CN117305368A - 一种快速制备t细胞的方法 - Google Patents
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Abstract
本发明涉及快速制备T细胞的方法,具体提供一种制备表达功能分子的T细胞的方法,包括步骤:1)PBMC复苏缓解1~4小时,2)由PBMC分选并活化CD3+T细胞10~36小时,3)使用含有功能分子的编码序列的逆转录病毒转导CD3+T细胞得到所述表达功能分子的T细胞。本发明方法大幅缩短表达功能分子的T细胞的总制备时长,制备得到的T细胞具有高活化状态、更高的T细胞干性、更优秀的扩增及肿瘤杀伤能力。
Description
技术领域
本发明属于生物领域,具体涉及免疫细胞治疗领域。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor,CAR)T细胞是一种细胞治疗产品,利用基因工程改造T细胞,使细胞表面表达识别肿瘤恶性细胞特异性抗原的CAR分子,引导CAR-T细胞与恶性细胞结合并启动T细胞杀伤功能。CAR分子序列结构包含特异性识别抗原的单链抗体序列、影响靶向效率的铰链区、固定CAR分子于细胞膜上的跨膜区、传递胞内信号的T细胞激活结构域及加强信号传递的胞内共刺激结构域。借由设计靶向不同抗原的单链抗体序列,能够制造出针对表达不同抗原的肿瘤恶性细胞。CAR基因序列由逆转录病毒载体携带并转导至患者自体或供者异体来源的活化T细胞中,经体外培养扩增后,回输患者体内。在体内,CAR-T细胞的单链抗体序列与其靶向的肿瘤恶性细胞抗原结合,引起胞内结构域下游信号传递,启动T细胞毒杀功能,达到清除肿瘤恶性细胞的效果。
CD19 CAR-T细胞作为第一个商业化的CAR-T细胞疗法,在美国已逐渐开始被广泛运用于治疗B-ALL、LBCL及FL,并有极高的市场收益。然而,高营收的背后是CAR-T产品的超高定价,在中国两项上市产品定价均约120万人民币,美国产品定价约为40万美元,非常规药物能企及。CAR-T产品的高定价来自其研发阶段付出的成本远高于一般传统药物,且生产技术复杂,高度要求生产人员及相关设备的准确性及稳定度。如此高昂的价格使产品难以触及有需求的患者。因此,为触及更多患者需思考如何降低定价,也就是从降低CAR-T细胞生产成本,且不影响CAR-T细胞质量开始。一般CAR-T细胞生产过程总时长约在10到25天之间,其中生产人力的调动及各式耗材试剂的使用所形成的成本不可小觑。因此,降低CAR-T细胞生产成本的根本方式,最直观者为缩短CAR-T细胞制备时长。缩短CAR-T细胞制备时长除了可以降低成本,更能避免患者在等待CAR-T细胞制备完成的期间发生疾病进展的情况。
现有技术的CAR-T细胞制备通常包含以下几个工序:一、患者或供者机采血采集:利用血细胞分离机自患者或供者血液中富集白细胞,低温运输至细胞制备中心;二、自单采血中分离PBMC:手动或自动方式分离富集白细胞中的PBMC,其中含淋巴细胞及单核细胞,去除大部分红细胞、多核细胞、血小板及血浆。三、自PBMC中分离及活化T细胞:利用共价偶联抗-CD3和/或抗-CD28抗体的磁珠或其他固体相界面与PBMC共同孵育后,磁性分离与磁珠结合的T细胞;四、CAR基因修饰T细胞:利用病毒载体或其他基因转导方式将CAR基因序列转入T细胞中,使其在细胞表面上表达CAR分子;五、CAR-T细胞体外扩增:将CAR-T细胞于细胞培养板、细胞培养瓶或细胞培养袋中进行扩增培养;六、CAR-T细胞灌装及冻存:收获CAR-T细胞并保存于液氮中备用。
以上多数工序可在一天内操作完成,但CAR-T细胞体外扩增工序为速率决定步骤,且根据细胞原材料的个体化差异及不同的所需剂量,扩增时间一般不低于三天,且可能长至十天以上。
发明内容
为了CAR-T细胞缩短制备时长,本发明提供了一种制备表达功能分子的T细胞的方法。所述方法包括步骤:
1)PBMC复苏缓解1~4小时,优选2-3小时,
