CN116814557A - 杂交瘤细胞株、抗人Axl的单克隆抗体及其应用 - Google Patents
杂交瘤细胞株、抗人Axl的单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明涉及杂交瘤细胞株、抗人Axl的单克隆抗体及其应用。所涉及的杂交瘤细胞株保藏号为CCTCC NO:C202317;该杂交瘤细胞株可分泌抗人Axl的单克隆抗体;所述述抗人Axl单克隆抗体可用于检测癌症组织中人Axl,以及对肿瘤生长具有明显抑制作用于,基于此,本发明的Axl单克隆抗体可被用于诊断和治疗表达Axl的肿瘤。
Description
技术领域
本发明涉及生物医药技术领域,更具体地,本发明公开了抗人Axl的单克隆抗体、相关杂交瘤细胞株及相关应用。所公开的抗人Axl的单克隆抗体可用于癌症免疫组化检测、肿瘤细胞杀伤和抑制肿瘤生长。
背景技术
Axl是一种受体酪氨酸激酶(基因Axl编码蛋白的GeneBank国际通用序列编号为:NP_068713.2),和Tyro3和Mer三种蛋白质属于TAM(Tyro3/Axl/Mer receptor tyrosinekinase,TAM)受体酪氨酸激酶家族,由相似的一个胞外区域、一个跨膜结构域和一个胞内激酶结构域蛋组成。由于其特有的受体激酶特性,TAM家族参与组织修复、凋亡清除和免疫调控等生理过程。
在胃癌中,研究显示175例胃癌组织中Axl蛋白高表达,且与胃癌的预后相关。高水平的Gas6和Axl表达与胃癌患者的总生存率降低相关,并且在胃癌细胞系中Gas6和Axl的表达上调。同时,Axl的异位表达可诱导上皮间充质转化,促进胃癌细胞的侵袭和增殖。敲除Axl可抑制上皮间充质转化,抑制胃癌细胞的增殖和侵袭。体内研究表明,抑制Axl对胃癌细胞的生长和肺转移有抑制作用。
上述研究结果显示,Axl的高表达和癌症的肿瘤生物学行为相关密切相关,可以作为潜在的肿瘤标志物候选分子。
发明内容
针对现有技术的缺陷或不足,本发明第一方面提供了一株杂交瘤细胞株,其保藏号为CCTCC NO:C202317。同时提供了该杂交瘤细胞株产生的抗人Axl的单克隆抗体,所述抗体能特异性识别人Axl蛋白。
所述杂交瘤细胞系已于2023年3月16日保藏在中国典型培养物保藏中心(CCTCC),保藏地址:湖北省武汉市武昌区八一路299号,中国.武汉.武汉大学,邮编:430072,保藏编号为:CCTCC NO:C202317,分类命名为Hybridoma cell line XA291 No.2。培养物名称是杂交瘤细胞株Axl。
本发明进一步提供了抗人Axl的单克隆抗体,所述抗体的重链可变区的CDR1序列为:SYGMS;CDR2序列为:TINDGGSYNYYPDSVRG;CDR3序列为:DYEDPYYAMDY;轻链可变区的CDR1序列为:TASSSVSSSYLH;CDR2序列为:STSSLAS;CDR3序列为:HQYHRSPYT。
更进一步,所述抗体的重链可变区氨基酸序列为:CEVQLVESGGDLVKPGGSLKLSCSASGFTFRSYGMSWVRQTPDKRLEWVATINDGGSYNYYPDSVRGRFTISRDNA KNTLYLQMSSLKSEDTAMYYCARDYEDPYYAMDYWGQGTSVTVSS;轻链可变区氨基酸序列为:QIVLTQSPAIMSASLGDRVTMTCTASSSVSSSYLHWYQQKPGSPPKLWIYSTSSLASGVPARFSGGGSGTSYSLTI SSMESEDAATYYCHQYHRSPYTFGGGTKVEIK。
虽然现有的商业化Axl抗体有十余种,由主流抗体试剂公司CST和Abcam
等提供,但是均属于基础研究用试剂,相对于现有Axl抗体,本发明的抗体具有合格的特异性并可用于癌症如胃癌免疫组化检测,本发明的Axl抗体经过大量组织标本的免疫组化验证并显示出可以通过对Axl的组织细胞表达水平进行检测来达到对胃癌恶性度及患者生存情况进行预后预判的目的。
