CN109609533B - 基于人源化cd276抗体的car慢病毒表达载体构建及其应用 - Google Patents
基于人源化cd276抗体的car慢病毒表达载体构建及其应用 Download PDFInfo
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Abstract
本发明涉及CAR慢病毒表达载体技术领域，尤其是基于人源化CD276抗体的CAR慢病毒表达载体构建及其应用，通过构建包含人源化CD276特异单链抗体(scFv)、CD8信号传导片段、以及CD28或41BB刺激信号分子的CAR序列，制备了靶向CD276的CAR(CD276‑28z‑CAR或CD276‑BBz‑CAR)表达质粒与CAR‑T细胞。本发明采用了人源化的抗CD276scFv作为CAR‑T细胞识别区，可以有效避免免疫排斥发生，有助于治疗效果改善。通过添加胞内刺激信号(CD28或41BB)，能够改善T细胞功能。此类转基因修饰的CAR‑T细胞具备更强、更持久的杀伤能力。
Description
技术领域
本发明涉及CAR慢病毒表达载体的技术领域，具体领域为基于人源化CD276抗体的CAR慢病毒表达载体构建。
背景技术
嵌合抗原受体(CAR)是一种人工合成的结构。利用转基因技术，将CAR序列转导入T细胞中，能够产生识别特异靶点、杀伤肿瘤细胞的CAR-T细胞。CAR-T细胞在血液***肿瘤中取得了良好的治疗效果，但在实体瘤中效果不佳。
CAR-T细胞治疗实体瘤面临的主要技术问题包括：
1、靶点抗原的表达是否广泛；2、CAR序列是否具有较强免疫原性，导致免疫排斥；3、CAR是否能将识别信号有效传递给T细胞。
发明内容
本发明的目的在于提供基于人源化CD276抗体的CAR慢病毒表达载体构建及其应用，以解决现有技术中CAR-T细胞治疗的问题。
CD276是在肿瘤中广泛表达，是较好的CAR-T细胞治疗靶点。本发明通过构建包含人源化CD276特异单链抗体(scFv)、CD8信号传导片段、以及CD28或41BB刺激信号分子的CAR序列，制备了靶向CD276的CAR(CD276-28z-CAR或CD276-BBz-CAR)表达质粒与CAR-T细胞。
CAR主要由3部分构成：胞外识别区、信号转导区和胞内信号区。其中，胞外识别区决定了CAR-T细胞杀伤的特异性，往往由scFv序列构成，但很多scFv序列是小鼠来源的，会造成较强的免疫排斥反应，导致CAR-T细胞治疗无效。而本发明选择了高亲和性识别CD276的人源化改造的scFv序列(anti-CD276scFv)，既能够有效避免免疫排斥，又能够高效识别靶细胞。胞内信号区也是决定CAR-T细胞治疗效果的重要结构，通过加入共刺激信号分子(CD28或41BB)，保证CAR-T细胞具备较强的杀伤功能与扩增能力。此外，信号转导区的结构选择也影响CAR-T细胞功能。利用CD8铰链区保证胞外识别区与信号区之间有了较好结合，保证了CAR-T细胞的功能。
为提高CAR-T细胞治疗效果，针对现有CAR结构缺陷，本发明对3个主要结构进行了优化，其技术方案具体如下：
基于人源化CD276抗体的CAR慢病毒表达载体构建方法，包括以下步骤：(1)CAR结构优化；(2)CAR序列合成及载体构建；
其中，步骤(1)优化后的CAR结构为CD276-28z-CAR结构或者CD276-BBz-CAR结构；CD276-28z-CAR编码蛋白序列如SEQ ID No:1所示；CD276-BBz-CAR编码蛋白序列如SEQ IDNo:7所示。
本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于：所述CD276-28z-CAR结构包括人CD8a分子信号肽、人源化CD276单链抗体、人CD8a分子柔性片段、人CD28分子跨膜区与胞内区和人CD3z分子胞内区；
其中，人CD8a分子信号肽序列如SEQ ID No:2所示；人源化CD276单链抗体序列如SEQ ID No:3所示；人CD8a分子柔性片段序列如SEQ ID No:4所示；人CD28分子跨膜区与胞内区序列如SEQ ID No:5所示；人CD3z分子胞内区序列如SEQ ID No:6所示。
本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于：所述CD276-BBz-CAR结构包括人CD8a分子信号肽、人源化CD276单链抗体、人CD8a分子柔性片段与跨膜区、人41BB分子胞内区和人CD3z分子胞内区；
其中，人CD8a分子信号肽序列如SEQ ID No:8所示；人源化CD276单链抗体如SEQID No:9所示；人CD8a分子柔性片段与跨膜区序列如SEQ ID No:10所示；人41BB分子胞内区序列如SEQ ID No:11所示；人CD3z分子胞内区序列如SEQ ID No:12所示。