2)由PBMC分选并活化CD3+T细胞10~36小时,优选15~24小时,
3)使用含有功能分子的编码序列的逆转录病毒转导CD3+T细胞得到所述表达功能分子的T细胞,所述转导包括共培养逆转录病毒和CD3+T细胞12-36小时,优选15-24小时。
在一个或多个实施方案中,所述功能分子是CAR。优选为含有抗CD19抗体或其抗原结合片段、铰链区、跨膜区、CD28胞内共刺激结构域、CD3-zeta信号转导结构域的CD19-28z。更优选地,所述抗CD19抗体或其抗原结合片段是抗人CD19抗体单链可变区。
在一个或多个实施方案中,所述编码序列构建在逆转录病毒质粒中。
在一个或多个实施方案中,步骤1)包括:将PBMC在培养基中以适合PBMC生长的条件培养1~4小时,优选2-3小时。
在一个或多个实施方案中,步骤1)中的培养基包括AIM-V、X-VIVO、DMEM、RPMI1640,优选为X-VIVO15。所述培养基还添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆中的一种或多种或全部。
在一个或多个实施方案中,适合PBMC生长的条件是约37℃及约5%CO2。
在一个或多个实施方案中,步骤2)包括:
2.1)在DPBS中,使用抗体由PBMC中分选CD3+T细胞,所述抗体包括抗CD3抗体,和
2.2)在培养基中以适合CD3+T细胞活化的条件孵育CD3+T细胞12-36小时,优选15-24小时。
在一个或多个实施方案中,所述抗体还包括抗CD28抗体。
在一个或多个实施方案中,步骤2)中的培养基含IL-2。所述培养基包括AIM-V、X-VIVO、DMEM或RPMI1640,优选为X-VIVO15。所述培养基还添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆中的一种或多种或全部。
在一个或多个实施方案中,适合CD3+T活化的条件是约37℃及约5%CO2。
在一个或多个实施方案中,所述抗体是标记的抗体。优选地,所述抗体偶联于固相载体上。更优选地,所述固相载体是磁颗粒。
在一个或多个实施方案中,步骤3)包括:在培养基中以适合逆转录病毒转导CD3+T细胞的条件共培养逆转录病毒和CD3+T细胞12-36小时,优选15-24小时。
在一个或多个实施方案中,步骤3)中的培养基含IL-2。所述培养基包括AIM-V、X-VIVO、DMEM或RPMI1640,优选为X-VIVO15。所述培养基还添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆中的一种或多种或全部。
在一个或多个实施方案中,适合逆转录病毒转导CD3+T细胞的条件是约37℃及约5%CO2。
在一个或多个实施方案中,步骤3)所述转导的转导MOI为0.1-2,优选0.5-1.5。
在一个或多个实施方案中,步骤3)中,CD3+T细胞的密度为1-5*106cells/mL,优选1*106cells/mL。
在一个或多个实施方案中,所述方法还包括去除固相载体的步骤。优选地,去除固相载体的步骤位于步骤3)之后。
在一个或多个实施方案中,所述方法还包括步骤:4)将步骤3)获得所述表达功能分子的T细胞冻存;具体包括:以氯化钠注射液洗涤T细胞,以冻存液重悬T细胞,程序降温,和液氮冻存。
本发明还提供由本发明方法制备获得的CAR-T细胞。
在一个或多个实施方案中,所述CAR为含有抗CD19、CD28胞内共刺激结构域、CD3-zeta胞内共刺激结构域的CD19-28z。
本发明优点:
-将PBMC复苏缓解时长缩短为2小时,不影响复苏后部分死细胞团块成絮,便于使用细胞滤网去除的特性;
-将CD3+T细胞的分选活化培养时长缩短为22-24小时,细胞可以得到适当的活化且不影响后续的病毒转导;
-细胞悬液不去除磁性颗粒,保留部分活化功能,直接进行病毒转导;
-去除CAR-T细胞体外扩大培养工序,在完成病毒转导时,去除磁珠后冻存CAR-T细胞,大幅缩短CAR-T细胞总制备时长。
