CN1059466C - 鉴定患有细胞异常性的个体的方法 - Google Patents
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- CN1059466C CN1059466C CN93112875A CN93112875A CN1059466C CN 1059466 C CN1059466 C CN 1059466C CN 93112875 A CN93112875 A CN 93112875A CN 93112875 A CN93112875 A CN 93112875A CN 1059466 C CN1059466 C CN 1059466C
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Abstract
本发明涉及鉴定异常细胞表面上的人白细胞抗原分子与酪氨酸酶衍生之肽的复合物。本发明的主题是这一研究结果在治疗和诊断上的应用。
Description
本发明涉及根据识别某些存在人白细胞抗原和由其表面上酪氨酸酶衍生之肽的复合物的异常细胞所导出的各种治疗方法学。此外,本发明还涉及鉴定那些根据以细胞异常性为特征之条件而诊断的个体的能力,其中所说个体的异常细胞存在该复合物、所呈现的肽及其分支。
哺乳动物免疫系统识别外来或异种并与之反应的过程是十分复杂的。该系统的一个重要方面是T细胞反应。该反应需要T细胞识别称为人白细胞抗原(“HLA”)或主要组织相容性抗原(“MHCs”)之细胞表面分子与肽的复合物,并与之反应。所说的肽衍生于被其中也存在HLA/MHC分子的细胞加工的大分子[参见Male et al.,Advanced Immunology(J.P.Lipincott Company,1987),特别是第6-10章)。T细胞与HLA/肽复合物的相互作用受到约束,需要有对HLA分子和肽的特定结合的特异的T细胞。如果不存在特异性T细胞,即使存在其对象复合物也不会有T细胞反应。同样,如果没有特异性复合物,而只存在T细胞也不会出现反应。该机制在自身免疫病理学中参予免疫系统对外来物质的反应,并参予对细胞异常的反应。目前,大量的研究工作已集中于使蛋白质加工成HLA结合肽的机制上(对此可参见Barinaga,Science 257:880(1992);Fremont et al.,Science 257:919(1992);Matsumura et al.,Science 257:927(1992);Latron et al.,Science 257:964(1992))。
也已证明T细胞识别细胞异常性的机制与肿瘤有关。例如,PCT申请PCT/US 92/04354(申请日1992年5月22日,公开日1992年11月26日,该文献列为本文的参考文献)公开了一家被加工成肽的基因家族,这些基因本身在细胞表面上表达,从而导致肿瘤细胞被特异性CTLs溶解。据说这些基因编码“肿瘤排斥抗原前体”或称“TRAP”分子,且由之衍生的肽被称为“肿瘤排斥抗原”或“TRAs”(有关这一基因家族的进一步信息参见Traversari et al.,Immunogenetics35:145(1992):van der Bruggen et al.,Science 254:1643(1991))。
美国专利申请序号938,334(其中公开的内容列为本文的参考文献)描述了与HLA-A1分子结合的九肽。该参考文献教导了特定肽对特定HLA分子的给定的已知特异性,故可期望某特定肽可与一种HLA分子结合,而不与其他分子结合。因为不同的个体具有不同的HLA表型,所以这一特异性是很重要的。作为结果,虽然鉴定一特定肽为特异性HLA分子的反应对象具有诊断和治疗意义,但这些只是对带有该特定HLA表型的个体相关的。因为细胞异常性并不限定于一种特定HLA表型,而且有针对性的治疗需要有关于某些在组织上异常细胞表型的知识,所以有必要就这一领域作更深入的研究。
