CN107384930B - 非编码免疫调节dna构建体 - Google Patents
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- CN107384930B CN107384930B CN201710517071.2A CN201710517071A CN107384930B CN 107384930 B CN107384930 B CN 107384930B CN 201710517071 A CN201710517071 A CN 201710517071A CN 107384930 B CN107384930 B CN 107384930B
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Abstract
本发明涉及核酸分子及其在调节免疫系统中的用途。提供了用于免疫调节的DNA构建体,其包含至少一个L‑构象的核苷酸。
Description
本发明是申请日为2011年12月23日,申请号为201180067799.0,标题为“非编码免疫调节DNA构建体”的专利申请的分案申请。
发明领域
本发明涉及核酸分子及其在调节免疫系统中的用途。
发明背景
新出现的一种对抗复杂疾病例如癌症、传染病、过敏反应和哮喘的策略是利用患者的免疫系统。已知免疫系统或其活性可以被特定的DNA序列调节。据Krieg等人报道(Nature 1995 374:6522 546-549),大部分已知的免疫修饰短DNA序列含有未甲基化的胞嘧啶鸟嘌呤基序(CG基序)。与原核生物或病毒相比,在真核生物的基因组中未甲基化的CG基序的出现基本上被抑制。因此,含有该基序的DNA分子已经进化为天然的“危险信号”并且引起免疫系统对原核病原体或病毒病原体的对抗。这可以在治疗上或在预防上用来治疗或预防传染病以及非传染病。
含有未甲基化CG基序的DNA构建体能够通过强烈刺激先天免疫系统的效应细胞从而引发相当的生理效应,所述效应细胞包括树突细胞、巨噬细胞、天然杀伤细胞(NK)和NKT细胞。未甲基化CG基序由先天免疫模式识别受体Toll样受体(TLR)9检测。尽管仍未完全了解其确切的识别机制,在阐明其通路上已经取得了显著进展(A.Krieg,Nat.Rev.DrugDisc.,5:471-484,2006)。假设在含有未甲基化CG的DNA构建体与受体结合后,应答细胞中多重信号级联被激活。通过特征表面分子的上调和细胞因子的分泌,诱导以Th1模式为主的适应性免疫。这样的构建体可以与例如抗体、化学疗法或放射疗法、疫苗或细胞因子组合使用。变应性疾病和哮喘主要是Th2介导的。通过增加Th1/Th2的比例,减弱Th2介导的应答,从而可以治疗或预防这些类型的疾病。
取决于所分析的特定细胞类型,表面分子包括,例如,CD40、CD69、CD80或CD86。不同细胞类型的细胞因子分泌也是特征性的;细胞因子包括,例如,巨噬细胞炎性蛋白质(MIP)-1α、MIP-1β、白细胞介素(IL)-6、IL-8、干扰素(IFN)-α、肿瘤坏死因子(TNF)-α、IFN-γ、单核细胞趋化蛋白(MCP)-1或IFN-γ诱导的10kDa蛋白(IP-10)。
为了预防或治疗疾病,已经证明疫苗接种是非常有效的方法。为了确保强烈而持续的免疫应答,能够刺激抗原呈递细胞例如树突细胞的佐剂通常和抗原一起施用,并且在这个用途上TLR9激动剂已经被证明是有效的免疫刺激剂。
不依赖于任何对未甲基化CG基序影响或调节免疫应答的机制的解释,已经开发了许多方法通过使用这样的基序调节免疫系统。WO 1998/018810公开了当含有未甲基化CG基序的免疫刺激性序列是单链的一部分时,其甚至更有效。然而,由于单链核酸的快速降解,施用开链的单链DNA分子是不可行的。因此,开发了用于保护含有未甲基化CG基序的单链或双链DNA构建体的不同方法。
为了实现对DNA核酸酶降解的抗性,核酸聚合物主链中的磷酸二酯键经常被修饰为硫代磷酸酯。这样的由硫代磷酸酯保护的核酸除了其刺激性活性稍微减少之外,最近几年的临床试验还显示硫代磷酸酯保护的毒性排除或严重限制了这样的核酸在药物组合物或药剂中的任何应用。
另一种保护含有CG基序的DNA序列的方法公开于例如EP 1 196 178。该文件公开了短的脱氧核糖核酸分子,其包含部分单链的、哑铃状的、共价闭合的包含CG基序的核苷酸残基序列(“dSLIM”)。根据EP 1 196 178的公开内容,CG基序位于所公开的分子的双链茎两端处的单链环内或位于所述双链茎内。单链发夹环保护双链茎免受细胞内外的DNA核酸酶的降解。
文件WO 2010/039137公开了免疫调节性寡核苷酸作为TLR介导的疾病的拮抗物,其在免疫刺激性基序的侧翼序列中和/或在寡核苷酸基序中具有一或多个化学修饰,所述寡核苷酸基序在无所述修饰时是免疫刺激性的。因此,WO 2010/039137所公开的寡核苷酸旨在抑制由TLR导致的免疫应答。
WO 2005/042018描述了新的所谓的C-型CpG寡核苷酸,其中c-型寡核苷酸由CpG序列以及富含GC的回文基序表征,所述CpG序列通常位于所述分子的5’或3’末端或其附近,所述富含GC的回文基序通常位于所述分子的另一末端或其附近。该文件公开了c-型DNA的回文序列的变体。
发明简述
针对本领域的状态,本公开的目的在于提供另一种免疫调节DNA构建体,其在转移入真核细胞后是稳定的且避免了有害的副作用。
本公开教导了用于免疫刺激的DNA构建体,其包含至少一个序列基序N1N2CGN3N4,其中N1N2和N3N4是C、G、A和T的任意组合,且C是脱氧胞苷,G是脱氧鸟苷,A是脱氧腺苷,T是脱氧胸苷,且其中所述构建体是包含至少一个L-构象核苷酸的线性单链或双链DNA序列。N1N2可以是选自包含GT、GG、GA、AT或AA的组的元件,N3N4是选自包含CT或TT的组的元件。
作为本公开的进一步的实施方式,提供了构建体,其中至少一个L-构象的核苷酸被包含在位于DNA单链5’-和/或3’-末端或其附近的末端5个核苷酸内。
本发明还提供了包括至少一个具有至少三个连续的脱氧鸟苷的G延伸(stretch)的DNA构建体,所述G延伸位于5’-和/或3’-末端附近,其中该G延伸可以位于两个根据权利要求1或2的序列基序之间。
根据权利要求1或2的两个序列基序之间的间隔可以是至少5个碱基,特别是当所述序列的元件没有脱氧鸟苷时。
进一步预期所述DNA序列是包含单链或双链DNA的线性开链DNA构建体,或是包含至少一个具有单链环的末端的线性DNA构建体。
上文定义的序列基序N1N2CGN3N4应位于所述DNA序列的单链和/或双链区域内。
作为进一步的实施方式,所述构建体包含分子间和/或分子内碱基对以及至少一个未配对的、单链的区域。
此外,提供了多聚体构建体,其中至少两个构建体相互连接,所述构建体包含分子间和/或分子内碱基对以及至少一个未配对的、单链的区域。
此外,所述构建体可以包含至少一个L-或D-构象的核苷酸,其被官能团修饰,所述官能团选自包含羧基、胺基、酰胺基、醛亚胺基、缩酮基、乙缩醛基、酯基、醚基、二硫基、硫醇基和醛基的组。
所述修饰的核苷酸可以连接至化合物,所述化合物选自包含肽、蛋白质、碳水化合物、抗体、合成分子、聚合物、微弹(micro projectile)、金属颗粒、纳米颗粒、微团、脂质载体、或固相的组。
本公开提供了DNA构建体,其具有在5’末端的第一G延伸和三个根据权利要求1或2的序列基序,其中至少5个碱基位于第一和第二基序之间,脱氧鸟苷除外,以及具有位于第二和第三序列基序之间的G延伸,且其中三个3’末端脱氧核苷酸中的两个是L-构象。
本公开的构建体可以用于治疗癌症或自身免疫病或用于调节免疫系统。
作为本公开的进一步的实施方式,提供了药物组合物,其包含上述的DNA构建体。所述药物组合物还可以包含化疗剂。
此外,提供了疫苗,其包含上述的DNA构建体。其中,可以包含所述DNA构建体作为佐剂。
发明详述
在本公开的含义中,线性开链DNA序列被指定为DNA构建体。所述DNA序列可以是单链的或是部分或完全双链的。术语DNA构建体不代表对相应DNA序列的长度限制。DNA构建体的单体单元是核苷酸。
DNA构建体可以合成制备或是部分或完全生物来源,其中生物来源包括基于遗传学方法制备DNA序列。
L-DNA或L-构象的核苷酸指的是核苷酸,其包含L-脱氧核糖而非天然存在的D-脱氧核糖作为糖残基。L-脱氧核糖是D-脱氧核糖的对映体(镜像)。部分或完全由L-构象核苷酸组成的DNA构建体可以是部分或完全单链或双链的;然而,L-构象的核苷酸不能与D-构象的核苷酸杂交(Hauser et al.,Nucleic Acid Res.2006 34:5101-11)。L-DNA与D-DNA同等地可溶和有选择性的。然而,L-DNA对天然存在的酶尤其是外切核酸酶的降解具有抗性,因此L-DNA被保护免受生物学降解(Urata et al.,Nucleic Acids Res.1992 20:3325-32)。因此,L-DNA可非常广泛地应用。
根据本公开,“茎”应当理解为DNA双链,其通过同一DNA分子内(其然后部分自我互补)或不同DNA分子内(其部分或完全互补)的碱基配对形成。分子内碱基配对指的是在同一分子内的碱基配对,而不同DNA分子之间的碱基配对称作分子间碱基配对。
在本公开的含义中,“环”应当理解为在茎结构内部或其末端的未配对的、单链的区域。“发夹”是茎和环的不同组合,其在同一DNA分子两个自我互补的区域杂交形成具有未配对环的茎时出现。哑铃状描述在茎区域侧翼两端都具有发夹的线性DNA构建体。因此,本公开上下文中的“线性DNA构建体”描述包含单链或双链DNA的线性开链DNA构建体或在双链DNA茎两端包含单链环的线性哑铃状DNA构建体。
术语“DNA末端”,无论其意指DNA单链的5’-或3’末端,指的都不仅仅是末端的核苷酸,也包含末端的5个核苷酸或甚至对各DNA末端来说的最后三个核苷酸。DNA末端的修饰涉及各核苷酸中的至少一个。
在本公开的含义中“G延伸”应理解为具有至少三个连续脱氧鸟苷的序列。
