CN101440131B - 抗人肿瘤坏死因子α的单克隆抗体的抗体可变区及其编码基因 - Google Patents
抗人肿瘤坏死因子α的单克隆抗体的抗体可变区及其编码基因 Download PDFInfo
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Abstract
特异性结合人肿瘤坏死因子-α的单克隆抗体的抗体可变区,含有具有特异性互补决定区的重链可变区和轻链可变区的至少之一。还提供了编码它的核酸分子、含有该核酸分子的重组载体和转化该重组载体的细胞。
Description
本申请是申请号为200480033769.8之中国专利申请的分案申请,原申请200480033769.8是根据专利合作条约的国际申请(PCT/KR2004/002964)进入中国国家阶段的国家申请。
发明领域
本发明涉及特异性结合人肿瘤坏死因子-α的单克隆抗体的抗体可变区、编码它的基因、含有该基因的重组载体和转化该重组载体的细胞。
发明背景
人肿瘤坏死因子-α(下文称作“hTNFα”)是由三个17kDa蛋白质亚基组成的同源三聚体(Eck MJ et al.,JBC 267:2119-2122,1992;Smith RA et al.,JBC 262:6951-6954,1987)。hTNFα是由单核细胞和巨噬细胞分泌的炎性细胞因子,在多种细胞反应如坏死和凋亡中作为信号传递物起作用(Beyaert R et al.,FEBS Lett.340:9-16,1994)。
hTNFα产生导致组织破坏的促炎作用,所述组织破坏例如分解软骨和骨(Saklatvala,Nature 322:547-549,1986),以及增加中性粒细胞和淋巴细胞的粘附(Pober et al.,K.Immunol.138:3319,1987)。此外,已知hTNFα在对抗感染性疾病和肿瘤的防御机制中起着重要作用(Fiers W,FEBSLett.285:199-212,1991)。
hTNFα参与炎性疾病、自身免疫病、细菌感染、癌症和退形性病变。在这些疾病中,hTNFα已经被当作特异性生理治疗类风湿性关节炎和节段性回肠炎(Crohn’sdisease)的有用靶蛋白。
同时,还提示使用hTNFα抑制剂来治疗类风湿性关节炎。已经报道hTNFα在分离自早期类风湿性关节的滑膜细胞中过表达(Buchan G et al.,Clin.Exp.Immunol.73:449-455,1988),上述滑膜细胞用抗hTNFα单克隆抗体处理时,与类风湿性关节炎病变相关的细胞因子下降(Butler DM et al.,Eur Cytokine Netw.6:225-230,1995)。
另外,已经发现抗hTNFα抗体或重组可溶性hTNFα受体抑制胶原诱导的小鼠关节炎模型中关节的炎症和破坏(Wpiquet PF et al.,Immunology 77:510-514,1992;Wooley PH et al.,J.Immunol.151:6602-6607,1993;Williams RO et al.,Immunology84:433-439,1995)。此外,观察到过表达hTNFα的转基因小鼠中诱导了炎性关节炎(Keffer J et al.,EMBO J.10:4025-4031,1991)。
这些结果表明hTNFα作为控制炎性细胞因子的直接或间接调节因子在类风湿性关节炎中起着重要作用。因此,需要开发对hTNFα具有高度选择性和反应性的单克隆抗体来治疗类风湿性关节炎。
发明内容
因此,本发明的一个目的是提供特异性结合hTNFα的单克隆抗体的抗体可变区。
本发明的另一个目的是提供编码特异性结合hTNFα的单克隆抗体的抗体可变区的基因;包含该基因的重组载体;和转化该重组载体的细胞。
根据本发明的一个方面,提供了特异性结合hTNFα的单克隆抗体的抗体可变区,其包含下列至少一种:
包含SEQ ID NO:9、10和11的氨基酸序列的重链可变区;和
包含SEQ ID NO:12、13和14的氨基酸序列的轻链可变区。
