首页|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

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

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

safetytoxicologyhuman epidermal growth factor2 chimeric antigen receptor T(HER2-CAR-T)cellscolony formation assayhemolysisvascular irritationorgan toxicity

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

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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

空军军医大学基础医学院免疫学教研室,陕西西安 710032

延安大学生命科学学院,陕西延安 716000

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

国家自然科学基金国家肿瘤生物学重点实验室自主课题陕西省自然科学基础研究计划

81972870CBSKL2022ZZ202022JM-485

2024

10.13423/j.cnki.cjcmi.009753

细胞与分子免疫学杂志
中国免疫学会,第四军医大学

细胞与分子免疫学杂志

CSTPCD北大核心
影响因子：0.817
ISSN：1007-8738
年,卷(期)：2024.40(4)
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