CN113234169B - 靶向cll1嵌合抗原受体及其应用 - Google Patents

靶向cll1嵌合抗原受体及其应用 Download PDF

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CN113234169B
CN113234169B CN202011461194.7A CN202011461194A CN113234169B CN 113234169 B CN113234169 B CN 113234169B CN 202011461194 A CN202011461194 A CN 202011461194A CN 113234169 B CN113234169 B CN 113234169B
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CN113234169A (zh
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李光超
罗敏
丁雯
周兆
王学俊
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Guangzhou Bio Gene Technology Co Ltd
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Abstract

本发明提供了靶向CLL1嵌合抗原受体及其应用，所述靶向CLL1嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；所述抗原结合结构域为抗CLL1抗体。本发明采用抗CLL1抗体作为抗原结合结构域构建嵌合抗原受体分子，靶向CLL1嵌合抗原受体对CLL1阳性肿瘤细胞具有特异性靶向作用，表达靶向CLL1嵌合抗原受体的免疫细胞体内外杀伤作用显著，与CLL1阳性肿瘤细胞共培养后分泌大量的细胞因子IFN‑γ，对CLL1阳性肿瘤细胞具有特异性清除作用。

Description

靶向CLL1嵌合抗原受体及其应用
技术领域
本发明属于生物医药技术领域，涉及靶向CLL1嵌合抗原受体及其应用。
背景技术
C型凝集素样分子1(CLL1)，又称C型凝集素域家族12成员A(CLEC12A)，是一种II型跨膜蛋白。研究表明，CLL1限制性地表达于造血细胞上，主要包括外周血和骨髓中髓系来源的细胞，比如单核细胞、树突状细胞、粒细胞以及大多数急性髓细胞白血病(AML)细胞。值得注意的是，虽然CLL1大量表达于外周血和骨髓中的髓细胞上，但是在外周组织中的髓系来源的细胞上不表达，比如组织巨噬细胞和组织树突状细胞均不表达CLL1。研究还发现，CLL1表达于AML干细胞(CD34+/CD38-)以及一小部分造血祖细胞(CD34+/CD38+或者CD34+/CD33+)上，但是在正常的造血干细胞(CD34+/CD38-或者CD34+/CD33-)上不表达。
由于这种特殊的表达模式，CLL1有望成为潜在的诊断和治疗AML的靶点。但目前鲜有靶向CLL1的免疫疗法报道。
发明内容
针对现有技术的不足和实际需求，本发明提供了靶向CLL1嵌合抗原受体及其应用，所述靶向CLL1嵌合抗原受体采用对CLL1具有结合能力的抗CLL1抗体为抗原结合结构域，不仅可以结合纯化的或游离的CLL1蛋白，还可以结合细胞表面的CLL1蛋白，表达所述靶向CLL1嵌合抗原受体的免疫细胞在肿瘤治疗领域具有重要应用前景。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了靶向CLL1嵌合抗原受体，所述靶向CLL1嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；
所述抗原结合结构域为抗CLL1抗体。
本发明中，采用对CLL1具有结合能力的抗CLL1抗体作为嵌合抗原受体的抗原结合结构域，使得嵌合抗原受体可以特异性结合CLL1阳性肿瘤细胞，实现对CLL1阳性肿瘤的特异性靶向作用。
优选地，所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列，SEQ ID NO:1和SEQ ID NO:2通过连接肽连接形成抗CLL1抗体19C1；
SEQ ID NO:1：
QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEIFPGSGSIKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVHYCARGGTYNDYSLFDYWGQGTTLTVSS；
SEQ ID NO:2：
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIFDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLELK。
优选地，所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列，SEQ ID NO:3和SEQ ID NO:4通过连接肽连接形成抗CLL1抗体23D7；
SEQ ID NO:3：
QVQLQQPGSDLVRPGASVKLSCKASGYTFTRYWMHWVKQRPGHGLEWIGYIYPGSGTSNYDEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTREARYTMDYWGQGTSVTVSS；
SEQ ID NO:4：
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPGLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSFPPTFGAGTKLELK。
优选地，所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，SEQ ID NO:5和SEQ ID NO:6通过连接肽连接形成抗CLL1抗体27H4；
SEQ ID NO:5：
EVQLQQSGPELVKPGASVKISCKASGYSFTGYHMHWVKQSHVKSLEWIGRINPYNGAASHNQKFKDKATLTVDKSSSTAYMELHSLTSEDSAVYYCARGWDYDGGYYAMDYWGQGTSVTVSS；
SEQ ID NO:6：
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIK。
优选地，所述抗原结合结构域包括SEQ ID NO:7和SEQ ID NO:8～10之一所示的氨基酸序列，SEQ ID NO:7和SEQ ID NO:8通过连接肽连接形成抗CLL1抗体H27H4L1，SEQ IDNO:7和SEQ ID NO:9通过连接肽连接形成抗CLL1抗体H27H4L2，SEQ ID NO:7和SEQ ID NO:10通过连接肽连接形成抗CLL1抗体H27H4L3；
SEQ ID NO:7：
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSS；
SEQ ID NO:8：
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIK；
SEQ ID NO:9：
DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSDNQKNYLAWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYTYPYTFGQGTKLEIK；
SEQ ID NO:10：
DIVMTQSPDSLAVSLGERATINCKSSQSLLYSDNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTYPYTFGQGTKLEIK。
优选地，所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列，SEQ ID NO:11和SEQ ID NO:12通过连接肽连接形成抗CLL1抗体1075.7；
SEQ ID NO:11：
DIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSS；
SEQ ID NO:12：
ENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLEL。
优选地，所述铰链区包括CD8α铰链区。
优选地，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区，优选为CD8α跨膜区。
优选地，所述信号传导结构域包括CD3ζ。
优选地，所述信号传导结构域还包括4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
优选地，所述靶向CLL1嵌合抗原受体还包括信号肽。
优选地，所述信号肽包括CD8α信号肽和/或IgGκ轻链信号肽。
作为优选技术方案，所述靶向CLL1嵌合抗原受体包括信号肽、抗CLL1抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
优选地，所述靶向CLL1嵌合抗原受体19C1-CAR包括SEQ ID NO:13所示的氨基酸序列；
SEQ ID NO:13：
MALPVTALLLPLALLLHAARPQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIFDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEIFPGSGSIKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVHYCARGGTYNDYSLFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
优选地，所述靶向CLL1嵌合抗原受体23D7-CAR包括SEQ ID NO:14所示的氨基酸序列；
SEQ ID NO:14：
MALPVTALLLPLALLLHAARPQIVLTQSPAIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPGLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSFPPTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQPGSDLVRPGASVKLSCKASGYTFTRYWMHWVKQRPGHGLEWIGYIYPGSGTSNYDEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTREARYTMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
优选地，所述靶向CLL1嵌合抗原受体27H4-CAR包括SEQ ID NO:15所示的氨基酸序列；
SEQ ID NO:15：
MALPVTALLLPLALLLHAARPDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKASGYSFTGYHMHWVKQSHVKSLEWIGRINPYNGAASHNQKFKDKATLTVDKSSSTAYMELHSLTSEDSAVYYCARGWDYDGGYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
优选地，所述靶向CLL1嵌合抗原受体H27H4-CAR包括SEQ ID NO:16所示的氨基酸序列；
SEQ ID NO:16：
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
优选地，所述靶向CLL1嵌合抗原受体1075.7-CAR包括SEQ ID NO:17所示的氨基酸序列；
SEQ ID NO:17：
MALPVTALLLPLALLLHAARPENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
第二方面，本发明提供了核酸分子，所述核酸分子包括第一方面所述的靶向CLL1嵌合抗原受体的编码基因。
优选地，所述核酸分子包括SEQ ID NO:18所示的核酸序列，为19C1-CAR的编码基因；
SEQ ID NO:18：
atggccctcccagtcacagctctgctgctcccactcgccctgctgctgcacgccgcacggcctcagatcgtcctcacccagtctccagccatcatgagcgcctctccaggcgagaaggtgacaatgacttgttccgcaagcagctcagttagttacatgtattggtaccagcagaaaccaggctcttctccaaggctcctgatcttcgacacttctaacctggcatccggagtcccagtgcggttcagcggcagcggcagtggaacatcttatagcctgactattagccggatggaggcagaagatgcagctacctactactgtcaacagtggtctagttatccactcacttttggtgcagggaccaagctggagctcaaaggtggaggaggatctggcggcggagggagtggaggaggcggctcacaggtccaactgcagcaatctggtgcagaactgatgaagcctggagcaagcgttaaaatctcttgcaaagcaacaggatacaccttttcttcctactggatagagtgggtgaaacaaagaccaggacacggactggaatggataggagagatctttcctggcagcggctcaatcaagtataacgagaagttcaaagggaaagccaccttcacagccgatacctcttctaatacagcctatatgcagctgtcatccctgaccagcgaagattctgctgttcactactgtgcacgcggaggaacatacaatgactactctctgtttgactattggggacagggcaccacactgactgtgagttctactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
优选地，所述核酸分子包括SEQ ID NO:19所示的核酸序列，为23D7-CAR的编码基因；
SEQ ID NO:19：
atggctctccctgttactgcactcctgctcccactggcactgctgctgcatgccgctcggccacaaatagttctgactcagagtcctgccattatgtcagcctctcctggagagaaggtcacaatgacttgctctgcctctagtagtgtgtcttacatatactggtaccagcagaagcctggttcttctcccggactgctgatctatgacacatccaatctggcttcaggcgttcccgtcagattcagcgggtctggatctggcacaagctattctctgaccatctcaagaatggaggctgaagatgctgctacttattattgccaacagtggtcttcctttccaccaaccttcggtgcaggtaccaagctcgaactcaaaggtggaggaggaagcggaggaggcggtagtggtggaggtgggtcccaagttcagctgcaacagcccggatctgatctggttcggcccggagctagcgtgaaactgtcttgcaaggctagcggatacactttcacccgctattggatgcactgggttaagcagcggccaggacacggactggagtggattggctatatctacccaggcagcgggacaagtaactacgatgagaaattcaagagtaaggctactctgactgtcgatacaagttcctcaaccgcttacatgcagctctcttcactcaccagcgaagacagtgctgtttattactgcaccagggaagctcggtacaccatggattattggggtcaaggaacttctgtgacagtgtcaagcactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
优选地，所述核酸分子包括SEQ ID NO:20所示的核酸序列，为27H4-CAR的编码基因；
SEQ ID NO:20：
atggctctgcctgttactgcactgctgctccctctggctctcctcctgcatgctgctcggcctgatatagtgatgtcccaaagcccatccagcctggccgtgtccgtcggcgaaaaggtgactatgagttgcaaatccagccaaagcctgctgtacagtgacaaccaaaagaactacctggcatggtaccagcagaagcctggacagtcaccaaagctcctcatctactgggctagcacaagggagagcggcgtcccagacaggtttactggcagcgggagtggcaccgatttcaccctgacaataagctctgtcaaggccgaagacctcgctgtgtactattgtcagcagtattatacctatccctatactttcggtggagggaccaaactcgagattaaaggaggtggcggctctggaggtggaggttccggcggtggcggtagtgaagtgcagctgcagcagagcgggcctgaactcgtgaaacctggtgcctccgttaaaatctcctgcaaggccagcggctactcattcacagggtatcacatgcattgggtgaagcagagccacgtcaaatcactggaatggatcggcaggattaatccatacaatggcgctgcttcacataaccagaagttcaaggacaaagccaccctgactgtcgataagtcatcaagtacagcatacatggagctgcattccctgactagcgaggacagcgctgtttactactgcgcacgcggctgggactacgacggtggctattacgccatggactactggggacaaggcaccagcgtcacagtttcaagtactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
优选地，所述核酸分子包括SEQ ID NO:21所示的核酸序列，为H27H4-CAR的编码基因；
SEQ ID NO:21：
atggatatgagggttcctgcacaactcctgggactcctcctgctctggctgagaggcgcaagatgtgatatccagatgacccagtctcctagtagcctgtctgcctccgtcggcgatcgggtgaccattacttgcaaatcctcacagagcctcctctactccgataatcagaagaactacctcgcctggtatcaacagaaaccagggaaggcacctaagctgctgatctactgggctagtacccgcgaatccggcgtccctagcaggttctctggcagcgggagcgggacagatttcaccctcactatctcctccctgcagcctgaagacttcgcaacttactactgtcagcagtattatacttacccatacactttcggacagggaacaaaactggaaattaaaggtggaggtggatctggtggcggtggcagtggcggaggcgggtctgaagtccaactggtgcagagcggtgcagaggtgaagaagcctggagcatcagtgaaggtgtcttgcaaagccagtggctacacattcactggatatcatatgcattgggttaggcaggcacccggccagcggctggagtggatgggaagaatcaacccttataatggcgctgcctctcacaatcaaaagtttaaggatcgggtcactatcactcgggacacttccgcaagcaccgcctatatggagctgagcagcctgcggagtgaagacacagcagtctactactgtgctcgcggatgggactatgacggcggttattatgccatggattactggggacagggcacactggtcaccgtgagcagcactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
优选地，所述核酸分子包括SEQ ID NO:22所示的核酸序列，为1075.7-CAR的编码基因；
SEQ ID NO:22：
atggctctgcctgttactgcactcctcctcccactggcactcctcctgcatgcagccaggcccgagaatgtgctgacacagtctccagccatcatgagcgcctctcccggtgaaaaggttactatgacctgtcgggcaagttcaaatgtgatctcctcttatgtgcactggtaccagcagcgctcaggtgcaagcccaaagctgtggatctattccacttctaacctggcctccggtgtcccagcccgcttttctggaagcgggtcaggcacctcatactccctcaccatatcaagtgtggaagctgaggatgcagctacttactactgccaacagtactctggttacccactgaccttcggagccgggacaaagctggaactgggaggaggcgggtccggcggtggagggtccggaggtggcgggtccgatatccagctgcaagagtcaggcccaggcctggtcaaaccttcccaaagcctgagtctcacctgttccgtgacaggttattccattactagcgcatattactggaactggataagacaattcccaggaaacaaactcgagtggatgggctacatctcatacgacgggcggaacaactataacccatccctgaagaatcggatttccatcactagagacacatccaagaaccagttctttctcaagctgaatagcgtgacaactgaggatacagcaacctactattgcgccaaggaaggagactatgatgttggcaactattatgcaatggactattggggacagggcacatcagtgaccgtgagcagcactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
