嵌合抗原受体T细胞治疗相关细胞因子释放综合征小鼠模型的建立

Establishment of a cytokine release syndrome associated with chimeric antigen receptor T cell treatment in SCID/Beige mice model

目的建立嵌合抗原受体T(CAR-T)细胞治疗相关细胞因子释放综合征(CRS)的小鼠模型。方法通过分子克隆及慢病毒转染技术,构建靶向人CD19分子的CAR-T细胞,采用流式细胞术检测CAR-T细胞转染效率,酶联免疫吸附试验(ELISA)法及流式细胞术检测CAR-T细胞特异性杀伤靶细胞的能力。通过尾静脉注射CAR-T细胞至荷瘤重症联合免疫缺陷裸鼠体内,小鼠分为磷酸缓冲液组、低负荷组(注射1×105个人淋巴瘤细胞Raji-Luc2细胞)和高负荷组(注射5×105个Raji-Luc2细胞)。采用动物活体成像法检测肿瘤治疗效果,ELISA法检测小鼠血清中细胞因子人白细胞介素2(IL-2)、人γ-干扰素(IFN-γ)、鼠IL-6、鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)的水平。结果经尾静脉注射人T细胞和T细胞+OKT-3抗体后,T细胞组和T细胞+OKT-3组小鼠的健康得分分别为(1.15±0.08)分和(2.90±0.15)分,差异有统计学意义(P<0.001)。T细胞+OKT-3组小鼠血清中人IL-2、人IFN-γ、人IL-15、鼠IL-6、鼠GM-CSF水平分别为(1064.00±50.14)、(1285.00±193.90)、(202.4±18.76)、(1478.00±289.20)和(350.70±42.27)pg/ml,均高于T细胞组[分别为(22.67±6.36)、(23.67±3.71)、(44.33±14.45)、(147.30±36.20)和(138.00±22.74)pg/ml,均P<0.05];OKT-3联合人T细胞引起小鼠血清中细胞因子人IL-2、人IFN-γ、鼠IL-6、鼠GM-CSF水平迅速升高,并伴有体温升高及体重下降。成功构建靶向CD19分子的CAR-T细胞,流式细胞术检测CAR-T细胞阳性率>30%。ELISA结果显示,在CD19抗原存在时,CAR-T19细胞与Raji、Nalm-6共孵育组细胞分泌IL-2和IFN-γ的水平分别为(561.00±37.07)、(680.30±71.27)、(369.00±25.71)和(523.00±26.31)pg/ml,均高于与K562共孵育组[分别为(55.00±20.53)和(64.00±7.55)pg/ml,均P<0.001]。小鼠成像实验显示,小鼠成瘤后第7天经尾静脉回输活化后的CAR-T19细胞,成瘤第13天,低负荷和高负荷组小鼠肿瘤荧光强度均低于接种肿瘤的第7天,高负荷组肿瘤荧光强度由144.00±24.69减少至5.02±2.35(P=0.005),低负荷组肿瘤荧光强度由58.47±9.36减少至3.48±1.67(P=0.004)。荷瘤小鼠注射CAR-T19细胞72 h后,血清中T细胞活化相关细胞因子人IL-2、人IL-15、人IFN-γ水平迅速增高,单核细胞相关因子鼠IL-16、鼠GM-CSF分泌增加,同时伴随有体温升高和体重降低等CRS典型特征。结论体外成功构建靶向CD19分子的CAR-T细胞,通过CAR-T细胞回输治疗验证了小鼠体内CRS现象的发生,为CAR-T细胞治疗相关CRS的发生机制及CRS预防策略提供了动物模型参考。

Objective To establish a cytokine release syndrome(CRS)mouse model related to CAR-T cell therapy and provide a research model for the clinical phenomena.Methods CAR-T cells targeting human CD19 molecule were constructed by molecular cloning and lentiviral transfection.Flow cytometry(FACS)was used to detect the transfection efficiency of CAR-T cells.The tumor-killing efficiency of CAR-T cells was detected by ELISA and flow cytometry.The CAR-T cells were injected into the tumor-bearing SCID/Beige mice through tail vein,and divided into phosphate buffered solution(PBS)group,low-burden group(1×105 Raji-Luc2 cells)and high-burden group(5×105 Raji-Luc2 cells).The tumor treatment effect was detected by animal in vivo imaging.Serum levels of cytokines including human IFN-γ,human IL-2,mouse IL-6,and mouse GM-CSF were measured by ELISA.The health status of the mice was evaluated by pathological examination.Results The health scores of T cell group and T cell+OKT-3 group were(1.15±0.08)and(2.90±0.15),respectively,after the injection of human T cell and T cell+OKT-3 antibody through tail vein,and the difference was statistically significant(P<0.001).The serum levels of human IL-2,human IFN-γ,human IL-15,mouse IL-6 and mouse GM-CSF in T cell+OKT-3 group were(1064.00±50.14),(1285.00±193.90),(202.4±18.76),(1478.00±289.20)and(350.70±42.27)pg/ml,respectively,higher than(22.67±6.36),(23.67±3.71),(44.33±14.45),(147.30±36.20),(138.00±22.74)pg/ml in T cell group(P<0.05).OKT-3 combined with human T cells caused a rapid increase in serum levels of human IL-2,human IFN-γ,mouse IL-6 and mouse GM-CSF,accompanied by an increase in body temperature and weight loss.CD19-targeting CAR-T cells were successfully constructed,and the positive rate of CAR-T cells was>30%detected by flow cytometry.ELISA results showed that in the presence of CD19 antigen,IL-2 and IFN-γsecreted by CAR-T19 cells co-incubated with Raji and Nalm were(561.00±37.07),(680.30±71.27),(369±25.71)and(523.00±26.31)pg/ml,respectively,higher than(55.00±20.53)and(64.00±7.55)pg/ml in the co-incubated with K562 group(P<0.001).Activated CAR-T19 cells were reinjected through the tail vein on the seventh day after tumor formation.Imaging experiments in mice showed that on the thirteenth day after tumor formation,the fluorescence intensities of tumors in the low-burden and high-burden groups were lower than on the seventh day of tumor inoculation,and the fluorescence intensity of tumors in the high-burden group decreased from 144.00±24.69 to 5.02±2.35(P=0.005).The fluorescence intensity of low burden group decreased from 58.47±9.36 to 3.48±1.67(P=0.004).The serum levels of T cell activation related cytokines IL-2,IL-15 and IFN-γincreased rapidly,and the secretion of monocyte related cytokines IL-16 and GM-CSF increased,accompanied by the typical characteristics of CRS such as increased body temperature and weight loss at 72 hours after injection of CAR-T19 cells.Conclusions CAR-T cells targeting CD19 molecule are successfully constructed,and CRS phenomenon is verified in tumor-bearing mice by CAR-T cell re-infusion,providing an animal model for the mechanism of CAR-T treatment-related CRS and CRS prevention strategies.