CN111983218A - 一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒 - Google Patents
一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种用于检测活细胞‑活细胞表面受体‑配体相互作用的试剂盒。该试剂盒包括三个部分:1)线性质粒载体,可用于插入待测配体DNA序列;2)应用于配体细胞表面展示的重组宿主细胞;3)可用于流式细胞技术检测细胞功能的荧光标记的抗体。本发明可以应用于原位检测活细胞‑活细胞相互作用,活细胞膜表面受体‑配体之间的相互作用以及待测配体的活性检测及分析。同时,在细胞免疫治疗方法的开发中,可以用于针对肿瘤相关抗原的嵌合抗原受体的评价和筛选。本发明的人工重组质粒及试剂盒系统可以解决受体‑配体体外构象不正确的影响,加快靶向CARs的获得,在免疫治疗上具有深远的意义。
Description
技术领域
本发明属于生物治疗技术领域,具体涉及一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒。
背景技术
CAR-T细胞免疫疗法,即嵌合抗原受体T细胞免疫疗法,是一种经临床验证高效的细胞免疫疗法。其原理是通过对患者体内的免疫细胞-T细胞进行基因改造,使其可以杀死体内的癌细胞。CAR-T细胞免疫疗法已在白血病、淋巴瘤、多发性骨髓瘤的治疗中展现出其惊人的效果。比如FDA批准上市的诺华(Novartis)的Kymriah,用于治疗25岁以下复发或难治性急性淋巴细胞白血病的患者,以及凯特(Kite)制药的Yescarta,用于治疗特定类型的大B细胞淋巴瘤成人患者。
CARs由T细胞受体(T cell receptor,TCR)的胞内信号区(如CD3ζ和CD28)、跨膜区以及胞外抗原结合区组成,而这个胞外区具有抗体单链可变区片段功能即识别肿瘤相关(tumor-associated antigen,TAA)的功能。其中肿瘤相关抗原(Tumor-associatedantigens,TAAs)是一类既表达于肿瘤细胞又存在于正常细胞和组织的抗原分子,在肿瘤组织中只表现出量的变化。
CARs通过与TAA结合,可通过由CD3或高亲和性受体FcεRI的胞内区使T细胞活化发挥效应功能。T细胞会活化增殖为CTL细胞。当CTL细胞再次遇到携带有同TAA的肿瘤细胞时,就会通过同样的机制与之结合,并分泌穿孔蛋白、粒酶及细胞因子协同作用杀死肿瘤细胞。CTL具有十分强大的杀伤肿瘤细胞的能力,理论上讲一个CTL可以杀死数十到上百个肿瘤细胞。
由于CAR-T使用其胞外单链抗体进行对肿瘤细胞表面相关抗原(TAA)或肿瘤特异性抗原(TSA)的靶向结合,进而通过其胞内结构域激活T细胞进行对肿瘤细胞的靶向杀伤,所以靶标抗原的选择对于这一特异性杀伤作用至关重要。而由于肿瘤细胞表面很难有TSA可供利用(CD19的特异性表达也是CAR-T在B系白血病和淋巴瘤等治疗方面如此成功的一个因素),导致一系列靶向TAA的CAR-T因为脱靶效应而出现严重副作用。因此,在使用肿瘤相关抗原作为CAR-T细胞疗法的靶点的同时又能降低其所引发的脱靶效应,是一个急需突破的瓶颈。
而针对TSA/TAA的有效靶向问题,需要根据不同的抗原表达情况和癌种的不同,选择不同的ScFv,例如,针对NY-ESO/CD19等特异性较高的抗原,可以采用高亲和力ScFv实现对CAR提供较强的结合力从而使其更迅速地激活启动的杀伤,而在正常组织中也会有微量表达的肿瘤特异性的TAA,适宜采用中等
亲和力的scFv以实现降低脱靶作用等副作用的发生。另外,针对微环境形成较为紧密和“顽固”的实体瘤,采用特殊策略辅助ScFv提高靶向性和免疫突触有效形成,将有助于CAR-T更好地发挥杀伤作用,与此同时,目前的双信号独立的CAR-T结构或者双特异性CAR-T结构,都需要在这一方面做一定改进和考虑。
发明内容
肿瘤相关抗原虽然在肿瘤细胞表面具有异于正常组织和细胞的高表达量,但是其作为细胞免疫治疗的靶点仍然会产生不同程度的脱靶效应,即靶向抗原分子但脱离靶组织,进而杀伤健康细胞。因此,使T细胞可以特异性识别病变的组织细胞,激活免疫反应的同时,减少脱靶效应是亟待解决的问题。
根据本发明的第一方面,提供一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒,该试剂盒包括线性质粒载体、宿主细胞和用于信号检测的荧光抗体三个组成部分,所述线性质粒载体包括胞外信号识别区、跨膜链接区和胞内信号转导区;所述胞外信号识别区包括基于不同识别域的配体,所述不同识别域包含单链抗体、纳米抗体、细胞因子和标签蛋白等配体识别区;所述的跨膜链接区用Hing TM表示,包含铰链区Hinge和跨膜结构TM,连接胞外信号识别区和胞内信号转导区。
