CN117327196A - 一种靶向psca和msln的双特异性嵌合抗原受体及其应用 - Google Patents
一种靶向psca和msln的双特异性嵌合抗原受体及其应用 Download PDFInfo
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Abstract
本发明涉及一种靶向PSCA和MSLN的双特异性嵌合抗原受体及其应用,所述嵌合抗原受体包含能够结合抗原的胞外结构域、跨膜结构域和至少一个胞内结构域,其中,所述胞外结构域包括抗PSCA和MSLN的抗原结合域。本发明的嵌合抗原受体有更小的临床副作用,可有效提高表达CAR的免疫细胞的扩增效率,有效提高肿瘤的治疗效果。
Description
技术领域
本发明涉及肿瘤的细胞免疫治疗领域,尤其涉及一种靶向PSCA和MSLN的双特异性嵌合抗原受体及其应用。
背景技术
目前,嵌合抗原受体(CAR)修饰的T细胞在治疗B细胞恶性肿瘤的临床试验中已取得了令人欣喜的结果,为CAR技术治疗复发难治性多发性骨髓瘤(MM)带来了曙光。多发性骨髓瘤是一种起源于骨髓中浆细胞的恶性疾病,而浆细胞是B淋巴细胞发育到最终功能阶段的细胞。
尽管CAR-T细胞疗法已取得明显的疗效,但仍然存在许多阻碍。目前广泛应用的CAR结构主要由以下部分组成:最外端的单克隆单链抗体序列,主要识别抗原,决定CAR-T细胞的靶向性;铰链区,连接单克隆抗体序列和跨膜序列;跨膜基序,使得CAR可以锚定在免疫细胞胞膜上;胞内信号传导序列,主要为一个或者多个共刺激分子胞内段以及CD3ζ链串联,为CAR-T细胞提供活化所需的第一、二信号。目前多认为铰链区仅仅是连接功能,是否对整个嵌合抗原受体能够起到增强作用鲜少研究。
发明内容
针对目前CAR-T技术治疗肿瘤中靶向不十分理想,以及肿瘤微环境影响CAR-T技术治疗效果的情况,本发明提供一种靶向PSCA和MSLN的双特异性嵌合抗原受体及其应用,本发明制备的嵌合抗原受体有更小的临床副作用与更高的安全性,可有效提高表达CAR的免疫细胞的扩增效率,有效提高肿瘤的治疗效果。
为达此目的,本发明采用以下技术方案:
一方面,本发明提供一种靶向PSCA和MSLN的双特异性嵌合抗原受体,所述包含至少一个胞外结构域、任选的跨膜结构域和至少一个胞内共刺激信号传导结构域,其中,所述胞外结构域包含抗PSCA和抗Mesothelin的抗原识别结合结构域。
根据本发明,所述胞外结构域为抗PSCA和抗MSLN的单链抗体或抗PSCA和抗MSLN的单域抗体,其中,对于抗PSCA抗原结合域和抗MSLN抗原结合域的顺序对于嵌合抗原的效果不会造成影响,连接顺序例如可以是抗PSCA抗原结合域-linker-抗MSLN抗原结合域,或抗MSLN抗原结合域-linker-抗PSCA抗原结合域,这样的顺序的调整都不会对嵌合抗原受体的效果产生影响。
本发明中,发明人发现通过将抗PSCA抗体和抗MSLN抗体采用linker连接后,得到的双特异性抗体制备成嵌合抗原受体,能够有效提高表达CAR的免疫细胞的扩增效率,还能有效避免发生靶点逃逸现象,提高肿瘤的治疗效果,不仅如此,发明人还发现抗PSCA抗原结合域和抗MSLN抗原结合域的连接顺序不会对嵌合抗元受体的效果产生影响。
根据本发明,所述所述胞外结构域和跨膜结构域是通过铰链区连接,所述铰链区V的核苷酸序列如SEQ ID NO.1所示或与其具有至少90%,优选95%序列同一性的变体。
根据本发明,所述SEQ ID NO.1所示的核苷酸序列如下:
GACTGCAATCCAGGGTTTCACTGCCTGGGGGCAGGATGCAGCATGTGTGAACAGGA
TTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACA
TTTAACGATCAGAAACGTGGCATCTGTCGACCCTGGACAAACTGTTCTTTGGATGGAAA
GTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCC
GACCTCTCTCCGGGAGCATCCTCTGTGACCCCGCCTGCCCCTGCGAGAGAGCCAGGAC
ACTCTCCGCAG.
