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Cancer vaccines aim at generating cytotoxic CD8+ T cells that kill cancer cells and confer durable tumor regression. Hereto, CD8+ peptide epitopes should be presented by antigen presenting cells to CD8+ T cells in lymphoid tissue. Unfortunately, in unformulated soluble form, peptide antigens are poorly taken up by antigen presenting cells and do not efficiently reach lymph nodes. Hence, the lack of efficient delivery remains a major limitation for successful clinical translation of cancer vaccination using peptide antigens. Here we propose a generic peptide nanoformulation strategy by extending the amino acid sequence of the peptide antigen epitope with 10 glutamic acid residues. The resulting overall anionic charge of the peptide allows encapsulation into lipid nanoparticles (peptide-LNP) by electrostatic interaction with an ionizable cationic lipid. We demonstrate that intravenous injection of peptide-LNP efficiently delivers the peptide to immune cells in the spleen. Peptide-LNP that co-encapsulate an imidazoquinoline TLR7/8 agonist (IMDQ) induce robust innate immune activation in a broad range of immune cell subsets in the spleen. Peptide-LNP containing the minimal CD8+ T cell epitope of the HPV type 16 E7 oncoprotein and IMDQ induces high levels of antigen-specific CD8+ T cells in the blood, and can confer protective immunity against E7-expressing tumors in both prophylactic and therapeutic settings.
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Ratones Endogámicos C57BL , Nanopartículas , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Animales , Nanopartículas/química , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/administración & dosificación , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Lípidos/química , Péptidos/química , Femenino , Proteínas E7 de Papillomavirus/inmunología , Quinolinas/farmacología , Quinolinas/química , Imidazoles/química , Imidazoles/farmacologíaRESUMEN
Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.
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Adyuvantes Inmunológicos , Pirimidinas , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Humanos , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/metabolismo , Animales , Ratones , Adyuvantes Inmunológicos/farmacología , Receptor Toll-Like 7/agonistas , Pirimidinas/farmacología , Pirimidinas/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Imidazoles/farmacología , Imidazoles/química , Células THP-1 , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , COVID-19/virología , COVID-19/inmunología , FN-kappa B/metabolismo , Femenino , Descubrimiento de Drogas/métodos , Inmunidad Innata/efectos de los fármacosRESUMEN
DNA vaccines represent an innovative approach for the immunization of diverse diseases. However, their clinical trial outcomes are constrained by suboptimal transfection efficiency and immunogenicity. In this work, we present a universal methodology involving the codelivery of Toll-like receptor 7/8 agonists (TLR7/8a) and antigen gene using TLR7/8a-conjugated peptide-coated poly(ß-amino ester) (PBAE) nanoparticles (NPs) to augment delivery efficiency and immune response. Peptide-TLR7/8a-coated PBAE NPs exhibit advantageous biophysical attributes, encompassing diminutive particle dimensions, nearly neutral ζ potential, and stability in the physiological environment. This synergistic approach not only ameliorates the stability of plasmid DNA (pDNA) and gene delivery efficacy but also facilitates subsequent antigen production. Furthermore, under optimal formulation conditions, the TLR7/8a-conjugated peptide coated PBAE NPs exhibit a potent capacity to induce robust immune responses. Collectively, this nanoparticulate gene delivery system demonstrates heightened transfection efficacy, stability, biodegradability, immunostimulatory effect, and low toxicity, making it a promising platform for the clinical advancement of DNA vaccines.
