cGAS‐STING pathway agonists are promising vaccine adjuvants

Adjuvants are of critical value in vaccine development as they act on enhancing immunogenicity of antigen and inducing long‐lasting immunity. However, there are only a few adjuvants that have been approved for clinical use, which highlights the need for exploring and developing new adjuvants to meet the growing demand for vaccination. Recently, emerging evidence demonstrates that the cGAS‐STING pathway orchestrates innate and adaptive immunity by generating type I interferon responses. Many cGAS‐STING pathway agonists have been developed and tested in preclinical research for the treatment of cancer or infectious diseases with promising results. As adjuvants, cGAS‐STING agonists have demonstrated their potential to activate robust defense immunity in various diseases, including COVID‐19 infection. This review summarized the current developments in the field of cGAS‐STING agonists with a special focus on the latest applications of cGAS‐STING agonists as adjuvants in vaccination. Potential challenges were also discussed in the hope of sparking future research interests to further the development of cGAS‐STING as vaccine adjuvants.


| INTRODUCTION
Vaccines are a crucial invention for preventing and controlling both infectious and noninfectious diseases, including cancers.Classical vaccines are originated from antiviral immunity. 1Modern vaccine developments have been largely depending on highly sophisticated and integrated vaccine development platforms 2 and new vaccines can be developed surprisingly quickly as evidenced by the recent developments of anti-COVID 19 vaccines. 3Maximizing vaccine efficacy while reducing potential risks has always been the top priority in the process of vaccine developments.Unlikely traditional inactivated or live-attenuated vaccines, modern vaccines increasingly focus on subunit vaccines and nucleic acid vaccines.Subunit vaccines are vaccines that contain purified parts of the pathogen that are antigenic and have well-defined target antigens and usually contain high-purity proteins or synthetic peptides.Thus, they compensate for the limitations of traditional vaccines and have become the most widely used kind of neoantigens tumor vaccines. 4The emergence of gene editing technology has led to the development of nucleic acid vaccines, specifically DNA and RNA vaccines.While DNA vaccines are more effective in stimulating the host immune responses, they carry a higher risk of gene mutation. 5On the other hand, mRNA vaccines have been successfully tested in clinical trials against SARS-CoV-2 infection (COVID-19) and tumors, marking a significant milestone in nucleic acid vaccine development. 2,6However, with molecular antigens becoming more well-defined and smaller, vaccines are consequently becoming less immunogenic compared to traditional inactivated or live-attenuated vaccines. 2,7In this regard, there is an urgent need for effective adjuvant systems to induce stronger and more long-lasting immune responses to target antigens.
Immunobiologically, the pattern recognition receptor (PRR) of innate immune cells recognizes pathogenassociated molecular patterns (PAMPs) from conserved microbial molecules and damage-associated molecular patterns (DAMPs) from dying or stressed cells.This recognition triggers intracellular signaling cascades and the expression of inflammatory mediators 4 (Figure 1).Various proteins from the PRR family, such as the endosomelocalized Toll-like receptor (TLR), the absent in melanoma2 (AIM2), and the enzyme cyclic GMP-AMP synthase (cGAS), are involved in DNA sensing.These proteins detect specific forms of DNA, such as unmethylated cytosineguanine (CpG)-containing DNA from the extracellular, cytosolic double-stranded DNA (dsDNA), and B-form DNA, respectively. 8Researchers are actively exploring different molecular structures that can serve as PAMP or DAMP to F I G U R E 1 Immune activation by pathogens.Immunotherapy induces immune responses by activating the host's innate immune defense mechanisms against target pathogens, thereby eliminating and preventing diseases.Innate immune defenses are initiated by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), the absent in melanoma2 (AIM2), retinoic acid-inducible gene (RIG-I)-like receptors, and nucleotide-binding oligomerization domain (NOD)-like receptors.These receptors recognize conserved structures of pathogens and damaged tissue or cells (PAMPs and DAMPs), including glycans, lipids, peptides, nucleic acids, and some small molecules.Recognition of PAMPs by PRRs activates intracellular signaling pathways and activates innate immune cells such as macrophages, dendritic cells (DCs) and natural killer cells (NKs), inducing the upregulated secretion of inflammatory cytokines, chemokines, interferons and costimulatory molecules.Different cytokines correspond to the distinct adaptive immune responses, including cellular (TH1) and antibody (TH2) responses, by T and B cells, respectively.[Color figure can be viewed at wileyonlinelibrary.com] appear to play a significant role in most cases. 12Therefore, it is important to develop strategies that allow selective modulation of cGAS-STING activity in different disease contexts to avoid the adverse effects of overactivation.

| OVERVIEW OF cGAS-STING PATHWAY
The cGAS-STING signaling axis can be briefly divided into two main components: cGAS and the dimerized receptor located on the endoplasmic reticulum (ER), known as STING.It has been demonstrated that cGAS functions as the DNA sensor upstream of STING. 27In the following discussion, we will focus on the role of cGAS-STING in modulating the major factors in immune cell that contribute to cellular immune responses.

| The activation of cGAS
cGAS, located on the inner leaflet of the plasma membrane, 28,29 comprises an unstructured, highly basic aminoterminal (N-terminal) domain and a spherical structural domain.The latter consists of two structural lobes, with one being evolutionarily conserved and the other facilitating interactions for nucleoside-triphosphate donor binding.
The active site is at the interface of the two structural lobes. 27,30,31In general, cGAS is activated by binding with dsDNA in a sequence-independent manner.It can also be activated by DNA-RNA hybrids, DNA viruses and retroviruses of both foreign and self-origin. 3,4,12,13,30,32,33Subsequently, cGAS dimerizes to form a stable and active conformation, with two DNA strands positioned between two cGAS proteins, catalyzing the formation of cGAMP. 34The N-terminal domain contributes to the formation of phase-separated condensates of dsDNA and cGAS. 35,36cGAMP, the second messenger, exhibits a high affinity for STING (Figure 2).

| The activation of STING and subsequent signal transmission
STING consists of a four-span transmembrane region, a cytosolic N-terminal segment, a connector domain and a ligand binding domain (LBD) on the C-terminal tail. 37Typically, STING on the ER membrane forms a homodimer, creating a V-shaped ligand binding pocket for cGAMP.Upon binding, cGAMP activates STING at the ER by forming an LBD/2′, 3′-cGAMP complex.This leads to a higher-order oligomerization of STING with open ends through side-by-side packing, 38 ultimately triggering the translocation of STING from the ER to the ER-Golgi intermediate compartment. 391][42] This process is driven by sulfated glycosaminoglycans. 43Palmitoylation of STING is proposed for TANK binding kinase 1 (TBK1) and IRF3 recruitment. 37,44e translocated STING formed STING-TBK1-IRF3 signalosome with recruited TBK1 and IRF3, allowing TBK1 to phosphorylate IRF3. 24Phosphorylated IRF3 dimerizes into a transcription factor complex, then translocates to the nucleus to induce the expression of type I IFNs and immune-stimulated genes (ISG), the most representative hallmark output signals of cGAS-STING activity. 3,12Critically, the NF-κB signaling pathway can also be activated by STING (Figure 2).

