CN117210410A - 一种t细胞、其用途及药物组合物 - Google Patents
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Abstract
本发明公开了一种T细胞、其用途及药物组合物。所述T细胞中ARHGAP11B蛋白和/或GREM1蛋白的表达量增加或活性增强。本发明的方法可以促进免疫细胞增殖、促进记忆性免疫细胞产生,并且增强免疫细胞抗肿瘤效应,包括对肿瘤杀伤能力、解决受试者肿瘤异质性、预防受试者中肿瘤复发,尤其是提高CAR‑T细胞抗肿瘤效应,从而增强ACT疗法的治疗效果。
Description
技术领域
本发明总体上涉及免疫学、细胞生物学、分子生物学和药学领域。
背景技术
过继细胞疗法(adoptive cell therapy, ACT)是指从患者体内分离出具有活性的免疫细胞,在体外进行遗传修饰(或不进行遗传修饰)和扩增,然后重新输入到患者体内以达到治疗疾病(主要是肿瘤)的目的。过继细胞疗法是治疗癌症的新的有效方法。过继细胞疗法主要包括TIL(肿瘤浸润淋巴细胞)、LAK(淋巴因子激活的杀伤细胞)、CIK(细胞因子诱导的杀伤细胞)、DC(树突状免疫细胞)、NK(自然杀伤细胞)、TCR-T(T细胞受体嵌合T细胞)和嵌合抗原受体(Chimeric antigen receptor,CAR)T疗法等。在ACT中,最常用的细胞类型是T细胞。T细胞是由来源于骨髓的淋巴干细胞,在胸腺中分化、发育成熟后,通过淋巴和血液循环而分布到全身的免疫器官和组织中发挥免疫功能。T细胞包括CD8+细胞、CD4+细胞、δ-γT细胞、调节性T细胞等。
T记忆干细胞(T memory stem cells, TSCM)是在人类和小鼠中发现的一种新型记忆T细胞亚群,既具有记忆T细胞的特性,同时又具有干细胞的特征。
ACT的有效性取决于过继的T细胞在患者体内的扩增能力和持久性。现有技术报道了高剂量的IL-2可以促进ACT后过继T细胞的扩增和持续存在,但是其具有治疗毒性,并提高了临床成本。
T细胞对特异性靶向肿瘤主要通过TCR和CAR技术来实现。CAR-T细胞疗法已发展成为全球新药研发的热点。该疗法通过基因编辑技术对于患者的淋巴T细胞进行体外修饰改造,使其具备特定抗原的识别功能,并在回输至患者体内后,能够靶向并杀伤携带相应抗原的肿瘤细胞。CAR的结构设计是决定肿瘤杀伤效果的关键因素。CAR的结构主要由特异性识别肿瘤相关抗原(Tumor associated antigen, TAA)的单链可变区结构域(Single chainvariable fragment, scFv)、铰链区、跨膜区和胞内信号传导区四个部分构成。scFv的形式一般为将单克隆抗体的重链可变区(VH)与轻链可变区(VL)通过多肽接头连接起来构成的抗原结合区。相较于TCR识别肿瘤抗原的复杂性,scFv识别肿瘤抗原不依赖于MHC,但是同时保留了特异性。铰链区通常由免疫球蛋白超家族组成,如IgG4、CD8、IgG1等。跨膜结构域的作用为连接CAR的胞外结构域和胞内激活信号结构域,一般由CD8、CD28或CD4等组成。而胞内信号域包含免疫受体酪氨酸活化基序(immune-receptor tyrosinebased activationmotifs,ITAM),通常由CD-3ζ链和协同共刺激信号分子41BB、CD28、ICOS、OX40等组成。CAR-T疗法目前在血液瘤治疗中已经取得了巨大成功,但由于实体瘤具有高度的异质性以及肿瘤微环境(Tumor microenvironment,TME)的阻碍,该疗法在实体瘤的应用进展则相对缓慢。
Gremlin1(GREM1,UniProtKB编号:O60565)基因最早于1998年在非洲爪蟾中发现,其编码的分泌型糖基化蛋白可以与骨形态发生蛋白(bone morphogenetic protein,BMP)特异性结合而发挥拮抗作用,抑制BMP配体与相应受体结合,从而参与调控胚胎时期骨组织、肾脏等重要器官的发育。GREM1基因在大部分器官和组织中表达为阴性或较弱,但在胃和皮肤中具有一定量的表达。
ARHGAP11B(UniProtKB编号:Q3KRB8)是与人类大脑新皮层发育、进化和扩张相关的人类特异性基因。研究发现,ARHGAP11B基因的一个C-G的单碱基突变,能够导致ARHGAP11B 的mRNA丢失55个核苷酸,因此阅读框的改变,从而产生了人类特有的、功能关键的47个氨基酸序列。ARHGAP11B基因通过抑制线粒体通透性转化孔的开放,增加线粒体Ca2+浓度,从而诱导谷氨酰胺分解代谢,这是基底祖细胞增殖所必需的。此外,在胎儿人脑新皮质中,ARHGAP11B基因优先表达于皮质神经祖细胞。它能够促进脑室下区基底祖细胞的扩增,在胎儿皮质发育过程中产生更多的神经元,从而在新皮层扩张中发挥关键作用。