第三方面，本发明提供了一种表达载体，所述表达载体包括第二方面所述的核酸分子。
优选地，所述表达载体为含有第二方面所述的核酸分子的病毒载体或非病毒载体。
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。
优选地，所述非病毒载体包括Piggybac转座子系统、Sleeping Beauty转座子系统或纳米载体中的任意一种。
第四方面，本发明提供了一种重组慢病毒，所述重组慢病毒由转染有第三方面所述的表达载体和辅助质粒的哺乳细胞制备得到。
第五方面，本发明提供了一种嵌合抗原受体免疫细胞，所述嵌合抗原受体免疫细胞表达第一方面所述的靶向CLL1嵌合抗原受体。
本发明中，表达靶向CLL1嵌合抗原受体的免疫细胞利用嵌合抗原受体的抗原结合结构域靶向CLL1阳性肿瘤细胞，发挥免疫细胞的杀伤功能分泌细胞因子IFN-γ，在不同效靶比下实现对CLL1肿瘤的杀伤作用。
优选地，所述嵌合抗原受体免疫细胞的基因组中整合有第二方面所述的核酸分子。
优选地，所述嵌合抗原受体免疫细胞包括第三方面所述的表达载体和/或第四方面所述的重组慢病毒。
优选地，所述免疫细胞包括T细胞、NK细胞或巨噬细胞中的任意一种。
优选地，所述T细胞包括αβΤ细胞和/或γδΤ细胞。
第六方面，本发明提供了一种药物组合物，所述药物组合物包括第五方面所述的嵌合抗原受体免疫细胞。
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。
第七方面，本发明提供了第一方面所述的靶向CLL1嵌合抗原受体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组慢病毒、第五方面所述的嵌合抗原受体免疫细胞或第六方面所述的药物组合物在制备恶性肿瘤治疗药物中的应用。
优选地，所述恶性肿瘤包括急性髓系白血病。
第八方面，本发明提供了一种治疗癌症的方法，所述方法包括向患者施用有效剂量的第五方面所述的嵌合抗原受体免疫细胞或第六方面所述的药物组合物，并同时、分开或依次施用一种或多种抗肿瘤药物。
优选地，所述癌症包括急性髓系白血病。
与现有技术相比，本发明具有如下有益效果：
(1)本发明采用抗CLL1抗体作为抗原结合结构域构建CAR分子，表达靶向CLL1CAR的T细胞依赖于CAR元件在不同的效靶比下对CLL1阳性肿瘤细胞发挥杀伤作用，其中，H27H4-CAR-T具有最优的杀伤功能；
(2)本发明的靶向CLL1CAR-T细胞与CLL1阳性肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，证明了CAR-T对CLL1肿瘤细胞的特异性杀伤功效。
附图说明
图1为不同肿瘤细胞对CLL1的表达情况；
图2为靶向CLL1的嵌合抗原受体的结构示意图；
图3A为含有H27H4CAR基因的慢病毒表达载体图谱，图3B为H27H4CAR在慢病毒表达载体中的位置；
图4为不同CAR-T细胞的CAR表达阳性率；
图5为靶向CLL1的CAR-T对Raji-CLL1细胞的杀伤率；
图6为靶向CLL1的CAR-T杀伤靶细胞的IFN-γ分泌情况。
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抗CLL1抗体的来源
本实施例选择抗CLL1抗体19C1、23D7、27H4、人源化27H4(H27H4)和1075.7(专利US8536310B2)作为抗原结合结构域用于构建CAR分子，其中，19C1包括SEQ ID NO:1～2所示的可变区、23D7包括SEQ ID NO:3～4所示的可变区、27H4包括SEQ ID NO:5～6所示的可变区、H27H4包括SEQ ID NO:7～8所示的可变区、1075.7包括SEQ ID NO:11～12所示的可变区。
实施例2肿瘤细胞对CLL1的表达
本实施例采用FITC anti-human CD371(CLL1)抗体(biolegend)与靶细胞Jurkat、KG-1a、共孵育后，流式检测靶细胞对CLL1的表达。
结果如图1所示，THP-1、U937和HL-60为CLL1阳性细胞，Jurkat和KG-1a为CLL1阴性细胞。
实施例3嵌合抗原受体的设计
本实施例设计靶向CLL1的嵌合抗原受体，结构示意图见图2，包括CD8α信号肽、与CLL1抗原特异性结合的单链抗体(Anti-CLL1scFv)、CD8α铰链区(Hinge)和跨膜区(Transmembrane)、4-1BB共刺激结构域和CD3ζ信号传导结构域，具体CAR分子为：
①19C1-CAR：CD8α信号肽、Anti-CLL1scFv(19C1)、CD8αHinge+TM、4-1BB和CD3ζ；
②23D7-CAR：CD8α信号肽、Anti-CLL1scFv(23D7)、CD8αHinge+TM、4-1BB和CD3ζ；
③27H4-CAR：CD8α信号肽、Anti-CLL1scFv(27H4)、CD8αHinge+TM、4-1BB和CD3ζ；
④H27H4-CAR：IgGκ轻链信号肽、Anti-CLL1scFv(H27H4)、CD8αHinge+TM、4-1BB和CD3ζ；
⑤1075.7-CAR：CD8α信号肽、Anti-CLL1scFv(1075.7)、CD8αHinge+TM、4-1BB和CD3ζ；
其中，CD8α信号肽的氨基酸序列如SEQ ID NO:23所示，核酸序列如SEQ ID NO:24所示，IgGκ轻链信号肽的氨基酸序列如SEQ ID NO:25所示，核酸序列如SEQ ID NO:26所示，CD8αHinge的氨基酸序列如SEQ ID NO:27所示，核酸序列如SEQ ID NO:28所示，CD8αTM的氨基酸序列如SEQ ID NO:29所示，核酸序列如SEQ ID NO:30所示，4-1BB的氨基酸序列如SEQID NO:31所示，核酸序列如SEQ ID NO:32所示，CD3ζ的氨基酸序列如SEQ ID NO:33所示，核酸序列如SEQ ID NO:34所示；
SEQ ID NO:23：
MALPVTALLLPLALLLHAARP；
SEQ ID NO:24：
atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgccct；
SEQ ID NO:25：
MDMRVPAQLLGLLLLWLRGARC；
SEQ ID NO:26：
atggatatgagggttcctgcacaactcctgggactcctcctgctctggctgagaggcgcaagatgt；
SEQ ID NO:27：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD；
SEQ ID NO:28：
accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat；
SEQ ID NO:29：
IYIWAPLAGTCGVLLLSLVITLYC；
SEQ ID NO:30：
atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc；
SEQ ID NO:31：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；
SEQ ID NO:32：
aagagaggcaggaagaagctgctgtacatcttcaagcagcccttcatgcgccccgtgcagacaacccaggaggaggacggctgcagctgtcggttcccagaggaggaggagggaggatgtgagctg；
SEQ ID NO:33：
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；
SEQ ID NO:34：
agggtgaagttttctcggagcgccgatgcaccagcatatcagcagggacagaatcagctgtacaacgagctgaatctgggcaggcgcgaggagtacgacgtgctggataagcggagaggcagagatcccgagatgggaggcaagccaaggaggaagaaccctcaggagggcctgtataatgagctgcagaaggacaagatggccgaggcctactctgagatcggcatgaagggagagcggagaaggggcaagggacacgatggcctgtatcagggcctgagcacagccaccaaggacacctacgatgcactgcacatgcaggccctgccacctagg。
实施例4靶向CLL1的嵌合抗原受体表达载体的构建
(1)根据实施例3设计的CAR分子，对CAR编码基因进行密码子优化、促进其在人源细胞中的高效表达，全基因合成CAR编码基因(广州艾基生物技术有限公司)；
(2)用EcoRI和BamHI双酶切全长CAR基因和空载体pCDH-EF1-MCS，于37℃水浴酶切30min后，使用1.5％琼脂糖凝胶进行DNA电泳，并使用琼脂糖凝胶纯化回收试剂盒(天根)对酶切产物进行纯化回收处理；
(3)配制表1所示的连接体系，对CAR基因片段和线性化pCDH-EF1-MCS进行22℃连接1h，将连接产物直接转化Stbl3大肠杆菌感受态细胞，取200μL转化产物涂布氨苄抗性LB平板，于37℃培养箱倒置培养过夜，次日早晨随机挑选3个单克隆进行菌落PCR鉴定，并将阳性克隆进行测序鉴定；
表1
成分 添加量
线性化pCDH-EF1-MCS载体 50ng
CAR基因 150ng
T4DNA连接缓冲液 2μL
T4DNA连接酶(NEB) 1μL
ddH<sub>2</sub>O 补齐至20μL
示例性地，构建的含有27H4CAR基因的慢病毒表达载体pBG-27H4如图3A和图3B所示。
实施例5慢病毒包装
本实施例采用四质粒系统对实施例4构建的慢病毒表达载体进行慢病毒包装，具体步骤如下：
(1)将慢病毒表达载体、辅助质粒gag/pol、Rev、VSV-G构成的四质粒系统与PEI转染试剂混合后，加至一定体积的无血清DMEM中，混匀放置15min；
(2)将上述混合液加至铺有293T细胞的T75细胞培养瓶中，轻轻混匀，于37℃、5％CO2细胞培养箱中培养6h；
(3)6h后更换新鲜培养基，继续培养，并加入10mM丁酸钠溶液；72h后收集慢病毒培养上清进行纯化检测。
实施例6 T细胞的获取与扩增
每位志愿者采集30mL全血，将外周血与生理盐水按1:1混合稀释；向离心管中加入Ficoll淋巴细胞分离液，缓缓加入稀释的外周血，1500rpm离心30min，轻轻吸取PBMC层移入另一离心管中；
用生理盐水多次洗涤PBMC，转入X-VIVO培养基(含50ng/mL OKT3，300IU/mL IL-2)中进行培养，PBMC分离后，用X-VIVO(含50ng/mL OKT3，300IU/mL IL-2)进行激活，2日后将培养基更换为含300IU/mL的X-VIVO进行扩大培养；而后每两天进行一次计数，并更换新鲜的含300IU/mL的X-VIVO培养基，将细胞浓度维持在(0.5～1)×106个/mL，连续观察10天。
实施例7 CAR-T细胞的制备
本实施例利用RetroNectin提高慢病毒对T细胞的感染效率，步骤如下：
