VVPs containing CD80 and CD58 costimulatory domains enhance T-cell activation and generate greater numbers of CAR T cells with increased in vitro and in vivo antitumor functionality. (A) VivoVec–induced CD4 and CD8 T-cell activation, as measured by CD25, 3 days after VVP addition, n = 3 PBMC donors and representative of 5 individual experiments. (B) Total CAR⁺ CD4 and CD8 T cells generated by VVPs in vitro, 7 days after vector addition. Data combined from 2 separate experiments with n = 3 PBMC donors for each. (C) CAR T-cell cytokine production (interferon gamma [IFN-γ], IL-2, and tumor necrosis factor-α [TNF-α]) upon Nalm6 coculture for 24 hours. Representative of 2 individual experiments, n = 3 PBMC donors. (D) CAR T-cell–Nalm6 in vitro serial stimulation assay, representative of 2 individual experiments, n = 3 PBMC donors. Arrows indicate restimulation with fresh Nalm6 cells. (E) Human T-cell activation, as measured by CD25 and CD71, in humanized NSG MHC I/II KO model 4 days after VivoVec administration at several particle doses, n = 5 to 9. (F) Percentage of CD19 CAR⁺ of human CD3⁺ cells and total human CD19 CAR⁺ cells per μL blood at day 11 after VivoVec particle administration, n = 5 to 9. (G) Representative flow plots for panel F. (H) Tumor burden over time as measured by Nalm6 bioluminescence, n = 5 to 9. Two-way analysis of variance (ANOVA) with Sidak multiple comparisons for panels A-B. Two-way ANOVA for panels C-D. Two-tailed unpaired student t test for panels E-F. ∗∗∗∗P < .0001; ∗∗∗P < .0002; ∗∗P < .002; and ∗P < .03. +Costim, VVP displaying anti-CD3scFv, CD80, and CD58 costimulatory ligands; MOI, multiplicity of infection; ns, not significant; TU, transducing units.