CN102167743A - 一种全人源抗egfr单克隆抗体、其制备方法及用途 - Google Patents
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Abstract
本发明涉及生物技术领域，更具体地，本发明公开了一种全人源抗EGFR单克隆抗体、其制备方法及用途。本发明构建了大容量的天然人源噬菌体抗体库，从中筛选获得了一株全人源抗EGFR抗体15H8，其重链可变区氨基酸序列为SEQ ID NO：6所示，轻链可变区氨基酸序列为SEQ ID NO：8所示。另外，本发明还公开了15H8抗体的制备方法以及编码15H8抗体的核苷酸序列、含有该核酸序列的表达载体和宿主细胞。本发明的15H8与其它抗EGFR抗体相比，具有更高的抗体亲和力，更强的EGFR高表达肿瘤细胞增殖抑制作用、凋亡诱导活性作用，可明显抑制肿瘤的增长，可用于制备抗肿瘤药物。
Description
技术领域
本发明涉及生物技术领域，更具体地，本发明公开了一种全人源单克隆抗体、其制备方法及用途。
背景技术
恶性肿瘤是当今社会严重危害人类健康的疾病，在各种疾病所致死亡中高居第二位。近年来，恶性肿瘤发病率呈明显上升趋势，而且恶性肿瘤治疗效果差，晚期转移率高，预后多不佳。目前临床上常规治疗方法如放、化疗和手术治疗虽然在很大程度上缓解了病痛，延长了生存时间，但这些方法均存在很大的局限性，其疗效难以进一步提高。
表皮生长因子受体(EGFR)的信号传导途径是癌症发生发展的重要影响因素，在肿瘤细胞生长、凋亡和转移等过程中发挥重要作用。目前在多种实体瘤如肺癌、头颈癌、结肠癌、脑肿瘤中均发现EGFR的过表达。大量实验证明，EGFR可以作为肿瘤治疗的一个有效靶点。EGFR是表皮生长因子受体家族中的4成员之一，是一种跨膜糖蛋白，分子量约为170kD，由胞外区、跨膜区及胞内区3部分组成。EGFR的胞外区分成4个亚区，是EGFR与配体(生长因子等)结合的部位；跨膜区由一个亲脂性多肽螺旋构成；胞内区含有保守的酪氨酸激酶磷酸化位点。在非活性状态下EGFR是单体，当EGFR受体与其配体结合后可形成同源或异源二聚体，受体胞内区发生自我磷酸化可以启动一系列胞内信号级联。EGFR对细胞增殖起重要作用。由于表皮生长因子受体(EGFR)信号转导途径在肿瘤细胞的增殖、损伤修复、侵袭及新生血管形成等方面起重要作用。
近年来靶向EGFR药物已成为肿瘤治疗的新热点。靶向EGFR药物主要有两类：一类是作用于受体胞内区的小分子酪氨酸激酶抑制剂(TKI)，主要包括吉非替尼、erlotinib等等；另一类是作用于受体胞外区的单克隆抗体(MAb)，包括西妥昔单抗(cetuximab)等。
西妥昔单抗(Cetuximab，商品名Erbitux)是抗人EGFR的嵌合型IgG1单克隆抗体，它于2006年被美国食品药品管理局批准为合用放射疗法治疗手术不能切除的头颈部鳞瘤。但Cetuximab是一个嵌合型抗体，仍未能达到人源化，且存在着亲和力不高的问题。
发明内容
本发明构建了大容量的天然人源噬菌体抗体库，从中筛选获得了一株全人源抗EGFR抗体15H8。
更具体地，本发明公开的全人源抗EGFR抗体，重链可变区氨基酸序列为SEQ ID NO：6所示，轻链可变区氨基酸序列为SEQ ID NO：8所示；
本发明公开的上述全人源抗EGFR抗体，重链氨基酸序列为SEQ ID NO：10所示，轻链氨基酸序列为SEQ ID NO：12所示；
本发明还公开了一种核苷酸序列，编码上述的全人源抗EGFR抗体；
本发明公开的上述核苷酸序列，其中编码全人源抗EGFR抗体重链可变区的核苷酸序列为SEQ ID NO：5所示，编码全人源抗EGFR抗体轻链可变区的核苷酸序列为SEQ ID NO：7所示；
