CN113528449A - 一种制备通用型人源化car19-dnt细胞的技术及其应用 - Google Patents
一种制备通用型人源化car19-dnt细胞的技术及其应用 Download PDFInfo
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Abstract
本发明提供了一种制备通用型人源化CAR19‑DNT细胞的技术及其应用。具体地,本发明提供了一种靶向CD19的通用型CAR‑T细胞,所述通用型CAR‑T细胞表达一外源的CAR构建物,所述CAR构建物具有如式I所示的结构,L‑scFv‑H‑TM‑C‑CD3ζ (式I)式中,L为无或信号肽序列;scFv为靶向CD19的抗体单链可变区序列;H为无或铰链区;TM为跨膜结构域;C为共刺激信号分子;CD3ζ为源于CD3ζ的胞浆信号传导序列。本发明提供的通用型人源化CAR19‑DNT细胞具有免疫原性低、无需基因编辑避免GvHD、安全性高、特异性高、杀瘤效果显著的优点,并且本发明所提供的构建方法可实现规模化生产,生产成本低。
Description
技术领域
本发明属于肿瘤免疫细胞治疗技术领域,具体涉及一种制备通用型人源化抗原嵌合受体CART-19细胞的技术方法及其应用。
背景技术
嵌合抗原受体T细胞(Chimeric Antigen Receptor T,CART)是目前最有发展前景的肿瘤免疫疗法之一,2017年已被美国FDA批准的2款CART细胞产品都是分离肿瘤患者自身的外周血单个核细胞(Peripheral Blood Mononuclear Cell,PBMC)细胞,通过基因工程嵌入特定的抗原受体(CAR),使得PBMC中T细胞的靶向性、杀伤活性和持久性增强,其对肿瘤细胞表面抗原的识别不依赖MHC的限制性。常见的CAR由胞外抗原结合区、跨膜区、以及胞内的T细胞受体的信号转导区(如CD3ζ和CD28)组成。胞外抗原结合区由单克隆抗体的轻链(VL)和重链(VH)组成,中间由铰链连接形成单链抗体(single chain fragment variable,scFv),能够识别特定的肿瘤抗原。CAR-T细胞技术已发展到第三代,第一代CAR由识别肿瘤表面抗原的单链抗体(scFv)抗原结合部位和免疫受体酪氨酸活化基序组成,胞外通过单链抗体(scFv)识别肿瘤标志分子,但是没有协同刺激信号,因此临床表现不佳,表现为T细胞体内存留时间短,细胞因子分泌能力低等问题;二代CART技术引入了共刺激信号序列,可提高T细胞的细胞毒活性、增殖能力以及体内存活时间、促进细胞因子的释放,在临床治疗急性淋巴细胞性白血病取得了成功;三代CART,在胞浆区串联排列了CD28、4-1BB两种协同刺激分子和CD3ζ分子,双共刺激的信号序列具有更好的效果。三代CART的结构示意图如图1所示。
目前自体CART细胞用于治疗靶向CD19的B细胞急性淋巴母细胞性白血病(B AcuteLymphoblastic cell Leukemia,B-ALL),取得了高达90%以上的完全缓解率;在治疗淋巴瘤上,完全缓解率也高达64%;CART技术在治疗这两种疾病时取得的效果是任何其他药物无法相比的。在我国,急性淋巴细胞性白血病占儿童急性白血病的80%,发病率为十万分之一,占所有成年人白血病发病率的20%。免疫细胞治疗是许多严重疾病的重要治疗手段,包括癌症、自体免疫疾病甚至严重病毒感染都有使用免疫细胞治疗成功的案例。
虽然靶向CD19 CART在临床试验中的效果非常显著,但也存在诸多缺陷,除了会导致细胞因子风暴等严重副反应外,还存在4大问题:首先,一些淋巴细胞数量较低或质量较差的晚期患者,由于没有足够数量的自体免疫细胞,失去了CART治疗的机会;其次,已有报道采用病人自体细胞制备的CART产品时,将病毒基因转导到外周血残存肿瘤细胞内,导致病人死亡;再者,目前大部分正在开发的通用型CART细胞产品,是通过基因编辑技术敲除可以引起移植物抗宿主病的基因,如TCR受体等,但敲除这些基因需要繁琐的构建过程和大规模测序,并且脱靶率高;最后,由于制备自体CART细胞产品是1对1的个体化治疗,制备成本昂贵,制备工艺要求复杂,导致产品价格昂贵,大多病人无法承受,增加社会医疗负担。
因此,本领域迫切需要开发一种源自健康供者外周血,无需复杂基因编辑方法敲除引起移植物抗宿主病的TCR基因,免疫原性低(不引起宿主抗移植物免疫应答,体内存留时间长)、安全性高、杀伤效果显著并且生产成本低的通用(即用)型CART细胞及其构建方法。
发明内容
鉴于现有相关技术的系列问题,本发明提供了一种制备通用型抗原嵌合受体CAR19-DNT细胞的技术方法及其应用。
本发明之目的是提供一种源自健康供者外周血,无需复杂基因编辑方法敲除引起移植物抗宿主病的TCR基因,免疫原性低(不引起宿主抗移植物免疫应答,体内存留时间长)、安全性高、杀伤效果显著并且生产成本低的通用(即用)型CART细胞及其构建方法。
在本发明的第一方面,提供了一种靶向CD19的通用型CAR-T细胞,所述通用型CAR-T细胞表达一外源的CAR构建物,所述CAR构建物具有如式I所示的结构,
L-scFv-H-TM-C-CD3ζ (式I)
式中,
L为无或信号肽序列;
scFv为靶向CD19的抗体单链可变区序列;
H为无或铰链区;
TM为跨膜结构域;
C为共刺激信号分子;
CD3ζ为源于CD3ζ的胞浆信号传导序列;
各“-”独立地表示连接上述各元件的连接肽或肽键。
在另一优选例中,所述靶向CD19的通用型CAR-T细胞是靶向CD19的通用型CAR-双阴性T细胞(CAR-DNT细胞)。
在另一优选例中,所述通用型CAR-T细胞中的TCR是敲除的或未敲除的。
在另一优选例中,所述通用型CAR-T细胞中的TCR未被敲除。
在另一优选例中,所述的L为选自下组蛋白的信号肽:CD8、GM-CSF、CD4、CD137、或其组合。
在另一优选例中,所述的L为CD8来源的信号肽。
在另一优选例中,所述scFv中包含具有如SEQ ID NO:6所示的氨基酸序列的重链可变区(VH)和具有如SEQ ID NO:5所示的氨基酸序列的轻链可变区(VL)。
在另一优选例中,所述scFv中还包含VH和VL之间的连接肽序列。
在另一优选例中,所述的连接肽序列具有如SEQ ID NO:7所示的氨基酸序列。
在另一优选例中,所述scFv具有如SEQ ID NO:4所示的氨基酸序列。
在另一优选例中,所述的H为选自下组蛋白的铰链区:CD8、CD28、CD137、或其组合。
在另一优选例中,所述的H为CD8来源的铰链区。
在另一优选例中,所述的TM为选自下组的蛋白的跨膜区:CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中,TM包括CD8来源的跨膜区,和/或CD28来源的跨膜区。
在另一优选例中,所述的C为选自下组的蛋白的共刺激信号分子:OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
在另一优选例中,C包括4-1BB来源的共刺激信号分子,和/或CD28来源的共刺激信号分子。
在另一优选例中,所述CAR构建物具有如SEQ ID NO:14所示的氨基酸序列。
在本发明的第二方面,提供了一种制备如本发明第一方面所述的靶向CD19的通用型CAR-DNT细胞的方法,包括步骤:
(i)提供一表达载体,所述表达载体中含有编码如式I所示的CAR构建物的核苷酸序列;
L-scFv-H-TM-C-CD3ζ (式I)
式中,
L为无或信号肽序列;
scFv为靶向CD19的抗体单链可变区序列;
H为无或铰链区;
TM为跨膜结构域;
C为共刺激信号分子;
CD3ζ为源于CD3ζ的胞浆信号传导序列;
各“-”独立地表示连接上述各元件的连接肽或肽键;
(ii)提供一DNT细胞培养液,所述细胞培养液中含有至少一个DNT细胞;将步骤(i)的表达载体转导入对所述DNT细胞,从而获得如本发明第一方面所述的靶向CD19的通用型CAR-DNT细胞;和
(iii)任选的检测步骤(ii)所获得的通用型CAR-DNT细胞。
在另一优选例中,所述的表达载体选自DNA、RNA,或其组合。
在另一优选例中,所述的表达载体选自下组:质粒、病毒表达载体、转座子,或其组合。
在另一优选例中,所述病毒表达载体选自下组:慢病毒载体、腺病毒载体、逆转录病毒载体,或其组合。
