CN114945376A - 从干细胞产生嵌合抗原受体修饰的t细胞以及其治疗用途 - Google Patents
从干细胞产生嵌合抗原受体修饰的t细胞以及其治疗用途 Download PDFInfo
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Abstract
描述了用于制备表达嵌合抗原受体(CAR)的T细胞或NK细胞的方法。所述方法需要:分离T细胞群,从T细胞生成诱导多能干细胞(iPSC),将编码CAR的核酸分子引入iPSC以产生CAR iPSC;并将CAR iPSC分化为CAR T细胞或CAR NK细胞。
Description
优先权要求
本申请要求于2019年11月5日提交的美国临时申请序列号62/931,125的权益。前述全部内容通过引用并入本文。
技术领域
本公开涉及从干细胞或祖细胞生成嵌合抗原受体修饰的T细胞和应用。
背景技术
嵌合抗原受体(CAR)T细胞疗法是一种癌症治疗,它遗传改变T细胞以重新定向和利用它们的癌症杀伤潜力。目前FDA批准的CAR T细胞产品是基于自体的,需要个性化的血液单采血液成分术(apheresis)和制造。衍生患者特异性CAR T细胞产品昂贵、费力且耗时,并面临许多后勤和监管挑战。
从诱导多能干细胞(iPSC)生成CAR T细胞对于生成“现成的(off-the-shelf)”CART细胞产品和克服这些挑战具有令人鼓舞的前景。iPSC几乎可以无限增殖,同时保持其多能性和谱系分化潜力。然而,T细胞发育的复杂性和CAR表达对T细胞分化的干扰为成功的iPSC衍生的CAR T细胞生成带来了挑战。
发明内容
本文尤其描述了用于制备和使用来自多能干细胞胚胎干细胞(ESC)或诱导多能干细胞(iPSC)的表达嵌合抗原受体(CAR)的表型确定的、功能性的和/或可扩增的T细胞或NK细胞的方法。本文所述的CAR T细胞和CAR NK细胞靶向在靶细胞的细胞表面上表达的特异性预先确定抗原,具有增强的功能潜力,增强癌症和/或靶定的疾病的存活和治疗,和/或具有细胞毒性潜力和抗肿瘤活性。本文所述的CAR T细胞和CAR NK细胞可以用作“现成的”细胞以施用于多个接受者,其跨越免疫原性屏障并至少减轻移植物抗宿主病(GVHD)的症状。
在一些实施方案中,幼稚和记忆T(Tn/mem)细胞衍生的iPSC是用于生成iPSC衍生的CAR T细胞的起始材料。在一些实施方案中,外周血单个核细胞(PBMC)、幼稚T(Tn)细胞、记忆T(Tmem)细胞、幼稚和记忆T细胞(Tn/mem)或其组合衍生的iPSC是用于生成iPSC衍生的CAR T细胞的起始材料。不受理论束缚,T细胞已经具有在发育过程中重排的TCR基因,并且T衍生的iPSC维持重排的TCR基因座,这对于在体外分化过程中的T细胞发育很重要。在一些实施方案中并且不受理论约束,Tn/mem是与终末分化的效应T细胞相比具有优质适应性的幼T细胞亚群。由于较少的表观遗传足迹,生成的Tn/mem衍生的iPSC也可以具有独特的特性。
在一些实施方案中,本文描述了用于制备CAR T细胞组合物的方法,该方法包括:
(a)分离外周血单个核细胞(PBMC)、幼稚T(Tn)细胞、记忆T(Tmem)细胞、幼稚和记忆T细胞(Tn/mem)或其组合的群体;
(b)从PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合生成诱导多能干细胞(iPSC);
(c)使iPSC与编码CAR的载体接触,从而产生CAR iPSC;和
(d)将CAR iPSC分化为CAR T细胞。
在一些实施方案中,本文描述了用于制备CAR NK细胞组合物的方法,该方法包括:
(a)分离外周血单个核细胞(PBMC)、幼稚T(Tn)细胞、记忆T(Tmem)细胞、幼稚和记忆T细胞(Tn/mem)或其组合的群体;
(b)从PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合生成诱导多能干细胞(iPSC);
(c)使iPSC与编码CAR的载体接触,从而产生CAR iPSC;和
(d)将CAR iPSC分化为CAR NK细胞。
在一些实施方案中,PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合是人的或分离自人血液。在一些实施方案中,PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合是CD14-、CD25-和CD26L+。
在一些实施方案中,PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合经重编程以生成iPSC。在一些实施方案中,通过使所述PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合与OCT3/4、OCT3、OCT4、SOX2、KLF4、L-MYC、C-MYC、LIN28或靶向TP53的短发夹RNA(shRNA-TP53)中的一种或多种接触而生成iPSC。在一些实施方案中,转导的细胞在补充有50U/mL IL-2、0.5ng/ml IL-15和CD3/CD28 Dynabeads(珠:细胞比率为1:1)的X-Vivo15培养基中培养。在一些实施方案中,转染后第一、两天或三天,添加等体积的含有bFGF和10μM Y27632的PSC培养基。在一些实施方案中,第三天、四天、五天、六天或七天,然后将培养基完全更换为PSC培养基。在一些实施方案中,培养iPSC细胞至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、26、27、28、29或30天。在一些实施方案中,挑取单个集落以进一步培养和评估。
在一些实施方案中,使iPSC与编码CAR的核酸或载体接触而生成CAR iPSC。在一些实施方案中,转导的CAR iPSC在单细胞分选和iPSC定殖之前培养至少2代。在一些实施方案中,将定殖的CAR IPSC扩增并入库用于分化。
在一些实施方案中,IPSC或CAR iPSC是遗传修饰的。在一些实施方案中,一种或多种基因被敲除、下调或上调。在一些实施方案中,一种或多种基因包括TRAC、TRBC、B2M、CIITA或其组合中的一种或多种。在一些实施方案中,TRAC、TRBC、B2M、CIITA被敲除。在一些实施方案中,TRAC、TRBC、B2M、CIITA被下调。在一些实施方案中,遗传修饰通过本文所述的方法和本领域已知的方法来实现。在一些实施方案中,遗传修饰方法包括基因编辑、同源重组、非同源重组、RNA介导的遗传修饰、DNA介导的遗传修饰、锌指核酸酶、大范围核酸酶、TALEN或CRISPR/CAS9。
在一些实施方案中,CAR iPSC分化成胚胎中胚层祖(EMP)细胞并进一步分化成CART细胞。在一些实施方案中,EMP细胞是CD56+和CD326-。
在一些实施方案中,表达CAR的iPSC分化成胚胎中胚层祖(EMP)细胞并进一步分化成CAR NK细胞。在一些实施方案中,EMP细胞是CD56+和CD326-。
在一些实施方案中,CAR iPSC分化成CD34+造血干细胞和祖细胞(HSPC)并进一步分化成CAR T细胞。
在一些实施方案中,CAR iPSC分化成CD34+HSPC并进一步分化成CAR NK细胞。
在一些实施方案中,使用基于纳米纤维基质的培养系统将CAR iPSC分化成CAR T细胞。
在一些实施方案中,使用基于纳米纤维基质的培养系统将CAR iPSC分化成CAR NK细胞。
在一些实施方案中,CAR对肿瘤、细胞表面标志物和/或毒素具有特异性。在一些实施方案中,CAR靶向以下的任何一种或多种:碳酸酐酶IX(CAIX)、癌胚抗原(CEA)、CDS、CD6、CD7、CD10、CD19、CD20、CD22、CD30、CD33、CD34、CD38、CD41、CD44、CD49f、CD56、CD74、CD123、CD133、CD138、CS1、氯毒素受体、巨细胞病毒(CMV)感染细胞的抗原(例如,细胞表面抗原)、上皮糖蛋白(EGP2)、上皮糖蛋白-40(EGP-40)、上皮细胞粘附分子(EpCAM)、受体酪氨酸蛋白激酶erb-B2、3、4、叶酸结合蛋白(FBP)、胎儿乙酰胆碱受体(AChR)、叶酸受体、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、人表皮生长因子受体2(HER-2)、人端粒酶逆转录酶(hTERT)、白细胞介素-13受体亚基α-2(IL-13Rα2)、轻链激酶插入域受体(KDR)、Lewis A(CA19.9)、Lewis Y(LeY)、LI细胞粘附分子(LICAM)、黑素瘤抗原家族A,1(MAGE-AI)、粘蛋白16(Muc-16)、粘蛋白1(Muc-1)、间皮素(MSLN)、NKG2D配体、癌症-睾丸抗原NY-ESO-1、癌胎抗原(h5T4)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、肿瘤相关糖蛋白72(TAG-72)、血管内皮生长因子R2(VEGF-R2)、维尔姆斯瘤蛋白(WT-1)或其组合。
在一些实施方案中,CAR是双特异性的。
在一些实施方案中,嵌合抗原受体包含:至少一个靶向域、间隔物、跨膜域、共刺激域和CD3ζ信号传导域。在一些实施方案中,CAR是1928z。
在一些实施方案中,本文描述的是包含iPSC衍生的CAR T细胞或CAR NK细胞的组合物。在一些实施方案中,包含iPSC衍生的CAR T细胞或CAR NK细胞的组合物具有增强的治疗特性。在一些实施方案中,iPSC衍生的CAR T细胞或CAR NK细胞表现出增强的功能活性,包括有力的细胞因子产生、细胞毒性和肿瘤生长的细胞抑制性抑制,例如,如减少肿瘤负荷的活性。
在一些实施方案中,包含CAR T细胞的组合物包含辅助T细胞、细胞毒性T细胞、记忆T细胞、幼稚T细胞、调节性T细胞、自然杀伤T细胞或其组合中的一种或多种。在一些实施方案中,包含CAR T细胞的组合物包含CD3+、CD5+、CD7+和TCRαβ+。在一些实施方案中,包含CAR T细胞的组合物包含CD8+CAR T细胞是CD8αβT细胞,其以抗原特异性方式针对肿瘤细胞具有强细胞毒性,并且可以有力地分泌细胞因子诸如IFNγ。在一些实施方案中,CAR T细胞具有主要的同质TCR表型。在一些实施方案中,包含CAR T细胞的组合物包含CD3+CD5+CD7+TCRαβ+CD8αβ+,CD3+CD5+CD7+TCRαβ+CD4+,CD62L+CD45RA+干记忆T细胞、CD62L-CD45RA-CD45RO+效应记忆T细胞和CD62L-CD45RA+效应T细胞,及其组合。
在一些实施方案中,本文描述了增加患有癌症的受试者的存活的方法,其包括施用包含本文所述的CAR T细胞或CAR NK细胞的组合物。
在一些实施方案中,本文描述了治疗患者中癌症的方法,其包括施用包含本文所述的CAR T细胞或CAR NK细胞的组合物。
在一些实施方案中,本文描述的是减少或改善患者中与癌症相关的症状的方法,其包括施用包含本文所述的CAR T细胞或CAR NK细胞的组合物。
在一些实施方案中,包含本文所述的CAR T细胞或CAR NK细胞的组合物是局部或全身施用的。在一些实施方案中,包含本文所述的CAR T细胞或CAR NK细胞的组合物通过单次或重复给药来施用。在一些实施方案中,将包含本文所述的CAR T细胞或CAR NK细胞的组合物施用于具有癌症、病原体感染、自身免疫性病症或同种异体移植物的患者。
在一些实施方案中,癌症选自血癌、B细胞白血病、多发性骨髓瘤、淋巴母细胞白血病(ALL)、慢性淋巴细胞白血病、非霍奇金淋巴瘤、卵巢癌、前列腺癌、胰腺癌、肺癌、乳腺癌和肉瘤、急性髓性白血病(AML)。
除非另有定义,本文使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。本文描述了用于本发明的方法和材料;也可以使用本领域已知的其他合适的方法和材料。材料、方法和实例仅是说明性的而不是限制性的。本文提及的所有出版物、专利申请、专利、序列、数据库条目和其他参考文献通过引用整体并入。在冲突的情况下,以本说明书(包括定义)为准。
本发明的其他特征和优点将从以下详细描述和附图以及从权利要求中显而易见。
附图说明
专利或申请文件含有至少一幅彩色绘图。在根据请求和支付必要的费用后,专利局将提供本专利或专利申请出版物的彩色附图副本。
