Challenges and Opportunities in the Clinical Development of STING Agonists for Cancer Immunotherapy.

Immune checkpoint inhibitors (ICI) have revolutionised cancer therapy. However, they have been effective in only a small subset of patients and a principal mechanism underlying immune-refractoriness is a 'cold' tumour microenvironment, that is, lack of a T-cell-rich, spontaneously inflamed phenotype. As such, there is a demand to develop strategies to transform the tumour milieu of non-responsive patients to one supporting T-cell-based inflammation. The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway is a fundamental regulator of innate immune sensing of cancer, with potential to enhance tumour rejection through the induction of a pro-inflammatory response dominated by Type I interferons. Recognition of these positive immune-modulatory properties has rapidly elevated the STING pathway as a putative target for immunotherapy, leading to a myriad of preclinical and clinical studies assessing natural and synthetic cyclic dinucleotides and non-nucleotidyl STING agonists. Despite pre-clinical evidence of efficacy, clinical translation has resulted into disappointingly modest efficacy. Poor pharmacokinetic and physiochemical properties of cyclic dinucleotides are key barriers to the development of STING agonists, most of which require intra-tumoral dosing. Development of systemically administered non-nucleotidyl STING agonists, or conjugation with liposomes, polymers and hydrogels may overcome pharmacokinetic limitations and improve drug delivery. In this review, we summarise the body of evidence supporting a synergistic role of STING agonists with currently approved ICI therapies and discuss whether, despite the numerous obstacles encountered to date, the clinical development of STING agonist as novel anti-cancer therapeutics may still hold the promise of broadening the reach of cancer immunotherapy.

Pulmonary Epithelial Cell-Derived Cytokine TGF-ß1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function.

Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-ß had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-ß1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-ß by expressing the receptor TGF-ßRII and ILC chemoactivity was enhanced by TGF-ß. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions.

Bisphenol A suppresses Th1-type immune response in human peripheral blood mononuclear cells in vitro.

Bisphenol A (BPA) is a widely used plasticizer, which came into focus because of its genotoxic and sensitizing potential. Besides its toxic properties, BPA is also well-known for its antioxidant chemical properties. This in vitro study investigated the interference of BPA with interferon-γ (IFN-γ)-induced tryptophan breakdown and neopterin production in human peripheral blood mononuclear cells (PBMC). The pro-inflammatory cytokine IFN-γ induces the conversion of the essential amino acid tryptophan into kynurenine via the enzyme indoleamine-2,3-dioxygenase (IDO-1). In parallel, GTP-cyclohydrolase produces neopterin, a marker of immune activation. A model system of phytohaemagglutinin (PHA)-stimulated PBMC was used to assess potential immunomodulatory properties of BPA. Treatment of cells with BPA [12.5-200µM] resulted in a significant and dose-dependent suppression of mitogen-induced tryptophan breakdown and neopterin formation along with a decrease of IFN-γ levels. Similar but less pronounced effects were observed in unstimulated cells. We postulate that the inhibitory effects of BPA on both T-cell activation and IDO-1 activity that we describe here may be critical for immune surveillance and is likely to influence T helper (Th) type 1/Th2 balance. Such immunosuppressive effects likely contribute to counteract inflammation. Further studies are required to address the in vivo relevance our in vitro findings.

IRF5 controls both acute and chronic inflammation.

