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CN110616188B - 一种通用型car-t细胞及其制备方法和应用 - Google Patents

一种通用型car-t细胞及其制备方法和应用 Download PDF

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CN110616188B
CN110616188B CN201810636386.3A CN201810636386A CN110616188B CN 110616188 B CN110616188 B CN 110616188B CN 201810636386 A CN201810636386 A CN 201810636386A CN 110616188 B CN110616188 B CN 110616188B
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杨选明
傅阳心
汪鑫
叶圣勤
张晓卿
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Shanghai Longyao Biotechnology Co ltd
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Abstract

一种通用型CAR‑T细胞，该CAR‑T细胞敲除CD3Delta、CD3Gamma、CD3 Epsilon和CD3 zeta中的一种或多种，并同时引入HSV‑TK基因。本发明还涉及一种上述CAR‑T细胞的制备方法，含有该CAR‑T细胞的制剂以及该CAR‑T细胞的应用。本发明采用脐血或者外周血来源的PBMC，首次利用CRISPR/Cas9敲除CD3达到避免GVHD的效果，从而构建通用型CAR‑T细胞，提高CAR‑T细胞治疗的易用性和适用范围，同时引入HSV‑TK基因，进一步提高通用型CAR‑T细胞的安全性。本发明所述的通用型CAR‑T细胞，避免移植物抗宿主反应(GVHD)小，大大增强扩增CAR‑T细胞治疗的便利性。

Description

一种通用型CAR-T细胞及其制备方法和应用
技术领域
本发明涉及细胞免疫治疗技术领域，尤其涉及一种通用型CAR-T细胞及其制备方法和应用。
背景技术
利用免疫学治疗手段来攻克肿瘤，一直是免疫学在转化医学方面应用的重要方向。随着各种组学(基因组学，蛋白组学等)的发展，肿瘤细胞由于突变产生的免疫原性得到了广泛的认可，这为肿瘤免疫治疗奠定了理论的基础。同时，随着肿瘤免疫学研究自身的积累，肿瘤免疫治疗近期取得了巨大的进步，一系列新的免疫治疗手段逐步进入临床。当前的肿瘤免疫学研究，奠定了T细胞杀伤在肿瘤免疫治疗中的中心地位，而嵌和抗原受体T细胞(CAR-T细胞)就是结合了抗体的靶向识别和T细胞的肿瘤杀伤功能，人工修饰生成的肿瘤杀伤细胞。
嵌和抗原受体T细胞的概念最早在1989年被Gross，Waks和Eshhar提出，他们将识别TNP的抗体表达在T细胞上，实现了抗原特异性的、非MHC限制的T细胞的活化和效应的增强，并提出了CAR-T技术在肿瘤治疗中的应用的概念。根据这一原理，将具有肿瘤特异性的抗体嵌入T细胞，将赋予T细胞新的杀瘤能力。之后，CAR-T技术被引入到抗肿瘤临床试验中，但早期的CAR-T细胞由于其胞内信号传递域仅含有第一信号，而且选择的肿瘤类型为实体瘤，最终的临床结果都不太理想。在2008年，Fred Hutchison肿瘤研究所等机构使用CAR-T对B细胞淋巴瘤来进行治疗，虽然治疗结果不太理想，但这一临床实验的关键在于证实了以表达CD20的B细胞作为靶点的CAR-T治疗是相对安全的。随后，在2010年NCI报道了一例B细胞淋巴瘤治疗成功的病例，利用针对CD19的CAR-T，病人的淋巴瘤得到控制，正常B细胞也被清除，血清Ig显著降低，为CAR-T治疗B细胞来源的淋巴瘤的有效性提供了理论和实际的支持。在2011年，美国宾夕法尼亚大学的的Carl June博士领导的团队将特异性识别CD19的CAR-T用于B细胞来源的慢性淋巴细胞白血病的治疗，显示了“治愈”的疗效，随后在复发难治性急性淋巴细胞白血病展开临床实验，也取得了良好的疗效。由于这一突破性的进展以及其他免疫调控手段的发展，Science杂志将肿瘤免疫治疗评为了2013年科技突破进展的第一名。这一成功在世界各国引起了广泛的影响，各国开始大量开展基于CAR-T的科学研究和肿瘤治疗的临床试验。