将30μg RetroNectin包被于6孔板内，置于37℃细胞培养箱保持2h；吸取RetroNectin，利用含2.5％BSA的Hank’s溶液封闭包被后的6孔板，置于37℃细胞培养箱保持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，2000g离心2h，弃上清；加入1×106个T细胞(CD3阳性>90％)，1000g离心10min，置于37℃、5％CO2和一定湿度的细胞培养箱内培养，第二日重复上述步骤。
慢病毒的加入量见表2。
表2
Figure BDA0002831864460000081
Figure BDA0002831864460000091
采用流式细胞仪检测T细胞表面CAR分子的表达，利用CLL1-Fc融合蛋白(Acrobiosystems公司)检测CAR表达，二抗采用FITC-Labeled anti-human Fc，未转染CAR的T细胞(T mock)作为阴性对照。
结果如图4和表3所示，19C1-CAR-T细胞的CAR表达阳性率为13.28％，23D7-CAR-T细胞的CAR表达阳性率为37.49％，27H4-CAR-T细胞的CAR表达阳性率为39.44％，H27H4-CAR-T细胞的CAR表达阳性率为33.59％，1075.7-CAR-T细胞的CAR表达阳性率为5.09％。
表3
T细胞类型 T mock 19C1 23D7 27H4 H27H4 1075.7
CAR表达阳性率 1.23％ 13.28％ 37.49％ 39.44％ 33.59％ 5.09％
实施例8 CAR-T细胞的杀伤功能
本实施例采用xCELLigence实时无标记动态分析技术(Real Time CellAnalysis，RTCA)对细胞杀伤效应进行全程自动化检测。
Figure BDA0002831864460000092
实时无标记细胞分析仪(RTCA)基于微电子阻抗技术，在E-plate板底整合有大量微金电极，当贴壁细胞粘附到微金电极上，细胞数量、直径以及贴壁能力均会影响微金电极间的电流传导，从而引起阻抗值的改变，这一改变极为精细且灵敏，在毒负效应下，细胞直接或间接影响阻抗值。因此，xCELLigence可以监测由分子靶标引起的细胞毒性效应。
步骤如下：
(1)用一定体积的1×Tether Buffer稀释anti-CD40，向E-Plate View 96孔板的每孔中加入50μL稀释好的anti-CD40作为包被液，室温避光孵育3h，每组设置三个复孔；
(2)弃包被液，用200μL PBS轻轻洗孔2次，向每个孔中加入50μL含2％FBS的1640培养基，将E-Plate View 96孔板放入xCELLigence仪中(仪器预先1h放入培养箱中)，37℃平衡1h，测量背景值；
(3)采用稳定过表达CLL1的Raji细胞(Raji-CLL1)作为靶细胞，制备靶细胞悬液并测定细胞密度，向每个孔中加入50000个细胞/50μL，每个孔的终体积为100μL，室温静置孵育30min，将E-Plate View 96孔板重新放回仪器中，操作软件进行数据收集，每5min检测一次电阻抗，检测时间为2h；
(4)分别制备不同效靶比的效应细胞悬液(即CAR-T细胞)和阴性对照细胞悬液(即未转染的T细胞)，向每孔中加入50μL稀释好的效应细胞悬液或阴性对照细胞悬液，空白对照组加入50μL培养基，阳性对照组加入50μL 1×Cytolysis Solution，室温静置孵育30min，使效应细胞均匀分布在固定的靶细胞上；
(5)将E-Plate View 96孔板重新放入仪器中，操作软件进行数据收集，每5min检测一次电阻抗，检测时间为16h，实验结束后保存数据并分析。
结果如图5所示，19C1-CAR-T、23D7-CAR-T、27H4-CAR-T、H27H4-CAR-T或1075.7-CAR-T在不同的效靶比(1:1、2:1、4:1)下与Raji-CLL1共孵育16h后，均能有效杀伤Raji-CLL1细胞，效靶比越大杀伤能力越强。其中，H27H4-CAR-T具有最强的杀伤能力，27H4-CAR-T的杀伤能力与H27H4-CAR-T相似，23D7-CAR-T次之，19C1-CAR-T和1075.7-CAR-T的杀伤能力偏弱，未转染CAR的T mock不能杀伤Raji-CLL1，说明anti-CLL1CAR-T的杀伤活性依赖于CAR元件。
实施例9 CAR-T细胞与肿瘤细胞共培养对IFN-γ的分泌
本实施例采用Human IFN-γELISA试剂盒(欣博盛)检测CAR-T细胞释放IFN-γ细胞因子的浓度，分析CAR-T与靶细胞共培养后对IFN-γ的分泌量，具体地，采用CLL1阳性细胞Raji-CLL1、HL60、U937作为阳性靶细胞，CLL1阴性细胞Raji作为阴性靶细胞。
将CAR-T细胞与不同的靶细胞按照效靶比为1:1共孵育16h，取上清用酶联免疫法(ELISA)检测培养液上清中IFN-γ的分泌量。检测原理采用双抗体夹心ELISA法，将抗人IFN-γ抗体包被于酶标板上，实验时样品或标准品中的人IFN-γ与包被抗体结合，游离的成分被洗去；依次加入生物素化抗人IFN-γ抗体和辣根过氧化物酶标记的亲和素，抗人IFN-γ抗体与结合在包被抗体上的人IFN-γ结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去；加入显色底物(TMB)，在辣根过氧化物酶的催化下呈现蓝色，加入终止液后变成黄色，用酶标仪在450nm波长处测OD值，IFN-γ浓度与OD450之间呈正比，通过绘制标准曲线计算样品中IFN-γ的浓度。
结果如图6所示，19C1-CAR-T、23D7-CAR-T、27H4-CAR-T、H27H4-CAR-T或1075.7-CAR-T与Raji-CLL1、HL60或U937共培养后释放大量IFN-γ，而与Raji细胞共培养后未见大量IFN-γ释放，说明靶向CLL1的CAR-T细胞的杀伤具有特异性。
综上所述，本发明的靶向CLL1CAR-T细胞在不同的效靶比下对CLL1阳性肿瘤细胞具有显著的杀伤作用，与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，在CLL1阳性肿瘤治疗领域具有应用前景。
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> 广州百暨基因科技有限公司
<120> 靶向CLL1嵌合抗原受体及其应用
<130> 20201208
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100 105
<210> 5
<211> 122
<212> PRT
<213> 人工序列
<400> 5
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
His Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Ala Ala Ser His Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Trp Asp Tyr Asp Gly Gly Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 6
<211> 113
<212> PRT
<213> 人工序列
<400> 6
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 7
<211> 122
<212> PRT
<213> 人工序列
<400> 7
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
His Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Ala Ala Ser His Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Trp Asp Tyr Asp Gly Gly Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 113
<212> PRT
<213> 人工序列
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 9
<211> 113
<212> PRT
<213> 人工序列
<400> 9
Asp Ile 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 Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Gln Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 10
<211> 113
<212> PRT
<213> 人工序列
<400> 10
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 11
<211> 123
<212> PRT
<213> 人工序列
<400> 11
Asp Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ala
20 25 30
Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Lys Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 12
<211> 107
<212> PRT
<213> 人工序列
<400> 12
Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Asn Val Ile Ser Ser
20 25 30
Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala Ser Pro Lys Leu Trp
35 40 45
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro
85 90 95
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105
<210> 13
<211> 486
<212> PRT
<213> 人工序列
<400> 13
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 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
20 25 30
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser
35 40 45
Ser Val Ser Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro
50 55 60
Arg Leu Leu Ile Phe Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
85 90 95
Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser
