CN110563846A - 一种特异性结合egfr的单克隆抗体及其抗癌用途 - Google Patents

一种特异性结合egfr的单克隆抗体及其抗癌用途 Download PDF

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CN110563846A
CN110563846A CN201910907276.0A CN201910907276A CN110563846A CN 110563846 A CN110563846 A CN 110563846A CN 201910907276 A CN201910907276 A CN 201910907276A CN 110563846 A CN110563846 A CN 110563846A
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彭菲
顾超
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Nuosa Union (Beijing) Biomedical Technology Co., Ltd
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Abstract

本发明提供了一种抗EGFR抗体，该抗体与EGFR分子具有很强的亲和力，可特异性结合抗原分子，具有较好的生物学的活性，并且能够用于癌症的治疗。

Description

一种特异性结合EGFR的单克隆抗体及其抗癌用途
技术领域
本发明涉及生物制药领域，具体的涉及一种特异性结合EGFR的单克隆抗体及其抗癌用途。
背景技术
表皮生长因子受体(epidermal growth factor receptor,EGFR,HER1,c-ErbB-1)是由1186个氨基酸残基构成,分子量为170kD的一种跨膜糖蛋白。EGFR分为胞外区、跨膜区和胞内区3部分。胞外区包含L-1/2两个富含亮氨酸序列和两个富含半胱氨酸序列CR-1/2,是由621个氨基酸残基构成的与配体结合的氨基端区。跨膜区是23个氨基酸残基构成的α螺旋。胞内区由近膜区(juxtamembraneregion,JM)、酪氨酸蛋白激酶区和C-末端构成,含有542个氨基酸残基。EGFR属于Ⅰ型酪氨酸激酶受体亚族(ErbB1-4),具有酪氨酸激酶的活性。
EGFR稳定的表达于许多上皮组织,以及间质和神经源性组织。不同器官发生的实体瘤也高表达EGFR,如头颈部癌、卵巢癌、宫颈癌、膀胱癌和食管癌等。转化生长因子-α(transforminggrowthfactor-α,TGF-α)和表皮生长因子(epidermalgrowthfactor,EGF)等生长因子是EGFR的内源性配体。这些配体与EGFR的结合导致EGFR二聚化,激活了受体胞内酪氨酸蛋白激酶的活性,使C-末端特异的酪氨酸残基磷酸化(如Tyr 1068,1086,1148,1173等位点),为细胞内信号转导因子提供结合位点,由此启动Shc,Grb2,RasM/APK,PI3K及JAKs/STATs等多条信号转导途径。EGFR通过介导这些通路调节正常细胞的生长和分化,增加肿瘤细胞的侵袭力、促进血管生成、抑制肿瘤细胞的凋亡。EGFR在肿瘤中的高度表达及其在肿瘤细胞生长、分化中起着重要作用的这些特点,使EGFR成为具有良好前景的肿瘤诊断和治疗的靶点。
目前用于EGFR靶向性治疗肿瘤的药物主要分为两类:EGFR单克隆抗体和小分子化合物酪氨酸激酶拮抗剂。酪氨酸激酶拮抗剂主要为小分子喹啉类化合物,能够竞争性抑制ATP与EGFR胞内酪氨酸激酶结构域的结合,进而影响酪氨酸残基磷酸化,抑制EGFR下游的信号转导。酪氨酸激酶拮抗剂的临床疗效有很大的个体差异,使治疗剂量的确立存在困难。EGFR单克隆抗体是与内源性配体竞争结合EGFR,通过抑制酪氨酸激酶的激活、促进EGFR内化等作用产生抗肿瘤效应。目前已有3种抗EGFR单克隆抗体上市,与其他化疗药相比,这些抗体作用特异性强,副作用小,在临床上取得了较好的疗效。
