CN106497931B - 一种针对Her3ECD的ssDNA适配体及其在诊断治疗HER3相关疾病中的应用 - Google Patents
一种针对Her3ECD的ssDNA适配体及其在诊断治疗HER3相关疾病中的应用 Download PDFInfo
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- CN106497931B CN106497931B CN201610928829.7A CN201610928829A CN106497931B CN 106497931 B CN106497931 B CN 106497931B CN 201610928829 A CN201610928829 A CN 201610928829A CN 106497931 B CN106497931 B CN 106497931B
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公布了一种通过指数富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment，SELEX)筛选得到的针对HER3胞外区蛋白(extracellular domain，ECD)高亲和力、高选择性的DNA适配体。本发明通过ELISA、细胞流式、激光共聚焦实验进一步证明该适配体无论是对游离Her3ECD还是对高表达Her3ECD蛋白的乳腺癌细胞都具有一定的特异性和亲和性，为HER3高表达的相关疾病的诊断治疗提供了新的思路。
Description
技术领域
本发明涉及生物技术领域，具体涉及与表皮生长因子HER3的胞外域(Her3ECD)特异性结合的ssDNA适配体及其在诊断治疗HER3相关疾病中的应用。
背景技术
HER3又称ErbB3，LCCS2，ErbB-3，c-erbB3，erbB3-S，MDA-BF-1，c-erbB-3，p180-ErbB3，p45-sErbB3，p85-sErbB3，是酪氨酸激酶表皮生长因子家族成员之一，大小为161kDa。人HER3基因定位于染色体12q13。该家族由4个成员组成，除HER3外，还有EGFR、HER2、HER4。每位成员又是由三个部分构成，分别是胞外区配体结合区、跨膜区、胞内激酶区。HER3的胞外区有神经调节蛋白结合位点但胞内没有激酶活性。因此，必须与该家族其他成员形成异源二聚体后才能激活MAPK、PI3K-Akt通路，参与包括乳腺癌、肺癌、头颈癌等多种肿瘤发生发展。
目前，国内外已有多家公司以HER3为靶点着手研发抗癌药物，HER3抑制剂包括Merrimack制药的seribantumab(MM-121)，目前处于III期临床阶段；葛兰素史克的GKS2849330，处于I期临床研发阶段；以及罗氏旗下基因泰克的HER3/EGFR双向抑制剂MEHD7945A，正处于研发中期。
靶向Her3ECD的抑制剂药物可以应用在HER3过表达的疾病模型中，包括：1)HER3在HER2介导的乳腺肿瘤中高表达。在HER2高表达肿瘤中敲低HER3表达后会引起肿瘤缩减，与敲低HER2效果抑制，而在该肿瘤模型中敲低EGFR则不引起肿瘤的消减，此外临床标本检测表明，在HER2高表达乳腺癌标本中，HER3高表达与HER2的高表达具有高度一致性，而EGFR与HER2的表达无此相关性，上述研究均强调了HER3在HER2致癌机制中的独特的、关键作用。2)HER3在黑色素瘤中高表达。HER3可能作为EGFR和/或HER4的协同作用分子，在黑色素瘤中显著高表达，且与疾病的恶性程度和预后差相关。3)HER3在肺癌中的高表达的发生率是18％-100％，尤其是对EGFR酪氨酸激酶抑制剂(tyrosine kinase inhibitor，TKI)吉非替尼(gefitinib)治疗耐药的非小细胞肺癌(non-small lung cell lung carcinomas，NSCLC)中，与治疗敏感的NSCLC比较，其HER3高表达率为100％，此外，在NSCLC患者中，HER3高表达与其脑转移、生存期短具有显著相关性。4)HER3在儿童骨肉瘤中显著高表达；5)HER3在某些类型的前列腺癌、结肠癌和卵巢癌中高表达(表达率在10-90％之间)，尽管其参与机制尚未阐明，但可以明确HER3高表达与其高转移、预后差密切相关。
