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CN112608902B - 一种第四代car-t细胞及其应用 - Google Patents

一种第四代car-t细胞及其应用 Download PDF

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CN112608902B
CN112608902B CN202011521237.6A CN202011521237A CN112608902B CN 112608902 B CN112608902 B CN 112608902B CN 202011521237 A CN202011521237 A CN 202011521237A CN 112608902 B CN112608902 B CN 112608902B
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汤朝阳
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秦乐
吴迪
冯世忠
冯嘉昆
杨乐旋
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Guangdong Zhaotai Cell Biotechnology Co ltd
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Abstract

本发明提供了一种第四代CAR‑T细胞及其应用，所述第四代CAR‑T细胞表达特异性结合抗原的嵌合抗原受体、复合型IL‑6和CCL19；所述复合型IL‑6和CCL19为NFAT调控型过表达。本发明采用NFAT调控启动子调控T细胞对复合型IL‑6和CCL19的表达，在识别肿瘤抗原后表达分泌HIL‑6和CCL19，显著提高了CAR‑T细胞的增殖能力和抗肿瘤效果，同时规避了全身性或者局部的炎症反应，降低了CAR‑T的副作用。

Description

一种第四代CAR-T细胞及其应用
技术领域
本发明属于生物医药技术领域，涉及一种第四代CAR-T细胞及其应用，尤其涉及一种条件性过表达复合型IL-6和CCL19的第四代CAR-T细胞及其应用。
背景技术
随着肿瘤免疫学理论和临床技术的发展，嵌合抗原受体T细胞疗法(Chimericantigen receptor T-cell immunotherapy，CAR-T)成为最有发展前景的肿瘤免疫疗法之一。目前，CAR-T细胞疗法已经广泛应用于治疗B细胞恶性肿瘤，靶向CD19的CAR-T细胞是CAR-T疗法治疗B细胞恶性肿瘤的先驱，为治疗B细胞恶性肿瘤提供了有效方案。
但是，CAR-T细胞疗法仍然存在多种问题。在白血病治疗方面，CAR-T细胞疗法存在复发率高的问题，促进CAR-T细胞的维持和记忆性T细胞的形成可能是解决该问题的技术突破口；此外，抗CD19的CAR-T细胞的临床治疗研究结果显示，患者对CAR-T细胞治疗的响应率与细胞因子释放综合征(CRS)的发生存在一定的正相关关系，而CRS又可能导致CAR-T细胞相关的脑病综合征(CRES)等副作用，严重限制了CAR-T细胞的临床应用；而在实体瘤治疗方面，CAR-T细胞疗效差，目前尚未报道相关的临床研究突破。
发明内容
针对现有技术的不足和实际需求，本发明提供了一种第四代CAR-T细胞及其应用，通过条件性过表达复合型IL-6和CCL19，不仅提高了CAR-T抗肿瘤作用，而且降低了其在治疗过程中的副作用。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种第四代CAR-T细胞，所述第四代CAR-T细胞表达特异性结合抗原的嵌合抗原受体、复合型IL-6和CCL19；
所述复合型IL-6和CCL19为NFAT调控型过表达。
本发明中，采用NFAT调控启动子调控T细胞对复合型IL-6和CCL19的表达，当CAR-T细胞处于静息状态时，复合型IL-6和CCL19不表达，当CAR-T细胞识别肿瘤抗原被激活后，NFAT调控启动子5×NFAT-RE-minP启动对复合型IL-6和CCL19的转录和翻译，复合型IL-6和CCL19表达，复合型IL-6刺激CAR-T细胞增殖、记忆分化、增强杀伤效果，CCL19将外周血免疫细胞最大程度被招募到肿瘤内部，两者相互配合共同实现对肿瘤的靶向促凋亡作用。
优选地，所述复合型IL-6为IL-6和IL-6Rα的复合物，所述复合型IL-6(HIL-6)包括SEQ ID NO:1所示的氨基酸序列；
SEQ ID NO:1：
PPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPARGGGSGGGGSVEPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM。
优选地，所述CCL19包括SEQ ID NO:2所示的氨基酸序列；
SEQ ID NO:2：
MALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLL IKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS。
优选地，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域和信号传导结构域。