| cGAS-STING pathway in innate immunity
cGAS functions as an innate immune sensor, recognizing cytosolic DNA of both foreign and self-origin, and stimulates the host's innate immune response. 12,457][48] STING is theoretically expressed in APCs, including DCs, macrophages, and B cells. 49These cells play a crucial role in clearing pathogens from the circulation by presenting their antigens on the surface through major histocompatibility complexes (MHCs).Consequently, DNA released into the cytosol of APCs activates the cGAS-STING pathway, with pathogen-contained DNA acting as a natural agonist for this pathway.Studies have confirmed that DCs can internalize and sense nuclear DNA from dying cells, activating cGAS-STING signaling pathway.Activation of the cGAS-STING pathway results in the massive production of type I IFNs, leading to DC maturation and upregulation of the expression of MHCII molecules, costimulatory molecules (CD80/CD86), and the maturation marker CD83.As a ligand, type I IFN activates the IFN receptor (IFNAR1/IFNAR2) and promotes antigen capture by DCs, processing, and presentation. 4,50Accumulated evidence confirms that natural killer (NK) cell-mediated tumor clearance is triggered by cyclic dinucleotides (CDNs) through the cGAS-STING signaling pathway in a CD8 + T cells-independent manner.CDNs activate STING, leading to the release of type I IFN.IFN can directly activate NK cells or indirectly activate them by inducing IL-15 and IL-15 receptors.Interestingly, DCs are also found to upregulate IL-15Rα when F I G U R E 2 Overview of cGAS-STING signaling pathway.Exogenous and endogenous double-stranded DNA (dsDNA) bind with cytosolic sensor cGAS in a sequence-independent manner, resulting in enzymatic activation of cGAS and synthesis of cGAMP in the presence of adenosine 5′-triphosphate (ATP) and guanosine 5′-triphosphate (GTP).cGAMP and cyclic dinucleotides (CDNs) from bacteria subsequently binds to stimulator of interferon genes (STING) at endoplasmic reticulum (ER), leading to profound conformational changes with the ligand-binding pocket closed.This triggers STING oligomerization, liberation from anchoring factors and ultimately translocation from ER to Golgi compartments where STING recruits TBK1 and IKK kinases.Phosphorylated interferon regulatory factor 3 (IRF3) and IκBα, respectively, form functional signalosomes and then translocate into nuclear to induce expression of INF-I and immune-stimulated genes (ISGs).Meanwhile, canonical nuclear factor kappa B (NF-κB) signaling is also activated.[Color figure can be viewed at wileyonlinelibrary.com] induced by CDNs, 51 cultivating an inflammatory environment for T cell priming and leading to subsequent humoral and cellular immunity.

| cGAS-STING pathway in adaptive immunity
The activation of STING leads to an upregulation of type I IFN, providing the third signal to CD8 + T cells in a signal transducer and activator of transcription 4 (STAT4)-dependent manner.Consequently, the proliferation of CD8 + T cells and the secretion of the chemoattractant CXCL10 are enhanced, inducing the recruitment of antigen-specific CD8 + T cells to inflammatory foci and tumors. 52,53As mentioned earlier, IFN also contributes to the maturation and activation of DCs and macrophages for antigen presentation, further enhancing the proportion of memory T cells. 54rthermore, studies have demonstrated that cGAS-STING activating adjuvants can control the differentiation of CD4 + T cells and induce specific immune responses.Critically, it has been reported that STING-activating adjuvants significantly enhance antibody titers by inducing germinal center (GC) formation in the spleen and promoting differentiation into memory B cells. 55Follicular helper T (Tfh) cells, the primary T helper cells in the GC, play a crucial role in this process. 56 summary, the cGAS-STING signal pathway serves as a bridge between innate immunity and adaptive immunity.It is undoubtedly an ideal target for tumor control and virus clearance, where both types of immune responses are highly desired.

| cGAS-STING PATHWAY AGONISTS AND THEIR DELIVERY STRATEGIES
Uniquely, the cGAS-STING pathway presents two tandem druggable candidate sites: cGAS and STING.
Consequently, agonists can be categorized into cGAS and STING agonists.Given the highly cooperative nature of the cGAS-STING pathway with other immune response pathways, cGAS-STING agonists can be further classified as direct and indirect agonists.In this section, we will review the current advancements in cGAS-STING pathway agonists and discuss their pharmacological effect (Figure 3).

Metal ions
Metal ion homeostasis is fundamental for life, and free Zn 2+ is primarily stored in the mitochondria and ER. 57Zinc ions play a crucial role in the cGAS-STING signaling pathway, modulating the immune response.Specifically, cGAS is inactive under quiescent conditions, and Zinc ions can significantly increase cGAS activity in physiological buffers.
In vivo, Zn 2+ contributes to cGAS activation by promoting cGAS phase transition in the presence of cytosolic DNA. 58,591][62] It has been demonstrated that Mn 2+ enhances the capture and presentation of antigens via the cGAS-STING pathway. 63Mn 2+ is released from intracellular membrane-enclosed organelles, presumably Golgi and mitochondria, accumulating in the cytosol.
Subsequently, Mn 2+ binds to cGAS, strengthening its sensitivity to dsDNA and increasing the cGAMP-STING binding affinity, leading to immune response activation.The liberated cytosolic Mn 2+ significantly lowers the detection limit to dsDNA by several orders of magnitude. 64Importantly, Mn 2+ is capable of inducing a type I-IFN response and cytokine production without any infection, suggesting that Mn 2+ acts as a robust innate immune stimulator. 65arrestin 2 β-arrestin 2 is extensively involved in various signaling pathways implicated in several diseases, including metabolic disorders and cancers.[66][67][68] Previous studies indicate that β-arrestin 2 can suppress the phosphorylation and degradation of IκB, subsequently inhibiting the activation and secretion of p65.69 Similarly, β-arrestin 2 enhances the DNA-binding ability of cGAS and promotes cGAS dimerization.This is attributed to the reduction in both cGAMP production and STING dimerization in the absence of β-arrestin 2. 70 Notably, viruses tend to evolve mechanisms to inhibit the expression of β-arrestin 2 by regulating its acetylation and ubiquitination or by enhancing the expression of negative regulators, thereby achieving immune evasion.Importantly, carvedilol has been found to block the degradation of β-arrestin 2 70 and upregulate the secretion of IFN-β, suggesting that this clinically proven drug may serve as an antiviral candidate.71