100 105 110
Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
130 135 140
Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser Val
145 150 155 160
Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr Trp Ile
165 170 175
Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu
180 185 190
Ile Phe Pro Gly Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe Lys Gly
195 200 205
Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Met Gln
210 215 220
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val His Tyr Cys Ala Arg
225 230 235 240
Gly Gly Thr Tyr Asn Asp Tyr Ser Leu Phe Asp Tyr Trp Gly Gln Gly
245 250 255
Thr Thr Leu Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
340 345 350
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
Gln Ala Leu Pro Pro Arg
485
<210> 14
<211> 482
<212> PRT
<213> 人工序列
<400> 14
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 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
20 25 30
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser
35 40 45
Ser Val Ser Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro
50 55 60
Gly Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
85 90 95
Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser
100 105 110
Ser Phe Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
130 135 140
Gln Leu Gln Gln Pro Gly Ser Asp Leu Val Arg Pro Gly Ala Ser Val
145 150 155 160
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Trp Met
165 170 175
His Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Tyr
180 185 190
Ile Tyr Pro Gly Ser Gly Thr Ser Asn Tyr Asp Glu Lys Phe Lys Ser
195 200 205
Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln
210 215 220
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg
225 230 235 240
Glu Ala Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
245 250 255
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
260 265 270
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
275 280 285
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
290 295 300
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
305 310 315 320
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
325 330 335
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
340 345 350
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
355 360 365
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 15
<211> 494
<212> PRT
<213> 人工序列
<400> 15
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 Ile Val Met Ser Gln Ser Pro Ser Ser Leu
20 25 30
Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln
35 40 45
Ser Leu Leu Tyr Ser Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
50 55 60
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
65 70 75 80
Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val
100 105 110
Tyr Tyr Cys Gln Gln Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gly Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
145 150 155 160
Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Ser Phe Thr Gly Tyr His Met His Trp Val Lys Gln Ser His Val Lys
180 185 190
Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Tyr Asn Gly Ala Ala Ser
195 200 205
His Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Ala Arg Gly Trp Asp Tyr Asp Gly Gly Tyr Tyr
245 250 255
Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 16
<211> 495
<212> PRT
<213> 人工序列
<400> 16
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser
35 40 45
Gln Ser Leu Leu Tyr Ser Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr
50 55 60
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser
65 70 75 80
Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
85 90 95
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
100 105 110
Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gln
115 120 125
Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu
145 150 155 160
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
165 170 175
Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly
180 185 190
Gln Arg Leu Glu Trp Met Gly Arg Ile Asn Pro Tyr Asn Gly Ala Ala
195 200 205
Ser His Asn Gln Lys Phe Lys Asp Arg Val Thr Ile Thr Arg Asp Thr
210 215 220
Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
225 230 235 240
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Trp Asp Tyr Asp Gly Gly Tyr
245 250 255
Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
260 265 270
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
305 310 315 320
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
325 330 335
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
340 345 350
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
355 360 365
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
370 375 380
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
385 390 395 400
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
405 410 415
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
420 425 430
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
