CN116426483A - Cd258蛋白在免疫治疗中的应用 - Google Patents
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- CN116426483A CN116426483A CN202111655106.1A CN202111655106A CN116426483A CN 116426483 A CN116426483 A CN 116426483A CN 202111655106 A CN202111655106 A CN 202111655106A CN 116426483 A CN116426483 A CN 116426483A
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Abstract
本发明涉及CD258蛋白或其功能突变体用于促进免疫细胞增殖的应用。
Description
技术领域
本发明涉及免疫学领域。具体地,本发明涉及CD258蛋白或其功能突变体的一种或多种在免疫治疗中的应用。
背景技术
嵌合抗原受体(Chimeric antigen receptor,CAR)T细胞疗法是近年来发展十分迅速的新一代肿瘤免疫治疗技术。CAR的结构主要由特异性识别肿瘤相关抗原((Tumorassociated antigen,TAA)的单链可变区结构域(Single chain variable 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,即VH-Linker-VL或VL-Linker-VH;铰链区通常由免疫球蛋白超家族组成,如IgG4、CD8、IgG1等;跨膜区一般由CD8、CD28或CD4等组成;胞内信号传导区主要由CD3ζ链和协同共刺激信号分子41BB、CD28、ICOS、OX40等组成。因此表达CAR的T细胞不需要通过抗原递呈机制,可直接识别TAA,具有MHC(Major histocompatibility complex)非依赖性的独特优势。
由于CAR-T技术在血液肿瘤治疗上表现出色,以CD19为靶点,CART治疗急性和慢性B淋巴细胞白血病更是取得惊人疗效,但对实体瘤的治疗临床疗效十分有限,亟需需新的解决方案。
Sirtuins是一类依赖于NAD+和核心区域高度保守的蛋白去乙酰化酶和ADP核糖基转移酶,在组蛋白的乙酰化/去乙酰化基因表达调控中起重要作用。哺乳动物中主要有七种Sirtuins蛋白亚型,即SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白,有时以“SIRT1-7”表示7种SIRT蛋白。SIRT1蛋白、SIRT6蛋白和SIRT7蛋白定位于细胞核,主要调节转录、末端着丝粒染色质结构、衰老以及代谢等生物学过程。此外,SIRT1蛋白在胞浆和线粒体中也存在。SIRT2蛋白主要定位于胞浆,与胞浆的微管蛋白相互作用,是调控细胞凋亡的重要因子之一;SIRT3蛋白、SIRT4蛋白和SIRT5蛋白定位于线粒体内,主要调节细胞能量代谢过程中关键蛋白的乙酰化修饰,并在细胞氧化磷酸化、三羧酸循环、脂肪酸有氧氧化及氨基酸降解等生物学过程中发挥着重要作用。
CD258蛋白又名肿瘤坏死因子超家族成员14(Tumor necrosisfactorsuperfamily member 14,TNFSF14)、T细胞上可诱导表达的、与单纯疱疹病毒糖蛋白竞争结合单纯疱疹病毒侵入受体的淋巴毒素同源类似物[Homologous to lymphotoxin,inducible expression,competes with herpes simplex virus(HSV)glycoprotein Dfor HSV entry mediator(HVEM),a receptor expressed on T lymphocytes,LIGHT]或HVEM配体(Herpesvirusentry mediator-ligand,HVEM-L)。CD258蛋白主要表达在活化的T细胞、B细胞、自然杀伤(Natural killer,NK)细胞、未成熟的树突状细胞(Immaturedendritic cells,im DC)和单核细胞表面,有3种存在形式:全长型包含240个氨基酸残基、相对分子质量29ku的2型跨膜糖蛋白;由204个氨基酸残基组成、缺少跨膜区并仅激活T淋巴细胞的非糖基化蛋白及细胞表面金属酶剪切作用下可溶性的CD258蛋白。CD258蛋白介导的信号通路与炎症性及自身免疫性疾病的发生、发展、移植物抗宿主病、病原体感染以及肿瘤的免疫调节有密切关系。
发明内容
本发明解决的技术问题为SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种在免疫治疗中的应用,具体表现为促进记忆性T细胞的形成,抑制免疫细胞免疫负调控蛋白的表达,增强免疫细胞细胞因子的释放,增强免疫细胞对肿瘤细胞的杀伤能力,以及调动机体自身抗肿瘤免疫反应,解决肿瘤异质性问题,预防肿瘤的复发。
具体地,一方面,本申请提供了一种促进免疫细胞增殖的方法,其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量。
一方面,本申请提供了一种促进记忆性免疫细胞产生的方法,其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量,从而促进所述免疫细胞分化为记忆性免疫细胞。
一方面,本申请提供了一种抑制免疫细胞分化的方法,其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量,从而抑制所述免疫细胞分化为分化型免疫细胞。
一方面,本申请提供了一种抑制免疫细胞免疫负调控蛋白表达的方法,其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量,从而抑制所述免疫细胞免疫负调控蛋白表达。在某些实施方式中,所述免疫负调控蛋白选自PD1、PDL1、TIM3和LAG3组成的组。
一方面,本申请提供了一种增强免疫细胞释放细胞因子的方法,其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
在某些实施方式中,所述细胞因子选自白介素、干扰素和/或肿瘤坏死因子。在某些实施方式中,所述细胞因子选自IL-2、IL4、IL6、IL7、IL10、IL12、TNF-α和/或IFNγ。
一方面,本申请提供了一种增强免疫细胞对肿瘤杀伤能力的方法,其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白蛋白或其功能突变体、SIRT1-7蛋白或其功能突变体的联合、以及SIRT1-7蛋白或其功能突变体与CD258蛋白或其功能突变体的联合的表达量上调。
一方面,本申请提供了一种解决肿瘤异质性的方法,所述方法包含:向受试者施用免疫细胞,其中所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
一方面,本申请提供了一种预防受试者中肿瘤复发的方法,所述方法包含:向受试者施用免疫细胞,其中所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
一方面,本申请提供了一种治疗有需要的受试者中的肿瘤的方法,其包含以下步骤:向所述受试者施用免疫细胞,其中所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
在某些实施方式中,所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
在某些实施方式中,所述方法包括体内方法和体外方法。
在本发明的一个或多个实施方式中,所述SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体表达量的上调。
在发明的一个或多个实施方式中,所述免疫细胞为淋巴细胞。在某些实施方式中,所述免疫细胞为T细胞、B细胞、自然杀伤(Natural killer,NK)细胞、未成熟的树突状细胞(Immature dendritic cells,im DC)、单核细胞和巨噬细胞。在某些实施方式中,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)。在某些实施方式中,所述TSCM为CCR7+和/或CD62L+。在某些实施方式中,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+。
在本申请中,术语“记忆性免疫细胞”通常是指具备免疫记忆的免疫细胞。所述免疫记忆可以指对某一抗原产生特异性识别及应答后,再次遇到同一抗原能够产生快速和强烈的免疫应答。在本申请中,所述记忆性免疫细胞可以包括记忆型T细胞。所述记忆型T细胞可以分为记忆性干细胞样T细胞(TSCM)和中央记忆性T细胞(TCM)。
在本申请中,术语“分化型免疫细胞”通常是指具有一定分化程度的免疫细胞。例如,所述分化型免疫细胞可以为具备一定分化程度的T细胞。在本申请中,所述分化型免疫细胞可以通过培养所述免疫细胞使其分化至一定程度而获得。例如,所述分化型免疫细胞可以包含调节性T细胞(Treg)。
在本申请中,术语“调节性T细胞”(Regulatory T cell,Treg)通常是指一群具有负调节机体免疫反应的淋巴细胞。所述调节性T细胞的分子标记可以为种转录因子Foxp3+或CD127-。在本申请中,所述调节性T细胞可被分为自然存在和诱导产生两类。其中自然存在为CD4+CD25+细胞,诱导产生为TR1细胞和TH3细胞。
在本申请中,术语“记忆性干细胞样T细胞”(T memory stem cells,TSCM)通常是指通常是指处于记忆性T细胞早期分化阶段,具有干细胞特征,具有较强的多向分化潜能的细胞。TSCM细胞在应答抗原刺激后,能够分化为中央记忆性T细胞(Central memory Tcells,TCM)、效应记忆性T细胞(Effector memory T cells,TEM)和效应性T细胞(EffectorT cells,TEF)。
在本申请中,术语“中央记忆性T细胞”(Central memory T cells,TCM)通常是指是幼稚T细胞(Naive T Cell)经过抗原激活后,产生的具有长期记忆性的T细胞。所述TCM的生物标记可以包含CD62L+和CD45RO+。所述中央记忆性T细胞能够通过淋巴屏蔽,回归淋巴结,同时处于被抗原激活的状态。
在某些实施方式中,所述免疫细胞选自经遗传修饰的免疫细胞,且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)或T细胞受体(TCR)。在某些实施方式中,所述经遗传修饰的免疫细胞为经遗传修饰的T细胞。
在某些实施方式中,所述TCR包含选自下组的亚基:TCRα、TCRβ、TCRγ和TCRδ。
在某些实施方式中,所述TCR的亚基包括特异性结合和/或识别肿瘤抗原的胞外域可变区。在某些实施方式中,胞外域可变区选自以下组:TCRα可变区片段Vα、TCRα可变区片段Jα、TCRβ可变区片段Vβ、TCRβ可变区片段Dβ和TCRβ可变区片段Jβ。
在某些实施方式中,所述胞外域可变区特异性结合和/或识别选自下组的靶标:MAGEA家族成员、CTA家族成员、HPV病毒和酪氨酸酶。