图1A-1E显示了Tn/mem iPSC衍生的CAR T细胞的表面标志物谱(profile)。
图2A-2B显示了Tn/mem iPSC衍生的CAR T/T细胞和常规PBMC衍生的CAR T或T细胞的TCR库(Repertoire)。细胞用IOTest Beta Mark TCR库试剂盒以及APC-抗CD3抗体染色。门控CD3+细胞以分析TCR Vβ库。
图3A-3D显示了在体外针对CD19+靶细胞具有有力抗原特异性细胞毒性的Tn/memiPSC衍生的1928z CAR T细胞。
图4显示了具有有力抗原特异性细胞因子产生的iPSC 1928CAR T。
图5A-5D显示了具有抗原特异性脱粒和活化的iPSC 1928CAR T。
图6A-6D显示了在体内具有有力抗肿瘤活性的iPSC CAR T细胞。
图7A-7D显示了Tn/mem iPSC HSPC衍生的CAR NK细胞和脐带血CD34+HSPC细胞衍生的NK细胞的表面标志物谱。
图8A-8B显示了iPSC衍生的CAR NK细胞针对不同肿瘤系的细胞毒性。
图9显示了iPSC CAR NK细胞针对肿瘤细胞的脱粒活性。
图10显示了由基于纳米纤维基质的共培养系统生成的iPSC CAR T细胞的表型。
图11A-11B显示了表达CAR的定殖iPSC系的表面标志物谱。
图12A-12B显示了iPSC衍生的CAR T细胞的表面标志物谱。A.没有REM扩增的情况下,第7周的iPSC CAR T表型;B:REM扩增后的表型。
图13A-13I显示了iPSC衍生的CD19-CAR T细胞的生成。(13A)PSC-ATO培养期间的事件(上)、细胞类型(中)和培养基条件(下)的示意图。参考在线STAR方法。(13B,13C)iPSCCD19-CAR T细胞与GFP+DLL4+MS5饲养细胞的七周类器官培养物用2%多聚甲醛固定,并用CD3(红色)和DAPI(蓝色)原位染色。白色标尺表示500μm(13B)和100μm(13C)的刻度。(13D)衍生自100万个模拟转导或表达CD19-CAR的iMP的分化T细胞的数量。描绘了三个独立实验的数据,以平均值±SEM作为棒。(13E)iPSC衍生的模拟T和CD19-CAR T细胞的扩增。如(13A)指示,扩增1x106个T细胞。描绘了三个独立实验的数据,以平均值±SEM作为棒。(13F)对常规(Conv.)与iPSC衍生的模拟转导(模拟)和表达CD19-CAR的T细胞的指示标志物进行代表性流式细胞术分析。表达每个标志物的细胞百分比显示在相关象限中,这些象限基于同种型对照染色绘制。(13G)在三个单独的实验中用指示标志物染色的细胞的百分比,以平均值±SEM作为棒。(13H)常规(Conv)与iPSC衍生的模拟T和CD19-CAR T细胞上转基因表达水平的比较。顶部,作为CAR表达标志物的EGFRt染色的代表性直方图,指示了平均荧光强度(MFI)。底部,描绘了三个独立实验的转基因MFI数据,以平均值±S.D.作为棒。*,P=0.0011,使用学生t检验。(13I)常规与iPSC衍生的模拟T和CD19-CAR T细胞的TCR Vβ库。
图14A-14F显示了iPSC CD19-CAR T细胞的基因和信号传导签名(signature)。(14A)主成分分析(PCA)和(14B)iPSC、常规(Conv.)模拟转导(模拟)或CD19-CAR T细胞、iPSC衍生的模拟T或CD19-CAR T细胞,或常规PBMC衍生的NK细胞的两个样品的全局转录谱的分层聚类。(14C)iPSC模拟T与Conv.模拟T细胞(左),或iPSC CD19-CAR T与Conv.CD19-CAR T细胞(右)的Vocano图。常规细胞中前五位上调基因用绿点突出显示,而iPSC衍生细胞中前五位上调基因用红点突出显示。(14D)T淋巴样相关基因、细胞毒性介质、抑制标志物和NK受体基因的z评分值的热图。(14E)亚硫酸氢盐转化的基因组DNA用作模板,使用EF1α启动子内的甲基化特异性引物(MSP)和非甲基化特异性引物(USP)进行PCR分析。(14F)通过亚硫酸氢盐测序测定EF1α启动子甲基化。从亚硫酸氢盐转化的基因组DNA中PCR扩增EF1α启动子的114-360bp区域,进行亚克隆,对每组6个克隆进行测序。在这个245bp区域中的23个CG位点中,每个克隆的甲基化CG位点的数量在每行的右侧指示。
图15A-15K显示了iPSC CD19-CAR T细胞的功能谱。(15A),iPSC衍生的模拟转导(Mock)或CD19-CAR T细胞与CD19+3T3细胞以4:1的效应物与靶物(E:T)比率共培养4小时后的明场图像。白色标尺表示100μm的比例。(15B-15E)当以指示的E:T比率共培养4h(15B,15E)或48h(15C,15D)时,iPSC CD19-CAR T细胞针对CD19+或CD19阴性/敲除(CD19KO)NALM6(15B,15C,15E)或Raji(15D)靶细胞的细胞毒活性。将裂解活性与iPSC衍生的模拟转导T细胞(MOCK,15B)或常规CD19-CAR T细胞(Conv.,15C)进行比较。描绘了复制培养物的平均值±S.D.值。*,P<0.001,通过(15E)中的双向ANOVA检验。(15F)iPSC衍生(iPSC)或常规(Conv.)CD19-CAR T细胞在以指示的E:T比率共培养4小时时针对患者衍生的ALL细胞的细胞毒活性。(15G)在Golgi Stop蛋白转运抑制剂存在下,在以1:1的E:T比率与指示的刺激物细胞(X轴标记)共培养5小时后,通过流式细胞术测量iPSC衍生的模拟转导(Mock)或CD19-CAR T细胞中的脱粒(即表面CD107,左)和细胞内IFN-g水平(右)。*,P<0.01通过学生t检验。(15H)在iPSC衍生的模拟T和未经刺激(无)或用CD19+或CD19阴性/敲除(CD19KO)NALM6以1:1的E:T比率刺激24小时的CD19-CAR T细胞之间比较活化标志物的流式细胞术分析。每个等值线图中指示了表达CD25或CD137/4-1BB的CD3+细胞的百分比,并根据同种型对照染色绘制门。(15I)通过对在以1:1的E:T比率与CD19+或CD19阴性/敲除(CD19KO)NALM6细胞共培养后24小时收获的上清液进行Bio-Plex分析,测量iPSC衍生或常规(Conv.)模拟T或CD19-CART细胞的细胞因子产生。*,P<0.001,通过学生t检验。(15J)在以1:2的E:T比率每2天经CD19+NALM6细胞再攻击,总共3次刺激后,iPSC衍生或常规(Conv.)CD19-CAR T细胞的T细胞耗竭标志物谱。细胞用抗PD-1、抗TIM-3、抗LAG-3染色,并通过流式细胞术测定CD3+细胞染色的无(0+)、一(1+)、二(2+)或所有三种(3+)标志物的百分比。(15K)ERK、磷酸化ERK、PLCγ、在Y782磷酸化的PLCγ、在Ser1248磷酸化的PLCγ、内源性CD3ζ、磷酸化内源性CD3ζ、CAR内的CD3ζ、CAR内的磷酸化CD3ζ或作为加载对照的GAPDH在单独培养60分钟或与为CD19+或CD19阴性(CD19KO)的NALM6肿瘤一起培养60分钟的指示T细胞中Western印迹分析。还检查了单独培养的肿瘤细胞作为对照。
图16A-16F显示iPSC CD19-CAR T细胞在体内表现出有力的抗肿瘤活性。(16A)使用腹膜内(i.p.)肿瘤模型的动物研究示意图。第-4天,用2.5x105ffluc+NALM6细胞i.p.接种NSG小鼠。然后在第0天和第3天,或是不处理,或是用6x106 iPSC衍生的模拟转导(模拟)或CD19-CAR T细胞i.p.处理小鼠;在接受iPSC CD19-CAR T细胞的一组中,还每周3次施用2x107辐照的NS0-hIL15细胞,持续3周。通过每周生物发光成像测定肿瘤负荷。(16B),i.p.肿瘤ffLuc通量随时间的几何平均值±95%CI。使用双向ANOVA检验:*,P=0.0008,**P<0.0001。(16C),i.p.异种移植小鼠的Kaplan-Meier存活分析。使用Mantel-Cox检验:*,P=0.0034,比较iPSC CD19-CAR T处理组与未处理组;**,P=0.0016,比较iPSC CD19-CAR T+NS0-hIL15处理组与iPSC CD19-CAR T处理组。(16D)使用静脉内(i.v.)肿瘤模型的动物研究示意图。第-4天,用2.5x105 ffluc+NALM6细胞i.v.接种NSG小鼠。然后在第0、3和6天,或是不处理,或是用5x106 iPSC衍生的CD19-CAR T细胞i.v.处理小鼠;在指示的情况下,每周3次施用2x107辐照的NS0-hIL15细胞,持续3周。其他对照组包括在第0天接受2x106供体匹配的Tn/mem衍生的模拟T的小鼠。通过每周生物发光成像测定肿瘤负荷。(16E),i.v.肿瘤ffLuc通量随时间的几何平均值±95%CI。使用双向ANOVA检验:*,P=0.0019,**,P=0.0002,***,P<0.0001。(16F)i.v.异种移植小鼠的Kaplan-Meier存活分析。使用Mantel-Cox检验:*,P=0.0035,比较iPSC CD19-CAR T处理组与未处理组。
图17A-17E显示iPSC从Tn/mem的衍生。(17A)代表性Tn/mem衍生iPSC的形态。在MEF饲养物上(上)或在无饲养物条件下(下)的iPSC的明场(左)和碱性磷酸酶染色(右)图像。黑色标尺表示200μm的刻度。(17B)从Tn/mem重编程的代表性克隆iPSC系的流式细胞术多能性标志物谱。显示了表达每个标志物的细胞的百分比;SSC,侧向散射。(17C)通过PCR检查iPSC克隆中整合的质粒DNA。使用对EBNA1具有特异性的引物作为质粒整合标志物,和FBX15作为加载对照。泳道1,H2O阴性对照;泳道2,阳性对照:用10ng含有EBNA1的附加型载体电穿孔的iPSC;泳道3-11,克隆iPSC系。(17D)代表性的模拟转导(上)和已经重新定殖、扩增和入库的CD19-CAR+(下)iPSC系的流式细胞仪谱。由于临床载体掺入与CD19-CAR共表达的EGFRt选择标志物,它被用于检测转基因表达系。(17E)使用Tn/mem衍生的CAR+iPSC进行畸胎瘤形成测定的代表性结果。黄色箭头:外胚层衍生组织(神经元玫瑰花结);白色箭头:中胚层衍生组织(肌肉、软骨和结缔组织);蓝色箭头:内胚层衍生组织(腺样组织)。白色标尺表示10mm(左小图)和200μm(H&E小图)的刻度。
图18A-18F显示了iPSC CD19-CAR T细胞的扩增表型。(18A)在iMO-ATO培养的第7周,来自iPSC CD19-CAR T细胞的类器官的H&E染色。(18B-18D),在REM扩增之前(18B)和之后(18C,18D)所得的iPSC CD19-CAR T细胞的代表性流式细胞仪谱。(18B,18C)表达每个标志物的细胞的百分比在相关象限中指示,这些象限基于同种型对照染色绘制。(18D)指示的T细胞系的单个参数直方图比较。灰色直方图,用同种型对照抗体染色的T细胞。(18E)起始PBMC(CD3门控)和Tn/mem细胞群(上)和长期培养的(35天)常规(Conv.)模拟转导(模拟)或CD19-CAR+T细胞(下)的TCR Vβ库。(18F)TCRB克隆性试剂盒对照(左)以及常规(Conv.)与iPSC衍生的模拟T细胞(中)和CD19-CAR T细胞(右)中TCRβ基因组重排的PCR片段分析。括号表示用于TCR基因组重排的170-210bp和285-325bp PCR片段分析的相关大小范围。
图19A-19C显示iPSC CD19-CAR T细胞的基因和信号传导签名。(19A)阳性选择相关基因的z分值热图,包括TCR重排和MHC基因(左小图)和报告的T细胞耗竭相关基因(Crawford et al.,2014;Gattinoni et al.,2011;Long et al.,2015)(右小图)。(19B)气泡图显示衍生自iPSC CD19-CAR T细胞与常规(Conv.)CD19-CAR T细胞的GSEA比较的最高上调或下调信号传导途径。(19C)EF1α启动子的序列,其中用正向(F1)和反向(R1)引物扩增的115-359bp区域含有23个CpG岛(用“+”号表示)。这些CpG岛的甲基化在图14F中描绘。
图20A-20B显示小鼠研究信息。来自i.p.(20A)或i.v.(20B)模型的NSG小鼠的生物发光图像在图16中描绘。红色“X”指示组/小鼠因疾病负荷而被安乐死。
