Bone Therapeutics

Abstract The profound benefit of immune checkpoint blockade for cancer therapy is restricted to limited subsets of patients with specific cancers. Many factors contribute to primary and acquired resistance to immune checkpoint blockade, including local accumulation of immunosuppressive metabolites such as the nucleoside adenosine and downstream adenosine receptor signaling. Pharmacological inhibition of adenosine generation and signaling are active areas of clinical investigation. Here we report a novel mechanism whereby adenosine suppresses anti-cancer immune responses by intracellular accumulation. We show that human T cells readily take up adenosine via equilibrative nucleoside transporter 1 (ENT1), suppressing proliferation and effector function by inhibition of de novo pyrimidine nucleotide synthesis in T cells. ENT1 expression increases upon T cell activation, suggesting uptake of extracellular adenosine is a mechanism to limit T cell responses in high adenosine environments. Quantitative mass spectrometry imaging reveals adenosine concentrations in human tumors up to the range of 100 µM - levels significantly greater than previously demonstrated and consistent with suppression of T cell responses. Deletion of ENT1 leads to potent control of tumor growth in syngeneic mouse models including KPC, a poorly immunogenic model of pancreatic ductal adenocarcinoma, and is associated with increased CD8+ T cell frequency, proliferation and cytokine production within tumors. Furthermore, ENT1 expression was observed by flow cytometry on human tumor-infiltrating CD8+ T cells. We have discovered and characterized EOS301984, a potent ENT1 antagonist that is currently being evaluated in patients with advanced solid tumors. EOS301984 blocks intracellular adenosine transport and restores pyrimidine levels in T cells activated in the presence of adenosine, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes in adenosine-rich environments. Finally, in a humanized mouse model of triple negative breast cancer resistant to anti-PD-1 blockade (MDA-MB-231), combination of EOS301984 with nivolumab synergistically led to control of tumor growth. Thus, ENT1 inhibition represents a novel approach to augment anti-cancer immune responses through restoring pyrimidine nucleotide synthesis in T cells suppressed by adenosine. Citation Format: Theodore J. Sanders, Christopher S. Nabel, Margreet Brouwer, Annelise L. Hermant, Lucas Chaible, Jean-Philippe Deglasse, Angelique Pabois, Wilfried Cathou, Aurore Smets, Michael Deligny, Joao Marchante, Quentin Dubray, Marie-Claire Lettelier, Chiara Martinoli, Reece Marillier, Olivier De Henau, Yvonne McGrath, Matthew G. Vander Heiden, Erica Houthuys. Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 734.

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3233.

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Background and study aims Endoscopic full-thickness resection (EFTR) is used to achieve R0 resection in difficult situations and as a way to overcome the limitations of endoscopic submucosal dissection. Multiple techniques have been described but adequate tools are still under evaluation. In this study, we evaluated the safety and feasibility of non-exposed endoscopic full-thickness resection using a novel endoscopic suturing device.Materials and methods Full-thickness resections of gastric predetermined lesions were performed on five pigs using the Endomina platform. After creating virtual lesion > 20 mm, sutures were placed around it using this triangulation platform. After tightening the knots, the bulging lesion, internalized into the gastric lumen, was cut with a needle knife.Results R0 resections of large lesions (42 to 60 mm) were achieved in all cases. One perforation occurred and prompted us to improve the procedure by shortening the sutures for more maneuverability and reinforcing the suture line before section. Procedure duration dropped by 50 % between the first case and the fourth case. Histological analysis confirmed successful full-thickness resection of all resected specimens.Conclusion EFTR using this triangulation platform seems feasible for lesions > 20 mm. Additional possible improvements were identified to simplify the procedure before moving to human trials.