Chitosan
3][74] A compelling alternative to alum is Chitosan, a biopolymer derived F I G U R E 3 Mechanism of agonists activating the cGAS-STING pathway.Metal ions and some proteins increase the sensing and binding efficiency of cGAS.Chitosan stimulates the intracellular release of DNA and thereby activates cGAS.The protein polyglutamine binding protein-1 (PQBP1) upregulates cGAMP production which leads to the activation of stimulator of interferon genes (STING) protein, while the recombinant STING proteins, such as STINGΔTM, are capable of triggering a direct activation on downstream signaling pathway and improving IFN production.Cyclic dinucleotides (CDNs) and some types of cyclic ionizable lipids directly activate the STING protein.Consequently, CDNs are encapsulated into ionizable lipids as a delivery strategy to address issues related to hydrophobicity and degradability.Nonnucleotide small molecule agonists induce a conformational change in the STING protein, leading to the activation of STING.[Color figure can be viewed at wileyonlinelibrary.com] from randomly distributed N-acetylated and deacetylated glucosamines, made from chitin. 73Chitosan exhibits significant potential as a vaccine adjuvant in mucosal settings, surpassing alum its ability to induce Th1 responses. 75portantly, studies have shown that Chitosan stimulates the intracellular release of DNA, activating cGAS and engaging the cGAS-STING pathway to promote the production of type I IFN and ISGs. 74This activation, in turn, mediates the activation and maturation of DCs, inducing robust cellular immunity.Numerous chitosan-based vaccine adjuvants have been reported, benefiting from their affordability, simple production, high biocompatibility and biodegradability.The mucoadhesive properties of chitosan make it an excellent candidate for orally and intranasally administered vaccines. 73However, a major drawback of chitosan is its low water solubility.Researchers have addressed this limitation by altering the characteristics or modifying chitosan to achieve compounds with improved water solubility.For instance, N, N, N-trimethylchitosan is a water-soluble chitosan derivative.
Additionally, chitosan can also be used as a solution or gel at different pH and concentrations by salification. 73,74her protein agonists G3BP1 increase of DNA-binding affinity.GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) plays a vital role in DNA sensing and the activation of cGAS.Accumulated evidence indicates that G3BP1 enhances the DNA binding of cGAS by promoting the formation of cGAS complexes. 75Concurrently, G3BP1 deficiency leads to inefficient cGAS-DNA binding, resulting in a decrease in cGAS-dependent IFN production.The binding of interferon stimulatory DNA (ISD) to cGAS promotes the assembly of intracellular G3BP1, after which protein kinase R and G3BP1 bind with cGAS to facilitate its DNA recognition. 76[79] TRIM21 contribution to cGAS detection in viral genomes.Tripartite motif-containing 21 (TRIM21), a member of the TRIM protein family of RING E3 ubiquitin ligases, is expressed in tumor cells. 80Functioning as an antibody-binding protein, TRIM21 controls viral infection by inducing post-entry neutralization and innate immune responses.
TRIM21 has been identified as a detector of antibody-bound virions, enhancing the sensing of antibody-bound DNA or RNA viruses by cytosolic nucleic acid sensors such as cGAS or RIG-I.Notably, TRIM21 facilitates the sensing of incoming genomes rather than progeny genomes, promoting a rapid immune response upon infection. 81,82CHC3 enhancement of cGAS sensing DNA and oligomerization.Zinc-finger (ZF) CCHC-type containing protein 3 (ZCCHC3), a CCHC-type ZF protein, acts as a co-sensor of cGAS for cytosolic dsDNA and DNA viruses in the innate immune response.ZCCHC3 directly binds to the nucleotidyl transferase (NTase) fold and the C-terminal region of cGAS, enhancing cGAS binding to dsDNA and playing a crucial role in cGAS activation during viral infection.
Additionally, ZCCHC3 promotes the self-association of cGAS, modulating cGAS oligomerization. 34As a co-sensor for the recognition of dsDNA by cGAS, the ZCCHC3-cGAS complex exhibits a much higher affinity for invading DNA viruses. 9,83BP1 enhancement of cGAS DNA sensing and oligomerization.Poly(rC)-binding protein 1 (PCBP1) is recruited to cGAS in a viral infection-dependent manner, promoting cGAS binding to its ligands through direct interaction with DNA. 84Specifically, the KH domains of PCBP1 interact with DNA, and the C-terminal fragment of cGAS interacts with PCBP1.Overexpression of PCBP1 enhances the binding of cGAS to dsDNA, while PCBP1 deficiency has the opposite effect. 84Furthermore, scientists have observed PCBP1, cGAS, and DNA forming punctate liquid-like structures, suggesting that PCBP1 is crucial for DNA-induced cGAS oligomerization and the liquid-phase condensation of cGAS.Altogether, PCBP1 serves as a crucial regulator for cGAS sensing of DNA, playing an essential player in the innate antiviral response. 84AN ET AL.
| 1775 Polyglutamine binding protein-1 (PQBP1) sensing of PAMP and enhancement of cGAMP production.PQBP1, predominantly expressed in lymphoid and myeloid cells, functions as an intracellular receptor in innate immune cells. 85Recent studies have revealed PQBP1 protein as a coreceptor for virus DNA, influencing the intensity and specificity of cGAS in DNA reorganization. 86,87For example, PQBP1 has been shown to directly target immunogenic HIV-1 DNA, demonstrating its ability to sense retroviral DNA. 87Notably, PQBP1 associates with cGAS independently of immune stimulation.The interaction between PQBP1 and STING has also been observed in the absence of cGAS, suggesting that PQBP1 acts as a bridge between cGAS and STING.
Moreover, PQBP1 has been reported to upregulate the production of cGAMP, 13 emphasizing its role in directly associating with PAMP and acting as a PRR.The formation of a complex between PQBP1 and cGAS ultimately initiates innate signaling through the cGAS-STING pathway. 88The ability of PQBP1 to recognize retroviral DNA and enhance cGAS-STING pathway activation positions it as a promising adjuvant in retroviral vaccines. 86reptavidin enhancement of DNA binding.Streptavidin, a secreted bacterial protein produced by the soil bacterium Streptomyces avidin, directly binds to cGAS, promoting DNA-induced cGAS activation and interferon-β production.The large size of cGAS prevents it from fitting into the biotin-binding pocket of streptavidin, while biotin cannot fit into the same binding pocket as cGAS. 89Interestingly, streptavidin selectively binds to full-length cGAS and promotes the activation of full-length cGAS, depending on the "YRNA" motif on streptavidin. 90

