CN101460178B - 用于tlr7和tlr8的稳定化的免疫调节性rna(simra)化合物 - Google Patents
用于tlr7和tlr8的稳定化的免疫调节性rna(simra)化合物 Download PDFInfo
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Abstract
本发明涉及稳定化的寡聚核糖核苷酸作为免疫调节剂在免疫治疗应用中的治疗用途。更具体而言,本发明提供基于RNA的寡聚核糖核苷酸,这样的寡聚核糖核苷酸拥有改善的核酸酶和核糖核酸酶稳定性并且具有经由TLR7和/或TLR8的免疫调节活性。
Description
发明背景
发明领域
本发明一般性地涉及使用寡聚核糖核苷酸作为免疫调节剂的免疫学和免疫治疗应用领域,更具体而言,本发明涉及稳定化的免疫调节RNA组合物和使用它们的方法。这些组合物和方法可以有效调节通过TOLL样受体8(TLR8)、TLR7和TLR8、以及TLR7(TLR7)的免疫应答。
相关领域概述
根据应答中涉及的细胞的亚群不同,免疫应答包括先天性和适应性应答。例如,经典的细胞介导的功能,例如迟发型超敏反应和细胞毒性T淋巴细胞(CTL)活化中涉及的T辅助(Th)细胞是Th1细胞;而充当B细胞活化的辅助细胞的Th细胞是Th2细胞。免疫应答的类型受到响应于抗原接触而产生的细胞因子和趋化因子的影响。细胞因子通过影响T辅助细胞1(Th1)和T辅助细胞2(Th2)的平衡为控制免疫应答提供一种手段,Th1和Th2的平衡直接影响发生的免疫应答类型。如果这个平衡朝向更多数量的Th1细胞,就会发生包括细胞毒性T淋巴细胞(CTL)活化的细胞介导的免疫应答。当这个平衡朝向更多数量的Th2细胞时,就会发生体液的或抗体免疫应答。每一种这样的免疫应答都会导致一组不同的细胞因子被Th1和Th2细胞分泌出来。Th1和Th2细胞分泌的细胞因子的不同可能是这两种亚群的不同生物学功能所致。
Th1细胞参与身体对抗原(例如病毒感染、细胞内病原体和肿瘤细胞)的先天性应答。对于抗原的最初应答可以是IL-12从抗原呈递细胞(例如活化的巨噬细胞和树突状细胞)中分泌出来以及与之伴随的Th1细胞活化。Th1细胞活化的结果是分泌某些细胞因子(例如IL-2,IFN-γ和其他细胞因子)和与之伴随的抗原特异性CTL的活化。已知Th2细胞响应于细菌、寄生物、抗体和变态反应原而活化,并且可能通过分泌某些细胞因子(例如IL-3,IL-4,IL-5,IL-6,IL-9,IL-10,IL-13和其他细胞因子)和趋化因子来介导身体的适应性免疫应答(例如IgE的产生和嗜酸性粒细胞的活化)。这之中某些细胞因子的分泌可能导致B细胞增殖和抗体产生。另外,这之中某些细胞因子可能会刺激或抑制其他细胞因子的释放(例如IL-10抑制Th1细胞分泌IFN-γ和树突状细胞分泌IL-12)。最终,Th1和Th2细胞的平衡以及响应于所选择的刺激的细胞因子和趋化因子的释放可以对身体的免疫系统如何应答疾病起重要作用。例如,IFN-α可能抑制丙型肝炎,而MIP-1α和MIP-1β(分别又称为CCL3和CCL4)可能抑制HIV-1感染。Th1/Th2免疫应答的最佳平衡可提供利用免疫系统治疗和预防多种疾病的机会。
在哺乳动物中,可以通过例如引入细菌或含有非甲基化CpG二核苷酸的合成DNA来诱导Th1免疫应答,这种免疫应答是特异性的寡核苷酸序列(例如非甲基化的CpG)被呈递给特定免疫细胞上的受体的结果,所述受体称作模式识别受体(PRRs)。这些PRR中的某一些是Toll-样受体(TLRs)。
Toll样受体(TLRs)与响应微生物感染的先天性免疫应答有紧密的关联。在脊椎动物中,已知一个包含十种蛋白的称为Toll-样受体(TLRs)的家族识别病原体相关性分子模式。在这十种蛋白之中,已知TLR3、7、8和9定位于细胞内的内体中,识别核酸(DNA和RNA)和小分子,如核苷和核酸代谢物。已知TLR3和TLR9分别识别存在于病毒DNA和细菌DNA和合成DNA中的核酸如dsRNA和非甲基化的CpG二核苷酸。已经表明细菌DNA活化免疫系统和抗肿瘤活性(Tokunaga T等人,J.Natl.Cancer Inst.(1984)72:955-962;Shimada S,et al.,Jpn.H cancer Res,1986,77,808-816;YamamotoS等人,Jpn.J.Cancer Res.,1986,79,866-73;Messina,J等人,J.Immunolo.(1991)147:1759-1764)。使用含有CpG二核苷酸的反义寡核苷酸的其它研究已经显示刺激了免疫应答(Zhao Q等人,Biochem.Pharmacol.1996,26,173-82)。后续的研究显示了TLR9识别存在于细菌DNA和合成DNA中的非甲基化的CpG基序(Hemmi H,等人,Nature.(2000)408:740-5)。对含有CpG的硫代磷酸酯寡核苷酸的其它修饰也可影响它们通过TLR9作为免疫应答调节剂发挥功能的能力(参见,例如Zhao等人,Biochem.Pharmacol.(1996)51:173-182;Zhao等人,Biochem Pharmacol.(1996)52:1537-1544;Zhao等人,Antisense Nucleic Acid Drug Dev.(1997)7:495-502;Zhao等人,Bioorg.Med.Chem.Lett.(1999)9:3453-3458;Zhao等人,Bioorg.Med.Chem.Lett.(2000)10:1051-1054;Yu等人,Bioorg.Med.Chem.Lett.(2000)10:2585-2588;Yu等人,Bioorg.Med.Chem.Lett.(2001)11:2263-2267;和Kandimalla等人,Bioorg.Med.Chem.(2001)9:807-813)。另外,通过结构活性关系研究已经鉴定了多种合成基序和新的基于DNA的结构,它们诱导的特异性免疫应答谱(profile)与非甲基化CpG二核苷酸产生的截然不同(Kandimalla ER,等人,Proc Natl Acad Sci USA.(2005)102:6925-30.Kandimalla ER,等人,Proc NatlAcad Sci USA.(2003)100:14303-8.Cong YP,等人,Biochem BiophysResCommun.(2003)310:1133-9.Kandimalla ER,等人,Biochem Biophys ResCommun.(2003)306:948-53.Kandimalla ER,等人,Nucleic Acids Res.(2003)31:2393-400.Yu D,等人,Bioorg Med Chem.(2003)11:459-64.Bhagat L,等人,Biochem Biophys ResCommun.(2003)300:853-61.Yu D,等人,NucleicAcids Res.(2002)30:4460-9.Yu D,等人,J Med Chem.(2002)45:4540-8.YuD,等人,Biochem Biophys Res Commun.(2002)297:83-90.Kandimalla ER,等人,Bioconjug Chem.(2002)13:966-74.Yu D,K等人,Nucleic AcidsRes.(2002)30:1613-9.Yu D,等人,Bioorg Med Chem.(2001)9:2803-8.Yu D,等人,BioorgMed Chem Lett.(2001)11:2263-7.Kandimalla ER,等人,BioorgMed Chem.(2001)9:807-13.Yu D,等人,Bioorg Med Chem Lett.(2000)10:2585-8,Putta MR,等人,Nucleic AcidsRes.(2006)34:3231-8)。然而,TLR7和TLR8的天然配体直至最近仍然未知。
已证明TLR7和8识别病毒单链RNA和合成单链RNA,以及小分子,包括多种核苷。(Diebold,S.S.,等人,Science v:303,1529-1531(2004)).Diebold等人(Science,v303:1529-1531(2004))证明IFN-α对于流感病毒的应答要求流感病毒基因组RNA的内体识别以及通过TLR7和MyD88的信号传导,他们还鉴定了ssRNA为一种TLR7配体。在人体中ssRNA被TLR8而不是TLR7识别,然而鼠TLR7能够识别ssRNA(Lund JM,等人,Proc NatlAcad SciUSA.2004Apr13;101(15):5598-603;Heil F,等人,Science.2004;303:1526-9;Diebold SS等人,Science.2004;30:1529-31;Triantafilou K等人,Eur J Immunol.2005 Aug;35(8):2416-23)。某些合成化合物:咪唑奎诺酮(imidazoquinolones)咪喹莫特(imiquimod)(R-837)和雷西莫特(resiquimod)(R-848)是TLR7和TLR8的配体(Hemmi H等人,(2002)NatImmunol3:196-200;Jurk M等人,(2002)Nat Immunol 3:499)。另外,某些鸟苷类似物,例如7-脱氮-G、7-硫杂-8-氧代G(TOG)、和7-烯丙基-8-氧代-G(洛索立宾(loxoribine)),已被证明在高浓度时可活化TLR7[Lee J等人,Proc Natl AcadSci USA.2003,100:6646-51]。然而,已知这些小分子,例如咪喹莫特,也通过其他受体发挥作用(Schon MP,等人,J.InvestDermatol.,2006,126,1338-47)。
缺乏任何已知的可供TLR7和TLR8识别的特异性ssRNA基序,而且刺激性ssRNA分子具有潜在的较广的范围,这两点暗示着TLR7和TLR8能够识别自体的以及病毒的RNA。最近已证明当某些富含GU的寡聚核糖核苷酸与N-[1-(2,3-二油酰氧基)丙基]-N,N,N三甲基铵乙基硫酸盐(DOTAP)或其他脂质剂复合时,具有免疫刺激性,并且通过TLR7和TLR8发挥作用(Heil等人,Science,303:1526-1529(2004);Lipford等人,WO03/086280;Wagner等人,WO98/32462)。然而,RNA分子已经被使用了很多年,例如作为核酶和siRNA,而且被作为核酶和siRNA使用的RNA包含GU而核苷酸。另外,许多这类RNA分子已被证明在脂质存在时通过TLR刺激引起免疫应答[Kariko等人,Immunity(2005)23:165-75;Ma Z等人,BiochemBiophysRes Commun.,(2005)330,755-9]。然而,这些RNA分子的不稳定性阻碍了在很多领域使用和应用这些分子的进展(例如预防和治疗人类疾病)。
包含核糖或脱氧核糖的寡核苷酸和寡脱氧核苷酸已被广泛使用于多种领域,包括但是不局限于诊断探测、PCR引发、基因表达的反义抑制、siRNA、适体、核酶和基于Toll-样受体(Toll-like receptors,TLRs)的免疫治疗剂。最近,许多出版物展示了寡脱氧核苷酸作为免疫调节剂的用途,以及在对多种疾病如变态反应、哮喘、自身免疫、癌症和感染性疾病的免疫治疗应用中,单独使用它们或将它们作为佐剂的用途。
很可能是由于DNA和RNA结构构象上的区别使得DNA寡核苷酸被TLR9识别而RNA寡核苷酸被TLR7和/或TLR8识别。然而,DNA和RNA的化学区别也使得DNA的化学稳定性和酶稳定性比RNA高得多。
RNA被普遍存在的细胞外核糖核酸酶(RNases)快速降解,这保证了极少的(如果有的话)自体ssRNA到达抗原呈递细胞。核酸外切酶对核酸的降解主要是3’-核酸酶消化,较小比例的降解是通过5’-核酸外切酶的作用。除了核酸外切酶消化,RNA还能够被核糖核酸酶的核酸内切酶活性降解。到目前为止,基于RNA的分子需要与脂质复合以提供核酸酶稳定性。
然而,虽然核糖核酸酶为预防自身免疫反应提供了一种关键的功能,它们也给为用于免疫疗法而设计的任何合成ssRNA分子造成了一个实质性的问题,因为它们会快速降解合成的和天然的ssRNA。为了克服这个障碍,人们尝试了将ssRNA封装入脂质体,用聚乙撑亚胺将其压缩(condense),或将其与例如N-[1-(2,3-二油酰氧基)丙基]-N,N,N三甲基铵乙基硫酸盐(DOTAP)的分子相复合以保护ssRNA分子不被降解。然而,这些保护措施只是对仍然不稳定的ssRNA所施加的第二手措施,而且这些保护措施对于ssRNA(天然的或合成的)的体内(in vivo)效力和免疫调节活性的影响尚不清楚。
因此,如何保留裸露的RNA,使它继续被识别为TLR7和/或TLR8的配体,同时改善它的稳定性使它能够成为在体内有用的分子,仍然是一个难题。从理想的角度考虑,可以通过设计本身稳定的基于RNA的分子来应对该难题,这些分子能够作为新型免疫治疗剂,这些治疗剂将有许多临床应用相关的用途,例如共同施用的时候增强疫苗的效果,或者当引起或增强免疫应答有益时,治疗和/或预防疾病,例如癌症、自身免疫病症、气道炎症、炎性病症、传染性疾病、皮肤病症、变态反应、哮喘或病原体引起的疾病。
发明简述
在第一个方面,本发明提供了一类新的稳定化的免疫调节性RNA(“SIMRA”)化合物(下文将进一步定义它们)以及它们在诱导和/或增强免疫应答方面的用途,从而满足了前述的需求。这些根据本发明的新化学实体提供了诱导和/或增强免疫应答的化合物,这些化合物显著更有效地诱导免疫应答而且显著更不易降解。根据本发明的方法能够修饰用于免疫治疗应用的SIMRA产生的细胞因子谱。
在第一个方面的一种实施方式中,本发明提供了一种作为TLR8激动剂的SIMRA化合物。
在第一个方面的另一种实施方式中,本发明提供了一种作为TLR8和TLR7激动剂的SIMRA化合物。
在第一个方面的进一步实施方式中,本发明提供了一种作为TLR7激动剂的SIMRA化合物。
在第一个方面的进一步实施方式中,本发明提供了一种作为佐剂的SIMRA化合物。
在第二个方面,本发明提供药物组合物。这些组合物包含本发明前三个方面中公开的任意一种组合物和可药用载体。
在第三个方面,本发明提供用于在脊椎动物中产生免疫应答的方法,所述方法包括对脊椎动物施用药学有效量的根据本发明的SIMRA化合物。
在第四个方面,本发明提供用于治疗患有可能受益于诱导或增强免疫应答的疾病或病症的脊椎动物的方法,所述疾病或病症例如癌症、自身免疫病症、气道炎症、炎性病症、感染性疾病、皮肤病症、变态反应、哮喘或病原体引起的疾病。这种方法包括对患有这类疾病或病症的患者施用药学有效量的根据本发明的SIMRA化合物。
在第五个方面,本发明提供用于预防脊椎动物中可能受益于诱导或增强免疫应答的疾病或病症的方法,所述疾病或病症例如癌症、自身免疫病症、气道炎症、炎性病症、感染性疾病、皮肤病症、变态反应、哮喘或病原体引起的疾病。这种方法包括对易患这类疾病或病症的脊椎动物施用药学有效量的根据本发明的SIMRA化合物。
在第六个方面,本发明提供用于分离能够产生细胞因子或趋化因子的细胞(例如免疫细胞、PBMC)的方法,在标准细胞培养条件下培养这些细胞,回体(ex vivo)用SIMRA处理这些细胞使得分离的细胞产生或分泌的细胞因子或趋化因子水平增加,并且对于需要细胞因子或趋化因子治疗的患者施用或再施用这些细胞以预防或治疗疾病。
附图简述
图1是用于本发明SIMRA化合物线性合成的合成方案。DMTr=4,4′-二甲氧三苯甲基;CE=氰乙基。
图2是用于本发明SIMRA化合物平行合成的合成方案。DMTr=4,4′-二甲氧三苯甲基;CE=氰乙基。
图3描述了适用于本发明的SIMRA化合物的线性合成的一组典型的烃基(alkyl)接头。
图4描述了适用于本发明的SIMRA化合物的平行合成的一组典型的小分子接头。
图5A显示了一种天然磷酸二酯RNA20-mer在1%人血清中于10分钟之内被完全降解。图5B显示同样序列的硫代磷酸酯骨架修饰的RNA在同样条件下相对稳定,当10分钟结束时有大约50%的完整寡聚物存在。图5C显示通过3’附接(3’attachment)的两个硫代磷酸酯骨架修饰的RNA更加稳定,10分钟之后有大约78%或更多的完整RNA存留,显示出该分子对抗核酸酶降解的增强的稳定性。图5D显示与没有5’接头或5’帽的RNA相比,附接5’端接头或加帽稍微增加了3’-3’端联结的硫代磷酸酯骨架修饰的RNA的稳定性,显示出主要的降解是从3’端发生的。图5E显示本发明的其它硫代磷酸酯骨架修饰的免疫调节化合物的稳定性。
图6描述了皮下施用本发明的SIMRA化合物2小时后,由ELISA测定的C57BL/6小鼠血清中IL-12的水平,它表明SIMRA化合物(例如SEQ IDNO11)可以在体内诱导IL-12的分泌。
图7描述了对小鼠施用了100mg/kg剂量的本发明的SIMRA化合物后,其血清中的细胞因子谱,表明SIMRA化合物(例如SEQ ID NO11)在体内施用后能诱导细胞因子的产生。
图8A和8B描述了用本发明的SIMRA化合物处理后的人PBMC的细胞因子/趋化因子谱,表明SIMRA化合物(例如Seq.ID 11)在人PBMC中能诱导细胞因子的分泌。
图9描述了在注射了肿瘤细胞的小鼠中,施用本发明的SIMRA化合物4小时后由ELISA测定的血清IL-12浓度,表明对带有肿瘤的动物施用SIMRA化合物后IL-12量的增加。
图10描述了施用本发明的SIMRA化合物后小鼠肿瘤模型中肿瘤结节的数量,表明在体内施用后,SIMRA化合物能减少肿瘤结节的数量。
图11A至11D描述了用本发明的SIMRA化合物处理过的人PBMC和pDC的细胞因子/趋化因子谱。图11A至11D表明与洛索立宾或7-脱氮-G相比,SIMRA化合物产生了更鲁棒、更清晰易辨的细胞因子/趋化因子谱。图11A至11D进一步表明对于SIMRA化合物骨架、接头、和/或加帽的修饰可导致SIMRA产生独特而清晰易辨的细胞因子/趋化因子谱。
图12描述了施用SIMRA化合物24小时后,食蟹猴血液学谱的变化,表明SIMRA化合物能够诱导针对选定的免疫细胞的作用。
图13A和13B描述了施用SIMRA化合物24小时后,用ELISA测量的食蟹猴血浆中细胞因子/趋化因子的浓度,显示出SIMRA化合物(例如SEQID NO11和30)能够在体内影响细胞因子/趋化因子谱。
图14描述了定量给药24小时后,与0小时相比,由流式细胞术测量的食蟹猴T调控细胞数、总T细胞数、单核细胞数、NK细胞数和B细胞数的变化,表明SIMRA化合物能在体内有效调节免疫应答。更具体而言,这些数据表明SIMRA化合物能够诱导产生针对选定的免疫细胞的效果。
