CN116478931A - 一种高亲和型抗肿瘤nk细胞及其制备方法和应用 - Google Patents
一种高亲和型抗肿瘤nk细胞及其制备方法和应用 Download PDFInfo
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- Plant Pathology (AREA)
Abstract
本发明属于生物技术领域，具体涉及一种高亲和型抗肿瘤NK细胞及其制备方法和应用；本发明设计haPD1高亲和型嵌合转换受体转染改造的NK92细胞，其中高亲和型嵌合转换受体包括haPD‑1胞外段、CD28跨膜段、DAP10胞内段、CD3ζ胞内段；本发明通过重组慢病毒载体的构建，慢病毒的包装以及慢病毒转染NK92细胞；制备获得haChR3‑NK92细胞。本发明haPD‑1作为CAR结构的胞外识别域，与肿瘤细胞上的PD‑L1特异性结合，以实现更强的肿瘤杀伤作用；共刺激分子CD28作为跨膜区，以将胞外的信息传递到胞内，将NK细胞表面的激活性受体NKG2D的接头蛋白DAP10融入胞内，另外再嵌入CAR设计常用的胞内段CD3ζ，共同促进NK细胞的激活；本发明的制备方法能够成功构建haChR3‑NK92细胞，能够实现目的质粒的表达，并应用在抗肿瘤药物研究中。
Description
技术领域
本发明属于生物技术领域，具体涉及一种高亲和型抗肿瘤NK细胞及其制备方法和应用。
背景技术
肿瘤免疫疗法是继手术、放疗、化疗之后的第四大肿瘤治疗手段，相较于其他形式的肿瘤免疫疗法，过继性细胞疗法具有更多的优势及更好的疗效，在这之中，嵌合抗原受体T细胞疗法（CAR-T）的发展最为迅猛。CAR-T在血液系统恶性肿瘤中效果显著，然而，实体瘤的效果则逊之，并且具有潜在的细胞因子风暴、神经毒性等副作用。此外，由于异源T细胞易引发移植物抗宿主病（graft-versus-host disease，GVHD），所以患者一般只能采用自体T细胞进行CAR修饰后再进行回输，这使临床CAR-T应用步骤繁琐且价格高昂。由于固有免疫系统中的自然杀伤细胞（natural killer cells，NK细胞）是一种天然抗肿瘤细胞，因此，一些研究人员开始尝试利用CAR结构修饰NK细胞（简称CAR-NK）。
相较于CAR-T疗法，NK细胞作为效应细胞具有很多优良特性。首先，不会像T细胞一样产生移植物抗宿主反应现象，有望成为通用型细胞治疗手段；其次，NK细胞不受抗原特异性组织相容性复合体（major histocompatibility complex，MHC）限制，具有更强的杀伤能力；再次，NK细胞表面CD16低亲和力分子可与靶细胞表面IgG抗体复合物介导ADCC作用；NK细胞也可通过Fas/FasL途径介导细胞凋亡，释放细胞因子。综合以上优点，使用NK细胞改造的嵌合抗原受体NK（CAR-NK）细胞将更有应用前景。
现有技术中，中国专利CN201911317011.1公开的一种抗肿瘤NK细胞及其制备方法和应用，其中嵌合抗原受体包括PD-1的胞外结构域片段、F2A肽片段、CD8a信号肽胞外区、识别结合HER2蛋白的单链抗体胞外可变区、CD8αLinker区、CD28跨膜及胞内协同共刺激区、胞内传递信号的CD3ζ片段；这种传统的ｓｃＦｖ因存在于胞外不能正常折叠从而无法正常发挥功能，需要增强亲和性避免此问题发生；另外经过国内外文献证实，采用CD8跨膜域、41BB胞内域和CD28跨膜域、DAP10胞内域的组合均可以增强抗肿瘤效应并提高在体内的存活时间，但需要优选出组合效应更强，且分化为中枢记忆表型的能力更强的重组片段；并且在NK细胞改造的研究过程中，发现NK细胞表面存在众多杀伤抑制受体（KIRs），在肿瘤微环境中可能会被诱导表达，从而抑制免疫细胞的杀伤功能，致使免疫细胞功能衰竭，此种抑制受体被称为免疫抑制检查点，如程序性死亡受体（Programmed Death 1，PD-1）与其配体PD-L1结合可抑制T细胞活化和增殖，下调T细胞免疫刺激性细胞因子的分泌，从而抑制T细胞免疫反应，促使T细胞凋亡；肿瘤细胞可高表达PD-L1，与T细胞表面PD-1结合后，抑制T细胞的杀伤活性，实现免疫逃逸。
