Figure 2.

ATG-101 activates 4-1BB signaling upon PD-L1 cross-linking. A and B, The 4-1BB agonistic activity of ATG-101 in NFκB luciferase assay. A, 293T-h41BB-NFκB-Luc cells were incubated with ATG-101 and CHO-hPDL1 (left) or CHO-mPDL1 (right) for 24 hours. Wild-type CHO cells as negative control. B, 4-1BB activation by ATG-101, bivalent PD-L1×4-1BB bsAb [ATG-101(1+1)], or isotype control antibodies in 293T-h41BB-NFκB-Luc cells with the presence of 293T-hPDL1. Tetravalent and bivalent ATG-101 activated 4-1BB signal pathway with EC₅₀ of 5.25 nmol/L and 15.44 nmol/L, respectively. C, Effect of ATG-101 on IL2 release by CD8⁺ T cells with the presence of PD-L1⁺ cell lines. PD-L1 densities (total antibody binding sites) on MCF7, RKO, NCI-H358, and 293T-hPDL1 cell surfaces were determined using QIFIKIT (left). All the indicated cells cocultured with CD8⁺ T cells and ATG-101 for 3 days, and supernatants were harvested for IL2 ELISA to assess T-cell activation (right). D, ATG-101–induced IFNγ release by human primary CD8⁺ T cells coculturing with varying proportions of PD-L1⁺ cells. HEK293-PDL1 cells were mixed with parental HEK293 at different proportions. The IFNγ release was detected by ELISA. E, The individual and mean expression pattern of surface markers and cytokine release of CD3⁺ T cells from three healthy donors through six rounds of CD3/CD28 activation. F, Individual and mean IL2, IFNγ, and TNFα production by T cells after six rounds of activation (from E), in response to ATG-101, parental PD-L1 antibody, parental 4-1BB antibody, or isotype control. In A, C, and D, data representative of three independent experiments. In A, B, C, and F, data are presented as means ± SEM. Statistical analysis used two-way ANOVA in C and one-way ANOVA in F. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.