CN111788302A - 修饰的t细胞及其用途 - Google Patents
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Abstract
本发明涉及其中TCR/CD3复合物表达被降低或消除的修饰的T细胞,及其制备方法。还包括用于过继性疗法和治疗疾病,例如癌症、感染或自身免疫性疾病的包含所述修饰的T细胞的药物组合物。
Description
技术领域
本发明涉及TCR/CD3复合物表达被降低或消除的修饰的T细胞,及其制备方法。还包括用于过继性疗法和治疗疾病,例如癌症、感染或自身免疫性疾病的包含所述修饰的T细胞的药物组合物。
背景技术
T细胞在控制肿瘤和病原体感染方面具有重要作用。过继性T细胞疗法是一种恢复免疫能力的新型有前途的治疗方法。靶向CD19的嵌合抗原受体(CAR)T细胞在B细胞白血病和淋巴瘤患者中获得了持久的缓解。然而,这种T细胞疗法的主要障碍在于从每个患者定制生产CAR T细胞。
这种患者特异性的自体范例是大规模应用CAR技术的主要限制因素,因为它需要由熟练的团队,用符合GMP要求的专门设施或对集中式加工系统进行大量投资进行。另外,由于CAR T产品制备所固有的延迟使得不能立即给药,从而损害对大多数重症患者有利的结果。此外,对于因先前化疗而淋巴细胞严重减少的患者而言,制备自体产品可能不可行。
为了克服这个障碍,“现成的”第三方产品可以用作通用的供体资源。在过继转移同种异体产品之前,必须防止由T细胞表面的T细胞受体(TCR)复合物引起的移植物抗宿主病(GvHD)。
研究表明,通过破坏α/βT细胞中的TCR组分(α或β链),TCR/CD3复合物的表达被破坏,导致同种异体T细胞的GvHD作用被消除(Laurent等,2015;Ren等,2016)。已经报道了其中α或β链被破坏的T细胞在治疗各种疾病例如癌症、感染或自身免疫疾病中的成功应用。例如,Cellectis报告称,用TCRα链敲除的CAR T细胞治疗小儿急性淋巴细胞白血病(ALL)患者成功消除了癌细胞并预防了GvHD(Waseem等,2017)。在此,发明人发现,除TCRα或β链外,破坏包括CD3γ链、CD3δ链和CD3ε链以及CD247ζ链在内的CD3组分也可以破坏T细胞中的TCR/CD3复合物,从而消除α/βT细胞的GvHD作用。令人惊讶地,发明人进一步观察到,破坏CAR T细胞中的CD3γ、CD3δ和CD3ε和CD247ζ增强了其中央记忆表型和肿瘤杀伤能力,特别是与TCRα或β链破坏的CAR T细胞相比。
发明内容
因此,在第一个方面,本发明涉及一种修饰的T细胞,其中通过抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达以破坏TCR/CD3复合物的表达水平。在进一步的实施方案中,根据本发明的修饰的T细胞进一步显示TCRα和/或β基因的表达被抑制或消除。
本发明还涉及包含根据本发明的修饰的T细胞的药物组合物。在进一步的实施方案中,所述药物组合物用于治疗或预防癌症、感染或自身免疫性疾病。
例如,可以用修饰的T细胞治疗的癌症包括但不限于急性淋巴细胞性白血病(ALL)、慢性淋巴细胞性白血病(CLL)、急性骨髓性白血病(AML)、乳腺癌、肺癌、结直肠癌、胃癌、胰腺癌、卵巢癌、转移性腺癌、肝转移瘤、肉瘤、骨肉瘤、神经母细胞瘤、黑色素瘤、间皮瘤、胶质母细胞瘤、神经胶质瘤、恶性神经胶质瘤、肝细胞癌、非小细胞肺癌(NSCLC)、神经节神经母细胞瘤、脑癌、肾癌和前列腺癌癌症。可以用修饰的T细胞治疗的传染病包括但不限于病毒、细菌、真菌和寄生虫引起的感染。可以用修饰的T细胞治疗的自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、Graves病、炎症性肠病、多发性硬化症、牛皮癣、类风湿关节炎、Addison病、综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血和系统性红斑狼疮。
在第二个方面,本发明涉及增强T细胞的中央记忆表型的方法,其包括通过在所述T细胞中抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达以破坏TCR/CD3复合物的表达水平。在进一步的实施方案中,根据本发明的方法进一步包括抑制或消除TCRα和/或β基因的表达。
