[关键词]
[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。
[Key word]
[Abstract]
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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[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)