附图说明
图1是Dash CAR-T细胞制备流程示意图。
图2是Dash CAR-T与一般CAR-T制备流程比较图。
图3是Dash CAR-T工艺与一般工艺的制备的CAR-T细胞性状检测结果。
图4是Dash CAR-T工艺与一般工艺的制备的CAR-T细胞功能检测结果。
图5是Dash CAR-T工艺与一般工艺的制备的CAR-T细胞的小鼠体内药效学研究结果。
具体实施方式
基于对缩短CAR-T细胞制备时长的需求,本发明提供了一种自PBMC开始在48小时内完成制备的CAR-T细胞工艺(以下称为Dash CAR-T)。
术语
CAR-T:嵌合抗原受体(Chimeric Antigen Receptor-T cell,CAR-T)T细胞是指经基因修饰后,能以MHC非限制性方式识别特定目的抗原,并且持续活化扩增的T细胞。2012年国际细胞治疗协会年会中指出生物免疫细胞治疗已经成为手术、放疗、化疗外的第四种治疗肿瘤的手段,并将成为未来肿瘤治疗必选手段。CAR-T细胞回输治疗是当前肿瘤治疗中最明确有效的免疫治疗形式。大量研究表明,CAR-T细胞可以有效的识别肿瘤抗原,引起特异性的抗肿瘤免疫应答,显著改善患者的生存状况。
PBMC:外周血单个核细胞(Peripheral Blood Mononuclear Cell,PBMC)是指外周血中的单个核细胞,主要包含淋巴细胞及单核细胞和其他少量细胞。为目前T细胞相关细胞治疗的重要原材料。
DPBS:DPBS全称Dulbecco's Phosphate-Buffered Saline,即杜氏磷酸缓冲盐溶液,主要成分包括NaCl、KCl、KH2PO4、Na2HPO4等,pH为7.2-7.4。DPBS是细胞的平衡盐溶液,通常用于重悬细胞及短时间保存细胞离体状态。根据是否含有钙镁离子将DPBS分为两种,与常规PBS不同的是DPBS磷酸盐的含量稍低。
培养基:本文所述培养基包括适用于免疫细胞(特别是CD3+细胞及其他种类白细胞)的任何培养基。包括但不限于AIM-V、X-VIVO、DMEM或RPMI1640。
X-VIVO:为Lonza X-VIVOTM15培养基的简称。适合T淋巴细胞的增殖,可用于CD3+细胞及其他种类白细胞的生长。
CTS Dynabeads CD3/28:CTSTMDynabeadsTMCD3/CD28适用于人类T细胞的体外分离、活化与扩增。Dynabeads上同时偶联抗-CD3和抗-CD28抗体,可提供T细胞活化与扩增所需的初级和协同刺激信号。
本文中,样品是包含PBMC的任何血液来源的样品,例如全血。优选地,样品是符合采集规程及运输方式的机采单采血。
本发明首先提供一种制备表达功能分子的T细胞的方法,包括步骤:1)PBMC复苏缓解1~4小时,优选2-3小时,2)由PBMC分选并活化CD3+T细胞10~36小时,优选15~24小时,和3)使用含有功能分子的编码序列的逆转录病毒转导CD3+T细胞得到所述表达功能分子的T细胞,所述转导包括共培养逆转录病毒和CD3+T细胞12-36小时,优选15-24小时。
本文所述“功能分子”可以为任何需要在T细胞中表达的蛋白或多肽,优选为CAR。本发明所述的CAR可以是本领域周知的各种CAR。CAR可依次包含结合肿瘤细胞膜抗原的多肽(如scFv)、铰链区、跨膜区和胞内信号区。可采用本领域周知的用于构建CAR的铰链区、跨膜区和胞内信号区来构建本发明的CAR。通常,结合肿瘤细胞膜抗原的多肽能够以中等亲和力结合肿瘤细胞广泛表达的膜抗原,该多肽通常插入有抗原表位,插入的位置选自如下3个位置中的任意1个、2个或3个:所述多肽的N端、所述多肽和所述铰链区之间和所述多肽内部。所述结合肿瘤细胞膜抗原的多肽为天然多肽或人工合成多肽;优选地,人工合成多肽为单链抗体或Fab片段。
