CN114634566A - 一种抗Claudin18_2的抗原结合片段、抗体及其应用 - Google Patents
一种抗Claudin18_2的抗原结合片段、抗体及其应用 Download PDFInfo
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Abstract
本发明提供一种抗Claudin18.2的抗原结合片段、抗体及其应用。所述抗原结合片段的重链可变区的CDR3包括SEQ ID NO.3所示的氨基酸序列；所述抗原结合片段的轻链可变区的CDR3包括SEQ ID NO.6所示的氨基酸序列。本发明提供的抗原结合片段和抗Claudin18.2抗体能够特异性的结合多种来源的Claudin18.2蛋白，且对于其他蛋白无结合作用，具有高度的特异性；同时，由所述抗体制备得到的嵌合抗原受体及CAR‑T细胞对稳定表达Claudin18.2蛋白的细胞具有明显的细胞毒性。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种抗Claudin18.2的抗原结合片段、抗体及其应用。
背景技术
紧密连接是细胞黏附形式的一种，主要存在于上皮细胞、内皮细胞间的连接复合体中，紧密连接分子由Occludin，Claudins蛋白和连接黏附分子3种完整的膜蛋白和闭合小环蛋白等外周胞浆蛋白组成。目前研究已证实，Claudin蛋白是细胞紧密连接的重要分子，其构成了细胞旁屏障，控制细胞间分子的流动。该分子具有四个跨膜结构域，NH2端和COOH端位于胞内，其异常表达可能导致上皮细胞、内皮细胞结构破坏、功能受损，在多种疾病的发病过程中起重要作用。
Claudin18(CLDN18)是该家族成员之一，其编码基因经可变剪接可形成两种亚型蛋白Claudin18.1(CLD18A1，GenBank：NM_016369)和Claudin18.2(CLD18A2，GenBank：NM_001002026)。其中，Claudin18.1主要表达于正常的肺组织；Claudin18.2作为一个高度特异性的细胞表面分子，在正常的组织中仅表达在分化的胃粘膜上表皮细胞(胃上皮短寿命细胞)上，不表达在胃干细胞上。在原发性胃癌及其转移后癌症类型中大部分都表达Claudin18.2分子，50％～80％的胃癌患者存在该靶点的表达，另外在胰腺癌，食管癌，卵巢癌，肺癌中也常常观察到Claudin18.2被激活表达，这些特点表明Claudin18.2是一个理想的肿瘤药物治疗靶点。
因此，提供一种特异性结合Claudin18.2的抗体，对于Claudin18.2表达阳性的肿瘤细胞引发的癌症而言，具有重要的治疗意义。
发明内容
鉴于现有技术中存在的问题，本发明的目的在于提供一种抗Claudin18.2的抗原结合片段、抗体及其应用。所述抗原结合片段和抗体能够特异性结合Claudin18.2蛋白，将其制备成嵌合抗原受体和CAR-T细胞，对表达Claudin18.2蛋白的靶细胞具有明显的细胞毒性。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供一种抗Claudin18.2的抗原结合片段，所述抗原结合片段的重链可变区的CDR3包括SEQ ID NO.3所示的氨基酸序列；所述抗原结合片段的轻链可变区的CDR3包括SEQ ID NO.6所示的氨基酸序列。
本发明中，所述抗原结合片段能够特异性结合Claudin18.2，含有所述抗原结合片段的抗体能够与多种来源的Claudin18.2蛋白，包括人源、鼠源和猴源等，且不结合Claudin18.1，特异性较高，对于以Claudin18.2为治疗靶点的药物或疫苗的研究具有重要的意义。
优选地，所述抗原结合片段的重链可变区的CDR1包括SEQ ID NO.1所示的氨基酸序列。
优选地，所述抗原结合片段的轻链可变区的CDR1包括SEQ ID NO.2所示的氨基酸序列。
优选地，所述抗原结合片段的重链可变区的CDR2包括SEQ ID NO.4所示的氨基酸序列。
优选地，所述抗原结合片段的轻链可变区的CDR2包括SEQ ID NO.5所示的氨基酸序列。
其中，相应序列如下表1所示：
表1
优选地，所述抗原结合片段的重链可变区的CDR1为SEQ ID NO.1所示的氨基酸序列，CDR2为SEQ ID NO.2所示的氨基酸序列，CDR3为SEQ ID NO.3所示的氨基酸序列；所述抗原结合片段的轻链可变区的CDR1为SEQ ID NO.4所示的氨基酸序列，CDR2为SEQ ID NO.5所示的氨基酸序列，CDR3为SEQ ID NO.6所示的氨基酸序列。
第二方面，本发明提供一种抗Claudin18.2抗体，包括如第一方面所述的抗原结合片段。
优选地，所述抗Claudin18.2抗体的重链可变区的氨基酸序列如SEQ ID NO.7所示，轻链可变区的氨基酸序列如SEQ ID NO.8所示。
