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Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

AB680 is a highly potent CD73 small molecule inhibitor discovered and developed by Arcus Biosciences, currently in clinical trials for the treatment of pancreatic cancer. Here, we report the develo...

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 μM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (Ki = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

AB928 is a potent and selective dual antagonist of the A2a and A2b receptors, which is currently in clinical trials. Here, we report the development of two scalable and practical syntheses of AB928...

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

Background The microenvironment of solid tumors is hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for cancer cells to survive and metastasize. The master transcriptional regulators of hypoxia-induced genes are the HIF proteins, consisting of three distinct oxygen-regulated α monomers (HIF-1α, -2α, and -3α). In normoxia, hydroxylation of HIF-2α allows for recognition by the pVHL E3-ubiquitin ligase complex and proteasomal degradation. Exposure to hypoxia, or VHL mutation or silencing, leads to HIF-2α stabilization, dimerization with HIF-1β/ARNT, and transcription of pro-tumorigenic gene sets in a variety of cancer and non-cancer cell types in the tumor microenvironment. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence suggests that inhibiting HIF-2α is an effective strategy to mitigate tumor growth, particularly in clear cell renal cell carcinoma (ccRCC), warranting further development of HIF-2α inhibitors and investigation into the role of HIF-2α in various cellular and combinatorial settings. Methods Using a suite of assays to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule inhibitors of HIF-2α. Results Optimized compounds inhibited HIF reporter transcription and VEGF secretion. Compounds that were biochemically confirmed to bind HIF-2α also inhibited HIF-2α-, but not HIF-1α-, mediated gene expression. Characterization of HIF-2α inhibition was expanded to human stromal and immune cell subsets. While compounds inhibited pro-angiogenic gene sets in endothelial cells, inhibiting HIF-2α in activated hypoxic T cells did not affect proliferation or cytokine secretion, suggesting that HIF-2α inhibitors would not impede T cell functionality in tumors. In contrast, in a M2-polarized macrophage model for suppressive tumor-associated macrophages, HIF-2α drove hypoxia-induced changes in the chemokine secretome that favored granulocytic rather than lymphocytic infiltration, an effect that was effectively reversed by HIF-2α inhibition. At the transcriptional level, mRNA-sequencing was used to define global gene sets impacted by HIF-2α inhibition in M2 macrophages. Additionally, in a set of liver, kidney, pancreatic, and colon cancer lines, CRISPR/Cas9-mediated gene editing was used to differentiate the transcriptomic profile driven by HIF-2α from that of HIF-1α or HIF-3α, allowing for the derivation of a HIF-2α-specific gene signature. Cancer cell and macrophage-derived signatures were applied to publicly available datasets to investigate cancer types, other than ccRCC, in which HIF-2α may play an important pathological role. Conclusions Collectively, these data support the development of our novel and selective HIF-2α inhibitors for the treatment of cancer and expand the indications that may benefit beyond ccRCC.