酪氨酸酶催化使酪氨酸转化为二羟基苯丙氨酸或“DOPA”反应,并且似乎是在单核细胞中得以选择性表达的(Muller et al.,EMBOJ7:2715(1988))。有关这种人类酶之cDNA的早期报导见于Kwon的美国专利4,898,814号。后来Bouchard等人(J.Exp.Med.169:2029(1989))的报导提供了稍微不同的顺序。因为已证明该酶的抑制剂与色素沉着性疾病有关,所以大量工作已深入到对这些抑制剂的研究。这方面的文献包括Jinbow的WO9116302,Mishima等人的美国专利5,077,059号和Nazzaropor的美国专利4,818,768号。本领域的技术人员将熟知那些提供相似材料的其他参考文献。
然而,这些参考文献均没有教导或提示酪氨酸酶可按与外来抗原或TRAP分子相似的方式受到处理,即现已发现在某些细胞异常如黑素瘤中,酪氨酸酶被加工,并且由之衍生的肽与某些异常细胞上的HLA分子形成复合物。这些复合物被溶胞性T细胞(“CTL”)识别,然后CTL溶解存在该复合物的细胞。这一令人惊异和不可预见的现象便构成了本发明的主题,下文将作更为详细的描述。
图1集中描述细胞溶解研究结果。具体地是:
图1A显示细胞系LB24的溶解;
图1B显示细胞系SK29-MEL的溶解;
图1C显示细胞系LB4.MEL的溶解;
图1D显示细胞系SK23.MEL的溶解;
图1E显示细胞系LE516.MEL的溶解;
图1F显示对NK靶K562所作的溶解研究;
图1G显示自体固有的EBV-B转化的细胞的溶解;
图1H显示在用HLA-A2的基因转化后,图1F中丢失变异体的溶解;
图1I显示自体固有的IEBV-β转化的细胞的溶解。
图2显示CTL IVSB的TNF释放研究结果。
图3描述CTL 210/9的TNF释放研究。
图4描述细胞溶解性T细胞克隆CTL-IVSB但不是细胞溶解性T细胞克隆CTL 2/9对肽YMNGTMSQV的识别。
图5显示肽YMNGTMSQV不被细胞溶解性T细胞克隆CTL 210/9所识别。
图6显示当对各种细胞,包括在其表面上呈现HLA-B44的细胞进行TNF释放分析时所获得的结果。
图7为SEQ ID NO:1。
实施例1
在下列实验中使用研究者多年前得到的黑素瘤细胞系29-MEL(文献中也称之为SK MEL-29)和LB24-MEL。
由病人AV和LB24-MEL体内(这些病人也分别是SK 29-MEL和LB24-MEL的来源)采得含单核血细胞的样品。使黑素瘤细胞系与含单核血细胞的样品接触。观察混合物中黑素瘤细胞系的溶解情况,这一细胞溶解现象表明样品中存在与存在于黑素瘤细胞的对肽的HLA分子复合物相特异的细胞溶解性T细胞(“CTL”)。
所使用的溶解检测法是由Herin等人(Int.J.Cancer 39:390-396(1987),其中公开的内容列为本文参考文献)建立的铬释放法。本文再次描述这一方法。体外生长靶黑素瘤细胞,然后将其以107细胞/ml浓度再次悬浮于添加了10mM HEPES及30%FCS的DMEM中,并在37℃下与200μCi/ml Na(51Cr)O4一起保温45分钟。用加有10mM HEPES的DMEM将标记的细胞洗三次。然后重新悬浮于加有10mM HEPES和10%FCS的DMEM中,并在加入含103个细胞的100μl等分样品后分加于96小井微量滴定板内。向100μl同样培养基中加入PBL样品,以一式两份进行检测。以100g将平板离心4分钟,并在5.5%CO2环境中37℃保温4小时。
再次离心此平板,收集100μl等分的上清液并进行细胞计算。按下列公式计算51Cr释放的百分率:其中ER观察的是实验组51Cr释放,SR是在200μl单一培养基中保温103个标记细胞测得的自发释放,且MR是向靶细胞内加入100μl 0.3%Triton X-100所测得的最大释放。
扩展并通过有限稀释法克隆那些显示了高CTL活性的单核血细胞样品,并使用同一方法学再次筛选之。
使用相同方法试验靶K562细胞。当使用EBV-B细胞时,只是将DMEM培养基换成添加有5%FCS的Hank’s培养基。
这些实验导致从病人AV中分离出CTL克隆“IVSB”和从病人LB24中分离出CTL克隆210/9。
图1分别在A、B、G和I框中给出了这些实验的结果。具体地说,从中可以看出两种CTL可溶解两个黑素瘤细胞系,而且看出没有溶解K562和EBV-B细胞系。
实施例2
对其他黑素瘤细胞系试验所述的CTL,以确定是否其他黑素瘤细胞系也共有它们的靶。按实施例1中所述的细胞溶解法研究细胞系LB4.MEL,SK23.MEL(也称为SK MEL-23)和LE516.MEL。图1的C、D和E框显示所述克隆溶解这些细胞系。
