-
Science Advances Apr 2023Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7...
Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7 (TLR7) and/or TLR8 on innate immune cells. Nevertheless, intravenous administration of IMDs causes severe immune-related toxicities, and attempts to improve their tissue-selective exposure while minimizing acute systemic inflammation have proven difficult. Here, using a library of R848 "bottlebrush prodrugs" (BPDs) that differ only by their R848 release kinetics, we explore how the timing of R848 exposure affects immune stimulation in vitro and in vivo. These studies led to the discovery of R848-BPDs that exhibit optimal activation kinetics to achieve potent stimulation of myeloid cells in tumors and substantial reductions in tumor growth following systemic administration in mouse syngeneic tumor models without any observable systemic toxicity. These results suggest that release kinetics can be tuned at the molecular level to provide safe yet effective systemically administered immunostimulant prodrugs for next-generation cancer immunotherapies.
Topics: Mice; Animals; Prodrugs; Toll-Like Receptor 7; Kinetics; Adjuvants, Immunologic; Neoplasms
PubMed: 37075115
DOI: 10.1126/sciadv.adg2239 -
PloS One 2021Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance...
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.
Topics: Animals; Chemokines; Cytokines; Dendritic Cells; Imidazoles; Interferon-alpha; Lupus Erythematosus, Systemic; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred NZB; Mice, Knockout; Survival Rate; Terpenes; Toll-Like Receptor 7; Toll-Like Receptor 9
PubMed: 33444326
DOI: 10.1371/journal.pone.0244439 -
Journal of Controlled Release :... Feb 2021Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly...
Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8 T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28 days (non-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 μg for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.
Topics: Animals; Hyperthermia, Induced; Imidazoles; Immunotherapy; Liposomes; Mice; Neoplasms
PubMed: 33189786
DOI: 10.1016/j.jconrel.2020.11.013 -
Inflammation & Allergy Drug Targets Dec 2012Asthma is an inflammatory disorder of the airways frequently characterized by an excessive Th2 adaptive immune response. Activation of Toll-like receptor (TLR)-7, a... (Review)
Review
Asthma is an inflammatory disorder of the airways frequently characterized by an excessive Th2 adaptive immune response. Activation of Toll-like receptor (TLR)-7, a single-stranded viral RNA receptor that is highly expressed in the airways, triggers a rapid innate immune response and favors a subsequent Th1 response. Because of this role in pulmonary immunoregulation, TLR7 has gained considerable interest as a therapeutic target in asthma. Synthetic TLR7 ligands, including the imidazoquinolines imiquimod (R837) and resiquimod (R848), and 8-hydroxyadenine derivatives have been developed for other clinical indications. TLR7 activation prevents ovalbumin-induced airway hyperreactivity, eosinophilic inflammation, goblet cell hyperplasia and airway remodeling in murine models of asthma. TLR7 activation also inhibits viral replication in the lung and prevents virus-induced airway hyperreactivity. Furthermore, it has recently been shown that stimulating TLR7 rapidly relaxes airway smooth muscle, dilating the airways. This bronchodilating effect, which occurs in seconds to minutes and depends on rapid production of nitric oxide, indicates that TLR7 can signal via previously unrecognized pathways. The effects of decreasing the allergic Th2 response, acting as an immediate bronchodilator, and promoting an antiviral immune environment, make TLR7 an attractive drug target. We examine the current understanding of TLR7 as a therapeutic target and its translation to asthma treatment in humans.
Topics: Animals; Anti-Asthmatic Agents; Asthma; Bronchodilator Agents; Disease Models, Animal; Drug Design; Humans; Mice; Molecular Targeted Therapy; Nitric Oxide; Th2 Cells; Toll-Like Receptor 7
PubMed: 23078048
DOI: 10.2174/187152812803589967 -
Frontiers in Immunology 2022Impairment of the innate immune function may contribute to the increased risk of bacterial and viral infections in people with HIV (PWH). In this study we aimed to...
INTRODUCTION
Impairment of the innate immune function may contribute to the increased risk of bacterial and viral infections in people with HIV (PWH). In this study we aimed to investigate the induced innate immune responses in PWH prior to and after initiation of combinational antiretroviral therapy (cART). Furthermore, we aimed to investigate if the induced innate immune responses before initiation of cART were associated with CD4+ T-cell recovery one year after initiating cART.
MATERIAL AND METHOD
The induced innate immune response was assessed by the TruCulture whole blood technique in 32 PWH before cART initiation and after 1, 6 and 12 months. To mimic bacterial and viral infections we used a panel of three stimuli (lipopolysaccharide (LPS), resiquimod (R848), and polyinosinic:polycytidylic acid (Poly I:C)) to stimulate the extracellular Toll-like receptor (TLR) 4 and the intracellular TLR7/8 and TLR3, respectively. The following cytokine responses were analyzed by Luminex 200: Tumor Necrosis Factor (TNF)-α, Interleukin (IL)-1b, IL-6, IL-8, IL-10, IL-12p40, IL17A, Interferon (IFN)-α, and IFN-γ.
