CN101265285A - 免疫刺激性核酸分子 - Google Patents

免疫刺激性核酸分子 Download PDF

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CN101265285A
CN101265285A CNA2007101300520A CN200710130052A CN101265285A CN 101265285 A CN101265285 A CN 101265285A CN A2007101300520 A CNA2007101300520 A CN A2007101300520A CN 200710130052 A CN200710130052 A CN 200710130052A CN 101265285 A CN101265285 A CN 101265285A
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immunostimulatory oligonucleotide
cpg
cell
dna
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阿撒尔·M·科瑞格
约尔·N·科里尼
丹尼斯·克林曼
乔治·韦纳
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Abstract

本发明涉及含有未甲基化的CpG二核苷酸的核酸序列,其可调节免疫反应,包括刺激Th1类型的免疫活化、细胞因子的产生、NK裂解活性、以及B细胞的增殖。这些序列也可以用作合成佐剂。

Description

免疫刺激性核酸分子
本申请是中国发明专利申请No.971993521的分案申请,其申请日为1997年10月30日,发明创造名称为“免疫刺激性核酸分子”。
本发明的研究部分地得到了美国国家卫生研究院的第R29-AR42556-01拨款的资助。美国政府有本发明的部分权利。
技术领域
本发明一般地涉及寡核苷酸,具体地讲,涉及那些免疫刺激性的、在序列中包括至少一个未甲基化CpG二核苷酸的寡核苷酸。
背景技术
在二十世纪七十年代,一些研究人员报道了高分子量DNA与细胞膜的结合(Lerner,R.A.et al.,1971.“Membran-associated DNA in the cytoplasm of diploid humanlymphocytes”.Proc.Natl.Acad.Sci.USA 68:1212;Agrawal,S.K.,R.W.Wagner,P.K.McAllister,and B.Rosenberg.1975.“Cell-surface-associated nucleic acid in tumorigenic cells madevisible with platinum-pyrimidine complexes by electronmicroscopy”.Proc.Natl.Acad.Sci.USA 72:928)。在1985年,Bennett等人首次提出证据证明了DNA与淋巴细胞的结合相似于配体与受体的相互作用,即结合是可饱和的、竞争性的、并导致DNA细胞内吞作用和降解为寡核苷酸(Bennett,R.M.,G.T.Gabor,and M.M.Merritt.1985.“DNA binding to humanleukocytes.Evidence for receptor-mediated association,internalization,and degradation of DNA”.J.Clin.Invest.76:2182)。类似于DNA,寡脱氧核糖核苷酸(ODN)也能以可饱和、不依赖序列、但依赖温度和能量的形式进入到细胞中(见Jaroszewski,J.W.,and J.S.Cohen.1991.“Cellular uptake ofantisense oligodeoxynucleotides”.Advanced Drug DeliveryReviews 6:235;Akhtar,S.,Y.Shoji,and R.L.Juliano.1992.“Pharmaceutical aspects of the biological stability andmembrane transport characteristics of antisenseoligonucleotides”.In:Gene Regulation:Biology of Antisense RNA and DNA.R.P.Erickson,and J.G.Izant,eds.RavenPress,Ltd.New York,pp.133:and Zhao,Q.T.Waldschmidt,E.Fisher,C.J.Herrera,and A.M.Krieg.,1994.“Stage specificoligonucleotide uptake in murine bone marrow B cellprecursors”.Blood,84:3660)。但是,还没有克隆化到负责DNA或ODN摄入的受体,而且,还不清楚ODN结合及细胞摄入是采取与高分子量的DNA相同或不同的机制。
淋巴细胞的ODN吸收已经被证明可由细胞活化来调节。用B细胞促分裂原LPS刺激的脾细胞在B细胞群体中显示了ODN吸收的显著提高,而用T细胞促分裂原Con A处理的脾细胞显示在T细胞中有ODN吸收的提高而在B细胞中没有(Krieg,A.M.,F.Gmelig-Meyling,M.F.Gourley,W.J.Kisch,L.A.Chrisey,and A.D.Steinberg.1991.“Uptake ofoligodeoxyribonucleotides by lymphoid cells is heterogeneousand inducible”.Antisense Research and Development 1:161)。
已经对一些多核苷酸作为生物反应修饰剂进行了广泛的研究。也许最好的例子是多聚(I,C),这是一种IFN生产的强力诱导物,也是巨噬细胞活化子和NK活性诱导物(Talmadge,J.E.,J.Adams,H.Phillips,M.Collins,B.Lenz,M.Schneider,E.Schlick,R.Ruffmann,R.H.Wiltrout,and M.A.Chirigos.1985.“Immunomodulatory effects in mice ofpolyinosinic-polycytidylic acid comlexed with poly-L-lysineand carboxymethylcellulose”.Cancer Res.45:1058;Wiltrout,R.H.,R.R.Salup,T.A.Twilley,and J.E.Talmadge.1985.“Immunomodulation of natural killer activity bypolyribonucleotides”.J.Biol.Resp.Mod.4:512;Krown,S.E.1986.“Interferons and interferon inducers in cancertreatment”.Sem.Oncol.13:207;and Ewel,C.H.,S.J.Urba,W.C.Kopp,J.W.Smith II,R.G.Steis,J.L.Rossio,D.L.Longo,M.J.Jones,W.G.Alvord,C.M.Pinsky,J.M.Beveridge,K.L.McNitt,and S.P.Creekmore.1992.“Polyinosinic-polycytidylicacid complexed with poly-L-lysine and carboxymethylcellulosein combination with interleukin-2 in patients with cancer:clinical and immunological effects”.Canc.Res.52:3005)。看起来,这种鼠NK活化可能仅仅是由于IFN-β分泌的诱导(Ishikawa,R.,and C.A.Biron.1993.“IFN induction andassociated changes in splenic leukocyte distribution”.J.Immunol.150:3713)。这种活化是对核糖为特异的,因为脱氧核糖没有效力。其强有力的体外抗肿瘤活性导致多种采用与多聚-L-赖氨酸和羧甲基纤维素复合的(用以减少由RNAse引起的降解)进行的临床研究(Talmadge,J.E.,et al.,1985.出处同上;Wiltrout,R.H.,et al.,1985.出处同上);Krown,S.E.,1986.出处同上);以及Ewel.C.H.,et al.,1992.出处同上)。不幸的是,毒性副作用由此大大妨碍了多聚(I,C)成为有用的治疗药剂。
在C8位发生溴或硫醇基取代的鸟嘌呤核糖核苷酸是B细胞促分裂原,并且可以代替“B细胞分化因子”(Feldbush,T.L.,and Z.K.Ballas.1985.“Lymphokine-like activity of8-mercaptoguanosine:indnction of T and B celldifferentiation”.J.Immunol.134:3204;and Goodman,M.G.1986.“Mechnism of snyergy between T cell signals andC8-substituted guanine nucleosides in humoral immunity:Blymphotropic cytokines induce responsiveness to8-mercaptoguanosine”.J.Immunol.136:3335)。8-巯基鸟苷和8-溴鸟苷还可以取代MHC限制型CTL产生的细胞因子需求(Feldbush,T.L.,1985.出处同上),增加大鼠NK活性(Koo,G.C.,M.E.Jewell,C.L.Manyak,N.H.Sigal,and L.S.Wicker.1988.“Activation of murine natural killer cells and macrophages by8-bromoguanosine”.J.Immunol.140:3249),以及与IL-2协同诱导鼠LAK产生(Thompson,R.A.,and Z.K.Ballas.1990.“Lymphokine-activated killer (LAK)cells.V.8-Mercaptoguanosine as an IL-2-sparing agent in LAKgeneration”.J.Immunol.145:3524)。这些C8取代的鸟苷的NK和LAK活性的增大看起来归因于它们对IFN的诱导(Thompson,R.A.,et al.,1990.出处同上)。近来,由分枝杆菌产生的5’三磷酸化胸苷被发现对人γδT细胞亚类有促细胞分裂作用(Constant,P.,F.Davodeau,M.A.Peyrat,Y.Poquet,G.Puzo,M.Bonneville,and J.-J.Fournie.1994.“Stimulation ofhuman γδT cells by nonpeptidic mycobacterial ligands”Science 264:267)。该篇报道指明有可能免疫系统已经进化出了对微生物核酸优先反应的方式。
一些观察结果暗示,某些DNA结构可能也具有活化淋巴细胞的潜力。例如,Bell等人曾经报道,在脾细胞上清液中的核小体蛋白质-DNA复合物(不是裸露的DNA)引起了B细胞的增殖和免疫球蛋白的分泌(Bell,D.A.,B.Morrison,and P.VanderBygaart.1990.“Immunogenic DNA-related factors”.J.Clin.Invest.85:1487)。在其它的情况下,裸露的DNA已经被报道具有免疫效应。例如,Messina等人最近曾报道说,多聚(dG)·(dC)和多聚(dG·dC)的260至800bp的片段对B细胞有促细胞分裂作用(Messina,J.P.,G.S.Gilkeson,and D.S.Pisetsky.1993.“The influence of DNA structure on the in vitrostimulation of murine lymphocytes by natural and syntheticpolynucleotide antigens”.Cell.Immunol.147:148)。Tokunaga等人已经报道了dG·dC可诱导γ-IFN和NK活性(Tokunaga,S.Yamamoto,and K.Namba.1988.“A synthetic single-strandedDNA,poly(dG,dC),induces interferon-α/b and-g,augmentsnatural killer activity,and suppresses tumor growth”Jpn.J.Cancer Res.79:682)。除了这些人造的均聚体序列之外,Pisetsky等人报道了纯的哺乳动物DNA没有可检测的免疫效应,但是,从某些细菌来的DNA则诱导了B细胞的活化和免疫球蛋白的分泌(Messina,J.P.,G.S.Gilkeson,and D.S.Pisetsky.1991.“Stimulation of in vitro murine lymphocyteproliferation by bacterial DNA”.J.Immunol.147:1759)。假设这些数据不是由一些罕见污染物所引起的,那么,这些研究则暗示,细菌DNA的特定结构或其它性质使其能够触发B细胞的活化。对分枝杆菌DNA序列的研究显示,含有某种回文序列的ODN可以活化NK细胞(Yamamoto,S.,T.Yamamoto,T.Kataoka,E.Kuramoto,O.Yano,and T.Tokunaga.1992.“Unique palindromic sequences in synthetic oligonucleotidesare required to induce INF and agument INF-meidatednatural killer activity”.J.Immunol.148:4072;Kuramoto,E.,O.Yano,Y.Kimura,M.Baba,T.Makino,S.Yamamoto,T.Yamamoto,T.Kataoka,and T.Tokunaga.1991.“Oligonucleotide sequences required fro natural killer cellactivation”.Jpn.J.Cancer Res.83:1128)。
还报道了其它一些硫代磷酸酯修饰的ODN诱导了体外或体内B细胞刺激(Tanaka,T.,C.C.Chu,and W.E.Paul.1992.“An antisense oligonucleotide complementary to a sequence inIg2b increases g2b germline transcripts,stimulates B cell DNAsynthesis,and inhibits immunoglobulin secretion”.J.Exp.Med.175:597;Branda,R.F.,A.L.Moore,L.Mathews,J.J.McCormack,and G.Zon.1993.“Immune stimulaiton by anantisense oligomer complementary to the rev gene of HIV-1”.Biochem.Pharmacol.45:2037;McIntyre,K.W.,K.Lombard-Gillooly,J.R.Perez,C.Kunsch,U.M.Sarmiento,J.D.Larigan,K.T.Landreth,and R.Narayanan.1993.“A sensephosphorothioate oligonucleotide directed to the initiationcodon of transcription factor NF-κB T65 causessequence-specific immune stimulation”.Antisense Res.Develop.3:309;and Pisetsky,D.S.,and C.F.Reich.1993.“Stimulation of murine lymphocyte proliferation by aphosphorothioate oligonucleotide with antisense activity forherpes simplex virus”.Life Sciences 54:101)。这些报道没有暗示在这些ODN中有可以解释这些效果的共同结构基元或序列因素。
cAMP效应元件结合蛋白质(CREB)和活化转录因子(ATF)或转录因子的CREB/ATF家族是普遍被表达的一类转录因子,其11个成员已经被克隆化了(reviewed in de Groot,R.P.,and P.Sassone-Corsi:“Hormonal control of gene expression:Multiplicity and versatility of cyclic adenosine3’,5’-monophosphate-responsive nucler regulators”.Mol.Endocrin.7:145,1993;Lee,K.A.W.,and N.Masson:“Transcriptional regulation by CREB and its relatives”.Biochim.Biophys.Acta 1174:221,1993)。它们都属于蛋白质的碱性区/亮氨酸拉链结构(bZip)类。所有的细胞似乎都表达一种或多种CREB/ATF蛋白质,但是,所表达的成员和对mRNA剪接的调节则似乎是组织特异性的。活化域的差异剪接可以确定某一具体的CREB/ATF蛋白质是转录抑制子还是活化子。许多的CREB/ATF蛋白质活化病毒转录,但是,有些缺少活化域的剪接变体则是抑制性的。CREB/ATF蛋白质可以作为均二聚体或异二聚体通过cAMP效应元件,即CRE来结合DNA,其中CRE的共有形式是未甲基化序列TGACGTC(如果CpG是甲基化的,则没有结合)(Iguchi-Ariga,S.M.M.,andW.Schaffner:″CpG methylation of the cAMP responsiveenhancer/promoter sequence TGACGTCA abolishes specificefactor binding as well as transcriptional activation″.Genes &Develop.3:612,1989.)。
CRE的转录活性在B细胞活化期间有提高(Xie,H.T.C.Chiles,and T.L.Rothstein:″Induction of CREB activity viathe surface Ig receptor of B cells″.J.Immunol.151:880,1993)。CREB/ATF蛋白质似乎通过CRE来对多种基因的表达进行调节,包括免疫学上重要的基因,例如fos、jun B、Rb-1、IL-6、IL-1(Tsukada,J.,K.Saito,W.R.Waterman,A.C.Webb,and P.E.Auron:″Transcription factors NF-IL-6 andCREB recognize a common essential site in the humanprointerleukin 1 gene″.Mol.Cell.Biol.14:7285,1994;Gray,G.D.,O.M.Hernadez,D.Hebel.M.Root,J.M.Pow-Sang,andE.Wickstrom:″antisense DNA inhibition of tumor growthinduced by c-Ha-ras oncogene in nude mice″.Cancer Res.53:577,1993),IFN-(Du,W.,and  T.Maniatis:″AnATF/CREB binding site protein is required for virus inductionof human interferon B  gene″.Proc.Natl.Acad.Sci.USA89:2150,1992),TGF-1(Asiedu,C.K.,L.Scott,R.K.Assoian,M.Ehrlich:″Binding of AP-1/CREB proteins and of MDBP tocontiguous sites downstream of the human TGF-B1 gene″.Biochim.Biophys.Acta 1219:55,1994),TGF-2,II类MHC(Cox,P.M.,and C.R.Goding:″An ATF/CREB bindingmotif is required for aberrant constitutive expression of theMHC class II DRa promoter and activation by SV40T-antigen″.Nucl.Acids Res.20:4881,1992),E-选择素,GM-CSF,CD-8,种系Ig恒定区基因,TCR V基因,以及增殖细胞核抗原(Huang,D.,P.M.Shipman-Appasamy,D.J.Orten,S.H.Hinrichs,and  M.B.Prystowsky:″Promoteractivity of the proliferating-cell nuclear antigen gene isassociated with inducible CRE-binding proteins in interleukin2-stimulated T lymphocytes:.Mol.Cell.Biol.14:4233,1994)。除了通过cAMP途径的活化,CREB还可以介导对细胞内Ca++浓度变化的转录应答(Sheng,M.,G.McFadden,and M.E.Greenberg:″Membrane depolarization and calcium inducec-fos transcription via phosphorylation of transcription factorCREB″.Neuron 4:571,1990)。
在CREB/ATF蛋白质的转录活化中,蛋白质-蛋白质之间的相互作用似乎极其重要。已经有一些发表的研究报道了在NFKB蛋白质和CREB/ATF蛋白质之间的直接性或间接性相互作用[Whitley,et al.,(1994)Mol.& Cell.Biol.14:6464;Cogswell,et al.,(1994)J.Immun.153:712;Hines,et al.,(1993)Oncogene 8:3189;and Du,et al.,(1993)Cell 74:887]。通过环AMP途径对CREB的活化需要蛋白质激酶A(PKA),其将CREB341在ser133磷酸化,并允许其与近来克隆的蛋白质CBP结合(Kwok,R.P.S.,J.R.Lundblad,J.C.Chrivia,J.P.Richards,H.P.Bachinger,R.G.Brennan,S.G.E.Roberts,M.R.Green,and R.H.Goodman:″Nuclear protein CBP is a coactivator forthe transcription factor CREB″.Nature 370:223,1994;Arias,J.,A.S.Alberts,P.Brindle,F.X.Claret,T.Smea,M.Karin,J.Feranmisco,and  M.Montminy:″Activation of cAMP andmitogen responsive genes relies on a common nuclear factor″.Nature 370:226,1994.)。CBP继而再与碱性转录因子TFIIB相互作用,导致转录提高。CREB还被报道与dTAFII 110相互作用,该dTAFII 110是一种TATA结合蛋白质相关因子,其结合可以调节转录(Ferreri,K.,G.Gill,and M.Montminy:″The cAMP-regulated transcription factor CREB interactswith a component of the TFIID complex″.Proc.Natl.Acad.Sci.USA 91:1210,1994)。除了这些相互作用之外,CREB/ATF蛋白质还可以特异性结合多种其它的核因子(Hoeffler,J.P.,J.W.Lustbader,and C.-Y.Chen:″Identification of multiplenuclear factors that interact with cyclic adenosine3′,5′-monohosphate response element-binding protein andactivating transcription factor-2 by protein-proteininteractions″.Mol.Endocrinol.5:256,1991),但是,这些相互作用中的大多数的生物学重要性还都是未知的。一般认为CREB作为均二聚体或与其它一些蛋白质的异二聚体形式与DNA结合。另人惊奇的是,CREB单体组成性地激活转录(Krajewski,Wl,and K.A.W.Lee:″A monomeric derivative ofthe cellular transcription factor CREB functions asconstitutive activator″.Mol.Cell.Biol.14:7204,1994)。
