CN114163537B - 分泌双特异性抗体的嵌合抗原受体t细胞及其制备方法和应用 - Google Patents

分泌双特异性抗体的嵌合抗原受体t细胞及其制备方法和应用 Download PDF

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CN114163537B
CN114163537B CN202111501944.3A CN202111501944A CN114163537B CN 114163537 B CN114163537 B CN 114163537B CN 202111501944 A CN202111501944 A CN 202111501944A CN 114163537 B CN114163537 B CN 114163537B
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万晓春
曹国政
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Shenzhen University Of Technology Preparatory
Shenzhen Institute of Advanced Technology of CAS
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Abstract

本发明公开了一种分泌双特异性抗体的嵌合抗原受体T细胞及其制备方法和应用。实验发现靶向GPC3同时分泌双特异性抗体的嵌合抗原受体T细胞，对GPC3和CD276阳性肿瘤细胞系有较强的杀伤活性，并且对GPC3阴性CD276阳性肿瘤细胞系有较强的杀伤活性，因此所述分泌双特异性抗体的嵌合抗原受体T细胞在肝细胞癌等的治疗中有较大的应用前景。

Description

分泌双特异性抗体的嵌合抗原受体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，具体涉及靶向GPC3同时分泌双特异性抗体的嵌合抗原受体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系统性疗法，比如化疗、放疗、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.,CombinationalImmunotherapy for Hepatocellular Carcinoma:Radiotherapy,Immune CheckpointBlockade 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。因此迫切需要的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，激活自身免疫系统，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的嵌合抗原受体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、前列腺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.Asingle 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。
欧洲专利EP3812398A2公开了“ANTI-CD3 ANTIBODIES,ANTI-CD123 ANTIBODIESAND BISPECIFIC ANTIBODIES SPECIFICALLY BINDING TO CD3 AND/OR CD123”，其指出CAR-T细胞与双特异性抗体联用可以解决肿瘤抗原异质性表达的难题，但双特异性抗体需要多次输注且会造成全身的毒性问题。
发明内容
为了解决以上的问题，本发明提供一种分泌双特异性抗体的嵌合抗原受体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，本发明提供了一种靶向GPC3同时包含双特异性抗体的嵌合抗原受体，该嵌合抗原受体命名为GPC3-BiTE CAR。
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、胞外铰链区、跨膜区、共刺激信号区、CD3ζ胞内区、剪切肽、IGHV3-2信号肽、靶向CD276的单链抗体、连接子(linker)I以及靶向CD3的单链抗体。
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。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。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。所述CD8α铰链区的氨基酸序列如SEQ.ID.NO.5所示。所述CD8α铰链区的编码基因的核苷酸序列如SEQ.ID.NO.6所示。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。所述CD8α跨膜区的氨基酸序列如SEQ.ID.NO.7所示。所述CD8α跨膜区的编码基因的核苷酸序列如SEQ.ID.NO.8所示。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。所述4-1BB胞内区的氨基酸序列如SEQ.ID.NO.9所示。所述4-1BB胞内区的编码基因的核苷酸序列如SEQ.ID.NO.10所示。所述CD3ζ胞内区的氨基酸序列如SEQ.ID.NO.11所示。所述CD3ζ胞内区的编码基因的核苷酸序列如SEQ.ID.NO.12所示。所述剪切肽为P2A；P2A的氨基酸序列如SEQ.ID.NO.13所示。所述P2A的编码基因的核苷酸序列如SEQ.ID.NO.14所示。所述IGHV3-2信号肽的氨基酸序列如SEQ.ID.NO.15所示。所述IGHV3-2信号肽的编码基因的核苷酸序列如SEQ.ID.NO.16所示。所述靶向CD276的单链抗体的氨基酸序列如SEQ.ID.NO.17所示。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.18所示。所述靶向CD3的单链抗体的氨基酸序列如SEQ.ID.NO.21所示。所述靶向CD3的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.22所示。所述嵌合抗原受体还包括标签蛋白的氨基酸序列，标签蛋白的氨基端通过连接子II与靶向CD3的单链抗体的羧基端相连。所述连接子I、连接子II均为G4S，G4S的氨基酸序列如SEQ.ID.NO.19所示。所述G4S的编码基因的核苷酸序列如SEQ.ID.NO.20所示。所述标签蛋白选自6×His、Myc、Flag、HA、Fc或GST蛋白。所述6×His蛋白的氨基酸序列如SEQ.ID.NO.23所示。所述6×His蛋白的编码基因的核苷酸序列如SEQ.ID.NO.24所示。所述嵌合抗原受体包括如SEQ.ID.NO.1所示的氨基酸序列。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，本发明提供了一种靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞，该T细胞包括上述嵌合抗原受体(即T细胞表面具有靶向GPC3同时包含双特异性抗体的嵌合抗原受体)。
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CD8α信号肽的编码基因、靶向GPC3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、CD3ζ胞内区的编码基因、剪切肽(例如P2A)的编码基因、IGHV3-2信号肽的编码基因、靶向CD276的单链抗体的编码基因、连接子I(例如G4S)的编码基因以及靶向CD3的单链抗体的编码基因。
优选的，所述嵌合抗原受体的编码基因还包括通过连接子II(例如G4S)的编码基因连接在靶向CD3的单链抗体的编码基因的3’端的标签蛋白(例如6×His蛋白)的编码基因。
优选的，所述CD8α信号肽的编码基因的核苷酸序列如SEQ.ID.NO.27所示。
优选的，所述CD8α信号肽的氨基酸序列如SEQ.ID.NO.28所示。
优选的，所述重组病毒载体及重组病毒中，靶向GPC3同时包含双特异性抗体的嵌合抗原受体的基因序列如SEQ.ID.NO.25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.25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CD8α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.26所示，具体如下：MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSHHHHHH
