CN112040960A - 修饰免疫细胞以增加活性 - Google Patents
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Abstract
组合物、制备经修饰的免疫细胞(如NK细胞)的方法以及使用经修饰的免疫细胞(如NK细胞)治疗癌症、病毒和微生物感染的方法。所述经修饰的CISH‑/‑NK细胞对细胞因子如IL‑2和/或IL‑15表现出高度敏感性,并维持扩增和抗肿瘤功能。
Description
相关申请的交叉引用
本申请要求2018年5月11日提交的美国临时申请第62/670,033号的优先权权益,所述申请以引用的方式并入本文中。
政府权益声明
本发明是在基金编号CA217885和CA203348下由美国国立卫生研究院支助进行的。政府拥有本发明的某些权利。
背景技术
天然杀伤(NK)细胞是先天免疫系统的关键部分,并且是淋巴细胞群在抗肿瘤和抗感染免疫方面的重要效应子。然而,肿瘤进展和慢性感染通常会造成NK细胞耗尽,导致效应子功能不佳,并限制NK细胞的抗肿瘤/感染潜力。导致肿瘤和慢性感染中NK细胞耗尽的确切机制尚不清楚。
NK细胞对异常细胞的检测是通过来自配体和细胞因子(如白介素15(IL-15))的激活和抑制信号来控制的。细胞因子诱导型含SH2的蛋白质(CIS)是NK细胞中IL-15信号传导的关键负调控因子,它由人的CISH基因编码。CISH是响应于IL-15而快速诱导的,并且CISH基因的缺失已显示出增加NK细胞对IL-15的敏感性。小鼠的最新研究已经表明,CIS是NK细胞介导的肿瘤免疫的有效抑制检查点。
NK细胞需要细胞因子如白介素2(IL-2)和IL-15来维持活性和功能,但是IL-2会造成全身性毒性。因此,仍然需要用于治疗癌症和其他疾病的临床NK细胞疗法,其无需细胞因子或仅需要低细胞因子剂量即可维持扩增和功能。
发明内容
本公开总体上提供用于在癌症治疗中使用经CISH-/-修饰的NK细胞的组合物和方法。经修饰的NK细胞表现出对IL-2和/或IL-15刺激的高度敏感性,并且可以通过低浓度细胞因子或生长因子如白介素来维持扩增和抗肿瘤功能。
根据本公开的一个方面,提供了一种经CISH-/-修饰的NK细胞,其可用作基于NK细胞的疗法的细胞来源,所述基于NK细胞的疗法用于治疗癌症和其他疾病或感染,具有优于未经修饰的天然NK细胞的改进的治疗效果。
根据本公开的一个方面,提供了一种用于制造CISH-/-NK细胞的方法。
根据本公开的另一方面,提供了一种CISH-/-NK细胞的细胞培养物,以及包含CISH-/-NK细胞的药物组合物。
附图说明
将通过参考以下详细描述(其阐述其中利用本公开的原理的说明性实施例)和附图获得对本公开的特征和优点的更好理解,其中:
图1A-图1C描绘了使用常规方法的CISH丧失对NK分化的影响。
图2A-图2B描绘了使用改良方法的CISH丧失对NK分化的影响。
图3A-图3B描绘了CISH-/-NK细胞扩增。
图4A-图4B描绘了来自incucyte杀伤测定法的结果。
图5A-图5C描绘了CISH-/-iPSC-NK细胞在细胞因子刺激后显示出较高的单细胞多功能反应。
图6A-图6C描绘了CISH-/-iPSC-NK细胞显示出增加的基础糖酵解和糖酵解能力。
图7A-图7C描述了CISH-/-iPSC-NK细胞在人白血病全身性肿瘤模型中介导更好的抗肿瘤活性。
具体实施方式
本说明书中提到的所有出版物、专利以及专利申请都以引用的方式并入本文中,其引用程度就如同每个单独的出版物、专利或专利申请特定地并且单独地指示以引用的方式并入一般。对出版物的引用旨在引用其最新版本。