在某些实施方式中,所述胞外域可变区特异性结合和/或识别选自下组的靶标:MAGEA3、MAGEA4、NY-ESO-1、MART1、HPV16-E6和黑素瘤抗原酪氨酸酶。
在某些实施方式中,所述CAR包含细胞内结构域,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。
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(优选核苷酸序列如SEQ ID NO:11所示,氨基酸序列如SEQ ID NO:83所示)、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:83所示的序列或与其具备至少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%同源性的序列,所述同源序列仍具备SEQ ID NO:83所示序列功能,或由其组成。
术语“信号传导结构域”通常是指是指选自CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(Fc Epsilon R1b)、CD79a、CD79b、FcγRIIa、DAP10和DAP12的蛋白质的功能性信号传导结构域。在本申请中,所述信号传导结构域可以包括:CD3ζ、CD3δ和CD3ε。
在某些实施方式中,所述共刺激结构域选自以下各项组成的组:CD27的共刺激结构域、CD28的共刺激结构域(优选核苷酸序列如SEQ ID NO:9所示,氨基酸序列如SEQ IDNO:81所示)和4-1BB的共刺激结构域(优选核苷酸序列如SEQ ID NO:10所示,氨基酸序列如SEQ ID NO:82所示)。在某些实施方式中,所述共刺激结构域包含下述任一项所示的序列或与其具备至少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%同源性的序列,或由其组成:SEQ ID NO:81和SEQ ID NO:82所述同源序列仍具备SEQ ID NO:81或82所示序列功能。
术语“共刺激结构域”通常是指选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、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、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。在本申请中,所述共刺激结构域可以包括:CD27、CD28和4-1BB。
在某些实施方式中,所述CAR包含铰链区。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(优选核苷酸序列如SEQ IDNO:7所示,氨基酸序列如SEQ ID NO:79所示)。在某些实施方式中,所述铰链区包含SEQ IDNO:79所示的序列或由其组成。
术语“铰链区”通常是指免疫球蛋白重链CHl和CH2功能区之间的区域。铰链区为位于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CAR包含跨膜区。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(优选核苷酸序列如SEQ ID NO:8所示,氨基酸序列如SEQ IDNO:80所示)、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 IDNO:8所示的序列或由其组成。
术语“跨膜区”通常是指跨膜区连接胞外抗原结合域和胞内信号域,一般由二聚体膜蛋白组成,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CAR包含靶向部分。在某些实施方式中,所述靶向部分为scFv。优选地,所述靶向部分选自针对GPC3的scFv,针对CD19的scFv,针对BCMA的scFv,针对MSLN的scFv,针对HER2的scFv。
在一些实施方案中,所述针对GPC3的scFv包含SEQ ID NO:35所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:36所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:37所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:38所示)和包含SEQ ID NO:39所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:40所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:41所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:42所示),更优选地,针对GPC3的scFv包含如SEQ ID NO:2所示的核苷酸序列或如SEQ ID NO:34所示的氨基酸序列,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%同源性的序列,或由其组成,所述同源序列仍具备SEQID NO:2或34所示序列功能。
在一些实施方案中,所述针对CD19的scFv包含SEQ ID NO:44所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:45所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:46所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:47所示)和包含SEQ ID NO:48所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:49所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:50所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:51所示),更优选地,针对CD19的scFv包含如SEQ ID NO:3所示的核苷酸序列或如SEQ ID NO:43所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ IDNO:3或43所示序列功能。
在一些实施方案中,所述针对BCMA的scFv包含SEQ ID NO:53所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:54所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:55所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:56所示)和包含SEQ ID NO:57所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:58所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:59所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:60所示),更优选地,针对BCMA的scFv包含的如SEQ ID NO:4所示核苷酸序列或如SEQ ID NO:52所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ IDNO:4或52所示序列功能。
在一些实施方案中,所述针对MSLN的scFv包含SEQ ID NO:62所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:63所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:64所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:65所示)和包含SEQ ID NO:66所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:67所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:68所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:69所示),更优选地,针对MSLN的scFv包含如SEQ ID NO:5所示的核苷酸序列或如SEQ ID NO:61所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ IDNO:5或61所示序列功能。
在一些实施方案中,所述针对HER2的scFv包含SEQ ID NO:71所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:72所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:73所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:74所示)和包含SEQ ID NO:75所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:76所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:77所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:78所示),更优选地,针对HER2的scFv包含如SEQ ID NO:6所示的核苷酸序列或如SEQ ID NO:70所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ IDNO:6或70所示序列功能。
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、BCMA、HER2、Mesothelin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序列:SEQ ID NO:2、3、4、5、6、34、43、52、61或70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%同源性的序列,或由其组成,所述同源序列仍具备SEQ IDNO:2、3、4、5、6、34、43、52、61或70所示序列功能。
