Insights on Droplet Digital PCR–Based Cellular Kinetics and Biodistribution Assay Support for CAR-T Cell Therapy

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1208/s12248-021-00560-6.