CN116375874B - 一种cd33抗体及其应用 - Google Patents
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Abstract
本发明涉及抗体技术领域，具体涉及一种抗CD33抗体及其应用。提供一种抗CD33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
技术领域
本发明涉及抗体技术领域，具体涉及一种抗CD33抗体及其应用。
背景技术
髓样细胞表面抗原CD33前体(CD33)，也称为Siglec-3，是在免疫细胞和造血细胞，包括未成熟和成熟的髓样细胞、树突状细胞和小神经胶质细胞上表达的1型免疫球蛋白样跨膜蛋白。CD33在早期多谱系造血祖细胞及骨髓单核细胞前体上表达。在多潜能造血干细胞中无表达。其表达在成熟粒细胞上下调，但保留于巨噬细胞、单核细胞及树突细胞上。除骨髓单核细胞性细胞外，Valent等人发现CD33亦可在人类肥大细胞及血液嗜碱性粒细胞上表达。
根据世界卫生组织的数据，癌症是全球第二大死亡原因，并且在2018年估计有960万人因此而死亡。急性骨髓性白血病(AML)是由克隆性增殖性成髓细胞的恶变引起的一类癌症，CD33已被广泛用作AML的治疗靶标。
本领域需要靶向CD33的安全且有效的抗体来诊断和治疗CD33相关状况，诸如癌症，阿尔茨海默病等。
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CD33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CD33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CD33的鼠源性单克隆抗体(mAb)，该抗体对CD33具有较高的亲和力，同时具有靶向CD33的能力。
具体地，本发明提供以下技术方案：
本发明提供抗CD33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CD33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CD33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CD33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CD33抗体或其抗原结合片段。
根据上述提供的抗CD33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CD33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CD33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***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CD33抗体或其抗原结合片段的功能，本发明提供所述抗CD33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CD33的试剂或试剂盒中的应用；
(2)在制备用于预防或治疗白血病的药物中的应用；
(3)在制备用于预防或治疗阿尔茨海默病的药物中的应用；
以上(1)中所述的检测CD33包括检测CD33的存在或其水平。
以上(2)中所述的白血病包括急性骨髓性白血病。
基于上述抗CD33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CD33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CD33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CD33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CD33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CD33的单克隆抗体，该抗体能够与CD33特异性结合，具有高度特异性和高结合能力，可用于抗D33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，该抗体还能为白血病，阿尔茨海默病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CD33抗体与原代人树突状细胞结合的数据。
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CD33蛋白对小鼠免疫，获得杂交瘤细胞并进行筛选，经ELISA初筛，筛选到1株对CD33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CD33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
实施例2、抗CD33抗体的表征
抗CD33抗体的表征涉及结合CHO细胞系上、在人原代巨噬细胞、人原代单核细胞、以及人原代树突细胞上表达的CD33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、2mM EDTA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CD33的抗体，均能结合表达有CD33的细胞上。
表2
进一步地，还表征了抗CD33抗体的表观亲和力常数。在BiaCore T200仪器上在25℃下以10Hz的速率采集SPR数据。使用BiaCore T200评价软件版本2.0执行数据分析。在固定有抗小鼠IgG的传感器芯片上捕集本发明抗CD33抗体，以及吉妥珠单抗。然后使20nM组氨酸标记的人CD33在捕集的抗CD33表面上流过(120s接触时间，30μL/min流速，300s缔合时间)。进行了一式两份的单一浓度试验并且通过空白(0nM抗原)样品进行分类。使用甘氨酸-HCl在循环之间再生成芯片表面(60s接触时间，30μL/min流速，60s稳定化时间)。使用1:1相互作用模型执行单一浓度动力学分析来提取每种抗体的缔合速率常数和解离速率常数(分别是ka和kd)。根据kd/ka比率计算表观亲和力常数(KD)，结果如表3所示，数据表明本发明的抗CD33抗体具有良好的亲和力，其具有很好的结合能力。
表3
| 抗体 | KD |
| 本发明抗CD33抗体 | 0.5nM |
| 吉妥珠单抗 | 1.3nM |
实施例3、本发明抗CD33抗体的人源化
本发明抗CD33抗体的人源化变体的制备，并表征其与CD33的结合。抗抗CD33抗体是通过将亲本小鼠抗体的CDR移植到序列最接近小鼠抗体的人种系框架上而人源化的。
通过流式细胞术评估本发明抗CD33抗体的人源化抗CD33抗体与原代树突状细胞的结合，将结合与包含抗CD33抗体的可变重链序列和可变轻链序列的抗CD33嵌合抗体(本发明可变区和人恒定区嵌合)的结合进行比较。从健康人供体的血液中分离单核细胞。将分离的单核细胞用100ng/mL GM-CSF和100ng/mL IL-4分化为树突状细胞。将树突状细胞与抗CD33抗体的稀释液在冰上于黑暗中孵育30分钟，然后与荧光缀合的抗人IgG二抗一起孵育30分钟。将细胞在FACS缓冲液(PBS+2％FBS，2mM EDTA)中洗涤两次，并且在BD FACS Canto上进行流式细胞术。使用FlowJo软件分析数据。在2μg/mL的抗体浓度下，通过流式细胞术测试抗CD33抗体与哺乳动物细胞的结合。结果如图1所示。
Claims (8)
1.一种抗CD33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SEQID 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CD33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CD33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CD33抗体的如下任一种应用：
（1）在制备用于检测CD33的试剂或试剂盒中的应用；
（2）在制备用于预防或治疗白血病的药物中的应用；
（3）在制备用于预防或治疗阿尔茨海默病的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述（2）中所述的白血病包括急性骨髓性白血病。
7.一种检测试剂，其特征在于，其含有权利要求1或2所述的抗CD33抗体。
8.一种药物组合物，其特征在于，其含有权利要求1或2所述的抗CD33抗体，以及药学上可接受的载剂。
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| CN104936617A (zh) * | 2012-05-18 | 2015-09-23 | 西雅图遗传学公司 | Cd33抗体及其在治疗癌症中的用途 |
| CA3149683A1 (en) * | 2019-06-26 | 2020-12-30 | Memorial Sloan Kettering Cancer Center | Anti-cd33 antibodies for treating cancer |
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| Fc-engineered anti-CD33 monoclonal antibody potentiates cytotoxicity of membrane-bound interleukin-21 expanded natural killer cells in acute myeloid leukemia;Rajeswaran Mani等;CYTOTHERAPY;第22卷(第7期);369-376 * |
| 重组载体免疫小鼠制备抗人 CD33 单克隆抗体;王卉等;细胞与分子免疫学杂志;第36卷(第2期);169-174 * |
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