CN101765610A - 免疫疗法的新型免疫抗原表位 - Google Patents
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- CN101765610A CN101765610A CN200880100806A CN200880100806A CN101765610A CN 101765610 A CN101765610 A CN 101765610A CN 200880100806 A CN200880100806 A CN 200880100806A CN 200880100806 A CN200880100806 A CN 200880100806A CN 101765610 A CN101765610 A CN 101765610A
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Abstract
本发明涉及可与任一类MHC复合物相结合且引发免疫反应的肿瘤相关抗原衍生肽的氨基酸序列,以及该肽在治疗疾病中的用途。
Description
技术领域
本发明涉及可与任一类MHC复合物相结合且引发免疫反应的肿瘤相关抗原衍生肽的新型氨基酸序列。
背景技术
是否能刺激免疫反应取决于是否提呈被宿主免疫***视为异物的抗原。发现肿瘤相关抗原的提呈增加了运用宿主免疫***干预肿瘤生长的可能性。对于癌症免疫疗法,目前正在探索利用免疫***中的体液和细胞免疫的各种机制。
细胞免疫反应的某些元素能特异性地识别和破坏肿瘤细胞。从肿瘤浸润细胞群或外周血中分离出的细胞毒性T细胞(CTL)表明,这些细胞对癌症的天然免疫防御中发挥了重要作用(Cheever et al.,Annals N.Y.Acad.Sci.1993690:101-112;Zeh HJ,Perry-Lalley D,Dudley ME,Rosenberg SA,Yang JC;J Immunol.1999,162(2):989-94;High avidity CTLs for two self-antigensdemonstrate superior in vitro and in vivo antitumour efficacy.)。特别是CD8阳性T细胞(TCD8+)在这种反应中发挥重要作用,TCD8+能识别细胞液8至10个源自蛋白或缺损核糖体产物(DRIPS)(Schubert U,Antón LC,Gibbs J,Norbury CC,Yewdell JW,Bennink JR.;Rapiddegradation of a large fraction of newly synthesized proteins by proteasomes;Nature 2000;404(6779):770-774)的残基中载有主要组织相容性复合体(MHC)的肽中所含的I类分子。人MHC分子也称为人白细胞-抗原(HLA)。
MHC分子有两类:MHC-I类分子,大部分细胞核提呈内源性蛋白DRIPS裂解生成的肽以及较大肽的细胞上都能发现此类分子。MHC-II类分子,主要发现于专职抗原提呈细胞(APC)及其在内吞作用过程中摄取并且随后进行加工的外源性蛋白提呈肽中(Cresswell P.Annu.Rev.Immunol.1994;12:259-93)。肽和MHC-I类分子的复合物由载有相应TCR的CD8阳性细胞毒性T淋巴细胞识别,肽和MHC-II类分子的复合物由载有相应TCR的CD4阳性辅助T细胞识别。本领域已熟知TCR、肽和MHC的化学计量数量丰富,比例为1∶1∶1。
CD4阳性辅助T细胞在安排抗肿瘤T细胞反应的效应子功能中发挥重要作用,因此,肿瘤相关抗原(TAA)衍生的CD4阳性T细胞表位的识别对开发能引发抗肿瘤免疫反应的药物可能非常重要(Kobayashi,H.,R.Omiya,M.Ruiz,E.Huarte,P.Sarobe,J.J.Lasarte,M.Herraiz,B.Sangro,J.Prieto,F.Borras-Cuesta,and E.Celis.2002.Identification of an antigenic epitope forhelper T lymphocytes from carcinoembryonic antigen.Clin.Cancer Res.8:3219-3225.,Gnjatic,S.,D.Atanackovic,E.M.Matsuo,A.Selvakumar,N.K.Altorki,R.G.Maki,B.Dupont,G.Ritter,Y.T.Chen,A.Knuth,and L.J.Old.2003.Survey of naturally occurring CD4+T-cellresponses against NY-ESO-1in cancer patients:Correlation with antibody responses.Proc.Natl.Acad.Sci.U.S.A.100(15):8862-7)CD4+T cells can lead to locally increased levels of IFN(Qin Z,Schwartzkopff J,Pradera F,Kammertoens T,Seliger B,Pircher H,Blankenstein T;A criticalrequirement of interferon gamma-mediated angiostasis for tumour rejection by CD8+T cells;Cancer Res.2003J;63(14):4095-4100)。
在没有炎症的情况下,MHC-II类分子的表达主要局限于免疫***细胞,尤其是专职抗原提呈细胞(APC),例如,单核细胞、单核细胞源性细胞、巨噬细胞、树突状细胞。令人吃惊的是,在肿瘤患者的肿瘤细胞中发现有MHC-II类分子的表达(Dengjel J,Nastke MD,Gouttefangeas C,Gitsioudis G,Schoor O,Altenberend F,Müller M,B,Missiou A,SauterM,Hennenlotter J,Wernet D,Stenzl A,Rammensee HG,Klingel K,S.;Unexpectedabundance of HLA class II presented peptides in primary renal cell carcinomas;Clin Cancer Res.2006;12:4163-4170)。
哺乳动物(如小鼠)模型显示,即使没有细胞毒性T淋巴细胞(CTL)效应细胞(如,CD8阳性T淋巴细胞),CD4阳性T细胞也能通过分泌干扰素-γ(IFNγ)抑制血管生成,从而抑制肿瘤的出现(Qin,Z.and T.Blankenstein.2000.CD4+T-cell-mediated tumour rejectioninvolves inhibition of angiogenesis that is dependent on IFN gamma receptor expression bynonhematopoietic cells.Immunity.12:677-686)。此外,研究还显示,CD4阳性T细胞可识别HLA-II类分子所提呈的肿瘤相关抗原中的肽,这样可通过诱导抗体(Ab)反应而阻止肿瘤进展(Kennedy,R.C.,M.H.Shearer,A.M.Watts,and R.K.Bright,2003.CD4+T lymphocytes play acritical role in antibody production and tumour immunity against simian virus 40large tumourantigen.Cancer Res.63:1040-1045)。与肿瘤相关肽和HLA-I类分子相结合相比较而言,迄今只对TAA的少量II类配体有过描述(www.cancerimmunity.org,www.syfpeithi.de)。
由于HLA-II类分子的组成性表达通常仅限于免疫***的细胞(Mach,B.,V.Steimle,E.Martinez-Soria,and W.Reith.1996.Regulation of MHC class II genes:lessons from a disease.Annu.Rev.Immunol.14:301-331),因此,直接从原发肿瘤中分离II类肽被认为是不可能的事。然而,Dengjel等人最近成功地在肿瘤中直接识别了MHC-II类抗原的表位(EP 04023546.7,EP 05019254.1;Dengjel J,Nastke MD,Gouttefangeas C,Gitsioudis G,Schoor O,Altenberend F,Müller M,B,Missiou A,Sauter M,Hennenlotter J,Wernet D,Stenzl A,Rammensee HG,Klingel K,S.;Unexpected abundance of HLA class II presentedpeptides in primary renal cell carcinomas;Clin Cancer Res.2006;12:4163-4170)。
对于触发(引发)细胞免疫反应的肽,它必须与MHC分子结合。这一过程依赖于MHC分子的等位基因以及肽氨基酸序列的特异性多态性。MHC-I类-结合肽的长度通常为8-10个氨基酸残基,并且在其与MHC分子相应结合沟槽相互作用的序列中通常包含两个保守残基(“锚”)。这样,每个MHC的等位基因都有“结合基序”,从而确定哪些肽能与结合沟槽特异性结合(Rammensee H.G.,Bachmann J.and Stevanovic,S;MHC Ligands andPeptide Motifs,Chapman&Hall 1998)。
在MHC-I类依赖性免疫反应中,肽不仅能与肿瘤细胞表达的某些MHC-I类分子结合,而且它们还必须能被T细胞负载的特异性T细胞受体(TCR)识别。
肿瘤特异性T淋巴细胞所识别的抗原,即它们的表位,可以是源自所有类蛋白的分子,如,酶、受体、转录因子等,这些分子在各个肿瘤的细胞中表达上调。此外,肿瘤相关抗原也可以只存在于肿瘤细胞中,例如,突变基因产物或其他开放阅读框架(ORF)或蛋白剪接产物中(Vigneron N,Stroobant V,Chapiro J,Ooms A,Degiovanni G,Morel S,van der Bruggen P,Boon T,Van den Eynde BJ.An antigenic peptide produced by peptide splicing in the proteasome,Science 2004Apr 23;304(5670):587-90.)。另一种重要的肿瘤相关抗原是组织特异性抗原,如,表达于不同类型肿瘤和健康睾丸组织中的癌睾丸(CT)抗原。
多种肿瘤相关抗原已经得到确定。此外,在识别其他肿瘤相关抗原方面也做了大量研究。有些肿瘤相关抗原,在本领域中也称作肿瘤特异性抗原,具有组织特异性。例子包括但(不仅限于)黑色素瘤的酪氨酸酶、***癌的PSA和PSMA、淋巴瘤中诸如bcr/abl的染色体交叉部位。但是,许多得到确定的肿瘤相关抗原出现于多种类型肿瘤中,有些抗原,如,实际上导致转化事件的致癌蛋白和/或肿瘤抑制基因(肿瘤抑制基因是Linehan WM,Walther MM,Zbar B关于肾癌综述The genetic basis of cancer of the kidney.J Urol.2003Dec;170(6Pt1):2163-72中所提及的)几乎出现于所有类型的肿瘤。例如,控制细胞生长和分化的正常细胞蛋白,如p53(一种肿瘤抑制基因)、ras、c-met、myc、pRB、VHL和HER-2/neu可累积突变,导致这些基因的表达上调,从而致癌(McCartey et al.Cancer Research 199815:582601-5;Disis et al.Ciba Found.Symp.1994,187:198-211)。这些突变蛋白也可是多种癌症的肿瘤特异性免疫反应的一个标靶。
对于被细胞毒性T淋巴细胞识别为肿瘤特异性抗原或相关性抗原以及用于治疗的蛋白,必须具备特殊的条件。该抗原应主要由肿瘤细胞表达,而正常健康组织根本不表达或表达数量较少。此外,必要时,各个抗原不仅出现于一种肿瘤中而且浓度(即,每细胞肽的拷贝数)高。肿瘤特异性抗原和肿瘤相关抗原通常是源于由于细胞周期调控或凋亡等功能在正常细胞转化为肿瘤细胞中直接受累的蛋白。另外,这些直接导致转化事件的蛋白的下游靶标可能会被上调,因此可能与肿瘤间接相关。这些肿瘤间接相关抗原也可能是预防接种方法的靶标(Singh-Jasuja H.,Emmerich N.P.,Rammensee H.G.,Cancer Immunol.Immunoether.2004Mar;453(3):187-95)。在这两种情况中,至关重要的是,都要存在抗原氨基酸序列的表位,所以这种来自肿瘤相关抗原的肽(“免疫原性肽”)可导致体外或体内T细胞反应。
基本上,任何能与MHC分子结合的肽都可能充当一个T细胞表位。诱导体外或体内T细胞反应的前提是存在有着相应TCR的T细胞并且没有这个特定表位的免疫耐受性。
因此,TAA是开发肿瘤疫苗的出发点。识别和标记TAA的方法基于对患者或健康受试者CTL的使用情况,或基于差别转录特性或肿瘤与正常组织肽差别表达模式情况(LemmelC.,Weik S.,Eberle U.,Dengjel J.,Kratt T.,Becker H.D.,Rammensee H.G.,Stevanovic S.Nat.Biotechnol.2004Apr.;22(4):450-4,T.Weinschenk,C.Gouttefangeas,M.Schirle,F.Obermayr,S.Walter,O.Schoor,R.Kurek,W.Loeser,K.H.Bichler,D.Wernet,S.Stevanovic,and H.G.Rammensee.Integrated functional genomics approach for the design of patient-individual antitumorvaccines.Cancer Res.62(20):5818-5827,2002.)。
然而,识别肿瘤组织或人肿瘤细胞株中过量或选择性表达的基因,不能提供免疫疗法中使用这些基因所转录抗原的准确信息。这是因为,有着相应TCR的T细胞必须要存在而且对这个特定表位的免疫耐受性需要不存在或达到最低水平,因此,这些抗原表位只有一部分适用于这种情况。因此,只选择那些蛋白过量或选择性表达的肽,并且这些肽与可发现功能性T细胞的MHC分子一起提呈,这一点非常重要。这种功能性T细胞被定义为“效应T细胞”,在特异性抗刺激后,可复制扩增并可执行效应功能。
辅助T细胞在安排抗肿瘤免疫方面的CTL效应子功能中发挥着重要作用。触发TH1细胞反应的辅助T细胞表位支持CD8阳性杀伤T细胞的效应子功能,其中包括直接作用于肿瘤细胞的细胞毒性功能(该类肿瘤细胞表面显示有肿瘤相关肽/MHC复合物)。这样,肿瘤相关T辅助细胞表位单独使用或与其他肿瘤相关肽结合使用可作为刺激抗肿瘤免疫反应疫苗化合物的活性药物成分。
由于CD8及CD4依赖型反应共同和协同促进抗肿瘤作用,因此,CD8+CTL(配体:MHC-I分子+肽表位)或CD4阳性CTL(配体:MHC-II类分子)对肿瘤相关抗原的识别和鉴定对开发肿瘤疫苗非常重要。因此,本发明的一个目标是为可与任一类MHC复合物结合的肽提供新型氨基酸序列。
附图说明
图1显示了ESI液相色谱质谱确定的结肠癌样本CCA707的肿瘤相关肽(TUMAP)PCN-002(图1a)、胶质母细胞瘤样本GB1006的TOP-002(图1b)、胶质母细胞瘤样本GB1006的PTP-001(图1c)、肾细胞癌样本RCC190的GAL-001(图1d)、胶质母细胞瘤样本GB1002的CHI-001(图1e)、胶质母细胞瘤样本GB1002的JAK-001(图1f)、非小细胞肺癌NSCLC库2中的AKR-001(图1g)、以及胰腺癌样本PC330的FNI-001(图1h),这些物质都以MHC-I类限制方式提呈。
图2显示了ESI液相色谱质谱确定的胃癌GC库2中的肿瘤相关肽(TUMAP)CEA-009(图2)、胃癌GC库1中的TGFBI-006(图2b)、胶质母细胞瘤样本GB6002的TGFBI-007(图2c)、胶质母细胞瘤样本GB1004的TGFBI-008(图2d)、非小细胞肺癌NSCLC库1中的TGFBI-009(图21e)、以及胶质母细胞瘤样本GB6002的TGFBI-010(图2f),这些物质都以MHC-II类限制方式提呈。
图3描述了编码胶质母细胞瘤相关肽PTP-001(图3a)和CHI-001(图3b)的两种基因的表达谱。这两种基因在正常组织不表达或表达很低,而在胶质母细胞瘤样本(GB1006T至GB1011T;NCH359T和NCH361T)中表达增加250多倍。
图4描述了选定肽对HLA-A*0201的结合亲和力,结果用EpI ELISA法根据Sylvester-Hvid,C,Kristensen,N,Blicher,T,Ferre,H,Lauemoller,SL,Wolf,XA,Lamberth,K,Nissen,MH,Pedersen,LO,和Buus,S;2002年在“Establishment of a quantitative ELISA capable ofdetermining peptide-MHC class I interaction,Tissue Antigens,59,251±-258”一文的叙述测得。仅对已知与MHC-I类分子结合的肽进行分析。HLA-DR结合肽的亲和力不能用这种测定法测得。
图5描述了微球促使的外周血ODC-001和NOX-001特异性CD8+淋巴细胞增殖的四聚体技术分析。
与抗CD28+高密度肿瘤抗原A*0201/ODC-001(上板)或抗CD28+高密度肿瘤抗原A*0201/NOX-001(下板)耦合的微球每周对每孔中健康HLA-A*0201的1×106CD8+浓缩PBMC+供体HD100进行刺激。经过三次体外刺激,所有的细胞用抗体CD8FITC+四聚体A*0201/NOX-001PE和A*0201/ODC-001APC进行染色。细胞在淋巴细胞群或CD8+淋巴细胞(右板)上得到门控,数字代表CD8+淋巴细胞群内四聚体+的百分比。
图6描述了5个刺激周期后干扰素γ酶联免疫斑点(IFNγELISPOT)检测到的TGFBI-004体外免疫原性。
TGFBI-004反复引发和再刺激细胞,然后以分别相关TGFBI-004(第1、2、3和4孔)和不相关(阴性对照)肽进行培养。IFNγELISPOT检测后,对ELISPOT阅读器(CTL,美国克利夫兰)进行分析。植物血凝素离子霉素作为阳性对照。数字表示阳性点计数。
图7描述了5个刺激周期后ICS法检测到的TGFBI-004体外免疫原性。
负载TGFBI-004的DC引发细胞,自体PBMC+TGFBI-004反复再刺激细胞。对于读出细胞分别以相关TGFBI-004(第1、2、3和4孔)和不相关(阴性对照)肽进行培养。除了细胞内IFNγ染色,细胞还用CD4-FITC和CD8-PerCP抗体染色。用四色流式细胞仪(BD生物科学公司,德国)进行分析。
图8描述了NOX-001肽体外重刺激后T细胞株产生IFNγ的ELISPOT分析。A.T细胞株7+源自供体HBC-154(分选的CD8+NOX-001四聚体+);B.T细胞株7-源自供体HBC-154(分选的CD8+NOX-001四聚体)。
分选的CD8+NOX-001四聚体+(A.)和分选的CD8+NOX-001四聚体-(B.)细胞在用不相关(MLA-001)(上孔)和相关(NOX-001)(下孔)肽(10μg/ml)重新刺激之后,用IFNγELISPOT法分析。数字表示阳性点计数。
图9显示了本发明肽对HLA-A*0201的亲和力。P116HLA-I类肽和病毒标志物HBV-001的解离常数(KD)用基于ELISA的测定法测得(见“实施例”)。
发明详述
在第一方面,本发明提出了一种肽,其包括选自SEQ ID No.1至SEQ ID No.29群组的一个序列、或与SEQ ID No.1至SEQ ID No.29具有80%同源性的其变体、或诱导与该肽发生T细胞交叉反应的一个变体。
在本发明中,“同源性”一词系指两个氨基酸序列之间的同一度,如肽或多肽序列。前文所述的“同源”是通过将理想条件下调整的两个序列与待比较序列进行比对后确定的。此处要比对的序列在两个序列最佳排列中可能有增加或删除(例如,序列缺口等)。此类序列同源性可通过使用ClustalW等算法创建一个排列而进行计算(Nucleic Acid Res.,22(22):46734680(1994).也可用使用一般序列分析软件,更具体地说,是Vector NTI、GENETYX或由公共数据库,如http://dragon.bio.purdue.edu/bioinfolinks/提供的分析工具。