A hypoxic environment is a common characteristic of solid tumors. Cancer cells adapt to hypoxia and become more aggressive by up-regulation of genes associated with metabolism, growth, proliferation, angiogenesis, and erythropoiesis. Hypoxia-inducible factors (HIFs) are the central driving force for the cellular response to hypoxia and regulate a vast array of these genes. HIFs are heterodimers composed of an oxygen-sensitive HIF-α subunit (HIF-1α, HIF-2α, and HIF-3α) and a constitutively expressed HIF-1β subunit. HIF-2α is constitutively synthesized and regulated in an oxygen-dependent manner. Proline residues present in the oxygen-dependent degradation domain of the HIF-2α subunit are hydroxylated and subject to ubiquitination via the von Hippel-Lindau (pVHL) E3 ligase complex for subsequent proteasomal degradation. Under hypoxia or pseudohypoxia, caused by loss of VHL function, this process is inhibited, allowing HIF-2α translocation to the nucleus where, in complex with HIF-1β/ARNT, it promotes transcription of various pro-tumorigenic gene sets. Inhibition of HIF-2α has been demonstrated to be an effective strategy to mitigate tumor growth in the clinic, particularly in clear cell renal cell carcinoma (ccRCC). Applying a pharmacophore mapping and structure-based design approach, we identified multiple novel series of small molecule HIF-2α inhibitors which avidly bind the HIF-2α PAS-B domain and disrupt dimerization with HIF-1β, preventing HIF-2α-mediated gene transcription. Interrogation of structure activity relationships and pharmacokinetic trends yielded highly optimized inhibitors, including AB521, which potently inhibits HIF-2α reporter transcription and VEGF secretion in a human ccRCC line. AB521 was confirmed to bind HIF-2α via multiple biochemical assays and inhibited HIF-2α-, but not HIF-1α-, mediated gene expression in a hepatocellular carcinoma cell line. Furthermore, AB521 is characterized by a favorable preclinical pharmacokinetic profile with high stability to human hepatocytes and no significant direct or time-dependent inhibition of the major CYP450 drug metabolizing enzymes. AB521 exhibits high bioavailability across preclinical species and is projected to possess a human pharmacokinetic profile suitable for once-daily dosing. In conclusion, we have discovered and extensively characterized a series of novel HIF-2α inhibitors, exemplified by AB521, which potently and selectively inhibits HIF-2α and possesses a favorable preclinical pharmacokinetic profile. The data described herein provides further support for the development of novel HIF-2α inhibitors for cancer therapy. Citation Format: Kenneth V. Lawson, Kelsey E. Sivick Gauthier, Artur K. Mailyan, Jeremy T. Fournier, Joel W. Beatty, Samuel L. Drew, Jarek Kalisiak, Balint Gal, Guillaume Mata, Zhang Wang, Xuelei Yan, Lixia Jin, Elaine Ginn, Dana Piovesan, Jennifer Au, Cesar A. Meleza, Stephen W. Young, Matthew J. Walters, Manmohan Leleti, Jay P. Powers. Discovery and characterization of AB521, a novel, potent, and selective hypoxia-inducible factor (HIF)-2α inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1206.

A protocol for the synthesis of hindered 4-aryl 2-aminopyrimidines from β–keto esters is described. The process employs trifluoroethanol as an essential additive to promote the guanidine condensation reaction, enabling the synthesis of 25 aryl- and heteroaryl substituted aminopyrimidines in good yields and high purities with no column chromatography. The conditions described herein are readily scalable and have been employed in the large-scale synthesis of the clinical A2a/A2bR antagonist AB928.

Introduction: High levels of immunosuppressive adenosine are found in the tumor microenvironment, reaching 50-100 μM in experimental models. Adenosine exerts its effects on immune cells primarily through the adenosine receptors A2aR/A2bR, which increase intracellular levels of cyclic AMP, leading to CREB phosphorylation (pCREB). We have previously shown that the dual A2aR/A2bR antagonist AB928 is capable of inhibiting adenosine-induced pCREB in healthy human volunteer (HV) blood lymphocytes. AB928 has also been shown to relieve adenosine-mediated T-cell suppression in vitro and exhibit combinatorial effects with standard-of-care chemotherapeutics in mouse syngeneic tumor models. Herein, we show that AB928 is capable of inhibiting NECA-induced gene expression changes and CREB phosphorylation in non-small cell lung carcinoma (NSCLC) patient whole blood (WB). Additionally, observations from our in vitro human studies showing the combinatorial effect of AB928 and α-PD-1 were reproduced in B16F10 syngeneic tumors. Methods: Human WB was stimulated with 5 μM of NECA and flow cytometry was used to quantify AB928-mediated inhibition of pCREB and CD3ζ phosphorylation. B16F10 tumors were treated with α-PD-1 +/- AB928 and gene expression was determined from excised mouse tumors using the nCounter PanCancer panel. Results: To ensure AB928 can successfully inhibit the high levels of intratumoral adenosine, we found that 5 μM NECA provides maximal stimulation and is significantly more potent (>20 fold) than adenosine in the pCREB assay. Additional experiments demonstrated that AB928 has comparable potency in NECA-stimulated WB from both HV and NSCLC patients. In addition to blocking downstream signaling, NanoString analysis showed that AB928 could prevent NECA-stimulated gene expression changes in NSCLC WB. We also found that NECA stimulation, alone or in combination with PD-1 inhibition, significantly reduced proximal TCR signaling, leading to reduced levels of CD3ζ phosphorylation at TYR142 (pTYR142). These reduced pTYR142 levels, with and without α-PD-1, could be significantly rescued by AB928, suggesting that blocking adenosine immunosuppression may provide additional benefit to PD-1 inhibition in tumors. Consistent with these results, AB928 was capable of suppressing growth (volume in mm3) of B16-F10 tumors both as a single agent (vehicle: 462 +/- 58; AB928: 292 +/- 55; p Conclusions: Collectively, these results support a role for AB928 in relieving adenosine-mediated immunosuppression by blocking A2aR/A2bR-induced signaling events, gene expression changes, and suppressing tumor growth in vivo. Citation Format: Daniel M. DiRenzo, Nell Narasappa, Dana Piovesan, Devika Ashok, Park Adam, Jenna L. Jeffrey, Manmohan R. Leleti, Joanne B.L. Tan, Lisa Seitz, Steve W. Young, Jay P. Powers, Matthew J. Walters. The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A10.

