Manufacturing of CD19 Specific CAR T-Cells and Evaluation of their Functional Activity in Vitro

EXPERIMENTAL STUDIES , , , , , ,

AV Petukhov1, VA Markova2, DV Motorin1, AK Titov1, NS Belozerova2, PM Gershovich2, AV Karabel’skii2, RA Ivanov2, EK Zaikova1, EYu Smirnov2, PA Butylin1, AYu Zaritskey1

1 VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341

2 Biocad Biotechnology Company, 34-A Svyazi str., Strel’na, Saint Petersburg, Russian Federation, 198515

For correspondence: Andrei Yur’evich Zaritskey, MD PhD, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341; Tel.: +7(812)702-68-28, Fax: +7(812)702-37-65; e-mail: zaritskey@gmail.com

For citation: Petukhov AV, Markova VA, Motorin DV, et al. Manufacturing of CD19 Specific CAR T-Cells and Evaluation of their Functional Activity in Vitro. Clinical oncohematology. 2018;11(1):1–9.

DOI: 10.21320/2500-2139-2018-11-1-1-9

ABSTRACT

Background. The most promising variant of adoptive immunotherapy of the B-line oncohematological diseases includes the use of cells with the chimeric antigen receptor (CAR T-cells), that showed extraordinary results in clinical studies.

Aim. To manufacture CAR T-cells for the clinical use and to study their cytotoxicity in vitro.

Methods. Human T-lymphocytes were transduced by the lentiviral vector containing anti-CD19-CAR, RIAD, and GFP genes. The T-cell transduction efficacy was assessed on the basis of GFP protein signal by flow cytometry. Propidium iodide was used to analyse the cell viability. Cytotoxic activity of the manufactured CAR T-cells was studied in the presence of the target cells being directly co-cultivated. Analysis of the number and viability of CAR T-cells and cytokine expression was performed by flow cytometry.

Results. The viability of the transduced T-cells and GFP expression reached 91.87 % and 50.87 % respectively. When cultured in the presence of IL-2 and recombinant CD19 (the target antigen), the amount of CAR-T after 120 h of the process was 1.4 times larger compared with the period of 48 h. In the cytotoxic test of co-cultivation CAR-T with the K562-CD19+ cells the percentage of CAR-T increased to 57 % and 84.5 % after 48 h and 120 h of exposure respectively. When cultured with the K562 cells (test line not expressing CD19) the number of CAR T-cells decreased to 36.2 % within 48 h while the number of K562 cells increased to 58.3 %. The viability of target cells in the experimental and control groups was 3.5 % and 36.74 % respectively. Comparison of IL-6 level in the control and experimental groups revealed that the differences are insignificant, as opposed to the level of other cytokines (IFN-γ, IL-2, TNF) which proved to be different in both groups.

Conclusion. The present work resulted in the production of anti-CD19 CAR T-cells with adequate viability. The in vitro model demonstrated their cytotoxicity. Manufacturing of CAR T-cells for clinical use is the first step of the development of adoptive immunotherapy in the Russian Federation.

Keywords: CAR T-cells, adoptive immunotherapy, acute lymphoblastic leukemia, non-Hodgkin’s lymphomas, lentiviral transduction, graft-versus-host reaction, сytokine release syndrome.

Received: September 15, 2017

Accepted: December 7, 2017

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