发明详述
本发明在以下实施例中进一步描述,这些实施例不限制权利要求中描述的本发明的范围。
实施例1-3的方法
DNA构建体
CD19靶向性CAR(1928zCAR)和IL13Rα2靶向性CAR构建体与目前在靶向B细胞白血病/淋巴瘤(clinicaltrials.gov#NCT01815749)(Wang,X.,et al.,Phase 1studies ofcentral memory-derived CD19 CAR T-cell therapy following autologous HSCT inpatients with B-cell NHL.Blood,2016.127(24):p.2980-90)和复发性/难治性GBM(clinicaltrials.gov#NCT02208362)(Brown,C.E.,et al.,Regression of Glioblastomaafter Chimeric Antigen Receptor T-Cell Therapy.N Engl J Med,2016.375(26):p.2561-9)的临床研究中使用的相同。
1928zCAR包含CD19scfv域、CD28z共刺激域、在CH2区内具有两个点突变(L235E和N297Q)的IgG4间隔物、作为安全开关的胞质截短的人表皮生长因子受体(huEGFRt)(Jonnalagadda,M.,et al.,Chimeric antigen receptors with mutated IgG4 Fcspacer avoid fc receptor binding and improve T cell persistence and antitumorefficacy.Mol Ther,2015.23(4):p.757-68;Urak,R.,et al.,Ex vivo Akt inhibitionpromotes the generation of potent CD19CAR T cells for adoptiveimmunotherapy.J Immunother Cancer,2017.5:p.26;Wang,X.,et al.,A transgene-encoded cell surface polypeptide for selection,in vivo tracking,and ablationof engineered cells.Blood,2011.118(5):p.1255-63)。
IL13Rα2CAR构建体由人GM-CSF受体α链前导肽、具有E13Y突变的人IL-13、具有2个点突变(L235E和N297Q)的IgG4间隔物、CD4跨膜域、人4-1BB共刺激域和人CD3ζ的胞质域构成。在一些实施方案中,还在构建体中引入截短的CD19以允许转导细胞的潜在富集和追踪(Brown,C.E.,et al.,Regression of Glioblastoma after Chimeric Antigen ReceptorT-Cell Therapy.N Engl J Med,2016.375(26):p.2561-9;Brown,C.E.,et al.,Optimization of IL13Ralpha2-Targeted Chimeric Antigen Receptor T Cells forImproved Anti-tumor Efficacy against Glioblastoma.Mol Ther,2018.26(1):p.31-44)。
Tn/mem分离
血液制品是根据COH IRB批准的方案从健康供体获得的,并且按照先前研究中描述的类似规程分离幼稚和记忆T(Tn/mem)细胞(例如Wang,X.,et al.,Phenotypic andfunctional attributes of lentivirus-modified CD19-specific human CD8+centralmemory T cells manufactured at clinical scale.J Immunother,2012.35(9):p.689-701)。简而言之,通过密度梯度离心在Ficoll-Paque(GE Healthcare)上分离PBMC,然后进行连续几轮的CliniMACS/AutoMACS(Miltenyi Biotec)消减以除去表达CD14的细胞和表达CD25的细胞,然后针对Tn/mem进行CD62L阳性选择。
从PBMC或Tn/mem生成iPS细胞
用与已发表方案类似的规程将PBMC或Tn/mem细胞重编程为iPS细胞(例如Okita,K.,et al.,An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral bloodcells.Stem Cells,2013.31(3):p.458-66)。简而言之,通过使用核转染器(Nucleofector)4D电穿孔装置(Lonza)用3μg质粒混合物对1-3百万个PBMC或幼稚和记忆T(Tn/mem)进行电穿孔。质粒混合物由编码OCT3/4、SOX2、KLF4、L-MYC、LIN28的附加型质粒和针对TP53的shRNA组成。转导的细胞在补充有50U/mL IL-2、0.5ng/ml IL-15和CD3/CD28 Dynabeads(珠:细胞比率为1:1)的X-Vivo15培养基中培养。转染后两天,添加等体积的含有bFGF和10μM Y27632的PSC培养基。然后在转染4天后将培养基完全更换为PSC培养基。在第20-30天显示iPSC集落,并挑取单个集落以进一步培养和评估。
CAR阳性iPS细胞和定殖的iPS细胞系的生成
iPS细胞通常在基质胶包被板中的cGMP级mTeSR1培养基(StemCellTechnologies)中培养。在慢病毒转导之前,用accutase处理解离iPS细胞,单个iPS细胞以每孔105个的密度接种在补充有1X cloneR和10μM Y27632(StemCell Technologies)的mTeSR1培养基中的12孔板中。过夜培养后,将编码CAR的临床级慢病毒与10μg/mL硫酸鱼精蛋白一起添加到培养物中以转导iPS细胞(感染复数[MOI]=1)。在进行单细胞分选和iPS定殖之前,将转导的细胞培养至少2代。将定殖的CAR阳性细胞扩增并入库用于分化使用。
iPSC基因编辑
某些基因,包括TRAC、TRBC、B2M、CIITA,通过CRISPR-Cas9基因编辑技术使用核糖核蛋白(RNP)复合物递送敲除。简而言之,将180pmol具有支架和基因特异性靶序列的化学修饰的向导RNA与50ul P3初级核转染器溶液(primary nucleofector solution)(Lonza)中的60pmol Truecut Cas9蛋白(Thermofisher Scientific)混合,并在室温下温育10分钟以形成RNP复合物。iPS细胞用Accutase处理解离。1x105单个iPS细胞在具有10μM Y27632的PBC中清洗,并通过在300g下离心3min进行旋下。小心除去上清液,将细胞重悬浮在50uL P3初级核转染器溶液中,然后与RNP复合物溶液合并。将合并的细胞悬浮液转移到皿中,并用Nucloefector 4D仪器(Lonza)进行电穿孔。电穿孔后,将500ul mTeSR1+1XcloneR添加到皿中并在培养箱中温育15分钟,然后转移到基质胶包被的6孔板中。将细胞在补充有1X clonR和10μM Y27632的mTeSR1培养基中培养2天。培养基更换为mTeSR1+cloneR。传代两次后,进行单细胞分选,将定殖的iPSC细胞冷冻保存。提取基因组DNA并进行靶向性PCR和测序以筛选编辑的集落。
人胚胎中胚层祖先(hEMP)的生成和分离
如前所述诱导中胚层定型(例如Montel-Hagen,A.,et al.,Organoid-InducedDifferentiation of Conventional T Cells from Human Pluripotent StemCells.Cell Stem Cell,2019.24(3):p.376-389e8;Chin,C.J.,et al.,Genetic TaggingDuring Human Mesoderm Differentiation Reveals Tripotent Lateral PlateMesodermal Progenitors.Stem Cells,2016.34(5):p.1239-50;Evseenko,D.,et al.,Mapping the first stages of mesoderm commitment during differentiation ofhuman embryonic stem cells.Proc Natl Acad Sci U S A,2010.107(31):p.13742-7)。简而言之,在Accutase(StemCell Technologies)处理后,将人多能干细胞(hPSC)作为单细胞悬液收获、清洗和计数。将细胞直接重悬浮于补充有rhActivin A(10ng/ml)(R&DSystems,Cat.338-AC-010)、rhBMP4(10ng/ml)(R&D Systems,Cat.314-BP-010)、rhVEGF(10ng/ml)(R&D Systems,Cat.298-VS-005)、rhFGF(10ng/ml)(R&D Systems,Cat.233-FB-025)和ROCK抑制剂Y-27632二氢氯化物(10μM)(Tocris,Cat.1254)的X-VIVO 15培养基中。将细胞以3ml中的每孔3x106个细胞铺板在基质胶包被的6孔板上。然后每天用补充有rhBMP4(10ng/ml),rhVEGF(10ng/ml和rhFGF(10ng/ml)的X-VIVO15更换培养基。在第3.5天,用PBS清洗细胞3次并用Accutase温育(每孔1mL,在37℃下10分钟)。使用细胞刮刀收获细胞,在PBS中清洗,并用抗体染色用于流式细胞术。CD326-CD56+hEMP通过在FACSARIA细胞分选仪(BD Biosciences,San Jose,CA)上FACS或通过CD56富集试剂盒(StemCellTechnologies)分离。通过EMO-ATO培养系统将CAR+/-Tn/mem iPSC分化为CAR T/T细胞(方案1A)
根据已发表的方案(例如,Montel-Hagen,A.,et al.,Organoid-InducedDifferentiation of Conventional T Cells from Human Pluripotent StemCells.Cell Stem Cell,2019.24(3):p.376-389e8),诱导具有或不具有CAR表达的Tn/memiPS细胞以分化为EMP(CD56+CD326-)细胞,然后进一步分化为T细胞。首先,通过聚集EMP细胞和MS5-hDLL4饲养细胞建立胚胎中胚层类器官(EMO)培养物。通过胰蛋白酶消化收获MS5-hDLL4细胞并重悬浮于由补充有10μM ROCK抑制剂Y-27632(StemCell Technologies)和10uM TGF-βRI抑制剂SB-431542(SB阻滞剂)的EGM2(Lonza)组成的造血诱导培养基中。在第-14天,将5x105个MS5-hDLL4细胞与0.5-1x104个纯化的hEMP/PSC-ATO在1.5mL Eppendorf管中合并,并在4℃的摇摆斗离心机中以300g离心5min。每管制备多个(最多12个)EMO。小心除去上清液,通过短暂涡旋重悬细胞沉淀物,并以每EMO 6μl的体积重悬浮于造血诱导培养基中。将6μl细胞作为EMO铺板于0.4μm Millicell transwell插入物(EMD Millipore)上,并置于每孔含有1mL造血诱导培养基的6孔板中。每2-3天用由具有SB-431542 10μM的EGM2组成的培养基完全更换培养基,持续1周。每2-3天更换该培养基。在第-7天,将培养基更换为具有造血细胞因子rhTPO 5ng/ml(Peprotech300-18)、rhFLT3L 5ng/ml(Peprotech,Cat.300-19)和rhSCF 50ng/ml(Peprotech,Cat.300-07)的EGM2+SB阻滞剂(10μM)。在第0天,启动PSC-ATO仅需将培养基更换为补充有10ng/ml rhSCF、5ng/ml rhFLT3L和5ng/mlrhIL-7的“RB27”。每3-4天完全更换培养基。分化培养5-7周后,通过向每个孔中添加MACS缓冲液(PBS/0.5%牛血清白蛋白/2mM EDTA)并通过用1mL“P1000”移液器移液短暂解聚ATO,然后通过50μm尼龙滤网(strainer)来收获PSC-ATO CAR T细胞或T细胞。