发明内容
本发明的目的在于利用合成生物学技术，设计新型靶向PD-L1高亲和型嵌合转换受体，转染改造NK92细胞，并利用haPD-1胞外域靶向识别、结合肿瘤表面的PD-L1配体，通过CD28跨膜域、DAP10胞内域、CD3ζ胞内域将激活性信号传递、活化及释放，增强NK92细胞抗肿瘤免疫活性。
为了实现上述目的，本发明采用以下技术方案：
一种高亲和型抗肿瘤NK细胞，其为haPD-1高亲和型嵌合转换受体转染改造的NK92细胞；所述haPD1高亲和型嵌合转换受体包括haPD-1胞外段、CD28跨膜段、DAP10胞内段、CD3ζ胞内段。
具体的，所述的haPD-1胞外段的核苷酸序列如 SEQ ID No.1所示。
具体的，所述的CD28跨膜段的核苷酸序列如 SEQ ID No.2所示。
具体的，所述的DAP胞内段的核苷酸序列如 SEQ ID No.3所示。
具体的，所述的CD3ζ胞内段的核苷酸序列如 SEQ ID No.4所示。
一种高亲和型抗肿瘤NK细胞的制备方法，包括如下步骤：
步骤一：重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH的构建；
通过全基因合成方法将haPD-1胞外段、CD28跨膜段、DAP胞内段、CD3ζ胞内段串联起来，获得haPD1-CD28-DAP10-CD3ζ片段；将haPD1-CD28-DAP10-CD3ζ片段亚克隆在pCDH-CMV-MCS-P2A-copGFP-T2A-Puro慢病毒载体上，获得重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH；
步骤二：慢病毒包装过程；
将步骤一获得的重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH提取出目的质粒haChR3；再将目的质粒haChR3、慢病毒包装辅助质粒psPAX2、慢病毒包装辅助质粒PMD2.G按照4:3:1的比例混合转染进293T细胞，以产出带有目的质粒的病毒；将转染后收获的病毒上清采用PEG浓缩法进行浓缩；
步骤三：慢病毒转染NK92细胞；
取浓缩好的带有目的质粒的病毒悬液侵染NK92细胞，可获得稳定表达haChR3-pCDH的NK92细胞株。
具体的，所述的重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH的核苷酸序列如SEQ ID No.8所示。
具体的，所述的haPD1-CD28-DAP10-CD3ζ片段的核苷酸序列如SEQ ID No.6；其氨基酸序列如 SEQ ID No.5。
通过上述制备方法得到的一种高亲和型抗肿瘤NK细胞，应用于抗肿瘤细胞药物研究；尤其是，其能够对SGC-7901胃癌细胞进行体外杀伤。
本发明的有益效果在于：
1、本发明获得的高亲和型抗肿瘤NK细胞，即haChR3-NK92细胞，利用haPD1高亲和型嵌合转换受体中，haPD-1胞外结构域片段（24-170aa）识别抗原，CD28跨膜段（153-179aa）与DAP10胞内段（70-93aa）协同共刺激区，CD3ζ（52-164aa）传递胞内信号，增强NK92细胞抗肿瘤免疫活性；其中haPD-1作为野生型PD-1的突变体，与PD-L1结合时的亲和性比野生型PD-1更高，有利于更好的识别靶向肿瘤细胞，尤其相较于传统的ｓｃＦｖ，本发明使用高亲和性haPD-1亲和性更强，传统的ｓｃＦｖ会因存在于胞外不能正常折叠从而无法正常发挥功能，而使用高亲和性haPD-1可以避免此问题发生；并且CD28跨膜段和DAP10胞内段联合使用，有利于提高细胞因子的分泌从而提升CAR-NK的抗肿瘤活性并延长在体内的存在时间；CD3ζ向胞内传递发挥抗癌作用的信号，赋予CAR-NK细胞CAR依赖性的杀伤能力。
