CN107058354A - 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞 - Google Patents
编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞 Download PDFInfo
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Abstract
编码一种表达于人T细胞表面的嵌合抗原受体蛋白的核酸，所述嵌合抗原受体蛋白包含顺序连接的胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别人表皮生长因子受体EGFR的第287‑302位氨基酸表位的单链抗体scFv(EGFR)。
Description
技术领域
本申请为CN201310108532.2的分案申请，原申请的申请日为2013年04月01日，发明名称为“编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的T淋巴细胞”。
本发明涉及肿瘤细胞治疗领域，更具体地，涉及对表达EGFRvIII或高表达EGFR的上皮来源的肿瘤的转基因T淋巴细胞治疗领域。
背景技术
表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)是一种跨膜糖蛋白，分子量为170KD，是原癌基因C-erbB-1(HER-1)的表达产物，广泛分布于人体各组织的细胞膜上[Alan Wells.Molecules in focus EGF receptor.Int J Biochem Cell Biol,1999,31:637-643.]。其在大多数肿瘤中(如非小细胞肺癌、膀胱癌、卵巢癌，乳腺癌、头颈部鳞癌、胶质细胞瘤、胰腺癌、食管癌、胃癌、前列腺癌等)过表达和(或)突变，与肿瘤的发生发展、恶性变、转移及预后密切相关[Jose B.Why the epidermal growth factor receptor？The rational for cancer therapy[J].Oncologist,2002,7(4):2-8.]。因此，EGFR是肿瘤治疗的一个重要靶点。另外，研究表明EGFR287-302表位仅在EGFRvIII或过表达EGFR的肿瘤中才暴露，而在正常组织中该表位隐匿[Gan HK,et.al.Targeting of aconformationally exposed,tumor-specific epitope of EGFR as a strategy forcancer therapy.Cancer Res,2012,72(12):2924-2930.]。提示EGFR287-302表位是靶向EGFR的相关肿瘤治疗的一个理想位点。
针对EGFR287-302表位的抗体已经被开发，其显示良好的肿瘤特异性杀伤作用。但抗体治疗存在着抗体在体内血液循环中半衰期的限制，一般来说，半衰期大多在23天以内。因此，持续给药和/或增大给药剂量是肿瘤抗体治疗所要求的，这导致患者治疗成本的增加，以及某些情况下甚至不得已终结治疗。另外，治疗性抗体作为异源蛋白，还有可能在体内产生过敏反应及针对该治疗性抗体的中和性抗抗体的风险。
T淋巴细胞在肿瘤免疫应答中的作用日益受到重视。基于T淋巴细胞的过继性免疫治疗在部分肿瘤中取得了一定的效果，并且该种免疫治疗方法可以克服抗体治疗的上述缺陷，但在大多数肿瘤的疗效仍不能令人满意[Grupp SA,et.al.Adoptive cellulartherapy.Curr Top Microbiol Immunol.2011,344:149-72.]。近年来，根据细胞毒性T淋巴细胞对靶细胞的识别特异性依赖于T细胞受体(T Cell Receptor,TCR)的发现，将针对肿瘤细胞相关抗原的抗体的scFv与T细胞受体的CD3ζ或FcεRIγ等胞内信号激活基序融合成嵌合抗原受体(Chimeric antigen receptor,CAR)，并将其通过如慢病毒感染等方式基因修饰在T细胞表面。这种CAR T细胞能够以主要组织相容性复合物(MajorHistocompatibility Complex,MHC)非限制性方式选择性地将T淋巴细胞重新定向到肿瘤细胞并特异性地杀伤肿瘤。CAR T细胞是肿瘤免疫治疗领域的一个新的免疫治疗策略[Schmitz M,et.al.Chimeric antigen receptor-engineered T cells foriImmunotherapy of Cancer.J Biomed Biotechnol,2010,doi:10.1155/2010/956304.]。
嵌合抗原受体包括胞外结合区，跨膜区和胞内信号区。通常胞外区包含能够识别肿瘤相关抗原的scFv，跨膜区采用CD8，CD28等分子的跨膜区，胞内信号区采用免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif,ITAM)如CD3ζ(即CD3zeta，简称Z)或FcεRIγ及共刺激信号分子CD28、CD137、CD134等的胞内信号区。
胞内信号区仅包含ITAM的为第一代CAR T细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD3ζ。该种CAR T细胞可以激发抗肿瘤的细胞毒性效应，但是细胞因子分泌比较少，并且在体内不能激发持久的抗肿瘤效应[Zhang T.et.al.Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a mannerinvolving multiple cytokines and cytotoxic pathways,Cancer Res 2007,67(22):11029–11036.]。
随后发展的第二代CAR T细胞加入了CD28或CD137(又名4-1BB)的胞内信号区，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-ITAM或scFv-TM-/CD137-ITAM。胞内信号区发生的B7/CD28或4-1BBL/CD137共刺激作用引起T细胞的持续增殖，并能够提高T细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR T在体内的存活周期和抗肿瘤效果[Dotti G.et.al.CD28costimulation improves expansion and persistence ofchimeric antigen receptor modified T cells in lymphoma patients.J ClinInvest,2011,121(5):1822-1826.]。
近些年发展的第三代CAR T细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-CD137-ITAM或scFv-TM-CD28-CD134-ITAM，进一步提高了CAR T在体内的存活周期和其抗肿瘤效果[Carpenito C.,et al.Control of large established tumorxenografts with genetically retargeted human T cells containing CD28andCD137domains.PNAS,2009,106(9):3360–3365.]。
尽管CAR T细胞在肿瘤免疫治疗中具有诱人的前景，但一些潜在的风险亦需要考虑。比如，由于某些/种正常组织低表达CAR所能识别的特异性抗原可能造成CAR T细胞对表达相应抗原的正常组织的损伤。如，针对肾细胞癌患者肿瘤细胞上表达的抗原碳酸酐酶IX(CAIX)是第一个用于临床的CAR T细胞过继治疗的案例，也是第一个报道含CAR细胞的脱靶效应的案例。病人在多次输入CAR T细胞后出现2-4级肝毒性。分析原因为肝胆管上皮细胞低表达CAIX，原临床试验被迫中断同时排除了病人治疗效果的任何评价。.[Stoter G.etal.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytesgenetically retargeted against carbonic anhydrase IX:first clinicalexperience.J clin oncol,2006,24(13):e20-e22.；Ngo MC.,et al.Ex vivo genetransfer for improved adoptive immunotherapy of cancer.Human MolecularGenetics,2011,R1–R7]。另外，CAR中过多的共刺激信号会降低效应细胞激活所需的阈值，使得基因修饰的T细胞在低水平抗原或没有抗原触发的条件下也可能会被活化，导致大量细胞因子的释放以致可能引发所谓的“细胞因子风暴”。这种信号外漏(singnal leakage)会导致脱靶细胞毒性，从而产生非特异性的组织损伤。例如，在采用针对Her2的第三代CAR临床治疗一个具有肝和肺转移的晚期结肠癌患者的过程中由于正常肺组织中低表达Her2而引发所谓的“细胞因子风暴”致病人猝死[Morgan RA.,et al.Report of a seriousadverse event following the administration of T cells transduced with achimeric antigen receptor recognizing Erbb2.Molecular Therapy,2010,18(4):843–851.]。
因此，本领域存在着对克服上述缺陷的CAR T淋巴细胞肿瘤治疗方案的强烈需求。
发明内容
