CN112125976B - 改造的铰链及其在构建car骨架中的应用 - Google Patents

改造的铰链及其在构建car骨架中的应用 Download PDF

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CN112125976B
CN112125976B CN202010932574.8A CN202010932574A CN112125976B CN 112125976 B CN112125976 B CN 112125976B CN 202010932574 A CN202010932574 A CN 202010932574A CN 112125976 B CN112125976 B CN 112125976B
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张巍
赵文旭
陈军
黄霞
赵永春
单娟娟
徐艳敏
张茜真
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Chongqing Precision Biotech Co ltd
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Abstract

本发明属于免疫治疗领域，具体涉及一种改造的铰链区及其在构建CAR骨架中的应用。本发明提供的铰链区能够延长CAR‑T细胞体内存续和/或提高CAR‑T细胞浸润肿瘤能力，所述铰链区氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示。本发明提供的铰链区结构能够延长CAR‑T细胞在体内存续时间，该铰链区结构组合的嵌合抗原受体能够更稳定的表达于T淋巴细胞，含有本发明铰链区的CAR‑T在体内存续时间更长、细胞浸润肿瘤能力显著增强，杀伤效果更好。

Description

改造的铰链及其在构建CAR骨架中的应用
技术领域
本发明属于免疫治疗领域，涉及一种改造的铰链区及其在构建CAR骨架中的应用，具体涉及一种能够延长CAR-T细胞体内存续和/或提高CAR-T细胞浸润肿瘤能力的铰链区及其在构建CAR骨架中的应用。
背景技术
嵌合抗原受体(chimeric antigen receptor，CAR)是模拟TCR功能的人工受体，包含抗原识别域、铰链区、跨膜区及胞内信号域。胞内信号域通常为CD3ζ链或FcRγ，或与一种或多种共刺激分子相连，如4-1BB(CD137)，CD28，ICOS(CD278)。肿瘤细胞表面的抗原(受体)与嵌合抗原受体的抗体(配体)结合时，通过铰链区和跨膜区将信号传递至胞内，胞内信号域将信号转化为活化信号，激活效应细胞，效应细胞增殖、产生细胞因子从而杀伤肿瘤细胞。
近年来，嵌合抗原受体T淋巴细胞(CAR-T)在肿瘤治疗中展现了显著的治疗效果尤其是治疗CD19阳性的恶性肿瘤方面。但是即使对于治疗效果显著地急性淋巴细胞白血病(ALL)，治疗后完全缓解中位时间一般均在8个月左右，仍有大量患者复发，这可能和CAR-T细胞在患者体内存续时间短相关，提高CAR-T体内存续时间对于CAR-T治疗疗效有重大意义。
虽然利用CAR-T进行实体瘤的研究也不断得到重视，但是大多数CAR-T治疗实体瘤的效果却并不令人满意，一方面也是因为CAR-T细胞在体内存续差，CAR-T细胞很快死亡影响杀伤有效性；另一方面大多数实体瘤会由于其在代谢、免疫逃逸、组织形成方面的多能性，在多方面限制免疫系统对其的控制和杀伤，导致CAR-T细胞难以浸润到实体瘤肿瘤组织内部，所以对于大的实体瘤CAR-T治疗收效甚微。延长CAR-T在体内存续性，促进CAR-T细胞在实体瘤组织浸润，能够有效杀伤肿瘤并抑制肿瘤细胞的复发，但是目前还没有有效延长CAR-T体内存续和促进CAR-T细胞肿瘤浸润的方法。
发明内容
有鉴于此，本发明的目的在于提供一种能够延长CAR-T细胞体内存续和/或提高CAR-T细胞浸润肿瘤能力的铰链区，含有本发明铰链区的CAR-T在体内存续时间更长、细胞浸润肿瘤能力显著增强，杀伤效果更好。
为实现上述目的，本发明的技术方案为：
能够延长CAR-T细胞体内存续和/或提高CAR-T细胞浸润肿瘤能力的铰链区，所述铰链区氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
所述铰链区的核苷酸序列如SEQ ID NO.20或SEQ ID NO.19所示。
肿瘤细胞表面的抗原(受体)与所述的嵌合抗原受体的抗体(配体)结合时，通过铰链区和跨膜区将信号传递至胞内，胞内信号域将信号转化为活化信号，激活效应细胞，效应细胞增殖、产生细胞因子及一系列活化反应从而杀伤肿瘤细胞。申请人通过对CAR结构铰链区改造的研究，发现铰链区结构与CAR-T的体内延续性和肿瘤浸润能力密切相关。
铰链区也称间隔区或hinge，连接CAR抗原识别区和跨膜区。铰链区应该有足够的灵活性，允许抗原结合区定向在不同的方向，以促进抗原识别。不同长度的铰链区对于CAR-T的稳定性起到不同作用，申请人通过多种对比组合意外发现铰链区对于CAR-T体内存续和肿瘤浸润具有重要影响；通过大量筛选工作，最终获得能够有效延长CAR-T体内存续和促进CAR-T细胞肿瘤浸润的铰链序列SEQ ID NO.1和SEQ ID NO.2并命名为：7H和G4HH2H3mt。
发明人共设计了5种铰链结构：7H、G4Hinge、G4HH3、G4HH2H3mt和DH，7H和G4HH2H3mt氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示，核苷酸序列分别如SEQ IDNO.20和SEQ ID NO.19所示。
对比目前常用的铰链结构G4h(G4HH2H3)和8H分别组合的CAR-T细胞CAR阳性率、CAR-T体内体外杀伤以及CAR-T体内存续和/或CAR-T细胞肿瘤浸润，发现包含7H或G4HH2H3mt铰链结构CAR的CAR-T体内存续时间更长，并且与含有G4HH2H3或8H铰链的CAR-T对比，CAR-T细胞浸润肿瘤能力显著增强，杀伤效果更好。
G4HH2H3氨基酸序列：
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
8H铰链氨基酸序列：
KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
延长CAR-T细胞体内存续和/或提高CAR-T细胞浸润肿瘤能力的铰链的改造方法，以G4HH2H3序列为模板以定点突变的方式进行铰链区改造。
本发明的目的之二在于提供一种得到上述铰链区的改造方法，所述方法的特征为，利用PCR介导的定点突变的方法，以G4HH2H3氨基酸序列为模板进行改造，G4HH2H3氨基酸序列如SEQ ID NO.3。
