CN1630819A - 肾细胞癌肿瘤标记 - Google Patents
肾细胞癌肿瘤标记 Download PDFInfo
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- CN1630819A CN1630819A CNA028068661A CN02806866A CN1630819A CN 1630819 A CN1630819 A CN 1630819A CN A028068661 A CNA028068661 A CN A028068661A CN 02806866 A CN02806866 A CN 02806866A CN 1630819 A CN1630819 A CN 1630819A
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Abstract
本发明涉及肿瘤标记,该标记可用于筛选、诊断、预测和鉴定肾细胞癌亚类。本发明也涉及所鉴定的抗原蛋白在免疫测定中的用途以及肿瘤标记用作免疫原以刺激免疫反应的用途。本发明还涉及所述肿瘤标记在制备针对肿瘤标记的抗体和抗体融合蛋白中的用途。
Description
发明领域
本发明涉及用于筛选、诊断和预测肾细胞癌(RCC)以及用于鉴定RCC亚类的肿瘤标记。本发明还涉及肿瘤标记作为免疫原激发免疫反应的用途和在制备针对肿瘤标记的抗体和抗体融合蛋白中的用途。
发明背景
与MHC I类相关的肽主要源于胞质蛋白的蛋白水解降解。在初始遍在蛋白化之后,这些蛋白由大的多催化蛋白酶复合体切割。不但有干扰素(IFN)-y-诱导亚基、低分子量蛋白(LMP)亚基LMP2,MECL1(LMP10)和LMP7,还有一些组成β-亚基,即Y、Z和X,也可以分别地形成蛋白酶复合体的蛋白水解活性位点。产生的肽被与抗原加工相关的转运蛋白(TAP)(由TAP1和TAP2亚基组成的异二聚体膜蛋白)从胞质转运到内质网(ER)。然后,在ER内肽加载到MHC I类分子上,这包含多步装配加工过程。新合成的MHC I类的重链(HCs)与ER定位的伴侣蛋白钙联接蛋白相连,然后结合β2-微球蛋白(β2-m),随后整入由伴侣蛋白钙联接蛋白、巯基氧化还原酶ERp57、TAP异二聚体和跨膜蛋白tapasin组成的大的MHC I类肽加载复合体中。另外,不但ER内而且胞质内的热休克蛋白也与肽结合并在其稳定和转运过程中扮演重要角色。然后稳定相连的MHC I类/肽复合体通过高尔基体外侧apparatus运输至细胞表面以呈递给CD8+T细胞。
在一些疾病例如,癌症、自身免疫疾病或者心血管失调中,正常或不正常的细胞蛋白的肽存在于细胞表面,这在健康个体的细胞表面没有发现。因此,这些肽或蛋白可以用作鉴别这些异常细胞的标记。而且,针对这些血清或其他体液内的肽或蛋白的抗体检测也可以用作危险指标或预测指标。
肾细胞癌(RCC)导致的死亡占所有癌症死亡的约5%。在出现时,超过50%的患者已经发展为局部晚期或转移性疾病,并且具有5年存活期的则小于20%。尽管RCC对传统的治疗方案有相对的抵抗性,但是对基于T细胞的免疫疗法对部分起作用。
在分化阶段或生理/病理生理过程中,蛋白质组可以用作研究在给定的条件培养的细胞中蛋白表达以及修饰模式的改变的重要工具(Pandey等,Nature 2000,405,837;Appella等,Exs.2000,88,1;Gevaert等.Electrophoresis 2000,21,1145)。
近来,蛋白质组学已用于搜寻不同来源肿瘤中诊断、预测和前兆参数(Alaiya等,Electrophoresis 2000,第21卷,第1210页;Unwin等,Electrophoresis 1999,第20卷,第3629页;Jungblut等,Electrophoresis 1999,第20卷,第2100页)。这些肿瘤标记(即与肿瘤相关的分子)可惯例地用于监测患者的疾病并可能在选择肿瘤患者对其使用特殊设计的免疫治疗策略时有进一步的帮助。
几个策略用于阐明这个治疗模式的潜在目标结构,包括2D-PAGE分离(Sarto等,Electrophoresis 1997,第18卷,第599页;Sarto等,Electrophoresis 1999,第20卷,第3458页)、SEREX分析(Scanlan等,Int.J.Cancer 1999,第83卷,第456页)、cDNA克隆表达(Boon等.Immunol.Today1997,第18卷,第267页)和扣除杂交步骤(Pitzer等,J.Cancer Res.Clin.Oncol.1999,第125卷,第487页)。
在WO99/00671中,对来自患者的血清进行双向凝胶电泳之后进行蛋白质印迹分析,鉴定出几个特异的β-微管蛋白同种型用作成神经细胞瘤的肿瘤标记。
在WO00/20586中,通过用来自癌症患者的抗血清对肾癌细胞中表达的核酸文库进行自身抗体筛选,鉴定出与肾癌相关的新抗原,可用作肿瘤标记。
可是,还需要另外的肿瘤标记用于开发适合于RCC癌症患者或其他癌症患者的疗法和诊断,以及用于鉴定RCC和区别RCC亚类的方法。
发明简述
因此,本发明的一个目的是提供新肿瘤标记。
更具体地,本发明涉及至少一个选自下列的蛋白作为肿瘤标记的用途:β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、细胞角蛋白、细胞角蛋白8(CK8)、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽(gluthathione)-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰-CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白(nucleophosmin)和波形蛋白。
特别地,本发明涉及至少一个选自下列的蛋白作为肾细胞癌的肿瘤标记的用途:β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、细胞角蛋白、细胞角蛋白8(CK8)、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰-CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白和波形蛋白。
另一个目的是提供在免疫测定中这些肿瘤标记的使用,免疫测定用于检测与个体血清中鉴定出的肿瘤标记特异结合的抗体的存在。
