CN109957016A - 一种靶向cd38的单链抗体、嵌合抗原受体t细胞及其制备方法和应用 - Google Patents
一种靶向cd38的单链抗体、嵌合抗原受体t细胞及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种靶向CD38的单链抗体,所述靶向CD38的单链抗体包括如SEQ ID NO:1所示的氨基酸序列,所述靶向CD38的单链抗体为人源化单链抗体。本发明还提供了包括所述靶向CD38的单链抗体的嵌合抗原受体T细胞,所述靶向CD38的嵌合抗原受体可以专一性地靶向表达CD38的肿瘤细胞,促进T细胞的扩增,高效且特异性的杀伤肿瘤细胞。此外,人源化单链抗体制得的靶向CD38的嵌合抗原受体T细胞具有更好的维持细胞的活力和杀伤力。本发明还提供了该靶向CD38的嵌合抗原受体T细胞的制备方法和应用。
Description
技术领域
本发明涉及医学生物领域,特别涉及一种靶向CD38的单链抗体、嵌合抗原受体T细胞及其制备方法和应用。
背景技术
恶性肿瘤(癌症)已成为威胁人类生命的元凶,发病率成上升趋势。CD38是一种单链Ⅱ型跨膜糖蛋白,主要表达于骨髓瘤、淋巴瘤等,在恶性浆细胞中表达量较高。CAR-T(嵌合抗原受体T细胞)技术是一种新型细胞疗法,它是将经过CAR改造的T细胞回输至人体,激活自身免疫***,对肿瘤细胞进行杀伤,被认为是目前最有效的恶性肿瘤的治疗方式之一。目前还未有靶向CD38的嵌合抗原受体T细胞的制备和研究。此外,CAR-T细胞的在体维持能力(per sistence)是CAR-T长期疗效的保证,但实际上CAR-T在回输后几天到几周就完全消失,而未能发挥长效持久的杀伤肿瘤细胞的能力。
发明内容
有鉴于此,本发明提供了一种靶向CD38的单链抗体,以及包括所述靶向CD38的单链抗体的嵌合抗原受体T细胞。所述靶向CD38的嵌合抗原受体可以专一性地靶向CD38,促进T细胞在患者体内扩增,高效且特异性地杀伤肿瘤细胞,同时带人源化单链抗体的靶向CD38的嵌合抗原受体T细胞能够持久地维持其自我更新活力和杀伤力。本发明还提供了一种靶向CD38的嵌合抗原受体T细胞的制备方法和应用。
第一方面,本发明提供了一种靶向CD38的单链抗体,所述靶向CD38的单链抗体包括如SEQ ID NO:1所示的氨基酸序列,所述靶向CD38的单链抗体为人源化单链抗体。
可选的,所述靶向CD38的单链抗体的编码基因包括如SEQ ID NO:2所示的核苷酸序列。
可选的,所述靶向CD38的单链抗体编码基因应该考虑简并碱基,即如SEQ ID NO:1所示的氨基酸序列的编码基因包括如SEQ ID NO:2所示的核苷酸序列,保护范围还应该保护与SEQ ID NO:2具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:1。
本发明第一方面提供的所述靶向CD38的单链抗体能够与CD38蛋白发生特异性结合,对表达CD38的肿瘤细胞有很好靶向性。此外,所述靶向CD38的单链抗体为人源化单链抗体,可以避免引起人机体的免疫反应。
第二方面,本发明提供了一种靶向CD38的嵌合抗原受体T细胞,包括靶向CD38的嵌合抗原受体CAR-CD38,所述CAR-CD38包括从氨基端到羧基端顺次连接的靶向CD38的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列,其中所述靶向CD38的单链抗体包括如SEQ ID NO:1所示的氨基酸序列。
其中,所述“从氨基端到羧基端顺次连接”具体为:所述靶向CD38的单链抗体的氨基酸序列的羧基端与所述铰链区的氨基酸序列的氨基端相连,所述胞外铰链区的氨基酸序列的羧基端与所述跨膜区的氨基酸序列的氨基端相连,所述跨膜区的氨基酸序列的羧基端与所述胞内信号区的氨基酸序列的氨基端相连。
本发明中,所述胞外铰链区用于促进所述靶向CD38的单链抗体与肿瘤上的CD38结合。可选的,所述胞外铰链区包括CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合。
进一步可选的,所述胞外铰链区为CD8α铰链区。
可选的,所述CD8α铰链区的氨基酸序列包括如SEQ ID NO:6所示的氨基酸序列。
可选的,所述CD8α铰链区的编码基因包括如SEQ ID NO:7所示的核苷酸序列。