具体情况下,所述线性质粒载体的核苷酸序列包括SEQ ID NO:1或该序列内一个或少数几个碱基突变的SEQ ID NO:1或与SEQ ID NO:1具有至少75%同源性的序列;该胞内信号转导区的氨基酸序列包括SEQ ID NO:2或该序列内一个或少数几个碱基突变的SEQ IDNO:2或与SEQ ID NO:2具有至少75%同源性的序列。
具体情况下,所述宿主细胞包括B淋巴细胞或T淋巴细胞来源的细胞。
根据本发明的第二方面,提供本发明的第一方面的试剂盒用于检测活细胞-活细胞表面受体-配体相互作用,包括以下步骤:
(1)人工重组质粒的构建,通过特异性引物扩增特定配体的基因片段,将目的基因片段连接到人工线性载体上,构建重组质粒;将构建的人工重组质粒导入大肠杆菌工程菌中扩增并提取质粒,得到足够拷贝数的重组质粒;
(2)将步骤(1)中构建的重组质粒通过慢病毒转染或者电转的细胞转染方法导入宿主细胞中,得到膜表面展示特定配体的重组宿主细胞;
(3)将扩增得到的重组宿主细胞重悬于缓冲液中,在细胞悬液中加入荧光抗体孵育,利用荧光标记的抗体检测染色细胞,通过流式细胞技术验证分析结果。
根据本发明的第三方面,提供本发明第一方面的试剂盒应用于CD19细胞表面抗原的识别验证,包括以下步骤:
(1)人工重组质粒的构建:通过特异性引物扩增FMC63-scFv目的基因片段,酶切连接到试剂盒提供的线性载体质粒上,转化大肠杆菌工程宿主菌中,提取质粒经过Sanger测序验证插入DNA序列的正确性;
(2)细胞转染:将带有特定配体基因的人工重组质粒导入宿主细胞中,通过流式技术验证质粒成功导入宿主细胞,获得表面展示FMC63-scFv的宿主细胞;
(3)细胞表面受体-配体识别验证:利用荧光标记的抗体检测效应细胞被激活的比例,将过表达CD19的肿瘤细胞作为靶细胞,表达嵌合抗原受体FMC-63-CAR的宿主细胞作为效应细胞,将靶细胞与效应细胞共培养,利用荧光抗体检测受体-配体在膜表面的相互作用。
本发明可以应用于原位检测活细胞-活细胞相互作用,活细胞膜表面受体-配体之间的相互作用以及待测配体的活性检测及分析。同时,在细胞免疫治疗方法的开发中,可以用于针对肿瘤相关抗原的CARs的评价和筛选。本发明的人工重组质粒及试剂盒系统可以解决受体-配体体外构象不正确的影响,加快靶向CARs的获得,在免疫治疗上具有深远的意义。
附图说明
图1表示本发明试剂盒中线性质粒载体的结构图。
图2表示空白重组细胞的荧光信号图。
图3表示CD19-antibody FMC-63成功表达在宿主细胞表面的荧光信号图。
图4表示基于细胞-细胞相互作用验证表达CD19-antibody FMC-63宿主细胞识别CD19抗原表达阴性的靶细胞K562的流式结果图。
图5表示基于细胞-细胞相互作用验证表达CD19-antibody FMC-63宿主细胞识别CD19抗原表达阴性的靶细胞Raji的流式结果图。
具体实施方式
术语定义
TAA是指肿瘤相关抗原(tumor-associated antigen,TAA),是非肿瘤细胞所特有的、正常细胞和其他组织上也存在的抗原,只是其含量在细胞癌变时明显增高。此类抗原只表现出量的变化,而无严格肿瘤特异性。如胚胎性抗原是其中的典型代表。TSA肿瘤特异性抗原(tumor specific antigen)指仅表达于某种肿瘤细胞表面而不存在于正常细胞上的新抗原,此类抗原乃通过肿瘤在同种系动物间的移植而被证实,故也称为肿瘤特异性移植抗原(tumor specific transplantation antigen,TSTA)或肿瘤排斥抗原(tumorrejection antigen,TRA)。化学或物理因素诱生的肿瘤抗原、自发肿瘤抗原和病毒诱导的肿瘤抗原等多属此类。
慢病毒(Lentivirus)载体是以HIV-1(人类免疫缺陷I型病毒)为基础发展起来的基因治疗载体。区别一般的逆转录病毒载体,它对分裂细胞和非分裂细胞均具有感染能力。慢病毒载体的研究发展得很快,研究的也非常深入。该载体可以将外源基因有效地整合到宿主染色体上,从而达到持久性表达。在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞,从而达到良好的的基因治疗效果。
