CN110099997A - 表达car和转录因子的细胞及其用途 - Google Patents
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Abstract
本发明提供了细胞,其包含编码嵌合抗原受体(CAR)的第一外源核酸分子和编码转录因子的第二外源核酸分子。
Description
发明领域
本发明涉及共表达嵌合抗原受体(CAR)和转录因子的细胞。转录因子的表达可以在体外和/或体内阻止或减少细胞的分化和/或耗竭。
发明背景
嵌合抗原受体是将单克隆抗体(mAb)的特异性移植到T细胞的效应功能的蛋白质。它们的通常形式是I型跨膜结构域蛋白,具有抗原识别氨基末端、间隔区、跨膜结构域和细胞内T细胞信号传导结构域。CAR技术使产生大量的对任何表面抗原具有特异性的T细胞成为可能。通过例如外周血T细胞群的离体病毒载体转导制备细胞。然后,表达CAR的经转导的细胞可以用于过继性免疫疗法用于治疗疾病。
在转导之前有必要激活T细胞并使其扩增以产生临床有效的治疗剂量的T细胞。现有的T细胞激活/扩增方法通常伴随大量的T细胞分化,并且可以导致短寿命效果,包括转移的T细胞的短寿命存活、缺乏持久性和缺乏体内扩增。迄今为止,CAR-T细胞免疫疗法的临床功效受到输注到患者中后T细胞扩增和持久性差的限制。因此需要改进的治疗性CAR-T细胞组合物,其在体内存活、扩增和持续存在。
附图说明
图1-说明T细胞分化的线性模型的示意图,显示了与每种细胞类型相关的表达标志物。APC-抗原呈递细胞;TCM-中央记忆T细胞;TEFF-效应T细胞;TEM-效应记忆T细胞;TN-初始T细胞;TSCM-T记忆干细胞。
图2-显示转导(第0天)和与表达CD19的靶细胞共培养24小时后6天(第7天)后的效应、效应记忆(EM)、中央记忆(CM)和初始细胞的比例的图表。T细胞未转导(NT)、用仅表达CAR的载体(HD37)转导或用表达CAR和转录因子FOXO1的载体(HD37-FOXO1)转导。分别分析CD4+和CD8+亚群。
图3-显示与表达CD19的靶细胞共培养24小时后6天CD4+和CD8+T细胞上CD27和CD62L的表达的图表。T细胞未转导(NT)、用仅表达CAR的载体(HD37)转导、用表达CAR和转录因子EOMES的载体(HD37-EOMES)转导或用表达CAR和转录因子FOXO1的载体(HD37-FOXO1)转导。
图4-显示与表达CD19的靶细胞共培养24小时后6天CD4+和CD8+T细胞上CD62L的表达的图表。T细胞未转导(NT)、用仅表达CAR的载体(HD37)转导或用表达CAR和转录因子Runx3和CBF beta的载体(HD37-Runx3_CBFbeta)转导。
图5-显示转导(第0天)和与表达CD19的靶细胞共培养24小时后6天(第7天)后的效应、效应记忆(EM)、中央记忆(CM)和初始细胞的比例的图表。T细胞未转导(NT)、用仅表达CAR的载体(HD37)转导、用表达CAR和转录因子BACH2的载体(HD37-BACH2)转导或用表达CAR和突变体形式的转录因子BACH2的载体(HD37-BACH2_S520A)转导。分别分析CD4+和CD8+亚群。
发明内容概述
为了使CAR-T细胞有效,重要的是其在体内持续存在并扩增,并且抵抗过度快速的分化和耗竭。CAR T细胞持久性和植入与施用的初始、中央记忆和T干细胞记忆T细胞的比例有关。
本发明人已发现,通过在细胞中共转染CAR与转录因子,可以阻止或减少细胞的分化和/或耗竭。这导致用于免疫疗法的细胞组合物中更大比例的初始、中央记忆和干细胞记忆细胞,以及体内细胞的更有效的持久性和扩增。
因此,在第一方面,本发明提供了细胞,其包含编码嵌合抗原受体(CAR)的第一外源核酸分子和编码转录因子的第二外源核酸分子。
转录因子可以阻止或减少细胞的分化和/或耗竭。
转录因子可以是效应记忆阻遏物,例如BLIMP-1。
或者,转录因子可以是中央记忆阻遏物,例如BCL6或Bach2。
转录因子可以是或包含Bach2或经修饰形式的Bach2,所述经修饰形式的Bach2具有减少的或去除的被ALK磷酸化的能力。例如,经修饰形式的Bach2可以参考如SEQ ID No.2所示的氨基酸序列在以下位置的一个或多个处包含突变:Ser-535、Ser-509、Ser-520。
转录因子可以是FOXO1。
转录因子可以是EOMES。
转录因子可以包含Runx3和/或CBF beta。
在第二方面,本发明提供了核酸构建体,其包含编码嵌合抗原受体(CAR)的第一核酸序列和编码如本发明的第一方面中所定义的转录因子的第二核酸序列。
核酸构建体可以具有以下结构:
CAR-coexpr-TF;或
TF-coexpr-CAR
其中:
CAR是编码CAR的核酸序列;
coexpr是使得CAR和转录因子能够共表达的核酸序列;并且
TF是编码转录因子的核酸序列。
核酸序列“coexpr”可以编码包含自切割肽的序列。
在第三方面,本发明提供了核酸序列的试剂盒,其包含编码嵌合抗原受体(CAR)的第一核酸序列和编码如本发明的第一方面中所定义的转录因子的第二核酸序列。
在第四方面,本发明提供了载体,其包含根据本发明的第二方面的核酸构建体。
在第五方面,本发明提供了载体的试剂盒,其包含第一载体,所述第一载体包含编码嵌合抗原受体(CAR)的第一核酸序列;和第二载体,所述第二载体包含编码如本发明的第一方面中所定义的转录因子的第二核酸序列。
在第六方面,本发明提供了用于制备根据本发明的第一方面的细胞的方法,其包括将以下各项引入到细胞中的步骤:根据本发明的第二方面的核酸构建体、根据本发明的第三方面的核酸序列的试剂盒、根据本发明的第四方面的载体或根据本发明的第五方面的载体的试剂盒。
细胞可以来自从受试者分离的样品。
在第七方面,本发明提供了药物组合物,其包含多个根据本发明的第一方面的细胞。
在第八方面,本发明提供了用于治疗和/或预防疾病的方法,其包括向受试者施用根据本发明的第七方面的药物组合物的步骤。
该方法可以包括以下步骤:
(i)从受试者分离含有细胞的样品;
(ii)用以下各项转导或转染细胞:根据本发明的第二方面的核酸构建体、根据本发明的第三方面的核酸序列的试剂盒、根据本发明的第四方面的载体或根据本发明的第五方面的载体的试剂盒;和
(iii)向受试者施用来自(ii)的细胞。
在第九方面,本发明提供了根据本发明的第七方面的药物组合物,其用于治疗和/或预防疾病。
在第十方面,本发明提供了根据本发明的第一方面的细胞在制备用于治疗和/或预防疾病的药物中的用途。
参考本发明的第八、第九和第十方面,该疾病可以是癌症。
发明详述
嵌合抗原受体(CAR)
本发明的细胞包含编码嵌合抗原受体(CAR)的外源核酸分子。
经典的CAR是嵌合I型跨膜蛋白质,其将细胞外抗原识别结构域(结合剂)连接至细胞内信号传导结构域(胞内域)。结合剂通常是衍生自单克隆抗体(mAb)的单链可变片段(scFv),但其可以基于包含抗体样抗原结合位点的其他形式。通常需要间隔区结构域以将结合剂从膜分离并使其具有合适的定向。使用的常见间隔区结构域是IgG1的Fc。取决于抗原,更紧凑的间隔区可以满足,例如来自CD8α的茎部,以及甚至仅仅是IgG1铰链。跨膜结构域将蛋白质锚定在细胞膜中并将间隔区连接至胞内域。
早期的CAR设计具有衍生自FcεR1或CD3ζ的γ链的细胞内部分的胞内域。因此,这些第一代受体传输免疫信号1,其足够触发T细胞对关联靶细胞的杀伤,但不能完全激活T细胞以增殖或存活。为了克服这一限制,构建了复合胞内域:将T细胞共刺激分子的细胞内部分融合至CD3ζ的细胞内部分,产生了第二代受体,其能够在抗原识别后同时传输激活和共刺激信号。最通常使用的共刺激结构域是CD28的共刺激结构域。这提供了最强力的共刺激信号-即免疫信号2,其触发T细胞增殖。已经描述了一些受体,其包括TNF受体家族胞内域,例如紧密相关的OX40和41BB,其传输存活信号。现在已经描述甚至更强力的第三代CAR,其具有能够传输激活、增殖和存活信号的胞内域。
可以使用例如逆转录病毒载体将编码CAR的核酸转移至T细胞。可以采用慢病毒载体。以这种方式,可以生成大量癌症特异性T细胞用于过继细胞转移。当CAR结合靶抗原时,这导致激活信号传输至上面有其表达的T细胞。因此CAR将特异性和T细胞的细胞毒性引导至表达靶定抗原的肿瘤细胞。
因此,CAR通常包含:(i)抗原结合结构域;(ii)间隔区;(iii)跨膜结构域;和(iii)包含信号传导结构域或者与信号传导结构域相关联的细胞内结构域。
抗原结合结构域
抗原结合结构域是CAR识别抗原的部分。本领域已知许多抗原结合结构域,包括基于抗体、抗体模拟物和T细胞受体的抗原结合位点的那些。例如,抗原结合结构域可以包含:衍生自单克隆抗体的单链可变片段(scFv);靶抗原的天然配体;对于靶标具有足够亲和力的肽;单结构域抗体;人工单结合剂,如Darpin(设计的锚蛋白重复蛋白);或来自T细胞受体的单链。
抗原结合结构域可以包含不基于抗体的抗原结合位点的结构域。例如,抗原结合结构域可以包含基于作为肿瘤细胞表面受体的可溶性配体(例如可溶性肽,如细胞因子或趋化因子)的蛋白质/肽的结构域;或膜锚定配体或受体的细胞外结构域,其结合对对应物在肿瘤细胞上表达。
抗原结合结构域可以基于抗原的天然配体。
抗原结合结构域可以包含来自组合文库的亲和肽或从头设计的亲和蛋白/肽。
间隔区结构域
CAR包含间隔区序列以将抗原结合结构域与跨膜结构域连接,并在空间上将抗原结合结构域与胞内域分开。柔性间隔区允许抗原结合结构域在不同方向上定向以促进结合。
跨膜结构域
跨膜结构域是跨越膜的CAR的序列。
跨膜结构域可以是在膜中热力学稳定的任何蛋白质结构。这通常是包含几个疏水残基的alpha螺旋。任何跨膜蛋白的跨膜结构域可以用于供应本发明的跨膜部分。蛋白质的跨膜结构域的存在和跨度可以由本领域技术人员使用TMHMM算法(http://www.cbs.dtu.dk/services/TMHMM-2.0/)确定。此外,鉴于蛋白质的跨膜结构域是相对简单的结构,即预测形成具有足以跨越膜的足够长度的疏水性alpha螺旋的多肽序列,也可以使用人工设计的TM结构域(US 7052906 B1描述了合成的跨膜组分)。
跨膜结构域可以衍生自CD28,其提供良好的受体稳定性。
胞内域
胞内域是CAR的信号传输部分。它可以是CAR的细胞内结构域的一部分或与其相关联。在抗原识别后,受体簇、天然CD45和CD148被排除在突触之外,并且信号被传输至细胞。最普遍使用的胞内域组分是含有3个ITAM的CD3-zeta的胞内域组分。这在抗原结合后传输激活信号至T细胞。CD3-zeta可能不提供完全有能力的激活信号,且可能需要另外的共刺激信号传导。例如,可以与CD3-Zeta一同使用嵌合CD28和OX40,以传输增殖/存活信号,或可以全部三个一起使用。
本发明的CAR或TanCAR的胞内域可以包含CD28胞内域和OX40和CD3-Zeta胞内域。
胞内域可以包含:
(i)含有ITAM的胞内域,例如来自CD3zeta的胞内域;和/或
(ii)共刺激结构域,例如来自CD28的胞内域;和/或
(iii)传输存活信号的结构域,例如TNF受体家族胞内域,如OX-40或4-1BB。
已经描述了许多系统,其中抗原识别部分在与信号传输部分分开的分子上,例如WO015/150771;WO2016/124930和WO2016/030691中描述的那些。因此,由本发明的细胞表达的CAR可以包含抗原结合组分,其包含抗原结合结构域和跨膜结构域;其能够与包含信号传导结构域的分开的细胞内信号传导组分相互作用。本发明的细胞可以包含CAR信号传导系统,其包含此类抗原结合组分和细胞内信号传导组分。
信号肽
本发明的细胞可以包含信号肽,使得当CAR在细胞内表达时,新生蛋白质被引导至内质网并随后被引导至其得以表达的细胞表面。
信号肽可以在分子的氨基末端处。
本发明的CAR可以具有通式:
信号肽-抗原结合结构域-间隔区结构域-跨膜结构域-细胞内T细胞信号传导结构域(胞内域)。
转录因子
本发明的细胞包含编码转录因子的外源核酸分子。
转录因子是通过与特定DNA序列结合来控制遗传信息从DNA到信使RNA的转录速率并调节包含该序列或与该序列相邻的基因的表达的蛋白质。
转录因子通过促进(作为激活物)或阻断(作为阻遏物)RNA聚合酶的募集起作用。
转录因子含有至少一个DNA结合结构域(DBD),其附接于DNA的增强子或启动子区域。取决于转录因子,相邻基因的转录可以上调或下调。转录因子还含有反式激活结构域(TAD),其具有其他蛋白质如转录辅调节物的结合位点。
转录因子使用多种机制用于调节基因表达,包括稳定或阻断RNA聚合酶与DNA的结合,或催化组蛋白的乙酰化或脱乙酰化。转录因子可以具有组蛋白乙酰转移酶(HAT)活性,其使组蛋白乙酰化,削弱DNA与组蛋白的缔合,并使DNA更易于转录,从而上调转录。或者,转录因子可以具有组蛋白脱乙酰酶(HDAC)活性,其使组蛋白脱乙酰化,加强DNA与组蛋白的缔合,并使DNA不易转录,从而下调转录。它们可以起作用的另一种机制是通过将辅激活物或辅阻遏物蛋白募集到转录因子DNA复合物中。
有两种机制类型的转录因子,一般转录因子或上游转录因子。
一般转录因子参与预起始复合物的形成。最常见的缩写为TFIIA、TFIIB、TFIID、TFIIE、TFIIF和TFIIH。它们普遍存在并与围绕所有II类基因的转录起始位点的核心启动子区域相互作用。
上游转录因子是结合起始位点的上游以刺激或阻遏转录的蛋白质。这些与特异性转录因子同义,因为它们根据基因附近存在的识别序列而显著变化。
特定转录因子的一些实例在下表中给出:
转录因子通常基于序列相似性并因此其DNA结合结构域的三级结构进行分类。
具有碱性结构域的转录因子包括:亮氨酸拉链因子(例如bZIP、c-Fos/c-Jun、CREB和植物G-box结合因子);螺旋-环-螺旋因子(例如普遍存在的(A类)因子;肌原性转录因子(MyoD);Achaete-Scute和Tal/Twist/Atonal/Hen);和螺旋-环-螺旋/亮氨酸拉链因子(例如bHLH-ZIP、c-Myc、NF-1(A、B、C、X)、RF-X(1、2、3、4、5、ANK)和bHSH)。
具有锌配位DNA结合结构域的转录因子包括核受体类型的Cys4锌指,如类固醇激素受体和甲状腺激素受体样因子;多种Cys4锌指,如GATA-因子;Cys2His2锌指结构域,如普遍存在的因子,包括TFIIIA、Sp1;发育/细胞周期调节物,包括Krüppel;具有NF-6B样结合特性的大因子;Cys6半胱氨酸-锌簇和交替组成的锌指。
具有螺旋-转角-螺旋结构域的转录因子包括具有同源结构域的那些;配对盒;叉头/翼螺旋;热激因子;色氨酸簇;和TEA(转录增强因子)结构域如TEAD1、TEAD2、TEAD3、TEAD4。