抗体重链可变区(VH)的氨基酸序列如下(SEQ ID NO.7)：
QVQLKQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRGYYGPYFDYWGQGTTLTVSS
抗体轻链可变区(VL)的氨基酸序列如下(SEQ ID NO.8)：
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNGGNQKNYLTWYQQKPG QPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYY YPYTFGGGTKLEIK。其中，下划线所示为互补决定区。
优选地，所述抗Claudin18.2抗体还包括恒定区。
优选地，所述抗Claudin18.2抗体修饰有糖基化基团。
其中，所述抗Claudin18.2抗体可以以单体或多聚体形式存在，若以多聚体形式存在，则其重链之一与轻链之一形成链间二硫键，另一重链与另一轻链形成链间二硫键，且其重链之一与另一重链形成两个链间二硫键。
同时，本发明还提供一种如第二方面所述的抗Claudin18.2抗体的制备方法，具体包括步骤如下：
(1)用DNA免疫BALB/c小鼠进行杂交瘤制备，选择鼠龄在7～8周BALB/C健康雌性小鼠5只进行免疫，达到一定免疫时间后，对免疫小鼠进行血清滴度检测，挑选血清滴度达到融合实验要求的小鼠脾细胞与骨髓瘤细胞SP2/0以适当的比例，在融合剂的作用下进行融合，制备杂交瘤单克隆细胞；
(2)利用选择性培养基R1640-HAT对杂交瘤细胞进行培养，7～10天后将其换为HT培养液培养3～4天，于第10～14天采用ELISA的方法对杂交瘤上清样品进行检测，获得阳性克隆；
(3)进行流式筛选实验，对其中与CLDN18.2结合，与CLDN18.1不结合的母克隆进行亚克隆，测序获得正确序列克隆。
第三方面，本发明提供一种核酸分子，所述核酸分子包括编码如第一方面所述的抗原结合片段或如第二方面所述的抗Claudin18.2抗体的DNA片段。
第四方面，本发明提供一种表达载体，所述表达载体包括如第三方面所述的核酸分子。
第五方面，本发明提供一种嵌合抗原受体(CAR)，所述嵌合抗原受体包括如第二方面所述的抗Claudin18.2抗体。
本发明中，包含所述嵌合抗原受体的CAR-T细胞，能够高表达抗Claudin18.2抗体，对于Claudin18.2表达阳性的细胞具有明显的细胞毒性。
优选地，所述嵌合抗原受体还包括信号肽(Leader)、铰链区(Hinge)、跨膜结构域(Transmembrane，TM)、共刺激域(ICD)和信号传导域。
优选地，所述信号肽包括CD8α信号肽和/或IgGκ轻链信号肽，优选为IgGκ轻链信号肽。
优选地，所述铰链区为CD8α、CD28、人IgGl、IgG2、IgG4和IgA的铰链区任意一种，优选为CD8α铰链区。
优选地，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区，优选为CD8α跨膜区。
优选地，所述信号传导结构域包括CD3ζ信号传导域。
优选地，所述信号传导结构域还包括共刺激域，例如4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
本发明中，所述靶向Claudin18.2嵌合抗原受体包括IgGκ轻链信号肽、抗Claudin18.2抗体(scFv)、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
本发明中，所述嵌合抗原受体包括IgGκ轻链信号肽序列、与Claudin18.2抗原特异性结合的抗体序列(8D2-scFv)、CD8a的铰链区、跨膜区序列、4-1BB共刺激域序列和CD3ζ信号传导域序列。
其中，IgGκ轻链信号肽的氨基酸序列(SEQ ID NO.9)为：
MDMRVPAQLLGLLLLWLRGARC；
CD8α铰链区(hinge)的氨基酸序列(SEQ ID NO.11)为：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
CD8α跨膜区(TM)的氨基酸序列(SEQ ID NO.13)为：
IYIWAPLAGTCGVLLLSLVITLYC
4-1BB胞内共刺激域(ICD)的氨基酸序列(SEQ ID NO.15)为：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3ζ信号传导域的氨基酸序列(SEQ ID NO.17)为：
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
第六方面，本发明提供一种宿主细胞，所述宿主细胞包括如第三方面所述的核酸分子、如第四方面所述的表达载体或如第五方面所述的嵌合抗原受体。
