CN113248622A - 一种靶向cll1和nkg2d配体的双靶点嵌合抗原受体及其应用 - Google Patents

一种靶向cll1和nkg2d配体的双靶点嵌合抗原受体及其应用 Download PDF

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CN113248622A
CN113248622A CN202011462878.9A CN202011462878A CN113248622A CN 113248622 A CN113248622 A CN 113248622A CN 202011462878 A CN202011462878 A CN 202011462878A CN 113248622 A CN113248622 A CN 113248622A
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李光超
罗敏
周兆
丁雯
王学俊
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Guangzhou Bio Gene Technology Co Ltd
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Abstract

本发明提供了一种靶向CLL1和NKG2D配体的双靶点嵌合抗原受体及其应用，所述双靶点嵌合抗原受体包括信号肽、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包括抗CLL1抗体和NKG2D。本发明构建同时靶向CLL1和NKG2D配体的CAR分子，实现了全面靶向急性髓系白血病细胞的效果，表达靶向CLL1和NKG2D配体双靶点CAR的CAR‑T细胞肿瘤清除作用显著，避免了抗原逃逸现象的发生，在肿瘤治疗领域具有重要意义。

Description

一种靶向CLL1和NKG2D配体的双靶点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，涉及一种靶向CLL1和NKG2D配体的双靶点嵌合抗原受体及其应用。
背景技术
在肿瘤免疫治疗领域，CAR-T是研究最火热的肿瘤免疫治疗方法，其在白血病、淋巴瘤、多发性骨髓瘤的治疗中展现出惊艳的效果。目前，以CD19为靶点的CAR-T产品研究相对深入，美国已批准的Kymriah和Yescarta均是以CD19为靶点的治疗血液肿瘤的CAR-T产品。从全球来看，CAR-T的研发管线迅速扩张，既包括新靶点的探索，如BCMA、CD123、CD33等，又包括新适应症的拓展，如由血液肿瘤向实体瘤的进阶。全球已有多家公司的项目推进到了临床阶段，预计未来将陆续有针对不同肿瘤的CAR-T产品问世。
急性髓系白血病(AML)是一种在治疗应答及生存中均具有异质性的疾病，其主要特征为骨髓与外周血中原始祖细胞和幼稚髓性细胞分化阻滞，使得处于不同分化阶段的原始髓细胞异常增殖并积累，而具有正常功能的红细胞、血小板和白细胞急剧减少。AML普遍发生于各个年龄阶段，老年人(>60岁)尤为常见，是成人急性白血病中最普遍的一种。AML具有原发性、难治性、易复发性及治疗致死性，是一种致命疾病，常用的治疗方法包括化学治疗和造血干细胞移植。
将CAR-T细胞疗法应用于AML治疗的关键是找到合适的识别抗原，提高CAR-T对恶性肿瘤细胞的靶向性，同时避免对非恶性髓样细胞造成伤害。为了选择性地靶向白血病髓系细胞，目前开发出CD33及CD123特异性CAR-T细胞，可能为AML患者提供了一种新的治疗机制。
CLL1是一种II型跨膜蛋白，表达于绝大多数AML患者的癌细胞和白血病干细胞(LSCs)上，在CD34阳性祖细胞上的表达很低，在正常造血干细胞上不表达。因此，CLL1蛋白是一个非常有希望的治疗AML的靶点。
值得注意的是，CD19 CAR-T细胞在AML等血液系统恶性肿瘤早期临床试验中显示了不俗的业绩，但治疗后复发及抗原逃逸仍是需要克服的难题。双特异性抗原靶点的CAR-T是一种新的治疗选择。根据目前已知的免疫逃逸机制和相关临床试验结果，减少CAR-T治疗后复发的概率以增加病患的生存率，将成为CAR-T研发的新方向之一。
NKG2D是固有免疫系统中的一个重要激活性受体，通过识别靶细胞表面诱导产生的配体来传递活化信号并激活免疫系统，从而对靶细胞发挥杀伤作用。所有NK细胞、CD8阳性T细胞、大多数NKT细胞、巨噬细胞和γδT细胞都表达NKG2D。NKG2D的配体主要有两类：一是MHC I类链相关A/B分子(MHC class I chain-related molecules A/B，MICA/B)，另一类是UL16结合蛋白(UL16 binding proteins，ULBPs)。NKG2D配体主要表达于大多数上皮源性肿瘤细胞上，如卵巢癌、结肠癌、白血病等，在正常细胞上表达极少。一般情况下，NKG2D配体在正常细胞中不表达或者表达量非常少，但是当细胞受到感染或者发生癌变时，NKG2D配体的表达量会大幅度上升，使得NKG2D配体成为一个潜在的CAR-T治疗靶点。CYAD-01(又名CAR-T NKG2D)是Celyad公司近期研发的一种靶向NKG2D配体的CAR-T细胞疗法，临床I期试验结果表明CYAD-01细胞疗法在治疗AML和多发性骨髓瘤(MM)时表现出良好的安全性和耐受性。
然而，目前鲜有共靶向NKG2D配体与AML抗原的CAR-T技术，以降低疾病复发率。
发明内容
针对现有技术的不足和实际需求，本发明提供了一种靶向CLL1和NKG2D配体的双靶点嵌合抗原受体及其应用，所述双靶点嵌合抗原受体同时靶向CLL1和NKG2D配体(NKG2DL)，表达所述双靶点嵌合抗原受体的CAR-T细胞有效而全面地消除白血病干细胞(LSCs)和肿瘤负荷，最大限度地减少了抗原逃逸现象。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种靶向CLL1和NKG2D配体的双靶点嵌合抗原受体，所述双靶点嵌合抗原受体包括信号肽、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包括抗CLL1抗体和NKG2D。
本发明中，构建同时靶向CLL1和NKG2DL抗原的双靶点嵌合抗原受体，实现了全面靶向急性髓系白血病细胞的效果，有效解决了肿瘤抗原异质性的问题，最大限度地减少了抗原逃逸现象。
优选地，所述NKG2D为NKG2D胞外段。
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区、抗CLL1抗体重链可变区和NKG2D胞外段。
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体重链可变区、抗CLL1抗体轻链可变区和NKG2D胞外段。
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段、抗CLL1抗体轻链可变区和抗CLL1抗体重链可变区。
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段、抗CLL1抗体重链可变区和抗CLL1抗体轻链可变区。
优选地，所述抗CLL1抗体H27H4包括SEQ ID NO:1所示的轻链可变区和SEQ ID NO:2所示的重链可变区；
SEQ ID NO:1：
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIK；
SEQ ID NO:2：
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSS。
优选地，所述抗CLL1抗体1075.7包括SEQ ID NO:3所示的轻链可变区和SEQ IDNO:4所示的重链可变区；
SEQ ID NO:3：
ENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLEL；
SEQ ID NO:4：
DIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSS。
优选地，所述抗CLL1抗体27H4包括SEQ ID NO:5所示的轻链可变区和SEQ ID NO:6所示的重链可变区；
SEQ ID NO:5：
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIK；
SEQ ID NO:6：
EVQLQQSGPELVKPGASVKISCKASGYSFTGYHMHWVKQSHVKSLEWIGRINPYNGAASHNQKFKDKATLTVDKSSSTAYMELHSLTSEDSAVYYCARGWDYDGGYYAMDYWGQGTSVTVSS。
优选地，所述NKG2D胞外段(NKG2D-ECD)包括SEQ ID NO:7所示的氨基酸序列；
SEQ ID NO:7：
LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV。
优选地，H27H4-NKG2D-CAR的抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区SEQ ID NO:1、抗CLL1抗体重链可变区SEQ ID NO:2和NKG2D胞外段SEQ ID NO:7。
优选地，NKG2D-H27H4-CAR的抗原结合结构域包括依次串联的NKG2D胞外段SEQ IDNO:7、抗CLL1抗体轻链可变区SEQ ID NO:1和抗CLL1抗体重链可变区SEQ ID NO:2。
优选地，1075.7-NKG2D-CAR的抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区SEQ ID NO:3、抗CLL1抗体重链可变区SEQ ID NO:4和NKG2D胞外段SEQ ID NO:7。
优选地，NKG2D-1075.7-CAR的抗原结合结构域包括依次串联的NKG2D胞外段SEQID NO:7、抗CLL1抗体轻链可变区SEQ ID NO:3和抗CLL1抗体重链可变区SEQ ID NO:4。
优选地，27H4-NKG2D-CAR的抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区SEQ ID NO:5、抗CLL1抗体重链可变区SEQ ID NO:6和NKG2D胞外段SEQ ID NO:7。
优选地，NKG2D-27H4-CAR的抗原结合结构域包括依次串联的NKG2D胞外段SEQ IDNO:7、抗CLL1抗体轻链可变区SEQ ID NO:5和抗CLL1抗体重链可变区SEQ ID NO:6。
优选地，所述抗CLL1抗体轻链可变区、抗CLL1抗体重链可变区和NKG2D胞外段之间通过连接肽连接。
优选地，所述信号肽包括CD8α信号肽。
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。
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。
作为优选技术方案，所述嵌合抗原受体H27H4-NKG2D-CAR包括SEQ ID NO:8所示的氨基酸序列；
SEQ ID NO:8：
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
作为优选技术方案，所述嵌合抗原受体NKG2D-H27H4-CAR包括SEQ ID NO:9所示的氨基酸序列；SEQ ID NO:9：
MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
作为优选技术方案，所述嵌合抗原受体1075.7-NKG2D-CAR包括SEQ ID NO:10所示的氨基酸序列；
SEQ ID NO:10：
MALPVTALLLPLALLLHAARPENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
作为优选技术方案，所述嵌合抗原受体NKG2D-1075.7-CAR包括SEQ ID NO:11所示的氨基酸序列；
SEQ ID NO:11：
MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGSTSGSGKPGSGEGSTKGENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
作为优选技术方案，所述嵌合抗原受体27H4-NKG2D-CAR包括SEQ ID NO:12所示的氨基酸序列；
SEQ ID NO:12：
MDMRVPAQLLGLLLLWLRGARCDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKASGYSFTGYHMHWVKQSHVKSLEWIGRINPYNGAASHNQKFKDKATLTVDKSSSTAYMELHSLTSEDSAVYYCARGWDYDGGYYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