核苷酸共价或非共价连接的“固相”指的是,但不限于,柱、基质、珠、玻璃(包括修饰的或功能化的玻璃)、二氧化硅或硅基材料(包括硅和修饰的硅)、塑料(包括聚丙烯、聚乙烯、聚苯乙烯和苯乙烯与其它材料的共聚物、丙烯酸酯、聚丁烯、聚氨基甲酸酯等)、尼龙或硝化纤维素、树脂、多糖、碳以及无机玻璃、金属、纳米颗粒、和塑料。因此,微孔板也在本公开的固相的范围内。
本公开的免疫调节指的是免疫刺激和免疫抑制。免疫刺激优选地指的是刺激免疫系统的效应细胞以便其增殖、迁移、分化或以任何其它方式活化。例如无共刺激信号时可以通过免疫刺激性DNA分子诱导B细胞增殖,而所述B细胞增殖通常需要来自辅助T细胞的共刺激信号。在另一方面,免疫抑制应当理解为降低免疫系统的活化或效力。通常故意诱导免疫抑制以便预防例如移植器官的排斥,治疗骨髓移植后的移植物抗宿主病,或用于治疗自身免疫病,例如类风湿关节炎或克罗恩氏病(Crohn’s disease)。
在这个背景下,免疫调节还可以指对免疫反应本质或特性的影响,其通过影响仍在发育或成熟的免疫反应或通过调节已经建立的免疫反应的特性。
本公开所用的术语“疫苗接种”指的是施用抗原性材料(疫苗)以产生对疾病的免疫。疫苗可以预防或改善许多病原体(例如病毒、真菌、原生动物寄生虫、细菌)感染的作用,以及预防或改善变应性疾病和哮喘以及肿瘤的作用。疫苗通常含有用于加强免疫应答的一或多种佐剂,例如,免疫刺激性核酸。通常认为疫苗接种是最有效且经济的预防传染病和其它疾病的方法。
所施用的材料可以,例如,是病原体(细菌或病毒)活的但弱化的形式、所述病原体的死亡的或灭活的形式、纯化的材料如蛋白质、编码抗原的核酸、或细胞例如肿瘤细胞或树突细胞。特别是,最近已经开发了DNA疫苗接种。DNA疫苗接种通过将编码抗原的DNA插入人或动物细胞(且表达,引起免疫系统识别)而生效。免疫系统中一些识别所述表达蛋白的细胞将发动对这些蛋白和表达这些蛋白的细胞的攻击。DNA疫苗的一个优势是其非常易于生产和储存。此外,相对于常规疫苗,DNA疫苗具有多种优势,包括诱导更宽范围的免疫应答类型的能力。
疫苗接种可以用作预防性方法,在疫苗接种的健康个体暴露于抗原后产生针对所述抗原的免疫性。或者,治疗性疫苗接种通过将经疫苗接种的患病个体的免疫系统导向抗原,可以导致所述免疫系统应答的加强。预防性和治疗性疫苗接种均可以应用于人类以及动物。
本公开所用术语“基因治疗”指的是对个体的细胞和/或生物学组织的暂时的或永久的遗传修饰(如基因的插入、替换或移除)以治疗疾病,例如肿瘤或自身免疫病。基因治疗最常见的形式涉及将功能基因插入到非特定的基因组位置以置换突变的基因,而其他的基因治疗形式涉及直接校正突变或修饰使病毒能够感染的正常基因或甚至将基因或基因片段转移进细胞内使其转录。
“自体基因治疗”指的是使用同一个体的组织或细胞。通过基因治疗修饰分离的细胞或组织并将其重新导入供体中。相反,“异体(allogenic)基因治疗”指的是使用来自受体之外的个体的细胞用于基因治疗。在遗传修饰后,所述异体细胞被导入到受体中。
术语“离体基因治疗”指的是这样的治疗方法,其中来自个体的细胞,如造血干细胞或造血祖细胞,离体进行遗传学修饰并随后被导入到待治疗的个体中。术语“体内基因治疗”指的是这样的治疗方法,其中来自个体的细胞,如造血干细胞或造血祖细胞,使用例如病毒载体或其它表达构建体在体内进行遗传学修饰。
基因治疗也可以被分为“生殖系基因治疗”和“体细胞基因治疗”。在“生殖系基因治疗”中,生殖细胞,即精子或卵子,被遗传学修饰。所述遗传变化通常整合至它们的基因组。因此,治疗引起的改变将是可遗传的且将传递至后代。该方法对于治疗遗传紊乱和遗传性疾病是有用的。在“体细胞基因治疗”中,将治疗性基因转移进入个体的体细胞中。任何修饰和作用将仅限于所述个体,且不会被该个体的子代和后代遗传。
术语“癌症”包括被治疗或预防的癌性疾病或肿瘤,其选自包括但不限于乳腺癌、黑色素瘤、皮肤肿瘤、淋巴瘤、白血病、胃肠肿瘤(包括结肠癌(colon carcinomas)、胃癌、胰腺癌、结肠癌症(colon cancer)、小肠癌)、卵巢癌、宫颈癌、肺癌、前列腺癌、肾细胞癌和/或肝转移的组。
本公开的自身免疫病包括类风湿关节炎、克罗恩氏病、全身性红斑狼疮(SLE)、自体免疫甲状腺炎、自身免疫性甲状腺炎、多发性硬化、格雷夫斯病(Graves’disease)、重症肌无力、乳糜泻和阿狄森氏病(Addison’s disease)。
本公开提供了包含至少一个CG基序和至少一个L-构象的核苷酸的线性开链DNA序列。由于部分/全部L-构象,所述DNA不能作为天然存在的D-构象特异性DNA降解酶的底物。由此,无需使用被证明具有毒性的硫代磷酸酯主链,而可以保护本发明的DNA构建体免受酶促降解。此外,所述DNA构建体仅仅由最小数目的核苷酸组成,这使得所述DNA构建体小且因此显著改善其被患者细胞的吸收。
含CG的DNA构建体的效果取决于其与TLR9的相互作用,而DNA-蛋白质相互作用取决于DNA和蛋白质两者的构象。由于单个分子的手性对于获得的聚合物的构象是决定性的,因此未知部分或全部L-构象的DNA分子是否能够结合并激活TLR9。实验数据证明这如此保护的DNA分子出人意料地适用于诱导免疫应答。如实施例和附图所示,单个核苷酸手性的至少部分改变仍然明显地允许结合并激活TLR9。因此,具有CG基序和L-构象核苷酸的DNA分子可以用于免疫调节。
令人惊奇的是,如在附图中可见,即使采用相同的核苷酸序列时,所诱导的刺激模式不同于EP 1 196 178(公开了哑铃状分子(“dSLIM”),其包含在所述分子两端处的单链环中或在双链茎中的CG基序)所公开的分子诱导的刺激模式。
所述DNA序列可以是单链的或是部分或完全双链的。这包括在相同分子内的(分子内)或在不同分子内的(分子间)或其任意组合的碱基配对。也可能所述构建体包含至少一个未配对的、单链的区域。作为进一步的实施方式,包括发夹结构。由于部分或全部L-构象,确保了所述构建体的更长的半衰期,原因是L-构象核苷酸不被降解。
也预期单链的或是部分或全部双链的至少两个分子可以相互连接从而形成多聚体构建体。因此所述多聚体构建体中并入至少和连接配偶体一样多的CG基序,其紧密地包装在一个分子中,并因此预期该多聚体构建体引发显著的免疫应答。获得的单链的或是部分或全部双链的多聚体构建体可以是共价闭合的,在其分子内包含L-构象核苷酸,或者是开放的多聚体构建体,其在5’-和/或3’-末端处或其附近包含L-构象核苷酸以保护其免受酶促降解。
根据本公开,所述一个或多个CG基序位于所述构建体的单链和/或双链区域内。正如EP 1 196 178中所公开的,CG基序无论是包含在所述分子的单链区域内还是双链区域内,其都能够引发免疫应答。
本公开还包括用官能团对至少一个L-或D-构象的核苷酸的化学修饰,所述官能团选自包含羧基、胺基、酰胺基、醛亚胺基、缩酮基、乙缩醛基、酯基、醚基、二硫基、硫醇基和醛基的组。这使得所述DNA构建体能通过例如吸附、共价键接或离子键接偶联至化合物,所述化合物选自包含肽、蛋白质、脂质、泡囊、微团、碳水化合物、抗体、合成分子、聚合物、微弹、金属颗粒、纳米颗粒或固相的组。根据各自的目的可以具体地选择所述修饰。所述构建体因此可以用于,例如,将其他分子运输至应答于所加入的CG基序的特定细胞。此外,通过这样的修饰有可能将所述构建体偶联至微弹,其可用于将所述构建体转移进入细胞。所述构建体也可以偶联至固相,例如微孔板。
偏向Th1的活化涉及NK细胞和细胞毒性T细胞的活化,而这些免疫应答可以用于癌症治疗。因为含有未甲基化CG基序的DNA构建体优选地导致Th1活化,因此本公开所述构建体可以用于治疗癌症。正在进行多项涉及将TLR9激动剂用于治疗癌症的临床试验。这样的分子已经有效地单独施用或与例如放射疗法、手术、化学疗法和冷冻疗法联合施用(Krieg,J.Clin.Invest.2007 117:1184-94)。由于本公开所述构建体的有效的免疫调节、小尺寸和稳定性,从这方面看能预期其是非常有优势的。此外,它们独特的免疫学谱将其与它优势较少的TLR9配体区分开,且该谱可用于癌症特异性治疗。
在另一方面,TLR9激动剂也参与调节性T细胞的产生并因此可以用于治疗自身免疫病。施用途径看起来是决定含有CG基序的DNA构建体的体内效果的一个变量(Krieg,J.Clin.Invest.2007 117:1184-94)。
已经证明这样的含有CG基序的DNA分子的免疫刺激作用能改善癌症治疗中标准治疗方法例如化学疗法的效力。因此,也提供了包含本公开所述构建体的药物组合物。再者,与现有技术中的TLR9激动剂相比,本公开所述构建体的优越性能使得该构建体成为治疗例如癌症、传染病、过敏反应和哮喘的疾病的有前景的工具。过敏反应和哮喘(主要是Th2-介导的)的治疗因此得益于对Th1活化的偏好性。
由于TLR9激动剂已经被证明是有效的疫苗佐剂,因此也提供了包含本公开所述DNA构建体的疫苗。本公开所述构建体仅包含用于TLR9刺激的相关序列且由于其L-核苷酸修饰而是稳定的。因此,可以避免由于无关序列而导致的副作用。所述分子较长的半衰期确保了高效的刺激,并因此预期强的免疫应答。
使用合成柱和各核苷酸(β-L-脱氧“NT”(n-bz)CED亚磷酰胺;“NT”代表腺苷、胞苷、鸟苷或胸苷)制备本公开所述DNA分子。所述DNA分子随后通过HPLC纯化。
为了揭示使用L-核糖而非D-核糖的DNA的效果,以下DNA分子用于此处描述的最初实验(表1)。
表1:非编码免疫调节DNA构建体和对照的序列。
使用表1所述序列的实验显示含有CG基序的L-核糖保护的线性序列能够刺激免疫系统且其诱导的免疫应答明显不同于EP 1 196 178公开的dSLIM诱导的免疫应答。因此,命名为ODN2216(GGGGGACGATCGTCGGGGGG;SEQ ID 6)的经修饰的序列和其修饰被用于研究结构差异的影响,如G-延伸的存在、长度和位置,CG基序间的间隔以及L-核糖核苷酸和CG基序、G-延伸之间的距离的各自影响。
