CN117625543A - 工程化Th9细胞及其用途 - Google Patents
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Abstract
本公开提供用于癌症疗法的工程化Th9细胞以及包含其的组合物。
Description
技术领域
本公开属于免疫治疗领域。更具体地，本公开涉及一种用于癌症疗法的工程化Th9细胞以及包含其的组合物。
背景技术
近年来，细胞治疗作为一种新兴的肿瘤治疗手段，已经在临床中展现出非常显著的疗效。细胞治疗主要是对免疫细胞进行体外扩增，然后再回输给肿瘤患者，以达到治疗目的。然而，这种疗法仍然面临细胞耗竭较快，在患者体内持续性不够等问题，导致疗效下降。因此，如何提高免疫细胞的持续性是该领域长期面临的挑战之一。
本公开通过筛选特定的免疫细胞亚型，并表达特定的细胞因子，能够降低免疫细胞的耗竭，进而提高其肿瘤抑制效果。
发明内容
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本公开所属领域的普通技术人员通常所了解的相同。
本公开的目的在于提供一种工程化的Th9细胞，其表达特异性识别Claudin18.2的细胞表面分子，例如TCR、CAR等。与常规CAR-T细胞相比，该工程化的Th9细胞耗竭速度更低，扩增能力更强，具有更好的肿瘤抑制效果。
工程化的Th9细胞
在第一个方面，本公开提供一种工程化的Th9细胞，其表达特异性识别Claudin18.2的细胞表面分子。
Th9细胞于2008年首次被发现，属于CD4+T细胞的一种新亚型，其特征在于能够分泌IL-9。目前，Th9细胞与肿瘤的关系还不明确，一方面IL-9表达失调会导致某些自主细胞增殖和淋巴细胞的恶性转化，对肿瘤生长起促进作用。另一方面，Th9细胞最近也被证实具有抗肿瘤作用。Th9细胞这种在肿瘤中的双重作用可能与肿瘤类型及局部肿瘤微环境的差异有关。本公开首次发现，用Th9细胞代替传统的T细胞(即，未经分选的含有各种亚群(例如CD4+T细胞和CD8+T细胞)的T细胞)来构建靶向Claudin18.2的CAR-T细胞，在体外杀伤和小鼠模型方面，均显示出强烈的肿瘤杀伤活性。
在一个实施方案中，细胞表面分子选自嵌合抗原受体、T细胞受体、T细胞融合蛋白或T细胞抗原耦合器。如本文所用，术语“细胞表面分子”是指在细胞表面表达的能够与Claudin18.2特异性结合的分子。此类表面分子一般包含能够与Claudin18.2特异性结合的抗原结合区、将表面分子锚定在细胞表面的跨膜结构域，以及负责信号传递的胞内结构域。常见的此类表面分子的实例包括例如T细胞受体(TCR)、嵌合抗原受体(CAR)、T细胞融合蛋白(TFP)或T细胞抗原耦合器(TAC)。
如本文所用，术语“T细胞受体”或“TCR”是T细胞表面的特征性标志，以非共价键与CD3结合形成复合物。抗原呈递细胞通过主要组织相容性复合体分子(MHC)将抗原肽呈递至T细胞并且结合至TCR复合物以诱发一系列胞内信号传导。TCR由分别形成异二聚体的六条肽链组成，其一般分为αβ型和γδ型。每条肽链包括恒定区和可变区，其中可变区负责结合特异性的抗原和MHC分子。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括抗原结合区(例如抗体的抗原结合部分)、跨膜结构域和胞内结构域(包含共刺激结构域和/或初级信号传导结构域)，各个结构域之间通过接头连接。CAR能够利用单克隆抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。非MHC限制性的抗原识别给予CAR细胞与抗原处理无关的识别抗原的能力，因此绕过了肿瘤逃逸的主要机制。此外，当在T细胞内表达时，CAR有利地不与内源性T细胞受体(TCR)的α链和β链二聚化。
如本文所用，术语“T细胞融合蛋白”或“TFP”是指由TCR各组分衍生的重组多肽，通常由TCR亚基和与其连接的抗原结合区组成并在细胞表面表达。其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内信号结构域。
如本文所用，术语“T细胞抗原耦合器”或“TAC”包括三个功能结构域：1肿瘤靶向结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAM)磷酸化作为T细胞活化的初始步骤。
如本文所用，“抗原结合区”是指可以与抗原结合的任何结构或其功能性变体。抗原结合区可以是抗体结构，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其功能性片段。例如，抗原结合区包括但不限于IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单域抗体、纳米抗体、双体、anticalin、DARPIN等，优选选自Fab、scFv、单域抗体和纳米抗体。在本公开中，抗原结合区可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。
