TMIC-84. Hypoxia reduction remodels the glioblastoma immune microenvironment to enhance therapeutic sensitivity

Glioblastoma (GBM) is characterized by an immunosuppressive tumor microenvironment (TME), largely orchestrated by anti-inflammatory glioma-associated macrophages/microglia (GAMs). Tumor hypoxia exacerbates this immunosuppression by activating the hypoxia-inducible factor (HIF) pathway in GAMs, promoting their polarization toward an anti-inflammatory phenotype that sustains a T cell–excluded, immune “cold” TME. This hypoxia-driven reprogramming severely impairs CD8⁺ T cell infiltration and effector function through metabolic suppression, contributing to resistance against chemotherapy and immune checkpoint blockade (ICB). We demonstrate that targeting tumor hypoxia with the hypoxia-activated prodrug evofosfamide (evo; TH-302; IMGS-101) markedly improves survival in both GL261 and CT2A murine GBM models (p<0.0001). Evo treatment significantly enhances CD8⁺ T cell infiltration (p<0.01) and restores effector function, as evidenced by increased granzyme B expression (p<0.001) and decreased expression of exhaustion markers TIM-3 and LAG-3 (p<0.001). CD8⁺ T cells isolated from evo-treated tumors display robust cytotoxicity against both antigen-specific (GL261-OVA) (p<0.001) and antigen-independent (CT2A) targets (p<0.0001), underpinned by enhanced metabolic fitness, including increased ATP production via both glycolysis and oxidative phosphorylation. Evo also reconfigures the myeloid compartment by disrupting microglial clustering within hypoxic niches, promoting a phenotypic shift toward pro-inflammatory GAMs (upregulation of CD80, MHC II; p<0.001) while suppressing M2-like markers (Arginase-1, PD-L1; p<0.01). These reprogrammed GAMs support greater CD8⁺ T cell proliferation (p<0.01) and survival (p<0.001) in co-culture systems. Notably, evo-mediated hypoxia alleviation sensitizes tumors to ICB and enables durable, curative responses when combined with intracranial anti–CTLA–4—, but not anti–PD-1—in ICB-refractory CT2A tumors. Together, these findings establish hypoxia as a central regulator of immune suppression in GBM and validate hypoxia-targeting strategies as powerful adjuncts to immunotherapy. Evofosfamide emerges as a potent immunomodulatory agent capable of reprogramming the GBM TME and unlocking new avenues for therapeutic intervention.