CN116514992A - 一种信号肽序列优化的靶向cd19的嵌合抗原受体及其应用 - Google Patents
一种信号肽序列优化的靶向cd19的嵌合抗原受体及其应用 Download PDFInfo
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Abstract
本发明公开了一种以CD19为靶点的嵌合抗原的信号肽优化及其用途,优化后的信号肽可显著提高以CD19受体为靶点的嵌合抗原受体在T细胞中的表达效率和稳定性,从而更好地发挥对靶细胞的杀伤作用。
Description
技术领域
本发明涉及生物医药领域,具体的,本发明涉及一种信号肽序列优化的靶向CD19的嵌合抗原受体及其应用。
背景技术
嵌合抗原受体T(Chimeric Antigen Receptor T,CAR-T)细胞技术是通过体外基因改造技术,将针对特异性抗原的单链抗体可变区(Single chain antibody variablefragments,scFvs)序列表达于患者的T细胞表面,进而靶向并杀伤肿瘤细胞。CAR的基础设计中包括一个肿瘤相关抗原结合区(通常来源于抗体抗原结合区域的scFv段)、一个胞外铰链区、一个跨膜区和一个胞内信号区。细胞膜外部分是从单克隆抗体中纯化的抗原靶向部分,该抗体由单链可变片段(scFv)(重链和轻链可变区的融合蛋白)组成,此外,scFvs中的信号肽对自身的表达尤为重要。
1975年,洛克菲勒大学细胞生物学教授Gunter Bloble在一项实验研究中提出了“信号假说”,以解决蛋白质合成后去向的问题。他认为新合成的蛋白质具有内源性信号,即信号肽(Signal peptide,SP)。信号肽是一段存在于前体蛋白N-端的短肽链,能够调节前体蛋白的折叠和转移过程。研究表明,在原核系统中,信号肽附着后,外源基因能够在原核表达系统中成功表达并分泌,如芽孢杆菌、乳酸杆菌、L型细菌、大肠杆菌等。除此之外,信号肽还广泛应用于真核表达系统,如昆虫杆状病毒表达系统和毕赤酵母表达系统。由此可见,信号肽在蛋白质的分泌过程中扮演着极其重要的角色,通过分子生物学技术,优化信号肽序列将对于提高外源蛋白的分泌量及活性具有重要的实际意义。
CAR结构中scFv的N端信号肽具有三重结构,并且与分泌蛋白的信号肽具有相同的特征:疏水性α螺旋核心(H区),其N端侧翼为极性氨基酸残基(N区)。C末端侧在裂解位点(C区)的位置-1和-3处含有螺旋断裂的脯氨酸和甘氨酸残基以及小的不带电荷的残基。scFv一旦与肿瘤抗原结合,它就会触发T细胞活化并导致细胞因子释放和T细胞增殖。研究表明,信号肽能促进CAR在T细胞膜表面的表达,此外,有研究报道,增加信号肽N端的正电荷数或增加信号肽疏水核心H区的疏水性或长度有利于提高信号肽的加工效率。而距离信号肽N端较近的保守片段以及附近某些保守序列氨基酸片段的突变会对前体蛋白和分泌通路的相关蛋白的亲和性产生较大影响。因此,对信号肽结构进行优化和选择是提高CAR表达水平的重要研究内容。
发明内容
针对现有技术的问题,本发明的目的在于提供一种信号肽序列优化的靶向CD19的嵌合抗原受体,其能够使得靶向CD19的嵌合抗原受体在T细胞中更加高效、稳定表达,携带靶向CD19的ScFv序列的CAR-T细胞可以有效的杀伤表面表达CD19的肿瘤细胞。
为了实现上述技术效果,本发明提供如下的技术方案:
本发明第一方面,提供一种信号肽序列优化的靶向CD19的嵌合抗原受体,其中,该嵌合抗原受体从N端到C端顺次拼接为:优化的信号肽、抗CD19抗体单链可变区、CD8hinge、CD28跨膜区、CD28胞内结构域、胞内共刺激域4-1BB和CD3ζ链。
在一种实施方式中,所述优化的信号肽分别为SP4(未突变)、SP4K(将酪氨酸Y突变为赖氨酸K)、SP4R(将酪氨酸Y突变为精氨酸R),其核苷酸序列分别选自如SEQ ID NO.2、4、6任一所示的序列。