| STING agonists
CDNs CDN, characterized as the major enzymatic product of cGAS, consists of various CDN families.During interaction, cGAMP binds to STING, inducing a conformational change from an "open" to a "closed" gesture, leading to subsequent oligomerization and activation. 37Natural CDN-based STING agonists include cGAMP, c-di-GMP and c-di-AMP, with cGAMP, especially its variants (3′3′-cGAMP, 2′3′-cGAMP, 3′5′-cGAMP, and 2′5′-cGAMP), 4 being common in triggering host immune responses, particularly mucosal immunity and antitumour effects. 91,92Studies show that 2′,3′-cGAMP interacts extensively with STING, inducing stronger type I IFNs responses, 93 and 2′5′-cGAMP alsoactivates human STING due to its 2′-5′ linkage. 94Additionally, c-di-GMP-adjuvanted vaccines induce a balanced Th1 and Th2 immune response, making it a promising adjuvant candidate for vaccine development. 95administration of c-di-GMP with peptide mixture demonstrates a potent antitumour effect, 96 and other CDNs like c-di-IMP and c-di-AMP 97,98 also exhibit powerful mucosal adjuvant properties. 99wever, these CDNs face limitations, such as poor pharmacokinetic profiles, hindering their clinical use as vaccine adjuvants. 95Challenges include their large molecular mass, strong polarity, susceptibility to enzymatic hydrolysis, and limitations in co-delivery systems. 100Structural modifications have been made to pharmacokinetic profiles, and some optimized CDNs have entered clinical trials. 101,102For instance, thio-, urea-, or thioureasubstitution of phosphodiester bonds enhances resistance to hydrolytic enzymes. 103Aduro's ADU-100, with a locked nucleic-acid structure on the ribose ring, shows enhanced cellular permeability and uptake compared to ADU-S100 and 2′,3′-cGAMP. 104,105Delivering specific vaccine components to the same cellular compartment as STING may improve overall potency of CDN-based adjuvants. 106Notably, human STING polymorphisms result in varying sensitivities to different CDNs.For example, people whose alleles encode histidine at position 232 are sensitive to noncanonical CDNs rather than canonical CDNs. 93,107For those unresponsive to canonical cGAMP, cGAMP can mediate signaling through specific STING alleles via noncanonical linkages.Nevertheless, it remains unknown whether 2′,5′-3′,5′ internucleotide phosphate bridges 106,108 are also required when activated by other CDNs with a relatively higher STING binding ability, such as c-di-GMP.Another task that should be carried on before clinical evaluation is to determine the adjuvant dose-dependent safety profile in toxicology models since the systemic cytokine profile has not been evaluated in previous studies of vaccines with CDN-based adjuvants. 106

Nonnucleotide small molecule agonists
In light of STING's pivotal role in various pathological conditions, research efforts have focused on developing small molecules to modulate STING activity.One such compound, 5, 6-dimethylxanthenone-4-acetic acid (DMXAA, ASA404, or vadimezan), initially held high expectations as a tumor vascular disrupting agent. 109Unfortunately, it failed in Phase III clinical trials due to its selective bonding to mouse STING (mSTING) rather than human STING (hSTING). 110,111This setback prompted scientists to design derivatives like 10-carboxymethyl-9-acridanone (CMA). 112,113DMXAA and CMA, which induce mSTING to form a "closed" conformation mimicking the cGAMP-STING complex.However, CMA faced similar challenges and did not progress.Surprisingly, scientists discovered that xanthenone α-mangostin shares the same backbone with DMXAA and can bind to both hSTING H232 and hSTING R232, inducing higher levels of IFN-β than 2′,3′-cGAMP. 114Further studies show that α-mangostin directly binds to the hSTING C-terminal domain (CTD) and potentiates the TBK1 and IRF3 phosphorylation. 114Despite this, Quan et al. 115 noted that α-mangostin is a weak and slow hSTING agonist that induces IFN-I peaking at 6 h, compared to 2′,3′-cGAMP's 2 h peaking.Subsequent efforts should focus on developing synthetic agonist compounds capable of binding to hSTING, improving stability, and resisting enzymatic degradation. 1057][118] In a unique mode of action, two ABZI molecules bind to one STING dimer, maintaining an "open" conformation, which sets them apart from other agonists.Notably, in 2018, Glaxo-SmithKline (GSK) introduced amino benzimidazole diABZIs, 117,119 marking the first potent, nonnucleotide-based STING agonist capable of activating both mSTING and hSTING.diABZIs, a dimerization of two ABZIs, keeps STING in the same conformation but exhibits a remarkable 400 times higher affinity than cGAMP. 120Recent developments by Kamelia et al. proposed an adjuvant composition comprising diABZI-based compounds and aluminum hydroxide, aluminum phosphate or aluminum oxyhydroxide.The diABZI based adjuvant significantly increased IFN-γ, IL-2, and TNF marker-positive CD4 + T cells. 121Additionally, diABZI-4 has demonstrated efficacy in inhibiting virus replication in cells as a diABZI analogue. 122,123Xie et al. 117 explored enhancing the efficacy of the STING agonist by increasing diABZIs asymmetry.They incorporated a furan ring into benzimidazole to replace one benzimidazole moiety in diABZIs, creating a more asymmetric structure named HB3089.Structural analysis revealed that HB3089-bound human STING closely resembled the STING mutant V147L, a constitutively activated mutant found in patients with SAVI with infantile-onset.Subsequent functional analysis confirmed that SAVI-associated mutations shared a novel mode of STING activation, where the connector of LBD and transmembrane domain (TMD) sensed the activation signal and controlled conformational changes in the LBD and TMD to activate STING.Currently, HB3089 has demonstrated the ability to stimulate both mSTING and hSTING, exhibiting high anticancer activity in various mouse tumor models without causing noticeable weight loss. 124ditionally, Chin et al. introduced an imidazole-pyridazine compound named SR-001, which acts as a prodrug of the active compound SR-012.Inside cells, SR-001 rapidly converted to SR-012, which then binds to STING. 120rough structural optimization, a difluoro-substituted compound, SR-717, was developed with cellular activity equivalent to SR-001 and demonstrated binding ability to common hSTING mutants and mSTING in vitro.Notably, two molecules of SR-717 binding to a STING dimer induce a closed conformation similar to cGAMP.Studies have reported that subcutaneous administration of SR-717 in mice promotes the activation of CD8 + T cells, NK cells and DCs in tumor-related tissues.Additionally, SR-717 enhances cross-immunity to antigens, showing promise as an immunomodulator with potential applications in cancer therapy and antiviral strategies. 120A-2, a benzothiophene compound, demonstrates a noncovalent dimerization before binding to STING, inducing a "closed" conformation. 125It competes with 2′,3′-cGAMP for STING binding, leading to the secretion of IFN-β.Notably, MSA-2 is a weak acid, and its concentration is higher in tumors than in nontumour tissues or plasma after administration.This property allows MSA-2 to accumulate cytokines in tumor tissues, making it a potential candidate for cancer therapy. 4,125Based on MSA-2, Feng et al. 126 designed BSP16 using a Se insertion strategy, resulting in enhanced activation of the cGAS-STING pathway compared to MSA-2.
In a more recent development, Feng et al. 127 discovered NVS-STG2, a molecular glue-like compound, through screening a subset of the Novartis chemical library.NVS-STG2 was found to modulate the formation of high-order STING oligomers through the TMD rather than the LBD.Cryo-electron microscopy revealed that the carboxylic acid of NVS-STG2 binds to R95 in STING with a salt bridge and R94 with electrostatic interactions. 127To address this limitation, an acid-ionizable iron nano-adjuvant library with superior pH sensitivity was developed.At pH 7.4 conditions, the iron nano-adjuvants formed spherical complexes.Under endosomal pH 5.4 conditions, amorphous aggregates were formed due to acid-triggered protonation of copolymers and dissociation of iron nano-adjuvants.This acid-ionizable iron nano-adjuvant effectively amplified the STING-IFNI response, facilitated efficient antigen uptake and cross-presentation by CD169 + APCs, and induced a robust antigen-specific CD8 + T-cell response.