Whereas the importance of macrophages in chronic inflammatory diseases is well recognized, there is an increasing awareness that neutrophils may also play an important role. In addition to the well-documented heterogeneity of macrophage phenotypes and functions, neutrophils also show remarkable phenotypic diversity among tissues. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. We have shown that the transcription factor IFN regulatory factor 5 (IRF5) polarizes macrophages toward an inflammatory phenotype. IRF5 is also expressed in other myeloid cells, including neutrophils, where it was linked to neutrophil function. In this study we explored the role of IRF5 in models of acute inflammation, including antigen-induced inflammatory arthritis and lung injury, both involving an extensive influx of neutrophils. Mice lacking IRF5 accumulate far fewer neutrophils at the site of inflammation due to the reduced levels of chemokines important for neutrophil recruitment, such as the chemokine (C-X-C motif) ligand 1. Furthermore we found that neutrophils express little IRF5 in the joints and that their migratory properties are not affected by the IRF5 deficiency. These studies extend prior ones suggesting that inhibiting IRF5 might be useful for chronic macrophage-induced inflammation and suggest that IRF5 blockade would ameliorate more acute forms of inflammation, including lung injury.

Targeting chemokine receptors in disease--a case study of CCR4.

Since their early 1990s, the chemokine receptor family of G protein-coupled receptors (GPCRs) has been the source of much pharmacological endeavour. Best known for their key roles in recruiting leukocytes to sites of infection and inflammation, the receptors present themselves as plausible drug targets for therapeutic intervention. In this article, we will focus our attention upon CC Chemokine Receptor Four (CCR4) which has been implicated in diseases as diverse as allergic asthma and lymphoma. We will review the discovery of the receptors and their ligands, their perceived roles in disease and the successful targeting of CCR4 by both small molecule antagonists and monoclonal antibodies. We will also discuss future directions and strategies for drug discovery in this field.

Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy.

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

CCL17/thymus and activation-regulated chemokine induces calcitonin gene-related peptide in human airway epithelial cells through CCR4.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a potent arterial and venous vasodilator. Increased airway epithelial cell expression of CGRP, together with increased CCL17 expression, was previously observed in a model of provoked asthma in atopic human subjects. OBJECTIVE: We sought to determine whether CCL17 induces CCR4-dependent CGRP synthesis and secretion by human airway epithelial cells. METHODS: Human airway epithelial cell lines (BEAS-2B and A549) and human primary airway cells were cultured with CCL17 or various other cytokines, and CGRP expression was measured by using RT-PCR, quantitative immunofluorescence, and enzyme immunoassay. CCR4 expression was determined in cultured cells by using flow cytometry and in bronchial biopsy specimens by using immunohistochemistry. RESULTS: CCL17 induced a several thousand-fold increase in CGRP mRNA expression and released peptide product from BEAS-2B and A549 cells in a time- and concentration-dependent fashion. Concentration-dependent CCL17-induced release of CGRP by primary human airway epithelial cells was also observed. Under comparable conditions, CCL17 induced greater CGRP release from BEAS-2B cells than either IL-13, a cytokine mixture (TNF-α, GM-CSF, and IL-1), or CCL22. CCR4 was expressed by BEAS-2B and A549 cells and internalized after ligation with CCL17. CCL17-induced CGRP release was inhibited by a specific anti-CCR4 blocking antibody. Bronchial biopsy specimens obtained from healthy volunteers and asthmatic patients before and after provoked asthma all exhibited CCR4 staining of equivalent intensity, indicating that the receptor is constitutively expressed. CONCLUSIONS: CCL17-induced, CCR4-dependent release of CGRP by human airway epithelial cells represents a novel inflammatory pathway and a possible target in patients with asthma and allergic disease.

Human T(H)2 cells respond to cysteinyl leukotrienes through selective expression of cysteinyl leukotriene receptor 1.

BACKGROUND: Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. OBJECTIVES: Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. METHODS: We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. RESULTS: We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after in vitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. CONCLUSIONS: These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.

Small molecule chemokine mimetics suggest a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3. EXPERIMENTAL APPROACH: As both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined. KEY RESULTS: The CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the '30s loop' (residues 30-40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues. CONCLUSION AND IMPLICATIONS: We provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.

A single nucleotide polymorphism in the CCR3 gene ablates receptor export to the plasma membrane.