在一个实施方案中，所述细胞表面分子包含结合Claudin18.2的抗体。在一个实施方案中，所述抗体为完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb或纳米抗体。优选地，所述抗体包含如SEQ ID NO：1、2、3所示的CDR-1、CDR-2和CDR-3。优选地，所述抗体与SEQ ID NO：4具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，所述细胞表面分子是包含结合Claudin18.2的抗体的嵌合抗原受体，所述嵌合抗原受体还包含跨膜结构域和胞内结构域，其中所述胞内结构域包含共刺激结构域和/或初级信号传导结构域。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合受体多肽与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本公开中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154或它们的任意组合。优选地，所述跨膜结构域选自CD8α、CD4、CD28或CD278的跨膜结构域。更为优选地，所述跨膜结构域为CD8α或CD28跨膜结构域，与SEQ ID NO:5、6、7所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本公开的嵌合抗原受体还可以包含位于抗体和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗体提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以源自全部或部分的天然分子，如源自全部或部分的CD8、CD4或CD28的胞外区，或源自全部或部分的抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。优选地，所述铰链区包含CD8α、CD28或I给G4的铰链区部分，与SEQ ID NO:17、18、19或20所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
本公开的共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其可以包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本公开的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的共刺激信号传导结构域：TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70或其任意组合。优选地，所述共刺激结构域选自4-1BB、CD28、CD27、OX40、CD278或其任意组合。更为优选地，所述共刺激结构域为4-1BB或CD28，与SEQ ID NO:8、9或10所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本公开的嵌合抗原受体包含的初级信号传导结构域可以是T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。初级信号传导结构域包含两种不同类型的细胞质信号序列：引发抗原依赖性初级活化的那些，以及以不依赖抗原的方式起作用以提供次级或共刺激信号的那些。初级细胞质信号序列可以包含许多免疫受体酪氨酸激活基序(ITAM)。本公开的初级信号传导结构域包括单不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b、CD66d或其任意组合。优选地，所述初级信号传导结构域包含CD3ζ的信号传导结构域。更为优选地，所述初级信号传导结构域与SEQID NO:11、12或13所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一些实施方案中，本公开的CAR还包含信号肽，所述信号肽选自以下蛋白的信号肽：CD8α、IgG1、GM-CSFRα、IgG4或其任意组合。优选地，所述信号肽包含CD8α或B2M的信号肽。更为优选地，所述信号肽与SEQ ID NO:14或15所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一些实施方案中，本公开的CAR还包含接头，其用于隔开任何本文中所述的结构域/区。例如，接头可位于信号肽与抗体之间、抗体的VH与VL之间、抗体与铰链区之间、铰链区与跨膜结构域之间、侧接共刺激结构域或在共刺激结构域的N-或C-区上、和/或在跨膜结构域与初级信号传导结构域之间。接头可以是长度为约6至约40个氨基酸、或长度为约6至约25个氨基酸的肽。
在一个实施方案中，本公开的工程化Th9细胞还包含至少一种MHC相关基因表达被抑制或沉默，例如使至少一种MHC基因的表达被抑制或沉默，或使与至少一种MHC基因相互作用或调控其表达的基因的表达被抑制或沉默。
在一个实施方案中，MHC相关基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA或其任意组合，优选选自HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA或其任意组合。
在一个实施方案中，本公开的工程化免疫细胞还包含至少一种TCR/CD3基因的表达被抑制或沉默。
在一个实施方案中，使至少一种TCR/CD3基因表达被抑制或沉默是指使选自以下的一个或多个基因的表达被抑制或沉默：TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ。
在一个实施方案中，除了MHC相关基因和任选的TCR/CD3基因，本公开的工程化Th9细胞还可以包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的，包括但不限于例如通过大范围核酸分子酶、锌指核酸分子酶、TALE核酸分子酶或CRISPR系统中的Cas酶介导DNA断裂、或通过反义寡核苷酸、RNAi、shRNA等技术使基因失活。
在一个实施方案中，本公开的工程化Th9细胞不与除T细胞、NK细胞之外的其他免疫细胞，例如巨噬细胞、树突细胞等组合使用。优选地，本公开的工程化Th9细胞不与任何其他免疫细胞组合使用。
