0 50 100 150 NK cells cytotoxicity 100 10 1 0.01 0.0001 control agonist concentration [uM ] viability of target cells Characterization of novel dual A 2A /A 2B adenosine receptor antagonists for cancer immunotherapy Michał Gałęzowski, Paulina Węgrzyn, Aneta Bobowska, Katarzyna Dziedzic, Joanna Szeremeta-Spisak, Marcin Nowogródzki, Grzegorz Satała, Alicja Obara, Iwona Łozińska-Raj, Przemysław Wyrębek, Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Magdalena Zastawna, Grzegorz Statkiewicz, Maciej Rogacki, Mateusz Świrski, Jakub Woyciechowski, Magdalena Ziembik, Karolina Grycuk, Kinga Michalik, Agnieszka Adamus, Karolina Wiatrowska, Natalia Lewandowska, Urszula Sierańska, Agata Stachowicz, Aniela Gołas, Olga Pierzchała, Krzysztof Brzózka, Mateusz Nowak Selvita S.A., Bobrzyńskiego 14, 30-348 Kraków, Poland Corresponding author: [email protected] B cells CD3 cells CD4 cells CD8 cells 12 h INTRODUCTION Project supported by the European Regional Development Funds under the Measure 1.1.1. Operational Program - Smart Growth (POIR.01.01.01-00- 0987/16- 00) SUMMARY Adenosine is the key immunometabolite responsible for immune tolerance in tumors. It is present in normal tissue in low concentrations, having various physiological functions. In the tumor, its concentration increases rapidly, as a result of overexpression of enzymes producing adenosine, additionally enhanced by hypoxia and inflammation. Adenosine inhibits the biological functions of T lymphocytes infiltrating the cancer tissue by binding to the A 2A receptor. The affinity to A 2B receptor is believed to attenuate the response of dendritic cells and other parts of innate system. Thus blocking simultaneously the effects mediated by both receptor subtypes with dual inhibitor seems to be a viable approach for cancer immunotherapy in comparison to A 2A inhibitors currently tested in clinical trials. We have discovered a novel series of potent and dual A 2A /A 2B adenosine receptors inhibitors. Our best compounds present high in vitro activity not only in low adenosine conditions but also in tumor like adenosine rich environment. We have observed rescue of adenosine-suppressed cytotoxicity of NK cells and reversal of NECA-induced CREB phosphorylation in in vivo murine model (up to 24 hours). Additionally the A 2B activity is key factor in restoring of the adenosine agonist-impaired functional activity of moDC (cytokine release assays). To sum up, presented compounds show improved pharmacological profile in comparison to A 2A inhibitors currently tested in clinical trials. SEL330-639 OUTPERFORMS COMPETITORS IN DOWNREGULATION IN VIVO OF CREB PHOSPHORYLATION SEL330-584 AND SEL330-639 RESTORE NK CELLS CYTOTOXIC FUNCTION SEL330-584 AND SEL330-639 ARE HIGHLY POTENT IN ADENOSINE-RICH TUMOR MICROENVIRONMENT A 2A EC 50 [nM] c 0.1 mM ADO A 2A EC 50 [nM] c 100 mM ADO A 2B EC 50 [nM] d 0.1 mM ADO A 2B EC 50 [nM] d 100 mM ADO SEL330-584 0.26 92 3.6 634 SEL330-639 0.25 4.2 6.4 1618 AZD4635 e,f 57.6 > 10000 188 >10000 CPI-444 e,f 13.8 >10000 138 >10000 Arcus Ex. I e,g 1.4 1962 1.9 120 Preladenant e,f 3.2 9222 >4000 >4000 Fig 2. SLV A 2A /A 2B antagonists outperform competitors in a) adenosine-low and b) adenosine-rich environment; c) assays used PC12 cell line; d) assays used A 2B overexpressing HEK293 cell line; e) data generated in Selvita with compound samples synthesized by Selvita; f) CPI‐444: Structure from AACR, April 2017 (#CT119); AZD4635: Structure from AACR, April 2017 (#2641); Preladenant purchased from MedChemExpress g) Example I from WO2018/136700A1 (synthesized and tested in Selvita) Fig. 3. 