所述抗CD19抗体单链可变区的氨基酸序列如SEQ ID NO.7所示,CD8hinge的核苷酸序列如SEQ ID NO.9所示,CD28跨膜区、CD28胞内结构域的氨基酸序列分别如SEQ IDNO.11、SEQ ID NO.13所示,胞内共刺激域4-1BB的氨基酸序列如SEQ ID NO.15所示,CD3ζ的氨基酸序列如SEQ ID NO.17所示;此外,抗CD19抗体单链可变区的核苷酸序列如SEQ IDNO.8所示,CD8hinge的核苷酸序列如SEQ ID NO.10所示,CD28跨膜区、CD28胞内结构域的核苷酸序列分别如SEQ ID NO.12、.14所示,胞内共刺激域4-1BB的核苷酸序列如SEQ IDNO.16所示,CD3ζ的核苷酸序列如SEQ ID NO.18所示。
在另一种实施方式中,所述嵌合抗原受体的核苷酸序列选自如SEQ ID NO.20、22、24任一所示的序列。
本发明的第二个方面,提供一种表达嵌合型抗原受体的免疫细胞,其特征在于,该细胞由如上述的嵌合抗原受体转染人或其他哺乳动物的免疫细胞获得。
在一种实施方式中,所述人或其他哺乳动物的免疫细胞选自脐带血、外周血或IPSC来源的T细胞、NK细胞、NKT细胞、αβT细胞、γδT细胞、CD4+T细胞或CD8+T细胞。优选的,所述人或其他哺乳动物的免疫细胞为外周血来源的T细胞。
本发明的第三个方面,提供一种将上述嵌合型抗原受体或表达嵌合型抗原受体免疫细胞用于制备治疗或预防肿瘤的药物中的应用。
在一种实施方式中,所述肿瘤为B细胞急性淋巴细胞白血病或其他以CD19作为治疗靶点的疾病。优选的,所述其他以CD19作为治疗靶点的疾病为B细胞淋巴瘤。
相对于现有技术,本发明取得了如下的有益的技术效果:
1、在本发明中,CD19嵌合抗原受体序列经过信号肽序列优化后,在T细胞中表达更加稳定、高效,如流式免疫荧光染色图结果所示,优化信号肽后,CAR的表达效率得到了明显的提高,对CD19阳性的B细胞白血病或淋巴瘤可起到更加良好的治疗作用;
2、本发明还提供表达嵌合型抗原受体的免疫细胞的制备方法,是将分离得到的免疫细胞激活2-10天后再感染表达嵌合抗原受体的慢病毒,这样,原有免疫细胞不会影响转染后的表达嵌合型抗原受体的免疫细胞的杀瘤效果,进一步地,表达嵌合型抗原受体的免疫细胞进行体外功能检测时,所选择的细胞系为细胞膜外高表达CD19靶点的细胞系,这样对表达嵌合型抗原受体的免疫细胞的杀瘤效果评价更为科学。
附图说明
图1为SP4和其突变型慢病毒活性滴度的结果示意图;
图2为SP4和其突变型的CAR-T细胞转染效率的结果示意图;
图3(中)为SP4和其突变型的CAR-T细胞对细胞系K562-CD19的体外杀伤结果示意图;
图3(右)为SP4和其突变型的CAR-T细胞对细胞系NALM6的体外杀伤结果示意图;
图3(左)为SP4和其突变型的CAR-T细胞对阴性细胞系K562的体外杀伤结果示意图;
图4A为SP4和其突变型的CAR-T细胞与细胞系NALM6的体外共孵育后细胞因子IFN-γ释放的结果示意图;
图4B为SP4和其突变型的CAR-T细胞与细胞系NALM6的体外共孵育后细胞因子TNF-α释放的结果示意图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:靶向CD19的嵌合抗原受体慢病毒载体(PTK-EF1α-SP4-CAR、PTK-EF1α-SP4K-CAR、PTK-EF1α-SP4R-CAR)的获得
1、SP4序列为分泌型蛋白本身特有的信号肽,根据发明人前期实验研究发现,与其他天然的信号肽序列相比,SP4能够显著增强CD19-CAR的功能(李帆等,《中国癌症杂志》,2022)。本发明在此基础上,为提高信号肽N端正电荷数,将序列中的酪氨酸分别突变为赖氨酸和精氨酸,命名为SP4K和SP4R。
2、采用OverlapPCR扩增分别以SP(SP4、SP4K、SP4R)、SCFV和CD8hinge-CD28TM+ICD-4-1BB-CD3ζ为模版,获得带有酶切位点EcoRI和BamHI的SP4-CAR、SP4K-CAR、SP4R-CAR片段,其中,SP(SP4、SP4K、SP4R)、抗CD19 SCFV、CD8hinge、CD28TM+ICD、4-1BB、CD3ζ的序列如下表1所示:
表1
获得SP4-CAR、SP4K-CAR、SP4R-CAR完整的CAR结构序列,如表2所示:
表2
3、将质粒PTK-EF1α-Kan(该质粒构建方法记载于发明人在先专利ZL201611246081.9中)使用EcoRI和BamHI限制性内切酶进行双酶切,产物经过0.8%的琼脂糖凝胶电泳,并割胶回收置于Eppendorf管内,用Axygen公司的琼脂糖凝胶回收试剂盒回收相应的片段,并测定产物的纯度和浓度。
4、将片段以1:2摩尔比加入Eppendorf管加入Exnase连接酶(Vazyme)与同源重组酶5×CE buffer,37℃反应0.5小时;将连接液取出10μL加入100μLDH5α感受态细胞冰浴30min后42℃热激90s,完成后加入500μLsoc培养基37℃、220rpm培养2小时;2小时后将Eppendorf管4000g离心1min移除400μL多余液体。将剩余液体涂布在LB平板37℃培养12小时;在平板上挑取单菌落,接种到5mL LB液体培养基中37℃、220rpm培养12小时。
5、用Axygen小提试剂盒提取质粒,获得质粒PTK-EF1α-SP4-CAR、PTK-EF1α-SP4K-CAR、PTK-EF1α-SP4R-CAR;送生工生物工程(上海)股份有限公司科技公司一代测序验证无误后,进行含质粒PTK-EF1α-SP4-CAR、PTK-EF1α-SP4K-CAR、PTK-EF1α-SP4R-CAR的大肠杆菌DH5α菌株保种。
实施例2、质粒的制备与测序
1、质粒的制备
将含质粒PTK-EF1α-SP4-CAR、PTK-EF1α-SP4K-CAR、PTK-EF1α-SP4R-CAR的大肠杆菌DH5α菌种分别接种至250mL含100μg/mL卡那霉素的LB培养液中,37℃、220rpm培养过夜。培养液在4℃于6000g离心20min,弃上清。
取出Endo Free plasmid mega kit(Qiagen)中的BuffersP1,向离心得到的大肠杆菌沉淀中加120mL提前预冷的Buffers P1,盖上离心瓶盖,剧烈振荡离心瓶使大肠杆菌沉淀在Buffers P1中完全分散。
向离心瓶中加120mLBuffers P2,盖上瓶盖放置在滚轴混匀仪上,慢慢提速至50rpm,彻底混匀后室温放置5min。
向离心瓶中加120mLBuffers P3,盖上瓶盖放置在滚轴混匀仪上,慢慢提速至滚轴混匀仪最大转速70rpm,彻底混匀直至呈白色不粘稠蓬松的混合液。在4℃于9000g离心15min。
向QIA filter Cartridge倒入50mLBuffer FW,将离心所得上清液倒入QIAfilter Cartridge中,轻轻地搅拌混匀。将混合液抽滤入已标记好对应的玻璃瓶中。
向每个玻璃瓶中加入20mL Buffer ER,上下颠倒混匀6次,在-20℃孵育30min。
将标记好的mega柱放入对应的架子上,向每个mega柱内加入35mL Buffers QBT平衡,重力作用使之流尽。
将玻璃瓶中的液体分批全部倒入对应标记的mega柱中,待柱中液体流尽后,向每个mega柱分批加入200mL Buffer QC进行清洗。待柱中液体流尽后,将废液收集盘中的废液倒入50mL洁净离心管内。
再向每个mega柱内加入40mL Buffer QN,使用50mL洁净离心管收集流出液,上下颠倒6次混匀,分装20mL至另一洁净已标记的50mL离心管内。
向每个50mL离心管加入14mL异丙醇(常温),上下颠倒6次混匀。在4℃于15000g离心50min。
超净工作台内吸尽上清,每管加入3.5mL Endotoxin-freewater漂洗,不要将底部沉淀冲散。在4℃于15000g离心30min。将Endo Free plasmid mega kit中的Buffer TE放入烘箱内预热。
