Abstract 2567: Enhancement of antitumor activity of DSP-6434, a novel TLR7 agonist through reduction of TLR tolerance

Abstract

Abstract Introduction: Systemic administration of the small molecule Toll-like receptor (TLR)-7 agonist DSR-6434 leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, clinical responses with systemically administered TLR7 agonists have been underwhelming, in part because activation of TLRs with small-molecule agonists can induce TLR tolerance; defined as a state of hyporesponsiveness to subsequent agonism. This study is undertaken to identify conditions to overcome TLR tolerance. Experimental procedures: To confirm the anti-tumor effect of DSR-6434, mice were inoculated with the mouse renal cell carcinoma cell line, Renca, at day 0, and then administrated DSR-6434 weekly from day 1, or twice weekly from day 6. To examine tolerance, wild-type or IFN-α/β receptor knockout (IFN-AR KO) mice were intravenously administrated DSR-6434 at intervals of 3, 7 or 10 days. Plasma samples were taken 2 hours after the second administration of DSR-6434 and IFN-α levels were measured. Splenocytes were isolated 24 hours following either single or sequential i.v. doses. To measure lymphocyte activation, CD69 expression was assessed and splenocyte-mediated cytotoxicity against YAC-1 target cells was determined. Bone-marrow derived pDC (BM-pDC) were also treated with DSR-6434 for 5 or 48 hours. Expression of TLR7 signaling-related genes was determined by real-time quantitative RT-PCR. Results: Weekly administration of DSR-6434 significantly reduced tumor burden when compared to vehicle-treated mice. By contrast, pre-inoculation and 2qw administration of DSR-6434 completely abolished anti-tumor activity. IFN-α induction was completely impaired following the second administration of DSR-6434 after 3 days, partially impaired after 7 days, and fully functional when the dosing interval was extended to 10 days. Sequential dosing of DSR-6434 reduced the frequency of activated splenocytes (defined as CD69+) and the level of cytotoxicity, compared with a single administration of DSR-6434. TLR7 tolerance was also observed in IFN-AR KO mice, suggesting this effect was independent of IFN signaling. Interestingly, TLR7 down-regulation was only observed after DSR-6434 dosing, while the expression of other TLR7-signaling related genes was unaltered. Conclusion: The dosing schedule of systemically administered TLR7 agonists significantly affect TLR tolerance and antitumor activity, offering a potential solution to overcome TLR tolerance . Furthermore, TLR7 expression on BM-pDC cells may serve as a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists. Citation Format: Erina Koga-Yamakawa, Masashi Murata, Simon J. Dovedi, Robert W. Wilkinson, Hiroki Umehara, Eiji Sugaru, Yuko Hirose, Hideyuki Harada, David T. Robinson, Philip J. Jewsbury, Setsuko Yamamoto, Chiang J. Li. Enhancement of antitumor activity of DSP-6434, a novel TLR7 agonist through reduction of TLR tolerance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2567. doi:10.1158/1538-7445.AM2014-2567