在另一优选例中,所述的表达载体是病毒表达载体,并且所述方法(ii)之前还包括步骤:将步骤(i)的病毒表达载体进行病毒包装。
在另一优选例中,所述病毒表达载体为逆转录病毒表达载体、慢病毒表达载体,优选地为慢病毒表达载体。
在另一优选例中,所述步骤(i)的病毒表达载体中所整合有的多核苷酸序列如SEQID NO:16所示。
在另一优选例中,在所述步骤(ii)中,包括将所述步骤(i)中的病毒表达载体与包装质粒psPAX2、pMD2.0G共转染入病毒包装宿主细胞。
在另一优选例中,所述步骤(i)中,所述的多核苷酸序列中含有如SEQ ID NO:15所示的编码式(I)中的scFv的核苷酸序列。
在另一优选例中,所述步骤(i)中,所述的多核苷酸序列中含有如SEQ ID NO:8所示的编码式(I)中的L的核苷酸序列。
在另一优选例中,所述步骤(i)中,所述的多核苷酸序列中含有如SEQ ID NO:9所示的编码式(I)中的H的核苷酸序列。
在另一优选例中,所述步骤(i)中,所述的多核苷酸序列中含有如SEQ ID NO:10所示的编码式(I)中的TM的核苷酸序列。
在另一优选例中,所述步骤(i)中,所述的多核苷酸序列中含有如SEQ ID NO:11所示的编码式(I)中的C的核苷酸序列。
在另一优选例中,所述步骤(i)中,所述的多核苷酸序列中含有如SEQ ID NO:12所示的编码式(I)中的CD3ζ的核苷酸序列。
在另一优选例中,所述病毒包装宿主细胞为哺乳动物细胞,优选地为293T细胞。
在另一优选例中,在所述步骤(ii)之前,还包括步骤:
(iia)从健康供体收集外周血样品;
(iib)去除所述外周血样品中的CD4+CD8+T细胞,从而获得DNT细胞;
(iic)在包被有CD3单克隆抗体的培养瓶中,用适合的培养基培养和扩增(iib)中获得的DNT细胞,从而获得步骤(ii)中所需的含DNT细胞的培养体系。
在另一优选例中,在所述步骤(iic)的培养基中,添加有选自下组的物质:庆大霉素、重组人白介素2、、重组人白介素7、重组人白介素12、重组人白介素15、自体血浆、AB血清,或其组合。
在另一优选例中,所述步骤(iic)的培养基中,不包含重组的或野生型的人白介素4。
在另一优选例中,所述步骤(iic)的培养基中,不包含AB血清。
在另一优选例中,在所述步骤(iic)的培养基中,所述庆大霉素的浓度为40-80单位/ml(优选为60单位/ml),所述重组人白介素2的浓度为150-1000IU/ml(优选为200-250IU/ml),,所述重组人白介素15的浓度为5-20ng/ml(优选为10ng/ml),所述重组人白介素7的浓度为1-5ng/ml(优选为2ng/ml),所述重组人白介素12的浓度为5-20ng/ml(优选为10ng/ml),所述自体血浆的浓度为3-25v%(优选为10-20v%),和/或所述AB血清的浓度为4-8v%(优选为6v%)。
在另一优选例中,在所述步骤(ii)中,所述DNT细胞培养液中的DNT细胞的浓度为1×106个细胞/ml至4×106个细胞/ml,较佳地为1×106个细胞/ml至2×106个细胞/ml,更佳地为0.5×106个细胞/ml至1×106个细胞/ml。
在另一优选例中,所述步骤(ii)中,所述病毒的MOI为0.1至10,更佳地为0.1至8,优选为5。
在另一优选例中,所述步骤(iii)的检测在步骤(ii)的转导之后的第7-14天后进行,优选地在第8天或第13天后。
在本发明的第三方面,提供了一种药物组合物,包括:
(a)如本发明第一方面所述的靶向CD19的通用型CAR-T细胞;和
(b)药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中,所述药物组合物是液态的药物组合物。
在另一优选例中,所述药物组合物是注射剂。
在另一优选例中,所述药物组合物中,所述靶向CD19的通用型CAR-T细胞的浓度为1×106-5×106个细胞/ml,较佳地为1×106-2×106个细胞/ml。
在本发明的第四方面,提供了一种如本发明第一方面所述的靶向CD19的通用型CAR-T细胞的用途,用于制备预防和/或治疗癌症的药物组合物或制剂。
在另一优选例中,所述肿瘤选自下组:血液肿瘤、实体瘤、或其组合。
在另一优选例中,所述肿瘤为血液肿瘤。
在另一优选例中,所述血液肿瘤选自下组:急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL),或其组合。
在另一优选例中,所述实体瘤选自下组:胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、肺鳞癌、肛门癌、头颈部肿瘤,或其组合。
在本发明的第五方面,提供了一种预防和/或治疗疾病的方法,包括步骤:向有所需要的对象施用如本发明第一方面所述的靶向CD19的通用型CAR-T细胞,或如本发明第三方面所述的药物组合物。
在另一优选例中,所述疾病为癌症或肿瘤。
在另一优选例中,所述需要的对象为人或非人哺乳动物。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了三代CART的结构。
图2显示了人源化的CD19 CAR构建体的结构示意图。
图3显示了CDR1结构域(V27G)发生突变的人源化CD19三维模型。
图4显示了培养第8天和第13天,使用流式细胞仪通过CD3、CD4、CD8抗体标记鉴定培养体系中DNT细胞比例,以及通过特异性抗人CD19可变区抗体标记检测DNT细胞的被携带抗体基因慢病毒感染的效率,由此计算细胞产品中CART细胞百分比。
图5显示了培养第8天和第13天,人源化CAR19-DNT分别对Hela和Hela-CD19靶细胞的体外特异杀瘤活性比较;其中5-A显示了效靶比为2:1时,用RTCA(Real-TimeCytotoxicity Assay)监测的实时杀伤情况;5-B显示了效靶比为2:1,添加效应细胞18小时后,用RTCA(Real-Time Cytotoxicity Assay)监测的实时定量杀伤情况。
具体实施方式
本发明人经过广泛而深入的研究,经过大量的筛选,首次开发了一种通用型抗原嵌合受体CAR19-DNT细胞的构建方法。实验证明,本发明提供的通用型抗原嵌合受体CART19-DNT细胞源自健康供者外周血,无需复杂基因编辑方法敲除引起移植物抗宿主病的TCR基因,免疫原性低(不引起宿主抗移植物免疫应答,体内存留时间长)、安全性高、特异性靶向人CD19抗原,杀瘤效果显著的优点,杀伤效果显著,并且本发明所提供的构建方法可实现规模化生产,生产成本低。在此基础上完成了本发明。
术语
为了可以更容易地理解本公开,首先定义某些术语。如本申请中所使用的,除非本文另有明确规定,否则以下术语中的每一个应具有下面给出的含义。
如本文所用,术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成,其将部分地取决于如何测量或测定值或组成。
如本文所用,术语“给予”、“施用”可互换使用,是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者,包括静脉内,肌内,皮下,腹膜内,脊髓或其它肠胃外给药途径,例如通过注射或输注。
如本文所用,术语“DNT细胞”是指双阴性T细胞(Double Negative T cells),是一种表型独特的TCRαβ+和/或TCRγδ+T淋巴细胞,表达CD3分子但不表达CD4、CD8、NK、iNKT等细胞的特征性表面分子(标志物),在人体免疫系统中发挥独特和多样化的生理作用。
双阴性T(Double Negative T,DNT)细胞是指外周血中正常存在的CD3+CD4-CD8-成熟T淋巴细胞亚群,约占外周血单个核细胞1-3%。DNT细胞表面表达CD3分子和αβ-或γδ-T细胞受体(T Cell Receptor,TCR),但不表达CD4和CD8分子,且对于恒定型自然杀伤T(invariant Nature Killer T,iNKT)细胞特异性αGalCer无反应,因此不同于常规的T细胞、NK细胞和NKT细胞。DNT细胞通过多种人体天然机制发挥细胞毒性、抗原呈递功能同时释放细胞因子/趋化因子,激活更广泛的免疫应答,这使其成为具备广泛应用前景的临床治疗候选药物。
