CN114539421B - 以HIV-1 gp120与CD20为双靶点的CAR-T细胞制备方法及其应用 - Google Patents

以HIV-1 gp120与CD20为双靶点的CAR-T细胞制备方法及其应用 Download PDF

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CN114539421B
CN114539421B CN202011291003.7A CN202011291003A CN114539421B CN 114539421 B CN114539421 B CN 114539421B CN 202011291003 A CN202011291003 A CN 202011291003A CN 114539421 B CN114539421 B CN 114539421B
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朱焕章
姜正涛
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Abstract

本发明属生物医学技术领域，涉及感染性疾病与肿瘤并发症免疫技术，具体涉及一种以HIV‑1 gp120与CD20为双靶点的CAR‑T细胞的制备方法及其应用。本发明还提供了一种基于广泛中和性抗体3BNC117的嵌合抗原受体与抗CD20嵌合抗原受体结合的慢病毒载体。本发明的嵌合型抗原受体包括：靶向HIV‑1 gp120的单链抗体ScFv、IgG4铰链区、CD8跨膜区、4‑1BB、白细胞抗原分化群3的ζ链。经试验表明，本发明的双靶型CAR‑T细胞可高效特异地清除HIV感染细胞和淋巴瘤细胞，用于制备治疗艾滋病相关淋巴瘤的药物和试剂，具有良好的应用前景。

Description

以HIV-1 gp120与CD20为双靶点的CAR-T细胞制备方法及其 应用
技术领域
本发明属于生物工程领域，涉及一种以HIV-1包膜蛋白gp120与CD20为双靶点的CAR-T细胞的制备方法及在制备抗艾滋病相关淋巴瘤的活细胞药物中的应用。
背景技术
据报道，艾滋病已经成为当今世界严重的公共卫生问题和社会问题。目前临床上采用的抗逆转录病毒疗法(Antiretroviral Therapy，ART)可以有效降低病人血浆病毒载量，却无法治愈艾滋病。同时艾滋病相关淋巴瘤仍然占HIV相关恶性肿瘤的50％以上，是艾滋病相关死亡的主要原因。因此迫切需要寻找一种新的疗法来同时治愈艾滋病和艾滋病相关淋巴瘤。
嵌合抗原受体T细胞疗法(chemical antigen receptor T cell therapy，CAR-Ttherapy)最早在上世纪90年代就开展了治疗HIV-1病人的临床实验。通过将可溶性CD4分子作为胞外抗原识别区来识别HIV env，并连接至T细胞IgG Fc片段作为胞内活化域来激活T细胞，可以使CD4-CAR基因修饰后的T细胞具有杀灭env表达细胞的能力。但是由于当时采用的第一代CAR载体仅含有一个胞内活化信号域，对T细胞活化程度不足而造成临床试验中抗病毒效果不佳。随着CAR-T疗法近几年在淋巴瘤治疗领域中取得的技术进步与巨大成功，CAR-T又逐步显示出在抗病毒治疗中的潜能。同时CD20是在治疗B细胞系恶性血液系统肿瘤时一个重要的靶点，并且已有多项以CD20为靶点的CAR-T治疗进入了临床试验，证实了CD20CAR-T治疗在复发难治性NHL患者中存在短期疗效及长期有效性的可能性。
因此，本申请的发明人拟提供，以HIV-1包膜蛋白gp120和CD20作为双靶点的CAR-T细胞同时靶向杀伤HIV-1感染细胞和B细胞系恶性血液系统肿瘤细胞，用于制备治疗艾滋病相关淋巴瘤患者的药物。
目前，利用CAR-T疗法分别靶向治疗HIV和淋巴瘤的研究已有报道。但是对双靶点同时靶向HIV-1gp120和CD20的研究未见报道。
发明内容
本发明的目的是构建一种用于同时靶向HIV-1gp120和CD20的双靶型CAR-T细胞的载体。
本发明的另一个目的是制备一种安全有效的、能够同时靶向gp120和CD20的双靶型anti-HIV CAR-T细胞及其在制药中的用途。
本发明在第二代CAR载体的基础上连接HIV-1广泛中和性抗体3BNC117来源的ScFv和靶向CD20的ScFv作为胞外抗原识别区，构建了3BNC117-CD20 CAR(3B-CD20 CAR)；其中3B-CD20 CAR携带有HIV-1广泛中和性抗体3BNC117来源的ScFv，可以中和90％HIV-1毒株，具有高效的特异性和亲和活性；同时携带有CD20 ScFv，可以靶向CD20靶点。其中CD20在临床水平被确认表达在大多数B细胞恶性肿瘤的特异性抗原，而在造血干细胞、血浆细胞和其他正常组织中不表达，是B细胞系血液系统肿瘤治疗的理想靶点。
本发明提供了一种嵌合型抗原受体，该嵌合型抗原受体包括：靶向HIV-1gp120的单链抗体ScFv、IgG4铰链区、CD8跨膜区、4-1BB、白细胞抗原分化群3的ζ链、中间通过EF1α连接、CD8引导肽、CD20-ScFv、CD8铰链区、CD8跨膜区、4-1BB、白细胞抗原分化群3的ζ链。
其中，靶向HIV-1gp120的单链抗体ScFv是能够识别艾滋病病毒HIV-1病毒gp120蛋白并与之结合的ScFv。所述单链抗体ScFv能够识别HIV病毒感染细胞表面的gp120，是通过串联针对HIV病毒感染细胞表面的gp120的抗体轻链、重链可变区而得。单链抗体ScFv作为整个CAR分子的胞外结合结构域，其氨基酸序列来源于3BNC117-pTRPE质粒。
本发明中，
IgG4铰链区即IgG4 hinge，是链接3BNC117 ScFv与CD8跨膜区的铰链分子，其序列可参见SEQ ID NO.5-6；
CD8跨膜区是链接嵌合抗原受体胞外区结构和胞内区结构的跨膜分子，其序列可参见SEQ ID NO.7-8；
4-1BB是一种胞内信号共刺激域，其序列可参见SEQ ID NO.9-10；
白细胞抗原分化群3的ζ链(CD3ζ)是一种胞内信号刺激域，其序列可参见SEQ IDNO.11-12；
CD8引导肽是一段引导信号肽段，其序列可参见SEQ ID NO.13-14；
CD20 ScFv是针对B细胞系恶性血液系统肿瘤理想靶点CDD20的特异性ScFv，参考已报道文献序列(PMID:27059623)，其序列可参见SEQ ID NO.15-16；
CD8铰链区是链接CD20-ScFv与CD8跨膜区的铰链分子，其序列可参见SEQ IDNO.17-18。
具体而言，本发明提供了一种治疗HIV感染的嵌合型抗原受体3B-CD20 CAR。该嵌合型抗原受体是通过N端到C端顺次拼接3BNC117单链抗体ScFv、IgG4 hinge、CD8跨膜区、4-1BB和白细胞抗原分化群3的ζ链CD3，在EF1a启动子后分别连接CD8 leader、CD20ScFv、CD8铰链区、CD8跨膜区、4-1BB和白细胞抗原分化群3的ζ链CD3得到的。
本发明还包括所述嵌合型抗原受体的编码序列。
所述的3B-CD20 CAR通过下述方法获得：
pCDH-CMV-MCS-EF1α-Puro质粒为骨架，使用内切酶切开MCS区；
以pTRPE-3BNC117-G4H-BBz质粒为模板，扩增出3BNC117 CAR片段；
将酶切产物与3BNC117 CAR片段连接；
得到阳性质粒pCDH-CMV-3BNC117-EF1α；