在第三个方面,本发明涉及增强T细胞的肿瘤杀伤能力的方法,其包括通过在所述T细胞中抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达以破坏TCR/CD3复合物的表达水平。在进一步的实施方案中,根据本发明的方法进一步包括抑制或消除TCRα和/或β基因的表达。
在第四个方面,本发明涉及消除T细胞的GvHD作用的方法,其包括通过在所述T细胞中抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达破坏TCR/CD3复合物的表达水平。在进一步的实施方案中,根据本发明的方法进一步包括抑制或消除TCRα和/或β基因的表达。
在一个实施方案中,可以使用本领域中的任何技术来抑制或消除靶基因的表达,包括但不限于基因突变、RNA介导的抑制、DNA基因编辑、RNA编辑、碱基编辑等。
在一个实施方案中,通过以下来实现根据本发明的TCR/CD3复合物的破坏:在选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因中引入基因突变,该基因突变导致所述选定基因的表达被抑制或消除。基因突变的实例包括但不限于敲除突变、截短突变、点突变、错义突变、取代突变、移码突变、插入突变、重复突变、扩增突变、易位突变或反向突变,以及导致相应基因活性降低或失活的任何其他基因突变。在靶基因中产生至少一个突变的方法是本领域众所周知的,包括但不限于随机诱变和筛选、定点诱变、PCR诱变、插入诱变、物理诱变、化学诱变和辐射。可以使用以下进行特异性的或随机的诱变,例如使用合适的物理或化学诱变剂,使用合适的寡核苷酸,使DNA序列进行PCR产生的诱变或其任意组合。物理和化学诱变剂的实例包括但不限于紫外线(UV)辐射、羟胺、N-甲基-N'-硝基-N-亚硝基胍(MNNG)、N-甲基-N'-亚硝基胍(NTG)O-甲基羟胺、亚硝酸、甲烷磺酸乙酯(EMS)、亚硫酸氢钠、甲酸和核苷酸类似物。当使用这样的试剂时,通常如下进行诱变:在合适的条件下,在选择的诱变剂存在下孵育待诱变的植物细胞或组织,然后选择表现出靶基因表达降低或不表达的突变体。
在一个实施方案中,通过RNA介导的抑制选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达水平来实现根据本发明的TCR/CD3复合物的破坏。特别地,所述RNA介导的对靶基因表达的抑制是通过以下来实现:将编码与靶基因的转录序列基本上相同或基本上互补的RNA分子或其片段的多核苷酸引入植物细胞,其中所述多核苷酸导致所述植物中靶基因的表达受抑制。本发明的范围还涵盖构建体,所述构建体包含编码与靶基因的转录序列基本上相同或基本上互补的RNA分子或其片段的多核苷酸,其中所述构建体的表达导致靶基因在所述植物中的表达受抑制。
本领域技术人员知道,根据本发明的多核苷酸不需要具有100%的序列互补性,但至少足以提供这样的RNA分子,所述RNA分子允许与从靶基因或靶基因的DNA转录的RNA杂交以形成双链体,从而允许基因沉默机制。因此,在实施方案中,将多核苷酸片段设计成与CD3γ、CD3δ、CD3ε和CD247ζ靶基因序列或从靶基因转录的信使RNA中的18个或更多个连续核苷酸的序列基本上相同或基本上互补。“基本上相同”是指当与靶基因或从靶基因转录的RNA中的18个或更多个连续核苷酸的序列相比时,具有100%的序列同一性或至少约83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98或99%同一性;“基本上互补”是指当与靶基因或从靶基因转录的RNA中的18个或更多个连续核苷酸的序列相比时,具有100%的序列互补性或至少约83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98或99%的序列互补性。在一些实施方案中,将多核苷酸分子设计成与给定靶基因的一个等位基因或一个家族成员具有100%的序列同一性或互补性。
