CN105087495B - 双嵌合抗原受体修饰的t淋巴细胞及其制备方法 - Google Patents
双嵌合抗原受体修饰的t淋巴细胞及其制备方法 Download PDFInfo
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Abstract
本发明属于生物技术领域,本发明涉及双嵌合抗原受体修饰的T淋巴细胞及其制备方法。本发明构建低亲和力嵌合抗原受体(chimeric?antigen?receptor,CAR-L)和高亲和力的嵌合抗原受体(CAR-H),分别识别两种肿瘤相关抗原,并且分别含有CD3ζ序列和共刺激分子信号序列(costimulatory?molecule,CM)序列,将它们同时转染至T淋巴细胞中,修饰后的T淋巴细胞只有同时识别两种肿瘤相关抗原才能被有效激活,增强了CAR-T细胞杀伤肿瘤的靶向性,降低对正常组织的损伤。
Description
技术领域
本发明属于生物技术领域,本发明涉及双嵌合抗原受体修饰的T淋巴细胞及其制备方法。
背景技术
过继性细胞免疫治疗(adoptivecellularimmunotherapy,ACI)是指将体外激活的自体或异体免疫效应细胞输注给患者,以杀伤患者体内的肿瘤细胞,是目前治疗恶性肿瘤的重要手段之一,已在多种实体瘤和血液肿瘤的临床治疗中取得较好疗效。其中,嵌合抗原受体(chimericantigenreceptor,CAR)修饰的T淋巴细胞技术是新近迅速发展的一种细胞治疗技术,通过基因工程技术修饰效应T淋巴细胞,克服肿瘤局部免疫抑制微环境和宿主免疫耐受状态,提高了抗肿瘤的靶向性、杀伤活性和持久性。
目前,大多数CAR由胞外抗原结合区、跨膜区和胞内信号转导区组成。胞外抗原结合区由来源于单克隆抗体的轻链(VL)和重链(VH)的可变区组成,中间由带韧性的铰链区连接形成单链抗体(singlechainfragmentvariable,scFv)。CAR通过将识别肿瘤抗原的scFv和胞内信号域“免疫受体酪氨酸活化基序(immunoreceptortyrosine-basedactivationmotifs,ITAM)”在体外进行基因重组,通过基因转染技术修饰患者的T淋巴细胞,使患者T淋巴细胞表达肿瘤抗原受体,经过纯化和大规模扩增修饰后的T淋巴细胞,称为嵌合抗原受体修饰的T淋巴细胞(CAR-T)。
目前CAR-T淋巴细胞技术发展到了第三代。第一代CAR由单链抗体通过跨膜区域与胞内信号传导区(ITAM)相连,ITAM通常为CD3ζ或FcεRIγ;第二代CAR的胞内信号转导区引入了共刺激分子(costimulatorymolecule,CM),主要为CD28分子;第三代CAR引入了双共刺激分子(CM1和CM2),主要为CD28分子加上CD134或CD137等。第一代CAR-T淋巴细胞研究较多,但是大多数试验在细胞扩增、体内存活时间、细胞因子分泌等方面还存在不足,没有达到预期的临床效果。研究表明,T淋巴细胞的完全活化有赖于双信号和细胞因子的作用。其中第一信号为特异性信号,由TCR识别抗原递呈细胞表面的抗原肽-MHC复合物所启动;第二信号为协同刺激信号,通过CD28/B7等重要的共刺激分子,促进IL-2合成,并使T淋巴细胞充分活化及免于凋亡。即使T淋巴细胞与抗原接触,如果没有协同刺激信号,细胞难以发挥正常功能。相应的,仅含有CD3ζ序列的嵌合抗原受体,如没有协同刺激信号2,也难以高效激活CAR-T淋巴细胞。因此,依照T淋巴细胞活化的双信号学说,第二和第三代CAR在嵌合抗原受体上加上如CD28、CD137等共刺激分子,以提高T淋巴细胞的细胞毒性、增殖活性,维持T淋巴细胞应答,延长T淋巴细胞存活时间等。研究证实第二代的CAR-T淋巴细胞在杀瘤活性和体内存活时间均优于第一代。目前第三代CAR-T淋巴细胞临床应用还比较少,其结构的构建、安全性和有效性还需进一步观察和优化。