本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于，所述步骤(2)具体包括：合成CAR编码序列，将获得的DNA序列通过酶切连接***到pCDH-EF1-MSC质粒中，构建慢病毒表达质粒pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz。
本发明所述的CAR慢病毒表达载体构建方法获得的基于人源化CD276抗体的CAR慢病毒表达载体。
采用本发明所述的基于人源化CD276抗体的CAR慢病毒表达载体制备CAR-T细胞，其方法包括以下步骤：(1)慢病毒包装；(2)T细胞纯化与感染；(3)T细胞CAR表达效率检测；(4)CAR-T细胞体外扩增；
其中，步骤(1)具体为，包装细胞293T铺板24小时后，将pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz表达质粒与包装质粒混合，利用磷酸钙转染试剂进行293T细胞转染；转染48小时后，收集上清，准备用于T细胞感染。
本发明所述的制备CAR-T细胞的方法，其步骤(2)具体为，人外周血经密度梯度离心后，分离外周血单个核细胞；利用T细胞分离试剂盒获得纯化的CD3+T细胞，再按照2个细胞加入1个磁珠的比例，加入适量CD3/CD28磁珠活化2天；2天后，加入病毒上清与polybrene孵育过夜；次日，离心清洗T细胞3次后，加入含1000U IL-2与5％胎牛血清的RPMI1640培养基扩增T细胞。
本发明所述的制备CAR-T细胞的方法，其步骤(3)具体为，T细胞感染3天后，利用流式细胞术对T细胞表面CAR的表达情况进行检测。
本发明所述的制备CAR-T细胞的方法，其步骤(4)具体为，计数1×106个T细胞进行感染，并在感染后3，7，10和14天时进行计数，检测CAR-T细胞增殖情况。
与现有技术相比，本发明的有益效果是：抗原表达强度与CAR-T细胞治疗效果紧密相关。本发明以肿瘤广泛高表达的CD276为靶点进行CAR-T细胞治疗，结果显示该抗原是较好的肿瘤治疗靶点。另外，本发明采用了人源化的抗CD276scFv作为CAR-T细胞识别区，可以有效避免免疫排斥发生，有助于治疗效果改善。通过添加胞内刺激信号(CD28或41BB)，能够改善T细胞功能。此类转基因修饰的CAR-T细胞具备更强、更持久的杀伤能力，有望提高肿瘤治疗效果。
附图说明
图1为CD276-28z-CAR结构示意图；
图2为CD276-BBz-CAR结构示意图；
图3为流式细胞术检测T细胞表达CAR表达；
图4为CAR-T细胞增殖情况；
图5说明CAR-T细胞能够特异杀伤CD276阳性细胞；
图6说明以CD276为靶点的CAR-T细胞的肿瘤杀伤效果更好；
图7说明CAR-T细胞能够控制肿瘤生长。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
1.为提高CAR-T细胞治疗效果，针对现有CAR结构缺陷，对3个主要结构进行了优化。具体包括CD276-28z-CAR结构和CD276-BBz-CAR结构。
(1)CD276-28z-CAR结构
如图1所示，CAR基本结构包括：人CD8a分子信号肽(Leading signal)、人源化CD276单链抗体(scFv)、人CD8a分子柔性片段(CD8Hinge)、人CD28分子跨膜区与胞内区(CD28)，人CD3z分子胞内区(CD3z)。
CD276-28z-CAR编码蛋白序列如下：(SEQ ID No:1所示)
MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*
其中，
人CD8a分子信号肽(Leading signal)序列：(SEQ ID No:2所示)
MALPVTALLLPLALLLHAARPGS
人源化CD276单链抗体(Humanized scFv)序列：(SEQ ID No:3所示)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK
人CD8a分子柔性片段(CD8Hinge)序列：(SEQ ID No:4所示)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
人CD28分子跨膜区与胞内区(CD28)序列：(SEQ ID No:5所示)
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
人CD3z分子胞内区(CD3z)序列：(SEQ ID No:6所示)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(2)CD276-BBz-CAR结构
如图2所示，CAR基本结构包括：人CD8a分子信号肽(Leading signal)、人源化CD276单链抗体(scFv)、人CD8a分子柔性片段与跨膜区(CD8Hinge TM)、人41BB分子胞内区(41BB)，人CD3z分子胞内区(CD3z)。
CD276-BBz-CAR编码蛋白序列如下：(SEQ ID No:7所示)
MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*
其中，
人CD8a分子信号肽(Leadingsignal)序列：(SEQ ID No:8所示)
MALPVTALLLPLALLLHAARPGS
人源化CD276单链抗体(Humanized scFv)序列：(SEQ ID No:9所示)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK
人CD8a分子柔性片段与跨膜区(CD8Hinge TM)序列：(SEQ ID No:10所示)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT
人41BB分子胞内区(41BB)序列：(SEQ ID No:11所示)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
人CD3z分子胞内区(CD3z)序列：(SEQ ID No:12所示)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
2.CAR序列合成及载体构建
CAR编码序列由上海生工公司合成，获得的DNA序列通过酶切连接***到pCDH-EF1-MSC质粒中，构建慢病毒表达质粒pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz。
3.慢病毒包装
包装细胞293T铺板24小时后，将pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz表达质粒与包装质粒(psPAX2与pMD2.G)混合，利用磷酸钙转染试剂进行293T细胞转染。转染48小时后，收集上清，准备用于T细胞感染。
4.T细胞纯化与感染
人外周血经密度梯度离心后，分离外周血单个核细胞。利用德国美天旎公司的T细胞分离试剂盒获得纯化的CD3+T细胞，再按照2个细胞加入1个磁珠的比例，加入适量CD3/CD28磁珠活化2天。2天后，加入病毒上清与polybrene(6μg/mL)孵育过夜。次日，离心清洗T细胞3次后，加入含1000U IL-2与5％胎牛血清的RPMI1640培养基扩增T细胞。
5.T细胞CAR表达效率
T细胞感染3天后，利用流式细胞术对T细胞表面CAR的表达情况进行检测。结果显示CAR表达阳性率达到～60％-70％(如图3)，证明CAR表达质粒构建及病毒包装成功。
6.CAR-T细胞体外扩增
计数1×106个T细胞进行感染，并在感染后3，7，10和14天时进行计数，检测CAR-T细胞增殖情况。如图4所示，CAR-T细胞均能够高效增殖(14天内扩增约50-60倍)，其中CAR-CD276-BBz细胞的增殖能力更好。
7.CAR-T细胞杀伤效果
T细胞感染14天后，计数T细胞与靶细胞，并利用细胞染料(eFluor670)对靶细胞进行标记。然后按照效靶比(效应细胞:靶细胞，E:T)1:1，1:5，1:20的比例，将T细胞(效应细胞)与CD276高表达靶细胞(Calu-3、KYSE70和SW620)，CD276低表达靶细胞(A549和H322)或CD276阴性靶细胞(95D)共孵育6小时。共孵育结束后，离心收集细胞，利用凋亡染色试剂盒标记细胞，然后流式细胞术分析靶细胞凋亡情况(如图5)。结果显示，CAR-T细胞对表达CD276的肿瘤细胞具有很强的杀伤能力，而对CD276阴性细胞的影响很小，说明该CAR-T细胞具有很强的特异性杀伤能力。
8.CD276特异CAR-T细胞具有较强杀伤能力
为了进一步验证CD276特异CAR-T细胞的杀伤能力，我们利用细胞活性实时检测***观察了CAR-T细胞对靶细胞A549(既表达CD276又表达Her2)的杀伤。CAR-T细胞(CAR-CD276-28z，CAR-CD276-BBz或CAR-Her2-28z)按照E:T＝1:10的比例分别加入到靶细胞中，然后对靶细胞的活性进行了5天的连续观察(如图6)。结果显示，与Her2特异CAR-T细胞相比，CD276靶向CAR-T细胞的肿瘤杀伤效果更好，说明以CD276为靶点能够提高CAR-T细胞治疗效果。
9.动物实验验证CAR-T细胞功能
免疫缺陷小鼠皮下接种5×106个Calu-3细胞10天后，经尾静脉分别注射5×106个CAR-T细胞或T细胞进行治疗。治疗0，3，7，14天时，利用小动物活体成像设备，观察肿瘤大小。如图7所示，在CAR-T细胞注射后7天(A)，肿瘤体积明显缩小，而且随着治疗时间的延长，CAR-T细胞治疗组的肿瘤体积越来越小(B)，提示CAR-T细胞具有良好的肿瘤杀伤能力。
由以上实验结果可知，抗原表达强度与CAR-T细胞治疗效果紧密相关。本发明以肿瘤广泛高表达的CD276为靶点进行CAR-T细胞治疗，结果显示该抗原是较好的肿瘤治疗靶点。另外，本发明采用了人源化的抗CD276scFv作为CAR-T细胞识别区，可以有效避免免疫排斥发生，有助于治疗效果改善。通过添加胞内刺激信号(CD28或41BB)，能够改善T细胞功能。此类转基因修饰的CAR-T细胞具备更强、更持久的杀伤能力，有望提高肿瘤治疗效果。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 郑州大学第一附属医院