本发明的第一方面涉及编码一种表达于T细胞表面的嵌合抗原受体蛋白的核酸，所述嵌合抗原受体蛋白包含顺序连接的胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别人表皮生长因子受体EGFR的第287-302位氨基酸(EGFR287-302)表位的单链抗体scFv(EGFR)。上述嵌合抗原受体蛋白的胞外结合区通过CD8铰链区与CD8或者CD28的跨膜区相连接，跨膜区后紧接胞内信号区。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明的编码嵌合抗原受体蛋白氨基酸序列的核酸密码子可以是简并的，即编码同一氨基酸序列的多种简并核酸序列都包含在本发明的范围之中。编码对应氨基酸的简并核酸密码子是本领域公知的。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％，最佳地至少90％或至少95％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与SEQ ID NO:2所示的成熟多肽有相同的生物学功能和活性
特异性识别人表皮生长因子受体EGFR的第287-302位氨基酸表位的单克隆抗体在中国专利文献CN102405235和CN101602808B中已有公开，其他已知和将来可知的特异性识别该表位的单克隆抗体也可以用于制备本发明的核酸所编码的嵌合抗原受体蛋白中的单链抗体。单链抗体可以根据上述文献公开的序列通过基因工程方法或化学合成方法制备。本发明中使用的术语“单链抗体(scFv)片段”指的是通过如下定义的抗体片段，其是包含通过接头(linker)连接的重链可变区(VH)和轻链可变区(VL)的重组蛋白，接头使得这两个结构域相关联，以最终形成抗原结合位点。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。本发明使用的单链抗体可单独或联合使用本领域已知的常规技术，例如氨基酸缺失、插入、取代、增加、和/或重组以及/或其他修饰方法作进一步修饰。根据一种抗体的氨基酸序列在其DNA序列中引入这种修饰的方法对本领域技术人员来说是众所周知的；见例如，Sambrook，分子克隆：实验手册，Cold SpringHarbor Laboratory(1989)N.Y.。所指的修饰优选在核酸水平上进行。上述单链抗体还可以包括其衍生物。上述单链抗体衍生物包括但不限于例如WO 89/09622中描述的嵌合抗体的产生的方法，EP-A10239400和WO90/07861中描述的人源化抗体产生的方法，WO91/10741,WO94/02602和WO96/33735中有描述的产生异种抗体例如小鼠中的人抗体的方法。本发明的术语“特异性识别”的意思是本发明的双特异性抗体不与或基本上不与目标抗原以外的任意多肽交叉反应。其特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。在本发明中，特异性识别优选通过流式细胞技术来确定，而具体情况下特异性识别的标准可由本领域一般技术人员根据其掌握的本领域常识来判断。
跨膜区可以选自CD8或CD28等蛋白的跨膜区。CD8或CD28是T细胞表面的天然标记物。人CD8蛋白是个异二聚体，由αβ或者γδ两条链组成，本发明的一个实施方案中，跨膜区选自CD8α或者CD28的跨膜区。此外，CD8α铰链区(hinge)是一个柔性区域，因此，CD8或CD28和跨膜区和铰链区可以用来连接嵌合抗原受体CAR的靶点识别结构域scFv和胞内信号区。
胞内信号区可以选自CD3ζ，FcεRIγ，CD28，CD137，CD134蛋白的胞内信号区，及其组合。CD3分子由五个亚单位组成，其中CD3ζ亚单位(又称CD3zeta，简称Z)含有3个ITAM基序，该基序是TCR-CD3复合体中重要的信号转导区。CD3δZ是突变的不具有ITAM基序的CD3ζ序列，在本发明实践中一般作为阴性对照的构建。FcεRIγ主要分布在肥大细胞和嗜碱性粒细胞表面，其含有一个ITAM基序，在结构、分布及功能上与CD3ζ类似。此外如前所述，CD28，CD137，CD134是共刺激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起T细胞的持续增殖，并能够提高T细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR T细胞在体内的存活周期和抗肿瘤效果。
本发明的核酸所编码的嵌合抗原受体蛋白可以是选自包含顺序连接的胞外结合区，
跨膜区和胞内信号区的如下的嵌合抗原受体蛋白：
scFv(EGFR)-CD8-CD3ζ，
scFv(EGFR)-CD8-CD137-CD3ζ，
scFv(EGFR)-CD28-CD28-CD3ζ，
scFv(EGFR)-CD28-CD28-CD137-CD3ζ，
及其组合，其中相关嵌合抗原受体蛋白中的第一个CD28代表其跨膜区，第二个CD28代表其胞内信号区。
在本发明的一个实施方案中，本发明的核酸具有如SEQ ID NO:1～4所述的序列。在本发明的另一个实施方案中，本发明的核酸是编码具有如SEQ ID NO:31～34之一的嵌合抗原受体蛋白的核酸。
本发明的第二方面包括包含上述编码一种表达于T细胞表面的嵌合抗原受体蛋白的核酸的载体。在一个具体实施方案中，本发明使用的载体是一种慢病毒质粒载体pPWT-eGFP。该质粒属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2；编码VSV-G蛋白的包膜质粒PMD2.G；及空载体pPWT-eGFP，其可以用于重组引入目的核酸序列，即编码CAR的核酸序列。空载体pPWT-eGFP(其本身为后续试验中的mock)中由延长因子-1α(elongation factor-1α，EF-1α)启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达。而包含编码CAR的目的核酸序列的重组表达载体pWPT-eGFP是通过由来自口蹄疫病毒(food andmouthvires disease，FMDV)的核糖体跳跃序列(ribosomal skipping sequence 2A)(简称F2A)实现eGFP与CAR的共表达的。
本发明的第三方面包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其他转染T细胞的病毒及其对应的质粒载体也可用于本发明。
在本发明的一个实施方案中，所述病毒是包含上述pWPT-eGFP-F2A-CAR重组载体(即含有scFv(EGFR)-CAR)的慢病毒。
本发明的第四方面包括一种转基因T淋巴细胞，其被转导有本发明的核酸或被转导有本发明的上述包含所述含有该核酸的重组质粒，或包含该质粒的病毒系统。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。基于非病毒的转导方法包括电穿孔法和转座子法。近期Amaxa公司研发的nucleofector核转染仪能够直接将外源基因导入细胞核获得目的基因的高效转导。另外，基于睡美人转座子(SleepingBeauty system)或PiggyBac转座子等转座子系统的转导效率较普通电穿孔有较大提高，将nucleofector转染仪与SB睡美人转座子系统联合应用已有报道[Davies JK.,etal.Combining CD19redirection and alloanergization to generate tumor-specifichuman T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res,2010,70(10):OF1-10.]，该方法既能够具有较高的转导效率又能够实现目的基因的定点整合。在本发明的一个实施方案中，实现嵌合抗原受体基因修饰的T淋巴细胞的转导方法是基于病毒如逆转录病毒或慢病毒的转导方法。该方法具有转导效率高，外源基因能够稳定表达，且可以缩短体外培养T淋巴细胞到达临床级数量的时间等优点。在该转基因T淋巴细胞表面，转导的核酸通过转录、翻译表达在其表面。通过对各种不同的培养的肿瘤细胞进行体外细胞毒实验证明，本发明的表面表达嵌合抗原受体的转基因T淋巴细胞具有高度特异性的肿瘤细胞杀伤效果(亦称细胞毒性)。因此本发明的编码嵌合抗原受体蛋白的核酸，包含该核酸的质粒，包含该质粒的病毒和转染有上述核酸，质粒或病毒的转基因T淋巴细胞可以有效地用于肿瘤的免疫治疗。
附图说明
图1显示的是作为示例的本发明的包含编码CAR序列的慢病毒载体pWPT-eGFP-F2A-CAR的结构示意图。
图2显示的是作为示例的包含在慢病毒载体中的本发明的CAR的不同区之间连接关系的示意图,其中由核糖体跳跃序列F2A连接的eGFP和svFv(EGFR)特异性嵌合抗原受体
图3显示的是MluI和SalI双酶切鉴定实施例1的慢病毒质粒的核酸电泳图。其中M1是DS2000分子量标记物(广州东盛生物科技有限公司)；M2是Hind III标记物(广州东盛生物科技有限公司)。泳道1-6分别是1:pWPT-eGFP；2:pWPT-eGFP-F2A-806-δZ；3:pWPT-eGFP-F2A-806-Z；4:pWPT-eGFP-F2A-806-BBZ；5:pWPT-eGFP-F2A-806-28Z；6:pWPT-eGFP-F2A-806-28BBZ；。
图4显示的是本发明实施例2的病毒感染CD8+T淋巴细胞后细胞表达的eGFP的流式细胞技术检测结果
图5显示的是本发明实施例2的表达不同嵌合抗原受体(CAR+)的CD8+T淋巴细胞的体外生长情况。图中显示在病毒感染后第14天，表达不同嵌合抗原受体的CD8+T体外扩增35-50倍。
图6显示的是用于本发明实施例3的各种肿瘤细胞系表面EGFR287-302表位的表达情况的流式细胞技术检测结果
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不能理解为用于限制本发明的范围。下面实施例中如未注明具体条件的实验方法，则按照常规条件如Sambrook等，“分子克隆：实验手册(New York：Cold Spring Harborlaboratory Press，1989)”中所述的条件，而在实施例中明确说明有试剂公司说明书时，则按照说明书所建议的条件进行。
实施例1.表达本发明的嵌合抗原受体的慢病毒质粒的构建
下表1解释了本发明示例的嵌合抗原受体各部分的连接顺序，该连接还可参见图2中所示。
表1
| 嵌合抗原受体 | 胞外结合区-跨膜区-胞内信号区1-胞内信号区2etc | 描述 |
| 806-δZ | scFv(EGFR)-CD8-CD3δzeta | 阴性对照 |
| 806-Z | scFv(EGFR)-CD8-CD3zeta | 第一代 |
| 806-BBZ | scFv(EGFR)-CD8-CD137-CD3zeta | 第二代 |
| 806-28Z | scFv(EGFR)-CD28-CD28-CD3zeta | 第二代 |