本发明的一个目的是提供免疫测定方法,该方法用于检测针对个体血清中肿瘤标记特异的抗体的存在。这些免疫测定可用于筛选、诊断、预测疾病。依照本发明,对个体样品中抗体水平的测定可用作RCC或其他肿瘤的早期诊断。而且,血清抗体水平的监测可用于预测疾病的进展。
本发明进一步的目的是肿瘤标记作为免疫原的用途,用来刺激个体内对肿瘤细胞的免疫反应以抑制肿瘤细胞生长或杀伤肿瘤细胞。
本发明的另一个目的是提供含有这些肿瘤标记的药物,用来刺激对肿瘤细胞的免疫反应以抑制肿瘤细胞生长和/或杀伤肿瘤细胞。
此外,本发明的另一方面是肿瘤标记在制备抗体或抗体融合蛋白中的用途。这些抗体或抗体融合蛋白可用作杀伤肿瘤细胞或抑制肿瘤生长的药物。
本发明的另一个目的是提供用免疫组织化学方法鉴定RCC和区别RCC亚类的方法和试剂盒。
基于以下的详述,对技术人员来说本发明的其他目的是显而易见的。
附图简述
图1:在高分子量筛选窗口检测到的目标(7%T/2.5%C凝胶)
图中所示是胶态考马斯染色2D凝胶(7%T/2.5%C)的一部分,代表来自约5×106未处理MZ1257RC细胞的全部裂解物的斑点模式。在第一维电泳中,蛋白聚集在非线性Immobiline DryStrip上(pH 3-10,NL;Amersham Pharmacia Biotech,Freiburg,德国)。对患者血清中用阳性免疫染色印迹所检测到的相关目标斑点用箭头示出。在相应的凝胶上通过肽质量指纹和/或部分测序分析鉴定这些目标斑点。
图2:在低分子量筛选窗口检测到的目标(16%T/2.5%C凝胶)
图中所示是胶态考马斯染色2D凝胶(16%T/2.5%C),代表来自约2.5×106未处理MZ1257RC细胞的全部裂解物的斑点模式。在第一维电泳中,蛋白聚集在非线性Immobiline DryStrip上(pH 3-10,NL;AmershamPharmacia Biotech,Freiburg,德国)。对患者血清中用阳性免疫染色印迹所检测到的相关目标斑点用箭头示出。在相应的凝胶上通过肽质量指纹和/或部分测序分析鉴定这些目标斑点。
图3:IFN-γ刺激细胞系MZ1257RC后,在低分子量筛选窗口检测到的目标(16%T/2.5%C凝胶)
图中所示是胶态考马斯染色2D凝胶(7%T/2.5%C),代表来自约2.5×106IFN-γ刺激(48小时)MZ1257RC细胞的全部裂解物的斑点模式。在第一维电泳中,蛋白聚集在非线性Immobiline DryStrip上(pH 3-10,NL;Amersham Pharmacia Biotech,Freiburg,德国)。对患者血清用阳性免疫染色印迹所检测到的相关目标斑点用箭头示出。在相应的凝胶上通过肽质量指纹和/或部分测序分析鉴定这些目标斑点。
图4:对CK8、stathmin和波形蛋白的正常肾组织和RCC免疫组织化学分析
用如实施例5所述的抗-CK8、抗-stathmin和抗-波形蛋白特异抗体进行正常肾组织、RCC透明细胞亚类(G2)和RCC嫌色亚类(G2)的免疫组织化学染色(400x,自左到右)。图中显示不但在RCC细胞透明亚类和嫌色亚类中而且在远端小管和集合管系统的上皮细胞中均存在CK8强阳性染色。图中显示远端小管系统中上皮细胞的stathmin中等到强烈阳性胞质染色;分散浸润炎症细胞和RCC细胞透明细胞亚类的阳性胞质染色;以及RCC细胞嫌色亚类的阴性反应。正常肾组织和RCC细胞透明细胞亚类的间质细胞对抗-波形蛋白染色是强烈的阳性胞质染色,而正常小管上皮细胞对其为阴性。发现了RCC细胞嫌色亚类中的波形蛋白弱表达。
发明详述
本发明目的的取得是基于以下意外发现:β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、β-微管蛋白、细胞角蛋白、CK8、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰-CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白和波形蛋白,优选膜联蛋白II、IV和V、stathmin、波形蛋白和B23/核仁磷酸蛋白在RCC患者体内是大多催化蛋白酶复合体蛋白水解降解的底物,因此,这些蛋白的肽是这些个体内与肾癌相关的抗原。因此,这些蛋白或其片段可用作肿瘤标记。
已经通过双向凝胶电泳(见图1到3)鉴定出本发明的蛋白,并随后通过患者血清免疫印迹检测到。从复制胶上切下免疫染色的蛋白斑点,进行凝胶消化,并用质谱分析。通过对来自患者的血清与对健康志愿者的血清的示差分析,以上提到的蛋白鉴定为RCC患者的肿瘤标记。
如本文所用,术语“肿瘤标记”根据本发明指蛋白β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、β-微管蛋白、细胞角蛋白、CK8、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰-CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白和波形蛋白,及它们的免疫原片段。
这些蛋白在RCC患者体内是免疫原性的发现,不但是研发监测此疾病不同药物治疗预测的方法的基础,还是研发RCC和其他癌症的诊断方法的基础。另外,这个发现提供了使用这些蛋白作为免疫原的方法,以刺激对肿瘤细胞的免疫反应。
因此,本发明提供β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、细胞角蛋白、细胞角蛋白8、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰-CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白和波形蛋白作为肿瘤标记的用途。
特别地,本发明提供β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、β-微管蛋白、细胞角蛋白、CK8、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰-CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白和波形蛋白作为肾细胞癌的肿瘤标记的用途。
可以用标准方法,包括层析(例如,离子交换、亲合和分子排阻柱层析)、离心、不同溶解度、电泳,或者用蛋白纯化的标准技术来分离和纯化这些蛋白。纯化的蛋白可用在设计用于检测个体样品中抗体存在的免疫测定中,或者可选择地,这些蛋白制剂可以用于如本文所述的免疫测定中。