RESULTS
At baseline PWH with nadir CD4+ T-cell count <350 cell/µL had lower levels of LPS-, R848-, and Poly I:C-induced IL-6 and IFN-γ, LPS- and R848-induced TNF-α and IL-12, LPS induced IL-1b, and R848-induced IL-10 than PWH with nadir CD4+ T-cell count >350 cells/µL. The majority (>50%) had induced cytokine concentrations below the reference intervals at baseline which was most pronounced for the LPS- and Poly I:C-induced responses. The induced responses in the whole population improved after 12 months of cART, and more PWH had induced cytokine concentrations within the reference intervals after 12 months. However, the majority of PWH still had LPS-induced INF-α, INF-γ and Poly I:C-induced TNF-α and IL-6 below the reference interval. The induced innate immune responses before cART initiation were not associated with the CD4+ T-cell recovery after 12 months of cART.
CONCLUSION
The innate immune response was impaired in PWH, with a more pronounced impairment in PWH with low nadir CD4+ T-cell count. Initiation of cART improved the innate immune response, but compared to the reference intervals, some impairment remained in PWH without viral replication.
Topics: Anti-HIV Agents; Antiretroviral Therapy, Highly Active; Cytokines; HIV Infections; Humans; Immunity, Innate; Interferon-alpha; Interleukin-10; Interleukin-6; Lipopolysaccharides; Poly I-C; Tumor Necrosis Factor-alpha; Virus Diseases
PubMed: 36059528
DOI: 10.3389/fimmu.2022.974767 -
International Journal of Nanomedicine 2021With the advance of screening techniques, there is a growing number of low-risk or intermediate-risk prostate cancer (PCa) cases, remaining a serious threat to men's...
PURPOSE
With the advance of screening techniques, there is a growing number of low-risk or intermediate-risk prostate cancer (PCa) cases, remaining a serious threat to men's health. To obtain better efficacy, a growing interest has been attracted to develop such emerging treatments as immunotherapy and focal therapy. However, few studies offer guidance on whether and how to combine these modalities against PCa. This study was designed to develop dual-functional nanoparticles (NPs) which combined photothermal therapy (PTT) with immunotherapy and determine the anti-tumor efficacy for PCa treatment.
METHODS
By a double emulsion technique, the drug nanocarrier, poly(lactic-co-glycolic acid) or PLGA, was applied for co-loading of a fluorescent dye, indocyanine green (ICG) and a toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) to synthesize PLGA-ICG-R848 NPs. Next, we determined their characteristic features and evaluated whether they inhibited the cell viability in multiple PCa cell lines. After treatment with PLGA-ICG-R848, the maturation markers of bone marrow-derived dendritic cells (BMDCs) were detected by flow cytometry. By establishing a subcutaneous xenograft model of mouse PCa, we explored both the anti-tumor effect and immune response following the NPs-based laser ablation.
RESULTS
With a mean diameter of 157.7 nm, PLGA-ICG-R848 exhibited no cytotoxic effect in PCa cells, but they significantly decreased RM9 cell viability to (3.9±1.0)% after laser irradiation. Moreover, PLGA-ICG-R848 promoted BMDCs maturation with the significantly elevated proportions of CD11c+CD86+ and CD11c+CD80+ cells. Following PLGA-ICG-R848-based laser ablation in vivo, the decreased bioluminescent signals indicated a significant inhibition of PCa growth, while the ratio of splenic natural killer (NK) cells in PLGA-ICG-R848 was (3.96±1.88)% compared with (0.99±0.10)% in PBS group, revealing the enhanced immune response against PCa.
CONCLUSION
The dual-functional PLGA-ICG-R848 NPs under laser irradiation exhibit the anti-tumor efficacy for PCa treatment by combining PTT with immunotherapy.
Topics: Animals; Antineoplastic Agents; Bone Marrow Cells; Cell Line, Tumor; Cell Survival; Dendritic Cells; Endocytosis; Humans; Imidazoles; Indocyanine Green; Lasers; Male; Mice, Inbred C57BL; Nanoparticles; Polylactic Acid-Polyglycolic Acid Copolymer; Prostatic Neoplasms; Mice
PubMed: 33880023
DOI: 10.2147/IJN.S301552 -
Clinical Cancer Research : An Official... Oct 2022DNMT3A mutations confer a poor prognosis in acute myeloid leukemia (AML), but the molecular mechanisms downstream of DNMT3A mutations in disease pathogenesis are not...
PURPOSE
DNMT3A mutations confer a poor prognosis in acute myeloid leukemia (AML), but the molecular mechanisms downstream of DNMT3A mutations in disease pathogenesis are not completely understood, limiting targeted therapeutic options. The role of miRNA in DNMT3A-mutant AML pathogenesis is understudied.
EXPERIMENTAL DESIGN
DNA methylation and miRNA expression was evaluated in human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. The treatment efficacy and molecular mechanisms of TLR7/8-directed therapies on DNMT3A-mutant AML were evaluated in vitro on human AML patient samples and in Dnmt3a/Flt3-mutant AML mice.
