Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Abstract

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies.

Significance: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397.

Trial registration: ClinicalTrials.gov NCT03762447.

Figure 1.

INCB086550 binds to PD-L1 and interrupts its interaction with PD-1 and also induces PD-L1 dimerization and internalization. A, Structure of INCB086550. B, INCB086550 antagonizes binding of PD-1 to cells expressing PD-L1. CHO PD-L1 cells were treated with increasing concentrations of INCB086550, with each concentration run in triplicate. Each image is a composite of several fields of view within a single well. Nuclei stained with Hoechst 33342 and PD-1/PE were pseudocolored blue and green, respectively. The percent inhibition relative to the DMSO control is graphed. C, CHO PD-L1 cells were incubated with 1 µmol/L of INCB086550, followed by incubation with 10 µg/mL of atezolizumab or durvalumab. Binding of atezolizumab to cells was detected with anti-human IgG, conjugated to AlexaFluor488. Data were significant (P < 0.05) between untreated and 86550 pretreated for both atezolizumab and durvalumab by the Student t test. D, SEC-MALS reveals an inhibitor-induced dimeric PD-L1 complex. SEC-MALS profiles of either 15 µmol/L of PD-L1 alone (gray) or complexed with 100 µmol/L INCB086550 (blue). E, CHO PD-L1 cells were preincubated with increasing concentrations of INCB086550 for 18 hours, followed by staining with anti–PD-L1 clone MIH1 or 28-8. Percent inhibition was calculated based on the reduction of the fluorescent signal with the antibodies; P < 0.05 using Student t test. F, CHO PD-L1 cells were treated with 1 µmol/L of INCB086550 or atezolizumab at 10 µg/mL for 18 hours. After fixation, cells were stained with anti–PD-L1 clone 28-8 (green) and DAPI (blue). Scale bar, 10 µm. G, Live-cell imaging of CHO PD-L1 cells stained with PD-L1 (green) and DAPI (blue) and treated with INCB086550. Images up to 285 minutes are shown. Scale bar, 10 µm. H, CHO PD-L1 cells were transduced with a fusion construct of human Golgi-resident enzyme (N-acetylgalactosaminyltransferase) and TagRFP (red). Cells were then treated with INCB086550 at 1 µmol/L for 18 hours, fixed and stained with anti–PD-L1 antibody (green). Nuclei were visualized with DAPI (blue). Scale bar, 10 µm.

Figure 2.

INCB086550 reduces available surface PD-L1 in various cell types and enhances T-cell activity. A, Isogenic MBT2 clones expressing different levels of PD-L1 were treated with various concentrations of INCB086550 for 16 hours. At the end of incubation, cells were harvested and stained for available surface human PD-L1, followed by flow-cytometric analysis. P < 0.05 for PD-L1–low clone as compared with PD-L1 medium and high clones by one-way ANOVA. B, Cells from pleural effusion fluids were left unstimulated or stimulated with 1 ng/mL of human IFNγ in the presence of various concentrations of INCB086550 for 20 hours. After the incubation, cells were stained and analyzed by flow-cytometric analysis. P < 0.05 by paired t test. C, MDA-MB-231 cells were treated with 250 nmol/L of INCB086550 for 18 hours. Residual compound was then removed with acid wash and cells were harvested at designated time points to determine levels of available PD-L1. Samples were run in triplicates, and the percentage of surface receptor was calculated using mean fluorescence intensity. Data were significant compared with time zero using one-way ANOVA with Dunnett multiple comparisons. D, Cynomolgus monkey and human whole blood was treated with INCB086550 and then stimulated with IFNγ for 20 hours. Cells were stained with anti–PD-L1 and anti-CD14 and flow-cytometric analysis was performed. E, T cells from normal human donors were mixed with CHO PD-L1 cells and treated with increasing concentrations of INCB086550 or atezolizumab at 10 nmol/L for 3 days. Human IFNγ levels were measured by Luminex. F, INCB086550 or atezolizumab was added to whole blood from normal human donors together with staphylococcal enterotoxin B and incubated for 5 days. IFNγ levels were measured by Luminex.

Figure 3.