基于此,本发明第二方面提供了的抗人Axl的单克隆抗体在制备检测人Axl非诊断试剂或试剂盒的应用。
本发明同时提供了所述抗人Axl的单克隆抗体制备癌症诊断试剂或试剂盒的应用。
所述试剂或试剂盒为免疫组化检测试剂或试剂盒,以对人Axl蛋白特异的抗体作为一抗或核心抗体,制备人Axl免疫组化检测试剂,制备出的试剂特异性好,灵敏度高,显色性好。免疫组织化学技术应用免疫学抗原抗体反应基本原理,通过抗体对体内蛋白质分子抗原的特异性识别,以及化学标记(荧光素、酶、金属离子、同位素等)的第二抗体的显色等作用来检测组织细胞内目标抗原(多肽和蛋白质)的表达量及其亚细胞定位。
以本发明抗人Axl单克隆抗体进行免疫组化检测时采用本发明的人Axl蛋白特异的抗体作为一抗或核心抗体,尤其可对癌症和癌旁组织中Axl的表达水平和定位进行检测。
本发明Axl单抗对肿瘤细胞和小鼠皮下肿瘤模型具有杀伤和抑制作用,是良好的肿瘤诊疗候选物。可以有效的对肿瘤细胞增殖有抑制作用。同时本发明的单抗对小鼠皮下肿瘤模型有明显的抑制作用,也充分说明了该Axl单抗对肿瘤的抑制作用,具备作为抗体药物的潜力,可能应用于各种缀合物抗体药物应用和后续的CART实验等临床治疗性应用。
基于此,本发明第三方面提供了所述抗人Axl的单克隆抗体用于制备抗肿瘤药物的应用。所述药物为用于肿瘤分子靶向治疗的药物或免疫治疗药物,所述分子靶向治疗的单克隆抗体与相应的生长因子受体结合,阻断细胞增殖信号的传导,从而抑制肿瘤细胞的增长;所述免疫治疗药物能够通过诱导免疫应答,来杀伤肿瘤细胞。
本发明所述肿瘤或癌症为表达Axl的肿瘤,例如胃癌、肠癌、乳腺癌、肺癌或肝癌。
附图说明
图1为本发明实施例1中小鼠抗人Axl单抗纯化结果;
图2为本发明实施例2中抗体纯化结果,其中1代表纯化后;2代表纯化前;
图3为本发明实施例3中本发明抗体与商品化抗体特异性对比情况;
图4为本发明实施例3中本发明抗体和商品化抗体对胃癌癌前病变(胃炎、肠化、上皮内瘤变)和胃癌样本IHC的识别结果对比;
图5为Axl表达和胃癌生存预后的相关性;
图6为本发明实施例4中Axl单抗对胃癌细胞的杀伤结果;
图7本发明实施例5中Axl单抗对小鼠皮下肿瘤生长的抑制作用。
具体实施方式
除非有特殊说明,本文中的科学与技术术语根据相关领域普通技术人员的认识理解。
以下通过实施例对本发明做进一步的阐述。实施例是对本发明进行详细说明,但本发明并不受这些的任何限定。
实施例1:本发明杂交瘤细胞株的制备
Axl抗原由北京义翘神州公司合成,根据人Axl序列(NP_068713.2)的DNA编码序列,在人细胞系内表达Axl胞外结构域1-499aa,C端融合HIS标签,经SDS-PAGE检测,其大小约为46.5kDa(图1),其氨基酸序列为:
MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVL DIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWP。
选取6-8周龄Balb/C雌性小鼠(来自南京集萃药康生物科技有限公司),前述Axl融合蛋白作为抗原,为获得高亲和力抗体,采用小剂量(约1μg)、长时程、多次免疫的方法,选择免疫10天后,采用间接ELISA方法检测血清效价(包被抗原为酶切并纯化的蛋白),效价达到1∶5万以上准备融合;
取经加强免疫的小鼠脾脏,研磨破碎,过滤洗涤之后,制成细胞悬液并计数;骨髓瘤SP2/0细胞生长至对数期,离心收集并洗涤,与脾细胞按1∶10或1∶5的比例混合,室温下采用PEG进行促融合,将融合后的细胞悬液加入含有饲养细胞的96孔板,置37℃,5%CO2,相对饱和湿度培养箱中培养;
在融合24小时后,采用RPMI培养液(购自Gibco公司)进行融合骨髓瘤细胞筛选,并进行连续克隆化;
