Figure 2.

CB NK cells are genetically modified to express the B7H3 CAR and maintain their expression of activating receptors

(A) Generation of CAR NK cells. (B and C) CB NK cells were transduced with a retroviral vector encoding for the B7H3 CAR. CD56⁺ NK cells (blue circular cells) selected from CB were rested overnight in recombinant human IL-15 (15 ng/mL) in Cell Genix Stem Cell Growth Medium (with 10% FBS). The next day (day 0), NK cells were stimulated with irradiated (100 Gy) clone 4 K562 cells (2:1 feeder cell:NK ratio) and IL-2 (200 IU/mL) and IL-21 (25 ng/mL). Activated NK cells were transduced with retroviral supernatants (red virus particles) on day +4 in retronectin-coated plates. Three days later (day +7), NK cells were stimulated again with irradiated clone 4 K562 cells. On day +12, CAR-transduced NK cells (blue cells with spikes) were collected for use. Media change with IL-2 (200 IU/mL) and IL-21 (25 ng/mL) was done every 2–3 days throughout the NK cells culture duration. Illustration created using Biorender.com. (B) Representative flow analysis of B7H3 CAR expression on CB NK cells. (C) Transduction efficiency of CB NK cells (n = 9). (D) Mean fold expansion of B7H3 CAR-NK cell products (n = 9). (E) The NK cell phenotypes after 10 days of ex vivo expansion of UT CB NK (black bar) cells versus B7H3 CAR (Red bar) expressing CB NK cells are shown (n = 8). Bar shows mean value. Individual point represents individual donor and error bars represent standard deviation.