Establishment of a Quantitative Polymerase Chain Reaction Assay for Monitoring Chimeric Antigen Receptor T Cells in Peripheral Blood

Wang, H.; Du, Xin; Chen, W.-H.; Jin, Lei; Xiao, Huizhong; Pan, Yue; Chen, H.; An, Ning; Zhang, Q.-X.

doi:10.1016/j.transproceed.2017.11.028

Cited by 17 publications

(10 citation statements)

References 23 publications

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“…Other approaches that target T cell signaling sequences in quantitative PCR assays, real-time (qPCR) [ 16 ] or endpoint PCR (dPCR) [ 23 – 25 ] are suitable for CAR T cell quantification. Here, we designed our own ddPCR assays based on 2 experimental CARs with known sequences, which were validated and tested in murine experiments.…”

Section: Discussionmentioning

confidence: 99%

“…Flow cytometry analysis is easy and rapid to perform and allows subcellular analysis among CAR T cell populations, but it needs to be performed on fresh samples, and the lack of sensitivity may impair analysis of long-term low-level CAR T cell persistence. Taking advantage of the presence of chimeric fusion nucleotide sequences coding for the CAR, molecular quantitative real-time PCR (qPCR) may be an ideal tool for quantitative determination of rare events, as it is already used in minimal residual disease quantification in oncohematology [ 15 ] or CAR T cell monitoring in peripheral blood [ 16 ]. More recently, droplet digital PCR (ddPCR), based on the multiplication of partitioned independent PCRs and Poisson statistics [ 17 ], has emerged as an easy, robust and reproducible molecular tool that may replace classical qPCR.…”

Section: Introductionmentioning

confidence: 99%

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Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients

et al. 2021

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Background Genetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer. Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells. Methods Based on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs. Results 28BBz and 28z ddPCR assays applied to 2 tumoral (acute myeloid leukemia (AML) or multiple myeloma (MM) xenograft humanized NSG mouse models allowed us to quantify the early expansion (up to day 30) of CAR T cells after injection. Interestingly, following initial expansion, when circulating CAR T cells were challenged with the tumor, we noted a second expansion phase. Investigation of the bone marrow, spleen and lung showed that CAR T cells disseminated more within these tissues in mice previously injected with leukemic cell lines. Finally, circulating CAR T cell ddPCR monitoring of R/R acute lymphoid leukemia or diffuse large B cell lymphoma (n = 10 for tisa-cel and n = 7 for axi-cel) patients treated with both approved CAR T cells allowed detection of early expansion, which was highly correlated with FC, as well as long-term persistence (up to 450 days), while FC failed to detect these events. Conclusion Overall, we designed and validated 2 ddPCR assays allowing routine or preclinical monitoring of early- and long-term circulating approved or experimental CAR T cells, including our own IL-1RAP CAR T cells, which will be evaluated in an upcoming phase I clinical trial.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients

et al. 2021

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“…Optimized CAR qPCR protocols have been developed to detect the anti-CD19 (clone FMC63) scFv. Wang et al (9) developed and validated TaqMan qPCR primers and probes for a ∼130 bp amplicon from the FMC63 nucleotide sequence. In their assay, qPCR was performed side-by-side against FMC63 and GAPDH to measure CAR copy number and genome copies, respectively.…”

Section: Real-time Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

The Chimeric Antigen Receptor Detection Toolkit

Huang

2020

Front. Immunol.

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Chimeric antigen receptor-T (CAR-T) cell therapy is a promising frontier of immunoengineering and cancer immunotherapy. Methods that detect, quantify, track, and visualize the CAR, have catalyzed the rapid advancement of CART cell therapy from preclinical models to clinical adoption. For instance, CAR-staining/labeling agents have enabled flow cytometry analysis, imaging applications, cell sorting, and high-dimensional clinical profiling. Molecular assays, such as quantitative polymerase chain reaction, integration site analysis, and RNA-sequencing, have characterized CAR transduction, expression, and in vivo CART cell expansion kinetics. In vitro visualization methods, including confocal and total internal reflection fluorescence microscopy, have captured the molecular details underlying CAR immunological synapse formation, signaling, and cytotoxicity. In vivo tracking methods, including two-photon microscopy, bioluminescence imaging, and positron emission tomography scanning, have monitored CART cell biodistribution across blood, tissue, and tumor. Here, we review the plethora of CAR detection methods, which can operate at the genomic, transcriptomic, proteomic, and organismal levels. For each method, we discuss: (1) what it measures; (2) how it works; (3) its scientific and clinical importance; (4) relevant examples of its use; (5) specific protocols for CAR detection; and (6) its strengths and weaknesses. Finally, we consider current scientific and clinical needs in order to provide future perspectives for improved CAR detection.

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“…To date, there are no available standardized methods for monitoring in vivo behaviors and targeting the efficacy of injected CAR T-cells. The most common (but limited) techniques used to identify CAR T-cells in the body are flow cytometry, biopsy/immunohistochemistry (IHC), enzyme-linked immunosorbent (ELISpot), and polymerase chain reaction (PCR) [9][10][11][12]. Unfortunately, none of these can monitor CAR T-cells within a live body.…”

Section: Introductionmentioning

confidence: 99%

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging

et al. 2020

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Establishment of a Quantitative Polymerase Chain Reaction Assay for Monitoring Chimeric Antigen Receptor T Cells in Peripheral Blood

Cited by 17 publications

References 23 publications

Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients

Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients

The Chimeric Antigen Receptor Detection Toolkit

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging

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