可选的,所述CD8α铰链区的编码基因应该考虑简并碱基,即如SEQ ID NO:6所示的氨基酸序列的编码基因包括如SEQ ID NO:7所示的核苷酸序列,保护范围还应该保护与SEQID NO:7具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQID NO:6。
本发明中,所述跨膜区用于固定所述靶向CD38的嵌合抗原受体CAR-CD38。可选的,所述跨膜区包括CD3跨膜区、CD4跨膜区、CD8跨膜区、CD28跨膜区中的一种或多种的组合。
进一步可选的,所述跨膜区为CD8跨膜区。
可选的,所述CD8跨膜区的氨基酸序列包括如SEQ ID NO:8所示的氨基酸序列。
可选的,所述CD8跨膜区的编码基因包括如SEQ ID NO:9所示的核苷酸序列。
可选的,所述CD8跨膜区的编码基因应该考虑简并碱基,即如SEQ ID NO:8所示的氨基酸序列的编码基因包括如SEQ ID NO:9所示的核苷酸序列,保护范围还应该保护与SEQID NO:9具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQID NO:8。
在本发明中所述胞内信号区用于提供T细胞活化的信号,维持T细胞的生存时间和激活T细胞增殖信号通路。可选的,所述胞内信号区包括4-1BB信号区、CD3ζ信号区、ICOS信号区、CD27信号区、OX40信号区、CD28信号区、IL1R1信号区、CD70信号区、TNFRSF19L信号区中的一种或多种的组合。
可选的,所述胞内信号区为从氨基端到羧基端顺次连接的CD27信号区和CD3ζ信号区。相应地,所述胞内信号区的编码基因包括从5’端到3’端顺次连接的CD27信号区的编码基因和CD3ζ信号区的编码基因。
可选的,所述CD27信号区的氨基酸序列包括如SEQ ID NO:10所示的氨基酸序列。所述CD27信号区作为共刺激信号。
可选的,所述CD27信号区的编码基因包括如SEQ ID NO:11所示的核苷酸序列。
可选的,所述CD27信号区的编码基因应该考虑简并碱基,即如SEQ ID NO:10所示的氨基酸序列的编码基因包括如SEQ ID NO:11所示的核苷酸序列,保护范围还应该保护与SEQ ID NO:11具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:10。
可选的,所述CD3ζ信号区的氨基酸序列包括如SEQ ID NO:12所示的氨基酸序列。
可选的,所述CD3ζ信号区的编码基因包括如SEQ ID NO:13所示的核苷酸序列。
可选的,所述CD3ζ信号区的编码基因应该考虑简并碱基,即如SEQ ID NO:12所示的氨基酸序列的编码基因包括如SEQ ID NO:13所示的核苷酸序列,保护范围还应该保护与SEQ ID NO:13具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:12。
可选的,所述CAR-CD38的氨基酸序列包括如SEQ ID NO:3所示的氨基酸序列。此时,如SEQ ID NO:12的氨基酸序列所示的CD3ζ信号区作为所述T细胞的抗原特异性信号(即,第一信号),如SEQ ID NO:10的氨基酸序列所示的CD27信号区作为所述T细胞的共刺激信号,在它们的共同作用下,T细胞在识别抗原后被完全活化。特别地,选用如SEQ ID NO:10的CD27信号区作为共刺激信号,可以更好地提升后续T细胞的扩增能力、杀伤活力。
可选的,所述CAR-CD38的编码基因包括如SEQ ID NO:4所示的核苷酸序列。
可选的,所述CAR-CD38的编码基因应该考虑简并碱基,即如SEQ ID NO:3所示的氨基酸序列的编码基因包括如SEQ ID NO:4所示的核苷酸序列,保护范围还应该保护与SEQID NO:4具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQID NO:3。
进一步地,所述CAR-CD38的编码基因包括如SEQ ID NO:5所示的核苷酸序列。
本发明第二方面提供的所述靶向CD38的嵌合抗原受体T细胞,包括靶向CD38的嵌合抗原受体CAR-CD38,该受体可供所述T细胞专一性地靶向表达CD38的肿瘤细胞,在CAR-CD38与CD38结合后,所述T细胞的胞内信号区被激活,促进T细胞在患者体内的扩增,并高效且特异性的杀伤肿瘤细胞,而对正常细胞几乎不会造成损伤。此外,靶向CD38的单链抗体为人源化单链抗体,这使得所述靶向CD38的嵌合抗原受体T细胞避免引起人机体的免疫反应,具有持久的在体维持能力(包括活力和杀伤力)。