| Transcriptional regulation agonists
Research conducted by Liao et al. explored the mechanisms underlying the transcriptional regulation of cGAS during inflammation.Surprisingly, they identified histone deacetylase 3 (HDAC3) as a key regulator of cGAS expression. 137Acetylation at K122 was found to degrades the nuclear translocation and DNA-binding activity of p65, a protein that binds to the cGAS promoter.HDAC3 was shown to reactivate p65 by deacetylating it at K122, increasing its nuclear accumulation.This sequential process resulted in the upregulation of cGAS transcription and enhanced cGAS-STING signaling.Moreover, HDAC3 was observed to function downstream of cGAS, as the knockdown or inhibition of HDAC3 significantly reduced cGAMP-stimulated phosphorylation of STING and IRF3 in microglia. 137This suggests that HDAC3 might play a role downstream of the cGAS-STING pathway, and further investigations are needed to elucidate the exact mechanism of HDAC3 regulation in the STING-TBK1-IRF3 complex.
Additionally, G10 was identified as a compound that activates IRF3 rather than the classical NF-κB pathway in human fibroblasts. 138Further examination revealed that G10 does not directly bind to STING but induces the expression of multiple IRF3-dependent antiviral effector genes and IFN, effectively blocking the replication of emerging α-virus species.Thus, G10 is considered a synthetic indirect hSTING activator, and its mechanism of action warrants further investigation.

| Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibitors
ENPP1 is a type II transmembrane glycoprotein that plays a role in downregulating STING signaling by hydrolyzing extracellular cGAMP. 103This activity of ENPP1 inhibits the activation of innate immunity, promoted tumor metastasis, facilitates immune evasion, and attenuates the tumor-killing effects of antitumour therapies like radiotherapy. 139Therefore, there is a need to develop ENPP1 inhibitors to counteract its negative regulation of the STING pathway.Several nucleotides and non-nucleotide ENPP1 inhibitors have been developed, including diadenosine poly-phosphonate derivatives, 140 methylene-triphosphate analogue, 141 imidazopyridine-and purinethioacetamide derivatives, 142 quinazolin-4-piperidine-4-methyl sulfamides and its derivatives, 143 lead phosphonate compounds. 144However, many of these inhibitors exhibit poor pharmacokinetic parameters (absorption, distribution, metabolism and elimination [ADME]) and short half-lives.A series of ENPP1 inhibitors based on thioguanine scaffolds, developed by Carozza et al., 144 shows promising results with a straightforward synthetic route, metabolic stability, excellent ADME parameters in vitro, minimal inhibition of cytochrome P450, and outstanding selectivity.Some of these inhibitors have been tested on LLC1 lung cancer homologous mouse models, demonstrating remarkable efficacy.
In summary, STING agonists in different formulations have been widely used as vaccine adjuvants against infectious diseases and cancers.A summary of their advancement is provided in Table 1.

| Biocompatible delivery strategies of adjuvants
Despite the discovery of numerous cGAS-STING pathway agonists with various mechanisms of action, their clinical applications as vaccine adjuvants face significant challenges.Barriers to the efficacy of exogenous CDNs include hydrophilicity, poor cellular targeting, rapid clearance, and inefficient cell membrane transport. 106,146,147ditionally, small-molecule STING agonists, when administered at a large scale locally or systemically, may cause off-target inflammation or autoimmunity. 148Therefore, it is crucial to develop suitable delivery systems for these agonists to alleviate or avoid these limitations.