在超净工作台内吸尽离心后的上清,于超净工作台内吹干(挥发残留的无水乙醇,时间在10min左右)。
在烘箱内拿出Buffer TE,在超净工作台内向每管加入1mL Buffer TE,用枪吹打10次后放入65℃烘箱,期间不间断地敲击管壁促使沉淀完全溶解。在4℃于4000g离心1min将管壁上的液体甩到管底后吹打混匀。
在超净工作台内将液体全部转移至无内毒素无热源无核酸酶对应标记的EP管中。吸出2μL,用微量分光光度计测质粒浓度,并标记在对应的EP管上,获得质粒PTK-EF1α-SP4-CAR、PTK-EF1α-SP4K-CAR、PTK-EF1α-SP4R-CAR。
2、目的基因测序
分别取20μL(500ng)质粒DNA,外送测序,根据原始种子序列,检查质粒生产所得产品的目的基因有无发生改变,稳定的工艺下,工作种子在进行发酵培养放大过程中,目的基因不会发生改变,可用于下一环节的生产和正确表达蛋白。
实施例3、PTK-EF1α-SP4-CAR、PTK-EF1α-SP4K-CAR、PTK-EF1α-SP4R-CAR慢病毒载体的制备与活滴检测
1、慢病毒载体的制备
在多层细胞培养瓶(Hyperflask)接入130.0~140.0×106数目的293T细胞(Takara),共560mL DMEM完全培养基(50mL胎牛血清、5mL Antibiotic-Antimycotic(100×),在37℃含5%CO2培养箱中培养24小时。分别将混有320μg质粒(PTK-EF1α-SP4-CAR/PTK-EF1α-SP4K-CAR/PTK-EF1α-SP4R-CAR∶BZ1质粒∶BZ2质粒∶BZ3质粒=12∶10∶6∶5,该三种质粒构建方法记载于发明人在先专利ZL201611246081.9中)的DMEM完全培养基加入960μgPEI管中,漩涡震荡,室温平衡10min。分别将上述35mL PEI与质粒的混合液与525mL DMEM完全培养基混匀,换入上述多层细胞培养瓶中。将多层细胞培养瓶置于37℃含5%CO2培养箱培养3天后,收集细胞培养上清液。
分别将上清液4000rpm(或3000g)离心30min后,向离心后上清中加入cryonase酶(Takara)置于4℃。6个小时后,使用0.22μm的滤膜对慢病毒上清液进行抽滤,4℃于30000g离心2.5h。去除上清,加入1mL T细胞培养基重悬沉淀。重悬后,留20μL做病毒活性滴度检测,剩余慢病毒浓缩液分装,标记为Lenti3-SP4-CAR、Lenti3-SP4K-CAR、Lenti3-SP4R-CAR并置于-80℃保存备用。
2、慢病毒载体活性滴度检测
原理:PE-Labeled Human CD19 Protein上标记有荧光素,而PE-Labeled HumanCD19 Protein能与CAR中ScFv特异性结合,通过流式细胞仪检测到的荧光信号间接反应了CAR在293T细胞中的表达情况。
方法:在6孔板中接入5.0×105个/孔293T细胞,慢病毒浓缩液每孔分别加入0.5μL、1μL、2μL、5μL、10μL,并设1个阴性对照。置于37℃含5%CO2培养箱内培养。三日后,用Versene溶液(Gibco)收集293T细胞送流式细胞学检测CAR阳性293T细胞比例,并换算得到Lenti3-SP4-CAR、Lenti3-SP4K-CAR、Lenti3-SP4R-CAR慢病毒浓缩液活性滴度。
目前的慢病毒浓缩液活性滴度在1×108~10×108(TU/mL)范围内,检测分析结果见图1和表2。表明各个慢病毒载体均能获得较高的活性滴度,可用于后续的嵌合抗原受体免疫细胞的制备。
表2慢病毒活性滴度检测分析结果
样品编号 | 活性滴度(TU/mL) |
Lenti3-SP4-CAR | (3.2±0.14)×108 |
Lenti3-SP4K-CAR | (3.6±0.10)×108 |
Lenti3-SP4R-CAR | (3.8±0.13)×108 |
实施例4、SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞的制备