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Nanopartículas , Péptidos , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Vacunas de ADN , Vacunas de ADN/inmunología , Vacunas de ADN/administración & dosificación , Receptor Toll-Like 8/inmunología , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/inmunología , Animales , Nanopartículas/química , Péptidos/química , Péptidos/inmunología , Humanos , Ratones , Femenino , Polímeros/química , Plásmidos/genética , Plásmidos/inmunología , Ratones Endogámicos C57BLRESUMEN
Tumor-associated macrophages (TAMs) constitute 50-80% of stromal cells in most solid tumors with high mortality and poor prognosis. Tumor-infiltrating dendritic cells (TIDCs) and TAMs are key components mediating immune responses within the tumor microenvironment (TME). Considering their refractory properties, simultaneous remodeling of TAMs and TIDCs is a potential strategy of boosting tumor immunity and restoring immunosurveillance. In this study, mannose-decorated poly(lactic-co-glycolic acid) nanoparticles loading with R848 (Man-pD-PLGA-NP@R848) were prepared to dually target TAMs and TIDCs for efficient tumor immunotherapy. The three-dimensional (3D) cell culture model can simulate tumor growth as influenced by the TME and its 3D structural arrangement. Consequently, cancer spheroids enriched with tumor-associated macrophages (TAMs) were fabricated to assess the therapeutic effectiveness of Man-pD-PLGA-NP@R848. In the TME, Man-pD-PLGA-NP@R848 targeted both TAMs and TIDCs in a mannose receptor-mediated manner. Subsequently, Man-pD-PLGA-NP@R848 released R848 to activate Toll-like receptors 7 and 8, following dual-reprograming of TIDCs and TAMs. Man-pD-PLGA-NP@R848 could uniquely reprogram TAMs into antitumoral phenotypes, decrease angiogenesis, reprogram the immunosuppressive TME from "cold tumor" into "hot tumor", with high CD4+ and CD8+ T cell infiltration, and consequently hinder tumor development in B16F10 tumor-bearing mice. Therefore, dual-reprograming of TIDCs and TAMs with the Man-pD-PLGA-NP@R848 is a promising cancer immunotherapy strategy.
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Imidazoles , Inmunoterapia , Manosa , Ratones Endogámicos C57BL , Nanopartículas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Macrófagos Asociados a Tumores , Animales , Imidazoles/administración & dosificación , Imidazoles/química , Receptor Toll-Like 8/agonistas , Inmunoterapia/métodos , Receptor Toll-Like 7/agonistas , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/efectos de los fármacos , Manosa/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Nanopartículas/administración & dosificación , Nanopartículas/química , Microambiente Tumoral/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Línea Celular Tumoral , Ratones , Neoplasias/terapia , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Femenino , Humanos , Glicoproteínas de MembranaRESUMEN
Toll-like receptor 7/8 (TLR-7/8) agonists serve as a promising class of pattern recognition receptors that effectively evoke the innate immune response, making them promising immunomodulatory agents for tumor immunotherapy. However, the uncontrollable administration of TLR-7/8 agonists frequently leads to the occurrence of severe immune-related adverse events (irAEs). Thus, it is imperative to strategically design tumor-microenvironment-associated biomarkers or exogenous stimuli responsive TLR-7/8 agonists in order to accurately evaluate and activate innate immune responses. No comprehensive elucidation has been documented thus far regarding TLR-7/8 immune agonists that are specifically engineered to enhance immune activation. In this feature article, we provide an overview of the advancements in TLR-7/8 agonists, aiming to enhance the comprehension of their mechanisms and promote the clinical progression through nanomedicine strategies. The current challenges and future directions of cancer immunotherapy are also discussed, with the hope that this work will inspire researchers to explore innovative applications for triggering immune responses through TLR-7/8 agonists.
