CN116426484A - 嵌合抗原受体t细胞群、组合物及其用途 - Google Patents
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Abstract
本公开描述了用于治疗癌症的嵌合抗原受体T细胞群、组合物及其用途。实施方案涉及经修饰以表达一种或多种分子的水平高于或低于未经修饰以表达一种或多种分子的细胞所表达的一种或多种分子的水平的细胞,其中所述一种或多种分子包括JUN、MYC、MYB、BCL2、BCLXL或其突变型,或Cavin3、ZBED2中的至少一种。
Description
序列表信息
本申请的序列表通过引用整体并入本文。
技术领域
本公开内容涉及用于扩增和维持修饰细胞(包括遗传修饰细胞)的组合物和方法,例如维持修饰细胞的数量,及其在治疗包括癌症在内的疾病中的用途。
背景技术
嵌合抗原受体(CAR)T细胞疗法在治疗白血病、淋巴瘤和骨髓瘤等血癌方面取得了重大进展。然而,该疗法面临许多挑战,例如物理屏障、肿瘤微环境免疫抑制、肿瘤异质性、靶点特异性以及体内细胞扩增以治疗实体瘤。
概要
实施方案涉及经修饰以表达一种或多种分子的水平高于或低于未经修饰以表达一种或多种分子的细胞所表达的一种或多种分子的水平的细胞,其中所述一种或多种分子包含JUN、MYC、MYB、BCL2、BCLXL、Cavin3和ZBED2中的至少一种。具体包括如下技术方案:
1.嵌合抗原受体(CAR)T细胞群,包含编码JUN、MYC、MYB、BCL2、BCLXL或其突变型,或小窝内膜相关蛋白3(CAVIN3)或含锌指床型2(ZBED2)的外源多核苷酸。
2.根据技术方案1所述的CAR T细胞群,包含编码CAVIN3或ZBED2的外源多核苷酸。
3.根据技术方案1所述的CAR T细胞群,所述外源多核苷酸包含SEQ ID NO:31-43之一的序列。
4.根据技术方案1-3所述的CAR T细胞群,与没有过表达CAVIN3和ZBED2的CAR T细胞相比,所述CAR T细胞具有增强的抗肿瘤作用,并且所述抗肿瘤作用表现为以下至少一种:减少细胞耗竭,增强细胞扩增,增强肿瘤生长抑制。
5.根据技术方案-41所述的CAR T细胞群,所述CAR T细胞的细胞耗竭水平低于未过表达CAVIN3和ZBED2的CAR T细胞的细胞耗竭水平,所述CAR T细胞的细胞扩增水平高于未过表达CAVIN3和ZBED2的CAR T细胞的细胞耗竭水平没有过表达CAVIN3和ZBED2的CAR T细胞,和/或CAR T细胞的肿瘤生长抑制作用大于没有过表达CAVIN3和ZBED2的CAR T细胞。
6.根据技术方案1-5所述的CAR T细胞群,结合肿瘤抗原的抗原结合域选自:TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1,CD33,EGFRvIII,GD2,GD3,BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2,Mesothelin,IL-11Ra,PSCA,PRSS21 VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、叶酸受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、前列腺酶、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2,gp100,bcr-abl,酪氨酸酶,EphA2,Fucosyl GM1,sLe,GM3,TGS5,HMWMAA,o-乙酰-GD2,叶酸受体β,TEM1/CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD99a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、HPV E6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺素、存活蛋白和端粒酶、PCTA-1/半乳糖凝集素8,MelanA/MART1、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2 ETS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 hsp70-2,CD79a,CD79b,CD72,LAIR1,FCAR,LILRA2,CD300LF,CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5和IGLL1。
7.根据技术方案1-6所述的CAR T细胞群,所述胞内信号域包含共刺激信号域,或初级信号域和共刺激信号域,其中所述共刺激信号域包含功能性信号域选自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的蛋白质,NKG2C,B7-H3,一种与CD83,CDS,ICAM-1,GITR,BAFFR,HVEM(LIGHTR),SLAMF7,NKp80(KLRF1),CD160,CD19,CD4,CD8alpha,CD8beta,IL2R beta特异性结合的配体,IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、CD11c ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(触觉)、CEACAM1、CRTAM、Ly9(CD229)、CD160 BY55),PSGL1,CD100(SEMA4D),CD69,SLAMF6(NTB-A,Ly 108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELLPG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D。
8.一种包含技术方案1~7中任意一项所述CAR T细胞群的组合物。
9.根据技术方案8的组合物,包括程序性死亡受体1(PD1)、细胞毒性T细胞相关抗原-4(CTLA-4)、B-和T-淋巴细胞衰减因子(BTLA)、T细胞免疫球蛋白粘蛋白3(TIM-3)、活化气泡蛋白3(LAG-3)、T细胞免疫球蛋白和ITIM结构域(TIGIT)、白细胞相关免疫球蛋白样受体-1(LAIR1)、自然保护细胞受体2B4(2B4)和CD 160;和/或
所述组合物包含结合第一抗原的第一CAR的第一细胞群和结合第二抗原的第二细胞群,其中所述的第二抗原是潜在的并且与第一抗原不同的,第一抗原包括白细胞(WBC)、其他潜在的细胞表面分子;和/或
所述细胞具有降低的内源TRAC基因,和/或所述细胞编码hTERT的基因序列或编码SV40LT的基因序列,以及其组合;和/或
所述细胞包括编码结合TGF-β1、TGF-β2、CTGF、αv/β3整联蛋白、α4/β7整联蛋白或α5/β11蛋白的scFv的多核苷酸、和/或包含IL-6、IFNγ、IL-7,IL-10或IL-12;和/或
所述细胞包括编码靶向一种或多种ECM分子合成的细胞因子抑制剂或细胞因子的多核苷酸、靶向一种或多种ECM分子降解的肽和/或靶向一种或多种ECM分子的信号传导的肽。
10.根据技术方案1~9中任意一项所述的细胞群或组合物在制备治疗实体瘤药物中的用途。
本发明内容不旨在标识所要求保护的主题的关键特征或必要特征,也不旨在用于限制所要求保护的主题的范围。
附图说明
具体实施方式参照附图进行描述。在不同的图中使用相同的附图标记表示相似或相同的项目。
图1显示表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的CAR表达和表型。
图2显示了在与底物细胞NALM6共培养后表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的细胞活化。
图3显示了在与底物细胞NALM6共培养后表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的细胞凋亡。
图4显示了在与底物细胞NALM6共培养后表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的细胞增殖。
图5A和5B(图5)显示表达CAVIN3或ZBED2的CAR T细胞的CAR表达和表型。
图6显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞活化。
图7显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞耗竭。
图8显示了与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞表型变化。
图9显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞因子释放。
图10显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的肿瘤抑制测定结果。
图11显示表达CAVIN3或ZBED2的CAR T细胞的细胞扩增。
具体实施方式
除非另外定义,否则本文使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。尽管可以在本公开的实践或测试中使用与本文描述的那些类似或等同的任何方法和材料,但是描述了优选的方法和材料。出于本公开的目的,以下术语定义如下。
本文使用冠词“一(a)”和“一个(an)”指的是一个或多于一个(即,指的是至少一个)该冠词的语法对象。举例来说,“元件”表示一个元件或多于一个元件。
“大约”是指数量,水平,值,数量,频率,百分比,尺寸,大小,数量,重量或长度相差多达20、15、10、9、8、7、6、5的数量,参考数量,水平,数值,数量,频率,百分比,尺寸,大小,数量,重量或长度的4%,3%,2%或1%。
如本文所用,术语“活化”是指已经充分刺激以诱导可检测的细胞增殖的细胞的状态。激活还可以与诱导的细胞因子产生和可检测的效应子功能相关联。术语“活化的T细胞”特别指正在进行细胞分裂的T细胞。
术语“抗体”以最广泛的含义使用,指单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段,只要它们表现出所需的生物活性或功能.本公开中的抗体可以多种形式存在,包括例如多克隆抗体;单克隆抗体;Fv、Fab、Fab'和F(ab')2片段;以及单链抗体和人源化抗体(Harlow等,1999,在:使用抗体:实验室手册,冷泉港实验室出版社,纽约;Harlow等,1989,在:抗体:实验室手册,冷Spring Harbor,纽约;Houston等,1988,Proc.Natl.Acad.Sci.USA 85:5879-5883;Bird等,1988,Science242:423-426)。
术语“抗体片段”是指全长抗体的一部分,例如抗体的抗原结合区或可变区。抗体片段的其他实例包括Fab、Fab'、F(ab')2和Fv片段;双体;线性抗体;单链抗体分子;和由抗体片段形成的多特异性抗体。
术语“Fv”是指包含完整抗原识别和结合位点的最小抗体片段。该片段由一个重链和一个轻链可变区结构域以紧密的非共价结合的二聚体组成。这两个结构域的折叠产生了六个高变环(H链和L链各有3个环),它们为抗原结合提供氨基酸残基,并赋予抗体抗原结合特异性。然而,即使是单个可变域(或仅包含三个对抗原特异的互补决定区(CDR)的Fv的一半)也具有识别和结合抗原的能力,尽管亲和力低于整个结合位点(二聚体)。
如本文所用,“抗体重链”是指以天然存在的构型存在于所有抗体分子中的两种类型的多肽链中的较大者。如本文所用,“抗体轻链”是指以天然存在的构型存在于所有抗体分子中的两种类型的多肽链中的较小者。κ和λ轻链是指两种主要的抗体轻链同种型。
术语“合成抗体”是指使用重组DNA技术产生的抗体,例如由噬菌体表达的抗体。该术语还包括通过合成编码抗体的DNA分子和表达DNA分子以获得抗体或获得编码抗体的氨基酸而产生的抗体。合成DNA是使用本领域已知的可用技术获得的。
术语“抗原”是指激发免疫反应的分子,其可能涉及抗体产生或特定免疫活性细胞的活化,或两者。抗原包括任何大分子,包括源自重组或基因组DNA的所有蛋白质或肽或分子。例如,包括编码蛋白质或肽的核苷酸序列或部分核苷酸序列的DNA,该蛋白质或肽引发免疫反应,并因此编码本文所用术语“抗原”。抗原不需要仅由基因的全长核苷酸序列编码。抗原可以从生物样品产生、合成或衍生,包括组织样品、肿瘤样品、细胞或生物流体。
如本文所用,术语“抗肿瘤作用”是指与肿瘤体积减少,肿瘤细胞数目减少,转移数目减少,肿瘤细胞增殖减少,减少有关的生物学作用。在肿瘤细胞存活率方面,具有肿瘤细胞的受试者的预期寿命增加,或与癌症状况相关的各种生理症状的改善。首先,肽,多核苷酸,细胞和抗体在预防肿瘤发生方面的能力也可以表现出“抗肿瘤作用”。
术语“自身抗原”是指被免疫系统错误地识别为外源的内源性抗原。自身抗原包括细胞蛋白,磷蛋白,细胞表面蛋白,细胞脂质,核酸,糖蛋白,包括细胞表面受体。
术语“自体”用于描述来自同一受试者的材料,稍后将其重新引入受试者。
术语“同种异体”用于描述源自同一物种的不同受试者的移植物。例如,供体受试者可能与受体受试者相关或不相关,但供体受试者具有与受体受试者相似的免疫系统标记。
术语“异种”用于描述源自不同物种的受试者的移植物。例如,供体受试者来自与受体受试者不同的物种,供体受试者和受体受试者可以在遗传上和免疫学上不相容。
术语“癌症”用于指以异常细胞的快速和不受控制的生长为特征的疾病。癌细胞可以局部或通过血流和淋巴系统扩散到身体的其他部位。各种癌症的实例包括乳腺癌,前列腺癌,卵巢癌,宫颈癌,皮肤癌,胰腺癌,结肠直肠癌,肾癌,肝癌,脑癌,淋巴瘤,白血病,肺癌等。
在整个说明书中,除非上下文另外要求,否则词语“包括”,“包括”和“包括”将被理解为暗示包括陈述的步骤或元件或步骤或元件的组,但不排除其他任何元件。步骤或元素或一组步骤或元素。
短语“由...组成”意味着包括并且限于在短语“由...组成”之后的任何事物。因此,短语“由...组成”表示列出的元素是必需的或强制性的,并且不存在其它元素可以存在。
短语“基本上由...组成”意在包括在该短语之后列出的任何元素,并且可以包括不干扰或有助于所列出元素的公开中指定的活动或动作的其他元素。因此,短语“基本上由……组成”表示所列要素是必需的或强制性的,但其他要素是可选的,并且可能存在也可能不存在,这取决于它们是否影响所列要素的活动或作用。例如,如果该元素不影响细胞的扩展、功能或表型,则该元素不是必需的并且被认为是可选的。
术语“互补”和“互补性”是指通过碱基配对规则相关的多核苷酸(即,核苷酸序列)。例如,序列“AGT”与序列“TCA”互补。互补性可以是“部分的”,其中只有一些核酸的碱基根据碱基配对规则匹配,或者可能是“完整的”“或”核酸之间的“总”互补性。核酸链之间的互补程度对核酸链之间的杂交效率和强度具有显着影响。
术语“对应于”是指(a)具有与参考多核苷酸序列的全部或一部分基本上相同或互补或编码与氨基酸序列相同的氨基酸序列的核苷酸序列的多核苷酸在肽或蛋白质中;或(b)具有与参考肽或蛋白质中的氨基酸序列基本上相同的氨基酸序列的肽或多肽。
术语“共刺激配体”是指抗原呈递细胞(例如,APC,树突状细胞,B细胞等)上的分子,其与T细胞上的同源共刺激分子特异性结合,从而提供除了通过例如TCR/CD3复合物与载有肽的MHC分子结合提供的主要信号外,还介导T细胞反应的信号,包括至少一种增殖,活化,分化和其他细胞反应。共刺激配体可以包括B7-1(CD80),B7-2(CD86),PD-L1,PD-L2、4-1BBL,OX40L,诱导型共刺激配体(ICOS-L),细胞间粘附分子(ICAM),CD30L,CD40,CD70,CD83,HLA-G,MICA,MICB,HVEM,淋巴毒素β受体,3/TR6,ILT3,ILT4,HVEM,CD7的配体,与Toll配体受体结合的激动剂或抗体和与B7-H3特异性结合的配体。共刺激配体还特别包括与存在于T细胞上的共刺激分子如CD27,CD28、4-1BB,OX40,CD30,CD40,PD-1特异性结合的激动剂或抗体,ICOS,淋巴细胞功能相关抗原1(LFA-1),CD2,CD7,LIGHT,NKG2C,B7-H3以及与CD83特异性结合的配体。
术语“共刺激分子”是指T细胞上的同源结合配偶体,其与共刺激配体特异性结合,由此介导T细胞的共刺激反应,例如增殖。共刺激分子包括MHC I类分子,BTLA和Toll样受体。
术语“共刺激信号”是指信号,其与主要信号(例如TCR/CD3连接)组合导致T细胞增殖和/或关键分子的上调或下调。
术语“疾病”和“病症”可以互换使用,也可以不同,因为特定的疾病或病症可能没有已知的病原体(因此病因尚未得到解决),因此尚未得到认可。它是一种疾病,但仅是一种不良状况或综合症,其中临床医生已识别出或多或少的特定症状。术语“疾病”是受试者的健康状态,其中受试者不能维持体内平衡,并且如果疾病没有得到改善,则受试者的健康继续恶化。相反,受试者的“疾病”是一种健康状态,其中动物能够维持体内平衡,但是其中动物的健康状态要比没有该疾病的情况下不利。如果不及时治疗,疾病不一定会导致动物健康状况进一步下降。
术语“有效”是指足以实现期望的,预期的或预期的结果。例如,治疗中的“有效量”可以是足以产生治疗或预防益处的化合物的量。
术语“编码”是指多核苷酸中特定核苷酸序列的固有特性,例如基因,cDNA或mRNA,以用作模板,用于在生物学过程中合成具有特定结构的其他聚合物和大分子。核苷酸序列(即rRNA,tRNA和mRNA)或确定的氨基酸序列以及由此产生的生物学特性。因此,如果对应于该基因的mRNA的转录和翻译在细胞或其他生物系统中产生该蛋白质,则该基因编码蛋白质。核苷酸序列与mRNA序列相同的编码链(除了用“U”代替“T”以外)通常都在序列表中提供,非编码链均用作模板用于基因或cDNA转录的“蛋白质”可以称为编码该基因或cDNA的蛋白质或其他产物。
术语“外源的”是指不是天然存在于野生型细胞或生物体中但通常通过分子生物学技术引入细胞的分子。外源多核苷酸的实例包括编码所需蛋白质的载体、质粒和/或人造核酸构建体。关于多核苷酸和蛋白质,术语“内源的”或“天然的”是指可以在给定的野生型细胞或生物体中发现的天然存在的多核苷酸或氨基酸序列。此外,从第一种生物体分离并通过分子生物学技术转移到第二种生物体的特定多核苷酸序列通常被认为是关于第二种生物体的“外源”多核苷酸或氨基酸序列。在特定的实施方案中,多核苷酸序列可以通过分子生物学技术“引入”到已经含有这种多核苷酸序列的微生物中,例如,以产生一个或多个另外的天然存在的多核苷酸序列的拷贝,从而促进编码的多肽。
术语“表达或过表达”是指例如由其启动子驱动的特定核苷酸序列转录和/或翻译成前体或成熟蛋白。“过表达”是指基因产物在转基因生物或细胞中的产量超过正常或非转化生物或细胞中的产量。如本文所定义,术语“表达”是指表达或过表达。
术语“表达载体”是指包含重组多核苷酸的载体,所述重组多核苷酸包括与待表达的核苷酸序列可操作地连接的表达控制(调控)序列。表达载体包括足够的顺式作用元件用于表达。其他表达元件可以由宿主细胞或在体外表达系统中提供。表达载体包括本领域已知的所有载体,例如粘粒,质粒(例如裸露或包含在脂质体中)和掺入重组多核苷酸的病毒(例如慢病毒,逆转录病毒,腺病毒和腺相关病毒)。
病毒可用于在体外和体内(在受试者中)将核酸递送到细胞中。可用于将核酸递送到细胞中的病毒的例子包括逆转录病毒,腺病毒,单纯疱疹病毒,牛痘病毒和腺伴随病毒。
还存在用于将核酸递送到细胞中的非病毒方法,例如电穿孔,基因枪,声穿孔,磁转染,以及使用寡核苷酸,脂复合物,树状聚合物和无机纳米颗粒。
术语“同源的”是指两个多肽之间或两个多核苷酸之间的序列相似性或序列同一性,当两个比较序列中的位置被相同的碱基或氨基酸单体亚基占据时,例如,如果两个多肽中的每一个中的位置。DNA分子被腺嘌呤占据,然后分子在该位置同源。两个序列之间的同源性百分比是两个序列共有的匹配或同源位置数除以相比的位置数×100的函数。例如,如果两个序列中10个位置中的6个是匹配的或同源的,那么这两个序列是60%同源的。举例来说,DNA序列ATTGCC和TATGGC具有50%的同源性。当两个序列比对以产生最大同源性时进行比较。
术语“免疫球蛋白”或“Ig”是指一类具有抗体功能的蛋白质。包括在此类蛋白质中的五个成员是IgA、IgG、IgM、IgD和IgE。IgA是存在于身体分泌物中的主要抗体,例如唾液、眼泪、母乳、胃肠道分泌物以及呼吸道和泌尿生殖道的粘液分泌物。IgG是最常见的循环抗体。IgM是大多数受试者在初级免疫反应中产生的主要免疫球蛋白。它是凝集、补体固定和其他抗体反应中最有效的免疫球蛋白,在防御细菌和病毒方面很重要。IgD是一种免疫球蛋白,没有已知的抗体功能,但可以作为抗原受体。IgE是一种免疫球蛋白,通过在暴露于过敏原时导致肥大细胞和嗜碱性粒细胞释放介质来介导即时超敏反应。
术语“分离的”是指基本上或基本上不含在其天然状态下通常伴随的组分的材料。该材料可以是细胞或大分子,例如蛋白质或核酸。例如,如本文所用,“分离的多核苷酸”是指已从天然存在状态下其侧翼的序列中纯化的多核苷酸,例如已从正常情况下的序列中去除的DNA片段。与片段相邻。或者,如本文所用,“分离的肽”或“分离的多肽”等是指肽或多肽分子从其天然细胞环境和与细胞的其他组分的结合体外分离和/或纯化.