近些年来，寡核苷酸适配体作为抗体分子的前景性替代分子，其研究较为引人注目。适配体是应用指数富集的配体系统进化技术(systematic evolution ofligands byexponential enrichment，SELEX)从单链随机寡核苷酸文库中筛选得到的能与靶标高特异性、高亲和力结合的配体。适配体具有靶分子范围广泛；高特异性与亲和性；与靶分子的结合条件可调控，易人工合成；稳定性好，易修饰；分子小，空间位阻小等优势。且作为低免疫原性的“抗体类似物”，适配体类靶向治疗药物已经应用于多种癌症、黄斑变性、血管性血友病、糖尿病等疾病的治疗，同时也可作为药物的靶向配体，开发适配体-药物偶联物(aptamer-drug conjugates，ApDCs)，协助药物在特定的组织位点释放。从目前研究及临床试验来看，适配体为靶向治疗提供了一个很好的平台。且有些药物已经通过FDA认证批准上市，在生物医药领域显示出巨大的应用前景。
目前针对Her3ECD的适配体研究较少，目前仅有2003年的文献报道针对Her3ECD具有较高亲和力的RNA适配体，但后续再无报道。基于HER3在多种癌症的发生、发展、耐药及预后中扮演着非常重要的角色，本发明可能应用在HER3相关癌症的诊断、治疗及相关研究中。
发明内容
本发明目的在于提供一种对Her3ECD具有高亲和力高选择性的ssDNA适配体。
本发明方法利用指数级富集配体的系统进化技术(SELEX技术)，选Her3ECD为靶蛋白。构建Her3ECD适配体-药物偶联物(ApDCs)递送系统，对其靶向性、抑瘤作用及相关机制进行研究，为同类药物的研发提供参考。
本发明的目的可以通过以下措施达到：本发明针对的靶蛋白Her3ECD抗原通过将Her3ECD质粒瞬时转染到293E细胞表达，蛋白大小为100kDa，三天后收集培养基并进行镍柱纯化得到。
本发明溶液中的适配体在靶目标分子存在的情况下能发生适应性的折叠，会形成发夹、茎环、假结、凸环、G-四聚体等特殊的三维空间结构，通过氢键、疏水堆积作用、范德华力等与靶目标分子紧密的结合，体现出较高的亲和力。
一方面，本发明提供了能特异性识别Her3ECD的ssDNA适配体。
在优选实施方案中，本发明所述的ssDNA适配体具有选自SEQ ID NO:1或SEQ IDNO:2所示的核苷酸序列。
本发明所述的适配体也可以是RNA，且文献报道RNA适配体一般显示出更高的亲和力，但RNA不如ssDNA稳定，在筛选及成药的过程中存在较大的困难。
本发明所述针对Her3ECD的适配体，经游离Her3ECD蛋白和两种HER3高表达乳腺癌细胞系MCF7、BT474验证，具有较好的特异性和亲和性。经ELISA、细胞流式、激光共聚焦实验证实了本发明适配体对Her3ECD高表达的乳腺癌细胞系具有较好的亲和性和特异性。
因此，另一方面，本发明提供了用于诊断Her3ECD高表达乳腺癌的试剂，其中含有本发明所述的ssDNA适配体。
另一方面，本发明提供了用于诊断Her3ECD高表达乳腺癌的DNA芯片，其上固定有本发明所述的ssDNA适配体。
另一方面，本发明提供了用于治疗Her3ECD高表达乳腺癌的药物组合物，其中含有本发明所述的ssDNA适配体。
另一方面，本发明提供了所述的ssDNA适配体在制备Her3ECD高表达的乳腺癌诊断试剂中的用途。
另一方面，本发明提供了所述的ssDNA适配体在制备用于Her3ECD高表达乳腺癌的靶向治疗药物中的用途。
本发明的适配体容易获得，并且能够大量合成，容易进行化学修饰改造，稳定性好，可通过糖苷2’位加以氟或氧甲基修饰，或连接高分子量的聚乙二醇或胆固醇，降低肾清除率，改善其药代动力学。稳定性修饰后适配体还可与纳米粒连接，实现对化疗药、核酸等药物的靶向递送。可见适配体无论在临床诊断还是药物研究等方面都有广泛应用。
本发明的有益效果：