通过EMO-ATO培养系统将CAR+/-Tn/mem iPSC分化为CAR NK/NK细胞(方案1B)
通过与方案1A(上文)类似的方案,在ATO培养的步骤中修改饲养细胞和细胞因子组合的情况下,诱导具有或不具有CAR表达的Tn/mem iPS细胞以分化为EMP(CD56+CD326-)细胞,然后进一步分化为CAR NK或NK细胞。简而言之,1B中的饲养细胞将使用MS5_DL1而不是MS5_DL4。从第0天起添加10ng/mL IL15以及其他细胞因子(10ng/ml rhSCF,5ng/mlrhFLT3L和5ng/ml rhIL-7)。在第28-50天收获Tn/mem iPSC CAR NK/NK细胞。
从iPSC细胞中生成和分离CD34+HSPC
通过使用STEMdiff造血试剂盒(StemCell Technologies),将iPS细胞分化为CD34+造血干细胞和祖细胞(HSPC)。简而言之,收获iPS细胞并将其作为小聚集体接种在mTeSR1培养基中。培养一天后,将培养基更换为分化培养基A以诱导细胞朝向中胚层样状态。在第2天,用新鲜培养基A更换半培养基。第3天,培养基更换为B,并在第5、7、10天更换半培养基以促进进一步分化为造血细胞。在第10-12天从培养上清液中收获造血祖细胞。使用CD34阳性富集试剂盒(Stemcell technologies)以富集CD34+HSPC细胞。
通过ATO培养系统将CAR+/-Tn/mem iPSC衍生的HSPC分化为CAR T/T细胞(方案2A)
使用公开的ATO培养系统{Montel-Hagen,2019#9;Seet,2017#19},将Tn/mem iPSC衍生的HSPC细胞分化为CAR T/T细胞。简而言之,通过胰蛋白酶处理收获MS5-hDLL4(或MS5-DLL1,如所指)细胞并重悬浮在无血清ATO培养基(‘RB27’)中,该培养基由RPMI 1640、4%B27补充剂(thermofisher scientific)、30uM L-抗坏血酸2-磷酸倍半镁盐水合物(Sigma-Aldrich)、1%青霉素-链霉素、1%Glutamax、5ng/ml rhFLT3L和5ng/ml rhIL-7组成。MS-hDLL4在1.5ml微量离心管中与富集的CD34+HSPC合并,并在4℃下在摇摆斗离心机中以300g离心5min。小心除去上清液,将细胞沉淀物以每ATO 6μl的体积重悬浮于ATO培养基中。将6μl细胞浆作为ATO铺板于0.4mm Millicell transwell插入物(EMD Millipore)上,并置于每孔含有1mL RB27的6孔板中。每3-4天完全更换培养基。培养数周后,通过向每个孔中添加MACS缓冲液(PBS/0.5%牛血清白蛋白/2mMEDTA)并通过用1mL移液管移液短暂解聚ATO,然后通过50μm尼龙滤网来收获生成的CAR T/T细胞。
通过ATO培养系统将CAR+/-Tn/mem iPSC衍生的HSPC分化为CAR NK/NK细胞(方案2B)
通过具有修改的与方案2A(上图)类似的方案,诱导具有或不具有CAR表达的Tn/memiPS细胞以分化为CAR NK或NK细胞。简而言之,2B中的饲养细胞是MS5_DL1而不是MS5_DL4。RB27中提供10ng/ml rhSCF和10ng/ml IL15以及其他细胞因子(5ng/ml rhFLT3L和5ng/ml rhIL-7)。在第28-50天收获Tn/mem iPSC CAR NK/NK细胞。
通过基于纳米纤维基质的培养系统将CAR+/-iPSC分化为CAR T/T细胞(方案3A)
在该方案中,当hMEP细胞在第-14天准备好时,通过胰蛋白酶消化收获MS5-hDLL4细胞,并对细胞悬液进行80Gy辐照。对于6孔板的每个孔,放置一个含有聚合物纳米纤维插入物(纳米纤维溶液,ECM基质)的Millicell transwell插入物(EMD Millipore,孔径0.4μm~3μm),并将2mL培养基添加到插入物外的孔中,将2.5x105 EMP细胞和5x106 MS5-DLL4细胞混合,重悬浮在250ul培养基中,并直接接种在纳米纤维基质插入物上。造血诱导培养基包含补充有10μM ROCK抑制剂Y-27632(StemCell Technologies)和10uM TGF-βRI抑制剂SB-431542(SB阻滞剂)的EGM2(Lonza)。每2-3天用由具有SB-431542 10μM的EGM2组成的培养基彻底更换培养基,持续1周。在第-7天,将培养基更换为具有造血细胞因子rhTPO 5ng/ml(Peprotech300-18)、rhFLT3L 5ng/ml(Peprotech,Cat.300-19)和rhSCF 50ng/ml(Peprotech,Cat.300-07)的EGM2+SB阻滞剂(10μM)。在第0天,将培养基更换为补充有10ng/ml rhSCF、5ng/ml rhFLT3L和5ng/ml rhIL-7的“RB27”。每3-4天完全更换培养基。分化培养5-7周后,通过向每个孔中添加MACS缓冲液(PBS/0.5%牛血清白蛋白/2mM EDTA)并通过用1mL移液器移液短暂解聚培养物,然后通过50μm尼龙滤网来收获产生的CAR T细胞或T细胞。
为了将iPSC分化为HSPC,然后进一步分化为CAR T或T细胞,将含有2.5x105富集的CD34+HSPC细胞和5x106辐照的MS-hDLL4细胞的250uL细胞混合物直接接种在2ml具有有5ng/ml rhFLT3L和5ng/ml rhIL-7的RB27培养基的6孔板中的纳米纤维基质插入物上。每2-3天完全更换培养基,持续5-7周。
还通过在纳米纤维板(纳米纤维溶液)中直接添加hMEP/HSPC细胞和辐照的MS5-DLL4细胞悬浮液并以300g离心3分钟来建立基于纳米纤维基质的共培养物。
通过在RB27培养基或基于甲基纤维素的半固体培养基中将EMP/HSPC和MS5-DLL4细胞与微粉化纳米纤维混合,然后接种到超低附着板中来制备基于纳米纤维基质的共培养物。
通过基于纳米纤维基质的培养系统将CAR+/-iPSC分化为CAR T/T细胞(方案3B)
通过具有修改的与方案3A(上图)类似的方案,诱导具有或不具有CAR表达的iPS细胞以分化成CAR T或T细胞。简而言之,3B中的饲养细胞将使用MS5_DLL1作为饲养细胞而不是MS5_DLL4。
从iPSC直接分化为CAR NK细胞或NK细胞,从第0天起添加10ng/mL IL15以及其他细胞因子(10ng/ml rhSCF、5ng/ml rhFLT3L和5ng/ml rhIL-7)。
从iPSC分化为HSPC,然后分化为CAR NK细胞或NK细胞,在RB27中提供10ng/mlrhSCF和10ng/ml IL15以及其他细胞因子(5ng/ml rhFLT3L和5ng/ml rhIL-7)。
CAR T细胞制备
用Dynabeads人T扩增物CD3/CD28(Invitrogen)以1:3(T细胞:珠)的比率刺激PBMC或Tn/mem并用慢病毒转导以在含有10%FCS与20μg/ml硫酸鱼精蛋白(APPPharmaceuticals)、50U/ml重组人IL-2(rhIL-2)和0.5ng/ml rhIL-15的X-VIVO15(Lonza)培养基中表达CAR。然后在相同的培养基和细胞因子条件下,在37℃、5%CO2下维持培养物。每隔一天提供细胞因子。在转导后第7天,使用DynaMag-50磁体(Invitrogen)从培养物中除去CD3/CD28Dynabeads。
流式细胞术
用Accutase(Innovative Cell Technologies)解离iPSC细胞,并重悬浮于具有1XCloneR补充剂(StemCell Technologies)的mTeSR1培养基中。如前所述收获并染色T细胞{Jonnalagadda,2015#3;Jonnalagadda,M.,et al.,Chimeric antigen receptors withmutated IgG4 Fc spacer avoid fc receptor binding and improve T cellpersistence and antitumor efficacy.Mol Ther,2015.23(4):p.757-68}。使用荧光染料缀合的针对SSEA3、SSEA4、TRA1-60、TRA1-81、CD30的抗体检查iPSC表型。使用荧光染料缀合的针对CD3、CD4、CD8a、CD8b、CD5、CD7、CD45、CD45RA、CD45RO、TCRab、TCRgd、CD16、CD56、CD27、CD28、NKP44、NKP46、NKG2A、NKG2D、CD178(FasL)、CD19的抗体检查T细胞表型。通过对截短的EGFR或截短的CD19进行染色来确定转基因表达。使用荧光染料缀合的针对CD45RO、CD45RA、CD62L的抗体分析记忆相关表型。所有样品均通过MacsQuant分析仪(MiltenyiBiotec)进行分析,并通过FlowJo v10进行处理。
TCR Vβ库表达分析
T细胞受体Vβ染色使用三色流式细胞术和IOTest Beta Mark TCR库试剂盒(Beckman Coulter)确定,该试剂盒由设计为鉴定24个不同TCR Vβ家族的单克隆抗体(mAb)组成。每组由三种不同的抗Vβ家族特异性mAb组成,这些mAb用异硫氰酸萤光素(FITC)、藻红蛋白(PE)标记或用FITC和PE双重标记。T细胞群也用APC抗CD3抗体共染色,并且门控CD3+群用于分析。
体外T细胞测定
为了测试细胞毒性和活性,将靶肿瘤细胞以指示的密度种植在96个圆底孔板中。然后将T细胞清洗并重悬浮于相同的培养基中并添加到靶细胞中。为了测试脱粒,在CD107a抗体和Golgistop蛋白转运抑制剂(BD Biosciences)存在下,将CAR T或对照T细胞与靶细胞温育5小时。共培养后,收获细胞、固定、透化并染色细胞内细胞因子。通过流式细胞术检查脱粒(CD107a染色)和细胞内细胞因子染色。对于细胞毒性测试,如所指示,共培养将持续4小时用于短期测定和48小时用于长期测定。共培养后,收获所有细胞并用指示的抗体染色,然后通过流式细胞术进行定量。
体外细胞因子产生测定
CAR T或T细胞与不同的靶细胞以1:1的效应物与靶物(E:T)比率共温育24小时。收集上清液并通过Bio-Plex阅读器(Bio-Rad)用细胞因子10-plex人组试剂盒(Invitrogen)检查细胞因子。
体内异种移植物研究
所有小鼠实验均获得了COH IACUC的批准。如前所述,使用6至8周龄的NOD/SCID/IL2R-/-(NSG)小鼠生成肿瘤异种移植模型(例如Urak,R.,et al.,Ex vivo Aktinhibition promotes the generation of potent CD19CAR T cells for adoptiveimmunotherapy.J Immunother Cancer,2017.5:p.26)。简而言之,在第0天,将ffLuc+NALM6细胞(1×106)腹膜内注射(ip)到NSG小鼠中。4天后,如每个实验所指示,用CAR T细胞或T细胞腹膜内处理小鼠。使用Xenogen IVIS 100通过体内生物光子成像测定肿瘤生长。还监测了小鼠的存活,根据美国兽医医学协会指南(American Veterinary Medical AssociationGuidelines)实施安乐死。
实施例4的方法
实施例4中使用的某些试剂和资源在本节末尾的表格中进行了描述。
小鼠
所有动物实验均按照希望之城动物研究委员会(City of Hope Animal ResearchCommittee)批准的方案进行。本研究使用来自杰克逊实验室(Jackson Laboratory)的6-8周龄NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ(NSG)小鼠。
