Background: CARs usually combine the antigen binding domain of a monoclonal antibody with the ζ signaling domain of the T cell receptor complex. CAR expression in T cells produces potent MHC-unrestricted effector function against tumor cells in vitro but limited T-cell persistence in vivo, likely due to their incomplete activation, as tumors do not express costimulatory molecules. Incorporation of costimulatory endodomains in the CAR (e.g. CD28) may enhance their benefit in vivo. Methods: We report a phase I trial of T cells redirected to CD19 given to patients with refractory/relapsed B-cell malignancies. Subjects simultaneously received 2 autologous T-cell products, both expressing CARs with identical CD19-specific exodomains. In one product, the endodomain contained only the ζ sequence (CAR.19ζ) while the second incorporated a costimulatory CD28 domain (CAR.19-28ζ). We generated the T-cell products by activating autologous peripheral blood mononuclear cells with OKT3 followed by transduction with retroviral vectors encoding either CAR.19ζ or CAR.19-28ζ and ex vivo expansion for a median of 14 days (range 6-18) in the presence of IL-2. Six subjects have been treated at 3 cell dose levels (0.2, 1 and 2×10⁸ cells/m²). Persistence of CAR+ T cells was assessed in blood by Q-PCR assays specific for each population. Results: All infusions were well tolerated. CAR.19-28ζ cells were detected at a low level after infusion, but progressively increased 7- to 63-fold, peaking at 1-2 wk, before declining to background levels over the ensuing 3 mo. By contrast, CAR.19ζ cells were barely detectable after infusion, showed no expansion and disappeared rapidly. Following treatment, 2 patients had stable disease for up to 6 mo and 4 had progressive disease. Conclusions: Infusion of both CAR.19ζ and CD19-28ζ T cells is safe at the doses used. Direct comparison of each cell product in individual patients showed that the CD28 endodomain enhances expansion and persistence of the CAR+ T cells. The limited clinical benefits suggest that additional modifications will be required and our approach will allow these changes to be systematically evaluated.