CN102372773A - 人膀胱癌肿瘤标志物及其抗体和应用 - Google Patents

人膀胱癌肿瘤标志物及其抗体和应用 Download PDF

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CN102372773A
CN102372773A CN2010102513846A CN201010251384A CN102372773A CN 102372773 A CN102372773 A CN 102372773A CN 2010102513846 A CN2010102513846 A CN 2010102513846A CN 201010251384 A CN201010251384 A CN 201010251384A CN 102372773 A CN102372773 A CN 102372773A
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bladder cancer
antibody
bcmab1
monoclonal antibody
human bladder
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CN102372773B (zh
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李翀
范祖森
张红莲
戴中华
唐海东
陈俊
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Abstract

本发明提供了一种新的人膀胱癌肿瘤标记物——异常糖基化的整合素AG-α3β1，和产生抗该肿瘤标记物的单克隆抗体的杂交瘤细胞，及其分泌的单克隆抗体BCMab1，BCMab1抗体识别的抗原表位为[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc。该单克隆抗体与人膀胱癌细胞系T24及人膀胱癌组织呈强阳性反应，而与人正常膀胱组织和其它非膀胱癌细胞无交叉反应。同时，该单克隆抗体在体外细胞培养和动物肿瘤模型中具有抑制膀胱癌细胞系T24增殖的功能。本发明还提供了包含单克隆抗体BCMab1体外诊断试剂盒和使用单克隆抗体BCMab1检测尿脱落细胞中肿瘤标记物含量的方法。

Description

人膀胱癌肿瘤标志物及其抗体和应用
技术领域
本发明属于肿瘤免疫学领域。具体地说本发明涉及一种新的人膀胱癌肿瘤标志物AG-α3β1及其制备(AG：异常糖基化(AberrantGlycosylation))，以及抗AG-α3β1的单克隆抗体BCMab1。细胞和组织学水平证明：AG-α3β1只表达于人膀胱肿瘤细胞膜上，BCMab1抗体不仅能够特异性识别AG-α3β1，而且在体外细胞水平和体内动物模型中能有效地抑制人膀胱肿瘤细胞的增殖。本发明还涉及一种竞争性ELISA检测人膀胱癌的方法。这种检测方法中的固定相抗原为本发明中的人膀胱癌肿瘤标志物AG-α3β1，检测抗体是本发明中的抗人膀胱癌整合素α3β1抗体BCMab1。
背景技术
膀胱癌是泌尿系统中最常见的肿瘤。尽管采用多种措施进行治疗，但仍有的患者要发生一次或多次复发，
Figure BSA00000226156300012
的患者发展成更高级别的肿瘤或发生转移，而且这些治疗方法还有明显的毒副作用。
膀胱癌在肿瘤切除后的预防复发是临床的重要课题。目前用于膀胱灌注预防复发的药物主要分以下几类：(1)抗肿瘤化疗药物，如丝裂霉素C等；(2)免疫增强剂，如卡介苗(BCG)等。(3)细胞因子，如干扰素等。以上几类药物可以单独使用或联合使用，虽在降低膀胱癌术后复发率上取得一些效果，但因特异性差、肿瘤多耐药性(MDR)等问题的存在，总体疗效并不理想，高达
Figure BSA00000226156300013
的患者要发生一次或多次复发，