CAR-T淋巴细胞临床应用的首要风险是脱靶效应,可导致针对正常组织的自身免疫反应,主要是由于目前已知的肿瘤特异性抗原较少,大多数CAR针对的是重要组织不表达或表达较少的肿瘤相关抗原。因此,如何提高CAR-T淋巴细胞的靶向性是目前临床应用面临的首要问题。
发明内容
本发明构建低亲和力嵌合抗原受体(chimericantigenreceptor,CAR-L)和高亲和力嵌合抗原受体(CAR-H),分别识别两种肿瘤相关抗原,并且分别含有CD3ζ序列和CM序列,将它们同时转染至T淋巴细胞中,修饰后的T淋巴细胞只有同时识别两种肿瘤相关抗原才能被有效激活,增强了CAR-T细胞杀伤肿瘤的靶向性,降低对正常组织的损伤。
本发明的第一方面是提供一种双嵌合抗原受体修饰的T淋巴细胞(BiCAR-T),所述T淋巴细胞表面表达两种嵌合抗原受体。
在本发明的一个实施方案中,所述嵌合抗原受体CAR-L是通过氨基端到羧基端顺次拼接所述的靶向EGBB2的单链抗体、CD8α铰链区及跨膜区、和CD3ζ链胞内区,所得到的嵌合抗原受体CAR-L的结构为ERBB2(scFv)-CD8α-CD3ζ,其氨基酸序列如SEQIDNO:1所示,其编码核苷酸序列如SEQIDNO:2所示。
在本发明的另一个实施方案中,所述嵌合抗原受体CAR-H是通过氨基端到羧基端顺次拼接所述的靶向EGFR的单链抗体、CD8α铰链区、CD28跨膜区及胞内区、和可诱导协同刺激分子(inducibleco-stimulator,ICOS)胞内区,所得到的嵌合抗原受体CAR-H的结构为EGFR(scFv)-CD8α-CD28-ICOS,其氨基酸序列如SEQIDNO:3所示,其编码核苷酸序列如SEQIDNO:4所示。
在本发明中,所述靶向EGBB2的单链抗体对靶向蛋白具有低亲和力,KD值>2nM;所述靶向EGFR的单链抗体对靶向蛋白具有高亲和力,KD值<0.5nM。
本发明的第二方面是所述双嵌合抗原受体修饰的T淋巴细胞及其相关嵌合抗原受体在制备抗肿瘤药物中的应用。
本发明的第三方面是提供所述双嵌合抗原受体修饰的T淋巴细胞的制备方法,具体步骤如下:
(1)通过PCR方法获得编码人源ERBB2单链抗体的核苷酸片段;
(2)通过PCR方法获得编码CD8α铰链区及跨膜区和CD3ζ链胞内区的核苷酸片段;
(3)将步骤(1)和(2)获得核苷酸片段依次装入载体pCDH-CMV-MCS;从而获得含有嵌合抗原受体CAR-L基因片段的慢病毒转移载体,命名为pCDH-ERBB2-CD3ζ(CAR-L);
(4)合成已知编码人源EGFR单链抗体的核苷酸片段;
(5)通过PCR方法获得编码CD8α铰链区、CD28跨膜区及胞内区、和ICOS胞内区的核苷酸片段;
(6)将步骤(4)和(5)获得核苷酸片段依次装入载体pCDH-CMV-MCS;从而获得含有嵌合抗原受体CAR-H基因片段的慢病毒转移载体,命名为pCDH-EGFR-CD28-ICOS(CAR-H);
(7)将慢病毒转移载体pCDH-EGFR-CD28-ICOS(CAR-H)和pCDH-ERBB2-CD3ζ(CAR-L)转染至293FT细胞;包装获得病毒颗粒,经离心浓缩后获得高滴度的慢病毒悬液;
(8)慢病毒悬液感染T淋巴细胞从而获得双嵌合抗原受体修饰的T淋巴细胞(BiCAR-T)。
具体实施方式
下面将进一步地详细说明本发明。需要指出的是,以下说明仅仅是对本发明要求保护的技术方案的举例说明,并非对这些技术方案的任何限制。本发明的保护范围以所附权利要求书记载的内容为准。
实施例1CAR-L表达质粒载体的构建
根据基因工程技术设计对ERBB2的胞外配体结合区RLD蛋白高特异性和低亲和性的ERBB2scFv抗体的核苷酸序列(核苷酸序列如SEQIDNo:5所示);且在两端加上限制性内切酶EcoRI位点。通过表达系统表达ERBB2scFv抗体,经ELISA测定其对RLD蛋白亲和力的KD值为2.14nM。
