Functional consequences of ZFN-mediated TCR knockout in CAR⁺ T cells. (A) Loss of responsiveness of TCR^neg CAR⁺ T cells to TCR stimulation. Dilution of PKH26 was measured 10 days after stimulation with aAPC loaded with OKT3 (top panel) or expressing CD19 (bottom panel). Flow cytometry data were gated on CAR⁺ T cells. Parental: CAR⁺ T cells without modification; no mRNA: mock electroporated CAR⁺ T cells; TRAC CD3^neg: CAR⁺ T cells electroporated with mRNA encoding ZFN pairs specific for TRAC, and depleted CD3^pos population; TRBC CD3^neg: CAR⁺ T cells electroporated with mRNA encoding ZFN pairs specific for TRBC, and depleted for CD3^pos population. (B) Redirected specificity of TCR^neg CAR⁺ T cells. Specific lysis by CAR⁺ T cells of an EL4 (mouse T-cell line) modified to express a truncated version of human CD19 (closed symbols) was measured by standard 4 hour ⁵¹Cr release assay. Specificity is shown by lack of lysis of CD19^neg (parental) EL4 cells (open symbols). CAR⁺ T cells were modified by ZFNs (TRAC and TRBC) or unmodified CAR⁺ T cells (parental and no mRNA). The error bars represent SD. (C) Cytotoxicity by TCR^neg CAR⁺ T cells against CD19⁺ primary B-cell tumors. Specific lysis by CAR⁺ T cells of B-cell malignances derived from patients was measured by 6 hour ⁵¹Cr release assay (effector: target ratio = 30:1). DLBCL: diffuse large B-cell lymphoma, CLL: chronic lymphocytic lymphoma, and MCL: mantle cell lymphoma. The error bars represent the standard deviation.