术语“基本上纯化的”是指基本上不含通常与其天然状态相关的组分的物质。例如,基本上纯化的细胞是指已经与其通常以其天然存在或天然状态相关联的其他细胞类型分离的细胞。在一些情况下,基本上纯化的细胞群是指同源的细胞群。在其他情况下,该术语仅指已经与天然状态下天然相关的细胞分离的细胞。在实施方案中,细胞在体外培养。在实施方案中,细胞不在体外培养。
在本公开的上下文中,使用下列普遍存在的核酸碱基的缩写。“A”指的是腺苷,“C”指的是胞嘧啶,“G”指的是鸟苷,“T”指的是胸苷,以及“U”指的是尿苷。
除非另有说明,“编码氨基酸序列的核苷酸序列”包括彼此简并形式并编码相同氨基酸序列的所有核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可以包括内含子,其程度使得编码蛋白质的核苷酸序列在一些版本中可以包含一个或多个内含子。
术语“慢病毒”是指逆转录病毒科的一个属。慢病毒在逆转录病毒中是独一无二的,它能够感染非分裂细胞;它们可以将大量遗传信息传递到宿主细胞的DNA中,因此它们是最有效的基因传递载体方法之一。此外,慢病毒的使用能够将遗传信息整合到宿主染色体中,从而稳定转导遗传信息。HIV、SIV和FIV都是慢病毒的例子。源自慢病毒的载体提供了在体内实现显着水平的基因转移的手段。
术语“调节”是指与没有治疗或化合物的情况下受试者的应答水平相比和/或与应答水平相比,在受试者中应答水平可检测地增加或减少。在其他相同但未经治疗的受试者中该术语包括干扰和/或影响天然信号或反应,从而在受试者,优选人中介导有益的治疗反应。
当核酸被置于与另一种核酸的功能关系中时,核酸被“有效连接”。例如,如果前序列或分泌前导序列的DNA表达为参与多肽分泌的前蛋白,则它与多肽的DNA可操作地连接;如果启动子或增强子影响序列的转录,则启动子或增强子与编码序列可操作地连接,或者如果核糖体结合位点的位置便于翻译,则其与编码序列可操作地连接。
术语“在转录控制下”是指与多核苷酸可操作地连接并且在与多核苷酸相关的正确位置和方向上的启动子,以控制(调节)RNA聚合酶的转录起始和多核苷酸的表达。
术语“过表达”的肿瘤抗原或肿瘤抗原的“过表达”意在表示来自患者特定组织或器官内的诸如实体瘤的疾病区域的细胞中肿瘤抗原的异常表达水平。达到该组织或器官在正常细胞中的表达水平。可以通过本领域已知的标准测定来确定具有以肿瘤抗原的过表达为特征的实体瘤或血液系统恶性肿瘤的患者。
实体瘤是通常不包含囊肿或液体区域的异常组织块。实体瘤可以是良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(例如肉瘤,癌和淋巴瘤)。实体瘤如肉瘤和癌的实例包括纤维肉瘤,粘液肉瘤,脂肪肉瘤,软骨肉瘤,骨肉瘤,滑膜瘤,间皮瘤,尤文氏瘤,平滑肌肉瘤,横纹肌肉瘤,结肠癌,淋巴恶性肿瘤,胰腺癌,乳腺癌,肺癌,卵巢癌,前列腺癌,肝细胞癌,鳞状细胞癌,基底细胞癌,腺癌,汗腺癌,甲状腺髓样癌,乳头状甲状腺癌,嗜铬细胞瘤皮脂腺癌,乳头状癌,乳头状腺癌,髓样癌,支气管癌,肾细胞癌,肝细胞癌,胆管癌,绒毛膜癌,肾母细胞瘤,宫颈癌,睾丸肿瘤,精原细胞瘤,膀胱癌,黑色素瘤和中枢神经系统肿瘤(如脑胶质瘤(如脑干胶质瘤和混合胶质瘤),胶质母细胞瘤(又称多形性胶质母细胞瘤),星形细胞瘤,中枢神经系统淋巴瘤,生殖细胞瘤,成神经管细胞瘤,神经鞘瘤opharyogioma,室管膜瘤,松果体,血管母细胞瘤,听神经瘤,少突神经胶质瘤,menangioma,神经母细胞瘤,视网膜母细胞瘤和脑转移瘤。
实体瘤抗原是在实体瘤上表达的抗原。在实施方案中,实体瘤抗原也在健康组织上以低水平表达。表1中提供了实体瘤抗原及其相关疾病肿瘤的实例。
表1
组合物的术语“肠胃外施用”包括例如皮下(sc),静脉内(iv),肌内(im),胸骨内注射或输注技术。
术语“患者”,“受试者”和“个体”等在本文中可互换使用,并且是指适用于本文所述方法的任何人或动物。在某些非限制性实施方案中,患者,受试者或个体是人或动物。在实施方案中,术语“受试者”旨在包括其中可引发免疫应答的活生物体(例如,哺乳动物)。受试者的实例包括人和动物,例如狗,猫,小鼠,大鼠及其转基因物种。
需要治疗或有此需要的受试者包括患有需要治疗的疾病,病症或病症的受试者。有此需要的受试者还包括需要治疗以预防疾病,病症或病症的受试者。
术语“多核苷酸”或“核酸”是指mRNA,RNA,cRNA,rRNA,cDNA或DNA。该术语通常是指长度至少为10个碱基的核苷酸的聚合形式,即核糖核苷酸或脱氧核苷酸,或任一类型核苷酸的修饰形式。该术语包括所有形式的核酸,包括单链和双链形式的核酸。
术语“多核苷酸变体”和“变体”等是指与参考多核苷酸序列显示基本序列同一性的多核苷酸或在下文定义的严格条件下与参考序列杂交的多核苷酸。这些术语还涵盖通过添加,缺失或取代至少一个核苷酸而与参考多核苷酸区分开的多核苷酸。因此,术语“多核苷酸变体”和“变体”包括其中一个或多个核苷酸已被添加或缺失或被不同核苷酸取代的多核苷酸。就这一点而言,本领域众所周知,可以对参考多核苷酸进行某些改变,包括突变,添加,缺失和取代,从而改变后的多核苷酸保留了参考多核苷酸的生物学功能或活性或具有增加的活性。与参考多核苷酸的关系(即优化的)。多核苷酸变体包括,例如,与参考多核苷酸序列具有至少50%(和至少51%至至少99%以及在例如90%,95%或98%之间的所有整数百分比)序列同一性的多核苷酸。本文所述。术语“多核苷酸变体”和“变体”还包括天然存在的等位基因变体和直系同源物。
术语“多肽”,“多肽片段”,“肽”和“蛋白质”在本文中可互换使用,是指氨基酸残基的聚合物以及其变体和合成类似物。因此,这些术语适用于氨基酸聚合物,其中一个或多个氨基酸残基是合成的非天然存在的氨基酸,例如相应天然存在的氨基酸的化学类似物,以及天然存在的氨基酸聚合物。在某些方面,多肽可以包括通常催化(即增加各种化学反应的速率)的酶促多肽或“酶”。
术语“多肽变体”是指通过添加,缺失或取代至少一个氨基酸残基而与参考多肽序列区分开的多肽。在实施方案中,多肽变体通过一个或多个取代而与参考多肽区别开,所述取代可以是保守的或非保守的。在实施方案中,多肽变体包含保守取代,并且就这一点而言,在本领域中众所周知,可以将一些氨基酸改变为具有广泛相似性质的其他氨基酸而不改变多肽活性的性质。多肽变体还涵盖其中一个或多个氨基酸已添加或缺失,或被不同氨基酸残基取代的多肽。
术语“启动子”是指被细胞的合成机器识别或被引入的启动多核苷酸序列特异性转录所需的合成机器的DNA序列。术语“表达控制(调节)序列”是指在特定宿主生物体中表达可操作连接的编码序列所必需的DNA序列。例如,适用于原核生物的控制序列包括启动子、任选的操纵子序列和核糖体结合位点。已知真核细胞利用启动子、聚腺苷酸化信号和增强子。
术语“结合”、“结合”或“与……相互作用”是指分子识别并粘附到样品或生物体中的第二分子,但基本上不识别或粘附到样品中的其他结构不相关的分子。如本文所用的关于抗体的术语“特异性结合”是指识别特定抗原但基本上不识别或结合样品中的其他分子的抗体。例如,特异性结合来自一种物种的抗原的抗体也可以结合来自一种或多种物种的抗原。但是,这种跨物种反应性本身不会改变抗体的特异性分类。在另一个例子中,特异性结合抗原的抗体也可以结合抗原的不同等位基因形式。然而,这种交叉反应本身不会改变抗体的特异性分类。在一些情况下,术语“特异性结合”或“特异性结合”可用于指抗体、蛋白质或肽与第二化学物质的相互作用,以表示相互作用取决于以下物质的存在:化学物种的特定结构(例如,抗原决定簇或表位);例如,抗体识别并结合特定的蛋白质结构而不是任何蛋白质。如果抗体对表位“A”具有特异性,则在含有标记的“A”和抗体的反应中含有表位A(或游离的、未标记的A)的分子的存在将减少与抗体结合的标记A的数量。
“统计上显着”是指结果不太可能是偶然发生的。统计显着性可以通过本领域已知的任何方法确定。常用的显着性度量包括p值,它是在原假设为真时观察到的事件发生的频率或概率。如果获得的p值小于显着性水平,则拒绝原假设。在简单的情况下,显着性水平定义为0.05或更小的p值。“减少的”或“减少的”或“较小的”量通常是“统计上显着的”或生理上显着的量并且可以包括约1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2、2.5、3、3.5、4、4.5、5、6、7、8、9、10、15、20、30、40或50次或更多次(例如,100、500、1000次)(包括所有1之间和之上的整数和小数点,例如1.5、1.6、1.7、1.8等)此处描述的数量或水平。
术语“刺激”是指由刺激分子(例如TCR/CD3复合物)与其同源配体结合而诱导的初级应答,由此介导信号转导事件,例如经由TCR/CD3复合物的信号转导。刺激可以介导某些分子的改变的表达,例如TGF-β的下调和/或细胞骨架结构的重组。
术语“刺激分子”是指T细胞上的分子,其特异性结合存在于抗原呈递细胞上的同源刺激配体。例如,衍生自刺激分子的功能信号传导结构域是与T细胞受体复合物相关的zeta链。刺激分子包括负责信号转导的结构域。
术语“刺激性配体”是指当存在于抗原呈递细胞(例如,APC,树突状细胞,B细胞等)上时可以与同源结合伴侣(称为“配体”)特异性结合的配体。在本文中称为“刺激性分子”)在细胞例如T细胞上,从而介导T细胞的初级应答,包括激活,免疫应答的起始,增殖和类似过程。刺激性配体在本领域中是众所周知的,并且尤其包括装载有肽,抗CD3抗体,超激动剂抗CD28抗体和超激动剂抗CD2抗体的MHC I类分子。
术语“治疗的”是指治疗和/或预防。通过抑制,减轻或消除疾病状态或减轻疾病状态的症状可获得治疗效果。
术语“治疗有效量”是指将引起研究人员、兽医、医生或其他临床医生正在寻找的组织、系统或受试者的生物学或医学反应的主题化合物的量。术语“治疗有效量”包括化合物的量,该量在施用时足以预防所治疗的病症或疾病的一种或多种体征或症状的发展或在一定程度上减轻一种或多种体征或症状。治疗有效量将根据化合物、疾病及其严重程度以及待治疗受试者的年龄、体重等而变化。
术语“治疗疾病”是指降低受试者经历的疾病或病症的至少一种体征或症状的频率或严重程度。
术语“转染的”或“转化的”或“转导的”是指将外源核酸转移或引入宿主细胞的过程。“转染的”或“转化的”或“转导的”细胞是已经用外源核酸转染,转化或转导的细胞。该细胞包括原代受试者细胞及其后代。
术语“载体”是指包含分离的核酸并且可用于将分离的核酸递送至细胞内部的多核苷酸。许多载体是本领域已知的,包括线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此,术语“载体”包括自主复制的质粒或病毒。该术语还包括促进核酸转移到细胞中的非质粒和非病毒化合物,例如聚赖氨酸化合物、脂质体等。病毒载体的例子包括腺病毒载体、腺相关病毒载体、逆转录病毒载体等。例如,慢病毒是复杂的逆转录病毒,除了常见的逆转录病毒基因gag、pol和env外,还包含其他具有调节或结构功能的基因。慢病毒载体是本领域众所周知的。慢病毒的一些例子包括人类免疫缺陷病毒:HIV-1、HIV-2和猿猴免疫缺陷病毒:SIV。慢病毒载体是通过多重减毒HIV毒力基因产生的,例如删除env、vif、vpr、vpu、nef基因,使载体具有生物学安全性。
范围:在整个本公开中,本公开的各个方面可以以范围格式给出。应当理解,范围格式的描述仅是为了方便和简洁,并且不应当被解释为对本公开的范围的硬性限制。因此,范围的描述应被认为具有具体公开的所有可能的子范围以及处于那个范围内的单个数值。例如,范围的描述诸如从1至6应被认为具有具体公开的子范围诸如从1至3、从1至4、从1至5、从2至4、从2至6、从3至6等,以及那个范围内的单个数值,例如,1、2、2.7、3、4、5、5.3和6。不管范围的宽度如何,这一点都是适用的。
“嵌合抗原受体”(CAR)分子是至少包括细胞外结构域,跨膜结构域和细胞质结构域或细胞内结构域的重组多肽。在实施方案中,CAR的结构域在相同的多肽链上,例如,嵌合融合蛋白。在实施方案中,结构域在不同的多肽链上,例如,结构域不连续。
CAR分子的胞外域包括抗原结合域。抗原结合域用于扩增和/或维持修饰的细胞,例如CAR T细胞,或用于杀死肿瘤细胞,例如实体瘤。在实施方案中,用于扩增和/或维持修饰细胞的抗原结合结构域结合WBC表面上的抗原,例如细胞表面分子或标志物。在实施方案中,WBC是GMP(粒细胞巨噬细胞前体)、MDP(单核细胞-巨噬细胞/树突细胞前体)、cMoP(普通单核细胞前体)、嗜碱性粒细胞、嗜酸性粒细胞、中性粒细胞、SatM(含分离核的非典型单核细胞)中的至少一种)、巨噬细胞、单核细胞、CDP(普通树突状细胞前体)、cDC(常规DC)、pDC(浆细胞样DC)、CLP(普通淋巴细胞前体)、B细胞、ILC(先天淋巴细胞)、NK细胞、巨核细胞、成髓细胞、早幼粒细胞、骨髓细胞、后骨髓细胞、带状细胞、淋巴母细胞、幼淋巴细胞、单核细胞、巨核细胞、原巨核细胞、巨核细胞、血小板或MSDC(骨髓源性抑制细胞)。在实施方案中,WBC是粒细胞、单核细胞和/或淋巴细胞。在实施方案中,WBC是淋巴细胞,例如B细胞。在实施例中,WBC是B细胞。在实施方案中,B细胞的细胞表面分子包括CD19、CD22、CD20、BCMA、CD5、CD7、CD2、CD16、CD56、CD30、CD14、CD68、CD11b、CD18、CD169、CD1c、CD33、CD38、CD138、或CD13。在实施方案中,B细胞的细胞表面分子是CD19、CD20、CD22或BCMA。在实施方案中,B细胞的细胞表面分子是CD19。
本文所述的细胞,包括修饰细胞,例如CAR细胞和修饰的T细胞,可以源自干细胞。干细胞可以是成体干细胞、胚胎干细胞,更特别地非人类干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞。修饰的细胞也可以是树突细胞、NK细胞、B细胞或T细胞,选自炎性T淋巴细胞、细胞毒性T淋巴细胞、调节性T淋巴细胞或辅助性T淋巴细胞。在实施方案中,修饰的细胞可以来自由CD4+T淋巴细胞和CD8+T淋巴细胞组成的组。在细胞的扩增和遗传修饰之前,可以通过多种非限制性方法从受试者获得细胞来源。T细胞可以从许多非限制性来源获得,包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、感染部位的组织、腹水、胸腔积液、脾组织和肿瘤.在实施方案中,可以使用本领域技术人员可用的和已知的任何数量的T细胞系。在实施方案中,修饰的细胞可以源自健康供体、诊断为癌症的患者或诊断为感染的患者。在实施方案中,修饰细胞是呈现不同表型特征的混合细胞群的一部分。
细胞群体是指两个或多个细胞组成的组。种群的细胞可以是相同的,从而种群是同质的细胞种群。群体的细胞可以是不同的,以使得该群体是细胞的混合群体或异质群体。例如,混合的细胞群可以包括包含第一CAR的修饰细胞和包含第二CAR的细胞,其中第一CAR和第二CAR结合不同的抗原。