需要有效地改善过继治疗中T细胞在体外及患者体内的扩增能力和持久性,以提高ACT疗法(包括CAR-T和TCR-T)的疗效。
发明内容
本发明的目的为提高ACT疗法中免疫细胞(尤其是T细胞)在体外及患者体内的扩增能力和持久性。
因此,本发明提供一种功能增强的免疫细胞、其用途及药物组合物。本发明通过遗传修饰使得所述免疫细胞中ARHGAP11B蛋白和/或GREM1蛋白的表达量增加或者蛋白活性增强,增强了免疫细胞的功能,改善了ACT的治疗效果。
根据本发明的一个方面,提供了一种T细胞,所述T细胞中ARHGAP11B蛋白和/或GREM1蛋白的表达量增加和/或活性增强。优选地,所述T细胞选自记忆性干细胞样T细胞(TSCM)。所述TSCM还具有选自下组的一种或多种性质:CCR7+和/或CD62L+、CD45RA+或CD45RA-、CD45RO+或CD45RO-、CD27+、CD28+、CD127+、CD122+、CD95+、CD3+、CD4+和CD8+。
所述ARHGAP11B蛋白、GREM1蛋白分别包括SEQ ID NO:22、SEQ ID NO:24所示的氨基酸序列,或所述ARHGAP11B蛋白、GREM1蛋白分别包括与SEQ ID NO:22、SEQ ID NO:24所示的序列具备至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列一致性的氨基酸序列。
所述T细胞还表达嵌合抗原受体(CAR)。优先地,所述CAR从N端到C端依次包括所述胞外抗原结合结构域、铰链区、跨膜结构域、胞内共刺激结构域和信号传导结构域。更优选地,所述铰链区包括SEQ ID NO:12所示氨基酸序列,所述跨膜结构域包括SEQ ID NO:14所示氨基酸序列,所述共刺激结构域包括SEQ ID NO:16所示氨基酸序列,所述信号传导结构域包括SEQ ID NO:18所示氨基酸序列;其中所述胞外抗原结合结构域为单链抗体(scFv)形式,所述scFv包含下述任一项所示的氨基酸序列:SEQ ID NO:4、6、8和10。
本发明的另一方面提供了所述免疫细胞在制备治疗或预防肿瘤的药物中的用途。优选地,所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
本发明还提供了一种药物组合物,包含所述的免疫细胞以及药学上可接受的载体。
本发明还提供了一种增强T细胞功能方法,所述方法为体内方法或体外方法,所述方法选自以下各项组成的组:
(1) 促进T细胞增殖的方法,(2) 促进TSCM产生的方法,和 (3) 增强T细胞抗肿瘤效应(包括对肿瘤杀伤能力、解决受试者肿瘤异质性、预防受试者中肿瘤复发的方法、治疗有需要的受试者中的肿瘤的方法、提高CAR-T细胞抗肿瘤效应)的方法,所述方法包含以下步骤:提高T细胞中ARHGAP11B蛋白和/或GREM1蛋白的表达量或活性。
本发明的方法可以促进免疫细胞增殖、促进记忆性免疫细胞产生,并且增强免疫细胞抗肿瘤效应,包括对肿瘤杀伤能力、解决受试者肿瘤异质性、预防受试者中肿瘤复发,尤其是提高CAR-T细胞抗肿瘤效应,从而增强ACT疗法的治疗效果。
附图说明
构成本申请的一部分的附图用来提供对本申请的进一步理解,本申请的示意性实施方式及其说明用于解释本申请。在附图中:
图1为ARHGAP11B、GREM1及其组合对T细胞增殖影响的示例图。
图2为ARHGAP11B、GREM1及其组合对CAR表达影响的示例图。
图3为ARHGAP11B、GREM1及其组合对CAR表达影响的统计图(n=3)。
图4为ARHGAP11B、GREM1及其组合对T细胞群中CD8+T细胞占比影响的示例图。
图5为ARHGAP11B、GREM1及其组合对T细胞群中CD8+T细胞占比影响的统计图(n=3)。
图6为ARHGAP11B、GREM1及其组合对CD3+T细胞中TSCM占比影响的示例图。
图7为ARHGAP11B、GREM1及其组合对CD3+T细胞中TSCM占比影响的统计图(n=3)。
图8为ARHGAP11B、GREM1及其组合对CD8+T细胞中TSCM占比影响的示例图。
图9为ARHGAP11B、GREM1及其组合对肿瘤细胞诱导的特异性CAR-T细胞扩增的影响。
本申请的其它特征和优点将在随后的具体实施方式部分予以详细说明。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施方式及各个实施方式中的特征可以相互组合。
除非另有说明,本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