被试验的细胞系已知为HLA-A2型,且结果提示CTL是对肽和HLA-A2的复合物特异的。这一提示可经试验已失去HLA-A2表达能力的SK 29-MEL的变异体得以证实。图1的F框中显示了这些结果。两克隆均不溶解丢失HLA的变异体。然而,当用SK29-MEL的HLA-A2基因转化此变异体并重新进行试验,则观察到细胞溶解作用。因此可以认为,所呈现的分子是HLA-A2。
实施例3
一旦鉴定出HLA分子,即进行进一步的研究以鉴定下文称为“肿瘤排斥抗原前体”或“TRAP”分子(其为所提出的肽的来源)的分子。
为此目的,从SK29-MEL的亚克隆的细胞系SK29-MEL.1中分离总RNA。按已知技术,使用Oligo-dT结合药盒分离RNA。一旦得到总RNA,即使用标准方法学将其转化成cDNA。然后将cDNA连接到EcoRI接头上并按制造商推荐的方法克隆到质粒pcDNA-I/Amp的EcoRI位点中。然后用电穿孔法将重组质粒转换到大肠杆菌JM101中(电穿孔条件:25微法,2500V1个脉冲)。
用氨苄青霉素(50μg/ml)选择被转化的细菌,然后分成700份,每份含200个克隆。各份代表了大约100个不同的cDNA,如分析所显示的,大约50%的质粒含有一插入段。将各份扩增至饱和,并按Maniatis等人(Molecular Cloning:A Laboratory Manual,Cold Spring Harbor,N.Y.,1982)所述方法,通过碱溶解、醋酸钾沉淀和苯酚提取分离质粒DNA。不使用铯梯度离心技术。
实施例4
然后将扩展的质粒转染到真核细胞中。以15,000个细胞/小井的浓度将COS-7细胞的样品(加在补充有10%胎牛血清的Dulbeco’s改进的Eagles培养基(“DMEM”)中)授种于平底组织培养微滴定板中。将细胞于37℃保温过夜,除去培养基后换成每小井30μl含有10%Nu血清、400μl/ml DEAE-葡聚糖、100μM氯喹、10ng质粒pcDNA-I/Amp-A2和100ng上述合并之cDNA库DNA的DMEM培养基。质粒pcDNA-I/Amp-A2含有来自SK29-MEL的HLA-A2基因。37℃保温4小时后,除去培养基并换成50μl含10%DMSO的PBS。2分钟后除去该培养基中并换成200μl添加了10%FCS的DMEM。
改变培养基后,将COS细胞于37℃保温48小时。弃去培养基,分别加入上述各CTL克隆的2000个细胞(在100μl含10%合并之人血清的Iscove培养基中)。当使用克隆210/9时,培养基中添加25U/ml IL-2。24小时后除去培养基,接Traversari等人(Immunogenetics 35:145-152(1992),其公开内容列入本文作为参考)所述方法对WEHI细胞进行检测,以确定TNF含量。
用IVSB试验的700个小井中,有696个显示每小井有0.6至4pg TNF。其余的4个小井则含有10至20pg/ml TNF。用CTL 210/9试验的类似小井显示出相似的、明显高的值。图2和3中给出了这些数据。
实施例5
选择出已鉴定为高生产菌株的4份合并物中的3份(编号为“123”、“181”和“384”),用于进一步的实验。具体地说,使细菌克隆化并试验由各集合物得到570个细菌菌落。由其中提取质粒DNA,按前述的相同方法将其转染到新的COS细胞样品中,并再次试验这些细胞的CTL 210/9和CTLIVSB刺激作用。在集合物123(“p123.B2”)中发现了阳性克隆,且在集合物384(“p384.L6”)发现了一个。对用cDNA和HLA-A2基因转染的COS细胞和只用HLA-A2转染的COS细胞进行比较试验,从而得到CTL识别被转染之细胞的令人信服的证据。通过对WEHI细胞所作试验来检测CTL上清液中的TNF释放。使用MTT检测存活WEHI细胞的光度。结果如下列表1所示:
表1
cDNA(123.B2) no cDNA+
+HLA-A2 DNA HLA-A2
Run 1 0.087 0.502
Run 2 0.108 0.562对于cDNA和HLA-A2转染,WEHI光密度的值相当24pg/ml TNF,而对照组则相当于2.3pg/ml。
