ABSTRACT
Ruzotolimod (Toll-like receptor 7 (TLR7) agonist, RG7854) is an oral, small molecule immuno-modulator activating the TLR 7 and is being evaluated in patients with CHB. As with other TLR7 agonists, the study drug-related adverse events of flu-like symptoms have been reported in some participants during phase I studies with ruzotolimod. An exploratory analysis of the relationship between pharmacokinetic (PK)/pharmacodynamic (PD) and flu-like symptoms was performed in participants from two phase I studies including both healthy volunteers and NUC-suppressed CHB patients who received either single or multiple ascending doses of orally administered ruzotolimod. Linear and logistic regression were used to explore potential relationships between dose, flu-like symptoms, PK, and PD. Generalized linear regression was performed to predict the probability of flu-like symptoms of all intensities at different RO7011785 (the active metabolite of the double prodrug ruzotolimod) PK exposure. This analysis showed that single or multiple doses of ruzotolimod at ⩾100 mg, the immune PD (IFN-α, neopterin, IP-10, and the transcriptional expression of ISG15, OAS-1, MX1, and TLR7) responses increase with the RO7011785 PK exposure, which increases linearly with the doses from 3 mg to 170 mg of ruzotolimod. The analysis also showed that the probability of flu-like symptoms occurrence increases with PD responses (IFN-α and IP-10). Dose reduction of ruzotolimod can be an effective way to reduce the magnitude of PD response, thus reducing the probability of study drug-related flu-like symptoms occurrence at all intensity in the participants who are highly sensitive to PD activation and intolerant to flu-like symptoms.
Subject(s)
Healthy Volunteers , Hepatitis B, Chronic , Toll-Like Receptor 7 , Humans , Toll-Like Receptor 7/agonists , Male , Adult , Female , Middle Aged , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/virology , Hepatitis B, Chronic/blood , Young Adult , Antiviral Agents/pharmacokinetics , Antiviral Agents/administration & dosage , Antiviral Agents/adverse effects , Influenza, Human/drug therapy , Influenza, Human/immunology , Dose-Response Relationship, Drug , Adolescent , Administration, Oral , Organic ChemicalsABSTRACT
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.
Subject(s)
Adjuvants, Immunologic , Pyrimidines , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Humans , Toll-Like Receptor 8/agonists , Toll-Like Receptor 8/metabolism , Animals , Mice , Adjuvants, Immunologic/pharmacology , Toll-Like Receptor 7/agonists , Pyrimidines/pharmacology , Pyrimidines/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Imidazoles/pharmacology , Imidazoles/chemistry , THP-1 Cells , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , COVID-19/virology , COVID-19/immunology , NF-kappa B/metabolism , Female , Drug Discovery/methods , Immunity, Innate/drug effectsABSTRACT
BACKGROUND: TLR7 is a key player in the antiviral immunity. TLR7 signaling activates antigen-presenting cells including DCs and macrophages. This activation results in the adaptive immunity including T cells and B cells. Therefore, TLR7 is an important molecule of the immune system. Based on these observations, TLR7 agonists considered to become a therapy weaponize the immune system against cancer. Radiation therapy (RT) is one of the standard cancer therapies and is reported to modulate the tumor immune response. In this study, we aimed to investigate the anti-tumor activity in combination of TLR7 agonist, DSP-0509, with RT and underlying mechanism. RESULT: We showed that anti-tumor activity is enhanced by combining RT with the TLR7 agonist DSP-0509 in the CT26, LM8, and 4T1 inoculated mice models. We found that once- weekly (q1w) dosing of DSP-0509 rather than biweekly (q2w) dosing is needed to achieve superior anti-tumor activities in CT26 model. Spleen cells from the mice in RT/DSP-0509 combination treatment group showed increased tumor lytic activity, inversely correlated with tumor volume, as measured by the chromium-release cytotoxicity assay. We also found the level of cytotoxic T lymphocytes (CTLs) increased in the spleens of completely cured mice. When the mice completely cured by combination therapy were re-challenged with CT26 cells, all mice rejected CT26 cells but accepted Renca cells. This rejection was not observed with CD8 depletion. Furthermore, levels of splenic effector memory CD8 T cells were increased in the combination therapy group. To explore the factors responsible for complete cure by combination therapy, we analyzed peripheral blood leukocytes (PBLs) mRNA from completely cured mice. We found that Havcr2low, Cd274low, Cd80high, and Il6low were a predictive signature for the complete response to combination therapy. An analysis of tumor-derived mRNA showed that combination of RT and DSP-0509 strongly increased the expression of anti-tumor effector molecules including Gzmb and Il12. CONCLUSION: These data suggest that TLR7 agonist, DSP-0509, can be a promising concomitant when used in combination with RT by upregulating CTLs activity and gene expression of effector molecules. This combination can be an expecting new radio-immunotherapeutic strategy in clinical trials.