术语“或”是指列举的可选择要素中的单个要素,除非上下文明确地另外指出。术语“和/或”是指所列举的可选择要素中的任意一个、任意两个、任意三个、任意更多个或其全部。
术语“包含”或“包括”指包括所述的要素、整数或步骤,但是不排除任意其他要素、整数或步骤。当使用“包含”或“包括”时,除非另有指明,否则也涵盖由所述及的要素、整数或步骤组成的情形。例如,当提及某组合或组合物“包括A和B”时,也旨在涵盖由A和B组成的组合或组合物。
本文中,术语“核酸分子”、“核酸”和“多核苷酸”可互换使用,指核苷酸聚合物。此类核苷酸聚合物可含有天然和/或非天然核苷酸且包括(但不限于) DNA、RNA和PNA。“核酸序列”或“核苷酸序列”指包含于核酸分子或多核苷酸中的核苷酸线性序列。
术语“载体”指可经工程改造以含有目的多核苷酸(例如目的多肽的编码序列)的核酸分子或可在宿主细胞中复制的核酸分子(例如,核酸、质粒、或病毒等)。载体可包括以下组件中的一个或更多个:复制起点、一或更多个调控目的多核苷酸的表达的调控序列(诸如启动子和/或增强子子)和/或一个或更多个可选择标记物基因(诸如抗生素抗性基因和可用于比色分析中的基因,例如β-半乳糖)。术语“表达载体”指用于在宿主细胞中表达目的多肽的载体。在某些实施方式中,本发明所述载体选自以下组:逆转录病毒载体、慢病毒载体和转座子质粒。
“宿主细胞”在本文中指可表达本文提供的CAR的哺乳动物免疫效应细胞,尤其是人细胞,例如T细胞或NK细胞。宿主细胞包括单个宿主细胞的后代,且后代可能由于自然、偶然或故意突变而不一定与原始母细胞完全一致(在形态或基因组DNA互补方面)。宿主细胞也包括在活体内经本文提供的核酸分子或表达载体转染的细胞。
提及药物组合物,所使用的术语“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质,这些物质适合于人和/或动物给药而无过度的不良副反应,同时适合于维持位于其中的药物或活性剂的活力。依照给药途径,可以施用本领域众所周知的各种不同的载体,包括,但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。本文所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组合物,例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下,或经吸入、透皮等途径给药。
提及氨基酸或核苷酸序列时,术语“序列一致性(sequence identity)”(也称为“序列同一性”)指两氨基酸或核苷酸序列(例如查询序列和参照序列)之间一致性程度的量,一般以百分比表示。通常,在计算两氨基酸或核苷酸序列之间的一致性百分比之前,先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置,两序列中的氨基酸残基或碱基相同,则认为两序列在该位置一致或匹配;两序列中的氨基酸残基或碱基不同,则认为在该位置不一致或错配。在一些算法中,用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中,还将缺口数量和/或缺口长度考虑在内。出于本发明的目的,可以采用公开的比对软件 BLAST (可在网页ncbi.nlm.nih.gov找到),通过使用缺省设置来获得最佳序列比对并计算出两氨基酸或核苷酸序列之间的序列一致性。
本发明所述的免疫细胞可为淋巴细胞,例如T细胞、B细胞、自然杀伤细胞、未成熟的树突状细胞、单核细胞和巨噬细胞;此外,所述T细胞选自记忆性干细胞样T细胞(TSCM)和/或中央记忆性T细胞(TCM);更优选地,所述TSCM为CCR7+和/或CD62L+,优选地,所述TSCM还具有选自下组的一种或多种性质:CD45RA+或CD45RA-、CD45RO+或CD45RO-、CD27+、CD28+、CD127+、CD122+、CD95+、CD3+、CD4+和CD8+。
本领域提高蛋白表达或其活性的方法均适用于本发明,不作特殊限制。例如可免疫细胞中导入ARHGAP11B和/或GREM1基因、mRNA或蛋白质中任意一种。本发明中,本领域常规的外源基因导入方法如构建质粒、慢病毒等均适用于本发明,不作特殊限制。
所述ARHGAP11B或其功能突变体、GREM1或其功能突变体可来源于人;并且所述ARHGAP11B蛋白、GREM1蛋白分别包含SEQ ID NO:22、SEQ ID NO:24所示的氨基酸序列(核苷酸序列分别为SEQ ID NO:21和SEQ ID NO:23),或与其具备至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%同源性的序列,或由其组成,并且所述同源序列仍具备SEQ ID NO:22或SEQ ID NO:24任一项所示序列功能。