按照Genebank(NCBI)中CD8α及CD3ζ基因序列设计合成嵌合抗原受体CAR-L的Hinge-TM-CD3ζ表达框,包括CD8α铰链区和跨膜区(aa135-205)及CD3ζ链胞内区(aa52-163),合成基因序列。设计引物,在引物两端分别加上限制性内切酶EcoRI、BamHI位点,PCR扩增上述基因序列。EcoRI、BamHI双酶切慢病毒转移质粒pCDH-CMV-MCS,置于37℃水浴中反应4h,反应结束后用PCR产物纯化试剂盒过柱回收。用SolutionI连接酶将Hinge-TM-CD3ζ片段插入到载体基因片段中。将连接体系全部加入装有DH5α感受态细胞,涂在氨苄青霉素抗性的LB平板上,正置约半小时待菌液基本被吸收,放入37℃培养箱倒置培养过夜。挑取平板上Amp筛选的单个克隆,按质粒DNA少量提取试剂盒操作说明扩增连接产物。提取质粒,用EcoRI、BamHI双酶切来鉴定,符合要求的命名为pCDH-CD3ζ。
EcoRI酶切质粒pCDH-CD3ζ,置于37℃水浴中反应4h,反应结束后用PCR产物纯化试剂盒过柱纯化回收。向酶切产物中加入1μLFastAP(使切开的载体末端去磷酸化),37℃水浴中反应10min,然后置于70℃电热烘干机中灭活酶10min,过柱纯化回收载体片段。用SolutionI连接酶将ERBB2scFv目的片段插入到载体基因片段中,4℃连接过夜。将连接体系放入70℃水浴灭活酶10min,置冰上冷却。将连接产物转化DH5α感受态细胞,经冰浴30min,42℃水浴热休克90s,再次立即置于冰上5min,加入LB液体培养基摇菌后,将菌液涂在氨苄青霉素(Amp+)抗性的LB平板上,37℃培养箱倒置培养过夜。次日观察平板上菌落生长情况。挑取平板上Amp筛选的单个克隆,进行菌落PCR鉴定,用1%琼脂糖凝胶电泳分析,筛选出扩增条带与预期相符的单克隆菌。将筛选出的单克隆余下的水溶菌液加到2.5mL含2.5μL氨苄青霉素的LB液体培养基中,过夜摇菌,扩大培养。次日,保存一部分菌液为15%的甘油菌于-100℃冰箱中。剩余菌液用质粒DNA少量提取试剂盒提取质粒。将提取的质粒进行DNA测序鉴定。经鉴定符合要求的含有嵌合抗原受体基因片段的慢病毒转移载体,命名为pCDH-ERBB2-CD3ζ(CAR-L);测序表明,编码CAR-L的各基因片段ERBB2scFv、铰链区、跨膜区和CD3ζ序列和连接正确,其核苷酸序列如SEQIDNO:2所示,其编码氨基酸序列如SEQIDNO:1所示。
实施例2CAR-H表达质粒载体的构建
根据基因工程技术设计对PEP3-KLH抗原高特异性和高亲和性的EGFRscFv抗体的核苷酸序列(核苷酸序列如SEQIDNo:6所示);且在两端加上限制性内切酶EcoRI位点。通过表达系统表达EGFRscFv抗体,经ELISA测定其对PEP3-KLH抗原亲和力的KD值为0.47nM。
根据Genebank(NCBI)中CD8α、CD28及ICOS基因序列设计合成CD8α铰链区、CD28跨膜区及胞内区、和ICOS胞内区基因序列。设计引物,在引物两端分别加上限制性内切酶EcoRI、BamHI位点,PCR扩增上述基因序列。将胶回收后的目的片段双酶切,置于37℃水浴中反应4h,使两端带有粘性末端,反应结束后取4μL上样经1%琼脂糖凝胶电泳分析,纯化获取两端带有粘性末端的Hinge-TM-CD28-ICOS目的片段。
EcoRI、BamHI双酶切慢病毒转移质粒pCDH-CMV-MCS,用PCR产物纯化试剂盒过柱回收。用SolutionI连接酶将Hinge-TM-CD28-ICOS片段插入到载体基因片段中,4℃连接过夜。将连接体系加入装有DH5α感受态细胞的EP管中,混匀后置于冰上30min,42℃水浴热休克90s,迅速置于冰上5min,加入600μLLB液体培养基(不含抗生素)放入37℃水平摇床(180r/min)摇菌1h,6000r/min离心2min,弃去上清450μL,剩余的轻轻吹打混匀后涂在氨苄青霉素(Amp+)抗性的LB平板上,正置约半小时,放入37℃培养箱倒置培养过夜。挑取平板上Amp筛选的单个克隆扩增,提取质粒,用EcoRI、BamHI双酶切来鉴定,符合要求的命名为pCDH-CD28-ICOS。