INCB086550 reduces tumor PD-L1 after in vivo administration. A, BALB/c nu/nu mice bearing MDA-MB-231 xenograft were treated orally with 15 or 200 mg/kg of INCB086550 once or twice with a 12-hour interval. Tumor samples were harvested at 24 hours after initial dose administration and processed into single-cell suspension to determine tumor PD-L1 expression by flow-cytometric analysis. Plasma samples were collected at 24 hours after initial dose administration to determine compound concentration by high-performance liquid chromatography (HPLC) assay. B, Balb/c nu/nu mice bearing MDA-MB-231 xenograft were dosed once with INCB086550 at 150 mg/kg. Plasma and tumor samples were collected at various time points to determine compound concentration by HPLC assay. Tumor samples were also processed into single-cell suspension to determine tumor PD-L1 expression by flow-cytometric analysis. C, MDA-MB-231 and HT-29 tumors engrafted in CD34⁺ humanized mice were analyzed for PD-L1 expression by IHC. MDA-MB-231 tumors were found to express abundant PD-L1. In contrast, HT-29 tumors were PD-L1–negative. Scale bar, 100 µm. MDA-MB-231 or HT-29 tumor–bearing humanized mice were dosed for 14 and 7 days, respectively, with 20 mg/kg INCB086550 twice daily. Plasma and tumor were collected 16 hours after the last dose and compound levels were analyzed.

Figure 4.

INCB086550 inhibits tumor growth in preclinical mouse models by reducing unoccupied cell-surface PD-L1 and increasing T-cell number and activation. Activity of orally dosed (p.o.) INCB086550 (2, 20, and 200 mg/kg b.i.d.) in C57BL/6 (A) or NSG (B) mice bearing established MC38-huPD-L1 tumors. C, Levels of unoccupied and occupied cell-surface PD-L1 on MC38-huPD-L1 tumors after dosing mice with INCB086550. P < 0.05 using Student t test. D, Percentage of intratumoral CD8⁺ T cells in MC38-huPD-L1 tumors at the end of the efficacy study measured by flow-cytometric analysis. P < 0.05 when compared with vehicle using one-way ANOVA. Activity of orally dosed INCB086550 in human CD34⁺ engrafted NSG (20, 60, and 200 mg/kg b.i.d.; E) or NSG (20 mg/kg; F) mice bearing established MDA-MB-231 tumors. Atezolizumab was included as a control at 5 mg/kg every 5 days in E. G, At the end of the efficacy study in E, levels of INCB086550 were measured in plasma and tumors were collected, 2, 4, 8, and 16 hours after the last dose. H, MDA-MB-231 xenograft-engrafted CD34⁺ humanized NSG mice were orally administered INCB086550 at 20 mg/kg b.i.d. for 31 days. Tumors from humanized mice from one donor were collected at the end of the study for RNA-seq analysis. The heat map displays a signature of 58 genes that increased by at least 1.2-fold with P < 0.05 in tumors from patients with melanoma who received nivolumab.

Figure 5.

PD-L1 engagement, immune activation, and tumor volume reduction observed in patients with cancer in a phase I trial of INCB086550. A, Dose-related decrease of free PD-L1 receptors in patient whole blood. The binding activity to PD-L1 on human blood monocytes was monitored in whole blood stimulated with exogenous IFNγ followed by the analysis of expression of PD-L1 by flow cytometry. Plasma samples were assessed on a targeted panel by immunoassay and on a panel of 1,104 analytes utilizing the Olink proximity extension assay platform. q.d., every day. On-treatment increases in CXCL9 (B), CXCL10 (C), IFNγ (D), and PD-L1 (E) were observed at cycle 1 day 8 and cycle 2 day 1 by immunoassay and confirmed by the Olink platform. F, Posttreatment increases in plasma concentrations of interferon-related cytokines and immune regulatory proteins including GZMH and GZMB were observed by the Olink platform. G, Clinical activity observed with INCB086550 in a 54-year-old female. Decrease in target lesion volume shown by CT scan at week 24 of INCB086550 therapy. Figure reproduced from Piha-Paul S, et al. Pharmacodynamic biomarkers demonstrate T-cell activation in patients treated with the oral PD-L1 inhibitor INCB086550 in a phase 1 clinical trial. Journal for ImmunoTherapy of Cancer 2020;8:doi: 10.1136/jitc-2020-SITC2020.0419 under the creative commons license (CC BY 4.0). Poster presented at SITC 2020.