A2B adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models

Background Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A2ARs. While blockade of the A2AARs subtype effectively rescues lymphocyte activity, with four A2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A2AAR/A2BAR dimers in tissues that coexpress the two receptor subtypes, where the A2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy. Methods We report the synthesis and functional evaluation of five potent A2BAR antagonists and a dual A2AAR/A2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models. Results We provide data for six novel small molecules: five A2BAR selective antagonists and a dual A2AAR/A2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A2AAR antagonist AZD-4635. We find that A2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules. Conclusions Our results demonstrate that A2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A2BAR blockade. Inhibition of A2BAR signaling restores T cell function and proliferation. Furthermore, A2BAR and dual A2AAR/A2BAR antagonists showed similar or better results than A2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A2BAR in the regulation of the immune system.


Annexin V viability analysis
Cells were washed with Annexin V binding buffer (BioLegend) then incubated with Alexa Fluor 647conjugated Annexin V (BioLegend) at room temperature for 15 minutes.For A2AAR and A2BAR expression profiling, cells were washed with BD Perm/Wash then incubated with BD CytoFix/CytoPerm at 4°C for 20 minutes.The cells were then washed and stained with PE-conjugated A2AAR mouse antibody (Clone 7F6-G5-A2; Santa Cruz Biotechnology, sc-32261 PE) at room temperature for 30 minutes.After washing twice to remove most A2AAR antibody possible, goat A2BAR primary antibody (Clone PA5-18422; Thermo Fisher Scientific) was incubated at 4°C overnight.The cells were subsequently washed and stained with Alexa Fluor 488-conjugated anti-goat secondary antibody (Thermo Fisher Scientific) at room temperature for 1 hour.The cells were then washed with FC buffer twice before acquiring on NovoCyte.A2AAR and A2BAR fluorescence minus one (FMO) staining were also prepared.For differential expression of ADO ectonucleotidases, day five patientderived sarcoma spheroids were digested and stained for CD45, CD73 and CD39 using the same procedures as cell surface staining (Supplementary Table 1).

Public database bioinformatic analysis
Normalised, batch-corrected, gene expression and DNA copy number of ADORA2B from The Cancer Genome Atlas (TCGA) Pan-Cancer Genome Atlas project (Pan-Can) were accessed and downloaded from USCS Xena Browser (https://xenabrowser.net).Fold change of transcript per million was visualized using Gene Expression Profiling Interactive Analysis (GEPIA) Browser (http://gepia.cancer-pku.cn/).DNA alteration frequency from TCGA Pan-Can were accessed and visualized in cBioPortal (https://www.cbioportal.org).
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Chromium (51Cr) release cytotoxicity assay
Chromium (51Cr) release assay was used to measure the autologous TILs-mediated cytotoxicity against the established patient-derived breast and cancer cell lines.Briefly, breast and sarcoma tumor cells were labelled with 51Cr (PerkinElmer) as target cells and seeded on 96-well V-bottom plate (Corning) at 5 000cells/well.Effector TILs were added at the indicated Effector: Target (E:T) ratios.The supernatants were collected carefully into LUMA plates (PerkinElmer) after 24 and 48 hours of co-culture.After drying overnight, radioactivity of the plate was read with MicroBeta2 (PerkinElmer).

Relative adenosine production assay
An ectonucleotidase CD73 assay was optimized to measure the relative ADO production based on competitive AMP blockade and presence of CD73+ cells [47,48].Day five patient-derived sarcoma spheroids were digested and split into triplicates in X-VIVO20 and 1% PS culture media with an excessive amount of AMP at 0.4mM (Sigma-Aldrich) incubated for 30 minutes.
After 1500 RPM was applied for three minutes, 25 μL supernatant were removed and mixed with 25 μL of 200 μM ATP (Sigma-Aldrich) in the same media.25 μL of this mixture was added to a white opaque OptiPlate (PerkinElmer) containing 25 μL Cell-Titer Glo reagent (Promega, US).Relative luminescence unit (RLU) can be read on SPARK 10M plate reader with an integration time of 100ms.

Molecular dynamics parameters.
A 25Å sphere centered on the center of geometry of the ligand is considered for MD simulations of each generated protein-ligand complex, in order to equilibrate it from the initial docking pose.Protein atoms in the boundary of the sphere (22-25Å outer shell) had a positional restraint of 20 kcal/mol/Å 2 , while solvent atoms were subject to polarization and radial restrains using the surface constrained allatom solvent (SCAAS) [1][2][3] model to mimic the properties of bulk water at the sphere surface.Atoms lying outside the simulation sphere are tightly constrained (200 kcal/mol/Å 2 force constant) and excluded from the calculation of non-bonded interactions.Long range electrostatics interactions beyond BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)  4 and all titratable residues outside the sphere were neutralized and histidine residues were assigned a hydrogen atom on the δ nitrogen solvent bond and angles were constrained using the SHAKE algorithm. 5The OPLS-AA/M force field was used, with compatible ligand parameters generated with the ffld server. 6The simulation sphere was warmed up from 0.1 to 298 K, during a first equilibration period of 0.61 nanoseconds, where an initial restraint of 25 kcal/mol/Å 2 imposed on all heavy atoms was slowly released for all complexes.Thereafter the system was subject to ten parallel replicates of unrestrained MD, of 0.5 ns length each.