EcoRI酶切质粒pCDH-CD28-ICOS,用PCR产物纯化试剂盒过柱纯化回收。向酶切产物中加入1μLFastAP(使切开的载体末端去磷酸化),37℃水浴中反应10min,然后置于70℃电热烘干机中灭活酶10min,过柱纯化回收载体片段。用SolutionI连接酶将EGFRscFv目的片段插入到载体基因片段中,4℃连接过夜。将连接体系放入70℃水浴灭活酶10min,置冰上冷却。将连接产物转化DH5α感受态细胞,经冰浴30min,42℃水浴热休克90s,再次立即置于冰上5min,加入LB液体培养基摇菌后,将菌液涂在氨苄青霉素(Amp+)抗性的LB平板上,37℃培养箱倒置培养过夜。次日观察平板上菌落生长情况。挑取平板上Amp筛选的单个克隆,进行菌落PCR鉴定,用1%琼脂糖凝胶电泳分析,筛选出扩增条带与预期相符的单克隆菌。将筛选出的单克隆余下的水溶菌液加到2.5mL含2.5μL氨苄青霉素的LB液体培养基中,过夜摇菌,扩大培养。次日,保存一部分菌液为15%的甘油菌液于-100℃冰箱中。剩余菌液用质粒DNA少量提取试剂盒提取质粒。将提取的质粒进行DNA测序鉴定。经鉴定符合要求的含有嵌合共刺激受体基因片段的慢病毒转移载体,命名为pCDH-EGFR-CD28-ICOS(CAR-H),其核苷酸序列如SEQIDNO:4所示,其编码氨基酸序列如SEQIDNO:3所示。
实施例3T淋巴细胞转染及表达检测
选择生长状态良好的293FT细胞进行传代培养,当细胞生长达70%融合时,弃培养基并以无血清DMEM培养基冲洗,将20μL脂质体LipofectamineTM2000加至500μL的无血清DMEM培养基,充分振荡后快速加入两种重组质粒CAR-L/CAR-H15μg、△8.210μg、VSV-G5μg,静置20min移至293FT细胞培养瓶中培养;6h后更换完全培养基培养,48h荧光显微镜下观察,72h后收集病毒上清,5000rpm低温离心10min,去除漂浮细胞及细胞碎片。ELISA法检测病毒滴度后置于-80℃保存。
抽取50mL健康志愿者的新鲜外周血,通过常规方法获得人外周血单个核细胞(PBMC)。用适量的MACS缓冲液(按1.5mL/107个PBMC)洗涤细胞一次,800r/min离心10min沉淀细胞,弃上清。用适量的MACS缓冲液重悬细胞(按80μL/107个PBMC)后,加入适量的antihuman-CD3免疫磁珠(按20μL/107个PBMC)混匀,4℃孵育15min。再加入适量的MACS缓冲液洗涤细胞(按1.5mL/107个PBMC),800r/min离心10min沉淀细胞,弃上清,用500μLMACS缓冲液重悬细胞。将MS分离柱放入MiniMACS分离器上,用500μLMACS缓冲液洗涤分离柱一次。加入细胞悬液于MS分离柱内,先流出来的细胞是未被磁珠标记的CD3-细胞。用500μLMACS缓冲液洗涤分离柱三次,从MiniMACS分离器上取下MS分离柱,放到15mL离心管上。加入1mLMACS缓冲液至分离柱上,迅速将滞留的细胞洗脱下来,洗脱液即为分离的CD3+T淋巴细胞。加入适量的MACS缓冲液,充分混匀后计数。1000r/min离心10min,弃上清。用含10%FBS的RPMI1640培养基重悬,调整细胞浓度至1×106/mL于6孔板中。将培养板置于37℃、5%CO2培养箱中培养。