表2总结了所使用的序列,以及与ODN2216相比(前两个和最后6个核苷酸用硫代磷酸酯修饰),它们对IFN-α和IP-10分泌的作用,其中粗体字母表示包含l-核糖的核苷酸,斜体字母指的是G-延伸而下划线字母指得是CG-基序。破折号将各序列置于合适位置以与CKm508相比较,但其不代表所述序列结构上或功能上的修饰。
表2:与ODN2216(ODN2216前两个和最后6个核苷酸用硫代磷酸酯修饰)相比,所指示的序列对IFN-α和IP-10分泌的作用。
用具有至少一个G延伸(特别是在3'末端处或其附近)的序列获得了好的结果。刺激还依赖于CG-基序的存在(CKm477),再次表明了所述刺激不是l-核糖而是CG基序的作用。
用ODN 2216修饰的序列获得的结果鉴定出了有利的结构组分,其被转移至CKm374的DNA序列。表3显示了将所述修饰的序列与EP 1 196 178公开的哑铃状dSLIM相比较的结果。同样地,粗体字母代表包含l-核糖的核苷酸,斜体字母指的是G-延伸,双下划线字母代表硫代磷酸酯修饰的核苷酸而下划线字母指的是GC-基序。
从表3的结果可获知,直接位于5’末端的G延伸看起来是有利的(对比CKm532和CKm499)。此外,在5’末端处使用4个而非3个脱氧鸟苷进一步增加了其对IFN-α和IP-10的刺激(对比CKm501和CKm532)。
在GC-基序之间添加额外的G-延伸看起来也是有益的(对比CKm532和CKm520)。第一个和第二个G-延伸之间的距离进一步影响所述DNA分子的效力。此外,仅在3'末端处或其附近使用包含l-核糖的脱氧核苷酸看起来赋予了所述DNA分子足够的稳定化程度。可以观察到预期的对IFN-α和IP-10的良好的刺激(见下文)。因为IL-8已经被证明是诱导新生血管发生,所以仅仅小量诱导IL-8分泌看起来是有益的。
明显地,G延伸的存在和小心选择的位置与含有l-核糖的脱氧核苷酸的稳定作用的组合使得可以制备优于dSLIM分子的刺激效率的DNA分子。
表3:与dSLIM相比,所指示的序列对IFN-α、IP-10和IL-8分泌的作用。
附图简述
本公开将通过实施例和附图进一步被阐明,但不被限于所公开的实施方式。其显示:
图1 酶消化后DNA构建体的琼脂糖凝胶电泳
图2 小鼠巨噬细胞细胞系刺激后的GFP强度。
图3 类浆细胞树突细胞(PDC)刺激后的MIP-1α浓度。
图4 PDC刺激后的MIP-1β浓度。
图5 PDC刺激后的IL-8浓度。
图6 PDC刺激后的IL-6浓度。
图7 PDC刺激后的IFN-α浓度。
图8 PDC刺激后的TNF-α浓度。
图9 外周血单个核细胞(PBMC)刺激后的MCP-1和IL-8浓度。
图10 PBMC刺激后活化的T细胞的频率。
图11,12 PBMC的IFN-α、IP-10和IL-8分泌。
图13 l-核糖修饰的末端脱氧核苷酸对ELAM9细胞刺激的作用。
图14 与未刺激状态相比,由CKm532和dSLIM对B细胞和PDC的免疫刺激。
附图详述
图1显示全部DNA构建体由来自T7噬菌体的T7-聚合酶消化后的凝胶。6μg的各DNA构建体与10单位的T7-聚合酶孵育(总反应体积:20μl)。经过0、1、2、5、30和1500分钟后,从样品中取出3μl的孵育混合物并用5μl含甲酰胺的Sanger染料稀释。所有的等分试样加样至3%的琼脂糖凝胶,并在100伏运行40分钟。
和T7-聚合酶孵育5分钟后,发现未修饰的DNA分子lin-30L2(泳带2)被完全消化,然而本发明所述构建体(CKm337;泳带3)以及dSLIM(泳带1)和硫代磷酸酯修饰的构建体CKm338(泳带4)和CKm339(泳带5)甚至在1500分钟的孵育后仍保持显著的存在。事实上,在全部测试的分子中,CKm337显示了最高的稳定性。由于其不足的稳定性,在进一步的研究中排除了Lin 30L2。
图2显示用不同的刺激性DNA构建体对ELAM9细胞的刺激。ELAM9细胞是TLR9-阳性鼠巨噬细胞(RAW264),其被人弹性蛋白启动子(hELAM)控制下的d1-eGFP稳定转染,所述人弹性蛋白启动子含有若干NFκB应答元件。在所述细胞接种1天后,其用所示DNA构建体(3μM)刺激7小时。通过流式细胞术测量GFP强度的几何平均值。
除了最后的T以外其他全部核苷酸为L-构象的DNA构建体(CKm336)没有刺激能力。然而,两端处均具有L-构象核苷酸的DNA构建体(CKm337)的确刺激了GFP表达。这是相当出人意料的,因为含有具有L-构象核苷酸的CG基序的DNA构建体是否能够结合并激活TLR9是未知的。此外,预期CKm337比dSLIM(EP 1 196 178公开的分子)更容易被细胞摄取,且比硫代磷酸酯修饰的构建体(CKm338和CKm339)具有更低的毒性。
图3至图8显示DNA构建体对pDC分泌炎症趋化因子和细胞因子的作用。使用来自Miltenyi,Diamond PDC试剂盒的组合分选方法从Ficoll纯化的PBMC中富集pDC:首先,使用来自Miltenyi’s试剂盒的pDC生物素-抗体混合物(Cocktail)从PBMC去除非-pDC,然后使用来自PDC Diamond试剂盒的CD304(BDCA-4)Diamond微球从细胞中正筛选pDC。将2.5x105/ml的PDC和10ng/ml的IL-3接种在培养基中(RPMI1640、10%胎牛血清、100单位/ml青霉素、100单位/ml链霉素、2mM谷氨酰胺,37℃,5%CO2),并用3μM的各构建体刺激2天。
为了测定细胞刺激后分泌的化合物的量,收集经刺激的细胞的澄清上清液并使用多重系统(来自eBioscience/Bender MedSystems的FlowCytomix)或ELISA分析。
出乎意料地,与使用dSLIM刺激相比,使用CKm337刺激的pDC显示出了对MIP-1α、-1β和IL-8分泌的类似的作用。使用lin CKm338和CKm339刺激后MIP-1α、-1β和IL-8分泌稍微提高(图3、4和5)。然而,全部的硫代磷酸酯修饰的构建体继承了上文所述的若干缺点。
关于IL-6分泌,dSLIM、CKm337和CKm338对pDC具有类似作用。CKm339稍微更有效(图6)。
值得注意的是,与全部其它线性构建体相比,CKm337对pDC的IFN-α分泌具有出乎意料地更强的作用(图7)。
dSLIM、CKm337、CKm338和CKm339全部对pDC的IFN-α分泌具有类似的作用(图8)。
通过Ficoll密度梯度从人血沉棕黄层分离PBMC。对于功能分析,通过以指定浓度(2-3μM)施加各化合物对培养基(RPMI1640、10%胎牛血清、100单位/ml青霉素、100单位/ml链霉素、2mM谷氨酰胺,37℃,5%CO2)中的106细胞/ml刺激2天。
图9(A和B)显示所示DNA构建体(各3μM)对PBMC分泌MCP-1和IL-8的作用。如根据使用pDC的实验所预期的,全部核苷酸为L-构象的DNA构建体(CKm336)施加至PBMC时没有刺激能力。然而,CKm337对激发MCP-1和IL-8两者的分泌是有效的。出乎意料地,与dSLIM相比,其对IL-8分泌的作用更强而对MCP-1分泌的作用更弱。
为了测定细胞亚群和其活化状态,使用选择性荧光团连接的抗体标记特征性表面标志物。以106细胞/染色组进行抗体染色;每组和多达4种不同的与荧光团连接的抗体一起孵育,最后重悬于400μl FACS缓冲液并通过流式细胞术分析至少100,000活细胞。用于测定T细胞和其中的活化细胞的门控策略是带有活化标志物CD69的CD3+/CD56-。
图10显示所示的DNA构建体(各2μM)对PBMC群体内活化T细胞频率的作用。全部5种构建体具有类似的刺激能力。T细胞不表达TLR9。因此,在用所述DNA构建体刺激后,PBMC群体内的细胞被活化,其进而能够活化T细胞。
序列的优化显示G-延伸的引入增强了寡核苷酸转染后的效力。所述效力还依赖于CG-基序之间的距离。通过使用在寡核苷酸3’-末端处的包含L-核糖的脱氧核苷酸,能够充分地保护线性DNA序列免受降解(比较表2和表3)。从表3可以得出,寡核苷酸CKm501(SEQ IDNO:33)、CKm527(SEQ ID NO:32)、CK 532(SEQ ID NO:31)和CKm534(SEQ ID NO:34)显示出意料之外的好结果。图11和12显示指定的DNA构建体对PBMC中分泌细胞因子IFN-α(上)、IP-10(中)和IL-8(下)的作用。实验操作如上文所述。
图11显示与哑铃状的dSLIM相比,CKm501和CKm527导致了IFN-α分泌水平升高且CKm527也增加了IP-10分泌。对于dSLIM,IL-8的分泌相当低,与CKm339相比更低,所述CKm339是哑铃状dSLIM的单链环的序列,其在两端用硫代磷酸酯修饰的脱氧核苷酸保护。
从图12可以得出,CKM532显示出显著且出人意料的对IFN-α和IP-10分泌的高诱导,但其显示出对IL-8分泌的相当低的诱导。因此,CKm532确认了结构元件G-延伸直接位于5’-末端处且有另一个G-延伸位于两个CG-基序(第二和第三GC-基序)之间,这看起来是有利的。表3中的CKm520和CKm532的比较,表明CKm532中G-延伸位于第二和第三CG-基序之间是IFN-α和IP-10分泌的预期增加的原因,而CKm520主要增加IL-8分泌。此外,仅用两个在3’末端处的包含L-核糖的脱氧核苷酸保护寡核苷酸似乎是足够的。
从图12中CKm532和CKm 534的比较可以得出,缩短5’-末端处的G-延伸导致效力降低。鉴于增加了的IFN-α和IP-10分泌和低的IL-8分泌,CKm532再次证明了所鉴别的结构组分的优势。
图13上部显示用所指示的DNA构建体刺激ELAM9细胞的结果,所述构建体包含具有不同程度L-核糖修饰的脱氧核苷酸。在图13下部的序列中,包含L-核糖的核苷酸以粗体字母表示。实验以一式两份进行(L-dSLIM032和L-dSLIM030)。