术语“肿瘤抗原”通常是指是指在肿瘤细胞内或由肿瘤细胞产生的抗原物质,其可能具有在宿主中触发免疫应答的能力。例如,肿瘤抗原可以是构成肿瘤细胞的一部分并且能够诱导肿瘤特异性细胞毒性T淋巴细胞的蛋白质,多肽,肽或其片段。肿瘤抗原肽可以是由于肿瘤细胞中的肿瘤抗原降解而产生的肽,并且可以通过结合HLA分子在细胞表面上表达后诱导或激活肿瘤特异性细胞毒性T淋巴细胞。在一些实施方案中,术语“肿瘤抗原”还可以指在癌细胞上专门或优先或差异表达和/或与癌细胞相关的生物分子(例如,蛋白质,碳水化合物,糖蛋白等)从而提供癌症优先或特异性的靶标。例如,与生物体中的任何其他细胞相比,优先表达可以为常规的优先表达,或者在生物体的特定区域内的优先表达(例如在特定器官或组织内)。例如,所述肿瘤抗原可以包括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、muthsp702、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中加入一种或多种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各项组成的组:针对B淋巴细胞表面抗原的抗体、抗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各项组成的组:抗CD3抗体、抗CD28抗体、抗4-1BB抗体、抗CD80抗体、抗CD86抗体、PHA、PMA和离子霉素。
在某些实施方式中,所述T细胞刺激因子为抗CD3抗体,且所述抗CD3抗体的浓度为1-10000ng/mL。在某些实施方式中,所述T细胞刺激因子为抗CD28抗体,且所述抗CD28抗体的浓度为1-10000ng/mL。
在某些实施方式中,所述方法还包括:向经分离的所述PBMC中加入一种或多种细胞因子。
在某些实施方式中,所述细胞因子为白细胞介素。
术语“白细胞介素”通常是指能够促进T和/或B淋巴细胞和/或造血细胞的发育和分化的分泌蛋白或信号分子。白介素可以由辅助CD4T淋巴细胞,以及通过单核细胞,巨噬细胞和内皮细胞合成。如本文所用,白介素(IL)可以包括IL-1,IL-2,IL-3,IL-4,IL-5,IL-6,IL-7,IL-8,IL-9,IL-10,IL-11,IL-12,IL-13,IL-14,IL-15,IL-16,IL-17,IL-18,IL-19,IL-20,IL-21,IL-22,IL-23,IL-24,IL-25,IL-26,IL-27,IL-28,IL-29,IL-30,IL-31,IL-32,IL-33,IL-34,IL-35和/或IL-36。如本文所用,术语“白细胞介素”可以包括全长白介素或片段(例如截短形式)或其变体,其基本上保持相应野生型白细胞介素的生物学活性(例如,具有生物活性至少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%,至少99%,或甚至100%的相应野生型白细胞介素的生物活性)。本文所用的白细胞介素可以来自任何哺乳动物物种。在某些实施方式中,白细胞介素来自选自人、马、牛、鼠、猪、兔、猫、狗、大鼠、山羊、绵羊和非人灵长类动物的物种。在某些实施方式中,白细胞介素可以是突变形式。例如,白细胞介素可以是超IL-2(也称为sIL2,参见Nature 484,529-533,2612),其可以通过修饰IL-2以增加其对IL-2Rβ的结合亲和力而获得。sIL-2中的突变主要是细胞因子的核心,分子动力学模拟表明,进化型突变使IL-2稳定,将IL-2Rβ结合位点中螺旋的灵活性降低到类似于优化的受体结合构象当绑定到CD25。与IL-2相比,sIL-2诱导细胞毒性T细胞的优异扩增,导致体内抗肿瘤反应改善,引起T调节细胞扩张少,肺水肿减少。例如,在本申请中,所述细胞因子可以包含IL-2、IL4、IL6、IL7、IL10、IL21、TNF-α和/或IFNγ。
在某些实施方式中,所述白细胞介素选自下组的一种或多种:IL2、IL21、IL7和IL15。在某些实施方式中,所述白细胞介素为IL2,且所述IL2的浓度为0.1-10000U/mL。在某些实施方式中,所述白细胞介素为IL21,且所述IL21的浓度为0.01-1000ng/mL。在某些实施方式中,所述白细胞介素为IL7,且所述IL7的浓度为0.01-1000ng/mL。在某些实施方式中,所述白细胞介素为IL15,且所述IL15的浓度为0.01-1000ng/mL。
在某些实施方式中,所述SIRT1-7或其功能突变体、CD258或其功能突变体来源于人。
SIRT1蛋白有2条核定位信号(SEQ ID NO:105所示的PLRKRPRR和SEQ ID NO:106所示的PPKRKKRK),优选的突变为任一氨基酸置换为A或缺失,更优选的突变体为SEQ ID NO:107所示的PLRKRPAA和SEQ ID NO:108所示的PPKRAAAA);优选的缺失突变为SEQ ID NO:105所示的PLRKRPRR和SEQ ID NO:106所示的PPKRKKRK的完全缺失;SIRT1蛋白有2条核出核信号(SEQ ID NO:109所示的LLLTDGLL和SEQ ID NO:110所示的VDLLIVI),优选的突变体为任一氨基酸置换为A或缺失,更优选的突变体为SEQ ID NO:111所示的AAATDGAA和SEQ ID NO:112所示的ADAAAAA(参见THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.282,NO.9,pp.6823–6832),优选的缺失突变为SEQ ID NO:109所示的LLLTDGLL和SEQ ID NO:110所示的VDLLIVI的完全缺失。通过对核定位信号和核出核信号的改变,调节蛋白在细胞质以及细胞核的表达量,进而调节蛋白的功能活性。
SIRT1序列为原始序列(UniProtKB编号:Q96EB6-1)截短体,由原始序列的小分子Sirtuin激活剂结合区(Small molecule sirtuin-activating compounds bindingdomain,SBD,原始序列第183-229位),去乙酰化酶区(Deacetylase domain,原始序列第229-516位)和C端调控区(C-terminal regulatory segment,CTR,原始序列第641-665位)依次拼接构成;SIRT2-7为原始序列,参见https://www.uniprot.org/uniprot/Q96EB6。
在某些实施方式中,所述SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白分别包含下述任一项所示的序列:SEQ ID NO:15-21或SEQ IDNO:86-92,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%,至少99%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:15-21或SEQ ID NO:86-92任一项所示序列功能。
在某些实施方式中,所述SIRT1-7蛋白的功能性突变体在所述SIRT1-7蛋白的选自下组的结构域进行突变:去乙酰化酶区(Deacetylase domain),小分子Sirtuin激活剂结合区(Small molecule sirtuin-activating compounds binding domain,SBD),和C端调控区(C-terminal regulatory segment,CTR)。
在某些实施方式中,所述SIRT1功能突变体包含下述任一项所示的序列:SEQ IDNO:22-30或SEQ ID NO:93-101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%,至少99%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:22-30或SEQ ID NO:93-101任一项所示序列功能。
在某些实施方式中,所述CD258蛋白包含SEQ ID NO:31或SEQ ID NO:102所示的序列或与其具备至少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%、至少99%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:31或SEQ ID NO:102所示序列功能。所述CD258蛋白功能突变体包括膜结合型CD258(优选序列如SEQ ID NO:32或SEQ ID NO:103所示)、分泌型CD258(优选序列如SEQ ID NO:33或SEQ ID NO:104所示)和CD258的胞内区。在某些实施方式中所述膜结合型CD258包括所述CD258蛋白水解位点(Proteolytic site)的缺失或定点突变。蛋白水解位点为SEQ ID NO:102所示序列的第82-83位的QL位点,优选地,所述缺失突变为CD258蛋白水解位点的完全缺失或在SEQ ID NO:102所示序列中第81-84位的EQLI缺失;所述置换突变为Q和/或L氨基酸置换为A,更优选地,QL氨基酸被置换为AA。通过对该蛋白水解位点进行置换突变或缺失,使该蛋白始终以膜结合形式表达在细胞膜上,减少分泌形式的表达(参见https://www.uniprot.org/uniprot/O43557)。
在某些实施方式中,所述SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体包含下述任一项所示的序列:SEQ ID NO:15-33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%,至少99%同源性的序列,所述同源序列仍具备SEQ ID NO:15-33任一项所示序列功能,优选包含下述任一项所示的序列:SEQ ID NO:86-104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%,至少99%同源性的序列,所述同源序列仍具备SEQ ID NO:86-104任一项所示序列功能。
另一方面,本申请提供了一种经遗传修饰的免疫细胞,其中所述遗传修饰使得该免疫细胞中SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体、以及其组合的表达量上调。
在某些实施方式中,所述表达量上调通过以下方式实现:
(1)通过向所述免疫细胞中添加增加所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的表达的激活剂以刺激所述免疫细胞自身增加所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的表达,优选所述激活剂选自以下各项组成的组:SRT2104(CAS号1093403-33-8,化学式C26H24N6O2S2);CAY10602(CAS号374922-43-7,化学式C22H15FN4O2S);OSS-128167(CAS号887686-02-4,化学式C19H14N2O6);
和/或
