Intratumoral delivery of the chitin-derived C100 adjuvant promotes robust STING, IFNAR, and CD8+ T cell-dependent anti-tumor immunity

Highlights

• •Intratumoral injection of the adjuvant C100 promotes potent anti-tumor immunity
• •Adjuvant functionality requires STING and IFNAR signaling and CD8⁺ T cells
• •C100 injection synergizes with systemic checkpoint blockade with anti-PD1
• •C100 triggers DNA damage to selectively activate the IFN arm of the cGAS-STING pathway

Summary

Stimulator of IFN genes (STING) is a promising target for adjuvants utilized in in situ cancer vaccination approaches. However, key barriers remain for clinical translation, including low cellular uptake and accessibility, STING variability necessitating personalized STING agonists, and interferon (IFN)-independent signals that can promote tumor growth. Here, we identify C100, a highly deacetylated chitin-derived polymer (HDCP), as an attractive alternative to conventional STING agonists. C100 promotes potent anti-tumor immune responses, outperforming less deacetylated HDCPs, with therapeutic efficacy dependent on STING and IFN alpha/beta receptor (IFNAR) signaling and CD8⁺ T cell mediators. Additionally, C100 injection synergizes with systemic checkpoint blockade targeting PD-1. Mechanistically, C100 triggers mitochondrial stress and DNA damage to exclusively activate the IFN arm of the cGAS-STING signaling pathway and elicit sustained IFNAR signaling. Altogether, these results reveal an effective STING- and IFNAR-dependent adjuvant for in situ cancer vaccines with a defined mechanism and distinct properties that overcome common limitations of existing STING therapeutics.

Introduction

The cGAS-STING signaling axis is a crucial regulator of type I interferon (IFN) responses and anti-tumor immunity. Upon recognition of tumor-derived, damaged double-stranded DNA (dsDNA), cGAS catalyzes the synthesis of the cyclic di-nucleotide (CDN) 2′3′-cGAMP, which serves as a second messenger that binds to and activates stimulator of IFN genes (STING). STING is an endoplasmic reticulum (ER)-associated protein that activates a TANK-binding kinase 1 (TBK1)-interferon regulatory factor 3 (IRF3)-dependent type I IFN response and nuclear factor κB (NF-κB)-mediated pro-inflammatory cytokine response. Type I IFNs stimulate the presentation of tumor antigens and the mobilization of anti-tumor immunity, in particular CD8⁺ cytotoxic T lymphocytes.¹ The role of STING-induced NF-κB activation in cancer therapy remains poorly defined, with both pro- and anti-tumorigenic roles suggested.^2,3,4

Despite the plethora of STING agonists that have been and are being developed, clinical translation has resulted in disappointingly modest efficacy, and to date, no cGAS-STING-targeted therapy has achieved clinical success. The small-molecule STING ligand DMXAA achieved robust anti-tumor immune responses in murine B16 melanoma models but failed in phase 3 trials (ClinicalTrials.gov: NCT00738387 and NCT00662597), likely due to its inability to bind human STING.⁵ Intratumoral (i.t.) administration of natural CDNs such as mammalian 2′3′-cGAMP and c-di-GMP, c-di-AMP, and 3′3′-cGAMP from prokaryotes was effective in mouse models of colon, brain, skin, pancreatic, breast, and B cell malignancies. However, intrinsic physicochemical characteristics such as their large size and electronegative charge render these molecules poorly membrane permeable and prone to rapid extracellular enzymatic degradation, resulting in low drug bioavailability in tumor tissues and rapid absorption into the plasma.^6,7

 Various approaches are under evaluation to address these limitations, such as the development of CDN drug delivery systems (e.g., exoSTING)⁸ and non-CDN small-molecule STING agonists with greater affinity for human STING (e.g., GSK3745417, ClinicalTrials.gov: NCT03843359).

To the best of our knowledge, there are no STING agonists under evaluation that do not result in the concomitant activation of the NF-κB pathway. In a tumor setting, the induction of pro-inflammatory signaling downstream of NF-κB could be detrimental for the establishment of anti-tumor immunity and possibly favor tumor progression. Intrinsic NF-κB signaling in tumor cells can favor proliferation, inhibit the induction of cell death pathways, upregulate coinhibitory receptor expression,⁹ and promote epithelial-mesenchymal transition (EMT)¹⁰ and angiogenesis in the tumor. The pro-inflammatory cytokines downstream of NF-κB activation can also have detrimental effects on anti-tumor immunity, promoting anergic responses. Indeed, inhibition of NF-κB signaling in tumor cells and i.t. myeloid cells favors tumor regression in pre-clinical models.¹¹ Therefore, therapeutic adjuvants that exclusively activate the type 1 IFN arm of the cGAS-STING pathway and not NF-κB may be of clinical benefit.

