GD2 CAR, CD22 CAR and ErbB2 CAR-encoding retroviral supernatants were produced via transient transfection of the 293GP cell line, as previously described20 (link). Briefly, 293GP cells were transfected via Lipofectamine 2000 (Life Technologies) with the plasmids encoding the CARs and the RD114 envelope protein. Supernatants were collected 48 and 72 hours post-transfection. CD19 CAR-encoding retroviral supernatant was harvested from the H3 producer cell clone.
Monocyte depeleted PBMCs were activated with anti-CD3/CD28 beads (Life Technologies) in a 3:1 bead:cell ratio with 40 IU/mL IL-2 for 3 days. Activated T cells were then retrovirally transduced on days 3 and 4 as previously described20 (link) using Retronectin (Takara) coated plates, and cultured in 300 IU/mL IL-2. anti-CD3/CD28 beads were removed on day 5. Media and IL-2 were changed every two days (Supplementary Fig. 4). Transduction efficiencies were routinely 80–90% for all CARs. T cells co-expressing the CD19 CAR and GD2 CAR were generated by transducing with a 50:50 mixture of both supernatants simultaneously.