DNA构建体
CD19靶向性嵌合抗原受体(CD19-CAR)构建体与我们目前在靶向B细胞白血病/淋巴瘤(clinicaltrials.gov#NCT02146924)[1,2]的临床研究中使用的相同。CD19-CAR包含衍生自FMC63 mAb的抗CD19 scFv域[3]、在CH2区内具有两个点突变(L235E和N297Q)的IgG4Fc间隔物、CD28跨膜域、CD28ζ共刺激域和CD3ζ信号传导域。然后,T2A核糖体跳跃序列[4]将该CAR序列与可以用作选择标志物和安全开关的截短的人表皮生长因子受体序列(huEGFRt)分开[5-7]。编码OCT3/4/shp53、SOX2/KLF4、L-MYC/LIN28和EBNA的附加型质粒是山中伸弥(Shinya Yamanaka)赠送的[8]。
Tn/mem分离
血液制品是根据COH IRB批准的方案从健康供体获得的,并且按照先前研究中描述的类似规程分离幼稚和记忆T(Tn/mem)细胞[9]。简而言之,人外周血单个核细胞(PBMC)通过Ficoll-Paque(GE Healthcare)上的密度梯度离心分离,然后进行连续几轮CliniMACS/AutoMACS(Miltenyi Biotec)消减以除去表达CD14和CD25的细胞,然后对Tn/mem细胞进行CD62L阳性选择。
从Tn/mem生成iPSC
通过从已发表的方案修改的无整合方法将Tn/mem细胞重编程为多能干细胞(iPSC)[8]。简而言之,使用人T细胞核转染器试剂盒和核转染器4D电穿孔装置(Lonza)用3μg质粒混合物对一百万个Tn/mem细胞进行电穿孔。质粒混合物由编码OCT3/4、SOX2、KLF4、L-MYC、LIN28的附加型质粒和针对TP53的shRNA组成[8]。转染的细胞在补充有10%FBS(HyClone)、50U/mL rhIL-2(Novartis Oncology)、0.5ng/mL rhIL-15(CellGenix)和Dynabeads人T-扩增物CD3/CD28(ThermoFisher Scientific)(珠与细胞的比率为1:1)的X-VIVO15培养基(Lonza)中培养。转染后两天,添加等体积的含有rhFGF-basic和10μM Y27632的多能干细胞(PSC)培养基[8]。然后在转染后4天将培养基完全更换为PSC培养基。iPSC集落在第20-30天可见,并在显微镜下挑取单个集落,用于在基质胶包被(Corning)板中的cGMP级mTeSR1培养基(StemCell Technologies)中进一步培养/扩增。
CAR阳性的克隆iPSC系的生成
在慢病毒转导之前,用Accutase(ThermoFisher Scientific)处理解离iPSC培养物,并将细胞以每孔105个的密度接种在补充有1X CloneR和10μM ROCK抑制剂Y-27632二氢氯化物(StemCell Technologies)的mTeSR1培养基中的12孔板中。过夜培养后,将编码CD19-CAR的cGMP慢病毒与10μg/mL硫酸鱼精蛋白(APP Pharmaceuticals)添加到培养物中以转导iPSC(感染复数[MOI]=1)。在通过流式细胞术进行单细胞分选和iPSC定殖之前,将转导的细胞培养至少两代。克隆CAR阳性细胞在基质胶包被板上的mTeSR1培养基中再次扩增,并入库用于随后的分化。
通过PCR的整合检测
EBNA1是所有附加型载体的共同组分[10]。为了检测用于从T细胞重编程iPSC的附加型质粒的基因组整合,使用如下引物进行PCR以从基因组DNA中扩增整合的EBNA组分:EBNA1_For:ATCAGGGCCAAGACATAGAGATG,EBNA1_Rev:GCCAATGCAACTTGGACGTT。无质粒整合的iPSC克隆未显示EBNA1信号。在多能干细胞上表达的FBX15在此用作看家基因,并通过以下引物进行扩增:FBX15_For:GCCAGGAGGTCTTCGCTGTA;FBX15_Rev:AATGCACGGCTAGGGTCAAA。
畸胎瘤形成测定
将200万个解离的iPSC悬浮于200uL培养基(100uL PBS(Irvine Scientific)和100uL基质胶)中,并皮下注射到NSG小鼠中。5-8周后,在PBS中收获畸胎瘤,在室温下在4%多聚甲醛(Boston BioProducts)中固定过夜,然后维持在70%乙醇中用于处理。将样品提交给希望之城组织学核心机构(City of Hope Histology Core Facility)进行切片和苏木精和伊红染色。切片用显微镜检查、解释并拍照。
通过PSC-ATO培养将Tn/mem衍生的CAR+iPSC分化为CAR+T细胞
图13A中概述了顺次分化方案的模式。首先,如前所述诱导中胚层定型[11-13]。简而言之,在Accutase处理后收获iPSC作为单细胞悬液,以1x106个细胞/mL重悬浮于含有10ng/mL rhActivin A(R&D Systems)、10ng/mL rhBMP4(R&D Systems)、10ng/mL rhVEGF(R&D Systems)、10ng/mL rhFGF(Peprotech)和10μM ROCK抑制剂Y-27632二氢氯化物(StemCell Technologies)的X-VIVO15培养基中。每孔有300万个细胞铺板在基质胶包被的6孔板中。然后每天用含有10ng/mL rhBMP4、10ng/mL rhVEGF和10ng/mL rhFGF的X-VIVO 15更换培养基。三天后(图13A中的第-14天),用PBS(Irvine Scientific)清洗细胞3次,并在37℃下用每孔1mL的Accutase温育5-7分钟。收获细胞,在含有1mM EDTA和2%FBS的PBS中清洗,并使用EasySep阳性选择试剂盒(StemCell Technologies)通过CD56富集分离CD56+CD326-人iPSC中胚层祖先(iMP)。进行流式细胞术以确认iMP的CD56+CD326-表型。
iPSC中胚层类器官(iMO)通过聚集iMP细胞和MS5-hDLL4饲养细胞生成。在第-14天,用胰蛋白酶收获MS5-hDLL4细胞并清洗到由具有10μM Y-27632和10μM TGF-βRI抑制剂SB-431542(StemCell Technologies)的EGM-2(Lonza)组成的造血诱导培养基中。使用40μm尼龙网滤网以除去聚集体后,将5x105MS5-hDLL4细胞与0.5-1x104纯化的iMP细胞在1.5mL微量离心管中合并,并在4℃下在摇摆斗离心机中以300x g离心5min。每管最多制备12个iMO。小心除去上清液后,MS5-hDLL4/iMP细胞沉淀物通过短暂脉冲涡旋以每iMO 6μl在造血诱导培养基(即具有10μM SB-431542的EGM-2)中重悬浮。在含有1.5mL造血诱导培养基的6孔板中,每孔将两个6μL细胞等分试样作为iMO接种在一个Millicell transwell插入物(MilliporeSigma)上。每2-3天完全更换培养基,持续1周。在第-7天,将培养基更换为具有10μM SB-431542加5ng/mL rhTPO(Peprotech)、5ng/mL rhFLT3L(Peprotech)和50ng/mLrhSCF(Peprotech)的EGM-2。
在第0天,开始人工胸腺类器官(ATO)T细胞分化阶段,转换为在RB27培养基中含有10ng/mL rhSCF,5ng/mL rhFLT3L,and 5ng/mL rhIL-7的无血清ATO培养基培养基,该RB27培养基由具有4%B27补充剂(ThermoFisher Scientific)、30μM L-抗坏血酸2-磷酸倍半镁盐水合物(Sigma)、1%GlutaMAX(ThermoFisher Scientific),1%青霉素-链霉素(Lonza),55uM 2-巯基乙醇(ThermoFisher Scientific)和1%MEM非必需氨基酸(ThermoFisherScientific)的RPMI 1640(Lonza)组成。每2-3天完全更换培养基。分化5-7周后,通过将1-2mL具有10%FBS的X-VIVO 15移液到每个transwell插入物的表面上并通过用P1000移液器反复抽吸解聚iMO-ATO来收获iMO-ATO衍生的CAR+T细胞。通过使解聚的细胞悬浮液通过40μm尼龙网滤网来分离单个细胞。用指示的抗体对回收细胞的等分试样进行染色,以通过流式细胞仪进行表型分析,并在具有微小修改的先前描述的快速扩增方法(REM)中培养其余细胞[14,15]。简而言之,在50mL含有10%FBS、20ng/mL抗CD3(Miltenyi Biotec)、50U/mLrhIL-2和10ng/mL rhIL-7的X-VIVO15培养基中,将1x106个iMO-ATO衍生的T细胞与50x106个γ-辐照(35Gy)PBMC和10x106个γ-辐照LCL细胞(80Gy)合并。REM培养物维持14天,每48小时更换一半体积的培养基。
免疫组织化学
PSC-ATO类器官用固定/透化溶液试剂盒(BD Biosciences)固定和透化,在透化缓冲液中用PE-抗CD3和DAPI染色15分钟,然后用清洗缓冲液冲洗3次。用BZ-X810荧光显微镜(Keyence)拍摄原位图像。
常规CD19-CAR T细胞的产生
在含有10%FBS、50U/mL rhIL-2和0.5ng/mL rhIL-15的X-VIVO15培养基中,用Dynabeads人T扩增物CD3/CD28以1:2(细胞:珠)的比率刺激PBMC(或Tn/mem,仅如图16D-F所指示的)。用临床级慢病毒转导细胞以用25μg/mL硫酸鱼精蛋白(APP Pharmaceuticals)表达CD19CAR。然后在相同的培养基和细胞因子条件下,在37℃、5%CO2下维持培养物。每隔一天提供新鲜的细胞因子。在转导后第7天,使用DynaMag-50磁体(ThermoFisherScientific)从培养物中除去CD3/CD28 Dynabeads。细胞在培养中扩增直至第17天或如指示收获。PBMC衍生的CAR+T细胞通过EasySep试剂盒用抗EGFRt抗体(StemCellTechnologies)进行富集,并用于表型表征和功能测定;用于体内测定的Tn/mem衍生的CAR+T细胞未富集,而是基于CAR+进行给与。
流式细胞术
用Accutase(ThermoFisher Scientific)解离iPSC,并重悬浮于具有1X CloneR补充剂(StemCell Technologies)的mTeSR1培养基中。使用荧光染料缀合的针对EGFR(以检测转基因)、SSEA3、SSEA4、TRA1-60、TRA1-81和CD30的抗体检查iPSC表型。如前所述收获和染色T细胞[5]。使用荧光染料缀合的针对CD3、CD4、CD8α、CD8β、CD5、CD7、TCRαβ、TCRγδ、CD16、CD56、CD27、CD28、NKP44、NKP46、NKG2A、NKG2D、CD178(FasL)和CD19的抗体检查T细胞表型。通过对截短的EGFR进行染色来确定CAR表达。用荧光染料缀合的针对CD45RO、CD45RA和CD62L的抗体评估记忆相关表型。
使用IOTest Beta Mark TCR库试剂盒(Beckman Coulter)进行T细胞受体Vβ染色,该试剂盒由设计为鉴定24个不同TCR Vβ家族的单克隆抗体(mAb)组成。每组由三种不同的抗Vβ家族特异性mAb组成,这些mAb用异硫氰酸萤光素(FITC)、藻红蛋白(PE)标记或用FITC和PE双重标记。T细胞群也用APC抗CD3抗体共染色,并且门控CD3阳性群用于分析。
在MacsQuant分析仪10(Miltenyi Biotec)或Fortessa(Becton Dickinson)流式细胞仪上采集数据,并使用FlowJo(v10.6.1)进行分析。
基于PCR的TCRβ克隆性测定
通过DNeasy试剂盒(Qiagen)提取基因组DNA并用作PCR模板。根据IdentiCloneTCRB+TCRG T细胞克隆性测定试剂盒(Invivoscribe)[16,17]的方案设置PCR检测,TCRB管A和B引物主混物(master mix)靶向TCRβ链基因座的可变区内的框架区和连接区。TCRB管C靶向TCRβ链基因座的多样性和连接区。样品控制大小梯主混物靶向多个基因并生成一系列扩增子以充当输入DNA的质量控制。引物是荧光标记的,并进行片段分析以检测PCR产物的片段大小,同时定期进行DNA琼脂糖凝胶检查。