以pCDH-CMV-3BNC117-EF1α质粒为模板，利用内切酶去除Puro片段；
以CD20-pUC57-Amp质粒(由金唯智公司全基因合成)为模板，扩增出含有CD8leader、CD20 ScFv、CD8铰链区、CD8跨膜区、4-1BB和白细胞抗原分化群3的ζ链CD3；
将酶切产物与所述的CD20-CAR片段连接；
得到阳性质粒3B-CD20 CAR。
pCDH-CMV-MCS-EF1α-Puro是第二代慢病毒骨架质粒，购自优宝生物。
pTRPE-3BNC117-G4H-BBz质粒：表达3BNC117-CAR的慢病毒载体质粒，来自于OttoO.Yang教授的馈赠并由本实验室保存。CD20-CAR序列由苏州金唯智生物科技有限公司全基因合成。
本发明的制备方法中，克隆序列可以采用PCR，人工合成，酶切等方法实现，拼接序列则可能采用酶切、退火、连接粘性末端等方法实现。
本发明中，所适用宿主包括各种真核生物。
本发明中，携带3BNC117-CAR和CD20-CAR的表达质粒系统应用的宿主细胞可以是静止细胞或者分裂细胞。
本发明中，携带3BNC117-CAR和CD20-CAR的表达质粒系统已经被证实可以制备同时抗HIV-1与CD20的CAR-T细胞。
本发明涉及的序列如下：
SEQ ID NO.1-4:
3BNC117-IgG4 Hinge-CD8跨膜区-4-1BB-CD3ζ氨基酸序列为：
MLLLVTSLLLCELPHPAFLLIPQVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVWGSGTQVTVSSASTKGPGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(SEQ ID NO.1)。
相应的核苷酸序列为：
ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCTGCCTTTCTGCTGATCCCCCAGGTGCAGCTGCTGCAGAGCGGAGCCGCCGTGACAAAGCCTGGCGCTTCTGTGCGGGTGTCCTGCGAGGCCAGCGGCTACAACATCCGGGACTACTTCATCCACTGGTGGCGGCAGGCCCCAGGCCAGGGACTGCAGTGGGTGGGATGGATCAACCCCAAGACCGGCCAGCCCAACAACCCCCGGCAGTTCCAGGGCCGGGTGTCCCTGACAAGACACGCCAGCTGGGACTTCGACACCTTCAGCTTCTACATGGACCTGAAGGCCCTGCGGAGCGACGATACCGCCGTGTACTTCTGCGCCAGACAGCGGAGCGACTACTGGGATTTCGACGTGTGGGGCAGCGGCACCCAGGTCACAGTGTCCAGCGCCAGCACAAAGGGACCTGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGCAGCGATATTCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACACCGTGACCATCACCTGTCAGGCCAACGGATACCTGAACTGGTATCAGCAGCGGAGAGGCAAGGCCCCCAAGCTGCTGATCTACGACGGCAGCAAGCTGGAACGGGGCGTGCCCAGCCGGTTCAGCGGCAGAAGATGGGGCCAAGAGTACAACCTGACCATCAACAACCTGCAGCCCGAGGATATTGCCACATACTTTTGCCAGGTGTACGAGTTCGTGGTGCCCGGGACCCGGCTGGATCTGAAGAGAACCGTGGCCGCTCCCGAGAGCAAATACGGGCCCCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQID NO.2)。
CD8 leader-CD20 ScFv-CD8 Hinge-CD8跨膜区-4-1BB-CD3ζ氨基酸序列为：
MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(SEQ IDNO.3)。
相应的核苷酸序列为：
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgGAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGCTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGACTATTACTGTGCAAGATCTAATTATTACGGTAGTAGCTACTGGTTCTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGACATTGTGCTGACCCAATCTCCAGCTATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAATTACATGGACTGGTACCAGAAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTTTTAATCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAgccctgagcaattccatcatgtacttcagccacttcgtgcccgtgtttctgcctgccaaacccaccacaacccccgctcctagaccccctacacccgctcccaccattgccagccaacctctgtccctgagacccgaagctagcaggcctgctgctggaggagccgtgcacaccaggggcctggacttcgcttgcgacATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO.4)。
SEQ ID NO.5-6
IgG4 Hinge氨基酸序列为：
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKD(SEQ ID NO.5)。
相应的核苷酸序列为：
GAGAGCAAATACGGGCCCCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGAT(SEQ ID NO.6)。
SEQ ID NO.7-8
CD8跨膜区氨基酸序列为：
IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO.7)。
相应的核苷酸序列为：
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO.8)。
SEQ ID NO.9-10
4-1BB氨基酸序列为：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.9)。
相应的核苷酸序列为：
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGA TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO.10)。
