Generation and nucleofection of primary CD19-CAR NK cells
NK cells were isolated from the peripheral blood of healthy donors and nucleofected with either pmaxGFP alone or SB transposons vectorized as MCs, which encode either a Venus fluorescent protein or a CD19-CAR in combination with a hyperactive SB100X transposase. The transposon MC vectors carrying CD19-CAR were flanked by ITRs and provided in the form of SNIM mRNA SB100X. Per sample, 1 × 10^6 NK cells were nucleofected with 2.5 µg of SB transposase and 1 µg MC DNA using the 4D Nucleofector (Lonza), P3 Primary Cell 4D Nucleofector X Kit S and L, and the DO-100 nucleofection program. After nucleofection, the cells were transferred to cultivation medium. Production of viral vectors and following transduction have been performed as previously described. The utilized transfer plasmid was provided by M. Hudecek.Transduction of IL-15 stimulated primary NK cells was performed by spinfection at 800 × g for 1 ½ h at 32 °C in presence of Vectofusin-1 (Miltenyi Biotec, 10µg/mL) as described previously.