| Biomembrane-based strategies
Nanocarriers can protect agonists such as CDNs from degradation and enhance drug accumulation at tumor sites by enhanced permeability and retention effect. 149On the one hand, cationic polymeric nanoparticles (NPs) or cationic liposomes can help CDNs achieve lysosomal escape through the "proton sponge effect."On the other hand, they also enhance the cellular uptake of CDNs through electrostatic adsorption to achieve full contact with cell membranes. 148Furthermore, it can increase ligand-modified nanocarrier uptake of target tissue via receptormediated endocytosis, reducing systemic toxicities. 148,150eng et al. reported an intracellular delivery system for cGAMP using NPs to promote innate immune activation and antitumour efficacy. 151,152Compared to soluble cGAMP, cGAMP-NP co-delivery was more effective in augmenting pro-inflammatory cytokines production, inducing the secretion of IFN-I and increasing CD8 + T cells.
Recently, a membrane-soluble, deformable oncology drug delivery vehicle was reported which conjugated CDN prodrug with PEG-phospholipid.Once being absorbed into cells, the CDN-PEG-lipid was cleavaged by peptidase in endosomes and the STING agonist was released. 153Dosta et al. 154 developed a covalent conjugate of CDNs with poly(β-amino ester) NPs through a cathepsin-sensitive linker, which demonstrated enhanced stability and prolonged half-life in vivo.In another study, a dual-delivery strategy of peptides and CDNs, combined with synthetic long peptide antigens, was constructed using polymer vesicles known as polymersomes.These polymersomes exhibit pH-responsive and membrane-destabilizing activity.The membrane-destabilizing block is enclosed within the polymersome bilayer and protected by a poly (ethylene glycol) corona under physiological pH conditions.Upon endocytosis and acidification, the NPs undergo rapid discomposition, releasing CDNs into the cytosol.The nanoSTING-vax system enhances intracellular uptake and cytosolic delivery of CDNs, thereby promoting CD8 + T cell responses to a variety of peptide antigens. 147,155tracellular vesicles (EVs) constitute a heterogeneous group of cell-derived membranous structures, including exosomes and microvesicles.Originated from the endosomal system, 156 EVs transport various "cargo" such as proteins and nucleic acids, influencing the function and behavior of recipient cells.Among them, exosomes exhibit cellular messenger properties and possess high biocompatibility, making them favorable as delivery carriers.A new autophagosome named Rafeesome is secreted in RAB22A-induced extracellular vesicle (R-EV), designed to package activated STING. 157These activated STING-containing EVs induce IFN-β expression in recipient cells, facilitating T cell infiltration to combat tumor cells.Codiak BioSciences recently reported positive data for its innovative engineered exosome therapy candidate, exoSTING (CDK-002), currently undergoing the first phase 1/2 clinical trial for monotherapy in solid tumors.exoSTING precisely delivers STING agonists via exosomes to tumor-resident APCs.Pharmacodynamic data suggests that exoSTING's intertumoral delivery is over 100 times more potent than previous clinical STING agonists, highlighting its potential to overcome the systemic toxicities associated with some STING agonists. 158

| Peptide-based strategies
Limited packaging capacity and storage instability pose challenges for lipid nanoparticles (LNPs) in encapsulating small hydrophilic molecules.Dosta et al. addressed this issue by modifying Polyamidoamine dendrimers with polypeptide moieties, serving a dual-purpose of enhancing the cationic nature of the complex and replacing external amines with biocompatible amino acids.This modification significantly attenuates toxicity and achieves higher CDNs complexation efficiency. 159ditionally, Yildiz et al. reported that complexing c-di-GMP with arginine peptides improves its delivery and immune stimulation in murine cells.They utilized various molar ratios of cationic polyarginine (nona-L-arginine) with anionic CDG to construct diverse molecular structures, enhancing the stability and cellular uptake of the complex. 160rthermore, Hartgerink et al. introduced a novel peptide hydrogel-based delivery system named STINGel to deliver CDNs, such as ADU-S100, based on the study of multidomain peptides.The peptide hydrogels facilitate drug delivery, remaining localized and undergoing complete cellular infiltration to maximize matrix-tissue interaction. 161Peptide sequences are designed to allow charged small molecule drugs to be incorporated through nanofiber cross-linking for extended release, potentially improving the efficacy of CDNs in challenging, refractory tumor models. 162,163

| Protein-based strategies
Antibody drug conjugates (ADCs) hold a significant market value due to their precise targeting ability and high biocompatibility.STING-based ADC drugs address the systemic toxicity drawbacks of small-molecule STING agonists and demonstrate targeted delivery into multiple tumor tissues. 162,164Notably, TAK-500 by Takeda 165 and XMT-2056 by Mersana 166 are both in phase 1 clinical trials.Meanwhile, JAB-X1800 by Jacobio Pharmaceuticals 167 is undergoing preclinical studies, and F-star Therapeutic has also disclosed their preclinical study on an ADC with STING agonist. 168TAK-500 is an ADC comprising a STING agonist conjugated to TAK-676, which will be tested as a single agent and in combination with pembrolizumab in adult patients with selected locally advanced or metastatic solid tumors. 169XMT-2056, an ADC developed by Mersana that targets human epidermal growth factor receptor 2 (HER2), provides targeted delivery of the STING agonist diABZI for potential treatment of multiple solid tumors.It is received Orphan Drug designation for treating gastric cancer by Food and Drug Administration (FDA), 170 showing increased CXCL10 induction.Preclinical data reveal that XMT-2056 exhibits potent STING activity with over a 100-fold improvement in activity compared to the free STING-agonist payload. 162,166cept for antibodies, other proteins can also be utilized to deliver agonists.Human heavy chain ferritin NPs disassemble into subunits when the pH is lowered to 2-3 and reassemble to an almost original form when the pH returns to physiological conditions. 169This expands its drug loading and delivery capacity, showing excellent biocompatibility and biodegradability. 170 Ultimately, elevated recruitment of immune cells and improved mRNA productions of pro-inflammatory cytokines lead to the effective inhibition of glioma growth and improved survival rates of glioma mice. 171nother elegant example is STING protein, which has a more efficient affinity with its agonist than other proteins.It can directly activate downstream signaling, by passing the need for endogenous STING expression.
Using an innovative approach, He et al. found that TM-deficient STING proteins form a self-assembled quaternary structure with cGAMP (cGAMP-STINGΔTM).Additionally, this ribonucleoprotein complex is found to efficiently trigger the cGAS-STING signaling pathway when delivered to cells independent of endogenous STING status. 172As a result, cGAMP-STINGΔTM effectively induces IFN production in STING-deficient cells, potently sitmulating the secretion of antigen-specific antibodies and T-cell responses and thereby improving efficiency in the treatment of melanoma and colon cancer. 173

