靶向EGFR家族成員分泌PD-1抗體的CAR-T細胞治療肺癌的實驗研究
發(fā)布時間：2018-10-10 17:19
【摘要】：肺癌是嚴重危害人類健康及影響人們生活質(zhì)量的重大疾病之一,在全世界范圍內(nèi)其發(fā)病率和死亡率占人類常見腫瘤的第三位。2015年預(yù)計有超過22萬新增病例被診斷為肺癌,而且同年死于肺癌的患者將超過15.8萬。目前臨床上肺癌常見的治療方法,包括外科手術(shù)切除治療,放射線治療,化療等并不能減少肺癌的發(fā)生率,肺癌患者的5年生存率并不高,因此我們迫切需要尋求新的治療方法。近年來,免疫治療作為癌癥治療的新方法,引起了研究者極大的興趣。癌癥靶向免疫療法,例如免疫檢查點抑制劑,已經(jīng)演化成第四種肺癌治療的策略,給癌癥患者帶來了新的希望。在腫瘤免疫細胞治療中,T細胞具有將腫瘤抗原作為外來物識別的能力并且能夠快速清除它們,盡管存在細胞毒性T細胞識別腫瘤相關(guān)抗原,腫瘤細胞也常常逃脫或逃避免疫系統(tǒng)的攻擊。機體產(chǎn)生的免疫反應(yīng)與一系列共刺激分子及共抑制分子緊密相關(guān),免疫檢查點是腫瘤細胞逃脫或逃避免疫系統(tǒng)攻擊的一種主要方式。目前已經(jīng)發(fā)現(xiàn)的抑制性免疫檢查點有細胞毒性T淋巴細胞相關(guān)抗原4(CTLA-4)、程序性細胞死亡蛋白1(PD-1)、淋巴細胞激活基因3(LAG-3)以及PD-1的兩個主要配體PD-L1和PD-L2等,它們能夠?qū)е滦?yīng)T淋巴細胞的耗竭和減少,減弱機體免疫系統(tǒng)產(chǎn)生的抗腫瘤反應(yīng)。隨著人們對免疫檢查點和抗腫瘤免疫反應(yīng)理解及認識的加深,生產(chǎn)免疫檢查點阻斷單抗是修復(fù)免疫系統(tǒng)和恢復(fù)T細胞抗腫瘤免疫反應(yīng)的有效方式。臨床實驗發(fā)現(xiàn)靶向程序性細胞死亡蛋白1或其配體PD-L1的單抗對大多數(shù)腫瘤都有臨床反應(yīng),包括惡性黑色素瘤、肺癌、卵巢癌等。盡管抗PD-1抗體阻斷治療對惡性黑色素瘤或非小細胞肺癌患者有很好的療效,但是其較高的生產(chǎn)成本和藥物不良反應(yīng)限制了它在臨床中的運用。本文構(gòu)建并檢測了靶向EGFR家族成員并且高水平穩(wěn)定表達PD-1抗體的Herin CAR-T細胞對幾種肺癌細胞株的體外及體內(nèi)殺傷作用。(1)構(gòu)建包含PD-1單鏈抗體可變區(qū)的重組質(zhì)粒,通過piggy Bac轉(zhuǎn)座子系統(tǒng)轉(zhuǎn)染T細胞,獲得能夠高水平穩(wěn)定表達PD-1抗體(5-10ug/ml)的T細胞(PIK-PD1),同時Western blotting實驗證明T細胞分泌的PD-1抗體包含正確的重鏈(50Kd)。(2)檢測PIK-PD1細胞分泌的PD-1抗體的功能,包括封閉活化T細胞表面表達的共抑制分子PD-1、Tim3以及LAG3,促進T細胞的增殖。通過流式檢測發(fā)現(xiàn)表達PD-1抗體的PIK-PD1細胞相比未電轉(zhuǎn)的對照T細胞而言能夠增強CD107?,記憶標志CD62L、CD45RO、CCR7的表達。(3)構(gòu)建表達PD-1抗體的Herin CAR-T細胞(PD1-Herin CAR-T),通過檢測其與腫瘤細胞共培養(yǎng)后細胞因子的分泌,發(fā)現(xiàn)IL-2、IL-4、IL-6、TNF-?和IFN-?的分泌量相比對照的Herin CAR-T細胞而言明顯要高。同時我們發(fā)現(xiàn)PD1-Herin CAR-T細胞能夠更有效的殺傷肺癌細胞株NCI-H1299,NCI-H460,NCI-H23和H446。(4)NCI-H460小鼠移植瘤模型中,分泌PD-1抗體的CAR-T細胞治療組相比PBS和Control T細胞治療組能夠更有效地抑制腫瘤的生長,且PD1-Herin CAR-T細胞在小鼠體內(nèi)能更好地增殖。本文首先構(gòu)建了能夠高水平穩(wěn)定表達PD-1抗體的T細胞,并且發(fā)現(xiàn)T細胞分泌的PD-1抗體能夠促進T細胞的增殖,細胞因子的分泌,封閉活化T細胞表面PD-1、LAG3、Tim3等抑制性免疫檢查點的表達。同時也構(gòu)建了表達PD-1抗體的Herin CAR-T細胞,發(fā)現(xiàn)PD1-Herin CAR-T細胞在體外和體內(nèi)實驗中能夠更有效的殺傷肺癌細胞株。
[Abstract]:Lung cancer is one of the major diseases that seriously endanger human health and affects people's quality of life. The incidence and death rate of lung cancer is the third place in the world. More than 220,000 new cases are expected to be diagnosed as lung cancer in 2015. And those who died in lung cancer in the same year will be more than 150,000. At present, there are common methods for treating lung cancer, including surgical resection, radiotherapy, chemotherapy, etc., which can not reduce the incidence of lung cancer, and the 5-year survival rate of lung cancer patients is not high, so we urgently need to seek new treatment methods. In recent years, immunotherapy as a new method for the treatment of cancer has aroused great interest in researchers. Cancer-targeted immunotherapy, such as an immunocheckpoint inhibitor, has evolved into a strategy for the treatment of a fourth lung cancer, bringing new hope to cancer patients. In tumor immune cell therapy, T cells have the ability to identify tumor antigens as foreign objects and can quickly clear them, even though there are cytotoxic T cells that recognize tumor-associated antigens, tumor cells often escape or evade attacks by the immune system. The immune response produced by the body is closely related to a series of co-stimulatory molecules and co-stimulatory molecules, which are one of the main ways in which tumor cells escape or evade immune system attacks. The inhibitory immune checkpoint, which has been found at present, has cytotoxic T lymphocyte associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (FBP-3), and two major ligands PD-L1 and PD-L2 of PD-1, which can lead to depletion and reduction of effector T lymphocytes, and the anti-tumor response generated by the immune system of the organism is weakened. With the understanding and understanding of immune checkpoint and anti-tumor immune response, the production of immune checkpoint blocking