本发明公开的上述核苷酸序列，其中编码全人源抗EGFR抗体重链的核苷酸序列为SEQ ID NO：9所示，编码全人源抗EGFR抗体轻链的核苷酸序列为SEQ ID NO：11所示；
本发明还公开了一种表达载体，含有上述的核苷酸序列，为pcDNA3.1/ZEO(+)或pcDNA3.1(+)；
本发明还公开了上述表达载体转化的宿主细胞，为CHO-K1细胞；
本发明最后公开的上述的全人源抗体在制备治疗抗肿瘤药物中的用途，肿瘤为EGFR高表达肿瘤，具体可为肺癌、头颈癌、结肠癌、脑肿瘤。
本发明利用得到的抗体进行了一系列实验，实验结果表明：与cetuximab(C225；西妥昔单抗)、中国专利申请号02815939.X申请日2002年6月13日发明名称为《表皮生长因子受体(EGFR)的人单克隆抗体》中公开的全人源抗EGFR抗体2F8、中国专利申请号200510024158.3申请日2005年3月2日发明名称《重组抗EGFR单克隆抗体》中公开的全人源抗体3C6比较起来，本发明得到的抗体具有更高的抗体亲和力，更强的EGFR高表达肿瘤细胞增殖抑制作用、凋亡诱导活性作用；体内抗肿瘤实验结果表明，本发明得到的抗体可以明显抑制肿瘤的增长。
附图说明
图1.抗EGFR抗体的凋亡实验结果(图1-1A431细胞，图1-2MDA-MB-231细胞，图1-3MCF-7细胞)；
图2.抗EGFR抗体的的生长抑制实验结果(图2-1A431细胞，图2-2MDA-MB-231细胞，图2-3MCF-7细胞)；
图3.抗EGFR抗体的体内抗肿瘤实验结果。
具体实施方式
以下实施例、实验例仅仅对本发明进行进一步的说明，不应理解为对本发明的限制。
实施例抗体的制备
(1)人抗体轻、重链恒定区基因的克隆
用淋巴细胞分离液(鼎国生物技术发展公司产品)分离健康人淋巴细胞，用Trizol试剂(Invitrogen公司产品)提取总RNA，根据文献(Cell，1980，22：197-207)和文献(NucleicAcids Research，1982，10：4071-4079)报道的序列分别设计引物采用RT-PCR反应扩增抗体重链和轻链恒定区基因。PCR产物经琼脂糖凝胶电泳纯化回收并克隆到pGEM-T载体(Promega公司产品)中，测序验证后确认获得了正确的克隆。SEQ ID NO：1和SEQ ID NO：2分别显示了重链恒定区(CH)的核苷酸序列和氨基酸序列。SEQ ID NO：3和SEQ ID NO：4分别显示了轻链恒定区(CL)的核苷酸序列和氨基酸序列。本例中的正确克隆记作pGEM-T/CH和pGEM-T/CL。
(2)cDNA的制备
收集50健康人的外周血各20ml，混合，用淋巴细胞分离液(医科院天津血研所生产)分离单个核细胞。用Trizol试剂(Invitrogen公司)从分离的人外周血淋巴细胞中提取细胞的总RNA。用cDNA反转录试剂盒(上海申能博彩生物科技有限公司)反转录出cDNA。以上步骤按照厂家提供的说明书进行。
(3)引物设计
参考文献(Immunotechnology，1998，3：271-278)设计并合成克隆人抗体重链可变区(VH)和轻链可变区(VL)基因的VHBack，VHFor，VLBack和VLFor引物。(VHBack，VHFor，VLBack和VLFor 的序列见Immunotechnology，1998，3：271-278)。其中在VHBack引物的5’端加上含有Sfi I位点的序列atg gcc cag ccg gcc atg gcc，在VHFor引物的5’端加上序列gcc agaacc acc gcc gcc gga gcc acc acc gcc，在VLBack引物的5’端加上序列tcc ggc ggcggt ggt tct ggc gga ggc gga tct，在VLFor引物的5’端加上含有Not I位点的序列atgcgg ccg c.
