CN114480504A - Claudin18.2报告基因CHO-K1稳转细胞株构建和应用 - Google Patents
Claudin18.2报告基因CHO-K1稳转细胞株构建和应用 Download PDFInfo
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Abstract
本发明公开了一种Claudin18.2报告基因CHO‑K1稳转细胞株构建,用含Claudin18.2基因的慢病毒转染CHO‑K1细胞,通过加入真核抗生素筛选和单克隆挑选,经过表达丰度检测,挑选得到合适的高表达Claudin18.2稳转细胞株单克隆;用含CMV‑luciferase基因的慢病毒感染所述Claudin18.2稳转细胞株单克隆,通过加入真核抗生素筛选和单克隆挑选,经过功能学评价得到合适的Claudin18.2报告基因稳转细胞株。并利用该报告基因稳转细胞株进行CDC杀伤的检测方法,利用该报告基因稳转细胞株进行ADCC的杀伤的检测方法作等。本发明中的Claudin18.2报告基因CHO‑K1稳转细胞株用于CDC,ADCC细胞杀伤测定的方法具有灵敏度高,不易受到外界干扰,所用方法的稳定性好。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种Claudin18.2报告基因CHO-K1稳转细胞株构建和应用。
背景技术
Claudin18.2(UniProt ID:P56856-2)是位于细胞膜上的一个跨膜蛋白,在多种肿瘤尤其是消化系统肿瘤及其转移瘤中发现高表达。Claudin18是紧密连接(tightjunction,TJs)蛋白,在正常上皮细胞与细胞黏附的重要功能中发挥重要作用,它们通过机械的方式连接细胞,形成上皮屏障,阻止细胞间的大分子运输,维持上皮细胞极性。
人类CLDN18基因的第一个外显子存在两个等位基因,分别表达Claudin18.2蛋白和Claudin18.1蛋白,Claudin18.1与claudin18.2虽然结构极为相似,但其在肿瘤中的表达量大不相同,在正常组织中,claudin18.1只在肺中表达,而claudin18.2在胃中有限表达;在肿瘤组织中,claudin18.1在肺中并没有发生明显的高表达,而claudin18.2在胃癌、食管癌、胰腺癌等癌种中表达发生上调,如当胃上皮组织发生恶性转化时,细胞极性的紊乱将导致细胞表面的claudin18.2蛋白表位暴露。在胃癌,食管癌的靶向治疗药物研发过程,靶向claudin18.2的抗体药物研发是目前研发的热点。目前尚未有靶向claudin18.2的药物上市,处于临床研发阶段的靶向claudin18.2的药物包括Zolbetuximab,LM-102,LM-302,AMG-910等十多个品中,其中有单抗,也有双特异抗体。靶向claudin18.2的药物研发前景广阔,竞争激烈,靶向Claudin18.2的生物学分析方法(包括活结合活性检测方法,Claudin18.2抗体介导补体依赖的细胞毒性的检测方法,抗Claudin18.2抗体依赖的细胞介导的细胞毒性作用的检测方法等)是靶向Claudin18.2药物研发重要的工具,有着极大的市场应用场景。
生物治疗药物的研发主要包括药物发现,临床前和临床研究,商业化生产三个大的阶段。在药物发现和商业化生产过程中,药物相对于靶点的生物学活性测定是必不可少的过程。通常情况下采用重组表达技术可以将Claudin18.2受体蛋白重组表达出来,通过Claudin18.2候选药物和Claudin18.2受体蛋白的结合活性实验利用ELISA技术来判断药物的生物学活性。天然状态下Claudin18.2在细胞膜表面为四次跨膜蛋白,通过常规的蛋白重组技术表达出具有活性的Claudin18.2难度较大。利用重组蛋白Claudin18.2进行Claudin18.2抗体的结合活性检测会有失真的风险。
