MDF protein comprising an anti-CD3 scFv fused to the CD58 extracellular domain and CD80 exhibit potentiated T-cell activation and transduction-enhancing properties. (A) VivoVec T-cell–binding assay. A total of 20 × 106 PBMCs cultured with VivoVec at MOI = 10 for 1 hour followed by flow cytometry for T-cell–associated cocal glycoprotein, n = 3 PBMC donors and representative of 2 individual experiments. (B) VivoVec–induced CD4 and CD8 T-cell activation, as measured by CD25, 3 days after vector addition, n = 3 PBMC donors and representative of 3 individual experiments. (C) Percent of CD4 and CD8 T cells expressing anti-CD19 CAR 7 days after VivoVec addition to PBMCs, n = 3 PBMC donors and representative of 3 individual experiments. (D) CAR T-cell cytokine production (IFN-γ, IL-2, and TNF-α) upon Nalm6 coculture for 24 hours, representative of 2 individual experiments, n = 3 PBMC donors. (E) CellTrace Violet (CTV) dilution on VivoVec–generated anti-CD19 CAR T cells 5 days after Nalm6 stimulation, representative of 2 independent experiments, n = 3 PBMC donors. (F) 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. Two-tailed unpaired student t test for panels A,E. Two-way ANOVA with Sidak multiple comparisons for panels B-C. Two-way ANOVA for panels D,F. ∗∗∗∗P < .0001; ∗∗∗P < .0002; and ∗P < .03.