2、本发明的制备方法能够成功构建高亲和型抗肿瘤NK细胞，即haChR3-NK92细胞，并且haChR3-NK92细胞在mRNA水平检测haPD-1，其mRNA水平表达正常； haChR3-NK92细胞在蛋白水平检测haPD-1，其蛋白水平表达正常； haChR3-NK92细胞在体外进行毒性试验，能够对癌症细胞进行体外杀伤。本制备方法工艺流程短，操作简单，容易实现，表达稳定性高，易于实现产业化。
3、本发明中的高亲和型抗肿瘤NK细胞，即haChR3-NK92细胞，应用于制备抗肿瘤药物，可靶向肿瘤细胞，还可增强细胞毒作用，免疫原性低，易响应激活，可异体回输，haChR3-NK92细胞肿瘤杀伤得到了显著提高，所得抗肿瘤药物药效好。
附图说明
图1为haPD1-CD28-DAP10-CD3ζ片段亚克隆在pCDH-CMV-MCS-P2A-copGFP-T2A-Puro慢病毒载体上获得重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH的示意图；
图2为慢病毒转染NK92细胞72h的荧光图（4X）；
图3为慢病毒转染NK92细胞嘌呤霉素富集14天后的荧光图（4X）；
图4为流式细胞术检测NK92、haChR3-NK92细胞的Flag和GFP表达情况；
图5为流式细胞术检测NK92、haChR3-NK92细胞的haPD-1和GFP表达情况；
图6为 NK92、haChR3-NK92细胞增殖水平示意图；
图7为 NK92、haChR3-NK92细胞对SCG-7901肿瘤细胞的杀伤效果图。
具体实施方式
本发明利用PD-L1在肿瘤细胞高表达这一特点，找到一种高亲和型PD-1（highaffinity PD-1，简称haPD-1）作为CAR结构的胞外识别域，可以将胞内抑制域替换为激活域向内传递正向激活信号；haPD-1与肿瘤细胞上的PD-L1特异性结合，以实现更强的肿瘤杀伤作用。
除抗原结合区外，通过基因修饰手段人为构建一条NK细胞活化通路还需融合跨膜区和胞内信号区；本发明采用共刺激分子CD28作为跨膜区，以将胞外的信息传递到胞内；而胞内信号区结构至关重要，研究表明利用NK自身相关激活元件设计的CAR预测能更好地在体内激活NK细胞，所以本发明将NK细胞表面的激活性受体NKG2D的接头蛋白DAP10融入胞内，另外再嵌入CAR设计常用的胞内段CD3ζ，共同促进NK细胞的激活。
本发明为了研究抗肿瘤细胞药物，通过如下步骤制备得到高亲和型抗肿瘤NK细胞，即haChR3-NK92细胞，具体步骤包括：
步骤一：重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH的构建
通过全基因合成的方法在南京金斯瑞生物科技有限公司合成含有haPD-1胞外段（24-170aa）435bp、CD28跨膜段（153-179aa）81bp、DAP胞内段（70-93aa）72bp、CD3ζ胞内段（52-164aa）的339bp串联起来，获得haPD1-CD28-DAP10-CD3ζ片段（简称haChR3）。将haPD1-CD28-DAP10-CD3ζ片段（简称haChR3）亚克隆在pCDH-CMV-MCS-P2A-copGFP-T2A-Puro慢病毒载体上，如图1所示，获得重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH（简称为重组慢病毒载体haChR3-pCDH），haPD1-CD28-DAP10-CD3ζ-pCDH的核苷酸序列如SEQ ID No.7所示。