435 440 445
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
450 455 460
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
465 470 475 480
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490 495
<210> 17
<211> 489
<212> PRT
<213> 人工序列
<400> 17
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 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met
20 25 30
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser
35 40 45
Asn Val Ile Ser Ser Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala
50 55 60
Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val
65 70 75 80
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
85 90 95
Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
130 135 140
Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser
145 150 155 160
Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ala Tyr
165 170 175
Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met
180 185 190
Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu Lys
195 200 205
Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu
210 215 220
Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
225 230 235 240
Lys Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro
260 265 270
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
275 280 285
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
290 295 300
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
305 310 315 320
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
325 330 335
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
340 345 350
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
355 360 365
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
370 375 380
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
385 390 395 400
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
405 410 415
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
420 425 430
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
435 440 445
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
465 470 475 480
Leu His Met Gln Ala Leu Pro Pro Arg
485
<210> 18
<211> 1461
<212> DNA
<213> 人工序列
<400> 18
atggccctcc cagtcacagc tctgctgctc ccactcgccc tgctgctgca cgccgcacgg 60
cctcagatcg tcctcaccca gtctccagcc atcatgagcg cctctccagg cgagaaggtg 120
acaatgactt gttccgcaag cagctcagtt agttacatgt attggtacca gcagaaacca 180
ggctcttctc caaggctcct gatcttcgac acttctaacc tggcatccgg agtcccagtg 240
cggttcagcg gcagcggcag tggaacatct tatagcctga ctattagccg gatggaggca 300
gaagatgcag ctacctacta ctgtcaacag tggtctagtt atccactcac ttttggtgca 360
gggaccaagc tggagctcaa aggtggagga ggatctggcg gcggagggag tggaggaggc 420
ggctcacagg tccaactgca gcaatctggt gcagaactga tgaagcctgg agcaagcgtt 480
aaaatctctt gcaaagcaac aggatacacc ttttcttcct actggataga gtgggtgaaa 540
caaagaccag gacacggact ggaatggata ggagagatct ttcctggcag cggctcaatc 600
aagtataacg agaagttcaa agggaaagcc accttcacag ccgatacctc ttctaataca 660
gcctatatgc agctgtcatc cctgaccagc gaagattctg ctgttcacta ctgtgcacgc 720
ggaggaacat acaatgacta ctctctgttt gactattggg gacagggcac cacactgact 780
gtgagttcta ctacgacccc tgcaccgcgg ccgcctactc ctgcacctac aatcgcaagt 840
cagccactga gtctcagacc cgaagcatgc cgccctgctg caggcggagc tgtccataca 900
cgcggactgg actttgcatg cgatatatac atctgggcac cactggccgg cacttgcggc 960
gtgctgctcc tgtccctcgt gattaccctg tactgcaaac gcggcaggaa gaagctcctg 1020
tatatcttta aacagccctt catgaggcca gtgcagacca ctcaagagga agacggttgt 1080
agctgccggt ttcccgagga agaagaggga ggctgcgagc tccgcgtgaa gttctcccgc 1140
tcagccgatg cacccgccta tcagcaaggg cagaaccagc tgtacaatga gctcaacctg 1200
ggaagaaggg aggaatatga cgttctggat aaacggcgcg gtcgcgatcc cgaaatgggt 1260
gggaagcctc gcaggaagaa tcctcaggaa gggctctaca atgagctgca gaaagacaaa 1320
atggcagagg cctattctga aatcggcatg aagggcgagc gccgcagagg caaaggacac 1380
gacggcctgt accagggcct gtctacagcc accaaggaca cctatgacgc tctccacatg 1440
caagccctgc caccaaggtg a 1461
<210> 19
<211> 1449
<212> DNA
<213> 人工序列
<400> 19
atggctctcc ctgttactgc actcctgctc ccactggcac tgctgctgca tgccgctcgg 60
ccacaaatag ttctgactca gagtcctgcc attatgtcag cctctcctgg agagaaggtc 120
acaatgactt gctctgcctc tagtagtgtg tcttacatat actggtacca gcagaagcct 180
ggttcttctc ccggactgct gatctatgac acatccaatc tggcttcagg cgttcccgtc 240
agattcagcg ggtctggatc tggcacaagc tattctctga ccatctcaag aatggaggct 300
gaagatgctg ctacttatta ttgccaacag tggtcttcct ttccaccaac cttcggtgca 360
ggtaccaagc tcgaactcaa aggtggagga ggaagcggag gaggcggtag tggtggaggt 420
gggtcccaag ttcagctgca acagcccgga tctgatctgg ttcggcccgg agctagcgtg 480
aaactgtctt gcaaggctag cggatacact ttcacccgct attggatgca ctgggttaag 540
cagcggccag gacacggact ggagtggatt ggctatatct acccaggcag cgggacaagt 600
aactacgatg agaaattcaa gagtaaggct actctgactg tcgatacaag ttcctcaacc 660
gcttacatgc agctctcttc actcaccagc gaagacagtg ctgtttatta ctgcaccagg 720
gaagctcggt acaccatgga ttattggggt caaggaactt ctgtgacagt gtcaagcact 780
acgacccctg caccgcggcc gcctactcct gcacctacaa tcgcaagtca gccactgagt 840
ctcagacccg aagcatgccg ccctgctgca ggcggagctg tccatacacg cggactggac 900
