Figure 4. PD-1 antibody ImmTAAI molecules are noncompetitive with PD-L1 for PD-1 binding and are additive with PD-L1 in inhibiting TCR complex signaling.

(A and B) SPR competition binding studies were conducted as described in Methods and Supplemental Figure 3. Response units (RU) were plotted over time to characterize the binding of H5- and CA949-ImmTAAI molecules to PD-1 in the presence of PD-L1–Fc (representative sensorgrams from 3 independent experiments). (C) Parental ECN90 (upper curves) or PD-L1 transduced ECN90 cells (lower curves) were pulsed with Melan-A activating peptide and titrations of PD-1 antibody ImmTAAI molecules added. Jurkat NFL Mel5 PD-1 cells were immediately added to run the PD-1 reporter assay (n = 2 and representative of 4 independent experiments). (D) Cell assay using PD-L1 transduced ECN90 cells was done as described above using titrations of PD-1 antibody ImmTAAI or PD-L1 ImmTAAI molecules. Relative NFAT activity is plotted against the concentrations where maximal inhibition was observed (n = 4 and representative of 4 independent experiments). (E) Titrations of soluble PD-1 antibody ImmTAAI molecules (with an irrelevant TCR) and a PD-1 blocking antibody (pembrolizumab) were tested in the ECN90–PD-L1: Jurkat NFL Mel5 PD-1 reporter assay (n = 2 and representative of 2 independent experiments). Data are plotted as mean ± SD and were compared by 1-way ANOVA and Dunnett’s multiple-comparison test. ***P ≤ 0.001.