Figure BSA00000226156300014
的患者发展成更高级别的肿瘤或发生转移。且以上各类药物分子量小，不但可非特异性地作用于全膀胱及尿道，还可经膀胱及尿道粘膜吸收，因而容易产生局部及全身的毒副作用。以疗效较为肯定的丝裂霉素C及卡介苗为例：使用卡介苗的患者90％有BCG膀胱炎，其它的副作用有血尿、皮疹、发热、关节炎、尿道狭窄等，还有少见而严重的肝炎及肺炎、致命性败血症等。丝裂霉素C的副作用相对较少，但仍有
Figure BSA00000226156300021
的化学性膀胱炎和过敏性反应，还可见尿道狭窄、骨髓抑制、膀胱壁钙化等副作用。
整合素(integrin)是一组二价阳离子依赖的细胞表面受体，其主要功能是与相应配体结合介导细胞与基质、细胞与细胞的粘附，进而影响细胞形态、基因表达与调控、细胞增殖分化、凋亡及肿瘤细胞的迁移、浸润和转移等。整合素α3亚单位(CD49c)可与β1亚单位(CD29)结合形成整合素α3β1，其配体为层粘连蛋白(LN)和Ⅳ型胶原蛋白(CollagenⅣ)。在上皮组织中，整合素α3β1主要参与细胞与上皮细胞基底膜的粘连，可调节信号转导，从而影响细胞的生物学特性。它在肿瘤细胞表面的异常修饰，被认为会导致恶性度的改变。
抗肿瘤靶向药物是一种具有特异性识别、杀伤肿瘤细胞的生物或化学分子(如单克隆抗体)。膀胱癌是一种人体腔内肿瘤，相当于一个人体的“肉试管”，而导向药物在体外对靶细胞具有高特异和强杀伤作用。寻找一种疗效好、副作用少的新的抗人膀胱癌的导向药物，在膀胱癌的治疗上具有重要意义。
在膀胱肿瘤的筛查中，膀胱癌的早期发现和正确的预后估计对临床治疗显得极为重要。近年新用于临床的分子标记，如核基质蛋白、膀胱肿瘤抗原和卫星不稳定性等，也同样受限于灵敏度和特异性低的不足。尿脱落细胞学分析和膀胱镜检查仍然是膀胱癌诊断和监测的金标准。所以，寻求有效、便利的膀胱癌的诊断方法是非常必要的。
发明内容
本发明利用人膀胱癌细胞系T24免疫小鼠，获得杂交瘤细胞。用ELISA的方法筛选到能与T24细胞高度特异结合的抗体BCMab1。免疫组织化学和免疫荧光证实，BCMab1抗体与人膀胱癌细胞系T24及人膀胱癌组织呈强阳性反应，与人正常膀胱组织和其它非膀胱癌细胞无交叉反应。
本发明利用BCMab1抗体采用免疫亲和层析的方法捕获该抗体识别的抗原，经过质谱分析和糖芯片鉴定为异常糖基化修饰的整合素α3β1，命名为AG-α3β1，其表位为糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc。用BCMab1抗体证实，AG-α3β1只表达于人膀胱肿瘤细胞上，并与膀胱癌的肿瘤分期和病理分级呈正相关。AG-α3β1可作为一种新的人膀胱癌肿瘤标记物。
本发明利用BCMab1抗体，处理体外培养的T24细胞和裸鼠移植瘤模型，发现BCMab1抗体能有效地抑制膀胱癌细胞系T24的增殖。BCMab1抗体可作为抗人膀胱癌的靶向药物。
本发明利用BCMab1抗体和AG-α3β1抗原制备成诊断试剂，即将人膀胱癌肿瘤标志物AG-α3β1作为固定相，与待测样品共同竞争AbBC1抗体，检测人尿液中的膀胱肿瘤细胞。
本发明的创新点在于：(1)研制了一种抗人膀胱癌的鼠单克隆抗体BCMab1，该抗体不仅能特异性地结合人膀胱癌组织，而且在体内或体外能有效抑制膀胱癌细胞系T24的增殖；(2)揭示了这种抗体识别的抗原表位AG-α3β1，为一种新的人膀胱癌肿瘤标记物，并证实了该表位只表达于人膀胱肿瘤细胞上，并与膀胱癌的肿瘤分期和病理分级呈正相关；(3)开发了一种高灵敏度的竞争ELISA方法用于人膀胱癌的检测。
发明详述
本发明提供了一种新的人膀胱癌肿瘤标记物——异常糖基化的整合素AG-α3β1，和产生抗该肿瘤标记物的单克隆抗体的杂交瘤细胞，及其分泌的单克隆抗体BCMab1，BCMab1抗体识别的抗原表位为[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc。该单克隆抗体与人膀胱癌细胞系T24及人膀胱癌组织呈强阳性反应，而与人正常膀胱组织和其它非膀胱癌细胞无交叉反应。同时，该单克隆抗体在体外细胞培养和动物肿瘤模型中具有抑制膀胱癌细胞系T24增殖的功能。本发明还提供了包含单克隆抗体BCMab1体外诊断试剂盒和使用单克隆抗体BCMab1检测尿脱落细胞中肿瘤标记物含量的方法。