图15描述了定量给药24小时后与0小时相比,由流式细胞术测量的食蟹猴免疫细胞上活化标志CD69的变化。图15表明SIMRA化合物能在体内有效活化不同的免疫细胞群。更具体而言,这些数据表明SIMRA化合物能够诱导产生针对选定的免疫细胞的效果。
优选实施方式详述
本发明涉及寡聚核糖核苷酸作为免疫调节剂用于免疫治疗应用的治疗用途。具体而言,本发明提供具有改善的体内稳定性并且通过单独的TLR7、TLR7和TLR8、或者单独的TLR8调节免疫应答的基于RNA的寡核苷酸(SIMRA化合物)。通过发起多种先天的和获得性的免疫应答机制,例如通过用稳定的TLR7和/或TLR8激动剂或者SIMRA化合物活化树突状细胞和其它抗原呈递细胞,因此产生的细胞因子谱可以致使病原体、被感染的细胞或者肿瘤细胞被破坏以及抗原特异性抗体和CTL应答的发生。本文通过提述并入本文引用的授权专利、专利申请和参考文献的全部内容,如同具体地和个别地写明将它们通过提述并入本文一样。本文通过提述并入本文引用的授权专利、专利申请和参考文献的全部内容,如同具体地和个别地写明将它们通过提述并入本文一样。在此处引用的任意文献的任意教导与本说明书不一致的情况下,本说明书应优先用于本发明的目的。
本发明提供增强SIMRA化合物引起的免疫应答的方法,所述SIMRA化合物用于免疫治疗应用,例如,但不限于,在成体和幼体的人类和兽医应用中的癌症、自身免疫病症、哮喘、呼吸系统变态反应、食物过敏以及细菌、寄生物和病毒感染的治疗。因此,本发明还提供对免疫治疗具有最佳水平的免疫调节效果的SIMRA化合物,以及制备和使用这些化合物的方法。此外,本发明的SIMRA化合物可以作为佐剂与可用于治疗所述疾病或症候而不降低该SIMRA化合物的预防和治疗疾病的免疫调节效果的试剂相结合。
定义
术语“2’-取代核糖核苷”或“2’-取代阿拉伯糖苷”一般地包括这样的核糖核苷或阿拉伯糖核苷,其中戊糖模块的2′位的羟基被取代产生2′-取代的或2′-O-取代的核糖核苷。在某些实施方式中,这些取代是用包含1-6个饱和或不饱和的碳原子的低级脂族烃基(hydrocarbyl)、卤素原子,或者用具有6-10个碳原子的芳基(aryl)取代,其中这种脂族烃基(hydrocarbyl)或芳基(aryl)可以是未取代的,或者可以是取代的,例如用卤素、羟基、三氟甲基、氰基、硝基、酰基、酰氧基、烷氧基、羧基、烷氧羰基或氨基取代。2′-O-取代核糖核苷或2′-O-取代-阿拉伯糖苷的实例包括但不限于2’-氨基、2’-氟代、2’-烯丙基、2’-O-烷基(2’-O-alkyl)、2’-炔丙基核糖核苷或阿拉伯糖核苷,2′-O-甲基核糖核苷或2′-O-甲基阿拉伯糖苷和2′-O-甲氧乙氧基核糖核苷或2′-O-甲氧乙氧基阿拉伯糖苷。
用来指方向时,术语“3”’一般地指多核苷酸或寡核苷酸上的某个区域或者位置,其处于在同一多核苷酸或寡核苷酸上另一个区域或者位置的3’一方(朝向糖的3’位置)。
用来指方向时,术语“5”’一般地指多核苷酸或寡核苷酸上的某个区域或者位置,其处于同一多核苷酸或寡核苷酸上另一个区域或者位置的5’一方(朝向糖的5’位置)。
术语“大约”一般地表示精确的数字并不是关键的。因此,寡聚核糖核苷酸中核糖核苷残基的数目并不是关键的,少1个或2个核糖核苷残基的寡聚核糖核苷酸,或者多1个到数个核糖核苷残基的寡聚核糖核苷酸被视作上述的每个实施方式的等同物。
术语“佐剂”一般地指这样的物质,当它被加入例如疫苗或抗原等免疫原性试剂中时,能提高或加强受体宿主在接触该混合物后对所述试剂的免疫应答。
术语“气道炎症”一般地包括,但不限于,由感染性变态反应原引起的呼气道炎症,包括哮喘。
术语“变态反应原”或“变应原”一般地指一种抗原或分子(通常为蛋白质)的抗原性部分,它在接触受试者时能引起变应性应答。在典型情况下,例如,通过水疱及潮红测试或任何本领域已知的方法显示受试者对变应原具有变应性。即使只有一小部分受试者在接触分子后表现变应性(例如IgE)免疫应答,这种分子仍被认为是变态反应原。
术语“变态反应”一般地包括,但不限于,食物变态反应(食物过敏)、呼吸系统变态反应和皮肤变态反应(皮肤过敏)。
术语“抗原”一般地指被抗体或T细胞抗原受体识别并选择性结合的物质。抗原可能包括但不局限于肽、蛋白质、核苷、核苷酸以及它们的组合。抗原可能是天然的或合成的,并且一般情况下诱导针对这种抗原的特异性的免疫应答。
术语“自身免疫病症”一般指“自身”抗原遭受免疫系统攻击的病症。
封闭3’或5’降解、或“帽”、或者“加帽”表示寡聚核糖核苷酸的3’或5’端附接于另一个分子(例如接头或其它非RNA核苷酸)以充分抑制核酸酶降解(例如3’核酸外切酶降解)。
术语“载体”通常情况下包括任意赋形剂、稀释剂、填充剂、盐、缓冲剂、稳定剂、增溶剂、油、脂类、包含脂类的囊泡、微球、脂质体包囊或本领域公知的用于药物制剂的其它材料。应了解载体、赋形剂或稀释剂的性质将取决于针对具体应用的施用途径。包含这些材料的可药用的制剂的制备描述于,例如,Remington’s Pharmaceutical Sciences,18thEdition,ed.A.Gennaro,Mack Publishing Co.,Easton,PA,1990。
术语“共同施用”一般指在时间上充分接近地施用至少两种不同的物质以调节免疫应答。优选地,共同施用指同时使用至少两种不同的物质。
术语“互补的”一般表示具有与核酸杂交的能力。这种杂交通常是互补链之间氢键键合(优选的是形成Watson-Crick或Hoogsteen碱基配对)的结果,虽然其它形式的氢键键合以及碱基堆积也可导致杂交。
术语“有效量”或者“足够量”一般指足以产生所希望的生物学效果,例如有益结果的量。因此,“有效量”或者“足够量”将取决于其被施用的情境。一个有效量可以分一次或多次施用。
术语“免疫调节性寡聚核糖核苷酸”一般指当对例如鱼类、禽类或哺乳动物等脊椎动物施用时能诱导或抑制免疫应答的寡聚核糖核苷酸。
术语“与...结合”或“与...组合”一般情况下表示在治疗同一患者的同一疾病的过程中,包括以任意顺序施用SIMRA化合物和有用于治疗疾病或症候而不降低所述SIMRA化合物的免疫调节作用的试剂,包括同时施用和以几秒到几天的时间间隔施用。这种组合治疗也可以包括施用SIMRA化合物多于一次,和/或独立地施用所述试剂多于一次。可能通过相同或不同的途径施用所述SIMRA化合物和所述试剂。
术语“个体”或“受试者”一般指例如人类的哺乳动物。哺乳动物一般包括但不限于人类、非人灵长类、大鼠、小鼠、猫、狗、马、牛(cattle)、奶牛(cows)、猪、羊和兔子。
术语“线性合成”是指从免疫调节性寡聚核糖核苷酸的一端开始,线性推进到另一端的合成。线性合成允许在免疫调节性寡聚核糖核苷酸中掺入相同或不同(就掺入的长度、碱基组成和/或化学修饰而言)的单体单元。
术语“修饰的核苷”一般情况下是指包括修饰的杂环碱基、修饰的糖模块或它们的组合的核苷。在一些实施方式中,修饰的核苷是如本文所述的非天然嘧啶或嘌呤核苷。就本发明而言,修饰的核苷、嘧啶或嘌呤类似物或非天然嘧啶或嘌呤可互换使用,并且指包含非天然碱基和/或非天然糖模块的核苷。就本发明而言,如果一个碱基不是鸟嘌呤、胞嘧啶、腺嘌呤或尿嘧啶,那么此碱基被认为是非天然的。在一些实施方式中,修饰的核苷是2′-取代核糖核苷、阿拉伯糖核苷或2′-脱氧-2′-取代-阿拉伯糖苷,可以将它们取代到寡聚核糖核苷酸的所选位置上以增强稳定性而不干扰TLR7或TLR8的活性。
术语“调节”(modulation)或“刺激”(stimulation)一般指例如应答增加或者应答性质上差别的变化,这种变化可以因应答的引起或增加而出现。
术语“接头”一般指能够通过糖、碱基或骨架共价或非共价键合而附接(attach)于寡聚核糖核苷酸的任意模块。接头可以用于附接两个或多个核苷或者可附接于寡聚核糖核苷酸的5’和/或3’末端核苷酸。接头既可以是非核苷酸接头也可以是核苷酸接头。
术语“非核苷酸接头”一般指除核酸联结以外的化学模块,它可以通过共价或非共价键合附接于寡聚核糖核苷酸。优选的是,这种接头的长度从大约2埃到大约200埃,而且可以是顺向或反向的。
术语“核苷酸联结”(nuleotide linkage)一般指相邻核苷之间通过这两个核苷的糖(例如3’-3’,2’-3’,2’-5’,3’-5’)而连接它们的化学联结,由磷酸酯、非磷酸酯、带电的或中性基团组成(例如磷酸二酯、硫代磷酸酯或二硫代磷酸酯)。
术语“回文顺序”一般表示自身互补或反向重复(也就是一段例如ABCDEE’D’C’B’A’的序列,其中A与A’、B与B’等等是能够形成通常的Watson-Crick碱基对的碱基)。在体内,这种序列可能形成分子内或分子间的双链结构。
术语“肽”一般指具有足以影响生物应答(例如抗体产生或细胞因子活性)的长度和组成的多肽,无论该肽是否为半抗原。术语“肽”可能包括修饰的氨基酸(是/否天然或非天然的发生的),这样的修饰包括但不限于磷酸化、糖基化、聚乙二醇化(pegylation)、脂质化和甲基化。
术语“PBMC”一般情况下指外周血单个核细胞。
术语“生理上可接受的”一般指不干扰SIMRA化合物的有效性,并且与生物系统例如细胞、细胞培养物、组织或生物体相容的材料。优选的,生物系统是活的生物,例如脊椎动物。
术语“SIMRA”一般指被TLR7和/或TLR8识别为配体的稳定化的免疫调节性RNA化合物,其中化合物可以包含单链RNA(ssRNA)和/或双链RNA(dsRNA),以及用以保护(稳定)其3’端的修饰(例如通过封闭3’降解或为3’端加帽或者通过连接两个或更多寡聚核糖核苷酸的3’端),条件是SIMRA在体内比未修饰的寡聚核糖核苷酸更加稳定且因而影响它的免疫调节能力。SIMRA可以包含修饰的寡聚核糖核苷酸。SIMRA化合物还可包括用于保护5’端以进一步提高寡聚核糖核苷酸稳定性的修饰(例如通过5’降解或5’端加帽)。SIMRA可以是线性或分支的,其中核酸是通过例如磷酸二酯、硫代磷酸酯或其他联结相连接的核糖核苷聚合体。SIMRA可以有一个嘌呤(腺嘌呤(A)或鸟嘌呤(G)或它们的衍生物(例如7-脱氮-G和ara-G))或嘧啶(胞嘧啶(C)或尿嘧啶(U),或它们的衍生物)碱基以及与其共价联结的一个核糖残基组成,或它们的衍生物组成。
术语“治疗”一般情况下指一种与其获得有益或期望效果的方法,这些效果可能包括缓和症状,延迟或改善疾病进展。
术语“病毒性疾病”一般情况下指有病毒作为病因的疾病,包括但不限于乙型肝炎、丙型肝炎、流行性感冒、获得性免疫缺陷综合征(AIDS)和带状疱疹。
在第一个方面,本发明提供SIMRA化合物。当前的发明者发现修饰免疫调节性寡聚核糖核苷酸以保护它的3’端(例如通过封闭3’降解、3’端加帽或者联接两个或更多寡聚核糖核苷酸的3’端)令人惊讶地影响其免疫调节能力。另外,已确定这种保护令人惊讶地提高了寡聚核糖核苷酸的稳定性,从而不再需要结合脂质或其他保护手段。进一步地,封闭5’降解或5’端加帽能够进一步改善寡聚核糖核苷酸的稳定性。
本发明显示了用新型SIMRA化合物活化TLR8并诱导免疫应答(例如细胞因子谱的变化)。此外,在这种人类TLR8活化性RNA(human TLR8activating RNA)中掺入某些化学修饰还能够活化TLR7,从而引起免疫应答和细胞因子/趋化因子谱的变化。因此,本发明者令人惊讶地发现,通过使用作为免疫调节性寡聚核糖核苷酸的一部分的经修饰的化学结构(包括修饰的碱基、修饰的糖、骨架、接头、联结和/或加帽),能够通过活化TLR8和/或TLR7来调节与之相关的细胞因子/趋化因子谱。
在一种实施方式中,本发明提供一种免疫调节性化合物,它包含至少两个基于RNA的寡核苷酸,这些寡核苷酸在它们的3’端处、或核苷间联接处、或者官能化的核碱基处或糖处与非核苷酸接头连接。所述发明实施方式可具有至少一个易接近的5’端。已确定此结构为SIMRA化合物提供了进一步的稳定性(例如抑制核酸外切酶活性)而无需结合脂质或其他保护。SIMRA的5’末端修饰方式不会阻止SIMRA化合物通过TLR7和/或TLR8调节免疫应答。
本发明的这个方面的另一个实施方式包含至少两个寡聚核糖核苷酸,其中免疫调节化合物的结构包括但不限于表1中化学式I-X所详述的结构。
表1:寡聚核糖核苷酸化学式I-X
域A、B、C和D的长度可以独立地为大约2个到大约35个核糖核苷酸,在一些实施方式中为从大约2到20个、或从大约2到12个、或从大约2到11个、或从大约2到8个核糖核苷酸。域A、B、C和D可以独立地为5’-3’或2’-5’RNA,它们可以有或无自身互补域、同聚(homo)或异聚(hetero)的核糖核苷酸序列或者接头。“n”可以是从1到无限制的数字。
“X”是给域A、B、C、和/或D加帽的接头,其可通过3’或5’联结、磷酸基团、非RNA核苷酸或者非核苷酸接头连接。其中非核苷酸接头可以是脂族的、芳香的、芳基的、环状的、手性的、非手性的、肽、碳水化合物、脂质、脂肪酸、单-、三-或六聚乙二醇或者杂环模块,或它们的组合。
在进一步的实施方式中,本发明提供包含至少两个由非核苷酸接头连接的寡聚核糖核苷酸的SIMRA化合物,其中免疫调节性寡聚核糖核苷酸的序列可以至少部分地自身互补。正如同本领域技术人员会认可的,寡聚核糖核苷酸的互补序列允许分子间氢键键合,从而给予寡聚核糖核苷酸二级结构。另外的寡聚核糖核苷酸能够结合在一起从而产生由根据本发明的寡聚核糖核苷酸组成的链或多聚体。
类似的考虑适用于有不同碱基序列的免疫调节性寡聚核糖核苷酸之间的分子间碱基配对。因此,当众多免疫调节性寡聚核糖核苷酸一起使用时,这些众多的免疫调节寡聚核糖核苷酸可以,但不需要,包括至少可以部分互补的序列。在一个实施方式中,所述众多免疫调节性寡聚核糖核苷酸中包括具有第一序列的免疫调节性寡聚核糖核苷酸和具有第二序列的免疫调节性寡聚核糖核苷酸,其中第一和第二个序列至少百分之五十互补。例如至少百分之五十互补的8-mer之间可以形成4,5,6,7或8个G-C,A-U和/或G-U摆动碱基对。这种碱基对可以但不必需包括位于互补的免疫调节寡聚核糖核苷酸任一末端的碱基。互补的程度可以依赖于免疫调节寡聚核糖核苷酸之间的比对,此比对可以包括或不包括单个或多个核苷突出(overhang)。在其他实施方式中,互补度为至少60%、至少70%、至少80%、至少90%或甚至100%。
正如同本领域技术人员会认可的,所描绘的免疫调节化合物可以因为域的互补性允许分子间氢键键合而具有二级结构。此外,可以从化学式I至X想到,更多联结的基于RNA的寡核苷酸(linked RNA-based oligonucleotides)能够通过分子间的氢键而结合,从而产生链或多聚体,其中可掺入任意数目的联结的基于RNA的寡核苷酸。
在另一个实施方式中,本发明提供一种免疫调节化合物,它包含至少两个基于RNA的寡核苷酸,所述寡核苷酸在它们的3’或5’端处连接,或通过核苷间联结、或者官能化的核碱基、或糖而连接于非核苷酸接头,且其中接头(例如帽)附接于至少一个5’端。已确定此结构为SIMRA化合物提供了进一步的稳定性(例如抑制核酸外切酶活性)。SIMRA的5’末端不会以这样的方式被修饰,以至于阻止SIMRA化合物通过TLR7和/或TLR8调节免疫应答。
在一些实施方式中,寡聚核糖核苷酸各自独立地具有大约2个到大约35个核糖核苷酸残基。因此在某些实施方式中寡聚核糖核苷酸可独自地长为2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34或35个核糖核苷酸。优选地,寡聚核糖核苷酸为从大约4个到大约30个核糖核苷酸残基,更优选地从大约4个到大约20个核糖核苷酸残基,或者从大约4个到大约11个核糖核苷酸残基。在一些实施方式中,免疫调节性寡聚核糖核苷酸包含具有从大约1个到大约18个,或从大约1个到大约11个,或从大约5个到大约14个核糖核苷酸残基的寡聚核糖核苷酸。在一些实施方式中,所述寡聚核糖核苷酸中的一个或多个具有11个核苷酸。在免疫调节性寡聚核糖核苷酸的语境中,优选的实施方式具有从大约1个到大约35个核糖核苷酸,优选地从大约5个到大约26个核苷酸,更优选地从大约13个到大约26个核糖核苷酸。优选地,免疫调节性寡聚核糖核苷酸包含至少一个磷酸二酯、硫代磷酸酯或二硫代磷酸酯核糖核苷酸间联结。
在优选的实施方式中,每个核糖核苷单位包括杂环碱基和呋喃戊糖基(pentofuranosyl)、海藻糖、阿拉伯糖、2′-脱氧-2′-取代阿拉伯糖、2′-O-取代阿拉伯糖或己糖糖基团。核糖核苷残基可以通过多种已知的核糖核苷间联结中的任一种彼此偶联。这些核糖核苷间联结包括但不限于,磷酸二酯、硫代磷酸酯、二硫代磷酸酯、烷基膦酸酯(alkylphosphonate)、烷基硫代膦酸酯(alkylphosphonothioate)、磷酸三酯、氨基磷酸酯、硅氧烷、碳酸酯、烷氧羰基(carboalkoxy)、乙酰胺化物(acetamidate)、氨基甲酸酯、吗啉代、borano、硫醚、桥联氨基磷酸酯、桥联亚甲基膦酸酯、桥联硫代磷酸酯、和砜核苷间联结。核糖核苷酸可能的缀合位点在下面的化学式XI中显示,其中的B表示杂环碱基。
化学式XI
本发明的SIMRA化合物可包括自然产生的核糖核苷、修饰的核糖核苷或它们的混合物。
在本发明中,新型的SIMRA化合物被人类TLR8识别,并且在这种人类TLR8活化RNA中掺入某些化学修饰能够使它们被TLR7识别并引起免疫应答。所述化学修饰包括但不限于鸟嘌呤类似物,例如7-脱氮-G,ara-G,6-硫代-G,肌苷,异-G(Iso-G),洛索立宾(loxoribine),TOG(7-硫代-8-氧代)-G,8-溴代-G,8-羟基-G,5-氨基间型霉素(aminoformycin)B,氧代间型霉素(Oxoformycin),7-甲基-G,9-对-氯苯基-8-氮杂-G,9-苯基-G,9-己基鸟嘌呤,7-脱氮-9-苯甲基-G,6-氯-7-脱氮鸟嘌呤,6-甲氧基-7-脱氮鸟嘌呤,8-氮杂-7-脱氮-G(PPG),2-(二甲氨基)鸟苷,7-甲基-6-硫代鸟苷,8-苄氧基鸟苷,9-脱氮鸟苷和1-(B-D-呋喃核糖基)-2-氧代-7-脱氮-8-甲基嘌呤。化学修饰还包括但不限于腺嘌呤类似物,例如9-苯甲基-8-羟基-2-(2-甲氧基乙氧基)腺嘌呤,2-氨基-N2-O-,甲基腺苷,8-氮杂-7-脱氮-A,7-脱氮-A,阿糖腺苷(Vidarabine),2-氨基腺苷,N1-甲基腺苷,8-氮杂腺苷和5-碘代杀结核菌素(Iodotubercidin)。化学修饰还包括但不限于胞嘧啶类似物。化学修饰还包括但不限于例如4-硫代-U的尿嘧啶类似物。
根据本发明的“免疫调节性寡聚核糖核苷酸”为SIMRA化合物,它包含至少两个寡聚核糖核苷酸,所述寡聚核糖核苷酸通过非核苷酸或核苷酸接头在它们的3’端或2’端处,或者官能化的核糖处,或者官能化的核碱基处相连接。下文列出了几个接头的实例。非共价联结包括,但不限于,静电相互作用、疏水性相互作用、π堆积相互作用和氢键键合。
在其他的实施方式中,所述非核苷酸接头是具有允许附接于所述寡聚核糖核苷酸的官能团的有机模块。所述附接优选地是通过稳定的共价联结。作为非限制性的实例,接头可以附接于核苷酸上的任意合适位置。在一些优选的实施方式中,接头附接于3′-羟基。在这类实施方式中,接头优选地包含羟基基团,该羟基基团优选地通过基于例如磷酸二酯、硫代磷酸酯、二硫代磷酸酯、甲基膦酸酯等基于磷酸酯的联结或非基于磷酸酯的联结(non-phosphate-based linkage)而附接于3′-羟基。