BACKGROUND: The chemokine receptor CCR3 orchestrates the migration of eosinophils, basophils, T(H)2 lymphocytes, and mast cells during the allergic response, with CCR3 blockade a potential means of therapeutic intervention. Non-synonymous single nucleotide polymorphisms (SNPs) within the ccr3 gene have previously been described, with little information regarding their effects on CCR3 function. OBJECTIVE: To characterize the effects of nonsynonymous SNPs within the ccr3 gene. METHODS: Site-directed mutagenesis was used to generate N-terminally tagged mutant CCR3 constructs corresponding to reported SNPs. Cell transfectants expressing either wild-type or mutant CCR3 were studied by flow cytometry, Western blotting, and confocal microscopy and examined for their ability to migrate to the CC chemokine ligand CCL11/eotaxin. RESULTS: An L324P mutant CCR3 protein corresponding to the previously identified T971C SNP was not expressed at the cell surface, and cells remained unresponsive to CCL11 in chemotaxis assays. Confocal microscopy confirmed that L324P-CCR3 had a predominantly intracellular distribution compared with wild-type CCR3. A L324A variant of CCR3 had an identical phenotype to the L324P mutant, suggesting that L324 per se is critical for successful trafficking of nascent CCR3 to the cell membrane. The processes involved appear to be specific for CCR3, because an identical mutation in the homologous receptor CCR1 had minor effects. CONCLUSION: Trafficking to the cell surface of nascent CCR3 is critically dependent on a C-terminal leucine residue, suggestive of specific mechanisms for CCR3 export. Manipulation of these mechanisms may suggest novel means of antagonizing CCR3 function in the treatment of allergy.

The role of the CCL2/CCR2 axis in mouse mast cell migration in vitro and in vivo.

Tissue-resident mast cells (MCs) are important in allergic diseases. In a mouse model of allergic airways inflammation, an increase in peribronchiolar MCs was associated with increased concentrations of the chemokine CCL2 in lung lavage. MC progenitors (MCps) arising in bone marrow (BM) are recruited to tissues by transendothelial migration, and we found that CCL2 is chemotactic for MCps in freshly isolated BM in vitro. Immature, but not mature, BM-derived MCs migrated in response to CCL2 when cultured in IL-3+stem cell factor (SCF) but not when cultured in IL-3 alone. However, the cells under both culture conditions expressed mRNA for CCR2, the receptor for CCL2, and bound the radiolabeled chemokine with similar affinities, highlighting SCF as a key mediator in coupling CCR2 to downstream events, culminating in chemotaxis. Immature BM-derived MCs from IL-3 +SCF cultures, when administered i.v., accumulated at skin sites injected with CCL2 in vivo. MCp recruitment to the allergen-sensitized/challenged lung was significantly reduced in CCR2(-/-) and CCL2(-/-) mouse strains. However, reconstitution studies of sublethally irradiated and BM-reconstituted mice indicated that BM cells and stromal elements could provide CCL2, whereas the CCR2 function resided with stromal elements rather than BM cells. These experiments revealed a new function of SCF in chemokine receptor coupling, but they suggest a complex role of the CCL2/CCR2 axis in recruiting MCps during pulmonary inflammation.

Elucidation of binding sites of dual antagonists in the human chemokine receptors CCR2 and CCR5.