优选地，所述抗原结合结构域结合肿瘤表面抗原，所述肿瘤表面抗原包括CD19、CD20、CD22、CD30、CEA、EGFR、BRAF、HER-2、Mesothelin、MUC1、PSCA、GPC3、TERT、PTEN、PD-1、PD-L1或VEGF中的任意一种，优选为CD19、MUC1、GPC3、Mesothelin、PSCA或HER2中的任意一种，进一步优选为Mesothelin。
优选地，所述抗原结合结构域为靶向Mesothelin的单链抗体。
优选地，所述跨膜结构域包括CD28跨膜结构域和/或CD8α跨膜结构域，优选为CD28跨膜结构域。
优选地，所述信号传导结构域包括CD28胞内结构域、CD3ζ、TLR2、4-1BB、TLR1、CD27、OX40或DAP10中的任意一种或至少两种的组合，优选为CD28胞内结构域、CD3ζ和TLR2的组合。
优选地，所述嵌合抗原受体还包括信号肽。
优选地，所述嵌合抗原受体由GM-CSF信号肽、Mesothelin抗原结合结构域、CD28跨膜结构域、CD28胞内结构域、CD3ζ和TLR2串联组成。
优选地，所述嵌合抗原受体包括如SEQ ID NO:3所示的氨基酸序列；
SEQ ID NO:3：
MLLLVTSLLLCELPHPAFLLSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSGILEQQGIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS。
第二方面，本发明提供了一种表达载体，所述表达载体包括嵌合抗原受体编码基因、NFAT调控启动子、复合型IL-6编码基因和CCL19编码基因。
优选地，所述嵌合抗原受体编码基因包括SEQ ID NO:4所示的核酸序列；SEQ IDNO:4：
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgagccaggtacagctgcagcagtcaggtccaggactcgtgacgccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataacgactatgcagtatctgtgaaaagtcgaatgagcatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctgtgtattactgtgcaagaggaatgatgacttactattacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggaattctaggatccggtggcggtggcagcggcggtggtggttccggaggcggcggttctcagcctgtgctgactcagtcgtcttccctctctgcatctcctggagcatcagccagtctcacctgcaccttgcgcagtggcatcaatgttggtccctacaggatatactggtaccagcagaagccagggagtcctccccagtatctcctgaactacaaatcagactcagataagcagcagggctctggagtccccagccgcttctctggatccaaagatgcttcggccaatgcaggggttttactcatctctgggctccggtctgaggatgaggctgactattactgtatgatttggcacagcagcgctgctgtgttcggaggaggcacccaactgaccgtcctctccggaattctagaacaacagggtattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgccaggccaaaaggaagcccaggaaagctcccagcaggaacatctgctatgatgcatttgtttcttacagtgagcgggatgcctactgggtggagaaccttatggtccaggagctggagaacttcaatccccccttcaagttgtgtcttcataagcgggacttcattcctggcaagtggatcattgacaatatcattgactccattgaaaagagccacaaaactgtctttgtgctttctgaaaactttgtgaagagtgagtggtgcaagtatgaactggacttctcccatttccgtctttttgatgagaacaatgatgctgccattctcattcttctggagcccattgagaaaaaagccattccccagcgcttctgcaagctgcggaagataatgaacaccaagacctacctggagtggcccatggacgaggctcagcgggaaggattttgggtaaatctgagagctgcgataaagtcc。
优选地，所述NFAT调控启动子5×NFAT-RE-minP包括SEQ ID NO:5所示的核酸序列；
SEQ ID NO:5：
ggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtagatctagactctagagggtatataatggaagctcgaattccagcttggcattccggtactgttggtaaaaagcttggcaatccggtactgttggtaaagccacc。