在一些实施方式中,非核苷酸接头是小分子、大分子或生物分子,包括,但不限于,多肽、抗体、脂类、抗原、变态反应原和低聚糖。在一些其它实施方式中,非核苷酸接头是小分子。就本发明而言,小分子是分子量小于1,000Da的有机模块。在一些实施方式中,小分子的分子量小于750Da。
在一些实施方式中,小分子是脂肪烃或芳烃,所述脂肪烃或芳烃可任选地一种或多种官能团,所述官能团包括但不限于羟基、氨基、硫醇、硫醚、醚、酰胺、硫代酰胺、酯、脲和硫脲,所述官能团或者位于连接寡聚核糖核苷酸的直链中,或者附接于其上。小分子可以是环状或非环状的。小分子接头的实例包括,但不限于,氨基酸、碳水化合物、环糊精、金刚烷、胆固醇、半抗原和抗生素。然而,为描述非核苷酸接头的目的,术语“小分子”意在不包括核苷。
在一些实施方式中,非核苷酸接头是烃基(alkyl)接头或氨基接头。烃基(alkyl)接头可以是分支化的或无分支的,环状的或非环状的,取代的或未取代的,饱和的或不饱和的,手性的,非手性的或者外消旋混合物。烃基(alkyl)接头可具有从大约2个到大约18个碳原子。在一些实施方式中,所述烃基(alkyl)接头具有从大约3个到大约9个碳原子。一些烃基(alkyl)接头包括一个或多个官能团,所述官能团包括但不局限于羟基、氨基、硫醇、硫醚、醚、酰胺、硫代酰胺、酯、脲和硫醚。所述烃基(alkyl)接头包括但不局限于1丙醇、1,2丙二醇、1,3丙二醇、1,2,3丙三醇、三乙二醇、六乙二醇、聚乙二醇接头(例如[-O-CH2-CH2-]n(n=1-9))、甲基接头、乙基接头、丙基接头、丁基接头或己基接头。在一些实施方式中,所述烃基(alkyl)接头可包括肽或氨基酸。
在一些实施方式中,非核苷酸接头可包括但不局限于表2所列的接头。
表2:典型性非核苷酸接头
表2:续
表2:续
表2:续
表2:续
在一些实施方式中,小分子接头是如式HO-(CH2)o-CH(OH)-(CH2)p-OH所示的甘油或者甘油同系物,其中o和p独立地是从1到大约6,从1到大约4或者从1到大约3的整数。在一些其它实施方式中,小分子接头是1,3-二氨基-2-羟基丙烷的衍生物。这类衍生物中的一些大约6,2到大约6或者2到大约4之间的整数。
本发明的一些非核苷酸接头容许超过两个如表1所描述的寡聚核糖核苷酸的附接。例如,小分子接头甘油具有3个可供寡聚核糖核苷酸共价附接的羟基。因此,本发明的一些免疫调节性寡聚核糖核苷酸包含超过两个寡聚核糖核苷酸(例如一个域C等等,另外的域包括上文为域A、B、C和D定义的寡聚核糖核苷酸),它们在其3′端与非核苷酸接头连接。
在本发明本方面进一步的实施方式中,SIMRA可包含三个或更多个寡聚核糖核苷酸,所述寡聚核糖核苷酸在它们的3’或5’端处、或通过核苷间联结、或者通过官能化的核碱基或糖与两个或更多表1所示的接头连接。本发明的此方面的寡聚核糖核苷酸可具有相同或不同的序列。本发明的此方面的接头可以相同或不同。
利用自动化合成仪和亚磷酰胺方法(如图1和2的示意图所示,在实施例中进一步描述),可以方便地合成本发明的免疫调节性寡聚核糖核苷酸。在一些实施方式中,通过线性合成方法合成免疫调节性寡聚核糖核苷酸(参见图1)。
另一种供选择的合成方式是“平行合成”,其中合成从中央接头模块向外进行(参见图2)。附接在固相支持物上的接头可以用于平行合成,如美国专利5,912,332所述。此外,可以使用通用的固相支持物(例如附接磷酸酯的可控孔径玻璃)。
免疫调节性寡聚核糖核苷酸的平行合成相对于线性合成具有几个优点:(1)平行合成容许掺入相同的单体单元;(2)与线性合成不同,所有的单体单元都是同时合成,因此合成步骤的数目和合成需要的时间与合成一个单体单元的相同;和(3)合成步骤的减少使最终的免疫调节性寡核苷酸产物的纯度和产量增加。
在依照线性合成或者平行合成规程进行的合成结束时,如果掺入了修饰核苷的话,可以方便地利用浓的氨溶液或根据亚磷酰胺供应商的推荐对免疫调节性寡聚核糖核苷酸进行脱保护。对产物免疫调节性寡聚核糖核苷酸可优选地利用反相HPLC纯化,脱三苯甲基,脱盐和透析。
表3a显示根据本发明的基于RNA的免疫调节性寡聚核糖核苷酸,它们是非限制性的和典型的。
表3a:稳定化基于RNA的免疫调节性寡聚核糖核苷酸序列实例
SIMRA#(SEQ ID NO.) | 序列和修饰 |
1 | 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ |
2 | 5’-CUGUGCUUCUC-X-CUCUUCGUGUC-5’ |
3 | 5’-UCUGUGCUUCU-X-UCUUCGUGUCU-5’ |
4 | 5’-UICAAICUUIC-X-CIUUCIAACIU-5’ |
5 | 5′-GUGUGUGUGUG-X-GUGUGUGUGUG-5′ |
6 | 5’-UGCUGCUU-X-UUCGUCGU-5’ |
7 | 5′-UGCUGCUUCUGUGU-X-UGUGUCUUCGUCGU-5′ |
8 | 5′-UGCUGCUUCUGUGUCUG-X-GUCUGUGUCUUCGUCGU-5′ |
9 | 5′-U1GCU1GCU1U1CU1G-X-GU1CU1U1CGU1CGU1-5′ |
10 | 5′-T1GCT1GCT1T1CT1G-X-GT1CT1T1CGT1CGT1-5′ |
11 | 5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’ |
12 | 5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′ |
13 | 5′-GUCCUUCAACU-X-UCAACUUCCUG-5′ |
14 | 5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’ |
15 | 5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′ |
16 | 5′-OUCCUUCAACU-X-UCAACUUCCUO-5′ |
17 | 5’-UOCUOCUUCUO-X-OUCUUCOUCOU-5’ |
18 | 5’-UICUICUUCUI-X-IUCUUCIUCIU-5’ |
19 | 5′-IUCCUUCAACU-X-UCAACUUCCUI-5′ |
20 | 5’-X2 UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’ |
21 | 5’-X2UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’ |
22 | 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ |
23 | 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ |
24 | 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ |
25 | 5’-UGCUGCUACUG-X-GUCAUCGUCGU-5’ |
26 | 5’-UGCUGCUUGUG-X-GUGUUCGUCGU-5’ |
27 | 5’-UGCUGCUGCUG-X-GUCGUCGUCGU-5’ |
28 | 5’-UGCUGCUUAUG-X-GUAUUCGUCGU-5’ |
29 | 5’-UG1CUG1CUUG1UG1-X-G1UG1UUCG1UCG1U-5’ |
30 | 5’-X2UGCUGCUUGUG-X-GUGUUCGUCGUX2-5 |
31 | 5’-UGCUGCUUCUG-X1-GUCUUCGUCGU-5’ |
32 | 5’-X7UGCUGCUUGUG-X-GUGUUCGUCGUX7-5’ |
33 | 5′-UGUUGUGUGAC-X-CAGUGUGUUGU-5′ |
34 | 5′-CUGGCGGCCUU-X-UUCCGGCGGUC-5′ |
35 | 5’-X3UG1CUG1CUUGUG1-X-G1UGUUCG1UCG1UX3-5’ |
36 | 5’-UGCUGCUUG2UG-X-GUG2UUCGUCGU-5’ |
37 | 5’-G2GCUGCUUGUG-X-GUGUUCGUCGG2-5’ |
38 | 5′-UGCUGCCUUUG-X-GUUUCCGUCGU-5′ |
39 | 5′-GUCCUUGCUUG-X-GUUCGUUCCUG-5′ |
40 | 5′-GUCCUUUGCUG-X-GUCGUUUCCUG-5′ |
41 | 5’-X3UGCUGCUGCUG-X-GUCGUCGUCGUX3-5’ |
42 | 5’-XUGCUGCUUGUG-X-GUGUUCGUCGUX-5’ |
43 | 5’-X7UGCUGCUGCUG-X-GUCGUCGUCGUX7-5’ |
44 | 5′-UUGCCCUUGCC-X-CCGUUCCCGUU-5′ |
45 | 5′-UUGCUGUUGCU-X-UCGUUGUCGUU-5′ |
46 | 5′-CUUUGGUGUGU-X-UGUGUGGUUUC-5′ |
47 | 5′-UUGGUUGUUUG-X-GUUUGUUGGUU-5′ |
48 | 5′-CUUUGGUGUGU-X-UGUGUGGUUUC-5′ |
49 | 5′-X3UUGGUUGUUUG-X-GUUUGUUGGUUX3-5′ |
50 | 5′-X3GUCCUUGCUUG-X-GUUCGUUCCUGX3-5′ |
51 | 5’-PUGCUGCUUGUG-X-GUGUUCGUCGUP-5’ |
52 | 5’-X4UGCUGCUUGUG-X-GUGUUCGUCGUX4-5’ |
53 | 5’-X5UGCUGCUUGUG-X-GUGUUCGUCGUX5-5’ |
54 | 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ |
55 | 5’-UG1CUG1CUUCUG1-X1-G1UCUUCG1UCG1U-5’ |
56 | 5’-X3UGCUGCUUGUG-X1-GUGUUCGUCGUX3-5’ |
57 | 5′-UG3CUGCUUCUG-X-GUCUUCGUCG3U-5′ |
58 | 5′-UGG4UGCUUCUG-X-GUCUUCGUG4GU-5′ |
59 | 5’-UUG1G1UUG1UUUG1-X-G1UUUG1UUG1G1UU-5’ |
60 | 5’-UG1CUG1CCUUU G1-X-G1UUUCCG1UCG1U-5’ |
61 | 5′-G1UCCUUG1CUU G1-X-G1UUCG1UUCCU G1-5′ |
62 | 5′-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5′ |
63 | 5’-UG1CUG1CUUCU G1-X8-G1UCUUCG1UCG1U-5’ |
64 | 5′-CUG1-X-G1UC-5’ |
65 | 5′-UUG1CUG1UUG1CU-X-UCG1UUG1UCG1UU-5′ |
66 | 5′-UG1CCUUG1AACU-X-UCAAG1UUCCG1U-5′ |
67 | 5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′ |
68 | 5′-UUCUG1CUUCUG1-X5-G1UCUUCG1UCUU-5′ |
69 | 5′-G1UCCUUCUCUG1-X-G1UCUCUUCCUG1-5′ |
70 | 5′-UG1UURUG1UG1AC-X-CAG1URUG1UUG1U-5′ |
71 | 5′-X2UUGGUUGUUUG-X-GUUUGUUGGUUX2-5′ |
72 | 5′-X2GUCCUUGCUUG-X-GUUCGUUCCUGX2-5′ |
73 | 5′-X6UUGGUUGUUUG-X-GUUUGUUGGUUX6-5′ |
74 | 5′-X6GUCCUUGCUUG-X-GUUCGUUCCUGX6-5′ |
75 | 5’-X2UGCUGCUUGUG-X8-GUGUUCGUCGUX2-5’ |
76 | 5′-UGCUGCUUCUGGACAUGUCCAG-3′ |
77 | 5′-UGCUGCUUCUGUGAUAUCACAG-3′ |
78 | 5′-UGCUGCUUCUGAAUUAAUUCAG-3′ |
79 | 5′-UGCUGCUUCUGGACUAGUCCAG-3′ |
80 | 5’-UGCUGCUUcugugauaucacag-3′ |
81 | 5′-AGUUGAAGGACUGCUGCUUCUG-3′ |
82 | 5′-GUCCUUCAACUCAGAAGCAGCA-3′ |
83 | 5′-AGUUGAAGGACX2UGCUGCUUCUG-3′ |
84 | 5′-GUCCUUCAACUX2CAGAAGCAGCA-3′ |
85 | 5’-CCCIIICCCX2CCCIIICCC-3′ |
86 | 5’-AGAAGCUUCUG-X-GUCUUCGAAGA-5’ |
87 | 5’-UGAAGCUUCUG-X-GUCUUCGAAGU-5’ |
88 | 5′-X6UUGGUUGUUUG-X-GUUUGUUGGUUX6-5′ |
89 | 5′-X6GUCCUUGCUUG-X-GUUCGUUCCUGX6-5′ |
90 | 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ |
91 | 5′-GUUUGCACAAC-X-CAACACGUUUG-5′ |
92 | 5′-GCACACUUGUU-X-UUGUUCACACG-5′ |
93 | 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ |
94 | 5′-CACUGUUGACA-X-ACAGUUGUCAC-5′ |
95 | 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ |
96 | 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ |
97 | 5′-AGCACAACUGU-X-UGUCAACACGA-5′ |
98 | 5’-UGCUGAGUGUU-X-UUGUGAGUCGU-5’ |
99 | 5’-AGUGUUUUCUG-X-GUCUUUUGUGA-5’ |
100 | 5’-UGCUGCUUCUGX2UGCUGCUUCUG-3’ |
101 | 5’-UGCUGCUUCUGX2UGCUGAGUGUU-3’ |
102 | 5’-AGUGUUUUCUGX2UGCUGCUUCUG-3’ |
103 | 5’-CAACGAACCCU-X-UCCCAAGCAAC-5’ |
104 | 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ |
105 | 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ |
106 | 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ |
107 | 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ |
108 | 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ |
109 | 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ |
110 | 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ |
111 | 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ |
112 | 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ |
113 | 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ |
114 | 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ |
115 | 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ |
116 | 5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ |
117 | 5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ |
118 | 5′-CCCIIICCCII-X-IICCCIIICCC-5′ |
119 | 5′-TTTTTTTTTTTTTTT-X-(G1UCUUCG1UCG1U)2-5′ |
120 | 3′-TTTTTTTTTTTTTTT-X-(G1UCUUCG1UCG1U)2-5′ |
121 | 5′-U1U1U1U1U1U1U1U1U1U1U1U1U1U1U1-X-(G1UCUUCG1UCG1U)2-5′ |
122 | 5’-CCIICCIICCC-X-CCCIICCIICC-5’ |
123 | 5’-CCIICCIICCX2CCIICCIICC-3’ |
I=肌苷;U1=dU;T1=核糖T;G1=7-脱氮-G;G2=ara-G;G3=6-硫代-G;G4=1-(β-D-呋喃核糖基)-2-氧代-7-脱氮-8-甲基嘌呤;O=洛索立宾;X=甘油;X1=1,3,5-戊三醇;X2=X3=C3接头或丙二醇;X4=三(乙二醇);X5=1,5戊二醇;X6=2’-脱氧-无碱基(abasic);X7=C3氨基接头;X8=顺,顺-环己烷三醇;X9=顺,反-环己烷三醇;下划线的=2’-O-Me-核糖核苷酸;P=硫代磷酸酯;小写字母=磷酸二酯骨架。
表3b显示根据本发明的一些寡聚核糖核苷酸可能形成的二级结构,他们是非限制性的和典型的。
表3b.一些SIMRA化合物可能形成的结构实例
在第二个方面,本发明提供包括本发明的SIMRA化合物和生理上可接受的载体的药物制剂。
在第三个方面,本发明提供在脊椎动物中产生TLR7和/或TLR8介导的免疫应答的方法,这种方法包括对脊椎动物施用本发明的SIMRA化合物。在一些实施方式中,所述脊椎动物是哺乳动物。在优选的实施方式中,对需要免疫刺激的脊椎动物施用所述SIMRA化合物。