根据本发明的另一个方面,提供了编码特异性结合hTNFα的单克隆抗体的重链可变区的核酸分子,其中所述的重链可变区包含SEQ ID NO:9、10和11的氨基酸序列。
根据本发明的另一个方面,提供了编码特异性结合hTNFα的单克隆抗体的轻链可变区的核酸分子,其中所述的轻链可变区包含SEQ ID NO:12、13和14的氨基酸序列。
具体实施方式
本发明涉及特异性结合hTNFα的单克隆抗体的抗体可变区及其编码基因。
为了制备特异性结合hTNFα的单克隆抗体,用重组hTNFα(Biosource PHC3011,Belgium)免疫小鼠。将从免疫小鼠获得的脾细胞与骨髓瘤细胞(Sp2/0-Agl4,ATCCCRL1581)融合,以制备杂交瘤细胞库(pool)。将这种杂交瘤细胞进行后续的克隆和选择程序,以提供多种单克隆抗体。在这些单克隆抗体中,选择特异性结合hTNFα的某些单克隆抗体进行进一步分析。结果,获得了杂交瘤细胞系TSK11,其产生特异性结合hTNFα并对TNFα表现出高度结合亲和力的单克隆抗体。
从杂交瘤细胞系TSK11提取总RNA,进行逆转录酶-聚合酶链式反应(RT-PCR),以合成单克隆抗体重链和轻链的cDNA分子。使用这种cDNA分子作为模板进行聚合酶链式反应(PCR),从而获得编码重链可变区并包括SEQ ID NO:5核苷酸序列的约470bp的cDNA分子;和编码轻链可变区并包括SEQ ID NO:6核苷酸序列的约450bp的cDNA分子。
分析这种重链和轻链可变区的互补决定区(CDR),结果发现重链可变区具有三个CDR,位于SEQ ID NO:7氨基酸序列中的31-35(SEQ ID NO:9)、50-66(SEQID NO:10)和99-106(SEQ ID NO:11)氨基酸位置处。同样,观察到轻链可变区具有三个CDR,位于SEQ ID NO:8氨基酸序列中的24-35(SEQ ID NO:12)、51-57(SEQ ID NO:13)和90-98(SEQ ID NO:14)氨基酸位置处。
因此,根据本发明一个实施方案的cDNA分子编码特异性结合hTNFα的单克隆抗体的重链可变区,其中所述的重链可变区包括SEQ ID NO:9、10和11的氨基酸序列。优选的是,本发明提供了编码含SEQ ID NO:7氨基酸序列的重链可变区的cDNA分子,更优选的是具有SEQ ID NO:5核苷酸序列的cDNA分子。
另外,根据本发明另一个实施方案的cDNA分子编码特异性结合hTNFα的单克隆抗体的轻链可变区,其中所述的轻链可变区包括SEQ ID NO:12、13和14的氨基酸序列。优选的是,本发明提供了编码含SEQ ID NO:8氨基酸序列的轻链可变区的cDNA分子,更优选的是具有SEQ ID NO:6核苷酸序列的cDNA分子。
编码重链可变区和轻链可变区的前述每一种cDNA分子可以插入到传统载体中以获得重组载体。在本发明的一个优选实施方案中,包括SEQ ID NO:5或6核苷酸序列的cDNA分子可以插入到pCR2.1-TOPO(Invitrogen Co.U.S.A)中,以制备含有重链可变区的重组载体pTSK11-Hv,或含有轻链可变区的重组载体pTSK11-Lv。
另外,可以将这种重组载体导入合适的宿主中,如诸如E.coli TOP10F的微生物。例如,用pTSK11-Hv或pTSK11-Lv转化E.coli TOP10F,以获得E.coli转化体,称作E.coli TOP 10F/pTSK11-Hv或E.coli TOP 10F/pTSK11-Lv,它们按照国际承认用于专利程序的微生物保藏布达佩斯条约的条款,于2003年8月26日保藏在KoreanCollection for Type Cultures(地址:#52,Oun-dong,Yusong-ku,Taejon 305-333,Republic of Korea),保藏编号是KCTC 10514BP或KCTC 10515BP。