CAR的结构由胞外抗原识别结构域，胞外铰链区，跨膜区和胞内的信号传导结构域组成。胞外的抗原识别域通常是由单链抗体组成，特异性识别肿瘤细胞膜表面分子，也可以是某些肿瘤特异抗原的配体或受体等。胞外铰链区是一段用于隔开抗原识别域和跨膜区的空间结构，其目的是为了提供合适的空间位置，使得胞外抗原识别域能够在识别抗原前后维持正确的结构并传导胞内信号。跨膜区是为了保证CAR分子在膜表面定位的结构域。胞内信号传导结构域是介导CAR信号传导的关键部分，通常是一个或几个第一信号(TCR和MHC-I-peptide复合物识别)，第二信号(共刺激受体和共刺激配体识别)的组合。第一代CAR只含有第一信号，第二代CAR有一个第一信号和一个第二信号，第三代CAR有一个第一信号和两个第二信号结构域。虽然CAR-T在针对B细胞来源的白血病治疗中获得了巨大的成功，但是其相对较高的复发率以及对实体瘤的低有效率是当前的重要挑战。因此发展新一代的高效CAR-T是目前临床所急需的。除了第三代CAR-T，目前还有其他新的CAR-T设计策略，即在第二代CAR-T的基础上引入了新的独立于CAR的调节分子，以进一步增强CAR-T的功能。
利用病人自体血液细胞制备的针对B细胞表面靶向分子CD19、CD20的CAR-T在B细胞白血病治疗中的应用已相对成熟，但整个流程复杂，耗时长，且对于一些特殊的如病情严重，细胞质量极差或艾滋病合并淋巴瘤的病人，自体免疫细胞不便于作为制备CAR-T的T细胞来源。虽然CAR-T在治疗B细胞来源的白血病取得了巨大的成功，但整个CAR-T治疗耗时较长，而且存在病人的异质性，有些病人由于自身细胞的缺陷不能有效的生产CAR-T细胞，这些局限了CAR-T的应用范围，发展通用型的CAR-T将很大程度上解决这些挑战。
当前临床使用的通用型CAR-T方案，都是通过CRISPR/Cas9或TALEN基因编辑手段敲除TCR来避免GVHD作用，尚未报道其他能有效避免GVHD作用的通用型CAR-T的制备方法，且尚未报道和CAR-T关闭开关联用。
发明内容
本发明的目的在于克服现有技术中的缺陷，提供一种通用型CAR-T细胞，利用敲除CD3Delta，CD3Gamma，CD3Epsilon，CD3zeta的方法来达到避免GVHD的效果，同时引入HSV-TK基因，当出现副作用，可利用临床已有的更昔洛韦治疗，去除CAR-T细胞。
为实现上述目的，本发明采用如下技术方案：
本发明的第一个目的是提供一种通用型CAR-T细胞，该CAR-T细胞敲除CD3Delta、CD3Gamma、CD3Epsilon和CD3zeta中的一种或多种。
为了进一步优化上述CAR-T细胞，本发明采取的技术措施还包括：
进一步地，所述CAR-T细胞引入HSV-TK基因。
进一步地，所述胞内信号传导结构域还包括CD3zeta、CD28、CD137、4-1BB、ICOS、OX40、IL-12、41BB、CD28、IL7R、IL2R中的至少一种。
进一步地，所述胞外抗原识别结构域为单链抗体或肿瘤特异抗原的配体或受体，所述单链抗体包括anti-CD19抗体、anti-CD20抗体、EGFR抗体、HER2抗体、EGFRVIII抗体，所述肿瘤特异抗原的配体或受体包括NKG2D；
进一步地，所述胞外铰链区选自CD8a或IgG的一段区域；所述跨膜区选自CD8a、CD28、CD137或CD3中的一个。
本发明的第二个目的是提供一种通用型CAR-T细胞的制备方法，其包括如下步骤：采用合适的基因敲除方法敲除CAR-T细胞CD3Delta、CD3Gamma、CD3Epsilon和CD3zeta中的一种或多种。
为了进一步优化上述通用型CAR-T细胞的制备方法，本发明采取的技术措施还包括：
进一步地，所述制备方法还包括如下步骤：对T细胞进行HSV-TK基因修饰，该步骤采用本领域常规技术手段实现。
进一步地，所述基因敲除方法为本领域中任一合适的方法，更优选为CRISPR/Cas9敲除方法。
进一步地，采用基因敲除方法制备通用型CAR-T细胞具体包括如下步骤：
步骤一、慢病毒载体的构建及病毒生产；
设计针对CD3Delta、CD3Gamma、CD3Epsilon、CD3zeta中的一种或多种的sgRNA，克隆到pLenti-CrisprV2，并和慢病毒包装质粒共同转染，预定时间后收取上清，过滤、离心浓缩病毒，制得plenti-CRISPRV2-sgRNA病毒；
步骤二、CD3阴性CAR-T细胞的制备；
将人PBMC经过分离纯化后，接种到具有合适刺激条件的培养板，培养预定时间后，感染CAR病毒和步骤一生产的plenti-CRISPRV2-sgRNA病毒，按照合适的刺激条件进行细胞扩增，所得细胞去除CD3阳性细胞，获得CD3阴性CAR-T细胞。