进一步根据上述改造方法获得铰链序列G4HH2H3mt，其特征在于G4HH2H3mt为G4HH2H3氨基酸序列在第15-18位和第79位进行突变和/或缺失。
本发明的目的之三在于提供一种嵌合抗原受体，包括上述的铰链区。所述铰链区为7H或G4HH2H3mt。
进一步，所述嵌合抗原受体还包含抗原识别区、跨膜区和胞内信号域。
进一步，所述嵌合抗原受体的抗原识别区可以识别肿瘤细胞表达抗原，包含但不限于PSCA、PSMA、CD19、BCMA、CD123、CD20、CD22、CEA、EGFR、EGFRVIII、GPC3或mesothelin抗原分子。
所述嵌合抗原受体可以识别的肿瘤细胞表达抗原包含但不限于以上抗原分子。
本发明的目的还在于提供一种靶向PSCA的嵌合抗原受体，包含抗人PSCA抗原的单链抗体、铰链区、跨膜区和胞内信号域；所述铰链区为7H或G4HH2H3mt，其氨基酸序列如SEQID NO.1或SEQ ID NO.2所示。
进一步，所述抗人PSCA抗原单链抗体的氨基酸序列如SEQ ID NO.4或SEQ ID NO.5所示。
进一步，所述跨膜区为CD28TM或CD8TM，所述CD28TM的氨基酸序列如SEQ ID NO:6，所述CD8TM的氨基酸序列如SEQ ID NO:7；所述胞内信号域为CD28和/或CD137和/或CD3，所述CD28的氨基酸序列如SEQ ID NO:8，所述CD137的氨基酸序列如SEQ ID NO:9，所述CD3的氨基酸序列如SEQ ID NO:10。
进一步，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.11或SEQ ID NO.12或SEQID NO.13或SEQ ID NO.14或SEQ ID NO.15或SEQ ID NO.16所示。
本发明的目的还在于提供一种所述的靶向PSCA的嵌合抗原受体的病毒载体的制备方法，包含以下步骤：
1)合成靶向PSCA的嵌合抗原受体的核苷酸序列：合成包含前导肽、抗人PSCA抗原的单链抗体、铰链区、跨膜区和胞内信号域的嵌合抗原受体核酸序列；所述前导肽核酸序列如SEQ ID NO.21所示；
2)构建表达嵌合抗原受体的病毒载体：设计引物，正向引物的核苷酸序列如SEQID NO:22所示，反向引物的核苷酸序列如SEQ ID NO:23所示，以所述嵌合抗原受体的基因序列为模板进行PCR扩增，得DNA片段；
将所述DNA片段的基因序列用限制性内切酶双酶切，同时用限制性内切酶酶切病毒表达载体pCDH-CAG，然后将酶切后的目的片段和病毒表达载体片段通过T4连接酶进行连接，获得表达嵌合抗原受体的病毒载体。所述病毒载体包含但不限于腺病毒、逆转录病毒和慢病毒。
作为一种优选，所述限制性内切酶为NheI和SalI。
进一步，步骤1)所述嵌合抗原受体的核苷酸序列如SEQ ID NO.17或SEQ ID NO.18所示。
进一步，在步骤2)之后，包装并纯化所述病毒载体，所述病毒载体为慢病毒载体。
本发明的目的还在于提供一种所述的制备方法所得的慢病毒载体。
在所述的方法下得到所述的慢病毒载体，这样的慢病毒载体转导的阳性细胞表达率高，在病人细胞培养过程中稳定，并且不会随着时间的推移导致CAR阳性率下降。用所述慢病毒载体感染的细胞，这样的细胞具备杀伤靶细胞的功能。
本发明的目的还在于提供一种所述的慢病毒载体感染的细胞，所述细胞包含但不限于T淋巴细胞、NK细胞和DC细胞。
进一步，所述慢病毒载体感染的细胞为T细胞。
本发明的目的还在于提供一种所述的慢病毒载体感染的CAR-T细胞在用于制备抗肿瘤药物中的应用。
进一步，所述的肿瘤细胞或组织能够表达PSCA。
本发明的目的还在于提供一种SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列作为铰链区在构建CAR骨架中的应用。
在某些实施例中嵌合抗原受体的铰链区可以为G4HH2H3序列改造的序列，如包含缺失G4HH2H3序列的H2H3段的氨基酸序列的铰链区的嵌合抗原受体；在某些实施例中，嵌合抗原受体的铰链区序列为G4HH2H3序列缺失H2段的序列，具体序列如表1所示。
表1，G4HH2H3铰链序列改造
Figure GDA0003506929910000061
在某些实施例中，包含所述铰链结构的嵌合抗原受体的抗原识别区可以是任何可以与PSCA抗原结合的多肽，例如可与PSCA特异结合的配体、双特异抗体、scFV、任选的交联的Fab、F(ab)2、单域抗体以及连接有His-标签或HA-标签的scFV。在某些实施例中特异性识别PSCA的抗体来源于7F5；某些实施例中抗原识别区氨基酸序列如下所示：
QVKLQESGGGLVQPGGSLKLSCVASGFTFSSYTMSWVRRTPEKRLEWVAYIHNGGGHTYYPDTIKGRFTISRDNAKNTLFLEMSSLKSEDTAMYYCTRRMYYGNSHWYFDVWGAGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAPKLLIYYTLKLNSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSKTLPWTFGGGTKVEIK
在某些实施例中特异性识别PSCA的抗体氨基酸序列如下所示：
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIK
在某些实施例中特异性识别PSCA的抗体氨基酸序列如下所示：
DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSS
在某些实施例中特异性识别PSCA的抗体氨基酸序列如下所示：
DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGGGGSGGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSS
本发明的有益效果在于：
1)本发明提供的铰链区结构能够延长CAR-T细胞在体内存续时间。
2)本发明提供的铰链区结构组合的嵌合抗原受体能够更稳定的表达于T淋巴细胞，具有更好的清除肿瘤细胞的能力，不仅可以维持靶向PSCA的嵌合抗原受体在病人细胞培养过程中的阳性率并且能够加强CAR-T的增殖和杀伤肿瘤的能力，对抗原阴性的细胞无毒副作用，能够用于肿瘤的靶向治疗。
3)包含本发明提供的铰链结构的CAR-T细胞浸润肿瘤组织的能力加强，可以有效对实体瘤进行杀伤。
附图说明
图1为不同铰链区及其组成的CAR结构图。
图2为不同铰链区组合的CAR表达率。
图3为不同铰链区组合的CAR-T细胞CAR平均荧光强度检测。
图4为不同铰链区组合的CAR-T细胞长期增殖。
图5为不同铰链区组合的CAR-T细胞杀伤率。
图6为不同铰链区组合的CAR-T细胞CAR表达稳定性检测。
图7为不同铰链区组合的CAR-T细胞体内杀伤效果对比。