最后,存在具有小沟接触的beta-支架因子,包括RHR(Rel同源区域)类;STAT;p53;MADS盒子;beta-Barrel alpha-螺旋转录因子;TATA结合蛋白;HMG盒子;异聚(Heteromeric)CCAAT因子;Grainyhead;冷激结构域因子;和Runt。
本发明的转录因子可以是组成型活性的或条件活性的,即需要激活。
转录因子可以是天然存在的或人工的。
T细胞分化的阻遏
激活后,T细胞分化成多种不同的T细胞亚型,如图1中所示。本发明人已经表明,受试者中CAR T细胞持久性和植入与向受试者施用的初始、中央记忆和干细胞记忆T细胞的比例有关。
本发明的细胞可以包含编码转录因子的外源核酸分子,其有效地增加用于向患者施用的组合物中初始、中央记忆和/或干细胞记忆T细胞的比例。
转录因子可以是例如中央记忆阻遏转录因子如BCL6或BACH2。中央记忆阻遏物抑制T细胞向效应记忆细胞的分化,使得它们保持为较低分化的T细胞亚型之一,如初始和干细胞记忆T细胞。它们阻断或降低T细胞通过图1中所示的各个阶段的分化速率,使T细胞群偏向更初始的表型。
或者,转录因子可以是效应记忆阻遏转录因子,如BLIMP-1。
BCL6
B细胞淋巴瘤蛋白(BCL6)是进化上保守的锌指转录因子,其含有N-末端POZ/BTB结构域。BCL6充当转录的序列特异性阻遏物,并且已显示调控B细胞的STAT依赖性白细胞介素4(IL-4)应答。它与几种辅阻遏复合物相互作用以抑制转录。
BCL6的氨基酸序列可从UniProt以登录号P41182获得,并如下面的SEQ ID No.1所示。
SEQ ID No.1-BCL6
MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSMYSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEGPEQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC
BCL6在以下氨基酸位置处包含六个锌指:518-541、546-568、574-596、602-624、630-652、658-681。
BACH2
广泛的复合物和帽和领(cap′n′collar)同源(Bach)2蛋白,也称为bric-a-brac和tramtrack,是92kDa的转录因子。经由碱性亮氨酸拉链结构域,它与肌肉腱膜纤维肉瘤(Maf)家族的蛋白质异源二聚体化。Bach2基因座位于超级增强子(Super Enhancer)(SE)中,并调节SE调节的基因的表达。SE对细胞谱系基因表达至关重要。在T细胞中,大多数SE调节的基因是细胞因子和细胞因子受体基因。在所有T细胞谱系中,Bach2是与SE相关联的主导性基因。
Bach2蛋白由72个磷酸化位点组成。在这些位点中,Ser-335由Akt靶标的共有序列(RXRXX(S/T)X)组成。11个位点(Ser-260、Ser-314、Thr-318、Thr-321、Ser-336、Ser-408、Thr-442、Ser-509、Ser-535、Ser-547和Ser-718)带有mTOR靶标的共有序列(脯氨酸位于+1位置处)。将Ser-535和Ser-509取代为Ala增加了Bach2的核定位,并增大了其靶基因的下调。
位点Ser-520已鉴定为用于磷酸化的Akt底物。将Ser-520取代为Ala也增加了Bach2的阻遏能力。eGFP融合至WT或突变的Bach2揭示了S520A Bach2的增大的核定位。T细胞激活后Bach2的磷酸化导致细胞质中的Bach2隔离。磷酸化位点处的突变使得Bach2对此类隔离具有抗性,并因此其对细胞核的定位增加。
本发明的细胞可以包含表达Bach2的变体的外源核酸分子,所述Bach2的变体与野生型蛋白质相比具有增加的核定位。参考如SEQ ID No.2所示的序列,该变体可以在Ser-535、Ser-520或Ser-509处具有突变。突变可以是取代,例如Ser至Ala取代。
Bach2结合在共有基序(5’-TGA(C/G)TCAGC-3’)上,其是AP-1家族识别的基序(5’-TGA(C/G)TCA-3’)的一部分。AP-1家族的转录因子参与诱导TCR激活的下游基因的表达。AP-1转录因子家族包括c-Jun、JunB和c-Fos。AP-1因子在TCR激活后被磷酸化,并随后调节参与效应分化的基因。Bach2通过与AP-1竞争结合在重叠基序上来阻遏那些基因的激活。
Bach2 mRNA的表达在初始CD8T细胞中高,并且在中央记忆(CD62L+KLRG1-)、效应(CD62L-KLRG1-)和终末分化的效应(CD62L-KLRG1+)细胞中逐渐下调。Bach2的缺乏导致终末分化的T细胞,并增加细胞凋亡。
Bac2的氨基酸序列可从UniProt以登录号Q9BYV9获得,并如下面的SEQ ID No.2所示。
SEQ ID No.2-Bach-2野生型
MSVDEKPDSPMYVYESTVHCTNILLGLNDQRKKDILCDVTLIVERKEFRAHRAVLAACSEYFWQALVGQTKNDLVVSLPEEVTARGFGPLLQFAYTAKLLLSRENIREVIRCAEFLRMHNLEDSCFSFLQTQLLNSEDGLFVCRKDAACQRPHEDCENSAGEEEDEEEETMDSETAKMACPRDQMLPEPISFEAAAIPVAEKEEALLPEPDVPTDTKESSEKDALTQYPRYKKYQLACTKNVYNASSHSTSGFASTFREDNSSNSLKPGLARGQIKSEPPSEENEEESITLCLSGDEPDAKDRAGDVEMDRKQPSPAPTPTAPAGAACLERSRSVASPSCLRSLFSITKSVELSGLPSTSQQHFARSPACPFDKGITQGDLKTDYTPFTGNYGQPHVGQKEVSNFTMGSPLRGPGLEALCKQEGELDRRSVIFSSSACDQVSTSVHSYSGVSSLDKDLSEPVPKGLWVGAGQSLPSSQAYSHGGLMADHLPGRMRPNTSCPVPIKVCPRSPPLETRTRTSSSCSSYSYAEDGSGGSPCSLPLCEFSSSPCSQGARFLATEHQEPGLMGDGMYNQVRPQIKCEQSYGTNSSDESGSFSEADSESCPVQDRGQEVKLPFPVDQITDLPRNDFQMMIKMHKLTSEQLEFIHDVRRRSKNRIAAQRCRKRKLDCIQNLECEIRKLVCEKEKLLSERNQLKACMGELLDNFSCLSQEVCRDIQSPEQIQALHRYCPVLRPMDLPTASSINPAPLGAEQNIAASQCAVGENVPCCLEPGAAPPGPPWAPSNTSENCTSGRRLEGTDPGTFSERGPPLEPRSQTVTVDFCQEMTDKCTTDEQPRKDYT
在位置520处具有S至A取代的突变体Bach2序列如SEQ ID No.3所示。S520A取代以粗体和下划线表示。
SEQ ID No.3-S520A Bach2突变体(对AKT不敏感)
MSVDEKPDSPMYVYESTVHCTNILLGLNDQRKKDILCDVTLIVERKEFRAHRAVLAACSEYFWQALVGQTKNDLVVSLPEEVTARGFGPLLQFAYTAKLLLSRENIREVIRCAEFLRMHNLEDSCFSFLQTQLLNSEDGLFVCRKDAACQRPHEDCENSAGEEEDEEEETMDSETAKMACPRDQMLPEPISFEAAAIPVAEKEEALLPEPDVPTDTKESSEKDALTQYPRYKKYQLACTKNVYNASSHSTSGFASTFREDNSSNSLKPGLARGQIKSEPPSEENEEESITLCLSGDEPDAKDRAGDVEMDRKQPSPAPTPTAPAGAACLERSRSVASPSCLRSLFSITKSVELSGLPSTSQQHFARSPACPFDKGITQGDLKTDYTPFTGNYGQPHVGQKEVSNFTMGSPLRGPGLEALCKQEGELDRRSVIFSSSACDQVSTSVHSYSGVSSLDKDLSEPVPKGLWVGAGQSLPSSQAYSHGGLMADHLPGRMRPNTSCPVPIKVCPRSPPLETRTRTSASCSSYSYAEDGSGGSPCSLPLCEFSSSPCSQGARFLATEHQEPGLMGDGMYNQVRPQIKCEQSYGTNSSDESGSFSEADSESCPVQDRGQEVKLPFPVDQITDLPRNDFQMMIKMHKLTSEQLEFIHDVRRRSKNRIAAQRCRKRKLDCIQNLECEIRKLVCEKEKLLSERNQLKACMGELLDNFSCLSQEVCRDIQSPEQIQALHRYCPVLRPMDLPTASSINPAPLGAEQNIAASQCAVGENVPCCLEPGAAPPGPPWAPSNTSENCTSGRRLEGTDPGTFSERGPPLEPRSQTVTVDFCQEMTDKCTTDEQPRKDYT
BLIMP-1
B淋巴细胞诱导的成熟蛋白1(BLIMP1)充当beta-干扰素(β-IFN)基因表达的阻遏物。该蛋白质特异性结合β-IFN基因启动子的PRDI(正调节结构域I元件)。
B淋巴细胞、T淋巴细胞、NK细胞和其他免疫系统细胞中Blimp-1蛋白的增加的表达引起通过分泌抗体的浆细胞的增殖和分化的免疫应答。Blimp-1也被认为是造血干细胞的“主要调节物”。
BLIMP-1参与控制抗体分泌细胞(ASC)的终末分化,并且在维持效应T细胞的稳态中具有重要作用。
BLIMP-1的氨基酸序列可从UniProt以登录号O75626获得,并如下面的SEQ IDNo.4所示。
SEQ ID No.4–BLIMP-1
MLDICLEKRVGTTLAAPKCNSSTVRFQGLAEGTKGTMKMDMEDADMTLWTEAEFEEKCTYIVNDHPWDSGADGGTSVQAEASLPRNLLFKYATNSEEVIGVMSKEYIPKGTRFGPLIGEIYTNDTVPKNANRKYFWRIYSRGELHHFIDGFNEEKSNWMRYVNPAHSPREQNLAACQNGMNIYFYTIKPIPANQELLVWYCRDFAERLHYPYPGELTMMNLTQTQSSLKQPSTEKNELCPKNVPKREYSVKEILKLDSNPSKGKDLYRSNISPLTSEKDLDDFRRRGSPEMPFYPRVVYPIRAPLPEDFLKASLAYGIERPTYITRSPIPSSTTPSPSARSSPDQSLKSSSPHSSPGNTVSPVGPGSQEHRDSYAYLNASYGTEGLGSYPGYAPLPHLPPAFIPSYNAHYPKFLLPPYGMNCNGLSAVSSMNGINNFGLFPRLCPVYSNLLGGGSLPHPMLNPTSLPSSLPSDGARRLLQPEHPREVLVPAPHSAFSFTGAAASMKDKACSPTSGSPTAGTAATAEHVVQPKATSAAMAAPSSDEAMNLIKNKRNMTGYKTLPYPLKKQNGKIKYECNVCAKTFGQLSNLKVHLRVHSGERPFKCQTCNKGFTQLAHLQKHYLVHTGEKPHECQVCHKRFSSTSNLKTHLRLHSGEKPYQCKVCPAKFTQFVHLKLHKRLHTRERPHKCSQCHKNYIHLCSLKVHLKGNCAAAPAPGLPLEDLTRINEEIEKFDISDNADRLEDVEDDISVISVVEKEILAVVRKEKEETGLKVSLQRNMGNGLLSSGCSLYESSDLPLMKLPPSNPLPLVPVKVKQETVEPMDP
FOXO1
叉头框蛋白O1(FOXO1),也称为横纹肌肉瘤中的叉头,是人中由FOXO1基因编码的蛋白质。FOXO1是转录因子,其在通过胰岛素信号传导调节糖异生和糖原分解中起重要作用,并且也是决定前脂肪细胞致力于脂肪生成的核心。它主要通过多个残基的磷酸化来调节;其转录活性取决于其磷酸化状态。
FOXO1的氨基酸序列可从UniProt以登录号Q12778获得,并如下面的SEQ ID No.5所示。
SEQ ID No.5–FOXO1
AEAPQVVEIDPDFEPLPRPRSCTWPLPRPEFSQSNSATSSPAPSGSAAANPDAAAGLPSASAAAVSADFMSNLSLLEESEDFPQAPGSVAAAVAAAAAAAATGGLCGDFQGPEAGCLHPAPPQPPPPGPLSQHPPVPPAAAGPLAGQPRKSSSSRRNAWGNLSYADLITKAIESSAEKRLTLSQIYEWMVKSVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIRVQNEGTGKSSWWMLNPEGGKSGKSPRRRAASMDNNSKFAKSRSRAAKKKASLQSGQEGAGDSPGSQFSKWPASPGSHSNDDFDNWSTFRPRTSSNASTISGRLSPIMTEQDDLGEGDVHSMVYPPSAAKMASTLPSLSEISNPENMENLLDNLNLLSSPTSLTVSTQSSPGTMMQQTPCYSFAPPNTSLNSPSPNYQKYTYGQSSMSPLPQMPIQTLQDNKSSYGGMSQYNCAPGLLKELLTSDSPPHNDIMTPVDPGVAQPNSRVLGQNVMMGPNSVMSTYGSQASHNKMMNPSSHTHPGHAQQTSAVNGRPLPHTVSTMPHTSGMNRLTQVKTPVQVPLPHPMQMSALGGYSSVSSCNGYGRMGLLHQEKLPSDLDGMFIERLDCDMESIIRNDLMDGDTLDFNFDNVLPNQSFPHSVKTTTHSWVSG
EOMES
EOMES,也称为脱中胚蛋白(Eomesodermin)和T-box脑蛋白2(Tbr2)是人中由EOMES基因编码的蛋白质。它是共享共同的DNA结合结构域(T-box)的保守蛋白质家族的成员。T-box基因编码控制基因表达的转录因子,参与发育过程的调节。Eomes本身控制放射状胶质细胞以及其他相关细胞的调节。也已发现Eomes在免疫应答中起作用,并且对于其在其他系统中的关联性存在一些松散的证据。
EOMES的氨基酸序列可从UniProt以登录号095936获得,并如下面的SEQ ID No.6所示。
SEQ ID No.6–EOMES