monoclonal antibody is an effective way to repair the immune system and restore the anti-tumor immune response of T cells. Clinical experiments show that the monoclonal antibody targeting programmed cell death protein 1 or its ligand PD-L1 has a clinical response to most tumors, including malignant melanoma, lung cancer, ovarian cancer, and the like. Although anti-PD-1 antibody blocking therapy has a good effect on patients with malignant melanoma or non-small cell lung cancer, their higher production costs and adverse drug reactions limit its clinical use. In this paper, we constructed and tested Herin CAR-T cells targeting EGFR family members and stably expressing PD-1 antibody in vitro and in vivo killing effects on several lung cancer cell lines. (1) A recombinant plasmid containing a variable region of PD-1 single chain antibody was constructed, and T cells were transfected with a pggy Bac transposon system to obtain a T cell (PIK-PD1) capable of stably expressing the PD-1 antibody (5-10ug/ ml) at a high level, while the Western blotting experiment demonstrated that the PD-1 antibody secreted by T cells contained the correct heavy chain (50Pin). (2) detecting the function of the PD-1 antibody secreted by PIK-PD1 cells, which comprises the following steps of: closing the co-inhibiting molecules PD-1, Ti3 and LAG3 expressed on the surface of the activated T cells, and promoting the proliferation of T cells. By streaming detection, it was found that the PIK-PD1 cells expressing the PD-1 antibody were able to enhance the CD107 compared to the control T cells that were not electrospun. Memory flag CD62L, CD45RO, CCR7 expression. (3) constructing Herin CAR-T cells expressing PD-1 antibody (PD1-Herin CAR-T), detecting cytokine secretion after co-culture with tumor cells, and discovering IL-2, IL-4, IL-6, TNF-? and IFN-? The amount of secretion was significantly higher in comparison with Herin CAR-T cells. At the same time, we found that PD1-Herin CAR-T cells were more effective in killing lung cancer cell lines NCI-H1299, NCI-H460, NCI-H23 and H446. (4) The CAR-T cell treatment group secreting PD-1 antibody can inhibit tumor growth more effectively than PBS and Control T cell treatment group, and PD1-Herin CAR-T cells can proliferate better in mice. In this paper, T cells capable of stably expressing PD-1 antibody can be stably expressed, and the PD-1 antibody secreted by T cells can promote the proliferation of T cells, the secretion of cytokines, and the expression of inhibitory immune checkpoints such as PD-1, LAG3, Ti3, and the like in the surface of the activated T cells. Meanwhile, Herin CAR-T cells expressing PD-1 antibody were also constructed, and PD1-Herin CAR-T cells were found to be more effective in killing lung cancer cell lines in vitro and in vivo experiments.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2
本文編號：2262629
[Abstract]:Lung cancer is one of the major diseases that seriously endanger human health and affects people's quality of life. The incidence and death rate of lung cancer is the third place in the world. More than 220,000 new cases are expected to be diagnosed as lung cancer in 2015. And those who died in lung cancer in the same year will be more than 150,000. At present, there are common methods for treating lung cancer, including surgical resection, radiotherapy, chemotherapy, etc., which can not reduce the incidence of lung cancer, and the 5-year survival rate of lung cancer patients is not high, so we urgently need to seek new treatment methods. In recent years, immunotherapy as a new method for the treatment of cancer has aroused great interest in researchers. Cancer-targeted immunotherapy, such as an immunocheckpoint inhibitor, has evolved into a strategy for the treatment of a fourth lung cancer, bringing new hope to cancer patients. In tumor immune cell therapy, T cells have