CN110028589A - 嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用 - Google Patents
嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种嵌合抗原受体、表达该嵌合抗原受体的NKG2D CAR‑NK细胞及其制备方法和应用，所述嵌合抗原受体，包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活NK细胞。本发明提供的CAR‑NK细胞，其以NKG2D配体为靶抗原，利用NKG2D CAR‑NK细胞特异性地杀伤肿瘤细胞。其可作为肿瘤类疾病的治疗药物，用于NKG2D分子的配体高表达的肿瘤的治疗，为肿瘤的预防和治疗提供了新的方法。
Description
技术领域
本发明涉及生物医药领域，具体涉及一种嵌合抗原受体、表达该嵌合抗原受体的NKG2D CAR-NK细胞及其制备方法和应用。
背景技术
嵌合抗原受体(chimeric antigen receptor，缩写CAR)修饰的免疫细胞使用遗传工程手段修饰免疫细胞使其表达外源性抗肿瘤基因。CAR基因主要包括细胞外识别域和细胞内信号转导结构域：前者用于识别肿瘤表面特异性分子和后者用于启动识别肿瘤表面分子后的免疫细胞应答，发挥细胞毒作用。其主要以T-细胞为载体，但CAR-T细胞***时会出现IL-6等细胞因子急剧升高，产生细胞因子风暴现象，存在靶向/脱靶毒性和神经毒性等问题，严重时会危及患者生命。并且，T细胞必须分离出体外(这一过程耗时长且费用高)。而且，由于T细胞是针对特定患者进行修改，但是一些患者可能无法采集T细胞，或者没有足够的时间等待T细胞的准备过程，虽然目前CAR-T在向通用型的CAR-T发展，但其实又增加了临床风险以及操作难度。此外，面对CAR-T的高额费用，这些局限性可能会导致一些有望受益的患者无法接受CAR-T免疫疗法。
自然杀伤(natural killer，缩写NK)细胞是非特异性免疫***的重要组成部分，先天免疫***反应的关键性介质细胞。NK细胞是一种广谱免疫细胞，具有快速发现和摧毁异常细胞(如癌症或病毒感染的细胞)的特异功能，而且不需要提前致敏或HLA配型，即可展示强大的溶解异常细胞的活性。使用免疫细胞(包括NK细胞)来治疗癌症是近年来新趋势，这种新疗法有望为对传统手术、化疗和放疗无效的肿瘤提供了新的治愈希望。
NKG2D是NK细胞的活化型受体，可识别MHC-I类分子，在天然免疫中发挥着重要的作用。NKG2D参与病毒感染细胞的识别及NK对肿瘤细胞的杀伤。NKG2D属于C型凝集素样受体家族，由于这个基因编码的受体存在于NKG2(naturalkillergroup2)复合体中。NKG2基因复合***于人的12号染色体上。NKG2D是II型跨膜蛋白，NKG2D需要通过跨膜区域的带电残基结合一些转接蛋白(adaptor proteins)完成信号转导。人NKG2D都能结合 10kDa的DNAX活化蛋白(DAP10)。DAP10在胞浆内含有YXXM基序 (Tyr-X-X-Meth)能够招募磷脂酰肌醇三羟基激酶(PI3K)和生长因子受体结合蛋白-2。所有的NK细胞、大多数NKT细胞、巨噬细胞都表达NKG2D。另外，NKG2D也存在于CD8+T细胞表面。在正常条件下，人和小鼠CD4+T 细胞不表达NKG2D。但是，在患者体内，表达NKG2D的CD4+T细胞主要聚集在肿瘤组织中。NKG2D可与许多不同的配体结合，这些配体属于主要组织相容性复合体I类(MHC-I)相关蛋白。人的NKG2D配体的另一家族是 MIC-A和MIC-B。MIC-A和MIC-B都具有多态性。目前，MIC-A有61个等位基因，MIC-B有30个等位基因。NKG2D分子的配体在正常细胞中不表达或者表达量非常少，但是当细胞受到感染或者发生癌变时，这些配体的表达量会大幅度上升。
目前用于治疗表达NKG2D肿瘤及相关疾病的CAR-NK细胞尚不存在，只有用于治疗表达NKG2D肿瘤及相关疾病的CAR-T细胞，但是其毒副作用大，成本高，如果能够开发研究一种NKG2D CAR-NK细胞，势必会推动肿瘤治疗领域的进展。
发明内容
有鉴于此，本发明提供了一种嵌合抗原受体、表达该嵌合抗原受体的NKG2D CAR-NK细胞及其制备方法和应用，该细胞能够特异性识别和杀伤肿瘤，具有更高效的肿瘤杀伤活性。
本发明的一方面提供一种嵌合抗原受体，所述嵌合抗原受体包含抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活NK细胞。
示例性地，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述 NKG2D配体特异性结合。
示例性地，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I) 分子、MIC-A和MIC-B中的一种或多种。
示例性地，所述的跨膜结构域选自CD28、CD3ε、CD45、CD4、CD5、 CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154 中的一种或多种；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、 CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB 和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和 CD3ζ胞内结构域。