作为优选技术方案，所述嵌合抗原受体NKG2D-27H4-CAR包括SEQ ID NO:13所示的氨基酸序列；
SEQ ID NO:13：
MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGSTSGSGKPGSGEGSTKGDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKASGYSFTGYHMHWVKQSHVKSLEWIGRINPYNGAASHNQKFKDKATLTVDKSSSTAYMELHSLTSEDSAVYYCARGWDYDGGYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。
第二方面，本发明提供了一种核酸分子，所述核酸分子包括第一方面所述的双靶点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:14所示的核酸序列，为27H4-NKG2D-CAR的编码基因；
SEQ ID NO:14：
atggatatgagggttcctgcacaactcctgggactcctcctgctctggctgagaggcgcaagatgtgacatcgttatgtcccagtctccatctagcctggcagttagcgtgggtgagaaggtgacaatgtcctgcaagtctagtcagtcactgctctacagtgacaatcagaagaactacctggcttggtatcagcagaaacctggccaatcacccaagctcctgatctactgggcttcaactagagagtctggagtcccagataggtttacaggcagcggatctgggaccgacttcacactcaccatctcttccgtgaaagccgaagacctcgccgtttactattgccagcaatactatacatatccttacacatttggcggcggaaccaagctcgagatcaaaggaggtggtggatctggaggaggtggatcaggtggcggaggctccgaagtccagctgcagcagagtggaccagaactggtgaagccaggagcatccgtgaagatctcttgcaaagcctctgggtacagcttcaccgggtatcatatgcactgggtcaaacagtcccatgtgaaatccctggagtggatcggcagaatcaatccctacaatggtgctgcctcccataaccagaaattcaaagataaggcaactctcactgtcgataagtcctcttctaccgcttacatggaactccattccctgacctctgaggatagcgccgtgtactactgtgctagagggtgggactatgatggagggtactacgcaatggactattggggccaagggacatccgtcaccgtgtctagcggatcaaccagcggaagcgggaagcctggttccggagaaggatcaactaaaggcctctttaaccaggaggtgcaaattccactgactgaatcctattgcggcccttgtcccaagaactggatttgttataagaacaactgttatcaattcttcgatgagtctaagaactggtatgaaagccaagcatcctgtatgtcccagaacgcttcactcctgaaggtctacagcaaagaagatcaggacctcctcaagctggtgaaatcatatcactggatgggactggtgcacatccctacaaacggctcatggcagtgggaggacggctccatcctgagcccaaatctgctcacaatcattgaaatgcagaagggtgattgcgctctctacgcaagctcttttaaaggctacatagagaactgctccactcccaatacatacatttgtatgcagcgcactgtgactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
优选地，所述核酸分子包括SEQ ID NO:15所示的核酸序列，为NKG2D-27H4-CAR的编码基因；
SEQ ID NO:15：
atggcactgcctgttacagcactgctcctccctctggctctcctgctgcacgcagcaagacctctctttaaccaggaggtgcaaattccactgactgaatcctattgcggcccttgtcccaagaactggatttgttataagaacaactgttatcaattcttcgatgagtctaagaactggtatgaaagccaagcatcctgtatgtcccagaacgcttcactcctgaaggtctacagcaaagaagatcaggacctcctcaagctggtgaaatcatatcactggatgggactggtgcacatccctacaaacggctcatggcagtgggaggacggctccatcctgagcccaaatctgctcacaatcattgaaatgcagaagggtgattgcgctctctacgcaagctcttttaaaggctacatagagaactgctccactcccaatacatacatttgtatgcagcgcactgtgggatcaaccagcggaagcgggaagcctggttccggagaaggatcaactaaaggcgacatcgttatgtcccagtctccatctagcctggcagttagcgtgggtgagaaggtgacaatgtcctgcaagtctagtcagtcactgctctacagtgacaatcagaagaactacctggcttggtatcagcagaaacctggccaatcacccaagctcctgatctactgggcttcaactagagagtctggagtcccagataggtttacaggcagcggatctgggaccgacttcacactcaccatctcttccgtgaaagccgaagacctcgccgtttactattgccagcaatactatacatatccttacacatttggcggcggaaccaagctcgagatcaaaggaggtggtggatctggaggaggtggatcaggtggcggaggctccgaagtccagctgcagcagagtggaccagaactggtgaagccaggagcatccgtgaagatctcttgcaaagcctctgggtacagcttcaccgggtatcatatgcactgggtcaaacagtcccatgtgaaatccctggagtggatcggcagaatcaatccctacaatggtgctgcctcccataaccagaaattcaaagataaggcaactctcactgtcgataagtcctcttctaccgcttacatggaactccattccctgacctctgaggatagcgccgtgtactactgtgctagagggtgggactatgatggagggtactacgcaatggactattggggccaagggacatccgtcaccgtgtctagcactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。
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。
优选地，所述表达载体为含有第二方面所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。
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CAR-T细胞，所述CAR-T细胞表达第一方面所述的双靶点嵌合抗原受体。
本发明中，采用靶向CLL1和NKG2D配体的双靶点嵌合抗原受体构建双特异性CAR-T细胞，实现了对白血病干细胞(LSCs)和肿瘤负荷的全面覆盖和清除，在降低肿瘤复发和解决肿瘤异质性方面具有显著效果。
优选地，所述CAR-T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CAR-T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。
第六方面，本发明提供了一种第五方面所述的CAR-T细胞的制备方法，所述方法包括将第一方面所述的双靶点嵌合抗原受体的编码基因导入T细胞的步骤。
第七方面，本发明提供了第一方面所述的双靶点嵌合抗原受体、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或第五方面所述的CAR-T细胞在制备疾病治疗药物中的应用。
优选地，所述疾病包括血液肿瘤。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的双靶点嵌合抗原受体同时靶向CLL1和NKG2D配体，与单靶点嵌合抗原受体相比，双靶点嵌合抗原受体对CLL1阳性细胞和/或NKG2D配体阳性细胞具有更高效的靶向活性，有效解决了肿瘤抗原异质性的问题，最大限度地减少了抗原逃逸现象；
(2)本发明的CLL1和NKG2D配体双特异性CAR-T靶向效率高、肿瘤杀伤作用强，对CLL1阳性和/或NKG2D配体阳性细胞均具有显著的杀伤效果，与CLL1阳性和/或NKG2D配体阳性细胞共孵育后释放大量IFN-γ，达到了全面清除急性髓系白血病细胞的效果，降低了疾病复发几率。
附图说明
图1A为靶向CLL1和NKG2DL的双靶点CAR的结构示意图，图1B为抗CLL1单靶点CAR和抗NKG2DL单靶点CAR的结构示意图；
图2A为pCDH-H27H4-NKG2D-CAR的载体图谱，图2B为局部pCDH-H27H4-NKG2D-CAR结构；
图3A为pCDH-NKG2D-H27H4-CAR的载体图谱，图3B为局部pCDH-NKG2D-H27H4-CAR结构，图3C为pCDH-NKG2D-27H4-CAR的载体图谱，图3D为局部pCDH-NKG2D-27H4-CAR结构，图3E为pCDH-27H4-NKG2D-CAR的载体图谱，图3F为局部pCDH-27H4-NKG2D-CAR结构；
图4A为CAR-T在慢病毒转染7天内的扩增曲线，图4B为第7天CAR-T的细胞数量，图4C为第7天CAR-T的细胞扩增倍数；
图5A为流式检测H27H4-CAR-T、H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T结合CLL1-Fc抗原的能力，图5B为流式检测27H4-CAR-T、27H4-NKG2D-CAR-T、NKG2D-27H4-CAR-T、1075.7-CAR-T、1075.7-NKG2D-CAR-T、NKG2D-1075.7-CAR-T结合CLL1-Fc抗原的能力，图5C为流式检测NKG2D-CAR-T、H27H4-CAR-T、H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T表达NKG2D的情况，图5D为流式检测27H4-CAR-T、27H4-NKG2D-CAR-T、NKG2D-27H4-CAR-T、1075.7-CAR-T、1075.7-NKG2D-CAR-T、NKG2D-1075.7-CAR-T表达NKG2D的情况，图5E为CD8CAR-T细胞、CD4CAR-T细胞对NKG2D的表达，；
图6A为H27H4-CAR-T、NKG2D-CAR-T、H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T的杀伤能力，图6B为27H4-CAR-T、NKG2D-CAR-T、27H4-NKG2D-CAR-T、NKG2D-27H4-CAR-T的杀伤能力，图6C为1075.7-CAR-T、NKG2D-CAR-T、1075.7-NKG2D-CAR-T、NKG2D-1075.7-CAR-T的杀伤能力；
图7A为H27H4-CAR-T、H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T分泌细胞因子IFN-γ的情况，图7B为27H4-CAR-T、27H4-NKG2D-CAR-T、NKG2D-27H4-CAR-T、1075.7-CAR-T、1075.7-NKG2D-CAR-T、NKG2D-1075.7-CAR-T分泌细胞因子IFN-γ的情况。
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嵌合抗原受体的设计
本实施例采用抗CLL1抗体H27H4(SEQ ID NO:1～2)、1075.7(SEQ ID NO:3～4)或27H4(SEQ ID NO:5～6)与NKG2D胞外段(NKG2D-ECD，SEQ ID NO:7)作为抗原结合结构域，结合CD8α信号肽(SEQ ID NO:16～17)或IgGκ轻链信号肽(SEQ ID NO:34～35)、CD8α铰链区(SEQ ID NO:18～19)和跨膜区(SEQ ID NO:20～21)、4-1BB共刺激结构域(SEQ ID NO:22～23)和CD3ζ信号传导结构域(SEQ ID NO:24～25)，构建靶向CLL1和NKG2D配体(NKG2DL)的双靶点CAR(SEQ ID NO:8～13)，结构示意图如图1A所示；