Abstract Extracellular adenosine is an immunosuppressive metabolite generated by sequential action of the two ectonucleotidases CD39 and CD73. Tissue hypoxia, which is frequently observed in neopleastic tissues, increases CD39 and CD73 expression, and is a major driver of extracellular adenosine accumulation. High levels of adenosine, predominantly signaling through the A2A receptor (A2AR), dampen innate and adaptive immune cell responses, leading to suppression of antitumor immunity. We therefore developed EOS100850, a potent and highly selective A2A receptor antagonist to treat a wide range of tumor types. With the aim to support selection of cancer indications for EOS100850, we used immunohistochemistry and computer-assisted image analysis to extensively characterize the expression and spatial distribution of markers of the adenosine pathway and the extent of lymphocyte infiltration in ten tumor types. First, 318 primary tumor specimens included in disease-specific tissue microarrays from nine cancer indications were screened for expression of CD39, CD73 and infiltration of A2AR+, CD8+ and Foxp3+ cells. CD39 and CD73 were expressed by immune cells, endothelial cells and other stromal cells as well as by tumor cells, and their expression was increased in hypoxic tumor regions. Expression levels of the two adenosine-generating enzymes were variable among the analyzed cancer types, with lung and gastric tumors displaying the highest levels of expression of CD39 (78% and 95% of samples with high CD39 expression, respectively) and gastric, uterine and lung cancers displaying the highest levels of CD73 (>70% of samples with high CD73 expression). A2AR staining was observed on different immune cell populations, and significantly correlated with CD8+ and Foxp3+ cell densities (p values <0.0001). A2AR+ immune cells were frequently detected in lung, gastric and head and neck tumors (>80% positive samples) but not in kidney and prostate tumors (<20% positive samples). To encompass the inherent heterogeneity of tumor tissues, the expression and spatial distribution of CD39, CD73, A2AR and lymphocyte populations were further analyzed in several lung, colorectal and breast primary tumor resections. These tumor types display high, medium and low levels of the adenosine cloud markers, respectively. CD39 expression as well as densities of A2AR+, CD8+ and Foxp3+ cells were all higher in the tumor surrounding stroma than in the center of the tumor in the three cancer types. Moreover, CD39 and A2AR were significantly correlated with CD8+ and Foxp3+ cell densities in the tumor center but not in the stroma. Altogether these data strongly support the relevance of targeting A2AR and the adenosine pathway in immuno-oncology, and pave the way to the identification of the cancer types that will benefit more from EOS100850. Citation Format: Noemie Wald, Chiara Martinoli, Marjorie Mercier, Annelise Hermant, Florence Nyawouame, Joao Marchante, Charlotte Moulin, Vanesa Bol, Stefano Crosignani, Veronique Bodo. Extensive characterization of the adenosine pathway in human solid tumors gives a hint on cancer indication selection for the A2A receptor antagonist EOS100850 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4149.

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant, orally administered agent to treat a wide range of tumor types. To monitor A2A receptor engagement by EOS100850 in clinical studies, we developed two pharmacodynamic assays that allow to quantify the inhibition of adenosine signaling in cancer patients dosed with this compound. The assays were developed and optimized to: 1) minimize the amount of patient blood required for each test; 2) minimize sample manipulation; 3) allow marker quantification in cryopreserved samples. The first assay is based on the quantification of phosphorylated cAMP response-element binding protein (pCREB) as a proximal readout of A2A receptor signaling. Whole blood is ex vivo stimulated with the A2A receptor agonist CGS-21608, or with dimethyl sulfoxide or phorbol-12-myristate-13 acetate and ionomycin as controls, and pCREB in CD4+ and CD8+ cells is analyzed by flow cytometry. After having confirmed that pCREB was rapidly induced after incubation with CGS-21680 (median fold-induction in 24 healthy donors was 5.4 and 4.0 in CD4+ and CD8+ cells, respectively), we showed that EOS100850 inhibited CGS-21608-induced pCREB in a dose-dependent manner, with an IC50 of 9.7 nM and 11.2 nM, respectively, for CD4+ and CD8+ T cells, and 83-87% signal inhibition in the presence of 40 nM EOS100850 (data from 10 donors). The second assay is based on the quantification of soluble mediators as a distal readout of A2A receptor signaling. Stimulation of whole blood with lipopolysaccharide (LPS) normally induces the release of innate cytokines and chemokines, but in the presence of the A2A receptor agonist CGS-21680, LPS activity is impaired. In this assay, whole blood is ex vivo stimulated with LPS in the presence or in the absence of CGS-21608 using TruCulture tubes (Myriad RBM), and secreted mediators are quantified using the human multianalyte profile immunoassay platform. After having confirmed that CGS-21680 induced a profound (>50%) alteration of cytokine secretion in LPS-stimulated blood, we showed that EOS100850 restored IFN-γ, IL-8, IL-10 and TNF-α secretion in a dose-dependent manner, with a complete rescue at 40-100 nM of compound (data from 3 donors). Both the assays were qualified in terms of precision, robustness and stability using blood of healthy volunteers, applied to blood of five cancer donors and are now ready to be used for the clinics. Citation Format: Chiara Martinoli, Margreet Brouwer, Erica Houthuys, Marjorie Mercier, Noemie Wald, Bruno Gomes, Stefano Crosignani, Veronique Bodo. EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4154.