SEQ ID NO.11-12
CD3ζ氨基酸序列为：
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.11)。
相应的核苷酸序列为：
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO.12)。
SEQ ID NO.13-14
CD8引导肽氨基酸序列为：
ALPVTALLLPLALLLHAARP(SEQ ID NO.13)。
相应的核苷酸序列为：
gccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg(SEQ IDNO.14)。
SEQ ID NO.15-16
抗CD20单链抗体氨基酸序列为：
EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK(SEQ ID NO.15)。
相应的核苷酸序列为：
GAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGCTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGACTATTACTGTGCAAGATCTAATTATTACGGTAGTAGCTACTGGTTCTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGACATTGTGCTGACCCAATCTCCAGCTATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAATTACATGGACTGGTACCAGAAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTTTTAATCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO.16)。
SEQ ID NO.17-18
CD8分子铰链区氨基酸序列为：
ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACD(SEQ ID NO.17。
相应的核苷酸序列为：
Gccctgagcaattccatcatgtacttcagccacttcgtgcccgtgtttctgcctgccaaacccaccacaacccccgctcctagaccccctacacccgctcccaccattgccagccaacctctgtccctgagacccgaagctagcaggcctgctgctggaggagccgtgcacaccaggggcctggacttcgcttgcgac(SEQ ID NO.18)。
另一方面，所述的嵌合型抗原受体可以用于制备基因修饰的CD3+T淋巴细胞，同时靶向HIV-1gp120和CD20。
本发明提供了一种基因修饰的CD3+T淋巴细胞，所述的CD3+T淋巴细胞表面表达上述的嵌合型抗原受体。
所述的CD3+T淋巴细胞的制备方法包括：
(1)将权利要求5所述的3B-CD20 CAR表达载体转染293T细胞获得慢病毒载体；
(2)使用步骤(1)获得的慢病毒载体转导CD3+T淋巴细胞。
较好的，所述的步骤(2)包括：
从外周血分离PBMC；
获得CD3+T细胞，经anti-CD3/28beads刺激；
在CD3+T细胞的培养环境中加入3B-CD20 CAR重组表达载体的慢病毒；
慢病毒感染24小时后换液(MOI＝20)。
在本发明的一个优选例中，基因修饰的CD3+T淋巴细胞由以下方法制备得到：
从外周血分离单个核细胞PBMC后再用磁珠阳选获得CD3+T细胞，经anti-CD3/28磁珠(细胞与磁珠比例为1:1)刺激24小时后，加入重组3B-CD20 CAR分子的慢病毒感染24小时后换液(MOI＝20)。从病毒感染后第四天开始，细胞计数并根据细胞状态和增殖情况补加培养基，细胞浓度调整至0.5x106/mL，并补充IL-2100U/mL，进一步扩增细胞直到满足回输的细胞数。
本发明在自主构建的HIV-1体外细胞模型中发现，3B-CD20 CAR-T细胞可以有效杀伤HIV-1细胞。同时在CD20+Raji细胞模型中发现，3B-CD20 CAR-T细胞可以有效杀伤CD20高表达的Raji细胞。
本发明提供一种双靶向型的CAR-T细胞，使得HIV-1感染细胞和CD20高表达细胞可以被高效，持久，特异地清除，为艾滋病相关淋巴瘤患者的治疗提供了新的思路。
本发明还提供了上述基因修饰的CD3+T淋巴细胞在制备抗HIV感染与B细胞系恶性血液系统肿瘤的活细胞药物中的应用。
本发明提供了一种同时靶向HIV-1包膜蛋白gp120和CD20的载体、受体、CAR-T细胞及其制备方法和应用。以及提供了一种基于广泛中和性抗体3BNC117的CAR-T疗法与基于CD20 ScFv CAR-T疗法结合的慢病毒载体。本发明还提供了一种新型的、双靶型的抗HIVCAR-T细胞的制法。经试验表明，本发明的CAR-T细胞可高效特异地清除HIV感染细胞，以及有效杀伤CD20高表达肿瘤细胞，在艾滋病相关淋巴瘤患者治疗中具有良好的应用前景。本发明中，携带3BNC117-CAR和CD20-CAR的表达质粒系统制备的CAR-T细胞已经被证实可以在体外特异性杀伤HIV-1细胞，效率高达70％-90％，同时可以高效杀伤Raji细胞，效率高达70％。本发明将HIV CAR-T疗法与淋巴瘤CAR-T疗法进行了结合，为艾滋病相关淋巴瘤患者治愈探索提供了一种可行的思路。
附图说明
图1.3B-CD20 CAR重组载体结构示意图。
图2.3B-CD20 CAR-T细胞表型鉴定，
其中，UTD是未经任何处理的T细胞作为对照，
纵坐标FS-A是前项散色光，横坐标CAR表示利用FITC-goat anti-human IgG流式抗体染色后，检测到CAR阳性率比例，
图中显示了，制备的3B-CD20 CAR-T细胞CAR分子表达效率约在90％。
图3.3B-CD20 CAR-T细胞靶向杀伤活性的检测，
纵坐标是杀伤百分比Specific lysis，单位％，指靶细胞被杀伤的比例，计算公式为
横坐标ratio是指效应细胞数量与靶细胞数量比例，
图中显示了，在不同共孵育比例下，3BD-CD20 CAR组对LEL6细胞和Raji细胞均具有杀伤作用。
图4.3B-CD20 CAR-T细胞细胞因子释放能力的检测，
纵坐标是三种细胞因子的浓度，
图中显示了，在与LEL6细胞或Raji共孵育后，3BD-CD20 CAR组TNF-α、IL-2、IFN-γ细胞因子释放量具有明显提升。***p<0.001。
具体实施方式
实施例1体外构建含有3BNC117和CD20双靶点的嵌合抗原受体表达载体
首先以pCDH-CMV-MCS-EF1α-Puro质粒为骨架，利用EcoRI和BamHI内切酶双酶切切开MCS区。随后以pTRPE-3BNC117-G4H-BBz质粒为模板，利用onestep-3BNC117-DNR-1F，onestep-3BNC117-DNR-1R引物PCR扩增出3BNC117 CAR片段。最终将酶切产物与PCR产物胶回收后进行Onestep同源重组连接，连接产物在DH5α感受态中转化后涂在Amp+的平板上筛选阳性克隆，阳性克隆扩大培养后抽提质粒并测序验证，得到阳性质粒pCDH-CMV-3BNC117-EF1α。