上述重组载体可以根据常规方法从转化体中恢复(J.Sambrook et al.,Molecularcloning Vol.1:1.25-1.28)。例如,转化体可以用溶液1(50mM葡萄糖,25mM Tris-HCl和10mM EDTA)处理以弱化其细胞膜,然后用溶液2(0.2N NaOH和1%SDS)处理以完全破坏细胞膜,变性所暴露的蛋白质和染色体。然后,通过进一步用溶液3(5M醋酸钾和醋酸)处理使重组载体之外的细胞组分聚集。然后,将所得的溶液进行离心以获得含重组载体的上清液。重组载体可以通过乙醇沉淀从上清液中回收。
本发明的抗体可变区可以包含下列至少一种:包含SEQ ID NO:9、10和11的氨基酸序列的重链可变区;和包含SEQ ID NO:12、13和14的氨基酸序列的轻链可变区。优选的是,抗体可变区可以包含具有SEQ ID NO:7氨基酸序列的重链可变区和具有SEQ ID NO:8氨基酸序列的轻链可变区中的至少之一。
抗hTNFα的人源化单克隆抗体可以通过将人抗体基因和编码含有SEQ ID NO:9-11所示CDR的重链可变区或编码含有SEQ ID NO:12-14所示CDR的轻链可变区的cDNA分子融合而得到。作为替代方案,人源化单克隆抗体可以将人抗体可变区替换为这种cDNA分子而得到。
如上所述,由于本发明的抗体可变区特异性结合hTNFα,它可以有效地用来中和并灭活hTNFα。
在下列实施例中进一步解释本发明。应理解这些实施例虽然指出了本发明的优选实施方案,但只是以说明方式给出。
实施例1:用hTNFα免疫小鼠
将30μg重组hTNFα(Biosource PHC3011,Belgium)溶解于150μl磷酸缓冲液(PBS)中,和150μl弗氏完全佐剂(Sigma F5881,U.S.A.)混合而乳化。将300μl所得乳液腹腔注射(i.p.)到6周龄雄性BALB/c小鼠体内。两周后,小鼠腹腔注射300μl含30μg hTNFα和150μl弗氏不完全佐剂(Sigma F5506,USA)的乳液混合物。第二次注射两周后,将溶解于150μl PBS中的30μg重组hTNFα静脉注射(i.v.)到小鼠体内。第3次注射10天后,小鼠静脉注射30μg的重组hTNFα的PBS溶液而加强免疫。
实施例2:产生抗hTNFα抗体的小鼠脾细胞的融合
将实施例中免疫的小鼠通过碳酸钾窒息而处死,然后从中摘除脾脏。从小鼠脾脏获得脾细胞,并与非分泌性骨髓瘤细胞Sp2/0(ATCC CRL1581)以10∶1比例混合。为了细胞融合,将预热到37℃的1ml 50%聚乙二醇(PEG 1500,Roche 783641)加到细胞混合物中。将融合细胞用含20%胎牛血清(FBS,JRH,12-10678P)、50μg/ml庆大霉素(Gibco-BRL,15750-060)、1×DMEM(JRH,56499-10L)和1×HAT补充物(0.1mM次黄嘌呤钠、0.4μM氨基蝶呤、16μM胸腺嘧啶;Gibco-BRL,31062-037)的生长培养基稀释,并以1.1×105细胞/孔的浓度、以0.2μl小份分到96孔板(Nunc,469949,Denmark)中。将融合细胞于37℃的湿化CO2孵箱中以5%CO2培养2~3周。
实施例3:产生抗hTNFα抗体的细胞系的筛选和克隆
一旦融合细胞形成集落,就取它们的上清液进行ELISA分析以确证抗体产生。为进行ELISA分析,将孔用1μg/ml hTNFα于4℃包被过夜,向每个孔中加入200μl0.5%酪蛋白-PBS溶液,然后在37℃进行反应1小时。接着,向每个孔中加入100μl上清液,在37℃进行反应2小时。然后,向每个孔中加入100μL以1∶1000比例稀释的偶联辣根过氧化物酶的山羊抗小鼠IgG(Bio-Rad,170-6516),于37℃保持1.5小时。最后,向每个孔中加入100μl辣根过氧化物酶底物溶液(Bio-Rad,172-1064),将这些孔于37℃保持3分钟以诱导显色。用ELISA酶标仪(Dinatec inc.,USA)测定410nm处的光吸收。
实施例4:产生抗hTNFα抗体的细胞系的选择
在实施例3克隆的细胞系中,获得了比获自hTNF-α免疫小鼠的阳性对照血清表现出更高光吸收的9个产生IgG的细胞系。在这些细胞系中,选择表现出最高光吸收的TSK11进行进一步分析。
实施例5:从杂交瘤细胞系TSK11获得的单克隆抗体的同种型分析