(2)通过向所述免疫细胞中转染包含编码所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的核酸的表达载体以增加所述免疫细胞中所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的量,优选地,所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体、和CD258蛋白或其功能突变体以单体形式存在或通过连接元件连接的缀合物的形式存在。在某些实施方式中,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,优选地,所述载体进一步包含CAR,更优选地,所述CAR通过连接元件连接调节单元,所述调节单元选自所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体、或其组合。
术语“2A序列”通常是指一段不依赖于蛋白酶的自剪切氨基酸序列。所述2A序列可以有助于转录产生两种蛋白。
在某些实施方式中,所述连接元件为2A序列,选自下列各项组成的组:T2A(优选核苷酸序列如SEQ ID NO:12所示,氨基酸序列如SEQ ID NO:84所示)、P2A(优选核苷酸序列如SEQ ID NO:13所示,氨基酸序列如SEQ ID NO:85所示)、F2A、E2A和IRES(优选核苷酸序列如SEQ ID NO:14所示)。在某些实施方式中,所述连接元件包含SEQ ID NO:84、85和14所示的序列或由其组成。
另一方面,本申请提供了一种组合物,其包含所述的经遗传修饰的免疫细胞。
在某些实施方式中,所述组合物还包含任选地药学上可接受的载体。
另一方面,本申请提供了所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体、或其组合在制备提高药物预防和/或治疗肿瘤的功效的试剂中的应用,优选地,所述药物为经遗传修饰的免疫细胞(优选CAR-T细胞)。
另一方面,本申请还提供了一种所述经遗传修饰的免疫细胞和/或所述的组合物在制备药物中的用途,其中所述药物用于治疗和/或预防肿瘤。
在某些实施方式中,所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
在某些实施方式中,所述经遗传修饰的免疫细胞选自淋巴细胞。在某些实施方式中,所述经遗传修饰的免疫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免疫细胞的步骤,其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用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。
附图说明
图1为不同CAR结构示意图;
图2为SIRT1蛋白促进肿瘤抗原诱导的特异性CAR-T细胞扩增;
图3为SIRT1蛋白抑制免疫负调控蛋白的表达;
图4为SIRT1蛋白促进CD3细胞中记忆性T细胞的增殖;
图5为SIRT1蛋白促进CD4、CD8细胞中记忆性T细胞的增殖;
图6-8为SIRT1蛋白功能突变体及其组合抑制免疫负调控蛋白的表达;
图9-10为SIRT1蛋白功能突变体及其组合促进CD3细胞中记忆性T细胞的增殖;
图11为SIRT1蛋白功能突变体及其组合促进CD4细胞中记忆性T细胞的增殖;
图12为SIRT1蛋白功能突变体及其组合促进CD8细胞中记忆性T细胞的增殖;
图13为SIRT1蛋白功能突变体及其组合促进T细胞的增殖;
图14为SIRT1蛋白功能突变体及其组合促进T细胞对免疫抑制微环境的抵抗能力;
图15为CD258蛋白及其功能突变体增强CAR-T细胞对肿瘤细胞的杀伤能力;
图16为CD258蛋白与SIRT1蛋白组合促进肿瘤抗原诱导的特异性CAR-T细胞扩增;
图17为CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(GPC3);
图18为CD258蛋白与SIRT1蛋白组合促进CD4、CD8细胞中记忆性T细胞的增殖(GPC3);
图19为CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(CD19);
图20为CD258蛋白与SIRT1蛋白组合促进CD8细胞中记忆性T细胞的增殖(CD19);
图21为CD258蛋白与SIRT1蛋白组合促进CD3、CD4细胞中记忆性T细胞的增殖(CD19);
图22为CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(MSLN);
图23为CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(MSLN);
图24为CD258蛋白与SIRT1蛋白组合增强CAR-T细胞对肿瘤细胞的杀伤能力;
图25为CD258蛋白与SIRT1蛋白组合增强CAR-T细胞抗肿瘤效果;
图26为CD258蛋白与SIRT1蛋白组合促进粒细胞和单核细胞体内增殖;
图27为CD258蛋白与SIRT1蛋白组合促进CAR-T细胞体内增殖以及Th1细胞因子的释放。
具体实施方式
以下通过实施例来进一步阐述本发明。但是应该理解,所述实施例只是举例说明的目的,并不意欲限制本发明的范围和精神。
本领域技术人员能够从下文的详细描述中容易地洞察到本发明的其它方面和优势。下文的详细描述中仅显示和描述了本发明的示例性实施方式。如本领域技术人员将认识到的,本发明的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地,本申请的附图和说明书中的描述仅仅是示例性的,而非为限制性的。
实施例1慢病毒载体的构建
所述CAR-T以靶向GPC3、CD19、Mesothelin(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-S1、GPC3-S2、GPC3-S3、GPC3-S6、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3、GPC3-C8、GPC3-C8A、GPC3-C8B、GPC3-S1A-C8A、CD19-CAR、CD19-S1A、CD19-C8A、CD19-S1A-C8A、MSLN-CAR、MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A、MSLN-S1A-C8A慢病毒。各CAR结构示意图如图1所示。
GPC3-CAR由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11依次拼接合成。
GPC3-S1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:15依次拼接合成。
GPC3-S2由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:16依次拼接合成。
GPC3-S3由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:17依次拼接合成。
GPC3-S6由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:20依次拼接合成。
GPC3-S1A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成。
GPC3-S1B由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:23依次拼接合成。
GPC3-S1B1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:24依次拼接合成。
GPC3-S1C由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:25依次拼接合成。
GPC3-S1C1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:26依次拼接合成。
GPC3-S1D由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:27依次拼接合成。
GPC3-S1D1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:28依次拼接合成。
GPC3-S1E由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:29依次拼接合成。
GPC3-S1E1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:30依次拼接合成。
GPC3-S1-S3由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:15、SEQ ID NO:13、SEQ ID NO:17依次拼接合成。
GPC3-S1A-S3由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:17依次拼接合成。
GPC3-C8由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成。
GPC3-C8A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成。
GPC3-C8B由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成。
GPC3-S1A-C8A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
CD19-CAR由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11依次拼接合成。
CD19-S1A由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成。
CD19-C8A由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成。
CD19-S1A-C8A由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
MSLN-CAR由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11依次拼接合成。
MSLN-S1A由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成。
MSLN-C8由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成。
MSLN-C8A由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成。
MSLN-C8B由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成。
MSLN-S1A-C8A由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
实施例2:慢病毒感染T细胞。
感染实验按照本领域技术人员已知的常规方法进行。简述感染步骤如下:
1.外周血单核淋巴细胞(PBMC)的获得,通过血液单采系统获得大于1×107的细胞。
2.抗人CD3/CD28抗体处理细胞培养皿。
