Transduction of CD25/71 allodepleted T cells with anti-CD19ζ TCR leads to efficient cytokine release in response to CD19 targets. (A) Schematic of the CD19R/pHR –SIN-SE. This consists of a self-inactivated (SIN) lentiviral construct, with a HIV central polypurine tract (cPPT), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a spleen focus-forming virus (SFFV) promoter. The CD19R transgene consists of a human immunoglobulin leader sequence (S), the variable domains of the CD19-specific murine monoclonal antibody FMC-63 assembled as a single-chain variable fragment (ScFv; V_L and V_H), connected by a linker (L), in frame with a sequence encoding the human IgG1 hinge and CH2-CH3 domain (Spacer), the human CD28 transmembrane domain (TM), and the cytoplasmic signaling domain of the human CD3ζ. (B) Anti-CD19ζ TCR-transduced CD25/71 allodepleted PBMCs demonstrate significantly enhanced IFNγ secretion to CD19⁺ targets (n = 6). CD19R-transduced or mock-transduced CD25/71 allodepleted PBMCs were cultured with CD19^+/− targets in an IFNγ ELISPOT assay. Transduced allodepleted PBMCs showed significantly enhanced IFNγ secretion to autologous and allogeneic LCLs, K562 cells stably transduced with a GFP-CD19⁺ transgene, Ramos, and 1° ALL blasts (*P < .05) compared with mock-transduced cells (mean ± SEM). (C) Anti-CD19ζ TCR-transduced CD25/71 allodepleted PBMCs demonstrate significantly enhanced granzyme B secretion to CD19⁺ targets (n = 6). CD19R-transduced or mock-transduced CD25/71 allodepleted PBMCs were cultured with CD19^+/− targets in a granzyme B ELISPOT assay. Transduced allodepleted PBMCs showed significantly enhanced granzyme B secretion to autologous and allogeneic LCLs, K562 cells stably transduced with a GFP-CD19⁺ transgene, Ramos, and 1° ALL blasts (*P < .05) compared with mock-transduced cells (mean ± SEM).