![]() The most common methods used for the monitoring of infused CAR-T cells in a clinical setting are flow cytometry and qPCR using CAR-specific primers. To clarify these side-effects, to elucidate non-response or relapse mechanisms and to develop CAR-T cell therapy, reliable detection methods are required. Additionally, tumor-targeting cells can also show an on target-off tumor effect, describing the attack of non-malignant tissue expressing the target antigen. Upon activation, CAR-T cells release cytokines, which can result in a potentially life-threatening cytokine release syndrome. Next to the impressive therapeutic potential, CAR-T cells can also induce significant toxicities. In the clinical setting, monitoring of transfused CAR-T cells is crucial to determine persistence and proliferation, the two key mechanisms which define the anti-tumor efficacy of CAR-T cells in vivo. The quality control assays have to be validated and require in vitro testing for the transduction efficiency of CAR-T cells and vector copy number per cell, which are according to current status performed by flow cytometry and by polymerase chain reaction (PCR). įor CAR-T cell manufacturing in the commercial as well as in the academic setting, stringent regulations were implemented to ensure quality, safety and efficacy. 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. First-generation CARs contain only a single CD3ζ intracellular domain from the TCR/CD3 receptor complex. This basic structure remains as a standard, but with progress in CAR development, different generations of CARs have evolved. 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. In the context of CAR-T cell production and clinical application, accurate and reproducible CAR detection methods are required. 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 (Kymriah™) and axicabtagene ciloleucel (Yescarta™), and has resulted in the initiation of more than 100 clinical trials with CD19.CAR-T cells listed in the database. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.Ĭhimeric 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. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. 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%). 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. 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). CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. ![]() Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy.
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