摘要
目的:设计并制备一种分别靶向B细胞表面抗原CD19和CD22的CAR-T细胞,检测其对肿瘤细胞的体内外杀伤效果。方法:将含有人源化CD19 Sc Fv的二代CAR分子和带有CD3ε链作为共刺激结构域的CD22 Sc Fv CAR分子以P2A自剪切肽连接,序列连接于慢病毒载体p LTR-CMV-MCS中,以HEK-293T细胞包装相应的慢病毒载体,感染健康志愿者提供的T细胞制备CAR-19-22-T细胞,同时以相同二代结构分别构建单靶向CAR-T细胞作为参照。构建表达荧光素酶、CD19和/或CD22的前列腺癌3M细胞(靶细胞)。将各种CAR-T细胞与靶细胞共同培养,采用荧光素酶化学发光法和ELISA法检测其对靶细胞的杀伤能力和细胞因子的分泌水平。通过尾静脉注射Raji-Luc细胞构建NOD-SCID免疫缺陷小鼠白血病模型,分别注射各组CAR-T细胞进行治疗并评估其疗效。结果:培养7 d的CAR-19-22-T细胞的CAR-19表达率为13.7%,CAR-22表达率为14.3%。CAR-19-22-T细胞在10∶1效靶比时,对3M-CD19-Luc、3M-CD22-Luc和3M-CD19-CD22-Luc细胞的杀伤率均显著高于T细胞[(78.1±14.4)%vs(11.1±4.3)%(、46.7±10.7)%vs(12.4±2.7)%(、90.5±4.3)%vs(14.3±3.7)%,均P<0.01];与3M-CD19-Luc、3M-CD22-Luc、3M-CD19-CD22-Luc靶细胞共培养后,CAR-19-22-T细胞IFN-γ、TNF-α和IL-2水平均显著低于CAR-19-T和CAR-22-T细胞(P<0.05或P<0.01)。CAR-19-22-T细胞对移植Raji-Luc细胞模型小鼠治疗效果明显,其生存期显著长于T细胞组(P<0.01),与CAR-19-T组和CAR-22-T组荷瘤小鼠比较差异均无统计学意义(均P>0.05)。结论:成功设计并制备了一种双靶点CAR-19-22-T细胞,其能够有效杀伤表达CD19和/或CD22抗原的肿瘤细胞,对Raji-Luc细胞的白血病模型小鼠有显著的治疗效果。
Objective:To design and prepare a bi-specific chimeric antigen receptor(CAR)-T cell targeting both CD19 and CD22 antigens on the surface of B lymphocytes and to detect its tumor-killing effects in vitro and in vivo.Methods:Second-generation CAR molecules containing CD19 Sc Fv(human originated)and CD22 Sc Fv(CD3εchain as costimulatory domain)were connected with P2A self-cleaving peptide.The synthesized fragment was packaged into lentivival vector p LTR-CMV-MCS.The lentiviruses were packaged with HEK-293T cells and infected with healthy human T cells to prepare CAR-19-22-T cells.Single targeted second-generation CAR-T cells were constructed with the same CD19 ScFv CAR molecules and CD22 ScFv CAR molecules respectively.Prostate cancer 3M cells expressing luciferase,CD19 and/or CD22 were constructed as target cells.Various CAR-T cells were co-cultured with target cells.Luciferase and ELISA methods were used to detect the target-cell killing ability of each group of CAR-T cells and their cytokine secretion.Mouse leukemia models were constructed by injecting Raji-Luc cells through the tail veins of the mice.Different groups of CAR-T cells were then injected respectively for treatment and the treatment efficacy was evaluated.Results:For CAR-19-22-T cells cultured for 7 days,the expression rates of CAR-19 and CAR-22 were 13.7%and 14.3%respectively.The killing rates of 3M-CD19-Luc,3M-CD22-Luc and 3M-CD19-CD22-Luc cells by CAR-19-22-T cells at 10∶1 efficiency target ratio were significantly higher than those of T cells[(78.1±14.4)%vs(11.1±4.3)%,(46.7±10.7)%vs(12.4±2.7)%,(90.5±4.3)%vs(14.3±3.7)%,all P<0.01].CAR-19-22-T cells co-cultured with 3M-CD19-Luc,3M-CD22-Luc or3M-CD19-CD22-Luc target cells secreted significantly lower amounts of IFN-γ,TNF-αand IL-2 than those for CAR-19-T or CAR-22-T(P<0.05 or P<0.01).CAR-19-22-T cells had significantly better treatment efficacy on mice infected with Raji-Luc cells.The survival time of these mice was significantly longer than that of the mice in the T-cell group(P<0.01)and
作者
李薇
曾伟杰
彭浩
刘灿
刘广花
肖非笛
曾桂芳
梁晓
蔡车国
胡隽源
周明
LI Wei;ZENG Weijie;PENG Hao;LIU Can;LIU Guanghua;XIAO Feidi;ZENG Guifang;LIANG Xiao;CAI Cheguo;HU Juanyuan;ZHOU Ming(Department of Hematology,Hunan Provincial People’s Hospital,Changsha 410005,Hunan,China;Shenzhen Beike Biotechnology Co.Ltd,Shenzhen 518000,Guangdong,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2022年第7期623-630,共8页
Chinese Journal of Cancer Biotherapy
基金
湖南省自然科学基金资助项目(No.2020JJ4403)
湖南省社会发展重点研发项目(No.2019SK2091)。