通过如下的ELISA分析确认从杂交瘤细胞系产生的单克隆抗体的同种型。向预先包被100μg/ml TSK11抗体的孔中加入100μl的小鼠MonoAb ID kit HRP溶液(Zymed,90-6550),然后,将孔板保持于室温下3分钟,以诱导显色。用ELISA酶标仪(Dinatec inc.,USA)测定410nm处的光吸收。结果发现TSK抗体含有IgG1型重链和κ型轻链。
实施例6:来自杂交瘤细胞系TSK11的单克隆抗体的体外结合亲和力
通过ELISA分析测定抗hTNFα的TSK11抗体的体外结合亲和力。这里,将每个孔用100μl浓度为4μg/ml的重组hTNFα包被。然后,在微管(Micro Tube)(AXYGEN,U.S.A.)中,将重组hTNFα用补充0.02%牛血清白蛋白的PBS稀释,以获得5×10-9M-1×10-10M的终浓度。将各浓度的hTNFα稀释物和30ng TSK11抗体混合,于37℃保持2小时。将混合溶液分到用重组hTNFα包被的每个孔中,于37℃孵育2小时。然后,向每个孔中加入100μl辣根过氧化物酶底物溶液(Bio-rad,172-1064),于37℃保持3分钟以诱导显色。用ELISA酶标仪(Dinatec inc.,USA)测定410nm处的光吸收。
根据Scatchard plot分析(Friguet E.etal.,J.of Immunological Method 77:305-319,1985)计算抑制竞争性ELISA中50%的最大结合所需的抗原浓度的倒数来确定表观亲和力。结果,TSK11对hTNFα的亲和力为1.95×10-9M(Kd)。
实施例7:从杂交瘤细胞系TSK11的RNA分离和cDNA合成
用RNeasy试剂盒(QIAGEN,U.S.A)从杂交瘤细胞系TSK11的1×108细胞中提取总RNA,并使用Thermotranscript试剂盒(GibcoBRL,U.S.A)进行cDNA合成。将作为模板的5μgRNA和0.5ng寡聚d(T)悬浮于蒸馏水中,并将终体积调至10μl。将混合物保持于65℃下5分钟以变性RNA,并冷却到室温以诱导引物退火。用于cDNA合成的RT-PCR反应溶液通过将1μl逆转录酶(1单位/μl)、2.5μl 0.1M DTT、2.5μl 10mM dNTP和1μl RNase抑制剂(1单位/μl)混合而制备,其终体积用蒸馏水调至25μl。RT-PCR反应于50℃进行1小时,通过将反应混合物于95℃加热5分钟而终止。
使用2μg的合成cDNA作为模板和扩增重链的一对引物(SEQ ID NO:1和2)或扩增轻链的另一对引物(SEQ ID NO:3和4)进行PCR。PCR反应溶液含有0.5μlAmpliTaq Gold聚合酶(5单位/μl,Perkin-Elmer Biosystem Co.,U.S.A)、1μl 10mMdNTP和5μl 25mM MgCl2,终体积用蒸馏水调至50μl。PCR条件如下:95℃初始变性5分钟,然后是94℃ 1分钟,55℃ 1分钟,72℃ 2分钟的30个循环,接着于72℃最终延伸10分钟。
将扩增的DNA进行1.5%琼脂糖凝胶电泳,将凝胶用100ml的0.5μg/ml溴化乙锭溶液染色20分钟。结果,鉴定到了两个扩增的DNA产物,对于重链在对应于约470bp的位置处,对于轻链在对应于约450bp的位置处,参照100bp的标准DNAladder(Lifetechnology Co.U.S.A.)。
实施例8:cDNA克隆
利用QIAquick Gel Extraction试剂盒(Qiagen,U.S.A.)从琼脂糖凝胶回收并纯化实施例7中扩增的470bp的重链DNA片段。将纯化的DNA片段亚克隆到载体pCR2.1-TOPO(Invitrogen Co.,U.S.A.)中,将所得的载体导入E.coli TOP10F(Invitrogen Co.,U.S.A.),以获得转化体(Cohen,S.N.et al.,Proc.Nat.Acad.Sci.69:2110,1972)。如此制备的E.coli转化体在补充100μg/ml氨苄青霉素的LB培养基中培养过夜。从培养的转化体中提取质粒DNA,并用限制酶EcoRI(BioLab Co.,USA)处理,以获得含470bp重链DNA片段的克隆TSK11-Hv。
