靶向催乳素受体的嵌合抗原受体T细胞制备条件的优化  

Optimal Preparation Conditions of Anti-PRLR Chimeric Antigen Receptor T Cells

作　　者：徐晨宵 朱建伟 张宝红 XU Chenxiao;ZHU Jianwei;ZHANG Baohong(Engineering Research Center of Cell&Therapeutic Antibody,School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240)

机构地区：[1]上海交通大学药学院,细胞工程及抗体药物教育部工程研究中心,上海200240

出　　处：《中国医药工业杂志》2022年第4期491-499,共9页Chinese Journal of Pharmaceuticals

摘　　要：嵌合抗原受体(CAR)修饰的T细胞疗法已成为恶性肿瘤治疗领域内的研究重点,催乳素受体(PRLR)也被证明其过表达会影响乳腺癌的发生与发展,是乳腺癌治疗的潜在靶点。本研究建立了三质粒系统[pHIV-绿色荧光蛋白(GFP)、psPAX2、pVSVG]包装慢病毒,并将其应用于新构建的靶向PRLR的CAR慢病毒表达载体,通过进一步优化慢病毒制备条件,测定慢病毒滴度,成功构建了靶向PRLR的CAR-T细胞。结果表明,制备慢病毒的最优条件为:PRLR-CAR∶psPAX2∶pVSVG质量比为3∶2∶1,且和转染试剂以质量比1∶2混合,经浓缩后滴度可达到1.88×10^(7)TU/ml。以此慢病毒感染人T细胞,并经终浓度0.5 μg/ml嘌呤霉素筛选2 d后可得到CD4阳性表达率约为55%,CD8阳性表达率约为35%的CAR-T细胞,且PRLR-CAR阳性表达率大于50%。构建的新型靶向PRLR的CAR-T细胞可为乳腺癌的免疫治疗提供参考。Chimeric antigen receptor(CAR)T cell therapy has become the focus of research in the field of malignant tumor treatment.Prolactin receptor(PRLR) is one of the potential targets for breast cancer treatment,and its overexpression will affect the occurrence and development of breast cancer.In this study,a three-plasmid system[pHIV-green fluorescent protein(GFP),psPAX2,pVSVG]was established to package lentivirus and applied to the newly constructed CAR lentiviral expression vector targeting PRLR.By further optimizing the preparation conditions of lentivirus and measuring the titer of lentivirus,CAR-T cells targeting PRLR were constructed successfully.The results showed that when the mass ratio of PRLR-CAR∶psPAX2∶pVSVG was 3∶2∶1,and mixed with transfection reagent at a mass ratio of 1∶2,the condition for the preparation of lentivirus was the optimality,and the titer could reach 1.88×10^(7) TU/ml after concentration.After infecting human T cells with the lentivirus and screening with a final concentration of 0.5 μg/ml puromycin for two days,CAR-T cells were obtained with the expression positive rates of CD4 and CD8 for 55% and 35%,respectively.And the expression positive rate of PRLR-CAR could be over 50%.The constructed novel CAR-T cells targeting PRLR can provide a reference for the immunotherapy of breast cancer.

关 键 词：乳腺癌 催乳素受体 嵌合抗原受体 T淋巴细胞 慢病毒 

分 类 号：R737.9[医药卫生—肿瘤]