本实施例还构建了抗CLL1单靶点CAR(SEQ ID NO:26～27、SEQ ID NO:28～29、SEQID NO:30～31)和抗NKG2DL单靶点CAR(SEQ ID NO:32～33)，结构示意图如图1B所示。
CAR分子的相关信息见表1。
表1CAR分子的相关信息
CAR名称 靶点 抗原结合区域 序列
NKG2D-CAR NKG2DL NKG2D-ECD SEQ ID NO:32
H27H4-CAR CLL1 H27H4-scFv SEQ ID NO:26
H27H4-NKG2D-CAR NKG2DL+CLL1 H27H4-scFv串联NKG2D-ECD SEQ ID NO:8
NKG2D-H27H4-CAR NKG2DL+CLL1 NKG2D-ECD串联H27H4-scFv SEQ ID NO:9
1075.7-CAR CLL1 1075.7-scFv SEQ ID NO:28
1075.7-NKG2D-CAR NKG2DL+CLL1 1075.7-scFv串联NKG2D-ECD SEQ ID NO:10
NKG2D-1075.7-CAR NKG2DL+CLL1 NKG2D-ECD串联1075.7-scFv SEQ ID NO:11
27H4-CAR CLL1 27H4-scFv SEQ ID NO:30
27H4-NKG2D-CAR NKG2DL+CLL1 27H4-scFv串联NKG2D-ECD SEQ ID NO:12
NKG2D-27H4-CAR NKG2DL+CLL1 NKG2D-ECD串联27H4-scFv SEQ ID NO:13
实施例2嵌合抗原受体表达载体的构建
(1)根据实施例1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2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2
成分 添加量
线性化pCDH-EF1-MCS载体 2μL(50ng)
CAR基因 10μL(150ng)
T4 DNA连接缓冲液 2μL
T4 DNA连接酶(NEB) 1μL
ddH<sub>2</sub>O 补齐至20μL
示例性地，靶向CLL1和NKG2DL的双靶点CAR慢病毒表达载体pCDH-H27H4-NKG2D-CAR和pCDH-NKG2D-H27H4-CAR图谱分别如图2A、图2B和图3A、图3B所示；pCDH-NKG2D-27H4-CAR和pCDH-27H4-NKG2D-CAR图谱分别如图3C、图3D和图3E、图3F所示。
实施例3慢病毒包装
本实施例采用四质粒系统对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。
包装的慢病毒滴度见表3。
表3
CAR慢病毒 滴度(TU/mL)
NKG2D-CAR 8.35E7
H27H4-CAR 1.56E8
H27H4-NKG2D-CAR 1.48E8
NKG2D-H27H4-CAR 1.52E8
1075.7-CAR 5.67E7
1075.7-NKG2D-CAR 1.54E8
NKG2D-1075.7-CAR 2.12E8
27H4-CAR 6.78E7
27H4-NKG2D-CAR 1.35E8
NKG2D-27H4-CAR 1.12E8
实施例4慢病毒转染T细胞和CAR-T细胞的扩增
采集30mL全血，将外周血与PBS缓冲液按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PBS缓冲液多次洗涤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；
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，得到成功转染慢病毒的T细胞。
实验组的初始感染细胞数为100万，对照组(Control)的起始细胞数为200万，每两天进行一次计数并更换含300IU/mL的X-VIVO，将细胞浓度维持在0.5×106～1×106/mL，连续观察计数7天，评估CAR-T细胞的扩增情况。
结果如图4A、图4B和图4C所示，各CAR-T组扩增良好，没有显著区别。
实施例5CAR-T细胞对CAR的表达
本实施例采用流式细胞仪检测抗CLL1单靶点CAR-T(H27H4-CAR-T、27H4-CAR-T、1075.7-CAR-T)、抗NKG2DL单靶点CAR-T(NKG2D-CAR-T)和同时靶向CLL1和NKG2DL的双靶点CAR-T(H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T、27H4-NKG2D-CAR-T、NKG2D-27H4-CAR-T、1075.7-NKG2D-CAR-T、NKG2D-1075.7-CAR-T)对CAR分子的表达。
利用融合Fc标签的CLL1蛋白(CLL-1Protein Fc Tag；CLA-H5266；ACROBiosystems公司)及对应的抗Fc二抗检测CAR-T结合CLL1的能力，未转染CAR的T细胞(Control T)作为阴性对照。
如图5A所示，慢病毒感染后第5天和第7天，H27H4-CAR-T、H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T能有效结合CLL1抗原蛋白，说明CAR-T成功表达H27H4，在第7天的H27H4阳性率分别为72.1％、95.7％、59.8％。
如图5B所示，未转导的T细胞(Mock T)和NKG2D-CAR-T不结合CLL1-Fc抗原蛋白，27H4-CAR-T的结合阳性率为58.5％，27H4-NKG2D-CAR-T的结合阳性率为68.5％，NKG2D-27H4-CAR-T的结合阳性率为65.3％；1075.7-CAR-T的结合阳性率为45.7％，1075.7-NKG2D-CAR-T的结合阳性率为39.5％，NKG2D-1075.7-CAR-T的结合阳性率为43.2％。可以看到，只有转导包含抗CLL1抗体scFv的CAR结构后，CAR-T才能有效结合CLL1-Fc抗原。
利用APC偶联的抗NKG2D抗体(biolegend)检测CAR-T表达NKG2D的情况，未转染CAR的T细胞(Control T)作为阴性对照。
如图5C所示，Control T本身表达NKG2D，在第7天的NKG2D阳性率为66.7％，NKG2D阳性细胞主要为CD8 T细胞，CD4 T细胞基本不表达NKG2D；慢病毒感染后第5天和第7天，NKG2D-CAR-T、H27H4-CAR-T、H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T均有不同程度的NKG2D表达率，在第7天的NKG2D阳性率分别为91.2％、41.4％、96.9％、76.1％。
如图5D所示，未转导的T细胞(Mock T)本身就有47.4％表达NKG2D，NKG2D-CAR-T对NKG2D的表达率为82.3％，27H4-CAR-T对NKG2D的表达率为43.6％，27H4-NKG2D-CAR-T对NKG2D的表达率为86.4％，NKG2D-27H4-CAR-T对NKG2D的表达率为81.3％；1075.7-CAR-T对NKG2D的表达率为42.5％，1075.7-NKG2D-CAR-T对NKG2D的表达率为67.9％，NKG2D-1075.7-CAR-T对NKG2D的表达率为63.8％。可以看到，激活的T细胞本身表达一定程度的NKG2D，当转导包含NKG2D胞外域的CAR结构后，NKG2D表达显著增加。
本实施例进一步检测了CAR-T细胞中CD4和CD8亚群细胞分别表达NKG2D的情况。结果如图5E显示，在未经转导的T细胞组中(T mock)，有42.71％的CD8阳性T细胞本身表达NKG2D，而在CD4阳性T细胞中没有检测到NKG2D的表达。当转导H27H4-NKG2D-CAR(其中含有NKG2D胞外域)慢病毒后，CD8阳性T细胞的NKG2D表达率为80.94％，显著高于未转导组的42.71％。而CD4阳性T细胞的NKG2D表达率达到75.39％，说明转导H27H4-NKG2D CAR慢病毒后，CD4亚群T细胞(本身不表达NKG2D)成功表达双靶点的CAR结构。
实施例6荧光素酶法检测CAR-T杀伤作用
本实施例首先采用FITC anti-human CD371(CLL1)抗体(biolegend品牌)和抗NKG2DL抗体(APC-NKG2D-Fc、anti-MICA、anti-ULBP2/5/6)检测靶细胞K562、H929、U937对CLL1和NKG2DL的表达情况。
结果如表4所示，K562细胞为CLL1阴性表达、NKG2DL阳性表达(用NKG2D-Fc、anti-MICA、anti-ULBP2/5/6都检出阳性)，H929细胞为CLL1阴性表达、NKG2DL阴性表达(anti-CLL1、NKG2D-Fc、anti-MICA、anti-ULBP2/5/6都检出阴性)，U937细胞为CLL1阳性表达、NKG2DL阳性表达(NKG2D-Fc、anti-ULBP2/5/6检出阳性)。
表4肿瘤靶细胞对CLL1和NKG2D配体(NKG2DL)的表达情况
K562 H929 U937 所用检测抗体
NKG2DL ++ - +++ NKG2D-Fc
MICA + - - anti-MICA
ULBP2/5/6 + - +++ anti-ULBP2/5/6
CLL1 - - +++ anti-CLL1
本实施例进一步利用荧光素酶法评估CAR-T细胞的杀伤能力，步骤如下：
将靶细胞按照每孔1×105个细胞的密度加入到黑色96孔板中(上海晶安，货号J09602)，按照E:T＝3接种CAR-T细胞，小心地将细胞振荡混匀后，置于细胞培养箱中孵育6h；
加入等量(90～100μL)荧光素酶底物试剂(Bright-Glo Luciferase AssaySystem，货号E2620)，等待至少2min；将96孔板去盖，用FLUOstar OMEGA仪器检测，根据以下公式计算杀伤效率。
杀伤率％＝(1-RLU实验组/RLU空白组)×100％
如图6A所示，H27H4-CAR-T只对CLL1阳性细胞U937具有杀伤作用，对CLL1阴性细胞(K562和H292)杀伤作用不明显，说明H27H4-CAR-T的特异性杀伤效果；NKG2D-CAR-T对NKG2D阳性细胞K562、U937具有杀伤效果，对H929也具有很强的杀伤作用，原因可能是H929存在其他类型NKG2D配体；两种双靶点CAR-T细胞H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T对K562、H929和U937均具有很强的杀伤作用。基于27H4和1075.7抗体scFv的CAR-T及其与NKG2D配体组成的双靶点CAR-T，在杀伤各个靶细胞时，也表现出与H27H4类似的杀伤效果，如图6B和6C所示。说明靶向CLL1和NKG2D配体的双靶点CAR-T都能更有效地杀伤CLL1阳性的肿瘤细胞。
实施例7CAR-T分泌细胞因子的情况
本实施例采用Human IFN-γELISA试剂盒(欣博盛)分析CAR-T在杀伤靶细胞的过程中对IFN-γ的分泌量，将CAR-T细胞与不同的靶细胞按3:1的效靶比共孵育21小时，取上清用酶联免疫法(ELISA)检测培养液上清中IFN-γ的分泌量。
如图7A所示，H27H4-CAR-T细胞与U937(CLL1表达阳性)共孵育后释放大量IFN-γ，与K562和H929(CLL1表达阴性)共孵育后未见大量IFN-γ释放，说明抗CLL1的H27H4-CAR-T细胞的杀伤具有特异性；NKG2D-CAR-T细胞与K562和H929细胞共孵育后都显著分泌IFN-γ，与U937细胞共孵育后分泌的IFN-γ较少，但实际显示有杀伤效果；双靶点H27H4-NKG2D-CAR-T、NKG2D-H27H4-CAR-T与三种靶细胞共孵育后均分泌较多的IFN-γ，说明双靶点CAR-T对肿瘤的杀伤具有普遍性；未经修饰的T Mock细胞未见大量IFN-释放。
如图7B所示，包含有NKG2D胞外域的CAR-T与三种靶细胞都能有效分泌IFN-γ；包含有抗CLL1单链抗体(27H4和1075.7)的CAR-T与CLL1阳性靶细胞U937都能分泌大量IFN-γ，而与表达阴性的K562和H929细胞共孵育后几乎不分泌IFN-γ。其中NKG2D-27H4-CAR和27H4-NKG2D-CAR在杀伤三种靶细胞时，都能分泌出较高水平的IFN-γ，表现出优秀的抗肿瘤能力。
综上所述，本发明的嵌合抗原受体同时靶向CLL1和NKG2D配体，构建的双特异性CAR-T细胞对白血病干细胞(LSCs)和肿瘤负荷具有更全面的靶向杀伤清除作用，有效解决了肿瘤抗原异质性的问题，最大限度地减少了抗原逃逸现象。
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> 一种靶向CLL1和NKG2D配体的双靶点嵌合抗原受体及其应用
<130> 20201210
<160> 35