本领域技术人员能评估特定肽变体诱导的T细胞是否可与该肽本身发生交叉反应(Fong,L,Hou,Y,Rivas,A,Benike,C,Yuen,A,Fisher,GA,Davis,MM,and Engleman,EG;2001,Altered peptide ligand vaccination with Flt3ligand expanded dendritic cells for tumorimmunotherapy,Proc.Natl.Acad.Sci.U.S.A,98,8809-8814)、(Zaremba,S,Barzaga,E,Zhu,M,Soares,N,Tsang,KY,and Schlom,J;Identification of an enhancer agonist cytotoxic T lymphocytepeptide from human carcinoembryonic antigen,Cancer Res.,1997,57,4570-4577;Colombetti,S,Fagerberg,T,Baumgartner,P,Chapatte,L,Speiser,DE,Rufer,N,Michielin,O,and Levy,F;,Impact of orthologous melan-A peptide immunizations on the anti-self melan-A/HLA-A2T cellcross-reactivity,J Immunol.,2006,176,6560-6567;Appay,V,Speiser,DE,Rufer,N,Reynard,S,Barbey,C,Cerottini,JC,Leyvraz,S,Pinilla,C,and Romero,P;Decreased specific CD8+T cellcross-reactivity of antigen recognition following vaccination with Melan-A peptide,Eur.J Immunol.,2006,36,1805-1814)。
表1显示了这些肽、各自SEQ ID NO以及亲本蛋白的信息。
表1:本发明中的肽
20号染色体开放阅读框42
C20orf42是参与肌动蛋白细胞骨架附着到细胞质膜和整合素介导细胞过程的粘着斑蛋白。C20orf42缺失导致丧失功能的突变,从而引起Kindler综合症,它是一种常染色体隐性遗传性皮肤病,其特点是皮肤起泡、逐步萎缩、对光敏感,偶尔也能致癌(Herz,C,Aumailley,M,Schulte,C,Schlotzer-Schrehardt,U,Bruckner-Tuderman,L,and Has,C;Kindlin-1is a phosphoprotein involved in regulation of polarity,proliferation,and motility of epidermalkeratinocytes,J Biol Chem.,2006,281,36082-36090)。最近,一名患有功能丧失突变的患者报告有严重的胃肠道疾病伴出血性肠炎(Sadler,E,Klausegger,A,Muss,W,Deinsberger,U,Pohla-Gubo,G,Laimer,M,Lanschuetzer,C,Bauer,JW,and Hintner,H;Novel KIND 1genemutation in Kindler syndrome with severe gastrointestinal tract involvement,Arch.Dermatol.,2006,142,1619-1624)。
在癌症方面,关于癌症相关基因表达的研究已对C20orf42进行了描述。结果发现,接受检查的70%结肠癌和60%肺癌(n=10)中C20orf42表达过量。Northern Blot法检测显示,正常组织表达仅限于神经肌肉组织(Weinstein,EJ,Bourner,M,Head,R,Zakeri,H,Bauer,C,and Mazzarella,R;URP 1:a member of a novel family of PH and FERM domain-containingmembrane-associated proteins is significantly over-expressed in lung and colon carcinomas,Biochim.Biophys.Acta,2003,1637,207-216)。此外,C20orf42已被确定为参与TGF-β介导的细胞迁移和肿瘤浸润的一种基因(Kloeker,S,Major,MB,Calderwood,DA,Ginsberg,MH,Jones,DA,and Beckerle,MC;The Kindler syndrome protein is regulated by transforming growthfactor-beta and involved in integrin-mediated adhesion,J.Biol.Chem.,2004,279,6824-6833)。
NADPH氧化酶同源物-1(NOX1)
NOX1是一种对生长因子有反应的酶,催化生成活性氧超氧化物(O2-)和过氧化氢(H2O2)。其表达最初发现于结肠、***、子宫和增殖的血管平滑肌细胞中(Suh,Y.A.etal.1999;Cell transformation by the superoxide-generating oxidase Mox1.Nature 401,79-82)。它的表达与一些生物反应相关,如:细胞增殖、血管生成、细胞信号通路激活(Harper,R.W.,Xu,C.,Soucek,K.,Setiadi,H.&Eiserich,J.P.A reappraisal of the genomic organization of humanNox1 and its splice variants.Arch.Biochem.Biophys.2005,435,323-330)。
NOX1在结肠中高表达,但其在结肠生理或病理学中的功能仍然知之甚少。对于正常组织,NOX1在回肠中表达低,在右结肠中表达中等,在左结肠中表达高。NOX1在腺瘤、高分化或低分化结肠腺癌样本中的表达没有统计学差异。NOX1在结肠上皮细胞的隐窝内和腔表面均呈高表达。总之,NOX1是组成型表达于结肠上皮细胞并与致瘤不直接相关的一种酶(Szanto,I.et al.Expression of NOX1,a superoxide-generating NADPH oxidase,in colon cancerand inflammatory bowel disease.J Pathol.2005,207,164-176)。
免疫组织化学表明,NOX1组成型表达于表面粘液细胞。腺瘤和高分化腺癌上调NOX1表达。核因子(NF)-κB主要在NOX1表达丰富的腺瘤和腺癌细胞中被激活,这表明NOX1可在结肠肿瘤中刺激NF-κB依赖型抗凋亡途径(Fukuyama,M.et al.Overexpression of a novelsuperoxide-producing enzyme,NADPH oxidase 1,in adenoma and well differentiatedadenocarcinoma of the human colon.Cancer Lett.2005,221,97-104)。
Wnt3a/β-Catenin信号传导被描述为可诱导NOX1表达(Petropoulos,H.&Skerjanc,I.S.Beta-catenin is essential and sufficient for skeletal myogenesis in P 19cells.J Biol Chem.2002,277,15393-15399)。
最近,活性氧分子表明可诱导内皮细胞凋亡,随后可诱导各种肿瘤细胞粘附分子的表达。这表明,通过阻止ROS的生成而预防肿瘤远处复发是可行的(Ten,KM,van der Wal,JB,Sluiter,W,Hofland,LJ,Jeekel,J,Sonneveld,P,and van Eijck,CH;The role of superoxide anionsin the development of distant tumour recurrence,Br.J Cancer,2006,95,14971503)。
增殖细胞核抗原(PCNA)
PCNA发现于细胞核中,是DNA聚合酶δ的辅助因子。编码蛋白质充当一个同源三聚体,帮助提高DNA复制过程中前导链的持续合成能力。因此,它在所有增殖细胞尤其是肿瘤细胞中表达,用作检测增殖的标志物。
DNA拓扑异构酶II
TOP2A和TOP2B编码一种DNA拓扑异构酶的异形体,这种酶能够控制和改变转录过程中DNA的拓扑状态。这种核酶参与诸如染色体凝集、染色单体分离以及减轻DNA转录和复制过程中产生的扭转应力等过程。DNA拓扑异构酶可催化两个双链DNA的短暂断裂和重新连接,这使得DNA链可穿过对方,从而改变DNA的拓扑结构。这种酶的两种异构体可能以基因复制产物的形式存在。该基因编码的α形式位于17号染色体处,β基因位于3号染色体处。
TOP2A是几种抗癌药物的靶标,这个基因的各种突变都可导致出现耐药。
TOP2A基因与HER-2癌基因(最常见的乳腺癌扩增癌基因)相邻,位于染色***置17q12-q21处,其扩增或删除频率在几乎90%的HER-2扩增原发性乳腺肿瘤中都相等(Jarvinen,TA and Liu,ET;Topoisomerase II alpha gene(TOP2A)amplification and deletion incancer-more common than anticipated,Cytopathology,2003,14,309-313)。另外,对于其他癌症,也报告了有TOP2A扩增。
没有TOP2A DNA复制和细胞***是不可能的。因此,它成为了众多抗癌治疗方案的主要靶标,即使杀伤细胞的确切机制仍难以解释(Kellner,U,Sehested,M,Jensen,PB,Gieseler,F,and Rudolph,P;Culprit and victim-DNA topoisomerase II,Lancet Oncol.,2002,3,235-243)。这种方法的成功与否受到自发耐药性的限制,药物引起的DNA损伤可增加肿瘤的恶性程度。最近的数据表明,TOP2A的扩增和缺失可证明该基因对TOP2A-抑制剂化疗的敏感或耐药,这取决于TOP2A处的具体基因缺陷。
目前尚不清楚TOP2B在癌症中的参是否与TOP2A相似或者这两种异构体之间是否存在重大差别。TOP2B至少可以增加TOP2A的一些活性(Sakaguchi,A and Kikuchi,A;Functional compatibility between isoform alpha and beta of type II DNA topoisomerase,J Cell Sci.,2004,117,1047-1054)。
癌胚抗原相关细胞粘附分子5
癌胚抗原(CEA=CEACAM5)是一种分子量为180kDa的高度糖基化膜蛋白分子,由三个C2Ig样重复单元组成,两侧含有一个N-端IgV样区域和一个C-端区域,其中包括糖磷脂酰肌醇连接区域(Hegde,P,Qi,R,Gaspard,R,Abernathy,K,Dharap,S,Earle-Hughes,J,Gay,C,Nwokekeh,NU,Chen,T,Saeed,AI,Sharov,V,Lee,NH,Yeatman,TJ,and Quackenbush,J;Identification of tumour markers in models of human colorectal cancer using a 19,200-elementcomplementary DNA microarray,Cancer Res.,2001,61,7792-7797)。
癌胚抗原CEA在胎儿发育过程中表达,但是在成人胃肠道上皮表达低。但是,CEA在人肿瘤中有较高比例的过量表达,其中包括90%的胃肠道癌、结直肠癌和胰腺癌,70%的非小细胞肺癌细胞和50%的乳腺癌(Thompson,JA,Grunert,F,and Zimmermann,W;Carcinoembryonic antigen gene family:molecular biology and clinical perspectives,J Clin LabAnal.,2005,5,344-366)。由于CEA在肿瘤细胞及血清中其分泌物呈高表达,已被广泛用作肿瘤标志物(Sikorska,H,Shuster,J,and Gold,P;Clinical applications of carcinoembryonicantigen,Cancer Detect.Prev.,12,321-3551988),并作为监测结直肠癌的标准血清标志物(Locker,GY,Hamilton,S,Harris,J,Jessup,JM,Kemeny,N,Macdonald,JS,Somerfield,MR,Hayes,DF,and Bast,RC,Jr.;ASCO 2006update of recommendations for the use of tumourmarkers in gastrointestinal cancer,J Clin Oncol,2006,24,5313-5327,)。
尽管CEA在肿瘤细胞中过量表达,但是癌症患者通常对这种抗原并未表现出免疫反应(Orefice,S,Fossati,G,Pietrojusti,E,and Bonfanti,G;Delayed cutaneous hypersensitivity reactionto carcinoembryonic antigen in cancer patients,Tumouri,1982,68,473-475)。由于CEA一般在体内表达水平低,因此免疫***通常对其具有耐受性。但是,在一系列的临床疫苗试验中,CEA的免疫原性已得到证明(Sarobe,P,Huarte,E,Lasarte,JJ,and Borras-Cuesta,F;Carcinoembryonic antigen as a target to induce anti-tumour immune responses,Curr.Cancer DrugTargets.,2004,4,443-454),特别是结直肠癌(CRC)(Mosolits,S,Ullenhag,G,and Mellstedt,H;Therapeutic vaccination in patients with gastrointestinal malignancies.A review of immunologicaland clinical results,Ann.Oncol.,2005,16,847-862),并且CEA是这种癌种试验中在最多疫苗平台上得到测试的肿瘤相关抗原(TAA)(von Mehren,M;Colorectal cancer vaccines:what weknow and what we don′t yet know,Semin.Oncol.,2005,32,76-84)。
CEA的几种细胞毒性和辅助T细胞表位已进行了描述(Crosti,M,Longhi,R,Consogno,G,Melloni,G,Zannini,P,and Protti,MP;Identification of novel subdominant epitopes on thecarcinoembryonic antigen recognized by CD4+T-cells of lung cancer patients,J Immunol.,2006,176,5093-5099;Novellino,L,Castelli,C,and Parmiani,G;A listing of human tumour antigensrecognized by T-cells:March 2004update,Cancer Immunol.Immunother.,2004,54,187-207;Ruiz,M,Kobayashi,H,Lasarte,JJ,Prieto,J,Borras-Cuesta,F,Celis,E,and Sarobe,P;Identification andcharacterization of a T-helper peptide from carcinoembryonic antigen,Clin Cancer Res.,2004,10,2860-2867),这使得各种以肽为基础的结直肠癌免疫试验成为可能(Babatz,J,Rollig,C,Lobel,B,Folprecht,G,Haack,M,Gunther,H,Kohne,CH,Ehninger,G,Schmitz,M,and Bornhauser,M;Induction of cellular immune responses against carcinoembryonic antigen in patients withmetastatic tumours after vaccination with altered peptide ligand-loaded dendritic cells,CancerImmunol.Immunother.,2006,55,268-276;Fong,L,Hou,Y,Rivas,A,Benike,C,Yuen,A,Fisher,GA,Davis,MM,and Engleman,EG;Altered peptide ligand vaccination with Flt3 ligand expandeddendritic cells for tumour immunotherapy,Proc.Natl.Acad.Sci.U.S.A,2001,98,8809-8814;Liu,KJ,Wang,CC,Chen,LT,Cheng,AL,Lin,DT,Wu,YC,Yu,WL,Hung,YM,Yang,HY,Juang,SH,and Whang-Peng,J;Generation of carcinoembryonic antigen(CEA)-specific T-cell responsesin HLA-A*0201and HLA-A*2402late-stage colorectal cancer patients after vaccination withdendritic cells loaded with CEA peptides,Clin Cancer Res.,2004,10,2645-2651;Matsuda,K,Tsunoda,T,Tanaka,H,Umano,Y,Tanimura,H,Nukaya,I,Takesako,K,and Yamaue,H;Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignanciesfollowing vaccination with CEA peptide-pulsed dendritic cells,Cancer Immunol.Immunother.,2004,53,609-616;Ueda,Y,Itoh,T,Nukaya,I,Kawashima,I,Okugawa,K,Yano,Y,Yamamoto,Y,Naitoh,K,Shimizu,K,Imura,K,Fuji,N,Fujiwara,H,Ochiai,T,Itoi,H,Sonoyama,T,Hagiwara,A,Takesako,K,and Yamagishi,H;Dendritic cell-based immunotherapy of cancer withcarcinoembryonic antigen-derived,HLA-A24-restricted CTL epitope:Clinical outcomes of 18patients with metastatic gastrointestinal or lung adenocarcinomas,Int.J Oncol.,2004,24,909-917;Weihrauch,MR,Ansen,S,Jurkiewicz,E,Geisen,C,Xia,Z,Anderson,KS,Gracien,E,Schmidt,M,Wittig,B,Diehl,V,Wolf,J,Bohlen,H,and Nadler,LM;Phase I/II combinedchemoimmunotherapy with carcinoembryonic antigen-derived HLA-A2-restricted CAP-1peptideand irinotecan,5-fluorouracil,and leucovorin in patients with primary metastatic colorectal cancer,Clin Cancer Res.,2005,11,5993-6001)。到目前为止,上述试验以及其他临床试验已经证实了CEA接种的安全性并提供了诱导这种抗原免疫反应的证据(von Mehren,M;Colorectal cancervaccines:what we know and what we don′t yet know,Semin.Oncol.,2005,32,76-84)。
