A Small Molecule Antagonist of PD-1/PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy

Abstract

Immune checkpoint inhibitors, such as monoclonal antibodies targeting programmed death 1 (PD-1) and programmed death ligand-1 (PD-L1), have achieved enormous success in the treatment of several cancers. However, monoclonal antibodies are expensive to produce, have poor tumor penetration, and may induce autoimmune side effects, all of which limit their application. Here, we demonstrate that PDI-1 (also name PD1/PD-L1 inhibitor 1), a small molecule antagonist of PD-1/PD-L1 interactions, shows potent anti-tumor activity in vitro and in vivo and acts by relieving PD-1/PD-L1-induced T cell exhaustion. We show that PDI-1 binds with high affinity to purified human and mouse PD-1 and PD-L1 proteins and is a competitive inhibitor of human PD-1/PD-L1 binding in vitro. Incubation of ex vivo activated human T cells with PDI-1 enhanced their cytotoxicity towards human lung cancer and melanoma cells, and concomitantly increased the production of granzyme B, perforin, and inflammatory cytokines. Luciferase reporter assays showed that PDI-1 directly increases TCR-mediated activation of NFAT in a PD-1/PD-L1-dependent manner. In two syngeneic mouse tumor models, the intraperitoneal administration of PDI-1 reduced the growth of tumors derived from human PD-L1-transfected mouse lung cancer and melanoma cells; increased and decreased the abundance of tumor-infiltrating CD8+ and FoxP3+ CD4+ T cells, respectively; decreased the abundance of PD-L1-expressing tumor cells, and increased the production of inflammatory cytokines. The anti-tumor effect of PDI-1 in vivo was comparable to that of the anti-PD-L1 antibody atezolizumab. These results suggest that the small molecule inhibitors of PD-1/PD-L1 may be effective as an alternative or complementary immune checkpoint inhibitor to monoclonal antibodies.

Keywords: PD-1/PD-L1; PD-1/PD-L1 inhibitor 1; PDI-1; T cell activation; immunotherapy; small molecule compound.

Figure 1

Computer modeling of predicted antagonism of PD-1/PD-L1 binding by PDI-1. (A, B) Ribbon representations of a predicted binding pocket for PDI-1 in human PD-1 (A) and human PD-L1 (B). Magnified views of the side-chain of Ser567 (A) and Phe19 (B) with PDI-1 are shown on the right. (C) PD-1/PD-L1 binding by PDI-1 in vitro, measured using a competitive binding ELISA assay. Data represent the mean ± SEM of three replicates. (D, E) Surface plasmon resonance assay of binding of PDI-1 to chip-bound purified human and mouse PD-1 (D) or PD-L1 (E).

Figure 2

PDI-1 restores T cell cytotoxicity and cytokine production. (A, B) Cytotoxicity assay of activated CD3+ T cells cultured for 18 h at an effector to target ratio of 5 to 1 with CFSE-labeled H1975, A549 (A) A375, or SK-MEL-2 (B) cells alone or in the presence of either PDI-1 (4 μM), nivolumab (1 μg/ml), or an isotype control Ab. At the end of the incubation, cells were labeled with PI and dead or dying tumor cells (CFSE+ PI+) were quantified by flow cytometry. (C, D) Cytokine production by activated CD3+ T cells incubated for 18 h, as described for (A, B). Culture supernatants were collected and levels of IFN-γ, TNF-α, perforin, and granzyme B were measured by ELISA assay. (E) Activated CD3+ T cells were cultured for 18 h with hPD-L1-TCR-HEK293T cells or TCR-HEK293 T cells in the presence or absence of PDI-1 (4 μM) or nivolumab (1 μg/ml). Dead/dying HEK293 cells were quantified by flow cytometry as described for (A, B). Data represent the mean ± SEM of three replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Figure 3

PDI-1 reverses inhibition of NFAT activity by PD-1/PD-L1. (A) Schematic of the principle underlying the luciferase reporter assay, in which luciferase expression in Jurkat cells was driven by the NFAT promoter. (B, C) hPD-1-NFAT-Jurkat cells were cultured alone or with hPD-L1-TCR-HEK293T cells (B) or TCR-HEK293T cells (C) overnight. PDI-1 or nivolumab was then added to the cells and luciferase activity was measured after 6 h. (D) hPD-1-NFAT-Jurkat cells were cultured with PDI-1 alone for 6 h and luciferase activity was measured. Data represent the mean ± SEM of three replicates. *p < 0.05; **p < 0.01; ns, not significant.