Subject(s)
Toll-Like Receptor 7 , Animals , Toll-Like Receptor 7/agonists , Mice , Cell Line, Tumor , Female , Lymphocyte Activation/drug effects , Mice, Inbred BALB C , Membrane Glycoproteins/agonists , Combined Modality Therapy , Humans , Mice, Inbred C57BL , Neoplasms/therapy , Neoplasms/immunology , Neoplasms/drug therapy , Disease Models, Animal , T-Lymphocytes/immunology , T-Lymphocytes/drug effectsABSTRACT
Regulation of neuroinflammation is critical for maintaining central nervous system (CNS) homeostasis and holds therapeutic promise in autoimmune diseases such as multiple sclerosis (MS). Previous studies have highlighted the significance of selective innate signaling in triggering anti-inflammatory mechanisms, which play a protective role in an MS-like disease, experimental autoimmune encephalomyelitis (EAE). However, the individual intra-CNS administration of specific innate receptor ligands or agonists, such as for toll-like receptor 7 (TLR7) and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2), failed to elicit the desired anti-inflammatory response in EAE. In this study, we investigated the potential synergistic effect of targeting both TLR7 and NOD2 simultaneously to prevent EAE progression. Our findings demonstrate that simultaneous intrathecal administration of NOD2- and TLR7-agonists led to synergistic induction of Type I IFN (IFN I) and effectively suppressed EAE in an IFN I-dependent manner. Suppression of EAE was correlated with a significant decrease in the infiltration of monocytes, granulocytes, and natural killer cells, reduced demyelination, and downregulation of IL-1ß, CCL2, and IFNγ gene expression in the spinal cord. These results underscore the therapeutic promise of concurrently targeting the TLR7 and NOD2 pathways in alleviating neuroinflammation associated with MS, paving the way for novel and more efficacious treatment strategies.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Nod2 Signaling Adaptor Protein , Toll-Like Receptor 7 , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/agonists , Nod2 Signaling Adaptor Protein/metabolism , Nod2 Signaling Adaptor Protein/genetics , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Mice , Mice, Inbred C57BL , Immunity, Innate/drug effects , Female , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord/drug effects , Membrane Glycoproteins/metabolism , Interferon Type I/metabolism , Signal Transduction/drug effectsABSTRACT
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.
Subject(s)
Mice, Inbred C57BL , Nanoparticles , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Animals , Nanoparticles/chemistry , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Cancer Vaccines/immunology , Cancer Vaccines/administration & dosage , Mice , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Lipids/chemistry , Peptides/chemistry , Female , Papillomavirus E7 Proteins/immunology , Quinolines/pharmacology , Quinolines/chemistry , Imidazoles/chemistry , Imidazoles/pharmacologyABSTRACT
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.