Design of dual antagonists for the chemokine receptors CCR2 and CCR5 will be greatly facilitated by knowledge of the structural differences of their binding sites. Thus, we computationally predicted the binding site of the dual CCR2/CCR5 antagonist N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzohepten-8-yl] carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium (TAK-779), and a CCR2-specific antagonist N-(carbamoylmethyl)-3-trifluoromethyl benzamido-parachlorobenzyl 3-aminopyrrolidine (Teijin compound 1) in an ensemble of predicted structures of human CCR2 and CCR5. Based on our predictions of the protein-ligand interactions, we examined the activity of the antagonists for cells expressing thirteen mutants of CCR2 and five mutants of CCR5. The results show that residues Trp98(2.60) and Thr292(7.40) contribute significantly to the efficacy of both TAK-779 and Teijin compound 1, whereas His121(3.33) and Ile263(6.55) contribute significantly only to the antagonistic effect of Teijin compound 1 at CCR2. Mutation of residues Trp86(2.60) and Tyr108(3.32) adversely affected the efficacy of TAK-779 in antagonizing CCR5-mediated chemotaxis. Y49A(1.39) and E291A(7.39) mutants of CCR2 showed a complete loss of CCL2 binding and chemotaxis, despite robust cell surface expression, suggesting that these residues are critical in maintaining the correct receptor architecture. Modeling studies support the hypothesis that the residues Tyr49(1.39), Trp98(2.60), Tyr120(3.32), and Glu291(7.39) of CCR2 form a tight network of aromatic cluster and polar contacts between transmembrane helices 1, 2, 3, and 7.

Small molecule receptor agonists and antagonists of CCR3 provide insight into mechanisms of chemokine receptor activation.

Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.

The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes.

Pharyngitis caused by Streptococcus pyogenes is one of the most common bacterial infections in humans and is also a starting point for invasive S. pyogenes infection. Here, we describe that tonsil fluid from patients with streptococcal pharyngitis contains high amounts of the interferon (IFN)-dependent CXC chemokine known as monokine induced by IFN- gamma (MIG)/CXCL9. Also in vitro, inflamed pharyngeal epithelium produced large amounts of MIG/CXCL9 in the presence of bacteria. The CXC chemokines MIG/CXCL9, IFN-inducible protein-10/CXCL10, and IFN-inducible T cell alpha -chemoattractant/CXCL11 all showed antibacterial activity against S. pyogenes, and inhibition of MIG/CXCL9 expression reduced the antibacterial activity at the surface of inflamed pharyngeal cells. S. pyogenes of the clinically important M1 serotype secrets the protein streptococcal inhibitor of complement (SIC), which inhibited the antibacterial activity of the chemokines. As exemplified by S. pyogenes pharyngitis, the data identify a novel innate defense mechanism against invasive bacteria on epithelial surfaces.

The role of interleukin-8 and its receptors in inflammatory lung disease: implications for therapy.

Neutrophils have been implicated in the pathogenesis of many inflammatory lung diseases, including the acute respiratory distress syndrome, chronic obstructive pulmonary disease and asthma. The CXC chemokine interleukin (IL)-8, is a potent neutrophil recruiting and activating factor and the detection of IL-8 in clinical samples from patients with these diseases has led clinicians to believe that antagonism of IL-8 may be a practicable therapeutic strategy for disease management. Work over the last decade has concentrated on both the molecular mechanisms by which IL-8 is produced in the inflammatory setting and also on the manner in which IL-8 activates the neutrophil. Expression of the IL-8 gene appears to be controlled by several components of the inflammatory milieu. Whilst lipopolysaccharide, IL-1beta and tumor necrosis factor-alpha are capable of augmenting IL-8 production, IL-10 is a potent inhibitor of IL-8 synthesis and appears to play an auto-regulatory role. Regulation of the IL-8 gene is under the control of nuclear factor kappaB which appears to be a primary target for corticosteroid-mediated repression of IL-8 production. IL-8 exerts is effects on neutrophils by binding with high affinity to two receptors on its cell surface, the chemokine receptors CXCR1 and CXCR2. These closely related receptors belong to the superfamily of G-protein coupled receptors, proteins that historically have proved amenable to antagonism by small molecules. The recent descriptions in the literature of highly potent small molecule antagonists of CXCR2 and their success in blocking in vivo trafficking of neutrophils suggest that antagonism of IL-8 at the receptor level is a viable therapeutic strategy. Clinical trials of such compounds will ultimately provide crucial information currently lacking and will define whether or not IL-8 blockade provides future therapy in pulmonary disease.