Fig. 4

HK010 selectively enhances the 4-1BB signaling activity of CD8+ T cells in a PD-L1-dependent manner. A PD-1/PD-L1 blocking activities of HK010 and control antibodies were determined using the luciferase reporter assay. A human IgG4 protein was used as a negative control, whereas the avelumab and anti-PD-L1 antibodies acted as positive controls. B The 4-1BB signaling activities of HK010 on HCC1954, HCC827, MDA-MB-231, and HT29 cells were compared with CHO-K1 using the HEK-293/NFκB-Luci/4-1BB reporter assay. C, D The 4-1BB agonist activities of HK010 and control antibodies were determined using the luciferase reporter assay. HEK-293/NFκB-Luci/4-1BB reporter cells were cocultured with HCC1954 cells (C) or cells with FcγR expression (D). E The IFN-γ secretion levels of human primary CD8+ T cells cocultured with HCC1954 cells in the HK010 and control antibody treatment groups were compared. F The IFN-γ secretion levels of human primary CD8+ T cells treated with HK010 and cocultured with the indicated ratios of PD-L1-expressing CHO-K1 cells were compared. G The IFN-γ secretion levels of human primary CD8+ T cells treated with HK010 and cocultured with different tumor cells were compared. H The IFN-γ secretion induced by HK010 was detected in the MLR assay. The red dashed line indicates the background value of the IgG4 isotype control. I The cytotoxicity of HK010 on HCC1954 cancer cells was determined by flow cytometry. J The cytotoxicities of HK010 on human monocytes and mDCs were determined by flow cytometry. The values are presented as the mean ± SD from one representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001