第三方面,本发明提供了一种重组病毒载体,所述重组病毒载体包括如第二方面所述的靶向CD38的嵌合抗原受体T细胞的CAR-CD38的编码基因。
可选的,所述CAR-CD38的编码基因包括如SEQ ID NO:4所示的核苷酸序列。
优选地,所述CAR-CD38的编码基因包括如SEQ ID NO:5所示的核苷酸序列。如SEQID NO:5所示的核苷酸序列与如SEQ ID NO:4所示的核苷酸序列相比,多了连接肽的编码基因。所述信号肽的编码基因可以较好地指导所述嵌合抗原受体CAR-CD38表达到细胞表面。
可选的,所述重组病毒载体中的病毒载体包括慢病毒载体、腺病毒载体或逆转录病毒载体。
进一步可选的,所述病毒载体为慢病毒载体。
本发明第三方面提供的所述重组病毒载体具有很高的感染效率以及转录效率,***的CAR-CD38编码基因片段能通过基因重组***宿主基因组,得到靶向CD38的嵌合抗原受体T细胞,使其持续、稳定地表达靶向CD38的嵌合抗原受体CAR-CD38。
第四方面,本发明提供了一种宿主细胞,所述宿主细胞包括如第三方面所述的重组病毒载体。
所述宿主细胞用于组装如第三方面所述的重组病毒载体,使其具有感染性。
可选地,所述宿主细胞可以包括HEK293T细胞、293细胞、293T细胞、293FT细胞、SW480细胞、u87MG细胞、HOS细胞、COS1细胞、COS7细胞等,但不限于此。
进一步可选的,所述宿主细胞为HEK293T细胞。
第五方面,本发明提供了一种靶向CD38的嵌合抗原受体T细胞的制备方法,包括:
(1)提供靶向CD38的嵌合抗原受体CAR-CD38的编码基因,包括从5’端到3’端顺次连接的信号肽的编码基因、靶向CD38的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因;所述靶向CD38的单链抗体为人源化单链抗体,所述靶向CD38的单链抗体的编码基因包括如SEQ ID NO:2所示的核苷酸序列;
(2)将所述CAR-CD38的基因序列***到pWPXLD载体中,得到pWPXLD-CAR-CD38重组质粒;
(3)将所述pWPXLD-CAR-CD38重组质粒与包膜质粒、包装质粒共转染宿主细胞,得到重组慢病毒;
(4)将所述重组慢病毒转染CD3阳性T淋巴细胞,获得靶向CD38的嵌合抗原受体T细胞。
上述“从5’端到3’端顺次连接”具体为:所述信号肽的编码基因序列的3’端与所述靶向CD38的单链抗体的编码基因序列的5’端相连,所述靶向CD38的单链抗体的编码基因序列的3’端与所述胞外铰链区的编码基因序列的5’端相连,所述胞外铰链区的编码基因序列的3’端与所述跨膜区的编码基因序列的5’端相连,所述跨膜区的编码基因序列的3’端与所述胞内信号区的编码基因序列的5’端相连。
在本发明中所述信号肽用于指导所述嵌合抗原受体CAR-CD38表达到细胞表面,所述信号肽在蛋白翻译成熟过程中被信号肽酶切割。
可选的,所述信号肽的氨基酸序列包括如SEQ ID NO:14所示的氨基酸序列。
可选的,所述信号肽的编码基因包括如SEQ ID NO:15所示的核苷酸序列。
可选的,所述信号肽的编码基因序列应该考虑简并碱基,即如SEQ ID NO:14所示的氨基酸序列的编码基因包括如SEQ ID NO:15所示的核苷酸序列,保护范围还应该保护与SEQ ID NO:15具有碱基简并性质的核苷酸序列,这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:14。
对于所述胞外铰链区、跨膜区、胞内信号区的具体选择及相应的编码基因序列,可参见本发明第二方面部分的描述,这里不再赘述。
可选地,所述CAR-CD38的编码基因包括如SEQ ID NO:5所示的核苷酸序列。如SEQID NO:5所示的核苷酸序列与如SEQ ID NO:4所示的核苷酸序列相比,多了所述连接肽的编码基因,但在所述嵌合抗原受体CAR-DR5表达到细胞表面时,所述信号肽在蛋白翻译成熟过程中被信号肽酶切割。因此,在翻译成的所述嵌合抗原受体CAR-CD38的氨基酸序列中并未带有如SEQ ID NO:14所示的氨基酸序列。
所述CAR-CD38的编码基因序列***到pWPXLD载体中BamHⅠ和EcoRⅠ酶切位点之间,且位于pWPXLD载体的延伸因子1α(EF1α)之后,以EF1α为启动子。所述CAR-CD38的基因序列***到pWPXLD载体时,所述CAR-CD38的基因序列的5’端还可加入起始密码子(如ATG)与pWPXLD载体中BamH1酶切位点相连,3’端还可加入终止密码子与pWPXLD载体中EcoR1酶切位点相连。