图8为不同铰链区组合的CAR-T细胞体内存续及肿瘤浸润能力的研究。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或按照制造厂商所建议的条件。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1铰链区改造
以IgG4 FC序列来源的G4HH2H3铰链区为模板，以定点突变的方式进行铰链区改造。改造的结果如图1所示：G4HH2H3mt为G4HH2H3在第15-17位和第79位进行突变，第18位缺失获得的改造序列；G4HH3为删除H2段序列；G4Hinge为删除H2H3段序列获得的改造。同时获得来源于人CD7和人IgD铰链区序列7H和DH。如图2为不同铰链区序列及改造位点。
实施例2含有不同铰链区的嵌合抗原受体病毒的构建
为了验证不同结构的铰链区对CAR-T体内延续性以及肿瘤浸润的影响，以靶向PSCA的CAR-T为例进行实验。设计如图1所示5种不同铰链结构的CAR。1合成含上述设计的铰链序列的靶向PSCA的嵌合抗原受体基因序列
合成含前导肽(又称信号肽)(简称LP)、抗人PSCA抗原的单链抗体，6组不同铰链区、CD28跨膜区(简称为TM)，以及CD28、CD137和CD3胞内信号域的CAR结构。
2构建表达嵌合抗原受体的慢病毒载体
设计如下引物，并将其由生物科技公司合成，具体引物如下：
引物1：5’-atcgctagcatggccctgccagtgaccgcc-3’，下划线为NheI限制性内切酶位点；
引物2：5’-ccaggtcgacttagcgagggggcagggcctg-3’，下划线为SalI限制性内切酶位点。
然后以上述所示序列为引物，上述合成的各嵌合抗原受体序列为模板进行PCR扩增，反应体系按KOD FX NEO DNA聚合酶(购自TOYOBO公司)说明书加样，将扩增产物鉴定后用回收试剂盒(Promega公司)进行DNA片段回收，具体方法见说明书，回收获得嵌合抗原受体，将DNA回收片段送生物科技公司测序。
将克隆获得的编码嵌合抗原受体的基因序列用限制性内切酶NheI和SalI(购自Thermo公司)双酶切，同时用限制性内切酶NheI和SalI酶切慢病毒表达载体pCDH-CAG(购至addgene Plasmid)，酶切反应按说明书进行。酶切产物经琼脂糖凝胶电泳分离后用琼脂糖凝胶DNA片段回收试剂盒进行DNA片段回收，然后将目的片段和载体片段通过T4连接酶(购自Promega公司)进行连接，获得表达嵌合抗原受体的慢病毒载体，命名为Lv-hinge。将慢病毒载体转化大肠杆菌TOP10，挑取单克隆培养12h后用质粒抽提试剂盒(Invitrogen公司)抽提质粒，具体方法见说明书。
按照如上方法分别构建7个慢病毒载体：
“scFv-8H hinge-CD28TM-CD28-CD137-CD3Z”；
“scFv-7H hinge-CD28TM-CD28-CD137-CD3Z”；
“scFv-DH hinge-CD28TM-CD28-CD137-CD3Z”；
“scFv-G4HH2H3 hinge-CD28TM-CD28-CD137-CD3Z”；
“scFv-G4HH2H3mt hinge-CD28TM-CD28-CD137-CD3Z”；
“scFv-G4HH3 hinge-CD28TM-CD28-CD137-CD3Z”；
“scFv-G4Hinge-CD28TM-CD28-CD137-CD3Z”。
3慢病毒的包装
本实施例包装慢病毒采用磷酸钙法，具体步骤见分子克隆实验指南(第三版，J.萨姆布鲁克等著)。
4慢病毒的纯化
将病毒上清收集在50ml离心管中，离心过滤，滤液在3000r/min离心10min至新的50ml离心管；根据病毒上清量，分别加入质量分数为50％的PEG6000和4M NaCl，再用医用盐水定容使PEG6000终浓度为8.5％，NaCl终浓度为0.3M，定溶后于4℃冰箱静置后于4℃、条件下离心并弃尽上清，用200μl DMEM培养基重悬病毒，1.5mlEP管分装，每管40μl，-80℃保存备用。
5慢病毒滴度测定
步骤1:病毒感染293T细胞
感染前铺板293T细胞，取已纯化的病毒1μl，用医用生理盐水稀释10倍，再向每孔细胞中加入1μl聚凝胺(Polybrene)溶液，然后分别加到293T细胞中，24h后用含10％FBS(wt)的DMEM培养基换液，感染72h后于1000r/min条件下离心5min以收集细胞，抽提基因组。
步骤2:抽提基因组
基因组抽提试剂盒为QIAamp DNA Blood Mini Kit购于Qiagen公司(货号511004)，按试剂盒说明书操作。
步骤3:qRT-PCR测定病毒滴度
反应体系如下：Premix Ex TaqTM II(2×)10μl，上游引物(GAG up)1μl，下游引物(GAG dn)1μl，抽提的基因组1μl，RNase-Free dH2O 7μl，每个样品、标准品至少3个重复孔。然后按如下程序进行扩增：95℃预变性30s，95℃变性5s，60℃退火30s，72℃延伸30s，反应结束后，用分析软件分析数据，根据标准曲线计算病毒滴度。
实施例3CAR转染T淋巴细胞能力检测
1人外周血单核细胞的分离
用采血管采集外周血约60ml，分装于50ml离心管，加入7.5ml羟乙基淀粉稀释；室温(18～25℃)自然沉降约30min，收集上层血浆，将收集的上层血浆离心后用生理盐水重悬沉淀，按体积比为1:1加到淋巴细胞分离液上，梯度离心；离心后，离心管由上至下分为：第一层：血浆层；第二层：环状乳白色淋巴细胞层；第三层：透明分离液层；第四层：红细胞层；取第二层白色淋巴细胞层，并用生理盐水洗涤2次，离心5min，生理盐水重悬细胞，加入含有10％FBS的RPMI1640完全培养基培养，得人外周血单核细胞。
2慢病毒载体感染T淋巴细胞
用含10％胎牛血清的RPMI 1640完全培养基培养新制备的单个核细胞PBMC，第1天进行PBMC活化；第3天进行慢病毒感染；加入5MOI对应的慢病毒载体，未感染的T淋巴细胞作为空白对照；24h后将培养基更换为含有500IU/ml重组人IL-2的RPMI 1640完全培养基，继续培养10-20天。获得表达包含抗原识别区、铰链区、跨膜区以及胞内信号域的嵌合抗原受体的CAR-T细胞，以改造的铰链区命名，命名为：PSCA-CAR-G4HH2H3、PSCA-CAR-G4HH2H3mt、PSCA-CAR-G4HH3、PSCA-CAR-G4Hinge、PSCA-CAR-7H、PSCA-CAR-8H以及PSCA-CAR-DH；为了作图时便于标记，以上CAR-T细胞在后续实施例及说明书附图中以G4HH2H3、G4HH2H3mt、G4HH3、G4Hinge、8H、7H以及DH简写。