上述内容中，haPD-1胞外段的核苷酸序列如 SEQ ID No.1所示，CD28跨膜段的核苷酸序列如 SEQ ID No.2所示，DAP胞内段的核苷酸序列如 SEQ ID No.3所示，CD3ζ胞内段的核苷酸序列如 SEQ ID No.4所示；haPD1-CD28-DAP10-CD3ζ片段（简称haChR3）的氨基酸序列如 SEQ ID No.5所示，核苷酸序列如SEQ ID No.6所示；pCDH-CMV-MCS-P2A-copGFP-T2A-Puro的核苷酸序列如SEQ ID No.7所示；haPD1-CD28-DAP10-CD3ζ-pCDH的核苷酸序列如SEQ ID No.8所示。
步骤二：慢病毒包装过程
（一）提取haChR3目的质粒
1、质粒转化
取出大肠杆菌TOP10感受态细胞，向TOP10感受态中分别加入1μg重组慢病毒载体haChR3-pCDH和慢病毒包装辅助质粒psPAX2（购买来源Addgene#12260）、PMD2.G（购买来源Addgene#12259），混合均匀，冰中孵育30min，42℃水浴锅热激90秒，再冰中孵育2min。加入800μL无抗LB培养基，37℃恒温培养箱中，180rpm/min，振荡培养1h，取100μL菌液涂到LB固体培养基上（含有氨苄抗生素Ampicillin，Amp，浓度为50μg/ml,按照1:1000的比例加入），37℃恒温培养箱倒置培养12h。
2、质粒提取
（1）分别挑取上述LB固体培养基中的单克隆菌落，接种到含Amp（浓度为50 μg/mL，接种比例为1:1000）的5mL新鲜无菌的LB液体培养基中，放入摇床37℃，220rpm/min，振荡培养8h；
（2）取上述菌液200 μL接种至含Amp的200 mL新鲜无菌LB培养基中，放至摇床，37℃，220rpm/min，振荡培养12h，直到OD600nm值达到1.5~2.0；
（3）按照OMEGA无内毒素质粒大量提取试剂盒（购买来源美国OmegaBio-Tek公司，产品货号为D6926-03）进行质粒抽提，用Nanodrop超微量分光光度计(购买来源，美国热电(上海)科技仪器有限公司，型号：NanoDrop2000)测取质粒浓度和纯度后，置于-20℃冰箱保存。
（二）慢病毒包装
先培养293T细胞（购买自美国ATCC公司），观察细胞密度到70%~80%时，更换15mL6%新鲜的含有6%FBS（购买自BI，货号为04-001-1ACS）的不含青霉素与链霉素的DMEM培养基（购买自Hyclone,货号为SH30022.01），细胞培养箱培养1h。采用磷酸钙试剂盒（购买自碧云天，货号为C0508）进行慢病毒包装，操作步骤为：将目的质粒haChR3，辅助质粒pSPAX2、PMD2.G按照4:3:1的比例混合加入（试剂盒提供的）CaCl2溶液中，吹打混匀，再将混合溶液逐滴加入BBS溶液中，吹打混匀，避免生产白色絮状物，室温孵育20min，将上述混合液加入293T细胞中，轻晃混匀，放入培养箱继续培养12h，吸弃上清，更换成15mL新鲜预热的含有10% FBS和1%双抗的DMEM培养基，并于48h、72h、96h收集病毒上清液。
（三）慢病毒浓缩
所得病毒上清3500g，4℃，离心10min，用0.45μm无菌滤膜过滤，去除细胞碎片。取过滤后的病毒上清，加入新鲜无菌PEG，4℃放置≥24h，期间上下颠倒1~2次。3000g、4℃离心30min，弃上清，用适量PBS重悬沉淀，分装保存于-20℃或-80℃，避免反复冻融。