对于实施例7中扩增的450bp的轻链DNA片段进行同样的程序,以获得E.coliTOP10F转化体。E.coli转化体在补充100μg/ml氨苄青霉素的LB培养基中培养过夜。从培养的转化体中提取质粒DNA,并用限制酶EcoRI(BioLab Co.,USA)处理,以获得含450bp轻链DNA片段的克隆TSK11-Lv。
实施例9:cDNA核苷酸测序
使用Wizard plus SV Minipreps DNA Purification System(Promega,U.S.A.)纯化实施例8中获得的克隆TSK11-Hv和TSK11-Lv,并进行核苷酸测序。
结果发现获得的重链DNA片段含有SEQ ID NO:5核苷酸序列和SEQ ID NO:7氨基酸序列。验证了属于克隆TSK11Hv的三个具体DNA片段(TSK11Hv1、TSK11Hv2和TSK11Hv3),它们的核苷酸序列相同。从克隆TSK11Hv获得的质粒载体命名为pTSK11-Hv。另外,具有质粒载体pTSK11-Hv的E.coli转化体命名为E.coli TOP10F/pTSK11-Hv,按照国际承认用于专利程序的微生物保藏布达佩斯条约的条款,于2003年8月26日保藏在Korean Collection for Type Cultures(地址:#52,Oun-dong,Yusong-ku,Taejon 305-333,Republic of Korea),保藏编号是KCTC10514BP。
另外,发现获得的轻链DNA片段含有SEQ ID NO:6核苷酸序列和SEQ ID NO:8氨基酸序列。验证了属于克隆TSK11Lv的两个具体DNA片段(TSK11Lv1和TSK11Lv3),它们的核苷酸序列相同。从克隆TSK11Lv获得的质粒载体命名为pTSK11-Lv。另外,具有质粒载体pTSK11-Lv的E.coli转化体命名为E.coliTOP10F/pTSK11-Lv,按照国际承认用于专利程序的微生物保藏布达佩斯条约的条款,于2003年8月26日保藏在Korean Collection for Type Cultures(地址:#52,Oun-dong,Yusong-ku,Taejon 305-333,Republic of Korea),保藏编号是KCTC10515BP。
根据从杂交瘤细胞系TSK11获得的单克隆抗体可变区的氨基酸序列分析(Harris.L.et al.,Protein Sci.4:306-310,1995;Kabat.E.A.et al.,Sequence ofproteins of immunological interest.5th Ed.,1991;Williams A.F.et al.,Annu.Rev.Immunol.6:381-406,1988),发现重链属于由Kabat定义的11类之外的混杂类,轻链属于κ6-型亚类。
重链中识别抗原的CDR发现位于氨基酸位置31-35(SEQ ID NO:9)、50-66(SEQID NO:10)和99-106(SEQ ID NO:11),轻链中识别抗原的CDR发现位于氨基酸位置24-35(SEQ ID NO:12)、51-57(SEQ ID NO:13)和90-98(SEQ ID NO:14)。
虽然描述并说明了本发明的实施方案,显而易见的是,可以在不背离本发明实质的情况下进行各种改变和改进,本发明实质仅受所附权利要求范围的限定。
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Claims (3)
1.特异性结合人肿瘤坏死因子-α的单克隆抗体的抗体可变区,其包含:
包含三个互补性决定区的重链可变区,所述三个互补性决定区分别具有SEQ IDNO:9、10和11的氨基酸序列;和
包含三个互补性决定区的轻链可变区,所述三个互补性决定区分别具有SEQ IDNO:12、13和14的氨基酸序列。
2.权利要求1的抗体可变区,其中所述重链可变区具有SEQ ID NO:7的氨基酸序列。
3.权利要求1的抗体可变区,其中所述轻链可变区具有SEQ ID NO:8的氨基酸序列。