除了在调节细胞转录中的重要作用之外,最近还证实了CREB/ATF蛋白质被某些感染性病毒和逆病毒所破坏,所述病毒需要该CREB/ATF蛋白进行病毒复制。例如,已知的最为强烈的哺乳动物启动子之一,即巨细胞病毒立即早期启动子,其含有对启动子功能必需的CRE 11个拷贝(Chang,Y.-N.,S.Crawford,J.Stall,D.R.Rawlins,K.-T.Jeang,and G.S.Hayward:″The palindromic series I repeats in the simiancytomegalovirus major immediate-early promoter behave asboth strong basal enhancers and cyclic AMP responseelements″.J.Virol.64:264,1990)。诱导众多启动子的腺病毒E1A蛋白质的至少一部分转录活化效应归因于其与CREB/ATF蛋白质的DNA结合域即ATF-2的结合,该结合域介导E1A可诱导转录活化(Liu,F.,and M.R.Green:″Promoter targeting by adenovirus E1a through interactionwith different cellular DNA-bin ding domains″.Nature 368:520,1994)。还已经暗示,E1A与CREB结合蛋白质CBP相结合(Arany,Z.,W.R.Sellers,D.M.Livingston,and R.Eckner:″E1A-associated p300 and CREB-associated CBP belong to aconserved family of coactivators″.Cell 77:799,1994)。人T嗜淋巴细胞病毒-I(HTLV-1),即造成人T细胞白血病和热带痉挛轻瘫的逆转录病毒,也需要CREB/ATF蛋白质来复制。在这种情况下,逆转录病毒产生蛋白质Tax,其与CREB/ATF蛋白质结合,将它们从其正常的细胞结合位点重导向存在于HTLV转录增强子中的不同DNA序列(侧翼为富含G和C的序列)(Paca-Uccaralertkun,S.,L.-J.Zhao,N.Adya,J.V.Cross,B.R.Cullen,I.M.Boros,and C.-Z.Giam:″In vitro selection ofDNA elements highly responsive to the human T-celllymphotropic virus type I transcriptional activator,Tax″.Mol.Cell.Biol.14:456,1994;Adya,N.,L.-J.Zhao,W.Huang,I.Boros,and  C.-Z.Giam:″Expansion of CREB′s DNArecognition specificity by Tax results from interaction withAla-Ala-Arg at positions 282-284 near the conservedDNA-binding domain of CREB″.Proc.Natl.Acad.Sci.USA91:5642,1994)。
本发明的概述
本发明基于的是如下发现,即某些含有未甲基化胞嘧啶-鸟嘌呤(CpG)二核苷酸的核酸能在受试者中活化淋巴细胞,并使受试者的免疫反应由Th2转变为Th1(例如通过诱导单核细胞以及其它细胞产生Th1细胞因子,包括IL-12、IFN-γ和GM-CSF)。依据这个发现,本发明在一方面涉及新的免疫刺激性核酸组合物。
在一个实施方案中,本发明提供了分离的免疫刺激性的含有CpG基元的核酸序列,其表达式为:
5′N1X1CGX2N23′
其中,至少一个核苷酸将连续的CpGs间隔开来;X1是腺嘌呤,鸟嘌呤,或胸腺嘧啶;X2是胞嘧啶或胸腺嘧啶;N是任何的核苷酸,且N1+N2是约0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该核酸序列的长度为约8-30个碱基。
在另一个实施方案中,本发明提供了分离的免疫刺激性的含有CpG基元的核酸序列,其表达式为:
5′N1X1X2CGX3X4N23′
其中,至少一个核苷酸将连续的CpGs间隔开来;X1 X2选自GpT、GpG、GpA、ApT和ApA;X3X4选自TpT或CpT;N是任何的核苷酸,且N1+N2是约0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该核酸序列的长度为约8-30个碱对。
在另一个实施方案中,本发明提供了刺激免疫活化的方法,其中将本发明的核酸序列提供给受试者,优选人。在优选的实施方案中,免疫活化产生主要是Th1形式的免疫活化。
在另一个实施方案中,本发明的核酸序列刺激细胞因子的生产。具体地讲,通过用本文中所述的核酸序列刺激免疫系统来产生细胞因子如IL-6、IL-12、IFN-γ、TNF-α和GM-CSF。在另一方面,本发明的核酸序列刺激天然杀伤细胞(NK)的裂解活性和B细胞的增殖。
在另一个实施方案中,本发明的核酸序列可用作人工佐剂用于哺乳动物如小鼠或人中抗体的产生。
在另一个实施方案中,通过抑制受试者对CpG介导的淋巴细胞活化的应答来治疗自身免疫紊乱。本发明提供了用内体酸化的抑制物如bafilomycin a,氯喹和莫能星来缓解自身免疫紊乱。具体地讲,用这种方式治疗系统性红斑狼疮。
本发明的核酸序列还可以用于治疗、预防或缓解其它的紊乱(例如,肿瘤或癌症,或者病毒性、真菌性、细菌性或寄生性感染)。此外,可以施用本发明的核酸序列刺激对疫苗的应答。进一步,通过将受试者的免疫反应从Th2改变为Th1,本发明的核酸序列可以用于治疗或预防气喘疾病。此外,本发明的核酸序列可以与特定的变应原一起施用给受试者,作为一种类型的脱敏疗法,用于治疗或预防与气喘相关的过敏反应的发生。
进一步,本发明所述核酸序列的诱导白血病细胞进入细胞循环的能力支持了其治疗白血病的应用,该治疗可通过提高慢性白血病细胞的敏感性随后进行常规消除性化疗,或者通过将所述核酸序列与其它的免疫治疗一起组合使用来实现。
本发明的其它的特点和优点将可以从下面的描述和权利要求书中更为明显地看出。
附图的简要说明
图1A-C用制图说明在消除了T细胞的脾细胞培养物中对各种DNA序列应答的剂量依赖性IL-6生产。
图1A是大肠杆菌DNA(l)和小牛胸腺DNA(n)序列和LPS(大肠杆菌和小牛胸腺DNA浓度的10×)(u)。
图1B是对照磷酸二酯寡脱氧核苷酸(ODN)5′ATGGAAGGTCCAGTGTTCTC3′(SEQ ID No:1)(n)和两种磷酸二酯CpG ODN5′ATCGACCTACGTGCGTTCTC3′(SEQ ID No:2)(u)和5′TCCATAACGTTCCTGATGCT3′(SEQ ID No:3)(l)。
图1C是对照硫代磷酸酯ODN5′GCTAGATGTTAGCGT3′(SEQ ID No:4)(n)和两种硫代磷酸酯CpG ODN 5′GAGAACGTCGACCTTCGAT3′(SEQ IDNo:5)(u)和5′GCATGACGTTGAGCT3′(SEQ ID No:6)(l)。数据代表一式三份实验的平均值±标准偏差。
图2图示由注射后1-8小时所确定的体内CpG DNA诱导的IL-6生产。数据代表从两只小鼠的血清的一式两份分析得到的平均值。对BALB/c小鼠(每组两只)静脉内注射100微升的PBS(o)或200微克的CpG硫代磷酸酯ODN5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)(n)或非-CpG硫代磷酸酯ODN 5′TCCATGAGCTTCCTGAGTCT3′(SEQ IDNo:8)(u)。
图3是放射性自显影图,其中显示了由反转录聚合酶链反应确定的肝、脾和胸腺中在进行如下处理后各个时期的IL-6mRNA表达情况:对BALB/c小鼠(每组两只)静脉内注射100微升的PBS,200微克的CpG硫代磷酸酯ODN5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)或非-CpG硫代磷酸酯ODN 5′TCCATGAGCTTCCTGAGTCT3′(SEQ IDNo:8)进行体内刺激。
图4A是图示抗IL-6对CpG诱导的IgM生产的剂量依赖性抑制。在如ELISA所测定培养物上清液中油指示浓度的中和性抗-IL-6(u)或同种型对照Ab(l)和IgM存在的情形下用CpG ODN 5′TCCAAGACGTTCCTGATGCT3′(SEQ ID No:9)刺激来自DBA/2小鼠的脾B细胞,有给出,在培养物上清液中的IgM水平有ELISA确定。在没有CpG ODN存在的情况下,抗-IL-6抗体对IgM分泌没有影响(n)。图4B图示用抗-IL-6和CpG S-ODN 5′TCCATGACGTTCCTGATGCT3′(SEQ IDNo:7)(u)培养或仅用抗-IL-6抗体培养(n)的脾B细胞中CpG-诱导的刺激指数。数据代表一式三份试验的平均值±标准偏差。
图5是条块图,说明在被不含启动子的CAT构建体(pCAT)、阳性对照质粒(RSV)、IL-6启动子-CAT构建体转染的WEHI-231细胞单独或者用所示浓度的CpG5′TCCATGACGTTCCTGATGCT3′(SEQ ID No:7)或非-CpG5′TCCATGAGCTTCCTGAGTCT3′(SEQ ID No:8)硫代磷酸酯ODN培养后细胞中的氯霉素、乙酰转移酶(CAT)的活性。数据代表一式三份试验的平均值。
图6是免疫刺激性的含有未甲基化CpG的核酸的免疫效果的总体图示,该核酸可以直接活化B细胞和单核细胞(包括巨噬细胞和树状细胞)。该免疫刺激性寡核苷酸不直接活化纯化的NK细胞,但可以使它们有能力应答IL-12,并且其IFN-γ生产有明显提高。通过诱导IL-12的产生以及随后NK细胞的IFN-γ分泌提高,该免疫刺激性核酸促进了Th1类型的免疫应答。没有发现高度纯化的T细胞的增殖或细胞因子分泌的直接激活。然而,由免疫刺激性寡核苷酸对Th1细胞因子分泌的诱导促进了细胞毒性淋巴细胞应答的产生。
图7是显示用大肠杆菌DNA处理的单核细胞中NFkB活化的时间的自显影图,显示被大肠杆菌(EC)DNA(含有未甲基化CpG基元)、对照(CT)DNA(不含未甲基化CpG基元)和脂多糖(LPS)所处理的单核细胞在不同的测量时间在接触后15分钟、30分钟的NFKB mRNA诱导。
图8A显示流式细胞仪的测定结果,其中使用小鼠B细胞,采用二氢若丹明123染料来确定反应性氧种类的水平。在该图的A图中仅用了染料的样品显示染料阳性细胞的背景值为28.6%。这个水平的反应性氧种类在用PMA和离子霉素即阳性对照(B图)处理20分钟的细胞中被大大提高到了80%。用CpG寡核苷酸(TCCATGACGTTCCTGACGTT,SEQ ID No.10)处理的细胞也显示了反应性氧种类水平的提高,即50%以上的细胞变为阳性(D图)。然而,用具有同样序列但CpGs交换(TCCATGAGCTTCCTGAGTGCT,SEQ ID No.11)的寡核苷酸处理的细胞则没有显示反应性氧种类水平的这种显著提高(E图)。
图8B显示流式细胞术研究的结果,其中使用小鼠B细胞,有氯喹存在,采用二氢若丹明123染料,以确定反应性氧种类的水平。氯喹将细胞中反应性氧种类的背景水平稍微降低了一些,使在A图中的未处理细胞仅有4.3%为阳性。氯喹完全消除了用CpG DNA处理的细胞中反应性氧种类的诱导(B图),但不降低用PMA和离子霉素处理的细胞中反应性氧种类的水平(E图)。
图9说明肺灌洗细胞计数对时间的作图,显示CpG和呼吸道暴露对肺部灌洗液的细胞计数效应。该图显示,当小鼠开始被注射了诱导Th2免疫应答的Schistosoma mansoni卵,“卵”、并且随后吸入Schistosoma mansoni卵抗原“SEA”后(空心环),则在肺部内有众多的炎性细胞。然而,当小鼠开始接受的是CpG寡核苷酸(SEQ ID No.10)以及卵时,则肺部内的炎性细胞并不因后续吸入SEA而增加(空心三角形)。
图10说明肺部灌洗嗜伊红粒细胞计数对时间的作图,显示CpG和呼吸道暴露对肺部灌洗液嗜伊红粒细胞数的效应。该图显示,当小鼠开始注射卵、并随后吸入SEA(空心环),则在肺部内有众多的嗜伊红粒细胞存在。然而,当小鼠开始接受的是CpG寡核苷酸(SEQ ID No.10)和卵时,则肺部内的炎性细胞就不因后来吸入SEA而增高(空心三角形)。
图11是显示CpG和呼吸道暴露肺部灌洗液中各成分的效应的条块图,说明分别暴露于仅仅盐水;卵,然后SEA;卵加SEQ ID No.11,然后SEA;卵加对照寡核苷酸(SEQ IDNo.11),然后SEA后,所诱导的对巨噬细胞、淋巴细胞、嗜中性细胞、嗜伊红粒细胞百分数的影响。当小鼠在初始暴露于卵之时给予对照寡核苷酸,则吸入SEA后对嗜伊红粒细胞向肺部的随后流入几乎没有影响。因此,当小鼠在第14天或第21天吸入卵时,它们在肺部内产生急性炎性反应。然而,如果在第0天和第7天开始接触抗原的时候将CpG寡核苷酸同卵一起提供给小鼠,则就完全消除了小鼠在第14天吸入卵抗原时嗜伊红粒细胞的增加。
图12是显示寡核苷酸剂量对总细胞数和嗜伊红粒细胞数的效应的一个条块图,说明对注射不同量的保护性寡核苷酸SEQ ID No.10响应的嗜伊红粒细胞计数。
图13显示CpG和呼吸道暴露对肺部灌洗中的IL-4的效应,其是响应于注射卵和随后的SEA(空心菱形);卵加SEQID No.10和随后的SEA(空心环);或盐水加随后的盐水(空心矩形)小鼠中白细胞介素-4(IL-4)的生成(pg/ml)随时间的作图。该图显示,所产生的炎性反应与肺部Th2细胞因子IL-4的水平相关。
图14是显示CpG和呼吸道暴露对肺部灌洗中的IL-12的效应的条块图,说明响应于注射盐水;卵和随后的SEA;或卵加SEQ ID No.10和随后的SEA,小鼠中白细胞介素-12(IL-12)的生成(pg/ml)随时间的作图。该图的结果显示,施用含有未甲基化CpG基元的寡核苷酸确实可以使肺的细胞因子应答重导向为产生IL-12,表明是Th1型的免疫应答。
图15是显示CpG和呼吸道暴露对肺部灌洗中的IFN-γ的效应的条块图,说明响应于注射盐水;卵和随后的SEA;或卵加SEQ ID No.10和随后的SEA小鼠中伽玛干扰素(IFN-γ)的生成(pg/ml)随时间的变化。该图的结果显示,施用含有未甲基化CpG基元的寡核苷酸也可以将肺的细胞因子应答重导向为IFN-γ产生,表明是Th1型的免疫应答。
本发明的详细描述
定义
在本文中,下面各个术语和词组所表达的意思定义如下:
术语“过敏原”指的是可以在易感受试者中产生过敏反应或气喘反应的物质。过敏原的名单是非常巨大的,可以包括花粉、昆虫毒物、动物皮、真菌孢子和药物(青霉素)等。天然的、动物的和植物的过敏原的实例包括下面各属特异性的蛋白质:犬(Canis familiaris),螨(Dermatophagoides farinae),Felis(Felisdomesticus),豚草(Ambrosia artemiisfolia),黑麦草(Loliumperenne或Lolium multiflorum),柳杉(Cryptomeria japonica),链格孢(Alternaria alternata),小蠹甲(Alder),桤木(Alnusgultinosa),桦木(Betula verrucosa),栎(Quercus alba),木犀榄(Olea europa),蒿(Artemisia vulgaris),车前(Plantago lanceolata),墙草(Parietaria officinalis或Parietaria judaica),小蠊(Blattellagermanica),蜂(Apis multiflorum),柏木(Cupressus sempervirens,Cupressus arizonica和Cupressus macrocarpa),刺柏(Juniperussabinoides,Juniperus virginiana,Juniperus communis和Juniperus ashei),Thuya(Thuya orientalis),扁柏(Chamaecyparisobtusa),大蠊(Periplaneta americana),冰草(Agropyron repens),黑麦(Secale cereale),小麦(Triticum aestivum),鸭茅(Dactylisglomerata),羊茅(Festuca elatior),早熟禾(Poa pratensis或Poacompressa),燕麦(Avena sativa),绒毛草(Holcus lanatus),黄花茅(Anthoxanthum odoratum),燕麦草(Arrhenatherum elatius),剪股颖(Agrostis alba),梯牧草(Phleum pratense),Phalaris(Phalaris arundinacea),雀稗(Paspalum notatum),高粱(Sorghum halepensis),以及雀麦(Bromus inermis)。
术语“过敏”指的是对某种物质(过敏原)所获得的过度敏感。过敏状况包括湿疹、过敏性鼻炎或鼻炎、枯草热、支气管气喘、荨麻疹、风疹、食物过敏,以及其它的特应性疾病。
术语“气喘”(asthma)指的是呼吸系统的疾病,其特征是发炎,气管变窄,以及气管对吸入物质的反应增加。气喘虽然不是必然的但也是经常地伴随着特应性或过敏性症状。
术语“免疫系统缺陷”指的是这样的疾病或紊乱,其中,个体的免疫系统不能正常发挥功能,或者其中增强个体的免疫应答可能比较有用,从而消除例如受试者中的肿瘤或癌症[例如脑、肺(包括小细胞和非小细胞)、卵巢、乳腺、前列腺、结肠肿瘤,以及其它的恶性瘤和肉瘤]或其它的感染。
感染性病毒的实例包括:Retroviridae[例如,人免疫缺陷病毒,如HIV-1(也称为HTLV-III,LAV或HTLV-III/LAV,或HIV-III;以及其它的分离物,例如HIV-LP)];小RNA病毒科(Picornaviridae)(例如,脊髓灰质炎病毒,甲肝病毒,肠病毒,人柯萨奇病毒,鼻病毒,艾柯病毒),嵌杯样病毒(Caliciviridae)(例如,造成肠胃炎的株系),披膜病毒(例如马脑炎病毒,风疹病毒),黄色病毒(Flaviridae)(例如,登革病毒(dengue viruses),脑炎病毒(encephalitis viruses),黄热病毒(yellow fever viruses)],冠形病毒[Coronaviridae,例如冠形病毒(coronaviruses)],棒状病毒[Rhabdoviridae,例如,疱疹性口腔炎病毒(vesicularstomatitis viruses),狂犬病毒(rabies viruses)],线状病毒(Filoviridae)(例如,依波拉病毒(ebola viruses)),副粘病毒(Paramyxoviridae),例如副流感病毒,腮腺炎病毒,麻疹病毒,呼吸道合胞病毒],正粘病毒(Orthomyxoviridae](例如流感病毒),Bungaviridae[例如,汉坦病毒(Hantaan viruses),bungaviruses,白蛉病毒(phleboviruses)和纳依罗病毒(Nairoviruses)],沙粒病毒病毒(Arena viridae)(例如,出血热病毒),呼吸道肠道病毒(Reoviridae)[例如,呼肠病毒(reoviruses),环状病毒(orbiviruses)和轮状病毒(rotaviruses)],双RNA病毒(Birnaviridae),嗜肝DNA病毒(Hepadnaviridae)[例如,乙肝病毒(Hepatitis B virus)],细小病毒(Parvoviridae](细小病毒属),乳多空病毒(Papovaviridae)(例如,乳头瘤病毒(papilloma viruses),多瘤病毒(polyoma viruses)),腺病毒科(Adenoviridae)(大部分腺病毒)],疱疹病毒(Herpesviridae)(例如单纯疱疹病毒(HSV)1和2,水痘带状疱疹病毒(varicellazoster virus),巨细胞病毒(CMV),疱疹病毒],痘病毒[天花病毒,痘苗病毒,痘病毒],和虹彩病毒(Iridoviridae)(例如,African swine fever virus],以及一些未分类的病毒[例如,海绵脑病的致病因子,丁型肝炎致病因子(被认为是乙型肝炎病毒的缺陷型变体),非甲非乙肝炎的致病因子(1类=内部传染,2类=肠胃外传染(如丙型肝炎),Norwalk病毒以及相关的病毒,和星状病毒)]。
感染性细菌的实例包括:幽门螺杆菌(Helicobacterpyloris),布氏疏螺旋体(Borelia burgdorferi),侵肺军团菌(Legionella pneumophilia),分枝杆菌(Mycobacteria sps.)(例如,结核分枝杆菌(M.Tuberculosis),鸟分枝杆菌(M.avium),胞内分枝杆菌(M.intracellulare),堪萨斯分枝杆菌(M.kansaii),戈登分枝杆菌(M.gordonae)),金黄色葡萄球菌(Staphylococcus aureus),淋病奈瑟氏球菌(Neisseriagonorrhoeae),脑膜炎奈瑟氏球菌(Neisseria meningitidis),单核细胞增多性李斯特氏菌(Listeria monocytogenes),酿脓链球菌[Streptococcus pyogenes(A组链球菌)],无乳链球菌[Streptococcus agalactiae(B组链球菌)],链球菌属(viridans组),粪链球菌(Streptococcus faecalis),牛链球菌(Streptococcus bovis),链球菌属(厌氧菌),肺炎链球菌(Streptococcus pneumoniae),致病性弯曲杆菌(Campylobactersp.,),肠球菌(Enterococcus sp.,),流感嗜血杆菌(Haemophilusinfluenzae),炭疽芽孢杆菌(Bacillus antracis),白喉棒杆菌(Corynebacterium diphtheriae),棒杆菌属(Corynebacteriumsp.,),红斑丹毒丝菌(Erysipelothrix rhusiopathiae),产气荚膜梭菌(Clostridium perfringers),破伤风梭菌(Clostridiumtetani),产气肠杆菌(Enterobacter aerogenes),肺炎克雷伯氏菌(Klebsiella pneumoniae),多杀巴斯德氏菌(Pasturellamultocida),拟杆菌(Bacteroides sp.,),具核梭杆菌(Fusobacterium nucleatum),念珠状链杆菌(Streptobacillusmoniliformis),梅毒螺旋菌(Treponema pallidium),极细密螺旋体(Treponema pertenue),钩端螺旋体(Leptospira)和伊氏放线菌(Actinomyces israelli)。
感染性真菌的实例包括:新型隐球酵母(Cryptococcusneoformans),荚膜组织胞浆菌(Histoplasma capsulatum),粗球孢子菌(Coccidioides immitis),皮炎芽酵母(Blastomycesdermatitidis),沙眼衣原体(Chlamydia trachomatis),白假丝酵母(Candida albicans)。其它的感染性有机体(例如,原生动物)包括:恶性疟原虫(Plasmodium falciparum)和鼠弓形虫(Toxoplasma gondii)。
术语“免疫刺激性核酸分子”指的是这样的核酸分子,它们含有未甲基化的胞嘧啶、鸟嘌呤二核苷酸序列(即,“CpG DNA”或含有胞嘧啶并接着鸟苷且由磷酸酯键所连接的DNA)并刺激脊椎动物淋巴细胞(例如,对所述细胞具有促分裂效应,或者诱导或提高其细胞因子表达)。免疫刺激性核酸分子可以是双链的或单链的。一般地,双链分子在体内更为稳定,而单链分子则具有提高的免疫活性。
本发明一方面提供了一种分离的核酸序列,其含有至少一个未甲基化CpG二核苷酸,其通式如下:
5′N1X1CGX2N23′
其中至少一个核苷酸将连续的CpGs间隔开来;X1是腺嘌呤,鸟嘌呤,或胸腺嘧啶;X2是胞嘧啶或胸腺嘧啶;N是任何核苷酸,并且N1+N2是约0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且所述核酸序列的长度为约8-30个碱基。
在一个实施方案中,其中的X1是胸腺嘧啶。
在另一个实施方案中,其中所述的X2是胸腺嘧啶。
在又一个实施方案中,所述的核酸序列是GTCG(T/C)T或是TGACGTT。
在又一个实施方案中,所述的核酸序列是TGTCG(T/C)T。
在又一个实施方案中,所述的核酸序列是TCCATGTCGTTCCTGTCGTT。
在又一个实施方案中,所述的核酸序列是TCCTGACGTTCCTGACGTT。
在又一个实施方案中,所述的核酸序列是TCGTCGTTTTGTCGTTTTGTCGTT。
本发明另一方面提供了一种分离的核酸序列,其含有至少一个未甲基化CpG二核苷酸,其通式如下:
5′N X1X2CGX3X4N3′
其中至少一个核苷酸将连续的CpGs分开;X1X2选自GpT、GpG、GpA、ApT和ApA;X3X4选自TpT或CpT;N是任何核苷酸,并且N1+N2是约0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且所述核酸序列的长度为约8-30个碱基。
在一个实施方案中,其中所述的分隔至少两个连续CpGs的核苷酸是胸腺嘧啶。
在又一个实施方案中,其中所述的X3和X4是胸腺嘧啶。
在又一个实施方案中,其中至少一个核苷酸具有磷酸主链修饰。
在又一个实施方案中,其中所述的磷酸主链修饰是硫代磷酸酯修饰或二硫代磷酸酯修饰。
在又一个实施方案中,其中所述的磷酸主链修饰发生在核酸的5′端。
在又一个实施方案中,其中所述的修饰发生在该核酸5′端的前两个核苷酸间连键(linkage)。
在又一个实施方案中,其中所述的磷酸主链修饰发生在该核酸的3′端。
在又一个实施方案中,其中所述的修饰发生在该核酸3′端的最后5个核苷酸间连键。
本发明还提供了一种刺激受试者的免疫活化的方法,其中所述的刺激主要是Th1类型的免疫活化,该方法包括向受试者施用前述的核酸序列。在一个实施方案中,其中所述的受试者是人。
本发明提供了一种刺激受试者的细胞因子生产的方法,该方法包括给受试者施用前述的核酸序列。
在一个实施方案中,其中所述的细胞因子选自包括IL-6、IL-12、IFN-γ、TNF-α和GM-CSF的一组。
在又一个实施方案中,其中所述的受试者是人。
在又一个实施方案中,其中所述的核酸序列选自包括下述序列的组中:
TCCATGTCGCTCCTGATGCT,
TCCATAACGTTCCTGATGCT,
TCCATGACGATCCTGATGCT
TCCATGGCGGTCCTGATGCT
TCCATGTCGGTCCTGATGCT
TCCATAACGTCCCTGATGCT
TCCATGTCGTTCCTGATGCT,以及
TCGTCGTTTTGTCGTTTTGTCGTT。
本发明还提供了一种刺激受试者的NK裂解活性的方法,该方法包括向受试者施用上述通式的核酸序列。
在一个实施方案中,其中所述的受试者是人。