本发明的Her3ECD高亲和力高特异性的适配体，可通过化学修饰改造，应用于HER3高表达的相关疾病诊断和治疗，可通过构建靶向递送系统在提高药效、减少毒副作用的基础上实现对化疗药、核酸类药物的靶向递送。所示HER3相关性疾病包括EGFR抗体治疗失败型肺癌、HER2阳性型抗体类药物耐药型乳腺癌、宫颈癌等。该目标适配体还可以用于与HER3相关的科学研究、或肿瘤等医学或药学的应用研究。
本发明所述序列如下：
7#GGGAGCTCAGAATAAACGCTCAAAGGGTCAAGCTGATTACACTTTGTCCACTATTGGGTCCTTCGACATGAGGCCCGGATC
13#GGGAGCTCAGAATAAACGCTCAAGGCTAACAGCACGCAACGGGGGGGAGTAATCGTGTCTGTTCGACATGAGGCCCGGATC
附图说明
图1图示的是本发明所述Her3ECD适配体的二级结构图；
图2图示的是细胞流式检测本发明所述适配体与MCF-7细胞的结合特异性；
图3细胞流式检测适配体与BT474细胞的结合实验；
定义
本发明中提到的参考著作、专利、专利申请及科学文献，构成本领域技术人员的已有知识，作为整体在此引入作为参考，与这些文献专门单个引入作为参考时的范围相同。在本申请所引入文献与本说明书特定含义之间如有任何抵触，都应该以后者为准。另外，本领域对词汇和短语定义的已有理解与本说明书中对该词汇或短语的专门解释而定下的定义之间如有抵触，都以后者为准。
在进一步阐述本发明之前，我们有必要认识到，本发明并不局限于描述的特定的实施方案，也就是说，在具体形式上可能存在着变化。还有一点需要提醒的是，由于本发明的范围受附加的权利要求书的限制，因此，本文使用的术语只是为了描述特定实施方案的目的，而不是为了限制本发明的目的。
另外应注意，如本说明书中所使用的，单数形式包括其所指对象的复数形式，除非清除且明确的限于一个所指对象。术语“或”可与术语“和/或”互换使用，除非上下文另有清除指明。
在某些实施方案中，适配体与其靶点之间的亲和力用KD(解离常数)来表征，它低于10-7M、10-8M、10-9M、10-10M、10-11M、或者约10-12M或更低。
具体实施方式
本发明公开了一种针对Her3ECD的适配体，及其相关亲和力验证实验证明该适配体具有较好的亲和性及特异性，将为HER3高表达的相关疾病的诊断治疗提供新的思路。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的应用以及通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施方案对本发明作进一步的详细说明。
实施例
实施例1.基于SELEX技术筛选Her3ECD ssDNA适配体
1.1构建随机单链DNA(即ssDNA)寡核苷酸文库5’-GGGAGCTCAGAATAAACGCTCAA(N38)TTCGACATGAGGCCCGGATC-3’，其中N38为38个碱基的随机序列，两端为序列固定的引物序列，引物upⅠ为5’-GGGAGCTCAGAATAAACGCTCAA-3’，引物downⅠ为5’-GATCCGGGCCTCATGTCGAA-3’，引物downⅡ为5’-生物素-GATCCGGGCCTCATGTCGAA-3’，库容量为1014以上，合成的随机单链DNA寡核苷酸文库按照1OD/管分装，冻干粉于-20℃保存备用。
1.2将Her3ECD质粒用PEI试剂(Invitrogen公司)瞬时转染293E细胞，三天后收取细胞培养上清经镍柱纯化获得带有His标签的靶蛋白，经考马斯亮蓝染色及WesternBlotting鉴定该抗原条带表达正确且纯度较好，可作为靶蛋白进行后续筛选。
1.3选用带有His标签的磁珠通过Binding Buffer(磷酸钠20mM，氯化钠50mM，咪唑5mM，PH＝7.4)固化蛋白，并加入相应PBST(K2HPO4 50mM，氯化钠150mM，Tween-20 0.05％，PH＝7.5)放于4℃冰箱保存。
