鼠抗人PD-L1功能性单克隆抗体的研制及其生物学特性的研究
发布时间：2018-10-24 08:33
【摘要】：Bretscher和Cohn在T细胞活化双信号模型的基础上提出“协同刺激信号”[1],如果缺少协同刺激分子提供的第二信号,将会导致T细胞的无反应性或特异性免疫耐受甚至凋亡。正性和负性协同刺激信号的调节及两者之间的平衡在机体免疫应答的整个过程中发挥着重要的调节作用[2-3]。PD-1/PD-L1是一对重要的负性协同刺激分子。人PD-L1基因定位在染色体9p24.2 ,编码290个氨基酸残基的I型跨膜蛋白[4,7,9]。PD-1/PD-L信号对淋巴细胞的增殖发挥负性调控功能,可防止过度的免疫损伤及自身免疫性疾病的发生,有利于维持机体的免疫自稳[6-7]。PD-L1组成性表达于单核/巨噬细胞以及树突状细胞(DC),并在活化后呈上调表达。同时,大量研究结果证实多种肿瘤细胞的胞膜和胞浆都有PD-L1的表达[8-9]。肿瘤细胞表面的PD-Ll在肿瘤细胞诱导特异性CTL凋亡的过程中发挥重要作用[9]。PD-1/PD-L1途径与肿瘤细胞免疫逃逸密切相关,阻断此途径可增强机体的抗肿瘤免疫应答[10-12]。因此,PD-L1分子成为近年来免疫学研究的热点,有望成为肿瘤、自身免疫性疾病和病毒感染性疾病的免疫干预治疗的有效靶分子。 本课题研究以高表达人PD-L1分子的基因转染细胞L929/PD-L1作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L1作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-L1单克隆抗体的杂交瘤细胞株命名为11G8 mAb,2G5 mAb和5C3 mAb,Western-blotting分析显示,3株抗体都可以特异性识别L929/PD-L1细胞上蛋白质,而不能识别L929/mock。抗原位点竞争抑制实验提示3株单抗与商品化的anti-PD-L1-PE mAb识别细胞抗原结合位点不同。对3株抗体生物学功能的研究结果提示,3株抗体均能够识别活化T细胞表达的PD-L1分子,且在体外能够显著促进T细胞的增殖,是具有较好效应的功能性单克隆抗体。 利用自主研制的sPD-L1 ELISA检测系统测定了健康人外周血、肺癌患者外周血及胸水血清中可溶性PD-L1的表达水平。结果显示,肺癌患者外周血中sPD-L1表达水平明显高于健康志愿者;肺癌患者胸水中sPD-L1的表达水平高于肺癌患者外周血;肺癌患者外周血中sPD-L1的表达与年龄、性别及肺癌III、IV期无明显相关性;腺癌患者、鳞癌患者外周血中sPD-L1表达水平明显高于小细胞癌患者。sPD-L1在肺癌患者外周血中异常高表达表明该因子进一步抑制了机体的免疫功能,使抗原特异性PD-1+ T细胞失能。因此,异常表达的sPD-L1参与了PD-1/PD-L1抑制途径的调节,增加了肿瘤细胞免疫逃逸的复杂性和多面性。 综上所述,本课题成功研制出3株特异性鼠抗人PD-L1功能型单克隆抗体,且这3株单抗可用于FACS、Western blot和ELISA检测等,这3株单抗具有促进T细胞增殖的功能;对健康人外周血、肺癌患者外周血及胸水血清中可溶性PD-L1的表达水平检测显示,sPD-L1在肺癌患者外周血中异常高表达。
[Abstract]:Bretscher and Cohn propose a 鈥渃o-stimulatory signal鈥漑1] on the basis of a T cell activation double signal model, which leads to non-reactive or specific immune tolerance or even apoptosis of T cells if a second signal provided by a co-stimulatory molecule is absent. The regulation of positive and negative co-stimulation signals and the balance between them play an important role in the whole process of body immune response[2-3]. PD-1/ PD-L1 is a pair of important negative co-stimulatory molecules. A human PD-L1 gene is positioned on chromosome 9p24. 2, an I-type transmembrane protein[4, 7, 9] encoding 290 amino acid residues, It is advantageous to maintain the immune homeostasis of the body[6-7]. PD-L1 constitutive expression is expressed in monocytes/ macrophages and dendritic cells (DC) and upregulated after activation. At the same time, a large number of studies confirmed that there were PD-L1 expression in the membrane and cytoplasm of multiple tumor cells[8-9]. PD-Ll of tumor cell surface plays an important role in tumor cell induction-specific CTL apoptosis[9]. The PD-1/ PD-L1 pathway is closely related to the immune escape of tumor cells, which can enhance the antitumor immune response of the organism[10-12]. Therefore, PD-L1 molecule has become the focus of immunological research in recent years and is expected to be an effective target molecule for immunotherapy of tumor, autoimmune diseases and viral infection diseases. In this study, L929/ PD-L1 gene transfected with high-expression human PD-L1 gene was used as immunogen, BALB/ c mice were immunized routinely, B lymphocyte hybridoma technique was used for cell fusion, L929/ PD-L1 was used as antibody screening cell, L929/ mock was used as control. The hybridoma cell lines secreting specific murine anti-human PD-L1 monoclonal antibodies were identified as 11G8 mAb, 2G5 mAb and 5C3 m by indirect immunofluorescence labeling and flow cytometry analysis. Ab, Western-blotting analysis showed that all three antibodies can specifically recognize proteins on L929/ PD-L1 cells without identifying L929/ mo Anti-PD-L1-PE mAb of 3 monoclonal antibodies and commercial anti-PD-L1-PE mAb were used to identify the binding sites of cell antigens. The results suggest that three antibodies can recognize PD-L1 molecules expressed by activated T cells, and can significantly promote the proliferation of T cells in vitro, which is a functional monoclonal antibody with better effect. Detection of soluble PD-L1 in serum of peripheral blood and thoracic water in peripheral blood and lung cancer patients using self-developed sPD-L1 ELISA system The results showed that the expression level of sPD-L1 in peripheral blood of lung cancer patients was significantly higher than that of healthy volunteers; the expression level of sPD-L1 in breast water of lung cancer patients was higher than that of patients with lung cancer; the expression of sPD-L1 in peripheral blood of lung cancer patients was not significantly correlated with age, sex and lung cancer III and IV. The expression level of sPD-L1 in peripheral blood of patients with adenocarcinoma and squamous cell carcinoma was significantly higher than that in patients with adenocarcinoma. The abnormal high expression of sPD-L1 in peripheral blood of patients with lung cancer shows that the factor further inhibits the immune function of the body, so that the antigen-specific PD-1 + Therefore, abnormal expression of sPD-L1 is involved in the regulation of PD-1/ PD-L1 inhibition pathway, which increases the complex immune escape of tumor cells. in conclusion, three specific murine anti-human PD-L1 functional monoclonal antibodies have been successfully developed, and that three monoclonal antibodies can be used for FACS, Western blot and ELISA detection, and the three monoclonal antibodies have the function of promoting T cell proliferation. The expression level of soluble PD-L1 in peripheral blood and chest water in peripheral blood and lung cancer patients showed that sPD-L1 was outside lung cancer patients.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2290875