术语“干细胞”是指具有自我更新能力和分化成其他种类细胞的能力的任何特定类型的细胞。例如,一个干细胞产生两个子代干细胞(如在体外培养胚胎干细胞时发生的那样)或一个干细胞和一个进行分化的细胞(如发生在造血干细胞中,其产生上升到血细胞)。不同类别的干细胞可以根据它们的来源和/或它们分化成其他类型细胞的能力程度进行区分。例如,干细胞可以包括胚胎干细胞(ES)(即,多能干细胞)、体细胞干细胞、诱导多能干细胞和任何其他类型的干细胞。
多能胚胎干细胞存在于胚泡的内部细胞团中,具有先天的分化能力。例如,多能胚胎干细胞有可能在体内形成任何类型的细胞。当长时间在体外生长时,ES细胞保持多能性,因为子代细胞保留了多谱系分化的潜力。
体干细胞可包括胎儿干细胞(来自胎儿)和成体干细胞(存在于各种组织中,例如骨髓)。这些细胞被认为具有低于多能ES细胞的分化能力-胎儿干细胞的能力大于成体干细胞的能力;它们显然只能区分为有限范围的细胞,并被描述为多能的。“组织特异性”干细胞通常仅产生一种类型的细胞。例如,胚胎干细胞可以分化成血液干细胞(例如,造血干细胞(HSC)),其可以进一步分化成各种血细胞(例如,红细胞,血小板,白细胞等)。
诱导的多能干细胞(即,iPS细胞或iPSC)可以包括通过诱导特定基因的表达而人工衍生自非多能细胞(例如,成年体细胞)的一种多能干细胞。诱导的多能干细胞在许多方面类似于天然多能干细胞,例如胚胎干(ES)细胞,例如某些干细胞基因和蛋白质的表达,染色质甲基化模式,倍增时间,胚状体形成,畸胎瘤形成,可行的嵌合体形成以及效价和可分化性。诱导的多能细胞可以从成年的胃,肝,皮肤和血细胞中获得。
在实施方案中,用于杀死肿瘤的抗原结合结构域结合肿瘤表面上的抗原,例如,肿瘤抗原或肿瘤标志物。肿瘤抗原是由肿瘤细胞产生的蛋白质,可引发免疫反应,尤其是T细胞介导的免疫反应。肿瘤抗原是本领域众所周知的,包括例如肿瘤相关MUC1(tMUC1)、神经胶质瘤相关抗原、癌胚抗原(CEA)、β-人绒毛膜促性腺激素、甲胎蛋白(AFP)、凝集素反应性AFP、甲状腺球蛋白,RAGE-1,MN-CA IX,人端粒酶逆转录酶,RU1,RU2(AS),肠羧基酯酶,muthsp70-2,M-CSF,前列腺酶,前列腺特异性抗原(PSA),PAP,NY-ESO-1、LAGE-1a、p53、前列腺素、PSMA、Her2/neu、存活、端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、中性粒细胞弹性蛋白酶、ephrinB2、CD22、胰岛素生长因子(IGF))-I、IGF-II、IGF-I受体、CD19和间皮素。例如,当肿瘤抗原为CD19时,其CAR可称为CD19 CAR(19CAR、CD19 CAR或CD19-CAR),为包含结合CD19的抗原结合域的CAR分子。
在实施方案中,CAR的胞外抗原结合域包括至少一种scFv或至少一种单域抗体。例如,一个CAR上可以有两个scFv。scFv包括通过柔性接头连接的靶抗原特异性单克隆抗体的轻链可变(VL)区和重链可变(VH)区。可以通过使用短连接肽连接轻链和/或重链可变区来制备单链可变区片段(Bird等,Science242:423-426,1988)。连接肽的实例是具有氨基酸序列(GGGGS)3(SEQ ID NO:30)的GS接头,其在一个可变区的羧基末端和另一可变区的氨基末端之间桥接约3.5nm。已经设计并使用了其他序列的接头(Bird等,1988,同上)。通常,接头可以是短的、柔性的多肽并且优选地由约20个或更少的氨基酸残基组成。单链变体可以重组或合成产生。对于scFv的合成生产,可以使用自动合成仪。对于scFv的重组生产,可以将含有编码scFv的多核苷酸的合适的质粒引入合适的宿主细胞中,所述宿主细胞可以是真核细胞,例如酵母、植物、昆虫或哺乳动物细胞,也可以是原核细胞,例如大肠杆菌。编码目的scFv的多核苷酸可以通过常规操作例如多核苷酸的连接来制备。可以使用本领域已知的标准蛋白质纯化技术分离得到的scFv。
本文描述的CAR分子的细胞质结构域包括一个或多个共刺激结构域和一个或多个信号结构域。共刺激和信号传导结构域用于响应抗原结合而传递信号并激活分子,例如T细胞。一个或多个共刺激结构域衍生自刺激分子和/或共刺激分子,并且信号结构域衍生自主要信号传导结构域,例如CD3ζ结构域。在实施方案中,信号传导结构域还包括衍生自共刺激分子的一个或多个功能信号传导结构域。在实施方案中,共刺激分子是激活对抗原的细胞应答所需的细胞表面分子(抗原受体或其配体除外)。
在实施方案中,共刺激结构域包括CD27,CD28,4-1BB,OX40,CD30,CD40,PD-1,ICOS,淋巴细胞功能相关抗原-1(LFA-1),CD2,CD7的细胞内结构域,LIGHT,NKG2C,B7-H3,与CD83特异性结合的配体,或其任何组合。在实施方案中,信号传导结构域包括衍生自T细胞受体的CD3ζ结构域。
本文描述的CAR分子还包括跨膜结构域。在CAR分子中掺入跨膜结构域使分子稳定。在实施方案中,CAR分子的跨膜结构域是CD28或4-1BB分子的跨膜结构域。
在细胞外结构域和CAR的跨膜结构域之间,可以掺入间隔结构域。如本文所用,术语“间隔结构域”通常是指用于将跨膜结构域连接至多肽链上的细胞外结构域和/或细胞质结构域的任何寡聚体或多肽。间隔结构域可包括至多300个氨基酸,优选10至100个氨基酸,最优选25至50个氨基酸。
目前,为了帮助CAR T细胞在体内扩增,需要表达抗原刺激CAR T细胞增殖,如DC疫苗/脂链整合抗原在DC表面/某些免疫细胞过表达的方法如肿瘤细胞表面抗原3-5。这种依赖TCR信号的长期放大方式可以大量刺激T细胞的扩增,但会导致细胞很快被耗尽和凋亡,使细胞处于功能失调状态。IL2/IL7/IL12/IL15/IL18/IL23/等细胞因子的表达是方法6-9之一。虽然IL12和IL18会迅速扩大细胞,但它们也会导致细胞快速分化并增加CAR-T细胞的耗竭和毒性,IL7/IL15的表达会使细胞保持在记忆状态,但不会导致细胞数量的良好扩展。目前还没有办法保持单元记忆状态并迅速扩展。
我们通过单细胞测序找到了抗原非依赖性扩增的源基因,这是过表达因子等常规方法无法实现的。本发明将细胞因子等信号的下游活化分子或持续活化受体与实体瘤CAR结合,在CAR中持续表达或诱导持续活化分子的表达,从而扩增T细胞。通过在CAR T中诱导表达替代细胞因子的作用,CAR T可以在无细胞因子或极少细胞因子的幼稚状态下进行扩增,增强其扩增效果,减少副作用。CAR T扩增到足够数量后,去除诱导因子,进一步控制其扩增。此外,本发明在实体瘤CAR中表达或敲除转录因子。允许CAR T在无细胞因子或极少细胞因子的情况下在幼稚状态下进行扩增,增强其扩增效果并减少副作用,并可对T细胞进行重新编程,使耗尽的T细胞恢复到幼稚状态。在实施例中,通过用多种病毒感染T细胞,可以从T细胞群中获得多基因表达复合T细胞,从而达到更强的扩增效果。
本公开描述了用于治疗癌症的组合物和方法。实施方案涉及经修饰以表达一种或多种分子的水平高于或低于未经修饰以表达一种或多种分子的细胞所表达的一种或多种分子的水平的细胞,其中所述一种或多种分子在单细胞谱分析中被鉴定。实施方案涉及经工程改造以表达抗原结合分子的修饰细胞,其中修饰细胞中一种或多种分子的表达和/或功能已被增强或降低(包括消除),其中一种或多种分子被鉴定为单个-细胞谱分析。在实施方案中,修饰的细胞包括与一种或多种分子的生物合成或运输途径相关的内源基因的破坏或外源基因的添加。实施方案涉及包含细胞群的药物组合物。
过表达分子的例子包括PLEKHN1、IL2、IL17F、IL31、IL3、CCL22、TNFSF4、PMCH、CCL1、XCL2、CCL17、TNIP3、IL5、G0S2、IL22、CSF2、CAVIN3、CCL4、CCL3、IFNGCL4、CCL3、IFNGCL4,ZBED2,CXCL9,CD40,FSCN1,GNLY,TPRG1,AL031777.3,LYN,LTA,CRTAM,GZMB,MIR155HG,MT2A,IL13,ELL2,RGS16,IER3,DUSP4,HLLM,NABPDU,PYCDA,PYC2A,BCAT1,NPM3,CCDC50,HSP90AB1,ODC1,TXN,PIM3,RAN,NAMPT,H2AFZ,PRDX1,LGALS1,MIF,EIF4G2,CCL5,TAGLN2,TRAF1,TUBB,CDC42,TUBA1B,,FTL,MA、TCP1、TPT1、CD70、PPIA、SEC61B、TMSB4X、PFN1、LDHA、HNRNPAB、BIRC3、NME2、TUBA1C、ATP5MC3、ANP32E、PTGES3、H3F3B、IL2RA、PKM、SH3GBRL1SB10、SERBP1、TPM3、BTF3、CFL1、MYL6、ATP5MC2、ACTB、OAZ1、S100A4和PGK1。
实施方案涉及多核苷酸的方法或用途,该方法包括提供包含载体基因组的病毒颗粒(例如,AAV、慢病毒或其变体),该载体基因组包含编码一个或多个分子的多核苷酸和编码抗原结合的多核苷酸分子,多核苷酸可操作地连接到赋予多核苷酸转录的表达控制元件;并且向受试者施用一定量的病毒颗粒,使得多核苷酸在受试者中表达,其中一种或多种分子在单细胞谱分析中被鉴定。在实施方案中,AAV制剂可以包括AAV载体颗粒、空衣壳和宿主细胞杂质,从而提供基本上不含AAV空衣壳的AAV产品。
实施方案涉及在有需要的受试者中引起或引发T细胞应答和/或治疗受试者的肿瘤的方法,该方法包括向受试者施用有效量的的组合物。实施方案涉及编码与修饰细胞的代谢相关的一种或多种分子的分离的核酸。
在实施方案中,一种或多种分子包含(过表达或诱导型过表达)JAK2、STAT1、STAT2、STAT3、STAT4、PRKCA、PRKCQ、MAP3K1、MAP3K4、MAP3K8、MAPK14、BRAF、SMAD5、IL2RB、IL12RB2、IL1 IL21R、IL7R、CD28、Cavin3、ZBED2、MYC、EGFR、HER2、HER3、HER4、VEGFR2、VEGFR3、PDGFRα、PDGFRβ、gp130、IL-23R、IL-7R、CRLF2、βc、GHR、THPOR、EPR CSF3R、TNFR1、TGFBR1、TGFBR2、ACVR1A、BMPR2 ACVR1B、CXCR1、CXCR2、CXCR3、CXCR4、CCR2和CCR5;和/或(敲除或敲低)TOX、TOX2、TOX4、NR4A2、NR4A3、一种或多种分子的功能变体、或一种或多种分子的功能片段。
在实施方案中,被修饰以表达一种或多种分子的细胞的水平高于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少JAK2、STAT1、STAT2、STAT3、STAT4、PRKCA、PRKCQ、MAP3K1、MAP3K4、MAP3K8、MAPK14、BRAF、SMAD5、IL2RB、IL12RB2、IL18R1、IL21R、IL7R、CD28之一。
在实施方案中,被修饰以表达一种或多种分子的细胞的水平高于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少Cavin3、ZBED2、MYC之一。
在实施方案中,被修饰以表达一种或多种分子的细胞的水平高于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少EGFR、HER2、HER3、HER4、VEGFR2、VEGFR3、PDGFRα、PDGFRβ、gp130、IL-23R、IL-7R、CRLF2、βc、GHR、THPOR、EPOR、LepR、CSF3R、TNFR1、TGFBR1、ACVR1、TGFBR2之一BMPR2 ACVR1B、CXCR1、CXCR2、CXCR3、CXCR4、CCR2和CCR5。
在实施方案中,被修饰以表达一种或多种分子的细胞以低于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少TOX、TOX2、TOX4、NR4A2、NR4A3之一。
在实施方案中,修饰的细胞包含抗原结合分子。在实施方案中,抗原结合分子是CAR,其包含抗原结合域、跨膜域和细胞内信号域。在实施方案中,结合肿瘤抗原的抗原结合域选自:TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2,Mesothelin,IL-11Ra,PSCA,PRSS21,VEGFR2,LewisY,CD24,PDGFR-beta,SSEA-4,CD20,叶酸受体α,ERBB2(Her2/neu),MUC1,EGFR,NCAM,Prostase,PAP,ELF2M,Ephrin B2,IGF-I受体,CAIX,LMP2,gp100,bcr-abl,酪氨酸酶,EphA2,岩藻糖基GM1,sLe,GM3,TGS5,HMWMAA,o-乙酰基-GD2,叶酸受体β,TEM1/CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD97,CD179a,ALK,聚唾液酸,PLAC1,NY-GloboH-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPV E6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺素、存活蛋白和端粒酶、PCTA-1/半乳糖凝集素8、MelanA/MART1、Ras突变体,hTERT,sar昏迷易位断点、ML-IAP、ERG(TMPRSS2 ETS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 hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY3、75、FCRL5和IGLL1。在实施方案中,细胞内信号传导域包含共刺激信号传导域,或初级信号传导域和共刺激信号传导域,其中共刺激信号传导域包含选自由CD27组成的组的蛋白质的功能性信号传导域,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-H3,一种配体与CD83、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8alpha、CD8beta、IL2R beta、IL2R gamma、IL7R alpha、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 1,ITGB7,TNFR2,TRANCE/RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1,CRTAM,Ly9(CD229),CD160(BY55),PSGL1,CD100),(SEMA)CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BL AME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D。