第七方面，本发明提供一种药物组合物，所述药物组合物包括第二方面所述的抗Claudin18.2抗体。
优选地，所述药物组合物还包括抗肿瘤药物。
本发明中，所述药物组合物也可以与其他的抗肿瘤药物联用，包括同时施用、分开施用或依次施用等方式。
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
第八方面，本发明提供一种如第一方面所述的抗原结合片段、第二方面所述的抗Claudin18.2抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的嵌合抗原受体、第六方面所述的宿主细胞或第七方面所述的药物组合物在制备癌症检测试剂和/或癌症治疗药物中的应用。
优选地，所述癌症包括Claudin18.2表达阳性的癌症。
优选地，所述癌症包括胃癌、食管癌、胰腺癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌或胆囊癌中的任意一种。
本发明所述的数值范围不仅包括上述列举的点值，还包括没有列举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。
与现有技术相比，本发明至少具有以下有益效果：
(1)本发明提供的抗原结合片段和抗Claudin18.2抗体能够特异性的结合多种种属(包括人、鼠和食蟹猴)来源的Claudin18.2蛋白，在后续动物模型的选择上有更多的选择；所述抗体与293T-Hu18.2结合的EC50值为2.303，与293T-Mu18.2的EC50值为7.331，与293T-RM18.2的EC50值为9.159，与MFC-Hu18.2的EC50值为2.727E-12；且所述抗体不与稳定表达人Claudin18.1的293T和鼠MFC细胞结合，通过膜蛋白阵列实验也可以看出8D2-scFv-hFc能特异性结合Claudin18.2，而不与其他非靶蛋白结合，说明该抗Claudin18.2抗体具有明显的特异性；
(2)本发明中提供了一种嵌合抗原受体8D2 CAR，该嵌合抗原受体通过慢病毒载体转入T细胞后，得到了表达8D2 CAR的CAR-T细胞，所述CAR-T细胞对稳定表达Claudin18.2蛋白的细胞具有明显的细胞毒性，因此，其对于Claudin18.2表达阳性的癌症，例如胃癌、食管癌、胰腺癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌和胆囊癌等，具有明显的治疗价值。
附图说明
图1为实施例2中构建的不同细胞系的流式检测结果图；
其中a图为293T-Hu18.1细胞、b图为MFC-Hu18.1细胞、c图为293T-Hu18.2细胞、d图为MFC-Hu18.2细胞、e图为NCI-N87-Hu18.2细胞、f图为MKN45-Hu18.2细胞、g图为293T-Mu18.2细胞、h图为293T-RM18.2细胞。
图2(A)为实施例3中抗体8D2与表达不同蛋白的293T细胞株的结合能力检测结果曲线图。
图2(B)为实施例3中抗体8D2与表达不同蛋白的鼠MFC细胞株的结合能力检测结果曲线图。
图3为实施例4中构建的嵌合抗原受体的结构示意图。
图4为实施例4中表达嵌合抗原受体的慢病毒表达载体的图谱。
图5为实施例4中慢病毒表达载体中嵌合抗原受体的图谱。
图6为实施例5中慢病毒感染T细胞前后流式细胞仪检测结果图，其中a图为感染前，b图为感染后。
图7为实施例6中8D2 CAR-T细胞在不同效靶比下对靶细胞的杀伤能力检测结果图。
图8为实施例7中用膜蛋白阵列验证8D2-scFv-hFc抗体的特异性相互作用结果图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案，但下述的实例仅仅是本发明的简易例子，并不代表或限制本发明的权利保护范围，本发明的保护范围以权利要求书为准。
以下实施例中，若无特殊说明，所以的试剂及耗材均购自本领域常规试剂厂商；若无特殊说明，所用的实验方法和技术手段均为本领域常规的方法和手段。
实施例1
本实施例中通过DNA免疫BALB/c小鼠制备杂交瘤单克隆细胞，并利用选择性培养基R1640-HAT对杂交瘤细胞进行培养，而后更换HT培养液培养，再采用ELISA的方法对杂交瘤上清样品进行检测，获得阳性克隆99个；
随后，进行流式筛选实验，对其中的7株母克隆(与Claudin18.2结合，与Claudin18.1不结合)进行亚克隆，获得亚克隆6株。
测序获得正确序列克隆8D2，即抗Claudin18.2抗体，经测序鉴定后发现，所述抗Claudin18.2抗体8D2的序列如下：
>8D2-VH(SEQ ID NO.7)：
QVQLKQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRGYYGPYFDYWGQGTTLTVSS；
