Figure 4.

DuoBody-CD40×4-1BB enhances T-cell activation in vitro. (A) CFSE-labeled human PBMCs were stimulated with 0.03 µg/mL anti-CD3 and incubated with DuoBody-CD40×4-1BB or control antibodies (0.2 µg/mL) for 4 days. CFSE dilution in CD8⁺ T cells was analyzed by flow cytometry. Expansion index of individual donors and mean±SD (n=7) is shown. (B) CD8⁺ T cells were electroporated with RNA encoding an HLA-A2/CLDN6-specific TCR, labeled with CFSE and cocultured with autologous DCs electroporated with CLDN6-encoding RNA in presence of the DuoBody-CD40×4-1BB or control antibodies (0.2 µg/mL; for combination of control antibodies 0.2 µg/mL of both antibodies was added) for 4 days. Proliferation was measured as described in (A). Expansion index of individual donors and mean±SD (n=9) is shown. ***P<0.001; *p<0.05; Friedman test. (C–D) Polyclonal (C) and antigen-specific T-cell proliferation assays (D) as described in A–B with increasing concentrations of DuoBody-CD40×4-1BB or isotype control antibody. Data shown are mean expansion index (±SD) of triplicate wells of one representative donor (n=12 for polyclonal assay, n=5 for antigen-specific assay). (E) Cytokine concentrations in supernatant taken after 48 hours from cultures as described in A–B. Data shown are mean concentration ±SD of triplicate wells from one representative donor (n=3 for polyclonal assay, n=5 for antigen-specific assay). (F–G) CD8⁺ T cells electroporated with a CLDN6-specific TCR were cocultured with CLDN6-expressing K562 cells for 48 hours. The percentage of IFN-γ⁺ cells or GzmB⁺CD107a⁺ cells (F), and expression levels of GzmB and CD107a normalized to the isotype control (G) were analyzed by flow cytometry. Data from individual donors and mean±SD (n=8) are shown. ***P<0.001; **p<0.01; One-way ANOVA with Dunnett’s multiple comparisons test (F) or paired t-test (G). ANOVA, analysis of variance; CFSE, carboxyfluorescein succinimidyl ester; DC, dendritic cell; IFN, interferon; PBMC, peripheral blood mononuclear cell.