Adenosine suppresses antitumor immune responses via A2a and A2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5′-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A2aR/A2bR antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 μM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.

The microenvironment of solid tumors is known to be hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for tumor cells to survive and metastasize. The master transcriptional regulator of hypoxia-induced genes is the Hypoxia-Inducible Factor (HIF), consisting of an oxygen-regulated alpha monomer, of which there are three isoforms (HIF-1α, HIF-2α, and HIF-3α), that can heterodimerize with a constitutively-expressed beta monomer (HIF-1β/ARNT) using Per-ARNT-SIM (PAS) protein-protein interaction domains. In normoxia, proline residues present in the oxygen-dependent degradation domain of the HIF-α subunits are hydroxylated, allowing for recognition by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex and subsequent proteasomal degradation. Upon exposure to low oxygen conditions or in the case of VHL mutation or silencing, HIF-α subunits accumulate in the cell and mediate transcription of various pro-tumorigenic gene sets. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence is mounting that suggests inhibiting HIF-2α is a valid approach to destroy tumor cells, particularly in clear cell renal carcinoma, warranting development of next-generation inhibitors. Using a suite of in vitro and in vivo assays designed to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule antagonists of HIF-2α. These compounds inhibited HIF-dependent reporter gene transcription as well as VEGF protein secretion in a human renal cell adenocarcinoma line. Compounds that were confirmed to bind the HIF-2α PAS-B domain by Microscale thermophoresis (MST) and Thermal shift assay (TSA) also significantly inhibited HIF-2α, but not HIF-1α, target gene expression in a hepatocellular carcinoma cell line (P Citation Format: Kelsey E Sivick Gauthier, Kenneth V Lawson, Dana Piovesan, Matthew J Walters, Ada Chen, Xiaoning Zhao, Cesar Meleza, Nikki Kimura, Tim Park, Steve Young, Anh Tran, Samuel L Drew, Lixia Jin, Manmohan Leleti, Elaine Ginn, Jay P Powers. A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C050. doi:10.1158/1535-7163.TARG-19-C050

Introduction: Adenosine, generated through the hydrolysis of extracellular adenosine monophosphate (AMP) by the ecto-nucleotidase CD73, is an important mechanism for immunosuppression in cancer development. Adenosine’s suppressive effects on immune cells are driven primarily through 2 of the 4 adenosine receptors, A2aR and A2bR. We have previously shown that adenosine-mediated suppression of T-cells can be blocked by the dual A2aR/A2bR antagonist, AB928. Herein, we show that AB928 is capable of relieving adenosine-mediated immune suppression using human in vitro cell cultures, advanced gene expression studies, and mouse syngeneic tumor models. Methods: The ability of AB928 to inhibit adenosine-mediated suppression of dendritic cell function in vitro was assessed using human monocyte-derived dendritic cells (moDC). Briefly, moDC were generated from freshly isolated CD14+ monocytes and differentiated with IL-4/GM-CSF for 7 days +/- adenosine/EHNA +/- AB928. Cells were then taken for NanoString analysis or placed in a mixed lymphocyte reaction (MLR) with CD4+ T-cells. Mouse syngeneic tumor studies were conducted using C57BL/6 mice inoculated with mouse mammary tumor AT3-OVA or melanoma B16-F10 cells. Tumors were subsequently treated with doxorubicin, oxaliplatin, or α-PD-1 +/- AB928. Results: Quantitative immunohistochemistry and analysis of public gene expression databases identified individual human tumor types that express high levels of adenosine processing enzymes. Most notably, non-small cell lung, renal, triple-negative breast, ovarian, colorectal, and gastroesophageal cancers were found to have the most favorable expression profiles for interventions targeting the adenosine pathway. Additionally, a high degree of correlation was found between transcript and protein measurements for CD73 (r2 = 0.87), illustrating the robust and reproducible nature of these techniques. In human in vitro cell cultures, moDC differentiated in the presence of adenosine showed a decreased ability to stimulate IFN-γ secretion from allogenic CD4+ T-cells in a MLR. This suppression was significantly reversed by addition of AB928. Next, multiplexed gene expression profiling using NanoString identified a cassette of 39 genes (>2.0 fold change, p Citation Format: Daniel DiRenzo, Dana Piovesan, Joanne Tan, Dillon H. Miles, Manmohan R. Leleti, Timothy Park, Ferdie Soriano, Bryan Handlos, Jenna L. Jeffrey, Ehesan U. Sharif, Brandon R. Rosen, Ulrike Schindler, Jay P. Powers, Matthew J. Walters. AB928, a dual antagonist of the A2aR and A2bR adenosine receptors, relieves adenosine-mediated immune suppression [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A162.