示例性地，所述嵌合抗原受体的结构为NKG2D-CD8α hinge-CD8TM-4-1BB-CD3ζ的融合蛋白，该融合蛋白能够特异性识别NKG2D 配体。
在本发明的一个具体实施方式中，所述NKG2D-CD8α hinge-CD8TM-4-1BB-CD3ζ融合蛋白氨基酸序列如SEQ ID NO：1所示或其同源序列等。
示例性地，所述同源序列与原序列的同源性约95％或以上、96％或以上、 97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
本发明的另一方面提供一种表达上述嵌合抗原受体的核苷酸序列。
示例性地，所述核苷酸序列如SEQ ID NO：2所示或其简并序列等。
示例性地，所述简并序列与原序列的同源性约95％或以上、96％或以上、 97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
本发明还提供一种NKG2D CAR-NK细胞，所述NKG2D CAR-NK细胞能够表达嵌合抗原受体，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。
示例性地，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述 NKG2D配体特异性结合。
示例性地，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I) 分子、MIC-A和MIC-B。
示例性地，所述的跨膜结构域选自CD28、CD3ε、CD45、CD4、CD5、 CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154 中的一种或多种；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、 CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB 和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和 CD3ζ胞内结构域。
示例性地，所述嵌合抗原受体的结构为 NKG2D-CD8αhinge-CD8TM-4-1BB-CD3ζ的融合蛋白。
示例性地，所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：1所示或其同源序列。
示例性地，所述同源序列与原序列的同源性约95％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
在本发明的一个优选实施方式中，所述 NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的编码核苷酸序列如SEQ ID NO：2所示或其同源序列。
示例性地，所述同源序列与原序列的同源性约95％或以上、97％或以上、 98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
在本发明的一个具体实施方式中，所述NKG2D CAR-NK细胞能够有效地杀伤和/或杀死肺癌细胞、乳腺癌细胞、结肠癌细胞、乳腺癌细胞和/或肺癌细胞等。
本发明还提供了上述NKG2D CAR-NK细胞的制备方法，其包括如下步骤：
(1)合成和扩增编码上述嵌合抗原受体的核苷酸序列，再将所述核苷酸序列克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染 293T细胞，包装和制备慢病毒；和
(3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到NKG2D CAR-NK 细胞。
示例性地，在步骤(1)中，合成和扩增编码所述 NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的核苷酸序列。
本发明还提供上述NKG2D CAR-NK细胞在制备治疗和/或预防癌症的药物中的应用。
示例性地，所述癌症为高表达NKG2D配体的肿瘤及相关疾病。
示例性地，所述癌症为乳腺癌、结肠癌或肺癌等。
本发明还提供了一种药物组合物，其包括有效治疗量的上述NKG2D CAR-NK细胞，以及任选地，药学上可接受的辅料。
示例性地，所述药物组合物用于治疗或预防肿瘤时，NKG2D CAR-NK 细胞与肿瘤细胞的效靶比为(0.5～1)：1。
示例性地，所述药物组合物的剂型为水剂。
本发明还提供一种上述NKG2D CAR-NK细胞用于治疗和/或预防癌症的方法。
示例性地，所述方法包括将有效治疗量的含有NKG2D CAR-NK细胞的药物组合物摄入患者体内。
示例性地，所述的癌症为高表达NKG2D配体的肿瘤及相关疾病。
示例性地，所述癌症为肺癌、乳腺癌或结肠癌。
示例性地，所述NKG2D CAR-NK细胞的给药量为(1～10)×106个/ 次，优选为(2.5～5)×106个/次。
示例性地，所述NKG2D CAR-NK细胞的摄入方法为瘤内注射、静脉注射、胸腔内注射或局部介入。
示例性地，所述NKG2D CAR-NK细胞的摄入方法为静脉注射。