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Receptor Toll-Like 7 , Receptor Toll-Like 8 , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Humanos , Inmunoterapia , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Inmunidad Innata/efectos de los fármacos , AnimalesRESUMEN
BACKGROUND: Combination therapy involving immune checkpoint blockade (ICB) and other drugs is a potential strategy for converting immune-cold tumors into immune-hot tumors to benefit from immunotherapy. To achieve drug synergy, we developed a homologous cancer cell membrane vesicle (CM)-coated metal-organic framework (MOF) nanodelivery platform for the codelivery of a TLR7/8 agonist with an epigenetic inhibitor. METHODS: A novel biomimetic codelivery system (MCM@UN) was constructed by MOF nanoparticles UiO-66 loading with a bromodomain-containing protein 4 (BRD4) inhibitor and then coated with the membrane vesicles of homologous cancer cells that embedding the 18 C lipid tail of 3M-052 (M). The antitumor immune ability and tumor suppressive effect of MCM@UN were evaluated in a mouse model of triple-negative breast cancer (TNBC) and in vitro. The tumor immune microenvironment was analyzed by multicolor immunofluorescence staining. RESULTS: In vitro and in vivo data showed that MCM@UN specifically targeted to TNBC cells and was superior to the free drug in terms of tumor growth inhibition and antitumor immune activity. In terms of mechanism, MCM@UN blocked BRD4 and PD-L1 to prompt dying tumor cells to disintegrate and expose tumor antigens. The disintegrated tumor cells released damage-associated molecular patterns (DAMPs), recruited dendritic cells (DCs) to efficiently activate CD8+ T cells to mediate effective and long-lasting antitumor immunity. In addition, TLR7/8 agonist on MCM@UN enhanced lymphocytes infiltration and immunogenic cell death and decreased regulatory T-cells (Tregs). On clinical specimens, we found that mature DCs infiltrating tumor tissues of TNBC patients were negatively correlated with the expression of BRD4, which was consistent with the result in animal model. CONCLUSION: MCM@UN specifically targeted to TNBC cells and remodeled tumor immune microenvironment to inhibit malignant behaviors of TNBC.
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Receptor Toll-Like 7 , Receptor Toll-Like 8 , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Animales , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Ratones , Femenino , Humanos , Línea Celular Tumoral , Microambiente Tumoral/efectos de los fármacos , Nanopartículas/química , Factores de Transcripción/metabolismo , Ratones Endogámicos BALB C , Proteínas de Ciclo Celular/metabolismo , Inmunoterapia/métodos , Epigénesis Genética/efectos de los fármacos , Proteínas que Contienen BromodominioRESUMEN
Covalently linking an adjuvant to an antigenic protein enhances its immunogenicity by ensuring a synergistic delivery to the immune system, fostering a more robust and targeted immune response. Most adjuvant-protein conjugate vaccines incorporate only one adjuvant due to the difficulties in its synthesis. However, there is a growing interest in developing vaccines with multiple adjuvants designed to elicit a more robust and targeted immune response by engaging different aspects of the immune system for complex diseases where traditional vaccines fall short. Here, we pioneer the synthesis of a dual-adjuvants protein conjugate Vaccine 1 by assembling a toll-like receptor 7/8 (TLR7/8) agonist, an invariant natural killer T cell (iNKT) agonist with a clickable bicyclononyne (BCN). The BCN group can bio-orthogonally react with azide-modified severe acute respiratory syndrome coronavirus-2 receptor-binding domain (SARS-CoV-2 RBD) trimer antigen to give the three-component Vaccine 1. Notably, with a mere 3 µg antigen, it elicited a balanced subclass of IgG titers and 20-fold more IgG2a than control vaccines, highlighting its potential for enhancing antibody-dependent cellular cytotoxicity. This strategy provides a practicable way to synthesize covalently linked dual immunostimulants. It expands the fully synthetic self-adjuvant protein vaccine that uses a single adjuvant to include two different types of adjuvants.
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Adyuvantes Inmunológicos , Vacunas contra la COVID-19 , COVID-19 , Células T Asesinas Naturales , SARS-CoV-2 , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/inmunología , SARS-CoV-2/inmunología , Animales , Células T Asesinas Naturales/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/farmacología , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/inmunología , Humanos , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/química , Ratones , COVID-19/prevención & control , COVID-19/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Antivirales/inmunología , Femenino , Adyuvantes de Vacunas/química , Adyuvantes de Vacunas/farmacología , Inmunoglobulina G/inmunologíaRESUMEN
Toll-like receptor (TLR) 7 plays an important role in recognizing virus-derived nucleic acids. TLR7 signaling in astrocytes and microglia is critical for activating immune responses against neurotrophic viruses. Neurons express TLR7, similar to glial cells; however, the role of neuronal TLR7 has not yet been fully elucidated. This study sought to determine whether resiquimod, the TLR7/8 agonist, induces the expression of inflammatory chemokines in SH-SY5Y human neuroblastoma cells. Immunofluorescence microscopy revealed that TLR7 was constitutively expressed in SH-SY5Y cells. Stimulation with resiquimod induced C-C motif chemokine ligand 2 (CCL2) expression, accompanied by the activation of nuclear factor-kappa B (NF-κB) in SH-SY5Y cells. Resiquimod increased mRNA levels of C-X-C motif chemokine ligand 8 (CXCL8) and CXCL10, while the increase was slight at the protein level. Knockdown of NF-κB p65 eliminated resiquimod-induced CCL2 production. This study provides novel evidence that resiquimod has promising therapeutic potential against central nervous system viral infections through its immunostimulatory effects on neurons.
