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

Abstract

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.

Quantification of cell-free DNA for the analysis of CD19-CAR-T cells during lymphoma treatment

“…Currently, clonal kinetics as well as in vivo distribution of CAR-T cells after re-infusion are topics of research. 16 , 35 , 36 Analyzing blood cells by flow cytometry and quantification of genomic DNA in peripheral blood mononuclear cells are the current standards to quantify CAR-T cells during treatment. Assay-specific limitations in qPCR analysis are adapted and under investigation.…”

“… 12 , 14 The handling of qPCR and ddPCR data is currently discussed, because multiple factors influence the amount and composition of cellular (genomic) DNA in patients' blood samples during CAR-T therapy. 15 , 16 …”

“…Following re-infusion, CAR-T cells distribute into various body areas and actively migrate into both tumor stroma and lymph nodes. 16 It is unknown whether loss of detectable CAR-T cells in blood samples corresponds to a loss of CAR-T cells due to apoptosis by cellular exhaustion or if it reflects cell sequestration in targeted tissues. Currently, studies investigating the biology of CAR-T cells following therapeutic administration are mainly focused on cellular samples.…”

Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

“…EC was added to all samples. DNA extraction from the calibration standard samples was conducted according to the method reported by Sugimoto et al (19), with minor modifications. DNA was extracted from 200 μL tissue homogenate or 50 μL blood using the KingFisher™ Flex system (MagMAX_Ultra2_200μL_Flex and MagMAXUltra 2TissueV).…”

“…Droplet digital (dd) PCR runs reactions to the endpoint in partitioned individual droplets (19)(20)(21). The positive and negative droplets are divided based on droplet fluorescence, and DNA copy number is determined based on Poisson statistics.…”