在又一个实施方案中,其中所述的核酸序列选自包括下述序列的组中:
TCGTCGTTGTCGTTGTCGTT,
TCCATGACGGTCCTGATGCT,
TCCATGACGATCCTGATGCT,
TCCATGACGCTCCTGATGCT,
TCCATGACGTTCCTGATGCT,
TCCATAACGTTCCTGATGCT,
TCCATCACGTGCCTGATGCT,
GGGGTCAACGTTGAGGGGGG,
TCGTCGTTTTGTCGTTTTGTCGTT,
TCGTCGTTGTCGTTTTGTCGTT,
GCGTGCGTTGTCGTTGTCGTT,
TGTCGTTTGTCGTTTGTCGTT,
TGTCGTTGTCGTTGTCGTT,以及
TCGTCGTCGTCGTT。
本发明还提供了一种刺激受试者的B细胞增殖的方法,该方法包括向受试者施用具有上述通式的核酸序列。
在一个实施方案中,其中所述的受试者是人。
在又一个实施方案中,其中所述的核酸序列选自包括下述序列的一组:
TCCTGTCGTTCCTTGTCGTT,
TCCTGTCGTTTTTTGTCGTT,
TCGTCGCTGTCTGCCCTTCTT,
TCGTCGCTGTTGTCGTTTCTT,
TCGTCGTTTTGTCGTTTTGTCGTT,
TCGTCGTTGTCGTTTTGTCGTT,以及
TGTCGTTGTCGTTGTCGTT。
本发明还提供了一种刺激受试者的免疫活化的方法,该方法包括向受试者施用具有上述通式的核酸序列,其中所述的核酸序列起佐剂的作用。
在一个实施方案中,其中所述的受试者是哺乳动物。
在另一个实施方案中,其中所述的核酸序列选自包括下述序列的组中:
TCCATGACGTTCCTGACGTT,
GTCG(T/C)T,以及
TGTCG(T/C)T。
本发明还提供了一种治疗患有气喘疾病的受试者的方法,该方法包括向该受试者施用处于药学可接受载体中的具有上文所示通式的核酸序列。在一个实施方案中,其中所述的受试者是人。在另一个实施方案中,其中所述的核酸序列是TCCATGACGTTCCTGACGTT。
本发明还提供了一种通过抑制CpG介导的淋巴细胞活化来治疗患有自身免疫疾病或其它的与CpG相关的疾病的受试者的方法,该方法包括向该受试者施用处于药学可接受载体中的内体酸化抑制物。在一个实施方案中,其中所述的受试者是人。在另一个实施方案中,其中所述的抑制物选自包括bafilomycin A、氯喹、和莫能菌素的组中。在又一个实施方案中,其中所述的抑制物的提供剂量小于约10μM。在又一个实施方案中,其中所述的疾病选自包括系统性红斑狼疮、脓毒症、炎性肠疾病、牛皮癣、龈炎、关节炎、克罗恩氏病(Crohn′sdisease)、格雷夫斯氏病(Grave′s disease)和气喘的组中。优选地,其中所述的疾病是系统性红斑狼疮。
在本发明的一个优选的实施方案中,提供了分离的免疫刺激性核酸序列,其含有如下所示的CpG基元:
5′N1X1CGX2N23′
其中,至少有一个核苷酸将连续的CpGs间隔开来;X1是腺嘌呤,鸟嘌呤,或胸腺嘧啶;X2是胞嘧啶或胸腺嘧啶;N是任何的核苷酸,且N1+N2是约0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该核酸序列的长度为约8-30个碱基。
在另一个实施方案中,本发明提供了分离的免疫刺激性核酸序列,其中含有下式所示的CpG基元:
5′N1X1X2CGX3X4N23′
其中,至少有一个核苷酸将连续的CpGs间隔开来;X1X2是选自GpT、GpG、GpA、ApT和ApA;X3X4是选自TpT或CpT;N是任何的核苷酸,且N1+N2是约0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该核酸序列的长度为约8-30个碱基。
优选的是,本发明的免疫刺激性核酸序列包括X1X2,它们选自GpT、GpG、GpA和ApA,且X3X4选自TpT、CpT和GpT(例如参见表5)。为了促进被摄入细胞中,含有CpG的免疫刺激性核酸分子优选为8-30个碱基的长度。然而,如果有足够的免疫刺激性基元存在,则任何长度的核酸(甚至数千个碱基对长)都可以是免疫刺激性的,因为这样的更大型的核酸在细胞内被降解为寡核苷酸。优选的合成寡核苷酸在其5′和/或3′端或附近不含有CCGG四元体或者多于一个的CCG或CGG三元体,和/或共有的促细胞分裂性CpG基元不是回文结构。用稳定化的寡核苷酸可以获得长久的免疫刺激,其中寡核苷酸中整合有磷酸酯主链的修饰。例如,当修饰为硫代磷酸酯或二硫代磷酸酯类的修饰时即是如此。另外,更具体地讲,磷酸酯主链修饰发生在核酸的5′端,例如在核酸5′端的最初两个核苷酸上。进而言之,磷酸酯主链修饰可以发生在核酸的3′端,例如在核酸3′端的最后5个核苷酸上。
优选地,所述免疫刺激性CpG DNA是寡核苷酸时,其长度为8-30个碱基。可选择地,CpG二核苷酸可以在质粒中大规模生产,当其被给予个体后,被降解为寡核苷酸。优选的免疫刺激性核酸分子[例如,用于提高疫苗的有效性,或在个体内通过刺激抗体(例如体液)应答来治疗免疫系统缺陷]具有相对高的针对B细胞、单核细胞和/或天然杀伤细胞反应的刺激指数(例如,细胞因子、增殖性、裂解性或其它的反应)。
本发明的核酸序列例如刺激受试者中的细胞因子产生。细胞因子包括但不限于IL-6、IL-12、IFN-γ、TNF-α、GM-CSF。这些序列的实例包括:TCCATGTCGCTCCTGATGCT(SEQID NO:42),TCCATGTCGTTCCTGATGCT(SEQ ID NO:43),和TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:56)。
本发明的核酸序列还可以用于刺激受试者例如人内天然杀伤细胞(NK)的裂解活性。这些序列的具体但非限制性的实例包括:
TCGTCGTTGTCGTTGTCGTT(SEQ ID NO:57),
TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:58),
TCGTCGTTGTCGTTTTGTCGTT(SEQ ID NO:59),
GCGTGCGTTGTCGTTGTCGTT(SEQ ID NO:),
TGTCGTTTGTCGTTTGTCGTT(SEQ ID NO:),
TGTCGTTGTCGTTGTCGTT(SEQ ID NO:60)和
TCGTCGTCGTCGTT(SEQ ID NO:61)。
本发明的核酸序列还可以刺激受试者例如人中B细胞增殖。具体的但非限制性的实例包括:
TCCTGTCGTTCCTTGTCGTT(SEQ ID NO:62),
TCCTGTCGTTTTTTGTCGTT(SEQ ID NO:63),
TCGTCGCTGTCTGCCCTTCTT(SEQ ID NO:64),
TCGTCGCTGTTGTCGTTTCTT(SEQ ID NO:65),
TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:66),
TCGTCGTTGTCGTTTTGTCGTT(SEQ ID NO:67),和
TGTCGTTGTCGTTGTCGTT(SEQ ID NO:68)。
在另一个方面,本发明的核酸序列可以用作佐剂,在哺乳动物中抗体生产期间使用。这种序列的具体但非限制性的实例包括TCCATGACGTTCCTGACGTT(SEQ ID NO:10),GTCG(T/C)T和TGTCG(T/C)T。进而言之,可以施用这些核酸序列,从而通过将受试者的免疫应答从Th2型改变为Th1型,以用于治疗或预防气喘疾病症状。代表性的序列包括TCCATGACGTTCCTGACGTT(SEQ ID NO:10)。
具体的免疫刺激性CpG DNA的刺激指数可以用各种免疫细胞检测来测定。优选地,免疫刺激性CpG DNA针对B细胞增殖的刺激指数为至少约5,优选至少约10,更优选地至少约15,最优选地至少约20,该刺激指数是通过在鼠B细胞培养物中掺入3H-尿苷而测定的,该B细胞培养物已在37℃下与20μM的ODN接触20小时,并且用1μCi的3H尿苷进行脉冲标记,并按照实施例1所述在4小时后收获和计数。对于体内的应用,例如通过刺激受试者中细胞介导的(局部)免疫反应用于治疗免疫系统缺陷,重要的是该免疫刺激性CpG DNA能够有效地诱导单核细胞的细胞因子分泌和/或天然杀伤细胞(NK)的细胞裂解活性。
优选的免疫刺激性CpG核酸应该可以导致产生至少约500pg/ml的TNF-α、15pg/ml的IFN-γ、70pg/ml的GM-CSF、275pg/ml的IL-6、200pg/ml的IL-12,这取决于治疗适应症,如按照实施例12中所述的检测法所测定的。其它优选的免疫刺激性CpG DNAs应能够产生至少约10%,更优选至少约15%,最优选至少约20%的YAC-1细胞特异性裂解,或至少约30%,更优选至少约35%,最优选至少约40%的2C11细胞特异性裂解,如通过实施例4中描述的检测法所测定的。
术语“核酸”或“DNA”指的是多个核苷酸{即,含有糖(如核糖或脱氧核糖)且其与磷酸酯基团和可互换的有机碱(其或者是取代的嘧啶[例如,胞嘧啶(C)、胸腺嘧啶(T)或尿嘧啶(U)]、或者取代的嘌呤[例如,腺嘌呤(A),鸟嘌呤(G)])相连接。在本文中,该术语指的是核糖核苷酸以及寡脱氧核糖核苷酸。该术语还应该包括多核苷(即,多核苷酸减去磷酸)以及含有有机碱的任何其它聚合物。核酸分子可以从已经存在的核酸来源获得(例如,基因组或cDNA),但是,优选合成性的(例如,由寡核苷酸合成所产生的)。
术语“核酸递送复合物”指的是与靶向装置[例如,对靶细胞(如B细胞和天然杀伤(NK)细胞)表面有较高亲和结合性和/或提高靶细胞的细胞摄入的分子]结合在一起(通过离子键或共价键结合,或包裹在其内)的核酸分子。核酸递送复合物的实例包括与下列物质结合的核酸:固醇(例如,胆固醇)、脂类(例如,阳离子脂质、病毒体、或脂质体)、或靶细胞特异性结合剂(例如,由靶细胞特异性受体所识别的配体)。优选的复合物必须在体内足够稳定,可以防止在被靶细胞内化之前被显著解偶联。然而,这些复合物应能够在细胞内在适宜的条件下被剪切,使核酸以功能性的形式被释放。
术语“回文序列”指的是反向的重复(即,如ABCDEE′D′C′B′A′这样的序列,其中A和A′是能够形成常规的Watson-Crick碱基对的碱基。在体内,这样的序列可能形成双链结构。
术语“稳定化的核酸分子”指的是对体内降解(例如,通过外切核酸酶或内切核酸酶的降解)具有相对抵抗性的核酸分子。稳定可以是长度或二级结构的函数。长度为数十至数百kb的含有未甲基化CpG的核酸分子对体内降解有相对的抵抗性。对于较短的免疫刺激性核酸分子,二级结构可以稳定和提高其效果。例如,如果核酸分子的3′端具有对上游区的自我互补性,使它可以回折并形成某种茎-环结构,那么该核酸分子就变为稳定化的,并因而表现出更多活性。
优选的本发明的稳定化核酸分子具有被修饰的主链。用于免疫刺激时,特别优选的稳定化核酸分子是硫代磷酸酯(即,核酸分子的至少一个磷酸氧被硫所取代)或二硫代磷酸酯修饰的核酸分子。更具体地讲,磷酸酯主链修饰发生在核酸的5′端,例如在核酸5′端的最初两个核苷酸处。进而言之,磷酸酯主链修饰可以发生在核酸的3′端,例如发生在核酸3′端的最后5个核苷酸处。除了稳定核酸分子外,正如本文中进一步描述的那样,硫代磷酸酯-修饰(包括二硫代磷酸酯-修饰)的核酸分子可以提高如本文所示含有未甲基化CpG二核苷酸的核酸分子的免疫刺激程度。题目为“硫代磷酸酯寡核苷酸类似物的免疫刺激”的国际专利申请公开第WO95/26204号也报道了硫代磷酸酯修饰的寡核苷酸的非序列特异性免疫刺激效果。如本文所描述的,含有未甲基化CpG、并具有硫代磷酸酯主链的核酸分子已经被发现优先活化B细胞活性,而含有未甲基化CpG并具有磷酸二酯主链的核酸分子已经被发现优先活化单核细胞(巨噬细胞、树状细胞和单核细胞)和NK细胞。带有优选的人基元的硫代磷酸酯CpG寡核苷酸也是单核细胞和NK细胞的强活化子。
其它的稳定化核酸分子包括:非离子性DNA类似物,例如烷基和芳基膦酸酯(其中带电的膦酸氧被烷基或芳基所取代),磷酸二酯和烷基磷酸三酯,其中带电的氧部分被烷基化。在任一末端或两末端含带有二醇例如四乙二醇或六乙二醇的核酸分子,已经显示对核酸酶降解有实质性抗性。
术语“受试者”指的是人或脊椎动物,包括狗、猫、马、牛、猪、绵羊、山羊、鸡、猴子、大鼠,和小鼠。
在本文中,术语“载体”指的是能够运送与之相连的另一核酸的核酸分子。优选的载体是能够对与之相连的核酸进行自我复制和表达的载体(例如,附加体)。能够指导与之可操作性相连的基因的表达的载体在本文中被称为“表达载体”。总体来说,在重组DNA技术中有用的表达载体经常都是“质粒”形式,其一般指的是双链DNA环,其在载体形式时不与染色体结合。在本说明书中,术语“质粒”和“载体”是可以相互替换使用的,因为质粒是最常用形式的载体。然而,本发明也意图包括可发挥等同功能以及后来为本领域所知的这种其它形式的载体。
某些含有未甲基化CpG的核酸在体内和体外显示具有B细胞 刺激活性
在根据随后的实施例1和2中描述的方案进行的对内源逆转录病毒序列具有特异性的两种反义寡核苷酸的淋巴细胞刺激效应的研究中,令人惊奇地发现了在24个“对照”(对于“反义”ODN图,包括各种杂乱的、有义的、和错配的对照)中的2个也介导了B细胞活化和IgM分泌,而其它的“对照”则不具有这样的效果。
这两个研究的结果表明,这种“对照”ODN的B细胞活化机制可能不涉及反义效果,1)通过与基因库(GenBank)所列的脊椎动物DNA序列相比较,没有显示出比那些没有刺激性的ODN更高的同源性,以及2)这两个对照在使用10μg的脾poly A+RNA的Northen印迹中没有显示出杂交。在不同的合成器上重合成这些ODN,或用聚丙烯酰胺凝胶电泳或高压液相色谱来充分提纯,都得到相同的刺激,排除了杂质的可能性。采用来自C3H/HeJ小鼠的B细胞也见到了类似的刺激,这排除了脂多糖(LPS)污染可能导致这些结果的可能性。
两个“对照”ODN所造成的B细胞活化与两个“反义”ODN造成的活化相类似这一事实提出了这样的可能性,即所有四个ODN对B细胞的刺激通过某种非反义机制进行的,该机制涉及在所有的其它非刺激性对照ODN中都不存在的序列基元。比较这些序列时发现,所有四个刺激性ODN都含有处于与非刺激性对照不同的序列环境中的CpG二核苷酸。
为了确定存在于刺激性ODN中的CpG基元是否负责所观察到的刺激作用,合成了超过300种ODN,它们的长度范围是5-42个碱基,并含有处于不同序列环境下的甲基化或未甲基化的CpG二核苷酸,或者不含有CpG二核苷酸。对这些ODN,包括两个原始的“对照”(即ODN 1和2)以及两个原始合成的“反义”物[ODN 3D和3M;Krieg,A.M.J.Immunol.143:2448(1989)],检查它们对脾细胞的体外影响(代表性序列在表1中给出)。含有CpG二核苷酸的几种ODN诱导了B细胞的活化和IgM的分泌;这种刺激的程度一般可以由可通过增加更多的CpG二核苷酸来提高(表1;比较ODN 2与2a或3D与3Da和3Db)。看起来刺激不是反义机制或杂质所致。ODN没有造成可检测的γδ或其它T细胞群体的增殖。
如果CpG二核苷酸发生突变,或如果CpG二核苷酸的胞嘧啶被5-甲基胞嘧啶取代(表1;ODN 1b,2b,3Dd,和3Mb),则促细胞分裂性ODN序列都一致地变为非刺激性的(表1;比较ODN 1与1a;3D与3Dc;3M与3Ma;以及4与4a)。CpG基元的部分甲基化造成刺激效果的部分损失(比较2a与2c,表1)。相对照而言,对其它胞嘧啶的甲基化没有减少ODN活性(ODN 1c,2d,3De和3Mc)。这些数据确认了CpG基元是ODN中存在的活化B细胞的必需元件。
在这些研究的过程中,变得更为清楚的是,在CpG二核苷酸侧翼的碱基在由ODN诱导的小鼠B细胞活化的确定中起着重要的作用。已确定最佳的刺激基元包括有以两个5′嘌呤(优选为GpA二核苷酸)和两个3′嘧啶(优选TpT或TpC二核苷酸)为侧翼的CpG。将此CpG基元更类似这种理想形态的ODN突变改善了刺激性(例如,表1,比较ODN 2与2e;3M与3Md),而那些扰乱该基元的突变则减弱了刺激性(见表1,比较ODN3D与3Df;4与4b,4c和4d)。另一方面,在CpG基元以外的突变没有减弱刺激性(例如,表1,比较ODN 1与1d;3D与3Dg;3M与3Me)。对于活化人细胞,最佳的侧翼碱基是稍有不同的(见表5)。
在那些实验过的ODNs中,长度小于8个碱基的ODN都没有刺激性(例如,表1,ODN 4e)。在所测试过的48个长度为8个碱基的ODN中,确定了具有强刺激性的序列是TCAACGTT(ODN 4),其含有自我互补的“回文序列”AACGTT。在对该基元的进一步优化中,发现在两个末端都有G的ODN具有更强的刺激性,当ODN被硫代磷酸酯修饰了末端的核苷酸间连键而具有对核酸酶的抗性后更是如此。ODN1585[5′GGGGTCAACGTTCAGGGGGG3′(SEQ ID NO:12)]中,前两个和最后5个核苷酸间连键都被硫代磷酸酯所修饰,该ODN使得小鼠脾细胞增殖平均提高了25.4倍,而相比之下,由ODN 1638所造成的增殖只增加了平均3.2倍,该ODN 1638与ODN 1585具有相同的序列,只是在两末端的10个G被10个A所取代。富含G的末端的效果是顺式的,将具有polyG末端但不含CpG基元的ODN与1638一起加入细胞,也不会导致增殖增加。对于长度大于8个碱基对的核酸分子,含有未甲基化CpG的非回文序列基元被发现是更具有刺激性的。
含有6碱基回文序列并在5′端有TpC二核苷酸的八聚ODN也是活性的(见表1,ODN 4b,4c)。在5′端的其它二核苷酸也减弱刺激性(例如,ODN 4f,对所有可能的16种二核苷酸都进行了测试)。有3′端二核苷酸存在并不足以补偿5′端二核苷酸的缺失(见表1,ODN 4g)。对回文序列的破坏就消除了八聚ODN的刺激性(例如,表1,ODN 4h),但是,在更长的ODN中,回文序列不是必须的。
表1:寡核苷酸对小鼠B细胞的刺激
Figure A20071013005200471
Figure A20071013005200481
′刺激指数是由至少三个单独实验得到的平均值和标准偏差,并且与没有加入ODN进行培养的小孔进行比较。
ND=未进行。
CpG二核苷酸用下划线标出。
黑点表示相同,短线表示缺失。
Z代表5甲基胞嘧啶。
表2:鉴定小鼠IL-6生产和B细胞活化的最优CpG基元
Figure A20071013005200482
黑点代表相同;CpG二核苷酸用下划线标出;ND=未进行
a该实验进行至少三次,得到相似结果。CH12.LX和脾B细胞的未受刺激的对照培养物的IL-6水平≤10pg/ml。未受刺激的培养物的IgM水平为547±82ng/ml。CpG二核苷酸用下划线标出,黑点代表相同。
b[3H]尿嘧啶吸收用对未受刺激的对照(2322.67±213.68cpm)而言的增加倍数表示(SI:刺激指数)。细胞用20μM各种CpG O-ODN刺激。数据代表一式三份的平均值±标准偏差。
c用ELISA测定。
对淋巴细胞活化的动力学的研究采用了小鼠脾细胞。在加入ODN的同时对细胞进行脉冲,4小时后立即收获,3H-鸟嘧啶掺入已经有2倍的增加。刺激在12-48小时达峰值,然后降低。24小时后,检测不到完整的ODN,这也许就是当纯化的B细胞在有或没有抗-IgM(亚促细胞分裂剂量)条件下与CpG ODN一起培养时发生随后刺激下降的原因,还发现在48小时后,这两种促细胞分裂原相组合造成增殖协同性地增加了约10倍。刺激的幅度是浓度依赖性的,并且在针对二者的优化条件下都一致地超过LPS的刺激幅度。含有抗核酸酶的硫代磷酸酯主链的寡核苷酸比未修饰的寡核苷酸强大约200倍。
采用细胞周期分析来确定由CpG-ODN活化的B细胞的比例。CpG-ODN在多于95%的B细胞中诱导了进入细胞周期。通过流式细胞术分拣成CD23-(边缘区)和CD23+(小泡区)亚群的脾B淋巴细胞对ODN诱导的刺激都有同样的反应,采用Percoll梯度通过分级分离的静息和活化B细胞群也是如此。这些研究表明,CpG-ODN诱导几乎所有的B细胞进入细胞周期。
免疫刺激性核酸分子阻断小鼠B细胞编程性细胞死亡
有些B细胞系,例如WEHI-231,应答于它们的抗原受体被抗-IgM的交联而被诱导经历生长停滞和/或编程性细胞死亡,[Jakway,J.P.et al.,″Growth regulation of the Blymphoma cell line WEHI-231 by anti-immunoglobulin,lipopolysaccharide and other bacterial products″J.Immunol.137:2225(1986);Tsubata,T.,J.Wu and T.Honjo:B-cellapoptosis indueced by antigen receptor corsslinking is blockedby a T-cell signal through CD40.″Nature 364:645(1993)]。用某些刺激物如LPS和CD40配体可以将WEHI-231细胞从这种生长停滞状态下恢复出来。含有CpG基元的ODN也被发现可以保护WEHI-231细胞不发生抗-IgM诱导的生长停滞,表明这种效果不需要辅助细胞群。随后的研究工作表明,CpG ODN诱导了Bcl-x和myc表达,它们可能对是免于编程性细胞死亡的保护作用的原因。再者,CpG核酸被发现可以阻断人细胞中的编程性细胞死亡。这种对编程性细胞死亡的抑制具有重要性,因为它将提高和延长CpG DNA的免疫活化。
鉴定诱导小鼠IL-6和IgM分泌以及B细胞增殖的为最优化 CpG基元
为了确定最优化的B细胞刺激性CpG基元是否与对IL-6分泌的最优化CpG基元相同,研究了一组ODN,它们的CpG二核苷酸侧翼的碱基被逐步取代。使用脾B细胞和CH12.LX细胞对该组ODN分析它们对B细胞增殖、Ig生产、IL-6分泌的影响。如表2所示,优化的刺激性基元含有未甲基化的CpG,侧翼为两个5′嘌呤和两个3′嘧啶。一般地说,5′嘌呤向3′嘧啶的突变或者3′嘧啶向嘌呤的突变都显著地降低这种效应。从5′嘌呤向C的变化是特别的有破坏性的,但是,从5′嘌呤向T的变化或3′嘧啶向嘌呤的变化所产生的影响就比较小。根据这些分析以及其它ODN的结果,确定了诱导IL-6分泌的优化CpG基元为TGACGTT,其与最佳的促细胞分裂和IgM诱导性CpG基元相同(表2)。该基元比任何所研究过的含有回文序列的序列都更具有刺激性(1639,1707和1708)。
细菌DNA中或寡核苷酸中的CpG基元对小鼠细胞因子分泌的 诱导
如在实施例9中所描述的那样,CpG DNA刺激后脾细胞分泌的IL-6的量用ELISA测定。在最初的研究发现T细胞对CpG DNA刺激的脾细胞生产的IL-6几乎没有或没有任何作用后没有任何作用后,不采用全脾细胞,而采用去除了T细胞的脾细胞培养物进行体外研究,如表3所示,在用大肠杆菌DNA培养的细胞中,IL-6的生产显著增加,而用小牛胸腺DNA培养的细胞则没有提高。为了确认用大肠杆菌DNA所观察到的IL-6生产的增加不是由于其它细菌产物的污染所造成的,在进行分析之前,对DNA用DNA酶进行消化。这种DNA酶的预处理消除了由大肠杆菌DNA诱导的IL-6的生产(表3)。此外,从LPS-非应答性C3H/HeJ小鼠来源的脾细胞响应于细菌DNA而产生相似水平的IL-6。为了研究由大肠杆菌DNA诱导的IL-6的分泌是否由细菌DNA中的未甲基化CpG二核苷酸所介导,检验了甲基化的大肠杆菌DNA和一组合成性ODN。如表3所示,CpG ODN显著地诱导了IL-6的分泌(ODN 5a,5b,5c),而CpG甲基化大肠杆菌DNA,或含有甲基化CpG的ODN(ODN 5f),或不含CpG的ODN(ODN 5d)则都没有发生诱导。在CpG二核苷酸以外位置的改变(ODN 5b),或者其它胞嘧啶的甲基化(ODN 5g)都没有减弱CpG ODN的效果。在含有三个CpG的ODN中单个CpG的甲基化导致了刺激性的部分降低(表3,比较ODN 5c和5e)。
表3:细菌DNA中或寡核苷酸中的CpG基元诱导小鼠IL-6分泌
  处理   IL-6(pg/ml)
  小牛胸腺DNA   ≤10
  小牛胸腺DNA+DNase   ≤10
  大肠杆菌DNA   1169.5±94.1
  大肠杆菌DNA+DNase   ≤10
  CpG甲基化大肠杆菌DNA   ≤10
  LPS   280.1±17.1
  培养基(没有DNA)   ≤10
  ODN
  5a SEQ ID NO:1 ATGGACTCTCCAGCGTTCTC   1096.4±372.0
  5b SEQ ID NO:2 ■■■■■AGG■■■■A■■■■■■■   1124.5±126.2
  5c SEQ ID NO:3 ■■C■■■■■■■G■■■■■■■■■   1783.0±189.5
  5d SEQ ID NO:4 ■■■■AGG■■C■■T■■■■■■■   ≤10
  5e SEQ ID NO:5 ■■C■■■■■■■G■■Z■■■■■■   851.1±114.4
  5f SEQ ID NO:6 ■■Z■■■■■■ZG■■Z■■■■■■   ≤10
  5g SEQ ID NO:7 ■■C■■■■■■■G■■■■■■Z■■   1862.3±87.26
来自DBA/2小鼠的去除了T细胞的脾细胞用磷酸二酯修饰的寡核苷酸(O-ODN)(20μM)、小牛胸腺DNA(50μg/ml)、大肠杆菌DNA(50μg/ml)(经过或不经过酶处理)、或LPS(10μg/ml)分别刺激24小时。数据代表一式三份实验的平均值±SD。CpG二核苷酸用下划线标出,黑点代表相同。Z表示5-甲基胞嘧啶。
CpG基元可以被用作人工佐剂
免疫应答的非特异性刺激物已知是佐剂。使用佐剂对于诱导针对可溶性抗原的强抗体应答是必需的(Harlow and Lane,Antibodies:A Laboratory manual,Cold Spring harbor,N.Y.Current Edition;通过在此引述而合并于本文)。佐剂的总体效应是强烈的,但它们的重要性不能被过分强调。佐剂的作用允许用更小剂量的抗原并产生更为一致的抗体应答。免疫应答的非特异性活化经常可以解释成功或未成功获得免疫应答之间的区别。应该在第一次注射的时候使用佐剂,除非有非常特殊的理由要求避免这样做。大多数的佐剂含有两种成分。一种成分是用来保护抗原不被快速分解代谢[例如,脂质体或合成的表面活性剂(Hunter et al.,1981)]。脂质体仅仅在免疫原被整合到外脂层时有效,内埋的分子是免疫系统看不见的。另一成分则是非特异性刺激免疫应答的物质。这些物质的作用是提高淋巴因子的水平。淋巴因子直接刺激抗原加工细胞的活性,并引起注射位置的局部发炎反应。早期的工作完全依赖于热杀死细菌(Dienes 1936)或脂多糖(LPS)(Johnson et al.,1956)。LPS是相当有毒的,通过对它的结构成分的分析,已发现其作为佐剂的大多数的性质存在于称作脂质A的部分。脂质A可以以多种合成的和天然的形式获得,其毒性远远小于LPS,但仍然保留了其母体LPS分子的大多数良好的佐剂性质。脂质A化合物经常通过脂质体递送。