除非另有指示,否则本发明的实施将采用分子生物学、微生物学、细胞生物学、生物化学和免疫学的常规技术(包括重组技术),这些都在本领域技术范围内。这类技术在如《分子克隆:实验室手册(Molecular Cloning:A Laboratory Manual)》,第二版(Sambrook等人,1989)冷泉港出版社(Cold Spring Harbor Press);《寡核苷酸合成(Oligonucleotide Synthesis)》(MJ.Gait编辑,1984);《分子生物学方法(Methods inMolecular Biology)》,胡玛纳出版社(Humana Press);《细胞生物学:实验笔记本(CellBiology:A Laboratory Notebook)》(J.E.Cellis编辑,1998)学术出版社(AcademicPress);《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编辑,1987);《细胞和组织培养简介(Introduction to Cell and Tissue Culture)》(J.P.Mather和P.E.Roberts,1998)普莱纽姆出版社(Plenum Press);《细胞和组织培养:实验室程序(Cell and TissueCulture:Laboratory Procedures)(A.Doyle、J.B.Griffiths和D.G.Newell编辑,1993-1998)约翰·威利父子(J.Wiley and Sons);《酶学方法(Methods in Enzymology)》(学术出版社公司(Academic Press,Inc.));《实验免疫学手册(Handbook of ExperimentalImmunology)(D.M.Weir和CC.Blackwell编辑);《哺乳动物细胞的基因转移载体(GeneTransfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编辑,1987);《分子生物学最新方案(Current Protocols in Molecular Biology)(F.M.Ausubel等人编辑,1987);《PCR:聚合酶链式反应(The Polymerase Chain Reaction)》,(Mullis等人编辑,1994);免疫学最新方案(Current Protocols in Immunology)》(J.E.Coligan等人编辑,1991);《分子生物学简明方案(Short Protocols in Molecular Biology)(威利父子(Wiley and Sons),1999);《免疫生物学(Immunobiology)》(CA.Janeway和P.Travers,1997);《抗体(Antibodies)》(P.Finch,1997);《抗体:一种实用方法(Antibodies:apractical approach)》(D.Catty编辑,IRL出版社(IRL Press),1988-1989);《单克隆抗体:实用方法(Monoclonal antibodies:apractical approach)(P.Shepherd和C.Dean编辑,牛津大学出版社(Oxford University Press),2000);《使用抗体:实验室手册(Usingantibodies:a laboratory manual)》(E.Harlow和D.Lane(冷泉港实验室出版社,1999);《抗体(The Antibodies)》(M.Zanetti和JD Capra编辑,哈伍德学术出版社(HarwoodAcademic Publishers),1995);以及《癌症:肿瘤学的原理和实践(Cancer:Principles andPractice of Oncology)》(V.T.DeVita等人编辑,J.B.Lippincott Company,1993)。
本发明涉及用于治疗人类受试者的疾病如癌症或由例如病毒或细菌引起的感染的方法,其包含对有需要的人类受试者施用有效量的药物组合物,所述药物组合物包含人CISH-/-自然杀伤(NK)细胞和药学上可接受的载剂。