本发明进一步提供对于本发明肿瘤标记的抗体检测和/或定量测量的方法,此抗体在生物样品中,象在患有RCC或其他疾病患者的血清或其他体液中;患者所患疾病是通过以细胞表面肿瘤标记片段的特异呈递为特征的。这些方法可以由许多方法中的任一个完成。这些方法包括免疫测定,它包括但不限于竞争性和非竞争性测定系统,使用的技术例如蛋白质印迹、放射免疫测定、ELISA(酶联免疫吸附测定)、“夹心”免疫测定、免疫沉淀测定、沉淀素反应、凝胶扩散沉淀素反应、免疫扩散测定、凝集测定、补体结合测定、免疫放射分析、荧光免疫测定、蛋白质A免疫测定,仅依此为例。
为了进行测定,将肿瘤标记固定在膜或底物上,或者用在液相中。合适的膜或底物例如能结合蛋白的表面例如聚苯乙烯微量滴定板的小孔或硝化纤维素膜。其他合适的体外测定对本领域技术人员是显而易见的。
例如,诊断和预测个体癌症的体外方法,包括通过免疫测定检测来自所述个体血清样品并且针对肿瘤标记蛋白的抗体的存在,可以用包括以下步骤的方法来实施:
(a)在膜或底物上固定至少一个肿瘤标记;
(b)使膜或底物与个体血清接触,和;
(c)检测个体血清样品中肿瘤标记特异抗体的存在。
为了检测血清样品中肿瘤标记的特异抗体,通常使用标记有可检测标记的第二抗体。可检测的标记可以是通过放射自显影检测的放射性同位素。就本发明目的而言,特别有用的同位素是3H、125I、131I、35S和14C。
第二抗体可以用荧光化合物标记。通过把免疫缀合物暴露于适当波长的光下,并检测因此产生的荧光来测定荧光标记抗体的存在。荧光标记化合物包括荧光素、异硫氰酸、罗丹明、藻红素、藻篮素、别藻篮素、邻苯二醛和荧光胺。
可选择地,用第二抗体与化学发光化合物偶联来对其进行可检测标记。通过检测在化学反应过程中出现的光的来检测带有化学荧光标记的免疫缀合物的存在。化学发光标记化合物的例子包括鲁米诺、异鲁米诺、芳族吖啶鎓酯、咪唑、吖啶鎓、草酸酯。
相似地,生物发光化合物可用于标记第二抗体。生物发光是在生物系统中发现的化学发光的一种,其中有催化活性的蛋白增加化学发光反应的效率。通过检测发光的存在测定生物发光蛋白的存在。用于标记的生物发光化合物包括荧光素、荧光素酶和acquorin。
可选择地,用第二抗体与酶相连来对第二抗体进行可检测标记。在合适底物存在下,抗体-酶缀合物温育时,酶部分与底物反应产生可检测的化学部分,例如,通过分光光度法、荧光测定法或目视法。用于可检测多特异免疫缀合物的酶的例子包括β-半乳糖苷酶、葡糖氧化酶、过氧化物酶和碱性磷酸酶。本领域技术人员知道能用于依据本发明的其他合适的标记。用本领域已知的标准技术可以完成标记部分与抗体的结合。
关于这方面的典型的方法学在Kennedy等,Clin.Chim.Acta 1976,第70卷,第1页;Schurs等,Clin.Chim.Acta 1977,第81卷,第1页;Shih等,Intl.J.Cancer 1990,第46卷,第1101页;Stein等,Cancer Res.1990,第50卷,第1330页中描述。
针对在血清或其他体液中本发明的肿瘤标记的抗体的检测和/或定量测量,可用在筛选RCC或其他疾病患病危险的个体中,患病危险个体是通过本发明肿瘤标记的免疫原性特性鉴定的。另外,抗体的测量可用于预测疾病的进展。
本发明也提供用于实施这些检测方法的试剂盒。这样的试剂盒可以包括用于实施上述诊断测定的所有必须成分。试剂盒将含有至少一个包括肿瘤标记的容器。试剂盒也含有包括抗体或其片段的第二容器,此抗体或其片段有对患者血清抗体的合适识别位点(例如抗人IgG抗体)和可检测标记,如上所述。
与RCC相关的肿瘤标记的鉴定为疾病的免疫治疗提供了基础。可以对患者免疫接种肿瘤标记以诱发能有利于杀死肿瘤细胞和/或抑制肿瘤细胞生长的免疫反应。可以用上述用来纯化蛋白的方法来制备肿瘤标记。
可选择地,可以对患者使用肿瘤标记的抗体,优选人源化抗体或抗体片段,以诱发有利于杀死肿瘤细胞和/或抑制肿瘤细胞的免疫反应。
术语“抗体片段”在本发明中的意思是指抗体的一部分例如F(ab′)2、F(ab)2、Fab′、Fab等。不管结构如何,抗体片段结合被完整抗体识别的相同抗原。该术语也包括结合特异抗原的合成的或基因工程多肽,例如由轻链可变区组成的多肽、由重链和轻链的可变区组成的“Fv”片段、重组单链多肽分子其中轻链和重链可变区通过肽接头连接(“scFv蛋白”)、以及由模拟超变区的氨基酸残基组成的最小识别单位。
术语“人源化抗体”指含有位于轻链和重链氨基酸可变区和恒定区的FR的抗体,可变区和恒定区来自人源,而超变区来自非人源。“FR”指抗体的框架区,并在可变区内发现。在这些区发生氨基酸的某些变更。
用本领域技术人员已知的方法可以制备本发明肿瘤标记的多克隆抗体(Green等,″Production of Polyclonal Antisera″:ImmunochemicalProtocols(Manson,ed.),pages 1-5(Humana Press 1992);Williams等,″Expression of foreign proteins in E.coli using plasmid vectors andpurification of specific polyclonal antibodies″DNA Cloning 2:ExpressionSystems,第二版,Glover等(eds.),第15页(Oxford University Press1995))。
通过使用佐剂,例如明矾(氢氧化铝)或弗氏完全或不完全佐剂,可以增强肿瘤标记的免疫原性。用于免疫接种的多肽也包括融合多肽,例如肿瘤标记或其部分与免疫球蛋白多肽或者与麦芽糖结合蛋白的融合多肽。多肽免疫原可以是全长分子或其一部分。如果多肽部分是“类半抗原”,那么这部分可以有益地与大分子载体(例如匙孔血蓝蛋白(KLH)、牛血清清蛋白(BSA)或破伤风类毒素)连接用来免疫接种。
用本领域技术人员已知的方法可以得到肿瘤标记的单克隆抗体(参看,例如,Kohler等,Nature 1975,第256卷:第495页;Coligan等.(eds.),CurrentProtocols in Immunology,第1卷,第2.5.1-2.6.7页(John Wiley & Sons1991);Picksley等,″Production of monoclonal antibodies against proteinsexpressed in E coli″in DNA Cloning 2:Expression Systems,第二版,Glover等.(eds.),第93页(Oxford University Press 1995))。
