Monitoring anti-CD19 chimeric antigen receptor T cell population by flow cytometry and its consistency with digital droplet polymerase chain reaction.
Monitoring anti-CD19 chimeric antigen receptor T cell population by flow cytometry and its consistency with digital droplet polymerase chain reaction.
复制标题
通过流式细胞术监测抗 CD19 嵌合抗原受体 T 细胞群及其与数字液滴聚合酶链反应的一致性
DOI：
10.1002/cyto.a.24676
复制
发表时间：
2023-01
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
抗CD19嵌合抗原受体（CAR19）T细胞疗法在复发性或难治性B细胞恶性肿瘤患者中产生了令人印象深刻的临床疗效。作为一种活的药物，监测CAR T细胞在体内的药代动力学是临床工作的重要组成部分，为评估治疗反应和相关副作用提供了有价值的信息。然而，没有关于CAR T细胞的检测和定量的指南。流式细胞术是监测CAR T细胞动力学的一种方便且常用的方法，但其性能仍有待验证。通过使用可检测最流行的CAR19构建体上独特表位的市售抗独特型抗体，我们评价了重要的性能参数，包括特异性、检测下限、定量下限和流式细胞术在CAR19 T细胞检测和定量中的精密度。然后在188对临床数据和细胞系实验中研究了流式细胞术和液滴数字PCR产生的结果之间的一致性。兔抗小鼠FMC63单克隆抗体对FCM检测CAR19阳性细胞具有高度特异性，临界值为0.05%。流式细胞术和ddPCR产生的结果在临床样品和细胞系中具有良好的相关性，但随着CAR19阳性细胞丰度的降低，相关性恶化。这在临床数据中淋巴细胞少于0.5%的情况下尤其明显，可能是由于液滴数字PCR和流式细胞术的精密度降低（由试验内和试验间变异系数表示）。我们证明，使用抗独特型抗体的流式细胞术是体内检测和定量CAR19 T细胞的可靠和稳健的方法，并且在监测CAR19 T细胞动力学方面与液滴数字PCR具有良好的一致性。Droplet Digital PCR在基因组水平上测量CAR载体拷贝数，而流式细胞术可以在蛋白质组水平上定量CAR蛋白。单克隆抗体R19 M在流式细胞术检测CAR 19阳性细胞方面具有较高的特异性和特异性。使用抗独特型抗体的流式细胞术是体内检测和定量CAR19 T细胞的可靠方法，在监测CAR19 T细胞动力学方面与ddPCR具有良好的一致性。
Anti‐CD19 chimeric antigen receptor (CAR19) T cell therapy has produced impressive clinical efficacy in patients with relapsed or refractory B‐cell malignancies. As a living drug, monitoring the pharmacokinetics of CAR T cells in vivo is an important part of clinical work, which provides valuable information for assessing therapeutic response and related side effects. However, no guidelines are available regarding the detection and quantification of CAR T cells. Flow cytometry is a convenient and commonly used method in monitoring CAR T cell kinetics, but its performance remains to be validated. By using a commercial anti‐idiotype antibody that detects unique epitopes on the most popular CAR19 construct, we evaluated important performance parameters, including specificity, lower limit of detection, lower limit of quantification, and precision of flow cytometry in the detection and quantification of CAR19 T cells. Consistency between the results generated by flow cytometry and droplet digital PCR was then investigated in 188 pairs of clinical data and in cell line experiments. Rabbit anti‐mouse FMC63 monoclonal antibody possesses high specificity in the detection of CAR19 positive cells by FCM with a cut‐off value of 0.05%. The results produced by flow cytometry and ddPCR were well correlated in the clinical samples and in cell lines, but the correlation deteriorated as the abundance of CAR19 positive cells decreased. This was especially evident with less than 0.5% of lymphocytes in clinical data, possibly due to reduced precision (indicated by intra‐ and inter‐assay coefficients of variability) of both droplet digital PCR and flow cytometry. We demonstrated that flow cytometry using anti‐idiotype antibody is a reliable and robust approach in the detection and quantification of CAR19 T cells in vivo and has good consistency with droplet digital PCR in monitoring CAR19 T cell kinetics. Droplet Digital PCR measures CAR vector copy number at the genomic level while flow cytometry can quantify the CAR protein at the proteomic level. The monoclonal antibody R19M possesses high specificity and specificity in the detection of CAR19 positive cells by FCM. Flow cytometry using anti‐idiotype antibody is a reliable approach to detecting and quantifying CAR19 T cells in vivo and has good consistency with ddPCR in monitoring CAR19 T cell kinetics.
登录
查看更多内容
影响因子：
12.4
作者：
Haas, Andrew R.;Tanyi, Janos L.;Beatty, Gregory L.
通讯作者：
Beatty, Gregory L.
DOI：
10.1158/1078-0432.ccr-18-0758
发表时间：
2018-12-15
期刊：
Clinical cancer research : an official journal of the American Association for Cancer Research
影响因子：
--
作者：
Mueller KT;Waldron E;Grupp SA;Levine JE;Laetsch TW;Pulsipher MA;Boyer MW;August KJ;Hamilton J;Awasthi R;Stein AM;Sickert D;Chakraborty A;Levine BL;June CH;Tomassian L;Shah SS;Leung M;Taran T;Wood PA;Maude SL
通讯作者：
Maude SL
影响因子：
48
作者：
Hindson, Christopher M.;Chevillet, John R.;Briggs, Hilary A.;Gallichotte, Emily N.;Ruf, Ingrid K.;Hindson, Benjamin J.;Vessella, Robert L.;Tewari, Muneesh
通讯作者：
Tewari, Muneesh
内源性参考基因对基因工程性低芥酸菜籽体事件的数字PCR评估的影响。
影响因子：
--
作者：
Demeke T;Eng M
通讯作者：
Eng M
影响因子：
168.9
作者：
Abramson, Jeremy S.;Palomba, M. Lia;Siddiqi, Tanya
通讯作者：
Siddiqi, Tanya