除去从阳性克隆中分离的质粒,按本领域已知技术测定其顺序。顺序分析表明,质粒插入段几乎完全相同于如Bouchard等人(J.Exp.Med.169:2029(1989),此公开列为本文的参考文献)所述的人酪氨酸酶的cDNA。因此,正常情况下存在的分子(即酪氨酸酶)可能是作为肿瘤排斥抗原前体并被加工以形成肿瘤排斥抗原肽,后者以与HLA-A2结合的形式存在于细胞表面上,从而刺激由CTL克隆产生的溶解作用。已鉴定之分子的核酸顺序如SEQ ID NO:1所示。
实施例6
Chomez等人(Immunogenetics 35:241(1992)报导的先期工作已显示,含有编码抗原肽之顺序的小的基因片段可导致该肽的表达。该文献(其全文列为本文参考文献)提示了上文和SEQ ID NO:1中所述人酪氨酸酶cDNA之小部分的克隆。使用实施例1-5中所述的方法学,用携带HLA-A2基因的各种cDNA片段共转染COS-7细胞,并进行TNF释放分析。这些实验导致鉴定大约400个碱基对的片段,当在共转染实验中使用时,该片段引发从上文所述细胞溶解性T细胞克隆CTL IVSB释放TNF,故表明它是对HLA-A2呈现细胞特异的。所用的该400碱基片段相当于SEQ ID NO:1中的碱基711至1152。推测出由该片段编码的氨基酸顺序,然后将该顺序与Hunt等人(Science 255:1261(1992)和Falk等人(Nature 351:290(1990))提供的资料相比较(上述公开均列入本文作为参考文献)。这些参考文献讨论了HLA-A2呈现肽的相符顺序。具体地说,Hunt讨论了总是在第二位上发现有Leu或Ile的九肽,其中Leu是“优势残基”。第九个残基被描述为总是带有脂族烃侧链的残基。Val是这个位置上的优势残基。Hunt讨论了第二位上的Leu的强信号和该位置上的Met、第6位上的Val、Leu、Ile或Thr之一以及第9位上的Val或Leu的中等信号,其中Val的信号特别强。在进行比较的基础上合成九肽,然后观察它们是否能致敏HLA-A2呈现细胞。为此使用了酪氨酸酶丢失变异体细胞系SK29-MEL 1.218和T202LB。向细胞系内加入各种浓度的被试样品,连同细胞溶解性T细胞克隆CTL IVSB或细胞溶解性T细胞克隆CTL 2/9。上述工作已经确定前者可溶解呈现HLA-A2的酪氨酸酶表达细胞,而后者则不能。
在加或不加抗HLA-A2抗体MA2.1(用于稳定空HLA-A2分子)的条件下,在含有51Cr的溶液中,将酪氨酸酶丢失的变异体37℃保温1小时。试验中,将细胞洗4次,然后与从100μM减至0.01μM的不同稀释度的肽保温。30分钟后,以40/1的E/T比例加入效应物细胞,4小时后收集100λ上清液并进行放射活性计数。
图4显示用九肽Tyr Met Asn GlyThr Met Ser Gln Val(SEQID NO:2)获得的结果。
下文称之为SEQ ID NO:2的这个肽相当于SEQ ID NO:1中所示酪氨酸酶之cDNA顺序的残基1129-1155。HLA-A2与该肽的复合物由CTL克隆CTL IVSB识别。
在平行实验中,由病人LB24得到的CTL克隆CTL210/9并不识别HLA-A2和SEQ ID NO:2之肽的复合物,尽管它识别HLA-A2和酪氨酸酶衍生之肽的复合物。因此,酪氨酸酶至少被加工成一种附加肽,当后者因HLA-A2分子的存在而呈现时即被CTL克隆所识别。
实施例7
在一项更深入的实验中,使用不编码SEQ IDNO:2所示肽的第二个基因片段。该片段由SEQID NO:1的碱基1处开始并终止在碱基1101处(即EcoRI-SphI片段)。以上文所述的同样方法对用该片段转染的COS-7细胞试验上述的细胞溶解性T细胞克隆CTL 210/9。还试验了CTL IVSB。这些结果显示LB24-CTL210/9可识别被该片段转染的HLA-A2细胞表面上的抗原,但CTL IVSB则不能。因此,第二种肿瘤排斥抗原肽衍生于酪氨酸酶。
实施例8
为了进一步限定由LB24-CTL 210/9识别的肿瘤排斥抗原,进行了下列实验。
将相当于SEQ ID NO:1的碱基451-1158的第二个片段连同HLA-A2的基因转染到COS细胞中,并进行TNF释放分析。该顺序引发从克隆SK29-CTL IVSB(20pg/ml),但不能从LB24-CTL 210/9(3.8pg/ml)释放TNF。这些结果进一步证实两个CTL克隆识别不同的肽,而且由LB24-CTL 210/9识别的肽必须是由1-451区域编码的。