本发明的嵌合抗原受体能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。该NKG2D CAR-NK细胞是通过将NKG2D受体用于CAR-NK细胞所构建。嵌合抗原受体和能够表达该嵌合抗原受体的NKG2D CAR-NK细胞以NKG2D配体为靶抗原，能够特异性地杀伤肿瘤细胞，其可作为肿瘤类疾病的治疗药物，用于NKG2D配体高表达的肿瘤的治疗，为肿瘤的预防和治疗提供了新的方法；并且通过对NK92细胞进行改造，装上CAR后，又大大增加了它的靶向性，并增加抗肿瘤靶点NKG2D，在提高靶向性地同时，大大地提高其抗肿瘤性能；也就是说本发明首次构建了NKG2D CAR-NK细胞，其通过对NK92细胞进行改造，装上CAR后不但增加了其靶向性，而且由于使用的是NK细胞，大大提高了其安全性，毒副作用小，成本低，通过CAR和NKG2D的配合大大提高了该细胞的抗肿瘤性能，并通过实验表明其抗肿瘤性能明显优于NK92细胞；此外，进一步扩展了使用CAR-NK细胞治疗和预防肿瘤的广谱性。
附图说明
图1所示为本发明实施例提供的慢病毒质粒载体PRRLSIN-NKG2D的结构示意图。
图2a-2b所示为本发明实施例提供的NKG2D CAR-NK流式检测CAR 细胞阳性率的结果图，其中，图2a为对照组；图2b为实验组。
图3a-3e所示为本发明实施例提供的利用常规流式方法检测不同肿瘤细胞中NKG2D配体MIC-A和MIC-B分子的表达的结果图。
其中，图3a为肺癌细胞A549的实验结果；图3b为肺癌细胞H1299的实验结果；图3c为乳腺癌细胞MCF-7的实验结果；图3d为乳腺癌细胞 MDMB-231的实验结果；图3e为结肠癌细胞SW480的实验结果。
图4a-4e所示为本发明实施例提供的NKG2D CAR-NK细胞杀伤不同肿瘤细胞的实验结果图。
其中，图4a为肺癌细胞A549的实验结果；图4b为肺癌细胞H1299的实验结果；图4c为乳腺癌细胞MCF-7的实验结果；图4d为乳腺癌细胞 MDMB-231的实验结果；图4e为结肠癌细胞SW480的实验结果。
图5a-b所示为本发明实施例提供的NKG2D CAR-NK细胞的用于治疗 NPG小鼠后的肿瘤生长曲线；其中，图5a为给药剂量为2.5×106个的小鼠肿瘤的生长曲线，图5b为给药剂量为5×106个的小鼠肿瘤的生长曲线。
图6所示为本发明实施例提供的NKG2D CAR-NK细胞用于治疗NPG 小鼠后的抑瘤结果。
图7所示为本发明实施例提供的NKG2D CAR-NK细胞用于治疗NPG 小鼠后的肿瘤重量的结果。
图8所示为本发明实施例提供的NKG2D CAR-NK细胞用于治疗NPG 小鼠后的肿瘤重量的结果。
上述图5a-图8中，PBS为PBS给药组，NK92-1为剂量为2.5×106的给药组，NK92-1为剂量为5×106的给药组，NKG2D CAR-NK-1为2.5×106的给药组，NKG2D CAR-NK-2为5×106的给药组，*表示P(显著性差异)﹤0.05。
具体实施方式
除非另有定义，本发明中使用的所有技术和科学术语具有与本发明所述技术领域的普通技术人员通常理解的相同含义。
具体而言，本发明所述的编码NKG2D-CD8αhinge-CD8TM-4-1BB-CD3ζ融合蛋白的核苷酸的序列是任何能够编码该融合蛋白的任何DNA序列，优选地，该序列为SEQ ID NO：2或其互补序列。另一方面，本发明所述的编码 NKG2D-CD8αhinge-CD8TM-4-1BB-CD3ζ融合蛋白的核苷酸的序列可为在严谨条件下与由SEQ ID NO：2的核苷酸序列进行杂交、且编码该融合蛋白的多核苷酸或其互补序列。
本文所述的“严谨条件”，可以为低严谨条件、中严谨条件、高严谨条件中的任一种，优选为高严谨条件。示例性地，“低严谨条件”可为30℃、5×SSC、 5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“中严谨条件”可为40℃、5×SSC、 5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“高严谨条件”可为50℃、5×SSC、 5×Denhardt液、0.5％SDS、52％甲酰胺的条件。本领域技术人员应当理解温度越高越能得到高同源性的多核苷酸。另外，本领域技术人员可以选择影响杂交的严谨度的温度、探针浓度、探针长度、离子强度、时间、盐浓度等多个因素形成的综合结果来实现相应的严谨度。
除此之外可杂交的多核苷酸还可以为，通过FASTA、BLAST等同源性检索软件用***设定的默认参数进行计算时，与编码序列号6的多核苷酸具有约 60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上同一性的多核苷酸。
核苷酸序列的同一性，可以使用Karlin及Altschul的算法规则BLAST(Proc.Natl.Acad.Sci.USA 87：2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873,1993)来确定。基于BLAST算法规则的程序BLASTN、BLASTX已被开发(Altschul SF,et al:JMol Biol 215:403,1990)。使用BLASTN分析碱基序列时，如使参数为score＝100、wordlength＝12；此外使用BLASTX分析氨基酸序列时，如使参数为score＝50、wordlength＝3；使用BLAST和Gapped BLAST程序时，采用各程序的***可设定默认参数值。
除非另有规定，“编码核苷酸”包括为彼此简并版本并编码相同的氨基酸序列的所有核苷酸序列。编码蛋白质的核苷酸序列可包括内含子。