可选的,所述包膜质粒为PMD2G,所述包装质粒为psPAX2,所述宿主细胞为HEK293T细胞。
所述包膜质粒PMD2G编码水疱性口炎病毒糖蛋白衣壳,所述水疱性口炎病毒糖蛋白衣壳可以协助重组慢病毒向细胞膜粘附,并保持重组慢病毒的感染性。
本发明所述重组慢病毒可以进一步含有来自其它病毒的被膜蛋白。例如,作为这种蛋白质,最好是来自感染人类细胞的病毒被膜蛋白。对这种蛋白质没有特别的限定,可例举出逆转录病毒的兼嗜性病毒手皮膜蛋白等,例如可以使用来自小鼠白血病病毒(MuMLV)4070A株的被膜蛋白。另外,也可以使用来自MuMLV 10Al的被膜蛋白。另外,作为疱疹病毒科的蛋白,可以举出例如,单纯性疱疹病毒的gB、gD、gH、gp85蛋白,EB病毒的gp350、gp220蛋白等。作为嗜肝病毒科的蛋白,可以例举出B型肝炎病毒的S蛋白等。所述被膜蛋白还可为麻疹病毒糖蛋白与其他单链抗体融合后形成。
重组慢病毒的包装通常采用瞬时转染或采用细胞系包装。瞬时转染时可以用作包装细胞使用的人类细胞株,例如包括293细胞、293T细胞、293FT细胞、293LTV细胞、293EBNA细胞及其他的从293细胞分离的克隆;SW480细胞、u87MG细胞、HOS细胞、C8166细胞、MT-4细胞、Molt-4细胞、HeLa细胞、HT1080细胞、TE671细胞等。也可以采用来源于猴子的细胞株,例如,COS1细胞、COS7细胞、CV-1细胞、BMT10细胞等。而且,通常采用的磷酸钙和PEI转染试剂,还有一些转染试剂如Lipofectamine2000、FuGENE和S93fectin也被经常使用。
重组慢病毒的包装也采用一些慢病毒包装细胞系,如使用最普遍的Env糖蛋白、VSVG蛋白或HIV-1gag-pol蛋白所产生的稳定细胞系。
为了安全起见,大规模使用的慢病毒载体***都是采用分割基因组的方法,即将起不同辅助功能的基因定位于不同的质粒。目前有四质粒***(编码gag-pol基因、Rev基因、VSVG基因、SIN转移基因分别位于四个不同的质粒)、三质粒***(去掉了编码Rev基因的质粒,在gag-pol质粒中gag-pol基因采用了在人细胞中偏爱性的密码子)和二质粒***(慢病毒载体包装所必需的辅助基因位于同一个质粒上,这些辅助基因是单一的基因序列;另一个则是转基因质粒)。也有超过四质粒***的慢病毒包装***在使用。
可选的,步骤(4)中,所述CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。
可选的,所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血和胎盘血等。进一步可选的,来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
具体地,所述CD3阳性T淋巴细胞的获得过程如下:先将外周血单个核细胞按一定比例加入CD3/CD28免疫磁珠,并孵育一段时间后,放入磁铁进行筛选,得到免疫磁珠包被的CD3阳性T淋巴细胞,去除磁珠后,获得CD3阳性T淋巴细胞。
第六方面,本发明提供了一种如第一方面所述的靶向CD38的单链抗体、如第二方面所述的或如第五方面所述的制备方法制得的靶向CD38的嵌合抗原受体T细胞、如第三方面所述的重组病毒载体或如第四方面所述的宿主细胞在制备预防、诊断和治疗恶性肿瘤药物中的应用。尤其适用于表达CD38的恶性肿瘤。例如,可用于骨髓瘤、淋巴瘤等恶性肿瘤的预防、诊断和治疗等。
所述应用具体为:提供了一种试剂盒,所述试剂盒包括第一方面所述的靶向CD38的单链抗体、如第二方面所述的靶向CD38的嵌合抗原受体T细胞、如第三方面所述的重组病毒载体、如第四方面所述的宿主细胞中的一种或多种。
本发明的优点将会在下面的说明书中部分阐明,一部分根据说明书是显而易见的,或者可以通过本发明实施例的实施而获知。
附图说明
图1为本发明实施例提供的pWPXLd-CAR-CD38重组质粒的质粒图谱。
具体实施方式
以下所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也视为本发明的保护范围。
实施例一
一种靶向CD38的嵌合抗原受体T细胞的制备方法,包括以下步骤:
(1)制备靶向CD38的嵌合抗原受体CAR-CD38的基因序列