本发明的嵌合抗原受体可针对如下抗原中的一种或多种:CD19、CD20、CEA、GD2、FR、PSMA、PMEL、CA9、CD171/L1-CAM、IL-13RL1、MART-1、ERBB2、NY-ESO-1家族蛋白、BAGE家族蛋白、GAGE家族蛋白、AFP、MUC1、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、IL-11R/、EGP-2、EGP-40、FBP、GD3、PSCA、FSA、PSA、HMGA2、胎儿型乙酰胆碱受体、LeY、EpCAM、MSLN、IGFR1、EGFR、EGFRvIII、ERBB3、ERBB4、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC、AFU、EBV-VCA、POA和PROGRP。优选地,所述CAR为含有抗人CD19抗体单链可变区、铰链区、跨膜区、CD28胞内共刺激结构域、CD3-zeta信号转导结构域的CD19-28z。本发明方法将PBMC复苏缓解时长缩短为2小时,将CD3+T细胞的分选活化培养时长缩短为22-24小时,并且省去了去除CAR-T细胞体外扩大培养工序,大幅缩短CAR-T细胞总制备时长。此外,发明人发现,根据本发明方法制备得到的CAR-T细胞具有高活化状态及更高的T细胞干性,可能促成更优秀的扩增及肿瘤杀伤能力。
以上结果很好的展示了Dash CAR-T细胞因T细胞干性强而具有的高度扩增能力及肿瘤杀伤能力。
本文所述“标记”的抗体是便于将抗体(例如抗CD3抗体和抗CD28抗体)和含表面抗原(例如CD3和CD28)的细胞的复合物与体系中的其他组分分离的物质,例如生物素、固相载体,其中抗CD3抗体和/或抗CD28抗体与这些标记偶联。优选的固相载体是磁颗粒,当然,本领域通常用于偶联抗体的其他固相载体也可用于本发明。
抗CD3抗体和抗CD28抗体可以是本领域已知的任何能结合CD3、CD28的抗体。优选的序列如SEQ ID NO:1和2所示。
在一个或多个实施方案中,步骤1)包括:将PBMC在培养基(添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆的X-VIVO15)中以适合PBMC生长的条件培养1~4小时,优选2-3小时。在一个或多个实施方案中,步骤2)包括:2.1)在DPBS中,使用抗体由PBMC中分选CD3+T细胞,所述抗体包括抗CD3抗体,和2.2)在培养基(添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆的X-VIVO15)中以适合CD3+T细胞活化的条件孵育CD3+T细胞12-36小时,优选15-24小时。在一个或多个实施方案中,步骤3)包括:在培养基(添加有乙酰半胱氨酸、GlutaMAX、HEPES、IL-2和人血浆的X-VIVO15)中以适合逆转录病毒转导CD3+T细胞的条件共培养逆转录病毒和CD3+T细胞12-36小时,优选15-24小时。在一个或多个实施方案中,所述方法还包括步骤:4)将步骤3)获得所述表达功能分子的T细胞冻存;具体包括:以氯化钠注射液洗涤T细胞,以冻存液重悬T细胞,程序降温,和液氮冻存。
本文中,“冻存液”指用于将其中的细胞冷冻保存的介质。本领域技术人员知晓适用于细胞,特别是免疫细胞(例如PBMC或CD3+T细胞)的冻存液组分。这些冻存液通常可商购。
本文中,“培养基”指用于培养细胞的介质。本领域技术人员知晓适用于细胞,特别是免疫细胞(例如PBMC或CD3+T细胞)的培养基组分。这些培养基通常可商购,例如X-VIVO15。
本文中用于转导细胞的“逆转录病毒”包括α逆转录病毒、β逆转录病毒、γ逆转录病毒、δ逆转录病毒、ε逆转录病毒;优选为γ逆转录病毒。本文中用于转导细胞的病毒不包括慢病毒。本文所述“逆转录病毒质粒”源自逆转录病毒的载体。
本发明还提供由本发明方法制备获得的CAR-T细胞。优选地,所述CAR为CD19-28z。