在实施例中,本发明涉及用于治疗人类受试者的癌症的方法,其中所述NK细胞衍生自人诱导的多能干细胞(iPSC)、胚胎干细胞(ESC)或外周血细胞。
在实施例中,本发明涉及用于治疗人类受试者的癌症或感染的方法,其中所述CISH-/-NK细胞对于受试者是自体的。
在实施例中,本发明涉及用于治疗人类受试者的癌症的方法,其中方法进一步包含向受试者施用有效量的细胞因子,如IL-2、IL-15或两者。
在实施例中,本发明涉及用于治疗人类受试者的癌症的方法,其中IL-2和/或IL-15的有效量小于天然NK细胞治疗所需的有效量。在实施例中,低浓度的IL-2在1和10U/ml或约5U/ml之间,而低浓度的IL-15在1和10ng/ml或约5ng/ml之间,这些浓度可以有效地维持CISH-/-NK细胞扩增和抗肿瘤功能。
可以在本发明中使用的细胞因子包括天然存在的、经修饰的和合成工程化的细胞因子和细胞因子样分子(如ALT-803或内克塔治疗公司(NEKTAR Therapeutics,Inc.)产品,如NKTR-358或NKTR-255)。细胞因子包括白介素,如IL-2、IL-12、IL-15、IL-18、IL-21。
在实施例中,本发明涉及用于治疗人类受试者的癌症的方法,其中癌症是造血系统肿瘤或实体瘤。
在实施例中,本发明涉及用于治疗人类受试者的疾病或感染的方法,其中与天然NK细胞相比,CISH-/-NK细胞对细胞因子刺激高度敏感,并且展示出改进的扩增、抗肿瘤功能和抗病毒功能。
在实施例中,本发明涉及药物组合物,所述药物组合物包含人CISH-/-NK细胞和至少一种药学上可接受的赋形剂。
在实施例中,本发明涉及药物组合物,其中与天然NK细胞相比,CISH-/-NK细胞对细胞因子刺激高度敏感,并展示出改进的扩增、抗肿瘤功能和抗病毒功能。
在实施例中,本发明涉及药物组合物,其中细胞因子刺激包含用白介素如IL-2和/或IL-15的刺激。在实施例中,低浓度的IL-2在1和10U/ml或约5U/ml之间,而低浓度的IL-15在1和10ng/ml或约5ng/ml之间,这些浓度可以有效地维持CISH-/-NK细胞扩增和抗肿瘤功能。
在实施例中,本发明涉及药物组合物,其中CISH-/-NK细胞衍生自诱导的多能干细胞、胚胎干细胞或外周血细胞。
在实施例中,本发明涉及用于生产CISH-/-NK细胞的方法,所述方法包含:从人诱导的多能干细胞(iPSC)或胚胎干细胞(ESC)中删除CISH基因;并且使用体外分化方案从CISH-/-iPSC衍生NK细胞。
在实施例中,本发明涉及用于生产CISH-/-NK细胞的方法,其中CISH基因的缺失是通过使用CRISPR系统(如CRISPR/Cas9系统)实现的。
在实施例中,本发明涉及用于生产CISH-/-NK细胞的方法,其中衍生步骤进一步包含将CISH-/-iPSC分化为>75%、>60%、>70%或>80%CD34+,然后分化为>75%、>60%、>70%或>80%CD45+和CD56+。
在实施例中,本发明涉及用于生产CISH-/-NK细胞的方法,其中第二分化在与Notch配体(例如与工程化为过表达Notch配体的OP9-DL4细胞)接触时发生。
在实施例中,本发明涉及用于生产CISH-/-NK细胞的方法,其中细胞培养物包含CISH-/-NK细胞。
定义
为了促进对本发明的理解,如本文使用的许多术语和缩写如下定义:
当介绍本发明或其(一个或多个)优选的实施例的要素时,冠词“一(a/an)”、“所述(the)”和“所述(said)”旨在表示存在一个或多个元素。术语“包含”、“包括”和“具有”旨在是包括性的并且意指可存在除所列要素外的附加要素。
当在两个或多个项目的列表中使用时,术语“和/或”表示所列出的项目中的任何一个可以单独使用或与所列出的项目中的任何一个或多个组合使用。例如,表述“A和/或B”旨在表示A和B中的任一个或两个,即单独的A、单独的B或组合的A和B。表述“A、B和/或C”旨在表示单独的A、单独的B、单独的C、组合的A和B、组合的A和C、组合的B和C或组合的A、B和C。