步骤三：慢病毒转染NK92细胞
取3.5×105个/孔NK92细胞接种于24孔板，按体积梯度加入浓缩过的含目的质粒的病毒液，每孔加入终浓度为8μg/mL的聚凝胺（翊圣生物，货号40804ES76），混匀，置于37℃培养箱中进行培养，12h后弃尽病毒上清，更换新鲜培养基，放入37℃细胞培养箱继续培养。转染72h后，在荧光显微镜下观察荧光的表达以检测转染效果，如图2所示，图2放大倍数为4×10，由图2可以看出，细胞成团生长，状态良好，可观察到少量细胞荧光。通过嘌呤霉素筛选，14d后在荧光显微镜下观察结果，如图3所示，图3放大倍数为4×10，由图3可以看出，细胞成团，状态良好，细胞荧光表达率高。因此，在成功构建了haChR3-NK92细胞之后，荧光显微镜检测稳转NK92细胞的荧光表达。5d后，转染过的NK92细胞用于后续试验。
试验一
采用流式细胞术检测NK92细胞、haChR3-NK92细胞的haPD-1、Flag、GFP表达，具体方式如下：
取嘌呤霉素富集14天后的haChR3-NK92细胞和NK92细胞各3×106个，300g，5min离心，弃上清，用含1%FBS的PBS（以下简称wash buffer）洗2次，在100μL体系下加入APC标记的小鼠抗Flag单抗（Biolegend，货号637307）和APC标记的小鼠抗人PD1单抗（Invitrogen公司，货号17-9969-42），冰上避光孵育30min，用wash buffer洗涤2次。加入400μL washbuffer重悬细胞，用流式细胞仪（型号BD FACSCantoII）进行检测分析，结果如图4、图5所示。
由图4可看出第三象限Flag、GFP均为阴性细胞，由于含有目的基因，第一象限可见haChR3-NK92中Flag表达率为99.2%。由图5可看出第三象限PD-1、GFP均为阴性细胞；由于含有目的基因，第一象限可见haChR3-NK92中PD-1表达率为98.9%。
试验二
利用CCK8检测haChR3-NK92细胞和NK92细胞的增殖情况，具体方式如下：
按照每孔1×104个分别取细胞haChR3-NK92和NK92细胞，300 g离心5 min后按照每孔100μL培养基重悬细胞沉淀，每种细胞设置4个副空，均匀铺到三个孔板。分别于24 h、48 h、72 h后取出一个孔板，每孔加10μL的CCK8检测液，同时其它未检测孔板加100μL培养基，检测液也随之递增。之后放置到37℃培养箱孵育2-3 h，用酶标仪检测各组OD值变化。通过CCK8增殖实验检测haChR3-NK92细胞的增殖速率与NK92细胞是否有差异，试验设置三个检测时间点。
结果如图6所示，24 h、48 h、72 h 三个检测时间点处haChR3-NK92和NK92细胞增殖速率无显著性差异，表示对NK92细胞的修饰改造仍然保持NK92细胞原有的增殖能力。
试验三
利用构建的haChR3-NK92细胞进行体外细胞毒性实验，具体方式如下：
1.细胞准备
取转入荧光素酶完成后的对数生长期的SGC-7901胃癌细胞，接种于96孔板中，保证每孔1×104个细胞，每种细胞设置3个副孔，置于5% CO2，37℃培养箱中过夜培养。
2.实验设计
实验设置4组，分别是靶细胞最大裂解组、靶细胞空白组、效应细胞（NK92细胞、haChR3-NK92细胞）杀伤实验组，每组设置三个副孔，每孔总体积100μL，效靶比为5:1。
3.荧光素酶检测
本实验采用ONE-GloTM Luciferase Assay System Promega进行检测。效应细胞与靶细胞置于5%CO237℃培养箱中共孵育12h。在预定的时间检测点前1h，从细胞培养箱中取出96孔板，在样品最大酶活性对照孔中加入Lysis Solution（10×），加入量为原有培养液体积的10%。加入Lysis Solution后，反复吹打混匀后继续放入培养箱中培养。达到共孵育时间后，加入检测液，在酶标仪上进行检测。