用PBS稀释抗人CD3及抗人CD28抗体(购自上海近岸科技公司),终浓度为1ug/ml,向细胞培养皿中加入稀释后的抗体混合液,使其铺满培养皿,室温孵育2小时。2小时后用PBS洗一次,备用。
3.对T细胞进行激活处理
将分离的PBMC用T淋巴细胞培养液(Xvivo15培养基+5%FBS+100U/mlIL2+20ng/mlIL21+20ng/mlIL7)进行重悬,使终浓度为1×106个细胞/ml,并放入步骤2中处理过的培养皿中培养,培养条件为37℃+5%CO2,培养时间为24小时。
4.对激活的T细胞进行感染
1)感染试剂配制。
取一定量的T细胞培养液,加入终浓度为1mg/ml的synperonic F108(Sigma公司),混匀,水浴锅加热至37℃待用。
2)培养板处理。
取1mg/ml抗人CD3抗体及0.5mg/ml抗人CD28抗体按1:1000体积比稀释至适量的PBS缓冲液中,并取retronectin(1mg/ml,Takara公司)试剂,按1:40体积比稀释至该PBS缓冲液中,混匀后均匀铺至细胞皿,室温孵育2小时。2小时后用PBS进行洗涤并待用。
3)慢病毒感染T细胞
用1)中所配感染试剂稀释已激活的T细胞,并按MOI=3加入慢病毒,并混匀。均匀铺在2)中所处理的培养皿中。
感染后监测细胞密度,使细胞维持在1×106个细胞/ml,一般14天,可扩增30-1000倍。
实施例3:SIRT1蛋白促进肿瘤抗原诱导的特异性CAR-T细胞扩增。
将表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞与辐照(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)按1:1的细胞个数比用Xvivo15培养基进共培养,每4天重新补加辐照的HepG2进行刺激,刺激3次。每次用台盼蓝染色进行计数,细胞增殖情况如图2A所示。结果显示,表达GPC3-S2、GPC3-S3或GPC3-S6的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
同样的,将表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞与辐照(X-RAD细胞辐照仪,辐照剂量为30Gy)的Huh7细胞(中科院细胞库购得)按1:1的细胞个数比用Xvivo15培养基进共培养,每4天重新补加辐照的Huh7进行刺激,刺激3次。每次用台盼蓝染色进行计数,细胞增殖情况如图2B所示。结果亦表明,表达GPC3-S2、GPC3-S3或GPC3-S6的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
实施例4:SIRT1蛋白抑制免疫负调控蛋白的表达。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况,结果如图3所示,表达GPC3-S2、GPC3-S3或GPC3-S6的T细胞中,同时表达两个负调控蛋白的细胞占比均显著低于GPC3-CAR对照组。
实施例5:SIRT1蛋白促进CD3细胞中记忆性T细胞的增殖。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图4所示,表达GPC3-S2、GPC3-S3或GPC3-S6的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例6:SIRT1蛋白促进CD4、CD8细胞中记忆性T细胞的增殖。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图5A所示,表达GPC3-S2、GPC3-S3或GPC3-S6的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
同样的,于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图5B所示,表达GPC3-S2、GPC3-S3或GPC3-S6的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例亦均显著高于GPC3-CAR对照组。
实施例7:SIRT1蛋白功能突变体及其组合抑制免疫负调控蛋白的表达。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况,结果如图6-8所示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3或GPC3-S1A-S3的T细胞中,同时表达两个负调控蛋白的细胞占比均显著低于GPC3-CAR对照组。
实施例8:SIRT1蛋白功能突变体及其组合促进CD3细胞中记忆性T细胞的增殖。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图9-10所示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例9:SIRT1蛋白功能突变体及其组合促进CD4细胞中记忆性T细胞的增殖。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图11所示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例10:SIRT1蛋白功能突变体及其组合促进CD8细胞中记忆性T细胞的增殖。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图12所示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例11:SIRT1蛋白功能突变体及其组合促进T细胞的增殖。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养11天,每2-3天用台盼蓝染色进行计数传代,细胞增殖情况如图13A所示。结果显示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
同样的,于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养7天,在第7天分别取细胞与Xvivo15培养基共孵育24h后,采用CCK8(MCE公司)检测T细胞增殖效果。结果如图13B显示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的增殖效果亦远高于对照组表达GPC3-CAR的T细胞。
实施例12:SIRT1蛋白功能突变体及其组合促进T细胞对免疫抑制微环境的抵抗能力。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养8天,在第8天分别取细胞与1640无葡萄糖、无血清培养基(Gibco公司)共孵育24h后,采用CCK8(MCE公司)检测T细胞增殖效果。结果如图14A显示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的增殖效果远高于对照组表达GPC3-CAR的T细胞。
同样的,于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞,共培养8天,在第8天分别取细胞与1640无葡萄糖、无血清培养基(Gibco公司)以及20mM乳酸(PH值从正常7.4降至6.5,Sigma公司)共孵育24h后,采用CCK8(MCE公司)检测T细胞增殖效果。结果如图14B显示,表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的增殖效果亦远高于对照组表达GPC3-CAR的T细胞。
实施例13:CD258蛋白及其功能突变体增强CAR-T细胞对肿瘤细胞的杀伤能力。
将表达GPC3-CAR、GPC3-C8、GPC3-C8A或GPC3-C8B的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用Xvivo15培养基共培养7天,采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果,结果如图15A所示,表达GPC3-C8或GPC3-C8A的T细胞对肿瘤细胞的杀伤效果远高于对照组表达GPC3-CAR的T细胞,其中表达GPC3-C8A的T细胞相较于表达GPC3-C8的T细胞具有更为优越的杀伤效果。
同样的,将表达GPC3-CAR、GPC3-C8、GPC3-C8A或GPC3-C8B的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用1640无葡萄糖、无血清(Gibco公司)以及20mM乳酸(PH值从正常7.4降至6.5,Sigma公司)培养基共培养7天,采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果,结果如图15B所示,表达GPC3-C8或GPC3-C8A的T细胞对肿瘤细胞的杀伤效果亦显著高于对照组表达GPC3-CAR的T细胞,表达GPC3-C8A的T细胞相较于表达GPC3-C8的T细胞亦具有更为优越的杀伤效果。
实施例14:CD258蛋白与SIRT1蛋白组合促进肿瘤抗原诱导的特异性CAR-T细胞扩增。
将表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞与辐照(X-RAD细胞辐照仪,辐照剂量为30Gy)的Huh7细胞(中科院细胞库购得)按1:1的细胞个数比用Xvivo15培养基共培养,每3-4天重新补加辐照的Huh7进行刺激,刺激4次。每次用台盼蓝染色进行计数,细胞增殖情况如图16所示。结果表明,表达GPC3-S1A、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
GPC3-S1A-C8A为GPC3、S1A和C8A依次通过2A序列偶联。
实施例15:CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(GPC3)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图17所示,表达GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例16:CD258蛋白与SIRT1蛋白组合促进CD4、CD8细胞中记忆性T细胞的增殖(GPC3)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图18A所示,表达GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
同样的,于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图18B所示,表达GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例亦均显著高于GPC3-CAR对照组。