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Quimiocina CCL2 , Quimiocina CXCL10 , Imidazoles , Interleucina-8 , Receptor Toll-Like 7 , Factor de Transcripción ReIA , Humanos , Línea Celular Tumoral , Quimiocina CCL2/genética , Quimiocina CCL2/biosíntesis , Quimiocina CXCL10/genética , Quimiocina CXCL10/biosíntesis , Imidazoles/farmacología , Interleucina-8/genética , Interleucina-8/biosíntesis , Neuroblastoma , Neuronas/efectos de los fármacos , Neuronas/metabolismo , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/genética , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/genética , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genéticaRESUMEN
Self-assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two-component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self-assembly in vitro. Single-stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length-dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small-molecule TLR7/8 agonist show that co-delivery of antigen and adjuvant results in a more than 20-fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo-loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.
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Nanopartículas , Nanopartículas/química , Animales , Ratones , Proteínas/química , Receptor Toll-Like 8/metabolismo , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/química , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 7/agonistasRESUMEN
BACKGROUND & AIMS: Selgantolimod (GS-9688) is a Toll-like receptor 8 (TLR8) agonist that suppresses HBV in vitro. In a phase II study, we evaluated the safety and efficacy of weekly selgantolimod treatment in virally suppressed individuals with chronic HBV taking oral antiviral treatment. METHODS: Forty-eight patients were randomized into two cohorts (hepatitis B e antigen [HBeAg]-positive and -negative [n = 24 each]) to receive oral selgantolimod 3 mg, 1.5 mg, or placebo (2:2:1) once weekly for 24 weeks while maintaining oral antivirals. The primary efficacy endpoint was the percentage of patients with a ≥1 log10 IU/ml decline in hepatitis B surface antigen (HBsAg) from baseline to week 24. Post-treatment, patients continued on oral antivirals for 24 weeks. RESULTS: The primary endpoint was reached by one participant, who was HBeAg-negative and received selgantolimod 1.5 mg. In contrast with placebo-treated patients (n = 9), only selgantolimod-treated patients (n = 39 total) had HBsAg declines greater than 0.1 log10 IU/ml at weeks 24 (18%, 7/39) and 48 (26%, 10/39), HBsAg loss (5%, 2/39 through 48 weeks), or HBeAg loss (16%, 3/19 through 48 weeks). The most common adverse events in selgantolimod-treated groups were nausea (46%), upper respiratory tract infection (23%), and vomiting (23%). Gastrointestinal disorders were mostly mild and transient. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40, IFN-γ, and IL-1RA, as well as rapid redistribution of some circulating immune cell subsets. CONCLUSION: Oral selgantolimod up to 3 mg once weekly for 24 weeks was generally safe and well tolerated and led to serologic changes associated with progression to durable cure in two individuals by week 48. GOV IDENTIFIER: NCT03491553. IMPACT AND IMPLICATIONS: The only robust criterion for stopping treatment in chronic hepatitis B is loss of hepatitis B surface antigen (known as functional cure), which is rare during nucleos(t)ide analogue therapy. It is likely that novel antiviral and immunomodulatory therapies will be needed to achieve finite functional cure. Selgantolimod is an oral Toll-like receptor 8 agonist that has shown antiviral activity in vitro as well as safety in a phase I clinical trial with weekly dosing. In this phase II study, selgantolimod therapy was associated with transient increases in serum cytokines, rapid redistribution of circulating immune cell subsets, modest reductions in HBsAg and HBeAg levels, and occasional loss of HBsAg (5%) and HBeAg (16%) among participants with chronic hepatitis B on nucleos(t)ide analogue therapy with viral suppression. Our results support continued development of selgantolimod as a component of a future hepatitis B cure regimen.