Chitin-derived polymers are incorporated in biomedical and pharmaceutical formulations, including prolonged- or controlled-release drug delivery systems,¹² wound dressings, and bone tissue engineering scaffolds, and have been investigated as vaccine adjuvants.¹³ We have previously demonstrated that the adjuvanticity of highly deacetylated chitin-derived polymers (HDCPs) relies on the activation of the cGAS-STING and NLRP3 inflammasome pathways via the induction of mitochondrial reactive oxygen species (mtROS) and DNA release.¹³ Subsequently we demonstrated that this ability to activate cGAS-STING and the NLRP3 inflammasome depended critically on the degree and pattern of polymer deacetylation, which controlled the extent of mtROS production.¹⁴

 In addition, these HDCPs exclusively drove type I IFN release in the absence of pro-inflammatory cytokine production.¹⁴

 Altogether, the results of this work have characterized a biocompatible polymer with a distinct means of cGAS-STING activation; therefore, this current study highlights the potential of HDCPs in the context of therapeutic cancer vaccines.

Immune checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1, are frequently employed as a standard treatment for various cancers. Yet, their effectiveness is limited in numerous cancers, prompting the exploration of combination approaches to improve the efficacy and expand the range of patients and tumor types suitable for such treatments. In situ vaccination with immunogenic compounds presents a promising avenue for addressing the limitations of checkpoint inhibitors, as it has the potential to transform “cold” tumors into “hot” ones by promoting the infiltration of tumor-specific T cells.^15,16

Here, we demonstrate the effectiveness of C100 (a chitin polymer deacetylated to <99.5%) as a cGAS-STING targeting i.t. therapeutic that can be used as a monotherapy or synergistically with checkpoint blockade. We have characterized the mechanism of action of this polymer, demonstrating exclusive activation of the IFN arm of STING signaling through the release of both nuclear and mitochondrial DNA (mtDNA), downstream of mitochondrial stress induction, circumventing the need for direct adjuvant-STING binding. Furthermore, we demonstrate that the therapeutic efficacy of C100 is dependent on STING and type I IFN signaling and identify CD8⁺ T cells as crucial mediators of C100-induced anti-tumor immunity. In summary, we characterize a STING-targeting i.t. therapeutic with advantages over alternative molecules and provide key insights into its mode of action.

Results

C100 is the most effective HDCP as an i.t. therapeutic

Pitfalls of current STING-based therapies contributing to poor clinical translation include rapid, transient, and excessive immune activation at the site of injection coupled to a reliance on direct binding to STING itself.^6,7,17,18 Given the distinctive profile of HDCPs as vaccine adjuvants that promote the STING pathway via induction of ROS and self-DNA-mediated cGAS-STING activation,^13,14 a therapeutic B16F10 tumor challenge model was used to investigate the anti-tumor potential of HDCPs when administered i.t. (Figure 1A). Tumor cell lines can render the cGAS-STING pathway defective by hypomethylating promoter regions of pathway components and decreasing protein expression or by blocking STING trafficking to sites required to promote the induction of IFN responses.¹⁹ As such, we firstly assessed the expression and functionality of cGAS and STING in B16F10 cells by immunoblot using DMXAA, a small-molecule STING agonist that directly binds and activates murine STING.¹⁷

 Both proteins were present under basal conditions (Figures S1A and S1B), and functional STING activity was demonstrated by its reduced expression after DMXAA treatment compared to the untreated control (Figure S1A). Following i.t. injection, C100 was the most potent adjuvant, with treatment significantly reducing tumor growth (Figures 1B and S1C) and enhancing survival compared to control mice (Figure 1C). To validate this efficacy in a second tumor model, i.t. injections were carried out following implantation of MC38 cells. As before, the expression and functionality of cGAS and STING proteins in the MC38 line were confirmed before tumor implantation (Figures S1D and S1E). Next, mice bearing MC38 tumors were treated in an identical manner to the B16 model. A more pronounced inhibition in growth and prolonged survival after C100 treatment was observed in MC38 compared to B16F10 tumors (Figures 1D, 1E, and S1F). This is in line with existing data showing increased responsiveness to immunotherapeutic interventions in MC38 tumors, owing to a higher degree of tumor immunogenicity.^20,21 In addition, C100 dose optimization studies were carried out in the B16 and MC38 tumor models, with a polymer dose of 100 μg exerting the most potent anti-tumor effects, outperforming the higher (Figure S1G) and lower doses (Figure S1H) tested i.t. Overall, these results indicate that C100 is an effective polymeric adjuvant for in situ vaccination against cancer and outperforms less deacetylated alternative polymers, in line with our previous observations that the degree of deacetylation is instructive in a chitin-derived polymer’s adjuvanticity.¹⁴