进一步地，所述步骤一的具体步骤包括：利用crispr.mit.edu设计针对CD3Delta、CD3Gamma、CD3Epsilon、CD3zeta的sgRNA，克隆到pLenti-CrisprV2；将测序正确的克隆无内毒素大提，和慢病毒包装质粒共同转染293X，48和72小时后收取上清，0.45uM过滤后，25000RPM离心2小时浓缩病毒，制得plenti-CRISPRV2-sgRNA病毒。
进一步地，培养分离纯化后的人PBMC的刺激条件为anti-hCD3和anti-hCD28，扩增细胞的刺激条件为每6天使用人工抗原呈递细胞或anti-hCD3/28刺激。
进一步地，所述步骤二的具体步骤包括：将人PBMC经过Stemcell T细胞分离试剂盒(阴性选择)纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染CAR病毒和步骤一生产的plenti-CRISPRV2-sgRNA病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，所得细胞用Stemcell T细胞阳性选择试剂盒去除CD3阳性细胞，即得CD3阴性20BBZ CAR-T细胞。
进一步地，所述步骤一中慢病毒包装质粒包括VSV-g，pMD Gag/Pol，RSV-REV，离心使用使用贝克曼超速离心机和SW28转头。
进一步地，所述人PBMC为脐血或者成人外周血来源单个核细胞。
本发明的第三个目的是提供一种含有上述CAR-T细胞或含有由上述方法制备的CAR-T细胞的制剂；进一步地，所述制剂还包括药用的稀释剂或赋形剂。
本发明的第四个目的是提供一种上述CAR-T细胞或由上述方法制备的CAR-T细胞在制备治疗或预防肿瘤药物中的应用。
进一步地，所述肿瘤包括实体瘤和非实体瘤，其中所述实体肿瘤包括但不限于，淋巴瘤、肾脏肿瘤、神经母细胞瘤、生殖细胞瘤、骨肉瘤、软骨肉瘤、软组织肉瘤、肝脏肿瘤、胸腺瘤、肺母细胞瘤、胰母细胞瘤、血管瘤等。
与现有技术相比，本发明具有以下有益效果：
本发明采用脐血或者外周血来源的PBMC，首次利用CRISPR/Cas9敲除CD3(CD3Delta，CD3Gamma，CD3Epsilon，CD3zeta)达到避免GVHD的效果，从而构建通用型CAR-T，提高CAR-T治疗的易用性和适用范围，同时引入HSV-TK，进一步提高通用型CAR-T的安全性。
本发明所述的通用型CAR-T，移植物抗宿主反应(GVHD)小，大大增强扩增CAR-T治疗的便利性。
附图说明
图1为本发明一实施例中所采用的20BBZ CAR分子的结构示意图；
图2为本发明一实施例中CD3阴性20BBZ CAR-T细胞表型分析的结果示意图；
图3为本发明一实施例中通过更昔洛韦调控CD3阴性20BBZ CAR-T细胞的存活的示意图；
图4为本发明一实施例中CD3阴性20BBZ CAR-T细胞和对照CAR-T细胞的肿瘤杀伤能力的示意图；
图5为本发明一实施例中CD3阴性20BBZ CAR-T细胞和对照CAR-T细胞的体内存活能力的示意图。
具体实施方式
一种通用型CAR-T细胞，该CAR-T细胞敲除CD3Delta，CD3Gamma、CD3Epsilon和CD3zeta，并同时引入HSV-TK基因。本发明还涉及一种上述CAR-T细胞的制备方法，含有该CAR-T细胞的制剂以及该CAR-T细胞的应用。
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
实施例1-CD3阴性20BBZ CAR-T细胞的制备
本实施例所述的CD3阴性20BBZ CAR-T细胞的制备包括以下步骤：
1.慢病毒载体pLenti-CrisprV2-sgRNA的构建及病毒生产
利用crispr.mit.edu设计针对CD3Delta、CD3Gamma、CD3Epsilon、CD3zeta的sgRNA，克隆到pLenti-CrisprV2；将测序正确的克隆无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol，RSV-REV)共同转染293X，48和72小时后收取上清，0.45uM过滤后，使用贝克曼超速离心机和SW28转头，25000RPM离心2小时浓缩病毒，以获得plenti-CRISPRV2-sgRNA病毒，用于后续CAR-T细胞的生产。