[Abstract]:Bretscher and Cohn propose a 鈥渃o-stimulatory signal鈥漑1] on the basis of a T cell activation double signal model, which leads to non-reactive or specific immune tolerance or even apoptosis of T cells if a second signal provided by a co-stimulatory molecule is absent. The regulation of positive and negative co-stimulation signals and the balance between them play an important role in the whole process of body immune response[2-3]. PD-1/ PD-L1 is a pair of important negative co-stimulatory molecules. A human PD-L1 gene is positioned on chromosome 9p24. 2, an I-type transmembrane protein[4, 7, 9] encoding 290 amino acid residues, It is advantageous to maintain the immune homeostasis of the body[6-7]. PD-L1 constitutive expression is expressed in monocytes/ macrophages and dendritic cells (DC) and upregulated after activation. At the same time, a large number of studies confirmed that there were PD-L1 expression in the membrane and cytoplasm of multiple tumor cells[8-9]. PD-Ll of tumor cell surface plays an important role in tumor cell induction-specific CTL apoptosis[9]. The PD-1/ PD-L1 pathway is closely related to the immune escape of tumor cells, which can enhance the antitumor immune response of the organism[10-12]. Therefore, PD-L1 molecule has become the focus of immunological research in recent years and is expected to be an effective target molecule for immunotherapy of tumor, autoimmune diseases and viral infection diseases. In this study, L929/ PD-L1 gene transfected with high-expression human PD-L1 gene was used as immunogen, BALB/ c mice were immunized routinely, B lymphocyte hybridoma technique was used for cell fusion, L929/ PD-L1 was used as antibody screening cell, L929/ mock was used as control. The hybridoma cell lines secreting specific murine anti-human PD-L1 monoclonal antibodies were identified as 11G8 mAb, 2G5 mAb and 5C3 m by indirect immunofluorescence labeling and flow cytometry analysis. Ab, Western-blotting analysis showed that all three antibodies can specifically recognize proteins on L929/ PD-L1 cells without identifying L929/ mo Anti-PD-L1-PE mAb of 3 monoclonal antibodies and commercial anti-PD-L1-PE mAb were used to identify the binding sites of cell antigens. The results suggest that three antibodies can recognize PD-L1 molecules expressed by activated T cells, and can significantly promote the proliferation of T cells in vitro, which is a functional monoclonal antibody with better effect. Detection of soluble PD-L1 in serum of peripheral blood and thoracic water in peripheral blood and lung cancer patients using self-developed sPD-L1 ELISA system The results showed that the expression level of sPD-L1 in peripheral blood of lung cancer patients was significantly higher than that of healthy volunteers; the expression level of sPD-L1 in breast water of lung cancer patients was higher than that of patients with lung cancer; the expression of sPD-L1 in peripheral blood of lung cancer patients was not significantly correlated with age, sex and lung cancer III and IV. The expression level of sPD-L1 in peripheral blood of patients with adenocarcinoma and squamous cell carcinoma was significantly higher than that in patients with adenocarcinoma. The abnormal high expression of sPD-L1 in peripheral blood of patients with lung cancer shows that the factor further inhibits the immune function of the body, so that the antigen-specific PD-1 + Therefore, abnormal expression of sPD-L1 is involved in the regulation of PD-1/ PD-L1 inhibition pathway, which increases the complex immune escape of tumor cells. in conclusion, three specific murine anti-human PD-L1 functional monoclonal antibodies have been successfully developed, and that three monoclonal antibodies can be used for FACS, Western blot and ELISA detection, and the three monoclonal antibodies have the function of promoting T cell proliferation. The expression level of soluble PD-L1 in peripheral blood and chest water in peripheral blood and lung cancer patients showed that sPD-L1 was outside lung cancer patients.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 ;Recombinant human B7-H4 expressed in Escherichia coli inhibits T lym-phocyte proliferation and IL-2 secretion in vitro[J];Acta Pharmacologica Sinica;2006年06期
2 邱玉华，张学光，，谢炜，朱学东;一种显著提高小鼠生产单抗腹水产量的新方法[J];中国免疫学杂志;1995年06期
本文编号：2290875
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