近来,寻找强有力的、副作用更可接受的佐剂的深入工作导致了新的合成佐剂的产生。在本发明中,我们提供了序列1826,即TCCATGACGTTCCTGACGTT(SEQ ID NO:10),它是包括含CpG的核酸的佐剂。该序列是强的免疫活化序列,并且是非常优秀的佐剂,其效力完全与弗氏佐剂相当或更为优越,但是又没有明显的毒性。
确定CpG基元对小鼠IL-6分泌的诱导水平
细菌DNA和CpG ODN以剂量依赖性方式诱导去除了T细胞的小鼠脾细胞中的IL-6生产,但是,脊椎动物DNA和非CpG ODN则不是(图1)。IL-6的生产在大约50μg/ml细菌DNA或40μM的CpG O-ODN达到平台。由细菌DNA和CpG ODN诱导的IL-6的最大水平分别是1-1.5ng/ml和2-4ng/ml。这些水平比在LPS刺激后所见到的(0.35ng/ml)要明显地高(图1A)。为了评价具有核酸酶抗性DNA主链的CpG ODN是否也可以诱导IL-6的生产,向去除了T细胞的小鼠脾细胞中加入S-ODN。CpG S-ODN也以剂量依赖型方式诱导IL-6的生产,水平类似于CpG O-ODN,而非CpG S-ODN则没有诱导IL-6的生产(图1C)。浓度为0.05μM的CpG S-ODN可以在这些细胞中诱导最大的IL-6生产。这个结果表明,核酸酶抗性的DNA主链修饰保留了CpG DNA对诱导IL-6分泌的序列特异性能力,并且在该检测系统中CpG S-ODN比CpG ODN的效力高出80倍以上。
CpG DNA在体内对小鼠IL-6分泌的诱导
为了评价细菌DNA和CpG S-ODN体内诱导IL-6分泌的能力,给BALB/c小鼠静脉注射内100μg大肠杆菌DNA、小牛胸腺DNA、CpG或非刺激性S-ODN,2小时后放血。在注射了大肠杆菌DNA的组中,血清内IL-6水平为约13ng/ml,而在注射了小牛胸腺DNA的组或注射了PBS的组中,血清内没有检测到IL-6(表4)。CpG S-ODN也诱导了体内IL-6分泌。在注射了CpG S-ODN的组中,血清内IL-6的水平为约20ng/ml。相比之下,在注射了非刺激性S-ODN的组中,在其血清内没有检测到IL-6(表4)。
表4.体内CpG DNA刺激诱导的小鼠IL-6分泌
  刺激物   IL-6(pg/ml)
  PBS   <50
  大肠杆菌DNA   13858±3143
  小牛胸腺DNA   <50
  CpG S-ODN   20715±606
  非-CpG S-ODN   <50
小鼠(每组2只)接受静脉内注射100μl的PBS、200μg的大肠杆菌DNA或小牛胸腺DNA、或500μg的CpG S-ODN或非-CpG对照S-ODN。注射后2小时对小鼠采血,按1∶10稀释,每种血清用ELISA测定IL-6。ELISA测定IL-6的灵敏度限度为5pg/ml。CpG S-ODN的序列是5′GCATGACGTTGAGCT3′(SEQ ID NO:48),非刺激性S-ODN的序列是5′TCTAGATGTTAGCGT3′(SEQ ID NO:49)。应该注意的是,虽然在序列48中有CpG,但如本文中所解释的,它太靠近3′端而不能引起刺激作用。数据代表一式两份实验的平均值±标准偏差。实验进行至少两次且结果类似。
体内CpG基元刺激后小鼠IL-6分泌的动力学
为了评价CpG DNA在体内诱导IL-6分泌的动力学,对BALB/c小鼠静脉注射CpG或对照非-CpG S-ODN。在注射了CpG S-ODN的组中,血清IL-6水平在1小时内就明显提高,并且在2小时达到峰值水平约9ng/ml(图2)。血清中IL-6蛋白质水平在4小时后快速下降,并在刺激后12小时时恢复到基础水平。与CpG DNA刺激的组相反,在注射了非刺激性S-ODN或PBS的组中,血清内IL-6没有明显提高(图2)。
CpG基元在体内诱导的IL-6mRNA表达的动力学和组织分布
如图2所示,在CpG DAN刺激后,血清IL-6水平迅速提高。为了调查这种血清IL-6的可能的组织起源以及CpGDNA刺激后体内IL-6基因表达的动力学,对BALB/c小鼠静脉内注射CpG或非CpG S-ODN,并在刺激后的不同时间点从肝、脾、胸腺和骨髓提取RNA。如图3A所示,注射CpG S-ODN后30分钟内,在肝、脾、和胸腺内的IL-6mRNA水平提高。肝内IL-6mRNA在注射后2小时达到峰值,随后迅速下降并在刺激后8小时回复到基础水平(图3A)。脾内IL-6 mRNA在刺激后2小时达到峰值,然后逐渐降低(图3A)。胸腺内IL-6mRNA在注射后1小时达到峰值,然后逐渐降低(图3A)。在注射CpG S-ODN后1小时内,骨髓内IL-6 mRAN明显提高,然后恢复到基础水平。作为对CpG S-ODN的应答,肝、脾和胸腺显示IL-6 mRNA表达的提高明显高于骨髓。
CpG DNA诱导的小鼠细胞因子表达的形式
在体内或在全脾细胞中,在最初的6个小时内,下列的白细胞介素IL-2、IL-3、IL-4、IL-5或IL-10都没有检测到其蛋白质水平的显著提高[Ilinman,D.M.et al.,(1996)Proc.Natl.Acad.Sci.USA 93:2879-2883]。然而,在注射CpG ODN的小鼠血清中,TNF-α的水平在30分钟内提高,IL-6的水平在2小时内显著地升高。在脾细胞中,在首先的2小时内也检测到IL-12和γ-干扰素(IFN-γ)mRNA的表达提高。
表5.CpG寡核苷酸诱导人PBMC细胞因子的分泌
  ODN   序列(5′-3′)   IL-61   TNF-α1   IFN-γ1   GM-CSF   IL-12
  512SEQ ID NO:31   TCCATGTCGGTCCTGATGCT 500 140 15.6 70 250
  1637SEQ ID NO:38   ■■■■■■C■■■■■■■■■■■■■   550   16   7.8   15.6   16
  1615SEQ ID NO:39 ■■■■■■G■■■■■■■■■■■■■ 600 145 7.8 45 145
  1614SEQ ID NO:40 ■■■■■■A■■■■■■■■■■■■■ 550 31 0 50 31
  1636SEQ ID NO:41   ■■■■■■■■■A■■■■■■■■■■   325   250   35   40   250
  1634SEQ ID NO:42   ■■■■■■■■■C■■■■■■■■■■   300   400   40   85   400
  1619SEQ ID NO:43 ■■■■■■■■■T■■■■■■■■■■ 275 450 200 80 450
  1618SEQ ID NO:44   ■■■■■■A■■T■■■■■■■■■■   300   60   15.6   15.6   62
  1639SEQ ID NO:45   ■■■■■AA■■T■■■■■■■■■■   625   220   15.6   40   220
  1707SEQ ID NO:46 ■■■■■■A■■TC■■■■■■■■■ 300 70 17 0 70
  1708SEQ ID NO:47   ■■■■■CA■■TG■■■■■■■■■   270   10   17   ND   10
黑点代表相同,CpG二核苷酸用下划线标出。
1由ELISA测定,采用了来自R&D System的Quantikine试剂盒(pg/ml)。在收集上清液和检测前,细胞培养在10%自身血清并加以给出剂量的寡脱氧核苷酸(12μg/ml),对TNF-α为4小时,对其它的细胞因子为24小时。数据代表相对于没有加入寡脱氧核苷酸的小孔而言细胞因子的提高水平。
CpG DNA诱导的人PBMC,特别是单核细胞的细胞因子分泌
采用了与研究小鼠细胞因子表达的同一组ODN,来确定人细胞是否也被CpG基元诱导表达细胞因子(或增殖),并确定有关的CpG基元。寡核苷酸1619(GTCGTT)是TNF-α和IFN-γ分泌的最好的诱导物,寡核苷酸1634中与其几乎相同的基元(GTCGCT)则紧随其后(表5)。在寡脱氧核苷酸1637和1614中的基元(GCCGGT和GACGGT)导致强烈的IL-6分泌,但对其它的细胞因子则相对没有诱导。因此,似乎人淋巴细胞同小鼠淋巴细胞一样,应答于CpG二核苷酸有差异地而分泌细胞因子,具体由其周围的碱基所决定。然而,对刺激小鼠细胞为最好的基元则不同于对人细胞最有效的基元。某些CpG寡脱氧核苷酸对活化人细胞是很差的(寡脱氧核苷酸1707、1708,它们分别含有形成回文的序列GACGTC和CACGTG)。
对DNA应答的细胞似乎是单核细胞,因为用L-亮氨酰-亮氨酸甲基酯(L-LME)处理细胞可以消除细胞因子的分泌,该物质对单核细胞有选择性毒性(对细胞毒性T淋巴细胞和NK细胞也有选择性毒性),并不影响B细胞的Ig分泌(表6)。经过L-LME处理而存活的细胞用锥虫蓝排斥法检测到有>95%的存活率,这表明,这些细胞缺少细胞因子应答并不简单反映所有细胞类型的非特异性死亡。应答大肠杆菌(EC)DNA的细胞因子分泌需要未甲基化CpG基元,因为EC DNA甲基化消除了这种作用(表6中倒数第二行)。LPS对DNA的污染并不能解释这些结果,因为在天然的和甲基化的DNA中污染水平是相同的,而且,加入两倍最高水平的污染LPS也没有产生效应(未示出)。
表6.CpG DNA诱导人PBMC的细胞因子分泌
  DNA   TNF-α(pg/ml)1   IL-6(pg/ml)   IFN-γ(pg/ml)   RANTES(pg/ml)
  EC DNA(50μg/ml)   900   12,000   700   1560
  EC DNA(5μg/ml)   850   11,000   400   750
  EC DNA(0.5μg/ml)   500   ND   200   0
  EC DNA(0.05μg/ml)   62.5   10,000   15.6   0
  EC DNA(50μg/ml)+L-LME2   0   ND   ND   ND
  EC DNA(10μg/ml)Methyl3   0   5   ND   ND
  CT DNA(50μg/ml)   0   600   0   0
1所有细胞因子的水平都用ELISA进行测定,采用的是R&D Systems的Quantikine试剂盒,如同前面的表中描述的一样。结果代表从不同供体来的PBMC的情况。
2对细胞用L-亮氨酰-L-亮氨酸甲基酯(M-LME)预处理15分钟,以确定在这样的条件下细胞因子的产生是否是来自单核细胞(或来自于其它的对L-LME敏感的细胞)。
3按照生产商的指示,对大肠杆菌DNA用2U/μg DNA的CpG甲基化酶(New England Biolabs)甲基化,以HpA-II和Msp-I酶解来确认甲基化。作为阴性对照,包括了含有在该实验条件下不能诱导可检测细胞因子生产的最高浓度大肠杆菌DNA中包含的最大量LPS两倍水平的样品。
ND=未进行。
用L-LME处理的PBMC中细胞因子生产的丧失这个现象暗示单核细胞也许对应答CpG DNA的细胞因子生产负责。为了更直接地验证这个假设,测试了CpG DNA对高度纯化的人单核细胞和巨噬细胞的效果。如同所假设的那样,CpG DNA直接活化人巨噬细胞对细胞因子IL-6、GM-CSF和TNF-α的生产,而非CpG DNA则不能(表7)。
表7.CpG DNA诱导纯化的人巨噬细胞中细胞因子的表达
  IL-6(pg/ml)   GM-CSF(pg/ml)   TNF-α(pg/ml)
  仅有细胞   0   0   0
  CT DNA(50μg/ml)   0   0   0
  EC DNA(50μg/ml)   2000   15.6   1000
IL-6在应答于CpG基元而诱导小鼠IgM生产中的生物学作
上述的动力学研究揭示了在CpG刺激后1小时内发生的IL-6分泌的诱导早于IgM分泌。因为对ODN诱导IL-6分泌的最佳CpG基元与IgM的一样(表2),所以检测了CpG基元是否独立诱导IgM和IL-6生产,或者IgM的生产依赖于早前的IL-6分泌。加入中和性抗-IL-6抗体抑制了由CpG ODN介导的剂量依赖型的体外IgM生产,但是对照抗体则不能(图4A)。相对照的是,加入IL-6则没有影响B细胞增殖的基础水平能够和CpG诱导的B细胞增殖(图4B)。
应答CpG DNA的IL-6启动子的转录活性提高
用CpG DNA刺激后的IL-6mRNA和蛋白质水平的提高可以是由于转录调节或转录后调节的结果。为了确定IL-6启动子的转录活性是否在与CpG ODN一起培养的B细胞中被上调,用IL-6启动子-CAT构建物(pIL-6/CAT)(Pottratz,S.T.etal.,17B-estradio Inhibits expression of humaninterleuking-6-promoter-reporter constructs by areceptor-dependent mechanism.J.Clin.Invest.93:944)转染应答CpG DNA产生IL-6的小鼠B细胞系WEHI-231,在用各种浓度的CpG或非CpG ODN刺激后进行CAT检测。如图5所示,CpG ODN诱导的CAT活性的提高是剂量依赖型的,而非CpGODN则没有诱导CAT活性。这就肯定了CpG诱导IL-6启动子的转录活性。
CpG ODN的B细胞活化对5′和3′硫代磷酸酯核苷酸间连键数 目的依赖性
为了确定ODN主链上的部分硫修饰是否足以提高B细胞的活化,测试了一系列具有相同的序列但在5′和3′端的S核苷酸间连键数目不同的ODN的效果。根据以前的对核酸酶降解ODN的研究,已确定需要在ODN的5′端有至少两个硫代磷酸酯连键才可以最佳地保护ODN不被细胞内外切核酸酶和内切核酸酶所降解。因此,只对含有两个5′硫代磷酸酯修饰的连键以及可变数目的3′修饰连键的嵌合性ODN进行了检测。
测试了这些ODN的淋巴细胞刺激效果,其中使用了三种浓度(3.3,10,和30μM),方法是测定在处理过的脾细胞培养物中RNA合成(由3H尿苷掺入)或DNA合成(有3H胸苷掺入)总水平(实施例10)。带有CpG基元的O-ODN(0/0硫代磷酸酯修饰)对脾细胞没有刺激,除非以的浓度至少10μM加入培养基中(实施例10)。然而,当这个序列在5′端被两个S连接修饰和在3′端被至少3个S连接修饰时,则在3.3μM的剂量时有显著的刺激表现出来。在这种低剂量的条件下,随着3′端被修饰碱基数目的增加,刺激水平显示逐渐增加,直到这种碱基数达到或超过6个,此时刺激指数开始下降。一般地讲,对脾细胞刺激的最佳3′S连键数目为5。在这些实验中测试的所有3种浓度中,S-ODN都比最佳嵌合性化合物的刺激性小。
CpG介导的淋巴细胞活化对主链修饰类型的依赖性
硫代磷酸酯修饰的ODN(S-ODN)比磷酸二酯修饰的ODN(O-ODN)具有远远更强的核酸酶抗性。因此,由S-ODN和S-O-ODN(即,嵌合性硫代磷酸酯ODN,其中的中央连键是磷酸二酯,但有2个5′和5个3′连键是硫代磷酸酯修饰的)所造成的比O-ODN更高的免疫刺激可能是前者对核酸酶的抗性的结果。为了确定ODN核酸酶抗性在CpG ODN的免疫刺激中的作用,测试了嵌合性ODN的刺激效果,在这些ODN中它们的5′端和3′端都因具有甲基膦酸酯(MP-)、甲基硫代磷酸酯(MPS-)硫代磷酸酯(S-)、或二硫代磷酸酯(S2-)核苷酸间连键变得有核酸酶抗性(实施例10)。这些研究显示,无论它们的核酸酶抗性如何,MP-O-ODN实际上都比O-ODN的免疫刺激性低。然而,通过用5′和3′MPS核苷酸间连键取代两个非桥连O分子而将MP和S的修饰结合起来,使得免疫刺激的水平恢复到稍微高于O-ODN所刺激的水平。
S-O-ODN比O-ODN具有远远更高的刺激性,甚至比S-ODN更具有刺激性,至少在高于3.3μM浓度时如此。在低于3μM浓度时,带有3M序列的S-ODN比相应的S-O-ODN更具刺激性,而带有3D序列的S-ODN则比相应的S-O-ODN的刺激性弱(实施例10)。在比较这两个序列的刺激性CpG基元时,注意到了3D序列与侧翼为2个5′嘌呤和2个3′嘧啶的CpG这样的基元完全吻合。然而,在ODN 3D中紧靠CpG的侧翼碱基不是优化的,其具有5′嘧啶和3′嘌呤。根据进一步的试验,发现免疫刺激所需要的序列对S-ODN比对S-O-ODN和O-ODN更为严谨。与优化CpG基元吻合性差的S-ODN对淋巴细胞的活化没有刺激或很小(例如,序列3D)。然而,与该基元吻合很好的S-ODN,特别是在紧靠CpG的侧翼位置,都比相应的S-O-ODN更具有刺激性(例如,序列3M,序列4和6),虽然在高浓度(大于3μM)时的峰值效应是S-O-ODN更强(实施例10)。
S2-O-ODN具有显著的刺激性,并且在每个测试浓度都引起比相应S-ODN或S-O-ODN显著更强烈的淋巴细胞活化。
在带有S或S2取代的CpG ODN中所见到的对B细胞刺激的升高可能是下述一种或所有效应所致:核酸酶抗性、增强的细胞摄入、增强的蛋白质结合和改变了的细胞内定位。然而,核酸酶抗性不会是唯一的解释,因为MP-O-ODN实际上比带有CpG基元的O-ODN的刺激性低。以前的研究已经显示淋巴细胞对ODN的吸收明显地受到主链化学结构的影响[Zhao etal.,(1993)Comparison of cellular binding and uptake ofantisense phosphodiester,phosphorothioate,and mixedphosphorethioate and methylphosphonate olignucleotides.(Andisense Research and Development 3,53-66;Zhao et al.,(1994)Stage specific oligonucleotide uptake in murine bonemarrow B cell precursors.Blood 84,3660-36666.)]。S-ODN有最高细胞膜结合和摄入,随后是S-O-ODN,O-ODN和MP-ODN。这种摄入的区别与免疫刺激的程度非常相关。
含有未甲基化CpG的寡核苷酸具有NK细胞刺激活性
为了确定含有CpG的寡核苷酸除了刺激B细胞外是否还对天然杀伤细胞(NK)具有刺激活性,进行了各种实验。如表8所示,用CpG ODN 1和3Dd培养的脾细胞中有明显的NK活性的诱导。相比之下,已用非CpG对照ODN处理的效应物中相对没有诱导。
表8.CpG寡核苷酸(ODN)诱导的NK活性
Figure A20071013005200641
含有CpG基元的DNA诱导的NK活性,而非CpG DNA则不
在37℃下对细菌DNA培养18小时,然后测定杀死K562(人)或Yac-1(小鼠)靶细胞在去除了B细胞的小鼠脾细胞中和在人PBMC细胞中诱导NK裂解活性,但是,脊椎动物的DNA没有这种效果(表9)。为了确定细菌DNA的刺激活性是否归因于其未甲基化CpG二核苷酸水平的提高,对含有未甲基化CpG二核苷酸、甲基化CpG二核苷酸、或没有CpG二核苷酸的50种以上的合成性ODN的活化性质进行了测验。结果如表9所示,显示了合成的ODN可以显著地刺激NK活性,只要它们含有至少一个未甲基化CpG二核苷酸即可。在CpG处于回文序列中的ODN(例如ODN1585,其含有回文序列AACGTT)与没有回文序列的ODN(例如ODN1613或1619)之间没有观察到明显的刺激效果差异,要注意的是,最佳的刺激一般见于那些CpG侧翼为2个5′嘌呤或5′GpT二核苷酸和2个3′嘧啶的ODN中。动力学实验表明NK活性的峰值出现在加入OND后的约18小时之时。数据指示小鼠NK应答依赖于在先的CpG DNA对单核细胞的活化,导致IL-12、THF-α、和IFN-α/b的产生(实施例11)。
表9.含有CpG基元的DNA诱导NK活性,而非CpG DNA则 没有
Figure A20071013005200651
在ODN序列中的CpG二核苷酸用下划线标出;Z代表甲基胞嘧啶。小写字母代表被核酸酶抗性的硫代磷酸酯修饰的核苷酸间连键,其在效价实验中显示了比非修饰的ODN强20倍以上,具体根据侧翼碱基而定。在某些ODN中采用多G末端(g),因为它们显著地提高了ODN摄入水平。
从所有这些研究中,得到了对CpG DNA的免疫效应的更为全面的理解,并将其总结于图6中。
CpG基元的免疫活化可能依赖于CpG侧翼的碱基,在ODN中存在的CpG的数量和间隔。虽然在理想的碱基环境下单一CpG可以是非常强和有用的免疫活化子,但在含有几个CpG并具有适宜的侧翼碱基和间隔的ODN中能见到更为优越的效果。对鼠B细胞的活化来讲,最佳的CpG基元是TGACGTT。
下面所进行的研究通过检验改变CpG二核苷酸的数目、间隔和侧翼碱基的效果而确定对刺激人细胞为最佳的ODN序列。
确定带有用于活化人NK细胞的最佳CpG基元的硫代磷酸酯 ODN
要想应用于临床,则ODN必须以受到保护而不被核酸酶降解的形式施用给受试者。用磷酸二酯ODN来达到这个目的的方法是本领域所熟知的,包括将其包被在脂质中或在递送系统中,例如,在纳米颗粒中。这种保护还可以利用对DNA的化学取代来达到,例如被修饰的DNA主链,包括核苷酸间连键为抗核酸酶的那些。有些修饰可以赋予另外的所需性质,例如提高细胞吸收。例如,磷酸二酯连键可以通过用硫替代非桥连氧原子中的一个来进行修饰,其构成硫代磷酸酯DNA。如果硫代磷酸酯DNA具有CpG基元,则其具有增强的细胞摄入(Krieg et al.,Antisense Res.Dev.6:133,1996)和改善的B细胞刺激。因为NK活化与体内佐剂效应强烈相关,所以确定那些可以活化人NK细胞的硫代磷酸酯ODN是十分重要的。
检验了不同的硫代磷酸酯ODNs-在各种碱基环境下含有CpG二核苷酸-对人NK细胞活化的效果(表10)。ODN 1840含有2拷贝的TGTCGTT基元,具有显著的NK裂解活性(表10)。为了进一步确定其它的对NK活化最佳的ODN,对大约100种含有不同数目和间隔的CpG基元的ODN进行了检测,并以ODN 1982作为对照。结果列于表11。
有效的ODNs以TC或TG作为5′端开始,然而,这个要求不是必须的。带有内部CpG基元的ODNs(例如,ODN 1840)一般是比其中GTCGCT基元紧接着5′TC的ODN更弱的刺激物(例如,ODN 1967和1968)。ODN 1968在其3′部分具有第二个GTCGTT基元,其始终比不含该第二个基元的ODN 1967更具有刺激性。然而,ODN 1967在实验1和3中比ODN 1968稍强,在实验2中则不是。ODN 2005具有第三个GTCGTT基元,其所诱导的NK活性平均比1968稍高。然而,由于在每个基元之间都加入了两个T而增加了各GTCGTT基元之间的间隔的ODN2006比ODN 2005和ODN 2007更为优越,后者只有一个基元添加了间隔性的2个T。在CpG基元之间的最小可接受的间隔是一个核苷酸,只要该ODN在3′有两个嘧啶(优选T)即可(如ODN 2015)。令人惊奇的是,用5′的T来把两个GTCGTT基元尾尾连接起来也创造了NK活性的合理强度的诱导物(例如,ODN 2016)。选择胸腺嘧啶(T)来分开相连的CpG二核苷酸不是绝对的,因为ODN 2002诱导相当的NK活化,虽然其中是由腺嘌呤(A)将其CpGs(即CGACGTT)间隔开来。还应该注意的是,不含CpG的ODN(例如ODN 1982),含有成串的CpGs的ODN,以及CpGs存在于坏序列环境中的ODN(例如,ODN 2010)都没有对NK活化的刺激效应。
表10.ODN诱导NK裂解活性(LU)
  ODN   序列(5′-3′)   LU
  仅有细胞   0.01
  1754   ACCATGGACGATCTGTTTCCCCTC   0.02
  1758   TCTCCCAGCGTGCGCCAT   0.05
  1761   TACCGCGTGCGACCCTCT   0.05
  1776   ACCATGGACGAACTGTTTCCCCTC   0.03
  1777   ACCATGGACGAGCTGTTTCCCCTC   0.05
  1778   ACCATGGACGACCTGTTTCCCCTC   0.01
  1779   ACCATGGACGTACTGTTTCCCCTC   0.02
  1780   ACCATGGACGGTCTGTTTCCCCTC   0.29
  1781   ACCATGGACGTTCTGTTTCCCCTC   0.38
  1823   GCATGACGTTGAGCT   0.08
  1824   CACGTTGAGGGGCAT   0.01
  1825   CTGCTGAGACTGGAG   0.01
  1828   TCAGCGTGCGCC   0.01
  1829   ATGACGTTCCTGACGTT   0.42
  18302   随机序列   0.25
  1834   TCTCCCAGCGGGCGCAT   0.00
  1836   TCTCCCAGCGCGCGCCAT   0.46
  1840   TCCATGTCGTTCCTGTCGTT   2.70
  1841   TCCATAGCGTTCCTAGCGTT   1.45
  1842   TCGTCGCTGTCTCCGCTTCTT   0.06
  1851   TCCTGACGTTCCTGACGTT   2.32
1裂解单位(LU)按照所述(8)进行。简言之,从正常的供体收集PBMC,用Ficoll离心,然后采用或不采用所示ODN(向培养物中加入的剂量为6μg/ml)培养24小时。然后确定它们的对51Cr-标记的K562细胞的裂解能力。给出的结果是从几个不同的正常人供体获得的典型的结果。2该寡核苷酸混合物含有在每个位置上从所有4种碱基中随机选择的碱基。
表11.带有良好基元的硫代磷酸酯CpG ODN诱导NK LU
  ODN1   序列(5′-3′)   实验1   实验2   实验3
  仅有细胞   0.00   1.26   0.46
  1840   TCCATGTCGTTCCTGTCGTT   2.33   ND   ND
  1960   TCCTGTCGTTCCTGTCGTT   ND   0.48   8.99
  1961   TCCATGTCGTTTTTGTCGTT   4.03   1.23   5.08
  1962   TCCTGTCGTTCCTTGTCGTT   ND   1.60   5.74
  1963   TCCTTGTCGTTCCTGTCGTT   3.42   ND   ND
  1965   TCCTGTCGTTTTTTGTCGTT   0.46   0.42   3.48
  1966   TCGTCGCTGTCTCCGCTTCTT   2.62   ND   ND
  1967   TCGTCGCTGTCTGCCCTTCTT   5.82   1.64   8.32
  1968   TCGTCGCTGTTGTCGTTTCTT   3.77   5.26   6.12