应理解,本文所描述的本发明的各方面和实施例包括“由…组成”和“主要由…组成”的各方面和实施例。
应当理解,范围格式的描述仅是为了方便和简洁,而不应被解释为对本发明范围的不灵活的限制。因此,应当认为范围的描述已经具体公开了所有可能的子范围以及所述范围内的各个数值。例如,对范围如从1到6的描述应被认为已经具体公开了如从1到3、从1到4、从1到5、从2到4、从2到6、从3到6等的子范围,以及所述范围内的单个数字,例如1、2、3、4、5和6。无论范围的广度如何,这都适用。值或范围在本文中可表达为“约”、从“约”一个特定值和/或到“约”另一个特定值。当表达这类值或范围时,所公开的其它实施例包括叙述的具体值,从一个特定值和/或到其它特定值。类似地,当通过使用先行词“约”将值表达为近似值时,应当理解,特定值形成另一个实施例。还将理解,其中存在公开的多个值,并且每个值在本文还被公开为除所述值本身之外的“约”特定值。在实施例中,“约”可用于意指例如在所叙述值的10%内、在所叙述值的5%内或在所叙述值的2%内。
如本文所用,“患者”或“受试者”是指待治疗的人类或动物受试者。
如本文所用,“增殖”或“扩增”是指细胞或细胞群体数目增加的能力。
如本文所用,含“纯化的细胞群”或“纯化的细胞组合物”的组合物意指至少30%、50%、60%、通常至少70%,并且更优选80%、90%、95%、98%、99%或更多的组合物中的细胞属于已鉴定类型。
如本文所用,“治疗有效”是指足以治疗或改善或以某种方式减轻与疾病如癌症或病状如感染相关联的症状的NK细胞的量。当参考一种方法使用时,方法足以有效地治疗或改善或以某种方式减轻与疾病或病状相关联的症状。例如,针对疾病的有效量是足以阻止或预防其发作的量;或如果疾病病理已开始,以减轻、改善、稳定、逆转或减慢疾病的进展,或以其他方式减轻疾病的病理后果。在任何情况下,有效量可以单剂量或分剂量给予。
如本文所用,术语“治疗”至少涵盖与患者的疾病或病状相关联的症状的改善,其中改善在广义上用于指至少参数值的降低,例如与所治疗病状相关联的症状。因此,“治疗”还包括疾病、病症、病理状况或与其相关联的至少症状被完全抑制(例如,防止发生)或停止(例如,终止)使得患者不再患有所述病状,或至少是表征所述病状的症状。
如本文所用,术语“药物组合物”是指药物可接受的组合物,其中组合物包含NK细胞,并且在一些实施例中进一步包含药学上可接受的载剂。在一些实施例中,药物组合物可以是组合。
如本文所用,术语“药学上可接受的”意指由联邦或州政府的监管机构批准的或在美国药典、除动物中(并且更具体地说人类和/或非人类哺乳动物中)安全使用的其他制剂之外的其他普遍认可的药典中列出的。
如本文所用,术语“药学上可接受的载剂”是指与NK细胞一起施用的赋形剂、稀释剂、防腐剂、增溶剂、乳化剂、佐剂和/或媒剂。这类载剂可为无菌液体,如水和油,包括石油、动物、植物或合成来源的那些,如花生油、大豆油、矿物油、芝麻油等;聚乙二醇;丙三醇;丙二醇或其它合成溶剂。抗菌剂,如苯甲醇或对羟苯甲酸甲酯;抗氧化剂,如抗坏血酸或亚硫酸氢钠;螯合剂,如乙二胺四乙酸;和用于调整张力的试剂,如氯化钠或右旋糖也可为载剂。用于生产组合物与载剂的组合的方法是本领域的普通技术人员已知的。在一些实施例中,措辞“药学上可接受的载剂”旨在包括任何和所有的与药物施用相容的溶剂、分散介质、包衣、等渗剂以及吸收延迟剂等。这类介质和试剂用于药学上活性物质的使用是在本领域中众所周知的。参见,例如,Remington,《药物科学与实践(The Science and Practice ofPharmacy)》,第20版(Lippincott,Williams&Wilkins 2003)。除非到任何常规介质或试剂与活性化合物不相容的程度,否则预期在组合物中的这类用途。