具体地,本发明的快速制备表达功能分子的T细胞(例如CAR-T)的方法如图1所示,包括:PBMC分离、PBMC复苏缓解、CD3+T细胞分选活化、逆转录病毒转导T细胞及CAR-T细胞灌装冻存。与一般CAR-T细胞的制备流程不同的改进点主要体现在缩短T细胞活化时长为22-24小时及删除CAR-T细胞体外扩增步骤。本制备流程适用的样本为符合采集规程及运输方式的机采单采血。
单采血的采集及运输。使用血细胞分离机(Spectra、SpectraFenwalTM/>或等同设备的标准机采设备),在临床中心病区进行白细胞及血浆的采集分离。单采血采集量约为20mL~200mL,血浆量约为20mL~200mL。将采集完成的单采血及血浆样本放置于2~8℃冷藏运输箱内,以冷链物流方式运输至细胞制备中心。本领域技术人员周知,根据《药品生产质量管理规范(2010年修订)》,将洁净区进行ABCD分级,A级:高风险操作区,如灌装区、放置胶塞桶和与无菌制剂直接接触的敞口包装容器的区域及无菌装配或连接操作的区域,应当用单向流操作台(罩)维持该区的环境状态。单向流系统在其工作区域必须均匀送风,风速为0.36-0.54m/s(指导值)。应当有数据证明单向流的状态并经过验证。在密闭的隔离操作器或手套箱内,可使用较低的风速。B级:指无菌配制和灌装等高风险操作A级洁净区所处的背景区域。C级和D级:指无菌药品生产过程中重要程度较低操作步骤的洁净区。本发明在细胞制备中心的B+A(即B级背景下的A级环境)或C+A(C级背景下的A级环境)环境下,开始进行以下的细胞处理步骤。
PBMC分离及血浆处理。本工序可以手动或自动化完成。手动方面,利用Ficoll密度梯度离心法将单采血缓慢加在Ficoll上,以离心机离心后,手动取出分离的PBMC层。自动化方面使用Sepax C-Pro细胞处理系统及对应的一次性使用封闭管路套件CT-90.1,同样利用Ficoll密度梯度离心法并执行仪器制造商开发的NeatCell程序以分离PBMC。最终将PBMC重悬于PBMC冻存液中并输出或转移至细胞冻存袋,细胞冻存袋在程序降温仪中进行冻存,最后转移至液氮罐中储存。装有供者血浆的血袋在56℃水浴锅中灭活30分钟后,以离心方式使血浆中的变性蛋白及其他杂质沉降到袋子底部。利用血液分浆夹将上清血浆转移至冻存袋中,于-80℃冻存备用。本工序目的在于分离出CAR-T细胞制备所需要的PBMC,去除单采血样本中残留的红细胞及白细胞中的多核细胞。处理过的血浆用以配制整个制备过程中所使用的培养基,培养基中含5%人血浆。
PBMC复苏缓解。将PBMC冻存袋放入37℃水浴中,来回晃动使其迅速均匀融化。将融化后的PBMC转移至培养基(添加有乙酰半胱氨酸、GlutaMAX、HEPES、人血浆的X-VIVO15)中,离心洗涤一次。将洗涤后PBMC以培养基重悬,置于细胞培养瓶或细胞培养袋中,放入二氧化碳培养箱,以37±1℃及5±0.5%CO2培养2小时。本工序目的在于使PBMC的活率及功能性得到恢复,去除凋亡细胞。
CD3+T细胞分选活化。自二氧化碳培养箱取出缓解培养完成的PBMC,将细胞悬液离心一次后,再以DPBS洗涤一次。洗涤后PBMC以DPBS重悬并经取样计数,再根据PBMC流式表型检测结果计算CD3+细胞数,补加DPBS于PBMC悬液中,调整CD3+细胞密度(1-50*106CD3+cells/mL)。加入以DPBS清洗过的共价偶联抗-CD3和抗-CD28抗体的磁珠(细胞数:磁珠数=1:1(CTSTMDynabeadsTMCD3/CD28),利用旋转混合仪使细胞磁珠共孵育(30-60分钟,室温)期间得到充分混合。孵育完成后将细胞置于DynaMagTM-5磁力架上,将与磁珠结合的CD3+细胞以磁性捕获并留滞在离心管中,弃去上清及CD3-细胞。加入含IL-2的培养基(添加乙酰半胱氨酸、GlutaMAX、HEPES、人血浆、白介素2的X-VIVO15)重悬CD3+细胞(1-5*106cells/mL)并转移至细胞培养瓶或细胞培养袋中。将细胞放入二氧化碳培养箱,在37±1℃及5±0.5%CO2的环境下活化培养22-24小时。本工序目的在于分选出用以转导病毒的CD3+T细胞,进一步去除其他细胞族群,并以CD3及CD28抗体共刺激T细胞,利于逆转录病毒的转导。