在第四个方面,本发明提供治疗性处理患有疾病或病症的患者的方法,这种方法包括对患者施用本发明的SIMRA化合物。在不同的实施方式中,待治疗的疾病或病症是癌症、自身免疫病症、感染性疾病、气道炎症、炎性病症、变态反应、哮喘或病原体引起的疾病。病原体包括细菌、寄生物、真菌、病毒、类病毒和朊病毒。
在第五个方面,本发明提供用于预防疾病或病症的方法,这些方法包括对患者施用本发明的SIMRA化合物。在多种实施方式中,要预防的疾病或病症是癌症、自身免疫病症、气道炎症、炎性病症、变态反应、哮喘或病原体引起的疾病。病原体包括细菌、寄生物、真菌、病毒、类病毒和朊病毒。
在第六个方面,本发明提供预防或治疗病症的方法,这些方法包括分离能够产生细胞因子或者趋化因子的细胞,包括但不局限于免疫细胞、T调节细胞、B细胞、PBMC、pDC和淋巴细胞;在标准细胞培养条件下培养上述细胞,在体外用SIMRA处理上述细胞以使得被分离的细胞产生或分泌的细胞因子和趋化因子的水平增加,和对需要细胞因子或趋化因子来预防或处理疾病的患者施用或再施用处理细胞。本发明的这方面将根据标准过继性细胞免疫治疗技术来产生活化免疫细胞。
在本发明这方面的一些实施方式中,能够产生细胞因子或者趋化因子的细胞可能从患有或不患有疾病或病症的受试者中分离。上述的分离可以包括鉴定和挑选,并可以使用标准的细胞分离程序,包括以下具体实施例中所阐明的那些程序。上述分离细胞将依照标准细胞培养程序使用标准细胞培养条件加以培养,上述程序和条件可以包括以下具体实施例中所阐明的培养程序和培养条件。在本发明的这个实施方式的另一方面,分离细胞将在至少一种SIMRA存在的情况下培养,SIMRA的量和培养的时间足以诱导、增加或提高细胞因子和/或趋化因子的产生和/或分泌(相比于所述一种或多种SIMRA不存在的情况)。上述的时间可以从若干分钟到若干小时、到若干天。上述的经分离及SIMRA处理的细胞可在对供体再施用后或施用于另一患者后发挥效用,其中上述供体或另一患者需要诱导、增加或提高细胞因子和/或趋化因子的产生和/或分泌。例如,对患有癌症、自身免疫病症、气道炎症、炎性病症、感染性疾病、变态反应、哮喘或病原体引起的疾病的供体的再次施用或对患有癌症、自身免疫病症、气道炎症、炎性病症、感染性疾病、变态反应、哮喘或病原体引起的疾病的另一患者的施用。上述的再施用或施用可以使用包括导管或注射或任何其他有效的途径的多种方法完成。本发明的这个方面还可以应用于具有有限的或不完全的免疫应答能力的患者或免疫受损的患者(例如受人类免疫缺损病毒感染的患者或骨髓移植的患者)。
在任何本发明的方法中,SIMRA化合物可以通过单独和/或与任何有用于治疗或预防所述疾病或症候且不降低所述SIMRA化合物的免疫调节作用的其它试剂联合施用来产生直接的免疫调节作用,从而以不同的方式起作用。在任何本发明的方法中,有用于治疗或预防疾病或症候的试剂或条件包括,但不限于,疫苗、抗原、抗体、优选单克隆抗体、细胞毒剂、变态反应原、抗生素、siRNA、反义寡核苷酸、TLR激动剂(例如TLR9和/或TLR3的激动剂)、化疗剂(传统的化学疗法和现代的靶向疗法)、靶向治疗剂、活化的细胞、肽、蛋白、基因治疗载体、肽疫苗、蛋白疫苗、DNA疫苗、佐剂,和共刺激分子(例如细胞因子、趋化因子、蛋白配体、反式激活因子、肽和包含修饰氨基酸的肽)、或者它们的组合。例如,在癌症的治疗中,预期可将SIMRA化合物与一种或多种化疗化合物、靶向治疗剂和/或单克隆抗体联合施用。或者,所述试剂可包括编码抗原或变态反应原的DNA载体。或者,可将SIMRA化合物与其他佐剂联合施用以提高特异性或对SIMRA化合物的免疫应答的幅度。
在任何本发明的方法中,单独施用SIMRA化合物或者将SIMRA化合物与任何其他试剂联合施用,可以通过任何合适的途径,包括但不限于非消化道、黏膜给药、口服、舌下、经皮、局部、吸入、鼻内、气溶胶、眼内、气管内、直肠内、阴道、通过基因枪、皮肤贴片或采用滴眼液或漱口剂形式。可使用已知的程序,以对减少疾病的症状或替代标记物有效的量和时间段进行SIMRA化合物的施用。例如,对治疗某种疾病和/或病症有效的SIMRA化合物量可以是减轻或减少症状,或延迟或改善肿瘤、癌症、或细菌、病毒或真菌感染所必需的量。作为疫苗佐剂使用的有效量可以是对增强受试者对疫苗或抗原的免疫应答有用的量。在施用调节针对共同施用的抗原的免疫应答的组合物(a composition that modulates an immuneresponse to aco-adminstered antigen)的背景下,SIMRA化合物和抗原的有效量是足够达到相比于单独施用该抗原所获得的免疫应答的期望的调节作用的量。对于任何具体的应用,有效量可以根据所治疗的疾病或症候、所施用的具体的寡核苷酸、受试者的体格、或疾病或条件的严重程度而变化。本领域普通技术人员可以根据经验确定具体的寡核苷酸的有效量而不需要进行过多的试验。
当全身施用时,优选施用足够剂量的治疗组合物以便SIMRA化合物的血液水平达到从大约0.0001微摩尔/升(micromolar)到大约10微摩尔/升。对于局部施用,比这低得多的浓度也可能是有效的,而高得多的浓度也可能是可耐受的。SIMRA化合物的总剂量优选为从大约0.001mg每个患者每天到大约200mg每千克体重每天。可能需要同时或连续地对个体施用治疗有效量的一种或多种本发明的治疗组合物作为单一治疗段落。
SIMRA化合物可任选地连接于一个或多个变态反应原和/或抗原(自身或外来的)、免疫原性蛋白,例如钥孔血蓝蛋白(KLH)、霍乱毒素B亚基或任意其它免疫原性载体蛋白。SIMRA还可以和其他化合物(如佐剂)联合施用,所述化合物包括但不限于,TLR激动剂(如TLR2的激动剂和TLR9的激动剂)、弗氏不完全佐剂、KLH、单磷酰类脂A(MPL)、明矾、和皂苷,包括QS-21和咪喹莫特,或它们的组合。
本发明这方面的方法可用于免疫系统的模型研究。这些方法还可用于人或者动物疾病的预防或者治疗。例如,这些方法可用于儿科用和兽用疫苗的应用。
下面的实施例旨在进一步说明本发明的特定的优选实施方式,而不是意图限定本发明的范围。
实施例
免疫调节性寡聚核糖核苷酸的合成
在DNA/RNA自动合成仪上用亚磷酰胺化学法化学合成免疫调节性寡聚核糖核苷酸。TAC基团保护的(除U外)2’-O-TBDMS RNA单体:A、G、C和U,购自Sigma-Aldrich。7-脱氮-G、肌苷和洛索立宾单体购自ChemGenesCorporation。0.25M的5-乙硫基-1H-四唑,PAC-无水Cap A和Cap B购自Glen Research。溶于二氯甲烷(DCM)的3%三氯乙酸(TCA)和5%的3H-1,2-苯并二硫醇-3-酮-1,1-二氧化物(Beaucage试剂)均自行配置。
免疫调节性寡聚核糖核苷酸依照标准RNA合成规程以1-2μM的规模合成。
剪切和碱基的脱保护
从固相载体上剪切下免疫调节性寡聚核糖核苷酸,将溶液进一步加热到65℃以去除外向环胺保护基团。经处理后的溶液在离心浓缩仪(Speedvac)中完全干燥。
离子交换高效液相色谱法纯化
免疫调节性寡聚核糖核苷酸由离子交换高效液相色谱法纯化。
色谱柱:Dionex DNAPac 100柱(22X250)
色谱柱加热器:ChromTech TL-105高效液相色谱色谱柱加热器,温度设为80℃。
缓冲液A:20mM Tris-HCl,pH 7.0,20%乙腈
缓冲液B:3.0M NaCl,20mM Tris-HCl,pH7.0,20%乙腈
流速:10ml/min
梯度:
0-2min:0%B
2-11min:0%B至35%B
11-41min:35%B至90%B
41-45min:100%B
将未提纯的免疫调节性寡聚核糖核苷酸溶液注射入高效液相色谱。执行上述的洗脱梯度并收集级分。将所有含有90%以上期望产物的级分混合,然后将溶液在旋转蒸发仪(Rotovap)中浓缩至几乎干燥状态。加入不含核糖核酸酶的水使得最终体积为10ml。
C-18反相色谱法脱盐
购自Waters的CC-18Sep-Pak色谱柱首先用10毫升的乙腈处理,然后用10ml的0.5M醋酸钠处理。将10ml的免疫调节性寡聚核糖核苷酸溶液上样到色谱柱。然后用15ml的水洗去盐。最后用1ml的50%乙腈水溶液洗脱免疫调节性寡聚核糖核苷酸。
将溶液置于离心浓缩仪中30分钟。剩余的溶液通过0.2μm滤膜过滤,然后冻干至干燥。然后将获得的固体重新溶解于水至预期的浓度。
在低于0℃的条件下保存最终的溶液。
毛细管电泳
仪器:Beckman5010
毛细管:62厘米ssDNA毛细管
样品准备:将0.2 OD的SIMRA化合物溶解于200ul的不含核糖核酸酶的水中。
注射:在5KV电压下电动注射5秒钟
电泳条件:在30℃,14KV的条件下50分钟
离子交换高效液相色谱分析
色谱柱:Dionex DNAPac护柱(22X250)
色谱柱加热器:Chrom Tech TL-105高效液相色谱色谱柱加热器,温度设为80℃。
缓冲液A:100mM Tris-HCl,pH8.0,20%乙腈
缓冲液B:2.0M LiCl,100mM Tris-HCl,pH8.0,20%乙腈
流速:2ml/min
梯度:
0-2min:0%B
2-10min:0%B至100%B
10-15min:100%B
聚丙烯酰胺凝胶电泳分析
将0.3 OD的免疫调节性寡聚核糖核苷酸上样到20%的聚丙烯酰胺凝胶中并在4瓦特的恒定功率下电泳大约5小时。凝胶在短波长紫外光下观察。
人细胞培养规程
人PMBC分离
利用聚蔗糖密度梯度离心法(Histopaque-1077,Sigma)从新鲜采集的健康志愿者的血液(CBR Laboratories,波士顿,马萨诸塞州)中分离外周血单个核细胞(PMBC)。
人pDC分离
利用聚蔗糖密度梯度离心法(Histopaque-1077,Sigma)从新鲜采集的健康志愿者的血液(CBR Laboratories,波士顿,马萨诸塞州)中分离外周血单个核细胞(PMBC)。用BDCA4细胞分离试剂盒(Milenyi Biotec)根据制造商的说明通过阳性选择从外周血单个核细胞中分离pDC。
细胞因子的酶联免疫吸附测定(ELISA)
将人PBMC以5 X 106细胞/ml接种在48孔平板中。将人pDC以1 X 106细胞/ml接种在96孔培养盘中。向细胞培养物中添加溶于DPBS(pH7.4;Mediatech)的SIMRA至终浓度为100.0μg/ml。然后将细胞在37℃孵育24小时,收集上清用于多元LUMINEX(LUMINEXMULTIPLEX)或ELISA分析。将实验进行一式3孔的重复。通过夹心ELISA分析上清中IFN-α、IL-6或TNF-α的水平。所需试剂,包括细胞因子抗体和标准品,均购自PharMingen。
用BIOSOURCE HUMAN MULTIPLEX细胞因子分析试剂盒在Luminex100仪器上进行多元LUMINEX分析。所得数据用AppliedCytometry Systems(Sacramento,CA)提供的StarStation软件进行分析。
分析表达TLR的HEK293细胞的规程
将HEK293/人TLR7或HEK293/人TLR8细胞(Invivogen,San Diego,CA)培养在5%CO2培养箱中的48孔平板中,其中每孔含250μl添加有10%热灭活的FBS的DMEM。
报道基因转化
在48孔板上250μl/孔补充了10%热灭活FBS的DMEM中培养稳定表达小鼠TLR9或人类TLR3、7或8的HEK293细胞(Invivogen,San Diego,CA),细胞在5%二氧化碳培养箱中培养。在80%汇合时,在培养基中存在4μl/ml脂质转染试剂(Lipofectamine)(Invitrogen,Carlsbad,CA)的条件下,将培养物用400ng/ml的SEAP(分泌形式的人类胚胎碱性磷酸酯酶)报道质粒(pNifty2-Seap)(Invitrogen)瞬时转染。在不含血清的培养基中分别稀释质粒DNA和脂质转染试剂,并在室温温育5分钟。温育之后,混合稀释的DNA和脂质转染试剂,再将混合物在室温温育20分钟。将含有100ng质粒DNA和1μl脂质转染试剂的25μl DNA/脂质转染试剂混合物添加至细胞培养平板的各个孔,继续培养4小时。
IMO处理
转染之后,培养基替换为新鲜的培养基,将SIMRA加入培养物并继续培养18个小时。在SIMRA处理的最后,从每个处理中取30μl培养物上清根据制造商的规程(Invivogen)来进行SEAP分析。
SEAP分析
简要地,将培养物上清与对磷酸硝基苯酯底物共同温育并用酶标仪在405nm测量所产生的黄色。数据显示为NF-κB活性与PBS对照相比的倍数增长。(Putta MR等人,NucleicAcids Res.,2006,34:3231-8)。
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
1 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 3.1
11 5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’ 4.8
12 5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′ 2.6
13 5′-GUCCUUCAACU-X-UCAACUUCCUG-5′ 4.3
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3和28.6μM
PBS 1 1
1 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 2.7 8
20 5’-X2 UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’ 3.12 8.5
21 5’-X2UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’ 8.1 14.7
22 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 0.6 1
R848 8.5
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3和28.6μM
PBS 1 1
1 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 9.2 64.5
23 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 1 4.1
24 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 6.7 33.6
25 5’-UGCUGCUACUG-X-GUCAUCGUCGU-5’ 7.6 32.9
26 5’-UGCUGCUUGUG-X-GUGUUCGUCGU-5’ 1.9 15.63
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3和28.6μM
PBS 1 1
1 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 4.13 14.5
21 5’-X2UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’ 8.6 22.9
24 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 12.7 27.3
26 5’-UGCUGCUUGUG-X-GUGUUCGUCGU-5’ 4.8 19
27 5’-UGCUGCUGCUG-X-GUCGUCGUCGU-5’ 2.8 12.4
28 5’-UGCUGCUUAUG-X-GUAUUCGUCGU-5’ 2.2 5
30 5’-X2UGCUGCUUGUG-X-GUGUUCGUCGUX2-5’ 17.9 27.9
R848 12.5
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
1 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 3.1
11 5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’ 4.8
12 5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′ 2.6
13 5′-GUCCUUCAACU-X-UCAACUUCCUG-5′ 4.3
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
31 5’-UGCUGCUUCUG-X1-GUCUUCGUCGU-5’ 4.2
32 5’-X7UGCUGCUUGUG-X-GUGUUCGUCGUX7-5’ 4.5
33 5′-UGUUGUGUGAC-X-CAGUGUGUUGU-5′ 7.0
34 5′-CUGGCGGCCUU-X-UUCCGGCGGUC-5′ 5.85
35 5’-X3UG1CUG1CUUGUG1-X-G1UGUUCG1UCG1UX3-5’ 9.2
36 5’-UGCUGCUUG2UG-X-GUG2UUCGUCGU-5’ 4.74
37 5’-G2GCUGCUUGUG-X-GUGUUCGUCGG2-5’ 4.4
38 5′-UGCUGCCUUUG-X-GUUUCCGUCGU-5′ 7.5
39 5′-GUCCUUGCUUG-X-GUUCGUUCCUG-5′ 8.7
405′-GUCCUUUGCUG-X-GUCGUUUCCUG-5′ 8.1
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 21.5μM
PBS 1
41 5’-X3UGCUGCUGCUG-X-GUCGUCGUCGUX3-5’ 13.7
42 5’-XUGCUGCUUGUG-X-GUGUUCGUCGUX-5’ 16.0
43 5’-X7UGCUGCUGCUG-X-GUCGUCGUCGUX7-5’ 15.2
44 5′-UUGCCCUUGCC-X-CCGUUCCCGUU-5′ 10.5
45 5′-UUGCUGUUGCU-X-UCGUUGUCGUU-5′ 12.9
46 5′-CUUUGGUGUGU-X-UGUGUGGUUUC-5′ 5.1
47 5′-UUGGUUGUUUG-X-GUUUGUUGGUU-5′ 17.4
48 5′-CUUUGGUGUGU-X-UGUGUGGUUUC-5′ 5.1
55 5’-UG1CUG1CUUCUG1-X1-G1UCUUCG1UCG1U-5’ 2.96