术语“组合”是指一种剂量单位形式的固定组合,或指用于联合施用的部分的试剂盒,其中NK细胞和组合伴侣(例如,下文所解释的另一种药物,也称为“治疗剂”或“共试剂”)可以同时或在时间间隔内分别施用。在一些情况下,组合伴侣表现出配合作用,例如协同作用。如本文所用,术语“共同施用”或“联合施用”等意在涵盖将所选择的组合伴侣施用于需要其的单个受试者(例如患者),并且旨在包括其中不一定通过相同的施用途径或在同一时间施用的治疗方案。如本文所用,术语“药物组合”意指通过混合或组合多于一种活性成分得到的产品,并且包括活性成分的固定和非固定组合。术语“固定组合”意指活性成分,例如化合物和组合伴侣,均以单一实体或剂量的形式同时施用于患者。术语“非固定组合”意指将活性成分例如化合物和组合伴侣都以单独实体的形式同时、合并地或连续地以无特定时间限制的形式施用于患者,其中这类施用在患者体内提供了治疗有效水平的两种化合物。后者也适用于鸡尾酒疗法,例如三种或多种活性成分的施用。
细胞因子诱导型含SH2的蛋白质(CIS)在调节人类自然杀伤(NK)细胞活化诱导的耗尽中起关键作用,并且与鼠类系统中的研究不同,CISH-缺失(CISH-/-)导致NK细胞活性降低。目前公开的人诱导的多能干细胞(iPSC)中的CISH-缺失的模型提供了进一步剖析人NK细胞发育、功能、激活、持久性和耗尽的CISH介导的调节的模型。在其他实施例中,CISH基因的缺失发生在人胚胎干细胞(hESC)中。在实施例中,T细胞衍生自CISH-/-iPSC或hESC。本文提供了调节免疫细胞如NK细胞或T细胞发育并抑制免疫细胞耗尽的组合物和方法。
本发明提供了可通过用Notch配体,如用过表达Notch配体的OP9-DL4细胞的培养层培养细胞,来防止或抑制CISH-/-NK耗尽。替代的Notch配体来源是已知的,并且包括细胞结合的或板结合的/无细胞的材料。
本公开部分基于基因组编辑工具,如可用于多种生物体(例如,用于添加、破坏或改变特异性基因的序列)的聚类的规则间隔的短回文重复序列(CRISPR)系统。CRISPR/Cas9系统基于两个要素。第一要素Cas9是具有用于第二要素的结合位点的核酸内切酶,所述第二要素是指导多核苷酸(例如,指导RNA)。指导多核苷酸(例如,指导RNA)基于序列同源性将Cas9蛋白引导至双链DNA模板。然后,Cas9蛋白切割所述DNA模板。通过将Cas9蛋白和适当的指导多核苷酸(例如,指导RNA)递送到细胞中,在所需位置切割生物体的基因组。用Cas9/gRNA复合物切割目标基因组序列后,两种替代的DNA修复机制之一可以恢复染色体的完整性:1)非同源末端连接(NHEJ),其产生DNA的一些碱基对(bp)在gRNA切割位点的插入和/或缺失,或2)同源导向修复(HDR),其可通过跨gRNA切割位点的附加“桥接”DNA模板纠正病变。本领域普通技术人员已知的CRISPR/Cas系统的其他方面在PCT公布第WO 2017/049266号中描述,所述公布的全部内容通过引用并入在此。本发明考虑了用于制备CISH-/-NK细胞的这些和其他众所周知的新技术,例如TALEN。本发明还考虑了用造血细胞如NK细胞、T细胞和其他免疫细胞的组合物、使用方法和制造方法。
实例
使用CRISPR/Cas9系统破坏了人诱导的多能干细胞(iPSC)中的CISH基因,并使用两阶段体外分化方案从CISH-/-iPSC中衍生NK细胞。使用WT或CISH-/-iPSC,分化为造血祖细胞的第一阶段是正常的(>80%CD34+细胞)。iPSC中CISH的缺失延迟了体外NK细胞分化的第二阶段(图1和图2)。具体而言,虽然使用WT iPSC在4周后NK细胞分化通常以>90%NK细胞完全完成,但CISH-/-iPSC衍生的细胞在4周时仅产生10%CD45+CD56+NK细胞,尽管到5周才>80%NK细胞。此后,CISH-/-iPSC衍生的NK细胞在表型上成熟,并显示出典型的NK表面标记表达,如CD94、CD16、NKG2D、NKp44、NKp46。