接着以构建得到的pCDH-CMV-3BNC117-EF1α质粒为模板，利用XmaI和SalI内切酶双酶切切除Puro片段。以CD20-pUC57-Amp质粒为模板，利用onestep-3BNC117-CD20-2F，onestep-3BNC117-CD20-2R引物PCR扩增出含有CD8引导肽，CD20 ScFv，CD8分子铰链区，CD8分子跨膜区，4-1BB，CD3ζ的CD20-CAR片段。将酶切产物与PCR产物胶回收后进行Onestep同源重组连接，连接产物在DH5α感受态中转化后涂在Amp+的平板上筛选阳性克隆，阳性克隆扩大培养后抽提质粒并测序验证，得到阳性质粒pCDH-CMV-3BNC117CAR-EF1α-CD20 CAR并命名为3BNC117-CD20 CAR(3B-CD20 CAR)(如图1所示)。
实施例23B-CD20 CAR-T细胞的制备与体外功能验证
为了获得表达3B-CD20 CAR的慢病毒颗粒，本发明将慢病毒骨架质粒，△8.91，VSVG三种质粒共转染进293T细胞，48h后收集病毒上清并过滤，超速离心浓缩后置于-80℃保存备用。
利用携带有3B-CD20 CAR元件的慢病毒感染健康人CD3+T淋巴细胞制备效应细胞。获得健康供体外周血后，利用淋巴细胞分离液分离得到PBMCs细胞，再利用磁珠法分选得到人CD3+T淋巴细胞。随后按照磁珠：细胞＝1:1的比例加入CD3/28活化磁珠。24h后利用表达3B-CD20 CAR的慢病毒，以MOI＝20静置感染的方式感染CD3+T细胞制备双靶型CAR-T效应细胞，并用未感染CD3+T细胞作为对照。
6天后通过Fluorescein(FITC)-conjugated AffiniPure F(ab')2Fragment GoatAnti-Human IgG(H+L)Antibody标记，结果显示，与UTD组相比3B-CD20 CAR组阳性率约为90％(图2)。
本发明在体外初步验证了其对HIV-1env+细胞模型LEL6靶细胞的杀伤作用，分别将未修饰的CD3+T细胞，3B-CD20 CAR-T细胞与LEL6细胞进行了共孵育，并用Jurkat细胞作为阴性对照，利用乳酸脱氢酶(lactate dehydrogenase，LDH)法检测了细胞杀伤效果。结果显示与UTD组相比，在1:1，5:1，10:1三种不同的共孵育比例下，3B-CD20 CAR组可以有效杀伤靶细胞，甚至在10:1共孵育比例时3B-CD20 CAR组均可以达到约90％的杀伤效果。而对于HIV-1env-的Jurkat细胞，效应细胞不会产生非特异性杀伤作用。
本发明在体外还验证了3B-CD20 CAR-T细胞对Raji细胞的杀伤能力，分别将未修饰的CD3+T细胞，3B-CD20 CAR-T细胞与Raji细胞进行了共孵育，并用Jurkat细胞作为阴性对照，利用乳酸脱氢酶(lactate dehydrogenase，LDH)法检测了细胞杀伤效果。结果显示与UTD组相比，在1:1，5:1，10:1三种不同的共孵育比例下，3B-CD20 CAR组可以有效杀伤靶细胞，甚至在10:1共孵育比例时3B-CD20 CAR组均可以达到约70％的杀伤效果。
Specific Lysis为杀伤百分比，计算公式为：
杀伤百分比越高，证实越多的靶细胞被杀灭(图3)。
为了进一步检测Anti-HIV CAR-T效应细胞的功能，本发明将该双靶型CAR-T效应细胞分别与Jurkat细胞(阴性对照)，LEL6靶细胞和Raji细胞按照10:1的比例进行了共孵育，并在24h后检测了IL-2，TNF-α，IFN-γ3种细胞因子的释放。结果显示在靶细胞的抗原刺激下，该双靶型CAR-T细胞均可以有效分泌细胞因子(图4)。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本领域技术的技术人员在本申请公开的技术范围内，可轻易想到的变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。
序列表
<110> 复旦大学
<120> 以HIV-1 gp120与CD20为双靶点的CAR-T细胞制备方法及其应用
<130> 20201118
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 679
<212> PRT
<213> Artificial Sequence
<400> 1
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Leu Gln Ser Gly Ala Ala
20 25 30
Val Thr Lys Pro Gly Ala Ser Val Arg Val Ser Cys Glu Ala Ser Gly
35 40 45
Tyr Asn Ile Arg Asp Tyr Phe Ile His Trp Trp Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Gln Trp Val Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro
65 70 75 80
Asn Asn Pro Arg Gln Phe Gln Gly Arg Val Ser Leu Thr Arg His Ala
85 90 95
Ser Trp Asp Phe Asp Thr Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu
100 105 110
Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp
115 120 125
Tyr Trp Asp Phe Asp Val Trp Gly Ser Gly Thr Gln Val Thr Val Ser
130 135 140
Ser Ala Ser Thr Lys Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
165 170 175
Leu Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn
180 185 190
Gly Tyr Leu Asn Trp Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu
195 200 205
Leu Ile Tyr Asp Gly Ser Lys Leu Glu Arg Gly Val Pro Ser Arg Phe
210 215 220
Ser Gly Arg Arg Trp Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu
225 230 235 240
Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val
245 250 255
Val Pro Gly Thr Arg Leu Asp Leu Lys Arg Thr Val Ala Ala Pro Glu
260 265 270
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
275 280 285