分别制备信号肽、靶向CD38的单链抗体、CD8α铰链区、CD8跨膜区、CD27信号区和CD3ζ信号区的编码基因,所述信号肽的编码基因序列如SEQ ID NO:15所示,所述靶向CD38的单链抗体的编码基因序列如SEQ ID NO:2所示,所述CD8α铰链区的编码基因序列如SEQID NO:7所示,所述CD8跨膜区的编码基因序列如SEQ ID NO:9所示,所述CD27信号区的编码基因如SEQ ID NO:11所示,所述CD3ζ信号区的编码基因如SEQ ID NO:13所示。
通过PCR的方法将上述信号肽、靶向CD38的单链抗体、CD8α铰链区、CD8跨膜区、CD27信号区和CD3ζ信号区的编码基因依次从5’端到3’端连接到一起,得到靶向CD38的嵌合抗原受体CAR-CD38的编码基因,所述CAR-CD38的编码基因序列如SEQ ID NO:5所示,其中所述靶向CD38的单链抗体为人源化单链抗体。
(2)构建pWPXLd-CAR-CD38重组质粒
将CAR-CD38的编码基因序列***到pWPXLD载体的BamH1和EcoR1酶切位点之间,并在pWPXLD载体EF1α之后,以EF1α为启动子。所述CAR-CD38的编码基因序列***到pWPXLD载体时,所述CAR-CD38的编码基因序列的5’端添加有起始密码子(如ATG)与pWPXLD载体中BamH1酶切位点相连,3’端还添加有终止密码子与pWPXLD载体中EcoR1酶切位点相连。然后转入大肠杆菌感受态细胞DH5α,进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列,成功构建如图1所示的pWPXLd-CAR-CD38重组质粒。
(3)重组慢病毒构建
将pWPXLd-CAR-CD38重组质粒、包装质粒psPAX2、包膜质粒pMD2G三者共转染入培养好的HEK293T细胞。第48h收获含病毒的上清,经0.45μm滤膜过滤,-80℃超低温冰箱中保存;第72h二次收获含病毒的上清,0.45μm滤膜过滤,与第48h收获的病毒上清合并后一起加入超速离心管中,逐一放入至Beckman超速离心机内,设置离心参数为25000rpm,离心时间为2h,离心温度控制在4℃;离心结束后,弃去上清,尽量去除残留在管壁上的液体,加入病毒保存液,轻轻反复吹打重悬;经充分溶解后,高速离心10000rpm,离心5min后,取上清荧光法测定滴度,病毒按照100μl,2×108个/mL分装,保存于-80℃超低温冰箱,得到重组慢病毒。
(4)靶向CD38的嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
抽取病人血液,送样至血液分离室;采集外周血单个核细胞,Ficoll离心分离后取中间层细胞;经PBS洗涤后,得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC,加入不含血清的基础培养基,配成细胞悬液;按磁珠与细胞的比例为3:1,加入CD3/CD28免疫磁珠,室温孵1-2h;采用磁铁对孵育好磁珠的细胞进行筛选;对筛选出的细胞去除磁珠,PBS洗涤,得到CD3阳性T淋巴细胞。
c)病毒转染法制备抗原特异性T淋巴细胞
取上述经过免疫磁珠分离法得到的CD3阳性T淋巴细胞,加入与CD3阳性细胞数相应的病毒滴度的所述重组慢病毒进行培养。
培养的第3天,进行细胞计数和换液,调整细胞浓度为1×106个/mL,接种,培养;培养的第5天,观察细胞状态,如果细胞密度增大,则稀释细胞浓度为1×106个/mL,检测细胞活性,继续培养。扩增培养到第9-11天,收集细胞,得到靶向CD38的嵌合抗原受体T细胞,并保存在回输专用的细胞冻存液中,以供患者回输用。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 深圳宾德生物技术有限公司
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ggatctgggg gaggcggaag cgaggtgcag ctgttggagt ctgggggagg cttggtacag 480
cctggggggt ccctgagact ctcatgtgca gtctctggat tcacctttaa cagctttgcc 540
atgagctggg tccgccaggc tccagggaag gggctggagt gggtctcagc tattagtggt 600
agtggtggtg gcacatacta cgcagactcc gtgaagggcc ggttcaccat ctccagagac 660
aattccaaga acacgctgta tctgcaaatg aacagcctga gagccgagga cacggccgta 720