实施例17:CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(CD19)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况,结果如图19所示,表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞中,同时表达两个负调控蛋白的细胞占比均显著低于CD19-CAR对照组。
实施例18:CD258蛋白与SIRT1蛋白组合促进CD8细胞中记忆性T细胞的增殖(CD19)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图20所示,表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于CD19-CAR对照组。
实施例19:CD258蛋白与SIRT1蛋白组合促进CD3、CD4细胞中记忆性T细胞的增殖(CD19)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图21A所示,表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于CD19-CAR对照组。
同样的,于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图21B所示,表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例亦均显著高于CD19-CAR对照组。
实施例20:CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(MSLN)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达MSLN-CAR、MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况,结果如图22所示,表达MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的T细胞中,同时表达两个负调控蛋白的细胞占比均显著低于MSLN-CAR对照组。
实施例21:CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(MSLN)。
于37℃,5%CO2细胞培养箱中体外培养实施例2中所得的分别表达MSLN-CAR、MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的T细胞,共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达,如图23所示,表达MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于MSLN-CAR对照组。
实施例22:CD258蛋白与SIRT1蛋白组合增强CAR-T细胞对肿瘤细胞的杀伤能力。
将表达GPC3-S1A或GPC3-S1A-C8A的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用Xvivo15培养基共培养7天,采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果,结果如图24A所示,表达GPC3-S1A-C8A的T细胞对肿瘤细胞的杀伤效果远高于对照组表达GPC3-S1A的T细胞。
同样的,将表达GPC3-CAR或GPC3-S1A-C8A的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用Xvivo15培养基共培养7天,采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果,结果如图24B所示,表达GPC3-S1A-C8A的T细胞对肿瘤细胞的杀伤效果亦远高于对照组表达GPC3-CAR的T细胞。
实施例23:CD258蛋白与SIRT1蛋白组合增强CAR-T细胞抗肿瘤效果。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×107个/只),14天后,测得小鼠瘤体积约200mm3。此时将小鼠分为5组,分别为T、GPC3-CAR和GPC3-S1A-C8A组,每组6-8只。然后通过尾静脉分别向T细胞组注射T细胞(9×105个/只),向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×105个/只或9×105个/只),向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×105个/只或9×105个/只)。于每周的周一和周四分别测量瘤体积大小,并记录小鼠死亡情况。结果如图25所示,GPC3-S1A-C8A组抑瘤效果显著高于其他对照组。
实施例24:CD258蛋白与SIRT1蛋白组合促进粒细胞和单核细胞体内增殖。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×107个/只),14天后,测得小鼠瘤体积约200mm3。此时将小鼠分为5组,分别为T、GPC3-CAR和GPC3-S1A-C8A组,每组6只。然后通过尾静脉分别向T细胞组注射T细胞(9×105个/只),向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×105个/只或9×105个/只),向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×105个/只或9×105个/只),于第7天从小鼠尾部取血50μl,用BD流式检测每组中单核细胞和中性粒细胞的细胞数以及细胞大小。结果如图26所示,GPC3-S1A-C8A组中单核细胞和中性粒细胞的细胞数以及细胞大小均远高于GPC3-CAR对照组。
实施例25:CD258蛋白与SIRT1蛋白组合促进CAR-T细胞体内增殖以及Th1细胞因子的释放。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×107个/只),14天后,测得小鼠瘤体积约200mm3。此时将小鼠分为5组,分别为T、GPC3-CAR和GPC3-S1A-C8A组,每组6只。然后通过尾静脉分别向T细胞组注射T细胞(9×105个/只),向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×105个/只或9×105个/只),向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×105个/只或9×105个/只),于第14天从小鼠尾部取血50μl,用BD流式检测每组中人CD3、CD4和CD8蛋白的表达。结果如图27A、图27B和图27C所示,在表达了CD3、CD4和CD8的组中,GPC3-S1A-C8A组中表达CD3、CD4或CD8蛋白的细胞占总细胞数的比例远高于GPC3-CAR对照组。
同样的,向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×107个/只),14天后,测得小鼠瘤体积约200mm3。此时将小鼠分为5组,分别为T、GPC3-CAR和GPC3-S1A-C8A组,每组6只。然后通过尾静脉分别向T细胞组注射T细胞(9×105个/只),向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×105个/只或9×105个/只),向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×105个/只或9×105个/只),于第7天从小鼠尾部取血50μl,用BD流式检测每组中人IL-2、IL4、IL6、IL10、TNF-α和IFN-γ细胞因子的表达。结果如图27D所示,GPC3-S1A-C8A组中IFN-γ细胞因子的表达显著高于GPC3-CAR对照组。
序列表:
SEQ ID NO.1:前导序列
前导序列的氨基酸序列:SEQ ID NO:105
SEQ ID NO.2:GPC3 scFv的核苷酸序列
GPC3 scFv的氨基酸序列:SEQ ID NO:34
(VL,SEQ ID NO:35:
下划线标注依次是CDR1(RSSQSLVHSNGNTYLH,SEQ ID NO:36)、CDR2(KVSNRFS,SEQID NO:37)和CDR3(SQNTHVPPT,SEQ ID NO:38);VH,SEQ ID NO:39:
下划线标注依次是CDR1(DYEMH,SEQ ID NO:40)、CDR2(ALDPKTGDTAYSQKFKG,SEQID NO:41)和CDR3(FYSYTYW,SEQ ID NO:42))
SEQ ID NO.3:CD19 scFv的核苷酸序列
CD19 scFv的氨基酸序列:SEQ ID NO:43
(VL,SEQ ID NO:44:
下划线标注依次是CDR1(RASQDISKYLN,SEQ ID NO:45)、CDR2(HTSRLHS,SEQ IDNO:46)和CDR3(QQGNTLPYT,SEQ ID NO:47);VH,SEQ ID NO:48:
下划线标注依次是CDR1(LPDYGVS,SEQ ID NO:49)、CDR2(VIWGSETTYYNSALKS,SEQID NO:50)和CDR3(HYYYGGSYAMDYW,SEQ ID NO:51))
SEQ ID NO.4:BCMA scFv的核苷酸序列
BCMA scFv氨基酸序列:SEQ ID NO:52
(VL,SEQ ID NO:53:
下划线标注依次是CDR1(RASESVSVIGAHLIH,SEQ ID NO:54)、CDR2(LASNLET,SEQID NO:55)和CDR3(LQSRIFPRT,SEQ ID NO:56);VH,SEQ ID NO:57:
下划线标注依次是CDR1(DYSIN,SEQ ID NO:58)、CDR2(WINTETREPAYAYDFRG SEQID NO:59)和CDR3(DYSYAMDYW,SEQ ID NO:60))
SEQ ID NO.5:MSLN scFv的核苷酸序列
MSLN scFv氨基酸序列:SEQ ID NO:61
(VL,SEQ I NO:62:
下划线标注依次是CDR1(TLRSGINVGPYRIY,SEQ ID NO:63)、CDR2(YKSDSDKQQGS,SEQ ID NO:64)和CDR3(MIWHSSAAV,SEQ ID NO:65);VH:SEQ ID NO:66
下划线标注依次是CDR1(SNSATWN,SEQ ID NO:67)、CDR2(RTYYRSKWYNDYAVSVKS,SEQ ID NO:68)和CDR3(GMMTYYYGMDVW,SEQ ID NO:69))
SEQ ID NO.6:HER2 scFv的核苷酸序列
HER2 scFv氨基酸序列:SEQ ID NO:70
(VL,SEQ ID NO:71:
下划线标注依次是CDR1(KASQDVYNAVA,SEQ ID NO:72)、CDR2(SASSRYT,SEQ IDNO:73)和CDR3(QQHFRTPFT,SEQ ID NO:74);VH,SEQ ID NO:75:
下划线标注依次是CDR1(NYGMN,SEQ ID NO:76)、CDR2(WINTSTGESTFADDFKG,SEQID NO:77)和CDR3(WEVYHGYVPYW,SEQ ID NO:78))
SEQ ID NO.7:CD8铰链区的核苷酸序列
CD8铰链区氨基酸序列:SEQ ID NO:79
SEQ ID NO.8:CD8跨膜区的核苷酸序列
CD8跨膜区的氨基酸序列:SEQ ID NO:80
SEQ ID NO.9:CD28共刺激结构域的核苷酸序列
CD28共刺激结构域的氨基酸序列:SEQ ID NO:81
SEQ ID NO.10:4-1BB的共刺激结构域的核苷酸序列
4-1BB的共刺激结构域的氨基酸序列:SEQ ID NO:82
SEQ ID NO.11:CD3ζ的信号传导结构域的核苷酸序列
CD3ζ的信号传导结构域的氨基酸序列:SEQ ID NO:83
SEQ ID NO.12:T2A的核苷酸序列
T2A的氨基酸序列:SEQ ID NO:84
SEQ ID NO.13:P2A的核苷酸序列
P2A的氨基酸序列:SEQ ID NO:85
SEQ ID NO.14:IRES的核苷酸序列
核糖体跳跃位点,不翻译成氨基酸
SEQ ID NO.15:S1的核苷酸序列
S1的氨基酸序列:SEQ ID NO:86
SEQ ID NO.16:S2的核苷酸序列
S2的氨基酸序列:SEQ ID NO:87
SEQ ID NO.17:S3的核苷酸序列
S3的氨基酸序列:SEQ ID NO:88
SEQ ID NO.18:S4的核苷酸序列
S4的氨基酸序列:SEQ ID NO:89
SEQ ID NO.19:S5的核苷酸序列
S5的氨基酸序列:SEQ ID NO:90
SEQ ID NO.20:S6的核苷酸序列
S6的氨基酸序列:SEQ ID NO:91
SEQ ID NO.21:S7的核苷酸序列
S7的氨基酸序列:SEQ ID NO:92
SEQ ID NO.22:S1A的核苷酸序列
S1A的氨基酸序列:SEQ ID NO:93
SEQ ID NO.23:S1B的核苷酸序列
S1B的氨基酸序列:SEQ ID NO:94
SEQ ID NO.24:S1B1的核苷酸序列
S1B1的氨基酸序列:SEQ ID NO:95
SEQ ID NO.25:S1C的核苷酸序列
S1C的氨基酸序列:SEQ ID NO:96
SEQ ID NO.26:S1C1的核苷酸序列
S1C1的氨基酸序列:SEQ ID NO:97
SEQ ID NO.27:S1D的核苷酸序列
S1D的氨基酸序列:SEQ ID NO:98
SEQ ID NO.28:S1D1的核苷酸序列
S1D1的氨基酸序列:SEQ ID NO:99
SEQ ID NO.29:S1E的核苷酸序列
S1E的氨基酸序列:SEQ ID NO:100
SEQ ID NO.30:S1E1的核苷酸序列
S1E1的氨基酸序列:SEQ ID NO:101
SEQ ID NO.31:C8的核苷酸序列
C8的氨基酸序列:SEQ ID NO:102
SEQ ID NO.32:C8A的核苷酸序列
C8A的氨基酸序列:SEQ ID NO:103
SEQ ID NO.33:C8B的核苷酸序列
C8B的氨基酸序列:SEQ ID NO:104
SIRT1蛋白核定位信号:SEQ ID NO:105
SIRT1蛋白核定位信号:SEQ ID NO:106
SIRT1蛋白核定位信号突变体:SEQ ID NO:107
SIRT1蛋白核定位信号突变体:SEQ ID NO:108
SIRT1蛋白核出核信号:SEQ ID NO:109
SIRT1蛋白核出核信号:SEQ ID NO:110
SIRT1蛋白核出核信号突变体:SEQ ID NO:111
SIRT1蛋白核出核信号突变体:SEQ ID NO:112
Claims (18)
1.一种方法,优选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)促进免疫细胞增殖的方法,(2)促进记忆性免疫细胞产生的方法,(3)抑制免疫细胞分化的方法,(4)抑制免疫细胞免疫负调控蛋白表达的方法,(5)增强免疫细胞释放细胞因子的方法,和(6)增强免疫细胞对肿瘤杀伤能力的方法,
所述方法包含以下步骤:上调所述免疫细胞中CD258蛋白或其功能突变体的表达量;
或者一种方法,所述方法选自以下各项组成的组:
(a)解决受试者肿瘤异质性的方法,(b)预防受试者中肿瘤复发的方法,和(v)治疗有需要的受试者中的肿瘤的方法,
所述方法包含向受试者施用免疫细胞,其中所述免疫细胞中CD258蛋白或其功能突变体的表达量上调。
2.权利要求1所述的方法,其中所述肿瘤选自肝癌、肺癌、白血病和间皮瘤,所述免疫负调控蛋白选自PD1、PDL1、TIM3和LAG3组成的组,所述细胞因子选自白介素、干扰素和/或肿瘤坏死因子,优选地,所述细胞因子选自IL-2、IL4、IL6、IL7、IL10、IL12、TNF-α和/或IFNγ;所述免疫细胞为淋巴细胞;优选地,所述免疫细胞为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。进一步优选地,所述T细胞选自记忆性干细胞样T细胞和/或中央记忆性T细胞,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+。
3.权利要求1-2任一项所述的方法,所述免疫细胞选自经遗传修饰的免疫细胞,且所述经遗传修饰的免疫细胞表达嵌合抗原受体或T细胞受体,优选地,所述经遗传修饰的免疫细胞为经遗传修饰的T细胞,优选地,所述方法包括分离并激活所述经遗传修饰的免疫细胞的步骤,其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用T细胞培养基,优选地,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。
4.权利要求1-3任一项所述的方法,所述TCR包含选自下组的亚基:TCRα、TCRβ、TCRγ和TCRδ,
优选地,所述TCR的亚基包括特异性结合和/或识别肿瘤抗原的胞外域可变区优选地,胞外域可变区选自以下组:TCRα可变区片段Vα、TCRα可变区片段Jα、TCRβ可变区片段Vβ、TCRβ可变区片段Dβ和TCRβ可变区片段Jβ,
优选地,所述胞外域可变区特异性结合和/或识别选自下组的靶标:MAGEA家族成员、CTA家族成员、HPV病毒和酪氨酸酶,
优选地,所述胞外域可变区特异性结合和/或识别选自下组的靶标:MAGEA3、MAGEA4、NY-ESO-1、MART1、HPV16-E6和黑素瘤抗原酪氨酸酶。
5.权利要求1-3任一项所述的方法,所述CAR包含细胞内结构域,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,
优选地,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(优选核苷酸序列如SEQ ID NO:11所示,氨基酸序列如SEQ ID NO:83所示)、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:83所示的序列或与其具备至少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%同源性的序列,所述同源序列仍具备SEQ ID NO:83所示序列功能,或由其组成,
优选地,共刺激结构域是指选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、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、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优选地,所述共刺激结构域选自:CD27、CD28和4-1BB,
更优选地,所述共刺激结构域选自以下各项组成的组:CD27的共刺激结构域、CD28的共刺激结构域(优选核苷酸序列如SEQ ID NO:9所示,氨基酸序列如SEQ ID NO:81所示)和4-1BB的共刺激结构域(优选核苷酸序列如SEQ ID NO:10所示,氨基酸序列如SEQ ID NO:82所示)优选地,所述共刺激结构域包含下述任一项所示的序列或与其具备至少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%同源性的序列,或由其组成:SEQ ID NO:81和SEQ ID NO:82,所述同源序列仍具备SEQ ID NO:81或82所示序列功能,
优选地,所述CAR包含铰链区,更优选地,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(优选核苷酸序列如SEQ ID NO:7所示,氨基酸序列如SEQ ID NO:79所示)优选地,所述铰链区包含SEQ ID NO:79所示的序列或由其组成,
优选地,所述CAR包含跨膜区优选地,所述跨膜区选自以下各种组成的组:CD8的跨膜区(优选核苷酸序列如SEQ ID NO:8所示,氨基酸序列如SEQ ID NO:80所示)、CD28的跨膜区和CD24的跨膜区优选地,所述跨膜区包含SEQ ID NO:80所示的序列或由其组成,
优选地,所述CAR包含靶向部分,优选所述靶向部分特异性结合和/或识别肿瘤抗原,
优选地,所述肿瘤抗原可以包括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,
更优选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,进一步优选地,所述靶向部分特异性结合和/或识别选自下组的靶标:CD19、BCMA、HER2、Mesothelin和GPC3。
6.权利要求5所述的方法,其中所述靶向部分为scFv,优选地,所述靶向部分选自针对GPC3的scFv,针对CD19的scFv,针对BCMA的scFv,针对MSLN的scFv,针对HER2的scFv,
优选所述针对GPC3的scFv包含SEQ ID NO:35所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:36所示),LCDR2(优选按照Kabat编号系统,序列如SEQID NO:37所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:38所示)和包含SEQID NO:39所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:40所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:41所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:42所示),更优选地,针对GPC3的scFv包含如SEQ ID NO:2所示的核苷酸序列或如SEQ ID NO:34所示的氨基酸序列,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%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:2或34所示序列功能;