RESULTS
miR-196b is hypomethylated and overexpressed in DNMT3A-mutant AML and is associated with poor patient outcome. miR-196b overexpression in DNMT3A-mutant AML is important to maintain an immature state and leukemic cell survival through repression of TLR signaling. The TLR7/8 agonist resiquimod induces dendritic cell-like differentiation with costimulatory molecule expression in DNMT3A-mutant AML cells and provides a survival benefit to Dnmt3a/Flt3-mutant AML mice. The small molecule bryostatin-1 augments resiquimod-mediated AML growth inhibition and differentiation.
CONCLUSIONS
DNMT3A loss-of-function mutations cause miRNA locus-specific hypomethylation and overexpression important for mutant DNMT3A-mediated pathogenesis and clinical outcomes. Specifically, the overexpression of miR-196b in DNMT3A-mutant AML creates a novel therapeutic vulnerability by controlling sensitivity to TLR7/8-directed therapies.
Topics: Animals; Bryostatins; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3A; DNA Modification Methylases; Humans; Immunity, Innate; Leukemia, Myeloid, Acute; Mice; MicroRNAs; Mutation; Toll-Like Receptor 7
PubMed: 35943291
DOI: 10.1158/1078-0432.CCR-22-1598 -
Advanced Healthcare Materials May 2021Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due...
Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8 T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8 T-cells across multiple tumors suggest the potential for treating metastatic cancer.
Topics: Animals; Breast Neoplasms; CD8-Positive T-Lymphocytes; Humans; Imidazoles; Immunity; Immunotherapy; Mice; Micelles; Nanoparticles; Polymers
PubMed: 33646600
DOI: 10.1002/adhm.202100008 -
Cancers Apr 2023Triple-negative breast carcinoma (TNBC) is one of the most aggressive types of solid-organ cancers. While immune checkpoint blockade (ICB) therapy has significantly...
Triple-negative breast carcinoma (TNBC) is one of the most aggressive types of solid-organ cancers. While immune checkpoint blockade (ICB) therapy has significantly improved outcomes in certain types of solid-organ cancers, patients with immunologically cold TNBC are afforded only a modest gain in survival by the addition of ICB to systemic chemotherapy. Thus, it is urgently needed to develop novel effective therapeutic approaches for TNBC. Utilizing the 4T1 murine model of TNBC, we developed a novel combination immunotherapeutic regimen consisting of intratumoral delivery of high-mobility group nucleosome binding protein 1 (HMGN1), TLR2/6 ligand fibroblast-stimulating lipopeptide (FSL-1), TLR7/8 agonist (R848/resiquimod), and CTLA-4 blockade. We also investigated the effect of adding SX682, a small-molecule inhibitor of CXCR1/2 known to reduce MDSC trafficking to tumor microenvironment, to our therapeutic approach. 4T1-bearing mice responded with significant tumor regression and tumor elimination to our therapeutic combination regimen. Mice with complete tumor regressions did not recur and became long-term survivors. Treatment with HMGN1, FSL-1, R848, and anti-CTLA4 antibody increased the number of infiltrating CD4+ and CD8+ effector/memory T cells in both tumors and draining lymph nodes and triggered the generation of 4T1-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes. Thus, we developed a potentially curative immunotherapeutic regimen consisting of HMGN1, FSL-1, R848, plus a checkpoint inhibitor for TNBC, which does not rely on the administration of chemotherapy, radiation, or exogenous tumor-associated antigen(s).
PubMed: 37190294
DOI: 10.3390/cancers15082366 -
BMC Veterinary Research Jan 2014Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod...
BACKGROUND
Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod (R848) is an imidazoquinoline compound with potent antiviral activity and functions through the TLR7/TLR8 MyD88-dependent signaling pathway. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of double-stranded RNA that induces the production of pro-inflammatory cytokines by the activation of NF-κB through TLR3. This study investigated the potential of R848 and poly(I:C) as an adjuvant 146S foot-and-mouth disease virus (FMDV) vaccine formulated with aluminum hydroxide (Al(OH)₃).
RESULTS
Antibody titers to FMDV and CD8+ T cells were markedly enhanced in mice immunized to 146S FMDV + Al(OH)₃ + R848 + poly(I:C) compared with mice immunized to FMDV + ISA206. IFN-γ secretion substantially increased compared with IL-4 secretion by splenic T cells stimulated with FMDV antigens in vitro, suggesting that R848, poly(I:C), and with Al(OH)₃ together biased the immune response toward a Th1-type direction.
CONCLUSIONS
These results indicated that the R848 and poly(I:C) together with Al(OH)₃ enhanced humoral and cellular immune responses to immunization with 146S FMDV antigens. Thus, this new vaccine formulation can be used for FMDV prevention.
Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Antibodies, Viral; Antibody Specificity; Female; Foot-and-Mouth Disease; Imidazoles; Mice; Mice, Inbred BALB C; Poly I-C; Spleen; T-Lymphocyte Subsets; Viral Vaccines
PubMed: 24386990
DOI: 10.1186/1746-6148-10-2