DNT细胞作为细胞免疫治疗候选药物的优点包括:
DNT细胞可用于多种肿瘤适应症,通过细胞表面的NKG2D和DNAM-1等受体选择性靶向AML、淋巴瘤、宫颈癌、肺癌等多种血液或者实体肿瘤上表达的相应配体,并分泌TNF-α、IFN-γ、Grazyme B、Peforin、IL-2、IL-4等发挥直接和间接的杀瘤活性。且可与PD-1、CTLA-4等免疫检查点抑制剂和化疗药联用,具有协同治疗效果。
DNT细胞杀死肿瘤细胞不受MHC(组织相容性复合物)分子限制,健康供者捐赠的异体DNT细胞输注到患者体内,对患者体内正常细胞无杀伤毒性且不影响造血干细胞的进一步分化,不会引起移植物抗宿主病(Graft versus Host Disease,GvHD),也没有宿主抗移植物(Host versus Graft)反应,可以使输入的异体DNT细胞在患者体内持续较长时间以发挥杀瘤活性。
健康捐赠者的DNT细胞,每ml外周血可高效扩增至5x108以上数量级,可收集冻存后用于多个病人治疗,使临床病人一经诊断明确就可实施治疗,免除自体CART产品所需要的产品制备等待期,成为真正意义上的从货架获得(off-the-shelf)的通用型(即用)细胞产品。结合DNA细胞具有的上述广谱抗瘤、安全性高、通用以及与其他免疫疗法联合使用等特性,本发明设计了构建一种靶向CD19的通用型人源化抗原嵌合受体CART-19产品,并以此衍生开发基于DNT细胞平台的系列无需基因编辑的通用性CART细胞产品。
嵌合抗原受体(CAR)
嵌合免疫抗原受体(Chimeric Antigen Receptors,CARs)由胞外抗原识别结构域,通常是scFv(single-chain variable Fragment),跨膜区以及胞内共刺激信号结构域组成。CARs的设计经历了以下过程:第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子,由于胞内只有一个活化结构域,因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌,而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应,所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子,如CD28、4-1BB、OX40、ICOS,与一代CARs相比功能有很大提高,进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134,发展成为三代和四代CARs。
CARs的胞外段可识别一个或多个特异性的抗原决定簇(epitopes),随后通过胞内结构域转导该信号,引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子,进而清除靶细胞。首先分离病人自体或异体(健康供者)的PBMC,激活并进行基因改造产生CAR的免疫细胞,随后注入病人体内,以非MHC限制的方式通过直接识别肿瘤细胞表面抗原对其进行特异性杀伤。
具体地,本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和/或ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子,而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间,或在CAR的胞浆结构域和跨膜结构域之间,可并入接头。
如本文所用,术语“接头”、“铰链区”可互换使用,通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸,优选地2至100个氨基酸和最优选地3至50个氨基酸。
本发明的CAR当在免疫细胞中表达时,能够基于抗原结合特异性进行抗原识别。当其结合肿瘤细胞上关联抗原时,导致肿瘤细胞死亡,患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和/或ζ链中的一个或多个的细胞内结构域融合。优选地,抗原结合结构域与CD28共刺激信号分子、4-1BB共刺激信号分子和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用,本发明嵌合抗原受体的基础结构包括:肿瘤相关抗原结合区,胞外铰链区,跨膜区和胞内信号区。肿瘤相关抗原的选择直接影响其对肿瘤的治疗效果。在本发明中,本发明的嵌合抗原受体靶向CD19。
CD19分子主要表达于早期的B细胞,是一个95KDa左右的B细胞系特有的跨膜糖蛋白。CD19在正常及恶性B淋巴细胞中均有表达,被视为B细胞发育过程中一个涵盖阶段较长的最为可靠的表面标记物。
在本发明的一个优选例中,选择抗CD19的单链抗体作为本发明CAR中靶向CD19的抗原结合结构域。
在一个优选的实施方式中,所述scFv的氨基酸序列如SEQ ID NO:4所示,可以高效的与CD19分子结合。
在本发明中,靶向CD19的抗原结合结构域还包括所述scFv的保守性变异体,指与本发明scFv的氨基酸序列相比,有至多10个,较佳地至多8个,更佳地至多5个,最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。在本发明中,所述添加、缺失、修饰和/或取代的氨基酸数量,优选为不超过初始氨基酸序列总氨基酸数量的40%,更优选为不超过35%,更优选为1-33%,更优选为5-30%,更优选为10-25%,更优选为15-20%。在本发明中,所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个,较佳地为1-3个,更佳地为1-2个,最佳地为1个。
对于绞链区和跨膜区(跨膜结构域),CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中,使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中,可选择跨膜结构域,或通过氨基酸置换进行修饰,以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域,从而最小化与受体复合物的其他成员的相互作用。
优选地,本发明的CAR构建物具有以下结构:CD8前导序列-CD19 scFv(VL-接头-VH)-CD8铰链区-CD8 TM-41BB-CD3-ζ。
在一个优选的实施方式中,本发明CAR的氨基酸序列如SEQ ID NO:14的第1-489位所示。需要特别注意的是,在本发明的靶向CD19的CAR的氨基酸序列中,对应于序列SEQ IDNO:14的第173位为甘氨酸(Gly)。
靶向CD19的通用型CAR-T细胞
如本文所用,术语“CAR19-DNT细胞”、“靶向CD19的通用型CAR-DNT细胞”、“本发明的CAR-T细胞”、“本发明的通用型CAR-T细胞”、可互换使用,均指本发明第一方面所述的表达具有式I结构的特异性靶向人CD19分子的嵌合抗原受体的通用型双阴性T细胞。
在本发明中,所述的靶向CD19的通用型CAR-DNT细胞中表达本发明的CAR构建物。
优选地,所述的靶向CD19的通用型CAR-DNT细胞中的TCR未被敲除。
制备方法
在本发明中,还提供了一种制备本发明的靶向CD19的通用型CAR-DNT细胞的方法,包括步骤:
(i)提供一表达载体,所述表达载体中含有表达本发明CAR构建物的核苷酸序列;
(ii)提供一DNT细胞培养液,所述细胞培养液中含有至少一个DNT细胞;将步骤(i)的表达载体转导入对所述DNT细胞,从而获得本发明的靶向CD19的通用型CAR-DNT细胞;和
(iii)任选的检测步骤(ii)所获得的通用型CAR-DNT细胞。
优选地,本发明制备方法中的表达载体是病毒表达载体,特别优选地是慢病毒表达载体。
值得注意的是,对于所述方法的步骤(ii)中的DNT细胞培养液的获得,需要以下步骤:
(iia)从健康供体收集外周血样品;
(iib)去除所述外周血样品中的CD4+CD8+T细胞,从而获得DNT细胞;
