MEDLINE/PubMed Journal Browser Search

Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

Title: [Construction and in vitro verification of a new humanized anti-CD19 CAR-T cells with high affinity].
Author: Zhang CX, Cheng H, Han X, Qi KM, Chen W, Wu QY, Cao J, Xu KL.
Journal: Zhonghua Xue Ye Xue Za Zhi; 2018 Jun 14; 39(6):465-470. PubMed ID: 30032561.
Abstract:
Objective: To construct humanized anti-CD19 chimeric antigen receptor T cells and investigate its ability to kill leukemia cells in vitro and in vivo. Methods: Humanized anti-human CD19 antibody with a high affinity was obtained based on mouse anti-human CD19 antibody (FMC63). Humanized CD19 CAR-T cells (hCART19) were constructed through transfection of lentivirus carrying a CAR sequence of humanized anti-CD19 scFv into human peripheral CD3(+) T cells. The ability of hCART19 to kill leukemia cells and secrete cytokines was detected by LDH release assay and ELISA. The in vivo tumor-killing effect of hCART19 was evaluated in a leukemia mouse model. Results: Several different humanized CD19 single-chain antibodies which were constructed by IMGT database were expressed in the eukaryotic expression vector and purified followed by acquiring humanized CD19 antibody (Clone H3L2) with similar binding ability to FMC63. Humanized CD19 CAR lentivirus vector was constructed and transfected into T cells to obtain hCART19 cells. The LDH release experiment confirmed that the killing rate of target cells was increased gradually along with the increased E/T ratio. When the ratio of E/T was 10∶1, the killing rate of target cells by hCART19 reached a maximum. When Raji cells were used as target cells, the hCART19 cells group had a significantly higher kill rate [(87.56±1.99)%] than the untransduced T cells group [(19.31±1.16)%] and the control virus transduced T cells group [(21.35±1.19)%](P<0.001). ELISA analysis showed that the secretion of IL-2 [ (10.56±0.88) pg/ml] and IFN-γ [ (199.02±12.66) pg/ml] in the hCART19 cells group were significantly higher than those in the untransduced T cells group [IL-2: (3.55±0.26) pg/ml; IFN-γ: (37.63±0.85) pg/ml] and the control virus transduced T cells group [IL-2: (2.92±0.32) pg/ml; IFN-γ: (52.07±3.33) pg/ml](P<0.001). The above experiments also showed similar results when CHO-K1-CD19 cells were used as target cells. Moreover, in a human leukemia xenograft animal model, the results showed that mice in the untransduced T cells group and the control virus transduced T cells group all died within 20 to 30 days, and the hCART19 cell group survived >40 days, which was more than the survival time of the other two groups of mice. The difference was statistically significant (χ(2)=11.73, P=0.008). Conclusion: Humanized CD19 CAR-T cells with anti-leukemic activity have been successfully constructed, which will lay a foundation for clinical studies in the future. 目的： 构建人源化抗CD19嵌合抗原受体T细胞（CAR-T），通过体外实验验证其杀伤白血病细胞的能力。 方法： 将人CD19的鼠源抗体（FMC63）进行了人源化改造，获得高亲和力的人源化CD19抗体；构建携带人源化CD19 CAR慢病毒载体，感染T细胞获得人源化CD19 CAR-T细胞（hCART19）；按不同效靶比将效应细胞[hCART19、未转染的T细胞（阴性组）及对照病毒转染的T细胞（对照组）]及靶细胞（CHO-K1-CD19及Raji细胞）混合培养，LDH释放实验及ELISA法检测hCART19杀伤白血病细胞的能力及细胞因子释放水平；白血病小鼠模型检测hCART19的杀瘤效果。 结果： LDH释放实验证实随着效靶比的不断增加，对靶细胞的杀伤率逐渐增加，当效靶比为10∶1时hCART19组的杀伤率最大，在Raji细胞中为（87.56±1.99）%，明显高于阴性组[（19.31±1.16）%]及对照组[（21.35±1.19）%]（P值均<0.001）。ELISA法检测显示Raji细胞作为靶细胞时，hCART19组IL-2水平[（10.56±0.88）pg/ml]及IFN-γ[（199.02±12.66）pg/ml]较阴性组[IL-2：（3.55±0.26）pg/ml；IFN-γ：（37.63±0.85）pg/ml]及对照组[IL-2：（2.92±0.32）pg/ml；IFN-γ：（52.07±3.33）pg/ml]明显升高（P值均<0.001）。以上实验在CHO-K1-CD19细胞作为靶细胞时也出现了相似的结果。给予白血病小鼠模型尾静脉分别注射hCART19、对照病毒转染的T细胞及未转染的T细胞，结果显示hCART19组小鼠存活时间>40 d，另外两组小鼠在20~30 d全部死亡，差异有统计学意义（χ(2)=11.73，P=0.008）。 结论： 成功构建了具有抗白血病活性的人源化CD19 CAR-T细胞，为下一步的临床研究奠定了基础。.

[Abstract] [Full Text] [Related] [New Search]