术语“慢病毒”指的是逆转录病毒科的属，其能够有效感染非周期性和有丝***后的细胞；它们可传递显著量的遗传信息进入宿主细胞的DNA，以便它们是基因传递载体的最有效的方法之一。
术语“启动子”被定义为开始多核苷酸序列的特异性转录需要的，由细胞的合成机器识别或引导合成机器的DNA序列。
术语“特异性结合”指识别特异性抗原但基本上不识别或结合样本中的其他分子。
术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。很多载体在本领域中是已知的，包括但不限于线性多核苷酸、与离子或两性分子化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语也应被解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，诸如例如聚赖氨酸化合物、脂质体等等。病毒载体的例子包括但不限于，腺病毒载体、腺伴随病毒载体、逆转录病毒载体等等。
术语“癌症”被定义为以畸变细胞的快速和失控生长为特征的疾病。癌症细胞可局部蔓延或通过血流和淋巴***蔓延至身体的其他部分。各种癌症的例子包括但不限于乳腺癌结肠直肠癌、肝癌、肺癌等等。
如本文所使用的，“包含”与“包括”、“含有”或“特征在于”同义，并且是包括在内的或开放性的，并且不排除另外的未陈述的元件或方法步骤。术语“包含”在本文中的任何表述，特别是在描述本发明的方法、用途或产品时，应理解为包括基本上由所述组分或元件或步骤组成和由所述组分或元件或步骤组成的那些产品、方法和用途。本文示例性描述的本发明适当地可以在不存在本文未具体公开的任何一种或多种元件、一种或多种限制的情况下进行实践。
本文已采用的术语和表述用作描述性而不是限制性术语，并且在此种术语和表述的使用中不预期排除所示和所述特征或其部分的任何等价物，但应认识到各种修饰在请求保护的本发明的范围内是可能的。因此，应当理解尽管本发明已通过优选实施方案和任选特征具体公开，但本领域技术人员可以采用本文公开的概念的修饰和变化，并且此类修饰和变化被视为在如由附加权利要求定义的本发明的范围内。
本文中出现的英文名称不区分大小写；NKG2D CAR-NK、NKG2D-CAR NK 表示的含义相同；NKG2D CAR-NK与NKG2D-CAR NK表示相同的含义，均表示抗NKG2D分子的CAR-NK细胞；NK-92、NK92均表示NK92细胞；CD8TM表示跨膜结构域。
本发明所述的“NK”为人体正常NK细胞或NKT细胞或NK细胞系，其包括NK-92细胞，YT细胞，NKL细胞，HANK-1细胞，NK-YS细胞，KHYG-1 细胞，SNK-6细胞和IMC-1细胞等。本发明的具体实施例中以NK-92细胞为例进行说明。
为更清楚地说明本发明，现结合如下实施例进行详细说明，但这些实施例仅仅是对本发明的示例性描述，并不能解释为对本申请的限制。
下述实施例中的材料来源：
NK-92细胞( CRL-2407)，肺癌细胞A549、乳腺癌MDMB-231 细胞、结肠癌细胞SW480、乳腺癌MCF-7细胞、HCC1187细胞和肺癌细胞 H1299均购自中国科学院上海生科院细胞资源中心。
NPG小鼠购自北京维通利华实验动物技术有限公司，SPF级，雌性，5-6 周，体重：18-20g，±20％。
实施例1慢病毒载体的制备
基因合成NKG2D-CD8TM-4-1BB-CD3ζ融合基因序列(其氨基酸序列如 SEQ ID NO：1所示，基因序列如SEQ ID NO：2所示)，通过酶切，将 NKG2D-CD8TM-4-1BB-CD3ζ融合基因序列转化连接到PRRSLIN载体中，基因上游为EP-1α启动子。载体转化Stbl3大肠杆菌菌株，氨苄青霉素筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，获得PRRLSIN-NKG2D慢病毒转染载体(如图1所示)。
实施例2慢病毒的制备
(1)转染前24小时，以每皿约8×106个将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度，且转染的细胞均匀分布于培养皿中。
(2)准备溶液A和溶液B
溶液A：6.25ml 2×HEPES buffer缓冲液(5个大皿一起包装的量，效果最好)。
溶液B：为分别加入以下质粒得到的混合物：112.5μg PRRLSIN-NKG2D (targetplasmid)；39.5μg pMD2.G(VSV-G envelop)；73μg pCMVR8.74(gag， pol，tat，rev)；625μl2M钙离子溶液。溶液A总体积：6.25ml。
充分混匀溶液B，在轻轻涡旋溶液A的同时，逐滴加入溶液B，得到A 和B的混合溶液，静置5-15分钟。再轻轻涡旋上述A和B的混合溶液，并将其逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。然后，(不要旋转培养皿)放置于培养箱中培养 16-18小时。更换新鲜培养基，继续培养，分别在48小时和72小时后收集含病毒的上清液。再将收集的上清液，于500g，25℃离心10分钟，PES膜 (0.45μm)过滤，得到过滤后的上清液。然后以70％乙醇消毒贝克曼库尔特 Ultra-clear SW28centrifuge tubes，并置于紫外灯下消毒30分钟，得到消毒后的离心管。再将已过滤的含慢病毒的上清液转移至消毒后的离心管中，并且在离心管底部小心铺上一层20％蔗糖(每8ml上清液加1ml蔗糖)。以 PBS平衡离心管，25000rpm(82，700g)，4℃离心2小时。小心取出离心管，倒掉上清液，倒置离心管去掉残余液体。再在倒掉残余液体的离心管中加入100μl PBS，密封离心管，在4℃放置2小时，每20分钟轻轻涡旋一次， 500g离心1分钟(25℃)，收集病毒上清，得到慢病毒。冰上冷却后，置于-80℃保存。