转化生长因子β诱导的(TGFBI)基因
TGFBI最先被认定为人肺腺癌细胞株的TGF-β诱导基因。它能编码分泌出的细胞外基质蛋白,该蛋白被认为作用于细胞粘附物和细胞外基质成分。
TGFBI是一种结直肠癌中升高最为明显的基因,也在腺瘤中高表达。定量PCR结果显示,它在未纯化的和纯化的肿瘤上皮细胞中均大大升高。因此,原位杂交实验表明,TGFBI在基质和上皮细胞等多种细胞类型中表达(Buckhaults,P,Rago,C,St,CB,Romans,KE,Saha,S,Zhang,L,Vogelstein,B,and Kinzler,KW;Secreted and cell surface genes expressed in benignand malignant colorectal tumours,Cancer Res.,2001,61,6996-7001)。
在一次对结直肠癌基因表达研究进行的荟萃分析中,TGFBI被确认为9个基因中唯一的一个不断上调的基因(TGFBI的4个研究)(Shih,W,Chetty,R,and Tsao,MS;Expressionprofiling by microarrays in colorectal cancer,Oncol.Rep.,2005,13,517-524)。
在人胰腺癌组织中,TGFBI mRNA水平比正常对照组织中增加32.4倍。原位杂交分析结果显示,TGFBI mRNA主要表达于胰腺肿瘤中的癌细胞(Schneider,D,Kleeff,J,Berberat,PO,Zhu,Z,Korc,M,Friess,H,and Buchler,MW;Induction and expression of betaig-h3inpancreatic cancer cells,Biochim.Biophys.Acta,2002,1588,1-6)。
在体外模型中,TGFBI被确定为一种促血管生成基因。此外,在另外几种肿瘤中,检测到TGFBI表达显著增强。TGFBI的反义寡核苷酸既阻止体外基因表达又阻止体外内皮细胞管形成,这表明TGFBI可在内皮细胞基质的相互作用中发挥着关键作用(Aitkenhead,M,Wang,SJ,Nakatsu,MN,Mestas,J,Heard,C,and Hughes,CC;Identification of endothelial cellgenes expressed in an in vitro model of angiogenesis:induction of ESM-1,(beta)ig-h3,and NrCAM,Microvasc.Res.,2002,63,159-171)。
蛋白酪氨酸磷酸酶受体型ζ-1(PTPRZ1)
PTPRZ1是受体型蛋白酪氨酸磷酸酶家族的成员,编码一次通过(single-pass)I型膜蛋白,该被编码蛋白含两个细胞质酪氨酸蛋白磷酸酶区域,一个α-碳酸酐酶区域和一个纤维连接蛋白III型区域。在以下细胞中该基因被诱导表达:胃癌细胞(Wu,CW,Li,AF,Chi,CW,and Lin,WC;Protein tyrosine-phosphatase expression profiling in gastric cancer tissues,CancerLett.,2006,242,95-103)、多发性硬化病变的再髓鞘化少突胶质细胞(Harroch,S,Furtado,GC,Brueck,W,Rosenbluth,J,Lafaille,J,Chao,M,Buxbaum,JD,and Schlessinger,J;A critical rolefor the protein tyrosine phosphatase receptor type Z in functional recovery from demyelinatinglesions,Nat.Genet.,2002,32,411-414)、以及组织缺氧情况下的人胚胎肾细胞(Wang,V,Davis,DA,Haque,M,Huang,LE,and Yarchoan,R;Differential gene up-regulation by hypoxia-induciblefactor-lalpha and hypoxia-inducible factor-2alpha in HEK293T-cells,Cancer Res.,2005,65,3299-3306)。
蛋白质和转录物都在胶质母细胞瘤细胞中过量表达,这促进了它们按络合点次序进行迁移(haptotactic migration)(Lu,KV,Jong,KA,Kim,GY,Singh,J,Dia,EQ,Yoshimoto,K,Wang,MY,Cloughesy,TF,Nelson,SF,and Mischel,PS;Differential induction of glioblastoma migrationand growth by two forms of pleiotrophin,J Biol Chem.,2005,280,26953-26964)。
此外,PTRPZ1在胶质母细胞瘤中经常发生基因组DNA水平的扩增(Mulholland,PJ,Fiegler,H,Mazzanti,C,Gorman,P,Sasieni,P,Adams,J,Jones,TA,Babbage,JW,Vatcheva,R,Ichimura,K,East,P,Poullikas,C,Collins,VP,Carter,NP,Tomlinson,IP,and Sheer,D;Genomicprofiling identifies discrete deletions associated with translocations in glioblastoma multiforme,Cell Cycle,2006,5,783-791)。
Janus激酶与微管相互作用的蛋白2(JAKMIP2)
JAKMIP2被确定为PAX3-FKHR众多已知或假定下游靶标之一,这些靶标在ARMS(小儿肺泡亚型横纹肌肉瘤)中高度过量表达(Lae,M,Ahn,E,Mercado,G,Chuai,S,Edgar,M,Pawel,B,Olshen,A,Barr,F,and Ladanyi,M;Global gene expression profiling of PAX-FKHRfusion-positive alveolar and PAX-FKHR fusion-negative embryonal rhabdomyosarcomas,J Pathol.,2007,212,143-151)。
纤维连接蛋白1(FN1)
纤维连接蛋白是一种高分子量糖蛋白,其中约含5%碳水化合物,可与跨越细胞膜的受体蛋白(称为整合素)结合。除了整合素外,它们还可与细胞外基质成分(如,胶原蛋白、纤维蛋白和肝素)结合。有几种纤维连接蛋白的异构体,所有这些异构体都是单一基因的产物。FN在维持正常细胞形态、细胞粘附、迁移、止血、血栓形成、伤口愈合、分化和增殖中都发挥着关键作用(Hynes,RO;Fibronectins,Sci.Am.,1987,254,42-51)。
通过用76-aa肽、III1-C(称为Anastellin,从纤维连接蛋白中第一个III型重复物中获得)处理可溶性纤维连接蛋白,从而形成聚合型纤维连接蛋白sFN。荷瘤小鼠体内研究表明,Anastellin或sFN全身使用抑制了肿瘤的生长、血管生成和转移(Yi,M and Ruoslahti,E;A fibronectin fragment inhibits tumour growth,angiogenesis,and metastasis,Proc.Natl.Acad.Sci.U.S.A,2001,98,620-624)。Anginex是一种含33个氨基酸的合成肽,最初设计为生成抗血管生成蛋白的β-折叠结构。研究已经表明,anginex可启动纤维连接蛋白的聚合,在缺乏血浆纤维结合蛋白的小鼠中没有活性(Akerman,ME,Pilch,J,Peters,D,and Ruoslahti,E;Angiostatic peptides use plasma fibronectin to home to angiogenic vasculature,Proc.Natl.Acad.Sci.U.S.A,2005,102,2040-2045)在一项研究中,研究者研究了FN在小鼠中对D-氨基半乳糖(GalN)/脂多糖(LPS)诱导暴发性肝功能衰竭的作用。结果表明,FN可保护小鼠免于GalN/LPS诱导的肝衰竭,而肝衰竭由于NF-κB激活受到抑制的机制产生,这可导致下调TNF-α和IL-10的下调以及Bcl-xL诱导的凋亡信号阻滞增加,其中GalN/LPS引起的肝细胞凋亡受到抑制(Qiu,Z,Kwon,AH,Tsuji,K,Kamiyama,Y,Okumura,T,and Hirao,Y;Fibronectin prevents D-galactosamine/lipopolysaccharide-induced lethal hepatic failure in mice,Shock,2006,25,80-87)。其他结果表明,FN可通过诱导COX-2基因表达和PGE2的生物合成,从而刺激人肺癌细胞增殖并减少体外凋亡(Han,S,Sidell,N,Roser-Page,S,and Roman,J;Fibronectin stimulates human lung carcinoma cell growth by inducing cyclooxygenase-2(COX-2)expression,Int.J Cancer,2004,111,322-331)。
纤维连接蛋白(FN)已被证实在器官形成和肿瘤形成过程中主要进行选择性剪接。外主体-B(ED-B)FN是一种此类剪接变体,正常成人组织通常不含该变体,它是肿瘤血管生成的一种推荐标志物(Khan,ZA,Caurtero,J,Barbin,YP,Chan,BM,Uniyal,S,and Chakrabarti,S;ED-B fibronectin in non-small cell lung carcinoma,Exp.Lung Res.,2005,31,701-711)。Mhawech等人的研究表明,EDB染色阳性的头颈部肿瘤患者的总生存期都有显著下降的趋势(Mhawech,P,Dulguerov,P,Assaly,M,Ares,C,and Allal,AS;EB-D fibronectin expression insquamous cell carcinoma of the head and neck,Oral Oncol.,2005,41,82-88)。
纤维连接蛋白表达调节血管生成和血管新生,并参与脑组织对缺血和癫痫的反应。与SWS正常皮肤的成纤维细胞相比,SWS(Sturge-Weber综合征)病灶部位成纤维细胞中纤维连接蛋白在的基因表达显著升高(p<0.05)(Comi,AM,Hunt,P,Vawter,MP,Pardo,CA,Becker,KG,and Pevsner,J;Increased fibronectin expression in sturge-weber syndrome fibroblastsand brain tissue,Pediatr.Res.,2003,53,762-769)。与良性卵巢肿瘤和正常卵巢相比,卵巢癌中的纤维连接蛋白含量明显增高。与未复发卵巢癌患者相比,复发性卵巢癌患者的纤维连接蛋白含量明显升高。表达的肿瘤衍生胞壁质水解酶和纤维连接蛋白对于卵巢肿瘤的生长非常重要(Demeter,A,Szirmai,K,Olah,J,Papp,Z,and Jeney,A;Elevated expression of matrixmetalloproteinase-9,and fibronectin concentration in recurrent epithelial ovarian cancer,Orv.Hetil.,2004,145,1617-1624)。在一项研究中,分析了1176个基因,FN是仅有的两个显著下调基因之一,这一事实强调了FN可作为乳腺癌转移癌的重要抑制基因这一假设(Urtreger,AJ,Werbajh,SE,Verrecchia,F,Mauviel,A,Puricelli,LI,Kornblihtt,AR,and Bal de Kier Joffe ED;Fibronectin is distinctly downregulated in murine mammary adenocarcinoma cells with highmetastatic potential,Oncol.Rep.,2006,16,1403-1410)。
在一份报告中,研究人员发现,3种可溶性纤维连接蛋白肽(RGD、CS-1和FN-C/H-V)都诱导肺成纤维细胞的凋亡。细胞凋亡的发生是由于粘附中断(失巢凋亡)。使用纤维连接蛋白小肽可促进成纤维细胞凋亡,这使我们有理由对抗纤维化疗法作进一步的研究(Hadden,HL and Henke,CA;Induction of lung fibroblast apoptosis by soluble fibronectin peptides,Am.J Respir.Crit Care Med,2000,162,1553-1560)。另一项研究表明,纤维连接蛋白(FN)刺激人非小细胞肺癌(NSCLC)的细胞增殖。研究显示,FN在NSCLC细胞中增加MMP-9蛋白和mRNA的表达,并提高了液化明胶的活性(Han,S,Ritzenthaler,JD,Sitaraman,SV,andRoman,J;Fibronectin increases matrix metalloproteinase 9expression through activation of c-Fosvia extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lungcarcinoma cells,J Biol Chem.,2006,281,29614-29624)。在一项研究中,研究人员研究了支配所有细胞黏附性的机制是否也能介导维生素D(VD)化合物的肿瘤抑制作用。引入干扰FN的小RNA造成FN的表达下调并削弱细胞对胶原蛋白I型基质的粘附力。这些研究结果突出了FN在调节甲状腺癌细胞粘附力中的意义,至少部分地影响了VD对癌细胞生长的作用(Liu,W,Asa,SL,and Ezzat,S;lalpha,25-Dihydroxyvitamin D3targets PTEN-dependentfibronectin expression to restore thyroid cancer cell adhesiveness,Mol.Endocrinol.,2005,19,2349-2357)。
肿瘤相关FN异构体的生成使得我们可以开发特定配体(如抗体),从而将治疗药物选择性地输送到肿瘤环境中。FN可作为生物分子干预的靶标,既可用于开发出阻断FN与整合素和细胞表面其他受体相互作用的抑制分子,又可开发出基于配体的靶向成像策略和治疗策略(Kaspar,M,Zardi,L,and Neri,D;Fibronectin as target for tumour therapy,Int.J Cancer,2005,118,1331-1339)。一项研究表明,以FN的一种重组CBD-HepII多肽(指定为CH50)体内表达进行治疗很强地抑制了肿瘤的生长、肿瘤浸润和肿瘤血管生成。运用CH50的基因疗法不仅延长了负荷肝癌小鼠的生存期,而且还抑制了肿瘤在脾脏中的生长和浸润能力以及向肝脏转移的能力。总而言之,这些发现表明,使用CH50在肝癌的基因治疗中是一种很有前途的用途(Liu,Y,Huang,B,Yuan,Y,Gong,W,Xiao,H,Li,D,Yu,ZR,Wu,FH,Zhang,GM,and Feng,ZH;Inhibition of hepatocarcinoma and tunour metastasis to liver by gene therapy withrecombinant CBD-HepII polypeptide of fibronectin,Int.J Cancer,2007121(1)184-92)。纤维连接蛋白(FN)具有一个隐蔽的功能性位点(第14III型重复基因的YTIYVIAL序列),阻止细胞粘附到细胞外基质。包含该位点的一种22-mer FN肽(称为FNIII14),在没有与整合素结合的情况下,就能抑制β-1整合素介导的粘附行为。研究表明,FNIII14能防止淋巴瘤细胞转移(Kato,R,Ishikawa,T,Kamiya,S,Oguma,F,Ueki,M,Goto,S,Nakamura,H,Katayama,T,and Fukai,F;A new type of antimetastatic peptide derived from fibronectin,Clin Cancer Res.,2002,8,2455-2462)。
表皮生长因子受体(EGFR)
EGFR在调节正常细胞的增殖、分化和生存中发挥着重要作用。由于这种原因,EGFR的状态往往在一系列人肿瘤中发生了改变,并通常与预后不良相关。在肿瘤细胞中,EGFR通过各种不同途径促进细胞成长和生存(Maehama,T and Dixon,JE;The tumour suppressor,PTEN/MMAC1,dephosphorylates the lipid second messenger,phosphatidylinositol 3,4,5-trisphosphate,J Biol Chem.,1998,273,13375-13378)。EGFR异常是胶质母细胞瘤最常见的分子畸变(Zawrocki,A and Biernat,W;Epidermal growth factor receptor in glioblastoma,FoliaNeuropathol.,2005,43,123-132)。
EGFR基因扩增和mRNA表达频繁发生于起源于星形细胞的高级别胶质瘤,并总是与EGFR蛋白的水平增加相关(Wong,AJ,Bigner,SH,Bigner,DD,Kinzler,KW,Hamilton,SR,and Vogelstein,B;Increased expression of the epidermal growth factor receptor gene in malignantgliomas is invariably associated with gene amplification,Proc.Natl.Acad.Sci.U.S.A,1987,84,6899-6903;Chaffanet,M,Chauvin,C,Laine,M,Berger,F,Chedin,M,Rost,N,Nissou,MF,andBenabid,AL;EGF receptor amplification and expression in human brain tumours,1992,Eur.JCancer,28,11-17)。.据报道,无基因扩增的蛋白过量表达发生于多达27%的GBM中,但是,恶性度较低的星形细胞瘤和少突胶质细胞瘤也报告发生无基因扩增的EGFR过量表达(Reifenberger,J,Reifenberger,G,Ichimura,K,Schmidt,EE,Wechsler,W,and Collins,VP;Epidermal growth factor receptor expression in oligodendroglial tumours,Am.J Pathol.,1996,149,29-35)。