按照人T淋巴细胞CD3/CD28免疫激活磁珠说明书激活T淋巴细胞。将分离出的CD3+T淋巴细胞以每孔1×106个铺于24孔板上。每孔加入25μL已预洗的磁珠,加入重组人IL-2使终浓度为30U/mL。将24孔板置于37℃、5%CO2培养箱中培养。每2-3d更换含有重组人IL-2的培养基。根据细胞生长密度进行传代。
当CD3+T淋巴细胞生长状态良好,密度为70%左右时可进行感染。将Polybrene加入24孔板中至终浓度为4μg/mL,同时加入慢病毒悬液CAR-L/CAR-H,第四天换液继续培养并冻存。慢病毒感染目的细胞,经有限稀释法,铺96孔板(80cells/plate),37℃、5%CO2至细胞克隆长出,挑选单克隆细胞鉴定阳性克隆,即获得双嵌合抗原受体修饰的T淋巴细胞(BiCAR-T)。
实施例4BiCAR-T体外杀瘤活性检测实验根据不同的靶细胞分为四个实验组:空白对照组(4T1细胞)、ERBB2组(转染了ERBB2的4T1细胞)、EGFR组(转染了EGFR的4T1细胞)、ERBB2-EGFR组(转染了ERBB2和EGFR的4T1细胞),设置效应细胞对照组和靶细胞对照组。调整效应细胞BiCAR-T(实施例3制备)和各组相应的靶细胞密度,分别为1×106/mL和1×107/mL,在各实验组中按效靶比5:1、10:1、20:1、40:1往96孔板加入效应细胞悬液和靶细胞悬液,总体积为200μL,放入37°C,5%C02培养箱中培养48h后每孔加入20uLCCK-8,继续孵育2h后上酶标仪检测,于450nm读取OD值,杀伤率=【1-(实验组OD值-效应细胞对照组OD值)/靶细胞对照组OD值】×100%。
具体结果如下:
杀伤率% | 空白对照组 | ERBB2组 | EGFR组 | ERBB2-EGFR组 |
效靶比 5:1 | 0.7±0.1 | 2.4±0.1 | 1.7±0.1 | 9.7±0.6 |
效靶比10:1 | 1.1±0.1 | 4.8±0.3 | 2.2±0.2 | 18.8±0.7 |
效靶比20:1 | 1.6±0.1 | 7.3±0.6 | 2.4±0.2 | 29.1±1.7 |
效靶比40:1 | 1.9±0.1 | 8.9±0.7 | 3.2±0.3 | 45.2±2.5 |
实施例5BiCAR-T分泌IFN-γ的检测
实验分组同实施例4,每组设3个复孔。取出已包被抗体的酶标板,设置TMB空白显色孔,依次加入0.1mL按一定倍数稀释的标准品和用样品稀释液稀释的样品。酶标板加上盖,37℃反应90min。反应后用自动洗板机吸去酶标板内的液体。将生物素抗人IFN-γ抗体工作液按每孔0.1mL依次加入(TMB空白显色孔除外),37℃反应60min,0.01MPBS洗涤3次。将ABC工作液按每孔0.1mL依次加入(TMB空白显色孔除外),37℃反应30min,0.01MPBS洗涤5次。按每孔90μL依次加入TMB显色液,37℃避光反应20-25min,按每孔0.1mL依次加入TMB终止液,此时蓝色立转黄色,用酶标仪在450nm测定OD值。样品OD值减去空白孔OD值后以OD值及标准品浓度为XY轴绘图,在标准曲线上查找IFN-γ浓度后乘以稀释倍数,计算样本中IFN-γ浓度;具体结果如下:
ng/ml | 空白对照组 | ERBB2组 | EGFR组 | ERBB2-EGFR组 |
效靶比 5:1 | 0.10±0.01 | 0.19±0.01 | 0.13±0.01 | 0.97±0.04 |
效靶比10:1 | 0.11±0.01 | 0.32±0.02 | 0.15±0.01 | 2.65±0.16 |
效靶比20:1 | 0.14±0.01 | 0.56±0.03 | 0.17±0.01 | 3.69±0.28 |
效靶比40:1 | 0.18±0.01 | 0.67±0.05 | 0.23±0.02 | 4.51±0.33 |
实施例6BiCAR-T体内杀瘤活性检测