优选地，所述复合型IL-6(HIL-6)编码基因包括SEQ ID NO:6所示的核酸序列；
SEQ ID NO:6：
ccccccgaggagccccagctctcctgcttccggaagagccccctcagcaatgttgtttgtgagtggggtcctcggagcaccccatccctgacgacaaaggctgtgctcttggtgaggaagtttcagaacagtccggccgaagacttccaggagccgtgccagtattcccaggagtcccagaagttctcctgccagttagcagtcccggagggagacagctctttctacatagtgtccatgtgcgtcgccagtagtgtcgggagcaagttcagcaaaactcaaacctttcagggttgtggaatcttgcagcctgatccgcctgccaacatcacagtcactgccgtggccagaaacccccgctggctcagtgtcacctggcaagacccccactcctggaactcatctttctacagactacggtttgagctcagatatcgggctgaacggtcaaagacattcacaacatggatggtcaaggacctccagcatcactgtgtcatccacgacgcctggagcggcctgaggcacgtggtgcagcttcgtgcccaggaggagttcgggcaaggcgagtggagcgagtggagcccggaggccatgggcacgccttggacagaatccaggagtcctccagctcgcggtggtggatcaggaggtggagggtcagtcgagccagtacccccaggagaagattccaaagatgtagccgccccacacagacagccactcacctcttcagaacgaattgacaaacaaattcggtacatcctcgacggcatctcagccctgagaaaggagacatgtaacaagagtaacatgtgtgaaagcagcaaagaggcactggcagaaaacaacctgaaccttccaaagatggctgaaaaagatggatgcttccaatctggattcaatgaggagacttgcctggtgaaaatcatcactggtcttttggagtttgaggtatacctagagtacctccagaacagatttgagagtagtgaggaacaagccagagctgtgcagatgagtacaaaagtcctgatccagttcctgcagaaaaaggcaaagaatctagatgcaataaccacccctgacccaaccacaaatgccagcctgctgacgaagctgcaggcacagaaccagtggctgcaggacatgacaactcatctcattctgcgcagctttaaggagttcctgcagtccagcctgagggctcttcggcaaatg。
优选地，所述CCL19编码基因包括SEQ ID NO:7所示的核酸序列；
SEQ ID NO:7：
atggccctgctactggccctcagcctgctggttctctggacttccccagccccaactctgagtggcaccaatgatgctgaagactgctgcctgtctgtgacccagaaacccatccctgggtacatcgtgaggaacttccactaccttctcatcaaggatggctgcagggtgcctgctgtagtgttcaccacactgaggggccgccagctctgtgcacccccagaccagccctgggtagaacgcatcatccagagactgcagaggacctcagccaagatgaagcgccgcagcagt。
优选地，所述表达载体包括病毒载体。
优选地，所述病毒载体包括慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种，优选为慢病毒载体。
第三方面，本发明提供了一种重组慢病毒，所述重组慢病毒采用第二方面所述的表达载体与包装辅助质粒共转染哺乳细胞制备得到。
第四方面，本发明提供了一种第一方面所述的第四代CAR-T细胞的制备方法，所述方法包括将第二方面所述的表达载体或第三方面所述的重组慢病毒导入T细胞的步骤。
第五方面，本发明提供了一种药物组合物，所述药物组合物包括第一方面所述的第四代CAR-T细胞。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第六方面，本发明提供了第一方面所述的第四代CAR-T细胞、第二方面所述的表达载体、第三方面所述的重组慢病毒或第五方面所述的药物组合物在制备肿瘤治疗药物中的应用。
优选地，所述肿瘤包括肝癌、肺癌、乳腺癌、肾母细胞瘤、神经胶质瘤、神经母细胞瘤、黑色素瘤、鼻咽癌、间皮质瘤、胰岛细胞瘤、视网膜母细胞瘤、胰腺癌、子宫肌瘤、宫颈癌或甲状腺癌中的任意一种或至少两种的组合。
与现有技术相比，本发明具有如下有益效果：
(1)本发明构建条件性表达复合型IL-6和CCL19的CAR-T细胞，CAR-T在识别肿瘤抗原后表达分泌复合型IL-6和CCL19，复合型IL-6增强CAR-T的扩增和抗肿瘤作用，CCL19将外周血免疫细胞最大程度被招募到肿瘤内部，两者相互配合共同实现对肿瘤的靶向促凋亡作用；