EGFR在脑瘤中扩增/过量表达对预后的影响目前尚有争议。有些作者并没有发现EGFR扩增/过量表达对患者的生存期有任何影响(Olson,JJ,Barnett,D,Yang,J,Assietti,R,Cotsonis,G,and James,CD;Gene amplification as a prognostic factor in primary brain tumours,Clin CancerRes.,1998,4,215-222;Newcomb,EW,Cohen,H,Lee,SR,Bhalla,SK,Bloom,J,Hayes,RL,andMiller,DC;Survival of patients with glioblastoma multiforme is not influenced by alteredexpression of p16,p53,EGFR,MDM2or Bcl-2genes,Brain Pathol.,1998,8,655-667;Waha,A,Baumann,A,Wolf,HK,Fimmers,R,Neumann,J,Kindermann,D,Astrahantseff,K,Blumcke,I,von,DA,and Schlegel,U;Lack of prognostic relevance of alterations in the epidermal growthfactor receptor-transforming growth factor-alpha pathway in human astrocytic gliomas,JNeurosurg.,1996,85,634-641),而另外一些作者则认为这些变化是一个消极的预后因素(Etienne,MC,Formento,JL,Lebrun-Frenay,C,Gioanni,J,Chatel,M,Paquis,P,Bernard,C,Courrdi,A,Bensadoun,RJ,Pignol,JP,Francoual,M,Grellier,P,Frenay,M,and Milano,G;Epidermal growth factor receptor and labelling index are independent prognostic factors in glialtumour outcome,Clin Cancer Res.,1998,4,2383-2390;Jaros,E,Perry,RH,Adam,L,Kelly,PJ,Crawford,PJ,Kalbag,RM,Mendelow,AD,Sengupta,RP,and Pearson,AD;Prognosticimplications of p53protein,epidermal growth factor receptor,and Ki-67labelling in brain tumours,Br.J Cancer,1992,66,373-385;Schlegel,J,Merdes,A,Stumm,G,Albert,FK,Forsting,M,Hynes,N,and Kiessling,M;Amplification of the epidermal-growth-factor-receptor gene correlates withdifferent growth behaviour in human glioblastoma,Int.J Cancer,1994,56,72-77;Zhu,A,Shaeffer,J,Leslie,S,Kolm,P,and El-Mahdi,AM;Epidermal growth factor receptor:an independentpredictor of survival in astrocytic tumours given definitive irradiation,Int.J Radiat.Oncol.BiolPhys.,1996,34,809-815)。
对于癌细胞上的EGFR分子有几种治疗方法。研究最为广泛的治疗方法包括:特异性抗体疗法(使用未修饰抗体或与毒素、脂质体或核素共轭形成的抗体)以及使用受体酪氨酸激酶抑制剂。有几种直接作用于EGFRwt的单克隆抗体。这些抗体的使用阻断了其配体(西妥昔单抗)的进入和/或受体(ABX-EGF)快速内化(Sridhar,SS,Seymour,L,and Shepherd,FA;Inhibitors of epidermal-growth-factor receptors:a review of clinical research with a focus on non-small-cell lung cancer,Lancet Oncol.,2003,4,397-406)。由于EGFRwt也在正常的细胞表面发生,因此副作用可能会限制其使用。
EGFR在头颈部鳞状细胞癌(HNSCC)中过量表达,其表达水平与生存期下降相关。阻断EGFR的疗法在临床试验中以及和标准疗法结合使用时显示的疗效都有限。EGFRvIII在头颈部鳞状细胞癌中表达,导致癌细胞生长加快并且对靶向型野生型EGFR产生抗性。EGFR靶向策略的抗肿瘤疗效可通过加入EGFRvIII特异性阻滞剂而得到提高(Sok,JC,Coppelli,FM,Thomas,SM,Lango,MN,Xi,S,Hunt,JL,Freilino,ML,Graner,MW,Wikstrand,CJ,Bigner,DD,Gooding,WE,Furnari,FB,and Grandis,JR;Mutant epidermal growth factorreceptor(EGFRvIII)contributes to head and neck cancer growth and resistance to EGFR targeting,Clin Cancer Res.,2006,12,5064-5073)。
另一种策略是有选择性地诱导过量表达EGF受体的胶质母细胞瘤细胞和其他癌细胞的死亡。使用包裹EGF受体的非病毒输送载体,使一种细胞凋亡的强激活剂-合成抗增殖型dsRNA(聚肌苷-胞嘧啶[poly IC])选择性地靶向作用于癌细胞。EGFR靶向性poly IC在体外和体内诱导靶细胞迅速凋亡。向肿瘤输送EGFR靶向性poly IC导致了预先建立的裸鼠模型中颅内肿瘤完全消退,而且对正常脑组织未产生明显的不良毒性作用。治疗一年后,受治小鼠仍然没有癌症并且健康状况良好(Shir,A,Ogris,M,Wagner,E,and Levitzki,A;EGFreceptor-targeted synthetic double-stranded RNA eliminates glioblastoma,breast cancer,andadenocarcinoma tumours in mice,PLoS.Med,2006Jan;3(1):e6.Epub 2005Dec 6)。
应用小型干扰RNA(siRNA)已成为调节基因表达的一种有效的高度特异性工具,并且广泛的致癌基因已被成功地压制。在EGFR表达水平各异的两种胶质瘤细胞株中(U373MG、LN18),siRNA介导了EGFR的下调。EGFR mRNA和蛋白质的表达下调了70-90%。但是,siRNA治疗对细胞增殖、迁移和EGFR耦合的信号级联的激活状态并没有抑制作用。按照这些结果,微阵列基因表达分析结果显示,虽然在表达模式上有特定的变化,但是只有很小的变化。总之,这些数据表明,EGFR的特异性下调对恶性胶质瘤的单药疗法来说,可能还不够(Vollmann,A,Vornlocher,HP,Stempfl,T,Brockhoff,G,Apfel,R,and Bogdahn,U;Effective silencing of EGFR with RNAi demonstrates non-EGFR dependent proliferation of gliomacells,Int.J Oncol.,2006,28,1531-1542)。
几项已经进行的临床研究显示了有希望的结果。例如:
h-R3为一人源化单克隆抗体,能识别EGFR外部区域,具有高亲和力,能抑制酪氨酸激酶的活化。为了评价h-R3在新诊断的高级别胶质瘤患者中的安全性、免疫原性和初步疗效,已经进行了I/II期临床试验(Ramos,TC,Figueredo,J,Catala,M,Gonzalez,S,Selva,JC,Cruz,TM,Toledo,C,Silva,S,Pestano,Y,Ramos,M,Leonard,I,Torres,O,Marinello,P,Perez,R,and Lage,A;Treatment of high-grade glioma patients with the humanized anti-epidermal growthfactor receptor(EGFR)antibody h-R3:report from a phase I/II trial,Cancer Biol Ther.,2006,5,375-379)。
EKB-569是表皮生长因子受体(EGFR)的一种有效的、低分子量、具选择性且不可逆的抑制剂,目前正在作为抗癌药物进行开发。在日本患者中,进行了一项I期剂量递增的研究。根据RECIST标准,这些患者病情稳定,但是X光射线检查发现到了肿瘤消退(Yoshimura,N,Kudoh,S,Kimura,T,Mitsuoka,S,Matsuura,K,Hirata,K,Matsui,K,Negoro,S,Nakagawa,K,and Fukuoka,M;EKB-569,a new irreversible epidermal growth factor receptortyrosine kinase inhibitor,with clinical activity in patients with non-small cell lung cancer withacquired resistance to gefitinib,Lung Cancer,2006,51,363-368)。
吉非替尼是一种表皮生长因子受体(EGFR)相关酪氨酸激酶的抑制剂,在常规化疗失败的非小细胞肺癌(NSCLC)患者中显示出疗效。另据报告,该药物对NSCLC的脑转移癌也有抗肿瘤作用。此外,EGFR突变都表明与NSCLC对吉非替尼敏感性有着很大的关系。吉非替尼对NSCLC脑转移癌的疗效也进行了评估,并且该疗效与EGFR突变之间的关系也进行了评价。吉非替尼似乎能有效地治疗一些患者的脑转移癌。这些数据表明了吉非替尼治疗脑转移癌的疗效与EGFR突变之间的关系(Shimato,S,Mitsudomi,T,Kosaka,T,Yatabe,Y,Wakabayashi,T,Mizuno,M,Nakahara,N,Hatano,H,Natsume,A,Ishii,D,and Yoshida,J;2006,EGFR mutations in patients with brain metastases from lung cancer:association with the efficacy ofgefitinib,Neuro.Oncol.,8,137-144)。
几丁质酶3样2(CHI3L2)
CHI3L2最初确定源自软骨细胞。它经常被描述为类风湿关节炎的靶抗原。CHI3L2与癌症的相关性尚未确定。几丁质酶3样蛋白表明可通过激活细胞外信号调节激酶和PKB介导的信号通路而刺激人体***细胞(例如成纤维细胞)的增殖(Recklies AD,White C,Ling H;The chitinase 3-like protein human cartilage glycoprotein 39(HC-gp39)stimulatesproliferation of human connective-tissue cells and activates both extracellular signal-regulatedkinase-and protein kinase B-mediated signalling pathways;Biochem J.2002;365:119-126)。在幽门螺杆菌诱导的小鼠胃癌模型中,发现几丁质酶3样蛋白大幅上调(Takaishi S,Wang TC;Gene expression profiling in a mouse model of Helicobacter-induced gastric cancer;Cancer Sci.2007(3):284-293)
双皮质素和CaM激酶-样2蛋白(DCAMKL2)
微管(MT)相关DCX蛋白在哺乳动物大脑皮层的发育中发挥着重要作用。有报告确认了一种蛋白激酶-双皮质素激酶2(DCAMKL2),其基因区域(DC)与DCX高度同源。DCAMKL2对MT有结合活性,该活性与其DC区域以及激酶区域介导的蛋白激酶活性有相关性,因此,在DCAMKL2结构中,这两个区域具有独立的功能。
DCAMKL2的过量表达可稳定MT的细胞骨架,从而抵抗冷诱导解聚作用。DCAMKL2的自体磷酸化大大降低了DCAMKL2对MT的亲和力。DCAMKL2和DCX mRNA均具有神经***特异性,并且在大脑皮层片层化期间表达。DCX在出生后下调,而DCAMKL2表达量丰富直至成年期,这表明DC序列在成熟神经***中具有先前发现的功能。在交感神经元中,DCAMKL2位于细胞体以及轴突和树突的终端部分。
在神经元生长锥附近,DCAMKL2可能扮演磷酸化依赖性开关的角色,可逆性地控制MT的动态。DCAMKL2的表达模式、功能活动、调节作用和位置表明,它与DC基因家族(DC区域编码基因)的其他成员一起或合作参与神经***发育的重要事件,也可能参与成熟神经***发生的这些事件。DCAMKL2包含两个独立的功能区域-MT结合和稳定区域(DC序列)以及具有蛋白磷酸转移酶的激酶区域。
有研究表明,DC序列在转导胞外诱化源及其胞内信号从而发生MT动力学改变中发挥着重要作用。特别是,根据其与MT以磷酸化调节的方式相互作用之能力以及进入轴突和树突终端部分之能力(MT的区域具有动态不稳定性),DCAMKL2应被视为细胞骨架快速重构的一种潜在候选介导子,这种重构的发生是对神经元信号传导事件的响应(Edelman,AM,Kim,WY,Higgins,D,Goldstein,EG,Oberdoerster,M,and Sigurdson,W;Doublecortin kinase-2,a novel doublecortin-related protein kinase associated with terminal segments of axons anddendrites,J Biol Chem.,2005,280,8531-8543)。
ATP敏感性内向整流钾通道10(KCNJ10)
内向整流钾通道(Kir)的主要作用是建立胶质细胞的细胞膜和强负静息膜电位(RMP)的高度的钾离子(K+)选择性,这两者是胶质细胞特有的生理特性。Kir的经典特性是,当RMP对钾离子的平衡电位(E(K))为负时,钾离子向内流动,但是有更多的正电位时,就会抑制向外电流。中枢神经***胶质细胞的特征是表达KCNJ10亚型,这是钾离子在胶质细胞膜内传导的主要方式并且在确定胶质细胞RMP中具有关键的作用。因此,Kir,特别是KCNJ10是胶质细胞功能的主要调节因子,而这又决定了神经元的兴奋性和轴突的传导性(Butt,AM and Kalsi,A;Inwardly rectifying potassium channels(Kir)in central nervous systemglia:a special role for Kir4.1in glial functions,J Cell Mol.Med,2006,10,33-44)。
星形胶质细胞钾和谷氨酸的缓冲能力下降会导致神经元过度兴奋以及突触传递异常。KCNJ10通路主要负责皮质星形胶质细胞的重要超极化,并有可能在钾缓冲中发挥重要作用。KCNJ10水平降低后,星形胶质细胞中的谷氨酸清除得到明显的抑制,这突出了膜超极化在这一过程中的作用(Kucheryavykh,YV,Kucheryavykh,LY,Nichols,CG,Maldonado,HM,Baksi,K,Reichenbach,A,Skatchkov,SN,and Eaton,MJ;,Downregulation of Kir4.1inwardrectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake bycultured cortical astrocytes,Glia 2006,55(3),274-281)。
由于KCNJ10在胶质细胞表面分布不均匀,所以,KCNJ10只能对中枢神经***细胞外钾离子进行空间缓冲。在各种人脑肿瘤(低、高级别的星形细胞瘤和少突胶质细胞瘤)中,观察到了KCNJ10移位,这表明胶质细胞的缓冲能力可能会受到影响,从而导致水侵入(细胞毒性水肿)(Warth,A,Mittelbronn,M,and Wolburg,H;Redistribution of the water channelprotein aquaporin-4and the K+channel protein Kir4.1differs in low-and high-grade human braintumours,Acta Neuropathol.(Berl),2005,109,418-426)。在脑损伤的星形胶质细胞中,KCNJ10表达也上调。于是,研究人员提出了这样的假说:在星形胶质细胞中,正常情况下AQP4将水分运输与KCNJ10介导的钾离子虹吸管运输结合,但是在病理状态下,AQP4促进了脑水肿液的流动,并且KCNJ10缓冲了增多的细胞外钾离子(Saadoun,S,Papadopoulos,MC,andKrishna,S;Water transport becomes uncoupled from K+siphoning in brain contusion,bacterialmeningitis,and brain tumours:immunohistochemical case review,J Clin Pathol.,2003,56,972-975)。
发明人用给定氨基酸序列的“变种”表示,一个或多个氨基酸残基等的侧链通过被另一个天然氨基酸残基的侧链或其他侧链取代而发生改变,这样,这种肽仍然能够以含有给定氨基酸序列的肽大致同样的方式与HLA分子结合。例如,一种肽可能被修饰以便至少维持(如果不提升的话)其与HLA-A或-DR等合适的MHC分子互动和结合,以及至少维持(如果不提升的话)其产生能识别和杀伤细胞(这些细胞表达含本发明中所定义的一种氨基酸序列的多肽)的激活CTL的能力。正如数据库中所述,HLA-A结合肽的某些位点通常为锚定残基,可形成一种与HLA结合槽的结合基序相称的核心序列。
这些不与T细胞受体互动的氨基酸残基可通过取代另一个几乎不影响T细胞反应并不妨碍与相关MHC结合的氨基酸而得到修饰。因此,除了特定限制性条件外,本发明中的肽可能为任何包括给定氨基酸序列或段或变体的肽(发明人所用的这个术语包括寡肽或多肽)。
MHC-II类提呈肽更为大家熟知,这些肽由含有一个“核心序列”,该序列含某种HLA特异性氨基酸基序,视情况还含有不干扰肽核心序列功能的N和/或C-端延伸区(即,它们被视为与肽和所有或部分能识别其天然配对物的T细胞克隆物之间的相互作用无关)。N-和/或C端可分别延伸1至10个氨基酸的长度。这些肽可直接用于加载MHC-II类分子或其序列可克隆入下文所述载体。由于这些肽可在细胞内形成较大肽加工过程的最终产物,因此,也可用长肽。本发明中肽的大小不限,但通常它们的分子量可能小于100000,优选为小于50000,更优选为小于10000,通常约为5000。对于氨基酸残基数目,本发明中的肽可能少于1000个残基,优选为少于500个残基,更优选为少于100个残基。因此,本发明还提出了总长度为8至100个、优选为8至30个、最优选为8至16个(即8、9、10、11、12、13、14、15或16个)氨基酸的肽或变体。
相应地,诱导T细胞与本发明中的肽发生交叉反应的天然或人工变体往往为长度可变的变体。表1中分别列举了长度为SEQ IDNO 11、12、21、24的天然变体的例子。
如果长度约为12个氨基酸残基的肽直接与MHC-II类分子结合,那么,两侧有核心HLA结合区、且不会对该肽与MHC-II类分子的结合槽特异性结合能力或该肽提呈至CTL的能力产生重大影响的残基则为优选。但是,正如上所述,应了解,可使用较大的肽(例如,核苷酸进行编码时),因为这些较大的肽可被适当的抗原提呈细胞分成片段。
MHC-I类表位(通常长度为8至10个氨基酸)也可能由肽从较长的肽或包含实际表位的蛋白中加工而产生。与MHC-II表位相似,两侧有结合区、不会对该肽与MHC-I类分子的结合槽特异性结合能力或该肽提呈至CTL的能力产生重大影响、也不会掩盖加工过程中暴露实际表位所需蛋白裂解位点的残基为MHC-I类表位的优选。
因此,本发明还提出了总长度为8至100个、优选为8至30个、最优选为8至16个(即8、9、10、11、12、13、14、15或16个)氨基酸的MHC-I类表位的肽或变体。
