T-cell expression of second-generation CARs

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T-cell expression of second-generation CARs T-cell expression of second-generation CARs. A, pictorial representations of 19z1 with second-generation CAR genes demonstrating the genetic fragments used to generate the costimulatory CARs. B, Western blot analysis of T cells retrovirally transduced with ... T-cell expression of second-generation CARs. A, pictorial representations of 19z1 with second-generation CAR genes demonstrating the genetic fragments used to generate the costimulatory CARs. B, Western blot analysis of T cells retrovirally transduced with CAR genes under reducing conditions. Membranes were probed with a monoclonal antibody specific to the cytoplasmic domain of the human ζ chain and show the expression of CARs at the expected molecular weights. The native T-cell ζ chain is indicated. Lane 1, 19z1; lane 2, 19-28z; lane 3, 19(DAPic)z; lane 4, 19z(DAPic); lane 5, 19(DAP)z; lane 6, 19(4-1BBic)z; lane 7, 19(4-1BB)z; and lane 8, 19(OX-40ic)z. C, NALM-6 tumor lysis by CAR+ T cells was assessed by standard 4 h 51Cr release assays. All T cells expressing CD19-targeted second-generation CARs lysed target tumor cells equally well when compared with T cells expressing the first-generation 19z1 CAR. Control T cells expressing the irrelevant Pz1 CAR did not significantly lyse NALM-6 tumors. Effector to target ratios (E:T Ratio) represent T cells normalized to the CD8+ CAR+ T-cell fraction. Data represent one of three different experiments using three different healthy donor T-cell populations with similar results. Renier J. Brentjens et al. Clin Cancer Res 2007;13:5426-5435 ©2007 by American Association for Cancer Research