CN116178557B - 一种靶向人类her2的单域抗体及其应用 - Google Patents
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Abstract
本发明公开了靶向HER2受体的单域抗体,使用HER2蛋白免疫羊驼后筛选获得特异性高的IDM3‑31,其序列如SEQ ID No.1所示。本发明公开的单域抗体IDM3‑31具有高度特异性和较高的亲和力,且结构简单,便于生产和常温保存,提高了检测HER2受体的准确度,为高效快速检测HER2受体提供了可替代的选择。
Description
技术领域
本发明涉及生物医学或生物技术应用领域,尤其涉及一种靶向HER2的单域抗体及其应用。
背景技术
HER2全称为人表皮生长因子受体-2,也称为ErbB2或Neu,是一种受体酪氨酸激酶(RTK),属于表皮生长因子受体(EGFR/ErbB)家族。编码HER2的基因定位于人类染色体17q21,属于原癌基因,产物为185kD的跨膜蛋白,简称p185,是由1255个氨基酸组成的,包含四个胞外结构域和一个跨膜结构域。HER2蛋白主要通过与HER家族中的其他成员(包括EGFR(HERl/erbBI)、HER3/erbB3、HER4/erbB4)形成异二聚体而与各自的配体结合,从而激活下游信号通路,目前暂未发现能与其直接结合的配体。
HER2蛋白介导的信号转导途径主要有Ras/Raf/MAPK/PI3K以及Akt等信号通路。HER2与配体结合后,主要通过引起受体的二聚化以及胞浆内酪氨酸激酶区的自磷酸化,激活酪氨酸激酶的活性;其具有特殊的开放结构,可以在没有特定配体参与的情况下激活自身,因而能够与自身或HER家族其他成员形成同源/异源二聚体,此外HER2还是HER家族成员异二聚体的首选伴侣,其参与形成的异二聚体活性常强于其它异二聚体。
HER2在多种癌症中都有高度表达,目前已知的乳腺癌、胃癌、肺癌、子宫内膜癌等多种类型的癌症患者都会进行HER2表达水平的检测。HER2高表达在在乳腺癌中的检出率约为15%-25%,在胃癌检出率约为20%,在肺癌中的检出率约2.5%,而在子宫内膜癌中的检出率可以达到18%-80%。
HER2的过表达可能导致受体酪氨酸激酶的持续增强激活从而引起一系列下游级联反应,如MAPK、PI3K-PKB/Akt、STAT等。Her2介导的信号通路还可以调节肿瘤相关基因的表达,从而促进肿瘤侵袭和转移。Her2的过表达在某些浸润性癌症尤其是浸润性乳腺癌的发生和发展中起重要作用,因此Her2已成为乳腺癌的重要生物学标志物和治疗靶点。
单克隆抗体曲妥珠单抗(Trastuzumab,赫赛汀/Herceptin)、帕妥珠单抗(Pertuzumab,帕捷特/Perjeta)、拉帕替尼(Lapatinib,泰立沙/Tykerb)、来那替尼(Neratinib,Nerlynx)、吡咯替尼(Pyrotinib)是特异性靶向HER2的抗体药物,主要用于HER2阳性的浸润或转移性乳腺癌患者的治疗,通过靶向结合HER2,抑制了HER2与其它HER家族成员的二聚化作用,从而抑制肿瘤生长;拉帕替尼、来那替尼和吡咯替尼在靶向抑制HER2的同时还可靶向抑制HER1(EGFR/ErbB1),通过减少EGFR和HER2的自磷酸化,抑制下游的MAPK和AKT信号通路,从而抑制EGFR或HER2过表达肿瘤细胞的增殖。
传统抗体大多只能在哺乳动物表达系统中表达,生产工艺复杂且成本高,抗体稳定性欠佳,免疫原性较高且组织穿透力低,这些综合因素往往导致抗体的治疗效果不佳。相较于传统的抗体,纳米单域抗体具有诸多优势,其可在哺乳动物表达系统和大肠杆菌以及昆重表达系统中进行表达,产生的抗体通常具有较高的水溶性且稳定性强。纳米单域抗体是目前已知的具有完整抗体活性的最小的抗体结构模式,其分子量通常为常规抗体的1/10,这就决定了其具有较高的组织穿透能力,和较短的半衰期,可轻松穿透血脑屏障,且血清清除速度较快,因而纳米单域抗体克服了传统抗体的诸多缺点,这也使其更加适合携带放射性同位素。纳米单域抗体可以快速而具体地穿透肿瘤组织结合靶标,而非结合纳米体可以快速从血液中去除并减少身体的辐射剂量。与传统抗体相比,纳米单域抗体作为示踪剂和靶向内部放疗药物具有更多优势。
