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100μL PBS缓冲液)、MOCK-T组(未转染的T细胞,剂量为100μL)和CAR-T低、中、高剂量组(分别每只计数1×10~6、5×10~6、10×10~6个CAR-T,每只一次性尾iv 100μL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。
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.1 s-luc multiple myeloma mouse model was established in NSG mice with severe immunodeficiency,they were randomly divided into extracellular fluid group(100μL PBS buffer per mouse),MOCK-T group(100μL untransfected T cells),low,medium and high dose groups of CAR-T(1×10~6,5×10~6,10×10~6 CAR-T per mouse,and100μL 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.
作者
荣斌
吴纯启
原野
尹纪业
瞿文生
李晓旭
范丹
贺小琼
王全军
RONG Bin;WU Chunqi;YUAN Ye;YIN Jiye;QU Wensheng;LI Xiaoxu;FAN Dan;HE Xiaoqiong;WANG Quanjun(School of Public Health,Kunming Medical University,Kunming 650504,China;National Beijing Center for Drug Safety Evaluation and Research,State Key Laboratory of Medical Countermeasures and Toxicology,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China)
出处
《药物评价研究》
CAS
2019年第5期822-827,共6页
Drug Evaluation Research
基金
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)