Figure 4

PDI-1 inhibits the growth of human PD-L1-transfected B16-F10 melanoma cells in a mouse model. (A–D) B16F10 murine melanoma cells were transfected with hPD-L1 and injected subcutaneously into the flanks of three groups of syngeneic C57BL/6J mice (n=11/group). The mice were then injected intraperitoneally once daily with vehicle, 4 mg/kg PDI-1, or 8 mg/kg PDI-1. (A, B) Tumor volumes were measured for up to 29 days. (B) Tumors were excised on day 29 and weighed. (C) Left: Representative images of H&E-stained tumor sections on day 29. Right: Fluorescent multiplex immunohistochemical staining of PD-L1, CD8a, and FoxP3 proteins in tumors excised on day 29. Nuclei were stained with DAPI. (D) Quantification of CD8a, PD-L1, and FoxP3 staining intensity in the images shown in (C). Data represent the mean ± SEM of three replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 5

PDI-1 inhibits the growth of human PD-L1-transfected KLN205 NSCLC cells in a mouse model. (A–E) KLN205 cells were transfected with hPD-L1 and injected subcutaneously into the flanks of three groups of syngeneic DBA/2 mice (n=8/group). The mice were then injected intraperitoneally once daily with vehicle, 2 mg/kg PDI-1, or 4 mg/kg PDI-1. (A) Representative photographs of tumors excised on day 33. (B) Tumor volumes measured for up to 33 days. (C) Tumor weights on day 33. (D) Left: Representative images of H&E-stained tumor sections on day 33. Right: Fluorescent multiplex immunohistochemical staining of PD-L1, CD8a, and FoxP3 proteins in tumors excised on day 33. Nuclei were stained with DAPI. (E) Quantification of CD8a, PD-L1, and FoxP3 staining intensity in the images shown in (D). Data represent the mean ± SEM of three replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 6

PDI-1 increases T cell-mediated anti-tumor responses in vivo. (A, B) Blood samples were collected on day 29 from hPD-L1-B16F10 tumor-bearing mice (A) or on day 33 from hPD-L1-KLN205 tumor-bearing mice, as described in Figures 4 and 5 . Serum levels of IFN-γ, TNF-α, IL-1α, and IL-1β were analyzed using a multiplex flow cytometry assay. (C) Flow cytometric analysis of CD3+ T cells in peripheral blood mononuclear cells (left) or dissociated tumor tissue (middle) on day 29 after injection of hPD-L1-B16F10 cells. Dot plots (right) show representative FACS analysis of CD3 + T cells derived from tumor specimens. (D) Flow cytometric analysis of CD3+, CD4+, and CD8+ T cells in peripheral blood or of intratumorally CD3+ T cells on day 33 after injection of hPD-L1-KLN205 cells. Dot plots show representative FACS analysis of CD3 + T cells derived from tumor specimens. Data represent the mean ± SEM of three replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Figure 7

PDI-1 treatment rapidly boosts the host immune response to hPD-L1-KLN205 NSCLC tumors. (A) Schematic of the experimental design. DBA/2 mice were injected subcutaneously with hPD-L1-KLN205 cells and administered vehicle, 2 mg/kg PDI-1, or 4 mg/kg PDI-1 by intraperitoneal injection. Mice were sacrificed on day 7 and peripheral blood and tumors were collected for analysis. (B) Representative images of H&E-stained tumor sections. (C) Fluorescent multiplex immunohistochemical staining of CD8a, PD-L1, and FoxP3 protein in excised tumors. Nuclei were stained with DAPI. (D) Serum levels of IFN-γ, TNF-α, IL-1α, and IL-1β were analyzed using a multiplex flow cytometry assay. (E, F) Flow cytometric analysis of CD3+, CD4+, and CD8+ T cells in peripheral blood (E) or of intratumoral CD3+ T cells (F) on day 7. Dot plots indicate representative FACS analysis of CD3 + T cells derived from tumor specimens. Data represent the mean ± SEM of three replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.