包含L-核糖的脱氧核苷酸的程度和位置对于ELAM9细胞的刺激有影响。完全L-构象的序列(CKm 336;SEQ ID NO:2)根本没有任何刺激作用,这与WO 2010/039137的公开一致。使用由在3’-和5’-末端处的包含L-核糖的脱氧核苷酸进行保护的包含CG-基序的寡核苷酸获得了好的效果,而在5’末端处的包含L-核糖的脱氧核苷酸的长延伸则适得其反(比较CKm489和CKm490)。此外,用包含L-核糖的脱氧核苷酸修饰CG-基序导致作用的丧失。因此,为了达到良好的刺激作用,CG-基序不应当包含L-核糖且在两端的L-核糖修饰的脱氧核苷酸的延长应当受到限制,即5’末端不超过8个末端脱氧核甘酸和最多最后一个CG-基序之后的3’末端脱氧核苷酸。
图14显示与未刺激状态相比,CKm532和dSLIM产生的免疫刺激。使用图10中所述实验应用的方案进行FACS实验并分别应用于B细胞(门控策略:CD19阳性,CD86作为活化标志物)和PDC(门控策略:谱系阴性,HLA-DR阴性,CD123阳性细胞,CD40和HLA-DR作为活化标志物)。显示的数据是基于对三种不同血沉棕黄层制备物的测量。
图14上显示通过标志物CD86证明的B细胞的刺激。显然,与dSLIM和未刺激状态相比,CKm532导致B细胞的刺激增加。这显示为B细胞成熟的增加,例如产抗体细胞,其是免疫刺激的重要特征。
图14下显示使用标志物HLA-DR检测的PDC的刺激。HLA-DR是MHC分子的一部分,也因此是免疫系统抗原呈递过程的一部分。与dSLIM或未刺激的细胞相比,CKm532再次显示了该免疫刺激特征的更强的增加。
总之,CKm337(D-DNA构建体,其两端具有L-构象的核苷酸)令人惊奇地对PBMC和分离的pDC两者均具有刺激作用,而全部核苷酸为L-构象的DNA构建体(CKm336)没有作用。显然,CKm337的构象仍然允许其与TLR9的结合,而CKm336空间上不能结合或刺激TLR9。
出乎意料地,相比于哑铃状的dSLIM和硫代磷酸酯修饰的寡核苷酸,由CKm337诱导的刺激模式与其它所有构建体相比都是独特的。CKm337诱导pDC分泌最高量的IFN-α。PBMC分泌IL-8弱于硫代磷酸酯修饰的分子,但强于dSLIM。相反,dSLIM诱导PBMC分泌更高量的MCP-1,但Ckm337与硫代磷酸酯修饰的分子相当。
通过在线性DNA分子的5’末端直接引入所谓的G-延伸使得可能增强用CKm337观察到的效果。此外,结果表明仅仅通过3’-末端处的包含L-核糖的脱氧核苷酸针对降解的保护对于稳定所述寡核苷酸来说是足够的。所鉴别的结构特征(G-延伸、CG-基序、CG-基序的间隔以及通过使用不同程度和位置的L-核糖修饰的脱氧核苷酸的保护)使得能够调节包含L-核糖的寡核苷酸的免疫刺激作用。看起来相当明显,本公开揭示了构建用于细胞或免疫系统靶向刺激的免疫调节DNA构建体的新工具。
多年来已知IFN-α为抗病毒细胞因子。其刺激Th1细胞发育,从而促进含有CG的DNA分子的作用。IFN-α还显示出在小鼠和人恶性肿瘤中的抗肿瘤活性并能够减少移植肿瘤细胞的致肿瘤性,其部分地是通过活化细胞毒性T细胞从而增加肿瘤细胞细胞溶解的可能性。IFN-α也增加NK细胞和巨噬细胞的活性,这两者对于抗肿瘤细胞毒性均是重要的(Brassardet al.,J.Leukoc.Biol.2002 71:565-81)。因此,用本公开的所述DNA构建体刺激后IFN-α量的增加预期对于治疗癌症是有益的。
最近已经证明IP-10是体内有效的抗血管生成(angiostatic)蛋白。因此,IP-10的诱导尤其在治疗肿瘤疾病中看起来也是有益的。
IL-8是促炎细胞因子,已知其能介导中性粒细胞活化以及从外周血向组织内的迁移。如已经在卵巢癌中被证明的,所获得的中性粒细胞浸润可能是抑制肿瘤生长的部分原因(Lee et al.,J.Immunol.2000 164:2769-75)。此外,IL-8还是对T细胞和嗜碱性粒细胞是趋化性的。因此,对于至少一些肿瘤类型的治疗或预防,含CG的DNA构建体引起的IL-8的选择性上调是有利的。在另一方面,已经明确IL-8引起血管发生,使得IL-8分泌的诱导可能适得其反。因此,通过本发明所述的不同的DNA分子对IL-8不同程度的诱导可能使得能够针对所需治疗效果改造所述分子。
已知MCP-1参与单核细胞/巨噬细胞募集至损伤和感染位点,并因此可能参与刺激宿主抗肿瘤应答。已经证明MCP-1可以活化单核细胞,使之在体外对几种人肿瘤细胞的细胞生长抑制更强(Zachariae et al.,J.Exp.Med.1990 171:2177-82)。因此,和IL-8类似,根据具体的肿瘤类型调节MCP-1的表达是有益的。
因此,诱导的特异性细胞因子对于治疗和预防不同的肿瘤类型是有益的。显然,未甲基化的CG-基序呈递给TLR9的具体状况决定了应答细胞中诱导的各自独特的刺激模式。
序列表
<110> 莫洛根股份公司
<120> 非编码免疫调节DNA构建体
<130> 80523WO
<160> 52
<170> PatentIn version 3.5
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<211> 30
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<213> Artificial Sequence
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<220>
<221> misc_feature
<223> Nucleotides 1-2 and 20-21: in L-conformation Nucleotides 3-6and 16-19: G-stretch Nucleotides 8-9, 12-13 and 15-16: CG-motif
<400> 12
ggggggacga tcgtcggggg gt 22
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm503
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 23-24: in L-conformation Nucleotides 3-7and 17-22: G-stretch Nucleotides 9-10, 13-14 and 16-17: CG-motif
<400> 13
aagggggacg atcgtcgggg ggaat 25
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm507
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 23-24: in L-conformation Nucleotides 3-7and 17-22: G-stretch Nucleotides 9-10, 13-14 and 16-17: CG-motif
<400> 14
ttgggggacg atcgtcgggg ggttt 25
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm459
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 3-5,15-16 and 18: G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 15
gggggacgat cgtcggaggg t 21
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm506
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 20-21: in L-conformation Nucleotides 3-5and 16-19: G-stretch Nucleotides 7-8, 11-12 and 15-16: CG-motif
<400> 16
gggggacgat cgtgcggggg gt 22
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm478
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 3-5and 15-16: G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 17
ggggggcgat cgtcggaagg t 21
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm480
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 3-5and 15-18: G-stretch Nucleotides 7-8 and 14-15: CG-motif
<400> 18
gggggacgat gctcgggggg t 21
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm462
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 3-5and 15-16: G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 19
gggggacgat cgtcggaagg t 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm505
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 3-5and 16-18 : G-stretch Nucleotides 7-8, 11-12 and 15-16: CG-motif