实施例3NKG2D CAR-NK细胞的制备
将NK-92细胞密度调整至2～3×105/ml，按体积比(V/V)慢病毒：细胞培养基＝1:5比例添加慢病毒(实施例2所制备的)，同时添加聚凝胺 8μg/ml。4h之后，补加等量的新鲜的完全培养基将NK-92细胞密度调整至 1×105/ml继续培养。次日，将所有的细胞离心，加入新鲜的培养基，继续培养。每隔1-2天进行补液，使维持细胞密度在2～3×105/ml。72h后进行CAR 抗体染色，同时流式分选NKG2D CAR NK-92阳性细胞并扩大培养。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。
利用流式检测NKG2D CAR NK-92细胞阳性率，流式检测结果如图2a 和图2b所示。图2a为对照组，为未转CAR分子的NK92细胞；图2b为实验组，为转CAR分子的NK-92细胞。图2a和图2b中，纵坐标SSC-H表示流式侧向散射数值，FSC-H表示流式前向散射数值，这两个参数主要用于圈定出两组用于分析的活细胞，如图上椭圆圈定处；APC-H表示用抗体染色后荧光强度，该荧光强度越强，表示与对照组相比NKG2D CAR NK-92细胞阳性比率越大。从图2a和图2b中可以看出，APC荧光标记的信号值显著升高，表明NK-92细胞成功表达出CAR分子，CAR-NK92阳性率为98.97％。
实施例4NKG2D CAR-NK细胞对体外肿瘤杀伤效果的评估
为了确定用于NKG2D CAR NK-92检测的肿瘤细胞，利用常规流式分析方法对不同肿瘤细胞进行NKG2D配体MIC-A和MIC-B分子的表达检测。其中，选取的肿瘤细胞分别为肺癌细胞A549、乳腺癌MDMB-231细胞、结肠癌细胞SW480、乳腺癌MCF-7细胞和肺癌细胞H1299，其实验结果如图 3a-e所示。
图3a为肺癌细胞A549的实验结果；图3b为肺癌细胞H1299的实验结果；图3c为乳腺癌细胞MCF-7的实验结果；图3d为乳腺癌细胞MDMB-231 的实验结果；图3e为结肠癌细胞SW480的实验结果。
从图3a-3e中可以看出，肺癌细胞A549、乳腺癌MDMB-231细胞和结肠癌细胞SW480低表达，表达率分别为0.19％、1％和0.12％；乳腺癌MCF-7 细胞中度表达，阳性率61.99％；肺癌细胞H1299高表达，表达率85.16％。
利用CCK-8方法(参见：Human Leukocyte Antigen-G Inhibits the Anti-TumorEffect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 inGastric Cancer,Rui Wan Zi-Wei Wang Hui Li,et al.)检测NKG2D CAR-NK 细胞对上述不同肿瘤细胞系的杀伤效果。实验操作方法如下：
1)提前一天将上述五种不同的实体瘤细胞(40000个)均匀的铺于24 孔中，待细胞贴壁；
2)第二天按照0.5:1和1:1的效靶比例，将NKG2D-CARNK92铺在24 孔中(分别为20000个和40000个细胞)，总体系为1ml，37度孵育4小时；
3)作用4小时后，将培养基舍去，加PBS轻轻地漂洗两次，加入CCK8 试剂200ul，37℃作用1-4小时；
4)450nm处读板计算细胞杀伤率。
NKG2D CAR-NK细胞体外肿瘤杀伤效果评估的实验结果如下表1及图 4a-4e所示。
表1NKG2D CAR-NK细胞体外肿瘤杀伤效果
图4a-4e中，纵坐标表示杀伤效率，横坐标表示效靶比。其中图4a为肺癌细胞A549的实验结果；图4b为肺癌细胞H1299的实验结果；图4c为乳腺癌细胞MCF-7的实验结果；图4d为乳腺癌细胞MDMB-231的实验结果；图4e为结肠癌细胞SW480的实验结果。
如表1及图4a-4e所示，针对高表达NKG2D配体的H1299细胞，在效靶比为0.5:1和1:1的条件下，NKG2D CAR-NK杀伤效果优于普通NK-92 细胞，在1:1效靶比条件下杀伤效果可达到80％。针对中度表达NKG2D配体的MCF-7细胞，NKG2D CAR-NK杀伤效果优于普通NK-92，1:1效靶比条件下杀伤效果可达到56％。针对其它低表达NKG2D配体的肿瘤细胞， NKG2DCAR-NK杀伤效果也优于普通NK-92细胞。即NKG2D CAR-NK的杀伤效果均优于普通的NK-92细胞，但对于低表达NKG2D配体的细胞的杀伤效果不如高表达该配体的肿瘤细胞效果明显。
实施例5NKG2D CAR-NK细胞对体内肿瘤抑制效果的评估
将HCC1187细胞连续培养，培养10代之前，收集处于对数生长期的细胞通过皮下注射接种于NPG小鼠背部皮下。每个点接种约2×106个细胞，接种体积为100μl左右。
1.具体给药设计：当肿瘤体积达到40-50mm3时进行分组，使用PBS重悬至给药设计的浓度，按设定的组别以相应的给药方式给药，给药组包括NKG2D CAR-NK细胞组、PBS对照组和NK-92组，分别以2.5×106个/次和5×106个/ 次的剂量给药，做两组平行试验。
