CN117567651A - 协同表达血管内皮黏附分子vcam1的嵌合抗原受体及其应用 - Google Patents
协同表达血管内皮黏附分子vcam1的嵌合抗原受体及其应用 Download PDFInfo
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Abstract
本发明涉及免疫疗法和基因工程技术领域,具体涉及一种协同表达血管内皮黏附分子VCAM1的嵌合抗原受体及其应用。本发明将血管内皮黏附分子VCAM1与抗CDH17的CAR分子构建到同一个慢病毒载体上,并使用可剪接的2A linker使两者相连,使得T细胞既表达抗CDH17的CAR,也表达血管内皮黏附分子VCAM1。通过抗CDH17的CAR‑T细胞共表达血管内皮黏附分子VCAM1,显著增强了CAR‑T的迁移能力,并帮助CAR‑T细胞向实体瘤迁移。因此,本发明的CAR‑T细胞对实体瘤展现出更强的杀伤功能。
Description
技术领域
本发明涉及免疫疗法和基因工程技术领域,更具体地说,它涉及一种协同表达血管内皮黏附分子VCAM1的嵌合抗原受体及其应用。
背景技术
CAR-T细胞疗法,即嵌合抗原受体T细胞疗法,是一种新型的细胞疗法,为许多传统疗法无法治疗的疾病带来了希望。然而,CAR-T在治疗实体肿瘤时仍面临一些挑战。首先,实体肿瘤具有复杂的微环境,包括免疫抑制因子、肿瘤细胞的抗原逃逸等,这使得CAR-T细胞难以有效地在肿瘤部位发挥作用。其次,CAR-T细胞的持久性和扩增能力也是关键因素,因为它们直接影响到CAR-T细胞对肿瘤的杀伤效果。此外,如何将CAR-T细胞高效地输送到肿瘤部位也是一个难点,因为现有的输送方法往往不能完全保证CAR-T细胞在肿瘤部位富集。
CDH17是一种在结肠癌中高表达的钙黏蛋白,研究表明其在肿瘤的发生和发展中起到了关键作用。因此,CDH17成为了一个理想的CAR-T靶点。通过靶向CDH17,CAR-T细胞可以更有效地识别和杀伤表达CDH17的结肠癌细胞,从而达到治疗的目的。近年来,针对CDH17的CAR-T细胞疗法已经在临床试验中取得了显著的成果。在一项早期临床试验中,研究人员对一部分结肠癌患者进行了CDH17 CAR-T细胞疗法,结果显示,部分患者的肿瘤明显缩小,生活质量得到显著改善。这一结果证明了CDH17 CAR-T细胞疗法的有效性和安全性。
血管内皮黏附分子VCAM1是一种在血管内皮细胞上表达的糖蛋白,它在免疫应答和炎症反应中起着关键作用。VCAM1可以与淋巴细胞表面的VLA-4受体相互作用,引导淋巴细胞穿越血管内皮层,从而到达炎症部位。这一特性使得VCAM1成为了一个有潜力的CAR-T细胞共表达分子,帮助CAR-T细胞在实体肿瘤中迁移和发挥杀伤作用。
综上所述,为了增强CAR-T细胞(即嵌合抗原受体T细胞)在实体肿瘤中的迁移能力和杀伤效果,本发明将VCAM1与CAR-T细胞共表达。通过基因工程技术,制备出同时表达CDH17受体和VCAM1的CAR-T细胞。通过靶向CDH17并共表达VCAM1,CAR-T细胞在治疗实体肿瘤方面展示了显著的优势。这种策略不仅能增强CAR-T细胞的迁移能力,还能帮助它们更有效地在实体肿瘤中发挥杀伤作用。未来的研究将进一步优化这种治疗方法,以期能在临床试验中取得更好的效果,为结肠癌患者提供一种更有效的治疗选择。
发明内容
本发明的目的是提供一种协同表达血管内皮黏附分子VCAM1的嵌合抗原受体及其应用。本发明通过将血管内皮黏附分子VCAM1与抗CDH17的CAR分子构建到同一个慢病毒载体上,中间用可剪接的2A linker相连,使得T细胞既表达抗CDH17的CAR(即嵌合抗原受体),也表达血管内皮黏附分子VCAM1。
抗CDH17的CAR-T细胞共表达血管内皮黏附分子VCAM1后,显著增强了CAR-T的迁移能力,并帮助CAR-T细胞向实体瘤迁移,从而可以对实体瘤展现出更强的杀伤功能。
本发明的上述技术目的是通过以下技术方案得以实现的:
一种协同表达血管内皮黏附分子VCAM1的嵌合抗原受体,所述的嵌合抗原受体包括有SEQ ID No:1所示的氨基酸序列和SEQ ID No:2所示的氨基酸序列。
优选地,SEQ ID No:1所示的氨基酸序列与SEQ ID No:2所示的氨基酸序列之间使用可剪接的2A linker相连。
优选地,所述的嵌合抗原受体具有下式I结构:
L-CDH17 scFv-H-TM-C-CD3ζ-2A-VCAM1(I)
式中,
各“-”为连接肽或肽键;L为信号肽序列;CDH17 scFv为针对CDH17肿瘤抗原的抗体,所述CDH17 scFv的氨基酸序列如SEQ ID NO:1所示;H为铰链区序列;TM为跨膜区序列;C为共刺激域序列;CD3ζ为信号传导域序列。
一种核酸分子,其编码上述的嵌合抗原受体。
优选地,所述的核酸分子包括有SEQ ID No:5所示的核苷酸序列和SEQ ID No:6所示的核苷酸序列。
一种载体,其包括有上述的核酸分子。
优选地,所述的载体选自DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子中的一种或多种组合。
一种细胞,其包括有上述的载体、或染色体中整合有外源的上述的核酸分子、或表达上述的嵌合抗原受体。
优选地,所述细胞为T细胞。