当然,本发明中的肽或变体能与人主要组织相容性复合体(MHC)I或II类分子结合。肽或变体与MHC复合物结合性可用本领域内的已知方法进行测试,例如,本发明实施例4中所述的方法或文献中所述的检测不同MHC-II类等位基因的方法(例如,Vogt AB,Kropshofer H,Kalbacher H,Kalbus M,Rammensee HG,Coligan JE,Martin R;Ligand motifs ofHLA-DRB5*0101and DRB1*1501molecules delineated from self-peptides;J Immunol.1994;153(4):1665-1673;Malcherek G,Gnau V,Stevanovic S,Rammensee HG,Jung G,Melms A;Analysis of allele-specific contact sites of natural HLA-DR17ligands;J Immunol.1994;153(3):1141-1149;Manici S,Stumiolo T,Imro MA,Hammer J,Sinigaglia F,Noppen C,SpagnoliG,Mazzi B,Bellone M,Dellabona P,Protti MP;Melanoma cells present a MAGE-3epitope toCD4(+)cytotoxic T cells in association with histocompatibility leukocyte antigen DR11;J Exp Med.1999;189(5):871-876;Hammer J,Gallazzi F,Bono E,Karr RW,Guenot J,Valsasnini P,Nagy ZA,Sinigaglia F;Peptide binding specificity of HLA-DR4molecules:correlation with rheumatoidarthritis association;.J Exp Med.1995181(5):1847-1855;Tompkins SM,Rota PA,Moore JC,Jensen PE;A europium fluoroimmunoassay for measuring binding of antigen to class II MHCglycoproteins;J Immunol Methods.1993;163(2):209-216;Boyton RJ,Lohmann T,Londei M,Kalbacher H,Halder T,Frater AJ,Douek DC,Leslie DG,Flavell RA,Altmann DM;Glutamic aciddecarboxylase T lymphocyte responses associated with susceptibility or resistance to type Idiabetes:analysis in disease discordant human twins,non-obese diabetic mice and HLA-DQtransgenic mice;Int Immunol.1998(12):1765-1776)。
在本发明的一个特别优选实施方案中,肽系由或基本系由根据SEQ ID NO 1至SEQ IDNO 29所选的氨基酸序列组成。
“基本由...组成”系指本发明的肽,除了根据SEQ ID No.1至SEQ ID No.29中的任一序列或其变体组成外,还含有位于其他N和/或C端延伸处的氨基酸,而它们不一定能形成作为MHC分子表位的肽。
但这些延伸区域对有效将本发明中的肽引进细胞具有重要作用。在本发明的一实施案中,本发明的肽为融合蛋白,含来自NCBI、GenBank登录号X00497的HLA-DR抗原相关不变链(p33,以下称为“Ii”链)的80个N-端氨基酸等(Strubin,M.,Mach,B.and Long,E.O.The complete sequence of the mRNA for the HLA-DR-associated invariant chain reveals apolypeptide with an unusual transmembrane polarity EMBO J.1984,3(4),869-872)。
此外,该肽或变体可进一步修饰以提高稳定性和/或与MHC分子结合,从而引发更强的免疫反应。肽序列的该类优化方法是本领域内所熟知的,包括,例如,反式肽键和非肽键的引入。
在反式肽键氨基酸中,肽(-CO-NH-)并未连接其残基,但是其肽键是反向的。这种逆向反向模拟肽(retro-inverso peptidomimetics)可通过本领域已知的方法制备,例如:Meziere等人在《免疫学杂志》(J.Immunol.1997,159,3230-3237)中所述的方法,以引用的方式并入本文。这种方法涉及制备包含骨架(而并非侧链)改变的模拟肽。Meziere等人(1997年)的研究显示,这些模拟肽有利于MHC和辅助性T细胞的反应。以NH-CO键替代CO-NH肽键的逆向反向肽大大地提高了抗水解性能。
非肽键为-CH2-NH、-CH2S-、-CH2CH2-、-CH=CH-、-COCH2-、-CH(OH)CH2-和-CH2SO-等。美国4897445号专利提出了多肽链中非肽键(-CH2-NH)的非固相合成法,该方法涉及按标准程序合成的多肽以及通过氨基醛和一种含NaCNBH3的氨基酸相互作用而合成的非肽键。
含上述序列的肽可与其氨基和/或羧基末端的其他化学基团进行合成,从而提高肽的稳定性、生物利用度、和/或亲和力等。例如,苄氧羰基、丹酰基等疏水基团或叔丁氧羰基团可加入肽的氨基末端。同样,乙酰基或9-芴甲氧羰基可能位于肽的氨基末端。此外,疏水基团、叔丁氧羰基团或氨基团都可能被加入肽的羧基末端。
另外,本发明中的所有肽都可能经合成而改变其空间构型。例如,可能使用这些肽的一个或多个氨基酸残基的右旋体,通常不是其左旋体。更进一步地,本发明中肽的至少一个氨基酸残基可被熟知的一个非天然氨基酸残基取代。诸如此类的改变可能有助于增加本发明肽的稳定性、生物利用度和/或结合作用。
同样,本发明中的肽或变体可在合成肽之前或之后通过特异氨基酸的反应而进行化学修饰。此类修饰的实施例为本领域所熟知,例如,在R.Lundblad所著的《Chemical Reagentsfor Protein Modification》(3rd ed.CRC Press,2005)中有概述,以参考文献的方式并入本文。虽然氨基酸的化学修饰方法无限制,但其包括(但不限于)通过以下方法修饰:酰基化、脒基化、赖氨酸吡哆基化、还原烷基化、以2,4,6-三硝基苯磺酸(TNBS)三硝基苯基化氨基团、通过将半胱氨酸过甲酸氧化为磺基丙氨酸而对羧基团和巯基进行氨基修饰、形成易变衍生物、与其他巯基化合物形成混合二硫化合物、与马来酰亚胺反应,与碘乙酸或碘乙酰胺羧甲基化、在碱性pH值下与氰酸盐甲氨酰化。在这方面,技术人员参考了《Current ProtocolsIn Protein Science》(Eds.Coligan et al.(John Wiley&Sons NY 1995-2000))中第15章所述的在蛋白质化学修饰相关的广泛方法。
简言之,修饰蛋白质的精氨酰残基等往往基于于邻二羰基化合物(如苯甲酰甲醛、2,3-丁二酮以及1,2-烯巳二酮)的反应而形成加合物。另一个实施例是丙酮醛与精氨酸残基的反应。半胱氨酸可在赖氨酸和组氨酸等亲核位点不作随同修饰的情况下就得到修饰。因此,有大量试剂可进行半胱氨酸的修饰。Pierce化学公司、Sigma-Aldrich公司以及其他公司的网站含有具体试剂的信息。
蛋白质中二硫键的选择性还原也很普遍。二硫键可在生物制药热处理中形成和氧化。伍德沃德氏试剂K可用于修饰特定的谷氨酸残基。N-(3-二甲氨基丙基)-N′-乙基-碳二亚胺可用于形成赖氨酸残基和谷氨酸残基的分子内交联。例如:焦碳酸二乙酯是修饰蛋白质组氨酸残基的试剂。组氨酸也可使用4-羟基-2-壬烯醛进行修饰。赖氨酸残基与其他α-氨基团的反应,例如,有利于肽结合到蛋白/肽的表面或交联处。赖氨酸聚是多(乙烯)乙二醇的附着点,也是蛋白质糖基化的主要修饰位点。蛋白质的蛋氨酸残基可通过碘乙酰胺、溴乙胺、氯胺T等被修饰。四硝基甲烷和N-乙酰基咪唑可用于酪氨酸残基的修饰。经二酪氨酸形成的交联可通过过氧化氢/铜离子完成。
对色氨酸修饰的最近研究中使用了N-溴代琥珀酰亚胺、2-羟基-5-硝基苄溴或3-溴-3-甲基-2-(2-硝苯巯基)-3H-吲哚(BPNS-粪臭素)。
当蛋白与戊二醛、聚乙二醇二丙烯酸酯和甲醛的交联用于配制水凝胶时,治疗性蛋白和含聚乙二醇的肽的成功修饰往往可延长循环半衰期。针对免疫治疗的***反应原化学修饰往往通过氰酸钾的氨基甲酰化实现。
一种肽或变体,其中肽被修饰或含非肽键,优选为本发明的实施方案。
一般来说,肽和变体(至少含氨基酸残基之间的肽联接)可使用Lu等人(1981)J.Org.Chem.46,3433)以及此处列出的参考文献所披露的固相肽合成Fmoc-聚酰胺模式进行合成。芴甲氧羰基(Fmoc)团对N-氨基提供临时保护。使用N,N-二甲基甲酰胺中的20%二甲基哌啶中对这种碱高度敏感的保护基团进行重复***。由于它们的丁基醚(在丝氨酸苏氨酸和酪氨酸的情况下)、丁基酯(在谷氨酸和天门冬氨酸的情况下)、叔丁氧羰基衍生物(在赖氨酸和组氨酸的情况下)、三苯甲基衍生物(在半胱氨酸的情况下)及4-甲氧基-2,3,6-三甲基苯磺酰基衍生物(在精氨酸的情况下),侧链功能可能会受到保护。只要谷氨酰胺和天冬酰胺为C-末端残基,侧链氨基功能保护所使用的是由4,4′-二甲氧基二苯基团。固相支撑基于聚二甲基丙烯酰胺聚合物,其由三个单体二甲基丙烯酰胺(骨架单体)、双丙烯酰乙烯二胺(交联剂)和N-丙烯酰肌胺酸甲酯(功能剂)组成。使用的肽-树脂联剂为酸敏感的4-羟甲基苯氧乙酸衍生物。所有的氨基酸衍生物均作为其预制对称酸酐衍生物加入,但是天冬酰胺和谷氨酰胺除外,它们使用被逆转的N,N-二环己基碳二亚胺/1-羟基苯并***介导的耦合程序而加入。所有的耦合和脱保护反应用茚三酮、硝基苯磺酸或isotin测试程序监测。合成完成后,用浓度为95%含50%清道夫混合物的三氟醋酸,从伴随去除侧链保护基团的树脂支承物中裂解肽。常用的清道夫混合物为乙二硫醇、苯酚、苯甲醚和水,准确的选择依据合成肽的氨基酸组成。此外,固相和液相方法结合使用对肽进行合成是可能的(例如,请参阅Bruckdorfer T,Marder O,Albericio F.From production of peptides in milligram amounts forresearch to multi-ton quantities for drugs ofthe future Curr Pharm Biotechnol.2004Feb;5(1):29-43以及本文引用的参考文献)。
三氟乙酸用真空中蒸发、随后用承载粗肽的二乙基***滴定进行去除。用简单萃取程序(水相冻干后,该程序制得不含清道夫混合物的肽)清除任何存在的清道夫混合物。肽合成试剂一般可从Calbiochem-Novabiochem(英国)公司(NG72QJ,英国)获得。
纯化可通过以下技术的任何一种或组合方法实现,如:再结晶法、体积排阻色谱法、离子交换色谱法、疏水作用色谱法以及(通常)反相高效液相色谱法(如使用乙腈/水梯度分离)。
肽分析可使用以下方法进行:薄层色谱法、电泳法、特别是,毛细管电泳法、固相萃取法(CSPE)、反相高效液相色谱法、酸水解后的氨基酸分析、快原子轰击(FAB)质谱分析法以及MALDI、ESI-Q-TOF质谱分析法。
另一方面,本发明提出了一种编码本发明中肽或变体的核酸(如多聚核苷酸)。多聚核苷酸可能为DNA、cDNA、PNA、CAN、RNA、mRNA和siRNA或其组合物,它们可为单链和/或双链、或多聚核苷酸的原生或稳定形式(如:具有硫代磷酸骨架的多聚核苷酸),并且只要它编码肽,就可能包含也可能不包含内含子。当然,多聚核苷酸只能编码加入天然肽键并含有天然氨基酸残基的肽。另一个方面,本发明提出了一种可根据本发明表达多肽的表达载体。
对于多核苷酸的可操作性连接,已经开发出多种方法,尤其是针对DNA,可通过向载体补充可连接性末端等方法进行连接。例如,可向DNA片段加入补充性均聚物轨道,之后DNA片段被***到载体DNA。然后,通过补充性均聚物尾巴的氢键结合,将载体和DNA片段结合,从而形成重组DNA分子。
含有一个或多个酶切位点的合成接头为DNA片段与载体连接提供了另一种方法。含各种限制性核酸内切酶的合成接头可通过多种渠道购得,其中包括从国际生物技术公司(International Biotechnologies Inc,New Haven,CN,美国)购得。
编码本发明多肽的DNA理想修饰方法利用Saiki等人(1988年)在《科学》(Science239,487-491)杂志上披露的聚合酶链反应方法。此方法可用于将DNA引入合适的载体(例如,通过设计合适的酶切位点),也可用于本领域已知的其它有用方法修饰DNA。
如果使用病毒载体,痘病毒载体或腺病毒载体为优选。
之后,DNA(或在逆转录病毒载体情况下,RNA)可能表达于合适的宿主,从而制成含本发明肽或变体的多肽。因此,可根据已知技术使用编码本发明肽或变体的DNA,用本文所述方法适当修饰后,构建表达载体,然后表达载体用于转化合适宿主细胞,从而表达和产生本发明中的多肽。这些方法包括下列美国专利中披露的方法:1984年4月3日授予Rutter等人的4440859号美国专利、1985年7月23日授予Weissman的4530901号美国专利、1986年4月15日授予Crowl的458200号美国专利、1987年6月30日授予Mark等人的4677063号美国专利、1987年7月7日授予Goeddel的4678751号美国专利、1987年11月3日授予Itakura等人的4704362号美国专利、1987年12月1日授予Murray的4710463号美国专利、1988年7月12日授予Toole、Jr.等人的4757006号美国专利、1988年8月23日授予Goeddel等人的4766075号美国专利、1989年3月7日授予Stalker的4810648号美国专利,这些专利号以参考文献的方式并入本文。
编码含本发明化合物多肽的DNA(或在逆转录病毒载体情况下,RNA)可能被加入到其它多种DNA序列,从而引入到合适的宿主中。同伴DNA将取决于宿主的性质、DNA引入宿主的方式、以及是否需要保持为游离体还是要相互结合。
一般来说,DNA可以适当的方向和正确的表达阅读框架附着到一种表达载体(如质粒)中。如有必要,该DNA可能与所需宿主所识别的相应转录和翻译调节控制核苷酸序列连接,尽管表达载体中一般存在此类控制功能。然后,该载体通过标准方法被引入宿主。一般来说,并不是所有的宿主都会被载体转化。因此,有必要选择转化过的宿主细胞。选择方法包括用任何必要的控制元素向表达载体***一个DNA序列,该序列对转化细胞中的可选择性属性(如抗生素耐药性)进行编码。
另外,有这种选择属性的基因可在另外一个载体上,该载体用来协同转化所需的宿主细胞。
然后,本发明中的重组DNA所转化的宿主细胞在本文中所述本领域技术人员熟悉的合适条件下培养足够长的时间,从而表达之后可回收的肽。
有许多已知的表达***,包括细菌(如大肠杆菌和枯草芽孢杆菌)、酵母(如酵母菌)、丝状真菌(如曲霉菌)、植物细胞、动物细胞及昆虫细胞。该***可优选为哺乳动物细胞,如来自ATCC细胞生物学库(Cell Biology Collection)中的结直肠癌或胶质母细胞瘤细胞。
一个典型的哺乳动物细胞载体质粒是从Pharmacia公司(Piscataway,新泽西,美国)获得的pSVL。一种可诱导型哺乳动物表达载体的例子是pMSG,也可以从Pharmacia公司获得。有用的酵母质粒载体是pRS403-406和pRS413-416,一般可从Stratagene CloningSystems公司(La Jolla,CA 92037,美国)获得。质粒pRS403、pRS404、pRS405和pRS406是酵母整合型质粒(YIp),并***了酵母可选择性标记物HIS3、TRP1、LEU2和URA3。pRS413-416质粒为酵母着丝粒质粒(Ycp)。其他与各种宿主细胞一起应用的载体和表达***是本领域熟知众所周知的。
本发明还涉及一种宿主细胞,其以本发明的多核苷酸载体构建转化而来。宿主细胞可为原核细胞,也可为真核细胞。在有些情况下,细菌细胞为优选原核宿主细胞,典型为大肠杆菌株,例如,大肠杆菌菌株DH5(从Bethesda Research Laboratories公司(Bethesda,MD,美国)获得)和RR1(从美国菌种保藏中心(ATCC,Rockville,MD,美国),ATCC编号31343获得)。首选的真核宿主细胞包括酵母、昆虫和哺乳动物细胞,优选为脊椎动物细胞,如:小鼠、大鼠、猴子或人成纤维细胞和结肠癌细胞株中的细胞。酵母宿主细胞包括YPH499、YPH500和YPH501,一般可从Stratagene Cloning Systems公司(La Jolla,CA92037,美国)获得。首选哺乳动物宿主细胞包括中国仓鼠卵巢(CHO)细胞为ATCC中的CCL61细胞、NIH瑞士小鼠胚胎细胞NIH/3T3为ATCC中的CRL 1658细胞、猴肾源性COS-1细胞为ATCC中的CRL 1650细胞以及人胚胎肾细胞的293号细胞。首选昆虫细胞为Sf9细胞,可用杆状病毒表达载体转染。
含本发明DNA结构的适当宿主细胞的转化可使用大家熟知的方法完成,通常取决于使用载体的类型。对于原核宿主细胞的转化,可参阅,如:Cohen等人在Proc.Natl.Acad.Sci.USA 1972,69,2110中以及Sambrook等人(1989)所著《Molecular Cloning,A LaboratoryManual》Cold Spring Harbor Laboratory,Cold Spring Harbor,NY中使用的方法。酵母细胞的转化在Sherman等人(1986)在Methods In Yeast Genetics,A Laboratory Manual,Cold SpringHarbor,NY中有描述。Beggs,Nature 1978,275,104-109中所述方法也很有用。对于脊椎动物细胞,转染这些细胞的试剂等,例如,磷酸钙和DEAE-葡聚糖或脂质体配方,可从Stratagene Cloning Systems公司或Life Technologies公司(Gaithersburg,MD 20877,美国)获得。电穿孔也可用于转化和/或转染细胞,是本领域用于转化酵母细胞、细菌细胞、昆虫细胞和脊椎动物细胞大家熟知的方法。
被成功转化的细胞(即含本发明DNA结构的细胞)可用本领域熟知的方法(如PCR)进行识别。另外,上清液存在的蛋白可使用抗体进行检测。
应了解,本发明中的某些宿主细胞用于制备本发明中的肽,例如细菌细胞、酵母细胞和昆虫细胞。但是,其他宿主细胞可能对某些治疗方法有用。例如,抗原提呈细胞(如树突状细胞)可用于表达本发明中的肽,使他们可以加载入相应的MHC分子中。因此,本发明提出了含本发明中核酸或表达载体的一种宿主细胞。
在一个优选实施方案中,宿主细胞为抗原提呈细胞,尤其是树突状细胞或抗原提呈细胞。目前,载有含***酸性磷酸酶(PAP)的重组融合蛋白正在针对用于治疗***癌(Sipuleucel-T)进行研究(Small EJ,Schellhammer PF,Higano CS,Redfem CH,Nemunaitis JJ,Valone FH,Verjee SS,Jones LA,Hershberg RM.;Placebo-controlled phase 3trial of immunologictherapy with sipuleucel-T(APC8015)in patients with metastatic,asymptomatic hormone refractoryprostate cancer;J Clin Oncol.2006;24(19):3089-3094;Rini BI,Weinberg V,Fong L,Conry S,Hershberg RM,Small EJ;Combination immunotherapy with prostatic acid phosphatase pulsedantigen-presenting cells(Provenge)plus bevacizumab in patients with serologic progression ofprostate cancer after definitive local therapy;Cancer.2006;107(1):67-74)。
另一方面,本发明提出了制备一种肽或及其变体的一种方法。该方法包括培养宿主细胞和从宿主细胞或其培养基中分离肽。
在另一个实施方案中,本发明中的肽、核酸或表达载体用于药物中。例如,肽或其变体可制备为静脉(i.v.)注射剂、皮下(s.c.)注射剂、皮内(i.d.)注射剂、腹腔(i.p.)注射剂、肌肉(i.m.)注射剂。肽注射的优选途径为s.c.、i.d.、i.p.、i.m.和i.v.注射。DNA注射的优选途径为i.d.、i.m.、s.c.、i.p.和i.v.注射。例如,给予50μg至1.5mg,优选为125μg至500μg的肽或DNA,这取决于具体的肽或DNA。上述剂量范围在以前的试验中成功使用(Brunsvig PF,Aamdal S,Gj ertsen MK,Kvalheim G,Markowski-Grimsrud CJ,Sve I,Dyrhaug M,Trachsel S,M,Eriksen JA,Gaudernack G;Telomerase peptide vaccination:a phase I/IIstudy in patients with non-small cell lung cancer;Cancer Immunol Immunother.2006;55(12):1553-1564;M.Staehler,A.Stenzl,P.Y.Dietrich,T.Eisen,A.Haferkamp,J.Beck,A.Mayer,S.Walter,H.Singh,J.Frisch,C.G.Stief;An open label study to evaluate the safety andimmunogenicity of the peptide based cancer vaccine IMA901,ASCO meeting 2007;Abstract No3017)。