在真实的生理环境下,Claudin18.2抗体类药物通过Fab结合肿瘤细胞,其Fc端结合血清的补体,发挥抗体介导补体依赖的细胞毒性(CDC)杀伤表达Claudin18.2的肿瘤细胞。Claudin18.2抗体类药物通过Fab结合表达Claudin18.2的肿瘤细胞,其Fc端结合NK细胞,发挥抗体依赖的NK细胞介导的细胞毒作用(ADCC)来杀伤表达Claudin18.2的肿瘤细胞。为表征Claudin18.2抗体类药物功能活性,CDC效应通常会选用人血清和靶细胞在Claudin18.2抗体类药物存在的情况下共孵育,通过检测靶细胞的内含物的释放如乳酸脱氢酶,通过乳酸脱氢酶的检测试剂盒(如商家Promega,货号J2380)来检测;ADCC效应通常会选用人外周血PBMC和靶细胞在Claudin18.2抗体类药物存在的情况下共孵育,通过检测靶细胞的内含物的释放如乳酸脱氢酶,通过乳酸脱氢酶的检测试剂盒来检测;采用靶细胞的内含物的释放如乳酸脱氢酶来表征ADCC效应或者CDC效应通常方法的灵敏度比较差,方法的信噪比窗口较小,容易受到死亡的PBMC本身的乳酸脱氢酶释放的干扰等缺点。
发明内容
本发明的目的提供一种Claudin18.2报告基因CHO-K1稳转细胞株构建和应用,解决上述现有技术问题中的一个或者多个。
根据本发明所提供的一种Claudin18.2报告基因CHO-K1稳转细胞株构建,用含Claudin18.2基因的慢病毒转染CHO-K1细胞,通过加入真核抗生素筛选和单克隆挑选,经过表达丰度检测,挑选得到合适的高表达Claudin18.2稳转细胞株单克隆;用含CMV-luciferase基因的慢病毒感染所述Claudin18.2稳转细胞株单克隆,通过加入真核抗生素筛选和单克隆挑选,经过功能学评价得到合适的Claudin18.2报告基因稳转细胞株。
在一些实施方式中,所述Claudin 18.2稳转细胞株单克隆的构建过程如下:Claudin 18.2慢病毒以MOI为20在24孔板中感染1E4个/孔的CHO-K1细胞,总体积为1000μL;第二天进行细胞换液,去除慢病毒;第三天消化细胞转入6孔板中并且加入500μL/mLHygromycin的F12培养基中进行加压培养。维持500μg/ml Hygromycin的F12培养基生长传代加压两代后,第10天将细胞以1个/孔铺在96孔板中;第30天将96孔板中观察到的单克隆转移到24孔板,经评价筛选后,转移到6孔板,T25,T75,T175培养得到单克隆。
在一些实施方式中,所述CMV-Luciferase报告基因稳转细胞株构建过程如下:CMV-Luc2慢病毒MOI为20在24孔板中感染1E5个/孔的CHO-K1
Claudin18.2细胞,总体积为1000μL;第二天进行细胞换液,去除慢病毒;第三天消化细胞转入6孔板中并且加入5μL/mLPuromycin和500μg/mL Hygromycin的F12培养基中进行加压培养。维持加压5μL/mL Puromycin和500μg/mL Hygromycin的F12培养基生长传代两代后,第10天将细胞以1个/孔铺在96孔板中;第28天将96孔板中观察到的单克隆转移到24孔板,经评价筛选后,转移到6孔板,T25,T75,T175培养得到单克隆。
Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定Claudin 18.2受体与抗体结合能力的方法。
Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定抗Claudin 18.2抗体介导的CDC效应的检测方法。
Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定抗Claudin 18.2抗体依赖的ADCC效应的检测方法。
Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定抗Claudin 18.2抗体的中和抗体的检测方法。