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115 120
<210> 7
<211> 135
<212> PRT
<213> 人工序列
<400> 7
Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly
1 5 10 15
Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe
20 25 30
Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser
35 40 45
Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu
50 55 60
Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro
65 70 75 80
Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn
85 90 95
Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala
100 105 110
Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr
115 120 125
Ile Cys Met Gln Arg Thr Val
130 135
<210> 8
<211> 648
<212> PRT
<213> 人工序列
<400> 8
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
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
275 280 285
Lys Gly Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
290 295 300
Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr
305 310 315 320
Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys
325 330 335
Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln
340 345 350
Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His
355 360 365
Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser
370 375 380
Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu
385 390 395 400
Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn
405 410 415
Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg
420 425 430
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
435 440 445
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
450 455 460
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
465 470 475 480
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
485 490 495
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
500 505 510
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
515 520 525
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
530 535 540
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
545 550 555 560
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
565 570 575
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
580 585 590
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
595 600 605
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
610 615 620
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
625 630 635 640
His Met Gln Ala Leu Pro Pro Arg
645
<210> 9
<211> 647
<212> PRT
<213> 人工序列
<400> 9
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 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Gly Ser Thr Ser
145 150 155 160
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile
165 170 175
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
180 185 190
Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asp Asn
195 200 205
Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
210 215 220
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Ser
225 230 235 240
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
245 250 255
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
260 265 270
Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly
275 280 285
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
290 295 300
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val
305 310 315 320
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr His Met
325 330 335
His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Arg
340 345 350
Ile Asn Pro Tyr Asn Gly Ala Ala Ser His Asn Gln Lys Phe Lys Asp
355 360 365
Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu
370 375 380
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
385 390 395 400
Gly Trp Asp Tyr Asp Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
405 410 415
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
420 425 430
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
435 440 445
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
450 455 460
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
465 470 475 480
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
485 490 495
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
500 505 510
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
515 520 525
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
530 535 540
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
545 550 555 560
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
565 570 575
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
580 585 590
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
595 600 605
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
610 615 620
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
625 630 635 640
Met Gln Ala Leu Pro Pro Arg
645
<210> 10
<211> 642
<212> PRT
<213> 人工序列
<400> 10
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 Gly Ser Thr Ser Gly Ser
260 265 270
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Leu Phe Asn Gln
275 280 285
Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys
290 295 300
Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser
305 310 315 320
Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser
325 330 335
Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val
340 345 350
Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser
355 360 365
Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile
370 375 380
Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys
385 390 395 400
Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln
405 410 415
Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
420 425 430
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
435 440 445
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
450 455 460
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
