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Re-constitution of anti-tumor T-cell responses by clinically-approved immune checkpoint inhibitors (ICIs) targeting CTLA4 or PD-1/PD-L1 represents a breakthrough cancer therapy. Nevertheless, a substantial number of patients do not benefit from these new therapeutic modalities chiefly due to local immunosuppression in the tumor microenvironment. In addition, the long circulation time of ICIs restricts options to modify dosing regimens for management of adverse effects. Oral small molecule inhibitors as next generation immune-oncology agents may - in contrast to antibodies - allow targeting of intracellular targets for a defined duration of time. This will permit a fine-tuning of efficacy versus tolerability in single-agent treatment as well as in combination with approved ICIs. The overexpression of indole dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) by many tumors results in increased metabolism of tryptophan (TRP) into kynurenine (KYN), which induces immunosuppression via activation of the aryl hydrocarbon receptor (AhR). Inhibition of AhR was proposed to restore T-cell function and induce tumor rejection. However, it was expected that identification of selective lead candidates for AhR inhibition would be challenging due to the known affinity of the AhR ligand binding site for polyaromatic ligands such as 2,3,7,8-tetrachlorodibenzodioxine (TCDD). A library of 4 million compounds was screened in a cell-based HTS campaign. A thorough hit reduction process was performed based on stringent filter parameters for lead-likeness. This process delivered a hit set of significant chemical diversity. Out of several compound classes with drug-like properties, 1,3-diaryl-pyrazin-6-one-5-carboxylic amides were selected as a preferred lead series. A comprehensive SAR exploration, including in vitro mechanistic and functional validation, was performed. Lead optimization was strongly emphasized on improving lipophilicity efficiency (LLE) to balance potency with a viable PK and CYP450 interaction profile. Several candidates suitable for in vivo profiling were identified and BAY-218 was advanced to in-depth pharmacodynamic and pharmacokinetic in vivo assessments. BAY-218 showed mono-therapeutic efficacy that was comparable to ICI treatment and further therapeutic improvement was achieved by combination with an anti-PD-L1 antibody. We were able to characterize 1,3-diaryl-pyrazin-6-one-5-carboxylic amides as a new and unprecedented class of AhR inhibitors as well as identify the key substitutions that contribute to the overall compound profile. Citation Format: Norbert Schmees, Ilona Gutcher, Ulrike Roehn, Horst Irlbacher, Detlef Stoeckigt, Benjamin Bader, Christina Kober, Lars Roese, Rafael Carretero, Iris Oezen, Ludiwg Zorn, Michael Platten, Ingo Volker Hartung, Bertolt Kreft, Hilmar Weinmann. Identification of BAY-218, a potent and selective small-molecule AhR inhibitor, as a new modality to counteract tumor immunosuppression [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 4454.