最终从11株杂交瘤细胞中筛选得到能够稳定分泌抗Axl单克隆抗体的杂交瘤细胞株,对Axl为阳性反应,对Axl为阴性反应的杂交瘤细胞株,均保藏于中国典型培养物保藏中心,地址:武汉市武昌珞珈山武汉大学,保藏号为:CCTCC NO:C202317。
实施例2:抗人Axl的单克隆抗体的制备
保藏编号为C202317杂交瘤细胞以106个/只的量,腹腔注射预先致敏8-10周龄BABL/C雌性小鼠(来自南京集萃药康生物科技有限公司),7-10天后,采集小鼠腹水,用间接ELISA方法检测单抗的效价;
腹水4℃、12000rpm离心15min,与2倍体积的醋酸缓冲液(0.06M,pH4.8)混合,逐滴加入正辛酸,室温搅拌30min,4℃静置其充分沉淀;
离心后取上清过滤,加入1/10体积的磷酸盐缓冲液(0.1M,pH7.4),并用2M NaOH调节pH至7.4;
采用硫酸铵沉淀将抗体沉淀,离心后取沉淀,用含有0.2mM EDTA磷酸盐缓冲液溶解,并透析过夜;
采用离子交换层析进行纯化,所用缓冲液:平衡缓冲液为20mM pH7.5的Tris-HCl缓冲液、洗脱缓冲液为含1.0M NaCl的20mM pH7.5的Tris-HCl缓冲液。NaCl浓度梯度洗脱结合的抗体蛋白,检测A280,收集洗脱的抗体蛋白并进行SDS-PAGE验证(图2);
抗体测序由北京义翘神州公司完成,其步骤为:杂交瘤细胞采用RPMI 1640(购自Gibco公司)加20%血清培养24h,离心,弃去上清,采用TriZol(购自Invitrigen)重悬细胞,106个细胞/mL;提取DNA后,采用RT-PCR扩增重链和轻链基因,克隆至T载体后进行DNA测序。
重链可变区:
CEVQLVESGGDLVKPGGSLKLSCSASGFTFRSYGMSWVRQTPDKRLEWVATINDGGSYNYYPDSVRGRFTISRDNA KNTLYLQMSSLKSEDTAMYYCARDYEDPYYAMDYWGQGTSVTVSS;CDR1序列:SYGMS;CDR2序列:TINDGGSYNYYPDSVRG;CDR3序列:DYEDPYYAMDY;
轻链可变区:
QIVLTQSPAIMSASLGDRVTMTCTASSSVSSSYLHWYQQKPGSPPKLWIYSTSSLASGVPARFSGGGSGTSYSLTI SSMESEDAATYYCHQYHRSPYTFGGGTKVEIK CDR1序列:TASSSVSSSYLH;CDR2序列:STSSLAS;CDR3序列:HQYHRSPYT。
实施例3:Axl免疫组织化学检测
1)材料来源:
胃癌切片和胃癌癌前切片有胃炎切片、肠上皮化生切片和上皮内瘤变切片均取自空军军医大学第一附属医院消化病医院组织标本库,常规脱蜡;;
一抗稀释液、辣根过氧化物酶(HRP)标记的通用二抗、二氨基联苯胺(DAB)底物、底物稀释液等购自中杉金桥;
商品化抗体1为CST Axl抗体8661;商品化抗体2为Abcam Axl抗体37861;商品化抗体3为sigma Axl抗体HPA037422;商品化抗体4为sigma Axl抗体HPA037423。
2)免疫组化检测试剂条件及样本组织中Axl表达与定位的检测方法:
方法为:组织切片脱蜡至水,抗原修复,內源过氧化物酶阻断,以1:200滴加实施例(1)制备得到的抗体作为一抗,4℃孵育过夜;缓冲液洗3次每次5min;滴加通用HRP酶标二抗,在室温下孵育30分钟;缓冲液洗3次每次5min。DAB显色,复染,脱水封片后显微镜下观察染色情况。
3)结果:
胃癌患者组织细胞中Axl在浆中呈不同程度的着色;和所有商品化Axl抗体相比,本发明的Axl单抗具有更好的Axl特异性染色,同时在癌旁非特异性染色较少(图3);
同时在胃癌癌前病变组织中,Axl单抗在胃癌癌前病变组织中显示阴性结果,在胃癌组织中阳性结果,可以区分胃癌癌前病变和胃癌;然而两种商品化抗体在癌前病变和胃癌组织中均有强阳性着色(图4)。
