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 (38 (link)). CAR T cells were prepared according to previously
reported protocols (50 ). Briefly,
splenocytes that were isolated from donor C57BL/6 mice were made into a
single-cell suspension and cultured in RPMI (HyClone) supplemented with 10% FBS,
50 μM 2-mercaptoethanol (Sigma-Aldrich), 1% PenStrep (Corning), 1%
Non-essential amino acids (NEAA) (Corning), 1% sodium pyruvate (Corning), human
interleukin-2 (IL-2; 50 U/ml; Novartis), and concanavalin A (2.5 ug/ml;
Sigma-Aldrich). In some experiments, CAR T cells were expanded using murine
IL-21 (30 ng/ml), IL-15 (5 ng/ml), and IL-7 (10 ng/ml). Retroviral supernatant
was produced from 293T cells cotransfected 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 1
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.
the CD28, 4–1BB, and CD3z moieties, in tandem with the scFv derived from
the human monoclonal antibody 139 and the marker Thy1.1 (38 (link)). CAR T cells were prepared according to previously
reported protocols (50 ). Briefly,
splenocytes that were isolated from donor C57BL/6 mice were made into a
single-cell suspension and cultured in RPMI (HyClone) supplemented with 10% FBS,
50 μM 2-mercaptoethanol (Sigma-Aldrich), 1% PenStrep (Corning), 1%
Non-essential amino acids (NEAA) (Corning), 1% sodium pyruvate (Corning), human
interleukin-2 (IL-2; 50 U/ml; Novartis), and concanavalin A (2.5 ug/ml;
Sigma-Aldrich). In some experiments, CAR T cells were expanded using murine
IL-21 (30 ng/ml), IL-15 (5 ng/ml), and IL-7 (10 ng/ml). Retroviral supernatant
was produced from 293T cells cotransfected 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 1
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.