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
355 360 365
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
370 375 380
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
405 410 415
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
420 425 430
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
435 440 445
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
450 455 460
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
465 470 475 480
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
485 490 495
Ser Leu Gly Lys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
500 505 510
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
515 520 525
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
530 535 540
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
545 550 555 560
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
565 570 575
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
580 585 590
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
595 600 605
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
610 615 620
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
625 630 635 640
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
645 650 655
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
660 665 670
Met Gln Ala Leu Pro Pro Arg
675
<210> 2
<211> 2040
<212> DNA
<213> Artificial Sequence
<400> 2
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atcccccagg tgcagctgct gcagagcgga gccgccgtga caaagcctgg cgcttctgtg 120
cgggtgtcct gcgaggccag cggctacaac atccgggact acttcatcca ctggtggcgg 180
caggccccag gccagggact gcagtgggtg ggatggatca accccaagac cggccagccc 240
aacaaccccc ggcagttcca gggccgggtg tccctgacaa gacacgccag ctgggacttc 300
gacaccttca gcttctacat ggacctgaag gccctgcgga gcgacgatac cgccgtgtac 360
ttctgcgcca gacagcggag cgactactgg gatttcgacg tgtggggcag cggcacccag 420
gtcacagtgt ccagcgccag cacaaaggga cctggcggcg gaggatctgg cggaggcgga 480
agtggcggag ggggcagcga tattcagatg acccagagcc ccagcagcct gagcgccagc 540
gtgggcgaca ccgtgaccat cacctgtcag gccaacggat acctgaactg gtatcagcag 600
cggagaggca aggcccccaa gctgctgatc tacgacggca gcaagctgga acggggcgtg 660
cccagccggt tcagcggcag aagatggggc caagagtaca acctgaccat caacaacctg 720
cagcccgagg atattgccac atacttttgc caggtgtacg agttcgtggt gcccgggacc 780
cggctggatc tgaagagaac cgtggccgct cccgagagca aatacgggcc cccctgcccc 840
ccttgccctg cccccgagtt cctgggcgga cccagcgtgt tcctgttccc ccccaagccc 900
aaggacaccc tgatgatcag ccggaccccc gaggtgacct gtgtggtggt ggacgtgtcc 960
caggaggacc ccgaggtcca gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 1020
aagaccaagc cccgggagga gcagttcaat agcacctacc gggtggtgtc cgtgctgacc 1080
gtgctgcacc aggactggct gaacggcaag gaatacaagt gtaaggtgtc caacaagggc 1140
ctgcccagca gcatcgagaa aaccatcagc aaggccaagg gccagcctcg ggagccccag 1200
gtgtacaccc tgccccctag ccaagaggag atgaccaaga accaggtgtc cctgacctgc 1260
ctggtgaagg gcttctaccc cagcgacatc gccgtggagt gggagagcaa cggccagccc 1320
gagaacaact acaagaccac cccccctgtg ctggacagcg acggcagctt cttcctgtac 1380
agccggctga ccgtggacaa gagccggtgg caggagggca acgtctttag ctgctccgtg 1440
atgcacgagg ccctgcacaa ccactacacc cagaagagcc tgagcctgtc cctgggcaag 1500
gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1560
atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1620
atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1680
gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1740
aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1800
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1860
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1920
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1980
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 2040
<210> 3
<211> 511
<212> PRT
<213> Artificial Sequence
<400> 3
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 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
20 25 30
Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
35 40 45
Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln
50 55 60
Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser
65 70 75 80
Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
85 90 95
Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
100 105 110
Ala Asp Tyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp
115 120 125
Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly
130 135 140
Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro
165 170 175
Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr
180 185 190
Met Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile
195 200 205
Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala
225 230 235 240
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro
245 250 255
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ala Leu Ser Asn Ser
260 265 270
Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro
275 280 285
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
290 295 300
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly
305 310 315 320
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
325 330 335
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
340 345 350
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
355 360 365
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
370 375 380
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
385 390 395 400
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln
405 410 415
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
420 425 430
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
435 440 445
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
450 455 460
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
465 470 475 480
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
485 490 495
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 510
<210> 4
<211> 1536
<212> DNA
<213> Artificial Sequence
<400> 4
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggaggtgc agctgcagca gtctggggct gagctggtga agcctggggc ctcagtgaag 120
atgtcctgca aggcttctgg ctacacattt accagttaca atatgcactg ggtaaagcag 180
acacctggac agggcctgga atggattgga gctatttatc caggaaatgg tgatacttcc 240
tacaatcaga agttcaaagg caaggccaca ttgactgcag acaaatcctc cagcacagcc 300
tacatgcagc tcagcagcct gacatctgag gactctgcgg actattactg tgcaagatct 360
aattattacg gtagtagcta ctggttcttc gatgtctggg gcgcagggac cacggtcacc 420
gtctcctcag gcagtactag cggtggtggc tccgggggcg gttccggtgg gggcggcagc 480