QLGEQLLVSSVNLPGAHFYPLESARGGSGGSAGHLPSAAPSPQKLDLDKASKKFSGSLSCEAVSGEPAAASAGAPAAMLSDTDAGDAFASAAAVAKPGPPDGRKGSPCGEEELPSAAAAAAAAAAAAAATARYSMDSLSSERYYLQSPGPQGSELAAPCSLFPYQAAAGAPHGPVYPAPNGARYPYGSMLPPGGFPAAVCPPGRAQFGPGAGAGSGAGGSSGGGGGPGTYQYSQGAPLYGPYPGAAAAGSCGGLGGLGVPGSGFRAHVYLCNRPLWLKFHRHQTEMIITKQGRRMFPFLSFNINGLNPTAHYNVFVEVVLADPNHWRFQGGKWVTCGKADNNMQGNKMYVHPESPNTGSHWMRQEISFGKLKLTNNKGANNNNTQMIVLQSLHKYQPRLHIVEVTEDGVEDLNEPSKTQTFTFSETQFIAVTAYQNTDITQLKIDHNPFAKGFRDNYDSSHQIVPGGRYGVQSFFPEPFVNTLPQARYYNGERTVPQTNGLLSPQQSEEVANPPQRWLVTPVQQPGTNKLDISSYESEYTSSTLLPYGIKSLPLQTSHALGYYPDPTFPAMAGWGGRGSYQRKMAAGLPWTSRTSPTVFSEDQLSKEKVKEEIGSSWIETPPSIKSLDSNDSGVYTSACKRRRLSPSNSSNENSPSIKCEDINAEEYSKDTSKGMGGYYAFYTT
RUNX3
Runx3或Runt相关转录因子3是含有runt结构域的转录因子的家族的成员。该蛋白质和beta亚基的异源二聚体形成与在许多增强子和启动子中发现的核心DNA序列5′-YGYGGT-3′结合的复合物,并且可以激活或阻抑转录。它还与其他转录因子相互作用。它起肿瘤阻抑物的作用,并且该基因在癌症中经常被删除或转录沉默。已经发现了编码该基因的不同同种型的多种转录物变体。
RUNX3的氨基酸序列可从UniProt以登录号Q13761获得,并如下面的SEQ ID No.7所示。
SEQ ID No.7-RUNX3
MRIPVDPSTSRRFTPPSPAFPCGGGGGKMGENSGALSAQAAVGPGGRARPEVRSMVDVLADHAGELVRTDSPNFLCSVLPSHWRCNKTLPVAFKVVALGDVPDGTVVTVMAGNDENYSAELRNASAVMKNQVARFNDLRFVGRSGRGKSFTLTITVFTNPTQVATYHRAIKVTVDGPREPRRHRQKLEDQTKPFPDRFGDLERLRMRVTPSTPSPRGSLSTTSHFSSQPQTPIQGTSELNPFSDPRQFDRSFPTLPTLTESRFPDPRMHYPGAMSAAFPYSATPSGTSISSLSVAGMPATSRFHHTYLPPPYPGAPQNQSGPFQANPSPYHLYYGTSSGSYQFSMVAGSSSGGDRSPTRMLASCTSSAASVAAGNLMNPSLGGQSDGVEADGSHSNSPTALSTPGRMDEAVWRPYPAAKRVKLD
CBF BETA
核心结合因子亚基beta(CBF beta)是异源二聚体核心结合转录因子的beta亚基,属于PEBP2/CBF转录因子家族,其主要调节对血细胞发生具有特异性的基因(例如RUNX1)和对骨发生具有特异性的基因(例如RUNX2)。beta亚基是非DNA结合调节亚基;它通变构增强alpha亚基与DNA结合,因为复合物与各种增强子和启动子(包括鼠白血病病毒、多瘤病毒增强子、T细胞受体增强子和GM-CSF启动子)的核心位点结合。可变剪接产生两种mRNA变体,每种变体编码独特的羧基末端。
CBF beta的氨基酸序列可从UniProt以登录号Q13951获得,并如下面的SEQ IDNo.8所示。
SEQ ID No.8-CBF beta
MPRVVPDQRSKFENEEFFRKLSRECEIKYTGFRDRPHEERQARFQNACRDGRSEIAFVATGTNLSLQFFPASWQGEQRQTPSREYVDLEREAGKVYLKAPMILNGVCVIWKGWIDLQRLDGMGCLEFDEERAQQEDALAQQAFEEARRRTREFEDRDRSHREEMEARRQQDPSPGSNLGGGDDLKLR
外源核酸分子
本发明提供了包含编码转录因子的外源核酸分子的细胞。词语“外源”意指核酸分子通过重组方法制备,并通过载体引入到细胞中。将细胞工程化以含有核酸分子并表达(或过表达)转录因子。
核酸
如本文所用,术语“多核苷酸”、“核苷酸”和“核酸”旨在彼此同义。
技术人员将会理解,由于遗传密码的简并性,许多不同的多核苷酸和核酸可以编码相同的多肽。另外,应当理解的是,技术人员可以使用常规技术进行核苷酸替换,其不影响此处描述的多核苷酸编码的多肽序列,以反映多肽在其中表达的任何特定宿主生物体的密码子使用。
根据本发明的核酸可以包含DNA或RNA。它们可以是单链或双链的。它们也可以是其中包括合成或修饰的核苷酸的多核苷酸。对寡核苷酸的许多不同类型的修饰是本领域已知的。这些包括甲基膦酸酯和硫代磷酸酯主链,在分子的3′和/或5′末端添加吖啶或多聚赖氨酸链。为了如本文所述的用途的目的,应当理解可以通过本领域可用的任何方法来修饰多核苷酸。可以进行此类修饰以增强感兴趣的多核苷酸的体内活性或寿命。
与核苷酸序列有关的术语“变体”、“同源物”或“衍生物”包括对序列的一种(或多种)核酸的任何取代、变异、修饰、置换、缺失或添加。
核酸构建体
本发明提供了核酸构建体,其包含编码嵌合抗原受体(CAR)的第一核酸序列和编码转录因子的第二核酸序列。
核酸构建体还可以包含使两种或更多种蛋白质的表达成为可能的核酸序列。例如,它可以包含编码两个核酸序列之间的切割位点的序列。切割位点可以是自切割的,使得当产生新生多肽时,它立即被切割成两种蛋白质而不需要任何外部切割活性。
已知各种自切割位点,包括口蹄疫病毒(FMDV)2a自切割肽,其具有以下序列:
SEQ ID NO:9
RAEGRGSLLTCGDVEENPGP
或
SEQ ID NO:10
QCTNYALLKLAGDVESNPGP
或者,共表达序列可以是内部核糖体进入序列(IRES)或内部启动子。
载体
本发明还提供了载体,或载体的试剂盒,其包含根据本发明的一种或多种核酸序列或构建体。此类载体可以用于将核酸序列或构建体引入到宿主细胞中,使得其表达由核酸序列或构建体编码的蛋白质。
载体可以是例如质粒或病毒载体,如逆转录病毒载体或慢病毒载体,或基于转座子的载体或合成mRNA。
载体可以能够转染或转导T细胞。
细胞
本发明提供了共表达CAR和转录因子的细胞。
细胞可以是细胞裂解性免疫细胞。
细胞裂解性免疫细胞可以是T细胞或T淋巴细胞,其是在细胞介导的免疫中发挥中心作用的淋巴细胞类型。通过细胞表面上T细胞受体(TCR)的存在,它们可以与其他淋巴细胞如B细胞和自然杀伤细胞(NK细胞)区分。存在多种类型的T细胞,如下文所总结。
辅助性T辅助细胞(TH细胞)在免疫学过程中辅助其他白细胞,包括B细胞成熟为浆细胞和记忆B细胞,以及激活细胞毒性T细胞和巨噬细胞。TH细胞在其表面上表达CD4。当TH细胞通过抗原呈递细胞(APC)的表面上的MHC II类分子得以呈递肽抗原时,TH细胞被激活。这些细胞可以分化成几种亚型之一,包括TH1、TH2、TH3、TH17、Th9或TFH,其分泌不同的细胞因子以促进不同类型的免疫应答。
细胞毒性T细胞(TC细胞或CTL)破坏病毒感染的细胞和肿瘤细胞,并且也涉及移植排斥。CTL在其表面处表达CD8。这些细胞通过与存在于所有有核细胞的表面上的MHC I类缔合的抗原结合而识别其靶标。通过调节性T细胞分泌的IL-10、腺苷和其他分子,可以使CD8+细胞失活至无反应性(anergic)状态,其阻止自身免疫性疾病,如实验性自身免疫性脑脊髓炎。
记忆T细胞是在感染已经消退之后长期持续的抗原特异性T细胞的子集。它们在再次暴露于其关联抗原后迅速扩增为大量效应T细胞,从而为免疫系统提供针对过去感染的“记忆”。记忆T细胞包含三种亚型:中央记忆T细胞(TCM细胞)和两种类型的效应记忆T细胞(TEM细胞和TEMRA细胞)。记忆细胞可以是CD4+或CD8+。记忆T细胞通常表达细胞表面蛋白CD45RO。
调节性T细胞(Treg细胞),以前称为抑制性T细胞,对于维持免疫耐受性至关重要。它们的主要作用是关闭T细胞介导的免疫以朝向免疫反应的结束,以及阻抑逃脱胸腺中阴性选择的过程的自身反应性T细胞。
已经描述了两种主要类型的CD4+Treg细胞,即天然存在的Treg细胞和适应性Treg细胞。
天然存在的Treg细胞(也称为CD4+CD25+FoxP3+Treg细胞)出现在胸腺中,并已经与发育中的T细胞和已经用TSLP激活的髓样(CD11c+)和浆细胞样(CD123+)树突状细胞之间的相互作用相关。天然存在的Treg细胞可以通过称为FoxP3的细胞内分子的存在与其他T细胞区分开来。FOXP3基因的突变可以阻止调节性T细胞发育,导致致命的自身免疫性疾病IPEX。
适应性Treg细胞(也称为Tr1细胞或Th3细胞)可以在正常免疫应答期间产生。
自然杀伤细胞(或NK细胞)是形成先天免疫系统的一部分的细胞裂解性细胞的类型。NK细胞以MHC非依赖性方式对来自病毒感染细胞的先天信号提供快速应答。
NK细胞(属于先天性淋巴样细胞群)被定义为大颗粒淋巴细胞(LGL),并构成从产生B和T淋巴细胞的共同淋巴样祖细胞分化的第三种细胞。已知NK细胞在骨髓、淋巴结、脾、扁桃体和胸腺中分化和成熟,在那里它们进入到循环中。
本发明的细胞可以是上述任何细胞类型。
在转录因子减少或抑制T细胞分化或耗竭的情况下,细胞可以优选为以下T细胞亚型之一:初始T细胞;干细胞记忆T细胞;和中央记忆T细胞。
本发明的细胞可以从患者自己的外周血(第1方)离体产生,或者在来自供体外周血的造血干细胞移植物(第2方)或来自不相关供体的外周血(第3方)的背景中产生。
或者,细胞可以衍生自诱导型祖细胞或胚胎祖细胞离体分化为例如T细胞。或者,可以使用保留其裂解功能并且可以充当治疗剂的永生化细胞系。
在所有这些实施方案中,可以通过包括用病毒载体转导、用DNA或RNA转染的许多方式之一引入编码CAR的DNA或RNA和转录因子来生成细胞。
本发明的细胞可以是来自受试者的离体细胞。细胞可以来自外周血单核细胞(PBMC)样品。细胞可以在用本发明的核酸序列或构建体转导之前激活和/或扩增,例如通过用抗CD3单克隆抗体处理。
本发明的细胞可以通过以下制备:
(i)从受试者或上文列出的其他来源分离含有细胞的样品;和
(ii)用根据本发明的核酸序列或构建体转导或转染细胞。
组合物
本发明还涉及含有多个本发明的细胞的药物组合物。药物组合物可以另外包含药学上可接受的载体、稀释剂或赋形剂。药物组合物可以任选地包含一种或多种另外的药学活性多肽和/或化合物。此类剂型可以是例如以适合用于静脉输注的形式。
治疗方法
本发明的细胞可以能够杀伤靶细胞,例如癌细胞。
本发明的细胞可以用于治疗感染,例如病毒感染。
本发明的细胞还可以用于控制致病性免疫应答,例如在自身免疫性疾病、过敏和移植物抗宿主排斥中。
本发明的细胞可以用于治疗癌性疾病,例如膀胱癌、乳腺癌、结肠癌、子宫内膜癌、肾癌(肾细胞)、白血病、肺癌、黑色素瘤、非霍奇金淋巴瘤、胰腺癌、前列腺癌和甲状腺癌。
本发明的细胞可以用于治疗:口腔和咽的癌症,包括舌、口和咽的癌症;消化系统的癌症,包括食管癌、胃癌和结肠直肠癌;肝和胆系(biliary tree)的癌症,包括肝细胞癌和胆管癌;呼吸系统的癌症,包括支气管癌和喉癌;骨和关节的癌症,包括骨肉瘤;皮肤的癌症,包括黑色素瘤;乳腺癌;生殖道的癌症,包括女性的子宫、卵巢和宫颈癌,男性的前列腺和睾丸癌;肾道(renal tract)的癌症,包括肾细胞癌和尿道(utterers)或膀胱的移行细胞癌;脑癌,包括胶质瘤、多形性胶质母细胞瘤和髓母细胞瘤;内分泌系统的癌症,包括甲状腺癌、肾上腺癌和多种内分泌新生物综合征相关的癌症;淋巴瘤,包括霍奇金淋巴瘤和非霍奇金淋巴瘤;多发性骨髓瘤和浆细胞瘤;急性和慢性白血病,髓样或淋巴样;以及其他和未指明的部位的癌症,包括神经母细胞瘤。
现在将通过实施例进一步描述本发明,这些实施例旨在帮助本领域普通技术人员实施本发明,而并不意图以任何方式限制本发明的范围。
实施例
实施例1–嵌合抗原受体(CAR)和转录因子(TF)共表达
双顺反子构建体在BW5T细胞中表达为单一转录物,其在2A位点自切割以产生嵌合抗原受体(CAR);和转录因子(TF)。还产生了缺乏2A位点和转录因子(“仅CAR”)或缺乏CAR和2A位点(“仅TF”)的对照构建体。
CAR是抗CD19CAR,其包含衍生自CD3zeta和衍生自共刺激受体41BB的胞内域。
测试构建体包含下表中所示的转录因子:
实施例2–表型测定
在不存在CAR抗原的情况下,T细胞表面上的各种CAR的表达可以影响那些T细胞的记忆状态。此外,CAR与其关联抗原的结合激活T细胞并导致进一步从更初始的中央记忆表型分化为更分化的效应记忆/效应表型。预期适当的转录因子/阻遏物的表达不同程度地阻止该CAR介导的分化。
将表达各种CAR-TF组合的T细胞与相关的仅CAR和仅TF的对照一起与CD19阳性SKOV3靶细胞共培养24小时,然后回收并培养T细胞直至第7天。在共培养的第0天和第7天通过流式细胞术分析以下记忆标志物,以观察表达使其偏向中央记忆的因子的细胞是否在转导后更初始,并且在用携带抗原的靶细胞刺激时保持更初始。
记忆标志物-CCR7、CD45RA、CD62L、CD27
FOXO1的数据显示在图2中。对于CD4+和CD8+亚群两者,FOXO1与CAR(HD37)的共表达在第0天和第7天产生更大比例的初始和中央记忆细胞(CM)。这表明FOXO1在转导后和与靶细胞共培养后均使细胞偏向初始/中央记忆表型。
图3显示了与靶细胞共培养24小时后6天的CD27和CD62L表达数据。转录因子EOMES引起CD4+和CD8+T细胞亚群两者中CD27的显著上调。FOXO1引起CD27的上调,尤其是在CD8+细胞上。转录因子FOXO1引起CD4+和CD8+亚群两者上CD62L的显著上调。CD62L是初始/中央记忆细胞的标志物,并且FOX01的记忆表型与CD62L水平相关:更初始和记忆细胞。CD27是完全分化的效应细胞以外的所有的标志物,因此可能表达EOMES的细胞主要是分化程度较低的效应记忆亚型,其不显示CD62L的显著上调。
如图4中所示,Runx3和CBFbeta两者的存在引起转导后(第0天)和24小时共培养后6天的CD62L的上调。
BACH2和BACH2突变体S520A的数据显示在图5中。BACH2和BACH2S520A两者在第0天和第7天都增加了初始和中央记忆细胞(CM)的比例。
在单独的测定中,将表达各种CAR-TF组合的T细胞与相关的仅CAR一起与CD19阳性SupT1靶细胞共培养。在共培养的第0天和第2、4和7天通过流式细胞术分析以下耗竭标志物的表达,以观察细胞在刺激时是否表达“耗竭”标志物至较低程度。