具体给药组如下述表2所示：
表2
2.小鼠肿瘤生长曲线和抑制率的计算
给药后观察动物NPG小鼠外观和行为，并测量体重，按下式计算肿瘤生长抑制率。
肿瘤体积(V，mm3)计算公式：V＝(长×宽2)/2
治疗组/对照组肿瘤体积比(T/C，％)＝(Td-T0)/(Cd-C0)×100％，Td、 Cd分别表示给药组与对照组最后一次测量时肿瘤的体积，T0、C0分别表示给药组与对照组分组时肿瘤的体积。
肿瘤生长抑制率(TGI，％)计算方式：TGI＝(1-T/C)×100％，具体抑制效果如下述表3，及图5a-b和图6所示。
表3
备注：a表示Mean±SD；b表示与Solution比较的P值；V0表示给药前的肿瘤体积，Vt表示给药后的肿瘤体积。
给药后的NPG小鼠的肿瘤生长曲线结果如图5a-b所示，从图中可以看出， NKG2DCAR-NK用于杀伤HCC1187成瘤小鼠后，肿瘤的体积明显小于NK92 组和PBS组，抑瘤率的结果如图6所示，NKG2D CAR-NK-1组和NKG2D CAR-NK-2组的抑瘤作用明显，从给药后第6天开始逐渐升高，直至试验结束；而NK92-1组和NK92-2组的抑瘤作用一直不明显。
3.肿瘤重量的变化
试验结束之后，将NPG小鼠安乐死，并取各组小鼠的肿瘤组织、拍照并称重，肿瘤的重量及肿瘤重量占体重的百分比(肿瘤重量百分比)，结果如图7-8 所示，从各组的肿瘤重量及肿瘤重量占体重的百分比上看，NK92-1组的肿瘤重量及肿瘤重量百分比最大，PBS组、NK92-1、NKG2D CAR-NK-1组和NKG2D CAR-NK-2组肿瘤重量及肿瘤重量百分比最大依次递减，且与给药后26天测量的肿瘤体积的结果相比一致性较好。
上述实验结果说明，本发明的NKG2D CAR-NK细胞的杀伤和抑瘤效果明显好于NK92组，本发明构建的NKG2D CAR-NK细胞通过增加的CAR和 NKG2D受体的配合明显提高了其靶向性，从而大大提高了其杀伤和抑瘤效果。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
序列表
<110> 阿思科力（苏州）生物科技有限公司
<120> 嵌合抗原受体、表达该嵌合抗原受体的NKG2D CAR-NK细胞及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 370
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> NKG2D CARNK-92
<400> 1
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Phe Asn Gln
1 5 10 15
Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys
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Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser
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Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser
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Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val
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Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser
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Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile
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Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys
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Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln
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Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
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Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
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Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
