499 Combination Immunotherapy to Tumor Draining Lymph Nodes Results in Decreased Immunosuppression and Improved Overall Survival in Murine Glioblastoma

INTRODUCTION: Clinical trials involving immune checkpoint blockade such as anti-PD-1 fail to improve survival compared to standard of care in glioblastoma (GBM). Despite the advent of new immunotherapies such as STING agonists and anti-CSF-1R, combination of these strategies is limited by necessitation of intracranial delivery (STING) or off-target side effects with systemic exposure (anti-CSF-1R + anti-PD-1). We have previously shown that delivery of anti-PD-1 to tumor draining lymph nodes via hydrogels decreases systemic exposure of anti-PD-1 and results in improved therapeutic response compared to systemic delivery. METHODS: CT-2A murine glioma cells were orthotopically implanted into C57BL/6J mice with treatment on day 7 via deep cervical injection of hydrogel polymers loaded with either PBS (control), STING, aCSF1r, aPD1, or a combination of all three immunomodulators. Flow cytometry was used for immunophenotyping and Kaplan-Meier survival curves were generated to assess therapeutic efficacy. RESULTS: Each immunomodulator resulted in reversal of a different component of immunosuppression. Compared to controls, aCSF1r resulted in the greatest decrease of immunosuppressive myeloid cells in the brain (P=0.0017) and cervical lymph nodes (P=0.0008), STING resulted in the greatest activation of intracranial dendritic cells (P=0.0001), and aPD1 resulted in the highest level of T cell activation of the monotherapies (P=0.0008) but also caused compensatory upregulation of regulatory T cells in the brain (P=0.0176). Combination therapy resulted in the greatest decrease in immunosuppression with increased T cell IFN-y production and dendritic cell activation, with resultant synergistic improvement in median and overall survival (P=0.0006). CONCLUSIONS: Triple combination therapy to tumor draining lymph nodes that specifically targets multiple components of immunosuppression is a safe and efficacious treatment that can be translated to clinical applications.