Abstract 2094: Conjugates of TLR9 and STING agonists achieved profound synergistic effects in vitro and in vivo
Authors: Song Yuntao , Li Anrong , Li Hui , Li Xianfeng , Huang Zeao , Yang Junbao , Ding Yi , Szu Grace 
Published: June 2022

Abstract
Introduction: Both Toll-like receptor 9 (TLR9) and STING pathways are two important pathways involved in immune activation. We reasoned that concurrent activation of TLR9 and STING pathways could activate immunity with an efficacy and safety profile that could not be achieved by activating either pathway alone, and we designed a series of conjugates where TLR9 and STING agonists were linked together by a releasable or stable linker. The conjugates have been evaluated in in vitro assays and in vivo mouse models in comparison with TLR9 agonist or STING agonist alone and combination of the two agonists. Methods: Cellular potency was assessed using THP-1 monocyte cells and human PBMC. In vivo efficacy was evaluated in mouse tumor models, where tumor cells were inoculated into either the right flank or both flanks of C57BL/6 or BALB/c mice, test compounds were injected intratumorally into the right flank three times at every three days when tumors reached a volume of 90-100 mm3, muDX400 was dosed via IP injection six times at every four days. In MC38 murine model, compounds-treated mice free from tumor for 30 days were re-challenged with MC38 tumor cells. Results: Conjugates CS-2554, 2571 and 2600 showed five to ten folds improvement in potency over the corresponding STING agonist alone or a mixture of the corresponding STING and TLR9 agonists (1/1molar ratio) in THP1 assay, a similar fold of increase in EC50 was observed in human PBMC assay, where induction of IFNα, IFNβ, TNFα and IL6 were measured. In MC38 murine model, CS-2554 at the dose of 9 ug/injection resulted in CR in all treated mice; whereas no CR was obtained when a mixture of an equivalent amount of the corresponding STING agonist and TLR9 agonist was used. The tumor-free mice remained tumor-free after re-challenge study. In MC38 dual tumor model, when combined with aPD-1, a mixture of 50 ug of TLR9 agonist and 5 ug of STING agonist resulted in CR in 38% treated mice, whereas the same level was achieved with only 4 ug of the corresponding conjugate CS-2571. Comparisons of a conjugate and either the corresponding agonist alone or the two in combination were also carried out in CT26 dual tumor model. On day 13, for the injected tumor, the tumor growth inhibition rate (TGI) of an equivalent amount of a TLR9 agonist, a STING agonist, a mixture of the two, and the corresponding conjugate CS-2600 was 29.65%, 31.3%, 61.00%, and 91.93%, respectively. For the distal tumor, only the conjugate CS-2600 showed significant inhibition of tumor growth with 57% TGI, whereas no tumor growth inhibition was observed with a TLR9 agonist, a STING agonist, or a mixture of the two. Conclusions: The conjugates harnessed significant synergy between TLR9 and STING pathways in vitro and in vivo and resulted in over ten times enhancement of potency in certain assays over either agent alone or a combination of the two. The preclinical studies of the conjugates support clinical evaluation of this class of novel compounds.
Citation Format: Yuntao Song, Anrong Li, Hui Li, Xianfeng Li, Zeao Huang, Junbao Yang, Yi Ding, Grace Szu. Conjugates of TLR9 and STING agonists achieved profound synergistic effects in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2094.