在实施方案中,修饰的细胞包含抗原结合分子,抗原结合分子是修饰的TCR(TCR)或TCR(TIL)。在实施方案中,TCR源自患者中自发发生的肿瘤特异性T细胞。在实施方案中,TCR结合肿瘤抗原。在实施方案中,肿瘤抗原包括CEA、gp100、MART-1、p53、MAGE-A3或NY-ESO-1。在实施方案中,TCR包括TCRγ和TCRδ链或TCRα和TCRβ链,或其组合。
在实施方案中,其中细胞是免疫效应细胞(例如,免疫效应细胞群)。在实施方案中,免疫效应细胞是DC、巨噬细胞、T细胞或NK细胞。在实施方案中,免疫效应细胞是T细胞。在实施方案中,T细胞是CD4+T细胞、CD8+T细胞或其组合。在实施方案中,细胞是人类细胞。
在实施方案中,一种或多种分子的增强表达和/或功能通过在重组DNA构建体中引入编码一种或多种分子和/或结合分子的核酸来实现,所述核酸存在于经修饰的细胞中的重组DNA构建体中。mRNA,或病毒载体。在实施方案中,核酸是未整合到修饰细胞的基因组中的mRNA。在实施方案中,核酸与氧敏感多肽结构域相关联。在实施方案中,氧敏感多肽结构域包含HIF VHL结合结构域。在实施方案中,核酸由包含转录调节剂的结合位点的启动子调节,所述转录调节剂调节细胞中治疗剂的表达和/或分泌。在实施方案中,转录调节剂是或包括Hifla、NFAT、FOXP3和/或NFkB。
实施方案涉及包含编码抗原结合分子的多核苷酸的修饰DC,所述抗原结合分子包含抗原结合结构域、跨膜结构域和细胞内信号传导结构域。一些实施方案涉及编码抗原结合分子的分离的核酸,其包含抗原结合结构域、跨膜结构域和细胞内信号传导结构域。一些实施方案涉及包含细胞群的药物组合物。一些实施方案涉及在有需要的受试者中增强T细胞应答和/或治疗受试者的肿瘤的方法,该方法包括向受试者施用有效量的组合物。一些实施方案涉及多核苷酸的方法或用途,该方法包括提供包含载体基因组的病毒颗粒(例如,AAV、慢病毒或其变体),载体基因组包含核酸,多核苷酸可操作地连接至表达控制元件赋予多核苷酸转录;并且向受试者施用一定量的病毒颗粒,使得多核苷酸在受试者中表达,其中一种或多种分子在癌细胞中过度表达,与免疫细胞的募集相关,和/或与自身免疫相关。在实施方案中,AAV制剂可以包括AAV载体颗粒、空衣壳和宿主细胞杂质,从而提供基本上不含AAV空衣壳的AAV产品。
在实施方案中,一种或多种分子的表达受一种或多种启动子调控。在实施方案中,多核苷酸包含包含转录调节剂的结合位点的启动子,所述转录调节剂调节细胞中一种或多种分子的表达和/或分泌。例如,转录调节剂是或包括Hif1a、NFAT、FOXP3和/或NFkB。例如,一种或多种分子包含至少一种与氧敏感多肽结构域相关的细胞因子,并且氧敏感多肽结构域包含HIF VHL结合结构域。
在实施方案中,多核苷酸可以整合到修饰细胞的基因组中,并且修饰细胞的后代也将表达多核苷酸,从而产生稳定转染的修饰细胞。在实施方案中,经修饰细胞可表达编码CAR的多核苷酸,但多核苷酸不整合到经修饰细胞的基因组中,使得经修饰细胞在有限时间段(例如,数天)内表达瞬时转染的多核苷酸,之后多核苷酸因细胞分裂或其他因素而丢失。例如,多核苷酸以重组DNA构建体、mRNA或病毒载体的形式存在于修饰细胞中,和/或多核苷酸是未整合到修饰细胞基因组中的mRNA。
目前治疗癌症最有效的方法是CAR T,但CAR T在实体瘤中存在几个问题:1.如何迁移到肿瘤细胞中;2.需要DC细胞提供一些特殊因子(如IL12);3.自身呈递新抗原的能力。如何完全激活DC细胞,之前在做CAR-DC之类的文章或者CAR T CAR分子,这两种细胞的激活是有很大区别的(T细胞主要靠TCR)。DC细胞的主要信号来源于TOLL like Receptor)。4.外周血中DC细胞比例低于T细胞,纯化培养困难。本发明使DC特异性识别肿瘤,增强DC细胞的功能,通过DC招募其他修饰和未修饰的免疫细胞,并通过DC释放各种细胞因子(IL12等),通过DC激活促进免疫系统功能。其他免疫细胞,从而增强免疫治疗的有效性。相关实验包括:1.nCAR-DC杀伤实验,2.tCAR-DC(TraditionCAR)杀伤实验,3.nCAR-DC+NT杀伤实验,4.tCAR-DC+NT杀伤实验,5.nCAR-DC+CAR-T杀伤实验,6.tCAR-DC+CAR T杀伤实验。本发明基于Toll-Like Receptor(TLR)设计的CAR,充分激活DC细胞,促进DC细胞识别肿瘤的功能。同时,我们也可以在DC细胞中表达适合CAR-T的CAR结构,从而激活DC细胞,促进DC细胞的功能。它也可以是CAR-DC和CAR T、TIL、TCR和其他细胞疗法的组合。信号域包括10个不同的TLR;TLR1-10(使用TLR9或Myd88的计划实验)。Myd88:髓系分化因子,与TLR分子的胞内结构域具有相同的TIR结构域,是TLR中的关键接头分子,在传递下游信息中起关键作用。TRIF可以产生IFN-β和含有TIR结构域的衔接蛋白。
在实施方案中,跨膜结构域包含下表2所列分子中的至少一种的跨膜结构域(例如,CD8 CD40)。
表2
分子 | 跨膜结构域序列 |
CD8b | SEQ ID NO:1 |
CD8a | SEQ ID NO:2 |
CD40 | SEQ ID NO:3 |
CD4 | SEQ ID NO:4 |
CD5 | SEQ ID NO:5 |
CD3zeta | SEQ ID NO:6 |
CD22 | SEQ ID NO:7 |
CD28 | SEQ ID NO:8 |
CD33 | SEQ ID NO:9 |
CD64 | SEQ ID NO:10 |
CD80 | SEQ ID NO:11 |
CD86 | SEQ ID NO:12 |
CD134 | SEQ ID NO:13 |
CD137 | SEQ ID NO:14 |
CD154 | SEQ ID NO:15 |
在实施方案中,细胞内信号域包含TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、MYD88、TRIF、TRAM和TIRAP中的至少一种的信号域。在实施方案中,细胞内信号结构域包含TLR9和MYD88中至少一种的信号结构域。在实施方案中,细胞内信号结构域包含下表3所列分子中的至少一种的信号结构域。
表3
分子 | 信令域序列 |
TLR1 | SEQ ID NO:16 |
TLR2 | SEQ ID NO:17 |
TLR3 | SEQ ID NO:18 |
TLR4 | SEQ ID NO:19 |
TLR5 | SEQ ID NO:20 |
TLR6 | SEQ ID NO:21 |
TLR7 | SEQ ID NO:22 |
TLR8 | SEQ ID NO:23 |
TLR9 | SEQ ID NO:24 |
TLR10 | SEQ ID NO:25 |
MYD88 | SEQ ID NO:26 |
TRIF | SEQ ID NO:27 |
TRAM | SEQ ID NO:28 |
TIRAP | SEQ ID NO:29 |
在实施方案中,细胞内信号结构域包含CD40和RAGE中的至少一种。
在实施方案中,抗原结合分子是CAR。在实施方案中,细胞内信号传导域包含共刺激信号传导域,或初级信号传导域和共刺激信号传导域,其中共刺激信号传导域包含选自由CD27组成的组的蛋白质的功能性信号传导域,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-H3,一种与CD83、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8alpha、CD8beta、IL2R beta、IL2R gamma、IL7R alpha、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 1,ITGB7,TNFR2,TRANCE/RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1,CRTAM,Ly9(CD229),CD160(BY55),PSGL1,CD100),(SEMA)CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BL AME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46或NKG2D结合的配体。
在实施方案中,结合肿瘤抗原的抗原结合域选自:TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-11Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-beta,SSEA-4,CD20,叶酸受体α,ERBB2(Her2/neu),MUC1,EGFR,NCAM,Prostase,PAP,ELF2M,Ephrin B2,IGF-I受体,CAIX,LMP2,gp100,bcr-abl,酪氨酸酶,EphA2、岩藻糖基GM1、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、聚唾液酸、PLAC1、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、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺素、存活蛋白和端粒酶、PCTA-1/半乳糖凝集素8、MelanA/MART1、Ras突变体,hTERT,肉瘤易位断点、ML-IAP、ERG(TMPRSS2 ETS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 hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY3、75、FCRL5和IGLL1。
在实施方案中,抗原结合分子进一步包含CD3 zeta结构域。在实施方案中,抗原结合分子是修饰的TCR。在实施方案中,TCR源自患者中自发发生的肿瘤特异性T细胞。在实施方案中,TCR结合肿瘤抗原。在实施方案中,肿瘤抗原包括CEA、gp100、MART-1、p53、MAGE-A3或NY-ESO-1。在实施方案中,TCR包括TCRγ和TCRδ链或TCRα和TCRβ链,或其组合。
在实施方案中,细胞是人类细胞。在实施方案中,一种或多种分子的增强表达和/或功能通过在重组DNA构建体中引入编码一种或多种分子和/或结合分子的核酸来实现,所述核酸存在于经修饰的细胞中的重组DNA构建体中。mRNA,或病毒载体。在实施方案中,核酸是未整合到修饰细胞的基因组中的mRNA。
实施方案涉及经修饰以表达一种或多种分子的水平高于或低于未经修饰以表达一种或多种分子的细胞所表达的一种或多种分子的水平的细胞,其中所述一种或多种分子在单细胞谱分析中被鉴定。
实施方案涉及经工程改造以表达抗原结合分子的修饰细胞,其中修饰细胞中一种或多种分子的表达和/或功能已被增强或降低(包括消除),其中一种或多种分子被鉴定为单个-细胞谱分析。
实施方案涉及多核苷酸的方法或用途,该方法包括提供包含载体基因组的病毒颗粒(例如,AAV、慢病毒或其变体),该载体基因组包含编码一个或多个分子的多核苷酸和编码抗原结合的多核苷酸分子,多核苷酸可操作地连接到赋予多核苷酸转录的表达控制元件;并且向受试者施用一定量的病毒颗粒,使得多核苷酸在受试者中表达,其中一种或多种分子在单细胞谱分析中被鉴定。
实施方案涉及包含细胞群的药物组合物。
实施方案涉及在有需要的受试者中引起或引发T细胞应答和/或治疗受试者的肿瘤的方法,该方法包括向受试者施用有效量的组合物。
实施方案涉及编码与修饰细胞的代谢相关的一种或多种分子的分离的核酸。
在实施方案中,修饰的细胞包括与一种或多种分子的生物合成或运输途径相关的内源基因的破坏或外源基因的添加。
在实施方案中,AAV制剂可以包括AAV载体颗粒、空衣壳和宿主细胞杂质,从而提供基本上不含AAV空衣壳的AAV产品。
在实施方案中,一种或多种分子包含(过表达或诱导型过表达)JAK2、STAT1、STAT2、STAT3、STAT4、PRKCA、PRKCQ、MAP3K1、MAP3K4、MAP3K8、MAPK14、BRAF、SMAD5、IL2RB、IL12RB2、IL1IL21R、IL7R、CD28、Cavin3、ZBED2、MYC、EGFR、HER2、HER3、HER4、VEGFR2、VEGFR3、PDGFRα、PDGFRβ、gp130、IL-23R、IL-7R、CRLF2、βc、GHR、THPOR、EPR CSF3R、TNFR1、TGFBR1、TGFBR2、ACVR1A、BMPR2 ACVR1B、CXCR1、CXCR2、CXCR3、CXCR4、CCR2和CCR5;和/或(敲除或敲低)TOX、TOX2、TOX4、NR4A2、NR4A3、一种或多种分子的功能变体、或一种或多种分子的功能片段。
在实施方案中,被修饰以表达一种或多种分子的细胞的水平高于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少JAK2、STAT1、STAT2、STAT3、STAT4、PRKCA、PRKCQ、MAP3K1、MAP3K4、MAP3K8、MAPK14、BRAF、SMAD5、IL2RB、IL12RB2、IL18R1、IL21R、IL7R、CD28之一。
在实施方案中,被修饰以表达一种或多种分子的细胞的水平高于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少Cavin3、ZBED2、MYC之一。
在实施方案中,被修饰以表达一种或多种分子的细胞的水平高于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少EGFR、HER2、HER3、HER4、VEGFR2、VEGFR3、PDGFRα、PDGFRβ、gp130、IL-23R、IL-7R、CRLF2、βc、GHR、THPOR、EPOR、LepR、CSF3R、TNFR1、TGFBR1、ACVR1、TGFBR2之一BMPR2 ACVR1B、CXCR1、CXCR2、CXCR3、CXCR4、CCR2和CCR5。
在实施方案中,被修饰以表达一种或多种分子的细胞以低于未被修饰以表达一种或多种分子的细胞表达的一种或多种分子的水平,一种或多种分子包含至少TOX、TOX2、TOX4、NR4A2、NR4A3之一。
在实施方案中,修饰的细胞包含抗原结合分子。
在实施方案中,抗原结合分子是CAR,其包含抗原结合域、跨膜域和细胞内信号域。