1)在培养过程中对培养至10天的已感染病毒的T细胞，离心弃尽上清以收集细胞；用含有体积分数1％胎牛血清的PBS溶液重悬细胞，并将细胞调整密度为1×106个/ml；将收集的细胞分别分装利用流式细胞术检测Protein-L阳性率，检测结果即表示培养第10天不同CAR组合在T淋巴细胞表达阳性率，同时分析流式检测MFI(平均荧光强度)可以获得CAR平均荧光强度的结果。
结果如图2所示，结果可见改造的铰链结构G4HH2H3mt、G4HH3、G4Hing、7H以及DH组合的CAR在T细胞表面表达阳性率与常规铰链G4HH2H3或8H组合的CAR差异不大。如图3表示不同铰链结构的CAR-T细胞表面CAR表达平均荧光强度，其中G4HH2H3、G4HH2H3mt或7H结构的CAR能够更高的表达于T细胞表面，表面平均荧光强度更高，预示CAR分子在细胞表面表达更多；而G4HH3、G4Hinge或DH结构的CAR表面平均荧光强度低。
2)分别感染获得CAR-T细胞后第5天、第10天以及第14天利用1)的检测方法检测不同铰链结构组合的CAR表达，检测在长时间培养后CAR-T细胞CAR表达的稳定性。
结果如图6所示：检测CAR的长期表达发现改造的结构与未改造结构G4HH2H3和8H对比，DH铰链结构的CAR表达稳定性差，在第5天后CAR阳性率显著下降并且随着培养时间延长下降更为显著，余下改造铰链结构与未改造铰链组合的CAR稳定性差异不大。
实施例4CAR-T细胞长期增殖能力检测
检测感染PSCA-CAR-T细胞在正常培养条件下的增殖；实施例3获得的CAR-T细胞，采用实施例3步骤2中的培养方法培养CAR-T细胞10天。PSCA抗原包被24孔板四度过夜，CAR-T细胞1*10^6/孔铺板细胞在PSCA抗原包被的24孔板，观察CAR-T细胞在抗原刺激后的存续时间。如图4所示分别在刺激后第3天，第6天，第9天以及第12天利用细胞计数仪统计CAR-T细胞数目并计算CAR-T细胞增殖倍数。依据体内实验的经验，每7天进行一次抗原刺激，图4结果在抗原刺激第3次后CAR-T细胞不再增殖，所以在刺激3次第15天后终止实验，进行CAR-T长期增殖倍数统计，长期增殖反应CAR-T细胞的存续。
实验如图4所示，G4HH2H3、G4HH2H3mt、7H以及8H铰链组合的CAR-T细胞长期增殖相对较快，CAR-T细胞体外存续能力更强；DH、G4Hinge和G4HH3长期增殖能力差。
实施例5CAR-T细胞对肿瘤细胞杀伤能力的影响
不同铰链结构的CAR-T对靶细胞的杀伤能力通过ACEA xCELLigence RTCA MP仪器完成，实验步骤依据仪器说明书进行；第一天将靶细胞(表达PSCA的肿瘤细胞)以2-5*10^4每孔铺板于仪器配备的96孔板中，附着于孔底的肿瘤细胞以电阻指数为数据每15分钟记录一次，24小时后依据预先设计的效靶比每孔铺入相应的CAR-T细胞，CAR-T细胞铺入后每隔15分钟记录一次电阻指数，通过电阻指数判断贴壁的靶细胞的增殖或者死亡情况。利用电阻指数分析结果公式为：CAR-T细胞杀伤率＝基线电阻指数-实时电阻指数。
Hela和RT4为PSCA高表达的肿瘤细胞系，T24为不表达PSCA的阴性对照细胞。
实验结果如图5所示：7H具有更好的杀伤能力，不仅对于RT4这种对T细胞较为敏感的肿瘤细胞杀伤力强，对于Hela这种对于T细胞不敏感的肿瘤细胞杀伤能力更与RT4相当，并且对阴性细胞不杀伤，特异性强。
综合上述实验，DH、G4Hinge和G4HH3铰链结构组合的CAR稳定性、长期增殖和体外杀伤能力较差不进行后续评价，后续分别以包含G4HH2H3、G4HH2H3mt、7H以及8H铰链结构的CAR-T细胞进行体内实验验证。
实施例6CAR-T细胞在动物模型中的抗肿瘤效果验证
建立人PSCA阳性肿瘤细胞系的小鼠移植瘤模型用于验证表达靶向PSCA的嵌合抗原受体的T淋巴细胞在动物模型中的抗肿瘤效果。
体内验证使用小鼠为NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl，简称NOG小鼠，由日本实验动物研究所(CIEA)的Mamoru Ito培育而成，为国际上CAR-T体内相关成瘤实验的最常见品系。体内验证使用的成瘤靶向细胞为前期体外验证使用的稳定表达萤火虫荧光素酶的PSCA阳性细胞系Hela(简称Hela-luc)。
通过前期体外实验发现改造的铰链G4HH2H3mt和7H结构具有更好的杀伤和体外存续能力，因此在动物实验中以这2种CAR-T细胞对比常用的G4HH2H3和8H为铰链的CAR-T细胞在小鼠体内杀伤肿瘤的能力。治疗注射的效应细胞为含有G4HH2H3mt、G4HH2H3、7H以及8H铰链结构的CAR-T细胞，对照为生理盐水组、未感染病毒的PBMC细胞。
成瘤后尾静脉注射效应CAR-T细胞5*10^5细胞/只鼠。注射CAR T细胞后每隔7天通过PerkinElmer公司的IVS活体成像系统拍照成像，显示肿瘤生长情况。期间每天观察小鼠存活情况并记录，结果见图7A图所示：包含G4HH2H3mt或7H铰链的CAR-T细胞在体内对肿瘤细胞均有较好的杀伤效果。进一步在实验终点处死小鼠后取出肿瘤组织进行称重并统计结果，结果如图7B图所示：与7A图结果相似，包含G4HH2H3mt或7H铰链的CAR-T细胞治疗的小鼠肿瘤重量更轻，尤其是7H铰链组合的CAR-T细胞治疗的小鼠肿瘤基本得到清除。
实施例7CAR-T细胞在体内存续和肿瘤浸润能力研究
CAR-T细胞体内延续和CAR-T细胞浸润肿瘤的能力，利用RT-PCR检测肿瘤组织的CAR基因拷贝数进行检测。
1.引物设计：
BBZ-HF：CAGAAGAAGAAGAAGGAGGATGTG；
BBZ-HR:TACTCCTCTCTTCGTCCTAGATTG。
2.肿瘤组织RNA提取
先在研钵中加入液氮，再将肿瘤组织剪成小块在液氮中磨成粉末，用液氮预冷的药匙取适量组织粉末加入已盛有Trizol液的EP管中，充分混合均匀。室温放置5min，然后加入200ml的氯仿，盖紧EP管并剧烈摇荡15秒钟。离心取上层水相于一新的EP管中，加入500ml异丙醇，温和颠倒混匀。室温放置10min离心。小心弃去上清液，加入1ml的75％乙醇，涡旋混匀，离心重复操作一次。弃去上清液，室温或真空干燥5～10min。用30ml DEPC处理过的水将RNA溶解，贮存于70％乙醇并保存于-70℃。
3.RT-PCR
进行RT-PCR，反应体系如下：
反应体系如下：Forward primer(10μM)0.5μl，Reverse primer(10μM)0.5μl，2×SYBRPremix Ex Taq II 10μl，Template 1ul，抽提的基因组1μl，RNase-Free dH2O 7μl，每个样品、标准品至少3个重复孔。然后按如下程序进行扩增：95℃2min，95℃15s，60℃1min，40个循环，反应结束后，用分析软件分析数据，分析结果如表3和图8所示。
表3为检测到的不同结构的CAR-T细胞治疗的肿瘤组织中的CAR拷贝数，图8为依据表3的结果绘制的图表。结果可见7H或G4HH2H3mt作为铰链区可以显著增强CAR-T浸润肿瘤的能力。
