Multimodal glioma immunotherapy combining TLR9-targeted STAT3 antisense oligodeoxynucleotides with PD1 immune checkpoint blockade

Therapeutic resistance in glioblastoma (GBM) is multifactorial and results from genetic heterogeneity, the immunoprivileged localization and the potently tolerogenic microenvironment. Signal transducer and activator of transcription 3 (STAT3) plays a key role in both glioma cell survival and immune evasion reinforcing GBM resistance.

Here, we describe a new cell-selective and double-stranded STAT3 antisense oligonucleotide (CpG-STAT3dsASO) for targeting human/mouse glioma cells and GAMs but not T-cells. The oligonucleotide safety and efficacy against orthotopic GBM was assessed in immunocompetent or immunodeficient mice.

CpG-STAT3dsASO injected intracranially/intratumorally was well-tolerated and reduced progression of human U251 GBM xenotransplants and mouse GL261 or neural cell-derived QPP8 gliomas. Unlike the single-stranded oligonucleotide, local CpG-STAT3dsASO administration did not trigger type-I IFN-dependent neurotoxicities in immunocompetent mice within the therapeutic dose range. CpG-STAT3dsASO activated intratumoral GAMs, such as dendritic cells, macrophages and microglia, thereby expanding CD4⁺ Th1 cells while reducing T_REG numbers. CpG-STAT3dsASO monotherapy did not have curative effects as it led to recruitment of only limited numbers of mostly exhausted effector CD8⁺ T-cells. However, when combined with systemic PD1 inhibition, CpG-STAT3dsASO/anti-PD1 treatments caused regression of GL261 as well as immunotherapy-resistant QPP8 gliomas and resulted in long-term survival of the majority of mice. The combination treatment boosted CD8⁺ effector T-cell activity, while promoting their intratumoral interaction with activated CD4⁺ Th1 cells and activated macrophages as indicated by spatial transcriptomics.

Our results suggest rationale for the GBM immunotherapy using CpG-STAT3dsASO to disrupt GAMs-dependent immune evasion, thereby restoring sensitivity to PD1-blockade and facilitating T-cell-mediated antitumor immune responses.