A quantitative method for measuring the transfection efficiencyof CD19-directed chimeric antigen receptor in target cells

BACKGROUND: Adoptive cell therapy (ACT) based on chimeric antigen receptors (CARs) expressed on the surface of T cells shows a remarkable clinical outcome, particularly for B-cell malignancies. However, toxicity and side effects of CD19-redirected CAR T cells have been observed concurrently in most cases due to cytokine release and tumor cell lysis. Therefore, strictly controlling the amount of valid T cells re-transfused to patients seems to be an important step in reducing toxicity and side effects of CAR T cells. Transfection efficiency via lentiviral particles varies widely in different cases. OBJECTIVES: The aim of this study was to accurately calculate and control the number of valid CAR T cells through ACT because the restriction antibiotics gene or the fluorescence gene are not suitable for tracking or screening for valid transfected T cells. MATERIAL AND METHODS: We expressed and purified a GFP-CD19 fusion protein as a probe to measure the expression efficiency of CD19-redirected CAR on the cell surface in adherent and suspension cell lines. RESULTS: We can precisely calculate the transfected efficiency of lentiviral particles by counting the number of GFP-labeled cells under a microscope, as well as calculate the percentage by comparing the number of GFP-labeled cells to total cells. CONCLUSIONS: We propose a method to control the number of valid cells in ACT and to reduce toxicity and side effects in clinical use - a convenient technique for monitoring the dosage of CAR T cells for patients.