实施例9
分析由cDNA片段1-451编码的酪氨酸酶衍生肽,看其是否为已知结合HLA-A2的相符顺序。合成相当于这些相符顺序的肽,并检验它们致敏HLA-A2呈现细胞的能力。为此目的,使用两个酪氨酸酶阴性黑素瘤细胞系(即NA8-MEL和用HLA-A2转染的MZ2-MEL 2.2),以及Salter等人(Immunogenetics 21:235-246(1985))所述的细胞系T2。
将细胞与51Cr和对HLA-A2特异的单克隆抗体MA.2.1于37℃一起保温50分钟,然后洗细胞(参见Bodmer et al.,Nature342:443-446(1989),其全文引入本文作为参考文献)。将靶细胞与各种浓度的肽,以及LB24-CTL克隆210/5或210/9一起保温。保温4小时后检测铬释放的百分数。
发现肽Met Leu Leu Ala ValLeu Tyr Cys Leu Leu(SEQID NO:3)是有活性的。
在本文总结的进一步的实验中,已显示CTL-IVSB可识别YMNGTMSQV,而不识别SEQID NO:3的肽。
结果总结于下列表2-4中:
表2
肽
YMNGTMSQV MLLAVLYCLL
(1120-1155) (25-54)SK29-CTL-TVSB + -LB24-CTL-210/5 - +LB24-CTL-210/9 - +
表3 *用Cr51和单-Ab MA2.1(抗-HLA-A2)与靶细胞一起保温50min,然后洗涤3次。再用各种浓度的肽与上述物质一起保温30min。按所指定(E∶T)的比率加入CTL细胞。在保温4小时后测定特定的Cr51的百分释放率。
表4
对由LB24-CTL 210/5和210/9或SK29-CTL IVSB识别的
酪氨酸酶肽的试验
(%Cr51比释放率))效应物细胞 肽 剂量 NA8-MEL* MZ2-2.2∶A2 T2LB24-CTL. MLLAVLYCLL 10μM 30 31 36210/5 (LAUS 17-5) 3 23 27 36
1 17 20 26(41∶1) 300nM 6 17 16
100 2 6 5
30 3 5 2
0 0 0 0LB24-CTL. MLLAVLYCLL 10μM 14 19 21210/9 (LAUS 17-5) 3 13 17 20
1 9 14 13(28∶1) 300nM 3 9 5
100 1 1 1
30 0 1 0
0 0 1 0SK29-CTL YMNGTMSQV 10μM 46 46 59IVS8 (MAINZ) 3 38 44 52
1 27 40 46(42∶1) 300nM 14 22 34
100 3 13 21
30 1 9 10
10 1 3 3
3 0 3 4
1 0 1 0
0 0 4 0spt.rel. 339 259 198max-spt 2694 1693 1206% 11 13 14
实施例10
使用CTL克隆22/31完成进一步的实验。前已显示该克隆溶解衍生于自体黑素瘤细胞MZ2-MEL的亚细胞系MZ2-MEL.43,但不溶解其他亚系如MZ2-MEL3.0和MZ2-MEL61.2,它也不溶解自体EBV转化的B细胞,或杀伤细胞系K562(参见Van den Eynde et al.,Int.J.Cancer 44:634-640(1989))。由MZ2-MEL.43呈现的抗原被称是抗原C。
在以前的包括本申请的在先申请中报导的工作中,发现酪氨酸酶基因编码由大多数HLA-A2黑素瘤细胞上的自体CTLs识别的抗原。用PCR扩增法检验该基因在细胞系MZ2-MEL的亚系上的表达。结果发现克隆MZ2-MEL.43是阳性的,而其他MZ2-MEL克隆如MZ2-MEL.3.0是阴性的。酪氨酸酶基因的表达与抗原MZ2-C的相互关系提示,MZ2-C可能是一种由酪氨酸酶衍生的、并由MZ2-MEL表达的HLA分子呈现的肿瘤排斥抗原。该细胞系不表达HLA-A2,这将表明如果一种酪氨酸酶衍生的肽是作为TRA呈现的,则可能还有第二种HLA分子卷入。
