CD30CAR-transduced EBV-CTLs specifically lyse CD30⁺ targets. (A) The results of a standard ⁵¹Cr-release assay of several CD30⁺ tumor-cell lines, at a CTL/tumor-cell ratio of 20:1, are shown. Bars represent the mean (± SD) of the EBV-CTLs generated from 8 donors and transduced with the CD30CAR (■) or an irrelevant CAR (□; P < .05). (B) Killing (shown is the percentage of lysis at 20:1 E/T ratio) of the CD30⁺ targets (■) by CD30CAR is inhibited by incubation with CD30 MAb (▒) but not by isotype control MAb (□) or by class I MHC MAb (▩), indicating that killing of CD30CAR is not MHC restricted. (*P < .05). Bars represent SD. (C) EBV-CTLs expressing the CD30CAR can eliminate CD30⁺ tumor cells in a long-term culture assay. EBV-CTLs obtained from healthy donors and transduced either with irrelevant CAR (left panels) or CD30CAR (right panels) were cocultured with the indicated CD30⁺ tumor-cell lines (ratio 5:1). After 5 to 7 days of culture, cells were collected and stained with CD3-PerCP and CD30-FITC to evaluate the growth of CD30⁺ tumor cells. No CD30⁺ cells were detectable after coculture with the CD30CAR⁺ EBV-CTLs, whereas CD30⁺ cells were detectable when tumor cells were cocultured with control CTLs. The phenotypes shown are representative of 4 performed experiments.