(4)噬菌体抗体库的构建及筛选
采用(2)中的cDNA和(3)中的引物，利用recombinant Phage antibody system试剂盒(Amersham Biosciences公司)构建噬菌体单链抗体库，然后用特异性抗原对文库进行淘选。抗体库构建和淘选方法参照recombinant Phage antibodysystem试剂盒说明书进行，用于淘选的特异性抗原“可溶性人EGFR”购自Sigma公司。经多次淘选抗体库后获得了一株抗人EGFR单链抗体15H8ScFv，测序后获得其基因序列。SEQ ID NO：5和SEQ ID NO：6分别显示了15H8ScFv重链可变区VH的核苷酸序列和氨基酸序列。SEQ ID NO：7和SEQ ID NO：8分别显示了15H8ScFv轻链可变区VL的核苷酸序列和氨基酸序列。
(5)全人源抗体在真核细胞中的表达
以15H8ScFv基因和pGEM-T/CH为模版，通过重叠PCR合成全人源抗体重链基因，反应条件为：95℃15分钟；94℃50秒，58℃50秒，72℃50秒，30个循环；72℃10分钟。并使此全人源抗体重链基因的5′端含有限制酶位点HindIII和信号肽基因序列，3′端含有翻译终止密码TAA和限制酶位点EcoR I。信号肽基因序列为(ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGA)。最后琼脂糖凝胶电泳分离PCR扩增产物，回收目的条带并克隆到pGEM-T载体(Promega公司产品)中，筛选阳性克隆测序。挑选测序正确的克隆用HindIII和EcoR I酶切，经琼脂糖凝胶电泳纯化回收全人源抗体重链片段15H8VHCH，与用HindIII和EcoR I酶切的质粒pcDNA3.1(+)(Invitrogen公司)进行连接，构建成全人源重链真核表达载体pcDNA3.1(+)(15H8VHCH)。
以15H8ScFv基因和pGEM-T/CL载体为模板，通过重叠PCR合成全人源化抗体轻链基因，反应条件为：95℃15分钟；94℃50秒，58℃50秒，72℃50秒，30个循环；72℃10分钟，得到PCR产物，其5′端含有限制酶位点HindIII和信号肽基因序列，，3′端含有翻译终止密码TAA和限制酶位点EcoR I。信号肽基因序列为(ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGA)。挑选测序正确的克隆用HindIII和EcoR I酶切，经琼脂糖凝胶电泳纯化回收全人源抗体轻链片段15H8VLCL，与用HindIII和EcoR I酶切的质粒pcDNA3.1/ZEO(+)(Invitrogen公司)载体进行连接，构建成全人源轻链真核表达载体pcDNA3.1/ZEO(+)(15H8VLCL)。
于3.5cm组织培养皿中接种3×105CHO-K1细胞(ATCC CRL-9618)，细胞培养至90％-95％融合时进行转染：取质粒10μg(质粒pcDNA3.1(+)(15H8VHCH)4μg，质粒pcDNA3.1/ZEO(+)(15H8VLCL)6μg)和20μlLipofectamine2000Reagent(Invitrogen公司产品)按Lipofectamine2000Reagent试剂盒说明书进行转染。转染进行24h后细胞换含600μg/ml G418(Invitrogen公司产品)和250μg/ml Zeocin(Invitrogen公司产品)的DMEM培养基筛选抗性克隆。取细胞培养上清用ELISA检测筛选高表达克隆：羊抗人IgG(Fc)(KPL公司)包被于ELISA板，4℃过夜，用2％BSA-PBS于37℃封闭2h，加入待测的抗性克隆培养上清或标准品Human myeloma IgG1，κ(Sigma)，37℃温育2h，加入HRP-羊抗人IgG(κ)((Southern Biotechnology Associates公司)进行结合反应，37℃温育1h，加入TMB显色液于37℃作用5min，最后用H2SO4终止反应，测A450值。将筛选得到的高表达克隆用无血清培养基扩大培养，用Protein A亲和柱(GE公司产品)分离纯化全人源抗体15H8。将纯化抗体用PBS进行透析，最后以紫外吸收法定量。SEQ ID NO：9和SEQ ID NO：10分别显示了全人源抗体15H8的重链核苷酸序列和氨基酸序列。SEQ ID NO：11和SEQ ID NO：12分别显示了全人源抗体15H8的轻链核苷酸序列和氨基酸序列。
实验例
2F8参照中国专利申请号02815939.X申请日2002年6月13日发明名称为《表皮生长因子受体(EGFR)的人单克隆抗体》中公开的方法制备；3C6参照中国专利申请号200510024158.3申请日2005年3月2日发明名称《重组抗EGFR单克隆抗体》中公开的方法制备
实验例1.抗EGFR抗体的凋亡实验
将人乳腺癌细胞A431(EGFR高表达，ATCC号：CRL 1555)、MDA-MB-231(EGFR中表达，ATCC号：HTB-27)及MCF-7(EGFR低表达，ATCC号：HTB-22)分别与不同稀释度的抗EGFR抗体(包括15H8，C225，2F8，3C6)在37℃培养20小时，洗细胞后用AnnexinV/PI试剂盒(BD公司产品)按照产品说明书检测早期凋亡细胞百分率。抗凋亡实验如图1所示：15H8抗体杀伤A431细胞的能力明显强于C225，2F8和3C6(在抗体浓度≥0.5nM的浓度的时候，P＜0.05，t检验，同样的结果也在MDA-MB-231细胞中得到证实(在抗体浓度≥0.5nM的浓度的时候，P＜0.05，t检验)。然而，在EGFR低表达的MCF-7细胞中，15H8抗体杀伤能力与C225，2F8和3C6差不多。这些结果显示了15H8抗体杀伤细胞的EGFR特异性，且其杀伤EGFR中高表达的细胞的能力要强于C225，2F8和3C6。
实验例2.抗EGFR抗体的生长抑制实验
将人乳腺癌细胞A431、MDA-MB-231及MCF-7细胞分别与不同稀释度的抗EGFR抗体(包括15H8，C225，2F8，3C6)在37℃进行孵育，第五日用MTT染色法染色读数后计算生长抑制率。生长抑制实验如图2所示：15H8抗体对A431细胞的生长抑制能力明显强于C225，2F8和3C6(在抗体浓度≥2.5nM的浓度的时候，P＜0.05，t检验)，同样的结果也在MDA-MB-231细胞中得到证实(在抗体浓度≥2.5nM的浓度的时候，P＜0.05，t检验)。然而，在EGFR低表达的MCF-7细胞中，15H8抗体对细胞的抑制能力与C225，2F8和3C6差不多。这些结果显示了15H8抗体对细胞生长抑制的的EGFR特异性，且其抑制EGFR中高表达的细胞的能力要强于C225，2F8和3C6。
实验例3.抗EGFR抗体的体内抗肿瘤实验
SCID小鼠(购自上海斯莱克公司)于0天时分别皮下接种高度表达表达EGFR的人乳腺癌细胞MDA-MB-231，待肿瘤长至0.2cm3时给荷瘤小鼠分别腹腔注射5，25mg/kg的各抗EGFR抗体，每周2次，连续治疗4周。定期观测各组小鼠体重和肿瘤大小的变化，共观察50天。评价抗EGFR抗体的抗肿瘤治疗效果。体内抗肿瘤实验如图3所示：15H8抗体对高表达EGFR的MDA-MB-231乳腺癌的生长抑制能力明显强于C225，2F8和3C6(在5mg/kg和25mg/kg的剂量时，第20、25、30、35、40、45、50天的时候，P＜0.05，Mann-Whitney检验)。
