Optimizing activation and culture conditions for the production of cord blood-derived CAR-T and natural killer cells.

Cord blood (CB) is gaining attention as a source of chimeric antigen receptor (CAR) T/NK cells. Many studies have focused on the therapeutic effects, but the methods of activating and culturing T/NK cells without excessive exhaustion during the initial stages of the production of CB-derived CAR-T/NK cells are yet to be established. The activation and culture conditions developed for peripheral blood (PB) CAR-T/NK cells have been used for CB-CAR-T/NK cells, but there are differences in the composition and maturity of PB and CB lymphocytes. Thus, this study aims to optimize activation and culture conditions for the production of CB-CAR-T/NK cells. We compared different doses of various cytokines and found that the balance between activation and exhaustion was best with stimulation with a CD3/CD28 agonist followed by 40 U/mL interleukin (IL)-7+ 40 U/mL IL-15 for PB-T cells; 40 U/mL IL-2+ 40 U/mL IL-7+ 40 U/mL IL-15 for CB-T cells; 40 U/mL IL-2+ 40 U/mL IL-15 for PB-NK cells; and 40 U/mL IL-2+ 200 U/mL IL-15 for CB-NK cells. Using killing assays, we confirmed that these cytokine protocols improved the anti-tumour effects. These results will be useful for the development of CB-CAR-T/NK-cell therapies and suggest the potential of these modalities.