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Antivirales , Hepatitis B Crónica , Receptor Toll-Like 8 , Humanos , Antivirales/uso terapéutico , Citocinas , Antígenos e de la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Hepatitis B Crónica/tratamiento farmacológico , Receptor Toll-Like 8/agonistas , Resultado del TratamientoRESUMEN
The innate immune system recognizes conserved features of viral and microbial pathogens through pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one type of PRR used by the innate immune system to mediate the secretion of proinflammatory cytokines and promote innate and adaptive immune responses. TLR family members TLR7 and TLR8 (referred to as TLR7/8 from herein) are endosomal transmembrane receptors that recognize purine-rich single-stranded RNA (ssRNA) and bacterial DNA, eliciting an immunologic reaction to pathogens. TLR7/8 were discovered to mediate the secretion of proinflammatory cytokines by activating immune cells. In addition, accumulating evidence has indicated that TLR7/8 may be closely related to numerous immune-mediated disorders, specifically several types of cancer, autoimmune disease, and viral disease. TLR7/8 agonists and antagonists, which are used as drugs or adjuvants, have been identified in preclinical studies and clinical trials as promising immune stimulators for the immunotherapy of these immune-mediated disorders. These results provided reasoning to further explore immunotherapy for the treatment of immune-mediated disorders. Nevertheless, numerous needs remain unmet, and the therapeutic effects of TLR7/8 agonists and antagonists are poor and exert strong immune-related toxicities. The present review aimed to provide an overview of the TLR family members, particularly TLR7/8, and address the underlying molecular mechanisms and clinical implications of TLR7/8 in immune-mediated disorders. The aim of the work is to discuss the underlying molecular mechanisms and clinical implications of TLR7/8 in immune-mediated disorders.
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Receptor Toll-Like 7 , Receptor Toll-Like 8 , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/fisiología , Receptores Toll-Like , Citocinas , Adyuvantes Inmunológicos , Inmunoterapia , InmunidadRESUMEN
Toll-like receptors (TLR)s are homo- or heterodimeric proteins, whose structure and function were widely described in the antigen presenting cells (APC), such as Dendritic cells (DC). Recently, the expression and the role of TLRs in fighting against pathogens, was described also in NK cells. Their activation and functional properties can be directly and indirectly modulated by agonists for TLRs. In particular CD56bright NK cells subset, that is the most abundant NK cell subset in tissues and tumor microenvironment (TME), was mostly activated in terms of pro-inflammatory cytokine production, proliferation and cytotoxicity, by agonists specific for endosomal TLR8. The interplay between DC and NK, that depends on both cell-to-cell contact and soluble factors such as cytokines, promote both DC maturation and NK cell activation. Based on this concept, a TLR based immunotherapy aimed to activate NK-DC axis, may modulate TME by inducing a pro-inflammatory phenotype, thus improving DC ability to present tumor-associated antigens to T cells, and NK cell cytotoxicity against tumor cells. In this mini-review, we report data of recent literature about TLRs on human NK cells and their application as adjuvant in cancer vaccines or in combined tumor immunotherapy.
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Neoplasias , Receptor Toll-Like 8 , Humanos , Receptor Toll-Like 8/agonistas , Receptores Toll-Like/metabolismo , Células Asesinas Naturales , Inmunoterapia , Células Dendríticas , Receptor Toll-Like 7/metabolismo , Microambiente TumoralRESUMEN
A growing body of preclinical evidence has led to the hypothesis that human Toll-like receptor 8 (hTLR8) activation in the tumor microenvironment (TME) could have potent anticancer effects through its action on monocytes, myeloid dendritic cells (mDCs), and natural killer (NK) cells. This has motivated the initiation of several clinical trials for chemical hTLR8 agonists in a variety of cancers. Concurrently, a growing number of synthetic antisense oligonucleotides (ASOs) are being developed as cancer therapeutics. We have recently reported that 2'-O-methyl (2'OMe)-modified ASOs can potentiate sensing of hTLR8 chemical agonists in a sequence-dependent manner. This suggests that select gene-targeting ASOs with anticancer activity may synergize with low-dose hTLR8 agonists in the TME. Here, we provide a detailed protocol to rapidly screen and identify such synthetic bifunctional oligonucleotides with synergistic activity on hTLR8 sensing.