然而目前针对HER2的靶向抗体药物缺乏切实有效的纳米单域抗体,因而开发出合适的靶向HER2的抗体药物具有重要的,在该领域缺乏令人满意的针对Her2的纳米体。因此,迫切需研究和应用意义,应积极开发针对Her2的新型有效的特异性纳米体药物。
发明内容
针对上述现有技术中的不足,本发明的目的是提供一种靶向HER2的纳米单域抗体,以解决上述现有技术存在的问题。
因为纳米单域抗体克服了传统抗体的缺点,具有分子量低、稳定性强、溶解性好、易表达、低免疫原性、渗透性和靶向性强,且生产成本低易表达纯化的特点,因而靶向HER2的纳米单域抗体无疑将拥有更广泛的适用性,低免疫原性和强靶向性意味着同计量抗体将拥有更低的机体负担和更高的治疗效果。
根据上述技术原理,本发明第一方面,公开了靶向新HER2的单域抗体VHH,所述单域抗体命名为IDM3-31,所述单域抗体IDM3-31序列如SEQ ID No.1所示。
本发明第二方面公开了一种编码上述第一方面公开的单域抗体的核酸。
本发明第三方面,公开了一种制备上述第一方面公开的两种单域抗体的方法,包括以下步骤:
(1)构建重组表达载体:根据单域抗体IDM3-31的氨基酸序列进行编码核酸的人工合成,获得编码两种单域抗体的核酸,将所获的核酸与表达载体连接,筛选获得阳性表达载体;
(2)将步骤(1)中阳性表达载体转化宿主细胞,诱导表达单域抗体IDM3-31;
(3)纯化所述单域抗体。
进一步的,步骤(1)中所述表达载体可以为真核表达载体或原核表达载体,
优选的,所述表达载体为真核表达载体时,所述表达载体选自:pFastBacDual、pCMV、p-NEO-BAN载体、pEGFP,增强型绦色荧光蛋白表达载体、pEGFT-Actin增强型绿色荧光蛋白/人肌动蛋白表达载体、pSV2表达载体、CMV4表达载体。
优选的,所述表达载体为原核表达载体时,所述表达载体选自:pBAD载体、T7载体、pET表达载体或其他带GST/His等的融合蛋白的表达载体。
进一步的,步骤(2)中所述宿主细胞选自原核宿主或真核表达宿主,优选的,当所述宿主细胞为真核时,选自CHO、COS、HEK-293;当所述宿主细胞为原核细胞时,选自大肠杆菌、谷氨酸棒杆菌。
本发明第四方面,公开了一种制备检HER2蛋白的快速检测试剂的方法,所述方法包括将单域抗体IDM3-31与常规的检测试剂使用的材料采用检测试剂常规制备方法加工制备并包装。
优选的,所述检测试剂包括检测试纸条。
有益效果
本发明提供了特异性靶向HER2的单域抗体IDM3-31,所述单域抗体由HER2蛋白免疫羊驼后筛选获得,由于仅有抗体的重链,因此具有较高的亲和力,且其结构简单,便于生产和保存,常温下可保存更久时间。并且本发明公开的单域抗体IDM3-31的靶向效果好,为HER2的高效快速诊断提供了可替代的选择对象。
附图说明
图1为单域抗体IDM3-31镍柱亲和层析电泳图(注:浓度较低,胶图上未显示出);其中IDM3-31(VHH+标签):26.8kDa,标签:13.35kDa,IDM3-31蛋白:13.48kDa。
图2为单域抗体IDM3-31经分子筛浓缩后的流出图。
图3为单域抗体IDM3-31对几种不同的细胞的West Blotting效果图。
图4为单域抗体IDM3-31的OctetRed亲和鉴定效果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合实施例对本发明的各实施方式进行详细的阐述。然而,本领域的普通技术人员可以理解,在本发明各实施方式中,为了使读者更好地理解本申请而提出了许多技术细节。但是,即使没有这些技术细节和基于以下各实施方式的种种变化和修改,也可以实现本申请所要求保护的技术方案。
实施例中使用的各个试剂等均为常规商业途径购买获得。
实施例1、单克隆抗体筛选
使用HER2蛋白免疫羊驼5-6次,采血后抽提RNA反转并构建羊驼免疫库,用HER2蛋白从免疫库中筛选出32个抗体,将抗体序列构建至Gal10-pSumo载体上进行表达(20℃,180rpm,IPTG=0.2mM),并用Ni柱进行纯化,纯化获得的蛋白经分子筛进一步分离纯化后,用除盐柱将蛋白Buffer置换为PBS,随后进行WB、ELISA和BioRed验证,最终筛选出特异性和亲和力较高的抗体IDM3-31。
单域抗体IDM3-31的氨基酸序列如下所示:
SQVQLVESGGGLVQAGHSLRLSCAASGDTFISTLAWFRRVSGKEREFVGAIRWSAGSTFYADSVKGQFTISRDNAKTTAYLQMNSLKPEDTAVYYCATSVIYRQNYAEAANYDAWGQGTQVTVSS。