简要地,单克隆抗体可以通过以下步骤获得:对小鼠注射含有一个或多个肿瘤标记的组合物,移取血清样品以证实抗体产物的存在,摘除脾脏以获得B-淋巴细胞,B-淋巴细胞与骨髓瘤融合产生杂交瘤,克隆杂交瘤,筛选产生针对抗原的抗体的阳性克隆,培养产生针对抗原的抗体的克隆,从杂交瘤培养物中分离抗体。
另外,本发明的抗-肿瘤标记的抗体可以来自人单克隆抗体。从已构建用以产生应答抗原挑战的特异人抗体的转基因小鼠可以获得人单克隆抗体。在这个技术中,将人重链和轻链基因座的元件引如入小鼠品系,此小鼠品系来自含有定向破坏的内源重链和轻链基因座的胚胎干细胞系。转基因小鼠可以合成对人体原特异的人抗体,并且此小鼠可用以产生分泌人抗体的杂交瘤。
从转基因小鼠中获得人抗体的方法由,例如,Green等,Nature Genet.1994 7,13;Lonberg等,Nature 1994,368,856;和Taylor等,Int.Immun.1994,6,579,描述。
通过各种已经建立好的技术可以从杂交瘤培养物中分离并纯化出单克隆抗体。这些分离技术包括用蛋白质-A Sepharose的亲合层析、分子排阻层析和离子交换层析(参见,例如Coligan 2.7.1-2.7.12页和2.9.1-2.9.3页;Baines等,″Purification of Immunoglobulin G(IgG)″in Methods inMolecular Biology,第10卷,第79-104页(The Humana Press,Inc.1992)。
为了特殊用途,可能需要制备抗-肿瘤标记抗体的片段。可以通过,例如,抗体的蛋白质水解来获得这些抗体片段。用传统方法对整个抗体用胃蛋白酶或者木瓜蛋白酶消化可以获得抗体片段。作为说明,抗体的胃蛋白酶酶切可以产生抗体片段从而提供5S片段,F(ab′)2。可以用巯基还原剂进一步切割这个片段以产生3.5S Fab′单价片段。可选择地,使用巯基基团的封闭基团完成切割反应,所述巯基基团由二硫键切割产生。作为选择,用胃蛋白酶酶切直接产生两个单价Fab片段和一个Fc片段。这些方法由,例如,Goldenberg,美国专利号4,331,647,Nisonoff等,Arch Biochem.Biophys.1960,89,230;Porter,Biochem.J.1959,73,119;Edelman等,inMethods in Enzymology第1卷,422页(Academic Press 1967),和Coligan2.8.1-2.8.10页和2.10.-2.10.4页,描述。
只要所得片段可以结合被完整抗体识别的抗原,那么则可以用切割抗体的其他方法,例如重链分离以形成单价轻-重链片段,片段的进一步切割或其他酶学、化学或基因技术。例如,Fv片段含有VH和VL链的结合。这个结合可以是非共价的,如Inbar等.Proc.Natl.Acad.Sci.美国1972,69,2659所述。可选择地,可变链可以通过分子间二硫键连接或者用化学试剂例如戊二醛交联(参见,例如Sandhu,Crit.Rev.Biotech.1992,12,437)。
Fv片段可以含有由肽接头连接起来的VH和VL链。通过构建含有编码VH和VL结构域的DNA序列(由寡核苷酸连接)的结构基因来制备这些单链抗原结合蛋白(scFv)。结构基因插入到随后转入寄主细胞(例如大肠杆菌)的表达载体中。重组宿主细胞合成有接头肽连接两个V结构域的单多肽链。生产scFv的方法由,例如,Whitlow等.Methods:A Companionto Methods in Enzymology 1991 2,97(也参见,Bird等,Science1988,242,423,Ladner等,美国专利号4,946,778),描述。
作为示例,通过将淋巴细胞体外暴露于肿瘤标记,并筛选噬菌体或相似载体内的抗体展示文库(例如,通过使用固定化的或标记的肿瘤标记)。抗体片段的另一个形式是编码单个互补决定区(CDR)的多肽。通过构建编码目的抗体CDR的基因可以获得CDR多肽(“最小识别单位”)。这些基因可以制备,例如,通过聚合酶链式反应从产生抗体的细胞的RNA来合成可变区(参见,例如Larrick等,Methods:A Companion to Methodsin Enzymology 1991,2,106;Courtenay-Luck,″Genetic Manipulation ofMonoclonal Antibodies″in Monoclonal Antibodies:Production,Engineering and Clinical Application,Ritter等(eds.),166页(CambridgeUniversity Press 1995),and Ward等,″Genetic Manipulation andExpression of Antibodies″in Monoclonal Antibodies:Principles andApplications,Birch等,(eds.),137页(Wiley-Liss,Inc.1995))。
可选择地,抗肿瘤标记的抗体可以来自“人源化”单克隆抗体。通过把来自鼠免疫球蛋白重链和轻链可变区的鼠互补决定区转入人可变区,可以生产人源化单克隆抗体。然后,人抗体的典型残基在鼠相应部位的框架区内被替换。来自人源化单克隆抗体的抗体组分的使用排除了与鼠恒定区免疫原性有关的潜在问题。克隆鼠免疫球蛋白可变区的通用技术由,例如,Orlandi等,Proc.Natl.Acad Sci.USA 1989,86,3833描述。生产人源化单克隆抗体的技术由,例如,Jones等,Nature 1986,321,522;Carter等;Proc.Natl.Acad.Sci.美国1992,89,4285;Sandhu,Crit.Rev.Biotech.1992,12,437;Singer等,J.Immun.1993,150,2844;Sudhir(ed.),AntibodyEngineering Protocols(Humana Press,Inc.1995);Kelley,″EngineeringTherapeutic Antibodies″in Protein Engineering:Principles and Practice,Cleland等(eds.),399-434页(John Wiley & Sons,Inc.1996);和Queen等,美国专利号5,693,762(1997),描述。
可选择地,可以对患者使用针对肿瘤标记蛋白的抗体融合蛋白进行治疗以诱发有助于杀伤肿瘤细胞和/或抑制肿瘤细胞生长的反应。