所述T细胞还表达CAR,即CAR-T细胞。CAR-T细胞指表达CAR的T细胞,通常采用编码CAR的表达载体转导T细胞获得。常用的表达载体为病毒载体,例如慢病毒表达载体。经嵌合抗原受体修饰的T细胞(CAR-T)不受主要组织相容性复合体的限制,具有特异性靶向杀伤活性和持久扩增的能力。除T细胞外,也可以用编码CAR的表达载体转化诸如NK细胞的其他淋巴细胞,获得表达该CAR的靶向杀伤性细胞。
嵌合抗原受体(CAR),也称为嵌合T细胞受体、嵌合免疫受体,为一种工程化的蛋白受体分子,其可将期望的特异性赋予免疫效应细胞,例如与特定肿瘤抗原结合的能力。嵌合抗原受体通常由胞外抗原结合结构域、跨膜结构域和胞内信号结构域构成。
所述CAR从N端到C端依次包括所述胞外抗原结合结构域、铰链区、跨膜结构域、以及胞内共刺激结构域和信号传导结构域。
所述CAR包含靶向部分,所述靶向部分特异性结合和/或识别肿瘤抗原,所述肿瘤抗原包括TSHR、CD19、CD123、CD138、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、TnAg、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-11Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、叶酸受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Prostase、PAP、ELF2M、肝配蛋白B2、I GF-I受体、CAIX、LMP2、gp100、bcr-abl、酪氨酸酶、EphA2、岩藻糖基GM1、sLe、GM3、TGS5、HMWMAA、邻乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPVE6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、ie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、prostein、存活蛋白和端粒酶、PCTA-1/Galectin 8、MelanA/MART1、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、细胞周期蛋白B1、MYCN、RhoC、TRP-2、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠羧基酯酶、mut hsp702、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5和IGLL1。
在某些实施方式中,所述靶向部分特异性结合和/或识别肿瘤抗原。在某些实施方式中,所述靶向部分特异性结合和/或识别选自下组的靶标:B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。在某些实施方式中,所述靶向部分特异性结合和/或识别选自下组的靶标:CD19、FcRH5、Mesothelin(MSLN)和GPC3。在某些实施方式中,所述靶向部分包含下述任一项所示的氨基酸序列:SEQ ID NO: 4、6、8和10(核苷酸序列SEQ ID NO:3、5、7和9)或与其具备至少80%同源性的序列。
多数情况下,抗原结合结构域为一段scFv序列,负责识别和结合特定的抗原。单链抗体(single chain fragment variable, scFv),是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠,来自重链和轻链的可变区通过非共价键相互作用形成Fv段,因而scFv能较好地保留其对抗原的亲和活性。
在某些实施方式中,所述铰链区选自以下各项组成的组:IgG4的铰链区、IgG1的铰链区和CD8的铰链区。术语“铰链区”通常是指免疫球蛋白重链CH1和CH2功能区之间的区域。铰链区为位于胞外抗原结合结构域(如scFv)和T细胞膜之间的一段区域。铰链区通常来源于IgG家族,例如,可来源于IgG1和IgG4,还可以来源于IgD和CD8。在本申请中,铰链区可以包含选自下组的部分:IgG4的铰链区、IgG1的铰链区和CD8的铰链区。在某些实施方式中,所述铰链区包含SEQ ID NO: 12所示的氨基酸序列(核苷酸序列SEQ ID NO:11)或与其具备至少80%同源性的序列。
