PD-L1特异性CAR基因的构建及CAR-T细胞的功能活性研究

发布时间：2018-03-14 23:28

本文选题：程序性死亡配体-1　切入点：嵌合抗原受体　出处：《浙江理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：继手术,放疗,化疗后,肿瘤的免疫疗法已成为第四种行之有效的治疗手段,近期因发展迅速,而广受关注。其中,嵌合抗原受体T细胞技术(Chimeric Antigen Receptors-T cell,CAR-T)是一种新兴的过继细胞疗法(Adoptive cell transfer therapy,ACT),该技术将患者的T细胞在体外修饰,活化和增殖后回输到患者体内,通过T细胞表达的特异性受体,靶向性的识别肿瘤细胞,并显示较强的杀伤活性和持久性。PD-L1(Programmed death ligand-1)是程序性死亡分子1(Programmed death-1,PD-1)的配体,主要诱导性的表达在上皮细胞(如肿瘤细胞)和免疫细胞(如肿瘤浸润淋巴细胞)等,在免疫应答负调控中发挥重要作用,PD-1/PD-L1信号通路的激活,有利于形成肿瘤微环境,使肿瘤细胞逃避免疫系统的免疫监视和免疫杀伤,而阻断该信号通路,在一定程度上,促进可识别肿瘤抗原并产生特异性反应的T细胞增殖,发挥其杀伤肿瘤细胞的作用。故本课题设计了PD-L1抗原特异CAR-T细胞,既可以阻断PD-1/PD-L1信号通路,提高T细胞介导的肿瘤免疫治疗,又可以特异性的杀死肿瘤细胞,并且构建的第三代CAR基因,可以增强杀伤的活性和持久性。本研究构建了PD-L1(73-739)基因的原核表达载体,分离纯化His-PD-L1融合蛋白,免疫BALB/c小鼠,利用细胞融合技术和有限稀释法,筛选获得2株能够稳定分泌PD-L1单克隆抗体的杂交瘤细胞株,杂交瘤细胞腹腔接种BALB/c雌性小鼠,通过收集腹水制备并纯化抗体,进行Western Boltting验证,利用ELISA法检测,优选其中一株杂交瘤细胞株,提取杂交瘤细胞总RNA,逆转录合成第一链cDNA,PCR扩增单克隆抗体单链可变区片段,克隆片段经人源化替换与共刺激信号分子CD28、CD137和胞内信号分子CD3-ζ链的基因组合,构建并合成第三代CAR基因,将合成的CAR基因构建于慢病毒载体pCDH-CMV-MCS-EF1-copGFP上,慢病毒载体与包膜载体pLP1、pLP2以及包被载体pLP-VSVG按照优化比例,利用脂质体3000共转染到293T细胞完成慢病毒包装,使用PEG浓缩法,获得有活性的抗PD-L1慢病毒,使用CD8+磁珠分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的CD8+T细胞,经过体外刺激,慢病毒感染CD8+T细胞,待其扩增5 d左右,进行CAR表达的测定,扩增20倍左右,利用非放射性细胞毒性杀伤CytoTox 96和ELISA法检测IFN-γ的分泌来测定CAR-T细胞的体外活性。本研究在成功制备了PD-L1单克隆抗体的基础上,进一步构建了PD-L1特异性的嵌合抗原受体pCDH-CMV-MCS-EF1-copGFP-CAR,转染293T细胞的效率可达到95.66%以上。包装的慢病毒的滴度可达到测定滴度的结果为5×10~7 TU/mL,可成功感染CD8+T细胞,CAR的表达率可达到22%以上。经过CAR-T细胞的体外活性测定,证明PD-L1特异性CAR-T细胞有一定的杀伤力,为进一步的体内试验研究和临床试验提供了重要的技术支撑。
[Abstract]:After surgery, radiotherapy and chemotherapy, immunotherapy for tumors has become an effective treatment for 4th types of cancer. Recently, because of its rapid development, it has attracted much attention. Chimeric Antigen Receptors-T cell (CAR-T) is a novel adoptive cell transfer therapeutic technique that modifies, activates and proliferates T cells in vitro and infuses them into the body of patients. Targeted recognition of tumor cells and their strong cytotoxicity and persistence. PD-L1 programmatic death ligand-1 are the ligands of programmed death molecule 1hp death-1 (PD-1). The main inducible expression in epithelial cells (such as tumor cells) and immune cells (such as tumor infiltrating lymphocytes) plays an important role in the negative regulation of immune response and the activation of PD-1 / PD-L1 signaling pathway, which is beneficial to the formation of tumor microenvironment. To prevent tumor cells from immune surveillance and killing in the immune system, and to block the signaling pathway, to some extent, to promote the proliferation of T cells that recognize tumor antigens and produce specific responses. Therefore, we designed PD-L1 antigen-specific CAR-T cells, which can block PD-1/PD-L1 signaling pathway, improve tumor immunotherapy mediated by T cells, and specifically kill tumor cells. In this study, the prokaryotic expression vector of PD-L1O73-739) gene was constructed, His-PD-L1 fusion protein was isolated and purified, BALB/c mice were immunized, and cell fusion technique and limited dilution method were used to immunize BALB/c mice. Two hybridoma cell lines which could stably secrete monoclonal antibody to PD-L1 were obtained. The hybridoma cells were inoculated intraperitoneally with BALB/c female mice. The antibody was prepared and purified by collecting ascites, and the antibody was verified by Western Boltting, and detected by ELISA method. One of the hybridoma cell lines was selected, the total RNAs were extracted from the hybridoma cells, and the single-strand variable region fragment of monoclonal antibody was amplified by reverse transcription synthesis of the first strand cDNA-PCR. The third generation CAR gene was constructed and synthesized by the combination of human substitution and costimulatory signal molecule CD28 + CD137 and intracellular signal molecule CD3- 味 chain. The synthesized CAR gene was constructed on the lentivirus vector pCDH-CMV-MCS-EF1-copGFP. Lentivirus vector, pLP1pLP2 and pLP-VSVG were cotransfected into 293T cells by liposome 3000 to complete the packaging of lentivirus. The active anti-lentivirus was obtained by PEG concentration method. CD8 magnetic beads were used to isolate CD8 T cells from peripheral blood mononuclear cells (PBMC). After stimulation in vitro, lentivirus was infected with CD8 T cells. After 5 days of expansion, the expression of CAR was detected and the expression of CAR was increased by about 20 times. CytoTox 96 and ELISA were used to detect the activity of CAR-T cells in vitro. In this study, monoclonal antibodies against PD-L1 were successfully prepared. The chimeric antigen receptor pCDH-CMV-MCS-EF1-copGFP-CARwas further constructed. The efficiency of transfection into 293T cells was over 95.66%. The titer of packaged lentivirus was 5 脳 107TU / mL, and the expression rate of car in CD8 T cells could be successfully infected with pCDH-CMV-MCS-EF1-copGFP-CAR. More than 22%. The activity of CAR-T cells was determined in vitro. It was proved that PD-L1 specific CAR-T cells had a certain cytotoxicity, which provided important technical support for further in vivo experimental research and clinical trials.
【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】：R730.51

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