在实施方案中,结合肿瘤抗原的抗原结合域选自:TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2,Mesothelin,IL-11Ra,PSCA,PRSS21,VEGFR2,LewisY,CD24,PDGFR-beta,SSEA-4,CD20,叶酸受体α,ERBB2(Her2/neu),MUC1,EGFR,NCAM,Prostase,PAP,ELF2M,Ephrin B2,IGF-I受体,CAIX,LMP2,gp100,bcr-abl,酪氨酸酶,EphA2,岩藻糖基GM1,sLe,GM3,TGS5,HMWMAA,o-乙酰基-GD2,叶酸受体β,TEM1/CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD97,CD179a,ALK,聚唾液酸,PLAC1,NY-GloboH-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPV E6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺素、存活蛋白和端粒酶、PCTA-1/半乳糖凝集素8、MelanA/MART1、Ras突变体,hTERT,sar昏迷易位断点、ML-IAP、ERG(TMPRSS2 ETS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 hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY3、75、FCRL5和IGLL1。
在实施方案中,细胞内信号传导域包含共刺激信号传导域,或初级信号传导域和共刺激信号传导域,其中共刺激信号传导域包含选自由CD27组成的组的蛋白质的功能性信号传导域,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-H3,一种配体与CD83、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8alpha、CD8beta、IL2R beta、IL2Rgamma、IL7R alpha、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 1,ITGB7,TNFR2,TRANCE/RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1,CRTAM,Ly9(CD229),CD160(BY55),PSGL1,CD100),(SEMA)CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BL AME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D
在实施方案中,修饰的细胞包含抗原结合分子,抗原结合分子是修饰的TCR(TCR)或TCR(TIL)。
在实施方案中,TCR源自患者中自发发生的肿瘤特异性T细胞。
在实施方案中,TCR结合肿瘤抗原。
在实施方案中,肿瘤抗原包括CEA、gp100、MART-1、p53、MAGE-A3或NY-ESO-1。
在实施方案中,TCR包括TCRγ和TCRδ链或TCRα和TCRβ链,或其组合。
在实施方案中,细胞是免疫效应细胞(例如,免疫效应细胞群)。
在实施方案中,免疫效应细胞是DC、巨噬细胞、T细胞或NK细胞。
在实施方案中,免疫效应细胞是T细胞。
在实施方案中,T细胞是CD4+T细胞、CD8+T细胞或其组合。
在实施方案中,细胞是人类细胞。
在实施方案中,一种或多种分子的增强的表达和/或功能通过在重组DNA构建体中引入编码一种或多种分子和/或结合分子的核酸来实现,所述核酸存在于经修饰的细胞中的重组DNA构建体中。mRNA,或病毒载体。
在实施方案中,核酸是未整合到修饰细胞的基因组中的mRNA。
在实施方案中,核酸与氧敏感多肽结构域相关联。
在实施方案中,氧敏感多肽结构域包含HIF VHL结合结构域。
在实施方案中,核酸由包含转录调节剂的结合位点的启动子调节,所述转录调节剂调节细胞中治疗剂的表达和/或分泌。
在实施方案中,转录调节剂是或包括Hifla、NFAT、FOXP3和/或NFkB。
实施方案涉及包含编码抗原结合分子的核酸的修饰DC,其包含抗原结合结构域、跨膜结构域和细胞内信号传导结构域。
实施方案涉及编码抗原结合分子的分离核酸,其包含抗原结合结构域、跨膜结构域和细胞内信号传导结构域。
实施方案涉及多核苷酸的方法或用途,该方法包括提供包含载体基因组的病毒颗粒(例如,AAV、慢病毒或其变体),该载体基因组包含核酸,该多核苷酸可操作地连接至赋予表达控制元件的表达控制元件多核苷酸的转录;并且向受试者施用一定量的病毒颗粒,使得多核苷酸在受试者中表达,其中一种或多种分子在癌细胞中过度表达,与免疫细胞的募集相关,和/或与自身免疫相关。
在实施方案中,AAV制剂可以包括AAV载体颗粒、空衣壳和宿主细胞杂质,从而提供基本上不含AAV空衣壳的AAV产品。
实施方案涉及包含细胞群的药物组合物。
实施方案涉及在有需要的受试者中增强T细胞应答和/或治疗受试者的肿瘤的方法,该方法包括向受试者施用有效量的组合物。
在实施方案中,跨膜结构域包括CD8或CD40的跨膜结构域。
在实施方案中,细胞内信号域包含TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、MYD88、TRIF、TRAM和TIRAP中的至少一种的信号域。
在实施方案中,细胞内信号结构域包含TLR9和MYD88中至少一种的信号结构域。
在实施方案中,细胞内信号结构域包含CD40和RAGE中的至少一种。
在实施方案中,抗原结合分子是CAR。
在实施方案中,细胞内信号传导域包含共刺激信号传导域,或初级信号传导域和共刺激信号传导域,其中共刺激信号传导域包含选自由CD27组成的组的蛋白质的功能性信号传导域,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-H3,一种配体与CD83、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8alpha、CD8beta、IL2R beta、IL2Rgamma、IL7R alpha、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 1,ITGB7,TNFR2,TRANCE/RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1,CRTAM,Ly9(CD229),CD160(BY55),PSGL1,CD100),(SEMA)CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BL AME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D。
在实施方案中,结合肿瘤抗原的抗原结合域选自:TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2,Mesothelin,IL-11Ra,PSCA,PRSS21,VEGFR2,LewisY,CD24,PDGFR-beta,SSEA-4,CD20,叶酸受体α,ERBB2(Her2/neu),MUC1,EGFR,NCAM,Prostase,PAP,ELF2M,Ephrin B2,IGF-I受体,CAIX,LMP2,gp100,bcr-abl,酪氨酸酶,EphA2,岩藻糖基GM1,sLe,GM3,TGS5,HMWMAA,o-乙酰基-GD2,叶酸受体β,TEM1/CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD97,CD179a,ALK,聚唾液酸,PLAC1,NY-GloboH-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPV E6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺素、存活蛋白和端粒酶、PCTA-1/半乳糖凝集素8、MelanA/MART1、Ras突变体,hTERT,sar昏迷易位断点、ML-IAP、ERG(TMPRSS2 ETS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 hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY3、75、FCRL5和IGLL1。
在实施方案中,抗原结合分子进一步包含CD3 zeta结构域。
在实施方案中,抗原结合分子是修饰的TCR。
在实施方案中,TCR源自患者中自发发生的肿瘤特异性T细胞。
在实施方案中,TCR结合肿瘤抗原。
在实施方案中,肿瘤抗原包括CEA、gp100、MART-1、p53、MAGE-A3或NY-ESO-1。
在实施方案中,TCR包括TCRγ和TCRδ链或TCRα和TCRβ链,或其组合。
在实施方案中,细胞是人类细胞。
在实施方案中,一种或多种分子的增强的表达和/或功能通过在重组DNA构建体中引入编码一种或多种分子和/或结合分子的核酸来实现,所述核酸存在于经修饰的细胞中的重组DNA,mRNA,或病毒载体的构建体中。
在实施方案中,核酸是未整合到修饰细胞的基因组中的mRNA。
实施方案涉及包含一种或多种编码MYD88的外源多核苷酸的淋巴细胞。在实施方案中,淋巴细胞包含编码CD40胞内结构域的外源多核苷酸。
实施方案涉及包含一种或多种编码MYC原癌基因、bHLH转录因子(MYC)、Caveolae相关蛋白3(CAVIN3)和/或含锌指床型2(ZBED2)的外源多核苷酸的淋巴细胞。
实施例涉及一种增强淋巴细胞抗肿瘤作用的方法,该方法包括:将一种或多种编码小窝窝内相关蛋白3(CAVIN3)和/或含锌指床型2(ZBED2)的外源多核苷酸引入淋巴细胞中以获得修饰的淋巴细胞;使修饰的淋巴细胞与修饰的淋巴细胞结合的肿瘤细胞接触;并且允许淋巴细胞发生抗肿瘤作用,其中与未过表达CAVIN3和ZBED2的淋巴细胞相比,抗肿瘤作用增强。
在实施方案中,外源多核苷酸包含编码SEQ ID NO:31-43之一的氨基酸。
在实施方案中,淋巴细胞是T细胞或NK细胞。
在实施方案中,抗肿瘤作用表现为细胞耗竭减少、细胞扩增增强和肿瘤生长抑制增强中的至少一种。
在实施方案中,修饰的淋巴细胞的细胞耗竭水平低于没有过表达CAVIN3和ZBED2的淋巴细胞的细胞耗竭水平。
在实施方案中,修饰的淋巴细胞的细胞扩增水平高于没有过表达CAVIN3和ZBED2的淋巴细胞的细胞扩增水平。
在实施方案中,修饰的淋巴细胞的肿瘤生长抑制大于没有过表达CAVIN3和ZBED2的淋巴细胞的肿瘤生长抑制。
在实施方案中,经修饰的淋巴细胞释放的细胞因子水平不显着高于没有过表达CAVIN3和ZBED2的淋巴细胞的水平。
在实施方案中,修饰的淋巴细胞的活化水平不显着高于没有过表达CAVIN3和ZBED2的淋巴细胞的活化水平。
在实施方案中,淋巴细胞包含抗原结合分子。
在实施方案中,淋巴细胞包含编码显性失活形式的抑制性免疫检查点分子或其受体的核酸序列,并且抑制性免疫检查点分子选自程序性死亡1(PD-1)、细胞毒性T淋巴细胞抗原4(CTLA-4)、B和T淋巴细胞衰减剂(BTLA)、T细胞免疫球蛋白粘蛋白3(TIM-3)、淋巴细胞活化蛋白3(LAG-3)、具有Ig和ITIM结构域(TIGIT)、白细胞相关免疫球蛋白样受体1(LAIRl)、自然杀伤细胞受体2B4(2B4)和CD 160。
在实施方案中,淋巴细胞包含编码hTERT的核酸序列或编码SV40LT的核酸,或其组合。
在实施方案中,淋巴细胞包含编码细胞因子的核酸序列。在实施方案中,细胞因子包括IL-6、IL-7、IL-15、IL-12或IFNγ。
在实施方案中,淋巴细胞包含包含结合白细胞(WBC)的细胞表面分子的CAR的第一T细胞群和包含结合实体瘤抗原的CAR的第二T细胞群。
例子
表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞
图1显示表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的CAR表达和表型,可以看到,均能在T细胞上检测到CAR表达,CD4/CD8分型正常。图2显示了在与底物细胞NALM6共培养后表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的细胞活化,可以看到,共培养24小时后,CAR-T细胞均能被正常激活。图3显示了在与底物细胞NALM6共培养后表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的细胞凋亡,可以看到,通过比较活细胞群的百分比来比较这些基因的抗凋亡能力:MYB-WT>JUN T231A>MYC S62D>JUN-DD>MYC-WT>BCL2>MYC S71F>MYC T58A>BCLXL>MYC S329D>MYC K51R。图4显示了在与底物细胞NALM6共培养后表达JUN、MYC、MYB、BCL2、BCLXL或以上突变型的CAR T细胞的细胞增殖,可以看到,MYB-WT,JUN T231A和BCLXL对细胞增殖上的增强比较明显,BCL2,JUN-DD,MYC S62D和MYC S71F对细胞的增殖有一定增强,其余gene对细胞的增殖方面的加强较弱。
表达CAVIN3或ZBED2的CAR T细胞
图5A和5B(图5)显示表达CAVIN3或ZBED2的CAR T细胞的CAR表达和表型。用含有载体CD19CAR和CAVIN3或ZBED2的慢病毒转导T细胞,并分析CAR表达和CAR T细胞的表型。如图5所示。CAVIN3和ZBED2不影响CAR的表达,表达CAVIN3或ZBED2的CD19 CAR T细胞耗竭水平低于未过表达CAVIN3和ZBED2的CD19 CAR T细胞。表达ZBED2的CAR T细胞表现出更多的幼稚和类似记忆的表型,预期细胞在体内外的持久性更高。总之,CAVIN3和ZBED2不影响CART细胞的功能,CAVIN3使CAR T细胞向效应T细胞分化,而ZBED2使细胞向记忆CAR T细胞分化。
图6显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞活化。表达CAVIN3或ZBED2的NT或CD19 CAR T细胞与NALM6细胞以1:1的比例共培养。CAR T细胞显示正常活化,未过表达CAVIN3和ZBED2的CD19 CAR T细胞的活化高于表达CAVIN3或ZBED2的CD19 CAR T细胞,表明CAVIN3或ZBED2的表达保护了细胞不被过度活化.
