5,6-dimethylxanthenone-4-acetic acid (DMXAA), a partial STING agonist, competes for human STING activation.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative-3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)-that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Machine Learning-Assisted Screening of Herbal Medicine Extracts as Vaccine Adjuvants.

Adjuvants are important vaccine components, composed of a variety of chemical and biological materials that enhance the vaccine antigen-specific immune responses by stimulating the innate immune cells in both direct and indirect manners to produce a variety cytokines, chemokines, and growth factors. It has been developed by empirical methods for decades and considered difficult to choose a single screening method for an ideal vaccine adjuvant, due to their diverse biochemical characteristics, complex mechanisms of, and species specificity for their adjuvanticity. We therefore established a robust adjuvant screening strategy by combining multiparametric analysis of adjuvanticity in vivo and immunological profiles in vitro (such as cytokines, chemokines, and growth factor secretion) of various library compounds derived from hot-water extracts of herbal medicines, together with their diverse distribution of nano-sized physical particle properties with a machine learning algorithm. By combining multiparametric analysis with a machine learning algorithm such as rCCA, sparse-PLS, and DIABLO, we identified that human G-CSF and mouse RANTES, produced upon adjuvant stimulation in vitro, are the most robust biological parameters that can predict the adjuvanticity of various library compounds. Notably, we revealed a certain nano-sized particle population that functioned as an independent negative parameter to adjuvanticity. Finally, we proved that the two-step strategy pairing the negative and positive parameters significantly improved the efficacy of screening and a screening strategy applying principal component analysis using the identified parameters. These novel parameters we identified for adjuvant screening by machine learning with multiple biological and physical parameters may provide new insights into the future development of effective and safe adjuvants for human use.

Anti-tumor immunity by transcriptional synergy between TLR9 and STING activation.

Agonists for TLR9 and stimulator of IFN genes (STING) offer therapeutic applications as both anti-tumor agents and vaccine adjuvants, though their clinical applications are limited; the clinically available TLR9 agonist is a weak IFN inducer and STING agonists induce undesired type 2 immunity. Yet, combining TLR9 and STING agonists overcame these limitations by synergistically inducing innate and adaptive IFNγ to become an advantageous type 1 adjuvant, suppressing type 2 immunity, in addition to exerting robust anti-tumor activities when used as a monotherapeutic agent for cancer immunotherapy. Here, we sought to decipher the immunological mechanisms behind the synergism mediated by TLR9 and STING agonists and found that their potent anti-tumor immunity in a Pan02 peritoneal dissemination model of pancreatic cancer was achieved only when agonists for TLR9 and STING were administered locally, and was via mechanisms involving CD4 and CD8 T cells as well as the co-operative action of IL-12 and type I IFNs. Rechallenge studies of long-term cancer survivors suggested that the elicitation of Pan02-specific memory responses provides protection against the secondary tumor challenge. Mechanistically, we found that TLR9 and STING agonists synergistically induce IL-12 and type I IFN production in murine APCs. The synergistic effect of the TLR9 and STING agonists on IL-12p40 was at protein, mRNA and promoter activation levels, and transcriptional regulation was mediated by a 200 bp region situated 983 bp upstream of the IL-12p40 transcription initiation site. Such intracellular transcriptional synergy may hold a key in successful cancer immunotherapy and provide further insights into dual agonism of innate immune sensors during host homeostasis and diseases.

B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice.

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

Type I and II interferons toward ideal vaccine and immunotherapy.

Introduction: Innate immunity is armed with interferons (IFNs) that link innate immunity to adaptive immunity to generate long-term and protective immune responses against invading pathogens and tumors. However, regulation of IFN production is crucial because chronic IFN responses can have deleterious effects on both antitumor and antimicrobial immunity in addition to provoking autoinflammatory or autoimmune conditions.Areas covered: Here, we focus on the accumulated evidence on antimicrobial and antitumor activities of type I and II IFNs. We first summarize the intracellular and intercellular mechanisms regulating IFN production and signaling. Then, we discuss the mechanisms modulating the dual nature of IFNs for both antitumor and antimicrobial immune responses. Finally, we review the detrimental role of IFNs for induction of autoinflammation and autoimmunity.Expert opinion: The current evidence suggests that the dual role of IFNs for antimicrobial and antitumor immunity is dependent not only on the timing, administration route, and dose of IFNs but also on the type of pathogen/tumor. Therefore, we think that combinatorial therapies involving IFN-inducing adjuvants and immune-checkpoint blockers may offer therapeutic potential, especially for cancer, whereas infectious, autoinflammatory or autoimmune diseases require fine adjustment of timing, dose, and route of the administration for candidate IFN-based vaccines or immunotherapies.

Cyclic GMP-AMP Triggers Asthma in an IL-33-Dependent Manner That Is Blocked by Amlexanox, a TBK1 Inhibitor.

Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro, and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33-/- mice, but also Sting-/-, Tbk1-/-, and Irf3-/-Irf7-/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Combination and inducible adjuvants targeting nucleic acid sensors.

Innate immune sensing of nucleic acids derived from invading pathogens or tumor cells via pattern recognition receptors is crucial for mounting protective immune responses against infectious disease and cancer. Recently, discovery of tremendous amounts of nucleic acid sensors as well as identification of natural and synthetic ligands for these receptors revealed the potential of adjuvants targeting nucleic acid sensing pathways for designing efficacious vaccines. Especially, current data indicated that unique adjuvants targeting TLR9 and stimulator of interferon genes (STING)-dependent cytosolic nucleic acid sensing pathways along with the combinations of already existing adjuvants are promising candidates for this purpose. Here, we review current vaccine adjuvants targeting nucleic acid sensors and their modes of action.

Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Circulating nano-particulate TLR9 agonist scouts out tumor microenvironment to release immunogenic dead tumor cells.

Recent evidence suggest that a ß-glucan derived from mushroom Schizophyllan(SPG) complexed with a humanized TLR9 agonistic CpG DNA, K3 (K3-SPG) is a promising vaccine adjuvant that induces robust CD8 T cell responses to co-administered antigen. However, it has not been investigated whether K3-SPG alone can act as an anti-cancer immunotherapeutic agent or not. Here, we demonstrate that intravenous injection of K3-SPG, but not CpG alone, is accumulated in the tumor microenvironment and triggered immunogenic cell death (ICD) of tumor cells by local induction of type-I interferon (IFN) as well as IL-12. Resultant innate immune activation as well as subsequent tumor-specific CD8 T cell responses were contributed the tumor growth suppression. This anti-tumor effect of K3-SPG monotherapy was also confirmed by using various tumor models including pancreatic cancer peritoneal dissemination model. Taken together, nano-particulate TLR9 agonist injected intravenously can scout out tumor microenvironment to provoke local innate immune activation and release dead tumor cells into circulation that may induce broader and protective tumor antigen-specific CD8 T cells.

Vaccine adjuvants as potential cancer immunotherapeutics.

Accumulated evidence obtained from various clinical trials and animal studies suggested that cancer vaccines need better adjuvants than those that are currently licensed, which include the most commonly used alum and incomplete Freund's adjuvant, because of either a lack of potent anti-tumor immunity or the induction of undesired immunity. Several clinical trials using immunostimulatory adjuvants, particularly agonistic as well as non-agonistic ligands for TLRs, C-type lectin receptors, retinoic acid-inducible gene I-like receptors and stimulator of interferon genes, have revealed their therapeutic potential not only as vaccine adjuvants but also as anti-tumor agents. Recently, combinations of such immunostimulatory or immunomodulatory adjuvants have shown superior efficacy over their singular use, suggesting that seeking optimal combinations of the currently available or well-characterized adjuvants may provide a better chance for the development of novel adjuvants for cancer immunotherapy.

Species-dependent role of type I IFNs and IL-12 in the CTL response induced by humanized CpG complexed with ß-glucan.

CpG oligodeoxynucleotide (ODN) is one of promising nucleic acid-based adjuvants. We recently improved its ability to enhance CD8(+) T-cell responses to coadministered protein antigen without conjugation or emulsion, by forming a nanoparticulate complex between CpG ODN (K3) and mushroom-derived ß-glucan schizophyllan (SPG), namely K3-SPG. Here, we sought to elucidate the cellular immunological mechanisms by which K3-SPG induce such potent CD8(+) T-cell responses to coadministered antigen. By focusing on two DC subsets, plasmacytoid DCs and CD8α(+) DCs, as well as the secreted cytokines, IFN-α and IL-12, we found that K3-SPG strongly activates mouse plasmacytoid DCs to secrete IFN-α and CD8α(+) DCs to secrete IL-12, respectively. Although a single cytokine deficiency had no impact on adjuvant effects, the lack of both type I IFN and IL-12 in mice resulted in a significant reduction of Th1 type immune responses and CD8(+) T-cell responses elicited by protein vaccine model. By sharp contrast, type I IFN, but not IL-12, was required for the production of IFN-γ by human PBMCs as well as antigen-specific CD8(+) T-cell proliferation. Taken together, K3-SPG may overcome the species barrier for CpG ODN to enhance antigen-specific CD8(+) T-cell responses despite the differential role of IL-12 between human and mice.

TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN.

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type-1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K-type) induces IFNs only weakly and STING ligands rather induce type-2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type-1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING-ligand cyclic GMP-AMP (cGAMP), culminating in NK cell IFN-γ (type-II IFN) production, is due to the concurrent effects of IL-12 and type-I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type-1 adjuvant, capable of inducing strong Th 1-type responses, as demonstrated by enhanced antigen-specific IgG2c and IFN-γ production, as well as cytotoxic CD8(+) T-cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen-free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type-II IFN inducer.