；
所述共刺激信号区选自4-1BB胞内区；
所述嵌合抗原受体的氨基酸序列如SEQ.ID.NO.1所示。
2.一种同时靶向GPC3和CD276的嵌合抗原受体T细胞，其特征在于：该T细胞包括如权利要求1所述的同时靶向GPC3和CD276的嵌合抗原受体。
3.一种重组病毒载体，其特征在于：该重组病毒载体包括如权利要求1所述的同时靶向GPC3和CD276的嵌合抗原受体的对应编码基因；所述重组病毒载体中，同时靶向GPC3和CD276的嵌合抗原受体的基因序列如SEQ.ID.NO.17所示。
4.一种同时靶向GPC3和CD276的嵌合抗原受体T细胞的制备方法，其特征在于：包括以下步骤：
1）将如权利要求1所述的同时靶向GPC3和CD276的嵌合抗原受体的对应编码基因***到pWPXLd载体中，得到重组质粒；
2）将所述重组质粒与包膜质粒、包装质粒共转染，得到重组慢病毒；
3）将所述重组慢病毒转染CD3阳性T淋巴细胞，经分离获得同时靶向GPC3和CD276的嵌合抗原受体T细胞。
5.一种治疗肝细胞癌的药物，其特征在于：该药物包括如权利要求2所述的同时靶向GPC3和CD276的嵌合抗原受体T细胞。
6.一种如权利要求2所述的同时靶向GPC3和CD276的嵌合抗原受体T细胞在制备抗肝癌药物中的应用。
CN202111501965.5A 2021-12-09 2021-12-09 同时靶向gpc3和cd276的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用 Active CN114163538B (zh)

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