(iic)在包被有CD3单克隆抗体的培养瓶中,用适合的培养基培养和扩增(iib)中获得的DNT细胞,从而获得步骤(ii)中所需的含DNT细胞的培养体系。
优选地,在所述步骤(iic)的培养基中,添加有选自下组的物质:庆大霉素、重组人白介素2、、重组人白介素7、重组人白介素12、重组人白介素15、自体血浆、AB血清,或其组合。
在另一优选例中,在所述步骤(iic)的培养基中,所述庆大霉素的浓度为40-80单位/ml(优选为60单位/ml),所述重组人白介素2的浓度为150-100IU/ml(优选为200-250IU/ml),,所述重组人白介素15的浓度为5-20ng/ml(优选为10ng/ml),所述重组人白介素7的浓度为1-5ng/ml(优选为2ng/ml),所述重组人白介素12的浓度为5-20ng/ml(优选为10ng/ml),所述自体血浆的浓度为3-25v%(优选为10-20v%),和/或所述AB血清的浓度为4-8v%(优选为6v%)。
在本发明的一个优选的实施方式中,所述步骤(iic)的培养基中,不包含重组的或野生型的人白介素4,并且不包含AB血清。
药物组合物
本发明提供了一种含有本发明第一方面所述的靶向CD19的通用型CAR-T细胞,以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中,所述药物组合物为液态制剂。优选地,所述制剂为注射剂。优选地,所述制剂中所述CAR-T细胞的浓度为1×105-1×108个细胞/ml,更优地1×105-1×106个细胞/ml。
在一个实施方式中,所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等;碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇;蛋白质;多肽或氨基酸诸如甘氨酸;抗氧化剂;螯合剂诸如EDTA或谷胱甘肽;佐剂(例如,氢氧化铝);和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明还提供了本发明第一方面的靶向CD19的通用型CAR-T细胞和本发明第三方面所述的药物组合物的用途,用于制备预防和/或治疗癌症的药物组合物或制剂,以及用于治疗/或预防癌症。
所述的癌症包括各个进展期的肿瘤,包括临床上I期到IV期肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤,例如白血病和淋巴瘤)或实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤,和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤,例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病,包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌、卵巢癌。
本发明的通用型CAR-T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地,哺乳动物为人。
对于离体免疫细胞制备,以下中的至少一项在将细胞施用进入哺乳动物前在体外发生:i)扩增细胞,ii)将编码CAR的核酸引入细胞,和/或iii)冷冻保存细胞。
离体细胞处理程序在本领域中是公知的,并在以下更完全地进行讨论。简单地说,细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体对上述细胞进行基因修饰(即体外转导或转染)。CAR-修饰的细胞可被施用于哺乳动物接受者,以提供治疗益处。哺乳动物接受者可为人,CAR-修饰的细胞相对于接受者可为自体,也可为同种异基因的、同基因的(syngeneic)。
除了就离体免疫细胞而言使用基于细胞的疫苗之外,本发明也提供了用于体内以增强针对患者中靶向抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法,其包括施用给需要的对象以有效量的本发明的通用型CAR-T细胞。
本发明的通用型CAR-T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-15、IL-17或其他细胞因子或细胞群结合施用。简单地说,本发明的药物组合物可包括如本文所述的靶细胞,与一种或多种药学或临床上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等;碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇;蛋白质;多肽或氨基酸诸如甘氨酸;抗氧化剂;螯合剂诸如EDTA或谷胱甘肽;佐剂(例如,氢氧化铝);和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定,如患者的病症的特征、疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时,待施用的本发明组合物的精确量可由医师确定,其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出:包括本文描述的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量,优选105至107个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等,New Eng.J.of Med.319:1676,1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象,治疗方案由医学领域技术人员确定。
对象组合物的施用可以任何方便的方式进行,包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内、胸膜内施用给患者。在另一个实施方式中,本发明的T细胞组合物优选通过i.v.静脉内注射施用。T细胞的组合物可被直接注入肿瘤,淋巴结或感染位置。(CART细胞产品以静脉输注为主,可采用直接注入肿瘤,淋巴结或感染位置)
在本发明的某些实施方式中,利用本文描述的方法或本领域已知的其他将T细胞扩增至治疗性水平的方法活化和扩增细胞,与任何数量的有关治疗形式结合(例如,之前、同时或之后)施用给患者,所述治疗形式包括但不限于用以下试剂进行治疗:所述试剂诸如抗病毒疗法、西多福韦、白细胞介素-2、IFN-γ、阿糖胞苷(也已知为ARA-C)等其它细胞毒化疗药物、检验点抑制剂,如PD-1抗体、抗CTLA-4抗体和抑制细胞因子风暴的药物,如抗IL-6受体的托珠抗体等其他治疗。在进一步的实施方式中,本发明的T细胞可与以下结合使用:化疗、辐射、免疫抑制剂,诸如,环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和,抗体或其他免疫治疗剂。在进一步的实施方式中,本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如,之前、同时或之后)而施用给患者。例如,在一个实施方式中,对象可经历高剂量化疗的标准治疗,之后进行外周血干细胞移植。在一些实施方式中,在移植后,对象接受本发明的扩增的免疫细胞的注入。在一个额外的实施方式中,扩增的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常,每次治疗或每个疗程,可将1×106个至1×1010个本发明的通用型CAR-DNT细胞,通过例如静脉回输的方式,施用于患者。
本发明的主要优点包括:
1)本发明中的CD19 CAR为人源化的嵌合抗原受体,与全小鼠原性嵌合抗原受体相比,具有免疫原性低,不易引起免疫排斥反应的优点。
2)本发明中的CAR19-DNT细胞为无需基因编辑而制备的通用型免疫细胞产品,工艺简单,安全性更好,DNT细胞杀伤肿瘤不需要TCR分子参与,不具有MHC限制性,也不会引起GvHD和HvG,可以作为一个通用型免疫细胞产品,避免了敲除TCR等基因所需要的繁琐构建过程以及大规模测序和脱靶效率高的问题。
