Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 2.1 (5-Year IF – 2.0)
Journal Citation Indicator (JCI) (2023) – 0.4
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Index Copernicus  – 171.00; MNiSW – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

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Advances in Clinical and Experimental Medicine

2019, vol. 28, nr 2, February, p. 159–164

doi: 10.17219/acem/90772

Publication type: original article

Language: English

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

Na Fang1,B,C, Niannian Zhong1,C, Tingxuan Gu1,B, Yahui Wang1,B, Xiangqian Guo1,E, Shaoping Ji1,A,D,F

1 Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, China

Abstract

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.
Conclusion. 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.

Key words

immunotherapy, CAR-T, CD19, examination, B-cell malignancy

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