>8D2-VL(SEQ ID NO.8)：
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNGGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYYYPYTFGGGTKLEIK。
实施例2
本实施例采用流式细胞术检测稳定表达Claudin18.2蛋白的细胞系构建情况，其步骤如下：
(1)Claudin18.1和Claudin18.2的表达载体的构建及慢病毒的制备通过基于PCR搭桥的基因合成技术全系列合成人Claudin18.1完整编码序列(GenBank：NM_016369，以下简称“Hu18.1”)、人Claudin18.2完整编码序列(GenBank：NM_001002026，以下简称“Hu18.2”)、小鼠Claudin18.2完整编码序列(GenBank：NM_001194921.1，以下简称“Mu18.2”)和猴Claudin18.2完整编码序列(GenBank：XM_001114708.4，以下简称“RM18.2”)；
酶切连接转化后挑取克隆PCR鉴定并测序确认获得正确的慢病毒载体质粒pCDH-Claudin18.1和pCDH-Claudin18.2；
将上述质粒分别与四质粒系统慢病毒载体包装所需的gag/pol、Rev、VSV-G载体共转染至293T细胞，于转染72h后收集Claudin18.1、Claudin18.2病毒液，浓缩分装后，-80℃保存；
(2)Claudin18.1，Claudin18.2外源表达稳定系的建立及流式检测
将上述收集的Claudin18.1和Claudin18.2病毒液分别加至铺于T75细胞培养瓶的293T细胞与小鼠胃癌细胞MFC(购自南京科佰，CBP60882)中；
另外，使用Claudin18.2病毒液分别感染人胃癌细胞NCI-N87(购自南京科佰，CBP60491)、人胃癌细胞MKN45(购自南京科佰，CBP60488)，分别用于构建293T-Hu18.1、MFC-Hu18.1、293T-Hu18.2、MFC-Hu18.2、NCI-N87-Hu18.2、MKN45-Hu18.2、293T-Mu18.2、293T-RM18.2细胞系，具体信息如下表2所示：
表2
经过嘌呤霉素连续筛选培养后，流式抗体检测上述细胞系构建情况，所得结果如图1，由图可知，本实施例中成功构建了293T-Hu18.1(a图)、MFC-Hu18.1(b图)、293T-Hu18.2(c图)、MFC-Hu18.2(d图)、NCI-N87-Hu18.2(e图)、MKN45-Hu18.2(f图)、293T-Mu18.2(g图)、293T-RM18.2(h图)细胞系。
实施例3
本实施例中通过荧光激活细胞分选仪(BECKMAN COULTER，CytoFLEX S FlowCytometer)分析抗体8D2与各细胞系的结合能力。
具体方法如下：
取对数生长期的293T-Hu18.1、MFC-Hu18.1、293T-Hu18.2、MFC-Hu18.2、NCI-N87-Hu18.2、MKN45-Hu18.2、293T-Mu18.2、293T-RM18.2肿瘤细胞接种到6cm平皿中，接种细胞密度为90％，37℃孵箱过夜培养；
胰酶消化细胞，200g×5min离心收集细胞；
以1×106/mL的浓度重悬于含质量分数为1％小牛血清的磷酸盐缓冲液(NBS PBS)中，按100μL/管的量加入流式专用管中；
200g×5min离心，弃上清，分别加入待测抗体8D2，同时以8K13作为同型对照，抗体终浓度为25、5、1、0.2μg/mL，每管加入100μL；
室温孵育30min，弃上清，加入1:20稀释的FITC荧光标记的羊抗人二抗(BioLegend，Cat.No 409306)，每管加入100μL，室温孵育30min；
弃上清，重悬于200μL 1％NBS PBS中，流式细胞仪检测，再应用流式细胞仪数据分析软件Flowjo 10分析数据；
经过流式细胞检测分析可知，抗体8D2可以特异识别稳定表达人Claudin18.2的293T、MKN45、NCI-N87和鼠MFC细胞，而不与稳定表达人Claudin18.1的293T和鼠MFC细胞结合；因此，表明8D2抗体能够特异识别人Claudin18.2蛋白；同时，抗体8D2也能与稳定表达鼠源及猴源Claudin18.2的细胞结合，表明8D2抗体也可特异识别鼠及猴来源的Claudin18.2蛋白。
其中，部分流式细胞分析结果如图2(A)和图2(B)所示；
由图2(A)所示，抗体8D2与293T-Hu18.2结合的EC50值为2.303，与293T-Mu18.2的EC50值为7.331，与293T-RM18.2的EC50值为9.159，而抗体8D2基本不与293T-Hu18.1结合；
由图2(B)所示，抗体8D2基本不与MFC-Hu18.1结合，其与MFC-Hu18.2的EC50值为2.727E-12。
