Genetically Engineered CAR T Cells

The EGFRvIII third generation MSGV1 retroviral CAR construct contains the CD28, 4-1BB, and CD3z moieties, in tandem with the scFv derived from the human monoclonal antibody 139, and the marker Thy1.1^{21 (link)}. Alternatively, the EGFRvIII specific 139 scFv was replaced with the FMC63 CD19 specific scFv. CAR T cells were prepared according to previously reported protocols⁵³ . Briefly, splenocytes were isolated from donor C57BL/6 CD45.2 or CD45.1 or IFNAR1 KO mice, made into a single cell suspension, and cultured in RPMI (HyClone) supplemented with 10% FBS, 50 μM 2-Mercaptoethanol (Sigma), 1% PenStrep (Corning), 1% NEAA (Corning), 1% Sodium Pyruvate (Corning), 50 U/mL human IL2 (Novartis) and 2.5 μg/mL Concanavalin A (Sigma). Retroviral supernatant was produced from 293T cells co-transfected with the MSGV1 retroviral plasmid and the helper plasmid pCL Eco (Imgenex) and T cells were transduced on RetroNectin-coated plates (Takara) 2 days after stimulation. Cells were split one day after transduction and used for in vitro analysis or in vivo administration on day 4 or 5. Transduced cells were identified by the expression of Thy1.1 and a representative gating scheme identifying murine CAR T cells is shown in Supplementary Fig. 10.