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Claims (9)
1.基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于，包括以下步骤：(1)CAR结构优化；(2)CAR序列合成及载体构建；
其中，步骤(1)优化后的CAR结构为CD276-28z-CAR结构或者CD276-BBz-CAR结构；CD276-28z-CAR编码蛋白序列如SEQ ID No:1所示；CD276-BBz-CAR编码蛋白序列如SEQ IDNo:7所示。
2.根据权利要求1所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于：所述CD276-28z-CAR结构包括人CD8a分子信号肽、人源化CD276单链抗体、人CD8a分子柔性片段、人CD28分子跨膜区与胞内区和人CD3z分子胞内区；
其中，人CD8a分子信号肽序列如SEQ ID No:2所示；人源化CD276单链抗体序列如SEQID No:3所示；人CD8a分子柔性片段序列如SEQ ID No:4所示；人CD28分子跨膜区与胞内区序列如SEQ ID No:5所示；人CD3z分子胞内区序列如SEQ ID No:6所示。
3.根据权利要求1所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于：所述CD276-BBz-CAR结构包括人CD8a分子信号肽、人源化CD276单链抗体、人CD8a分子柔性片段与跨膜区、人41BB分子胞内区和人CD3z分子胞内区；
其中，人CD8a分子信号肽序列如SEQ ID No:8所示；人源化CD276单链抗体如SEQ IDNo:9所示；人CD8a分子柔性片段与跨膜区序列如SEQ ID No:10所示；人41BB分子胞内区序列如SEQ ID No:11所示；人CD3z分子胞内区序列如SEQ ID No:12所示。
4.根据权利要求1-3任一所述的基于人源化CD276抗体的CAR慢病毒表达载体构建方法，其特征在于，所述步骤(2)具体包括：合成CAR编码序列，将获得的DNA序列通过酶切连接***到pCDH-EF1-MSC质粒中，构建慢病毒表达质粒pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz。
5.根据权利要求4所述的CAR慢病毒表达载体构建方法获得的基于人源化CD276抗体的CAR慢病毒表达载体。
6.采用权利要求5所述的CAR慢病毒表达载体制备CAR-T细胞的方法，其特征在于，包括以下步骤：(1)慢病毒包装；(2)T细胞纯化与感染；(3)T细胞CAR表达效率检测；(4)CAR-T细胞体外扩增；
其中，步骤(1)具体为，包装细胞293T铺板24小时后，将pCDH-EF1-CAR-CD276-28z或pCDH-EF1-CAR-CD276-BBz表达质粒与包装质粒混合，利用磷酸钙转染试剂进行293T细胞转染；转染48小时后，收集上清，准备用于T细胞感染。
7.根据权利要求6所述的制备CAR-T细胞的方法，其特征在于：步骤(2)具体为，人外周血经密度梯度离心后，分离外周血单个核细胞；利用T细胞分离试剂盒获得纯化的CD3+T细胞，再按照2个细胞加入1个磁珠的比例，加入适量CD3/CD28磁珠活化2天；2天后，加入病毒上清与polybrene孵育过夜；次日，离心清洗T细胞3次后，加入含1000U/mL IL-2与5％胎牛血清的RPMI1640培养基扩增T细胞。
8.根据权利要求7所述的制备CAR-T细胞的方法，其特征在于：步骤(3)具体为，T细胞感染3天后，利用流式细胞术对T细胞表面CAR的表达情况进行检测。
9.根据权利要求8所述的制备CAR-T细胞的方法，其特征在于：步骤(4)具体为，计数1×106个T细胞进行感染，并在感染后3，7，10和14天时进行计数，检测CAR-T细胞增殖情况。
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| CN111518834B (zh) * | 2020-04-07 | 2022-10-18 | 湖南光琇医院有限公司 | Car-t细胞及其制备方法和药物 |
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| CN114437218B (zh) * | 2021-01-12 | 2022-09-30 | 北京门罗生物科技有限公司 | 靶向cd276的嵌合抗原受体以及包含其的免疫细胞 |
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