下划线分别为两个单域抗体的CDR,按照IMGT规则编号。
抗体筛选具体步骤如下所示:
(1).根据人HER2蛋白基因序列人工合成编码基因,异源表达HER2蛋白,纯化获得HER2蛋白备用;
(2).使用5mg/kg的HER2免疫羊驼,连续免疫5-6次,每次间隔24h;采血抽提RNA;
(3).将上述步骤(2)获得的RNA反转录构建文库,使用大肠杆菌表达系统进行表达;发酵培养后,收集菌体,破碎,纯化回收目的蛋白;
(4).对抗体结合能力进行筛选。
其中,步骤(3)中,包括如下操作:
1)大肠杆菌表达系统自带His标签,将反转录获得的cDNA插入携带6×His Tag和Sumo的表达载体上,转化感受态大肠杆菌,筛选阳性转化子。
2)将阳性转化子培养诱导表达:当菌液生长至OD=0.6时,加入终浓度为20nM的IPTG(20℃,180rpm),诱导表达16-20小时后收菌。
3)离心收集沉淀菌体,将菌体破碎后用Ni柱纯化回收目的蛋白,用PAGE胶鉴定纯化,结果如图1所示。进一步使用分子筛纯化获得纯的单域抗体IDM3-31,结果如图2所示。共筛选获得32个单域抗体,其他效果差的单域抗体效果未展示。
步骤(4)包括如下步骤:
分别使用SKBR3(HER2阳性细胞)、BT549(HER2阴性细胞)、构建的HER2稳转株、p95HER2(构建的p95HER2稳转株)、一抗(纯化的C蛋白1mg/ml,按1:150稀释)、二抗(Rabbitanti-Camelid VHH-HRP(Genscrip)(1:5000))对获得的32个单域抗体进行West Blotting鉴定,确定抗体的特异性和敏感性。结果如图3所示,由图3可以看出,筛选获得的单域抗体IDM3-31仅对SKBR3和HER2有反应,而对截短的HER2以及其他蛋白并不结合。由此可知IDM3-31对HER2全长蛋白具有较高的特异性。
灵敏度效果如图4和表1所示。
表1、单域抗体IDM3-31的OctetRed亲和鉴定数据
Loading Sample ID | Sample ID | KD(M) | kdis(1/s) | kon(1/Ms) | RMax | FullR^2 | Conc.(nM) |
Protein-C | IDM3-31 | 1.885E-07 | 0.000775 | 4111 | 0.5929 | 0.893 | 1000 |
由表1可知,单域抗体IDM3-31对HER2具有较高亲和力,灵敏度高。
Claims (9)
1.一种靶向人类HER2受体的单域抗体C3-31,其特征在于,所述单域抗体的氨基酸序列如SEQ ID No.1所示。
2.一种编码权利要求1所述单域抗体的核酸。
3.一种含有权利要求2所述核酸的表达载体。
4.根据权利要求3所述的表达载体,其特征在于,所述载体为真核表达载体或原核表达载体,当载体为真核表达载体时,所述载体包括:pFastBacDual、pCMV、p-NEO-BAN载体、pEGFP,增强型绦色荧光蛋白表达载体、pEGFT-Actin增强型绿色荧光蛋白/人肌动蛋白表达载体、pSV2表达载体、CMV4表达载体中的一种;当载体为原核表达载体时,所述载体包括:pBAD载体、T7载体、pET表达载体或其他带GST/His等的融合蛋白的表达载体中的一种。
5.一种含有权利要求2所述的核酸或权利要求3-4任一项所述表达载体的工程宿主细胞。
6.根据权利要求5所述的宿主细胞,其特征在于,所述宿主细胞为真核细胞或原核细胞,当宿主为真核细胞时,所述宿主细胞包括:CHO、COS、HEK-293中的一种;所述宿主细胞为原核细胞时,所述宿主细胞包括:大肠杆菌或谷氨酸棒杆菌。
7.一种制备权利要求1所述单域抗体的方法,其特征在于,所述方法包括:利用权利要求2所述的核酸或权利要求3-4任一项所述的载体或权利要求5-6任一项所述的宿主细胞进行表达获得权利要求1所述的单域抗体。
8.权利要求1所述单域抗体或权利要求2所述的核酸或权利要求3-4任一项所述的载体或权利要求5-6任一项所述的宿主细胞制备检测人类HER2试剂盒的用途。
9.一种检测人类HER2的试剂盒,其特征在于,所述试剂盒包含权利要求1所述的单域抗体和其他配套试剂。
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