Abstract Adenosine is a potent immunosuppressive metabolite that is often found elevated in the extracellular tumor microenvironment (TME). We determined concentrations of extracellular adenosine in different patient-derived xenografts (PDX) from 7 different histological types, in which extracellular median adenosine concentrations were shown to range from 0.5 to 45 µM, with the overall median adenosine concentration being about 4.5 µM. Adenosine concentration in the non-tumorous subcutaneous space in mice was measured at about 0.5 µM. Adenosine in the TME is generated mainly by the concerted action of the ectonucleotidases CD39 and CD73. The expression of these enzymes across various cancer types was evaluated by flow cytometry in dissociated human tumor biopsies. CD73 and CD39 were strongly expressed by multiple tumor-infiltrating T cell types. Tumor-associated myeloid cells mostly expressed CD39 and high frequencies of EpCAM+ tumor cells strongly expressed CD73. These data strongly suggest that adenosine levels could be further increased compared to non-immune-infiltrated PDX.Adenosine activates 4 G protein-coupled receptor subtypes, of which the adenosine A2A receptor in particular suppresses innate and adaptive immune cell responses leading to suppression of anti-tumor immunity. Among the 4 adenosine receptors, we confirmed the A2A receptor as the main adenosine receptor expressed in CD4+ and CD8+ T cells, natural killer cells, monocytes, and dendritic cells. Stimulation of these immune cell subsets further increased A2A receptor expression. A2B receptor was expressed at very low levels in stimulated CD4+ and CD8+ T cells and in monocytes and immature DCs. A1 and A3 receptors were hardly detected in these subsets of immune cells. EOS100850, a highly potent and selective A2A receptor antagonist, was characterized in various in vitro functional assays. A2A receptor activation by a selective agonist suppressed priming of mouse OVA-specific OT1 T cells and subsequent antigen-specific CD8+ T cell cytotoxicity in a co-culture assay of effector CD8+ T cells and target cancer cells. This A2A receptor-mediated immune suppression was potently and dose-dependently reversed by EOS100850. In a mixed lymphocyte reaction between human dendritic cells and T cells, EOS100850 blocked the adenosine-dependent inhibition of T cell proliferation and secretion of IFNg, TNFa and IL-2 in a dose-dependent manner. In conclusion, extracellular adenosine as well as adenosine pathway components were strongly present across multiple tumor types, and across multiple tumor-associated cell types. In addition, EOS100850, a highly potent A2A receptor antagonist, reversed A2A receptor-mediated suppression of T cell priming, cytotoxicity, cytokine production and proliferation. Citation Format: Erica Houthuys, Paola Basilico, Veronique Bodo, Margreet Brouwer, Michel Detheux, Gregory Driessens, Bruno Gomes, Annelise Hermant, Catherine Hoofd, Florence Lambolez, Xavier Leroy, Reece Marillier, Chiara Martinoli, Marjorie Mercier, Florence Nyawouame, Shruthi Prasad, Ariane Scoumanne, Stefano Crosignani. EOS100850 potently restores adenosine A2Areceptor-dependent suppression of T cell function in the adenosine rich tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3278.

