Early transduction of EBVSTs with GD2.CAR vector. PBMCs were stimulated four times with irradiated autologous LCLs on days 0, 9, 16 and 23 of culture. They were transduced with the GD2.CAR retroviral vector either on day 3 or day 19. GD2.CAR transgene levels were measured by flow cytometry on (A) day 3- early transduced (ET) or (B) day 19 late-transduced (LT) EBVSTs at the end of each stimulation. Data represent the mean +/− sd of 2 donors. (C) The transduction efficiency of ET or LT cells was compared with patient-derived EBVSTs (transduced on day ~19) from the previous clinical trial  using real-time PCR analysis for the retroviral vector. Day 19-transduced (LT) EBVSTs and patient lines demonstrated similar transduction efficiencies while Day 3-transduced (ET) EBVSTs attained a much higher transgene expression. Transgene PCR was performed 5 to 8 days post-transduction. (D) Specific killing was determined in a 6-hour 51Chromium-release assay using the GD2 expressing neuroblastoma cell line, LAN-1 (top), and autologous LCL (bottom).