更具体地，本发明的一个目的是提供一种人膀胱癌肿瘤标志物AG-α3β1，其为异常糖基化的整合素α3β1，其特征在于带有作为抗原表位的糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc。糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc的结构式如下：
Figure BSA00000226156300041
在本发明的一个优选实施方案中，所述整合素α3β1的α3亚单位的氨基酸序列如SEQ ID No：1所示，所述整合素α3β1的β1亚单位的氨基酸序列如SEQ ID No：2所示，优选所述异常糖基化是处在α3亚单位的第740位的氨基酸T(苏氨酸)上。
本发明的另一个目的是提供针对本发明所述的人膀胱癌肿瘤标志物AG-α3β1的抗体，其能够特异性识别所述作为抗原表位的糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc，所述抗体为多克隆抗体或单克隆抗体，优选单克隆抗体。
在一个优选实施方案中，所述的抗体是抗人膀胱癌整合素α3β1的单克隆抗体BCMab1，该单克隆抗体由保藏号为CGMCC No.3845的杂交瘤细胞株分泌。
本发明的另一个目的是提供一种用于治疗人膀胱癌的药物组合物，其包含本发明上述的抗体。
在一个优选实施方案中，所述的药物组合物还包含抗肿瘤化疗药物，如丝裂霉素C；免疫增强剂，如卡介苗(BCG)；和/或细胞因子，如干扰素。
本发明的另一个目的是提供一种缀合物，其中所述抗人膀胱癌整合素α3β1的抗体BCMab1与选自以下组成的组的物质缀合：生物标记物、抗肿瘤药物、毒素和放射性活性剂。
本发明的另一个目的是提供一种用于检测或治疗人膀胱癌的试剂盒，其包含本发明上述的抗体。
在一个优选实施方案中，在所述的试剂盒中，所述检测是通过酶联免疫吸附法进行，优选所述酶联免疫吸附法是竞争酶联免疫吸附法，其中将本发明上述的人膀胱癌肿瘤标志物AG-α3β1作为固定相，与待测样品共同竞争本发明上述的抗体。在一个优选实施方案中，所述待测样品是含尿脱落细胞的人尿液。
本发明还有的另一个目的是提供分泌抗人膀胱癌整合素α3β1的抗体BCMab1的杂交瘤细胞株，其保藏号为CGMCC No.3845。
附图说明
图1.BCMab1单抗对人膀胱癌组织切片的免疫组化染色；
图2.BCMab1单抗对人正常膀胱粘膜组织切片的免疫组化染色；
图3.过碘化氧化实验(过碘酸氧化实验原理：过碘酸在酸性条件下能氧化糖蛋白中糖的羟基而不改变多肽链结构))。在图3中，膀胱癌细胞T24经过碘酸处理，糖抗原结构被破坏，BCMab1抗体无法识别，没有出现棕色的阳性反应。抗-MUC1抗体(商购自Novocastra Laboratories Ltd，UK)是已知的只结合糖结构的抗体，在这里作为对照。抗-CK20抗体(Novocastra Laboratories Ltd，UK)是已知的只结合蛋白多肽的抗体，在这里也作为对照；
图4.过碘化氧化实验(斑点杂交实验，结果与图3类似)。将膀胱癌细胞T24裂解后，点在膜上，经过碘酸处理，糖抗原结构被破坏，BCMab1抗体无法识别，没有出现阳性反应；
图5.糖芯片检测结果；
图6.BCMab1抗体抑制肿瘤细胞的增殖；
图7.整合素α3亚单位的氨基酸序列；和
图8.整合素β1亚单位的氨基酸序列。
具体实施方式
下文将参考实施例详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例1：BCMab1单克隆抗体的制备和纯化
(1)杂交瘤的制备