如本文所用,术语“抗体融合蛋白”指基本上由本发明肿瘤标记的抗体或其片段和与免疫球蛋白或其片段直接或者通过接头或间隔物融合的治疗剂组成的融合分子。适合于这些融合蛋白的治疗剂的例子包括免疫调制剂和毒素,例如(但不限于)细胞因子例如IL-1、IL-2、IL-4、IL-6、IL-7、IL-10、IL-13、IFNs、IFNα或CSFs。
通过制备融合蛋白的每个组分并把它们化学缀合,用本领域技术人员已知的方法可以制备融合蛋白。可选择地,用已知的技术可以产生在适当读框内编码融合蛋白的两个组分的多核苷酸,并用如在EP 0659439中描述的方法表达。
在本发明的一个实施方案中,使用含有纯化的肿瘤标记(癌症患者对该标记已出现了抗体)的一个或混合物的免疫原来诱发免疫反应。
在本发明的另一个实施方案中,可以采用针对本发明肿瘤标记的抗体或抗体片段,以诱发有利于杀伤肿瘤细胞和/或抑制肿瘤细胞生长的反应。
本发明的肿瘤标记、其混合物或者抗体、抗体片段或抗体融合蛋白可以对患有RCC或其他疾病的患者直接使用或者用在含有所述化合物和药学上可接受的稀释剂、载体或赋形剂的药物组合中,这些患者的特征在于肿瘤标记片段在细胞表面上的特异呈递。
如本文所用,术语“药学上可接受的载体”是指惰性、无毒的固体或液体填充剂、稀释剂或包裹材料,它们没有对活性化合物或患者产生不利反应。合适的、优选的液体载体是本领域已知的,例如无菌水、盐水、葡萄糖溶液、糖溶液、乙醇、二元醇和油,该油包括石油、动物、植物或合成来源的油,例如花生油、大豆油和矿物油。
根据本发明的制剂可以以单位剂量用药,所述制剂含有传统无毒药学上可接受的载体、稀释剂、添加剂和介质,只要它们是肠胃外用药惯用的即可。
可以用许多方法使用以上的制剂;包括(但不限于)经口、皮内、肌内、腹膜内、静脉和皮下。
术语“肠胃外”在本文中包括皮下、静脉内、关节内和气管内注射及输注技术。其他用药方法例如口服或局部用药是合适的。肠胃外组合物最优选静脉内用药,可以根据已知方法大剂量注射或连续输注。
当将本发明的化合物配制为片剂、胶囊或粉末形式时,可以使用通常的载体和赋形剂例如碳酸镁、碳酸钙、碳酸氢钠、硬脂酸镁、硬脂酸钙、滑石粉、乳糖、微晶纤维素、甲基纤维素、羧甲基纤维素钠淀粉和无水二氧化硅,润滑剂例如水合蓖麻油、硬脂酸镁、十二烷基硫酸钠、糖、果胶、糊精、西黄蓍胶、低熔点蜡、可可油、藻酸盐、明胶、聚乙烯吡咯烷酮、聚乙二醇、季铵化合物等,还可以使用粘合剂例如淀粉、葡萄糖、阿拉伯胶和甘露醇。用本领域已知的方法可以对片剂或胶囊进行包衣。
口服液体制剂可以是水或油混悬液、溶液、乳剂、糖浆或酏剂的形式,也可以制成干燥产物,在使用前用水或其他合适载体复制。这些液体制剂可含有传统的添加剂像悬浮剂、乳化剂、非水载体和防腐剂。
局部用药可以是水或油混悬液、溶液、乳剂、胶凝剂或优选乳剂软膏的形式。
在含有肿瘤标记的组合物和组合中,优选肿瘤标记与合适助剂一起制成的制剂,由此可以增强对蛋白抗原的免疫反应。合适的助剂包括(但不限于)矿物胶,如氢氧化铝、表面活性物质例如溶血卵磷脂、多聚醇、聚阴离子、肽、油乳剂,和可能有用的人佐剂例如BCG(卡介苗)和(短棒状杆菌)。
根据本发明的单位剂量可包括每日所需根据本发明化合物的量,或其多个亚剂量以组成所需的剂量。对于给定患者(哺乳动物,包括人)的最佳治疗可接受剂量或剂量比取决于技术人员所知的各种因素,例如所使用的具体活性化合物的活性、年龄、体重、基本健康状况、性别、饮食、用药的时间和途径、清除率、治疗目的,即是用于治疗还是用于预防以及疾病的性质。
因此,在被治疗患者(体内),组合物和组合中的本发明活性化合物的药物有效日剂量在约0.01到100mg/kg体重之间,优选0.1到10mg/kg体重之间。根据使用形式,单剂量可含有0.01到10mg活性化合物。
本发明的肿瘤标记、抗体和抗体融合蛋白也可以与其他化学治疗剂结合一起使用。根据本发明,可以用于与本发明的化合物结合的化学治疗剂包括有抗瘤作用的试剂,例如,阻止瘤细胞发育、成熟或扩散,直接作用在肿瘤细胞上,例如抑制细胞生长或产生细胞毒效应,非间接通过例如生物反应调节机制起作用。根据本发明的化学治疗试剂优选天然或合成的化学化合物,但不排除生物分子,例如蛋白、抗体、趋化因子、细胞因子、多肽等。大量在商业使用、临床评估、临床前研发中可以得到的化学治疗剂也包括在本发明中。
化学治疗剂的例子包括烷化剂,例如,氮芥、乙烯亚胺化合物、烷基磺酸酯和其他有烷化作用的化合物例如亚硝基脲、顺铂、达卡巴嗪;抗代谢物,例如,叶酸、嘌呤或嘧啶拮抗物;有丝分裂抑制剂,例如,长春花生物碱和鬼臼毒素的衍生物;有细胞毒性的抗生素和喜树碱衍生物。优选的化学治疗试剂或化学疗法包括氨磷汀(羟乙基)、顺铂、达卡巴嗪(DTIC)、放线菌素D、氮芥(氮芥子气)、链佐星、环磷酰胺、卡莫司汀(BCNU)、洛莫司汀(CCNU)、多柔比星、盐酸多柔比星脂质体、吉西他滨(健择)、道诺霉素、道诺霉素脂质体(枸橼酸道诺霉素脂质体)、丙卡巴肼、丝裂霉素、阿糖胞苷、依托泊苷、氨甲喋呤、5-氟尿嘧啶(5-FU)、长春碱、长春新碱、博来霉素、紫杉醇、多西他赛(泰索帝)、阿地白介素、天冬酰胺酶、白消安、卡铂、克拉屈滨、喜树碱、CPT-11、10-羟基-7-乙基喜树碱(SN38)、达卡巴嗪、氟尿苷、氟达拉滨、羟基脲、异环磷酰胺、伊达比星、美钠、α-干扰素、β-干扰素、伊立替康、米托蒽醌、托泊替康、亮丙立德、甲地孕酮、美法仑、巯基嘌呤、普卡霉素、米托坦、培门冬酶、喷司他丁、哌泊溴烷、普卡霉素、链佐星、他莫西芬、替尼泊苷、睾内酯、硫鸟嘌呤、噻替派、尿嘧啶芥子气、长春瑞滨、苯丁酸氮芥及它们的组合。
本发明进一步提供了基于下述发现用免疫组织化学方法鉴定RCC和区别RCC亚类的方法和试剂盒。
根据组织学特征,RCC分为不同的亚类,最常见的为透明细胞、嫌色细胞、嗜色细胞和癌细胞亚类。鉴定RCC不同亚类的方法在Thoenes等(Path.Res.Pract.1986,181,125)和Srkel及van der Berg(World J.Urol.1995,13,153)中已描述。
已经发现,在正常肾和不同RCC亚类中三个选择免疫原性蛋白的表达模式不同。在一系列外科摘除不同亚类RCC病灶和自体正常肾上皮中用免疫组织化学分析了CK8、stathmin和波形蛋白的表达模式。如图4所示,收集管系统的上皮细胞以及近端和远端小管系统上皮细胞显示CK8的细胞膜强烈阳性染色,而所有的正常肾组织上皮细胞显示波形蛋白的阴性染色。与之相反,在36%和72%外科摘除病灶中不同RCC亚类分别显示对CK8和波形蛋白中等强烈阳性染色(图4、表1)。
表1:在不同RCC亚类中CK8和波形蛋白的表达
CK8 波形蛋白 | ||||||||||