本发明的一个重要方面是得出了一种体外激活CTL的方法。该方法包括将CTL与载有抗原的人I或II类MHC分子进行体外连接,这些分子在合适的抗原提呈细胞表面表达足够的一段时间从而以抗原特异性方式激活CTL。抗原是本发明中的一种肽。优选情况是足够量的抗原与抗原提呈细胞一同使用。
当MHC-II类表位用作一种抗原时,CTL为CD4阳性辅助细胞,优选为TH1型。MHC-II类分子可表达于任何合适细胞的表面,优选为不自然表达MHC-II类分子的细胞(在这种情况下,转染细胞表达此类分子)。或者,如果该细胞自然表达MHC-II类分子,则该细胞在抗原加工或抗原提呈途径中有缺陷。这样,表达MHC-II类分子的细胞有可能在激活CTL之前就有可能被选定肽抗原几乎完全启动。
抗原提呈细胞(或刺激因子细胞)通常在其表面有一个MHC-II类分子,优选为其本身基本上不能以选定抗原加载所述MHC-II类分子。MHC-II类分子可很容易在体外载入选定抗原。
优选情况是,哺乳动物细胞的TAP肽转运载体缺乏或水平下降或功能降低。缺乏TAP肽转运载体的适合细胞包括T2、RMA-S和果蝇细胞。TAP表示转运载体相关的抗原加工。
人体肽载入的缺陷细胞株T2从属美国菌种保藏中心(ATCC,12301Parklawn Drive,Rockville,Maryland 20852,美国)目录号CRL1992;果蝇细胞株Schneider 2号株从属ATCC目录CRL 19863;小鼠RMA-S细胞株Karre和Ljunggren(985)在《实验医学杂志》(J.Exp.Med.162,1745)中有过描述。
优选情况是,宿主细胞在转染前不表达MHC-I类分子。优选情况是,刺激因子细胞表达对T细胞共刺激起到重要作用的分子,如,B7.1、B7.2、ICAM-1和LFA 3中的任一种分子。
大量MHC-II类分子和共刺激分子的核酸序列可从GenBank和EMBL数据库中公开获得。
同样,当MHC-I类表位用作一种抗原时,CTL为CD8阳性辅助细胞。MHC-I类分子可表达于任何合适细胞的表面,优选为不自然表达MHC-I类分子的细胞(在这种情况下,转染细胞表达此类分子)。或者,如果该细胞自然表达MHC-I类分子,则该细胞在抗原加工或抗原提呈途径中有缺陷。这样,表达MHC-I类分子的细胞有可能在激活CTL之前就有可能被选定肽抗原几乎完全启动。
如果抗原提呈细胞受到转染而表达这种表位,则优选的细胞包括一个表达载体,该载体有能力表达含SEQ ID NO 1至SEQ ID NO 29的肽或其变体氨基酸序列。
可使用其他一些方法来体外生成CTL。例如,可使用下述方法:Peoples等人在Proc.Natl.Acad.Sci.USA 1995,92,432-436中、Kawakami等人在(1992)J.Immunol.148,638-643中使用自体肿瘤浸润淋巴细胞生成CTL。Plebanski等人在(1995)Eur.J.Immunol.25,1783-1787中,使用自体外周血淋巴细胞(PLB)制得CTL。Jochmus等人在(1997)J.Gen.Virol.78,1689-1695中描述了用肽或多肽脉冲处理树突状细胞或通过与重组病毒感染而制成自体CTL。Hill等人在(1995)J.Exp.Med.181,2221-2228中、Jerome等人在(1993)J.Immunol.151,1654-1662中使用B细胞制成自体CTL。此外,用肽或多肽脉冲处理或用重组病毒感染的巨噬细胞可用于配制自体CTL。Walter等人在(Walter S,Herrgen L,Schoor O,Jung G,Wernet D,Buhring HJ,Rammensee HG,Stevanovic S.Cutting edge:predetermined avidity ofhuman CD8T-cells expanded on calibrated MHC/anti-CD28-coated microspheres.J Immunol.2003Nov 15;171(10):4974-8)中描述了通过使用人工抗原提呈细胞体外启动T细胞,这也是生成作用于所选肽的T细胞的一种合适方法。
也可用同种异体细胞制得CTL,实例方法在WO 97/26328中有详细描述,以参考文献方式并入本文。例如,除了果蝇细胞和T2细胞,也可用其他细胞来提呈肽,如CHO细胞、杆状病毒感染的昆虫细胞、细菌、酵母、牛痘感染的靶细胞。此外,也可使用植物病毒(例如,参阅Porta等人在《病毒学》(1994,202,449-955)中描述了将豇豆花叶病毒开发为一种提呈外来肽的高产***。
被激活的CTL直接针对本发明中的肽,有助于治疗。因此,本发明的另一方面提出了用本发明前述方法制得的激活CTL。
按上述方法制成的激活CTL将会有选择性地识别异常表达含SEQ ID NO 1至29氨基酸序列的多肽的一种细胞。
优选情况是,CTL通过与其含HLA/肽复合物的TCR相互作用(如,结合)而识别该细胞。CTL是杀伤患者靶细胞方法中有用的细胞,其中患者的靶细胞异常表达含本发明中氨基酸序列的多肽。此类患者给予有效量的激活CTL。给予患者的CTL可能源自患者,并按上述方法激活(即,它们为自体CTL)。或者,CTL不是源自该患者,而是来自另一个人。当然,供体优选为健康人。“健康人”系指一个人一般状况良好,优选为免疫***合格,更优选为无任何可很容易测试或检测到的疾病。
根据本发明,CD4阳性CTL的靶细胞可为肿瘤细胞(有时表达MHC-II类抗原)和/或肿瘤周围的基质细胞(癌细胞)(有时也表达MHC-II类抗原;(Dengjel,J,Nastke,MD,Gouttefangeas,C,Gitsioudis,G,Schoor,O,Altenberend,F,Muller,M,Kramer,B,Missiou,A,Sauter,M,Hennenlotter,J,Wernet,D,Stenzl,A,Rammensee,HG,Klingel,K,and Stevanovic,S;Unexpected Abundance of HLA Class II Presented Peptides in Primary Renal Cell Carcinomas,Clin Cancer Res.,2006,12,4163-4170))。
发明的CTL可用作治疗性组合物中的活性成分。因此,本发明也提出了一种杀伤患者靶细胞的方法,其中患者的靶细胞异常表达含本发明中氨基酸序列的多肽。该方法包括给予患者上述有效量的CTL。
我们所用的“异常表达”的意思包括,与正常表达水平相比,多肽过量表达,或该基因在源自肿瘤的组织中未表达,但是在该肿瘤中却表达。“过量表达”系指多肽水平至少为正常组织中的1.2倍;优选为至少为正常组织中的2倍,更优选为至少5或10倍。
CTL可用本领域已知的方法制得(如,上述方法)。
CTL继转移方案为本领域所熟知的方案并可在以下参考文献中找到,例如:(Rosenberg,SA,Lotze,MT,Muul,LM,Chang,AE,Avis,FP,Leitman,S,Linehan,WM,Robertson,CN,Lee,RE,Rubin,JT,et al.,A progress report on the treatment of157patients with advanced cancer usinglymphokine-activated killer cells and interleukin-2or high-dose interleukin-2alone,N.Engl.J.Med.,1987,316,889-897;Rosenberg,SA,Packard,BS,Aebersold,PM,Solomon,D,Topalian,SL,Toy,ST,Simon,P,Lotze,MT,Yang,JC,Seipp,CA,et al.;Use of tumor-infiltrating lymphocytesand interleukin-2in the immunotherapy of patients with metastatic melanoma.A preliminary report,N.Engl.J Med,1988,319,1676-1680;Dudley,ME,Wunderlich,JR,Robbins,PF,Yang,JC,Hwu,P,Schwartzentruber,DJ,Topalian,SL,Sherry,R,Restifo,NP,Hubicki,AM,Robinson,MR,Raffeld,M,Duray,P,Seipp,CA,Rogers-Freezer,L,Morton,KE,Mavroukakis,SA,White,DE,and Rosenberg,SA;Cancer regression and autoimmunity in patients after clonal repopulation withantitumor lymphocytes,Science,2002,298,850-854;Yee,C,Thompson,JA,Byrd,D,Riddell,SR,Roche,P,Celis,E,and Greenberg,PD;Adoptive T cell therapy using antigen-specific CD8+T cellclones for the treatment of patients with metastatic melanoma:in vivo persistence,migration,andantitumor effect of transferred T cells,Proc.Natl.Acad.Sci.U.S.A,2002,99,16168-16173;Dudley,ME,Wunderlich,JR,Yang,JC,Sherry,RM,Topalian,SL,Restifo,NP,Royal,RE,Kammula,U,White,DE,Mavroukakis,SA,Rogers,LJ,Gracia,GJ,Jones,SA,Mangiameli,DP,Pelletier,MM,Gea-Banacloche,J,Robinson,MR,Berman,DM,Filie,AC,Abati,A,andRosenberg,SA;Adoptive cell transfer therapy following non-myeloablative but lymphodepletingchemotherapy for the treatment of patients with refractory metastatic melanoma,J.Clin.Oncol.,2005,23,2346-2357);revierwed in(Gattinoni,L,Powell,DJ,Jr.,Rosenberg,SA,and Restifo,NP;Adoptive immunotherapy for cancer:building on success,Nat.Rev.Immunol.,2006,6,383-393)and(Morgan,RA,Dudley,ME,Wunderlich,JR,Hughes,MS,Yang,JC,Sherry,RM,Royal,RE,Topalian,SL,Kammula,US,Restifo,NP,Zheng,Z,Nahvi,A,de Vries,CR,Rogers-Freezer,LJ,Mavroukakis,SA,and Rosenberg,SA;Cancer Regression in Patients After Transfer of GeneticallyEngineered Lymphocytes,Science,2006,314(5796):126-129)。
本发明的任一分子(即肽、核酸、表达载体、细胞,激活CTL、T细胞受体或编码核酸)都有益于治疗疾病,其特点在于细胞逃避免疫反应的打击。因此,本发明的任一分子都可用作药剂或用于制造药剂。这种分子可单独使用也可与本发明中的其他分子或已知分子联合使用。
药剂优选为一种疫苗。该疫苗可直接给到患者的受影响器官,也可全身给药,或体外应用到来自患者或其细胞株的细胞(随后再将这些细胞注入到患者中),或体外用于从来自患者的免疫细胞的一个细胞亚群(然后再将细胞重新给予患者)。如果核酸体外注入细胞,可能有益于细胞转染,以共同表达免疫刺激细胞因子(如白细胞介素-2)。肽可完全单独给药,也可与免疫刺激佐剂相结合(见下文)、或与免疫刺激细胞因子联合使用、或以适当的输送***给药(例如脂质体)。该肽也可共轭形成一种合适的载体(如钥孔虫戚血蓝蛋白(KLH)或甘露)到合适的载体(参阅WO 95/18145及Longenecker等(1993)Ann.NY Acad.Sci.690,276-291)。该肽也可进行标记、或是一种融合蛋白或是杂合分子。本发明中的肽预期会刺激CD4或CD8CTL。但是,有反向CD阳性T细胞提供帮助时,刺激更为有效。因此,对于刺激CD4CTL的MHC-II类表位,一种杂合分子的融合伙伴或片段提供了刺激CD8阳性T细胞的适当表位。另一方面,对于刺激CD8CTL的MHC-I类表位,一种杂合分子的融合伙伴或片段提供了刺激CD4阳性T细胞的适当表位。CD4-和CD8刺激表位为本领域所熟知、并包括本发明中确定的表位。
本发明的一方面,疫苗包括至少一个肽,优选为2至50个、更优选为2至25个、再优选为2至15个、最优选为2、3、4、5、6、7、8、9、10、11、12或13个本发明中的肽或者其他肽。肽可能从一个或多个特定TAA获得,并且可能与MHC-I类和/或II类分子结合。
优选情况是,当本发明的肽用于疫苗或本发明的药剂中时,它们以盐的形式存在(如但不局限于醋酸盐或氯盐)。实施例7中描述了对疫苗IMA-910(含本发明的肽)的研究,并描述了使用以盐形式或颗粒大小存在的肽制得疫苗。
多聚核苷酸可完全单独给药,可用合适的载体或输送***给药。核酸可能为DNA、cDNA、PNA、CAN、RNA也可能为其组合物。这种核酸的设计和引入方法为本领域所熟知。例如,下列文献中有其概述:S.Pascolo:Vaccination with messenger RNA Methods MolMed 2006,127;23-40;R.Stan,JD Wolchok and AD Cohen DNA vaccines against cancer HematolOncol Clin North Am 2006,3;613-636or A Mahdavi and BJ Monk Recent advances in humanpapillomavirus vaccines Curr Oncol Rep 2006,6,465-472。多核苷酸疫苗很容易制备,但这些载体诱导免疫反应的作用模式还没有彻底了解。合适的载体和输送***包括病毒DNA和/或RNA,如基于腺病毒、牛痘病毒、逆转录病毒、疱疹病毒、腺相关病毒或含一种以上病毒元素的混合病毒的***。非病毒输送***包括阳离子脂质体和阳离子聚合物,是DNA输送所属领域内熟知的***。也可使用物理输送***,如通过“基因枪”。肽或核酸编码的肽可以是一种融合蛋白,例如,含刺激T细胞进行上述CDR的表位。
本发明的药剂也可能包括一种或多种佐剂。佐剂是那些非特异性地增强或加强免疫反应的物质(例如,通过CTL和辅助T(TH)细胞介导的对一种抗原的免疫应答,因此被视为对本发明的药剂有用。适合的佐剂包括(但不仅限于)1018ISS、铝盐、Amplivax、AS15、BCG、CP-870,893、CPG7909、CyaA、dSLIM、GM-CSF、IC30、IC31、Imiquimod、ImuFact IMP321、IS Patch、ISS、ISCOMATRIX、JuvImmune、LipoVac、MF59、单磷酰脂A、Montanide IMS1312、Montanide ISA 206、Montanide ISA 50V、Montanide ISA-51、OK-432、OM-174、OM-197-MP-EC、ONTAK、载体***、PLG微粒、resiquimod、SRL172、病毒颗粒和其他病毒样颗粒、YF-17D、VEGF trap、R848、β-葡聚糖、Pam3Cys、源自皂角苷、分支杆菌提取物和细菌细胞壁合成模拟物的Aquila公司的QS21刺激子,以及其他专有佐剂,如:Ribi′s Detox。优选佐剂如:弗氏佐剂或GM-CSF。前面一对一些树突状细胞特异性免疫佐剂(如MF59)及其制备方法进行了描述(Dupuis M,Murphy TJ,Higgins D,Ugozzoli M,van Nest G,Ott G,McDonald DM;Dendritic cells internalize vaccineadjuvant after intramuscular injection;Cell Immunol.1998;186(1):18-27;Allison AC;The mode ofaction of immunological adjuvants;Dev Biol Stand.1998;92:3-11)。也可使用细胞因子。一些细胞因子直接影响树突状细胞向淋巴组织迁移(如,TNF-α),加速树突状细胞成熟为T淋巴细胞的有效抗原提呈细胞(如,GM-CSF、IL-1和IL-4)(美国5849589号专利,以其完整引用形式并入本文),并充当免疫佐剂(如,IL-12)(Gabrilovich DI,Cunningham HT,Carbone DP;IL-12and mutant P53peptide-pulsed dendritic cells for the specific immunotherapyof cancer;J Immunother Emphasis Tumor Immunol.1996(6):414-418)。
据报告,CpG免疫刺激寡核苷酸可提高佐剂在疫苗中的作用。如果没有理论的约束,CpG寡核苷酸可通过Toll样受体(TLR)(主要为TLR9)激活先天(非适应性)免疫***从而起作用。CpG引发的TLR9活化作用提高了对各种抗原的抗原特异性体液和细胞反应,这些抗原包括肽或蛋白抗原、活病毒或被杀死的病毒、树突状细胞疫苗、自体细胞疫苗以及预防性和治疗性疫苗中的多糖结合物。更重要的是,它会增强树突状细胞的成熟和分化,导致TH1细胞的活化增强以及细胞毒性T淋巴细胞(CTL)生成加强,甚至CD4T细胞帮助的缺失。甚至有疫苗佐剂的存在也能维持TLR9活化作用诱发的TH1偏移,这些佐剂如:正常促进TH2偏移的明矾或弗氏不完全佐剂(IFA)。CpG寡核苷酸与以下其他佐剂或配方一起制备或联合给药时,表现出更强的佐剂活性,如微粒、纳米粒子、脂肪乳或类似制剂,当抗原相对较弱时,这些对诱发强反应尤为必要。他们还能加速免疫反应,使抗原剂量减少约两个数量级,在有些实验中,对不含CpG的全剂量疫苗也能产生类似的抗体反应(Arthur M.Krieg,Therapeutic potential of Toll-like receptor 9activation,Nature Reviews,DrugDiscovery,5,JUNE 2006,471-484)。美国6406705B1号专利对CpG寡核苷酸、非核酸佐剂和抗原结合使用促使抗原特异性免疫反应进行了描述。市售CpG TLR9拮抗剂为Mologen公司(德国柏林)的dSLIM(双干环免疫调节剂),这是本发明药物组合物的优选成分。也可使用其他如TLR结合分子,如:RNA结合TLR7、TLR8和/或TLR9。
其他有用的佐剂例子包括(但不限于)化学修饰性CpG(如CpR、Idera)、Poly(I:C),如:AmpliGen、非CpG细菌性DNA或RNA以及免疫活性小分子和抗体,如:环磷酰胺、舒尼替单抗、贝伐单抗、西乐葆、NCX-4016、西地那非、他达拉非、伐地那非、索拉非尼、XL-999、CP-547632、帕唑帕尼、ZD2171、AZD2171、抗-CTLA4和SC58175,这些药物都可能有治疗作用和/或充当佐剂。技术人员无需过度进行不当实验就很容易确定本发明中有用的佐剂和添加剂的数量和浓度。
优选佐剂为dSLIM、BCG、OK432、ALDARA、PeviTer和JuvImmune。