| 806-28BBZ | scFv(EGFR)-CD28-CD28-CD137-CD3zeta | 第三代 |
1.核酸片段的扩增
(1)scFv(EGFR)序列的扩增
scFv(EGFR)序列的扩增以本实验室构建的单链双功能抗体核苷酸806/CD3或hu7B3/CD3为模板，模板的序列分别参见中国专利申请201210094008.x中的SEQ ID NO：10和11。扩增所采用的引物对为上游引物5’-gacatcctgatgacccaatctccatcctc-3’(SEQ IDNO:5)和下游引物5’-tgcagagacagtgaccagagtcccttgg-3’(SEQ ID NO:6)用于扩增806scFv(EGFR)；以及上游引物5’-gatattcagatgacccagagcccg-3’(SEQ ID NO:7)和下游引物5’-gctgctcacggtcaccagggtg-3’(SEQ ID NO:8)用于扩增hu7B3scFv(EGFR)。在两种情况下，目的扩增条带大小均为720bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行25个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
阴性对照scFv(CD19)的序列根据GenBank的FMC63-28Z(HM852952.1)序列确定，序列由上海锐劲生物技术有限公司通过全基因合成获得。
(2)嵌合抗原受体其他部分的核酸序列
嵌合抗原受体蛋白的其他部分及连接这些部分的铰链区的扩增如下：加1mlTrizol(Invitrogen公司)于1×107健康人外周血单个核细胞(上海市血液中心提供)中裂解细胞后，采用酚-氯仿法抽提总RNA，采用ImProm-IITM逆转录试剂盒(promaga公司)逆转录制备cDNA。以上述制备的cDNA为模板，分别：
(a)以上游引物5’-cactgtctctgcaaccacgacgccagcg-3’(SEQ ID NO:9)和下游引物5’-ggtgataaccagtgacaggag-3’(SEQ ID NO:10)扩增获得CD8α铰链区-跨膜区，PCR扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：58℃，30s；延伸：68℃，30s；进行25个循环，然后总延伸68℃，10min。条带理论大小为198bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(b)以上游引物5’-cactgtctctgcaaccacgacgccagcg-3’(SEQ ID NO:11)和下游引物5’-gaggtcgacctacgcgggggcgtctgcgctcctgctgaacttcactctggtgataaccagtg-3’(SEQ IDNO:12)扩增获得CD8α铰链区-跨膜区-delta Z(δZ)，PCR扩增条件同上。条带理论大小为234bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(c)以上游引物5’-ttttgggtgctggtggtggttgg-3’(SEQ ID NO:13)和下游引物5’-gctgaacttcactctggagcgataggctgcgaag-3’(SEQ ID NO:14)扩增获得CD28跨膜区-胞内信号区片段，PCR扩增条件同上，条带理论大小为465bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(d)以上游引物5’-aaacggggcagaaagaaactc-3’(SEQ ID NO:15)和下游引物5’-cagttcacatcctccttc-3’(SEQ ID NO:16)扩增获得CD137胞内区，PCR扩增条件同上,条带理论大小为126bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(e)以上游引物5’-cactggttatcaccagagtgaagttcagcaggagc-3’(SEQ ID NO:17)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:18)扩增获得CD3zeta信号区，PCR扩增条件同上，条带理论大小为339bp，扩增产物经琼脂糖电泳确认与理论大小一致。
2.核酸片段的拼接
(a)以上游引物5’-accacgacgccagcgccg-3’(SEQ ID NO:19)和下游引物5’-cacccagaaaataataaag-3’(SEQ ID NO:20)拼接获得CD8α铰链区-CD28跨膜区，拼接条件：CD8α铰链区(50ng)+CD28跨膜区(50ng)预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行5个循环，然后总延伸68℃，10min，补充DNA聚合酶及上下游引物后PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，30s；退火：60℃，30s；延伸：68℃，30s，进行25个循环，然后总延伸68℃，10min。理论大小为216bp。扩增产物经琼脂糖电泳确认与理论大小一致。
(b)以上游引物5’-agagtgaagttcagcaggagcgcag-3’(SEQ ID NO:21)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:18)拼接扩增获得4-1BB胞内信号区和CD3zeta，即为BBZ，拼接和PCR扩增条件同上。条带理论大小为465bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(c)以上游引物5’-cactgtctctgcaaccacgacgccagcg-3’(SEQ ID NO:22)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:18)将等摩尔的CD8α铰链区-跨膜区与CD3zeta(约50ng)拼接及PCR获得CD8-CD3zeta(即CD8-Z)，拼接和PCR扩增条件同上。理论大小为537bp。扩增产物经琼脂糖电泳确认与理论大小一致。
(d)以上游引物5’-cactgtctctgcaaccacgacgccagcg-3’(SEQ ID NO:23)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:18)将CD8α铰链区-跨膜区与BBZ拼接及PCR获得目的片段：CD8-CD137-CD3zeta(即CD8-BBZ)，拼接和PCR扩增条件同上。理论大小为663bp。扩增产物经琼脂糖电泳确认与理论大小一致。
(e)以上游引物5’-accacgacgccagcgccg-3’(SEQ ID NO:24)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:18)将CD8铰链区-CD28跨膜区与Z采用同上的拼接和PCR扩增获得目的片段：CD8铰链区-CD28跨膜区-28Z胞内区)，拼接和PCR扩增条件同上。条带理论大小为678bp，扩增产物经琼脂糖电泳确认与理论大小一致。
(f)以上游引物5’-accacgacgccagcgccg-3’(SEQ ID NO:19)和下游引物5’-cgaggtcgacctagcgagggggcagggcctgcatg-3’(SEQ ID NO:18)将CD8铰链区与PCR获得的CD28跨膜区-胞内信号区片段与BBZ拼接，获得目的片段：CD8铰链区-CD28TM-28BBZ，其理论大小为804bp，拼接扩增产物经琼脂糖电泳确认与理论大小一致。
(g)以等摩尔的上述扩增得到的分别带有铰链区和跨膜区的δZ、Z及28BBZ核酸片段，分别与等摩尔的单链抗体核酸序列scFv806或者scFvCD19(质量约为50ng)进行拼接并PCR，条件同上述片段拼接和PCR扩增，以图2所示的模式拼接获得编码嵌合抗体806-δZ、806-Z、806-BBZ、806-28Z及806-28BBZ的核酸序列。
3.质粒载体的构建
本实施例使用的载体系统属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2；编码VSV-G蛋白的包膜质粒PMD2.G及基于空载体pPWT-eGFP的编码目的基因CAR的重组表达载体。
在空载体pPWT-eGFP中，延长因子-1α(elongation factor-1α，EF-1α)的启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达，而在编码目的基因CAR的重组表达载体中通过来自口蹄疫病毒的核糖体跳跃序列(food and mouthvirus disease，FMDV,ribosomal skipping sequence,F2A)实现eGFP与目的基因CAR的共表达。F2A是来自口蹄疫病毒的2A(或称为“自剪切多肽2A”)的一段核心序列，具备2A的“自剪切”功能，可以实现上游和下游基因共表达。2A由于其剪切效率高、上下游基因表达平衡性高及自身序列短小的优点为构建基因治疗多顺反子载体提供了一种有效的可行策略。尤其在基于嵌合抗原受体基因修饰T淋巴细胞的免疫治疗中多应用该序列实现目的基因与GFP或者eGFP的共表达，通过检测GFP或者eGFP即可间接检测CAR的表达。
本实施例构建了由F2A相连的eGFP与特异性CAR共表达的慢病毒表达载体，统称为pWPT-eGFP-F2A-CAR。拼接eGFP-F2A-CAR各部分的方法如下：
通过引物拼接的方法获得包含F2A(66bp)-CD8α信号肽(63bp)及与上游eGFP和下游CAR拼配的少许核酸(约18bp)序列的片段，理论大小为165bp，引物分别为：
5’-attcaaagtctgtttcacgctactagctagtccg-3’(SEQ ID NO:25)
5’-gtgaaacagactttgaattttgaccttctgaagttggcaggagacgttgagtccaac-3’(SEQID NO:26)
5’-agcggcaggagcaaggcggtcactggtaaggccatgggcccagggttggactcaacgtc-3’(SEQID NO:27)