+++ | ++ | + | - | RCC亚类 | G | N | +++ | ++ | + | - |
3 | 5 | 7 | 2 | 透明细胞 | 1 | 17 | 11 | 2 | 2 | 2 |
3 | 1 | 7 | 6 | 透明细胞 | 2 | 17 | 14 | 1 | 0 | 2 |
2 | 3 | 9 | 3 | 透明细胞 | 3 | 17 | 15 | 2 | 0 | 0 |
8 | 9 | 23 | 11 | ∑ | 51 | 40 | 5 | 2 | 4 | |
16% | 18% | 45% | 22% | % | 78% | 10% | 4% | 8% | ||
2 | 1 | 1 | 1 | 嫌色细胞 | 1 | 5 | 0 | 1 | 3 | 1 |
2 | 1 | 4 | 1 | 嫌色细胞 | 2 | 8 | 0 | 0 | 0 | 8 |
4 | 2 | 5 | 2 | ∑ | 13 | 0 | 1 | 3 | 9 | |
31% | 15% | 38% | 15% | % | 0% | 8% | 23% | 69% |
在全部不同RCC亚类的64个RCC病灶(透明细胞类:51;嫌色:13)中,用抗-CK8和抗波形蛋白单克隆抗体进行免疫组织化学分析进行(根据Srkel和van der Berg(World J.Urol.1995,13,153)的组织病理学分类)。结果由实施例中打分系统概括(+++=强烈,++=中等,+=弱,-=非常弱或无阳性染色)
在16%和18%的RCC病灶中检测到细胞膜的强烈或中等阳性CK8染色,在45%所分析的肿瘤中显示CK8的弱表达(图4)。在RCC透明和嫌色细胞中发现CK8和波形蛋白表达的不同频率(表1)。78%和10%RCC透明细胞分别显示强烈或中等阳性胞质波形蛋白染色。在4%这个RCC亚类中发现波形蛋白的弱表达。与之相反,在31%和15%所分析的病灶中RCC嫌色亚类显示CK8的强烈或中等阳性染色,而在38%这个RCC亚类中检测到CK8的弱表达。仅有8%和23%的嫌色RCC分别显示中等或弱波形蛋白胞质染色(图4;表1)。观察到的CK8和波形蛋白共表达好像经常发生在RCC中,尤其是其透明细胞亚类。因此,两个蛋白的组合表达可用作RCC透明细胞检测的诊断标记。
RCC病灶和正常肾组织的染色显示不同stathmin表达模式(表2)。虽然抗-stathmin抗体染色少于10%的近端和远端小管系统的上皮细胞,但扁平peritumoral tubules的内皮细胞、炎症细胞和上皮细胞显示强烈阳性胞质染色。与之相反,RCC透明细胞的肿瘤细胞在10%和33%中分别显示仅有中等或弱阳性stathmin染色,而57%的这个RCC亚类完全缺乏stathmin表达(图4;表2)。在60%的所分析病灶中RCC嫌色亚类显示stathmin弱阳性染色,而其余40%是阴性stathmin染色(图4;表2)。
表2:RCC亚类中stathmin表达
Stathmin | ||||||
+++ | ++ | + | - | RCC亚类 | G | n |
0 | 1 | 3 | 3 | 透明细胞 | 1 | 7 |
0 | 1 | 1 | 5 | 透明细胞 | 2 | 7 |
0 | 0 | 3 | 4 | 透明细胞 | 3 | 7 |
0 | 2 | 7 | 12 | ∑ | 21 | |
0% | 10% | 33% | 57% | % | ||
0 | 0 | 3 | 2 | 嫌色细胞 | 1 | 5 |
0 | 0 | 3 | 2 | 嫌色细胞 | 2 | 5 |
0 | 0 | 6 | 4 | ∑ | 10 | |
0% | 0% | 60% | 40% | % |
在全部31RCC中,用抗-atathmin单克隆抗体通过免疫组织化学法分析不同亚类病灶和等级。根据实施例中描述的打分系统进行定量分析。(+++=强烈,++=中等,+=弱,-=非常弱或无阳性染色)。
这些结果显示CK8、波形蛋白和/或stathmin的共表达可用作RCC亚类的诊断标记。
因此本发明提供用抗-CK 8、抗-波形蛋白和/或抗stathmin抗体对肾上皮细胞组织样品进行免疫组织化学染色来鉴定RCC和区别RCC亚类的方法。
原则上,所述方法包括以下步骤:
a)把来自怀疑患有RCC个体的肾上皮组织样品与选自由CK8、抗-波形蛋白和抗-stathmin(第一抗体)组成的组中的至少一个抗体在保证所述抗体与组织样品结合的情况下温育;
b)将第一抗体与含有如上述第一抗体结合亲合识别位点和可检测标记的第二抗体在保证抗体与第一抗体结合的情况下接触;
c)检测第二抗体与第一抗体的结合
d)将步骤C)检测的组织样品与根据步骤a)到c)处理的参考样品对比,此参考样品来自患有RCC个体的透明细胞、嫌色、嗜色或癌细胞亚类。可以使用参考样品的RCC亚类鉴定由,例如,Thoenes等,(Path.Res.Pract.1986,181,125)和Srkel and van der Berg(World J.Urol.1995,13,153)所述。
本发明还提供含有免疫组织化学方法鉴定RCC和区别RCC亚类的组分的试剂盒。
这些组分至少是:
A)抗-CK8、抗-波形蛋白和/或抗-stathmin抗体;
B)直接针对第一抗体的、带有可检测标记的第二抗体。
实施例
实施例1
细胞培养与IFN-γ处理
MZ1257RC和MZ1940RC代表充分定义的、鉴定为肾细胞癌(RCC)透明细胞类型的人源细胞系(Seliger,B.等,Cancer Res.1996,第56卷,第1756-60页),而其相应的正常肾组织MZ2733RC和MZ2733NN近来建立自有初级RCC透明细胞类型的患者。所有的RCC细胞系维持在添加有10%胎小牛血清、2mM谷氨酰胺和100U/ml青霉素/100μg/ml链霉素的DMEM中。
在300U/ml重组IFN-γ(Imukin,Boehringer Ingelheim,Ingelheim,德国)存在下,对MZ1257RC细胞用IFN-γ进行处理48小时。
血清样品:
所有血清样品分离自人静脉血样品,经每个个体同意后收集自诊断为肾细胞癌的患者或来自正常志愿捐献者。
实施例2:
双向凝胶电泳
样品制备:
细胞系扩增到每批细胞数为5×107到1×108,然后用胰酶消化(trypsination)收获。细胞沉淀在磷酸盐缓冲液(PBS)中洗3~4次,之后贮存在无菌低温管中,将干燥细胞沉淀以5×106或1×107细胞/管的诸份储存在液氮中待用。在裂解缓冲液(7M尿素、2M硫脲,0.2M丙磺酸二甲基苯甲铵(NDSB)、1%二硫苏糖醇(DTT)、4%3-[(3-cholamidopropyl)二甲基-ammino]-1-丙-磺酸盐(CHAPS)、0.5%pharmalytes和微量染料溴酚蓝)中重悬细胞沉淀。裂解物用超声处理(超声波浴中3×4分),然后用在微量离心机中离心澄清(90分钟,15℃,13,000rpm)。