(2)本发明构建的条件性表达IL-6和CCL19的CAR-T细胞具有更强的肿瘤抑制作用，可以显著抑制肿瘤的增殖和体积，同时不引起或加剧全身性或者局部的炎症反应，降低了CAR-T的副作用。
附图说明
图1为不同CAR-T细胞分泌HIL-6和CCL19的结果；
图2为不同CAR-T细胞的增殖情况；
图3为不同CAR-T细胞对T细胞的招募能力；
图4为不同CAR-T细胞的杀伤效率。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1重组慢病毒载体的构建
通过全基因合成以下核酸分子：
①由GM-CSF信号肽、Mesothelin scFv、CD28跨膜结构域、CD28胞内结构域、CD3ζ、TLR2和2A肽的核酸序列串联形成的CAR分子；
②由5×NFAT-RE-minP启动子(SEQ ID NO:5)、HIL-6(SEQ ID NO:6)和CCL19(SEQID NO:7)串联形成的条件性分子；
将①克隆至慢病毒表达载体pwpxld-eGFP中，得到CAR-pwpxld-eGFP；
将①克隆至慢病毒表达载体pwpxld-tCD19中，随后将②反向克隆至pwpxld-tCD19的末端，得到CAR-HIL-6/CCL19-pwpxld-tCD19。
实施例2慢病毒包装
采用293T细胞制备重组慢病毒，当293T细胞铺100mm培养皿板底达80～90％时，进行慢病毒包装：
病毒包装前2h，更换培养基为含1％胎牛血清的DMEM，加入量为6mL/100mm培养皿；
配制如表1所示的质粒混合液；
表1
试剂 剂量
重组慢病毒载体 4.5μg
pMD2.G 1.5μg
psPAX2 6μg
将36μg PEI加至另一500μL opti-MEM培养基内，混匀，室温静置5min；
将表1所示的质粒混合液与PEI混合，吹打混匀，室温静置25～30min；
将上述混合液逐滴加至培养在100mm培养皿中的293T细胞上；
培养6h后，更换培养基为含1％胎牛血清的DMEM，加入量为7mL/100mm培养皿；
包装后24h、48h和72h，收取病毒上清，同时向293T细胞补加培养基，加入量为7mL/100mm培养皿；
1000g离心10min，0.45μm滤器过滤，得到重组慢病毒，4℃保存待用。
实施例3 T细胞激活和慢病毒转染
通过Ficoll密度梯度法分离血液中的单个核细胞，采用红细胞裂解液裂解去除红细胞后，通过MACS Pan-T磁珠分选出T细胞，采用培养基AIM-V加5％FBS、100U/mL青霉素和0.1mg/mL链霉素稀释至浓度为2.5×106个/mL待用；利用包被CD2、CD3和CD28抗体的磁珠(美天旎)刺激T细胞，磁珠与T细胞比例为1:2，T细胞密度为5×106个/mL/cm2，T细胞在37℃、5％CO2培养箱中培养刺激48h；
去除T细胞中的磁珠，将激活后的T细胞在300g下离心5min，去上清，用新鲜培养基重悬，分别加入不同的重组慢病毒、8μg/mL的polybrene和300IU/mL的IL-2，病毒加入量为MOI＝10，37℃、5％CO2培养箱中培养24h后，300g离心5min，去上清，用含300IU/mL IL-2的新鲜培养基重悬细胞，37℃、5％CO2培养箱培养；
将CAR-T细胞密度维持在1×106个/mL左右，每2～3天进行一次半量换液；培养10天后进行后续实验。
本实施例制备的CAR-T细胞为Mesothelin CAR-T和HIL-6/CCL19-CAR-T。
实施例4 CAR-T细胞对HIL-6和CCL19的分泌
取两种CAR-T细胞各2×106个，用新鲜的T细胞培养基培养24小时，收集细胞上清；同时取2×106个CAR-HIL-6/CCL19-T，与等量的Mesothelin阳性细胞K562-Mesothelin-GL共培养24h，收集细胞上清；采用ELISA试剂盒(R&D system)检测每种细胞对HIL-6和CCL19的分泌情况。
如图1所示，Mesothelin CAR-T细胞的培养上清中没有HIL-6和CCL19，HIL-6/CCL19-CAR-T细胞的培养上清中也没有HIL-6和IL-7，与K562-Mesothelin-GL共培养后，CAR-HIL-6/CCL19-T分泌HIL-6和CCL19，说明HIL-6/CCL19-CAR-T细胞对HIL-6和CCL19的表达受到肿瘤抗原调控。
实施例5 CAR-T细胞的体外增殖实验
取两种CAR-T细胞各2×106个，用新鲜的T细胞培养基培养；同时取2×106个两种CAR-T细胞，与等量的Mesothelin阳性细胞K562-Mesothelin-GL共培养，每天进行计数，连续培养7天，分析CAR-T细胞的增殖情况。
结果图2所示，Mesothelin CAR-T由于不分泌HIL-6，对CAR-T细胞没有促进增殖的作用；HIL-6/CCL19-CAR-T在没有肿瘤抗原的情况下也不分泌HIL-6，对CAR-T细胞同样没有促进增殖的作用，反而由于HIL-6/CCL19的影响，增殖能力不如Mesothelin CAR-T；HIL-6/CCL19-CAR-T与K562-Mesothelin-GL共培养，分泌HIL-6促进CAR-T细胞的增殖。
实施例6 CAR-T细胞的体外迁移实验
取两种CAR-T细胞各2×106个，于Transwell装置下层培养24h，同时取2×106个两种CAR-T与等量K562-Mesothelin-GL于下层共培养24h，取CFSE标记的野生T细胞于上层培养5h，用流式细胞仪检测下层细胞中CFSE标记的T细胞的数量。