技术效果:通过检测报告基因翻译出来的蛋白或者酶的活性,报告基因如荧光素酶基因(luciferase)经常用于表征细胞的活性。在萤火虫素酶检测方法中,萤火虫素酶以ATP,氧气和萤火虫素luciferin为底物,催化化学反应,释放出可以被仪器检测的光,化学发光的强弱能够代表萤火虫素酶多少。萤火虫素酶报告基因检测方法相对于其他荧光类报告基因方法有极低的背景信号,有极高的灵敏程度,检测到的萤火虫素酶的浓度下限能够达到10–11M数量级别,被广泛应用于细胞的活性检测。本发明构建Claudin18.2稳转细胞株,同时额外插入自主表达荧光素酶基因组成了Claudin18.2报告基因稳转细胞株,荧光素酶基因能够在CHO-K1细胞内自主表达非分泌型的荧光素酶,当细胞活性好细胞膜完整时,荧光素酶不会释放,检测细胞上清将得不到信号值,当细胞被杀伤,细胞膜破碎时,荧光素酶将会从细胞内释放,检测细胞上清将得较高的信号值,信号值大小和细胞被杀伤的个数成正相关。因此可以通过检测荧光素酶的量来表征细胞是否被Claudin18.2抗体介导的CDC效应或者ADCC效应杀伤,从而用于Claudin18.2抗体类药物ADCC和CDC活性测定的方法,进一步涉及到Claudin18.2抗体的中和抗体的测定方法。鉴于荧光素酶检测方法的高灵敏性,利用该细胞株进行杀伤类方法的检测有极高的灵敏程度。
由于luciferase来源于萤火虫,真核细胞不表达,只有工程改造的Claudin18.2报告基因CHO-K1稳转细胞株才会表达,luciferase只在Claudin18.2报告基因CHO-K1稳转细胞株胞内表达。只有当Claudin18.2报告基因CHO-K1稳转细胞株被杀伤时,细胞膜破碎后才会有释放到细胞上清中,在ADCC的实验过程中,PBMC细胞和Claudin18.2报告基因CHO-K1稳转细胞株同时存在的情况下,检测细胞上清的luciferase表达就能表征Claudin18.2报告基因CHO-K1稳转细胞的被杀伤情况,相对于乳酸脱氢酶释放的检测方法,此方法不会受到死亡的PBMC的干扰,方法的特异性好,抗干扰能力强。
采用Claudin18.2报告基因稳转细胞株进行靶向Claudin18.2的结合活性检测方法,抗Claudin18.2抗体介导补体依赖的细胞毒性(CDC assay)的检测方法,抗Claudin18.2抗体依赖的细胞介导的细胞毒性作用(ADCC assay)的检测方法具有通用性的特征,该Claudin18.2报告基因稳转细胞株可以对所有的抗Claudin18.2的候选药物进行测试,方法的稳定性好,灵敏程度高。
在临床前和临床研究中评估动物/人血液样品中抗药抗体对候选药物的影响即中和抗体检测是药物安全和有效性的一部分,中和抗体检测方法是将药物发现过程中活性检测方法进行条件的调整转换而来,其检测体系和活性检测方法基本一致,构建Claudin18.2报告基因稳转细胞株利用其化学发光报告基因检测系统进行抗Claudin18.2抗体类药物的中和抗体的检测是较优的选择。
附图说明
图1为表达Claudin 18.2单克隆细胞株转染效率评估分析;
图2为CMV-Luciferase报告基因稳转细胞株的筛选结果;
图3为CL25稳表细胞株用于CDC检测的方法开发的实验结果
图4为CL25稳表细胞株用于ADCC检测的方法开发的实验结果。
具体实施方式
下面结合说明书附图,对本发明进行进一步详细的说明。
实施例1表达Claudin 18.2稳转细胞株构建过程
Claudin 18.2慢病毒(金唯智定制)以MOI为20在24孔板中感染1E4个/孔的CHO-K1细胞(ATCC,货号CCL-61),总体积为1000μL;第二天进行细胞换液,去除慢病毒;第三天消化细胞转入6孔板中并且加入500μL/mL Hygromycin的F12培养基中进行加压培养。维持500μg/ml Hygromycin的F12培养基生长传代加压两代后,第10天将细胞以1个/孔铺在96孔板中;第30天将96孔板中观察到的单克隆转移到24孔板,经评价筛选后,转移到6孔板,T25,T75,T175培养得到单克隆,克隆号CA03。
实施例2表达Claudin 18.2单克隆细胞株转染效率评估
