Fig. 2
ATOR-1015 generates clustering of cells and induces T-cell activation. (a) ATOR-1015, the combination of monotargeting anti-CTLA-4 and anti-OX40 antibodies or IgG1 control was added to a 1:1 mix of PKH26-labelled CTLA-4-expressing HEK cells and PKH67-labelled OX40-expressing CHO cells. The formation of cell complexes was determined by flow cytometry. Data show one representative experiment out of three. (b) ATOR-1015 blocks CTLA-4 and promotes T-cell activation in a CTLA-4 blockade reporter assay. Jurkat T cells engineered to express CTLA-4 with an IL-2 dependent luciferase reporter were co-cultured with Raji cells expressing CD80/CD86 and an artificial T cell receptor activator. Serially diluted ATOR-1015 or IgG1 control was added. Upon addition of ATOR-1015, binding of CD80/CD86 to CTLA-4 was prevented, and co-stimulation of T cells via CD28 increased, leading to a luminescent signal. Data presented as fold induction over media control based on relative light units (RLU) (n = 2 independent experiments). (c) OX40-mediated T-cell activation upon ATOR-1015 crosslinking to immobilized CTLA-4. Plates were coated with CTLA-4Fc (5 μg/ml) and suboptimal levels of anti-CD3 (OKT3, 3 μg/ml). CD3+ T cells (100,000 cells/well) were added along with ATOR-1015, the combination of monotargeting antibodies and IgG1 control. After 72 h of incubation, levels of IFN-γ were measured in the supernatants by ELISA (n = 8 donors). (d) OX40-mediated T-cell activation upon ATOR-1015 crosslinking to CTLA-4 on cells. HEK cells expressing CTLA-4 (30,000 cells/well) were irradiated and allowed to adhere overnight. CD4+ T cells (100,000 cells/well) were added along with anti-CD3 (UCHT1) beads, ATOR-1015, the combination of monotargeting antibodies or IgG1 control. After 72 h of incubation, levels of IL-2 were measured in the supernatants by ELISA (n = 6 donors). (e) FcγRI-expressing CHO cells (100,000 cells/well) were irradiated and allowed to adhere overnight. CD4+ T cells (100,000 cells/well) were added along with anti-CD3 (OKT3) beads, ATOR-1015, the combination of monotargeting antibodies or IgG1 control. After 72 h of incubation, levels of IL-2 were measured in the supernatants by ELISA (n = 8 donors). All data presented as mean ± SEM. Statistical analysis (in C-E) was performed using the Mann Whitney, two-tailed test (**, p < 0.01; ***, p < 0.001)