Introduction: Extracellular adenosine (ADO), present at high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T-cell, natural killer (NK) cell, and dendritic cell (DC) activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Here we present the preclinical characterization of AB680, a novel, highly potent, reversible and selective small-molecule inhibitor of CD73, currently in preclinical development as a potential antitumor agent. The link between CD73 levels present in different tissues, efficacy in mouse tumor models, plasma and tumor exposure, and projected human pharmacokinetic (PK) profile can be combined to provide an expected AB680 dosing strategy for the upcoming first-in-human clinical trial. Methods: The potency of AB680 against human CD73 was determined using CHO-CD73 cells, blood CD8+ T-cells, recombinant human CD73, and serum/plasma using either malachite green assay, AMP-Glo assay, or LCMS/MS. The selectivity of AB680 against related ecto-nucleotidases was also assessed using similar methods. Quantitation of soluble CD73 in mouse and human serum, and mouse tumor homogenates, was performed via in-house developed and validated ELISA or Western blot methods. Syngeneic mouse tumor models were established to assess the efficacy of AB680 at multiple doses. AB680 levels in plasma and tumor associated with each dosing regimen were determined via LCMS/MS. The potency of AB680 in an intratumoral setting was determined using various biochemical methods. The effects of AB680 on syngeneic tumor volumes were assessed in prophylactic and therapeutic settings. The PK properties of AB680 were evaluated in multiple preclinical species and a projected human dosing schedule for AB680 was determined via allometric scaling. Results: AB680 is a highly potent, reversible and selective inhibitor of CD73 activity (IC50 < 0.01 nM on human CD8+ T-cells), which retains its high potency in the presence of human serum. Inhibition of AMP hydrolysis by AB680 completely reversed ADO-mediated suppression of CD4+ and CD8+ T-cell effector function, as measured by cytokine secretion and proliferation. The in vivo efficacy of AB680, as measured by its ability to restrict tumor growth at doses as low as 10 mg/kg once daily, is reflective of the following parameters: 1) plasma half-life, 2) partitioning from blood to tumor compartment, 3) potency against soluble CD73 in plasma or serum, and 4) potency against membrane-bound CD73. The PK properties of AB680 in rodent and non-rodent species are characterized by very low clearance and long half-lives. The combination of in vitro potency against mouse and human soluble and membrane-bound CD73, in vivo tumor growth regulation observed in mouse models, quantification of mouse and human CD73 levels, and the projected human PK profile for AB680 have resulted in predicted human PK properties (projected half life: 4-14 days) suitable for intravenous dosing on a schedule consistent with typical monoclonal antibody dosing cycles. Conclusions: AB680 is a highly potent and selective small-molecule inhibitor of CD73 which can mitigate AMP and ADO-mediated tumor immunosuppression by potently blocking the production of ADO. AB680 exhibits a unique projected human PK profile suitable for parental administration and is expected to enter clinical development in 2018. Citation Format: Joanne B.L. Tan, Jie Chen, Elaine Ginn, Devika Ashok, Amy E Anderson, Jesus Banuelos, Kristen Zhang, Amber Pham, Timothy Park, Ada Chen, Xiaoning Zhao, Kenneth K.V. Lawson, Jenna Jeffreys, Jarek Kalisiak, Manmohan R. Leleti, Matthew J. Walters, Jay P. Powers. Preclinical pharmacokinetic and pharmacodynamic characterization of AB680, a small-molecule CD73 inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A157.