体外T细胞功能测定
清洗效应细胞(iPSC CD19-CAR T、iPSC模拟T、常规CD19-CAR T或常规模拟T细胞),重悬于含有50U/mL rhIL-2和0.5ng/mL rhIL-15的新鲜培养基中,并且在96孔U底板中,以指示的效应物与靶标(E:T)的比率与指示的肿瘤细胞共培养4小时或48小时。然后通过计数活的(即DAPI阴性)表达GFP的肿瘤细胞,通过流式细胞术常规评估细胞毒性活性;对于原代ALL细胞,计数了DAPI-/CD19+细胞。或者,对于基于萤光素酶的细胞毒性测定,在每个时间点,以0.14mg/mL的最终浓度将D-萤光素钾盐(PerkinElmer)添加到每个孔中,并将板在37℃下温育10分钟。在用萤光素温育后,小心混合每个培养板的内容物,并用多通道移液器将其转移到不透明的96孔U底板中。用Cytation 3读板器(Biotek)读取生物发光通量。对于每个肿瘤系,使用单独的肿瘤细胞的重复孔来生成内部MIN(0%存活率)和MAX(100%存活率)参考,用于计算百分比溶解;通过在添加萤光素前十分钟添加SDS至终浓度1%以获得MIN[18]。
为了评估T细胞活化,在CD107a抗体和GolgiStop蛋白转运抑制剂(BDBiosciences)存在下,iPSC衍生或常规CAR T或模拟T细胞与指示的肿瘤细胞以1:1的E:T比温育5小时。然后收获细胞、固定、透化和染色细胞内细胞因子。然后通过流式细胞术检查CD3门控细胞上的脱粒(CD107a染色)和细胞内细胞因子染色(例如IFNγ)。收集不具有GolgiStop的类似共培养物用于对CD3门控细胞上的表面活化标志物CD25和CD137/4-1BB进行染色,通过流式细胞术评估。
为了进一步表征细胞因子的产生,iPSC衍生或常规CAR T或模拟T细胞与指示的NALM6肿瘤细胞在不添加细胞因子的培养基中以1:1的E:T比共温育24小时。收集上清液并通过Bio-Plex阅读器(Bio-Rad)用细胞因子10-Plex人组试剂盒(ThermoFisherScientific)量化细胞因子水平。收获了类似的共培养物,用于流式细胞术分析CD3门控细胞上的表面活化标志物CD25和CD137/4-1BB。
对于体外重复攻击测定,将105个CAR T细胞与4X105 CD19+NALM6细胞以1:4的E:T比共培养,并每隔一天用4X105 NALM6细胞再攻击3次。然后用表面耗竭标志物PD-1、TIM-3和LAG-3以及T细胞标志物对细胞进行染色。通过流式细胞术在CD3门控细胞上评估每个耗竭标志物[19]。
RNA和蛋白质分析
用Quick-RNA Microprep试剂盒(ZymoResearch)提取RNA,并用DNase I处理。RNA深度测序由希望之城整合基因组学核心机构(City of Hope Integrative Genomics CoreFacility.Briefly)进行。简而言之,根据制造商推荐的方案,使用KAPA mRNA HyperPrep试剂盒(Roche)制备了链RNA-seq文库。使用Qubit定量试剂盒(ThermoFisher Scientific)对文库进行量化,并加载到HiSeq 2500测序平台(Illumina)上进行单端51-bp测序。使用Illumina实时分析(RTA)v1.18.64完成碱基识别(base calling)。
对于western印迹进行的蛋白质分析,收获的细胞在RIPA缓冲液(ThermoFisherScientific)中裂解,并用BCA蛋白质测定试剂盒(ThermoFisher Scientific)对蛋白质提取物进行定量。Bolt Mini Gel System(ThermoFisher Scientific)用于凝胶电泳和蛋白质转移。使用抗p44/42MAPK(Erk1/2)和抗磷酸化p44/42MAPK(Erk1/2)(Thr202/Tyr204);抗PLCγ1、抗磷酸化PLCγ1(Tyr783)和抗磷酸化PLCγ1(Ser1248);抗CD3ζ和抗磷酸化CD3ζ(Y142);和抗磷酸化ZAP70以询问CAR T和T细胞信号传导途径(有关抗体的详细信息,参见资源表)。
RNAseq数据的生物信息学分析
为了分析RNAseq数据,使用基于PCA算法的R包“DESeq2”(v.3.10)实现PCA的二维可视化。使用分层聚类方法由Cluster(v.3.0)和JavaTreeView(v.1.1.6r4)生成z评分的热图。使用R包“edgeR”(v.3.28.0)[20]进行差异表达基因(DEG)分析。推导DEG的流程涉及分位数调整的条件最大似然(qCML)和准似然(QL)F检验。使用R包“ggplot2”(v.3.2.1)采集气泡图。在GSEA(v.4.0.3)上运行基因集富集分析(GSEA)算法[21,22]。生物信息学软件包的资源列于“关键资源”表。
亚硫酸氢盐转化、PCR和测序
通过DNeasy试剂盒(Qiagen)制备基因组DNA。使用EZ DNA Methylation-Lightning试剂盒(ZymoResearch)用亚硫酸氢钠处理500ng基因组DNA以转化非甲基化的胞嘧啶。根据制造商的方案进行反应。进行甲基化特异性PCR,并扩增来自亚硫酸氢盐转化的iPSC CD19-CAR T细胞和常规CD19-CAR T细胞的gDNA的EF1a启动子的245bp PCR片段。将PCR片段亚克隆到pCR4-TOPO载体(ThermoFisher Scientific)中,并通过Sanger测序对每组的六个克隆进行测序。将测序结果与原始和推定的甲基化序列进行比对,以确定CG位点的甲基化状态。
动物研究
所有小鼠实验均按照希望之城机构动物护理和使用委员会(City of HopeInstitutional Animal Care and Use Committee)批准的方案进行。使用如前所述的6至8周龄NOD/SCID/IL2R-/-(NSG)小鼠(杰克逊实验室)生成肿瘤异种移植模型[6]。简而言之,在第0天,将ffLuc+NALM6细胞(2.5×105)腹膜内(i.p.)或静脉内(i.v.)注射至NSG小鼠。4天后,然后用iPSC衍生或常规CAR T或模拟T细胞处理小鼠,如每个实验所述。每周3次用20x106辐照(80Gy)人hIL-15分泌抚育细胞(IL15-NS0)i.p.注射指示组中的小鼠[14]。图16A和16D的参考示意图用于在每个肿瘤模型中注射T细胞。每周使用Xenogen IVIS 100通过体内生物光子成像测定肿瘤生长。还监测小鼠的存活,根据美国兽医医学协会指南实施安乐死。
实施例1
在本实施例中,来自健康供体T细胞亚群的iPSC系——幼稚和记忆T细胞(Tn/mem)通过使用iPSC重编程附加型载体的无整合方法生成(如以上和如例如Okita,K.,et al.,Anefficient nonviral method to generate integration-free human-inducedpluripotent stem cells from cord blood and peripheral blood cells.Stem Cells,2013.31(3):p.458-66中所述)。用临床级慢病毒转导所得iPS细胞以表达CD19特异性CAR(CD19CAR)或其他CAR。对单个细胞进行分选、定殖和筛选,以生成同质的CAR+iPSC细胞库。
通过使用EMO-ATO培养系统,如上文方案1A所述,成功生成了Tn/mem iPSC衍生的CART细胞。产生的iPSC CD19CAR T细胞具有常规的T细胞表面标志物表型,具有CD3+CD5+CD7+TCRαβ+CD8αβ+和CD3+CD5+CD7+TCRαβ+CD4+(图1A)。扩增的细胞由经典的CD62L+CD45RA+干记忆T细胞、CD62L-CD45RA-CD45RO+效应记忆T细胞和CD62L-CD45RA+效应T细胞组成(图1C)。Tn/mem iPSC 1928zCAR T细胞中的CAR表达水平低于从来自同一供体的PBMC细胞生成的CAR T细胞。值得注意的是,这些细胞不表达NK细胞特异性标志物NKP46和CD16(图1D),这与单层共培养产生的T细胞不同(如Themeli,M.,et al.,Generation of tumor-targetedhuman T lymphocytes from induced pluripotent stem cells for cancertherapy.Nat Biotechnol,2013.31(10):p.928-33中所述)。iPSC CD19CAR T细胞表达相似水平的泛细胞毒性受体分子NKG2D、更高水平的NKP44,并且负表达NKG2和B细胞谱系标志物CD19(图1E)。
基于流式细胞术的TCR Vβ库表达测定证明Tn/mem iPSC 1928zCAR T细胞仅显示一种类型的TCR库(图2A-2B)。这种表型类似于TCR转基因表达诱导的等位基因表达效应(Brady,B.L.,N.C.Steinel,and C.H.Bassing,Antigen receptor allelic exclusion:anupdate and reappraisal.J Immunol,2010.185(7):p.3801-8),它具有生成纯抗原特异性T细胞以减少不需要的移植物抗宿主效应(GvH)的潜在应用。因此,在一些实施方案中,本文公开的CAR T细胞减少至少一种与GvH相关的症状。
Tn/mem iPSC 1928zCAR T细胞在两周内稳健扩增(约100倍),与其CD19敲除对照细胞相比,针对CD19+靶细胞(诸如CD19+3T3细胞、亲代肿瘤细胞NALM6和Raji)显示出有力的抗原特异性细胞毒性(图3A-3C)。iPSC衍生的CD19CAR T细胞的体外细胞毒性效力优于从同一供体生成的常规PBMC衍生的CAR T细胞(图3D)。
iPSC衍生的CD19CAR T细胞也表现出高效的脱粒和活化表型(图4)。检查了CD19+阳性癌细胞攻击后的细胞因子谱。Tn/mem iPSC 1928zCAR T细胞可以有力地分泌Th1细胞因子IFNγ和TNFα。Tn/mem iPSC 1928zCAR T细胞在没有CD19抗原攻击的情况下在静态状态下分泌较低水平的GMC-SF、IFNγ和TNFα。预期在体内具有较少的细胞因子释放综合征。
在植入有NALM6细胞的NSG小鼠模型中检测了Tn/mem iPSC 1928zCAR T细胞的体内抗肿瘤活性。Tn/mem iPSC 1928zCAR T细胞显著消除了植入的肿瘤细胞并提高了小鼠的存活。Tn/mem iPSC 1928zCAR T细胞和IL15分泌抚育细胞IL15_NS0的组合进一步改善了治疗作用(图6A-6D)。
实施例2
如上所述生成了Tn/mem iPSC HSPC衍生的CAR NK细胞,例如,如方案2B中所述。生成的CAR NK细胞展示了CD3-CD56+NKP46+的典型NK标志物谱(图7A-7D)。它们还表达了NKG2D、NKP44和低水平的CAR。
Tn/mem iPSC HSPC衍生的1928zCAR NK细胞是功能性的,并且以抗原依赖性和抗原非依赖性方式对一组肿瘤细胞系表现出有力的细胞毒性(图8A-8B)。当与肿瘤细胞共培养时,它们还显示出有力的脱粒活性(图9)。
实施例3
如上所述,例如,如方案3A中所述,还由基于纳米纤维基质的培养生成了iPSC1928zCAR T细胞。生成的1928zCAR T细胞也展示了具有CD3+CD8αβ+或CD3+CD4+的常规T细胞表型(图10)。
如图11A-11B所示,定殖的iPSC系表达BBzCD19-CAR和28zCLTX-CAR并且不具有阶段特异性胚胎抗原4(SSEA-4)的高表达。iPSC衍生的BBzCD19-CAR T和28zCLTX-CAR T细胞的细胞表面表达显示于图12A-12B(12A:没有REM扩增的情况下,第7周的iPSC CAR T表型;B:REM扩增后的iPSC CAR T表型)。
实施例4
本实施例还显示了使用iPSC分化来生成具有规范T细胞表型和CAR T功能的CAR T细胞。本实施例中引用的出版物列在实施例末尾。
概要
