Nivolumab (anti-human PD-1) binds PD-1 with high affinity (KD 2.6 nmol/l by Scatchard analysis to polyclonally activated human T cells) and blocks its interactions with both B7-H1 and B7-DC. It effectively inhibits the interaction between PD-1 and its ligands. In vitro assays demonstrated the ability of nivolumab to potently enhance T-cell responses and cytokine production in the mixed lymphocyte reaction and superantigen or cytomegalovirus stimulation assays. Nivolumab inhibits the interaction between PD-1 and its ligands, PD-L1 and PD-L2, with IC50 values of 2.52 and 2.59 nmol/L, respectively, as shown by surface plasmon resonance. In a study using FACS to evaluate ligand binding to PD-1 expressed on CHO cells, the IC50 values for nivolumab-mediated inhibition of PD-1 binding to PD-L1 or PD-L2 were similar (1.04 and 0.97 nmol/L, respectively). Nivolumab binds specifically to PD-1 and not to other immunoglobulin superfamily proteins, such as CD28, CTLA-4, ICOS, and BTLA. Nivolumab can, at very low concentrations (~1.5 ng/mL), enhance T-cell reactivity in the presence of a T-cell receptor stimulus. However, nivolumab had no stimulatory effect in the absence of antigen or T-cell receptor stimulus. Specifically, there was no significant release of inflammatory cytokines, including IFNγ, TNFα, IL-2, IL-4, IL-6, or IL-10, from unstimulated whole blood after coincubation with nivolumab. Nivolumab does not cause nonspecific lymphocyte activation.