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Oligonucleótidos Antisentido , Receptor Toll-Like 8 , Humanos , Oligonucleótidos Antisentido/genética , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/agonistas , Oligonucleótidos/genética , Secuencia de Bases , Adyuvantes Inmunológicos , Marcación de GenRESUMEN
BACKGROUND: Viral infections during pregnancy can have deleterious effects on mothers and their offspring. Monocytes participate in the maternal host defense against invading viruses; however, whether pregnancy alters monocyte responses is still under investigation. Herein, we undertook a comprehensive in vitro study of peripheral monocytes to characterize the differences in phenotype and interferon release driven by viral ligands between pregnant and non-pregnant women. METHODS: Peripheral blood was collected from third-trimester pregnant (n = 20) or non-pregnant (n = 20, controls) women. Peripheral blood mononuclear cells were isolated and exposed to R848 (TLR7/TLR8 agonist), Gardiquimod (TLR7 agonist), Poly(I:C) (HMW) VacciGrade™ (TLR3 agonist), Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 agonist), or ODN2216 (TLR9 agonist) for 24 h. Cells and supernatants were collected for monocyte phenotyping and immunoassays to detect specific interferons, respectively. RESULTS: The proportions of classical (CD14hiCD16-), intermediate (CD14hiCD16+), non-classical (CD14loCD16+), and CD14loCD16- monocytes were differentially affected between pregnant and non-pregnant women in response to TLR3 stimulation. The proportions of pregnancy-derived monocytes expressing adhesion molecules (Basigin and PSGL-1) or the chemokine receptors CCR5 and CCR2 were diminished in response to TLR7/TLR8 stimulation, while the proportions of CCR5- monocytes were increased. Such differences were found to be primarily driven by TLR8 signaling, rather than TLR7. Moreover, the proportions of monocytes expressing the chemokine receptor CXCR1 were increased during pregnancy in response to poly(I:C) stimulation through TLR3, but not RIG-I/MDA-5. By contrast, pregnancy-specific changes in the monocyte response to TLR9 stimulation were not observed. Notably, the soluble interferon response to viral stimulation by mononuclear cells was not diminished in pregnancy. CONCLUSIONS: Our data provide insight into the differential responsiveness of pregnancy-derived monocytes to ssRNA and dsRNA, mainly driven by TLR8 and membrane-bound TLR3, which may help to explain the increased susceptibility of pregnant women to adverse outcomes resulting from viral infection as observed during recent and historic pandemics.
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Leucocitos Mononucleares , Monocitos , Embarazo , Humanos , Femenino , Receptores de Lipopolisacáridos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 3 , Receptores de IgG , InterferonesRESUMEN
Glioblastoma, the most common and aggressive primary brain tumor type, is considered an immunologically "cold" tumor with sparse infiltration by adaptive immune cells. Immunosuppressive tumor-associated myeloid cells are drivers of tumor progression. Therefore, targeting and reprogramming intratumoral myeloid cells is an appealing therapeutic strategy. Here, we investigate a ß-cyclodextrin nanoparticle (CDNP) formulation encapsulating the Toll-like receptor 7 and 8 (TLR7/8) agonist R848 (CDNP-R848) to reprogram myeloid cells in the glioma microenvironment. We show that intravenous monotherapy with CDNP-R848 induces regression of established syngeneic experimental glioma, resulting in increased survival rates compared with unloaded CDNP controls. Mechanistically, CDNP-R848 treatment reshapes the immunosuppressive tumor microenvironment and orchestrates tumor clearing by pro-inflammatory tumor-associated myeloid cells, independently of T cells and NK cells. Using serial magnetic resonance imaging, we identify a radiomic signature in response to CDNP-R848 treatment and ultrasmall superparamagnetic iron oxide (USPIO) imaging reveals that immunosuppressive macrophage recruitment is reduced by CDNP-R848. In conclusion, CDNP-R848 induces tumor regression in experimental glioma by targeting blood-borne macrophages without requiring adaptive immunity.