  19792   TCCATGTZGTTCCTGTZGTT   1.32   ND   ND
  1982   TCCAGGACTTCTCTCAGGTT   0.05   ND   0.98
  1990   TCCATGCGTGCGTGCGTTTT   2.10   ND   ND
  1991   TCCATGCGTTGCGTTGCGTT   0.89   ND   ND
  2002   TCCACGACGTTTTCGACGTT   4.02   1.31   9.79
  2005   TCGTCGTTGTCGTTGTCGTT   ND   4.22   12.75
  2006   TCGTCGTTTTGTCGTTTTGTCGTT   ND   6.17   12.82
  2007   TCGTCGTTGTCGTTTTGTCGTT   ND   2.68   9.66
  2008   GCGTGCGTTGTCGTTGTCGTT   ND   1.37   8.15
  2010   GCGGCGGGCGGCGCGCGCCC   ND   0.01   0.05
  2012   TGTCGTTTGTCGTTTGTCGTT   ND   2.02   11.61
  2013   TGTCGTTGTCGTTGTCGTTGTCGTT   ND   0.56   5.22
  2014   TGTCGTTGTCGTTGTCGTT   ND   5.74   10.89
  2015   TCGTCGTCGTCGTT   ND   4.53   10.13
  2016   TGTCGTTGTCGTT   ND   6.54   8.06
1PBMC基本如本文所述。结果是6个分别的实验的代表;每个实验代表不同的供体。2这是甲基化形式的ODN 1840;Z=5-甲基胞嘧啶,LU是裂解单位,ND=未进行,CpG二核苷酸用下划线标出。
确定带有人B细胞增殖活化的最佳CpG基元的硫代磷酸酯 ODN
CpG ODN诱导B细胞增殖的能力是其佐剂潜力的良好指标。具有强佐剂效应的ODN确实一般都诱导B细胞增殖。为了确定由于诱导B细胞增殖的最佳CpG ODN是否与诱导NK细胞活性的ODN相同,测试了相似的一组ODN(表12)。表现出最为一致的刺激性的ODN是ODN 2006(表12)。
表12.硫代磷酸酯CpG ODN对人B细胞增殖的诱导
Figure A20071013005200701
1细胞=手术中收集的并储存于-70℃的人脾细胞,或从正常供体采集的并经过Ficoll离心的PBMC细胞。细胞培养在96孔U底微滴板上,其中加入或不加入所示的ODN(向培养物中加入的剂量为6μml)。N=12个实验。细胞培养4-7天,用1μCi的3H胸苷脉冲18小时,然后收获并闪烁计数。刺激指数=不加入ODN的小孔中的cpm与在整个培养过程中用所示ODN刺激的小孔的cpm的比率(一旦建立培养物后就没有再进一步加入ODN)。ND=未进行。
确定诱导人IL-12分泌的硫代磷酸酯ODN
CpG ODN诱导IL-12分泌的能力是其佐剂潜力的良好指标,特别从其诱导Th1免疫应答这种高度依赖IL-12的能力的角度来讲更是如此。因此,检验了某一组硫代磷酸酯ODN诱导人PBMC体外分泌IL-12的能力(表13)。这些实验显示,在某些人PBMC细胞中,大多数CpG ODN可以诱导IL-12的分泌(例如,实验1)。然而,其它的供体只应答少数的CpG ODN(例如,实验2)。ODN 2006是大多数受试者中IL-12分泌的一致诱导物(表13)。
表13.硫代磷酸酯CpG ODN诱导人IL-12分泌
1PBMC采集于正常供体并用Ficoll离心,然后以106细胞/孔培养在96孔微滴板中,其中加入或不加入所示的ODN,如果在培养物中加入ODN,其剂量为6μg/ml。24小时后收获上清液,用ELISA如方法部分所述检测IL-12的水平。对每个实验建立标准曲线,其代表不同的供体。
确定B细胞和单核细胞/NK细胞特异性的寡核苷酸
如图6所示,CpG DNA可以直接活化高度纯化的B细胞和单核细胞。CpG DNA活化这些种类细胞的机制中有很多相似之处。例如,两者都需要NFkB活化,对此在下面给予进一步解释。
在对CpG DNA的不同免疫效应的进一步研究中,发现有多于一种类型的CpG基元。具体地讲,寡核苷酸1668带有最佳的小鼠B细胞基元,它是B细胞和天然杀伤细胞(NK)活化的强诱导物,而寡核苷酸1758是B细胞的弱活化物,但仍然诱导很好的NK应答(表14)。
表14.不同的CpG基元刺激最佳的小鼠B细胞和NK活
  ODN   序列   B细胞活化1   NK活化2
  1668   TCCATGACGTTCCTGATGCT(SEQ ID NO:44)   42,849   2.52
  1758   TCTCCCAGCGTGCGCCAT(SEQ ID NO:55)   1,747   6.66
  无   367   0.00
CpG二核苷酸用下划线标出,寡核苷酸是合成的,并有硫代磷酸酯修饰的主链,以改善其核酸酶抗性。1按照实施例1所述,用200nM浓度的寡脱氧核苷酸培养48小时后的3H胸苷摄入来进行测定。2按照裂解单位来测定。
免疫刺激性核酸的目的论基础
脊椎动物DNA是高度甲基化的,并且CpG二核苷酸是不足表达的。然而,刺激性CpG基元在微生物基因组DNA中是常见的,但是在脊椎动物DNA中就很少见。此外,细菌DNA已经被报道可以诱导B细胞增殖和免疫球蛋白(Ig)的生产,而哺乳动物DNA则不行[Messina,J.P.et al.,J.Immunol.147:1759(1991)]。在实施例3中进一步描述的实验中,用CpG甲基化酶对细菌DNA的甲基化被发现可消除促细胞分裂原性,这就证实了CpG状态的差异是细菌DNA引起B细胞刺激的原因。这个数据支持了下面的结论:即存在于细菌DNA中的未甲基化CpG二核苷酸负责了细菌DNA的刺激效应。
就目的论而言,看起来CpG基元造成的淋巴细胞活化代表了免疫防御机制,其可以区分细菌DNA和宿主DNA。宿主DNA由于编程性细胞死亡而普遍存在于很多发炎的解剖学组织和区域中,但它通常不诱导或很少诱导淋巴细胞活化,因为存在CpG抑制和甲基化。然而,含有未甲基化CpG基元的细菌DNA的存在可以准确地在感染的解剖学区域引起淋巴细胞活化,这则是有益的。这种新的活化途径提供了T细胞依赖性抗原特异性B细胞活化的快速替代途径。由于CpG途径通过抗原受体与B细胞活化相协同,带有细菌抗原特异性的抗原受体的B细胞将通过其细胞膜Ig接受一个活化信号以及从细菌DNA来的第二个信号,并从而倾向于有区分地被活化。这个途径与其它的B细胞活化途径的相互关系提供了使用多克隆抗原来诱导抗原特异性应答的生理学机制。
然而,B细胞活化似乎不是完全非特异性的。带有细菌产物特异性的抗原受体的B细胞可以通过细胞膜Ig接受一个活化信号,以及从细菌DNA来的第二信号,从而更强烈地启动抗原特异性免疫应答。如同其它的免疫防御机制,对细菌DNA的应答可能在某些环境下具有不期望的后果。例如,对自身抗原的自身免疫应答也将倾向于由细菌感染优先刺激,因为自身抗原还可以向由细菌DNA刺激的自身反应性B细胞提供第二活化信号。确实,细菌感染对自身免疫的诱导是临床观察中常见的。例如,自身免疫性疾病系统性红斑狼疮,其本身是1)以产生抗-DNA抗体为特征,2)由抑制DNA甲基转移酶的药物所诱导[Cornacchia,E.J.et al.,J.Clin.Invest.92:38(1993)],以及3)与降低的DNA甲基化有关[Richardson,B.,L.et al.,Arth.Rheum35:647(1992)],所以,它可能至少部分地由DNA特异性B细胞通过由CpG基元提供的刺激信号的活化来启动,以及细菌DNA同抗原受体的结合来启动。
此外,特征在于对免疫系统的大量非特异性活化而导致高度发病率和死亡率的脓毒病,可能由从死亡细菌释放后的、达到足以直接活化许多淋巴细胞的浓度的细菌DNA和其它产物来启动。脓毒病综合症中CpG DNA作用的进一步证据可见于Cowdery,J.等人的文献[Cowdery,J.,et al.,(1996)The Journalof Immunolgy 156:4570-4575]。
与通过其表面Ig受体来刺激B细胞的抗原不同的是,CpG-ODN不诱导任何可检测到的Ca2+流,蛋白质酪氨酸磷酸化的改变,或IP3的产生。以带有或不带有CpG基元的缀合FITC的ODN进行的流式细胞术按照Zhao,Q等人所述进行[Antisense Research and Development 3:53-66(1993)],其中显示了相等的膜结合,细胞摄入,外流,以及胞内定位。这就暗示也许不存在对CpG ODN特异的细胞膜蛋白质。不同于经过细胞膜来起作用,这些数据暗示的是含有未甲基化CpG的寡核苷酸需要细胞摄入来发挥活性:与固体Teflon支持物共价连接的ODN是非刺激性的,在抗生物素蛋白小珠或抗生物素蛋白涂布的培养皿上固定化的生物素化ODN也一样。与FITC或生物素缀合的CpG ODN保留了全部的促细胞分裂性质,表明没有立体阻碍。
最近的数据表明,转录因子NFkB作为CpG效应的直接或间接介导物而介入其中。例如,用CpG DNA处理B细胞或单核细胞15分钟内,NFkB结合活性的水平被提高了(图7)。然而,那些不含CpG基元的DNA不能引起这种提高。此外,还发现的是,NFkB活化的两个不同的抑制物,即PDTC和胶毒素,完全封闭了CpG DNA对淋巴细胞的刺激,正如B细胞增殖或单核细胞的细胞因子分泌的测定所显示的,这暗示两种细胞类型都需要NFkB活化。
NFkB能够被活化的机制可能有几种。它们包括通过对各种蛋白质激酶的活化,或通过反应性氧类的产生。没有发现在CpG DNA处理B细胞或单核细胞后立即诱导蛋白质激酶活化的任何证据,而且蛋白质激酶A、蛋白质激酶C,以及蛋白质酪氨酸激酶的抑制物都对CpG诱导的活化没有任何影响。然而,CpG DNA在B细胞和单核细胞中都快速诱导反应性氧类的生产,如按照Royall,J.A.和Ischiropoulos,H.(Archives ofBiochemistry and Biophysics 302:348-355(1993))所述用敏感性荧光染料二氢若丹明123检测所证实的。然而,这些反应性氧类产生的抑制物完全封闭了NFkB的诱导以及随后的CpGDNA对细胞增殖和细胞因子分泌的诱导。
下一个问题是,CpG DNA如何快速地导致了反应性氧的产生。本发明人的先前的研究证实了寡核苷酸和质粒或细菌DNA被细胞摄入到内体中。这些内体迅速地在细胞内被酸化。为了确定这个酸化步骤是否在CpG DNA活化反应性氧类的机制中起重要作用,用内体酸化的特异性抑制物封闭该酸化步骤,包括氯喹、莫能菌素、和bafilomycin等,它们通过不同的机制工作。图8A显示了流式细胞术的研究结果,其中采用了小鼠B细胞以及二氢若丹明123染料来确定反应性氧类的水平。该图内A小图中仅仅含有染料的样品显示了细胞对染料的阳性背景水平为28.6%。正如所预期的,反应性氧类的这个水平在用PMA和离子霉素处理20分钟的细胞(即阳性对照)内被大大地提高到80%(B小图)。用CpG寡核苷酸处理的细胞也显示反应性氧类型水平的提高,使大于50%的细胞变为阳性(D小图)。然而,用除了CpG被改变之外序列完全相同的寡核苷酸处理的细胞没有显示反应性氧类型水平的这种显著提高(E小图)。
在有氯喹存在时,这些结果非常不同(图8B)。氯喹将细胞中反应性氧类型的背景水平稍微的降低了一些,使A小图中的未处理细胞只有4.3%为阳性。氯喹完全消除了用CpG DNA处理的细胞中的反应性氧类型的诱导(B小图),但是对用PMA和离子霉素处理的细胞中反应性氧类型的水平没有减弱(E组)。这就证明,与PMA加上离子霉素不同,用CpG DNA处理B细胞后反应性氧类型的产生需要DNA在内体中经历酸化步骤。这是淋巴细胞活化的全新的机制。氯喹、莫能菌素、以及bafilomycin还似乎可以封闭CpG DNA对NFkB的活化,以及对随后的增殖和细胞因子分泌的诱导。
CpG DNA的慢性免疫活化以及自身免疫疾病
CpG DNA对B细胞的活化与通过B细胞受体的信号有协同作用。这就提出了这样的可能,即DNA特异性B细胞可能通过细菌DNA与其抗原受体的共同结合以及共刺激性的CpG介导的信号的刺激来活化。此外,CpG DNA诱导B细胞变为抗编程性细胞死亡的,这种机制被认为在防止对自身抗原如DNA的免疫应答中是重要的。确实,对bDNA的暴露可以启动抗-DNA的抗体的产生。既然CpG DNA具有促进自身免疫的这种潜在能力,那么值得注意的是,患有自身免疫疾病系统性红斑狼疮的患者都具有持续的升高水平的循环血浆DNA,其中富含低甲基化CpG。这个发现暗示在狼疮疾病发病机理中CpG DNA造成的慢性免疫活化所可能扮演的角色。
治疗狼疮疾病的一类有效药剂是抗疟疾药物,例如氯喹。虽然这些药物的治疗机制还不清楚,但是已知的是它们可抑制内体的酸化。CpG DNA对淋巴细胞的活化不是通过与细胞表面受体的结合所介导的,而是需要细胞吸摄入实现的,这种细胞摄入通过吸收性内吞到酸化的氯喹敏感性胞内区室而发生。这就暗示一种假说,即CpG DAN对淋巴细胞的活化可能与酸化的内体一起发生,并且甚至可能是依赖于pH的。为了检验这个假说,采用了DNA酸化的特异性抑制物来确定B细胞或单核细胞是否可能在酸化被抑制的条件下对CpG DNA产生应答。
被检测到的对CpG DNA应答的最早淋巴细胞活化现象是反应性氧类型(ROS)的产生,其在原代脾细胞以及B细胞和单核细胞中都是在5分钟内被诱导出来的。内体酸化的抑制物,包括氯喹、bafilomycin A,以及莫能菌素,它们具有不同的作用机制,可封闭CpG诱导的ROS产生,但是对由PMA、或者与CD40或IgM的连接等所介导的ROS的产生没有影响。这些研究显示,ROS的产生是通过多种途径的淋巴细胞活化中一种所共有的现象。这种ROS的产生一般不依赖于内体的酸化,而酸化仅仅是针对应答CpG DNA的ROS反应所需要的。应答CpG的ROS生产并不被NFkB抑制物胶霉毒素所抑制,确认其不是NFkB活化的继发现象。
为了确定CpG DNA的内体酸化是否也是其它免疫刺激效果所必需的,进行了各种实验。LPS和CpG DNA都诱导相似快速的NFκB活化,原癌基因mRNA水平的增加,以及细胞因子分泌。DNA对NFκB的活化依赖于CpG基元,因为其不被用CpG甲基酶处理的bDNA诱导,也不被其中碱基发生改变而破坏了CpG的ODN所诱导。采用特异性抗体的超迁移实验表明,被活化的NFκB复合物包括p50和p65组分。并非出人意料的是,在LPS或CpG处理的细胞中的NFκB活化伴随有IκBα和IκBβ的降解。然而,内体酸化的抑制物选择性地阻断了所有的CpG诱导的细胞活化现象,但没有抑制LPS诱导的任何细胞活化现象。值得注意的是,已经确定很低浓度的氯喹(<10μM)抑制了CpG介导的淋巴细胞活化,因为该剂量远远低于抗疟疾活性和其它被报道的免疫效应所需要的剂量(例如,100-1000μM)。这些实验支持了pH依赖性信号机制在介导CpG DNA的刺激效果中所起到的作用。
表15.内体酸化或NFkB活化的抑制物对CpG诱导的 TNF-α和IL-12表达的特异性阻断
Figure A20071013005200791
表15图例说明了IL-2和TNF-α检测:小鼠单核细胞系J774(对IL-12为1×105细胞/毫升,对TNF-α为1×106细胞/毫升),在有给出浓度的所示抑制物或没有抑制物的条件下培养2小时,然后用2μM CpG 寡核苷酸(ODN)1826(TCCATGACGTTCCTGACGTT SEQ ID NO:10)或LPS(10μg/ml)刺激,对TNF-α为4小时,对IL-12为24小时,在此期间收集上清液。对上清液用ELISA检测IL-12或TNF-α(pg/ml),操作参见以前的文献[A.K.Krieg,A.-K.Yi,S.Matson,T.J.Waldschmidt,G.A.Bishop,R.Teasdale,G.Koretzky and D.Klinman,Nature 374,546(1995);Yi,A.-K.,D.M.Klinman,T.L.Martin,S.Matson and A.M.Krieg,J.Immunol,157:5394-5402(1996);Krieg,A.M.,J.Lab.Clin.Med.,128,128-133(1996)]。用不含有CpG基元的ODN培养的细胞没有诱导细胞因子的分泌。在IL-6的检测中也见到了相似的CpG应答的特异性抑制,也见于采用原代脾细胞或B细胞系CH12.LX和WEHI-231的实验中。2.5μg/ml氯喹相当于<5μM。NFkB活化的其它抑制物,包括PDTC和钙蛋白酶抑制物I和II,都给出了与所示抑制物相似的效果。给出的数据代表在10个不同的实验中所得到的结果。
CpG基元的过量免疫活化可能对自身免疫疾病系统性红斑狼疮的发病有作用,其伴随着升高水平的循环低甲基化CpGDNA。氯喹和有关的抗疟疾化合物是对系统性红斑狼疮和其它一些自身免疫疾病的有效治疗药物,虽然它们的作用机制尚不明了。我们对极低浓度氯喹特异性抑制CpG介导的淋巴细胞活化的能力的确定,暗示了其有益效果一种可能的新机制。值得注意的是,狼疮复发经常被认为是微生物感染启动的。在感染的组织中存在的bDNA水平可能足以诱导局部发炎应答。结合CpG DNA作为脓毒综合症和其它疾病的介导物的可能作用,我们的研究暗示作为内体酸化抑制物的抗疟疾药物的新型可能医药用途。
CpG诱导的ROS生产可能是细胞活化的偶然结果,或者是介导这种活化的信号。ROS的清除物N-乙酰基-L-半胱氨酸(NAC)可阻断CpG诱导的NFkB活化,细胞因子产生,以及B细胞增殖,暗示在这些途径中ROS生产的起因角色。这些数据与先前支持ROS在活化NFkB中的作用的证据相吻合。WEHI-231B细胞(5×105细胞/毫升)在有或没有氯喹[5μg/ml(<10μM)]或胶霉毒素(0.2μg/ml)条件下预培养30分钟。然后对细胞等份试样按照上述在RPMI培养基中培养10分钟,其中添加或不添加1μM CpG ODN(1826)或非CpG ODN(1911),或乙酸肉豆蔻佛波酯(phorbol myristate acetate,PMA)加离子霉素(iono),然后对细胞用二氢若丹明123染色并用流式细胞术分析胞内ROS的产生,具体操作按照以前所述的进行[A.K.Krieg,A.-K.Yi,S.Matson,T.J.Waldschmidt,G.A.Bishop,R.Teasdale,G.Koretzky and D.Klinman,Nature 374,546(1995);Yi,A.-K.,D.M.Klinman,T.L.Martin,S.Matson and A.M.Krieg,J.Immunol,157:5394-5402(1996);Krieg,A.M.,J.Lab.Clin.Med.,128,128-133(1996)]。J774细胞,一种单核细胞系,显示了相似的pH依赖性CpG诱导的ROS应答。与之相对照,CpG DNA没有诱导胞外ROS的产生,也没有任何可检测的嗜中性粒细胞ROS。氯喹的这些浓度(以及与其它内体酸化抑制物使用的那些浓度)使用荧光素缀合的ODN如Tonkinson等人所述证实了防止了内化CpG DNA的酸化[Tonkinson et al.,Nucl.Acids Res.22,4268(1994);A.M.Krieg,In:DeliveryStrategies for Antisense Oligonucleotide Therapeutics.Editor,S.Akhtar,CRC Press,Inc.,pp.177(1995)]。在高于抑制内体酸化所需的浓度下,观察到了非特异性的抑制效果。每个实验都进行至少三次并得到相似结果。
虽然已知NFkB对基因表达是重要的调节物,但其在对CpG DNA的转录应答中的作用却始终不清楚。为了确定是否这种NFkB活化是否是CpG介导的基因表达的诱导是必需的,在有或没有二硫代氨基甲酸吡咯烷(PDTC)这种IkB磷酸化抑制物存在的条件下用CpG DNA活化细胞。这些NFkB活化的抑制物完全阻断了CpG诱导的原癌基因和细胞因子mRNA和蛋白质的表达,显示NFkB在这些现象中作为介导物的基本作用。在这些研究中所用的实验条件下,没有任何一种抑制物减弱了细胞的生存力。J774,一种小鼠单核细胞系,在有5μg/ml小牛胸腺(CT)、大肠杆菌(EC)、或甲基化大肠杆菌(mEC)DNA(用CpG甲基化酶按照所述进行甲基化4)或者0.75μM的CpG寡核苷酸(ODN 1826,表15)或非CpGODN(TCCATGAGCTTCCTGAGTCT;ODN 1745)存在下培养1小时,然后,对细胞进行裂解,制备核酸提取物。对含有共有NFkB位点的双链ODN进行5′放射性标记,用作EMSA的探针,具体操作按照以前所述进行[J.D.Dignam,R.M.Lebovitz and R.G.Roeder,Nucliec Acids Res.11,1475(1983);M.Briskin,M.Damore,R.Law,G.Lee,P.W.Kincade,C.H.Sibley,M.Kuehl and R.W all,Mol.Cell.Biol.10,422(1990)]。p50/p65异二聚体的位置通过用p65和p50的特异性抗体以超变动方法确定[Santa Cruz Biotechnology,Santa Cruz,CA]。用J774细胞建立了氯喹对CpG诱导的但不是LPS诱导的NFkB活化的抑制。将细胞在有或没有氯喹(20μg/ml)存在的条件下预培养2小时,然后按照上述用EC DNA、CpG ODN、非CpGODN或LPS(1μg/ml)刺激1小时。在B细胞系WEHI-231和原代脾细胞中见到了相似的氯喹敏感性CpG诱导的NFkB活化。这些实验都进行三次,氯喹的浓度范围是2.5至20μg/ml,得到了相似的结果。
还明白了的是,CpG刺激的mRNA表达需要B细胞和单核细胞中的内体酸化以及NFkB活化。J774细胞(2×106细胞/毫升)在有或没有氯喹[2.5μg/ml(<5μM)]或N-甲苯磺酰基-L-苯丙氨酸氯甲酮(TPCK,50μM)这种丝氨酸/苏氨酸蛋白酶抑制物的存在下进行培养2小时,后者可防止IkB蛋白水解并因此阻断NFkB活化。然后对细胞进行刺激3小时,使用的是大肠杆菌DNA(EC,50μg/ml)、小牛胸腺DNA(CT,50μg/ml)、LPS(10μg/ml)、CpG ODN(1826,1μM)、或对照非CpGODN(1911,1μM)。WEHI-231B细胞(5×105细胞/毫升)在有或没有胶霉毒素(0.1μg/ml)或双胶霉毒素(0.1μg/ml)存在的条件下培养2小时,然后用CpG ODN(1826)或对照非CpGODN(1911,TCCAGGACTTTCCTCAGGTT)以0.5μM刺激8小时。在这两种情况下,细胞被收集后,用RNAzol按照厂商的说明书来制备RNA。进行多探针Rnase保护检测,操作按照以前所述的进行[A.-K.Yi,P.Hornbeck,D.E.Lafrenz andA.M.Krieg,J.Immunol.,157,4918-4925(1996)]。将可比量的RNA上样到每条泳道内,用核糖体μRNA作为上样对照(L32)。对这些实验重复三次,得到相似的结果。
这些结果表明,淋巴细胞对CpG DNA的应答是通过涉及胞内ROS的pH依赖性产生的新途径进行的。该依赖pH的步骤可能是CpG DNA的转运或加工步骤,ROS的产生,或某些其它事件。ROS被广泛地认为是多种类型细胞中的信号途径的第二信使,但是在此之前还没有被证明可以在B细胞中介导刺激性信号。
推测在内体之中或其附近有一种蛋白质,它特异性识别含有CpG基元的DNA,并导致反应性氧类型的产生。为了探测细胞的胞质中可能特异性结合CpG DNA的蛋白质,进行了电泳迁移率变动分析(EMSA),其中使用5′放射性标记的带有或不带有CpG基元的寡核苷酸。发现有一条带似乎代表与具有CpG基元的单链寡核苷酸特异性结合、但不与缺少CpG基元的寡核苷酸或含有已被甲基化了的CpG基元的寡核苷酸结合的蛋白质。这种结合活性在加入过量的含有NFkB结合位点的寡核苷酸时被封阻断。这就暗示NFkB或相关的蛋白质是与刺激性CpG寡核苷酸结合的蛋白质或蛋白质复合体的组成部分。
在NFkB被强烈活化的时间点没有发现CREB/ATF蛋白质的任何活化。因而,这些数据没有提供NFkB蛋白质实际上结合CpG核酸的证据,而是说明了CpG活性在某种方式上需要该蛋白质。可能的是,CREB/ATF或相关的蛋白质与NFkB蛋白质或其它的蛋白质以某种方式相互作用,这样可以解释CREB蛋白质的结合基元与最佳CpG基元之间显著的相似性。还可能的是寡核苷酸结合CREB/ATF或相关的蛋白质,并且这导致NFkB的活化。
另外,很可能的是,CpG核酸可以与TRAF蛋白质中的一种相结合,该蛋白质在CD40被交联时与CD40的胞质区结合,并介导NFkB活性。这样的TRAF蛋白质的例子包括TRAF-2和TRAF-5。
制备免疫刺激性核酸的方法
在本发明中,核酸可以使用本领域熟知的任何数目的方法从头合成。例如,可以采用b-氰乙基亚磷酰胺法[S.L.Beaucageand M.H.Caruthers,(1986)Tet.Let.22:1859],核苷H-膦酸酯法[Caregg et al.,(1986)Tet.Let.27:4051-4054;Froehler et al.,(1986)Nucl.Acid.Res.14:5399-5407;Caregg et al.,(1986)Tet.Let.27:4055-4058,Gaffney et al.,(1988)Tet.Let.29:2619-2622]。这些化学合成可以用市售的各种自动化寡核苷酸合成仪完成。另外,也可以用已知的方法从已经存在的核酸序列(例如,基因组DNA或cDNA)中制备寡核苷酸,所述技术例如有那些利用限制性酶,核酸外切酶或核酸内切酶的技术。
为了用于体内,核酸优选对降解(例如,核酸外切酶或核酸内切酶的降解)相对有抗性。次级结构,例如茎-环结构,可以稳定核酸而不被降解。另外,核酸的稳定化可以通过磷酸酯主链修饰来实现。优选的稳定化核酸具有至少部分的硫代磷酸酯修饰的主链。硫代磷酸酯可以用自动化技术采用亚磷酰胺或H-膦酸酯法来化学合成。芳基和烷基膦酸酯可以通过如美国专利第4,469,863号所描述的方法来制备,并且,烷基磷酸三酯(其中带电的氧基团如美国专利第5,023,243号和欧洲专利第092,574号所述被烷基化)可以用市售的试剂以自动化固相合成法来制备。进行其它的DNA主链修饰和替换的方法已经被描述了[Uhlmann,E.and Peyman,A.(1990)Chem.Rev.90:544;Goodchild,J.(1990)Bioconjugate Chem.1:165]。带有CpG基元的2′-O-甲基核酸也引起免疫活化,乙氧基修饰的CpG核酸也如此。事实上,还没有发现哪些主链修饰可以完全消除CpG效应,虽然用5-甲基C取代C可以极大地削弱之。