进行研究以鉴定哪种HLA分子可使抗原C出现于CTL22/31上。为此目的,从MZ2-MEL.43cDNA库中分离已知是细胞表面上的HLA分子,即HLA-A29、HLA-B37、HLA-B44.02和HLA-C克隆10,然后克隆到表达载体pcDNAI/Amp中。然后再用这些构建体之一或含有HLA-A1加上编码酪氨酸酶之cDNA(SEQID NO:1)的构建体转染受体COS 7细胞。按上述方法进行共转染。1天后加入CTL22/31,24小时后按Traversari等人(文献同上)所述方法通过检验对WEHI-164-13的细胞毒性,以测定TNF释放率。图6显示,只有在用HLA-B44和酪氨酸酶基因转染的细胞存在下才能由CTL22/31释放TNF。由此可以得出的结论是,HLA-B44呈现酪氨酸酶衍生的肿瘤排斥抗原。
前述实验证明,酪氨酸酶被加工作为肿瘤排斥抗原前体,而导致加工产生之肿瘤排斥抗原与至少某些异常细胞如带有HLA-A2或HLA-B44表型之黑素瘤细胞上的分子的复合物的形成。该复合物可被CTLs识别,并使其呈现细胞溶解。这一观察结果具有治疗和诊断上的价值,它构成了本发明的特征。就治疗应用来说,观察到产生了对呈现上述复合物的异常细胞特异的CTLs,从而提示了有关的各种治疗方法。其中之一是给在组织(issue)上有这种表型之异常细胞的个体使用对复合物特异的CTLs。开发这些CTSs的体外应用是本领域技术人员易于实现的。具体的说,使细胞如血细胞的样品与呈现复合物并能激发产生对增殖特异之CTL的细胞接触。靶细胞可以是转染体,如上述这样类型的COS细胞。这些转染体在其表面上呈现所需的复合物,并且当与所研究的CTL结合时即刺激其增殖。为使本领域技术人员能够制得这些CTL,已按照布达佩斯条约将含有有用基因即pcDNA-1/Amp1(HLA-A2)和p123.B2(人酪氨酸酶)的载体保藏在Institut Pasteur,登记号分别是I1275和I1276。COS细胞,如本发明所用的这些细胞同其他适用的宿主细胞一样可从很多途径得到。
为了详述称为继承性转移的治疗方法学(Greenberg,J.Immunol.136(5):1917(1986);Reddel et al.,Science 257:238(7-10-92);Lynch et al.,Eur.J.Immuno1.21:1403-1410(1991);Kast et al.,Cell 59:603-614(11-17-89)),使呈现所需复合物的细胞与导致对其特异的CTLs增殖的CTLs结合。然后将已增殖的CTLs输给具有特征在于某些异常细胞呈现特定复合物之细胞异常性的个体。然后CTLs溶解异常细胞,从而达到预期的治疗目的。
上述治疗方法是基于设想至少某些个体的异常细胞呈现一种或多种HLA/酪氨酸酶衍生的肽的复合物。这一设想可以很容易地得以确定。例如使用上文讨论的转染体鉴定并分离CTLs后,即可与病人的异常细胞样品一起来确定体外溶解作用。如观察到细胞溶解,那么在这种疗法中即可使用特异性CTLs来缓解与异常细胞相关的病理状况。一种较少涉及的方法学是使用标准检测法检测异常细胞的HLA表型特征,并使用例如PCR法通过扩增来确定酪氨酸酶的表达情况。多数不同的HLA分子呈现由酪氨酸酶衍生的TRAs这一事实增加了适于进行本文所讨论之治疗方法的个体的数目。
继承性转换(adoptive transfer)并不是仅有的按照本发明建立的治疗方式。也可使用许多方法在体内激发CTLs。已在上文所述工作的基础上,详细阐述了使用表达复合物之非增殖性细胞的方法。该方法中使用的细胞可以是正常表达复合物的细胞,例如经过照射的黑素瘤细胞或用为呈现复合物所必需的一种或两种基因转染的细胞。Chen等人(Proc.Natl.Acad.Sci.USA 88:110-114(January 1991)举例说明了这种方法,显示了表达HPVE7肽的被转化细胞在治疗中的应用。可使用各种类型的细胞。同样,可使用携带一种或两种有用因基的载体。特别可取的是病毒或细菌载体。在这些系统中,例如由牛痘病毒或细菌BCG携带有用基因,并由这些材料“感染”宿主细胞。导致呈现有用复合物的细胞被自体CTLs识别,然后增殖。使酪氨酸酶本身与佐剂合用以有利于掺入HLA-A2呈现细胞可获得相似效果。然后加工该酶以产生作为HLA分子结合对象的肽。
上述的讨论涉及到“异常细胞”和“细胞异常性”。这些术语是以其最宽的解释使用的,并且涉及到被研究的细胞至少表现了一种特性(借以表明它们不同于它们的特异类型的正常细胞)的任何状态。异常特性的例子包括形态学和生物化学改变,例如包括黑素、自身免疫病等的肿瘤在内的细胞异常性。