耗竭标志物-PD1、Tim3、Lag3
细胞在CAR-表达(经由RQR8转导标志物)和各种T细胞以及根据感兴趣的亚群的T细胞亚群标志物(CD3和CD8)上门控细胞。
实施例3–功能测定
随着细胞逐渐分化,它们在体外获得更强的裂解靶细胞的能力并分泌更多的IFN-γ,但是具有较低的增殖能力。本研究调查偏向表型是否会改变这些能力。
将SupT1细胞(CD19阴性)工程化为CD19阳性,产生靶阴性和阳性细胞系。用SupT1细胞或SupT1.CD19细胞1:1刺激(chanllenge)转导的和未转导的T细胞以及用对照构建体转导的T细胞。在24和72小时后通过FACS分析靶细胞的杀伤。还使用Incucyte测定监测杀伤,所述Incucyte测定涉及在表达相应CAR抗原的单层荧光标记的贴壁细胞上共培养转导的T细胞。CAR介导的细胞毒性导致荧光靶细胞数量的减少,其可以随时间连续监测,从而允许测量CAR介导的细胞毒性的动力学。
为了监测细胞因子释放,在刺激后48小时取样上清液,并使用细胞因子珠阵列测定下列细胞因子:IL2、IL4、IL6、IL10、TNF-a、IFN-g、GzmB。
为了测量增殖,用荧光染料Cell Trace Violet标记T细胞20分钟。标记后,以1:1(靶细胞:转导的T细胞)的比例建立共培养物。CAR介导的T细胞增殖导致Cell Traceviolet荧光的减少,因为染料在连续的子细胞世代之间分配。可以在共培养的第4天和第7天通过流式细胞术测量该稀释发生的程度,以及因此增殖的程度。
在上述说明书中提及的所有出版物通过引用并入本文。在不脱离本发明的范围和精神的情况下,本发明的所描述的方法和系统的各种修改和变化对于本领域技术人员将是显而易见的。虽然已经结合具体的优选实施方案描述了本发明,但是应当理解,所要求保护的本发明不应该过度限于此类具体实施方案。实际上,对于分子生物学或相关领域的技术人员显而易见的用于实施本发明的所述模式的各种修改旨在在所附权利要求书的范围内。
序列表
<110> 奥托路斯有限公司
<120> 表达CAR和转录因子的细胞及其用途
<130> P111277PCT
<150> GB 1621889.3
<151> 2016-12-21
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 706
<212> PRT
<213> 人
<400> 1
Met Ala Ser Pro Ala Asp Ser Cys Ile Gln Phe Thr Arg His Ala Ser
1 5 10 15
Asp Val Leu Leu Asn Leu Asn Arg Leu Arg Ser Arg Asp Ile Leu Thr
20 25 30
Asp Val Val Ile Val Val Ser Arg Glu Gln Phe Arg Ala His Lys Thr
35 40 45
Val Leu Met Ala Cys Ser Gly Leu Phe Tyr Ser Ile Phe Thr Asp Gln
50 55 60
Leu Lys Cys Asn Leu Ser Val Ile Asn Leu Asp Pro Glu Ile Asn Pro
65 70 75 80
Glu Gly Phe Cys Ile Leu Leu Asp Phe Met Tyr Thr Ser Arg Leu Asn
85 90 95
Leu Arg Glu Gly Asn Ile Met Ala Val Met Ala Thr Ala Met Tyr Leu
100 105 110
Gln Met Glu His Val Val Asp Thr Cys Arg Lys Phe Ile Lys Ala Ser
115 120 125
Glu Ala Glu Met Val Ser Ala Ile Lys Pro Pro Arg Glu Glu Phe Leu
130 135 140
Asn Ser Arg Met Leu Met Pro Gln Asp Ile Met Ala Tyr Arg Gly Arg
145 150 155 160
Glu Val Val Glu Asn Asn Leu Pro Leu Arg Ser Ala Pro Gly Cys Glu
165 170 175
Ser Arg Ala Phe Ala Pro Ser Leu Tyr Ser Gly Leu Ser Thr Pro Pro
180 185 190
Ala Ser Tyr Ser Met Tyr Ser His Leu Pro Val Ser Ser Leu Leu Phe
195 200 205
Ser Asp Glu Glu Phe Arg Asp Val Arg Met Pro Val Ala Asn Pro Phe
210 215 220
Pro Lys Glu Arg Ala Leu Pro Cys Asp Ser Ala Arg Pro Val Pro Gly
225 230 235 240
Glu Tyr Ser Arg Pro Thr Leu Glu Val Ser Pro Asn Val Cys His Ser
245 250 255
Asn Ile Tyr Ser Pro Lys Glu Thr Ile Pro Glu Glu Ala Arg Ser Asp
260 265 270
Met His Tyr Ser Val Ala Glu Gly Leu Lys Pro Ala Ala Pro Ser Ala
275 280 285
Arg Asn Ala Pro Tyr Phe Pro Cys Asp Lys Ala Ser Lys Glu Glu Glu
290 295 300
Arg Pro Ser Ser Glu Asp Glu Ile Ala Leu His Phe Glu Pro Pro Asn
305 310 315 320
Ala Pro Leu Asn Arg Lys Gly Leu Val Ser Pro Gln Ser Pro Gln Lys
325 330 335
Ser Asp Cys Gln Pro Asn Ser Pro Thr Glu Ser Cys Ser Ser Lys Asn
340 345 350
Ala Cys Ile Leu Gln Ala Ser Gly Ser Pro Pro Ala Lys Ser Pro Thr
355 360 365
Asp Pro Lys Ala Cys Asn Trp Lys Lys Tyr Lys Phe Ile Val Leu Asn
370 375 380
Ser Leu Asn Gln Asn Ala Lys Pro Glu Gly Pro Glu Gln Ala Glu Leu
385 390 395 400
Gly Arg Leu Ser Pro Arg Ala Tyr Thr Ala Pro Pro Ala Cys Gln Pro
405 410 415
Pro Met Glu Pro Glu Asn Leu Asp Leu Gln Ser Pro Thr Lys Leu Ser
420 425 430
Ala Ser Gly Glu Asp Ser Thr Ile Pro Gln Ala Ser Arg Leu Asn Asn
435 440 445
Ile Val Asn Arg Ser Met Thr Gly Ser Pro Arg Ser Ser Ser Glu Ser
450 455 460
His Ser Pro Leu Tyr Met His Pro Pro Lys Cys Thr Ser Cys Gly Ser
465 470 475 480
Gln Ser Pro Gln His Ala Glu Met Cys Leu His Thr Ala Gly Pro Thr
485 490 495
Phe Pro Glu Glu Met Gly Glu Thr Gln Ser Glu Tyr Ser Asp Ser Ser
500 505 510
Cys Glu Asn Gly Ala Phe Phe Cys Asn Glu Cys Asp Cys Arg Phe Ser
515 520 525
Glu Glu Ala Ser Leu Lys Arg His Thr Leu Gln Thr His Ser Asp Lys
530 535 540
Pro Tyr Lys Cys Asp Arg Cys Gln Ala Ser Phe Arg Tyr Lys Gly Asn
545 550 555 560
Leu Ala Ser His Lys Thr Val His Thr Gly Glu Lys Pro Tyr Arg Cys
565 570 575
Asn Ile Cys Gly Ala Gln Phe Asn Arg Pro Ala Asn Leu Lys Thr His
580 585 590
Thr Arg Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Glu Thr Cys Gly
595 600 605
Ala Arg Phe Val Gln Val Ala His Leu Arg Ala His Val Leu Ile His
610 615 620
Thr Gly Glu Lys Pro Tyr Pro Cys Glu Ile Cys Gly Thr Arg Phe Arg
625 630 635 640
His Leu Gln Thr Leu Lys Ser His Leu Arg Ile His Thr Gly Glu Lys
645 650 655
Pro Tyr His Cys Glu Lys Cys Asn Leu His Phe Arg His Lys Ser Gln
660 665 670
Leu Arg Leu His Leu Arg Gln Lys His Gly Ala Ile Thr Asn Thr Lys
675 680 685
Val Gln Tyr Arg Val Ser Ala Thr Asp Leu Pro Pro Glu Leu Pro Lys
690 695 700
Ala Cys
705
<210> 2
<211> 841
<212> PRT
<213> 人
<400> 2
Met Ser Val Asp Glu Lys Pro Asp Ser Pro Met Tyr Val Tyr Glu Ser
1 5 10 15
Thr Val His Cys Thr Asn Ile Leu Leu Gly Leu Asn Asp Gln Arg Lys
20 25 30
Lys Asp Ile Leu Cys Asp Val Thr Leu Ile Val Glu Arg Lys Glu Phe
35 40 45
Arg Ala His Arg Ala Val Leu Ala Ala Cys Ser Glu Tyr Phe Trp Gln
50 55 60
Ala Leu Val Gly Gln Thr Lys Asn Asp Leu Val Val Ser Leu Pro Glu
65 70 75 80
Glu Val Thr Ala Arg Gly Phe Gly Pro Leu Leu Gln Phe Ala Tyr Thr
85 90 95
Ala Lys Leu Leu Leu Ser Arg Glu Asn Ile Arg Glu Val Ile Arg Cys
100 105 110
Ala Glu Phe Leu Arg Met His Asn Leu Glu Asp Ser Cys Phe Ser Phe
115 120 125
Leu Gln Thr Gln Leu Leu Asn Ser Glu Asp Gly Leu Phe Val Cys Arg
130 135 140
Lys Asp Ala Ala Cys Gln Arg Pro His Glu Asp Cys Glu Asn Ser Ala
145 150 155 160
Gly Glu Glu Glu Asp Glu Glu Glu Glu Thr Met Asp Ser Glu Thr Ala
165 170 175
Lys Met Ala Cys Pro Arg Asp Gln Met Leu Pro Glu Pro Ile Ser Phe
180 185 190
Glu Ala Ala Ala Ile Pro Val Ala Glu Lys Glu Glu Ala Leu Leu Pro
195 200 205
Glu Pro Asp Val Pro Thr Asp Thr Lys Glu Ser Ser Glu Lys Asp Ala
210 215 220
Leu Thr Gln Tyr Pro Arg Tyr Lys Lys Tyr Gln Leu Ala Cys Thr Lys
225 230 235 240
Asn Val Tyr Asn Ala Ser Ser His Ser Thr Ser Gly Phe Ala Ser Thr
245 250 255
Phe Arg Glu Asp Asn Ser Ser Asn Ser Leu Lys Pro Gly Leu Ala Arg
260 265 270
Gly Gln Ile Lys Ser Glu Pro Pro Ser Glu Glu Asn Glu Glu Glu Ser
275 280 285
Ile Thr Leu Cys Leu Ser Gly Asp Glu Pro Asp Ala Lys Asp Arg Ala
290 295 300
Gly Asp Val Glu Met Asp Arg Lys Gln Pro Ser Pro Ala Pro Thr Pro
305 310 315 320
Thr Ala Pro Ala Gly Ala Ala Cys Leu Glu Arg Ser Arg Ser Val Ala
325 330 335
Ser Pro Ser Cys Leu Arg Ser Leu Phe Ser Ile Thr Lys Ser Val Glu
340 345 350
Leu Ser Gly Leu Pro Ser Thr Ser Gln Gln His Phe Ala Arg Ser Pro
355 360 365
Ala Cys Pro Phe Asp Lys Gly Ile Thr Gln Gly Asp Leu Lys Thr Asp