细胞毒性（%）=(RLU min– RLU sample)/( RLU min– RLU max)×100.结果如图7所示，在5:1的效靶比下，对于SGC-7901细胞，亲本NK92细胞杀伤约18%，haChR3-NK92细胞杀伤约为89%，杀伤力增加约4倍，说明改造后的haChR3-NK92细胞细胞毒性显著增加。
Claims (10)
1.一种高亲和型抗肿瘤NK细胞，其特征在于，其为haPD-1高亲和型嵌合转换受体转染改造的NK92细胞；所述haPD-1高亲和型嵌合转换受体包括haPD-1胞外段、CD28跨膜段、DAP10胞内段、CD3ζ胞内段。
2.根据权利要求1所述的一种高亲和型抗肿瘤NK细胞，其特征在于，所述的haPD-1胞外段的核苷酸序列如 SEQ ID No.1所示。
3.根据权利要求1所述的一种高亲和型抗肿瘤NK细胞，其特征在于，所述的CD28跨膜段的核苷酸序列如 SEQ ID No.2所示。
4.根据权利要求1所述的一种高亲和型抗肿瘤NK细胞，其特征在于，所述的DAP胞内段的核苷酸序列如 SEQ ID No.3所示。
5.根据权利要求1所述的一种高亲和型抗肿瘤NK细胞，其特征在于，所述的CD3ζ胞内段的核苷酸序列如 SEQ ID No.4所示。
6.一种高亲和型抗肿瘤NK细胞的制备方法，其特征在于，包括如下步骤：
步骤一：重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH的构建；
通过全基因合成方法将haPD-1胞外段、CD28跨膜段、DAP胞内段、CD3ζ胞内段串联起来，获得haPD1-CD28-DAP10-CD3ζ片段；将haPD1-CD28-DAP10-CD3ζ片段亚克隆在pCDH-CMV-MCS-P2A-copGFP-T2A-Puro慢病毒载体上，获得重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH；
步骤二：慢病毒包装过程；
将步骤一获得的重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH提取出目的质粒haChR3；再将目的质粒haChR3、慢病毒包装辅助质粒psPAX2、慢病毒包装辅助质粒PMD2.G按照4:3:1的比例混合转染进293T细胞，以产出带有目的质粒的病毒；将转染后收获的病毒上清采用PEG浓缩法进行浓缩；
步骤三：慢病毒转染NK92细胞；
取浓缩好的带有目的质粒的病毒悬液侵染NK92细胞，可获得稳定表达haChR3-pCDH的NK92细胞株。
7.根据权利要求6所述的一种高亲和型抗肿瘤NK细胞的制备方法，其特征在于，所述的重组慢病毒载体haPD1-CD28-DAP10-CD3ζ-pCDH的核苷酸序列如SEQ ID No.8所示。
8.根据权利要求6所述的一种高亲和型抗肿瘤NK细胞的制备方法，其特征在于，所述的haPD1-CD28-DAP10-CD3ζ片段的核苷酸序列如SEQ ID No.6；其氨基酸序列如 SEQ IDNo.5。
9.权利要求1~7中任一项所述的一种高亲和型抗肿瘤NK细胞，或权利要求6~8中任意一项所制备的一种高亲和型抗肿瘤NK细胞，其特征在于，应用于抗肿瘤细胞药物研究。
10.根据权利要求8所述的一种高亲和型抗肿瘤NK细胞，其特征在于，其应用于SGC-7901胃癌细胞的体外杀伤。
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