为了体内给药,核酸可以结合有能导致与靶细胞[例如,B细胞,单核细胞和天然杀伤细胞(NK)]表面以高亲和力结合和/或靶细胞的细胞摄入增加的分子,从而形成“核酸递送复合物”。核酸可以是离子性地或共价地与适宜的分子通过本领域所熟知的技术相结合。可以使用多种偶联或交联剂,例如,蛋白质A,碳二亚胺,N-琥珀酰亚氨基-3-(2-吡啶二硫基)丙酸盐/酯(SPDP)。核酸还可以用熟知的技术包埋在脂质体或病毒体中。
免疫刺激性核酸分子的医疗应用
根据其免疫刺激性质,可以将含有至少一个未甲基化CpG二核苷酸的核酸分子给受试者体内给药来治疗“免疫系统缺陷”。或者,含有至少一个未甲基化CpG二核苷酸的核酸分子可以同来自免疫系统缺陷疾病的受试者的淋巴细胞(例如,B细胞,单核细胞或NK细胞)离体接触,然后可将活化的淋巴细胞植回到受试者体内。
正如本文所描述的,作为对含有未甲基化CpG的核酸分子的应答,有更多数量的脾细胞分泌了IL-6、IL-12、IFN-γ、IFN-α、IFN-β、IL-1、IL-3、IL-10、TNF-α、TNF-β、GM-CSF、RANTES、甚至还有其它的物质。在B细胞、CD4+T细胞和单核细胞中发现了IL-6表达的提高。
免疫刺激性核酸分子还可以同疫苗一起施用给受试者,以促进其免疫系统并从而产生对疫苗的更好的应答。优选的是,将免疫刺激性核酸分子与疫苗一起或稍微提前一点施用给受试者。任选地常规的佐剂可以同疫苗一起施用,该疫苗最低限度地由抗原组成,因为常规的佐剂可以通过提高抗原的吸收来进一步改善疫苗接种的效果。
如果疫苗是DNA疫苗,则至少有两个成分决定其效力。首先,由疫苗编码的抗原决定免疫应答的特异性。其次,如果质粒的主链含有CpG基元,其就作为疫苗的佐剂起作用。因此,CpG DNA起着有效的“危险信号”的作用,并引起免疫系统对该区域内的新抗原产生强烈的反应。这种作用方式似乎是CpG DNA对树枝状细胞和其它的“专业”抗原呈递细胞的局部刺激性效应以及对B细胞的共同刺激效应的结果。
免疫刺激性寡核苷酸和含有未甲基化CpG的疫苗直接活化淋巴细胞并共同刺激抗原特异性应答,它们与常规的佐剂(例如,铝沉淀物)完全不同,常规佐剂在单独注射时是惰性的,并被认为是通过吸附抗原将其更有效地呈递给免疫细胞而起作用。进而言之,常规佐剂只对某些抗原有效,仅仅诱导抗体(体液)免疫应答(Th2),并且在诱导细胞免疫应答(Th1)方面很差。对于许多的致病物,体液应答对防御仅有很小的保护作用,有时甚至是有害的。
此外,免疫刺激性寡核苷酸可以在化疗或免疫治疗之前、之中或之后给药,以提高肿瘤细胞对后续的化疗或免疫治疗的反应性,或通过诱导恢复性细胞因子如GM-CSF来加速骨髓的恢复。CpG核酸还提高天然杀伤细胞的裂解活性和依赖抗体的细胞毒性(ADCC)。对NK活性和ADCC的诱导在癌症免疫治疗单独或者与其它治疗结合时同样是有益的。
所述的免疫刺激性核酸分子的另外的一种应用是对过敏的去敏化治疗,其中过敏一般由针对无害过敏原的IgE抗体产生所导致的。由未甲基化CpG核酸诱导的细胞因子主要是属于“Th1”类型的,其最主要的特点是细胞免疫应答,并与IL-12和IFN-γ有关。另一种主要类型的免疫应答被称为“Th2”免疫应答,其更多地与抗体免疫应答以及IL-4、IL-5和IL-10的产生有关。一般来讲,似乎所有的过敏疾病都是由Th2型免疫应答介导的,自身免疫疾病是由Th1免疫应答介导的。根据免疫刺激性核酸分子将受试者的免疫应答从Th2(其与IgE抗体的产生和过敏有关)转换为Th1应答(其有抗过敏反应的保护作用)的能力,可以单独或者与过敏原相结合地向受试者施用有效剂量的免疫刺激性核酸(或含有核酸地载体),以便治疗或防止过敏。
含有未甲基化CpG基元的核酸还可能在治疗气喘中有重要的治疗用处。Th2细胞因子,特别是IL-4和IL-5,都是在气喘病人的呼吸道中水平增加。这些细胞因子促进了气喘发炎反应的重要的方面,包括IgE同种型转换,嗜伊红粒细胞趋化性和活化,以及肥大细胞生长。Th1细胞因子,特别是IFN-γ和IL-12,可以对Th2克隆的形成和Th2细胞因子的生产进行抑制。
如同在下面的实施例12中所具体描述的那样,含有未甲基化CpG基元的寡核苷酸(即,TCCATGACGTTCCTGACGTT,SEQ ID NO:10)可以在小鼠气喘模型中防止炎性细胞浸润和嗜伊红细胞增多化,而对照寡核苷酸(TCCATGAGCTTCCTGAGTCT SEQ ID NO:11,)却不能够。进而言之,对嗜伊红细胞性发炎的抑制与对Th2应答的抑制和对Th1应答的诱导相关。
在治疗应用中,可以将有效量的适宜的免疫刺激性核酸分子自己或者配制成递送复合物地形式通过任何能够让寡核苷酸被适宜的靶细胞(例如,B细胞和单核细胞)摄入的方式向受试者给药。优选的给药途径包括口服和透皮给药(例如通过贴片)。其它的给药途径实例包括注射(皮下、静脉内、肠胃外、腹膜内、胸内,等)。注射可以一次给足,也可以连续输注。
核酸自己或作为核酸递送复合物的形式都可以与药理学可接受的载体一起给药。在本文中,术语“药理学可接受的载体”指的是那些能够同核酸或核酸递送复合物一起给药并使核酸发挥其指定功能的物质。这样的载体的例子包括溶液、溶剂、分散介质、延迟剂、乳液,等等。这些介质作为药理学活性物质的用途是本领域熟知的。任何其它的适合用于核酸的常规载体也都在本发明所包括的范围之内。
术语核酸分子的“有效量”指的是对实现所需要的生物学效果所必需的或充足的用量。例如,对含有至少一个未甲基化CpG的核酸用于治疗免疫系统缺陷来讲,有效量是对消除肿瘤、癌症、或者细菌、病毒、或真菌感染所必需的量。对于用作疫苗佐剂来讲,有效量是可用于加强受试者对疫苗的免疫应答的用量。治疗气喘的“有效量”可以是能将与气喘相关的Th2型免疫应答转变为Th1型应答的用量。对于任何具体应用的有效量,可以根据诸多因素来决定,例如,需要治疗的疾病或状态,具体施用的核酸(例如,未甲基化CpG基元的数目或它们在核酸中的位置),治疗受试者的身材,或状况的严重性。本领域的技术人员不需要进行过度实验就可以确定特定寡核苷酸的有效用量。
本发明将由下面的实施例进一步阐述,这些实施例不从任何角度限制本发明。在本申请全文中所提到的文献(包括文献材料,授权的专利,公开的专利申请,和共悬未决的专利申请)都通过引用而明确地并入本文。
实施例
实施例1:ODN对B细胞总RNA合成以及细胞周期的影响
从不带有特定病原体的6-12周龄的DBA/2或BXSB小鼠(饲养在Iowa大学动物实验中心,没有发现明显的品系差异)的脾中获得B细胞,用抗-Thy-1.2和补体消除T细胞,对淋巴细胞M离心(Cedarlane Laboratories,Hornby,Ontario,Canada)(此后称为“B细胞”)。B细胞中含有少于1%的CD4+或CD8+细胞。将8×104个B细胞一式三份加入96孔微滴板中,每孔中含有100μl的RPMI,其中含有10%的FBS(在65℃下热灭活30分钟),50μM的2-巯基乙醇,100U/ml的青霉素,100μg/ml链霉素,以及2mM的L-谷氨酸。在开始培养时加入20μM的ODN,在37℃下进行20小时,细胞用1μCi的3H尿苷脉冲,4小时后收获细胞并计数。全脾细胞用20μM的ODN培养48小时后,对分泌Ig的B细胞用ELISA点检测法计数。表1给出了有关的结果,数据代表与未用ODN培养的细胞比较而言的刺激指数。3H胸苷掺入检测显示了相似的结果,但是有从降解的ODN释放的胸苷造成的一些非特异性抑制[Matson.S and A.M.Krieg(1992)Nonspecific suppression of3H-thymidine incorporation by control oligonucleotides.Antisense Research and Development 2:325]。
实施例2:ODN对B细胞生产IgM的影响
从新杀死的小鼠获得的脾制备单细胞悬浮液,用抗-Thyl,抗-CD4,和抗-CD8以及补体处理,具体方法见Leibson等人的文献[Leibson et al.,J.Exp.Med.154:1681(1981)]。按照DeFranco等人的程序[DeFranco et al.,J.Exp.Med.155:1523(1982)]从不连续Percoll梯度的63-70%条带中分离静息B细胞(T细胞污染<0.2%)。按照上述用30μM的ODN或20μg/ml的LPS培养它们48小时。活跃分泌IgM的B细胞数在此时间点为最大,正如ELISpot检测法所确定的[Klinman,D.M.et al.,J.Immunol.144:506(1990)]。在这个检测中,B细胞在抗-Ig涂布的微滴板上培养6小时。它们所产生的Ig(>99%的IgM)用磷酸酶-标记的抗-Ig检测[Southern Biotechnology Associated,Birmingham,AL]。各B细胞产生的抗体通过加入BCIP(SigmaChemical Co.,St.Louis MO)来观察,在有磷酸酶存在时,BCIP形成不溶性兰色沉淀。将产生20-40点/孔的细胞稀释度用于确定样品中抗体分泌性B细胞的总数。所有的检测都一式三份进行(数据给出在表1)。在有些实验中,培养物上清液用ELISA来检测IgM,显示了响应于CpG-ODN有相似的提高。
实施例3:细菌DNA对B细胞的刺激
不加入DNA、或加入50μg/ml的a)Micrococcuslysodeikticus;b)NZB/N小鼠脾;c)NFS/N小鼠脾基因组DNA对DBA/2B细胞培养48小时后,然后用3H胸苷脉冲4小时,再收获细胞。对一式双份的DNA样品用DNASE I在37℃下酶解30分钟,然后加入细胞培养物。用ELISA-点检测法确定了在48小时的时候,大肠杆菌DNA也诱导了IgM分泌性B细胞数目提高了8.8倍。
不加入任何添加物,或加入50μg/ml的LPS,或20μM的ODN 1、1a、4、或4a对DBA/2B细胞进行培养。培养细胞并在第4、8、24和48小时收集细胞。对BXSB细胞按照实施例1所述进行培养,其中分别采用5、10、20、40或80μM的ODN1、1a、4、4a或LPS。在这个实验中,没加入ODN的小孔中为3833cpm。每个实验都进行至少三次,得到了相似的结果。重复的三个小孔的标准偏差小于5%。
实施例4:ODN对天然杀伤细胞(NK)活性的影响
10×106个C57BL/6脾细胞培养在2ml的RPMI中(按照实施例1进行补充),其中加入或不加入40μM的CpG或非-CpG ODN,培养48小时。洗过细胞后,在短期51Cr释放检测中作为效应细胞,检测中使用了YAC-1和2C11这两种NK敏感性靶细胞系[Ballas,Z.K.et al.(1993)J.Immunol.150:17]。效应细胞以各种浓度加至V底微滴板中0.2ml内含有的10451Cr标记的靶细胞,在37℃温度于5%的CO2下培养4小时。然后离心,对上清液的等份试样计数放射活性。特异性裂解百分数的计算是,在有效应细胞存在时的51Cr释放减去靶细胞单独培养时的51Cr释放,与用2%乙酸进行细胞裂解后释放的总计数减去细胞单独培养时释放的51Cr cpm之间的比值。
实施例5:对CpG硫代磷酸酯ODN的体内研究
对小鼠称重,腹腔内注射0.25ml的无菌PBS或给定剂量的溶于PBS中的硫代磷酸酯ODN。24小时后,收获脾细胞,洗过之后染色,进行流式细胞术测定,使用缀合藻红蛋白的6B2,来门控B细胞,以及缀合生物素的抗Ly-6A/E或抗-Iad(Pharmingen,San Diego,CA)或抗-Bla-1[Hardy,R.R.et al.,J.Exp.Med.159:1169(1984)]。每种条件都实验了两只小鼠,每只小鼠单独进行分析。
实施例6:硫代磷酸酯ODN对B细胞刺激的效价
用硫代磷酸酯ODN培养B细胞,该ODN具有对照ODN1a的序列,或CpG ODN 1d的序列,或是3Db的序列,20小时后,加入3H尿苷脉冲,或44小时后加入3H胸苷脉冲,然后收获细胞并测定cpm。
实施例7:挽救B细胞免于编程性细胞死亡
在存在或不存在LPS或对照ODN 1a或CpG ODN 1d和3Db的条件下将WEHI-231细胞(5×104个/孔)于37℃下培养1小时,然后加入抗-IgM(1μ/ml)。细胞继续培养20小时,然后加入2μCi/孔的3H胸苷脉冲4小时。在这个实验中,没有ODN的或抗-IgM的细胞有90.4×103cpm的3H胸苷掺入。表1中给出的磷酸二酯ODN也给予了相似的保护,虽然由于ODN的降解有一些非特异性抑制。每个实验都进行至少3次,得到相似结果。
实施例8:体内诱导小鼠IL-6
DBA/2雌性小鼠(2月龄)腹膜内注射500g CpG或对照硫代磷酸酯ODN。在注射后的不同时间点对小鼠采血。每个时间点研究2只小鼠。用ELISA测定IL-6,IL-6的浓度通过与用重组IL-6制备的标准曲线进行比较来计算。用与标准曲线比较来计算。该检测的灵敏度为10pg/ml。8小时后的水平无法检测到。
实施例9:系统性诱导小鼠IL-6转录
小鼠和细胞系.DBA/2,BALB/c,和C3H/HeJ小鼠,5-10周龄,用作淋巴细胞来源。所有的小鼠都来自于The JacksonLaboratory(Bar Harbor,ME),并在Iowa大学的动物饲养中心内在无特异性病原体条件下培育和饲养。小鼠B细胞系CH12.LX由G.Bishop博士(Iowa大学,Iowa City)慷慨提供。
制备细胞.对小鼠断颈处死。从小鼠的脾无菌制备单细胞悬浮液。使用抗-Thy-1.2和补体制备消除了T细胞的小鼠脾细胞,并对淋巴细胞M(Cedarlane Laboratories,Hornby,Ontairo,Canada)离心,具体如文献所述进行[Krieg,A.M.et al.,(1989)A role for endogenous retroviral sequences in theregulation of lymphocyte activation.J.Immunol.143:2448]。
ODN和DNA.磷酸二酯寡核苷酸(O-ODN)和主链修饰的硫代磷酸酯寡核苷酸(S-ODN)得自于Iowa大学DNA中心机构或Operon Technologies(Alameda,CA)。大肠杆菌DNA(菌株B)和小牛胸腺DNA购自于Sigma公司(St.Louis,MO)。所有的DNA和ODN都通过用酚∶氯仿∶异戊醇(25∶24∶1)和/或乙醇沉淀进行纯化。在使用前,对大肠杆菌DNA和小牛胸腺DNA煮沸10分钟,随后冰上冷却5分钟,使它们成为单链。对于某些实验,大肠杆菌DNA和小牛胸腺DNA都用DNase I(2U/μg DNA)在含有5mM MgCl2的1X SSC中在37℃下消化2小时。为了对大肠杆菌DNA中的CpG二核苷酸内的胞嘧啶甲基化,将大肠杆菌DNA用CpG甲基化酶(M.SssI;2U/μg DNA)于NE缓冲液2(补充160μM的S-腺苷酰甲硫氨酸)中处理,37℃下过夜孵育。对甲基化DNA的纯化按照上述进行。甲基化的效率通过Hpa II消化,随后凝胶电泳分析来证实。所有的酶都购自于New England Biolabs(Beverly,MA)。由鲎检测法确定的,在ODN中的LPS水平低于12.5ng/mg,大肠杆菌DNA和小牛胸腺DNA中含有少于2.5ng的LPS/mgDNA。
细胞培养物.所有的细胞都在5% CO2的湿润气氛下在RPMI-1640培养基中于37℃培养24小时(对于IL-6生产)或5天(对于IgM生产),该RPMI-1640中补充了10%(v/v)的热灭活胎牛血清(FCS),1.5mM L-谷氨酰胺(50μg/ml),CpG或非CpG磷酸二酯ODN(O-ODN)(20μM),硫代磷酸酯ODN(S-ODN)(0.5μM),或大肠杆菌DNA(50μg/ml)或小牛胸腺DNA(50μg/ml)。刺激物浓度的选择按照滴定法的初步研究来决定。在某些情况下,细胞用CpG O-ODN以及各种浓度(1-10μg/ml)的抗小鼠IL-6的中和性大鼠IgG1抗体(杂交瘤MP5-20F3)或针对大肠杆菌b-半乳糖苷酶的对照大鼠IgG1单克隆抗体(杂交瘤GL113;ATCC,Rockville,MD)(20)处理5天。在培养结束时,对培养物上清液组分用ELISA按照下述进行分析。
体内诱导IL-6和IgM.对BALB/c小鼠静脉内注射PBS,小牛胸腺DNA(200μg/100μl PBS/小鼠),大肠杆菌DNA(200μg/100μl PBS/小鼠),或者CpG或非CpG S-ODN(200μg/100μl PBS/小鼠)。小鼠(2只/每组)在不同的时间点在后眼窝穿刺采血并折颈处死。取出肝,脾,胸腺和骨髓,从这些器官中用RANzol B(Tel-Test,Friendswood,TX)根据厂商的说明制备RNA。
ELISA.平底Immun 1板(Dynatech Laboratories,Inc.,Chantilly,VA)用100μl/孔抗-小鼠IL-6单克隆抗体(MP5-20F3)(2μg/ml)或抗小鼠IgM μ-链特异性(5μg/ml;Sigma,St.Louis,MO)在碳酸盐-碳酸氢盐pH9.6缓冲液(15nM Na2CO3,35mMNaHCO3)中4℃过夜进行包被。然后,用TPBS(0.5mMMgCl2·6H2O,2.68mM KCl,1.47mM KH2PO4,0.14M NaCl,6.6mM K2HPO4,0.5%Tween 20)洗该板,用TPBS中的10%FCS室温下封闭该板2小时,然后再次洗该板。培养物上清、小鼠血清、重组的小鼠IL-6(Pharmingen,San Diego,CA)或纯化的小鼠IgM(Calbiochem,San Diego,CA)都用10%FCS进行适宜的稀释,在一式三份的小孔内在室温下培育6小时,洗该板,将生物素化大鼠抗-小鼠IL-6单克隆抗体(MP5-32C11,Pharmingen,San Diego,CA)(1μg/ml,在10%FCS中)或生物素化的抗-小鼠Ig(Sigma,St.Louis,MO)以100μl/孔的剂量加入到各个小孔中,室温下培育45分钟,然后用TPBS洗。加入以1∶4000的稀释度在10%FCS(100μl/孔)中稀释的缀合了辣根过氧化物酶(HRP)的抗生物素蛋白(Bio-rad Laboratories,Hercules,CA),室温下培育30分钟。洗板后,加入邻苯二胺二盐酸盐(OPD,Sigma,St.Louis MO),0.05M磷酸盐-柠檬酸盐缓冲液,pH 5.0显色30分钟。加入0.67N的H2SO4终止反应,用微滴板读数仪(Cambridge Technology,Inc.,Watertown,MA)在490-600nm处读板。结果在图1和2中给出。
RT-PCR.有义引物、反义引物、以及IL-6的内部寡核苷酸探针都用公开的序列来合成[Montgomery,R.A.and M.S.Dallman(1991),Analysis of cytokine gene expression duringfetal thymic ontogeny using the polymerase chain reaction(J.Immunol.)147:554]。cDNA合成以及IL-6的PCR都基本按照Montgomery和D allman所述的方法进行[Montgomery,R.A.and M.S.Dallman(1991),Analysis of cytokine geneexpression during fetal thymic ontogeny using the polymerasechain reaction(J.Immunol.)147:554],其中采用了来自Perkin-Elmer公司(Hayward,CA)的RT-PCR试剂。扩增30个循环后采用凝胶电泳及随后unblot分析方法[Stoye,J.P.etal.,(1991)DNA hybridization in dried gels with fragmentedprobes:an improvement over blotting techniques,Techniques3:123]分析样品。简言之,凝胶在变性缓冲液(0.05M NaOH,1.5M NaCl)中于室温下杂交30分钟,然后在复性缓冲液(1.5M NaCl,1M Tris,pH 8)中培育30分钟,再用双蒸水洗30分钟。干燥凝胶后,在47℃预杂交2小时,杂交缓冲液(5X SSPE,0.1%SDS)中含有10μg/ml的变性鲑鱼精子DNA。对凝胶用2×106cpm/ml的针对IL-6的g-[32P]ATP末端标记的内部寡核苷酸探针[(5′CATTTCCACGATTTCCCA3′)SEQ ID NO:56]在47℃过夜杂交,室温下洗4次(2X SSC,0.2%SDS),然后放射性自显影。结果在图3中给出。
细胞增殖检测.DBA/2小鼠脾B细胞(5×104个细胞/100μl/孔)在37℃下用培养基、CpG或非CpG S-ODN(0.5μM)或O-ODN(20μM)处理24小时。最后4小时加入[3H]胸苷或[3H]尿苷(1μCi/孔)对细胞进行脉冲。[3H]掺入量用液体闪烁分析仪(Packard Instrument Co.,Downers Grove,IL)测定。
转染和CAT检测.WEHI-231细胞(107细胞)用20μg对照或人IL-6启动子-CAT构建物(由阿肯色大学的S.Manolagas慷慨提供)[Pottratz,S.T.et al.,(1994)17B-estradiol inhibitsexpression of human interleukin-6 promoter-reporterconstructs by a receptor-dependent mechanism.J.Clin.Invest.93:944]在250mV和960μF下进行电穿孔。电穿孔后,对细胞用各种浓度的CpG或非-CpG ODN刺激。转染16小时后,用溶液检测法来测定氯霉素乙酰转移酶(CAT)的活性[Seed,B.and J.Y.Sheen(1988)A single phase-extraction assay forchloramphenical acetyl transferase activity.Gene 76:271]。结果在图5中给出。
实施例10:寡脱氧核苷酸修饰 决定了CpG基元的B细胞刺激程度
采用标准程序在Applied Biosystem公司(AppliedBiosystems Inc.,Foster City,CA)的型号为380A、380B、或394的DNA合成仪上合成ODN[Beacage and Caruthers(1981)Deoxynucleoside phosphoramidites--A new class of keyintermediates for deoxypolynucleotide syntheses.TetrahedronLetters 22,1859-1862]。磷酸二酯ODN用标准的β-氰基乙基亚磷酰胺化学方法合成。硫代磷酸酯连键用元素硫氧化亚磷酸连键来引入,而不是采用标准的碘氧化法。四种常见的核苷亚磷酰胺购自于Applied Bisystem公司。所有含磷酸二酯和硫代磷酸酯的ODN都用浓氨水在55℃处理12小时来去保护。对ODN用凝胶排阻层析提纯并在使用前冻至干燥。二硫代磷酸酯连键用脱氧核苷S-(b-苯甲酰基巯基乙基)吡咯烷硫代亚磷酰胺来引入[Wiesler,W.T.et al.,(1993)In Methods in Molecular Biology:Protocols for Oligonucleotides and Analogs-Synthesis andProperties,Agrawal,S.(ed),Humana Press,191-206.]。含有ODN的二硫代酯用浓氨水在55℃处理12小时,然后用反向HPLC提纯来去保护。
为了合成在所需要的核苷酸间连键位置含有甲基硫代膦酸酯或甲基膦酸酯以及磷酸二酯的寡聚物,采用了两种不同的合成途径。这两种途径的主要合成区别是采用二烷基氨基甲基核苷磷化氢时的偶连,而对甲基硫代膦酸酯采用了氧化试剂。为了合成任一种衍生物,对二烷基氨基甲基核苷磷化氢延长了其缩合时间,因为偶连动力学比较缓慢[Jager and Engels,(1984)Synthesis of deoxynucleoside methylphophonates via aphosphonamidite approach.Tetrahedron Letters 24,1437-1440]。在偶连步骤完成后,对甲基磷酸二酯用硫化试剂处理[5%元素硫,100mM的N,N-二甲基氨基吡啶,存在于二硫化碳/吡啶/三乙胺中],连续四次的450秒处理,每次都产生了甲基硫代膦酸酯。为了产生硫代膦酸酯连键,对甲基磷酸二酯用标准的氧化试剂处理(0.1M碘,在四氢呋喃/2,6-二甲基吡啶/水中)。
对硅胶结合的寡聚物用蒸馏吡啶/浓氨水1∶1(v/v)在4℃下处理4天。上清液真空干燥后,溶解于水中,在G50/50 Sephadex柱上色谱。
在本文中,O-ODN指的是磷酸二酯ODN;S-ODN指的是完全硫代磷酸酯修饰的ODN;S-O-ODN指的是嵌合性ODN,其中的中央连键是磷酸二酯,但是2个5′和5个3′连键是硫代磷酸酯修饰的;S2-O-ODN指的是嵌合性ODN,其中的中央连键是磷酸二酯,但2个5′和5个3′连键是二硫代磷酸酯修饰的;MP-O-ODN是嵌合性ODN,其中的中央连键是磷酸二酯,但2个5′和5个3′连键是甲基膦酸酯修饰的。所研究的ODN序列(CpG二核苷酸用下划线标出)包括:
3D(5″GAGAACGCTGGACCTTCCAT),(SEQ ID NO.14);
3M(5′TCCATGTCGGTCCTGATGCT),(SEQ ID NO.31);
5(5′GGCGTTATTCCTGACTCGCC),(SEQ ID NO.57);
6(5′CCTACGTTGTATGCGCCCAGCT),(SEQ ID NO.58)。
这些序列基本上代表了在这些研究过程上所测验过的数百种CpG和非-CpG的ODN。
小鼠.DBA/2或BXSB来自于The JacksonLaboratory(Bar Harbor,ME),饲养在特定的物病原体条件下,用5-10周龄的小鼠作淋巴细胞来源,得到基本相同的结果。
细胞增殖检测.对于细胞增殖检测,将小鼠脾细胞(5×104个细胞/100μl/孔)于37℃在5%CO2润湿箱中培养于RPMI-1640中,该培养基中补充以10%(v/v)热灭活胎牛血清(对O-ODN实验加热到65℃,对仅仅使用修饰的ODN的实验加热到56℃),1.5μM L-谷氨酰胺,50μM的2-巯基乙醇,100U/ml青霉素和100μg/ml链霉素,按照给出的要求培养24小时或48小时。每个小孔中加入1μCi的3H尿苷或胸苷(按给出的要求进行),继续培养4小时后,收获细胞。对过滤物用闪烁仪计数。一式三份小孔的标准偏差小于5%。结果在图6-8中给出。
实施例11:NK活性的诱导
磷酸二酯ODN购自于Operon Technologies公司(OperonTechnologies,Alameda,CA)。硫代磷酸酯ODN购自于Iowa大学的DNA中心机构,或购自于Midland Certified ReagentCompany(Midland,TX)。大肠杆菌(菌株B)DNA和小牛胸腺DNA购自于Sigma公司(Sigma,St.Louis,MO)。所有的DNA和ODN都用酚∶氯仿∶异戊醇(25∶24∶1)萃取和/或乙醇沉淀来纯化。在ODN中的LPS水平低于12.5ng/mg,大肠杆菌DNA和小牛胸腺DNA含有低于2.5ng LPS/mg DNA,由鲎检测方法确定。