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773.

INTRODUCTION: Extracellular adenosine triphosphate (ATP) is efficiently hydrolyzed to adenosine by ecto-nucleotidases CD39 and CD73, which converts adenosine-monophosphate (AMP) into adenosine (ADO). ADO suppresses immune responses including those of T cells, natural killer (NK) cells, and dendritic cells (DC) through activation of A2aR and A2bR receptors. Treatment of cancer cells with platinum-based and anthracycline chemotherapy has been shown to induce immunogenic cell death (ICD), characterized by increased extracellular ATP levels, and upregulation of CD39 and CD73, leading to the enhanced generation of adenosine. METHODS: The ability of AB928 to inhibit adenosine-mediated suppression of immune cell function was assessed using human CD4 and CD8 cells. BALB/c mice were inoculated with AT-3-OVA tumors, which express the model antigen ovalbumin, and subsequently treated with chemotherapy or AB928 alone, or a combination of both. The growth rate and immune composition of the tumors were assessed (flow cytometry). RESULTS: Consistent with the ability of adenosine to suppress immune function, AB928 inhibited the ability of adenosine to suppress CD4 or CD8 T cell activation. Tumor-bearing mice that have been treated with either doxorubicin (DOX, an anthracycline) or oxaliplatin (OX) exhibit increased expression of CD39 and CD73. Established AT-3-OVA tumors treated with AB928 alone had a small but significant decrease in their growth rate; similarly, tumors treated with chemotherapy exhibited a reduction in growth rate. Consistent with the ICD hypothesis, treatment of AT-3-OVA-bearing mice with DOX or OX resulted in an increase in OVA-specific CD8 T cells in the tumor. Concurrent treatment with AB928 and chemotherapy resulted in significantly reduced tumor growth rates, when compared to chemotherapy alone. Analysis of the tumor-infiltrating cell populations showed a significant increase in OVA-specific CD8 T cells, relative to those treated with chemotherapy alone. CONCLUSIONS: Treatment of tumor-bearing mice with a combination of AB928, a dual A2aR and A2bR antagonist, and ICD-inducing chemotherapy results in increased tumor antigen-specific CD8 T cell responses and significantly reduced tumor growth. AB928 is currently undergoing clinical evaluation. Citation Format: Matt J. Walters, Dana Piovesan, Joanne Tan, Daniel DiRenzo, Fangfang Yin, Dillon Miles, Manmohan R. Leleti, Tim Park, Ferdie Soriano, Eshan Sharif, Ulrike Schindler, Jay P. Powers. Combining adenosine receptor inhibition with AB928 and chemotherapy results in greater immune activation and tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5556.