tttgcatgcg atatatacat ctgggcacca ctggccggca cttgcggcgt gctgctcctg 960
tccctcgtga ttaccctgta ctgcaaacgc ggcaggaaga agctcctgta tatctttaaa 1020
cagcccttca tgaggccagt gcagaccact caagaggaag acggttgtag ctgccggttt 1080
cccgaggaag aagagggagg ctgcgagctc cgcgtgaagt tctcccgctc agccgatgca 1140
cccgcctatc agcaagggca gaaccagctg tacaatgagc tcaacctggg aagaagggag 1200
gaatatgacg ttctggataa acggcgcggt cgcgatcccg aaatgggtgg gaagcctcgc 1260
aggaagaatc ctcaggaagg gctctacaat gagctgcaga aagacaaaat ggcagaggcc 1320
tattctgaaa tcggcatgaa gggcgagcgc cgcagaggca aaggacacga cggcctgtac 1380
cagggcctgt ctacagccac caaggacacc tatgacgctc tccacatgca agccctgcca 1440
ccaaggtga 1449
<210> 20
<211> 1485
<212> DNA
<213> 人工序列
<400> 20
atggctctgc ctgttactgc actgctgctc cctctggctc tcctcctgca tgctgctcgg 60
cctgatatag tgatgtccca aagcccatcc agcctggccg tgtccgtcgg cgaaaaggtg 120
actatgagtt gcaaatccag ccaaagcctg ctgtacagtg acaaccaaaa gaactacctg 180
gcatggtacc agcagaagcc tggacagtca ccaaagctcc tcatctactg ggctagcaca 240
agggagagcg gcgtcccaga caggtttact ggcagcggga gtggcaccga tttcaccctg 300
acaataagct ctgtcaaggc cgaagacctc gctgtgtact attgtcagca gtattatacc 360
tatccctata ctttcggtgg agggaccaaa ctcgagatta aaggaggtgg cggctctgga 420
ggtggaggtt ccggcggtgg cggtagtgaa gtgcagctgc agcagagcgg gcctgaactc 480
gtgaaacctg gtgcctccgt taaaatctcc tgcaaggcca gcggctactc attcacaggg 540
tatcacatgc attgggtgaa gcagagccac gtcaaatcac tggaatggat cggcaggatt 600
aatccataca atggcgctgc ttcacataac cagaagttca aggacaaagc caccctgact 660
gtcgataagt catcaagtac agcatacatg gagctgcatt ccctgactag cgaggacagc 720
gctgtttact actgcgcacg cggctgggac tacgacggtg gctattacgc catggactac 780
tggggacaag gcaccagcgt cacagtttca agtactacga cccctgcacc gcggccgcct 840
actcctgcac ctacaatcgc aagtcagcca ctgagtctca gacccgaagc atgccgccct 900
gctgcaggcg gagctgtcca tacacgcgga ctggactttg catgcgatat atacatctgg 960
gcaccactgg ccggcacttg cggcgtgctg ctcctgtccc tcgtgattac cctgtactgc 1020
aaacgcggca ggaagaagct cctgtatatc tttaaacagc ccttcatgag gccagtgcag 1080
accactcaag aggaagacgg ttgtagctgc cggtttcccg aggaagaaga gggaggctgc 1140
gagctccgcg tgaagttctc ccgctcagcc gatgcacccg cctatcagca agggcagaac 1200
cagctgtaca atgagctcaa cctgggaaga agggaggaat atgacgttct ggataaacgg 1260
cgcggtcgcg atcccgaaat gggtgggaag cctcgcagga agaatcctca ggaagggctc 1320
tacaatgagc tgcagaaaga caaaatggca gaggcctatt ctgaaatcgg catgaagggc 1380
gagcgccgca gaggcaaagg acacgacggc ctgtaccagg gcctgtctac agccaccaag 1440
gacacctatg acgctctcca catgcaagcc ctgccaccaa ggtga 1485
<210> 21
<211> 1488
<212> DNA
<213> 人工序列
<400> 21
atggatatga gggttcctgc acaactcctg ggactcctcc tgctctggct gagaggcgca 60
agatgtgata tccagatgac ccagtctcct agtagcctgt ctgcctccgt cggcgatcgg 120
gtgaccatta cttgcaaatc ctcacagagc ctcctctact ccgataatca gaagaactac 180
ctcgcctggt atcaacagaa accagggaag gcacctaagc tgctgatcta ctgggctagt 240
acccgcgaat ccggcgtccc tagcaggttc tctggcagcg ggagcgggac agatttcacc 300
ctcactatct cctccctgca gcctgaagac ttcgcaactt actactgtca gcagtattat 360
acttacccat acactttcgg acagggaaca aaactggaaa ttaaaggtgg aggtggatct 420
ggtggcggtg gcagtggcgg aggcgggtct gaagtccaac tggtgcagag cggtgcagag 480
gtgaagaagc ctggagcatc agtgaaggtg tcttgcaaag ccagtggcta cacattcact 540
ggatatcata tgcattgggt taggcaggca cccggccagc ggctggagtg gatgggaaga 600
atcaaccctt ataatggcgc tgcctctcac aatcaaaagt ttaaggatcg ggtcactatc 660
actcgggaca cttccgcaag caccgcctat atggagctga gcagcctgcg gagtgaagac 720
acagcagtct actactgtgc tcgcggatgg gactatgacg gcggttatta tgccatggat 780
tactggggac agggcacact ggtcaccgtg agcagcacta cgacccctgc accgcggccg 840
cctactcctg cacctacaat cgcaagtcag ccactgagtc tcagacccga agcatgccgc 900
cctgctgcag gcggagctgt ccatacacgc ggactggact ttgcatgcga tatatacatc 960
tgggcaccac tggccggcac ttgcggcgtg ctgctcctgt ccctcgtgat taccctgtac 1020
tgcaaacgcg gcaggaagaa gctcctgtat atctttaaac agcccttcat gaggccagtg 1080
cagaccactc aagaggaaga cggttgtagc tgccggtttc ccgaggaaga agagggaggc 1140
tgcgagctcc gcgtgaagtt ctcccgctca gccgatgcac ccgcctatca gcaagggcag 1200
aaccagctgt acaatgagct caacctggga agaagggagg aatatgacgt tctggataaa 1260
cggcgcggtc gcgatcccga aatgggtggg aagcctcgca ggaagaatcc tcaggaaggg 1320
ctctacaatg agctgcagaa agacaaaatg gcagaggcct attctgaaat cggcatgaag 1380
ggcgagcgcc gcagaggcaa aggacacgac ggcctgtacc agggcctgtc tacagccacc 1440
aaggacacct atgacgctct ccacatgcaa gccctgccac caaggtga 1488
<210> 22
<211> 1470
<212> DNA
<213> 人工序列
<400> 22
atggctctgc ctgttactgc actcctcctc ccactggcac tcctcctgca tgcagccagg 60
cccgagaatg tgctgacaca gtctccagcc atcatgagcg cctctcccgg tgaaaaggtt 120
actatgacct gtcgggcaag ttcaaatgtg atctcctctt atgtgcactg gtaccagcag 180
cgctcaggtg caagcccaaa gctgtggatc tattccactt ctaacctggc ctccggtgtc 240
ccagcccgct tttctggaag cgggtcaggc acctcatact ccctcaccat atcaagtgtg 300
gaagctgagg atgcagctac ttactactgc caacagtact ctggttaccc actgaccttc 360
ggagccggga caaagctgga actgggagga ggcgggtccg gcggtggagg gtccggaggt 420
ggcgggtccg atatccagct gcaagagtca ggcccaggcc tggtcaaacc ttcccaaagc 480
ctgagtctca cctgttccgt gacaggttat tccattacta gcgcatatta ctggaactgg 540