| Neutrophils (NEs)-based strategies
]174 Unlike NEs cytopharmaceuticals with cargos inside the cells, Hao et al. 175  protein subunit vaccine with CDNs as adjuvants, demonstrating protective immunity against Mycobacterium tuberculosis in mice comparable to the live-attenuated vaccine bacillus Calmette-Guérin.Notably, the protein subunit vaccine achieved superior protection through the induction of Th1 and Th17 immune responses in a T celldependent manner when administered intranasally. 178In another study by Su et al., a series of adjuvants for HBV vaccines were investigated, revealing that c-di-AMP exhibited potent immune stimulatory effects on DC.This adjuvant even caused complete clearance of HBV antigens in HBV-carrier mice, showcasing its potential in developing effective vaccines against HBV. 179wever, the widespread distribution of cGAS-STING agonists from the injection site throughout the entire organism raises concerns about potential toxic effects.Therefore, it is imperative to design an appropriate delivery system that co-delivers CDNs with antigens in a targeted or slow and continuous manner to enhance the safety of vaccination. 4,30Indeed, studies have reported that CDN-adjuvanted influenza vaccines, in the form of microneedles with sustained local release of influenza antigens, significantly reduce systemic toxicity and achieve higher efficacy compared to vaccines without CDN adjuvant. 180Furthermore, Lirussi et al. discovered that neonatal vaccination with cyclic di-adenosine monophosphate (CDA) + ovalbumin (OVA) leads to higher antigen-specific IgG titers compared to vaccinations with monophosphoryl lipid A or CpG as adjuvants.According to their study, CDA elicits more Th1 and Tfh cytokines and higher antigen-specific IgG titers than R848, considered the "gold standard" neonatal adjuvant.This suggests that CDA has promising potential as an adjuvant in vaccines for newborns, potentially reducing antigen load or the required vaccine doses to achieve protective immunity. 181imilarly, to enhance cellular uptake, Batty et al. encapsulated cGAMP into microparticles (MPs) composed of acetylated dextran (Ace-DEX), a biocompatible polymer that degrades in the endo/lysosome, releasing the cargo inside.The adjuvant solution is ionized and atomized in a capillary tube, and as the solvent evaporates, the remaining micron and nano-sized particles are electrospray Ace-DEX particles with encapsulated cGAMP.This approach resulted in a significantly more robust interferon production. 182portantly, STING agonists have demonstrated increased therapeutic effects in antiviral vaccines during the COVID-19 pandemic.As discussed in the previous section, the activation of STING matures DCs and T cells, triggering potent humoral immune responses and increasing antibody titers, contributing to the efficacy of vaccineinduced virus clearance. 53,54,56An et al. 183 developed an intranasal subunit vaccine composed of liposomal cGAMP and lyophilized S protein, eliciting comprehensive immune responses, including mucosal immunity in the lung and other tissues.Additionally, S protein and cGAMP were combined with a mucoadhesive nano delivery platform, intranasally administered to stimulate profound mucosal immunities in the upper respiratory tract.This approach resulted in a total concentration of antibodies and T cells, reducing viral shedding and prohibiting the virus from invading deep into the host respiratory system. 184,185Apart from cGAMP, other small molecular STIGN agonists could also function as adjuvants for antiviral vaccines.As adjuvants, the STING agonist CF501 was combined with human IgG Fc-conjugated receptor binding domain (RBD) from the original SARS-CoV-2 strain, inducing highly potent and durable protective immunity responses without apparent biological toxicity. 123,186As mentioned earlier, Mn 2+ NPs can enhance retention and cellular uptake, leading to prolonged activation of the cGAS-STING pathway after injection.Zhang et al. 187 found that NP manganese was the most potent adjuvant in stimulating immune responses to SARS-CoV-2 protein-based subunit vaccines, as evidenced by the concentration of RBD-specific IgG and specific neutralizing antibodies of SARS-CoV-2 and variants in mouse models.Zhou et al. sequentially prepared a nanoparticle (nano-MnP) with long-term stability, distinct pH-sensitivity, and biodegradability, employing them as STING agonist adjuvants in the bivalent COVID-19 protein vaccine based on S1-WT and S1-Omicron proteins, eliciting a broad-spectrum immunity. 188

| cGAS-STING agonist as a cancer vaccine adjuvant
Vaccination has emerged as a promising treatment option for many types of cancer.Despite profound progress, it still has limitations and falls short of being ideal.In light of the inadequate immunogenicity of antigens, the search for available adjuvants remains a potent driving force for developing antitumour vaccines.However, safe and effective immunotherapeutic adjuvants are still an unmet medical need in cancer.0][191] Among these, the cGAS-STING pathway agonist is considered a promising adjuvant for anticancer vaccines.
For example, Corrales et al. 192 found that the synthetic CDN (ML RR-S2 CDA) induced lasting immunemediated antitumour activity in B16-F10 melanoma cell-triggered lung metastases.Furthermore, Nakamura et al. 193 demonstrated that the c-di-GMP-loaded liposomes showed potent antitumour activity mediated by NK cells in a B16-F10 lung metastasis model.Another study also pointed out that liposomes encapsulating cGAMP with phosphatidylserine are taken up by APC, activating the STING pathway and CD8 + T cell cross-stimulation.When synergized with radiation therapy, this combination can elicit antitumour activity in a B16-OVA melanoma lung metastasis model. 194Moreover, a recent study reported the application of SR-717 agonist as a novel adjuvant for a recombinant protein vaccine against coronavirus, claiming to potentiate both body fluid and cellular immune reactions induced by the vaccine and contribute to the preparation of neutralizing antibodies. 195 addition to small molecules, the STING protein can also serve as an adjuvant carrier.He et al. recently   reported their study of a fully functional platform for the delivery of cGAMP that utilizes STING proteins lacking transmembrane components (STINGΔTM) to self-assemble with cGAMP into the cell membrane under physiological conditions.The combination of the antigen epitope, adjuvant cGAMP, and STINGΔTM as a carrier has been reported to effectively trigger the STING signaling pathway in cell lines with or without STING, consequently resulting in the stimulation of IFN-I secretion, activation of APCs, antigen-specific T-cell responses, and the transformation to the inguinal lymph nodes.These results demonstrate the potential of this platform in stimulating draining lymph node trafficking and T cells priming of peptide vaccines. 196reover, mutations in STING have been studied as a new adjuvant.Tse et al. elucidated that the active mutation STINGV155M from a patient with SAVI could induce antigen-specific CD8 + T cell responses through the strong activation of NF-kB, IRF3, and IRF7 as an adjuvant in mRNA vaccines.Building on their previous study, they designed an LNP-encapsulated mRNA vaccine with STINGV155M as an adjuvant.Surprisingly, they found that the antigen/ STINGV155M mass ratio (5:1) enhances the capacity of antigen-specific CD8 + T cell responses, thus increasing the efficacy of mRNA vaccines for tumors in murine tumor models and the murine lung metastasis model. 197 has been reported that layered double hydroxide (LDH) depletes excessive H+ and neutralizes the tumor's acidic extracellular and intracellular endo/lysosomal microenvironment. 14 enhancing antigen presentation and stimulating CTLs, which helped overcome primary, distant and metastatic tumors. 198As for manganese, the OVA/MnO 2 nano-vaccine induced more potent cellular immunity with enhanced T cell proliferation capabilities and the upregulation of TNF-α and IFN-g, exhibiting potent preventive and therapeutic antitumour efficacies. 199ong various PRR families that have been identified, TLRs are the most extensively studied.The combination of STING agonists with TLR agonists has also become a research hotspot with some progress. 200Hu et al. designed a complex Pam3CSK4-CDGSF based on STING and TLR1/2 agonists to be co-administered with the antigen OVA.
By conjugating CDGSF to Pam3CSK4, the complex enhanced the antigen-specific immune response, resulting in the induction of both humoral immunity and cellular immunity for tumor treatment. 201Besides, it has been reported that the combination of cGAMP with the TLR9 agonist CpG led to increased IL-12 production, 160,202 and liposomal c-di-GMP with MPL (an FDA-approved TLR4 agonist) resulted in improved humoral and cellular immunity. 148llier et al. co-delivered cGAMP with TLR7/8 agonist R848 and found that cGAMP/R848 Ace-DEX MPs upregulated the production of IFN-γ, IL-2, IL-6, and TNF, induced high overall IgG1 and IgG2c titers, and enhanced Th2-type responses by dual activation of the immune system.Thus, cGAMP/R848 Ace-DEX MPs are an excellent adjuvant system stimulating solid immune responses when administered at shallow doses. 203In another study, a pH-sensitive liposome has been developed to co-encapsulate both the TLR9 ligands oligodeoxynucleotides (ODNs) expressing unmethylated CpG motifs (CpG-ODNs) and cGAMP, which increases the production of type I and type II IFNs from splenocytes and elevates IL-12 and IL-6 production from bone marrow-derived macrophages and bone marrow-derived dendritic cells.Consequently, it reduces tumor growth by approximately 70%. 204Remarkably, Temizoz et al. 205 found that the combined treatment of TLR9 and STING ligands achieved a 100% survival rate in mice rechallenged with Pan02 tumors.Notably, OncoNano Medicine is investigating ONM-501, a therapeutic cancer vaccine that uses a synthetic polymer nanoparticle (PC7A NP) serving as the STIGN agonist to encapsulate tumor antigens.Preclinical data demonstrated that the nano-vaccine restrained tumor growth and prolonged the survival of animals.Especially the combination of the PC7A nano-vaccine and an anti-PD-1 antibody showed promising synergy with a 100% survival rate over 60 days in a TC-1 tumor model. 206Moreover, ONM-500, a vaccine consisting of HPV-specific antigens and a STING adjuvant, is also regarded as a potential treatment for HPV-related cancers.The antigens and the STING agonists were packaged into intradermally delivered pH-sensitive micelles, and the antigens were delivered to APCs in the lymph node, significantly inhibiting tumor growth, which has been observed in the preclinical study. 207Another vaccine adjuvant under trial is NBTXR-3, which has been previously developed into a complex consisting of hafnium oxide crystal NPs for the potential use as a radiosensitizer in the treatment of cancer.

