Abstract
Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.
Highlights
Chimeric antigen receptor T (CAR-T) cell therapy constitutes an innovative and promising approach for the treatment of cancer that has fundamentally changed the field of immunotherapy [1]
The unparalleled 40% complete response rates in relapsed/refractory B-cell leukemia and lymphoma patients have resulted in the approval of commercially available CAR-T cell products, such as tisagenlecleucel (KymriahTM) and axicabtagene ciloleucel (YescartaTM) [2,3], and has resulted in the initiation of more than 100 clinical trials with CD19.CAR-T cells listed in the database ClinicalTrials.gov
The aim of this study is to compare CAR-T cell frequencies measured by four different CAR-T cell detection reagents with flow cytometry, as well as to compare the two most commonly used techniques: flow cytometry and quantitative polymerase chain reaction (PCR)
Summary
Chimeric antigen receptor T (CAR-T) cell therapy constitutes an innovative and promising approach for the treatment of cancer that has fundamentally changed the field of immunotherapy [1]. In the context of CAR-T cell production and clinical application, accurate and reproducible CAR detection methods are required [4]. The basic structure of the chimeric antigen receptor comprises three main components: (1) the extracellular antigen-specific domain derived from an antibody’s single chain variable fragment (scFv); (2) the transmembrane domain; and (3) the intracellular domain that mediates downstream signaling. This basic structure remains as a standard, but with progress in CAR development, different generations of CARs have evolved. Firstgeneration CARs contain only a single CD3ζ intracellular domain from the TCR/CD3 receptor complex. Second-generation CARs carry an added co-stimulatory domain, such as CD28 or 4-1BB, and third-generation CAR-T cells include two co-stimulatory molecules within their CAR constructs [5,6]
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