Combination with as PD-1–blocking antibody increases AMG 701 cytotoxicity in vitro. (A) Expression of PD-1 on CD3⁺ T cells from TDCC assays with cell lines and E:T ratios as indicated, assessed by flow cytometry at different times. Data are mean ± standard deviation (SD) of 2 technical replicates. Curves are representative of 2 human pan T-cell donors. (B) Specific cytotoxicity against KMS12BM_PDL1 (left panel) and U266B1_PDL1 (right panel) cultured for 24 hours 1:1 with CD3/CD28-activated human pan T cells and increasing concentrations of AMG 701, with (closed triangles with dark-shaded curves; mustard or magenta) or without (open triangles with light-shaded curves; yellow or lavender) 10 µg/mL PD-1–blocking antibody. Data are mean ± SD of 2 technical replicates. Curves are representative of 5 pan T-cell donors. (C) EC₅₀ of AMG 701, with or without anti–PD-1–blocking antibody, calculated in panel B, n = 5 T-cell donors, KMS12BM_PDL1 (P = .01); U266B1_PDL1 (P = .0008); paired Student t test. Data are mean ± SD. (D) Specific cytotoxicity against KMS12BM_PDL1 and U266B1_PDL1 cultured for 96 hours at 1:5 E:T ratio with resting T cells and increasing concentrations of AMG 701, with (closed triangles with dark-shaded curves; mustard or magenta) or without (open triangles with light-shaded curves; yellow or lavender) 10 µg/mL PD-1–blocking antibody. Data are mean ± SD of 2 technical replicates, representing 1 of 2 assays. (E) Concentration of IFN-γ in supernatants collected from panel D at 72 hours, quantified by an enzyme-linked immunosorbent assay.