在某些实施方式中,所述跨膜区选自以下各种组成的组:CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。术语“跨膜区”通常是指连接胞外抗原结合域和胞内信号域的跨膜区段,一般来自二聚体膜蛋白,主要包括CD3ζ、CD4、CD8、CD28等,能将CAR结构锚定于T细胞膜上。跨膜区不同的设计能影响导入的CAR基因的表达。在本申请中,所述跨膜区可以包含选自下组的部分:CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。在某些实施方式中,所述跨膜区包含SEQ ID NO:14所示的氨基酸序列(核苷酸序列SEQ ID NO:13)或与其具备至少80%同源性的序列。
在某些实施方式中,所述细胞内结构域包括信号传导结构域和/或共刺激结构域。
胞内信号传到结构域通常包括免疫受体酪氨酸活化基序(ITAM),例如来源于CD3ζ分子的信号传导结构域,负责激活免疫效应细胞,产生杀伤作用。另外,嵌合抗原受体还可在氨基端包括负责新生蛋白在细胞内定位的信号肽,以及在抗原结合结构域和跨膜结构域之间包括铰链区。除了信号传导结构域,胞内信号结构域还可包括例如来源于4-1BB分子的共刺激结构域。
在某些实施方式中,所述信号传导结构域选自以下各项组成的组:CD3ζ的信号传导结构域、CD3δ的信号传导结构域和CD3ε的信号传导结构域。在某些实施方式中,所述信号传导结构域包含SEQ ID NO:18所示的氨基酸序列(核苷酸序列SEQ ID NO:17)或与其具备至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%一致性的序列。
在某些实施方式中,所述共刺激结构域包含选自下组的部分:CD27的共刺激结构域、CD28的共刺激结构域和4-1BB的共刺激结构域。在某些实施方式中,所述共刺激结构域部分包含下述任一项所示的氨基酸序列:SEQ ID NO:16(核苷酸序列SEQ ID NO:15)或与其具备至少80%同源性的序列。
术语“共刺激结构域”通常是指来自以下一个或多个的蛋白质的功能性信号传导结构域:CD27、CD28、41BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、NKG2C、B7-H3、特异结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D。在本发明中,所述共刺激结构域可以来自:CD27、CD28或41BB。
在某些实施方式中,本申请提供的CAR还可包括前导序列(如信号肽)。
在某些实施方式中,所述ARHGAP11B、GREM1、或者GREM1与ARHGAP11B联合与CAR通过连接元件相连。在某些实施方式中,所述连接元件包含选自下组的部分:T2A、P2A、F2A、E2A和IRES。在某些实施方式中,所述元件部分包含下述任一项所示的氨基酸序列:SEQ IDNO:20(核苷酸序列SEQ ID NO:19)或与其具备至少80%同源性的序列。
根据某些实施方式,所述免疫细胞可拥有制备治疗或预防肿瘤的药物。根据某些实施方式,所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
根据某些实施方式,所述药物组合物,包含所述的免疫细胞以及药学上可接受的载体。
根据某些实施方式,本申请提供了一种促进T细胞增殖的方法,其包含以下步骤:使所述T细胞中ARHGAP11B、GREM1以及GREM1与ARHGAP11B联合的表达量上调。
根据某些实施方式,本申请提供了一种促进记忆性T细胞产生的方法,其包含以下步骤:使所述T细胞中ARHGAP11B、GREM1以及GREM1与ARHGAP11B联合的表达量上调,从而促进所述T细胞分化为记忆性T细胞。
根据某些实施方式,本申请提供了一种提高CAR-T细胞抗肿瘤效应的方法,其包含以下步骤:使所述T细胞中ARHGAP11B、GREM1、以及GREM1与ARHGAP11B联合的表达量上调。
在某些实施方式中,所述方法还包括以下步骤:分离获得外周血单核细胞PBMC、CD3+T淋巴细胞、CD8+T淋巴细胞、CD4+T淋巴细胞或调节T细胞。
在某些实施方式中,所述方法还包括:向经分离的所述PBMC加入一种或多种T细胞刺激因子。在某些实施方式中,所述T细胞刺激因子选自以下组:B淋巴细胞表面抗原抗体、TNF抗体、细胞内聚酯和抗生素。在某些实施方式中,所述T细胞刺激因子选自以下组:CD3抗体、CD28抗体、4-1BB抗体、CD80抗体、CD86抗体、PHA、PMA和离子霉素。在某些实施方式中,所述T细胞刺激因子包括CD3抗体,且所述CD3抗体的浓度为1-10000 ng/mL。在某些实施方式中,所述T细胞刺激因子包括CD28抗体,且所述CD28抗体的浓度为1-10000 ng/mL。
