Department of Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International Co, 125 Binney Street, Cambridge, Massachusetts, 02142, USA.
Department of Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International Co, 35 Landsdowne Street, Cambridge, Massachusetts, 02139, USA.
AAPS J. 2021 Mar 2;23(2):36. doi: 10.1208/s12248-021-00560-6.
Characterizing in vivo cellular kinetics and biodistribution of chimeric antigen receptor T (CAR-T) cells is critical for toxicity assessment, nonclinical and clinical efficacy studies. To date, the standardized assay to characterize CAR-T cell distribution, expansion, contraction, and persistence profiles is not readily available. To overcome this limitation and increase comparability among studies, we have established a universal protocol for analysis. We established a duplexing ddPCR protocol for the CAR-T transgene and reference gene to normalize the genomic DNA input prepared from mouse blood and tissues. The high-throughput gDNA extraction method enabled highly reproducible gDNA extraction while eliminating labor-intensive steps. The investigational CAR-T cells were intravenously injected into immunodeficient mice bearing human colorectal cancer xenografts. The blood and tissue samples were collected to measure the cellular kinetics by ddPCR and flow cytometry. The standard curves were linear throughout the calibration range with acceptable intra- and inter-day precision and accuracy. The gDNA recovery study performed by spiking in the exo-gene plasmid DNA or CAR-T cells revealed that the recovery ranged from 60 to 100% in blood and tissue homogenates. The use of both units of copy/μg gDNA and copy/μL blood met the current regulatory requirement and allowed for a systematic understanding of CAR-T cell expansion and a direct comparison with the flow cytometry data. A standardized ddPCR assay, including automated gDNA extraction procedures, has been established for evaluating cellular kinetics and biodistribution in CAR-T cell therapies.
表征嵌合抗原受体 T（CAR-T）细胞的体内细胞动力学和生物分布对于毒性评估、非临床和临床疗效研究至关重要。迄今为止，尚无用于表征 CAR-T 细胞分布、扩增、收缩和持久性特征的标准化检测方法。为了克服这一限制并增加研究之间的可比性，我们已经建立了一种通用的分析方案。我们建立了一种用于 CAR-T 转基因和参照基因的双 ddPCR 方案，以标准化从小鼠血液和组织中制备的基因组 DNA 输入。高通量 gDNA 提取方法能够实现高度可重复的 gDNA 提取，同时消除了劳动密集型步骤。研究用 CAR-T 细胞被静脉注射到携带人结直肠癌异种移植物的免疫缺陷小鼠中。采集血液和组织样本，通过 ddPCR 和流式细胞术测量细胞动力学。标准曲线在整个校准范围内呈线性，具有可接受的日内和日间精密度和准确度。通过在外源性质粒 DNA 或 CAR-T 细胞中掺入进行的 gDNA 回收研究表明，在血液和组织匀浆中的回收率范围为 60%至 100%。使用拷贝/μg gDNA 和拷贝/μL 血液这两个单位既满足了当前的监管要求，又允许系统地了解 CAR-T 细胞的扩增，并与流式细胞术数据进行直接比较。已经建立了一种标准化的 ddPCR 检测方法，包括自动化的 gDNA 提取程序，用于评估 CAR-T 细胞治疗中的细胞动力学和生物分布。
