Fig. 3

ATOR-1015 induces Fcγ receptor signaling and depletion of target-expressing cells. (a, b) CHO cells expressing CTLA-4 and OX40 were cultured together with FcγRIIIa (F158 or V158 variant) reporter cells with an NFAT response element driving expression of a firefly luciferase. ATOR-1015, monotargeting anti-CTLA-4 and anti-OX40 antibodies alone or in combination and IgG1 control were added and Fcγ receptor activation was quantified through the luciferase produced and measured as luminescence. Data presented as fold induction based on relative light units (RLU) over media control (n = 2 independent experiments). (c) The expression of CTLA-4 and OX40 on the cell surface of freshly isolated and activated (stimulated for 48 h with anti-CD3/CD28 beads) Tregs determined by flow cytometry. Histogram plots from one representative donor are shown. (d) The expression of CTLA-4 and OX40 on the cell surface of freshly isolated and activated Tregs. Mean fluorescence intensity (MFI) was measured by flow cytometry (n = 5 donors). (e) FcγRIIIa reporter cells (V158 variant) were cultured together with activated Tregs. FcγRIIIa activation was detected as described above (n = 5 donors). (f) Activated Tregs as target cells were cultured together with allogeneic NK cells as effector cells (effector:target cell ratio 15:1) in the absence or presence of antibodies. After 4 h, target cell lysis was measured based on lactate dehydrogenase (LDH) release (n = 7 donors). All data presented as mean ± SEM. Statistical analysis (in D-F) was performed using the Mann Whitney, two-tailed test (**, p < 0.01; ***, p < 0.001)