PBLs used in the study were obtained from patients under institutional review board approved clinical protocols at the Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD. All patients gave informed consent for sample procurement. PBLs were stimulated with AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 5% human AB serum (Valley Biomedicals, Winchester, VA), 300 IU/ml IL2 (Aldesleukin Proleukin
®, Novartis, Basel, Switzerland) and 50 ng/ml OKT3 (MuromonAB-CD3, Orthoclone OKT3, Ortho Biotech, Raritan, NJ) for two days before transduction with retroviral supernatants expressing various CAR constructs. MSGV1-based retroviral vectors expressing CARs against tumor-associated antigens ERBB2, VEGFR2, CSPG4, and CD19 were previously described in detail [
5‐
8]. Anti-EGFRvIII and PSCA CARs are based on a human and a humanized mouse antibody respectively. Vector production and PBL transduction was also previously described in detail [
5,
7]. Briefly, 2 ml of viral vector supernatants expressing the various CARs were diluted with an equal volume of AIM-V medium supplemented with 5% human AB serum and added into one well of 6-well plates previously coated with RetroNectin (Takara Bio Inc., Otsu, Japan). Plates were then loaded with retroviral supernatants by centrifugation at 32°C, 2000 g for 2 hours (Sorvall Legend RT, Newtown, CT). Transduction was carried out by removing the vector supernatant and adding 2 × 10
6 activated PBLs per well, centrifugation at 1000 g for 10 minutes and the plates were then incubated at 37°C with 5% CO
2. The transduction procedure was repeated on the following day. Five to seven days after transduction, PBLs were used for FACS analysis using protein-L and other antibodies as described below.