Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration.

嵌合抗原受体（CAR）T细胞被认为是转基因生物（GMO），构成基因治疗药物。因此，严格控制用于临床应用的CAR T细胞的生产。需要合适的方法来评估CAR T细胞产品中的载体拷贝数（VCN）并监测患者的CAR T细胞频率。定量聚合酶链反应（qPCR）是VCN评估的首选方法。然而，尚未描述具有高再现性的标准化程序。在这里，我们报告基于单拷贝基因（SCG）的双链体（DP）-qPCR分析（SCG-DP-PCR），以确定CAR T细胞产品中的VCN。已在海德堡大学医院对具有良好生产规范（GMP）级CAR T细胞的患者进行临床试验的框架内，对SCG-DP-PCR进行了验证并与绝对标准曲线法（ACM）进行了比较。从方法上讲，SCG-DP-PCR显示出优于ACM的技术优势，并最大限度地减少了数学分析。作为一种高度可重复的方法，SCG-DP-PCR可用于对接受CAR T细胞或其他GMO治疗的患者的临床随访，并可能取代已建立的VCN定量方法。这项工作将使临床医生能够评估患者体内的VCN以及CAR T细胞频率，作为决定后续疗法（包括重复施用CAR T细胞）的基础。SCG-DP-PCR显示了优于ACM的技术优势，并最大限度地减少了数学分析。作为一种高度可重复的方法，SCG-DP-PCR可用于对接受CAR T细胞或其他GMO治疗的患者的临床随访，并可能取代已建立的VCN定量方法。这项工作将使临床医生能够评估患者体内的VCN以及CAR T细胞频率，作为决定后续疗法（包括重复施用CAR T细胞）的基础。SCG-DP-PCR显示了优于ACM的技术优势，并最大限度地减少了数学分析。作为一种高度可重复的方法，SCG-DP-PCR可用于对接受CAR T细胞或其他GMO治疗的患者的临床随访，并可能取代已建立的VCN定量方法。这项工作将使临床医生能够评估患者体内的VCN以及CAR T细胞频率，作为决定后续疗法（包括重复施用CAR T细胞）的基础。