10 mM NECA (potent pan-adenosine agonist) simulates Hi-Ado conditions and suppresses the cytotoxicity of NK delivered cell line against target tumor cells. SEL330-475 outperforms competitors in restoration of NK cytotoxicity. Representative data show. CPI‐444: Structure from AACR, April 2017 (#CT119), synthesized by Selvita; AZD4635: Structure from AACR, April 2017 (#2641) synthesized by Selvita. Fig 5. a) SEL-330-639 and Arcus Example I from WO2018/136700A1 (synthesized in Selvita) dual antagonists inhibit CREB phosphorylation in surrogate PK/PD assay. Antagonists were administrated to C57BL/6 mice (50mg/kg for SEL- 330-639 and 100mg/kg for Arcus Ex. I). Blood was collected at three timepoints 30min, 12h and 24h. NECA (10 mM) was used as CREB activator via the adenosine receptors. The level of CREB phosphorylation was detected in different immune cells via flow cytometry analysis, and compared with control group (no antagonist). The most significant changes are visible in B cells and CD8+ cells subpopulations; b) Disconnection between PK and PD plots shows unsurmountable mode of action of SEL330-639. a) b) SEL330-584 SEL330-639 CPI-444 AZD4635 Fig 1. Immunosuppressive activities of adenosine in cancer’ adapted from: Allard et al.; Curr. Opin. Pharmacol. 29:7-16 SEL330-584 AND SEL330-639 RESTORE moDC CELLS FUNCTION BY A 2B ACTIVITY b) a) c) d) A 2A R A 2B R - 0.1 mM -1 mM - 10 mM 2000 2500 3000 3500 4000 4500 TNF secretion by mature moDC NECA DMSO pg/ml 200 400 600 800 1000 IL-12 secretion by mature moDC NECA DMSO pg/ml - 0.1 mM -1 mM - 10 mM TNFa secretion by mature moDC IL-12 secretion by mature moDC Concentration [mM] b) a) SEL330-584 SEL330-639 CPI-444 AZD-4635 Preladenant Arcus Ex. I Low-Ado (0.1mM) Hi-Ado (100mM) Fig. 4. Restoration of adenosine suppression of a) TNFa and b) IL-12 production in mature moDCs is observed only for A 2B antagonists (Selvita dual antagonists and LAS101057 – selective A 2B antagonist) neither for selective A 2A inhibitors (Preladenant, CPI-444, AZD-4635). Human monocyte derived dendritic cells (moDCs) were differentiated in culture with GM-CSF and IL-4 for 6 days. Then cells were pretreated with A 2A /A 2B selective or dual receptor antagonists and maturated by LPS stimulation in presence of 10 mM NECA (Hi-Ado conditions). The effect was measured by TNFa and IL-12 production after 24h. Modulation of A 2A and A 2B receptors expression during maturation of dendritic cells. After maturation the level of A 2A receptor is downregulated (c), while the A 2B level is slightly upregulated (d). CPI‐444: Structure from AACR, April 2017 (#CT119), synthesized by Selvita; AZD4635: Structure from AACR, April 2017 (#2641) synthesized by Selvita. Preladenant (selective A 2A antagonist) and LAS101057 (selective A 2B antagonist) were purchased from MedChemExpress. Concentration [mM] Concentration [mM] 1500 2000 2500 3000 3500 4000 MFI 4000 4500 5000 5500 6000 6500 MFI 4000 4500 5000 5500 6000 6500 MFI 4000 5000 6000 7000 MFI 10 mM NECA 0 50 100 10 1 0.1 0.01 0.001 % of control 0 50 100 10 1 0.1 0.001 0.00001 % of control 10 mM NECA 10 mM NECA 10 mM NECA 0 4 8 12 16 20 24 10 100 1000 0 25 50 75 100 PK/PD 50m g/kg SEL330-639 Tim e (h) Free fraction conc. (nM ) pCREB % inhibition CD8 + A 2A HiAdo EC 90