the ability to identify tumor antigens as foreign objects and can quickly clear them, even though there are cytotoxic T cells that recognize tumor-associated antigens, tumor cells often escape or evade attacks by the immune system. The immune response produced by the body is closely related to a series of co-stimulatory molecules and co-stimulatory molecules, which are one of the main ways in which tumor cells escape or evade immune system attacks. The inhibitory immune checkpoint, which has been found at present, has cytotoxic T lymphocyte associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (FBP-3), and two major ligands PD-L1 and PD-L2 of PD-1, which can lead to depletion and reduction of effector T lymphocytes, and the anti-tumor response generated by the immune system of the organism is weakened. With the understanding and understanding of immune checkpoint and anti-tumor immune response, the production of immune checkpoint blocking monoclonal antibody is an effective way to repair the immune system and restore the anti-tumor immune response of T cells. Clinical experiments show that the monoclonal antibody targeting programmed cell death protein 1 or its ligand PD-L1 has a clinical response to most tumors, including malignant melanoma, lung cancer, ovarian cancer, and the like. Although anti-PD-1 antibody blocking therapy has a good effect on patients with malignant melanoma or non-small cell lung cancer, their higher production costs and adverse drug reactions limit its clinical use. In this paper, we constructed and tested Herin CAR-T cells targeting EGFR family members and stably expressing PD-1 antibody in vitro and in vivo killing effects on several lung cancer cell lines. (1) A recombinant plasmid containing a variable region of PD-1 single chain antibody was constructed, and T cells were transfected with a pggy Bac transposon system to obtain a T cell (PIK-PD1) capable of stably expressing the PD-1 antibody (5-10ug/ ml) at a high level, while the Western blotting experiment demonstrated that the PD-1 antibody secreted by T cells contained the correct heavy chain (50Pin). (2) detecting the function of the PD-1 antibody secreted by PIK-PD1 cells, which comprises the following steps of: closing the co-inhibiting molecules PD-1, Ti3 and LAG3 expressed on the surface of the activated T cells, and promoting the proliferation of T cells. By streaming detection, it was found that the PIK-PD1 cells expressing the PD-1 antibody were able to enhance the CD107 compared to the control T cells that were not electrospun. Memory flag CD62L, CD45RO, CCR7 expression. (3) constructing Herin CAR-T cells expressing PD-1 antibody (PD1-Herin CAR-T), detecting cytokine secretion after co-culture with tumor cells, and discovering IL-2, IL-4, IL-6, TNF-? and IFN-? The amount of secretion was significantly higher in comparison with Herin CAR-T cells. At the same time, we found that PD1-Herin CAR-T cells were more effective in killing lung cancer cell lines NCI-H1299, NCI-H460, NCI-H23 and H446. (4) The CAR-T cell treatment group secreting PD-1 antibody can inhibit tumor growth more effectively than PBS and Control T cell treatment group, and PD1-Herin CAR-T cells can proliferate better in mice. In this paper, T cells capable of stably expressing PD-1 antibody can be stably expressed, and the PD-1 antibody secreted by T cells can promote the proliferation of T cells, the secretion of cytokines, and the expression of inhibitory immune checkpoints such as PD-1, LAG3, Ti3, and the like in the surface of the activated T cells. Meanwhile, Herin CAR-T cells expressing PD-1 antibody were also constructed, and PD1-Herin CAR-T cells were found to be more effective in killing lung cancer cell lines in vitro and in vivo experiments.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2
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