蛋白定量
根据Ramagli和Rodriguez描述的实验流程进行蛋白定量,该流程允许即使在大量尿素存在下也能使用原始Bradford方法。简略地,澄清的裂解物的2.5μl~10μl等分试样调整到终体积为10μl,一式两份,然后每个样品与10μl 0.1M HCl混合。随后每个样品中加入80μl ddH2O,样品再次混合。每个复份样品(100μl)中加入3.5ml 1∶3的稀染料试剂混合液(Bio-Rad蛋白分析染料试剂浓缩液),用轻轻的涡旋来混合混合物。5分钟后,用试剂空白(10μl裂解缓冲液,如上述处理)在塑料样品杯中测595nm的光吸收作为对照。
上样/等电聚焦和带(strip)平衡
用新裂解缓冲液调整每个裂解液的终体积为350μl,其中340μl转入IPGphor strip固定器(Amersham Pharmacia Biotech)。ImmobilineDryStrip(pH 3-10,NL,18cm,Amersham Pharmacia Biotech)再水合与样品上样是在一个步骤中进行的。把DryStrips加到裂解物90分钟后,用400μl Immobiline DryStrip覆盖液体覆盖吸收样品的带。于20℃在IPGphor单元(Amersham Pharmacia Biotech)上进行等电聚集,用以下参数,于0V再水合2小时,30V 10小时,500V 1小时,1000V 1小时,5000V 1小时,8000V 4~5小时,如果目标蛋白(低分子量组分)是在16%T/2.5%C SDS-PAGE凝胶(最后一步是8000v4小时)的第二向分离则加至36.000~38.000伏小时或者如果样品裂解物在7%T/2.5%CSDS-PAGE凝胶上分离高分子量组分(最后一步是8000V 5小时)则加至44.000~46.000伏小时。所有步骤按步并在一固定模式下进行。聚焦的带贮存在-80℃或者直接进行带平衡步骤,它的实施是通过在添加有1.5%DTT的10ml平衡缓冲液(50mM Tris-HCl pH8.8,6M尿素,30%甘油,2%SDS)中温育15分钟,之后在添加有4.8%jodacetamide的10ml平衡缓冲液中温育15分钟。
第二向SDS-PAGE:
用Hoefer ISO-DALT系统(Amersham Pharmacia Biotech)进行SDS-PAGE分离,并在聚丙烯酰胺/哌嗪二丙烯酰胺(PDA)PAGE凝胶上电泳。凝胶混合物含有375mM Tris//HCl pH8.8,5mMNa2S2O4和4%甘油但不含十二烷基磺酸钠(SDS)。新平衡好的Immobiline DryStrip转到完全漂洗PAGE凝胶表面。通过把带包埋在含有微量标记染料(7%T/2.5%C凝胶用溴酚蓝,16%T/2.5%C凝胶用溴酚蓝加二甲苯青FF)的1%软熔化琼脂糖中使带固定。在SDS-PAGE电泳缓冲液(2.5mM Tris,192mM甘氨酸,0.1%SDS)中严格温度控制(<20℃)下电泳凝胶直到染料前端到达凝胶的末端(16%T/2.5%C凝胶是直到二甲苯青FF染料前端跑出凝胶)。在低压(恒压50V 1小时)下进行从等电聚焦(IEF)带到凝胶的样品初始转移,而分离则是在恒定高压下(100~140V)进行。
凝胶染色:
用胶态考马斯蓝对凝胶染色。所有凝胶在传统扫描仪(HewlettPackard ScanJet 6100C)上分辨率为600dpi下扫描,并存为TIFF图片。用作蛋白质印迹分析的凝胶或者含有对其用质谱分析的蛋白斑点的凝胶,只用胶态考马斯蓝染色溶液(10%硫酸铵、2%磷酸、0.1%考马斯亮蓝G-250、20%甲醇)染色,因此跳过了初始固定步骤及其后的在H2O(dd)中漂洗使之脱色。
实施例3
免疫印迹:
为了免疫印迹分析,胶态考马斯蓝预染的2-D PAGE凝胶用ISO-DALT槽印迹系统(Amersham Pharmacia Biotech)印在ImmobilonP膜上,用添加20%甲醇的SDS-PAGE电泳缓冲液作为转移缓冲液,并且每次转移用500伏小时。随后印迹膜在封闭溶液(140mM NaCl,10mMTris/HCl pH7.4,0.4%Tween20,5%低脂奶粉和10%马血清)中温育1小时,在Tris缓冲盐(TBS:140mM NaCl、10mM Tris/HCl pH7.4)中漂洗两次,然后与在抗体温育缓冲液(TBS,0.1%Tween20,2%低脂奶粉)中1∶20稀释的对照或患者血清4℃温育过夜。然后在TBS、0.4%Tween20中洗膜3次(每次10分钟),在室温下与辣根过氧化物酶(HRP)-缀合第二单克隆抗体溶液(20ml/膜,兔抗人IgG,在抗体温育缓冲液中以1∶1000稀释)温育0.5~1小时。用TBS、0.4%Tween20洗3次之后,根据制造商的说明用化学发光检测试剂盒(Lumi-Light蛋白质印迹底物,RocheMolecular Biochemicals,Mannheim)进行斑点显色,并记录在科学图象胶片(Kodak X-Omat Blue XB-1)上。把图象胶片与相应的凝胶印重叠预形成与点匹配的信号。
实施例4:
质谱
为了质谱分析,从胶态考马斯蓝染色的复份胶上切下免疫染色蛋白斑点。每个样品转入无菌微量反应管中,30℃在50mM NH4CO3/乙腈(60%/40%)中温育凝胶切片30分钟,随后取出并丢弃产生的上清液。然后凝胶切片真空干燥,在-80℃贮存直至进一步使用。为了凝胶消化,每个样品在25~40μl含有0.1μg/ml修饰的胰蛋白酶(Promega,Madison,WI,美国)的50mM NH4HCO3中浸泡1小时。收集上清,加入25μl新NH4HCO3,然后样品37℃温育过夜。通过在抽提缓冲液(H2O/三氟乙酸(TFA);50%/50%,v/v)中温育20分钟两次,然后在含有乙腈/TFA;50%/50%,v/v的缓冲液中温育20分钟两次,完成肽抽提。浓缩来自每个样品的上清至终体积约25~50μl/样品,然后根据制造商的操作流程用ZipTips(Millipore)脱盐。产生的洗脱物1μl等分试样加在MALDI基质上,用Perseptive Biosystems Voyager RP-DE仪器(Perseptive Biosystems,Framington,MA)直接进行肽指纹分析。
实施例5
用作免疫组织化学的患者和组织样品