在某些实施方式中,所述方法还包括:向经分离的所述PBMC加入一种或多种细胞因子剂。在某些实施方式中,所述细胞因子剂包括白细胞介素。在某些实施方式中,所述白细胞介素包括选自下组的一种或多种:IL2、IL21、IL7和IL15。
在某些实施方式中,所述经遗传修饰的T细胞表达嵌合抗原受体(CAR)。在某些实施方式中,所述方法包括分离并激活所述经遗传修饰的T细胞的步骤,其中所述激活包括向经分离的所述经遗传修饰的T细胞施用T细胞培养基。
在某些实施方式中,所述T细胞培养基选自以下组中的一种或多种:DMEM培养基、1640培养基、MEM培养基、X-VIVO培养基和干细胞培养基。
本申请至少部分地基于发明人偶然发现,ARHGAP11B蛋白和/或GREM1蛋白的表达量上调可增强免疫细胞的功能,包括(1) 促进免疫细胞增殖的方法,(2) 促进记忆性免疫细胞产生的方法,因此ARHGAP11B蛋白和/或GREM1蛋白可增强免疫细胞抗肿瘤效应(包括对肿瘤杀伤能力、解决受试者肿瘤异质性、预防受试者中肿瘤复发以及提高CAR-T细胞抗肿瘤效应,从而治疗有需要的受试者中的肿瘤。
下面将参考附图并结合实施方式来详细说明本申请。
本发明使用的试剂盒耗材信息如表1所示。
表1本发明使用的试剂、耗材信息
实施例1 慢病毒载体的构建
所述CAR-T以靶向GPC3、CD19、FcRH5、MSLN为例,人工合成包含CAR结构的片段,并构建到慢病毒载体(LV100A,System Biosciences公司),随后依照其说明书记载的方式转染获得慢病毒,分别得到GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1、GPC3-ARHGAP11B-GREM1、CD19-CAR、CD19-ARHGAP11B、CD19-GREM1、CD19-ARHGAP11B-GREM1、FcRH5-CAR、FcRH5-ARHGAP11B、FcRH5-GREM1、FcRH5-ARHGAP11B-GREM1、MSLN-CAR、MSLN-ARHGAP11B、MSLN-GREM1、MSLN-ARHGAP11B-GREM1转染的慢病毒。各CAR结构示意如表2所示。
表2 CAR结构示意
实施例2:慢病毒载体的病毒液的制备与滴度检测
将293T细胞以1*10e6 细胞/孔的细胞量接种至六孔板中,并将培养基补至2ml,放入细胞培养箱中培养过夜。
24小时后,吸取200 μl的Opti-MEM培养基加入1.5 ml的EP管中,并将实施例1中获得CAR的慢病毒重组质粒、包装质粒psPAX2和包膜质粒pMD2G,按照4:2:3的比例,用转染试剂(FuGENE)共转染至293T细胞中。
转染24h后,将细胞培养基更换为半完全培养基;转染48h后,收集1 ml的细胞培养上清液,并用预热好的半完全培养基补液1 ml;转染72h后,收取全部的培养上清液,与转染48h时收集的1 ml的细胞培养上清液进行充分混匀,于室温下以2000 g的转速离心10min,收集上层清液,从而获得慢病毒载体病毒液。
实施例3:慢病毒滴度的检测
将293T细胞以1*10e5 细胞/孔的细胞量接种入六孔板内,并加入3 ul上述实施例2中获得的病毒,再加入终浓度为8 μg/ml的聚凝胺(polybrene),吹打混匀后将细胞放入培养箱继续培养;病毒转导72h后,收集细胞进行相应抗体染色后,以流式细胞仪( BDCantoII)检测转导后细胞中的阳性率,并计算病毒滴度,病毒滴度均大于5*105TU/ml。病毒滴度=接种细胞总数×孔阳性率/加入病毒体积(TU/ml)。
实施例4:T细胞的体外激活、转导与培养
Day 0复苏与激活:取健康供者的新鲜单采血,通过FICOLL离心液分离得到外周血单核细胞(PBMC)。在PBMC中加入T细胞激活磁珠(Invitrogen,Human T-Activator CD3/CD28)进行CD3+T细胞的激活,并补加完全培养基将细胞培养密度调节至1*10e6 细胞/mL,并进一步转入细胞培养箱中继续培养。
Day 1 T细胞转导:取激活后的细胞以5*10e5细胞/孔的细胞量接种到12孔板中;从-80℃冰箱取出实施例2中制备的病毒,在室温下融化,按照MOI为3的量加入至含T细胞的孔内,再加入终浓度为8μg/mL的聚凝胺,吹打混匀;将12孔板放入甩板机中,在配平后以1200rpm的转速进行室温离心1小时,之后放回细胞培养箱中继续培养。
Day 2去病毒:从细胞培养箱中取出12孔板,将所有细胞悬液转移至15ml离心管内,以400 g的转速离心5min;弃去上层清液后,使用适量新鲜T细胞完全培养基重悬后,重新接种至12孔板内。