优选的，所述重组病毒载体为pWPXLd慢病毒载体。
第四方面，本发明提供了一种靶向GPC3分泌双特异性抗体的嵌合抗原受体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靶向GPC3同时包含双特异性抗体的嵌合抗原受体GPC3-BiTE CAR的基因序列，所述GPC3-BiTE CAR的基因序列包括如SEQ.ID.NO.2所示的核苷酸序列(例如GPC3-BiTE CAR的基因序列如SEQ.ID.NO.25所示)；2)将所述GPC3-BiTECAR的基因序列插入到pWPXLd载体中，得到重组质粒pWPXLd-GPC3-BiTE CAR；3)将所述重组质粒pWPXLd-GPC3-BiTE CAR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)，经分离获得靶向GPC3分泌双特异性抗体的嵌合抗原受体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。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。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，该药物包括上述靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞。
本发明的有益效果体现在：
本发明中所提出的嵌合抗原受体T细胞在靶向GPC3的同时分泌双特异性抗体(CD276的单链抗体与CD3的单链抗体的串联蛋白复合物)，不仅对同时表达GPC3和CD276的肿瘤细胞有杀伤作用，同时招募非转导T细胞对GPC3阴性CD276阳性的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-BiTE CAR的编码基因的结构示意图。
图2为GPC3-BiTE CAR在T细胞上的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-BiTE CAR-T组为靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞。
图3为HEK293T上清中BiTE表达的Western blot检测结果；其中：Control组为单靶点靶向GPC3的嵌合抗原受体的编码基因转染HEK293T细胞后收集的上清；BiTE组为靶向GPC3同时包含双特异性抗体的嵌合抗原受体的编码基因转染HEK293T细胞后收集的上清。
图4为靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞对Huh7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-BiTE CAR-T组为靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞。
图5为靶向GPC3分泌双特异性抗体的嵌合抗原受体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-BiTE CAR-T组为靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞；**表示差异显著，ns表示无显著差异。
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)靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞的制备
(1)制备靶向GPC3同时包含双特异性抗体的嵌合抗原受体的基因序列
参见图1，靶向GPC3同时包含双特异性抗体的嵌合抗原受体GPC3-BiTE CAR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)、靶向GPC3单链抗体(GPC3-scFv)、CD8α铰链区(CD8αhinge)、CD8α跨膜区(CD8αTM)、4-1BB胞内区(4-1BB)、CD3ζ胞内区(CD3-zeta)、P2A、IGHV3-2信号肽(IGHV3-2 SP)、靶向CD276的单链抗体(CD276-scFv)、连接子(G4S)以及靶向CD3的单链抗体(CD3-scFv)。所述基因序列中还包括通过另一连接子(G4S)连接在靶向CD3的单链抗体的编码基因3’端的标签蛋白6×His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.27所示、靶向GPC3单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.4所示、CD8α铰链区的编码基因的核苷酸序列如SEQ.ID.NO.6所示、CD8α跨膜区的编码基因的核苷酸序列如SEQ.ID.NO.8所示、4-1BB胞内区的编码基因的核苷酸序列如SEQ.ID.NO.10所示、CD3ζ胞内区的编码基因的核苷酸序列如SEQ.ID.NO.12所示、P2A的编码基因的核苷酸序列如SEQ.ID.NO.14所示、IGHV3-2信号肽的编码基因的核苷酸序列如SEQ.ID.NO.16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.18所示、位于靶向CD3的单链抗体的编码基因5’端及3’端的两个连接子(G4S)的编码基因的核苷酸序列如SEQ.ID.NO.20所示、靶向CD3的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.22所示、6×His蛋白的编码基因的核苷酸序列如SEQ.ID.NO.24所示。通过在6×His蛋白的编码基因的3’端连接终止密码子，即得到所述嵌合抗原受体GPC3-BiTE CAR的基因序列，具体如SEQ.ID.NO.25所示。
所述嵌合抗原受体GPC3-BiTE CAR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-BiTE CAR
将合成的嵌合抗原受体GPC3-BiTE CAR的基因序列插入到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-BiTE CAR用于后续实验。
(3)重组慢病毒构建
将重组质粒pWPXLd-GPC3-BiTE CAR、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。
PBMC的分离：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分离法得到的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CD3阳性T淋巴细胞中嵌合抗原受体GPC3-BiTE CAR有15％的表面表达，表明嵌合抗原受体T细胞制备成功。继续培养48小时，收集嵌合抗原受体T细胞用于杀伤实验分析或保存在细胞冻存液中，放置于程序降温盒中-80℃保存24小时，然后转移至液氮灌长期保存。
(二)Western blot检测BiTE表达
将靶向GPC3的嵌合抗原受体GPC3 CAR的编码基因和靶向GPC3同时包含双特异性抗体的嵌合抗原受体GPC3-BiTE CAR的编码基因通过质粒在6孔细胞培养板中瞬时转染HEK293T细胞，48小时后收集上清进行Western blot检测。Western blot实验操作按标准流程进行，使用anti-his-tag-HRP抗体进行免疫显色。结果如图3所示，BiTE组检测到约55kDa的蛋白条带(对应双特异性抗体)，Control组在相应位置没有条带，结果说明BiTE(具体指双特异性抗体)可以成功分泌，这表明以上收集的BiTE组的嵌合抗原受体T细胞为靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞。
(三)使用RTCA系统分析靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞对肝癌细胞系的杀伤活性
3.1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细胞)、GPC3-BiTE CAR-T组(加入靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞)，同时设置对照组(control，培养基组)
