CAR-NK therapy: NK cell expansion exposed to HEK 293T cell line

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.