1.4ssDNA文库与靶蛋白结合：取1nmol ssDNA文库用100μL PBST溶解，95℃变性5min，冰浴10min后，加入到10mL上述步骤(3)中与靶蛋白连接的磁珠中，于室温旋转结合30min，然后利用磁力架弃上清，磁珠用PBST缓冲液洗涤3次，弃上清，用Elution buffer(磷酸钠20mM，氯化钠50mM，咪唑500mM，PH＝7.4)将磁珠上的蛋白进行洗脱，具有一定亲和性的ssDNA会结合在蛋白上，将洗脱液当做QPCR模板进行扩增，引物选择upⅠ和downⅡ，程序：预变性95℃15min，PCR反应：95℃10s变性；58℃20s退火；72℃30s延伸。(注明：做过相应实验比较将蛋白核酸混合物进行qPCR或者将结合的核酸沉淀回收再进行qPCR，通过较为灵敏的银染图鉴定，两者差别很小，且考虑到核酸回收过程中存在大量损失，后直接采取磁珠洗脱产物直接作为qPCR模板使用。)
1.5链霉亲和-生物素磁珠分离dsDNA为ssDNA:取链霉亲和-生物素磁珠用对应Buffer(Tris-Hcl(PH＝7.5)10mM，EDTA 1mM，氯化钠1M，Tween-20 0.01％-0.1％)洗涤适量磁珠两次，加入qPCR产物室温混匀结合30～60min，Buffer洗涤三次弃净上清，加入50μl0.1mol/L新鲜配制的NaOH，于37℃孵育15min，使得dsDNA解链。将含有ssDNA的50μl NaOH溶液直接作为下一轮的筛选文库，或加入到1ml PBST中，用10mmol/ml的磷酸二氢缓冲液将pH调节至7.5后保存。
1.6不相关蛋白反筛：取不相关蛋白进行筛选，与正筛的区别是，我们留取与不相关蛋白未结合的缓冲液体系。将该体系通过加入等体积的酚：氯仿：异戊醇(25:24:1)抽提两次(静置5min，离心12000rpm 5min)，加入等体积遇冷的冰乙醇，混匀后冰上静置30min，12000rpm离心10分钟，弃上清，70％乙醇漂洗1遍，弃上清，晾干沉淀。加入ddH2O溶解作为模板进行qPCR。
1.7qPCR溶解曲线结合银染条带比对，确认多次筛选后文库的准确性。
1.8筛选到最后的序列经PCR扩增纯化，酶连克隆测序得到多条适配体，分析其二级结构、自由能、同源性，选出几条备选序列。
实施例2.Her3ECD ssDNA适配体亲和力鉴定
以Her3ECD包被酶标板，设置不同浓度梯度，依次加入1.25、2.5、5、10、50、100及200pmol蛋白，100μl包被液稀释，4℃包被过夜；1％BSA 37℃封闭2h；加入10pmol 5’端生物素修饰Her3ECD适配体，37℃孵育1h；PBST洗涤5遍，加入SA-HRP抗体(1:8000)，37℃孵育1h；PBST洗涤5遍，加入TMB底物显色。其中以GPC3、BSA为阴性对照。检测结果显示筛选的Her3ECD适配体与其余两种蛋白不结合，数值与Blank相一致，进一步证明该适配体针对Her3ECD具有较高的特异性与亲和力。拟合亲和力曲线得出适配体的KD值，其中7#，13#的KD值分别为116nM、98nM，其序列分别为SEQE ID NO:1、SEQE ID NO:2，其二级结构见图1。
实施例3.Her3ECD ssDNA细胞结合特异性鉴定
细胞流式：选经NRG1提前4h刺激的乳腺癌细胞系MCF-7(MCF7细胞属于NRG1刺激阳性细胞，经刺激后膜表面HER3表达提高)及未经刺激的乳腺癌细胞系BT474进行适配体的亲和性特异性验证，选购买商品化的HER3抗体做阳性对照，选低表达HER3的人胚胎细胞系293T作为阴性对照。取消化的细胞PBS洗两遍，重悬计数，取1×106个，用100μl PBS重悬，分别取5’端FAM标记的适配体100pmol以及APC标记的商品化的HER3抗体与细胞避光孵育1h，PBS洗两遍，2％多聚甲醛固定，分别用细胞流式仪检测细胞阳性率。
实验结果显示，商品化HER3抗体与细胞的亲和力在95％，适配体结合乳腺癌细胞系MCF-7(图2流式检测仪：BD，美国biosciences)及BT474(图3流式检测仪：Guava，德国Merck Millipore)的阳性率均在75％左右，两者均对293T表现出极低的亲和性(5％左右)，体现出适配体本身良好的亲和力及特异性。
实施例4.Her3ECD ssDNA靶向入胞