High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant, orally administered agent to treat a wide range of tumor types. To monitor A2A receptor engagement by EOS100850 in clinical studies, we developed two pharmacodynamic assays that allow to quantify the inhibition of adenosine signaling in cancer patients dosed with this compound. The assays were developed and optimized to: 1) minimize the amount of patient blood required for each test; 2) minimize sample manipulation; 3) allow marker quantification in cryopreserved samples. The first assay is based on the quantification of phosphorylated cAMP response-element binding protein (pCREB) as a proximal readout of A2A receptor signaling. Whole blood is ex vivo stimulated with the A2A receptor agonist CGS-21608, or with dimethyl sulfoxide or phorbol-12-myristate-13 acetate and ionomycin as controls, and pCREB in CD4+ and CD8+ cells is analyzed by flow cytometry. After having confirmed that pCREB was rapidly induced after incubation with CGS-21680 (median fold-induction in 24 healthy donors was 5.4 and 4.0 in CD4+ and CD8+ cells, respectively), we showed that EOS100850 inhibited CGS-21608-induced pCREB in a dose-dependent manner, with an IC50 of 9.7 nM and 11.2 nM, respectively, for CD4+ and CD8+ T cells, and 83-87% signal inhibition in the presence of 40 nM EOS100850 (data from 10 donors). The second assay is based on the quantification of soluble mediators as a distal readout of A2A receptor signaling. Stimulation of whole blood with lipopolysaccharide (LPS) normally induces the release of innate cytokines and chemokines, but in the presence of the A2A receptor agonist CGS-21680, LPS activity is impaired. In this assay, whole blood is ex vivo stimulated with LPS in the presence or in the absence of CGS-21608 using TruCulture tubes (Myriad RBM), and secreted mediators are quantified using the human multianalyte profile immunoassay platform. After having confirmed that CGS-21680 induced a profound (>50%) alteration of cytokine secretion in LPS-stimulated blood, we showed that EOS100850 restored IFN-γ, IL-8, IL-10 and TNF-α secretion in a dose-dependent manner, with a complete rescue at 40-100 nM of compound (data from 3 donors). Both the assays were qualified in terms of precision, robustness and stability using blood of healthy volunteers, applied to blood of five cancer donors and are now ready to be used for the clinics. Citation Format: Chiara Martinoli, Margreet Brouwer, Erica Houthuys, Marjorie Mercier, Noemie Wald, Bruno Gomes, Stefano Crosignani, Veronique Bodo. EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4154.

Abstract T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory receptor expressed by lymphocytes, preferentially CD8+ T cells, NK, as well as by regulatory T cells (Treg). CD226 (DNAM-1), a co-stimulatory receptor also expressed on NK and T cells competes with TIGIT for PVR binding but with a lower affinity. Co-expression of TIGIT and CD226 receptors on T and NK effector cells suggests a role of these molecules in the fine control of cell activation. In cancer, TIGIT expression is upregulated on conventional and even more on regulatory T cells and co-expression with exhaustion markers such as PD-1 is frequent, giving a strong rationale for blocking TIGIT as a therapeutic approach to reverse T or NK cell dysfunction linked with cancer progression. Antagonistic anti-TIGIT antibodies were selected using a synthetic yeast display library of fully human antibodies and characterized for their binding and functional properties. EOS884448 anti-TIGIT mAb displays a strong affinity for recombinant and native TIGIT expressed on human primary T cells. It competes with CD155 for binding to human TIGIT and restores cytokine production when human primary T cells are suppressed in presence of CD155. To further explore its potency, EOS884448 was produced in a mammalian system on different human isotypes with different Fc effector functions. In the hIgG1 format, EOS884448 demonstrated preferential depletion of Treg cells in vitro when spiked in PBMC from healthy volunteers. Interestingly, the preferential depletion of Treg was also observed in PBMCs from cancer patients that harbour higher level of expression of TIGIT on Tregs but also on conventional CD4+ and CD8+ T cell populations. In vivo, two different isotypes of surrogate mouse anti-TIGIT mAb were used to evaluate the anti-tumor efficacy in the CT26 syngeneic murine model. Interestingly, only the ADCC enabling mIgG2a isotype was able to induce strong antitumor efficacy in monotherapy and in combination with an anti-PD1, resulting in a complete regression of pre-established tumors in the majority of animals. Anti-tumor efficacy was associated with an increased activity of conventional CD8+ and CD4+ T cells but also with Treg depletion within the tumor microenvironment, reaffirming the in vitro data generated on human PBMCs. The importance of depleting Treg using an a-TIGIT Ab isotype with effector functions was further confirmed in CT26 tumor bearing mice where the a-TIGIT mIgG1 isotype only shows efficacy when Treg are previously depleted using an anti-CD4 antibody. In summary, in vitro and in vivo data demonstrate for the first time the potential for a-TIGIT mAb EOS884448 to promote antitumor immunity by preferential depletion of Treg cells and activation of conventional T cells, which supports the rationale for its clinical evaluation. Citation Format: XAVIER LEROY, Catherine Hoofd, Julia Cuende, Sofie Denies, Virginie Rabolli, Julie Preillon, Florence Lambolez, Shruthi Prasad, Marjorie Mercier, Florence Nyawouame, Véronique Bodo, Noémie Wald, Grégory Driessens, Michel Detheux. a-TIGIT antagonist antibody EOS884448 shows dual mechanism of action by restoration of T cell effector functions and preferential depletion of Treg [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 LB-114.