Overcoming high level adenosine-mediated immunosuppression by DZD2269, a potent and selective A2aR antagonist

Abstract

Background: Adenosine is a potent immunosuppressant whose levels in the tumor microenvironment (TME) are often much higher than those in normal tissues. Binding of adenosine to its receptor A2aR activates a cascade of genes and leads to immunosuppression. In addition, immune checkpoint blockage markedly increases A2aR expression in T cells, which could dampen their anti-tumor response. Several A2aR antagonists are under clinical development, but with limited clinical benefit reported so far. These A2aR antagonists showed much diminished activity at high adenosine levels found in TME, which may explain their clinical underperformance. We report the discovery and early clinical development of DZD2269, a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Adenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction. Our data support further clinical development of DZD2269 in patients with cancer.

Methods: The selectivity of DZD2269 for adenosine receptors was tested in engineered cell lines, and its efficacy in blocking A2aR signaling and reversing adenosine-mediated immunosuppression was assessed in human T cells and peripheral blood mononuclear cells (PBMCs). The anti-tumor effects of DZD2269 were evaluated in multiple syngeneic mouse models as a single agent as well as in combination with chemotherapy, radiotherapy, or immune checkpoint inhibitors. A phase 1 study in healthy volunteers (NCT04932005) has been initiated to assess safety, pharmacokinetics (PK) and pharmacodynamics (PD) of DZD2269.

Results: Adenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction.

Conclusion: DZD2269 is a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Clinical development of DZD2269 in patients with cancer is warranted (NCT04634344).

Keywords: A2aR; Adenosine; DZD2269; Immunosuppression.

Conflict of interest statement

DZD2269 is an investigational drug of Dizal Pharmaceuticals, and currently in clinical evaluation. All authors are or have been employees and shareholders of Dizal Pharmaceuticals.

© 2022. The Author(s).

Figures

Fig. 1

Ex vivo modulation of pCREB and IFN-γ in response to NECA. (A) NECA regulates CREB phosphorylation on CD3 + T cells in whole blood samples. Data from test samples from three different donors and plotted using GraphPad Prism. (B) NECA affects IFN-γ secretion in human PBMCs activated by anti-CD3/CD28 antibodies. The experiment was repeated at least 3 times, and the data shown here are from one representative experiment

Fig. 2

In vitro selectivity of DZD2269 against adenosine receptors and its role in relieving NECA-induced immunosuppression. (A) Effects of DZD2269 on 10 µM NECA-induced cAMP accumulation in CHO-A2aR and CHO-A2bR cells and calcium increase in CHO-A1R and CHO-A3R cells. (B) The ability of A2aR antagonists to block CREB phosphorylation in CD4 + T cells was measured by flow cytometry after stimulation of whole blood with 10 µM NECA. (C-E) DZD2269 relieved the inhibition of 10 µM NECA on the secretion of IL-2 (C), IFN-γ (D) and TNF-α (E) by activated PBMCs. (F) DZD2269 relieved the inhibition of DC maturation by 10 µM NECA. Each data point represents the mean of at least two technical replicates, and error bars represent SD. Each experiment was repeated at least three times, and the data shown here are from one representative experiment

Fig. 3

Anti-tumor efficacy of DZD2269 in animal models in the presence or absence of T cells (A) B16F10 cells were inoculated in the back of C57BL/6 mice, and the mice were treated with DZD2269. Statistically significant difference was calculated by using two-way ANOVA (**** P:

Fig. 4

DZD2269 enhances the efficacy of radiotherapy (RT) in syngeneic mouse in vivo models. (A-B) RM-1 tumor cells were irradiated with 20 Gy radiation and were co-cultured anti-CD3/CD28 antibodies activated mouse splenocytes. (A) IFN-γ in supernatant was measured 72 h after co-culture. (B) IL-2 in supernatant was measured 24 h after co-culture. Significance Statistically significant difference was calculated by using one way ANOVA (*: p 

Fig. 5

DZD2269 synergizes with anti-PD-1 antibody or oxaliplatin in syngeneic mouse in vivo model. (A) Prostate RM-1 tumor-bearing mice were treated with 10 mg/kg anti-PD-1 antibody, with or without DZD2269 (3 mg/kg). (B) Pancreatic Pan02 tumor-bearing mice were treated with 10 mg/kg oxaliplatin with or without DZD2269 (3 mg/kg). bid: twice daily, qw: once a week. tiw: three times a week. Statistically significant difference was calculated by using two-way ANOVA (**: p 

Fig. 6

Correlation of DZD2269 plasma concentration (PK) and pCREB inhibition (PD) on CD4 + T Cell. Whole blood samples were collected at pre-dose, 2 h, and 24 h after single dose of DZD2269, samples were stimulated with or without 10 µM NECA. pCREB inhibition was calculated for each PD time point and plotted against PK at the corresponding time point. pCREB data from placebo-treated subjects (red dot) were also included in the analysis to enable assessment of biological and technical assay variation in PD assay. The dashed black line indicates the 90% pCREB inhibition