Peripheral blood mononuclear cells (PBMCs) derived from healthy donors (Cellular Technology Limited, Cleveland, OH) were stimulated with mitomycin C-treated K562 cells (JCRB, Osaka, Japan) expressing anti-CD3 mAb (clone OKT3)-derived single-chain variable fragment (scFv) and CD80 on the cell surface at an effector to target ratio of 7:1 (4 (link)). Stimulated T cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and recombinant IL-2 (100 IU/ml, Nipro, Osaka, Japan). Unstimulated T cells were maintained in the presence of recombinant IL-7 (10 ng/ml, PeproTech, Waltham, MA). When indicated, naïve T cells were enriched by magnetically isolating CD45RO− cells using CD45RO MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany).
To generate CAR-T cells, T cells were retrovirally transduced with CD19- or GD2-targeting CAR genes two days after stimulation. We used PG13 packaging cells for retrovirus production. The CAR constructs consisted of a single-chain variable fragment (scFv) derived from the clone FMC63 (targeting CD19) (24 (link)) or 14g2a with E101K high-affinity mutation (targeting GD2) (25 (link)) linked to CD28 and CD3z signaling domains.
To generate CAR-T cells, T cells were retrovirally transduced with CD19- or GD2-targeting CAR genes two days after stimulation. We used PG13 packaging cells for retrovirus production. The CAR constructs consisted of a single-chain variable fragment (scFv) derived from the clone FMC63 (targeting CD19) (24 (link)) or 14g2a with E101K high-affinity mutation (targeting GD2) (25 (link)) linked to CD28 and CD3z signaling domains.