以人膀胱癌细胞系T24(ATCC：HTB-4)免疫Balb/C小鼠(商购自北京维通利华实验动物技术有限公司)，以1×107个细胞/ml PBS的剂量进行腹腔注射。2周后对小鼠进行再次免疫，注射体积和方法不变。待小鼠血清效价达到要求后准备进行细胞融合，融合前三天对小鼠进行加强免疫。在免疫小鼠的同时准备小鼠骨髓瘤细胞Sp2/0(ATCC CRL-1772)。
将致敏的B淋巴细胞与骨髓瘤细胞融合(《实用免疫学》，杨廷彬主编，长春出版社，1994年12月出版)，用HAT培养基(购于Invitrogen公司，HAT系次黄嘌呤(hypoxantin)、氨基蝶呤(aminopterin)和胸腺嘧啶脱氧核苷(thymidin)三种物质各英文首字之缀列，HAT培养基也就是指含有这三种物质的细胞培养基)进行选择性培养(以小鼠腹腔巨噬细胞为饲养细胞)。
接着，用ELISA方法检测杂交瘤细胞培养上清：以T24细胞(1×105个/ml)包被96孔板，每孔100μl，37℃培养过夜。细胞贴壁后，加4％多聚甲醛室温固定10分钟，洗涤3次，加待检上清100μl，37℃孵育1小时。洗涤3次，加酶标二抗(抗小鼠IgG-HRP)(商购自北京中杉金桥生物技术有限公司)，37℃孵育1小时。洗涤3次，加TMB(北京中杉金桥生物技术有限公司)50μl显色，室温静置5分钟，加终止液50μl。结果用酶标仪测定波长450nm的OD值，OD值高于阴性对照2倍以上者可视为阳性。
然后，将选出的阳性杂交瘤细胞克隆化培养(有限稀释法，以小鼠腹腔巨噬细胞为饲养细胞)。经过2-3轮克隆化培养，获得稳定的能够产生高效价单抗的杂交瘤细胞克隆。将杂交瘤细胞克隆扩大培养，并冻存保种。
本发明中的一种阳性杂交瘤细胞为抗人膀胱癌单克隆抗体杂交瘤细胞株，该杂交瘤细胞株于2010年5月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC，中国，北京)，保藏号为CGMCC No.3845。
(2)BCMab1单抗的制备和纯化
将上述杂交瘤细胞BCMab1接种至Balb/C小鼠腹腔，制备腹水，再从腹水中提取单抗。单抗BCMab1的纯化：采用Protein G亲和层析法。首先制备Protein G亲和层析柱(购于“GE”公司)，用PBS(磷酸盐缓冲液)平衡柱子后，取含BCMab1单抗的腹水过柱，然后用PBS洗柱子，至OD值接近于零，以0.2M的甘氨酸-HCl溶液(pH 2.8)洗脱，收集洗脱液，测定各收集管的OD值，保留峰值区的洗脱液，洗脱液经透析浓缩后20℃冻存。
实施例2：BCMab1单抗的鉴定
在实施例1中制备的BCMab1单抗，按常规方法对人膀胱癌组织切片(获自北京大学第三医院)进行免疫组化染色，结果如图1、2所示。结果表明，人膀胱癌组织经BCMab1单抗、二抗(抗小鼠IgG-HRP)及DAB底物(北京中杉金桥生物技术有限公司)染色后呈阳性反应，而人正常膀胱组织经BCMab1单抗、二抗及底物染色后呈阴性反应。
用BCMab1单抗，按常规方法对膀胱癌细胞T24和其它细胞进行流式细胞仪检测，以及用免疫组化检测人正常组织。结果如表1所示。结果表明，BCMab1单抗与T24细胞呈强阳性反应，与其它非膀胱癌细胞无交叉反应，与人正常组织无交叉反应。
表1
流式细胞术与免疫组化检测抗人膀胱癌单抗BCMab1对多种细胞与组织的免疫反应
Figure BSA00000226156300071
实施例3：AG-α3β1抗原的制备