根据本发明,所述所述胞外结构域和跨膜结构域是通过铰链区连接,所述铰链区V的氨基酸序列如SEQ ID NO.2所示或与其具有至少90%,优选95%序列同一性的变体。
在具体的实施例中,所述氨基酸序列如SEQ ID NO.2所示:
DCNPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLD GKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ.
在本发明中,发明人意外发现通过上述铰链区能够显著提高嵌合抗原受体的扩增效率,有效提高肿瘤的治疗效果。
在具体的实施例中,所述序列同一性可以为90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%的变体。
根据本发明,所述抗PSCA和抗MSLN的抗原结合域的氨基酸序列如SEQ ID NO.2-3所示,所述SEQ ID NO.3的核苷酸序列如下:
DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGS
SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEF
VPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSEPKSCDKTH
TCPPC.
所述SEQ ID NO.4的核苷酸序列如下:
SQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSK
WYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQG
TTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIY
WYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYC
MIWHSSAAVFGGGTQLTVLSGILEQQG.
根据本发明,所述跨膜结构域为CD28跨膜结构域和/或CD8α跨膜结构域。
根据本发明,所述胞内结构域还包括胞内共刺激信号传导域和/或CD3ζ信号传导域。
根据本发明,所述共刺激信号传导结构域为人4-1BB胞内区、人CD28胞内区、人CD27胞内区、人OX40胞内区、人CD30胞内区、人CD40胞内区或人OX40胞内区中的任意一种或至少两种的组合,优选为人4-1BB胞内区。
根据本发明,所述胞外结构域和跨膜结构域是通过铰链区连接的,所述铰链区包含IgG1铰链区和/或CD8α铰链区。
根据本发明,所述双特异性嵌合抗原受体还包括信号肽,所述信号肽为能够指导嵌合抗原受体跨膜转移的信号肽都是可行的,本领域技术人员可以根据需要选择本领域常规的信号肽,所述嵌合抗原受体还包括信号肽,优选为CD8α信号肽或Secretory信号肽。
本发明的双特异性嵌合抗原受体还包括启动子,所述启动子可以为EFα或任何一种高表达的启动子,本领域技术人员可以根据实际情况进行选择,在此不做特殊限定,启动子的存在不会对本发明的双特异性嵌合抗原受体的性能产生影响。
根据本发明,所述双特异性嵌合抗原受体包括信号肽、结合PSCA和MSLN抗原的胞外结构域、铰链区、跨膜结构域、共刺激信号传导结构域和DAP10-CD3ζ-TLR2信号传导结构域串联而成。
根据本发明,所述双特异性嵌合抗原受体为DAP10-CD3ζ-TLR2信号肽,结合PSCA和MSLN抗原的胞外结构域,V铰链区,CD8α跨膜结构域,4-1BB信号传导结构域和CD3ζ信号传导结构域串联而成。
第二方面,编码如第一方面所述的嵌合抗原受体的核酸。
第三方面,本发明提供一种病毒载体,包括第二方面所述的核酸。