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CN1280190A (zh) * | 2000-07-14 | 2001-01-17 | 中国人民解放军第四军医大学 | 重组的抗人TNFα基因工程抗体及其制备方法 |
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US6280825B1 (en) | 1988-12-07 | 2001-08-28 | Laminating Technologies, Inc. | Method of making a composite of paper and plastic film and composites |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
CN101712720A (zh) * | 1996-02-09 | 2010-05-26 | 艾博特生物技术有限公司 | 结合人TNFα的人抗体 |
GB0013810D0 (en) * | 2000-06-06 | 2000-07-26 | Celltech Chiroscience Ltd | Biological products |
DE602005026571D1 (de) * | 2004-12-29 | 2011-04-07 | Yuhan Corp | Tumornekrosefaktor-alpha spezifische humanisierte antikörper |
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- 2004-11-16 WO PCT/KR2004/002964 patent/WO2005047329A1/en active Application Filing
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0492448B1 (en) * | 1990-12-28 | 2000-03-15 | PHARMACIA & UPJOHN S.p.A. | Monoclonal antibodies against human tumor necrosis factor alpha |
CN1280190A (zh) * | 2000-07-14 | 2001-01-17 | 中国人民解放军第四军医大学 | 重组的抗人TNFα基因工程抗体及其制备方法 |
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CN1882611A (zh) | 2006-12-20 |
KR100772800B1 (ko) | 2007-11-01 |
EP1692179B1 (en) | 2010-09-29 |
KR20050047182A (ko) | 2005-05-20 |
US20050124041A1 (en) | 2005-06-09 |
CN101440131A (zh) | 2009-05-27 |
US7109320B2 (en) | 2006-09-19 |
JP2007530012A (ja) | 2007-11-01 |
US7196177B2 (en) | 2007-03-27 |
EP1692179A4 (en) | 2007-10-31 |
EP1692179A1 (en) | 2006-08-23 |
ATE482980T1 (de) | 2010-10-15 |
US20060147452A1 (en) | 2006-07-06 |
JP4563397B2 (ja) | 2010-10-13 |
DE602004029394D1 (de) | 2010-11-11 |
WO2005047329A1 (en) | 2005-05-26 |
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