人膀胱癌细胞系T24培养于含10％胎牛血清的RPMI-1640培养基中(购于Invitrogen公司)，1×108个T24细胞用0.25％胰酶消化后，PBS洗涤3次，加入100μg BCMab1单抗，4℃孵育2小时，PBS洗涤3次，用1ml三去污裂解液(50mM Tris-HCl，pH 8.0；150mM NaCl；0.02％叠氮钠；0.1％SDS；100μg/ml PMSF；1μg/ml Aprotinin；1％NP-40；0.5％脱氧胆酸钠)裂解30分钟，12,000g离心10分钟，取上清，过Protein G亲和层析柱。然后用PBS洗柱子，至OD值接近于零，以0.2M的甘氨酸-HCl溶液(pH2.8)洗脱，收集洗脱液，测定各收集管的OD值，保留峰值区的洗脱液，进行质谱分析，证实为人整合素α3β1(见表2)。用过碘酸氧化实验(DaigoTsubokawa，Yukinobu Goso，Akira Sawaguchi，et al.A monoclonal antibody，PGM34，against 6-sulfated blood-group H type 2antigen，on the carbohydratemoiety of mucin.FEBS J.2007Apr；274(7)：1833-48)，结合BCMab1抗体的糖芯片(美国，The Consortium for Functional Glycomics)检测数据，BCMab1抗体识别的表位是人整合素α3β1上的糖链，该表位是[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc(见图3、4和5)，并且该糖链是处在α3亚单位的第740位的氨基酸T(苏氨酸)上(STSS中的T上)。由于BCMab1抗体只特异性地结合人膀胱癌组织，表明该抗原为异常糖基化修饰的整合素α3β1(AG-α3β1)，其表位[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc只表达于膀胱癌组织细胞。经鉴定，[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc的结构式如下：
Figure BSA00000226156300081
表2
BCMab1抗原的质谱鉴定
实施例4：BCMab1抗体抑制肿瘤细胞的增殖
将T24细胞按每孔1×103个细胞的密度接种于96孔板中，常规培养至70-80％的密度，用无血清培养基饥饿24小时。加入100μg/mL的BCMab1抗体，恢复血清继续培养48小时，同时以同型的小鼠IgG作为对照。细胞掺入[3H]-TdR(中国原子能科学研究院同位素研究所合成)孵育4小时，用液闪仪LKB1219(瑞典LKB公司)计数标定[3H]-TdR的量，结果见图6，与对照组相比，BCMab1抗体处理组体显著地抑制了肿瘤细胞的增殖。
实施例5：膀胱癌免疫诊断试剂的制备
利用本发明中所述AG-α3β1抗原和BCMab1抗体，本发明开发出了一种高灵敏度的竞争ELISA方法，检测人尿液中膀胱肿瘤。主要方法是用AG-α3β1抗原作为固定相和标准品，BCMab1抗体作为检测抗体，结合辣根过氧化酶(HRP)标记的山羊抗小鼠IgG的二抗，检测正常人和膀胱癌病人尿液中的膀胱癌AG-α3β1抗原。实验证实本发明的膀胱癌免疫诊断试剂与已有技术相比具有以下积极效果：(1)非损伤性，直接检测尿液体，避免了膀胱镜检查给病人带来的不便与痛苦。(2)灵敏度高，特异性强，远高于目前的膀胱肿瘤标记物(如：核基质蛋白、细胞角蛋白、透明质酸等)的检测和尿脱落细胞的病理学检查。(3)特异性强、灵敏度高，采用竞争ELISA法检测细胞中的膀胱癌抗原，避免了细胞病理学方法本身无法克服的人为性较高和特异性及灵敏度较低的缺点。(4)方便快捷，适合于早期膀胱癌患者的筛查和肿瘤复发的检测。
具体实验方法如下：
1)样品来源及临床资料：健康志愿者和膀胱癌患者的尿液均来自北京大学第三医院。
2)具体步骤：
i)ELISA板包被抗原：将AG-α3β1抗原用PBS稀释至1μg/mL，50μg/孔4℃包被平板过夜。
ii)封闭非特异结合位点：200μg/孔2％的BSA/PBS，37度孵育2小时。
iii)样品孵育：待测样品为新鲜尿液，上样量50μL，健康志愿者的尿液取代样品作为阴性对照。同时每孔加入1μg/mL的BCMab1抗体50μL，每个样品设两次重复，37度孵育1小时。
iv)PBST洗涤5次。
