282 Tumor-Targeting TLR9 Agonist Promotes Antitumor Immune Activity in Intracranial Glioma Model

INTRODUCTION: Immunotherapies involving toll-like receptor 9 (TLR9) agonists can promote anti-tumor immune responses in various preclinical models. However, the adoption of systemically-administered TLR9 agonists for glioblastoma (GBM) is limited by poor trafficking to the tumor and an immunosuppressive tumor microenvironment (TME). Conjugating tumor-targeting peptides represents a potential solution to these issues and may unleash the efficacy of TLR9 agonists in GBM. METHODS: Syngeneic GL261-luc glioma cells were orthotopically implanted in 6–8-week-old female C57BL/6J mice. Mice were intraperitoneally injected with either vehicle (phosphate-buffered saline), 200 ug anti-programmed cell death protein 1 (PD1) antibody, 250 ug PIP-CpG V3, or both anti-PD1 and PIP-CpG V3, on days 10, 12, and 14 post-tumor implantation. Mice were either monitored for survival or euthanized on day 17 for immunological assays. RESULTS: Systemic administration of PIP-CpG V3 monotherapy and combination therapy with anti-PD1 + PIP-CpG V3 increased the median survival of GL261 tumor-bearing mice (PIP-CpG V3: p = 0.049, combination: p = 0.012). Flow cytometry analysis of tumor-bearing brains revealed significantly increased infiltration of CD8+ T cells (PIP-CpG V3: p < 0.0001, combination: p < 0.0001) and CD11b+ MHCII+ CD11c+ dendritic cells (DCs) (PIP-CpG V3: p < 0.0001, combination: p < 0.0001) in groups that received PIP-CpG V3, relative to vehicle mice. Additionally, PIP-CpG V3 therapy induced upregulation of various immune activation markers (CD69, CD86, PD-L1) across different immune cell subsets, including DCs, NK cells, B cells, and other myeloid cells in tumor-bearing brains. CONCLUSIONS: PIP-CpG V3 improved survival of GL261 tumor-bearing mice, both by itself and in combination with anti-PD1. Flow cytometry immunophenotyping indicates that PIP-CpG V3 can remodel the glioma microenvironment, resulting in a higher proportion of CD8+ T cells and DCs as well as upregulation of activation markers on diverse immune subsets.