目前该靶点的单抗主要有如下几种产品：爱必妥(西妥昔单抗)：由Imclone公司与BMS联合开发的一种嵌合型IgG1单克隆抗体，可竞争性结合EGFR细胞外结构域，用于治疗转移性头颈癌、非转移性头颈癌、转移性结直肠癌、非小细胞肺癌等。2016年全球销售规模达8.8亿欧元。Vectibix(帕尼单抗)：由安进推出的全人源化单克隆抗体，与EGFR有高度亲和性，用于治疗转移性结直肠癌，是第一个结直肠癌生物靶向治疗生物药，与西妥昔单抗相比有更好的免疫耐受性。2016年全球销售规模达6.1亿美元。Portrazza(耐昔妥珠单抗)：由礼来于2015年11月推出的人源化单抗，获批治疗转移性鳞状非小细胞肺癌。
Cetuximab,panitumumab和nimotuzomab这3种抗EGFR单克隆抗体具有靶向性强、毒副作用相对较小的特点。这些单克隆抗体与放化疗结合在临床上用于治疗EGFR阳性肿瘤已经取得了一定的疗效,为肿瘤患者带来了新的曙光。目前在我国抗EGFR单克隆抗体的临床应用还不够广泛,费用也相对较高。抗EGFR单克隆抗体未能单独用于肿瘤的治疗,还需要与放疗或化疗联合应用；而且在临床用于治疗肿瘤的类型也较少,这些在一定程度上限制了抗EGFR单克隆抗体在临床上的使用。
基于现有技术的现状，在本领域还缺乏针对EGFR具有更好结合特性同时活性仍较高的国产化的单克隆抗体。
发明内容
本发明一方面，提供一种EGFR胞外蛋白片段。本领域公知，EGFR胞外段蛋白全长由1～621个氨基酸残基构成，其中EGFR胞外段蛋白的L2结构域含有配基结合域，独立的L2结构域仍具有与配体结合能力，且亲和力与EGFR相近。因此构建EGFR氨基酸序列为从55～525个氨基残基，包含了完整的L2结构域，并在N端添加His标签，表达与纯化后得到EGFR胞外段蛋白，一方面可用于研究EGF与EGFR结合的特异性强度等，另一方面可通过阻止EGF与EGFR结合而抑制EGFR信号并起到抑制肿瘤生长的作用。
本发明另外提供一种具体的，EGFR单克隆抗体，轻链可变区的氨基酸序列如SEQID NO:1所示，重链可变区的氨基酸序列如SEQ ID NO:5所示。
更具体的，EGFR单克隆抗体中，重链恒定区为同种型人CH的恒定区，轻链恒定区为人CL的恒定区。
更具体的所述序列如下所示：
3G1重链序列：
EVQLQQSGPELVKPGASVKISCKASQZSYFFGHWTWVKQRPGQGLECQVGWSSLNKKDDYRACSSTKATLTADTSSSTAYMQVSSLTSEDSAVYFCARQSTGNYSQDRCQWGQGTSVTVSS(SEQ ID NO.:1)。
3G1轻链序列：
DIVMTQSPSSLTVTAGEKVTMSCTGYEDLASYYKTGGVSTWYQQKPGQPPKLLIYAAWGSPGGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCCPSYQLFGSFGGGTKLEIKR(SEQ ID NO.:2)。
进一步的，本发明还提供一种如上述抗EGFR单克隆抗体的编码基因。
进一步的，抗EGFR单克隆抗体编码基因的表达载体。
更进一步的，本发明提供了EGFR抗体在制备抗肿瘤药物中的应用。
更进一步的，本发明提供一种药物组合物，其含有3G1所示的抗体药物以及合适的载体。
另外一方面，本发明提供一种检测试剂盒，其特性在于所述试剂盒能够特异性的检测人EGFR蛋白，以及配套的说明书及辅助试剂。
一种免疫偶联物，其特征在于，该免疫偶联物含有：
(a)载体部分，所述载体部分含有本发明所述的抗体；和(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
一种多核苷酸，其特征在于，它编码本发明所述的抗体。
一种载体，其特征在于，它含有前述的多核苷酸，并且能够用于表达所述的多核苷酸。
一种遗传工程化的宿主细胞，其特征在于，它含有前述的载体。
有益效果
本发明提供了一种新型的抗EGFR抗体，该抗体与EGFR分子具有很强的亲和力，可特异性结合抗原分子，具有较好的生物学的活性，并且能够用于相应的疾病的治疗。
附图说明
图1为SDS-PAGE电泳图，图中条带为EGFR蛋白。
图2为抗EGFR抗体的RTV图。
具体实施方式
本文所提供的实施例代表优选的实施方案，为示例性的，并且不旨在为对本发明的范围的限制。
实施例1 EGFR蛋白表达
以人血DNA为模板，PCR扩增EGFR基因片段，其基因引物:
F1 5-CCTGCACATATGCTGGAGGAAAAGAAAGTT-3，
