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CN116333137A - 一种cd103抗体及其应用 - Google Patents

一种cd103抗体及其应用 Download PDF

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CN116333137A
CN116333137A CN202310397371.7A CN202310397371A CN116333137A CN 116333137 A CN116333137 A CN 116333137A CN 202310397371 A CN202310397371 A CN 202310397371A CN 116333137 A CN116333137 A CN 116333137A
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黄启宽
朱国振
刘国卿
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Ningbo Xining Testing Technology Co ltd
Shanghai Jinghan Biotechnology Co ltd
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Abstract

本发明涉及抗体技术领域，具体涉及一种抗CD103抗体及其应用。提供一种抗CD103抗体，其重链可变区的互补决定区CDR1具有如SEQ ID NO.1所示的氨基酸序列，CDR2具有如SEQ ID NO.2所示的氨基酸序列，CDR3具有如SEQ ID NO.3所示的氨基酸序列。轻链可变区的互补决定区CDR1具有如SEQ ID NO.4所示的氨基酸序列，CDR2具有如SEQ ID NO.5所示的氨基酸序列，CDR3具有如SEQ ID NO.6所示的氨基酸序列。

Description

一种CD103抗体及其应用
技术领域
本发明涉及抗体技术领域，具体涉及一种抗CD103抗体及其在疾病的诊断、预后、检测和治疗中应用。
背景技术
整合素αE亚单位即CD103，与整合素β7组成αEβ7。整合素αEβ7高表达在肠道黏膜中的白细胞上，包含上皮内淋巴细胞、树突状细胞、肥大细胞和T调节细胞。整合素αEβ7通过与钙粘蛋白的相互作用介导淋巴细胞与肠道上皮细胞的黏附，使得淋巴细胞可以驻留在肠道内，而这种黏附主要依赖于整合素αE亚基内的α-I域的金属离子依赖的黏附位点。
CD103被用来作为组织驻留记忆(TRM)状态的分子标志，而CD103在T细胞上的表达，预示了其在肿瘤组织中的归巢和驻留的能力。肿瘤中CD8+T细胞、Vδ1T细胞和Vδ2T细胞多为CD103+TRM表型。值得注意的是，正常肺组织中也有许多CD8+T细胞、部分Vδ1T和CD4+T细胞表现为CD103+TRM表型。
本领域需要一种抗CD103的抗体，其能够用于检测CD103并且对疾病，特别地，对癌症特异性淋巴细胞的表达特征的了解，和对应癌症靶标的治疗和预防。
发明内容
有鉴于此，第一方面，本发明提供了本发明的目的之一是提供一种抗CD103抗体。本发明的另一目的是提供该抗体的应用及其相关产品。
为实现上述目的，本发明通过杂交瘤技术筛选到一株抗CD103特异性的杂交瘤，制备小鼠腹水并纯化抗体，得到能够特异性结合CD103的鼠源性单克隆抗体(mAb)，该抗体对CD103具有较高的亲和力，同时具有靶向CD103的能力。
具体地，本发明提供以下技术方案：
本发明提供抗CD103抗体或其抗原结合片段，其重链可变区的互补决定区CDR1具有如SEQ ID NO.1所示的氨基酸序列，CDR2具有如SEQ ID NO.2所示的氨基酸序列，CDR3具有如SEQ ID NO.3所示的氨基酸序列。轻链可变区的互补决定区CDR1具有如SEQ ID NO.4所示的氨基酸序列，CDR2具有如SEQ ID NO.5所示的氨基酸序列，CDR3具有如SEQ ID NO.6所示的氨基酸序列。
优选地，所述抗CD103抗体或其抗原结合片段的重链可变区具有如SEQ ID NO.7所示的氨基酸序列。轻链可变区具有如SEQ ID NO.8所示的氨基酸序列。
进一步优选地，所述抗CD103抗体为鼠源单克隆抗体。
本发明中，所述抗原结合片段可选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体、双特异抗体或多特异抗体。
本发明提供含有所述抗CD103抗体或其抗原结合片段的双特异抗体或多特异抗体。
基于上述抗体，本发明提供一种核酸分子，其编码所述抗CD103抗体或其抗原结合片段。
根据上述提供的抗CD103抗体或其抗原结合片段的氨基酸序列，本领域技术人员可以确定编码所述抗CD103抗体或其抗原结合片段的核酸分子的核苷酸序列，并根据宿主细胞的密码子偏好性选择不同的核酸分子的核苷酸序列。
本发明还提供一种生物材料，其含有编码所述抗CD103抗体或其抗原结合片段的核酸分子，所述生物材料为表达盒、载体或宿主细胞。
以上所述的表达盒是指将所述核酸分子的上游或下游连接用于转录、翻译的调控元件得到的重组核酸分子。
以上所述的载体是指可将核酸分子插入其中的一种核酸运载工具，包括但不限于质粒、人工染色体、噬菌体、动物病毒等，可以为表达载体，也可以为克隆载体或非复制型载体。
以上所述的宿主细胞可以为微生物细胞(如：大肠杆菌、酵母细胞等)或动物细胞(如：昆虫细胞、CHO细胞、BHK细胞，HEK293细胞等用于表达、制备抗体的细胞)，其中，动物细胞为不能够繁殖成为动物个体的细胞。
基于上述抗CD103抗体或其抗原结合片段的功能，本发明提供所述抗CD103抗体或其抗原结合片段、所述核酸分子或所述生物材料的如下任一种应用：
(1)在制备用于检测CD103的试剂或试剂盒中的应用；
(2)在制备用于预防或治疗癌症的药物中的应用；