统计结果如图3所示，CAR-HIL-6/CCL19-T与肿瘤细胞共培养后可以招募到更多的T细胞，说明CAR-HIL-6/CCL19-T条件性表达CCL19，只有在识别肿瘤细胞后才会分泌CCL19，发挥招募作用，避免了炎症副反应。
实施例7体外检测CAR-T细胞对肿瘤细胞的杀伤功能
取WT、Mesothelin CAR-T和HIL-6/CCL19-CAR-T细胞各2×106个，与肿瘤细胞K562-Mesothelin-GL按E:T为4:1、2:1、1:1、1:2、1:4、1:8、1:16的比例混合，加入到96孔板中，每组设置3个复孔，250g离心5min后，置于37℃、5％CO2培养箱共培养18h；
18h后，向96孔板中加入100μL/孔的荧光素酶底物(1×)，将细胞重悬混匀，立即通过多功能酶标仪测定RLU(relative light unit)，测定时间为1秒，利用荧光素酶(Luciferase)定量杀伤效率评估方法，体外比较WT、Mesothelin CAR-T和CAR-HIL-6/CCL19-T对K562-Mesothelin-GL的杀伤作用，杀伤比例计算公式如下：
100％×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)
结果如图4所示，CAR-HIL-6/CCL19-T对K562-Mesothelin-GL的体外杀伤效率显著高于WT和Mesothelin CAR-T，在E:T很小的情况下，即肿瘤靶细胞远大于效应T细胞，CAR-HIL-6/CCL19-T也能表现出较强的肿瘤杀伤活性。
综上所述，本发明的第四代CAR-T细胞在识别肿瘤抗原后表达分泌HIL-6和CCL19，显著提高了CAR-T细胞的增殖能力和抗肿瘤效果，同时规避了全身性或者局部的炎症反应，降低了CAR-T的副作用。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 广东昭泰体内生物医药科技有限公司
<120> 一种第四代CAR-T细胞及其应用
<130> 202012
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 407
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Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
145 150 155 160
Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala
165 170 175
Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile
180 185 190
Asn Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser
195 200 205
Pro Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln
210 215 220
Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala
225 230 235 240
Asn Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala
245 250 255
Asp Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly
260 265 270
Gly Thr Gln Leu Thr Val Leu Ser Gly Ile Leu Glu Gln Gln Gly Ile
275 280 285
Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly
290 295 300
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe
305 310 315 320
Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val
325 330 335
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
340 345 350
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
355 360 365
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
370 375 380
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg
385 390 395 400
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
405 410 415
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
420 425 430
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