实施例4
本实施例中构建了抗Claudin18.2的嵌合抗原受体(8D2 CAR)及其表达载体。
(1)序列设计
该嵌合抗原受体包括IgGκ轻链信号肽序列(Leader)，与Claudin18.2抗原特异性结合的抗体序列(8D2-scFv)，CD8a的铰链区(Hinge)和跨膜区序列(Transmembrane)，4-1BB共刺激域序列和CD3ζ信号传导域序列；
具体结构如图3所示；其中，各部分的氨基酸序列及核苷酸序列如下表3所示：
表3
(2)构建抗Claudin18.2的嵌合抗原受体表达载体
首先，全基因合成8D2 CAR序列，用EcoRI和BamHI双酶切全基因合成的8D2 CAR和空载体，于37℃水浴中酶切30min后，使用1.5％的琼脂糖凝胶进行DNA电泳，然后使用天根的琼脂糖凝胶试剂盒纯化回收处理；
而后，连接pCDH-EF1载体与8D2 CAR基因片段，具体连接体系如下表4所示：
表4
| 试剂 | 使用量 |
| pCDH-EF1载体 | 2μL(50ng) |
| 8D2 CAR基因 | 10μL(150ng) |
| T4 DNA Ligase Buffer | 2μL |
| T4 DNA Ligase(NEB) | 1μL |
| ddH<sub>2</sub>O | 5μL |
| 总量 | 20μL |
于22℃连接1h，连接产物直接转化Stbl3大肠杆菌感受态细胞，取200μL转化产物涂布氨苄抗性的LB平板，LB平板于37℃的培养箱中倒置培养过夜；
次日早晨随机挑选3个单克隆进行菌落PCR鉴定，将阳性克隆送样测序，获得抗Claudin 18.2的嵌合抗原受体慢病毒表达质粒，即为pCDH-EF1-L8D2-CAR，Claudin18.2载体图谱见图4，其中，嵌合抗原受体的序列如图5所示。
实施例5
本实施例中对慢病毒表达载体进行慢病毒包装、构建CAR-T细胞并检测慢病毒对T细胞的感染效率。
(1)使用四质粒系统进行慢病毒包装
四质粒系统分别表达慢病毒载体包装所需的gag/pol、Rev、VSV-G及工程稳定的单链抗体构成的人工嵌合抗原受体，将四质粒进行瞬时转染293T细胞，总质量为10μg；
将上述质粒加入至无血清的DMEM中，混匀后放置15分钟，将上述混合液加入至铺有293T细胞的T75培养瓶中，轻轻混匀，于37℃、5％CO2细胞培养箱培养6h；
6h后更换新鲜培养基，继续进行培养，并且加入10mM的丁酸钠溶液，72h后收集慢病毒的培养上清进行纯化检测。
(2)CAR-T细胞的扩增
采30mL的全血，将外周血与生理盐水1:1进行稀释，在离心管内加入Ficoll，缓缓加入稀释后的外周血，1500rpm离心30min轻轻吸取PBMC层移入另一离心管内；
用生理盐水洗涤PBMC多次，转入CAR-T细胞培养基(含50ng/mL的OKT3，300IU/mL的IL-2)中进行培养；
PBMC分离后，需要用含50ng/mL的OKT3，300IU/mL的IL-2的CAR-T细胞培养基进行激活；
2日后将培养基更换成含300IU/mL的CAR-T细胞培养基进行扩大培养；
而后每两天进行一次计数并更换含300IU/mL的CAR-T细胞培养基，并且将细胞浓度维持在0.5×106～1×106/mL，连续观察10天；
(3)慢病毒感染T细胞
利用RetroNectin提高慢病毒对T细胞的感染效率，将30μg的RetroNectin，包被于6孔板内，放于37℃细胞培养箱2h；
吸取RetroNectin，利用含2.5％BSA的Hank’s溶液封闭包被后的6孔板，放于37℃细胞培养箱0.5h；
吸取封闭液，利用含2％Hepes的Hank’s溶液洗涤6孔板，加入X-VIVO培养基，加入适量的慢病毒溶液，2000g，离心2h；
弃上清，加入1×106的T细胞，1000g，离心10min，于37℃、5％CO2细胞培养箱内培养，第2日重复上述过程；感染5天后测定8D2 CAR的表达，利用FITC-Protien L与8D2 CAR结合，通过流式细胞仪检测8D2 CAR的表达；
所得结果如图6所示，结果显示，感染前CD3阳性率为98.52％(a图)，8D2CAR的表达阳性率为51.77％(b图)。
实施例6
本实施例中采用8D2 CAR进行细胞毒性检测实验，并空白对照NC(未转染的T细胞)作为对照。
利用LDH检测CAR-T细胞对293T-Hu18.1(稳定表达人Claudin18.1的293T细胞)，293T-Hu18.2(稳定表达人Claudin18.2的293T细胞)和293T-Mu18.2(稳定表达鼠Claudin18.2的293T细胞)细胞的毒性。
本实施例中采用的检测方法参照CN104877032A中记载的检测方法进行，包括步骤如下：
将细胞离心后，用无血清无酚红的DMEM培养基洗涤后计数；