ataagacaat tcccaggaaa caaactcgag tggatgggct acatctcata cgacgggcgg 600
aacaactata acccatccct gaagaatcgg atttccatca ctagagacac atccaagaac 660
cagttctttc tcaagctgaa tagcgtgaca actgaggata cagcaaccta ctattgcgcc 720
aaggaaggag actatgatgt tggcaactat tatgcaatgg actattgggg acagggcaca 780
tcagtgaccg tgagcagcac tacgacccct gcaccgcggc cgcctactcc tgcacctaca 840
atcgcaagtc agccactgag tctcagaccc gaagcatgcc gccctgctgc aggcggagct 900
gtccatacac gcggactgga ctttgcatgc gatatataca tctgggcacc actggccggc 960
acttgcggcg tgctgctcct gtccctcgtg attaccctgt actgcaaacg cggcaggaag 1020
aagctcctgt atatctttaa acagcccttc atgaggccag tgcagaccac tcaagaggaa 1080
gacggttgta gctgccggtt tcccgaggaa gaagagggag gctgcgagct ccgcgtgaag 1140
ttctcccgct cagccgatgc acccgcctat cagcaagggc agaaccagct gtacaatgag 1200
ctcaacctgg gaagaaggga ggaatatgac gttctggata aacggcgcgg tcgcgatccc 1260
gaaatgggtg ggaagcctcg caggaagaat cctcaggaag ggctctacaa tgagctgcag 1320
aaagacaaaa tggcagaggc ctattctgaa atcggcatga agggcgagcg ccgcagaggc 1380
aaaggacacg acggcctgta ccagggcctg tctacagcca ccaaggacac ctatgacgct 1440
ctccacatgc aagccctgcc accaaggtga 1470
<210> 23
<211> 21
<212> PRT
<213> 人工序列
<400> 23
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
<210> 24
<211> 63
<212> DNA
<213> 人工序列
<400> 24
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cct 63
<210> 25
<211> 22
<212> PRT
<213> 人工序列
<400> 25
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys
20
<210> 26
<211> 66
<212> DNA
<213> 人工序列
<400> 26
atggatatga gggttcctgc acaactcctg ggactcctcc tgctctggct gagaggcgca 60
agatgt 66
<210> 27
<211> 45
<212> PRT
<213> 人工序列
<400> 27
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> 28
<211> 135
<212> DNA
<213> 人工序列
<400> 28
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 29
<211> 24
<212> PRT
<213> 人工序列
<400> 29
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> 30
<211> 72
<212> DNA
<213> 人工序列
<400> 30
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 31
<211> 42
<212> PRT
<213> 人工序列
<400> 31
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> 32
<211> 126
<212> DNA
<213> 人工序列
<400> 32
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60
acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120
gagctg 126
<210> 33
<211> 112
<212> PRT
<213> 人工序列
<400> 33
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 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> 34
<211> 336
<212> DNA
<213> 人工序列
<400> 34
agggtgaagt tttctcggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60
tacaacgagc tgaatctggg caggcgcgag gagtacgacg tgctggataa gcggagaggc 120
agagatcccg agatgggagg caagccaagg aggaagaacc ctcaggaggg cctgtataat 180
gagctgcaga aggacaagat ggccgaggcc tactctgaga tcggcatgaa gggagagcgg 240
agaaggggca agggacacga tggcctgtat cagggcctga gcacagccac caaggacacc 300
tacgatgcac tgcacatgca ggccctgcca cctagg 336

Claims (14)

1.靶向CLL1嵌合抗原受体，其特征在于，所述靶向CLL1嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；
所述抗原结合结构域为抗CLL1抗体；
所述抗原结合结构域包括如SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列；或者
所述抗原结合结构域包括如SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列；或者
所述抗原结合结构域包括如SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；或者
所述抗原结合结构域包括如SEQ ID NO:7和SEQ ID NO:8～10之一所示的氨基酸序列；或者
所述抗原结合结构域包括如SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列；
所述靶向CLL1嵌合抗原受体包括信号肽、抗CLL1抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ；
所述靶向CLL1嵌合抗原受体包括如SEQ ID NO:13所示的氨基酸序列；或者
所述靶向CLL1嵌合抗原受体包括如SEQ ID NO:14所示的氨基酸序列；或者
所述靶向CLL1嵌合抗原受体包括如SEQ ID NO:15所示的氨基酸序列；或者
所述靶向CLL1嵌合抗原受体包括如SEQ ID NO:16所示的氨基酸序列；或者
所述靶向CLL1嵌合抗原受体包括如SEQ ID NO:17所示的氨基酸序列。
2.核酸分子，其特征在于，所述核酸分子包括权利要求1所述的靶向CLL1嵌合抗原受体的编码基因；
所述核酸分子包括如SEQ ID NO:18所示的核酸序列；或者
所述核酸分子包括如SEQ ID NO:19所示的核酸序列；或者
所述核酸分子包括如SEQ ID NO:20所示的核酸序列；或者
所述核酸分子包括如SEQ ID NO:21所示的核酸序列；或者
所述核酸分子包括如SEQ ID NO:22所示的核酸序列。
3.一种表达载体，其特征在于，所述表达载体包括权利要求2所述的核酸分子。
4.根据权利要求3所述的表达载体，其特征在于，所述表达载体为含有权利要求2所述的核酸分子的病毒载体或非病毒载体。
5.根据权利要求4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；
所述非病毒载体包括Piggybac转座子系统、Sleeping Beauty转座子系统或纳米载体中的任意一种。
6.一种重组慢病毒，其特征在于，所述重组慢病毒由转染有权利要求4或5所述的表达载体和辅助质粒的哺乳细胞制备得到。
7.一种嵌合抗原受体免疫细胞，其特征在于，所述嵌合抗原受体免疫细胞表达权利要求1所述的靶向CLL1嵌合抗原受体。
8.根据权利要求7所述的嵌合抗原受体免疫细胞，其特征在于，所述嵌合抗原受体免疫细胞的基因组中整合有权利要求2所述的核酸分子。
9.根据权利要求8所述的嵌合抗原受体免疫细胞，其特征在于，所述嵌合抗原受体免疫细胞包括权利要求4或5所述的表达载体和/或权利要求6所述的重组慢病毒。
10.根据权利要求9所述的嵌合抗原受体免疫细胞，其特征在于，所述免疫细胞包括T细胞、NK细胞或巨噬细胞中的任意一种。
11.根据权利要求10所述的嵌合抗原受体免疫细胞，其特征在于，所述T细胞包括αβΤ细胞和/或γδΤ细胞。
12.一种药物组合物，其特征在于，所述药物组合物包括权利要求7-11任一项所述的嵌合抗原受体免疫细胞。
13.根据权利要求12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。
14.权利要求1所述的靶向CLL1嵌合抗原受体、权利要求2所述的核酸分子、权利要求4或5所述的表达载体、权利要求6所述的重组慢病毒、权利要求7-11任一项所述的嵌合抗原受体免疫细胞或权利要求12或13所述的药物组合物在制备恶性肿瘤治疗药物中的应用；
所述恶性肿瘤为急性髓系白血病。
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GB2207722.6A GB2606869A (en) 2020-12-11 2020-12-22 CLL1-Targeting chimeric antigen receptor and application thereof
KR1020227011658A KR20220084040A (ko) 2020-12-11 2020-12-22 Cll1을 표적으로 하는 키메라 항원 수용체 및 그의 응용
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