Background: Agonists of the Stimulator of Interferon Genes (STING) pathway have great potential in cancer immunotherapy. Activation of STING in tumor cells and/or antige- presenting cells (APCs) can induce type I Interferon production, leading to the induction of innate and adaptive immune response. We recently reported the discovery of the cyclic dinucleotide SB 11285 as a potent, first-in-class, STING agonist. Herein, we report SB 11 and SB 12 as unique linkage analogs of SB 11285.
Methods: (a) Synthesis: Several linkage analogs of SB 11285 were synthesized by standard solution-phase phosphoramidite chemistry and screened for STING agonistic activity using the previously described SZ14 reporter cell lines, and SB 11 and SB 12 were identified as lead compounds., (b) Binding affinity: Differential scanning fluorimetry was used to determine binding affinity of SB 11 and 12 to wtSTING, with 2´,3´-cGAMP being used as a positive control. (c) Induction of IRF3 and NF-KB: wtTHP-1 or R232-THP1 or RAW cells carrying the respective reporter constructs were treated with SB 11 and 12 or controls for 22hrs and induction of IRF3 and NF-KB was determined as % fold-change in luminescence compared to vehicle-treated cells. (d) Induction of cytokines: PBMCs and mBMDCs were treated with SB 11 and 12 at different doses, and cell supernatants were analyzed for IFN-β, TNF-α, and RANTES by ELISA or multiplexing Luminex assays. (e) In vivo studies: Both SB 11 and 12 were tested for antitumor efficacy in CT26 syngeneic mouse tumor model after intravenous (i.v.) administration at 3mg/kg on days 1,5,9,14.
Results: (i) SB 11 and 12 demonstrated high binding affinity to human wild-type and mouse STING that is evident by 16° thermal shift. (ii) SB 11 and 12 showed potent induction of (a) STING-dependent IRF3 (EC50: 0.8 and 39.7 nM) and NF-κB (EC50: 9 and 5265 nM) signaling induction in wtTHP-1 cells; (b) IRF3 (EC50: 2.6 and 317 nM) and NF-κB (EC50: 83 and 3783 nM) induction in R232-THP-1 cells; (c) IRF3 (EC50: 67 and 46 nM) in RAW macrophages. (iii) Both SB 11 and SB 12 induced STING dependent secretion of IFN-β (~10 ng/ml) in mBMDCs. (iv) In the CT26 colon carcinoma syngeneic mouse models, both SB 11 and 12 showed potent antitumor activity when administered by i.v. route with 98 and 97% TGI and 91 and 79% TGD, respectively.
Conclusion: SB 11 and SB 12 showed excellent safety and antitumor activity in syngeneic mouse model when administered by i.v. route. SB 11 and SB 12 were shown to cause STING-dependent activation of IRF3 and NF-κB signaling, as well as the induction of type I IFN signature and ISGs. Further preclinical studies of systemically administered analogs SB 11 and SB 12 are in progress.
Citation Format: Shenghua Zhou, Sreerupa Challa, Diane Shmidt, Leena Suppiah, Vishal Nair, Anjaneyulu Sheri, Geeta Meher, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Radhakrishnan Iyer. Development of SB 11 and SB 12, structurally unique linkage analogs of SB 11285 as STING agonists for Immuno-oncology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B53.