435 440 445
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
450 455 460
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
465 470 475 480
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
485 490 495
Ala Leu His Met Gln Ala Leu Pro Pro Arg Gln Ala Lys Arg Lys Pro
500 505 510
Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr
515 520 525
Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu
530 535 540
Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe
545 550 555 560
Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys
565 570 575
Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu
580 585 590
Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu
595 600 605
Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys
610 615 620
Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys
625 630 635 640
Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp
645 650 655
Val Asn Leu Arg Ala Ala Ile Lys Ser
660 665
<210> 4
<211> 1995
<212> DNA
<213> 人工序列
<400> 4
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
agccaggtac agctgcagca gtcaggtcca ggactcgtga cgccctcgca gaccctctca 120
ctcacctgtg ccatctccgg ggacagtgtc tctagcaaca gtgctacttg gaactggatc 180
aggcagtccc catcgagagg ccttgagtgg ctgggaagga catactacag gtccaagtgg 240
tataacgact atgcagtatc tgtgaaaagt cgaatgagca tcaacccaga cacatccaag 300
aaccagttct ccctgcagct gaactctgtg actcccgagg acacggctgt gtattactgt 360
gcaagaggaa tgatgactta ctattacggt atggacgtct ggggccaagg gaccacggtc 420
accgtctcct caggaattct aggatccggt ggcggtggca gcggcggtgg tggttccgga 480
ggcggcggtt ctcagcctgt gctgactcag tcgtcttccc tctctgcatc tcctggagca 540
tcagccagtc tcacctgcac cttgcgcagt ggcatcaatg ttggtcccta caggatatac 600
tggtaccagc agaagccagg gagtcctccc cagtatctcc tgaactacaa atcagactca 660
gataagcagc agggctctgg agtccccagc cgcttctctg gatccaaaga tgcttcggcc 720
aatgcagggg ttttactcat ctctgggctc cggtctgagg atgaggctga ctattactgt 780
atgatttggc acagcagcgc tgctgtgttc ggaggaggca cccaactgac cgtcctctcc 840
ggaattctag aacaacaggg tattgaagtt atgtatcctc ctccttacct agacaatgag 900
aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag tcccctattt 960
cccggacctt ctaagccctt ttgggtgctg gtggtggttg ggggagtcct ggcttgctat 1020
agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 1080
ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1140
cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1200
agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1380
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1440
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1500
caggccctgc cccctcgcca ggccaaaagg aagcccagga aagctcccag caggaacatc 1560
tgctatgatg catttgtttc ttacagtgag cgggatgcct actgggtgga gaaccttatg 1620
gtccaggagc tggagaactt caatcccccc ttcaagttgt gtcttcataa gcgggacttc 1680
attcctggca agtggatcat tgacaatatc attgactcca ttgaaaagag ccacaaaact 1740
gtctttgtgc tttctgaaaa ctttgtgaag agtgagtggt gcaagtatga actggacttc 1800
tcccatttcc gtctttttga tgagaacaat gatgctgcca ttctcattct tctggagccc 1860
attgagaaaa aagccattcc ccagcgcttc tgcaagctgc ggaagataat gaacaccaag 1920
acctacctgg agtggcccat ggacgaggct cagcgggaag gattttgggt aaatctgaga 1980
gctgcgataa agtcc 1995
<210> 5
<211> 255
<212> DNA
<213> 人工序列
<400> 5
ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt 60
ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt 120
ggaggaaaaa ctgtttcata cagaaggcgt agatctagac tctagagggt atataatgga 180
agctcgaatt ccagcttggc attccggtac tgttggtaaa aagcttggca atccggtact 240
gttggtaaag ccacc 255
<210> 6
<211> 1221
<212> DNA
<213> 人工序列
<400> 6
ccccccgagg agccccagct ctcctgcttc cggaagagcc ccctcagcaa tgttgtttgt 60
gagtggggtc ctcggagcac cccatccctg acgacaaagg ctgtgctctt ggtgaggaag 120
tttcagaaca gtccggccga agacttccag gagccgtgcc agtattccca ggagtcccag 180
aagttctcct gccagttagc agtcccggag ggagacagct ctttctacat agtgtccatg 240
tgcgtcgcca gtagtgtcgg gagcaagttc agcaaaactc aaacctttca gggttgtgga 300
atcttgcagc ctgatccgcc tgccaacatc acagtcactg ccgtggccag aaacccccgc 360
tggctcagtg tcacctggca agacccccac tcctggaact catctttcta cagactacgg 420
tttgagctca gatatcgggc tgaacggtca aagacattca caacatggat ggtcaaggac 480
ctccagcatc actgtgtcat ccacgacgcc tggagcggcc tgaggcacgt ggtgcagctt 540
cgtgcccagg aggagttcgg gcaaggcgag tggagcgagt ggagcccgga ggccatgggc 600
acgccttgga cagaatccag gagtcctcca gctcgcggtg gtggatcagg aggtggaggg 660
tcagtcgagc cagtaccccc aggagaagat tccaaagatg tagccgcccc acacagacag 720
ccactcacct cttcagaacg aattgacaaa caaattcggt acatcctcga cggcatctca 780
gccctgagaa aggagacatg taacaagagt aacatgtgtg aaagcagcaa agaggcactg 840
gcagaaaaca acctgaacct tccaaagatg gctgaaaaag atggatgctt ccaatctgga 900
ttcaatgagg agacttgcct ggtgaaaatc atcactggtc ttttggagtt tgaggtatac 960