Subject(s)
Imidazoles , Immunotherapy , Mannose , Mice, Inbred C57BL , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Tumor-Associated Macrophages , Animals , Imidazoles/administration & dosage , Imidazoles/chemistry , Toll-Like Receptor 8/agonists , Immunotherapy/methods , Toll-Like Receptor 7/agonists , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/drug effects , Mannose/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Tumor Microenvironment/drug effects , Dendritic Cells/immunology , Dendritic Cells/drug effects , Cell Line, Tumor , Mice , Neoplasms/therapy , Neoplasms/immunology , Neoplasms/drug therapy , Female , Humans , Membrane GlycoproteinsABSTRACT
Despite the groundbreaking impact of immune checkpoint blockade (ICB), response rates in non-small cell lung cancer remain modest, particularly in immune-excluded or immune-desert microenvironments. Toll-like receptor 7 (TLR7) emerges as a latent target bridging innate and adaptive immunity, offering a promising avenue for combination therapies to augment ICB efficacy. Here, we explored the anti-tumor activity of the novel oral TLR7 agonist TQ-A3334 and its potential to enhance anti-programmed death ligand 1 (PD-L1) therapy through a combination strategy in a syngeneic murine lung cancer model. Oral administration of TQ-A3334 significantly alleviated tumor burden in C57BL/6J mice, modulated by type I interferon (IFN), and exhibited low toxicity. This therapy elicited activation of both innate and adaptive immune cells in tumor tissue, particularly increasing the abundance of CD8+ TILs through type I IFN pathway and subsequent CXCL10 expression. In vitro examinations validated that IFN-α-stimulated tumor cells exhibited increased secretion of CXCL10, conducive to the promoted trafficking of CD8+ T cells. Furthermore, combining TQ-A3334 with anti-PD-L1 treatment exceeded tumor control, with a further increase in CD8+ TIL frequency compared to monotherapy. These findings suggest that TQ-A3334 can mobilize innate immunity and promote T cell recruitment into the tumor microenvironment; a combination of TQ-A3334 and anti-PD-L1 antibodies can intensify the sensitivity of tumors to anti-PD-L1 therapy, which demonstrates significant potential for treating poorly immune-infiltrated lung cancer.
Subject(s)
B7-H1 Antigen , Immune Checkpoint Inhibitors , Interferon Type I , Lung Neoplasms , Mice, Inbred C57BL , Toll-Like Receptor 7 , Toll-Like Receptor 7/agonists , Animals , Interferon Type I/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , B7-H1 Antigen/metabolism , B7-H1 Antigen/antagonists & inhibitors , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Mice , Humans , Cell Line, Tumor , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Administration, Oral , Drug Synergism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Membrane Glycoproteins/agonists , Membrane Glycoproteins/metabolism , Signal Transduction/drug effects , Female , Immunity, Innate/drug effects , Adaptive Immunity/drug effectsABSTRACT
Leishmaniasis, caused by Leishmania (L.) species, remains a neglected infection. Therapeutic vaccination presents a promising strategy for its treatment. In this study, we aimed to develop a therapeutic vaccine candidate using Leishmaniaantigens (SLA) and Toll-like receptor (TLR) 7/8 agonist (R848) encapsulated into the poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Moreover, TLR1/2 agonist (Pam3CSK4) was loaded onto the NPs. The therapeutic effects of these NPs were evaluated in L. major-infected BALB/c mice. Footpad swelling, parasite load, cellular and humoral immune responses, and nitric oxide (NO) production were analyzed. The results demonstrated that the PLGA NPs (SLA-R848-Pam3CSK4) therapeutic vaccine effectively stimulated Th1 cell responses, induced humoral responses, promoted NO production, and restricted parasite burden and lesion size.Our findings suggest that vaccination with SLA combined with TLR1/2 and TLR7/8 agonists in PLGA NPs as a therapeutic vaccine confers strong protection againstL. majorinfection. These results represent a novel particulate therapeutic vaccine against Old World cutaneous leishmaniasis.
