CART免疫疗法中慢病毒拷贝数检测的实验研究

Detection of Lentivirus Copy Number in the Chimeric Antigen Receptor T Cell Immumotherapy

目的：利用特异探针检测慢病毒拷贝数,在CART免疫治疗中准确区分携带不同靶点的CART细胞在体内的增殖情况。方法：采用分子克隆技术构建含CD19和CD22CAR结构的慢病毒重组质粒;通过流式和ELISA技术检测两种CART细胞在体外与Raji共培养时的杀瘤效率;通过TaqMan技术检测293T细胞及病人基因组中的病毒拷贝数。结果：CD19和CD22的慢病毒载体成功构建,并收集具有高滴度的病毒颗粒;体外研究结果显示感染病毒的T细胞（CD19-CART和CD22-CART）分别与Raji共培养组的杀瘤效率和IL-6释放水平均显著（P〈0.05）或极显著（P〈0.01）高于对照组,在293T细胞中,转染了病毒与重组质粒组的慢病毒拷贝数均显著高于未转染组（P〈0.05）;CD19-CART和CD22-CART序贯回输20min后,均可在体内检测到102-104个病毒拷贝,到第10天显著上升（P〈0.05）,随后变化平稳,且均高于初始回输水平。结论：跟踪监测病毒拷贝数,能确定特定CART细胞在体内的存续时间,为选择合适的治疗时机提供实验依据。

Objective： To detect lentivirus copy number by using specific probes, and to accurately dis- tinguish the chimeric antigen receptor T （CART） cells that target different antigens. Methods： Lentivirus re-plasmids containing CD19/CD22 CAR element were constructed by molecular cloning technology. The tumor killing efficiency and cytokine IL-6 level in coculture of CART and Raji cells were determined by FACS （fluorescent activated cell sorter） and enzyme linked immu- nosorbent assay, respectively. Lentivirus copy numbers in 293T cell and patients＂ genomes were detected using TaqMan real-time fluorescent quantitative PCR technology. Results： The re-plas- mids pTK881-CD19scfv and pTK881-CD22scfv were successfully constructed, and infectious vi- rus particle was collected. Preclinical study revealed that the tumor killing efficiency and IL-6 level in coculture of CD19-CART/ CD22-CART and Raji cells were significantly higher than control groups （P〈0.05 or P〈0.01）. In 293T cell, the lentivirus copy numbers transfected with virus and plasmid groups were significantly higher than untransfected group （P〈0.05）. In addition,we were tracking to detect the virus copy numbers in leukemia patient （sequential transfusion of CD19-CART and CD22-CART）, and found that the copy numbe was range from 102 to 104, which significantly increased in 10 d （P〈0.05） and followed by a subsequent stable changes, but they were higher than the initial transfusion level. Conclusion. Lentivirus copy number detection can be used for evaluating the existing time of CART cell, and further to provide evidence for the clinical treatment.