根据合同通过Veterans Affairs由National CancerInstitute(Bethesda,MD)获得不含有病毒的4-6周龄DBA/2小鼠、C57BL/6(B6)小鼠,以及先天无胸腺的BALB/C小鼠。C57BL/6SCID小鼠饲养在Iowa大学动物饲养站的SPF封闭饲养设施中。
人外周血单核淋巴细胞(PBMC)按照前述获得[Ballas,Z.K.et al.,(1990)J.Allergy Clin.Immunol.85:453;Ballas,Z.K.andW.Rasmussen(1990)J.Immunol.145:1039;Ballas,Z.K.andW.Rasmussen(1993)J.Immunol.150:17]。人或小鼠的细胞按5×106个细胞/孔的数量放在24孔微滴板中,培养于37℃、5% CO2润湿气氛下[Ballas,Z.K.et al.,(1990)J.Allergy Clin.Immunol.85:453;Ballas,Z.K.and W.Rasmussen(1990)J.Immunol 145:1039;and Ballas,Z.K.and W.Rasmussen(1993)J.Immunol.150:17],其中仅加入培养基,或以所示浓度加入CpG或非CpG ODN,或加入大肠杆菌DNA或小牛胸腺DNA(50μg/ml)并在37℃培养24小时。所有的培养物在18小时后收获,将这些细胞,按照以前的描述在标准的4小时51Cr-释放检测中用作针对K562(人)或YAC-1(小鼠)靶细胞的效应细胞。在计算裂解单位(LU)时,1LU定义为实现30%特异性裂解所需要的细胞数。在指示时,在开始培养的时候,加入中和性抗IFN-β抗体(Lee Biomolecular,San Diego,CA),或IL-12(C15.1,C15.6,C17.8,和C17.15;来自于GiorgioTrinchieri博士,The Wistar Institute,Philadelphia,PA),或它们的同型型对照,至浓度为10μg/ml。在加入抗-IL-12时,同时加入4种单克隆抗体(MAB)(或同型型对照)各10μg。重组的人IL-2的使用浓度为100U/ml。
实施例12:在小鼠气喘模型中防止炎性细胞浸润 和嗜伊红粒细胞增多症的发展
6-8周龄的C56BL/6小鼠(来自The Jackson Laboratory,Bar Harbor,ME)在第0天和第7天腹膜内注射5000个血吸虫卵(Schistosoma mansoni eggs)进行免疫。这些血吸虫卵含有抗原[血吸虫卵抗原(SEA)],其诱导Th2免疫应答(例如产生IgE抗体)。IgE抗体的产生已知是气喘的重要诱因。
然后,对免疫的小鼠用寡核苷酸(30μg在200μl盐水中,腹膜内注射)处理,这些寡核苷酸中要么含有未甲基化CpG基元(即,TCCATGACGTTCCTGACGTT;SEQ ID NO.10),或不含这种基元(即,对照物,TCCATGAGCTTCCTGAGTCT;SEQ ID NO.11)。可溶性SEA(10μg在25μl盐水中)在第14天和第21天鼻内滴入给药。盐水用作对照。
在呼吸道攻击后的不同时间点处死小鼠。进行全肺灌洗,收集呼吸道和肺泡的炎性细胞。用ELISA测定灌洗液的细胞因子水平。从全肺中分离RNA,进行Northern分析和RT-PCR研究,使用了CsCl梯度。对肺部组织充气并灌注4%低聚甲醛来进行组织学检查。
图9显示的是,小鼠开始被腹膜内注射血吸虫卵,然后吸入血吸虫卵抗原(空心环)时,则在肺部有很多的炎性细胞。然而,当小鼠在开始的时候与血吸虫卵一起接受了含有未甲基化CpG基元的核酸,则在随后吸入血吸虫卵抗原(空心三角形)后,肺部炎性细胞没有增多。
图10显示的是,当测定肺部灌洗液中存在的嗜伊红粒细胞时获得相同结果。嗜伊红粒细胞是与气喘非常密切相关的炎性细胞类型。
图11显示,当小鼠在开始暴露于血吸虫卵的时候用对照寡核苷酸进行处理,则在吸入了SEA后,对随后的嗜伊红粒细胞向肺部的流入没有影响。因此,当小鼠在第14天或第21天吸入血吸虫卵后,它们发生了肺部的急性炎性反应。然而,在第0天和第7天开始接触抗原的时候将CpG寡核苷酸与血吸虫卵一起提供给小鼠的话,则几乎彻底消除了小鼠在第14天吸入血吸虫卵抗原时所造成的嗜伊红粒细胞的增高。
图12显示,很低剂量的寡核苷酸(<10μg)就可以提供这样的保护。
图13显示,所发生的炎性应答与肺部Th2细胞因子IL-4的水平相关。
图14显示,施用含有未甲基化CpG基元的寡核苷酸确实能够将肺部的细胞因子应答改变为IL-12的生产,表明Th1类型的免疫应答。
图15显示,施用含有未甲基化CpG基元的寡核苷酸还可以将肺部的细胞因子应答改变为IFN-γ的生产,表明Th1类型的免疫应答。
实施例13:CpG寡核苷酸诱导人PBMC分泌细胞因子
对Ficoll Hypaque用标准离心从全血中制备人PBMC。细胞(5×105/ml)在96孔微滴板内,在与10%自体血清中,用CpG或对照寡脱氧核苷酸(对磷酸二酯寡核苷酸为24μg/ml,对核酸酶抗性硫代磷酸酯寡核苷酸为6μg/ml)一起培养,对TNF-α而言为4小时,对其它的细胞因子而言为24小时,然后收获上清并用ELISA检测,使用Quantikine试剂盒,或使用来自R&D Systems(pg/ml)的试剂,或使用来自Biosource的细胞因子ELISA试剂盒(对于IL-12检测)。各检测都按照厂商的说明书进行。表6提供了这些实验的数据,以比没有加入寡脱氧核苷酸的小孔高出的细胞因子水平来表示。
本领域的技术人员将能够认识到或采用不超过常规的实验可以确定本文所述本发明具体实施方案的众多等同物。这些等同物都意图包括在本发明的权利要求书中。
序列表
<110>艾奥华大学研究基金会
     美国政府,其由健康与人类服务部部长作为代表
<120>免疫刺激性核酸分子
<130>C1039.70005CN01
<140>200710130052.0
<141>2007-07-25
<150>US 08/738,652
<151>1996-10-30
<150>PCT/US97/19791(97199352.1)
<151>1997-10-30
<160>104
<170>PatentIn version 3.3
<210>1
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>1
atggaaggtc cagtgttctc                                     20
<210>2
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>2
atcgacctac gtgcgttctc                                     20
<210>3
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>3
atcgactctc gagcgttctc                                     20
<210>4
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>4
atggaggctc catcgttctc                                        20
<210>5
<211>19
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>5
gagaacgtcg accttcgat                                         19
<210>6
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(3)..(3)
<223>其中n是5-甲基胞嘧啶
<220>
<221>修饰的碱基
<222>(10)..(10)
<223>其中n是5-甲基胞嘧啶
<220>
<221>修饰的碱基
<222>(14)..(14)
<223>其中n是5-甲基胞嘧啶
<400>6
atngactctn gagngttctc                                        20
<210>7
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>7
tccatgacgt tcctgatgct                                        20
<210>8
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>8
tccatgagct tcctgagtct                                        20
<210>9
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>9
tccaagacgt tcctgatgct                                        20
<210>10
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>10
tccatgacgt tcctgacgtt                                        20
<210>11
<211>21
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>11
tccatgagct tcctgagtgc t                                      21
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<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>12
ggggtcaacg ttcagggggg                                        20
<210>13
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>13
gctagacgtt agcgt                                            15
<210>14
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(7)..(7)
<223>其中n是5-甲基胞嘧啶
<400>14
gctagangtt agcgt                                            15
<210>15
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(13)..(13)
<223>其中n是5-甲基胞嘧啶
<400>15
gctagacgtt agngt                                            15
<210>16
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>16
gcatgacgtt gagct                                            15
<210>17
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>17
atggaaggtc cagcgttctc                                        20
<210>18
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>18
atcgactctc gagcgttctc                                        20
<210>19
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(3)..(3)
<223>其中n是5-甲基胞嘧啶
<220>
<221>修饰的碱基
<222>(10)..(10)
<223>其中n是5-甲基胞嘧啶
<220>
<221>修饰的碱基
<222>(14)..(14)
<223>其中n是5-甲基胞嘧啶
<400>19
atngactctn gagngttctc                                          20
<210>20
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(3)..(3)
<223>其中n是5-甲基胞嘧啶
<400>20
atngactctc gagcgttctc                                            20
<210>21
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(18)..(18)
<223>其中n是5-甲基胞嘧啶
<400>21
atcgactctc gagcgttntc                                            20
<210>22
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>22
atggaaggtc caacgttctc                                           20
<210>23
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>23
gagaacgctg gaccttccat                                            20
<210>24
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>24
gagaacgctc gaccttccat                                            20
<210>25
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>25
gagaacgctc gaccttcgat                                            20
<210>26
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>26
gagcaagctg gaccttccat                                            20
<210>27
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(6)..(6)
<223>其中n是5-甲基胞嘧啶
<400>27
gagaangctg gaccttccat                                            20
<210>28
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(14)..(14)
<223>其中n是5-甲基胞嘧啶
<400>28
gagaacgctg gacnttccat                                            20
<210>29
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>29
gagaacgatg gaccttccat                                            20
<210>30
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>30
gagaacgctc cagcactgat                                            20
<210>31
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>31
tccatgtcgg tcctgatgct                                            20
<210>32
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>32
tccatgctgg tcctgatgct                                            20
<210>33
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(8)..(8)
<223>其中n是5-甲基胞嘧啶
<400>33
tccatgtngg tcctgatgct                                            20
<210>34
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(12)..(12)
<223>其中n是5-甲基胞嘧啶
<400>34
tccatgtcgg tnctgatgct                                            20
<210>35
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>35
tccatgacgt tcctgatgct                                            20
<210>36
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>36
tccatgtcgg tcctgctgat                                            20
<210>37
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>37
atggactctc cagcgttctc                                          20
<210>38
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>38
tccatgccgg tcctgatgct                                          20
<210>39
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>39
tccatggcgg tcctgatgct                                          20
<210>40
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>40
tccatgacgg tcctgatgct                                           20
<210>41
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>41
tccatgtcgatcctgatgct                                            20
<210>42
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>42
tccatgtcgc tcctgatgct                                           20
<210>43
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>43
tccatgtcgt tcctgatgct                                             20
<210>44
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>44
tccatgacgt tcctgatgct                                             20
<210>45
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>45
tccataacgt tcctgatgct                                             20
<210>46
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>46
tccatgacgt ccctgatgct                                             20
<210>47
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>47
tccatcacgt gcctgatgct                                             20
<210>48
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>48
gcatgacgtt gagct                                                     15
<210>49
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>49
gctagatgtt agcgt                                                     15
<210>50
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>E0
ggggtcaagt cttgacgggg                                                20
<210>51
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>51
gctagacgtt agtgt                                                     15
<210>52
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(7)..(7)
<223>其中n是5-甲基胞嘧啶
<220>
<221>修饰的碱基
<222>(7)..(7)
<223>其中n是甲基胞嘧啶
<400>52
gctagangtt agtgt                                                 15
<210>53
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(8)..(8)
<223>其中n是甲基胞嘧啶
<400>53
tccatgtngt tcctgatgct                                            20
<210>54
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(14)..(14)
<223>其中n是5-甲基胞嘧啶
<400>54
atcgactctc gagngttctc                                              20
<210>55
<211>18
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>55
tctcccagcg tgcgccat                                                18
<210>56
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>56
tcgtcgtttt gtcgttttgt cgtt                                         24
<210>57
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>57
tcgtcgttgt cgttgtcgtt                                              20
<210>58
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>58
tcgtcgtttt gtcgttttgt cgtt                                         24
<210>59
<211>22
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>59
tcgtcgttgt cgttttgtcg tt                                           22
<210>60
<211>19
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>60
tgtcgttgtc gttgtcgtt                                                19
<210>61
<211>14
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>61
tcgtcgtcgt cgtt                                                14
<210>62
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>62