取1×106的293T-Hu18.1、293T-Hu18.2和293T-Mu18.2细胞各50μL，铺板于96孔板内，作为靶细胞；
按靶细胞：效应细胞＝1:1/4/8分别加入未转染的T细胞和各CAR-T细胞；
于37℃、5％CO2和一定湿度的细胞培养箱内培养12h。加入裂解液作为阳性对照，而后250g离心5min。每孔取100μL培养上清，加入新的96孔板内；加入20μL反应液，放于暗室中反应20～30min，酶标仪590nm进行检测。
根据公式计算溶解百分率：
细胞毒性(％)＝[(实验孔-培养基背景孔)-(效应细胞自发LDH释放孔-培养基背景孔)-(靶细胞自发LDH释放孔-培养基背景孔)]/[(靶细胞最大LDH释放孔-体积校正孔)-(靶细胞自发LDH释放孔－培养基背景孔)]×100％。
结果如图7所示，8D2 CAR-T细胞能特异性的杀伤稳定表达人Claudin18.2的293T细胞(293T-Hu18.2)和稳定表达鼠Claudin18.2的293T细胞(293T-Mu18.2)，而对稳定表达人Claudin18.1的293T细胞(293T-Hu18.1)几乎没有杀伤能力。
实施例7
本实施例中用膜蛋白阵列(Membrane Proteome Array)验证抗体的非靶点结合的相互作用。
首先，表达抗体融合蛋白8D2-scFv-hFc，8D2-scFv-hFc是8D2的单链抗体序列融合人IgG1的Fc段；膜蛋白质组阵列(MPA)是一个分析特异性抗体和其他配体靶向人膜蛋白的平台，可用于确定抗体靶点的特异性；
将含有约6000个膜蛋白克隆(占人膜蛋白组的94％以上)的质粒分别转染到HEK-293T细胞(ATCC，CRL-3216)；或QT6细胞(ATCC，CRL-1708)；384孔细胞培养板(Corning，3764)，18000个细胞/孔；
孵育36小时后，试验抗体以预先确定的浓度加入膜蛋白组阵列基质板，使用流式细胞仪直接检测抗体8D2-scFv-hFc与约6000种膜蛋白表达细胞的结合。因此，所有的靶蛋白都具有天然构象和适当的翻译后修饰。
膜蛋白阵列结果如图8所示：8D2-scFv-hFc能特异性结合人Claudin18.2，而不与其他非靶蛋白结合，其中FCGR1A、FCGR2B、FCGR3B是IgG Fc受体。
综上所述，本发明提供的抗Claudin18.2抗体能够特异性的结合多种来源的Claudin18.2蛋白，包括人源、鼠源和猴源，且对于其他蛋白基本上不具有结合能力，具有高度特异性，同时本发明中提供的CAR和含有该CAR的T细胞对于表达Claudin18.2蛋白的细胞具有明显的细胞毒性；因此，本发明提供的抗Claudin18.2抗体对于以Claudin18.2蛋白为靶点的疾病具有特异性的治疗作用。
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 广州百暨基因科技有限公司
<120> 一种抗Claudin18_2的抗原结合片段、抗体及其应用
<130> 20201214
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> 人工合成
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1 5 10
<210> 2
<211> 10
<212> PRT
<213> 人工合成
<400> 2
Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
1 5 10
<210> 3
<211> 10
<212> PRT
<213> 人工合成
<400> 3
Arg Gly Tyr Tyr Gly Pro Tyr Phe Asp Tyr
1 5 10
<210> 4
<211> 17
<212> PRT
<213> 人工合成
<400> 4
Lys Ser Ser Gln Ser Leu Leu Asn Gly Gly Asn Gln Lys Asn Tyr Leu
1 5 10 15
Thr
<210> 5
<211> 7
<212> PRT
<213> 人工合成
<400> 5
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工合成
<400> 6
Gln Asn Asp Tyr Tyr Tyr Pro Tyr Thr
1 5
<210> 7
<211> 119
<212> PRT
<213> 人工合成
<400> 7
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Tyr Tyr Gly Pro Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 8
<211> 113
<212> PRT
<213> 人工合成
<400> 8
