Tumor-reactive DMF5 or gp100(154) alpha and beta TCR chain RNA electroporated into PBLs confer high reactivity to melanoma tumor antigens. (A) Ten-day anti-CD3–stimulated donor PBLs were electroporated with in vitro–transcribed RNA encoding paired DMF4, DMF5, or gp100(154) TCR alpha and beta chains, or GFP control. Cells were cocultured for 18 hours with T2 cells pulsed with peptide, HLA-A*02⁺ melanomas mel624⁺ or mel526⁺, or HLA-A*02⁻ melanomas mel888⁻ or mel938⁻. IFN-γ in the supernatant was detected by ELISA. (B) Structure of the MSGV-based γ-retroviral vectors DMF4 and gp100(154), incorporating an IRES and DMF5 with a furin 2A ribosomal skip sequence, allowing for dual gene expression.