本发明的优选药剂为具有抗癌活性的药剂。癌种可能是转移癌,也可能不是转移癌,尤其是,口腔癌、咽癌,消化道癌、结肠癌、直肠癌、***癌、呼吸道癌、乳腺癌、***、***癌、外阴癌、子宫体癌、卵巢癌、男性生殖道癌、尿道癌、骨癌、软组织癌、卡波西肉瘤、皮肤黑色素瘤、眼黑色素瘤、非黑色素瘤眼癌、脑癌、中枢神经***癌、甲状腺癌、其他内分泌腺癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、骨髓瘤。本发明方法治疗的肿瘤疾病最优选为结直肠癌、肺癌、乳腺癌、胰腺癌、***癌、胃癌、肾癌、胃肠间质瘤或胶质母细胞瘤。
由于本发明的肽从胶质母细胞瘤、结直肠癌、胰腺癌、肺癌、肾癌或胃癌中分离而得,如果要治疗的癌症为胶质母细胞瘤、结直肠癌、胰腺癌、肺癌、肾癌或胃癌,则本发明的药剂会特别有用。
本发明中的肽除了用于治疗癌症,也可用于诊断。由于肽由胶质母细胞瘤产生,并且这些肽确定不在正常组织中出现,因此这些肽可用于诊断癌症是否存在。
含本发明肽的组织切片有助于病理师诊断癌症。用抗体、质谱或其它本领域内已知的方法检测本发明的某些肽可使病理师判断该组织为恶性的、炎症还是一般病变。本发明肽的提出使得能对病变组织进行分类或进一步分成子类。
对病变标本中本发明肽的检测使得能对免疫***治疗方法的利益进行判断,特别是如果T淋巴细胞已知或预计与作用机制有关。MHC表达的缺失是一种机制,充分说明了哪些受感染的恶性细胞逃避了免疫监视。因此,本发明肽的提出表明,分析过的细胞并没有利用这种机制。
本发明的肽可用于分析对本发明肽发生的淋巴细胞反应(如T细胞反应),或对本发明肽发生的抗体反应,或分析本发明中与MHC分子络合的肽。这些淋巴细胞反应可以作为预后指标,决定是否采取进一步的治疗。这些反应也可以用作免疫疗法中的替代指标,旨在以不同方式诱导淋巴细胞反应,如接种蛋白疫苗、核酸、自体材料、淋巴细胞过继转移。基因治疗中,对本发明肽发生的淋巴细胞反应可以在副作用的评估中考虑。淋巴细胞反应监测也可能成为移植疗法复查中的一种有价值的工具,如,用于检测移植物抗宿主和宿主抗移植物疾病。
本发明中的肽可用于生成和开发出针对MHC/肽复合物的特定抗体。这些抗体可用于治疗,将毒素或放射性物质靶向作用于病变组织。这些抗体的另一用途是为了成像之目的(如PET)将病变组织作为放射性核素的靶标。这可有助于检测小转移灶或确定病变组织的大小和准确位置。此外,可用这些肽在活检样本的基础上验证病理师对癌症的诊断。
在另一个方面,本发明涉及一个套件,包括(a)一个容器,包含上述溶液或冻干粉形式的药物组合物;(b)可选项,第二个容器,含冻干粉剂型的稀释剂或重组溶液;及(c)可选项,(i)使用溶液或(ii)重组和/或使用冻干粉剂型的说明书。该套件还步包括一个或多个(iii)缓冲剂,(iv)稀释剂,(v)过滤液,(vi)针,或(v)注射器。容器优选为瓶子、西林瓶、注射器或试管;也可为多用途容器。药物组合优选为冻干粉剂。
本发明中的套件优选包含一种置于合适容器中的冻干制剂以及重组和/或使用说明。适当的容器包括,例如瓶子、西林瓶(如双室瓶)、注射器(如双室注射器)和试管。该容器可能由多种材料制成,如玻璃或塑料。优选情况是,套件和/或容器上有说明,表明重组和/或使用的指示。例如,标签可能表明冻干剂型重组为上述肽浓度。该标签可进一步表明制剂用于皮下注射。
存放制剂的容器可使用多用途西林瓶,使得可重复给予(例如,2-6次)重组剂型。该套件可进一步包括装有合适稀释剂(如碳酸氢钠溶液)的第二个容器。
稀释液和冻干制剂混合后,重组制剂中的肽终浓度优选为至少0.15mg/mL/肽(=75μg),不超过3mg/mL/肽(=1500μg)。该套件还可包括商业和用户角度来说可取的其他材料,包括其他缓冲剂、稀释剂,过滤液、针头、注射器和带有使用说明书的包装插页。
本发明中的套件可能有一个单独的容器,其中包含本发明所述的药物组合物制剂,该制剂可有其他成分(例如,其他化合物或及其药物组合物),也可无其他成分,或者每种成分都有其不同容器。
优选情况是,本发明的套件包括与本发明的一种制剂,包装后与第二种化合物(如佐剂(例如GM-CSF)、化疗药物、天然产品、激素或拮抗剂、抗血管生成剂或抑制剂、凋亡诱导剂或螯合剂)或其药物组合物联合使用。该套件的成分可进行预络合或每种成分在给予患者之前可放置于单独的不同容器。该套件的成分可以是一种或多种溶液,优选为水溶液,更优选为无菌水溶液。该套件的成分也可为固体形式,加入合适的溶剂后转换为液体,最好放置于另一个不同的容器中。
治疗套件的容器可能为西林瓶、试管、烧瓶、瓶子、注射器、或任何其他指装载固体或液体的工具。通常,当成分不只一种时,套件将包含第二个西林瓶或其他容器,使之可以单独定量。该套件还可能包含另一个装载药用液体的容器。优选情况是,治疗套件将包含一个设备(如,一个或多个针头、注射器、滴眼器、吸液管等),使得可注射本发明的药物(本套件的组合物)。
本发明的药物配方适合以任何可接受的途径进行肽给药,如口服(肠道)、鼻内、眼内、皮下、皮内、肌内,静脉或经皮给药。优选为皮下给药,最优选为皮内给药,也可通过输液泵给药。
为了本发明之目的,所有参考文献均以完整引用的形式并入本文。
实施例
1.合成与结构
肽通过使用Fmoc化学以标准、广为接受的固相合成法合成。制备液HPLC纯化之后,进行离子交换纯化程序从而加入生理相容性反离子(醋酸或氯化物)。最后,在冻干后制得白色至类白色固体。在制造过程中由于技术原因IMA-CCN-001以氯化盐形式存在之外,其它所有TUMAP都作为醋酸盐给药。
2.细胞表面提呈的肿瘤相关肽(TUMAP)的识别
组织样本
患者肿瘤组织和健康组织由几个不同的临床试验点提供(见下表)。所有患者在手术前都获得了书面知情同意。手术后立即用液态氮对组织进行冷休克处理,在分离TUMAP前储存于-80℃下。
从组织样本中分离HLA肽
根据方案略加修改,使用HLA-A*02特异性抗体BB7.2或HLA-A、-B、-C特异性抗体W6/32、CNBr活化的琼脂糖凝胶、酸处理和超滤方法以固体组织的免疫沉淀法获得了冷休克组织样本的HLA肽库(Falk,K.,Rotzschke,O.,Stevanovic,S.,Jung,G.and Rammensee,H.G.Allele-specific motifs revealed by sequencing of self-peptides eluted from MHCmolecules.Nature 351,290-296(1991);Seeger,F.H.et al.The HLA-A*6601peptidemotif:prediction by pocket structure and verification by peptide analysis.Immunogenetics 49,571-576(1999))。
用电喷雾-液相色谱质谱(ESI-LCMS)检测TUMAP
获得HLA肽库根据其疏水性用反相色谱(CapLC,Waters)分离,洗脱肽用装备电喷雾源的四极杆-飞行时间串联质谱(Q-TOF Ultima,Waters)进行了分析。肽库被载到C18预处理柱进行浓缩和脱盐。载入后,C18预处理柱排成一行,用填充5μm C18反相材料(Dionex)的熔炼石英微毛细管柱(75μm内径×250mm)进行分离。溶剂A为4mM醋酸铵/水。溶剂B为2M浓度为80%乙腈/水中的醋酸铵。这两种溶剂均用甲酸将pH值调整为3.0。对15%至60%的溶剂B在90分钟内进行二元梯度色谱法分析,分流***将应用流量从5μl/min降低至200nl/min。金镀膜玻璃毛细管(PicoTip,New Objective)用于引入到微电喷雾源。TOF分析仪的积分时间为1.9秒,扫描间延迟时间为0.1秒。随后,用碰撞诱导解离(CID)质谱(ESI-LCMS/MS)揭示肽序列。生成的天然TUMAP的片段模式与合成序列相同参考肽的片段模式进行比较后,确保了识别TUMAP的序列。
图1和图2显示了从肿瘤组织中MHC-I类相关TUMAP(图1a-1h)和MHC-II类相关TUMAP(图2a-2f)的典型表达谱。
3.编码本发明肽的基因的表达谱
确定为由MHC分子提呈于肿瘤细胞表面的肽有可能能够诱导对其来源组织识别有高特异性的T细胞。为了最大限度地降低这些肽接种所诱导的自身免疫的风险,发明人主要采用从过量表达于肿瘤细胞上(与大多数正常组织相比)的蛋白中所获得的肽。
理想的肽来源于对该肿瘤独一无二且不出现于其他组织中的蛋白中。为了确定来源于理想表达基因中的肽,确定的肽被分别分配到蛋白和基因中,从中获得基因并生成基因表达谱。
RNA来源与制备
手术切除组织标本由几个不同的临床中心(参见表2)在获得每名患者的书面知情同意后提供。
手术后立即在液态氮中速冻肿瘤组织标本,之后在液态氮中用杵臼匀浆。使用TRIzol(Invitrogen公司,Karlsruhe,德国),再接着用RNeasy(QIAGEN公司,Hilden,德国)进行清理,从这些样本中制备总RNA;这两种方法都按制造商的方案进行应用。
健康人体组织中的总RNA从商业途径获得(Ambion公司,Huntingdon,英国;Clontech公司,海德堡,德国;Stratagene公司,阿姆斯特丹,荷兰;BioChain公司,Hayward,CA,美国)。混合数个人(2至123个人)的RNA,从而使每个人的RNA得到等加权。白细胞从4个健康志愿者的血液样本中分离获得。
所有RNA样本的质量和数量都在Agilent 2100Bioanalyzer分析仪(Agilent公司,Waldbronn,德国)上使用RNA 6000Pico LabChip Kit试剂盒(Agilent公司)进行评估。
微阵列实验
所有肿瘤和正常组织的RNA样本都使用Affymetrix Human Genome(HG)U133A或HG-U133Plus 2.0Affymetrix寡核苷酸芯片(Affymetrix公司,Santa Clara,CA,美国)进行基因表达分析。所有步骤都根据Affymetrix手册进行(http://www.affymetrix.com/support/technical/manual/expression manual.affx)。简言之,如手册中所述,使用SuperScript RTII(Invitrogen公司)以及oligo-dT-T7引物(MWG Biotech公司,Ebersberg,德国)从5-8μg RNA中合成双链cDNA。用BioArray High Yield RNATranscript Labelling Kit(ENZO Diagnostics公司,Farmingdale,NY,美国)进行U133A测定或用GeneChip IVT Labelling Kit(Affymetrix公司)进行U133Plus 2.0测定,之后用链霉亲和素-藻红蛋白和生物素化抗链霉素蛋白抗体(Molecular Probes公司,Leiden,荷兰)进行破碎、杂交和染色,这样完成体外转录。用Agilent 2500A GeneArray Scanner(U133A)或Affymetrix Gene-Chip Scanner 3000(U133Plus 2.0)对图像进行扫描,用GCOS软件(Affymetrix公司)在所有参数默认设置情况下对数据进行分析。为了实现标准化,使用了Affymetrix公司提供的100种管家基因(housekeeping gene)(http://www.affymetrix.com/support/technical/mask_files.affx)。相对表达值用软件给定的signallog ratio进行计算,正常样本的值任意设置为1.0。
本发明所有肽的表达谱显示,每种基因在肿瘤组织中呈高表达,而在正常组织中不表达或表达很低。
图3显示了胶质母细胞瘤特异性肽PTP-001(基因:PTPRZ1,图3a)和CHI-001(基因:CH3L2,图3b)的基因表达谱。
4.用电喷雾-液相色谱质谱(ESI-LCMS)再检测其他肿瘤样本中的TUMAP
用实施例1中的方法确定的TUMAP用质谱法在结直肠癌样本上进行了***搜索。
获得HLA肽库根据其疏水性用反相色谱(CapLC,Waters)分离,洗脱肽用装备电喷雾源的四极杆-飞行时间串联质谱(Q-TOF Ultima,Waters)进行了分析。肽库被载到C18预处理柱进行浓缩和脱盐。载入后,C18预处理柱排成一行,用填充5μm C18反相材料(Dionex)的熔炼石英微毛细管柱(75μm内径×250mm)进行分离。溶剂A为4mM醋酸铵/水。溶剂B为2M浓度为80%乙腈/水中的醋酸铵。这两种溶剂均用甲酸将pH值调整为3.0。对15%至60%的溶剂B在90分钟内进行二元梯度色谱法分析,分流***将应用流量从5μl/min降低至200nl/min。金镀膜玻璃毛细管(PicoTip,New Objective)用于引入到微电喷雾源。TOF分析仪的积分时间为1.9秒,扫描间延迟时间为0.1秒。对于已定义肽的检测,根据已知分子量和肽在色谱***中的停留时间,在ESI-LCMS实验中进行了高灵敏度筛选。因此,在选择前体中,应用一个数据清单,其中含先前确定的肽(单电荷和/或双电荷)的m/z值。随后,用碰撞诱导解离(CID)质谱(ESI-LCMS/MS)揭示序列。生成的天然TUMAP的片段模式与合成序列相同参考肽的片段模式进行比较后,确认了TUMP的序列。使用企业内标准-丰富内源性HLA-A*02肽(从DDX5获得的YLLPAIVHI)的强度和存留时间,评价了HLA纯化产量和分析***的可复制性,包括保留时间的稳定性。因此,这些实验中为了检测先前确认的TUMAP所用的结直肠癌样本纳入标准在LCMS/MS实验中(企业内标双电荷信号,YLLPAIVHI)每次扫描的最小强度设定为650次,以确保成功分离HLA肽以及分析***的性能。
表2显示了不同阶段结肠和直肠癌样本以及源自这两种原发性肿瘤转移癌的分析结果。大部分样本中都发现了所有HLA-A*02TUMAP。HLA-DR TUMAP一般很少需要再次检测。这是因为每个核心序列都可能存在HLA-II类肽、几种长度可变的变体。ODC-001作为阳性对照物,它是一种先前确定的TUMAP(M Diehl,博士论文1998年,Tuebingen大学)并已知在很多结肠肿瘤中提呈。
表2重新检测结直肠癌样本中的TUMAP
n.a.:未作分析
5.HLA-I类限制肽与HLA-A*0201结合
根据Sylvester-Hvid提出的方法(Sylvester-Hvid,C,Kristensen,N,Blicher,T,Ferre,H,Lauemoller,SL,Wolf,XA,Lamberth,K,Nissen,MH,Pedersen,LO,and Buus,S;Establishmentof a quantitative ELISA capable of determining peptide-MHC class I interaction,Tissue Antigens,2002,59,251-258)以及制造商的ELISA EpI Kit手册,使用ELISA EpI Kit(从丹麦哥本哈根大学医学微生物学及免疫学研究所Soeren Buus处获得)对HLA结合力进行测定。
制备肽溶液
肽溶解于浓度为10mg/ml的DMSO+0.5%TFA溶液(Merck公司,Darmstadt,德国)中。本次测定中所用的肽最高浓度为200μM,因此,储备溶液在肽稀释缓冲液(含0.1%Lutrol-F68和10mg/l酚红的PBS)中按1∶50的比例稀释,终溶液为100μl。用肽稀释缓冲液进行5倍系列稀释。
HLA-A*0201/肽复合物的重折叠
根据手册,用PBS溶液混合3倍pH缓冲液(pH 6.6)、Lutrol-F68、人β2m、重组HLA-A*0201(所有都包含于ELISA EpI Kit),制备2倍浓缩的HLA-A*0201溶液。
对于重折叠过程,在96孔板(Nunc公司,Rochester NY,美国)中混合15μl肽系列稀释液和15μl的2倍浓缩MHC混合液并在18℃下培养48小时。
用ELISA法定量分析复合物
用包被缓冲液(pH 9.6)中的5μg/ml w6/32抗体包被Maxisorp板(Nunc公司,Rochester,NY),在4℃下培养24小时,并用PBS溶液中的5%脱脂奶粉(Merck公司,Darmstadt,德国)在4℃下封闭过夜。
用PBS(SMP/PBS)中的2%脱脂奶粉将MHC复合标准液(ELISA EpI Kit)的浓度稀释为10nM。配制3.16倍系列稀释液,并转移到被包被和封闭的Maxisorp板中。用2%SMP/PBS将肽-MHC复合物稀释10倍,然后转到同一个Maxisorp板中,并在4℃下培养2小时。将兔抗-hβ2m抗体(ELISA EpI Kit)加入到2%SMP/PBS中,按1∶2500比例稀释,并在4℃下培养1小时。扩增缓冲液(HRP偶联的羊抗兔聚合物)和小鼠血清(两者均由ELISA EpIKit提供)在2%SMP/PBS中稀释,然后加入板中,在室温下培养30分钟。加入显色缓冲液(四甲基联苯胺,TMB;ELISA EpI Kit)增加了,显色板在室温下避光培养30分钟。加入0.2M硫酸(VWR公司,Darmstadt,德国),停止反应。使用VERSAmax ELISA-Reader阅读器(Molecular Devices公司,Sunnyvale,CA,美国)在OD450nm条件下读板。
数据用Excel和Graphpad 3.0解读。
结果如图4所示。较低的KD值反映了对HLA-A*0201较高的亲和力。结合亲和力可扩展至大约40倍的范围,但大多数肽都具有相似的结合亲和力,在10倍以内(C20-001,ODC-001,PCN-001,TOP-001)。与大多数纳入的配体相比,MUC-001的亲和力大约低了10倍,但是用作肾癌的疫苗时,MUC-001仍能诱导T细胞反应(Wierecky,J,Muller,MR,Wirths,S,Halder-Oehler,E,Dorfel,D,Schmidt,SM,Hantschel,M,Brugger,W,Schroder,S,Horger,MS,Kanz,L,and Brossart,P;Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients,Cancer Res.,2006,66,5910-5918)。另一方面,NOX-001具有稍高的结合亲和力和TGFBI-001是最强的结合剂,与大多数肽相比,其KD值要低100倍。
从绝对值来看,大多数具有强结合力的肽观察到的KD值为0.01和0.1nM之间。在成功测试的肾细胞癌疫苗IMA901包含的肽中也观察到了相似的结合力(H.Singh-Jasuja,S.Walter,T.Weinschenk,A.Mayer,P.Y.Dietrich,M.Staehler,A.Stenzl,S.Stevanovic,H.Rammensee,J.Frisch;Correlation of T-cell response,clinical activity and regulatory T-cell levelsin renal cell carcinoma patients treated with IMA901,a novel multi-peptide vaccine;ASCOMeeting 2007Poster#3017;M.Staehler,A.Stenzl,P.Y.Dietrich,T.Eisen,A.Haferkamp,J.Beck,A.Mayer,S.Walter,H.Singh,J.Frisch,C.G.Stief;An open label study to evaluate thesafety and immunogenicity of the peptide based cancer vaccine IMA901,ASCO meeting 2007;Poster#3017).。因此,本发明中肽的结合特性与那些在体外显示出诱导T细胞反应的肽十分相似。
6.MHC-I类提呈肽的体外免疫原性
CD8+T细胞体外启动