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Gly
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Tyr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 9
<211> 22
<212> PRT
<213> 人工合成
<400> 9
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys
20
<210> 10
<211> 66
<212> DNA
<213> 人工合成
<400> 10
atggatatga gggttcctgc acaactcctc ggtctgctgc tgctctggct gagaggagcc 60
agatgt 66
<210> 11
<211> 45
<212> PRT
<213> 人工合成
<400> 11
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 12
<211> 135
<212> DNA
<213> 人工合成
<400> 12
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 13
<211> 24
<212> PRT
<213> 人工合成
<400> 13
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 14
<211> 72
<212> DNA
<213> 人工合成
<400> 14
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 15
<211> 42
<212> PRT
<213> 人工合成
<400> 15
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 16
<211> 126
<212> DNA
<213> 人工合成
<400> 16
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60
acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120
gagctg 126
<210> 17
<211> 112
<212> PRT
<213> 人工合成
<400> 17
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 18
<211> 336
<212> DNA
<213> 人工合成
<400> 18
agggtgaagt tttctcggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60
tacaacgagc tgaatctggg caggcgcgag gagtacgacg tgctggataa gcggagaggc 120
agagatcccg agatgggagg caagccaagg aggaagaacc ctcaggaggg cctgtataat 180
gagctgcaga aggacaagat ggccgaggcc tactctgaga tcggcatgaa gggagagcgg 240
agaaggggca agggacacga tggcctgtat cagggcctga gcacagccac caaggacacc 300
tacgatgcac tgcacatgca ggccctgcca cctagg 336
Claims (10)
1.一种抗Claudin18.2的抗原结合片段，其特征在于，所述抗原结合片段的重链可变区的CDR3包括SEQ ID NO.3所示的氨基酸序列；
所述抗原结合片段的轻链可变区的CDR3包括SEQ ID NO.6所示的氨基酸序列。
2.根据权利要求1所述的抗原结合片段，其特征在于，所述抗原结合片段的重链可变区的CDR1包括SEQ ID NO.1所示的氨基酸序列；
优选地，所述抗原结合片段的轻链可变区的CDR1包括SEQ ID NO.2所示的氨基酸序列；
优选地，所述抗原结合片段的重链可变区的CDR2包括SEQ ID NO.4所示的氨基酸序列；
优选地，所述抗原结合片段的轻链可变区的CDR2包括SEQ ID NO.5所示的氨基酸序列。
3.根据权利要求1或2所述的抗原结合片段，其特征在于，所述抗原结合片段的重链可变区的CDR1为SEQ ID NO.1所示的氨基酸序列，CDR2为SEQ ID NO.2所示的氨基酸序列，CDR3为SEQ ID NO.3所示的氨基酸序列；
所述抗原结合片段的轻链可变区的CDR1为SEQ ID NO.4所示的氨基酸序列，CDR2为SEQID NO.5所示的氨基酸序列，CDR3为SEQ ID NO.6所示的氨基酸序列。