370 375 380
Tyr Thr Pro Phe Thr Gly Asn Tyr Gly Gln Pro His Val Gly Gln Lys
385 390 395 400
Glu Val Ser Asn Phe Thr Met Gly Ser Pro Leu Arg Gly Pro Gly Leu
405 410 415
Glu Ala Leu Cys Lys Gln Glu Gly Glu Leu Asp Arg Arg Ser Val Ile
420 425 430
Phe Ser Ser Ser Ala Cys Asp Gln Val Ser Thr Ser Val His Ser Tyr
435 440 445
Ser Gly Val Ser Ser Leu Asp Lys Asp Leu Ser Glu Pro Val Pro Lys
450 455 460
Gly Leu Trp Val Gly Ala Gly Gln Ser Leu Pro Ser Ser Gln Ala Tyr
465 470 475 480
Ser His Gly Gly Leu Met Ala Asp His Leu Pro Gly Arg Met Arg Pro
485 490 495
Asn Thr Ser Cys Pro Val Pro Ile Lys Val Cys Pro Arg Ser Pro Pro
500 505 510
Leu Glu Thr Arg Thr Arg Thr Ser Ser Ser Cys Ser Ser Tyr Ser Tyr
515 520 525
Ala Glu Asp Gly Ser Gly Gly Ser Pro Cys Ser Leu Pro Leu Cys Glu
530 535 540
Phe Ser Ser Ser Pro Cys Ser Gln Gly Ala Arg Phe Leu Ala Thr Glu
545 550 555 560
His Gln Glu Pro Gly Leu Met Gly Asp Gly Met Tyr Asn Gln Val Arg
565 570 575
Pro Gln Ile Lys Cys Glu Gln Ser Tyr Gly Thr Asn Ser Ser Asp Glu
580 585 590
Ser Gly Ser Phe Ser Glu Ala Asp Ser Glu Ser Cys Pro Val Gln Asp
595 600 605
Arg Gly Gln Glu Val Lys Leu Pro Phe Pro Val Asp Gln Ile Thr Asp
610 615 620
Leu Pro Arg Asn Asp Phe Gln Met Met Ile Lys Met His Lys Leu Thr
625 630 635 640
Ser Glu Gln Leu Glu Phe Ile His Asp Val Arg Arg Arg Ser Lys Asn
645 650 655
Arg Ile Ala Ala Gln Arg Cys Arg Lys Arg Lys Leu Asp Cys Ile Gln
660 665 670
Asn Leu Glu Cys Glu Ile Arg Lys Leu Val Cys Glu Lys Glu Lys Leu
675 680 685
Leu Ser Glu Arg Asn Gln Leu Lys Ala Cys Met Gly Glu Leu Leu Asp
690 695 700
Asn Phe Ser Cys Leu Ser Gln Glu Val Cys Arg Asp Ile Gln Ser Pro
705 710 715 720
Glu Gln Ile Gln Ala Leu His Arg Tyr Cys Pro Val Leu Arg Pro Met
725 730 735
Asp Leu Pro Thr Ala Ser Ser Ile Asn Pro Ala Pro Leu Gly Ala Glu
740 745 750
Gln Asn Ile Ala Ala Ser Gln Cys Ala Val Gly Glu Asn Val Pro Cys
755 760 765
Cys Leu Glu Pro Gly Ala Ala Pro Pro Gly Pro Pro Trp Ala Pro Ser
770 775 780
Asn Thr Ser Glu Asn Cys Thr Ser Gly Arg Arg Leu Glu Gly Thr Asp
785 790 795 800
Pro Gly Thr Phe Ser Glu Arg Gly Pro Pro Leu Glu Pro Arg Ser Gln
805 810 815
Thr Val Thr Val Asp Phe Cys Gln Glu Met Thr Asp Lys Cys Thr Thr
820 825 830
Asp Glu Gln Pro Arg Lys Asp Tyr Thr
835 840
<210> 3
<211> 841
<212> PRT
<213> 人工序列
<220>
<223> 突变体Bach2序列
<400> 3
Met Ser Val Asp Glu Lys Pro Asp Ser Pro Met Tyr Val Tyr Glu Ser
1 5 10 15
Thr Val His Cys Thr Asn Ile Leu Leu Gly Leu Asn Asp Gln Arg Lys
20 25 30
Lys Asp Ile Leu Cys Asp Val Thr Leu Ile Val Glu Arg Lys Glu Phe
35 40 45
Arg Ala His Arg Ala Val Leu Ala Ala Cys Ser Glu Tyr Phe Trp Gln
50 55 60
Ala Leu Val Gly Gln Thr Lys Asn Asp Leu Val Val Ser Leu Pro Glu
65 70 75 80
Glu Val Thr Ala Arg Gly Phe Gly Pro Leu Leu Gln Phe Ala Tyr Thr
85 90 95
Ala Lys Leu Leu Leu Ser Arg Glu Asn Ile Arg Glu Val Ile Arg Cys
100 105 110
Ala Glu Phe Leu Arg Met His Asn Leu Glu Asp Ser Cys Phe Ser Phe
115 120 125
Leu Gln Thr Gln Leu Leu Asn Ser Glu Asp Gly Leu Phe Val Cys Arg
130 135 140
Lys Asp Ala Ala Cys Gln Arg Pro His Glu Asp Cys Glu Asn Ser Ala
145 150 155 160
Gly Glu Glu Glu Asp Glu Glu Glu Glu Thr Met Asp Ser Glu Thr Ala
165 170 175
Lys Met Ala Cys Pro Arg Asp Gln Met Leu Pro Glu Pro Ile Ser Phe
180 185 190
Glu Ala Ala Ala Ile Pro Val Ala Glu Lys Glu Glu Ala Leu Leu Pro
195 200 205
Glu Pro Asp Val Pro Thr Asp Thr Lys Glu Ser Ser Glu Lys Asp Ala
210 215 220
Leu Thr Gln Tyr Pro Arg Tyr Lys Lys Tyr Gln Leu Ala Cys Thr Lys
225 230 235 240
Asn Val Tyr Asn Ala Ser Ser His Ser Thr Ser Gly Phe Ala Ser Thr
245 250 255
Phe Arg Glu Asp Asn Ser Ser Asn Ser Leu Lys Pro Gly Leu Ala Arg
260 265 270
Gly Gln Ile Lys Ser Glu Pro Pro Ser Glu Glu Asn Glu Glu Glu Ser
275 280 285
Ile Thr Leu Cys Leu Ser Gly Asp Glu Pro Asp Ala Lys Asp Arg Ala
290 295 300
Gly Asp Val Glu Met Asp Arg Lys Gln Pro Ser Pro Ala Pro Thr Pro
305 310 315 320
Thr Ala Pro Ala Gly Ala Ala Cys Leu Glu Arg Ser Arg Ser Val Ala
325 330 335
Ser Pro Ser Cys Leu Arg Ser Leu Phe Ser Ile Thr Lys Ser Val Glu
340 345 350
Leu Ser Gly Leu Pro Ser Thr Ser Gln Gln His Phe Ala Arg Ser Pro
355 360 365
Ala Cys Pro Phe Asp Lys Gly Ile Thr Gln Gly Asp Leu Lys Thr Asp
370 375 380
Tyr Thr Pro Phe Thr Gly Asn Tyr Gly Gln Pro His Val Gly Gln Lys
385 390 395 400
Glu Val Ser Asn Phe Thr Met Gly Ser Pro Leu Arg Gly Pro Gly Leu
405 410 415
Glu Ala Leu Cys Lys Gln Glu Gly Glu Leu Asp Arg Arg Ser Val Ile
420 425 430
Phe Ser Ser Ser Ala Cys Asp Gln Val Ser Thr Ser Val His Ser Tyr
435 440 445
Ser Gly Val Ser Ser Leu Asp Lys Asp Leu Ser Glu Pro Val Pro Lys
450 455 460
Gly Leu Trp Val Gly Ala Gly Gln Ser Leu Pro Ser Ser Gln Ala Tyr
465 470 475 480
Ser His Gly Gly Leu Met Ala Asp His Leu Pro Gly Arg Met Arg Pro
485 490 495
Asn Thr Ser Cys Pro Val Pro Ile Lys Val Cys Pro Arg Ser Pro Pro
500 505 510
Leu Glu Thr Arg Thr Arg Thr Ser Ala Ser Cys Ser Ser Tyr Ser Tyr
515 520 525
Ala Glu Asp Gly Ser Gly Gly Ser Pro Cys Ser Leu Pro Leu Cys Glu
530 535 540
Phe Ser Ser Ser Pro Cys Ser Gln Gly Ala Arg Phe Leu Ala Thr Glu
545 550 555 560
His Gln Glu Pro Gly Leu Met Gly Asp Gly Met Tyr Asn Gln Val Arg
565 570 575
Pro Gln Ile Lys Cys Glu Gln Ser Tyr Gly Thr Asn Ser Ser Asp Glu
580 585 590
Ser Gly Ser Phe Ser Glu Ala Asp Ser Glu Ser Cys Pro Val Gln Asp
595 600 605
Arg Gly Gln Glu Val Lys Leu Pro Phe Pro Val Asp Gln Ile Thr Asp
610 615 620
Leu Pro Arg Asn Asp Phe Gln Met Met Ile Lys Met His Lys Leu Thr
625 630 635 640
Ser Glu Gln Leu Glu Phe Ile His Asp Val Arg Arg Arg Ser Lys Asn
645 650 655
Arg Ile Ala Ala Gln Arg Cys Arg Lys Arg Lys Leu Asp Cys Ile Gln
660 665 670
Asn Leu Glu Cys Glu Ile Arg Lys Leu Val Cys Glu Lys Glu Lys Leu
675 680 685
Leu Ser Glu Arg Asn Gln Leu Lys Ala Cys Met Gly Glu Leu Leu Asp
690 695 700
Asn Phe Ser Cys Leu Ser Gln Glu Val Cys Arg Asp Ile Gln Ser Pro
705 710 715 720
Glu Gln Ile Gln Ala Leu His Arg Tyr Cys Pro Val Leu Arg Pro Met
725 730 735
Asp Leu Pro Thr Ala Ser Ser Ile Asn Pro Ala Pro Leu Gly Ala Glu
740 745 750
Gln Asn Ile Ala Ala Ser Gln Cys Ala Val Gly Glu Asn Val Pro Cys
755 760 765
Cys Leu Glu Pro Gly Ala Ala Pro Pro Gly Pro Pro Trp Ala Pro Ser
770 775 780
Asn Thr Ser Glu Asn Cys Thr Ser Gly Arg Arg Leu Glu Gly Thr Asp
785 790 795 800
Pro Gly Thr Phe Ser Glu Arg Gly Pro Pro Leu Glu Pro Arg Ser Gln
805 810 815
Thr Val Thr Val Asp Phe Cys Gln Glu Met Thr Asp Lys Cys Thr Thr
820 825 830
Asp Glu Gln Pro Arg Lys Asp Tyr Thr
835 840