SEQUENCE LISTING
<110>百迈博药业有限公司
<120>一种全人源抗EGFR单克隆抗体、其制备方法及用途
<160>12
<170>PatentIn version 3.2
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<213>人抗体重链恒定区(CH)的核苷酸序列
<400>1
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggaaga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tcccggtaaa 990
<210>2
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Set Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
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<213>人抗体轻链恒定区(CL)的核苷酸序列
<400>3
actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120
aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180
aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240
cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300
ttcaacaggg gagagtgt 318
<210>4
<211>106
<212>PRT
<213>人抗体轻链恒定区(CL)的氨基酸序列
<400>4
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210>5
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<213>全人源抗体15H8重链可变区核苷酸序列
<400>5
gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120
ccagggaagg gactggaata tgtttcagct attagtagta atgggggtag cacatattat 180
gcaaactctg tgaagggcag attcaccatc tccagagaca attccaagaa cacgctgtat 240
cttcaaatgg gcagcctgag agctgaggac atggctgtgt attactgtgc gagagaaggg 300
tatagcagca gctggtacca caactggttc gactcctggg gccaaggaac cctggtcacc 360
gtctcctca 369
<210>6
<211>123
<212>PRT
<213>全人源抗体15H8重链可变区氨基酸序列
<400>6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val
35 40 45
Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asn Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Tyr Ser Ser Ser Trp Tyr His Asn Trp Phe Asp Ser
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210>7
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<213>全人源抗体15H8轻链可变区核苷酸序列
<400>7
gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcttg gatagtgatg atggaaacac ctatttggac 120
tggtacctgc agaagccagg gcagtctcca cagctcctga tctatacgct ttcctatcgg 180
gcctctggag tcccagacag gttcagtggc agtgggtcag gcactgattt cacactgaaa 240
atcagcaggg tggaggctga ggatgttgga gtttattact gcatgcaacg tatagagttt 300
ccttctctgt acacttttgg ccaggggacc aagctggaga tcaaacgt 348
<210>8
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<212>PRT
<213>全人源抗体15H8轻链可变区氨基酸序列
<400>8
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
85 90 95
Arg Ile Glu Phe Pro Ser Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu
100 105 110
Glu Ile Lys Arg
115
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<213>全人源抗体15H8的重链核苷酸序列
<400>9
gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120
ccagggaagg gactggaata tgtttcagct attagtagta atgggggtag cacatattat 180
gcaaactctg tgaagggcag attcaccatc tccagagaca attccaagaa cacgctgtat 240
cttcaaatgg gcagcctgag agctgaggac atggctgtgt attactgtgc gagagaaggg 300
tatagcagca gctggtacca caactggttc gactcctggg gccaaggaac cctggtcacc 360
gtctcctcag ctagcaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420
acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480
acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540
cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600
acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga 660
gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720
ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtcc ccgggtaaa 1359
<210>10
<211>453
<212>PRT
<213>全人源抗体15H8的重链氨基酸序列
<400>10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val
35 40 45
Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asn Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Tyr Ser Ser Ser Trp Tyr His Asn Trp Phe Asp Ser
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys
450
<210>11
<211>666
<212>DNA
<213>全人源抗体15H8的轻链核苷酸序列
<400>11
gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcttg gatagtgatg atggaaacac ctatttggac 120
tggtacctgc agaagccagg gcagtctcca cagctcctga tctatacgct ttcctatcgg 180
gcctctggag tcccagacag gttcagtggc agtgggtcag gcactgattt cacactgaaa 240
atcagcaggg tggaggctga ggatgttgga gtttattact gcatgcaacg tatagagttt 300
ccttctctgt acacttttgg ccaggggacc aagctggaga tcaaacgtac tgtggctgca 360
ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 420
gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 480
gccctccaat cgggtaactc ccaggagagt gtcacagagc aggacagcaa ggacagcacc 540
tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca caaagtctac 600
gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt caacagggga 660
gagtgt 666
<210>12
<211>222
<212>PRT
<213>全人源抗体15H8的轻链氨基酸序列
<400>12
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
85 90 95
Arg Ile Glu Phe Pro Ser Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu
100 105 110
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
130 135 140
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
145 150 155 160
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
165 170 175
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
180 185 190
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
195 200 205
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
Claims (10)
1.一种全人源抗EGFR抗体，其重链可变区氨基酸序列如SEQ ID NO：6所示，轻链可变区氨基酸序列如SEQ ID NO：8所示。
2.权利要求1所述的全人源抗EGFR抗体，其重链氨基酸序列如SEQ IDNO：10所示，轻链氨基酸序列如SEQ ID NO：12所示。
3.一种核苷酸序列，编码权利要求1～2任一所述的全人源抗EGFR抗体。
4.权利要求3所述的核苷酸序列，其中编码全人源抗EGFR抗体重链可变区的核苷酸序列如SEQ ID NO：5所示，编码全人源抗EGFR抗体轻链可变区的核苷酸序列如SEQ ID NO：7所示。
5.权利要求4所述的核苷酸序列，其中编码全人源抗EGFR抗体重链的核苷酸序列如SEQ ID NO：9所示，编码全人源抗EGFR抗体轻链的核苷酸序列如SEQ ID NO：11所示。
6.一种表达载体，含有权利要求3～5任一所述的核苷酸序列，为pcDNA3.1/ZEO(+)或pcDNA3.1(+)。
7.一种转化权利要求6所述的表达载体的宿主细胞，为CHO-K1细胞。
8.权利要求1～2任一所述的全人源抗EGFR抗体制备治疗抗肿瘤药物中的用途。
9.权利要求8所述的用途，其中肿瘤为EGFR高表达肿瘤。
10.权利要求9所述的用途，其中EGFR高表达肿瘤可为肺癌、头颈癌、结肠癌、脑肿瘤。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995025167A1 (en) * | 1994-03-17 | 1995-09-21 | Merck Patent Gmbh | Anti-egfr single-chain fvs and anti-egfr antibodies |
| CN101029082A (zh) * | 2006-03-03 | 2007-09-05 | 广州枫岛医药科技有限公司 | 一种人源化抗egfr单克隆抗体的重组制备方法 |
| CN101058609A (zh) * | 2006-05-26 | 2007-10-24 | 神州细胞工程有限公司 | 人源抗体及其表达 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995025167A1 (en) * | 1994-03-17 | 1995-09-21 | Merck Patent Gmbh | Anti-egfr single-chain fvs and anti-egfr antibodies |
| CN101029082A (zh) * | 2006-03-03 | 2007-09-05 | 广州枫岛医药科技有限公司 | 一种人源化抗egfr单克隆抗体的重组制备方法 |
| CN101058609A (zh) * | 2006-05-26 | 2007-10-24 | 神州细胞工程有限公司 | 人源抗体及其表达 |
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