许多RNA介导的抑制方法是本领域已知的。在RNA介导的抑制方法中使用的RNA分子的非限制性实例包括但不限于反义RNA、miRNA、siRNA和长的非编码RNA。反义RNA是单链RNA,与细胞中转录的信使RNA(mRNA)链互补。当反义RNA在细胞中表达时,它会与特定的信使RNA分子结合并使之失活。siRNA是双链RNA分子,长度为20-25个碱基对。当分离成单链并整合到活性RISC复合物中后,它与其靶标mRNA碱基配对并诱导靶标mRNA的裂解,从而阻止将其用作翻译模板。miRNA是通常约21个核苷酸的小RNA,能够通过与靶蛋白的mRNA结合来调节靶基因表达,从而导致靶蛋白mRNA的去稳定化或翻译抑制,最终导致靶蛋白mRNA的降低。目标蛋白。选择和设计用于基因抑制的siRNA和miRNA的方法是本领域众所周知的。长的非编码RNA(长ncRNA或IncRNA)是长于200个核苷酸的非蛋白质编码转录本(Perkel,BioTechniques,54(6):301-304(2013))。与在不同物种中表现出强烈保守性的许多小RNA相比,长ncRNA通常缺乏强烈的保守性。根据长ncRNA在基因组中与蛋白质编码基因的接近程度,可以将其分为五类:有义、反义、双向、内含子和基因间,其通过多种机制调节基因表达,例如通过基因转录(例如,通过基因特异性转录调节和基础转录机制调节)、转录后调节(例如,通过mRNA剪接,翻译和siRNA指导的基因调控)或通过表观遗传调控。
在一个实施方案中,通过使用核酸酶来基因编辑选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因来实现根据本发明的TCR/CD3复合物的破坏。核酸酶的非限制性实例包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、基于转录激活子样效应子的核酸酶(TALEN)和用于成簇规则间隔的短回文重复序列(CRISPR/Cas)系统的Cas酶。在一个优选的实施方案中,通过CRISPR/Cas系统获得根据本申请的修饰的T细胞。
在微生物物种中常见的大范围核酸酶具有独特的特性,即具有非常长的识别序列(>14bp),因此使其天然具有很高的特异性。但是,实际上没有机会找到对特定DNA序列起作用所需的确切的大范围核酸酶。为了克服这一挑战,已经使用诱变和高通量筛选方法来创建识别独特序列的大范围核酸酶变体。其他人已经能够融合各种大范围核酸酶并产生识别新序列的杂合酶。还有其他人尝试以一种称为合理设计的大范围核酸酶的方法来改变大范围核酸酶的DNA相互作用氨基酸,以设计序列特异性的大范围核酸酶。
锌指核酸酶(ZFN)以模块方式识别目标DNA:每种蛋白质由至少三个锌指结构域组成,并且单个锌指结构域与3bp序列相互作用,使其成为理想的可编程序列特异性DNA结合蛋白
在2011年,TALEN成为ZFN的竞争替代物。与锌指不同,TALE蛋白中的每个重复结构域都识别一个碱基。可以将四个不同的重复结构域混合并匹配以创建新的DNA结合蛋白,这些蛋白可以与FokI结构域连接以创建一类新的可编程靶标DNA核酸酶。这些分子能够在特定的基因组位点进行精确的靶向和切割,以生成双链断裂(DSB),然后进行非同源末端连接(NHEJ)或同源定向修复(HDR)介导的修复,从而实现精确的基因组编辑。
使用ZFN和TALEN的研究已导致重要的科学发现和治疗方法的发展。实际上,使人原代T细胞中的HIV共受体C-C趋化因子受体5型(CCR5)失效的基于ZFN的HIV治疗目前正在临床试验中,并显示出了巨大的希望。但是,这些基于蛋白质的基因组工程系统对靶标DNA序列的识别取决于蛋白质序列。因此,对于每个特定的靶标DNA序列,都需要繁琐复杂的蛋白质工程和优化,并且将许多这些蛋白质递送到细胞中以同时进行多重遗传操作具有挑战性。由于这些困难,它们在大规模基因组操作或基因筛选中的使用受到限制。