2.CD3阴性20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒(阴性选择)纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染20BBZ(其结构如图1所示，其采用的抗体胞外抗原识别结构域为anti-CD20抗体)和plenti-CRISPRV2-sgRNA病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，所得细胞用Stemcell T细胞阳性选择试剂盒去除CD3阳性细胞，即获得CD3阴性20BBZ CAR-T细胞(CD3-U-CAR-T，简称U-CAR-T细胞)，以用于后续实验和表型分析显示，其结果如图2所示。由图可知，所得U-CAR-T细胞为CAR阳性和CD3阴性。
实施例2-通过更昔洛韦调控U-CAR-T细胞的存活
将实施例1中步骤2所得的U-CAR-T细胞和对照CAR-T接种到96孔板，加入所示浓度的更昔洛韦，48小时后比较存活的CAR-T数目，其结果如图3所示。由图可知，更昔洛韦能够调控U-CAR-T的存活，可以在U-CAR-T引起副作用的条件下迅速从体内清除U-CAR-T，提高安全性。
实施例3-比较U-CAR-T和对照CAR-T的肿瘤杀伤能力
将实施例1中步骤2所得的U-CAR-T和对照CAR-T细胞，接种到96孔板，按照CAR-T：肿瘤细胞比例1:1加入Raji肿瘤细胞，24、48小时后比较肿瘤细胞的存活比例，其结果如图4所示。由图可知，U-CAR-T和对照CAR-T相比有着相似的肿瘤杀伤能力。
实施例4-比较U-CAR-T和对照CAR-T的体内存活能力
将106Raji肿瘤细胞通过静脉接种到B-NDG小鼠，6天后给予107U-CAR-T和对照CAR-T治疗，观察小鼠的存活率，其结果如图5所示。由图可知，U-CAR-T和对照CAR-T都延长了小鼠的存活时间。
由上述实施例可知，本发明通过敲除CD3构建的通用型CAR-T，其移植物抗宿主反应(GVHD)小，大大增强扩增CAR-T治疗的便利性；同时引入HSV-TK，通过更昔洛韦的调控可迅速从体内清除U-CAR-T，进一步提高通用型CAR-T的安全性。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (9)

1.一种通用型CAR-T细胞，其特征在于，所述CAR-T细胞敲除CD3Delta、CD3Gamma、CD3Epsilon和CD3 zeta；所述CAR-T细胞引入HSV-TK基因。
2.根据权利要求1所述的通用型CAR-T细胞的制备方法，其特征在于，包括如下步骤：采用合适的基因敲除方法敲除CAR-T细胞CD3Delta、CD3Gamma、CD3Epsilon和CD3 zeta。
3.根据权利要求2所述的通用型CAR-T细胞的制备方法，其特征在于，还包括如下步骤：对T细胞进行HSV-TK基因修饰。
4.根据权利要求2所述的通用型CAR-T细胞的制备方法，其特征在于，采用基因敲除方法制备通用型CAR-T细胞具体包括如下步骤：
步骤一、慢病毒载体的构建及病毒生产；
设计针对CD3Delta、CD3Gamma、CD3 Epsilon、CD3 zeta的sgRNA，克隆到pLenti-CrisprV2，并和慢病毒包装质粒共同转染，预定时间后收取上清，过滤、离心浓缩病毒，制得plenti-CRISPRV2-sgRNA病毒；
步骤二、CD3阴性CAR-T细胞的制备；
将人PBMC经过分离纯化后，接种到具有合适刺激条件的培养板，培养预定时间后，感染CAR病毒和步骤一生产的plenti-CRISPRV2-sgRNA病毒，按照合适的刺激条件进行细胞扩增，所得细胞去除CD3阳性细胞，获得CD3阴性CAR-T细胞。
5.根据权利要求4所述的通用型CAR-T细胞的制备方法，其特征在于，培养分离纯化后的人PBMC的刺激条件为anti-hCD3和anti-hCD28，扩增细胞的刺激条件为每6天使用人工抗原呈递细胞或anti-hCD3/28刺激。
6.根据权利要求4所述的通用型CAR-T细胞的制备方法，其特征在于，所述步骤一中慢病毒包装质粒包括VSV-g，pMD Gag/Pol，RSV-REV；离心使用贝克曼超速离心机和SW28转头。
7.根据权利要求4或5所述的通用型CAR-T细胞的制备方法，其特征在于，所述人PBMC为脐血或者成人外周血来源单个核细胞。
8.一种含有权利要求1所述的通用型CAR-T细胞的制剂。
9.一种如权利要求1所述的通用型CAR-T细胞在制备治疗或预防肿瘤药物中的应用。
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