ctagagtacc tccagaacag atttgagagt agtgaggaac aagccagagc tgtgcagatg 1020
agtacaaaag tcctgatcca gttcctgcag aaaaaggcaa agaatctaga tgcaataacc 1080
acccctgacc caaccacaaa tgccagcctg ctgacgaagc tgcaggcaca gaaccagtgg 1140
ctgcaggaca tgacaactca tctcattctg cgcagcttta aggagttcct gcagtccagc 1200
ctgagggctc ttcggcaaat g 1221
<210> 7
<211> 294
<212> DNA
<213> 人工序列
<400> 7
atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60
agtggcacca atgatgctga agactgctgc ctgtctgtga cccagaaacc catccctggg 120
tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180
gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240
cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagt 294

Claims (18)

1.一种第四代CAR-T细胞，其特征在于，所述第四代CAR-T细胞表达特异性结合抗原的嵌合抗原受体、复合型IL-6和CCL19；
所述复合型IL-6和CCL19为NFAT调控型过表达；
所述复合型IL-6为IL-6和IL-6Rα的复合物，所述复合型IL-6为SEQ ID NO:1所示的氨基酸序列；
所述CCL19为SEQ ID NO:2所示的氨基酸序列；
所述嵌合抗原受体包括抗原结合结构域、跨膜结构域和信号传导结构域；
所述抗原结合结构域为靶向Mesothelin的单链抗体；
所述靶向Mesothelin的单链抗体为SEQ ID NO:3所示的氨基酸序列中的第21位到第287位氨基酸。
2.根据权利要求1所述的第四代CAR-T细胞，其特征在于，所述跨膜结构域包括CD28跨膜结构域和/或CD8α跨膜结构域。
3.根据权利要求2所述的第四代CAR-T细胞，其特征在于，所述跨膜结构域为CD28跨膜结构域。
4.根据权利要求1所述的第四代CAR-T细胞，其特征在于，所述信号传导结构域包括CD28胞内结构域、CD3ζ、TLR2、4-1BB、TLR1、CD27、OX40或DAP10中的任意一种或至少两种的组合。
5.根据权利要求4所述的第四代CAR-T细胞，其特征在于，所述信号传导结构域为CD28胞内结构域、CD3ζ和TLR2的组合。
6.根据权利要求1所述的第四代CAR-T细胞，其特征在于，所述嵌合抗原受体还包括信号肽。
7.根据权利要求1所述的第四代CAR-T细胞，其特征在于，所述嵌合抗原受体由GM-CSF信号肽、Mesothelin抗原结合结构域、CD28跨膜结构域、CD28胞内结构域、CD3ζ和TLR2串联组成。
8.根据权利要求7所述的第四代CAR-T细胞，其特征在于，所述嵌合抗原受体包括如SEQID NO:3所示的氨基酸序列。
9.一种表达载体，其特征在于，所述表达载体包括嵌合抗原受体编码基因、NFAT调控启动子、复合型IL-6编码基因和CCL19编码基因；所述嵌合抗原受体编码基因为SEQ ID NO:4所示的核酸序列；
所述NFAT调控启动子为SEQ ID NO:5所示的核酸序列；
所述复合型IL-6编码基因为SEQ ID NO:6所示的核酸序列；
所述CCL19编码基因为SEQ ID NO:7所示的核酸序列。
10.根据权利要求9所述的表达载体，其特征在于，所述表达载体包括病毒载体。
11.根据权利要求10所述的表达载体，其特征在于，所述病毒载体包括慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。
12.根据权利要求11所述的表达载体，其特征在于，所述病毒载体为慢病毒载体。
13.一种重组慢病毒，其特征在于，所述重组慢病毒采用权利要求9-12中任一项所述的表达载体与包装辅助质粒共转染哺乳细胞制备得到。
14.一种权利要求1-8任一项所述的第四代CAR-T细胞的制备方法，其特征在于，所述方法包括将权利要求9-12中任一项所述的表达载体或权利要求13所述的重组慢病毒导入T细胞的步骤。
15.一种药物组合物，其特征在于，所述药物组合物包括权利要求1-8任一项所述的第四代CAR-T细胞。
16.根据权利要求15所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
17.权利要求1-8任一项所述的第四代CAR-T细胞、权利要求9-12中任一项所述的表达载体、权利要求14所述的重组慢病毒或权利要求15或16所述的药物组合物在制备肿瘤治疗药物中的应用。
18.根据权利要求17所述的应用，其特征在于，所述肿瘤包括肝癌、肺癌、乳腺癌、肾母细胞瘤、神经胶质瘤、神经母细胞瘤、黑色素瘤、鼻咽癌、间皮质瘤、胰岛细胞瘤、视网膜母细胞瘤、胰腺癌、子宫肌瘤、宫颈癌或甲状腺癌中的任意一种或至少两种的组合。
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