5’-ctcctgccgctggccttgctgctccacgccgccaggccggacatcctgatgacccaatc-3’(SEQID NO:28)
引物拼接条件为预变性：94℃，4min；变性：94℃，20s；退火：50℃，20s；延伸：68℃，30s，进行25个循环，然后总延伸68℃，10min。扩增产物(经琼脂糖电泳确认与理论大小一致。
以上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3’(SEQ ID NO:29)和下游引物5’-gctactagctagtccggacttgtacagctcgtccatg-3’(SEQ IDNO:30)扩增目的基因eGFP，以pWPT-eGFP空载体为模板，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：56℃，40s；延伸：68℃，40s，进行25个循环，然后总延伸68℃，10min，理论大小为735bp，扩增产物经琼脂糖电泳确认与理论大小一致。
以上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3’(SEQ ID NO:29)和下游引物5’-gaggtcgacctacgcgggggcgtctgcgctcctgctgaacttcactctggtgataaccagtg-3’(SEQ ID NO:12)采用上述获得的等摩尔的F2A-CD8α信号肽片段、eGFP及806-δZ(约80ng)拼接获得eGFP-F2A-806-δZ，拼接条件为预变性：94℃，4min；变性：94℃，40s；退火：62℃，40s；延伸：68℃，140s，扩增5个循环后，补充合适体积的DNA聚合酶及上下游引物后PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：62℃，40s；延伸：68℃，140s。理论大小为1861bp，扩增产物经琼脂糖电泳确认与理论大小一致。
以上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3’(SEQ ID NO:29)和下游引物5’-gaggtcgacctagcgagggggcagggcctgcatgtgaag-3’(SEQ IDNO:18)采用上述获得的等摩尔的F2A和CD8α信号肽片段、eGFP及806-Z、806-BBZ、CD19-BBZ、806-28Z及806-28BBZ(约80ng)分别拼接。拼接条件为预变性：94℃，4min；变性：94℃，40s；退火：62℃，40s；延伸：68℃，140s，扩增5个循环后，补充合适体积的DNA聚合酶及上下游引物后PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：62℃，40s；延伸：68℃，140s。
获得eGFP-F2A-806-Z，eGFP-F2A-806-BBZ，eGFP-F2A-806-28Z和eGFP-F2A-806-28BB Z理论大小分别为2164bp，2290bp，2305bp，2431bp，扩增产物经琼脂糖电泳确认与理论大小一致。其中在开放阅读框的上下游引入MluI和SalI酶切位点。上述获得的目的基因eGFP-F2A-CAR由MluI和SalI双酶切，连入同样双酶切的pWPT载体中，构建成功的表达各嵌合抗原受体的慢病毒载体经MluI和SalI酶切鉴定(图3)及序列测定正确后进行慢病毒包装。
如前所述，eGFP-F2A-CAR转录为一条mRNA，但翻译为eGFP和抗EGFR287-302嵌合抗原受体两个蛋白，在CD8α信号肽的作用下，抗EGFR287-302嵌合抗原受体将定位在细胞膜上。
4.质粒转染293T包装慢病毒
以6×106的密度接种培养至第6～10代的293T细胞(ATCC：CRL-11268)于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染。培养基为含10％胎牛血清(PAA公司)的DMEM(PAA公司)，次日，在转染前约2小时更换培养液为无血清DMEM。
转染的步骤如下：
4.1将20μg空质粒pWPT-eGFP(mock对照)或20μg目的基因质粒pWPT-eGFP-F2A-CAR，分别与15μg包装质粒PAX2：和6μg包膜质粒pMD2.G：，溶入500μL MillQ水中，混匀，
4.2逐滴加入62μL 2.5M CaCl2(Sigma公司)，以1200rpm/min vortex混匀，
4.3最后逐滴加入500μL 2×HeBS(280mM NaCl,10mM KCl,1.5mM Na2HPO4·2H2O,12mM葡萄糖，50mM Hepes(Sigma公司)，pH7.05,0.22μM过滤除菌)，1200rpm/min vortex混匀10s，
4.4立即逐滴加入培养皿中，轻轻摇匀，37℃，5％CO2，培养4～6h后，更换为含10％胎牛血清的DMEM。
在转染次日观察转染效率(即呈绿色荧光的细胞比例)，～80％的阳性转染效率即为转染实验成功。在转染48h或72h后，使用0.45μm滤器(Millipore公司)过滤收集病毒，然后采用Beckman Optima L-100XP超速离心机28000rpm，4℃离心2小时，弃离心上清，离心所得沉淀用1/10～1/50原液体积的Quantum 007培养液(PAA公司)进行重悬，以100μL/管分装冻存于-80℃，以待病毒滴定或感染T淋巴细胞。
5.测定包装有mock或者eGFP-F2A-CAR的慢病毒滴度
第一天，以1×105/mL接种293T细胞于96孔培养板，100μL/孔，37℃，5％CO2培养，培养液为含10％胎牛血清的DMEM。第二天，弃50μL/孔培养上清，补加50μL/孔新鲜上述培养液，并含终浓度为6μg/mL的polybrene，37℃，5％CO2孵育30min。加10μL/孔的病毒原液或1μL/孔的病毒浓缩液，5倍稀释，4个梯度，两个复孔，37℃，5％CO2培养。感染48h后，流式细胞仪检测eGFP，以阳性率为5～20％的细胞数为宜，计算滴度(U/mL)＝阳性率×稀释倍数×100×104。磷酸钙转染法包装病毒的滴度约0.5～2×106U/mL，经浓缩后所测的病毒滴度约为2×107U/mL。
实施例2.重组慢病毒感染CD8+T淋巴细胞
由健康人外周血通过密度梯度离心法获得人外周血单个核细胞(上海市血液中心提供)，外周血单个核细胞通过CD8+T淋巴细胞磁珠(Stem Cell Technologies)负性分选方法获得CD8+T淋巴细胞，分选后的CD8+T淋巴细胞进行流式细胞检测CD8+T淋巴细胞的纯度，以CD8+T淋巴细胞的阳性率≥95％为宜进行下一步操作。以约1×106/mL密度加入Quantum007淋巴细胞培养基液(PAA公司)培养并以细胞：磁珠比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)和终浓度100U/mL的重组人IL-2(上海华新生物高技术有限公司)刺激培养24h。然后以MOI≈5用上述重组慢病毒感染CD8+T淋巴细胞。感染后的细胞每隔一天采用5×105/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度100U/mL的重组人IL-2。
感染的CD8+T淋巴细胞在培养第7天时通过流式细胞检测各不同嵌合抗原受体表达，由于eGFP与CAR共表达，检测eGFP的阳性细胞即为表达嵌合抗原受体的阳性细胞(图4)。以未感染的T淋巴细胞作为阴性对照，表达不同嵌合抗原受体的病毒感染CD8+T淋巴细胞其阳性率如下表所示。该阳性率结果表明通过慢病毒感染的方法能够获得一定阳性率的CAR+T淋巴细胞。
表2
| 转染有下列CAR的CD8+T淋巴细胞 | CD8+T淋巴细胞eGFP阳性率 |
| 806-δZ | 38.8％ |
| 806-Z | 62％ |
| 806-BBZ | 45％ |
| 806-28Z | 78％ |
| 806-28BBZ | 33.8％ |
CD8+T淋巴细胞在分别感染包装有不同嵌合抗原受体的病毒后，以细胞密度为5×105/ml隔天传代培养、计数、并对传代的细胞培养液补加IL-2(终浓度为100U/ml)，培养第14天约有35～55倍的扩增(见图5)，表明表达不同嵌合抗原受体的CD8+T淋巴细胞在体外能够进行一定数量的扩增，为后续体外毒性试验及体内试验提供了保证。
实施例3.检测EGFR287-302表位在上皮来源肿瘤细胞系中的暴露情况
使用流式细胞检测方法通过荧光激活细胞分选仪(FACSCalibur,来自BectonDickinson)检测几种上皮来源的肿瘤细胞表面EGFR287-302表位的暴露情况。使用的材料包括：
(1)本实验室构建的识别该位点的单克隆抗体CH12(构建方法参见中国专利CN101602808B,实施例1-4)作为为一抗(终浓度20μg/ml，100μL/样品)，
(2)FITC标记的羊抗人IgG为二抗(AOGMA公司)，
表位暴露情况的具体检测方法如下：
1.取对数生长期的如表3所列各肿瘤细胞接种到6cm平皿中，接种细胞密度约为90％，37℃孵箱过夜培养。
2.使用10mM的EDTA消化细胞，200g×5min离心收集细胞。以～1×107/mL的浓度重悬于含1％小牛血清的磷酸盐缓冲液(1％NBS/PBS)中，按100μl/管的量加入流式专用管中。
3.200g×5min离心，弃上清。
4.实验组分别加入待测抗体CH12，同时一个对照组加入无关抗体作为阴性对照，另一个对照组为不加抗体的PBS空白对照。各抗体的终浓度均为20μg/ml，每管加入100ul。冰浴，45分钟。
5.每管加入2ml 1％NBS/PBS，以200g×5min离心，共二遍。
6.弃上清，加入1:50稀释的FITC标记的羊抗人IgG，每管加入100ul。冰浴，45分钟。
7.每管加入2ml 1％NBS/PBS，以200g×5min离心，共二遍。
8.弃上清，重悬于300ul 1％NBS PBS中，流式细胞仪检测。
9.应用流式细胞仪数据分析软件WinMDI 2.9分析数据。
