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Supplementary Data from Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

Supplementary Figure from Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell–mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti–PD-1, to improve cancer patient outcomes.

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell–mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti–PD-1, to improve cancer patient outcomes.

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.

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.

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.

Cells in the solid tumor microenvironment are frequently exposed to hypoxic conditions, necessitating molecular adaptations for survival. Of particular importance are transcriptional changes mediated by heterodimeric Hypoxia-Inducible Factor (HIF) proteins that consist of an oxygen-regulated α monomer (either HIF-1α, -2α, and -3α) coupled to a constitutively expressed β monomer (HIF-1β/ARNT). In normal oxygen conditions, HIF-2α is degraded following ubiquitination by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex. Exposure to hypoxia, VHL mutation, or epigenetic silencing of pVHL leads to HIF-2α stabilization and transcription of pro-tumorigenic gene sets in both cancer and non-cancer cells. Inhibition of HIF-2α has been shown clinically to be an effective strategy to mitigate tumor growth, particularly in patients suffering from VHL disease or clear cell renal cell carcinoma (ccRCC), a cancer that has a particularly high prevalence of pVHL dysfunction. Applying a pharmacophore mapping and structure-based design approach, we identified a novel and potent small molecule HIF-2α inhibitor, AB521. AB521 avidly binds the HIF-2α PAS-B domain, preventing HIF-2α-mediated gene transcription. AB521 is characterized by a favorable preclinical pharmacokinetic profile and is projected to be suitable for once-daily dosing in humans. When delivered orally in mice, AB521 significantly regressed established 786-O xenograft tumors and decreased pharmacodynamic markers associated with HIF-2α in a dose-dependent manner. In vitro, AB521 potently inhibited HIF-2α-specific luciferase reporter transcription under high-serum conditions, VEGF protein secretion, colony formation in soft agar, and did not exhibit off-target cytotoxicity in 786-O cells. AB521 selectively inhibited HIF-2α-, but not HIF-1α-, mediated gene expression in hypoxic Hep3B hepatocellular carcinoma cells. AB521 also inhibited the transcriptional activity of endogenous HIF-2α in relevant human primary cell types, including endothelial cells and pro-tumorigenic M2-polarized macrophages. Importantly, inhibiting HIF-2α did not impact functionality of activated hypoxic human T cells, suggesting that AB521 would be favorable combination partner for I-O therapeutic agents. Indeed, expression of CD73, the primary enzyme responsible for synthesis of the immunosuppressive metabolite adenosine, was highly correlated with hypoxic signatures across several indications in publicly available bioinformatic datasets, suggesting combinations with adenosine pathway antagonists in ccRCC and beyond. In summary, AB521 is a novel and selective HIF-2α inhibitor with potent anti-tumor activity. Clinical evaluation of this molecule is expected to begin in the latter part of 2021. Citation Format: Kelsey E. Sivick Gauthier, Dana Piovesan, Soonweng Cho, Kenneth V. Lawson, Patrick G. Schweickert, Alejandra Lopez, Suan Liu, Timothy Park, Artur Mailyan, Jeremy T. A. Fournier, Joel W. Beatty, Samuel L. Drew, Jarek Kalisiak, Balint Gal, Guillaume Mata, Zhang Wang, Brandon R. Rosen, Clayton Hardman, Matthaw P. Epplin, Kai Yu, Karl T. Haelsig, Lixia Jin, Elaine Ginn, Jennie Au, Cesar A. Meleza, Joel Tencer, Amber Pham, Hyock J. Kwon, Stephen W. Young, Manmohan Leleti, Jay P. Powers, Matthew J. Walters. AB521 potently and selectively inhibits pro-tumorigenic gene transcription by Hypoxia-Inducible Factor (HIF)-2α in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P206.

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.

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

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

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.

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.