<400> 20
gggggacgat cgtgcggggg t 21
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm476
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 20-21: in L-conformation Nucleotides 3-5and 16-17 : G-stretch Nucleotides 8-9, 12-13 and 15-16: CG-motif
<400> 21
gggggaacga tcgtcggaag gt 22
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm464
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 15-18:G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 22
ggcccccgat cgtcgggggg t 21
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm476
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 20-21: in L-conformation Nucleotides 3-5and 16-17: G-stretch Nucleotides 8-9, 12-13 and 15-16: CG-motif
<400> 23
gggggaacga tcgtcggaag gt 22
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm460
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 4-5,15-16 and 18: G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 24
ggaggacgat cgtcggaggg t 21
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm475
<220>
<221> misc_feature
<223> Nucleotides 1-7 and 17-21: in L-conformation Nucleotides 15-16:G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 25
cccccccgat cgtcgggggg gt 22
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm477
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 20-21: in L-conformation Nucleotides 3-5and 16-19: G-stretch
<400> 26
gggggagcat gctgcggggg gt 22
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm463
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 5 and15-16: G-stretch Nucleotides 7-8, 11-12 and 14-15: CG-motif
<400> 27
ggaagacgat cgtcggaagg t 21
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm504
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 19-20: in L-conformation Nucleotides 3-5and 15-18: G-stretch Nucleotides 11-12 and 14-15: CG-motif
<400> 28
gggggagcat cgtcgggggg t 21
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm510
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 18-19: in L-conformation Nucleotides 3-5,14-15 and 17: G-stretch Nucleotides 6-7, 10-11 and 13-14: CG-motif
<400> 29
gggggcgatc gtcggagggt 20
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm509
<220>
<221> misc_feature
<223> Nucleotides 1-6 and 19-23: in L-conformation Nucleotides 3-5and 17-18: G-stretch Nucleotides 9-10, 13-14 and 16-17: CG-motif
<400> 30
ccccccctcg atcgtcgggg gggt 24
<210> 31
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm532
<220>
<221> misc_feature
<223> Nucleotides 32-33: in L-conformation Nucleotides 1-4 and 21-24:G-stretch Nucleotides 13-14, 20-21 and 25-26: CG-motif
<400> 31
ggggtcatta aacgttcttc ggggcgttct tttt 34
<210> 32
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm527
<220>
<221> misc_feature
<223> Nucleotides 33-34: in L-conformation Nucleotides 1-4, 21-24 and26-30: G-stretch Nucleotides 13-14, 20-21 and 25-26: CG-motif
<400> 32
ggggtcatta aacgttcttc ggggcggggg ttttt 35
<210> 33
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm501
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 1-3 and 20-23:G-stretch Nucleotides 12-13, 19-20 and 24-25: CG-motif
<400> 33
gggtcattaa acgttcttcg gggcgttctt ttt 33
<210> 34
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm534
<220>
<221> misc_feature
<223> Nucleotides 32-33: in L-conformation Nucleotides 1-3 and 21-24:G-stretch Nucleotides 13-14, 20-21 and 25-26: CG-motif
<400> 34
gggtcattaa aacgttcttc ggggcgttct tttt 34
<210> 35
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm520
<220>
<221> misc_feature
<223> Nucleotides 35-36: in L-conformation Nucleotides 1-4 and 28-32:G-stretch Nucleotides 13-14, 20-21 and 27-28: CG-motif
<400> 35
ggggtcatta aacgttcttc gttcttcggg ggttttt 37
<210> 36
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm535
<220>
<221> misc_feature
<223> Nucleotides 29-30: in L-conformation Nucleotides 17-20 and 22-26: G-stretch Nucleotides 9-10, 16-17 and 21-22: CG-motif
<400> 36
tcattaaacg ttcttcgggg cgggggtttt t 31
<210> 37
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm528
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 1-3 and 20-23:G-stretch Nucleotides 13-14, 19-20 and 24-25: CG-motif
<400> 37
gggtcattaa aacgttctcg gggcgttctt ttt 33
<210> 38