本实施例表明,从诱导多能干细胞(iPSC)无限生成嵌合抗原受体(CAR)T细胞可以用于开发“现成的”CAR T细胞免疫疗法。然而,实现高效地将iPSC定向分化为规范αβT细胞谱系以及维持CAR表达和功能具有挑战性。下面描述的是q连续的3D类器官系统有助于从CAR工程化iPSC生成T细胞,并对产品赋予具有常规CAR T细胞特性。iPSC从富集的CD62L+幼稚和记忆子集(Tn/mem)重编程,然后进行CAR转导、单细胞分选和定殖。通过3D类器官培养诱导T细胞定向分化是明显的,因为所得的CD19-CAR T细胞(iPSC CD19-CAR T细胞)主要是CD3/CD5/CD7/TCRαβ/CD8αβ阳性和TCRγδ阴性。虽然与常规衍生的CAR T细胞相比,iPSCCD19CAR T细胞由于EF1α启动子的高甲基化表现出较低的CAR表达水平,它们表现出在细胞因子释放中的更好的抗原特异性和更稳健的TCR/CAR信号传导。与由供体匹配的PBMC生成的常规CD19-CAR T细胞相比,扩增的iPSC CD19-CAR T细胞显示出相当的抗原特异性活化、脱粒、细胞毒性和细胞因子分泌,并且它们维持衍生自初始克隆的TCR的同质表达。iPSCCD19-CAR T细胞在体内也表现出抗肿瘤活性,延长了CD19+人肿瘤异种移植小鼠的存活。总之,这些方法从iPSC中生成高功能的常规CAR T细胞,以支持“现成”制造策略的开发。
结果
产生具有常规T细胞表型的iPSC衍生的CAR T细胞
iPSC克隆衍生自原代CD62L+幼稚和记忆T细胞(Tn/mem),该T细胞群已被提出在CAR T细胞治疗中具有卓越的持久性并改善临床结果(McLellan and Ali Hosseini Rad,2019;Morgan and Schambach,2018;Popplewell et al.,2018;Samer K.Khaled,2018;Zahet al.,2020)。通过编码KLF4、SoX2、OCT-4、C-MYC和LIN28的附加型质粒以及P53 shRNA转导和重编程从健康人供体的外周血中富集的Tn/mem细胞(Okita et al.,2013),并筛选和表征了多个无整合的iPSC克隆(图17A-17E)。通过碱性磷酸酶染色和干细胞标志物SSEA3、SSEA4、TRA1-60、TRA1-81和CD30的检查证实iPSC克隆的多能性(图17A-17B),EBNA PCR证明iPSC克隆是无整合的(图17C)。用编码CD19靶向性CAR的临床级慢病毒转导合格的克隆(Popplewell et al.,2018;Samer K.Khaled,2018),并且CAR+细胞通过流式细胞仪进行单细胞分选、定殖、扩增和入库。经模拟转导的和CAR表达性的克隆两者均维持干细胞标志物表达(图17D)。亲本iPSC和CD19-CAR+iPSC克隆通过畸胎瘤形成测定进一步测试,以确认它们产生外胚层、内胚层和中胚层胚层的多能性潜能(图17E)。为了指导表达CD19-CAR的iPSC分化为表达CD19-CAR的T细胞,修改了Montel-Hagen等人(Montel-Hagen et al.,2019)的改良胚胎和诱导多能干细胞人工胸腺类器官(PSC-ATO)系统。首先,CD19-CAR+iPSC在前三天在无饲养物条件下培养以诱导中胚层分化(图13A)。然后通过磁性选择CD56+细胞富集CD56+CD326-iPSC中胚层祖细胞(iMP),并通过与MS5-hDLL4饲养细胞的iPSC中胚层类器官培养(iMO)分化成造血祖细胞(14天),然后进行T细胞定型和分化(另外5-7周)(图13A)。成熟类器官培养物的原位染色和成像证明了具有CD3+T细胞和GFP+MS5-DLL4饲养细胞的异质组织样结构(图13B、图13C和图18A)。CD19-CAR+iMP的细胞产量与模拟转导的iMP的细胞产量相当(图13D),并且PSC-ATO分化的iPSCT细胞,无论是经模拟转导的还是CD19-CAR+,均可以使用改进的快速扩增方法(REM)高效地扩增到临床相关数量(Wang et al.,2011b),在2周内扩增约75倍(图13E)。然后收获所得扩增的iPSC CD19-CAR T细胞用于表型表征和扩增。PSC-ATO分化和扩增的iPSC CD19-CAR T细胞表现出CD3/CD5/CD7/TCRαβ/CD8αβ阳性、NKG2A/NKP46/CD16/CD19阴性表型。作为常规CAR T细胞表型和功能的基准,我们利用了使用标准CD3/CD28珠刺激规程从同一供体生成的PBMC衍生的CD19-CAR T细胞,并证明iPSCCAR T细胞在表型上与常规CD19-CAR T细胞的CD8+亚群相似(图13F、图13G、图18B、图18C和图18D)。此外,与常规CD19-CAR T细胞相似,iPSC CD19-CAR T细胞由处于不同分化阶段的群体,包括幼稚或干细胞样T细胞以及基于CD62L、CD45RA和CD45RO谱的记忆T细胞组成(图13F-13G)。与常规CD19-CAR T细胞相比,它们还表达相似水平的FasL,但更高的CD56、NKG2D和NKP44水平(图18C-18D)。有趣的是,iPSC CD19-CAR T细胞似乎比常规CD19-CAR T细胞表达更少的CAR/转基因(图1H)。通过流式细胞术(图13I和图18E)和gDNA的PCR(图18F)进行的TCR库分析表明,iPSC模拟转导的T细胞和CD19-CAR+T细胞保留了它们的克隆TCR,而常规T细胞是高度多克隆的。
iPSC衍生的CAR T细胞的转录谱
使用批量RNA深度测序分析以探索iPSC CD19-CAR T细胞与来自同一供体的常规PBMC衍生的CD19-CAR T细胞之间的差异。来自同一供体的NK细胞也用于比较。主成分分析(PCA)表明,iPSC模拟T和iPSC CD19-CAR T细胞显示出与来自同一供体的常规模拟转导T细胞、CD19-CAR T细胞或NK细胞相似的转录谱(PC评分约7%方差),但与iPSC有显著区别(PC评分约84%方差)(图14A)。全局转录谱的分层聚类显示iPSC衍生的T细胞与常规衍生的T细胞比与NK细胞更相似(图14B)。观察最显著分化的基因,观察到与常规CD19-CAR T细胞相比,iPSC CD19-CAR T细胞表达更低水平的IL-13、HLA-DR、IL7R、CCR4和CD74,但表达更高水平的DLL1、FOSL2、TXK、REG4、和IFITM2(图14C)。对选定的功能相关基因集的评估揭示,与常规CD19-CAR T细胞相比,iPSC CD19-CAR T细胞表达更高水平的T淋巴细胞基因CD3E、CD3D、CD8、LCK和ZAP70,以及更低水平的CD4、GATA3、BCL11B和LEF1基因(图14D)。对于细胞毒性介质基因,与常规CD19-CAR T细胞相比,iPSC CD19-CAR T细胞表达更多的GNLY和PRF1,但更少的GZMB。对于T细胞抑制基因,iPSC CD19-CAR T表达更少的CTLA4、PD1和TIGIT,但更多的LAG3和TIM3(图14D)。iPSC CD19-CAR T细胞不表达NK细胞签名基因,这与常规CD19-CAR T细胞相似(图14D)。iPSC衍生的T细胞也表现出比常规T细胞低水平的MHC基因,并且没有表现出偏向耗竭表型的基因签名(图19A)。此外,基因集富集分析显示了与常规CD19-CAR T细胞相比,iPSC CD19-CAR T细胞中的上调的缺氧和下调的MYC靶基因签名(图19B),这可能与3D类器官培养中的缺氧微环境有关,并表明独特的代谢签名,其代表与常规的CAR T细胞相比稳定状态中更低的活化(Palazon et al.,2017;Pavlacky and Polak,2020;Wang etal.,2011a)。
如流式细胞术所示,iPSC CD19-CAR T细胞表达的CAR转基因水平比常规CAR T细胞低得多(图13H)。然而,CAR转导的定殖iPSC中的CAR表达水平相当高,并且与模拟转导的iPSC明显可区分(图17D),表明随后的CAR下调可能由细胞分化期间的转录或翻译调节介导。在此,与许多基于慢病毒的CAR T平台一样(Porter et al.,2011;Programs,2019),CAR转基因表达由含有许多CpG岛的EF1α启动子驱动(图19C)。因此,我们研究了在表达CD19-CAR的iPSC向表达CD19-CAR的T细胞的细胞分化过程中,CpG富集的EF1α启动子是否可能被甲基化,并导致CAR的转录下调。使用亚硫酸氢盐转化的基因组DNA作为模板,通过亚硫酸氢盐特异性PCR检查甲基化状态表明,与衍生自同一供体的常规CD19-CAR T细胞相比,iPSCCD19-CAR T细胞中的EF1α启动子甲基化状态显著增强(图14E)。在对含有23个CpG位点的EF1α启动子的245bp区域进行进一步的亚硫酸氢盐测序分析后,证实了这种高甲基化(图14F)。这些数据表明EF1α启动子高甲基化发生在T细胞从iPSC分化过程中,导致iPSC CD19-CAR T细胞中下调的CAR表达。
综上所述,iPSC CAR T细胞总体上具有与常规CAR T细胞相似的RNA表达签名,同时在稳定状态下的活性状态相对较低,这伴随着分化过程中转基因启动子高甲基化导致的较低CAR表达水平。
iPSC衍生的CAR T细胞的功能分析
我们接下来评估了REM扩增的iPSC CD19-CAR T细胞在体外裂解CD19表达性靶物的效应物功能。iPSC CAR T细胞介导了有力的针对CD19+3T3细胞(图15A)、NALM6细胞(图15B-15C)和Raji细胞(图15D)的CAR定向溶细胞活性,但不针对它们的CD19阴性对应物。我们使用PBMC衍生的CD19-CAR T细胞作为比较对照,该细胞是通过临床相关程序产生的,并且没有经过REM扩增。重要的是,iPSC CD19-CAR T细胞的杀伤活性与来自同一供体的常规PBMC衍生的CD19-CAR T细胞相当或更优,如通过iPSC CD19-CAR T细胞在低E:T比下对CD19+NALM6细胞表现出更有力的裂解活性所证明(图15E),并显示针对患者衍生的原代CD19+B-ALL细胞表现出相当的细胞毒性(图15F)。在CD19+肿瘤细胞刺激后,iPSC CD19-CAR T细胞也表现出有力的脱粒、细胞内IFNγ的表达、活化标志物CD25和CD137/4-1BB的表面表达以及抗原依赖性方式的Th1细胞因子释放(图15G-I)。有趣的是,在没有抗原刺激的情况下,来自iPSC CD19-CAR T细胞的上清液中GM-CSF和IFN-γ的水平远低于常规CD19-CAR T细胞中的水平(图15I),这与较低的基础ERK蛋白磷酸化水平一致(图15K),并且表明CAR紧张性信号传导的水平较低。此外,在用CD19+肿瘤细胞的系列攻击后,与常规CD19-CAR T细胞相比,iPSC CD19-CAR T细胞展示出PD-1、TIM-3和LAG-3表达降低,表明更少的耗竭表型。(图15J)。
接下来,我们探索了与亲本CD19+或CD19 KO NALM6细胞共培养时的CAR T细胞信号传导。iPSC CD19-CAR T细胞以与常规CD19-CAR T细胞相当的抗原特异性方式显示ERK1/2Thr202/Thr204和PLCγSer1248磷酸化(图15K)。然而,抗原刺激的iPSC CD19-CAR T细胞中的PLCγY783、ZAP70和内源性CD3ζ磷酸化水平高于抗原刺激的常规CD19-CAR T细胞,这支持了有力的细胞毒性活性。有趣的是,CD19-CAR T细胞中的内源性CD3ζY142和CAR相关的CD3ζ磷酸化均受到与CD19阴性NALM6细胞共培养的抑制,表明癌细胞的免疫抑制作用(图15K)。Western印迹分析还证实,iPSC CD19-CAR T细胞表达的CAR转基因水平远低于常规CAR T细胞(图15K),这与流式细胞术数据(图15H)一致。这也可以解释它们在不存在抗原的情况下如通过ERK磷酸化(图15K)和细胞因子分泌(图15I)测量的较低活化水平,因为已经显示较低的CAR表达有利于较低的紧张性(tonic)信号传导(Eyquem et al.,2017)。
与使用临床相关方法扩增的常规CAR T细胞相比,所公开的iPSC CD19-CAR T细胞产生的产品具有相当或更优的体外效应活性。
iPSC衍生的CAR T细胞的抗肿瘤功效