3.2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细胞)、GPC3 CAR-T组(对应嵌合抗原受体GPC3 CAR的基因序列与GPC3-BiTE CAR相比，区别在于缺少图1中P2A以后的基因结构；CAR-T细胞生产方式一致，GPC3 CAR表达阳性率约为30％)、GPC3-BiTE CAR-T组(加入靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞)，同时设置对照组(control，培养基组)。
3.3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电阻系统中，24小时后收集按不同的效应细胞(例如靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞)与靶细胞的比例(E:T)20:1、10:1，把相应细胞数目的效应细胞悬液100μL加入到靶细胞中，每组设置2个复孔，然后放入RTCA电阻系统中，在一定时间后进行杀伤活性分析。
3.4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-BiTE CAR-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4)，靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞与未转导病毒的CD3阳性T细胞相比(杀伤30小时)，对Huh7细胞有更强的杀伤活性，杀伤效应在效靶比(即E:T)为20:1时达到最优。
另外实验结果还表明(图5)，靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞比单靶点靶向GPC3的嵌合抗原受体T细胞(杀伤20小时)，对SK-HEP-1-CD276细胞系有更强的杀伤活性。
总之，本发明构建的靶向GPC3同时分泌双特异性抗体的嵌合抗原受体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活性，克服了GPC3异质性表达导致的肿瘤抗原逃逸的问题。因此所述靶向GPC3同时分泌双特异性抗体的嵌合抗原受体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> 分泌双特异性抗体的嵌合抗原受体T细胞及其制备方法和应用
<160> 28
<210> 1
<211> 1005
<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 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
290 295 300
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
405 410 415
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
420 425 430
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
435 440 445
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
450 455 460
Pro Arg Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
465 470 475 480
Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val
485 490 495
Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly
500 505 510
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
515 520 525
Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala
530 535 540
Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser
545 550 555 560
Ala Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg
565 570 575
Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp
580 585 590
Glu Asp Thr Ala Val Tyr Tyr Cys Gly Arg Gly Arg Glu Asn Ile Tyr
595 600 605
Tyr Gly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
610 615 620
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
625 630 635 640
Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val
645 650 655
Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr
660 665 670
Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu
675 680 685
Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser
690 695 700
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
705 710 715 720
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro
725 730 735
Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly
740 745 750
Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly
755 760 765
Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg
770 775 780
Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
785 790 795 800
Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys
805 810 815
Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala
820 825 830
Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
835 840 845
Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln
850 855 860
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
865 870 875 880
Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala
885 890 895
Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala
900 905 910
Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr
915 920 925
Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val
930 935 940
Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
945 950 955 960
Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
965 970 975
Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
980 985 990
Asn Arg Gly Gly Gly Gly Ser His His His His His His
995 1000 1005
<210> 2
<211> 3018
<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
gtgagcagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 780
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 960
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1020
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1080
agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1200
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380
caggccctgc cccctcgcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa 1440
gagaatcctg gaccgatgga gtttgggctg agctggcttt ttcttgtggc tattttaaaa 1500
ggtgtccagt gtgaggtaca actggtggag tctggggggg gcctggttca gcctgggggc 1560
tctctgagac tgagctgtgc tgcctctggc ttcaccttca gcagctttgg catgcactgg 1620
gtgagacaag cccctggcaa gggcctggag tgggtggcct acatcagctc tgacagctct 1680
gccatctact atgctgacac agtgaagggc agattcacca tcagcagaga caatgccaag 1740
aacagcctgt acctgcagat gaacagcctg agagatgagg acacagctgt gtactactgt 1800
ggcagaggca gagagaacat ctactatggc agcagactgg actattgggg ccaaggcaca 1860
acagtgacag tcagctctgg gggtggagga tctggaggtg ggggctctgg gggtggggga 1920
tctgacattc agctgacaca gagccctagc ttcctgtctg cctctgtggg ggacagagtg 1980
accatcacct gcaaggcttc tcagaatgtg gacaccaatg tggcctggta tcagcagaag 2040
cctggcaagg cccccaaggc cctgatctac tctgctagct acagatactc tggggtgcct 2100
agcagattct ctggctctgg ctctggcaca gacttcaccc tgaccatcag cagcctgcag 2160
cctgaggact ttgccaccta ctactgtcag cagtacaaca actacccctt cacctttggc 2220
caaggcacta agctggaaat caagggcggg ggcggcagcc aagtgcagct gcagcaaagc 2280
ggcgctgagc tggctagacc cggcgctagc gtgaagatga gctgcaaggc tagcggctac 2340
accttcacaa gatacaccat gcactgggtg aagcagagac ccggccaagg cctggagtgg 2400
atcggctaca tcaaccctag cagaggctac accaactaca atcagaagtt caaggacaag 2460
gccaccctga ccaccgacaa gagcagcagc accgcctaca tgcagctgag cagcctgaca 2520
agcgaggaca gcgccgtgta ctactgcgct agatactacg acgaccacta cagcctggac 2580
tactggggcc aaggcaccac cctcaccgtg agctccggcg ggggcggcag cgggggcggg 2640
ggcagcggcg gcgggggcag ccaaatcgtg ctgacacaga gccccgccat catgagcgct 2700
agccccggcg agaaggtgac catgacctgc agcgctagca gcagcgtgag ctacatgaac 2760
tggtatcagc agaagagcgg cacaagcccc aagagatgga tctacgacac aagcaagctg 2820
gctagcggcg tgcccgccca cttcagaggc agcggcagcg gcacaagcta tagcctgacc 2880
atcagcggca tggaggccga ggacgccgcc acctactact gtcagcagtg gagcagcaac 2940
cccttcacct tcggctccgg cacaaagctc gagatcaaca gaggcggggg cggctcccac 3000
catcaccatc accactga 3018
<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
<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> 45
<212> PRT
<213> CD8α hinge
<400> 5
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> 6
<211> 135
<212> DNA
<213> CD8α hinge
<400> 6
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 7
<211> 24
<212> PRT
<213> CD8α TM
<400> 7
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> 8
<211> 72
<212> DNA
<213> CD8α TM
<400> 8
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 9
<211> 42
<212> PRT
<213> 4-1BB
<400> 9
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> 10
<211> 126
<212> DNA
<213> 4-1BB
<400> 10
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 11
<211> 112
<212> PRT
<213> CD3-zeta
<400> 11
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> 12
<211> 336
<212> DNA
<213> CD3-zeta
<400> 12
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> 13
<211> 19
<212> PRT
<213> P2A
<400> 13
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 14
<211> 57
<212> DNA
<213> P2A
<400> 14
gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccg 57
<210> 15
<211> 19
<212> PRT
<213> IGHV3-2 SP
<400> 15
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys
<210> 16
<211> 57
<212> DNA
<213> IGHV3-2 SP
<400> 16
atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgt 57
<210> 17
<211> 244
<212> PRT
<213> CD276-scFv
<400> 17
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> 18
<211> 732
<212> DNA
<213> CD276-scFv
<400> 18
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> 19
<211> 5
<212> PRT
<213> G4S
<400> 19