已有研究发现胃癌组织样本中肿瘤细胞中Axl表达阴性患者的生存预后明显好于Axl表达阳性患者(图5)。结合上述实验结果表明,采用本发明提供抗体为核心的免疫组化检测方法,能很好地检测胃癌组织细胞中Axl的表达量和表达位置,便于从免疫组化图中直接判读Axl在癌组织细胞中的定位和表达情况,并以此为依据判别胃癌的个体化异质性及其生存预后。
实施例4:Axl单抗对肿瘤杀伤研究
细胞杀伤试验:分别取对数生长期的胃癌细胞SGC7901、AGS和GES(永生化胃粘膜上皮细胞)接种至96孔板(每孔5×103个),正常培养48h;每一种细胞分别设置Axl单抗组、小鼠抗结核单抗(购自上海生工生物有限公司)对照组和空白对照组,Axl单抗和抗结核单抗用含1.5%胎牛血清的RPMI-1640培养液稀释成400μg/ml、200μg/ml、100μg/ml、50μg/ml和25μg/ml;洗细胞一次,加入含不同浓度抗体的培养液,置CO2培养箱中继续培养,倒置显微镜下观察细胞状态,不同浓度的Axl加入胃癌细胞48-72h,显微镜下可发现细胞从48h开始,出现折光性变差、贴壁不牢等明显的凋亡特征,甚至出现死亡。
图6显示当Axl浓度达到100μg/ml时,72h MTT法检测证实存活的胃癌细胞SGC7901和AGS明显减少(P<0.05),抗结核抗体对照并无此杀伤作用,任何浓度的Axl单抗对永生化胃粘膜上皮细胞GES无明显杀伤作用。
实施例5:Axl单抗对胃肿瘤生长抑制研究而
为了研究Axl单抗作为潜在的癌症治疗剂,通过7901细胞系在裸鼠中建立皮下肿瘤种植模型检测肿瘤生长抑制,所用小鼠来自南京集萃药康生物科技有限公司;
在肿瘤长至100mm3大小后,向小鼠腹腔注射10mg/kg Axl单抗,每周两次,持续4周;相对于对照未用药组,Axl单抗明显的抑制小鼠皮下肿瘤的生长,如图7所示,因此,Axl单抗在小鼠体内表现出较强的抗肿瘤活性。
综合上述,对细胞和小鼠体内肿瘤的杀伤和抑制生长的作用,Axl单抗表现出了良好的肿瘤治疗作用,是非常有前景的可以作为靶向表达Axl的肿瘤的诊断和治疗的候选物。
上述实施例只为说明本发明的技术构思及特点,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。
Claims (9)
1.杂交瘤细胞株,该杂交瘤细胞株的保藏编号为CCTCC NO:C202317。
2.权利要求1所述杂交瘤细胞株产生的抗人Axl的单克隆抗体。
3.抗人Axl的单克隆抗体,其特征在于,该抗体的重链可变区的CDR1序列为:SYGMS;CDR2序列为:TINDGGSYNYYPDSVRG;CDR3序列为:DYEDPYYAMDY;轻链可变区的CDR1序列为:TASSSVSSSYLH;CDR2序列为:STSSLAS;CDR3序列为:HQYHRSPYT。
4.根据权利要求3所述的抗体,其特征在于,该抗体的重链可变区氨基酸序列为:CEVQLVESGGDLVKPGGSLKLSCSASGFTFRSYGMSWVRQTPDKRLEWVATINDGGSYNYYPDSVRGRFTISRDNA KNTLYLQMSSLKSEDTAMYYCARDYEDPYYAMDYWGQGTSVTVSS;轻链可变区氨基酸序列为:QIVLTQSPAIMSASLGDRVTMTCTASSSVSSSYLHWYQQKPGSPPKLWIYSTSSLASGVPARFSGGGSGTSYSLTI SSMESEDAATYYCHQYHRSPYTFGGGTKVEIK。
5.权利要求2、3或4所述抗体用于制备检测人Axl非诊断试剂或试剂盒的应用。
6.权利要求2、3或4所述的抗人Axl的单克隆抗体用于制备癌症诊断试剂或试剂盒的应用。
7.根据权利要求6所述的应用,其特征在于,所述癌症为胃癌、肠癌、乳腺癌、肺癌或肝癌。
8.权利要求2、3或4所述的抗人Axl的单克隆抗体用于制备抗肿瘤药物的应用。
9.根据权利要求8所述的应用,其特征在于,所述肿瘤为胃癌、肠癌、乳腺癌、肺癌或肝癌。
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