465 470 475 480
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
485 490 495
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
500 505 510
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
515 520 525
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
530 535 540
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
545 550 555 560
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
565 570 575
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
580 585 590
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
595 600 605
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
610 615 620
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
625 630 635 640
Pro Arg
<210> 11
<211> 642
<212> PRT
<213> 人工序列
<400> 11
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 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Gly Ser Thr Ser
145 150 155 160
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Asn
165 170 175
Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys
180 185 190
Val Thr Met Thr Cys Arg Ala Ser Ser Asn Val Ile Ser Ser Tyr Val
195 200 205
His Trp Tyr Gln Gln Arg Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr
210 215 220
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
225 230 235 240
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu
245 250 255
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro Leu Thr
260 265 270
Phe Gly Ala Gly Thr Lys Leu Glu Leu Gly Gly Gly Gly Ser Gly Gly
275 280 285
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Gln Glu Ser Gly
290 295 300
Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Ser Val
305 310 315 320
Thr Gly Tyr Ser Ile Thr Ser Ala Tyr Tyr Trp Asn Trp Ile Arg Gln
325 330 335
Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly
340 345 350
Arg Asn Asn Tyr Asn Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg
355 360 365
Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr
370 375 380
Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Lys Glu Gly Asp Tyr Asp Val
385 390 395 400
Gly Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
405 410 415
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
420 425 430
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
435 440 445
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
450 455 460
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
465 470 475 480
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
485 490 495
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
500 505 510
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
515 520 525
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
530 535 540
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
545 550 555 560
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
565 570 575
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
580 585 590
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
595 600 605
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
610 615 620
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
625 630 635 640
Pro Arg
<210> 12
<211> 648
<212> PRT
<213> 人工序列
<400> 12
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 Val Met Ser Gln Ser Pro Ser Ser
20 25 30
Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser 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 Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser
65 70 75 80
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
85 90 95
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala
100 105 110
Val Tyr Tyr Cys Gln Gln Tyr Tyr Thr Tyr Pro Tyr Thr Phe Gly Gly
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 Gln Gln Ser Gly Pro Glu
145 150 155 160
Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
165 170 175
Tyr Ser Phe Thr Gly Tyr His Met His Trp Val Lys Gln Ser His Val
180 185 190
Lys Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Tyr Asn Gly Ala Ala
195 200 205
Ser His Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys
210 215 220
Ser Ser Ser Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp
225 230 235 240
Ser 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 Ser Val Thr Val Ser Ser
260 265 270
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
275 280 285
Lys Gly Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
290 295 300
Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr
305 310 315 320
Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys
325 330 335
Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln
340 345 350
Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His
355 360 365
Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser
370 375 380
Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu
385 390 395 400
Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn
405 410 415
Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg
420 425 430
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
435 440 445
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
450 455 460
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
465 470 475 480
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
485 490 495
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
500 505 510
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
515 520 525
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
530 535 540
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
545 550 555 560
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
565 570 575
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
580 585 590
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
595 600 605
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
610 615 620
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