Tumor cells co-opt multiple pathways in order to evade attack by infiltrating immune cells. One such mechanism is the upregulation of indole-2,3-dioxygenase (IDO1) and/or tryptophan-2,3-dioxygenase (TDO2), both of which are first-step, rate-limiting enzymes degrading tryptophan to the immunosuppressive metabolites kynurenine (KYN) and kynurenic acid (KA). KYN and KA bind and activate the aryl hydrocarbon receptor (AhR), which is expressed in many cell types and is well known for its immunosuppressive effects. Targeting of the AhR with an inhibitor may therefore provide a novel immunotherapeutic approach for enhancing anti-tumoral immune responses and treating cancer. Here we describe the identification and functional immune characterization of BAY-218, a novel, selective and potent AhR small molecule inhibitor. Mechanistically, BAY-218 inhibited AhR nuclear translocation, dioxin response element (DRE)-luciferase reporter expression and AhR-regulated target gene expression induced by both exogenous and endogenous AhR ligands. In vitro, BAY-218 rescued TNFα production from KA-suppressed LPS-treated primary human monocytes. Furthermore, BAY-218 enhanced T cell cytokine production in a human mixed lymphocyte reaction (MLR) and a mouse antigen-specific bone-marrow-derived dendritic cell (BMDC)-OT-I T cell co-culture. In the MLR, BAY-218 increased anti-PD1 antibody-mediated IL-2 and IFNγ secretion, while an IDO inhibitor did not, indicating that BAY-218 is able to block AhR activation mediated by ligands outside of the IDO-KYN pathway. In vivo, BAY-218 enhanced anti-tumoral immune responses and reduced tumor growth in the syngeneic mouse tumor models CT26 and B16-OVA. FACS analysis of leukocytes infiltrating B16-OVA tumors demonstrated that administration of BAY-218 increased the frequency of tumor-infiltrating CD8+ T cells and NK cells while decreasing GR1+ myeloid cells and CD206+M2 macrophages. Furthermore, BAY-218 enhanced therapeutic efficacy of an anti-PD-L1 antibody in the CT26 model. In summary, AhR inhibition with BAY-218 stimulates pro-inflammatory monocyte and T cell responses in vitro and drives anti-tumor immune responses, resulting in decreased tumor growth, in vivo. Thus, inhibiting AhR represents a novel immunotherapeutic approach for blocking AhR-mediated tumor-associated immunosuppression. Citation Format: Ilona Gutcher, Christina Kober, Lars Roese, Julian Roewe, Norbert Schmees, Florian Prinz, Matyas Gorjanacz, Ulrike Roehn, Benjamin Bader, Horst Irlbacher, Detlef Stoeckigt, Rafael Carretero, Katharina Sahm, Iris Oezen, Hilmar Weinmann, Ingo V. Hartung, Bertolt Kreft, Michael Platten. Blocking tumor-associated immune suppression with BAY-218, a novel, selective aryl hydrocarbon receptor (AhR) inhibitor [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 1288.

The second messenger diacyl glycerol (DAG) plays a key role in T cell receptor downstream signaling and thereby in T-cell activation. Diacylglycerol kinase zeta (DGK-ζ) is a lipid kinase that can down-modulate T-cell activation by catalyzing the conversion of DAG to phosphatidic acid, thereby acting as a ligand independent, intracellular immune checkpoint. Inhibition of DGK- ζ offers the potential to enhance T cell priming against suboptimal tumor antigens and to overcome multiple immune-suppressive mechanisms in the tumor microenvironment. Bayer AG in collaboration with the German Cancer Research Center (DKFZ) have developed BAY 2965501, a highly selective, orally available DGK-ζ inhibitor which shows a unique binding mode and good DMPK profile. The compound enhances the reactivity of human and mouse T-cells, both in the priming and the effector phase, and overcomes the inhibitory impact of suppressive factors, such as adenosine and prostaglandin E2. Oral dosing of BAY 2965501 shows T cell dependent efficacy in syngeneic, murine tumor models. Furthermore, this results in reactivation of exhausted T cells in vivo. Preclinical safety studies showed only mild findings, so that good clinical tolerability is expected. Collectively, the preclinical data support first in human (FiH) phase I testing of therapeutic potential. A FiH trial with BAY 2965501 in patients with advanced solid tumors including NSCLC, gastric/GEJ AdCa, ccRCC, and melanoma patients, is currently enrolling patients (NCT05614102). This study will evaluate the safety, tolerability, maximum tolerated or administered dose, pharmacokinetics, pharmacodynamics, and tumor response profile of BAY 2965501. Citation Format: Rienk Offringa, Catherine Olesch, Frederik Cichon, Mareike Grees, Norbert Schmees, Ulrike Roehn, Florian Prinz, Judith Guenther, Detlef Stoeckigt, Michael Erkelenz, Thi Thanh Uyen Nguyen, Ulf Boemer, Nicolas Werbeck, Marc Kunze, Kirstin Petersen, Dennis Kirchhoff. BAY 2965501: A highly selective DGK- ζ inhibitor for cancer immuno-therapy with first-in-class potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND04.