CRISPR技术源自II型CRISPR系统。II型CRISPR系统将来自入侵DNA的序列整合到细菌宿主基因组内以阵列编码的CRISPR重复序列之间。来自CRISPR重复序列阵列的转录物被加工成CRISPR RNA(crRNA)(Deltcheva等,2011),每个都包含一个从入侵DNA转录的可变序列(即“原间隔子”序列),以及部分CRISPR重复序列。每个crRNA与称为反转录CRISPR RNA(tracrRNA)(Deltcheva等,2011)的第二RNA杂交,这两个RNA与Cas9 DNA核酸内切酶形成复合物(Jinek等,2012)。crRNA的原间隔子编码部分将Cas9引导至互补的靶标DNA序列,并且如果与称为原间隔子相邻基序(PAM)的短序列相邻,则切割DNA。化脓性链球菌的II型CRISPR系统已用于诱导序列特异性双链断裂(DSB)和靶向基因组编辑。2012年,Jinek等人首次证明化脓性链球菌的Cas9蛋白(SpCas9)可以与tracrRNA-crRNA RNA复合体结合,在体外通过crRNA和靶标DNA之间的Watson-Crick碱基配对在靶标DNA序列处诱导DSB(Jinek等,2012)。这项研究还表明,指导Cas9结合并切割特定的DNA序列不需要RNA复合物。通过使用设计的单向导RNA(sgRNA)可以轻松实现该过程。
几项研究报道了使用不同方案成功使用CRISPR/Cas9系统破坏T细胞中B2m、PD1、CTLA4、CCR5、CXCR4、Lag3等的效率,范围从7%到90%以上。除了天然II型CRISPR外,近年来还发现了新型V型CRISPR。迄今为止,经过实验测试的V型CRISPR系统包括使用以下效应蛋白,这些效应蛋白已被重新命名为Cas12a-e:Cas12a(也称为Cpf1;V-A亚型)、Cas12b(也称为C2c1;V-B亚型)、Cas12c(也称为C2c3;V-C亚型)、Cas12d(也称为CasY;V-D亚型)和Cas12e(也称为CasX;V-E亚型),它们在进化上都与Cas9不同。设计包含针对靶基因的特异性sgRNA的CRISPR/Cas9系统并将其递送的方法是本领域已知的。例如,可以通过用编码Cas和sgRNA的质粒转染,通过非整合病毒例如腺病毒和腺病毒相关病毒(AAV),通过Cas核糖核蛋白(RNP)或通过电穿孔来递送系统。
还可以使用Argonaute(Ago)蛋白来实现基因编辑。所有RNA干扰途径使用小的单链RNA(ssRNA)分子将Argonaute(Ago)家族蛋白引导至互补的ssRNA靶标:RNA引导的RNA干扰。Daan C等人证明,嗜热栖热菌(Thermus thermophilus)的Ago(TtAgo)是外源DNA摄入和传播的屏障。尽管与其真核同类物在结构上同源,TtAgo在宿主防御中通过DNA引导的DNA干扰发挥作用。2017年,伊利诺伊斯的一个小组声称使用另一种来自嗜热古细菌(Pyrococcusfuriosus)的Argonaute蛋白(PfAgo)作为人工限制性酶和向导DNA来编辑DNA(Enghiad等,2017)。
在一个实施方案中,通过RNA编辑选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的转录物来实现根据本发明的TCR/CD3复合物的破坏。RNA编辑是转录后的过程,通过该过程,细胞机器可以对RNA分子内的特定核苷酸序列进行离散变化,从而增强RNA和蛋白质的多样性(Gott和Emeson,2000)。RNA编辑可能涉及核碱基修饰,例如由胞苷脱氨酶介导的胞苷向尿苷的转化,或涉及作用于RNA的腺苷脱氨酶(ADAR)的腺苷向肌苷的转化,以及非模板核苷酸的添加和插入。也有报道称,包含Cas13a的CRISPR系统用于靶向敲除内源转录本,敲除水平与RNA干扰相当,且特异性更高(Abudayyeh等,2017)。
在一个实施方案中,根据本申请的T细胞是T细胞、CAR T细胞、TCR T细胞、病毒特异性T细胞、NTK细胞、肿瘤浸润淋巴细胞、造血干细胞或多能干细胞。
提供以下定义以更好地定义本发明并指导本领域普通技术人员实施本发明。除非另有说明,否则术语应根据相关领域的普通技术人员的常规用法来理解。
如本文所用,术语“TCR”或“T细胞受体”是指在T细胞或T淋巴细胞的表面上发现的分子,其负责将抗原片段识别为与主要组织相容性复合物(MHC)分子结合的肽。TCR由两条不同的蛋白质链组成(即,它是一个异二聚体)。在95%的人T细胞中,TCR由一条α链和一条β链(α/βT细胞)组成,而在5%的人T细胞中,TCR由γ链和δ链组成(γ/δT细胞)。术语“TCR T细胞”是指表达转基因TCR的T细胞。