1、CAR-T细胞制剂制备:
采集健康供者外周血100mL,采用Ficoll淋巴细胞分离液分离单个核细胞。计数后,使用适量CD3 MicroBeads,human(美天旎)分选CD3阳性细胞,并以1.0~2.0×106个/mL密度在T细胞完全培养液(OpTmizerTMCTSTMT-Cell Expansion Basal Medium,OpTmizerTMCTST-Cell Expansion Supplement(Invitrogen),500IU/mL的IL-2(双鹭药业))中培养,同时按每106个细胞加入25μL Dynabeads HumanT-Activator CD3/CD28(Invitrogen)活化T细胞。
24小时(Day1)后,按MOI为5分别加入Lenti3-SP4-CAR、Lenti3-SP4K-CAR、Lenti3-SP4R-CAR慢病毒载体进行转导,混匀后置于CO2培养箱孵育,4小时后补加适量的T细胞完全培养基进行培养。
慢病毒转导24小时后将转导后的细胞换入新鲜T细胞完全培养液,并调整活细胞密度为1.0-2.0×106/mL,继续培养扩增10~20天,每天进行观察和计数,并根据计得的细胞数量进行补液扩大培养,始终保持细胞培养密度为1.0-2.0×106/mL。
2、SP4CAR-T、SP4KCAR-T、SP4RCAR-T细胞转导效率检测
取1.0×106个转导后T细胞,与1μL PE-Labeled Human CD19 Protein室温孵育30分钟,生理盐水清洗两次后,通过流式细胞仪检测PE荧光信号,测量PE阳性细胞比率,反映了CAR-T细胞在总细胞中的比率。SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞转导效率检测结果如图2和表3所示。表3表明成功制备了CAR-T细胞且SP4K/SP4R CAR-T细胞的CAR的表达效率显著高于SP4-CAR-T细胞的CAR的表达效率,此结果说明,不同的信号肽经过优化后对于 嵌合抗原受体的表达影响存在差异,SP4K和SP4R信号肽的优化可以显著提高CD19嵌合抗原 受体在T细胞的表达。
表3 CAR-T细胞转导效率检测结果
编号 | 转导类型 | 表达效率 |
1 | SP4CAR-T | 33.9% |
2 | SP4KCAR-T | 35.5% |
3 | SP4RCAR-T | 36.8% |
实施例5、SP4CAR-T、SP4KCAR-T、SP4RCAR-T体外功能检测
1.体外杀瘤检测:
采用钙黄绿素检测法分别对T、SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞进行体外杀瘤功能检测。
靶细胞为K562(CD19阴性细胞系)、K562-CD19、NALM6肿瘤细胞系。取适量的靶细胞,在1×106/mL的细胞悬液(PBS,5%胎牛血清)加入钙黄绿素-乙酰羟甲基酯(Calcein-AM)至终浓度25μM,培养箱中孵育30min。常温,洗两遍后将细胞重悬至0.5×105/mL,96孔板中每孔加入0.5×105/mL个细胞,按25∶1的效靶比分别加入普通T细胞、SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞,37℃孵育2~3小时。孵育完成后取上清,测量其中钙黄绿素的荧光强度,并根据自发释放对照和最大释放对照,计算靶细胞裂解百分数。
普通T细胞、SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞对CD19高表达K562-CD19细胞系体外杀伤裂解结果如图3(中)所示,对CD19高表达NALM6细胞系(人急性淋巴细胞白血病细胞系)体外杀伤裂解结果如图所3(右)所示,对CD19不表达K562细胞系的体外杀伤裂解结果如图3(左)所示,结果显示,SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞靶向性裂解能力与T细胞相比均有提高,说明其具有对K562-CD19、NALM6细胞系的体外杀伤性功能。并且,在与K562-CD19、NALM6细胞系共孵育体系中,SP4R-CAR-T细胞的靶向性裂解能力要明显高于SP4-CAR-T、SP4K-CAR-T细胞。由以上体外杀瘤结果可知,优选SP4构建的SP4R-CAR-T细胞,用于治疗肿瘤。