56 5’-X3UGCUGCUUGUG-X1-GUGUUCGUCGUX3-5’ 9.14
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
57 5′-UG3CUGCUUCUG-X-GUCUUCGUCG3U-5′ 9.16
58 5′-UGG4UGCUUCUG-X-GUCUUCGUG4GU-5′ 2.29
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
86 5’-AGAAGCUUCUG-X-GUCUUCGAAGA-5’ 1.42
87 5’-UGAAGCUUCUG-X-GUCUUCGAAGU-5’ 1.54
表达人类TLR8的HEK293细胞中NF-kB活性的倍数变化
序号 序列 21.5μM
PBS 1.0
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 9.7
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 6.1
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 7.0
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 8.1
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 4.8
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 2.8
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 3.6
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 3.8
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 4.5
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 3.6
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 8.8
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 2.6
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 2.5
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 3.1
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 2.5
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 10.7
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 6.1
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 10.6
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 6.3
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 1.3
表达人类TLR7的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
1 5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’ 1.1
11 5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’ 9.3
12 5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′ 11.5
13 5′-GUCCUUCAACU-X-UCAACUUCCUG-5′ 1.8
洛索立宾(465μM) 7.26
R848 11.5
表达人类TLR7的HEK293细胞中NF-kB活性的倍数变化
序号 序列 14.3μM
PBS 1
59 5’-UUG1G1UUG1UUUG1-X-G1UUUG1UUG1G1UU-5’ 2.311
60 5’-UG1CU G1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 3.160
61 5′-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5′ 2.603
62 5′-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5′ 2.274
63 5’-UG1CUG1CUUCU G1-X8-G1UCUUCG1UCG1U-5’ 2.887
64 5′-CUG1-X-G1UC-5’ 2.104
65 5′-UUG1CUG1UUG1CU-X-UCG1UUG1UCG1UU-5′ 1.454
66 5′-UG1CCUUG1AACU-X-UCAAG1UUCCG1U-5′ 1.411
67 5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′ 2.330
69 5′-G1UCCUUCUCUG1-X-G1UCUCUUCCUG1-5′ 2.377
用ELISA确定的人类PBMC测定中的IFN-α(pg/ml)
序号 序列 21.5μM
PBS 0
61 5′-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5′ 1092.2
62 5′-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5′ 491.4
63 5’-UG1CUG1CUUCUG1-X8-G1UCUUCG1UCG1U-5’ 1803.8
65 5′-UUG1CUG1UUG1CU-X-UCG1UUG1UCG1UU-5′ 1046.9
66 5′-UG1CCUUG1AACU-X-UCAAG1UUCCG1U-5′ 455.3
67 5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′ 1275.4
68 5′-UUCUG1CUUCUG1-X5-G1UCUUCG1UCUU-5′ 466.3
69 5′-G1UCCUUCUCUG1-X-G1UCUCUUCCUG1-5′ 618.1
70 5′-UG1UURUG1UG1AC-X-CAG1URUG1UUG1U-5′ 1125.9
用ELISA确定的人类PBMC测定中的IFN-α(pg/ml)
序号 序列 7.15μM
PBS 0
60 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 907
用多元Luninex确定的人类PBMC分析中的IFN-α(pg/ml)
序号 序列 7.15μM
PBS 46.4
63 5’-UG1CUG1CUUCUG1-X8-G1UCUUCG1UCG1U-5’ 101.7
67 5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′ 133.6
68 5′-UUCUG1CUUCUG1-X5-G1UCUUCG1UCUU-5′ 79.1
用多元Luninex确定的人类PBMC测定中的IFN-α(pg/ml)
序号 序列 14.3μM
培养基 0
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 4381
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 4160
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 1117
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 158
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 41
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 1042
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 2394
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 9
用多元Luninex测定的人类PBMC分析中的IFN-α(pg/ml)
序号 序列 7.2μM
培养基 0
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 459
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 898
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 394
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 478
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 694
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 1326
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 153
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 1130
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 889
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 773
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 65
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 1106
用多元Luninex确定的人类pDC分析中的IFN-α(pg/ml)
序号 序列 7.2μM
培养基 0
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 9486
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 7992
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 12305
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 10144
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 12572
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 10584
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 15426
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 13035
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 8815
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 11210
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 141
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 6480
用多元Luninex确定的人类PBMC测定中的IL-10水平(pg/ml)
序号 序列 7.15μM
PBS 57.2
63 5’-UG1CUG1CUUCUG1-X8-G1UCUUCG1UCG1U-5’ 2502.4
67 5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′ 2895.2
68 5′-UUCUG1CUUCUG1-X5-G1UCUUCG1UCUU-5′ 2603.1
用多元Luninex确定的人类PBMC测定中的IL-10水平(pg/ml)
序号 序列 14.3μM
培养基 13
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 7713
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 6780
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 5457
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 7152
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 8675
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 1230
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 2378
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 6324
用多元Luninex确定的人类PBMC测定中的IL-10水平(pg/ml)
序号 序列 7.2μM
培养基 7
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 184
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 242
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 126
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 88
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 169
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 285
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 392
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 189
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 270
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 183
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 417
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 331
用多元Luninex确定的人类PBMC分析中的IL-12水平(pg/ml)
序号 序列 14.3μM
培养基 39
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 6551
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 4305
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 7915
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 6440
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 4701
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 8065
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 10226
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 1944
用多元Luninex确定的人类PBMC分析中的IL-12水平(pg/ml)
序号 序列 7.2μM
培养基 25
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 1410
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 1405
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 750
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 671
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 875
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 2749
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 2742
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 1110
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 1428
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 1126
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 3034
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 2055
