Abstract LB116: Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors
Authors: Temme Achim , Hagstotz Alexander , Schau Isabell , Seifert Michael , Sürün Duran , Zimmermann Stefan , Buchholz Frank , Rupp Luise , Wehner Rebekka , Schmitz Marc , Schackert Gabriele , Eyüpoglu Ilker Y. 
Published: April 2024

Abstract
In solid cancers, an immunosuppressive tumor microenvironment (TME) restrains immunotherapy. Induction of immunogenic cell death in tumor cells and simultaneously programming the TME from immunosuppressive to inflammatory employing dsRNA agonists for endosomal Toll-like receptor 3 (TLR3) represents an appealing in situ immunomodulation strategy. However, uptake of dsRNA by tumor cells is limited and systemic administration is linked to adverse side effects. Here we describe an exceptional therapeutically effect of systemically delivered TLR3 agonist RIBOXXOL using a recently developed antibody-guided nanoparticle-system designated “Rapid Inducer of Cellular Inflammation and Apoptosis” (RICIA) for targeting epidermal growth factor variant III (EGFRvIII) on tumor cells. We suggest that systemically applied monotherapy with RICIA might be effective in experimental solid tumors. This includes, (i) preferential enrichment of the immune-stimulatory RIBOXXOL and (ii) subsequent induction of a type-I interferon response in tumor cells expressing TLR3, and (iii) synergistic inflammation of the TME through TLR3 agonist thereby expediting anti-tumor immune responses. The effects of RICIA-treatment were comprehensively assessed in vitro and in murine syngeneic epidermal growth factor receptor variant III (EGFRvIII)-positive SMAvIII gliomas. Our in vitro studies confirm uptake of RICIA by EGFRvIII-mediated endocytosis in human and murine glioma cells and furthermore demonstrate TLR3-triggered type-I interferon release and mRNA signatures indicative of an innate immune response against virus. We show in the SMAvIII tumor model that monotherapy with picomolar amounts of RICIA induces regression of subcutaneous (s.c.) tumors leading to increase in survival time and results in complete tumor clearance in a significant fraction of mice accompanied by immune memory when compared to RICIAs equipped with an unspecific control antibody. Mechanistically, RICIA treatment reshapes the immunosuppressive tumor environment by mimicking viral infection leading to increased expression of the interferon beta (IFN-β) gene (Ifnb1), by subsequent induction of an inflammatory state characterized by RNA-expression signatures indicating induction of apoptosis, interleukin 1beta (IL-1β) signaling, and programming immunosuppressive M2 TAMs to activated pro-inflammatory M1 TAMs. In addition, a single stereotactic injection of RICIA into established intracranial glioma significantly improves mean survival time of treated mice and is able to induce complete tumor clearance thus showing therapeutic potency of RICIA in a relevant pre-clinical setting for glioma. Thus, we show that targeted activation of TLR3 represents a promising therapeutic strategy for immune-modulation of tumors with the prospect to elicit long-lasting anti-tumor immune responses.
Citation Format: Achim Temme, Alexander Hagstotz, Isabell Schau, Michael Seifert, Duran Sürün, Stefan Zimmermann, Frank Buchholz, Luise Rupp, Rebekka Wehner, Marc Schmitz, Gabriele Schackert, Ilker Y. Eyüpoglu. Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB116.