鼠抗人PD-L1功能性單克隆抗體的研制及其生物學(xué)特性的研究
發(fā)布時(shí)間：2018-10-24 08:33
【摘要】：Bretscher和Cohn在T細(xì)胞活化雙信號(hào)模型的基礎(chǔ)上提出“協(xié)同刺激信號(hào)”[1],如果缺少協(xié)同刺激分子提供的第二信號(hào),將會(huì)導(dǎo)致T細(xì)胞的無(wú)反應(yīng)性或特異性免疫耐受甚至凋亡。正性和負(fù)性協(xié)同刺激信號(hào)的調(diào)節(jié)及兩者之間的平衡在機(jī)體免疫應(yīng)答的整個(gè)過(guò)程中發(fā)揮著重要的調(diào)節(jié)作用[2-3]。PD-1/PD-L1是一對(duì)重要的負(fù)性協(xié)同刺激分子。人PD-L1基因定位在染色體9p24.2 ,編碼290個(gè)氨基酸殘基的I型跨膜蛋白[4,7,9]。PD-1/PD-L信號(hào)對(duì)淋巴細(xì)胞的增殖發(fā)揮負(fù)性調(diào)控功能,可防止過(guò)度的免疫損傷及自身免疫性疾病的發(fā)生,有利于維持機(jī)體的免疫自穩(wěn)[6-7]。PD-L1組成性表達(dá)于單核/巨噬細(xì)胞以及樹(shù)突狀細(xì)胞(DC),并在活化后呈上調(diào)表達(dá)。同時(shí),大量研究結(jié)果證實(shí)多種腫瘤細(xì)胞的胞膜和胞漿都有PD-L1的表達(dá)[8-9]。腫瘤細(xì)胞表面的PD-Ll在腫瘤細(xì)胞誘導(dǎo)特異性CTL凋亡的過(guò)程中發(fā)揮重要作用[9]。PD-1/PD-L1途徑與腫瘤細(xì)胞免疫逃逸密切相關(guān),阻斷此途徑可增強(qiáng)機(jī)體的抗腫瘤免疫應(yīng)答[10-12]。因此,PD-L1分子成為近年來(lái)免疫學(xué)研究的熱點(diǎn),有望成為腫瘤、自身免疫性疾病和病毒感染性疾病的免疫干預(yù)治療的有效靶分子。 本課題研究以高表達(dá)人PD-L1分子的基因轉(zhuǎn)染細(xì)胞L929/PD-L1作為免疫原,常規(guī)免疫BALB/c小鼠,采用B淋巴細(xì)胞雜交瘤技術(shù)進(jìn)行細(xì)胞融合,以L(fǎng)929/PD-L1作為抗體篩選細(xì)胞,L929/mock為對(duì)照細(xì)胞,經(jīng)間接免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析、反復(fù)篩選和多次克隆化培養(yǎng),篩選出分泌特異性鼠抗人PD-L1單克隆抗體的雜交瘤細(xì)胞株命名為11G8 mAb,2G5 mAb和5C3 mAb,Western-blotting分析顯示,3株抗體都可以特異性識(shí)別L929/PD-L1細(xì)胞上蛋白質(zhì),而不能識(shí)別L929/mock�？乖稽c(diǎn)競(jìng)爭(zhēng)抑制實(shí)驗(yàn)提示3株單抗與商品化的anti-PD-L1-PE mAb識(shí)別細(xì)胞抗原結(jié)合位點(diǎn)不同。對(duì)3株抗體生物學(xué)功能的研究結(jié)果提示,3株抗體均能夠識(shí)別活化T細(xì)胞表達(dá)的PD-L1分子,且在體外能夠顯著促進(jìn)T細(xì)胞的增殖,是具有較好效應(yīng)的功能性單克隆抗體。 利用自主研制的sPD-L1 ELISA檢測(cè)系統(tǒng)測(cè)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的表達(dá)水平。結(jié)果顯示,肺癌患者外周血中sPD-L1表達(dá)水平明顯高于健康志愿者;肺癌患者胸水中sPD-L1的表達(dá)水平高于肺癌患者外周血;肺癌患者外周血中sPD-L1的表達(dá)與年齡、性別及肺癌III、IV期無(wú)明顯相關(guān)性;腺癌患者、鱗癌患者外周血中sPD-L1表達(dá)水平明顯高于小細(xì)胞癌患者。sPD-L1在肺癌患者外周血中異常高表達(dá)表明該因子進(jìn)一步抑制了機(jī)體的免疫功能,使抗原特異性PD-1+ T細(xì)胞失能。因此,異常表達(dá)的sPD-L1參與了PD-1/PD-L1抑制途徑的調(diào)節(jié),增加了腫瘤細(xì)胞免疫逃逸的復(fù)雜性和多面性。 綜上所述,本課題成功研制出3株特異性鼠抗人PD-L1功能型單克隆抗體,且這3株單抗可用于FACS、Western blot和ELISA檢測(cè)等,這3株單抗具有促進(jìn)T細(xì)胞增殖的功能;對(duì)健康人外周血、肺癌患者外周血及胸水血清中可溶性PD-L1的表達(dá)水平檢測(cè)顯示,sPD-L1在肺癌患者外周血中異常高表達(dá)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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 邱玉華，張學(xué)光，，謝煒，朱學(xué)東;一種顯著提高小鼠生產(chǎn)單抗腹水產(chǎn)量的新方法[J];中國(guó)免疫學(xué)雜志;1995年06期
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