3)本发明中所提供的构建及扩增CAR-DNT的方法可实现规模化生产,降低生产成本,与1对1的个体化治疗相比,该通用型CAR-DNT细胞产品,大大降低生产成本,减轻病人负担。
4)本发明中提供的CAR-DNT细胞,不受患者来源限制,可大规扩增来源于健康捐赠者的免疫细胞,细胞活性更好且可即时为患者提供治疗,解决了现在自体CART细胞治疗过程中肿瘤中晚期患者淋巴细胞数量低、质量差以及数量不足等问题。
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用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
本发明使用了第二代CD19-CAR,在VH的CDR1区中包含突变V27G的人源化CD19scFv(克隆11),图2显示了人源化的CD19 CAR构建体的结构示意图。以下通过实施例对本发明作进一步的说明,但本发明并不限于这些具体实施方式。
实施例1:构建人源化的anti-CD19 FMC63 scFv-41BB-CD3ζ
本发明在慢病毒载体的XbaI和EcoRI位点间插入了人源化的CD19-ScFv-CAR结构,该结构在XbaI和EcoRI克隆位点之间包含人源化CD19 ScFv-41BB-CD3ζ的插入片段。
1.1人源化CD19抗体:VH和VL和scFv的序列
本发明从小鼠CD19 FMC63 scFv克隆中获得了人源化的CD19 scFv,选择把其CDR1突变为人源化的scFv(克隆11)。人源化CD19 scFv的结构为:VL-连接肽-VH。连接肽序列为GSTSGSGKPGSGEGSTKG(SEQ ID NO.:7)。
核苷酸序列中粗体为人源化CD19 VL的序列(SEQ ID NO:1);常规字体为VH的核苷酸序列(SEQ ID NO:2,粗体标记为编码G的突变(ggc));中间的斜体为(SEQ ID NO:3)编码连接肽序列GSTSGSGKGPGSGEGSTKG的核苷酸序列(SEQ ID NO:7)。
人源化CD19 scFv(SEQ ID NO:4):粗体为VL,规则字体为VH,下划线为CDR区,突变的氨基酸位置为VH(SEQ ID NO:6)的第27位,显示为大号斜体字G(VH的V27G)
氨基酸序列中,粗体为VL的氨基酸序列(SEQ ID NO:5);常规字体为VH的氨基酸序列(SEQ I D NO:6);斜体为连接肽的氨基酸序列(SEQ ID NO:7)。
本发明通过3D模型(图3)和抗原抗体结合实验,发现对人源化CD19抗体VH的CDR1区域突变可以改善抗体与CD19抗原的结合能力。
1.2人源化的CD19-CAR序列
人源化CD19-CAR结构如图2所示。使用EF1a启动子的慢病毒载体克隆人源化scFvCAR序列。
以下核苷酸序列是人源化的CD19ScFv-CD8 hinge-TM8-41BB-CD3ζ的CD8前导序列。CAR结构包括人CD8信号肽,人源化CD19 scFv(VL-铰链–VH),CD8铰链,CD8跨膜,41BB共刺激结构域和CD3ζ激活结构域(图2)。CD8 leader sequence-CD19 scFv(VL-Linker–VH)-CD8 hinge-CD8 TM-41BB-CD3-zeta:
<CD8前导>
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG(SEQID NO.:8)
<人源化CD19(VL-linker-VH),clone 11scFv>(粗体标记为编码V27G的突变序列ggc)
<CD8铰链>
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ IDNO.:9)
<CD8 TM>
ATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO.:10)
<4-1BB共刺激结构域>
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO.:11)
<CD3ζ>
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAG(SEQ ID NO.:12)
<EcoRI restriction site>
gaattc(SEQ ID NO.:13)
人源化CD19-4-1BB-CD3-CAR蛋白的氨基酸序列如下(有关构建体结构,请参见图3),其中CDR1 VH中的突变以粗体下划线标识:
人源化CD19-4-1BB-CD3-CAR蛋白的核苷酸序列如下:
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGgatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcgcgagccaggatattagcaaatatctgaactggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatcataccagccgcctgcatagcggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgccagcagggcaacaccctgccgtatacctttggcggcggcaccaaagtggaaattaaa
ggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggccaggtgcagctgcaggaaagcggcccgggcctggtgaaaccgagcgaaaccctgagcctgacctgcaccgtgagcggcggcagcctgccggattatggcgtgagctggattcgccagccgccgggcaaaggcctggaatggattggcgtgatttggggcagcgaaaccacctattataacagcgcgctgaaaagccgcgtgaccattagcgtggataccagcaaaaaccagtttagcctgaaactgagcagcgtgaccgcggcggataccgcggtgtattattgcgcgaaacattattattatggcggcagctatgcgatggattattggggccagggcaccctggtgaccgtgagcagc
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
ATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAG(SEQ ID NO.:16)
实施例2:慢病毒包装
293T细胞培养于37℃,5%CO2孵箱内,培养基为DMEM+10%FBS。第2天待细胞达到90%的汇合度时,用表达质粒以及包装质粒psPAX2、pMD2.0G共转,将以适合摩尔比混合的质粒加入培养器皿内,轻晃、混匀,放入孵箱。48-72小时后,可以收获病毒,离心去除漂浮死亡的293T细胞,然后将含病毒培养基过滤,浓缩,纯化,分装,-80℃冻存以及测定滴度。
实施例3:CAR-19DNT细胞的制备
3.1采集人体外周血样品
从健康供体收集30-400ml外周血到含肝素钠管中。
3.2 CAR19-DNT细胞的制备和检测
3.2.1 CAR19-DNT细胞的制备
方法一:
第0天,按照厂商说明书,利用试剂盒(Stem Cell TechnologiesInc),通过与RBC的Rosetting去除CD4+、CD8+T细胞。血液样品与抗人CD4和CD8去除试剂标记在室温下孵育20分钟,然后血液在50ml离心管与等体积Ficoll-Hypaque的密度梯度分层。在2500rpm离心25分钟后,在Ficoll和血浆的界面收集已去除PBMC中CD4+和CD8+的细胞,即DNT细胞,用0.9%生理盐水洗涤1次。将获得的DNT细胞在75cm2培养瓶中以1-6×106个/ml细胞在AIM-V培养基中37℃和5%CO2培养,所述的培养瓶已用抗人CD3单克隆抗体(克隆OKT3)(5-20μg/ml)包被,所述在AIM-V培养基包含了庆大霉素(60单位/ml)、重组人白介素2(250IU/ml)、重组人白介素15(10ng/ml)、重组人白介素7(2ng/ml)、重组人白介素12(10ng/ml)、自体血浆(20v%)。