tatttctgtg cgaaagataa gattctctgg ttcggggagc ccgtctttga ctactggggc 780
cagggaaccc tggtcaccgt ctcctcagcc tccaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960
gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020
aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 1080
agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 1140
gagcctgcct gctccccccg cgtgaaattc agccgcagcg cagatgctcc agcctacaag 1200
caggggcaga accagctcta caacgaactc aatcttggtc ggagagagga gtacgacgtg 1260
ctggacaagc ggagaggacg ggacccagaa atgggcggga agccgcgcag aaagaatccc 1320
caagagggcc tgtacaacga gctccaaaag gataagatgg cagaagccta tagcgagatt 1380
ggtatgaaag gggaacgcag aagaggcaaa ggccacgacg gactgtacca gggactcagc 1440
accgccacca aggacaccta tgacgctctt cacatgcagg ccctgccgcc tcgg 1494
<210> 6
<211> 45
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
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> 7
<211> 135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 8
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
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> 9
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 10
<211> 46
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro Ala Glu
1 5 10 15
Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro
20 25 30
Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro
35 40 45
<210> 11
<211> 138
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 60
agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 120
gagcctgcct gctccccc 138
<210> 12
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
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> 13
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60
tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120
cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180
gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240
agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300
tatgacgctc ttcacatgca ggccctgccg cctcgg 336
<210> 14
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
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> 15
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
Claims (10)
1.一种靶向CD38的单链抗体,其特征在于,所述靶向CD38的单链抗体包括如SEQ IDNO:1所示的氨基酸序列,所述靶向CD38的单链抗体为人源化单链抗体。
2.如权利要求1所述的靶向CD38的单链抗体,其特征在于,所述靶向CD38的单链抗体的编码基因包括如SEQ ID NO:2所示的核苷酸序列。
3.一种靶向CD38的嵌合抗原受体T细胞,其特征在于,包括靶向CD38的嵌合抗原受体CAR-CD38,所述CAR-CD38包括从氨基端到羧基端顺次连接的靶向CD38的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列,其中所述靶向CD38的单链抗体包括如SEQ IDNO:1所示的氨基酸序列。
4.如权利要求3所述的靶向CD38的嵌合抗原受体T细胞,其特征在于,所述胞外铰链区包括CD8α铰链区,所述跨膜区包括CD8跨膜区,所述胞内信号区包括从氨基端到羧基端顺次连接的CD27信号区和CD3ζ信号区。
5.如权利要求4所述的靶向CD38的嵌合抗原受体T细胞,其特征在于,所述CAR-CD38的氨基酸序列包括如SEQ ID NO:3所示的氨基酸序列。
6.如权利要求3或4所述的靶向CD38的嵌合抗原受体T细胞,其特征在于,所述CAR-CD38的编码基因包括如SEQ ID NO:4所示的核苷酸序列。
7.一种重组病毒载体,其特征在于,所述重组病毒载体包括如权利要求3-6任一项所述的靶向CD38的嵌合抗原受体T细胞的CAR-CD38的编码基因序列。
8.一种宿主细胞,其特征在于,所述宿主细胞包括如权利要求7所述的重组病毒载体。
9.一种靶向CD38的嵌合抗原受体T细胞的制备方法,其特征在于,包括:
(1)提供靶向CD38的嵌合抗原受体CAR-CD38的编码基因,包括从5’端到3’端顺次连接的信号肽的编码基因、靶向CD38的单链抗体的编码基因、CD8α铰链区的编码基因、CD8跨膜区的编码基因、CD27信号区的编码基因和CD3ζ信号区的编码基因;所述靶向CD38的单链抗体为人源化单链抗体,所述靶向CD38的单链抗体的编码基因包括如SEQ ID NO:2所示的核苷酸序列;
(2)将所述CAR-CD38的编码基因***到pWPXLD载体中,得到pWPXLD-CAR-CD38重组质粒;
(3)将所述pWPXLD-CAR-CD38重组质粒与包膜质粒、包装质粒共转染宿主细胞,得到重组慢病毒;
(4)将所述重组慢病毒转染CD3阳性T淋巴细胞,获得靶向CD38的嵌合抗原受体T细胞。
10.一种如权利要求1-2任一项所述的靶向CD38的单链抗体、如权利要求3-6任一项所述的或如权利要求9所述的制备方法制得的靶向CD38的嵌合抗原受体T细胞、如权利要求7所述的重组病毒载体或如权利要求8所述的宿主细胞在制备预防、诊断和治疗恶性肿瘤药物中的应用。
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CN113801228A (zh) * | 2020-06-17 | 2021-12-17 | 常州费洛斯药业科技有限公司 | 靶向cd38的单链抗体、全人源嵌合抗原受体、制备方法和应用 |
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CN110157679A (zh) * | 2018-02-12 | 2019-08-23 | 深圳宾德生物技术有限公司 | 一种靶向性t淋巴细胞及其制备方法和应用 |
CN110157679B (zh) * | 2018-02-12 | 2022-05-27 | 深圳宾德生物技术有限公司 | 一种靶向性t淋巴细胞及其制备方法和应用 |
CN113801228A (zh) * | 2020-06-17 | 2021-12-17 | 常州费洛斯药业科技有限公司 | 靶向cd38的单链抗体、全人源嵌合抗原受体、制备方法和应用 |
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