Development of a novel TLR8 agonist for cancer immunotherapy

Abstract

Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase 1 clinical trials.

Keywords: Cancer; Immunotherapy; Innate immunity; TLR8.

Fig. 1

In vitro profiles of DN052. (a) Differentiated parameters of DN052. The molecular weight (MW) of DN052 and motolimod are shown. The detailed chemical structure and synthesis of DN052 were described in our published patent 10,669,252. (b) DN052 was potent and selective in cell-based assays. DN052 was evaluated for its agonistic effect on TLR8 and selectivity over TLR4, 7 and 9 in HEK-Blue™ TLR cells and the data was presented as EC50 (50% effective concentration). The potential effect of DN052 on cell proliferation was tested in HEK-Blue™ TLR7 and 8 cells in parallel and the data was presented as CC50 (50% cytotoxicity concentration). Motolimod was used as a reference compound. The agonists (LPS-EK, R848 and ODN2006) known to activate each of the TLRs were used as positive controls in TLR4, 7 and 9 assays, respectively. DN052 was highly selective for TLR8 over TLR4, 7 and 9. DN052 and motolimod had little effect on cell proliferation

Fig. 2

Comparison of PK with different dosing routes. DN052 and motolimod were iv, ip, sc or po administered to Sprague Dawley rats, respectively. Overall, sc appeared to have better PK than the other dosing routes for DN052. DN052 showed superior PK profile than motolimod

Fig. 3

DN052 was more efficacious than motolimod and well-tolerated as a single agent in various mouse tumor models. DN052 treatment resulted in significant tumor growth inhibition in CT26 colon cancer (a, b), EMT6 breast cancer (c, d) and HL-60 AML (e, f) models. DN052 more strongly suppressed tumor growth than motolimod under the same conditions

Fig. 4

DN052 further reduced tumor growth when combined with other anti-cancer agents in mouse tumor models. Combination of DN052 and cyclophosphamide, WEE1 inhibitor AZD-1775, sorafenib or αPD-1 increased tumor suppression in several mouse tumor models: CT26 colon cancer model (a), EMT6 breast cancer model (b), H22 liver cancer model (c) and MC38 colon cancer model (d)