从Day 3开始,每隔1天进行细胞计数和细胞传代,传代密度5*10e5细胞/mL。如图1所示,培养到Day 11时,四组实验组的T细胞都得到了显著的扩增。与各组Day 0时候相比,GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1、GPC3-ARHGAP11B-GREM1组的T细胞分别扩增了413、1599、1011、1798倍。实验结果表明,ARHGAP11B、GREM1、ARHGAP11B与GREM1的联合表达均能显著提高T细胞体外增殖的能力。
实施例5:利用流式细胞术分析检测CAR-T细胞中GPC3的表达
取实施例4中培养至Day 9的CAR-T细胞,离心弃去上层清液,以PBS缓冲液离心洗涤两次后,分别加入0.5μg带有His标签的GPC3蛋白和30μl的PBS缓冲液,涡旋混匀后,在4℃下孵育60min。孵育结束后,用PBS缓冲液离心洗涤一次,再分别加入1.5μl的His(APC)抗体和30μl的PBS缓冲液,避光条件下4℃孵育30分钟。孵育结束后,用PBS缓冲液离心洗涤一次,加入300μl的PBS缓冲液,涡旋混匀后以流式细胞仪进行检测,并用对测试结果进行分析。如图2所示,GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1、GPC3-ARHGAP11B-GREM1组中表达GPC3细胞占比率分别为64.0%、85.7%、73.7%和75.3%。对三次平行实验的结果进行统计分析,结果表明各组的CAR转导效率均在70%左右,且组间无显著性差异(图3)。这些实验结果表明,ARHGAP11B、GREM1、ARHGAP11B与GREM1的联合表达均不会影响CAR的转导效率。
实施例6:利用流式细胞术分析检测CAR-T细胞中CD8+T细胞的比例
取实施例4中培养至Day 9的CAR-T细胞,离心弃去上层清液,用PBS缓冲液离心洗涤两次,分别加入1.0 μl的CD8(Percp-Cy5.5)抗体和30 μl的PBS缓冲液,涡旋混匀后,避光条件4℃下孵育30 min。孵育结束后,用PBS缓冲液离心洗涤一次,加入300 μl的PBS缓冲液,涡旋混匀后以流式细胞仪进行检测,并用FlowJo软件对测试结果进行分析。如图4所示,GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1、GPC3-ARHGAP11B-GREM1组中CD8+T细胞的占比分别为62.6%、66.1%、65.4%和62.4%。对三次平行实验的结果进行统计分析,结果表明各组CD8+T细胞的占比均在65%左右,组间无显著性差异(图5)。这表明ARHGAP11B、GREM1、ARHGAP11B与GREM1的联合表达不会影响CD8+T细胞的占比。
实施例7:利用流式细胞术分析检测CAR-T细胞中CD3+T细胞中TSCM的比例
取实施例4中培养至Day 11的CAR-T细胞,离心弃去上层清液,用PBS缓冲液离心洗涤两次,分别加1.0 μl的CD3(Pecy7)抗体、1.5 μl的CD13RO(APC-CY7)抗体、1.5 μl的CD62L(FITC)抗体和30 μl的PBS缓冲液,涡旋混匀后,避光条件4℃下孵育30分钟。孵育结束后,用PBS缓冲液离心洗涤一次,加入300 μl的PBS缓冲液,涡旋混匀后以流式细胞仪进行检测,并用FlowJo软件对测试结果进行分析。如图6所示,GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1、GPC3-ARHGAP11B-GREM1组CD3+T细胞中TSCM的占比分别为16.2%、23%、14.2%和34.0%。对三次平行实验的结果进行统计分析,与对照GPC3-CAR组相比,GREM1组中CD3+T细胞中TSCM的占比处于同一水平,ARHGAP11B组以及GREM1与ARHGAP11B联合表达组中CD3+T细胞中TSCM的占比显著提升(图7),*代表P<0.05,ns为无显著差异。这些结果表明,GREM1不会影响CD3+T细胞中TSCM的占比,而ARHGAP11B以及GREM1与ARHGAP11B联合表达可以显著提高CD3+T细胞中TSCM的占比,且GREM1与ARHGAP11B的联合表达对于CD3+T细胞中TSCM的占比影响更为显著。
实施例8:利用流式细胞术分析检测CAR-T细胞中CD8+T细胞中TSCM的比例