如本文所用,术语“CD3”是指有助于激活细胞毒性T细胞(CD8+初始T细胞)和T辅助细胞(CD4+初始T细胞)的T细胞共受体。它由蛋白质复合物组成,包含四条不同的链。在哺乳动物中,复合物包含一条CD3γ链、一条CD3δ链和两条CD3ε链。
如本文所用,“TCR/CD3复合物”是涉及GvHD作用的蛋白质复合物,并且由可变的TCR受体α和β链以及三个二聚体信号模块CD3δ/ε、CD3γ/ε、CD247ζ/ζ或ζ/η组成(见图1)。每个亚基跨膜结构域中的可电离残基形成相互作用的极性网络,将复合物结合在一起。由于TCR的胞质尾非常短,不太可能参与信号传导,因此这些信号分子对于将信号从触发的TCR传到细胞中至关重要。当TCR与抗原肽和MHC(肽/MHC)结合时,通过信号传导(即,由相关酶、共受体、特异性衔接子分子以及激活或释放的转录因子介导的一系列生化事件)激活T淋巴细胞。
术语“DNA基因编辑”是指一种基因工程,其中在活生物体的基因组中插入、删除、修饰或替换DNA。在2018年,进行此类编辑的常用方法是使用工程核酸酶或“分子剪刀”。这些核酸酶在基因组中的期望位置产生位点特异性双链断裂。诱导的双链断裂通过非同源末端连接(NHEJ)或同源重组(HR)修复,从而导致靶向突变(“编辑”)。截至2015年,已使用了四个工程化核酸酶家族:大范围核酸酶、锌指核酸酶(ZFN)、基于转录激活子的基于效应子的核酸酶(TALEN)和成簇的规则间隔的短回文重复(CRISPR/Cas9)系统。
最初“CRISPR”被描述为含有短的重复的碱基序列的原核DNA片段。在回文重复中,两个方向上的核苷酸序列相同。每个重复之后是先前暴露于外源DNA(例如病毒或质粒)的间隔DNA的短片段。CRISPR基因座通常由以下组成:CRISPR相关(Cas)基因的簇集和签名CRISPR阵列--一系列由与外部遗传元件(原间隔子)中的序列相对应的可变序列(间隔子)隔开的重复序列(直接重复)。尽管Cas基因被翻译成蛋白质,但大多数CRISPR阵列首先被转录为单个RNA,然后再加工成较短的CRISPR RNA(crRNA),从而指导某些Cas酶的溶核活性以降解目标核酸。
术语“CRISPR/Cas系统”是指赋予对外源遗传元件(诸如质粒和噬菌体中存在的那些)的抗性并提供一种形式的获得性免疫的原核免疫系统。通常,CRISPR/Cas系统至少包含Cas内切核酸酶和向导RNA。带有间隔区序列的RNA可以帮助Cas(CRISPR相关)蛋白识别并切割外源DNA。
术语“向导RNA”或“gRNA”通常是指一种RNA分子,其将Cas核酸内切酶引导至靶基因座并与靶基因座内的互补序列特异性杂交,从而在该核酸内切酶的作用下引起靶基因座的双链断裂。gRNA包括但不限于crRNA、sgRNA和其他嵌合向导RNA,例如caRNA、csRNA、catRNA。术语“单向导RNA”或“sgRNA”是指通过将crRNA和tracrRNA分子融合为“单向导RNA”而设计的人工工程化RNA,当与Cas9蛋白结合使用时,它可以找到并切割向导RNA特异性的DNA靶标。
在典型的CRISPR系统中,向导RNA是crRNA,其通常包含直接重复序列和间隔子序列。“直接重复序列”是指在CRISPR基因座内由可变序列(间隔子)隔开的重复序列。“间隔子”是指插入到由入侵的病毒或质粒DNA产生的CRISPR基因座(称为“原型间隔子”)中的病毒DNA。野生型Cas9的间隔序列为20bp长,而野生型Cpf1的crRNA的全长间隔子为24bp。在随后的入侵中,crRNA会将Cas蛋白引导至入侵的原间隔子序列。但是,除非有相邻的PAM序列,否则Cas蛋白不会切割原间隔子序列。细菌CRISPR基因座中的间隔子不包含PAM序列,因此不会被核酸酶切割。但是,入侵病毒或质粒中的原间隔子包含PAM序列,因此将被Cas内切核酸酶切割。为了编辑基因,合成向导RNA以执行识别在3'端具有PAM序列的基因序列的功能。
术语“碱基编辑”是指一种新的基因组编辑技术,该技术通过利用与脱氨酶融合的催化死Cas蛋白(dCas),将目标基因组位点处的特定DNA碱基直接且不可逆地转化为另一种碱基。重要的是,在DNA的情况下,这不需要双链断裂(DSB)就可以实现。由于许多遗传性疾病源于点突变,因此这项技术对人类健康和疾病的研究具有重要意义(Landrum,M.J.等,2015)。