CISH是NK细胞介导的肿瘤免疫中有效的细胞内抑制检查点。人iPSC衍生的NK细胞中CISH基因的缺失使NK细胞对细胞因子高度敏感,从而增强了它们对肿瘤的细胞毒性(图3A-图4B)。与未经修饰的人NK细胞相比,当用作用于治疗癌症、病毒和微生物感染的适应性细胞疗法的细胞来源时,CISH敲除的人NK细胞对人类患者具有更好的持久性和抗肿瘤、抗病毒和抗微生物作用。
创建的CISH-/-人iPSC-NK细胞对IL-2/IL-15刺激表现出高度敏感性,以及在低浓度的IL-2(5U/ml)和IL-15(5ng/ml)的情况下维持扩增和抗肿瘤功能的能力(图3A-图3B)。CISH-/-iPSC-NK细胞可以在低浓度的IL-2(5U/ml)和IL-15(5ng/ml)的情况下在体外维持扩增和细胞毒性功能超过3周。
与现有技术相比,基因修饰的iPSC衍生的NK细胞具有更好的抗肿瘤作用,因为它们在体内可扩增并持续长于未修饰的NK细胞。现有的NK细胞疗法使用未修饰的NK细胞,它们是从外周血获得的NK细胞(PB-NK细胞)或未修饰的iPSC衍生的NK细胞,它们通常需要施用大剂量的IL-2和/或IL-15来维持扩增和抗肿瘤功能。但是,据临床数据报道,高浓度的IL-2和/或IL-15具有高毒性。因此,由于仅需低剂量的Il-2和/或L-15或其他细胞因子维持扩增和抗肿瘤功能即可减轻IL-2和/或IL-15引起的毒性,因此CISH-/-iPSC衍生的NK细胞可有益地用于NK细胞疗法。
CISH-/-iPSC衍生的NK细胞显示出改进的单细胞多功能性。图5A示出了使用Isoplexis 32-plex免疫细胞因子反应组,5种效应细胞因子(粒酶B、IFNγ、MIP-1α、穿孔素、TNFα)进行的单细胞细胞因子产生分析,这些细胞因子涉及细胞毒性功能。图5B示出了分泌图5A所示的两种或更多种细胞因子的样品的百分比。图5C示出了多功能性是通过多功能强度指数(PSI)进行测量的,涵盖了以下主要类别的32个主要免疫学相关分子的预先指定组:稳态/增殖、炎症、趋化、调节和免疫效应子。CAR-T细胞的多功能性(通过Isoplexis32-plex进行测量,与我们使用的测定法相同)与临床结果呈正相关。与未修饰的野生型NK细胞相比,CISH-/-iPSC-NK细胞的多功能性增加说明了更好的抗肿瘤活性。
CISH-/-iPSC-NK细胞显示出增加的基础糖酵解和糖酵解能力。图6A示出了使用Seahorse XF糖酵解速率测定试剂盒测量的细胞外酸化率(ECAR)。图6B示出了基础糖酵解速率的量化。图6C示出了糖酵解能力的量化。细胞外酸化率(ECAR)是葡萄糖代谢率的指标。该数据表明CISH-/-iPSC-NK细胞具有改进的葡萄糖代谢,这可能是CISH-/-iPSC-NK细胞功能改进的机制(据报道,葡萄糖代谢的改进有助于功能增强)。
CISH-/-iPSC-NK展示出更好的体内抗肿瘤活性NSG小鼠用表达萤火虫荧光素酶基因的5x106 Molm13细胞进行腹腔接种。肿瘤移植后1天,不治疗小鼠或用10x106 WT-iPSC-NK或CISH KO-iPSC-NK细胞治疗。通过每周注射IL-2持续3周来支持NK细胞,并且每周进行IVIS成像以追踪肿瘤负荷。图7A示出了IVIS图像。图7B示出了每组的存活率曲线。该数据表明,CISH-/-iPSC-NK细胞在异种移植肿瘤模型中具有改进的抗肿瘤活性。
在实施例中,CISH-/-iPSC衍生的NK细胞用作NK细胞疗法的改进的治疗细胞来源。
在实施例中,CISH-/-iPSC衍生的NK细胞在体外扩增以获得足够数量的细胞用于作为癌症、病毒和微生物疾病以及其他病状的治疗方案的一部分来施用。
在实施例中,以与使用未修饰的外周血NK细胞的基于NK细胞的疗法的先前临床工作相似的方式,将CISH-/-iPSC衍生的NK细胞施用于患者。在实施例中,与用wtNK细胞的常规疗法相比,使用低浓度的细胞因子刺激,如用IL-2和IL-15。在实施例中,低浓度的IL-2在1和10U/ml或约5U/ml之间,而低浓度的IL-15在1和10ng/ml或约5ng/ml之间,这些浓度可以有效地维持CISH-/-NK细胞扩增和抗肿瘤功能。