第1天,将制备获得的DNT细胞培养过夜后,加入MOI为5的慢病毒感染过夜,随后根据细胞状态每天或者隔天补加AIM-V培养基;
第5天,DNT细胞已充分活化,增殖旺盛,用新鲜的AIM-V增殖培养基(含60单位/ml庆大霉素、500IU/ml重组人白介素2和重组人白介素15(10ng/ml)、重组人白介素7(2ng/ml)、重组人白介素12(10ng/ml)、自体血浆(10v%)将DNT细胞调整成1-3×106个细胞/ml浓度继续扩增培养;
第7天,传至175cm2培养瓶,以1-3×106个/ml细胞在AIM-V培养基中继续培养3天,所述AIM-V培养基包含了庆大霉素(60单位/ml)、重组人白介素2(250IU/ml)、重组人白介素15(10ng/ml)、重组人白介素12(10ng/ml)和50ng/ml抗人CD3单克隆抗体。
第10天,将175cm2培养瓶细胞传入培养袋中,以1-3×106个/ml细胞在AIM-V培养基中继续培养至第14天,所述AIM-V培养基包含了庆大霉素(60单位/ml)、重组人白介素2(500IU/ml)、重组人白介素15(10ng/ml)、重组人白介素12(10ng/ml)和100ng/ml抗人CD3单克隆抗体。
第14天,收获CAR-19DNT细胞,用250ml尖底离心瓶收集细胞,900×g离心10分钟,再以含2.5%人血白蛋白的生理盐水(又称“溶媒”)洗涤。将收集到的CAR-19-DNT细胞以0.1-1×107个细胞/mL的浓度冻存在液氮中,此为CAR19-DNT细胞制剂成品,质检合格后可供临床使用。
方法二:
第0天,按照厂商说明书,利用试剂盒(Stem Cell TechnologiesInc),通过与RBC的Rosetting去除CD4+、CD8+T细胞。血液样品与抗人CD4和CD8去除试剂标记在室温下孵育20分钟,然后血液在50ml离心管与等体积Ficoll-Hypaque的密度梯度分层。在2500rpm离心25分钟后,在Ficoll和血浆的界面收集已去除PBMC中CD4+和CD8+的细胞,即DNT细胞,用0.9%生理盐水洗涤1次。将分离纯化的DNT细胞(磁珠:DNT细胞=1:1)与CD3/CD28磁珠混匀,在75cm2培养瓶中以1-6×106个/ml细胞在AIM-V培养基中37℃和5%CO2培养,所述在AIM-V培养基包含有庆大霉素(60单位/ml)、重组人白介素2(500IU/ml)、重组人白介素15(2ng/ml)、重组人白介素7(2ng/ml)、重组人白介素12(10ng/ml)和自体血浆(20v%)。
第1天,将制备获得的DNT细胞培养过夜后,加入MOI为5的慢病毒感染过夜,随后根据细胞状态每天或者隔天补加AIM-V培养基;
第5天,将培养瓶中的DNT细胞吹打均匀后过磁力架去除CD3/CD28磁珠,用新鲜的AIM-V增殖培养基(含60单位/ml庆大霉素、500IU/ml重组人白介素2和组人白介素15(10ng/ml)、重组人白介素7(2ng/ml)、重组人白介素12(10ng/ml)、自体血浆(10v%)将DNT细胞调整成1-3×106个细胞/ml浓度继续扩增培养;
第7天,传至175cm2培养瓶,以1-3×106个/ml细胞在AIM-V培养基中继续培养3天,所述AIM-V培养基包含了庆大霉素(60单位/ml)、重组人白介素2(500IU/ml)、重组人白介素15(10ng/ml)、重组人白介素12(10ng/ml)。
第10天,将175cm2培养瓶细胞传入培养袋中,以1-3×106个/ml细胞在AIM-V培养基中继续培养至第14天,所述AIM-V培养基包含了庆大霉素(60单位/ml)、重组人白介素2(500IU/ml)、重组人白介素15(10ng/ml)、重组人白介素12(10ng/ml)。
第14天,收获CAR-19DNT细胞,用250ml尖底离心瓶收集细胞,900×g离心10分钟,再以含2.5%人血白蛋白的生理盐水(又称“溶媒”)洗涤。将收集到的CAR19-DNT细胞以0.1-1×107个细胞/mL的浓度冻存在液氮中,此为CAR19-DNT细胞制剂成品,质检合格后可供临床使用。
3.2.2 CAR-19DNT细胞表型及阳性率检测
第8天和第13天分别进行DNT表型及CAR19-DNT阳性率检测,此时可获得高纯度的DNT细胞(纯度达到85%以上,如图4)以及hCD19-CAR阳性较高的CAR19-DNT细胞,使用流式细胞仪通过抗人的CD3抗体、CD4抗体、CD8抗体标记对培养体系中DNT细胞进行鉴定(图4),同时用抗人源化CD19的FMC63-CD19抗体标记进行转导阳性率检测(图4)。
实施例4:细胞杀伤实验
通过慢病毒感染的方法建立表达CD19分子的Hela细胞(Hela-CD19的稳定细胞系)做为靶细胞,采用不表达CD19的Hela细胞()作为靶细胞对照;培养第8天和第13天分别收集的未转导CAR-CD19的DNT细胞和已转导人源化CAR-CD19的CD19CAR-DNT细胞作为效应细胞,效靶比为2:1,用RTCA(Real-Time Cytotoxicity Assay)实时监测杀伤情况。
结果如图5所示:与未转导CD19-CAR的DNT细胞相比,转导了人源化CD19CAR的CAR19-DNT细胞特异性杀伤Hela-CD19细胞,上述两种细胞在杀伤CD19阴性的Hela细胞上没有显著差异。由此表明,人源化CAR19-DNT细胞特异性靶向表达CD19抗原的Hela-CD19靶细胞(图5)。5-A显示效靶比为2:1时,用RTCA监测的实时动态杀伤情况;达到最大杀伤时间时(18小时),RTCA监测的实时定量杀伤结果如图5-B所示。定量杀伤结果显示培养第8天和第13天CAR19-DNT细胞对Hela-CD19的特异性杀伤分别高达98.52%和98.10%(图5-B),且最大杀伤效应随着杀伤时间的延长可持续保持(图5-A)。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
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<110> 浙江瑞加美生物科技有限公司
<120> 一种制备通用型人源化CAR19-DNT细胞的技术及其应用
<130> P2019-2103
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 321
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca ggatattagc aaatatctga actggtatca gcagaaaccg 120
ggcaaagcgc cgaaactgct gatttatcat accagccgcc tgcatagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag ggcaacaccc tgccgtatac ctttggcggc 300
ggcaccaaag tggaaattaa a 321
<210> 2
<211> 360
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg 60
acctgcaccg tgagcggcgg cagcctgccg gattatggcg tgagctggat tcgccagccg 120
ccgggcaaag gcctggaatg gattggcgtg atttggggca gcgaaaccac ctattataac 180
agcgcgctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg 240
aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgaa acattattat 300
tatggcggca gctatgcgat ggattattgg ggccagggca ccctggtgac cgtgagcagc 360
<210> 3
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54
<210> 4
<211> 245