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm339
<220>
<221> misc_feature
<223> Nucleotides 6-7 and 20-23: G-stretch Nucleotides 12-13, 19-20and 24-25: CG-motif Nucleotides 1-2 and 29-30: phosphothioate bonds
<400> 38
tcattggaaa acgttcttcg gggcgttctt 30
<210> 39
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm498
<220>
<221> misc_feature
<223> Nucleotides 34-35: in L-conformation Nucleotides 6-7 and 27-33:G-stretch Nucleotides 12-13, 19-20 and 26-27: CG-motif
<400> 39
tcattggaaa acgttcttcg ttcttcgggg gggttt 36
<210> 40
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm536
<220>
<221> misc_feature
<223> Nucleotides 26-27: in L-conformation Nucleotides 1-3 and 16-19:G-stretch Nucleotides 8-9, 15-16 and 20-21: CG-motif
<400> 40
gggaaaacgt tcttcggggc gttctttt 28
<210> 41
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm499
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 6-8 and 20-23:G-stretch Nucleotides 12-13, 19-20 and 24-25: CG-motif
<400> 41
tcattgggaa acgttcttcg gggcgttctt ttt 33
<210> 42
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm533
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 1-3, 15-17 and20-23: G-stretch Nucleotides 12-13, 19-20 and 24-25: CG-motif
<400> 42
gggtcattaa acgtgggtcg gggcgttctt ttt 33
<210> 43
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm500
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 9-11 and 20-23: G-stretch Nucleotides 12-13, 19-20 and 24-25: CG-motif
<400> 43
tcattaaagg gcgttcttcg gggcgttctt ttt 33
<210> 44
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm521
<220>
<221> misc_feature
<223> Nucleotides 23-24: in L-conformation Nucleotides 1-3 and 14-17:G-stretch Nucleotides 6-7, 13-14 and 18-19: CG-motif
<400> 44
gggaacgttc ttcggggcgt ctttt 25
<210> 45
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm502
<220>
<221> misc_feature
<223> Nucleotides 21-22: in L-conformation Nucleotides 1-3 and 12-15:G-stretch Nucleotides 4-5, 11-12 and 16-17: CG-motif
<400> 45
gggcgttctt cggggcgtct ttt 23
<210> 46
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm374
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 29-30: in L-conformation Nucleotides 6-7and 20-23: G-stretch Nucleotides 12-13, 19-20 and 24-25: CG-motif
<400> 46
tcattggaaa acgttcttcg gggcgttctt t 31
<210> 47
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm497
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 6-7 and 27-30:G-stretch Nucleotides 12-13, 19-20 and 26-27: CG-motif
<400> 47
tcattggaaa acgttcttcg ttcttcgggg ttt 33
<210> 48
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm524
<220>
<221> misc_feature
<223> Nucleotides 31-32: in L-conformation Nucleotides 1-3 and 21-24:G-stretch
<400> 48
gggtcattaa agcttcttgc ggggcttctt ttt 33
<210> 49
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm525
<220>
<221> misc_feature
<223> Nucleotides 1-2 and 29-30: in L-conformation Nucleotides 6-7and 21-24: G-stretch
<400> 49
tcattggaaa agcttcttgc ggggcttctt t 31
<210> 50
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm526
<220>
<221> misc_feature
<223> Nucleotides 25-26: in L-conformation Nucleotides 1-3 and 15-18:G-stretch Nucleotides 7-8, 14-15 and 19-20: CG-motif
<400> 50
gggaaacgtt cttcggggcg ttctttt 27
<210> 51
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm537
<220>
<221> misc_feature
<223> Nucleotides 33-34: in L-conformation Nucleotides 1-4, 16-18,21-24 and 26-30: G-stretch Nucleotides 13-14, 20-21 and 25-26: CG-motif
<400> 51
ggggtcatta aacgtgggtc ggggcggggg ttttt 35
<210> 52
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic oligodeoxynucleotide, CKm538
<220>
<221> misc_feature
<223> Nucleotides 30-31: in L-conformation Nucleotides 1-4, 9-12, 16-19 and 23-27: G-stretch Nucleotides 8-9, 15-16 and 22-23: CG-motif
<400> 52
ggggaaacgg ggttcggggt tcgggggttt tt 32
Claims (13)
1.一种用于免疫调节的非编码线性开链DNA构建体,所述构建体由SEQ ID NO:7-25和27中任一项所示的序列组成。
2.根据权利要求1所述的构建体,其由SEQ ID NO:7-18中任一项所述的序列组成。
3.根据权利要求1所述的构建体,其由SEQ ID NO:7-16中任一项所述的序列组成。
4.根据权利要求1所述的构建体,其由SEQ ID NO:7所述的序列组成。
5.根据权利要求1所述的构建体,其由SEQ ID NO:8所述的序列组成。
6.根据权利要求1所述的构建体,其由SEQ ID NO:9所述的序列组成。
7.根据权利要求1所述的构建体,其由SEQ ID NO:10所述的序列组成。
8.根据权利要求1所述的构建体,其由SEQ ID NO:11所述的序列组成。
9.根据权利要求1所述的构建体,其由SEQ ID NO:12所述的序列组成。
10.根据权利要求1所述的构建体,其由SEQ ID NO:13所述的序列组成。