625 630 635 640
His Met Gln Ala Leu Pro Pro Arg
645
<210> 13
<211> 647
<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 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Gly Ser Thr Ser
145 150 155 160
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile
165 170 175
Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly Glu Lys
180 185 190
Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asp Asn
195 200 205
Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
210 215 220
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
225 230 235 240
Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
245 250 255
Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr
260 265 270
Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
275 280 285
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
290 295 300
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
305 310 315 320
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr His Met
325 330 335
His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile Gly Arg
340 345 350
Ile Asn Pro Tyr Asn Gly Ala Ala Ser His Asn Gln Lys Phe Lys Asp
355 360 365
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
370 375 380
Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
385 390 395 400
Gly Trp Asp Tyr Asp Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
405 410 415
Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
420 425 430
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
435 440 445
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
450 455 460
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
465 470 475 480
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
485 490 495
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
500 505 510
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
515 520 525
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
530 535 540
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
545 550 555 560
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
565 570 575
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
580 585 590
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
595 600 605
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
610 615 620
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
625 630 635 640
Met Gln Ala Leu Pro Pro Arg
645
<210> 14
<211> 1947
<212> DNA
<213> 人工序列
<400> 14
atggatatga gggttcctgc acaactcctg ggactcctcc tgctctggct gagaggcgca 60
agatgtgaca tcgttatgtc ccagtctcca tctagcctgg cagttagcgt gggtgagaag 120
gtgacaatgt cctgcaagtc tagtcagtca ctgctctaca gtgacaatca gaagaactac 180
ctggcttggt atcagcagaa acctggccaa tcacccaagc tcctgatcta ctgggcttca 240
actagagagt ctggagtccc agataggttt acaggcagcg gatctgggac cgacttcaca 300
ctcaccatct cttccgtgaa agccgaagac ctcgccgttt actattgcca gcaatactat 360
acatatcctt acacatttgg cggcggaacc aagctcgaga tcaaaggagg tggtggatct 420
ggaggaggtg gatcaggtgg cggaggctcc gaagtccagc tgcagcagag tggaccagaa 480
ctggtgaagc caggagcatc cgtgaagatc tcttgcaaag cctctgggta cagcttcacc 540
gggtatcata tgcactgggt caaacagtcc catgtgaaat ccctggagtg gatcggcaga 600
atcaatccct acaatggtgc tgcctcccat aaccagaaat tcaaagataa ggcaactctc 660
actgtcgata agtcctcttc taccgcttac atggaactcc attccctgac ctctgaggat 720
agcgccgtgt actactgtgc tagagggtgg gactatgatg gagggtacta cgcaatggac 780
tattggggcc aagggacatc cgtcaccgtg tctagcggat caaccagcgg aagcgggaag 840
cctggttccg gagaaggatc aactaaaggc ctctttaacc aggaggtgca aattccactg 900
actgaatcct attgcggccc ttgtcccaag aactggattt gttataagaa caactgttat 960
caattcttcg atgagtctaa gaactggtat gaaagccaag catcctgtat gtcccagaac 1020
gcttcactcc tgaaggtcta cagcaaagaa gatcaggacc tcctcaagct ggtgaaatca 1080
tatcactgga tgggactggt gcacatccct acaaacggct catggcagtg ggaggacggc 1140
tccatcctga gcccaaatct gctcacaatc attgaaatgc agaagggtga ttgcgctctc 1200
tacgcaagct cttttaaagg ctacatagag aactgctcca ctcccaatac atacatttgt 1260
atgcagcgca ctgtgactac gacccctgca ccgcggccgc ctactcctgc acctacaatc 1320
gcaagtcagc cactgagtct cagacccgaa gcatgccgcc ctgctgcagg cggagctgtc 1380
catacacgcg gactggactt tgcatgcgat atatacatct gggcaccact ggccggcact 1440
tgcggcgtgc tgctcctgtc cctcgtgatt accctgtact gcaaacgcgg caggaagaag 1500
ctcctgtata tctttaaaca gcccttcatg aggccagtgc agaccactca agaggaagac 1560
ggttgtagct gccggtttcc cgaggaagaa gagggaggct gcgagctccg cgtgaagttc 1620
tcccgctcag ccgatgcacc cgcctatcag caagggcaga accagctgta caatgagctc 1680
aacctgggaa gaagggagga atatgacgtt ctggataaac ggcgcggtcg cgatcccgaa 1740
atgggtggga agcctcgcag gaagaatcct caggaagggc tctacaatga gctgcagaaa 1800
gacaaaatgg cagaggccta ttctgaaatc ggcatgaagg gcgagcgccg cagaggcaaa 1860
ggacacgacg gcctgtacca gggcctgtct acagccacca aggacaccta tgacgctctc 1920
cacatgcaag ccctgccacc aaggtga 1947
<210> 15
<211> 1944
<212> DNA
<213> 人工序列
<400> 15
atggcactgc ctgttacagc actgctcctc cctctggctc tcctgctgca cgcagcaaga 60
cctctcttta accaggaggt gcaaattcca ctgactgaat cctattgcgg cccttgtccc 120
aagaactgga tttgttataa gaacaactgt tatcaattct tcgatgagtc taagaactgg 180
tatgaaagcc aagcatcctg tatgtcccag aacgcttcac tcctgaaggt ctacagcaaa 240
gaagatcagg acctcctcaa gctggtgaaa tcatatcact ggatgggact ggtgcacatc 300
cctacaaacg gctcatggca gtgggaggac ggctccatcc tgagcccaaa tctgctcaca 360
atcattgaaa tgcagaaggg tgattgcgct ctctacgcaa gctcttttaa aggctacata 420
gagaactgct ccactcccaa tacatacatt tgtatgcagc gcactgtggg atcaaccagc 480
ggaagcggga agcctggttc cggagaagga tcaactaaag gcgacatcgt tatgtcccag 540
tctccatcta gcctggcagt tagcgtgggt gagaaggtga caatgtcctg caagtctagt 600
cagtcactgc tctacagtga caatcagaag aactacctgg cttggtatca gcagaaacct 660
ggccaatcac ccaagctcct gatctactgg gcttcaacta gagagtctgg agtcccagat 720
aggtttacag gcagcggatc tgggaccgac ttcacactca ccatctcttc cgtgaaagcc 780
gaagacctcg ccgtttacta ttgccagcaa tactatacat atccttacac atttggcggc 840