逆转录病毒转导T细胞。首先在37℃环境下以RetroNectin工作液包被细胞培养瓶或细胞培养袋,包被完成后将培养瓶或培养袋中上清弃去。将活化培养完成的CD3+T细胞经一次洗涤后,以含IL-2培养基(添加乙酰半胱氨酸、GlutaMAX、HEPES、人血浆、白介素2的X-VIVO15)重悬。洗涤后细胞经取样计数,计算转导MOI,MOI应为0.1-2且最终病毒转导中的细胞密度为1-5*106cells/mL。将细胞悬液及病毒液(MOI=1,细胞密度1*106cells/mL)共同放入RetroNectin包被完成的培养瓶或培养袋中,在37±1℃及5±0.5%CO2的二氧化碳培养箱中培养16-24小时。本工序目的为将CAR基因转导入T细胞,实现CAR-T细胞制备。
CAR-T细胞灌装冻存。完成病毒转导后,首先利用DynaMagTM-5磁力架去除细胞悬液中残留的磁珠,再以氯化钠注射液(0.9%)洗涤CAR-T细胞三次并以冻存液(含DMSO、氯化钠注射液、复方电解质注射液、葡萄糖注射液、人血白蛋白)重悬,经取样计数后,分装CAR-T细胞于冻存袋中,再运输至程序降温仪。待降温完毕,转移至液氮中长期保存。降温程序是本领域技术人员的常规知识,示例性降温程序包括以下步骤:1)仪器预冷至20摄氏度;2)以1摄氏度每分,降至-6摄氏度;3)以25摄氏度每分,降至-50摄氏度;4)以10摄氏度每分,回温至-14摄氏度;5)以1摄氏度每分,降至-45摄氏度;6)以10摄氏度每分,降至-90摄氏度。
Dash CAR-T工艺与现有技术的工艺相比具有以下优势。在PBMC复苏缓解方面,原有制程需22±2小时而Dash CAR-T仅需缓解培养2小时。CD3+T细胞分选活化方面,DashCAR-T工艺将活化培养时长缩短至22-24小时,其他操作步骤未改变。病毒转导方面,DashCAR-T工艺中先不磁性去除细胞悬液中的磁珠,直接进行病毒转导;而原有制程中为先去除磁珠再病毒转导。Dash CAR-T工艺中无CAR-T细胞扩大培养工序,在病毒转导完成后,直接进行CAR-T细胞灌装冻存。CAR-T细胞灌装冻存方面,由于还未去除磁珠,在Dash CAR-T工艺中,先以磁力架去除磁珠后再行灌装冻存,其他步骤与原有制程相同。如图2所示。
以下将以具体实施例的方式对本发明作进一步说明。应理解,这些实施例仅仅是阐述性的,并非用于限制本发明的范围。实施例中所用到的方法和试剂,除非另有说明,否则为本领域的常规方法和试剂。
实施例
实施例1,Dash CAR-T工艺及原有制程制备的CAR-T细胞的活率、直径、扩增倍数、流式表型及CAR感染率
以Dash CAR-T工艺制备Dash CAR-T细胞,并比较Dash CAR-T细胞与原有制程生产的CAR-T细胞的差异。Dash CAR-T制备:将冻存PBMC以37℃水浴复苏后,转移至培养基中,离心洗涤一次。洗涤后PBMC以培养基重悬,并以5×106个活细胞/毫升的细胞密度,接种于培养瓶中。将带有细胞悬液的培养瓶置于37℃,5%CO2培养箱中,缓解培养2小时。
将完成缓解培养的PBMC自培养箱中取出,转移至离心管中,以DPBS离心洗涤一遍后,以DPBS将细胞密度调整为1×107个CD3+细胞/毫升。将共价偶联抗-CD3和抗-CD28抗体的磁珠加入细胞悬液中,在旋转混合仪上共同于室温孵育30分钟。将孵育完成的细胞磁珠悬液转移至磁力架上,磁性分选出CD3+细胞。将分选完成的细胞以含IL-2培养基调整为1×106个CD3+细胞/毫升,置于培养瓶中,在37℃,5%CO2培养箱中,活化培养22小时。
将活化完成的细胞自培养箱中取出,以含IL-2培养基离心洗涤一次后,调整细胞密度为1×106个活细胞/毫升。将细胞悬液与病毒液混合以进行CAR基因转导,MOI=1。将细胞悬液转移至RetroNectin包被的细胞培养袋中,在37℃,5%CO2培养箱中,病毒转导24小时。