agcgacattg tgctgaccca atctccagct atcctgtctg catctccagg ggagaaggtc 540
acaatgactt gcagggccag ctcaagtgta aattacatgg actggtacca gaagaagcca 600
ggatcctccc ccaaaccctg gatttatgcc acatccaacc tggcttctgg agtccctgct 660
cgcttcagtg gcagtgggtc tgggacctct tactctctca caatcagcag agtggaggct 720
gaagatgctg ccacttatta ctgccagcag tggagtttta atccacccac gttcggaggg 780
gggaccaagc tggaaataaa agccctgagc aattccatca tgtacttcag ccacttcgtg 840
cccgtgtttc tgcctgccaa acccaccaca acccccgctc ctagaccccc tacacccgct 900
cccaccattg ccagccaacc tctgtccctg agacccgaag ctagcaggcc tgctgctgga 960
ggagccgtgc acaccagggg cctggacttc gcttgcgaca tctacatctg ggcgcccttg 1020
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1080
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1140
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1200
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1260
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1320
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1380
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1500
gacgcccttc acatgcaggc cctgccccct cgctaa 1536
<210> 5
<211> 230
<212> PRT
<213> Artificial Sequence
<400> 5
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys Asp
225 230
<210> 6
<211> 690
<212> DNA
<213> Artificial Sequence
<400> 6
gagagcaaat acgggccccc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60
agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120
gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180
gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240
acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300
tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360
gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420
accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480
gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540
gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600
gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660
aagagcctga gcctgtccct gggcaaggat 690
<210> 7
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 7
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> 8
<211> 72
<212> DNA
<213> Artificial Sequence
<400> 8
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 9
<211> 42
<212> PRT
<213> Artificial Sequence
<400> 9
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> 10
<211> 126
<212> DNA
<213> Artificial Sequence
<400> 10
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 11
<211> 112
<212> PRT
<213> Artificial Sequence
<400> 11
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 12
<211> 339
<212> DNA
<213> Artificial Sequence
<400> 12
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339
<210> 13
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 13
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro
20
<210> 14
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 14
gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60
<210> 15
<211> 246
<212> PRT
<213> Artificial Sequence
<400> 15
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys
85 90 95
Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp
100 105 110
Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly
115 120 125
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu
130 135 140
Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr
145 150 155 160
Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln
165 170 175
Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn
180 185 190
Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr
210 215 220
Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly
225 230 235 240
Thr Lys Leu Glu Ile Lys
245
<210> 16
<211> 738
<212> DNA
<213> Artificial Sequence
<400> 16
gaggtgcagc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatg 60
tcctgcaagg cttctggcta cacatttacc agttacaata tgcactgggt aaagcagaca 120
cctggacagg gcctggaatg gattggagct atttatccag gaaatggtga tacttcctac 180
aatcagaagt tcaaaggcaa ggccacattg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac atctgaggac tctgcggact attactgtgc aagatctaat 300
tattacggta gtagctactg gttcttcgat gtctggggcg cagggaccac ggtcaccgtc 360
tcctcaggca gtactagcgg tggtggctcc gggggcggtt ccggtggggg cggcagcagc 420
gacattgtgc tgacccaatc tccagctatc ctgtctgcat ctccagggga gaaggtcaca 480
atgacttgca gggccagctc aagtgtaaat tacatggact ggtaccagaa gaagccagga 540
tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 600
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 660
gatgctgcca cttattactg ccagcagtgg agttttaatc cacccacgtt cggagggggg 720
accaagctgg aaataaaa 738
<210> 17
<211> 66
<212> PRT
<213> Artificial Sequence
<400> 17
Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe
1 5 10 15
Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
20 25 30
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser
35 40 45
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
50 55 60
Cys Asp
65
<210> 18
<211> 198
<212> DNA
<213> Artificial Sequence
<400> 18
gccctgagca attccatcat gtacttcagc cacttcgtgc ccgtgtttct gcctgccaaa 60
cccaccacaa cccccgctcc tagaccccct acacccgctc ccaccattgc cagccaacct 120
ctgtccctga gacccgaagc tagcaggcct gctgctggag gagccgtgca caccaggggc 180
ctggacttcg cttgcgac 198

Claims (8)

1.一种嵌合型抗原受体，其特征在于，该嵌合型抗原受体包括3BNC117-CAR和CD20-CAR；
3BNC117-CAR包括靶向HIV-1gp120的单链抗体ScFv、IgG4铰链区、CD8跨膜区、4-1BB、白细胞抗原分化群3的ζ链，其氨基酸序列如SEQ ID NO.1所示；
CD20-CAR包括CD8前导肽、靶向CD20的单链抗体ScFv、CD8铰链区、CD8跨膜区、4-1BB、白细胞抗原分化群3的ζ链，其氨基酸序列如SEQ ID NO.3所示。
2.权利要求1所述的嵌合型抗原受体的编码核酸。
3.一种以HIV-1gp120与CD20作为双靶的慢病毒载体，其特征在于，所述的载体包括权利要求1所述的嵌合型抗原受体的编码核酸；
所述的权利要求1所述的嵌合型抗原受体的编码核酸通过下述方法获得：
pCDH-CMV-MCS-EF1α-Puro质粒为骨架，使用内切酶切开MCS区；
以pTRPE-3BNC117-G4H-BBz质粒为模板，扩增出3BNC117 CAR片段；
将酶切产物与3BNC117 CAR片段连接；
得到阳性质粒pCDH-CMV-3BNC117-EF1α；
以pCDH-CMV-3BNC117-EF1α质粒为模板，利用内切酶去除Puro片段；
以CD20-pUC57-Amp质粒为模板，扩增出含有抗CD20嵌合抗原受体片段；
将酶切产物与所述的CD20嵌合抗原受体片段连接；
得到阳性质粒3BNC117-CD20 CAR。
4.权利要求1所述的嵌合型抗原受体的应用，其特征在于，所述的嵌合型抗原受体用于制备基因修饰的CD3+T淋巴细胞，所述的基因修饰的CD3+T淋巴细胞识别HIV-1gp120和/或CD20。
5.一种基因修饰的CD3+T淋巴细胞，其特征在于，所述的CD3+T淋巴细胞表面表达权利要求1所述的嵌合型抗原受体。
6.权利要求5所述的CD3+T淋巴细胞的制备方法，其特征在于，所述的制备方法包括：
(1)将权利要求3所述的3BNC117-CD20 CAR表达载体转染293T细胞获得慢病毒载体；
(2)使用步骤(1)获得的慢病毒载体转导CD3+T淋巴细胞。
7.根据权利要求6所述的制备方法，其特征在于，所述的步骤(2)包括：
从外周血分离PBMC；
获得CD3+T细胞，经anti-CD3/28刺激；
在CD3+T细胞的培养环境中加入重组有权利要求3所述的慢病毒载体的慢病毒；
慢病毒感染24小时后换液。
8.权利要求5所述的基因修饰的CD3+T淋巴细胞的用途，其特征在于：所述基因修饰的CD3+T淋巴细胞在制备抗艾滋病和/或抗B细胞系血液系统肿瘤的活细胞药物中的应用。
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