图7显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞耗竭。表达CAVIN3或ZBED2的NT或CD19 CAR T细胞与NALM6细胞以1:1的比例共培养。如图所示。如图7所示,表达CAVIN3或ZBED2的CAR T细胞的细胞耗竭水平低于未过表达CAVIN3和ZBED2的CAR T细胞。因此,CAVIN3或ZBED2的表达有助于减少CAR T细胞的耗竭,促进CAR T细胞的持久性。
图8显示了与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞表型变化。表达CAVIN3或ZBED2的NT或CD19 CAR T细胞与NALM6细胞以1:1的比例共培养。共培养后,虽然NT或CAR T细胞向效应细胞表型分化,但与未过表达CAVIN3和ZBED2的CAR T细胞相比,CAR T细胞CAVIN3或ZBED2保持了更多的细胞表型(更少的效应T细胞)。
图9显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的细胞因子释放。表达CAVIN3或ZBED2的NT或CD19 CAR T细胞与NALM6细胞以1:1的比例共培养。共培养后,测量了CAR T细胞释放的细胞因子。如图所示。如图9所示,这三种类型的CAR T细胞在与NALM6共培养后显示出相似的细胞因子释放水平。
图10显示了在与底物细胞NALM6共培养后表达CAVIN3或ZBED2的CAR T细胞的肿瘤抑制测定结果。表达CAVIN3或ZBED2的NT或CD19 CAR T细胞与NALM6细胞以1:1的比例共培养。共培养不同时间后,分析肿瘤细胞群(NALM6)。如图所示。如图10所示,CAR T细胞CAVIN3或ZBED2的抑癌水平高于未过表达CAVIN3和ZBED2的CAR T细胞。
图11显示表达CAVIN3或ZBED2的CAR T细胞的细胞扩增。将表达CAVIN3或ZBED2的NT或CD19 CAR T细胞与含有IL2的TEXMACS一起培养,并在第1、5、7、10、13和15天计数。如图11所示,CAR T细胞CAVIN3或ZBED2的细胞扩增水平高于未过表达CAVIN3和ZBED2的CAR T细胞。
表4
身份标识 | SEQ ID NO: | 身份标识 | SEQ ID NO: |
CD8b | SEQ ID NO:1 | TLR8 | SEQ ID NO:23 |
CD8a | SEQ ID NO:2 | TLR9 | SEQ ID NO:24 |
CD40 | SEQ ID NO:3 | TLR10 | SEQ ID NO:25 |
CD4 | SEQ ID NO:4 | MYD88 | SEQ ID NO:26 |
CD5 | SEQ ID NO:5 | TRIF | SEQ ID NO:27 |
CD3zeta | SEQ ID NO:6 | TRAM | SEQ ID NO:28 |
CD22 | SEQ ID NO:7 | TIRAP | SEQ ID NO:29 |
CD28 | SEQ ID NO:8 | 链接 | SEQ ID NO:30 |
CD33 | SEQ ID NO:9 | Cavin3 | SEQ ID NO:31 |
CD64 | SEQ ID NO:10 | ZBED2 | SEQ ID NO:32 |
CD80 | SEQ ID NO:11 | MYC | SEQ ID NO:33 |
CD86 | SEQ ID NO:12 | MYB | SEQ ID NO:34 |
CD134 | SEQ ID NO:13 | BCL2 | SEQ ID NO:35 |
CD137 | SEQ ID NO:14 | BCLXL | SEQ ID NO:36 |
CD154 | SEQ ID NO:15 | JUN-DD | SEQ ID NO:37 |
TLR1 | SEQ ID NO:16 | MYC K51R | SEQ ID NO:38 |
TLR2 | SEQ ID NO:17 | MYC T58A | SEQ ID NO:39 |
TLR3 | SEQ ID NO:18 | JUN T231A | SEQ ID NO:40 |
TLR4 | SEQ ID NO:19 | MYC S62D | SEQ ID NO:41 |
TLR5 | SEQ ID NO:20 | MYC S329D | SEQ ID NO:42 |
TLR6 | SEQ ID NO:21 | MYC S71F | SEQ ID NO:43 |
TLR7 | SEQ ID NO:22 |
本说明书中引用的所有出版物,专利和专利申请均通过引用整体并入本文,如同每个单独的出版物,专利或专利申请被具体和单独地指示为通过引用并入。虽然已经根据各种实施例描述了前述内容,但是本领域技术人员将认识到,在不脱离其精神的情况下可以进行各种修改,替换,省略和改变。
序列表
<110> 创新细胞治疗控股有限公司
创新细胞治疗公司
<120> 嵌合抗原受体T 细胞群、组合物及其用途
<160> 28
<170> SIPOSequenceListing 1.0
<210> 16
<211> 185
<212> PRT
<213> TLR1(homo sapiens )
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<212> PRT
<213> TLR3(homo sapiens )
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Ser Val His
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<211> 187
<212> PRT
<213> TLR4(homo sapiens )
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Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala
100 105 110
Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg
115 120 125
Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu
130 135 140
Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg
145 150 155 160
Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly
165 170 175
Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile
180 185
<210> 20
<211> 198
<212> PRT
<213> TLR5(homo sapiens )
<400> 20
Thr Lys Phe Arg Gly Phe Cys Phe Ile Cys Tyr Lys Thr Ala Gln Arg
1 5 10 15
Leu Val Phe Lys Asp His Pro Gln Gly Thr Glu Pro Asp Met Tyr Lys
20 25 30
Tyr Asp Ala Tyr Leu Cys Phe Ser Ser Lys Asp Phe Thr Trp Val Gln
35 40 45
Asn Ala Leu Leu Lys His Leu Asp Thr Gln Tyr Ser Asp Gln Asn Arg
50 55 60
Phe Asn Leu Cys Phe Glu Glu Arg Asp Phe Val Pro Gly Glu Asn Arg
65 70 75 80
Ile Ala Asn Ile Gln Asp Ala Ile Trp Asn Ser Arg Lys Ile Val Cys
85 90 95
Leu Val Ser Arg His Phe Leu Arg Asp Gly Trp Cys Leu Glu Ala Phe
100 105 110
Ser Tyr Ala Gln Gly Arg Cys Leu Ser Asp Leu Asn Ser Ala Leu Ile
115 120 125
Met Val Val Val Gly Ser Leu Ser Gln Tyr Gln Leu Met Lys His Gln
130 135 140
Ser Ile Arg Gly Phe Val Gln Lys Gln Gln Tyr Leu Arg Trp Pro Glu
145 150 155 160
Asp Phe Gln Asp Val Gly Trp Phe Leu His Lys Leu Ser Gln Gln Ile
165 170 175
Leu Lys Lys Glu Lys Glu Lys Lys Lys Asp Asn Asn Ile Pro Leu Gln
180 185 190
Thr Val Ala Thr Ile Ser
195
<210> 21
<211> 189
<212> PRT
<213> TLR6(homo sapiens )
<400> 21
Tyr Leu Asp Leu Pro Trp Tyr Leu Arg Met Val Cys Gln Trp Thr Gln
1 5 10 15
Thr Arg Arg Arg Ala Arg Asn Ile Pro Leu Glu Glu Leu Gln Arg Asn
20 25 30
Leu Gln Phe His Ala Phe Ile Ser Tyr Ser Glu His Asp Ser Ala Trp
35 40 45
Val Lys Ser Glu Leu Val Pro Tyr Leu Glu Lys Glu Asp Ile Gln Ile
50 55 60
Cys Leu His Glu Arg Asn Phe Val Pro Gly Lys Ser Ile Val Glu Asn
65 70 75 80
Ile Ile Asn Cys Ile Glu Lys Ser Tyr Lys Ser Ile Phe Val Leu Ser
85 90 95
Pro Asn Phe Val Gln Ser Glu Trp Cys His Tyr Glu Leu Tyr Phe Ala
100 105 110
His His Asn Leu Phe His Glu Gly Ser Asn Asn Leu Ile Leu Ile Leu
115 120 125
Leu Glu Pro Ile Pro Gln Asn Ser Ile Pro Asn Lys Tyr His Lys Leu
130 135 140
Lys Ala Leu Met Thr Gln Arg Thr Tyr Leu Gln Trp Pro Lys Glu Lys
145 150 155 160
Ser Lys Arg Gly Leu Phe Trp Ala Asn Ile Arg Ala Ala Phe Asn Met
165 170 175
Lys Leu Thr Leu Val Thr Glu Asn Asn Asp Val Lys Ser
180 185
<210> 22
<211> 189
<212> PRT
<213> TLR7(homo sapiens )
<400> 22
His Leu Tyr Phe Trp Asp Val Trp Tyr Ile Tyr His Phe Cys Lys Ala
1 5 10 15
Lys Ile Lys Gly Tyr Gln Arg Leu Ile Ser Pro Asp Cys Cys Tyr Asp
20 25 30
Ala Phe Ile Val Tyr Asp Thr Lys Asp Pro Ala Val Thr Glu Trp Val
35 40 45
Leu Ala Glu Leu Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe
50 55 60
Asn Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu
65 70 75 80
Glu Asn Leu Ser Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val
85 90 95
Met Thr Asp Lys Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala Phe Tyr
100 105 110
Leu Ser His Gln Arg Leu Met Asp Glu Lys Val Asp Val Ile Ile Leu
115 120 125
Ile Phe Leu Glu Lys Pro Phe Gln Lys Ser Lys Phe Leu Gln Leu Arg
130 135 140
Lys Arg Leu Cys Gly Ser Ser Val Leu Glu Trp Pro Thr Asn Pro Gln
145 150 155 160
Ala His Pro Tyr Phe Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp
165 170 175
Asn His Val Ala Tyr Ser Gln Val Phe Lys Glu Thr Val
180 185
<210> 23
<211> 193
<212> PRT
<213> TLR8(homo sapiens )
<400> 23
His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu
1 5 10 15
Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr
20 25 30
Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp
35 40 45
Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn
50 55 60
Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile
65 70 75 80
Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe
85 90 95
Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe
100 105 110
Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile
115 120 125
Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu
130 135 140
Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro
145 150 155 160
Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr
165 170 175
Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln
180 185 190
Tyr
<210> 24
<211> 193
<212> PRT
<213> TLR9(homo sapiens )
<400> 24
Gly Trp Asp Leu Trp Tyr Cys Phe His Leu Cys Leu Ala Trp Leu Pro
1 5 10 15
Trp Arg Gly Arg Gln Ser Gly Arg Asp Glu Asp Ala Leu Pro Tyr Asp
20 25 30
Ala Phe Val Val Phe Asp Lys Thr Gln Ser Ala Val Ala Asp Trp Val
35 40 45
Tyr Asn Glu Leu Arg Gly Gln Leu Glu Glu Cys Arg Gly Arg Trp Ala
50 55 60
Leu Arg Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Lys Thr Leu
65 70 75 80
Phe Glu Asn Leu Trp Ala Ser Val Tyr Gly Ser Arg Lys Thr Leu Phe
85 90 95
Val Leu Ala His Thr Asp Arg Val Ser Gly Leu Leu Arg Ala Ser Phe
100 105 110
Leu Leu Ala Gln Gln Arg Leu Leu Glu Asp Arg Lys Asp Val Val Val
115 120 125
Leu Val Ile Leu Ser Pro Asp Gly Arg Arg Ser Arg Tyr Val Arg Leu
130 135 140
Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Leu Trp Pro His Gln Pro
145 150 155 160
Ser Gly Gln Arg Ser Phe Trp Ala Gln Leu Gly Met Ala Leu Thr Arg
165 170 175
Asp Asn His His Phe Tyr Asn Arg Asn Phe Cys Gln Gly Pro Thr Ala
180 185 190
Glu
<210> 25
<211> 214
<212> PRT
<213> TLR10(homo sapiens )
<400> 25
Cys Leu His Phe Asp Leu Pro Trp Tyr Leu Arg Met Leu Gly Gln Cys
1 5 10 15
Thr Gln Thr Trp His Arg Val Arg Lys Thr Thr Gln Glu Gln Leu Lys
20 25 30
Arg Asn Val Arg Phe His Ala Phe Ile Ser Tyr Ser Glu His Asp Ser
35 40 45
Leu Trp Val Lys Asn Glu Leu Ile Pro Asn Leu Glu Lys Glu Asp Gly
50 55 60
Ser Ile Leu Ile Cys Leu Tyr Glu Ser Tyr Phe Asp Pro Gly Lys Ser
65 70 75 80
Ile Ser Glu Asn Ile Val Ser Phe Ile Glu Lys Ser Tyr Lys Ser Ile
85 90 95
Phe Val Leu Ser Pro Asn Phe Val Gln Asn Glu Trp Cys His Tyr Glu
100 105 110
Phe Tyr Phe Ala His His Asn Leu Phe His Glu Asn Ser Asp His Ile
115 120 125
Ile Leu Ile Leu Leu Glu Pro Ile Pro Phe Tyr Cys Ile Pro Thr Arg
130 135 140
Tyr His Lys Leu Lys Ala Leu Leu Glu Lys Lys Ala Tyr Leu Glu Trp
145 150 155 160
Pro Lys Asp Arg Arg Lys Cys Gly Leu Phe Trp Ala Asn Leu Arg Ala
165 170 175
Ala Ile Asn Val Asn Val Leu Ala Thr Arg Glu Met Tyr Glu Leu Gln
180 185 190
Thr Phe Thr Glu Leu Asn Glu Glu Ser Arg Gly Ser Thr Ile Ser Leu
195 200 205
Met Arg Thr Asp Cys Leu
210
<210> 26
<211> 172
<212> PRT
<213> MYD88(homo sapiens )
<400> 26
Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser
1 5 10 15
Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg
20 25 30
Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr
35 40 45
Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu
50 55 60
Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly
65 70 75 80
Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu
85 90 95
Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp
100 105 110
Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro
115 120 125
Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu
130 135 140
Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg
145 150 155 160
Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile
165 170
<210> 27
<211> 712
<212> PRT
<213> TRIF(homo sapiens )
<400> 27
Met Ala Cys Thr Gly Pro Ser Leu Pro Ser Ala Phe Asp Ile Leu Gly
1 5 10 15
Ala Ala Gly Gln Asp Lys Leu Leu Tyr Leu Lys His Lys Leu Lys Thr
20 25 30
Pro Arg Pro Gly Cys Gln Gly Gln Asp Leu Leu His Ala Met Val Leu
35 40 45
Leu Lys Leu Gly Gln Glu Thr Glu Ala Arg Ile Ser Leu Glu Ala Leu
50 55 60
Lys Ala Asp Ala Val Ala Arg Leu Val Ala Arg Gln Trp Ala Gly Val
65 70 75 80
Asp Ser Thr Glu Asp Pro Glu Glu Pro Pro Asp Val Ser Trp Ala Val
85 90 95
Ala Arg Leu Tyr His Leu Leu Ala Glu Glu Lys Leu Cys Pro Ala Ser
100 105 110
Leu Arg Asp Val Ala Tyr Gln Glu Ala Val Arg Thr Leu Ser Ser Arg
115 120 125
Asp Asp His Arg Leu Gly Glu Leu Gln Asp Glu Ala Arg Asn Arg Cys
130 135 140
Gly Trp Asp Ile Ala Gly Asp Pro Gly Ser Ile Arg Thr Leu Gln Ser
145 150 155 160
Asn Leu Gly Cys Leu Pro Pro Ser Ser Ala Leu Pro Ser Gly Thr Arg
165 170 175
Ser Leu Pro Arg Pro Ile Asp Gly Val Ser Asp Trp Ser Gln Gly Cys
180 185 190
Ser Leu Arg Ser Thr Gly Ser Pro Ala Ser Leu Ala Ser Asn Leu Glu
195 200 205
Ile Ser Gln Ser Pro Thr Met Pro Phe Leu Ser Leu His Arg Ser Pro
210 215 220
His Gly Pro Ser Lys Leu Cys Asp Asp Pro Gln Ala Ser Leu Val Pro
225 230 235 240
Glu Pro Val Pro Gly Gly Cys Gln Glu Pro Glu Glu Met Ser Trp Pro
245 250 255
Pro Ser Gly Glu Ile Ala Ser Pro Pro Glu Leu Pro Ser Ser Pro Pro
260 265 270
Pro Gly Leu Pro Glu Val Ala Pro Asp Ala Thr Ser Thr Gly Leu Pro
275 280 285
Asp Thr Pro Ala Ala Pro Glu Thr Ser Thr Asn Tyr Pro Val Glu Cys
290 295 300
Thr Glu Gly Ser Ala Gly Pro Gln Ser Leu Pro Leu Pro Ile Leu Glu
305 310 315 320
Pro Val Lys Asn Pro Cys Ser Val Lys Asp Gln Thr Pro Leu Gln Leu
325 330 335
Ser Val Glu Asp Thr Thr Ser Pro Asn Thr Lys Pro Cys Pro Pro Thr
340 345 350
Pro Thr Thr Pro Glu Thr Ser Pro Pro Pro Pro Pro Pro Pro Pro Ser
355 360 365
Ser Thr Pro Cys Ser Ala His Leu Thr Pro Ser Ser Leu Phe Pro Ser
370 375 380
Ser Leu Glu Ser Ser Ser Glu Gln Lys Phe Tyr Asn Phe Val Ile Leu
385 390 395 400
His Ala Arg Ala Asp Glu His Ile Ala Leu Arg Val Arg Glu Lys Leu
405 410 415
Glu Ala Leu Gly Val Pro Asp Gly Ala Thr Phe Cys Glu Asp Phe Gln
420 425 430
Val Pro Gly Arg Gly Glu Leu Ser Cys Leu Gln Asp Ala Ile Asp His
435 440 445
Ser Ala Phe Ile Ile Leu Leu Leu Thr Ser Asn Phe Asp Cys Arg Leu
450 455 460
Ser Leu His Gln Val Asn Gln Ala Met Met Ser Asn Leu Thr Arg Gln
465 470 475 480
Gly Ser Pro Asp Cys Val Ile Pro Phe Leu Pro Leu Glu Ser Ser Pro
485 490 495
Ala Gln Leu Ser Ser Asp Thr Ala Ser Leu Leu Ser Gly Leu Val Arg
500 505 510
Leu Asp Glu His Ser Gln Ile Phe Ala Arg Lys Val Ala Asn Thr Phe
515 520 525
Lys Pro His Arg Leu Gln Ala Arg Lys Ala Met Trp Arg Lys Glu Gln
530 535 540
Asp Thr Arg Ala Leu Arg Glu Gln Ser Gln His Leu Asp Gly Glu Arg
545 550 555 560
Met Gln Ala Ala Ala Leu Asn Ala Ala Tyr Ser Ala Tyr Leu Gln Ser
565 570 575
Tyr Leu Ser Tyr Gln Ala Gln Met Glu Gln Leu Gln Val Ala Phe Gly
580 585 590
Ser His Met Ser Phe Gly Thr Gly Ala Pro Tyr Gly Ala Arg Met Pro
595 600 605
Phe Gly Gly Gln Val Pro Leu Gly Ala Pro Pro Pro Phe Pro Thr Trp
610 615 620
Pro Gly Cys Pro Gln Pro Pro Pro Leu His Ala Trp Gln Ala Gly Thr
625 630 635 640
Pro Pro Pro Pro Ser Pro Gln Pro Ala Ala Phe Pro Gln Ser Leu Pro
645 650 655
Phe Pro Gln Ser Pro Ala Phe Pro Thr Ala Ser Pro Ala Pro Pro Gln
660 665 670
Ser Pro Gly Leu Gln Pro Leu Ile Ile His His Ala Gln Met Val Gln
675 680 685
Leu Gly Leu Asn Asn His Met Trp Asn Gln Arg Gly Ser Gln Ala Pro
690 695 700
Glu Asp Lys Thr Gln Glu Ala Glu
705 710
<210> 28
<211> 374
<212> PRT
<213> TRAM(homo sapiens )
<400> 28
Met Ala Ile Arg Lys Lys Ser Thr Lys Ser Pro Pro Val Leu Ser His
1 5 10 15
Glu Phe Val Leu Gln Asn His Ala Asp Ile Val Ser Cys Val Ala Met
20 25 30
Val Phe Leu Leu Gly Leu Met Phe Glu Ile Thr Ala Lys Ala Ser Ile
35 40 45
Ile Phe Val Thr Leu Gln Tyr Asn Val Thr Leu Pro Ala Thr Glu Glu
50 55 60
Gln Ala Thr Glu Ser Val Ser Leu Tyr Tyr Tyr Gly Ile Lys Asp Leu
65 70 75 80
Ala Thr Val Phe Phe Tyr Met Leu Val Ala Ile Ile Ile His Ala Val
85 90 95
Ile Gln Glu Tyr Met Leu Asp Lys Ile Asn Arg Arg Met His Phe Ser
100 105 110
Lys Thr Lys His Ser Lys Phe Asn Glu Ser Gly Gln Leu Ser Ala Phe
115 120 125
