CAR-NK THERAPY: NK CELL EXPANSION EXPOSED TO HEK 293T CELL LINE



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Abstract

Abstract

While creating chimeric antigen receptor (CAR) NK cells, it is necessary to conduct a stage of these immune cell enrichment. Feeder cells are most often used in methods for the effective NK cell expansion. The human embryonic kidney cell line containing the SV40 T-antigen (HEK 293T) is most often used for research purposes in various areas, since it is easily subjected to genetic modifications. This property indicates the potential for modifying HEK 293T cells to express tumor antigens or proinflammatory cytokines, which can be used to activate and enrich NK cells. In this work, we assessed the effect of unmodified HEK 293T cell culture on cytotoxicity and expression of NK and NKT cell activation markers in long-term cultivation in the presence of non-irradiated autologous feeder cells. The study used peripheral blood mononuclear cells collected from healthy volunteer donors. Proliferation was stimulated using antibodies against CD3 and CD28 receptors or mitomycin C-treated HEK 293T cell culture. Cell proliferation was assessed by direct cell counting added with trypan blue dye. Cytotoxicity was determined on HG3, T47D-HER2+, K562 target cultures. Flow cytometry with labeled monoclonal antibodies was used to analyze the expression of surface receptors. Four different methods for lymphocyte activation using HEK 293T were proposed. We found that when using the HEK 293T cell line, an increased percentage of CD3-CD56+ cells in the population was observed in all activation modes, as well as increased expression of NK cell activation markers - NKp30 and NKG2D, in addition, the proportion of CD16+ and CD3+CD4+ lymphocytes increased relative to activation with monoclonal antibodies alone. Of the proposed options for coincubation of lymphocytes with HEK 293T feeder cells, the most effective NK cells expansion was described for the protocol involving the use of the HEK 293T cell line once before the onset of incubation without proliferation additionally stimulated with monoclonal antibodies. This approach resulted in higher proportion of CD56+ lymphocytes reaching to 60% as early as on day 4 of cultivation. Thus, HEK 293T cells stimulate NK cells division, therefore, they can be used as feeder cells in a CAR NK cell product development.

About the authors

Polina Fedorova

Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University);
Federal State Budgetary Scientific Institution "Research Institute of Vaccines and Serums them. I.I. Mechnikov ");
Research Institute of Experimental Therapy and Diagnostics of Tumor, NN Blokhin National Medical Center of Oncology

Email: ppolite@mail.ru
ORCID iD: 0000-0001-7478-8783
Scopus Author ID: 57222573207

assistant of microbiology, virology and immunology departmenta, junior researcher of laboratory of applied virologyb; laboratory research assistant of laboratory of cellular immunityc.  Academic degree, academic title: none.

aFederal State Autonomous Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University);

bFederal State Budgetary Scientific Institution "Research Institute of Vaccines and Serums them. I.I. Mechnikov ");

cResearch Institute of Experimental Therapy and Diagnostics of Tumor, NN Blokhin National Medical Center of Oncology.

Россия, Russia, 119991, Moscow, Trubetskaya str, 8-2; Russia, 105064, Moscow, Malyj Kazennyj lane, 5A; Russia, 115522, Moscow, Kashyrskoe Sh., 24.

Irina Chikileva

Research Institute of Experimental Therapy and Diagnostics of Tumor, NN Blokhin National Medical Center of Oncology

Email: irinatchikileva@mail.ru
ORCID iD: 0000-0003-0769-1695
SPIN-code: 3649-7321

PhD (in biology), senior researcher of laboratory of cellular immunityc

cResearch Institute of Experimental Therapy and Diagnostics of Tumor, NN Blokhin National Medical Center of Oncology

Россия, Russia, 115522, Moscow, Kashyrskoe Sh., 24.

Mikhail V. Kiselevskiy

Research Institute of Experimental Therapy and Diagnostics of Tumor, NN Blokhin National Medical Center of Oncology

Author for correspondence.
Email: kisele@inbox.ru
ORCID iD: 0000-0002-0132-167X
SPIN-code: 8687-2387

PhD, MD (Medicine), Professor, Head of the Laboratory of Cell Immunity, Professor, Biomedical Engineering Research and Education Center

Россия, Moscow; Moscow

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