结果如图6所示，在脑胶质瘤细胞系U87中的检测不到EGFR287-302表位的暴露，而在外源过表达EGFR的U87-EGFR(本实验自行构建和保种，构建方法根据：Wang H.,et al.,Identification of an Exon 4-Deletion Variant of Epidermal Growth FactorReceptor with Increased Metastasis-Promoting Capacity.Neoplasia,2011,13,461–471.)及过表达EGFRvIII的U87-EGFRvIII(本实验室自行构建和保种，构建方法根据：WO/2011/035465)中能够检测到EGFR287-302表位，此外在3个胰腺癌细胞系PANC-1、CFPAC-1和BxPC-3中均能检测到EGFR287-302表位的暴露。
表3
| 细胞名称 | 来源 | 性质 |
| U87 | ATCC HTB-14 | 低表达EGFR |
| U87-EGFRvIII | 本实验自行构建和保种 | 过表达EGFRvIII |
| U87-EGFR | 本实验室自行构建和保种 | 过表达EGFR |
| PANC-1 | ATCC CRL-1469 | 过表达EGFR |
| CFPAC-1 | ATCC CRL-1918 | 过表达EGFR |
| BxPC-3 | ATCC CRL-1687 | 过表达EGFR |
实施例4.表达嵌合抗原受体细胞的体外毒性效果实验
体外毒性实验使用的材料如下：
靶细胞分别为上表所示的6种细胞。效应细胞为体外培养12天的FACS检测嵌合抗原受体表达的阳性细胞记为嵌合抗原受体阳性(CAR+)的CD8+T淋巴细胞,
效靶比视情况分别为3:1，1:1和1:3或者5:1，2.5:1和1:1，靶细胞数量为10000/孔，根据不同效靶比对应效应细胞。各组均设4个复孔，取四个复孔的平均值。检测时间为第18h或20h。
其中各实验组和各对照组如下：
各实验组：各靶细胞+表达不同嵌合抗原受体的CD8+T淋巴细胞，
对照组1：靶细胞最大释放LDH，
对照组2：靶细胞自发释放LDH，
对照组3：效应细胞自发释放LDH。
检测方法：采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。该方法是基于比色法的检测方法，可替代51Cr释放法。CytoTox检测定量地测量乳酸脱氢酶(LDH)。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放出的LDH培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
细胞毒性计算公式为：
实验结果表明：
本发明的表达scFv(EGFR)-806-Z CAR+的CD8+T淋巴细胞和806-28BBZ CAR+的CD8+T淋巴细胞对肿瘤细胞U87-EGFRvIII表现出非常显著的细胞毒性，分别高达55.5％和85％。
此外上述本发明的CD8+T淋巴细胞的细胞毒性是高度肿瘤特异性的，因为相对于对暴露EGFR287-302表位的肿瘤细胞U87-EGFRvIII的高细胞毒性的同时，作为对比的是其显示出对不暴露EGFR287-302表位的肿瘤细胞U87很低的细胞毒性，在两种情况下均小于2％。同时，作为实验结果可靠性证据的空白对照的空质粒(mock)转染的T细胞和作为阴性对照的评价原始T细胞内效应分子影响的嵌合抗原受体806-δZ转基因T细胞也均对U87和U87-EGFRvIII表现出几乎相同的很低的细胞毒性％。上述实验是在效靶比为5：1，作用时间20h时测定的。
表4
此外，在不同效靶比的情况下，在作用时间18h时测定的本发明的表达scFv(EGFR)-806-Z CAR+的CD8+T淋巴细胞，和806-28BBZ CAR+的CD8+T淋巴细胞对暴露EGFR287-302表位的肿瘤细胞U87-EGFR和U87-EGFRvIII和对三个胰腺癌细胞系PANC-1，CFPAC-1和BxPC-3的细胞毒性作用均呈现出效靶比梯度的依赖性，如下表所示，效靶比越高，细胞毒性越高。
表5
在效靶比3：1时，嵌合抗原受体806-28BBZ CAR+的CD8+T淋巴细胞对U87-EGFR的细胞毒性高达98％，对U87-EGFRvIII的细胞毒性高达81％，对三个胰腺癌细胞系PANC-1，CFPAC-1和BxPC-3的细胞毒性分别为65％、40％和70％。
而作为阴性对照的评价非特异性scFv在嵌合抗原受体中的影响的嵌合抗原受体CD19-BBZ CAR+的CD8+T淋巴细胞对上述三个胰腺癌细胞系的细胞毒性％都小于10％，并且不表现出效靶比梯度依赖性。
序 列 表
<110> 科济生物医药(上海)有限公司
<120> 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的T淋巴细胞
<130> 158558F1
<160> 34
<170> PatentIn version 3.3
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gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc 60
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gggaaatcat ttaagggcct gatctatcat ggaaccaact tggacgatga agttccatca 180
aggttcagtg gcagtggatc tggagccgat tattctctca ccatcagcag cctggaatct 240
gaagattttg cagactatta ctgtgtacag tatgctcagt ttccgtggac gttcggtgga 300
ggcaccaagc tggaaatcaa acgtggtgga ggcggttcag gcggaggtgg ctctggcggt 360
ggcggatcgg ccgatgtgca gcttcaggag tcgggaccta gcctggtgaa accttctcag 420
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tggatccggc agtttccagg aaacaagctg gagtggatgg gctacataag ttatagtggt 540
aacactaggt acaacccatc tctcaaaagt cgaatctcta tcactcgaga cacatccaag 600
aaccaattct tcctgcagtt gaattctgtg actattgagg acacagccac atattactgt 660
gtaacggcgg gacgcgggtt tccttattgg ggccaaggga ctctggtcac tgtctctgca 720
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 780
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 840
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ggacgcgggt ttccttattg gggccaaggg actctggtca ctgtctctgc aggtggaggc 360
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accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 780
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cgttttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
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aacacctttt tcctgcagct gaacagcctg cgtgcggaag ataccgcggt gtattattgc 660
gcgcgcctgg gacgcggctt ccgctactgg ggccagggca ccctggtgac cgtgagcagc 720
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 780
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cgttttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgcgttcag tacgcccagt tcccatatac atttggccag 300
ggcaccaaag tggaaattaa acgttcaggt ggaggcggtt caggcggagg tggctctggc 360
ggtggcggat cggatgtgca gctggtggaa agcggcggcg gcctggtgca gccgggcggc 420
agcctgcgtc tgagctgcgc ggtgagcggc tatagcatta ccagcgatta tgcgtggaac 480
tggattcgtc aggcgccggg caaaggcctg gaatggctgg gctatattag ctatcgcggc 540
cgcaccagct ataacccgag cctgaaaagc cgtattagca ttacccgtga taacagcaaa 600
aacacctttt tcctgcagct gaacagcctg cgtgcggaag ataccgcggt gtattattgc 660
gcgcgcctgg gacgcggctt ccgctactgg ggccagggca ccctggtgac cgtgagcagc 720
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 780
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 840
gacttcgcct gtgatttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 900
ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 960
cacagtgact acatgaacat gactccccgc cgccccgggc caacccgcaa gcattaccag 1020