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Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
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Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
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Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
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Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
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Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
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Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
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Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
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Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
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Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
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Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
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Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
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Pro Arg
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<210> 2
<211> 1113
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> NKG2D CARNK-92
<400> 2
gattacaagg atgacgacga taagggtggt ggtggttctt tcaaccaaga agttcaaatt 60
cccttgaccg aaagttactg tggcccatgt cctaaaaact ggatatgtta caaaaataac 120
tgctaccaat tttttgatga gagtaaaaac tggtatgaga gccaggcttc ttgtatgtct 180
caaaatgcca gccttctgaa agtatacagc aaagaggacc aggatttact taaactggtg 240
aagtcatatc attggatggg actagtacac attccaacaa atggatcttg gcagtgggaa 300
gatggctcca ttctctcacc caacctacta acaataattg aaatgcagaa gggagactgt 360
gcactctatg cctcgagctt taaaggctat atagaaaact gttcaactcc aaatacgtac 420
atctgcatgc aaaggactgt gaccacgacg ccagcgccgc gaccaccaac accggcgccc 480
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 540
gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 600
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 660
aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 720
gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 780
aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 840
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 900
cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 960
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1020
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1080
gcccttcaca tgcaggccct gccccctcgc taa 1113
Claims (10)
1.一种嵌合抗原受体，其特征在于，包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活NK细胞；优选地，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述NKG2D配体特异性结合；更为优选地，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I)分子、MIC-A和MIC-B。
2.根据权利要求1所述的嵌合抗原受体，其特征在于，所述的跨膜结构域选自CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域；
更优选地，所述嵌合抗原受体的结构为NKG2D-CD8αhinge-CD8TM-4-1BB-CD3ζ的融合蛋白；进一步优选地，所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：1所示或其同源序列；所述同源序列与原序列的同源性优选为95％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
3.一种编码权利要求1或2所述嵌合抗原受体的核苷酸序列；优选地，所述的核苷酸序列如SEQ ID NO：2所示或其简并序列；更为优选地，所述同源序列与原序列的同源性约95％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
4.一种NKG2D CAR-NK细胞，其表达嵌合抗原受体，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活该NK细胞；优选地，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述NKG2D配体特异性结合；更为优选地，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I)分子、MIC-A和MIC-B。
5.根据权利要求4所述的NKG2D CAR-NK细胞，其特征在于，所述的跨膜结构域选自CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域；
更为优选地，所述嵌合抗原受体的结构为NKG2D-CD8αhinge-CD8TM-4-1BB-CD3ζ的融合蛋白；进一步优选地，所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：1所示或其同源序列；所述同源序列与原序列的同源性优选为95％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
6.根据权利要求5所述的NKG2D CAR-NK细胞，其特征在于，所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的编码核苷酸序列如SEQ ID NO：2所示或其同源序列；优选地，所述同源序列与原序列的同源性约95％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
7.根据权利要求4至6中任一项所述的NKG2D CAR-NK细胞，其特征在于，所述NKG2DCAR-NK细胞能够有效地杀伤和/或杀死肺癌细胞、乳腺癌细胞、结肠癌细胞、乳腺癌细胞和/或肺癌细胞。
8.根据权利要求4至7中任一项所述NKG2D CAR-NK细胞的制备方法，其包括如下步骤：
(1)合成和扩增编码权利要求1或2所述的嵌合抗原受体的核苷酸序列或权利要求3所述的核苷酸序列，并将所述的核苷酸序列克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染细胞，包装和制备慢病毒；和
(3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到NKG2D CAR-NK细胞；
优选地，在步骤(1)中，合成和扩增编码所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的核苷酸序列。
9.一种药物组合物，其包含有效治疗量的权利要求4至7中任一项所述的NKG2D CAR-NK细胞，和/或权利要求8所述的方法制备的NKG2D CAR-NK细胞；优选地，还包括药学可接受的辅料；更为优选地，所述药物组合物用于治疗或预防癌症时，所述NKG2D CAR-NK细胞与肿瘤细胞的效靶比为(0.5～1)：1；进一步优选地，所述药物组合物的剂型为水剂。
10.根据权利要求4至7中任一项所述的NKG2D CAR-NK细胞，和/或权利要求8所述的方法制备的NKG2D CAR-NK细胞在制备用于治疗和/或预防癌症的药物中的应用；优选地，所述的癌症为高表达NKG2D配体的肿瘤及相关疾病；所述癌症优选为肺癌、乳腺癌或结肠癌。
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