表3:回输CAR-T细胞45天后，不同铰链结果的CAR-T体内存续时间
Figure GDA0003506929910000171
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
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<120> 改造的铰链及其在构建CAR骨架中的应用
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225 230 235 240
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
245 250 255
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
275 280 285
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val
305 310 315 320
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
340 345 350
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
450 455 460
Lys Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
465 470 475 480
Leu Ser Leu Val Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys
485 490 495
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
500 505 510
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
515 520 525
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
530 535 540
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
545 550 555 560
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
565 570 575
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
580 585 590
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
595 600 605
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
610 615 620
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
625 630 635 640
Leu Pro Pro Arg
<210> 16
<211> 644
<212> PRT
<213> Artificial
<400> 16
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser
100 105 110
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser Glu Val Gln
115 120 125
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
130 135 140
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile
165 170 175
Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe Gln Gly Arg
180 185 190
Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
195 200 205
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys Thr Gly
210 215 220
Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys
225 230 235 240
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
245 250 255
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
275 280 285
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val
305 310 315 320
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
340 345 350
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
450 455 460
Lys Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
465 470 475 480
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
485 490 495
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
500 505 510
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
515 520 525
Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
530 535 540
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
545 550 555 560
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
565 570 575
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
580 585 590
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
595 600 605
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
610 615 620
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
625 630 635 640
Leu Pro Pro Arg
<210> 17
<211> 1488
<212> DNA
<213> Artificial
<400> 17
gatattcagc tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgca gcgcgagcag cagcgtgcgc tttattcatt ggtatcagca gaaaccgggc 120
aaagcgccga aacgcctgat ttatgatacc agcaaactgg cgagcggcgt gccgagccgc 180
tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa 240
gattttgcga cctattattg ccagcagtgg agcagcagcc cgtttacctt tggccagggc 300
accaaagtgg aaattaaagg cagcaccagc ggcagcggca aaccgggcag cggcgaaggc 360
agcaccaaag gcagcgaagt gcagctggtg gaaagcggcg gcggcctggt gcagccgggc 420
ggcagcctgc gcctgagctg cgcggcgagc ggctttaaca ttaaagatta ttatattcat 480
tgggtgcgcc aggcgccggg caaaggcctg gaatgggtgg cgtggattga tccggaaaac 540
ggcgataccg aatttgtgcc gaaatttcag ggccgcgcga ccattagcgc ggataccagc 600
aaaaacaccg cgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtattat 660
tgcaaaaccg gcggcttttg gggccagggc accctggtga ccgtgagcag cgcgccgccg 720
cgcgcgagcg cgctgccggc gccgccgacc ggcagcgcgc tgccggatcc gcagaccgcg 780
agcgcgctgc cggatccgcc ggcggcgagc gcgctgccgt tttgggtgct ggtggtggtg 840
ggcggcgtgc tggcgtgcta tagcctgctg gtgaccgtgg cgtttattat tttttgggtg 900
cgcagcaaac gcagccgcct gctgcatagc gattatatga acatgacccc gcgccgcccg 960
ggcccgaccc gcaaacatta tcagccgtat gcgccgccgc gcgattttgc ggcgtatcgc 1020
agcgtgaaac gcggccgcaa aaaactgctg tatattttta aacagccgtt tatgcgcccg 1080
gtgcagacca cccaggaaga agatggctgc agctgccgct ttccggaaga agaagaaggc 1140
ggctgcgaac tgcgcgtgaa atttagccgc agcgcggatg cgccggcgta tcagcagggc 1200
cagaaccagc tgtataacga actgaacctg ggccgccgcg aagaatatga tgtgctggat 1260
aaacgccgcg gccgcgatcc ggaaatgggc ggcaaaccgc gccgcaaaaa cccgcaggaa 1320
ggcctgtata acgaactgca gaaagataaa atggcggaag cgtatagcga aattggcatg 1380
aaaggcgaac gccgccgcgg caaaggccat gatggcctgt atcagggcct gagcaccgcg 1440
accaaagata cctatgatgc gctgcatatg caggcgctgc cgccgcgc 1488
<210> 18
<211> 2064
<212> DNA
<213> Artificial
<400> 18
gatattcagc tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgca gcgcgagcag cagcgtgcgc tttattcatt ggtatcagca gaaaccgggc 120
aaagcgccga aacgcctgat ttatgatacc agcaaactgg cgagcggcgt gccgagccgc 180
tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa 240
gattttgcga cctattattg ccagcagtgg agcagcagcc cgtttacctt tggccagggc 300
accaaagtgg aaattaaagg cagcaccagc ggcagcggca aaccgggcag cggcgaaggc 360
agcaccaaag gcagcgaagt gcagctggtg gaaagcggcg gcggcctggt gcagccgggc 420
ggcagcctgc gcctgagctg cgcggcgagc ggctttaaca ttaaagatta ttatattcat 480
tgggtgcgcc aggcgccggg caaaggcctg gaatgggtgg cgtggattga tccggaaaac 540
ggcgataccg aatttgtgcc gaaatttcag ggccgcgcga ccattagcgc ggataccagc 600
aaaaacaccg cgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtattat 660
tgcaaaaccg gcggcttttg gggccagggc accctggtga ccgtgagcag cgaaagcaaa 720
tatggcccgc cgtgcccgcc gtgcccggcg ccgccggtgg cgggcccgag cgtgtttctg 780