选取最优克隆CA03在添加500μL/mL Hygromycin的F12培养基继续培养,同时培养未转染的CHO-K1细胞。胰酶消化计数后,取1E6细胞至1.5mL离心管,1000rpm离心5min后,弃上清,留约100ul上清重悬细胞,加入5μL Anti-18.2-362Antibody,终浓度5μg/ml,混匀的过程中尽量减少气泡的产生,4℃孵育30min,取出加入适量PBS离心1000rpm 5min,弃上清,重复此操作2次,剩余100μL的样品,加入5μL Goat pAb to Hu IgG(PE),终浓度5μg/mL,4℃孵育30min,取出加入适量PBS离心1000rpm 5min,弃上清,重复此操作2次,400μL PBS重悬细胞,使用流式细胞仪检测。设置使用未转染的CHO-K1作为阴性对照,进行CA03克隆Claudin18.2阳性率分析,结果如图1。
结果表明克隆号CA03细胞相对于未转染的CHO-K1有较高的荧光信号值,说明CA03有高丰度的Claudin18.2的表达,同时说明克隆号CA03细胞可抗Claudin18.2抗体有效结合,可用于抗Claudin18.2抗体的结合活性检测。
实施例3 CMV-Luciferase报告基因稳转细胞株构建过程
CMV-Luc2慢病毒(和元生物定制)MOI为20在24孔板中感染1E5个/孔的CHO-K1Claudin18.2(CA03)细胞,总体积为1000μL;第二天进行细胞换液,去除慢病毒;第三天消化细胞转入6孔板中并且加入5μL/mLPuromycin和500μg/mL Hygromycin的F12培养基中进行加压培养。维持加压5μL/mL Puromycin和500μg/mL Hygromycin的F12培养基生长传代两代后,第10天将细胞以1个/孔铺在96孔板中;第28天将96孔板中观察到的单克隆转移到24孔板,经评价筛选后,转移到6孔板,T25,T75,T175培养得到单克隆,克隆号CL25。
实施例4 CMV-Luciferase报告基因稳转细胞株的筛选
取CMV-Luciferase稳转并经5μL/mL Puromycin和500μg/mL Hygromycin加压培养后的细胞,胰酶消化后计数,60μL(3E4个)/孔加至96孔细胞培养板中,然后添加20μL(10μg/mL)的抗Claudin18.2抗体或者20μL细胞培养液作为阴性对照,再20μL人血清,移液枪混匀后。5%CO2 37℃培养箱孵育5H,500g室温离心5min后,小心吸取20μL上清至白色不底透微孔板,加入100μL Bright-Lumi II Luciferase试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测。
单克隆的检测结果见图2。
由结果显示,不同的克隆有不同的信号值及信噪比,选取最优克隆CL25冻存及功能学实验。
实施例5 CL25稳表细胞株用于CDC检测的方法开发
使用F12培养基+10%灭活FBS的完全培养基培养至对数生长的CL25稳表细胞株,80μL(3E4个)/孔加至96孔细胞培养板中,抗Claudin18.2抗体使用培养基配置到初始工作浓度2μg/mL,然后用培养基连续2倍稀释至3.9ng/mL,10μL/孔添加至培养板中,随后加入10μL个体血清并混匀。5%CO2 37℃培养箱孵育5H,500g室温离心5min后,小心吸取20μL上清至白色不底透微孔板,加入100μL Bright-Lumi II Luciferase试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测。
结果剂量曲线见图3。实验结果表明方法的信噪比有60,有区分度的剂量曲线,其EC50约为91.8ng/mL,可以用于抗Claudin18.2抗体的CDC检测。
实施例6 CL25稳表细胞株用于ADCC检测的方法开发