术语“CAR”或“嵌合抗原受体”是指将任意特异性移植到免疫效应细胞(例如T细胞)上的工程化受体。通常,这些受体用于将单克隆抗体的特异性移植到T细胞上,逆转录病毒载体可促进其编码序列的转移。受体之所以称为嵌合体,是因为它们由不同来源的部分组成。
术语“CAR T细胞”或“嵌合抗原受体T细胞”是指具有嵌合抗原受体的工程化T细胞,其对选定的靶标具有预定的特异性。CAR T细胞一旦遇到靶标(例如癌细胞),就会通过诸如以下机制来破坏癌细胞:广泛刺激细胞增殖,增加细胞对其他活细胞的毒性程度(即,细胞毒性)以及通过使免疫系统细胞分泌的且对生物体内其他细胞有影响的因子产量增加。
术语“中央记忆T(TCM)细胞”是指表达CD45RO、C-C趋化因子受体7型(CCR7)和L-选择蛋白(CD62L)的T细胞。中央记忆T细胞也具有中等至高表达的CD44。这种记忆亚群通常在淋巴结和外周循环中发现。TCM细胞被认为具有与记忆细胞干细胞相关的某些特性。由于称为STAT5的重要转录因子的高磷酸化水平,TCM细胞显示出自我更新的能力。在小鼠中,与最终分化的效应细胞相比,TCM细胞已在几种不同的模型系统中显示出针对病毒、细菌和癌症的出色保护。据报道,在CAR T细胞中,更年轻的中央记忆表型与增强的持久性和更广泛的增殖能力有关。因此,增强中央记忆表型对于CAR T细胞的性质将是有益的,特别是当所述CART细胞用于治疗疾病时。
术语“截短的sgRNA”或“较短的sgRNA”是指靶标互补性区域长度小于20个核苷酸的较短的sgRNA,可在不牺牲中靶基因组编辑效率的情况下将某些脱靶位点处的不希望的突变降低5,000倍或更多。研究表明,使用具有17、18或19个互补核苷酸的间隔子序列的较短或截短的sgRNA不会降低平台的靶向范围,因为具有17、18或19个互补核苷酸的靶位点每个都会以与具有20个互补核苷酸的那些相同的频率出现在随机DNA中。
附图说明
图1:示出了TCR/CD3复合物的结构。
图2:分别用靶向TCRα链、CD247ζ链、CD3ε链、CD3γ链和CD3δ链的sgRNA破坏TCR/CD3复合物。
图3:TCRα链、CD247ζ链、CD3ε链、CD3γ链和CD3δ链敲除的T细胞的中央记忆表型。
图4:TCRα链、CD247ζ链、CD3ε链、CD3γ链和CD3δ链敲除的T细胞的细胞毒性。n=3,通过学生T检验,*P<0.05。
具体实施方式
仅出于说明的目的提供以下实施例,而不以任何方式限制本发明的保护范围。
实施例1.CAR T细胞中的TCR/CD3复合物破坏
分别将由T7启动子驱动的编码靶向TCRα、CD247ζ、CD3ε、CD3γ和CD3δ的sgRNA的序列克隆进BH-MSG载体。
进行RNA体外转录(IVT)之前,将Cas9和sgRNA质粒线性化。将IVT RNA存储于-80℃的无核酸酶的小瓶中,用于单次使用。用mMESSAGE mMACHINE T7ULTRA试剂盒(LifeTechnologies,AM1345,Carlsbad,CA)体外转录Cas9 mRNA。用HiScribeTM T7 High YieldRNA Synthesis试剂盒(NEB)转录sgRNA。
为制备修饰的CAR T细胞,通过Ficoll-PaqueTM PREMIUM(GE healthcare)用白细胞分离术从健康志愿者供体分离原代人CD4和CD8 T细胞。然后,先用CD3/CD28磁珠激活T细胞1填,再用表达CAR的慢病毒转导。之后,用OPTI-MEM洗涤CAR T细胞3次,并重悬于OPTI-MEM(Invitrogen)中,终浓度为1-3×108个细胞/ml。在第3天,用BTX Agile Pulse Max电转仪(Harvard Apparatus BTX)在360V、1ms下将20ug Cas9 mRNA电转进细胞,并在第4天电转进分别靶向TCRα、CD247ζ、CD3ε、CD3γ和CD3δ的各种sgRNA。每2天分离细胞。用空载体电转的细胞用作对照(Mock)。