Subject(s)
Antigens, Protozoan , Leishmaniasis Vaccines , Leishmaniasis, Cutaneous , Mice, Inbred BALB C , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Animals , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/drug therapy , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Antigens, Protozoan/immunology , Nanoparticles/chemistry , Leishmaniasis Vaccines/immunology , Mice , Female , Nitric Oxide/metabolism , Imidazoles/pharmacology , Imidazoles/chemistry , Th1 Cells/immunology , Leishmania major/immunology , Toll-Like Receptors/agonists , Humans , Toll-Like Receptor 7/agonists , Toll-Like Receptor AgonistsABSTRACT
Infections during pregnancy are associated with increased risk for adult neuropsychiatric disease, such as major depressive disorder, schizophrenia, and autism spectrum disorder. In mouse models of maternal immune activation (MIA), different toll-like receptors (TLRs) are stimulated to initiate inflammatory responses in mother and fetus. The goal of this study was to determine sex-dependent aspects of MIA using a TLR7/8 agonist, Resiquimod (RQ), on neurodevelopment. RQ was administered to timed-pregnant mice on embryonic day (E) 12.5. At E15, maternal/fetal plasma cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Maternal cytokines interleukin (IL)-6 and IL-10 were higher while tumor necrosis factor (TNF)-α and IL-17 were lower in pregnant dams exposed to RQ. Fetal cytokines (E15) were altered at the same timepoint with fetal plasma IL-6 and IL-17 greater after RQ compared to vehicle, while IL-10 and TNF-α were higher in male fetuses but not female. Other timed-pregnant dams were allowed to give birth. MIA with RQ did not alter the female to male ratio of offspring born per litter. Body weights were reduced significantly in both sexes at birth, and over the next 5 weeks. Offspring from RQ-injected mothers opened their eyes 5 days later than controls. Similarly, female offspring from RQ-injected mothers exhibited pubertal delay based on vaginal opening 2-3 days later than control females. On the behavioral side, juvenile and adult male and female MIA offspring exhibited less social-like behavior in a social interaction test. Anhedonia-like behavior was greater in MIA adult female mice. This study provides support for sex-dependent influences of fetal antecedents for altered brain development and behavioral outputs that could be indicative of increased susceptibility for adult disorders through immune mechanisms. Future studies are needed to determine neural cellular and molecular mechanisms for such programming effects.
Subject(s)
Prenatal Exposure Delayed Effects , Toll-Like Receptor 7 , Animals , Female , Pregnancy , Male , Mice , Prenatal Exposure Delayed Effects/immunology , Toll-Like Receptor 7/agonists , Behavior, Animal/drug effects , Imidazoles/pharmacology , Cytokines/blood , Mice, Inbred C57BLABSTRACT
Hepatitis B virus (HBV) infection, as a serious global public health issue, is closely related to the immune dysfunction. Herein, thirty-seven 1-(indolin-1-yl)-2-(thiazol-4-yl)ethan-1-one derivatives were prepared as potential immunomodulatory anti-HBV agents. Anti-HBV activity evaluation confirmed compound 11a could significantly suppress the HBV DNA replication in both wild and resistant HBV stains, with IC50 values of 0.13 µM and 0.36 µM, respectively. Preliminary action mechanism studies showed that 11a had an inhibitory effect on cellular HBsAg secretion and could effectively activate TLR7, thereby inducing the secretion of TLR7-regulated cytokines IL-12, TNF-α and IFN-α in human PBMC cells. SPR analysis confirmed that 11a could bind to TLR7 protein with an affinity of 7.06 µM. MD simulation predicted that 11a could form tight interactions with residues in the binding pocket of TLR7. Physicochemical parameters perdition and pharmacokinetic analysis indicated that 11a displayed relatively favorable drug-like properties. Considering all the results, compound 11a might be a promising lead for developing novel immunomodulatory anti-HBV agents.