tttccgccga aaccgaaaga taccctgatg attagccgca ccccggaagt gacctgcgtg 840
gtggtggatg tgagccagga agatccggaa gtgcagttta actggtatgt ggatggcgtg 900
gaagtgcata acgcgaaaac caaaccgcgc gaagaacagt ttcagagcac ctatcgcgtg 960
gtgagcgtgc tgaccgtgct gcatcaggat tggctgaacg gcaaagaata taaatgcaaa 1020
gtgagcaaca aaggcctgcc gagcagcatt gaaaaaacca ttagcaaagc gaaaggccag 1080
ccgcgcgaac cgcaggtgta taccctgccg ccgagccagg aagaaatgac caaaaaccag 1140
gtgagcctga cctgcctggt gaaaggcttt tatccgagcg atattgcggt ggaatgggaa 1200
agcaacggcc agccggaaaa caactataaa accaccccgc cggtgctgga tagcgatggc 1260
agcttttttc tgtatagccg cctgaccgtg gataaaagcc gctggcagga aggcaacgtg 1320
tttagctgca gcgtgatgca tgaagcgctg cataaccatt atacccagaa aagcctgagc 1380
ctgagcctgg gcaaattttg ggtgctggtg gtggtgggcg gcgtgctggc gtgctatagc 1440
ctgctggtga ccgtggcgtt tattattttt tgggtgcgca gcaaacgcag ccgcctgctg 1500
catagcgatt atatgaacat gaccccgcgc cgcccgggcc cgacccgcaa acattatcag 1560
ccgtatgcgc cgccgcgcga ttttgcggcg tatcgcagcg tgaaacgcgg ccgcaaaaaa 1620
ctgctgtata tttttaaaca gccgtttatg cgcccggtgc agaccaccca ggaagaagat 1680
ggctgcagct gccgctttcc ggaagaagaa gaaggcggct gcgaactgcg cgtgaaattt 1740
agccgcagcg cggatgcgcc ggcgtatcag cagggccaga accagctgta taacgaactg 1800
aacctgggcc gccgcgaaga atatgatgtg ctggataaac gccgcggccg cgatccggaa 1860
atgggcggca aaccgcgccg caaaaacccg caggaaggcc tgtataacga actgcagaaa 1920
gataaaatgg cggaagcgta tagcgaaatt ggcatgaaag gcgaacgccg ccgcggcaaa 1980
ggccatgatg gcctgtatca gggcctgagc accgcgacca aagataccta tgatgcgctg 2040
catatgcagg cgctgccgcc gcgc 2064
<210> 19
<211> 684
<212> DNA
<213> Artificial
<400> 19
gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cgccggtggc gggcccgagc 60
gtgtttctgt ttccgccgaa accgaaagat accctgatga ttagccgcac cccggaagtg 120
acctgcgtgg tggtggatgt gagccaggaa gatccggaag tgcagtttaa ctggtatgtg 180
gatggcgtgg aagtgcataa cgcgaaaacc aaaccgcgcg aagaacagtt tcagagcacc 240
tatcgcgtgg tgagcgtgct gaccgtgctg catcaggatt ggctgaacgg caaagaatat 300
aaatgcaaag tgagcaacaa aggcctgccg agcagcattg aaaaaaccat tagcaaagcg 360
aaaggccagc cgcgcgaacc gcaggtgtat accctgccgc cgagccagga agaaatgacc 420
aaaaaccagg tgagcctgac ctgcctggtg aaaggctttt atccgagcga tattgcggtg 480
gaatgggaaa gcaacggcca gccggaaaac aactataaaa ccaccccgcc ggtgctggat 540
agcgatggca gcttttttct gtatagccgc ctgaccgtgg ataaaagccg ctggcaggaa 600
ggcaacgtgt ttagctgcag cgtgatgcat gaagcgctgc ataaccatta tacccagaaa 660
agcctgagcc tgagcctggg caaa 684
<210> 20
<211> 108
<212> DNA
<213> Artificial
<400> 20
gcgccgccgc gcgcgagcgc gctgccggcg ccgccgaccg gcagcgcgct gccggatccg 60
cagaccgcga gcgcgctgcc ggatccgccg gcggcgagcg cgctgccg 108
<210> 21
<211> 63
<212> DNA
<213> Artificial
<400> 21
atggcactgc cagtgaccgc cctgctgctg cccctggcac tgctgctgca cgcagctcgg 60
cct 63
<210> 22
<211> 30
<212> DNA
<213> Artificial
<400> 22
atcgctagca tggccctgcc agtgaccgcc 30
<210> 23
<211> 31
<212> DNA
<213> Artificial
<400> 23
ccaggtcgac ttagcgaggg ggcagggcct g 31

Claims (7)

1.靶向PSCA的嵌合抗原受体，其特征在于，所述嵌合抗原受体的氨基酸序列如SEQ IDNO.14所示。
