The Defined TLR3 Agonist, Nexavant, Exhibits Anti-Cancer Efficacy and Potentiates Anti-PD-1 Antibody Therapy by Enhancing Immune Cell Infiltration.

Nexavant was reported as an alternative to the TLR3 agonist of Poly(I:C) and its derivatives. The physicochemical properties, signaling pathways, anti-cancer effects, and mechanisms of Nexavant were investigated. The distinctive characteristics of Nexavant compared to that of Poly(I:C) were demonstrated by precise quantification, enhanced thermostability, and increased resistance to RNase A. Unlike Poly(I:C), which activates TLR3, RIG-I, and MDA5, Nexavant stimulates signaling through TLR3 and RIG-I but not through MDA5. Compared to Poly(I:C), an intratumoral Nexavant treatment led to a unique immune response, immune cell infiltration, and suppression of tumor growth in various animal cancer models. Nexavant therapy outperformed anti-PD-1 antibody treatment in all the tested models and showed a synergistic effect in combinational therapy, especially in well-defined cold tumor models. The effect was similar to that of nivolumab in a humanized mouse model. Intranasal instillation of Nexavant led to the recruitment of immune cells (NK, CD4+ T, and CD8+ T) to the lungs, suppressing lung metastasis and improving animal survival. Our study highlighted Nexavant's defined nature for clinical use and unique signaling pathways and its potential as a standalone anti-cancer agent or in combination with anti-PD-1 antibodies.

STING activation counters glioblastoma by vascular alteration and immune surveillance.

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

The cGAS-STING pathway is a therapeutic target in a preclinical model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and the incidence of HCC is increasing. Recently, cancer immunotherapy has emerged as an efficient treatment against some cancers. Here we have used a mouse model of mutagen-induced HCC to explore the therapeutic usefulness of targeting the DNA-activated STING pathway in HCC. STING-deficient mice exhibited unaltered initial development of HCC, but had higher number of large tumors at late stages of disease. In the liver of STING-deficient HCC mice, we observed reduced levels of phospho-STAT1, autophagy, and cleaved caspase3. These responses were activated in the liver by treatment with a cyclic dinucleotide (CDN) STING agonist. Importantly, CDN treatment of mice after HCC development efficiently reduced tumor size. Initiation of CDN treatment at an even later stage of disease to allow HCC detection by MR scanning revealed that the majority of tumors regressed in response to CDN, but new tumors were also detected, which were unresponsive to CDN treatment. Overall, the modulation of the STING pathway affects the development of HCC, and holds promise for a use as a treatment of this disease, most likely in combination with other immunomodulatory treatments such as PD1 inhibitors or with standard of care.

The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells.

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice.

In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2'3'-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2'3'-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice in vivo, without stimulating significant inflammatory gene expression. Systemic treatment with 2'3'-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections in vivo.

Human monocyte recognition of adenosine-based cyclic dinucleotides unveils the A2a Gαs protein-coupled receptor tonic inhibition of mitochondrially induced cell death.

Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cells in vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a Gαs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic. In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34(+) cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a Gαs protein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.

In vivo studies of dialkynoyl analogues of DOTAP demonstrate improved gene transfer efficiency of cationic liposomes in mouse lung.

A novel set of dialkynoyl analogues of the cationic, gene delivery lipid DOTAP (1) was synthesized. Structure-activity studies demonstrate that replacement of the cis-double bonds of DOTAP with triple bonds in varying positions alters both the physical properties of the resultant cationic liposome-DNA complexes and their biological functionalities, both in vitro and in vivo. Particularly, in vivo studies demonstrate that pDNA transfection of mouse lung endothelial cells with lead analogue DS(14-yne)TAP (4):cholesterol lipoplexes exhibits double the transfection level with less associated toxicity relative to the well-established DOTAP:cholesterol system. In fact, 4:cholesterol delivers up to 3 times the dose of pDNA in mice than can be tolerated by DOTAP, leading to nearly 3 times greater marker-gene expression. X-ray diffraction studies suggest that lipoplexes containing analogue 4 display increased stability at physiological temperatures. Our results thus suggest that analogue 4 is a potentially strong candidate for the gene therapy of lung tumors.

Synthesis and formulation of neoglycolipids for the functionalization of liposomes and lipoplexes.

Novel carbohydrate-based agents for the stabilization of ternary liposome:mu:DNA (LMD) nonviral vector systems are described. LMD vector systems comprise plasmid DNA (pDNA; D,7.5 kb) expressing a reporter gene (in this instance beta-galactosidase expressing gene) that is precondensed with the adenoviral core peptide mu (mu, M; MRRAHHRRRRASHRRMRGG) and then further packaged by means of DC-Chol:DOPE (3:2; m/m) cationic liposomes. Final optimized lipid:mu:pDNA ratio is typically 12:0.6:1 (w/w/w). We report the synthesis of a series of nine neoglycolipids prepared by coupling completely unprotected sugar monomers or oligomers (mannose, glucose, galactose, glucuronic acid, maltose, lactose, maltotriose, maltotetraose, and maltoheptaose) through their reducing-residue termini to an aminoxy-functionalized cholesterol-based lipid. Characterization of these novel neoglycolipids by (1)H NMR reveals that the coupling reaction has a major configurational preference for the beta-anomer. Unusually, even mannose coupling results in a neoglycolipid product with a predominantly beta-anomeric conformation (>85%). Formulation of neoglycolipids into LMD vector systems by incubation of LMD particles with neoglycolipid micelles results in the formation of a range of potential stabilized-LMD (sLMD) vector systems. Those potential sLMD systems prepared with longer chain neoglycolipids are found to have enhanced stabilities, with respect to aggregation in high ionic strength buffers, and enhanced transfection efficacies in comparison to the transfection properties of the naked first generation LMD vector system (i.e., gene delivery and expression). By contrast, when LMD vector systems are incubated with poly(ethylene glycol) DSPE-PEG micelles, resulting PEG-LMD vector systems are very stable with respect to colloidal instablility and aggregation in high ionic strength buffers and in serum, but are completely refractory to transfection. These data suggest that oligosaccharides could represent an alternative to PEG as a stealth polymer able to stabilize synthetic nonviral vector systems in some fluids but without impairing transfection efficiency. Furthermore, sLMD systems prepared with longer chain neoglycolipids appear to have sufficient useful characteristics to form the basis of viable second-generation LMD vector systems after further development.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.