用多元Luninex确定的人类PBMC测定中的IP-10水平(pg/ml)
序号 序列 7.15μM
PBS 0
63 5’-U G1CUG1CUUCUG1-X8-G1UCUUCG1UCG1U-5’ 132.3
67 5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′ 122.7
68 5′-UUCUG1CUUCUG1-X5-G1UCUUCG1UCUU-5′ 13.9
用多元Luninex确定的人类PBMC测定中的IP-10水平(pg/ml)
序号 序列 14.3μM
培养基 28
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 398
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 358
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 679
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 613
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 318
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 263
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 245
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 140
用多元Luninex确定的人类PBMC测定中的IP-10水平(pg/ml)
序号 序列 7.2μM
培养基 28
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 835
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 847
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 587
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 661
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 696
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 943
110 5’-UG1CUGXCCUUUG1-X-G1UUUCCG1UCG1U-5’ 927
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 796
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 827
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 815
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 659
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 890
用多元Luninex确定的人类pDC测定中的IP-10水平(pg/ml)
序号 序列 7.2μM
培养基 28
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 166
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 257
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 128
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 180
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 138
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 348
110 5’-U G1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 416
111 5’-U G1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 144
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 230
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 171
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 89
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 126
用多元Luninex确定的人类PBMC测定中的IL-8水平(pg/ml)
序号 序列 14.3μM
培养基 273
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 4078
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 13018
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 5898
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 6386
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 8506
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 5776
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 4494
用多元Luninex确定的人类PBMC测定中的IL-8水平(pg/ml)
序号 序列 7.2μM
培养基 1982
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 46124
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 88695
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 113574
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 33359
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 155855
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 90944
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 106354
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 129362
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 1397924
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 71610
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 208436
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 103725
用多元Luninex确定的人类PBMC测定中的MCP-1水平(pg/ml)
序号 序列 14.3μM
培养基 12
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 3010
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 3533
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 3188
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 2770
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 3589
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 521
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 997
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 2308
用多元Luninex确定的人类PBMC测定中的MCP-1水平(pg/ml)
序号 序列 7.2μM
培养基 79
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 192086
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 361681
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 160970
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 139299
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 294514
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 109354
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 218045
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 183109
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 278467
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 210977
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 149298
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 112087
用多元Luninex确定的人类PBMC测定中的MCP-1α水平(pg/ml)
序号 序列 14.3μM
培养基 21
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 19586
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 5037
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 16677
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 3575
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 566
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 24329
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 39964
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 25
用多元Luninex确定的人类PBMC分析中的MCP-1α水平(pg/ml)
序号 序列 7.2μM
培养基 31
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 8103
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 11628
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 4511
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 3858
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 6507
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 17164
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 15559
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 7714
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 11119
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 9111
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 20355
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 16284
用多元Luninex确定的人类PBMC测定中的MCP-1β水平(pg/ml)
序号 序列 14.3μM
培养基 175
90 5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′ 42262
92 5′-GCACACUUGUU-X-UUGUUCACACG-5′ 42685
93 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 39680
95 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′ 48949
96 5′-CAACGACCUGU-X-UGUCCAGCAAC-5′ 47724
116 (5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′ 38198
117 (5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′ 44528
118 5′-CCCIIICCCII-X-IICCCIIICCC-5′ 49838
用多元Luninex确定的人类PBMC测定中的MCP-1β水平(pg/ml)
序号 序列 7.2μM
培养基 77
104 5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’ 18921
105 5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’ 30590
106 5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’ 13947
107 5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’ 12919
108 5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’ 14264
109 5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’ 35365
110 5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’ 38605
111 5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’ 11093
112 5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’ 24994
113 5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’ 17191
114 5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’ 18002
115 5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’ 24605
小鼠模型中基于RNA的寡核苷酸的体内抗癌活性
BALB/c小鼠被分为包含三只小鼠的组。静脉(i.v)注射培养的CT26.CL25细胞(4 x105个细胞/小鼠)。然后对小鼠皮下(s.c.)施用根据本发明的基于RNA的寡核苷酸(SIMRA化合物)或对照,剂量是50mg/kg。第一剂施用4小时后,从小鼠体内取出血清并通过ELISA确定IL-12水平。结果在图9显示。小鼠在静脉施用CT26.CL25细胞后24小时、72小时和144小时进一步接受皮下施用。在第14天处死小鼠并收集肺脏。图10显示在肺脏中发现的肿瘤结节数量。
非人类灵长动物中基于RNA的寡核苷酸的体内免疫应答
食蟹猴被分为3组,每组4只(盐水组2只)。然后对食蟹猴皮下(s.c.)施用根据本发明的基于RNA的寡核苷酸(SIMRA化合物)或对照,剂量是5mg/kg。其他剂量(例如1mg/kg)也可能产生想要的效果。在施用后8、16和24小时从食蟹猴中取出血清并确定免疫应答中细胞因子和趋化因子的水平及变化。结果在图12-15中显示。
等同物
尽管为了清楚和理解的目的,已经对上述发明进行了一定程度的详细描述,但通过阅读本文本领域技术人员应当了解在形式和细节方面可以做出不脱离本发明和附属权利要求实质范围的各种变化。
序列表
<110>艾德拉药物股份有限公司(Idera Pharmaceuticals,Inc.)