电转后,立即将细胞放入1ml预热的培养基中,并在IL-2(300IU/ml)存在下,于37℃、5%CO2下培养。为富集细胞,于4℃下将用Auto MACS缓冲液洗涤的CAR T细胞与CD3微珠(Miltenyi Biotec,130-050-101)一起孵育15分钟。洗涤2次后,将细胞通过LD柱(MiltenyiBiotec),收集通过级分以待后用。
实施例中所用的sgRNA的间隔子序列选自下表1。
表1.靶向TCRα、CD247ζ、CD3ε、CD3γ和CD3δ基因的sgRNA的间隔子序列
实施例2.修饰的CAR T细胞中TCR/CD3复合物的表达
据报道,破坏α/βT细胞中的TCR组分(α或β链)可导致TCR/CD3复合物表达的丧失,从而阻止同种异体T细胞的GvHD作用。我们假设TCR/CD3复合物的完整性需要TCR组分、CD3组分和CD247ζ的完全组装。
因此,为了测试CD3组分和CD247ζ的破坏是否可能导致TCR/CD3复合物的破坏,我们通过CRISPR基因编辑敲除了CD3组分(包括CD3γ、CD3δ和CD3ε)和CD247ζ。
根据实施例1中描述的方法获得修饰的CAR T细胞。提取修饰的CAR T细胞的基因组DNA,对位于靶位点两侧的PCR产物进行Sanger测序,以确认在DNA链上的靶标编辑。还通过TIDE(跟踪Indel,DE composition)软件分析了结果。证实了TCRα、CD247ζ、CD3ε、CD3γ和CD3δ基因中的基因组破坏和插入(数据未显示)。
接下来,通过流式细胞术用APC抗CD3抗体(目录号555335,BD Biosciences)染色来测量TCR/CD3表达。
图2显示了流式细胞仪的结果,其中以CD3阴性细胞的数量表示TCR/CD3表达。如图2所示,在Mock电转组中几乎没有CD3阴性细胞群,但用针对TCRα链、不同CD3组分和CD247ζ的CRISPR观察到了有效的基因消除。这些结果表明,敲除CD3组分和CD247ζ也可以消除TCR/CD3复合物在T细胞中的表达。
实施例3.测定修饰的CAR T细胞的表型
为了测量CD3组分和CD247ζ破坏的T细胞的中央记忆表型,通过流式细胞术测定CD45RO和CD62L的表达。结果如图3所示。
令人惊讶地,我们发现CD247ζ、CD3ε和CD3γ敲除的CAR T细胞比TCRα敲除的CAR T细胞表现出更多的CD45RO和CD62L双阳性中央记忆表型。具体地,在模拟T细胞群中大约有31.9%的CD45RO和CD62L双阳性中央记忆细胞。在TCRα基因敲除的细胞中,CD45RO和CD62L双阳性细胞群减少至13.1%。然而,与TCRα敲除相比,CD247ζ、CD3ε和CD3γ破坏的T细胞包含更高数量的CD45RO和CD62L双阳性中央记忆细胞,其水平相当于Mock T细胞。还注意到,CD3δ敲除导致与TCRα相当的中央记忆表型。
已知更年轻的CAR T细胞中央记忆表型与持久性增强和更广泛的增殖能力有关,因此对于延长CAR T细胞的能力将是有益的。因此,该数据表明,CD247ζ、CD3ε和CD3γ破坏比TCRα破坏更有利于制备有效的通用CAR T细胞。
实施例4测定修饰的CAR T细胞的细胞毒性
为了检测TCR/CD3破坏是否会影响CAR T细胞的效应子功能,将修饰的CAR T细胞与Nalm6靶细胞进行共培养。具体地,通过改良版本的基于萤光素酶的CTL测定检测修饰的CART细胞的细胞毒性,其中制备并使用了Nalm6肿瘤细胞。将所得的Nalm6细胞以1X105个细胞/mL的浓度重悬在R10培养基中,并与修饰的CAR T细胞在37℃孵育过夜。然后,将100μL的混合物转移至96孔黑色发光计板。接下来,添加100μL的底物,并立即测定发光。结果如图4所示。
令人惊讶地,发现修饰的CAR T细胞在靶标特异性杀伤中表现出显著差异。尤其是,尽管Mock和TCRα链敲除的CAR T细胞之间没有显著差异,但CD247ζ、CD3ε和CD3δ敲除的CAR T细胞显示出显著更高的体外裂解能力,而CD3γ敲除的CAR T细胞表现出与TCRα敲除的CART细胞相当的杀伤能力。
综上所述,所有这些数据表明,CD3γ、CD3δ和CD3ε以及CD247ζ破坏导致与TCR组分(TCRα链或β链)破坏同样有效的TCR/CD3复合物消融,同时表现出更高的中央记忆表型和增强的目标肿瘤的杀伤能力。