<210> 4
<211> 825
<212> PRT
<213> 人
<400> 4
Met Leu Asp Ile Cys Leu Glu Lys Arg Val Gly Thr Thr Leu Ala Ala
1 5 10 15
Pro Lys Cys Asn Ser Ser Thr Val Arg Phe Gln Gly Leu Ala Glu Gly
20 25 30
Thr Lys Gly Thr Met Lys Met Asp Met Glu Asp Ala Asp Met Thr Leu
35 40 45
Trp Thr Glu Ala Glu Phe Glu Glu Lys Cys Thr Tyr Ile Val Asn Asp
50 55 60
His Pro Trp Asp Ser Gly Ala Asp Gly Gly Thr Ser Val Gln Ala Glu
65 70 75 80
Ala Ser Leu Pro Arg Asn Leu Leu Phe Lys Tyr Ala Thr Asn Ser Glu
85 90 95
Glu Val Ile Gly Val Met Ser Lys Glu Tyr Ile Pro Lys Gly Thr Arg
100 105 110
Phe Gly Pro Leu Ile Gly Glu Ile Tyr Thr Asn Asp Thr Val Pro Lys
115 120 125
Asn Ala Asn Arg Lys Tyr Phe Trp Arg Ile Tyr Ser Arg Gly Glu Leu
130 135 140
His His Phe Ile Asp Gly Phe Asn Glu Glu Lys Ser Asn Trp Met Arg
145 150 155 160
Tyr Val Asn Pro Ala His Ser Pro Arg Glu Gln Asn Leu Ala Ala Cys
165 170 175
Gln Asn Gly Met Asn Ile Tyr Phe Tyr Thr Ile Lys Pro Ile Pro Ala
180 185 190
Asn Gln Glu Leu Leu Val Trp Tyr Cys Arg Asp Phe Ala Glu Arg Leu
195 200 205
His Tyr Pro Tyr Pro Gly Glu Leu Thr Met Met Asn Leu Thr Gln Thr
210 215 220
Gln Ser Ser Leu Lys Gln Pro Ser Thr Glu Lys Asn Glu Leu Cys Pro
225 230 235 240
Lys Asn Val Pro Lys Arg Glu Tyr Ser Val Lys Glu Ile Leu Lys Leu
245 250 255
Asp Ser Asn Pro Ser Lys Gly Lys Asp Leu Tyr Arg Ser Asn Ile Ser
260 265 270
Pro Leu Thr Ser Glu Lys Asp Leu Asp Asp Phe Arg Arg Arg Gly Ser
275 280 285
Pro Glu Met Pro Phe Tyr Pro Arg Val Val Tyr Pro Ile Arg Ala Pro
290 295 300
Leu Pro Glu Asp Phe Leu Lys Ala Ser Leu Ala Tyr Gly Ile Glu Arg
305 310 315 320
Pro Thr Tyr Ile Thr Arg Ser Pro Ile Pro Ser Ser Thr Thr Pro Ser
325 330 335
Pro Ser Ala Arg Ser Ser Pro Asp Gln Ser Leu Lys Ser Ser Ser Pro
340 345 350
His Ser Ser Pro Gly Asn Thr Val Ser Pro Val Gly Pro Gly Ser Gln
355 360 365
Glu His Arg Asp Ser Tyr Ala Tyr Leu Asn Ala Ser Tyr Gly Thr Glu
370 375 380
Gly Leu Gly Ser Tyr Pro Gly Tyr Ala Pro Leu Pro His Leu Pro Pro
385 390 395 400
Ala Phe Ile Pro Ser Tyr Asn Ala His Tyr Pro Lys Phe Leu Leu Pro
405 410 415
Pro Tyr Gly Met Asn Cys Asn Gly Leu Ser Ala Val Ser Ser Met Asn
420 425 430
Gly Ile Asn Asn Phe Gly Leu Phe Pro Arg Leu Cys Pro Val Tyr Ser
435 440 445
Asn Leu Leu Gly Gly Gly Ser Leu Pro His Pro Met Leu Asn Pro Thr
450 455 460
Ser Leu Pro Ser Ser Leu Pro Ser Asp Gly Ala Arg Arg Leu Leu Gln
465 470 475 480
Pro Glu His Pro Arg Glu Val Leu Val Pro Ala Pro His Ser Ala Phe
485 490 495
Ser Phe Thr Gly Ala Ala Ala Ser Met Lys Asp Lys Ala Cys Ser Pro
500 505 510
Thr Ser Gly Ser Pro Thr Ala Gly Thr Ala Ala Thr Ala Glu His Val
515 520 525
Val Gln Pro Lys Ala Thr Ser Ala Ala Met Ala Ala Pro Ser Ser Asp
530 535 540
Glu Ala Met Asn Leu Ile Lys Asn Lys Arg Asn Met Thr Gly Tyr Lys
545 550 555 560
Thr Leu Pro Tyr Pro Leu Lys Lys Gln Asn Gly Lys Ile Lys Tyr Glu
565 570 575
Cys Asn Val Cys Ala Lys Thr Phe Gly Gln Leu Ser Asn Leu Lys Val
580 585 590
His Leu Arg Val His Ser Gly Glu Arg Pro Phe Lys Cys Gln Thr Cys
595 600 605
Asn Lys Gly Phe Thr Gln Leu Ala His Leu Gln Lys His Tyr Leu Val
610 615 620
His Thr Gly Glu Lys Pro His Glu Cys Gln Val Cys His Lys Arg Phe
625 630 635 640
Ser Ser Thr Ser Asn Leu Lys Thr His Leu Arg Leu His Ser Gly Glu
645 650 655
Lys Pro Tyr Gln Cys Lys Val Cys Pro Ala Lys Phe Thr Gln Phe Val
660 665 670
His Leu Lys Leu His Lys Arg Leu His Thr Arg Glu Arg Pro His Lys
675 680 685
Cys Ser Gln Cys His Lys Asn Tyr Ile His Leu Cys Ser Leu Lys Val
690 695 700
His Leu Lys Gly Asn Cys Ala Ala Ala Pro Ala Pro Gly Leu Pro Leu
705 710 715 720
Glu Asp Leu Thr Arg Ile Asn Glu Glu Ile Glu Lys Phe Asp Ile Ser
725 730 735
Asp Asn Ala Asp Arg Leu Glu Asp Val Glu Asp Asp Ile Ser Val Ile
740 745 750
Ser Val Val Glu Lys Glu Ile Leu Ala Val Val Arg Lys Glu Lys Glu
755 760 765
Glu Thr Gly Leu Lys Val Ser Leu Gln Arg Asn Met Gly Asn Gly Leu
770 775 780
Leu Ser Ser Gly Cys Ser Leu Tyr Glu Ser Ser Asp Leu Pro Leu Met
785 790 795 800
Lys Leu Pro Pro Ser Asn Pro Leu Pro Leu Val Pro Val Lys Val Lys
805 810 815
Gln Glu Thr Val Glu Pro Met Asp Pro
820 825
<210> 5
<211> 654
<212> PRT
<213> 人
<400> 5
Ala Glu Ala Pro Gln Val Val Glu Ile Asp Pro Asp Phe Glu Pro Leu
1 5 10 15
Pro Arg Pro Arg Ser Cys Thr Trp Pro Leu Pro Arg Pro Glu Phe Ser
20 25 30
Gln Ser Asn Ser Ala Thr Ser Ser Pro Ala Pro Ser Gly Ser Ala Ala
35 40 45
Ala Asn Pro Asp Ala Ala Ala Gly Leu Pro Ser Ala Ser Ala Ala Ala
50 55 60
Val Ser Ala Asp Phe Met Ser Asn Leu Ser Leu Leu Glu Glu Ser Glu
65 70 75 80
Asp Phe Pro Gln Ala Pro Gly Ser Val Ala Ala Ala Val Ala Ala Ala
85 90 95
Ala Ala Ala Ala Ala Thr Gly Gly Leu Cys Gly Asp Phe Gln Gly Pro
100 105 110
Glu Ala Gly Cys Leu His Pro Ala Pro Pro Gln Pro Pro Pro Pro Gly
115 120 125
Pro Leu Ser Gln His Pro Pro Val Pro Pro Ala Ala Ala Gly Pro Leu
130 135 140
Ala Gly Gln Pro Arg Lys Ser Ser Ser Ser Arg Arg Asn Ala Trp Gly
145 150 155 160
Asn Leu Ser Tyr Ala Asp Leu Ile Thr Lys Ala Ile Glu Ser Ser Ala
165 170 175
Glu Lys Arg Leu Thr Leu Ser Gln Ile Tyr Glu Trp Met Val Lys Ser
180 185 190
Val Pro Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala Gly Trp
195 200 205
Lys Asn Ser Ile Arg His Asn Leu Ser Leu His Ser Lys Phe Ile Arg
210 215 220
Val Gln Asn Glu Gly Thr Gly Lys Ser Ser Trp Trp Met Leu Asn Pro
225 230 235 240
Glu Gly Gly Lys Ser Gly Lys Ser Pro Arg Arg Arg Ala Ala Ser Met
245 250 255
Asp Asn Asn Ser Lys Phe Ala Lys Ser Arg Ser Arg Ala Ala Lys Lys
260 265 270
Lys Ala Ser Leu Gln Ser Gly Gln Glu Gly Ala Gly Asp Ser Pro Gly
275 280 285
Ser Gln Phe Ser Lys Trp Pro Ala Ser Pro Gly Ser His Ser Asn Asp
290 295 300
Asp Phe Asp Asn Trp Ser Thr Phe Arg Pro Arg Thr Ser Ser Asn Ala
305 310 315 320
Ser Thr Ile Ser Gly Arg Leu Ser Pro Ile Met Thr Glu Gln Asp Asp
325 330 335
Leu Gly Glu Gly Asp Val His Ser Met Val Tyr Pro Pro Ser Ala Ala
340 345 350
Lys Met Ala Ser Thr Leu Pro Ser Leu Ser Glu Ile Ser Asn Pro Glu
355 360 365
Asn Met Glu Asn Leu Leu Asp Asn Leu Asn Leu Leu Ser Ser Pro Thr
370 375 380
Ser Leu Thr Val Ser Thr Gln Ser Ser Pro Gly Thr Met Met Gln Gln
385 390 395 400
Thr Pro Cys Tyr Ser Phe Ala Pro Pro Asn Thr Ser Leu Asn Ser Pro
405 410 415
Ser Pro Asn Tyr Gln Lys Tyr Thr Tyr Gly Gln Ser Ser Met Ser Pro
420 425 430
Leu Pro Gln Met Pro Ile Gln Thr Leu Gln Asp Asn Lys Ser Ser Tyr
435 440 445
Gly Gly Met Ser Gln Tyr Asn Cys Ala Pro Gly Leu Leu Lys Glu Leu
450 455 460
Leu Thr Ser Asp Ser Pro Pro His Asn Asp Ile Met Thr Pro Val Asp