11.根据权利要求1所述的构建体,其由SEQ ID NO:14所述的序列组成。
12.根据权利要求1所述的构建体,其由SEQ ID NO:15所述的序列组成。
13.根据权利要求1所述的构建体,其由根据SEQ ID NO:16所述的序列组成。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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GBGB1021867.5A GB201021867D0 (en) | 2010-12-23 | 2010-12-23 | Non-coding immunomodulatory DNA construct |
GB1021867.5 | 2010-12-23 | ||
PCT/EP2011/074033 WO2012085291A1 (en) | 2010-12-23 | 2011-12-23 | Non-coding immunomodulatory dna construct |
CN2011800677990A CN103370417A (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna构建体 |
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CN2011800677990A Division CN103370417A (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna构建体 |
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CN201710516200.6A Pending CN107299101A (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna 构建体 |
CN2011800677990A Pending CN103370417A (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna构建体 |
CN201710517071.2A Active CN107384930B (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna构建体 |
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CN201710516200.6A Pending CN107299101A (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna 构建体 |
CN2011800677990A Pending CN103370417A (zh) | 2010-12-23 | 2011-12-23 | 非编码免疫调节dna构建体 |
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US (2) | US20140010830A1 (zh) |
EP (1) | EP2655623B2 (zh) |
JP (1) | JP6027542B2 (zh) |
KR (2) | KR102022031B1 (zh) |
CN (3) | CN107299101A (zh) |
AU (1) | AU2011347095B2 (zh) |
BR (1) | BR112013015816B1 (zh) |
CA (1) | CA2822377C (zh) |
DK (1) | DK2655623T3 (zh) |
ES (1) | ES2618783T5 (zh) |
GB (1) | GB201021867D0 (zh) |
IL (1) | IL227105A (zh) |
MX (1) | MX343918B (zh) |
PL (1) | PL2655623T3 (zh) |
RU (1) | RU2583291C2 (zh) |
SG (1) | SG191328A1 (zh) |
WO (1) | WO2012085291A1 (zh) |
ZA (1) | ZA201304999B (zh) |
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GB201021867D0 (en) | 2010-12-23 | 2011-02-02 | Mologen Ag | Non-coding immunomodulatory DNA construct |
AR095882A1 (es) | 2013-04-22 | 2015-11-18 | Hoffmann La Roche | Terapia de combinación de anticuerpos contra csf-1r humano con un agonista de tlr9 |
GB2514591A (en) * | 2013-05-30 | 2014-12-03 | Mologen Ag | Predictive biomarker for cancer therapy |
AR097584A1 (es) | 2013-09-12 | 2016-03-23 | Hoffmann La Roche | Terapia de combinación de anticuerpos contra el csf-1r humano y anticuerpos contra el pd-l1 humano |
TR201908550T4 (tr) | 2014-06-04 | 2019-07-22 | Exicure Inc | Profilaktik veya terapötik uygulamalar için lipozomal sferik nükleik asitler tarafından immün modülatörlerin çok değerlikli teslimi. |
US11213593B2 (en) | 2014-11-21 | 2022-01-04 | Northwestern University | Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates |
US10347146B2 (en) * | 2014-12-23 | 2019-07-09 | International Business Machines Corporation | Managing answer feasibility |
LU92821B1 (en) | 2015-09-09 | 2017-03-20 | Mologen Ag | Combination comprising immunostimulatory oligonucleotides |
GB2542425A (en) | 2015-09-21 | 2017-03-22 | Mologen Ag | Means for the treatment of HIV |
WO2018036852A1 (en) | 2016-08-25 | 2018-03-01 | F. Hoffmann-La Roche Ag | Intermittent dosing of an anti-csf-1r antibody in combination with macrophage activating agent |
EP3558360A1 (en) | 2016-12-22 | 2019-10-30 | F. Hoffmann-La Roche AG | Treatment of tumors with an anti-csf-1r antibody in combination with an anti-pd-l1 antibody after failure of anti-pd-l1/pd1 treatment |
MX2019012295A (es) | 2017-04-14 | 2020-02-07 | Tollnine Inc | Polinucleotidos inmunomoduladores, conjugados de anticuerpos de los mismos y metodos para su uso. |
EP3392345A1 (en) | 2017-04-22 | 2018-10-24 | Mologen AG | Biomarker for small cell lung cancer therapy |
US11433131B2 (en) | 2017-05-11 | 2022-09-06 | Northwestern University | Adoptive cell therapy using spherical nucleic acids (SNAs) |
JP2020531555A (ja) * | 2017-08-31 | 2020-11-05 | モロゲン・アーゲー | 腫瘍微小環境のモジュレーションのためのtlr−9アゴニスト |
US11806364B2 (en) | 2017-09-28 | 2023-11-07 | Industry-Academic Cooperation Foundation, Yonsei University | Method for producing myeloid-derived suppressor cells, myeloid-derived suppressor cells produced thereby, and methods thereof |