Gly Gly Gly Gly Ser
1 5
<210> 20
<211> 15
<212> DNA
<213> G4S
<400> 20
ggcgggggcg gcagc 15
<210> 21
<211> 241
<212> PRT
<213> CD3-scFv
<400> 21
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
130 135 140
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser
145 150 155 160
Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser
165 170 175
Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
180 185 190
Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
195 200 205
Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
210 215 220
Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn
225 230 235 240
Arg
<210> 22
<211> 723
<212> DNA
<213> CD3-scFv
<400> 22
caagtgcagc tgcagcaaag cggcgctgag ctggctagac ccggcgctag cgtgaagatg 60
agctgcaagg ctagcggcta caccttcaca agatacacca tgcactgggt gaagcagaga 120
cccggccaag gcctggagtg gatcggctac atcaacccta gcagaggcta caccaactac 180
aatcagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240
atgcagctga gcagcctgac aagcgaggac agcgccgtgt actactgcgc tagatactac 300
gacgaccact acagcctgga ctactggggc caaggcacca ccctcaccgt gagctccggc 360
gggggcggca gcgggggcgg gggcagcggc ggcgggggca gccaaatcgt gctgacacag 420
agccccgcca tcatgagcgc tagccccggc gagaaggtga ccatgacctg cagcgctagc 480
agcagcgtga gctacatgaa ctggtatcag cagaagagcg gcacaagccc caagagatgg 540
atctacgaca caagcaagct ggctagcggc gtgcccgccc acttcagagg cagcggcagc 600
ggcacaagct atagcctgac catcagcggc atggaggccg aggacgccgc cacctactac 660
tgtcagcagt ggagcagcaa ccccttcacc ttcggctccg gcacaaagct cgagatcaac 720
aga 723
<210> 23
<211> 6
<212> PRT
<213> 6×His
<400> 23
His His His His His His
1 5
<210> 24
<211> 18
<212> DNA
<213> 6×His
<400> 24
caccatcacc atcaccac 18
<210> 25
<211> 3081
<212> DNA
<213> 嵌合抗原受体（含CD8α SP）
<400> 25
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatgtgg 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 gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1080
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140
aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440
atgcaggccc tgccccctcg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc 1500
gaagagaatc ctggaccgat ggagtttggg ctgagctggc tttttcttgt ggctatttta 1560
aaaggtgtcc agtgtgaggt acaactggtg gagtctgggg ggggcctggt tcagcctggg 1620
ggctctctga gactgagctg tgctgcctct ggcttcacct tcagcagctt tggcatgcac 1680
tgggtgagac aagcccctgg caagggcctg gagtgggtgg cctacatcag ctctgacagc 1740
tctgccatct actatgctga cacagtgaag ggcagattca ccatcagcag agacaatgcc 1800
aagaacagcc tgtacctgca gatgaacagc ctgagagatg aggacacagc tgtgtactac 1860
tgtggcagag gcagagagaa catctactat ggcagcagac tggactattg gggccaaggc 1920
acaacagtga cagtcagctc tgggggtgga ggatctggag gtgggggctc tgggggtggg 1980
ggatctgaca ttcagctgac acagagccct agcttcctgt ctgcctctgt gggggacaga 2040
gtgaccatca cctgcaaggc ttctcagaat gtggacacca atgtggcctg gtatcagcag 2100
aagcctggca aggcccccaa ggccctgatc tactctgcta gctacagata ctctggggtg 2160
cctagcagat tctctggctc tggctctggc acagacttca ccctgaccat cagcagcctg 2220
cagcctgagg actttgccac ctactactgt cagcagtaca acaactaccc cttcaccttt 2280
ggccaaggca ctaagctgga aatcaagggc gggggcggca gccaagtgca gctgcagcaa 2340
agcggcgctg agctggctag acccggcgct agcgtgaaga tgagctgcaa ggctagcggc 2400
tacaccttca caagatacac catgcactgg gtgaagcaga gacccggcca aggcctggag 2460
tggatcggct acatcaaccc tagcagaggc tacaccaact acaatcagaa gttcaaggac 2520
aaggccaccc tgaccaccga caagagcagc agcaccgcct acatgcagct gagcagcctg 2580
acaagcgagg acagcgccgt gtactactgc gctagatact acgacgacca ctacagcctg 2640
gactactggg gccaaggcac caccctcacc gtgagctccg gcgggggcgg cagcgggggc 2700
gggggcagcg gcggcggggg cagccaaatc gtgctgacac agagccccgc catcatgagc 2760
gctagccccg gcgagaaggt gaccatgacc tgcagcgcta gcagcagcgt gagctacatg 2820
aactggtatc agcagaagag cggcacaagc cccaagagat ggatctacga cacaagcaag 2880
ctggctagcg gcgtgcccgc ccacttcaga ggcagcggca gcggcacaag ctatagcctg 2940
accatcagcg gcatggaggc cgaggacgcc gccacctact actgtcagca gtggagcagc 3000