在实施例中,CISH-/-iPSC衍生的NK细胞作为难治性恶性肿瘤的治疗方案的一部分施用于患者,如但不限于治疗难治性癌症,血液系统恶性肿瘤和实体瘤两者。
方法
造血作用和NK分化I:
CISH KO hiPSC首先分化为造血祖细胞,然后分化为NK细胞1,2。简而言之,在第6天EB内出现CD34+细胞后,EB被转移到NK细胞分化中。简而言之,将造血祖细胞转移到NK细胞分化培养基中,所述NK细胞分化培养基含有以下的2:1混合物:Dulbecco修饰的Eagle培养基/Ham F12(马萨诸塞州沃尔瑟姆的赛默飞世尔科技(Thermo Fisher Scientific,Waltham,MA),11965092,11765054)、2mM L-谷氨酰胺(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,25030081)、1%青霉素/链霉素(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,15140122),25μMβ-巯基乙醇(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,21985023)、20%热灭活的人血清AB(美国纽约州的科宁(Corning,NY,U.S.),MT35060CI)、5ng/ml亚硒酸钠(马萨诸塞州伯灵顿的默克密理博(Merck Millipore,Burlington,MA),S5261)、50μM乙醇胺(MP生物医疗(MP Biomedicals),ICN19384590)、20mg/mL抗坏血酸(马萨诸塞州伯灵顿的默克密理博,A4544)、白介素3(IL-3;明尼苏达州明尼阿波利斯的R&D系统(R&D Systems Minneapolis,MN),203-IL);仅适用于第一周),干细胞因子(SCF;明尼苏达州明尼阿波利斯的R&D系统,7466-SC)、白介素15(IL-15;R&D系统,247-ILB)、Fms样酪氨酸激酶3配体(FLT3L;明尼苏达州明尼阿波利斯的R&D系统,308-FK)和IL-7(IL-7;明尼苏达州明尼阿波利斯的R&D系统,207-IL)。然后将细胞在这些条件下放置21天,每周接受一次培养基更换。
NK分化II:
在NK分化培养基中放置21天(NK分化I)后,收集悬浮细胞,并与基质细胞OP9-DL4(OP9细胞过表达DL4,Notch配体)一起转移到6孔板中持续14天,每周接受一次培养基更换,直到它们已经发展成CD45+CD56+CD33-CD3-细胞,如通过流式细胞术测定。
扩增
分化后,使用经辐照的K562-IL21-4-1BBL3,4扩增NK细胞。简而言之,去除非贴壁细胞并通过流式细胞术分析以确定CD56+NK细胞的纯度。然后将这些细胞用2:1aAPC(以10,000Gy照射)刺激成NK细胞,处于350,000个NK细胞/mL培养基,所述培养基含有RPMI 1640(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,11875085)、2mM L-谷氨酰胺(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,25030081)、1%青霉素/链霉素(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,15140122)、1%非必需氨基酸(NEAA;马萨诸塞州沃尔瑟姆的赛默飞世尔科技,11140050)和10%标准FBS或10%人血清AB(马萨诸塞州沃尔瑟姆的赛默飞世尔科技,10100147)。这补充有50-100U/ml IL2(Prometheus,65483011607)。
参考文献
