荣斌,吴纯启,原野,尹纪业,瞿文生,李晓旭,范丹,贺小琼,王全军.NSG小鼠多发性骨髓瘤模型评价BCMA-CAR-T的药效和毒性及RNAscop在检测CAR-T分布中的应用[J].药物评价研究,2019,42(5):822-827
NSG小鼠多发性骨髓瘤模型评价BCMA-CAR-T的药效和毒性及RNAscop在检测CAR-T分布中的应用
NSG mouse multiple myeloma model was used to evaluate in vivo efficacy and toxicity of BCMA-CAR-T cells and application of RNAscop in the detection of CAR-T distribution
投稿时间:2018-12-20  
DOI:10.7501/j.issn.1674-6376.2019.05.003
中文关键词:  BCMA-CAR-T;多发性骨髓瘤;生物发光成像;药效;毒性;RNAscope;γ-干扰素
英文关键词:BCMA-CAR-T;nonclinical study;bioluminescent imaging;efficacy;toxicity;RNAscope
基金项目:重大新药创制科技重大专项(2018ZX09711003-007);重大新药创制科技重大专项(2017ZX09201008-001-002);重大新药创制科技重大专项(2018ZX09201017-003);重点仪器研发计划(ZDYZ2015-2);转基因生物新品种培育重大专项(2016ZX08011007)
作者单位E-mail
荣斌 昆明医科大学 公共卫生学院, 云南 昆明 650504
军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850 
 
吴纯启 军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850  
原野 军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850  
尹纪业 军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850  
瞿文生 军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850  
李晓旭 军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850  
范丹 昆明医科大学 公共卫生学院, 云南 昆明 650504  
贺小琼 昆明医科大学 公共卫生学院, 云南 昆明 650504 hexqcn@aliyun.com 
王全军 军事科学院军事医学研究院, 抗毒药物与毒理学国家重点实验室, 国家北京药物安全评价研究中心, 北京 100850 wangquanjunbeijing@163.com 
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中文摘要:
      目的 选择合适的小鼠模型,评价以B细胞成熟抗原为靶点的嵌合抗原受体T细胞(BCMA-CAR-T)治疗产品的抗多发性骨髓瘤作用及毒性,探讨RNAscope在CAR-T治疗中的应用。方法 在免疫缺陷严重的NSG小鼠中建立MM.1s-luc多发性骨髓瘤模型,随机分为细胞外液组(每只小鼠100 μL PBS缓冲液)、MOCK-T组(未转染的T细胞,剂量为100 μL)和CAR-T低、中、高剂量组(分别每只计数1×106、5×106、10×106个CAR-T,每只一次性尾iv 100 μL细胞悬液)。采用活体成像技术检测CAR-T治疗肿瘤的消退效果;酶联免疫吸附实验检测血浆中γ-干扰素(IFN-γ)水平;观察小鼠一般状态、体温、体质量;治疗后14 d对小鼠进行解剖和采样,利用RNAscope技术检测CAR-T在小鼠组织的特异性分布。结果 成功建立了多发性骨髓瘤NSG小鼠移植肿瘤模型;在输注BCMA-CAR-T后3、7、12 d,生物发光成像显示,CAR-T组小鼠平均光子强度显著低于细胞外液、MOCK-T组(P<0.05),中、高剂量组的治疗效果明显高于低剂量组(P<0.05);CAR-T组的IFN-γ分泌水平显著高于细胞外液、MOCK-T组(P<0.05);记录CAR-T治疗组小鼠的一般情况,未见明显不良反应;RNAscope检测到CAR-T在输注14 d后少量特异性分布于骨髓和脾脏。结论 本研究验证了BCMA-CAR-T抗多发性骨髓瘤药效明显,安全可靠,RNAscop技术可应用于CAR-T分布检测。
英文摘要:
      Objective Choose the appropriate mouse model, evaluate BCMA-CAR-T anti-tumor effects, and explore the application of RNAscope in CAR-T cell therapy. Methods MM. 1s-luc multiple myeloma mouse model was established in NSG mice with severe immunodeficiency, they were randomly divided into extracellular fluid group (100 μL PBS buffer per mouse), MOCK-T group (100 μL untransfected T cells), low, medium and high dose groups of CAR-T (1×106, 5×106, 10×106 CAR-T per mouse, and 100 μL cell suspension of one-off tail iv). In vivo imaging was performed to detect the tumor regression effect of CAR-T treatment. The level of interferon-gamma (IFN-gamma) in plasma was detected by ELISA. General clinical observation, body temperature, body weight monitoring were performed after CAR-T was given once. Mice were dissected and sampled 14 days after infusion. CAR-T distribution in the spleen and bone marrow of mice was detected by RNAscope technology, and the specific distribution of CAR-T in various tissues was detected. Results Successfully established many found myeloma NOD/SCID mice transplanted tumor model, At 3, 7 and 12 days after BCMA-CAR-T infusion, bioluminescence imaging showed that the average photon intensity of mice in CAR-T group was significantly lower than that in extracellular fluid and MOCK-T group (P<0.05), and the therapeutic effect in medium and high dose groups was significantly higher than that in low dose groups (P<0.05); the level of IFN-gamma secretion in CAR-T group was significantly higher than that in extracellular fluid and MOCK-T group (P<0.05); the general situation of mice in CAR-T group was recorded, but no significant difference was observed. Adverse reactions:RNA scope detected that CAR-T was localized in bone marrow and spleen 14 days after infusion. Conclusion The BCMA-CAR-T has been proven to be safe and reliable by some non-clinical evaluation methods. RNAscop technology can be applied to CAR-T cell distribution detection.
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