所述针对CD19的scFv包含SEQ ID NO:44所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:45所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:46所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:47所示)和包含SEQ ID NO:48所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:49所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:50所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:51所示),更优选地,针对CD19的scFv包含如SEQ ID NO:3所示的核苷酸序列或如SEQ ID NO:43所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:3或43所示序列功能;
所述针对BCMA的scFv包含SEQ ID NO:53所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:54所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:55所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:56所示)和包含SEQ ID NO:57所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:58所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:59所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:60所示),更优选地,针对BCMA的scFv包含的如SEQ ID NO:4所示核苷酸序列或如SEQ ID NO:52所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:4或52所示序列功能;
所述针对MSLN的scFv包含SEQ ID NO:62所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:63所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:64所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:65所示)和包含SEQ ID NO:66所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:67所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:68所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:69所示),更优选地,针对MSLN的scFv包含如SEQ ID NO:5所示的核苷酸序列或如SEQ ID NO:61所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:5或61所示序列功能;
所述针对HER2的scFv包含SEQ ID NO:71所示轻链可变区包含的LCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:72所示),LCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:73所示)和LCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:74所示)和包含SEQ ID NO:75所示的重链可变区包含的HCDR1(优选按照Kabat编号系统,序列如SEQ ID NO:76所示),HCDR2(优选按照Kabat编号系统,序列如SEQ ID NO:77所示)和HCDR3(优选按照Kabat编号系统,序列如SEQ ID NO:78所示),更优选地,针对HER2的scFv包含如SEQ ID NO:6所示的核苷酸序列或如SEQ ID NO:70所示的氨基酸序列或与其具备至少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%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:6或70所示序列功能。
7.权利要求1-6任一项所述的方法,其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,优选地,所述方法还包括:向经分离的所述PBMC中加入一种或多种T细胞刺激因子,优选地,所述T细胞刺激因子选自以下各项组成的组:针对B淋巴细胞表面抗原的抗体、抗TNF抗体、细胞内聚酯和抗生素优选地,所述T细胞刺激因子选自以下各项组成的组:抗CD3抗体、抗CD28抗体、抗4-1BB抗体、抗CD80抗体、抗CD86抗体、PHA、PMA和离子霉素,更优选地,所述T细胞刺激因子为抗CD3抗体,且所述抗CD3抗体的浓度为1-10000ng/mL,或所述T细胞刺激因子为抗CD28抗体,且所述抗CD28抗体的浓度为1-10000ng/mL。
8.权利要求1-7任一项所述的方法,其中所述方法还包括:向经分离的所述PBMC中加入一种或多种细胞因子,优选地,所述细胞因子为白细胞介素,更优选地,所述白细胞介素为IL-1,IL-2,IL-3,IL-4,IL-5,IL-6,IL-7,IL-8,IL-9,IL-10,IL-11,IL-12,IL-13,IL-14,IL-15,IL-16,IL-17,IL-18,IL-19,IL-20,IL-21,IL-22,IL-23,IL-24,IL-25,IL-26,IL-27,IL-28,IL-29,IL-30,IL-31,IL-32,IL-33,IL-34,IL-35和/或IL-36;更优选地,所述白细胞介素为IL2,且所述IL2的浓度为0.1-10000U/mL,或所述白细胞介素为IL21,且所述IL21的浓度为0.01-1000ng/mL,或所述白细胞介素为IL7,且所述IL7的浓度为0.01-1000ng/mL,或所述白细胞介素为IL15,且所述IL15的浓度为0.01-1000ng/mL。
9.权利要求1-8任一项所述的方法,其中所述CD258或其功能突变体来源于人。
10.权利要求1-9任一项所述的方法,其中所述CD258蛋白包含SEQ ID NO:31或SEQ IDNO:102所示的序列或与其具备至少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%、至少99%同源性的序列,或由其组成,所述同源序列仍具备SEQ ID NO:31或SEQ ID NO:102所示序列功能,
所述CD258蛋白功能突变体选自以下各项组成的组:
(1)在SEQ ID NO:102所示序列中第82-83位的Q和L的一种或两种置换为A(优选QL氨基酸被置换为AA)获得的功能突变体,所述功能突变体使该蛋白始终以膜结合形式表达在细胞膜上,减少分泌形式的表达,
(2)在SEQ ID NO:102所示序列中第82-83位的Q和L缺失,或在SEQ ID NO:102所示序列中第81-84位的EQLI缺失获得的功能突变体,所述功能突变体使该蛋白始终以膜结合形式表达在细胞膜上,减少分泌形式的表达,
(3)如SEQ ID NO:32-33所示的序列或如SEQ ID NO:103-104所示的序列,
(4)CD258的胞内区。
11.经遗传修饰的免疫细胞,其中所述遗传修饰使得所述免疫细胞中CD258蛋白或其功能突变体的表达量上调。
12.权利要求1所述的方法或权利要求12所述的经遗传修饰的免疫细胞,其中所述表达量上调通过以下方式实现:
通过向所述免疫细胞中转染包含编码所述CD258蛋白或其功能突变体的表达载体以增加所述免疫细胞中所述CD258蛋白或其功能突变体的量,优选地,所述CD258蛋白或其功能突变体以单体形式存在或通过连接元件连接的缀合物的形式存在。
13.权利要求11或12所述的经遗传修饰的免疫细胞,其中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,优选地,所述载体进一步包含CAR,更优选地,所述CAR通过连接元件连接调节单元,所述调节单元为CD258蛋白或其功能突变体的一种或多种。
14.权利要求11-13任一项所述的经遗传修饰的免疫细胞,其中所述连接元件选自下列各项组成的组:T2A(优选核苷酸序列如SEQ ID NO:12所示,氨基酸序列如SEQ ID NO:84所示)、P2A(优选核苷酸序列如SEQ ID NO:13所示,氨基酸序列如SEQ ID NO:85所示)、F2A、E2A和IRES(优选核苷酸序列如SEQ ID NO:14所示),优选地,所述连接元件包含SEQ ID NO:84、85和14所示的序列或由其组成。
15.权利要求11-14任一项所述的经遗传修饰的免疫细胞,其中所述免疫细胞包含选自以下各项组成的组的片段:
GPC3-C8,由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成,
GPC3-C8A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成,
GPC3-C8B,由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成,
CD19-C8A,由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成,
MSLN-C8,由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成。
MSLN-C8A,由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成,和
MSLN-C8B,由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成。
16.组合物,其包含权利要求11-15任一项所述的经遗传修饰的免疫细胞,任选地,所述组合物还包含药学上可接受的载体。
17.权利要求10所述方法中涉及的所述CD258蛋白或其功能突变体在制备提高药物预防和/或治疗肿瘤的功效的试剂中的应用,优选地,所述药物为经遗传修饰的免疫细胞(优选CAR-T细胞)。
18.权利要求11-15任一项所述的经遗传修饰的免疫细胞和/或权利要求16所述的组合物在制备药物中的用途,其中所述药物用于治疗和/或预防肿瘤,优选地,所述肿瘤选自肝癌、肺癌、白血病和间皮瘤,优选地,所述经遗传修饰的免疫细胞选自淋巴细胞,优选地,所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。
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