一种制备协同表达血管内皮黏附分子VCAM1的CAR-T细胞的方法,其包括如下步骤:将血管内皮黏附分子VCAM1与抗CDH17的CAR分子构建到同一个慢病毒载体上,并使用可剪接的2A linker使两者相连,然后将该慢病毒载体转染到T细胞中;所述血管内皮黏附分子VCAM1包括有如SEQ ID No:2所示的氨基酸序列;所述抗CDH17的CAR分子包括有如SEQ IDNo:1所示的氨基酸序列。
综上所述,本发明具有以下有益效果:本发明制备的协同表达血管内皮黏附分子VCAM1的CAR-T细胞不仅可以提高对实体瘤的治疗效果,还可以增强CAR-T细胞的迁移能力,有助于减少肿瘤转移和复发的风险。此外,本发明还提供了该协同表达血管内皮黏附分子VCAM1的CAR-T细胞在治疗其他免疫相关疾病中的应用,拓展了CAR-T细胞疗法的应用范围。
附图说明
图1是本发明中CDH17的结构示意图;
图2是本发明中CAR的结构示意图;
图3是本发明中CAR-T的扩增倍数图;
图4是本发明中流式检测CAR-T细胞的表达情况图I;
图5是本发明中流式检测CAR-T细胞的表达情况图II;
图6 是本发明中Transwell评估CAR-T的迁移情况图(***P≤0.001);
图7是本发明中CAR-T的杀伤率检测图(*P≤0.05)。
具体实施方式
为使本发明的目的、特征和优点能够更加明显易懂,下面结合附图对本发明的具体实施方式做详细的说明。附图中给出了本发明的若干实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。
实施例1:嵌合抗原受体的设计
本实施例构建了抗CDH17的嵌合抗原受体(CDH17 CAR)、共表达血管内皮黏附分子VCAM1的抗CDH17的(CDH17-VCAM1 CAR),其结构见图1-图2。嵌合抗原受体包括一段CD8α的信号肽序列(Leader),抗CDH17的单链抗体序列(anti-CDH17scFv),CD8α的铰链区(Hinge)和跨膜区序列(Transmembrane),4-1BB共刺激域序列和CD3ζ信号传导域序列,其结构见图2。
其中抗人CDH17抗体序列(Anti-CDH17 scFv)的氨基酸序列来源于专利WO2023107558A1中07-0663-h7scfv序列(SEQ ID No:1),具体如下所示:
DIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSS
VCAM1氨基酸序列(SEQ ID NO:2),具体如下所示:
MPGKMVVILGASNILWIMFAASQAFKIETTPESRYLAQIGDSVSLTCSTTGCESPFFSWRTQIDSPLNGKVTNEGTTSTLTMNPVSFGNEHSYLCTATCESRKLEKGIQVEIYSFPKDPEIHLSGPLEAGKPITVKCSVADVYPFDRLEIDLLKGDHLMKSQEFLEDADRKSLETKSLEVTFTPVIEDIGKVLVCRAKLHIDEMDSVPTVRQAVKELQVYISPKNTVISVNPSTKLQEGGSVTMTCSSEGLPAPEIFWSKKLDNGNLQHLSGNATLTLIAMRMEDSGIYVCEGVNLIGKNRKEVELIVQEKPFTVEISPGPRIAAQIGDSVMLTCSVMGCESPSFSWRTQIDSPLSGKVRSEGTNSTLTLSPVSFENEHSYLCTVTCGHKKLEKGIQVELYSFPRDPEIEMSGGLVNGSSVTVSCKVPSVYPLDRLEIELLKGETILENIEFLEDTDMKSLENKSLEMTFIPTIEDTGKALVCQAKLHIDDMEFEPKQRQSTQTLYVNVAPRDTTVLVSPSSILEEGSSVNMTCLSQGFPAPKILWSRQLPNGELQPLSENATLTLISTKMEDSGVYLCEGINQAGRSRKEVELIIQVTPKDIKLTAFPSESVKEGDTVIISCTCGNVPETWIILKKKAETGDTVLKSIDGAYTIRKAQLKDAGVYECESKNKVGSQLRSLTLDVQGRENNKDYFSPELLVLYFASSLIIPAIGMIIYFARKANMKGSYSLVEAQKSKV
Anti-CDH17 CAR氨基酸序列(SEQ ID NO:3),具体如下所示:
MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
共表达血管内皮黏附分子VCAM1的抗CDH17的(CDH17-VCAM1 CAR)氨基酸序列(SEQID NO:4),具体如下所示:MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRVSSIISSSKLHWYQQKPGKAPKPLIYGTSTLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSNYPFTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKISCKVSGYTFTDHTIHWMRQAPGQGLEWIGYIFPRDDIVVYAQKFQGRATLTADKSTSTAYMELSSLRSEDTAVYYCARPPYYYSRNFYFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GSGATNFSLLKQAGDVEENPGPMPGKMVVILGASNILWIMFAASQAFKIETTPESRYLAQIGDSVSLTCSTTGCESPFFSWRTQIDSPLNGKVTNEGTTSTLTMNPVSFGNEHSYLCTATCESRKLEKGIQVEIYSFPKDPEIHLSGPLEAGKPITVKCSVADVYPFDRLEIDLLKGDHLMKSQEFLEDADRKSLETKSLEVTFTPVIEDIGKVLVCRAKLHIDEMDSVPTVRQAVKELQVYISPKNTVISVNPSTKLQEGGSVTMTCSSEGLPAPEIFWSKKLDNGNLQHLSGNATLTLIAMRMEDSGIYVCEGVNLIGKNRKEVELIVQEKPFTVEISPGPRIAAQIGDSVMLTCSVMGCESPSFSWRTQIDSPLSGKVRSEGTNSTLTLSPVSFENEHSYLCTVTCGHKKLEKGIQVELYSFPRDPEIEMSGGLVNGSSVTVSCKVPSVYPLDRLEIELLKGETILENIEFLEDTDMKSLENKSLEMTFIPTIEDTGKALVCQAKLHIDDMEFEPKQRQSTQTLYVNVAPRDTTVLVSPSSILEEGSSVNMTCLSQGFPAPKILWSRQLPNGELQPLSENATLTLISTKMEDSGVYLCEGINQAGRSRKEVELIIQVTPKDIKLTAFPSESVKEGDTVIISCTCGNVPETWIILKKKAETGDTVLKSIDGAYTIRKAQLKDAGVYECESKNKVGSQLRSLTLDVQGRENNKDYFSPELLVLYFASSLIIPAIGMIIYFARKANMKGSYSLVEAQKSKV
Anti-CDH17 scFv核苷酸序列(SEQ ID NO:5),具体如下所示:
GATATACAAATGACACAAAGCCCATCTTCACTTAGCGCTTCAGTCGGGGATAGGGTGACTATCACTTGCCGGGTAAGCTCCATTATAAGCAGCTCCAAGTTGCACTGGTACCAGCAGAAACCCGGCAAGGCGCCAAAACCGCTTATTTACGGGACTTCCACACTGGCAAGTGGAGTGCCCTCCCGGTTTAGTGGCTCTGGCTCTGGCACTGACTACACTCTGACCATCTCTTCACTCCAGCCTGAGGATTTCGCTACTTACTACTGCCAGCAGTGGAGCAACTACCCATTCACCTTTGGTCAGGGGACAAAGCTCGAAATCAAAGGAGGCGGCGGGTCAGGGGGCGGAGGATCTGGAGGGGGTGGTTCTCAGGTCCAACTCGTGCAAAGCGGAGCCGAGGTAAAGAAGCCAGGATCTAGTGTCAAGATCTCATGCAAGGTATCTGGCTACACGTTTACAGACCACACTATACACTGGATGAGGCAGGCTCCTGGGCAGGGACTCGAATGGATTGGCTATATATTTCCACGGGACGACATCGTGGTCTACGCCCAGAAGTTTCAGGGAAGGGCTACACTTACTGCCGATAAGTCAACGTCCACCGCCTATATGGAGCTGTCCTCCCTCAGGAGCGAGGACACAGCAGTCTACTATTGCGCCCGACCCCCATACTACTACTCTCGGAATTTCTACTTTGATTATTGGGGACAGGGGACAACTCTTACCGTGAGTAGT
VCAM1核苷酸序列(SEQ ID NO:6),具体如下所示:
ATGCCTGGGAAAATGGTTGTCATTTTGGGAGCAAGTAATATCCTGTGGATCATGTTTGCCGCCTCACAGGCTTTTAAGATCGAGACCACACCAGAATCCAGGTACTTGGCCCAGATTGGTGATAGCGTCAGCCTTACATGCTCCACAACCGGGTGTGAGAGCCCTTTCTTTTCTTGGCGGACCCAAATCGATTCTCCTCTGAATGGGAAGGTCACTAATGAGGGAACGACCAGCACCCTGACTATGAACCCCGTGAGCTTCGGCAATGAACATAGCTATCTTTGTACTGCAACATGTGAGTCACGAAAGTTGGAGAAGGGCATCCAAGTCGAGATCTATTCCTTCCCCAAGGACCCTGAGATCCATTTGAGCGGTCCTTTGGAGGCCGGAAAGCCCATAACCGTAAAGTGCAGCGTGGCTGATGTGTATCCATTTGACCGGCTGGAAATCGACCTTCTCAAGGGAGACCACTTGATGAAGTCTCAAGAATTTCTTGAAGACGCCGACAGAAAGAGCCTGGAGACTAAAAGTCTTGAGGTGACCTTTACTCCCGTCATCGAAGATATTGGCAAAGTGCTGGTCTGTCGGGCCAAACTGCACATCGACGAAATGGACTCAGTGCCAACCGTTCGGCAAGCAGTAAAAGAGCTCCAGGTGTACATCAGTCCGAAGAACACCGTGATCTCAGTTAACCCTTCCACAAAGCTGCAAGAGGGAGGCTCAGTGACTATGACTTGTTCCAGCGAAGGCCTGCCTGCACCCGAGATCTTCTGGTCCAAAAAGCTGGACAACGGTAATTTGCAACATCTGTCAGGTAACGCTACGCTGACCCTTATCGCAATGAGGATGGAGGATAGTGGGATCTACGTGTGCGAGGGAGTGAATCTGATAGGAAAAAATAGAAAGGAGGTGGAGCTGATAGTGCAGGAGAAGCCCTTTACAGTCGAGATTTCACCAGGCCCCAGGATTGCTGCTCAGATCGGGGATTCTGTGATGTTGACTTGCAGTGTTATGGGGTGTGAGTCCCCTAGCTTCTCATGGCGAACCCAGATTGACAGTCCACTGAGTGGGAAAGTGCGGAGCGAAGGTACGAACTCAACACTGACTCTTTCTCCCGTGTCCTTCGAGAATGAACACTCATATCTGTGTACTGTTACCTGCGGCCATAAGAAGCTCGAGAAGGGCATACAGGTGGAGCTGTATTCCTTCCCTCGCGACCCCGAAATTGAGATGTCTGGCGGACTTGTGAATGGAAGTAGTGTGACAGTTAGTTGTAAGGTGCCTAGCGTGTACCCACTCGATAGACTCGAGATAGAGCTTCTGAAGGGAGAGACTATTCTGGAGAACATCGAGTTCCTGGAGGACACCGACATGAAAAGCCTCGAGAACAAGTCCCTCGAGATGACATTCATCCCAACCATAGAGGATACCGGGAAGGCACTCGTGTGTCAGGCTAAACTTCACATTGACGATATGGAGTTCGAGCCCAAACAGAGACAGAGTACTCAGACATTGTATGTAAATGTGGCCCCTCGAGATACTACAGTACTGGTTAGCCCTTCATCTATCTTGGAGGAGGGTAGCAGCGTGAATATGACCTGTCTCAGCCAAGGATTCCCTGCCCCTAAAATCCTGTGGTCACGACAGCTCCCCAACGGTGAGCTCCAACCTCTTTCTGAGAATGCAACGTTGACTCTCATCAGCACTAAGATGGAAGACAGCGGGGTTTACCTGTGTGAAGGCATCAATCAGGCGGGAAGATCCCGCAAAGAAGTGGAACTCATTATCCAGGTCACTCCTAAGGACATAAAGCTCACCGCCTTTCCAAGTGAGTCAGTGAAGGAAGGAGATACAGTGATTATTTCTTGCACATGCGGCAACGTCCCCGAGACATGGATCATCCTCAAAAAGAAAGCCGAGACAGGTGATACTGTGCTGAAATCCATTGACGGCGCCTACACTATAAGGAAGGCCCAGCTGAAAGATGCAGGCGTATACGAGTGTGAGTCTAAGAACAAAGTCGGGTCACAGTTGAGGTCTTTGACACTGGATGTGCAGGGCAGGGAAAATAACAAGGATTACTTCAGCCCTGAGTTGCTGGTTCTCTACTTTGCTAGTTCCCTGATTATACCAGCCATAGGTATGATCATCTATTTTGCCAGGAAAGCTAATATGAAGGGGTCTTACAGCTTGGTGGAGGCACAGAAATCTAAAGTG
Anti-CDH17 CAR核苷酸序列(SEQ ID NO:7),具体如下所示:
ATGGCCCTGCCCGTTACAGCTCTGTTGTTGCCCCTGGCACTCCTGTTGCATGCCGCCAGACCTGATATTCAGATGACACAGTCACCCAGTAGCCTGTCTGCCAGTGTGGGTGACAGGGTGACCATCACCTGTAGAGTGAGCAGTATCATATCATCATCTAAGCTGCATTGGTACCAGCAAAAACCAGGAAAGGCCCCCAAGCCACTGATTTACGGAACATCAACCCTCGCCTCCGGCGTGCCCTCTAGGTTTTCAGGATCTGGTAGCGGCACTGACTACACTCTGACTATCAGCTCCCTGCAGCCCGAGGATTTTGCAACATATTACTGTCAGCAATGGAGCAACTATCCCTTTACTTTCGGGCAGGGCACTAAGCTCGAGATAAAAGGTGGAGGTGGTTCAGGTGGGGGAGGTTCAGGAGGGGGAGGTTCACAGGTGCAGCTCGTTCAGTCAGGAGCCGAGGTGAAAAAGCCCGGCTCCAGCGTGAAAATTTCATGCAAAGTCAGTGGCTACACTTTCACCGATCATACCATACACTGGATGAGACAGGCTCCCGGACAGGGACTGGAGTGGATCGGTTATATATTTCCTCGCGACGACATCGTGGTTTATGCACAGAAGTTCCAAGGCAGGGCTACCCTGACCGCAGACAAGTCCACCTCTACCGCCTACATGGAACTCAGCTCTCTCAGGAGTGAGGACACGGCTGTATACTATTGCGCCCGCCCTCCCTATTACTATAGCAGGAACTTTTACTTTGATTATTGGGGCCAGGGGACAACATTGACCGTGAGTAGCACCACAACCCCAGCTCCTAGACCTCCAACCCCAGCTCCAACAATAGCTAGTCAGCCACTGAGTCTCAGACCTGAGGCTTGTAGGCCAGCGGCAGGAGGGGCAGTGCATACCAGAGGCCTGGATTTCGCCTGCGACATTTACATCTGGGCGCCTTTGGCGGGAACCTGCGGAGTGCTCTTGCTGAGCCTCGTGATTACTTTGTACTGCAAGCGCGGAAGGAAGAAGCTGCTGTATATCTTCAAGCAGCCATTCATGAGACCGGTTCAGACGACCCAGGAGGAAGATGGTTGCTCCTGTCGCTTCCCCGAGGAAGAAGAAGGAGGCTGCGAGTTGCGCGTGAAATTCAGTAGGTCTGCAGACGCTCCAGCCTACCAACAGGGGCAAAACCAGTTGTATAATGAGCTCAACCTCGGGAGGAGAGAAGAATACGATGTGCTGGATAAACGGAGAGGGCGCGATCCTGAAATGGGAGGAAAACCTAGGCGCAAGAATCCCCAGGAAGGCCTCTACAACGAACTGCAGAAGGATAAGATGGCCGAGGCATACTCAGAAATAGGCATGAAGGGAGAACGAAGACGGGGCAAGGGGCATGATGGCCTGTATCAAGGCCTGTCTACCGCAACTAAGGATACCTACGATGCGCTCCATATGCAGGCCCTTCCCCCTCGG
共表达血管内皮黏附分子VCAM1的抗CDH17的(CDH17-VCAM1 CAR)核苷酸序列(SEQID NO:8),具体如下所示:ATGGCCTTGCCCGTGACCGCTCTCTTGCTCCCCCTTGCATTGCTCCTCCACGCCGCCAGGCCAGACATTCAGATGACCCAATCACCCTCTAGCCTGAGCGCTAGTGTTGGCGATAGGGTCACCATCACGTGTCGGGTGAGCTCTATCATCTCTTCATCCAAGCTGCACTGGTACCAACAGAAGCCGGGCAAGGCCCCTAAGCCTCTGATATATGGTACATCCACTCTGGCTAGCGGGGTGCCATCTAGGTTTTCCGGCTCCGGGAGTGGGACAGATTACACCCTGACCATATCAAGCCTCCAGCCAGAGGACTTTGCTACATACTACTGCCAGCAGTGGAGCAACTACCCGTTTACATTCGGACAGGGGACGAAACTCGAAATCAAGGGAGGCGGCGGATCTGGAGGAGGGGGAAGTGGTGGGGGCGGTTCTCAGGTCCAATTGGTACAATCTGGCGCTGAAGTGAAGAAACCCGGAAGCAGTGTCAAGATTAGCTGCAAAGTCTCAGGCTACACCTTTACTGACCACACTATCCACTGGATGAGGCAGGCCCCCGGACAGGGATTGGAGTGGATCGGGTACATATTTCCTCGAGATGACATCGTGGTGTATGCTCAGAAATTTCAGGGCAGGGCTACCCTGACGGCTGATAAAAGCACAAGCACAGCCTATATGGAACTTAGTTCACTTAGGAGTGAGGACACCGCCGTGTATTATTGCGCGCGCCCCCCTTACTACTACAGCCGGAACTTTTACTTCGACTATTGGGGGCAGGGCACAACACTGACCGTCTCTTCCACCACAACACCCGCTCCTCGGCCTCCTACACCTGCTCCAACCATAGCGAGTCAACCGCTGTCTCTCAGGCCAGAGGCCTGTAGACCCGCTGCAGGAGGGGCTGTGCATACTCGGGGCCTGGATTTTGCATGCGATATATACATCTGGGCCCCTTTGGCTGGTACATGTGGGGTGCTCTTGCTGAGTCTGGTGATTACCCTGTACTGCAAACGGGGACGAAAAAAGCTGCTGTACATCTTCAAGCAACCGTTTATGCGCCCCGTGCAGACAACCCAGGAGGAGGATGGTTGTAGCTGTCGCTTCCCCGAGGAAGAGGAGGGTGGTTGTGAATTGAGGGTGAAATTTAGCAGGTCCGCCGATGCACCTGCGTACCAACAGGGGCAAAACCAGCTGTACAACGAATTGAATCTGGGACGCCGCGAAGAGTATGACGTGCTTGACAAACGAAGGGGACGAGACCCAGAAATGGGCGGCAAACCCAGAAGGAAGAACCCCCAGGAGGGCCTGTACAACGAGTTGCAGAAGGACAAAATGGCCGAAGCCTACTCCGAGATCGGCATGAAGGGCGAAAGACGACGCGGCAAAGGTCACGACGGACTTTACCAAGGGCTCAGCACCGCCACAAAGGATACTTATGACGCCCTCCACATGCAGGCCCTTCCTCCCAGAGGAAGCGGGGCAACTAATTTCTCTCTTCTCAAGCAGGCCGGTGACGTTGAGGAGAACCCTGGCCCTATGCCCGGTAAAATGGTTGTCATACTGGGCGCTTCTAATATCCTGTGGATCATGTTTGCAGCAAGCCAAGCCTTCAAGATTGAAACGACCCCCGAATCTCGGTACCTGGCGCAGATCGGAGATTCTGTGTCTCTGACTTGTAGTACAACCGGGTGTGAGAGCCCCTTTTTTAGCTGGCGCACTCAGATCGACAGCCCCTTGAATGGGAAGGTAACCAACGAAGGGACTACGTCAACACTGACAATGAACCCAGTGTCTTTCGGCAATGAGCATTCTTACCTCTGCACAGCAACTTGTGAGAGCAGGAAATTGGAAAAGGGTATTCAGGTGGAGATATATAGCTTCCCGAAAGACCCAGAAATCCACCTGTCCGGGCCTCTCGAGGCTGGAAAGCCCATCACGGTCAAGTGTTCAGTTGCTGACGTCTATCCCTTCGACAGACTGGAAATCGATTTGCTTAAGGGAGATCACCTGATGAAAAGCCAGGAGTTTCTTGAGGATGCGGACCGAAAGAGCCTGGAAACAAAAAGCCTGGAGGTCACTTTTACTCCAGTCATTGAGGACATCGGAAAAGTTCTGGTGTGTAGAGCGAAACTGCACATAGACGAAATGGACTCCGTGCCAACTGTGAGGCAAGCTGTCAAAGAGCTCCAAGTGTACATATCTCCAAAAAACACCGTTATTTCCGTGAACCCCAGTACCAAACTGCAAGAAGGTGGTAGCGTTACCATGACTTGTTCCTCTGAAGGCCTGCCAGCCCCCGAGATATTTTGGAGTAAGAAACTGGATAATGGCAATCTGCAGCACCTTAGCGGCAATGCGACACTCACACTTATTGCGATGAGAATGGAGGACTCAGGCATCTATGTCTGCGAAGGCGTGAATCTCATTGGGAAGAACAGGAAAGAGGTTGAGCTGATCGTGCAAGAAAAGCCCTTTACCGTTGAAATCTCCCCCGGCCCTAGAATCGCGGCTCAAATCGGCGACTCAGTCATGCTGACATGCAGCGTGATGGGCTGTGAGTCTCCTTCTTTCTCTTGGCGAACCCAAATTGACTCTCCCCTCAGCGGTAAGGTGAGGTCTGAGGGTACCAATTCCACACTCACACTGAGTCCAGTCTCATTTGAGAACGAACATTCCTATCTGTGCACTGTCACTTGTGGGCATAAAAAATTGGAGAAGGGTATTCAGGTTGAACTCTACAGCTTTCCACGCGACCCTGAGATCGAGATGTCAGGAGGTCTCGTTAACGGTTCCTCTGTCACTGTGTCATGCAAGGTGCCATCCGTGTATCCTCTTGACCGCCTGGAAATCGAACTTTTGAAGGGCGAAACGATTCTTGAGAACATTGAGTTCCTGGAAGATACCGACATGAAGTCATTGGAGAACAAATCCCTGGAAATGACTTTTATTCCCACCATTGAGGATACAGGAAAGGCCCTGGTCTGTCAGGCCAAACTTCATATCGATGACATGGAGTTCGAACCCAAGCAGAGGCAGTCCACCCAAACTTTGTATGTCAACGTGGCTCCAAGAGACACCACGGTACTGGTGAGCCCAAGTTCTATCCTCGAAGAGGGGAGCTCCGTCAACATGACTTGCCTCAGCCAGGGGTTCCCCGCTCCTAAGATCTTGTGGAGCAGGCAGCTGCCCAATGGAGAGCTCCAGCCTCTGTCCGAGAACGCCACTCTGACACTGATCTCCACCAAAATGGAGGACTCAGGTGTGTACCTTTGCGAGGGCATTAATCAGGCCGGACGATCTCGGAAAGAGGTAGAACTCATCATACAAGTCACACCTAAAGACATAAAATTGACCGCCTTTCCTTCCGAGTCCGTTAAAGAGGGGGACACTGTGATCATCTCTTGCACATGTGGCAACGTTCCAGAAACCTGGATCATCCTGAAGAAGAAGGCCGAGACTGGTGACACAGTGCTGAAGAGTATTGATGGGGCTTACACCATCAGGAAAGCTCAGCTGAAAGACGCCGGAGTCTATGAGTGCGAGAGTAAGAATAAGGTTGGGTCACAACTGAGGTCACTCACGCTGGACGTCCAGGGGCGCGAAAACAACAAAGATTATTTCTCACCCGAGTTGCTGGTGCTGTACTTCGCTAGCAGTTTGATCATACCTGCCATTGGCATGATCATCTATTTCGCCAGGAAAGCCAACATGAAGGGCTCATATTCCCTTGTGGAGGCTCAGAAATCCAAAGTC
具体各部分序列如下:
L(leader)为选自下组的蛋白的信号肽:CD8、GM-CSF、CD4、CD137、或其组合;优选为CD8α信号肽,其氨基酸序列(SEQ ID NO:40)为:MALPVTALLLPLALLLHAARP。
H(Hinge)为选自下组的蛋白的铰链区:CD8、CD28、CD137、或其组合;优选为CD8α铰链区,其氨基酸序列(SEQ ID NO:42)为:TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。