4. 2 |
Indirect agonists 4.2.1 | Adjuvant that promote the production of intracellular reactive oxygen species (ROS) Iron oxide nanoparticles (IONPs) have been investigated for their potential as adjuvants in cancer immunotherapy.IONPs are able to promote T cell immunity by inducing ferroptosis of cancer cells and repolarization of tumorassociated macrophages. 129-133Chen et al. 134 developed an acid-ionizable iron nano-adjuvant library containing IONPs and the STING agonist MSA-2, creating a potent and versatile vaccine platform for personalized cancer immunotherapy.The immunostimulatory effects of IONPs were evaluated in HEK293 cells expressing human STING variants (hSTINGR232 and hSTINGH232).The study observed a significant production of IFN-β in response F I G U R E 4 Structures of compounds as stimulator of interferon genes (STING) agonists.(A) ADU-S100 is the first atypical cyclic dinucleotides (CDNs) to enter clinical trial for solid tumor and lymphoma.It induces a more potent IFN response than cGAMP.(B) DMXAA and CMA are STING agonists with different structural scaffolds, but they also have essentially the same activation conformation upon binding to mouse STING (mSTING) rather than human STING (hSTING).α-Mangostin has the same skeletal structure with DMXAA, but it has been confirmed to be an hSTING agonist.(C) diABZIs is the dimerization of two amidobenzimidazoles (ABZIs).Glaxo-SmithKline identified ABZI, the first potent, nonnucleotide, agonist of mSTING and hSTING, by screening for small molecules competing with 3H-cGAMP for binding to STING CTDs.(D) SR-717 is a lead from nonnucleotide and a cGAMP-mimetic small molecule.(E) Benzo[b]selenophene (BSP) compound BSP16 was discovered as a novel STING agonist based on MSA-2.(F) A class of compounds named NVS-STGs were discovered through functional screens to be hSTING agonists, among which NVS-STG2 induces the high-order oligomerization of hSTING by binding transmembrane domain of STING.[Color figure can be viewed at wileyonlinelibrary.com] to MSA-2, indicating an amplification effect of the STING cascade induced by IONPs, even in the presence of human STING variants.Interestingly, experiments revealed that IONPs induced the secretion of pro-inflammatory cytokines in an iron concentration-dependent manner but IONPs alone cannot activate the IFN-1 response even at high concentrations.Surprisingly, Chen et al. discovered that IONPs induced a STING-independent immunostimulatory effect by augmenting the production of intracellular ROS and triggering NF-κB activation.Moreover, this is further enhanced by the phosphorylation of IRF3 in an MSA-2-dependent manner, eventually amplifying STING activation and IFN-I production.Systemic administration of a mixture of IONPs and STING agonists faced challenges due to suboptimal co-delivery to secondary lymphatic organs where immunomodulation is coordinated.
With this technology, Wang et al. successfully delivered SR-717 into an orthotopic brain tumor model, consequently upregulating the expression of STING signaling-related proteins.
developed a tumor-penetrating neotype NE cytopharmaceutical with liposomal STING agonists conjugated on the surface of NEs through a reduction-click engineering method, which effectively activates the STING pathway and reinvigorates the tumor environment by converting macrophages and NEs to antitumour phenotypes. 175-1775 | VACCINATION WITH cGAS-STING AGONISTS AS ADJUVANTS 5.1 | cGAS-STING agonist as an antiviral vaccine adjuvant The cGAS-STING pathway plays a crucial role in triggering immune responses to eliminate infections.Recent research has extensively explored the role of cGAS-STING pathway-associated agonists in various infectious diseases.Van Dis et al. observed that CDN-adjuvanted vaccines could stimulate CD4 + T cells, enhancing protection against tuberculosis by infiltrating macrophage lesions in the lungs.Subsequent studies led to the development of a Zhang et al. synthesized a Zn 2+ doped LDH (Zn-LDH)-based immunomodulating adjuvant.The Zn-OH binds in Zn-LDH hydrolyzed in the acidic TME and upregulated the pH of the tumor tissues to release Zn 2+ , contributing to the activation of the tumor-resident immune cells.Then, the tumor-associated antigens released from dead tumor cells are captured by Zn-LDH and transported to tumor-associated draining lymph nodes.In contrast, dead tumor cells were phagocytosed, thereby List of cGAS-STING agonists under development.145 | 1781 T A B L E 1 (Continued) | 1785