取实施例4中培养至Day 11的CAR-T细胞,离心弃去上层清液,用PBS缓冲液离心洗涤两次,分别加1.0 μl的CD8(Percp-Cy5.5)抗体、1.5 μl的CD13RO(APC-CY7)抗体、1.5 μl的CD62L(FITC)抗体和30 μl的PBS缓冲液,涡旋混匀后,避光条件4℃下孵育30分钟。孵育结束后,用PBS缓冲液离心洗涤一次,加入300 μl的PBS缓冲液,涡旋混匀后以流式细胞仪进行检测,并用FlowJo软件对于测试结果进行分析。如图8所示,GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1、GPC3-ARHGAP11B-GREM1组CD8+T细胞中TSCM的占比分别为19.7%、28.8%、16.0%和43.4%。实验结果表明,GREM1不会影响CD8+T细胞中TSCM的占比,而ARHGAP11B以及GREM1与ARHGAP11B联合表达可以显著提高CD8+T细胞中TSCM的占比,且GREM1与ARHGAP11B的联合表达对于CD8+T细胞中TSCM的占比影响更为显著。
将表达GPC3-CAR、GPC3-ARHGAP11B、GPC3-GREM1和GPC3-ARHGAP11B-GREM1的CAR-T细胞与辐照后(X-RAD细胞辐照仪,辐照剂量为30Gy)的 HepG2细胞(中科院细胞库购得)在T细胞培养基中按1:1的效靶比进行共培养。当靶细胞全部凋亡时,重新补加辐照的HepG2进行刺激,共刺激3次。每次以台盼蓝染色法进行细胞计数。三次实验的细胞增殖情况如图9所示,表达GPC3-ARHGAP11B、GPC3-GREM1的两组,T细胞的扩增倍数略高于对照GPC3-CAR组,而CAR-ARHGAP11-BGREM1组的T细胞扩增倍数要远高于对照GPC3-CAR组。
因此,本发明的方法可以促进免疫细胞增殖、促进记忆性免疫细胞产生,并且增强免疫细胞抗肿瘤效应,包括对肿瘤杀伤能力、解决受试者肿瘤异质性、预防受试者中肿瘤复发,尤其是提高CAR-T细胞抗肿瘤效应,从而增强ACT疗法的治疗效果。
以上所述仅为本申请的较佳实施方式而已,并不用以限制本申请,凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
Claims (10)
1.一种T细胞,其中,所述T细胞中ARHGAP11B蛋白和/或GREM1蛋白的表达量增加和/或活性增强。
2.根据权利要求1所述的T细胞,其中,所述T细胞选自记忆性干细胞样T细胞(TSCM)。
3.根据权利要求2所述的T细胞,其中,所述TSCM具有选自下组的一种或多种性质:CCR7+、CD62L+、CD45RA+或CD45RA-、CD45RO+或CD45RO-、CD27+、CD28+、CD127+、CD122+、CD95+、CD3+、CD4+和CD8+。
4.根据权利要求1-3任一项所述的T细胞,其中,所述ARHGAP11B蛋白、GREM1蛋白分别包括SEQ ID NO:22、SEQ ID NO:24所示的氨基酸序列,或所述ARHGAP11B蛋白、GREM1蛋白分别包括与SEQ ID NO:22、SEQ ID NO:24所示的序列具备至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列一致性的氨基酸序列。
5.根据权利要求1-3任一项所述的T细胞,其中,所述T细胞表达嵌合抗原受体(CAR)。
6.根据权利要求5所述的T细胞,其中,所述CAR从N端到C端依次包括胞外抗原结合结构域、铰链区、跨膜结构域、胞内共刺激结构域和信号传导结构域;
其中,所述铰链区包括SEQ ID NO:12所示氨基酸序列,所述跨膜结构域包括SEQ IDNO:14所示氨基酸序列,所述共刺激结构域包括SEQ ID NO:16所示氨基酸序列,所述信号传导结构域包括SEQ ID NO:18所示氨基酸序列;所述胞外抗原结合结构域为单链抗体(scFv)形式,所述scFv包含下述任一项所示的氨基酸序列:SEQ ID NO: 4、6、8和10。
7.权利要求1-6任一项所述的T细胞在制备治疗或预防肿瘤的药物中的用途;优选地,所述肿瘤选自肝癌、肺癌、白血病或间皮瘤。
8.一种药物组合物,包含权利要求1-6任一项所述的T细胞,以及药学上可接受的载体。
9.一种增强T细胞功能方法,所述方法为体内方法或体外方法,所述方法选自以下各项组成的组:
(1) 促进T细胞增殖的方法,(2) 促进TSCM产生的方法,
所述方法包含以下步骤:提高T细胞中ARHGAP11B蛋白和/或GREM1蛋白的表达量和/或活性。
10.根据权利要求9所述的增强T细胞功能方法,所述方法还包括向所述T细胞中加入一种或多种细胞因子剂;优选地,所述细胞因子剂包括白细胞介素。
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