虽然在不存在抗原的情况下降低的CAR表达导致较少的活化(图14I和图15E),并且可能解释了在抗原攻击存在下较少的耗竭表型(图15J),iPSC CD19-CAR T细胞在体外似乎仍表现出稳健的抗原特异性细胞毒活性(图14A-F)。然而,为了更好地评估这些T细胞的抗肿瘤活性,我们接下来使用表达萤火虫萤光素酶的NALM6细胞在小鼠异种移植模型中进行体内治疗测定,以允许生物发光成像(图20A-20B中提供的图像)。在腹膜内(i.p.)肿瘤模型中,iPSC CD19-CAR T细胞的i.p.施用显著延迟了肿瘤进展(图16B)并显著延长了小鼠存活(P=0.004)(图16C)。iPSC CD19-CAR T细胞与人IL15分泌抚育细胞(NS0-hIL15)的组合进一步增强了这种治疗作用,导致5只小鼠中有3只完全治愈(图20A)。iPSC CD19-CAR T细胞的治疗益处也在更具侵袭性的静脉内(i.v.)小鼠肿瘤模型中得到证实(图16E-16F),再次显示显著改善的小鼠存活(P=0.0035)。总之,通过PSC-ATO培养系统从表达CAR的Tn/mem细胞产生的iPSC CD19-CAR T细胞在体内表现出有力的抗肿瘤功效。
讨论
使用胸腺外培养系统生成T细胞和CAR T细胞(无论它们是否单层或3D类器官共培养)是一种挑战(Maeda et al.,2016;Montel-Hagen et al.,2019;Vizcardo et al.,2018;Vizcardo et al.,2013;Zhao et al.,2007)。首次报道的由单层共培养系统生成的iPSC CAR T细胞显示出先天样表型(即CD8αα+),以及与常规CAR T细胞相比的不太有效的抗原特异性细胞毒性和细胞因子分泌(Themeli et al.,2013)。我们修改和优化了3D类器官培养系统,该系统促进了成熟和功能性CD3+CD8αβ+和CD3+CD4+常规T细胞和TCR转基因T细胞的生成(Montel-Hagen et al.,2019),并且我们首次证明了成功生成具有常规T细胞表型和CAR T细胞功能的iPSC CAR T细胞。具体而言,通过使用经过基因修饰以表达CAR的Tn/mem衍生的iPSC,以及用于驱动分化的PSC-ATO培养系统,生成了表达常规CD5+CD7+TCRαβ+TCRγδ-CD8αβ+T细胞表型的iPSC CAR T细胞,表现出有力的细胞毒性杀伤,以及与来自同一供体的常规CAR T细胞相当的Th1细胞因子分泌活性。此类改进验证了iPSC用于生成治疗性CAR T细胞产品的潜在效用。
同样有利的是,我们的Tn/mem衍生的iPSC CAR T细胞显示出更同质的单克隆TCR库,这与ESC衍生的T细胞中的多克隆表型不同(Montel-Hagen et al.,2019;Nishimura etal.,2013)。即使使用末端分化的效应T细胞来生成iPSC,也导致再生的CD8αβT细胞,其通过额外的TCR重排而失去其抗原特异性,只有在iPSC的TCR转导时才诱导TCR稳定性(Minagawaet al.,2018)。这显示从分化程度较低的Tn/mem群开始可能对再分化过程中的TCR重排具有独特作用,这可能与预先存在的TCR基因座的等位基因排斥效应有关,也可能不相关(Brady et al.,2010)。无论如何,选择已知和/或无害TCR的Tn/mem衍生的iPSC克隆以最小化与制造“现成的”iPSC CAR T细胞产品有关的潜在移植物抗宿主毒性。本文所述的所有公开的iPSC CAR T细胞和iPSC CAR NK细胞均可以照此使用。
我们的iPSC CAR T细胞上MHC的较低表达水平和CD8的优势也可能与从Tn/mem衍生的iPSC克隆开始的独特作用有关,或者可能与培养系统中缺乏胸腺上皮细胞有关(Vizcardo et al.,2018)。虽然低MHC表达对于在过继转移后减少T细胞介导的排斥和促进iPSC CAR T细胞的持久性可能是可取的,改善CD4+和CD8+群之间的平衡可能很重要,因为最近已经显示CD4+CAR T细胞可以在过继免疫细胞疗法中发挥重要作用(Wang et al.,2018)。通过在分化过程中操纵培养条件或通过基因编辑操纵谱系选择途径,可以获得更平衡的CD4/CD8谱系分化(Singer et al.,2008)。
我们首次证明iPSC CAR T细胞中CAR表达降低与EF1α启动子的高甲基化有关。虽然已知启动子甲基化调节基因表达(Hofmann et al.,2006),分化诱导的CAR转基因启动子高甲基化代表了一种可能调节CAR表达的新机制。降低CAR表达可能比以前认为的更可取,因为据报道,CAR的最佳基础低表达可以减少紧张性信号传导并维持CAR T细胞功能(Eyquem et al.,2017)。在我们的研究中,没有抗原刺激的iPSC CD19-CAR T细胞的基础pERK磷酸化水平和基础GM-CSF/IFNγ分泌水平远低于在常规CD19-CAR T细胞中看到的水平。相反,在遭遇抗原时,通过pERK、PLCγ(Y782)和ZAP70磷酸化测量的iPSC CD19-CAR T细胞的细胞信号传导以及Th1细胞因子分泌与常规CD19-CAR T细胞相当或更高,尽管CAR表达较低。有趣的是,虽然NALM6肿瘤细胞抑制了内源性和CAR相关CD3ζ序列的磷酸化(图15K),抗CD3或抗原包被珠尚未报道这种现象(Salter et al.,2018;Sun et al.,2020),针对CD19+NALM6肿瘤观察到的细胞毒活性(图15E)表明iPSC CD19-CAR T细胞比常规CD19-CART细胞更好地克服了这种抑制。这些数据一起表明iPSC CAR T细胞可能表现出对安全性和有效性均有益的抗原特异性谱。
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实施例4中的方法的参考文献:
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其他实施方案
应当理解,虽然本发明已经结合其详细描述进行了描述,但前述描述旨在说明而不是限制由所附权利要求的范围限定的本发明的范围。其他方面、优点和修改在以下权利要求的范围内。
Claims (27)
1.用于制备表达嵌合抗原受体(CAR)的T细胞或NK细胞的组合物的方法,所述方法包括:
(a)分离外周血单个核细胞(PBMC)、幼稚T(Tn)细胞、记忆T(Tmem)细胞、幼稚和记忆T细胞(Tn/mem)或其组合的群体;
(b)从所述PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合生成诱导多能干细胞(iPSC);
(c)使所述iPSC与编码所述CAR的载体接触,从而产生CAR iPSC;和
(d)将所述CAR iPSC分化为CAR T细胞或CAR NK细胞。
2.权利要求1所述的方法,其中所述PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合是人的或分离自人血液。
3.权利要求1所述的方法,其中所述PBMC细胞是CD14-、CD25-和CD26L+。
4.权利要求1所述的方法,其中通过使所述PBMC、Tn细胞、Tmem细胞或Tn/mem细胞或其组合与OCT3/4、OCT3、OCT4、SOX2、KLF4、L-MYC、C-MYC、LIN28或靶向TP53的短发夹RNA(shRNA-TP53)中的一种或多种接触而生成所述iPSC。
5.权利要求1所述的方法,其中所述iPSC是遗传修饰的。
6.权利要求5所述的方法,其中所述遗传修饰包括敲除一种或多种基因,其中所述一种或多种基因包括TRAC、TRBC、B2M、CIITA或其组合中的一种或多种。
7.权利要求5所述的方法,其中所述遗传修饰方法包括基因编辑、同源重组、非同源重组、RNA介导的遗传修饰、DNA介导的遗传修饰、锌指核酸酶、大范围核酸酶、TALEN或CRISPR/CAS9。
8.权利要求1所述的方法,其中将所述CAR iPSC分化为CAR T细胞或CAR NK细胞的步骤包括将所述表达CAR的iPSC分化为胚胎中胚层祖(EMP)细胞和将所述EMP分化为CAR T细胞。
9.权利要求8所述的方法,其中所述EMP细胞是CD56+和CD326-。
10.权利要求1所述的方法,其中将所述CAR iPSC分化成CAR T细胞或CAR NK细胞的步骤包括将所述表达CAR的iPSC分化成胚胎中胚层祖(EMP)细胞和将所述EMP分化成CAR NK细胞。
11.权利要求10所述的方法,其中所述EMP细胞是CD56+和CD326-。
12.权利要求1所述的方法,其中将所述CAR iPSC分化为CAR T细胞或CAR NK细胞的步骤包括将所述CAR iPSC分化为CD34+造血干细胞和祖细胞(HSPC)以及将所述HSPC分化为CAR T细胞。
13.权利要求1所述的方法,其中将所述CAR iPSC分化为CAR T细胞或CAR NK细胞的步骤包括将所述CAR iPSC分化为CD34+HSPC和将所述HSPC分化为CAR NK细胞。
14.权利要求1所述的方法,其中将所述CAR iPSC分化成CAR T细胞的步骤包括使用基于纳米纤维基质的培养系统。
15.权利要求1所述的方法,其中将所述CAR iPSC分化成CAR NK细胞的步骤包括使用基于纳米纤维基质的培养系统。
16.前述权利要求中任一项所述的方法,其中所述CAR对肿瘤和/或毒素具有特异性。
17.前述权利要求中任一项所述的方法,其中所述CAR靶向以下的任何一种或多种:碳酸酐酶IX(CAIX)、癌胚抗原(CEA)、CDS、CD6、CD7、CD10、CD19、CD20、CD22、CD30、CD33、CD34、CD38、CD41、CD44、CD49f、CD56、CD74、CD123、CD133、CD138、CS1、氯毒素受体、巨细胞病毒(CMV)感染细胞的抗原(例如,细胞表面抗原)、上皮糖蛋白(EGP2)、上皮糖蛋白-40(EGP-40)、上皮细胞粘附分子(EpCAM)、受体酪氨酸蛋白激酶erb-B2、3、4、叶酸结合蛋白(FBP)、胎儿乙酰胆碱受体(AChR)、叶酸受体、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、人表皮生长因子受体2(HER-2)、人端粒酶逆转录酶(hTERT)、白细胞介素-13受体亚基α-2(IL-13Rα2)、轻链激酶插入域受体(KDR)、Lewis A(CA19.9)、Lewis Y(LeY)、LI细胞粘附分子(LICAM)、黑素瘤抗原家族A,1(MAGE-AI)、粘蛋白16(Muc-16)、粘蛋白1(Muc-1)、间皮素(MSLN)、NKG2D配体、癌症-睾丸抗原NY-ESO-1、癌胎抗原(h5T4)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、肿瘤相关糖蛋白72(TAG-72)、血管内皮生长因子R2(VEGF-R2)、维尔姆斯瘤蛋白(WT-1)或其组合。
18.前述权利要求中任一项所述的方法,其中所述CAR是双特异性的。
19.前述权利要求中任一项所述的方法,其中所述嵌合抗原受体包含:至少一个靶向域、间隔物、跨膜域、共刺激域和CD3ζ信号传导域。
20.组合物,其包含前述权利要求中任一项所述的iPSC衍生的CAR T细胞或CAR NK细胞。
21.权利要求20所述的组合物,其中所述CAR T细胞包括辅助T细胞、细胞毒性T细胞、记忆T细胞、幼稚T细胞、调节性T细胞、自然杀伤T细胞或其组合中的一种或多种。
22.权利要求20所述的组合物,其中所述CAR T细胞包含CD3+、CD5+、CD7+和TCRαβ+。
23.增加患有癌症的受试者的存活的方法,其包括向所述患者施用权利要求20-22中任一项所述的组合物。
24.治疗患者中的癌症的方法,其包括向所述患者施用权利要求20-22中任一项所述的组合物。
25.减轻或改善患者中与癌症相关的症状的方法,其包括向所述患者施用权利要求20-22中任一项所述的组合物。
26.权利要求22-24中任一项所述的方法,其中局部或全身施用所述组合物。
27.权利要求22-24中任一项所述的方法,其中通过单次或重复给药来施用所述组合物。
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US20220362300A1 (en) | 2022-11-17 |
WO2021092252A1 (en) | 2021-05-14 |
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