CARs由T细胞受体(T cell receptor,TCR)的胞内信号区(如CD3ζ和CD28)、跨膜区以及胞外抗原结合区组成,而这个胞外区具有抗体单链可变区片段功能即识别特定肿瘤抗原(tumor-associated antigen,TAA)的功能。CARs一旦与TAA结合,可通过由CD3或高亲和性受体FcεRI的胞内区使T细胞活化发挥效应功能。T细胞会活化增殖为CTL细胞。当CTL细胞再次遇到携带有同TAA的肿瘤细胞时,就会通过同样的机制与之结合,并分泌穿孔蛋白、粒酶及细胞因子协同作用杀死肿瘤细胞。
CAR-T是通过基因转导技术,把识别肿瘤相关抗原的单链抗体和T细胞活化序列的融合蛋白表达到T细胞表面。从只携带有CD3ζ胞内激活结构域的一代CAR,逐渐演化到携带CD28/4-1BB/OX40/ICOS等胞内共刺激结构域和CD3ζ胞内激活结构域的二代CAR(携带一个共刺激结构域),三代CAR(携带两个共刺激结构域)。CTL细胞细胞是指毒性T淋巴细胞(cytotoxic lympho-cyte,CTL),是白细胞的亚部,是一种特异T细胞,专门分泌各种细胞因子参与免疫作用。对某些病毒、肿瘤细胞等抗原物质具有杀伤作用,与自然杀伤细胞构成机体抗病毒、抗肿瘤免疫的重要防线。
这类相互作用原理是细胞表面的展示的分子之间的相互作用,发生在细胞-细胞之间,需要一种能够基于细胞相互作用的细胞膜表面的受体-配体相互作用的检测方法。
部分材料来源说明与此:
去内毒素小提质粒试剂盒(Omega公司,D6950-02)
DNA片段回收试剂盒(Taraka公司,9761)
胶回收试剂盒(Taraka公司,9762)
SfiI(NEB公司,R0123S)
大肠杆菌DH5α感受态细胞(天根公司,CB101)
DMEM(Life,11966025)
FBS(Gibco,10099141)
polybrene(Merck,TR-1003-G)
实验案例一人工重组质粒的构建
通过特异性引物扩增特定配体的基因片段,然后将目的基因片段连接到人工线性载体上,构建重组质粒,导入大肠杆菌工程菌中扩增并提取质粒得到足够拷贝数的重组质粒;
具体步骤包括:
1、目的基因片段的合成
设计引物PCR扩增CD19抗体基因FMC63,PCR产物回收试剂盒回收FMC63的DNA片段;取2μg CD19的抗体FMC-63基因片段,加入1μl SfiI进行酶切,50℃水浴酶切2h后胶回收酶切产物。
2、重组质粒的构建
取本试剂盒提供的线性载体质粒和FMC-63sfiI酶切产物各50ng,16℃连接1h;取2μl连接产物转入100μl大肠杆菌DH5α感受态细胞中,冰上孵育30min,42℃热激45s,冰上再放5min,加入2ml的LB培养基,37℃培养1h;离心收集菌体,加入100μl的培养基重悬,全部涂布到含有氨苄青霉素的LB平板中,37℃培养过夜。
3、重组质粒的验证
随机挑选3个单克隆到含有3ml LB培养基及100ng/μl氨苄青霉素的14ml培养管中,37℃过夜培养;离心收集过夜生长的菌体,用去内毒质粒小提试剂盒进行质粒提取,Sanger测序法验证序列的正确性。
实验结果:Sanger测序结果分析线性载体质粒中插入正确的目的基因片段,即构建CD19antibody-FMC63重组质粒成功。
实验案例二重组宿主细胞的构建
将实验案例一中构建的人工重组质粒通过脂质体或者电转的细胞转染方法导入宿主细胞中,得到含有特定配体基因的重组宿主细胞;
具体步骤包括:
1、转染前1天将0.5~2×105细胞接种于24孔培养板,并加入500ul不含抗生素的完全培养基。
2、准备复合物
(1)将0.8ug DNA稀释于50ul无血清无抗生素的培养液中轻轻混匀。
(2)将2ul Lipofectamine2000稀释于50ul无血清无抗生素的培养液中,轻轻混匀,室温孵育5分钟。
(3)5分钟后将它们混合,并轻轻混匀,室温孵育20分。
3、吸去培养板的培养基,用PBS或无血清培养基(最好)清洗细胞2次。
4、将复合物(总体积100ul)加入培养孔,前后摇动培养板使其分布均匀。
5、将细胞放入培养箱孵育4~6h后,更换含血清培养液去除复合物(也可不用)。
6、24~48h后可以观察转入基因表达情况。
实验结果:如图2所述,未转染本发明提供的线性载体质粒的空白宿主细胞没有绿色荧光信号;如图3所示,成功表达CD19-antibody FMC-63的宿主细胞表现明显的绿色荧光信号。
实验案例三CD19表面抗原-抗体的识别检测
利用流式细胞术检测CAR的表达以及宿主细胞的激活
1.将抗原过表达的Raji细胞/抗原无表达的K562细胞与转入FMC-63的重组质粒的宿主细胞在37℃混合共培养48h。