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Glioma , Nanopartículas , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Humanos , Adyuvantes Inmunológicos , Glioma/tratamiento farmacológico , Macrófagos , Linfocitos T , Receptor Toll-Like 7/agonistas , Microambiente Tumoral , Receptor Toll-Like 8/agonistasRESUMEN
BACKGROUND: Natural killer (NK) cells exhibit a selective deficiency of IFN-γ production in chronic hepatitis B (CHB). Toll-like receptor 8 (TLR8) agonists could induce IFN-γ production in immune cells, although their effects on the deficiency in NK cells remain unclear. AIMS: To investigate TLR8 expression in NK cells and the effect of TLR8 agonists in patients with CHB METHODS: We enrolled 32 patients with CHB and 19 healthy controls to assess TLR8 expression and IFN-γ production in NK cells. The sorted NK cells and monocytes were co-cultured to compare the extent of IFN-γ and IL-10 production after TLR8 agonist ssRNA40 stimulation. The synergic effect of NK cells and monocytes was assessed by blocking IL-12 and IL-18. We recruited another 22 patients with CHB undergoing nucleotide analogue (NA) therapy to explore the impact of antiviral treatment on the ssRNA40-mediated response of NK cells. RESULTS: In patients with CHB, TLR8 expression in NK cells was up-regulated, accompanied by insufficient IFN-γ production. The enhanced IFN-γ secretion by ssRNA40 in NK cells depended on monocyte-derived IL-12 and IL-18. NK cells displayed an imbalanced response to ssRNA40 in patients with CHB with a weak increase in IFN-γ despite a higher IL-10 production. The response was improved in patients with CHB undergoing NA therapy. CONCLUSIONS: In patients with CHB, targeting TLR8 partially rescues the IFN-γ insufficiency in NK cells. However, NK cells show an inhibitory response to TLR8 agonist stimulation. TLR8 agonist combined with NA may enhance the antiviral effect of NK cells.
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Hepatitis B Crónica , Monocitos , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/metabolismo , Interferón gamma/metabolismo , Interferón gamma/farmacología , Interleucina-10 , Interleucina-12/metabolismo , Interleucina-12/farmacología , Interleucina-12/uso terapéutico , Interleucina-18 , Células Asesinas Naturales/metabolismo , Monocitos/metabolismo , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/metabolismo , Receptor Toll-Like 8/uso terapéuticoRESUMEN
Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)-PEG-Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant. Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centers during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.
Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra la Influenza , Gripe Humana , Animales , Humanos , Ratones , Adyuvantes Inmunológicos/farmacología , Anticuerpos Antivirales , Inmunoglobulina G , Gripe Humana/prevención & control , Interleucina-4 , Nanogeles , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistasRESUMEN
BACKGROUND: Stereotactic body radiotherapy (SBRT) has been increasingly used as adjuvant therapy in pancreatic ductal adenocarcinoma (PDAC), and induces immunogenic cell death, which leads to the release of tumor antigen and damage-associated molecular patterns. However, this induction often fails to generate sufficient response to overcome pre-existing tumor microenvironment (TME) immunosuppression. Toll-like receptor (TLR) 7/8 ligands, such as R848, can amplify the effect of tumor vaccines, with recent evidence showing its antitumor effect in pancreatic cancer by modulating the immunosuppressive TME. Therefore, we hypothesized that the combination of R848 and SBRT would improve local and systemic antitumor immune responses by potentiating the antitumor effects of SBRT and reversing the immunosuppressive nature of the PDAC TME. METHODS: Using murine models of orthotopic PDAC, we assessed the combination of intravenous TLR7/8 agonist R848 and local SBRT on tumor growth and immune response in primary pancreatic tumors. Additionally, we employed a hepatic metastatic model to investigate if the combination of SBRT targeting only the primary pancreatic tumor and systemic R848 is effective in controlling established liver metastases. RESULTS: We demonstrated that intravenous administration of the TLR7/8 agonist R848, in combination with local SBRT, leads to superior tumor control compared with either treatment alone. The combination of R848 and SBRT results in significant immune activation of the pancreatic TME, including increased tumor antigen-specific CD8+ T cells, decreased regulatory T cells, and enhanced antigen-presenting cells maturation, as well as increased interferon gamma, granzyme B, and CCL5 along with decreased levels of interleukin 4 (IL-4), IL-6, and IL-10. Importantly, the combination of SBRT and systemic R848 also resulted in similar immunostimulatory changes in liver metastases, leading to improved metastatic control. CD8+ T cell depletion studies highlighted the necessity of these effector cells at both the local and hepatic metastatic sites. T cell receptor (TCR) clonotype analysis indicated that systemic R848 not only diversified the TCR repertoire but also conditioned the metastatic foci to facilitate entry of CD8+ T cells generated by SBRT therapy. CONCLUSIONS: These findings suggest that systemic administration of TLR7/8 agonists in combination with SBRT may be a promising avenue for metastatic PDAC treatment.