INTRODUCTION: CD73 converts extracellular adenosine monophosphate (AMP), derived from adenosine triphosphate, into adenosine (ADO). ADO suppresses immune responses, including those of T cells, natural killer (NK) cells and dendritic cells, via A2aR and A2bR receptors. Activation of these receptors results in increased intracellular levels of cyclic AMP (cAMP) and phosphorylation of the cAMP response element-binding protein (CREB). Monitoring the degree of pCREB induced by activation of the Aa2R and A2bR receptors in peripheral blood allows for a specific measurement of target engagement by AB928. In mice, the pCREB assay, coupled with changes in immune cell infiltrate and tumor growth rates, allows for a fuller understanding of the drug's PK/PD relationship. METHODS: Mouse and human whole blood was stimulated with the adenosine agonist NECA (5'-N-Ethylcarboxamidoadenosine) following in vitro spike in or in vivo dosing with AB928. Multi-color phospho-flow cytometry was used to assess levels of pCREB on immune cells. AB928 levels in plasma samples were determined using LC-MS-MS after protein precipitation. RESULTS: A2aR mRNA was the most prevalent adenosine receptor in CD4+ and CD8+ T cells. NK cells also express primarily A2aR, although A2bR was also detected. Dendritic cells and CD14+ monocytes expressed both A2aR and A2bR. NECA stimulation of mouse whole blood resulted in a significant increase in the levels of pCREB within CD8+ T cells with an EC50 of 100 nM, while in human whole blood the EC50 was higher at 700 nM. AB928 (100 nM) exhibited comparable shifts in the NECA dose response between human and murine whole blood, 15 and 16-fold, respectively. Due to the high level of adenosine in the tumor microenvironment, doses of AB928 associated with plasma levels that significantly inhibited 5 μM NECA in the whole-blood pCREB assay were selected for mouse efficacy studies. At these doses, significant reductions in tumor growth were noted. In human whole blood, approximately 90 nM AB928 was required to inhibit 50% of the 5 μM NECA-induced pCREB signal. Inhibition was observed on both CD4+ and CD8+ T cells. NECA-induced pCREB elevations were observed in CD14+ cells less frequently than T cells; however, when the signal was observed, AB928 inhibited it. The human pCREB assay is being utilized to monitor the PD responses in an ongoing clinical trial. Preliminary PK/PD results from this study will be presented. CONCLUSIONS: Systemic extent of receptor (A2aR and A2bR) occupancy by the novel dual antagonist AB928 can be assessed in humans and mice based on the extent of receptor-mediated CREB phosphorylation in blood lymphocytes. AB928 retains much of its inherent potency when competing against high concentrations of adenosine under physiologically relevant conditions (i.e., whole blood). Citation Format: Lisa Seitz, Devika Ashok, Manmohan R. Leleti, Jay P. Powers, Brandon Rosen, Dillon Miles, Lixia Jin, Adam Park, Tim Park, Steve Young, Ferdie Soriano, Aimee Rieger, Ulrike Schindler, Joyson Karakunnel, Matt J. Walters. Pharmacokinetic-pharmacodynamic relationship for AB928, a dual antagonist of the A2aR and A2bR adenosine receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3769.

Introduction: In the tumor micro-environment, extracellular ATP is sequentially hydrolyzed to adenosine by the ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→adenosine). Adenosine, through activation of the A2a receptor (A2aR), is a potent inhibitor of T-cell activation, resulting in an immunosuppressed phenotype. Thus, inhibition of A2aR has recently generated great interest in immuno-oncology. We present the characterization of a novel, selective, and highly potent small molecule antagonist of A2aR which is slated to enter the clinic in 2017. Methods: The cellular potency of A2aR antagonists was assessed as a function of decreased cAMP levels in CHO cells stably over-expressing hA2aR, a Gs coupled receptor, following stimulation with the agonist NECA. Experiments were conducted in the presence and absence of human serum. Selectivity against the Gi-coupled receptor A1R was assessed similarly as a function of cAMP elevation in CHO cells stably expressing hA1R, following pretreatment with forskolin and stimulation with NECA. The ability of AB928 to reverse adenosine-mediated immune suppression (25 μM) of human or mouse CD8+ T-cells was determined using standard CD3/CD28 activation conditions. CD25 expression and cytokine release were measured by flow cytometry and ELISA, respectively. The pharmacokinetic characteristics of AB928 were assessed in rodent and non-rodent species to facilitate calculation of a projected human dose. Results: AB928 represents a novel series of potent and selective compounds designed to inhibit adenosine-mediated A2aR activation. This molecule is different from most known A2aR antagonists in that it does not cross the blood brain barrier. AB928 inhibited NECA-mediated A2aR activation with a potency of Conclusions: AB928 is a potent, selective and peripherally restricted antagonist of the A2aR receptor which is slated to enter clinical development in 2017. Citation Format: Matt J. Walters, Joanne B. Tan, Annette Becker, Fangfang Yi, Tim Park, Manmohan R. Leleti, Brandon Rosen, Ehesan Sharif, Laurent Debien, Steve Young, Wan Hsin Lim, Stefan Garrido-Shaqfeh, Juan C. Jaen, Jay P. Powers. Characterization of the potent and selective A2aR antagonist AB928 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2017-4572

