Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

Figure 2

Overview of PD-1-blocking monoclonal antibodies and DARPin® proteins. (a) Schematic of the PD-1-binding reagents: two conventional monoclonal antibodies (mAbs) of IgG4 subtype (nivolumab and pembrolizumab) and two DARPin® proteins with monovalent (DARPin-1) or bivalent (DARPin-2) PD-1 binding. (b) Assessment of PD-1 binding using a titration of PD-1-targeting reagents with a PD-1-expressing cell line (transfected HEK293). PD-1 binding was measured by mean fluorescence intensity (MFI) using flow cytometry. Half-effective concentrations (EC50 values) are presented for each PD-1-binding reagent—nivolumab (nivo) in circles, pembrolizumab (pembro) in reversed triangles, DARPin-1 in squares, and DARPin-2 in diamonds. (c) PD-1/PD-L1 blockade bioassay (Promega) () was performed using PD-1+ effector T-cells and PD-L1-expressing aAPC/CHO-K1 cells in the presence of dose titrations of PD-1-targeting reagents. Increased luminescence indicates recovered T-cell activation as PD-1 engagement with PD-L1 interferes with the T-cell receptor-mediated transcription of the reporter gene luciferase. (d) Mixed lymphocyte reaction was performed using CD4+ T-cells and allogeneic dendritic cells generated from healthy donors in the presence of PD-1-targeting mAbs (nivolumab, ; pembrolizumab, ), DARPin® proteins ( for both), or corresponding controls (human IgG4 and negative control DARPin® protein, NCD, respectively, for both). The levels of IFN-γ in the supernatants were analyzed using ELISA, and results are presented for indicated concentrations. (e) Fold induction of IFN-γ release from T-cells isolated from the ascites or tumor from ovarian cancer patients () after 48 h incubation with α-CD3 (OKT-3) and different concentrations of PD-1-targeting mAbs (), DARPin® proteins (), or corresponding controls (IgG4 and NCD, and , respectively, both used at 100 nM). The response was normalized based on the response with only α-CD3, which was set to 1 (dashed line). The results are presented as a median with interquartile range, and concentrations are indicated in the figure.

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