收集对数生长期的U251肿瘤细胞,计数细胞数量后调整细胞浓度,每只小鼠右侧背部皮下接种1×107个细胞。待肿瘤体积达到300-400mm3时,计数BiCAR-T(实施例3制备)浓度调整至2×108个/mL,尾静脉注射小鼠100μL/只,隔一天注射一次,共三次;对照组注射未进行双嵌合抗原受体修饰的T淋巴细胞。接种肿瘤30天后,用游标卡尺测量肿瘤的长与宽,计算肿瘤体积并统计生存率。
另外,设计以下对比例用于对比试验,具体如下:
对比例1:仅包含CAR-L(实施例1制备)的T细胞,给药量同实施例3制备的BiCAR-T细胞;
对比例2:仅包含CAR-H(实施例2制备)的T细胞,给药量同实施例3制备的BiCAR-T细胞;
具体结果如下:
对照组 | BiCAR-T细胞组 | 对比例1 | 对比例2 | |
肿瘤体积(mm3) | 1214±120 | 376±36 | 745±65 | 1094±99 |
生存率(%) | 0 | 76.5% | 37.8% | 5.3% |
本发明内容仅仅举例说明了要求保护的一些具体实施方案,其中一个或更多个技术方案中所记载的技术特征可以与任意的一个或多个技术方案相组合,这些经组合而得到的技术方案也在本申请保护范围内,就像这些经组合而得到的技术方案已经在本发明公开内容中具体记载一样。
SEQUENCELISTING
<110>深圳市茵冠生物科技有限公司
<120>双嵌合受体修饰的T淋巴细胞及其制备方法
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Claims (5)
1.一种双嵌合抗原受体修饰的T淋巴细胞,所述T淋巴细胞表面表达嵌合抗原受体CAR-L和嵌合抗原受体CAR-H;其特征在于,
所述嵌合抗原受体CAR-L是通过氨基端到羧基端顺次拼接靶向ERBB2的单链抗体、CD8α铰链区及跨膜区、CD3ζ链胞内区,所述嵌合抗原受体CAR-L的氨基酸序列如SEQIDNO:1所示;
所述嵌合抗原受体CAR-H是通过氨基端到羧基端顺次拼接靶向EGFR的单链抗体、CD8α铰链区、CD28跨膜区及胞内区及ICOS胞内区,所述嵌合抗原受体CAR-H的氨基酸序列如SEQIDNO:3所示。
2.根据权利要求1所述的双嵌合抗原受体修饰的T淋巴细胞,其特征在于,所述嵌合抗原受体CAR-L的编码核苷酸序列如SEQIDNO:2所示。
3.根据权利要求1所述的双嵌合抗原受体修饰的T淋巴细胞,其特征在于,所述嵌合抗原受体CAR-H的编码核苷酸序列如SEQIDNO:4所示。
4.权利要求1-3任一项所述的双嵌合抗原受体修饰的T淋巴细胞在制备抗肿瘤药物中的应用。
5.一种制备权利要求1-3任一项所述的双嵌合抗原受体修饰的T淋巴细胞的方法,具体步骤如下:
(1)通过PCR方法获得人源编码低亲和力ERBB2单链抗体的核苷酸片段;
(2)通过PCR方法获得编码CD8α铰链区和跨膜区以及CD3ζ链胞内区的核苷酸片段;
(3)将步骤(1)和(2)获得核苷酸片段依次装入载体pCDH-CMV-MCS;从而获得含有嵌合抗原受体CAR-L基因片段的慢病毒转移载体,命名为pCDH-ERBB2-CD3ζ;
(4)通过PCR方法获得已知编码高亲和力EGFR单链抗体的核苷酸片段;
(5)通过PCR方法获得编码CD8α铰链区、CD28跨膜区和胞内区以及ICOS胞内区的核苷酸片段;
(6)将步骤(4)和(5)获得核苷酸片段依次装入载体pCDH-CMV-MCS;从而获得含有嵌合抗原受体CAR-H基因片段的慢病毒转移载体,命名为pCDH-EGFR-CD28-ICOS;
(7)将慢病毒转移载体pCDH-EGFR-CD28-ICOS和pCDH-ERBB2-CD3ζ转染至293FT细胞;包装获得病毒颗粒,经离心浓缩后获得高滴度的慢病毒悬液;
(8)慢病毒悬液感染T淋巴细胞从而获得双嵌合抗原受体修饰的T淋巴细胞。
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