Tyr Leu Phe Ala Cys Val Trp Gly Thr Phe Ile Leu Ile Ser Glu Asn
130 135 140
Tyr Ile Ser Asp Pro Thr Ile Leu Trp Arg Ala Tyr Pro His Asn Leu
145 150 155 160
Met Thr Phe Gln Met Lys Phe Phe Tyr Ile Ser Gln Leu Ala Tyr Trp
165 170 175
Leu His Ala Phe Pro Glu Leu Tyr Phe Gln Lys Thr Lys Lys Glu Asp
180 185 190
Ile Pro Arg Gln Leu Val Tyr Ile Gly Leu Tyr Leu Phe His Ile Ala
195 200 205
Gly Ala Tyr Leu Leu Asn Leu Asn His Leu Gly Leu Val Leu Leu Val
210 215 220
Leu His Tyr Phe Val Glu Phe Leu Phe His Ile Ser Arg Leu Phe Tyr
225 230 235 240
Phe Ser Asn Glu Lys Tyr Gln Lys Gly Phe Ser Leu Trp Ala Val Leu
245 250 255
Phe Val Leu Gly Arg Leu Leu Thr Leu Ile Leu Ser Val Leu Thr Val
260 265 270
Gly Phe Gly Leu Ala Arg Ala Glu Asn Gln Lys Leu Asp Phe Ser Thr
275 280 285
Gly Asn Phe Asn Val Leu Ala Val Arg Ile Ala Val Leu Ala Ser Ile
290 295 300
Cys Val Thr Gln Ala Phe Met Met Trp Lys Phe Ile Asn Phe Gln Leu
305 310 315 320
Arg Arg Trp Arg Glu His Ser Ala Phe Gln Ala Pro Ala Val Lys Lys
325 330 335
Lys Pro Thr Val Thr Lys Gly Arg Ser Ser Lys Lys Gly Thr Glu Asn
340 345 350
Gly Val Asn Gly Thr Leu Thr Ser Asn Val Ala Asp Ser Pro Arg Asn
355 360 365
Lys Lys Glu Lys Ser Ser
370
<210> 29
<211> 221
<212> PRT
<213> TIRAP(homo sapiens )
<400> 29
Met Ala Ser Ser Thr Ser Leu Pro Ala Pro Gly Ser Arg Pro Lys Lys
1 5 10 15
Pro Leu Gly Lys Met Ala Asp Trp Phe Arg Gln Thr Leu Leu Lys Lys
20 25 30
Pro Lys Lys Arg Pro Asn Ser Pro Glu Ser Thr Ser Ser Asp Ala Ser
35 40 45
Gln Pro Thr Ser Gln Asp Ser Pro Leu Pro Pro Ser Leu Ser Ser Val
50 55 60
Thr Ser Pro Ser Leu Pro Pro Thr His Ala Ser Asp Ser Gly Ser Ser
65 70 75 80
Arg Trp Ser Lys Asp Tyr Asp Val Cys Val Cys His Ser Glu Glu Asp
85 90 95
Leu Val Ala Ala Gln Asp Leu Val Ser Tyr Leu Glu Gly Ser Thr Ala
100 105 110
Ser Leu Arg Cys Phe Leu Gln Leu Arg Asp Ala Thr Pro Gly Gly Ala
115 120 125
Ile Val Ser Glu Leu Cys Gln Ala Leu Ser Ser Ser His Cys Arg Val
130 135 140
Leu Leu Ile Thr Pro Gly Phe Leu Gln Asp Pro Trp Cys Lys Tyr Gln
145 150 155 160
Met Leu Gln Ala Leu Thr Glu Ala Pro Gly Ala Glu Gly Cys Thr Ile
165 170 175
Pro Leu Leu Ser Gly Leu Ser Arg Ala Ala Tyr Pro Pro Glu Leu Arg
180 185 190
Phe Met Tyr Tyr Val Asp Gly Arg Gly Pro Asp Gly Gly Phe Arg Gln
195 200 205
Val Lys Glu Ala Val Met Arg Tyr Leu Gln Thr Leu Ser
210 215 220
<210> 30
<211> 15
<212> PRT
<213> 链接(人工序列)
<400> 30
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 31
<211> 261
<212> PRT
<213> Cavin3(homo sapiens )
<400> 31
Met Ala Gly Ser Ala Leu Gly Ala Gly Pro Val Pro Gly Ala Pro Ala
1 5 10 15
Gly Gly Pro Val His Ala Val Thr Val Val Thr Leu Leu Gly Leu Leu
20 25 30
Ala Ser Met Leu Gly Thr Leu Ala Gly Ala Gly Gly Gly Leu Ala Ala
35 40 45
Ala Gly Gly Gly Leu Ala Gly Ser Val Ala Ala Ile Gly Ser Gly Leu
50 55 60
Gly Ala Leu Ser Ala Ser His Ala Thr Thr Ser Ala Thr Leu Ala Gly
65 70 75 80
Leu Leu Ala Leu Ala Gly Ala Val Ser Ser His Ala Ala Ala Ala Gly
85 90 95
Gly Ala Ala Val Ala Ala Ala Ala Gly Val Gly Ala Leu Gly Ala Ala
100 105 110
His Gly Leu Leu Val Ala Ala Gly Leu Leu His Val Leu Leu Pro Leu
115 120 125
Gly Gly Gly Gly Val Pro Ala Ser Ala Pro Gly Leu Ala Pro Gly Pro
130 135 140
Leu Gly Pro Ala Ala Gly Ser Gly Leu Gly Pro Gly Gly Leu Gly Ala
145 150 155 160
Gly Val Gly Gly Ser Ser Ala Gly Gly Pro Val Gly Ser Ala Ala Gly
165 170 175
Ala Leu Ala Ala Thr Gly Leu Gly Leu Val Gly Ser Leu Ala Ala Ala
180 185 190
Leu Ser Gly Ala Leu Gly Pro Ala Ala Pro Pro Pro Thr Pro Val Leu
195 200 205
Pro Pro Ala Leu Gly Pro Gly Ala Ser Ala Gly Ala Gly Pro Gly Ala
210 215 220
Gly Pro Ala Leu Gly Pro Thr Leu Gly Pro Gly Pro Pro Gly Ala Thr
225 230 235 240
Gly Gly Ala Pro Gly Ala Pro Gly Ala Ala Gly Gly Ala Leu Leu Gly
245 250 255
Met Gly Ser Val Ala
260
<210> 32
<211> 218
<212> PRT
<213> ZBED2(homo sapiens )
<400> 32
Met Met Ala Ala Gly Ala Gly Gly Gly Gly Gly Thr Met Met Leu Ala
1 5 10 15
Leu Gly Ala Leu Gly Met Leu Gly Gly Gly Gly Ile Ser Gly Thr Gly
20 25 30
Gly Leu Val Gly Pro Pro Val Ser Ala Met Pro Thr Pro Met Pro His
35 40 45
Ala Leu Gly Thr Ala Pro Ser Gly Ala Thr Gly Thr Pro His Leu Ala
50 55 60
Pro Ala Ala Ala Gly His His Pro Ala Gly Thr Ala Thr Cys Ala Leu
65 70 75 80
Cys Gly Ala Gly Val Ser Ala Gly Pro Gly Val Ala Val Gly Thr Thr
85 90 95
Ala Leu Thr Leu His Leu Leu Ser Met His Ala Gly Gly Leu Gly Leu
100 105 110
Ser Gly His Gly Gly Ala Gly Gly Ala Gly Ala Pro Ala Pro His Gly
115 120 125
Pro Gly Leu Pro Thr Gly Ile Gly Gly Ala Thr Gly Ala Leu Leu Gly
130 135 140
Gly Val Gly Thr Met Ala Leu Thr Ala Ser Gly Ala Gly Leu Gly Val
145 150 155 160
Leu Ala Ala Gly Ala Ala Val Gly Thr Ala Gly Ala Ala Val Gly Leu
165 170 175
Ala Gly Ala Ala Leu Gly Gly Val Gly Ala Ala Ile Leu Gly Met Leu
180 185 190
Thr Leu Val Ala Ala Gly Leu Gly Ala Cys Gly Ala Gly Leu Gly Leu
195 200 205
Pro Ala Ala Val His Pro Pro His Pro Val
210 215
<210> 33
<211> 439
<212> PRT
<213> MYC(homo sapiens )
<400> 33
Met Pro Leu Ala Val Ser Pro Thr Ala Ala Ala Thr Ala Leu Ala Thr
1 5 10 15
Ala Ser Val Gly Pro Thr Pro Thr Cys Ala Gly Gly Gly Ala Pro Thr
20 25 30
Gly Gly Gly Gly Gly Ser Gly Leu Gly Pro Pro Ala Pro Ser Gly Ala
35 40 45
Ile Thr Leu Leu Pro Gly Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser
50 55 60
Ala Ala Ser Gly Leu Cys Ser Pro Ser Thr Val Ala Val Thr Pro Pro
65 70 75 80
Ser Leu Ala Gly Ala Ala Ala Gly Gly Gly Gly Ser Pro Ser Thr Ala
85 90 95
Ala Gly Leu Gly Met Val Thr Gly Leu Leu Gly Gly Ala Met Val Ala
100 105 110
Gly Ser Pro Ile Cys Ala Pro Ala Ala Gly Thr Pro Ile Leu Ala Ile
115 120 125
Ile Ile Gly Ala Cys Met Thr Ser Gly Pro Ser Ala Ala Ala Leu Leu
130 135 140
Val Ser Gly Leu Leu Ala Ser Thr Gly Ala Ala Ala Leu Ala Ser Gly
145 150 155 160
Ser Pro Ala Pro Ala Ala Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175
Thr Leu Gly Ala Leu Ser Ala Ala Ala Ser Gly Cys Ile Ala Pro Ser
180 185 190
Val Val Pro Pro Thr Pro Leu Ala Ala Ser Ser Ser Pro Leu Ser Cys
195 200 205
Ala Ser Gly Ala Ser Ser Ala Pro Ser Pro Ser Ser Ala Ser Leu Leu
210 215 220
Ser Ser Thr Gly Ser Ser Pro Gly Gly Ser Pro Gly Pro Leu Val Leu
225 230 235 240
His Gly Gly Thr Pro Pro Thr Thr Ser Ser Ala Ser Gly Gly Gly Gly
245 250 255
Gly Ala Gly Gly Gly Ile Ala Val Val Ser Val Gly Leu Ala Gly Ala
260 265 270
Pro Gly Leu Ala Ser Gly Ser Gly Ser Pro Ser Ala Gly Gly His Ser
275 280 285
Leu Pro Pro His Ser Pro Leu Val Leu Leu Ala Cys His Val Ser Thr
290 295 300
His Gly His Ala Thr Ala Ala Pro Pro Ser Thr Ala Leu Ala Thr Pro
305 310 315 320
Ala Ala Leu Ala Val Leu Leu Ala Ser Val Ala Val Leu Ala Gly Ile
325 330 335
Ser Ala Ala Ala Leu Cys Thr Ser Pro Ala Ser Ser Ala Thr Gly Gly
340 345 350
Ala Val Leu Ala Ala Thr His Ala Val Leu Gly Ala Gly Ala Ala Ala
355 360 365
Gly Leu Leu Ala Ser Pro Pro Ala Leu Ala Ala Gly Ile Pro Gly Leu
370 375 380
Gly Ala Ala Gly Leu Ala Pro Leu Val Val Ile Leu Leu Leu Ala Thr
385 390 395 400
Ala Thr Ile Leu Ser Val Gly Ala Gly Gly Gly Leu Leu Ile Ser Gly
405 410 415
Gly Ala Leu Leu Ala Leu Ala Ala Gly Gly Leu Leu His Leu Leu Gly
420 425 430
Gly Leu Ala Ala Ser Cys Ala
435
<210> 34
<211> 640
<212> PRT
<213> MYB(homo sapiens )
<400> 34
Met Ala Arg Arg Pro Arg His Ser Ile Tyr Ser Ser Asp Glu Asp Asp
1 5 10 15
Glu Asp Phe Glu Met Cys Asp His Asp Tyr Asp Gly Leu Leu Pro Lys
20 25 30
Ser Gly Lys Arg His Leu Gly Lys Thr Arg Trp Thr Arg Glu Glu Asp
35 40 45
Glu Lys Leu Lys Lys Leu Val Glu Gln Asn Gly Thr Asp Asp Trp Lys
50 55 60
Val Ile Ala Asn Tyr Leu Pro Asn Arg Thr Asp Val Gln Cys Gln His
65 70 75 80
Arg Trp Gln Lys Val Leu Asn Pro Glu Leu Ile Lys Gly Pro Trp Thr
85 90 95
Lys Glu Glu Asp Gln Arg Val Ile Glu Leu Val Gln Lys Tyr Gly Pro
100 105 110
Lys Arg Trp Ser Val Ile Ala Lys His Leu Lys Gly Arg Ile Gly Lys
115 120 125
Gln Cys Arg Glu Arg Trp His Asn His Leu Asn Pro Glu Val Lys Lys
130 135 140
Thr Ser Trp Thr Glu Glu Glu Asp Arg Ile Ile Tyr Gln Ala His Lys
145 150 155 160
Arg Leu Gly Asn Arg Trp Ala Glu Ile Ala Lys Leu Leu Pro Gly Arg
165 170 175
Thr Asp Asn Ala Ile Lys Asn His Trp Asn Ser Thr Met Arg Arg Lys
180 185 190
Val Glu Gln Glu Gly Tyr Leu Gln Glu Ser Ser Lys Ala Ser Gln Pro
195 200 205
Ala Val Ala Thr Ser Phe Gln Lys Asn Ser His Leu Met Gly Phe Ala
210 215 220
Gln Ala Pro Pro Thr Ala Gln Leu Pro Ala Thr Gly Gln Pro Thr Val
225 230 235 240
Asn Asn Asp Tyr Ser Tyr Tyr His Ile Ser Glu Ala Gln Asn Val Ser
245 250 255
Ser His Val Pro Tyr Pro Val Ala Leu His Val Asn Ile Val Asn Val
260 265 270
Pro Gln Pro Ala Ala Ala Ala Ile Gln Arg His Tyr Asn Asp Glu Asp
275 280 285
Pro Glu Lys Glu Lys Arg Ile Lys Glu Leu Glu Leu Leu Leu Met Ser
290 295 300
Thr Glu Asn Glu Leu Lys Gly Gln Gln Val Leu Pro Thr Gln Asn His
305 310 315 320
Thr Cys Ser Tyr Pro Gly Trp His Ser Thr Thr Ile Ala Asp His Thr
325 330 335
Arg Pro His Gly Asp Ser Ala Pro Val Ser Cys Leu Gly Glu His His
340 345 350
Ser Thr Pro Ser Leu Pro Ala Asp Pro Gly Ser Leu Pro Glu Glu Ser
355 360 365
Ala Ser Pro Ala Arg Cys Met Ile Val His Gln Gly Thr Ile Leu Asp
370 375 380
Asn Val Lys Asn Leu Leu Glu Phe Ala Glu Thr Leu Gln Phe Ile Asp
385 390 395 400
Ser Phe Leu Asn Thr Ser Ser Asn His Glu Asn Ser Asp Leu Glu Met
405 410 415
Pro Ser Leu Thr Ser Thr Pro Leu Ile Gly His Lys Leu Thr Val Thr
420 425 430
Thr Pro Phe His Arg Asp Gln Thr Val Lys Thr Gln Lys Glu Asn Thr
435 440 445
Val Phe Arg Thr Pro Ala Ile Lys Arg Ser Ile Leu Glu Ser Ser Pro
450 455 460
Arg Thr Pro Thr Pro Phe Lys His Ala Leu Ala Ala Gln Glu Ile Lys
465 470 475 480
Tyr Gly Pro Leu Lys Met Leu Pro Gln Thr Pro Ser His Leu Val Glu
485 490 495
Asp Leu Gln Asp Val Ile Lys Gln Glu Ser Asp Glu Ser Gly Ile Val
500 505 510
Ala Glu Phe Gln Glu Asn Gly Pro Pro Leu Leu Lys Lys Ile Lys Gln
515 520 525
Glu Val Glu Ser Pro Thr Asp Lys Ser Gly Asn Phe Phe Cys Ser His
530 535 540
His Trp Glu Gly Asp Ser Leu Asn Thr Gln Leu Phe Thr Gln Thr Ser
545 550 555 560
Pro Val Ala Asp Ala Pro Asn Ile Leu Thr Ser Ser Val Leu Met Ala
565 570 575
Pro Ala Ser Glu Asp Glu Asp Asn Val Leu Lys Ala Phe Thr Val Pro
580 585 590
Lys Asn Arg Ser Leu Ala Ser Pro Leu Gln Pro Cys Ser Ser Thr Trp
595 600 605
Glu Pro Ala Ser Cys Gly Lys Met Glu Glu Gln Met Thr Ser Ser Ser
610 615 620
Gln Ala Arg Lys Tyr Val Asn Ala Phe Ser Ala Arg Thr Leu Val Met
625 630 635 640
<210> 35
<211> 239
<212> PRT
<213> BCL2(homo sapiens )
<400> 35
Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met
1 5 10 15
Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala
20 25 30
Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile
35 40 45
Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp
50 55 60
Pro Val Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr
85 90 95
Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe
100 105 110
Ala Glu Met Ser Ser Gln Leu His Leu Thr Pro Phe Thr Ala Arg Gly
115 120 125
Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp
130 135 140
Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu
145 150 155 160
Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp
165 170 175
Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn
180 185 190
Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro
195 200 205
Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala
210 215 220
Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Tyr Leu Gly His Lys
225 230 235
<210> 36
<211> 233
<212> PRT
<213> BCLXL(homo sapiens )
<400> 36
Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys
1 5 10 15
Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu
20 25 30
Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro
35 40 45
Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala
50 55 60
Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp Ala Arg Glu Val
65 70 75 80
Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu Ala Gly Asp Glu
85 90 95
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu
100 105 110
His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu Gln Val Val Asn
115 120 125
Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe
130 135 140
Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gln
145 150 155 160
Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp
165 170 175
His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp Asp Thr Phe Val
180 185 190
Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gln Glu
195 200 205
Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val
210 215 220
Leu Leu Gly Ser Leu Phe Ser Arg Lys
225 230
<210> 37
<211> 331
<212> PRT
<213> JUN-DD(人工序列)
<400> 37
Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala
1 5 10 15
Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro Lys
20 25 30
Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser
35 40 45
Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Leu Thr Asp Pro
50 55 60
Asp Val Gly Leu Leu Lys Leu Ala Asp Pro Glu Leu Glu Arg Leu Ile
65 70 75 80
Ile Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln
85 90 95
Phe Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu
100 105 110
Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro
115 120 125
Ser Val Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala
130 135 140
Pro Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser
145 150 155 160
Ala Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe
165 170 175
Asn Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala
180 185 190
Ala Gly Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln Gln Gln Pro Pro
195 200 205
His His Leu Pro Gln Gln Met Pro Val Gln His Pro Arg Leu Gln Ala
210 215 220
Leu Lys Glu Glu Pro Gln Thr Val Pro Glu Met Pro Gly Glu Thr Pro
225 230 235 240
Pro Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys Ala Glu
245 250 255
Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Lys Arg
260 265 270
Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys
275 280 285
Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln
290 295 300
Val Ala Gln Leu Lys Gln Lys Val Met Asn His Val Asn Ser Gly Cys
305 310 315 320
Gln Leu Met Leu Thr Gln Gln Leu Gln Thr Phe
325 330
<210> 38
<211> 439
