The specific TLR3-agonist TL-532 induces life-long anti-tumor autovaccination, cross-immunity against unrelated cancers and reverses resistance to immune checkpoint inhibitors

Abstract Background: Toll-Like Receptor 3 (TLR3) is an innate immunity receptor that triggers inflammation by recognizing double stranded RNAs. In the past decades, TLR3-agonists have been used to fight cancer as inducer of type-I interferons and inducer of apoptosis specifically in tumor cells but not in normal cells. No TLR3-agonist has reached the market to date due to lack of manufacturing reproducibility, specificity, activity and/or due to toxicity. In this context, a new family of synthetic, specific and fully defined TLR3-agonist has been rationally designed (WO2019211492). Here we report the preclinical proof-of-concept of TL-532 in non-muscular invasive bladder cancer (NMIBC). Methods: In-vitro 2D and 3D cultures of J82 and RT4 NMIBC versus normal urothelial cells, were used. Freshly resected bladder tumors from patients were sliced and cultured ex vivo for 24 to 48h. In vivo experiments were performed using the ICI-resistant murine syngeneic/ectopic bladder cancer model MBT2, treated by intra- and peri-tumoral injections of TL-532. Results: We demonstrated in vitro, ex vivo and in vivo that TL-532 induced tumor cell death specifically by apoptosis, while it was well tolerated by normal cells, representing a broad in vitro safety margin. Ex vivo and in vivo, this tumor cell death was associated with a tumor microenvironment switch, evidenced by increases in antitumor biomarker secretion (IFN-α, IFN-λ1, IFN-γ, CCL5, CXCL9, CXCL10, CX3CL10), decreases in protumor biomarkers CCL22, sFAS and was associated in vivo with the recruitment and activation of conventional dendritic cells (cDCs) and cytotoxic T-lymphocytes (CTLs) at the tumor site. In vivo activity of TL-532 led to substantial tumor growth inhibition (88%) and delay (370%), translating in 35% complete response (CR) rate and 5.3-fold median survival benefit. Interestingly, among these CRs, 62% (13/21) showed life-long tumor auto-vaccination after 3 consecutive rechallenges at 3, 10 and 30 months. Remarkably, 54% (6/11) of the mice autovaccinated against bladder cancer also demonstrated cross-immunity against an unrelated and poorly immunogenic, syngeneic osteosarcoma cancer cell model (LM8). In addition, TL-532 treatment appeared to decrease the expression of the immune checkpoint PD-L1 on tumor cells ex-vivo and in cDCs in vivo and demonstrated a remarkable ability to reverse the anti-PD-L1 tumor-resistance when combined with the ICI leading to doubling of CR rate. Conclusion: We identified TL-532 as spearhead of a new rationally designed TLR3-agonist family. In monotherapy, it demonstrated substantial tolerance and promising anti-cancer and autovaccinal activity, which included unrelated cancers. TL-532 also demonstrated its remarkable ability to overcome ICI tumor-resistance, thus increasing the clinical landscape for ICI combination treatment. Citation Format: Marc Bonnin, Saïd Ourfali, Mathilde Boucard-Jourdin, Clémence Perret, Chloé Renoux, Nelly Vey, Marc Colombel, Bettina Werlé, Sylvain Thierry. The specific TLR3-agonist TL-532 induces life-long anti-tumor autovaccination, cross-immunity against unrelated cancers and reverses resistance to immune checkpoint inhibitors [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 2085.