465 470 475 480
Pro Gly Val Ala Gln Pro Asn Ser Arg Val Leu Gly Gln Asn Val Met
485 490 495
Met Gly Pro Asn Ser Val Met Ser Thr Tyr Gly Ser Gln Ala Ser His
500 505 510
Asn Lys Met Met Asn Pro Ser Ser His Thr His Pro Gly His Ala Gln
515 520 525
Gln Thr Ser Ala Val Asn Gly Arg Pro Leu Pro His Thr Val Ser Thr
530 535 540
Met Pro His Thr Ser Gly Met Asn Arg Leu Thr Gln Val Lys Thr Pro
545 550 555 560
Val Gln Val Pro Leu Pro His Pro Met Gln Met Ser Ala Leu Gly Gly
565 570 575
Tyr Ser Ser Val Ser Ser Cys Asn Gly Tyr Gly Arg Met Gly Leu Leu
580 585 590
His Gln Glu Lys Leu Pro Ser Asp Leu Asp Gly Met Phe Ile Glu Arg
595 600 605
Leu Asp Cys Asp Met Glu Ser Ile Ile Arg Asn Asp Leu Met Asp Gly
610 615 620
Asp Thr Leu Asp Phe Asn Phe Asp Asn Val Leu Pro Asn Gln Ser Phe
625 630 635 640
Pro His Ser Val Lys Thr Thr Thr His Ser Trp Val Ser Gly
645 650
<210> 6
<211> 684
<212> PRT
<213> 人
<400> 6
Gln Leu Gly Glu Gln Leu Leu Val Ser Ser Val Asn Leu Pro Gly Ala
1 5 10 15
His Phe Tyr Pro Leu Glu Ser Ala Arg Gly Gly Ser Gly Gly Ser Ala
20 25 30
Gly His Leu Pro Ser Ala Ala Pro Ser Pro Gln Lys Leu Asp Leu Asp
35 40 45
Lys Ala Ser Lys Lys Phe Ser Gly Ser Leu Ser Cys Glu Ala Val Ser
50 55 60
Gly Glu Pro Ala Ala Ala Ser Ala Gly Ala Pro Ala Ala Met Leu Ser
65 70 75 80
Asp Thr Asp Ala Gly Asp Ala Phe Ala Ser Ala Ala Ala Val Ala Lys
85 90 95
Pro Gly Pro Pro Asp Gly Arg Lys Gly Ser Pro Cys Gly Glu Glu Glu
100 105 110
Leu Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
115 120 125
Ala Thr Ala Arg Tyr Ser Met Asp Ser Leu Ser Ser Glu Arg Tyr Tyr
130 135 140
Leu Gln Ser Pro Gly Pro Gln Gly Ser Glu Leu Ala Ala Pro Cys Ser
145 150 155 160
Leu Phe Pro Tyr Gln Ala Ala Ala Gly Ala Pro His Gly Pro Val Tyr
165 170 175
Pro Ala Pro Asn Gly Ala Arg Tyr Pro Tyr Gly Ser Met Leu Pro Pro
180 185 190
Gly Gly Phe Pro Ala Ala Val Cys Pro Pro Gly Arg Ala Gln Phe Gly
195 200 205
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Gly Ser Ser Gly Gly Gly
210 215 220
Gly Gly Pro Gly Thr Tyr Gln Tyr Ser Gln Gly Ala Pro Leu Tyr Gly
225 230 235 240
Pro Tyr Pro Gly Ala Ala Ala Ala Gly Ser Cys Gly Gly Leu Gly Gly
245 250 255
Leu Gly Val Pro Gly Ser Gly Phe Arg Ala His Val Tyr Leu Cys Asn
260 265 270
Arg Pro Leu Trp Leu Lys Phe His Arg His Gln Thr Glu Met Ile Ile
275 280 285
Thr Lys Gln Gly Arg Arg Met Phe Pro Phe Leu Ser Phe Asn Ile Asn
290 295 300
Gly Leu Asn Pro Thr Ala His Tyr Asn Val Phe Val Glu Val Val Leu
305 310 315 320
Ala Asp Pro Asn His Trp Arg Phe Gln Gly Gly Lys Trp Val Thr Cys
325 330 335
Gly Lys Ala Asp Asn Asn Met Gln Gly Asn Lys Met Tyr Val His Pro
340 345 350
Glu Ser Pro Asn Thr Gly Ser His Trp Met Arg Gln Glu Ile Ser Phe
355 360 365
Gly Lys Leu Lys Leu Thr Asn Asn Lys Gly Ala Asn Asn Asn Asn Thr
370 375 380
Gln Met Ile Val Leu Gln Ser Leu His Lys Tyr Gln Pro Arg Leu His
385 390 395 400
Ile Val Glu Val Thr Glu Asp Gly Val Glu Asp Leu Asn Glu Pro Ser
405 410 415
Lys Thr Gln Thr Phe Thr Phe Ser Glu Thr Gln Phe Ile Ala Val Thr
420 425 430
Ala Tyr Gln Asn Thr Asp Ile Thr Gln Leu Lys Ile Asp His Asn Pro
435 440 445
Phe Ala Lys Gly Phe Arg Asp Asn Tyr Asp Ser Ser His Gln Ile Val
450 455 460
Pro Gly Gly Arg Tyr Gly Val Gln Ser Phe Phe Pro Glu Pro Phe Val
465 470 475 480
Asn Thr Leu Pro Gln Ala Arg Tyr Tyr Asn Gly Glu Arg Thr Val Pro
485 490 495
Gln Thr Asn Gly Leu Leu Ser Pro Gln Gln Ser Glu Glu Val Ala Asn
500 505 510
Pro Pro Gln Arg Trp Leu Val Thr Pro Val Gln Gln Pro Gly Thr Asn
515 520 525
Lys Leu Asp Ile Ser Ser Tyr Glu Ser Glu Tyr Thr Ser Ser Thr Leu
530 535 540
Leu Pro Tyr Gly Ile Lys Ser Leu Pro Leu Gln Thr Ser His Ala Leu
545 550 555 560
Gly Tyr Tyr Pro Asp Pro Thr Phe Pro Ala Met Ala Gly Trp Gly Gly
565 570 575
Arg Gly Ser Tyr Gln Arg Lys Met Ala Ala Gly Leu Pro Trp Thr Ser
580 585 590
Arg Thr Ser Pro Thr Val Phe Ser Glu Asp Gln Leu Ser Lys Glu Lys
595 600 605
Val Lys Glu Glu Ile Gly Ser Ser Trp Ile Glu Thr Pro Pro Ser Ile
610 615 620
Lys Ser Leu Asp Ser Asn Asp Ser Gly Val Tyr Thr Ser Ala Cys Lys
625 630 635 640
Arg Arg Arg Leu Ser Pro Ser Asn Ser Ser Asn Glu Asn Ser Pro Ser
645 650 655
Ile Lys Cys Glu Asp Ile Asn Ala Glu Glu Tyr Ser Lys Asp Thr Ser
660 665 670
Lys Gly Met Gly Gly Tyr Tyr Ala Phe Tyr Thr Thr
675 680
<210> 7
<211> 424
<212> PRT
<213> 人
<400> 7
Met Arg Ile Pro Val Asp Pro Ser Thr Ser Arg Arg Phe Thr Pro Pro
1 5 10 15
Ser Pro Ala Phe Pro Cys Gly Gly Gly Gly Gly Lys Met Gly Glu Asn
20 25 30
Ser Gly Ala Leu Ser Ala Gln Ala Ala Val Gly Pro Gly Gly Arg Ala
35 40 45
Arg Pro Glu Val Arg Ser Met Val Asp Val Leu Ala Asp His Ala Gly
50 55 60
Glu Leu Val Arg Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu Pro
65 70 75 80
Ser His Trp Arg Cys Asn Lys Thr Leu Pro Val Ala Phe Lys Val Val
85 90 95
Ala Leu Gly Asp Val Pro Asp Gly Thr Val Val Thr Val Met Ala Gly
100 105 110
Asn Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn Ala Ser Ala Val Met
115 120 125
Lys Asn Gln Val Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg Ser
130 135 140
Gly Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn Pro
145 150 155 160
Thr Gln Val Ala Thr Tyr His Arg Ala Ile Lys Val Thr Val Asp Gly
165 170 175
Pro Arg Glu Pro Arg Arg His Arg Gln Lys Leu Glu Asp Gln Thr Lys
180 185 190
Pro Phe Pro Asp Arg Phe Gly Asp Leu Glu Arg Leu Arg Met Arg Val
195 200 205
Thr Pro Ser Thr Pro Ser Pro Arg Gly Ser Leu Ser Thr Thr Ser His
210 215 220
Phe Ser Ser Gln Pro Gln Thr Pro Ile Gln Gly Thr Ser Glu Leu Asn
225 230 235 240
Pro Phe Ser Asp Pro Arg Gln Phe Asp Arg Ser Phe Pro Thr Leu Pro
245 250 255
Thr Leu Thr Glu Ser Arg Phe Pro Asp Pro Arg Met His Tyr Pro Gly
260 265 270
Ala Met Ser Ala Ala Phe Pro Tyr Ser Ala Thr Pro Ser Gly Thr Ser
275 280 285
Ile Ser Ser Leu Ser Val Ala Gly Met Pro Ala Thr Ser Arg Phe His
290 295 300
His Thr Tyr Leu Pro Pro Pro Tyr Pro Gly Ala Pro Gln Asn Gln Ser
305 310 315 320
Gly Pro Phe Gln Ala Asn Pro Ser Pro Tyr His Leu Tyr Tyr Gly Thr
325 330 335
Ser Ser Gly Ser Tyr Gln Phe Ser Met Val Ala Gly Ser Ser Ser Gly
340 345 350
Gly Asp Arg Ser Pro Thr Arg Met Leu Ala Ser Cys Thr Ser Ser Ala
355 360 365
Ala Ser Val Ala Ala Gly Asn Leu Met Asn Pro Ser Leu Gly Gly Gln
370 375 380
Ser Asp Gly Val Glu Ala Asp Gly Ser His Ser Asn Ser Pro Thr Ala
385 390 395 400
Leu Ser Thr Pro Gly Arg Met Asp Glu Ala Val Trp Arg Pro Tyr Pro
405 410 415
Ala Ala Lys Arg Val Lys Leu Asp
420
<210> 8
<211> 187
<212> PRT
