The cGAS/STING pathway initiates an innate immune response when DNA is detected in the cytosol. DNA bound cGAS synthesizes cyclic dinucleotides which bind and activate the adaptor STING, leading to downstream secretion of Type I interferons and other pro-inflammatory NFκB pathway cytokines. In the mouse, the STING driven innate immune response is central to immune based clearance of various tumors and this has triggered a significant effort focused on the discovery of human STING agonists for the treatment of cancer. This report uses an in vitro kinase assay to show that G10, a previously identified STING pathway activator is actually a weak but direct STING agonist and identifies other more potent leads.
IDO1/TDO* mediate substantial immunosuppressive effects through the metabolism of tryptophan (Trp) to kynurenine (Kyn). The consequent decrease in Trp suppresses T cell activity by multiple mechanisms, including the activation of GCN2 and mTOR pathways. Additionally, increased levels of Kyn further enhance the effect of Trp metabolism by engagement of aryl hydrocarbon receptor and potentially enhancing the number and activity of regulatory T cells. Taken together, expression of IDO1 and TDO in the tumor micro-environment dampens tumor-specific effector T cell response, and elevated expression of IDO1/TDO correlates with reduced survival of cancer patients. IDO1 selective inhibitors have already demonstrated clinical anti-tumor activity for certain tumor types. Therefore, targeting the Trp/Kyn pathway via simultaneous inhibition of IDO1 and TDO enzymes has the potential to bring enhanced benefit to cancer patients by relieving immunosuppression in a wide variety of tumor types. We have discovered a novel, highly potent, small molecule IDO1/TDO dual inhibitor, RG70099, with favorable preclinical oral bioavailability and safety profile. RG70099 potently inhibits both enzymes in cell based assays (IDO1 IC50: <100nM while TDO IC50: <100nM) and in preclinical in vivo model systems, a single oral administration of RG70099 efficiently prevented the formation of Kyn by ∼90% at plasma level. Furthermore, RG70099 efficiently penetrates into IDO1+ tumors and tumor draining lymph nodes where it reduced Kyn levels by more than 95%. We evaluated the inhibitory activity of the molecule in the TDO+ U87MG mouse tumor model. Twice-a-day administration of RG70099 reduced Kyn concentration in TDO+ tumors by ∼90% while pure IDO1 inhibitors failed to modulate Kyn levels in this setting. Studies of IDO1/TDO dual inhibition in pre-clinical immunocompetent animal models, and whether TDO inhibition in tumors that express IDO1 and TDO will provide additional benefits have been initiated. Prevalence analysis performed by IHC on several conditions indicates that both proteins are highly expressed either on tumor cells or immune cells with important differences among tumor types suggesting the potential for improved efficacy and differentiation of dual IDO1/TDO inhibitors. Our data show for the first time that a dual inhibition of IDO1 and TDO significantly reduces Kyn levels in preclinical tumor models. RG70099 is a potent, dual-selective IDO1 and TDO small molecule inhibitor with favorable pharmaceutical and pharmacokinetic properties that has the potential to relieve immunosuppression by both IDO1 and TDO and activate anti-tumor immune responses for a broad range of cancer types. *IDO1: Indoleamine 2,3-Dioxygenase 1; TDO: Tryptophan 2,3-Dioxygenase Citation Format: Gabor Gyulveszi, Christine Fischer, Massimiliano Mirolo, Martin Stern, Luke Green, Maurizio Ceppi, Haiyan Wang, Beatrice Bürgi, Andreas Staempfli, Wolfgang Muster, Robert van Waterschoot, Andreas Gloge, Hadassah Sade, Irina Klaman, Gabriele Hoelzlvimmer, Arjun Surya, Monali Banerjee, Ritesh Shrivastava, Sandip Middya, Dharmendra Yadav, Sourav Basu, Gonzalo Acuna. RG70099: A novel, highly potent dual IDO1/TDO inhibitor to reverse metabolic suppression of immune cells in the tumor micro-environment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-085.
BACKGROUND Innate immune signaling mediated through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into cold or non-inflamed tumors leading to significant regression (Woo et al., 2015). Comprehensive data from one of our STING agonists was presented at the AACR Tumor Immunology meeting in 2017. Here we describe CRD5500, a versatile next generation compound that can be dosed either by intra-tumoral route, systemically (IV or SC) or as an antibody drug conjugated to Trastuzumab (ADC). The ADC approach allows for tumor targeting through systemic delivery with low systemic exposure of the payload and should result in a higher Therapeutic Index (TI) in the clinic. METHODS We have described the discovery of small molecule activators of human STING that generate potent anti-tumor innate and adaptive responses in recently published patents. CRD 5500 was evaluated in (1) in reporter gene assays on HEK293T cells containing stably transfected hSTING polymorphs, (2) by immunoblots to confirm pSTING, pTBK1, pIRF3, (3) by real-time PCR and ELISA monitoring induction of Type I cytokines in human tumor cell lines, isolated human PBMCs and dendritic cells (hMo-DC). Direct STING binding was demonstrated by (1) Cellular Thermal Shift Assays (CETSA) and (2) in vitro kinase assay. Anti-tumor activity of CRD5500 was demonstrated by multiple routes in syngenic tumors containing human STING. Libraries of linkers were used to permit conjugation of CRD5500 with Trastuzumab. Prior to conjugation, penultimate constructs (Pre-Ab) were screened for their ability to preferentially release CRD5500 in tumor homogenate with reduced plasma exposure. Antibody drug conjugates were synthesized at Abzena, UK and were tested in HER2 expressing cells to assess release of free payload . RESULTS Treatment of an isolated cell free system containing purified recombinant STING and TBK1 leads to STING phosphorylation confirming that CRD-5500 is a direct STING binder. Evidence of direct binding also comes from CETSA. CRD-5500 activates all five common hSTING variants with agonist EC50 comparable to CDNs in the pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumor cell lines or hPBMC with CRD5500 leads to phosphorylation of IRF3, TBK1 and STING. CRD-5500 causes the maturation of hDCs and the release of innate and adaptive inflammatory cytokines such as IFNβ and TNFα from hDCs and hPBMCs. In vivo administration of CRD5500 IT or systemically caused tumor regression in CT26 syngeneic tumors containing human STING. CRD5500 demonstrated the abscopal effect and when combined with check point blockade dramatically reduced tumor mass. Interestingly, when Pre-Ab constructs that were not conjugated to antibody were delivered IV, they combined with an anti-CTLA4 antibody to eliminate tumors in the hSTING.CT26 syngenic tumor model. Conjugation with Trastuzumab was demonstrated and the ADC thus formed was screened in HER2 expressing cell lines. CONCLUSIONS The ability of hSTING agonists to generate Type 1 IFN anti-tumor immune responses makes them a promising therapeutic option either as a single agent or in combination with other therapies. CRD5500 is a small molecule STING agonist that can be delivered in multiple formats making it well suited for further development as an anti-cancer agent. Citation Format: Monali Banerjee, Sourav Basu, Sandip Middya, Ritesh Shrivastava, Rajib Ghosh, David C. Pryde, Dharmendra Yadav, Gopal Bhattacharya, Thanilsana Soram, Kavita Puniya, Ritika Raina, Vijay Kadam, Sujay Garai, Priti Sharma, Anuj Singh, Vaibhav Shinde, Nidhi Rawat, Anindita Middya, Arghyotri Sinha, Sunita Chandel, Ganesh Narisipuram, Abhisek Chatterjee, Nagaswamy Mane, Arjun Surya. CRD5500: A versatile small molecule STING agonist amenable to bioconjugation as an ADC [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 LB-061.
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