Chemistry
All starting materials, reagents and solvents were purchased and used without further purification.After extraction from aqueous phases, the organic solvents were dried over anhydrous sodium sulfate.The reactions were monitored by thin-layer chromatography (TLC) on 2.5 mm Merck silica gel GF 254 strips, and each purified compound showed a single spot; unless stated otherwise, UV light and/or iodine vapor were used to detect compounds.The reactions were performed in coated Kimble vials on a PLS (6×4) Organic Synthesizer with orbital stirring.Purification of isolated products was carried out by column chromatography (Kieselgel 0.040-0.063mm, E. Merck).
The purity and identity of all tested compounds were established by a combination of HPLC, high resolution mass spectrometry and NMR spectroscopy.Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected.The NMR spectra were recorded on Bruker AM300 spectrometer.Chemical shifts are given as  values against tetramethylsilane as internal standard and J values are given in Hz.High-resolution mass spectra (HRMS) were obtained on an Autospec Micromass spectrometer.NMR and HRMS reports of novel compounds are shown at the end of Spectroscopical and analytical data section.The unequivocally assignation of the different regioisomers was assisted by NOE experiments.Routinely purity control was performed by analytical HPLC using a Water Breeze™ 2 (binary pump 1525, detector UV/Visible 2489, 7725i Manual Injector Kit 1500 Series) using an Luna® Silica 100 Å, 4.6 mm × 150 mm, 5 µm column with gradient elution using hexane/isopropyl alcohol mixture in different percentages as mobile phase.The purity of all tested compounds was determined to be 95%.
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S5
The purity and identity of all tested compounds were established by a combination of HPLC, high resolution mass spectrometry and NMR spectroscopy.Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected.The NMR spectra were recorded on Bruker AM300 spectrometer.Chemical shifts are given as δ values against tetramethylsilane as internal standard and J values are given in Hz.A copy of the NMR spectra is found in the supplementary data file.High-resolution mass spectra (HRMS) were obtained on an Autospec Micromass spectrometer.
HRMS reports of novel compounds are shown in Supporting information.The unequivocally assignation of the different regioisomers was assisted by NOE experiments.Routinely purity control was performed by analytical HPLC using a Water Breeze™ 2 (binary pump 1525, detector UV/Visible 2489, 7725i Manual Injector Kit 1500 Series) using a Luna® Silica 100 Å, 4.6 mm × 150 mm, 5 µm column with gradient elution using hexane/isopropyl alcohol mixture in different percentages as mobile phase.The purity of all tested compounds was determined to be ≥95%.

Synthetic procedures
Procedure for the Biginelli synthesis of 3,4-dihydropyrimidin-2(1H)-ones SY1AF-30 7 and SY1AF-80 7 A mixture of the urea 1a or thiourea 1b (7.5 mmol), 2-furancarboxaldehide 1a or 3thiophenecarboxaldehyde 1b (5 mmol), isopropyl 3a or ethyl 3b acetoacetate (5 mmol) and ZnCl2 (0.5 mmol) in 3 mL of THF in coated Kimble vials was stirred with orbital stirring at 80ºC for 12h.After completion of the reaction, as indicated by TLC, the reaction mixture was poured onto crushed ice and stirred for 5-10 minutes.The solid separated was filtered under suction, washed with ice-cold water (20 mL), and then purified either by recrystallization or column chromatography on silica gel.A mixture of cyanamide 4 (2 mmol), 2-furanecarboxaldehyde 2a (1 mmol), isopropyl acetoacetate 3a, sodium acetate (1 mmol), and concentered hydrochloric acid (0.5 mL) in 7 mL of ethanol in a coated Kimble vial was stirred by orbital stirring at 80ºC for 8h.After completion of the reaction, as indicated by TLC, the reaction mixture was poured onto crushed ice and stirred for 10 min.
The solid was filtered under suction, washed with ice-cold water (20 mL) and then purified either by recrystallization or column chromatography on silica gel. 9mixture of 2-aminobenzimidazole 5 (7.5 mmol), 2-furanecarboxaldehyde 2a (5 mmol), isopropyl acetoacetate 3a (5 mmol), and 2-chloroacetic acid (0.05 mmol) in 3 mL of THF in coated Kimble vials was stirred with orbital stirring at 80ºC for 12h.After completion of the reaction, as indicated by TLC, the reaction mixture was poured onto crushed ice and stirred for 10 min.The solid was filtered under suction, washed with ice-cold water (20 mL) and then purified either by recrystallization or column chromatography on silica gel.BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Table 1 . List of cell surface antibodies used in flow cytometry. A.
A2AAR & A2BAR expression profiling B. CFSE Proliferation and Annexin V viability assay C. ADO ectonucleotidase expression D. Spheroid infiltrated TIL phenotyping E. Extracellular ADO uptake assay.F. IFNy and Perforin cytokine production assay G. Breast tumor resection phenotyping.
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