本发明还提供了鉴定CTLs之靶前体的方法。当靶细胞是肿瘤细胞时,这些前体被称为肿瘤排斥抗原,但必须指出的是,当有异常性特征的细胞不是肿瘤时,也有时将该分子误称为肿瘤排斥抗原。该方法主要包括鉴定作为上述类型的细胞溶解性T细胞之靶的细胞。一旦鉴定出这样的细胞,即将总RNA转化成cDNA库,然后将此cDNA转染到能够呈现与相关HLA分子形成复合物之抗原的细胞样品中。使转染体与上文讨论的CTL接触,然后便可观察到CTL的击靶现象(细胞溶解和/或TNF产生)。然后处理这些被溶解的转化体以除去cDNA并进行顺序分析,从而能够以这种方法鉴定出导致异常状态的前体,如肿瘤排斥抗原前体。
本发明的其他方面对于本领域技术人员来说是很清楚的,在此不必重复。
所用的术语和措词是作为说明中的术语而不是为限制目的使用的,在这些术语和措词的使用中,无意排除所显示并描述的任何等同特征或其部分,应理解到,在本发明的范围内进行各种形式的改变是可能的。
Claims (11)
1、鉴定适于用对HLA-A2分子和由SEQ ID NO:3的氨基酸序列组成的酪氨酸酶衍生肽的复合物特异的治疗剂进行治疗的候选者的方法,该方法包括:
(i)使得自病人的异常细胞样品与对所说的复合物特异的胞溶性T细胞接触,以及
(ii)确定至少一部分所述异常细胞样品的溶解作为适于进行所说的治疗的候选者的指征。
2、一种治疗患有细胞异常的病人的药物组合物,其包括能激发针对在其表面上呈递HLA-A2分子和由SEQ ID NO:3的氨基酸序列组成的酪氨酸酶衍生肽的复合物的细胞产生胞溶性T细胞反应的制剂,所述的制剂的量足以激发对在其表面呈递所述复合物的异常细胞的反应。
3、权利要求2的药物组合物,其中所说的制剂包括编码人酪氨酸酶的载体。
4、权利要求3的药物组合物,其中所说的制剂还包括编码HLA-A2的载体。
5、权利要求3的药物组合物,其中所说的载体还编码HLA-A2。
6、权利要求2的药物组合物,其中所说的制剂是在其表面呈递所说复合物的非增殖性细胞样品。
7、对HLA-A2和由SEQ ID NO:3的氨基酸序列组成的酪氨酸酶衍生肽的复合物特异的分离的胞溶性T细胞。
8、权利要求7的胞溶性T细胞在制备治疗患者的细胞异常的药物中的应用,其中所述细胞异常的特征在于在异常细胞的表面呈递HLA-A2分子和由SEQ ID NO:3的氨基酸序列组成的酪氨酸酶衍生肽的复合物。
9、权利要求8所述的应用,其中所述的胞溶性T细胞是自身的。
10、鉴定在其表面上呈递HLA-A2分子和由SEQ ID NO:3的氨基酸序列组成的酪氨酸酶衍生肽的复合物的异常细胞的方法,包括使异常细胞样品与权利要求7的对所述复合物特异的胞溶性T细胞接触,并确定所述异常细胞的溶解以作为呈递所述复合物的细胞的指征。
11、由SEQ ID NO:3的氨基酸序列组成的分离的肽。
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US5519117A (en) * | 1992-12-22 | 1996-05-21 | Ludwig Institute For Cancer Research | Isolated, tyrosinase derived peptides and uses thereof |
US5843648A (en) * | 1995-01-10 | 1998-12-01 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | P15 and tyrosinase melanoma antigens and their use in diagnostic and therapeutic methods |
US5821122A (en) * | 1995-06-07 | 1998-10-13 | Inserm (Institute Nat'l De La Sante Et De La Recherche . .) | Isolated nucleic acid molecules, peptides which form complexes with MHC molecule HLA-A2 and uses thereof |