ggaaccaagc tcgagatcaa aggaggtggt ggatctggag gaggtggatc aggtggcgga 900
ggctccgaag tccagctgca gcagagtgga ccagaactgg tgaagccagg agcatccgtg 960
aagatctctt gcaaagcctc tgggtacagc ttcaccgggt atcatatgca ctgggtcaaa 1020
cagtcccatg tgaaatccct ggagtggatc ggcagaatca atccctacaa tggtgctgcc 1080
tcccataacc agaaattcaa agataaggca actctcactg tcgataagtc ctcttctacc 1140
gcttacatgg aactccattc cctgacctct gaggatagcg ccgtgtacta ctgtgctaga 1200
gggtgggact atgatggagg gtactacgca atggactatt ggggccaagg gacatccgtc 1260
accgtgtcta gcactacgac ccctgcaccg cggccgccta ctcctgcacc tacaatcgca 1320
agtcagccac tgagtctcag acccgaagca tgccgccctg ctgcaggcgg agctgtccat 1380
acacgcggac tggactttgc atgcgatata tacatctggg caccactggc cggcacttgc 1440
ggcgtgctgc tcctgtccct cgtgattacc ctgtactgca aacgcggcag gaagaagctc 1500
ctgtatatct ttaaacagcc cttcatgagg ccagtgcaga ccactcaaga ggaagacggt 1560
tgtagctgcc ggtttcccga ggaagaagag ggaggctgcg agctccgcgt gaagttctcc 1620
cgctcagccg atgcacccgc ctatcagcaa gggcagaacc agctgtacaa tgagctcaac 1680
ctgggaagaa gggaggaata tgacgttctg gataaacggc gcggtcgcga tcccgaaatg 1740
ggtgggaagc ctcgcaggaa gaatcctcag gaagggctct acaatgagct gcagaaagac 1800
aaaatggcag aggcctattc tgaaatcggc atgaagggcg agcgccgcag aggcaaagga 1860
cacgacggcc tgtaccaggg cctgtctaca gccaccaagg acacctatga cgctctccac 1920
atgcaagccc tgccaccaag gtga 1944
<210> 16
<211> 21
<212> PRT
<213> 人工序列
<400> 16
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> 17
<211> 63
<212> DNA
<213> 人工序列
<400> 17
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cct 63
<210> 18
<211> 45
<212> PRT
<213> 人工序列
<400> 18
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> 19
<211> 135
<212> DNA
<213> 人工序列
<400> 19
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 20
<211> 24
<212> PRT
<213> 人工序列
<400> 20
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> 21
<211> 72
<212> DNA
<213> 人工序列
<400> 21
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 22
<211> 42
<212> PRT
<213> 人工序列
<400> 22
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> 23
<211> 126
<212> DNA
<213> 人工序列
<400> 23
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60
acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120
gagctg 126
<210> 24
<211> 112
<212> PRT
<213> 人工序列
<400> 24
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> 25
<211> 336
<212> DNA
<213> 人工序列
<400> 25
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
<210> 26
<211> 495
<212> PRT
<213> 人工序列
<400> 26
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> 27
<211> 1488
<212> DNA
<213> 人工序列
<400> 27
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> 28
<211> 489
<212> PRT
<213> 人工序列
<400> 28
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro 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> 29
<211> 1470
<212> DNA
<213> 人工序列
<400> 29
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> 30
<211> 494
<212> PRT
<213> 人工序列
<400> 30
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> 31
<211> 1485
<212> DNA
<213> 人工序列
<400> 31
atggctctgc ctgttactgc actgctgctg cctctggctc tgctcctcca cgcagctaga 60
cctgacatcg ttatgtccca gtctccatct agcctggcag ttagcgtggg tgagaaggtg 120
acaatgtcct gcaagtctag tcagtcactg ctctacagtg acaatcagaa gaactacctg 180
gcttggtatc agcagaaacc tggccaatca cccaagctcc tgatctactg ggcttcaact 240
agagagtctg gagtcccaga taggtttaca ggcagcggat ctgggaccga cttcacactc 300
accatctctt ccgtgaaagc cgaagacctc gccgtttact attgccagca atactataca 360
tatccttaca catttggcgg cggaaccaag ctcgagatca aaggaggtgg tggatctgga 420
ggaggtggat caggtggcgg aggctccgaa gtccagctgc agcagagtgg accagaactg 480
gtgaagccag gagcatccgt gaagatctct tgcaaagcct ctgggtacag cttcaccggg 540
tatcatatgc actgggtcaa acagtcccat gtgaaatccc tggagtggat cggcagaatc 600
aatccctaca atggtgctgc ctcccataac cagaaattca aagataaggc aactctcact 660
gtcgataagt cctcttctac cgcttacatg gaactccatt ccctgacctc tgaggatagc 720
gccgtgtact actgtgctag agggtgggac tatgatggag ggtactacgc aatggactat 780
tggggccaag ggacatccgt caccgtgtct agcactacga 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> 32
<211> 379
<212> PRT
<213> 人工序列
<400> 32
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 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr
20 25 30
Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn
35 40 45
Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln
50 55 60
Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys
65 70 75 80
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly
85 90 95
Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser
100 105 110
Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp
115 120 125
Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser
130 135 140
Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro
145 150 155 160
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
165 170 175
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
180 185 190
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
195 200 205
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
210 215 220
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
225 230 235 240
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
245 250 255
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
260 265 270
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
275 280 285
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
290 295 300
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
305 310 315 320
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
325 330 335
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
340 345 350
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
355 360 365
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
370 375
<210> 33
<211> 1140
<212> DNA
<213> 人工序列
<400> 33
