新型全人源LAG3单克隆抗体的体外抗肿瘤作用及其机制初探  

Preliminary study on the anti-tumor effect and mechanism of a novel fully human anti-LAG3 monoclonal antibody in vitro

作　　者：张陈 刘平 于晓杰 刘剑飞 钦可为 吴成林 周丽君 ZHANG Chen;LIU Ping;YU Xiaojie;LIU Jianfei;QIN Kewei;WU Chenglin;ZHOU Lijun(Naval Clinical College,Anhui Medical University,Hefei 230032,Anhui,China;Central Laboratory,the Sixth Medical Center of the Chinese PLA General Hospital,Beijing 100048,China;College of Otolaryngology Head and Neck Surgery,the Sixth Medical Center of the Chinese PLA General Hospital,Beijing 100048,China;Department of General Practice,the Sixth Medical Center of the Chinese PLA General Hospital,Beijing 100048,China)

机构地区：[1]安徽医科大学海军临床学院,安徽合肥230032 [2]中国人民解放军总医院第六医学中心中心实验室,北京100048 [3]中国人民解放军总医院第六医学中心耳鼻咽喉头颈外科医学部,北京100048 [4]中国人民解放军总医院第六医学中心全科医学科,北京100048

出　　处：《中国肿瘤生物治疗杂志》2022年第5期419-425,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.32170933,No.31470897);海军总医院创新培育基金资助项目(No.CXPY201817)。

摘　　要：目的:构建LAG3+Jurkat细胞与肿瘤细胞的共培养模型,探讨新型全人源LAG3单克隆抗体(LAG3 mAb)的体外抗肿瘤作用及其可能的机制。方法:使用PHA刺激Jurkat细胞模拟TIL,通过ELISA法检测Jurkat细胞的IL-2分泌水平来评估细胞活化程度,通过FCM、免疫荧光法和WB法检测活化后Jurkat细胞的LAG3表达水平及肿瘤细胞(胃癌HGC-27、MGC-803细胞和NSCLC A549细胞)中LAG3配体MHCⅡ类分子(MHC-Ⅱ)的表达水平。构建活化的LAG3+Jurkat细胞与肿瘤细胞的共培养模型,CCK-8法评估在不同效靶比时LAG3+Jurkat细胞杀伤肿瘤细胞的效率及抗LAG3 mAb对杀伤效率的影响,ELISA法检测共培养体系上清液中细胞因子IL-2、IL-10和TNF-α的分泌水平。结果:2μg/mL PHA刺激48 h对Jurkat细胞无明显毒性(P>0.05),且能够活化Jurkat细胞使其分泌IL-2(P<0.01)并诱导LAG3表达(P<0.01),获得活化的LAG3+Jurkat细胞。MGC-803和A549细胞显著表达MHC-Ⅱ(P<0.01),但HGC-27细胞不表达(P>0.05)。选用效靶比10∶1构建LAG3+Jurkat细胞与肿瘤细胞的共培养模型,抗LAG3 mAb能够有效增强Jurkat细胞对两种MHC-Ⅱ+肿瘤细胞的杀伤作用(均P<0.05),并且MHC-Ⅱ+靶细胞组共培养上清液中细胞因子IL-2、IL-10和TNF-α水平均显著升高(均P<0.01)。结论:成功构建LAG3+Jurkat细胞与肿瘤细胞体外共培养模型,抗LAG3 mAb可能通过阻断LAG3/MHC-Ⅱ相互作用提高LAG3+Jurkat细胞对肿瘤细胞MGC-803和A549的杀伤作用,并且该作用可能与共培养体系中细胞因子IL-2、IL-10和TNF-α分泌水平升高有关。Objective: The co-culture model of LAG3+Jurkat cells and tumor cells was constructed to investigate the anti-tumor effect and mechanism of a novel fully human anti-LAG3 monoclonal antibody in vitro. Methods: Jurkat cells were stimulated with PHA to simulate TIL, and the secretion of IL-2 was detected by ELISA to evaluate the degree of Jurkat cell activation. Meanwhile, FCM,Immunofluorescence and WB assays were employed to detect the expression of LAG3 in activated Jurkat cells and MHC class Ⅱmolecule(MHC-Ⅱ), a LAG3 ligand, in HGC-27, MGC-803 and A549 tumor cells. The co-culture model of activated LAG3+Jurkat cells and tumor cells was constructed, and CCK-8 assays were employed to detect the killing efficiency of LAG3+Jurkat cells against tumor cells at different effector-target ratios and the effect of the anti-LAG3 antibody. The secretion levels of cytokines IL-2, IL-10 and TNF-α in supernatant of co-culture system were detected by ELISA. Results: After 48 h treatment, 2 μg/mL PHA exhibited no obvious cytotoxicity to Jurkat cells(P>0.05), but could significantly induce IL-2 secretion(P<0.01) and LAG3 expression(P<0.01), indicating activated LAG3+Jurkat cells were acquired. MGC-803 and A549 cells significantly expressed MHC-Ⅱ(P<0.01), but HGC-27 cells did not express MHC-Ⅱ(P>0.05). The co-culture model of LAG3+Jurkat cells and tumor cells was constructed at a effector-target ratio of10∶1. The anti-LAG3 antibody could effectively enhance the killing efficiency of Jurkat cells against MHC-Ⅱ+tumor cells(P<0.05).Further analysis revealed that the secretion levels of cytokines IL-2, IL-10 and TNF-α were increased in the co-culture supernatant of MHC-Ⅱ+target cell group(all P<0.01). Conclusion: A co-culture model of LAG3+Jurkat cells and tumor cells was successfully constructed in vitro. The anti-LAG3 antibody might increase the killing effect of Jurkat cells against MGC-803 and A549 tumor cells through blocking LAG3/MHC-Ⅱ interaction, which may be related to the increased secretion levels of cytokin

关 键 词：肿瘤 T细胞 JURKAT细胞 淋巴细胞活化基因3 全人源单克隆抗体 MHCⅡ类分子 细胞因子 

分 类 号：R730.51[医药卫生—肿瘤] R73-362[医药卫生—临床医学]