序列表
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Claims (17)
1.一种修饰的T细胞,其中通过抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达以破坏TCR/CD3复合物的表达水平。
2.权利要求1所述的修饰的T细胞,其中所述修饰的T细胞进一步显示TCRα和/或β基因的表达被抑制或消除。
3.权利要求1或2所述的修饰的T细胞,其中所述T细胞是T细胞、CAR T细胞、TCR T细胞、病毒特异性T细胞、NTK细胞、肿瘤浸润淋巴细胞、造血干细胞或多能干细胞。
4.一种药物组合物,其包含权利要求1-3任一项所述的修饰的T细胞。
5.权利要求1-3任一项所述的修饰的T细胞或权利要求4所述的药物组合物在制备用于治疗或预防癌症、感染或自身免疫性疾病的药物中的用途。
6.一种增强T细胞中央记忆表型的方法,包括通过在所述T细胞中抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达以破坏TCR/CD3复合物的表达水平。
7.一种增强T细胞的肿瘤杀伤能力的方法,其包括通过在所述T细胞中抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达以破坏TCR/CD3复合物的表达水平。
8.一种消除T细胞的GvHD作用的方法,其包括通过在所述T细胞中抑制或消除选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的表达破坏TCR/CD3复合物的表达水平。
9.权利要求6-8任一项所述的方法,进一步包括抑制或消除TCRα和/或β基因的表达。
10.权利要求6-8任一项所述的方法,其中所述破坏TCR/CD3复合物的表达水平通过基因突变、RNA介导的抑制、RNA编辑、DNA基因编辑或碱基编辑来实现。
11.权利要求10所述的方法,其中所述基因编辑方法涉及使用选自以下的核酸酶:大范围核酸酶、ZFN、TALEN和Cas酶。
12.权利要求11所述的方法,其中所述核酸酶是Cas9酶。
13.权利要求11所述的方法,其中所述Cas酶用于包含至少一种sgRNA的CRISPR/Cas系统,所述sgRNA包含选自SEQ ID NO:1-40的间隔子序列,或与选自SEQ ID NO:1-40的间隔子序列具有至少17个核苷酸相同或互补的截短的间隔子序列。
14.根据权利要求6-13任一项所述的方法获得的T细胞。
15.包含至少一种sgRNA的CRISPR/Cas系统,所述sgRNA包含选自SEQ ID NO:1-40的间隔子序列,或与选自SEQ ID NO:1-40的间隔子序列具有至少17个核苷酸相同或互补的截短的间隔子序列,其中所述CRISPR/Cas系统在T细胞种的表达导致所述T细胞中的TCR/CD3复合物表达被破坏。
16.一种包含多核苷酸的构建体,所述多核苷酸编码与选自CD3γ、CD3δ、CD3ε和CD247ζ的至少一种基因的转录序列基本上相同或基本上互补的RNA分子或其片段,其中所述构建体在T细胞种的表达导致所述T细胞中的TCR/CD3复合物表达被破坏。
17.权利要求16所述的构建体,其中所述RNA分子是反义RNA、miRNA、siRNA或长的非编码RNA。
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PETROS CHRISTOPOULOS等: "A Novel Thymoma-Associated Immunodeficiency with Increased Naive T Cells and Reduced CD247 Expression", 《JOURUAD OF IMMUUODOGY》, vol. 194, no. 7, pages 3045 - 3053, XP055710995, DOI: 10.4049/jimmunol.1402805 * |
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