靶向人表皮生长因子受体2嵌合抗原受体T(HER2-CAR-T)细胞毒性作用的体内外验证  

In vitro and in vivo validation of cytotoxicity of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells

作　　者：张仪婷 席文锦 王鹏举 赵晓娟 郑瑞 梁思辛 蒙若彤 阎博 杨安钢 ZHANG Yiting;XI Wenjin;WANG Pengju;ZHAO Xiaojuan;ZHENG Rui;LIANG Sixin;MENG Ruotong;YAN Bo;YANG Angang(Department of Biochemistry and Molecular Biology,Basic Medical Science Academy,Air Force Medical University,Xi’an 710032;Department of Immunology,Basic Medical Science Academy,Air Force Medical University,Xi’an 710032;College of Life Science,Yan’an University,Yan’an 716000,China)

机构地区：[1]空军军医大学基础医学院生物化学与分子生物学教研室,陕西西安710032 [2]空军军医大学基础医学院免疫学教研室,陕西西安710032 [3]延安大学生命科学学院,陕西延安716000

出　　处：《细胞与分子免疫学杂志》2024年第4期289-295,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81972870);国家肿瘤生物学重点实验室自主课题(CBSKL2022ZZ20);陕西省自然科学基础研究计划(2022JM-485)。

摘　　要：目的 对靶向人表皮生长因子受体2嵌合抗原受体T(HER2-CAR-T)细胞进行毒理学评估,为其后期HER2-CAR-T细胞治疗的临床上评估提供安全性依据。方法 利用重组慢病毒载体制备HER2-CAR-T细胞,采用软琼脂集落形成实验,观察HER2-CAR-T细胞集落形成情况并对集落形成率进行统计分析;将HER2-CAR-T细胞悬液与兔红细胞悬液共孵,通过直接观察法以及酶标仪检测法评估红细胞溶解情况;对雄性新西兰兔耳缘静脉注射HER2-CAR-T细胞制剂,通过组织切片染色观察HER2-CAR-T细胞对动物血管的刺激作用;以pMD 2.G载体上的水泡性口炎病毒囊膜糖蛋白(VSV-G)基因为目标序列,利用荧光定量PCR进行慢病毒载体安全性的验证;取接受HER2-CAR-T细胞输注的小鼠心、肝、肺、肾,HE染色观察其病变情况。结果 成功制备了HER2-CAR-T细胞,这些细胞在体外不具备软琼脂集落形成能力,HER2-CAR-T细胞制剂对新西兰兔红细胞不产生溶血现象。新西兰兔耳缘静脉输注HER2-CAR-T细胞后未发现明显血管刺激反应,也未检测到VSV-G的特异性扩增,治疗组小鼠心、肝、肺、肾组织未见明显病变。结论 所制备的HER2-CAR-T细胞具有可靠安全性。Objective To evaluate the toxicology of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T(HER2-CAR-T)cells and to provide a safety basis for the clinical evaluation of HER2-CAR-T cell therapy.Methods The recombinant lentiviral vector was used to generate HER2-CAR-T cells.Soft agar colony formation assay was used to observe the colony formation of HER2-CAR-T cells,and the colony formation rate was statistically analyzed.The HER2-CAR-T cell suspension was co-incubated with rabbit red blood cell suspension,and the hemolysis of red blood cells was evaluated by direct observation and microplate reader detection.The HER2-CAR-T cell preparation was injected into the ear vein of male New Zealand rabbits,and the stimulating effect of HER2-CAR-T cells on the blood vessels of the animals was observed by staining of tissue sections.The vesicular stomatitis virus envelope glycoprotein(VSV-G)gene of pMD 2.G vector was used as the target sequence,and the safety of the lentiviral vector was verified by real-time fluorescence quantitative PCR.The heart,liver,lung,and kidney of mice receiving HER2-CAR-T cell infusion were collected,and the lesions were observed by HE staining.Results The HER2-CAR-T cells were successfully prepared.These cells did not exhibit soft agar colony formation ability in vitro,and the HER2-CAR-T cell preparation did not cause hemolysis in New Zealand rabbit red blood cells.After the infusion of HER2-CAR-T cells into the ear vein of New Zealand rabbits,no obvious vascular stimulation response was found,and no specific amplification of VSV-G was detected.No obvious lesions were found in the heart,liver,lung and kidney tissues of the treatment group.Conclusion The prepared HER2-CAR-T cells have reliable safety.

关 键 词：安全性 毒理学 靶向人表皮生长因子2嵌合抗原受体T(HER2-CAR-T)细胞 集落形成实验 溶血 血管刺激性 器官毒性 

分 类 号：R457.2[医药卫生—治疗学] S481.1[医药卫生—临床医学] R730.51[农业科学—农药学] R392.12[农业科学—植物保护]