INTRODUCTION: In the tumor micro-environment (TME) Adenosine (ADO) dampens the immune response towards cancer cells by inhibiting the cytotoxic activity of effector cells and promoting the proliferation of immunosuppressive cells. Extracellular ADO is generated by the two ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→ADO). In immune cells ADO signals primarily through the G-protein coupled receptor A2aR. Inhibition of ADO generation and signaling have been shown to be promising therapeutic approaches for the treatment of cancer. METHODS: Potent and highly selective small molecule inhibitors for A2aR, CD73 and CD39 have been designed and synthesized by the Medicinal Chemistry Department at Arcus Biosciences. Enzymatic assays: CD39 and CD73 activity was assayed by quantitating hydrolysis of ATP or AMP, respectively. Inorganic phosphate was detected using a colorimetric (malachite green) protocol. ADO receptor assays: The potency of A2aR antagonists and selectivity towards related receptors was determined by measuring cAMP elevation in stably-expressing CHO cells following NECA (ADO mimic) stimulation. CD8+ T cell assays: Activation, proliferation and effector function of CD8+ T cells were quantified following compound treatment in the presence and absence of ATP. Mixed Lymphocyte Reaction (MLR): Cytokine levels were determined in an MLR assay after compound treatment in the presence and absence of ATP. Tumor models: CT26 and B16F10 syngeneic tumor models were used to assess the therapeutic effect of the inhibitors. RESULTS: Potent and highly selective small molecules have been generated to block either ADO generation or ADO signaling. The therapeutic potential of these molecules was assessed in CD8+ T cell assays, MLR assays and in various tumor models. CD73 inhibition blocked the conversion of AMP to ADO. CD39 inhibition blocked the conversion of ATP to AMP and A2aR inhibition abolished the increases in intracellular cAMP following ADO stimulation. The compounds are highly selective relative to related enzymes/receptors, as well as a large panel of unrelated targets. Inhibition of any one of the three targets robustly reversed adenosine-mediated inhibition of proliferation, CD25 expression, and IFN-γ and granzyme B production by human CD8+ T-cells. Robust inhibition of tumor growth in combination with anti-PD1 antibody was observed for several of these compounds. CONCLUSIONS: Highly potent and selective inhibitors of each of the 3 molecular targets involved in the ATP/adenosine pathway have been identified. Their ability to interfere with the extracellular generation of ADO and the immune-suppressive effects of ADO, as well as their effects on experimental tumor biology, have been demonstrated in various in vitro and in vivo models. Citation Format: Ulrike Schindler, Joanne B. Tan, Matt Walters, Annette Becker, Fangfang Yin, Ada Chen, Yu Chen, Wan Hsin Lim, Steve Young, Manmohan Leleti, Jay Powers, Juan C. Jaen. Small-molecule inhibitors of CD73, CD39 and A2aR: Three anti-cancer targets in the ATP/adenosine signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2640. doi:10.1158/1538-7445.AM2017-2640

Introduction: The intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, depends on the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to form ADO). For this reason, a number of anti-CD73 antibodies are being advanced into clinical trials; however, to date there have been few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Ecto-nucleotidase activity was calculated using the Malachite green assay after 50-min incubation with 25µM AMP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; hCD73/CD8 T cells. Stable over-expression: hCD73/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. Human CD8 T cells enriched from buffy coats or leukopaks were pre-treated with varying concentrations of CD73 inhibitors prior to addition of 50 μM AMP + 10 μM EHNA and activated with T cell Activation/Expansion kit (Miltenyi). In some experiments, exogenous recombinant human IL12p70 (1-10 ng/mL) was added to the culture. Activation (CD25) and effector functions (Granzyme B and IFNγ) were measured by flow cytometry. CT26 cells were implanted into the shaved right flank of 7-8 week old Balb/c mice and measured three times a week starting at 7 days. Mice were enrolled into 4 cohorts and dosed according to the following conditions when tumour volume reached ~100 mm3. Group 1: 1% HPMC (sc/QD) + 2A3 (10 mg/kg; IP/Q3D) Group 2: A000830 (30 mg/kg; sc/QD) + 2A3 (10 mg/kg; IP/Q3D) Group 3: 1% HPMC (sc/QD) + RMP1-14 (10 mg/kg; IP/Q3D) Group 4: A000830 (30 mg/kg; sc/QD) + RMP1-14 (10 mg/kg; IP/Q3D) For interim analysis, single cell suspension was generated from tissues, blocked (clone 2.4G2), and stained with antibodies. For intracellular FOXP3 staining, samples were fixed and stained using FOXP3/Transcription Factor Staining Buffer Set. Non-specific blocking was performed with 20% normal rat serum prior to addition of anti-mouse FOXP3 antibody. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 and A001190 with the following IC50 values in overexpression systems: A000830: Mouse IC50 (1 nM); Human IC50 (3 nM) A001190: Mouse IC50 (n.d.); Human IC50 (0.03 nM) A000830 and A001190 also blocked AMP hydrolysis by freshly isolated human CD8 T cells at potencies comparable to the over-expression systems. In in vitro models of AMP/ADO-driven inhibition of human CD8+ T-cell activation, A000830 and A001190 showed robust rescue of CD25 expression and granzyme B production. Complete rescue of IFNγ; production was achieved by adding exogenous IL12. In vivo, A000830 was well-tolerated in mice, resulting in sustained plasma concentrations above IC90. Therapeutic dosing of A000830 to these mice in combination with an α-PD1 antibody resulted in robust CT26 tumor growth inhibition, greater than either treatment alone. Conclusions: Cumulatively, these data provide the initial characterization of a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Joanne BL Tan, Ada Chen, Manmohan Leleti, Annette Becker, Erick Lindsey, Jaroslaw Kalisiak, Jay P. Powers, Steve Young, Ulrike Schindler, Juan C. Jaen. Small-molecule inhibitors of ecto-nucleotidase CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumor models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B46.