2.体外细胞因子检测:
取适量的靶细胞,在1×106/mL的细胞悬液(PBS,5%胎牛血清)常温,洗两遍后将细胞重悬至0.5×105/mL,96孔板中每孔加入0.05×105/mL个靶细胞,按25∶1的效靶比加入T、SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞,200g离心30秒,37℃孵育18小时。孵育完成后取上清,测量其中IFN-γ和TNF-α的浓度。
普通T细胞、SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞与CD19高表达NALM6肿瘤细胞系体外孵育后IFN-γ和TNF-α分泌结果如图4所示,与杀瘤结果一致,IFN-γ和TNF-α的浓度结果显示,SP4-CAR-T、SP4K-CAR-T、SP4R-CAR-T细胞分泌的IFN-γ和TNF-α与普通T细胞相比均显著提高,进一步说明其对急性淋巴细胞白血病的体外杀伤性功能。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (9)
1.一种信号肽优化的靶向CD19的嵌合抗原受体,其特征在于,从N端到C端顺次拼接为:优化的信号肽、抗CD19抗体单链可变区、CD8hinge、CD28跨膜区、CD28胞内结构域、胞内共刺激域4-1BB和CD3ζ链;所述优化的信号肽的核苷酸序列选自如SEQ ID NO.2、4、6任一所示的序列。
2.如权利要求1所述的嵌合抗原受体,其特征在于,所述抗CD19抗体单链可变区的氨基酸序列如SEQ ID NO.7所示,CD8hinge的氨基酸序列如SEQ ID NO.9所示,CD28跨膜区、CD28胞内结构域的氨基酸序列分别如SEQ ID NO.11、SEQ ID NO.13所示,胞内共刺激域4-1BB的氨基酸序列如SEQ ID NO.15所示,CD3ζ的氨基酸序列如SEQ ID NO.17所示。
3.如权利要求1或2所述的嵌合抗原受体,特征在于,抗CD19抗体单链可变区的核苷酸序列如SEQ ID NO.8所示,CD8hinge的核苷酸序列如SEQ ID NO.10所示,CD28跨膜区、CD28胞内结构域的核苷酸序列分别如SEQ ID NO.12、.14所示,胞内共刺激域4-1BB的核苷酸序列如SEQ ID NO.16所示,CD3ζ的核苷酸序列如SEQ ID NO.18所示。
4.如权利要求1或2所述的嵌合抗原受体,其特征在于,所述嵌合抗原受体的氨基酸序列选自SEQ ID NO.19、21、23任一所示的序列,所述嵌合抗原受体的核苷酸序列选自SEQ IDNO.20、22、24任一所示的序列。
5.一种表达嵌合型抗原受体的免疫细胞,其特征在于,该细胞由权利要求1-4任一所述的嵌合抗原受体转染人或其他哺乳动物的免疫细胞获得。
6.如权利要求5所述的免疫细胞,其特征在于,所述人或其他哺乳动物的免疫细胞选自脐带血、单采血、外周血或IPSC来源的T细胞、NK细胞、NKT细胞、αβT细胞、γδT细胞、CD4+T细胞或CD8+T细胞。
7.如权利要求6所述的免疫细胞,其特征在于,所述人或其他哺乳动物的免疫细胞为外周血来源的T细胞。
8.一种将如权利要求1-4任一所述的嵌合型抗原受体、权利要求5或6所述的表达嵌合型抗原受体免疫细胞用于制备治疗或预防肿瘤的药物中的应用。
9.如权利要求8所述的应用,其特征在于,所述肿瘤为B细胞急性淋巴细胞白血病或淋巴瘤。
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