Asunto(s)
Carcinoma Ductal Pancreático , Imidazoles/farmacología , Neoplasias Hepáticas , Neoplasias Pancreáticas , Radiocirugia , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/radioterapia , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/radioterapia , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Radiotherapy (RT) has been shown to cause immunogenic cell death (ICD) of cancer cells, which promote the release of tumor-associated antigens, and trigger the cancer-immunity cycle (CIC). However, ICD induced by RT usually does not occur in hypoxic tumor cells due to their resistance to radiation. Moreover, RT also induces programmed death ligand 1 (PD-L1) upregulation on tumor cells, which has an inhibitory effect on T lymphocytes. Therefore, therapy based on CIC must selectively target the restricted steps of antitumor immunity. Herein, the authors design a versatile three-in-one assembling nanoparticle that can simultaneously execute these obstacles. The amphiphilic peptide drug conjugate NIA-D1, containing the hydrophobic radio-sensitizer 2-(2-nitroimidazol-1-yl) acetic acid (NIA), a peptide substrate of matrix metalloproteinase-2, and a hydrophilic PD-L1 antagonist D PPA-1, is constructed and co-assembled with hydrophobic Toll-like receptor (TLR) 7/8 agonist R848 to form nanoparticle NIA-D1@R848. The NIA-D1@R848 nanoparticles combined with RT can trigger the apoptosis of tumor cells and initiate the CIC. In the presence of R848, it promotes the maturation of dendritic cells, which together with protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 blockade to relieve T cell suppression, and amplify the antitumor immune cycle. In conclusion, a functionalized three-in-one nanoparticle NIA-D1@R848 is successfully constructed, which can induce strong systemic antitumor immune response.
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Nanopartículas , Neoplasias , Receptor Toll-Like 8/agonistas , Adyuvantes Inmunológicos , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Humanos , Inmunidad , Inmunoterapia , Metaloproteinasa 2 de la Matriz , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Péptidos/uso terapéutico , Receptor Toll-Like 7RESUMEN
Toll-like receptor (TLR) agonists are potent immune-stimulators that hold great potential in vaccine adjuvants as well as cancer immunotherapy. However, TLR agonists in free form are prone to be eliminated quickly by the circulatory system and cause systemic inflammation side effects. It remains a challenge to achieve precise release of TLR7/8 agonist in the native form at the receptor site in the endosomal compartments while keeping stable encapsulation and inactive in nontarget environment. Here, we report a pH-/enzyme-responsive TLR7/8 agonist-conjugated nanovaccine (TNV), which responds intelligently to the acidic environment and cathepsin B in the endosome, precisely releases TLR7/8 agonist to activate its receptor signaling at the endosomal membrane, stimulates DCs maturation, and provokes specific cellular immunity. In vivo experiments demonstrate outstanding prophylactic and therapeutic efficacy of TNV in mouse melanoma and colon cancer. The endosome-targeted responsive nanoparticle strategy provides a potential delivery toolbox of adjuvants to advance the development of tumor nanovaccines.