F2 5-CCCTGACGCAGAGAATCGAGCTCTAAGAA-3，98℃预变性10min后开始循环，然后98℃变性1min，62℃退火1min，72℃延伸2min，共进行32个循环；然后用Nde I和Xho I对EGFR基因片段和pET28a质粒酶切得到相同粘性末端的产物；最后用连接酶连接过夜得到EFGR-pET28a载体，转入DH5α克隆菌株表达，选取阳性单克隆载体双酶切测序鉴定，测序正确将重组质粒转入BL21工程菌中表达。诱导BL21表达EGFR，将50μL菌液+0.1％Kana(100mg/mL)接种到5mL LB管中，培养12h；将200μL菌液+50μL Kana接种50mL小锥形瓶中，培养12h；最后，接种10mL菌液+500μL Kana接种到500mL大锥形瓶中，菌体A值达到一定值，用ITPG诱导ED-EGFR表达。菌液8000g离心10min，菌体质量(g)与破碎液体积(mL)比例1∶8～10，匀浆。将得到菌液在超声破碎仪上破碎，工作时间3s，暂停5s，振幅65％，直到所有菌体破碎完全，8000g离心40min，得到上清液。用5个柱体积的平衡缓冲液(150mmol/L NaCl，20mmol/L PB，1mmol/LPMSF)平衡镍亲和层析柱，待平衡液冲洗至基线上样，用3个柱体积平衡液洗去未结合到柱上的蛋白；用洗脱液(500mmol/L NaCl，20mmol/L PB，500mmol/L咪唑)洗脱，收集280紫外吸光值曲线上升时的样品，直到恢复到基线停止收集，将得到的EGFR用SDS-PAGE分析纯度。从图1的结果可以看出，得到了纯度较高的靶标蛋白。
实施例2 EGFR单克隆抗体的制备
以实施例1制备得到的EGFR片段为免疫原,免疫BALB/c小鼠,腹腔注射(100mg/只),每隔21d免疫1次,共免疫3次。于融合前4～5d,再次腹腔注射50mg/只以加强免疫,取小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0按常规方法进行细胞融合,用含1％HAT、10％小牛血清的DMEM完全培养基培养杂交瘤细胞,4d后半量换液,10d后改用含1％HT、10％小牛血清的DMEM完全培养基,2周后用含10％小牛血清的DMEM完全培养基培养。吸取杂交瘤培养上清用间接免疫荧光标记法和流式分析进行筛选后获得阳性克隆。适时换液并及时按上述方法进行复筛。选择分泌抗体效价高、呈单个克隆生长、形态良好的抗体分泌阳性率大于99％的杂交瘤细胞3G1继续亚克隆培养,并及时液氮冻存。经过序列鉴定，其重链可变区序列如SEQID NO：1所示，其轻链可变区如SEQ ID NO：2所示。
实施例3单克隆抗体的性质鉴定
Ig亚类的鉴定采用快速定性试纸法,具体操作按使用说明书进行。单克隆抗体的制备采用本领域常用的体内诱生腹水法,8～l0 d后收获腹水,离心取上清分装,保存于-80℃。应用ProteinG亲和层析法纯化单抗,按照Pharmacia公司提供的单抗纯化方案进行。应用间接免疫荧光标记法测定纯化后抗体的效价。结果显示：快速定性试纸法鉴定表明,3G1重链为IgGl,轻链则为κ。采用腹水诱生方法生产单克隆抗体,腹水纯化后单抗蛋白含量在10mg/ml左右。免疫荧光法分析结果表明,纯化后单抗的效价为1∶50000以上。
实施例4抗体结合特性鉴定
利用Fortibio测定抗体结合及解离常数。利用OctetRED(Fortebio,USA)仪器，测定抗3G1抗体亲和力。PBS稀释抗体浓度为10μg/mL，包被AHC传感器；PBS为对照，稀释3G1浓度为0.1μg/mL、1μg/mL、3μg/mL、10μg/mL、测定抗3G1抗体与EGFR蛋白相互作用的结合、解离曲线；利用GlobeFitting拟合曲线，计算两者相互作用的解离常数。结果，本发明获得的单克隆抗体其解离常数达到了0.17nM。
实施例5 EGFR抗体对裸小鼠皮下移植瘤的生长抑制作用
人表皮细胞癌A431细胞，购置于美国典型细胞中心(ATCC)，该细胞株为EGFR高表达表达的人肿瘤细胞株。BALB/cA裸小鼠，雌性，40-45日龄，体重18±2g，购自上海灵畅生物科技有限公司；每组动物数6只。
受试药EGFR抗体采用pH6.0的PBS溶解，剂量为O.5mg/dose剂量组；阳性对照药Erbitux的剂量为O.5mg/dose。
1、小鼠接种肿瘤细胞
收集体外培养的人表皮细胞癌A431细胞，将细胞悬液浓度调整为2.5*105个/ml；在无菌条件下，接种200ul细胞悬液于裸小鼠背部皮下，用游标卡尺测量裸小鼠皮下移植瘤直径，待肿瘤生长至100-200mm3后，将动物随机分组。