根据本发明,所述病毒载体为慢病毒载体和/或逆转录病毒载体,优选为慢病毒载体。
第四方面,本发明提供一种T细胞,采用如第一方面所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达。
根据本发明,所述转染的方式为通过病毒载体和/或真核表达质粒转染到T细胞,优选为通过病毒载体转染到T细胞。
本发明中,所述T细胞具有良好的靶向杀伤作用,同时能够释放低剂量免疫因子,具备低毒性高免疫杀伤反应性质。
第五方面,本发明提供一种重组慢病毒,将包含如第三方面所述的病毒载体与包装辅助质粒gag/pol、Rev和VSV-G共转染哺乳细胞得到的重组慢病毒。
根据本发明,所述哺乳细胞为293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。
第六方面,本发明提供一种组合物,所述组合物包括如第一方面所述的嵌合抗原受体和/或如第五方面所述的重组慢病毒。
第七方面,本发明提供如第一方面所述的嵌合抗原受体、如第二方面所述的核酸、如第三方面所述的病毒载体、如第四方面所述的T细胞、如第五方面所述的重组慢病毒或如第六方面所述的组合物在制备嵌合抗原受体T细胞、免疫细胞或在肿瘤治疗药物中的应用。
根据本发明,所述肿瘤包括肝癌、肺癌、乳腺癌、胃癌、肾母细胞瘤、神经胶质瘤、神经母细胞瘤、黑色素瘤、鼻咽癌、间皮质瘤、胰岛细胞瘤、视网膜母细胞瘤、胰腺癌、子宫肌瘤、宫颈癌或甲状腺癌中的任意一种或至少两种的组合。
与现有技术相比,本发明具有如下有益效果:
(1)本发明通过将嵌合抗原受体中的胞外结构域设置为抗PSCA和抗MSLN的抗原结合域,不仅能够有更小的临床副作用与更高的安全性,可有效提高表达CAR的免疫细胞的扩增效率,有效提高肿瘤的治疗效果。
(2)本发明的构建的包含抗PSCA和抗MSLN的双特异性嵌合抗原受体的细胞对特异性肿瘤细胞具有显著提高的清除杀伤作用,具有良好的治疗效果。
附图说明
图1为PSCA-V-MSLN CAR的体外扩增效率;
图2为MSLN-V-PSCACAR的体外扩增效率;
图3为PSCA-H-MSLN CAR的体外扩增效率;
图4为WT的体外扩增效率;
图5为CAR-T细胞对胃癌细胞的杀伤功能。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果,以下结合附图并通过具体实施方式来进一步说明本发明的技术方案,但本发明并非局限在实施例范围内。
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
本实施例构建了抗PSCA和抗MSLN的双特异性嵌合抗原受体,该嵌合抗原受体包括DAP10-CD3ζ-TLR2信号肽,结合PSCA和MSLN抗原的胞外结构域,V铰链区,CD8α跨膜结构域,4-1BB信号传导结构域和CD3ζ信号传导结构域串联而成。
实施例2:构建抗PSCA的嵌合抗原受体表达载体
(1)全基因合成PSCA-V-MSLN CAR、MSLN-V-PSCACAR、PSCA-H-MSLN CAR、WT序列,用EcoRI和BamHI双酶切全基因合成的CAR和空载体,于37℃水浴中酶切30min后,使用1.5%的琼脂糖凝胶进行DNA电泳,然后使用天根的琼脂糖凝胶试剂盒纯化回收处理;
(2)pLVX-EF1-MCS载体与CAR基因片段的连接:
连接体系如下:
组件 | 添加量(μl) |
pLVX-EF1-MCS载体 | 2(50ng) |
CAR基因 | 10(150ng) |
T4DNA连接缓冲液 | 2 |
T4DNA连接酶(NEB) | 1 |
ddH2O | 5 |
总共 | 20 |