在一个实施方案中，本公开的工程化的Th9细胞还表达一种或多种外源性的细胞因子，所述细胞因子选自IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、TSLP、XCL1、XCL2、CXCL9、CXCL10、CXCL11、CXCL12、CX3CL1、CCL2、CCL3、CCL4、CCL5、CCL7、CCL8、CCL12、CCL13、CCL19、CCL21。优选地，所述细胞因子选自TSLP、IL7、XCL1和XCL2。更优选地，所述细胞因子是TSLP，或IL7和XCL1的组合，或IL7和XCL2的组合，或IL7、XCL1和XCL2的组合。
组合物
本公开还提供一种组合物，其包含本公开所述的工程化Th9细胞作为活性剂，和一种或多种药学上可接受的赋型剂。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本公开期望的药物组合物。用于本公开的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本公开的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本公开的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimersodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreateglucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如IL-2，趋化因子比如IL-8、血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。
用途
本公开还提供上述工程化Th9细胞或药物组合物在制备治疗/预防/诊断癌症、感染或自身免疫性疾病药物中的用途。
在一个实施方案中，所述疾病是与Claudin18.2表达有关的癌症。例如，所述癌症包括但不限于：食管癌、胃肠癌、胰腺癌、甲状腺癌、结直肠癌、肾癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌、胃食管交界处(GEJ)腺癌、头颈癌、膀胱癌、乳腺癌、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸癌、生殖细胞癌、骨癌、皮肤癌、胸腺癌、胆管癌、胆囊癌、黑素瘤、间皮瘤、淋巴瘤、骨髓瘤(例如多发性骨髓瘤)、肉瘤、神经胶质母细胞瘤、白血病、畸胎瘤、成神经细胞瘤、神经胶质瘤、直肠癌、子宫内膜癌、肾上腺癌、脑癌、结肠癌、头颈癌、淋巴结癌、耳鼻喉(ENT)癌，优选选自胃癌、胃食管交界处(GEJ)腺癌、食管癌、胃肠癌、胰腺癌、肺癌等。
下面将参考附图并结合实施例来详细说明本公开。需要说明的是，本领域的技术人员应该理解本公开的附图及其实施例仅仅是为了举例，并不能对本公开构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1示出了各种CAR-T细胞中的CAR表达水平。
图2示出了各种CAR-T细胞的扩增水平。
图3示出了各种CAR-T细胞的LAG3表达水平。
图4示出了各种CAR-T细胞的IL9释放水平。
图5示出了各种CAR-T细胞对靶细胞的杀伤活性。
图6示出了经各种CAR-T细胞处理的胃癌小鼠的体重变化。
图7示出了经各种CAR-T细胞处理的胃癌小鼠的肿瘤负荷变化。
具体实施方式
实施例1.制备CAR-T细胞
1.1构建载体
构建MSCV-cCAR质粒，其包含编码以下蛋白的核酸序列：Claudin18.2-VHH(SEQ IDNO：4)、CD8α铰链区(SEQ ID NO：19)、CD8α跨膜区(SEQ ID NO：7)、41bb胞内域(SEQ ID NO：10)和CD3ζ胞内域(SEQ ID NO：13)。
在上述MSCV-cCAR质粒中进一步包含编码IL-7(SEQ ID NO：26)和XCL1(SEQ IDNO：27)的核酸序列，其通过T2A(SEQ ID NO：21)的编码序列连接，获得MSCV-71CAR质粒。
在上述MSCV-cCAR质粒中进一步包含通过T2A(SEQ ID NO：21)连接的TSLP(SEQ IDNO：22)，获得MSCV-TSCAR质粒。
1.2制备CAR-T细胞
将PBMC用 CD4+T cell分离试剂盒(Miltenyi Biotec)分选后，获得初始CD4+T细胞。在含有10ng/ml IL-4、1ng/ml TGF-β1、10μg/ml anti-human IFNγ的X-VIVO15培养基中培养10天，获得Th9细胞。
将MSCV-cCAR质粒、MSCV-71CAR质粒和MSCV-TSCAR质粒包装入逆病毒后，分别转染激活的T细胞或Th9细胞，获得六种细胞：cCAR-T细胞、71CAR-T细胞、TSCAR-T细胞、cCAR-Th9细胞、71CAR-Th9细胞和TSCAR-Th9细胞。未转染病毒的T细胞或Th9细胞作为阴性对照(NT或NTh9)。
取2×105个CAR-T细胞，用Anti-Camelid VHH-iFluor 488(金斯瑞)作为流式检测抗体，通过流式细胞术检测CAR的表达水平，结果如图1所示。可以看出，各种CAR-T细胞上的CAR均可有效表达。
实施例2.CAR-T细胞的功能验证
此外，通过细胞计数检测CAR-T细胞的增殖能力，结果如图2所示。可以看出，CAR-Th9细胞的增殖能力均高于CAR-T细胞，其中，71CAR-Th9细胞的增殖能力最强，TSCAR-Th9和cCAR-Th9细胞次之。
通过流式细胞术检测LAG3的表达水平，从而评估细胞的耗竭状态，结果如图3所示。可以看出，与CAR-T细胞相比，CAR-Th9细胞的LAG3表达水平显著降低，表明CAR-Th9的耗竭水平更低，能够持续的时间更长。