使用F12培养基+10%灭活FBS的完全培养基培养至对数生长的CL25稳表细胞株,100μL(2E4个)/孔加至96孔细胞培养板中培养过夜,抗Claudin18.2抗体使用培养基配置到初始工作浓度60μg/mL,然后用培养基连续3倍稀释至0.082μg/mL,40μL/孔添加至培养板中,复苏一支冻存的PBMC(上海澳能生物技术有限公司),培养基重悬,4E5/孔加至培养板并混匀。5%CO2 37℃培养箱孵育5H,500g室温离心5min后,小心吸取50μL上清至白色不底透微孔板,加入100μL Bright-Lumi II Luciferase试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测。
结果剂量曲线结果见图4。实验结果表明方法的信噪比可达19倍,不同剂量有区分度明显的信号值,可用于ADCC效应的检测。
以上所述仅是本发明的优选方式,应当指出,对于本领域普通技术人员来说,在不脱离本发明创造构思的前提下,还可以做出若干相似的变形和改进,这些也视为发明保护之内。
Claims (7)
1.Claudin18.2报告基因CHO-K1稳转细胞株构建,其特征在于,用含Claudin18.2基因的慢病毒转染CHO-K1细胞,通过加入真核抗生素筛选和单克隆挑选,经过表达丰度检测,挑选得到合适的高表达Claudin18.2稳转细胞株单克隆;用含CMV-luciferase基因的慢病毒感染所述Claudin18.2稳转细胞株单克隆,通过加入真核抗生素筛选和单克隆挑选,经过功能学评价得到合适的Claudin18.2报告基因稳转细胞株。
2.根据权利要求1所述的Claudin18.2报告基因CHO-K1稳转细胞株构建,其特征在于,所述Claudin 18.2稳转细胞株单克隆的构建过程如下:Claudin 18.2慢病毒以MOI为20在24孔板中感染1E4个/孔的CHO-K1细胞,总体积为1000μL;第二天进行细胞换液,去除慢病毒;第三天消化细胞转入6孔板中并且加入500μL/mL Hygromycin的F12培养基中进行加压培养。维持500μg/ml Hygromycin的F12培养基生长传代加压两代后,第10天将细胞以1个/孔铺在96孔板中;第30天将96孔板中观察到的单克隆转移到24孔板,经评价筛选后,转移到6孔板,T25,T75,T175培养得到单克隆。
3.根据权利要求1所述的Claudin18.2报告基因CHO-K1稳转细胞株构建,其特征在于,所述CMV-Luciferase报告基因稳转细胞株构建过程如下:CMV-Luc2慢病毒MOI为20在24孔板中感染1E5个/孔的CHO-K1 Claudin18.2细胞,总体积为1000μL;第二天进行细胞换液,去除慢病毒;第三天消化细胞转入6孔板中并且加入5μL/mLPuromycin和500μg/mLHygromycin的F12培养基中进行加压培养。维持加压5μL/mL Puromycin和500μg/mLHygromycin的F12培养基生长传代两代后,第10天将细胞以1个/孔铺在96孔板中;第28天将96孔板中观察到的单克隆转移到24孔板,经评价筛选后,转移到6孔板,T25,T75,T175培养得到单克隆。
4.权利要求1-6任一项所述的Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定Claudin 18.2受体与抗体结合能力的方法。
5.权利要求1-6任一项所述的Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定抗Claudin 18.2抗体介导的CDC效应的检测方法。
6.权利要求1-6任一项所述的Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定抗Claudin 18.2抗体依赖的ADCC效应的检测方法。
7.权利要求1-6任一项所述的Claudin18.2报告基因CHO-K1稳转细胞株构建用来测定抗Claudin 18.2抗体的中和抗体的检测方法。
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