Kunz Alexander, Gern Ulrike, Schmitt Anita, Neuber Brigitte, Wang Lei, Hückelhoven-Krauss Angela, Michels Birgit, Hofmann Susanne, Müller-Tidow Carsten, Dreger Peter, Schmitt Michael, Schubert Maria-Luisa
Department of Internal Medicine V (Hematology/Oncology/Rheumatology), University Hospital Heidelberg, Heidelberg, Germany.
German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ)/National Center for Tumor Diseases (NCT), Heidelberg, Germany.
Mol Ther Methods Clin Dev. 2020 Feb 20;17:448-454. doi: 10.1016/j.omtm.2020.02.003. eCollection 2020 Jun 12.
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细胞被视为转基因生物（GMO），并构成基因治疗药物产品。因此，用于临床应用的CAR T细胞制造受到严格监管。需要有适当的方法来评估CAR T细胞产品中的载体拷贝数（VCN）以及监测患者体内的CAR T细胞频率。定量聚合酶链反应（qPCR）是评估VCN的首选方法。然而，尚未描述具有高重现性的标准化程序。在此，我们报告一种基于单拷贝基因（SCG）的双重（DP）-qPCR检测方法（SCG-DP-PCR），用于确定CAR T细胞产品中的VCN。在海德堡大学医院对采用药品生产质量管理规范（GMP）级CAR T细胞治疗患者的一项临床试验框架内，对SCG-DP-PCR进行了验证，并与绝对标准曲线法（ACM）进行了比较。在方法学上，SCG-DP-PCR显示出优于ACM的技术优势，并将数学分析降至最低。SCG-DP-PCR作为一种高度可重现的方法，可用于接受CAR T细胞或其他GMO治疗患者的临床随访，并可能取代现有的VCN定量方法。这项工作将使临床医生能够评估患者的VCN以及CAR T细胞频率，作为决定后续治疗（包括重复给予CAR T细胞）的依据。
