Loss of HLA-A expression on primary CD19-specific CAR⁺T cells genetically edited with ZFNs. (A) Disruption of HLA-A2 in CAR⁺T cells by electro-transfer of mRNA encoding ZFNs. T cells from a HLA-A2⁺ donor were electroporated and propagated to express CD19-specific CAR (CD19RCD28). These T cells were re-electroporated with 2.5 μg of each mRNA encoding the heterodimeric FokI domain variants of the HLA-A-specific ZFNs (ZFN-L-EL and ZFN-R-KK). HLA-A2 expression was analyzed after culturing at 30°C for 3 days, followed by 37°C for 1 day. Enrichment of the HLA-A2^neg population was performed by paramagnetic selection. (B) HLA-A^neg CAR⁺T cells evade lysis by HLA-A2-restricted CTL. Pools of the indicated CAR⁺T cells were pulsed with serial dilutions of cognate peptide before being used as targets in a CRA. CTL clone GAS2B3-5, which is specific for C19ORF48/A2, was added at an effector-to-target ratio of 20:1. (C) ZFN-modified HLA^neg CAR⁺ T cells maintain desired antigen-specific cytotoxicity. Redirected specificity for CD19 by HLA-A^neg T cells expressing CD19RCD28CAR was demonstrated using the mouse T-cell line EL4 genetically modified to express a truncated variant of human CD19. Expression of introduced human CD19 on EL4 was 100%. (D) HLA^neg CAR⁺ T cells maintain cytotoxicity against CD19⁺malignant cells. Cytotoxicity of HLA-A^neg CD19-specificCAR⁺ T cells was evaluated against CD19⁺ cell lines (NALM-6 and Daudi) and primary lymphoma cells derived from patients. Data shown are from an effector-to-target ratio of 10:1. Primary cells from DLBCL are diffuse large B-cell lymphoma, and those from MCL are mantle cell lymphoma.