2.混合培养的细胞重悬于100微升FACS缓冲液(含2mM EDTA和0.5%BSA的PBS)(EDTA,即Ethylenediaminetetraacetic acid)中,在细胞悬液中加入荧光标记的单克隆抗体(1:500稀释,Cell Signaling,2276S),冰上4℃孵育15分钟。
3.清洗细胞一次后重悬于100微升FACS缓冲液。
4.CytoFLEX(购自:Beckman Coulter)流式细胞仪用于获取染色细胞,CytExpert用于分析结果。
实验结果:将CD19抗原表达阴性的人慢性髓原白血病细胞K562和CD19抗原表达阳性的人源Burkitt’s淋巴瘤细胞Raji作为靶细胞,分别与转入可以识别CD19的FMC-63重组质粒的效应宿主细胞共同培养后,用荧光抗体检测FMC63识别能力,如图4、5所示,流式细胞术分析结果显示转入FMC-63质粒的重组细胞能够较好地识别高表达CD19抗原的Raji细胞,不能识别CD19表达阴性的K562细胞。
序列表
<110> 常州费洛斯药业科技有限公司
<120> 一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒
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gaccttctca agttggcggg agacgtggag tccaacccag ggcccgctag catggtgagc 840
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 900
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Claims (5)
1.一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒,该试剂盒包括线性质粒载体、宿主细胞和用于信号检测的荧光抗体三个组成部分,所述线性质粒载体包括胞外信号识别区、跨膜链接区和胞内信号转导区;所述胞外信号识别区包括基于不同识别域的配体,所述不同识别域包含单链抗体、纳米抗体、细胞因子和标签蛋白等配体识别区;所述的跨膜链接区用Hing TM表示,包含铰链区Hinge和跨膜结构TM,连接胞外信号识别区和胞内信号转导区。
2.根据权利要求1所述的试剂盒,所述线性质粒载体的核苷酸序列包括SEQ ID NO:1或该序列内一个或少数几个碱基突变的SEQ ID NO:1或与SEQ ID NO:1具有至少75%同源性的序列;该胞内信号转导区的氨基酸序列包括SEQ ID NO:2或该序列内一个或少数几个碱基突变的SEQ ID NO:2或与SEQ ID NO:2具有至少75%同源性的序列。
3.根据权利要求1所述的试剂盒,所述宿主细胞包括B淋巴细胞或T淋巴细胞来源的细胞。
4.根据权利要求1-3之一所述的试剂盒用于检测活细胞-活细胞表面受体-配体相互作用,包括以下步骤:
(1)人工重组质粒的构建,通过特异性引物扩增特定配体的基因片段,将目的基因片段连接到人工线性载体上,构建重组质粒;将构建的人工重组质粒导入大肠杆菌工程菌中扩增并提取质粒,得到足够拷贝数的重组质粒;
(2)将步骤(1)中构建的重组质粒通过慢病毒转染或者电转的细胞转染方法导入宿主细胞中,得到膜表面展示特定配体的重组宿主细胞;
(3)将扩增得到的重组宿主细胞重悬于缓冲液中,在细胞悬液中加入荧光抗体孵育,利用荧光标记的抗体检测染色细胞,通过流式细胞技术验证分析结果。
5.一种采用权利要求1-3之一所述的试剂盒应用于CD19细胞表面抗原的识别验证,包括以下步骤:
(1)人工重组质粒的构建:通过特异性引物扩增FMC63-scFv目的基因片段,酶切连接到试剂盒提供的线性载体质粒上,转化大肠杆菌工程宿主菌中,提取质粒经过Sanger测序验证插入DNA序列的正确性;
(2)细胞转染:将带有特定配体基因的人工重组质粒导入宿主细胞中,通过流式技术验证质粒成功导入宿主细胞,获得表面展示FMC63-scFv的宿主细胞;
(3)细胞表面受体-配体识别验证:利用荧光标记的抗体检测效应细胞被激活的比例,将过表达CD19的肿瘤细胞作为靶细胞,表达嵌合抗原受体FMC-63-CAR的宿主细胞作为效应细胞,将靶细胞与效应细胞共培养,利用荧光抗体检测受体-配体在膜表面的相互作用。
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