4.一种抗Claudin18.2抗体，其特征在于，所述抗Claudin18.2抗体包括如权利要求1～3任一项所述的抗原结合片段；
优选地，所述抗Claudin18.2抗体的重链可变区的氨基酸序列如SEQ ID NO.7所示，轻链可变区的氨基酸序列如SEQ ID NO.8所示；
优选地，所述抗Claudin18.2抗体还包括恒定区；
优选地，所述抗Claudin18.2抗体修饰有糖基化基团。
5.一种核酸分子，其特征在于，所述核酸分子包括编码如权利要求1～3任一项所述的抗原结合片段或如权利要求4所述的抗Claudin18.2抗体的DNA片段。
6.一种表达载体，其特征在于，所述表达载体包括如权利要求5所述的核酸分子。
7.一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包括如权利要求4所述的抗Claudin18.2抗体。
所述嵌合抗原受体还包括信号肽、铰链区、跨膜结构域和信号传导域；
优选地，所述信号肽包括CD8α信号肽和/或IgGκ轻链信号肽，优选为IgGκ轻链信号肽；
优选地，所述铰链区包括CD8α、CD28、人IgGl、IgG2、IgG4或IgA中的铰链区任意一种，优选为CD8α铰链区；
优选地，所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区，优选为CD8α跨膜区；
优选地，所述信号传导域包括CD3ζ信号传导域；
优选地，所述信号传导结构域还包括共刺激域。
8.一种宿主细胞，其特征在于，所述宿主细胞包括如权利要求5所述的核酸分子、如权利要求6所述的表达载体或如权利要求7所述的嵌合抗原受体。
9.一种药物组合物，其特征在于，所述药物组合物包括权利要求4所述的抗Claudin18.2抗体；
优选地，所述药物组合物还包括抗肿瘤药物；
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
10.如权利要求1～3任一项所述的抗原结合片段、权利要求4所述的抗Claudin18.2抗体、权利要求5所述的核酸分子、权利要求6所述的表达载体、权利要求7所述的嵌合抗原受体、权利要求8所述的宿主细胞或权利要求9所述的药物组合物在制备癌症检测试剂和/或癌症治疗药物中的应用；
优选地，所述癌症包括Claudin18.2表达阳性的癌症；
优选地，所述癌症包括胃癌、食管癌、胰腺癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌或胆囊癌中的任意一种。
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| US18/039,678 US20240050472A1 (en) | 2020-12-16 | 2020-12-22 | Anti-claudin18.2 antigen-binding fragment or antibody, and use thereof18255119 |
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| CN109206524A (zh) * | 2018-09-25 | 2019-01-15 | 山东兴瑞生物科技有限公司 | 抗Claudin18A2嵌合抗原受体、其修饰的T细胞及T细胞制备方法和用途 |
| CN110606891A (zh) * | 2018-06-17 | 2019-12-24 | 上海健信生物医药科技有限公司 | 一种针对人cldn18.2的新型抗体分子, 抗原结合片段及其医药用途 |
| WO2020200196A1 (zh) * | 2019-04-01 | 2020-10-08 | 江苏恒瑞医药股份有限公司 | 抗Claudin18.2抗体及其应用 |
| CN111944048A (zh) * | 2019-05-16 | 2020-11-17 | 启愈生物技术(上海)有限公司 | 抗cldn抗体及其药物组合物和检测方法 |
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| CN112888458A (zh) * | 2018-09-30 | 2021-06-01 | 佧珐药业有限公司 | Cldn18的抗体和化疗药物的联合治疗 |
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| WO2020228806A1 (zh) * | 2019-05-16 | 2020-11-19 | 齐鲁制药有限公司 | 针对密蛋白18a2的抗体及其应用 |
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