<120>用于TLR7和TLR8的稳定化的免疫调节性RNA(SIMRA)化合物
<130>IDR-043US1
<140>11/697,422
<141>2007-04-06
<150>60/790,466
<151>2006-04-07
<150>60/863,926
<151>2006-11-01
<150>60/827,835
<151>2006-10-02
<160>127
<170>PatentIn version 3.3
<210>1
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>1
ugcugcuucu g 11
<210>2
<211>11
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<400>2
cugugcuucu c 11
<210>3
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>3
ucugugcuuc u 11
<210>4
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(6)..(6)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(10)..(10)
<223>肌苷
<400>4
uncaancuun c 11
<210>5
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>5
gugugugugu g 11
<210>6
<211>8
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<400>6
ugcugcuu 8
<210>7
<211>14
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<400>7
ugcugcuucu gugu 14
<210>8
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<220>
<223>人工序列描述:合成寡核苷酸
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ugcugcuucu gugucug 17
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<223>人工序列描述:合成寡核苷酸
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<221>修饰的_碱基
<222>(1)..(1)
<223>dU
<220>
<221>修饰的_碱基
<222>(4)..(4)
<223>dU
<220>
<221>修饰的_碱基
<222>(7)..(8)
<223>dU
<220>
<221>修饰的_碱基
<222>(10)..(10)
<223>dU
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ugcugcuucu g 11
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<223>riboT
<220>
<221>修饰的_碱基
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<223>riboT
<220>
<221>修饰的_碱基
<222>(7)..(8)
<223>riboT
<220>
<221>修饰的_碱基
<222>(10)..(10)
<223>riboT
<400>10
tgctgcttct g 11
<210>11
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<212>RNA
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<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
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ugcugcuucu g 11
<210>12
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<212>RNA
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guccuucaac u 11
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guccuucaac u 11
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<222>(2)..(2)
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<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
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ugcugcuucu g 11
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<400>15
guccuucaac u 11
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<212>RNA
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<223>人工序列描述:合成寡核苷酸
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<223>洛索立宾
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nuccuucaac u 11
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<212>RNA
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<221>修饰的_碱基
<222>(5)..(5)
<223>洛索立宾
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>洛索立宾
<400>17
uncuncuucu n 11
<210>18
<211>11
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
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<221>修饰的_碱基
<222>(2)..(2)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>肌苷
<400>18
uncuncuucu n 11
<210>19
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>肌苷
<400>19
nuccuucaac u 11
<210>20
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>2’-O-Me-核糖-U
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>2’-O-Me-核糖-G
<400>20
ugcugcuucu g 11
<210>21
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>21
ugcugcuucu g 11
<210>22
<211>11
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>2’-O-Me-核糖-U
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>2’-O-Me-核糖-G
<400>22
ugcugcuucu g 11
<210>23
<211>11
<212>RNA
<213>人工序列
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<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(8)..(8)
<223>2’-O-Me-核糖-U
<400>23
ugcugcuucu g 11
<210>24
<211>11
<212>RNA
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<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(9)..(9)
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<400>24
ugcugcuucu g 11
<210>25
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<212>RNA
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<223>人工序列描述:合成寡核苷酸
<400>25
ugcugcuacu g 11
<210>26
<211>11
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<400>26
ugcugcuugu g 11
<210>27
<211>11
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<400>27
ugcugcugcu g 11
<210>28
<211>11
<212>RNA
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<220>
<223>人工序列描述:合成寡核苷酸
<400>28
ugcugcuuau g 11
<210>29
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(9)..(9)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>29
ugcugcuugu g 11
<210>30
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>30
ugcugcuugu g 11
<210>31
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>31
ugcugcuucu g 11
<210>32
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>32
ugcugcuugu g 11
<210>33
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>33
uguuguguga c 11
<210>34
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>34
cuggcggccu u 11
<210>35
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>35
ugcugcuugu g 11
<210>36
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(9)..(9)
<223>ara-G
<400>36
ugcugcuugu g 11
<210>37
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>ara-G
<400>37
ggcugcuugu g 11
<210>38
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>38
ugcugccuuu g 11
<210>39
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>39
guccuugcuu g 11
<210>40
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>40
guccuuugcu g 11
<210>41
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>41
ugcugcugcu g 11
<210>42
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>42
ugcugcuugu g 11
<210>43
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>43
ugcugcugcu g 11
<210>44
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>44
uugcccuugc c 11
<210>45
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>45
uugcuguugc u 11
<210>46
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>46
cuuuggugug u 11
<210>47
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>47
uugguuguuu g 11
<210>48
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>48
cuuuggugug u 11
<210>49
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>49
uugguuguuu g 11
<210>50
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>50
guccuugcuu g 11
<210>51
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>phoso-U
<400>51
ugcugcuugu g 11
<210>52
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>52
ugcugcuugu g 11
<210>53
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>53
ugcugcuugu g 11
<210>54
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>54
ugcugcuugu g 11
<210>55
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>55
ugcugcuucu g 11
<210>56
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>56
ugcugcuugu g 11
<210>57
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>6-thio-G
<400>57
ugcugcuucu g 11
<210>58
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(3)..(3)
<223>1(beta-D-呋喃核糖基)-2-氧代-7-脱氮-8-甲基-嘌呤
<400>58
ugnugcuucu g 11
<210>59
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(3)..(4)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>59
uugguuguuu g 11
<210>60
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>60
ugcugccuuu g 11
<210>61
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>61
guccuugcuu g 11
<210>62
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(8)..(8)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>62
guccuuugcu g 11
<210>63
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>63
ugcugcuucu g 11
<210>64
<211>3
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(3)..(3)
<223>7-脱氮-G
<400>64
cug 3
<210>65
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(3)..(3)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(6)..(6)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(9)..(9)
<223>7-脱氮-G
<400>65
uugcuguugc u 11
<210>66
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<400>66
ugccuugaac u 11
<210>67
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>67
uucugcuucu g 11
<210>68
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>68
uucugcuucu g 11
<210>69
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>69
guccuucucu g 11
<210>70
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(9)..(9)
<223>7-脱氮-G
<400>70
uguuruguga c 11
<210>71
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>71
uugguuguuu g 11
<210>72
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>72
guccuugcuu g 11
<210>73
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>73
uugguuguuu g 11
<210>74
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>74
guccuugcuu g 11
<210>75
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>75
ugcugcuugu g 11
<210>76
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>76
ugcugcuucu ggacaugucc ag 22
<210>77
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>77
ugcugcuucu gugauaucac ag 22
<210>78
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>78
ugcugcuucu gaauuaauuc ag 22
<210>79
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>79
ugcugcuucu ggacuagucc ag 22
<210>80
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>80
ugcugcuucu gugauaucac ag 22
<210>81
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>81
aguugaagga cugcugcuuc ug 22
<210>82
<211>22
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>82
guccuucaac ucagaagcag ca 22
<210>83
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>83
aguugaagga c 11
<210>84
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>84
guccuucaac u 11
<210>85
<211>9
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(4)..(6)
<223>肌苷
<400>85
cccnnnccc 9
<210>86
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>86
agaagcuucu g 11
<210>87
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>87
ugaagcuucu g 11
<210>88
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>88
uugguuguuu g 11
<210>89
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>89
guccuugcuu g 11
<210>90
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>90
ucugaauuca g 11
<210>91
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>91
guuugcacaa c 11
<210>92
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>92
gcacacuugu u 11
<210>93
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>93
cacuguugag a 11
<210>94
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>94
cacuguugac a 11
<210>95
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>95
aacuguugac c 11
<210>96
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>96
caacgaccug u 11
<210>97
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>97
agcacaacug u 11
<210>98
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>98
ugcugagugu u 11
<210>99
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>99
aguguuuucu g 11
<210>100
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>100
ugcugcuucu g 11
<210>101
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>101
ugcugcuucu g 11
<210>102
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>102
aguguuuucu g 11
<210>103