tcctgtcgtt ccttgtcgtt                                          20
<210>63
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>63
tcctgtcgtt ttttgtcgtt                                          20
<210>64
<211>21
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>64
tcgtcgctgt ctgcccttct t                                        21
<210>65
<211>21
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>65
tcgtcgctgt tgtcgtttct t                                        21
<210>66
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>66
tcgtcgtttt gtcgttttgt cgtt                                        24
<210>67
<211>22
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>67
tcgtcgttgt cgttttgtcg tt                                          22
<210>68
<211>19
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>68
tgtcgttgtc gttgtcgtt                                              19
<210>69
<211>19
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>69
ggggtcaacg ttgacgggg                                              19
<210>70
<211>18
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>70
catttccacg atttccca                                               18
<210>71
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>71
ggcgttattc ctgactcgcc                                               20
<210>72
<211>22
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>72
cctacgttgt atgcgcccag ct                                            22
<210>73
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>73
accatggacg atctgtttcc cctc                                          24
<210>74
<211>18
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>74
taccgcgtgc gaccctct                                                  18
<210>75
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>75
accatggacg aactgtttcc cctc                                           24
<210>76
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>76
accatggacg agctgtttcc cctc                                           24
<210>77
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>77
accatggacg acctgtttcc cctc                                        24
<210>78
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>78
accatggacg tactgtttcc cctc                                        24
<210>79
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>79
accatggacg gtctgtttcc cctc                                        24
<210>80
<211>24
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>80
accatggacg ttctgtttcc cctc                                        24
<210>81
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>81
cacgttgagg ggcat                                                  15
<210>82
<211>15
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>82
ctgctgagac tggag                                                 15
<210>83
<211>12
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>83
tcagcgtgcg cc                                                    12
<210>84
<211>17
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>84
atgacgttcc tgacgtt                                               17
<210>85
<211>17
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>85
tctcccagcg ggcgcat                                               17
<210>86
<211>18
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>86
tctcccagcg cgcgccat                                              18
<210>87
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>87
tccatgtcgt tcctgtcgtt                                        20
<210>88
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>88
tccatagcgt tcctagcgtt                                       20
<210>89
<211>21
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>89
tcgtcgctgt ctccgcttct t                                     21
<210>90
<211>19
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>90
tcctgacgtt cctgacgtt                                        19
<210>91
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>91
tccaggactt tcctcaggtt                                       20
<210>92
<211>19
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>92
tcctgtcgtt cctgtcgtt                                     19
<210>93
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>93
tccatgtcgt ttttgtcgtt                                    20
<210>94
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>94
tccttgtcgt tcctgtcgtt                                    20
<210>95
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<220>
<221>修饰的碱基
<222>(8)..(8)
<223>其中n是5-甲基胞嘧啶
<220>
<221>修饰的碱基
<222>(17)..(17)
<223>其中n是5-甲基胞嘧啶
<400>95
tccatgtngt tcctgtngtt                                    20
<210>96
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>96
tccaggactt ctctcaggtt                                            20
<210>97
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>97
tccatgcgtg cgtgcgtttt                                            20
<210>98
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>98
tccatgcgtt gcgttgcgtt                                            20
<210>99
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>99
tccacgacgt tttcgacgtt                                            20
<210>100
<211>21
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>100
gcgtgcgttg tcgttgtcgt t                                          21
<210>101
<211>20
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>101
gcggcgggcg gcgcgcgccc                                            20
<210>102
<211>21
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>102
tgtcgtttgt cgtttgtcgt t                                          21
<210>103
<211>25
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>103
tgtcgttgtc gttgtcgttg tcgtt                                      25
<210>104
<211>13
<212>DNA
<213>人工序列
<220>
<223>合成的寡核苷酸
<400>104
tgtcgttgtc gtt                                                   13

Claims (72)

1. 一种免疫刺激性寡核苷酸,其中包含两个或多于两个的未甲基化CpG二核苷酸,其序列包括:5’N1X1X2CGTTN23’,其中所述免疫刺激性寡核苷酸包含一个或多个具有磷酸主链修饰的核苷酸间连键,其中一个或多个核苷酸将连续的CpGs分开;X1X2是GpT或ApT;N1是任何包括5’TC或TG的核苷酸,并且N1+N2为约0-26个碱基,条件是N1和N2均不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该免疫刺激性寡核酸的长度为8-30个碱基。
2. 一种免疫刺激性寡核苷酸,其中包含两个或多于两个的未甲基化CpG二核苷酸,其用来治疗、预防或改善受试体的病变,其序列包括:5’N1X1X2CGTTN23’,其中一个或多个核苷酸将连续的CpGs分开;X1X2是GpT或ApT;N1是任何包括5’TC或TG的核苷酸,并且N1+N2是约0-26个碱基,条件是N1和N2均不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该免疫刺激性寡核酸的长度为8-30个碱基。
3. 根据权利要求1或2的免疫刺激性寡核苷酸,其中N1包括5’TC。
4. 根据权利要求1或2的免疫刺激性寡核苷酸,其中包含被胸腺嘧啶分开的两个或多于两个的连续CpGs。
5. 根据权利要求3的免疫刺激性寡核苷酸,其中GTCGT基元紧跟在5’TC后。
6. 根据权利要求2的免疫刺激性寡核苷酸,其中进一步包含抗原。
7. 根据权利要求1或2的免疫刺激性寡核苷酸,其中所述寡核苷酸是合成的。
8. 根据权利要求2的免疫刺激性寡核苷酸,其中所述寡核苷酸包括一个或多个具有磷酸主链修饰的核苷酸间连键。
9. 根据权利要求1或8的免疫刺激性寡核苷酸,其中所述磷酸主链修饰是硫代磷酸酯修饰或二硫代磷酸酯修饰。
10. 根据权利要求9的免疫刺激性寡核苷酸,其中每个核苷酸间连键是硫代磷酸酯连键。
11. 根据权利要求1或2的免疫刺激性寡核苷酸,其中寡核苷酸包括GTCGCT或GTCGTT基元。
12. 根据权利要求1或2的免疫刺激性寡核苷酸,其不是回文的。
13. 一种免疫刺激性寡核苷酸,其具有如下序列:
TCGTCGTTTTGTCGTTTTGTCGTT。
14. 一种免疫刺激性寡核苷酸,其具有序列
TCGTCGTTGTCGTTTTGTCGTT,
TCCATGTCGTTCTGTTCGTT,
TCCTGTCGTTCCTGTCGTT,
TCCTGTCGTTCCTTGTCGTT,
TCCTTGTCGTTCCTGTCGTT,
TCCTGTCGTTTTTTGTCGTT,
TCGTCGCTGTTGTCGTTTCTT,
TCGTCGTTGTCGTTGTCGTT,
TCGTCGTTTTGTCGTTTTGTCGTT,
TCGTCGTTGTCGTTTTGTCGTT,
TGTCGTTGTCGTTGTCGTTGTCGTT,
TGTCGTTGTCGTTGTCGTT,
TCGTCGTCGTCGTT或
TGTCGTTGTCGTT。
15. 根据权利要求13或14的免疫刺激性寡核苷酸,其中所述寡核苷酸包含一个或多个具有磷酸主链修饰的核苷酸间连键。
16. 根据权利要求15的免疫刺激性寡核苷酸,其中所述磷酸主链修饰是硫代磷酸酯修饰或二硫代磷酸酯修饰。
17. 根据权利要求15的免疫刺激性寡核苷酸,其中每个核苷酸间连键是硫代磷酸酯连键。
18. 一种组合物,其中包含权利要求1、2、8、13或14中任何一项所述免疫刺激性寡核苷酸,并结合有固醇、阳离子脂质、病毒粒子或脂质体,以形成核酸递送复合物。
19. 一种组合物,其中包含权利要求1、2、8、13或14中任何一项所述免疫刺激性寡核苷酸和药理学可接受的载体。
20. 一种组合物,其基本上由权利要求1、2、8、13或14中任何一项所述免疫刺激性寡核苷酸组成。
21. 权利要求1、2、8、13或14中任何一项所述免疫刺激性寡核苷酸在制备用于通过刺激受试体的免疫系统来治疗、预防或改善该受试体内病变的药物中的应用。
22. 根据权利要求21的应用,其中所述药物用来治疗或预防受试体的癌症。
23. 根据权利要求21的应用,其中所述药物用于诱导受试体内的细胞毒性淋巴细胞应答。
24. 根据权利要求21的应用,其中所述药物用于治疗或预防受试体的病毒性,真菌性,细菌性,或寄生虫性感染。
25. 根据权利要求21的应用,其中所述药物用于刺激受试体内对疫苗的应答。
26. 根据权利要求21的应用,其中所述药物用作为刺激受试体的免疫活化的佐剂。
27. 根据权利要求21的应用,其中所述药物用于治疗或预防受试体内的过敏。
28. 根据权利要求21的应用,其中所塑药物用于治疗或预防受试体内的气喘。
29. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备治疗或预防气喘的药物中的应用,其中所述免疫刺激性寡核苷酸不与所施用的过敏原一起使用。
30. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于治疗或预防气喘的口服药物中的应用。
31. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于治疗或预防过敏的药物中的应用,其中所述免疫刺激性寡核苷酸不与所施用的过敏原一起使用。
32. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于治疗或预防过敏的口服药物中的应用。
33. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于治疗或预防湿疹,过敏性鼻炎,鼻炎,枯草热,风疹或食物过敏的药物中的应用。
34. 根据权利要求29-33中任何一项的应用,其中所述免疫刺激性寡核苷酸具有包含通式如下表示的CpG基元的序列:
5′N1X1CGX2N23′
其中一个或多个核苷酸将连续的CpGs分开;X1是腺嘌呤,鸟嘌呤,或胸腺嘧啶;X2是胞嘧啶或胸腺嘧啶;N是任何核苷酸,并且N1+N2是0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且该寡核苷酸的长度为8-30个碱基。
35. 根据权利要求29-33中任何一项的应用,其中所述免疫刺激性寡核苷酸包含通式如下表示的CpG基元:
5′N1X1X2CGX3X4N23′
其中一个或多个核苷酸将连续的CpGs分开;其中X1X2是GpT,GpG,GpA,ApT,或ApA,X3X4是TpT或CpT;N是任何核苷酸,并且N1+N2是0-26个碱基,条件是N1和N2不含有CCGG四元体或多于一个的CCG或CGG三元体;并且所述免疫刺激性寡核苷酸的长度为8-30个碱基。
36. 根据权利要求35中的应用,其中X1X2是GpT,GpG,GpA,或ApA,X3X4是TpT,CpT或GpT。
37. 根据权利要求34的应用,所述药物用来治疗气喘,其中所述免疫刺激性寡核苷酸是:
(a)GTCG(T/C)T或TGACGTT;
(b)TGTCG(T/C)T。
38. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸的长度为8-30个碱基。
39. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在5’端有TC或TG。
40. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸包括被一个或多个胸腺嘧啶分开的两个或更多个连续CpG。
41. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸不包括回文CpG基元。
42. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸是磷酸二酯寡核苷酸。
43. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸包括磷酸主链修饰。
44. 根据权利要求43的应用,其中所述磷酸主链修饰包括硫代磷酸酯修饰的连键。
45. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸是核糖核苷酸或脱氧核糖核苷酸。
46. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸是合成的。
47. 根据权利要求29-33中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸是稳定化的。
48. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于通过增强抗体依赖性细胞毒性效果来治疗癌或肉瘤的药物中的应用。
49. 根据权利要求48的应用,其中癌是脑、肺、卵巢、乳腺、前列腺或结肠肿瘤。
50. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备与化疗一起施用或化疗之后施用以增加恶性细胞对后来的化疗或免疫治疗的反应的药物中的应用。
51. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于与免疫治疗一起施用、之前施用或之后施用以增加恶性细胞对后来的化疗或免疫治疗的反应的药物中的应用。
52. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于与化疗或免疫治疗一起施用、之前施用或之后施用以加快骨髓的恢复的药物中的应用。
53. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于治疗乙型肝炎病毒感染的药物中的应用。
54. 包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸在制备用于治疗丙型肝炎病毒感染的药物中的应用。
55. 根据权利要求29-33或48-54中任何一项的应用,其中所述免疫刺激性寡核苷酸是权利要求1、2、8、13或14中任何一项所述的免疫刺激性寡核苷酸。
56. 根据权利要求29-33或48-54中任何一项的应用,其中所述药物包含固醇、阳离子脂质、病毒体或脂质体,以形成核酸递送复合物或药理学可接受的载体。
57. 根据权利要求29-33或48-54中任何一项的应用,其中所述药物是供人类使用的。
58. 根据权利要求29-33或48-54中任何一项的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服、经皮或通过皮下注射、静脉注射、非肠道注射、腹膜内注射或胸内注射而施用。
59. 根据权利要求22的应用,其中所述药物是供人类使用的。
60. 根据权利要求22的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸是经口服、经皮或通过皮下注射、静脉内注射、胃肠外注射、腹膜内注射或胸内注射而施用。
61. 根据权利要求23的应用,其中所述药物是供人类使用的。
62. 根据权利要求23的应用,其中包括一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服、经皮或通过皮下注射、静脉内注射、胃肠外注射、腹膜内注射或胸内注射而施用。
63. 根据权利要求24的应用,其中所述药物是供人类使用的。
64. 根据权利要求24的应用,其中包括一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服、经皮或通过皮下注射、静脉内注射、胃肠外注射、腹膜内注射或胸内注射而施用。
65. 根据权利要求25的应用,其中所述药物是供人类使用的。
66. 根据权利要求25的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服的、经皮或通过皮下注射、静脉内注射、胃肠外注射、腹膜内注射或胸内注射而施用。
67. 根据权利要求26的应用,其中所述药物是供人类使用的。
68. 根据权利要求26的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服、经皮或通过皮下注射、静脉内注射、胃肠道外注射、腹膜内注射或胸内注射而施用。
69. 根据权利要求27的应用,其中所述药物是供人类使用的。
70. 根据权利要求27的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服、经皮或通过皮下注射、静脉内注射、胃肠外注射、腹膜内注射或胸内注射。
71. 根据权利要求28的应用,其中所述药物是供人类使用的。
72. 根据权利要求28的应用,其中包含一个或多个未甲基化CpG二核苷酸的免疫刺激性寡核苷酸经口服、经皮或通过皮下注射、静脉内注射、胃肠外注射、腹膜内注射或胸内注射而施用。
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CN112263675A (zh) * 2013-07-19 2021-01-26 财团法人卫生研究院 CpG寡脱氧核苷酸、包含其的免疫组合物及制备组合物并通过其刺激免疫反应的方法
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