aaccccttca ccttcggctc cggcacaaag ctcgagatca acagaggcgg gggcggctcc 3060
caccatcacc atcaccactg a 3081
<210> 26
<211> 1026
<212> PRT
<213> 嵌合抗原受体（含CD8α SP）
<400> 26
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 Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
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 Ala Thr Asn Phe Ser Leu Leu Lys Gln
485 490 495
Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser
500 505 510
Trp Leu Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln
515 520 525
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
530 535 540
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His
545 550 555 560
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile
565 570 575
Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg
580 585 590
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met
595 600 605
Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Gly Arg Gly
610 615 620
Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp Gly Gln Gly
625 630 635 640
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
645 650 655
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe
660 665 670
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
675 680 685
Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
690 695 700
Ala Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val
705 710 715 720
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
725 730 735
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
740 745 750
Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
755 760 765
Lys Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
770 775 780
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
785 790 795 800
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly
805 810 815
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
820 825 830
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
835 840 845
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
850 855 860
Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu
865 870 875 880
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly
885 890 895
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu
900 905 910
Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr
915 920 925
Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln
930 935 940
Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys
945 950 955 960
Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr
965 970 975
Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr
980 985 990
Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly
995 1000 1005
Thr Lys Leu Glu Ile Asn Arg Gly Gly Gly Gly Ser His His His His
1010 1015 1020
His His
1025
<210> 27
<211> 63
<212> DNA
<213> CD8α SP
<400> 27
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 28
<211> 21
<212> PRT
<213> CD8α SP
<400> 28
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)

1.一种靶向GPC3同时包含双特异性抗体的嵌合抗原受体，其特征在于：该嵌合抗原受体包括靶向GPC3的单链抗体、胞外铰链区、跨膜区、共刺激信号区、CD3ζ胞内区、剪切肽、IGHV3-2信号肽、靶向CD276的单链抗体、连接子以及靶向CD3的单链抗体；
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.17所示，所述靶向CD3的单链抗体的氨基酸序列如SEQ.ID.NO.21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；
所述剪切肽为P2A；
所述嵌合抗原受体的氨基酸序列如SEQ.ID.NO.1所示。
2.一种靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞，其特征在于：该T细胞包括如权利要求1所述的靶向GPC3同时包含双特异性抗体的嵌合抗原受体。
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所述的靶向GPC3同时包含双特异性抗体的嵌合抗原受体的对应编码基因；所述重组病毒载体中，靶向GPC3同时包含双特异性抗体的嵌合抗原受体的基因序列如SEQ.ID.NO.25所示。
4.一种靶向GPC3分泌双特异性抗体的嵌合抗原受体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所述的靶向GPC3同时包含双特异性抗体的嵌合抗原受体的对应编码基因插入到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，经分离获得靶向GPC3分泌双特异性抗体的嵌合抗原受体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所述的靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞。
6.一种如权利要求2所述的靶向GPC3分泌双特异性抗体的嵌合抗原受体T细胞在制备抗肝癌药物中的应用。
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