Subject(s)
Antiviral Agents , Hepatitis B virus , Toll-Like Receptor 7 , Humans , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Hepatitis B virus/drug effects , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/agonists , Structure-Activity Relationship , Molecular Structure , Dose-Response Relationship, Drug , Indoles/chemistry , Indoles/pharmacology , Indoles/chemical synthesis , Thiazoles/chemistry , Thiazoles/pharmacology , Thiazoles/chemical synthesis , Virus Replication/drug effects , Microbial Sensitivity Tests , Animals , Hep G2 CellsABSTRACT
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.
Subject(s)
Nanoparticles , Peptides , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Vaccines, DNA , Vaccines, DNA/immunology , Vaccines, DNA/administration & dosage , Toll-Like Receptor 8/immunology , Toll-Like Receptor 8/agonists , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/immunology , Animals , Nanoparticles/chemistry , Peptides/chemistry , Peptides/immunology , Humans , Mice , Female , Polymers/chemistry , Plasmids/genetics , Plasmids/immunology , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Toll-Like Receptor 7 , Toll-Like Receptor 8 , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Humans , Immunotherapy , Neoplasms/drug therapy , Neoplasms/immunology , Immunity, Innate/drug effects , AnimalsABSTRACT
Toll-like receptor (TLR)-7 agonists are immunostimulatory vaccine adjuvants. A systematic structure-activity relationship (SAR) study of TLR7-active 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine led to the identification of a potent hTLR7-specific p-hydroxymethyl IMDQ 23 with an EC50 value of 0.22 µM. The SAR investigation also resulted in the identification of TLR7 selective carboxamide 12 with EC50 values of 0.32 µM for hTLR7 and 18.25 µM for hTLR8. In the vaccination study, TLR7-specific compound 23 alone or combined with alum (aluminum hydroxide wet gel) showed adjuvant activity for a spike protein immunogen in mice, with enhanced anti-spike antibody production. Interestingly, the adjuvant system comprising carboxamide 12 and alum showed prominent adjuvant activity with high levels of IgG1, IgG2b, and IgG2c in immunized mice, confirming a balanced Th1/Th2 response. In the absence of any apparent toxicity, the TLR7 selective agonists in combination with alum may make a suitable vaccine adjuvant.
Subject(s)
Adjuvants, Immunologic , Toll-Like Receptor 7 , Toll-Like Receptor 7/agonists , Structure-Activity Relationship , Animals , Humans , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/chemical synthesis , Mice , Female , Alum Compounds/pharmacology , Alum Compounds/chemistry , Mice, Inbred BALB C , Imidazoles/chemistry , Imidazoles/pharmacology , Imidazoles/chemical synthesisABSTRACT
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.
Subject(s)
Toll-Like Receptor 7 , Toll-Like Receptor 8 , Triple Negative Breast Neoplasms , Tumor Microenvironment , Animals , Triple Negative Breast Neoplasms/drug therapy , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Mice , Female , Humans , Cell Line, Tumor , Tumor Microenvironment/drug effects , Nanoparticles/chemistry , Transcription Factors/metabolism , Mice, Inbred BALB C , Cell Cycle Proteins/metabolism , Immunotherapy/methods , Epigenesis, Genetic/drug effects , Bromodomain Containing ProteinsABSTRACT
TLR7/8 agonists are versatile immune stimulators capable of treating various diseases such as viral infections, autoimmune, and cancer. Despite the structural similarity of TLR7/8, their immune stimulation mechanisms and time-course responses significantly differ. In this study, a new series of TLR7-selective agonists was synthesized utilizing the economical building block 2,6-dichloropurine. Compound 27b showed the most potent activity on hTLR7 with an EC50 of 17.53 nM and demonstrated high hTLR7 selectivity (224 folds against TLR8). 27b effectively stimulated the secretion of proinflammatory cytokines in mouse macrophages and enhanced intranasal vaccine efficacy against influenza A virus in vivo. Assessment of humoral and mucosal antibody titers confirmed that 27b elevates IgG and IgA levels, protecting against both homologous and heterologous influenza viral infections. These findings suggest that 27b is a promising candidate as a vaccine adjuvant to prevent viral infections or as a robust immunomodulator with prolonged activity for treating immune-suppressed diseases.