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>103
caacgaaccc u 11
<210>104
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>104
guccuugcuu g 11
<210>105
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(1)
<223>7-脱氮-G
<400>105
guccuugcuu g 11
<210>106
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>106
uucugcuucu g 11
<210>107
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>107
uucugcuucu g 11
<210>108
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>108
uucugcuucu g 11
<210>109
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(7)..(7)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>109
guccuugcuu g 11
<210>110
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>110
ugcugccuuu g 11
<210>111
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>111
ugcugccuuu g 11
<210>112
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(8)..(8)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>112
guccuuugcu g 11
<210>113
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(1)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(8)..(8)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>113
guccuuugcu g 11
<210>114
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>114
ugcugcuugu g 11
<210>115
<211>10
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>115
gcugccuuug 10
<210>116
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>116
ugcugcuugu g 11
<210>117
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>117
ugcugcuugu g 11
<210>118
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(4)..(6)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(10)..(11)
<223>肌苷
<400>118
cccnnncccn n 11
<210>119
<211>15
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>119
tttttttttt ttttt 15
<210>120
<211>15
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>120
tttttttttt ttttt 15
<210>121
<211>15
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(1)..(15)
<223>dU
<400>121
uuuuuuuuuu uuuuu 15
<210>122
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(3)..(4)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(7)..(8)
<223>肌苷
<400>122
ccnnccnncc c 11
<210>123
<211>10
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(3)..(4)
<223>肌苷
<220>
<221>修饰的_碱基
<222>(7)..(8)
<223>肌苷
<400>123
ccnnccnncc 10
<210>124
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<220>
<221>修饰的_碱基
<222>(2)..(2)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(5)..(5)
<223>7-脱氮-G
<220>
<221>修饰的_碱基
<222>(11)..(11)
<223>7-脱氮-G
<400>124
ugcugcuucu g 11
<210>125
<211>11
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>125
cagaagcagc a 11
<210>126
<211>10
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>126
ccguugacag 10
<210>127
<211>10
<212>RNA
<213>人工序列
<220>
<223>人工序列描述:合成寡核苷酸
<400>127
acacgcgugu 10
Claims (32)
1.一种TLR8和/或TLR7的激动剂,其中包含稳定化的免疫调节性RNA化合物,其中所述稳定化的免疫调节性RNA包含至少两个寡聚核糖核苷酸,所述寡聚核糖核苷酸通过3’附接而连接,且其中所述激动剂不与脂质结合。
2.根据权利要求1的激动剂,其中寡聚核糖核苷酸在它们的3’端直接相互连接。
3.根据权利要求1的激动剂,其中寡聚核糖核苷酸的3’端与非核苷酸接头相连接。
4.根据权利要求3的激动剂,其中的非核苷酸接头是烃基(alkyl)接头或氨基接头,其中的烃基接头或氨基接头是任选地分支化的或无分支的,环状的或非环状的,取代的或未取代的,饱和的或不饱和的,手性的,非手性的或者外消旋混合物。
5.根据权利要求4的激动剂,其中的烃基接头具有2个至18个碳原子。
6.根据权利要求4的激动剂,其中的烃基接头具有3至9个碳原子。
7.根据权利要求4的激动剂,其中的烃基接头选自化合物1,2,3-丙三醇、甘油、1,2,4-丁三醇、2-羟甲基-1,3-丙二醇、1,1,1-三(羟甲基)乙烷、2-氨基-2-(羟甲基)1,3-丙二醇、三(羟甲基)硝基甲烷、1,1,1-三(羟甲基)丙烷、1,2,6-己三醇、1,3,5-己三醇、1,3,5-戊三醇、3-甲基-1,3,5-戊三醇、1,2,3-庚三醇、2-(羟甲基)1,4-丁二醇、1,3-二(羟甲基)苯酚、1,3,5-三(羟甲基)苯、1,3-二(羟基乙氧基)-2-羟基-丙烷、1,3-二(羟基丙氧基)-2-羟基-丙烷,D-半乳醛,1,3,5-三(2-羟乙基)氰尿酸或1,3,5-三(4-羟苯基)苯。
8.根据权利要求1的激动剂,其中至少一个寡聚核糖核苷酸包含修饰的寡聚核糖核苷酸。
9.根据权利要求8的激动剂,其中修饰的寡聚核糖核苷酸包含7-脱氮-G,ara-G、6-硫代-G、肌苷、异-G、洛索立宾、(7-硫代-8-氧)-G、8-溴-G、8-羟基-G、5-氨基间型霉素B,氧代间型霉素、7-甲基-G、9-对-氯苯基-8-氮杂-G、9-苯基-G、9-己基鸟嘌呤、7-脱氮-9-苯甲基-G、6-氯-7-脱氮鸟嘌呤、6-甲氧基-7-脱氮鸟嘌呤、8-氮杂-7-脱氮-G、2-(二甲氨基)鸟苷、7-甲基-6-硫基鸟苷、8-苄氧基鸟苷、9-脱氮鸟苷、9-苯甲基-8-羟基-2-(2-甲氧基乙氧基)腺嘌呤、2-氨基-N2-O-、甲基腺苷、8-氮杂-7-脱氮-A、7-脱氮-A、阿糖腺苷、2-氨基腺苷、N1-甲基腺苷、8-氮杂腺苷、5-碘代杀结核菌素、1-(B-D-呋喃核糖基)-2-氧代-7-脱氮-8-甲基嘌呤以及4-硫代-U,或它们的组合。
10.根据权利要求1-9中任意一项的激动剂,其中所述寡聚核糖核苷酸化合物进一步包含5’帽。
11.根据权利要求10的激动剂,其中所述5’帽是非核苷酸接头。
12.根据权利要求1的激动剂,其中所述稳定化的免疫调节性RNA化合物选自下组:
5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’
5’-CUGUGCUUCUC-X-CUCUUCGUGUC-5’
5’-UCUGUGCUUCU-X-UCUUCGUGUCU-5’
5’-UICAAICUUIC-X-CIUUCIAACIU-5’
5′-GUGUGUGUGUG-X-GUGUGUGUGUG-5′
5’-UGCUGCUU-X-UUCGUCGU-5’
5′-UGCUGCUUCUGUGU-X-UGUGUCUUCGUCGU-5′
5′-UGCUGCUUCUGUGUCUG-X-GUCUGUGUCUUCGUCGU-5′
5′-T1GCT1GCT1T1CT1G-X-GT1CT1T1CGT1CGT1-5′
5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’
5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′
5′-GUCCUUCAACU-X-UCAACUUCCUG-5′
5’-UG1CUG1CUUCUG1-X-G1UCUUCG1UCG1U-5’
5′-G1UCCUUCAACU-X-UCAACUUCCUG1-5′
5′-OUCCUUCAACU-X-UCAACUUCCUO-5′
5’-UOCUOCUUCUO-X-OUCUUCOUCOU-5’
5’-UICUICUUCUI-X-IUCUUCIUCIU-5’
5′-IUCCUUCAACU-X-UCAACUUCCUI-5′
5’-X2 UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’
5’-X2UGCUGCUUCUG-X-GUCUUCGUCGUX2-5’
5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’
5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’
5’-UGCUGCUUCUG-X-GUCUUCGUCGU-5’
5’-UGCUGCUACUG-X-GUCAUCGUCGU-5’
5’-UGCUGCUUGUG-X-GUGUUCGUCGU-5’
5’-UGCUGCUGCUG-X-GUCGUCGUCGU-5’
5’-UGCUGCUUAUG-X-GUAUUCGUCGU-5’
5’-UG1CUG1CUUG1UG1-X-G1UG1UUCG1UCG1U-5’
5’-X2UGCUGCUUGUG-X-GUGUUCGUCGUX2-5
5’-UGCUGCUUCUG-X1-GUCUUCGUCGU-5’
5’-X7UGCUGCUUGUG-X-GUGUUCGUCGUX7-5’
5′-UGUUGUGUGAC-X-CAGUGUGUUGU-5′
5′-CUGGCGGCCUU-X-UUCCGGCGGUC-5′
5’-X3UG1CUG1CUUGUG1-X-G1UGUUCG1UCG1UX3-5’
5’-UGCUGCUUG2UG-X-GUG2UUCGUCGU-5’
5’-G2GCUGCUUGUG-X-GUGUUCGUCGG2-5’
5′-UGCUGCCUUUG-X-GUUUCCGUCGU-5′
5′-GUCCUUGCUUG-X-GUUCGUUCCUG-5′
5′-GUCCUUUGCUG-X-GUCGUUUCCUG-5′
5’-X3UGCUGCUGCUG-X-GUCGUCGUCGUX3-5’
5’-XUGCUGCUUGUG-X-GUGUUCGUCGUX-5’
5’-X7UGCUGCUGCUG-X-GUCGUCGUCGUX7-5’
5′-UUGCCCUUGCC-X-CCGUUCCCGUU-5′
5′-UUGCUGUUGCU-X-UCGUUGUCGUU-5′
5′-CUUUGGUGUGU-X-UGUGUGGUUUC-5′
5′-UUGGUUGUUUG-X-GUUUGUUGGUU-5′
5′-CUUUGGUGUGU-X-UGUGUGGUUUC-5′
5′-X3UUGGUUGUUUG-X-GUUUGUUGGUUX3-5′
5′-X3GUCCUUGCUUG-X-GUUCGUUCCUGX3-5′
5’-PUGCUGCUUGUG-X-GUGUUCGUCGUP-5’
5’-X4UGCUGCUUGUG-X-GUGUUCGUCGUX4-5’
5’-X5UGCUGCUUGUG-X-GUGUUCGUCGUX5-5’
5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’
5’-UG1CUG1CUUCUG1-X1-G1UCUUCG1UCG1U-5’
5’-X3UGCUGCUUGUG-X1-GUGUUCGUCGUX3-5’
5′-UG3CUGCUUCUG-X-GUCUUCGUCG3U-5′
5′-UGG4UGCUUCUG-X-GUCUUCGUG4GU-5′
5’-UUG1G1UUG1UUUG1-X-G1UUUG1UUG1G1UU-5’
5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’
5′-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5′
5′-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5′
5’-UG1CUG1CUUCUG1-X8-G1UCUUCG1UCG1U-5’
5′-CUG1-X-G1UC-5’
5′-UUG1CUG1UUG1CU-X-UCG1UUG1UCG1UU-5′
5′-UG1CCUUG1AACU-X-UCAAG1UUCCG1U-5′
5′-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5′
5′-UUCUG1CUUCUG1-X5-G1UCUUCG1UCUU-5′
5′-G1UCCUUCUCUG1-X-G1UCUCUUCCUG1-5′
5′-UG1UURUG1UG1AC-X-CAG1URUG1UUG1U-5′
5′-X2UUGGUUGUUUG-X-GUUUGUUGGUUX2-5′
5′-X2GUCCUUGCUUG-X-GUUCGUUCCUGX2-5′
5′-X6UUGGUUGUUUG-X-GUUUGUUGGUUX6-5′
5′-X6GUCCUUGCUUG-X-GUUCGUUCCUGX6-5′
5’-X2UGCUGCUUGUG-X8-GUGUUCGUCGUX2-5’
5′-UGCUGCUUCUGGACAUGUCCAG-3′
5′-UGCUGCUUCUGUGAUAUCACAG-3′
5′-UGCUGCUUCUGAAUUAAUUCAG-3′
5′-UGCUGCUUCUGGACUAGUCCAG-3′
5’-UGCUGCUUcugugauaucacag-3′
5′-AGUUGAAGGACUGCUGCUUCUG-3′
5′-GUCCUUCAACUCAGAAGCAGCA-3′
5′-AGUUGAAGGACX2UGCUGCUUCUG-3′
5′-GUCCUUCAACUX2CAGAAGCAGCA-3′
5’-CCCIIICCCX2CCCIIICCC-3′
5’-AGAAGCUUCUG-X-GUCUUCGAAGA-5’
5’-UGAAGCUUCUG-X-GUCUUCGAAGU-5’
5′-X6UUGGUUGUUUG-X-GUUUGUUGGUUX6-5′
5′-X6GUCCUUGCUUG-X-GUUCGUUCCUGX6-5′
5′-UCUGAAUUCAG-X-GACUUAAGUCU-5′
5′-GUUUGCACAAC-X-CAACACGUUUG-5′
5′-GCACACUUGUU-X-UUGUUCACACG-5′
5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′
5′-CACUGUUGACA-X-ACAGUUGUCAC-5′
5′-AACUGUUGACC-X-CCAGUUGUCAA-5′
5′-CAACGACCUGU-X-UGUCCAGCAAC-5′
5′-AGCACAACUGU-X-UGUCAACACGA-5′
5’-UGCUGAGUGUU-X-UUGUGAGUCGU-5’
5’-AGUGUUUUCUG-X-GUCUUUUGUGA-5’
5’-UGCUGCUUCUGX2UGCUGCUUCUG-3’
5’-UGCUGCUUCUGX2UGCUGAGUGUU-3’
5’-AGUGUUUUCUGX2UGCUGCUUCUG-3’
5’-CAACGAACCCU-X-UCCCAAGCAAC-5’
5’-G1UCCUUG1CUUG1-X8-G1UUCG1UUCCUG1-5’
5’-G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1-5’
5’-UUCUG1CUUCUG1-X8-G1UCUUCG1UCUU-5’
5’-UUCUG1CUUCUG1-X-G1UCUUCG1UCUU-5’
5’-X2UUCUG1CUUCUG1-X-G1UCUUCG1UCUUX2-5’
5’-X2G1UCCUUG1CUUG1-X-G1UUCG1UUCCUG1X2-5’
5’-UG1CUG1CCUUUG1-X-G1UUUCCG1UCG1U-5’
5’-UG1CUG1CCUUUG1-X9-G1UUUCCG1UCG1U-5’
5’-G1UCCUUUG1CUG1-X-G1UCG1UUUCCUG1-5’
5’-G1UCCUUUG1CUG1-X9-G1UCG1UUUCCUG1-5’
5’-X6UGCUGCUUGUG-X-GUGUUCGUCGUX6-5’
5’-X5GCUGCCUUUG-X9-GUUUCCGUCGUX5-5’
(5′-UGCUGCUUGUG)2-X-CCGUUGACAG-3′
(5′-UGCUGCUUGUG)2-X-ACACGCGUGU-3′
5′-CCCIIICCCII-X-IICCCIIICCC-5′
5′-TTTTTTTTTTTTTTT-X-(G1UCUUCG1UCG1U)2-5′
3′-TTTTTTTTTTTTTTT-X-(G1UCUUCG1UCG1U)2-5′
5′-U1U1U1U1U1U1U1U1U1U1U1U1U1U1U1-X-(G1UCUUCG1UCG1U)2-5′
5’-CCIICCIICCC-X-CCCIICCIICC-5’
5’-CCIICCIICCX2CCIICCIICC-3’
其中I为肌苷;U1为dU;T1为核糖T;G1为7-脱氮-G;G2为ara-G;G3为6-硫代-G;G4为1-(β-D-呋喃核糖基)-2-氧代-7-脱氮-8-甲基嘌呤;O为洛索立宾;X为甘油;X1为1,3,5-戊三醇;X2为C3接头或丙二醇;X3为C3接头或丙二醇;X4为三(乙二醇);X5为1,5戊二醇;X6为2’-脱氧-无碱基;X7为C3氨基接头;X8为顺,顺-环己烷三醇;X9为顺,反-环己烷三醇;下划线的为2’-O-Me-核糖核苷酸;且P为硫代磷酸酯。
13.一种药物组合物,其包含根据权利要求1-12中任意一项的激动剂和可药用载体。
14.根据权利要求1-12中任一项的激动剂在制备用于脊椎动物中产生免疫应答的药物中的用途。
15.根据权利要求1-12中任一项的激动剂在制备用于治疗性处理患有癌症、自身免疫病症、炎性病症、皮肤病症、变态反应、哮喘或病原体引起的疾病的脊椎动物的药物中的用途。
16.根据权利要求15的用途,其中所述炎性病症是气道炎症。
17.根据权利要求15的用途,其中所述病原体引起的疾病是感染性疾病。
18.根据权利要求15的用途,其中所述药物是与一种或多种化疗化合物联用的药物。
19.根据权利要求15的用途,其中所述药物是与靶向治疗剂联用的药物。
20.根据权利要求15的用途,其中所述药物是与抗体联用的药物。
21.根据权利要求15的用途,其中所述药物是与DNA疫苗联用的药物。
22.根据权利要求15的用途,其中所述药物是与疫苗联用的药物。
23.根据权利要求15的用途,其中所述药物是与抗原联用的药物。
24.根据权利要求1-12中任一项的激动剂在制备用于预防脊椎动物中的癌症、自身免疫病症、炎性病症、皮肤病症、变态反应、哮喘或病原体引起的疾病的药物中的用途。
25.根据权利要求24的用途,其中所述炎性病症是气道炎症。
26.根据权利要求24的用途,其中所述病原体引起的疾病是感染性疾病。
27.根据权利要求24的用途,其中所述药物是与一种或多种化疗化合物联用的药物。
28.根据权利要求24的用途,其中所述药物是与靶向治疗剂联用的药物。
29.根据权利要求24的用途,其中所述药物是与抗体联用的药物。
30.根据权利要求1-12中任一项的激动剂在制备用于在脊椎动物中产生免疫应答的药物中的用途。
31.根据权利要求1的激动剂,其中所述寡聚核糖核苷酸各自独立的长度为8至17个核糖核苷酸。
32.根据权利要求1的激动剂,其中所述寡聚核糖核苷酸的序列是相同的。
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- 2007-04-06 WO PCT/US2007/008739 patent/WO2007117686A2/en active Application Filing
- 2007-04-06 MX MX2008012993A patent/MX2008012993A/es active IP Right Grant
- 2007-04-06 KR KR1020087027357A patent/KR101221589B1/ko not_active IP Right Cessation
- 2007-04-06 CA CA2648585A patent/CA2648585C/en active Active
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- 2007-04-06 ES ES07755117.4T patent/ES2564303T3/es active Active
- 2007-04-06 AU AU2007235231A patent/AU2007235231B2/en not_active Ceased
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Also Published As
Publication number | Publication date |
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US20080171712A1 (en) | 2008-07-17 |
US20130202584A1 (en) | 2013-08-08 |
JP2014031373A (ja) | 2014-02-20 |
US20140308300A1 (en) | 2014-10-16 |
US9243050B2 (en) | 2016-01-26 |
CA2648585A1 (en) | 2007-10-18 |
AU2007235231A1 (en) | 2007-10-18 |
AU2007235231B2 (en) | 2012-04-12 |
CA2648585C (en) | 2017-07-25 |
ES2564303T3 (es) | 2016-03-21 |
EP2021008A2 (en) | 2009-02-11 |
JP5761911B2 (ja) | 2015-08-12 |
MX2008012993A (es) | 2008-12-18 |
KR20090029187A (ko) | 2009-03-20 |
KR101221589B1 (ko) | 2013-01-15 |
HK1127727A1 (zh) | 2009-10-02 |
WO2007117686A3 (en) | 2008-06-26 |
JP5669897B2 (ja) | 2015-02-18 |
EP2021008B1 (en) | 2015-12-02 |
JP2009533348A (ja) | 2009-09-17 |
US8106173B2 (en) | 2012-01-31 |
EP2021008A4 (en) | 2010-10-13 |
US8759310B2 (en) | 2014-06-24 |
CN101460178A (zh) | 2009-06-17 |
KR20120115412A (ko) | 2012-10-17 |
WO2007117686A2 (en) | 2007-10-18 |
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