US10685050B2 (en) * | 2018-04-23 | 2020-06-16 | Adobe Inc. | Generating a topic-based summary of textual content |
KR20200138597A (ko) | 2019-06-01 | 2020-12-10 | 서미주 | 클렌징 마스크팩 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070179101A1 (en) * | 2004-02-27 | 2007-08-02 | Harukazu Kitagawa | Immunostimulatory oligonucleotide that induces interferon alpha |
Family Cites Families (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6207646B1 (en) | 1994-07-15 | 2001-03-27 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
PL187107B1 (pl) * | 1996-10-16 | 2004-05-31 | Icn Pharmaceuticals | L-nukleozydy monocykliczne, kompozycja farmaceutyczna zawierająca te związki oraz ich zastosowanie |
US6251666B1 (en) † | 1997-03-31 | 2001-06-26 | Ribozyme Pharmaceuticals, Inc. | Nucleic acid catalysts comprising L-nucleotide analogs |
DE19935756A1 (de) † | 1999-07-27 | 2001-02-08 | Mologen Forschungs Entwicklung | Kovalent geschlossenes Nukleinsäuremolekül zur Immunstimulation |
WO2001040515A1 (en) * | 1999-11-12 | 2001-06-07 | Isis Pharmaceuticals, Inc. | Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages |
DE60132471T2 (de) † | 2000-09-26 | 2009-01-15 | Idera Pharmaceuticals, Inc., Cambridge | Modulation der immunostimulatorischen aktivität von immunostimulierenden oligonukleotidanaloga durch positionelle chemische veränderungen |
CA2452458A1 (en) * | 2001-07-03 | 2003-01-16 | Isis Pharmaceuticals, Inc. | Nuclease resistant chimeric oligonucleotides |
US7276489B2 (en) * | 2002-10-24 | 2007-10-02 | Idera Pharmaceuticals, Inc. | Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends |
WO2003035836A2 (en) † | 2001-10-24 | 2003-05-01 | Hybridon Inc. | Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5' ends |
DE10211558A1 (de) * | 2002-03-15 | 2003-10-09 | Noxxon Pharma Ag | Neue Formen RNAi |
US8158768B2 (en) * | 2002-12-23 | 2012-04-17 | Dynavax Technologies Corporation | Immunostimulatory sequence oligonucleotides and methods of using the same |
EP1605973B1 (en) * | 2003-03-26 | 2012-09-26 | Cytos Biotechnology AG | Packaging of immunostimulatory oligonucleotides into virus-like particles: methods of preparation and uses |
CN101454451A (zh) | 2003-10-30 | 2009-06-10 | 科勒制药有限公司 | 具有增强免疫刺激能力的c类寡核苷酸类似物 |
WO2005076824A2 (en) | 2004-02-03 | 2005-08-25 | The Regents Of The University Of California | Methods of treating irritable bowel syndrome |
NZ552522A (en) † | 2004-06-15 | 2009-04-30 | Idera Pharmaceuticals Inc | Immunostimulatory oligonucleotide multimers |
US7879992B2 (en) * | 2005-01-31 | 2011-02-01 | Isis Pharmaceuticals, Inc. | Modification of MyD88 splicing using modified oligonucleotides |
AU2006301230A1 (en) † | 2005-10-12 | 2007-04-19 | Cancer Research Technology Ltd. | Methods and compositions for treating immune disorders |
CN104278037B (zh) † | 2005-10-12 | 2020-09-15 | 艾德拉药物股份有限公司 | 基于变异应答调制Toll样受体的免疫调节寡核苷酸(IRO)化合物 |
AU2007260775A1 (en) * | 2006-06-13 | 2007-12-21 | Bayhill Therapeutics, Inc. | Methods and immune modulatory nucleic acid compositions for preventing and treating disease |
US20080260820A1 (en) * | 2007-04-19 | 2008-10-23 | Gilles Borrelly | Oral dosage formulations of protease-resistant polypeptides |
US9109012B2 (en) * | 2007-05-29 | 2015-08-18 | Nature Technology Corporation | Vectors and method for genetic immunization |
WO2009035554A2 (en) * | 2007-09-07 | 2009-03-19 | University Of Florida Research Foundation, Inc. | Superior structure stability and selectivity of hairpin nucleic acid probes with an l-dna stem |
CN101820908A (zh) * | 2007-10-09 | 2010-09-01 | 科利制药公司 | 包含改性糖部分的免疫刺激寡核苷酸类似物 |
WO2010039137A1 (en) * | 2008-10-02 | 2010-04-08 | Idera Pharmaceuticals, Inc. | Immune regulatory oligonucleotide (iro) compounds to modulate toll-like receptor based immune response |
EP2246433A1 (de) * | 2009-04-30 | 2010-11-03 | Mologen AG | Concatemere zur Immunmodulation |
GB201021867D0 (en) † | 2010-12-23 | 2011-02-02 | Mologen Ag | Non-coding immunomodulatory DNA construct |
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2010
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070179101A1 (en) * | 2004-02-27 | 2007-08-02 | Harukazu Kitagawa | Immunostimulatory oligonucleotide that induces interferon alpha |
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