TM为选自下组的蛋白的跨膜区:CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154;优选为CD8α跨膜区,其氨基酸序列(SEQ ID NO:44)为:IYIWAPLAGTCGVLLLSLVITLYC。
C为选自下组的共刺激域:CCD28、4-1BB、GITR、ICOS-1、CD27、OX-40、DAP10;优选为4-1BB,其氨基酸序列(SEQ ID NO:46)为:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。
CD3ζ信号传导域的氨基酸序列(SEQ ID NO:48)为:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
2A linker为2A肽,其氨基酸序列包括但不限于以下几种:
P2A (e.g., sequence: ATNFSLLKQAGDVEENPGP)、
E2A (e.g., sequence: QCTNYALLKLAGDVESNPGP)、
F2A (e g., sequence: VKQTLNFDLLKLAGDVESNPGP)、
T2A (e g., sequence :EGRGSLLTCGDVEENPGP)。
实施例2:构建的嵌合抗原受体表达载体
(1)根据CAR基因的蛋白理论序列,优化CAR基因,使其能够在人细胞中高效表达,通过密码子优化及全基因合成方法制备CAR基因,进行全基因合成;
(2)用EcoRI和BamHI双酶切全基因合成的CAR基因和空载体pCDH-EF1-MCS,于37℃水浴中酶切30min后,使用1.5%的琼脂糖凝胶进行DNA电泳,然后使用天根的琼脂糖凝胶试剂盒纯化回收处理;
(3)pCDH-EF1-MCS载体与CAR基因片段的连接,连接体系如表1所示:
表1 pCDH-EF1-MCS载体与CAR基因片段的连接体系表
组件 | 添加量(μl) |
pCDH-EF1-MCS载体 | 2(50ng) |
CAR基因 | 10(150ng) |
T4 DNA 连接缓冲液 | 2 |
T4 DNA 连接酶(NEB) | 1 |
dd H2O | 5 |
总共 | 20 |
于22℃连接1h,连接产物直接转化Stbl3大肠杆菌感受态细胞,取200μl转化产物涂布氨苄抗性的LB平板,LB平板于37℃的培养箱中倒置培养过夜。次日早晨随机挑选3个单克隆进行菌落PCR鉴定,将阳性克隆送样测序。
实施例3:慢病毒包装
分别对实施例中的慢病毒表达载体进行慢病毒包装,采用四质粒系统,具体步骤如下:
(1)四质粒系统分别表达慢病毒载体包装所需的gag/pol、Rev、VSV-G及本发明构建的CAR表达载体:将四质粒进行瞬时转染293T细胞,DNA含量为2μg/mL;
(2)将上述质粒与PEI转染试剂混合,加入至一定体积的无血清的DMEM中,混匀后放置15分钟,将上述混合液加入至铺有293T细胞的T75培养瓶中,轻轻混匀,于37℃、5%CO2细胞培养箱培养6h;
(3)6h后更换新鲜培养基,继续进行培养,并且加入10mM的丁酸钠溶液,72小时后收集慢病毒的培养上清进行纯化检测。
实施例4:慢病毒感染T细胞
分离PBMC后,用含50ng/mL的OKT3,300IU/ml的IL-2的X-VIVO进行激活,2日后将培养基更换成含300IU/mL的X-VIVO进行扩大培养;利用RetroNectin提高慢病毒对T细胞的感染效率,将30μg的RetroNectin,包被于6孔板内,放于37℃细胞培养箱2h;吸取RetroNectin,利用含2.5%BSA的Hank’s溶液封闭包被后的6孔板,放于37℃细胞培养箱0.5h;吸取封闭液,利用含2%Hepes的Hank’s溶液洗涤6孔板,加入X-VIVO培养基,加入适量的慢病毒溶液,2000×g,离心2h;弃上清,加入1×106的激活后的PBMC细胞,1000×g,离心10min,于37℃、5%CO2和一定湿度的细胞培养箱内培养。每两天进行一次计数并更换含300IU/mL的X-VIVO,并且将细胞浓度维持0.5×106-1×106/mL,连续培养8天。用CountstarIC1000 自动细胞计数仪计数,评估CAR-T细胞的扩增情况。结果发现,各CAR-T组扩增良好,未转导T细胞、VCAM1-T细胞、CDH17 CAR-T和CDH17-VCAM1 CAR-T细胞在培养10天后分别扩增41.00、56.65、68.50和70.50倍,见图3所示。
实施例5:CAR的表达及其功能评估
采用流式细胞仪检测CAR-T细胞表面CAR分子的表达及其与对应抗原蛋白的结合能力,利用APC-anti-CD3抗体标记T细胞群,然后用CDH17蛋白(ACRO Biosystems公司;货号CA7-H5258)检测CAR表达阳性率。结果显示:未转导T细胞、VCAM1-T细胞、CDH17 CAR-T和CDH17-VCAM1 CAR-T细胞的CAR表达率分别为3.3%、5.0%、84.36%和81.27%(相比未转导T细胞,***P≤0.001,见图4所示),表明CAR-T能有效识别CDH17抗原。
另外,未转导T细胞不表达嵌合受体,VCAM1-T细胞只表达VCAM1。未转导T细胞、VCAM1-T细胞、CDH17 CAR-T和CDH17-VCAM1 CAR-T细胞的VCAM1表达率分别为15.34%、72.69%、16.89%和78.36%,见图5所示。