ccctatgccc caccacgcga cttcgcagcc tatcgctcca aacggggcag aaagaaactc 1080
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1140
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1200
aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1260
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1320
gggggaaagc cgcagagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1380
gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1440
gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1500
cacatgcagg ccctgccccc tcgctag 1527
<210> 5
<211> 29
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 5
gacatcctga tgacccaatc tccatcctc 29
<210> 6
<211> 28
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 6
tgcagagaca gtgaccagag tcccttgg 28
<210> 7
<211> 24
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 7
gatattcaga tgacccagag cccg 24
<210> 8
<211> 22
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 8
gctgctcacg gtcaccaggg tg 22
<210> 9
<211> 28
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 9
cactgtctct gcaaccacga cgccagcg 28
<210> 10
<211> 21
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 10
ggtgataacc agtgacagga g 21
<210> 11
<211> 28
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 11
cactgtctct gcaaccacga cgccagcg 28
<210> 12
<211> 62
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 12
gaggtcgacc tacgcggggg cgtctgcgct cctgctgaac ttcactctgg tgataaccag 60
tg 62
<210> 13
<211> 23
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 13
ttttgggtgc tggtggtggt tgg 23
<210> 14
<211> 34
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 14
gctgaacttc actctggagc gataggctgc gaag 34
<210> 15
<211> 21
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 15
aaacggggca gaaagaaact c 21
<210> 16
<211> 18
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 16
cagttcacat cctccttc 18
<210> 17
<211> 35
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 17
cactggttat caccagagtg aagttcagca ggagc 35
<210> 18
<211> 35
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 18
cgaggtcgac ctagcgaggg ggcagggcct gcatg 35
<210> 19
<211> 18
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 19
accacgacgc cagcgccg 18
<210> 20
<211> 19
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 20
cacccagaaa ataataaag 19
<210> 21
<211> 25
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 21
agagtgaagt tcagcaggag cgcag 25
<210> 22
<211> 28
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 22
cactgtctct gcaaccacga cgccagcg 28
<210> 23
<211> 28
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 23
cactgtctct gcaaccacga cgccagcg 28
<210> 24
<211> 18
<212> DNA
<213> atificial
<400> 24
accacgacgc cagcgccg 18
<210> 25
<211> 34
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 25
attcaaagtc tgtttcacgc tactagctag tccg 34
<210> 26
<211> 57
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 26
gtgaaacaga ctttgaattt tgaccttctg aagttggcag gagacgttga gtccaac 57
<210> 27
<211> 59
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 27
agcggcagga gcaaggcggt cactggtaag gccatgggcc cagggttgga ctcaacgtc 59
<210> 28
<211> 59
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 28
ctcctgccgc tggccttgct gctccacgcc gccaggccgg acatcctgat gacccaatc 59
<210> 29
<211> 52
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 29
cttacgcgtc ctagcgctac cggtcgccac catggtgagc aagggcgagg ag 52
<210> 30
<211> 37
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 30
gctactagct agtccggact tgtacagctc gtccatg 37
<210> 31
<211> 419
<212> PRT
<213> ARTIFICIAL
<220>
<223> CDS,不包含信号肽
<400> 31
Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly
1 5 10 15
Asp Thr Val Ser Ile Thr Cys His Ser Ser Gln Asp Ile Asn Ser Asn
20 25 30
Ile Gly Trp Leu Gln Gln Arg Pro Gly Lys Ser Phe Lys Gly Leu Ile
35 40 45
Tyr His Gly Thr Asn Leu Asp Asp Glu Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Asp Val Gln Leu
115 120 125
Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Ser Leu Ser Leu
130 135 140
Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn
145 150 155 160
Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile
165 170 175
Ser Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile
180 185 190
Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn
195 200 205
Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys Val Thr Ala Gly
210 215 220
Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
225 230 235 240
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
245 250 255
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