UA68327C2 (en) * | 1995-07-04 | 2004-08-16 | Gsf Forschungszentrum Fur Unwe | A recombinant mva virus, an isolated eukaryotic cell, infected with recombinant mva virus, a method for production in vitro of polypeptides with use of said cell, a method for production in vitro of virus parts (variants), vaccine containing the recombinant mva virus, a method for immunization of animals |
US6951917B1 (en) | 1995-09-26 | 2005-10-04 | The United States Of America As Represented By The Department Of Health And Human Services | MHC-class II restricted melanoma antigens and their use in therapeutic methods |
US7501501B2 (en) | 1995-09-26 | 2009-03-10 | The United States Of America As Represented By The Secretary Department Of Health And Human Services | MHC-Class II restricted melanoma antigens and their use in therapeutic methods |
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US7019112B1 (en) | 1996-03-19 | 2006-03-28 | University Of Virginia Patent Foundation | Peptides recognized by melanoma-specific A1-, A2- and A3-restricted cytoxic lymphocytes, and uses therefor |
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US20030138808A1 (en) * | 1998-02-19 | 2003-07-24 | Simard John J.L. | Expression vectors encoding epitopes of target-associated antigens |
US20030215425A1 (en) * | 2001-12-07 | 2003-11-20 | Simard John J. L. | Epitope synchronization in antigen presenting cells |
AU2003303082B2 (en) | 2002-01-30 | 2009-07-02 | Dana-Farber Cancer Institute, Inc. | Compositions and methods related to TIM-3, a Th1-specific cell surface molecule |
US8188243B2 (en) | 2004-06-23 | 2012-05-29 | Board Of Regents Of The University Of Texas System | Methods and compositions for the detection of biological molecules using a two particle complex |
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