实施例6:Transwell评估CAR-T的迁移能力
使用Transwell评估CAR-T细胞的迁移能力的具体步骤如下:
(1)在24孔板中每孔接种8×10E4个细胞,常规条件培养24小时;
(2)弃培养基,用PBS洗涤细胞2次,用消化液消化细胞,继而用无血清培养液重悬细胞,计数,将细胞密度调为30万/ml;
(3)将transwell培养小室放入24孔板中,共分2组,每组3个复孔,一组在小室下层分别加600μl无血清培养基、另一组加600μl含10%胎牛血清的培养液,小室内则加入100μl含3× 10E4个细胞的单细胞悬液,常规培养12小时;
(4)取出transwell小室,用甲醇固定15分钟,0.1%结晶紫染色20分钟,用棉签小心擦去微孔膜上层细胞,PBS清洗两次;
(5)细胞计数:在荧光显微镜下给微孔膜下层细胞拍照,每孔拍9个视野,计数。
培养一段时间以后,一部分细胞穿过transwel小室,迁移到孔膜下层,在荧光显微镜下可看到蓝紫色的细胞。通过以上步骤,可以评估CAR-T细胞的迁移能力。
结果显示:相比未转导T细胞和CDH17 CAR-T细胞,共表达VCAM1分子的VCAM1-T细胞和CDH17-VCAM1 CAR-T细胞的迁移能力明显提升,见图6所示。
实施例7:CAR-T功能评估
肿瘤细胞杀伤效率由基于荧光素酶的细胞杀伤检测方法(Luciferase-basedcytotoxicity assay)进行评估。用过表达CDH17和荧光素酶基因的人结肠癌细胞SW480(SW480-CDH17-Luc)作为靶细胞,进行杀伤试验。
首先,将1×104个SW480-CDH17-Luc接种于96孔平底黑板上,每孔100μL培养基,置于37℃,5%CO2细胞培养箱中培养18~20小时。第二天,以效应细胞:靶细胞=1:1的比例加入嵌合受体修饰T细胞至含有靶细胞的孔中,置于37℃,5%CO2细胞培养箱中继续培养6小时,共培养结束后使用微孔板发光检测仪检测靶细胞的荧光素酶活力值。细胞杀伤率的计算公式如下所示,细胞杀伤率(%)=(未转导T细胞组荧光素酶活力值-实验组荧光素酶活力值)/未转导T细胞组荧光素酶活力值×100。
结果显示,CDH17 CAR-T细胞和CDH17-VCAM1 CAR-T细胞都能杀伤SW480-CDH17-Luc,杀伤率分别为29.00%和52.00%。共表达血管内皮黏附分子VCAM1的CAR-T细胞(CDH17-VCAM1 CAR-T)比经典CAR-T(CDH17 CAR-T)的杀伤能力显著提高(*P≤0.05),见图7所示。
综上所述,共表达血管内皮黏附分子VCAM1的CAR-T细胞(CDH17-VCAM1 CAR-T)能特异性识别CDH17抗原;相比经典的CAR-T(anti-CDH17 CAR-T),CDH17-VCAM1 CAR-T的迁移能力更强,杀伤力显著提升。本发明提供的共表达血管内皮黏附分子VCAM1的CAR-T细胞在实体瘤的治疗中具有极大的临床转化价值。
以上所述仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种协同表达血管内皮黏附分子VCAM1的嵌合抗原受体,其特征在于,所述的嵌合抗原受体包括有SEQ ID No:1所示的氨基酸序列和SEQ ID No:2所示的氨基酸序列。
2.根据权利要求1所述的嵌合抗原受体,其特征在于,SEQ ID No:1所示的氨基酸序列与SEQ ID No:2所示的氨基酸序列之间使用可剪接的2A linker相连。
3.根据权利要求1所述的嵌合抗原受体,其特征在于,所述的嵌合抗原受体具有下式I结构:
L-CDH17 scFv-H-TM-C-CD3ζ-2A-VCAM1(I)
式中,
各“-”为连接肽或肽键;L为信号肽序列;CDH17 scFv为针对CDH17肿瘤抗原的抗体,所述CDH17 scFv的氨基酸序列如SEQ ID NO:1所示;H为铰链区序列;TM为跨膜区序列;C为共刺激域序列;CD3ζ为信号传导域序列。
4.一种核酸分子,其特在在于,其编码权利要求1所述的嵌合抗原受体。
5.根据权利要求4所述的核酸分子,其特征在于,所述的核酸分子包括有SEQ ID No:5所示的核苷酸序列和SEQ ID No:6所示的核苷酸序列。
6.一种载体,其特征在于,所述的载体包括有权利要求5所述的核酸分子。
7.根据权利要求6所述的载体,其特征在于,所述的载体选自DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子中的一种或多种组合。
8.一种细胞,其特征在于,所述的细胞包括有权利要求6所述的载体、或染色体中整合有外源的权利要求4所述的核酸分子、或表达权利要求1所述的嵌合抗原受体。
9.根据权利要求8所述的细胞,其特征在于,所述细胞为T细胞。
10.一种制备协同表达血管内皮黏附分子VCAM1的CAR-T细胞的方法,其特征在于,将血管内皮黏附分子VCAM1与抗CDH17的CAR分子构建到同一个慢病毒载体上,并使用可剪接的2A linker使两者相连,然后将该慢病毒载体转染到T细胞中;所述血管内皮黏附分子VCAM1包括有如SEQ ID No:2所示的氨基酸序列;所述抗CDH17的CAR分子包括有如SEQ ID No:1所示的氨基酸序列。
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