在96孔圆底板中以2×105个细胞/100μl的浓度分别加入NT细胞和各种CAR-T细胞，然后在各孔中以1×104个细胞/100μl的浓度分别加入靶标NUGC4-Claudin18.2细胞(表达human Claudin18.2的NUGC4细胞)或非靶标NUGC4细胞。于37℃培养14h后，收集培养物上清液，并用Human IL-9DuoSet ELISA试剂盒(R&D)检测培养物上清液中IL-9的表达水平。结果如图4所示。可以看出，与CAR-T细胞相比，CAR-Th9细胞与靶标细胞共培养后释放的IL9水平显著升高，这更有助于招募体内其他免疫细胞。
以1×104个细胞/100μl/孔的浓度将靶细胞NUGC4-Claudin18.2-luci细胞铺入96孔板中，然后以20：1、10：1、5：1和1:1的效靶比将各CAR-T细胞和CAR-Th9细胞铺入到96孔板进行共培养，8小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图5所示。可以看出，CAR-T细胞和CAR-Th9细胞的体外杀伤活性相当。
实施例3.CAR-T细胞的肿瘤抑制效果验证
在健康NPI小鼠的左前肢腋下部位，经皮下接种4×106个NUGC4-Claudin18.2胃癌细胞。将接种了胃癌细胞的小鼠随机分为5组，每组6只。待肿瘤体积生长至100mm3时，向各组小鼠经尾静脉注射PBS或1×106个71CAR-T细胞、TSCAR-T细胞、71CAR-Th9细胞以及TSCAR-Th9细胞。监测小鼠的体重和肿瘤体积变化，直至实验结束。
小鼠的体重变化如图6所示。可以看出，施用CAR-T细胞后，各组小鼠的体重与对照组相比没有显著差异，且在观察周期中，当小鼠肿瘤未超过1500mm3时，小鼠行动活泼，毛色正常，这表明，施用CAR-T细胞不会对小鼠有明显的毒副反应。
小鼠的肿瘤体积变化如图7所示。可以看出，71CAR-Th9细胞的抗肿瘤作用显著优于71CAR-T细胞，TSCAR-Th9细胞显著优于TSCAR-T细胞，表明与常规的T细胞相比，Th9细胞搭载的靶向Claudin18.2的CAR具有更好的肿瘤抑制效果。
需要说明的是，以上仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。本领域技术人员理解的是，凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
Claims (17)
1.一种工程化的Th9细胞，其表达特异性识别Claudin18.2的细胞表面分子。
2.根据权利要求2所述的Th9细胞，其还表达一种或多种外源性的细胞因子，所述细胞因子选自TSLP、IL7、XCL1和XCL2。
3.根据权利要求2所述的Th9细胞，其中所述细胞因子是TSLP。
4.根据权利要求2所述的Th9细胞，其中所述细胞因子是IL7和XCL1的组合，或IL7和XCL2的组合，或IL7、XCL1和XCL2的组合。
5.根据权利要求1所述的Th9细胞，其中所述细胞表面分子选自嵌合抗原受体、T细胞受体、T细胞融合蛋白或T细胞抗原耦合器。
6.根据权利要求1所述的Th9细胞，其中所述细胞表面分子包含结合Claudin18.2的抗体。
7.根据权利要求6所述的Th9细胞，其中所述抗体为完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb或纳米抗体。
8.根据权利要求6所述的Th9细胞，其中所述抗体包含如SEQ ID NO：1、2、3所示的CDR-1、CDR-2和CDR-3。
9.根据权利要求6所述的Th9细胞，其中所述抗体与SEQ ID NO：4具有至少90％同一性。
10.根据权利要求1所述的Th9细胞，其中所述细胞表面分子是包含结合Claudin18.2的抗体的嵌合抗原受体，所述嵌合抗原受体还包含跨膜结构域和胞内结构域，其中所述胞内结构域包含共刺激结构域和/或初级信号传导结构域。
11.根据权利要求10所述的Th9细胞，其中所述共刺激结构域选自以下蛋白质的共刺激信号传导结构域：LTB、CD94、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134、CD137、CD270、CD272、CD276、CD278、CD357、DAP10、DAP12、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM、ZAP70或其任意组合。
12.根据权利要求10所述的Th9细胞，其中所述初级信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b、CD66d或其任意组合。
13.根据权利要求10所述的Th9细胞，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154或其任意组合。
14.根据权利要求1所述的Th9细胞，其中所述Th9细胞的至少一种MHC相关基因和/或至少一种TCR/CD3相关基因的表达被抑制或沉默。
15.根据权利要求14所述的Th9细胞，所述MHC相关基因选自HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA或其任意组合，优选HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA或其任意组合；所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ或其任意组合。
16.一种药物组合物，其中包含权利要求1-15任一项所述的Th9细胞和一种或多种药学上可接受的载体和/或赋型剂。
17.权利要求1-15任一项所述Th9细胞或权利要求16所述药物组合物在制备治疗/预防/诊断癌症的药物中的用途。
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