Background Targeting the cGAS-STING (cyclic GMP-AMP synthase-Stimulator of Interferon Genes) pathway with cyclic dinucleotides (CDNs) offers a promising immunotherapeutic strategy for lymphocyte-depleted, myeloid cell-enriched solid tumors like Glioblastoma (GBM). However, this approach is constrained by the limited bioavailability of CDNs and the presence of immune-evasive mechanisms in the tumor microenvironment (TME), particularly those driven by the master transcriptional regulator STAT3 (Signal Transducer and Activator of Transcription 3).

Methods To activate the cGAS-STING pathway and simultaneously inhibit STAT3-driven immune suppression, we developed bimodal Spherical Nucleic Acids (SNAs) by conjugating 15 nm gold nanoparticles with cGAS-activating stem-loop oligonucleotides containing a palindromic STAT3 decoy sequence.

Results In vitro, our first-in-class bimodal SNAs effectively activated cGAS and inhibited STAT3 in reporter cell lines. To test anti-glioma in vivo efficacy, we administered bimodal SNAs and their unimodal counterpart, cGAS-agonist ISD⁴⁵-SNAs (45 base pair Interferon Stimulating DNA) intra-tumorally, in SB28-bearing female and male wild-type mice. A two-dose monotherapy with bimodal SNAs significantly improved median survival in female mice (n=10, p value =0.0012) and resulted in long-term survivors (3/10 mice, >60 days), while a monofunctional cGAS-agonistic SNA architecture lacking STAT3 decoy capabilities (ISD⁴⁵-SNAs) provided a modest benefit with no long-term survivors. To define immune activation in the TME following bimodal SNAs and ISD⁴⁵-SNA treatment in female mice, we performed high-dimensional spectral flow cytometry analysis. ISD⁴⁵-SNAs significantly increased the pro-inflammatory cytokines TNF alpha and IFN gamma, the expression of activation markers CD40, 4-1BBL on tumor-associated myeloid cells (TAMCs), dendritic cells and B cells, respectively, and increased cytotoxicity of natural killer (NK) and CD4, CD8 T-cells abundance and activation. However, this inflamed TME resulted in the compensatory expansion of exhausted adaptive immune cells, causing immune evasion. In contrast, the bimodal SNAs prevented immune cell exhaustion, reduced immunosuppressive TAMCs, and increased the infiltration and activity of adaptive immune cells for durable anti-tumor responses in females. Interestingly, male mice showed no survival benefit as bimodal SNA treatment increased TAMCs and decreased CD8 T-cell abundance, resulting in an exhausted immune microenvironment.

Conclusions Our study demonstrates the potential of bimodal SNAs as a novel single-entity nucleic acid cGAS-STING and STAT3 targeted therapy. These SNAs effectively activate innate and adaptive immunity in the TME, achieving a sustained anti-tumor effect in checkpoint-resistant GBM in female animal subjects. To further enhance survival benefits, we propose the exploration of combining these bimodal SNAs with immune checkpoint inhibitors for the development of a novel combinatorial immunotherapeutic strategy.