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
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 Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser 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 Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser
130 135 140
Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Val
180 185 190
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
195 200 205
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
225 230 235 240
Val Thr Val Ser Ser
245
<210> 5
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
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 Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser 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 Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 6
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 7
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 8
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 9
<211> 135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 10
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atctacatct gggcgcccct ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 11
<211> 126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 12
<211> 342
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgctaat ag 342
<210> 13
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaattc 6
<210> 14
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
130 135 140
Lys Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
145 150 155 160
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Pro
165 170 175
Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
180 185 190
Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala
195 200 205
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
210 215 220
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro
260 265 270
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
275 280 285
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
290 295 300
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
305 310 315 320
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
325 330 335
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
340 345 350
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
355 360 365
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
370 375 380
Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
385 390 395 400
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
405 410 415
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
420 425 430
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
435 440 445
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
465 470 475 480
Leu His Met Gln Ala Leu Pro Pro Arg
485
<210> 15
<211> 735
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca ggatattagc aaatatctga actggtatca gcagaaaccg 120
ggcaaagcgc cgaaactgct gatttatcat accagccgcc tgcatagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag ggcaacaccc tgccgtatac ctttggcggc 300
ggcaccaaag tggaaattaa aggctccacc tctggatccg gcaagcccgg atctggcgag 360
ggatccacca agggccaggt gcagctgcag gaaagcggcc cgggcctggt gaaaccgagc 420
gaaaccctga gcctgacctg caccgtgagc ggcggcagcc tgccggatta tggcgtgagc 480
tggattcgcc agccgccggg caaaggcctg gaatggattg gcgtgatttg gggcagcgaa 540
accacctatt ataacagcgc gctgaaaagc cgcgtgacca ttagcgtgga taccagcaaa 600
aaccagttta gcctgaaact gagcagcgtg accgcggcgg ataccgcggt gtattattgc 660
gcgaaacatt attattatgg cggcagctat gcgatggatt attggggcca gggcaccctg 720
gtgaccgtga gcagc 735
<210> 16
<211> 1473
