靶向EGFR家族成员分泌PD-1抗体的CAR-T细胞治疗肺癌的实验研究
发布时间：2018-10-10 17:19
【摘要】：肺癌是严重危害人类健康及影响人们生活质量的重大疾病之一,在全世界范围内其发病率和死亡率占人类常见肿瘤的第三位。2015年预计有超过22万新增病例被诊断为肺癌,而且同年死于肺癌的患者将超过15.8万。目前临床上肺癌常见的治疗方法,包括外科手术切除治疗,放射线治疗,化疗等并不能减少肺癌的发生率,肺癌患者的5年生存率并不高,因此我们迫切需要寻求新的治疗方法。近年来,免疫治疗作为癌症治疗的新方法,引起了研究者极大的兴趣。癌症靶向免疫疗法,例如免疫检查点抑制剂,已经演化成第四种肺癌治疗的策略,给癌症患者带来了新的希望。在肿瘤免疫细胞治疗中,T细胞具有将肿瘤抗原作为外来物识别的能力并且能够快速清除它们,尽管存在细胞毒性T细胞识别肿瘤相关抗原,肿瘤细胞也常常逃脱或逃避免疫系统的攻击。机体产生的免疫反应与一系列共刺激分子及共抑制分子紧密相关,免疫检查点是肿瘤细胞逃脱或逃避免疫系统攻击的一种主要方式。目前已经发现的抑制性免疫检查点有细胞毒性T淋巴细胞相关抗原4(CTLA-4)、程序性细胞死亡蛋白1(PD-1)、淋巴细胞激活基因3(LAG-3)以及PD-1的两个主要配体PD-L1和PD-L2等,它们能够导致效应T淋巴细胞的耗竭和减少,减弱机体免疫系统产生的抗肿瘤反应。随着人们对免疫检查点和抗肿瘤免疫反应理解及认识的加深,生产免疫检查点阻断单抗是修复免疫系统和恢复T细胞抗肿瘤免疫反应的有效方式。临床实验发现靶向程序性细胞死亡蛋白1或其配体PD-L1的单抗对大多数肿瘤都有临床反应,包括恶性黑色素瘤、肺癌、卵巢癌等。尽管抗PD-1抗体阻断治疗对恶性黑色素瘤或非小细胞肺癌患者有很好的疗效,但是其较高的生产成本和药物不良反应限制了它在临床中的运用。本文构建并检测了靶向EGFR家族成员并且高水平稳定表达PD-1抗体的Herin CAR-T细胞对几种肺癌细胞株的体外及体内杀伤作用。(1)构建包含PD-1单链抗体可变区的重组质粒,通过piggy Bac转座子系统转染T细胞,获得能够高水平稳定表达PD-1抗体(5-10ug/ml)的T细胞(PIK-PD1),同时Western blotting实验证明T细胞分泌的PD-1抗体包含正确的重链(50Kd)。(2)检测PIK-PD1细胞分泌的PD-1抗体的功能,包括封闭活化T细胞表面表达的共抑制分子PD-1、Tim3以及LAG3,促进T细胞的增殖。通过流式检测发现表达PD-1抗体的PIK-PD1细胞相比未电转的对照T细胞而言能够增强CD107?,记忆标志CD62L、CD45RO、CCR7的表达。(3)构建表达PD-1抗体的Herin CAR-T细胞(PD1-Herin CAR-T),通过检测其与肿瘤细胞共培养后细胞因子的分泌,发现IL-2、IL-4、IL-6、TNF-?和IFN-?的分泌量相比对照的Herin CAR-T细胞而言明显要高。同时我们发现PD1-Herin CAR-T细胞能够更有效的杀伤肺癌细胞株NCI-H1299,NCI-H460,NCI-H23和H446。(4)NCI-H460小鼠移植瘤模型中,分泌PD-1抗体的CAR-T细胞治疗组相比PBS和Control T细胞治疗组能够更有效地抑制肿瘤的生长,且PD1-Herin CAR-T细胞在小鼠体内能更好地增殖。本文首先构建了能够高水平稳定表达PD-1抗体的T细胞,并且发现T细胞分泌的PD-1抗体能够促进T细胞的增殖,细胞因子的分泌,封闭活化T细胞表面PD-1、LAG3、Tim3等抑制性免疫检查点的表达。同时也构建了表达PD-1抗体的Herin CAR-T细胞,发现PD1-Herin CAR-T细胞在体外和体内实验中能够更有效的杀伤肺癌细胞株。
[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.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
本文编号：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.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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