<213> 人
<400> 8
Met Pro Arg Val Val Pro Asp Gln Arg Ser Lys Phe Glu Asn Glu Glu
1 5 10 15
Phe Phe Arg Lys Leu Ser Arg Glu Cys Glu Ile Lys Tyr Thr Gly Phe
20 25 30
Arg Asp Arg Pro His Glu Glu Arg Gln Ala Arg Phe Gln Asn Ala Cys
35 40 45
Arg Asp Gly Arg Ser Glu Ile Ala Phe Val Ala Thr Gly Thr Asn Leu
50 55 60
Ser Leu Gln Phe Phe Pro Ala Ser Trp Gln Gly Glu Gln Arg Gln Thr
65 70 75 80
Pro Ser Arg Glu Tyr Val Asp Leu Glu Arg Glu Ala Gly Lys Val Tyr
85 90 95
Leu Lys Ala Pro Met Ile Leu Asn Gly Val Cys Val Ile Trp Lys Gly
100 105 110
Trp Ile Asp Leu Gln Arg Leu Asp Gly Met Gly Cys Leu Glu Phe Asp
115 120 125
Glu Glu Arg Ala Gln Gln Glu Asp Ala Leu Ala Gln Gln Ala Phe Glu
130 135 140
Glu Ala Arg Arg Arg Thr Arg Glu Phe Glu Asp Arg Asp Arg Ser His
145 150 155 160
Arg Glu Glu Met Glu Ala Arg Arg Gln Gln Asp Pro Ser Pro Gly Ser
165 170 175
Asn Leu Gly Gly Gly Asp Asp Leu Lys Leu Arg
180 185
<210> 9
<211> 20
<212> PRT
<213> 口蹄疫病毒
<400> 9
Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu
1 5 10 15
Asn Pro Gly Pro
20
<210> 10
<211> 20
<212> PRT
<213> 口蹄疫病毒
<400> 10
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 11
<211> 10
<212> DNA
<213> 人工序列
<220>
<223> C/EBP转录因子识别序列
<400> 11
attgcgcaat 10
<210> 12
<211> 15
<212> DNA
<213> 人工序列
<220>
<223> NF-1转录因子识别序列
<220>
<221> misc_feature
<222> (6)..(10)
<223> n是a, c, g或t
<400> 12
ttggcnnnnn gccaa 15
<210> 13
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> Akt靶标的共有序列
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa可以是任何天然存在的氨基酸
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa可以是任何天然存在的氨基酸
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa可以是Ser或Thr
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa可以是任何天然存在的氨基酸
<400> 13
Arg Xaa Arg Xaa Xaa Xaa Xaa
1 5
Claims (23)
1.细胞,其包含编码嵌合抗原受体(CAR)的第一外源核酸分子和编码转录因子的第二外源核酸分子。
2.根据权利要求1的细胞,其中所述转录因子阻止或减少所述细胞的分化和/或耗竭。
3.根据权利要求1或2的细胞,其中所述转录因子是效应记忆阻遏物。
4.根据权利要求3的细胞,其中所述转录因子是BLIMP-1。
5.根据权利要求1或2的细胞,其中所述转录因子是中央记忆阻遏物。
6.根据权利要求5的细胞,其中所述转录因子是BCL6或Bach2。
7.根据权利要求6的细胞,其中所述转录因子是Bach2或包含Bach2。
8.根据权利要求5的细胞,其中所述转录因子包含经修饰形式的Bach2,其具有减少的或去除的被ALK磷酸化的能力。
9.根据权利要求8的细胞,其中所述转录因子包含经修饰形式的Bach2,其参考如SEQID No.2所示的氨基酸序列在以下位置的一个或多个处具有突变:Ser-535、Ser-509、Ser-520。
10.核酸构建体,其包含编码嵌合抗原受体(CAR)的第一核酸序列和编码如前述权利要求的任一项中所述的转录因子的第二核酸序列。
11.根据权利要求10的核酸构建体,其具有以下结构:
CAR-coexpr-TF;或
TF-coexpr-CAR
其中:
CAR是编码所述CAR的核酸序列;
coexpr是使得所述CAR和所述转录因子能够共表达的核酸序列;并且
TF是编码所述转录因子的核酸序列。
12.根据权利要求11的核酸构建体,其中coexpr编码包含自切割肽的序列。
13.核酸序列的试剂盒,其包含编码嵌合抗原受体(CAR)的第一核酸序列和编码如权利要求1至9的任一项中所定义的转录因子的第二核酸序列。
14.载体,其包含根据权利要求10至12中任一项的核酸构建体。
15.载体的试剂盒,其包含第一载体,所述第一载体包含编码嵌合抗原受体(CAR)的第一核酸序列;和第二载体,所述第二载体包含编码如权利要求1至9的任一项中所定义的转录因子的第二核酸序列。
16.用于制备根据权利要求1至9中任一项的细胞的方法,其包括将以下各项引入到细胞中的步骤:根据权利要求10至12中任一项的核酸构建体、根据权利要求13的核酸序列的试剂盒、根据权利要求14的载体或根据权利要求15的载体的试剂盒。
17.根据权利要求16的方法,其中所述细胞来自从受试者分离的样品。
18.药物组合物,其包含多个根据权利要求1至9中任一项的细胞。
19.用于治疗和/或预防疾病的方法,其包括向受试者施用根据权利要求18的药物组合物的步骤。
20.根据权利要求19的方法,其包括以下步骤:
(i)从受试者分离含有细胞的样品;
(ii)用以下各项转导或转染所述细胞:根据权利要求10至12中任一项的核酸构建体、根据权利要求13的核酸序列的试剂盒、根据权利要求14的载体或根据权利要求15的载体的试剂盒;和
(iii)向所述受试者施用来自(ii)的所述细胞。
21.根据权利要求19或20的方法,其中所述疾病是癌症。
22.根据权利要求18的药物组合物,其用于治疗和/或预防疾病。
23.根据权利要求1至9中任一项的细胞在制备用于治疗和/或预防疾病的药物中的用途。
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GBGB1621889.3A GB201621889D0 (en) | 2016-12-21 | 2016-12-21 | Cell |
PCT/GB2017/053834 WO2018115865A1 (en) | 2016-12-21 | 2017-12-20 | Cell expressing a car and a transcription factor and its use |
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EP (1) | EP3559213A1 (zh) |
JP (2) | JP7149273B2 (zh) |
CN (1) | CN110099997A (zh) |
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CN114805596A (zh) * | 2021-01-22 | 2022-07-29 | 华东师范大学 | 一种以磷脂酰肌醇聚糖3为靶点的嵌合抗原受体及其应用 |
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US20220127570A1 (en) | 2018-05-03 | 2022-04-28 | Cafa Therapeutics Limited | Immune effector cell and use thereof |
US20210363217A1 (en) * | 2018-06-19 | 2021-11-25 | Autolus Limited | Cell |
AU2020235395A1 (en) | 2019-03-08 | 2021-09-02 | Autolus Limited | Compositions and methods comprising engineered chimeric antigen receptor and modulator of CAR |
US20220169687A1 (en) | 2020-11-10 | 2022-06-02 | Kyverna Therapeutics, Inc. | Method for treating disease using foxp3+cd4+ t cells |
CN116997563A (zh) | 2021-01-27 | 2023-11-03 | 莱尔免疫制药股份有限公司 | 改良的免疫细胞疗法 |
WO2022165419A1 (en) | 2021-02-01 | 2022-08-04 | Kyverna Therapeutics, Inc. | Methods for increasing t-cell function |
TW202246511A (zh) | 2021-02-25 | 2022-12-01 | 美商萊爾免疫藥物股份有限公司 | 靶向ny-eso-1之增強免疫細胞療法 |
AU2022227686A1 (en) | 2021-02-25 | 2023-07-27 | Lyell Immunopharma, Inc. | Ror1 targeting chimeric antigen receptor |
EP4298230A1 (en) | 2021-02-25 | 2024-01-03 | Lyell Immunopharma, Inc. | Codon-optimized nucleotide sequences encoding an ap-1 transcription factor |
EP4347826A1 (en) | 2021-06-02 | 2024-04-10 | Lyell Immunopharma, Inc. | Nr4a3-deficient immune cells and uses thereof |
CA3234822A1 (en) | 2021-10-28 | 2023-05-04 | Suman Kumar VODNALA | Methods for culturing cells expressing ror1-binding protein |
WO2023077032A1 (en) | 2021-10-28 | 2023-05-04 | Lyell Immunopharma, Inc. | Methods for culturing cells expressing c-jun |
WO2023212566A1 (en) * | 2022-04-25 | 2023-11-02 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods for preventing t cell exhaustion |
WO2023225665A1 (en) | 2022-05-19 | 2023-11-23 | Lyell Immunopharma, Inc. | Polynucleotides targeting nr4a3 and uses thereof |
WO2024020429A1 (en) | 2022-07-22 | 2024-01-25 | Lyell Immunopharma, Inc. | Immune cell therapy |
WO2024064952A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells overexpressing c-jun |
WO2024064958A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
WO2024077174A1 (en) | 2022-10-05 | 2024-04-11 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
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CN114805596A (zh) * | 2021-01-22 | 2022-07-29 | 华东师范大学 | 一种以磷脂酰肌醇聚糖3为靶点的嵌合抗原受体及其应用 |
CN114805596B (zh) * | 2021-01-22 | 2023-07-14 | 华东师范大学 | 一种以磷脂酰肌醇聚糖3为靶点的嵌合抗原受体及其应用 |
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JP2020511115A (ja) | 2020-04-16 |
EP3559213A1 (en) | 2019-10-30 |
GB201621889D0 (en) | 2017-02-01 |
US20200030379A1 (en) | 2020-01-30 |
WO2018115865A1 (en) | 2018-06-28 |
AU2017380449B2 (en) | 2023-01-05 |
JP2022145846A (ja) | 2022-10-04 |
CA3047621C (en) | 2024-01-02 |
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