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatattc agatgaccca gagcccgagc agcctgagcg cgagcgtggg cgatcgcgtg 120
accattacct gccgcgcgag ccaggatatt agcaaatatc tgaactggta tcagcagaaa 180
ccgggcaaag cgccgaaact gctgatttat cataccagcc gcctgcatag cggcgtgccg 240
agccgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctgcag 300
ccggaagatt ttgcgaccta ttattgccag cagggcaaca ccctgccgta tacctttggc 360
ggcggcacca aagtggaaat taaaggctcc acctctggat ccggcaagcc cggatctggc 420
gagggatcca ccaagggcca ggtgcagctg caggaaagcg gcccgggcct ggtgaaaccg 480
agcgaaaccc tgagcctgac ctgcaccgtg agcggcggca gcctgccgga ttatggcgtg 540
agctggattc gccagccgcc gggcaaaggc ctggaatgga ttggcgtgat ttggggcagc 600
gaaaccacct attataacag cgcgctgaaa agccgcgtga ccattagcgt ggataccagc 660
aaaaaccagt ttagcctgaa actgagcagc gtgaccgcgg cggataccgc ggtgtattat 720
tgcgcgaaac attattatta tggcggcagc tatgcgatgg attattgggg ccagggcacc 780
ctggtgaccg tgagcagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840
atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900
gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cctggccggg 960
acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020
aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080
gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc ccctcgctaa tag 1473
Claims (10)
1.一种靶向CD19的通用型CAR-T细胞,其特征在于,所述通用型CAR-T细胞表达一外源的CAR构建物,所述CAR构建物具有如式I所示的结构,
L-scFv-H-TM-C-CD3ζ (式I)
式中,
L为无或信号肽序列;
scFv为靶向CD19的抗体单链可变区序列;
H为无或铰链区;
TM为跨膜结构域;
C为共刺激信号分子;
CD3ζ为源于CD3ζ的胞浆信号传导序列;
各“-”独立地表示连接上述各元件的连接肽或肽键。
2.如权利要求1所述的靶向CD19的通用型CAR-T细胞,其特征在于,所述靶向CD19的通用型CAR-T细胞是靶向CD19的通用型CAR-双阴性T细胞(CAR19-DNT细胞)。
3.如权利要求1所述的靶向CD19的通用型CAR-T细胞,其特征在于,所述通用型CAR-T细胞中的TCR未被敲除。
4.如权利要求1所述的靶向CD19的通用型CAR-T细胞,其特征在于,所述CAR构建物具有如SEQ ID NO:14所示的氨基酸序列。
5.一种制备如权利要求1所述的靶向CD19的通用型CAR-DNT细胞的方法,其特征在于,包括步骤:
(i)提供一表达载体,所述表达载体中含有编码如式I所示的CAR构建物的核苷酸序列;
L-scFv-H-TM-C-CD3ζ (式I)
式中,
L为无或信号肽序列;
scFv为靶向CD19的抗体单链可变区序列;
H为无或铰链区;
TM为跨膜结构域;
C为共刺激信号分子;
CD3ζ为源于CD3ζ的胞浆信号传导序列;
各“-”独立地表示连接上述各元件的连接肽或肽键;
(ii)提供一DNT细胞培养液,所述细胞培养液中含有至少一个DNT细胞;将步骤(i)的表达载体转导入对所述DNT细胞,从而获得如权利要求1所述的靶向CD19的通用型CAR-DNT细胞;和
(iii)任选的检测步骤(ii)所获得的通用型CAR-DNT细胞。
6.如权利要求5所述的方法,其特征在于,所述表达载体为慢病毒表达载体。
7.如权利要求5所述的方法,其特征在于,所述步骤(i)的病毒表达载体中所整合有的多核苷酸序列如SEQ ID NO:16所示。
8.一种药物组合物,其特征在于,包括:
(a)如权利要求1或2所述的靶向CD19的通用型CAR-T细胞;和
(b)药学上可接受的载体、稀释剂或赋形剂。
9.一种如权利要求1或2所述的靶向CD19的通用型CAR-T细胞的用途,其特征在于,用于制备预防和/或治疗癌症的药物组合物或制剂。
10.如权利要求9所述的用途,其特征在于,所述肿瘤选自下组:血液肿瘤、实体瘤、或其组合。
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CN202010314336.0A CN113528449A (zh) | 2020-04-20 | 2020-04-20 | 一种制备通用型人源化car19-dnt细胞的技术及其应用 |
EP21792496.8A EP4141118A1 (en) | 2020-04-20 | 2021-04-14 | Technique for preparing universal humanised car19-dnt cells and application therefor |
CN202180029612.1A CN115708414A (zh) | 2020-04-20 | 2021-04-14 | 一种制备通用型人源化car19-dnt细胞的技术及其应用 |
US17/920,375 US20230279107A1 (en) | 2020-04-20 | 2021-04-14 | Technique for preparing universal humanised car19-dnt cells and application therefor |
JP2022563458A JP2023522112A (ja) | 2020-04-20 | 2021-04-14 | 汎用型ヒト化car19-dnt細胞を製造する技術およびその使用 |
PCT/CN2021/087311 WO2021213235A1 (zh) | 2020-04-20 | 2021-04-14 | 一种制备通用型人源化car19-dnt细胞的技术及其应用 |
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CN110734931A (zh) * | 2019-11-18 | 2020-01-31 | 山东省齐鲁细胞治疗工程技术有限公司 | 一种靶向CD19的人源化scFv嵌合抗原受体T细胞及制备方法、应用 |
CN110964121B (zh) * | 2019-12-20 | 2022-09-16 | 山东省齐鲁细胞治疗工程技术有限公司 | 一种分泌il-7、il-15因子的cd19-car-t细胞及其应用 |
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WO2023078425A1 (zh) * | 2021-11-05 | 2023-05-11 | 浙江瑞加美生物科技有限公司 | 一种体外高效稳定转导抗原嵌合受体双阴性t细胞的方法 |
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