260 265 270
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
275 280 285
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
290 295 300
Ile Thr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
305 310 315 320
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
325 330 335
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
340 345 350
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
355 360 365
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
370 375 380
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
385 390 395 400
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
405 410 415
Pro Pro Arg
<210> 32
<211> 508
<212> PRT
<213> artificial
<220>
<223> CDS，不包含信号肽
<400> 32
Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly
1 5 10 15
Asp Thr Val Ser Ile Thr Cys His Ser Ser Gln Asp Ile Asn Ser Asn
20 25 30
Ile Gly Trp Leu Gln Gln Arg Pro Gly Lys Ser Phe Lys Gly Leu Ile
35 40 45
Tyr His Gly Thr Asn Leu Asp Asp Glu Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Asp Val Gln Leu
115 120 125
Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Ser Leu Ser Leu
130 135 140
Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn
145 150 155 160
Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile
165 170 175
Ser Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile
180 185 190
Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn
195 200 205
Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys Val Thr Ala Gly
210 215 220
Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
225 230 235 240
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
245 250 255
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
260 265 270
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val
275 280 285
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
290 295 300
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
305 310 315 320
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
325 330 335
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
340 345 350
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
355 360 365
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
370 375 380
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
385 390 395 400
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
405 410 415
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
420 425 430
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys
435 440 445
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
450 455 460
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
465 470 475 480
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
485 490 495
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505
<210> 33
<211> 419
<212> PRT
<213> artificial
<220>
<223> CDS，不包含信号肽
<400> 33
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asp Ile Asn Ser Asn
20 25 30
Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile
35 40 45
Tyr His Gly Lys Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly Gly
100 105 110
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu
115 120 125
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
130 135 140
Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
145 150 155 160
Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Tyr Ile
165 170 175
Ser Tyr Arg Gly Arg Thr Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile
180 185 190
Ser Ile Thr Arg Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn
195 200 205
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly
210 215 220
Arg Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
245 250 255
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
260 265 270
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
275 280 285
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
290 295 300
Ile Thr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
305 310 315 320
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
325 330 335
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
340 345 350
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
355 360 365
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
370 375 380
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
385 390 395 400
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
405 410 415
Pro Pro Arg
<210> 34
<211> 508
<212> PRT
<213> ARTIFICIAL
<220>
<223> CDS，不包含信号肽
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asp Ile Asn Ser Asn
20 25 30
Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile
35 40 45
Tyr His Gly Lys Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly Gly
100 105 110
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu
115 120 125
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
130 135 140
Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
145 150 155 160
Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Tyr Ile
165 170 175
Ser Tyr Arg Gly Arg Thr Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile
180 185 190
Ser Ile Thr Arg Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn
195 200 205
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly
210 215 220
Arg Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
245 250 255
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
260 265 270
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val
275 280 285
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
290 295 300
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
305 310 315 320
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
325 330 335
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
340 345 350
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
355 360 365
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
370 375 380
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
385 390 395 400
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
405 410 415
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
420 425 430
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys
435 440 445
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
450 455 460
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
465 470 475 480
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
485 490 495
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505
Claims (16)
1.编码一种表达于人T细胞表面的嵌合抗原受体蛋白的核酸，所述嵌合抗原受体蛋白包含顺序连接的胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别人表皮生长因子受体EGFR的第287-302位氨基酸表位的单链抗体scFv(EGFR)。
2.如权利要求1所述的核酸，其中跨膜区选自包含CD8或CD28的跨膜区和铰链区的序列。
3.如权利要求1或2所述的核酸，其中胞内信号区选自CD3ζ，FcεRIγ，CD28，CD137，CD134的胞内信号区序列，及其组合。
4.如权利要求3所述的核酸，其中所述嵌合抗原受体蛋白是选自包含顺序连接的胞外结合区，跨膜区和胞内信号区的如下的嵌合抗原受体蛋白：
scFv(EGFR)-CD8-CD3ζ，
scFv(EGFR)-CD8-CD137-CD3ζ，
scFv(EGFR)-CD28-CD28-CD3ζ，
scFv(EGFR)-CD28-CD28-CD137-CD3ζ，
及其组合，其中相关嵌合抗原受体蛋白中的第一个CD28代表其跨膜区，第二个CD28代表其胞内信号区。
5.如权利要求1-4任一所述的核酸，所述核酸选自编码具有SEQ ID NO:31～34之一序列的嵌合抗原受体蛋白的核酸。
6.如权利要求1-4任一所述的核酸，所述核酸编码与SEQ ID NO:31～34所示的序列竞争结合EGFR的第287-302位的氨基酸序列。
7.如权利要求1-4任一所述的核酸，所述单链抗体含有SEQ ID NO:31或32中第1-240所示的氨基酸序列或者含有SEQ ID NO:33或34中第1-240所示的氨基酸序列。
8.核酸或核酸变体，其编码具有下组中任一所述的氨基酸序列：
序列(1)，与SEQ ID NO:31具有至少80％相似性的氨基酸序列；
序列(2)，与SEQ ID NO:32具有至少80％相似性的氨基酸序列；
序列(3)，与SEQ ID NO:33具有至少80％相似性的氨基酸序列；
序列(4)，与SEQ ID NO:34具有至少80％相似性的氨基酸序列。
9.如权利要求8所述的核酸或核酸变体，其编码含有下组中任一所述序列的氨基酸序列：
序列(1)，与SEQ ID NO:31具有至少90％相似性的氨基酸序列；
序列(2)，与SEQ ID NO:32具有至少90％相似性的氨基酸序列；
序列(3)，与SEQ ID NO:33具有至少90％相似性的氨基酸序列；
序列(4)，与SEQ ID NO:34具有至少90％相似性的氨基酸序列。
10.如权利要求1-7任一所述的核酸，具有SEQ ID NO:1～4之一所示的序列或具有SEQID NO:1～4之一所示的序列的变体序列。
11.如权利要求10所述的核酸，所述变体序列选自下组中的核酸序列：
序列(1)，与SEQ ID NO:1具有至少80％相似性的氨基酸序列；
序列(2)，与SEQ ID NO:2具有至少80％相似性的氨基酸序列；
序列(3)，与SEQ ID NO:3具有至少80％相似性的氨基酸序列；
序列(4)，与SEQ ID NO:4具有至少80％相似性的氨基酸序列。
12.包含权利要求1-11之一所述的核酸的载体。
13.如权利要求12所述的载体，该载体来自慢病毒质粒pWPT-eGFP。
14.包含权利要求1-11任一所述的核酸或包含有权利要求12或13所述载体的病毒。
15.一种转基因T淋巴细胞，其被转导有权利要求1-11任一所述的核酸或权利要求12或13所述的载体或权利要求14所述的病毒。
16.一种转基因T淋巴细胞，其表面表达一种嵌合抗原受体，所述嵌合抗原受体由权利要求1-11任一所述的核酸编码表达。
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| CN201710375055.4A CN107058354A (zh) | 2013-04-01 | 2013-04-01 | 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞 |
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| CN201710375055.4A CN107058354A (zh) | 2013-04-01 | 2013-04-01 | 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞 |
| CN201310108532.2A CN104087607B (zh) | 2013-04-01 | 2013-04-01 | 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞 |
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Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170159025A1 (en) * | 2014-05-14 | 2017-06-08 | Carsgen Therapeutics Limited | Nucleic acid for coding chimeric antigen receptor protein and t lymphocyte for expression of chimeric antigen receptor protein |
| CN109504697A (zh) * | 2017-09-15 | 2019-03-22 | 上海恒润达生生物科技有限公司 | 一种靶向人源化mesothelin的嵌合抗原受体的方法和用途 |
| WO2019149279A1 (zh) | 2018-02-02 | 2019-08-08 | 科济生物医药（上海）有限公司 | 细胞免疫治疗的组合 |
| WO2019170147A1 (zh) | 2018-03-09 | 2019-09-12 | 科济生物医药（上海）有限公司 | 用于治疗肿瘤的方法和组合物 |
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| CN104087607B (zh) | 2017-06-20 |
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