于22℃连接1h,连接产物直接转化Stbl3大肠杆菌感受态细胞,取200μl转化产物涂布氨苄抗性的LB平板,LB平板于37℃的培养箱中倒置培养过夜。次日早晨随机挑选3个单克隆进行菌落PCR鉴定,将阳性克隆送样测序。
抗PSCA和抗MSLN的双特异性嵌合抗原受体慢病毒表达载体pLVX-PSCA-V-MSLNCAR、pLVX-PSCA-H-MSLN CAR;抗MSLN和抗PSCA的双特异性嵌合抗原受体慢病毒表达载体pLVX-MSLN-V-PSCACAR。
实施例3:慢病毒包装
分别对实施例中的慢病毒表达载体进行慢病毒包装,采用四质粒系统,具体步骤如下:
(1)四质粒系统分别表达慢病毒载体包装所需的gag/pol、Rev、VSV-G及本发明工程稳定的单链抗体构成的人工嵌合抗原受体:将四质粒进行瞬时转染293T细胞,总质量为10μg;
(2)将上述质粒加入至一定体积的无血清的DMEM中,混匀后放置15分钟,将上述混合液加入至铺有293T细胞的T75培养瓶中,轻轻混匀,于37℃、5%CO2细胞培养箱培养6h;
(3)6h后更换新鲜培养基,继续进行培养,并且加入10mM的丁酸钠溶液,72小时后收集慢病毒的培养上清进行纯化检测。
实施例4:CAR-T细胞的扩增
每位志愿者采30ml的全血,将外周血与生理盐水1:1进行稀释,在离心管内加入Ficoll,缓缓加入稀释后的外周血,1500rpm离心30min轻轻吸取PBMC层移入另一离心管内;
用生理盐水洗涤PBMC多次,转入X-VIVO培养基(含50ng/mL的OKT3,300IU/mL的IL-2)中进行培养,PBMC分离后,需要用含50ng/mL的OKT3,300IU/ml的IL-2的X-VIVO进行激活,2日后将培养基更换成含300IU/mL的X-VIVO进行扩大培养,而后每两天进行一次计数并更换含300IU/mL的X-VIVO,并且将细胞浓度维持在0.5×106-1×106/mL,连续观察15天,每2天补充一次新鲜培养基,调整细胞密度;每2~3天进行一次细胞计数,流式检测CART细胞的百分比,同时检测CD4+、CD8+CAR-T细胞的比例直到体外培养结束,结果如图1-4所示。
如图1-4所示,体外培养过程中,PSCA-V-MSLN CAR、MSLN-V-PSCACAR、PSCA-H-MSLNCAR和WT细胞的数量均得到提高,PSCA-V-MSLN CAR、MSLN-V-PSCA CAR、PSCA-H-MSLN CAR增加的主要是CD4+T细胞,PSCA-V-MSLN CAR、MSLN-V-PSCA CAR增加量明显高于PSCA-H-MSLNCAR,而WT增加的主要是CD8+T细胞,
实施例6:体外检测CAR-T细胞对胃癌细胞的杀伤功能
将实施例3制备的PSCA-V-MSLN CAR、MSLN-V-PSCACAR、PSCA-H-MSLN CAR和WT分别与1×104个肿瘤细胞BGC823按E:T为4:1、2:1、1:1、1:2、1:4、1:8的比例混合,加入到96孔板中,每组设置3个复孔,250g离心5min后,置于37℃、5%CO2培养箱共培养18h;18h后,向96孔板中加入100μL/孔的荧光素酶底物(1×),将细胞重悬混匀,立即通过多功能酶标仪测定RLU(relative light unit),测定时间为1秒,利用荧光素酶(Luciferase)定量杀伤效率评估方法,体外比较PSCA-V-MSLN CAR、MSLN-V-PSCACAR、PSCA-H-MSLN CAR和WT对BGC823的杀伤作用,杀伤比例计算公式如下:
100%×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)
结果如图5所示,可以看出,相对于PSCA-H-MSLN和WT,PSCA-V-MSLN CAR、MSLN-V-PSCACAR对胃癌细胞的杀伤作用更强.