为了免疫组织化学分析,RCC和相应正常肾上皮的外科切除组织样品是随机来自经历根部(radical)肾切除的患者的。根据Thoenes和其同事(Thoenes等,Path.Res.Pract.1986,181,125;Strkel和van der Berg,World J.Urol.1995,13,153)提出的标准进行每个肿瘤的组织病理学分类。这些数据包括性别、疾病的阶段、肿瘤入侵和根据TNM(肿瘤结转移)系统的淋巴结介入。总共,在切除时不但收集64个自体肾样品而且收集包括51个透明细胞癌和13个嫌色癌的64个初级肾肿瘤。组织样品用福尔马林固定并用石蜡包埋。
免疫组织化学
用单克隆抗体抗-人细胞角蛋白8(clone βH11,DAKO,Hamburg,德国,1∶25稀释),抗波形蛋白单克隆抗体(clone V9,DAKO,1∶40稀释)和抗stathmin(B37545,Calbiochem,美国,1∶500稀释)进行免疫组织化学染色。为了抗原提取,在微波炉中将连续切片在柠檬酸缓冲液中分别温育8和6分钟,之后有一个Tris缓冲盐洗涤步骤,并再与正常猪血清(1∶10稀释)温育10分钟。室温下玻片与初级抗体温育1小时。如所述(DAKODiagnostika GmbH,Hamburg,德国)用LASB(标记链酶抗生物素生物素)-过氧化酶试剂盒和AEC(氨基-9-乙基咔唑)进行检测。省略初级抗体作阴性对照。
根据以下得分进行每个肿瘤定量分析:
%阳性肿瘤细胞/样品 | 得分 | 分类 |
<5 | - | 阴性 |
>5和<25 | + | 弱阳性 |
>26和<50 | ++ | 中等阳性 |
>50 | +++ | 强烈阳性 |
Claims (18)
1、至少一种选自下列的蛋白作为肿瘤标记的用途:β-肌动蛋白、γ-肌动蛋白、α-微管蛋白、细胞角蛋白、细胞角蛋白8、细胞骨架原肌球蛋白、F-肌动蛋白加帽蛋白、hsp27、hsp60、hsp70、hsp90、grp78(BIP)、gp96、谷光甘肽-S-转移酶、谷光甘肽合成酶、超氧化物歧化酶、硫氧还蛋白过氧化物酶、PA28α、遍在蛋白巯基酯酶、丙糖磷酸异构酶、醛糖还原酶、烯酰--CoA水合酶、α-烯醇化酶、膜联蛋白II、IV和V、stathmin、烟酰胺-N-甲基转移酶、B23/核仁磷酸蛋白和波形蛋白。
2、权利要求1中限定的蛋白和/或β-微管蛋白作为肾细胞癌的肿瘤标记的用途。
3、用来诊断和预测个体内癌症的体外方法,该方法包括用免疫测定的方法检测抗体的存在,此抗体来自所述个体的血清样品并且是如权利要求1或2中限定的、在血清中存在的肿瘤标记蛋白的抗体。
4、权利要求3的方法,其中免疫测定包括以下步骤:
(a)在膜或底物上固定根据权利要求1的蛋白;
(b)使所述膜或底物与个体的血清样品接触,和;
(c)检测个体血清样品中肿瘤标记特异抗体的存在。
5、权利要求4的方法,其中用对所述血清抗体的外源标记的抗体来检测血清样品中的肿瘤标记特异抗体。
6、根据权利要求3到5中任一项的方法,其中所述个体患有肾细胞癌。
7、适合于实施权利要求3到6中任一项的方法的诊断试剂盒,该试剂盒含有至少一种或多种如权利要求1或2中限定的肿瘤标记。
8、至少一种如权利要求1或2中限定的肿瘤标记在制备刺激个体的免疫反应的药物中的用途。
9、至少一种与至少一种如权利要求1或2限定的肿瘤标记发生免疫特异结合的抗体或其片段在制备有利于杀伤肿瘤细胞和/或抑制肿瘤细胞生长的反应的药物中的用途。
10、权利要求9的用法,其中所述抗体或其片段是抗体融合蛋白。
11、药物组合物,该组合物含有至少一种如权利要求1或2中限定的肿瘤标记和任选的药学上可接受的载体、稀释剂或赋形剂。
12、药物组合物,该组合物含有至少一种与至少一种如权利要求1或2中限定的肿瘤标记免疫特异结合的抗体或其片段和任选的药学上可接受的载体、稀释剂或赋形剂。
13、权利要求12的药物组合物,其中所述抗体或其片段是抗体融合蛋白。
14、根据权利要求11到13中任一项的药物组合物,该组合物还含有化疗剂。
15、药物包装,该包装在第一容器中含有根据权利要求11到13中任一项的药物组合物以及在第二容器中含有包括化疗剂的药物组合物,这两种组合物同时给药或交替给药。
16、至少一种选自下列的抗体在用免疫组织化学方法鉴定RCC和区别RCC亚类中的用途:抗--细胞角蛋白8、抗-波形蛋白和抗-stathmin抗体。
17、鉴定RCC和区别RCC亚类的方法,该方法包括以下步骤:
a)把来自怀疑患有RCC个体的肾上皮组织样品与选自抗--细胞角蛋白8、抗-波形蛋白和抗-stathmin(第一抗体)的至少一种抗体在保证所述抗体与组织样品结合的情况下温育;
b)将第一抗体与含有对上述第一抗体具有结合亲合性识别位点并具有可检测标记的第二抗体在保证第二抗体与第一抗体结合的情况下接触;
c)检测第二抗体与第一抗体的结合;和
d)将步骤c)检测的组织样品与来自患有RCC的个体的透明细胞、嫌色细胞、嗜色细胞或癌细胞亚类的参考样品对比。
18、鉴定RCC和区别RCC亚类的试剂盒,该试剂盒包括:
a)至少一种根据权利要求16的抗体(第一抗体),和
b)至少一种对抗第一抗体的载有可检测标记的第二抗体。
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BR0208603A (pt) | 2004-03-02 |
HUP0303749A2 (hu) | 2004-03-01 |
PL363009A1 (en) | 2004-11-15 |
SK12872003A3 (sk) | 2004-02-03 |
MXPA03009018A (es) | 2004-02-12 |
RU2003130645A (ru) | 2005-04-10 |
EP1373900A2 (en) | 2004-01-02 |
HUP0303749A3 (en) | 2005-09-28 |
ZA200308487B (en) | 2005-01-31 |
KR20030086345A (ko) | 2003-11-07 |
WO2002082076A3 (en) | 2003-09-04 |
WO2002082076A2 (en) | 2002-10-17 |
JP2004531713A (ja) | 2004-10-14 |
CA2442957A1 (en) | 2002-10-17 |
CZ20032787A3 (en) | 2004-03-17 |
US20040096916A1 (en) | 2004-05-20 |
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