<212> PRT
<213> MYC K51R(人工序列)
<400> 38
Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr
1 5 10 15
Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr
20 25 30
Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp
35 40 45
Ile Trp Arg Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser
50 55 60
Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe
65 70 75 80
Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala
85 90 95
Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn
100 105 110
Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile
115 120 125
Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu
130 135 140
Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly
145 150 155 160
Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175
Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser
180 185 190
Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys
195 200 205
Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu
210 215 220
Ser Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu
225 230 235 240
His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln
245 250 255
Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala
260 265 270
Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser
275 280 285
Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300
His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro
305 310 315 320
Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile
325 330 335
Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu
340 345 350
Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn
355 360 365
Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu
370 375 380
Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr
385 390 395 400
Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
405 410 415
Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu
420 425 430
Gln Leu Arg Asn Ser Cys Ala
435
<210> 39
<211> 439
<212> PRT
<213> MYC T58A(人工序列)
<400> 39
Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr
1 5 10 15
Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr
20 25 30
Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp
35 40 45
Ile Trp Lys Lys Phe Glu Leu Leu Pro Ala Pro Pro Leu Ser Pro Ser
50 55 60
Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe
65 70 75 80
Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala
85 90 95
Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn
100 105 110
Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile
115 120 125
Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu
130 135 140
Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly
145 150 155 160
Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175
Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser
180 185 190
Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys
195 200 205
Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu
210 215 220
Ser Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu
225 230 235 240
His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln
245 250 255
Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala
260 265 270
Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser
275 280 285
Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300
His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro
305 310 315 320
Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile
325 330 335
Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu
340 345 350
Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn
355 360 365
Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu
370 375 380
Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr
385 390 395 400
Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
405 410 415
Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu
420 425 430
Gln Leu Arg Asn Ser Cys Ala
435
<210> 40
<211> 331
<212> PRT
<213> JUN T231A(人工序列)
<400> 40
Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala
1 5 10 15
Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro Lys
20 25 30
Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser
35 40 45
Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro
50 55 60
Asp Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile
65 70 75 80
Ile Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln
85 90 95
Phe Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu
100 105 110
Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro
115 120 125
Ser Val Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala
130 135 140
Pro Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser
145 150 155 160
Ala Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe
165 170 175
Asn Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala
180 185 190
Ala Gly Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln Gln Gln Pro Pro
195 200 205
His His Leu Pro Gln Gln Met Pro Val Gln His Pro Arg Leu Gln Ala
210 215 220
Leu Lys Glu Glu Pro Gln Ala Val Pro Glu Met Pro Gly Glu Thr Pro
225 230 235 240
Pro Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys Ala Glu
245 250 255
Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Lys Arg
260 265 270
Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys
275 280 285
Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln
290 295 300
Val Ala Gln Leu Lys Gln Lys Val Met Asn His Val Asn Ser Gly Cys
305 310 315 320
Gln Leu Met Leu Thr Gln Gln Leu Gln Thr Phe
325 330
<210> 41
<211> 439
<212> PRT
<213> MYC S62D(人工序列)
<400> 41
Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr
1 5 10 15
Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr
20 25 30
Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp
35 40 45
Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Asp Pro Ser
50 55 60
Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe
65 70 75 80
Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala
85 90 95
Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn
100 105 110
Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile
115 120 125
Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu
130 135 140
Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly
145 150 155 160
Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175
Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser
180 185 190
Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys
195 200 205
Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu
210 215 220
Ser Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu
225 230 235 240
His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln
245 250 255
Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala
260 265 270
Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser
275 280 285
Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300
His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro
305 310 315 320
Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile
325 330 335
Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu
340 345 350
Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn
355 360 365
Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu
370 375 380
Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr
385 390 395 400
Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
405 410 415
Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu
420 425 430
Gln Leu Arg Asn Ser Cys Ala
435
<210> 42
<211> 439
<212> PRT
<213> MYC S329D(人工序列)
<400> 42
Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr
1 5 10 15
Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr
20 25 30
Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp
35 40 45
Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser
50 55 60
Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe
65 70 75 80
Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala
85 90 95
Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn
100 105 110
Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile
115 120 125
Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu
130 135 140
Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly
145 150 155 160
Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175
Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser
180 185 190
Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys
195 200 205
Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu
210 215 220
Ser Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu
225 230 235 240
His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln
245 250 255
Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala
260 265 270
Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser
275 280 285
Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300
His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro
305 310 315 320
Ala Ala Lys Arg Val Lys Leu Asp Asp Val Arg Val Leu Arg Gln Ile
325 330 335
Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu
340 345 350
Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn
355 360 365
Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu
370 375 380
Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr
385 390 395 400
Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
405 410 415
Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu
420 425 430
Gln Leu Arg Asn Ser Cys Ala
435
<210> 43
<211> 439
<212> PRT
<213> MYC S71F(人工序列)
<400> 43
Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr
1 5 10 15
Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr
20 25 30
Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp
35 40 45
Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser
50 55 60
Arg Arg Ser Gly Leu Cys Phe Pro Ser Tyr Val Ala Val Thr Pro Phe
65 70 75 80
Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala
85 90 95
Asp Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn
100 105 110
Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile
115 120 125
Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu
130 135 140
Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly
145 150 155 160
Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175
Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser
180 185 190
Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys
195 200 205
Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu
210 215 220
Ser Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu
225 230 235 240
His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln
245 250 255
Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala
260 265 270
Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser
275 280 285
Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300
His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro
305 310 315 320
Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile
325 330 335
Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu
340 345 350
Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn
355 360 365
Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu
370 375 380
Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr
385 390 395 400
Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
405 410 415
Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu
420 425 430
Gln Leu Arg Asn Ser Cys Ala
435
Claims (10)
1.嵌合抗原受体(CAR)T细胞群,其特征在于,包含编码JUN、MYC、MYB、BCL2、BCLXL或其突变型,或小窝内膜相关蛋白3(CAVIN3)或含锌指床型2(ZBED2)的外源多核苷酸。
2.根据权利要求1所述的CAR T细胞群,其特征在于,包含编码CAVIN3或ZBED2的外源多核苷酸。
3.根据权利要求1所述的CAR T细胞群,其特征在于,所述外源多核苷酸包含SEQ IDNO:31-43之一的序列。
4.根据权利要求1所述的CAR T细胞群,其特征在于,与没有过表达CAVIN3和ZBED2的CAR T细胞相比,所述CAR T细胞具有增强的抗肿瘤作用,并且所述抗肿瘤作用表现为以下至少一种:减少细胞耗竭,增强细胞扩增,增强肿瘤生长抑制。
5.根据权利要求1所述的CAR T细胞群,其特征在于,所述CAR T细胞的细胞耗竭水平低于未过表达CAVIN3和ZBED2的CAR T细胞的细胞耗竭水平,所述CAR T细胞的细胞扩增水平高于未过表达CAVIN3和ZBED2的CAR T细胞的细胞耗竭水平没有过表达CAVIN3和ZBED2的CAR T细胞,和/或CAR T细胞的肿瘤生长抑制作用大于没有过表达CAVIN3和ZBED2的CAR T细胞。
6.根据权利要求1所述的CAR T细胞群,其特征在于,结合肿瘤抗原的抗原结合域选自:TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1,CD33,EGFRvIII,GD2,GD3,BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2,Mesothelin,IL-11Ra,PSCA,PRSS21VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、叶酸受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、前列腺酶、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2,gp100,bcr-abl,酪氨酸酶,EphA2,Fucosyl GM1,sLe,GM3,TGS5,HMWMAA,o-乙酰-GD2,叶酸受体β,TEM1/CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD99a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、HPV E6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺素、存活蛋白和端粒酶、PCTA-1/半乳糖凝集素8,MelanA/MART1、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2 ETS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 hsp70-2,CD79a,CD79b,CD72,LAIR1,FCAR,LILRA2,CD300LF,CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5和IGLL1。
7.根据权利要求6所述的CAR T细胞群,其特征在于,所述胞内信号域包含共刺激信号域,或初级信号域和共刺激信号域,其中所述共刺激信号域包含功能性信号域选自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的蛋白质,NKG2C,B7-H3,一种与CD83,CDS,ICAM-1,GITR,BAFFR,HVEM(LIGHTR),SLAMF7,NKp80(KLRF1),CD160,CD19,CD4,CD8alpha,CD8beta,IL2R beta特异性结合的配体,IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、CD11c ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(触觉)、CEACAM1、CRTAM、Ly9(CD229)、CD160 BY55),PSGL1,CD100(SEMA4D),CD69,SLAMF6(NTB-A,Ly 108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELLPG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D。
8.一种包含权利要求1~7中任意一项所述CAR T细胞群的组合物。
9.根据权利要求8的组合物,其特征在于,包括程序性死亡受体1(PD1)、细胞毒性T细胞相关抗原-4(CTLA-4)、B-和T-淋巴细胞衰减因子(BTLA)、T细胞免疫球蛋白粘蛋白3(TIM-3)、活化气泡蛋白3(LAG-3)、T细胞免疫球蛋白和ITIM结构域(TIGIT)、白细胞相关免疫球蛋白样受体-1(LAIR1)、自然保护细胞受体2B4(2B4)和CD 160;和/或
所述组合物包含结合第一抗原的第一CAR的第一细胞群和结合第二抗原的第二细胞群,其中所述的第二抗原是潜在的并且与第一抗原不同的,第一抗原包括白细胞(WBC)、其他潜在的细胞表面分子;和/或
所述细胞具有降低的内源TRAC基因,和/或所述细胞编码hTERT的基因序列或编码SV40LT的基因序列,以及其组合;和/或
所述细胞包括编码结合TGF-β1、TGF-β2、CTGF、αv/β3整联蛋白、α4/β7整联蛋白或α5/β11蛋白的scFv的多核苷酸、和/或包含IL-6、IFNγ、IL-7,IL-10或IL-12;和/或
所述细胞包括编码靶向一种或多种ECM分子合成的细胞因子抑制剂或细胞因子的多核苷酸、靶向一种或多种ECM分子降解的肽和/或靶向一种或多种ECM分子的信号传导的肽。
10.根据权利要求1~9中任意一项所述的细胞群或组合物在制备治疗实体瘤药物中的用途。
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