Subject(s)
Administration, Intranasal , Drug Design , Influenza Vaccines , Purines , Toll-Like Receptor 7 , Toll-Like Receptor 7/agonists , Animals , Mice , Humans , Influenza Vaccines/immunology , Influenza Vaccines/administration & dosage , Purines/pharmacology , Purines/chemistry , Adjuvants, Vaccine/pharmacology , Adjuvants, Vaccine/chemistry , Structure-Activity Relationship , Mice, Inbred BALB C , Female , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/immunology , Cytokines/metabolism , RAW 264.7 Cells , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/chemical synthesis , Adjuvants, Immunologic/chemistryABSTRACT
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.
Subject(s)
Adjuvants, Immunologic , COVID-19 Vaccines , COVID-19 , Natural Killer T-Cells , SARS-CoV-2 , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/immunology , SARS-CoV-2/immunology , Animals , Natural Killer T-Cells/immunology , COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , Toll-Like Receptor 8/agonists , Toll-Like Receptor 8/immunology , Humans , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/chemistry , Mice , COVID-19/prevention & control , COVID-19/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/immunology , Female , Adjuvants, Vaccine/chemistry , Adjuvants, Vaccine/pharmacology , Immunoglobulin G/immunologyABSTRACT
The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.
Subject(s)
Antiviral Agents , Enterovirus D, Human , Toll-Like Receptor 7 , Virus Replication , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/metabolism , Humans , Virus Replication/drug effects , Enterovirus D, Human/drug effects , Antiviral Agents/pharmacology , Indoles/pharmacology , Enterovirus Infections/virology , Immunity, Innate/drug effects , Cell Line , Virus Internalization/drug effects , PteridinesABSTRACT
Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.
Subject(s)
Fish Proteins , Ictaluridae , Imidazoles , Imiquimod , Immunity, Innate , Lysosomes , Toll-Like Receptor 7 , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/genetics , Imidazoles/pharmacology , Ictaluridae/immunology , Lysosomes/metabolism , Fish Proteins/genetics , Fish Proteins/metabolism , Signal Transduction , Humans , Aminoquinolines/pharmacology , Poly I-C/immunologyABSTRACT
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.
Subject(s)
Chemokine CCL2 , Chemokine CXCL10 , Imidazoles , Interleukin-8 , Toll-Like Receptor 7 , Transcription Factor RelA , Humans , Cell Line, Tumor , Chemokine CCL2/genetics , Chemokine CCL2/biosynthesis , Chemokine CXCL10/genetics , Chemokine CXCL10/biosynthesis , Imidazoles/pharmacology , Interleukin-8/genetics , Interleukin-8/biosynthesis , Neuroblastoma , Neurons/drug effects , Neurons/metabolism , NF-kappa B/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/genetics , Toll-Like Receptor 8/agonists , Toll-Like Receptor 8/genetics , Transcription Factor RelA/metabolism , Transcription Factor RelA/geneticsABSTRACT
Pattern recognition receptors (PRRs) play a critical role in the innate immune response, and toll-like receptor 7 (TLR7) is an important member of PRRs. Although several TLR7 agonists are available, most of them are being tested clinically, with only one available on the market. Thus, it is imperative to develop new TLR7 agonists. In this study, we designed and synthesized three kinds of quinazoline derivatives and five kinds of pyrrolo[3,2-d]pyrimidine derivatives targeting TLR7. The antiviral efficacy of these compounds was evaluated in vitro and in vivo. Our findings indicated that four kinds of compounds showed exceptional antiviral activity. Furthermore, molecular docking studies confirmed that compound 11 successfully positioned itself in the pocket of the TLR7 guanosine loading site with a binding energy of -4.45 kcal mol-1. These results suggested that these compounds might be potential antiviral agents.