atggcactgc ctgttacagc actgctcctc cctctggctc tcctgctgca cgcagcaaga 60
cctctcttta accaggaggt gcaaattcca ctgactgaat cctattgcgg cccttgtccc 120
aagaactgga tttgttataa gaacaactgt tatcaattct tcgatgagtc taagaactgg 180
tatgaaagcc aagcatcctg tatgtcccag aacgcttcac tcctgaaggt ctacagcaaa 240
gaagatcagg acctcctcaa gctggtgaaa tcatatcact ggatgggact ggtgcacatc 300
cctacaaacg gctcatggca gtgggaggac ggctccatcc tgagcccaaa tctgctcaca 360
atcattgaaa tgcagaaggg tgattgcgct ctctacgcaa gctcttttaa aggctacata 420
gagaactgct ccactcccaa tacatacatt tgtatgcagc gcactgtgac tacgacccct 480
gcaccgcggc cgcctactcc tgcacctaca atcgcaagtc agccactgag tctcagaccc 540
gaagcatgcc gccctgctgc aggcggagct gtccatacac gcggactgga ctttgcatgc 600
gatatataca tctgggcacc actggccggc acttgcggcg tgctgctcct gtccctcgtg 660
attaccctgt actgcaaacg cggcaggaag aagctcctgt atatctttaa acagcccttc 720
atgaggccag tgcagaccac tcaagaggaa gacggttgta gctgccggtt tcccgaggaa 780
gaagagggag gctgcgagct ccgcgtgaag ttctcccgct cagccgatgc acccgcctat 840
cagcaagggc agaaccagct gtacaatgag ctcaacctgg gaagaaggga ggaatatgac 900
gttctggata aacggcgcgg tcgcgatccc gaaatgggtg ggaagcctcg caggaagaat 960
cctcaggaag ggctctacaa tgagctgcag aaagacaaaa tggcagaggc ctattctgaa 1020
atcggcatga agggcgagcg ccgcagaggc aaaggacacg acggcctgta ccagggcctg 1080
tctacagcca ccaaggacac ctatgacgct ctccacatgc aagccctgcc accaaggtga 1140
<210> 34
<211> 22
<212> PRT
<213> 人工序列
<400> 34
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> 35
<211> 66
<212> DNA
<213> 人工序列
<400> 35
atggatatga gggttcctgc acaactcctg ggactcctcc tgctctggct gagaggcgca 60
agatgt 66

Claims (10)

1.一种靶向CLL1和NKG2D配体的双靶点嵌合抗原受体，其特征在于，所述双靶点嵌合抗原受体包括信号肽、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包括抗CLL1抗体和NKG2D。
2.根据权利要求1所述的双靶点嵌合抗原受体，其特征在于，所述NKG2D为NKG2D胞外段；
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区、抗CLL1抗体重链可变区和NKG2D胞外段；
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体重链可变区、抗CLL1抗体轻链可变区和NKG2D胞外段；
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段、抗CLL1抗体轻链可变区和抗CLL1抗体重链可变区；
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段、抗CLL1抗体重链可变区和抗CLL1抗体轻链可变区；
优选地，所述抗CLL1抗体包括SEQ ID NO:1所示的轻链可变区和SEQ ID NO:2所示的重链可变区；
优选地，所述抗CLL1抗体包括SEQ ID NO:3所示的轻链可变区和SEQ ID NO:4所示的重链可变区；
优选地，所述抗CLL1抗体包括SEQ ID NO:5所示的轻链可变区和SEQ ID NO:6所示的重链可变区；
优选地，所述NKG2D胞外段包括SEQ ID NO:7所示的氨基酸序列；
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区SEQ ID NO:1、抗CLL1抗体重链可变区SEQ ID NO:2和NKG2D胞外段SEQ ID NO:7；
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段SEQ ID NO:7、抗CLL1抗体轻链可变区SEQ ID NO:1和抗CLL1抗体重链可变区SEQ ID NO:2；
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区SEQ ID NO:3、抗CLL1抗体重链可变区SEQ ID NO:4和NKG2D胞外段SEQ ID NO:7；
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段SEQ ID NO:7、抗CLL1抗体轻链可变区SEQ ID NO:3和抗CLL1抗体重链可变区SEQ ID NO:4；
优选地，所述抗原结合结构域包括依次串联的抗CLL1抗体轻链可变区SEQ ID NO:5、抗CLL1抗体重链可变区SEQ ID NO:6和NKG2D胞外段SEQ ID NO:7；
优选地，所述抗原结合结构域包括依次串联的NKG2D胞外段SEQ ID NO:7、抗CLL1抗体轻链可变区SEQ ID NO:5和抗CLL1抗体重链可变区SEQ ID NO:6。
3.根据权利要求1或2所述的双靶点嵌合抗原受体，其特征在于，所述抗CLL1抗体轻链可变区、抗CLL1抗体重链可变区和NKG2D胞外段之间通过连接肽连接。
4.根据权利要求1-3任一项所述的双靶点嵌合抗原受体，其特征在于，所述信号肽包括CD8α信号肽；
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；
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；
优选地，所述嵌合抗原受体包括SEQ ID NO:8～13之一所示的氨基酸序列。
5.一种核酸分子，其特征在于，所述核酸分子包括权利要求1-4任一项所述的双靶点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:14～15之一所示的核酸序列。
6.一种表达载体，其特征在于，所述表达载体包括权利要求5所述的核酸分子；
优选地，所述表达载体为含有权利要求5所述的核酸分子的病毒载体；
优选地，所述表达载体为含有权利要求5所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。
7.一种重组慢病毒，其特征在于，所述重组慢病毒由转染有权利要求6所述的表达载体和辅助质粒的哺乳细胞制备得到。
8.一种CAR-T细胞，其特征在于，所述CAR-T细胞表达权利要求1-4任一项所述的双靶点嵌合抗原受体；
优选地，所述CAR-T细胞的基因组中整合有权利要求5所述的核酸分子；
优选地，所述CAR-T细胞包括权利要求6所述的表达载体和/或权利要求7所述的重组慢病毒。
9.一种权利要求8所述的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1-4任一项所述的双靶点嵌合抗原受体的编码基因导入T细胞的步骤。
10.权利要求1-4任一项所述的双靶点嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的表达载体、权利要求7所述的重组慢病毒或权利要求8所述的CAR-T细胞在制备疾病治疗药物中的应用；
优选地，所述疾病包括血液肿瘤。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114031690A (zh) * 2021-12-03 2022-02-11 广州百暨基因科技有限公司 靶向ccr1和nkg2d配体的嵌合抗原受体及其应用
CN114702596A (zh) * 2022-03-11 2022-07-05 南京凯地医疗技术有限公司 靶向人cd33和nkg2dl的嵌合抗原受体细胞及其制备方法和应用
CN117736340A (zh) * 2023-12-20 2024-03-22 青岛麦迪赛斯医疗技术有限公司 一种靶向cll-1和nkg2dl的嵌合抗原受体nk细胞的构建及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406516A (zh) * 2015-01-26 2017-11-28 塞勒克提斯公司 用于癌症免疫治疗的抗CLL1特异性单链嵌合抗原受体(scCAR)
CN110028589A (zh) * 2018-02-07 2019-07-19 阿思科力(苏州)生物科技有限公司 嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用
CN110981970A (zh) * 2019-12-25 2020-04-10 华夏源（上海）细胞基因工程股份有限公司 一种靶向nkg2d配体和cd19的双靶点嵌合抗原受体及其表达载体和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406516A (zh) * 2015-01-26 2017-11-28 塞勒克提斯公司 用于癌症免疫治疗的抗CLL1特异性单链嵌合抗原受体(scCAR)
CN110028589A (zh) * 2018-02-07 2019-07-19 阿思科力(苏州)生物科技有限公司 嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用
CN110981970A (zh) * 2019-12-25 2020-04-10 华夏源（上海）细胞基因工程股份有限公司 一种靶向nkg2d配体和cd19的双靶点嵌合抗原受体及其表达载体和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VERMA, BIKASH: "A NKG2D-based CAR-T therapy in a multinational phase I dose escalation and expansion study targeting multiple solid and hematologic tumor types.", 《JOURNAL OF CLINICAL ONCOLOGY》 *
邓新娜: "NKG2D嵌合抗原受体T细胞对人结肠癌细胞杀伤作用的体内外研究", 《万方学位论文》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114031690A (zh) * 2021-12-03 2022-02-11 广州百暨基因科技有限公司 靶向ccr1和nkg2d配体的嵌合抗原受体及其应用
CN114702596A (zh) * 2022-03-11 2022-07-05 南京凯地医疗技术有限公司 靶向人cd33和nkg2dl的嵌合抗原受体细胞及其制备方法和应用
CN114702596B (zh) * 2022-03-11 2023-02-14 南京凯地医疗技术有限公司 靶向人cd33和nkg2dl的嵌合抗原受体细胞及其制备方法和应用
CN117736340A (zh) * 2023-12-20 2024-03-22 青岛麦迪赛斯医疗技术有限公司 一种靶向cll-1和nkg2dl的嵌合抗原受体nk细胞的构建及应用
CN117736340B (zh) * 2023-12-20 2024-07-26 青岛麦迪赛斯医疗技术有限公司 一种靶向cll-1和nkg2dl的嵌合抗原受体nk细胞的构建及应用

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