2、分组给药
受试药EGFR单克隆抗体O.5mg/dose，阳性对照药Erbitux按O.5mg/dose剂量给药，空白对照组给等量受试药PBS缓冲液。
动物随机按上述3组给药，每周腹腔注射给药两次，连续给药6周。
3、测量及称重
整个实验过程中，每周2次测量移植瘤直径，同时称量小鼠体重；肿瘤体积(tumorvolume,TV)的计算公式为:TV＝1/2XaXb2，其中a,b分别表示长、宽；根据测量的结果计算出相对肿瘤体积(relative tumor volume)RTV，计算公式为:RTV＝Vt/V0，其中V0为分笼给药时(即do)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。
4、评价指标
抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下:
T/C(％)＝(TRTV/CRTV)X100％
TRTV治疗组RTV；其中，CRTV:阴性对照组RTV。
疗效评价标准:T/C(％)>40％为无效；T/C(％)<＝40，并经统计学处理p<0.05为有效。结果如下：
表1 3G1抗体对人表皮细胞癌裸小鼠移植瘤的42天生长抑制结果图
实验结果显示:从图亲可以看出，EGFR抗体在O.5mg/dose剂量组给药1周左右，移植瘤即明显缩小；在给药14天后，荷瘤小鼠的肿瘤体积较给药初期变小。给药42天后，其T/C值为8.5，显示了较好的抑制效果。在整个实验过程中，裸小鼠体重无明显下降，无死亡。阳性对照药Erbitux在给药剂量O.5mg/dose时，对人表皮细胞癌A431裸小鼠移植瘤的生长也呈现出显著的抑制作用，其T/C值为13.0；结果表明，EGFR抗体对EGFR高表达的人表皮细胞癌A431裸小鼠皮下移植瘤的生长抑制作用非常显著。并且在同等给药剂量下，受试药EGFR抗体的体内抗肿瘤作用比Erbitux效果还有提高的表现。
应当理解的是，本发明在其应用上并不一定局限于在以下说明中所描述和/或在附图中所说明的组件的构造和布置的细节。本发明能够具有除所述和以不同方式实践或进行的那些实施方案之外的实施方案。而且，应理解本文所采用的短语和术语以及摘要出于描述目的并且不应视为限制性的。
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<110> 北京岳昊科技发展有限公司
<120> 一种特异性结合EGFR的单克隆抗体及其抗癌用途
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Claims (10)

1.一种EGFR单克隆抗体，其特征在于：轻链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的氨基酸序列如SEQ ID NO:2所示。
2.一种如权利要求1所述的EGFR单克隆抗体的编码基因。
3.含有权利要求2所述的EGFR单克隆抗体编码基因的表达载体。
4.权利要求1所述的EGFR抗体在制备抗肿瘤药物中的应用。
5.一种药物组合物，其含有权利要求1所述的抗体以及合适的药物载体。
6.一种检测试剂盒，其特性在于含有权利要求1所述的抗体；同时所述试剂盒能够特异性的检测人EGFR蛋白，以及配套的说明书及辅助试剂。
7.一种免疫偶联物，其特征在于，该免疫偶联物含有：
(a)载体部分，所述载体部分含有权利要求1所述的抗体；和(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
8.一种多核苷酸，其特征在于，它编码权利要求1所述的抗体。
9.一种遗传工程化的宿主细胞，其特征在于，它含有权利要求3的载体，并且能够表达所述的抗体。
10.一种治疗癌症的方法，其特征在于：对患者施用权利要求1所述的抗体。
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EP2727943A1 (en) * 2012-11-05 2014-05-07 MAB Discovery GmbH Trispecific antibodies against human EGFR, HER2 and HER3
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