Reprogramming of experimental glioma microenvironment with TLR3 agonist improves function of cytotoxic T-lymphocytes and correlates to increased overall survival in a syngeneic glioma model
Alexander Hagstotz (Dresden), Luise Rupp (Dresden), Marc Schmitz (Dresden; Heidelberg), Ilker Yasin Eyüpoglu (Dresden; Heidelberg), Achim Temme (Dresden; Heidelberg)
High-grade gliomas (HGG) are primary CNS tumors resistant to current therapies, including immunotherapy. Tumor recurrence is driven by therapy-resistant traits and an immunosuppressive tumor microenvironment (TME). This study evaluates the efficacy of a nanoparticle-based (neo)adjuvant immunotherapy delivering the Toll-like receptor 3 (TLR3) agonist RIBOXXOL to epidermal growth factor receptor variant III (EGFRvIII)-expressing glioma cells. Conceptually, TLR3-agonist delivery should reprogram the TME, promote immunogenic cell death, and reinvigorate or restore anti-tumor immunity.
Anti-EGFRvIII RICIA nanoparticles were assembled by conjugating EGFRvIII-specific single-chain antibody fragments, neutravidin, and biotinylated RIBOXXOL. The effects of RICIA treatment were evaluated in TLR3-expressing reporter cells as well as wildtype and TLR3-deficient murine SMAvIII glioma cells. Cytokine production and immune activation were assessed by multiplex bead assays, flow cytometry, and ELISA. Tumor growth and survival after RICIA treatment were investigated in syngene subcutaneous (s.c.) and intracranial (i.c.) SMAvIII-FfLuc-VM/Dk glioma mouse models. In s.c. tumors, transcriptional alterations within the post-therapeutic TME were analyzed, and the phenotype of tumor-infiltrating lymphocytes (TILs) was evaluated by immunohistochemistry.
Anti-EGFRvIII RICIA treatment induced TLR3-dependent sterile inflammation, leading to enhanced IFN-β secretion and cellular inflammation in SMAvIII cells. In vivo, RICIA-treated s.c. gliomas exhibited significant tumor growth inhibition, with inflammatory reprogramming of the TME, including a decrease of the immunosuppressive M2 macrophage phenotype and an increase in GrzB+ PD-1- CD8+ TILs. Long-term experiments demonstrated significantly prolonged survival, with 25% of treated mice achieving complete glioma regression and protective immune memory confirmed by tumor rejection upon rechallenge. Furthermore, RICIA treatment of orthotopic glioma resulted in tumor growth inhibition, extended survival, and complete tumor regression in 13% of cases.
Anti-EGFRvIII RICIA represent a tumor cell-selective, immunomodulatory strategy that induces TLR3-dependent glioma cell death and reprogramming of the TME thereby enhancing CTL responses. These findings highlight the therapeutic potential of RICIA for the treatment of HGG.
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