以上(1)中所述的检测CD103包括检测CD103的存在或其水平。
在一个具体的实施方案中，所述存在或其水平可以是机体内的成像。
以上(2)中所述的癌症包括毛细胞白血病、HCLv、肠和肠外淋巴瘤、肠病相关T细胞淋巴瘤、T淋巴细胞白血病/淋巴瘤、T细胞幼淋巴细胞白血病、成人T细胞白血病/淋巴瘤、蕈样肉芽肿、间变性大细胞淋巴瘤ALCL、皮肤T细胞淋巴瘤，和母细胞浆细胞样树突状细胞肿瘤。
基于上述抗CD103抗体或其抗原结合片段，本发明提供一种抗体偶联物，其为由所述抗CD103抗体或其抗原结合片段与载体或药物偶联得到，或者由所述抗CD103抗体或其抗原结合片段与经化学或生物标记得到。
以上所述的载体可为任意可与蛋白质偶联的载体或药物载体。
以上所述的化学标记包括同位素、胶体金、荧光素、生物素标记等。
以上所述的生物标记包括蛋白标记、酶标记等。
本发明提供一种检测试剂，其含有所述抗CD103抗体或其抗原结合片段或含有所述抗体偶联物。
以上所述的试剂盒还可包含免疫学检测、流式细胞检测等所需的其它试剂。
本发明提供一种药物组合物，其含有所述抗CD103抗体或其抗原结合片段或含有所述抗体偶联物。
以上所述的药物组合物还可包含药学上可接受的载体或赋形剂。所用载体或赋形剂的类型可根据药物组合物的剂型和给药方式进行选择。所述药物组合物还可包含具有其它功效的抗体或药物活性成分。
本发明的有益效果在于：本发明提供一种针对CD103的单克隆抗体，该抗体能够与CD103特异性结合，具有高度特异性和高结合能力，可用于抗CD103的免疫荧光染色和流式细胞染色等检测，即使在较高稀释度时，仍然具有较好的检测效果。同时，该抗体还能为CD103阳性的肿瘤的治疗都提供了有效的潜在选择的工具。
附图说明
图1为示出了显示本发明的抗CD103抗体与CHO-hCD103结合的数据；
图2为示出了显示本发明的抗CD103抗体与CD103阳性T细胞结合的数据。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例1、杂交瘤细胞和单克隆抗体的筛选和制备
使用制备的编码CD103蛋白(整合素αE)的质粒对小鼠免疫，获得杂交瘤细胞并进行筛选，经ELISA初筛，筛选到1株对CD103具有结合能力的单克隆抗体。将杂交瘤注射入小鼠腹腔内，约10天后收获小鼠腹水，采用Protein G进行纯化，获得纯化单克隆抗体，浓度为2mg/ml。
进一步地，培养杂交瘤细胞，提取细胞总RNA，RT-PCR扩增抗体重链及轻链基因，最后选择阳性克隆进行测序，得到抗体重链及轻链全长的序列。
经测序，抗CD103的单克隆抗体的重链可变区的互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO.1-3所示，轻链可变区的互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO.4-6所示。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，如表1所示。
表1
Figure BDA0004178058110000051
实施例2、本发明抗CD103抗体的细胞ELISA
中国仓鼠卵巢CHO细胞在DMEM/F12(Gibco)、1％PenStrep(Gibco)、5％NCBS(Biowest)中生长,并在37℃和5％CO2的潮湿环境中培养。MCF7细胞在EMEM(ATCC)、1％PenStrep(Gibco)、10％FBS(Gibco)中生长,并在37℃和5％CO2的潮湿环境中培养。CHO-hCD103细胞系是通过用pCI-neo转染编码人类整合素AlphaE(UniProt P38570)的pcDNA3.1(+)-hygro载体进入CHO细胞而获得的。
CD103阳性T细胞如下产生：通过Ficoll-Paque密度梯度离心从健康志愿者身上分离出人外周血单个核细胞。然后使用MagniSortTM人CD8 T细胞富集试剂盒对CD8阳性T细胞进行阴性选择。随后，用10pg/mL PHA、6000U/mL IL-2和10ng/mL重组TGFP刺激细胞,并在RPMI(10％FCS和青霉素/链霉素(100U/mL))中培养。将细胞培养至少10天以获得>80％CD103阳性的CD8细胞。
将CHO-hCD103和CD103阳性T细胞(30,000个细胞/孔)接种在96孔板中。随后,将连续稀释的本发明抗CD103抗体和同种型对照添加到96孔板的每个孔中,并在37℃下孵育1小时。用PBS洗涤孔并与兔抗小鼠/IgG-HRP(1:4000)在37℃孵育1小时。接下来,用PBS洗涤孔并添加TMB底物(KPL)。通过添加1M HCl溶液停止颜色反应,并通过酶标仪(ThermoScientific)测量吸光度，本发明抗体对这些细胞系的结合数据如图1和2所示，从数据中可以看出，本发明的抗CD103抗体对于CD103阳性的细胞具有良好的结合效果。
实施例3、本发明抗CD103抗体的流式细胞术
抗CD103抗体的表征涉及结合CHO细胞系上、CHO-hCD103，和CD103阳性T细胞上表达的CD103的能力。收获细胞，在96孔板中以106个/ml铺板，并且在冰中在含有2％FBS、2mMEDTA和10μg/ml抗体和Fc阻断试剂的100μl PBS中孵育1小时。洗涤细胞两次，并且在冰上在含有2％FBS、2mM EDTA和5μg/ml PE偶联的二抗的100μlPBS中孵育30分钟。在冷PBS中洗涤细胞两次，并且在BD FACS Canto上通过流式细胞术进行分析。使用FlowJo软件版本10.0.7执行数据分析和平均荧光强度(MFI)值的计算。具体的阳性细胞结合百分比和平均荧光强度在表2当在列出。从表2中可以看出，本发明筛选的抗CD103的抗体，均能结合表达有CD103的细胞上。
表2
Figure BDA0004178058110000061
Figure BDA0004178058110000071