综上所述,本发明采用特定的嵌合抗原受体的铰链区,显著提高了CAR-T细胞、尤其是CD4+CAR-T细胞的体外扩增效率,构建的包含抗PSCA和抗MSLN的双特异性嵌合抗原受体的细胞对特异性肿瘤细胞具有显著提高的清除杀伤作用。
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。
Claims (10)
1.一种靶向PSCA和MSLN的双特异性嵌合抗原受体,其特征在于,其包含至少一个胞外结构域、任选的跨膜结构域和至少一个胞内共刺激信号传导结构域,其中,所述胞外结构域包含抗PSCA和抗Mesothelin的抗原识别结合结构域。
2.根据权利要求1所述的双特异性嵌合抗原受体,其特征在于,所述胞外结构域和跨膜结构域是通过铰链区连接,所述铰链区V的核苷酸序列如SEQ ID NO.1所示或与其具有至少90%,优选95%序列同一性的变体;
优选地,所述氨基酸序列如SEQ ID NO.2所示或与其具有至少90%,优选95%序列同一性的变体;
优选地,所述胞外结构域为抗PSCA和抗MSLN的单链抗体或抗PSCA和抗MSLN的单域抗体;
优选地,所述抗PSCA和抗MSLN抗体采用linker连接;
优选地,所述抗PSCA和抗MSLN的抗原结合域的氨基酸序列如SEQ ID NO.3-4所示。
3.根据权利要求1或2所述的双特异性嵌合抗原受体,其特征在于,所述跨膜结构域为CD28跨膜结构域和/或CD8α跨膜结构域;
优选地,所述胞内结构域还包括胞内共刺激信号传导域和/或CD3ζ信号传导域;
优选地,所述共刺激信号传导结构域为人4-1BB胞内区、人CD28胞内区、人CD27胞内区、人OX40胞内区、人CD30胞内区、人CD40胞内区或人OX40胞内区中的任意一种或至少两种的组合,优选为人4-1BB胞内区;
优选地,所述双特异性嵌合抗原受体还包括信号肽,优选为CD8α信号肽或Secretory信号肽。
4.根据权利要求1-3中任一项所述的双特异性嵌合抗原受体,其特征在于,所述双特异性嵌合抗原受体包括信号肽、结合PSCA和MSLN抗原的胞外结构域、铰链区、跨膜结构域、共刺激信号传导结构域和DAP10-CD3ζ-TLR2信号传导结构域串联而成;
优选地,所述双特异性嵌合抗原受体为CD8α信号肽,结合PSCA和MSLN抗原的胞外结构域,V铰链区,CD8α跨膜结构域,4-1BB信号传导结构域和DAP10-CD3ζ-TLR2信号传导结构域串联而成。
5.编码如权利要求1-4任一项所述的双特异性嵌合抗原受体的核酸。
6.一种病毒载体,其特征在于,包括如权利要求5所述的核酸;
优选地,所述病毒载体为慢病毒载体和/或逆转录病毒载体,优选为慢病毒载体。
7.一种T细胞,其特征在于,采用如权利要求1-4中任一项所述的双特异性嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达;
优选地,所述转染的方式为通过病毒载体和/或真核表达质粒转染到T细胞,优选为通过病毒载体转染到T细胞。
8.一种重组慢病毒,其特征在于,将包含如权利要求6所述的病毒载体与包装辅助质粒共转染哺乳细胞得到的重组慢病毒;
优选地,所述哺乳细胞为293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。
9.一种组合物,其特征在于,所述组合物包括如权利要求1-4中任一项所述的双特异性嵌合抗原受体和/或如权利要求8所述的重组慢病毒。
10.如权利要求1-4中任一项所述的双特异性嵌合抗原受体、如权利要求8所述的重组慢病毒或如权利要求9所述的组合物在制备嵌合抗原受体T细胞、免疫细胞或在肿瘤治疗药物中的应用;
优选地,所述肿瘤包括肝癌、肺癌、乳腺癌、胃癌、肾母细胞瘤、神经胶质瘤、神经母细胞瘤、黑色素瘤、鼻咽癌、间皮质瘤、胰岛细胞瘤、视网膜母细胞瘤、胰腺癌、子宫肌瘤、宫颈癌或甲状腺癌中的任意一种或至少两种的组合。
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