1.Knorr,D.A.,Ni,Z.,Hermanson,D.,Hexum,M.K.,Bendzick,L.,Cooper,L.J.,Lee,D.A.&Kaufman,D.S.(2013).从人多能干细胞临床规模衍生自然杀伤细胞用于癌症治疗(Clinical-scale derivation of natural killer cells from human pluripotentstem cells for cancer therapy).《干细胞转化医学(Stem Cells Transl Med)》2,2013。
2.Zhu,H.&Kaufman,D.S.(2019).一种从人多能干细胞生产临床规模的自然杀伤细胞的改进方法(An improved method to produce clinical scale natural killercells from human pluripotent stem cells).bioRxiv,2019。
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Claims (20)
1.一种用于治疗人类受试者的疾病或感染的方法,其包含向有需要的人类受试者施用有效量的药物组合物,所述药物组合物包含人CISH-/-自然杀伤(NK)细胞和药学上可接受的载剂。
2.根据权利要求1所述的方法,其中所述NK细胞衍生自诱导的多能干细胞、胚胎干细胞或外周血细胞。
3.根据权利要求1所述的方法,其中所述CISH-/-NK细胞对于所述受试者是自体的。
4.根据权利要求1所述的方法,其中所述方法进一步包含向所述受试者施用有效量的一种或多种细胞因子。
5.根据权利要求4所述的方法,其中所述细胞因子是IL-2和/或IL-15,并且所述有效量小于用天然NK细胞治疗所需的有效量。
6.根据权利要求1所述的方法,其中所述疾病是造血系统癌症或实体瘤。
7.根据权利要求1所述的方法,其中所述感染是由病毒或微生物引起的。
8.根据权利要求1所述的方法,其中与天然NK细胞相比,所述CISH-/-NK细胞对细胞因子刺激高度敏感并且展示出改进的扩增、抗肿瘤功能和抗病毒功能。
9.一种药物组合物,其包含人CISH-/-NK细胞和至少一种药学上可接受的赋形剂。
10.根据权利要求9所述的药物组合物,其中所述CISH-/-NK细胞衍生自诱导的多能干细胞、胚胎干细胞或外周血细胞。
11.根据权利要求9所述的药物组合物,其中所述CISH-/-NK细胞衍生自诱导的多能干细胞。
12.根据权利要求9所述的药物组合物,其中与天然NK细胞相比,所述CISH-/-NK细胞对细胞因子刺激高度敏感并且展示出改进的扩增、抗肿瘤功能和抗病毒功能。
13.根据权利要求12所述的药物组合物,其中所述细胞因子刺激包含用Il-2和/或IL-15的刺激。
14.一种用于产生人CISH-/-NK细胞的方法,其包含:
从人诱导的多能干细胞(iPSC)、人胚胎干细胞(ESC)或人外周血细胞(PBC)中删除CISH基因;和
使用体外分化方案从CISH-/-iPSC、ESC或PBC中衍生NK细胞。
15.根据权利要求14所述的方法,其中所述CISH基因的缺失是通过使用CRISPR系统实现的。
16.根据权利要求14所述的用于产生CISH-/-NK细胞的方法,其中所述衍生步骤进一步包含将所述CISH-/-iPSC、ESC或PBC分化为>80%CD34+,然后分化为>80%CD45+和CD56+。
17.根据权利要求16所述的方法,其中所述第二次分化在与Notch配体接触时发生。
18.根据权利要求17所述的方法,其中所述Notch配体由OP9-DL4细胞提供。
19.一种细胞培养物,其包含人CISH-/-NK细胞。
20.根据权利要求19所述的细胞培养物,其中与天然NK细胞相比,所述CISH-/-NK细胞对细胞因子刺激高度敏感并且展示出改进的扩增、抗肿瘤功能和抗病毒功能。
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