Claims (9)

1.一种抗CD103抗体，其特征在于，其重链可变区的互补决定区CDR1的氨基酸序列如SEQ ID NO.1所示，CDR2的氨基酸序列如SEQ ID NO.2所示，CDR3的氨基酸序列如SEQ IDNO.3所示；轻链可变区的互补决定区CDR1的氨基酸序列如SEQ ID NO.4所示，CDR2的氨基酸序列如SEQ ID NO.5所示，CDR3的氨基酸序列如SEQ ID NO.6所示。
2.根据权利要求1所述的抗CD103抗体，其特征在于，其重链可变区的氨基酸序列如SEQID NO.7所示；轻链可变区的氨基酸序列如SEQ ID NO.8所示。
3.核酸分子，其特征在于，其编码权利要求1或2所述的抗CD103抗体。
4.生物材料，其特征在于，其含有权利要求3所述的核酸分子，所述生物材料为表达盒、载体或宿主细胞。
5.权利要求1或2所述的抗CD103抗体或权利要求3所述的核酸分子或权利要求4所述的生物材料的如下任一种应用：
(1)在制备用于检测CD103的试剂或试剂盒中的应用；
(2)在制备用于预防或治疗癌症的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述(2)中所述的癌症包括毛细胞白血病、HCLv、肠和肠外淋巴瘤、肠病相关T细胞淋巴瘤、T淋巴细胞白血病/淋巴瘤、T细胞幼淋巴细胞白血病、成人T细胞白血病/淋巴瘤、蕈样肉芽肿、间变性大细胞淋巴瘤ALCL、皮肤T细胞淋巴瘤，和母细胞浆细胞样树突状细胞肿瘤。
7.一种抗体偶联物，其特征在于，其为由权利要求1或2所述的抗CD103抗体与载体或药物偶联得到，或者由权利要求1或2所述的抗CD103抗体与经化学或生物标记得到。
8.一种检测试剂，其特征在于，其含有权利要求1或2所述的抗CD103抗体或权利要求7所述的抗体偶联物。
9.一种药物组合物，其特征在于，其含有权利要求1或2所述的抗CD103抗体或权利要求7所述的抗体偶联物，以及药学上可接受的载剂。
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