激光共聚焦：商品化的HER3抗体与适配体理论上都与细胞内源表达HER3ECD抗原结合，两者共孵育形成靶位竞争，但是也可以通过共定位说明其与细胞内源抗原结合特性。将乳腺癌细胞系MCF-7提前种于24孔板大小圆形玻片上，实验4h前加入NRG1刺激MCF-7细胞，PBS洗两遍去除残留培养基，加入2％多聚甲醛固定细胞，PBS洗两遍，加入FAM标记的适配体与商品化的兔源HER3抗体避光共孵育1h，PBS洗两遍，加入针对抗体的PE标记的羊抗兔IgG二抗孵育30min，PBS洗两遍，加入300nM DAPI染核3min，PBS洗两遍，加入防淬灭剂倒置于盖玻片上，指甲油封片，激光共聚焦显微镜观察。结果显示，FITC-适配体在细胞质和细胞核中均有分布，PE-HER3商品化抗体在主要分布胞浆中，其次核仁中可见，但是除核仁以外的细胞核中分布较少。
上述结果表明，本发明的适配体和商品化抗体具有共定位，提示两者均与内源性HER3抗原结合后入胞。值得注意的是，适配体除与该抗体共定位外，在细胞核仁中高表达，其核仁定位机制可能与入胞后微观分布机制与抗体不同造成，具体机制有待进一步阐明。
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<110> 中国人民解放军军事医学科学院放射与辐射医学研究所 军科正源（北京）药物研究有限责任公司
<120> 一种针对Her3ECD的ssDNA适配体及其在诊断、治疗HER3相关疾病中的应用
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<222> (1)..(81)
<400> 1
gggagctcag aataaacgct caaagggtca agctgattac actttgtcca ctattgggtc 60
cttcgacatg aggcccggat c 81
<210> 2
<211> 81
<212> DNA
<213> 人工序列
<220>
<221> gene
<222> (1)..(81)
<400> 2
gggagctcag aataaacgct caaggctaac agcacgcaac gggggggagt aatcgtgtct 60
gttcgacatg aggcccggat c 81
Claims (7)
1.一种特异性识别HER3胞外域的ssDNA适配体，其核苷酸序列如SEQ ID NO:1或SEQ IDNO:2所示。
2.权利要求1所述的ssDNA适配体的经修饰的产物，所述修饰选自通过糖苷2’位加以氟或氧甲基、连接高分子量聚乙二醇或胆固醇、或连接纳米粒。
3.一种HER3胞外域高表达的乳腺癌诊断试剂，其中含有权利要求1所述的ssDNA适配体。
4.一种HER3胞外域高表达的乳腺癌诊断用DNA芯片，其上固定有权利要求1所述的ssDNA适配体。
5.一种HER3胞外域高表达乳腺癌的靶向治疗药物，其中含有权利要求1所述的ssDNA适配体。
6.权利要求1所述的ssDNA适配体在制备HER3胞外域高表达的乳腺癌诊断试剂中的用途。
7.权利要求1所述的ssDNA适配体在制备用于HER3胞外域高表达乳腺癌的靶向治疗药物中的用途。
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Non-Patent Citations (2)
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| Aptamer based strategy for cytosensing and evaluation of HER-3 on the surface of MCF-7 cells by using the signal amplification of nucleic acid-functionalized nanocrystals;Shaoping Lv等;《Analytica Chimica Acta》;20130220;第772卷;第26-32页 * |
| Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3;Chi-hong B. Chen等;《PNAS》;20030805;第100卷(第16期);第9226-9231页 * |
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