转导完成的Dash CAR-T细胞先以磁力架去除悬液中的磁珠,再以氯化钠注射液离心洗涤三次,重悬于冻存液中,并转移至冻存袋中,以程序降温仪进行冻存,并于液氮中长期保存。
原有制程CAR-T细胞的制备操作工序时长不同,其中,PBMC缓解24小时,CD3+细胞活化培养48小时,病毒转导后先去除磁珠并转移至培养瓶中进行扩大培养5天,再进行冻存。
Dash CAR-T细胞和原有制程的CAR-T细胞在冻存前进行检测,结果如图3所示。Dash CAR-T工艺所生产出的CAR-T细胞在与产品质量高度相关的指标,包括CAR感染率、细胞活率、CD3比例、CD19比例等均与原有制程生产的CAR-T细胞的质量差异小。总细胞群体中的CD14及CD16+/CD56+的表达比例趋近皆0%。细胞活化相关的指标,如CD25、CD69及细胞直径在两种制程的结果差异大。由图可见,Dash CAR-T细胞较原有制程CAR-T细胞的活化状态更高,尤其是CD25及CD69的表达有巨大的差别。CD45RO-/CCR7+象征细胞的族群比例也有明显差异,其中,Dash CAR-T细胞仍有近80%/>细胞而原有制程的CAR-T细胞中/>细胞比例已低于20%。CD45RO-/CCR7+所代表的/>细胞为具有高度干性的T细胞,T细胞干性与细胞扩增及肿瘤杀伤能力相关。根据此体外实验结果可以发现,DashCAR-T细胞具有高活化状态及更高的T细胞干性,可能促成更优秀的扩增及肿瘤杀伤能力。
实施例2,Dash CAR-T工艺的培养终点的CAR-T细胞功能检测
将冻存后的Dash CAR-T细胞和原有制程CAR-T细胞在37℃水浴中复苏,以含IL-2培养基离心洗涤一次后重悬,接种于培养瓶中。在37℃,5%CO2培养箱中缓解培养48小时,使CAR-T细胞功能得到良好恢复后再进行功能检测。
功能检测是以CAR-T细胞与表达相应靶点的靶细胞共同培养,取CAR-T细胞检测CD107a表达,以及取靶细胞检测被杀伤比例。CD107a分子是毒杀型T细胞脱颗粒的敏感标志,直接反映细胞杀伤活性水平,而Dash CAR-T细胞的CD107a表达与原有制程CAR-T细胞相近。如图4所示,在细胞杀伤试验中,效靶比20:1至1.25:1的范围内,Dash CAR-T细胞与原有制程CAR-T细胞表现出相似的杀伤能力。
实施例3,Dash CAR-T细胞体内药效学研究
图5显示以Nalm-6-luciferase-GFP人B淋巴白血病细胞在NOG小鼠中建立CD19Dash CAR-T细胞体内药效学研究模型。实验组别及剂量包含:溶媒组、1×106个未转导CAR基因的T细胞、1×106个BCMA CAR+细胞、1×106个原有制程CAR+细胞、5×106个原有制程CAR+细胞、1×106个Dash CAR-T工艺CAR+细胞。每组五只小鼠,于回输前1天及回输后每7天进行活体荧光成像及小鼠外周血中T细胞比例分析。由活体荧光影像中可以看出,回输后第7天的Dash CAR-T细胞组的肿瘤荧光已几乎不可见,而相同剂量的原有制程CAR-T细胞则仍有部分肿瘤细胞留存,直到回输后14天才能清除。五倍剂量的原有制程CAR-T细胞在第7天也几乎完全清除肿瘤,显示Dash CAR-T细胞仅需五分之一的原有制程CAR-T细胞剂量即可达到明显的肿瘤完全杀伤效果。回输后第14天,1×106个原有制程CAR+细胞将肿瘤完全清除,但到了回输后21天又有明显复发。5×106个原有制程CAR+细胞组及1×106个Dash CAR-T工艺CAR+细胞组的肿瘤清除效果持续至回输后第21天,但原有制程CAR+细胞组已有肿瘤复发的迹象。比较总荧光通量可以发现Dash CAR-T组别自回输后第14天起,其平均总荧光通量维持最低值;此外,Dash CAR-T组别的小鼠外周血T细胞扩增倍数远高于其他各组,至回输后第21天已扩增超过100倍。以上结果很好的展示了Dash CAR-T细胞因T细胞干性强而具有的高度扩增能力及肿瘤杀伤能力。