为了用载有肽-MHC复合物(pMHC)和抗-CD28抗体的人工抗原提呈细胞(aAPC)进行体外刺激,我们首先运用标准密度梯度分离介质(PAA公司,德国)从新鲜HLA-A*02+血沉棕黄层中分离出PBMC(外周血单核细胞)。血沉棕黄层从Tübingen或Katharinenhospital Stuttgart的血库获得。分离出的PBMC在T细胞培养基(TCM)中培养过夜,在体外填装,包括RPMI-Glutamax(Invitrogen公司,卡尔斯鲁厄,德国)并补充10%热灭活人AB血清(PAA公司,科尔贝,德国),100U/ml青霉素/100μg/ml链霉素(Cambrex公司,韦尔维耶,比利时),1mM丙酮酸钠(CC Pro公司,Neustadt,德国)和20μg/ml庆大霉素(Cambrex公司)。CD8+淋巴细胞根据制造商的说明使用CD8+MACS阳性选择套件(Miltenyi公司,Bergisch Gladbach,德国)进行分离。获得的CD8+T细胞培养,直到TCM补充了2.5ng/ml的IL-7(PromoCell公司,海德堡,德国)和10U/ml的IL-2(Chiron公司,慕尼黑,德国)后才使用。pMHC/抗-CD28涂层珠的生成、T细胞的刺激和读出方法如前所述(Walter,S,Herrgen,L,Schoor,O,Jung,G,Wernet,D,Buhring,HJ,Rammensee,HG,and Stevanovic,S;Cutting edge:predetermined avidity of human CD8Tcells expanded on calibrated MHC/anti-CD28-coated microspheres,J.Immunol.,2003,171,4974-4978)并作微小改动。简言之,缺乏跨膜域和在重链羧基端生物素化的生物素化重组HLA-A*0201分子用以下所述方法制成(Altman et al.(Altman,JD,Moss,PA,Goulder,PJ,Barouch,DH,Heyzer-Williams,MG,Bell,JI,McMichael,AJ,and Davis,MM;Phenotypic analysis ofantigen-specific T lymphocytes,Science,1996,274,94-96)。纯化的共刺激小鼠IgG2a抗人CD28抗体9.3(Jung,G,Ledbetter,JA,and Muller-Eberhard,HJ;Induction of cytotoxicity inresting human T lymphocytes bound to tumor cells by antibody heteroconjugates,Proc Natl AcadSci USA,1987,84,4611-4615)使用制造商(Perbio公司,波恩,德国)推荐的N-羟基琥珀酰亚胺生物素进行化学生物素化处理。所用珠为5.60μm的大链霉抗生物素蛋白包裹的多聚苯乙烯颗粒(Bangs Labooratories,伊利诺伊州/美国)。作为阳性和阴性对照的pMHC分别为A*0201/MLA-001(从Melan-A/MART-1中修饰制得的肽ELAGIGILTV)和A*0201/DDX5-001(从DDX5中获得的YLLPAIVHI)或A*0201/HBV-001(FLPSDFFPSV)。
800.000珠/200μl包裹于96孔板,以600ng生物素抗CD28+200ng相关生物素pMHC(高密度珠)或2ng相关+200ng不相关(pMHC库)MHC(低密度珠)存在。在37℃下,在含5ng/ml IL-12(PromoCell)的200μl TCM中共培养1×106CD8+T细胞与2×105的清洗涂层珠3至4天,从而启动刺激。之后,一半培养基与补充80U/ml IL-2的新鲜TCM进行交换,并且在37℃下持续3至4天。这种刺激性周期总共进行3次。最后,在四色流式细胞仪(BD公司)上用荧光MHC四聚体(如Altman,JD,Moss,PA,Goulder,PJ,Barouch,DH,Heyzer-Williams,MG,Bell,JI,McMichael,AJ,and Davis,MM;Phenotypic analysis of antigen-specific T lymphocytes,Science,1996,274,94-96)+抗体CD8-FITC克隆SK1(BD公司,海德堡,德国)进行四聚体分析。肽特异性细胞以占总CD8+T细胞的百分比形式进行计算。四聚体分析结果用FCS Express软件(De Novo Software公司)进行评估。特定四聚体+CD8+淋巴细胞的体外填装用适当的门控技术以及与阴性对照刺激组比较而进行检测。如果健康供体中的至少一个可评价的体外刺激孔在体外刺激后发现含有特异性CD8+T细胞株(即该孔包含至少1%特定四聚体+细胞,并且特定四聚体+的百分比至少为阴性对照刺激中位数的10倍),则检测给定抗原的免疫原性。
本发明中的肽连同要与之比较的具有已知免疫原性的肽一起测试。代表性染色显示了NOX-001和ODC-001特异性T细胞的生成,如图5所示。结果总结见表3。
表3:与疫苗肽比较,本发明肽的体外免疫原性
抗原 | 是否检测免疫抗原性 |
TGFBI-001 | 是 |
NOX-001 | 是 |
PCN-001 | 是 |
top-001 | 是 |
C20-001 | 是 |
ODC-001 | 是 |
CCN-001 | 是 |
PTP-001 | 是 |
CHI-001 | 是 |
JAK-001 | 是 |
表3a:本发明肽的免疫原性
抗原 | 阳性供体/得到测试的供体 | 阳性孔/得到测试的孔 |
IMA-HBV-001 | 7/16(44%) | 10/107(9%) |
IMA-TGFBI-001 | 3/4(75%) | 4/22(18%) |
IMA-NOX-001 | 3/5(60%) | 9/60(15%) |
IMA-PCN-001 | 3/4(75%) | 4/42(10%) |
IMA-top-001 | 2/5(40%) | 7/72(10%) |
抗原 | 阳性供体/得到测试的供体 | 阳性孔/得到测试的孔 |
IMA-C20-001 | 1/5(20%) | 1/60(2%) |
IMA-ODC-001 | 1/5(20%) | 1/60(2%) |
IMA-HBV-001 | 2/5(40%) | 10/54(19%) |
IMA-CEA-004 | 4/4(100%) | 50/60(83%) |
IMA-CCN-001 | 5/5(100%) | 42/54(78%) |
IMA-MET-001 | 4/6(67%) | 30/72(42%) |
发明人所做的体外免疫原性结果总结如上。所示结果通过刺激高密度珠CD8+细胞而获得。由于不同批次的血清可能会大大影响免疫原性的结果,所以只评价了那些只使用同一批次血清的化验结果。
7.MHC-II类提呈肽的体外免疫原性
辅助T细胞在协助CTL激活和维持肿瘤细胞的免疫反应中发挥着重要作用。因此,MHC-II类肽被列入IMA910。TGFBI-004是IMA910中三个II类肽之一,对其体外免疫原性潜力进行了检测,结果证明它既是特异性CD4+T细胞也是CD8+T细胞的一种诱导物。使用自体***刺激的实验显示生成了CD4+和功能性CD8+T淋巴细胞。
测试原理
特异性人CD4+和CD8+细胞的启动和扩增使用自体DC、通过单核细胞缺乏的PBMC启动以及使用自体PBMC进行重新刺激而进行体外检测。简言之,为了生成抗原特异性CD4+T细胞,健康供体的单核细胞缺乏的PBMC(HLA基因型I类:A1/A25/B8/B18和II类:DQB1*02/DQB1*06/DRB1*03/DRB1*15/DRB3/DRB)使用肽脉冲击自体DC进行了刺激并且使用了自体PBMC+肽进行了再刺激。作为一个读出***,短期重刺激后的IFNγ产量用ELISPOT和流式细胞术进行了评估。在ELISPOT和细胞内IFNγ染色+CD4-FITC和CD8-PerCPT刺激8次之后,对T细胞进行了分析以确定特异性T细胞亚群中产生IFNγ细胞的百分比。在本实验中,对不同孔中的TGFBI-004所刺激的细胞,进行汇集、使用读出的不相关肽进行培养并作为阴性对照。
树突状细胞(DC)的生成
人DC从DC培养基中培养的单核细胞获得,培养基包括RPMI 1640-Glutamax/25mMHepes(Invitrogen公司,德国)和10%自体血浆//100U/ml青霉素和100μg/ml链霉素。首先,血沉棕黄层和血浆经离心健康供体血液而获得(Tübingen血库)。然后,PBMC使用标准密度梯度分离法(淋巴细胞分离液,PAA公司,奥地利)从血沉棕黄层中分离,并在DC培养液中悬浮以确定总细胞数。洗涤100-120Mio PBMC,在15ml X-Vivo 20培养基(BioWhittaker公司,比利时)中重新悬浮,并转移至细胞培养瓶。在37℃下待2小时后,含外周血白细胞(PBL)的培养液被清除,粘附单核细胞用10ml PBS洗涤两次并在用100ng/ml GM-CSF和30ng/ml IL-4(ImmunoTools公司,德国)或20ng/ml(R&D systems公司,德国)在10ml DC培养液中培养6天。在第3天和第5天,加入100ng/ml GM-CSF和30ng/ml IL-4(Immunotools公司)或20ng/ml IL-4(R&D Systems公司,德国)。第7天,未成熟的DC用10ng/ml TNF-α(R&D Systems公司,德国)和20μg/ml多聚(IC)(Sigma Aldrich公司,德国)或100ng/ml LPS刺激24小时。其余的PBMC以及获得的PBL进行等分和冷冻。
特异性T细胞体外启动
为了生成CD4+T细胞,用2×105自体DC对3百万PBMC/PBL进行刺激。DC在第8天收集(参见第3.1章,“DC的生成”)。含5mM乙二胺四乙酸的PBS用于获得尽可能多细胞之目的(包括粘附细胞)。DC培养液洗涤后,确定细胞数。为了装载肽,DC在1ml DC培养液中再悬浮并用25μg/ml肽在37℃下培养)、Padre和CMV。解冻自体PBMC/PBL、用DC培养液洗涤(至少两次)并在24孔板中展开,密度为1ml中3Mio细胞/ml。然后,将载有肽的DC加入(如含肽的1ml悬浮液)到入板的PBMC/PBL并在37℃下培养7天。启动后,首先用已接受照射(30Gy;Gammacell 1000Elite照射仪,Nordion International公司,加拿大)。为此目的,每孔加入5×105CTL和2、5×106PBMC。使用肽对PBMC进行冲击如前所述(针对DC的方法)。第一次重新刺激的第1天,加入IL-2(R&D Systems公司,德国)和IL-7使最终浓度分别为2ng/ml和5ng/ml。此后,在每个第2天和第7天,向培养液加入IL-2和IL-7。7天后进行第二次重新刺激,但这次只向培养的CTL加入肽(不加入PBMC)。重刺激7天为一周期用负载肽的PBMC和单肽交替加入。在第8次刺激之后,用细胞内IFNγ和IFNγELISPOT法进行分析。
结果
启动对相关肽特异性反应的CD4+T细胞株是可能的(图6和图3)。可通过ELISPOT法在4个细胞株中检测到其中2个有T细胞反应,而使用ICS法的检测结果表明,有四分之三的T细胞株TGFBI-004特异性IFNγ产生CD4+和/或CD8+细胞。因此,用上述实验***的测试中,TGFBI-004能引发供体的CD4+和CD8+T细胞反应。根据这一有前景的结果,很可能这种肽具有免疫原性,并有能力诱导T细胞反应。
8.NOX-001和TGFBI-001功能验证举例
IMA910疫苗中所含肽的免疫原性使用immatics`TUMAP验证平台(immaticsbiotechnologies公司,Tübingen,德国)进行了论证。对特异性T细胞的诱导说明了肽有能力成功地成功激活免疫***。由于只有激活的T细胞具有高亲合力和功能性时才有可能发生有效的抗肿瘤免疫反应,因此,我们通过检测其产生IFNγ或杀死肿瘤细胞株的能力从而研究了TUMAP启动其高亲合力和功能性T淋巴细胞的能力。由于NOX-001和TGFBI-001能体外诱导CTL的高亲合力,因此选择这两种肽进行深入验证。结果证明,高亲合力前体T细胞对人体肽和NOX-001能产生的CD8+T细胞株都有作用。
测试原理
为了对IMA910肽免疫原性和特异性T细胞的特性更加深入的认识,选定NOX-001和TGFBI-001这两种肽作进一步的评估。为此目的,在immatics biotechnologies公司(Tübingen,德国)进行了实验(细胞分选在Tübingen大学的Dr.Bühring实验室进行)。
T细胞株依据其被高密度或低密度抗原激活的能力大小,可分为高或低亲合力T细胞株。如先前研究所示,(Walter,S,Herrgen,L,Schoor,O,Jung,G,Wernet,D,Buhring,HJ,Rammensee,HG,and Stevanovic,S;Cutting edge:predetermined avidity of human CD8T cellsexpanded on calibrated MHC/anti-CD28-coated microspheres,J.Immunol.,2003,171,4974-4978),与低亲合力CD8+T细胞相比,人高亲合力CTL可使用较少肽进行激活而成功获得。研究还表明,细胞以这种方式扩增可更有效地识别抗原表达细胞株,从而构成了开发治疗策略的一个可能的主要工具。
为了确定肽生成高亲合力CTL株的能力,用含低密度pMHC(肽-MHC-复合体)涂层珠和含IL-12与IL-2的抗-CD28抗体对分离出来的人CD8+细胞进行反复刺激从而使之启动和扩增。经过三次刺激后,激活T细胞的一部分以四聚体染色和流式细胞术法检测。之后,根据其抗原特异性,对每个供体的四聚体阳性细胞进行储存,以pMHC-四聚体和人抗CD8-FITC抗体进行染色,最后在FACSAria上进行FACSA分类。分类过的细胞在照射过的饲养细胞、细胞因子及丝裂素中培养和扩增。为了读出高亲合力抗原特异性细胞的生成情况,进行四聚体法染色。为了确定它们的功能,在以相应的肽和肿瘤细胞株重刺激这些细胞后,用ELISPOT法测定IFNγ的产量,并在死/活细胞染色的基础上,使用细胞毒性测定法检查肿细胞株的杀伤情况。
特异性CD8+T细胞株的生成
如上所述,我们应用载有肽-MHC复合体(pMHC)和抗-CD28抗体的人工刺激抗原提呈细胞(aAPC)进行了体外刺激。与上述方法唯一的区别就是:进行刺激用的珠装载有2ng相关+200ng不相关库内(pMHC)MHC(低密度珠),而不是200ng相关MHC(高密度珠)。因此,高亲合力T细胞的产生主要是为了对肽进行更深入的验证。经过三次刺激后,激活T细胞的一部分以四聚体染色和流式细胞术法检测。如果健康供体中的至少一个可评价的体外刺激孔在体外刺激后发现含有特异性CD8+T细胞株(即该孔包含至少1%特定四聚体+细胞,并且特定四聚体+的百分比至少为阴性对照刺激中位数的10倍),则检测给定抗原的免疫原性。之后,根据其抗原特异性,对每个供体的四聚体阳性细胞进行储存,以相应的pMHC-四聚体和人抗CD8-FITC抗体克隆SK1进行染色,最后在FACSAria(BD Biosciences公司,德国)上进行FACSA分类。分类过的细胞在含有5×105细胞/ml照射过的新鲜异源PBMC、5×104细胞/ml照射过的LG2-EBV细胞、150U/ml IL-2(Chiron公司,慕尼黑,德国)和0.5μg/ml PHA-L(Roche Diagnostics公司,Mannheim,德国)的T细胞培养基中培养(培养基为RPMI-Glutamax,补充有10%热灭活人AB血清、100U/ml青霉素、100μg/ml链霉素、1mM丙酮酸钠和20μg/ml庆大霉素)。这些细胞在含150U/ml IL-2的T细胞培养基中扩展。为了读出高亲合力抗原特异性细胞的生成情况,如前述方法进行四聚体法染色并在四色FACSCalibur上进行了分析(BDBiosciences公司,德国)。
功能性测试
为了确定它们的功能,在以相应的肽重刺激这些细胞后,用ELISPOT法(IFNγELISPOT Set,BD公司,德国)评估IFNγ的产量。此外,细胞介导的特异性CTL的细胞毒性使用LIVE/DEAD(死/活)细胞介导的细胞毒性套件(L7010,Invitrogen公司,德国)杀死肿瘤细胞株而进行了研究。除非另有说明,否则上述两种测定方法都根据制造商的指示进行。
结果
用低密度pMHC aAPC成功启动后显示,NOX-001和TGFBI-001这两种肽具有体外免疫原性。对于NOX-001以及TGFBI-001特异性T细胞株可以用FACS建立,从而证明高亲合力CD8+T细胞前体存在于健康供体中。
此外,对于NOX-001,可确定一个T细胞株,ELISPOT法也证明了其功能,因为在用这种肽重刺激后,NOX-001特异性表达IFNγ(图8)。
9.将本发明中的HLA-I类限制肽与HLA-A*0201结合
本分析的目的是评价HLA-I类肽CHI-001、DCA-001、JAK-001和PTP-001对HLA-A*0201等位基因编码的MHC分子的亲和力。所有的肽对HLA-A*0201的亲和性都可与大家熟知的对照肽HBV-001进行比较,解离常数(KD)范围为0.05到1.6nM。
测试原理
稳定HLA/肽复合物包括三种分子:HLA重链、β-2微球蛋白(b2m)和肽类配体。变性重组HLA-A*0201重链分子的活性便可进行保存,使之功能与“空载HLA-A*±0201”相当。被稀释为包含b2m和相应肽的水缓冲液后,这些分子以完全肽依赖方式迅速有效地折叠。这些可用的分子用于基于ELISA的检测方法中,来衡量肽和HLA-I类分子相互作用间的亲和力(Sylvester-Hvid C,Kristensen N,Blicher T,Ferre H,Lauemoller SL,Wolf XA,Lamberth K,Nissen MH,Pedersen LO,Buus S.Establishment of a quantitative ELISA capable ofdetermining peptide-MHC class I interaction.Tissue Antigens 2002,59,251-258)。
纯化的重组HLA-A*0201分子与b2m、不同等级剂量的肽一起培养。从头折叠HLA/肽复合物的量用定量ELISA法测定。解离常数(KD值)使用从校准HLA/肽复合物稀释液中记录的标准曲线进行计算。
结果
结果如图9所示。较低的KD值反映了HLA-A*0201具有较高的亲和力。所有的肽对HLA-A*0201的亲和性都可与大家熟知的对照肽HBV-001进行比较,解离常数(KD)范围为0.05到1.6nM。
序列表
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Claims (22)
1.一种肽,其包括选自SEQ ID No.1至SEQ ID No.29群组的一个序列、或与SEQ ID No.1至SEQ ID No.29具有80%同源性的其变体、或诱导与该肽发生T细胞交叉反应的一个变体。
2.根据权利要求1所述的肽,其中该肽总长度为8至100个氨基酸、优选为8至30个氨基酸,最优选为8至16个氨基酸。
3.根据权利要求1或2任一项中所述的肽,其具有与主要组织相容性复合体(MHC)I或II类分子结合的能力。
4.根据权利要求1至3任一项中所述的肽,其中该肽系由或基本系由根据SEQ ID NO 1至SEQ ID NO 29所选的氨基酸序列组成。
5.根据权利要求1至5任一项中所述的肽,其中该肽被修饰或包含非肽键。
6.根据权利要求1至5任一项中所述的肽,其中该肽为融合蛋白,特别是含HLA-DR抗原相关不变链(Ii)的N-端氨基酸。
7.一种核酸,其编码根据权利要求1至5任一项中所述的肽。
8.根据权利要求7中所述的核酸,其可能为DNA、cDNA、PNA、CAN、RNA,也可能为其组合物。
9.根据权利要求7或8中所述的一种能表达核酸的表达载体。
10.根据权利要求1至6任一项中所述的一种肽、根据权利要求7或8任一项中所述的一种核酸、或根据权利要求9中所述的一种表达载体。
11.根据权利要求7或8中所述的一种宿主细胞或根据权利要求9中所述的一种表达载体。
12.根据权利要求11中所述的宿主细胞,该宿主细胞为抗原提呈细胞,尤其是树突状细胞或抗原提呈细胞。
13.根据权利要求1至6任一项中所述的一种制备肽的方法,该方法包括根据权利要求11中所述的培养宿主细胞,从宿主细胞或其培养基中分离出肽。
14.一种体外制备激活的细胞毒性T淋巴细胞(CTL)的方法,该方法包括将CTL与载有抗原的人I或II类MHC分子进行体外连接,这些分子在合适的抗原提呈细胞表面表达足够的一段时间从而以抗原特异性方式激活CTL,其中所述抗原为权利要求1至6任一项中所述的肽。
15.根据权利要求14中所述的方法,其中抗原通过与足够量的含抗原提成细胞的抗原结合被载入表达于合适抗原提呈细胞表面的I或II类MHC分子。
16.根据权利要求14中所述的方法,其中该抗原提呈细胞包括一个表达载体,该载体有能力表达含SEQ ID NO 1至SEQ ID NO 29的肽或所述变体氨基酸序列。
17.根据权利要求14至16任一项中所述的按其方法制成的激活细胞毒性T淋巴细胞(CTL),该淋巴细胞会有选择性地识别一种细胞,该细胞异常表达含权利要求1至4任一项中给定氨基酸序列的多肽。
18.一种杀灭患者中靶向细胞的方法,其中靶向细胞异常表达一种多肽,该多肽含权利要求1至4任一项中给定的氨基酸序列;一种给药方法,其中包括给予患者权利要求14至17任一项中定义的有效量细胞毒性T淋巴细胞(CTL)。
19.根据权利要求1至6任一项中所述的一种肽的用途、根据权利要求7至8中所述的一种核酸的用途、根据权利要求9中所述的一种表达载体的用途、根据权利要求11或12中所述的一种细胞的用途、根据权利要求17中所述的一种作为药剂或制造药剂的激活细胞毒性T淋巴细胞的用途。
20.根据权利要求19中所述的用途,其中药剂系指一种疫苗。
21.根据权利要求19或20中所述的用途,其中药剂有抗癌活性。
22.根据权利要求21中所述的用途,其中所述癌细胞为胶质母细胞瘤细胞、结直肠癌细胞、胰腺癌细胞、肺癌细胞、肾癌细胞或胃癌细胞。
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