Introduction: Intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, requires the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to ADO). For this reason, various anti-CD73 antibodies are being advanced into clinical trials; however, there are few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Enzymatic assays: Ecto-nucleotidase activity was calculated based on the amount of inorganic phosphate (malachite green assay; absorbance at 620 nm) produced after 50-min incubation with 25μM AMP or ATP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; mCD73/E771 cells. Stable over-expression: hCD73/CHO; hCD39/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. CD8+ T cell functional assays: Human (enriched from buffy coats or leukopaks) and mouse (isolated from spleen of C57BL/6 mice) CD8+ T cells were pretreated with CD73 inhibitor or buffer control. One hour later, cells were stimulated (anti-CD3/CD28; +/- 50 μM AMP) in the presence of ADO deaminase inhibitor (10 μM). 3 days after stimulation, cell activation (CD25), proliferation (CFSE) and effector function (IFN-γ and granzyme B) were quantified by flow cytometry. Tumor model: CT26 cells were subcutaneously implanted into Balb/c mice. When tumor volumes were ∼100 mm3, mice were enrolled into cohorts (1: vehicle control; 2: A000830; 3: PD-1 Ab; 4: A000830 + PD-1 Ab) of ≥13 mice each. Interim immune phenotyping was performed when Group 1 tumor volumes were 800-1,000 mm3. Single-cell suspensions were generated from tumors and spleens (gentleMACS). 106 cells were blocked using purified CD16/32 antibodies (clone 2.4G2) and stained with the appropriate antibody cocktails. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 (IC50 against human and mouse CD73 of 1.0 nM and 3 nM, respectively). A000830 blocked ADO generation from AMP by human ovarian cancer cells (SKOV-3) with IC50: 0.2 nM and >10,000-fold selectivity relative to other ecto-nucleotidases (CD39, NTPDase2, NTPDase3, NTPDase8) and a large panel of unrelated enzymes, receptors and ion channels. In in vitro models of ADO-driven inhibition of human and mouse CD8+ T-cell activation, A000830 showed robust rescue of proliferation, CD25 expression, and IFNγ and granzyme B production. A000830 was easily administered (and well tolerated) to mice, resulting in sustained plasma concentrations at least 1,000 times higher than its potency against mouse CD73. In the spleen of CT26 tumor-bearing Balb/c mice, CD39 expression was found mainly on natural killer (NK) cells and myeloid-lineage cells, while in the tumor CD39 was highly expressed on myeloid cells (myeloid-derived suppressor cells (MDSC) and dendritic cells (DC)) as well as CD4+ and CD8+ T cells. CD73 expression was limited (spleen and tumor) to NK cells and CD4+ and CD8+ T cells. Therapeutic dosing of A000830 to these mice (tumors ∼100 mm3 in size) in combination with anti-PD1 antibody produced robust tumor growth inhibition, greater than either treatment alone. This effect was associated with increased CD8:Treg ratios in tumors (but not spleens). Conclusions: We describe a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human and mouse T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Juan C. Jaen, Jay P. Powers, Ada Chen, Manmohan R. Leleti, Jarek Kalisiak, Ulrike Schindler, Joanne Bl Tan, Steve Young, Shin-Heng Chiou, Laurent Debien. Small-molecule inhibitors of CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR10.