2.权利要求1所述嵌合抗原受体的病毒载体的制备方法，其特征在于，包括以下步骤：
1)合成靶向PSCA的嵌合抗原受体的核苷酸序列：合成包含前导肽、抗人PSCA抗原的单链抗体、铰链区、跨膜区和胞内信号域的嵌合抗原受体的核酸序列；所述前导肽核酸序列如SEQ ID NO.21所示；所述铰链区的核苷酸序列如SEQ ID NO.19所示；
2)构建表达嵌合抗原受体的病毒载体：设计引物，正向引物的核苷酸序列如SEQ IDNO:22所示，反向引物的核苷酸序列如SEQ ID NO:23所示，以所述嵌合抗原受体的核苷酸序列为模板进行PCR扩增，得DNA片段；
将所述DNA片段的基因序列用限制性内切酶双酶切，同时用限制性内切酶酶切病毒表达载体pCDH-CAG，然后将酶切后的目的片段和病毒表达载体片段通过T4连接酶进行连接，获得表达所述嵌合抗原受体的病毒载体，所述病毒载体包含但不限于腺病毒、逆转录病毒和慢病毒载体。
3.根据权利要求2所述的制备方法，其特征在于，步骤1)所述嵌合抗原受体中，编码抗人PSCA抗原的单链抗体、铰链区、跨膜区和胞内信号域的核苷酸序列如SEQ ID NO.18所示。
4.根据权利要求3所述的制备方法，其特征在于，在步骤2)之后，包装并纯化所述病毒载体，所述病毒载体为慢病毒载体。
5.权利要求4所述慢病毒载体感染的细胞，其特征在于，所述细胞包含但不限于T细胞或NK细胞或DC细胞。
6.权利要求5所述的细胞，其特征在于，所述细胞为T细胞。
7.权利要求5或6所述的细胞在用于制备治疗表达PSCA的肿瘤的药物中的应用。
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Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2899779T3 (es) 2014-12-05 2022-03-14 Memorial Sloan Kettering Cancer Center Receptores antigénicos quiméricos dirigidos a receptor acoplado a proteína G y usos de los mismos
CN111902159A (zh) 2017-11-01 2020-11-06 朱诺治疗学股份有限公司 对b细胞成熟抗原（bcma）具有特异性的嵌合抗原受体
SG11202003501XA (en) 2017-11-01 2020-05-28 Juno Therapeutics Inc Antibodies and chimeric antigen receptors specific for b-cell maturation antigen
CN110950954B (zh) * 2018-09-26 2021-06-25 重庆精准生物技术有限公司 抗cd123人源化单链抗体及其应用
CN110950965B (zh) * 2018-09-26 2021-10-15 重庆精准生物技术有限公司 抗人cd123的嵌合抗原受体及其应用
CN110964112B (zh) * 2018-09-30 2023-05-02 重庆精准生物技术有限公司 增强抗psca嵌合抗原受体活性的人源化抗体及其应用
TW202021981A (zh) * 2018-11-01 2020-06-16 美商奇諾治療有限公司 G蛋白偶合受體c類第5群成員d（gprc5d）特異性嵌合抗原受體
CN110452294B (zh) 2019-08-06 2020-08-07 复旦大学 五种铰链区及其嵌合抗原受体和免疫细胞
CN113493522B (zh) * 2020-04-03 2023-04-28 重庆精准生物技术有限公司 双特异性嵌合抗原受体及其应用
CN113493521B (zh) * 2020-04-03 2023-05-05 重庆精准生物技术有限公司 靶向cd19和cd123的双靶点嵌合抗原受体及其应用
IL298346A (en) * 2020-05-22 2023-01-01 Chongqing Prec Biotech Co Ltd A fusion protein for reversal of the tumor microenvironment and its use
US20240150456A1 (en) * 2021-04-02 2024-05-09 Nanjing Legend Biotech Co., Ltd. Engineered immune cells and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107033248A (zh) * 2017-05-03 2017-08-11 重庆精准生物技术有限公司 识别癌胚抗原的嵌合抗原受体及其应用
CN107226867A (zh) * 2017-07-25 2017-10-03 重庆精准生物技术有限公司 抗人cd19抗原的嵌合抗原受体及其应用
CN107312091A (zh) * 2017-05-02 2017-11-03 重庆精准生物技术有限公司 靶向人cd19抗原的人源化单克隆抗体
CN109306014A (zh) * 2017-07-27 2019-02-05 上海细胞治疗研究院 一种靶向间皮素的嵌合抗原受体修饰t细胞及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112016024957A2 (pt) * 2014-04-25 2017-10-24 Bluebird Bio Inc métodos aperfeiçoados para fabricação de terapias celulares adotivas
CN105949325B (zh) * 2016-07-08 2019-07-16 重庆精准生物技术有限公司 包含cd27胞内结构域的嵌合抗原受体、慢病毒载体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107312091A (zh) * 2017-05-02 2017-11-03 重庆精准生物技术有限公司 靶向人cd19抗原的人源化单克隆抗体
CN107033248A (zh) * 2017-05-03 2017-08-11 重庆精准生物技术有限公司 识别癌胚抗原的嵌合抗原受体及其应用
CN107226867A (zh) * 2017-07-25 2017-10-03 重庆精准生物技术有限公司 抗人cd19抗原的嵌合抗原受体及其应用
CN109306014A (zh) * 2017-07-27 2019-02-05 上海细胞治疗研究院 一种靶向间皮素的嵌合抗原受体修饰t细胞及其用途

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