v)加入辣根过氧化物酶(HRP)标记的山羊抗小鼠IgG抗体(Sigma公司)，100μL/孔，37℃孵育1小时。
vi)PBST洗涤5次。
vii)加入底物(四甲基联苯胺，H2O2)100μl/孔，室温避光孵育10-20分钟；加入2M H2SO450μL中止反应；在酶标仪上测定OD值(450nm)。
3)结果分析：根据阴性OD值/OD临界值≈2.1，得到OD临界值≈阴性OD值/2.1，根据OD临界值，可判断其灵敏度和特异性。
Figure BSA00000226156300111
临床灵敏度可用来衡量某种试验检测出有病者的能力，灵敏度是将实际有病的人正确地判定为真阳性的比例。
本实验灵敏度＝82/(82+18)×％＝82％。
临床特异度是衡量试验正确地判定无病者的能力，特异度是将实际无病的人正确地判定为真阴性的比例。
本实验特异度＝93/(7+93)×％＝93％。
尽管本发明的具体实施例已经描述如上，但是可以知道本发明可以进行除了上述说明以外的实践。本发明的保护范围不受说明书的限制。
Figure ISA00000226156500011
Figure ISA00000226156500021
Figure ISA00000226156500031
Figure ISA00000226156500041
Figure ISA00000226156500051
Figure ISA00000226156500061
Figure ISA00000226156500071
Figure ISA00000226156500081
Figure ISA00000226156500091

Claims (10)

1.一种人膀胱癌肿瘤标志物AG-α3β1，其为异常糖基化的整合素α3β1，所述异常糖基化的整合素α3β1的特征在于整合素α3β1带有作为抗原表位的糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc，
其中糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc的结构式如下：
2.根据权利要求1所述的人膀胱癌肿瘤标志物AG-α3β1，其中所述整合素α3β1的α3亚单位的氨基酸序列如SEQ ID No：1所示，所述整合素α3β1的β1亚单位的氨基酸序列如SEQ ID No：2所示，优选所述异常糖基化是处在α3亚单位的第740位的氨基酸T(苏氨酸)上。
3.针对根据权利要求1或2所述的人膀胱癌肿瘤标志物AG-α3β1的抗体，其能够特异性识别所述作为抗原表位的糖结构[3OSO3]Galb1-4(Fuca1-3)[6OSO3]GlcNAc，所述抗体为多克隆抗体或单克隆抗体，优选单克隆抗体。
4.根据权利要求3所述的抗体，其为抗人膀胱癌整合素α3β1的单克隆抗体BCMab1，该单克隆抗体由保藏号为CGMCC No.3845的杂交瘤细胞株分泌。
5.一种用于治疗人膀胱癌的药物组合物，其包含根据权利要求3或4所述的抗体，优选还包含抗肿瘤化疗药物，如丝裂霉素C；免疫增强剂，如卡介苗(BCG)；和/或细胞因子，如干扰素。
6.一种缀合物，其中所述抗人膀胱癌整合素α3β1的抗体BCMab1与选自以下组成的组的物质缀合：生物标记物、抗肿瘤药物、毒素和放射性活性剂。
7.一种用于检测或治疗人膀胱癌的试剂盒，其包含根据权利要求3或4所述的抗体。
8.根据权利要求7所述的试剂盒，其中所述检测是通过酶联免疫吸附法进行，优选所述酶联免疫吸附法是竞争酶联免疫吸附法，其中将权利要求1或2所述的人膀胱癌肿瘤标志物AG-α3β1作为固定相，与待测样品共同竞争根据权利要求3或4所述的抗体。
9.根据权利要求7或8所述的试剂盒，其中所述待测样品是含尿脱落细胞的人尿液。
10.分泌抗人膀胱癌整合素α3β1的抗体BCMab1的杂交瘤细胞株，其保藏号为CGMCC No.3845。
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