以上结果表明,Dash CAR-T工艺制成的CAR-T细胞在细胞活率、CD3+细胞比例、非T细胞族群及CAR感染率方面与原有制程的CAR-T细胞无明显差异;但在细胞活化状态及T细胞干性部分,Dash CAR-T细胞均高于原有制程来源的CAR-T细胞,因此使Dash CAR-T细胞在细胞扩增、细胞杀伤能力及小鼠模型药效试验的表现均与原有制程CAR-T细胞具有高度可比性,甚至高于原有制程CAR-T细胞。总体上,本专利的关键点除了能够缩短CAR-T细胞制程,达到降低成本的作用之外,更生产出杀伤效果更佳的CAR-T,并在体外功能学及体内药效学方面均得到验证。
Claims (10)
1.一种制备表达功能分子的T细胞的方法,包括步骤:
1)PBMC复苏缓解1~4小时,优选2-3小时,
2)由PBMC分选并活化CD3+T细胞10~36小时,优选15~24小时,
3)使用含有功能分子的编码序列的逆转录病毒转导CD3+T细胞得到所述表达功能分子的T细胞,所述转导包括共培养逆转录病毒和CD3+T细胞12-36小时,
优选地,所述功能分子是CAR。
2.如权利要求1所述的方法,其特征在于,步骤1)包括:将PBMC在培养基中以适合PBMC生长的条件培养1~4小时,
优选地,
步骤1)中的培养基包括AIM-V、X-VIVO、DMEM、RPMI1640,优选为X-VIVO15,和/或
所述培养基还添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆中的一种或多种或全部,和/或
适合PBMC生长的条件是约37℃及约5%CO2。
3.如权利要求1或2所述的方法,其特征在于,步骤2)包括:
2.1)在DPBS中,使用抗体由PBMC中分选CD3+T细胞,所述抗体包括抗CD3抗体,和
2.2)在培养基中以适合CD3+T细胞活化的条件孵育CD3+T细胞12-36小时,优选15-24小时。
4.如权利要求3所述的方法,其特征在于,
所述抗体还包括抗CD28抗体,和/或
步骤2)中的培养基含IL-2,和/或
所述培养基包括AIM-V、X-VIVO、DMEM或RPMI1640,优选为X-VIVO15,和/或
所述培养基还添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆中的一种或多种或全部,和/或
所述抗体是标记的抗体;优选地,所述抗体偶联于固相载体上。
5.如权利要求1或2所述的方法,其特征在于,步骤3)包括:在培养基中以适合逆转录病毒转导CD3+T细胞的条件共培养逆转录病毒和CD3+T细胞12-36小时,
优选地,
步骤3)中的培养基含IL-2,和/或
所述培养基包括AIM-V、X-VIVO、DMEM或RPMI1640,优选为X-VIVO15,和/或
所述培养基还添加有乙酰半胱氨酸、GlutaMAX、HEPES和人血浆中的一种或多种或全部,
适合逆转录病毒转导CD3+T细胞的条件是约37℃及约5%CO2。
6.如权利要求5所述的方法,其特征在于,
步骤3)所述转导的MOI为0.1-2,和/或
步骤3)中,CD3+T细胞的密度为1-5*106cells/mL。
7.如权利要求4所述的方法,其特征在于,所述方法还包括去除固相载体的步骤,位于步骤3)之后。
8.如权利要求1或2所述的方法,其特征在于,所述方法还包括步骤:4)将步骤3)获得所述表达功能分子的T细胞冻存,
优选地,步骤4)包括:以氯化钠注射液洗涤T细胞,以冻存液重悬T细胞,程序降温,和液氮冻存。
9.由权利要求1-8中任一项所述的方法制备获得的CAR-T细胞。
10.如权利要求9所述的所述CAR-T细胞,其特征在于,所述CAR为含有抗CD19抗体或其抗原结合片段、铰链区、跨膜区、CD28胞内共刺激结构域、CD3-zeta信号转导结构域的CD19-28z。
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